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' Jr 1.. “’4'. ‘III..IQ'\HI~I I *‘abs O - .lll t..QI‘II. ‘h' lg I- I .‘|‘ .Ol‘l‘ i O. . . .I‘ I .‘ \l.‘ .lI‘I-‘II- I a a." ‘0’. IL... L Q o. O . J t _ .o .. l . I. 1‘.‘.I1 fin I w‘ . cut! 1..-...- oh 90‘. .I :35 l|| ‘1”an ‘I' o 9 ‘I‘I‘ ll .. .-n I... . A; I‘D? VI .. O ‘ 1| 9 .II D . n .I . a Q . 0. Cl. ‘- It ‘ 'A T ... ‘ I‘l ‘4!“ o . II o I .r...‘ ‘0. N \ '. I." ' u "’3 Qh'.‘ " -n-ll|l ‘u'l ‘- ‘ . .:l-“p.!. o 'I“ ix H ._ a . I! -v... _ _ - i... - . :flai....tl\.i\.l ~. .1. AH. . -- awn. Msaxfinmmmw xmwf.._v.v\\ .. 'Hwfiwll“ lg”! . . - 3&9 tdr -15? .I " .. - “uh-“n!!! 11.1....1." u {All-k This is to certify that the dissertation entitled Photoperiod, a signal for stimulation of prolactin secretion and body growth in cattle presented by Denis Petitclerc has been accepted towards fulfillment of the requirements for Ph. D. degree in Animal Science fl/flJ Major protes(or Date MSU is an Affirmative Action/Equal Opportunity Institution 0‘ 12771 MSU LIBRARIES fl ‘3, 2m, BEIURNINQ_MATERIALS: Piace in book drop to remove this checkout from your record. FINES wiii be charged if book is returned after the date stamped beiow. I . 4: 4' :1: .. :7! A”: :1)" :3 ‘ "‘5': U! ' ‘ “M U . - - - - PHOTOPERIOD A SIGNAL FOR STIMULATION OF PROLACTIN SECRETION AND BODY GROWTH IN CATTLE By Denis Petitclerc A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1983 ABSTRACT PHOTOPERIOD A SIGNAL FOR STIMULATION OF PROLACTIN SECRETION AND BODY GROWTH IN CATTLE By Denis Petitclerc Increasing ambient temperatures and duration 'of daily light increased concentrations (secretion) of prolactin (PRL) in serum of cattle. Ambient temperature and photoperiod account for most but not all seasonal variation in PRL. Blinding abolished photoperiod-induced changes in PRL, however, seasonal changes in PRL persisted. An endogenous annual rhythm in PRL secretion is hypothesized. Changing photoperiod from 8L:16D to 16L:8D or to 6L:8D:2L:8D increased PRL concentrations 418%. Changing photoperiod from 8L:16D to 6L:14D:2L:2D increased PRL only 173%. Thus, a continuous block of 16 h of light is unnecessary to stimulate PRL secretion. A photosensitive period of PRL secretion 14 to 16 h after dawn (or 8 to 10 h after dusk) is postulated. Concentrations of PRL increased between 14 and 18 h after dawn (6 to 10 h after dusk) after switching photoperiod from 16L:8D to 8L:16D but not from 8L:16D to 16L:8D. Thus, absence of light unmasks the photosensitive period of PRL secretion. Thyrotropin—releasing hormone induced release of PRL is greater 3 h after dawn (11 h after dusk) than after 9, 15 and 21 h in animals given 16L:8D but not 8L:16D. The photosensitive period of PRL secretion is associated with dusk, not dawn. Denis Petitclerc Photoperiod of 16L:8D increased body growth rates 10-18% in heifers fed LOW or HIGH planes of nutrition. The increase growth rates were due to increased feed efficiency. HIGH heifers had greater concentrations of PRL and less growth hormone (GH) than LOW heifers, but photoperiod did not affect GH. Heifers given 16L:8D or fed LOW plane reached puberty at a smaller weight. 16L:8D enhanced carcass weight gain (9.8%) and protein percentage (11.096) in rib sections of HIGH, but not LOW heifers. HIGH plane increased fat percentage 24% over that in LOW heifers. HIGH plane reduced concentrations and total DNA in mammary parenchymal tissue. Photoperiod did not influence mammary development of peripubertal heifers. Management of photoperiod is a promising practice for increasing efficiency of growing cattle. DEDICATION I dedicate this thesis to my wife, Angela T. Petitclerc, and my friend, Larry T. Chapin. Their support, their demand for quality, their joviality and Angie's love have instilled in me a drive for hard work, greatness and knowledge. Through them and with them, I have searched for truth and perfection. ACKNOWLEDGEMENTS To this country, this institution and the American people, I wish to express my deepest and most sincere appreciation for providing me training, funds, facilities and the friendship needed for my graduate study. I will never forget you and wish and hope to be your friend forever. Merci pour tout ce que vous m'avez permis d'obtenir et pour l'enrichessement personel acquis. To my major professor, Dr. H. Allen Tucker, my indebtedness for his guidance, help, patience and leadership in refining my skills for science is incommensurable. One word can desribe him, quintessence. And all I can say is Merci. To Larry T. Chapin, I am forever indebted. Your tremendous help, Larry, in every step throughout my research program will always be remembered. Sans toi, je n'am'ais pas accompli tout ce qu'il m'a ete possible de realiser. To Dr. EM. Convey, a great deal of gratitude is extended for helping me throughout my graduate program by his advice, because of his joviality and his love for science and for his friendship. Merci. To the members of my guidance committee, Drs. R.S. Emery, J. Meites and D.R. Romsos, I wish to express my appreciation for their helpful criticisms of this thesis. A special thanks is addressed to Dr. Clyde Anderson for his help during the statistical analysis of data. Finally, I extend my gratitude to the maple of this department and to all my fellow graduate students who have in many ways made my stay in this country so enjoyable. A special thanks is addressed to Evette Williams for typing this thesis. iii TABLE OF CONTENTS List of tables ............................................................................... List of figures ...................................................................... . IntPOduction 00.....0........ ....................................... . ....................... . ..... Review of literature ...... . ................. . ..................................... .. 1.0) Introduction .................... . ................................................. 2.0) Photoperiod, its geophysical pattern ........................... i 3.0) Biological clocks .............. .. ......... 3.1) Historical Milestones ........................ . ........................ 3.2) .Bunning's hypothesis ..... ............ ...... 4.0) Entrainment to light ........ ........ ...... 4.1) Definitions ....... 4.2) Circadian sensitivity to light 4.3) Mechanism of light entrainment 4.4) Circadian activity rhythm of hamster: an example ...................................................... 5.0) Photoperiod and time measurement 5.1) Photoperiod and seasonality 5.2) Hourglass model 5.3) Coincidence model 5.4) Evidence for a circadian rhythm of photoresponsiveness 5.4.1) Resonance experiments ...... . ............. . .......... 5.4.2) T-experiments 6.0) Anatomy of circadian rhythmicity 6.1) Input pathways ...... . ..... 6.1.1) Extraretinal perception. 6.1.2) Retinal perception 6.2) Rhythm generators 6.3) Output pathways ............. 6.3.1) Diurnal rhythmicity of pineal gland 6.3.2) Seasonal reproductive cycles iv 14 16 16 l6 l7 18 22 23 24 25 26 29 29 32 Chapter 1. Effect of blinding on photoperiod and seasonal variations in secretion of prolactin in cattle ..... 39 Introduction ....................................................................... .. 40 materials and methOdS 0......0... ......... ............0.....0..........0.....0..00.. 42 ReSUIts .........00....0.0.0.0...........0..0...0...0....00..00.......0........000...0.....0 45 Discussion ................................................................................ 56 Chapter 2. Evidence for a diurnal rhythm of photosensitivity on the regulation of secretion of prolactin in prepubertal bulls .. ............................ 59 Introduction ............................ . .................................. ..' ....... 60 Materials and methods . .............. . .......... ..... . ........ . ..... .. 61 Results ..... . ........ .. ....... ......... .... 63 Discussion ............. .............................. .. ............................. 73 Chapter 3. Diurnal pattern of prolactin release in prepubertal bulls exposed to short- or long-day photoperiods .. ....... .. ................ . .......................... 75 Introduction .. ..................... . ..................... . ................... . ......... . 76 Materials and methods ........................................................ 78 Results ...... . ...... 81 Discussion ...................... ................... ..... ....... . ......... 96 Chapter 4. Body growth, growth hormone, prolactin and puberty responses to photoperiod and plane of nutrition in Holstein heifers .. ........................................... 100 IntrOduction ..... O 000000000000 0......0 ...... ....00. ................. .0 ....... ......000.. 101 Materials and methods ........ ...... . ....................... . ................. . 103 ReSUItS ......00................00.00.....0.........0..00.....0.....0........0.......0.0..0 107 DiSCUSSion .0.0.....00.0.0.......0.......0..........O...0......0.............0........O... 119 Chapter 5. Carcass composition and mammary development responses to photoperiod and plane of nutrition in HOlStein heifers ......0.....0................0...0...0.0..0.0 ....... . ...... 123 IntPOduction ......0..0.0.. ...... .00....00.........O.....0.0........O...00......0........ 124 Materials and methws ......0.....00.00....0....0...0.0.0....0...0...0...00...0..0. 124 Results ..................... . ............................................. . ....... 127 Discussion ......O...0.....0 00000 ..0.00.0...0.........O.....00...0.........O....0..O..OO.. 146 Conclusion .. .................................................................................... 149 Bibliography ........... ........... ................................................ 152 V LIST OF TABLES Table ‘ Page 1 Split plot analysis of seasonal variation in concentrations of prolactin in sera of blind and sighted steers. 55 2 Area under the curve (ng ml-1 min) of prolactin release after TRH injection (16.5 pg/loo kg body weight) 3, 9, 15 and 21 h after dawn in calves exposed to 8L:16D or 16L:8D photo- periods. 95 3 Feed composition and ingredients in the diet for each plane of nutrition. 104 4 Average daily dry matter intake expressed as percent of body weight. 110 5 Growth performance of heifers fed low or high plane of nutrition and exposed to 8 or 16 h of light per day. 123 vi LIST OF FIGURES Prolactin in sighted or blind bulls (n=7). All bulls were exposed to 8L:16D for 9 weeks. At week 10, one group (n=4) was switched abruptly to 16L:8D for 6 weeks, and the other group (n=3) was maintained on 8L:16D. All bulls were blinded on week 4. Blood was collected during a 24-h interval at the end of weeks 3, 9 and 15. Prolactin concentrations in sera of sighted (week 3) and blind (week 9) bulls (n=7) exposed to 8L:16D for 9 weeks. Data are adjusted for ambient temperature variation recorded at 30-min intervals. Prolactin in blind bulls after iv injection of TRH (33 ug/100 kg body weight) at time 0 during the 15th week of exposure to 8L:16D (n=3) or 6th week of exposure to 16L:8D (n=4). Pooled SEM were 20.7 and 15.1 ng/ml, respectively. Seasonal variation of prolactin in sighted (n=4) and blind (n=4) steers. Each point re resents the mean of 36 samples. Ambient temperatures ( C) averaged -2 (Feb), 5 (April), 15 (June), 30 (Aug), 16 (Oct) and 10 (Dec) at the time of sampling. Basal prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (open symbols), then switched to 16L:8D or 6L:8D:2L:8D for 6 additional weeks (closed symbols). Onset of lights during the primary period of lighting was 0700 h. Pooled SE of means during weeks 6 and 12 were 0.4 and 3.3 ng/ml, respectively. TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (open symbols), then switched to 16L:8D or 6L:8D:2L:8D for 6 additional weeks (closed symbols). TRH was injected at time 0 (0930 to 1000 h). Onset of lights during the primary period of lighting was 0700 h. Pooled SE of mean concentrations of prolactin measured between -15-0, 4-15 and 20-45 min appear above response curves. Basal prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (open symbols), then switched to 6L:8D:2L:8D or 6L:14D:2L:2D for 6 additional weeks (closed symbols). Onset of lights during the primary period of lighting was 0700 h. Pooled SE during week 6 was 0.9 ng/ml; during week 12, pooled SE for 6L:14D:2L:2D and 6L:8D:2L:8D were 4.6 and 5.8 ng/ml, respectively. vii 47 49 51 53 65 67 69 10 12 l3 14 15 TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (open symbols), then switched to 6L:8D:2L:8D or 6L:l4D:2L:2D for 6 additional weeks (closed symbols). TRH was injected at time 0 (1100 h). Onset of lights during the primary period of lighting was 0700 h. Pooled SE of mean concentrations of prolactin measured between -30—0, 5- 15 and 20-60 min appear above response curves. Basal prolactin in serum of prepubertal bulls after an abrupt change from 8L:16D to 16L:8D (open circles) or 16L:8D to 8L:16D (open squares). Initial time when both groups were first exposed to a different photoperiod was 1630 h of day 1. Open horizontal bars indicate day light times and closed bars night times. Onset of lights was 0800 h. Basal prolactin in serum of prepubertal bulls between 2000 and 0200 h on the day prior (day 0) and l, 2 and 4 days after switching photoperiod from 8L:16D to 16L:8D (open circles) or 16L:8D to 8L:16D (open squares). Open horizontal bars indicate day light times and closed bars night times. Onset of lights was 0800 h. TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 16L:8D. TRH was injected (time 0) 3 (open triangles) and 15 h (open circles) after dawn. Onset of lights was 0300 h. TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 16L:8D. TRH was injected (time 0) 3 (dash line), 9 (solid line), 15 (dotted line) and 21 h (dashed- dotted line) after dawn. Onset of lights was 0800 h. TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D. TRH was injected (time 0) 3 (dash line) 9 (solid line), 15 (dotted line) and 21 h (dashed- dotted line) after dawn. Onset of lights was 0800 h. L-DOPA-induced inhibition of prolaction release in serum of prepubertal bulls exposed for 6 weeks to 16L:8D. L-DOPA was injected (time 0) 3 (Open circles) and 15 h (open triangles) after dawn. Onset of lights was 0300 h. Changes in body weight of Holstein heifers in response to photoperiod and plane of nutrition. Each point represents the mean of 14 or 15 animals. Pooled SE of means is 3.6 kg. Average daily gains throughout the experiment are shown on the right for each treatment. viii Page 71 83 85 87 89 91 93 108 16 17 18 19 20 21 22 23 24 25 26 Changes in basal growth hormone concentrations in Holstein heifers as influenced by photoperiod and plane of nutrition. Each point represents the mean of 120 to 130 samples from 10 animals. Pooled SE of means is 1.0 ng/ml. Changes in basal prolactin concentrations in Holstein heifers as influenced by photoperiod and plane of nutrition. Each point represents the mean of 120 to 130 samples from 10 animals. Pooled SE of means is 1.1 ng/ml. Effects of photoperiod and plane of nutrition on body weight at puberty (pooled SE = 17.7 kg) and interval from start of experiment to puberty (pooled SE = 1.3 week). Each bar represents the mean of 14 or 15 animals. Percentage of water in 9,10, 11th rib section of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error- - 14.11 (11: 4 or 5). Percentage of fat in 9,10, 11th rib section of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error - 24. 96 (11: 4 or 5). Percentage of protein in 9, 10, 11th rib section of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 1.08 (n = 4 or 5). Mammary parenchymal tissue weight of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 966.4 (11 = 4 or 5). Concentration of DNA in mammary parenchyma of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = .0872 (n = 4 or 5). Concentration (of fat in mammary parenchyma of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 28.06 (11 = 4 or 5). Total DNA in mammary parenchyma of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 6952.0 (11 = 4 or 5). Total fat in mammary parenchyma of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 345.3 (11 = 4 or 5). ix 113 115 117 130 132 134 136 138 140 142 144 INTRODUCTION Milk production, reproduction, growth rate and ability to synthesis protein are major factors affecting the efficiency of output in the cattle industry. In recent years, these traits of cattle production were observed to be influenced by photoperiod. Forbes et al. (1975, 1979a) and Peters et al. (1978, 1980) reported that 16 h of light daily stimulates growth rate of sheep and dairy cattle by 10 to 2096. Furthermore, in comparison with daily light exposures of less than 12 h, 16 h of light per day stimulates milk production of dairy cattle approximately 1096 (Peters et al., 1978, 1981; Bodurov, 1979). Thus, management of photoperiod to improve growth rate and milk production is a potentially promising practice that may enhance output of the cattle industry. Comprehension of the mechanism of photoperiod action in cattle is still in its infancy. Photoperiodic induction of growth and milk production is probably the result of modification of neuro-endocrine events set in motion by some time- measuring device or biological clock. Of all the hormones measured in cattle, prolactin (PRL) is the most responsive to changes in photoperiod (Tucker and Ringer, 1982). Increasing daily light from 8 to 16 h increases secretion of PRL several folds (Bourne and Tucker, 1975; Leining et al., 1979). No cause-effect relationship between changes in PRL secretion and stimulation of growth and lactation has yet been established, and this issue will not be addressed directly in this thesis. Nevertheless, changes in PRL secretion are highly correlated with changes in photoperiod. Thus, PRL can be used as a bioassay to monitor photoperiod changes. In this thesis, general objectives were: first, to study the mechanism of photoperiod action by monitoring changes in PRL concentrations in response to various photoperiods and treatments in prepubertal Holstein bulls; concentrations in serum were used as an index of PRL secretion. The second objective was to investigate the effects of photoperiod and plane of nutrition on body growth of Holstein heifers. Specifically, the pathway involved in the transmission of photic signals to the anterior pituitary is examined. Evidence for a diurnal rhythm of photosensitivity on the regulation of PRL secretion are presented.“ In addition, diurnal rhythmicity of PRL release after an abrupt change in photoperiod or after injection of either thyrotropin-releasing hormone or L-dihydrophenylalanine is investigated. Finally, effects of photoperiod and plane of nutrition on body growth, carcass composition, mammary gland development, growth hormone and PRL secretion, and puberty of Holstein heifers are studied. REVIEW OF LITERATURE In the first book of Moses commonly called Genesis, it is written that God said, "Let there be lights in the firmament of the heaven to separate the day from the night; and let them be for SIGNS and for SEASONS, and for DAYS, and for YEARS; and let them be for lights in the firmament of the heaven to give light upon the earth and it was so. And God made the two great lights; the greater light to rule the day, and the lesser light to rule the night". 1.0) Introduction In this review of literature, I will examine how photoperiod has been used by biological systems to measure time. Does anybody really know what time it is? Does anybody really care? (Chicago, 1968). The review. of literature pertaining to the photoperiod and seasonal responses of PRL secretion and body growth of cattle will be presented in each of the specific chapters of this thesis. The geophysical cycle of daylight and darkness provides a source of reliable information between an organism and its environment. And through'this channel of communication, daily, seasonal and latitudinal information is transmitted to the living system. Photoperiodism is the study of the adaptive mechanisms by which living systems exploit this source of temporal information (Beck, 1980). Survival of each species in a world where food supply and predator activity are often cyclic depends on the appropriate anticipation and timing of behavioral and internal metabolic adjustments with respect to the daily events in the environment. The precise scheduling of behavioral and physiological events is one of the most critical services performed by the circadian timing system (Moore-Ede et al., 1982). Commenting on the old saying "The early bird catches the worm", Aschoff et a1. (1971) said that it is the circadian system that wakes up the bird, often before dawn. The circadian system is sensitive to certain specific environmental variables capable of acting as circadian time cues. Aschoff (1951, I954) coined the term Zeitgeber, from the German meaning "time-giver", to describe such periodic environmental cycles. Photoperiod is the principal Zeitgeber for a host of circadian rhythms (Rusak and Zucker, 1979; Elliott and Goldman, 1981). The annual cycle of seasonal fluctuations in the environment constitutes major problems and/or opportunities to most organisms; and a focal point in their evolutionary response to these challenges has been the necessity to anticipate season reliably (Pittendrigh and Minis, 1964). Clear adaptive advantages exist in restricting part of the life cycle of an organism to the Optimal time of the year, a fact particularly true in temperate regions where the climate varies markedly. To survive winter, insects enter diapause, birds often migrate to warmer regions ' whilst many mammals increase their fat storage, hibernate and change their haircoat. In contrast, spring and summer offer an abundance of food, and so animals reproduce to give birth of their offspring during these favorable times of the year (Follett, 1982). Unfortunately, the relevant physiological and behavioral modifications involved do not take place instantaneously (Pittendrigh, 1954). Hence, the animals must anticipate the proper time of the year. Time concepts are very much involved in the subject of photoperiodism. Biological time measurements are needed and can be detected in cells, tissues, organisms and populations of organisms. The fundamental time measuring unit is the living cell, plant or animal; the fundamental unit of time being measured and utilized is the cycle of daylight and darkness, the photoperiod (Beck, 1980). 2.0) Photoperiod, its geophysical pattern In the vast literature on photoperiodism, the term photoperiod is often used ambiguously. Sometimes, it is used to denote the entire cycle of illumination and darkness; other times, to represent only the lighted portion of the cycle. In this text, photoperiod will be used to define a cycle composed of a period of light and a period of darkness. The daylight portion of the photoperiod will be referred to as the photophase, the dark portion as the scotophase. The term daylength will be synonymous with photophase in reference to the photophases of natural or artificial 24-h photoperiods. The yearly revolution of the earth around the sun and its daily rotation on its axis determine the light-dark patterns of exposure. The true period of rotation of the earth is not 24 h but 23 h and 56 min. Because the earth is revolving around the sun in the same direction as its rotation, it must then turn for an extra 4 min each day to complete a rotation. If the polar axis of the earth was precisely perpendicular to the plane of its orbit around the sun, the photoperiod would consist of 12 h of daylight and 12 h of darkness everywhere on earth and throughout the entire year. But, the axis of the earth is at an angle of 23.5 degrees and this results in seasonal changes of daylength (and nightlength). These seasonal changes form a photoperiodic pattern over the entire surface of the globe, so that the daylength at any geographical point is determined by the latitude of that point and the date of observation (Beck, 1980). 3.0) Biological clocks The physiological system responsible for measuring time and synchronizing the internal proCesses of an organism with the daily events in its environment is known as the circadian timing system (Moore-Ede et al., 1982). This timing system can be likened to a biological clock. The word circadian (Latin: circa = about; dies = day) was coined by Halberg (1959) to describe the approximately 24- h cycles that are endogenously generated by the organism. 3.1) Historical Milestones Alexander the Great, in the fourth century B.C., documented the daily movements of leaves and flower petals. However, these diurnal rhythms were interpreted then as passive responses to a cyclic environment. Centuries elapsed before the persistence of rhythms was first demonstrated in the absence of a cyclic component of the environment, photoperiod. M. Marchand in a brief communication de L'Academie des Sciences de Paris, reported how Jean Jacques d'Ortous de Mairan (1729) had studied the leaf movement of a sensitive heliotrope plant. This plant opens its leaves and pedicels during the day and folds them at night. When it was moved to a place where the sunlight could not reach it, it was observed that the plant still opened its leaves during the day and closed them for the entire night. Later, Duhamel DuMonceau (1758) and Zinn (1759) showed that this rhythm of leaf movement persisted under constant photoperiod and environmental temperature. Yet, another century passed before the first demonstration that circadian rhythms persisted (free-run) with their own endogenous period when not synchronized by the light-dark cycle. Augustin de Candolle (1832) reported the daily leaf rythms of Mimosa pudica not only persisted in constant dark but, the leaves opened one or two hours earlier everyday and displayed a periodicity of 22 to 23 h. The plant had "an inherent tendancy to show periodic movements". In mammals, the endogenous nature of circadian rhythms was first reported by William Ogle (1866) from observations of the daily body temperature rhythm of man. He concluded that the rhythm was not directly dependent on any obvious environmental influences nor on the sleep-wake cycle of the subjects. However, the first conclusive demonstration of the endogenous nature of the mammalian body temperature rhythm was made by Simpson and Galbraith (1906). In their study, the rectal and axillary temperature of 5 monkeys was recorded every 2 h, day and night for 60 days; temperatures were high during the photophase and low during the scotophase. The body temperature rhythm persisted under constant light or darkness with an endogenous periodicity. Furthermore, this rhythm gradually inverted when the photoperiod was reversed; thus, the rhythm could be synchronized (entrained) by the 24—h light-dark cycle. Later, Richter (1922) demonstrated the endogenous nature of circadian wheel-running activity rhythms of rats. Johnson (1939) subsequently suggested that "the animal (rat) had an exceptionally substantial and durable self-winding and self-regulating physiological clock, the mechanism of which remains to be worked out". 3.2) Bunning's hypothesis Bunning's hypothesis is usually evoked to explain photoperiodic time measurement in vertebrates. In 1936, Bunning reported that light could synchronize the endogenous rhythm of plant leaf movements and control the photoperiodic response of flowering (Bunning, 1960). From his observations conducted on the rhythmic motions of seedling bean leaves (Phasoleus multiflorus), he described two phases in the daily rhythm of leaf movement: 'a tension phase of 12 h normally occurring during the photophase when leaves were upward, and a relaxation phase of 12 h when leaves were downward during the scotophase. This oscillation of tension and relaxation was thought to be phase- regulated by dawn. And, depending on the daylength, the environmental light might or might not coincide with the beginning of the relaxation phase; for example, under long daylengths (> 13 hours), light would-invade the relaxation phase of the endogenous oscillator. The coincidence or absence of coincidence of light with the relaxation phase would determine a typical response of the organism to photoperiod. Thus, in his hypothesis, Bunning searched for a single explanation of both phase regulation of overt rhythms such as leaf movements in plants and locomotor activities in mammals, and photoperiodic induction of such phenomena such as plant flowering, insect diapause and seasonal reproduction in mammals (Bunning, 1960). Recently, our knowledge of the mechanism of entrainment of rhythm by light-dark cycles has been incorporated into the model of photoperiodic time measurement in vertebrates. A more detailed discussion of photoperiodic time measurement will be presented later but, first, I will discuss the entrainment properties of light. 4.0) Entrainment to light For the biological clockwatcher, rhythms are driven by and, hence, are overt manifestations of some metabolic process or system pacemaker capable of continued self-generated oscillations. The major action of photoperiod is not to generate the rhythmicity but to entrain the internal pacemaker(s) to a period of precisely 24 h (Elliott and Goldman, 1981). For photoperiod (or any Zeitgeber) to entrain the circadian system, it must in each cycle reset the phase of an otherwise free-running rhythm by an amount of time that corrects for the difference between the period of the time cue (photoperiod) and that of the rhythm. This is achieved through a circadian rhythm of sensitivity of the organism to the time cue (Moore-Ede et al., 1982). 10 4.1) Definitions Before reviewing the mechanism of entrainment of endogenous rhythms to light, the definition and explanation of certain terms will be useful for better comprehension of this discussion. The definitions of these terms have been taken from texts written by Beck (1980), Elliott and Goldman (1981) and Moore-Ede et al., (1982). A rhythm is a regularly recurring cyclic fluctuation. A reference point in a rhythmic system is symbolized by the Greek letter phi ( ); it is an arbitrary. point that can be observed consistently such as the onset of activity in locomotor rhythms or the mean time of adult emergence in eclosion rhythms. Most organisms appear to be capable of exhibiting a variety of endogenous rhythms; that is, organisms are able to generate rhythms even when they are prevented from receiving rhythmic cues from their environment. The time between two successive point of an endogenous rhythm measures the period of the rhythm symbolized by tau ( rt ) and represents the length of time required to pass through one complete cycle. The process of achieving synchrony between internal (endogenous) and external (environmental) rhythms is called entrainment. The environmental cue that is employed for entrainment is called a Zeitgeber as previously described (Aschoff, 1951, 1954). The period of a Zeitgeber is symbolized by the letter T. When an endogenous rhythm is entrained by a Zeitgeber, T of the endogenous rhythm equals T of the Zeitgeber. The time between the beginning of the photoperiod and any arbitrary (P of a circadian cycle is called phase angle and symbolized by psi (111 ). In a 24-h cycle, a phase angle of 15 degrees is the equivalent of l h. However, 11) is usually expressed in time units rather than degrees. The temporal relationship between any two points of an endogenous rhythm can also be expressed in terms of phase angle. Perturbations, in the form of light or temperature pulses, may cause to occur earlier (phase advance), later (phase delay) or no phase shift at all on the time axis. A phase advance is measured by a decrease 11; or by definition a + A9 and visa versa for a phase delay. The observed relationship between the time of a pulse and the phase shift obtained can be conveniently plotted as a phase response curve (PRC). (De Coursey, 1960a, b; Pittendrigh, 1960). Most of the curves derived for mammalian species have been obtained with light pulses (10-15 min in length) applied to an animal maintained in constant dark or just released into constant dark after being entrained by a light-dark cycle. 4.2) Circadian sensitivity to light Kleinhoonte (1932) was first to observe in plants a daily change in sensitivity to light (Moore-Ede et al., 1982). Later, in mammals, Rawson (1956) observed a similar phenomenon but did not quantify it precisely. He noticed that, when the lights were temporarily turned on to feed mice maintained in constant dark, a phase-shift in their locomotor activity rhythm was introduced at certain phases of their subjective night but not at other times (Moore-Ede et al., 1982). It was, however, Pittendrigh and Bruce (1957; Pittendrigh, 1958) and, Hasting and Sweeney (1958) who first precisely quantified phase shifts in response to light pulses using Drosophila and the unicellular algae, Gonyaula. In mammals, De Coursey (1960a,b) did the first detailed examination of the PRC of wheel-running activity of flying squirrels (Glaucomys volans) in response 12 to 10-min light pulses given at hourly intervals throughout their subjective day or night. From such studies, very similar phase shifts were observed in all species whether, they were single-celled algae or primates, and whether they were nocturnally or diurnally active (Moore-Ede et al., 1982). Firstly, responsiveness to light is confined mostly to the subjective night in both nocturnal and diurnal species (although there are little data from diurnal mammals). Secondly, at the time when the animal is normally exposed to light (in mid—subjective day), light has no effect on the circadian system. Thirdly, the PRC documents how the sun rising in the morning (during the animal's subjective day) tends to produce a phase advance (so an animal would start activity earlier), whereas light falling at dusk (late subjective day) causes a phase delay (so that the animal will continue activity longer). Thus, a natural daily light-dark cycle is constantly nudging the circadian clock of animals forward in the morning and backward in the evening with each 24—h day (Moore-Ede et al., 1982). 4.3) Mechanism of light entrainment Synchrony between an endogenous rhythm and the daily light-dark cycle is achieved when the T of the rhythm equals T of the Zeitgeber. Thus, when the natural period of the rhythm is shorter than 24 h, the entrained rhythm must phase-delay; in contrast, when it is longer than 24 h, it must phase-advance. These phase-changes are obtained through a circadian rhythm of sensitivity to light pulses of the organism. A daily resetting can be achieved by a short light pulse every 24 h provided the pulses falls on the portion of the PRC producing a phase-shift of the required magnitude (amount of time) and direction (phase- advance, phase delay or no phase-shift) (Moore-Ede et al., 1982). 13 For example, let us examine an endogenous rhythm with a 26-h period that is entrained to a single l-h pulse per day. This signifies that a stable phase- relationship will be established when the light pulse' will cause a 2-h phase- advance per day. The initial pulse might induce a phase-advance, phase-delay or no phase-shift at all depending on its position of occurrence on the PRC. Imagine that no phase-shift occurred the first day. The second day, since the pacemaker period is 26 h and the light-pulse recurs every 24 h, the same phase- point on the PRC will occur 2 h later with respect to light-pulse (phase-delay). Consequently, the light-pulse on the second day will hit the curve 2 h earlier. Assume this produces a 1 h phase-advance of the pacemaker. On the third day, the) light pulse will hit the PRC l h earlier; this is the net result of the 2-h phase- delay between the rhythm period and the light pulse period plus the l-h phase- advance induced by the light pulse. Thus, on the third day, this pulse might produce a 2-h phase advance so that entrainment is obtained. Because the period of the pacemaker and the Zeitgeber for that cycle are now the same, the 2-h phase-advance will occur with each subsequent cycle. By a series of successive approximations, therefore, a steady state is gradually reached. Two pulses of light per cycle provide a more stable entrainment than a single pulse (Moore-Ede et al., 1982) and are usually referred to as skeleton photoperiods. They provide the temporal framework of a full photoperiod and mimic the influence of light-dark transitions that occur naturally at dawn and dusk. Each pulse instantaneously resets the pacemaker PRC and the net phase- shift of the two pulses gives the total phase-shift needed per cycle for entrainment. However, skeleton photoperiods are often misleading for the animals as far as discerning between long and short day lengths. Two pulses 14 given to mimic 16 h of light (L):8 h of dark (D) are often interpreted as 8L:16D (Pittendrigh and Daan, 1976). Consequently, full light-dark cycles insures that day or night are unambiguous. However, the transition from dark to light and light to dark conveys much of the information for stable entrainment (Moore-Ede et al., 1982). 4.4) Circadian activity rhythm of hamsters: an example The Syrian hamster is a burrowing, nocturnally active rodent. In nature, these animals spend much of the day sleeping in their underground burrows. At dusk, they emerge from their burrows to forage for food and mate. In the laboratory, the clock system that times nocturnal activity is usually studied by monitoring wheel-running behavior (locomotion). Locomotor activity onset is a most regular and precisely timed feature of the rhythm. On a 14-h photoperiod (14L:10D), hamsters begin vigorous wheel- running approximately 20 min after dark and the phase angle difference ( 1p ) measured between activity onset and the onset of photoperiod (beginning of daylength) is stable at 9.7 h. The active phase uSually lasts 6 to 10 h (Elliott and Goldman, 1981). Hamsters released into constant dark from prolonged entrainment to cycles of 24 h (14L:10D or 6L:18D) free-run with T ranging from 23.9 to 24.37 h with a population average of 24.12 h (i 0.04 h standard error of mean) (Elliott, 1981). In order to simplify the discussion of phase relationships, a circadian time (CT) scale has been adopted; one full circadian cycle (360°) with period ‘1' is divided into 24 circadian hours, each lasting 1/24 h of real time. The activity onset is designated CT 12 and marks the beginning of the subjective night (CT 12 15’ to 24). The half cycle before onset of activity is designated subjective day (CT 0 to 12) (Elliott, 1981). The phase of a circadian rhythm free-running in constant dark can be shifted to an earlier or a later time by a single brief pulse of light. The phase shift is seen as a displacement on the time scale. Both the magnitude (amount of time) and direction (advance: positive values; or delay: negative values) of the phase shift response (Ad) ) to a standard pulse are characteristic of the particular phase 6 or circadian time (CT) at which the pulse is given (Elliott, 1981). This dependence is summarized in a PRC (DeCoursey, 1964; Daan and Pittendrigh, 1976). Phase delays ( —A ) are elicited by light in the late subjective day and early subjective night (CT 10 to 16). Phase advances ( +A<1> ) peak in the middle of the subjective night (CT 18) and continue with diminishing magnitude into the early subjective day (CT 2). In the remainder of the subjective day, light pulses have little or no effect on the phase of the rhythm (Elliott, 1981). The magnitude of the phase shift varies among species (Daan and Pittendrigh, 1976) but the general shape of the curve remains. The PRC describes a circadian rhythm of sensitivity to light. This rhythm of sensitivity is used to mediate the entrainment of a circadian rhythm (such as wheel-running activity in hamsters) to the light- dark cycle (Pittendrigh and Minis, 1964; Elliott, 1981). Thus, if ‘I' is known and one has measured a PRC for a standard light pulse, then it is possible to predict the circadian time at which the light pulse must fall to entrain the rhythm to different T‘s. In the next section, I will present how these properties of circadian rhythms have been used to develop models for time measurement. 16 5.0) Photoperiod and time measurement 5.1) Photoperiod and seasonality Rowan (1925) was the first to report the influence of photoperiod as a primary environmental signal regulating the seasonal reproductive cycle in birds. Then, Bissonette (1932) and Baker and Ranson (1932) demonstrated the involvement of photoperiod on the seasonality of reproductive activity in two species of mammals, ferrets and field voles (as cited by Elliott, 1976). They showed that estrus could be induced in winter, well in advance of the breeding season, by increasing day length exposure. Later, Yeates (1949) induced estrus out of season in sheep by reducing daylength exposure (as cited by Elliott, 1976). Conversely, anestrus could be induced during the breeding season by increasing daylength exposure. Since these pioneer investigations, a great deal of attention has been directed toward answering a fundamental question: how do animals measure length of the day (or the night), how do animals measure time? The major theories concerning time measurement came largely from work with plants and insects (Elliott, 1976) but lately, they have been tested in birds (Hamner, 1963) and mammals (Elliott et al., 1972). ‘There are two general classes of hypotheses of time measurement; one is based on some form of an hourglass or interval timer, the other assumes the involvement of an endogenous circadian rhythm of photosensitivity (coincidence model). 5.2) Hourglass model The principal feature of this model is the involvement of some photochemical process occurring during the light (or the dark) period which is reversed in the other portion of the light-dark cycle (Follett, 1973). If enough of 17 some hypothetical reaction product accumulates as a result of the organism's exposure to a long day (or night), then a threshold is exceeded and a photoperiodic effect is observed. The proponents of hourglass mechanisms stress the importance of a critical duration of light or dark (Jenner and Engels, 1952; Farner et al., 1953; Wolfson, 1960) or a critical ratio of light:dark in each cycle (Bissonette, 1930, 1931). The time measuring process is thought to begin anew with each cycle of light and dark; it is driven by the environmental cycle and lacks endogenous rhythmicity (Elliott, 1976). This model might be widespread in invertebrates and insects (Beck, 1980) but most experiments in mammals are best interpreted on the basis of the second hypothesis (Follett, 1973; Stetson et al., 1975). 5.3) Coincidence model In its modern version, the hypothesis of Bunning assumes that photoperiodic measurement of time is based on a circadian rhythm of responsiveness to light with two qualitatively distinct halves: during the first half (subjective day), the organism is non-responsive to light; during the second half of the cycle (subjective night), the organism becomes responsive to light. Photoperiodic response (inhibition or stimulation) by long days occurs when light extends into the second portion of the circadian rhythm of photoresponsiveness or photosensivity (Elliott and Goldman, 1981). In Bunning's model, the light sensitive phase of the oscillation is assumed to occur at a fixed time after dawn regardless of the duration of the photoperiod (Bunning, 1960; Elliott and Goldman, 1981). Subsequently, Pittendrigh and Minis (1964, 1971) developed a new model that resembles the model of Bunning but also incorporated the property of 18 entrainment of rhythms by light, the external coincidence model. In this model, it is postulated a photoinducible phase ( M) in the circadian rhythm of photoresponsiveness and light has a dual role: first, it entrains all the circadian rhythms of the organism including the circadian of photoresponsiveness; and second, it acts as an inducer of the photoperiodic response when (Pi is illuminated. Thus, the temporal coincidence of light and (b i depends on the entrainment of the circadian rhythm of photoresponsiveness by the light-dark cycle and not only on the duration of the daylength (Elliott and Goldman, 1981). For a long time, it was considered that the circadian timing system consisted of a single self-sustained oscillator. However, there is now compelling evidence which will be examined later for a multiple oscillator arrangement. These putative oscillators control many oscillations whose mutual phase relationships may have a significant impact on a variety of physiological functions (Pittendrigh, 1960, 1974; Aschoff and Wever, 1976; Moore-Ede et al., 1982). This has led to an alternative model, the internal coincidence model (Pittendrigh, 1974, 1981). In this model, light serves only as an entraining agent controlling the phase of the many endogenous rhythms within the organism. When the phase relationships of these rhythms enter into the proper configuration, photoperiodic induction occurs (Pittendrigh, 1981). But now, let us consider the experimental evidence for the participation of a circadian rhythm of photoresponsiveness in the control of seasonal cycles. 5.4) Evidence for a circadian rhythm of photoresponsiveness In practice, few experimental designs can adequately test the involvement of a circadian rhythm of photoresponsiveness in daylength measurement. That 19 photoperiodic induction depends not upon the absolute duration of light or dark, nor of the light-dark ratio but rather upon light being received during a unique period of the circadian rhythm of photoresponsiveness usually requires the use of exotic light-dark schedules. Some of these tests most elegantly and successfully applied are presented below. 5.4.1) Resonance experiments The so called resonance experiment introduced by Nanda and Hamner (1958) is one of the most powerful tests to indicate a circadian basis for photoperiodic measurement of time. In this test, a short photophase (e.g., 6 h) is coupled to different durations of scotophase to generate photoperiods with different T's such as 24, 36, 48, 60 and 72 h. Thus, an animal exposed to T's of 36 or 60 h would receive 6 h of light followed by 30 or 54 h of dark in repeating 36 (6L:30D) or 60 (6L:54D) h cycles. On a 24 h scale, there would be alternation of light in the morning followed by light in the evening one day (6L:30D) or 2 days later (6L:54D). Photoperiods of 6L:18D, 6L:42D or 6L:66D present light only in the morning every 1, 2 or 3 days. - Hamner (1963) was first to upset the cherished concept that time measurement was not made based on a hour-glass mechanism but on a system involving circadian rhythmicity. Using a resonance experimental design, he found that testicular growth of the house finch ( Carpodacus mexicanus) occurs with 6L:6D, 6L:30D and 6L:54D photoperiods but not with 6L:18D, 6L:42D and 6L:66D. Later, similar resonance experiments were performed to study the growth of testis in voles (Grocock and Clarke, 1974) and in Golden hamsters 20 (Elliott et al., 1972; Stetson et al., 1975) and yielded comparable results to those of the house finch. In hamsters, 12.5 h of light daily is the minimum daylength required for photoperiodic induction and maintenance of gonadal activity (Hoffman et al., 1965; Elliott, 1976; Gaston and Menaker, 1967). Hamsters exposed to 6L:18D or 6L:42D photoperiods are incapable of maintaining the mature size of their testes or seminal vesicles. Moreover, the locomotor activity of these hamsters in typical of short day exposure. Photostimulation of testicular recrudescence and maintenance, and the pattern of locomotor activity typical of long-day photoperiods is, however, obtained with 6L:30D or 6L:54D photoperiods (Stetson et al., 1975; Elliott and Goldman, 1981). Thus, these results provided evidence of involvement of a circadian rhythm of photoresponsiveness for photoperiodic measurement of time in hamsters. In many animals, a period of sensitivity to the light cycle is followed by a period of insensitivity (Turek and Campbell, 1979). In golden hamsters, exposure to short days induces gonadal regression but, after prolonged exposure (25 weeks), a spontaneous gonadal recrudescence will occur (Reiter, 1972; Turek et al., 1975). Reproductive function can be restored earlier by exposure to long photoperiods. However, long-day exposures fulfill another important role. In order to become responsive again to the inhibitory effects of short days, hamsters must be exposed to long days for about 11 to 20 weeks (Stetson et al., 1977). The physiological state of insensitivity to a particular light cycle is referred to as the refractory condition (Farner and Follett, 1966). Using the resonance paradigm, Stetson et al., (1976) showed that the termination of refractoriness was induced by photoperiods of 14L:10D,' 6L:30D and 6L:54D but 21 not with 6L:18D or 6L:42D. Thus, the discrimination of long from short days in terminating the refractory period is based on a circadian rhythm of photosensitivity. In order to obtain a measurable change after photoperiodic induction whether it is gonadal growth in vertebrates, diapause in insects or flowering in plants, it is usually necessary to repeat light-dark schedules for several cycles. In resonance experiments, this can lead to complex problems of entrainment (Follett et al., 1981). In fact, it has been impossible to show rhythmicity using resonance schedules longer than 72 h (Follett et al., 1974). The transfer of white-crowned sparrows and Japanese quail from short (8L:16D) to long (20L:4D) day photoperiods cause a significant increase in luteinizing hormone (LH) levels within 24 h of exposure to long days (Nicholls and Follett, 1974; Follett et al., 1974). This finding suggested another test of the original hypothesis of Bunning (1960), the single pulse technique. In this technique, birds are transferred from short days (8L:16D) into darkness, then pulsed with single 8-h light period at different times thereafter (Follett et al., 1974). If a circadian rhythm of photoresponsiveness is involved in photoperiodic induction of LH secretion, high levels of LH should be seen only during periodically recurring intervals. In this system, the inductiveness occurred only when the light period coincided with an underlying rhythm of photoresponsiveness whose period was about 24 h (Follett et al., 1974; Follett, 1978). The rhythm persisted in constant darkness without damping for 4 complete cycles. This provides evidence that the photoperiodic induction of LH secretion involves a self-sustained circadian oscillation (Follett, 1978). Unfortunately, however, the pulses were too long to indicate the precise duration of the photoinducible phase 22 and the free-running period of the circadian rhythm of photoresponsiveness (Follett et al., 1981). 5.4.2) T-experiments The description of the precise location of the photoinducible phase can be mapped in T—experiments which deliberately exploit the entraining action of light on circadian rhythms. Entrainment of a circadian rhythm to different periods (T) implies that a single light pulse must fall at a unique circadian time (CT) to cause the necessary phase-shif t adjustment (see section 4.4). By varying T within the range of entrainment, one could describe in detail the photoperiodic effects of brief light pulses applied at numerous discrete phases of the circadian system. In the hamster (Elliott, 1976; Elliott, 1981), the position of the photoinducible phase was mapped in T-experiments utilizing 15-min or l-h pulses of light with different periods between pulses. CT of the light pulse was determined by monitoring the rhythm of wheel-running activity which presumably bears a constant phase relationship with the circadian rhythm of photoresponsiveness (Elliott, 1981). Ten weeks after transfer to constant darkness, hamsters pulsed with light at CT times l3, 14, 15, 16 or 18 had significantly larger testes than controls receiving no pulses. Pulses at CT 10, 12, 21 or 24 failed to alter the time course of gonadal regression in constant darkness (Elliott, 1981). The dead zone of the hamster PRC lies between CT 2 and 10. That is, light falling in this region can not cause a phase-shift in the locomotor activity and, therefore, can not be used in the T-cycle paradigm (Elliott, 1981). Nevertheless, this portion of the circadian cycle is known to be non-responsive to 23 photoperiodic stimulation being lighted anyway under standard non-stimulatory photoperiods such as 6L:18D or 10L:14D (Elliott, 1976). Thus, the photoperiodic clock of the hamster can be described in terms of a circadian rhythm of photoresponsiveness entrained by the daily light-dark cycle. These data give support to the coincidence models, but they can not differentiate between the external and the internal versions. Pittendrigh (1981) has recently offered the soundest experimental evidence in favor of the internal coincidence model. He reported that the circadian system of time of emergence in Drosophila involved a distinct slave oscillator as well as a pacemaker (master oscillator). The phase relationship of the slave oscillator to the pacemaker not I only varies as a function of the photoperiod but also changes with the cycle length (T). The signal for eclosion behavior is given by the slave oscillator which is coupled to and driven by the pacemaker. The effectiveness of the signal depends of the phase relationship between these two oscillators, the master and the slave. Much more research will be needed to discriminate between the internal and external model, however. Up to now, nothing has been said about the physiological mechanisms of neural and endocrine events involved in the transfer of photoperiodic information from the circadian system to the target system. The next section discusses this matter. 6.0) Anatomy of. circadian rhythmicity Since photoperiod influences the majority of circadian and seasonal rhythms~ observed in mammals (Pittendrigh and Minis, 1964; Follett, 1978; Turek and Campbell, 1979; Elliot and Goldman, 1981), the description of the anatomical pathway involved in the transduction of photic signals is important. For more 24 information concerning invertebrates, amphibians, reptiles and birds, the reader is referred to Rusak and Zucker (1979). The physiological system responsible for circadian rhythmicity must contain at least three major components: first, an input pathway for entrainment; second, a circadian pacemaker that generates the rhythm; and third, an output pathway for the expression of the rhythm. Thus, for photoperiodic entrainment to occur, a photoreceptor must be coupled to a circadian pacemaker; and to generate a rhythm output, the, pacemaker must be coupled to the target tissue that generates the overt rhythm (Takahaski and Zatz, 1982). Such coupling is likely to be achieved by a variety of neurophysiological mechanisms (Rusak and Zucker, 1979) involving neural and endocrine effectors. 6.1) Input pathways 6.1.1) Extraretinal perception Presumably, extraocular photoreceptors do not play a role for entrainment of circadians rhythms in mammals (Rusak and Zucker, 1979). Unfortunately, these experimental data were obtained with blind adult mammals under artificial lighting of intensity far inferior (40-100 times) to direct sunlight. Natural daylight can penetrate many compartments of the body, even deep subcortical structures such as the ventral hypothalamus of sheep (Van Brunt et al., 1964). That the eyes are the only photoreceptors for entrainment, therefore, must remain tentative (Rusak and Zucker, 1979) until further research using blind animals exposed to natural sunlight intensity confirms this conclusion. 25 6.1.2) Retinal perception In adult mammals, the only functional site of light reception is the retina of the eye (Richter, 1965). The visual pathway conveying the information about light-dark cycles involved in entrainment is separate from those involved in other aspects of vision. In brief, the visual processes from ganglion cells form the optic nerves that enter the optic chiasm. Then, four neural tracts leave the chiasm bilaterally: the largest is the primary Optic tract and terminates in the superior colliculus, pretectal nuclei and lateral geniculate nuclei; the superior accessory Optic tract runs along’ the primary Optic tract and terminates in the midbrain tegmentum; the inferior accessory optic tract runs along the medial forebrain bundle and eventually ends in the medial terminal nuclei of the midbrain tegmentum; and finally, the retinohypothalamic tract connects with the suprachiasmatic nuclei of ,the hypothalamus (Moore, 1974). The retinohypothalamic tract projects to the suprachiasmatic nuclei (SCN) (Rusak and Zucker, 1979; Moore-Ede et al., 1982). The common feature of the mammalian retinohypothalamic tract includes a bilateral projection that rises out of the chiasm and enters the SCN from its ventro—lateral aspect to terminate preferentially in the ventral and caudal portions of the nuclei. Interrestingly, projections from the raphe nuclei (Aghajanian et al., 1969; Moore et al., 1978) and the lateral geniculate nuclei (Ribak and Peters, 1975) also terminate in the ventral portion of the SCN. However, direct cuts of both the primary optic tracts and the accessory Optic system failed to disrupt entrainment by light-dark cycles (Moore and Eichler, 1972; Stephan and Zucker, 1972). This unexpected 26 observation led to the hypothesis of a retinohypothalamic pathway conveying photic information to the pacemaker. Thus, the eyes are transducers of the circadian timing system (Moore-Ede et al., 1982). Unfortunately, the distribution, receptive fields and discharge properties of the retinal ganglion cells for entrainment of circadian rhythms are unknown (Rusak and Zucker, 1979). 6.2) Rhythm generators The existence of an endogenous circadian rhythm at the cellular level was first demonstrated by Schweiger et a1. (1965) using the very large unicellular and uninuclear green alga acetabularia sp. The rhythm of photosynthesis was phase- regulated by photoperiod and persisted under continuous dim light. Cells that were enucleated continued to display rhythmicity (endogenous) but were no longer sensitive to phase regulation by photoperiod. After a series of nuclear exchange experiments, it was concluded that the rhythm was endogenously expressed in the cytoplasm but its phase control by photoperiod was associated with the nucleus. In mammals, Richter (1967) was first to report elimination of rhythms in eating, drinking, wheel-running and activity of rats bearing lesions in the ventral medial portion of the hypothalamus. In search of the specific locus of the rhythm generator, Moore and Eichler (1972) and Stephan and Zucker (I972) lesioned the terminal nuclei of the retinohypothalamic tracts and observed that the circadian rhythmicity and entrainment by photoperiod of drinking behavior, locomotor activity and adrenal corticosterone rhythms were abolished. These nuclei were located in the SCN. So far, the numerous rhythms disrupted after- 27 SCN lesions are the following: wheel-running activity, drinking, feeding, sleep, body temperature, adrenal corticosterone, pineal N-acetyltransferase activity, ovulation, estrous cyclicity and photoperiodic measurement of time (Rusak and Morin, 1976; Stetson and Watson-Whitmyre, I976; Ibuka et al., 1977; Stephan and Nunez, 1977; Klein and Moore, 1979; Van Den P01 and Powley, 1979). Thus, the SCN could be considered as a possible site for generation of circadian rhythmicity; to meet the criterion of a pacemaker, it must be able to self-sustain circadian oscillations. Recently, Inouye (1982), and Inouye and Kawamura (1979, 1982) observed that circadian neural activity in the SCN after surgical isolation of the SCN from the rest of the brain persisted for at least 34 cycles, whereas rhythmicity disappeared outside the hypothalamic island. However, neural isolation experiments do not preclude entrainment of rhythmicity by hormonal influences. In most free-running situations, the various circadian physiological variables within an individual are internally synchronized with one another. That is, they possess identical periods and stable phase relationships. This phenomenon of internal synchronization suggested that the circadian timing system consisted of a single self-sustained oscillator. However, there is now compelling evidence in favor of a multiple oscillator arrangement which could account for all the various behaviors of the circadian timing system. In man, the 'sleep-wak'e cycle and the body temperature rhythm dissociate from each other and free-run with different periods after a long period of time under constant environment (Aschoff and Wever, 1976; Moore-Ede et al., 1982; Winfree, 1982). This phenomenon is called spOntaneous internal desynchronization. Other phenomona such as transient or forced internal desynchronization of the 28 circadian rhythm, splitting of rhythms into multiple components as well as persistence of rhythmicity under in vitrO conditions have added evidence to the multi-oscillator theory. For more details the reader is referred to Moore-Ede et al. (1982). Moreover, in studies of monkeys (Fuller et al, 1981; Takahashi and Zatz, 1982) abolition of drinking rhythm, wheel-running activity rhythm and melatonin rhythm occurs after destruction of the SCN, whereas the body temperature and cortisol rhythms are maintained. So, these results provide evidence for a circadian oscillator located outside the SCN. This oscillator is probably coupled to the SCN and the light-dark cycle. The organization of such multioscillator systems is complex. In man, Kronauer et a1. (1982) designed a mathematical model using two mutually coupled oscillators. Pacemaker X drove the rhythm of REM sleep, core body temperature, plasma cortisol concentration and urinary potassium excretion. Pacemaker Y drove the rest-activity cycle and the rhythms of slow-wave sleep, skin temperature, plasma growth hormone concentration and urinary calcium excretion. They considered that the SCN contained the Y pacemaker and that the X pacemaker was located outside the SCN (Fuller et al., 1981, Kronauer et al., 1982; Moore-Ede et al.,1982). The rest- activity cycle and core body temperature rhythms were utilized as marker outputs for the separate pacemakers. With this very simple model, all the stages of internal desynchronization could be readily replicated by steadily increasing only one variable, the period of the Y pacemaker (SCN). The mathematical model also made possible examination of how Zeitgeber inputs are coupled to the pacemakers. The analyses indicated that, in humans, the predominant target of environmenal information is the pacemaker Y. Since that pacemaker appears to correspond to the SCN, and this nucleus in other mammals receives a major input 29 of light-dark cycle information via the retinohypothalamic tract, the model conforms very well with the physiological evidence (Moore-Ede et al., 1982). 6.3) Output pathways 6.3.1) Diurnal rhythmicity of pineal gland. In 1960, Fiske et al. reported that pineal glands Of rats maintained under constant dark weighed more than those of rats under constant light. Quay (1963, 1964) demonstrated that serotonin and melatonin content of pineal glands of rats varied reciprocally during the light-dark cycle: high serotonin and low melatonin during the day and visa versa during the dark. In 1965, Axelrod et al. showed that the enzyme activity of hydroxyindol-O-methyltransferase (HIOMT) involved in the methylation of acetylserotonin to form melatonin was increased by constant dark and decreased by constant light over a period of a week. Furthermore, the enzyme exhibited a daily change (1.3- to 3-fold) with its maximum occurring in the dark. Then, Klein and Weller ( 1970) discovered that the key regulating enzyme involved in the conversion of serotonin to melatonin, N- acetyltransferase, exhibited a daily rhythm in its activity that was even more dramatic (30- to 70-fold) than the rhythms observed in HIOMT activity, serotonin or melatonin. The rhythm in pineal N-acetyltransferase activity is not only apparent in a light-dark cycle of birds and mammals, it also persists in constant dark (Binkley, 1976) and in blind rats (Klein and Moore, 1979), but it is obliterated by constant light exposure (Klein, 1979). However, the SCN is necessary for generation of the N-acetyltransferase rhythm in blind rats and is responsible for the tonic elevation of HIOMT activity in blind animals. Neural regulation of the pineal gland in rats involves the retinohypothalamic tract, SCN, tuberal 30 hypothalamus, medial forebrain bundle, spinal cord, superior cervical ganglion and nerve terminal on the pineal gland (Klein and Moore, 1979). In chickens, the superior cervical ganglion is not necessary for the expression of pineal rhythm during light-dark cycles, and the role of the SCN has not been studied yet (Binkley, 1976). Adrenergic compounds such as norepinephrine (NE) are released at the nerve terminal on the pineal gland and stimulate the activity of N- acetyltransferase in pineal gland even in organ culture (Klein et al., 1970).. A diurnal variation in sensitivity Of the pineal gland to adrenergic agents (Binkley et al., 1973; Romero and Axelrod, 1975) was observed such that the gland is refractory (insensitive) during the day and supersensitive during the night. Constant light, reserpine or superior cervical ganglionectomy (all reducing the amount of norepinephrine reaching pineal gland 6-receptors produces supersensitivity whereas exposure to NE or mimetics Of NE produces subsensitivity (Deguchi and Axelrod, 1973; Holz et al., 1974; Romero and Axelrod, 1975; Binkley, 1976). In mammals, light acts through retinohypothalamic projection to the SCN and has two apparently independent modes of action. First, it entrains the pineal circadian rhythm with the environmental lighting (Klein and Weller, 1970; Moore and Klein, 1974; Moore and Traynor, 1976); and in absence of the light signal, the rhythms in N-acetyltransf erase, serotonin and melatonin free-run and eventually go out of phase with the photoperiod. Several days are needed to shift the rhythm when animals are shifted from one photoperiod to another (Klein, 1979). However, a much faster effect of light is to cause a rapid decrease in pineal N- acetylserotonin transferase activity and blood melatonin, and increase in blood serotonin typical of day values when light is presented briefly at night (Klein and 31 Weller, 1972; Minneman et al., 1974; Rollag and Niswender, 1976). Furthermore, unexpected light-dark transitions produced a rise in N-acetyltransferase activity only at certain times relative to the previous light-dark cycle; that is, there is a period during the light time when rats are refractory to the initiation of N- acetylserotonin transf erase activity by dark (Binkley et al., 197 3). SO far, a daily rhythm in concentrations of melatonin in blood, urine and cerebrospinal fluid has been detected in a large number of mammals, man, birds and reptiles (Klein, 1979). Most impressive is the fact that melatonin is always elevated at night irrespective of the life style of the animals. Thus, in both night-active or day-active animals, melatonin is at a high level at night (Klein, 1979). Even in blind rats for up to 60 days, free-running pineal rhythms are maintained with undamped high amplitudes during their subjective night. Furthermore, melatonin rhythm maintained a stable phase-relationship with the rhythm of locomoter activity of the rats (Pohl and Gibbs, 1978). Thus, melatonin secretion by the pineal gland may play a role in entrainment of rat circadian rhythms. In fact, free-running activity rhythms of rats maintained under constant dim'light can be entrained by daily injection of melatonin only when the time of the daily injection coincides with the onset of activity (Redman et al., 1983). When injections are stopped, the activity rhythm begins free-running again. Thus, melatonin secretion may represent a dusk signal for the activity rhythm. The integrity of the SCN is vital for the expression of circadian rhythms in rodents. Electral stimulation of SCN causes phase-shifts and period changes in the free-running feeding rhythms of rats and activity rhythms of hamsters. Interestingly, the PRC for SCN electrical stimulation appears to parallel that for 32 light pulses (Rusak and Groos, 1982). This evidence is consistent with the hypothesis that the SCN are the dominant light-entrained oscillators and the neural activity in the SCN regulates phase and period of rodent circadian rhythms (Rusak and Groos, 1982) such as melatonin secretion and wheel-running activity. The physiological evidence presented here suggests that the pineal gland has an overt rhythm driven and phase-regulated by the SCN and the light-dark cycles. In such a scheme, it seems reasonable to hypothesize a hierarchical system (Moore-Ede et al., 1976) controlling circadian rhythms in vertebrates. The SCN may function as a pacemaker generating a circadian rhythm of neural activity; this rhythm is entrained by photic signals and regulates the activity of other rhythms through an integrative mechanism. 6.3.2) Seasonal reproductive cycles In 1969, Reiter and Fraschini stated "the profession of the pineal is to regulate reproduction while its avocation is to influence the function of the other endocrine organs"; Regulation of Seasonal reproductive cycles can be manifestated in a number of ways. In the absence of the pineal gland, the sexual organs of male hamsters and rams continue to maintain their size and to produce viable gametes regardless Of the season (Reiter, 1973/74; Lincoln, 1979). Pinealectomized female ferrets and ewes continue to exhibit estrous and anestrous periods (Herbert, 1972; Karsch et al., 1981; Bittman et al., 1983) that are no longer in synchrony with the seasons (Herbert, 1972; Herbert et al., 1978). However, these pinealectomized animals can not be induced into estrus by photoperiod treatment during their natural anestrous periods. Thus, the pineal 33 can no longer be ignored as a potential regulator of reproductive physiology, especially when photoperiod manipulations are part of the experimental paradigm (Reiter, 1980). Under natural conditions, the reproductive organs of hamsters experience a marked waxing and waning depending of the time of the year. The annual reproductive cycle of hamsters has been divided into four phases: the inhibition phase, the sexually quiescent phase, the restoration phase and the sexual active phase (Reiter, 1980). During the inhibition phase, there are at least two primary requirements. First, photoperiod must fall below 12.5 h Of light to lead to reproductive collapse (Hoffman et al., 1965; Gaston and Menaker, 1967: Elliott, 1976). Second, the pineal must be present. In a number of species, pinealectomy blocks the inhibitory effects of short days on gonadal activity (Czyba et al., 1964; Hoffman and Reiter, 1965; Farrar and Carke, 1976; Thorpe and Herbert, 1976; Reiter, 1980). Furthermore, pinealectomized hamsters seem to be incapable of prolonged deep hibernation if their reproductive organs and associated hormones are at summertime levels (Frehn and Liu, 1970; Smit-Vis, 1972). Thus, under short days, a pineal factor somehow manages to suppress reproduction either directly or indirectly, whereas under long days, either the secretory pattern of this factor is altered or there is a change in the target tissue and the hamster grows its gonads (Follett, 1982). During the sexually quiescent phase, hamsters are physiologically and behaviorly incapable of reproduction. The requirements for this phase are the same as for the inhibition phase: the animals must be exposed to short days and the pineal gland must be intact (Reiter, I980). Testicular regression during the quiescent phase can be reversed by either transferring the animals back to long 34 days (Morin et al., 1977; Turek, 1977) or removing the pineal gland of hamsters maintained on short days (Turek and Ellis, 1980). Long-day exposure is often analogous to a physiological pinealectomy. During the restoration phase, the reproductive capacity is restored regardless of the photoperiodic condition of the animals and the regeneration is spontaneous or endogenous. Hamsters with involuted gonads after surgical blinding (Reiter, 1969 1980) or exposure to short days (Frehn and Liu, 1970; Hoffman, 1973; Turek et al., 1975) will spontaneously regenerate their gonads _ after a certain period of dormancy. Testes which have Spontaneously recrudesced remain large for at least 80 weeks on short days (Reiter, 1972). Furthermore, after the reproductive organs attain a fully functional status, they will not regress in response to total light deprivation or short day exposure even after being previously exposed to long days for up to 11 weeks (Reiter, 1980). Finally, the hamsters initiate the sexually active phase of the reproductive cycle. The refractoriness period, initiated during the restoration phase, also extends into the sexually active phase. This seems to negate role for long days in the annual reproductive cycle of hamsters (Reiter, 1980). Nevertheless, a potential role of long days has been acknowledged by Reiter (1972) and Stetson et a1. (1976). Long-day exposure for more than 11 weeks interrupts the summer refractoriness and consequently keeps the various phases of the annual cycle in synchrony with the seasons. The seasonal reproductive cycle is associated with a gamut of endocrine changes all geared to render the animals sexually competent or incompetent. The reader is referred to Turek and Campbell (1979) and Reiter (1980) for a discussion of changes involved in LH, FSH, LHRH, PRL and steroid hormone 35 secretion. Recently, Steger et a1. (1982) reported the temporal alterations in neuroendocrine function during photoperiod-induced testicular atrophy and recrudescence in golden hamsters. In summary, testicular weight fell within 7 weeks of short-day exposures and remained low until 19 weeks when spontaneous recrudescence occurred. Plasma LH, FSH and PRL were undetectable after 7.5 weeks of exposure to short days, but LH and FSH returned to basal levels by week 15 and PRL by week 20.5. Hypothalamic LHRH content increased concurrently with. the fall in LH and FSH which may suggest a decrease in LHRH release. Conversely, increasing gonadotropin levels in plasma and testicular recrudescence were associated with a decrease in hypothalamic LHRH content. Similarly, hypothalamic norepinephrine turnover was low at week 10 but subsequently increased over these values at week 15. The authors concluded that the endocrine changes associated with photoperiod-induced testicular regression and recrudescence were secondary to changes in hypothalamic function. The physiological mechanisms involved in the photosexual response at the . level of the hypothalamo-hypophyseal level is not well documented. A steroid- independent mechanism has been observed in sparrows (Mattocks et al., 1976), mares (Garcia and Ginther, 1976) and rams (Pelletier and Ortavant, 1975) where photic-induced changes in circulating gonadotropins occur in castrated as well as intact animals. At least in sheep, presence of the pineal gland is essential for photic-induced changes during the anestrous period of the seasonal cycle (Karsch et al., 1981; Lincoln, 1979; Lincoln et al., 1982). In addition to the steroid- independent mechanism, a steroid—dependent mechanism has been described in the photoperiodic control of neuroendocrine-gonadal activity of hamsters. Hoffman (1973) hypothesized that in photoperiodic species, sensitivity of the 36 gonadotropin control centers to steroid feedback was altered by photoperiod. Experimental work with the ram (Pelletier and Ortavant, I975), ewe (Legan et al., 1977), hamster (Tamarkin et al., 1976; Turek, 1977; Ellis and Turek, 1979) and quail (Davies et al., 1976) showed more sensitivity to administration of exogenous steroids during non-stimulatory photoperiods than when exposed to stimulatory photoperiods. Furthermore, in hamsters, the spontaneous recrudescence of gonads under non-stimulatory photoperiods involves a spontaneous decrease in steroid feedback sensitivity of the neuroendocrine axis (Ellis et al., 1979). Again, while the pineal gland did not appear to mediate the effects of steroid hormones on pituitary gonadotropin release during exposure to stimulatory long days, it is surely involved with the increased sensitivity of the hypothalamo—pituitary axis to steroid feedback during exposure to non-stimulatory short days in castrated male hamster. In fact, testosterone treatment reduces testes and gonadotropin levels in hamsters with intact pineals but not in pinealectomized animals maintained on 6L:18D (Turek, 1979; Turek and Ellis, 1980). Two general hypothesis could explain these results. First, the pineal gland could be producing a substance(s) during exposure to short days Which directly or indirectly increases the responsiveness to the inhibitory effects of testosterone. On the other hand, the sensitivity of the pineal gland itself could be altered by steroid hormones such as testosterone which may induce the pineal to produce a putative antigonadotropin substance (Turek, 1979). Melatonin can no longer be doubted as an antigonadotropic agent secreted by the pineal gland (Reiter, 1980, 1981). The single most important factor in terms of melatonin injection to mediate its antigonadotrophic actions is the time of injection during the light:dark cycle. For example, in hamsters kept under 37 14L:10D (long days), afternoon but not morning injections inhibit the reproductive system (Tamarkin et al., 1976; Reiter et al., 1976). The changes induced by melatonin injections were as effective as reducing day length to less than 12 h daily. Paradoxically, depots .of melatonin placed under the skin such that the indole is continuously released can actually duplicate the effect of pinealectomy on the reproductive system (Reiter, 1980). Moreover, subcutaneous reserves of melatonin prevent regression of reproductive organs by short days (Hoffman, 1974; Reiter et al., 1974), and also negate the antigonadotropic action of afternoon melatonin injections (Reiter et al., 1977). The seemingly paradoxical influences of melatonin has led Reiter et a1. (1978) to the formulation of the down regulation hypothesis of melatonin action. Presumably, after release, melatonin interacts with its receptors. And as part of this interaction, the receptors become transiently desensitized (down regulated) to additional melatonin (Reiter et al., 1978). Theoretically, this hypothesis may explain the antigonadotropic activity efficacy of late afternoon injections of melatonin because the receptors have regained their sensitivity. On the other hand, morning injections of melatonin after long night is ineffective because the receptors are down-regulated as a result of high melatonin exposure during the previous night. Similar reasoning can be applied to the results obtained when melatonin is continuously available (Reiter, 1981). Thus, melatonin could be the hormone that accounts for the inhibition phase of the annual reproductive cycle in hamster. Once the sexual organs are in the quiescent phase, melatonin injections (Tamarkin et al., 1976), similar to short days, maintain the quiescence of the organs for a period of time. However, “if melatonin injections are continued for 38 an extended period of time (Tamarkin et al., 1976), the gonads eventually recrudesce to their fully mature condition. Once the gonads have regrown, they remain refractory to melatonin injections (Bittman, 1978; Reiter et al., 1979), until the refractoriness is interrupted by exposure of the animals to long days for periods of 10 to 22 weeks. The number of parallels between the responses of hamsters to short days and to melatonin injections strongly suggest a causal relationship to the seasonal cycle of reproduction (Reiter, 1980). However, even if melatonin is found to be the pineal hormone that mediates the seasonal changes in reproductive status, it does not undercut the significance of work to isolate and identify other active pineal factors (Reiter, 1980). These constituents may mediate other subtle daily and seasonal changes in activity rhythms, sleep-wake cycles, hormonal secretion, reproduction, hibernation and so on that are synchronized by the circadian timing system and the light-dark cycle. CHAPTER 1 Effect of blinding on photoperiod and seasonal variations in secretion of prolactin in cattle. 39 INTRODUCTION Secretion of prolactin varies seasonally in cattle (Koprowski and Tucker, 1973; Schams and Reinhardt, 1974; Lacroix et al., 1977), sheep (Pelletier, I973; Ravault, 1976) and goats (Buttle, 1974); prolactin secretion is greatest in summer and lowest in winter. Increasing daily light from 8 to 16 h increases secretion Of prolactin several fold (Bourne and Tucker, 1975; Forbes et al., 1975; Ravault and Ortavant, 1977; Leining et al., 1979). The pathway involved in the transmission of photic signals to the anterior pituitary is not well-known. Photic signals impinging upon the retina of the eyes are transferred over a circuitous connection of neurons to the pineal gland (Moore, 1978; Reiter, 1980) involving retinohypothalamic fibers, suprachiasmatic nuclei and superior cervical ganglia. Light suppresses and darkness increases secretion of melatonin from the pineal gland. In turn, melatonin may affect secretion of the anterior pituitary gland. In fact, pinealectomy or superior cervical ganglionectomy of sheep, goats and cattle abolishes photoperiod-induced increases in secretion of prolactin (Buttle, 1977; Lincoln, 1979; Barrell and Lapwood, 1979; Peters et al., 1979; Brown and Forbes, 1980). Although blinding increases secretion of prolactin into blood of rats (Blask and Reiter, 1975), it is not established if the eyes are essential to mediate photoperiod-induced changes in secretion of prolactin. In the present study, this question was examined in cattle. Seasonal variation in secretion of prolactin has been attributed to changing photoperiods (Hart, 1975; Ravault, 1976; Barrel and Lapwood, 1978/79; Munro et al., 1980). However, ambient temperatures were not cOntrOlled in those studies 40 41 and temperature markedly affects secretion of prolactin in cattle (Tucker and Wettemann, 1976); within hours of changing ambient temperature from 21 to 32°C or from 21 to 4.500, concentrations of prolactin increased 113% or decreased 8096, respectively. Furthermore, ambient temperatures at freezing or below inhibit the ability of 16 h photoperiod to stimulate secretion Of prolactin (Peters and Tucker, 1978; Peters et al., 1980). The last Objective of the present study was to characterize the seasonal variation in secretion of prolactin in blind cattle. MATERIALS AND METHODS Management of Animals and Blood. In experiment 1, animals were housed in ambient temperature and light-controlled chambers as previously described (Leining et al., 1979). They were fed 92 M a complete pelleted diet, alfalfa hay and trace mineralized salt with free access to water. In experiment 2, animals were enclosed in pens outdoors with free access to a covered, three- sided shelter. These animals were fed daily 1 kg of grain concentrate with free access to alfalfa hay, trace mineralized salt and water. In both experiments, blood was collected from a polyvinyl cannula inserted in a jugular vein. Blood samples were allowed to clot for 2 to 6 h at room temperature. Then, samples were kept at 4°C for approximately 24 h before centrifugation at 2000g for 20 to 30 min. Sera were decanted and stored at - 20°C until assayed for prolactin (Koprowski and Tucker, 1971). Experiment 1. The objectives of this experiment were to study the effect of surgical blinding on secretion of prolactin in prepubertal bulls exposed to 8 h of cool-white fluorescent light (L):16 h of dark (D) and to compare the effect of 8L:16D and 16L:8D photoperiods on secretion of prolactin in blind prepubertal bulls. For these purposes, two groups of Holstein bulls (2 to 3 months of age) were exposed for 9 weeks to 8 h of cool-white fluorescent light daily (lights on at 0700 h). At the beginning of week 4, eyes and a narrow rim of tissue containing the eyelashes were removed surgically under general anesthesia induced with sodium thiamylal (Surital, Parke-Davis) and maintained by 2-brOmo-2-chloro—l, 1, 42 43 l trifluoroethane (Halothane, Halocarbon Laboratories, Inc.) and oxygen. Gelfoam (Upjohn Company) was inserted into the orbital cavities and eyelids were sutured together. Between weeks 10 and 15, one group (n=4) was exposed to 16 h of light daily (lights on at 0300 h) while the other group (n=3) was maintained on 8 h of light per day (lights on at 0700 h). At the end Of weeks 3, 9 and 15, concentrations of prolactin were determined in serum collected every 30 min for 24 h. At week 15, following the 24 h sampling period, all animals were injected intravenously with thyrotropin-releasing hormone (TRH, 33 ug/100kg body weight) and blood samples were collected at frequent intervals for 60 additional min. Ambient temperature was recorded each time a blood sample was collected. 1 Experiment 2. The goal of this experiment was to determine seasonal variation in secretion of prolactin in blind and sighted steers. Four blind bulls were randomly selected from animals used in Experiment 1 and matched with sighted bulls of similar age and weight. Two months before beginning the experiment, all animals were castrated. On a single day in October, December, February, April, June and August, a jugular vein of all animals was cannulated at 0700 h. Blood collection started at 1100 h and continued at 30-min intervals for 4 consecutive h. Statistical Analysis. To minimize heterogeneity of variance of PRL means, statistical analyses of serum PRL were conducted on data transformed to natural logarithms. These transformed data were analyzed by least-squares split-plot analysis of variance (Gill and Hafs, 1971). Logarithmic transformation was only for statistical inferences. Means and standard errors reported are untransformed. In Experiment 2, linear regression for ambient temperature and »44 photoperiod were introduced as covariates into the statistical model in an attempt to account for monthly variation in secretion of prolactin. The average temperature during sampling was used as the covariate. RESULTS Experiment 1. Concentrations of prolactin were similar between both groups of bulls averaging 30 (sighted) and 53 (blind) ng/ml of serum at weeks 3 and 9 (Fig. l). The increase in prolactin between weeks 3 and 9 was significant (P< 0.01). Ambient temperatures increased from an average 16.5 and 15.500 in the two chambers at week 3 to 18.2 and l8.6°C at week 9. Using ambient temperatures at weeks 3 (sighted) and 9 (blind) as a covariate, least squares- means of prolactin in serum were similar (P>0.10) in sighted (38 ng/ml) and blind (45 ng/ml) animals (Fig. 2). At week 15, prolactin averaged 66 and 62 ng/ml of serum (P>0.10) in blind bulls exposed to 8L:16D or 16L:8D, respectively (Fig. 1). Blind bulls exposed to 8L:16D or 16L:8D released similar quantities Of prolactin into serum following administration of TRH at week 15 (Fig. 3). There was substantial variation in concentrations of prolactin among samples collected within animal at each week, but there was no obvious diurnal rhythm present (Fig. 1). Experiment 2. At every month tested, concentrations of prolactin were virtually identical in blind and sighted steers averaging overall 27 and 28 ng/ml of serum, respectively (Fig. 4; P>0.10). However, prolactin in sera of blind and sighted steers varied among months (P<0.01) from minimums of 11 and 9 ng/ml in December to maximums of 77 and 96 ng/ml in August, respectively. Split-plot analysis of seasonal variation in concentrations of prolactin in serum of blind and sighted steers is shown in Table I. Introduction of ambient temperature and 45 46 photoperiod as covariates into the model reduced the variance associated with month by 9196; nonetheless, variance associated with month remained significant (P<0.01). Figure 1. 47 Prolactin in sighted or blind bulls (n=7). All bulls were exposed to 8L:16D for 9 weeks. At week 10, one group (n=4) was switched abruptly to 16L:8D for 6 weeks, and the other group (n=3) was maintained on 8L:16D. All bulls were blinded on week 4. Blood was collected during a 24-h interval at the end of weeks 3, 9 and 15. 118 80- SIGHTED, WEEK 3 0—0 8L: I60 60'- D--D 8L3IGD - . ° 40- 00 o. 'o . .0 o . .. . '3 '3 O. I.. .: I .. .3 O. .3 ' . O A O i i i J i l J l J J J L S fiao E \ 8'60 Z:40 '9 .1 20_ 0—0 8L:160 ESE D--D 8L:I60 O J J 4 l l l i i i J J Al 80 60 4o— 0—0 8L3 ISO 20- D--D|6L=80 J i J J J i J i i J l i i 1100 l500 I900 2300 0300 0700 l|00 HOURS 49 Prolactin concentrations in sera of sighted (week 3) and blind (week 9) bulls (n=7) exposed to 8L:16D for 9 weeks. Data are adjusted for ambient temperature variation recorded at 30-min intervals. Figure 2. 50 mmDOI OO_ _ OOmO COMO 00mm _ 023m 00:15 Din. owFIQw 09an 010 00m. 000. 00. _ _ _ _ _ o lo. (wmes Iw/DU) NLLOV‘IOHd Figure 3. 51 Prolactin in blind bulls after iv injection of TRH (33 pg/100 kg body weight) at time 0 during the 15th week of exposure tO 8L:16D (n=3) or 6th week of exposure to 16L:8D (n=4). Pooled SEM were 20.7 and 15.1 ng/ml, respectively. 52 woo l woo 1 N00 I N00 1 _mOI .001 PROLACTIN (ng / ml serum) mo... olo Elmo V DID .mrumo _ __________ _ Iwo 151.010 0 m _0 G No mm wo mo 2:2C._.mm Figure 4. 53 Seasonal variation of prolactin in sighted (n=4) and blind (n=4) steers. Each point re resents the mean of 36 samples. Ambient temperatures C) averaged -2 (Feb), 5 (April), 15 (June), 30 (Aug), 16 (Oct) and 10 (Dec) at the time of sampling. IOO S 80 G) U) 5» 5 so Z |._. U 4 .1 40 0 CE C1. 20 5’4 O—-O SIGHTED D—D BLIND D \ 7. 1 J I 1 J 1 FEB APR JUNE AUG OCT DEC 55 TABLE 1. SPLIT-PILOT ANALYSIS OF SEASONAL VARIATION IN .mNCENTRA- TIONS OF PROLACTIN-INSERA OF BLIND AND SIG'I‘I'ED SI'EERS. Souroel ..d.f. . 14.8. F Blind vs Sighted (Trt) 1 0.304 2.11 Error a 6 0.144 Month 5 4.823 56.67** Photoperiod 1 2.498 28.58** Remainder of Month 4 . 0.975 ll.16** Month x Trt 5 0.075 0.89 Error b 30 0.085 Tenperaturez 1 7.060 80.78** Remainder of Error b 29 0.087 **P < .01 lIndented rows indicated further partitioning for analysis with covariates. 2111s to missing value, temperature was partitioned fran error b instead of month. DISCUSSION Increasing daily light from 8 to 16 h daily markedly increases basal as well as TRH-induced secretion Of prolactin in sighted cattle (Bourne and Tucker, 1975; Leining et al., 1979) and in sham-pinealectomized steers (Peters et al., 1979). However, blinding abolishes (Fig. l and 2) and pinealectomy substantially reduces or abolishes (Peters et al., 1979) the expected increases in basal or TRH-induced release of prolactin. The effect of pinealectomy in cattle supports similar observations made in pinealectomized sheep (Lincoln, 1979; Barrel and Lapwood, 1979; Brown and Forbes, 1980) and superior crevical ganglionectomized goats (Buttle, 1977). Thus, blinding or pinealectomy renders cattle essentially nonphotoperiodic, at least in terms of prolactin secretion. Blinding per se reportedly increases secretion of prolactin in rats (Blask and Reiter, 1975), and we Observed a 7796 increase in concentrations of prolactin 6 weeks after blinding Of prepubertal bulls. Although ambient temperatures in the chambers on the day of bleeding ranged within at 1°C, average temperatures increased 2.400 during the first 6 weeks after blinding. Since increasing ambient temperatures are known to stimulate prolactin secretion in cattle (Tucker and Wettemann, 1976), this could account for the increase in secretion of prolactin after blinding. Indeed, adjusting concentrations of prolactin using ambient temperature as a covariate resulted in similar concentrations in sighted and blind bulls. Furthermore, prolactin concentrations were similar between blind and sighted steers at every month tested throughout the year. Thus, blinding does 56 57 not seem to increase prolactin secretion in cattle. 0n the other hand, the increase between weeks 3 and 9 could have been associated with aging. But, this is doubtful since Lacroix et a1. (1977) did not Observe changes in average concentrations of prolactin in male calves between birth and 12 months. Several authors have suggested that photoperiod is the primary climatic variable that regulates seasonal variation in secretion of prolactin in sheep and goats (Hart, 1975; Ravault, 1976; Ravault and Ortavant, 1977; Barrel and Lapwood, 1978/79; Munro et al., 1980). In the present studies, however, seasonal rhythms in secretion of prolactin persisted in nonphotoperiodic blind steers. Similar seasonal rhythms persisted in pinealectomized steers (Peters et al., 1979). Furthermore, the seasonal variations in concentrations of prolactin in blind and pinealectomized steers were not different from those of their respective controls. Similar seasonal rhythmic patterns in prolactin secretion were observed in sheep rendered nonphotoperiodic by pinealectomy or removal of sympathetic innervation to the pineal gland (Buttle, 1977; Lincoln, 1979; Munro et al., 1980). Persistency of seasonal pattern of secretion of prolactin in these nonphotoperiodic cattle and sheep may reflect a response to another climatic variable associated with changing seasons. Since increasing ambient temperatures increase secretion of prolactin (Tucker and Wettemann, 1976) and decreasing ambient temperatures reduce the ability of 16L:8D to stimulate secretion of prolactin (Peters and Tucker, 1978; Peters et al., 1981), this variable would be the most Obvious candidate. Indeed, in the present study, ambient temperature accounted for a large amount of the seasonal variation in secretion of prolactin (Table 1). However, when the data were adjusted by covariance for variations in temperature and photoperiod, significant variation in monthly 58 secretion of prolactin persisted. Similarly, significant month effects on secretion of prolactin remained in sham-pinealectomized and pinealectomized steers after adjusting for temperature and photoperiod effects (Petitclerc et al., 1983). I believe this constitutes evidence for the existence of an endogenous annual rhythm in the secretion of prolactin in cattle. Alternatively, persistency of seasonal variation 'in prolactin secretion may represent a response to an environmental variable other than ambient temperature or photoperiod. Previously, Koprowski et a1. (1972) Observed diurnal variations in secretion of prolactin in cattle with greatest concentrations at 1600 h and lowest occurring between 0400 and 1000 h. However, in that experiment, lighting was continuous and ambient temperatures were not controlled. In the present study (Exp. 1), when ambient temperatures were restricted to 1- 1°C on the day of blood sampling, there was no diurnal pattern in secretion of prolactin regardless of photoperiod. This contrasts with the sharp increases noted in serum prolactin in sighted sheep when lights are initially turned off (Barrel and Lapwood, 1978; Lincoln, 1979; Ravault and Ortavant, 1977). I conclude that blinding per se does not increase prolactin secretion but abolishes the effects of photoperiod on secretion of prolactin in bulls. Ambient temperature and photoperiod account for most but not all of the seasonal variation in secretion of prolactin in steers. I hypothesize there is an endogenous annual rhythm in secretion of prolactin in cattle. CHAPTER 2 Evidence for a diurnal rhythm of .photosensivity on the regulation of secretion of prolactin in prepubertal bullsl. 1The author of this chapter acknowledges and thanks P.A. Harkins for her work in collecting blood samples and running prolactin assay for the first experiment. 59 INTRODUCTION According to the hypothesis of Bunning (Bunning, 1960), animals measure time from the degree of coincidence between a daily endogenous rhythm and the exogenous rhythm of photoperiod. Pittendrigh and Minis (1964) suggested that light entrains the proposed cycle of endogenous photosensitivity, and if the period of light coincides with the endogenous rhythm of photosensitivity, physiological responses occur. Increasing exposure of calves from a single continuous block of 8 h of light to 16 h of light per day increased concentrations of prolactin in serum 4 to 8 fold (Bourne and Tucker, 1974; Leining et al., 1979). Similar Observations have been made in sheep (Ravault and Ortavant, 1977). Hence, in terms of secretion of prolactin in ruminants, there should be an endogenous daily rhythm in sensitivity to light. Indeed, a photoperiod of 7L:9D:1L:7D [light (L), dark (D)] was as effective as 16L:8D in stimulating secretion of prolactin in adult rams and ewes (Ravault and Ortavant, 1977; Thimonier et al., 1978). Insertion of a pulse of light at other times during the scotoperiod was ineffective in stimulating secretion Of prolactin. Furthermore, in sheep, there is a marked diurnal increase in secretion of prolactin at the beginning of the scotoperiod (Ravault and Ortavant, 1977; Barrel and Lapwood, 1978; Lincoln, 1979). Thus, there is good evidence in sheep of a photosensitive phase for secretion of prolactin. The objective of the present study was to determine if a photosensitive phase for secretion of prolactin occurs in cattle, 8 Species in which there is no Obvious pattern of changes (other than random) in the daily secretion of prolactin (Chapter 1). 60 MATERIALS AND METHODS Management of Animals and Blood. Prepubertal Holstein bulls approximately 6 weeks of age at the beginning of the experiments were housed at 20 i 1°C. Cool-white fluorescent lamps emitting a median of 500 lux at eye level of calves were used. Calves were fed E Elihu—m a complete pelleted ration, alfalfa hay and trace mineralized salt with free access to water. Experiment 1. Two groups of four bulls each were exposed daily to 8L:16D for 6 weeks. Following this control period, one group was subjected to 16L:8D while the other received 6L:8D:2L:8D for 6 additional weeks (through week 12 of experiment). Arbitrary dawn for the 8-, 16- and 6-h intervals of light was 0700 h each day. The 2 h interval of light occurred l4-16 h after arbitrary dawn. Experiment 2. During the first 6 weeks of this experiment, two groups of four bulls were maintained on 8L:16D. After this exposure, one group was exposed to 6L:8D:2L:8D while the other received 6L:14D:2L:2D for 6 additional weeks (through week 12 of experiment). Arbitrary dawn for the 8-and 6-h intervals of light was 0700 h. The 2-h intervals of light occurred 14-16 or 20-22 h after arbitrary dawn. Collection of Blood. Blood was collected from a polyvinyl cannula inserted into a jugular vein approximately 15 h before collection of blood samples. At the end of weeks 6 and 12 in both experiments, animals were bled every 30 min for 24 consecutive h. Thyrotropin-releasing hormone (TRH, 33 jug/100 kg body weight) was administered at 0930—1000 (Experiment I) or at 1100 (Experiment 2) h 61 62 following each 24 h sampling period, and a series of blood samples were collected for an additional 45 (Exp. 1) or 60 (Exp. 2) min. Blood was allowed to clot for 2 to 6 h at approximately 21° C, then stored at 4° C for approximately 24 h before centrifugation at 2000 g for 20 to 30 min. Sera were decanted and stored at -20° C until assayed for prolactin (Koprowski and Tucker, 1971). Statistical AnaLLsis. Analyses were performed on data transformed to natural logarithms to reduce heterogeneity of variance Of prolactin means. The transformed data were analysed as a double split-plot analysis (Gill and Hafs, 1971). Means and standard errors presented are not transformed. RESULTS Experiment 1. At the end of 6 weeks of 8L:16D, basal prolactin was similar in both groups of calves averaging 8.3 and 8.5 ng/ml of serum (Fig. 5). Following 6 weeks of 16L:8D or 6L:8D:2L:8D, basal prolactin increased (P<0.01) to averages of 42.0 and 37.3 ng/ml of serum, respectively. These latter concentrations were not different from each other (P>0.10). There was no apparent diurnal pattern of change in secretion of prolactin. At week 6, prolactin averaged 11.8 ng/ml during intervals of light and 12.4 ng/ml during intervals of dark. At week 12, prolactin averaged 43.7 and 52.6 ng/ml during light, and 38.5 and 47.2 ng/ml during dark for animals exposed to 16L:8D and 6L:8D:2L:8D, respectively. There were no differences in concentrations of prolactin during light versus dark periods (P>OJOL Greater quantities of prolactin were released following TRH when bulls were exposed to 16L:8D or 6L:8D:2L:8D then when exposed to 8L:16D (Fig. 6; P<0.05). However, neither peak height nor shape of the response curve following TRH differed between the two groups of bulls within weeks 6 or 12 (P>0.10). Experiment 2. Basal prolactin averaged 11.4 and 13.0 ng/ml of serum after 6 weeks of 8L:16D in the two groups of bulls (Fig. 7). Subsequently, prolactin increased (P<0.05) to averages of 49.0 and 22.5 ng/ml following 6 weeks of 6L:8D:2L:8D or 6L:14D:2L:2D, respectively. Concentration of prolactin in bulls given 6L:8D:2L:8D was greater (P<0.05) than for bulls maintained on 6L:14D:2L:2D. Diurnal variation in secretion of prolactin was not apparent. 63 64 Prolactin averaged 8.8 ng/ml during light periods and 8.2 ng/ml during dark periods at week 6. At week 12, prolactin averaged 38.7 and 23.7 ng/ml during the light periods, and 36.7 and 22.2 ng/ml during dark periods for animals under 6L:8D:2L:2D and 6L:14D:2L:2D, respectively. Concentrations of prolactin during light periods did not differ from concentrations during dark periods in any treatment group (P >0.10). TRH-induced release of prolactin was greater (P<0.05) in bulls given 6L:8D:2L:8D (4.9 fold) or 6L:14D:2L:2D (2.5 fold) than in bulls given 8L:16D (Fig. 8). Release of prolactin after TRH tended to be greater (P<0.10) in bulls given 6L:8D:2L:8D than in bulls exposed to 6L:14D:2L:2D. Figure 5. 65 Basal prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (open symbols), then switched to 16L:8D or 6L:8D:2L:8D for 6 additional weeks (closed symbols). Onset of lights during the primary period of lighting was 0700 h. Pooled SE of means during weeks 6 and 12 were 0.4 and 3.3 ng/ml, respectively. 3.85 02.... w OONO Omvo OONO Ommm OO_N 0mm. 000. 0mm. 00: OOmO _ _ q _ a _ _ . . O O~«m 4 O O O O O m.u4«\O.«. O 4 O OOOtOfirO O 4 O I o .- I 4 . a4O «4 .O4J4anOag4 «.O;’OO4I\O4 O44t4oolO444OO4OIO_ . 4 ‘O4. 0 C 4 ‘ O. 4 C { 4 JON ”/4 \4/4. 4/4 4 \/\v« 44 3...]. On .(4 4/ \4\/4 444/44‘.44/O¢ J 4 4 \ l 4 4/ 4 />\ K «\«4 4.0m, 4/ 4 4 1 4 4 4 I ION 10m omfim. 4114 I omfimnomfim III. 10m 093m 414 " 0.10 am. .5 too. (WW/9N) 'ltid Figure 6. 67 TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (open symbols), then switched to 16L:8D or 6L:8D:2L:8D for 6 additional weeks (closed symbols). TRH was injected at time 0 (0930 to 1000 h). Onset of lights during the primary period of lighting was 0700 h. Pooled SE of mean concentrations of prolactin measured between ~15-0, 4-15 and 20-45 min appear above response curves. AmmSEEV m2; omflm. 4IIu4 / omflmuom “.5 ell. A 8.16 olo o mum—“4m Clio ON— o ‘1' (WW/9N) 'IHd o 2‘: 00. cm. OON Figure 7. 69 Basal prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (open symbols), then switched to 6L:BD:2L:8D or 6L:14D:2L:2D for 6 additional weeks (closed symbols). Onset of lights during the primary period of lighting was 0700 h. Pooled SE during week 6 was 0.9 ng/ml; during week 12, pooled SE for 6L:14D:2L:2D and 6L:8D:2L:8D were 4.6 and 5.8 ng/ml, respectively. 3.505 m2; 000. OOmO Om _ mO OOMO OMOO OONN 0mm. OON. Om: OON. _ _ . _ . _ _ _ C omfimuom Lo 4|...4 4 QNDNOSLO I 8.15 ollo 093m OIIO O 9 ON on 00 On OO Ox. Om IOm 1 OO_ (1W/9N)"|I:id Figure 8. 71 TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D (Open symbols), then switched to 6L:8D:2L:8D or 6L:14D:2L:2D for 6 additional weeks (closed symbols). TRH was injected at time 0 (1100 h). Onset of lights during the primary period of lighting was 0700 h. Pooled SE Of mean concentrations of prolactin measured between -30-0, 5- 15 and 20-60 min appear above response curves. 323580 22:. m m- 9.. mm- fl mm mm 00 mm mm m. _ omimnom Jo 4||4 omjmuoifim YII. ominm <14 007.6 o...|o l I l O O O 91 9 0° (WW/0N) 188 1 O S. 100_ 100. 1 00m DISCUSSION Results of this study confirm previous reports that increasing duration of daily light from 8 to 16 h increases basal secretion and TRH-induced release of prolactin several fold in ruminants (Bourne and Tucker, 1975; Ravault and Ortavant, 1977; Leining et al., 1979). In addition, I have now established that light does not need to be supplied as a continuous block of 16 h since 6L:8D:2L:8D is as effective as 16L:8D in stimulating secretion of prolactin. This constitutes evidence that bull calves, similar to sheep (Ravault and Ortavant, I977; Thimonier et al., 1978), possess a diurnal rhythm of sensitivity in terms of prolactin response to light. However, in cattle, this diurnal rhythm in photosensitivity appears to be unrelated to actual diurnal change in secretion of prolactin since they do not express any diurnal pattern of change in secretion of prolactin. In contrast, definitive increases in serum prolactin occur in sheep when lights are initially turned off (Ravault and Ortavant, 1977; Barrel and Lapwood, 1978; Lincoln, 1979). The period of photosensitivity in adult sheep is a discrete period between 16 and 17 h after dawn (Ravault and Ortavant, 1977; Thimonier et al., 1978). In contrast, results of the present study provide evidence that the period of photosensitivity for secretion of prolactin in prepubertal cattle extends at least from 14 to 22 h after dawn. It should be noted, however, that insertion of a block of light between hours 20 and 22 after arbitrary dawn is much less effective in stimulating secretion of prolactin than insertion of light 14 to 16 h after dawn. I 73 74 speculate that the cycle of photosensitivity is waning by 20 to 22 h after arbitrary dawn. Ravault et a1. (1981) presented evidence in sheep that the phase of photosensitivity for secretion of prolactin may be related to onset of darkness, not to dawn. They Observed greatest secretion of prolactin when a 1-h pulse of light was inserted 9 h after onset of darkness. Similarly, I Obtained a greater increase in secretion of prolactin in bulls when a 2-h pulse of light was given 8 h _ as compared with 14 h after onset of dark. According to this concept, duration of uninterrupted dark rather than light is the critical feature of a day in photoperiodic phenomena (Pittendrigh and Minis, 1964). Additional studies will be required to determine the precise location of the photosensitive phase of secretion of prolactin in cattle. CHAPTER 3 Diurnal pattern of prolactin release in prepubertal bulls exposed to short- or long-day photoperiods 75 INTRODUCTION Photoperiodic stimulation of physiological phenomena Often takes many weeks or months before noticeable changes are observed. In cattle, 6 to 8 weeks elapse before significant increases in growth rate can be detected in animals shifted from 8 to 16 h of light daily (Peters et al., 1980); similarly, it takes 7 to 10 days before significant increases are Observed in basal concentrations Of prolactin (PRL) (Leining et al., 1979; Stanisiewski et al., 1983). In birds, on the other hand, photoperiodic stimulation of luteinizing hormone (LH) secretion is established very rapidly (Follett et al., 1977). On the very first day following a shift from 8 h of light (L):16 h of dark (D) to 20L:4D, a significant increase in concentrations of LH is detectable 20 to 22 h after onset of light. This increased concentration of LH is maintained until another rise occurs on day 2. Successive increases occur for several days. Soon, however, these incremental changes are lost and gonadotropin secretion remains consistently high (Follett, 1978). In cattle, the early temporal changes in PRL secretion after a sudden change in photoperiod have not been studied. Overt stimulation of body functions by photoperiod is probably the result of modification in neuroendocrine events set in motion by some photoperiod- measuring device or biological clock. The hypothesis that photoperiodic-time measurement is based on an endogenous circadian rhythm of photosensitivity was advanced by Bunning (1960). Later, Pittendrigh and Minis (1964) suggested that light entrains the proposed cycle of endogenous photosensitivity, and if light 76 77 coincides with the period of photosensitivity, a physiological response occurs. In fact, a skeleton photoperiod of 6L:8D:2L:8D is as effective as 16L:8D in stimulating PRL secretion in cattle (Chapter 2). Similar Observations have been made in sheep (Ravault and Ortavant, 1977). This diurnal rhythm of photosensitivity appears to be unrelated to an overt diurnal rhythm in secretion of PRL since cattle maintained for prolonged periods on a repeating photoperiod do not express diurnal rhythmicity (Chapter 2). In contrast, definitive increases in serum PRL occur in sheep when lights are initially turned Off (Barrel and Lapwood, 1978; Lincoln, 1979). In this present research, I have examined early temporal changes in PRL secretion after an abrupt change in photoperiod and diurnal responsiveness of animals given a PRL-releasing or inhibiting factor when exposed to different photoperiods. MATERIALS AND METHODS Management of Animals and Blood. In each experiment, prepubertal Holstein bull calves (1-3 days of age) were housed in a light-controlled chamber for 6 to 8 weeks and exposed to 8L:16D photoperiod (lights on at 0700 h). Calves were individually penned in a 1.2 m wide X 1.8 m long stall. Cool-white fluOrescent lamps emitting a median of 210 lux at eye level of the calves were used. All calves were weaned from milk at approximately 6 weeks of age. After weaning, groups of four bulls each were relocated into one of two temperature- and light-controlled chambers as previously described (Leining et al., 1979). They were fed a_d_ liiitfl a complete pelleted diet, alfalfa hay and trace mineralized salt with free access to water. Blood was collected from a polyvinyl cannula inserted in a jugular vein 1 day prior to collection. Blood samples were allowed to clot for 2 to 6 h at room temperature; then, they were kept at 4° C for approximately 24 h before centrifugation at 2000g for 20 to 30 min. Sera were decanted and stored at -20° C until assayed for PRL (Koprowski and Tucker, 1971). Experiment 1. The Objective of this experiment was to study early diurnal changes in secretion of PRL following an abrupt change in photoperiod. For this purpose, two groups of bulls (n = 4) were exposed for 7 weeks to 8 or 16 h of cool- white fluOrescent light daily (lights on at 0800 h). Then, on a single day, the light period was shifted from 8 to 16 or from 16 to 8 h of light per day. For each group, these new photoperiods were maintained for four consecutive days. Collection of blood started at 1500 h the day before changes in photoperiod were 78 79 initiated (day 0). Samples were taken at 30-min intervals for 35 consecutive h until 0200 h of day 1. Blood collection resumed at 30—min intervals between 2000 and 0200 h on days 2 and 4. Experiment 2. In this trial, we measured concentrations of PRL after injections of thyrotropin-releasing hormone (TRH) at two times of the day in animals exposed for prolonged periods to 16L:8D. To accomplish this goal, seven bull calves were exposed to 16L:8D for 6 weeks (lights on at 0300 h). Then, on a single day, all animals received intravenously TRH (33 pg/100 kg body weight) 3 and 15 h after onset of lights (0600 and 1800 h). Blood samples were collected 120, 90, 60, 30, 15, 5 min and just before injection, and at 4, 6, 8, 10, 15, 20, 25, 30, 45 and 60 min after injection of TRH. E_xperiment 3. In view of results obtained in Experiment 2, we decided to investigate further the diurnal variation of TRH-induced release of PRL in bull calves. Two groups of four animals each were subjected to either 8 or 16 h of light daily (lights on at 0800 h) for 6 weeks. Then, each calf received on a single day of four consecutive days a single rapid intravenous injection Of a sub- maximal dose of TRH (16.5 ug/100 kg body weight) 3, 9,15 and 21 h after Onset of lights. A 4 X 4 Latin square design balanced for residual effects (Gill, 1978) was used such that, on any given day, each of the four injection times were tested but each, calf received TRH only once a day, and one day was allowed between each injection. During each challenge, blood samples were collected 30, 15, 10, 5 min and just prior to injection, and at 4, 6, 8, 10, 15, 20, 25, 30, 45 and 60 min after' injection of TRH. 80 Experiment 4. In this experiment, we studied the effects of L- dihydrophenylalanine (L-DOPA), the immediate precursor of dopamine, on PRL release at two different times of the day in animals exposed to 16L:8D. L-DOPA was used at a dose of 1.5 mg/100 kg body weight. This sub-maximal dose was determined in a preliminary trial to suppress basal PRL levels 20 to 40%. After 6 weeks exposure to 16L:8D (lights on at 0300 h), on a single day, all animals (n = 8) received intravenously L-DOPA 3 and 15 h after onset of lights (0600 and 1800 h). Samples of blood were collected as described in Experiment 2 plus one additional time 10 min before and three additional times 90, 120 and 180 min after L-DOPA injection. Statistical Analysis. Analyses were performed on data transformed to natural logarithms to reduce heterogeneity of variance of PRL means. The transformed data were analyzed by split-plot analysis (Gill and Hafs, 1971) in all experiments. Least-squares means reported, however, are untransformed. In Experiments 2 to 4, the area under the response curve of PRL was estimated according to the following calculations (Vines et al., 1976). Briefly, the average ' baseline concentration (ng/ml) of PRL prior to TRH or L-DOPA was calculated for each animal each time a challenge was performed. This value was subtracted within animal from subsequent hormone concentrations measured at each post- treatment sampling time. Adjusted values (ng/ml) and their respective times (min) were fitted to a three-degree least-squares polynomial curve. The area under this PRL response curve was integrated and expressed in ng ml.1 min. These areas were subjected to analysis of variance. Differences among least- squares means were tested by the Bonferroni procedure (Gill, 1978). RESULTS Experiment 1. Beginning at 1500 h on day 0 and extending through 1600 h on day l, PRL averaged 22.0 and 36.4 ng/ml of serum (P<0.05) in animals exposed to 8 or 16 h of light daily, respectively (Fig. 9). Beginning at 1630 h on day 1 (initial time when both groups were first exposed to a different photoperiod) and extending through 0200 h, there was no observable change in mean concentrations of PRL in animals switched from 8L:16D to 16L:8D (Fig. 9). Moreover, concentrations of PRL did not change in this group (P>0.05) through day 4 (Fig. 10) even though there seemed to be a greater pulsatile. activity in these animals during that last day. These data are in sharp contrast to those obtained in animals switched from 16 to 8 h of light daily where during the dark period on day 1 (Fig. 9), PRL decreased (P<0.05) from 50.1 ng/ml (1630 h) to a minimum of 21.6 ng/ml (2100 h) and then increased (P<0.01) to a maximum of 57.3 ng/ml (0200 h). The diurnal pattern in the secretory profile of PRL secretion reoccurred on days 2 and 4 (Fig. 10). PRL reached peak values of 57.3, 62.7 and 78.9 at 0200, 0030 and 0100 h, on days I, 2 and 4, respectively; these values were consistently greater (P<0.01) than respective values on day 0. However, PRL concentrations between 2000 and 0200 h averaged 33.4, 35.5, 37.2 and 41.2 ng/ml on days 0, l, 2 and 4, respectively (P>0.10). Experiment 2. Peak PRL concentrations attained after TRH injections were significantly greater (P<0.001) when performed 3 h after dawn (268.9 ng/ml) 81 82 than at 15 h after onset of lights (149.1 ng/ml) in calves exposed to 16L:8D (Fig. 11). Similarly, area under the curve Of PRL was somewhat greater at 3 versus 15 h after dawn (data not shown). Experiment 3. A greater amount (P<0.01) of PRL (peak and area under the curve) was released 3 h after dawn in animals exposed to 16L:8D as compared with release at 9, 15 and 21 h after onset of lights (Fig. 12, Table 2). However, TRH-induced release of PRL did not vary diurnally in animals exposed to 8L:16D (Fig. 13, Table 2). At every time tested during the day, TRH-induced release of PRL was greater (P<0.01) for animals subjected to 16 as compared with 8 h of light daily (Fig. 12-13, Table 2). Experiment 4. There was no significant (P>0.25) difference in the ability of L-DOPA to inhibit PRL secretion (P<0.05) at 3 or 15 h after dawn as indicated by area under the curve of PRL in calves exposed to 16L:8D (Fig. 14). Maximal inhibition (26.2 and 28.3%) was similar (P>0.25) in both treatments and occurred 6 min after injection of L-DOPA. Figure 9. 83 Basal prolactin in serum of prepubertal bulls after an abrupt change from 8L:16D to 16L:8D (Open circles) or 16L:8D to 8L:16D (open squares). Initial time when both groups were first exposed to a different photoperiod was 1630 h of day 1. Open horizontal bars indicate day light times and closed bars night times. Onset of lights was 0800 h. é:: ms:h TI 1 20 "x 0 >40 I.” .OOVN OOON OOO_ CON. Como OOVO OO§N OOON COO. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 0 Ii 0'0 Omen—9 n 010 0911.0 10. O _ u . . . - O O. 0 ll... .00... O OOOOOOIO O _ O 00 Gig ON .0. "H. II 0 u 0.. O O I Oil 0 I _O O m w 10M .00 O :- nwD flu DD 0 _ ( loo “ Kuuo _ 10m aluomtnm __ olnomfimy '(wmes |LlJ/OU) NIlOV‘IOézId 85 Figure 10. Basal prolactin in serum of prepubertal bulls between 2000 and 0200 h on the day prior (day 0) and l, 2 and 4 days after switching photoperiod from 8L:16D to 16L:8D (open circles) or 16L:8D to 8L:16D (open squares). Open horizontal bars indicate day light times and closed bars night times. Onset of lights was 0800 h. CE 0.2:. OONO OOVN OONN OOON OONO OOVN OONN OOON O _ — q — ‘ u _ _ — _ _ q q l 1. Iii. ill .U olo omgm. - olo 81.6. - o. l 1 ON IU/O 1 0/ IR D\CI0 J on U \ 4 1 CV \Q\Q\D |\1\ KNOW _ L - on 01009.00 0.1.0 007.00 — _ q i— q — — — 4 _ _ d d O I J olo om ..m. olo co. ..m .o. é . 8 D \< / \UID \ l on 010/0\0/010\ 0 / \Rox - ob - oo _ >40 0 >40 0x010 .mx R00 010 007.6 0 oulc om. HQ Hos I.I.IIII.I ,Iil....il 1..-.1U (uumas IUJ/bu) NIiDV‘IOtId 87 Figure 11. TRH-induced release Of prolactin in serum of prepubertal bulls exposed for 6 weeks to 16L:8D. TRH was injected (time 0) 3 (Open triangles) and 15 h (open circles) after dawn. Onset of lights was 0300 h. 1.2502: om om oo om om o. O 001 00. .li _ _ _ _ Esau 12.6 E m. 01.0 :38 .26 E m 4114 ____ON 89 Figure 12. TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 16L:8D. TRH was injected (time 0) 3 (dash line) 9 (solid line), 15 (dotted line) and 21 h (dashed- dotted line) after dawn. Onset of lights was 0800 h. PROLACTIN (ng /ml serum) 240 220 200 |80 |60 I40 |20 100 m 0 O) O .15 O N O O l6L38D ..‘m‘fi’vlfiar-Vfw'iii - 1 TRH l l I l J l l l l l _J_J -3O -|5 '50 IO 20 3O 4O 50 60 TIME (min.) 91 Figure 13. TRH-induced release of prolactin in serum of prepubertal bulls exposed for 6 weeks to 8L:16D. TRH was injected (time 0) 3 (dash line) 9 (solid line), 15 (dotted line) and 21 h (dashed- dotted line) after dawn. Onset of lights was 0800 h. 1 O 9 BLIIBD l l l l O O O 0 CD £0 <1' (\I (wmas Iw/fw) NLLDV‘IOHd -30 TIME (min) 93 Figure 14. L-DOPA-induced inhibition of prolaction release in serum Of prepubertal bulls ex osed for 6 weeks to 16L:8D. L-DOPA was injected (time 0) 3 open circles) and 15 h (open triangles) after dawn. Onset of lights was 0300 h. 2.25 02; 09 00. 0: ON. . Om ON 0_ O 00.. 00.. ONT. _ _ _ 3T_ _ _ 14.4% _ _ om <¢OQIJ I00 .0 O. H HO O O 44 ..O. O O O <\<<\ /OO 10.? W O \q u <\ / O \ «/ O u \ O \4/ 1 «a. / Jon. W O \O/ 4’ / U \ O 4/ D r // / \ / \ 4 <00 w . A“ « 55% .25 z m. 4|..4 mo. \ \ E8125 in I ..o» m \ \ ( 4x 95 TABLE 2. AREA UNDER THE (CURVE (ng m1‘1 min) OF PROLACTIN RELEASE AFTER TRH INJECTION (16.5 ug/lOO kg body weight) 3, 9, 15, and 21 h AFTER DAWN m CALVES EXPOSED .TO 8L:16D PHOIOPERIODS. Photoperiod . Time of injection (h) 3 9 15 21 8L:16D 2185 ‘ 2109 1914 1750 16L: 8D 5718 . 3906 4173 . 3864 DISCUSSION Recently, a diurnal rhythm of photosensitivity in PRL secretion was reported in cattle (Petitclerc et al., 1983; Chapter 2). Insertion of a daily pulse of light 14 to 16 h after dawn (6L:8D:2L:8D) was as effective in stimulating PRL secretion as a continuous block Of 16 h of light (16L:8D). In the present study, a clear diurnal pattern of PRL release was observed between 14 and 18 h after dawn (2200 and 0200 h) in animals switched from 16 to 8 h of light daily; this release of PRL coincides with the photosensitive period of PRL secretion. However, this effect was not Observed in animals switched from 8 to 16 h of light daily. Thus, it was the absence of light which unmasked the photosensitive period of PRL secretion in animals previously exposed to 16L:8D. The relative sluggishness of photoperiodic stimulation of PRL secretion, as Observed from biweekly blood sampling schedule (Leining et al., 1979; Stanisiewski et al., 1983), is again confirmed in the present study. After 4 days, of exposure from 8 to 16 h of light daily, there was still no significant increase in basal PRL concentrations even though the secretory profile of PRL showed enhanced pulsatile activity on the fourth day. Nevertheless, basal PRL concentrations would be expected to increase within 7 to 10 days and attain maximal values after 3 to 6 weeks exposure to 16L:8D (Leining et al., 1979; Stanisiewski et al., 1983). These data on PRL secretion in cattle are in sharp contrast with results obtained in birds concerning photoperiodic stimulation of LH secretion (Follett et al., 1977). On the very first day after switching from 96 97 8L:16D to 20L:4D, there is a significant increase in LH concentrations between 20 and 22 h after dawn. But, as pointed by Follett (1978), birds in contrast with mammals do not require participation Of the pineal gland for transmission of the photic signal to the pituitary gland. Thus, in mammals, changes in photoperiod might involve alterations in the secretory activity of the pineal gland first; then, the pineal gland relays the photic signal to the pituitary gland and thereby alters secretion of PRL. Indeed, pinealectomized cattle and sheep are unable to respond to changes in photoperiod (Bittman et al., 1983; Petitclerc et al., 1983). Such a hierarchical system with one more step, the pineal gland, in the transmission of photic signal to the pituitary might explain the relative slowness of the mammalian system. For the first time in cattle, I report that L-DOPA inhibits PRL secretion, but this effect did not vary diurnally in animals exposed to 16L:8D. TRH is known to stimulate release of PRL in cattle (Vines et al., 1976; Convey et al., 1973). However, the effect of a standard dose of TRH on PRL release is markedly enhanced when cattle are exposed to 16L:8D or 6L:8D:2L:8D as compared with 8L:16D (Leining et al., 1979; Chapter 2). In the present study, I have confirmed these observations. Furthermore, I Observed a diurnal rhythm of responsiveness to PRL release after TRH challenge in animals exposed to 16L:8D but not in animals given 8L:16D; a greater release was obtained at 3 h than at 9, 15 and 21 h after dawn. Recently, Ravault et al. (1981) presented in sheep evidence in favor Of a dusk hypothesis for photoperiodic control of PRL secretion. They observed greatest secretion of PRL when a l-h pulse of light was inserted 9 to 10 h after onset of darkness. In this model, the phase of photosensitive period for PRL secretion was related to dusk, not to dawn. In our 98 experiments concerning diurnal variation of TRH-induced release of PRL, greatest responsiveness to TRH occured 3 h after dawn or the equivalent of 11 h after dusk in animals exposed to 16L:8D. Thus, in terms of a dawn hypothesis, maximal release of PRL after TRH challenge did not coincide with the photosensitive period which occurs 14 to 16 h after dawn; but, in terms of the dusk hypothesis, maximal release occurred very close to the photosensitive period as postulated to be present 9 to 10 h after onset of dark. Consequently, I believe these results are evidence in favor of a dusk hypothesis for photoperiodic control of PRL secretion. Concerning animals exposed to 8L:16D, TRH challenges occurred 1, 7, l3 and 19 h after dusk, respectively; thus, none of these challenges were close enough to the photosensitive phase to produce maximal release of PRL after TRH. The mechanism implicated for the enhanced TRH-induced release of PRL under 16L:8D and its diurnal rhymicity is not known. Diurnal secretions from the pineal gland could modulate these effects at the pituitary gland or hypothalamus levels. In fact, administration of an adrenergic receptor blocker (Gala et al., 1978) or sectioning of the pituitary stalk (Norman et al., 1980) results in a potentiation of TRH-induced release of PRL. Therefore, a decrease in dopamine input on the pituitary, a known prolactin-inhibiting factor (Weiner and Ganong, 1978), could account for increased secretion and enhanced TRH-induced release of PRL in animals exposed to 16L:8D photoperiod. On the other hand, ,a greater amount of TRH reaching the pituitary gland, an increased number of TRH receptors or a greater content of PRL in the pituitary gland could also explain these results. Clearly, more research will be needed to understand the neuroendocrine events involved in the control of PRL secretion by photoperiod. 99 Chronic injections of TRH cause release of PRL, growth hormone and thyrotropin, and increase growth rate of wether lambs and dairy heifers (Davis et al., 1976, 1977; McGuffey et al., 1977). In view of the effect of photoperiod and time of day on TRH-induced release of PRL, these factors should probably be considered in the use of TRH as a growth-promoting factor. CHAPTER 4 Body growth, growth hormone, prolactin and puberty responses to photoperiod and plane of nutrition in Holstein heifers. 100 INTRODUCTION A daily photoperiod of 16 h of light (L) and 8 h of dark (D) applied for 4 months increased body growth of heifers 11 to 17% over that of heifers exposed to natural duration photoperiods of 9 to 12 h daily (Peters et al., 1978), 8L:16D or 24L:0D (Peters et al., 1980). Although heifers exposed to 16L:8D eat more, their efficiency in converting feed into body mass is greater than heifers given less than 12 h of light daily. In these previous experiments, animals were fed ad libitum a high plane of nutrition. However, no information is available concerning the effects of photoperiod on heifers fed a lower plane of nutrition. Thus, our first objective was to determine if 8 or 16 h of light per day affected growth rate of heifers fed high or low planes of nutrition. Prolactin (PRL) and growth hormone (GH) may control body growth (Bates et al., 1964; Purchas et al., 1971). Serum PRL increases three to seven-fold in calves when duration of light is shifted from 8 to 16 h per day (Bourne and Tucker, 1975; Leining et al., 1979). Furthermore, concentrations of serum PRL increase and GH decrease as energy intake and body growth rate increase (Bassett et al., 1971; Forbes et al., 1975, 1979a; Sejrsen et al., 1983). Therefore, our second Objective was to study the effects of photoperiod and plane of nutrition on concentrations of PRL and GH in serum. . PRL appears to participate in the maturational process that leads to onset of estrous cyclicity (Ojeda et al., 1980). Hyperprolactinemia (Advis and Ojeda, 1978) or injection of PRL into the median eminence (Clemens et al., 1969) leads to precocious puberty in the female rat. Puberty is also dependent on plane of 101 102 nutrition. For example, faster growth rates induce earlier onset of estrous cyclicity in cattle (Swanson, 1978). However, a positive relationship exists between average daily gain and body weight at first estrus (Swanson, 1978). Considering the effects of photoperiod on growth rate and prolactin secretion, our last goal was to determine if photoperiod and plane of nutrition affected body weight at puberty. MATERIALS AND METHODS Management of Animals and Blood. Sixty prepubertal Holstein heifers were assigned on the basis of body weight (average 156 kg at beginning of experiment) to one of four treatment groups arranged as a 2 X 2 factorial. Heifers were housed unrestrained in separate pens and no supplemental heat was provided. Main effects were photoperiod and plane of nutrition. Photoperiods were 8L:16D or 16L:8D. Lights came on at 0700 h each day in all groups. Planes of nutrition were designed to produce average body weight gains Of approximately 0.7 (LOW) or > I kg/day (HIGH). Diets for both planes of nutrition contained the same ingredients but differed in composition (table 3). Each diet was fed as a complete mix once a day at 1300 h. HIGH groups of heifers were fed _a_q 119131311 a ration greater in protein and energy content than LOW heifers. Total orts/group for heifers on the HIGH plane of nutrition were weighed daily. Daily feed intake per group of LOW heifers was restricted with no orts and was similar for heifers subjected to either 8 or 16 h of light daily; average dry matter intake per heifer ranged from 3.99 to 4.73 kg/day during the experiment. The experiment started on January 25 and the first month of the trial was used to adjust feed intake to the desired growth rate for LOW heifers. Water was freely available. Approximately every month, heifers were deprived of water for 16 h, then body weight was measured between 0800 and 1000 h. The last body weight for the growth trial was measured for all heifers on June 12 after 138 days. 103 104 TABLE3. FEEDCINPOSITIONAMDJNGREDIENTSINTHEDIEI'FOREAQi PLANE OF NUTRITION . Plane of nutrition Item Low High Canposition Dry matter (is) 32.5 40.3 Protein (5%) 12.5 13.5 Energy (Ma-a1 ME/kg) 2.59 2.80 Ingredients (75, DM basis) Alfalfa-brute haylage (IFN 3-08—147) 9.7 9.7 Corn silage (IFN 3—28-250) 73.8 44.9 High moisture corn (lFN—4-20-770) 6.9 33.9 Supplement (42% crude protein) 9.7 11.6 105 On February 2, March 18 and June 5, an indwelling cannula was inserted into a jugular vein of five heifers initially selected at random from each treatment group. The same heifers were bled each time. The following day, starting at 0700 h, blood samples were collected at 30-min intervals and discarded until 1000 h to condition animals to the sampling procedure. Thereafter, 10 ml of blood was collected at 30-min intervals for 6 consecutive h. Sufficient feed was provided to all groups on the day before collection of blood, so that feed was continuously present during collection. On the day of blood collection, feeding was delayed until after all blood samples were collected. Average ambient temperatures during collection of blood in February, March and June were 4.3, 8.6 and 22.3 C, respectively. Blood samples were allowed to clot for 2 to 6 h at 20 C, then stored overnight at 4 C. The following day, sera were obtained by centrifugation at 2000g for 30 min. Sera were decanted and stored at 20° C until assayed for PRL (Koprowski and Tucker, 1971) and GH (Purchas et - al., 1970). Starting at 205 kg body weight, progesterone concentration in serum from a single sample of blood from the tail vessel was monitored biweekly as an index of onset of estrous cyclicity (puberty) in all heifers. Concentrations of progesterone greater than 1 ng/ml indicated the existence of a functional corpus luteum. Body weight at puberty was extrapolated from the regression of the body growth curve of each animal. Animals (26 of 60) that had not reached puberty by the end of growth trial (day 138) were maintained under photoperiod and nutritional treatments until detection of puberty in all heifers. Measurements of body weights of these prepubertal heifers were continued at monthly intervals until detection of puberty. 106 Statistical Anaysis. Body weight, serum PRL and GH changes were analyzed by split-plot analyses of variance (Gill and Hafs, 1971). Body weight on day 28 was used as a covariate in the analysis of body weight changes during the remainder of the experiment. To minimize heterogeneity of variance of PRL and GH means, statistical analyses were conducted on data transformed to natural logarithm. Least-square means were tested by Bonferroni procedure (Gill, 1978). Means for monthly dry matter intake/100 kg body weight of heifers on the HIGH plane of nutrition were calculated from monthly total intake and average body weight per group. Body weight at puberty and days on experiment to reach puberty were subjected to analyses of variance. RESULTS Weight Gain. Body weight Of all heifers averaged 186 kg after 28 days on the experiment (February 22). Body weights increased to 252, 268, 296 and 308 kg on day 138 (June 12) for 8L:16D, LOW; 16L:8D, LOW; 8L:16D, HIGH; and 16L:8D, HIGH heifers, respectively (figure 15). Average daily gains (ADG) of heifers subjected to 16L:8D were 18 and 10% greater (P<0.001) than. those of heifers exposed to 8L:16D in the LOW and HIGH groups, respectively. There was no photoperiod by plane of nutrition interaction (P>0.10). Dpv Matter Intake. Statistical comparisons of dry matter intake between photoperiod or nutritional treatments were not possible because animals were fed in groups, not individually. Average daily dry matter intakes throughout the experiment, expressed in percentage of body weight for heifers exposed to either 8L:16D or 16L:8D were 1.94 and 1.86% for LOW heifers, and 2.76 and 2.85% for HIGH heifers (table 4). During the last 3 months of the experiment, animals exposed to 16L:8D and on HIGH nutrition consumed 6.7% more dry matter/d than HIGH heifers given 8L:16D. Nevertheless, feed to gain ratio during the trial remained in favor of HIGH animals exposed to 16L:8D (5.97) as compared with HIGH animals given 8L:16D (6.07). Dry matter intake per day per heifer on LOW nutrition was similar in both photoperiod groups and ranged from 3.99 to 4.73 kg/day throughout the trial. Yet, feed to gain ratio in these heifers was smaller for those exposed to 16L:8D (6.79) than for those subjected to 8L:16D (6.95). 107 108 Figure 15. Changes in body weight of Holstein heifers in response to photoperiod and plane of nutrition. Each point represents the mean of 14 or 15 animals. Pooled SE of means is 3.6 kg. Average daily gains throughout the experiment are shown on the right for each treatment. 00. _ ON. 00_ O0 00 00 ON _ . _ _ _ _ O0. OON ONN O¢N 00N OOm NNOD \0 \\ 00u00_ D \ \ am: A .5 .. omm \ \D O mm.oO\ \\ 8513225 :9: III I \\ :2:th 9.20 260 (6») 04wa APOB TABLE4. AVERAGEDAEYDRYMATIERINTAKEEXPRESSEDASPERCENT OF mDY WEIGIT. - - Plane of nutrition Days 8L:16D 16L:8D 8L:16D 16L:8D 0—28 - - 3.33 3.34 29—59 2.07 2.01 3.18 3.16 60-83 2.09 2.01 2.77 2.94 84-111 1.93 1.86 2.50 2.65 112-138 1.95 1.85 2.37 2.56 0—138 1.94 1.86 2.76 2.85 aDrmeatter intake for both groups was restricted to similar amount daily and ranged from 3.99 to 4.73 kg of dry matter per day per heifer throughout the experiment. bDry matter intake was provided §g_1ibitum. Serum GH and PRL. Days of bleeding did not influence (P>0.10) concentrations of GH in serum (figure 16). Photoperiod neither affected nor interacted with days of bleeding to change concentrations of GH in serum (P>0.10). Nevertheless, averaging across all days of bleeding, there was a tendency (P<0.10) for GH to be higher in animals given 8L:16D (10.3 ng/ml) in comparison with 16L:8D (9.2 ng/ml). On the other hand, there was a significant interaction between planes of nutrition and days of bleeding (P<0.05). During the last bleeding (day 133), greater (P<0.05) concentrations (ng/ml) of GH. were found in animals on the LOW nutrition (10.9) compared with HIGH nutrition treatment (8.5). Month significantly affected (P<0.001) serum PRL (figure 17) with greater concentrations (ng/ml) at the end of the experiment (80.1) than on day 10 (22.7) or day 53 (29.9). Averaging across all days of bleeding, PRL in serum was greater (P<0.001) under 16L:8D (41.2 ng/ml) than 8L:16D (34.9 ng/ml). However, there was a significant interaction between photoperiod and days of bleeding (P<0.01). Plane of nutrition affected (P<0.001) PRL concentration in serum. Averaging across the three sampling times, PRL was greater (P<0.001) for animals on the HIGH nutrition (42.0 ng/ml) than for animals on LOW nutrition (34.4 ng/ml). There was no interaction between plane of nutrition and days of bleeding (P>0.10) even though greater (P<0.05) concentrations (ng/ml) of PRL were found in animals on the HIGH nutrition (93.6) when compared with animals on the LOW nutrition (68.6) on day 133. Puberty. Heifers tended to reach puberty at a smaller weight when exposed to 16L:8D (P<0.19) or to the LOW plane of nutrition (P<0.19) in comparison 112 with 8L:16D or HIGH nutrition, respectively (figure 18). Nevertheless, the interval from the start of the experiment to puberty was shortened for animals on HIGH nutrition (R0.01) or under 16L:8D photoperiod (P<0.07). 113 Figure 16. Changes in basal growth hormone concentrations in Holstein heifers as influenced by photoperiod and plane of nutrition. Each point represents the mean of 120 to 130 samples from 10 animals. Pooled SE of means is 1.0 ng/ml. 0><0 0.0. ON. 00. O0 00 00 ON 0 J _ _. _ q q _ coztSc ..o 96:. no.1 III I 0 D 6.35: .0 6:03 26.. II. 0 III, a III 0 1|— H I//OL O_ H // w / W O 0- __ m 3 . . _ . . . . \l 802 ll 1 m 0 0071.0 III W .III-nIII-IIIIIIIIIIl-D II. D11. // .. m .... / m // m. 115 Figure 17. Changes in basal prolactin concentrations in Holstein heifers as influenced by photoperiod and plane of nutrition. Each point represents the mean of 120 to 130 samples from 10 animals. Pooled SE of means is 1.1 ng/ml. 0><0 00. ON_ 00. 00 00 0.» ON 0 _ _ _ _ _ _ _ I. ON OII\\\\\ \D\ I 0.4 \ , \ sorts: I 00 \ .6 96.6 no... III \\ 2.62.1356 96.6 26.. III I 00 0\ ON I 0.9 I 00 I 00 (wnJeSIw/5U)NI10V'IOHd Figure 18. 117 Effects of photoperiod and plane of nutrition on body weight at puberty (pooled SE = 17.7 kg) and interval from start Of experiment to puberty (pooled SE = 1.3 week). Each bar represents the mean of 14 or 15 animals. Weight of Puberty (kg) N N N N N N N N 0‘ b ()1 O) \i (I) O O O O O O O lj j I l 1 0l" J [—0 225 l 03: J r-—. 921':- j Interval From Start of Experiment to Puberty (wk) - _ _ — — m N O m 4:. (D 00 o m IIIIIIIIIFII 00f" ] l—O .95 J 001 J I—-- _ SIJ I—-°_ DISCUSSION Our results confirm previous findings (Peters et al., 1980) that a photoperiod of 16L:8D increases growth rate of Holstein heifers over that Of heifers exposed to 8L:16D when they are fed ad libitum a high plane of nutrition. Furthermore, for the first time in cattle, I have demonstrated that the 16L:8D photoperiod will stimulate body growth rates when animals are fed a relatively lower plane of nutrition. Similar Observations have been reported by Forbes et al. (1975, 1979b) in studies with lambs. Therefore, stimulation of body growth by manipulating photoperiod could prove to be a useful management tool in cattle production. Also, in the present study, I have confirmed previous investigations in cattle (Peters et al., 1980) that the increased growth rate in heifers exposed to 16L:8D and fed ad libitum a high plane of nutrition was associated with greater feed consumption. Increased intakes commenced approximately 8 weeks after initiation Of 16L:8D photoperiod. Nevertheless, in both studies, feed to gain ratios were lowest for heifers given 16 h of light daily, suggesting greater efficiency in converting feed into body weight gain. This premise is further strenghtened by data from the animals fed the LOW plane of nutrition where both photoperiod groups were given identical amounts of feed daily; nevertheless, LOW heifers on 16L:8D grew faster than LOW heifers exposed to 8L:16D. This latter observation does not support the hypothesis that increased body growth rate associated with 16L:8D was due only to increased gut fill. 119 120 Similar Observations on feed consumption and feed efficiency were made by Forbes et al. (1975, 1979b) and Schanbacher and Crouse (1980) in studies with lambs. Thus, a 16L:8D photoperiod may stimulate growth rate by increasing feed intake and efficiency. . Homeorhesis is defined as the coordination of metabolism of body tissues in support of a dominant developmental or physiological process (Bauman and Currie, 1980; Bauman et al., 1981). Bauman et a1. (1981) advanced the hypothesis that GH and PRL serve as chronic coordinators of nutrient partitioning among tissues such as between adipose tissue and muscle. In the present study, we Observed discernable effects of plane of nutrition and photoperiod on GH and PRL concentrations in serum. By the end of the experiment, animals on HIGH nutrition had greater concentrations of PRL and less GH in comparison with animals on LOW nutrition. Thus, high concentrations Of PRL and low GH were associated with faster growth rates. When animals were exposed to 16L:8D, levels of PRL were increased without significant change in GH. Yet, these animals grew faster than animals under 8L:16D. In this physiological comparison, fast growth rates were associated with high levels of PRL only. Similar observations have been made in lambs (Forbes et al., 1979b). Collectively, it suggests a possible role of PRL as a homeorhetic factor involved in the photoperiodic regulation of growth. In fact, active immunization against PRL (Ohlson et al., 1981) or inhibition of PRL secretion with 2-Br-a-ergocryptine decreased body weight gain and feed intake in sheep (Eisemann et al., 1981). As ambient temperatures decrease, the ability of 16 h photoperiod to increase prolactin concentrations is reduced (Peters and Tucker, 1978), yet the 121 increased rates of gain persist in cold environments (Peters et al., 1978, 1980). These data should be interpreted cautiously, however, . because neither temperature nor prolactin was measured continuously. It is possible that temporary increases in prolactin due to unrecorded sporadic fluctuations in temperature may have been sufficient to accelerate anabolic processes. A negative relationship exists between concentrations of GH and rate of body growth (Purchas et al., 1971). My data support these observations. For example, heifers on HIGH nutrition were the fastest growing heifers, but they had smaller concentrations of GH than LOW plane heifers. PRL varies with seasons in cattle (Koprowski and Tucker, 1973; Schams and Reinhardt, 1974). In the present experiment, days of bleeding variations in concentrations of PRL were observed in the face of constant photoperiod. The concentrations of PRL increased from a minimal value in February to a maximum in June. On the other hand, there was no days of bleeding variation in G'H concentrations. Similar observations have been reported by Peters et al., 1978. Based on previous observations (Peters et al., 1978), I believe that the major part of the days of bleeding variation in PRL may have been due to the temperature increment among bleeding days. Swanson (1978) showed a positive relationship between average daily gain and body weight at first estrus. These results were confirmed in the present experiment. Compared with the LOW plane of nutrition, animals on the HIGH plane of nutrition reached puberty at a heavier weight. However, this relationship is not always maintained. For example, within each plane of nutrition, heifers under 16L:8D grew faster, but reached puberty at a smaller weight than heifers exposed to. 8L:16D. This supports previous observations 122 that 16L:8D may accelerate onset of puberty (Peters et al., 1978). The PRL changes associated with photoperiod might be involved in hastening onset of puberty in cattle. Nevertheless, no cause and effect relationship can be established at this time. In conclusion, photoperiod may prove to be a useful management tool in cattle production. Growth rate and feed efficiency are increased when animals are exposed to 16 h of light daily. Furthermore, these animals may reach puberty at an earlier age and lighter weight. Further data will be needed to establish a role for PRL and GH in mediating these effects of photoperiod. CHAPTER 5 Carcass composition and mammary development responses to photoperiod and plane of nutrition in Holstein heifers. 123 INTRODUCTION Previous research has indicated that 16 h of light (L): 8 h of dark (D) daily, in comparison with 8L:16D, increases body growth rate and feed efficiency of cattle and sheep fed restricted or ad libitum planes of nutrition (Peters et al., 1978,1980; Forbes et al., 1981; Chapter 4). Furthermore, carcass measurements in sheep suggest that longer periods of daily light stimulate growth of non-fat tissue (Forbes et al., 1981). However, the effect of photoperiod on carcass composition, as estimated from quantitative chemical analysis, has not been reported. Because 16 h of light daily and (or) an ad libitum plane of nutrition increase growth rate and lower age at first parturition, rearing costs of heifers would be expected to be reduced under such management conditions. But, heifers fed a high plane of nutrition (> 1 kg body weight per day) have reduced growth of mammary secretory tissue (Swanson, 1960; Pritchard et al., 1972; Sejrsen et al., 1982) and decreased subsequent yields of milk (Swanson, 1960; Gardner et al., 1977; Little and Kay, 1979). This harmful effect of high plane of nutrition on growth of mammary parenchymal tissue occurs before, but not after puberty (Sejrsen et al., 1982). Thus, the accelerated body growth rate obtained under a 16L:8D photoperiod would be beneficial in raising dairy heifers only if it does not retard development of mammary parenchymal tissue. In this study, I examined the effects of photoperiod and plane of nutrition on carcass composition and mammary development of peripubertal Holstein heifers. 124 MATERIALS AND METHODS Management of Animals. The design was a 2 X 2 factorial with two photoperiods (8L:16D or 16L:8D) and two planes of nutrition. Planes of nutrition were designed to produce average body weight gains of approximately 0.7 (LOW) or greater than 1.0 kg/day (HIGH). Feeding and management of these animals have been described (Chapter 4). Briefly, HIGH heifers were fed ad libitum a ‘ diet greater in protein and energy contents than LOW heifers. Daily feed intake per group of LOW heifers was restricted with no orts and identical for the two groups of LOW heifers subjected to either 8 or 16 h of light daily. From four original groups of 15 animals each (Chapter 4), five prepubertal Holstein heifers per treatment group were selected on the basis of body weight (average 155 kg) at the start of experiment on January 25. Animals were maintained on treatments until they averaged approximately 340 kg body weight and completed at least 2 estrous cycles. Estrous cyclicity was monitored by twice weekly rectal palpation of ovaries and biweekly determination Of concentrations Of progesterone in serum; a concentration Of progesterone greater than 1 ng/ml was indicative of a previous ovulation. Heifers were slaughtered in the luteal phase of an estrous cycle. The luteal phase was confirmed at slaughter by size Of follicles and presence of a corpus luteum. Choice of slaughter date represented a compromise between weight of the animal and stage of the estrous cycle. The latter was standardized because stage of estrous markedly affects mammary development (Sinha and Tucker, 1969). 124 A 125 Initial carcass weight of these animals was estimated by assuming a 50% yield of the body weight determined after deprivation Of water for 16 h at the beginning of the experiment. The assumption of 50% carcass yield at the beginning of the experiment should not affect carcass weight gain among treatments since body weights were similar among treatment groups at the beginning of the experiment. At slaughter, live body weight of heifers was determined without deprivation of water. After slaughter, the carcasses were chilled for approximately 24 h at 4 C and weighed. Then, the 9, 10, ll rib section was cut from the left side of the chilled carcass according to the method of Hankins and Howe (1946) and stored in vacuum-sealed bags at - 20 C until further analysis. Later, rib sections were deboned, ground and a sample was analyzed for water, lipid and protein content. Fat and protein content was determined on dry sample with a Goldfisch-ether extraction and macro-Kjeldahl apparatus, respectively (AOAC, 1965). Mammary glands were trimmed of skin and teats, and left halves were stored at -200 C. Subsequently, the frozen left half was sawed into 1 cm slices. The mammary parenchymal tissue of each slice was dissected at 4° C from surrounding adipose and connective tissue. Deoxyribonucleic acid (DNA) and lipid contents of mammary parenchymal tissue were measured according to the procedure described by Tucker (1964). The DNA content was used as an index of cell numbers. Statistical analysis. Data were subjected to analysis of variance. Bonferroni t-test was used to compare means (Gill, 1978). Body weight at slaughter when used as a covariate did not have any significant effect on carcass composition. One animal in the group subjected to 8L:16D on the HIGH nutrition 126 had a cystic follicle for a major portion of the trial. Values for the amounts of mammary parenchymal tissue (g/100 kg body weight) and total DNA (mg/100 kg body weight) deviated 2.5 and 3.1 times the standard deviation from the average of the group including data from this animal. Consequently, mammary and carcass data from this animal were deleted. RESULTS Growth Performance. Growth variables characterizing performance of the animals in each of the four treatment groups are depicted in table 5. Heifers on the HIGH nutrition were slaughtered at slightly heavier body (P=0.09) and carcass weights (P=0.l) than heifers on LOW nutrition. Number of days to reach slaughter weight of approximately 340 kg was greater for heifers on LOW versus HIGH nutrition (P<0.001). However, HIGH heifers under 8L:16D were slaughtered at a later date than HIGH heifers given 16L:8D (R0.01). Average body weight gain and estimated carcass weight gain of heifers on HIGH nutrition was greater (R0.002) than heifers on the LOW plane. Within LOW or HIGH planes of nutrition, photoperiod did not affect live body weight gains (P>.25). Similarly, photoperiod did not affect estimated carcass weight gain in heifers fed the LOW plane of nutrition. But, estimated carcass weight gain of heifers subjected to 16L:8D on HIGH nutrition was greater (R0.05) than heifers on HIGH nutrition under 8L:16D. Composition of Carcass. Percentage of water (9-10-llth rib section) was smaller (R0.05) for heifers on HIGH nutrition (56.3%) as compared with LOW nutrition (60.1%; figure 19) On the other hand, percentage of fat in 9-10-llth rib section was greater (R0.05) for heifers on HIGH (25.2%) versus LOW nutrition (20.3%) (figure 20). There was no effect of photoperiod (P>0.25) on percentage of water or fat in the rib section. However, there was a photoperiod by plane of nutrition interaction (R0.01) on percentage of protein in the rib section (figure 127 128 TABLES. GKIATI'HPERFORMANCEOFHEIFERSFEDIIMORHIGIPIANE OFNUTRITIONANDEDCPOSED'IOBORIGHOURSOFIJGII‘ PER DAY . ,. low plane of High plane of nutrition nutrition Item 8L:16D 16L:80 8L:16D 16L:8D N 5 5 4 5 Body wt at beginning of exp., kga 155.5 155.2 154.0 155.8 Body wt at slaughter, kgb 337.1‘5l 334.1d 359.8e 348.5e Carcass wt at f . f slaughter, kg 169.1 171.4 190.09 189.39 Days from slaughter 233h 236h 2061 1813' Body wt gain, kg/d .78k .76k 1.001 1.061 Carcass wt gain? kg/d . 39m . 40m . 55n . 61° aBody weight determined after deprivation of water for 16 h. bBody weight determined at slaughterhouse without deprivation of water . c’Assumes 50% yield in carcass weight at beginning of experiment. d vs e; P = .09 f vs 9; P = .10 h VS i, j; P <.001 1 vs j; P <.01 r VS 1; P <.002 10 vs 11, o; P <.002 nvso P<.05 ‘O 129 21). There was no effect Of photoperiod (16L:8D vs 8L:16D) on protein content (P>0.25) among heifers on LOW nutrition (15.5 vs 16.1 %). But, among heifers on HIGH nutrition, 8L:16D reduced (R0.05) percentage of protein in comparison with 16L:8D (14.6 vs 16.2%). Furthermore, heifers exposed to 8L:16D on HIGH nutrition had a lower (R0.05) percentage of protein in the rib sections as compared with heifers under 8L:16D on LOW nutrition (14.6 vs 16.1%). Composition of Mammary Gland. Heifers on HIGH nutrition had similar amounts of mammary parenchymal tissue as heifers on LOW nutrition (94.8 vs 114.7 g/100 kg body wt, P = .19) (figure 23, 24). However, DNA and fat concentrations in parenchymal tissue Of heifers on HIGH nutrition (1.77 mg/g and 50.0%) were smaller (P<0.10 and R0.03) than for heifers on LOW nutrition (1.92 mg/g and 54.8%) (figure 23, 24). These effects of HIGH nutrition only tended to reduce (R031) total DNA in parenchymal tissue (170.2 mg/100 kg body weight) in comparison with heifers on LOW nutrition (227.4 mg/100 kg body weight) (figure 25). Total amount of fat within the parenchymal tissue was less (R0.07) for heifers on HIGH nutrition (47.4 mg/100 kg body weight) versus LOW nutrition (63.7/100 kg body weight) (figure 26). Similarly, total parenchymal tissue weight, DNA and fat uncorrected for body weight was less for heifers on HIGH nutrition (335 g, 602 mg and 168 g, respectively) than for heifers on LOW nutrition (385 g, 763 mg and 214 g, respectively). Photoperiod did not influence (p>.25) any measurements of mammary development (figures 22-26). 130 Figure 19. Percentage of water in 9, 10, 11th rib section of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 14.11 (n = 4 or 5). 62 57 WOIOI' (o/o) 52 BL: I60 I6Lr80 |6L38D BLIIGD LOW PLANE HIGH PLANE 132 Figure 20. Percentage of fat in 9, 10, 11th rib section of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 24.96 (n = 4 or 5). F01 (‘70) 28 l8 8LI|6D |6L380 16L:8D 8L=I6D * LOW PLANE HIGH PLANE 134 Figure 21. Percentage of protein in 9, 10, 11th rib section Of heifers fed a low or high plane Of nutrition and exposed to 8 or 16 h Of light per day. Mean square error = 1.08 (n = 4 or 5). Protein (0/0) | 8 l 6 | 4 I 2 I O 8L3IGD |6L380 I6L38D 8L3I6D LOW PLANE HIGH PLANE 136 Figure 22. Mammary parenchymal tissue weight of heifers fed a low or high plane Of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 966.4 (11 = 4 or 5). PARENCHYMAL TISSUE (g/IOO kg BW) I50 5 o 01 O 8L3I6D |6L38D 8L3I6D I6L380 LOW PLANE HIGH PLANE 138 Figure 23. Concentration of DNA in mammary parenchyma of heifers fed a low or high plane Of nutrition and exposed to 8 or 16 h of light per day. Mean square error = .0872 (n = 4 or 5). DNA (mg/g) 2.0 |.8 |.6 -I6L180 8LII6D — mug IIJI; LOW PLANE HIGH PLANE 140 Figure 24. Concentration of fat in mammary parenchyma of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 28.06 (11 = 4 or 5). FAT ("/6) 60 50 40 8LII6D I6L38D - a -00 I“. 03 r- 00 O lIllI- LOW PLANE HIGH PLANE 142 Figure 25. Total DNA in mammary parenchyma of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h Of light per day. Mean square error = 6952.0 (11 = 4 or 5). 300 PARENCHYMAL DNA (mg/I00 kg BW) N O 0 '03 I" 00 0 III- LOW PLANE HIGH PLANE 144 Figure 26. Total fat in mammary parenchyma of heifers fed a low or high plane of nutrition and exposed to 8 or 16 h of light per day. Mean square error = 345.3 (11 = 4 or 5). PARENCHYIVIAL FAT (g/l00 kg BW) |00 01 O 8L1I6D 16L:8D -— 81.2160I O) I" (I) U IIIIJ lllll- LOW PLANE HIGH PLANE DISCUSSION Daily photoperiods of 16L:8D increased by 10 to 16% the rate of body growth of cattle fed a low or a high plane of nutrition (Chapter 4). This accelerated growth rate is accompanied by an increased efficiency in converting feed into body mass. In contrast, live weight gains of the subsample of heifers from our previous report (Petitclerc et al, 1983), and used in the present study, were not affected by photoperiod. This discrepency may be associated with the limited numbers of animals slaughtered in this experiment and the fact that measurement of body weights was not based on weights after deprivation of drinking water. In the present study, we have evidence that a photoperiod of 16L:8D enhances gain in carcass weight (9.8%) and percentage of protein (11.0%) in the 9, 10, 11th rib section of heifers fed a HIGH plane Of nutrition, but not in heifers fed a LOW plane of nutrition. Animals on the LOW plane of nutrition were slaughtered at a later time (September 15 and 18 for 8L:16D and 16L:8D groups, respectively) than heifers fed the high plane of nutrition (July 25 and August 25 for 16L:8D and 8L:16D groups, respectively). Thus, time on experiment before slaughter could have contributed to this discrepancy between planes of nutrition and the response to photoperiod. In other words, animals on the experiment for longer periods might have become refractory to photoperiod stimulation. Indeed, refractoriness to photoperiod is a phenomenon that has been Observed in many species Of birds (Follett, 1978) and mammals (Reiter, 1980). 146 147 Many researchers (Winchester and Howe, 1955; Waldman et al., 1971; Prior et al., 1977; Ferrel et al., 1978) have observed that, at a constant weight, there is an increase of fat storage in cattle fed higher levels Of nutrition. In the present study, we have again confirmed these Observations. After adjusting for an 11% difference in body weight at slaughter, heifers on the HIGH plane of nutrition had 24% more fat in the 9, 10, ll rib section than heifers fed the LOW plane of nutrition. Waldman et al. (1971) reported that carcass protein percent of Holstein steers fed a limited plane of nutrition (about 70% of 5111M feed intake as in the present study) was 12.3% higher than for steers fed an 951m plane of nutrition. In our study, this effect of high plane of nutrition was also Observed in heifers exposed to 8L:16D, but not for heifers given 16L:8D. Thus, 16L:8D probably counteracted the decreased carcass protein percent caused by high plane of nutrition. In confirmation of earlier work by Pritchard et a1. (1972) and Sejrsen et al. (1982), we also Observed that DNA concentration and total DNA of mammary parenchymal tissue tended to be higher in peripubertal heifers fed LOW versus HIGH plane of nutrition. The decreased amount of parenchymal tissue in the mammary gland of peripubertal heifers raised on high plane of nutrition may explain the reduced yield of milk during their subsequent Iactations (Sejrsen, 1978). Sejrsen, et a1. (1982) reported increased fat accretion in the extraparenchymal mammary tissue (fat pad) in peripubertal heifers fed a high plane of nutrition. However, within the parenchyma, fat was reduced in heifers fed a high plane of nutrition. This agrees with our data. The increased fat 148 within the mammary parenchyma for heifers on the low plane of nutrition probably reflects the method of dissection used to separate parenchyma from fat pad. As the ductular tissue invades the fat pad, a proportionately larger amount of adipose tissue is included with the parenchymal tissue. This would explain why mammary parenchymal tissue of LOW plane heifers contains more fat than that of HIGH plane heifers because of a greater growth of the ductular tissue. Photoperiod did not affect total amount nor composition of parenchymal tissue in the mammary gland. We expected greater growth of the mammary parenchyma for the animals exposed to 16L:8D based on palpation Observations of the mammary gland (D. Petitclerc, L.T. Chapin and H.A. Tucker, unpublished data). Further research using more animals might be needed to detect significant differences due to photoperiod. In summary, level of nutrition and photoperiod affect carcass composition at specific slaughter weights. Level of nutrition, but not photoperiod, affected development and composition of the mammary parenchymal tissue of peripubertal heifers. CONCLUSION In this thesis, experiments were designed to study the mechanism of photoperiod action by monitoring changes in PRL secretion in response to various photoperiods and treatments in prepubertal Holstein bulls. Furthermore, I have investigated the effects of photoperiod and plane Of nutrition on body growth and mammary development of peripubertal Holstein heifers. Evidence presented in this dissertation indicates that blinding per se does not affect PRL secretion but abolishes the ability of 16L:8D to increase concentration Of PRL in bulls. Thus, the eyes are essential for transmission of photic signs to the pituitary gland at least in terms of PRL secretion. However, seasonal changes in PRL persisted in blind steers. Ambient temperature and photoperiod accounted for most but not all of the seasonal variation of PRL. Thus, an endogenous annual rhythm in secretion of PRL in cattle is hypothesized. In addition, I have established that light does not need to be supplied as a continuous block of 16 h since 6L:8D:2L:8D. is as effective as 16L:8D in stimulating secretion of PRL. This constitutes evidence for a diurnal rhythm of photosensitivity of PRL secretion present 14 to 16 h after dawn (or 8 to 10 h after dusk). However, in contrast with sheep, this diurnal rhythm of photosensitivity in cattle appears to be unrelated to actual diurnal change in secretion of PRL since cattle do not express such diurnal rhythmicity. Nevertheless, a clear diurnal pattern of PRL release was observed between 14 to 18 h after dawn (6 to 10 h after dusk) l, 2 and 4 days after switching photoperiod from 16L:8D to 8L:16D but not from 8L:16D to 16L:8D. This release of PRL coincided with the 149 150 photosensitive period of PRL secretion. Thus, it was the absence of light which unmasks the photosensitive period of PRL secretion in animals previously exposed to 16L:8D. Furthermore, I observed a diurnal rhythm of responsiveness to TRH-induced release of PRL in animals exposed to 16L:8D but not to 8L:16D; a greater release was obtained 3 h after dawn (11 h after dusk) as compared with 9, 15 and 21 h after dawn. From these results, I hypothesize that the photosensitive period of PRL secretion is associated with length of dark period or dusk, not dawn. In this thesis, I observed that,'in comparison with 8L:16D, a photoperiod of 16L:8D increased body growth rates 10-18% in heifers fed 8 LOW (average daily gain approximately .7 kg/day) or a HIGH (average daily gain > I kg/day) plane Of nutrition. Heifers of both photoperiod treatments on the LOW plane of nutrition were given identical amounts of feed daily. Therefore, 16L:8D increases growth rate by increasing efficiency of feed conversion into body mass. Furthermore, 16L:8D enhances carcass weight gain (9.8%) and protein percentage (11.0%) in the 9, 10, and 11th rib section of HIGH but not LOW heifers. In addition, 16L:8D heifers reached puberty at a smaller weight. Thus, management of heifers is a potentially promising practice that may enhance output of the cattle industry. Photoperiod did not affect total weight nor composition of mammary parenchymal tissue in prepubertal heifers. On the other hand, HIGH plane of nutrition tended to reduce DNA concentration and total DNA of mammary parenchymal tissue. Recently, I observed that 16L:8D increases total weight, DNA concentration and total DNA of mammary parenchymal tissue of pre—and postpubcrtal heifers (D. Petitclerc, R.D. Kineman, S.A. Zinn and H.A. Tucker, unpublished results). But, HIGH plane of nutrition does not influence total 151 weight nor composition of mammary parenchymal tissue of postpubertal heifers. Therefore, it is hypothesized that the type of response to photoperiod and plane of nutrition is dependent on stage of puberty. 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