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"1'3." 5 . ,0 c'nnwhbfir-un. )1 .;I;: 234:5 . y. ‘73:]:- ’C' 5... 5 .v- 4:.“ .5. ,. , 4.. t I'" 5}” I...” .. .. . ,,_ :‘1-H;‘\~Kl. .- “r! ‘ L. 5:: L‘v::-~ LN?" r “x -; lung: .1. ...- a...” 5-... .5. -.- a": .5. .. x 4 '2‘. ;'1.',.::.-;.'..:M.\y .~~r u u m- '3?va “1'7““... “5.1!.- n ‘31.: 5 .' 931685176 RSI TY LIBRAFH lES l\lllll\llllllllllllll Will \H 3 12930 LIBRAR‘ 6‘ Michigan State W l This is to certify that the thesis entitled EFFECT OF PHOTOPERIOD ON MAMMARY DEVELOPMENT AND SERUM HORMONE CONCENTRATIONS IN PREGNANT DAIRY HEIFERS presented by Jacqueline Anne Newbold has been accepted towards fulfillment of the requirements for Master of Science of t. d . Anima? Science egree 1n Major professor [hue August 10, 1989 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE meal] l l li—T MSU Is An Affirmative Action/Equal Opportunity Institution EFFECT OF PHOTOPERIOD ON MAMMARY DEVELOPMENT AND SERUM HORMONE CONCENTRATIONS IN PREGNANT DAIRY HEIFERS BY Jacqueline Anne Newbold A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science. 1989 oil» (147 \0 i ABSTRACT EFFECT OF PHOTOPERIOD ON MAMMARY DEVELOPMENT AND SERUM HORMONE CONCENTRATIONS IN PREGNANT DAIRY HEIFERS. BY Jacqueline Anne Newbold From 128 d of pregnancy, heifers were exposed to 16 h light, 8 h dark (16L:8D) or 8L:160 until 35 d before calving. Photoperiod had no effect on weights and percentages of fat or parenchyma of the mammary gland, or amount of fat, DNA or RNA in mammary parenchyma. Serum prolactin was 1.7-fold greater under 16L:8D than under 8L:16D. For both treatments, serum melatonin concentrations were 2.4-fold greater during dark than light periods, but photoperiod did not affect the total amount of melatonin to which heifers were exposed during 24 h. ID1 a second experiment, the peri-parturient prolactin surge was 1.6-fold greater in heifers exposed to 16L:8D than in those exposed to 8L:160. Duration of the surge, and its timing relative to calving, were not affected by photoperiod. In conclusion, mammary development is not responsive to photoperiod during gestation; however, increased exposure to light increases the amplitude and magnitude of the peri- parturient prolactin surge. ACKNOWLEDGEMENTS I wish to express my. appreciation of all my friends and fellow graduate students who helped me during the past two years. I would particularly like to thank Dr. H.A. Tucker, firstly for the opportunity to study in the United States and, secondly, for the enormous amount of help and advice he has given me. My thanks also to the rest of my guidance committee, Drs. M. Allen, N.K. Ames and R.A. Merkel, for their assistance and helpful comments. My special thanks go to Larry Chapin and Trudy Hughes for their infinate patience in answering my never-ending questions. I also wish to thank my fellow graduate students and everyone else who made my stay in The United States of America so enjoyable. ii TABLE OF CONTENTS LIST OF TABLESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO ....... Vi LISTOF FIGURESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0.. Vii INTRODUCTIONOOOOOOOOOOO0.0000000000000000000000.0.00... 1 LITERATURE REVIEW (a). MAMMARY DEVELOPMENT UNTIL PARTURITION.......... 4 HORMONES AND MAMMARY DEVELOPMENT 1. ESTROGEN AND PROGESTERONE........... ........ 6 2. PROI—ACTINOOOOOOOOOOOOOOOOOOOOOOO. ........... 9 3 0 GROWTH HORMONE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 11 4. OTHER HORMONES.................... .......... 12 (b) O LACTOGENESIS. O O O O O O O O O O O O ..... O ....... O ........ 14 HORMONES AND LACTOGENESIS. ................ ..... 15 (C). PHOTOPERIOD AND HORMONES ................ . ...... 19 1. PROIACTINOOOOOOOOOOOOOOO ......... O 0000000000 20 2. mmTONINOOOOOOOO00.0.0.0...00.0.0000...O... 21 3. OTHER HORMONES.. ..... ....... ................ 23 (d). PHOTOPERIOD AND MAMMARY DEVELOPMENT ............ 25 EXPERIMENT ONE (a). EXPERIMENTAL OBJECTIVES..... .................... 27 (b). MATERIAL AND METHODS.................... ........ 27 iii (C) - (d). l. TREATMENTS AND MANAGEMENT...._ ................ 27 2O BLOOD SAMPLINGOOOOOOOOOO‘OOOOO 00000000000000 OO 29 3. SLAUGHTER AND ANALYSIS OF CARCASSES .......... 30 4. ANALYSIS OF MAMMARY GLANDS..... .............. 30 5. STATISTICAL ANALYSIS............. ............ 32 RESULTS 0000000000 OOOOOOOOOOOOO 000000000000000000 33 l. MAMMARY DEVELOPMENT GROSS MAMMARY TISSUE COMPOSITION... ........ .. 33 CHEMICAL COMPOSITION OF PARENCHYMA ........... 35 HISTOLOGY................... ................. 36 2. HEIFER GROWTH, CARCASS AND FETAL CHARACTERISTICS. ................... 36 3. SERUM HORMONES PROLACTIN..... ............................... 38 MELATONIN .................................... 43 PROGESTERONE......... ............. . .......... 47 DISCUSSIONOOOOOOOOOOOOOOOOOOOOOOOOOO 0000000000 OO 47 1. MAMMARY DEVELOPMENT .......... ... ............. 47 2. HEIFER GROWTH, CARCASS AND FETAL CHARACTERISTICS........... .......... 57 3. SERUM HORMONES......... ..................... . 58 EXPERIMENT TWO. (a). (b). (c) . EXPERIMENTAL OBJECTIVES ......................... 64 MATERIAL AND METHODS O O O O O O O O O O O O O OOOOOOOOOOOOOOO 65 1. TREATMENTS AND MANAGEMENT .................... 65 2. BLOOD SAMPLING ............................... 66 3. STATISTICAL ANALYSES ......................... 67 RESULTS ...... . ....................... . .......... 68 1. SERUM PROLACTIN -INITIAL PRE-SURGE BASAL CONCENTRATIONS.... ..... . ......... 68 2. PERI-PARTURIENT PROLACTIN SURGE .............. 69 3 O SERUM MELATONIN. O O O O O O O O O O O O O O O O ..... O OOOOOOO 74 iv (d)O DISCUSSIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOO 78 1. PERI-PARTURIENT PROLACTIN SURGE.............. 78 2. ENTRAINMENT TO PHOTOPERIOD.............. ..... 82 3. RESPONSE TO PERI-PARTURIENT PROLACTIN SURGE..,84 SUMMARY AND CONCLUSIONS..... ......... ........... ........ 87 APPENDIX SOURCES OF VARIATION AND DEGREES OF FREEDOM IN STATISTICALMODEISOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 90 LIST OF REFERENCES........ ......... . .......... . ......... 95 LIS Table 1. Effect of photoperiod on gross tissue composition of the mammary gland 2. Effect of photoperiod parenchymal tissue in pregnant heifers. 3. Effect of photoperiod tissue in the mammary 4. Effect of photoperiod and carcass and fetal 5. Effect of photoperiod concentrations in pre 6. Effect of photoperiod prolactin on predicte pregnancy. 7. Effect of photoperiod surge of serum prolac 8. Effect of photoperiod concentrations in pre T OF TABLES in pregnant heifers. on chemical composition of the mammary gland of on histology of parenchymal gland of pregnant heifers. on pregnant heifer growth characteristics. on serum melatonin gnant heifers. on initial pre-surge basal d d 270 and d 271 of on the peri-parturient tin in pregnant heifers. on serum melatonin gnant heifers. vi Page 34 46 69 72 77 LIST OF FIGURES. Figure Page 1. Serum prolactin concentrations in pregnant 40 heifers exposed to either 16L:80 or 8L:16D. Each point represents the mean for one heifer during one of three 24-h bleeds. Each heifer was represented three times. Pooled SE = .14 ng/ml of serum. 2. Serum prolactin concentrations in pregnant 42 heifers exposed to 16L:80 or 8L:160. Each point represents one treatment mean during one of three 24-h bleeds. Stage of pregnancy averaged 160, 197 and 219 d for bleeds 1, 2 and 3, respectively. Pooled SE = .14 ng/ml of serum. 3. Serum melatonin concentrations in pregnant 45 heifers exposed to 16L:8D or 8L:16D. Each point represents one treatment mean at 2-h intervals for one of three 24-h bleeds. Stage of pregnancy averaged 160, 197 and 219 d at bleeds l, 2 and 3, respectively. The hatched bar represents darkness. Pooled SE = 15.69 pg/ml of serum. 4. Serum progesterone concentrations in pregnant 49 heifers exposed to 16L:8D or 8L:160. Each point represents mean for one heifer during one of three 24-h bleeds. Each heifer was represented three times. Pooled SE= .28 ng/ml of serum. 5. Serum progesterone concentrations in pregnant 51 heifers exposed to 16L:8D or 8L:16D. Each point represents one treatment mean during one of three 24-h bleeds. Stage of pregnancy averaged 160, 197 and 219 d at bleeds 1, 2 and 3, respectively. Pooled SE = .28 ng/ml of serum. . vii Serum prolactin concentrations during the peri-parturient period in pregnant heifers exposed to 16L:8D or 8L:16D. Prolactin concentrations are shown centered relative to peak values. Points represent treatment means at 4-h intervals. Serum melatonin concentrations in pregnant heifers exposed to 16L:8D or 8L:16D. Each point represents one treatment mean at 2-h intervals for 48 h.The hatched bar represents darkness. Pooled SE = 15.46 pg/ml of serum. viii 71 76 INTRODUCTION For the individual dairy farmer, profit has always been related to cow productivity and efficiency. Cow productivity may be increased by manipulating genotype or cow environment, which includes nutrition, housing, photoperiod and many other factors. Photoperiod influences milk yield. For example, lactating cows exposed to 16 hours (h) light and 8 h dark (16L:8D) yielded 6 to 10% more milk than cows exposed to 8L:16D (Peters gt g;., 1981). Such photoperiodic effects may become increasingly important, since they offer a means of increasing milk yield without the use of exogenous hormones which are perceived as dangerous by some consumers. Changing the lightzdark cycle from 8L:16D to 16L:8D increases the rate of live-weight gain (Peters gt g;., 1978; Petitclerc gt g;., 1983), improves feed conversion efficiency (Peters gt g]_.., 1980; Petitclerc e_t 521;” 1983) and increases the proportion of protein and decreases the proportion of fat in heifer carcasses (Zinn gt _a_]_._., 1986). While the biological mechanisms underlying these effects are still unknown, it has been postulated that 2 photoperiod-induced increases in serum prolactin‘(Tucker gt gl., 1984) and (or) decreases in melatonin concentrations (Zinn gt 3-11., 1988) may be potential mediators of some of the responses outlined above. Increasing the photoperiod from 8 to 16 h per day (d) increases mammary growth in non-pregnant pre- and post- pubertal heifers (Petitclerc gt _l., 1985). Heifers exposed to 16L:8D have increased weight of mammary parenchymal tissue and increased total deoxyribonucleic acid (DNA, an index of cell numbers) compared with heifers exposed to 8L:16D. Since mammary epithelial cell numbers are major determinants of milk yield (Tucker, 1987) it seems probable that increased parenchymal tissue would lead to increased milk yield. However, the link between photoperiod- induced mammary growth and milk yield has not yet been established. . The majority of mammary growth occurs during pregnancy (Tucker, 1969). Despite this, effects cmf photoperiod on gestational mammary growth have not been investigated. While variations in photoperiod influence some hormones known to be important in mammary development, clear, causative links have not been established. It first step would be to study the relationship between photoperiod, serum hormone concentrations and mammary development. Coincident with mammary development during late pregnancy is lactogenesis: this, too, is controlled by hormones which could be influenced by photoperiod. A peri-parturient surge 3 of prolactin is an important stimulator of lactogenesis. However direct effects of photoperiod on this surge and, perhaps, on subsequent milk yield, have not been established. Knowledge of interactions between photoperiod, mammary growth, lactogenesis and milk yield is, therefore, far from complete. These interactions were studied in two experiments. In EXperiment 1, the effects of photoperiod on serum hormone concentrations and mammary development during pregnancy were studied, while the effect of photoperiod on the peri-parturient surge of prolactin was studied in Experiment 2. LITERATURE REVIEW (a). MAMMARY DEVELOPMENT UNTIL PARTURITION Primordial thickening of the ventrolateral surface of the ectoderm of cattle appears very early in embryonic life. Serial changes in this thickened area form the mammary bud, a ball of epithelial cells from which develop the structures responsible for milk secretion in the mature gland (Anderson, 1985). Subsequently, the mammary’ bud differentiates into primary and secondary sprouts during growth of the fetus. At birth, the mammary gland consists of an immature duct system and mature non-glandular tissue, called stroma, which is formed from the mesoderm layer and consists of connective tissue, smooth mmscle, blood, lymph and adipose cells (Tucker, 1969). In heifers, mammary growth becomes allometric (i.e., rate of mammary growth exceeds that of the rest of the body) by the second or third month of age, well in advance of puberty (Williams and Turner, 1961). This allometric growth of mammary tissue and DNA is more than three times faster than total body growth and continues for several estrous cycles after onset of puberty. Mammary growth then returns to an isometric pattern until conception (Sinha and Tucker, 1969). 5 Failure of the mammary gland to grow allometrically after the first few estrous cycles may be related to the asynchrony between estrogen and progesterone secretion observed during normal estrous cycles (Tucker, 1987). Allometric growth of the mammary gland recommences during pregnancy. At conception, alveoli begin to form and replace the lipid of the mammary fat pad (Tucker, 1969). Mammary growth is limited by the amount of fat pad present (Hoshino, 1964) . Throughout pregnancy mammary parenchymal tissue and DNA increase exponentially at approximately 25% per month (Swanson and Poffenbarger, 1979). Most of the increase in total mammary cell numbers during pregnancy is associated with proliferation of parenchymal tissue, which is mainly comprised of secretory cells but has some connective tissue, smooth muscle and blood vessels rather than stroma, although some synthesis of collagen is needed in early pregnancy to form a framework for epithelial cell growth (Paape and Sinha, 1971). 'In most species, including cattle, mammary cell numbers continue to increase during early lactation. Akers g g. (1981) found a 65% increase in mammary cell numbers between 10 d before and 10 d after parturition. In goats, mammary growth increased through at least 5 d after parturition (Anderson gt g;., 1981). Mammary cell numbers decline during lactation as milk yield decreases. In rats and goats the decline in total mammary DNA precedes, and is responsible in part for the decline in milk yield (Knight and Peaker, 6 1984; Knight gt 1., 1984). These changes in the mammary gland during the productive life of the animal are mediated by changes in several hormones. HORMONES AND MAMMARY DEVELOPMENT 1_. ESTROGEN m PROGESTERONE Estrogen and progesterone are the major hormones responsible for mammary growth. Ovariectomy prevents both pre-pubertal and gestational allometric growth in all species studied (Cowie, 1949). Ovariectomized heifers given estradiol-17B and progesterone at rates of either 800 ug estrogen and 200 mg progesterone or 400 ug estrogen and 100 mg progesterone three times a week for 20 weeks had tightly packed alveolar cells similar tx: those found in normal 5-month pregnant heifers (Sud gt g;., 1968). Estrogen may be a more important stimulus to mammary growth than progesterone, since during the. first few post-pubertal estrous cycles, while the mammary gland is still growing allometrically, maximum growth occurred during estrus (Sinha and Tucker, 1969) when estrogen concentrations are greatest. Conversely, mammary growth was lowest during the luteal phase when estrogen concentrations are minimal but serum progesterone concentrations are elevated (Sinha and Tucker, 1969). Concentrations of estrogen and progesterone remain high throughout pregnancy and are thought to cause exponential .7 mammary growth by decreasing the time required to complete each cell division in parenchymal tissues (Bresciani, 1965). Estrogen is important for stimulating ductular growth while progesterone stimulates lobular-alveolar development (Turner, 1939). Estrogen can act either locally or systemically to cause mitosis of the mammary epithelium (Haslam, 1988). Both hormones stimulate DNA synthesis at the' terminal end buds of the mammary gland, although only progesterone induces DNA synthesis in ductal epithelium. Progesterone is also responsible for differentiation of terminal buds into alveoli (Bresciani, 1968). Estrogen receptors are found in mammary tissue only in mature animals. Their synthesis is induced In! estradiol- 176 (Muldoon, 1979). Prolactin gradually converts these estrogen receptors from the 48 to the 88 form throughout gestation so that by late pregnancy and during lactation the receptor exists almost completely in the 88 form (Muldoon, 1979). This change in receptor type is thought to be associated with the greater responsiveness of the mammary epithelial cells found at lactogenesis (Muldoon, 1981). Progesterone is bound to its receptors in the mammary tissue during gestation. However, receptors disappear during lactation before reappearing at involution (Capuco gt a” 1982) . Lactogenesis depends upon a sudden decrease in progesterone concentrations immediately prior to parturition (Tucker, 1981). Estrogen concentrations increase rapidly about 1 month before parturition and then 8 decrease shortly after parturition. These two hormones are not responsible for growth during early lactation because ovariectomy of goats immediately before parturition did not prevent lactational mammary growth (Cowie gt gl., 1965). In attempts to stimulate mammary gland development in ovariectomized heifers using exogenous hormones, the two hormones which elicited the greatest response were estrogen and progesterone (Cowie gt g;., 1965; Sud gt g;,, 1968). However, these two hormones alone or in combination failed to produce complete mammary development as observed in peri-parturient animals. For example, Sykes and Wrenn (1951) found that steroids alone produced abnormal growth of the mammary ducts, alveoli and lobules, whereas a combination of diethylstilbestrol, progesterone and pituitary extracts produced histologically normal mammary development. Collier gt g1. (1977a) increased milk production in cows which had previously failed to lactate following estrogen and progesterone treatment by injecting reserpine, a drug known to increase prolactin concentrations. The anterior pituitary hormones, growth hormone and prolactin, are therefore needed to synergize with the ovarian hormones to produce maximal mammary development (Lyons, 1958). 2 . PROLACTIN Prolactin acts synergistically with progesterone to stimulate lobular-alveolar development during allometric mammary growth (Lyons, 1958). Sinha and Tucker (1969) observed that pituitary prolactin concentrations in heifers increased at the start of the pubertal allometric growth phase and reached a maximum at the end. However, Tucker gt g1. (1973) and Vines gt g1. (1976) found no difference in serum prolactin concentrations in heifers between 3 and 5 months of age. During pregnancy, serum prolactin concentrations remain relatively low and stable (Oxender gt a_l., 1972) before increasing sharply 1 to 2 d before, and declining again 1 to 2 d after, parturition (Akers, 1985). This prolactin surge is an important stimulator of -lactogenesis. Because prolactin concentrations are low during the greater part of pregnancy, it seems unlikely that prolactin drives the allometric mammary growth phase during gestation. However, prolactin is thought to be important in activating the estrogen receptor, causing estrogen to bind to DNA, at least in the mouse mammary gland (Muldoon, 1987). Prolactin concentrations are also related to estrogen concentrations. For example, prolactin synthesis and secretion within pituitary cells is stimulated by estrogen (Chen and Meites, 1970) . In rabbits, progesterone limits prolactin receptor numbers (Djiane and Durand, 1977); whether this is also true in cattle is unknown. 10 Prolactin has no effect on milk yield during an established lactation, and serum concentrations of prolactin are not highly correlated with milk secretion rate. Use of 2-bromo-a-ergokryptin (C3154), an ergot alkaloid which reduces prolactin concentrations, did not reduce milk secretion in cattle during an established lactation (Smith gt a_l., 1974). Inaddition, Plaut gt g1. (1987) administered pituitary-derived prolactin to cows before and after peak milk production but failed to stimulate milk yield. However, indirect evidence suggests that prolactin could be important during lactation. For example, mammary uptake of prolactin is greatest during early lactation when milk yields are highest (Beck gt g1.,1979) as are the clearance and secretion rates of prolactin (Akers gt gl_._, 1980). In lactating rats, the number of prolactin binding sites in mammary tissue increased during early lactation and then declined along with milk yield (Bohnet gt g;., 1977). Several environmental factors affect both serum prolactin concentrations and milk yield. Increasing the photoperiod from 8 to 16 h per d increased serum prolactin concentrations by 50 to 80% in lactating cows (Peters _e_t g” 1978, 1981). Milking-associated stimuli, for example washing the mammary gland, caused an immediate 2.8-fold increase in serum prolactin concentrations, which decreased to basal concentrations about 30 min after milking (Koprowski and Tucker, 1973) . These milking-induced surges 11 reach a maximum amplitude at 8 weeks of lactation and then decrease until there is no response after 32 weeks (Koprowski and Tucker, 1973) . The importance of these milking-associated prolactin surges has yet to be determined, because increasing the amplitude of these surges using thyrotropin—releasing-hormone (TRH) or decreasing them by administration of C8154 does not alter milk yield (Karg _e_t gl_., 1972; Hart, 1973; Beck gt g_l_., 1979). Decreased ambient temperature reduces both basal and TRH- stimulated prolactin concentrations in serum of cows (Wettemann and Tucker, 1974). Ambient temperatures below 1 C suppress prolactin concentrations in animals exposed to 16L:8D to values found in heifers exposed to natural short- day photoperiod (Peters and Tucker, 1978). Cold days, therefore, reduce the effects of long-day stimulation of prolactin concentrations. 3. GROWTH HORMONE Growth hormone (somatotrop in) , the other anterior pituitary hormone needed for full mammary devel opment , synergi zes with estrogen to stimulate mammary duct growth (Lyons, 1958). Administration of exogenous somatotropin daily for 15.4 weeks increased mammary parenchyma in heifers by 46% over control values and decreased extraparenchymal tissue and mammary gland weight (SejrSen gt gl., 1986). Overfeeding pre-pubertal heifers can lead to decreased 12 milk production due to decreased mammary parenchyma. This effect is thought to be related to growth hormone because overfeeding decreases serum growth hormone concentrations (Sejrsen g gt, 1983). Concentrations of serum growth hormone remain stable throughout gestation (Oxender gt g1., 1972) before increasing sharply at parturition. Serum concentrations remain high during early lactation but decline as lactation progresses (Koprowski and Tucker, 1973). Unlike prolactin, growth hormone can markedly stimulate milk production during an established lactation. Milk yield is increased by 16 to 18% using pituitary-derived growth hormone and between 23 and 41% using recombinant growth hormone (Machlin, 1973; Bauman gt g1” 1985). Growth hormone receptors have not been found in the mammary gland, suggesting an indirect mechanism for these effects (Keys, 1985), possibly via effects on gluconeogenesis and adipose tissue lipolysis (Akers, 1985). Growth hormone releasing-factor also stimulates milk production during lactation, probably by increasing concentrations of growth hormone (Enright gt _1., 1986). 4. OTHER HORMONES Corticosteroids are required in small amounts for full mammary development (Lyons, 1958). Although exogenous hydrocortisone acetate can maintain total mammary cell numbers in rats, metabolic activity per cell is not 13 increased unless prolactin is also administered (Tucker, 1969). Cortisol binding sites increase 300% in the mammary gland during the second half of pregnancy in mice (Chomczynski and Zwierzchowski, 1976) and remain elevated throughout lactation (Gorewit, 1972). Glucocorticoid concentrations are relatively stable throughout pregnancy but increase markedly prior to parturition (Tucker, 1979). Placental lactogens are important for mammary growth in many species. These can be extracted from the placenta and when injected into hypophysectomized, ovariectomized rats they stimulate lobular-alveolar development (Ray g g_l. , 1955). However, placental lactogens have little cm' no role to play in mammary development in cattle, where they appear unable to cross the placenta into the maternal circulation (Schellenberg and Friesen, 1982). The placenta also produces estrogens and, in some species, not including cattle, progesterone (Thorburn gt gL, 1977). Fetal placentae could be the major source of mammotrophic hormones after mid-pregnancy in the majority of species (Desjardins gt _a_l., 1968). In fact, placental hormones could explain why weight of the mammary gland is positively correlated_ with the number of fetuses in some species, including rats and goats (Desjardins gt gtt, 1968; Hayden gt git, 1979). The role of thyroid hormones in mammogenesis is unclear, because a thyroidectomized animal can become pregnant and lactate (Tucker, 1985). However, Vonderhaar and Greco (1979) 14 observed that hypothyroidism in rats retarded ductal and lobular-alveolar growth in the mammary gland. Insulin stimulates mitosis in the mammary gland _i-Il vitro (Topper and Freeman, 1980) . However, serum insulin concentrations decrease during gestation in cattle, suggesting that insulin is not limiting for normal mammary growth i vivo (Grigsby gt al., 1974). Insulin is thought to be essential for lactation because casein synthesis can not be initiated by prolactin in vitro unless insulin is in the culture medium (Bolander gt glt, 1981). Relaxin is another hormone with little mammogenic activity pg; 55, but it is capable of synergizing with other mammogenic hormones to stimulate mammary growth in rats and mice (Harness and Anderson, 1977). Its role in cattle has yet to be determined. (13) . LACTOGENESIS Lactogenesis is a series of events in which mammary cells are converted from a non-secretory to a secretory state. It can be divided into two stages (Hartmann, 1973). The first, which occurs about 3 weeks before parturition in cattle, consists of enzymatic and cytological differentiation of alveolar cells and limited secretion of an extra-cellular-type fluid. The second stage, occurring 1 to 4 d before parturition, consists of a rapid increase in lactose secretion, with a concurrent increase in milk production (Hartmann, 1973). 15 HORMONES AND LACTOGENESIS Marked changes occur in blood serum concentrations of many hormones during the third trimester of gestation. The first detectable hormonal change is an increase in serum estrogen concentrations starting about 10 to 20 d before parturition and reaching a peak 1 to 4 d before parturition (Hunter e_t g1” 1977), before returning to basal concentrations by 48 h after parturition (Robertson, 1974). While serum estrone concentrations dominate during this surge, both isomers of estradiol (17a and 178) increase during this time (Erb, 1977) . Exogenous estrogens increase serum prolactin concentrations, prolactin receptors and glucocorticoid concentrations (Schams and Karg,1972; Sheath g g_1_. , 1978). These effects could be mediated by prostaglandin-F20: (PGFZa ) , since high estrogen concentrations also act on the uterine endometrium to stimulate prostaglandin production. PGFZa is luteolytic in cattle (Britt e_t gl_., 1981) and may play a role in lactogenesis by destroying the corpus luteum and causing a decrease in progesterone concentration. Exogenous PGF2a also increases prolactin, growth hormone and glucocorticoid concentrations in serum of heifers (Louis gt git, 1974). Progesterone inhibits lactogenesis during gestation by suppressing the normal peri-parturient synthesis of lactose, casein and a-lactalbumin (Kuhn, 1969; Turkington and Hill, 1969). There is a large decrease in serum progesterone 2 d before parturition, with a nadir of about 1 ng/ml at l6 parturition (Chew gt git, 1977). This causes a large increase in synthesis of mammary enzymes and. secreted proteins. Progesterone binds to its own receptor in mammary tissue and also competes with glucocorticoids for binding to the glucocorticoid receptor (Capuco _e_t glt, 1982) . During gestation, progesterone inhibits the lactogenic effects of glucocorticoids by binding to their receptor (Dj iane and Durand, 1977), and reduces the ability of estrogen to bind to it's own receptor (Bohnet gt gt” 1977). Prolactin triggers up-regulation of the prolactin receptor (Houdebine gt g;., 1983) by increasing the concentration of receptors on the Golgi and plasma membranes of epithelial cells in mammary glands of cows during pregnancy and early lactation (Holcomb gt g;., 1976; Houdebine gt g1., 1983). Progesterone -prevents these effects (Djiane and Durand, 1977). Thus, in cows, when serum concentrations of progesterone decrease, prolactin concentrations start to rise, about 2 d before parturition, until a peak (285 ng/ml) is reached at about 12 tx: 24 h before parturition. Prolactin then decreases linearly, returning to basal concentrations (90 ng/ml) 2 d after parturition (Ingalls gt g;., 1973; Chew gt gl., 1979). The prolactin surge is an important stimulus for lactogenesis and is necessary for full structural differentiation of the mammary alveolar epithelium (Akers gt glt, 1981b). This differentiation involves enlargement of the alveoli and induction of cell polarity, with the nucleus 17 increasing in size and becoming situated adjacent to the lumen. Secretory products appear within the cell and lumina (Mills and Topper, 1970). Prolactin also causes swelling of the Golgi membranes and formation of rough endoplasmic reticulum (Akers gt _aL, 1981b). This leads to increased casein mRNA synthesis and thus increased casein production (Teyssot gt _a_l_., 1981). Casein forms micelles within Golgi vacuoles (Hallowes gt g;., 1973). Elevated prolactin concentrations also increase synthesis of fatty acids (Collier gt g;., 1977), lactose (Delouis and Denamur, 1972) and a-lactalbumin (Goodman e_t g1., 1983). Alpha- lactalbumin is a major milk protein and is also rate- limiting to production of lactose synthetase, the enzyme responsible for lactose synthesis (Akers, 1985). Estradiol, insulin and cortisol synergize with prolactin to increase a- lactalbumin production in a dose-related manner (Vondehaar gt al., 1973; Goodman gt al., 1983; Nagamatsu and Oka, 1983). The peri-parturient prolactin surge is essential for lactogenesis in some species, for example rats (Kuhn,1977) and humans (Rolland and Schellekens, 1978) . When the surge is inhibited in cattle with CB154, subsequent milk yield decreases by 11.4 kg/d compared with controls (Akers 53 gl_., 1981a). Similar results were also obtained in cows by Schams gt _a]._. (1972), Johke and Hodate (1978) and in goats by Davies gt Q; (1983). Akers gt git (1981a) suggested that this inhibition of the peri-parturient 18 prolactin surge reduced mammary secretory cell activity (RNA concentration, RNA/DNA ratio) rather than cell numbers (DNA concentration). Milk yield continued to increase despite suppression of prolactin secretion (Akers e_t gt.,1981a). This .is thought to be a result of low circulating prolactin concentrations taking more time to increase receptor numbers (Akers gt g;.,1981a). Growth hormone concentration follows a pattern similar to prolactin, but peaks 24-h later at time of parturition, and returns to a basal concentration 4 d after parturition. The role of growth hormone in lactogenesis is unclear, although it appears to synergize with adrenocorticotropin and prolactin (Cowie, 1969). Exogenous glucocorticoids given to cattle with mature mammary glands quickly caused copious milk secretion (Tucker and Meites, 1965). Glucocorticoid concentrations increase just prior to or during parturition. However, it is not known whether this surge is necessary for lactogenesis or Only a result of the stress of parturition (Smith gt g1., 1973; Hunter gt g;., 1977). Glucocorticoids are bound to corticoid-binding-globulin (CBG) in blood and in this form the hormones are inactive. Concentrations of CBG decrease during the peri-parturient period which allows an increase in free glucocorticoids (Gala and Westphal, 1965). Glucocorticoids are important in alveolar cell differentiation of the rough endoplasmic reticulum and Golgi apparatus (Mills and Topper, 1970). Mehta _et a_l_. 19 (1980) concluded that cortisol synergizes with prolactin to initiate lactogenesis, because it was needed to stimulate expression of the casein gene in cultured mouse mammary glands. In ovariectomized rats, removal of the adrenal glands reduced synthesis of RNA and casein .and other proteins in mammary tissue (Davies and Liu, 1969). This cortisol-induced differentiation is a prerequisite of the subsequent induction of casein synthesis by prolactin. It appears, therefore, that cortisol synergizes with prolactin to initiate lactation. (C). PHOTOPERIOD AND HORMONES Increasing exposure to light from 8L:16D to 16L:8D increases milk production in cows (Peters e_t g_l_., 1978; Stanisiewski and Tucker, 1986; Phillips and Schofield, 1989). Peters g _a_t. (1981) found this response to be independent of the stage of lactation at which the extended light period was introduced. However,the observed increase in milk yield could be explained by a concomitant increase in dry matter intake (Peters gt g1” 1981). Milk fat percentage was unaffected by increased exposure to light (Peters g g]_.., 1978; 1981) . Mechanisms controlling this response in milk yield to photoperiod are not known. Those hormones which are responsive to changing photoperiod could be the mediators of these photoperiodic effects. 20 l. PROLACTIN Exposure to 16L:8D increased serum concentrations of prolactin 4 to 8 fold compared with animals under 8L:16D (Bourne and Tucker, 1975; Petitclerc gt, gl., 1983; Stanisiewski gt g1., 1984). Leining gt g1. (1979) found that increasing daily light from 16 to 20 h had no further effect on peak prolactin concentrations; in fact, continuous light for 3 to 4 weeks reduCed prolactin concentrations to those found during 8L:16D. Stanisiewski gt a_l. (1988) reported that exposure to continous light for 3 to 4 weeks increased prolactin concentrations, but only 1x) values found in 16L:8D. Furthermore, this response was slower than the response following introduction ‘to 16L:8D. Therefore, to achieve and maintain high concentrations of prolactin in serum a 4 to 8 h dark period each day is needed. Leining _e_t fl. (1979) reported that serum prolactin concentrations start to increase about 1 week after an abrupt change from 8L;16D to 16L:8D and reached a maximum at 4 to 6 weeks. This increase in prolactin concentration under long days is not indefinite. After about 12 weeks, bulls became refractory to long-day photoperiod and serum prolactin concentrations begin to return toward those found under 8L:16D (Stanisiewski gt gl., 1987). In contrast, the response of serum prolactin concentration to changes in ambient temperature is almost instantaneous (Wettemann and Tucker, 1974). Temperatures below 1.<: can prevent the photoperiod-induced rise in 21 prolactin concentrations (Peters and Tucker, 1978), but do not inhibit milking-induced prolactin release (Peters gt gl., 1981). The effects of both photoperiod and temperature on the peri-parturient surge of prolactin are unknown; in the one experiment in which the peri-parturient prolactin surge ‘was studied, photoperiod and temperature were confounded (Chew gt g1., 1979). Prolactin secretion in cattle is sensitive to a broad spectrum of light. "Vita-lite" fluorescent (simulates natural sunlight), incandescent, high pressure sodium or mercury vapor lamps and red and blue fluorescent lights were as effective as cool-white fluorescent light in stimulating prolactin concentrations in serum (Leining g gl., 1979; Stanisiewski-gt gl., 1984). It is thought that changes in prolactin concentrations in response to photoperiodic changes are mediated through the eye, since increasing the light from 8 to 16 h daily does not affect prolactin secretion in blind animals (Petitclerc gt g;., 1981). 2. MELATONIN Melatonin is synthesized by the pineal gland and secreted in a circadian pattern, concentrations being lowest during periods of light and greatest during - darkness (Cardinali, 1981). The retina of the eye perceives darkness and sends a signal to the suprachiasmatic nuclei (SCN). The SCN neurons then convey impulses to the pineal gland, via 22 the upper thoracic spinal cord and sympathetic nervous system, to stimulate :melatonin. secretion (Reitery 1988). Light is a potent suppressor of this stimulus. Serum melatonin concentrations increased 1.6 to 5.1 fold during darkness compared with light in bull calves (Stanisiewski gt gl., 1988). Continuous light prevented the nocturnal increase in melatonin in three of four calves (Stanisiewski gt g;., 1988). Melatonin does not appear to be causally related to prolactin in cattle because pinealectomy, although destroying the response of melatonin to photoperiod, did not affect the long-day induced increase in prolactin concentrations (Stanisiewski gt _at., 1988). Recent evidence appears to contradict this. Feeding melatonin in order to mimic short days reduced serum prolactin concentrations (110 ng/ml) compared with heifer calves exposed to 16h light (145 ng/ml, p<.001, E.Sanchez, personal communication). Feeding melatonin during the middLe of a 16 h light period mimicked short days in post-pubertal beef heifers. Fat concentrations in the 9-10-11 rib, Longissimus dorsi muscle and carcass were increased and protein concentrations in both the 9-10-11 rib and carcass were decreased (Zinn gt gl., 1988). However, when heifers approaching maturity were used, melatonin had no effect on fat accretion (Zinn gt g1., 1988). Mhatre gt a_l_. (1984) observed that administering melatonin to rats reduced plasma estrogen concentrations and 23 mammary development. However, it is not known . whether this was a direct effect of melatonin on manunary growth or an indirect effect mediated by estradiol. The effect of daylength on serum melatonin concentrations and mammary development in pregnant heifers has not been investigated. 3. OTHER HORMONES Several other hormones have been measured in cattle subjected to different photoperiods. No effects of photoperiod have been found on serum concentrations of growth hormone (Peters gt g1” 1980, 1981; Petitclerc gt g;., 1983; Zinn gt g1., 1986), insulin, thyrotropin or thyroxin (Leining gt g1” 1980; Tucker gt _l_., 1984). The effect of photoperiod on glucocorticoid concentrations is unclear. Leining gt _a_l_,_ (1980) reported decreased glucocorticoid concentrations when photoperiod was increased from 8 to 16 h per day in pre-pubertal animals. The reverse was also true: glucocorticoid concentrations increased 118% when photoperiod was decreased. However, it is possible that the interval between collection of samples in this experiment was too long to detect, and account for, the diurnal rhythm of cortisol secretion, because others failed to find any effect of photoperiod on glucocorticoid concentrations (Peters gt a_l., 1980, 1981; Zinn e_t a_l., 1986). Photoperiod had no effect on timing, amplitude or pattern of the pre-ovulatory surges of LH or FSH 24 (Rzepkowski e_t _a_l_., 1982). Bourne and Tucker (1975) and Stanisiewski _t g1. (1987) also noted that differing photoperiods did not alter LH concentration in bull calves. However, McNatty gt _a_l. (1984) reported a higher LH peak frequency in cattle during summer compared with winter months. Follicles were larger, contained more granulosa cells and corpora lutea were heavier and secreted more progesterone during winter compared with summer. The increased. progesterone concentrations could offer an explanation for the lower LH peak frequency in the winter, since progesterone inhibits LH pulse frequency (Goodman gt gl., 1981). In contrast, Rhodes gt g;. (1982) found heavier corpora lutea during summer than winter. Release of progesterone from winter corpora lutea in LH-challenged cultures was reduced compared with corpora lutea removed from cattle during. summer months. This increased progesterone secretion during the summer could also help to explain the fact that heifers under long days attain puberty earlier than those under 8 h of light (Peters gt g;., 1977; Hansen gt g;., 1983; Petitclerc gt gl., 1983). Testosterone concentrations were also increased in pre- pubertal bull calves exposed to 16L:8D compared with calves under 8L:16D (Stanisiewski gt gt, 1987). This could be associated with earlier onset of puberty in male cattle. In conclusion, it is clear that many of the hormones known to play a role in mammary development are influenced by variations in photoperiod. 25 (d). PHOTOPERIOD AND MAMMARY DEVELOPMENT. There have been few studies of the direct effect of photoperiod on mammary development. Burns (1987) reported a circadian rhythm in the mammary gland response to prolactin in rats. Ovine prolactin was injected into rats at the beginning or in the middle of either a 12 h light or 12 h dark period. Mammary glands were heavier when prolactin was injected at the start of the light period, compared with the middle of the light period or the dark period. This indicates an effect of prolactin on the mammary gland mediated by photoperiod. Burns (1987) concluded that sensitivity of prolactin receptors to prolactin increased in the light. Mhatre (gt .gtt (1984) found positive correlations among photoperiod, serum prolactin concentration and mammary development in rats. Continuous lighting increased serum prolactin concentration and mammary DNA concentration and number of alveolar buds compared with rats on a 10L:14D cycle. When C8154 or melatonin were administered to rats under a 24L:0D regimen, there was a decrease in total DNA and number of mammary buds relative to controls. Petitclerc gt g1. (1985) exposed pre- and post-pubertal heifers to 16L:8D and observed 40 and 30% increases in mammary parenchyma weight relative to heifers given 8L:16D. Total parenchymal DNA also increased under long days by 68% and 35% in pre- and post- pubertal heifers, respectively. However, photoperiod did not influence concentrations of DNA 26 or fat in parenchymal tissue. In contrast, extraparenchymal tissue weight was 12 to 35% less in heifers given 16L:8D than in heifers exposed to 8L:16D. Therefore, in comparison with 8L:16D, 16L:8D stimulated mammary parenchymal tissue to grow into the fat pad of pre- and post-pubertal heifers (Petitclerc gt gl., 1985). Finally, recent work involving the feeding of melatonin to pre-pubertal dairy heifers to mimic short days also reduced mammary parenchyma development compared with heifers exposed to 16L:8D (E.Sanchez, personal communication). This suggests that 16L:8D might not stimulate mammary parenchymal growth, but that increased nocturnal elevation of melatonin under 8L:16D might have an inhibitory effect. There are no reports concerning effects of photoperiod on mammary gland development in pregnant dairy cattle. Such effects were studied in Experiment 1. EXPERIMENT ONE (a). EXPERIMENTAL OBJECTIVES The effect of photoperiod on mammary growth during the allometric mammary growth phase of pregnancy has never been studied, and the hormonal links between photoperiod and mammary development have not been established. Therefore, the first objective of the first experiment was to assess the effect of a 16L:8D photoperiod versus 8L:16D on secretory cell numbers and fat content of mammary glands. Any effects of photoperiod could be mediated by changes in serum concentrations of one or more hormones. A second objective was to determine the effect of photoperiod on serum concentrations of prolactin, melatonin and progesterone. This has not previously been studied in pregnant heifers. (b) . MATERIAL AND METHODS. 1 . TREATMENTS AND MANAGEMENT Twenty Holstein dairy heifers were purchased and then divided into five blocks of four according to stage of pregnancy (calculated from breeding date provided by 27 28 farmers). All animals were exposed to 8L:16D for an acclimation period. Since heifers at the time of assignment to treatment were at different stages of pregnancy the acclimation period began at 48 to 100 d of pregnancy and lasted 80 to 28 d, respectively. However, average duration of the acclimation period was not different between photoperiod treatments. Beginning at 128 d of pregnancy heifers were moved at random, within a block, to one of four pens. Two pens received a photoperiod of 16L:8D and two received 8L:16D (treatments). Lights came on at 0700 11 each day' in all pens and light intensity, measured at heifer eye level, was 298 (SE = 8.6) lux. Heifers were bedded on straw and fed, on a fresh-weight basis, 67.7% corn silage, 27% alfalfa haylage, 5% soybean meal and .3% vitamin-mineral supplement, at a rate of 9 kg dry matter. head'1.d'1. Heifers were weighed at 14-d intervals throughout the experiment, and the amount offered adjusted to maintain .7 kg live-weight gain/d. Heifers were fed once daily immediately after lights were turned on at 7 am. Water was freely available at all times. Live weights of heifers were recorded before feeding on three consecutive days at the start (initial body weight, BW) and end of the experiment, and live-weight gain (kg/d) was calculated by difference. Heifers were slaughtered at 248 (SE = 1.1) d of pregnancy. Two heifers (one from each light treatment) aborted and were removed from the experiment. 29 2. BLOOD SAMPLING Three times during the experiment blood was collected for 24 h. Each 24 h period is defined as a bleed. Heifers averaged 160, 197 and 219 d of gestation (range of i 26 d). On the day before blood collection, all heifers were fitted with indwelling jugular polyvinyl cannulas and halter-tied to a fence in their respective pens with access to feed and water. At 0700 h on the following day, blood samples were drawn and discarded at 20-min intervals for a pre-sampling period of 2 h. This was an attempt to accustom the animals to the sampling procedure and thus minimize stress effects on prolactin concentrations during the sampling period. After this pre-sampling period, blood was collected every 30 min for 6 h for prolactin analysis. Blood samples were collected at 2-h intervals for the remainder of the 24 IL Melatonin was measured in samples collected at 2-h intervals for the 24-h period, and progesterone was quantified in samples collected at 8-h intervals for the 24-h period. Cannulas were filled with an anticoagulant (35 g/l sodium citrate) between samplings. Red. lamps. were ‘used 'to facilitate. blood collection during dark periods. The light intensity of these lamps was less than 11 lux at a distance of .5 m, and approximately 1 min was required to collect each sample. Blood samples were allowed to clot for approximately 2 h at room temperature before storage for 24 h at 4 C. After centrifugation at 3000 rpm for 30 min, serum was decanted 30 and frozen at -20 C until assayed for prolactin (Koprowski and Tucker, 1971), melatonin (Webley gt gt., 1985) and progesterone (Spicer gt g1., 1981). 3. SLAUGHTER AND ANALYSIS OF CARCASSES Heifers were weighed and transported to the abattoir approximately 1 h before slaughter. They were stunned with a captive bolt, killed by exsanguination and mammary glands removed from the carcass and weighed. Samples of tissue from one quarter were taken for histological studies while the remainder of the mammary gland was frozen. The fetus 'was removed from. placental membranes and weighed. Crown-rump length of the fetus was measured and used as a supplemental index of stage of pregnancy. Perirenal fat and kidneys were removed from the carcass on the day of slaughter and weighed. The carcass was then weighed and chilled overnight at 0 C. The following morning the left half of the carcass was quartered between the 12th and 13th ribs and the area of the Longissimus dorsi muscle measured. Depth of subcutaneous fat on the cut surface of the 12th rib was measured at a point two-thirds of the maximum width of the Lpngissimus dorsi, measuring from the ventral surface of the carcass. 4. ANALYSIS OF THE MAMMARY GLANDS The whole frozen glands were sliced into 10 mm slices using a band saw and skin, teats and supramammary lymph 31 nodes were removed. Slices were dissected into parenchyma and extraparenchymal fat. These tissues were then weighed. For each animal, parenchyma was mixed and ground twice through an 8 mm plate followed by once through a 4 mm plate using a meat grinder (Toledo Co., Ohio), with thorough mixing between each grinding. Sub-samples were collected, composited and analyzed for DNA, RNA (Tucker, 1964) and ether-extracted fat (soxlet analysis). The right rear quarter was sliced at a 900 angle to the medial suspensory ligament and an imaginary line drawn parallel to this ligament, beginning at the teat attachment. Two samples of parenchymal tissue were taken 3 cm either side of the mid-point of this imaginary line (Akers gt git, 1981b) . Tissue was immersed in fixative (Karnovsky, 1965) for 6 to 7 h before being washed three times with .2 M sodium phosphate buffer containing 80 g/l sucrose. Tissue was stored in this buffer until dehydrated in a series of alcohol baths and embedded in JB-4 plus resin (Polysciences Inc., Warrington, PA). Samples were sliced into 2 um sections using a JB-4 ultramicrotome (Polysciences Inc., Warrington, PA) and stained with toluidine blue (10 ml/l toluidine blue in 10 g/l sodium borate). Four sections from one tissue sample (chosen at random from the two samples taken) from each heifer were examined at a magnification of 400 X (A.0.Spencer Microscope Inc.). Tissue types were counted at 64 intersections of an ocular planimeter, for three fields of view, chosen at random. Tissue was defined 32 as either stromal tissue, epithelial cells or alveolar lumen. This procedure gave 768 observations for each animal. 5. STATISTICAL ANALYSES Mammary gland composition, live-weight gain and data obtained at time of slaughter were analyzed by analysis of variance (Gill, 1978), with main effects of treatment (photoperiod), block and pen within treatment (Appendix 1a). Initial body weight (BW) (on d 128 of pregnancy) was tested as a covariate for all indices of mammary development and carcass and fetal characteristics, but was excluded from the model if non-significant (P>.1). Prolactin and progesterone concentrations were analyzed using a split-plot design with repeated measurements over time (Appendix 1b). Melatonin was analyzed as a split-split plot design with repeated measurement, the extra split-plot being light and dark periods within each day (Gill, 1986, Appendix 1c). For the purpose of calculating area under the curve, the dark period was defined as lasting from 1 h after lights were switched off to 1 h before lights were switched on. Similarly, the light period was considered to have commenced 1 h after lights were switched on and ended l h before lights were switched off. In other words, lights were switched on or off in the middle of 2-h periods. These periods were not included in the calculation of exposure to melatonin during the light or dark period, but were included in the calculation of exposure to melatonin during the whole 33 24-h period. Light and dark periods were compared using the Bonferroni 't' test (Gill, 1986). Stage of pregnancy at time of first bleed was tested as a covariate in the analysis of hormone concentrations. Ambient temperature at the time of collection of each blood sample was also tested as a covariate in the analysis of prolactin concentrations. (C) . RESULTS L; MAMMARY DEVELOPMENT GROSS MAMMARY TISSUE COMPOSITION There was no effect of photoperiod (16L:8D vs 8L:16D) on total mammary gland weight (parenchyma, extraparenchyma, teats, skin and supramammary lymph nodes), total mammary parenchymal weight and proportion of parenchyma in the mammary gland (Table 1). However, there was a numerical trend in total mammary gland weight (P=.12) with heifers given 8L:16D having heavier mammary glands than 16L:8D. Although the effect of treatment on weight of extra- parenchymal fat was statistically non-significant (P=.54), heifers given 8L:16D had 41% more extraparenchymal fat than heifers exposed to 16L:8D. There was a significant pen(within treatment) effect (P=.03) which necessitated the use of pen(within treatment) as the unbiased error term rather than block x pen(within treatment) as in the original model (Appendix 1a). The power and sensitivity of the test 34 TABLE 1. Effect of photoperiod on gross tissue composition of the mammary gland in pregnant heifers Treatment 16L:8D 8L:160 531 P Mammary gland weight (g) 11393.5 14068.1 1046.27 .12 Extra-parenchymal fat: (g) 3148.9 4442.9 153.09 .542 (% gland weight) 34.4 40.4 3.30 .53 Parenchyma: (9) 6399.4 7278.2 985.57 .44 (% gland weight) 65.6 59.6 3.30 .53 1 Pooled SE. 2 P value adjusted for significant covariate (initial BW). 35 of treatment differences was, therefore, reduced. Initial BW was also significant when used as a covariate and further reduced the difference in extraparenchymal fat between treatments. CHEMICAL COMPOSITION OF PARENCHYMA Treatment (16L:8D vs 8L:16D) had no effect on DM, fat or nucleic acid concentration in parenchyma (Table 2). Total amount of nucleic acids in the parenchyma was also not affected by photoperiod (Table 2). TABLE 2. Effect of photoperiod on chemical composition of parenchyma in the mammary gland of pregnant heifers TREATMENT 16L:8D 8L:16D SE1 p Dry matter (g/100g wet weight) 33.4 31.5 1.10 .16 Fat (g/100g wet weight) 11.3 9.3 1.70 .44 DNA (9) 26.1 29.8 4.04 .32 DNA (mg/g wet weight) 4.05 4.16 .120 .83 RNA (g) 18.7 21.9 3.03 .28 RNA (mg/g wet weight) 2.87 2.96 .064 .35 1 Pooled SE. 36 HISTOLOGY Relative amounts of stromal tissue, epithelial cells and alveolar lumen in parenchyma were not affected by photoperiod (Table 3). TABLE 3. Effect of photoperiod on histology of parenchyma in the mammary gland of pregnant heifers TREATMENT 16L:8D 8L:16D SE1 P Stromal cells (%) 38.9 36.7 3.59 .35 Epithelial cells (%) 26.1 26.4 .69 .79 Alveolar lumen (%) 35.0 ' 36.9 3.35 .29 1 Pooled SE. 2. HEIFER GROWTH, CARCASS AND FETAL CHARACTERISTICS. Rate of live-weight gain tended to be greater for heifers given 16L:8D compared with 8L:16D (Table 4). This difference in live-weight gain between treatments was not a function of initial BW, which was not significant when tested as a covariate. However, there was no significant difference between treatments in carcass weight when initial BW was used as a covariate (Table 4). Subcutaneous fat, 37 TABLE 4. Effect of photoperiod on pregnant heifer growth and carcass and fetal characteristics Treatment 16L:8D 8L:16D SE1 P Heife; growth: Initial body weight (kg) 481 Final body weight (kg) 592 Live-weight gain (kg/d) .93 Catcass characteristics: Carcass weight (kg) 282 Perirenal fat (kg) 14.0 Subcutaneous fat (mm) 3.9 Longissimgs ggtgi area (cm ) 65.9 Fetal characteristics: Crown-rump length (mm) 978 Fetal weight (kg) 31.9 525 615 .75 304 15.0 69.9 989 34.1 10.5 .02 7.5 .32 .057 .11 2.4 .912 .79 .452 .34 .152 1.73 24 14.8 .38 1.49 12 1 Pooled SE. 2 P value adjusted for significant covariate (initial BW). 38 perirenal fat and Longissimus dorsi area did not differ between photoperiod treatments (Table 4). Although treatment effects on fetal characteristics were non-significant,there was a trend (P=.12) for greater fetal weight in heifers given 8L:16D than 16L:8D (Table 4). This was not a function of initial BW, which was not significant when tested as a covariate. 3. SERUM HORMONES Stage of pregnancy (based on farmer-provided breeding dates and on fetal crown-rump length) may confound effects of treatment on serum hormone concentrations. Therefore stage of pregnancy was tested as a covariate in the initial model for all serum hormones (Appendix 1b). Stage of pregnancy was found not to be significant (P>.05) for any of the serum hormones and so was dropped from the model. PROLACTIN Mean prolactin concentrations (pooled across the three bleeds) was greater for heifers exposed to 16L:8D (103.5 ng/ml) than for heifers given 8Ial6D (61.5 ng/ml, SE = 8.58, P=.02). There was no effect of stage of pregnancy on prolactin concentrations because stage of pregnancy was tested as a'covariate and found to be non-significant (P>.05; Figure 1). However, there was a significant bleed effect (P=.044), with prolactin concentrations increasing from bleed 1 to bleed 3 (Figure 2). Although ambient Figure 1. 39 Serum prolactin concentrations in pregnant heifers exposed to either 16L:8D ( E] ) or 8L:16D ( + ). Each point represents the mean for one heifer during one of three 24-h bleeds. Each heifer was represented three times. Pooled SE = 8.58 ng/ml of serum. 40 + . +53J [:1 a a 04 U3!— El + l D '. \ '2 D +EI+ + '3 I: + 3+ + Cl if [:1 94+ {:1 1+ [:1 KPH-1+ +’. if a a + . 43:1 + L 1 J J L J C C C C C O O In C IO C ID a N N *4 F" (In: /Bu) unoeIOJd mules 160 180 200 220 240 260 Stage of pregnancy (d) 140 120 Figure 2. 41 Serum. prolactin concentrations in pregnant heifers exposed to 16L:8D ( D ) or 8L:16D ( + ). Each point represents one treatment mean during one of three 24-h bleeds. Stage of pregnancy averaged 160, 197 and 219 d for bleeds 1, 2 and 3 respectively. Pooled SE = 8.58 ng/ml of serum. 42 140 )- 120 L 20- l l l l O O O O O O CO ‘d‘ v-i __ (1m /Fu) unoeIOJd mnxas Bleed 43 temperature also increased from bleed 1 to bleed 3, the correlation coefficient between individual prolactin concentrations and temperature was only .05 (P=.18). There was more variation in individual prolactin concentrations in 16L:8D heifers (cv = 31.4%) than in those under 8L:16D (cv = 19.6%). MELATONIN In terms of pattern of concentrations, all groups of heifers responded similarly to the light cycle, with an increase in melatonin concentrations in dark periods compared with light (Figure 3). Total exposure to melatonin (area under the curve) was also greater during the dark periods compared with light within each treatment (Table 5). There was no effect of treatment on the total amount of melatonin heifers were exposed to each day (total area under curve over 24 h) or on daily mean serum melatonin concentrations (Table 5). Due to the difference between treatments in duration of dark and light periods, total exposure to melatonin during dark periods was greater for heifers given 8L:16D than 16L:8D but greater for 16L:8D than 8L:16D during light periods (Table 5). During the dark period, mean melatonin concentrations were greater in heifers exposed to 16L:8D than 8L:16D (Table 5). Heifers under 16L:8D also had numerically greater mean serum melatonin concentrations during light periods. Differences between treatments in Figure 3. 44 Serum melatonin concentrations in pregnant heifers exposed. to (a) 16L:8D or (b) 8L:16D. Each point represents one treatment mean at 2- h intervals for one of three 24-h bleeds. Stage of pregnancy averaged 160, 197 and 219 d at bleeds 1, 2 and 3, respectively. The hatched bar represents darkness. Pooled SE = 15.69 pg/ml of serum . Serum melatonin (pg/m1) 240 200 160 120 40 240 200 100 120 00 40 o. 0 4 012102024 45 r WIIIIIIII1IIWIIIIIIITl—I—I’IT I’m-4': + I ++++ Time (h) Bleed l Bleed 2 1. ' '//////A m 04012102024 04012102024 I—TTIIIIITII {VFW ++++' m Bleed 3 46 TABLE 5. Effect of photoperiod on serum melatonin concentrations in pregnant heifers Treatment 16L:8D 8L:16D SE1 p Over 24 h: Area un er curve (pg.ml' .h) 2100.7 1925.0 259.30 .75 Mean concentration (pg/ml) 117.8 78.1 15.69 .15 During dark periods: Area un er curve (pg.ml- .h) 1033.0 1510.8 132.20 .003 Mean concentration (pg/ml) 171.5 104.5 18.76 .003 During light periods: Area un er curve (pg.ml‘ .h) 825.2 298.1 132.20 .003 Mean concentration (PG/ml) 64.1 51.7 18.76 >.1 1 SE = pooled SE. 47 melatonin concentrations during the dark period account for the lack of difference in total amount of melatonin secreted over 24 h. Serum concentrations of melatonin tended to decline from bleed l to bleed 3 in heifers exposed to 16L:8D (Figure 3). However, this effect was not significant (P>.1). PROGESTERONE There was no relationship between stage of pregnancy and serum progesterone concentrations (Figure 4) . Serum progesterone concentrations, averaged across all three bleeds, were not affected by treatment (mean = 3.6 ng/ml for 16L:8D and 3.8 ng/ml for 8L:16D, pooled SE = .28, P=.56). There was an unexplained bleed x treatment interaction (P=.01, Figure 5). Further analysis (Bonferroni-t test) showed no treatment effect on progesterone concentrations within any particular 24-h bleed, although the slope from bleed 1 to bleed 2 was different across treatments (P=.003). (d) . DISCUSSION L. MAMMARY DEVELOPMENT Total synthetic capacity of the mammary gland is a function of the number of epithelial cells and the secretory action of these cells. We used DNA and RNA as indices of cell numbers and activity and neither was affected by Figure 4. 48 Serum progesterone concentration in pregnant heifers exposed to 16L:8D ( D ) or 8L:16D ( + ). Each point represents mean for one heifer during one of three 24-h bleeds. Each heifer was represented three times. Pooled SE = .28 ng/ml of serum. 49 o + :1 o (:1 +++EZEZU ‘3 + + 3:! 1+ 13' '=- a. 'PCIC] _F ++C-'H:1 BL +03. +1 I: a; + +D @311 1 DEW-1++ + g + E] g + +5143 L_ .1 1 l I I0 ‘2' CO N «H (1m /Bu) auoxeqsefioxd mnxes 160 180 200 220 240 260 Stage of pregnancy ((1) 140 120 Figure 5. 50 Serum progesterone concentration in pregnant heifers exposed to 16L:8D ( D ) or 8L:16D ( + ). Each point represents one treatment mean during one of three 24-h bleeds. Stage of pregnancy averaged 160, 197 and 219 d at bleeds 1, 2 and 3, respectively. Pooled SE = .28 ng/ml of serum. 51 .N‘ \ Bleed L 4 co IQ m 02 45- 4 35L (1m /Bu) euomqsefioxd mnxas 52 photoperiod. These findings in pregnant heifers contrast with those of Petitclerc gt a_IL. (1985) who studied the effect of photoperiod on pre- and post-pubertal heifers. They used the same photoperiod treatments, light intensities and barn as used in the present study. In the study of Petitclerc gt gt. (1985) total parenchymal weight of the mammary gland was 40% (pre-pubertal) or 30% (post- pubertal) greater in heifers exposed to 16L:8D than in heifers given 8Ia16D. E.Sanchez (unpublished observations) fed melatonin to pre-pubertal heifers during a 16L:8D photoperiod, to mimic melatonin secretion observed under 8L:16D. Parenchymal DNA was less (P<.005) when melatonin was fed (1.8 mg/g parenchyma) compared with vehicle-fed controls (2.3 mg/g parenchyma). There are several possible explanations for discrepancies between our results in pregnant heifers and those of Petitclerc gt gt. (1985) and E. Sanchez (personal communication) ‘using ‘virginal. heifers. JMammary' growth is exponential during' pregnancy (approximately 25% /month), with the majority occurring in the last trimester (Swanson and Poffenbarger, 1979). At this time, cell division may be proceeding at the maximal rate, and therefore, increased photoperiod cannot stimulate growth further. Secondly, photoperiod may be a more effective stimulant of mammary growth in virginal than in pregnant heifers. In our experiment, heifers were slaughtered 35 d before expected parturition, by which time they may have been 53 approaching maturity with respect to mammary growth. A third explanation is suggested by results of Petitclerc gt a_l_. (1984), who reported an experiment with pubertal heifers in which photoperiod had no effect on mammary growth. They attributed this to the fact that heifers started on treatment during the allometric phase of mammary growth, instead of during the isometric phase (as in the later study of Petitclerc gt gt. (1985) in which 16L:8D stimulated mammary growth). No hypothesis was offered to explain this difference. In the experiment reported here, mammary growth would have been allometric at the start of the trial. Perhaps during normal allometric growth the mammary gland is less responsive to external mammogenic factors, such as photoperiod. Prolactin is necessary for normal mammary gland development. Sykes and Wrenn ( 1951) found complete mammary development when estradiol, progesterone and pituitary extracts were given to heifers. Similar results were obtained in goats by Cowie gt gt. (1966) . However, Tucker (1987) suggested that prolactin, although necessary for full structural differentiation of the mammary gland, may not limit the rate of mammary growth during pregnancy. Our data showed that although 16L: 8D increased prolactin concentrations in heifers compared with 8L:16D, these increased concentrations of prolactin did not affect mammary development, It is unclear whether differences in mammary growth found by Petitclerc gt gt. (1985) were 54 related to prolactin, since hormone concentrations were not measured. The function of the increased prolactin in response to long days during pregnancy observed in the present study is an enigma. Physiological effects of melatonin could be due to total amount secreted, distribution of total melatonin secretion among light and dark periods, mean concentration within light or dark periods, or duration of elevated or depressed melatonin concentrations. For example, in sheep (Karsch gt gt., 1988) and hamsters (Goldman gt gt,, 1984), duration of the nocturnal rise in melatonin concentration, rather than concentration of melatonin during dark periods, is the cue for resumption of reproductive activity. In the present experiment, duration of exposure to melatonin and distribution of melatonin secretion among light and dark periods varied between treatments, but had no effect on mammary growth. This contrasts with results of E.Sanchez (unpublished observations), who fed melatonin to pre- pubertal heifers under 16L:8D, thereby increasing the time heifers were exposed to elevated melatonin concentrations. Heifers supplemented with melatonin had more extraparenchymal fat, but less parenchyma, than heifers under 16L:8D without supplemental melatonin. The effect of total daily exposure to melatonin on mammary development was not tested in the present experiment, because photoperiod had no effect on the total amount of melatonin secreted. Values obtained for the indices of mammary development 55 measured in the present experiment are consistent with other studies, so there is little reason to suggest that the animals were atypical or the execution of the techniques inadequate. For example, Swanson and Poffenbarger (1979) found total parenchyma weight to be 6843 g at 8 months of pregnancy, while Akers gt gt. (1981a) found 9200 g at 8.5 months. These values compare with an overall mean of 6839 g at 8 months in Experiment 1. Similarly, Akers gt gt. (1981a) reported total amounts of DNA and RNA in parenchyma of 28 g and 24 g, respectively, in agreement with values of 28 g and 20 g reported here. Proportions of stroma, epithelial cells and alveolar lumen in parenchyma in Experiment 1 were similar to those reported by Akers gt gt. (1981b). There is a close relationship between heifer size and mammary gland weight. Across species, Linzell (1972) found a correlation of .99 between total mammary gland weight (including' extraparenchymal fat) and BW. In the present study, heifers were not blocked according to BW, and initial BW was greater for'8L:16D heifers (525 kg) than 16L:8D heifers (481 kg, P=.02). The numerical difference between treatments in extraparenchymal fat weight was removed when initial BW was used as a covariate. Furthermore, there were no 1differences in extraparenchymal fat between .treatments when results were expressed as a percentage of total gland weight. Thus, the large numerical difference in extraparenchymal fat weight between treatments was due simply to variation in heifer size. Future experiments of 56 this type should use initial BW as a criterion when assigning' animals. to ‘treatments. The numerical trend in total mammary gland weight (P=.12) with heifers exposed to 8L:16D having heavier mammary glands than 16L:8D is not due to differences in amounts of skin, hair, secretions or lymph nodes because these components of the total mammary gland, expressed as percentage of total gland weight were not different between treatments (16.2% and 16.7% for 16L:8D and 8L:16D, respectively). This difference is therefore probably a function of the numerically larger amounts of both parenchyma and extraparenchyma found in heifers exposed to 8L:16D. Since stage of pregnancy is a major determinant of mammary gland composition, it was important to slaughter heifers at the‘same stage of pregnancy. Slaughter dates were based on breeding dates provided by the farmer selling the heifer. An error in recording the date, or conception at an earlier insemination, could have resulted in heifers being slaughtered at different stages of pregnancy. This could have obscured real treatment effects. Crown-rump length was variable, but no fetus was a significant outlier (Gill, 1978), confirming that breeding dates given by farmers were reasonably accurate. It is, therefore, unlikely that variation in either initial BW or stage of pregnancy at slaughter caused real effects of photoperiod on mammary development to pass undetected in this experiment. 57 _2_,_. HEIFER GROWTHI CARCASS AND FETAL CHARACTERISTICS. The increase in live-weight gain in heifers exposed to 16L:8D compared with 8L:16D (P=.1l) agrees with Peters e_t gt. (1980) and Petitclerc gt gt.(l983a). Petitclerc gt gt. (1983a) found improved feed conversion efficiency in 16L:8D compared with 8L:16D heifers fed either gct libitum or a restricted diet. However, feed intake was not measured in the present study, so it is unclear whether live-weight gain differences reflect increased nutrient intake (n: more efficient nutrient utilization. Zinn gt gt. (1986) found increased fat accretion in the carcasses of pre-pubertal heifers exposed to 8L:16D. This may be the result of increased duration of elevated melatonin concentrations in 8L:16D heifers, since increased fat deposition was also seen in growing heifers fed melatonin to mimic a 8L:16D photoperiod (Zinn gt gt., 1988). In the present study, photoperiod did not affect the weight of perirenal fat or depth of subcutaneous fat. Similarly, Zinn gt Q. (1988) found no effect of photoperiod on carcass fat when melatonin was fed to post-pubertal heifers approaching maturity with respect to fatness. There were no effects of photoperiod on Longissimus dorsi area in either my experiment or in work of Zinn gt gt. (1986). Heifers exposed to 8L:16D tended to produce heavier fetuses than heifers exposed to 16L:8D. Heifers under 8L:16D were larger and grew more slowly than 16L:8D heifers 58 and may have been diverting more nutrients towards fetal growth than live-weight growth. 3 . SERUM HORMONES Prolactin concentrations were relatively constant throughout the period of the present experiment, and were approximately 70% greater in cattle given 16L:8D than 8L:16D. This difference is consistent with results of Stanisiewski gt gt. (1984) and Petitclerc gt gt. (1989), who reported increases of 104% and 136%, respectively, in pre- pubertal bulls in 16L:8D compared with 8L:16D. However, concentrations in the present study were, on average, 71% higher than the average of those reported by Stanisiewski gt gt. (1984). The average temperature in the pens during bleeds 1, 2 and 3 was 28.5 C, 33.4 C and 30.4 C, respectively (range 22.5 to 37.0 C). Stanisiewski gt _a_l. maintained ambient temperatures at 20 C. Such high temperatures in the present study may have elevated serum prolactin concentrations for both treatments (Wettemann and Tucker, 1974). However, despite evidence of a link between temperature and prolactin concentrations, (Wettemann and Tucker, 1974; Peters gt gt, 1978) the correlation in the present experiment was only .05. The general stability of the high ambient temperatures and the marked variability of prolactin (range across all samples = 8.8 to 435 ng/ml) probably explains the low correlation observed 59 between temperature and prolactin in the present experiment. Some of the increased variability in prolactin concentrations could be due, in part, to stress, caused by high temperatures. Collier gt g_. (1982) found increased stress (as measured by rectal temperature, respiration rate and heart rate) in pregnant cows maintained at 37.5 C compared-with those at 29.8 C. The response of prolactin concentrations to stress varies greatly between individual animals (Tucker, 1971). Thus, one might expect increased variability in prolactin concentrations at high temperatures. The diurnal rhythm in serum melatonin concentration observed for all heifers in all bleeds was consistent with other findings in cattle (Hedlund gt gt., 1977; Martin gt g” 1983; Stanisiewski gt gt., 1988) and all other vertebrates studied (Underwood and Goldman, 1987). In the present study, concentrations of melatonin during dark periods was greater for heifers given 16L:8D than 8L:16D. In agreement, Stanisiewski gt gt. (1988) also reported greater melatonin concentrations in dark periods in bull calves given 16L:8D compared with 8L:16D, and Idncoln gt gt. (1982) found a similar effect in sheep. However, the cause of this treatment difference in serum melatonin concentrations during different length dark periods is unclear. It is also not known whether the treatment difference in mean melatonin concentrations during different length dark periods occurs as soon as different photoperiods 60 are introduced, or whether it develops gradually over several light-dark cycles (entrainment). Effects of photoperiod on melatonin concentrations are unlikely to involve differences in release of melatonin stored in the pineal gland because melatonin is released from the pineal gland immediately after synthesis (Reiter, 1988). A possible explanation of differences in melatonin concentrations during different length dark periods is that effects of light on melatonin secretion interact with an endogenous rhythm. Underwood and Goldman (1987) discussed the existence of two circadian clocks driving daily fluctuations of N-acetyltransferase, a pineal enzyme controlling melatonin synthesis. The duration, and possibly the amplitude, of the melatonin cycle may be controlled partly by the phase-relationship between these cycles. This, in turn, may be determined by the light-dark cycle to which the animal is exposed. While there is little evidence for such endogenous cycles in' cows, this could explain the entrainment of an animal to a light-dark cycle and the effect of photoperiod on melatonin concentrations during the dark period. Response to melatonin could be mediated by amplitude or duration of the melatonin cycle. Reproductive cycling in sheep is initiated during short-day photoperiods when the amplitude of the nocturnal melatonin surge is reduced and the duration is increased compared with a long-day photoperiod. Kennaway e_t_ g_l. (1982) fed melatonin to 61 anestrous sheep under 16L:8D, thereby increasing both the duration and amplitude of the nocturnal melatonin surge. Reproductive cycles commenced suggesting that increased duration, rather than decreased amplitude, of the nocturnal melatonin surge during short days is the important trigger. Heifers exposed to 8L:16D did not maintain elevated serum melatonin concentrations for the entire 16 h of the dark period. Serum concentrations began to decline after approximately 14 h in the dark. This has also been reported in pre-pubertal bulls (Stanisiewski gt gt., 1988), sheep and cats (Reiter, 1988) and albino rats (Johnson gt gt., 1982). According to Reiter (1988), this is due to down-regulation of the fi-adrenergic receptors of the pinealocyte membrane. This decreases the stimulatory effect of norepinephrine on melatonin secretion. In the present experiment, such down- regulation was not apparent in heifers under 16L:8D, because elevated melatonin concentrations were maintained for the full 8 h of the dark period. Thus, down regulation must come into play after 8 but before 16 h of darkness. Melatonin concentrations reported in the literature vary according to assay procedures used. However, concentrations reported in the present study (57.9 and 138.0 pg/ml for light and dark periods, respectively) also differ from those previously found in our laboratory, using assay procedures similar to those used here. Stanisiewski gt gt. (1988) found light and dark means of 10.8 and 47.6 pg/ml, and B.Buchanan (unpublished data) found 11.2 and 48.0 pg/ml. 62 Both of these studies involved pre-pubertal animals. Age could be important because Stanisiewski (unpublished data) found concentrations up to 400 pg/ml in steers that were similar in age to that of heifers used in the present study. However, in both humans (Reiter, 1988) , rats and hamsters (Reiter, 1980), the ,nocturnal increase :h1 serum concentrations of melatonin decreases with age. There is little evidence that pregnancy affects serum concentrations of melatonin. Concentrations of serum progesterone (mean = 3.7 ng/ml, SE=.28) were similar to those reported by other workers during pregnancy (e.g. 2.3 ng/ml, Chew gt _a_l_., 1977) . Progesterone is one of the major determinants of mammary development. Since there was no effect of photoperiod on mammary development, it is not surprising that there was no difference in serum progesterone. Rhodes gt gt. (1982) challenged cmltures of corpora lutea tissue with LH and found more progesterone secretion from corpora lutea taken from cycling cows in summer than in winter. However, such seasonal changes are not necessarily mediated by photoperiod. The significant treatment x bleed interaction (progesterone concentrations greater for 8L:16D and lower for 16L:8D during bleed 2 than bleed 1, Figure 5) is difficult to explain. However, the variation from bleed to bleed was small (approximately .5 ng variation). Since all animals were bled on the same dates, rather than at the same 63 stages of pregnancy or periods on trial, statistically significant effects of bleed or bleed x treatment interactions probably have little biological meaning. EXPERIMENT TWO (a). EXPERIMENTAL OBJECTIVES Previous studies in our laboratory suggest that the peri-parturient surge of prolactin is an important stimulant of lactogenesis. Akers gt gt. (1981a) blocked the peri-parturient surge of prolactin in dairy cows and reported reductions in subsequent milk yield of 11.4 kg/d for the first 10 d of lactation. Photoperiod is known to affect prolactin concentrations in non-pregnant heifers. Our first objective, therefore, was to determine the effect of photoperiod on the magnitude (total prolactin measured), amplitude (increase in prolactin concentration over basal value) and duration of the peri-parturient prolactin surge. Current definition of the peri-parturient prolactin surge is imprecise, since it is based on infrequent measurements of prolactin concentration. A second objective, therefore, was to obtain a better description of the peri- parturient prolactin surge, by measuring serum prolactin concentration at frequent intervals in the days immediately before calving. To verify that heifers were entrained to their 64 65 respective photoperiod treatments, melatonin and prolactin concentrations were measured before onset of the peri- parturient prolactin surge. (b). MATERIAL AND METHODS t._ TREATMENTS AND MANAGEMENT Nine heifers from the Michigan State University Dairy Cattle Research Center and 16 from the Kellogg Biological Station (KBS) had their estrous cycles synchronized with prostaglandin F20, and were bred in early July, 1988. Twelve heifers which conceived to this breeding were used for the trial, remaining with their respective herds until transferred to a light-controlled barn 56 d prior to expected calving date. Within source of origin, heifers were assigned randomly to one of four pens. All heifers were exposed to 8L:16D for a 21 d pre-treatment period. Thirty five days before expected calving date, the photoperiod in two pens was changed to 16L:8D, while the other two pens remained on 8L:16D. This resulted in 6 heifers per treatment. Light intensities within each pen were the same as in Experiment 1. Heifers were fed a ration designed for pregnant dairy heifers (77% corn silage, 14.6% alfalfa silage, 4.8% ground shelled corn, 3.2% soybean meal, .4% vitamin-mineral mix, on a fresh-weight basis) at 10.8 kg DM head‘1.d‘1. 66 Heifers remained on treatment until approximately 2 11 after calving, when they were separated from their calves and hOused with the rest of the University dairy herd, under continuous light. Animals from KBS were returned to their (own herd 48 h after calving. _2_._ BLOOD SAMPLING Fourteen days before expected calving date (d 269 of gestation), a polyvinyl chloride cannula was inserted into the jugular vein of each heifer. Cannulae were coated with a heparin complex to reduce fibrin accumulation (TDMAC Heparin, Polysciences Inc., Warrington, PA) and sterilized with ethylene oxide. After cannulation, heifers were haltered and tied to a feed-fence with free access to feed and water. Beginning the following morning, blood samples (10 ml) were collected at 2-h intervals for 48 h (d 270 and 271 of gestation). These data are defined as the initial pre-surge basal concentrations. A dim red light was used to aid collection of blood during dark periods. Concentrations of melatonin (Webley gt gt., 1985) and prolactin (Koprowski and Tucker, 1971) in these samples were used to establish whether animals had become entrained to photoperiod. Thereafter, blood samples were collected at 4-h intervals until 48 h after calving. Samples covering the period 72 h before, to 48 h after, calving were used to define the 67 peri-parturient prolactin surge and immediate pre- and post-surge basal values. Serum was prepared as in Experiment 1 and stored at -20 C until analysis. L STATISTICAL ANALYSES Prolactin concentrations from 72 h before to 48 h after calving were used to define the following characteristics: (a) immediate pre-surge basal concentration, (b) immediate post-surge basal concentration, (c) area under the surge, (d) peak concentration (maximum prolactin concentration attained during the surge), and (e) surge amplitude (surge peak minus immediate pre-surge basal concentration). Beginning and end-points of the surge were defined using a pulse analysis computer program (PULSAR, Merriam and Wachter, 1982). Area under the surge (above immediate pre- surge basal concentration), bounded by the defined start and end points, was calculated using the trapezoidal rule. In both treatments, immediate post-surge basal concentrations were greater than immediate pre-surge concentrations. The increase in basal prolactin concentration after the surge could be due to the change in environment associated with moving the cattle 2 h after calving. Elevated immediate post-surge basal concentrations could affect the definition of the peri-parturient prolactin surge. Therefore, the area under the curve was calculated for both the entire surge and the surge truncated at calving. The effect of 68 photoperiod treatment on the immediate pre- and post-surge basal prolactin concentration was evaluated using the Bonferroni t-test (Gill, 1978). Statistical models used to analyze derived characteristics of the peri-parturient prolactin surge are shown in Appendix 1d and 1g. Mean serum concentrations of melatonin during d 270 and 271 of jpregnancy (initial. pre-surge. concentrations), and concentrations and area under the curve during light and dark periods, were analyzed as a split-split plot design with repeated measures over time (Gill, 1986; Appendix 1e). Within. light. and. dark; periods, treatments were compared using the Bonferroni-t test (Gill, 1986). Prolactin concentrations measured at 2-h intervals during the first 48 h after cannulation (initial pre-surge basal concentrations) were analyzed as a split-plot design with repeated measurement over time (Appendix 1f). (C). RESULTS 1. SERUM PROLACTIN - INITIAL PRE-SURGE BASAL CONCENTRATIONS Two heifers, one on each treatment, calved either during, or immediately after, the first 48 11 of the bleed (predicted d 270 and 271 of pregnancy). Since concentrations of prolactin for these animals during this period reflected the rising prolactin concentrations of the peri-parturient surge, their data were excluded from calculation of the 69 basal concentration. Results of the remaining 10 animals confirm that heifers responded to photoperiod (Table 6). TABLE 6. Effect of photoperiod on initial pre-surge basal prolactin (ng/ml) on predicted d 270 and d 271 of pregnancy Treatment 16L:8D 8L:16D SE1 P Prolactin (ng/ml) 27.8 13.8 2.46 .01 1 Pooled SE. gt PERI-PARTURIENT PROLACTIN SURGE All heifers exhibited a surge of prolactin between 72 h before and 48 h after calving, with prolactin concentrations beginning to increase approximately 17.5 h before peak values were reached (33 h before calving) (Figure 6). In comparison with 8L:16D exposure to 16L:8D resulted in higher peak prolactin concentrations, increased amplitude of the prolactin surge and greater exposure to prolactin (area under the curve) (Figure 6, Table 7). However, duration of the surge, and timing of the peak relative to both the start of the surge and calving, were unaffected by photoperiod (Table 7). Figure 6. 70 Serum prolactin concentrations during the peri- parturient period in pregnant heifers exposed to to 16L:8D ( E] ) or 8L:16D ( + ). Prolactin concentrations are shown centered relative to peak values. Points represent treatment means at 4-h intervals. C = calving. 71 A5 Room 3 0333.“ 052. «4m mm we 0% mm... «4N m: w 9 ml 0H! «4N1 Mm...) fl _ — A 1 o ...T...“_.:iT.n: m .1...+.. . <5 J OOH ..- ..o :7 .. ..... .... oom a ...+...+.. ... .- r. L. 5 1 com 5 l co“4 : L com. ([111 /Bu) unomoxd mules 72 TABLE 7. Effect of photoperiod on the peri-parturient surge of serum prolactin in pregnant heifers Treatment 16L:8D 8L:16D SE1 P Maximum prolactin 466.8 262.2 24.5 <.001 concentgation (DQ/ml) Surge amplitude3 439.7 243.6 24.19 <.001 (mg/ml) Area un er curve4 (ng.ml' h) Total surge 10627 6410 1095.9 .04 Surge truncated at calving 7229 4183 757.0 .03 Duration of prolactin surge (h) 68.0 64.0 4.87 .84 Timing of peak: Interval from start of surge to peak (h) 20.7 14.7 3.68 .25 Interval from peak to calving (h) 13.3 18.0 2.27 .16 Pooled SE. #UNH concentrations. Maximum prolactin concentration attained during surge. Surge peak minus immediate pre-surge concentrations. Area under the surge above immediate pre-surge 73 Immediate pre-surge basal concentrations (27.1 and 29.6 ng/ml for 16L:8D and 8L:16D, respectively) were lower (P=.05) than post-surge concentrations (5545 and 56.5 ng/ml for 16L:8D and 8L:16D, respectively, pooled SE = 7.45). Elevation of immediate post-surge basal prolactin concentrations compared with immediate pre-surge values could be due to a number of factors such as stress (calving, removal of calf, new housing, changed feed, handling, milking), change in temperature (average increase when moved to milking barn = 5.5 C), or change in photoperiod (housed under constant light after calving). An attempt was made to account for effects of temperature, since it is well-known that a change in temperature elicits an immediate response in serum prolactin concentration (see Literature Review). However, correlations between ambient temperature and initial pre-surge basal concentrations of prolactin were only .38 and .2 for 16L:8D and 8D:16L, respectively. Temperature also was not a Significant covariate (P>.1) in analysis of initial pre-surge basal concentrations of prolactin. It was, therefore, considered inappropriate to adjust post-surge prolactin concentrations for temperature. Area under the curve for both the total surge and for the surge truncated at calving was greater for heifers given 16L:8D compared with 8L:16D (Table 7). There was no effect (P>.1) of photoperiod on either immediate pre-surge basal prolactin concentrations (27.1 and 29.6 ng/ml, 16L:8D and 8L:16D, respectively) or immediate 74 post-surge basal concentrations (55.5 and 56.5 ng/ml, 16L:8D and 8L:16D, respectively, pooled SE = 7.45). gt SERUM MELATONIN Melatonin concentrations were measured at 2—h intervals for 48 h beginning at predicted d 270 of pregnancy (initial pre-surge basal concentrations). One heifer (2409) calved during the second 24-h period and was immediately moved to the milking barn (continuous light). Melatonin concentrations during the second 24-h period were greater for this heifer (420 pg/ml) compared with the other animals (99 pg/ml). Data for 2409 for the second 24-h period were, therefore, excluded from the analysis. All heifers responded similarly to photoperiod, with greater melatonin concentrations in dark periods than light periods (Figure 7). Although duration of the dark period was longer for heifers under 8L:16D than for heifers exposed to 16L:8D, there was no difference between treatments in the total amount of melatonin heifers were exposed to over 24 h (area under curve, Table 8). This was due to numerically greater serum concentrations of melatonin during both dark and light periods for heifers given 16L:8D than 8L:16D. Area under the melatonin curve was greater during light periods for 16L:8D than for 8L:16D. During dark periods, the area under the curve was greater for 8L:16D than 16L:8D (Table 8). Thus, a higher proportion of total exposure to Figure 7. 75 Serum melatonin concentrations in pregnant heifers exposed. to (a) 16L:8D or (b) 8L:16D. Each point represents one treatment mean at 2-h intervals for 48 h. The hatched bar represents darkness. Pooled SE = 15.46 pg/ml of serum. Serum melatonin (ng/ ml) 200 100 120 100 120 60 76 m @522 I I I I I I I I I I I I T I T I T T r I I I I I /// W1. 8 12 10 20 24 4 0 12 10 20 24 4 8 Time of day (h) 77 Table 8. Effect of photoperiod on serum melatonin concentrations in pregnant heifers Treatment 16L:8D 8L:16D SEl p Over 24 h: Area un er curve (pg.ml- .h) 2025 2112 44.2 .89 Mean concentration (pg/m1) 99.0 86.5 15.46 .48 During dark periods: Area un er curve (pg.ml- .h) 711 1547 188.1 .003 Mean concentration (pg/ml) 118.0 107.7 16.21 >.1 During light periods: Area un er curve (pg.ml .h) 1122 390 188.1 .003 Mean concentration (pg/ml) 80.0 65.3 16.21 >.1 l Pooled SE. 78 melatonin occurred during the light period for heifers given 16L:8D (61%), and a higher proportion occurred during the dark for 8L:16D (80%). The nocturnal elevation in serum melatonin was maintained for the full 8 h of the dark period for 16L:8D heifers. However, in heifers exposed to 8L:16D melatonin concentrations began to decline about 2 h before lights were switched on (Figure 7). (d). DISCUSSION tt BERI-PARTURIENT PROLACTIN SURGE Any lactogenic effect of the peri-parturient surge of prolactin could be a response to one or more of several characteristics of that surge, such as total amount of prolactin estimated from area under the curve, duration of exposure to elevated prolactin, peak prolactin concentration attained, or the increase over basal concentration (peak amplitude). Exposure to 16L:8D increased all of these characteristics of the prolactin surge, except surge duration, relative to 8L:16D. Results of Experiment 2 contrast with those of Chew gt gt, (1979), who found that, while peak prolactin concentration during the surge was greater in summer than winter, there was no difference in amplitude of the surge. However, results of our experiment are more precise, since 79 the study of Chew gt _t. (1979) involved confounding effects of seasonal fluctuations in temperature, changes in housing and diet, and variation in photoperiod around calving (depending whether cows calved indoors or outside). These factors were either held constant (diet, housing) or were less variable (temperature) in our study. . Other workers have reported seasonal, possibly photoperiodic, effects on the response of prolactin concentrations to other stimuli. Vines gt a_l_. (1977) observed greater concentrations of prolactin, in response to exogenous thyrotropin releasing hormone, in pre-pubertal cattle in summer than winter. In goats, the increase in serum concentrations of prolactin induced by milking is greater in August than October. This milking-induced elevation in prolactin may be maintained by keeping the goat in a long-day photoperiod (Hart gt gt., 1975). In contrast, in cows, Koprowski and Tucker (1973) found that the prolactin response to milking was less in summer than winter. Prolactin concentrations did not return to pre-surge values after the peri-parturient surge in the present study. This is normal in some species, such as sheep (Davies e_t gt., 1971), rats (Amenomoni e_t a_l., 1970) and goats (Johke gt g” 1971). However, in cows, Ingalls gt gt. (1973) and Akers gt _a_l. (1981a) reported that prolactin returned to pre-surge concentrations by 48 h after parturition. Chew gt gt. (1979), in a study involving 176 cows, found that, in 80 some individuals, prolactin remained elevated until at least 60 h after parturition. This could also be the case in the present experiment. There are, however, other explanations for the difference between pre- and post-surge prolactin concentrations in the present study. Immediately after calving, heifers were separated from their calves and moved to another barn, where ambient temperature was, on average, 5.5 C higher than .in the light-controlled barn. Increased temperature is known to increase serum prolactin concentrations (Wettemann and Tucker, 1974). Heifers also encountered. changed. diet, new social interactions and a milking parlor. Stress associated with these changes may have prevented prolactin concentrations returning to 'the immediate pre-surge baseline (Raud gt gt., 1971, Tucker, 1971). Koprowski and 'Fucker (1973) found that serum prolactin increased 2.8-fold in response to stimuli associated with milking during the first 8 weeks of lactation, returning to basal concentrations by 30 to 35 min after milking (Tucker, 1971). Prolactin concentrations in some of the 4-h blood samples taken after calving may have been. affected. by 'this response. In contrast, continuous light, to which heifers in the present study were exposed after calving, decreases prolactin concentration in bull calves, relative to 16L:8D (Leining gt gt., 1979). Because all these factors are confounded, it is impossible to assess their individual importance. However, such confounding of immediate post-surge basal prolactin concentrations does not 81 invalidate the results because the effect of photoperiod on the peri-parturient surge truncated at calving was similar to the effect on the peri-parturient surge calculated from 72 h before to 48 h after calving. Several of the characteristics of the prolactin surge found in the present study differ from earlier findings. Peak prolactin (467 ng/ml) was greater than that reported by Akers _e_t g. (1981, 194 ng/ml in multiparous cows under 16L:8D). This difference could be due, firstly, to assay reagents and, secondly, to sampling frequency and number of animals. Akers gt gt. (1981) collected blood from four cows at 12-h intervals, and so peak prolactin concentration was defined less precisely than in the present study. Estimates of the timing of the prolactin surge also vary. Akers gt gt. (1981) estimated. that. the jpeak. occurred at parturition, while Ingalls gt gt. (1973) suggested 24 h and Chew gt gt. (1979) 12 h before parturition. Much of this variation may be due to imprecision in the sampling procedures. By collecting blood at 4-h intervals, the present study provided a more precise definition of the peri-parturient prolactin surge than previously has been reported. The lack of photoperiod effect on either immediate pre-surge basal prolactin concentrations or immediate post- surge prolactin concentrations contrasts with the photoperiod effect on initial pre-surge basal prolactin concentrations. Variability ixx basal prolactin concentrations may increase near the start of the peri- 82 parturient prolactin surge. The absence of a photoperiod effect on basal prolactin concentrations immediately before the peri-parturient prolactin surge is unlikely to be related to temperature, which was similar between treatments (3.8 C for 16L:8D, 4.0 C for 8L:16D) throughout the immediate pre-surge period. Conclusions from Experiment 2 are only valid if the animals used were responsive (entrained) to changes in light conditions. Evidence for this is provided by serum concentrations of melatonin and prolactin before onset of the peri-parturient prolactin surge. 2. ENTRAINMENT IQ PHOTOPERIOD Prolactin. concentrations. during' the first 48 h of blood sampling (initial pre-surge basal concentrations) show that heifers had responded to photoperiod treatment. The 2- fold increase for 16L:8D over 8L:16D was similar to that in Experiment 1 (1.7-fold). However, absolute serum prolactin concentrations ‘were.:much lower in Experiment 2 (overall means were 82.5 and 20.8 ng/ml in Experiments 1 and 2, respectively). This could, in part, be due to the different ambient temperatures involved (overall means were 30.8 and 7.5 C in Experiments 1 and 2, respectively). Serum prolactin concentrations in Experiment 2 concur with those found by Peters and Tucker (1978) during winter (mean of 31.4 ng/ml). In both Experiments 1 and 2, prolactin concentrations were 83 more variable for heifers under 16L:8D than those under 8L:16D. Serum concentrations of melatonin increased in response to onset of darkness, confirming that heifers were entrained to photoperiod. Mean serum concentrations of melatonin, and total exposure to melatonin (area under curve) were similar for both experiments (Tables 5 and 8). However, in Experiment 1, the majority (55%) of daily exposure to melatonin in 16L:8D heifers occurred during the dark period, while in Experiment 2, only 39% occurred during the dark period (Tables 5 and 8'). In Experiment 1, heifers exposed to 16L:8D had greater serum melatonin concentrations in the: dark. period. than. heifers exposed to 8L:16D. In Experiment 2, there was no significant difference between treatments in melatonin concentrations during dark periods. Results of Stanisiewski e_t_ g. (1988) are similar to Experiment 1. The difference between Experiments 1 and 2 may reflect, in part, disruption of the normal melatonin cycle by other hormonal changes occurring around parturition. Heifer 2409, which calved during the period over which melatonin concentrations were measured, had high serum concentrations of melatonin after calving (420 pg/ml, compared with a mean of 99 pg/ml before calving for other heifers). 84 3. RESPONSE IQ PERI-PARTURIENT PROLACTIN SURGE The practical significance of the effect of photoperiod on the peri-parturient prolactin surge depends on subsequent differences in milk yield and (or) composition. However, there were insufficient numbers of animals in Experiment 2 to measure -the lactational response to photoperiodic manipulation of the peri-parturient prolactin surge. An increase in serum prolactin concentration around parturition is necessary for full structural differentiation of the mammary gland (Akers gt gt., 1981b) and for the production of casein mRNA, a-lactalbumin and fatty acid and lactose synthetase (Akers g gt., 1981a). Removal of the peri-parturient surge drastically reduces subsequent milk production (Akers e_t_ a” 1981a). However, it remains unclear which characteristics of the surge are responsible for this lactogenic response. If effects of prolactin on cell differentiation are additive, (i.e., the more prolactin secreted, the greater the response), greater yield of milk and milk components would be expected from heifers exposed to 16L:8D during late pregnancy than those under 8L:16D. Akers g a_l_. (1981a) inhibited the prolactin surge, using a synthetic ergot drug, CB154, and then infused prolactin (CB154 plus prolactin) to mimic the surge. During the 6 d of this artificial surge, serum prolactin reached a peak of 311 ng/ml, compared with 194 ng/ml in untreated control heifers. However, there was 85 no difference in milk yield for the first 10 d of lactation between controls and cows given CB154 and prolactin. Akers gt gt. (1981a) only infused prolactin into three cows, and, due to variability in time of calving, two received their artificial peak 48 and 60 h before calving, and the third, at parturition. Thus, at 15.5 h before calving (time of prolactin peak in our experiment), prolactin concentrations in the experiment of Akers gt gt. (1981a) could have been similar for treated and control animals. If timing of the peak of prolactin in relation to calving is important, this could account for the lack of milk yield response in the study of Akers gt gt. (1981a). Increasing the duration of the surge seems to be unimportant in the initiation of lactogenesis, since Akers gt a_l. (1981) found no difference in milk yield between animals infused with prolactin for 6 d and control animals, which had surges lasting only 3 d. Amplitude of the prolactin surge (increase over basal) could be an important stimulator of lactogenesis. However, to my knowledge, this has not been tested. In the present study, amplitude of the prolactin surge was greater for heifers under 16L:8D than 8L:16D. In the present study, photoperiod influenced prolactin concentrations (both before and during the peri-parturient surge) and melatonin concentrations (duration of nocturnal elevation and concentration during dark periods). Any effects of photoperiod on lactogenesis could, therefore, be 86 mediated by either prolactin or melatonin. Effects of melatonin on lactogenesis have not been studied. Further experiments could involve direct study of the effect of photoperiod during pregnancy on subsequent lactation. Now that the peri-parturient prolactin surge has been precisely defined, blocking of the natural surge and administration of exogenous prolactin could be used to determine which elements of the surge are responsible for any lactogenic effect. SUMMARY AND CONCLUSIONS Effects of photoperiod on mammary growth and hormone concentrations during pregnancy were studied in two experiments. The objective of the first experiment was to compare mammary development in heifers reared under 16 h light, 8 h dark (16L:8D) or 8L:16D. Eighteen heifers were blocked according to stage of pregnancy and, within blocks, assigned randomly to one of four pens. Treatment photoperiods were imposed at 128 d of pregnancy (two pens per treatment) and heifers were slaughtered at 248 d of pregnancy. Photoperiod had no effect (P>.1) on mammary development (mammary’ gland. weight, concentrations of extraparenchymal fat and parenchymal tissue, concentrations of dry matter and fat and total amounts of nucleic acids in parenchymal tissue, or distribution of cell types). Heifer rate of gain tended (P=.1) to be greater in heifers exposed to 16L:8D than in heifers exposed to 8L:16D, but there were no effects on heifer carcass composition. Fetal weight was numerically greater for 8L:16D than 16L:8D. Venous blood samples were collected at 30-min intervals for 6 h (for prolactin determination) or at 2-h and 8-h intervals for 24 I1 (fer' melatonin and progesterone determinations, respectively) at approximately 160, 197 and 87 88 219 d of gestation. Serum prolactin concentration was 1.7- fold greater in 16L:8D heifers than 8L:16D heifers (P=.02). There was a rise in serum melatonin in dark periods for both treatment groups. However, total exposure to melatonin was not. different. between ‘treatments (P>.1), due to greater melatonin concentration in dark periods in 16L:8D than 8D:16L heifers (P<.003). Photoperiod had no effect (P>.1) on serum concentrations of progesterone. In Experiment 2, 12 heifers, expected to calve on the same date, were used to study effects of photoperiod on the peri-parturient surge in serum prolactin concentration. Heifers were assigned randomly to four pens. Two pens received 16L:8D and two 8L:16D from 35 C! before expected calving' date. Fourteen.'days before calving ‘venous blood samples were collected at 2-h intervals for 48 h (initial pre-surge basal concentrations). Serum :melatonin and prolactin concentrations were used to establish whether heifers were entrained to their respective photoperiods. Thereafter, blood samples were collected at 4-h intervals until 48 h after calving. Blood samples from 72 h before to 48 h after calving were analyzed for prolactin. During the initial pre-surge period, serum prolactin concentrations were 100% greater for heifers under 16L:8D than heifers under 8L:16D (P=.01). Serum melatonin concentration increased for all heifers at onset of dark periods, confirming that all heifers were entrained to photoperiod. As in Experiment 1, there was no effect of 89 photoperiod on total exposure to melatonin. Heifers under 16L:8D were exposed to greater amounts of prolactin ie. total area under the surge during the peri- parturient surge (P=.04) , increased amplitude of the surge (P<.001) and a greater concentration at the peak of the surge (P=<.001) than heifers under 8L:16D. However, duration of the surge and its timing, relative to both the start of the surge and calving, were unaffected by photoperiod. In conclusion, melatonin concentrations and responsiveness to light changes were similar to those observed in non-pregnant cattle. Despite increased duration of the nocturnal rise in melatonin concentrations in heifers exposed to 8L:16D, total daily melatonin secretion was unaffected by photoperiod. Mammary growth and composition during pregnancy were not responsive to increased serum prolactin. concentratione However, exposure to 16L:8D increased serum prolactin concentration relative to heifers exposed to 8L:16D, both before and during the peri- parturient prolactin surge. Effects of photoperiod on the peri-parturient. prolactin surge could influence 'milk production in the subsequent lactation. APPENDIX APPENDIX 1 Soutces of variation and degrees of freedom in statistical models Note: in the following tables, each error term (E E2, or E4) is used to test effects which precede th in the model. (a). Experiment 1: mammary gland, fetal and carcass characteristics Source of variation df Initial body weighta 1 Treatment 1 Block 4 Treatment x block 4 Pen (tgeatment) 2 E1=Residual 5 Total 17 a Initial BW tested as covariate. It was deleted if non- significant (P>.1). b. Block x pen(treatment) The pen(treatment) term was significant (P<.05) in the analysis of extra-parenchymal fat weight. The above model was, therefore, modified to: Source of variation df Treatment 1 Block 4 Treatment x block 4 E1= Pen (treatment) 2 Total 11 90 91 (b). Experiment 1: serum prolactin and progesterone concentrations 0. H) Source of variation Treatment Blocka Treatment x block Pen (treatment) El= Block H pen(treatment) Bleed Treatment x bleed Block x bleed Treatment x block x bleed Bleed x pen(treatment) E2= Residualc Total 0>NJ>G>mbONJmtO¢bbrd (Ill--| a. Group of heifers at similar stage of pregnancy. Stage of pregnancy averaged 160, 197 and 219 d for bleeds 1, 2 and 3, respectively. b. Mean of individual samples collected during 24 h. C. Block x bleed x pen(treatment) Stage of pregnancy and temperature were tested as covariates in the model. They were excluded if non-significant (P>.05). 92 (c). Experiment 1: serum melatonin concentrations 0. H) Source of variation Treatment Block Treatment x block Pen (treatment) E1= Block x pen (treatment) Bleed Treatment x bleed Block x bleed Treatment x block x bleed Bleed x pen (treatment) E2= Block x bleed x pen (treatment) 1 Perioda Treatment x period Block x period Treatment x block x period Period x pen (treatment) E3= Block x period x pen (treatment) Bleed x period Treatment x bleed x period Block x bleed x period Treatment x block x bleed x period Bleed x period x pen (treatment) E4= Residualb Total poooowwoxweeHr-ampoooowmmmper- I'" OH \IN a. Light and dark periods within each bleed. b. Block x bleed x period x pen(treatment) Stage of pregnancy was tested as a covariate and excluded when found to be non-significant (P>.05). (d). Experiment 2: characteristics of peri-parturient prolactin surge Source of variation df Treatment 1 Pen (treatment) 2 El= Heifer x pen (treatment) 8 Total 11 93 (e). Experiment 2: serum melatonin concentrations 0.. H) Source of variation Treatment Pen(treatment) E1= Heifer(treatment x pen) Daya Treatment x day Pen x day(treatment) E2= Heifer x day(treatment x pen) Period Treatment x period Pen x period(treatment) E3= Heifer x period(treatment x pen) Day x period Treatmegt x day x period E4: Residual Total UIKDHHNNHHQNHHmNH .p. a. Each day is a 24 h period of blood collection b. Heifer x day x period x pen(treatment) (f). Experiment 2: basal serum prolactin concentrations Source of variation df Treatment 1 Pen(treatment) 2 E1= Heifer x pen(treatment) 6 Total 9 94 (g). 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