• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Section I: Review of literatur...
 Section II: Hormonal patterns during...
 Section III: Experiment 1: thermal...
 Section IV: Summary and conclu...
 Appendix
 References
 Biographical sketch






Title: Interrelationships of certain thermal and endocrine phenomena and reproductive function in the female bovine /
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00097546/00001
 Material Information
Title: Interrelationships of certain thermal and endocrine phenomena and reproductive function in the female bovine /
Physical Description: xi, 118 leaves : ill. ; 28 cm.
Language: English
Creator: Gwazdauskas, Francis Charles, 1943-
Publication Date: 1974
Copyright Date: 1974
 Subjects
Subject: Cattle -- Reproduction   ( lcsh )
Reproduction   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1974.
Bibliography: Bibliography: leaves 108-117.
Additional Physical Form: Also available on World Wide Web
Statement of Responsibility: by Francis Charles Gwazdauskas.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00097546
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000317372
oclc - 08797839
notis - ABU4193

Downloads

This item has the following downloads:

PDF ( 5 MBs ) ( PDF )


Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
        Page iii
    Table of Contents
        Page iv
        Page v
    List of Tables
        Page vi
    List of Figures
        Page vii
        Page viii
    Abstract
        Page ix
        Page x
        Page xi
    Introduction
        Page 1
        Page 2
    Section I: Review of literature
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    Section II: Hormonal patterns during heat stress from PGF2a injection through estrus and ovulation and flowing adrenal stimulation by acth in heifers
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
    Section III: Experiment 1: thermal changes of the bovine uterus following administration of estradiol-17B
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
    Section IV: Summary and conclusions
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
    Appendix
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
    References
        Page 108
        Page 109
        Page 110
        Page 111
        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
        Page 117
    Biographical sketch
        Page 118
        Page 119
        Page 120
        Page 121
Full Text








INTERRELATIONSHIPS OF CERTAIN THERMAL AND ENDOCRINE PHENOMENA
AND REPRODUCTIVE FUNCTION IN THE FEMALE BOVINE








BY

FRANCIS CHARLES GWAZDAUSKAS


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL. OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY








UNIVERSITY OF FLORIDA


1974












ACKNOWLEDGEMENTS


The author is sincerely grateful to Dr. W. W. Thatcher, Chairman

of the Supervisory Committee, for his guidance, assistance, encourage-

ment, patience and friendship during the study.

Gratitude is expressed to Dr. C. J. Wilcox for his invaluable

assistance with statistical analyses and preparation of this manuscript.

The author is grateful to Dr. R. M. Abrams for the close association

and assistance throughout this endeavor. A word of thanks is due Drs.

D. H>-Barron, F. W. Bazer, D. Caton and H. H. Head for suggestions and

moreover for their assistance in projects and the authors' increase in

knowledge as members of the Supervisory Committee.

A special thanks goes out to Dr. R. B. Becker for his encourage-

ment and friendship throughout the entire study. The writer is indebted

to Drs. P. S. Kalra, C. A. Kiddy and M. J. Paape for their assistance

during different phases of the experiments. Thanks are given to Mr.

J. P. Boggs, Mr. A. L. Green and Mr. J. E. Lindsey for their help in the

barn and with cattle handling; to Mr. M. Casey, Mrs. D. Clark, Mrs. L.

Owens and Miss N. Baldwin for laboratory assistance and Miss L.

Buzzerd for clerical assistance.

Gratitude is expressed to R. W. Adkinson, J. R. Chenault, H.

Roman, L. C. Fernandez, R. Eley, J. M. Knight, E. Muljono, E. G. Benya,

L. W. Whitlow, S. Chakriyarat and J. L. Kratz who as fellow graduate


- ii -










students assisted technically and encouraged the author academically.

The author wishes to express his gratitude and appreciation to

his wife, Judy, for her constant understanding and encouragement during

the course of his studies.


- iii -













TABLE OF CONTENTS


Page No.
ACKNOWLEDGEMENTS ii

LIST OF TABLES vi

LIST OF FIGURES vii

ABSTRACT ix

INTRODUCTION 1

SECTION I 3

REVIEW OF LITERATURE 3

Influences of Thermal Stress on Reproductive
Performance 3

Prostaglandins 10

Hormone Relationships of Uterine Blood Flow and
Temperature 18

SECTION II 21

HORMONAL PATTERNS DURING HEAT STRESS FROM PGF2a
INJECTION THROUGH ESTRUS AND OVULATION AND FOLLOWING
ADRENAL SIMULATION BY ACTH IN HEIFERS 21

Introduction 21

Materials and Methods 23

Results and Discussion 27

SECTION III 60

EXPERIMENT 1: THERMAL CHANGES OF THE BOVINE UTERUS
FOLLOWING ADMINISTRATION OF ESTRADIOL-178 60

Introduction 60


- iv -








Table of Contents (continued):

Materials and Methods

Thermocouple Preparation and Calibration

Surgical Techniques and Experimental Protocol

Results and Discussion

EXPERIMENT-2: THERMAL CHANGES OF THE BOVINE UTERUS
FOLLOWING PGF2a INJECTION THROUGH ESTRUS AND OVULATION

Introduction

Materials and Methods

Results and Discussion

SECTION IV

SUMMARY AND CONCLUSIONS

APPENDIX

LIST OF REFERENCES

BIOGRAPHICAL SKETCH


Page No.

61

61

62

64


72

72

72

74

89

89

95

108

118












LIST OF TABLES


Table Page No.

1 Physiological parameters of heifers in environmental 28
chambers at 21.3 C and 32.0 C.

2 Simple correlations between hormone measurements. '37

3 Physical characteristics of plasma in heifers at 49
21.3 C and 32.0 C.

4 Overall least squares analyses of variance for 96
hormones in heifers at 21.3 C and 32.0 C.

5 Plasma progestins (ng/ml) following PGF2a injection. 97

6 Plasma estradiol (pg/ml) following PGF2a injection. 98

7 Plasma estrone (pg/ml) following PGF2a injection. 99

8 Plasma LH (ng/ml) following PGF2a injection. 100

9 Plasma prolactin (ng/ml) following PGF2a injection. 101

10 Plasma corticoids (ng/ml) following PGF2a injection. 102

11 Plasma corticoids (ng/ml) prior to and following 103
200 IU ACTH.

12 Plasma progestins (ng/ml) prior to ACTH injection. 104

13 Simple correlations between hormones and temperatures. 105

14 Analysis of variance for aortic and uterine temperatures. 106

15 Hormonal and temperature measurements for G665 (G) and 107
JN15 (J).


- vi -












LIST OF FIGURES


Figure

1 Evaluation of thermal stress on transitory hormonal
changes in the bovine during the period of
luteal regression, estrus and ovulation:
Experimental design.


2 Sequential changes in
at 21.3 C or 32.0
the LH peak.

3 Sequential changes in
at 21.3 C or 32.0
the LH peak.

4 Sequential changes in
at 21.3 C or 32.0
PGF2a injection.

5 Sequential changes in
21.3 C or 32.0 C.

6 Sequential changes in
at 21.3 C or 32.0
PGF2a injection.


plasma progestins in heifers
C synchronized to the time of


plasma estradiol in heifers
C synchronized to the time of


plasma estrone in heifers
C synchronized to the time of


plasma LH in heifers at


plasma prolactin in heifers
C synchronized to the time of


7 Sequential changes in plasma corticoids synchronized
to the time of the LH peak using pooled means of
heifers at 21.3 C and 32.0 C.

8 Transitory changes in plasma corticoids following
injection of 200 IU ACTH in heifers at 21.3 C
and 32.0 C.

9 Uterine and aortic temperature prior to and following
IV injection of 12 ml sterile physiological saline.

10 Uterine and aortic temperature prior to and following
IV injection of 3 mg estradiol-17B.

11 ATuterus-aorta prior to and following either 12 ml
saline or 3 mg estradiol-17B.


- vii


Page No.

24







List of Figures (continued):

Figure Page No.

12 Uterine and aortic temperature prior to and after 70
injection of estradiol-17B from continuous
recording.

13 Uterine and aortic temperatures prior to and following 75
PGF2a injections in G665.

14 Uterine and aortic temperatures prior to and following 76
PGF2a injections in JN15.

15 Changes in ATua following PGF2a injections. 77

16 Uterine and aortic temperatures, LH and estradiol in 82
G665 and air temperatures.

17 Uterine and aortic temperatures, LH and estradiol in 83
JN15 and air temperatures.

18 Circadian uterine, aortic and air temperature changes. 85

19 Changes in AT associated with endogenous LH and 87
estradioluEcncentrations.


- viii -







Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirei nts
for the Degree of Doctor of Philosophy

INTERRELATIONSHIPS OF CERTAIN THERMAL AND ENDOCRINE PHENOMENA
AND REPRODUCTIVE FUNCTION IN THE FEMALE BOVINE

By

Francis Charles Gwazdauskas

December, 1974

Chairman: W. W. Thatcher
Major Department: Animal Science

Ten normally cycling Holstein heifers were assigned to one of two

environmental treatment groups (21.3 C, 59% RH or 32.0 C, 67% RH). PGF2a

was used to cause luteal regression and synchronize estrus. Least-squares

analyses were conducted to characterize treatment, animal and within-

animal time trends in plasma progestins, estradiol, estrone, LH,

prolactin and corticoids.

Environmental treatment (32.0 C) evoked a 1.49 C increase in

rectal temperature and a 3.59 C increase in skin temperatures. Length

of estrus was shorter (P<.10) for the 32.0 C heifers. Two of four

heifers at 21.3 C inseminated were pregnant at 40 days compared to none

of five at 32.0 C.

Average progestin concentration between treatments were not differ-

ent (P>.10; .53 ng/ml at 21.3 C compared to .65 ng/ml at 32.0 C). Mean

estradiol concentrations were significantly (P<.10) lower in 32.0 C

heifers (3.45 pg/ml compared to 2.96 pg/ml). There was a significant

elevation (P<.05) of estrone due to heat stress (1.55 pg/ml compared to

1.85). No significant differences (P>.10) were found in mean LH con-

centrations between heifers at 21.3 C or 32.0 C. Preovulatory peak LH


- ix -







concentrations were 32.2 and 33.2 ng/ml plasma, respectively. All

animals had a preovulatory LH surge, suggesting that hyperthermia did

not prevent the triggering mechanism for LH release. Mean prolactin

(14.51 ng/ml at 21.3 C compared to 14.78 ng/ml at 32.0 C) and corticoid

(8.01 ng/ml at 21.3 C compared to 7.76 ng/ml at 32.0 C) concentrations

were not different between temperature treatments (P>.10).

In an attempt to determine if plasma dilution may have occurred,

total protein concentration and osmolality were measured. There was

no difference (P>.10) in total protein concentration or osmolality

between treatment groups. The affinity (K ) of cortisol for CBG was not

different between treatments (P>.10); however, the binding capacity of

CBG for cortisol was reduced (P<.05) in the 32.0 C heifers.

Results of this experiment showed only subtle thermal effects on

estradiol and estrone plasma concentrations and no effects on LH, pro-

gestins, corticoids and prolactin. Apart from possible hormonal involve-

ment with duration of estrus, heat stress did not appear to affect the

hormonal mileau in peripheral plasma associated with corpus luteum re-

gression, follicle growth and ovulation.

Eight days following ovulation in the last heifer, 200 IU ACTH was

injected, IV, into the 10 heifers. The 32.0 C heifers responded with

significantly lower (P<.10) corticoid concentrations. The 6th order re-

gression response curves were not parallel (P<.01) suggesting that the

hot group response was earlier to reach a peak (75 compared to 105 min.),

had a lower magnitude (73.5 compared to 100.2 ng/ml corticoids) and was

of shorter duration (4 compared to 5 hr.).

Because the first experiment did not specifically consider environ-

mental and hormonal effects on uterine temperature it was necessary to

x -







document possible estrogen induced uterine thermal changes. In the second

experiment thermocouples were placed into the uterine serosa and saphenous

artery of four dairy heifers. Injection of 3 mg estradiol-178 caused a

.25 C decrease (P<.01) in the difference between uterine and aortic

temperature (ATu-a) by 2.5 hr. postinjection. In contrast, there was no

significant change (P>.10) in the ATu-a after injection of saline.

The final experiment was an attempt to document and evaluate changes in

uterine temperature during the period of luteal regression, follicle

growth and ovulation induced by PGF2a under conditions of a mild heat

stress. Thermocouples were placed into the uterine serosa and aortic

blood vessel of four dairy cattle. PGF2a caused an immediate drop in

uterine and aortic temperatures, and a decrease in the AT of almost
u-a
.4 C at 45 min. postinjection. The two cows, in which thermocouples

remained operational for the duration of the study, had monophasic daily

uterine and aortic temperature rhythms. However, both temperatures lagged

about 6 hr. behind air temperature changes. Uterine temperatures reached

40 C for periods of up to 6 hr. Failure to detect an association between

ATa and hormonal measurements may have been due to a time lag association.

Not until the preovulatory surge of LH was there an appreciable rise in

ATua (P<.01), and this occurred at a time when estradiol was decreasing.

The mild environmental heat stress may have contributed to the high

uterine and aortic blood temperatures.





Chairman


- xi -












INTRODUCTION


Reduced reproductive efficiency occurs during the hot seasons of

the year in many parts of the United States. Lowered conception rate

due to heat stress occurs over a prolonged period of the year in

Florida and represents a major production problem to dairymen. A 12

year study in the University of Florida dairy herd revealed a con-

ception rate per service of less than 40% (Gwazdauskas, Thatcher and

Wilcox, 1975). Economically, poor reproductive performance under con-

ditions of thermal stress decreases heifer replacement availability

and long term milk production and increases calving interval and

culling rate.

Before systems for reproductive management can be developed to

counter these adverse effects of heat stress, several fundamental

questions must be answered. Among these are:

1. How does a standard heat stress alter hormonal and phy-

siological responses during the normal estrous cycle? The objective

of the first experiment (Section II) was to characterize hormonal

changes progestinss, estradiol, estrone, LH, prolactin and corticoids),

rectal temperature, plasma protein concentration and osmolality and

plasma cortisol binding capacity (CBC) in heifers subjected to a

standard heat stress (21.3 compared to 32.0 C). In addition, plasma


- 1 -







- 2 -


corticoids in response to ACTH were measured to evaluate possible

thermal stress effects on adrenal responsiveness.

2. What are the factors influencing uterine temperature?

Can estradiol, which is known to have a marked effect on uterine blood

flow and metabolism, alter uterine temperature? What are the changes

in uterine temperature during the period of luteal regression, follicle

growth and ovulation under conditions of mild heat stress? In Section

III a series of experiments were designed in an attempt to answer these

questions.

Prostaglandin F2a (PGF2 ) causes luteal regression in the bovine

and has enabled the researcher to use it efficiently to control endo-

crine and physiological changes near the time of estrus and ovulation.

Such a compound maximizes use of experimental facilities over short

periods of time without adverse chronic alterations of normal bovine

physiology. In answering questions one and two above, PGF2a was used

to synchronize the hormonal events associated with corpus luteum re-

gression, estrus and ovulation.












SECTION I

REVIEW OF LITERATURE


Stress is defined as a condition harmful to an organism, which

results from inability of the organism to maintain a constant internal

environment (Taber, 1961). Factors involved in altering homeostasis

include trauma, surgical operations, restraint, extreme cold or heat,

intense solar radiation, social stress due to peck order, nutritional

stress and internal stress caused by pathogens or toxins (Hafez, 1968;

Guyton, 1966). The purpose of the initial review section is to report

on the effects of thermal stress on reproductive performance with major

emphasis on hormonal or endocrine aspects.


Influence of Thermal Stress on Reproductive Performance

Hot environments may exert their depressive effect on fertility

via the gonads, accessory sex glands, uterine environment, gametes or

endocrine system (Hafez, 1959; Ulberg and Burfening, 1967). Reproductive

behavior has been shown to be altered by heat stress, in that estrous

duration was shorter (Branton et al., 1957; Gangwar, Dranton and Evans,

1965; Hall et al., 1959), there was an increased frequency of quiet

ovulations (Labhsetwar et al., 1963) and anestrus (Bond and McDowell,

1972) and a reduction in estrous intensity (Gangw:ar, Branton and Evans,

1965).


-3-






- 4 -


High temperatures exert direct effects on fertilized ova grown

in vitro (Alliston et al., 1965). Fertilized ova grown through first

cell division at 40 C in vitro had a lower rate of embryo survival than

those grown at 38 C when returned to synchronized pseudopregnant recipient

rabbits. There were no morphological.differences between ova in dif-

ferent media. As the period of culture at 40 C was delayed to second

cell division, differences in post-implantation death losses disappeared.

An environment of 32.2 C and 65% Relative Humidity (RH) did not inhibit

estrus or alter ovulation rate in sheep (Alliston and Ulberg, 1961).

However, an increase in embryo death was detected when embryos (2-32

cell stage) were transferred from donor ewes kept at 32.2 C to recipient

ewes at 21.1 C ambient temperature. Embryo survival was highest when

both donor and recipient ewes were maintained at 21.1 C, indicating that

damage to the early embryo was most likely to occur in uteri of ewes

kept at 32.2 C. Heat stressing one or both parents of a mouse embryo

affected the rate of thymidine, uridine and guanine incorporation into

nucleic acids during pre-implantation development, which may lead to

altered DNA and RNA synthesis and subsequent embryonic mortality (Sheean,

Durrant and Ulberg, 1974).

Due to limitations of facilities and methodology, most investi-

gations of the effects of heat stress on hormonal balance and their

relationships to reproductive performance have monitored only one or

two hormonal responses. Therefore pooling results from different

laboratories by combining data from different animals within and between

species can be misleading when an effort is made to develop a hypothesis

on how thermal stress affects reproduction. Stott, Thomas and Glenn







- 5 -


(1967) found progesterone to be elevated on the day of estrus in

thermally stressed cows. Heifers maintained at 32 C and 21 C for

72 hr. beginning at the onset of estrus had conception rates of 0 and

48%, respectively, according to a report by Dunlap and Vincent (1971).

Associated with the decreased fertility was an elevated plasma progestin

concentration of only .42 ng/ml plasma (Mills et al., 1972). In con-

trast, Stott and Wiersma (1973) reported depressed plasma progestin

levels during chronic heat stress in the bovine.

Evidence of detrimental progesterone effects on embryo cleavage

stages has been reported by Dickman (1970). Fertilized ova were

transferred on day 4 of pregnancy to pseudopregnant rats which had

been ovariectomized on day 2. When transfer was preceded by 2, 3, 4,

5 or 6 days of progesterone treatment in pseudopregnant rats, 49, 38,

13, 2.5 and 2% of the transferred morula developed into fetuses.

However, when blastocysts were transferred, there was a 62.5% fetal

survival. Overstimulation with progesterone apparently interfered

with embryonic development. Johnsson et al. (1974) reported a 60 to

75% reduction in fertility in ewes receiving a single injection of

progesterone on days 0, 1, 2 or 3 or daily injections on days 1 to 4.

The progesterone given before day 4 may have affected embryo transport

through the oviduct or directly altered it and therefore inhibited or

abolished its ability to cope with the 'luteolysin' and prevent corpus

luteum regression during pregnancy.

Progesterone, superimposed on estradiol administration in ewes,

caused a prompt decrease in uterine blood flow (Greiss and Anderson,

1970). Extreme or prolonged limitation of blood flow to the vicinity







- 6 -


of the embryo can result in fetal death. Blockage of blood supply to

the uterus one day post coitum in the mouse was most detrimental to

implantation rate (Senger et al., 1967). These observations may have

been the result of the uterine coagulation procedure since necrosis of

the tissue was detected (F. W. Bazer, personal communication). However,

coagulation of blood vessels to one uterine horn resulted in 51% fewer

embryos migrating to the uterus by 4 days after mating in mice. At 10

days post mating there were fewer live fetuses on the coagulated side

compared to the control side (57% compared to 73%). Therefore, reduced

blood flow may be responsible for failure of embryo transport to the

uterus and increased fetal death rate (Bazer, Ulberg and LeMunyan, 1969).

Thus, if heat stress increased plasma progesterone levels, an altered

blood flow may be a factor associated with reduced fertility.

Heat stress has been shown to cause elevated plasma corticoid

levels in the bovine within 4 hr. of exposure which suggests increased

adrenal activity (Christison et al., 1970). Other workers reported

that plasma corticoids decreased during chronic heat stress in cattle

(Alvarez and Johnson, 1973; Christison and Johnson, 1972; Rhynes and

Ewing, 1973) and that corticoid turnover rates decreased (Christison

and Johnson, 1972). However, chronic hyperthermia resulted in elevated

epinephrine and norepinephrine, though corticoids were depressed, which

suggested a decreased sensitivity to physiological actions of

catecholamines (Alvarez and Johnson, 1973). Shayanfar (1973) compared

adrenal responsiveness to adrenocorticotropin (ACTH) in cows exposed

to ambient temperatures of greater or less than 21.1 C. At ambient

temperatures above 21.1 C, plasma corticoid response to ACTH was slower,







- 7 -


peak levels were lower and the response was of shorter duration. Yousef

and Johnson (1967) reported a 30 to 40% increase in heat production fol-

lowing injections of hydrocortisone acetate to cattle at 35 C ambient

temperature. Thus, during prolonged thermal stress, depressed plasma

corticoids and lowered adrenal responsiveness to ACTH may be indicative

of altered adrenal function.

Madan and Johnson (1971) have reported that the preovulatory peak

of plasma LH and basal plasma LH concentrations were lower in heifers

maintained at 33.5 C and 55% RH compared to those at 18.2 C and 55% RH.

These results support the hypothesis that thermal stress may alter

secretion or metabolism of various hormones associated with reproductive

function. However, Riggs, Alliston and Wilson (1974) detected a breed

difference in response of the pre-ovulatory surge of LH to heat stress

in gilts. Heat stressed pigs of the Duroc breed had no difference in

magnitude of the preovulatory plasma LH surge compared to controls,

whereas pigs of the Hampshire breed had a three to sixfold increase in

the preovulatory peak of LH compared to their controls. It appears

that species and breeds may,therefore,respond differently to thermal

stress and that inferences among species and breeds must be reviewed

with caution.

Koprowski and Tucker (1973), Schams and Reinhart (1974) and

Thatcher (1974) found elevated peripheral plasma prolactin concentrations

during the hot months of the year, suggesting that photoperiod and

temperature modulate prolactin release. At a constant day length,

calves exposed to 27 C had significantly higher plasma prolactin con-

centrations than those exposed to 10 C, whereas at 27 C prolactin levels







- 8 -


were only slightly higher than at 21 C (Wetteman and Tucker, 1974).

However, Karg and Shams (1974) found a positive correlation between

day length and basal prolactin concentrations in male and female cattle.

Relkin (1972) demonstrated that changes in light:dark ratios for rats

altered pituitary content and plasma concentrations of prolactin. It

would appear that this question has yet to be resolved in cattle, i.e.

whether photoperiod or temperature is the primary factor affecting

plasma prolactin concentrations.

These various studies suggest that thermal stress may alter

circulating plasma concentrations of certain pituitary, adrenal and

ovarian hormones. Such excesses or deficiencies of these hormones may

influence certain reproductive phenomena and account for lowered

fertility.

Environmental factors play an important role in bovine fertility.

Seasonal depressions in conception rate due to heat stress effects on

the male can be eliminated through A.I. (artificial insemination) in

which semen from bulls can be collected and frozen during cooler times

of the year. Under these circumstances Stott (1961) still found a

seasonal depression in breeding efficiency of cows which paralleled

high climatic temperatures in Arizona and California. Thus, this

experiment indicated that altered reproductive efficiency in the female

was the major contributor to summer depression of fertility.

In Florida, a year-long study was conducted to relate climatic,

rectal and uterine temperatures, plasma corticoid and progesterone

concentrations, breed, service number, time of service, sire and age to

conception rate (Gwazdauskas, Thatcher and Wilcox, 1973). Significant







- 9 -


effects due to environmental temperature on the day after insemination,

rectal and uterine temperatures at insemination, sire and days post-

partum were detected on conception rate. Deleting environmental

temperature from the statistical model revealed significant effects of

uterine temperature the day after insemination on fertility. Relationships

between uterine temperatures at insemination or the-day after insemination

with fertility were intriguing. Uterine temperature the day after in-

semination appeared to be positively associated with environmental

temperature on that day. Their inverse associations with fertility

may reflect direct detrimental thermal effects on early cleavage and

development of the embryo. In contrast, the association of uterine

temperature at insemination with fertility might be related to certain

physiological (uterine blood flow and vaginal thermal conductance) and

hormonal changes occurring at estrus that may be associated with proper

timing of insemination to achieve maximal fertility.

In a subsequent study with 12 years of data, effects on conception

rate of age of cow, inseminator, service sire, month, year, breed and

21 climatological variables were evaluated (Gwazdauskas, Wilcox and

Thatcher, 1975). Age, inseminator, sire and breed had significant

effects on conception. Maximum temperature the day after insemination,

rainfall the day of insemination, minimum temperature the day of insem-

ination, solar radiation the day of insemination and minimum temperature

the day after insemination were the five highest ranking climatological

variables associated with fertility. The most potent environmental

variable, maximum temperature the day after insemination, had a

significant curvilinear relationship with fertility. As maximum







- 10 -


temperature increased from 21.1 to 35 C, conception rates declined from

40 to 31%. Month effects were found to have a significant relationship

with fertility when climatological measurements were deleted from

statistical models. This agreed with most previous research (Stott,

1961; Hafez, 1959). In no case were month effects significant when

climatological measurements were included in the model, suggesting that

month effects may have represented climatological factors to a greater

degree than nutritional and management factors. This work clearly

showed the importance of ambient environmental conditions at the time

of insemination and fertilization on bovine fertility.

Alterations of peripheral plasma estrogen concentrations during

periods of thermal stress have not been reported previously. Therefore,

speculation as to estrogenic effects on uterine blood flow and estrogen

participation in pre-ovulatory LH release cannot be reviewed here.

Physiological and endocrine factors controlling thermal properties of

the uterus at estrus and ovulation need clarification. In addition,

effects of stressful ambient temperatures on these'factors and uterine

temperature need further study as they relate to fertility.


Prostaglandins


Prostaglandins (PG), unsaturated 20 carbon fatty acids containing

a cyclopentane ring and two alipahtic side chains, were first dis-

covered in extracts of human and sheep seminal vesicles during the

1930's. The first report of their activity before identification was

shown when fresh semen was placed into the human uterus and caused the







- 11 -


uterus.to contract or relax (Kurzok and Lieb, 1930). The luteolytic

effects of PGF2a were reviewed extensively by Inskeep (1973),and

effects in cattle well documented by Hafs et al. (1974)-and Chenault

(1973). Injection of PGF2a-Tham Salt in the bovine does not drastically

alter the normal sequential hormonal patterns leading to estrus and

ovulation. In addition, fertility of cattle to the PGF2a induced

ovulation is apparently the same as in a normal spontaneous ovulation

of control cattle (Lauderdale et al., 1974). Therefore, PGF2a can be

used as an experimental tool to synchronize estrus and investigate

physiological and hormonal changes under conditions the researcher

wishes to impose. This would be beneficial in large animal research

where animal numbers may be few and biological events (the estrous cycle

and pregnancy) of long duration. The intention of this portion of the

review on prostaglandins is to recapitulate various effects of prosta-

glandins on the circulatory system and reproductive tract. Such

knowledge is essential in evaluating effects of PGF2 in the following

experiments.

The role of the autonomic nervous system in controlling uterine

contractility and blood flow has been discussed by Shabanah et al.

(1964). The parasympathetic system generates uterine contractions and

causes vasodilation. The excitatory (a) action of the catecholamines

is manifested chiefly on the circular fibers whereas the inhibitory

(W) action influences the whole myometrium. Acetylcholine causes

vasodilation, especially of smaller blood vessels (Koelle, 1970).

Epinephrine is a vasopressor. Vasoconstriction occurs markedly in the

venous system, as well as smaller arterioles and precapillary sphincters.







- 12 -


Norepinephrine increases peripheral vascular resistance due to veno-

constriction (Innes and Nickerson, 1970). Estrogens govern the

parasympathetic acetylcholinee) activity and are responsible for the

basic contractile mechanism of the uterus (Shabanah et al., 1964),

whereas progesterone influences the sympathetic activity (epinephrine

and norepinephrine). Morris (1967) reviewed the sympathetic vaso-

constrictor action on the uterine vascular bed. Epinephrine and nore-

pinephrine reportedly cause a decrease in uterine blood flow with a

concomitant increase in arterial pressure, suggesting increased

vascular resistance in the uterus. Isoproteranol also acts as a

vasoconstrictor. These vasoconstrictor actions appeared to be due to

increased vascular resistance because myometrial tension changes were

negligible.

Clegg (1966) reported that prostaglandins produce two types of

effects on smooth muscle. They produce direct short-lived actions such

as stimulation of the isolated uterus or relaxation of the isolated

tracheal chain preparation. Alternately, they potentiated long-term

effects of other stimulants when given in low doses. An example of an

indirect long-term effect of prostaglandins (PGF and PGE series) is

depression of responses of various isolated smooth muscle preparations

to sympathomimetic substances (epinephrine, norepinephrine, phenylephrine

and isopropylnoradrenaline).

Different classes of prostaglandins have various effects on smooth

muscle and blood pressure and have been reviewed by Bergstrom et al.

(1968). PGE's and PGF2a cause contraction of uterine myometrium in rats

and guinea pigs. However, in humans, the PGE's decrease tonus, frequency







- 13 -


and amplitude of spontaneous contractions of the myometrium. There is

an increase in sensitivity of the rat uterus to PGF2a following estrogen

treatment (Anggard and Bergstrom, 1963). PGF2a also has been shown to

stimulate and increase tone of the rabbit fallopian tube in vivo

(Bergstrom et al., 1968; Horton and Main, 1965), whereas PGE1 causes

relaxation. Isolated strips of human myometrium have a regular motility

pattern. This motility in the nonpregnant myometrium was inhibited by

PG's A, B and E; however, PG's Fla and F2 stimulated contractions. The

sensitivity of the myometrium was highest late in the menstrual cycle

and during pregnancy. PGF2. also increased motility of the human oviduct

in vivo. Intra-uterine application of PGF2a or intravenous infusion

increased the motility of the non-pregnant human uterus (Eliasson, 1973).

Following intramuscular injection of 10 or 20 mg PGF2a in non-

pregnant women, no cardiovascular changes were observed but there was

pain at the injection site and increased uterine activity within

minutes. The uterine contractility lasted 2 to 3 hr. (Karim et al.,

1971). Within 1 to 6 hr. after vaginal insertion of PGE2 or PGF2,
10 to 12 women had menstrual-like uterine bleeding. This bleeding

was preceded by a marked increase in uterine contractions which started

within 10 min., peaked between 60 to 90 min., and lasted about 4 hr.

PGE and PGF2a induced uterine activity that was similar to that recorded

for the non-pregnant uterus during the time of the menstrual flow.

Contractions measured between 50 to 200 mm Hg and occurred every 1 to 2

min. (Karim, 1971).

In non-pregnant dogs, PGE1 infused into the uterine artery reduced

perfusion pressure. The dilator effect of PGE1 was seen at doses as







- 14 -


little as 20 pg/ml blood. Such potent vasodilatory effects of PGE1 were

not seen in pregnant dogs near term even with large doses. PGF2, on the

other hand, had little effect on vascular smooth muscle in dogs, but

potentiated responses to sympathetic nerve stimulation, occasionally

in parallel with increased responses to norepinephrine. PGF2a appears

to work primarily on nerve terminals in the dog uterus since there was

a greater effect on neurogenically induced vasoconstrictor responses

than to responses of norepinephrine itself (Clark et al., 1972).

In a review by Brody (1973) PGF2a was reported to influence effector

response to sympathetic nerve stimulation. Vasoconstrictor action in

cutaneous and muscle vessels was facilitated by PGF2a without any change

in responsiveness of the vessels to norepinephrine, suggesting that PGF2a

facilitated liberation of the adrenergic transmitter. This specificity

was not found in venous smooth muscle when PGF2a facilitated responses

to both sympathetic nerve stimulation and to norepinephrine. Thus,

PGF2a venoconstrictor action was dependent upon integrity of sympathetic

innervation.
No changes in cholinergic vasodilator nerves were noted in the

presence of prostaglandins (Brody and Kadowitz, 1974). Responses of

uterine vessels to norepinephrine were potentiated at PGF2a concentra-

tions which had no effect on uterine vascular resistance.
Recently, Ryan et al. (1974) showed that, in the dog,PGE1

redistributed the blood flow from the myometrium to the endometrium.

Therefore, PGE1 maybeavasodilator intermediate in an estrogen induced

uterine hyperemic response. To test this hypothesis estrogen was in-

jected into rats causing a visible intense hyperemia and a doubling of







- 15 -


uterine blood volume. In comparison, rats pre-treated with indomethacin,

a prostaglandin inhibitor, failed to show a large increase in uterine

blood volume. In conflict with the observation that PGF2a was a vaso-

constrictor was the finding of elevated uterine PGF content following

estrogen treatment which could be inhibited by indomethacin pre-

treatment. Except for this latter observation, the PGF series appears

to be associated with vasoconstrictor actions and reduced blood flow

to the uterus.

The cardiovascular actions of PGF2a also are complicated because

of quantitative species variation. Anggard and Bergstrom (1963) reported

that intravenous injection of PGF2a into cats caused increased right

ventricular pressure and a decreased systemic blood pressure. Intra-

arterial injections into muscles caused increased blood flow through

that area, i.e. vasodilation. PGF2a perfused into rabbit hindquarters

also caused tissue vasodilation. Horton and Main (1965) reported that

PGF2a or PGE1 injected intravenously in rabbits caused a fall in arterial

blood pressure. A review by Bergstrom et al. (1968) contrasts these re-

sults with the pressor action of prostaglandins in the rat, dog and spinal

chick. In dogs the pressor action of PGF2a is accompanied by an

increase in cardiac output and right atrial pressure, but the calculated

peripheral resistance was unchanged. It appeared as if there were a

decrease in venous capacitance because when a pressure stabilizer was

put into the venous side, it caused a shift of blood into the stabilizer

reservoir. Ducharme et al. (1968) reported similar results and also

found that, in the dog, PGF2a had little effect on'femoral arterial

pressure or small artery pressure but caused an increase in small vein






- 16 -


pressure when administered to an innervated limb. Abolishing the
sympathetic chain to the limb eliminated the venoconstrictor activity

of PGF2a. They found no real change in myocardial contractility. Thus

the pressor action was due to an increased venous return.

Horton (1969) found PGF to be weakly dilatory on arterioles.

In some species (rat and dog) they act as a venoconstrictor, thus in-

creasing venous return.and cardiac output. Neither PGE1 or PGFla

injected close-arterially released catecholamines from the adrenals

of anesthetized cats, but PGE1 did so in dogs. PGF2. injected intra-

arterially caused no constant change in blood pressure or in baroreceptor

discharge frequency. Moreover, intravenous injections caused a transient

rise in arterial pressure which was associated with an increase in
baroreceptor discharge. It appeared that the increased discharge fre-

quency was secondary to the pressure rise because in animals where the

blood pressure fell slightly, so did discharge frequency. PGF2a in-

jections into the carotid artery resulted in a variable response on

chemoreceptor discharges. Intravenously injected PGF2. caused a small

increase, decrease or no change at all in blood pressure (McQueen and

Belmonte, 1974). Therefore, the authors suggested that direct action

was on pressure changes not by way of baroreceptors. These actions

may be related to the rapid disappearance of prostaglandins as only

5 to 10% of the injected PGF2a was detected 1 min. later and negligible

PGF2a was found at 90 sec. (Raz, 1972). Also, more than 95% of injected

prostaglandins were removed during one circulation through the pulmonary

vascular bed (Ferreira and Vane, 1967).

Various investigators observed actions of prostaglandins on







- 17 -


respiratory smooth muscle. Main (1964) has shown that PG's E1, E2, E3

and Fla relaxed tracheal muscle in vitro in rabbit, guinea pig (also
Puglisi, 1972), ferret, pig, sheep, cat and monkey preparations. Except

fn the cat, they decreased lung resistance to inflation in vivo (also

Anggard and Bergstrom, 1963). PGF2a has similar biological activity to

PGFla, so these observations should hold for its actions. This con-

clusion was confirmed in a cat-trachea preparation by Horton and Main

(1965), in which PGF2a inhibited acetylcholine produced contractions.

In the dog the action of PGF2a was a reduction in dynamic lung compliance

and alveolar ventilation (Horton, 1969).

Investigations on systemic actions of prostaglandin in the bovine

are very limited to date. Lewis and Eyre (1972) reported that PGE1

and E2 lowered systemic blood pressure in calves, but PGF2a caused a

pressor response. Furthermore, pulmonary arterial pressure and abdominal

venous pressure were raised by the three substances. PGF2a caused con-

traction of the pulmonary artery and vein and produced an increase in

heart rate. Also noted was an increase in respiratory volume produced

by PGF2 Anderson et al. (1972) also concurred that PGF2a increased

pulmonary arterial pressure, but they found a drop in cardiac output

and essentially no change in femoral arterial pressure, left ventricle

and diastolic pressure, heart rate, blood gases and pH.

There is a definite need for more study on actions of prostaglandins

to determine their roles in physiological functions in the bovine.

Additional work is needed because of the contradictory results obtained

among and within species,






- 18 -


Hormone Relationships of Uterine Blood Flow and Temperature


The uterus responds to cyclic hormonal changes during the estrous

or menstrual cycles. Blood levels of estrogen and progesterone are in-

volved with this phenomena. In the human, the first half of the cycle

is associated with rapid growth of the uterine vascular elements and is

under estrogenic control. This is a period of tissue repair and pro-

liferation. The latter half of the cycle is characterized by glandular

secretary activity and elaboration of vascular elements under the con-

trol of estrogen and progesterone (Reynolds, 1949).

One of the principal characteristics of the uterus following

estrogen administration is its bright red color. The degree of redness

suggests a high level of oxygen saturation of the blood and there is

a high rate of blood flow. In the presence of an active corpus luteum

(progestational influence), the uterus is bluish in color. Oxygen

consumption is low and blood flow is sluggish. Oxytocin causes intense

muscular spasms within the uterus without affecting the rate of blood

flow, whereas vasopressin causes relaxation of uterine musculature but

a constriction of its vasculature (Reynolds, 1949).

Uterine hyperemia following injection of estrogen has been

estimated in ewes by direct collection of uterine venous blood (Huckabee

et al., 1970), by flow meters (Greiss and Anderson, 1970; Rosenfeld et

al., 1973) and microspheres (Rosenfeld et al., 1973). Endogenous estrogens

produced during the estrous cycle appear to have similar effects on uterine

blood flow. Patterns of change in plasma estradiol concentrations

(Scaramuzzi, Caldwell and Moor, 1970) are very similar to records of






- 19 -


uterine blood flow changes in the ewe (Greiss and Anderson, 1970;

Huckabee et al., 1968, 1970). Specificity of estrogen actions on

uterine blood flow have been shown by local injection of estrogen into

one uterine horn artery. An increase in blood flow was measured only

in that uterine artery (Resnik et al., 1974).

Progesterone injected into ovariectomized ewes did not alter

uterine blood flow, whereas progesterone superimposed on estradiol in-

jections caused a decrease in uterine blood flow rates (Greiss and

Anderson, 1970). Estrogens did not appear to affect systemic blood-
pressure (Huckabee bt al., 1968, 1970; Resnik et al., 1974), but caused

a fall in the coefficient of oxygen utilization [(AV)02 X 100] in the
A02
uterus. However, due to the higher uterine blood flow there was

essentially no change in oxygen uptake of the uterus (Huckabee et al.,

1968, 1970). Thus a dissociation between uterine metabolic rate and the

rate of blood flow might be reflected in temperature differences between

the uterus and aortic blood. In sheep a decrease in the temperature

difference between the uterine cavity and aortic blood provided a con-

venient method for monitoring increased uterine blood flow changes

following estrogen injection. A rise in blood flow resulted in a

lowered uterine temperature (Abrams et al., 1970a, 1971).. The actions

of estrogen to lower uterine temperature may be mediated through its inter-

action with acetylcholine to cause vasodilation (Shabanah et al., 1964)

or through the release of uterine histamine which was shown to be

involved in a rapid onset of hyperemia and water imbibition '( Jensen
and DeSombre, 1972). Lowering the rate of uterine heat production is

unlikely because of the many metabolic activities induced by estrogens






- 20 -


(Talwar and Segal, 1971; Jensen and DeSombre, 1972).

In cattle, plasma estrogens increasedprior to estrus and declined

precipitously during estrus (Chenault et al., 1973; Henricks, Dickey

and Hill, 1971). These changes may have distinct thermal effects on

the uterus. Greiss and Anderson (1969) reported increased uterine blood

flow associated with the onset of estrus in sheep, which could cause a

drop in uterine temperature (Abrams et al., 1970a,1971; Caton et al.,

1974) and thus be related to an optimal time for insemination to achieve

maximal fertility. However, the thermal response of the bovine uterus

to estrogen has not been documented.

Although several hormonal changes due to hyperthermia have been
documented in the bovine there is a sparcity of results related to a

multiplicity of hormonal responses to a controlled thermal stress in

which such sources of variation due to breed, age, animal and time

responses are evaluated. Uterine blood flow and temperature relation-

ships have been reported in sheep in response to estrogen injections,

but have not been reported in the bovine. Also, changes in utetine

temperature during phases of the estrous cycle under conditions of mild

heat stress have not been reported.











SECTION II

HORMONAL PATTERNS DURING HEAT STRESS FROM PGF2a INJECTION
THROUGH ESTRUS AND OVULATION AND FOLLOWING ADRENAL
STIMULATION BY ACTH IN HEIFERS


Introduction


Lowered conception rate due to heat stress occurs over a pro-

longed period of the year in tropical and subtropical climates. Before

systems for reproductive management can be developed to counter these

adverse effects of heat stress, a more complete understanding of

endocrine and physiological changes within the same animals must be

made. We need to know how a standard heat stress alters hormonal and

physiological responses during the normal estrous cycle, and determine

if chronic heat stress alters adrenal responsiveness to an IV injection

of ACTH.

Due to limitations of facilities and methodology, most inves-

tigations of heat stress effects on hormonal balance and their

relationships to reproductive performance have monitored only one

or two hormonal responses. Therefore, pooling results from different

laboratories and from different animals within and between species

can be misleading when an effort is made to develop a hypothesis on

how thermal stress affects reproduction.


- 21 -






- 22 -


Reduced fertility in hot environments was associated with elevated

body temperature (Dunlap and Vincent, 1971; Gwazdauskas, Thatcher and

Wilcox, 1973). Hot climates may exert their depressive effects on

fertility by acting on the gonads, uterine environment, endocrine
system or gametes (Hafez, 1959; Ulberg and.Burfening, 1967). Seasonal

Infertility has been attributed primarily to the bovine female (Stott,

1961). Studies on hormonal alterations due to thermal stress have

failed to document interrelationships of more than two different hormones

in the same animals. -Plasma progestin changes have been documented by

Mills et al. (1972), Abilay and Johnson (1973) and Abilay, Johnson and

Seif (1973); changes in plasma corticoid levels have been reported by

Lee, Roussel and Beatty (1973), Christison and Johnson (1972), Abilay

and Johnson (1973), Shayanfar (1973) and Miller and Alliston (1974a).

Plasma LH changes have been reported by Madan and Johnson (1971) and

Miller and Alliston (1973) and seasonal changes in prolactin have been

detected by Koprowski and Tucker (1973), Schams and Reinhart (1974) and

Thatcher (1974). Such hormonal alterations may be causative agents

contributing to suppressed estrous manifestation and depressed fertility

under hot climatic conditions.

There are essentially no studies designed to test specific effects

of heat stress environments on a multiplicity of hormonal responses with-

in the same animal. Such a study is needed in which variations due to

breed, age, animal (among and within) and hormonal interrelationships

are considered in evaluating thermal stress effects.
Objectives of this study were to characterize changes in peripheral

plasma concentrations of LH, progestins, estradiol, estrone, prolactin







- 23 -


and cor.ticoids after an intramuscular (IM) injection of PGF2-Tham Salt

(PGF2a) under controlled environmental temperatures (21.3 C compared to

32.0 C), and to determine if chronic heat stress alters adrenal responsive-

ness to an intravenous (IV) injection of ACTH (200 IU).


Materials and Methods

Ten normally cycling Holstein heifers at the USDA, Agricultural

Research Center, Beltsville, Maryland, were assigned alternately, based

on age, to one of two treatment groups (figure 1). All heifers were

placed in one of two environmental chambers at 21.3 C, 59% RH for 2

weeks. On day 9 of this adaptation period, 8 of 10 heifers in the luteal

phase of the estrous cycle received 30 mg PGF2 a (IM) to cause luteal

regression. This injection allowed all heifers to be in the luteal

phase of the cycle when PGF2a was injected 12 days later. PGF2a

effectively regresses the bovine corpus luteum and synchronizes estrus.

Lauderdale et al. (1973, 1974), Louis et al. (1974) and Chenault et al.

(1974) reported that fertility at the synchronized estrus, and induced

hormonal changes resulting in estrus and ovulation appeared normal in

PGF2, treated cattle. Thus, it was felt that PGF2a treatment could be

utilized as a tool to best control reproductive status of the heifers

and maximize efficient use of the chambers.

On day 14, the environment of one chamber was adjusted to 32.0 C,

67.2% RH. On day 20, all heifers were fitted with indwelling polyvinyl

jugular catheters (V-7; Bolab Inc., Derry, N.H.) and PGF2a (30 mg, IM)

apGF2a-Tham Salt was graciously supplied by Dr. J. W. Lauderdale, Upjohn
Co., Kalamazoo, Michigan.







- 24 -


10 Heifers
at 21.3 C

I----


Day 1


30 mg
PGF2a

I~--


Day 9


5 Heifers (21.3 C)
5 Heifers (32.0 C)


Day 14


Catheterize
sample every 6 hr.


Day 20


30 mg -- 6 hr. sample
PGF2a


4 hr. samples


Day 23 ovulation


8 Days
post ovulation


Evaluation of thermal stress on transitory hormonal changes
in the bovine during the period of luteal regression, estrus
and ovulation: Experimental design.


Day 21


200 IU
ACTH


Figure 1.


"


-~--






- 26 -


was given on day 21. Such treatment would allow for monitoring of

hormonal responses associated with corpus luteum regression, follicle

growth and ovulation under two different environments (21.3 C compared

to 32.0 C).

Blood samples (50 ml) were collected from jugular catheters at

-18, -12 and 0 hr. pre-PGF2a (day 21), at 6 hr. intervals for 48 hr.

post-injection, every 4 hr. thereafter until ovulation, and then twice

daily until the last heifer ovulated (figure 1). All blood was col-

lected into heparinized syringes, placed immediately into an ice bath,

centrifuged at 12,000 g for 10 min. at 4 C, and plasma stored at -20 C

until analyzed for progestins, LH, estradiol, estrone, corticoids, pro-

lactin, protein concentration, osmolarity and cortisol binding capacity.

Beginning 48 hr.post-PGF2a injection, animals were checked visually

for estrus at 4 hr. intervals. Heifers were artificially inseminated 12

hr. after onset of estrus, and ovulation determined by rectal palpation

of an ovarian ovulatory crypt. Palpations were performed at 4 hr.

intervals following cessation of estrus.

Chamber temperatures and relative humidities were recorded con-

tinuously (Honeywell Recorder, Washington, Pa.), and temperature of

each chamber verified daily with a tele-thermometer [Model 46 TUC,

Yellow Springs Instrument Co., Inc. (YSI), Yellow Springs, Ohio; air

temperature probe T 2620 (YSI)]. Rectal temperatures were monitored

daily (tele-thermometer probe T 2600, YSI). Skin temperatures taken

on the shoulder, rump and approximately 5 to 8 cm lateral to the vulva

(surface temperature probe T 2630, YSI) also were monitored daily during

the serial blood collection period. Thermister probes were calibrated







- 26 -


against a Bureau of Standards Certified Thermometer in a well-stirred,

insulated water bath held at 35 to 40 C. Data collected from the various

probes were corrected for constant temperature differences above and

below the certified thermometer readings.

Plasma samples were pooled within heifers (after the drop of the

preovulatory LH peak to basal levels and having less than 3 pg/ml

estradiol) to determine total protein concentration (Lowry method),

osmolality, (freezing point depression, Fiske Osmometer, Model G-61,

Fiske Ass., Inc., Bethel, Conn.), cortisol binding capacity and cortisol

association constants (Pegg and Keane, 1969; Shayanfar, 1973) for each

heifer.

In the second phase of the trial adrenal responsiveness to ACTH

was tested. Eight days after the last heifer ovulated, all heifers

received 200 IU ACTH (Porcine ACTH, Sigma Chemical Co., St. Louis, Mo.).

Blood samples (50 ml) were collected from indwelling jugular catheters

at -2, -1, 0 hr. pre-injection, 15, 30, 45, 60 min. and hourly thereafter

up to 12 hr. postinjection.

LH and prolactin were assayed in plasma at two dilutions using

the double antibody radioimmunoassay (RIA) of Niswender et al. (1969).

Guinea pig antibovine LH serum (Oxender, Hafs and Edgerton, 1972) was

supplied by Dr. H. D. Hafs of Michigan State University and revalidated

with NIH-LH-B7 for measuring plasma LH in our laboratory (Troconiz,

1973). Guinea pig anti-bovine prolactin serum (Koprowski and

Tucker, 1971) was donated by Dr. H. A. Tucker of Michigan State

University and revalidated for measuring plasma prolactin with NIH-P-B3

prolactin (Chakriyarat, 1974 personal communication). Plasma progestins,







- 27 -


estradiol and estrone were measured by RIA procedures described by

Abraham et al. (1971) and Hotchkiss et al. (1971), respectively.

The antiprogesterone antibody was a gift from Dr. K. Kirton of the

Upjohn Co., and the estrogen antibody was donated by Dr. V. L. Estergreen

of Washington State University. Extraction, purification and quantita-

tive procedures were validated in our laboratory by Chenault et al.

(1973, 1974, 1975). Plasma corticoids were extracted, isolated and

quantified by competitive protein binding (Gwazdauskas, Thatcher and

Wilcox, 1972, 1973). The only modification was use of a .2 ml dextran

coated charcoal suspension (100 mg Dextran, Type 60 C, Sigma Chemical

Co., St. Louis, Mo.; 1 gm Norit A, Sigma Chemical Co. and 100 ml deionized

water) instead of 80 mg florisil for adsorption of free steroid in the

competitive protein binding assay.

An extensive series of least-squares analyses was conducted to

characterize treatment, animal and within-animal time trends in plasma

LH, progestins, estradiol, estrone, prolactin and corticoids. Other

response variables were analyzed by analysis of variance.


Results and Discussion

Averages and standard deviations for chamber conditions, rectal

and skin temperatures, and events associated with estrus are shown in

table 1. Based on Christison and Johnson's (1972) criteria for a

moderate heat stress condition (rectal temperature increase of .5 C)

climatic conditions of our study exerted a greater than moderate heat

stress since rectal temperatures of heifers in the 32.0 C chamber were







- 28 -


Table 1. Physiological parameters of heifers in environmental
chambers at 21.3 C and 32.0 C.


Air temperature

Relative humidity

Rectal temperature

Shoulder temperature

Vulval temperature

Rump temperature

PGF2 to.LR peak

PGF2a to ovulation

Estrus length


21.3a

58.9

38.75

32,60

33.68

33.29

94.4

118.4

21.0


+ .75c

+ 3.3C

+ .23c

+ .97c

+ .71c

+ .89c

-+ 26.2e

+ 23.9e

+ 3.8f


32.0

67.2

40.24

36.54

36.83

36.97

72.8

96.0

16.0


,48d

3.5d

.33d

.47d

.68d

.45d

23.2e

24.8e

3.7e


C


C**

C**

C**

C**

hr.

hr.

hP.


a(4 + SD)
**(P<.01)
-b(P<.10)
c(n=26), d(n=23), e(n=5), f(n=4)






- 29 -


1.49 C greater than heifers in the 21.3 C chamber. Skin temperatures

also were significantly (P<.01) elevated in the hot chamber. Visual

appraisal of the data showed there was no tendency for skin or rectal

temperatures to decline during chronic exposure to the heat stress of

our experiment which would have suggested adaptation. Indices of

physiological response to PGF2a (duration of time between PGF2a injection

and the LH peak and time between PGF2a injection and ovulation) were not

different between treatments (P>.10). The interval from the LH peak to

ovulation was approximately 24 hr. for both groups (24 hr. in 21.3 C;

23.2 hr. in 32.0 C). This interval is similar to that reported by

Chenault et al. (1973, 1975). Arije, Wiltbank and Hopwood (1974), and

Christenson, Echternkamp and Laster (1974) for unsynchronized animals

and the PGF2a induced interval reported by Chenault et al. (1974) and

Hafs et al. (1974). If the trend for the heat stress group to have

shorter intervals from PGF2a injection to LH peak and ovulation is real

(table 1), we may have failed to detect differences due to small numbers

(n=5 each) and appreciable variation. In this study, if hyperthermia

affected endocrine-physiological interactions, it did not appear to

alter time between the LH peak and ovulation.

Length of estrus was significantly shorter (P<.0.) for the heat

stressed heifers and was comparable to the 14 hr. duration of estrus re-

ported by Gangwar, Branton and Evans (1965) for Holstein heifers in hot

natural summer climatic conditions of Louisiana. Two of four heifers

inseminated in the 21.3 C chamber were pregnant at 40 days compared to

none of 5 in the 32.0 C chamber. Though there were small numbers of

animals inseminated in this trial, the percentage of successful pregnancies






- 30 -


was comparable to that of the Dunlap and Vincent (1971) environmental

chamber study. This related well to observations that thermal stress

in this study interfered with the overall reproductive process in

heifers. Thus, the environmental condition did affect body temperature,

duration of estrus and probably overall fertility. Whether hormonal re-

sponse under these conditions varied was. of utmost interest.

Pre-PGF2a injection plasma samples were analyzed by analysis of

variance to detect possible differences in progestins, estradiol, estrone,

LH, prolactin and corticoids due to temperature, sampling time, temper-

ature X sampling time interaction and animals within temperature treatment.

Progestins (X = 3.21 ng/ml, 21.3 C; 7 = 3.16 ng/ml, 32.0 C) were not in-

fluenced by temperature or sampling time. However,a significant (P<.05)

temperature by time interaction suggested different progestin concentra-

tions at different times of sampling in each treatment chamber. Plasma

progestins appeared to decline in the heat stressed group with pro-

gressive sampling, whereas in the cool group they did not change

(Appendix, table 12). Also, there was significant (P<.01) variability
in progestin concentrations among heifers in each chamber. This would
suggest that there is considerable variation in progestin secretion from

heifers during the luteal phase of the cycle. Animal variability in

pre-injection plasma estradiol concentrations (X = 3.02 pg/ml, 21.3 C;

2.32 pg/ml, 32.0 C) was significant (P<.01). Estrone (X = 3.04 pg/ml,

21.3 C; X = 3.25 pg/ml, 32.0 C), LH (X = .53 ng/ml, 21.3 C; X = 182 ng/ml,

32.0 C) and prolactin (X = 12.73 ng/ml, 21.3 C; X = 15.32 ng/ml, 32.0 C)

pre-PGF2a plasma concentrations were not influenced by temperature, time

of sampling, temperature X time interaction or animals within temperature






- 31


treatment (P>.10). Variability in plasma corticoids was significant

due to sampling time (P<.01) and treatment X sample time interaction

(P<.01). However, there was no difference due to main effects of

temperature (9.0 ng/ml, 21.3 C; 9.55 ng/ml, 32.0 C).

A logical physiological reference to analyze the data is the

preovulatory surge of LH. All animals had a preovulatory LH surge, and

each hormone was analysed initially to determine time relationships

with the LH surge. Least squares statistical models were selected

based on tests of significance of higher order terms (time) in the

regression analyses and visual appraisal of the graphs.

Figure 2 shows the progestin responses at 21.3 C and 32.0 C.

Data for the regression analyses have been synchronized to the LH peak

for analysis. The statistical model included treatment, heifer within

treatment and time trends (Appendix, table 4). The progestin time trend

for heifers at 21.3 C was best described by the equation Y (progestin,

ng/ml) = 1.533 + 1.160X .344X2 + .034X3 .0014X4 + .00002X5 (P<.01)

where X = .1 hr., whereas the time trend for the 32.0 C heifers showed

a significant (P<.G1) curvilinear relationship best described by a third

order equation: Y = 7.923 1.123X + .061X2 .0010X3

Tests for heterogeneity of regression were significant (P<.01),

suggesting that the 5th order regression curves for each treatment were

not parallel. This observation implies that there was a different time

response to treatments. We feel that this difference was probably due

to heifers in the 21.3 C chamber having their LH peak approximately

22 hr. later from the start of blood sampling than the heifers main-

tained at 32.0 C (table 1). Therefore, on the average cool heifers had










-J
_I




0&



Cz5r
l-5

U]


-144 -120


-96 -72 --48 -24 0 24 43 72 97
HOURS


FIGURE 2,


SEQUENTIAL CHANGES IN PLASMA PROGESTINS IN HEIFERS AT
SYNCHRONIZED TO THE .TIE OF THE LH PEAK.


21,3 C OR 32,0 C


-21.- C (.2= .5 2)

---- 2.0 C ( .)



LH
PE A



-







- 33 -


a longer plateau of low progestins prior to the LH peak. PGF2a caused

a drop in progestin concentration by 18 hr. after injection in all

heifers (Appendix, table 5). Apparently, factors controlling the pre-

ovulatory release of LH in cool heifers were slightly delayed (~ 24 hr.)

due either to treatment effects or chance. Thus, due to a shorter interval

between PGF2a and the LH peak for the 32.0 C heifers, one would expect

higher progestins because of shorter trough duration. Also, the number

of observations earlier than -120 hr. was very small. Significant

(P<.01) among heifer differences were detected within treatments, but

differences among treatments were not significant [(P>:10) Appendix,

table 4). This finding is in direct conflict with Stott, Thomas and

Glen (1967), who reported elevated progesterone on day of estrus in

thermally stressed cows. However, Stott and Wiersma (1973) more recent-

ly reported a depression in plasma progestins due to chronic thermal

stress.

The progestin RIA has a sensitivity of 25 pg or .025 ng/ml plasma.

Coefficients of Variation (C.V.) for progestins after accounting for

variability due to treatment, heifer in treatment and time trends were

115% (cool) and 99% (hot). Acute increases of 2.5 (Gwazdauskas,

Thatcher and Wilcox, 1972) and 1.5 ng progesterone per ml plasma (Wagner,

Strohbehn and Harris, 1972) for a period of 2 hr. were detected follow-

ing 200 IU and 100 IU ACTH injections, respectively. If thermal stress

had elicited an adrenal release of progesterone we would have been able

to detect it. Mills et al. (1972) detected a significant elevation of

only .47 ng/ml progestins in heifers thermally stressed for 72 hr. at

the onset of estrus. Therefore, with overall progestin levels of






- 34 -


.147 + .095 ng/ml (X + SD) the day of estrus and day after estrus and

no differences due to temperature, we cannot conclude that chronic

heat stress caused adrenal hyperprogesterone response. Our findings

support Miller and Alliston (1974b), who found no difference in plasma

progesterone during the bovine estrous cycle when twice daily measure-

ments were made in control (17-21 C) or heat stress (21-34 C)

environments. Progestins do not appear to be elevated during the

period between luteal regression and ovulation.

Data for regression analyses of estradiol were separated into

two periods and independently analyzed to characterize time trends.

The two periods were from -144 hr. to time 6f the LH peak (including

LH peak time) and from the LH peak to +96 hr. (also including the LH

peak time). Pre and post LH peak time trends for estradiol were best
2 3
described by Y (estradiol, pg/ml) = 4.53 .482X .045X + .0069X

(P<.05) and ?post = 45860.38 12165.711X + 1285.829X2 67.674X3 +
1.773X4 .019X5 (P<.05) for the cool heifers and Y = 5.67 1.279X

+ .092X2 (P<.01) and Yp = 490.168 73.503X + 3.647X2 .060X3
post
(P<.01) for the hot heifers (figure 3). Plasma estradiol was depressed

(P<,10) in the 32.0 C chamber (peak estradiol:10.4 pg/ml plasma for

21.3 C heifers compared to 7.2 pg/ml plasma for 32.0 C heifers; Appendix,

tables 4 and 6).

Lower plasma estradiol may have contributed to the shorter periods

of estrus seen in the 32.0 C heifers. However, these lower concentra-

tions of estradiol were adequate to elicit estrous behavior and LH

release causing a subsequent ovulation. The lower estradiol may reflect

altered production, secretion, clearance or receptor binding under







U!4


-J
a.
-,,,


/


LH
PEAK


* -.144 -120 -96


-72 -48


-24 0 24


48 72


FIGURE 3.


HOURS
SEQUENTIAL CHANGES IN PLASfMA ESTRADIOL IN HEIFERS AT 21.3 C OR 32.0 C
SYNCHRONIZED TO THE TIME 01- THE LH PEAK.


21.3 C
32.OC


96


,,1.,_,:,, rbYILTO~C~-~II-C ly- II.-I---tUMUI(P---~-r-~U~-P-? --1






- 36 -


conditions of hyperthermia in cattle. Significant among heifer vari-

ability (P<.0l) was detected in the 32.0 C heifers (Appendix, table 4).

Whether slightly altered plasma estradiol would affect such factors as

uterine and oviductal blood flow, temperature of the reproductive tract,

tract motility, gamete and embryo transport that may contribute to poor

fertility under heat stress is not known. Results of the present study

reveal only a subtle effect of heat stress on plasma estradiol.
Unlike the time responses of estradiol, estrone showed no apparent

association with onset of estrus or LH peak when data were synchronized

to the time of the LH peak (r-0, estrone:estradiol; table 2). Hansel

(1971) reported an estrone peak between days 13 to 16 of the bovine

estrous cycle and suggested that this elevation in estrone may be related

to corpus luteum regression. Therefore, data were analyzed from the

time of the PGF2o injection (figure 4; Appendix, tables 4 and 7). Time
A
responses were best characterized by Y (estrone, pg/ml) = 2.671 + 1.488X

.763X2 + .120X3 .0077X4 + .00018X5 (P<.01) for the 21.3 C group and
^2 3 4 5
S= 2.298 + 1.828X .781X + .145X3 .0072X + .00017X (P<.05) for

the 32.0 C heifers. The significant elevation (P<.05) of estrone

(Appendix, tables 4 and 7) due to heat stress is best seen following

PGF2. through 72 hr. (figure 4). There was no evidence that these

curves were not parallel (5th order, P>;10) suggesting that in both

treatments estrone followed the same decline post-PGF2 However, estrone

levels were higher through 72 hr. in heat stressed heifers. The slight

rise in estrone prior to PGF2a injection to +12 hr. may be related to a

luteolytic action as suggested by Hansel (1971), although heifers were

only day 9 of the estrous cycle at time of PGF2c injection.







- 37 -


Table 2. Simple correlations between

Progestins Estradiol

LH -.14* .45**

Progestins -.14*

Estradiol

Estrone

Corticoids


hormone

Estrone

.03

.40**

.00


measurementsa

Corticoids

-.02

.14*

.11*

.09


Prolactin

-.01

-.07

.02

-.06

.22**


an=291
*(P<.05), >.11
**(P<.01), >.15











S"\ 2 1.3 C -- R2 .39
32.00--- -,R2 .27


Al


;IE P PG F2c<
Cc'




-18 0 24 48 72 96 120 144
HOURS

FIGURE 4, -SEQUENTIAL CHANGES IN PLASMA ESTRONE IN HEIFERS AT 21,3 C OR 32.0 C
SYNCHRONIZED TO THE TI'E OF PGF2, INJECTION,







- 39 -


Chenault et al. (1974) and Henricks et al. (1974) reported

estrone to vary greatly within and among animals after PGF2a injections.

The C.V.'s for estrone in this study after accounting for heifer and

time variability were 65.3 and 74.0% for cool and hot groups, respectively.

Plasma estrone concentrations were less than 5 pg/ml and agree with the

work of Echternkamp and Hansel (1973). However, they reported that

estrone was slightly elevated at estrus in one cow. This observation

was difficult to support since there was no statistical analysis or re-

port of estrone variability.

Because LH increases above basal levels for only about 10 hr.

(Chenault et al., 1975), LH data were separated into four time periods

to analyze, independently, both basal and preovulatory peak concentra-

tions. These periods were: (1) from -148 to 8 hr. prior to the LH

peak (-8 hr.); (2) -8 hr. to the peak of LH; (3) LH peak to +8 hr.

and (4) +8 hr. to +96 hr. (figure 5). Average LH concentrations for

period (1) were 1.80 + 1.21 ng/ml plasma (X + SD; n=102) for the cool

heifers (21.1 C) compared to 1.75 + 1.27 ng/ml (n=74) for the heat

stressed heifers (32.0 C). A linear increase (P<.01) in LH occurred

between -8 hr. to the LH peak (peak LH 32.19 ng/ml for cool heifers

compared to 33.17 ng/ml for hot heifers), and then dropped linearly

(P<.01) to basal levels by +8 hr. (.43 ng/ml cool compared to .49

ng/ml hot). Basal levels were defined as any LH concentration within

three standard deviations of the mean for all samples (n=169) up to

-8 hr. (1.75 + 1.22 ng/ml). During the initial period, two heifers in

the cool chamber had sporadic peaks (>3 S.D.) of LH that occurred

between -72 and -12 hr. prior to the LH peak (Appendix, table 8).






OVULATI O0
ESTR US





c
il 21.3 C ---
f!s.0C -


- -


-144 -120


-~3 -72 -403 -~


0 24 40 72


OURS


SEQUENTIAL CHANGES IN PLASMA LH IN HEIFERS AT EITHER 21,3 C OR 32,0 C,


K20


irc~~r~.~c~rararrt4~rzrrr;rrrsr31rp~mr


FIGURE 5,







- 41 -


The preovulatory surge of LH remained above basal levels for 10.4 and

9.6 hr. for the 21.3 C and 32.0 C groups, respectively. LH concentra-

tions in this study agree with those of Henricks, Dickey and Niswender

(1970) and Snook, Saatman and Hansel (1971). Unlike results of Madan

and Johnson (1971) and Miller and Alliston (1973), we found no

significant difference in LH levels in response to heat stress (Appendix,

table 4). Our contradictory results under conditions of thermal stress

may be due to frequency of sampling (twice daily; Miller and Alliston,

1973), sensitivity of experimental design in which among animal variability

was considered in the present study, duration of the LH peak (~ 10 hr.)

or breed differences (Madan and Johnson, 1971). Riggs, Alliston and

Wilson (1974) detected a difference in the preovulatory LH surge during

heat stress between Hampshire and Duroc gilts. Such differences may

exist between the study of Madan and Johnson (1971) in which Guernsey

cattle were used and in our study where only Holstein heifers were

used.

All animals had a preovulatory LH surge, suggesting that hyper-

thermia did not prevent the triggering mechanism for LH release.

Although estradiol levels in peripheral plasma were slightly depressed

in the 32.0 C heifers, it does not appear that these lowered estradiol

concentrations altered LH release.

Plasma prolactin was analyzed initially in the same manner as

estradiol (pre- and post peak of LH). However, there was no change in

prolactin associated with estrus or the LH peak as previously reported

by Swanson and Hafs (1971). Absence of any association between prolactin

and estrus is supported by work of Hoffman et al. (1974) in which an






- 42 -


inhibitor of prolactin secretion caused no estrous cycle disorders.

Also, Wetteman and Hafs (1973) were unable to find elevated prolactin

on the day of estrus.

Since there were no detectable changes in prolactin associated

with the LH peak, data were analyzed further relative to time of PGF2,

injections. Hafs et al. (1974) reported that prolactin increased

immediately following PGF2a injection (within 1 hr. and lasting for

4 hr.). However, an increase in our study was not detected because

the first blood samples were not taken until 6 hr. after injection. No

differences were detected between the 21.3 C and 32.0 C treatment groups

(P>.10; Appendix, tables 4 and 9). Time trends of prolactin for the

21.3 C and 32.0 C treatment groups were described by the following

equations: Y (prolactin, ng/ml) = 14.50 1.707X + .371X2 .018X3 and

Y = 14.28 + 2.738X .733X2 + .066X3 .002X4 (3?.0 C) (figure 6). The

4th order time curves were not parallel (P<.005). Prolactin C.V. after

accounting for heifers and the above time equations was 49.4% (21.3 C)

and 26.3% (32.0 C). During the initial 42 hr. (-18 to 24 hr.), the

prolactin response in the cool chamber appeared to decline. This

observation may be due to a lowering of stress-induced prolactin secretion

with more sampling (Tucker, 1971). Apparently heifers in the 32.0 C

chamber could not adjust to sampling as quickly since prolactin increased

and remained elevated until 24 hr. after PGF2 However, this is

questionable since trends are very subtle and the curves account for

little of the variability (figure 6).

An increase in plasma prolactin due to heat stress was antici-

pated in the present study based on reports of Koprowski and Tucker (1973),













- -
-


21.3 C R2 .07
32.0 ---- ,R2= .lI


PGF2c


-18


FIGURE 6,


0 24 48 72 96


120


HOURS
SEQUENTIAL CHANGES IN PLASMA PROLACTIN IN HEIFERS AT 21,3 C OR 32,0 C
SYNCHRONIZED TO THE TIME OF PGF2, INJECTION,


20


-i
144
144


;------- --- ----- -ra~F~ur~arr~-a90a~~a~i~oy-- ----~-.-~~rm






- 44 -


Schams and Reinhart (1974) and Thatcher (1974), in which seasonal

changes of plasma prolactin were detected (high during summer). Wetteman

and Tucker (1974), using twice daily sampling, detected only slight

differences (P<.10) in serum prolactin in 3 mo. old calves exposed either

to 21 or 27 C temperatures under a 12 hr. per day light regime. The

induced release of prolactin following injection of thyrotropin releasing

hormone (TRI) was twice as great in calves exposed to 27 C as calves

at 10 C. Furthermore, they suggested that these results, which are

opposite those seen in lactating cows following the milking stimulus,

may be due to differences in anterior pituitary responsiveness of 3 m6.

old calves at different temperatures.

In our study, no differences in average plasma prolactin concentra-

tions were detected between heifers at 21.3 C or 32.0 C (figure 6;

Appendix, tables 4 and 9). Thus at a constant 14 hr. light 10 hr.

dark regime an environmental temperature of 32.0 C caused no increase

in prolactin compared to controls at 21.3 C. Perhaps other factors

control the seasonal increase in prolactin previously reported (Koprowski

and Tucker, 1973; Schams and Reinhart, 1974; Thatcher, 1974). Karg and

Schams (1974) reported a positive correlation of day length and basal

prolactin levels in cattle. Relkin (1972) showed that changes in light:

dark ratios for rats altered pituitary prolactin content and plasma

prolactin concentrations. This effect is seen after only 4 to 8 hr. of

exposure to different lighting regimes. Photoperiod may be a factor

influencing pituitary prolactin secretion. Under Florida conditions

seasonal temperature changes also are correlated with increasing periods

of day length (Gwazdauskas, unpublished observations). Thus, temperature






- 45 -


and photoperiod effects are confounded in evaluating seasonal effects

on plasma prolactin concentrations. Under controlled environmental

conditions of the present study, temperature seemed unimportant in

eliciting a major change in prolactin secretion.

Figure 7 shows the plasma corticoid response when data were

synchronized to the LH peak. Statistical analysis revealed no dif-

ferences (P>.10) between treatment means (Appendix, tables 4 and 10).

Furthermore, we were unable to detect any individual treatment time

trends after looking-at regressions up to the 5th order. After account-

ing for corticoid variability due to treatment, heifers within

treatment and time trends (up to the 5th order), there was a 65% C.V.

for plasma corticoid concentrations. Our data, with blood samples

taken at 4 hr. intervals at least 2 days prior to estrus, do not sup-

port Miller and Alliston's (1974a)finding of increased corticoids

early the day of estrus (twice daily sampling). Nor does it support

a report which showed lower plasma corticoids in dairy cows during

summer months in Arizona (Stott and Wiersma, 1973). However, Arizona

climatic conditions of high temperature and low humidity may be dif-

ferent from our study with high humidity and high temperature. Our

results show numerous episodic peaks during the day. Wagner and

Oxenreider (1972) also reported episodic peaks of plasma corticoids

when measured at 30 min. intervals throughout the day. They also noted

diurnal corticoid variation, but we were unable to detect any time of

day differences (P>.10) when data were analysed at 4 hr. intervals.

Due to large variability in plasma corticoid levels, a large treatment

difference in corticoid concentrations would be needed to detect a












POOLED MEANS -


-144 -120


-96 -72 -48 -24 0 24 48 72 96
HOURS


FIGURE 7,


SEQUENTIAL CHANGES IN PLASMA CORTICOIDS SYNCHRONIZED TO THE
LH PEAK USING POOLED MEANS OF HEIFERS AT 21,3 C AND 32,0 C,


TIME OF THE


20


U)
_J

_J



(3


t--
0
0






- 47 -


significant difference. We failed to detect any differences in plasma

corticoid associated with estrus, ovulation or heat stress when

monitoring plasma concentrations at 4 hr. intervals.

When time was removed from the model and each hormone considered

as a dependent variable, plasma LH had a negative association with

progestins (r = -.14, P<.05; table 2). Progestins also were negatively

related to estradiol (r = -.14, P<.05). In the overall model LH was

positively related to estradiol (r = .45, P<.01). These observations

are consistent with findings of Chenault et al. (1975) and support

their hypothesis that progestins may be inhibiting estradiol biosynthesis

and LH release. The significant relationship between plasma corticoids

and prolactin (r = .22, P<.01) may be related to the stress response of

both hormones (Gwazdauskas, Thatcher and Wilcox, 1972; Koprowski and

Tucker, 1973).

Increases in plasma and blood volumes due to heat stress have

been reported in cattle (Bianca, 1965), and chronic exposure to heat

resulted in decreased hematocrit. Changes in plasma electrolyte

concentrations due to thermal stress are reported to be slight (T. N.

Wegner, personal communication). Cattle in tropical areas had a

higher body water content in summer than in winter months (Thompson,

1973). Such factors as plasma volume and dilution may influence

interpretation of hormonal responses to a controlled heat stress. For

example, a difference in plasma corticoid concentration was not detected

in our study. However, a heat stress induced increase may have been

undetectable due to a possible plasma dilution response of these heifers.

Conversely, a decrease in estradiol concentration may have occurred







- 48 -


due to plasma dilution and not necessarily decreased secretion. Such

criticisms apply to all other hormonal responses in this study. Thus,

it was of interest to measure plasma total protein concentration and

plasma osmolality to determine if a possible plasma dilution had occurred.

Plasma samples from 8 to 96 hr.. after the LH peak and having less

than 3 pg/ml estradiol were pooled within heifer for evaluation. There

was no difference (P>.10) in total protein concentration or plasma

osmolality between heifers at 21.3 C and 32.0 C (table 3). These re-

sults suggest that no appreciable plasma dilution had occurred.

However, we have no measurement of total plasma volume of heifers for

this study.

A Corticosteroid Binding Globulin (CBG) of plasma has been re-

ported for various species (Seal and Doe, 1965) and also is present in

the bovine (Lindner, 1964). Such a protein acts as a corticoid carrier

molecule through the blood. Although total plasma protein concentration

(table 3) did not vary between treatments, certain alterations of pro-

tein composition may have occurred. Although plasma corticoid

concentrations did not differ between treatments, their potential

biological effectiveness would be appreciably altered if the concentra-

tion of plasma CBG differed.

Utilizing the procedure of Pegg and Keane (1969), the association

constant (Ka) and cortisol binding capacity of CBG were determined on

pooled samples (within heifer) of each experimental heifer. The

association constant did not vary due to treatment (P>.10; table 3).

An average experimental Ka of 1.86 X 10' M-1 was indicative of a protein

with an intermediate affinity for the cortisol ligand. It is a protein







- 49 -


Table 3. Physical characteristics of plasma
at 21.3 C and 32.0 C.


21.3 C


in heifers


32.0 C


Protein
(mg/ml)

Osmolality
(milliosmoles/kg H20)

Corticoids
(ng/ml)

Cortisol Binding Capacity
(ng/ml)

Association Constant
(Ka X 107 M-1)


76.38 -+


8.52a


259.65 + 11.65


6.7 + 1.2


118.93 + 48.02


1.52 +


75.50 + 9.05


268.70 + 10.41


5.9 + 1.0


55.86 + 11.19*


2.20 + .69


a (I + SD)
*(P<-.05)







- 50 -


with an association constant higher than a low affinity protein such as

human serum albumin (Ka = 1 X 10' M-1) but lower than the Ka for a

tissue receptor protein such as the cortisol mammary gland receptor

(Ka = 5 X 108 M-1; Tucker, Larson and Gorski, 1971). It was not expect-

ed that thermal stress would alter the physical chemical properties of

the CBG protein (Ka) but perhaps may alter the amount (capacity) of CBG

per ml of plasma. Indeed there was a significant difference (P<.05) in

cortisol binding capacity (ng/ml) between treatments (table 3). Thus

under experimental conditions for quantifying cortisol binding capacity

at 4 C, plasma of heat stressed heifers had a 53% lower capacity to

bind cortisol. This suggested that under such conditions, plasma from

hyperthermic heifers contained a decreased concentration of CBG.

Therefore, under environmental temperatures of 32.0 C at a body tempera-

ture of 40.24 C both the concentration of plasma CBG and cortisol bound

CBG (product) would be less if the rate constant for the forward reaction

was not different at an elevated body temperature of 1.5 C (table 1).

With this reasoning, heifers exposed to a thermal stress characteristic

of our experiment would have a greater percent free cortisol compared

to CBG bound cortisol at a constant total cortisol concentration. As

previously described, total plasma corticoid concentrations did not

vary between treatments (6.7 compared to 5.9 ng/ml; table 3).

Under other stressful conditions a lowered corticoid binding

capacity has been reported for various species. In human burn patients

a slightly lowered cortisol binding capacity has been reported (Mortensen

et al., 1972), in which decreased capacity was inversely related to burn

area. By analogy, lactation can be considered a stress in the sense







- 51 -


that reproductive efficiency is lower during this period. Lactation

inhibits the onset of estrous cycling in rats nursing 6 or 12 pups

compared to post-parturient rats which do not lactate (Tucker and

Thatcher, 1968). Early weaning of calves from their dams increased the

occurrence of estrus and increased pregnancy rates in beef and dairy

cattle (Laster, Glimp and Gregory, 1973). Troconiz (1973) has reviewed

the cystic ovary condition in dairy cattle. High milk producing cows

had a greater incidence of cystic ovaries and therefore a greater fre-

quency of reproductive problems. In rats nursing 12 pups, CBG activity

was lower in comparison to rats nursing only four pups (Westphal, 1970).

Such a nursing intensity will delay occurrence of normal estrous cycles

(Tucker and Thatcher, 1968). Thus under conditions of lactational

stress (relative to reproductive performance) CBG activity was depressed,

The liver is the reported source of CBG (Guyton, 1966) and the

thyroid gland is reported to exert a controlling influence on CBG

activity. Gala and Westphal (1966) showed that TSH stimulated CBG

activity in hypophysectomized rats and was primarily responsible for

regulation of CBG levels. In cattle under conditions of high environ-

mental temperatures, thyroid activity was depressed (Johnson and Yousef,

1966). If the hormonal control of CBG production is grossly comparable

between rats and cattle, then lower CBG binding capacity of heifers

detected in our study would be expected.

Hypothyroid patients have a slower turnover of cortisol. Both

bound and free steroid fraction disappearance rates were slower than

normal or hyperthyroid patients (Beisel et al., 1964). In our study

the amount of free hormone would have a greater biological role in the







- 52 -


heat stress group due to the lower binding capacity. This may result in

a lower level of ACTH secretion due to greater negative feedback inhibi-

tion. In the bovine, corticoid turnover rates were depressed during

chronic heat stress (Christison and Johnson, 1972). This also suggests

a longer biological life for the circulating corticoid allowing a greater

ACTH negative feedback since less corticoid is also bound to transcortin.

However, the amount of free cortisol in our study, estimated by extra-

polation at 4 C, was not different (P>.10) between the 21.3 C heifers

(1.39 ng/ml) and 32.0 C heifers (1.43 ng/ml).

Clarification is needed in this area as to the physiological role

of bound and free steroids because of conflicting reports between species

and stress situations. Our finding that corticoids were not elevated

during chronic heat stress, irrespective of the binding capacities, may

be advantageous to the cow in that heat production has been shown to

increase 30 to 40% at 35 C when hydrocortisone acetate was administered

(Yousef and Johnson, 1967).

In the second phase of the experiment, 8 days following ovulation

in the last heifer, 200 IU ACTH was injected, IV, into 10 heifers. The

ACTH was given while heifers were in the luteal phase of the estrous

cycle or at a time when a progesterone increases in peripheral plasma

due to ACTH injection (Gwazdauskas, Thatcher and Wilcox, 1972; Wagner,

Strohbehn and Harris, 1972) may not have a detrimental effect on the

developing embryo (Johnsson et al., 1974). Figure 8 shows the corticoid

response curves following ACTH injection. The 32.0 C group responded

with significantly lower (P<.10) corticoid concentrations. The 6th

order regression curves were not parallel (P<.01) suggesting that the










U)

CL



(D




O
0
0

F--
0
0-


100

90

80

70

60.

50-
l-
40-

30-

20-


-2 -I 0 I 2 3 4 5 6 7 8 9 10 II 12


FIGURE 8.


TRANSITORY
IN HEIFERS


CHANGES
AT 21,3


IN PLASMA
C OR 32,0


HOURS
CORTICOIDS FOLLOWING INJECTION
C,


OF 200 IU ACTH


ACTH


Vi


32.OC x---,R=.70
21.3Co ---,R2P .67






- 54 -


hot group response was earlier to reach a peak (75 min. compared to

105 min.), had a lower magnitude (73.5 compared to 100.2 ng/ml corticoid)

and was of shorter duration (4 hr. compared to 5 hr.; Appendix, table 11).

This response is comparable to that reported by Shayanfar (1973) in

which lactating cows exposed to environmental temperatures above 21.1 C

responded the same way. The cool heifer response was best described by:

Y (corticoids, ng/ml) = -521.387 + 4648.999X 12253.643X2 + 13609.684X3

- 5942.539X4 + 4.326X5 + 464.541X6 (P<.01), whereas the hot heifer re-
^2
sponse was best characterized by Y = -613.342 + 6525.433X 23576.092X

+ 41551.725X3 38756.632X4 + 18359.263X5 3477.023X6 (P<.01).

The apparent reduced ability of the adrenal to secrete and/or

synthesize corticoids following ACTH stimulation during heat stress may

be related to a chronic lower level of endogenous ACTH secretion. The

lower plasma cortisol binding capacity in the 32.0 C heifers may provide

a greater amount of free corticoid to exert a feedback inhibition on

endogenous ACTH secretion. In addition, there is also a lower level of

corticoid turnover and secretion during chronic heat stress (Christison

and Johnson, 1972). As a result the degree of chronic endogenous ACTH

secretion maybe less, causing a reduction of responsive adreno-cortical

tissue. These conditions may result in a lower adrenal corticoid increase

in response to a pharmacological challenge with ACTH. A reduced level

of adrenal function during heat stress would be advantageous to the animal

calorigenically. Corticoid secretion did not appear to be higher in the

heat stressed group since resting corticoid levels were not greater than

controls. However, a possible decreased adrenal secretion rate was not

reflected by a lower plasma corticoid concentration. It was not until







- 55 -


a response to ACTH was evaluated that adrenocortical function appeared

to be depressed.

To determine the significance of this apparent reduction in

adrenal responsiveness to ACTH due to hyperthermia plasma ACTH levels

need to be determined in the bovine under different physiological stress

situations. Other possibilities include determining effects of stress

on ACTH receptors, more definitive studies on corticoid-CBG binding

properties in relation to thermal stress and possible steroidogenic-

enzyme alterations in the adrenal.

Pre-ACTH plasma progestin and corticoid concentrations were test-

ed to detect differences in levels due to heat stress and pregnancy

status (Appendix, tables 11 and 12). The analyses include temperature,

pregnancy status and heifers nested in temperature-pregnancy status.

There were no statistically significant differences (P>.10) either in

hormone concentrations due to temperature or pregnancy status. However,

significant among animal variability (P<.05) was found in progestin

levels. These results agree with the pre-PGF2a treatment hormonal

values in the first phase of this experiment (Page 30). Our observa-

tions conflict with a summer seasonal depression in corticoid and

progestin concentrations reported by Stott and Wiersma (1973). The

present study also did not confirm their finding of higher progestins

in fertile cows on day 15 of pregnancy or the estrous cycle. This

period of corpus luteum function is comparable to our study (heifers

were between estrous cycle days 9-13).

In summary, environmental treatment of 32.0 C evoked a 1.49 C

increase in rectal temperature and a 3 to 4 C increase in skin






- 56 -


temperatures. The time durations between PGF2a injection and the LH

peak and the period between PGF2a and ovulation were not different

(P>.10) between treatments. Length of estrus was shorter (P<.10) for

the heat stressed heifers. Two of four heifers inseminated in the 21.3

C chamber were pregnant at 40 days compared to none of five in the 32.0

C chamber. Thus, the environmental condition did affect body temperature,

duration of estrus and overall fertility.

Preinjection plasma samples showed no differences (P>.10) in

any of the hormonal measurements due to the main effect of temperature.

Average progestin concentration between treatments was not different

(P>.10). However the 5th order response curves were not parallel (P<.01)

indicating a different time response between treatments. Progestin

concentrations declined in a similar manner in both groups following

PGF2a injection. Heifers in the 21.3 C group, on the average, had a

LH surge about 24 hr. later than heifers in the 32.0 C group. This

24 hr. time lag would account for the difference in time responses when

data were synchronized to time of LH peak. Mean estradiol concentra-

tions were significantly (P<.10) lower in the heat stressed heifers.

The lower plasma estradiol may have contributed to the shorter estrous

periods seen in the 32.0 C heifers. However, these lower concentrations

of estradiol were adequate enough to elicit estrous behavior and trigger

LH release causing a subsequent ovulation.

Estrone showed no apparent association with the onset of estrus

or LH peak when the data were synchronized to the time of the LH peak.

There was a significant elevation (P<.05) of estrone due to heat stress

but there was no evidence that estrone time trends following PGF2a were







- 57 -


not parallel (P>.10) suggesting that in both treatments estrone follow-

ed a similar decline postinjection. No significant differences (P>.10)

were found in mean LH concentrations between heifers at 21.3 C or 32.0

C. Preovulatory peak LH concentrations were 32.2 ng/ml and 33.2 ng/ml

plasma for the 21.3 C and 32.0 C heifers, respectively. All animals

had a preovulatory LH surge, suggesting that hyperthermia did not pre-

vent the triggering mechanism for LH release.

There was no change in prolactin associated with estrus or the

LH peak, therefore prolactin was analyzed relative to time of PGF2a

injection. Mean prolactin concentrations were not different between

treatments (P>.10). The 4th order time curves were not parallel (P<.005).

Heifers in the 21.3 C chamber had a decline in plasma prolactin after

the initial sampling as compared to increased prolactin concentrations

in the 32.0 C heifers during this early blood sampling period. The

summer seasonal increase in plasma prolactin reported by various

researchers may be more related to photoperiod effects. There was no

difference (P>.10) between treatment means in plasma corticoid concentra-

tions. Furthermore, we were unable to detect any individual treatment

time trends after looking at regressions up to the 5th order. Plasma

corticoid C.V. was 65% after accounting for variability due to treat-

ment, heifers within treatment and time trends up to the 5th order.

In an attempt to determine if plasma dilution may have occurred,

total protein concentration and osmolality were measured. There was no

difference (P>.10) in total protein concentration or osmolality between

treatment groups. However, no measurement of total plasma volume was

made. Cortisol binding capacity of CBG and its association constants






- 58 -


(Ka) were determined. The affinity (Ka) of cortisol for CBG was not

different between treatments (P>.10); however,the binding capacity

of CBG for cortisol was significantly (P<.05) reduced in the 32.0 C

heifers. This observation suggested that under experimental conditions

(4 C) for determining the binding capacity of cortisol, the hyperthermic

heifers may have had a decreased concentration of CBG.

ACTH (200 IU) was injected, IV, into 10 heifers. The 32.0 C

heifers responded with a significantly lower (P<.10) corticoid concen-

tration. The 6th order regression response curves were not parallel

(P<.01) suggesting that the hot group response was earlier to reach a

peak (75 min. compared to 105 min.), had a lower magnitude (73.5 compared

to 100.2 ng/ml corticoids) and was of shorter duration (4 hr. compared

to 5 hr.). Adrenal responsiveness was significantly less in heifers

maintained at 32 C.

Results of this experiment show only subtle thermal effects on

plasma concentrations of estradiol and estrone and no effects on LH,

progestins, corticoids and prolactin. Apart from possible hormonal

involvement with duration of estrus, heat stress does not appear to af-

fect the hormonal milieu associated with corpus luteum regression,

follicle growth and ovulation. The significance of possible lowered

adrenal response in hot environments may be related to a state of

lowered heat production. Since corticoids are known to be calorigenic

(Yousef and Johnson, 1967) a lowered adrenal responsiveness in hyper-

thermic heifers might be physiologically advantageous.

The experiment described in this section has not specifically

considered the possible environmental and hormonal effects on uterine







59 -


temperature. It was of prime importance to characterize uterine

thermal changes during the period of luteal regression, follicle growth

and ovulation under conditions of a mild heat stress, and to document

possible estrogen induced uterine thermal changes.











SECTION III

EXPERIMENT I: THERMAL CHANGES OF THE BOVINE UTERUS FOLLOWING
ADMINISTRATION OF ESTRADIOL-17B


Introduction



The first experiment (Section II) indicated that a thermal stress

increased body temperature, suppressed fertility and caused a slight de-

crease in endogenous estradiol secretion. Furthermore, we reported

previously that uterine temperatures both on day of and day after

insemination were inversely related to fertility (Gwazdauskas, Thatcher

and Wilcox, 1973). This directly indicated that temperature of the

uterus was closely associated with fertility.

Other factors in addition to environmental temperature may in-

fluence uterine temperatures. For example estrogen administration was

shown to increase uterine blood flow in sheep (Huckabee et al., 1970;

Greiss and Anderson, 1970; Rosenfeld et al., 1973; and Resnik et al.,

1974). This uterine hyperemia may have caused heat to be dissipated

from the uterus, thus cooling the uterine cavity (Abrams.et al., 1970a).

Uterine blood flow changes in sheep were monitored following estrogen

injections by looking at differences in temperature between the uterus

and aorta. A rise in blood flow rate resulted in a lower uterine

temperature (Abrams et al.,o1970a).


- 60 -







- 61 -


Although uterine temperature and estrogen relationships have

been foundin sheep, this phenomena has not been examined in the bovine.

Objectives of this study were to determine if uterine-aortic temperature

differences exist in the bovine, and if such differences change following

injection of Estradiol-178.


Materials and Methods


Thermocouple Preparation and Calibration

Lengths of 36 gauge, nylon coated, copper constantan wire

(Revere Corp., Wallingford, Conn.) were pulled through polyvinyl

tubing (V5-V7; Bolab Inc., Derry, N. H.) for measurements of uterine

and blood temperatures. The terminal thermojunctions to be placed in

the saphenous artery then were pulled through a larger polyvinyl tube

(V-12) for additional support. The ends of all thermojunctions were

heat-sealed in the polyvinyl by pushing them through a siliconized,

narrowbore glass tubing which was being heated on a soldering iron.

After sealing, ends were coated with liquid tygon (U. S. Stoneware Co.).

Stranded, untinned copper extension wires (Leads and Northrup, #27-32-36,

Philadelphia, Pa.) were soldered to divided copper wires leading to the

thermojunctions. All extension wires led either to a millivolt

potentiometer (#8686, Leads and Northrup, Philadelphia, Pa.; limits

of error of recording system + .075 C) or to a strip chart recorder

(Hewlett-Packard, M 7100B; limits of error of recording system j+ .03 C).

Most, but not all of the potentials from the aortic-ice water thermo-

couple were suppressed by known amounts before being amplified and

recorded.







- 62 -


Calibration of the thermocouples was made routinely by use of a

Bureau of Standards Certified Thermometer in a well-stirred, insulated

water bath held at intervals between 36 to 40 C. The thermocouple

readings were 0.05 to 0.075 C above the certified thermometer reading,

so all data collected were corrected for these constants.


Surgical Techniques and Experimental Protocol

Four 2-year-old heifers with histories of regular estrous cycles

were used in these experiments. Prior to surgery, heifers were placed

on a 48 to 72 hr. feed and water fast. Heifers were anesthetized with

2 to 4 g sodium thiopental (Abbott Laboratories, North Chicago, Ill.)

dissolved in saline (2 g/20 ml) while standing and restrained. They

were placed onto a portable operating table, tracheotomized and maintained

under surgical anesthesia with methoxyfluorane (Pitman-Moore, Washington

Crossing, N. J.). After removal of hair, the abdominal and inguinal

regions were scrubbed thoroughly with germicidal soap and rinsed with

70% alcohol.

A 15 cm longitudinal midventral incision was made through the

abdominal wall at the cranial margin of the mammary gland. A sharpened

stainless steel cannula was carried into the abdominal cavity through

this midventral incision and pressed through the abdominal wall in the

flank area. All thermojunctions and approximately 2.5 m of extension

wires were drawn through the cannula leaving the remainder of the 3 m

of extension wire and connectors coiled up in a canvas pack. The

cannula was removed from the abdominal cavity by sliding it over the

thermojunctions and withdrawing it through the midline incision. The







- 63 -


pack subsequently was attached to the flank with one or two stainless

steel pins passed through a flap of skin.

The uterus was elevated so that the junction of the uterine horns

with the uterine body could be visualized. Using small scissors and

straight forceps, a 3 to 4 cm tunnel was made under the serosa in the

medial aspect of one uterine horn about 1 cm from the bifurcation. A

thermojunction was inserted into this tunnel and tied in place with

000 silk thread. The extension wires were secured by two to three

additional ties through the serosa along the uterine horn.

Thermojunctions for aortic blood temperatures were routed through

the midventral incision, tunneled under the skin to the inguinal area

where the saphenous artery was exposed. These thermojunctions then

were inserted into the saphenous artery, passed 70 to 75 cm upward to

the abdominal aorta and extension wires fixed with silk suture at the

point of entry into the vessel. Incisions were closed in layers.

Thermocouple placements were confirmed prior to their surgical removal

7 to 10 days after completion of the experiment.

Twenty-four hr. prior to intravenous (IV) injection either of

3 mg Estradiol-17 (Progynon-Schering Corp., Bloomfield, N. J.) or 12

ml of .9% sterile saline, heifers were fitted with polyvinyl catheters

(V-7) by jugular venipuncture. Catheters were'filled with heparin

solution (15 U/ml of .9% saline), capped with a brad and the external

catheter placed in an adhesive tape pouch glued to the neck with branding

cement (Electro Cote Co., Minneapolis, Minn.).

On the day of injection heifers were placed in a stanchion barn

on rubber comfort mats at least 2 hr. prior to recording temperatures.






- 64 -


Each of the four heifers received an estradiol injection, and two of the

heifers also received two saline injections each. Thus there were a

total of four estradiol and four saline experiments. All heifers received

treatment during the luteal phase of the cycle. Recordings were made

from the millivolt potentiometer at 15-min. intervals beginning 1 hr.

prior to IV injection either of Estradiol-17g or saline and ending 6 hr.

after the injections. A pre-experimental control period of 1 hr. was used

to determine a steady state level of uterine temperature. Repeatability

of triplicate measurements at each time was 0.92 for aortic temperature

(mV) with a C.V. of 0.07% (n=66). Repeatability and C.V. for

ATuterus-aorta (V) were 0.99 and 6.44%, respectively.

Initially, an additional thermocouple was placed in the uterine

lumen as well as in the uterine serosa. Prior to and following estrogen

injection the temperatures at both reference points were identical. To

avoid any possible complication due to presence of an intrauterine object,

all subsequent animals were fitted only with a uterine serosa thermo-

junction. In a separate experiment, temperatures were recorded

continuously before and after an injection of Estradiol-173. The major

statistical technique to analyse time changes was least squares as de-

scribed by Harvey (1960). Statistical models were selected based on

tests of significance of the higher order terms in the regression

analyses and visual appraisal of the graphs.


Results and Discussion

The uterine and aortic temperature response following intravenous

injection of 12 ml.saline is shown in figure 9. The slight increase in







OC 12 ml
38.4 SALINE

UTERUS o
38.3 0

38.2- o0 o --- o
0[ o Y = 38.13 +- .0'223X

8T A
38.0 AORTA A
A A '
37. 8 A .

37.8- A A ^A A A

Y-= 37.80 + .0210X
37.7 A
o, A = actual means


S. 0 I 2 3 4 5 6
HOURS
FIGURE 9, UTERINE AND AORTIC TEMPERATURE PRIOR TO AND FOLLOWING IV INJECTION OF 12 ML
STERILE SALINE,







- 66 -


both mean temperatures (~ 0.2 C) which occurred during the 7 h experi-

ment may be related to the normal rhythmic rise in body temperature in

cattle during the day (Bianca, 1968). The greater variability in both

temperatures 4 to 6 hr. after saline injection could have been because

of blood temperature changes induced by some restlessness due to long

confinement in the stanchions. In spite of these changes in uterine

and aortic temperatures, temperature differences between the two were

quite stable during the experiment, indicating that the ratio between

uterine heat production and uterine heat loss had remained unchanged.

Relationships of time (X) and uterine (Y ) and aortic (Y ) temperatures

are shown in figure 9. There was no evidence of curvilinearity; fitting

the two equations accounted for 36 and 37% of the within-heifer vari-

ability in Yu and Ya, respectively. There was no evidence that the

two slopes were not parallel, which suggested that the saline vehicle

had no depressive effect either on uterine or aortic temperature.

Effects of Estradiol-17 on uterine and aortic temperatures are

illustrated in figure 10. The initial fall in uterine temperature of

slightly more than 0.3 C compares favorably with the response noted

previously in sheep (Abrams et al., 1970a). The slight rise in uterine

temperature between 4 to 6 hr. post injection was undoubtedly due to

the rise in blood temperatures as noted in control experiments.

Uterine changes were curvilinear (P<.01) as indicated by the equation

(R = 0.18). A significant quadratic equation (P<.01; R = 0.04) best

describes the aortic temperature response. Why aortic temperature fell

initially is not known. Increased respiratory evaporative heat loss or

sweating may have been responsible. Estrogens are known to be potent









UTERUS


38.9

38.8

38.7

38.6

38.5


39.06 -.2783X + .0304X



o ,


A A


AORTA
!AORTA


S, A = actual means


A
Y = 38.53 -
A


.120X + .0157X2


-1i 0 I 2


FIGURE 10.


3
HOURS


UTERINE AND AORTIC TEMPERATURE PRIOR TO AND FOLLOWING IV INJECTION OF 3 MG
ESTRADIOL-173,


3 mg
E2-17/3
Y,


o u


38.2

38.1







- 68 -


vasodilators of skin blood vessels (Reynolds and Foster, 1940), and to

the extent that heat loss was promoted by this increased skin blood flow,

a lowered temperature may result. One may propose that estrogens could

have had a subtle effect of the thermoregulatory "set point" (Hammel

et al., 1963) which resulted in activating one or more heat loss

mechanisms. However, the decrease in aortic temperature was only about

.1 C.

When the difference in temperature between uterine serosa and

aorta was examined the result of Estradiol-17P administration was obvious

(figure 11). The decrease in ATuterusaorta (AT ) of 0.25 C was de-

scribed by a highly significant (P<.01) curvilinear trend over time.

The ATa began to plateau at approximately 2.5 hr. post estrogen

injection and remained depressed for the duration of the recording period,

although both uterine and aortic temperature started to rise 4 to 5 hr.

post-injection. There was no significant change (P>.10) in ATua follow-

ing saline injection.

Figure 12 is a plot in 30 sec. intervals taken from a continuous

recording of temperatures of one heifer prior to and following Estradiol-

178 injection. In this animal the estrogen effect on uterine temperature

was noted within 1 hr. Rapid oscillations in temperature of the uterine

tracing were considerably dampened by the heat capacity of the uterine

tissue.

A consistent finding in the estrogen experiments was the decrease

in the temperature difference between the uterus, as represented by the

subserosal temperature and the blood df the abdominal aorta. Such a

decrease in AT could occur as a result of a lowered rate of uterine






.50r


SALINE


y =.34 + .0038X


0 0
o


30 -


ESTRADIOL 17/3
A
y = .68 -.309X


E2 -17/3
or A =actual means
-SALINE


-- .0571X2-.0035X5


O
o0



0


DO

LU
I-
c-


2.7
R = .78


- -. ......---.-. S, .4


FIGURE 11, ATuterus-aorta
ESTRADIOL-17,


HOURS
PRIOR TO AND FOLLOWING EITHER 12 [iL SALINE OR 3 MG


A A


.20




.10




0


.40-

/








C

38.8 T, RUS



38.6 -



38.4 -



38.2 -

LT0
AORTA
38.0 -

ESTRADIOL
3 mg (IV)
37. 8 I



-15 0 15 30 45 60 75 90 105 115
MINUTES
FIGURE 12. UTERINE AND AORTIC TEMPERATURE PRIOR TO AND AFTER INJECTION OF ESTRADIOL-17$
-FROM CONTINUOUS RECORDING,







- 71 -


heat production, a possibility which appears remote in view of the many

cellular metabolic activities induced by estrogens (Talwar and Segal,

1971). A more reasonable explanation for the lowered AT is the
u-a
augmented rate of heat loss resulting from the marked estrogen induced

elevation in uterine blood flow. Endogenous estrogens released during

the estrous cycle in ewes are known to be associated with elevated

uterine blood flow rate (Greiss and Anderson, 1970) and increased

vaginal blood flow as inferred from a significant rise in vaginal thermal

conductance in cattle (Abrams et al., 1973). Thus, there is reason to

believe that comparable cyclic, blood flow-induced changes in temperature

of the reproductive tract may occur during the estrous cycle in the

bovine. High uterine temperatures at the time of artificial insemination

are associated with diminished fertility (Gwazdauskas, Thatcher and

Wilcox, 1973). Elevated environmental temperature is thought to

suppress fertility by acting directly on the developing embryo and/or

through altering maternal endocrine function (Vincent, 1972).

Findings in the first experiment indicated that plasma estradiol

of heat stressed heifers was lower during the pre-estrous period.

Results of the present study indicate that a pharmacological injection

of Estradiol-170 can significantly decrease uterine temperatures. In

the final experiment, attempts were made to evaluate changes in uterine

temperature during the period of luteal regression (decreasing progesterone),

follicle growth (increasing estradiol) and ovulation under conditions of

a mild heat stress.












EXPERIMENT 2: THERMAL CHANGES IN THE BOVINE UTERUS FOLLOWING
PGF2a INJECTION THROUGH ESTRUS AND OVULATION


Introduction



The first experiment (Section II) indicated that a thermal stress

increased body temperature, suppressed fertility and caused a slight

decrease in endogenous estradiol secretion. Next, an effect of exogenous

Estradiol-17B on uterine temperature was documented. In this final

experiment, estrus was synchronized by PGF2a and an attempt was made to

evaluate changes in uterine temperature and aortic blood temperature

with plasma estradiol and LH under conditions of mild heat stress. Such

an experiment would closely mimic responses of animals under normal

field conditions and provide additional insight into factors controlling

uterine temperature under conditions of poor reproductive efficiency.


Materials and Methods

Thermocouple preparation and calibration were the same as described

in the previous experiment with the exception that all thermocouples were

made in triplicate for each location. During the experiment, extension

wires led to a recording potentiometer (9835 A, D-C Microvolt Amplifier

and Speedomax G, Model S6000 Recorder, Leeds and Northrup, Philadelphia,

Pa.; limits of error of the recording system + .0125 C). Surgical


- 72 -







- 73 -


techniques were identical except cattle were anesthetized with 3 g

sodium thiamylal (Surital-Park Davis, Detroit, Michigan) dissolved

in saline (3 g/20 ml) and were maintained under surgical anesthesia

with halothane (Fluothane-Ayerst Laboratories, Inc., New York, N. Y.).

Blood samples were collected prior to PGF2a injection (0 hr.), at

6 hr. intervals for 48 hr. and every 4 hr. until 24 hr. after visual

detection of estrus. Measurements of LH and estradiol were by methods

previously cited. Three cycling first lactation dairy cows between 60

to 90 days postpartum and one cycling heifer were given 30 mg PGF2a-

Tham Salt (IM). All animals were between days 9 to 15 of the estrous

cycle at the time of injection. Each animal had a functional corpus

luteum at the time of surgery, 4 to 5 days earlier. At the time of

PGF2a injection each animal maintained a uterine-aortic temperature

difference (AT u) greater than .3 C during the previous 2 days and a
u-a
palpable corpus luteum. A second injection of PGF2a (10 mg) was given

to 3 of the 4 cows at 21 hr. after the first injection. This was done

to insure complete luteal regression. Cow aortic temperatures and AT
u-a
were monitored continually from 5 hr. prior to the initial PGF2a

injection until 24 hr. after the detection of estrus. Twice daily,

recordings were temporarily interrupted for 90 min. (0800 and 2000 hr.)

for estrous checks and exercise. Temperatures in the heifer were re-

corded continually for 15 min. prior to PGF2a injection until 6 hr.

postinjection.

Rectal palpations were made 2 days postinjection to confirm

corpus luteum regression, and again approximately 24 hr. after visual

appraisal of estrous behavior to detect occurrence of ovulation.







- 74 -


Thermocouple placement was verified 4 days after estrus by surgical

examination of the reproductive tract. At ovariectomy confirmation

of luteal regression, ovulation and new corpus luteum formation was

verified by direction.


Results and Discussion


Regression of the corpus luteum, as determined by rectal palpation,

occurred in all four animals. The three cows, at the time of ovariectomy,

had newly formed corpora lutea near the area where the old corpus luteum

had regressed. Two of the cows were detected in estrus while the thermo-

couples remained functional. The usual life span of thermocouples was

about 2 weeks. However, due to mechanical failure the thermocouples in

one cow lasted only 7 days, and the heifer was not detected in estrus

during this period. At the time of ovariectomy and recovery of thermo-

couples, confirmation of ovulation was made on the basis of a newly

formed corpus luteum.

The immediate effects of PGF2a on uterine and aortic temperatures

of two cows and ATu-a of all four animals are shown in figures 13, 14
u-a
and 15. To simplify and assimilate the continuous recordings, points

at 15 min. intervals were taken to describe the data. Following the 30

mg injection of PGF2,, the ATu-a dropped .4 C (P<.01) from approximately

.54 C to .16 C at 45 min. postinjection (figure 15). A similar drop

(P=.10) in ATua occurred following the 10 mg PGF2a injection. However,

the magnitude of the decline was only about .15 C which occurred 30 min.

postinjection. The lower ATa before injection of PGF2. (10 mg) and
u-a 2a







40.0


uterus
uterus


, II

/I

S /
I j


1%
I


a,
1


T
aorta


39.5




39.0





38.5


PGF2
2a


!, ,\,, ,.


',I


35.0 F


25.0 -- -
I I t I i


---I I I I a A


1000

FIGURE 13.


1400


Tair


1800


UTERINE AND AORTIC
IN G665,


2200 0200 0600 1000 1400 1800
HOURS OF DAY
TEMPERATURES PRIOR TO AND FOLLOWING PGF2, INJECTIONS


PGF^

JY


'0t

VI


38.0 "


t s t t I t I I I I



















t r

I I/ i
aort1
aortA


1%
9'
I


\t



PGF2,


38.0 L
35.0


25.0
I I 1 t I I


T
air

1 t I t I


0200
HOURS OF CAY


FIGURE 14, UTERINE AND AORTIC TEMPERATURES PRIOR TO AND FOLLOWING PGF2. INJECTIONS
IN JN15,


40.0 r-


39.5 -


PGF2a


I


39.0 1-


I
,.l,~
3%'


-38.5 -


1000


1400


1800


2200


0600


1000


1400


1800


_~ I I __ ___ __ _____


s 1_4_ 1 1 I














0-
.6 "




( 5 -



.4


I
U3




f-








-2


I


PGF


/
/


I'
I '
I
\
v


o- MG PG5c (M=4)

- --- 10 NG PGF (Nt3)


FIGURE 15. CHANGES IN ATu-a


HOURS
FOLLOWING PGF2 INJECTIONS.







- 78 -


smaller decline may be related both to time and hormonal status after

first injection and also dose of PGF2 These observations were not

anticipated because various researchers (Bergstrom et al., 1968; Brody

and Kodowitz, 1974; Clark et al., (1972) have reported a vasoconstrictor

effect of PGF2 A vasoconstrictor action would tend to decrease blood

flow through the uterus and therefore elevate the ATua. The marked

drop in blood temperature might be attributed to an increased respiratory

evaporative heat loss. Indeed, an increased respiratory rate was de-

tected shortly after PGF2a injection but not quantified. Sweating is

negligible in cattle, so in order for heat to be eliminated by way of

respiratory evaporative heat loss there has to be a tremendous increase

in lung ventilation (Brody, 1945). Also, Lewis and Eyre (1972) report-

ed increased respiratory volume following PGF2a administration to

calves. However, if aortic temperature did fall, a decrease in the

ATua would not occur unless there was selective PGF2a action on the

uterus to increase heat loss or decrease heat production.

If one uses the thermal balance equation, Q=FcAT, then theoretical

heat production, Q, and uterine blood flow, F, can be calculated based

on the ATu-a changes.

Q = rate of uterine heat production (cal/gm tissue-min.)

F = rate of uterine blood flow (gm blood/gm tissue-min.),

density of blood taken as 1 gm/ml

c = specific heat of blood (.87 cal/gm blood-C)

ATa = temperature difference between the uterus and
aortic blood (C); (adapted from Abrams et al., 1970b).
aortic blood (C); (adapted from Abrams et al., 1971b).







- 79 -


The major assumption is that all heat loss from uterine tissue

is by way of the uterine veins. Therefore, in order to calculate Q,

uterine blood flow during the luteal phase of the estrous cycle needs

to be obtained. Assuming no species differences, then.we can use for

cattle the value of 119 ml blood/kg-min. for uterine blood flow in

sheep (Huckabee et al., 1968).

Theoretically, at ATua = .55 C just prior to PGF2a injection

in the present experiment:

Q = FcAT

Q = .119 gm blood/gm tissue-min. X .87 cal/gm blood-C X .55 C =

.057 cal/gm-min.

By contrast, the uterine heat production rate, Q, calculated 45 min.

post-PGF2a injection when ATu-a = .16 C was determined to be:

Q = .119 X .87 X .16 =

.017 cal/gm-min.

This theoretical calculation would suggest a 3.4 fold decrease in

uterine heat production in response to PGF2a.

Lowered ATua in response to PGF2a could also be explained by an

increase in heat loss. One mechanism of heat loss would be an increase

in uterine blood flow. Be rearranging the equation, the theoretical

blood flow, before and after PGF2a, can be calculated based on oxygen

consumption data for a 350 kg Jersey cow (oxygen consumption 3.26 ml/

kg-min.; Brody, 1945). Assuming no differences between oxygen con-

sumption (per kg) of various organs of the body (in sheep the oxygen

consumption of the total body as well as the uterus is approximately

5 ml/kg-min.), oxygen consumption of uterine tissue of 3.26 ml/kg-min.







- 80 -


multiplied by a calorific value of 4.8 cal/ml oxygen (based on an assumed

R.Q. of .8; Brody, 1945) would give a calculated heat production of

3.26 ml/kg-min. X 4.8 cal/ml = .0156 cal/gm-min.

Rearranging the original Equation:

F Q
cAT

then at ATua = .55 C (pre-PGF2a injection):

F .0156 cal/gm-min.
.87 cal/gm blood-C X .55 C

.0326 gm/gm-min.

or

32.6 ml blood/kg-min.

and at ATua = .16 C (post-PGF2a injection):

F .0156
.87 X .16

.122 gm/gm-min.

or

112 ml blood/kg-min.

The 3.4 fold increase in theoretical uterine blood flow calculated

above would be comparable to changes reported by various researchers

(Huckabee et al., 1970; Abrams et al., 1970a) following estrogen in-

jections. However, the time course of maximum PGF2a response (45.min.)

was of shorter duration and had a more rapid onset than an estrogen in-

duced decrease in ATa (Section III, Experiment 1). These observations
u-a
indicate the need for more definitive experiments in the bovine to pin-

point the cause of the lowered ATu-a in response to PGF2 A basic

question is whether there is an increase in uterine blood flow or a







- 81 -


decrease in heat production.

Figures 13, 14, 16 and 17 show individual cow aortic and uterine

temperature changes pre- and post PGF2a injection (figures 13 and 14)

and at the time of the LH peak (figures 16 and 17). Both cows appeared

to exhibit circadian changes in aortic and uterine temperatures. The

range of aortic temperatures (37.9 to 41.0 C) within the two cows is

comparable to body temperatures reported by Bligh and Harthoorn (1965)

in African cattle. They reported that maximum and minimum body tempera-

tures were closely associated with sunset and sunrise, respectively.

In the latter study thermistors were implanted 8 cm into the dorsal

caudal neck region to record deep body temperature. Aortic temperature

patterns in our study showed that maximum daily deep body temperature

occurred close to midnight, whereas minimal body temperature occurred

between 0800 and 1200 hr. Although cows were turned out to exercise

at 0800 and 2000 hr., their aortic temperatures returned to pre-

turnout baselines within 2 hr. after their return to the barn. Also,

there appears to be a 4 to 6 hr. lag behind barn air temperature in

maximum and minimum body temperature.

The uterine and aortic temperatures were highly correlated

(Appendix, table 13) but no correlation between AT and either uterine
u-a
or aortic temperature was detected. This might suggest that there was

no change in uterine blood flow and uterine heat production. However,

these correlations were based on data throughout the entire experiment

and any possible increases in ATua at higher body temperatures (figures

16 and 17) may have been undetected statistically. Visual appraisal of

figures 16 and 17 do show a widening of the AT at maximum daily
u-a







- 82 -


40.5


40.0


39.5


39.0


38.5


38.0


35.0 T


25.0 -


Estrus


F2c
2a
*^LY


PGF
2a
v


i rd t,'dU I


0


L__^---^---^---_^4
2400 1200 2400 1200 2400 120
HOURS OF DAY


0 2400 1200


FIGURE 16. UTERINE AND AORTIC TEMPERATURES, LH AND
ESTRADIOL IN G665 AND AIR TEMPERATURES,


I --* \ %
.4 '
.4"
V


Tuterus


'V
p.
4.


\ I
t /
I
1


* PC

E
) 40


-o
30
n3
20,
' 20


1n


o-.. Js-


1I


* s







- 83 -


40.5

T
40.0 uterus
/ 1I- / /




I -" aorta \
l \ r I





38.5 -1

38.0 -
35.0

25.0


PGF2


E
2 40-


" 30
4--
u" 20
lj


IGF2a


Estradi
0


o


0 a


2400
2400


1200 2400 1200 2400 1200
HOURS OF DAY


FIGURE 17.


UTERINE AND AORTIC TEMPERATURES, LH AND ESTRADIOL
IN JN15 AND AIR TEMPERATURES,


Estrus


/
Q -~







- 84 -


uterine and aortic temperatures.

Figure 18 shows the significant (P<.01) curvilinear (2nd order)

time trends for uterine and aortic temperatures when data were pooled

across days for each time of blood sampling. The data representing

each individual sampling point is the average of individual 15 min.

points + 2 or 3 hr. from time of the blood sample. Also only tempera-

tures preceding turn out of cows (0800 and 2000 hr.) were used in

obtaining an average for the 0800 and 2000 hr. blood sampling times.

The air temperature plot is comprised of average values across the

individual days. Uterine temperature and aortic temperature were not

influenced by barn air temperature (P>.10; Appendix, table 13). The

uterine temperature trend throughout the day was best described by
2
Y(uterine temperature, C) = 40.04 .143X + .007X (P<.01) where X:= hr.,
whereas aortic temperature was best characterized by Y(aortic tempera-

ture, C) = 39.50 .125X + .006X2 (P<.O0; Appendix, table 14). The
body temperature lag of about 6 hr. behind air temperature (1600 hr.-

peak air temperature compared to 2400 hr. body temperature peak) is

best seen in figure 18.

Possible explanations for the time delay could be:

1) Thermal inertia of the cow. For example, ambient temperature

will increase more rapidly than body temperature because of the mass and

heat capacity of the large mammal.
2) Inherent circadian rhythm of body temperature which may be

independent of environmental temperature and cued to external events

such as light-dark cycles, feeding regimen, presence of barn personnel

and other factors which were uncontrolled in this experiment.









40.5 -
C
40.0

T R2 = .35
39.5 Tuterus, R2 35

39.0 ------

Taortic R2 = .45

33.0


31.0 Tair


29.0


27.0

0400 0800 1200 1600 2000 '2400
HOURS OF DAY
FIGURE 18. CIRCADIAN UTERINE, AORTIC AND AIR TEMPERATURE CHANGES.







- 86 -


Correlated responses between concurrent temperature measurements

(uterine temperature, aortic temperature and air temperature) are low

due to the time delay phenomena.

Of interest in this experiment is the observation that uterine

temperatures during the day reached 40 C for periods of up to 6 hr. as

ambient temperature fluctuated near 30 C (figures 16 and 17). Tempera-

tures of this magnitude (40 C) are damaging to embryo development at

the 1 to 4 cell stages's (Alliston et al., 1965). In JN15 (figure 17)

this increased uterine temperature was at the time when artificial

insemination would normally have been performed.

We failed to detect an association between concurrent measurements

of AT with estradiol or LH (Table 13). Based upon Experiment 1 there
u-a
was a 2.5 hr. delay between injection of a pharmacological dose of

estradiol and the minimum ATu-a From PGF2a injection through the

LH surge (figure 19) estradiol concentrations fluctuated considerably

as did the AT (figures 16 and 17). Not until the massive LH dis-
u-a
charge was there an appreciable rise in ATa at a time when estradiol
u-a
was decreased (figure 19).

Findings of this experiment indicate that uterine and aortic

temperatures followed a daily circadian rhythm, and,because of a time

lag in these temperatures behind air temperatures, correlations between

body temperatures and ambient temperature were negligible. Failure to

detect an association between AT and hormonal measurements may be
u-a
due to the time lag, also, The mild heat stress (which by definition

occurs in cattle anytime ambient temperatures exceed 30 C), to which

these cows were subjected to, may have contributed to the high uterine

and aortic blood temperatures. Uterine temperatures periodically












5

I-
< R2 = .76

.4


-30


25 o



% I
I i ler















HOURS
SI I H AD EST L
Fo 1% I

4 )S % -





-84 -72 -60 -48 -36 -24 -12 0 +12 +24
HOURS
FIGURE 19. CHANGES IN ATu-a ASSOCIATED WITH ENDOGENOUS LH AND ESTRADIOL CONCENTRATIONS.







88 -


exceeded 40 C (Appendix, table 15). Since survival rate of fertilized

ova exposed to 40 C for 3 hr. is seriously decreased, these observations

may be of some practical significance in improving reproductive per-

formance in a hot climate.












SECTION IV

SUMMARY AND CONCLUSIONS


Ten normally cycling Holstein heifers at the USDA, Agricultural

Research Center, Beltsville, Maryland, were assigned to one of two en-

vironmental treatment groups (21.3 C, 59% RH or 32.0 C, 67% RH).

PGF2a-Tham Salt (PGF2c) was used to cause corpus luteum regression and

synchronize estrus. Blood samples were collected prior to PGF2a

injection and at 6 or 4 hr. intervals following injection through

ovulation. Plasma samples were analysed to determine concentrations of

progestins, estradiol, estrone, LH, prolactin, corticoids, total protein

concentration, osmolality, cortisol binding capacity and cortisol

association constants. In the second phase of this first experiment

adrenal responsiveness to ACTH (200 IU) was tested by quantification'of

corticoid concentrations in plasma prior to and up to 12 hr. following

injection of ACTH. Least-squares analyses were conducted to characterize

treatment, animal and within-animal time trends in plasma progestins, estra-

diol, estrone, LH, prolactin and corticoids. Other response variables were

analyzed by analysis of variance.

Environmental treatment of 32.0 C evoked a 1.49 C increase in

rectal temperature and a 3.59 C increase in skin temperatures. Time

durations between PGF2x injection to LH peak and ovulation were not

different (P>.10) between treatments. Length of estrus was shorter


- 89 -




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs