EFFECT OF LEVEL OF NITROGEN ON
GROWTH AND REPRODUCTIVE
PHYSIOLOGY OF YOUNG
BULLS AND RAMS
THOMAS N. MEACHAM
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
The author wishes to express his sincere appreciation to
Dr. T. J. Cunha, chairman of his supervisory committee, and
Drs. A. C. Warnick and J. F. Hentges, Jr., for the many hours spent
in advising and assisting in the conduction of these experiments and
the preparation of this dissertation. He is most grateful to Dr.
R. L. Shirley and Dr. T. W. Stearns for their help and advice with his
To his fellow graduate students, D. D. Hargrove, D. L. Huffman,
R. E. Deese and J. H. Meade, Jr., go the author's appreciation for
their many hours of help in the collection of these data.
The patience and understanding of his wife, Carol, are sincerely
TABLE OF CONTENTS
ACKNOWLEDGMENTS ... . . . . . . . . . . ii
LIST OF TABLES . . . . . . . . . . . . iv
LIST OF FIGURES ......................... vii
INTRODUCTION . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . ...... 5
EXPERIMENT I . . . . . . . . . . . 15
Experimental Procedures . . . . . . . .. 15
Results and Discussion . . . . . . . . 25
EXPERIMENT II . . . . . .... . . . . . ... 55
Experimental Procedures . . . . . . . . 55
Results and Discussion . . . . . . . . 61
GENERAL DISCUSSION AND CONCLUSIONS . . . . . . . 85
SUMMARY . . . . . . . . . . . . . . 89
LITERATURE CITED . . . . . . . 92
APPENDIX .............. ..... ...... 98
LIST OF TABLES
1 Composition of Experimental Rations--Experiment I . .. 18
2 Vitamins Added to Ration Per Ton of Feed . . . 19
5 Mineral Composition of the Trace Mineralized Salt Fed
During the Experiment . . . . . . . . 19
4 Mean Interval to First Mount, Interval to First Mate
and Libido Score by 28-Day Periods . . . ... 57
5 Summary of the Semen Exhaustion Study for Individual
Bulls and Treatment Groups by Days and Total
Production .. . . . . . . . . . . 46
6 Quantitative Evaluation of Testes and Epididymis from
the Two Groups of Bulls . . . . . . . . 48
7 Quantitative Evaluation of the Seminal Vesicles,
Prostate and Cowper's Glands from the Two Groups
of Bulls--Experiment I . . . . . . . .. 49
8 Summary of Mean Gland and Organ Weights Taken at
Time of Slaughter. .. . . . . . . . 50
9 Composition of the Purified Rations Fed the Rams in
Experiment II . . . . . . . . . . 58
10 Summary of Libido Data for the Three Treatment Groups
of Rams by 14-Day Periods .. . . . .. 75
11 Quantitative Evaluation of the Testes and Epididymis
from the Three Groups of Rams . . . . ... 80
12 Quantitative Evaluation of the Seminal Vesicles and
Cooper's Glands from the Three Groups of Rams . . 81
15 Summary of the Mean Gland and Organ Weights Taken at
Time of Slaughter from the Three Groups of Rams . . 85
14 Biological Assay of Adenophypophyses from the Three
Treatment Groups of Rams . .. . . . . 84
LIST OF TABLES--Continued
15 Summary of Total Feed, Crude Protein Per Cwt. and TDN
Per Cwt. Consumption Per Day for the Two Treatment
Groups by Periods--Bulls . . . . .... ... 99
16 Summary of the Initial and 28-Day Period Weights of
Individual Bulls and Treatment Groups (Pounds) . .. 100
17 Summary of Blood Hemoglobin, Hematocrit and Serum
Protein Values for Individual Bulls and Treatment
Groups by 28-Day Periods . . . . . . .. 101
18 Summary of the Semen Volume, Motility and the Total
Sperm Cell Production for Individual Bulls and
Treatment Groups by Periods . . . . . . .. 103
19 Summary of Individual Ram and Treatment Group Mean
Feed Consumption by 14-Day Periods . . . . .. 105
20 Summary of Individual Ram and Treatment Group Mean
Body Weights by 14-Day Periods . . . . .... 106
21 Summary of Individual Ram and Treatment Group Mean
Blood Hematocrit, Hemoglobin and Serum Protein
Values by 28-Day Periods . . . . . . .. 107
22 Summary of Individual Ram and Treatment Group Mean
Semen Volume, Motility and Total Sperm Production
Values for 14-Day Periods . . . . . .... 109
25 Analysis of Variance Mean Squares for Semen Volume,
Motility and Total Sperm Cell Production in
Experiment I, Phase I . . . . . . .... 11
24 Analysis of Variance Mean Squares for Blood Hemoglobin,
Hematocrit, Serum Protein, Libido Scores, Intervals
to First Mount and Interval to First Mate--
Experiment I, Phase I . . . . . . .... ll
25 Analysis of Variance Mean Squares for Semen Volume,
Motility and Total Sperm Cell Production--
Experiment I, Phase II . . . . . . .... 112
26 Analysis of Variance Mean Squares for Blood Components
and Libido Evaluations--Experiment I, Phase II . .. 112
LIST OF TABLES--Continued
27 Analysis of Variance Mean Squares for Blood Hemoglobin,
Hematocrit and Serum Protein--Experiment II,
Phases I and II . . . . . . . .... ... 113
28 Analysis of Variance Mean Squares for Semen and
Libido Criteria--Experiment II, Phase I . . . .. 114
29 Analysis of Variance Mean Squares for Semen and
Libido Criteria--Experiment II, Phase II . . .. 114
LIST OF FIGURES
1 Effect of Protein Deficiency on Daily Total Feed
Intake of Bulls . . . . . . . ... .. 24
2 Effect of Protein Deficiency on Crude Protein and
TDN Intake Per Cwt. Daily of Bulls .......... 25
3 Cumulative Average Daily Gains of Bulls . . . ... 27
4 Effect of a Low Protein Ration on Bull Growth ..... 30
5 Effect of Protein Deficiency on Average Hemoglobin
Levels of Bulls .................... 32
6 Effect of Protein Deficiency on Average Hematocrits
of Bulls . . . .. . . . . . . 534
7 Effect of Protein Deficiency on Average Serum Protein
Level of Bulls . . . . . . . . ... 36
8 Effect of Low Protein Intake on Mean Semen Volume
of Bulls ... . . .. . .. .. .. . .. 39
9 Effect of Low Protein Intake on Mean Sperm Motility
of Bulls ...................... .. . 41
10 Effect of Low Protein Intake on Total Sperm Production
of Bulls . .. .. .. ... . .... .. . . .. 43
11 Effect of a Protein Deficiency on the Testes and
Seminal Vesicles of Bulls . . . . . . .. 52
12 Effect of a Protein Deficiency on the Cowper' s and
Prostate Glands of Bulls . . . . . . .. 54
13 Effect of Source and Level of Nitrogen on Feed Intake
of Rams . . . . . . .... . .. .. . 62
14 Effect of Source and Level of Nitrogen on Cumultaive
Average Daily Gain of Rams . . . . . .... .64
15 Effect of Source and Level of Nitrogen on Ram Growth . 67
LIST OF FIGURES--Continued
16 Effect of Source and Level of Nitrogen
Hemoglobin Values for Rams . .
17 Effect of Source and Level of Nitrogen
Hematocrit Levels of Rams .. ...
18 Effect of Source and Level of Nitrogen
Serum Protein Values of Rams . .
19 Effect of Level and Source of Nitrogen
Semen Volume of Rams . . . .
20 Effect of Level and Source of Nitrogen
Semen Motility of Rams . . .
21 Effect of Source and Level of Nitrogen
Cell Production of Rams . . .
. . .
Maximum production and profit in any livestock breeding operation
depends to a large extent on reproductive efficiency. The role of the
female in achieving this efficiency has been the object of consider-
able study in the past. The part of the male, while of equal importance,
has received little or no attention in efforts to improve reproductive
efficiency in breeding programs.
Protein, one of the most essential nutrients for growth and
development, is frequently a limiting factor in the feeding value of
pasture and roughage. Since pasture and roughage usually make up a
major portion of the ration fed to both mature and growing bulls and
rams, these animals may be subjected to a marginal or deficient protein
intake. This situation is particularly acute during periods of re-
stricted plant growth when the protein content of forages may be reduced
Several studies have been conducted recently to ascertain the
effect of reduced protein intake on the growth and reproductive per-
formance of heifers and cows. These studies indicate that when the
protein intake is reduced to 50 percent of the recommended level,
retarded growth and impairment of estrus and ovulation result. A recent
study by the author (Meacham, 1960) has revealed that the reproductive
performance of the bull, while more resistant than that of the heifer
or cow, is adversely affected by a low protein intake. With these
facts in mind, the experiments reported herein were conducted to
provide additional information concerning the effect of source and
level of nitrogen and protein on the growth, blood composition, and
reproductive performance of young bulls and rams.
The literature contains little information concerning the
effect of a protein deficiency on bulls or rams. In recent years,
several workers have studied the effect of reduced feed and energy
intake on the growth and reproduction of dairy bulls and boars, but
little work has been done on a protein deficiency in the male of any
of the farm animals.
In an extensive review of the relationship of nutrition to
fertility Reid (1949) reports that reduced protein intake results in
decreased appetite, inanition and loss of weight in laboratory animals
as well as large animals. The accompanying depressing effect on the
pituitary gland adversely affects the secretion of gonadotrophic
hormones which regulate the reproductive processes. Supplementation
with protein has improved the reproduction rate in beef cows (Guilbert
and Hart, 1951) and increased the testicular development and activity
in the rat (Horn, 1955). In view of these findings, a definite need
for protein for normal reproduction does exist.
Dietary Protein and Growth
A number of workers have investigated the problem of how much pro-
tein is required for normal growth of beef calves. Winchester et al. (1957)
stated that 0.59 pounds of digestible protein (IP) daily would maintain
calves for six months with little or no gain in weight. Blaxter and
Mitchell (1948) estimated that 1.1 pounds of IP were needed daily for
dairy calves for adequate growth. Armsby (1917), the National Research
Council (NRC) committee for beef cattle in 1958, Morrison (1956), and
Guilbert and Loosli (1951) reported EP values for young calves falling
in the range of 0.50 to 0.82 pounds of EP per day for normal growth.
Darlow et al. (1946), the NRC committee for beef cattle (1958),
Lofgreen et al. (1951) and Morrison (1956) gave DP requirements for
older calves of 0.8 to 0.9 pounds daily. Both the NRC (1958) and
Morrison (1956) recommended 1.2 pounds of DP per day for young bulls.
This increased protein requirement for bulls over that for steers and
heifers is in agreement with the work of Speer et al. (1957) who
indicated that young boars may have a higher protein requirement for
optimum growth than do barrows or gilts.
The protein requirements for lambs are quite varied. The NRC
committee for sheep (1957) recommends 0.17 to 0.18 pounds IP for grow-
ing rams and 0.17 to 0.20 pounds per day for fattening lambs. Morrison
(1956) gives a range of 0.19 to 0.26 pounds of DP per day for growing
rams and a similar range for fattening lambs. Gallup et al. (1952),
Ross et al. (1954) and Bush et al. (1955) reported the optimum EP
level to be 0.20 to 0.22 pounds per day for fattening lambs. Very
few research data are available related to the protein requirements
of young rams.
Oltjen et al. (1959, 1960 and 1961), Meacham et al. (1961),
Repp et al. (1955) and Gallup et al. (1952) have all reported on the
use of urea in replacing a part or all of the protein in rations for
growing lambs and rams. Slen and Whiting (1955) studied the effect of
supplemental urea in rations for gestating and lactating ewes. In all
cases, urea was found to be a satisfactory source of nitrogen although
it was usually not as effective as the conventional protein sources.
As the percentage of nitrogen supplied by urea increased, the perform-
ance of the animals tended to decrease. Repp et al. (1955) stated
that when 15 to 30 percent of ration nitrogen was supplied as urea
lamb growth was essentially the same as that when all protein-nitrogen
was used. When urea was used as the sole source of nitrogen, Oltjen
et al. (1959) and Meacham et al. (1961) found that weight gains were
lower than those of lambs and rams fed protein sources of nitrogen.
Dietary Protein and Blood Composition
Hemoglobin. The normal hemoglobin values for beef cattle
reported by various workers are quite varied. Long et al. (1952) gave
a range of 8.0 to 15.0 grams (gm.) of hemoglobin per 100 milliliters
(ml.) of blood. Dukes (1955), Byers et al. (1952), Blucker (1952),
Flipse (1957) and Davis et al. (1958) reported normal hemoglobin values
for cattle that fell in the range given by Long et al. (1952). Kehar
and Murty (1945) reported a marked decrease in hemoglobin levels in
Zebu cattle fed protein deficient rations for short periods of time.
The protein deficiency reduced the hemoglobin levels from 11.0 to 8.9
gm. per 100 ml. of blood. A low protein intake lowered the hemoglobin
levels in young bulls from 11.9 to 8.01 gm. per 100 ml. of blood
(Meacham, 1960). Bedrak (1958) and Cruz et al. (1961) have reported
similar decreases in hemoglobin levels in beef heifers and cows fed
protein deficient rations.
Dukes (1955) gives the normal hemoglobin level of sheep as
12.4 gm. per 100 ml. of blood. Weir et al. (1948) reported an average
hemoglobin level of 10.16 gm. per 100 ml. of blood for young lambs.
Klosterman et al. (1950) stated that mature ewes had normal hemoglobin
of 11 gn. per 100 ml. of blood. Klosterman et al. (1948), Weir et al.
(1948) and Klosterman et al. (1950) all reported that low protein
intake in both ewes and lambs will result in a lowered hemoglobin level.
Hematocrit. A fairly wide range of values for normal hemato-
crits is found in the literature for cattle. Maynard.and Loosli (1956)
give a range of 30 to 45 percent of the blood as packed cells or
hematocrit. Blucker (1952) reported 31 to 38 percent as normal for
Hereford cattle. Dukes (1955) states that a normal hematocrit for
cattle as a whole is around 40 percent. Davis et al. (1958) reported
that Florida cattle have a hematocrit of 46 percent. Blincoe and
Brody (1951) give a range of 36 to 46 percent as normal for several
Kehar and Murty (1945) and Meacham (1960) found that low protein
rations reduced the hematocrit of beef cattle from about 45 percent to
40 and 27 percent for Zebu and Hereford and Angus cattle respectively.
Low protein rations similarly reduced the hematocrit of beef heifers
as reported by Bedrak (1958) and Cruz et al. (1961).
The average hematocrit for sheep is considerably lower than
that for cattle. Dukes (1955) gives a figure of 32 percent as normal
Serum Protein. The normal serum protein level in the blood
of cattle is 7.60 percent according to Dukes (1955). Gardner (1950)
gives a range of 8.0 to 11.0 percent serum protein in cattle. Crook-
shank and Sims (1955) give a lower range of 6.44 to 9.10 percent for
serum protein levels in cattle.
Kehar and Murty (1945) in their work with Zebu.cattle state
that adequately fed cattle have a serum protein level of 8.60 percent
compared to 7.32 percent in protein deficient animals. Beef bulls
fed low protein rations had serum protein levels of 6.07 percent as
compared to 7.63 percent serum protein in control bulls (Meacham,1960).
Other workers have reported similar decreases in serum protein per-
centages in beef heifers fed low protein rations (Bedrak 1958, and
Cruz et al.,1961).
Klosterman et al. (1950) reported a range of 6.9 to 7.3 per-
cent serum protein in the blood of adequately fed pregnant ewes. Dukes
(1955) gives a normal serum protein value of 5.58 percent for sheep.
Klosterman et al. (1948) report that low protein intake in ewes will
lower the serum protein in both the blood of ewes and the blood of
Dietary Protein and Reproduction
With the exception of a few amino acid and protein studies
with the rat and the human, the literature is practically devoid of
information concerning the effect of a protein deficiency on reproduc-
tion in the male. A preliminary study with bulls by the author (Meacham,
1960) provides the only information available concerning a protein
deficiency and reproduction, in males, with large animals. This work
indicated that such a deficiency had adverse effects on growth and
reproduction as shown by semen quality and libido.
Keller et al. (1946) reported that young male and female rats
fed a tryptophan deficient diet for as little as three days manifested
sterility in both sexes. Later work by Berg and Rohse (1947) however,
failed to confirm the work of Keller et al. The former workers were
unable to produce sterility in the young rats on a similar deficient
diet. Shettles (1960) in a recent report indicated that tryptophan,
as well as lysine and arginine, was essential for spermatogenesis
in man. The argininedeficient diet resulted in decreased sperm con-
centration and lowered motility in human semen. Leathem (1956)
reported that mature rats fed a protein free diet exhibited normal
spermatogenesis for 30 days. After 90 days, testes weights had
decreased 50 percent and complete atrophy had occurred in some cases.
Spermatogenesis was sharply reduced or stopped entirely. Working with
immature male rats, Horn (1955) found that a protein free diet in-
hibited testes maturation and spermatogenesis. Replacing the protein
in the diet brought about increased size and activity of the testes
with a corresponding increase in sperm production.
An early indication of a protein deficiency is the loss of
appetite and subsequent decrease in feed intake. With this in mind,
the work done on restricted energy and feed intake might well be
pertinent to the problem of protein deficiencies and reproduction.
Numerous workers have studied the effect of restricted feed
intake or energy intake on the reproductive performance of laboratory
and large animals. Friedman and Turner (1959) stated that an inhibi-
tion of spermatogenesis occurred when male rats were restricted to
feed intakes 25 to 50 percent below that of self-fed controls. When
feed intake was reduced to the point where body growth was halted,
testes atrophied and spermatogenesis was ultimately halted. Studies
with the male reviewed by Reid (1949) revealed a similar deficiency
situation when the energy intake was reduced 15 to 30 percent of the
self-fed controls. Talbert and Hamilton (1955), however, reported
that initiation of sperm production was not influenced by a reduction
of growth rate to one-third that of normal young male rats. In con-
trast to these data, Teitelbaum and Gantt (1956) reported an increase
in sperm production in dogs subjected to short periods of starvation.
When the dogs were returned to a normal ration, the level of sperm
output dropped back to normal, pre-starvation levels. Chang and
Sheaffer (1956) took exception to these data and reported that the
sperm they were measuring had been produced before the starvation
period was started and thus had not been affected by the treatment.
It appears from these studies that the initiation and maintenance of
spermatogenesis is impaired by a reduction in either feed or energy
intake severe enough to stop body growth. Apparently, as long as the
animal is able to grow, attainment of puberty and spermatogenesis are
not materially affected.
Studies with large animals have indicated fairly similar
effects from reduced energy and total feed intake on reproduction.
Jones et al. (1942, 1945) report that young dairy bulls restricted
to a roughage ration after the first seven months of age reached
puberty later and grew at a slower rate than bull calves supplemented
with concentrates and vitamins. There was no difference in the fer-
tility of the two groups of bulls at the normal breeding age of 24
to 40 months. The roughage-fed bulls did, however, reach puberty at
a later date than the supplemented bulls. These workers concluded
that any ration which permitted normal growth to 24 months of age
would be adequate for normal reproductive development. This is in
agreement with statements made by Walton (1949) and Rice and Andrews
(1951) that rations sufficient for normal body growth were also ade-
quate for reproduction. Reid (1949) reported essentially the same
results in a study where young dairy bulls were grown out on a reduced
Baker et al. (1953, 1955a, 1955b) and Flipse (1957) report
that the first ejaculate with viable sperm was obtained at 9 to 10
months of age with dairy bulls. Flipse et al. (1955) stated that young
dairy bulls restricted to 70 percent of the energy recommended by the
(NRC) committee for dairy cattle were delayed 12 to 14 weeks in reach-
ing puberty. They found that feeding 130 percent of the energy
recommended did not hasten the onset of puberty materially. Semen
concentration and motility were also adversely affected by the restric-
tion of the energy intake to 70 percent.
[Bratton (1953, 1957) and Bratton et al. (1959) have published
an extensive study conducted at Cornell University in which three
levels of total digestible nutrients(TDN) were fed to young dairy bulls.
The bulls were started at one week of age and the study was continued
over an 80-week period. These workers report that feeding 60 percent
of the recommended TDN allowance for dairy heifers delayed the onset
of spermatogenesis eight weeks beyond that of the 100 percent TDN
group. Feeding 160 percent of the allowance hastened the onset by
six weeks. It was found in the study that essentially the same total
amount of TDN was required to raise a bull to sperm-producing age
regardless of the feeding level. Those bulls on the high level merely
reached puberty at an earlier age. The non-return rates of females
bred by artificial insemination from the three groups of bulls were
identical, all 74 percent. This would indicate that the fertility of
the bulls was not influenced by the level of TDN feeding. Histo-
logical investigations were made of the tissue from the testes of
these bulls slaughtered at various intervals during the study. This
work indicated that the delay in sperm production in the low TDN level
bulls was due to a retarded differentiation of the seminiferous
tubules and interstitial tissue in the testes.
Davies et al. (1957) conducted a total feed intake deficiency
experiment with identical twin beef calves. These workers studied
the androgenic activity and the appearance of spermatozoa in the semen
of deficient and control calves. One twin from each pair was assigned
to the deficient group and the other to a normally-fed control group.
The appearance of fructose and citric acid in the semen was considered
an indication of the onset of testosterone production. The normally-
fed bulls began secretion of fructose and citric acid at five months
of age while the deficient bulls were delayed an average of four months
beyond this time with testosterone production not starting until the
bulls were nine months old. The low feed intake delayed the start of
spermatogenesis, but to a lesser extent. Sperm were observed in the
ejaculate obtained by electrical stimulation at 9.5 and 10.5 months
of age for the control and the deficient bulls respectively. Histo-
logical studies indicated that the differentiation of the seminiferous
tubules and interstitial tissue was delayed in the testes of the
deficient bulls. This accounted for both the retarded hormone secre-
tion and the delayedspermatogenesis. Shirley et al. (1960) reported
a marked decrease in fructose and citric acid concentrations in semen
obtained from protein deficient bulls. There was a corresponding
decline in spermaogenesis and libido in these same bulls (Meacham,
Mann and Walton (1953) reduced the feed intake of one mature
Dexter bull to 50 percent of the required amount and later to a near
starvation level for 25 weeks. This drastic reduction in feed intake
was without effect on sperm concentration, motility or sperm morphology.
The volume of semen produced was, however, reduced somewhat.
Hulet et al. (1956) studied the effect of roughage versus
roughage and concentrates feeding on the fertility of rams. They
found no significant differences in the fertilizing capacity of the
semen from the two groups of rams. Matousek et al. (1959) investigated
the effect of high and low energy rations on various semen character-
istics in mature rams. Rams fed a bulky, low energy ration produced
semen equal to that of rams fed concentrates with respect to volume,
motility, sperm concentration, fructose content and pH.
Theorizing from the data available in the literature, it seems
that once sexual maturity is attained, spermatogenesis and fertility
are not easily affected by restricting the energy or total feed intake
of large animals. Work by Dutt and Barnhart (1959) with young boars
indicates that perhaps a different situation exists with the boar.
These workers were unable to affect the concentration, motility or
morphology of the semen from young boars fed as little as 50 percent
of the NRC requirements for TDN. The volume of semen produced by the
boars on the 50 percent level was significantly smaller than those
on the 70 to 100 percent levels. Puberty, libido and fertilizing
capacity of the semen were not influenced by the low levels of TDN
intake. Steverman et al. (1961) studied the effect of reduced feed
intake in mature boars. Three levels of feed intake were used;
a level supplying 120 to 150 percent of the NRC requirements, a level
just meeting these requirements and a level providing only 50 to 75
percent of the required intake. The results of this study were quite
similar to those reported above. The volume of semen produced by the
boars on the low level was reduced somewhat, but there was no differ-
ence in total sperm production, motility or fertility between any of
the three groups. Some decline in sperm concentration was noted after
the boars on the low intake level had reached a point of lethargy.
Conflicting reports appear in the literature concerning the
role played by quality of protein in reproduction. Reid (1949)
reviewed work by Russian workers who reported beneficial responses in
semen quality from dairy bulls fed animal protein sources. These
workers fed bulls, in an Artificial Insemination Center, blood meal
and fish meal and found an increase in sperm concentration and motility.
Branton et al. (1948) and Flipse et al. (1956) attempted to duplicate
this favorable response from animal protein sources. Flipse and co-
workers fed both liquid and dried skim milk and soybean and linseed
meal to young dairy bulls. They reported that the source of protein
had no effect on growth, age at puberty, motility or morphology of
the sperm cells. There was an increase in sperm concentration with
the liquid skim milk, but the milk reduced the total volume of semen
produced. Branton and his co-workers fed corn gluten meal, dried
skim milk, and soybean meal to young dairy bulls. They were unable
to show any advantage attributable to the animal protein sources in
growth rate, age at puberty or semen quality. Apparently, it is
questionable whether or not animal protein feeds offer an advantage
in well-balanced rations for young breeding bulls. Flipse and
Almquist (1954) investigated the effect which dehydrated young grass
had on the reproductive efficiency of dairy bulls. They reported
no differences in the non-return rates of females for the treated and
control bulls. Hultnas (1959), in his work with dairy bulls in
Sweden, stated that part of the variation in libido, total sperm per
ejaculate and percent abnormals could be due to changes in forage
quality. He associated the decline in reproductive performance with
seasons of the year when the protein content of the forage was low.
In this experiment 12 weaned Angus bull calves were used.
The average weight and age at the start of the experiment was 590
pounds and approximately 315 days of age. The bulls were purchased
from the M&M ranch at Loxahatchee, Florida. The bulls were brought
to the Purebred Beef Unit shortly after weaning and fed on pasture for
approximately two and a half months before the experiment was initiated.
Assignment to the treatment groups was accomplished in the
following manner. The bulls were exposed to an "estrogenized" heifer
of suitable size. As the bulls attained puberty, indicated by suc-
cessfully mating the heifer, they were assigned to a control and a
protein deficient group. Every third bull to mate the heifer was
placed in the control group and the other two bulls were placed in
the low protein group. This procedure was followed in an effort to
obtain bulls of the same physiological age in both treatment groups.
The average weights of the four control bulls and the eight low
protein bulls were 420 and 575 pounds, respectively, when the exper-
iment was started on October 8, 1960.
The experiment was conducted at the South Experimental Barn
at the Purebred Beef Unit. The barn contained individual feeding pens
equipped with feed boxes and waterers. The bulls had access to two
small lots on each side of the barn at night. Water was available
to the animals in these lots.
Scales, working pens and chutes were available at the main
barn a short distance away. A stanchion was constructed in one of
the small lots by the barn to secure a heifer for libido checking.
Feedstuffs and Rations
The composition of the experimental rations, their crude pro-
tein analysis and estimated total digestible nutrients (TDN) content
are given in table 1. Ground corn cobs and shuck meal was used to
supply roughage in the rations. Molasses was included in the rations
to improve their palatability and insure a uniform blending of the
fine textured ingredients with the coarser materials in the rations.
The concentrate portion of the rations was mixed and bagged
at the beginning of the trial for the entire experimental period.
This was done to obtain rations as uniform as possible throughout the
study. The molasses and corn cob and shuck meal were mixed with the
concentrates periodically as needed during the trial. Vitamins A, D
and E were mixed in the concentrate portion along with the trace
mineralized salt and bone meal. The vitamin and mineral mixtures are
given in tables 2 and 5, respectively.
The average crude protein content of the control and low pro-
tein rations were 13.75 and 1.61 percent, respectively. Protein
analyses were made on each lot of feed mixed during the experiment.
Estimated TDN content was 67.41 and 73.45 percent for the control and
low protein rations, respectively. A higher energy level in the low
protein ration was used to counteract in part the reduction in total
feed intake which occurs on a low protein ration.
Feeding and Management. The bulls were fed once daily in the
morning in individual feeding stalls. The bulls received 2 percent
of their body weight of the mixed ration daily throughout the exper-
iment. The animals remained in the stalls until late afternoon. At
this time they were turned out into the small lots adjacent to the barn
for the night.
Feed refused was weighed back daily and the net feed intake
recorded.- Feed consumption was summarized for each bull weekly and
for the treatment groups every 28 days.
Termination of the experiment was to occur when four of the
eight protein deficient bulls had died or been sacrificed.
Body Weights. The bulls were weighed every 14 days in the
morning before feeding. The feed intake for the following two-week
period was based on these weights. Average daily gains (ADG) per
weigh period and cumulative daily gains were summarized for each
14-day period and for each 28-day period.
Blood Evaluation. Blood samples were taken initially and at
intervals of 28 days for the first two months. After this, bleedings
were done every 14 days for the remainder of the experiment.
COMPOSITION OF EXPERIMENTAL RATIONS--EXPERIMENT I
Control Ration Low Protein Ration
Ingredient C in Ration S in Ration
Granulated Corn Starch 16.00 42.00
Granulated Corn Sugar 13.00 27.00
Standard Cane Molasses 3.00 3.00
Corn Cob and Shuck Meal 25.00 25.00
Yellow Corn Meal 16.00
Cottonseed Meal, 41% 25.00
Trace Mineralized Salt 1.00 1.00
Steamed Bone Meal 1.00 2.00
Crude Proteinb 13.75 1.61
Estimated TDNc 67.41 73.45
aVitamins were added in corn starch carrier to each ration
at the time the concentrate portion was mixed.
bActual crude protein analysis of the rations at the Nutrition
CTDN estimated from Morrison's Feeds and Feeding ingredient
VITAMINS ADDED TO RATION PER TON OF FEED
Vitamin International Units
Vitamin Aa 1,800,000
Vitamin D2b 250,000
Vitamin Ec 3,468
aVitamin A used was the Dawe's Laboratories "Fixtay";
10,000 IU of vitamin A per gram.
bVitamin D2 was supplied by Dawe's Laboratories Dry
D2; 4 million USP units of vitamin D2 per pound.
cVitamin E was obtained from the American Cyanamid
Co.; 20,000 IU of vitamin E per pound.
MINERAL COMPOSITION OF THE TRACE MINERALIZED SALT
FED DURING THE EXPERIMENT
Mineral in Mixture
Salt (NaC1) 98.800
Inert Material 0.625
Blood was drawn from the jugular vein and analyzed for the
following components: blood hemoglobin, hematocrit and serum pro-
tein. Hemoglobin values were determined by the method of Cohen
and Smith (1919). Hematocrit values were obtained by the Wintrobe
method using the Wintrobe tubes and centrifuging the blood for 50
minutes at 3,000 rpm. Serum protein concentration was determined
by the Kjeldahl method as described by the Association of Official
Agricultural Chemists (1950).
The blood data were summarized for each 14-day period and by
treatment groups every 28 days.
Libido Evaluation. The bulls were checked for libido by
exposing each bull individually to an "estrogenized" heifer secured
in a stanchion. Each bull was allowed a maximum of ten minutes with
the heifer. Records were kept of the amount of interest displayed
by the bull, the number of mounts made and the number of matings
completed. The following system of scoring the interest displayed
was used: 1, no interest; 2, interest but did not mount; 3, mounted
without an erection; 4, mounted with an erection; and 5, mated the
Libido was checked in the described manner every 14 days.
The data were summarized for each 14-day and 28-day period by indi-
Vidual and treatment group.
Semen Evaluation. Semen was collected routinely throughout
the study to detect and evaluate changes in the various semen char-
acteristics. Semen was collected by means of an electro-ejaculator.
The criteria studied were semen volume, motility, sperm
concentration and total sperm production. Volume of semen was
determined directly by collecting the semen in calibrated tubes.
Motility was evaluated to the nearest 5 percent by microscopic
examination of the cells with progressive movement. A blood hema-
cytometer was used to determine the number of sperm per cubic
Single samples of semen were collected from each bull for
the first four months and duplicate samples collected for the final
two months of the experiment.
Midway through the experiment on January 5, 1961, semen was
collected and frozen for use in a fertility study. Ampules of semen
containing equivalent numbers of motile sperm were prepared from each
of 10 bulls (four control and six low protein animals). Two of the
low protein bulls were not included due to the poor quality of the
semen collected from them. An average of 24 ampules from each con-
trol bull and 21 from each low protein bull were used to inseminate
210 two-year-old heifers at the Deseret Farms of Florida, Melbourne,
Florida. The heifers were palpated to determine the number pregnant
on July 20, 1961.
On January 26 and 27, 1961, a semen exhaustion study was
conducted. Four ejaculates from each bull on each of the two days
were collected. Two collections were made in the morning and two
in the afternoon on each day. Evaluation of the semen characteristics
was made for each of the collections.
Histological Investigation. Tissue samples from the testes,
epididymis, prostate, Cowper's Gland and the seminal vesicles were
taken and fixed in formalin solution for histological studies.
Samples were taken from the four low protein bulls that were sacrificed
prior to the termination of the experiment.
The eight bulls, four control and four low protein, remain-
ing at the end of the experiment were slaughtered at the Experiment
Station Meats Laboratory. Tissue samples were obtained from these
bulls at this time.
The procedures of Gurr (1956) were followed for fixing,
imbedding, slicing and staining with eosin-hematoxylin stains.
Microscopic evaluations were made (with a calibrated eye piece) of
the size and development of the secretary tissues in the secondary
sex glands. The ducts of the epididymis were studied for differences
in size and sperm content. The testes were examined for the relative
size and development of the seminiferous tubules, germinal epithelium
and secretary tissue.
Additional slaughter data collected were: size and weight
of the testes, weights of the adeno hypophysis, the secondary sex
glands, the thyroid and the adrenal glands.
Statistical Analysis. The blood, libido, and semen data were
summarized for 28-day periods to facilitate statistical analysis.
The data were then divided into two phases; phase I which included
all 28-day periods in which all eight of the low protein bulls were
represented and phase II where the number of protein deficient bulls
varied due to death losses. There were five periods in phase I for
the semen data and four periods for the libido and blood data.
Phase II contained the final two periods for the three criteria
analyzed. The two phases were analyzed separately.
The Analysis of Variance technique for repeated-measurement
experiments as described by Danford et al. (1960) was used.
Results and Discussion
The low crude protein content of the deficient ration had
an immediate effect on the appetite of the low protein bulls. There
was a marked difference in consumption between the two treatment
groups at the end of the first 28 days. At this time, the control
and low protein bulls were consuming 7.54 and 4.17 pounds of feed
per head per day, respectively. The same pattern continued to hold
true throughout the experiment. Figure 1 illustrates this effect
of a protein deficiency on feed intake. The average feed intake for
the control and the low protein bulls was 9.78 and 3.28 pounds
daily, respectively, for the experimental period.
Considering the feed intake on a body weight basis, the
differences are not as great. The average total feed intake per
100 pounds (cwt.) of body weight (table 15, in Appendix) was 1.86
and 1.13 pounds daily for the control and the low protein bulls,
respectively. In figure 2 the changes in crude protein and TDN
intake per cwt. of body weight daily are shown. The control bulls
S ----- LOW PROTEIN
- - - -
1 2 3 4 5 6 7
Figure 1. Effect of protein deficiency on daily total feed intake of bulls.
--- LOW PROTEIN
I I I I I I p
1 2 3 4 5 6 7
Effect of protein deficiency on crude protein and TDN intake per cwt.
daily of bulls.
consumed about 1.25 pounds of TDN per cwt. of body weight fairly
consistently during the trial. This was not unexpected since the
controls were fed according to body weight. The TDN intake by the
low protein bulls, on the other hand, declined steadily even on a
body weight basis. The low protein bulls were losing appetite faster
than body weight.
The intake of protein, however, was fairly stable throughout
for both groups. Again, this would be expected for the control
bulls due to the method of feeding. The lack of any appreciable
change in protein intake, in spite of the marked change in total
feed intake, was due to the very low protein level in the deficient
These data indicate rather conclusively the definite depress-
ing effect of low protein levels on feed intake. This response is
in good agreement with the work of Guilbert and Hart (1951) and
Reid (1949) which demonstrated a similar effect with low protein
rations in both large and small animals. The results compare closely
with, and hence tend to confirm, earlier work by the author (Meacham,
1960) with low protein rations for beef bulls.
Body Weight Changes
The immediate reduction in feed intake by the low protein
bulls was reflected in a corresponding loss of body weight. The
pattern of weight changes is shown in figure 5. The control bulls
gained rather slowly the first 28-day period and then maintained
- - -
- -------- --
1 2 3 4 5 6 7
Figure 3. Cumulative average daily gains of bulls.
an ADG of about a pound a day for the remaining periods. The ADG for
the 182 days of the experiment for the control bulls was 1.1 pounds.
The low protein bulls lost weight continuously throughout the exper-
imental period with the greatest losses occurring in the early part
of the study. The weight losses were less severe during the final
periods, due possibly to a lack of any additional tissue that could
readily be mobilized. The low protein bulls had a negative daily
gain of 0.71 pounds for the 182 days. The individual bull weights
for the seven periods are summarized in table 16 in the Appendix.
The control bulls gained an average of 200 pounds and the low
protein bulls lost an average of 150 pounds during the trial. The
ranges in body weight changes were losses of 115 to 145 pounds for
the low protein bulls and gains of 190 to 210 pounds for the control
bulls. As indicated by these relatively close ranges, the bulls dis-
played a very uniform response to the experimental rations.
Figure 4 illustrates the effect of the low protein ration on
the growth, weight and general appearance of the bulls.
Four of the low protein bulls died or were sacrificed shortly
before dying. The death of the fourth bull terminated the experiment
in accordance with the experimental plan. The four bulls survived
120, 132, 169 and 182 days respectively. The cause of death was
primarily due to starvation.
Figure 4. Effect of a low protein ration on bull growth.
A low protein bull at the end of the experiment.
Hemoglobin. The effect of the low protein ration and reduced
feed intake on the hemoglobin levels of the bulls is shown in figure 5.
There was no significant difference between the two groups for the
first four 28-day periods. As indicated in figure 5, a definite
spread occurred in the mean hemoglobin values during the final two
periods. Statistical analysis of the data indicated a significant
(P < .01) difference due to treatment in these final periods. The
significant interaction (P < .05) between treatments and periods sig-
nified that the increasing difference between the means was due to
an increase for the controls and a decrease in hemoglobin values for
the low protein bulls.
The final hemoglobin values 13.00 and 7.91 grams of hemoglobin
per 100 ml. of blood for the control and low protein bulls, respec-
tively, fit the normal range for hemoglobin reported by Long et al.
(1952) of 8.0 to 13.0 g. per 100 ml. of blood. In view of this, it
can not be concluded that the protein deficiency produced a partic-
ularly severe anemic condition in these low protein bulls. The mean
hemoglobin value for the low protein bulls in this experiment compares
quite closely with that reported by the author (Meacham, 1960) for
similar protein deficient bulls.
Individual and group hemoglobin values are summarized for
each 28-day period in table 17 in the Appendix. The Analysis of
Variance mean squares for the blood components are given in tables
24 and 26 in the Appendix.
P 8.0 .
7.0 ---LOW PROTEIN
o I I I I I I
1 2 3 4 5 6
Figure 5. Effect of protein deficiency on average hemoglobin levels of bulls.
Hematocrits. The pattern of changes in the mean hematocrit
values for the two groups of bulls followed that of the hemoglobin
values as might be expected. Figure 6 illustrates these changes
graphically. Again no significant differences due to treatment
occurred during the first four 28-day periods. There was, however,
a significant (P < .01) period x treatment interaction. The values
for the low protein bulls tended to decrease and those for the control
bulls to increase during this phase.
In periods five and six the spread between the two groups
widened markedly. The mean hematocrit values for the control and low
protein groups in the final period were 54.54 and 33.17 percent,
respectively. This difference was significant (P < .01). Signif-
icant (P < .05) effects due to period and period x treatment inter-
action were found during this final phase also. The increasing and
decreasing trends for the control and low protein groups, respectively,
continued to manifest themselves.
As was found with the hemoglobin analysis, the mean hematocrit
level for the low protein group was still within the normal range
reported in the literature. In spite of the significant difference
(P < .01) between treatments, the packed cell volumes for the surviving
bulls on the low protein ration had not reached a critical level.
Hematocrit levels were higher for both the control and the low
protein groups compared to those reported earlier (Meacham, 1960) for
bulls under similar nutritional conditions. Both the control and the
low protein group means were approximately 20 percent higher in the
Figure 6. Effect of protein deficiency on average hematocrits of bulls.
present study. The magnitude of the difference between the treatment
groups, however, was similar. In both experiments, the low protein
groups had mean hematocrit levels 40 percent lower than the controls.
Table 17 in the Appendix summarizes the individual and mean
hematocrit values for each 28-day period.
Serum Protein. As shown in figure 7, the percent protein in
the serum appeared to be more sensitive to the low protein intake by
the bulls than the two other blood components. A significant difference
(P < .01) occurred between the two ration groups during the first four
periods. The significant period effect (P < .01) and a lack of a sig-
nificant period x treatment interaction indicated that both groups
declined in percent protein in the serum. Final mean values for the
control and low protein bulls were 6.76 and 5.34 percent, respectively.
The control value is in the lower end of the normal range reported by
Crookshank and Sims (1955) and is lower than the normal value of 6.70
percent given by Dukes (1955). The control and low protein group means
are both lower than corresponding serum protein values obtained by the
author (Meacham, 1960) in an earlier study similar to the present one.
The reduced protein level in the blood of the low protein bulls in this
study would be expected considering the severely restricted dietary
protein intake and diminished tissue protein supplies, but the com-
paratively low serum protein levels for the control group cannot readily
be explained. It is possible that the lower feed intake by the control
bulls in the present experiment may have influenced this somewhat.
The individual and mean serum protein values are summarized in
table 17 in the Appendix.
5.0 LOW PROTEIN
i 1 ,I I
1 2 5 4 5 6
Figure 7. Effect of protein deficiency on average serum protein
level of bulls.
Statistical analysis of the libido data revealed a significant
difference due to treatment only in the interval to first mating
(P < .05) during phase I. The interval to first mount and libido score
were not affected sufficiently to overcome the wide variation within
treatment groups. These values are summarized by treatment and
period in table 4.
MEAN INTERVAL TO FIRST MOUNT, INTERVAL TO FIRST MATE
AND LIBIDO SCORE BY 28-DAY PERIODS
Interval to Interval to Libido
Period First Mount First Mate Score
(min.) (min.) (1-5)
Low Low Low
Control Protein Control Protein Control Protein
1 2.0 0.7 5.1 4.4 4.4 4.7
2 0.4 0.6 5.2 7.1 4.6 4.4
5 0.5 3.8 2.2 9.3 4.9 3.2
4 0.3 3.4 3.8 9.3 4.8 3.5
5a 0.7 2.8 3.8 10.0 4.9 5.2
aThree of the low protein bulls died during the fifth period.
In the final period, both the libido scores and the interval
to first mate for the low protein bulls were significantly (P < .01)
lower than the controls. Only two of the low protein bulls mated the
heifer during the final period. Mean libido score and interval to
first mate (minutes) for the control and low protein bulls were 4.9
and 3.8 and 3.2 and 10, respectively, during the second phase.
The increasing physical weakness of the low protein bulls
probably influenced the increase in time required to mount and mate.
The significant (P < .01) period effect and treatment x period inter-
action in the first phase indicated that the control bulls tended to
increase in libido while the low protein bulls decreased rapidly.
There was considerable variation in libido among the bulls in
both treatment groups. One control bull mated the heifer only three
times in the entire experiment. One of the low protein bulls mated
the heifer in the preliminary period, was assigned to a treatment
group, and then failed to mate again.
In general, the lack of libido seemed to be closely associated
with the physical condition of the bulls. As the bulls lost weight
and became weak, libido decreased and eventually reached the point
where they displayed little or no interest in the heifer. It would
be difficult, therefore, to separate a possible endocrine disturbance
from a simple state of physical weakness.
Analysis of Variance mean squares for the various libido
criteria are given in tables 24 and 26 in the Appendix.
Volume. The volume of semen produced by the two groups of
bulls was quite varied as shown in figure 8. This variation was
noted both between bulls within groups and within bulls between
collections. There were, however, significant differences in volume
between the two groups in both the first (P < .05) and the second
phase (P < .01). The within group variation was not as great in the
5.0- \ />
4.0 CONTROL N "
LOW PROTEIN N ".
1 2 5 4 5 6 7
Figure 8. Effect of low protein intake on mean semen volume of bulls.
second phase as in the first. The significant period effect (P < .01)
and interaction between period and treatment (P < .05) during the five
periods indicated that while the semen production for both groups tended
to decline, the low protein bulls were more severely affected.
In phase II, the significant (P < .01) treatment effect and
lack of a period or period x treatment interaction of significance
was indicative of an uncomplicated treatment effect. The low protein
bulls produced a smaller volume of semen in both periods.
The semen volume, motility and total sperm cell production
figures for individual bulls and treatment groups are given by
periods in table 18 in the Appendix. Analysis of Variance mean
squares for the various semen characteristics are presented in tables
25 and 25 in the Appendix.
Motility. As indicated in figure 9, motility values were
extremely variable. The variation within treatment groups precluded
assigning any statistical significance to the differences between
means. There was, however, a definite trend, particularly during the
latter part of the experiment, for the motility values of the control
bulls to be higher than those of the low protein bulls. Mean per-
centages for the control and low protein bulls were 70 and 24,
respectively, during the final period of the study.
The sharp decline in motility in the first part of the exper-
iment was due in part to low environmental temperatures. The semen
was collected in an exposed area where it was subjected to appreciable
cold shock. Changes in the collection technique whereby the semen
--0 ---- LOW PROTEIN
0 - -
1 2 3 4 5 6 7
Figure 9.- Effect of low protein intake on mean sperm motility of bulls.
was protected from the cold weather improved the motility ratings
of both treatment groups. This drop and subsequent rise in percent
motility is indicated in figure 9 between periods two and five. The
significant period effect (P < .01) during phase I substantiates this
cold weather effect.
A significant treatment x period interaction (P < .01)
indicates the motility of the low protein bulls declined more rapidly
and did not recover as fast or as completely as the motility of the
control bulls. It would appear from this that the treatment was
influencing sperm motility even though there was no significant
Total Sperm Production. During the first two 28-day periods,
both treatment groups had similar increases in total sperm production
as shown in figure 10. After the second period the low protein bulls
produced progressively fewer sperm per ejaculate. Their production
dropped from 1,877 x 106 in the second period to 337 x 106 sperm per
ejaculate in the final period. The control bulls had a total sperm
count average of 1,079 x 106 at the second period and a mean count
of 1,767 x 106 at the final period. Due to the variation and fluc-
tuation between bulls and between periods, the difference between
treatment means was not statistically significant until the final
two periods. At this time, the control bulls produced significantly
(P < .05) more sperm per ejaculate than did the low protein bulls.
There were no significant period or interaction effects in either
part of the experiment.
o 1700- \/
300 LOW PROTEIN
1 2 3 4 5 6
Effect of low protein intake on total sperm production of bulls.
The marked difference in total cell production during the
final periods was not unexpected in light of the significant drop
in semen volume that occurred during these same periods.
It is noteworthy that even though the low protein bulls had
become so emaciated and weak that four of them had been sacrificed
by the end of the experiment, only one of the bulls had stopped
producing semen completely. The one low protein bull failed to pro-
duce semen for two consecutive collections 'just before death. As
indicated in figure 10, there was a marked reduction in sperm produc-
tion, but spermatogenesis was not completely halted.
Fertility Study. The 210 heifers used in the study were
inseminated with an ampule of semen from an experimental bull at
their first detected estrus during the breeding season. Heifers that
returned in heat were rebred with semen from a commercial insemination
The results of the insemination, as determined by rectal
palpation approximately 60 days after the insemination period, were
rather disappointing. Only 68 of the 210 heifers were pregnant at
this time, Of these 68 pregnancies, 29 of them were attributed to
insemination with semen from the experimental animals. The remaining
39 pregnancies were considered to have resulted from the reinsemina-
tions with the commercial semen. This distinction was made on the
basis of fetal. size at the time of palpation.
Many of the heifers did not become pregnant regardless of the
source of semen or number of inseminations and others were inseminated
three or four times before conceiving. These difficulties, plus the
fact that bulls were turned in with the breeding herd on the week-ends
during the breeding season, precluded an accurate evaluation of the
fertility data obtained. With the beforementioned problems in mind,
an approximate fertility percentage was calculated. This percentage
was based on the number of heifers considered pregnant with semen
from each group of experimental bulls divided by the total number of
heifers inseminated with semen from the respective treatment groups
that were pregnant at the time of palpation. These percentages were
49 and 34 for the control and low protein bulls, respectively. These
data are subject to question and the confounding factors involved
need to be kept in mind when considering the apparent differences
between the two groups.
Semen Exhaustion Study. The exhaustion study was conducted
for two days during the last part of phase I. The data from this
study are summarized in table 5.
The total volume of semen and the total number of sperm cells
produced on the first day, averaged by groups, were 24.5 ml. and
4555 x 106 cells and 17.2 ml. and 1190 x 106 cells for the control
and low protein bulls, respectively. On the second day, correspond-
ing values were 15.0 ml. and 1564 x 106 cells and 11.0 ml. and 568
x 106 cells for the control and low protein bulls, respectively.
The control bulls produced a greater amount of semen contain-
ing more sperm cells than did the low protein bulls on both days. Both
the control and the low protein bulls had a marked reduction on the
SUMMARY OF THE SEMEN EXHAUSTION STUDY FOR INDIVIDUAL BULLS AND
TREATMENT GROUPS BY DAYS AND TOTAL PRODUCTION
Treatment Groups First Daya Second Daya Total
and Animal No. Vol.b TCPc Vol.b TCPc Vol.b TCP
6 18.4 2,784 12.3 1,898 30.7 4,682
9 43.9 11,824 20.3 1,787 64.2 13,611
11 19.6 1,344 15.6 122 35.2 1,466
14 16.0 2,260 12.0 2,450 28.0 4,710
Av. 24.5 4,553 15.0 1,564 39.5 6,117
4 22.9 2,136 26.4 1,472 49.2 3,608
8 23.2 88 6.4 0 29.6 88
10 29.2 2,872 12.0 960 41.2 3,832
12 18.7 132 4.0 0 22.7 132
13 14.6 568 8.0 144 22.6 712
15 9.6 0 12.0 0 21.6 0
16 11.2 2,324 10.8 683 22.0 3,00
17 8.4 1,404 8.4 1,289 16.8 2,695
Av. 17.2 1,190 11.0 568 28.2 1,759
aEach observation is the sum of four determinations.
bVolume of semen in milliliters.
cTotal cells production per ejaculate times 106.
second day of the study. One of the low protein bulls did not pro-
duce sperm cells on the first day and three of the bulls in the low
protein group failed to produce sperm on the second day.
Examination of the semen for abnormal sperm cells did not
indicate any treatment effect. The percentages of abnormal sperm
in the semen of the two treatment groups were quite similar and fairly
normal for bulls of this age.
It is evident from these data that there was a definite
difference in spermatogenic capacity between the two treatment
groups. It was possible to collect considerably more total sperm
from the control bulls as compared to the low protein bulls. This
is in agreement with earlier work by the author with bulls under
similar experimental conditions (Meacham, 1960).
The results of the histological investigation are given in
tables and 7. The objective measurement of the thickness of the
epithelial tissue of the organs and glands revealed a marked and
consistent reduction in the tissues from the low protein bulls.
The Cowper's Gland presented the greatest difference and the spermatic
ducts of the epididymis the smallest. The diameter of the semini-
ferous tubules of the testes and the ducts in the epididymis were
approximately 30 percent larger in the control bulls than in the
low protein bulls. The amount of interstitial cells in the control
bull testes was greater than that in the low protein bulls.
QUANTITATIVE EVALUATION OF TESTES AND EPIDIDMIS FROM THE TWO GROUPS OF BULLS
Treatment Epithelium Tubule Inter- Epithelium Duct Sperm
Group and Thickness Diameter Sperma stitialb Thickness Diameter Content
Animal No. (mm x 10-2) (mm) Content Tissue (mm x 10-2) (mm)
6 5.4 .21 2 2 6.6 .55 2
9 4.8 .20 2 5 5.4 .51 5
11 3.0 .24 1 3 6.6 .28 2
14 4.8 .19 1 5 6.0 .30 5
Av. 5.0 .20 1.5. 3.7 6.1 .51 2.5
4 2.4 .15 1 2 4.8 .22 1
8 1.2 .15 1 1 3.6 .17 1
10 -_c 4.8 .50 2
12 1.8 .14 1 1 4.2 .22 2
15 5.6 .15 1 2 5 6 .14 5
15 2.4 .15 1 1 2.4 .21 1
16 4.8 .15 1 2 6.0 .23 1
17 1.8 .12 1 2 4.2 .20 1
Av. 2.6 .155 1 1.6 4.2 .21 1.5
aSperm content scored from 1 to 5 with
bInterstitial tissues scored from 1 to
cTestis sample from this bull was lost
1, no sperm, and 5, abundant.
5, with 1, sparse, and 5, abundant.
QUANTITATIVE EVALUATION OF THE SEMINAL VESICLES, PROSTATE AND COOPER'S
GLANDS FROM THE TWO GROUPS OF BULLS--EXPERIMENT I
Seminal Vesicles Prostate Cowpers
Treatment Epithelium Ratio of Epithelium Epithelium
Group and Thickness Secretory Tissuea Thickness Thickness
Animal No. (mm x 10-2)c to Total Area (mm x 10-2) (mm x 10-2)
6 3.6 2 1.2 1.8
9 3.0 2 1.2 2.4
11 3.0 2 1.2 --b
14 3.0 2 __-b 1.8
Av. 3.1 2 1.2 2.0
4 1.8 2 .6 1.2
8 1.2 3 --_b .6
10 1.2 1 --b 1.2
12 2.4 3 .6 .6
13 1.8 3 .6 .6
15 1.8 3 -__b .6
16 1.8 5 .6 .6
17 1.8 3 --.b .8
Av. 1.7 2.6 .6 .78
aScale is based on values from 1 to 3, 1 being a narrow ratio of
secretary tissue to total area and 3 being a wide ratio.
bSamples lost in processing.
cMeasurements were made with a calibrated eyepiece and converted
Perhaps the most notable difference was in the germinal
epithelium of the seminiferous tubules. The numbersof layers of germ
cells in the low protein bulls were greatly reduced and the existing
layers were characterized by a general disorganization. The germinal
epithelium of the tubules of the control bulls on the other hand,
was well developed and had numerous and orderly layers of developing
sperm cells. / These findings are in very good agreement with the
sperm production records of the two groups of bulls as noted by the
routine semen analysis. The reduction in size and secretary capacity
of the secondary sex glands in the low protein bulls was also con-
sistent with the reduced volume of semen produced by these bulls.
These differences are illustrated in figures 11 and 12.
The gross weights of these various organs and glands were
obtained at the time of slaughter and are summarized in table 8.
SUMMARY OF MEAN GLAND AND ORGAN WEIGHTS
TAKEN AT TIME OF SLAUGHTER
Control Low Protein
Gland or Organa Per Cwt. Per Cwt.
Mean Body Weight Mean Body Weight
Testes (4)b 197.2 31.81 (8) 67.0 27.35
Epididymis (4) 15.6 2.52 (8) 6.4 2.62
Seminal Vesicle (4) 26.0 4.19 (8) 8.3 5.38
Cowper's Gland (3) 6.3 1.03 (7) 1.4 .57
Adrenal Gland (4) 6.8 1.10 (6) 4.0 1.63
Thyroid Gland (2) 22.8 3.52 (6) 11.9 4.86
aProstate gland tissue was taken, but the direction was
bNumber in parenthesis is the number of samples represented
in the mean.
Figure 11. Effect of a protein deficiency on the testes and
seminal vesicles of bulls.
A and B: Sections of the testis from a control
and a low protein bull, respectively
C and D: Sections of the seminal vesicle from a
control and a low protein bull, respectively.
( x 645 )
II- - *
*- *. *
* P lt .
~ -a -
f ",, r *.: ".
p 5. ... .I
*rj~ atW 55/
Figure 12. Effect of a protein deficiency on the Cowper' s and
prostate glands of bulls.
A and B: Sections of the Cowper's gland from a control
and a low protein bull, respectively.
C and D: Sections of the prostate gland from a control
and a low protein bull, respectively.
( x 645 )
* .. ".S-, r,
a' i V -. -" ". a ' . "
- C.. A ,
' . .I,
^ .--a a *e;*C! 10W-' n^ rp
S.*m *-, -
-, b kt. . r "v'-" "
*: 7 -r :w... *4 .*.
. .. . .- ;
..** ." 1 i
I.:, at r1
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, j -P : ": t \, *" "',
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s'- -"r :"Y 5 ..* ,-.
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*. " -
+,+' , ) -A
"* * .,
Eighteen Dorset or Hampshire x Rambouilet crossbred rams
approximately 11 months old and weighing an average of 99 pounds were
used in this experiment. The rams were purchased from Auburn Univer-
sity in Alabama, and brought to the Experiment Station as three-month-
old weaned lambs.
The rams were used in preliminary studies with purified rations
prior to the start of the present experiment. The rams were all placed
on a basal purified ration several weeks before the experiment was
initiated to minimize any effect of previous treatment.
During the preliminary feeding period, three semen collections
were made. The average total sperm production value for each ram
from these three collections was used as the basis for assignment to
treatment groups. The 18 rams were ranked according to total sperm
production and the top, middle and low ramspulled out. The remaining
15 rams were then stratified into three uniform groups of five rams
each. A protein-nitrogen ration, a urea-nitrogen ration and a nitrogen-
free ration were assigned at random to the three groups of rams. The
three rams pulled out during the ranking were then added to the group
receiving the nitrogen-free ration. In this manner two groups of five
rams and a group of eight rams were obtained that were relatively
uniform in total sperm production.
At the start of the experiment on September 1, 1960, the
average initial weights of the three groups were 97.6, 92.5 and 100.2
pounds for the urea-nitrogen, protein-nitrogen and nitrogen-free rams,
The rams were maintained in individual stalls in metal metab-
olism racks under a shed roof at the Experiment Station Sheep Unit.
Each stall was equipped with a feed and a water container. Platform
scales were available at the unit. Pens and chutes for working the
rams were adjacent to the shed area. A small, portable stanchion was
constructed to secure an "estrogenized" ewe for libido checking.
Feedstuffs and Rations
Purified ingredients were used in the experimental rations.
Drackett C-1 assay protein, a purified soybean protein, was used as
the protein source and urea as the non-protein source of nitrogen.
The composition of the two control rations and the nitrogen-free
ration is given in table 9. The levels of Drackett protein and urea
were calculated to supply approximately 11 percent crude protein in
each control ration. The methionine analogue was added to the control
rations as a source of sulfur containing amino acid. The total pro-
tein content was 11.54 and 11.05 for the Drackett and urea rations,
respectively. The corn sugar and corn starch levels were increased
in the nitrogen-free ration to replace the nitrogen source. Chemical
analysis indicated essentially no nitrogen in the nitrogen-free ration.
Four percent corn oil was added to all three rations to reduce the
dustiness and improve the texture and palatability.
The mineral mixture used in the rations is shown in table 9.
The mixture containing the 13 essential mineral elements was essentially
that reported by Oltjen et al. (1959).
Vitamins A, D, E and choline in a corn sugar carrier were
added to each ration. Table 9 gives the vitamins and the levels
at which they were fed.
The rations were mixed in 100-pound lots in a stainless steel
mixer. The mixed rations were stored under refrigeration at approx-
imately 400 F.
Management and Feeding. The rams were fed twice daily all
the feed they would clean up by the next feeding. Feeding was done
at 7:00 a.m. and 5:00 p.m. Feed refused was weighed back daily at
the evening feeding. Fresh water was available at all times. The
rams were maintained in the individual stalls at all times except
when removed to collect experimental data.
Feed consumption data were summarized individually every seven
days and by treatment groups for 28-day periods.
Weighing. The rams were weighed every seven days on platform
scales. Individual and group average daily gains were summarized for
COMPOSITION OF THE
FED THE RAMS
Corn Starch, granulated
Urea (262 Protein)
Methionine (Hydroxy analogue)
aHydrolyzed wood pulp, Solka Floc, was used as a source of
bThe mineral mixture supplied the following amounts of the
13 essential minerals per 100 pounds of feed: CaHP04*2H20, 1444.2 gm.;
K2C03'1~H20, 950.7 gm.; MgSO43H20, 316.9 gm.; NaC1, 227 gm.; FeSO4
2H20, 59.8 gm.; Na2B407lOH20, 5.6 gm.; MnSO4*H20, 2.9 gm.; ZnS04-7H20,
2.0 gm.; KI, 0.88 gi.; CuSO4, 1.18 gm.; 2CoC03*5Co(0H)2, 0.008 gm.;
MoOg, 0.025 gm.; Corn sugar carrier, 1568.90 gm.
CThe vitamin mixture supplied 100,000 IU of vitamin A palmitate,
15,000 ICU of vitamin Dg, 200,000 units of vitamin E, 200 gm. of choline
chloride (254 in carrier) and corn sugar carrier to make four pounds per
100 pounds of feed. The vitamins were furnished by Chas. Pfizer and Co.,
Terre Haute, Indiana.
each seven and 28-day period. The weighing was done in the morning
before the rams were fed.
Blood Evaluation. Procedures followed in the blood evaluation
were similar to those used in Experiment I. Blood samples were obtained
at 28-day intervals in Experiment II.
Libido Studies. Libido evaluations were conducted in the same
manner as in Experiment I.
Semen Evaluation. The procedures followed in the routine
semen evaluation were basically the same as those of Experiment I.
Fertility and semen exhaustion studies were not conducted in Exper-
iment II. Single ejaculates per ram were collected in the second
SHistological Investigations. The same procedures used in
Experiment I were employed in Experiment II. The prostate gland,
however, was not used in these investigations in Experiment II.
Adenohypophysis Assay. The gonadotrophic activity of the
adenohypophyses from the three groups of rams was determined by the
chick testes weight technique. The gonadotrophic assay was performed
using day-old White Leghorn chicks injected with a total of 10 mg.
of dried adenohypophysis powder in a saline solution.
Due to the small number of chicks that could be used on
individual glands, the powder from rams within ration groups was
pooled. Twelve, 14 and 15 chicks were used for the urea, Drackett
and nitrogen-free groups, respectively. The 10 mg. of dried powder
in 0.75 ml. of 0.9 percent saline solution was injected subcutaneously
in three injections 12 hours apart. Four groups of 10 chicks each
received 250, 500, 750 and 1000 IU of A standard gonadotrophin'.
An additional group of 10 chicks received saline injections only and
a control group of 10 chicks received no injections.
Sixteen hours after the last injections, the chicks were
weighed and sacrificed. The testes were removed and wet weights of
both testes were taken. The degree of stimulation from the adenohypo-
physis powder was determined by comparing the testes weights of the
test chicks with those of the chicks injected with saline only. An
approximation of the potency of the adenohypophysis powder from the
treatment groups was estimated from the curve established with the
testes weights of the chicks injected with the graded doses of the
Statistical Analysis. The blood, semen, and libido data were
subjected to the same statistical analysis used in Experiment I. In
this experiment, however, there were only four periods in phase I and
a single period in phase II. The analysis used for phase II involved
comparisons of values for the various criteria for the four surviving
rams in the nitrogen-free group.
Results and Discussion
The purified rations fed in the second experiment were not
consumed readily by any of the treatment groups. One ram in the
Drackett control group and one in the urea control group would not
eat the ration and had to be removed from the experiment. The
Drackett control, urea control and the nitrogen-free groups consumed
an average of only 1.60, 1.46 and 0.61 pounds of feed per day,
respectively, during the experiment. There was a great deal of var-
iation in feed intake between rams in the control groups as indicated
in table 19 in the Appendix. Feed intake in the nitrogen-free group
was more uniform, possibly due to the rather severe restriction
brought about by the lack of nitrogen. The reason for the limited
feed intake of the two control rations is not immediately apparent.
It may have been related to the bulky nature of the ration or to some
environmental factor associated with the experiment.
While the two control groups consumed fairly similar amounts
of feed the lack of nitrogen had a marked and immediate effect on the
appetite of the rams fed the nitrogen-free ration. Intake dropped off
the first week of feeding, and continued to decline throughout the
study. The feed intake pattern for the three treatment groups is
given in figure 15. The effect of a complete absence of nitrogen
was even more severe than that found with the low protein ration in
Experiment I and earlier work reported by the author (Meacham, 1960).
Four of the eight rams on the nitrogen-free ration died or were
N - - -
I -I I__J I -II
1 2 3 4 5 6 7
Figure 13. Effect of source and level of nitrogen on feed intake of rams.
sacrificed an average of 78 days after the start of the study. In
Experiment I, the low protein bulls survived 120 days on the low
protein ration before the first bull died. The nitrogen-free rams
consumed an average of 40 percent of the amount consumed by the con-
trol rams whereas the low protein bulls in Experiment I ate only 33
percent as much as the control bulls.
There was little difference in feed intake between the two
sources of nitrogen, Drackett protein and urea. The average intake
of the urea rams tended to be lower during the first part of the study.
This was due in part to the performance of one ram in this group that,
after starting on the ration, became very erratic in feed consumption
and finally went completely off-feed. This ram, no. 19, was subse-
quently sacrificed after 84 days on the experiment.
This feed consumption response is somewhat at variance with
that observed in earlier work with these rations by the author
(Meacham et al., 1961) and work by others (Oltjen et al., 1959) with
these same sources of nitrogen. The performance of the protein-
nitrogen was usually much better than that of the non-protein-nitrogen.
Body Weight Changes
The average daily gains of the treatment groups reflected
very closely their respective feed intakes. Figure 14 shows the
cumulative ADG for the three groups. The Drackett control group had
an average gain of 0.09 pounds daily for the experimental period.
The urea control group lost considerable weight during the second
period and then gradually recovered, finishing the experiment with a
Effect of source and level of nitrogen on cumulative average daily gain
_ .- -
cumulative ADG of 0.06 pounds. As was the case with feed intake for
the urea control group, ram no. 19 tended to hold the group average
down. This ram lost 42 pounds in the 84 days he was on the experiment.
The other rams in the group held their initial weight or gained
slightly. These individual weight changes are summarized by treat-
ment group and period in table 20 in the Appendix.
The nitrogen-free group lost weight consistently throughout
the study. This was not unexpected in view of their reduced feed
intake. The four rams surviving the 98-day experimental period lost
an average of 37 pounds or over one third of their initial weight.
The weight losses for all eight rams in this group tended to be
fairly uniform. The cumulative ADG for this group was -.58 pounds.
Figure 15 illustrates the appearance of these rams near the
end of the experiment.
Hemoglobin. The effect of the three experimental rations on
blood hemoglobin levels in the rams is shown in figure 16. The mean
hemoglobin value for the nitrogen-free rams dropped rapidly during
the second and third periods and then tended to level off. The two
control groups, however, increased slightly during the second period
and then dropped during the third period with a similar leveling off
for the remainder of the study.
While the hemoglobin of the two control groups tended to be
higher than that of the nitrogen-free group during the experiment,
Figure 15. Effect of source and level of nitrogen on ram growth.
A, B and C: A representative Drackett control, urea
control and nitrogen-free ram, respectively, at the
end of the experiment.
_- UREA CONTROL
S .- NITROGEN-FREE
Figure 16. Effect of source and level of nitrogen on mean
hemoglobin values for rams.
aPeriod 4 only seven days.
the differences were not statistically significant. The lack of
statistical significance was due primarily to the wide variation in
hemoglobin levels within treatment groups.
Initial and final mean hemoglobin values for the Drackett
control, urea control and nitrogen-free groups were 11.06 and 11.09,
12.02 and 10.05 and 12.74 and 8.74 grams of hemoglobin per 100 ml. of
blood, respectively. It would appear that had the nitrogen-free rams
survived longer, real differences might have developed.
Individual and treatment group blood values for the five
periods are summarized in table 21 in the Appendix.
Hematocrit. As indicated in figure 17, hematocrit values for
the three treatment groups fluctuated markedly. Statistical analysis
of the data revealed no significant differences due to treatment or
period. The urea-control and the nitrogen-free groups tended to fluc-
tuate together and both were somewhat lower than the Drackett controls
during the last part of the study.
Mean hematocrit values for the initial and final periods were
44.06 and 42.87, 44.22 and 37.80 and 48.34 and 55.88 percent, respec-
tively, for the Drackett control, urea control and the nitrogen-free
groups. All groups, however, were well above the normal hematocrit
figure of 32 percent reported by Dukes (1955).
Serum Protein. Serum protein values varied somewhat between
periods during the experiment, but practically no difference was ob-
served between treatment groups. This situation is shown graphically
in figure 18. Statistical'analysis of the serum protein data failed
/ /\ \\
,/ / \ \
Figure 17. Effect of source and level of nitrogen on mean
hematocrit levels of rams.
Period 4 only seven days.
I I_ r ---
6.0 UREA CONTROL
01 1 a I I.
Initial I 2 3 4a
Figure 18. Effect of source and level of nitrogen on mean
serum protein values of rams.
aPeriod.4 is a seven-day period.
to indicate any treatment effect also. Apparently, the nitrogen-free
rams were maintaining blood protein levels through tissue breakdown.
The initial and final serum protein values for the three groups were
7.77 and 7.36, 7.81 and 7.44,and 7.78 and 6.77 percent for the Drackett
control, the urea control and the nitrogen-free groups, respectively.
Analysis of Variance mean squares for the various blood com-
ponents are given in table 27 in the Appendix.
The three dietary treatments had very little effect on the
libido of the rams. No significant differences were noted for the
interval to first mount, interval to first mating or libido score.
As indicated in table 10, the Drackett control and the nitrogen-free
rams tended to have shorter mean intervals to first mount and mating
and higher libido scores than the urea control rams. Two of the four
rams in the urea group, nos. 19 and 14, displayed little or no interest
in the estrogenized ewe throughout the trial. No. 14 did mate the ewe
once near the end of the study. These two rams were undoubtedly
responsible for most of the difference between the libido performance
of the urea control group and the other two groups. One of the
nitrogen-free rams also showed no interest in the ewe during the ex-
Comparing the effect of low protein rations on the libido of
bulls with the effect of nitrogen-free rations on rams, reveals a pos-
sible species difference. In Experiment I and earlier work by the
author (Meacham, 1960) libido was adversely affected in the protein
SUMMARY OF LIBIDO DATA FOR THE THREE TREATMENT GROUPS OF RAMS
BY 14-DAY PERIODS
Criteria and Periods
Treatment Groups-- 1 2 3 4 5 6
Interval to First Mount, min.a
Drackett Control 1.0 .5 .8 .5 .5 .5
Urea Control 5.9 5.2 5.2 5.6 5.0 .8b
Nitrogen-free 1.8 1.6 1.7 1.6 1.6 2.9c
Interval to First Mating, min.a
Drackett Control 1.4 1.1 1.5 .9 2.8 .9
Urea Control 5.3 6.1 8.0 8.0 3.8 4.6b
Nitrogen-free 2.2 1.9 .9 7.2 5.5 5.9c
Drackett Control 5.0 5.0 5.0 5.0 4.8 5.0
Urea Control 3.5 5.0 2.8 3.0 4.2 4.7b
Nitrogen-free 4.5 4.6 4.5 4.4 4.6 4.2c
aRams allowed a total of 10 minutes with the ewe; rams
mating in 10 minutes received a value of 10.
bMean of three animals only in period 6.
cMean of four animals only in period 6.
dLibido score based on a 1 to 5 scale; 1, no interest;
as described on page 20.
not mounting or
5, mated ewe,
depleted bulls, while a complete lack of nitrogen had little or no
effect on the libido of these rams. Even the four rams that did not
finish the experiment mated the ewe at the last libido check before
they died or were sacrificed.
The obvious physical weakness of the nitrogen-free rams did not
have the same deterring effect on the libido that was observed in the
low protein bulls in Experiment I.
Analysis of Variance mean squares for the various libido criteria
are given in table 28 in the Appendix.
Volume. The mean volume of semen produced by the urea control and
nitrogen-free groups was quite similar until the latter part of the study
when the nitrogen-free group produced slightly smaller amounts. There
was an appreciable drop in volume during period 2, followed by a more uni-
form production for both groups. These changes are shown in figure 19.
The mean for the Drackett control group, on the other hand, fluctuated
widely, being both above and below the other two groups.
Statistical analysis of these data indicated no significant
difference between treatment groups. There was a significantly
greater volume (P < .01) produced in period 1 as compared to the
other periods. Lack of a significant interaction indicated that the
higher volume in period 1 was consistent for all groups. Initial and
final semen volume figures for the three groups were 1.28 and 1.38,
1.85 and 1.12, and 1.45 and 1.12 ml. per ejaculate for the Drackett
control, urea control and nitrogen-free groups, respectively.
- - UREA CONTROL
- -.- NITROGEN-FREE
1 2 5 4 5 6
Effect of level and source of nitrogen on mean semen volume
Semen volume, motility and total cell production figures for
individual rams and treatment groups are summarized in table 22 in
Motility. Mean estimates of the percent motile sperm for the
three groups of rams are shown in figure 20. There was a character-
istic decline for all groups as the experiment progressed. This
decline was due primarily to the progressively colder environmental
temperatures as the experiment went into the winter months. The semen
collections were made in an exposed area and the cold shock had a
marked influence on the motility scores.
In view of this environmental effect, the motility values are
not too meaningful except from a relative standpoint. The average
motility for the nitrogen-free group tended to drop more than that
of the control group, but the differences were not statistically
significant. There was a significant period effect as would be ex-
pected. The motility of all groups was lower during the latter part
of the experiment. A great deal of variation was observed within
rams between collections and within treatment groups for any one
collection. This would tend to preclude assigning any statistical
significance to the apparent differences also.
Total Sperm Cell Production. Subjecting the total sperm cell
production data to a statistical analysis revealed no significant
differences due to treatment or period. Again, the small number of
animals involved and the considerable variation within treatment
groups tend to minimize the treatment effects.
1 2 3 4 5 6
Effect of level and source of nitrogen on mean semen motility
The pattern of total sperm cell production for the groups is
presented in figure 21. While there was considerable fluctuation in
the two control groups, the nitrogen-free rams had a fairly uniform
production. The larger number of animals in this group could explain
part of this uniformity. The total cell production of the urea control
group appeared to be consistently higher than the others and the nitro-
gen-free group generally lower than the two control groups. This was
particularly true during the last part of the study.
The average total cells per ejaculate for the experimental
period for the three treatment groups was 2609, 4660, and 1665 times
106 sperm for the Drackett control, the urea control and the nitrogen-
free rams, respectively. In spite of the lack of statistical evidence,
there appears to be some treatment effect on the total cell production,
particularly when the urea control and nitrogen-free groups are
Analysis of Variance mean squares for the semen characteristics
are given in table 28 in the Appendix.
Microscopic examination of the reproductive organs and tissues
did not reveal any marked differences due to treatments. Measurements
of the epithelial tissue, tubule diameters and secretary areas of the
tissues studied are given in tables 11 and 12. There was some reduc-
tion in the number of layers of germ cells in the seminiferous tubules
of the testes from the nitrogen-free rams. Cellular disorganization
was observed in the seminal vesicles of several of these nitrogen-free
source and level of nitrogen on total cell production
 I, I
QUANTITATIVE EVALUATION OF THE TESTES AND EPIDIDYMIS FROM THE THREE GROUPS OF RAMS
Treatment Epithelium Tubule Inter- Epithelium Duct Sperm
Group and Thickness Diameter Sperma stitialb Thickness Diameter Content
Animal No. (rmm x 10-2) (rmm) Content Tissue (mm x 10-2) (mm)
2 3.0 .10 1 3 6.0 .20 1
5 4.8 .17 4 2 6.0 .28 5
9 6.0 .17 3 1 7.2 .35 5
17 4.2 .13 1 1 6.0 .22 5
Av. 4.5 .14 2.2 1.7 6.5 .26 3.5
4 3.6 .17 4 2 7.2 .28 2
13 3.6 .14 1 2 6.5 .27 4
14 4.8 .13 1 2 7.8 .32 5
19 3.6 .11 1 4 4.8 .28 1
Av. 3.9 .14 1.8 2.2 6.6 .29 3.0
1 4.8 .11 1 3 4.8 .22 1
6 3.6 .10 1 5 4.8 .20 1
7 3.6 .10 1 4 7.2 .28 2
8 4.8 .11 1 5 6.0 .22 1
15 4.8 .12 1 3 6.6 .25 1
16 4.8 .12 1 2 4.8 .24 1
18 4.2 .10 1 4 4.2 .24 1
20 6.0 .12 1 2 6.6 .28 2
Av. 4.6 .11 1 3.2 5.6 .24 1.2
aSperm content scored from 1 to 5: 1, no sperm, and 5, abundant.
bInterstitial tissue scored from 1 to 5; 1, sparse, and 5, abundant.
QUANTITATIVE EVALUATION OF THE SEMINAL VESICLES AND
FROM THE THREE GROUPS OF RAMS
Seminal Vesicles Cowpers Glands
Treatment Epithelial Rate of Epithelial
Group and Thickness Secretory Tissuea Thickness
Animal No. (mm x 10-2) to Total Area (mm x 10-2)
2 1.8 1 1.2
5 1.2 5 1.5
9 5.0 5 1.2
17 5.0 2 1.2
Av. 2.2 2.2 1.3
4 2.1 .2 1.8
15 1.8 1 1.5
14 1.8 2 1.8
19 1.8 2 0.9
Av. 1.9 1.7 1.5
1 1.2 2 .9
6 1.2 1 .6
7 1.8 3 --b
8 1.8 1 1.5
15 2.1 1 --b
16 1.5 2 .6
18 1.8 1 .9
20 1.8 1 .9
Av. 1.6 1.6 .9
aScale is based on values from 1 to 5, 1 being a wide ratio of
secretory tissue to total area and 5 being a narrow ratio.
"Samples lost in processing.
Absence of any major differences in these testes and secondary
glands is not completely without explanation. In fact, it would be
difficult to explain differences had they occurred. Both libido and
secondary sex gland development and activity are controlled by testos-
terone secretion from the testes primarily. Since the nitrogen-free
ration had little or no effect on libido, differences of any magnitude
would not be expected in these glands.
The results here are somewhat different from those obtained in
Experiment I. With the low protein bulls, both libido and histological
differences were observed. Again, it is possible that a species dif-
ference may exist.
The gross weights of these glands and organs were obtained at
time of slaughter. The gross weights, along with the weights per cwt.
of body weight, are summarized by treatment groups in table 13. Very
little difference is noted between treatment groups when the weights
per cwt. of body weight are considered.
The results of the adenohypophysis assay are given in table 14.
The Drackett control and the nitrogen-free groupswere identical in
gonadotrophic activity according to the assay. The urea control group
was slightly lower than the other groups. When the extremely wide
ranges in chick testesweights for any one group are considered, it
must be concluded that there was no real difference between the three
treatment groups in gonadotrophic activity.
SUMMARY OF THE MEAN GLAND AND ORGAN WEIGHTS TAKEN AT TIME OF SLAUGHTER
FROM THE THREE GROUPS OF RAMS
Drackett Control Urea Control Nitrogen-free
Gland or Organ Per Cwt. Per Cwt, Per Cwt.
Mean Body Weight Mean Body Weight Mean Body Weight
Testes 221.97 220.0 168.12 163.00 102.47 163.00
Seminal Vesicle 8.10 8.0 7.70 7.50 4.77 7.22
Cowper's Gland 2.70 2.6 3.22 3.10 1.85 2.80
Pituitary Gland .76 .75 .65 .65 .51 .81
Thyroid Gland 3.62b 5.60 4.07c 4.00 3.77 6.00
aMean of four samples only.
bMean of three samples only.
CMean of two samples only.
This lack of treatment effect is quite comparable with the
lack of treatment effect on libido, reproductive tissue histology and
semen production. The gonadotrophic hormones, Follicle Stimulating
Hormone (FSH) and Leutinizing Hormone (LH), from the adenohypophysis
regulate semen production and testosterone production which in turn
regulates libido and activity of the secondary sex glands.
BIOLOGICAL ASSAY OF ADENOHYPOPHYSES FROM
THE THREE TREATMENT GROUPS OF RAMS
Ram Treatment Groups
aStandard used was unfractionated sheep pituitary powder
obtained from Abbott Laboratory.
GENERAL DISCUSSION AND CONCLUSIONS
Before discussing the effects of the experimental treatments
on the growth and reproductive performance of the bulls and rams, it
is necessary to consider the fact that a confounding multiple defi-
ciency condition existed in both experiments. The depressing effect
of low protein and nitrogen-free rations on feed intake created defi-
ciencies of energy, vitamins, minerals and other dietary factors, as
well as a nitrogen or protein deficiency. It should be stated, how-
ever, that these deficient rations would have supplied the required
amounts of these other nutrients had they been consumed at the same
level as the control rations. The basic cause of this multiple defi-
ciency is therefore, a protein or nitrogen deficiency.
Comparing the effects of the low protein ration on the per-
formance of the bulls in Experiment I with those of the nitrogen-free
ration in Experiment II on ram performance reveals several notable
similarities and several striking differences.
The immediate response in both experiments to a low level or
absence of nitrogen was a pronounced drop in feed intake. As a result
of this reduction in feed intake, losses in body weight rapidly
developed. This response is in good agreement with earlier work by
the author (Meacham, 1960) and that of Guilbert and Hart (1951).
Under the conditions of these experiments, it may be concluded that
low levels or complete absence of nitrogen will result in marked
reduction in feed intake and losses in body weight.
In Experiment II, it appeared the source of nitrogen, whether
it was of a protein or non-protein source, did not have an appreciable
effect on feed intake or weight gains. These findings are not in com-
plete agreement with the work of other investigators (Meacham et al.,
1961, and Oltjen et al., 1959) who reported that a protein source of
nitrogen was more effective than a non-protein source. It is possible,
however, that the conditions of the experiment did not permit full
manifestation of the potential differences.
The experimental rations affected the blood composition of
the bulls and rams to differing extents. Bulls receiving the low
protein ration in Experiment I had significantly lower hemoglobin,
hematocrit and serum protein levels by the end of the experiment than
the control animals. The rams receiving the nitrogen-free ration in
Experiment II did not, on the other hand, differ significantly from
the control rams in these blood components. The nitrogen-free rams
did tend to be lower in all three components by the end of the trial.
The differences in length of experimental periods, 98 days in Exper-
iment II and 182 days in Experiment I, undoubtedly played a part in
this differential response. It should be pointed out, however, that
in both experiments hemoglobin and hematocrit levels for all treatment
groups were near the normal values reported in the literature. The
differences observed in Experiment I were usually due to increases in
the control levels rather than marked decreases in the low protein
group. The serum protein levels at the end of the first experiment
for the low protein bulls were slightly below the normal levels
reported in the literature.
The two sources of nitrogen used in Experiment II were fairly
comparable in maintaining normal blood composition. The urea tended
to be slightly less effective in this respect than the protein source,
but the differences were not statistically significant.
The effect of nitrogen deprivation on libido presented one
of the more striking differences between the two experiments. The low
protein ration in Experiment I brought about a significant decline in
libido whereas the nitrogen-free ration had very little effect on the
libido of the rams in Experiment II. Keeping in mind the small num-
bers of animals involved, there may be a species difference in the
effect of a nutritional stress on the libido of rams and bulls.
This lack of effect on the libido of the rams was confirmed
by a similar lack of differences in the histology of the primary and
secondary sex glands and in the gonadotrophic activity of the adenohy-
pophysis of the three groups of rams. The histological changes ob-
served in the reproductive tissues of the low protein bulls in turn
confirmed the differences observed in the libido of these bulls.
The lack of protein had rather distinct effects on these tissues.
No real difference was obtained between the two sources of
nitrogen in the histology study with the rams. On the basis of these
limited data, it appears that non-protein nitrogen can be effectively
utilized for these reproductive tissues.
A comparison of the semen production in the two experiments
revealed a rather similar treatment response. In both cases, the
deprived group tended to be lower in volume, motility and total sperm
cell production. The differences in volume and total cell production
were significant for the bulls in Experiment I, but not for the rams
in Experiment II. The nitrogen-free rams were sufficiently low by
the end of the study to indicate a possible treatment effect had the
numbers in each group been larger.
The results of these experiments are basically in agreement
with the limited data available in the literature. Mann and Walton
(1953) stated that severe starvation resulted in only reduced volume
of semen per ejaculate in a mature bull. Dutt and Barnhart (1959) and
Steverman et al. (1961) working with boars and Baker et al. (1953) and
Flipse (1957) working with dairy bulls, all reported little effect on
semen characteristics other than semen volume in animals restricted
in feed intake.
In Experiment II, the non-protein-nitrogen had no adverse
effect on any semen characteristic. Moreover, the total cell pro-
duction for this group was generally higher than that of the other
two groups. On the basis of these data, it could be concluded that
the ram is able to utilize non-protein nitrogen adequately for sperm-
atogenesis and semen production.
Two experiments were conducted to study the effect of source
and level of nitrogen on the growth and reproductive performance of
young bulls and rams.
In Experiment I, 12 bulls averaging 390 pounds were allowed
to two groups of four and eight bulls each as they reached puberty.
The four bulls received a ration adequate in all nutrients. The
eight bulls were fed a ration containing 1.61percent CP, but other-
wise adequate. Feed intake, weight changes, blood composition,
semen characteristics, libido and reproductive tissue histology were
The low protein ration depressed appetite severely. Average
daily feed consumption was 9.78 and 3.28 pounds for the control and
low protein bulls, respectively.
At the end of the 182-day experiment, the control bulls had
gained 200 pounds and the low protein bulls had lost an average of
130 pounds. Four of the low protein bulls died or were sacrificed
during the experiment. Average survival time was 151 days.
The low protein bulls had final blood hemoglobin, hematocrit
and serum protein levels significantly (P < .01) lower than the
Libido was adversely affected by the low protein ration. The
low protein bulls had significantly longer intervals to first mating
and lower libido scores (P < .01).
Semen volume (P < .01) and total sperm cells per ejaculate
(P < .05) were significantly lower for the low protein bulls. Sperm
motility was quite varied and not significantly affected by treatments.
Artificial insemination of heifers revealed no treatment effect
on semen fertility.
The semen exhaustion study indicated reduced spermatogenic
capacity in the low protein bulls. The controls produced greater
volumes of semen containing greater numbers of sperm.
Histological investigation of the reproductive organs and
glands revealed marked differences in size and development. The thick-
ness of the epithelium in the glands and organs of the low protein
bulls was greatly reduced. Tubule diameters in the testes and epididy-
mis were similarly reduced. The control bulls tended to have greater
amounts of interstitial tissue.
In Experiment II, 16 crossbred rams weighing 99 pounds were
assigned to three purified rations. Two groups of four rams each
received control rations, one containing protein-nitrogen and the other
non-protein-nitrogen. Eight rams received a nitrogen-free ration.
The experimental period was 98 days. The same criteria studied in
Experiment I were studied in Experiment II.
The nitrogen-free ration brought about marked decreases in
feed intake. Average intakes were 1.60, 1.46 and 0.61 pounds daily
for the Drackett control, urea control and nitrogen-free rams,
ADG were 0.09, 0.06 and -.58 pounds for the Drackett control,
urea control and nitrogen-free groups, respectively. Four of the
nitrogen-free rams died or were sacrificed during the experiment.
Mean survival time was 78 days. One urea control ram died after
Blood components were not significantly affected by the treat-
ments. All groups were within the normal ranges.
The libido criteria were not significantly different for any
of the treatment groups.
Semen volume, motility and total sperm production were not
affected significantly by the treatments. The urea control group
tended to have the higher total sperm production average and the
nitrogen-free group the lower average.
Histological examination of the reproductive tissues revealed
very little treatment effect. There was some reduction of epithelium
in the seminiferous tubules of the nitrogen-free ram testes.
The gonadotrophic activity of the adenohypophyses of the rams
was not affected by the treatments.
The differential response to the deficient rations of the bulls
and rams was due to the shorter experimental period for the rams, 98
versus 182 days. The depleted rams would die before libido, blood
components and tissue histology were affected.
Armsby, H. P. 1917. The Nutrition of Farm Animals. Macmillan Book
Company, New York, N.Y.
Association of Official Agricultural Chemists. 1950. Official Methods
of Analysis. Eighth Edition.
Baker, F. N., N. L. Van Demark and G. W. Salisbury. 1955. A year's
study of the semen characteristics and libido of young bulls
subjected to various frequenciesof ejaculation. Journal of
Animal Science, 12:942.
1955a. Growth of Holstein bulls and its relation to sperm
production. Journal of Animal Science. 14:5 75.
1955b. The effect of frequency of ejaculation on sperm
production. Journal of Dairy Science. 38:1000.
Bedrak, E. 1958. Effect of protein intake on weight changes, blood
constituents and reproduction in beef heifers. Ph.D. disser-
tation. Department of Animal Husbandry, University of Florida.
Berg, C. P. and W. G. Rohse. 1947. Is sterility induced in growing
rats on a tryptophan deficient diet? Science. 105:96.
Blaxter, K. L. and H. H. Mitchell. 1948. Protein requirements of
ruminants. Journal of Animal Science. 7:25.
Blincoe, C. and S. Brody. 1951. Environmental physiology with special
reference to domestic animals. XVII. The influence of tem-
perature on blood composition of cattle. University of Missouri
Research Bulletin No. 488.
Blucker, J. D. 1952. Mineral and management studied with range beef
cattle. Master of Science Thesis, Oklahoma Agricultural and
Mechanical College, Stillwater, Oklahoma.
Branton, C., R. W. Bratton and G. W. Salisbury. 1948. Semen produc-
tion and fertility of dairy bulls fed rations containing
proteins of plant and animal sources. Journal of Dairy