ENDOCRINE AND REPRODUCTIVE CHANGES IN DAIRY HEIFERS AS AFFECTED BY GROWTH RATE AND MELENGESTROL ACETATE Thesis for the Degree of Ph. D. MICHEGANSTATE UNIVERSITY DONALD EDWARD PRITCHARD 1970 LIBRARY rues": Michigan State University This is to certify that the thesis entitled Endocrine and Reproductive Changes in Dairy Heifers as Affected by Growth Rate and Melengestrol Acetate presented by Donald Edward Pritchard has been accepted towards fulfillment of the requirements for Ph.D. degree in Dairy fiwg {3M Mprofess Date December 22, 1969 0-169 Imolua av " ‘ me a sans f. ET 800K atrium me ' ‘ was“ among T mum“. IIIIAI _ ABSTRACT ENDOCRINE AND REPRODUCTIVE CHANGES IN DAIRY HEIFERS AS AFFECTED BY GROHTH RATE AND HELENCESTROL ACETATE BY DONALD E. PRITCHARD This studv was conducted to determine the effects of a normal and hioh level of nutrition alone or with the synthetic nroqestanen melenoes- trol acetate (VGA) on body orowth, levels of certain anterior pituitary hormones in the pituitary and blood, develonment of the reproductive tract and mammary oland, and subsequent reproductive and lactational performance of l40 holstein heifers. Heifers were raised under uniform conditions from 2 weeks to 2.5 months of aqe at which time they were ran- domly assigned to 14 groups consisting of lo heifers each. MGA was fed beoinninq at 2.5 months of age or after first estrus. One hundred heifers were slauqhtered either at 2.5 months of age, at first estrus. or at breeding size, while 40 heifers fed a roughaqe ration only between prea- nancy diagnosis and parturition were kept to obtain data on breedino and lactational performances. Heifers fed the hioh level of nutrition exhibited first estrus at a significantly vounoer (P<0.0l) ane than those fed the normal level (7.5 :_ o.l vs 8.7 :_0.2 months), but there was no sionificant difference (P>0.10) in body weight (255 i 4 vs 250 i 5 kn) or withers heiqht (l08.6 i 0.6 vs l09.2 :_0.7 cm) at first estrus. These data emphasize that first estrus is Donald E. Pritchard associated more with physical size than with calendar age. At breeding size (l20 cm withers height), heifers fed the high level of nutrition were ll.4 i.0'4 months old while those fed the normal level were l2.5 :_0.2 months old (P<0.0l). ”GA fed with either the normal or high levels of nutrition at the rate of 0.45 mg per heifer per day did not significantly afféct the ages at breeding size, indicating that MGA did not affect skeletal growth. However, HGA increased body weight gains, but only when fed with the high level of nutrition (P<0.0l). Heifers fed the high level of nutrition with FDA gained faster (P<0.05) after about 5.5 months of age than heifers fed the high level alone. The time from first estrus to breeding size (about 3.5 months) was not significantly different (P>O.l0) for heifers fed the two nutritional levels without or with NRA, indicating that level of nutrition or addition of MFA did not affect rate of skeletal growth after first estrus. Uterine weights, nucleic acids concentrations, and epithelial cell heights were not affected bv level of nutrition, but these parameters indi- cated that uterine hypertrOphv was associated with MFA feeding. Ovarian weights were not affected by nutritional level, but more large diameter fellicles were present on the ovaries of heifers fed HGA. Level of nutri- tion had no effect on narenchvmal tissue weights or nucleic acids concen- trations and contents of the mammary gland. MGA did not affect mammary parenchymal tissue weights but caused significantly greater (P<0.0l) concentrations and contents of nucleic acids in the heifers at breeding size. Paired adrenal weights were not significantly different (P>0.l0) fer groups fed the two levels of nutrition without or with ”GA. However, at breeding size, MGA caused a significant decrease (P<0.05) in the width of the glucocorticoid producing fasciculata zone of the cortex. Ho large differences in pituitarv weights or pituitary or plasma concentrations of Donald E. Pritchard LH, FSH, and prolactin resulted at first estrus or breeding size from feeding the two levels of nutrition without or with ”CA. In all treatment groups, correlation coefficients between pituitary concentration and plasma concentration of LH and prolactin, and between pituitary content and plaSma concentration of these two hormones were not significant (P>0.05). The interval between MGA withdrawal and occurrence of estrus was considerably longer, though not significantly different (P>0.l0). for heifers fed MGA from 2.5 months than fbr those that received MGA after first estrus only (l9.7 vs 7.7 days). However, once estrous cycles commenced thev were of normal length (l7-24 days) fer all HGA treated animals. while heifers fed the high level of nutrition without or with MCA were younger at breeding size than those fed the normal level with- out or with MGA, there were no significant differences (P>0.l0) among treatment groups in ages at conception or services required per conception. At parturition, the level of nutrition or ”CA fed prior to conception produced no significant differences (P>0.l0) in body weights or withers heights of the dams, birth weights of the calves, or in the subjective dystocia ratings. Birth weights of the calves sired by the two bulls were not significantly different (P>0.l0). There were no significant differences (P>0.lO) between the treatment groups in actual milk production weights fer the first 60 days of lactation or extended 305 day milk production values. ENDOCRINE AND REPRODUCTIVE CHANGES IN DAIRY HEIFERS AS AFFECTED BY GROWTH RATE AND MELENGESTROL ACETATE By Donald Edward Pritchard A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy l970 Dedicated to my grandfather, Oliver Edward, and to my father, Earl Stanley, whose dreams and sacrifices encouraged and permitted me to obtain my college education. ii IT'S ALL IN THE STATE OF MIND If YOU think you are beaten, you are; If you think you dare not, you don't; If you think you'd like to win, but you can‘t; It's almost a "cinch" you won't; If you think you'll lose, you've lost; For out in the world you'll find Success begins with a fellow's will - It's all in the state of mind. FULL many a race is lost Ere even a race is run, And many a coward fails Ere even his work's begun. Think big and your deeds will grow Think small and you fall behind. Think that you can, and you will; It's all in the state of mind. If YOU think you are outclassed, you are; You've got to think big to rise; You've got to be sure of yourself before You can ever win a prize. Life's battle doesn't always go To the stronger or faster man; But sooner or later, the man who wins Is the fellow who thinks he can. Author Unknown. iii AUTOBIOGRAPHICAL SKETCH of Donald Edward Pritchard Born in Aurora, Illinois, on March 9, l942, I grew up on a dairy- grain farm about one hour‘s drive west of Chicago, Illinois. My primary and secondary education was obtained from the Hinckley-Big Rock Consolidated School District. I attended the University of Illinois from September, 1960, to Harch, 1950, and received a 8.5. and H.S. in dairy science during that time. In Harch of 1966 I was awarded a graduate research assistantship bv the Department of Dairy, Hichigan State University. This position allowed me to study for my Ph.D.. which was completed in December, l9b9. iv ACKNOWLEDGMENTS In acknowledging all those who have assisted me during my Ph.D. program, I first must thank the Department of Dairy here at Michigan State University. Without its interest and financial assistance, my studying for a doctoral degree would not have been possible. My adviser, Dr. Louis Boyd, and Dr. Harold Hafs both receive my sincerest appreciation for their advice, support, assistance, and encouragement. Were it not for them, I would have terminated my program the first year. I also thank Drs. Paul Reineke, John Gill, and Karl Wright for their advice and guidance, and for serving on my graduate committee. My research program required the assistance of numerous people. Dr. R. G. Zimbelman and his staff in the TUCO products division of the Upjohn Company were most generous in helping to finance the study and in supplying the melengestrol acetate. Mr. Dennis Armstrong, the M. S. U. dairy herd manager, was most helpful in procuring the heifers and in providing the facilities and labor to house and care for them. Drs. H. A. Tucker and J. T. Huber deserve recognition for the many hours of advice and assistance they rendered in conducting various aspects of the study. And the technical assistance and loyal dedication of Marianne Holfelner is appreciated. I am also indebted to my student contemporaries for their advice and many hours of assistance provided in taking the monthly body measurements, observing the heifers for estrus, and slaughtering the heifers. Members of the group included Roger Purchas, Bill Thatcher, A”: Hackett, Robert Nettemann, Lloyd Swanson, Jim Koprowski, Linda Miller, V Mayne Oxender, and Dean Peterson. The gifts of purified LH and the antibody to LH from Drs. Hiswender and Hidglev, and the hormones from Sguibb Laboratories and the Endocrin- ology Study Section of the National Institutes of Health are rec0gnized and appreciated. vi TABLE OF CONTENTS Dedication It's All In The State Of Mind . Autobiographical Sketch . Acknowledgements List of Tables . List of Figures. List of Appendices. INTRODUCTION REVIEH 0F LITERATURE . A. Growth Rate of Dairy Heifers. Influence of Nutritional Level. Effect of Gonadal Steroids . Effect on Age At Puberty. . Effect on Conception and Dystocia. Ohm-th-J Performance . B Reproductive Tract Development . 1. Changes Associated with Nutrition and Age . 2. Effects of Ovarian Steroids. . . C. Mammary Gland Development. l. Influence of Nutritional Level. 2. Changes Associated with Age. 3. Effects of Steroids D. The Adrenal Glands. l. Structure and Function vii Effect on Subsequent Lactational Performance : Effect of Age at First Calving. on Lactational Page ii iii iv xii xiii domain 4> Ad l2 l4 l4 IS 18 l8 T9 20 2l 2l E. 2. Effects of Ovarian Hormones The Gonadotropins and Prolactin 1. Concentrations Associated with Age . 2. Effects of Ovarian Steroids MATERIALS AND METHODS. an> E. F. Experimental Design . . Management of Experimental Animals Slaughter Procedures . . . Assays of Anterior Pituitary Hormones l. Homogenization of the Anterior Pituitaries . 2. Follicle Stimulating Hormone (FSH) Bioassay. 3 Prolactin Radioimmunoassay. . 4. Luteinizing Hormone (LH) Radioimmunoassay. Determination of Nucleic Acids in the Uterus and Mammary Gland. . Histological Technique RESULTS AND DISCUSSIONS . A. Body Growth . 1 From 2 Weeks to 2.5 Months. . . 2 From 2.5 Months to First Estrus . . 3. From First Estrus to Breeding Size . 4 Body Size at Slaughter . . . Reproductive Tract Changes . l Uterine Weight. 2 Uterine Nucleic Acids . 3. Uterus Epithelial Cell Height 4 Ovarian Changes . Mammary Gland Changes l. Mammary Gland Weight. . 2. Mamnary Gland Nucleic Acids Adrenal Gland Changes. l. Adrenal Weights . . 2. Adrenal Cortex Histology Changes in the Pituitary Weight and Hormones . l. Pituitary Weight . viii Page Page 2. Hormones in the Anterior Pituitary. . . . . . 76 3. Hormones in the Blood Plasma . . . . . 79 4. Plasma to Pituitary Hormone Content Ratios . . 82 5. Correlations Between Anterior Pituitary and Plasma Hormone Values. . . . . . . . . . . . . 84 F. Data on the Bred Heifers. . . . . . . . . . . 85 l. Estrous Cycle Data . . . . . . . . . . . 85 2. Breeding Data . . . . . . . . . . . . . 86 3. Parturition Data . . . . . . . . . . . . 90 4. Calving Data by Sire. . . . . . . . . . . 92 5. Milk Production Data. . . . . . . . . . . 92 G. Nitrogen Balance Trials . . . . . . . . . . . 95 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 99 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . 104 APPENDICES . . . . . . . . . . . . . . . . . . llZ ix LIST OF TABLES Table Page 1. Experimental treatments beginning at 2.5 months of age for T40 Holstein heifers. . . . . . . . . . . 29 2. Description of nutrition levels . . . . . . . . . 30 3. Composition of experimental grain mixes. . . . . . . 30 4. Growth from 2 weeks to 2.5 months of age for Holstein heifers . . . . . . . . . . . . . . . . 43 5. Growth to first estrus for Holstein heifers fed differ- ent nutrition treatments. . . . . . . . . . . 44 6. Growth to first estrus for Holstein heifers fed differ- ent nutrition treatments but not slaughtered at first estrus. . . . . . . . . . . . . . . . . 47 7. Growth to breeding size (120 cm withers height) for Holstein heifers fed different nutrition treatments. . 49 8. Body weights by age for Holstein heifers fed differ— ent nutrition treatments but not slaughtered at first estrus. . . . . . . . . . . . . . . 5] 9. Withers heights by age for Holstein heifers fed differ- ent nutrition treatments but not slaughtered at first estrus. . . . . . . . . . . . . . . 54 lo. Age and body size at slaughter of Holstein heifers fed different nutrition treatments. . . . . . . . . 56 ll. Body size by age for Holstein heifers fed different nutrition treatments and slaughtered at first estrus. . . . . . . . . . . . . . . . . 57 l2. Uterine measurements of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months or first estrus. . . . . . . . . . . . . . 60 I3. Uterine measurements of Holstein heifers fed different nutrition treatments and slaughtered at breeding size . . . . 51 Table Page 14. Ovarian measurements of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size. . . . . . . . . 65 l5. Mammary gland measurements of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size. . . . . 67 16. Adrenal gland measurements of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size. . . . . 7T 17. Pituitary weights of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size. . . . . . . . . 75 TB. Hormones in the anterior pituitary of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size. . . . . 77 19. Hormones in the blood plasma of Holstein heifers fed different.nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size. . . . . 80 20. Plasma to pituitary hormone ratios of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size. . . . . 83 2l. Age and body size at breeding size and first breeding of Holstein heifers fed different nutrition treat- ments . . . . . . . . . . . . . . . . . 87 22. Age and conception data for Holstein heifers fed differ- ent nutrition treatments. . . . . . . . . . . 88 23. Some observations at parturition of Holstein heifers fed different nutrition treatments prior to conception . . 9T 24. Calving data by sire . . . . . . . . . . . . . 93 25. Milk production data of Holstein heifers fed different nutrition treatments prior to conception . . . . . 94 26. Nitrogen balance data of Holstein heifers fed different nutrition treatments . . . . . . . 97 xi LIST OF FIGURES Figure l. Body weights by age for Holstein heifers fed different nutrition treatments but not slaughtered at first estrus. . . . . . . . 2. Photographs at breeding size of Holstein heifers fed normal level, high level, and high level plus MGA nutrition treatments from 2.5 months of age 3. Rear view photographs of mammary glands on Holstein heifers at breeding size that were fed normal level, high level, and high level plus MGA nutrition treat- ments from 2. 5 months of age . . . xii Page 52 55 7O LIST OF APPENDICES Appendix I. II. III. IV. Age, body weight, and withers height of individual heifers at 2.5 months, first estrus, breeding size, first breeding, or slaughter . . . . . . . Weight, nucleic acids, and cell height of the uterus, and ovarian weight and number of follicles fer individual heifers . . . . . . . . . . . . Height and nucleic acids of the mammary gland, weight and cortex zone widths of the adrenals fer indivi- dual heifers . . . . . . . . . . . . . . Pituitary weight and anterior pituitary levels of LH, FSH, and prolactin; plasma levels of LH and prolac- tin, and plasma to pituitary content ratios for LH and prolactin fer individual heifers . . . . . . Age at first breeding and conception, number of services, body size at parturition, dystocia rating, calf weight, sire,and sex, and first lactation milk yield fbr individual heifers . . . . . . . . . . . xiii Page ll3 l27 l37 I47 l62 INTRODUCTION During this century mankind has made tremendous progress in all facets of science. Industrial production has reached levels that at one time were unimaginable. Space technology has now attained man's age old dream of flying to the moon. Eradicating many diseases and controlling others that have plagued man for centuries have extended our life expectancy by decades. Equally important as these areas is the progress that has occurred in the agricultural industry. Mechanized farming, croo yields per acre, production per animal, and output per man make the agriculture of yester-years appear strangled with inefficienty. Because of gigantic advancements in food production, spurred by Land Grant universities, approximately 95 percent of our population is free to pursue other industrial and technical endeavors. Dairymen have also shared in the benefits of science. Besides having automated equipment for handling the feed and waste products, milk is now removed from the cow, transported, processed, and never exposed to the air until the consumer pours it from a container. Feed additives which provide a nonprotein source of nitrogen and estrous cycle control are examples of progress in dairy production. And surely artificial insem- ination and the use of frozen semen have been a real bonanza to genetic progress. We now know how to feed cows, how to milk cows, how to manage cows, and how to breed cows to increase their milk producing ability. But l 2 while these research findings and applied procedures have been filtered out to dairymen at various rates over time, little progress has resulted in having dairy heifers freshen at an earlier age. Official records show that, on the average, heifers today are about 30 months of age at first parturition, which is a few months older than what Eckles found in a 1915 survey. Many dairymen still feed and breed their heifers in the same manner they did many years ago. This means that heifers are about two and a half years old before they become a productive unit. Needless to say, this time period is wasteful and costly. It should be reduced by feeding heifers so they will grow faster and breeding them according to body size rather than age. Recent studies by Sinha (1967) indicate that mammary gland develop- ment, as measured by deoxyribpnucleic acid (DNA) determinations, was about the same for both 9- and 16-month old heifers. This finding suggests that at least in terms of maximal mammary development before conception, there would be no advantage to delay breeding of heifers beyond 9 months of age. However, Desjardins (1966) and Hackett (1968) found that uterine nucleic acids approximately doubled between 12 and 17 months of age in Holstein heifers. This fact suggests that the repro- ductive tract of lZ-month old Holstein heifers is not fully developed and perhaps not completely ready to support pregnancy. However, epithelial cell heights of all portions of the tubular genitalia did not change greatly between the two ages and this characteristic may be more important than the DNA changes. This finding on epithelial cell heights implies that the reproductive tract of 12-month old heifers may be capable of supporting pregnancy successfully. Thus, armed with the desire to have heifers calve sooner and the previous observations on mammary gland and reproductive tract development 3 as an incentive, this study was initiated. My immediate purposes were to determine if Holstein heifers could grow to the usual breeding size (320-385 kg) by 12 months of age, and to study the physiological effects of rapid growth on certain endocrine, mammary gland, and reproductive tract changes at the beginning, at puberty, and at the end of this growing period. My ultimate aim was to determine if growing heifers as fast as possible and then breeding them according to body size rather than age is a feasible and practical approach to raising herd replacements. With these goals driving me, I enthusiastically proceeded to conduct this study, a combination of basic and practical research. REVIEW OF LITERATURE A. Growth Rate of Dairy Heifers 1. Influence of Nutritional Level Numerous studies have been conducted to determine the influence of underfeeding and overfeeding on growth rate of dairy heifers. The work of Eckles (1915), which was later expanded upon by Eckles and Swett (1918), is classic in this field. Eckles divided 4O heifers into two groups; one of which received a heavy ration from birth to first calving and the other group a light ration. The heavy ration consisted of whole milk during the first 6 months and all the grain and hay the animals would consume up to first calving. The light - fed group received skim milk during the first 6 months, and hay or pasture only from that age to first calving. His conclusions are generally still acceptable today: a) the heavy ration accelerated skeletal growth, especially during the period of most rapid development; b) later in the growing period , heifers receiving the heavy ration became excessively fat; c) the animals receiving the light ration grew less rapidly',but continued growing for a longer period of time; and d) the level of nutrition fed growing heifers had a greater effect upon body weight than upon the rate of skeletal growth. Reed et. a1., (1924) compared an all roughage ration with roughage plus grain for heifers after 6 months of age,and found satisfactory development of Holstein heifers only when grain was included in the ration. Herman and Ragsdale (T946) overfed growing heifers and noted 4 5 that they were characterized by a heavy, course build which in dairy heifers is objectionable and costly. Hansson (1956),in a series of experiments in Sweden, fed heifers at levels ranging from 51 to 124 per- cent of the Swedish normal feeding program. Heifers receiving the highest levels of feeds gained more than twice as much as the heifers fed the lowest levels during the period of l to 19 months of age. The great retardation in rate of growth of heifers on the extremely low level of nutrition had no serious effect on growing capacity after the level of feeding was increased. Crichton et. al., (l959,1960a)fed heifers for 44 weeks on either a high or low plane of nutrition, then reversed the nutrition level on half of the heifers in each group until 2 months before parturition. In heifers kept continuously on the restricted nutritional level, they noted that late maturing characters such as live weight and heart girth were affected most while height and length which are earlier maturing charac- ters were affected least. Height was more affected than length in the heifers which had their rations reversed from a high to a low nutritional plane. Using identical twin heifers, Swanson (1957,1967) has for several years studied the effects of nutritional level on growth. His findings concur with those of other investigators who have used unrelated animals. Cornell workers raised dairy heifers at various nutritional levels in an extensive study of the causes and prevention of reproductive failures in dairy cattle. Sorensen et_gl;, (1959) and Reid gt_gl;, (1964) reported on the growth rate of heifers included in the experiment. The low and high levels of feed consumption were 61 and 129 percent of the medium level which amounted to 93 percent of the total digestible nutrients recommended by Morrison (1956). After 80 weeks. heifers on 6 the low nutrient level weighed about 350 pounds less and were about 10 and 30 centimeters shorter in height and length, respectively, than heifers on the medium nutrient level. Meanwhile, heifers on the high nutrient level were only about 200 pounds heavier and 3.5 centimeters taller and longer than the medium level group. These findings indicate that at feeding levels used in this study, low nutrient intake retards growth more than high nutrient intake accelerates it. One of the most recent studies evaluating the influence of nutrition level on growth in Holstein heifers was reported by Gardner and Garcia (1966). Starting at 6 weeks of age, 24 heifers were fed grain and alfalfa hay free choice, while 24 control heifers were limited to II pounds grain per day and alfalfa hay free choice. All heifers were changed to roughage only after pregnancy verification. Heifers fed grain free choice grew 40 percent faster than controls in body dimensions. Evidence from this study tends to negate the notion that heifers fed rations of high caloric value utilize excess calories for fattening rather than growth. Host of the studies cited in this section were reviewed in detail by Schultz (1969). 2. Effect of Gonadal Steroids To my knowledge, no one has attempted to stimulate growth in dairy heifers with gonadal steroids. So the literature review which follows de- scribes the growth stimulatory effects of gonadal steroids in beef cattle. From such studies one can obtain indications of the responses that might result if dairy heifers were given the gonadal steroids. An excellent review was presented by Casida gt_al:, (1959) in a publication by the National Research Council (NRC) of the National Academy of Sciences. 7 The following evidence on the effects of testosterone and the estrogen- like compounds was obtained from this NRC publication. Use of testosterone to stimulate growth in heifers has proven to be effective only in certain studies. Apparently,intramuscular injections of about 1 mg per kg of body weight per week are required to cause an increase in feed efficiency and rate of gain. The detriments to using testosterone in heifers are that it produces a marked masculine behavior and appearance, effective results require intramuscular administration, and the cost per animal is greater than for the synthetic estrogens or progestagens. The estrogen-like compound used most often as a growth promoter has been diethylstilbestrol (DES). Although it is most effective in steers, it does increase weight gain in heifers by about 0.01 to 0.35 pounds daily. Other orally active estrogens which have been used are dienestrol and hexestrol. They increase rate of gain to approximately the same degree as DES. Estrogens, like testosterone, also improve feed efficiency. Despite the beneficial effects of estrogens, they do cause undesirable effects. Relaxation of the lumbar ligaments, producing the typical nymphomaniac stance, is objectional to many cattlemen. Furthermore, extreme hyperemia and swelling of the external genitalia, an increased incidence of vaginal prolapse, and mammary development and teat growth nay result from the estrogens. While numerous studies have been conducted with various estrogens and androgens to improve the performance of feedlot heifers, little attention had been given until the early 1960's to the possible use of progestagens. Perhaps this is because the progestagens were not consi- dered anabolic. Only recently have potent and orally active synthetic progestagens become available for growth promotion. Raun et al., (1965) 8 were among the first to study the effects of a synthetic progestagen on growth in cattle. In their study, heifers fed chlormadinone acetate (CAP) gained 13.3 percent faster than the control heifers. Bloss et_914, (1966) obtained significantly greater weight gains and feed efficiencies from feeding 0.35 to 0.50 mg melengestrol acetate (MGA) daily to beef heifers. Burroughs gt_§l;, (1966) found that MGA improved live weight gains by 15 percent over controls. But, Newland and Henderson (1966) and Young gt_gl;, (1969) reported no beneficial effects on growth rate from feeding MGA. Still, the unpublished summaries of over 100 trials conducted by the Upjohn Company, the developer of MGA, in cooperation with universi- ties and feedlots throughout the country show an increase of at least 10 percent in weight gain by MGA heifers over controls (Zimbelman, 1968). These findings lend credence to the effectiveness of the progestogen as a growth stimulant. 3. Effect on Age at Puberty That age at sexual maturity is influenced to a considerable extent by the ration is an accepted fact among animal husbandrymen (Casida, 1959, Reid, 1960). Eckles (1915) noted that heifers receiving a heavy ration mature sexually at an age from 2 to ‘4 months younger than those receiving a light ration. In his study, heavy fed Holstein heifers had their first estrus at an average age of 8.7 months, while light fed heifers exhibited first estrus at 12.4 months of age. These ages are somewhat younger than those observed by other workers. Reed gt_gl;, (1924) observed first estrus at 18.5 months for heifers fed an all forage ration from 6 months of age, while heifers that received grain in addition to forage exhibited first estrus at 13 months of age. The heifers of Hansson (1956) that were fed 43, 62, 81, or 119 percent of 9 the normal growing feed requirements, exhibited first estrus at 13.3, 12.5.10.9 and 10.6 months of age, respectfully. Feeding the normal ration allowed first estrus to occur at 10.4 months of age. In the study of Crichton gt_al;, (1959), heifers fed the high plane of nutri- tion exhibited first estrus when 12.4 months old, while the low plane heifers were delayed until 15.8 months. The group that was switched from a low to a high nutrition plane at 44 weeks of age showed first estrus at 14.7 months, whereas the group that went from the high to the low level was retarded to 18.4 months. It is interesting to note that going from a high to a low nutrition level slows the attainment of sexual maturity more than does a continual low nutrition level. Sorensen gt_§l;, (1959) found striking differences in the average age at first estrus in heifers fed three levels of nutrition. Fifteen heifers on the high feeding level came into estrus at 8.7 months of age, whereas 10 heifers on the medium feeding level averaged 11.4 months. Only 3 of the 5 heifers on the low feeding level showed estrus before they were slaughtered at 80 weeks of age, and they averaged 15.5 months of age. Gardner and Garcia (1966) increased growth rate through accelerated feeding to the extent that the heifers exhibited first estrus when they were 7.7 months old. Control heifers were 9.7 months old at first estrus. Desjardins (1966) fed 24 heifers for a normal growth rate and observed first estrus at an average age of 6.9 months. Since there is such variation in age at first estrus among the studies cited, it is apparent that differences in nutrition levels and /or accuracy and method of detecting first estrus existed among the experiments. It is important to remember, as Sorensen gt;al;, (1959) stated, that there is more of a tendency for heifers to come in first estrus at a given skeletal growth rather than at a certain weight or age. 10 4. Effect on Conception and Dystocia Few studies using different nutrition levels for rearing heifers have reported the effects on conception and dystocia. Reid (1960) cited a New Zealand report which stated that heifers fed a high level of energy while growing required more services per conception than heifers on a low nutritional plane. He also stated that in the Cornell study the percentage of heifers conceiving at first service was 79, 68, and 58 for the low, medium and high nutritional levels, respectively. Thus, this study and others cited by him suggest that nutrition level affects either fertilization rate or embryonic mortality. However, other studies have shown no difference attributable to feeding level on conception rate (Eckles, 1915, Reed gt_gl;, 1924. Joubert, 1954, Reid gt_al;, 1964, Hibbs and Conrad, 1965, Gardner and Garcia, 1966). The number of services required per conception in all of these studies ranged from about 1.0 to 2.0. Since the literature contains differing opinions, no conclusion can be made on the nutrition level effect on conception. It is not possible or correct to relate feeding level during the growing period to dystocia at first parturition. Rather, it is more a matter of relating size of heifer at parturition to dystocia. Heifer size at parturition may, in turn, be related to feeding level during rearing. It is well established that if a heifer does not have sufficient skeletal size at parturition, she will encounter a certain degree of dystocia (Wickersham and Schultz, 1963, Swanson and Hinton, 1964, Reid_et_al:, 1964, Hibbs and Conrad, 1965, and Gardner and Garcia, 1966). Thus, it is important that heifers be fed adequately before 11 parturition to ensure the skeletal growth necessary to eliminate or minimize dystocia. 5. Effect on Subsegyent Lactational Performance Ultimately, the ability of a dairy heifer to produce milk (and progeny) determines her value. Thus, if factors other than genetic potential, such level of nutrition during the growing period, influence milk production they should be considered by dairymen. Eckles (1915) considered heifers receiving a heavy ration until first parturition to be slightly inferior in milk production to those receiving a light ration. Turner (1932) concluded that the most efficient milk production would be obtained by breeding heifers to calve at 20 to 24 months of age. This would mean that heifers should be fed so they would grow large enough by parturition to minimize calving problems. In a study by Herman and Ragsdale (1946), milk production of heifers which received the "rapid growth ration" until parturition was disap- pointing to them and remained so for the second and third lactations. Swanson and co-workers in a series of papers (Swanson and Spann, 1954, Swanson, 1957, 1960, 1967, Swanson gt_al;, 1967) concluded that fattened heifers or heavy feeding until first parturition will result in lower milk production for the first two lactations than that of normal and light fed animals. In their studies, the light fed heifers produced the most milk. Hansson (1956) also found that as the level of feeding until first parturition increased from 60 to 80, 100, 120, or 140 percent of the normal recommended level, the average yield of 4 percent fat corrected milk (FCM) for all lactations declined. Those heifers reared at the 12 60 percent feeding level were actually the best milk producers. Crichton et_gl;.(l960b)raised heifers at a high or low nutrition level for the first 44 weeks of age, at which time half of each group was switched to the other nutrition level. They found that feeding these rations until first parturition resulted in no differences in milk production among the groups over 3 lactations. This finding does not agree with Swanson's contention that overfeeding during the growth period results in a lowered level of production. But, it supports his finding that heifers reared on below standard feed levels milk just as well as control and heavy fed heifers. Reid gt_al;, (1964) also found no differences in milk production during the first four lactations of Icows reared on a low, medium, or high nutrition level until first parturition. Gardner and Garcia (1966) found that heifers fed for accelerated growth until conception produced about 2200 fewer pounds of milk than the controls during the first lactation, but both groups produced at the same level in the second lactation. These first lac— tation results could be a result of age at calving as discussed in the next section; the accelerated heifers were 19.7 months and the controls were 36.7 months old. Reviews of this topic are presented by Burt (1956) and Schultz (1969). 6. Effect of Ag; at First Calving on Lactational Performance The effect of age at first calving upon subsequent lactational performance has been studied for several years. Eckles (1915) was one of the first to make such a study. He found that Jerseys and Holsteins bred to calve at 20 to 24 months of age produced slightly less milk and butterfat in the first lactation than heifers bred to calve at 30 to 34 l3 months of age. Turner (l932) examined official breed association records for age at first parturition and the subsequent production yield. He found an increase of about l400 pounds in average yearly milk yield of Holstein heifers calving at 30 months of age as compared to 24 months. Delaying first calving until after 30 months of age resulted in practi- cally no additional increase. However, Turner stated that because of the additional costs incurred by delaying first calving beyond 24 months of age, the most efficient milk production would be obtained by breeding heifers to calve at 20 to 24 months of age. Nickersham and Schultz (l963) noted that the average first lactation (305 day, 4% FCM) yields of heifers which calved at about 20, 24, and 28 months of age were not significantly different, although the oldest age group produced about l500 pounds more milk. Hibbs and Conrad (l965) and Gardner and Garcia (1966) also found that heifers freshening at about 20 months of age produced less milk the first lactation than heifers which were about 27 months old at first calving. Thus, the findings of these researchers show most conclusively that as the age at first freshening increases up to about 30 months, the first lactation yield also increases. However, in evaluating the effects of early calving on production, a truer picture is obtained if lifetime production is examined rather than production during the first lactation only. A l953 English Milk Marketing Board study, as cited by Salisbury and VanDemark (l96l), showed that after five lactations there was little difference in total milk production between heifers freshening for the first time at 24 or 36 months of age. Chapman and Dickerson (l936) and Hansson (l94l, as cited by Salisbury and VanDemark, l96l) determined the amount of butter- fat produced to a specified age and found the cows calving at an early age produced considerably more than those that calved at an older age. 14 And Salisbury and VanDemark (l96l) presented lifetime milk production data showing that the later-calving cows never catch up with the earlier calversin total milk produced to any particular age. From these studies it appears quite conclusive that although earlier calving heifers pro- duce less milk their first lactation, total yield during their productive life, or to any specified age, will be greater than that for later calving heifers. 8. Reproductive Tract Development l. ChanggsAssociated with Nutrition and Agg_ Sorensen gt_gl;. (l959) slaughtered Holstein heifers at l, l6, 32, 48, 64, and 80 weeks of age after they had been on either a low, medium, or high nutritional plane. The most striking changes in the reproductive organs were those that took place in the uterus at puberty. The weight of the uterus and the length of the oviducts, uterus, and vagina increased greatly at about the time of first estrus. These increases occurred between l6 and 32 weeks in the high plane heifers, 32 and 48 weeks in the medium plane heifers, and 48 and 64 weeks in the low plane heifers. Once estrous cycles were initiated, uterine growth continued at a slower rate in all groups. As expected, the degree of uterine epithelial develop- ment reflected the degree of sexual maturity. At a given age, the high plane heifers had the thickest endometrium and most endometrial glands, fOllowed by the medium plane heifers and the low plane heifers. The height of the surface epithelium increased from approximately 14 to 36 microns at first estrus. Marked increases in ovarian weight occurred at about the time of first estrus in the heifers fed the high (8.7 mo.) and med- ium planes (ll.4 mo.) of nutrition. Although mature ovarian fbllicles developed earlier and the onset of estrus and ovulation occurred earlier in heifers on the 15 high level of feeding, ovarian function after first estrus was not affected by age of heifers or their nutritional level. Desjardins and Hafs (l969) slaughtered Holstein heifers at monthly intervals from birth through l2 months of age. They determined nucleic acids, protein, endometrial cell height, weight, and length of the tubu- lar genitalia as indices of growth and function. Relative to the values at birth, uterine weight, ribonucleic acid (RNA), and protein increased more rapidly after 6 months than before this age. The relative increase in DNA to lo months was only about two-thirds as great as the increases in uterine weight and RNA, suggesting hyper- trophy of uterine cells concurrent with hyperplasia. Uterine epithelial cell height was stimulated at birth and then regressed. It did not return to the value at birth (20.9 microns) until 9 months of age (24.3 microns) but by l2 months had increased to 33.0 microns. The increase in endometrium thickness did not occur until about 2 months after first estrus and 3 months after changes in uterine weight, RNA, and protein content. Ovarian weight increased nearly four times more rapidly than body weight from birth to 5 months, but plateaued from 5 to 8 months. From 8 to l2 months of age, growth rate of the ovaries was comparable to that for the body. No follicles were visible on the ovaries at birth, but by 4 months of age the number of small and large follicles reached a maximum, after which it decreased to 8 months, and then remained relatively constant thereafter. Since stage of estrous cycle at slaughter was not constant in this experiment, it is possible that differences in stages of the estrous cycle among the age groups contributed considerable variation to the reproductive criteria observed. l6 2. Effects of Ovarian Steroids Certain physiological effects of ovarian steroids on the reproduc- tive processes are well established. Estrogens, secreted primarily by ovarian follicles, cause growth and vascularization of the uterus, while progesterone, secreted primarily by ovarian corpora lutea, promotes growth of the uterine endometrium and glands and suppresses estrous cycles. Hisaw and Hisaw (l96l) discussed the effects of estrogens and progesterone on the reproductive tract. The subject of estrous sychronization has been reviewed by Ulberg (l955), Hansel (l959), and Lamond (l964). The fact that estrous cycles in cattle can be regulated with progesterone was demonstrated by Ulberg gt_al;, (l95l) and Ulberg and Lindley (l960). They found that estrus and ovulation could be inhibited by daily injections of as little as l2.5 mg progesterone. Estrus occurred 2.5 to 9.5 days after the l4-day injection period. An injection of 0.5 to l0.0 mg of estradiol benzoate 3 days after the last injection of progesterone reduced the variation in the onset of estrus. Conception rate, however, was reduced by the progesterone injections with the higher dosages being more detrimental. During the past l0 years orally active progesterone analogues have been developed since progesterone itself is inactivated when administered orally. Pincus and Merrill (l96l) described some of the earliest work on oral progestagens developed to inhibit ovulation in women. The first synthetic progestagen studied quite extensively in cattle was medroxyprogesterone acetate (MAP) (Barnes §E_él;; l959, Hansel and Malven, l960, Hansel gt_al;, l961, Hansel, l96l, Nelms and Combs, l96l, Zimbelman, l96l, Collins, l96l, Anderson gt_al;) l962, and Zimbelman, l963). These studies showed that estrus and ovulation were inhibited during the oral administration period. After withdrawal, l7 heifers came into estrus, ovulated, and most of the studies showed conception rate to be nearly normal after cycle synchronization. Zimbelman (l963), however, reported a first service conception rate of El percent. After two services it was 76 percent while that of- controls after two services was 74 percent. Another compound which has received some attention is chlormadinone acetate (CAP). Wagner gt_al;, (l963) and Van Blake gt_al;, (l963) found this synthetic hormone to be extremely potent in inhibiting estrus and ovulation in cattle when fed for l5 to 20 days. Heifers came into estrus 4 to 6 days after the drug was withdrawn. Although conception rates were somewhat reduced at first service, the percent of heifers pregnant after two services was the same for treated and controls. An orally active synthetic progestagen presently being studied quite extensively is melengestrol acetate (MGA). It is an analogue of medroxyprogesterone acetate with enhanced capacity to promote endometrial proliferation, maintain pregnancy, and delay estrus activity (Duncan §t_al;, l964). Zimbelman and Smith (l966a) found the minimal effective oral dose required to inhibit estrus and ovulation in cattle to be about 0.4 mg daily, while that for MAP was l80 mg orally daily (Zimbelman, l963). For progesterone given subcutaneously, the minimal effective dose was l2.5 mg daily (Ulberg et_gl;, l95l). These dosage level ddfferences emphasize the potency of MGA. Zimbelman and Smith (l966a) reported that conception rate at first dnsemination averaged 42 percent for the various dose levels used, but after two services it was 82 percent. In other studies, Zimbelman and Smith (l966a) and O'Brien gt_gl;_(l968) found that 0.4 mg MGA daily for l8 days caused increased ovarian weights due to an increased incidence of a de- tectable follicle which increased in size with time on MGA. l8 Follicular fluid weight also increased, a finding also reported by Young gt_gl;_(l969). Zimbelman and Smith (l966a) concluded from the cervical mucous fern patterns and increased adrenal gland weights that the follicles were secreting estrogen even though estrus and ovulation were inhibited by MGA treatment. Zimbelman (l966) reported that MGA caused elevated pituitary luteinizing hormone (LH) content, suggesting that LH was not being released. However, there was no effect on follicle stimulating hormone (FSH) content in MGA fed heifers which would agree with the increased follicle size and inhibited LH release. This finding would seem to explain the increased incidence of large persistent follicles which do not ovulate in MGA fed cattle. A search of the literature revealed only one study on the histology of the reproductive tract after progestagen administration. Smallwood and Sorensen (l969) administered MAP to heifers in an effort to determine some of the possible causes of lowered conception rate at first service. While they could make no definite conclusions, they noted that cystic follicles were found in several heifers and the surface epithelium of the uterus was separated from the stratum compactum in numerous cases. Perhaps these findings explain part or all of the lowered conception rate observed at first service after progestagen administra— tion. Certainly additional study of this problem is needed. C. Mammary Gland Development l. Influence of Nutritional Level Although Herman and Ragsdale (1946) did not measure the effect of nutrition on the mammary gland directly, they observed that the heavy fed heifers had a great deal of fat deposition in the udder before freshening. Swanson and his associates have studied this topic more extensively than l8 Follicular fluid weight also increased, a finding also reported by Young gt_al;_(l969). Zimbelman and Smith (l966a) concluded from the cervical mucous fern patterns and increased adrenal gland weights that the follicles were secreting estrogen even though estrus and ovulation were inhibited by MGA treatment. Zimbelman (l966) reported that MGA caused elevated pituitary luteinizing hormone (LH) content, suggesting that LH was not being released. However, there was no effect on follicle stimulating hormone (FSH) content in MGA fed heifers which would agree with the increased follicle size and inhibited LH release. This finding would seem to explain the increased incidence of large persistent follicles which do not ovulate in MGA fed cattle. A search of the literature revealed only one study on the histology of the reproductive tract after progestagen administration. Smallwood and Sorensen (l969) administered MAP to heifers in an effort to determine some of the possible causes of lowered conception rate at first service. While they could make no definite conclusions, they noted that cystic follicles were found in several heifers and the surface epithelium of the uterus was separated from the stratum compactum in numerous cases. Perhaps these findings explain part or all of the lowered conception rate observed at first service after progestagen administra- tion. Certainly additional study of this problem is needed. C. Mammarnyland Development l. Influence of Nutritional Level Although Herman and Ragsdale (l946) did not measure the effect of nutrition on the mamnary gland directly, they observed that the heavy fed heifers had a great deal of fat deposition in the udder before freshening. Swanson and his associates have studied this topic more extensively than l9 anyone else (Swanson and Spann, l954, Swanson, l957, l960, Swanson and Hinton, l964, Swanson, l967, Swanson §t_al;, l967). They conclude that fattening heifers causes large fat deposits in the mammary gland. The fat deposits, which are different from the normal fat pad, inhibit the development of the lobule-alveolar system, and in turn, lower the milk producing ability of the fattened heifers. Cross sections of udders from fattened animals showed incomplete development of the lobule-alveo- lar system. Sorensen gt_gl;, (l959) attempted to quantify the mammary development in heifers fed a low, medium, or high nutrient intake by using the method of Swett (l947). They found that udder development measured by this method was markedly affected by the level of feeding, with higher development ratings being associated with higher levels of feeding. The size of the excised mammary glands was closely related to development and feeding level at l6 weeks of age. 2. Changes Associated with Age Presentations by Folley (l952), Cowie and Folley (l96l), and Raymaud (l96l) review mammary gland development during embyronic and fetal stages of life as well as growth of the mammary gland after birth. Based on gross observation and histology, it is generally accepted that the bovine mammary gland grows in size up to the time of puberty due to fat infiltration and ductular development. No appreciable lobule-alveolar growth occurs prepuberally. After puberty and especially during the last half of pregnancy, ductular and lobule-alveolar growth is greatly accelerated. Only the study of Sinha and Tucker (l969) was found in which quantitative measurements were made of the changes in the bovine mammary gland with age. They measured changes in mammary gland weight, nucleic acids, lipid, and collagen between birth and l2 months of age. and 20 during the various stages of the estrous cycle in l6-month old heifers. Deoxyribonucleic acid content increased little between birth and 2 months of age. But between the second and third months, DNA content increased l5-fold and continued to increase almost linearly until 9 months of age. Between 9 and l2 months DNA content did not change. Mammary RNA and hydroxyproline (measure of collagen) followed patterns similar to mammary DNA from birth to l2 months of age. But, hydroxyproline values were more variable and the changes were not as great as for the nucleic acid changes. Mammary DNA and RNA values in the cycling l6- months old heifers were greatest on the day of estrus and lowest on day 20 of the cycle. Per l00 kg body weight, the mammary DNA value of l6- month old heifers was no greater than the value for 9- month old heifers, suggesting that a major portion of puberal mammary growth was largely completed by 9 months of age. 3. Effects of Steroids The importance of ovarian hormones in growth of the mammary gland has been known and accepted for many years. The early work of Turner and coworkers (l939) clearly showed that estrogens stimulate duct growth, whereas a combination of estrogen and progesterone are needed for lobule-alveolar development. Following this initial report, numerous researchers attempted to develop the mammary gland and initiate lactation with exogenous hormones. Most investigators have used estro- gen or a combination of estrogen and progesterone and have attained varying degrees of success (Turner, 1939, Folley, l952, Cowie and Folley, l96l, Jacobsohn, l96l). Sud gt_al;, (1968) for example, obtained mammary development in open heifers similar to that in 5- month Pregnant heifers by injecting either 200 mg progesterone and 800 ug EStradiol-l7 Beta, or l00 mg progesterone and 400 ug estradiol-l7 Beta 2l three times weekly for 20 weeks. The effects of adrenal steroids on mammary development have been studied quite extensively in mice and rats (Jacobsohn, l96l). In general, most studies have shown that duct growth will result from low level adrenal steroid injections, but estrogens are usually necessary to obtain lobule-alveolar development. Kumaresan gt_al;, (l967) injected corticosterone during pregnancy in rats and found a 23 percent increase in DNA and a 52 percent increase in RNA over that of pregnant controls, but subsequent lactational performance was not tested. Apparently no corticoid studies have been conducted on mammary gland development in cattle. Mammary development has been observed, but never’studied quantitatively, in several experiments involving diethylstibestrol administration (Casida gt_g1;, l959). Also, at least one of the synthetic progestagens, melengestrol acetate, was observed to cause mammary development in cattle (Young gt_a1., l969). However, no studies were found which quantified the mammary development in cattle caused by the synthetic estrogens and progestagens. D. The Adrenal Glands l. Structure and Function Structurally, the adrenal is a compound gland composed of an inner medulla and an outer cortex of different embryological origin (Turner. l960). The medulla is ectodermal in origin and secretes amine hormones, while the cortex is derived from mesoderm and secretes steroid hormones. Neural innervation of the medulla regulates its secretions, but the cortex is practically devoid of nerves and is regulated by other body hormones. The cortex is composed of three zones; the zona glomerulosa, 22 zona fasciculata, and zona reticularis, from the exterior to the interior, respectively. The zona glomerulosa is little affected by hypophysectomy and secretes mineralocorticoids that are involved in regulating electro- lyte metabolism. The fasciculata and reticularis are highly dependent on an jgsjtu_pituitary and secrete glucocorticoids which regulate carbo- hydrate metabolism. Desjardins (1966) and Macmillan (1967) studied changes in adrenal weight and cortex zone widths from birth to 12 months of age in Holstein heifers and bulls. Height of the paired adrenal glands increased lin- early from birth to 'H) months of age with only slight changes thereafter in both heifers and bulls. Differences in average adrenal weights for the two sexes were usually less than a gram. However, the width of the zona glomerulosa was consistently greater in bulls than heifers from 2 to 11 months of age. Although the combined width of the zonas fasciculata and reticularis was greater at most ages in bulls than in heifers, the pro- portional differences were not as great as the differences between the sexes in the width of the zona glomerulosa. The monthly increase in width of the zona glomerulosa in bulls was quite erratic, but showed a general increase from birth to 12 months of age. However, in heifers the glomerulosa width declined from birth to» 6 months and then increased to a value at 12 months which was similar to the value at birth. The monthly increase in combined fasciculata-reticularis width was erratic in both heifers and bulls, but increased in both sexes by about 50 percent from birth to 12 months of age. Detailed discussions of the histological structure of the adrenal are presented by Elias (1948), Weber et_a1;, (1950), and Nicander (1952). 2. Effects of Ovarian Hormones It has been known for many years that ovarian hormones affect the 23 adrenal glands. Ellison and Burch (1936), for example, found increased adrenal weights and increased width of the fasciculata and reticularis zones of the cortex in animals that had received estrogen. The synthetic estrogens, notably diethylstilbestrol, also cause adrenal weight increase, as noted by Clegg and Cole (1954), Cahill et_al;, (1956), and Casida gt_al;, (1959). Turner (1960) speculated that estrogens are acting by blocking the synthesis of adrenal corticoids. Since the lowered level of corticoids in the blood would act to stimulate increased pituitary adrenocorticotrophin (ACTH) release, adrenal hypertrophy would result. Guyton (1961) offered a different theory on the action of estrogens. He thought the estrogens were acting directly on the pituitary to cause ACTH release, rather than indirectly as Turner (1960) postulated. Both concur, though, that the pituitary is necessary to produce an estrogen effect on the adrenals. At least one synthetic progestagen, melengestrol acetate, also affects the adrenals. Duncan gt_gl;, (1964) found that adrenal weights of male rats fed MGA decreased. But, Zimbelman and Smith (1966b) and 81055 §t_al;, (1966) found that adrenal weights of intact heifersincreased following long-term MGA administration. vaiously, more investinations are needed on the relationship between progestagens and the adrenals. E. The Gonadotropins and Prolactin 1. Concentrations Associated with Age The literature is almost devoid of studies on the changes in pituitary and blood concentrations of gonadotropins and prolactin with advancing age in the bovine. In 1935 Bates gt_al;, reported the potency of pituitary Prolactin in seven classes of cattle; embryos, veal calves, adult steers, adult bulls, and either open, early pregnant, or late pregnant cows. 24 Prolactin content was highest in embryos by two to three fold. Values for steers and bulls were greater than for nonpregnant cows, while the value for cows increased as the stage of pregnancy advanced. Reece and Turner (1937) also reported values for pituitary prolactin in cattle of various ages. They found that total pituitary content and the content per gram of anterior pituitary increased steadily with age in females. Values for bulls were generally lower than those for heifers, while lactating cows had higher values than dry cows. Desjardins §t_gl;) (1966) found similar prolactin concentrations in five 12-month old Holstein heifers and 42 nonpregnant mature Holstein cows. Sinha and Tucker (1969) reported that pituitary prolactin values showed no significant differences between birth and 12 months of age in Holstein heifers killed at monthly intervals. However, the values were somewhat elevated at 3 and 4,and 8 and 9 months of age. These elevated values at 3 and 4 months corresponded to the times when there was a shift to an increased rate of mammary growth, and the latter increase occurred when mammary growth was the greatest. Bates gt_al;, (1935) found that FSH potency was the lowest in steers, about 25 percent greater in embryos, bulls, and nonpregnant cows, 40 percent greater in veal calves and cows in late pregnancy, and 75 per— cent greater in early pregnant cows than in steers. From a recent study of Holstein heifers from birth to 12 months of age, Desjardins and Hafs (1968) found that anterior pituitary concentration of FSH was greatest at 1 month of age; it declined at 2 months and fluctuated only slightly there- after to 12 months of age. Pituitary LH concentration was considerably greater than that for FSH. LH levels increased rapidly from 1 to 3 months, fluctuated considerably between 3 and 6 months, had a greatly increased peak value at 7 months, and then fluctuated at prepeak values 25 from 8 through 12 months. The peak at 7 months occurred at the time of puberty in the heifers. Hackett and Hafs (1969) measured pituitary FSH and LH at various stages of the estrous cycle in 16-month old heifers. Averaging all values of the cycle showed FSH concentration to be only about one-fourth and LH about one-third the values found by Desjardins and Hafs (1968) in lZ-month old heifers. Desjardins §t_gl;, (1966) also measured pituitary LH and FSH levels in 42 nonpregnant mature cows. The level of pituitary LH which they found was only one-third that reported by Desjardins and Hafs (1968) in heifers at 12 months of age. In contrast, pituitary FSH concentration was similar in the 12-month old heifers and cows. 2, Effects of Ovarian Steroids While an interaction of the estrogens and progesterone with pituitary gonadotropins and prolactin definitely exists, knowledge of specific action is far from complete. One fairly definite fact is that the estro- gens cause an increase in content and release of pituitary prolactin (Meites, 1966). Beyond this, the relationships are much more conditional. The effects obtained in various experiments have been dependent, among other things, on the dosage levels of estrogens and progesterone, length of the injection period, age of test animals, and the species used in the studies. Thus, results have varied from an inhibitory effect, to no effect, to a stimulatory effect (Greep, 1961, and Flerko, 1966). Consequently, only a general scheme of the mechanism of interaction thought to exist between the ovarian hormones and the pi- tuitary gonadotropins and prolactin are presented. According to current concepts, a feedback mechanism operates whereby the pituitary release of FSH and LH is controlled by the levels of estrogen and progesterone in the circulation (Turner, 1960). Very low levels of 26 estrogens, coming from the immature follicles or extragonadal sources, stimulate the pituitary (probably by way of the hypothalamus) to augment its release of FSH. When the blood estrogen level becomes high, indi- cating that the ovarian follicle is mature, it acts to inhibit further FSH release and promotes an increase in the rate of LH release (cyclic LH release) above its usual continuous secretion level. Apparently this continual release of LH is needed along with FSH for significant estrogen production by the follicles. Under the influence of rising LH titers, the follicle matures, lutein changes occur in the walls of the mature follicle, and some progesterone along with large quantities of estrogens are secreted. The increasing levels of LH are in some manner involved in promoting ovulation. Once ovulation occurs, there is an immediate fall in the level of circulating estrogens. The ruptured follicle becomes transformed into a corpus luteum either spontaneously, or under the influence of LH or prolactin, depending on the species, and commences to secrete progesterone. Further release of LH above the base line secretion level is prevented by the high levels of progesterone. FSH, however, is released in quantities sufficient to cause follicle growth midway through the cycle. Although estrogen is secreted by these follicles, since the follicles do not grow to the mature ovulatory size because LH is lacking, estrogen production is not great enough to promote the behavioral signs of estrus. since the level of LH is insufficient to promote follicle maturation and ovulation, the follicle regresses in size and becomes atretic. Progesterone level remains high until the corpus luteum begins degenerating in function and structure. When this occurs, cyclic LH release is again possible and the estrous cycle is repeated (Turner, 1960). Even though the advent of synthetic estrogens such as diethylstil- 27 bestrol occurred several years ago, their effect on pituitary gonadotro- pins and the hypothalamus remains unknown. But, this void of knowledge with regard to the progestagen, melengestrol acetate, has been filled by the findings of Zimbelman (1966). He found no consistent effect of MGA on pituitary FSH, and therefore concluded that the corpus luteum was more effective than MGA in the control of follicular development. In intact pregnant heifers, MGA caused an increase in pituitary LH which was interpreted to mean that MGA inhibited LH release. However, this effect of MGA on pituitary LH was not evident in either bilaterally ovariectomized heifers with a low LH content or in unilaterally ovariec- tomized heifers with increased follicular development in the absence of a corpus luteum. Zimbelman (1966) concluded that these results are consistent with the concept of two hypothalamic centers for LH release, but only the center controlling cyclic LH release appears affected by MGA. Furthermore, it would seem necessary to conclude that a low level of LH release occurs during MGA treatment to allow enlarged follicle development and estrogen production. Additional studies are required to confirm and further clarify the effects of progestagens on the pituitary and hypothalamus. MATERIALS AND METHODS A. Experimental Design This experiment was designed to determine the effects of two levels of nutrition fed alone and in combination with the synthetic progestagen melengestrol acetate (MGA), during the period of most rapid postnatal development in Holstein heifers. Parameters measured were body growth, levels of certain anterior pituitary hormones in the pituitary and blood, development of the reproductive tract and mammary gland, and subsequent reproductive and lactational performance. For the study 140 heifers were purchased when less than 2 weeks old. They were fed and handled similarly for the first 2.5 months to be assured they were all growing well. According to previous random- ized assignments, at 2.5 months of age the heifers were divided into 14 groups of 10 heifers each. A designation of the treatments each group received is shown in Table 1. Two levels of nutrition were fed (Tables 2 and 3); a normal level designed to allow for a normal growth rate, and a high level formulated and fed to promote a maximal growth rate. Besides designating the nutritional level, the group to which each heifer was assigned also determined if and when she would receive MGA, and if and when she would be slaughtered. MGA was fed at the rate of 0.30 mg per heifer per day starting at 2.5 months of age or after first estrus to determine its prepuberal versus only postpuberal effects. One hundred heifers in designated groups were slaughtered either at 2.5 months of age, first estrus or at breeding size to obtain measure— 28 29 TABLE l.--Experimental treatments beginning at 2.5 months of age for 140 Holstein heifers. Nutrition treatments Age at Groupa Level MGA slaughter 1 Normal None -b 2 High None - 3 High From 2.5 mo. - 4 High From first estrus - 5 Normal None Breeding size 6 High None Breeding size 7 High From 2.5 mo. Breeding size 8 High From first estrus Breeding size 9 Normal From 2.5 mo. Breeding size 10 Normal None First estrus 11 High None First estrus 12c High From 2.5 mo. First estrus 13d High From first estrus First estrus 14 Normal None 2.5 mo. aEach group contained 10 heifers. bHeifers in groups 1-4 were not slaughtered. They were bred and retained for reproductive, dystocia, and subsequent lactational perform- ance studies. cHeifers were slaughtered when their group 11 pairmates were slaugh— tered. dGroup included for statistical balance. group 11. Received same ration as 30 TABLE 2.--Description of nutrition levels. Normal level High level (Per day) (Per day) 0.9 kg of a 12% protein grain mix free choice to a maximum of 4.5 kg per day of a 20% protein grain mix Free choice for both levels: (1; Corn silage 2 Alfalfa-grass hay (3) Trace mineralized salt TABLE 3.--Composition of experimental grain mixes. Ingredient Amount per 100 kg 12% protein 20% protein mix le Ground shelled corn (kg) 83.3 61.7 Soybean meal (50% protein) (kg) 9.7 29.3 Molasses (kg) 5 7 Dicalcium phosphate (kg) 1 1 Trace mineralized salt (kg) 1 1 Vitamin A (IU) 660,000 660,000 Vitamin D (IU) 880,000 880,000 Auromwcin (mg) 22,000 4,400 K 31 ments of the various parameters at those physiological ages. Forty heifers were kept to obtain data on reproductive efficiency, dystocia, and lactational performance. To obtain experimental design balance to the groups slaughtered at first estrus, a group (Group 13) destined to receive the high nutritional level without MGA until first estrus and then MGA in addition thereafter was included. However, since this groups was slaughtered at first estrus, they never received MGA and consequently received the same feeding treatment up to slaughter as the heifers fed the high level without MGA (Group 11). In analyzing the results, data from these two groups were combined. Because heifers fed MGA would be in a proestrus hormonal condition when they were slaughtered 48 hours after MGA withdrawal, heifers that had not been fed MGA were slaughtered 17 to 20 days after an estrus so they would be in a similar hormonal condition. Since heifers fed MGA from 2.5 months of age were not expected to exhibit estrous cycles, such heifers fed the high nutritional level plus MGA and scheduled for slaughter at first estrus were paired by body weight at 2 weeks of age with heifers that were to be fed the high nutritional level without MGA. Thus, when a heifer fed the high level without MGA was slaughtered after her first estrus, her high level pair-mate fed MGA was also slaugh— tered. To measure growth rate, height at the withers and body weight were taken once a month in the morning before the heifers were fed. Height and weight were also recorded on the day before slaughter and at parturi- tion. The criterion for breeding size was that heifers be 120 cm tall at the withers. After a heifer had reached 118 cm at the withers, height measurements were taken every 2 weeks to obtain a more precise estimate of 32 the date she reached 120 cm at the withers. In each of the groups kept for breeding, five randomly selected heifers were bred artificially to Zeldenrust Royal Pontiac, registration number 1397753, and 5 to Wis Symbol, registration number 1189593. These two bulls were used since a previous study by Boyd and Hafs (1965) had shown that Pontiac sired calves that weighed about 5 kg more at birth than those sired by Symbol, and we wished to determine if this difference in calf size at birth would be reflected in the degree of dystocia encountered by the dams. 8. Management of Experimental Animals Holstein heifers from production tested dams and registered sires were purchased when less than 2 weeks old from dairymen near Madison, Wisconsin. They were transported by truck to the M.S.U. dairy barn, and were examined and treated if necessary by a veterinarian upon arrival. Individual health record sheets were kept for each heifer. They were weighed on the second day after arrival. Calves were purchased in two lots of 40 and two lots of 30. Lot 1 arrived on April 12, 1967; lot 2 on July 8, 1967; lot 3 on September 27, 1967; and lot 4 on April 12, 1968. Heifers in lots 1 and 2 were randomly assigned at 5 per group to groups 1 - 8. Those in lots 3 and 4 were, likewise, assigned to groups 9 - 14 (see Table 1). From arrival until 2.5 months of age, the calves were kept in individual 4' by 6' pens. By 3 weeks of age, iwie heifers received 8 kg whole milk per day and were fed water, excellent Quality alfalfa hay, and a 16 percent protein grain mixture free choice. Grain and hay consumption increased gradually, so that by 2.5 months, When switched to the nutritional treatments, the heifers were consuming abOut 2.3 kg of grain and 0.9 kg of hay per day. At 2.5 months of age 33 the heifers were moved to loose housing dry lot facilities and penned communally according to the nutritional treatments. Commencing when the heifers were about 5.0 months old, they were observed for estrus signs twice daily at approximately 8:00 a.m. and 5:00 p.m. Any prOestrus signs such as mucous discharge from the vulva, or bawling and general restlessness, as well as the often observed post-estrus bleeding were recorded. A heifer was recorded in estrus when she would stand to be mounted by other heifers. She was also considered in estrus if she would not stand but displayed estrus symptoms such as swollen and inflamed vulva, attempted to mount other heifers, and general uneasiness. Starting at 6 months of age the ovaries of the heifers were palpated per rectum each month to detect corpora lutea, as evidence of ovulation. Unexpectedly, some heifers receiving 0.30 mg MGA per day showed signs of ovulation as per rectal palpation. When this happened, each heifer in the entire group was then increased to 0.45 mg daily. Heifers fed MGA from 2.5 months of age and slaughtered at first estrus received 0.30 mg daily for about 3.5 months, and the 0.45 mg level for about 0.6 months. Heifers that had been fed MGA from 2.5 months of age to breeding size received the lower dose for about 5.4 months and the higher dose for about 4.2 months. Meanwhile, those heifers fed MGA only after first estrus received the 0.30 mg dose for about 0.8 months and the 0.45 mg dose for about 3.8 months. When heifers receiving MGA reached breeding size the drug was withdrawn from their ration and they were fed only the high nutrition level. Heifers that were not slaughtered at breeding size were bred artificially in the late afternoon if first observed in estrus that norning, and in the morning of the following day if first observed in 34 estrus in the afternoon. Heifers on the high level of nutrition were switched to the normal level when they were diagnosed pregnant by palpa- tion, i.e. about 50 - 60 days after conception. As the experiment progressed, it became obvious that conception rate was lower than expected. Therefore, heifers on the high nutritional level which failed to conceive by the fifth service were switched to the normal level on the day of the fifth service to prevent excessive fattening. A heifer was bred a maximum of 10 times to the bull she was previously assigned, and if still not pregnant she was bred an eleventh time to a different bull. Any heifer that did not become pregnant to the eleventh service was slaughtered and the reproductive tract examined macroscopically and microscopically to determine possible causes for the infertility. Pregnant heifers were sold to Driggs Dairy at Palmyra, Michigan, where they were kept in a dry lot loose housing barn along with the regular herd heifers through the remainder of pregnancy. They received corn silage and hay free choice. After parturition they were placed in a free stall barn with the regular milking herd and milked in a parlor. Corn silage, alfalfa haylage, and alfalfa-grass hay were fed free choice, and either 2.3 or 7 kg of a 14 percent protein grain mixture per day was fed each cow, depending on her level of milk production. The heifers were weighed and measured at the withers about 10 days before the expected day of calving and within 3 days after calving. At parturition, weight and sex of the calf, health and condition of the dam and calf and a subjective rating of dystocia were recorded. Dystocia was rated from 1 to 4 according to increasing degree of difficulty at parturition. A rating of 1 indicated the heifer had a normal delivery; 2 indicated the heifer encountered a more than normal amount of straining but did 35 not require assistance; 3 indicated that assistance was required to deliver the calf; and 4 indicated the calf was born dead due to the difficult delivery, or that a Cesarean section was required to deliver the calf. Daily milk weights for the first 60 days of lactation were used to estimate each heifer's milk producing ability. C. Slaughter Procedures On the day of slaughter, heifers were transported about 8 miles to the Van Alstine Packing Company near Okemos, Michigan at approximately 6:30 a.m. Usually, slaughter began at about 7:00 a.m., and was completed by 8:30 a.m. The procedure followed at slaughter was as follows: (1) 0n the afternoon prior to slaughter each heifer was weighed and height at withers recorded. A sample of urine, usually less than 500 ml, was collected and stored at -15°C. until analyzed for estrogens. (2) At slaughter each animal was stunned in the forehead With a captive- bolt gun and exsanguinated. Two litres of mixed venous and arterial blood were collected in a cold heparinized glass jar and immediately stored at 4°C. It was later centrifuged and the plasma frozen in 10 m1 samples for hormonal analyses. (3) Within 10 minutes after stunning, the top of the skull was sawed off, the brain displaced, and the pituitary and hypothalamus dissected free. The whole pituitary was weighed and then the two lobes were weighed separately. The anterior pituitary was placed in a polyethylene bag on Dry Ice. The hypothalamus, median eminence and pituitary stalk were diced, immersed in a minimum volume of 0.1N hydrochloric acid and placed on Dry Ice. 88th the anterior pituitary and the hypothalamus were stored at -15 C. until analyzed for hormone content and releasing factors, respectively. (4) The mammary gland was removed and halved down the medial suspensory ligament. A representative sample of the right rear quarter was placed in Bouin's fixative for subsequent histological examination. The two halves were wrapped separately in heavy paper and within one and one half hours were stored at !-15°C. for.1ater nucleic acid analyses. (5) The reproductive tract was removed and the ovaries were dissected from the rest of the tract and weighed. Follicles were measured for surface diameter and recorded as ranging from 4-9mn, lO-lSnm, 16-20mm,>20mm in size. Size of corpora lutea, presence of ovula- tion points and any other noteworthy observations were also recorded. The ovary containing the largest follicle was bisected through the follicle. A section of the follicle wall was placed in cold 2 % 36 glutaraldehyde fixative for 4 hours and then stored at 4°C. in 0.1M phosphate buffer until further preparation for electron microscopy studies. After dissecting the uterus from the rest of the tract, it was weighed. A section of the right horn was put in Bouin's fixative for histological examination, and a 20- to 30—gram piece from the left horn was placed in 0.25 M sucrose and put on Dry Ice until stored at -15°C. for later nucleic acid analyses. (6) The adrenal and thyroid glands were removed, weighed, and a sample of each placed in Bouin's fixative for later histological examina- tion. The adrenal section was taken midway down the lobe on the right adrenal gland. The remaining adrenal tissue was put in 0.25 M sucrose, held on Dry Ice and later stored at -15°C. until analyzed for corticoid content. 0. Assays of Anterior Pituitary Hormones l. Homogenization of the Anterior Pituitaries The anterior pituitaries which had been stored at -15°C. were partially thawed at room temperature, weighed to the nearest 0.1 mg, diced, and homogenized in a Servall Omni-mixer in 10 ml of cold 0.85% saline for about 2 minutes. The volume of the homogenate was adjusted to a final concentration of 50 mg anterior pituitary equivalent per ml. The homogenate was centrifuged in a Servall Superspeed centrifuge type SS-l for 15 minutes and the supernatant fluid frozen in plastic vials for later FSH, LH, GH, and prolactin analyses. 2. Follicle Stimulating Hormone (FSH) Bioassay Anterior pituitary FSH content was measured by the immature rat ovarian weight augmentation assay of Steelman and Pohley (1953). Because bovine pituitaries contain little FSH relative to LH or relative to pituitary FSH potencies in other species (Macmillan, 1967), doses of 40 and 80 mg equivalents of anterior pituitary tissue were used whenever possible. Sixteen pituitaries had to be assayed using doses of 30 and 60 mg equivalents, and 6 pituitaries using 25 and 50 mg equivalents due to a lack of pituitary material. The 40-80 mg level unknowns were compared ‘bo 40 and 80 pg levels of ovine NIH-FSH-SS, shile the 30-60 and 25—50 mg 37 level unknowns were compared to 30 and 60 ug levels of ovine NIH-FSH-SS. The unknowns were also compared to a 20 IU dose of human chorionic gonadotropin (Squibb Follutein Chorionic Gonadotropin). Female Sprague-Dawley rats (from Spartan Research Animals, Haslett, Michigan) were injected subcutaneously between 7 and 8 a.m., 12 and l p.m., and 5 and 6 p.m. on days 22,23, and 24 of age. Besides receiving either the unknown or known amounts of FSH, each rat was also injected with 20 IU of human chorionic gonadotropin. For each assay, five female rats were used at each unknown dose level and seven rats for each standard dose level. The total dose was given over a three day period in 9 injections. Between 8 and 11 a.m. on day 25 of age, the rats were killed, both ovaries removed, trimmed, and weighed. Later, the potencies were estimated by the slope ratio procedure of Bliss (1952). 3. Prolactin Radioimmunoassay Plasma and anterior pituitary prolactin levels were measured by a radioimmunoassay technique developed by Dr. H. A. Tucker and J. A. Koprowski from our laboratory. The methods were essentially those of Niswender gt_gl;, (1968, l969a, 1969b). Briefly, the procedure consisted of the following steps: (1) Antibodies to NIH-B] prolactin were prepared in guinea pigs by emulsifying approximately 2 mg of hormone in 2 ml of 0.85 percent NaCl and 2 ml of Freund's complete adjuvant. Each guinea pig received this mixture subcutaneously once every 2-3 weeks except that incomplete adjuvant was used after the initial injection. Serum was collected via heart puncture at 2- to 3-week intervals after the ninth week. (2) A sheep (wether) was immunized with guinea pig gamma globulin. Forty to 50 mg gamma globulin (Fraction II, Pentex Inc., Kankakee, Illinois)were emulsified in 2.5 ml 0.85 percent NaCl and 2.5 ml Freund's complete adjuvant. Subcutaneous injections of gamma globulin emulsified in incomplete adjuvant were continued at 3- week intervals for 9 weeks and serum was collected at monthly intervals thereafter. (3) The methods used in radioiodination and in the radio-immunoassays were essentially those of Niswender et al., (1969b) except that 1251 was used. 38 (4) Cross reactivity of the prolactin antibody with other hormones was checked against NIH-bovine LH, TSH, GH, and ovine FSH. Positive responses did not occur at levels up to 100 mug of GH per tube and up to 1000 mug of each of the other three hormones per tube. Minimal and maximal amounts of prolactin actually measured on a per tube basis were 0.2 and 4 mug. Thus, the assay was specific at the levels used. The inmunoassay produced parallel dose response curves between the standards and bovine sera or pituitary extracts. (5) The plasma samples were diluted 1:6 while the anterior pituitary homogenates were diluted 1:500 with 1 percent bovine serum albumin in phosphate buffered saline. 4. Luteinizing Hormone (LH) Radioimmunoassay Anterior pituitary and blood plasma levels of LH were determined by the radioimmunoassay procedure of Niswender et al., (1969b), employing a few minor changes. It was essentially the same basic procedure described for the prolactin radioimmunoassay. While the plasma samples were not diluted, the pituitary homogenates were diluted 1:7500 with phosphate buffered saline containing 1 percent egg white albumin. Those changes in the procedure included: (1) using 1251 for radioiodination (2) using purified bovine LH (LER 1072-2) (3) using goat anti-rabbit gamma globulin from Nutritional Biochemicals Corporation as the second antibody Drs. Niswender and Midqley (1968) supplied the LH antiserum and the purified bovine LH. E. Determination of Nucleic Acids in the Uterus and Mammary_§land The nucleic acid content of the uteri and mammary glands was determined using the procedure of Schmidt and Thannhauser (1945) as modified by Tucker (1964). An outline of the procedure was: (1) If tissue frozen, thaw at room temperature. (2) Dissect mammary parenchymal tissue from hide and fat and weigh it. (3) Grind parenchymal tissue with a meat grinder to obtain a represen- tative sample for analyses. (4) Dice the uterus to obtain a representative sample for analyses. 39 Suspend in cold distilled water at a 1:20 dilution. Homogenize for 2 minutes in a Waring blender at top speed. (8) Put duplicate 2 m1 samples into 16 ml plastic centrifuge tubes, add 10 ml 95% ethyl alcohol, stopper, and shake for 12-18 hours. (9) Centrifuge at 17,000 rpm for 20-30 minutes in a Sorvall with a SM-24 rotor and discard supernatant. (10) Add 5 ml anhydrous ether, centrifuge for 10-15 minutes, and discard supernatant. (11) Dry tubes until tissue forms a hard pellet. (12) Add 10 ml methanolzchloroform (2:1), stopper, and shake for 18-24 ours. (l3 Centrifuge for 10-15 minutes and discard supernatant. {lg Add 10 ml anhydrous ether, stopper, and shake for 18-24 hours (5} Weigh out 15 to 20 grams of the tissue. Centrifuge for 10-15 minutes and discard supernatant. 16) For steps 16 and 17, keep tubes in ice water when not in centrifuge. Add 5 ml ice-cold 10% trichloracetic acid, mix well, centrifuge for 5-10 minutes, and discard supernatant. Repeat this step. (17) Add 5 ml ice—cold ethanol saturated with sodium acetate, mix well, centrifuge for 5-10 minutes, and discard supernatant. (18) Add 2 ml 1N potassium hydroxide, mix well, stopper, and store at 37°C. for 15 hours. (19) Cool tubes in ice-water, add 0.3 ml ice-cold 6N hydrochloric acid and 5 ml ice-cold 10% perchloric acid, mix well, centrifuge for 10-15 minutes, pour supernatant into 25 m1 calibrated test tube. (20) Add 5 ml ice-cold 5% perchloric acid, mix well, centrifuge for 10-15 minutes, pour supernatant into 25 m1 calibrated test tube from step 19. Repeat this step. Keep all tubes cold. (21) Bring volume of calibrated test tube up to 20 ml with 5% per- chloric acid, mix well, take 3 m1 and combine with 3 ml fresh «(1 hour old) orcinal reagent. (22) Cap tube with a marble and heat in a water bath for 30 minutes at 100°C. Allow to cool. (23) Read optical density at 670 mu on a Beckman DB spectrophotometer. Adjust to 0 optical density with a mixture of 3 ml 5% perchloric acid and 3 m1 orcinal reagent that has been heated to 100°C. for 30 minutes. The optical densities obtained are compared with a standard curve prepared with highly purified yeast RNA. (24) To the precipitate in step 20, add 5 m1 ice-cold 5% perchloric acid, mix well, heat in a water bath for 15 minutes at 70°C., cool to 5 C., centrifuge for 10-15 minutes, pour supernatant into 25 m1 calibrated test tube. (25) Add 5 ml ice-cold 5% perchloric acid to precipitate, mix well, centrifuge for lO-15 minutes, pour supernatant into calibrated test tube from step 24. Repeat this step. (26) Bring volume of calibrated test tube up to 25 ml with 5% perchloric acid, mix well, and read optical density at 268 mu on a Beckman DB spectrophotometer. Adjust to 0 optical density with 5% perchloric acid. The optical densities obtained are compared with a standard curve prepared with highly polymerized DNA. 16 convertthe optical density values obtained into mg of DNA and RNA in the entire mammary gland or uterus, the following mathematical steps were 40 used: Slope of the x Final diluted x Optical x Total weight x 1 standard curve volume density of the organ 10 F. Histological Techniqpe After fixed in Bouin's fluid for 48 or more hours, the adrenal and uterine tissues were cleared by placing them in the following solutions in the order listed: 50, 70, 80, and 95 percent ethanol, 95 percent ethanol: methyl salicylate (1:1), and methyl salicylate. Tissues were left in each solution for at least 24 hours. Infiltrating the tissues with melted paraffin followed, With the tissues being in the paraffin baths for at least 3 hours (1 and 1/2 hours in each of two baths). The tissues were then transferred to molds and imbedded in paraffin blocks. Sectioning the tissue blocks into 8- to 10-micron sections with a micro- tome, and placing the sections on a warmed glass slide coated with egg white albumin fellowed. After drying for 48 hours, the slides were stained using the following steps: 15 min. in Xylene; 5 min. in 95% EtOH; 5 min. in 95% EtOH; 5 min. in 70% EtOH; 5 min. in 35% EtOH; 5 to 20 minutes in Harris' Hematosylin depending on the type of tissue; 2 minutes in dis- tilled water; 5 min. in 35% EtOH; 5 min. in 70% EtOH; a few seconds in acidulated 70% EtDH if over stained; 15 to 45 seconds in alkalinated 70% EtOH if desire greater differentiation; 2 to 3 min. in Eosin; 5 to 10 minutes in 95% EtOH; 10 min. in xylene; put a few drops of Permount on the slide, add a cover slip and allow to dry. The uterine slides were examined at 400 power and the endometrial cell height measured with a calibrated occular micrometer. Nearly all the adrenal slides contained complete cross sections. These were projected by means of a Bioscope with a 2 power ocular onto paper on a table 122 (ml 41 below the slide. Tracing the medulla and the three cortex zones on the paper was quite easy with this apparatus. Ten locations around the medulla on the tracing from each slide which typified the cortex structure were selected. Lines perpendicular to the medulla were drawn and the cortex zone widths measured at these locations. The values obtained were then corrected for the distance projected and converted into millimeters. RESULTS AND DISCUSSION A. Body Growth 1. From 2 Weeks to 2.5 Months Group means for the body measurements taken at 2 weeks and at 2.5 months of age are presented in Table 4. The overall mean and its standard error for weight at 2 weeks of age was 46 :_1 kg. At 2.5 months it was 96 :_1 kg and the withers height was 86.6 :_0.3 cm. Although variation in weight existed among the group values at each age, all heifers started on the treatments at 2.5 months of age at about the same weight and height. 2. From 2.5 Months to First Estrus That the different levels of nutrition fed without MGAT influenced body growth up to the time of first estrus is indicated by the data in Table 5 and Appendix I. At first estrus, the 30 normal level heifers were 8.7 i 0.2 months old with a range of 6.0 - 11.5 months, while the 60 high level heifers were 7.5 :_0.1 months old with a range of 5.7 - 10.1 months. The age and body size values recorded at the estimated time of first estrus for the 30 heifers fed the high level plus MGA from 2.5 months are shown in Table 5 and will be discussed later. The ages at first estrus for the normal and high level heifers were significantly different (P<0.01) and agree with the findings of previous studies that heifers fed a higher than normal nutritional level will be younger at first estrus. 42 43 TABLE 4.-—Growth from 2 weeks to 2.5 months of age for Holstein heifers. Weight Withers height Groupa 2 weeks 2.5 months 2.5 months ---------- (kq)------------- (cm) 1 50 97 86.2 2 48 95 86.6 3 47 96 86.6 4 46 99 84.1 5 48 98 86.8 6 49 101 86.8 7 48 92 87.7 47 98 I 86.1 9 45 97 89.2 10 42 90 85.0 11 41 93 86.9 12 44 96 86.8 13 44 94 88.1 14 52 95 85.9 Mean :_SE 46 + l 96 + 1 86.6 :_0.3 aTen heifers in each group. 44 .Apo.ovav mm=_a> 28:50 as» Eocc pcaamccwu s_pcmuwc_cmemo .mmpmsgwma ucmEpmmeg saw; we mzepmm pmcwe pm mm:_m>n .meoegm ugmvcmum L_m;u use memos mew mm:_m>m m.o + m.o~ PF._ o._.H m.~o_ o_ + mom N.o + e.~ om .oe m.N sate n qoz + saw: 0.0.“ _.NN mo.F 0.0.“ o.mo_ a.“ mmN F.o.w m.e 00 Low: m.o.u N.mm umm.o 5.0.“ N.mo_ m.“ 0mm .um.o.u 5.x om Faseoz AEUV Amxv AEUV Amxv Aoev unmww; ammo agave; agave: mm< memwwmc acmEpwmeu mcmzowz e? apwao memcoez Langsz co_pwep=z mmmmcucH magumm umgwm ou mgpcoe m.m Eoeu mscumm umgwm a.mucmsumogu cowuweuzc “cmemmmwu we» memevm; :wmumpo: com maepmm pmewe op cuzoeo--.m m4m<~ 45 However, the ages in this study were younger than those in most of the previous studies cited in the literature review (Eckles, 1915, Reed et_al;, 1924, Hansson, 1956, Crichton gt_gl;, l959, Sorensen gt_a1;, 1959, Gardner and Garcia, 1966). A multitude of factors such as climate, inheritance, improved rations or better methods of detecting estrus could have caused the age differencesbetween this and previous studies. The heifers in this study had earlier communal contact in loose housing pens than heifers in some of the previous studies which may have prompted the development of estrus behavioral patterns at an earlier age. Although age at first estrus was different for heifers fed the two levels of nutrition, body weight and height at withers (Table 5) did not differ significantly at first estrus (P>0.lO). These findings substan— tiate the contention that heifers exhibit first estrus at a relatively con- stant physiological age, as indicated by body size, rather than at a certain calendar age. That heifers fed the normal level of nutrition were signifi- cantly older but not heavier or taller at first estrus implies that the high level heifers grew faster than the normal level heifers from 2.5 months to first estrus. This in fact did occur as indicated by the nonsignificant difference (P>0.lO) in withers height increase and the significant difference (P>0.01) in daily gain from 2.5 months to first estrus for heifers fed the two levels of nutrition without MGA (Table 5). The normal level heifers gained 0.83 kg per day which is very close to the value of 0.87 kg for daily gain by Holstein heifers from 70 to 260 days of age as reported by Matthews and Fohrman (1954) in the Beltsville growth standards. Morrison (1959) gives values in his growth standards which calculate to be 0.84 kg gain per day between 2.5 and 8.7 months of age. Thus, by these two standards the normal level heifers in this study grew at a normal rate up to first estrus. 46 Also presented in Table 5 are growth values for the 30 heifers receiving the high level plus MGA from 2.5 months. Since the progestagen inhibited estrous cycles in these heifers, the values shown in Table 5 were taken when the high level without MGA pairmates exhibited their first estrus. Thus, these data are estimations of relative values which might have existed at first estrus and are not absolute values. Heifers fed the normal level plus MGA from 2.5 months did not have contemporary normal level without MGA pairmates, so estimated first estrus values were not available. Although the high level plus MGA values in Table 5 for weight and daily gain were not significantly different (R>0.10) from the high level without MGA values, analysis of the data on a monthly basis revealed that after 5.5 months of age the heifers fed MGA gained significantly faster (P<0.05) than those not receiving the drug. This finding supports the contention of Zimbelman (1968), that MGA causes greater weight gains beginning 1 to 2 months before first estrus. Since MGA apparently increases weight gains through the action of the estrogens from the persistent ovarian follicles (Zimbelman and Smith, 1966b), this action implies that the ovaries commence a certain degree of activity before first estrus. I think this is probably an acceptable explanation, as puberty evolves over a period of time rather than occurring suddenly (Donovan and van der Werff ten Bosch, 1965). Data extracted from Table 5 for the heifers that were not slaughtered at first estrus are shown in Table 6 and Appendix I. Although com- bined with the high level values in Table 5, the data for heifers fed the high level up to first estrus but designated to also receive MGA after first estrus are presented separately in this table. Though 47 .A_o.ovmv mmapm> emzpo mg» Eoem ucmemeewu xppcmu_mwcm_mu .mmumscwma pcmsummgp now; we mzepmm pmeww um mm:_m>n .mgoe—Lm Umecwpm mecp U20 mcme mLm mmzpm>m m H a: o; H 0.8 8; 90 + :2 e + SN 3 + E 8 258 per: 8: 0.lO) from the normal level value and indicates no significant growth advantage from feeding MGA with a normal nutritional level. Body weights at 120 cm withers height (Table 7) for the heifers fed MGA along with the high nutrition level were significantly greater than for heifers fed the other nutrition treatments (P<0.01). This confirms the claim that MGA will increase weight gains by about 10 percent in feedlot heifers fed a heavy concentrate ration. However, those heifers fed the normal level plus MGA treatment weighed the same and were the same age at 120 cm withers height as the normal level heifers. Thus, no increase in body weight or rate of skeletal growth due to MGA occurred when a low level of grain was fed. Furthermore, MGA did not increase the rate of skeletal growth when fed with the high nutrition level; heifers fed the high level reached 120 cm at the withers at the 49 .Amo.ovav ucmEpmmeu magpmm pmcwe Eocm cmzp mmmp xpucmueewcmwmm .Ammo.ovav mucmspmmgu can» mmmp x_ucmormpcumn .Apo.ovmv mpcmspmmgp car; new . cmgp emgmmem apucmupepcmpmu .Apo. ovmv mucmsummeu can» Lmummgm xpucmupwpcmpmu .mgoegm ueoucmpm mecp can memes mew mm:_m>m m_._ m.o.H m.N_ m.H NN_ N.m um.“ “mm m.o + e.__ om mstpmm “were 502% 0.lO) among the treatments (3.4 to 3.7 months). This implies that the normal level heifers grew just as fast after first estrus as those fed the high level without MGA. And indeed this did occur as evidenced by the weight and height changes and the daily gains during this period (Table 7). Analysis of the data comprising Table 8 and shown in Figure 1 revealed that the body weight curves for heifers receiving the normal level plus MGA and the high level plus MGA were linear. But, the curves for heifers fed the normal and high levels without MGA were not linear (P<0.01). To explain these results becomes difficult since they do not agree with the Morrison (1959) or Beltsville (1954) standard growth curves, or the values obtained by Sorenson gt_algg (1959) for heifers receiving a high nutritional level. According to these studies cited, Holstein heifers gain in body weight at a linear rate through at least the first 12 months of life. The monthly weights shown in Table 8 and Figure 1 reveal that the normal level without MGA heifers started gaining at a faster rate after about 6.5 months of age. Meanwhile, heifers fed the high level without MGA declined in their rate of gain after about 8 months of age. The cause (or causes) for this phenomenon is unknown. Apparently MGA compensated for the decline in rate of gain by high 51 TABLE 8.--Body weights by age for Holstein heifers fed different nutri- tion treatments but not slaughtered at first estrus.a ---------------- Nutrition treatment-------------------------------- High + MGA High + MGA from Normal + MGA Normal High from 2.5 mo. first estrus from 2.5 mo. Age Weight Weight Weight Weight Age Weight (m0) (kg) (kg) (k9) (kg) (m0) (kg) 0.5 49 49 48 46 0.5 45 (10) 2.5 96 96 100 95 2.5 97 (10) 3.0 106 110 109 105 3.3 117 (10) 3.4 114 120 117 118 4.3 140 (10) 4.4 133 150 150 150 5.3 168 (10) 5.4 155 185 185 184 6.3 197 (10) 6.3 174 212 213 215 7.3 217 (10) 7.9 221 265 276 273 8.2 '245 (10) 8.7 248 291 300 298 9.2 275 (5) 9.8 268 310 (18) 328 (19) 326 (18) 9.7 284 (10) 10.7 292 (19) 331 (17) 356 (18) 357 (18) 11.1 325 (5) 11.7 315 (18) 356 (15) 381 (15) 391 (14) 12.1 352 (8) 12.5 344 (17) 380 (12) 403 (12) 415 (12) 12.9 382 (5) 13.6 374 (16) 405 (12) 436 (11) 448 (10) in Values are means. aTwenty heifers at each value except for values followed by number parentheses. 52 420-;- High + MGA from 395’ first estrus ‘ High + MGA from " , ’0 370T 2.5 months .- .33,” O. ’ Ji.d’ I ' ’I 345- I” ’3. ’ q 1’ ’c 1’ 320+ ”“1"“ + "GA M 1’ a . , "l ..‘ ”I D I 0' 1 .. r ' / 295 High + MGA , .3" I/ . I ’ ormal I .°. I’d / 32 ' 3. if I”’ :3. 707' ngh --—-?_______’.h // ,0 Morrison 4) ’7': ’, ,I’ “Standard .c I’o'. ’ 0 I” 3245 (- I '. ’0 I’ C) .0 ’I ” 3 “.0 ”’ ’I gzzor l’ ’0’ .- no .I’ ’ ,r I I ,’ 1951- High and High + ’0 7” . , , I’ l’ I, Ef’. 170- p’ I I’ I” _ a”, I: ‘45 . ’d ’ I, ’ I ’/ 120- -0 la! / x . ' 1’ 95 " 1’ . I I” I 70%;" 2 3 4 5 6 7 8 9 IO II I2 I3 ’ Age (months) Figure l.--Body weights by age for Holstein heifers fed different nutrition treatments but not slaughtered at first estrus. 53 level heifers as indicated in Tables 6 and 7 and Figure l. Heifers receiving the high level and the high level plus MGA after first estrus gained nearly the same amount between 2.5 months and first estrus (169 kg vs 165 kg, respectively), but between first estrus and breeding size, a nearly equal time period for the two groups, heifers receiving MGA gained about 50 percent more (P<0.025) than heifers receiving just the high level (127 kg vs 87 kg, respectively). Between 2.5 and 4.4 months of age, heifers fed the high level without MGA had a faster rate of skeletal growth than those fed the normal level without MGA. As the data in Table 9 show, after about 4.4 months of age heifers on all nutritional treatments increased in withers height at the same rate. From these findings, it is apparent that nutritional level has little influence on rate of skeletal growth. Photographs taken at breeding size which are representative of the heifers fed the normal level, high level, and high level plus MGA treat- ments are shown in Figure 2. 4. Body Size at Slaughter The ages, body weights, and withers heights at slaughter for heifers on the various nutritional treatments are presented in Table 10 and Appendix I. Study of the first estrus slaughter data and the monthly body size of heifers slaughtered at this physiological age (Table 11) shows the growth stimulating effect of the high nutritional level. MGA did not produce a further increase in weight or height, although as noted earlier, after about 5.5 months of age, heifers fed the high level plus MGA did gain faster than the high level heifers. Although heifers fed the normal level without MGA and slaughtered at first estrus were older (P<0.01), body weight and height at the withers were the same for heifers on the normal and high levels without MGA (P>0.lO). Heifers fed 54 TABLE 9.—-Withers heights by age for Holstein heifers fed different nutri- tion treatments but not slaughtered at first estrus.a ---------------- Nutrition treatment—------------------------------ High + MGA High + MGA after Normal + MGA Normal High from 2.5 mo. first estrus from 2.5 mo. Age Height Height Height Height Age Height (m0) (cm) (cm) (cm) (cm) (m0) (cm) 2.5 86.5 86.7 87.2 85.1 2.5 89.2 (10) 3.0 88.5 89.0 88.7 87.7 3.3 91.9 (10) 3.4 89.3 (10) 91.3 (10) 90.6 (10) 90.9 (10) 4.3 95.7 (10) 4.4 91.9 94.9 94.3 94.0 5.3 99.9 (10) 5.4 96.2 99.3 98.7 98.7 6.3 104.0 (10) 6.3 99.6 (10) 103.2 102.5 103.2 7.3 107.3 (10) 7.9 105.3 108.5 108.4 108.8 8.2 110.4 (10) 8.7 108.9 112.1 111.9 112.0 9.2 112.4 (5) 9.8 112.0 114.7 (18) 114.7 (19) 114.5 (18) 9.7 114.1 (10) 10.7 114.7 (19) 117.6 (17) 117.6 (18) 117.4 (18) 11.3 116.5 (8) 11.7 117.7 (18) 119.6 (15) 119.8 (15) 119.0 (14) 12.1 118.2 (8) 12.5 119.3 (17) 120.8 (12) 121.2 (12) 120.7 (12) 12.9 120.1 (5) 13.6 121.5 (16) 122.5 (12) 122.6 (11) 122.8 (10) aTwenty heifers at each value except for values followed by number in parentheses. Values are means. 55 Fm>mF _mELoz Figure 2.--Photographs at breeding size of Holstein heifers fed normal level, high level, and high level plus MGA nutrition treat- ments from 2.5 months of age. .A_o.ovav mpcmspmmep now; new . Eocm pcmemmwwn x_ucmuwmwcmwmm .Ammo.ovav mpCmEpaatp quz + sue; new not; asp 20c mm=_a> 502» “cmtmccwe s_scauwce=msm .AoF.0vavmo.ov mms_m> emcee as» 502$ pcmcmeewu »_p=auwcw=mwmw .Apo.ovmv mmspm> emgpo esp Eoem ucmcmeewu heucmuwwwcmwmn . .mgmmwm; om Low mew gown; mm:_m> mzepmm umewe “casummep saw; pamuxm .memewm; op Low meoeem vgmncmpm gems“ use memes mew mm:_m>m + I ¢.o v.omp mm_ + «mm m.o + q._P 1 1 1 1 1 mzepmm pme_m Levee 0.lO) in age, weight, or withers height. This again points to the interaction between MGA and the high level of grain as discussed previously. Data on body composition, length and dry weight of the right cannon bone, and thyroid gland weights were collected on the 100 heifers that were slaughtered. Also, acinar cell heights and plasma bound iodine determinations were obtained on certain treatment groups. These data arms presented by Roger W. Purchas (1970) as part of his Ph.D. thesis. 3- Reproductive Tract Changes 1 . Uterine Weight Uterine weight increased about four fold from 2.5 months of age to 59 first estrus (Table 12 and Appendix II). Per 100 kg body weight the uteri more than doubled in weight during this period. Although some uterine weight change is associated with body growth, the majority occurs shortly before first estrus (Desjardins and Hafs, 1969). This would seemingly imply that the ovaries begin steroid hormone secretion before first estrus, since it is accepted that ovarian steroids regulate uterine growth. The various nutritional treatments imposed upon the heifers after 2.5 months of age had no apparent influence on uterine weight at first estrus. However, at breeding size (Table 13 and Appendix II), uteri from heifers fed the normal level and normal level plus MGA weighed significantly less (P<0.05) than uteri from heifers fed the high level and high level plus MGA. But uterine weight per 100 kg body weight revealed no significant effect (P>0.lO) of the various nutri- tional treatments. Thus, the uteri of heifers fed the high level and high level plus MGA weighed more because the hiefers were heavier. 2. Uterine Nucleic Acids Uterine nucleic acids (DNA and RNA) increased in total amounts from 2.5 months to first estrus (Table 12 and Appendix II), reflecting the increase in uterine weight during this time period. However, per gram of uterus the picture is different. DNA concentration (mg DNA/g uterus) declined significantly (P<0.01) from 2.5 months to first estrus for all treatments. The value for heifers fed the high level plus MGA declined the most, and it was significantly different (P<0.01) from the values for heifers fed the normal or high level without MGA. Since uterine Weights of heifers fed MGA were not different from those of heifers not 'Fied MGA, hypertrophy of the uterine cells is suggested. Mean- VVItile, RNA concentration (mg RNA/g uterus) values for all treatment 60 .Amo.oVav pamEummeu Eoee acmemew_n appcmuwmwcmwme .AFo.oV¢V pcmspmmeu see; ucmgmmmwn zpucwuwwwcmvmm . mo.ovav mmzpw> mzeumm umewe esp can» emummcm xpucmu_ewcmwm Po.0vav mmzpm> magumm “mew; mg» cage empmmem x_pcmuw$?cmwm .mmpmegwma ucmEpomgu sow; eo mseumm “mew; pm mm:_m> n u .mcacmm; ON Lo; mew goes; mm:_m> ucmEummep saw; pamoxm .mgmewm; op gee meogem nemucmum ewmgp use memos mam mmapm>m m._.w a.mm N._.H m.m~ m._.u m.m~ o.F.H o.m_ Asv “some; F_mu No.o.u o_._ ceo.o.H _m.o mo_.o.u m~.o mo.o.H we.o <20\ cegp mmep .Amo.ovev e:_e> acespeegu :02» weep .Amo.ovev mpceEeeegu new; new Peggec esp gee mmepe> can» weep .Amo.0vev mucespeegp megume peg?» geume gasp mme_ .Amo.0ve v mmepe> gecpe —_e cesu mmm_ .Amo.ovev mecmseeegp cecp mme_ .mgeewez op gee mgegge egeecepm gwenu e20 memes ege me2_e>e xpeemegggceem xgecaeggg00gmm 2_0000_gg00emg 2gecmeecg00gm sgeemeeg_0eemm 2_20aeeggc0_mw seecaeeggcmgm0 g.N.H 0.20 0.0.“ 0.00 20.2.“ N.g~ 0.2.“ 0.e0 g0.0.H 0.00 200 e20g02 __00 00.0.“ 00._ 0_.0.H 20.2 g00.0.H <0.0 00.0.“ 00._ g000.0.fl 0g.0 <20\<20 Q0090 00H00 10H0.05) the ratio over the value at 2.5 months and the other treatment values at first estrus. These data, like the DNA data, impli- cate hypertrophy of uterine cells due to MGA. This contention is further supported by the nucleic acid data at breeding size (Table 13 and Appendix II). Uterine DNA concentrations at breeding size were lowest in all treatment groups fed MGA, and singificantly less (P<0.05) in heifers fed the normal level plus MGA than in those fed the normal or high level. But unlike the first estrus data, RNA concentrations were elevated significantly (P<0.05) in heifers fed the high level plus MGA. There was no effect on RNA concentration in heifers fed the normal level plus MGA. As at first estrus, the RNA/DNA ratios were greater in heifers fed MGA; the normal level plus MGA value being greater (P<0.05) than the normal level value, and the high level plus MGA values being greater (P<0.05) than the high level value. Thus, the stimulatory effect of MGA on uterine cell size appears quite conclusive, as measured by nucleic acids content. 63 The high level of nutrition produced no significant change in DNA and RNA concentrations nor in the RNA/DNA ratios from the values for normal level heifers slaughtered at breeding size. Values for these normal level heifers slaughtered at about 13 months of age were similar to those of 17-month old heifers slaughtered by Hackett and Hafs (1969) on day 18 of the estrous cycle. Although the data are similar, the observations do not agree with the conclusions of Hackett and Hafs (1969), because from the standpoint of uterine nucleic acids, 12— to 13— month old Holstein heifers appear as mature at that age as they will be by 17 months of age. 3. Uterus Epithelial Cell Height Height of the uterus epithelial cells increased by 50 percent between 2.5 months and first estrus in heifers of all treatment groups (Table 12 and Appendix II). No treatment effect was detected at first estrus. The values obtained agree very closely with those of Desjardins and Hafs (1969) for heifers of comparable ages. Values at breeding size (Table 13 and Appendix II) for normal level heifers were less than reported values for llaand lZ-month old heifers(Desjardins and Hafs, 1969); but they were greater than values of Hackett and Hafs (1969) for l71month old heifers. Animal variation most likely explains these differences since the heifers in all three studies were treated similarly, and slaugh— tered at the same stage of the estrous cycle. A treatment effect on the epithelial height at breeding size is suggested by the data in Table 13 and Appendix II. Although the normal level plus MGA heifers had taller uterine epithelial cells than the normal and high level heifers, the difference was not significant (P>0.lO). But uterine cell height of heifers fed the high level plus MGA was significantly taller (P<0.05) than those of heifers fed the normal 64 and high level. Thus, it appears that MGA produced an increase in the uterine epithelial cell height. Whether this increase was due to the direct progestational action of MGA on the epithelium or to the indirect action through the ovary and thus an estrogenic effect is not clear. Perhaps there was synergism between the two possible routes of action. The data of Hackett and Hafs (1969) on Holstein uterine epithelial height during the estrous cycle suggest that estrogen was the principal effector. In their study epithelial height was greatest on the day of estrus and at the time of the mid-cycle follicle. Still, this could mean that both hormones are required, since minimal amounts of progesterone would also be present at these times. 4. Ovarian Changes Ovarian weight increased approximately 50 percent from 2.5 months to first estrus, and also from first estrus to breeding size (Table 14 and Appendix II). Values at all comparable ages were higher than those reported by Desjardins and Hafs (1969). No treatment effect existed at first estrus, but at breeding size ovaries of heifers fed the high level of nutrition without MGA weighed significantly less (P<0.025) than those of heifers fed the other nutritional treatments. This may have resulted from animal variation, or more likely from an unknown cause. MGA did not cause a significant (P>0.lO) increase in ovarian weights which agrees with the results of Zimbelman and Smith (1966b). However, they found that MGA caused a significant increase (P:0.05) in the follicular fluid weight, which was not measured in this study. Average number of folli- cles by size (Table 14) showed that heifers fed MGA had a higher inci— dence of larger follicles. This also confirms Zimbelman and Smith's (1966b) data. It therefore appears that MGA increases ovarian follicle size, and the follicles in turn secrete estrogens (perhaps at elevated 65 TABLE l4.--0varian measurements of Holstein heifers fed different nutri- tion treatments and slaughtered at 2.5 months, first estrus, (N' breeding size. Paired No. Follicles by size Nutrition Time of ovarian treatment slaughter weight a 4-9 10—15 16-20 :>20 Total (9) -------- (mn) --------- Normal 2.5 months 6.8 :_l.4 2.0 0.6 0 0 2.6 Normal First estrus 10.0 i 0.9 3.9 0.7 0.1 0 4.6 High First estrus 11.4 :_0.8 2.0 0.8 0.3 0 3.1 High + MGA b from 2.5 mo. First estru5' 10.2 :_1.1 3.9 0.4 0.5 0.3 5.1 Normal Breeding size 15.1 :_0.8 1.5 1.3 0.5 0 3.3 Normal + MGA from 2.5 mo. Breeding size 15.4 :_0.9 2.2 0.7 0.5 0.7 4.1 High Breeding size 12.9_+_o.7¢ 3.3 1.1 0.4 0.1 4.9 High + MGA from 2.5 mo. Breeding size 15.4 :_l.8 1.4 0.3 1.0 0.7 3.4 High + MGA after - first estrus Breeding size 17.6 :_1.3 3.7 0.3 0.6 1.0 5.6 a Values are means and their standard errors for 10 heifers, except high treatment first estrus values which are for 20 heifers. bValues at first estrus of high treatment pairmates. cSignificantly less than values for all other treatments at breeding size (P<0.025). 66 levels from normal) which according to Bloss et al., (1966) cause the body weight increase observed in feedlot heifers. C. Mammarnyland Changes l. Mammary Gland Weight The left halves of the mamnary glands were used to obtain the diff- erent parameters shown in Table 15 and Appendix III. Weight of the dissected parenchymal tissue increased about ten fold between 2.5 months of age and first estrus. But between first estrus and breeding size the value increased only about two fold. No detectable difference (P>0.20) in mammary gland weight due to nutritional treatment existed at either slaughter age because of the large variation within each treatment group. Although mammary weights of heifers in this study were considerably larger than those obtained by Sinha and Tucker (1969) from heifers of comparable ages, the same growth pattern existed. That is, starting at 2 to 3 months of age up to about 8 or 9 months of age, or near the time of first estrus, the mammary gland grew at a greatly accelerated rate. During this time it had an allometric growth pattern in comparison to the body's rate of growth. Before and after this time interval, the mammary gland grew at about the same rate as the body, or isometrically. 2. Mammary Gland Nucleic Acids At first estrus heifers fed the high level of nutrition without MGA had less total mammary DNA (P<0.05) than heifers fed the other two nutri- tional treatments (Table 15 and Appendix III). This might be expected since the mammary glands, though not significantly smaller, actually weighed the least. But per gram of tissue, no difference in the DNA values was detectable, although the value for the group fed MGA was the largest, suggesting somewhat greater cell numbers. This indicates no nutritional 67 0eeexe .0gee002 o0 gee 002000 0205505 .Aom.oVe V 020500020 <0: + Pegge: gee 02000 2020 0000 xpueeeeeeceem .Aeo.ovev 0020200020 2020 0000 heuceeeeecmeme e .Aeo.0vev 0020500020 <0: + 2002 gee 000000 2020 0000 0000000eeceeme .Amo.ovev 00205000g0 gespe gee 002000 2020 0000 xepceeeeeceeme .mepeegeee 020500020 2002 ee megume «mgwe 00 00200>e .mgeeees om gee 0g0 20023 00200> 022000 0020e 000000020 2002 020 ee 00>.02 peep gee 0gegge 02002000 gees» 020 02002 ege 00200>0 00.0.0 00.0 0.0.0 0.0 000.0 0000 0.0.0 0.0 000.0 0000 000.0 000 0000 00000020 002000 00200 20we0 .1 1. 1. 1. .1 .1 <02 + 2 02 00.0 + 00.0 2.0 + 0.0 002 + 0000 0.0 + 0.0 000 + 0000 00 + 000 0000 02200020 .00 0.0 0M20 11 .1 11 1. .1 .1 <0: + 2 0: 00.0.0 00.0 00.0.0 0.0 0000.0 0000 00.0.0 0.0 0000.0 0000 00.0 000 0000 02000020 2002 00.0 + 00.0 0.0 + 0.0 000 + 0002 0.0 + 0.0 000 + 0000 00 + 000 0000 02200020 .00 0.0 0020 1 1. .1 1 1 1. <02 + 000202 00.0 + 00.0 00.0 + 0.0 00000 + 0000 00.0 + 0.0 0000. + 0000 00 + 000 0000 00000020 000202 099000 89090 0:00: N9090 00000 0003203802: gee0fit 1. 1. 1. 1. 1. 1. <02 + 2 02 00.0.0 00.0 00.0.0 0.0 00.0 000 0.0.0 0.0 000.0 000 00.0 000 002000 00202 2002 00.0 + 00.0 00.0 + 0.2 00 + 000 0.0 + 0.0 00 + 000 00 + 000 002000 00202 200202 07908; 99090 2000 @9090 0000 0000 .000 0002 g0\00v 2000 g0\002 2000 200 <20 \ <22 020002 200200000 020000020 <22 <20 000000000 00 0020 000002002 0.e~em meeeeegn ge .ngume pmgee .mgpces m.~ 00 eegeugmzeem 020 00205000g0 seepeguec ucegeeeee ewe mgeeees :eeumee: ee 002020220005 02000 020520211.m0 mgm<0 68 treatment effect on cell concentration up to this age. In a reversal pattern, total RNA values were not significantly affected by the nutri- tional treatments, but RNA concentration was; heifers fed MGA had a significantly (P<0.01) greater RNA concentration. Therefore, it would appear that protein synthesis had been stimulated by MGA. Although this fact is suggested, RNA to DNA ratios revealed no detectable differences (P>0.20). Thus it would seem that MGA fed with the high level of nutrition from 2.5 months of age to first estrus had stimulated cellular growth to a certain extent and most likely protein synthesis on a cellular basis. Effects of MGA on nucleic acids become quite conclusive after studying data obtained when the heifers reached breeding size (Table 15 and Appendix III). In each of the two groups fed the high level plus MGA, 7 of the 10 heifers exhibited mammary proliferation, whereas only 5 of 10 heifers in the group fed the normal level plus MGA showed a response. Total mammary DNA of heifers fed the high level plus MGA was about 60 percent greater (P<0.01) than the value for heifers fed just the high level, while the value for heifers fed the normal level plus MGA was about 30 percent greater (P<0.20) than the value for heifers fed just the normal level. No apparent beneficial effect resulted from com— mencing MGA feeding at 2.5 months of age rather than after first estrus. Total RNA showed trends similar to those for DNA, with the value being about 78 percent greater (P<0.01) when MGA was given with the high level of nutrition than when it was not, and about 38 percent greater (P<0.20) when given with the normal level than when it was not. Examining the data on a concentration basis led to the same findings. Both DNA and RNA per gram of tissue were significantly greater (P<0.01) for the groups fed MGA, indicating more cells and more protein synthesis 69 per gram of tissue. RNA to DNA ratios were not significantly different, implying the same degree of protein synthesis occurring per cell for the heifers on the various nutritional treatments. The values obtained were about 50 percent greater than the value reported by Sinha and Tucker (1969) for 12-month old Holstein heifers. A difference among the treatment groups in the type of mammary protein being synthesized was suggested by obser- vations at slaughter. Most of the heifers fed the normal or high level had only a small quantity of a nearly clear fluid in the mammary gland, while in the glands of most of the heifers fed MGA there was a rather large volume of a cloudy, milky-looking substance. Whether this secretion and the nucleic acids changes were caused by progesterone and estrogen activity or by corticoid activity of MGA, or by certain pituitary hormones, or by synergism of several of these hormones is not known. Certain structural differences were also observed in the glands. The parenchymal tissue appeared pinker and the duct system seemed more developed in mammary glands of heifers fed MGA than in the glands of heifers not fed MGA. Examination of the histological sections revealed that the degree of ductular development was greater in heifers fed MGA. However, no satis- factory method was found to quantitatively measure the development. Representative rear view pictures of the mammary glands as they appeared at breeding size on heifers fed the normal level, high level, and high level plus MGA are shown in Figure 3. D. Adrenal Gland Changes l. Adrenal Weights Adrenal weights increased about 80 percent from 2.5 months to first estrus as shown in Table 16 and Appendix III. There was no significant effect (P>0.10) of the various nutritional treatments on actual adrenal weights or weight per 100 kg body weight at first estrus. By the time the ' Normal level High level High level plus MGA Figure 3.——Rear view photographs of mammary glands on Holstein heifers at breeding size that were fed normal level, high level, and high level plus MGA nutrition treatments from 2.5 months of age. . 00.0v2vmo.ov 00205000g0 ge20e gee 00200> 2020 ge000gm 0002eeeee2meme Mmo.oVev 00205000g0 <02 + 2me2 gee 00200> 2020 ge000gm 0002000e02meme .Amo.oVev 02espeeg0 2020 geueegm 0eu2eeeee2meme .meueegeee 0202000g0 2002 ee 022000 umgee 00 00200>2 .mgeeee2 om gee 020 2ee23 00200> 02g000 00gee pceeueegu 2002 peeexe .mgeeee2 oe gee mgegge 0g002000 gee20 020 02002 ege 00200>0 00.0.0 00.0 00.0.0 00.0 00.0.0 00.0 00.0.0 20.0 2.0.0 0.00 0000 00000020 002000 0MMfie 2mwwm + . 00.0.0 00.0 00.0.0 00.. 00.0.0 00.0 00.0.0 00.0 0.0.0 0.00 0020 02000020 .00 0.0 0020 .1 .1 .1 1. 1. <0: + 2002 00.0.0 00.0 200.0.0 00.2 00.0.0 00.0 000.0.0 00.0 0.0.0 0.00 0000 02000020 . . 2002 00.0 + 00.0 00.0 + 00.0 00.0 + 00.0 00.0 + 00.0 0.0 + 0.00 0000 00000020 00 0 0 0022 .1 .1 .1 1. 1. <0: + eeegez 000.0 + 00.0 000.0 + 00.0 00.0 + 00.0 000.0 + 00.0 0.0 + 0.02 0000 02000020 000202 00.0.0 00.0 00.0.0 00.0 00.0.0 00.0 20.0.0 00.. 0.0.0 0.00 002000 00202 .00 0.0 002g 00 .1 1. .1 1. .1 2 <02 + 2002 00.0.0 00.0 00.0.0 00.0 00.0.0 00.0 00.0.0 00.0 0.0.0 0.2. 002000 00202 2002 00.0 + 00.0 000.0 + 00.0 00.0 + 00.0 00.0 + 00.0 0.0 + 0.00 002000 00202 000202 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 0.0 0 0.0 .00 0.0 000202 1111-----1111111111120021111111111-111111 2002 202 megeezeeuem 00002000002 emceegeeeeu 20003 02mewz g002m2000 02msueegu ~02mgee 002e~ xepgee ee 20003 xepgeu eegeee ee 0000 2e000g022 0.0000 m2weeeg2 ge .022000 pmgee .0202ee 0.0 00 00200202000 020 00202000g0 2e0002022 02egeee00 ewe 0geeee2 2ee0mee: ee 002esmg20002 e200m 0020g0<11.0_ ~2m<0 72 heifers had grown from first estrus to breeding size the adrenals increased another 2 to 4 grams in weight, depending on the nutritional treatment. Again no significant differences were detected among nutritional treatments in the actual adrenal weights at breeding size. But, when expressed per 100 kg body weight, adrenals of the heifers fed the high level plus MGA weighed significantly less (P<0.0l) than those of heifers fed the high level. These adrenal weight data contrast with Zimbelman and Smith's (1966b) data. They found that long term MGA administration increased adrenal weights when compared with controls. This they took as further supportive evidence that the ovarian follicles of MGA fed heifers were secreting estrogens. Such evidence is not available from this study. (Adrenal weights obtained in this study agree fairly well with those of Desjardins and Hafs (l966) for heifers of comparable ages. 2. Adrenal Cortex Histology Total width of the cortex increased between 0.2 and 0.3 mm from 2.5 months of age to first estrus for all nutritional treatment groups (Table l6 and Appendix III). No significant difference (P>0.l0) among the group values existed at first estrus. But this was no longer true at breeding size, as heifers fed MGA had adrenal cortex widths that were significantly smaller (P<0.05) than their normal and high level controls (Table l6 and Appendix III). Perhaps this observation was reflected in the decreased per unit weights as discussed in the previous section. From the data on widths of the various cortex zones at breeding size (Table 16 and Appendix III), it is evident that MGA decreased the fasci— culata zone width (P<0.05) in both the normal level plus MGA and high level plus MGA heifers. Also, the reticularis zone width of the normal level plus MGA heifers was significantly less (P<0.05) than for the normal level heifers. The width of the glomerulosa zone did not change due to 73 nutritional treatments or with increasing size of the heifers. Desjardins (l966) found this also, although his values were somewhat smaller. Since the fasciculata and reticularis zones secrete glucocorticoids, and since MGA has glucocorticoid activity (Duncan et_gl;, l964), it is tempting to speculate that MGA acts directly on the adrenal cortex to decrease the fasciculata zone width, or that perhaps MGA causes a reduction in the release of ACTH from the anterior pituitary. Preventing ACTH release could be by direct action on the anterior pituitary or via affecting release of corticotropin releasing factor from the hypothalamus. Because of the lowered circulating levels of ACTH to stimulate the fasciculata and reticularis zones, these zones would decrease somewhat in width and per— haps function. Data supporting this last statement about a functional decline are presented by Roger w. Purchas (l970). He measured the adrenal and plasma corticoids of the heifers fed the normal level, high level, and high level plus MGA and slaughtered at breeding size. Heifers fed MGA had significantly lower (P<0.05) cortisol concentrations in both the adrenals and plasma than the heifers fed either the normal or high nutritional levels without MGA. One might then conclude that MGA, through a positive feedback mechanism, causes the glucocorticoid secreting adrenal cortex zones to decrease in both width and function. However, Purchas (1970) found a nonsignificant correlation between adrenal cortex width and adrenal corticoid content which would not support such a conclusion. Why MGA caused a decreased fasciculata width when fed with both levels of nutrition, but a decreased reticularis width when fed with only the normal level is not known. Perhaps there is an interaction between level of nutrition and MGA in the manner by which MGA affects the adrenal cortex. 74 E. Changes in the Pituitary Height and Hormones l. Pituitary Weight Weights of the total pituitary and its two parts as influenced by body size and treatments are shown in Table l7 and Appendix IV. Weight of the total pituitary increased about 50 percent between 2.5 months and first estrus, with the nutritional treatments producing no detectable difference (P>0.05). Between first estrus and breeding size there was only a 20 to 30 percent increase in total pituitary weight in all nutri- tional treatment groups. Again no significant differences existed (P>0.l0) between the treatment values, although values for heifers fed MGA were the largest. These observations agree with those of Zimbelman (l966) who reported that MGA does not significantly affect pituitary weights. Total pituitary weight values obtained in this study also agree closely with those reported by Desjardins (1966) for heifers of comparable ages. Anterior pituitary weight changes followed the same pattern of the total pituitary (Table l7 and Appendix IV). However, at breeding size, heifers fed the high level plus MGA had significantly heavier (P<0.025) anterior pituitaries than heifers fed the high level without MGA. Even per 100 kg body weight this difference was still significant (P<0.l0). The value for heifers fed the normal level plus MGA was not significantly different (P>0.l0) from that of the normal level without MGA. Thus, it appears that feeding heifers a high level of nutrition plus MGA resulted in enlarged anterior pituitaries when compared with values for high level ones. There appears to be no simple explanation for the elevated anterior pituitary weights. Zimbelman (1966) found elevated levels of LH in the pituitaries of heifers fed MGA. So, an immediate explanation for the increased anterior pituitary weights is that they are associated with increased LH content. However, as will be discussed in the next section, 75 TABLE l7.—-Pituitary weights of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size.a Nutrition Time of Total Anterior Posterior treatment slaughter pituitary pituitary pituitary ----------------- (g)------------------- Normal 2.5 mo. 0.99 :_0.08 76 :_0.06 0.18 :_0.02 Normal First estrus l.64 : 0.l0 .26 :_0.09 0.33 :_0.0l High First estrus l.48 :_o.04 .10 :_o.03 0.34 :_o.o1 High + MGA b from 2.5 mo. First estrus l.47 :_0.07 ll i_0.07 0.33 + 0.01 Normal Breeding size 1.72 :_0.l0 .35 + 0.07 0.34 :_0.03 Normal + MGA from 2.5 mo. Breeding size l.80 :_0.08 .39 + 0.07 0.37 :_0.02 High Breeding size l.74 :_0.07 .26 + 0.05 0.40 :_0.03 High + MGA from 2.5 mo. Breeding size 1.92 + 0.14 .54 + 0.13C 0.35 + 0.02 High + MGA c after first Breeding size l.93 + 0.10 5l + 0.08 0.34 + 0.02 estrus " T' " aValues are means and their standard errors for l0 heifers, except the high treatment first estrus values which are bValues at first estrus of high treatment pairmates. for 20 heifers. cSignificantly greater than value for high treatment (P<0.025). 76 in this study anterior pituitary LH values were not affected by MGA. Since the fasciculata zone of the adrenals was reduced in width as already discussed, perhaps ACTH release was inhibited and the pituitary build up of ACTH was reflected in the anterior pituitary weight. Posterior pituitary weights nearly doubled from 2.5 months of age to first estrus (Table 17 and Appendix IV), but values at breeding size were not changed from those at first estrus. Furthermore, the different nutritional treatments produced no differences in posterior pituitary weights at first estrus or breeding size. Since the posterior pituitary is derived from neural tissue which undergoes mainly prenatal growth, this finding might have been expected. Still, Desjardins (l966) found that heifer posterior pituitary weights generally increased from l to l2 months of age, suggesting more postnatal growth than observed in this study. 2. Hormones in the Anterior Pituitary Data on the pituitary gonadotropins and prolactin are presented in the study while growth hormone data are presented by Roger N. Purchas (l970). Levels of the gonadotropins and prolactin in the anterior pituitary are shown in Table l8 and Appendix IV. LH concentration values generally increased from 2.5 months to first estrus, with some additional increase within a nutritional treatment from first estrus to breeding size. The values at first estrus did not differ significantly (P>0.l0) among the nutritional treatment groups. Breeding size values also showed little effect of nutritional treatment, except the value for heifers fed the normal level without MGA was slightly greater (P<0.l0) than the other treat- ment values. Since all heifers were slaughtered at a similar stage of the estrous cycle, this difference was not expected. The LH concentration values were generally smaller than those reported by Desjardins (1966) and Zimbelman (1966). But the fact that they used an LH bioassay whereas a 77 mmzpm> masamm pme_m pcmEpmmgp sow; uamuxm .AoP.ovav mucmsummgp can» smummso XFucmuwmwcmwmm .Amo.0vav mm=~m> ucmsumwsp smguo may cusp mmmF »_pcmuwmwcmwmu .Ao_.ovmv mmzpm> pcmspmmg» emspo mg» can» memmsm »_»:muvewcmwmu .mmmemen acmEummsp saw; mo magpmm pms_m um mmzpw>n .msmmwm; om Lo; wee gown: .msmwwm; op com mgogsm ugmucmpm gems“ use mcmme use mm:_m>m mm.“ mm? New.“ mmm ee_.u PN~_ Fo.o.u Pp.o e_.o.H mm.o o_.o.H eN.F eNWm meeeeeem assume “mete eewee 1. .1 .1 .1 1. .1 «oz + e e: mm + mom New + _mo_ mmm + emem _o.o + e_.o ow.o + N~.o oe.o + eN.F mN_m meweeeem .oe m.~ emse .1 1. .1 .. .1 .1 <9: + e *1 NF.” om_ m__.w mes omm.w Fmom No.0.” mp.o PF.O.H om.o mm.o.w we.. eNem meeemeem ewe: mm + com eo__ + ooe Awe + mmm_ emo.o + m".o eso.o + m~.o m_.o + ee._ seem meweeeem .oe m.~ ease 1. . 11 11 .1 1. 11 0.l0) in content due to the nutritional treatments were detected at first estrus or breeding size. FSH concentration values decreased markedly from 2.5 months to first estrus, with no significant (P>0.l0) nutritional treatment differences present at first estrus (Table 18 and Appendix IV). Desjardins (l966) also found a precipitous drop in FSH concentration between 2 and 3 months of age. Perhaps this FSH decline is associated in some manner with the gradual processes involved in sexual maturation and the occurrence of first estrus. From first estrus to breeding size, the values remained relatively the same, with perhaps a slight increase within a particular nutritional treatment. While the values at breeding size are about one half the magnitude of Zimbelman's (l966) values for pregnant heifers, the two sets of data agree that MGA does not affect pituitary FSH concentration. However, there is one exception in that heifers at breeding size which were fed the normal level plus MGA had significantly smaller (P<0.05) pituitary FSH concentrations than all other nutritional treatment values at both breeding size and first estrus. This finding would suggest a depressing interaction effect of the normal level and MGA on pituitary FSH concen- tration. Such an interaction does not seem plausible. Due to increased concentration, the pituitary FSH content at 2.5 months was as large as the first estrus values. Values at breeding size were about 35 percent greater than the first estrus values. No nutritional 79 treatment produced significantly different values (P>0.lO) at first estrus or breeding size, except for the normal level plus MGA effect, as already discussed regarding concentration changes. Pituitary prolactin concentration values were unaffected by the nutritional treatments or body size when comparing the values at 2.5 months and first estrus (Table l8 and Appendix IV). Sinha and Tucker (l969) also found no appreciable difference in pituitary prolactin values in heifers of comparable ages. However, at breeding size heifers fed the normal level and normal level plus MGA had slightly larger (P<0.l0) prolactin concentrations than heifers fed the high level plus MGA. Since the value for heifers fed the high level without MGA was also somewhat lower than that of heifers fed the normal level or normal level plus MGA, an effect of nutritional level is implied. However, analysis of the data showed such an effect did not exist (P>0.l0). Maybe heifers fed the nor- mal level and normal level plus MGA were experiencing elevated pituitary prolactin levels concomitant with changes in mammary biochemical parameters, as proposed by Sinha and Tucker (1969) for lZ-month old heifers in their study. Pituitary prolactin content values nearly doubled from 2.5 months to first estrus. But from first estrus to breeding size only about a 25 percent increase resulted. No nutritional treatment effect was detectable at either age though the normal level and normal level plus MGA values were the largest. Thus, the concentration differences observed at breeding size were not significantly reflected in total pituitary prolactin content. 3. Hormones in the Blood Plasma LH concentration in the blood plasma of heifers fed the normal level without MGA did not differ at 2.5 months and first estrus (Table l9 and Appendix IV). However, heifers fed the high level and high level plus MGA 80 TABLE l9.-—Hormones in the blood plasma of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size.a Nutrition Time of Concentration Contentg treatment slaughter LH Prolactin LH Prolactin ------ (ng/ml)------ -----(ug)--—------- Normal 2.5 mo. 2.3 :_0.3 58 :_9 8 :_l l96 1.33 Normal First estrus 2.4 1 0.2c 92 1160' 21 1 2 852 1153d High First estrus 3.0 1 0.5 42 110 25 13 335 1 94 High + MGA d from 2.5 mo. First estrusb 3.8 1 0.3 37 1 7 33 1 3h 315 1 68 Normal Breeding size 2.5 1 0.2 112 112f 32 1 21419 1151f Normal + MGA . from 2.5 mo. Breeding size 3.3 1 0.363 52 111 41 1 41 755 1124 High Breeding size 2.8 :_0.2 8l :_l6 36 :_3 l035 :_2l9 High + MGA from 2.5 mo. Breeding size 2.6 :_0.2 95 :_l7 36 :_3 l34l :_253 High + MGA after first estrus Breeding size 3.2 1 0.3e 79 112 43 1 5“ 1075 1170 aValues are means and their standard errors for l0 heifers, except high treatment first estrus values which are for 20 heifers. cValues at first estrus of high treatment pairmates. Significantly less than the other treatment values (P<0.05). dSignificantly greater than the other treatment values and the 2.5 month value (P<0.01). eSignificantly greater than the normal and high + MGA from 2.5 mo. treatment values (P<0.05). Significantly greater than the normal + MGA treatment value (P<0.l0). gEstimated by assuming plasma volume to be 3.5 percent of the heifer's slaughter weight. Significantly greater than the other treatment values P<0.l0 . iSignificantly greater than the normal treatment value P<0.l0 . 8l had plasma LH concentrations which were singificantly greater at first estrus (P<0.05) than the value of heifers fed the normal level without MGA. Furthermore, the value for heifers fed the high level plus MGA was signifi- cantly greater (P<0.0l) than first estrus values for heifers fed the normal or high level without MGA. This suggests an effect of high level of nutrition which is augmented by MGA. Values at breeding size further suggest that heifers fed MGA had elevated plasma LH concentrations, as two of the three groups fed MGA had elevated LH levels (P<0.05). Since heifers fed MGA had not consumed the drug for 48 hours prior to slaughter, perhaps during this time the drug lost its inhibitory action on cyclic LH release as Zimbelman (1966) proposed, thereby explaining the elevated blood levels. Pituitary LH concentration values, however, did not indicate LH release, as discussed previously. Furthermore, all heifers were supposedly slaughtered in a similar hormonal condition, which would not support this explanation. As an approximation of the total LH content in the blood, plasma volume, as estimated at 3.5 percent of a heifers's slaughter weight, was multiplied by the concentration values. Granted this procedure is sub- Ject to error, it provided an estimate of the total blood LH content. With this mathematical calculation, it was found, as shown in Table 19 and Appendix IV, that heifers slaughtered at first estrus which had been fed MGA, and two of the three groups slaughtered at breeding size which had received MGA had greater total plasma LH contents (P<0.lO) than heifers which had not been fed the drug. These data simply reflect the plasma concentration data, and indicate that more LH was available to the end organs in the heifers fed MGA. However, since all heifers were slaugh- tered at the same stage of the estrous cycle, the effects of MGA on LH values at other times in the estrous cycle are not known. 82 Plasma prolactin data as shown in Table l9 present a different pic- ture from that of the LH plasma concentration. Data at first estrus reveal that heifers fed the normal level without MGA had the largest concentration (P<0.0l) of prolactin. This occurrence might coincide with a period of rapid mammary gland growth. Sinha and Tucker (l969) suggest this explanation for their data on pituitary prolactin concentration of 9-month old heifers, the approximate age of normal level heifers at first estrus in this study. At breeding size the concentration value of heifers fed the normal level without MGA was significantly greater (P<0.025) than the value of heifers fed the normal level plus MGA. This finding suggests an inhibitory effect of MGA on plasma prolactin concentration when administered only with the normal nutritional level. Such an action does not seem plausible. It may be that the elevated value at breeding size of heifers fed the normal level without MGA was associated in some way with mammary gland development. Such an explanation supports Sinha and Tucker's (l969) pituitary prolactin data on lZ-month old heifers. Total prolactin content in the blood, like LH content, reflects the concentration data already discussed. That the heifers fed the normal level without MGA had the largest value (P<0.01) at first estrus, and a value significantly greater (P<0.025) than that of heifers fed the normal Ilevel plus MGA at breeding size suggests that prolactin was performing, or at least associated with, some function in heifers fed the normal nutritional level. Whether this function concerned mammary growth or something else is not known. 4. Plasma to Pituitary Hormone Content Ratios To obtain an indication of the release to storage ratio for LH, total plasma content was divided by the total anterior pituitary content. As shown in Table 20 and Appendix IV, the ratio value increased from two to 83 TABLE 20.--Plasma to pituitary hormone ratios of Holstein heifers fed different nutrition treatments and slaughtered at 2.5 months, first estrus, or breeding size.a Nutrition Time of treatment slaughter LHb Prolactinc Normal 2.5 months 7.64 1_0.96 2.41 1_0.43 Normal First estrus 12.22 11.97d 5.52 10.666 High First estrus 23.29 :_4.82 2.73 :_0.60 High + MGA from 2.5 mo. First estrus l9.ll 1_2.15 2.16 1_0.42 Normal Breeding size 13.85 11.97d 7.65 11.45f Normal + MGA from 2.5 mo. Breeding Size 23.97 i_4.09 4.17 :_0.94 High Breeding size 23.72 1 5.32 6.52 11.51 High + MGA ' from 2.5 mo. Breeding size 19.84 i 5.10 7.46 :_1.47 High + MGA after first estrus Breeding size 25.25 + 3.63 8.14 + 1.43 aValues are means and their standard errors for 10 heifers, except high treatment first estrus values which are for 20 heifers. bug plasma LH + mg pituitary LH. cug plasma prolactin 9 U9 pituitary prolactin. dSignificantly less than the other treatment values (P<0.lO). eSignificantly greater than the other treatment values (P<0.01). fSignificantly greater than the normal + MGA treatment value (P<0.lO). 84 three fold between 2.5 months and first estrus, but then remained about the same within a nutritional treatment between first estrus and breeding size. This suggests that after first estrus occurs, the plasma and pituitary LH contents establish a certain ratio which does not change, at least up to breeding size. The data suggest a nutritional level effect on the LH ratio. At both first estrus and breeding size the value of heifers fed the normal level without MGA was significantly smaller (P<0.10) than the other nutritional treatment values, suggesting that a lower ratio is associated with a normal level of nutrition. However, when MGA was fed with the normal level, the hormone content ratio was comparable to that of heifers fed the high nutritional level, without or with MGA, which does not fit the ratio-nutritional level hypothesis. Plasma-pituitary prolactin ratios (Table 20) show that the first estrus value of heifers fed the normal level without MGA was significantly greater (P<0.01) than the other nutritional treatment values. But, at breeding size heifers fed the normal level without MGA had a value which was significantly greater (P<0.10) than those fed the normal level plus MGA. The ratio values for each nutritional treatment increased from first estrus to breeding size, which was different from the LH data. Also, the prolactin ratios show generally the reverse patterns of the LH ratios. suggesting different regulatory pathways for the two hormones. 5. Correlations Between Anterior Pituitary and Plasma Hormone Values To study pituitary and plasma hormone data and find a singificant pattern existing between them would be an endocrinologist's desire. Such findings were hoped for in this study. However, when pituitary LH and prolactin concentrations and also total pituitary contents were correlated with their respective plasma concentration values for the various nutri- tional treatments at the three different slaughter ages, no significant 85 correlations existed (P>0.05). In fact, of the 36 coefficients calculated only the one between pituitary and plasma prolactin concentrations for heifers fed the high level of nutrition plus MGA after first estrus and slaughtered at breeding size approached significance. It was a positive correlation suggesting that plasma prolactin concentration changed in the same direction as pituitary prolactin concentration. So because of the nonsignificant correlations, no apparent conclusions can be made which relate pituitary and plasma hormone levels. F. Data on the Bred Heifers l. Estrous Cycle Data Lengths of the estrous cycles were recorded for all heifers kept beyond first estrus and not fed MGA. They ranged from 17 to 24 days, the usually accepted normal range, for all heifers except one. She consis- tently had cycles that ranged from 29 to 32 days in length. A few heifers developed cystic corpora lutea which resulted in abnormally long cycles. If the cysts did not spontaneously recover in 30 to 60 days, the heifers were given a 5000-IU injection of human chorionic gonadotropin intramuscularly to hasten recovery. This treatment seemed beneficial. Cycle lengths were not affected by the normal and high nutritional levels, and the first cycle was of the same length as all subsequent ones. When heifers fed the high level plus MGA reached breeding size, MGA was withdrawn from their ration. Those that had received MGA from 2.5 months averaged 19.7 days after withdrawal before estrus occurred. Four heifers were in estrus on the second or third day after withdrawal, while the other six did not exhibit estrus until 12, 21, 27, 28, 35, and 64 days after withdrawal. 0f the heifers that did not receive MGA until after first estrus, six were in estrus on the second or third day, while the 86 other four went 5, 10, 16, and 30 days from withdrawal to first estrus. The 10 heifers had a mean interval of 7.7 days between withdrawal and estrus. This value, however, was not significantly different (P>0.lO) from the value of 19.7 days for heifers fed MGA from 2.5 months. Clearly, some of the long cycles could have resulted from silent or missed estrual periods, but an effect from prepuberal MGA administration is suggested. Still, once heifers started cycling, the cycles were of normal duration. Perhaps commencing long term MGA administration prepuberally results in a longer carryover effect in certain heifers than the approximately 2 to 7 days observed by Zimbelman and Smith (1966a) after a 16-day administration period. Or maybe the pathways for eliminating MGA from the body are not as functional in certain heifers as in others. And it is possible that the hypothalamus requires a longer recovery time in certain heifers. Whatever the cause or causes, the situation demands further investigation. 2. Breeding Data At first breeding, heifers fed the normal level without MGA were significantly older (P<0.05) than heifers fed the other nutritional treat- ments (Table 21 and Appendix I). Also, heifers fed the high level without MGA were significantly younger (P<0.05) at first breeding than those fed the high level plus MGA. Since this significant age difference did not exist at breeding size (Table 21), it apparently resulted from the interval between MGA withdrawal and first breeding as discussed in the previous section. The time interval between breeding size and first breeding (Table 21 and Appendix I) for heifers fed a normal or high level was caused by the time lapse after reaching breeding size until the heifers were observed in estrus and bred. Because of this time interval, withers heights at first breeding exceeded 120 cm. Withers height at first breeding, however, did not differ among the nutritional treatment groups, but body 87 TABLE 21.--Age and body size at breeding size and first breeding of Holstein heifers fed different nutrition treatments.a First breeding Age at Nutrition breeding Treatment size Age Weight Height ---------- (m0)-------t--- (kg) (cm) Normal 12.5 1 0.3b 13.11 0.4C 363 1 9 121.2 1 0.2 High 11.0 1 0.2 11.3 1 0.2c 358 111 121.0 1 0.3 High + MGA , i d from 2.5 mo. 11.4 1 0.4 12.2 1 0.4 401112 121.3 1 0.4 High + MGA after first estrus 11.7 1 0.4 12.11 0.4 410 1 6d 120.7 1 0.2 aValues are means and their standard errors for 10 heifers. bSignificantly different from the other treatment values (P<0.01). CSignificantly different from the other treatment values (P<0.05). ( dSignificantly different from the normal and high treatment values P<0.0] . weight revealed the stimulatory affect of MGA as noted previously. Heifers fed the normal or high level without MGA weighed the same at first breeding. As emphasized previously in the section on growth, that heifers fed the normal level were bred by about 13 months of age is most exciting. If dairy farmers could be challenged to feed their heifers at a level similar to the normal nutritional level in this study, the dairy industry would benefit greatly. 88 Breeding data for only the heifers that conceived are shown in Table 22 and Appendix V. Due to apparent infertility, only 36 of the 40 heifers conceived. One heifer fed the high level plus MGA after first estrus finally conceived to the eleventh service when she was about 2 years old. Because of her age, she was excluded from the data in Table 22. The four infertile heifers were bred 10 times to one bull and TABLE 22.--Age and conception data for Holstein heifers fed different nutrition treatments.a Age at Age at Age Services Nutrition Number breeding first at per f treatment heifers size breeding conceptionf conception Mmfl 9 12610H13310N147107 23107 mm 9 1091051L310f134107 32109 High + MBA from 2.5 mo. 10 11.4 1_0.4 12.2 1_0.4 14.7 1_0.7 3.4 1_0.7 High + MGA . after first estrus 7 12.2 :_0.4 12.6 :_0.4 14.6 i_0.8 3.0 1 0.8 aValues are means and their standard errors. bSignificantly different from the high and high + MGA from 2.5 mo. treatment values (P<0.01). cSignificantly different from the high + MGA after first estrus treatment values (P<0.01). dSignificantly different from the other treatment values (P<0.01). eSignificantly different from the high + MGA treatment values (P>0.05). fNo significant differences in the values (P>0.lO). 89 an eleventh time to a different bull. Since they were still not pregnant, they were slaughtered. All four had some reproductive tract disorder which could have caused the infertility. Two cases of chronic endometri- tis, one extremely fibrous endometrium, and one case where an ovary was surrounded by fibrous tissue may have caused the infertility. Although age at first breeding differed among the nutritional treatments, age at conception (Table 22 and Appendix V) did not differ significantly (P>0.lO). This was the result of more services per concep- tion, though the difference was not significant (P>0.lO), for heifers fed the high level and high level plus MGA. Because of the lack of a significant difference in services per conception among the nutritional treatments, one must conclude the high level and high level plus MGA had no detrimental effect on conception. This agrees with the results of Reid gt_al;, (1964) and several others as cited in the literature review. However, since heifers fed the various nutritional treatments were of different ages at first breeding but not at conception, an effect of nutritional level on conception is strongly implicated. Perhaps the increased amount of pelvic area fat observed in heifers fed the high level and high level plus MGA that were slaughtered at breeding size also existed in heifers that were bred, and thereby in some manner affected fertility. Since heifers fed the normal level required more services than expected, perhaps the outbreak of IBR in the herd when the hefiers were about 9 months old increased the services required per conception by all nutritional treatment groups. However, heifers were vaccinated for IBR and seemingly recovered within a month with no after effects. Although heifers fed MGA with the high level of nutrition required as many services per conception and as many heifers conceived to the first service as did heifers fed the high level, data from our laboratory using rabbits 90 (Pritchard gt_gl1, 1969) and that data of Quinlivan and Robinson (1969) with ewes suggest that MGA may inhibit sperm transportation at the first service after withdrawal of the progestagen. 3. Parturition Data 0f the 36 heifers that conceived, parturition data were available on only 34 of them. Observations at parturition are shown in Table 23 and Appendix V. In studying the data, it should be remembered that all weights, withers heights measurements, and subjective ratings of dystocia were made by the workmen at Driggs Dairy. Although some reservation may exist as to accuracy, the data are worthy of examination. Suffice it to say at this point that the nutritional treatments produced no significant differences (P>0.lO) in the data for any category listed in Table 23 and Appendix V. From the dams' weights before and after parturition and dystocia ratings, it appears that the smaller heifers encountered more difficulty at parturition. However, these heifers were not any smaller in skeletal size as indicated by withers heights. Correlating the dams' weights before and after parturition with dystocia ratings indicated no signifi- cant correlation (P>0.05). Thus, size of dam had no apparent effect on calving difficulty. Calf birth weight and dystocia rating data hint that the heavier calves were associated with a more difficult parturition. This in fact was true, for the correlation between these two parameters was highly significant (P<0.01). When birth weights of the calves are expressed as a percentage of both the pre- and postpartum dam weights, the data suggest that when calf weight as a percent of the prepartum dam weight increases, the calving difficulty rating increases. However, only the correlation coefficient between calf weight as a percent of postpartum dam weight and dystocia rating was significant (P<0.05). Thus, one can 91 .mgoeem eeeecepm even“ use mcees wee mm=Fe> .Ao_.onav agomeueu ace Low waspe> pcmEueeeu ecu c? mmocmemewwe acerewcmrm oze eoo.o m mmo.o “00.0 m “No.0 eoo.o m aeo.o 500.0.“ eeo.o peeve: Eaeeeaemea moo.o + mwo.o moo.o + _uo.o voo.o + mmo.o moo.o.H omo.o azure: Eaaeeamea see , peeve: epeu em“ ee New em we“ oe mm“ mm “Dev peeve: e_eu m.e1+ 5., «.01+ m._ e.e1+ e._ m.o1+ e.e meeeee eeeoeuxe 8.0 + e.ee_ m.o + e.e~_ _._ + e.mep m.o + a.e~_ Asuv peeve; meeeeez oeum oee efium ewe mpHm wee e_um oee waxy eoeeeeseeea sueee “seem: om + mom my + Rpm op + oee a_ + eom A xv eoeeeeseeea eeoeee Hem_ez eeumee adueee “emcee Reneee A8v8< a o_ e m maeeee; e6 geese: magpmm umgw$ Lmuwm .OE m.N EOL$ cow: FwELoz :owmewLQ emmao osom11.mm u4m0.lO). 95 dix III) indicated the heifers fed MGA were commencing pregnancy with considerably more parenchymal tissue, it was anticipated that this difference would result in greater milk production after parturition. But since no significant differences (P>0.lO) existed among the nutri— tional treatment groups in the actual first 60 day milk production weights or in the estimated 305 day production values, such an occurrence did not happen. Perhaps the mammary gland growth that resulted from feeding MGA before the heifers were bred regressed during the first few months of pregnancy. Since MGA was not fed during pregnancy, the hormonal stimulus required for mammary proliferation was perhaps not sufficient the first several months after conception. Consequently, the enlarged glands may have regressed in development, and therefore heifers fed all nutritional treatments entered the latter half of pregnancy with the same degree of gland development. Although the milk production values in Table 25 and Appendix V were not significantly different (P>0.lO), the data hint that heifers fed the normal level were better milk producers. The lower milk production by heifers fed the high level of nutrition supports the conten— tion of Swanson (1960) that feeding above normal nutritional levels during growth and pregnancy results in lowered milk production. Since heifers in this study were not fed at an elevated level during pregnancy, this may explain why we did not get the dramatic difference in milk production that Swanson obtained. Perhaps it is the feeding of high nutritional levels during pregnancy that is associated with lowered milk production. 6. Nitrogen Balance Trials Unpublished data of the Upjohn Company show that feedlot heifers fed MGA gain faster than controls, but the increase is due partly to protein deposition and not solely to the accumulation of depot fat. Knowing this, 96 it was decided to conduct nitrogen balance trials on several of the heifers used in this study to ascertain if indeed heifers fed MGA were retaining more protein from their ration. Postpuberal cycling heifers ranging from 7 to 13 months of age were placed in metabolism stalls a few days after an estrus and allowed to acclimate for 5 to 10 days. Heifers of comparable age and size fed MGA were put in the stalls at the same time. They were fed their regular level of grain plus corn silage and hay ad lib. Data were collected for a 7-day period on the amount of feed consumed, and feces and urine excreted. Samples of the feed offered, feed not eaten, feces, and urine were collected and analyzed for nitrogen content by the Kjeldahl method. The nitrogen values were then converted to protein equivalent. The data obtained in Trial 1 are shown in Table 26. Heifers fed the normal level plus MGA retained about the same amount of protein daily as heifers fed the normal level without MGA. These data support the growth data which showed little or no stimulatory effect of MGA on weight increase when fed with the normal level of nutrition. But, data for heifers fed the high level plus MGA are very perplexing. At least during the collection period, heifers fed the high level plus MGA retained considerably less protein from the daily ration than heifers fed just the high level. This seemingly conflicts with growth and carcass evaluation data. Also, heifers fed the high level plus MGA excreted more protein in the urine as well as more urine than heifers fed the high level without MGA. To obtain further data on this phenomenon, an experiment was designed in which each of four heifers was to be fed both the normal and high nutritional levels, without and with MGA. Four regular university herd heifers about 12 months old with the stage of their estrous cycles unknown were placed in the metabolism stalls and allowed 2 weeks to .mceme wee mmape>e 97 _eee 0.e~ New Nee eke 0e0. <0: + :00: 0e0e e.~e ewe 0ee 00e e0e_ gee: _N0_ 000e, 0.0_ 0__ eoe 000 00_F <02 + :00: e000, e.~e e0e 00m e00 ee__ 50,: NNOP eoee e.ee me, Pee eee 0ee <0: + _eeeoz eeee 0.eP- 00.1 000 eee eee _eseoz ee0_ 0Ne~ e.ee 0e~ 00m Pee _ee <02 + Pesto: meme e._e 0pm _ep 00m 00s Peseoz 0N0? A00 1111111111111-11-11111A00-111111---1111111111 mE=_o> emceepme emceeume cempoea :Pmuoga vessecoo ucmsuemep geese: wcwgz cwmuoga N :wmuosa mcwga pmumu :wwuogm comuwgusz gmwwm: .N _eeee aee Lea eeee _.0_ e0 0N0 see 000 5.0 0 .02 e.~ Eeee emceepmg emceeume cemuoea cemuoea emszmcou mm< memeem; unusuemep wave: cemuoea N :wmuoga wave: Peume cemuoga Lucasz coeueeusz e00 000 _ _eeee a.mvcmEummLp cowuvaac Hcmmemmfi Um$ mgwmwwz Cmem—OI $0 aunt mucwpmn cmmoguw211.0N m4m0.lO) in body weight (255 :_4 vs 250 :_5 kg) or withers height (108.6 :_0.6 vs 109.2 :_0.7 cm). These data emphasize that first estrus is associated more with physical maturity than with calendar age. At breeding size (120 cm withers height), heifers fed the high level of nutrition were 11.4 :_0.4 months old while those fed the normal level were 12.5 :_0.2 months old (P<0.01). MGA fed at the rate of 0.45 mg per heifer per day with either the normal or high levels of nutrition did not significantly affect the ages at breeding size, indicating that MBA did not 99 100 affect skeletal growth. However, MFA increased body weight gains, but only when fed with the high level of nutrition (P<0.01). Heifers fed the high level of nutrition with MGA\hained faster (P<0.05) after about 5.5 months of age than heifers fed the high level alone. At breeding size, heifers that had been fed the high level plus MBA weighed about 35 kg more than heifers fed either the normal level, normal level plus MBA, or high level of nutrition. The time from first estrus to breeding size (about 3.5 months) was not significantly different (P>0.lO) for heifers fed either level of nutri- tion without or with MGA, indicating that level of nutrition or addition of MGA did not affect rate of skeletal growth after first estrus. Uterine weights for the various nutrition groups at first estrus or at breeding size were not significantly different (P>0.lO) when expressed per 100 kg body weight. Uterine nucleic acids concentrations at both first estrus and breeding size showed no significant differences (P>0.lO) in the normal and high nutritional level values. But, when ”CA was fed with both the normal and high levels, DNA concentration was generallv lower and RNA concentration generally higher than when the compound was not fed. These data implicate uterine hypertrophy associated with VGA feeding. Also suggestive of uterine hypertrOphy were the increased uterine epithe- lial cell heights, but only at breeding size, in heifers fed MGA. Ovarian weights were not affected by the nutritional treatments, but more large diameter fellicles were present on the ovaries of heifers fed MFA. Heights of the dissected parenchymal tissue from one half of the nemmary gland were not affected by the nutritional treatments at either first estrus or breeding size. Nucleic acids concentrations at first estrus suggested an elevated DNA value and showed a significantly increased (P<0.01) RNA value in heifers fed NRA. At breeding size both DNA and RNA 101 concentration values were significantly increased (P>0.01) in heifers fed MGA. MGA fed with the normal level of nutrition increased total mammary DNA content about 30 percent and RNA content about 38 percent over the values fer heifers fed the normal level alone. This stimulatory action of MGA was twice as great when it was fed with the high level of nutrition as compared to when it was fed with the normal level. Neither the paired adrenal weights nor the weights expressed per 100 kg body weight were affected significantly (P>0.lO) by the various nutritional treatments. However, at breeding size, HGA caused a signifi- cant decrease (P<0.05) in the width of the glucocorticoid producing fas- ciculata zone of the cortex. Since MGA is known to have glucocorticoid activity, some direct or indirect regulatory action of MGA on the adrenal cortex is suggested. The various nutritional treatments produced no significant differences (P>0.lO) in total, anterior, or posterior pituitary weights at first estrus, but heifers fed the high level of nutrition plus MGA had signifi- cantly larger (P<0.10) anterior pituitary weights per 100 kg body weight at breeding size than the other nutritional treatment values. No large dramatic differences in pituitary of plasma concentrations of LH, FSH, and prolactin resulted at first estrus or breeding size from feeding the various nutritional treatments. Correlations between pituitary concentration and plasma concentration, and between pituitary content and plasma concentration for LH and prolactin fer all nutritional treatments at the three slaughter times were not signi- ficant (P>0.05). The interval from MGA withdrawal until the heifers came in estrus was considerably longer, though not significantly so (P>0.lO), for heifers that received the compound from 2.5 months than for those that received 102 MGA only after first estrus (19.7 vs 7.7 days). Still, once estrous cycles commenced, they were of normal length (17-24 days) fer all MGA treated animals. Heifers that were bred and conceived were 14.7 :_0.7, 13.4 :_0.7, 14.7 :_0.7, and 14.6 :_0.8 months old at conception for normal level, high level, high level plus MGA from 2.5 months, and high level plus MBA from first estrus nutritional treatment groups, respectively. The number of services required per conception was 2.3 I 0.7, 3.2 :_0.9, 3.4 :_0.7, and 3.0 I 0.8 for the preceding resoective nutritional treatments. These different nutritional treatment values for age at conception or services per concep- tion were not significantly different (P>0.lO). At parturition no significant differences (P>0.lO) were found in body weights, withers heights or subjective dystocia ratings of the dams fed the various nutritional treatments prior to conception. Calf birth weights were not significantly different (P>0.lO) fer the two sires or the various nutritional treatments fed the dams prior to conception. But, the positive correlations between calf birth weight and dystocia rating, and the calf birth weight as a percentage of the dam's postpartum weight and dystocia rating were significant (P<0.05). Neither the actual milk production weights for the first 60 days of lactation nor the extended 305 day values were significantly different (P>0.lO) for heifers fed the various nutritional treatments prior to conception. Preliminary nitrogen balance trials revealed no effect on protein retention when MGA was fed with the normal level of nutrition. This agreed with weight gain data. But, feeding “GA with the high level of nutrition resulted in increased urine protein loss and a reduction in the amount of protein retained dailv from the ration when compared to high level controls. 103 These data do not agree with growth and slaughter data. So, from the data accumulated in this study, certain general conclu- sions can be made. rirowth rate is definitely affected by the level of nutrition; a high level accelerates weight gain but has little influence on rate of skeletal growth; and heifers fed a normal level, that is corn silage and hay free choice plus a small amount of grain daily, can grow to breeding size by 13 months of age. The reproductive tract is suffi- ciently mature at this age to permit breeding of the heifers. HGA does not affect skeletal growth and it accelerated weight gains only when fed with a high level of grain. No gross effect on hormone levels resulted from feeding MGA or the high level of nutrition. If conception rate were better than obtained in this study, feeding growing heifers a high level of nutri- tion may be advantageous. But, from this exneriment, since all heifers were the same age at first parturition, it is obvious that the high level is not practical. 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APPENDICES 112 113 APPENDIX I.--Age, body weight, and withers height of individual heifers at 2.5 months, first estrus, breeding size, first breeding, or slaughter. 2.5 Months First Estrus Group Heifer Withers Withers No. No. Weight Height Age Weight Height (kg) (cm) (m0) (kg) (cm) 1 206 74 80.0 8.0 209 102.0 209 77 84.0 11.5 318 118.0 212 120 94.0 8.8 270 108.5 215 77 87.0 9.5 209 109.0 232 85 87.0 10.7 232 105.5 250 118 88.0 8.5 270 114.0 273 85 84.0 9.2 225 109.5 275 94 84.0 8.3 234 107.0 284 118 87.0 7.7 234 108.0 286 121 87.5 8.9 280 111.5 Mean i SE 97:6 86.2:1.1 9.1:0.4 248:11 lO9.3:1.4 2 203 91 89.0 6.6 227 104.0 205 93 84.0 9.7 293 116.0 207 89 84.5 7.0 236 102.5 210 98 90.0 7.1 266 111.0 213 103 85.0 7.8 277 109.0 257 82 85.0 6.6 234 105.0 268 126 87.0 9.0 302 112.0 270 85 81.0 10.1 311 116.0 283 100 88.0 8.8 277 114.0 288 81 92.0 8.8 277 116.0 Mean i SE 95:4 86.6:1.0 8.1:0.4 270:9 110.6:1.6 114 Breeding Size First Breeding Withers Age Weight Age Weight Height (m0) (k9) (m0) (kg) (cm) 14.0 382 15.2 426 122.5 13.0 370 13.0 370 120.0 12.1 375 12.6 393 121.0 12.6 323 13.2 336 121.0 12.8 341 13.5 365 122.0 11.2 332 12.0 342 121.5 14.0 323 15.0 357 122.0 12.2 350 12.8 359 121.0 11.4 314 12.3 329 121.0 11.3 355 11.8 348 120.0 12.5:O.3 346:8 13.1i0.4 363:9 121.2i0.2 10.4 343 10.5 347 120.5 10.7 316 11.0 325 121.0 12.0 364 12.2 381 121.0 10.3 341 11.0 355 122.0 11.3 370 12.0 402 122.0 11.7 414 11.7 414 120.0 11.4 361 11.8 372 121.0 11.5 352 11.5 352 120.0 10.7 321 10.7 318 120.0 9.8 300 11.0 314 123.0 \ 11.0:0.2 348:10 11.3:0.2 358:11 121.0:0.3 115 2.5 Months First Estrus Group Heifer Withers Withers No. No. Weight Height Age Weight Height (kg) (cm) (m0) (kg) (cm) 3 208 98 87.0 6.5 239 105.0 211 96 91.5 7.0 282 108.5 220 86 85.5 7.7 234 103.0 231 106 85.5 8.5 361 115.5 236 87 85.0 6.8 227 103.5 259 91 87.0 9.0 323 115.0 261 95 88.0 9.1 334 113.0 269 84 85.5 8.6 275 109.5 272 105 85.0 9.3 343 117.0 280 112 86.5 6.7 230 106.0 Mean : SE 96:3 86.6:O.6 7.9:O.3 290:17 109.6:1.6 4 218 121 90.0 7.0 273 111.0 222 77 91.5 6.2 245 107.0 225 82 91.0 6.1 232 106.0 235 105 85.0 7.8 275 109.0 239 101 82.0 8.3 291 107.0 249 92 85.0 8.1 250 106.0 258 102 86.0 8.3 245 108.5 274 105 75.0 8.7 243 104.0 281 89 76.0 9.4 284 109.5 287 112 80.0 8.5 225 105.5 Mean : SE 99:4 84.1:1.9 7.8:0.3 256:7 107.3:O.7 116 Breeding Size First Breeding nmuu -' . Withers Age Weight Age Weight Height (m0) (k9) (m0) (kg) (cm) 10.1 368 10.7 383 121.0 10.3 380 10.7 393 121.0 14.0 416 14.0 416 120.0 12.0 455 13.0 491 122.0 11.8 377 12.8 401 121.0 10.6 370 13.3 409 125.0 11.4 384 12.0 390 121.0 11.8 345 12.8 357 120.5 11.7 395 12.0 406 120.5 9.9 357 10.5 365 121.0 11.4:0.4 385:10 12.2:O.4 401:12 121.3:0.4 10.3 368 10.5 376 120.5 10.0 389 10.1 398 120.5 10.0 366 10.7 384 121.5 11.5 409 12.5 425 122.5 12.0 420 12.5 425 121.0 11.6 417 12.0 439 120.0 13.3 398 13.3 401 120.0 13.0 420 13.0 423 120.0 13.3 409 13.5 409 120.0 12.4 405 13.0 420 121.0 ll.7:0.4 400:6 12.1:O.4 410:6 120.7:0.2 117 2.5 Months First Estrus Group Heifer Withers Withers No. No. Weight Height Age Weight Height (kg) (cm) (m0) (kg) (cm) 5 221 102 90.0 10.8 255 113.0 226 120 89.0 8.0 234 107.5 229 93 85.0 10.9 298 113.5 238 90 87.0 8.0 234 104.0 242 88 89.0 8.8 241 104.0 251 114 79.0 8.8 248 109.5 263 111 92.0 6.0 211 105.0 264 88 87.0 10.3 266 116.0 266 89 86.0 9.1 293 115.0 278 89 84.0 10.2 236 111.0 Mean : SE 98:4 86.8i1.1 9.1iO.5 252:9 109.8i1.4 6 204 96 93.0 7.6 280 111.0 214 85 82.0 8.3 259 104.5 217 100 84.5 6.5 202 102.5 223 90 ‘ 92.5 7.5 305 109.0 241 118 88.0 7.6 270 104.5 254 94 81.0 10.0 261 108.0 255 126 89.0 8.0 259 116.5 262 77 88.0 9.5 323 119.0 279 102 81.0 6.6 202 100.0 285 118 89.5 6.1 232 105.0 Mean : SE 101:5 86.8i1.4 7. 8:0. 4 259i12 108.0i1.9 118 Breeding Size Slaughter Withers Age Weight Age Weight Height (m0) (k9) (m0) (kg) (cm) 12.0 336 13.7 366 123.0 13.5 368 13.8 366 121.0 13.0 398 13.3 382 121.0 13.3 377 14.0 382 122.0 13.5 350 13.5 350 120.0 13.0 364 14.0 393 121.5 11.5 361 11.5 361 120.0 12.0 302 12.0 302 120.0 11.0 341 11.0 341 120.0 14.0 341 15.0 370 121.5 12.7:0.3 354:8 13.2:0.4 361:8 121.0:0.3 10.6 339 11.2 361 121.0 14.3 405 14.3 404 120.0 12.7 348 12.7 348 120.0 10.7 382 11.8 391 121.0 12.0 368 12.5 377 120.5 16.5 373 16.5 373 118.0 9.7 323 10.0 316 121.0 9.7 325 10.2 336 121.5 12.1 382 12.1 382 119.5 9.3 314 9.8 323 121.0 11.8:0.7 356:10 12.1:0.6 361:8 120.3:0.3 119 2.5 Months First Estrus Group Heifer Withers Withers No. No. Weight Height Age Weight Height (kg) (cm) (m0) (kg) (cm) 7 202 119 89.0 7.5' 289 106.0 219 96 85.5 6.3 198 103.5 230 103 85.5 8.5 305 109.0 237 87 87.0 7.5 261 106.0 240 92 88.0 7.6 280 103.0 253 98 85.0 6.0 195 100.0 256 102 90.0 9.7 368 115.0 260 80 81.0 6.5 150 97.0 265 65 90.0 10.0 364 120.5 282 80 96.0 8.0 311 118.5 Mean : SE 92:5 87.7:1.3 7.8:O.4 272:23 107.8:2.5 8 201 90 85.0 7 9 280 106.0 233 95 85.5 7 3 261 105.0 234 102 89.0 7 8 268 107.0 243 89 88.0 7 5 264 106.0 246 91 86.5 7.3 270 106.5 271 104 84.0 7.8 286 110.5 276 95 84.0 7.9 248 107.0 277 78 80.5 8 9 275 109.5 289 107 88.0 6 9 241 113.0 290 127 91.0 5 9 248 110.5 Mean : SE 98:4 86.1:O.9 7.5:O.2 264:5 108.1:O.8 120 Breeding Size Slaughter 5 Withers ' Age Weight Age Weight Height (mo) (k9) (m0) (kg) (cm) 10.7 384 11.3 404 121.5 10.5 355 11.8 366 122.0 11.7 423 12.0 425 120.5 7 11.5 370 12.0 389 120.0 13.0 464 13.5 473 120.5 11.2 380 11.5 389 121.0 11.5 420 11.5 420 120.0 13.0 359 13.2 368 120.5 9.5 345 10.5 375 121.0 9.3 357 9.3 357 120.0 11.2:0.4 386:12 11.7:O.4 397:11 120.7:0.2 12.3 400 12.3 400 120.0 12.1 409 12.1 409 120.0 10.1 332 11.0 345 121.0 10.3 350 11.3 386 121.5 10.2 361 11.0 386 121.0 11.0 389 11.0 389 120.0 13.5 407 13.5 407 120.0 13.3 450 13.3 450 120.0 9.0 316 9.3 318 121.0 8.5 332 8.9 345 121.0 11.0:0.5 375:14 11.4:0.5 383:12 120.5:0.2 121 2.5 Months Breeding Size Group Heifer Withers No. No. Weight Height Age Weight (kg) (cm) (m0) (k9) 9a 294 102 89.0 12.3 393 305 100 91.0 12.3 373 312 95 87.0 13.3 389 318 105 89.0 13.0 375 322 85 84.0 13.7 370 344 88 89.0 12.8 343 357 105 92.0 12.2 339 364 94 93.0 10.7 302 368 94 89.0 11.9 350 369 100 89.0 9.3 298 Mean : SE 97:2 89.2:0.8 12.1:O.4 353:11 aNo contempory pairmates not fed MGA, so no first estrus values. 2.5 Months First Estrus Group Heifer Withers Withers No. No. Weight Height Age Weight Height (kg) (cm) (m0) (kg) (cm) 10 292 108 86.0 6.4 232 T 106.5 293 114 86.0 6.1 227 107.0 300 85 86.0 8.6 282 111.0 311 73 82.0 8.9 277 111.0 320 88 84.5 8.4 257 106.0 354 85 85.0 9.4 255 110.0 355 100 85.5 8.5 261 108.0 363 71 84.0 9.1 255 111.0 366 90 83.0 7.7 248 108.0 373 85 88.0 7.4 227 107.0 Mean : SE 90:4 85.0:O.5 8.0:O.3 252:6 108.5:0.6 122 Slaughter Withers Age Weight Height (m0) (kg) (cm) 13.0 404 123.0 12.8 382 121.5 13.7 395 121.0 13.0 379 120.0 13.7 370 119.0 12.8 343 119.0 12.8 348 121.0 10.7 302 119.0 12.6 361 121.0 9.6 304 120.0 12.5:0.4 359:11 120.4:0.4 Slaughter Withers Age Weight Height (m0) (kg) (cm) 7.0 234 107.5 6.7 239 108.0 9.2 273 112.0 9.5 286 112.0 9.0 261 107.0 10.0 268 115.0 9.1 275 112.0 9.7 261 112.5 8.3 252 109.0 8.0 227 108.0 8.7:0.3 258:6 110.3:0.8 123 .31 2.5 Months First Estrus ' Group Heifer Withers Withers No. No. Weight Height Age Weight Height (kg) (cm) (m0) (kg) (cm) 11 298 84 83.5 5.9 182 99.5 301 87 86.0 5.9 207 105.0 j 308 86 82.0 7.1 248 107.0 309 87 84.0 7.3 216 105.0 315 91 84.5 5.7 214 102.5 347 87 87.0 6.6 234 109.0 350 112 95.0 6.5 252 111.0 358 98 89.5 6.9 241 108.5 352 107 91.0 7.2 261 110.0 371 89 87.0 6.1 220 107.0 Mean : SE 93:3 86.9:1.2 6.5:O.2 227:8 106.4:1.1 12 291 83 85.5 5.9 191 104.0 295 98 85.0 5.7 216 107.0 297 109 87.0 5.7 234 107.0 302 89 83.5 7.1 264 107.5 307 93 89.0 7.3 223 105.0 353 96 86.0 6.2 214 102.5 359 106 87.5 7.0 270 107.5 360 108 89.0 6.5 234 104.0 362 90 86.5 6.9 225 106.5 365 89 89.0 7.2 255 111.5 Mean : SE 96:3 86.8:O.6 6.5:O.2 233:8 106.2:O.8 124 Slaughter Withers Age Weight Height (m0) (kg) (cm) 6.5 189 101.5 6.5 214 107.0 7.7 259 109.0 7.9 227 106.0 6.3 223 104.0 7.2 243 111.0 7.1 266 112.0 7.5 266 110.0 7.8 250 112.0 6.7 232 109.0 7.1:0.2 237:8 108.1:1.l 6.5 198 105.0 6.3 227 109.5 6.3 245 108.0 7.7 277 109.0 7.9 243 106.0 6.8 225 104.0 7.6 289 108.5 7.1 241 105.0 7.5 227 108.0 8.0 270 113.0 7.2:0.2 244:9 107.6:O.8 125 2.5 Months First Estrus Group Heifer Withers Withers No. No. Weight Height Age Weight Height (kg) (cm) (ma) (kg) (cm) 13 299 86 87.0 7.1 259 113.0 303 75 83.5 10.1 343 118.0 317 120 96.0 6 4 255 112.0 319 81 81.0 6 4 214 104.0 321 96 85.0 7.4 245 106.0 349 103 89.5 6.4 232 108.5 351 121 96.0 7.8 307 118.0 356 110 92.0 6 2 243 110.0 361 78 86.5 6 9 227 112.5 367 75 85.0 8.0 230 108.0 Mean : SE 94:6 88.1:1.6 7.3:O.4 255:13 111.0:1.5 14 296 87 84.0 310 89 82.5 313 92 85.5 314 102 87.0 316 93 83.0 342 125 95.0 343 93 87.0 345 107 88.0 346 80 81.0 348 86 86.0 Mean : SE 95:4 85.9:1.2 126 Slaughter Withers Age Weight Height (1110) (kg) (cm) 7.7 270 115.0 10.7 343 119.0 7.0 264 113.0 7.0 236 106.0 8.0 248 108.0 7.0 243 110.5 8.4 318 119.0 6.8 250 112.0 7.5 239 114.5 8.6 236 109.0 7.9:O.4 265:12 112.6:1.4 127 APPENDIX II.--Weight, nucleic acids, and cell height of the uterus, and ovarian weight and number of follicles for individual heifers. Uterus Group Heifer No. No. Weight DNA RNA “1 (9) (m9) (mg/9) (m9) (mg/9) 5a 221 129.0 583.7 4.5 494.6 3.8 226 160.0 527.7 3.3 560.8 3.5 229 123.6 567.0 4.6 428.7 3.5 238 138.6 611.8 4.1 438.9 3.2 242 123.5 513.1 4.1 392.5 3.2 1 251 119.8 545.0 4.5 277.5 2.3 s” 263 121.0 525.5 4.3 422.6 3.5 264 112.9 637.0 5.6 347.2 3.1 266 194.7 706.7 3.6 710.5 3.6 278 145.0 643.7 4.4 362.8 2.5 Mean : SE 136.8:7.8 586.1:19.9 4.3:0.2 443.6:38.6 3.2:0. 6a 204 181.7 690.2 3 8 619.6 3.4 214 131.1 543.1 4 1 454.7 3.5 217 140.5 705.1 5 0 470.9 3.3 223 149.5 674.5 4 5 571.4 3.8 241 134.5 521.2 3.9 444.6 3.3 254 164.0 796.6 4.9 515.6 3.1 255 149.3 688.5 4.6 466.1 3.1 262 174.5 488.2 2 8 720.1 4.1 279 156.5 670.3 4 3 493.9 3.2 285 135.4 624.4 4 6 447.6 3.3 Mean : SE 151.7:5.5 640.2:30.3 4.2:0.2 520.5:28.6 3.420. 128 Uterus Ovaries Epithelial Cell Paired No. Follicles RNA/DNA Height Weight 4-9 10-15 16-20 >20 (u) (mm) 0.85 26.4 16.4 1 1 1.06 26.4 15.0 1 l 0.76 26.4 12.7 1 1 0.72 24.5 13.4 1 6 1 0.76 26.4 20.2 1 1 0.51 26.4 12.2 2 1 1 0.80 30.2 12.9 3 0.54 24.5 13.7 2 1.01 28.3 15.0 1 0.56 -- 18.0 5 1 1 0.76:0.06 26.6:0.6 15.1:0.8 1.5 1.3 0.5 O 0.90 28.3 14.4 1 1 0.84 26.4 12.2 2 3 0.67 22.6 13.5 4 l 0.85 20.7 16.6 3 2 0.85 34.0 14.0 3 2 0.65 24.5 14.0 2 1 0.68 28.3 10.8 3 1 1.47 28.3 9.4 2 1 0.74 34.0 11.2 7 1 0.72 24.5 12.6 7 l 1 0.84:0.08 27.2:1.4 12.9:0.7 3.3 1.1 0.4 0.1 129 Uterus Group Heifer ' ._. No. No. Weight DNA RNA "1 f (9) (11191 ngf 91 T1191 (mg/91 7a 202 104.2 436.5 4.2 468.8 4.5 219 199.6 522.0 2.6 868.5 4.3 230 137.4 665.6 4.8 599.5 4.4 237 98.9 504.6 5.1 469.7 4.7 J 240 193.7 650.2 3.4 1072.4 5.5 .1: 253 166.2 589.6 3.5 817.6 4.9 256 205.4 632.3 3.1 1001.7 4.9 260 174.0 486.8 2.8 461.0 2.6 265 117.3 414.9 3.5 481.0 4.1 282 156.8 572.4 3.6 482.2 3.1 Mean : SE 155.4:12.5 547.5:27.9 3.7:0.3 672.2:77.0 4.3:0.3 8a 201 179.5 834.8 4.6 896.0 5.0 233 167.2 629.8 3.8 772.9 4.6 234 134.3 589.4 4.4 515.0 3.8 243 171.0 720.3 4.1 941.4 5.5 246 243.4 800.2 3.3 998.1 4.1 271 162.4 527.8 3.2 653.4 4.0 276 137.5 612.0 4.4 359.8 2.6 277 142.0 660.4 4.6 394.5 2.8 289 158.2 413.8 2.6 439.5 2.8 290 162.0 649.1 4.0 702.7 4.3 Mean : SE l65.8:9.8 643.8:39.1 3.9:0.2 667.3:74.0 4.0:0.3 130 Uterus Ovaries Epithelial Cell Paired No. Follicles ‘ F“' RNA/DNA Height Weight 4-9 10-15 16-20 >20 (u) (mm) 1.07 28.3 12.5 3 l ’ 1.66 37.7 14.3 1 i 0.90 28.3 14.8 2 l 1 5 0.93 24.5 13.6 3 l 1 i 1.65 39.6 29.1 3 ;0 1.39 37.7 13.5 2 1 l l 1.58 37.7 15.8 1 1 0.95 32.1 8.4 1 1 1.16 18.9 20.0 1 1 2 0.84 24.5 11.6 2 1 1 1.21:0.10 30.9:2.2 15.4:1.8 1 4 0.3 l O 0 7 1.07 28.3 14.6 3 l l 1.23 -- 21.1 4 1 1 1.38 26.4 19.5 5 1 1.31 30.2 18.7 2 1 l 1 1.25 30.2 20.9 3 1 l 1.24 49.1 12.1 4 1 1 0.59 20.8 18.2 1 1 2 0.60 28.3 21.3 10 1 1.06 30.2 10.0 1 1 1.08 39.6 19.9 4 l 1 1.08:0.09 31.5:2.7 17.6:1.3 3.7 0.3 0.6 1.0 131 Uterus Group Heifer No. No. Weight DNA RNA (91 ‘TMBIF (mg/91’ (n01 Elmo/91 9a 294 95.5 353.6 3.7 299.1 3.1 305 144.3 300.4 2.1 350.5 2.4 312 111.2 547.5 4.9 287.6 2.6 318 104.3 432.1 4.1 304.6 2.9 322 107 0 400.7 3.7 402.3 3.8 344 157.3 455.4 2.9 635.0 4.0 357 120.8 427.1 3.5 430.5 3.6 364 153.7 518.7 3.4 685.0 4.5 368 149.4 520.8 3.5 678.3 4.5 369 149 5 638.5 4.3 458.2 3.1 Mean : SE 129.3:7.5 459.5:31.4 3.6:0.2 450.4:48.6 3.4:0.2 | 10b 292 142.9 558.3 3 9 639.0 4.5 293 79.3 370.7 4 7 420.6 5.3 300 124.8 548.0 4 4 303.2 2.4 311 138.0 694.5 5 0 794.8 5.8 320 161.3 852.5 5.3 542.6 3.4 354 152.5 644.2 4.2 353.3 2.3 355 110.0 607.2 5 5 238.2 2.2 363 153.3 775.6 5 1 309.3 2.0 366 120.3 615.3 5 1 272.5 2.3 373 86.1 330.8 3 8 318.5 3.7 Mean : SE 126.9:8.9 599.7:51.1 4.7:0.2 419.2:57.6 3.4:0.4 132 Uterus Ovaries Epithelial Cell Paired No. Follicles RNA/DNA Hei ht Weight 4-9 10-15 16-20 >20 (H) (AND 0.85 32.1 13.4 2 l l 1.17 30.2 14.1 1 l 1 0.53 34.0 18.9 1 l 0.70 32.1 18.7 5 1 1 1.00 28.3 17.7 2 l 1.39 22.0 12.4 3 l 1.01 29.1 17.2 3 1 1 1.32 22.6 10.3 1 1 1.30 25.4 16.9 1 l 0.72 ‘ 30.2 15.0 6 2 1 1.00:0.09 28.6:1.3 15.5:0.9 2.2 0.7 0.5 0.7 1.14 28.3 9.6 3 1 1.13 20.8 3.3 2 0.55 24.5 9.0 6 l 1.14 24.5 13.9 4 1 0.64 17.0 9.8 5 1 0.55 28.3 8.4 5 l 0.39 28.3 9.9 1 1 0.40 30.2 10.5 4 1 0.44 32.1 13.8 5 1 0.96 20.8 11.4 4 0.73:0.10 25.5:1.5 10.0:0.9 3.9 0.7 0.1 0 133 Uterus Group Heifer No. No. Weight DNA RNA ‘"1 , (9) (m9) (mg/9) (mg) (mg/g) 11b 298 114.0 480.7 4.2 418.5 3.7 301 94.3 562.8 6.0 406.4 4.3 308 114.6 482.3 4.2 379.4 3.3 309 107.4 454.1 4.2 514.0 4.8 , 315 200.8 764.9 3.8 839.3 4.2 g V 347 152.6 675.6 4.4 516.7 3.4 ,0-v 350 93.4 511.4 5.5 285.2 3.0 352 119.2 632.9 5.3 387.1 3.2 358 121.4 628.3 5.2 458.5 3.8 371 98.8 439.7 4.4 245.1 2.5 Mean : SE 121.7:10.3 563.3:34.2 4.7:0.2 445.0:51.7 3.6:0. 12b 291 35.0 133.3 3.8 140.7 4.0 295 104.5 484.6 4.6 483.8 4.6 297 118.8 521.6 4.4 458.6 3.9 302 114.6 422.0 3.7 443.8 3.9 307 102.6 293.4 2.9 418.1 4.1 353 135.8 384.7 2.8 497.2 3.7 359 150.5 460.5 3.1 485.1 3.2 360 119.2 520.5 4.4 457.1 3.8 362 104.5 267.1 2.6 262.5 2.5 365 182.8 419.3 2.3 612.7 3.3 Mean : SE 116.8:12.0 390.7:39.5 3.5:0.3 426.0:41.8 3.7:0. 134 Uterus Ovaries Epithelial Cell Paired No. Follicles RNA/DNA Height Weight 4-9 10-15 16-20 >20 (u) (mm) 0.87 26.4 5.5 2 0.72 28.3 10.1 5 1 0.79 20.7 16.3 2 1 1.13 22.6 12.1 3 l 1.10 37.7 20.3 1 0.76 22.6 12.7 1 l 0.56 24.5 12.5 5 1 0.60 26.4 10.7 3 l 0.73 22.6 12.5 1 1 0.56 30.2 10.5 4 0.78:0.06 26.2:1.6 12.3:1.2 2 3 1.0 0 l 0 1.06 18.9 6.7 4 1 1.00 18.9 5.6 l 0.88 35.8 8.7 5 1 1 1.05 30.2 14.1 1 1 1 1.42 24.5 11.1 1 l 1.29 26.4 8.5 11 0.99 -- 14.4 4 1 1 0.88 24.5 13.4 6 l 0.98 28.3 6.6 1 1 1.46 20.7 13.1 5 2 1.10:0.07 25.4:1.9 710.2:1.1 3.9 0.4 0.5 0.3 135 Uterus Group Heifer No. No. Weight DNA RNA -1 (9) 169) Ins/917’ (mgl' (mg/91 13b 299 148.2 559.8 3.8 366.2 2.5 303 188.0 351.9 1.9 518.1 2.7 317 92.2 595.6 6.5 439.2 4.8 319 86.5 342.7 4.0 431.8 5.0 321 93.3 364.5 3.9 574.4 6.2 g 349 64.3 288.5 4.5 216.3 3.4 - 351 139.2 685.5 4.9 311.2 2.2 356 180.3 558.8 3.1 398.6 2.2 361 105.4 658.2 6.2 510.5 4.8 367 136.0 566.2 4.2 433.7 3.2 Mean : SE 123.3:13.1 497.2:45.9 4.3:0.4 420.0:33.2 3.7:0.4 14C 296 25.4 136.4 5 4 80.2 3.1 310 28.9 130.4 4 5 118.9 4.1 313 29.9 156.3 5 2 119.5 4.0 314 24.7 134.9 5 5 101.0 4.1 316 31.1 192.1 6.2 140.8 4.5 342 42.2 224.2 5.3 184.1 4.4 343 27.9 131.5 4 7 108.7 3.9 345 27.0 144.6 5 4 117.5 4.3 346 26.4 123.1 4 7 96.0 3.6 348 32.0 165.0 5 2 132.9 4.1 Mean : SE 29.6:1.5 153.9:10.l 5.2:0.1 119.9:9.0 4.0:O.1 aSlaughtered at breeding size. bSlaughtered at first estrus. cSlaughtered at 2.5 months of age. 136 Uterus Ovaries Epithelial Cell Paired No. Follicles RNA/DNA Height Weight 4-9 10-15 16-20 >20 (H) (mm) 0.65 26.4 19.1 1 1 1.47 37.7 10.7 1 0.74 32.1 9.7 1 1 1.26 22.6 11.7 2 l 1.58 30.2 7.5 4 1 0.75 20.8 8.4 3 2 0.45 32.1 11.9 1 0 71 24.5 9.4 2 l 0.78 28.3 7.1 3 2 0.77 18.9 10.0 5 0.92:0.12 27.4:1.8 10.6:1.l 1 8 0.7 0.6 0.1 0.59 18.9 3.7 2 1 0.91 15.1 3.8 l 0.76 20.7 9.6 2 0.75 11.3 8.4 1 0.73 15.1 3.2 l 2 0.82 18.9 16.6 1 0.83 15.1 2.5 2 1 0.81 15.1 10.1 5 1 0.78 18.9 6.2 2 0.81 -- 3.4 3 1 0.78:0.03 l6.6:1.0 6.8:l.4 2 0 0.6 0 0 137 eucepw xgessez mo.owmm.o ~.ow_.m «.mmmwp.mwmp N.ow¢.~ e.m¢~wn.oomp eewmmm mm o cemz mm.o <.P a.mmo e.m e._o~p wee mmm mm.o -o.~ o.e¢m_ v.m e.owm_ com mum em.o e._ o.~om ~.F e.¢¢m Pom mom No.0 ¢.F p.mmo m.p m.omm ewe mmm mm.o o.~ m.P¢~_ ¢.m m.ommm mmw «mm mu.o N., m.mmm ~.~ m.om- omm Fem N_._ N.N o.ome m.~ a.mmm Rom mum mm.P m.m m.~mm~ m.~ m._mom om“ Rpm mw.o o.~ o.memp m.~ ~.¢om~ emm epm No.P ~.~ F.meop N.~ e.m~o_ eue vow no op.owmm.o m.owp.~ ~.mo~wm.m¢ep ~.ow¢.~ o.empwn.mmep mehuon mm A see: _m.o N.F ¢.m¢m e.~ m.mmwp mmm mmm «0., m._ N.mmm e._ “.mmm mew mew em.o N.N m.omm_ ~.m “.mmmp mam emu we.P ~.¢ m.meom N.m o.meom mew mew _m.o e.p m.Fo¢_ N.m «.mmmm Fem _mm N~.P P.~ m.¢mmp ~._ a.mmep “mm New em.o ¢._ N.m¢_P e._ o.m~mp mom mmm o~.o M., u.¢mmp m.P m._mn_ omm mum mo._ m.~ N.eem_ w.m P.mmm— New omw em.o n.~ m.o~m m.N o.¢mep mac —- am Am\msv Amev Am\mev Away Amv ee:_ cow e—ecmcee we» we weave: mace xepcou 000 eeeeez 000 .eee_0 seeEEee wee e0 weede uee_000 eee 0:000:11.Hee xH0ze00< mo.owom.o mp.ownm.P _o.owmm.o mp.owom.m m.owm.mp 138 m~.o NN.P _m.o Fm.F ¢.- No.0 om._ mm.o mv.m m.o_ om.o oo._ “N.o nn._ o.m_ m¢.o mp.— mm.o mo.N m.P— m¢.o mw.o mm.o vo.F ¢.m~ o¢.o No._ um.o m¢.~ ¢.m_ om.o om.o mm.o mw.P m.m~ em.o mo.~ m~.o om.~ m.¢p mm.o oo.m mm.o Fm.m m.m_ _m.o N¢.P Fm.o ¢N.N m.m— no e0.0u0e.0 00.0nee._ e0.0w0e.0 ep.0u00.~ 0.0he.e. no.0 om.p mm.o mm.m m.¢— mm.o n~.P mN.o mo.m m.mp o¢.o NN.~ Fm.o mm.p o.mp oe.o No.— m¢.o ~m.P a.m— ou.o mm.p m~.o no.m a.mp _m.o —m.o om.o mm.p a.m— No.o m¢.F P¢.o m¢.P “.mp Fw.o P¢.F mm.o _m.~ m.F~ m¢.o 0N.F om.o No.m o.m~ 00.0 no._ NN.o mm.— w.pp nm E: 2: eecepsoeemm eueezueomem emopaseeo_o spew: “nave: .oz11 xeusou emeeea macaw eeeeez eeeN encepw _ecmev< 139 00.0w00.0 0,000.0 0.00000.0000 000.0 0.000e0.0000 00.0000 00 0 2002 0... 0.0 ..000. 0.0 0.00.. 000 000 00.0 ..0 0.000. 0.0 ..000. 0.0 000 00.0 0.0 0.0000 0.0 0.0000 000. 000 00.0 0.0 0.000. 0.0 0.0000 000 000 00.0 0.0 0.0000 0.0 0.00.0 000 .00 00.0 0.0 ..0000 0.0 0.0000 000 000 00.. 0.0 0.000. 0.0 0.000. 000 000 00.0 0.0 0.0000 0.0 0.0000 000 000 00.. 0.0 0.0000 0.0 0.0.00 000. 000 00.0 0.0 0.000. 0.0 0.0000 0.0 .00 e0 00.0w00.0 000.0 0.00.00.0000 000.0 0.000e0.0000 000000 00 w 2002 00.0 0.0 0.0000 0.0 0..0e0 000 000 00.0 0.0 0.0000 ..0 0.0000 0.0 000 00.0 0.0 0.0000 0.0 ..0.00 000 000 00.0 0.0 0.000. 0.0 0.00.0 000 000 00.0 0.0 0..000 0.0 0.0000 000 000 00.0 0.0 0.0000 0.0 0.0000 000 000 00.. 0.0 ..000. 0.0 0.000. 0.0 000 00.0 0.0 0.0000 0.0 0.0000 0.0 000 00.. 0.0 0.0000 0.0 0.000. 000 0.0 00.. 0.0 0.0000 0.0 ..0.0. .00 000 00 .0000. .00. .0000. .00. .0. 0200220 <20 020 020.02 .02 .02 000.0: aaoeo eece.w xgeesez 140 0o.ow¢m.o mo.ow~... 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H.OHm._F mm H com: m._mp _mm omm N H._P o._. mam o.m~_ HmH mac _ H.o_ “.op «mm o.om_ __m mum N m.~F m._p mom o.m~_ «pm 0mm P H... N.._ “mu -- - - m o.m_ o.~F m_~ a.mmp mom mew a o.HF o.P_ o_~ o.-_ mse «mm H a.m. ~.~_ ~o~ o.~m_ cue o~m m m.m_ o.__ mo~ o.m~_ wme sum 0 m.o_ m.op HON HH .m—Pugmmcw mmzoumn umgmugmaopm emu .oz gmmwmzm m.OH~.m~P HFHooH HPHHom H.OHm.~ H.0H~.HF H.0Hm.m_ mm H cam: o.H~P Pam mme H m.m_ m.~_ Haw m.m~_ NMH «me _ m.~_ m.~P mum o.Pm. aFH HRH _ a.mp a.mp HRH o.-F MHH - N m.~_ o.~_ omN o.m~. “me mew m m.o_ m.m_ mmm o.m~_ Nae Noe _ “.mp «.m. HFN o.omp NHH omm _ m.~_ c.~_ ~_~ m.~mp mHH amm N “.m. a.m. mom o._mp omm mum . ~.m_ ~.m_ new HP Asuv Aux: :54: :05: :02: cowuwgaggom cowy_gngma cowuwgaugma mwup>gmm cowuamucou mcwummgm .oz .oz H< Hgmwm: LHHH< HLOHmm .oz H< mm< HHLHH Lamwmz usage HLHHHH3 Hgmwmz H;m_mz H< ma< .mgmwwm; ”mauw>_u:_ go; Umex x__s cowpmuump ngwk new .xwm ucm .mgwm .Hcmwmz wpmu .acwumg «wuoamxu .cowpwgapgma um mNHm xuon .mmuw>gmm we Lungs: .cowuamucou ucm mc_ommgn ngwm an mm<--.> xHozmma< 163 NHHHHH HmFHHHOH NHOH H.OHH.F mam HHNH x mm mH H Hmm HHOH z m: PH P mam Hmmm H m: on _ __N_ mmpm H mm mm _ II II I a”. I I moo_ PHNH z m: HLHHHEHHQ _ HRH mHHm z m: on P was Humm z m: um - moo. «man 2 am an N HHHmmoH mmpHHm.H HHHH m.oHH.~ mum 3585 RE 3585 ... m: mm _ OH~. onH H m: Hm F “Hm OHmm : Hm mH _ mmm oooH H m: mw F mmop mmpH 2 mm mm H HHHP a.mH z _HHEHW H. HH m one. m_HH z Ha Pm H How mmHm «_Hsmu am mH m HHoP HHPH HPHz HH_H=o¢ PHHHH Hm H :mxv Aux: Aux: H_m_> H_Hz HFHH> H_Hz HHHQ H_Hu Hmwwo: HHHHHH HHQ om HHQ won me xmm Ho ogHm :HLHH H_Hu H_uoHHHd HHLHH FHHHHH HHHHEHHHM .xv:um ms“ EoLH umu=~uxm we: mgm om .u_o mgpcos NN mm; mcm HHch m>wmu=ou Ho: urn NNN .oz gmwpm: .mpwugmmcw mmamumn cmgmugm=HHm mmN new mpN .oz mgmmpm:u 164 m.0Ho.mNp ONHooH oNHmom m.OHo.m m.0Hm.H_ H.0Hm.NH mm H cum: a.mNF one mmH m N.HH o.mF NmN m.NNP mHH ewe _ m.mp m.m_ _mN a.mNp opH one N . m.Hp a.mp HNN m.oNH FPH NmH N o.HH m.mp mmN o.NmF HoH com o a.mp o.N_ mHN m.mNp awe mNm H m.N_ m.NH mmN m.Pmp com HP: 0 o.HF N.op mNN UH m.OHH.mNP wHHmmH aHHNPm N.OHH.m N.oHN.H_ H.OHN.NF mm H cam: o.omp mmH . m o.m_ m.o_ owN m.mNP mNH opm N H.N~ o.NH NNN a.mNH mmH NmH H a.mp m.NF moN a.mNF __H mmH H o.Np o.N_ HoN o.Nm— mmH one H m.m~ m.m_ mmN m.mN_ oHH me m a.m— w.N_ mmN o.mNH mom mNo m m.mp a.mp _mN o.HN_ mHH mNH N m.HF o.H— oNN o.om_ omm mom N m.o_ N.op _HN m.mm_ mom mam o N.mp N.o~ NON m Asuv :Hxv Aux: :02: figs: cowuwgaugma coppwgaugma coHHHgapgwa mmuw>gmm cowunoucoo mcvummgm .oz .oz an “nova: gou$< «Lemma oz an mm< ngHH Lompm: azogw HHHHHHH HHHHHI HHHHHI HH HHH omHHooH HHNHHHOH HHHH m.on.H HNH NHNm z HH mm H Nm._ HHHH H m: - _ Hpm comm H HH mm _ HmoP NHHH H HH mm H 329:8: H 339—.35 ... 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