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Nutritional and Management Strategies to Enhance the Reproductive Performance of the Cowherd

Permanent Link: http://ufdc.ufl.edu/UFE0022797/00001

Material Information

Title: Nutritional and Management Strategies to Enhance the Reproductive Performance of the Cowherd
Physical Description: 1 online resource (155 p.)
Language: english
Creator: Cooke, Reinaldo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: beef, management, nutrition, performance, reproduction
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Four experiments were conducted to evaluate nutritional and management strategies to enhance the reproductive performance of forage-fed Brahman-crossbred females. Experiment 1 compared performance and physiological responses of replacement heifers consuming an energy-based supplement daily or 3 times per week at similar weekly amounts. Daily supplementation resulted in a normalized mRNA expression pattern of genes associated with nutritional metabolism and growth, reduced daily variation in plasma concentrations of urea nitrogen, glucose, and insulin, and increased hepatic mRNA expression and plasma concentrations of IGF-I. These beneficial physiological effects of daily supplementation were translated into the greater BW gain and hastened attainment of puberty and pregnancy detected in daily-fed heifers compared to heifers supplemented thrice weekly. Experiment 2 evaluated physiological responses of brood cows offered supplements similar to those of Experiment 1. Daily supplementation resulted in a normalized pattern of gluconeogenic enzyme mRNA expression, reduced variation in plasma concentrations of glucose and insulin, and improved cow nutritional status as observed by increasing plasma IGF-I concentrations. Experiment 3 evaluated the effects of acclimation to handling procedures on growth, temperament, physiological responses, and reproductive performance of replacement heifers. Acclimation reduced growth rates because of the additional exercise that heifers were exposed to, but reduced plasma concentrations of cortisol and hastened onset of puberty. Experiment 4 evaluated the effects of acclimation to human interaction on temperament, physiological responses, and pregnancy rates of brood cows. Acclimation did not influence temperament and concentrations of plasma cortisol and acute phase proteins. Nevertheless, reproductive performance of cows appeared to be influenced by acclimation, although additional evaluations are still required. Further, measurements associated with cow temperament, acute phase response, and energy status influenced the probability of cows to become pregnant during the breeding season. In summary, daily supplementation of energy-based supplements enhances performance, reproduction, and physiological responses of forage-fed Brahman-crossbred females. Acclimation to human handling may be an additional alternative to enhance performance of Brahman-crossbred females because strategies that improve cattle disposition and reduce the physiological responses to handling stress appear to be beneficial to the reproductive function of replacement heifers and brood cows.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Reinaldo Cooke.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Arthington, John D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022797:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022797/00001

Material Information

Title: Nutritional and Management Strategies to Enhance the Reproductive Performance of the Cowherd
Physical Description: 1 online resource (155 p.)
Language: english
Creator: Cooke, Reinaldo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: beef, management, nutrition, performance, reproduction
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Four experiments were conducted to evaluate nutritional and management strategies to enhance the reproductive performance of forage-fed Brahman-crossbred females. Experiment 1 compared performance and physiological responses of replacement heifers consuming an energy-based supplement daily or 3 times per week at similar weekly amounts. Daily supplementation resulted in a normalized mRNA expression pattern of genes associated with nutritional metabolism and growth, reduced daily variation in plasma concentrations of urea nitrogen, glucose, and insulin, and increased hepatic mRNA expression and plasma concentrations of IGF-I. These beneficial physiological effects of daily supplementation were translated into the greater BW gain and hastened attainment of puberty and pregnancy detected in daily-fed heifers compared to heifers supplemented thrice weekly. Experiment 2 evaluated physiological responses of brood cows offered supplements similar to those of Experiment 1. Daily supplementation resulted in a normalized pattern of gluconeogenic enzyme mRNA expression, reduced variation in plasma concentrations of glucose and insulin, and improved cow nutritional status as observed by increasing plasma IGF-I concentrations. Experiment 3 evaluated the effects of acclimation to handling procedures on growth, temperament, physiological responses, and reproductive performance of replacement heifers. Acclimation reduced growth rates because of the additional exercise that heifers were exposed to, but reduced plasma concentrations of cortisol and hastened onset of puberty. Experiment 4 evaluated the effects of acclimation to human interaction on temperament, physiological responses, and pregnancy rates of brood cows. Acclimation did not influence temperament and concentrations of plasma cortisol and acute phase proteins. Nevertheless, reproductive performance of cows appeared to be influenced by acclimation, although additional evaluations are still required. Further, measurements associated with cow temperament, acute phase response, and energy status influenced the probability of cows to become pregnant during the breeding season. In summary, daily supplementation of energy-based supplements enhances performance, reproduction, and physiological responses of forage-fed Brahman-crossbred females. Acclimation to human handling may be an additional alternative to enhance performance of Brahman-crossbred females because strategies that improve cattle disposition and reduce the physiological responses to handling stress appear to be beneficial to the reproductive function of replacement heifers and brood cows.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Reinaldo Cooke.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Arthington, John D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022797:00001


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NUTRITIONAL AND MANAGEMENT STRATEGIES TO ENHANCE THE REPRODUCTIVE PERFORMANCE OF THE COWHERD By REINALDO FERNANDES COOKE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Reinaldo Fernandes Cooke 2

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To my wife Flavia, and also to my entire family. 3

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ACKNOWLEDGMENTS Firstly, I would like to thank my wife, Flavia Cooke, and my family for all their love, support and patience. I also would like to thank Dr. John Arthington for the opportunity, guidance, and excellent mentorship. I would lik e to thank the members of my committee (Dr. Cliff Lamb, Dr. Matt Hersom, Dr. Joel Yelich, Dr. Ramon Littell, and Dr. Peter Hansen) for their support with my research and classwork. Other faculty members not involved in my committee (such as Dr. Jos Eduardo Portela Santos, Dr. Wi lliam Thatcher, Dr. Charles Staples, Dr. Joo Vendramini, and Dr. Richard Miles) were fundame ntal for the accomplishment of my goals. Last but not least, I would like to thank Dr. Alan Ea ly for all the help and guidance during the period that I spent working in his lab. I would like to thank all the staff who assisted me with my research projects, with special thanks to Mr. Austin Bateman, Ms. Idania Alvarez, Mrs. Joyce Hayen, and Mrs. Andrea Dunlap. My former advisor from Brazil, Dr. Jos Luiz Mo raes Vasconcelos, is also very important to my academic and research career, since he is the one that introduced me to the scientific world. Special thanks are extended to my fellow graduate students and friends, particularly Brad Austin, Dr. Jeremy Block, and Davi Araujo for all thei r friendship and collaboration during my research program. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................8LIST OF FIGURES .......................................................................................................................10ABSTRACT ...................................................................................................................... .............11CHAPTER 1 INTRODUCTION ................................................................................................................ ..132 LITERATURE REVIEW .......................................................................................................14Summary of the Endocrine Control of Reproduction in Cattle ..............................................14Onset of Puberty in Heifers .............................................................................................15Reproductive Efficiency of Mature Cows .......................................................................17Supplementation Programs for Gr azing Cow-Calf Operations ..............................................19Frequency of Supplementation ........................................................................................21Energy Metabolism and Reproduction ............................................................................24Glucose ....................................................................................................................... .....25Insulin ....................................................................................................................... .......28Insulin-like Growth Factor I ............................................................................................30Progesterone .................................................................................................................. ..35Temperament, Acclimation, and Reproduction of Cattle .......................................................36Assessment of Temperament in Beef Cattle ...................................................................36Stress and Excitable Temperament .................................................................................39The Hypothalamic-Pituitary-Adrenal axis ......................................................................39Physiologic Responses to Excitable Temperament in Cattle ..........................................42Temperament and Performance of Beef Cattle ...............................................................46Temperament and Reproduction of Beef Cattle ..............................................................48Cattle Acclimation ...........................................................................................................51Novel Strategies to Enhan ce the Reproductive Efficiency of Cow-Calf Operations .............523 EFFECTS OF SUPPLEMENTATION FREQUENCY ON PERFORMANCE, REPRODUCTIVE, AND METABOLIC RE SPONSES OF BRAHMAN-CROSSBRED FEMALES ....................................................................................................................... .......54Introduction .................................................................................................................. ...........54Materials and Methods ...........................................................................................................55Animals ....................................................................................................................... .....55Experiment 1 ............................................................................................................55Experiment 2 ............................................................................................................56 5

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Diets ......................................................................................................................... ........56Experiment 1 ............................................................................................................56Experiment 2 ............................................................................................................57Sampling ...................................................................................................................... ....57Experiment 1 ............................................................................................................57Experiment 2 ............................................................................................................58Blood Analysis ................................................................................................................59Tissue Analysis ................................................................................................................60Tissue Collection and RNA Extraction ....................................................................60Real-time RT-PCR ...................................................................................................60Statistical Analysis .......................................................................................................... 61Experiment 1 ............................................................................................................61Experiment 2 ............................................................................................................62Results and Discussion ........................................................................................................ ...63Experiment 1 .................................................................................................................. .63Experiment 2 .................................................................................................................. .704 EFFECTS OF ACCLIMATION TO HANDLING ON PERFORMANCE, REPRODUCTIVE, AND PHYSIOL OGICAL RESPONSES OF BRAHMANCROSSBRED HEIFERS ........................................................................................................85Introduction .................................................................................................................. ...........85Materials and Methods ...........................................................................................................86Animals ....................................................................................................................... .....86Diets ......................................................................................................................... ........87Acclimation Procedure ....................................................................................................88Sampling ...................................................................................................................... ....88Blood Analysis ................................................................................................................90Statistical Analysis .......................................................................................................... 91Results and Discussion ........................................................................................................ ...925 EFFECTS OF TEMPERAMENT AND ACCLIMATION ON PERFORMANCE, PHYSIOLOGICAL RESPONSES, AND PREGNANCY RATES OF BRAHMANCROSSBRED COWS ..........................................................................................................108Introduction .................................................................................................................. .........108Materials and Methods .........................................................................................................109Animals and Diets .........................................................................................................109Acclimation Procedure ..................................................................................................110Sampling ...................................................................................................................... ..111Breeding Season ............................................................................................................111Blood Analysis ..............................................................................................................112Statistical Analysis ........................................................................................................11 3Results and Discussion ........................................................................................................ .114 6

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LIST OF REFERENCES .............................................................................................................130BIOGRAPHICAL SKETCH .......................................................................................................155 7

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LIST OF TABLES Table Page 3-1 Ingredient composition and nutrient profile of the supplem ent offered in Exp. 1 and 2 ...743-2 Primer sets used for qua ntitative real-time RT-PCR .........................................................753-3 Blood urea nitrogen and plasma glucose c oncentrations of heifers offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 18.2 kg of DM per heifer ...................................................................................................763-4 Plasma concentrations of insulin and IG F-I of heifers offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 18.2 kg of DM per heifer .....................................................................................................................773-5 Expression of hepatic genes associated with nutritional metabolism and growth of heifers offered an energy supplement base d on fibrous byproducts daily or 3 times weekly, at a weekly rate of 18.2 kg of DM per heifer .......................................................783-6 Blood urea nitrogen and plasma insulin c oncentrations of cows offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 20.3 kg of DM per cow ......................................................................................................793-7 Expression of hepatic genes associated with nutritional metabolism and status of cows offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 20.3 kg of DM per cow ..........................................................803-8 Plasma IGF-I concentrations of cows offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 20.3 kg of DM per cow .............814-1 Average daily gain, pregnancy rates, a nd concentrations of plasma ceruloplasmin, haptoglobin, and IGF-I of heifers exposed or not to handling acclimation procedures ....994-2 Pearson correlation coefficients among ADG, plasma measurements, and temperament of heifers ....................................................................................................1004-3 Temperament measurements of heifers exposed or not to handling acclimation procedures .................................................................................................................... ....1014-4 Pearson correlation coefficients among m easurements of temperament and plasma cortisol concentra tions of heifers .....................................................................................1024-5 Effects of breed on performance, temperament, and physiologic parameters of replacement heifers ..........................................................................................................1035-1 Mean BW and BCS of Brahman-crossbre d cows exposed or not to acclimation procedures .................................................................................................................... ....121 8

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5-2 Body weight change, temperament measur ements, and plasma concentrations of cortisol, haptoglobin, and IGF-I of Brahman-crossbred cows exposed or not to acclimation procedures ....................................................................................................1225-3 Body weight, BCS, and plasma ceruloplasmi n concentrations of Brahman-crossbred cows exposed or not to acclimation procedures ..............................................................1235-4 Pearson correlation coefficients am ong measurements of temperament and concentrations of plasma cortisol, ce ruloplasmin and haptoglobin of Brahmancrossbred cows during yr 1 of the study ..........................................................................1245-5 Pearson correlation coefficients am ong measurements of temperament and concentrations of plasma cortisol, ce ruloplasmin and haptoglobin of Brahmancrossbred cows during yr 2 of the study ..........................................................................125 9

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LIST OF FIGURES Figure Page 3-1 Proportion of prepubertal heifers by week during Exp. 1 ..................................................82 3-2 Proportion of non-pregnant heifers by week during the 60-d breeding season of Exp. 1..........................................................................................................................................83 3-3 Expression of muscle myostatin, as rela tive fold change, of heifers in Exp. 1 .................84 4-1 Body weight of heifers exposed or no t to handling acclimation procedures ...................104 4-2 Puberty attainment of heifer s exposed or not to handling acclimation procedures .........105 4-3 Plasma cortisol concentrations of heifer s exposed or not to handling acclimation procedures .................................................................................................................... ....106 4-4 Plasma progesterone concentra tions of prepubertal heifers exposed or not to handling acclimation procedures ....................................................................................................107 5-1 Pregnancy rates during the breeding seas on of Brahman x British and Braford cows exposed or not to acclimation procedures ........................................................................126 5-2 Effects of BCS and plasma IGF-I con centrations on the probability of Brahmancrossbred cows to become pregnant ................................................................................127 5-3 Effects of temperament score and plasma cortisol concentrations on the probability of Brahman British and Brafor d cows to become pregnant. ........................................128 5-4 Effects of plasma ceruloplasmin and hapt oglobin concentrations on the probability of Brahman-crossbred cows to become pregnant. ...............................................................129 10

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NUTRITIONAL AND MANAGEMENT STRATEGIES TO ENHANCE THE REPRODUCTIVE PERFORMANCE OF THE COWHERD By Reinaldo Fernandes Cooke December 2008 Chair: John D. Arthington Major: Animal Sciences Four experiments were conducted to evaluate nutritional and manage ment strategies to enhance the reproductive performanc e of forage-fed Brahman-crossbred females. Experiment 1 compared performance and physiological respon ses of replacement heifers consuming an energy-based supplement daily or 3 times per week at similar weekly amounts. Daily supplementation resulted in a normalized mRNA e xpression pattern of genes associated with nutritional metabolism and growth, reduced daily variation in pl asma concentrations of urea nitrogen, glucose, and insu lin, and increased hepatic mRNA expression and plasma concentrations of IGF-I. These beneficial phys iological effects of da ily supplementation were translated into the greater BW gain and hasten ed attainment of puberty and pregnancy detected in daily-fed heifers compared to he ifers supplemented thrice weekly. Experiment 2 evaluated physiological res ponses of brood cows offered supplements similar to those of Experiment 1. Daily supplem entation resulted in a normalized pattern of gluconeogenic enzyme mRNA expression, reduced vari ation in plasma concentrations of glucose and insulin, and improved cow nutritional status as observed by increasing plasma IGF-I concentrations. 11

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12 Experiment 3 evaluated the effects of accl imation to handling procedures on growth, temperament, physiological responses, and reproductive performance of replacement heifers. Acclimation reduced growth rates because of the additional exercise that heifers were exposed to, but reduced plasma concentrations of cortisol and hastened onset of puberty. Experiment 4 evaluated the effects of acclim ation to human interaction on temperament, physiological responses, and pregnancy rates of brood cows. Acclimation did not influence temperament and concentrations of plasma cor tisol and acute phase proteins. Nevertheless, reproductive performance of cows appeared to be influenced by acclimation, although additional evaluations are still required. Fu rther, measurements associated with cow temperament, acute phase response, and energy status influenced the probability of cows to become pregnant during the breeding season. In summary, daily supplementation of ener gy-based supplements enhances performance, reproduction, and physiological responses of forage-fed Br ahman-crossbred females. Acclimation to human handling may be an additional alternative to enhance performance of Brahman-crossbred females because strategies that improve cattle disposition and reduce the physiological responses to handling stress appear to be beneficial to the re productive function of replacement heifers and brood cows.

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CHAPTER 1 INTRODUCTION The beef cattle industry in Florida is main ly constituted by grazing cow-calf operations. According to the USDA census (USDA, National Agricultural Statistics Service, 2002), Florida contains approximately 983,000 beef cows, ranking 11th nationally, and leads the country in number of operations with more than 2,500 cows. The two most important fa ctors that affect the profitability of cow-calf operations are reproduction and nutrition (Hess et al, 2005). Reproduction efficiency of the herd is optimal when replacement heifers attain puberty as yearlings and calve at 2 yr of age (Bagley, 1993) and brood cows are able to become pregnant early during the annual breeding s eason (Rae, 2006). Nutrition is th e environmental factor that most influences reproductive efficiency of cattl e (Bagley, 1993; Diskin et al., 2003); therefore the cowherd should be always maintained at adequate planes of nourishment. In southern Florida, where the majority of the states beef he rd resides, cattle have a high Brahman influence, and beef females containing Brah man-breeding typically reach pubert y at older ages (Martin et al., 1992) and exhibit excitable temperament (Hear nshaw and Morris, 1984; Fordyce et al., 1988; Voisinet et al., 1997), which may further comp romise their performance and reproductive function (Plasse et al., 1970). A dditionally, the forages grown in the state usually do not have adequate nutrient density to meet the require ments of developing heif ers and brood cows (Moore et. al., 1991). Consequently, management strate gies to offset the genetic and nutritional disadvantages encountered in typical Florida co w-calf operations are required to optimize the reproductive efficiency of the cowherd. 13

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CHAPTER 2 LITERATURE REVIEW Summary of the Endocrine Control of Reproduction in Cattle The mechanisms associated with acquisiti on, resumption, and mainte nance of reproductive ability in cattle are mediated by the hypothalamic-pituitary-ovaria n axis (Hess et al., 2005). The hypothalamus synthesizes and releases GnRH via ne urosecretory neurons to the pituitary portal blood system, which transports GnRH to the anterior pituitary gland, resulting in synthesis and secretion of gonadotropins (LH and FSH) to the circulation (Anderson et al., 1981; Hess et al., 2005). In the ovaries, recruitment and growth of follicles are stimulated by FSH until follicle deviation is reached (Ginther et al., 1996). Following deviation, the maturation and consequent ovulation of the dominant follicle are mediated by LH (Ginther et al., 2001; Yavas et al., 2000). Estradiol is mainly produced by the ovarian foll icles, and regulates gonadotropin synthesis and release both positively and negatively (Kesner et al., 1981). Estradiol has been shown to feedback negatively onto the hypothalamus and s uppress GnRH synthesis prior to puberty and during late gestation in cattle (Day et al ., 1984; Yavas and Walton, 2000). Conversely, synthesis of estradiol increases significantly as follicles grow, influencing follicle deviation (Ginther et al., 2000) and eventually reaching a threshold that s timulates GnRH synthesis, increases the number of GnRH receptors in the pitu itary gland (Gregg et al., 1990; He ss et al., 2005), and induces the LH surge required for ovulation (Kesner et al., 1981). Progesterone (P4), produced by the corpus luteum formed following ovulation, suppresses GnRH release and consequent gonadotropin synthesis in the brain; therefore the corpus luteum needs to be regressed for the ovulation process to be repeated, or preserved for maintenance of pregnancy (Hess et al., 2005). Additionally, P4 appears to be required for the onset of normal estr ous cycles in cattle because transient increases in blood P4 concentrations were detected prior to puberty in heifers (G onzalez-Padilla et al., 14

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1975) and prior to the first ovulation following parturition in matu re cows (Werth et al., 1996; Looper et al., 2003). The same endocrine mechanisms that control reproduction are present in Bos taurus and B. indicus females. However, Brahman-influenced cows have a decreased responsiveness in gonadotropin release to GnRH and estradiol s timuli (Griffin and Randel, 1978; Rhodes and Randel, 1978), indicating that B. indicus females may have a decreased sensitivity to GnRH and estradiol in the brain compared to B. taurus females, which may lead to differences in reproductive ability between species. Onset of Puberty in Heifers The inclusion of replacement he ifers into the cowherd is one of the most important factors for the overall efficiency of cow-calf operations (Bagley, 1993), and strategies to maximize the number of heifers conceiving ear ly during their first breeding s eason has been shown to improve the profitability of these operations (Leismeister at al., 1983). Ideally, replacement heifers should be able to conceive at 14 to 16 mo of age, and consequently calve at approximately at 2 yr of age (Schillo et al., 1992). Conception rates have been shown to be greater during the third estrus compared to the pubertal estrus (Byerley et al., 1987), thus replacement heifers should attain puberty at 12 mo of age to experience optimal reproductive performance during the beginning of their first breeding season (Schillo et al., 1992; Bagley, 1993). Puberty is attained when a heifer expresses he r first estrous behavior and ovulates a fertile oocyte followed by a luteal phase (Larson, 2007). The physiological proce sses associated with follicle growth to preovulatory stages are pr imarily regulated by gradual increases in LH secretion (Kinder et al., 1995). A lthough heifers are cap able of synthesizi ng significant amounts of LH prior to puberty (Schilo et al., 1982), ba sal quantities of estradiol secreted by ovarian 15

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follicles during the prepubertal phase inhibit the LH surge required for ovulation (Day et al., 1984). As puberty approaches, the number of estr adiol receptors available in the hypothalamus and anterior pituitary decreases (Kinder et al., 1987), resulting in reduced negative feedback of estradiol on LH secretio n. Synthesis and pulse frequency of LH are thus enhanced, and reach the threshold necessary to stimulat e ovulation (Schillo et al., 1992) As commented previously, P4 also seems to be required for the establishment of puberty in heifers. Although the major sources of this hormone are the corpus luteum and th e pregnant placenta (Hoffmann and Schuler, 2002), significant increases in blood concentr ations of P4 are detected in heifers 2 wk prior to the onset of puberty (Gonzalez-Padilla et al ., 1975). The adrenal gland and lu teal structures found within the ovary were credited as the prepubertal s ources for this hormone (Gonzalez-Padilla et al., 1975; Berardinelli et al., 1979). Progesterone seem s to stimulate the onset of puberty by priming the hypothalamic-pituitary-ovarian axis (Looper et al., 2003) and suppressing estradiol receptors in the hypothalamus, resulting in enhanced LH secretion (Anderson et al., 1996). Breed type highly influences the age at which beef heifers attain puberty. In the case of B. indicus vs. B. taurus, Martin et al. (1992) compiled data fr om several experiments and reported that Brahman-influenced heifers reach puberty at older ages and greater body size compared to B. taurus heifers. However, the same breed effect was not observed for pregnancy rates after heifers were exposed to their first breeding seas on (Gregory et al., 1979), indicating that after reaching puberty, heifers with Brahman influen ce have similar capability of conceiving as B. taurus heifers. The differences observed for age at puberty between B. indicus and B. taurus heifers may be attributed to the nutritional a nd environmental differences among the regions of the world where these breed types were devel oped and were adapted to (Frisch and Vercoe, 1984; Rodrigues et al., 2002). However, even when heifers of similar age from both breed types 16

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are reared in the same locality and have similar growth rates, B. taurus heifers still reach puberty at younger ages and lighter BW (Rodrigues et al., 2002). The same authors reported that the physiological processes related to onset of puberty were similar between breed types but occurred later for B. indicus, leading to the differen ces in age at puberty. Reproductive Efficiency of Mature Cows The major objective of cow-calf operations is to produce one calf per cow annually. The average gestation length of Brah man-crossbred cattle is 286 d (R eynolds et al., 1980); therefore brood cows should be able to become pregnant within 80 d after calvi ng in typical Florida operations. However, nursing cows experience a period of ovarian inactivity following parturition that may last 60 d or more (Yav as and Walton, 2000), when they are unable to ovulate and consequently conceive The length of this period, de nominated postpartum interval, significantly determines the li kelihood of cows becoming pre gnant during the breeding season (Wiltbank, 1970). Consequently, one of the main objectives to be accomplished in order to maximize the reproductive efficiency of cowcalf operations is to minimize the postpartum interval of the cowherd. During late pregnancy, circulating concentra tions of gonadotropins are reduced in cows because the constant production of estradiol and P4 by maternal tissues impairs the synthesis of GnRH by the hypothalamus (Short et al., 1990; Lucy, 2003), and thus depletes pituitary reserves of LH and FSH (Wettemann et al., 2003). However, LH reserves and follicular growth are reestablished weeks prior to the resumption of cyclicity in most beef cows (Yavas and Walton, 2000; Day, 2004), but postpartum anes trous is sustained because estr adiol still exerts a negative feedback on gonadotropin synthesis (Short et al ., 1990) and is not produced by the dominant follicle in sufficient amounts to trigger th e LH surge required for ovulation (Day, 1994). 17

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Consequently, the first postpartum ovulation hi ghly depends on sufficien t production of estradiol by the dominant follicle to signal the preovul atory gonadotropin surge (Day et al., 2004), whereas alternatives to alleviate the estradiol negative feedback, stimulate GnRH delivery to the pituitary, and anticipate the ovulat ory LH surge are options to decrease the postpartum interval of beef cows. Similar to the processes associated with onset of puberty, transient incr eases in P4 prior to the first postpartum ovulation seems to infl uence the endocrine ch anges required by the reproductive system of mature cows to attain no rmal and fertile estrous cycles (Kinder et al., 1987). During postpartum anestrous, P4 concentrati ons are depressed beca use the major sources of P4 production functional co rpus luteum and pregnant pl acenta (Hoffmann and Schuler, 2002) are absent. Transient increases in P4 conc entrations are typically observed in mature cows within 10 d prior to the first observed postpartum estrus (Humphrey et al., 1983; Werth et al., 1996; Looper et al., 2003), and th is P4 may have originated from luteinized follicles (Corah et al., 1974; Rawlings et al., 1980), or short-lived corpus lu teum tissue originating from undetected ovulations (Perry et al., 1991). Looper et al. (2003) reported normal estrous cycles for 81% of cows detected with transient increases in P4 concentrations during the anestrous period compared with 36% for cows without P4 incr ease. Werth et al. (1996) detected transient increases in P4 concentrations prior to first pos tpartum estrus in 70% of cows from their study, and reported greater conception rate s to AI at the first estrus for those cows (76%) compared with cows that did not experience transient P4 increases (41%). Circulating P4 is critical for the establishm ent and maintenance of pregnancy (Spencer and Bazer, 2002). Progesterone produced by the corpus luteum originated from the ovulated dominant follicle suppresses gonadotropin synthesi s, release, and cons equent ovulation during 18

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gestation (Hess et al., 2005). More importantl y, P4 prepares the uterine environment for conceptus growth and development, and modulates the release of hormone s that may regress the corpus luteum and thus disrupt pregnancy (Bazer et al., 1998). Consequently, the corpus luteum has to be maintained so circulating P4 levels are adequate for maintena nce of gestation, while circulating P4 are required in certain levels to prevent cor pus luteum regression. Several researchers have reported that bl ood concentrations of P4 in cattle prior to or after breeding have been positively associated with conception rates. Folman et al. (1990) reported that plasma progesterone concentrations prior to AI were highly correlated (r = 0.87) with conception rates in dairy cows. Fonseca et al. (1983) indicated that conception rates to AI increased by approximately 13 % for each ng/mL increase in average blood P4 concentration during the 12-d period preceding AI. Robinson et al (1989) reported that dairy co ws treated with exogenous P4 from d 5 to 12 or d 10 to 17 following AI had in creased pregnancy rates compared to untreated cows (60 vs. 30%, respectively). Research by Lopez-Gatius et al. (2004) indicated that supplementation of exogenous P4 to pregnant dairy cows from d 40 to 68 after AI decreased the risk of pregnancy loss by approximately 60%. Th ese findings are also supported by other studies (Wiltbank et al., 1956; Xu et al., 1997; Starbuck et al., 2001), indicating the importance of P4 for the establishment and maintenance of pregnancy in cattle. Supplementation Programs for Gr azing Cow-Calf Operations The cow-calf industry in Florida relies on tropical forages as the main source of feed for beef cattle. Bahiagrass ( Paspalum notatum ) is the primary pasture fora ge available in the state, covering an area of approximately 1 million ha (Chambliss and Sollenberger, 1991). Other grasses such as limpograss (Hermathria altissima), bermudagrass (Cynodon dactylon ), and stargrass ( Cynodon spp.) are also commonly used as a fora ge source for cattle (Arthington and 19

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Brown, 2005). In Florida, temperature and droug ht may limit tropical forage production during the fall and winter (Sollenberger and Chambliss, 1991), but methods to conserve summer forage surplus and offer it during periods of shortage are typical alternatives to provide adequate amounts of forage to cattle th roughout the year. Nevertheless, tropical forages do not always have the adequate nutrient composition and quality to meet the requirements of developing heifers and lactating brood cows, even when thes e animals consume sufficient amounts of forage DM (Moore et al., 1991; Pate, 1991). Therefore, deficiencies in forage quality or quantity must be corrected by nutrient supplementation to mainta in cattle at adequate levels of nourishment. Supplementation programs need to be designed according to the nutritional requirements of the animal to be supplemented, inadequacies of the consumed forage, and economic viability (DelCurto et al., 2000; Kunkle et al., 2000). Moore et al. (1991) co mpiled the nutritional analysis of 637 samples of forages commonly grown in Fl orida (bahiagrass, berm udagrass, digitgrass, stargrass, and limpograss) and reported that most of these grasses contained between 5 to 7% CP (DM basis), and 48 to 51% TDN (DM basis). Deve loping heifers require at least 55% TDN and 8.5% CP of diet DM to sust ain adequate growth rates ( 0.5 kg/d), whereas lactating brood cows require approximately 60% TDN and 11% CP of diet DM (NRC, 1996). Therefore, nutritional programs focusing on energy and protein supplement ation are often require d in typical Florida cow-calf operations to maintain the he rd in adequate levels of nutrition. Intake of low-quality forages can be increa sed by protein supplementation (Kster el al., 1996; Kunkle et al., 2000); however it does not result in adequate energy intake by the animal (Bowman and Sanson, 1996). Supplemental energy is re quired in areas where energy availability from grazed forages is limited (Caton and Dhuyvetter, 1997), and energy-based supplements containing adequate amounts of protein has been shown to improve the performance of cattle 20

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grazing low-quality forages (Kunkle et al., 2000; Bodine and Purv is, 2003). Research has also shown that energy intake positively influences th e performance and reproductive efficiency of brood cows and developing heifers (Mass, 1987; Schillo et al., 1992; Roberts et al., 1997). Consequently, nutritional program s emphasizing energy-based suppl ements are required by cowcalf operations based on low-quality forages to maintain the performance of the cowherd at satisfactory levels. Up to 63% of annual production costs in beef cow/calf operations are associated with cattle feeding, including forage production and feed purchase (Miller et al., 2001). Additional expenses associated with feeding, such as fuel and labor also contribute significa ntly to these production costs. Therefore, choosing feedstuffs that are economically viable and adop tion of strategies that minimize costs associated with the supplementation may contribute positively to the profitability of cow-calf operations. Several feed stuffs are widely available in Florida to constitute energybased supplements, such as soybean hulls, citrus pulp, sugarcane molasses, and more recently distillers grains. Determining which feedstuff to use depends on economics, management of operation, and also on the effects of the feed on animal performance. This later factor is important and has been thoroughly investigated pr eviously, but will not be further addressed in this document. Nevertheless, energy feedstuffs that favor propionate synthesis in the rumen should be supplemented because they have been shown to improve the performance of beef cattle (Pate, 1983; Ciccioli et al ., 2005; Cooke et al., 2007a). Frequency of Supplementation Supplementing cattle infrequently, such as once or 3x weekly instead of daily, is a typical strategy to decrease costs of production because the expenses associated with labor, fuel, and equipment are reduced. Kunkle et al. (2000) compiled results from several experiments 21

PAGE 22

comparing different supplementa tion frequencies of protein-base d feeds for cattle grazing lowquality forages, and reported that supplementing pr otein as infrequently as once a wk instead of daily did not alter cattle performance. Wettemann and Lusby (1994) reported similar performance measures among range cows supplem ented 6x/wk or 3x/wk with a protein-based supplement. Similarly, Huston et al. (1999) re ported no differences in BW and BCS changes among beef cows offered cottonseed meal daily, 3x/wk, or once a w k, at a weekly rate of 5.6 kg of DM. Bohnert et al. (2002a; 2002b) reported that st eers consuming low-quality forages and offered protein-based supplements as infrequently as once every 6 d had similar performance, DMI, nutrient utilization, and ruminal microbial efficiency compared to steers supplemented daily. Farmer et al. (2001) repor ted that mature cows and steer s consuming dormant forages and offered protein-based supplements at a weekly rate of 11.2 and 5.6 kg of DM, respectively, had a linear improvement in performance (cows), forage intake and diet digestibility (steers) as supplementation frequency increased. However, the authors stated that changes in performance were small enough to support that reduced supplem entation frequency of pr otein-based feeds is an acceptable management alternativ e to decrease costs of production. In contrast to protein-base d supplements, decreasing the s upplementation frequency of energy-based feeds to cattle consuming low-quality forages has been shown to be detrimental to animal performance. Research by Chase and Hi bberd (1989) reported that cattle consuming lowquality forages and offered corn-based supplements daily instead of every other day, at weekly rates of 9.8 or 14.0 kg (as-fed), experienced s light improvements in DM digestibility and consequent OM intake. The aut hors concluded that cattle offere d corn-based supplements daily utilized their diet more effici ently than cattle supplemented in frequently. Beaty et al. (1994) reported that steers consuming wheat straw and offered grain-based supplements (sorghum in 22

PAGE 23

addition to soybean meal) daily instead of 3x/ wk, at a weekly rate of 14.0 kg of DM, had increased digestible DMI but decreased DM dige stibility. The same authors also reported that mature cows grazing dormant tallgrass prairie and supplemented similarly as the steers experienced alleviated BW and BCS losses during the calving period but had similar pregnancy rates if supplemented daily instead of 3x/wk. In their review, Kunkle et al. (2000) reported several research results indicating improved pe rformance of cattle offered energy-based feeds daily instead of infrequently, and attributed this effect to improved ruminal function and consequent forage intake of da ily-fed cattle. It is important to note that all these studies comparing supplementation frequency of energybased feeds utilized supplements containing high-starch ingredients. These supplements are known to have negative effects on rumen health and forage intake and digestibility in cattle even if offered daily (Sans on et al., 1990; Olson et al., 1999). This negative impact is associated with decreased rumina l pH and activity of cellulotic enzymes (Martin et. al., 2001), impaired bacterial attachment to fibrous material (Hiltner and Dehority, 1983), and an increase in lag time for digestion (Mertens and Loften, 1980). Conversely, energy supplements based on lo w-starch ingredients, such as fibrous byproducts, has been shown to not depress forage intake (Bowman and Sanson, 2000) and maintain animal performance at the same levels as when high-starch supplements are fed (Sunvold et al., 1991; Horn et al., 1995). However, few studies have evaluated supplementation frequency of low-starch feeds to cattle consuming low-quality forages. Loy et al. (2007) reported that foragefed heifers offered supplements based on distillers grains daily or in alternate days, at a weekly rate of 10.5 kg of DM, had similar mean forage intake (1.69 vs. 1.66% of BW, respectively), rumen pH (6.12 vs. 6.17, respectively), and in situ rate of NDF disappearance (4.09 vs. 4.01% per h, respectively). Cooke et al. (2007b) repor ted that steers offered supplements based on 23

PAGE 24

citrus-pulp daily had similar mean forage DMI (1.36 vs. 1.29% of BW, respectively), but improved ADG (0.30 vs. 0.18 kg/d, respectively) comp ared to steers offered similar supplements 3x/wk, at a weekly rate of 16.1 kg of DM per steer for both treatments. Both studies also reported a treatment day interaction on forage DMI because forage intake of infrequently supplemented cattle was reduced on days that su pplements were offered, but greater on the remaining days compared to that of cattle s upplemented daily. Nevertheless, this outcome is likely attributed to a substitu tion effect (Caton and Dhuyvetter, 1997; Bodine and Purvis, 2003) rather than to compromised ruminal func tion. Cooke et al. (2007b) attributed the ADG differences detected between cattle supplemented daily or 3x/wk to beneficial effects of frequent supplementation on concentrations of blood hormones and metabolites associated with energy intake and metabolism, such as glucose, insulin and IGF-I. These same substances have been positively associated with reproductive function of cows and heifers (Schilo et al., 1992; Hess et al., 2005); therefore frequent suppl ementation of low-starch energy feeds is also expected to benefit cattle reproduction. Energy Metabolism and Reproduction Energy is the primary nutrient c onsideration for optimal repro ductive performance of cattle (Mass, 1987). Attainment of puberty can be hasten ed by increasing energy intake and consequent BW gain (Schillo et al., 1992). Cows with ade quate energy status have decreased postpartum interval (Richards et al., 1986; Looper et al., 1997) and greater pregnancy rates (Wiltbank et al., 1962; Dunn et al., 1969; Bellows and Short, 1978) compared to cows with inadequate energy status. These beneficial effects of energy on reproduction can be attributed, at least in part, to plasma hormones and metabolites that are influen ced positively by energy intake and by levels of body energy reserves, such as glucose, insulin, and IGF-I (Wettemann et al., 2003). 24

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Glucose Glucose is a small, polar, and water-sol uble monosaccharide (Nussey and Whitehead, 2001), essential for maintenance and productive functions of ruminants (Huntington, 1997; Reynolds, 2005). Previous research indicated that blood glucose concentrations in beef cattle are positively associated with feed intake and rates of BW gain (Vizcarra et al., 1998; Hersom et al., 2004). Cooke et al. (2007a) report ed greater mean glucose concen trations (83.3 vs. 74.7 mg/dL, respectively) and ADG (0.40 vs. 0.30 kg/d, respectiv ely) for grazing heifers consuming citrus pulp-based supplements instead of iso-caloric and iso-nitrogenous supplements based on sugarcane molasses. On the other hand, Cooke et al. (2007b) reported th at forage-fed steers supplemented daily with a citrus pulp-based supplement had greater ADG (0.30 vs. 0.18 kg/d, respectively) but lesser mean glucose concen trations (66.0 vs. 76.2 mg/dL, respectively) compared to steers offered supplements 3x/wk, but at similar weekly amounts. Metabolic fates of glucose include generation of ATP via glycolys is and TCA cycle, and generation of NADPH through the hexose monophosphate shunt (Hunti ngton, 1997). Cattle consuming forage-based diets ferment the majority of the dietary car bohydrates to VFA in the rumen, and often obtain less than 10% of their glucose requirements from the diet (Y oung, 1977). Consequently, foragefed cattle depend significantly on liver gluconeogenesis to m eet their metabolic glucose requirements (Young, 1977; Huntington, 1997). The major substrate for liver gluconeogenesis in cattle is propionate or iginated from rumen fermentation (Reynolds et al., 1994; Reynolds, 2005) followed by other substrates originated from digestion processes such as lactate, carbon skeletons from glucogenic aminoacids deamination, and glycerol originated fr om TAG breakdown (Huntington, 1997). Following digestion processes, these subs trates are removed by the liver from the portal blood flow that 25

PAGE 26

drains the gut (Huntington, 1997). In the liver, pr opionate is oxidized into succinyl-CoA, a TCA cycle intermediate, and then converted to oxa loacetate (Nelson and C ox, 2005). Aminoacids can be either degraded into TCA cycle intermediate s, pyruvate or oxaloacetate, whereas lactate is converted into pyruvate as part of the Cori cycle (Nelson and C ox, 2005). The initial steps of the gluconeogenesis pathway are the conversion of pyruvate into oxaloacetate, catalyzed by the enzyme pyruvate carboxylase (PC), and oxaloac etate into phosphoenolpy ruvate, catalyzed by phosphoenolpyruvate carboxykinase (PEPCK; Nelson and Cox, 2005). These two enzymes have been shown to highly regulate the rate of gluconeogenesis in ma mmals, including cattle (Drackley et al., 2006). Furthermore, PEPCK has tw o isozymes of similar activities and kinetic properties, compartmentalized to the cell m itochondria (PEPCK-M) a nd cytosol (PEPCK-C; Agca et al., 2002) Research with cattle has shown that activ ity and mRNA expression of PC and PEPCK influence positively glucose synthesis via gluconeogenesis. The availability of gluconeogenic precursors was positively associated with mRNA e xpression of PC and PEPCK-C in the liver of dairy cattle (Greenfield et al. 2000; Hammon et al., 2003; Karcher et al., 2007), whereas mRNA expression of these enzymes in hepatic tissue was correlated with enzyme activity (Greenfield et al. 2000; Agca et al. 2002) and consequently glucose synthesis (Bra dford and Allen, 2005). Karcher et al. (2007) reported that transiti on dairy cows supplemented with monensin had increased hepatic expression of PEPCK-C because of enhanced propionate synthesis in the rumen, although no differences in blood glucose c oncentrations were detected compared to nonsupplemented cows. Expression of PEPCK-C and PC were observed to be inhibited directly by elevated plasma concentrations of glucose a nd insulin (Agca et al. 2002; Hammon et al., 2003). Conversely, PEPCK-M has been shown not to be regulated by mRNA levels and also not 26

PAGE 27

responsive to hormones and substrates (Greenfi eld et al., 2000; Agca et al., 2002), although the activity of this enzyme may be responsible for up to 61% of glucose synthesis in ruminant hepatocytes (Aiello and Armentano, 1987), and Hammon et al. (2003) re ported that neonatal calves fed colostrum had greater PEPCK-M mRNA expression compared to cohorts fed milkbased formulas. Glucose is the main source of energy for the central nervous system; consequently inadequate availability of glucose may re duce the synthesis and release of GnRH and gonadotropins by the brain and impair reproduction. Previous studies have shown that ruminants administered glucose antagonists experienced in duced anestrus, anovulation (McClure et al., 1978), and impaired LH secretion (F unston et al., 1995; Bucholtz et al., 1996; Rutter and Manns, 1987). In cattle, the hypothalamus may recogni ze low-blood glucose concentrations by a threshold-dependent fashion, given that GnRH secretion is impaired when glucose availability is inadequate, but resumed when glucose levels are adequate (Hess et al., 2005). On the other hand, Wettemann and Bossis (2000) indicated that glucose concentrations in blo od are probably not a major factor controlling follicular growth and ovulation because glucose concentrations in anestrous heifers were similar to those observed in cycling heifers at least two follicular waves before resumption of cyclicity. Blood glucose concentrations in ru minants are fairly stable, and this fact may partially explain the lack of a direct relations hip between circulating glucose concentrations and reproductive function reported by Wettemann and Bossis (2000). Further, the positive effects of increased circulating gl ucose through enhanced gluconeogenesis on reproduction of cattle may be associated with improvements in energy status and concentrations of other blood metabolites and hor mones instead of the increase in glucose availability itself (Randel, 1990; Hess et al., 2005). 27

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Insulin Bovine insulin is a small peptide hormone containing 51 aminoacid residues and a molecular weight of 5.7 kDa (Smith, 1966). Insuli n is synthesized within the pancreas and initially stored as a pro-hormone in secretory gr anules in the pancreatic beta-cells (Nelson and Cox, 2005). Several stimuli trigge r the conversion of proinsulin into active insulin, and consequent insulin release into the bloodstream (Nelson and Cox, 2005). The main effects of insulin are on liver, muscle and adipose tissu es, where it increases an abolic processes and consequently decreases tissue catabolism (Nusse y and Whitehead, 2001). Insulin directly induces the uptake of glucose by muscle and fat tissue by recruiting intracellular GLUT4 transporters and increasing their cell-surface expression, conseq uently enhancing cell ATP production, glycogen synthesis in muscle, and lipogenesis in adi pose tissues (Nussey and Whitehead, 2001; Nelson and Cox, 2005). In the liver, insulin promotes glycogenesis but does not modulate glucose uptake by the hepatocytes via GLUT2 transporter. Insulin also modulates cellular uptake of amino acids and some electrolytes (Austgen et al., 2003). Although insulin is secreted in pulses approximately every 10 min, insulin secretion is mainly stimulated by high blood glucose concentrations (Nussey and Whitehead, 2001), therefore maintaining metabolic homeostasis because blood glucose is maintained within constant concentrations. Additionally, insulin secretion is also stimulated by gastrointestin al hormones and neural/p aracrine mechanisms associated with feed intake (Nussey and Whitehead, 2001). Because of the interaction betw een insulin and glucose metabolism, the effects of insulin on performance and reproduction of cattle have been extensively inve stigated. Similar to glucose, blood insulin concentrations have been positively associated with feed intake and rates of BW gain in beet cat tle (Vizcarra et al., 1998; Bossis et al ., 2000; Lapierre et al., 2000). Cooke 28

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et al. (2007a) reported greater mean insulin co ncentrations (0.89 vs. 0.75 ng/mL, respectively) and ADG (0.40 vs. 0.30 kg/d, respectively) for grazing heifers consuming citrus pulp-based supplements instead of iso-caloric and isonitrogenous supplements based on sugarcane molasses. Conversely, Cooke et al. (2007b) report ed that forage-fed steers supplemented daily with a citrus pulp-based supplement had gr eater ADG (0.30 vs. 0.18 kg/d, respectively) but inferior mean insulin concentrations (0.46 vs 0.60 ng/mL, respectively) compared to steers offered supplements 3x/wk. In terms of reproduc tion, Arias et al. (1992) reported that GnRH release from perifused hypothalamic fragments of female rats was dramatically increased when glucose and insulin were added simultaneously to the perifusion medium compared to those containing no supplemental glucos e, insulin, or both. On the other hand, Hileman et al (1993) reported that short-term intracer ebroventricular administ ration of insulin failed to increased LH secretion in lambs, suggesting that insulin alone may not act as a nutritional signal regulating LH secretion. Glucose uptake by brai n cells are dependent on glucos e transporters GLUT1, GLUT3, and GLUT8 (Maher et al., 1994; Olson and Pe ssin, 1996; Sankar et al., 2002), and GLUT3 is often considered the main neuronal glucose trans porter because of its relative abundance in the brain (Maher et al., 1994). These transporters, es pecially GLUT3, have been shown to be rather insensitive to insulin; therefore brain cells ar e considered insulin unr esponsive, and glucose uptake in neurons rely on other mechanisms (H eidenreich et al., 1989). Conversely, Livingstone et al. (1995) indicated that th e hypothalamic neurons may also express GLUT4 and therefore absorb glucose via insulin stimuli. Nevertheless, lack of insulin sensitivity by the majority of brain cells might explain why LH secretion in lambs was not increased by insulin injections (Hileman et al., 1993), and GnRH release from perifused hypothalamic fragments were not increased by addition of only insulin to pe rifusion medium (Ari as et al., 1992). 29

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Other studies, however, reported a positive effect of insulin on reproductive efficiency of cattle. Sinclair et al. (2002) reported that postpartum anestrous beef cows with low plasma insulin concentrations (< 5 mIU/ L) failed to ovulate the first domi nant follicle in response to restricted calf access compared to cows with moderate plasma insulin (5 to 8 mIU/L), and attributed this result to an inadequate responsiven ess of the dominant follicle to the LH surge that frequently accompanies calf removal. Gong et al. (2002) fed lactating dair y cows with diets to stimulate (propiogenic ingredients) or maintain insulin concentrations at normal levels (acetogenic ingredients) from d 0 to d 50 post-calving, a nd reported that gonadotropin concentrations, milk yield, and changes in BW and BCS were similar between treatments, but cows fed insulin-stimulating diets had greater plasma insulin concentrations (0.40 vs. 0.27 ng/mL) and reduced postpartum interval (34 vs. 48 d) compared to cows fed the acetogenic diet. Therefore, it can be speculated that insulin infl uences reproductive efficiency of cattle at the ovarian level by modulating the activity of gonado tropins rather than influencing gonadotropin synthesis by the brain. Insulin has been shown to be a potent stimulator of estradiol synthesis in the ovary of cattle (Spicer and Echternkamp, 1995), and estradiol is required for the ovulatory LH surge (Kesner et al., 1981) and also synthesis of LH receptors in the membranes of granulosa cells within the dominant follicle (Bao and Garverick, 1998). Consequently, cows with low insulin concentrations may have impaired LH surg e, and/or reduced numbers of LH receptors in the dominant follicle, failing to ovu late even if the LH surge is present (Diskin et al., 2003). Insulin-like Growth Factor I Bovine IGF-I is a single chain polypeptide hormone th at structurally resembles proinsulin (Gluckman et al., 1987). The IGF -I molecule contains 70 amino acid residues with a molecular weight of approximately 7.6 kDa (McGuire et al., 1992; Etherton, 2004), and is synthesized by 30

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most, if not all, body tissues (Le Roith et al., 2001) Liver is the main source of circulating IGF-I (DErcole et al., 1984), and IGF -I synthesized by other tissues mainly exert autocrine and paracrine effects (McGuire et al., 1992; Johnson et al., 1998). S ynthesis of IGF-I is mainly regulated by GH (McGuire et al., 1992), and its secretion into the ci rculation occurs in a constant pattern (Thissen et al., 1994). The majority of IGF-I found in blood or other body fluids is bound with high affinity to one of six different IGFBPs, which vary in length from 201 to 289 aminoacid residues, and molecular weight from 24 to 44 kDa (Thissen et al., 1994; Beattie et al., 2006). These IGFBPs serve mainly as circulatory transport vehicles, retard IGF-I degradation, and modulate the actions of IGF-I in target cells by enhancing or blocking IGF-I activity (Le Roith et al., 2001). In the circulation, more th an 90% of IGF-I is bound to IGFPB-3 (Martin and Baxter, 1992), whereas the remainder of circulat ing IGF-I is either free (< 5 %) or bound to IGFBP-1, IGFBP-2, or IGFBP-4 (Clemmons, 1991) The complex formed by IGF-I and IGFBP3 also contains an acid-labile unit, and has a molecular weight of approximately 150 kDa, whereas IGFBP-1 and IGFBP-2 associate directly with IGF-I into smaller complexes (30 to 40 kDA; Thissen et al., 1994). Due to its large mo lecular size, the IGFBP3/IGF-I complex cannot leave the circulatory system and reach target ti ssues. Therefore, IGF-I is released from this complex and associates with IGFBP-1 and IG FBP-2 in the circulation, forming smaller complexes that can cross the capillary endothelium and deliver IGF-I to targ et tissues (Thissen et al., 1994; Le Roith et al., 2001). The presence of IGFBP-5 and IGFBP-6 is not abundant in the blood; however, IGFBP-5 has been associated with bone and muscle development because of its role in cell survival, different iation, and apoptosis (Beattie et al., 2006; Dayton and White, 2008). The major anabolic effects of IGF-I are inherent to prot ein and carbohydrate metabolism, and also cell replication and differentiation (Jones and Clemmons, 1995; Le Roith et al., 2001). 31

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At target tissues, IGF-I binds to its receptor, which is 60% alike the insulin receptor at the aminoacid level (Ulrich et al., 1986), and initiates a cascade of cellular ev ents beginning with IRS-1 phosphorylation (Jones and Cle mmons, 1995). As a result, anabolic effects are stimulated via pathways such as PI3 kinase and MAPK kina se, whereas catabolism is inhibited via the BAD phosphorylation (Le Roith et al., 2001). Other processes such as cell function, cell secretory activity, and also immune response are influe nced positively by IGF-I (Jones and Clemmons, 1995). To fuel all of these activit ies, IGF-I has insulinlike stimulatory actions on most cells with IGF-I receptors, stimulating gluc ose and aminoacid uptake, and al so glycogen synthesis (Jacob et al., 1989; Dimitriadis et al., 1992). In beef cattle, feed intake and BW gain have been associated positively with IGF-I (Bossis et al., 2000; Armstrong et al., 2001; Ra usch et al., 2002), but negatively with GH concentrations (Ellenberger et al. 1989; Granger et al., 1989; La pierre et al., 2000). Therefore, IGF-I synthesis in cattle at ade quate levels of nutrition relies on additional mechanisms besides direct GH stimuli. Insulin facilitates IGF-I synt hesis in the liver by enha ncing binding of GH to hepatic GH receptors (Houston and ONeill, 1991; McGuire et al., 1995; Molento et al., 2002), whereas concentrations of insulin typically correlate positively with IG F-I concentrations in cattle (Keisler and Lucy, 1996; Webb et al., 200 4; Cooke et al., 2007a). Other studies have shown that IGF-I synthesis is also regulated by thyroid horm ones. Hypothyroid animals have impaired IGF-I synthesis (Burns tein et al., 1979), whereas he patic GH binding is enhanced by thyroid hormones, implicating thei r ability to potentiate IGF-I synthesis via GH stimuli (Barash and Posner, 1989; Hochberg et al., 1990). Additionally, aminoacid restriction, especially tryptophan, decreased IGF-I synthe sis in rats with adequate gl ucose, insulin, and GH levels (Maiter et al., 1989; Phillips et al., 1991). Thes e nutritional and physiolo gical modulations of 32

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IGF-I synthesis mainly occur at a pretranslational stage because ch anges in IGF-I concentrations are typically preceded by changes in hepati c IGF-I mRNA expression (Thissen et al., 1994). Recent studies indicated that IGF-I is a major metabolic signal regulating reproduction in cattle (Wettemann and Bossis, 2000). Research with developing beef heifers reported that IGF-I, but not glucose and insulin, was positively asso ciated with ADG and reproductive performance (Cooke et al. 2007a; Cooke et al., 2008a). Grange r et al. (1989) reported that plasma IGF-I concentrations were negatively associated with age at puberty, and Bossis et al. (1999) reported that feed restriction of cycling heifers lead s to significant decreases in plasma IGF-I and culminates with onset of anestrous. Cooke et al (2007a) reported similar ADG, but greater mean IGF-I concentrations for beef heifers that r each puberty (120 vs. 99 ng/mL) and conceive (123 vs. 111 ng/mL) as yearlings compared those that remain pre-pubertal and/or non-pregnant. In mature beef cows, blood concentrations of IGF-I were increased for those that resumed estrous cycles within 20 wk postpartum compared to cows that remained in anestrous (Roberts et al., 1997). Because of their close asso ciation with IGF-I, the IGFBPs have also been shown to influence performance and reproduction of cattl e. Blood concentrations and mRNA expression of IGFBP3 are associated positively with nutrient in take and rates of BW gain in cattle (Thissen et al., 1994; Vestergaard et al., 1995; Rausch et al., 2002). On the other hand, IGFBP2 is believed to decrease the bioavailability and co nsequently conserve IGF-I when the hormone synthesis is reduced (Jones and Clemmons, 199 5; Armstrong et al., 2001). Ruminants in poor nutritional status have increas ed blood concentrations and mRNA expression of IGFBP2 in several tissues (Vandehaar et al., 1995; Armstrong and Benoit, 1996; Armstrong et al., 2001). Roberts et al. (1997) reported that serum con centrations of IGFBP-2 were decreased whereas serum concentrations of IGFBP-3 were increased in beef cows that resumed estrus by 20 wk 33

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postpartum compared with anestrous cows. Based on these and other observations, Hess et al. (2005) postulated that IGF-I and IGFBP3 are posi tive signals and nutritional mediators into the reproductive axis. The effects of IGF-I on cattle reproduction, more specifically with in the hypothalamuspituitary-ovarian axis, are believ ed to be via autocrine, paracrine and endocrine mechanisms. Wettemann et al. (2003) complied results from st udies conducted with different mammals, and reported that IGF-I receptors were detected in the hypothalamus and pituitary, indicating that GnRH and gonadotropin secretion are potentially modulated by IGF-I. In their review, Diskin et al. (2003) also cited studies indi cating that IGF-I infl uences hypothalamic and pituitary functions of mammals. Conversely, Rutter et al. (1989) re ported that increases in IGF-I via glucose supplementation to beef cows at adequate plan es of nutrition did not influence LH pulsatile pattern. Consequently, a large po rtion of IGF-I effects on reproduc tion of cattle may rely on its effects within the ovary. Receptors for IGF-I a nd also IGF-I mRNA were detected in several ovarian cells, such as granulos a, thecal, and luteal cells (Spicer and Echternkamp, 1995; Armstrong and Benoit, 1996; Bao and Garverick, 1998). Previous research demonstrated that IGF-I enhances the responsiveness of ovarian ce lls to FSH and LH, stimulating cell mitosis, follicle growth, and steroidogenesis (Spicer and Stewart, 1996; Stewart et al., 1995; Armstrong et al. 2001). The ovulation proce ss appears to also be dependent on IGF-I stimulus. Roche (2006) indicated that follicle size, LH pulsatility, and systemic concentra tions of IGF-I are the factors responsible for the successful ovulation of the dominant follicle. Recent data by Ginther et al. (2001) indicated that the dominant follicle contains greater free IGF-I concentrations (12 vs. 4 ng/mL) and reduced IGFBP-2 concentration (492 vs. 648 ng/mL) within its fluid compared to the second-largest follicle after deviation. Further, follicle diameter, estradiol and free IGF-I 34

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concentrations were significantly and positivel y correlated with each other, and negatively correlated with IGFBP2 concentrat ions within the dominant follicl e postdeviation (Ginther et al., 2001). Others also reported greater concentrations of total IGF-I and de creased or undetectable concentrations of IGFBP2, -4, and -5 in domin ant follicles compared to subordinate follicles (Echternkamp et al., 1994; de la Sota et al., 199 6; Mihm et al., 1997). Therefore, IGF-I seems to play an important role in follicle responsivene ss to gonadotropins and consequently in follicle growth and steroid synthesis, which are requi sites for successful ovulation and consequent fertility of cattle. Recent data from dairy cattle indicated bene ficial effects of IGF-I on early pregnancy. Moreira et al. (2002) reported that addition of IGF-I to in vitro embryo cultures increased the rate of embryo development to the blastocyst stage and increased the number of blastocyst cells. Further, Jousan and Hansen (2004) reported that addition of IGF-I to in vitro cultures of embryos exposed to heat shock increased total embryo ce ll number and alleviated cell apoptosis. These positive effects of IGF-I on embryonic development al lows for greater secretion of interferon tau by the embryo (Bilby et al., 2006), and this protein has been shown to regul ate secretion of PG by the uterine endometrium and influence positivel y the establishment and maintenance of early pregnancy in cattle (T hatcher et al., 2001). Progesterone Another mechanism by which nutrition affect s reproduction in cattle is by altering hepatic P4 metabolism. As commented previously, P4 is required for adequate attainment of puberty, resumption of estrous cycles, and also establishment and maintenance of pregnancy (Gonzalez-Padilla et al., 1975; Spencer and Bazer, 2002; Looper et al., 2003). Research has shown that infrequent intake of large amounts of feed decreases circulatin g P4 concentrations in 35

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beef and dairy cattle. Vasconcelos et al. (2003) reported that P4 concentrations of lactating pregnant Holstein cows provided 10 0 % of daily diet at once or ha lf of the diet every 12 h were decreased by 1 h and remained depressed unt il 8 to 9 h after feeding compared to P4 concentrations of cows fed a quarter of diet every 6 h or unfed cows. Cooke et al. (2007a) reported that plasma P4 concentrations of grazing pubertal heifer s offered energy-based supplements 3 times weekly decreased by approximately 11% on supplementation days compared to non-supplementation days, whereas forage-fed cows similarly supplemented had decreased P4 concentrations 4 h after supplements were offered. These re ductions in circulating P4 concentrations after large meal consumption can be attributed to increases in hepatic blood flow and consequent steroid metabolism. After f eed intake, liver blood flow is intensified to transport digested nutrients from the gut th rough the liver and on to the rest of the body (Huntington, 1990; Sangsritavong et al., 2002). In the liver, P4 is inactivated or catabolized mainly by enzymes of the subfamilies cytochro me P450 2C and cytochrome P450 3A, and the resultant metabolites (21-hydroxyprogesterone and 6 -hydroxyprogesterone, respectively; Murray 1991, 1992) are eliminated primarily in th e urine (Steimer, 2003). Therefore, infrequent offering of large amounts of feed to cattle may be detrimental to their reproductive function. During periods when P4 is fundamental, feed intake should be managed properly to avoid sudden decreases in circul ating P4 concentrations. Temperament, Acclimation, and Reproduction of Cattle Assessment of Temperament in Beef Cattle For over a century, the word temperament has been used to define the behavioral responses of cattle when exposed to human handling (P ott, 1918; Fordyce et al., 1988). As cattle temperament worsens, their response to human contact or any othe r handling procedures 36

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becomes more agitated and/or aggressive. W ithin the beef cattle industry, some producers do select cattle for temperament mainly for safety reasons, whereas the productive and economic implications of this trait are still not we ll established (Voisine t et al., 1997a). Cattle temperament can be assessed and ev aluated by diverse techniques. Burrow and Corbet (2000) described and categorized these into non-restrained and restrained techniques. Within the non-restrained techniques, cattle temper ament is evaluated by their fear or aggressive response to man when they are free to move w ithin the evaluation area. Examples of these techniques are the chute exit veloci ty and the pen score. Exit velo city quantifies the speed of an individual animal immediately after it leaves the squeeze chute by measuring the time required for the animal to travel a pre-determined distance. The pen score evaluates the behavioral response of an individual animal when it enters a small pen and interacts with a person standing inside the pen. Typically in a 15 scale, the pen score increases as the animal response becomes more aggressive toward the person. The restrain ed techniques evaluate cattle temperament when these are physically restricted, such as in the squeeze chute (Burrow and Corbet, 2000). The major problem with these techniques is that cattle with excitable te mperament may freeze when restrained, and consequently not express their true behavior during these assessments (Burrow and Corbet, 2000). An example of the re strained techniques is the chute score, also denominated crush score. Cattle ar e individually restrained in th e chute and scored in a 1-5 or other scale according to its be havior, where chute score increa ses as shaking and struggling intensifies (Voisinet et al., 1997a; Arthington et al., 2008). Other methods to assess cattle temperament have also been reported, such as evaluation of the behavioral responses of small groups of cattle confined to a pen (Hammond et al., 1996), evaluation of chute score without squeezing the animal in the chute (Curley et al ., 2006), assessment of the amount of human force 37

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required to move the animal into the chute, and also the estimation of the animal speed when it enters the chute (Baszcza k et al., 2006). However, the first three methods reported herein (chute score, pen score, and exit velocity) have been shown to be repeatable within animals (Boivin et al., 1992; Grandin, 1993; Curley et al., 2006); theref ore reliable to quantify cattle temperament, and also relatively simple to carry out during cattle hand ling procedures. Cattle temperament is influenced by several factors such as sex, age, and horn status (Fordyce et al., 1988; Voisinet et al., 1997a), but none of these char acteristics has been shown to affect cattle temperament as much as breed type. Several studies indicated that cattle with high Brahman influence have more excitable temperament compared to B. taurus cattle. Hearnshaw and Morris (1984) evaluated beha vioral responses similar to th e chute score method of calves sired by B. indicus (Brahman, Braford, and Africander) or by B. taurus (Hereford, Simmental, and Friesian), and reported that the mean te mperament score of Brahman-sired calves was greater than that of B. taurussired calves (1.96 vs. 1.05, respectiv ely). Further, Brahman-sired calves had greater scores compared to Brafordor Africander-sired calves (1.95, 1.38, and 1.25, respectively), whereas similar temperament scores were detected among B. taurus breeds. Fordyce et al. (1988) reported that Brahman Shorthorn (50:50 breed composition) steers and non-lactating mature cows were more temperamental than Short horn cohorts. Voisinet et al. (1997a) reported that yearling st eers and heifers with Brahman breeding (Braford, Red Brangus, and Simbrah) had greater temperament scores compared to Angus and Angus-crosses (3.46 vs. 1.80 on a 5-point scale, respectively). These data indicate that Brahman-crossbred cattle are potentially difficult to be controlled and handle d, which can bring serious management problems to cattle operations especially if these are ba sed on extensive grazing systems where cattle have infrequent interaction with humans, such as the cow-calf operati ons in Florida. 38

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Stress and Excitable Temperament Stress response can be characterized as th e reaction of an anim al to factors that potentially influence its homeos tasis (Moberg, 2000), whereas animals that are unable to cope with these factors are classified as stresse d or distressed (Dobson and Smith, 2000; Moberg, 2000). Based on this concept, the agitated and/or aggressive res ponses expressed by cattle with excitable temperament when exposed to human in teraction and other hand ling procedures can be attributed to their inability to cope with thes e situations and therefore classified as a stress response. In addition to altered behavior, these animals may also experience changes in their neuroendocrine and nervous systems (Moberg, 2000) The neuroendocrine re sponse to stress is mainly mediated by the hypothala mus-pituitary-adrenal axis (Dobson and Smith, 2000), and the hormones produced within this axis influence seve ral aspects in cattle, such as growth, immune response, and reproductive function (Fe ll et al., 1999; Dobson et al., 2001). The Hypothalamic-Pituitary-Adrenal Axis The neuroendocrine stress response begins wh en the central nervous system of animals, including cattle, perceives a thr eat to homeostasis and activates the secretion of corticotrophinrelease hormone (CRH) by the neurosecretory cells of the hypothalamus into the pituitary portal blood system (Matteri et al., 2000; Smagin et al ., 2001). The sympathetic nervous system is also stimulated by stressors, further stimulating CRH secretion by the hypothalamus and culminating in the synthesis of catecholamines by adrenal gl ands (OConnor et al., 200 0). In the posterior pituitary, CRH stimulates vasopressin (VP) release and then acts in synergy with VP to stimulate ACTH synthesis by corticotrophs in the anterior pituitary (Minton et al., 1994; OConnor et al., 2000). Secretion of ACTH via CRH stimulus is al so enhanced by the presence of cytokines and other inflammatory mediators produced during peri ods of infection or inflammation (Carroll and 39

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Forsberg, 2007). Release of ACTH into the periph eral circulation occurs in a pulsatile fashion with a circadian rhythm and stimulates steroidog enesis in the adrenal glands (OConnor et al., 2000; Stewart, 2003). Further, ACTH secretion an d consequent adrenal steroidogenesis are the greatest in the morning, decrea ses throughout the day, and reach nadir levels in the evening (Thun et al., 1981; Arthington et al., 1997). The adrenal glands cons ist of two distinct regions: the adrenal cortex and adrenal medulla. Three ty pes of steroids are synthesized by the adrenal cortex: glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, and sexual steroids such as androgens and P4 (Brown, 1994). The adrenal cortex synthesizes these steroids from cholesterol mainly in response to ACTH although research has shown that circulating forms of CRH and VP also regulate cortisol sy nthesis (Carroll et al., 1996). Conversely, the adrenal medulla is not regulated by ACTH, and synthesizes catech olamines (mainly epinephrine and norepinephrine) directly in response to sympathetic nervous system stimuli (Carroll and Forsberg, 2007). Mineralocorticoids, more specifically aldosterone, are required to maintain adequate mineral balance within the body (Brown, 1994). Glucocorticoids and catecholamines elicit several biologic effects in the orga nism of stressed animals in or der to prepare and sustain their behavioral responses to stre ss, usually designated as the fight or flight response. Cathecolamines are rapidly released from the adrenal medulla when animals are exposed to stressors because of their nervous stimulus natu re. Within seconds, epinephrine increases heart rate and oxygen flow to tissues, whereas norep inephrine mainly induces vasoconstriction to increase blood pressure (Brown, 1994; Carro ll and Forsberg, 2007). Further, epinephrine stimulates breakdown of liver and muscle glycogen into glucose and stimulates mobilization of fatty acids from adipose reserves by increasing enzymatic activity in these tissues (Nelson and 40

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Cox, 2005). Both catecholamines regulate CRH, AC TH, and cortisol secretion, whereas their synthesis is in turn facilitat ed by cortisol (Plotsky et al., 1989; Carroll and Forsberg, 2007). Cortisol also degrades liver reserves and muscle and adipose tissues to increase the availability of energy to the animal. However, cortisol m odulates enzyme synthesis instead of enzyme activity; therefore ta rget cells are impacted in a chronic manner (Nelson and Cox, 2005). Besides fatty acid mobilization, cortisol s timulates protein breakdown in muscle tissues and enhances the mobilization of the resultant aminoacids to the liver where they serve as substrate for gluconeogenesis (Carroll and Forsberg, 2007). Furthe r, cortisol has been shown to suppress the immune response by decreasing synthesis of cyt okines. This process is required to prevent autoimmune diseases; however, if cortisol concentrations are chr onically elevated such as during prolonged stressful situations, it can lead to cases of im munosuppresion (Kelley, 1988). As commented previously, the adrenal cortex is capable of producing sexual hormones because these are intermediates or by-products in the synthesi s of mineralocorticoids and glucocorticoids. According to Stewart (2003), the first step of steroidogenesis in the adrenal gland is the uptake of cholestero l by the mitochondria of adrenal co rtex cells, and this process is mediated by the steroidogenic acute regulatory protein. In the mitochondria, cholesterol is converted into pregnolone by the enzyme chol esterol desmolase. Pregnolone can be either converted into P4 by the enzyme -hydroxysteroid dehydrogenase, or converted into 17hydroxypregnolone by the enzyme 17 -hydroxylase. Both P4 and 17-hydroxypregnolone can be converted into 17-hydroxyprogesterone and direct ed towards cortisol or androstenedione synthesis, whereas P4 only can be directed towards aldosterone synthesis. Further, androstenedione can be converted into estrad iol in tissues by the enzymes aromatase and 17 hydroxysteroid (Bulun and Adashi, 2003). Therefore, elevated secretion of ACTH may stimulate 41

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accumulation and secretion of sexual steroids by th e adrenal gland concomitantly with increased synthesis of glucocorticoids and mineralocort icoids, and this process may influence the reproductive functions of animal s under stressful situations. Physiologic Responses to Excitable Temperament in Cattle Several studies were conducted to evaluate the effects of excitable temperament on the physiologic responses of livestock, more specifically within the hypothalamic-pituitary-adrenal axis. Stahringer et al. (1990) reported that temperament scor es of Brahman heifers were positively correlated with mean cortisol con centrations (r = 0.65). Hammond et al. (1996) reported that Brahman heifers had greater temperam ent scores (scale 1 to 5) and plasma cortisol concentrations compared to Angus heifers during the summer (3.0 vs. 2.0 for temperament scores, and 39.4 vs. 28.4 ng/mL for cortisol) and fall (2.5 vs. 2.2 for temperament scores, and 36.9 vs. 18.7 ng/mL for cortisol). Fell et al. (1999) reported that plasma co rtisol concentrations were greater for beef steers classified as nervous compared to those classified as calm prior to weaning (156.5 vs. 95.1 nmol/L), after weaning (127.8 vs. 84.3 nmol/L), and at feedlot entry following truck transportation for 350 km (397.8 vs. 229.7 nmol/L). Curley et al. (2006) collected blood samples and assessed pen score, exit velocity, and chute score of 66 yearling Brahman bulls on three different occasions (60 d apart), and repor ted that only pen score and exit velocity were positively correlated with plasma cortisol concentrations (r 0.25 and 0.26, respectively). Curley et al. (2008) stratified Brahman heifers by ex it velocity and classified the fastest ones as temperamental (mean exit velo city = 3.14 m/s), and the slowest ones as calm (mean exit velocity = 1.05 m/s). The authors admi nistered heifers from both groups with bovine CRH, collected blood samples every 15 min from -6 h to 6 h post-challenge, and reported that temperamental heifers had greater plasma ACTH and cortisol concentrations prior to and 42

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following CRH challenge compared to calm heifer s. The same authors administered ACTH to the same heifers 14 d after the CRH challenge, collected blood samples in a similar schedule, and reported greater serum cortisol concentrations fo r temperamental heifers prior to and during the last 2 h following ACTH challenge These data suggest that catt le with excitable temperament experience stimulated hypothalamus-pituitary-ad renal axis function when exposed to humans and handling procedures, resulting in increase d production and circulatin g concentrations of cortisol. This outcome is one of the reasons why cortisol is commonly considered the paramount to the physiologic stress re sponse (Sapolsky et al., 2000). The effects of excitable temperament on the synthesis of sexual steroids by the adrenal gland have been poorly evaluated thus far. Neve rtheless, a fair amount of studies have reported that stressors such as chute restraining, heat, an d ACTH challenge stimulate the synthesis of P4 by the adrenal gland. Roman-Ponce et al. (1981) reported that lacta ting dairy cows with restricted access to shade during the summer in Fl orida had greater P4 co ncentrations during the estrous cycle in addition to increased mean cortis ol concentrations compared to cows with free access to shade. Hollenstein et al. (2005) restrain ed or not (control) ovariectomized Brown Swiss cows in a squeeze chute for 2 h, and reported that restrained cows had greater mean cortisol (33.8 vs. 3.9 ng/mL, respectively) and mean P4 concentrations ( 1.2 vs. 0.3 ng/mL, respectively) compared to control cows duri ng the restraining period. Simila rly, Thun et al. (1998) reported that Brown Swiss cows in estrus experienced consistent increases in plasma P4 and cortisol concentrations during chute restraining for an 8-h period, whereas the e ffects of restraining on plasma concentrations of epinephrine and norep inephrine were inconsis tent, indicating that plasma cortisol concentrations may be a more reliable indicator of stress in cattle than cathecolamines. Yoshida and Nakao (2006) re ported that administ ration of ACTH to 43

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ovariectomized Friesian cows in 4 different doses (3, 6, 12, and 25 UI) linearly increased plasma P4 and cortisol concentrations within 6 h after challenge, whereas peak concentrations of P4 and cortisol across treatments were positively correlat ed (r = 0.70). Research by Willard et al. (2005) reported that administration of ACTH (1.0 IU per kg of BW) to pregnant Brahman heifers resulted in a 1.5 fold-increase in plasma P4 c oncentrations 15 min after challenge, whereas P4 concentrations remained constant for heifers administered saline. C onversely, other studies indicated that administration of ACTH may decrease circulating P4 concentrations of cattle by impairing corpus luteum function. Da Rosa and Wagner (1981) reported that heifers administered ACTH daily had greater plasma P4 concentrations on d 3 and 4 after estrus detection, reduced P4 concentrations during midcycle (d 8 to 19 after estrus detection), and greater plasma corticoid concentrations thr oughout the estrous cycle compared to cows administered saline. Da Rosa and Wagner (1981) al so reported that admini stration of corticoids to adrenalectomized heifers increased plasma co rticoid concentrations and decreased plasma P4 concentrations during the estrous cycle compared to ACTH or saline administration. Similar results were reported by Wagner et al. (1972), indicating that ACTH challenge increased plasma P4 concentrations early in the estrous cycle wh en the corpus luteum is not fully functional by stimulating adrenal P4 production, whereas elevat ed cortisol through ACTH treatment decreased corpus luteum function and consequent luteal P4 synthesis during mid-estrous cycle. In conclusion, research efforts indicate that stimul ation of the hypothalamus-pituitary-adrenal axis increases substantially the production of P4 by the adrenal gland but decreases luteal P4 synthesis, and these events may impact the reproductive function of cattle. Fear responses in cattle with excitable te mperament may also trigger their acute phase response, although no research thus far associated temperament scores directly with circulating 44

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concentrations of acute phase proteins. The acute phase response is a component of the innate immune system and is stimulated by the proinflammatory cytokines IL-1, IL-6, and tumor necrosis factor which are secreted by macrophages and ot her cells in response to external and internal stimuli such as infec tions, inflammations, and trauma (M urata et al., 2004; Petersen et al., 2004). Upon stimulation, these cytokines elic it two major acute re sponses: increased body temperature (fever) and hepatic synthesis of the acute phase pr oteins (Carroll and Forsberg, 2007). Increases in body temperature, which is regulated by proinflammatory cytokines through the stimulation of PGE2, serve mainly to accelera te enzymatic and cellular processes associated with the immune response, and also to decreas e the survival rates of pathogens (Carroll and Forsberg, 2007). In the liver parenchyma, the pr oinflammatory cytokines stimulate hepatocytes to produce acute phase proteins such as ceruloplasmin and ha ptoglobin, which play important roles in tissue protection, repair and reestablishment of homeostas is (Murata et al., 2004; Tizard, 2004; Carroll and Forsberg, 2007). In cattle, the ac ute phase response is stimulated by stressful management procedures such as transporta tion, vaccination, and wean ing (Arthington et al., 2003; Arthington et al., 2005; Cooke et al., 2007c). Recent studies have also indicated that animals subjected to physical or physiologic stressors such as exposure to novel environments and body restraint experience substantial increases in IL-1 and IL -6 synthesis, and consequent stimulation of the acute phase response and hyp othalamic-pituitary-adre nal axis (Turnbull and Rivier, 1999). More specifical ly, IL-1 may cross the blood br ain barrier and stimulate CRH secretion by the hypothalamus, wher eas IL-1 and IL-6 are potent st imulators of ACTH synthesis by the pituitary and consequent co rtisol production by the adrenal cort ex. In turn, cortisol exerts a negative feedback onto cytokine activity to prevent unnecessary inflammation, as commented previously (Kelley, 1988; OConnor et al., 2000; Carroll and Forsberg, 2007). Still, hepatic 45

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synthesis of acute phase proteins was stimulated by administration of glucocorticoids to mature cows and ACTH to piglets (Yoshino et al., 1993; Burger et al., 1998), and in vitro by addition of glucocorticoids to cultures of bovine liver sli ces (Higuchi et al., 1994) Therefore, cattle exhibiting excitable temperament when exposed to humans and handling procedures may also experience elevated concentrations of acute pha se proteins because of the close association between the hypothalamic-pituitary-adrena l axis and the acute phase response. Temperament and Performance of Beef Cattle The effect of temperament on BW gain of feedlot cattle has been evaluated. Burrow and Dillon (1997) detected a negative association between daily BW gain, final liveweight, and carcass weight with exit velocity of Brahman Shorthorn steers and heifers. Voisinet et al. (1997) classified B. taurus and B. indicus B. taurus steers and heifers by temperament (scale 1 to 5), and reported that B. taurus cattle with the calmest temp erament (score 1) had a 0.19 kg/d increase in ADG compared to cohorts with the greatest temperament scor es (score 3), whereas B. indicus -crosses with greater temperament scores (4 and 5) had an approximate decrease of 0.10 kg/d in ADG compared to cohorts with low temp erament scores (1 and 2). Fell et al. (1999) reported greater ADG for Angus Hereford steers with moderate to low chute scores and exit velocity (1.04 kg/d) compared to cohorts with hi gh chute scores and exit velocity (1.46 kg/d). Nkrumah et al. (2007) indicated that chute exit velocity of B. taurus -crosses was negatively correlated with ADG (r = -0.26) and DMI (r = -0 .35), but not with feed conversion (r = 0.03). Similarly, Brown et al. (2004) repor ted that chute exit velocity of Bonsmara bulls was negatively correlated with ADG (r = -0.25) and DMI (r = -0.34), but not with feed efficiency measurements. These data suggest that cattle with excitable temperament experience reduced performance compared to cattle with good temperament, and this effect can be attrib uted to reduced feed 46

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intake in temperamental animals. Further, cat tle with excitable temperament may also have altered metabolism and greater partitioning of nutrients towards physio logic and behavioral responses to stressors compared to calm animals, which results in further decreases in nutrient availability to sustain their main tenance and growth requirements. There is a nutrient requirement for the synthesis and release of hormones and metabolites within the hypothalamus-pituitary-adrenal axis and acute pha se response (Carroll and Forsberg, 2007). Additionally, corticoids, cathecolamines and proinflammatory cytokines stimulate degradation of muscle and adipose tissues to release energy and also aminoacids that are directed towards gluconeogenesis or synthesis of the acute phase proteins by the liver (Johnson et al., 1997; Nelson and Cox, 2005; Carroll and Forsberg, 2007), which may further contribute to the decreased growth rates of temperamental catt le. Elevated circulating concentrations of glucocorticoids and proinflammato ry cytokines also reduces circ ulating IGF-I concentrations, perhaps to shift nutrients from growth to the cont rol of infection and/or inflammation, and also to support the fight or f light response. Studies have shown that cattle receiv ing endotoxin or corticoid challenges have decreased pituitary GH synthesis because of stimulated somatostatin release by the hypothalamus, in a ddition to a reduced number of tissue receptors for GH, causing impaired IGF-I synthesis in th e liver and many other parts of the body (Elsasser et al., 1997; Maciel et al., 2001). Associati ng these physiological events with animal performance, studies conducted by Fell et al. (1999) and Qiu et al. (2007) indicated th at plasma concentrations of cortisol and acute phase protei ns, respectively, were negatively correlated with BW gain of cattle. Therefore, the physiologi cal and behavioral responses e xpressed by temperamental cattle when exposed to human or handling procedures may act as a nutrient sink and impair the 47

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growth-promoting effects of IGF-I, resulting in decreased performance compared to cattle with good temperament. Temperament and Reproduction of Beef Cattle Because of the proximity in the endocrine control of both stress and reproductive axis (hypothalamic-pituitary-adrenal vs hypothalamic-pituitary-ovarian, respectively), the effects of several types of stressors on cat tle reproduction have been inves tigated. Besides the deleterious effects on nutritional status and circulating concentrations of IGF-I, the hormones and metabolites associated with stress directly a ffect the hypothalamic-pituitary-ovarian axis. Diseases and infections (Dobson et al., 2001), excessive heat (Hansen and Arechiga, 1999), and even transportation (Edwards et al., 1987; Harri ngton et al., 1995) were reported to decrease reproductive performance of cattle. Plasse et al. (1970) classified 2-y r-old Brahman heifers according to temperament (1 = calm, 2 = moderate, and 3 = nervous) and reproductive performance (heifers with poor reproductive pe rformance received the greatest scores), and reported that temperament and reproductive scores were positively correlated (r = 0.40), whereas temperament score was negatively correlated with length of estrus (r = -0.33). The authors suggested that consideration of temperament in selection programs might have a positive influence on the reproductive efficiency of the cowherd. Nevertheless, the detrimental effects of excitable temperament on reproductive performan ce and function of cattle still require further investigation. Within the hypothalamus, elevated concentr ations of CRH appear to inhibit GnRH secretion by neurosecreto ry cells in mammals (Moberg et al ., 1991). These events were further verified in livestock by Battaglia et al. (1998). These authors eval uated plasma concentrations of CRH, vasopressin, and GnRH in pituitary portal bl ood of ewes administered endotoxin or saline, 48

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and reported that endotoxin chal lenge potently stimulated CRH and vasopressin secretion and suppressed GnRH release into the pituitary portal blood system. Additionally, the authors reported decreased LH pulsatile secretion in ew es administered endotoxin as a consequence of suppressed GnRH secretion. Othe r studies reported that ACTH and cortisol may decrease LH synthesis by the pituitary even when GnRH secretion is adequate. Li and Wagner (1983) reported that continuous administ ration of ACTH to intact heif ers, or glucocorticoids to adrenalectomized heifers, decreased LH releas e following GnRH challenge compared to heifers infused with saline. The same authors repor ted that addition of glucocorticoids to in vitro cultures of pituitary cells decrea sed basal or GnRH-stimulated LH secretion compared to cultures containing no glucocorticoids, and attributed these outcomes to de leterious effects of glucocorticoids on cellular mechanisms required fo r adequate LH synthesis and release. Dobson et al. (2000) reported that daily ACTH administration to dairy and beef heifers beginning of d 15 of the estrous cycle increased plasma cortisol and disrupted LH pulsatile secretion, resulting in decreased estradiol concen trations, delayed LH surge, and cons equent late or failed ovulation. The negative effects of stress hormones on follicular estradiol synthesis are associated to impaired LH pulsatility and consequent retarded follicular growth, and may further compromise the estradiol signaling in the hypothalamus-pituitary for onset of estrus, LH surge, and consequent ovulation (Smith a nd Dobson, 2002). Hein and Allrich (1992) administered ACTH or vehicle to dairy heifers 30 h follo wing corpus luteum regression by PGF2 treatment and reported that ACTH-treated heifers experien ced delayed peak of estradiol, delayed onset of estrus (63 vs. 44 h, respectively), and greater peak concentrations of serum cortisol (91.0 vs. 23.5 ng/mL, respectively) and P4 (4.1 vs. 1.0 ng/mL, respectivel y) compared with vehicle-treated heifers. The authors suggested that elevated P4 concentrations likely originated from the adrenal gland 49

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delayed the endocrine signaling for onset of estr us and ovulation. The same authors administered ACTH or vehicle to ovariectomi zed dairy cows 10 h following estradiol benzoate treatment to induce estrus, and reported that ACTH-treated cows experienced decreased occurrence of estrus (16% vs. 91% of cows exhibiting estrus, respectiv ely), reduced peak concentrations of estradiol (36.9 vs. 47.5 pg/mL, respectively), and increased peak concentrati ons of serum cortisol (80.5 vs. 43.4 ng/mL, respectively) and P4 (3.0 vs. 0.7 ng/mL ), indicating that stress hormones may also affect the sensitivity of the brain to estrogens (Battaglia et al. 1999). Lastly, glucocorticoids are high ly involved in parturition. Th e fetus produces significant amounts of cortisol when uterin e space becomes limited, stimulati ng the conversion of maternal circulating P4 to estradiol and also synthesis of PGF2 by the placenta, which triggers a cascade of events that lead to pregna ncy termination (Senger, 2003). Therefore, elevated levels of glucocorticoids during pregnancy may cause abortions in cattle. Administration of exogenous glucocorticoids to pregnant beef cows during late-gestation terminated pregnancy mainly by reducing adrenal and placental P4 synthesis (Johnson et al. 1981). Gi ri et al. (1990) reported that beef and dairy cows at different stages of ge station (first, second, and third trimesters) and administered endotoxin challenges for a 6 h period experienced significant increases in plasma cortisol and PGF2 concentrations, resulting in a 55% aborti on rate within 96 h post-challenge. Focusing on early pregnancy loss, Geary et al. (2 005) reported that processing beef heifers, but not cows, through an animal handling facility 13 d after AI was detrimental to pregnancy rates, and attributed these outcomes to deleterious e ffects of handling stress on maintenance of the early pregnancy. Merrill et al. ( 2003) reported that beef cows tran sported via semi-truck for 4 h at moderate air temperature 14 d after AI experi enced greater plasma cortisol concentrations immediately after transporting ( 29 vs. 18 ng/mL, respectively), a nd also numerically decreased 50

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pregnancy rates to AI (69 vs. 76 %, respect ively) compared to non-transported cows. Additionally, cattle exposed to heat stress have in creased incidence of early pregnancy loss, and this effect may be attributed to the elevated temp eratures that the conceptu s is exposed to, and/or to the maternal neuroendocrine responses to heat stress (Hansen and Arechiga, 1999). Nevertheless, further research is required to address the effect s of stress responses on pregnancy maintenance of cattle, especially because the mech anisms by which increased stress, cortisol, or both, might affect PGF2 or initiate pregnancy loss are yet to be establis hed (Merrill et al. 2007). In conclusion, excitable temperament may affect negatively the attainment of puberty, fertility, and maintenance of pregnancy in cattle by im pairing synthesis and secretion of hormones associated with reproductive function, su ch as GnRH, gonadotropins, and steroids. Cattle Acclimation One alternative to alleviate the detrimen tal effects of excitable temperament on productive traits of cattle is to adapt them to humans and handling procedures. However, a limited amount of research has been conducted thus far to address this question. Fordyce et al. (1987) indicated that Brahmancrossbred steers frequently ex posed to handling procedures exhibited calmer temperament compared with cohorts with no handli ng experiences. Crookshank et al. (1979) reported that seru m cortisol concentrations decr eased significantly in calves (transported or not) that were handled and bled several times instead of only once within 16 d after transportation, indicating that calves became more familiarized and hence less excited during handling procedures. Andrad e et al. (2001) subjected Brah man cows to an acclimation process which consisted of restraining cows tw ice weekly during a 19-d period in the squeeze chute for 10 min, and reported that plasma cortis ol concentrations decreased from d 1 to d 19 (3.92 vs. 0.99 ng/mL, respectively), indicating that cows became acclimated to chute restraining. 51

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Curley et al. (2006) assessed exit velocity and cortisol concentrations of yearling Brahman bulls on three separate occasions (d 0, 60, and 120), and reported that mean exit velocity and plasma cortisol concentrations decreased from d 0 to d 120, indicating that cattle became acclimated with humans over the data collection period as reflected by both temperament and endocrine measurements. Echternkamp (1984) compared plasma concentrations of cortisol, LH, and P4 of ovariectomized Hereford cows acclimated to physical restrain for blood collection with those of cohorts with no previous acclimation. The author reported that acclimated cows had decreased mean concentration of cortisol (5.7 vs. 66.1 ng/mL, respectively) and increased mean LH concentration (8.1 vs. 4.1 ng/mL, respectively) and pulsatility (4.3 vs. 1.3 pulses/4 h, respectively) compared to non-acclimated cows. Fu rther, cortisol and P4 concentrations were negatively correlated with LH concentrations (r = -83 and -35, respec tively) within cows, whereas cortisol and P4 were positively correl ated (r = 0.62), suggesting that cattle acclimation may alleviate their neuroendocrine response to handling and consequently enhance their gonadotropin secretory activity and subseque nt reproductive response. In conclusion, acclimation procedures are options to improve endoc rine and behavioral re sponses of cattle with excitable temperament to management events involving human interaction and handling, which may result in beneficial effects on production traits such as BW gain and reproductive performance. Novel Strategies to Enhance the Reproduc tive Efficiency of Cow-Calf Operations Energy supplementation is often required in Florida grazing cow-calf operations to compensate for the reduced nutrient density of fo rages, and thus maintain cattle at adequate levels of nutrition. These supplements are usually offered 3 times or once weekly in order to reduce labor costs. However, recen t data indicate that nutritiona l status of cattle is improved 52

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when supplements are offered daily instead of 3 times weekly. Therefore, offering energy-based supplements to developing heifers or mature co ws on a daily basis may be an alternative to enhance their feed efficiency and reproductive performance, whereas the positive outcomes from daily feeding may at least compensate for th e extra expenses associated with labor. Another disadvantage encountered in typical Fl orida cow-calf operati ons is the excitable temperament of cattle because of their significan t Brahman breeding. Alternatives to ameliorate cattle temperament will likely bring beneficial e ffects to productive and reproductive traits of these animals. Acclimation of cattle to human in teraction and handling procedures alleviate their behavioral and neuroendocrine responses to th ese practices, and are expected to allow for hastened development of heifers and enhanced reproductive efficiency of mature cows. To address these nutritional and manageme nt assumptions, four experiments were conducted to evaluate the effects of supplementation frequency and acclimation to human handling on performance, physiological responses and reproductive performance of Brahmancrossbred females. Results from the experiment s are reported and discussed in the following chapters. 53

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CHAPTER 3 EFFECTS OF SUPPLEMENTATION FREQUENCY ON PERFORMANCE, REPRODUCTIVE, AND METABOLIC RE SPONSES OF BRAHMAN-CROSSBRED FEMALES Introduction Energy supplementation is a common practice in cow-calf systems because BW gain and reproductive function of beef females are influenc ed positively by energy intake (Schillo et al., 1992; Roberts et al., 1997). The la bor expenses associated with supplement feeding contribute significantly to the fixed costs of cattle operations (Miller et al., 2001); therefore offering supplements 3 times or once weekly instead of dail y are typical strategies to decrease costs of production. However, reducing the supplementati on frequency of energy feeds to cattle consuming low-quality forages can be detrimen tal to their performance (Kunkle et al., 2000). Supplementation frequency can affect performance of beef females by many mechanisms, including the modulation of blood concentrations of hormones and metabolites. Infrequent feed intake reduces circulating progest erone (P4) concentrations (Vasconcelos et al., 2003) and consequently may be detrimental to puberty attainment and pregnancy establishment (Gonzalez-Padilla et al., 1975; Spencer and B azer, 2002). Blood concentrations of glucose, insulin, and IGF-I are affected positively by increased supplementation frequency (Cooke et al., 2007b), and these substances are associated with BW gain and reproductive function of cattle (Schillo et al., 1992; Spicer and Echternka mp, 1995). Based on these observations, we hypothesized that beef females consuming low-qua lity forages would benefit if supplemented daily instead of 3 times weekly with an energy supplement. Two experiments were conducted to investigat e the effects of supplementation frequency on Brahman-crossbred females. Experiment 1 ev aluated BW gain, concen trations of plasma Reprinted with permission from the Journal of Animal Science (2008, 86:2296-2309) 54

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metabolites and hormones, mRNA expression of liver and muscle genes associated with metabolism and growth, and reproductive performance of heifers. Experiment 2 assessed plasma metabolites and hormone concentrations, and mRNA expression of hepatic genes associated with nutritional metabolism of mature cows. Materials and Methods Both experiments were conducted at the Univ ersity of Florida IFAS, Range Cattle Research and Education Center, Ona. The firs t experiment was conducted from September to December, 2006 and was divided into a sampling phase (September and October) and a breeding phase (November and December). The second expe riment was conducted during the months of October and November, 2006. The animals utilized in these experiments were cared for in accordance with acceptable practices as outline d in the Guide for the Care and Use of Agricultural Animals in Agricult ural Research and Teaching (FA SS, 1999), and the experimental protocols were reviewed and approved by the University of Florida, Institutional Animal Care and Use Committee (No. E659 and SOP0064). Animals Experiment 1. Fifty-six Brahman Angus heifers (initial BW SD = 228 28 kg; initial age SD = 293 29 d) were utilized in this experiment. For the sampling phase (d 0 to d 45), heifers were stratified by initial BW a nd age, and randomly allocated to 14 pens (4 heifers/pen) on d -11. Pens were assigned rando mly to receive an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. Pen was considered the experimental unit (7 pens/treatment), and each pen consisted of 2 ha of bahiagrass ( Paspalum notatum ) pasture. Heifers were adapte d to assigned treatments from d -11 to d -1. For the breeding phase (d 46 to d 10 7), heifers were re-allocated by treatment into 2 55

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bahiagrass pastures and exposed to Angus bulls. During both sampling and breeding phase, heifers were not rotated among pastures. Experiment 2. Twelve non-lactating, non-pregnant multiparous Brahman British cows (BW SD = 553 50 kg; average age = 6 2 yr) were stratified by BW and age, housed in individual pens, and randomly assigned to receive S3 or S7, at a weekly rate of 20.3 kg of DM per cow. Cow was considered the experimental un it (6 cows/treatment). Prior to the beginning of the experiment, with the purpose of acquiring co ws with similar and substantial plasma P4 concentrations on d 0 of the study, cows received a 100 g treatment of GnRH (Cystorelin; Merial Ltd., Duluth, GA) and received a contro lled internal drug releasing device containing 1.38 g of P4 (CIDR; Pfizer Animal Health, New York, NY) on d -18, PGF2 treatment (25 mg; Lutalyse; Pfizer Animal Health, New York, NY) and CIDR removal on d -12, and a second GnRH treatment (100 g) on d -10. On d -4, cows received another PGF2 treatment (25 mg) and received 2 CIDR that remained in cows throughout the experimental period (d 0 to 17). Transrectal ultrasonography examinations (7.5 MHz transducer, Aloka 500V, Wallingford, CT) were performed immediately and 48 h after second GnRH (d -8) and PGF2 (d -4) treatments to verify ovulation and corpus luteum regression, resp ectively. All cows utilized in this experiment responded to the hormonal treatment, and were adapte d to assigned treatments from d -8 to d 0. Diets Experiment 1. Forage and supplement samples were analyzed for nutrient content by a commercial laboratory (Dairy One Forage Laborat ory, Ithaca, NY). All samples were analyzed by wet chemistry procedures for concentra tions of CP, ADF, and NDF, whereas TDN was calculated using the equation proposed by Weiss et al. (1992). Pasture qual ity was estimated at 54% TDN and 8.8% CP (DM basis) from sample s collected at the beginning and during the 56

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experiment. The pastures utilized in this experi ment were not fertilized prior to or during the experimental period. Stargrass ( Cynodon nlemfuensis ) hay was offered in amounts to ensure ad libitum access when pasture availability was limited. Hay quality was estimated at 53% TDN and 7.7% CP (DM basis) from samples collected at the beginning of the experiment. A complete commercial mineral/vitamin mix (14% Ca, 9% P, 24% NaCl, 0.20% K, 0.30% Mg, 0.20% S, 0.005% Co, 0.15% Cu, 0.02% I, 0.05% Mn, 0.004% Se, 0.3% Zn, 0.08% F, and 82 IU/g of vitamin A) and water were offered for ad libitum consumption throughout the experiment. Random samples of the supplement were also co llected during the expe riment. Composition and nutritional profile of the supplement are described in Table 3-1. Heifers were offered supplement at 0700, daily (S7) or on Mondays, Wednesdays, and Fridays (S3). Experiment 2. Forage and supplement samples were analyzed for nutrient content similarly as in Exp. 1. Stargrass hay was offe red for ad libitum consumption throughout the entire experiment, and hay quality was estima ted at 51% TDN and 6.0% CP (DM basis) from samples collected at the beginning of the e xperiment. Cows had free access to a complete mineral mix (similar to Exp. 1) and water. A random sample of the supplement was also collected at the beginning of the experime nt. Composition and nutritional profile of the supplement are described in Table 3-1. Cows we re offered supplement at 0800, daily (S7) or on Mondays, Wednesdays, and Fridays (S3). Sampling Experiment 1. Heifers were weighed on 2 consecutive days to determine both full and shrunk (after 16 h of feed and wate r restriction) BW before the star t (d -12 and d -11) and at the end of the experiment (d 107 and d 108). Blood samples were collected weekly (Wednesday) throughout the entire experiment to determine ons et of puberty using plasma P4 concentrations. 57

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Heifers were considered pubertal once plasma P4 concentrations were greater than 1.5 ng/mL for 2 consecutive weeks (Cooke et al., 2007a). During the sampling phase, in addition to th e weekly collections, blood samples were obtained once per day during 4 consecutive days every other week, starting at 4 h after supplement was offered to determine concentr ations of glucose, blood urea nitrogen (BUN), insulin, IGF-I, and P4. These samples were collected from d 0 to d 3, d 14 to d 17, d 28 to d 31, and d 42 to d 45, which were classified as pe riods (PR1, PR2, PR3 and PR4, respectively). Periods began on Monday and ended on Thursday. Heifers within pen were assigned randomly for either muscle or liver biopsying on d 35 or 36 of the experiment (Monday or Tuesday). Biopsy procedures began 4 h after supplement was offered. As a result, 2 liver and 2 muscle samples were obtained from each pen for quantitative real-time RT-PCR assessment of IGF-I, IGFBP-3, pyruvate carboxylase (PC), cytosolic phosphoenolpyruvate carboxykinase (P EPCK-C), mitochondrial PEPCK (PEPCK-M), and cyclophilin mRNA expression in liver sample s, and IGF-I, IGFBP-3, IGFBP-5, myostatin, and cyclophilin mRNA expression in muscle samples. During the breeding phase, each treatment group was exposed to 2 mature Angus bulls at the same time (1:14 bull:heifer ra tio), and bulls were rotated w eekly between groups to account for potential bull effects. Heif er pregnancy status was verifi ed by detecting a fetus with transrectal ultrasonography (5.0 MHz transducer, Aloka 500V) 70 d after the end of the experiment. Date of conception was estimated retrospectively by subtra cting gestation length (286 d; Reynolds et al., 1980) from the calving date. Experiment 2. During a 3-wk period, blood samples were collected immediately prior to and 4, 8, 24, 28, and 32 h after the first supplement feeding of the week in which cows from both 58

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treatments were offered supplements (Mondays ; d 1, 8, and 15). Blood samples were analyzed for concentrations of glucose, BUN, insulin, IGF-I, and P4. Liver samples were collected on d 15 and 16 vi a needle biopsy, concurrently with blood samplings at 4 and 28 h after first supplement feeding of wk 3, to determine the mRNA expression of IGF-I, IGFBP-3, PC, PEPCK-C, PE PCK-M, and cyclophilin via quantitative realtime RT-PCR. Blood Analysis Blood samples were collected via jugular ve nipuncture during Exp. 1 and from coccygeal vein or artery during Exp. 2 into commercial blood collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin, placed on ice immediately, and centrifuged at 2,400 g for 30 min for plasma collection. Plasma was frozen at -20C on the same day of collection. Glucose and BUN concentrations were determ ined using quantitative colorimetric kits G7521 and B7551, respectively (Poi nte Scientific, Inc., Canton, MI). A double antibody RIA was used to determine concentrations of insulin (Malven et al., 1987; Ba dinga et al., 1991), and IGF-I (Badinga et al., 1991). Th e extraction procedure used in the IGF-I assay was modified from Badinga et al. (1991) by using an ethanol:a cetone:acetate ratio of 6: 3:1. Concentrations of P4 were determined using Coat-A-Count solid phase 125I RIA kit (DPC Dia gnostic Products Inc., Los Angeles, CA). The intraand inter-assay CV for Exp. 1 were, respectively, 4.1 and 2.7% for glucose, 3.8 and 5.3% for BUN, 8.8 and 7.9% for insulin, 8.9 and 11.8% for IGF-I, and 4.8 and 5.9% for P4. The intraand inter-assay CV for Exp. 2 were, respectively, 4.8 and 6.4% for glucose, 3.8 and 9.5% for BUN, 12.3 and 12.0% fo r insulin, 8.6 and 5.1% for IGF-I, and 6.7 and 6.1% for P4. 59

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Tissue Analysis Tissue Collection and RNA Extraction Liver and LM biopsies were performed by trained personnel following the techniques de scribed by Arthington and Corah (1995). Immediately after collection, liver and muscle samples (average 100 mg of tissue; wet weight) were placed in 1 mL of RNA stabilization solution (RNAlater; Ambion, Inc., Austin, TX), maintained at 4C for 24 h, and stored at -20C. Total RNA was extracted from tissue sample s using TRIzol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA). Quantity and quality of isolated RNA were assessed via UV absorbance at 260 nm and 260/280 nm ratio, re spectively (GeneQuant spectrophotometer; Amersham Pharmacia Biotech, Cambridge, UK). Extracted RNA was stored at -80C until further processing. Real-Time RT-PCR Extracted RNA from liver and muscle samples (2.5 and 1.0 g, respectively) were incubated at 37oC for 30 min in the presence of RNase-free DNase (New England Biolabs, Inc., Ipswich, MA) to remove contaminant genomic DNA. After inactivating the DNase (75oC for 15 min), samples were reverse-tr anscribed using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems; Foster City, CA). Realtime PCR was completed using the SYBR Green PCR Master Mix (Applied Biosystems) and specific primer sets (25 ng/mL; Table 3-2) with a 7300 Real-Time PCR System (Applied Biosystems). Following incubation at 95C for 10 min, 40 cycles of denaturation (95oC for 15 sec) and annealing/synthesis (60oC for 2 min) were completed. Each RNA sample was analyzed in triplicate, and the absence of genomic c ontamination was verified by including a fourth reaction lacking exposure to the reverse transcri ptase. At the end of each PCR, amplified products were subjected to a di ssociation gradient (95C for 15 sec, 60C for 30 sec, and 95C 60

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for 15 sec) to verify the amplification of a single product by denaturation at the anticipated temperature. A portion of the amplified produc ts were purified with the QIAquick PCR purification kit (Qiagen Inc.; Va lencia, CA) and sequenced at the University of Florida DNA Sequencing Core Facility to verify the specifi city of amplification. All amplified products represented only the genes of interest. Responses were quantified base d on the threshold cycle (CT), the number of PCR cycles required for target amplification to re ach a pre-determined threshold. All CT responses from genes of interest were no rmalized to cyclophilin CT examined in the same sample and run at the same time as the targets. Results ar e expressed as relative fold change (2CT), as described by Ocn-Grove et al. (2008). Internal RNA standard samples were include d in each assay (PCR plate). The inter-assay CV for Exp. 1 was 0.7% for liver IGF-I, 0.6% for liver IGFBP-3, 0.6% for PC, 0.6% for PEPCKC, 0.8% for PEPCK-M, 0.7% for muscle IGF-I, 0.5% for muscle IGFBP-3, 0.9% for IGFBP-5, and 0.7% for myostatin. The inter-assay CV for Exp. 2 was 0.9% for liver IGF-I, 1.0% for liver IGFBP-3, 0.9% for PC, 1.1% for PEPCK-C, and 1.2% for PEPCK-M. Statistical Analysis Experiment 1. Performance, physiological, and gene expression data were analyzed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC) and Satterthwaite approximation to determine the denominator df for the tests of fixed effects. Gene expression data were further tested for normality with the Shapiro-Wilk te st from the UNIVARIATE procedure of SAS, and results indicated that all data were distributed normally (W 0.90). The model statement used for hormone and metabolite analysis contained th e effects of treatment, period, day(period), and the interactions of treatment period and treatment day(period). Data were analyzed using 61

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heifer(pen) and pen(treatment) as random variables. The model statement used for ADG contained only the effect of treatment. Data were analyzed using pen(treatment) as the random variable. The model statement used for gene expression analysis contained the effects of treatment, day, and the interaction. Results are re ported as least square m eans and were separated using LSD. Puberty and pregnancy data were analyzed with survival analysis (LIFETEST procedure of SAS) by regressing the proportion of prepubertal or non-pregnant heifers on week of the study or breeding season, respectively. Differences between treatment survival curves were determined by the Wilcoxon test. For all analysis, signif icance was set at P 0.05, tendencies were determined if P > 0.05 and 0.10, and results are reported according to treatment effects if no interactions were significant, or according to the highest-order interaction detected. Experiment 2. Data were analyzed using the PROC MIXED procedure of SAS and Satterthwaite approximation to determine the denominator df for the tests of fixed effects. Gene expression data also were tested for normality with the Shapiro-Wilk test from the UNIVARIATE procedure of SAS, an d results indicated that all da ta were distributed normally (W 0.90). The model statement used for hormone a nd metabolite analysis contained the effects of treatment, week, time(week), and the intera ctions of treatment week and treatment time(week). Data were analyzed using cow(trea tment) and cow(treatment) week as random variables. The model statement used for gene expression analysis contained the effects of treatment, day, and the interaction. The random variable was cow(treatment). Additionally, mRNA expression of PEPCK-C, PEPCK-M, a nd PC were analyzed jointly. This model statement contained the effects of treatment, day, enzyme, and the resultant interactions, whereas cow(treatment) was the random va riable. Significance was set at P 0.05, tendencies were 62

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determined if P > 0.05 and 0.10. Results reported are least squa re means, were separated using LSD, and are reported according to treatment eff ects if no interactions were significant, or according to the highest-ord er interaction detected. Results and Discussion Experiment 1 Heifers provided S7 had greater ( P = 0.03) ADG compared to S3 heifers (0.41 vs. 0.33 kg/d, respectively; SEM = 0.02), concurring with st udies reporting increased BW gain of cattle fed low-quality forages and offered energy supplem ents daily vs. 3 times weekly (Kunkle et al., 2000; Cooke et al., 2007b). Reproductive perfor mance was also affected by treatments. Attainment of puberty (Figure 3-1) and pr egnancy (Figure 3-2) were hastened (P = 0.03 and 0.02, respectively) in S7 heifers comp ared to S3 heifers. In a revi ew article, Kunkle et al. (2000) indicated that decreased supplementation freque ncy of energy supplements containing highstarch ingredients is detrimen tal to cattle performance because of impaired rumen function, forage intake, and digestibility. However, different outcomes were observed in cattle supplemented with low-starch energy byproducts. L oy et al. (2007) reported similar mean forage intake, rumen pH, and in situ forage NDF disappearance of b eef heifers supplemented with distillers grains daily or on alternate days. Cooke et al. (2007 b) reported that beef steers offered citrus pulp-based supplements daily had similar mean forage DMI but improved ADG compared to steers offered the same supplement 3 times w eekly, and attributed the differences in ADG to beneficial effects of daily supplementation on concentrations of hormones and metabolites associated with energy intake. A treatment x day(period) in teraction was detected ( P < 0.01) for BUN analysis (Table 33). During the initial half of PR2, and thr oughout PR3 and PR4, S3 heifers had greater ( P 0.05) 63

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BUN concentrations compared to S7 heifers duri ng the days that only S7 heifers were offered supplements, but decreased (P < 0.01) BUN concentrations during the days that both treatment groups were offered supplements. Concentrations of BUN are positively associated with intake of rumen-degradable protein, le vels of ruminal ammonia, and ruminal protein:energy ratio (Hammond, 1997). The reductions in BUN concentrati ons detected in S3 heifers on days that both treatment groups were supplemented reflected a decreased ruminal protein:energy ratio created in these heifers because they were offered a greate r amount of supplemental energy compared to S7 heifers. Conversely, increased BUN concentrations of S3 heifers during days that only S7 heifers were supplemented reflec ted an increased ruminal protein:energy ratio because S3 heifers did not receive any supplemental energy but likely maintained a significant ruminal N supply due to urea-recycling (Lapierre and Lobley, 2001). This biphasic pattern of BUN concentrations detected in S3 heifers was also reported by others (Bohnert et al., 2002) and illustrates the greater daily variation of energy and protein intake of S3 heifers compared to S7 heifers. Heifers from both treatments frequently had BUN concentrations above the optimal level (11 and 15 mg/dL; Byers and Moxon, 1980), indicati ng that rumen-degradable protein and thus CP were consumed in excess by these animals. Further, the sharp increases in BUN detected for S3 heifers during non-supplementation days may ha ve influenced negatively their performance because excessive rumen ammonia concentrations requires additional energy to be metabolized into urea by the liver (Reynolds, 1992). A treatment x day(period) in teraction was detected ( P < 0.01) for glucose and insulin analysis (Table 3-3 and 3-4, respectively) A day(period) effect was detected (P < 0.01) for glucose concentrations of S3 heifers throughout the experimental period, whereas a tendency ( P = 0.06) for a day(effect) in gluc ose concentrations of S7 heif ers was detected only during PR4. 64

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Similarly, a day(period) effect was detected (P < 0.01) for insulin concentrations of S3 heifers throughout the experimental peri od, whereas it was significant ( P = 0.02) for S7 heifers only during PR4. In addition, glucose and insulin concentrations were ty pically greater for S3 heifers compared to S7 heifers on the days that only S7 heifers were supplemented, but not on days that both treatment groups were supplemented (Tab le 3-3 and 3-4). Significant differences ( P 0.05) were detected on Wednesday during PR1, Tues day during PR2, Tuesday and Thursday during PR3, and Tuesday during PR4 for glucose, a nd on Tuesday during PR2, PR3, and PR4 for insulin. Concentrations of plasma glucose and insulin are influen ced positively by rate of nutrient intake (Vizcarra et al., 1998), therefore the day(period) effects observed in this experiment reflected the differences in nutrient intake patte rn between treatments. During the experimental period, S3 heifers received and readily consumed (within 2 h) bolus amounts of supplements on 3 d of the week, but had low-quality forage as th e only source of feed available on the remaining days. Conversely, S7 heifers received and consum ed within 1 h smaller portions of supplements on a daily basis. A treatment x day interaction was detect ed for mRNA expression of liver PC ( P = 0.02) and PEPCK-C ( P < 0.01; Table 3-5). These interactions were detected because a day effect ( P < 0.01) was significant for these transcripts in S3 he ifers but not in S7 heifers. Additionally, the mRNA expressions of PC and PEPCK-C were greater ( P < 0.01 and P = 0.02, respectively) for S3 heifers compared to S7 heifers when both treatment groups were supplemented (d 35), but were similar or greater (PEPCK-C; P = 0.04) for S7 heifers when only these were supplemented (d 36). Treatment effects on liver PEPCKM mRNA expression differed from the other gluconeogenic transcripts because S7 heifers tended ( P = 0.08) to have a greater mRNA expression of liver PEPCK-M compared to S3 heif ers (Table 3-5). The treatment effects detected 65

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on mRNA expression of liver PC and PEPCK-C also reflect differences in the nutrient intake pattern between S3 and S7 heifers. Expression of these enzymes was associated positively with enzymatic activity and consequent glucose synthe sis in cattle (Greenfiel d et al., 2000; Agca et al., 2002; Bradford and Allen, 2005). Given that the availability of nutrients originating from ruminal fermentation rapidly increase in forage -fed ruminants after supplementation (Seoane and Moore, 1969; Rihani et al., 1993; Farmer et al., 2001) and that expression of liver enzymes associated with gluconeogenesis are quickly a ltered (She et al., 1999; Massillon et al., 2003) and increased when precursors are av ailable (Greenfield et al., 2 000; Karcher et al., 2007), the greater mRNA expression of PC and PEPCK-C detected in S3 heif ers compared to S7 heifers when both treatment groups were offered supplem ents can be attributed to their greater supplement consumption on that day. Accordin gly, mRNA expression of these enzymes was significantly reduced in S3 heif ers but not in S7 heifers when only S7 heifers received supplements. The reason to why PC and PE PCK-C expression was altered by 4 h postsupplementation whereas plasma glucose and insu lin responses to supplement consumption were not detected until the next day in S3 heifers are likely due to the time required for PC and PEPCK-C mRNA to be translated into the active enzymes and substantially change the magnitude of glucose synthesis and release by th e liver. Previous studie s also reported that plasma glucose concentrations of forage-f ed developing heifers (Cooke et al., 2007a) and yearling steers (Cooke et al., 2007b) offered s upplements based on low-starch energy byproducts 3 times weekly were greater at 28 vs. 4 h after supplementation. Liver PEPCK-M may be responsible for as much as 61% of glucose s ynthesis in ruminant hepatocytes (Aiello and Armentano, 1987), although it is considered c onstitutive and not responsive to hormones and nutritional state (Greenfield et al., 2000; Agca et al., 2002). Ne vertheless, the tendency for 66

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greater mRNA expression of liver PEPCK-M in S7 heifers compared to S3 heifers may be an indicator of their improved energy status and contributed to their in creased performance. Concentrations of IGF-I were typically increased for S7 heif ers compared to S3 heifers, and there was a treatment x day(period) interaction ( P < 0.01; Table 3-4). Si gnificant differences ( P 0.05) were detected on Mondays (PR1, PR 2, PR3, and PR4), and Wednesday of PR4. Furthermore, mean IGF-I concentrations tended ( P = 0.10) to be greater for S7 vs. S3 heifers (190 and 161 ng/mL, respectively; SEM = 12). Nu tritional status, growth, and reproductive performance of beef cattle are influenced positively by circulat ing IGF-I (Roberts et al., 1997; Diskin et al., 2003; Cooke et al ., 2007a). Therefore, the treatm ent effects detected for ADG and reproductive performance in this experiment can be attributed, at least to some degree, to the greater concentrations of IGF-I frequently detected for S7 compared to S3 heifers. Although several studies reported a positive association betw een IGF-I concentrations and nutrient intake (Ellenberger et al., 1989; Bossis et al., 1999; Lapie rre et al., 2000), treatment differences in IGF-I concentrations were detected de spite the similarity in overall supplement intake between S3 and S7 heifers. Thus, it remains possible that such differences resulted from treatment effects on forage intake, but this explanation is unlikely be cause previous research efforts reported similar mean forage intake of cattle offered low-starch supplements daily or 3 times weekly (Cooke et al., 2007b; Loy et al., 2007). In addition, increases in plasma IGF-I concentrations due to enhanced nutrient or feed intake are usually accompanied by increased mean glucose and insulin concentrations (Bossis et al., 1999; Lapierre et al., 2000; Hersom et al., 2004), and nutrients originated from low-quality forages do not contribu te significantly to hepatic IGF-I synthesis and release (Cooke et al., 2007a). 67

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Heifers provided S7 had greater ( P = 0.04) expression of liver IGF-I mRNA compared to S3 heifers (Table 3-5). A treatment x day interaction was detected ( P = 0.04) for liver IGFBP-3 because a day effect (P < 0.01) was detected for IGFBP3 mR NA expression in S3 but not in S7 heifers, whereas IGFBP3 mR NA expression was greater ( P = 0.03) for S3 heifers compared to S7 heifers when both treatment groups were supplemented (d 35) but similar when only S7 heifers were supplemented (d 36). The major sour ce of circulating IGF-I synthesis is the liver (DErcole et al., 1984). Blood concentrations of IGF-I are associat ed with IGF-I mRNA expression in hepatic cells (Thissen et al., 1994), and this relationship is supported by the present experiment. The availability of energy substrat es has a positive influence on the expression of liver IGF-I mRNA and consequent translation into the circulati ng protein (McGui re et al., 1992; Thissen et al., 1994). Increased hepatic IGF-I mRNA expression and plasma IGF-I concentrations detected in S7 heifers may be attri buted to a greater availab ility of substrates for metabolic and physiologic processe s in these heifers due to an improved efficiency of nutrient metabolism. By consuming small quantities of supplement on a daily basis, S7 heifers were perhaps capable of retaining and processing these nutrients more efficiently than S3 heifers, although further research efforts are required to evaluate this assumption. Treatment effects on liver IGFBP3 mRNA expression differed from those detected for IGF-I, although IGFBP3 expression and synthesis are stimulated by circul ating IGF-I (Thissen et al., 1994). Nevertheless, the day effect detected for expression of liver IGFBP-3 mRNA only in S3 heifers may be an additional indicator of the daily variation of nutrient intake an d consequent availability of substrates for metabolic processes in these heifers. A treatment x day interaction was detected ( P = 0.05) for muscle myostatin mRNA expression (Figure 3-3). A day effect ( P < 0.01) was detected in S3 he ifers but not in S7 heifers. 68

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Further, myostatin mRNA expression was greater ( P = 0.04) for S3 heifers compared to S7 heifers when both treatment groups were supplemented (d 35) but similar when only S7 heifers were supplemented (d 36). Myostatin is a growth factor expressed in skeletal muscle of developing and mature animals that negatively influences muscle growth (Lee and McPherron, 2001; Dayton and White, 2008). Myostati n is believed to impair glucose uptake in muscle tissues by decreasing the activity of insulin-dependent GLUT4, resulting in insulin resistance and impaired muscle tissue development (Strassman et al., 2002; Antony et al., 2007). The treatment effects detected for myostatin mRNA expression i ndicate that muscle tissues of S7 heifers were developing in a more constant pattern compared to those of S3 heifers, possibly due to treatment differences on nutrient intake and availability. No further treatment diffe rences were detected within mRNA expression of muscle genes (data not shown). Nevertheless, the lack of treatment effects on muscle IGF-I and IGFBP may indicate th at within the IGF-I sources, circulating IGF-I was likely the major contributor for heifer body growth. Supporting our findings, Johnson et al. (1996) reported that muscle grow th is significantly stimulated by circulating IGF-I. Conversely, IGF-I synthesized by skeletal muscle in growing cattle is implicated as an important and often essential autocrine and paracrine mediator of tis sue development and differentiation (McGuire et al., 1992; Johnson et al., 1998), and research with mice suggested that IGF-I synthesized in muscle tissues exerts a more important role in muscle growth compared to hepatic-originated IGF-I (Sjogren et al., 19 99; Yakar et al., 1999). In summary, offering an energy supplement based on fibrous byproducts daily instead of 3 times weekly to developing heif ers resulted in a normalized mR NA expression pattern of genes associated with nutritional metabolism and growth, reduced daily variation in plasma concentrations of BUN, glucose, and insulin, and improved heifer nutritional status as reflected 69

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by increased hepatic mRNA expression and elevated plasma concentrations of IGF-I. These beneficial metabolic effects were translated into greater BW gain and hastened attainment of puberty and pregnancy; therefor e, daily supplementation of high-energy byproducts enhances development and performance of Brahman-crossb red heifers consuming low-quality forages. Experiment 2 A treatment effect and a treatment x tim e(week) interaction were detected ( P = 0.03 and P < 0.01, respectively) for BUN (Table 3-6) Cows receiving S7 had increased BUN concentrations compared to S3 cows during wk 2 and 3 (Table 3-6), with significant differences ( P < 0.05) detected at 4 and 8 h after the first supp lement feeding of wk 2, and at 0, 4, 8, 24 and 32 h after the first supplement feeding of wk 3. Additionally, S7 cows had greater mean BUN concentrations compared to S3 cows (9.2 vs. 7.9 mg/dL, respectively; SEM = 0.36). These results differed from Exp. 1 because S7 co ws likely had greater mean ruminal ammonia concentrations compared to S3 cows, BUN concen trations increased in a similar pattern for S3 and S7 cows after supplement consumption, and were typically at adequate levels for both treatments (8 mg/dL; Hammond, 1997). The differe nces in BUN responses between Exp. 1 and 2 may be explained by the reduced amount of supplement and thus rumen-degradable protein consumed by cows vs. heifers in relation to their BW (0.5 and 1.0% of BW on a daily basis, respectively). Cows were offered supplements at a daily rate of 0.5% of BW to avoid energy overfeeding that may hinder the detection of trea tment effects (Cooke et al., 2007a), whereas this rate is adequate to maintain brood cows at a moderate positive energy balance (NRC, 1996). A treatment x time(week) interaction was detected ( P = 0.01) for insulin (Table 3-6) because a time(week) effect was detected fo r insulin concentrations of S3 cows ( P < 0.01) but not S7 cows, and treatment differences were detected at 0 h ( P = 0.03) and 48 h ( P = 0.05) after 70

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the first supplement feeding of wk 2 (Table 3-6). Similarly, a significant time(week) effect was detected for glucose concentrations of S3 cows ( P < 0.01) but not S7 cows, although a treatment x time(week) interaction was not significant ( P = 0.12; data not shown). Within the gluconeogenic enzymes, a treatment x day interaction was detected ( P < 0.01) only for PC mRNA expression. Similar to Exp. 1, PC mRNA expression was numerically greater for S3 cows compared to S7 cows when both trea tment groups were supplemented (d 15), but numerically greater for S7 cows when only these were offered supplements (d 16; Table 3-7). When data from all 3 gluconeogenic enzymes are analyzed jointly, S3 cows tended ( P = 0.09) to have increased mRNA expression of these transc ripts on d 15 (2.49 vs. 2.07 relative fold change, respectively; SEM = 0.17), but reduced ( P = 0.03) mRNA expression of these transcripts on d 16 (1.48 vs. 2.03 relative fold change, respectively; SEM = 0.17) compared to S7 cows (treatment x day interaction, P < 0.01). Further, a day effect was obser ved in the combined mRNA expression of gluconeogenic enzymes for S3 cows ( P < 0.01), but not for S7 cows. These results support those reported in Exp. 1 particularly because su pplement intake behavior was similar in both experiments, and indicate that concentrations of glucose and insulin, and expression of gluconeogenic enzymes transcripts varied significantly within 32 h after supplementation in S3 cows, but remained generally constant for S7 co ws during the same time period due to treatment differences in nutrient intake pattern. Different from Exp. 1, treatment effects detected for PEPCK-M mRNA expression were similar to thos e detected for PC and PEPCK-C, indicating that mRNA expression of this transcript was also influenced by nutrient intake pattern. A treatment x week interaction was detected (P = 0.02) for IGF-I (Table 3-8) because concentrations of this hormone increased for S7 cows ( P < 0.01) but not for S3 cows with the advance of the experiment. As a consequence, S7 cows tended ( P = 0.09) to have greater IGF-I 71

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concentrations compared to S3 cows on wk 3 (T able 3-8), indicating that nutritional status was improving for S7 cows compared to S3 cows with the advance of the experiment. No treatment effects were detected in the expression of liver IGF-I and IGFBP-3 mRNA (Table 3-7). However, liver IGF-I mRNA expression was numerica lly increased for S7 cows compared to S3 cows, and the lack of statistical significance is most likely related to the reduced number of animals utilized in this experiment. No differences were detected for plasma P4 concentrations (data not shown) between treatments. A week effect was detected ( P < 0.01) because P4 concentrations decreased linearly for both treatments from the first week to the last week of the experiment, and this effect can be associated with the decrease in P4 release from CIDR with advancing time (6.62, 4.54, and 2.68 ng/mL for wk 1, 2, and 3, respectively; P < 0.01; SEM = 0.37). Infrequent intake of large amounts of feed decreases circulating concentrati ons of P4 in cattle (Vasconcelos, et al., 2003, Cooke et al., 2007a), and therefore may impair reproductive performance because P4 is required for adequate attainment of puberty (GonzalezPadilla et al., 1975) and establishment of pregnancy (Spencer and Bazer, 2002). Cooke et al (2007a) reported that b eef heifers and cows consuming low-quality forages and offered suppl ements based on low-starch energy byproducts 3 times weekly at a daily rate of 1.0% of BW ha d reduced plasma P4 concentrations on days that supplements were offered vs. days that supplemen ts were not offered. In the present experiment, cows were offered moderate amounts of supplem ent (0.5% of BW on a daily basis) to avoid energy overfeeding; therefore this lower level of supplementation likely prevented plasma P4 concentrations from changing sign ificantly. Conversely, heifers fr om Exp. 1 were supplemented at 1.0% of BW on a daily basis and treatment ef fects on P4 concentrations were not evaluated due to the reduced number of pubertal heifers during the sampling phase. Consequently, further 72

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research is required to address the effects of supplementation frequenc y on circulating P4 metabolism of developing heifers. In summary, offering an energy supplement based on fibrous byproducts daily instead of 3 times weekly to mature cows resulted in a normalized pattern of gluconeogenic enzymes mRNA expression, reduced variation in plasma concentrations of gl ucose and insulin, and improved cow nutritional status as observed by increasing plasma IGF-I concentrations. Therefore, it could be postulat ed that Brahman-crossbred cows consuming low-quality forages and supplemented daily with high-energy byprod ucts would experience improved performance and reproductive efficiency compared to cows supplemented 3 times weekly, although further research is required to address this matter. 73

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Table 3-1. Ingredient composition and nutrient profile of the suppl ement offered in Exp. 1 and 2. Item Concentration Ingredients, as-fed basis Wheat middlings, % 66.67 Soybean hulls, % 26.93 Blackstrap molasses, % 3.75 Cottonseed meal, % 2.65 Nutrient profile1, DM basis DM, % 88.9 TDN, % 62.5 NEm, Mcal/kg2 1.35 NEg, Mcal/kg2 0.77 CP, % 22.2 Rumen degradable protein3, % 16.6 Non-fiber carbohydrates, % 32.1 NDF, % 39.4 ADF, % 18.5 Ca, % 0.31 P, % 0.94 1 Values obtained from a commercial laborator y wet chemistry analysis (Dairy One Forage Laboratory, Ithaca, NY); TDN calcu lated as described by Weiss et al. (1992). 2 Calculated with the equations proposed by the NRC (1988). 3 Estimated using the NRC model (2001). 74

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Table 3-2. Primer sets used for quantitative real-time RT-PCR. Target gene Primer sequence1 Product size Accession no IGF-I Forward 5CTC CTC GCA TCT CTT CTA TCT -3 236 bp NM_001077828 Reverse 5ACT CAT CCA CGA TTC CTG TCT -3 IGFBP-3 Forward 5AAT GGC AGT GAG TCG GAA GA -3 194 bp NM_174556.1 Reverse 5AAG TTC TGG GTG TCT GTG CT -3 IGFBP-5 Forward 5TCC GAG ATG GCA GAG GAG -3 289 bp XM_600908.3 Reverse 5GGT CAC AGT TGG GCA GGT -3 Pyruvate carboxylase Forward 5CCA ACG GGT TTC AGA GAC AT -3 184 bp NM_177946.3 Reverse 5TGA AGC TGT GGG CAA CAT AG -3 Cytosolic phosphoenolpyruvate carboxykinase Forward 5CAA CTA CTC AGC CAA AAT CG -3 115 bp NM_174737.2 Reverse 5ATC GCA GAT GTG GAC TTG -3 Mitochondrial phosphoenolpyr uvate carboxykinase Forward 5GCT ACA ACT TTG GGC GCT AC -3 168 bp XM_583200 Reverse 5GTC GGC AGA TCC AGT CTA GC -3 Myostatin Forward 5GTG TTG CAG AAC TGG CTC AA -3 215 bp NM_001001525 Reverse 5CAG CAT CGA GAT TCT GTG GA -3 Cyclophilin Forward 5GGT ACT GGT GGC AAG TCC AT -3 259 bp NM_178320.2 Reverse 5GCC AT C CAA CCA CTC AGT CT -5 1 Primers sequences obtained from: IGF-I a nd IGFBP-5, Wall et al. (2005); IGFBP-3, Li et al. (2007); PC and PEPCK-C, Dr Erin Conner, BARC/USDA; PEPCK-M, myostatin and cyclophilin, Jellyfish Sequen ce Analysis Software (Field Scientific LLC, Lewisburg, PA). 75

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1 Days at which heifers from both treatme nts were offered supplements are underlin ed. Supplements were offered at 0700 h. 2 Samples were collected from d 0 to d 3, d 14 to d 17, d 28 to d 31, and d 42 to d 45 of the study, which were classified as pe riods 1 to 4, respectively. Periods bega n on Monday and ended on Thursday. 3 Treatmen t compar ison within pe rio d and d ay. Table 3-3. Blood urea nitrogen (BUN) and plasma glucose concentra tions (mg/dL) of heifers (Exp. 1) offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. 1,2 76

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1 Days at which heifers from both treatme nts were offered supplement s are underlined. Supplements were offered at 0700 h. 2 Samples were collected from d 0 to d 3, d 14 to d 17, d 28 to d 31, and d 42 to d 45 of the study, which were classified as pe riods 1 to 4, respectively. Periods began on Monday and ended on Thursday. 3 Treatmen t compar ison within pe rio d and d ay. Table 3-4. Plasma concentrations (ng/mL) of insulin and IGF-I of heifers (Exp. 1) o ffered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. 1,2 77

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78 1 Heifers from both S3 and S7 groups were supplemented on d 35, whereas only S7 heifers were supplemented on d 36 of the study. Tissue collection star ted 4 h after supplement feeding time. 2 Values are expressed as relative fo ld change (Ocn-Grove et al., 2008). 3 PC = Pyruvate carboxylase; PECK-C = cytosolic phosphoenolpyruvate carboxykinase ; PEPCK-M = mitochondrial PEPCK. 4 Treatment comparison within sampling day. Table 3-5. Expression of hepatic genes associated with nut ritional metabolism and growth of heifers (Exp. 1) offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. 1,2

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79 Table 3-6. Blood urea nitrogen (BUN) and plasma insulin concentrations of cows (Exp. 2) offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 20.3 kg of DM per cow. 1 1 Cows from both treatments were offered supplements after blood was sampled at 0 h. 2 Treatment comparison within week and hour.

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Table 3-7. Expression of hepatic genes associated with nut ritional metabolism and status of cows (Exp. 2) offered an energy supplement based on fibrous byproducts daily (S 7) or 3 times weekly (S3), at a weekly rate of 20.3 kg of DM per cow. 1,2 1 Cows from both S3 and S7 groups were supplemented on d 15, whereas only S7 cows were supplemented on d 16 of the study. Liver samples were colle cted 4 h after suppl ement feeding time. 2 Values are expressed as relative fo ld change (Ocn-Grove et al., 2008). 3 PC = Pyruvate carboxylase; PECK-C = cytosolic phosphoenolpyruvate carboxykinase; PEPCK-M = mitochondrial PEPCK 4 80

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Table 3-8. Plasma IGF-I concentrations (ng/mL) of cows (Exp. 2) offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 20.3 kg of DM per cow. 1 Item Wk 1 Wk 2 Wk 3 SEM S7 196 201 244 18 S3 203 18 191 196 P1 0.79 0.71 0.09 1 Treatment comparison within week. 81

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0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 12345678910111213141516Proportion of prepubertal heifersWeek of study S7 S3 Figure 3-1. Proportion of prepubertal heifers by week during Exp. 1. Survival curves represent heifers offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a w eekly rate of 18.2 kg of DM per heifer. Heifers were considered pubertal once plasma progester one concentrations we re greater than 1.5 ng/mL for 2 consecutive weeks, and puberty a ttainment was declared at the first week of elevated progesterone. A tr eatment effect was detected ( P = 0.03). 82

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0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 8910111213141516Proportion of non-pregnant heifersWeek of study S7 S3 Figure 3-2. Proportion of non-pregna nt heifers by week during the 60-d breeding season of Exp. 1. Survival curves represent heifers offe red an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3) at a weekly rate of 18.2 kg of DM per heifer. Date of conception was estimated retrospectively by s ubtracting gestation length (286 d; Reynolds et al ., 1980) from the calving date A treatment effect was detected (P = 0.02). 83

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0 1 2 3 4 5 6 7 S3 and S7 supplemented (Day 35) Only S7 supplemented (Day 36)Myostatin expression, relative fold change S7 S3*Figure 3-3. Expression of muscle myostatin, as rela tive fold change (Oc n-Grove et al., 2008), of heifers (Exp. 1) offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. Heifers from both S3 and S7 groups were supplemented on d 35, whereas only S7 heifers were supplemented on d 36 of the study. Tissue co llection started 4 h after supplement feeding time. A treatment x day interaction was detected ( P = 0.05). A day effect was detected for S3 heifers ( P < 0.01), but nor for S7 heifers. Treatment comparison within days: P = 0.04. 84

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CHAPTER 4 EFFECTS OF ACCLIMATION TO HANDLING ON PERFORMANCE, REPRODUCTIVE, AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED HEIFERS Introduction Development of replacement heifers is one of the most important components of the cowcalf production system (Bagley, 1993). Management strategies that maximize the number of heifers conceiving by 15 mo of age improve th e profitability of cow-calf operations because heifers that calve as 2-yr ol ds wean more and heavier calve s during their productive lives (Lesmeister et al., 1973). Because conception rate s are greater during the third estrus compared with the pubertal estrus (Byerl ey et al., 1987), replacement heifers should be managed to attain puberty at 12 mo of age so they can conceive by 15 mo of age (Schillo et al., 1992; Bagley, 1993). Age at puberty is influenced by breed type and heifers containing Brahman breeding typically reach puberty after 15 mo of age (Plasse et al., 1968 ; Rodrigues et al., 2002). In addition to this genetic effect, Brahman-crossbre d heifers are often described as temperamental, and this trait is expect ed to further negatively influence thei r reproductive functi on (Plasse et al., 1970). Cattle with excitable temperament experience stimulated secretion and circulating concentrations of ACTH and co rtisol (Curley et al., 2008). Thes e hormones directly impair the mechanisms responsible for puberty establishment a nd fertility of heifers, such as synthesis and release of GnRH and gonadotropins (Li a nd Wagner, 1983; Dobson et al., 2000). However, acclimation of beef females to handling has been reported to alleviate these negative physiological effects of temperament on repr oduction (Echternkamp, 1984). Based on these previous observations, we hypothesized that Brahman-crossbred heifers exposed to handling acclimation procedures after weaning would e xperience improved temperament, alleviated adrenal steroidogenesis, and enha nced reproductive performance. The objectives of the present 85

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experiment were to compare growth, temperament, plasma measurements associated with stress response, puberty attainment and pregnancy rates of Brahman Angus and Braford heifers exposed or not to acclimation procedures. Materials and Methods This experiment was conducted over 2 yr (2006 and 2007) at the University of Florida IFAS, Range Cattle Research and Education Center Ona. Each experimental period was divided into a sampling phase (d 0 to 130) and a breedin g phase (d 131 to 191). The animals utilized were cared for in accordance with acceptable practic es as outlined in the Guide for the Care and Use of Agricultural Animals in Agricultu ral Research and Teaching (FASS, 1999). Animals A total of 37 Braford (37.5% Brahman + 62.5% Hereford) and 43 Brahman Angus (approximately 25% Brahman) heifers weaned at approximately 7 mo of age were assigned to the experiment. Twenty heifers from each breed t ype were utilized in yr 1, whereas 23 Brahman Angus and 17 Braford heifers were utilized in yr 2. Within 30 d after weaning, heifers were initially evaluated (morning of d 0 and 10) for BW, puberty status, and temperament score (d 10 only). Puberty was assessed via trans-rectal ultrasonography (7.5 MHz transducer, Aloka 500V, Wallingford, CT) to verify ovarian activity and pl asma progesterone (P4) concentrations. Heifers were considered pubertal if a corpus luteum with volume greater than 2.15 cm3, and plasma P4 concentrations greater than 1.5 ng/ mL (Cooke et al., 2007a) were conc urrently detected in one or both evaluations. Corpus luteum volume was calcula ted using the formula for volume of a sphere (V = 4/3 [D/2]3, where D is the maximum luteal diameter). On d 11, heifers were stratified by breed, puberty status, temperament score, BW and age, and randomly assigned to receive or not (control) the acclimation treatment. Heifer was considered the experimental unit (40 86

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heifers/treatment). Across years and breeds, heif er mean BW and age at the beginning of the experiment were ( SD), respectively, 253 27 kg and 269 26 d. Diets During both sampling and breeding phases, heifer s were be maintained on separate 6 ha limpograss ( Hemarthria altissima ) pastures according to treatment. The pastures utilized in this experiment were not fertilized prior to or dur ing the experimental pe riod, and heifers were rotated among pastures every 2 wk. Heifers re ceived a blend of soybean hulls and cottonseed meal (75:25 as fed-basis) 3 times weekly, at a ra te to provide a daily amount of 2.7 kg of DM per heifer. Stargrass ( Cynodon nlemfuensis ) hay was offered in amounts to ensure ad libitum access when pasture availability was limited. A comple te commercial mineral/vitamin mix (14% Ca, 9% P, 24% NaCl, 0.20% K, 0.30% Mg, 0.20% S, 0.005% Co, 0.15% Cu, 0.02% I, 0.05% Mn, 0.004% Se, 0.3% Zn, 0.08% F, and 82 IU/g of vitamin A) and water were offered for ad libitum consumption throughout the experiment. Forage and supplement samples were collected at the beginning and during the experiment, and analyz ed for nutrient content by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY). All samples were analyzed by wet chemistry procedures for concentrations of CP, ADF, and NDF, whereas TDN was calculated using the equation proposed by Weiss et al. (1992). During yr 1, nutrient composition (DM basis) was estimated to be 54% TDN and 9.3% CP in pasture samples, 52% TDN and 11.9% CP in hay samples, 60% TDN and 11.3% CP in soybean hull samples, and 69% TDN and 49.4% CP in cottonseed meal samples. During yr 2, nutrient composition (DM basis) was estimated to be 54% TDN and 9.6% CP in pasture samples, 54% TDN and 13.6% CP in hay samples, 60% TDN and 11.7% CP in soybean hull samples, and 67% TDN and 47.0% CP in cottonseed meal samples. 87

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Acclimation Procedure Acclimated heifers were exposed to a hand ling process for 4 wk (d 11 to 39 of the experiment). During this period, he ifers were gathered in the past ure and obliged to walk to the handling facility 3 times weekly (Mondays, Wednesdays, and Fridays). The total distance traveled by heifers during each of the acclimation events was approximately 2 km (round-trip). During the first wk of acclimation, heifers were processed through the ha ndling facility but not restrained in the squeeze chute. During the s econd wk, heifers were processed through the handling facility and experienced a rapid chute restraining (5 s) On the third and fourth wk, heifers were similarly processed as in wk 2 but were restrained in the squeeze chute for longer periods (30 s). During the initial three wks, heifers were allowed to return to the pastures immediately after processing, wher eas during the fourth wk heif ers remained in the handling facility for 1 h and then returned to pasture. Sampling During the sampling phase, heifer puberty stat us and BW were assessed in the morning of d 40 and 50, 80 and 90, and 120 and 130, in additi on to the initial evaluation (d 0 and 10). Heifer puberty status was determined by trans-rectal ultrasonogra phy (7.5 MHz transducer, Aloka 500V) to verify ovarian activity and bl ood samples collected for analysis of P4 concentrations. Heifers were cons idered pubertal once a corpus lu teum with volume greater than 2.15 cm3, and plasma P4 concentrations greater than 1.5 ng/mL were concurrently detected in one or both evaluations performed on a 10-d in terval. Corpus luteum volume was calculated using the formula for volume of a sphere (V = 4/3 [D/2]3, where D is the maximum luteal diameter). Heifer BW gain during the sampling phase was calculated by averaging the values 88

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obtained in both 10-d interval asse ssments. Further, heifer shrunk (after 16 h of feed and water restriction) BW was collected on d 1 and 192 for calculation of heifer ADG during the study. Blood samples collected throughout the sampling phase for puberty assessment were also analyzed for plasma IGF-I concentrations to evaluate heifer energy status. On d 10 and 40, 2 extra blood samples were collected from each heif er in addition to the morning collection. These samples were collected in 3 h-intervals to account for the circadian rhythm of adrenal steroidogenesis (Thun et al., 1981; Arthington et al., 1997), and analyzed for plasma concentrations of cortisol, P4, ceruloplasmin a nd haptoglobin. Heifers remained in the handling facility during the intervals between blood collections. Heifer temperament scores were obtain ed in the morning of d 40, following blood collection and ultrasonography exam to evaluate treatment eff ects. Heifer temperament was assessed by pen score, chute score, and exit velocity. Chute score was assessed by a single technician based on a 5-point scale, where 1 = calm, no movement, and 5 = violent and continuous struggling. For pen score assessment, heifers exited the ch ute and entered a pen containing a single technician, and were assigned a scor e on a 5-point scale, where 1 = unalarmed and unexcited, and 5 = very excited and aggressive toward the technician in a manner that requires evasive action to a void contact between the technician and heifer. Exit velocity was assessed by determining the speed of the heifer exiting the squeeze chute by measuring rate of travel over a 1.5-m distance with an infrared sensor (FarmTek In c., North Wylie, TX). Further, within each assessment day (d 10 and 40), heifers were divided in quintiles according to their chute exit velocity, and assigned a score from 1 to 5 (exit score; 1 = slowest heifers; 5 = fastest heifers). Individual temperament scores were calculated by averaging heifer chute score, pen score, and exit score. 89

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During the breeding phase (d 131 to 191), each treatment group was exposed to one mature Angus bull (1:20 bull to heifer ratio), a nd bulls were rotated be tween groups every other wk to account for potential bull effects. Heifer pr egnancy status was verified by detecting a fetus with transrectal ultrasonography (5.0 MHz tran sducer, Aloka 500V) 70 d after the end of the breeding season. Blood Analysis Blood samples were collected via jugular ve nipuncture into commercial blood collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin, placed on ice immediately, and centrifuged at 2,400 g for 30 min for plasma collection. Plasma was frozen at -20C on the same day of collection. Concentrations of P4 and cortisol were determined using Coat-A-Count solid phase 125I RIA kits (DPC Diagnostic Products Inc., Los A ngeles, CA). A double antibody RIA was used to determine concentrations and IGF-I (Badinga et al., 1991; Cooke et al., 2007a). Concentrations of ceruloplasmin were determined according to pr ocedures described by Demetriou et al. (1974). Concentrations of haptoglobin were determin ed by measuring haptoglobin/hemoglobin complex by the estimation of differences in peroxidase activity (Makimura and Suzuki, 1982), and results are expressed as arbitrary units from the absorption reading at 450 nm 100. All samples were analyzed in duplicates. Across years, the intraand interassay CV were, respectively, 9.2 and 6.9% for cortisol, 6.3 and 7.3% for P4, 5.1 and 3.5% for ceruloplasmin, 8.8 and 9.8% for haptoglobin, and 4.5 and 4.6% for IGF-I. A ssay sensitivity was 0.1 ng/mL for P4, 0.5 g/dL for cortisol, and 10 ng/mL for IGF-I. 90

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Statistical Analysis Growth, temperament, and physiological data were analyzed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC) and Sa tterthwaite approximation to determine the denominator df for the tests of fixed effects. The model statement used for analysis of hormones and metabolites, heifer BW, and temperament contained the effects of treatment, breed, time variables, and appropriate interactions. Data were analyzed using heifer(breed treatment yr) as random variable. Further, temperament and phys iological data were ad justed covariately to measurement obtained prior to acclimation peri od (d 10). The model statement used for ADG contained the effect of treatment, breed, and yr. Data were also analyzed using heifer(breed treatment yr) as random variable. Results ar e reported as least square means and were separated using LSD. Puberty data were analy zed with the GLM and LOGISTIC procedure of SAS. The model statement contained the effects of treatment, breed, time of estimated puberty establishment, year, and the appropriate interac tions. Pregnancy data were analyzed with the GLM procedure of SAS, and the model statement c ontained the effects of treatment, breed, year, and the appropriate interactions. Pearson co rrelations were calcul ated among ADG, mean concentrations of hormones and metabolites, an d mean temperament measurements of heifers during the study. These correlation coefficients were determined across years, breeds, and treatments with the CORR procedure of SAS. For all analysis, significance was set at P 0.05 and tendencies were determined if P > 0.05 and 0.10. Results are reported according to treatment effects if no interactions were significant, or according to the highest-order interaction detected. 91

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Results and Discussion No interactions containing the effects of tr eatment, breed, and year were detected for measurements of heifer growth, plasma m easurements, temperament, and reproductive performance; therefore results were combined between breeds and year. Further, no treatment time(day) interaction was detected for the anal ysis of plasma measurements on d 10 and 40; therefore results regarding trea tment effects were combined among samples collected within each day. Acclimated heifers had reduced ( P < 0.01) ADG compared with control heifers (0.50 vs. 0.58 kg/d respectively; SEM = 0.02; Table 4-1). Given that both tr eatment groups were provided similar pastures and supplements during the stud y, treatment effects on ADG can be attributed to the additional exercise that acclimated heifer s were exposed to during the acclimation period. During each acclimation event, heifers had to walk n early 2 km in addition to the activity inside the handling facility, whereas cont rol heifers remained on their past ure. According to the Cornell Net Carbohydrate and Protein System for Cattle model (ver. 5.0.40), acclimated heifers required approximately 0.55 Mcal of ME during each of th e acclimation events. Supporting this rationale, heifers from both treatments had similar BW prio r to the acclimation peri od, but control heifers had numerically greater BW compared with acclimated heifers immediately following the acclimation period (treatment day interaction; P < 0.01; Figure 4-1). A treatment effect was detected ( P < 0.01) for puberty atta inment. Although age at puberty in cattle is highly determined by BW an d growth rate (Schillo et al., 1992), heifers exposed to acclimation procedures reached pube rty sooner than control heifers despite their reduced ADG (Figure 4-2). Research with humans and rodents reported that prepubertal females exposed to exercise regimens attained puberty at older ages compared with control cohorts 92

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(Warren, 1980; Manning and Bronson, 1991). These au thors attributed the delayed onset of puberty to suppressive effects of exercise a nd consequent additional energy consumption on GnRH and gonadotropin synthesi s and secretion. Pregnancy ra tes, however, were similar between treatments (Table 4-1). Nevertheless, treatment effects on pregnancy reported in this document were evaluated using heifer pregnancy status obtained via tr ansrectal ultrasonography 70 d after the end of the breeding season. This an alysis did not account for the time during the breeding season in which heifers became pregnant, wh ereas it is expected that acclimated heifers conceived earlier compared with control heifers, particularly because of treatment effects on puberty attainment. As soon as the calving season for heifers utilized in yr 2 is concluded, date of conception will be estimated retrospectively by s ubtracting gestation leng th (286 d; Reynolds et al., 1980) from the calving date, and treatment ef fects on pregnancy will be re-analyzed and reported as pregnancy attainment ove r time during the breeding season. Treatment effects were detected (P < 0.05) for concentrations of cortisol and P4. Acclimated heifers had reduced ( P < 0.01) cortisol concentrations compared with control heifers after the acclimation period (3.8 vs. 5.1 g/dL; SEM = 0.17; Figure 43). Within prepubertal heifers, P4 concentrations were reduced ( P = 0.03) in acclimated heif ers compared with control heifers after the acclimation period (0.52 vs. 0.78 ng/mL; SEM = 0.08; Figure 4-4). Previous research indicated that acclimation of cattle to handling procedures was an alternative to prevent elevated concentrations of co rtisol in response to handli ng stress (Crookshank et al., 1979; Andrade et al., 2001; Curley et al ., 2006). The same rationale can be extrapolated to treatment effects on prepubertal P4 concentrations. Although the major sources of P4 synthesis in cattle the corpus luteum and the pregnant placenta (Hoffmann and Schuler, 2002) are absent in prepubertal heifers, their adrenal gland is also capable of produci ng significant amounts of P4 as 93

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an intermediate of cortisol synthesis via ACTH stimuli (Gon zalez-Padilla et al., 1975; Brown, 1994). Supporting this rationale, conc entrations of cortisol and P4 were positively correlated in prepubertal heifers herein (Table 4-2), and also in prev ious research efforts evaluating the effects of ACTH administration to ova riectomized heifers and cows (Bage et al., 2000; Yoshida and Nakao, 2005). In accordance with previous studies indicating that the adrenal gland synthesizes steroids in a circadian rhythm (Thun et al., 1981; Arthington et al., 1997), a time effect was detected (P < 0.01) for the analysis of both cortisol and prepubertal P4. Concentrations of cortisol were the greatest ( P < 0.01) in the morning collection for heifers from both treatments (4.9 vs. 3.74 and 4.06 g/dL, respectively; SEM = 0.16). Similarly, P4 concentrations of prepubertal heifers were greater ( P < 0.01) in the morning collection compared with the subsequent collections (1.01 vs. 0.54 a nd 0.53 ng/mL, respectively; SEM = 0.08). No treatment effects were detected for c oncentrations of IGF-I, ceruloplasmin, and haptoglobin (Table 4-1), although previous studies reported that elevated concentrations of corticoids can stimulate synthesi s of acute phase proteins (Yoshi no et al., 1993; Higuchi et al., 1994) and reduce circulating concentr ations of IGF-I (Maciel et al ., 2001) in cattle Nevertheless, positive correlations were detected ( P < 0.05; Table 4-2) between c oncentrations of IGF-I and heifer ADG, concentrations of ceruloplasmin and haptoglobin, and heifer temperament and concentrations of ceruloplasmin (Table 4-2). Additionally, both heifer ADG and IGF-I concentrations were negatively correlated with heifer temperament and ceruloplasmin concentrations (Table 4-2). These results support previous studie s reporting that IGF-I is a key factor for heifer growth and its synthesis can be reduced during the acute phase response (Elsasser et al., 1997; Cooke et al., 2007a), and also suggests that cattle with excitable temperament experience reduced concentrations of IGF-I and stimulated acute phase response, 94

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which contributes to their decreased BW gain compared to cattle with calmer temperament (Voisinet et al., 1997; Fell et al., 1999; Qiu et al., 2007). No treatment effects were detected for temperament scores (Table 4-3), but acclimated heifers had reduced chute score (P < 0.01) compared with control heifers after the acclimation period (Table 4-3). Further, all measurements of temperament were positively correlated to each other, and also to cortisol concentrations ( P < 0.01; Table 4-4). Fordy ce et al. (1987) indicated that Brahman-crossbred steers frequently expos ed to handling procedur es exhibited calmer temperament compared with cohorts with no hand ling experiences. Curley et al. (2006) reported that frequent handling of Brahma n bulls decreased their exit velo city, but not their pen and chute score. The positive correlations detected am ong measurements of temperament and cortisol concentrations reported herein we re also described by others (Sta hringer et al. 1990; Fell et al., 1999; Curley et al., 2006), indicat ing that these three measurements of cattle behavior during handling can be used as indicators of temperamen t and also denote the amount of stress that the animal is experiencing (Thun et al., 1998; Sapols ky et al., 2000). However, pen score and exit velocity are classified as non -restrained techniques to evaluate cattle temperament, whereas chute score belongs to the restrained tec hniques category (Burrow and Corbet, 2000). An important flaw within restrain ed techniques is that cattle wi th excitable temperament may freeze when restrained, and consequently no t express their true behavior during these assessments (Burrow and Corbet, 2000). Therefore, treatment effects detected in the present study for chute score should be interpreted wi th caution, even though this measurement of temperament was positively correlated with cortis ol and non-restrained assessments (Table 4-4). Breed effects were detected ( P < 0.05; Table 4-5) for ADG and puberty attainment. Brahman Angus heifers had greater ( P = 0.02) ADG compared with Braford heifers during the 95

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study (0.55 vs. 0.49 kg/d, respectivel y; SEM = 0.02). A greater ( P < 0.01) number of Brahman Angus heifers attained puberty during the sampling phase compar ed with Braford heifers (80.6 vs. 38.8% pubertal heifers, resp ectively; SEM = 6.9). No further breed effects were detected (Table 4-5). These results support previous da ta from our research group (Arthington et al., 2004) reporting inferior ADG and re productive performance of Braford heifers compared with Brahman Angus heifers. The Braford heifers u tilized herein and by Arthington et al. (2004) were originated from a straightbred Braford co wherd that has been sharing similar genetics for more than 20 yr, which likely resulted in accumu lative inbreeding. Martin et al. (1992) indicated that inbreeding can be detrim ental to cattle growth and ag e at puberty. Therefore, breed differences in ADG and puberty attain ment reported herein can be attr ibuted, at least in part, to accumulative inbreeding effects within Braford heif ers, and also to the retained heterosis of Brahman Angus heifers (Koger, 1980). The main hypothesis of the present experiment was that acclimation to human handling would alleviate the detrimental effects of ex citable temperament on reproductive function of Brahman-crossbred heifers, and allow acclimated heifers to reach puberty earlier than nonacclimated cohorts. Plasse et al. (1970) repor ted that excitable temperament influences negatively the reproductive perfor mance of beef females. Cattle with high Brahman influence typically exhibit excitable te mperament (Hearnshaw and Morris, 1984; Fordyce et al., 1988; Voisinet et al., 1997) and experience stimulated function of the hypothala mic-pituitary-adrenal axis when exposed to humans and handling pr ocedures, resulting in increased production and circulating concentrations of AC TH and consequently cortisol (S tahringer et al., 1990; Curley et al., 2008). These hormones have been shown to di rectly impair the physiological mechanisms required for puberty attainment in heifers, part icularly synthesis and release of LH by the 96

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anterior pituitary (Li and Wa gner, 1983; Dobson et al., 2000), whereas methods to acclimate cattle to handling procedures we re reported to be successful in reducing circulating cortisol concentrations (Crookshank et al., 1979; Andrad e et al., 2001) and increasing pulsatility and mean concentrations of LH (Echternkamp, 1984) Supporting this rationale and our hypothesis, acclimated heifers in the present study had reduced cortisol concentrations and hastened onset of puberty compared with non-acclimated cohorts Nevertheless, the mechanisms by which acclimation procedures hastened puberty attainment regardless of decreased rates of BW gain remain unclear. Based on our hypothesis, it can be speculated that reduced cortisol concentrations in acclimated heif ers facilitated the in itiation of the physiol ogical events required for puberty attainment, particularly increased LH pulse frequency and c onsequent first ovulatory LH surge (Smith and Dobson, 2002). Although concen trations of cortisol were only evaluated when heifers were handled and restrained for bl ood collection, one can speculate that acclimated heifers also had reduced cortisol concentrations compared to control heifers on a daily basis given that heifers from both groups were often exposed to brief human interaction, particularly because of feeding and traffic of personne l/vehicles within the research station. Another mechanism responsible for treatmen ts effects on puberty attainment can be attributed to prepubertal P4 synthesis by the adrenal gland. Approximately 55% of the Brahman Angus and 86% of the Braford heifers from th e present study experienced P4 concentrations above 1.0 ng/mL before reaching puberty, whereas th e greatest prepubertal P4 value detected for both breeds was 2.8 ng/mL. These results indicate th at elevated production of P4 by the adrenal gland is a common occurrence in prepubertal Brahman-crossbred heifers during handling practices. This response was likely due to he ifer temperament, as positive correlations ( P < 0.01; Table 4-2) were detected between concentra tions of P4 and cortisol (r = 0.50), P4 and 97

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temperament score (r = 0.45), and cortisol a nd temperament score (r = 0.58). Progesterone appears to be a required stimulus for puberty esta blishment in heifers give n that it suppresses the number of estradiol receptors in the hypothalamus and primes th e hypothalamic-pituitary-ovarian axis toward enhanced synthesis and pulsatile secretion of LH (Anders on et al., 1996; Looper et al., 2003). Transient increases in circulating concentr ations of P4 were detected in beef heifers 2 wk prior to the onset of puberty (Gonzalez-Padilla et al., 1975), and the or igin of this hormone was attributed to the adrenal gl and and/or luteal structures found within the ovary (GonzalezPadilla et al., 1975; Berardinelli et al., 1979). Base d on this information, it can be theorized that acclimated heifers experienced transient increases in adrena l steroidogenesis and thus P4 synthesis during the acclimation proc ess, particularly during the initial weeks when heifers were still unfamiliar with the acclimation events. Transient increases in adrenal P4 synthesis during the early phases of the acclimation process, co mbined with alleviated cortisol synthesis as acclimation advanced, may have contributed to hastened puberty attainment of acclimated heifers compared with control cohorts. Neverthe less, further research is required to properly address this assumption. In conclusion, results from this experiment indicate that acclimation of Brahmancrossbred heifers to handling procedures and human interaction reduced ADG because of the additional exercise that heifer s were exposed to, but alleviat ed adrenal steroidogenesis and hastened onset of puberty. Therefore, acclimati on of Brahman Angus and Braford replacement heifers to human handling after weaning may be an alternative to enhance their reproductive development, and increase the efficiency of he ifer development programs in cow-calf operations containing Brahman-influenced cattle. 98

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Table 4-1. Average daily gain, pregnancy rates, and concentrations of plasma ceruloplasmin, haptoglobin, and IGF-I of heifers exposed or not (control) to handling acclimation procedures. Item Acclimated Control SEM P -value ADG, kg/d 1 0.50 0.58 0.016 < 0.01 Pregnancy rate, % 2 68 69 7.8 0.47 Ceruloplasmin, mg/dL 3 27.3 25.7 0.69 0.11 Haptoglobin, 450 nm 100 3 1.52 1.27 0.169 0.29 IGF-I, ng/mL 3 216 215 6.2 0.86 1 Calculated using initial (d 1) and final (d 192) shrunk BW. 2 Pregnant heifers / total heif ers during the breeding season. 3 Least squares means adjusted covariately to values obtained prior to acclimation period. 99

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100 1 Upper row = correlation coefficients. Lower row = P values. 2 Prepubertal heifers only (n = 71) Table 4-2. Pearson correlation coeffi cients among ADG, plasma measurem ents, and temperament of heifers. 1

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Table 4-3. Temperament measurements of heif ers exposed or not (control) to handling acclimation procedures. 1 Item Acclimated Control SEM P -Value Chute score 2 1.37 1.84 0.091 < 0.01 Pen score 3 2.85 2.72 0.137 0.51 Exit velocity, m/s 4 2.91 2.74 0.148 0.43 Temperament score 5 2.46 2.48 0.096 0.93 1 Least squares means adjusted covariately to values obtained prior to acclimation period. 2 Assessed by a single technician based on a 5-po int scale, where 1 = calm, no movement, and 5 = violent and conti nuous struggling. 3 Heifers exited the chute and entered a pen cont aining a single technici an, and were assigned a score on a 5-point scale, where 1 = unalarmed and unexcited, and 5 = very excited and aggressive toward the technician in a manner that requires evasive action to avoid contact between the technici an and heifer. 4 Assessed by determining the speed of the heifer exiting the squeeze chute by measuring rate of travel over a 1.5-m distance with an infrared sensor (FarmTek Inc., North Wylie, TX). 5 Calculated by averaging heifer chute score, ex it score, and pen score. Exit score was calculated by dividing chute exit velocity resu lts into quintiles, and assigning heifers with a score from 1 to 5 (exit score; 1 = slowest heif ers; 5 = fastest heifers). 101

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Table 4-4. Pearson correlation coefficients amon g measurements of temperament and plasma cortisol concentrations of heifers. 1 Item Cortisol Chute score Exit velocity Chute score 0.44 < 0.01 Exit velocity 0.55 0.46 < 0.01 < 0.01 Pen score 0.48 0.40 0.69 < 0.01 < 0.01 < 0.01 1 Upper row = correlation coefficients. Lower row = P values. 102

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Table 4-5. Effects of breed on performance, temperament, and physiologic parameters of replacement heifers. 1 Item Brahman Angus Braford SEM P -Value ADG, kg/d 2 0.55 0.49 0.016 0.02 Puberty rate, % 3 81 39 6.9 < 0.01 Pregnancy rate, % 4 71 67 7.8 0.39 Temperament score 5 2.47 2.62 0.121 0.50 Cortisol, g/dL 4.01 4.45 0.200 0.12 IGF-I, ng/mL 200 206 6.4 0.48 Ceruloplasmin, mg/dL 25.2 26.5 0.67 0.18 Haptoglobin 450 nm 100 2.14 1.74 0.315 0.35 1 Values reported are least square means from all samples/assessments obtained throughout the study 2 Calculated using initial (d 1) and final (d 192) shrunk BW. 3 Estrous cycling heifers / total heif ers during the experimental period. 4 Pregnant heifers / total heif ers during the breeding season. 5 Calculated by averaging heifer chute score, ex it score, and pen score. Exit score was calculated by dividing exit velocity results in to quintiles, and assigning heifers with a score from 1 to 5 (exit score; 1 = slowest heifer s; 5 = fastest heifers). 103

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200 240 280 320 360 400 d 0 and 10d 80 and 90d 120 and 130BW, kg Acclimated Control A 200 240 280 320 360 400 d 0 and 10d 40 and 50d 80 and 90d 120 and 130BW, kg Acclimated Control B Figure 4-1. Body weight of heifer s exposed or not (control) to handling acclimation procedures (d 11 to 39). Panel A reports results combined from both yr, whereas Panel B reports results from yr 1. In yr 2, the scale wa s not functional during d 40 and 50; therefore data combined among yr do not include BW values obtained on d 40 and 50 in yr 1. A treatment x day effect was detected in both analyses ( P < 0.01; SEM = 4.6 and 6.0 for panels A and B, respectively). Heifers from both treatments had similar BW prior to the acclimation period (d 10 to 40), but control heifer had numerical greater BW compared with acclimated heifers during the remainder of the study. 104

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0 10 20 30 40 50 60 70 80 90 100 d 0 and 10d 40 and 50d 80 and 90d 120 and 130Pubertal heifer, % Acclimated Control Figure 4-2. Puberty attainment of heifers exposed or not (c ontrol) to handling acclimation procedures (d 11 to 39). Heifers were cons idered pubertal once a corpus luteum and plasma P4 concentrations greater than 1.5 ng/mL were concurrently detected in one or both evaluations performed on a 10-d interval. A treatment effect was detected (P = 0.02; SEM = 6.5). 105

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0 1 2 3 4 5 6 7 Pre-acclimation (d 10)Post-acclimation (d 40)Plasma cortisol, g/dL Acclimated Control Figure 4-3. Plasma cortisol concentrations of heifers exposed or not (control) to handling acclimation procedures (d 11 to 39). Sample s collected on d 10 served as covariate, therefore results reported for d 40 are cova riately adjusted least square means. Acclimated heifers had reduced ( P < 0.01; SEM = 0.17) concentrations of cortisol compared to control heifers on d 40. 106

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0.0 0.2 0.4 0.6 0.8 1.0 1.2 Pre-acclimation (d 10)Post-acclimation (d 40)Plasma progesterone, ng/mL Acclimated Control Figure 4-4. Plasma progesterone con centrations of prepubertal heifers exposed or not (control) to handling acclimation procedures (d 11 to 39). Samples collected on d 10 served as covariate, therefore results reported for d 40 are covariately adjusted least square means. Acclimated heifers had reduced (P = 0.03; SEM = 0.08) concentrations of progesterone compared to control heifers on d 40. 107

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CHAPTER 5 EFFECTS OF TEMPERAMENT AND ACCLIMATION ON PERFORMANCE, PHYSIOLOGICAL RESPONSES, AND PREGNANCY RATES OF BRAHMANCROSSBRED COWS Introduction The major objective of cow-calf production systems is to produce one calf per cow annually. Ovulation of a competent oocyte determin es the length of the postpartum interval and also the fertility of beef cows during the breed ing season (Short et al., 1990). Follicle size and LH pulsatility are major factors responsible for a successful ovulation (R oche, 2006); therefore, alternatives to stimulate GnRH delivery to the pituitary, and antic ipate/enhance the ovulatory LH surge are options to maximize reproductive performance of beef cows (Day, 1994). Brahman-crossbred cows, however, may experience impaired pituitary sensitivity to GnRH (Griffin and Randel, 1978), which may compromise their ability to resume estrous cycles and conceive. In addition, Brahman-crossbred ca ttle are often described as temperamental (Hearnshaw and Morris, 1984; Fordy ce et al., 1988; Voisinet et al., 1997), and this trait is expected to further negatively in fluence their reproductive functi on (Plasse et al., 1970). Cattle with excitable temperament ofte n experience stimulated secretion and circulating concentrations of ACTH and cortisol (Curley et al., 2008). These hormones directly impair synthesis and release of GnRH and gonadotropins (Li and Wagner, 1983; Dobson et al., 2000). Nevertheless, acclimation of beef females to human interacti on and handling has been reported to alleviate these negative physiological effects of excita ble temperament on repr oduction (Echternkamp, 1984). Based on this information, we hypothesized that Brahman-crossbred cows exposed to acclimation procedures would experience impr oved temperament, reduced circulating concentrations of cortisol, and increased repr oductive performance compared to non-acclimated cohorts. The objectives of the present experiment were to compare BW and BCS, measurements 108

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of temperament, concentrations of plasma hor mones and metabolites, and pregnancy attainment in Brahman British and Braford cows e xposed or not to acclimation procedures. Materials and Methods This experiment was conducted over 2 yr (2006 and 2007) at the University of Florida IFAS, Range Cattle Research and Education Center Ona. Each experimental period was divided into a acclimation phase (August to January) an d a breeding season (January to April). The animals utilized were cared for in accordance w ith acceptable practices as outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Animals and Diets A total of 160 Braford (37.5% Brahman + 62.5% Hereford) and 235 Brahman British (mostly Angus, and containing approximately 25% of Brahman breeding) co ws were assigned to the experiment. Initial mean BW and age across breeds and yr were ( SD), respectively, 545 71 kg and 6 3 yr. Approximately 45 d after weaning in yr 1 (August 2006), cows were evaluated for BW, BCS (Wagner et al., 1988) and temperament, stratified by these measurements in addition to breed and age, a nd randomly allocated to 14 groups. Braford cows were divided into 8 groups of approximately 20 cows each, whereas Brahman British cows were divided into 6 groups of approximately 40 cows each. Groups were assigned randomly to receive or not (control) the acclimation treatment (7 groups/treatment). In yr 2, cows were reevaluated within 45 d after weaning (August 2007) for BW, BCS, and temperament, and were stratified and divided into 14 groups similarly as in yr 1. However, cows were re-allocated into groups in order that they received the same treatment assigned in yr 1. Within acclimated cows, 3 109

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Braford and 19 Brahman British cows were culled prior to the beginning of yr 2 and replaced by cohorts not previously assigned to the experiment. During both years, groups were maintained on separate bahiagrass ( Paspalum notatum ) pastures and were rotated among pastures weekly. Stargrass ( Cynodon nlemfuensis) was offered in amounts to ensure ad libitum access when pa sture availability was limited. From early December to the end of the breeding season, cows received a blend of sugarcane molasses and urea (97:3 as fed-basis) 3 times weekly, at a rate to provide a daily amount of 1.8 kg of DM per cow. Nutrient composition of sugarcane molasses was estimated to be (DM basis) 79.2% of TDN and 12.3% of CP (SDK Laboratorie s, Hutchinson, KS). A complete commercial mineral/vitamin mix (14% Ca, 9% P, 24% NaCl, 0.20% K, 0.30% Mg, 0.20% S, 0.005% Co, 0.15% Cu, 0.02% I, 0.05% Mn, 0.004% Se, 0.3% Zn, 0.08% F, and 82 IU/g of vitamin A) and water were offered for ad libitum consumption throughout the experiment. Acclimation Procedure The acclimation process was applied from August to January, and cows for both treatment groups calved during this period (from October to December of each yr). During the acclimation period, acclimated groups were vis ited twice weekly. Duri ng each visit, a person walked among cows for 15 min, and hand-offered approximately 50 g of range cubes per cow. Range cubes consisted of (as-fed basis) 66.7% of wheat middlings, 27.0% of soybean hulls, 3.7% of blackstrap molasses, and 2.6% of cottonseed meal. Nutrient composition of range cubes was estimated to be (DM basis) 62.5% of TDN and 22.2% of CP (Dairy One Forage Laboratory, Ithaca, NY). The amount of range cubes offered during each acclimation visit provided no meaningful nutritional c ontribution to cows (approximately 0.5 and 1.0 % of TDN and CP daily requirements, respectively). 110

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Sampling At the beginning of the acclimation phase (August) and breeding season (January), one blood sample was collected from each cow in addition to BW, BCS, and temperament evaluation. Blood samples were analyzed for plasma concentrations of cortisol, ceruloplasmin, haptoglobin, and IGF-I (pre-breeding samples only). Cow temperament was assessed by pen score, chute score, and exit ve locity. Chute score was assessed by a single technician based on a 5-point scale, where 1 = calm, no movement, and 5 = violent and continuous struggling. For pen score assessment, cows exited the chute and en tered a pen containing a single technician, and were assigned a score on a 5-point scale, where 1 = unalarmed and unexcited, and 5 = very excited and aggressive toward the technician in a manner that requires evasive action to avoid contact between the technician and cow. Exit ve locity was assessed by determining the speed of the cow exiting the squeeze chute by measuring rate of travel over a 1.5-m distance with an infrared sensor (FarmTek Inc., North Wylie, TX). Further, within each assessment day, cows were divided in quintiles according to their chute exit velocity, and assigned a score from 1 to 5 (exit score; 1 = slowest cows; 5 = fastest cows). Individual temp erament scores were calculated by averaging cow chute score, pen score, and exit score. Breeding Season During yr 1 and 2, each Braford group was exposed to one mature Angus bull (1:20 bull to cow ratio) for 90 d. Bulls were not rotated among groups, but were submitted to and approved by a breeding soundness evaluation (Chenoweth and Ball, 1980) before the breeding season. During yr 1, Brahman British cows were assigned to an estrus synchronization + fixed-time AI protocol at the beginning of the bree ding season (d 0). Cows received a 100 g treatment of GnRH (Cystorelin; Merial Ltd., Duluth, GA) and we re inserted with a controlled internal drug 111

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releasing device containing 1.38 g of progesterone (CIDR; Pfizer Animal Health, New York, NY) on d 0, received a PGF2 treatment (25 mg; Lutalyse; Pfizer Animal Health, New York, NY), CIDR and calf removal on d 6, followed by calf return and fixed-time AI on d 8. Fifteen days after AI, Brahman British groups were e xposed to two mature Angus or Brangus bulls (1:20 bull to cow ratio) for 90 d. Bulls were not rotated among groups, but were submitted to and approved by a breeding soundness evaluation (Chenoweth and Ball, 1980) before the breeding season. During yr 2, Brahman British cows were only exposed to natural breeding by mature bulls similarly as in yr 1. Mean days post-partum at the beginning of the breeding season were ( SD), respectively, 72 23.8 and 98 22.6 d for Br aford and Brahman British cows during yr 1, and 48 24.2 and 22 21.0 d for Braford a nd Brahman British cows during yr 2. Cow pregnancy status was verified by detecti ng a fetus via rectal palpation 90 d after the end of both breeding seasons. Pregnancy rates to AI in Brahman British cows were evaluated by detecting a fetus with tr ansrectal ultrasonography (5.0 MH z transducer, Aloka 500V, Wallingford, CT) 50 d after AI. For yr 1 only, date of conception was esti mated retrospectively by subtracting gestation lengt h (286 d; Reynolds et al., 1980) from the calving date. Blood Analysis Blood samples were collected via jugular ve nipuncture into commercial blood collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin, placed on ice immediately, and centrifuged at 2,400 g for 30 min for plasma collection. Plasma was frozen at -20C on the same day of collection. Concentrations of cortisol were dete rmined using a Coat-A-Count solid phase 125I RIA kit (DPC Diagnostic Products Inc., Los Angele s, CA). A double antibody RIA was used to determine concentrations and IGF-I (Badinga et al., 1991; Cooke et al., 2007a). Concentrations 112

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of ceruloplasmin were determined according to pr ocedures described by Demetriou et al. (1974). Concentrations of haptoglobin were determin ed by measuring haptoglobin/hemoglobin complex by the estimation of differences in peroxidase activity (Makimura and Suzuki, 1982), and results are expressed as arbitrary units from the abso rption reading at 450 nm 100. Across years, the intraand interassay CV were, respectiv ely, 9.5 and 7.2% for cortisol, 5.6 and 6.3% for ceruloplasmin, 8.4 and 9.7% for haptoglobin, and 5.8 and 6.3% for IGF-I. Assay sensitivity was 0.5 g/dL for cortisol and 10 ng/mL for IGF-I. Statistical Analysis Performance, temperament, and physiological data were analyzed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC) a nd Satterthwaite approximation to determine the denominator df for the tests of fixed effects. The model statement used for analysis of hormones and metabolites, BW, BCS, and temperament contained the effects of treatment, breed, time variables, and appropriate interactions. Data we re analyzed using group(breed treatment yr) as random variable. Results are reported as least square means and were separated using LSD. Pregnancy data for yr 1 were analyzed with th e GLM and LOGISTIC procedure of SAS, and the model statement contained the effects of treatmen t, breeds, estimated time of conception, and the appropriate interactions. Further, cow days pos tpartum at the beginning of the breeding season was included into the analysis as covariate; ther efore, pregnancy results for yr 1 are reported as covariately adjusted means and were separate d using LSD. Pregnancy data for yr 2 were analyzed with the GLM procedure of SAS. Th e model statement contained the effects of treatment, breed, year, and appropriate interac tions, and did not include days postpartum as covariate so potential carry over effects of treatments across yr could be accounted for. Results are also reported as least square means and were separated using LSD. Th e probability of cows 113

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to become pregnant during the breeding season was evaluated according to BCS, temperament score, and concentrations of plasma metabo lites and hormones obtained at the beginning of breeding. The GLM procedure of SAS was utilized to determine if each individual measurement influenced pregnancy rates linearly, quadraticall y, and/or cubically. If multiple continuous order effects were significant, the effect with the greatest F-value was selected. The LOGISTIC procedure was utilized to determine the inter cept and slope(s) values according to maximum likelihood estimates from the significant effect selected, and the probabi lity of pregnancy was determined according to the equation: Probability = (e logistic equation) / (1 + e logistic equation). Logistic curves were constructed according to the mi nimum and maximum values detected for each individual measurement. Pearson correlations we re calculated, within yr and collection day, among plasma and temperament measurements. Thes e correlation coefficients were determined across breeds and treatments with the CORR procedure of SAS. For all analysis, significance was set at P 0.05 and tendencies were determined if P > 0.05 and 0.10. Results are reported according to treatment effects if no interactions were significant, or according to the highestorder interacti on detected. Results and Discussion A treatment breed yr in teraction was detected ( P = 0.05) for mean BW (Tables 5-1). During yr 2, acclimated Braford cows had greater ( P = 0.01) mean BW compared to control cohorts (557 vs. 527 kg of BW, respectively; SEM = 7.8). However, no treatment effects were detected (P = 0.38) for BW change during the acclim ation period of both yr (Table 5-2). A treatment breed yr interaction was also detected (P = 0.02) for mean BCS and BCS change (Table 5-1). During yr 1, acclimated Braford cows had reduced (P = 0.04) mean BCS compared to control cohorts (5.71 vs. 6.06 of BCS, resp ectively; SEM = 0.114). Conversely, acclimated 114

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Braford cows had greater ( P = 0.05) mean BCS compared to c ontrol cohorts during yr 2 (6.04 vs. 5.71 of BCS, respectively; SEM = 0.116). Acclim ated Brahman British cows had reduced ( P < 0.01) BCS change compared to control cohorts du ring the acclimation period in yr 2 (-0.30 vs. 0.86 of BCS; SEM = 0.119). These results may sugge st that the acclimation process influenced cow nutritional status and resulted in the treat ment effects detected for mean BW and BCS. Nevertheless, the detection of treatment effects was inconsistent between breeds and yr. Further, mean BCS of cows from both breeds and treatments during yr 1 and yr 2 were always adequate (Kunkle et al., 1994), and treatment effects detected for mean BCS and BCS changes, although statistically significant, can be considered marg inal and insufficient to affect performance of cows with BCS greater than 5 (Kunkle et al., 1994; Cooke et al ., 2008b). Further supporting this rationale, no treatment effects were detected fo r IGF-I concentrations (Table 5-2), given that circulating concentrations of IG F-I are positively associated with nutritional status of cattle (Ellenberger et al., 1989; Bossis et al., 1999; Lapi erre et al., 2000). To ensure that acclimation effects were not confounded with nutritional effects, only 50 g of range cubes were offered per cow during each of the acclimation events. No treatment effects were detected for temper ament score and concentrations of cortisol and haptoglobin (Table 5-2). Fu rther, no treatment effects were detected for individual measurements of cow temperament (Table 5-2). A treatment day interaction was detected ( P = 0.04) for ceruloplasmin (Table 53). Acclimated cows tended ( P = 0.08) to have decreased ceruloplasmin concentrations compared to co ntrol cows at the beginning of the acclimation period (15.8 vs. 16.6, respectively; SEM = 0.30), but similar ( P = 0.82) at the beginning of the breeding season (14.2 vs. 14.1; SEM = 0.30). Previous research indicated that acclimation of cattle to human interaction was an alternative to improve temperament and prevent elevated 115

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cortisol concentrations in response to excitabl e temperament and handling stress (Fordyce et al., 1987; Andrade et al., 2001; Curley et al., 2006). Based on these studies, we hypothesized that Brahman-crossbred cows would benefit from acc limation particularly because of their common excitable temperament during ha ndling practices (Hearnshaw and Morris, 1984; Fordyce et al., 1988; Voisinet et al., 1997). This rationale can be extrapolated to ceruloplasmin and haptoglobin concentrations because the acute phase response is stimulated by elevated concentrations of corticoids (Yoshino et al., 1993; Higuchi et al., 1994). However, cows from both treatments had similar temperament, and concentrations of cortisol and acute phase proteins after the acclimation period. This lack of treatment effects may indicate that the acc limation process failed to improve cow temperament and the physiological responses associated with this trait. However, the sampling schedule of the present experiment perhaps hindered the detection of treatment effects on cortisol concentrations. Beca use of the large number of animals utilized, in addition to the extensive grazing conditions th at they were maintained, cows from both treatments were sampled for blood without a ny specific order throughout the day during all collection days. Adrenal steroidogenesis occurs in a circadian rhythm in cat tle, being highest in the morning, decreasing during the day, and reachi ng nadir levels in the evening (Thun et al., 1981; Arthington et al., 1997). Therefore, the in fluence of diurnal variation in cortisol concentrations may have impaired the detection of treatment effects for this hormone. Nevertheless, positive correlati ons were often detected ( P < 0.05), across breeds and treatments, between concentrations of ceruloplasmin a nd haptoglobin, and between measurements of temperament and cortisol concentrations (Table 5-4 and 5-5). Ceruloplas min and haptoglobin are correlated because both proteins are components of the acute phase response in cattle (Carroll and Forsberg, 2007). Positive correlations among measurements of temperament and cortisol 116

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concentrations were also descri bed by others (Stahringer et al 1990; Fell et a., 1999; Curley et al., 2006), suggesting that cattle with excitable temperament e xperience elevated concentrations of cortisol, whereas chute score, exit velocity, and pen score can be used as indicators of cattle temperament and also denote th e amount of stress that the animal is experiencing during handling practices (Thun et al ., 1998; Sapolsky et al., 2000). During yr 1 of the experiment, a treatment breed interaction was detected for pregnancy attainment ( P < 0.01; Figure 5-1). Acclimated Braford cows became pregnant earlier and at a greater number ( P < 0.01) during the breedi ng season compared to control cohorts. Conversely, no treatment effects were detected for Brahman British cows in pregnancy rates to fixed-time AI (6.8 and 9.8 % of pregnant cows / total cows for acclimated and control treatments, respectively; P = 0.45; SEM = 2.85) and also accumula tive (AI + bull breeding) pregnancy during the breeding season ( P = 0.40; Figure 5-1). Acclimation increased reproductive performance of Braford cows during yr 1 of the e xperiment, but the reasons for this effect cannot be clarified because of the similar treatment responses observed among the other measurements evaluated. The lack of similar tr eatment effects for pregnancy of Brahman British cows during yr 1 of the experiment can be attributed, at l east in part, to the estrus synchronization + fixedtime AI protocol, which resulted in unexpected low pregnancy rates and likely influenced reproductive function of cows during the remainde r of the breeding season. Brahman British cows were assigned to a fixed-tim e AI breeding prior to bull exposur e to evaluate if acclimation would alleviate the detrimental effects of exci table temperament on reproduction when cattle had to be handled frequently. The protocol utilized herein was selected because calf removal is an alternative to stimulate ovulati on instead of GnRH administration (Williams et al., 1983; Pursley et al., 1995), and exogenous GnRH was expected to bypass the effects of excitable temperament 117

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on the physiologic mechanisms associated w ith fertility, more specifically gonadotropin secretory activity. The protocol utilized he rein was based on a previous study reporting satisfactory pregnancy rates of B. indicus B. taurus cows exposed to a 7 d CIDR treatment followed by 48 h calf removal and fixed-time AI (Vasconcelos et al ., 2008); therefore the reasons for the reduced pregnancy rates to AI obtained herein are unknown, but this outcome likely influenced treatment effects on re productive performance of Brahman British. In contrast to yr 1, a treatment breed interaction was detected ( P = 0.03) for pregnancy rates during yr 2 because acclimated Braford cows had reduced (P = 0.04) pregnancy rates compared to control cohorts (76.3 vs. 88.1 pregna nt cows / total cows, respectively; SEM = 4.06), whereas no treatment effects were detected (P = 0.41) for Brahman British cows (89.3 vs. 85.3 pregnant cows / total cows for acclimated and control treatments, respectively; SEM = 3.39). Nevertheless, treatment effects on pregna ncy during yr 2 were evaluated using cow pregnancy status assessed by rectal palpation 90 d after the end of the breeding season. This analysis did not account for the time during the breeding season in which cows became pregnant, whereas cows that become pregnant earlier have greater chance to re -breed during the next breeding season (Reynolds, 1967; Wiltbank, 1970). Therefore, treatment effects on estimated pregnancy attainment during yr 2 are required for better evaluation of acclimation effects on reproductive performance of Brahman-crossbred cows, and as soon as the calving season for these cows is concluded, date of conception will be estimated and treatment effects on pregnancy will be re-analyzed and reported similarly as pregnancy rates for yr 1. Breed comparisons were not performed for physiologic, reproductive, and performance measurement because Braford and Brahman British cows were maintained at different management scenarios, such as grazing systems, stocking rates, and gr oup sizes. Day effects, 118

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however, were evaluated (Table 5-3). Cows from both breed types had reduced BW ( P < 0.01) and BCS ( P < 0.01) at the beginning of breeding season compared to the beginning of the acclimation period (Table 5-3). Cows calved du ring the acclimation period and were nursing young calves at the onset of breeding, which likel y stimulated mobilization of body reserves and tissues to sustain lactation during this period (NRC, 1996). The probability of cows to become pregnant according to measurements obtained at the beginning of the breeding season, was evaluated w ithin each yr because mean days post-partum across breeds at the onset of breeding differed ( P < 0.01) from yr 1 to yr 2 (88 vs. 34 d, respectively; SEM = 1.5). Plasma IGF-I concentr ations and cow BCS aff ected quadratically ( P < 0.01) the probability of pregnancy during both yr (Figure 5-2). These results indicate that reduced or excessive energy stat us is detrimental to reproducti ve performance of cattle, as reported by others (Armstrong et al., 2001; Bilby et al., 2006; Cooke et al., 2008b). Cow temperament score and plasma cortisol concentr ations affected the pr obability of pregnancy linearly ( P = 0.03) during yr 1, and quadratically ( P < 0.01) during yr 2 (Figure 5-3). These results indicate and support our hypothesis that excitable temperament and elevated cortisol concentrations, partially explaine d by cow temperament (Table 5-4 and 5-5), are detrimental to reproductive function of cows. Concurring with our findings, Plasse et al. (1970) reported that excitable temperament influences negatively th e reproductive performance of beef females. Additionally, as observed in yr 2, reduced cortisol c oncentrations and temperament score during the early postpartum period ma y denote health disorders th at negatively affect cattle reproduction, such as lethargy, lameness (Sprec her et al., 1997), and immunosuppresion (Goff, 2006). Plasma concentrations of ceruloplasmin and haptoglobin affected the probability of pregnancy linearly during yr 1 (P < 0.01 and = 0.04, respectively) and yr 2 ( P = 0.01; Figure 5119

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4), indicating and supporting previous data reporting that the acute phase response is detrimental to reproductive function of livesto ck (Peter et al., 1989; Battaglia et al., 2000; Williams et al., 2001). In conclusion, results from this experiment indicate that acclimation of Brahmancrossbred cows to human interaction did not influence temperament and concentrations of plasma cortisol and acute phase proteins. Neve rtheless, reproductive pe rformance of Braford cows was enhanced during yr 1. Measurements and physiologic responses associated with cow temperament, acute phase response, and energy st atus influenced the pr obability of cows to become pregnant during the breeding season. Ther efore, management strategies that improve cow disposition, enhance their immune status, and maintain the cowherd at adequate levels of nutrition are required for optimal reproductive performance of Brahma n-crossbred cows, and consequent productivity of cow-calf opera tions containing thes e types of cattle. 120

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Table 5-1. Mean BW and BCS of Brahman-cro ssbred cows exposed or not (control) to acclimation procedures. Year 1 Year 2 Item Brahman British Braford SEM Brahman British Braford SEM BW, kg Acclimated 517 519 7.7 514 556 8.0 Control 506 523 7.8 514 526 7.9 P2 0.32 0.72 0.97 0.01 BCS1 Acclimated 5.7 5.7 0.12 5.8 6.0 0.12 Control 5.5 6.1 0.11 5.7 5.7 0.12 P2 0.21 0.04 0.91 0.05 BCS change3 Acclimated -1.07 -0.66 0.116 -0.30 -0.59 0.123 Control -1.09 -0.82 0.115 -0.86 -0.44 0.119 P2 0.89 0.35 < 0.01 0.39 1 Emaciated = 1, obese = 9; Wagner et al., 1998 2 Treatment comparison within breed and year. 3 Calculated using BCS collected at the begi nning of acclimation period and breeding season. 121

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122 Table 5-2. Body weight change, temperament measurements, and plasma concentrations of cortisol, haptoglobin, and IGF-I of Brahman-crossbred cows exposed or not (control) to acclimation procedures. Item Acclimated Control SEM P BW change, kg 1 -43 -50 5.4 0.38 Chute Score 2 1.98 1.96 0.034 0.59 Pen Score 3 2.48 2.58 0.043 0.12 Exit Velocity, m/s 4 2.12 2.08 0.049 0.57 Temperament score 5 2.50 2.49 0.042 0.94 Cortisol, g/dL 3.33 3.36 0.122 0.88 Haptoglobin, 450 nm 100 1.96 2.12 0.275 0.68 IGF-I, ng/mL 133 123 5.7 0.21 1 Calculated using BW collected at the beginning of the acclimation period and breeding season. 2 Assessed by a single technician based on a 5-po int scale, where 1 = calm, no movement, and 5 = violent and conti nuous struggling. 3 Cows exited the chute and entered a pen contai ning a single technicia n, and were assigned a score on a 5-point scale, where 1 = unalarmed and unexcited, and 5 = very excited and aggressive toward the technician in a manner that requires evasive action to avoid contact between the technician and cow. 4 Assessed by determining the speed of the cow exiting the squeeze chute by measuring rate of travel over a 1.5-m distance with an infrared sensor (FarmTek Inc., North Wylie, TX). 5 Calculated by averaging cow chute score, exit sc ore, and pen score. Exit score was calculated by dividing exit velocity results in to quintiles, and assigning cows w ith a score from 1 to 5 (exit score; 1 = slowest cows; 5 = fastest cows).

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Table 5-3. Body weight, BCS, and plasma ceruloplasmin concentrations of Brahman-crossbred cows exposed or not (control) to acclimation procedures.1 Item Beginning of acclimation Beginning of breeding SEM Treatment effects Ceruloplasmin, mg/dL Acclimated 15.8 14.2 0.31 Control 16.6 14.2 0.30 P2 0.08 0.82 Day effects3 BW 546a 498b 3.3 BCS4 6.1b 5.4b 0.05 1 Day effects are reported across trea tments (treatment day effect; P > 0.10) 2 Treatment comparison within day. 3 Values with different superscrip ts within measurement differ (P < 0.01) 4 Emaciated = 1, obese = 9; Wagner et al., 1998 123

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1 Upper row = correlation coefficients. Lower row = P values. Table 5-4. Pearson correlation coefficients among measurements of temperament and concentr ations of plasma cortisol, ceruloplasmin and haptoglobin of Brahman-crossbred cows during yr 1 of the study. 1 124

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125 Table 5-5. Pearson correlation coefficients among measurements of temperament and concentr ations of plasma cortisol, ceruloplasmin and haptoglobin of Brahman-crossbred cows during yr 2 of the study. 1 1 Upper row = correlation coefficients. Lower row = P values.

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0 10 20 30 40 50 60 70 80 90 100 123456789101112131415 Week of the breeding season Acclimated Control Panel A 0 10 20 30 40 50 60 70 80 90 100 123456789101112 Week of the breeding season Acclimated Control Accumulative pregnancy attainment, % Panel B Figure 5-1. Pregnancy attainment (pregnant cows / total cows ) during the breeding season of Brahman x British (Panel A) and Braford (Panel B) cows exposed or not (control) to acclimation procedures during yr 1 of the study. Brahman x British cows were assigned to timed-AI during wk 1, and expos ed to bulls for a 90-d breeding season beginning in wk 4. Braford cows were only exposed to a 90-d bull-breeding. Date of conception was estimated retrospectively by subtracting gestat ion length (286 d; Reynolds et al., 1980) from the calving date. Values reported are least square means adjusted covariately to cow days postpar tum at the beginning of the breeding season. A treatment effect was detected for Braford cows (P < 0.01), but not for Brahman x British cows ( P = 0.48). 126

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0 10 20 30 40 50 60 70 80 90 100 123456789 BCS Year 1 Year 2 0 10 20 30 40 50 60 70 80 90 100 050100150200250300350 Plasma IGF-I, ng/mL Year 1 Year 2 Probability of pregnancy, % Figure 5-2. Effects of BCS (emaciated = 1, obese = 9; Wagner et al., 1988) and plasma IGF-I concentrations, assessed at the beginning of the breeding season, on the probability of Brahman-crossbred cows to become pregnant A quadratic effect was detected during yr 1 and 2 for BCS (P < 0.01) and plasma IGF-I (P = 0.02 and < 0.01, respectively). 127

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0 10 20 30 40 50 60 70 80 90 100 12345 Temperament Score Year 1 Year 2 0 10 20 30 40 50 60 70 80 90 100 0123456789 Plasma cortisol, g/dL Year 1 Year 2 Probability of pregnancy, % Figure 5-3. Effects of temperament score and plas ma cortisol concentrations, assessed at the beginning of the breeding season, on the probability of Brahman British and Braford cows to become pregnant. For temperament score, a linear effect ( P = 0.03) and a quadratic effect ( P < 0.01) were detected for both breeds during yr 1 and 2, respectively. For plasma cortisol, a linear effect was detected ( P = 0.04) for Braford cows during yr 1, whereas a quadr atic effect was detected ( P = 0.02) for both breeds during yr 2. Temperament score was calcula ted by averaging cow chute score, exit score, and pen score. 128

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0 10 20 30 40 50 60 70 80 90 100 0510152025303540 Plasma ceruloplasmin, mg/dL Year 1 Year 2 Figure 5-4. Effects of plasma ceruloplasmin and haptoglobin concentratio ns, assessed at the beginning of the breeding season, on the pr obability of Brahman-crossbred cows to become pregnant. A linear effect was de tected during yr 1 and 2 for plasma ceruloplasmin ( P < 0.01 and = 0.01, respectiv ely) and haptoglobin ( P = 0.04 and = 0.01, respectively). 0 10 20 30 40 50 60 70 80 90 100 01234567891 Plasma haptoglobin, 450 nm 100 0 Year 1 Year 2 Probability of pregnancy, % 129

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155 BIOGRAPHICAL SKETCH Reinaldo Fernandes Cooke was born in So Paulo, Brazil, in 1979. He is the oldest son of Nelson de Burgos Cooke and Rosangela C. F. Cooke. Reinaldo obtained his B.S. in animal sciences from So Paulo State University (UNESP) in 2003. In 2004, Reinaldo moved to Gainesville, FL, to begin his Mast er of Science program at the University of Florida in animal sciences, advised by Dr. John Arthington. His resear ch focused on beef cattle nutrition. In spring 2006, Reinaldo was awarded with the M. S. degree in animal sciences and immediately began his Ph.D. studies at the same inst itution. During his Ph.D training, Reinaldo evaluated nutritional and management strategies to improve the repr oductive performance of beef heifers and cows. After graduation, Reinaldo hopes to pursue a career in academia, and is currently applying for beef cattle nutrition positions in different parts of the U.S.