Season of the Year Influences Concentration and Pattern of Gonadotropins and Testosterone in Circulation of the Bovine Male'

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1 BIOLOGY OF REPRODUCTION 49, (199) Season of the Year Influences Concentration and Pattern of Gonadotropins and Testosterone in Circulation of the Bovine Male' T.T. STUMPF, M.W. WOLFE, 4 M.S. ROBERSON, 5 RJ. KIITOK, and J.E. KINDER Department of Animal Science, University of Nebraska-Lincoln, Lincoln, Nebraska ABSTRACT Serum concentrations of LH vary with season of the year in ovariectomized beef and dairy cows. The objective of the present study was to determine whether concentrations and profile of the gonadotropins and testosterone (T) in circulation vary with season of year in bovine males of a composite breed type (beef). Five INTACT and five gonadectomized (GNX) males that were mo of age at the initiation of the study were used. All of the males utilized were maintained on pasture throughout the year at the latitude of 41 0 N. Blood samples were collected in a serial regimen (10-min intervals for 4 h) at the spring and fall equinox and the summer and winter solstice. Concentrations of LH were quantified in all samples, and concentrations of T were determined in all samples of the INTACT group. Concentrations of FSH and 17[-estradiol (E ) were quantified in pooled samples for all animals, while concentrations of T were determined in pooled samples for males from the GNX group. Pulse frequency of LH or T and mean concentration of FSH did not vary with season of the year. A seasonal effect on mean concentration of LH (p < 0.01) occurred in males of the GNX group, with mean concentrations of LH being greatest during the spring equinox and lowest during the winter solstice. Season had an effect on pulse amplitude of LH (p < 0.01), mean concentration of T (p < 0.01), and pulse amplitude of T (p < 0.05) in males from the INTACT group. Pulse amplitude of LH was greatest at the spring equinox in INTACT males, while the greatest mean concentration and pulse amplitude of T occurred at the summer solstice in INTACT males. Scrotal circumference tended to vary (p = 0.06) with season in INTACT males and followed the same pattern as T. In summary, the profile of LH and T in circulation varies with season of the year in the bovine male. INTRODUCTION Season of the year influences circulating concentrations of LH, FSH, and testosterone (T) [1-5] as well as scrotal circumference [6, 7] in rams. Furthermore, circulating concentrations of gonadotropins [8,9] and T [9, 10] have been shown to vary with season of year in stallions. Breeding patterns of seasonal-breeding species can be accentuated by the changing sensitivity of the hypothalamic-pituitary axis to gonadal steroids, which results in changes in gonadotropin concentrations in circulation [11]. Interestingly, season of the year affects secretion of LH in both dairy and beef cows (nonseasonal breeder) that have been ovariectomized [1-14]. Season of the year has also been reported to influence the conception rate of Israeli-Friesian dairy cows [15] and the postpartum interval of anestrus in dairy cows [16] and Angus beef cows both north and south of the equator [17,18]. In Holstein bulls, season of the year influences the number of sperm cells per ejaculate [19, 0]. However, there are contradictory reports in the literature about the influence of season on circulating LH [1-] and T [1,, 4-7] Accepted July 6, 199. Received April 15, 199. 'Paper is published as paper no. 9691, Journal Series Nebraska Agricultural Research Division. 'Correspondence: James E. Kinder, A4j Animal Science, University of Nebraska, Lincoln, NE Current address: Department of Animal Science, University of Missouri, Columbia, MO 'Current address: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 'Current address: Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, OR in beef and dairy bulls. Secretory profiles of LH and T have not been evaluated over extended periods of time (e.g., the 4 h used in present study) in the bovine male. The working hypothesis for this study was that season of the year affects the profile of LH in circulation and that this is reflected in seasonal variation in the profile of circulating T. Experimental Design MATERIALS AND METHODS Ten bovine males of a composite breed type (1/4 Angus, 1/4 Red Poll, 1/4 Hereford, 1/4 Pinzgauer), mo of age at the initiation of the study, were used. Five males were INTACT and five had been gonadectomized (GNX) at 8 mo of age. This study was conducted at the beef physiology unit located at the University of Nebraska Agricultural Research and Development Center (41 N latitude). Blood samples were collected serially (at 10-min intervals for 4 h) from all males within 1 wk of the winter solstice, summer solstice, spring equinox, and fall equinox. Dates of blood collection were 17 December, 0 March, June, and 1 September. Blood was collected via indwelling jugular catheters and allowed to clot at room temperature; serum was then stored at 4C. Samples were centrifuged within 48 h of collection; serum was decanted, frozen, and stored at -0 0 C until assayed. Scrotal circumference and testis length were monitored on each date of blood collection. Body weight was also recorded on these days. Bulls were maintained on pasture throughout the study. Supplemental hay was provided dur- 1089

2 1090 STUMPF ET AL. ing the winter months; mineral supplement was also provided during the experimental period. Hormone Analysis Concentrations of LH in serum were determined in all samples by double-antibody RIA [8, 9]. The assay utilized rabbit antiserum against ovine LH (TEA-RaOLH #5), highly purified iodinated ovine LH (LER-1056-C) as labeled hormone, and NIH-LH-B7 as standard. All samples collected during the experiment for an individual bull were analyzed in a single assay. Assays for a male from each treatment were conducted at the same time. Intra- and interassay coefficients of variation for LH were.0 and 1.8%, respectively. Assay sensitivity was 7 pg/ml of serum. Concentrations of FSH and 17-estradiol (E ) for each animal were determined in pools of serum formed from combining aliquots of each of the 144 samples collected within each 4-h bleeding period. Serum concentrations of FSH were determined in the pooled samples by doubleantibody RIA [9,0]. The assay utilized rabbit antiserum against ovine FSH AD-RaOFSH #17-6,7,9) and highly purified iodinated ovine FSH (LER-1976-A) as labeled hormone and standard. Concentrations of FSH were determined in a single assay with an intraassay coefficient of variation of.0%, and the assay sensitivity was 190 pg/ml of serum. Concentrations of E were ascertained in a single assay that had an intraassay coefficient of variation of.5% [9, 1]. Assay sensitivity was 1.5 pg/ml. Serum concentrations of T were determined in all samples collected from males in the INTACT group []. Serum concentrations of T were determined in the pooled samples of males from the GNX group. All samples from an individual INTACT male were analyzed in a single assay, whereas all pooled samples from all males in the GNX group were analyzed in a single assay. Intra- and interassay coefficients of variation for T assays were 1.5 and 16.4%, respectively. Assay sensitivity was 18 pg/ml of serum. The profiles of LH and T in circulation for animals from the INTACT group were characterized by determination of mean concentration, frequency, duration, and amplitude of hormone pulses. Variables of the hormone profiles in circulation were determined through the use of Pulsar Software [] modified for the IBM-PC by Gitzen and Ramirez (Urbana, IL). Statistical Analysis An analysis of variance was performed via the General Linear Models procedure of the Statistical Analysis System [4]. Data from animals in the INTACT and GNX groups were analyzed independently for the main effect of season. Mean differences among seasons were tested by Fisher's Least Significance Difference [4]. Means presented from this analysis are least-squares means. Pearson correlation coefficients [4] were calculated to determine relationships between testicular or body weight measurements and concentrations of steroid hormones. RESULTS Hormone Profiles of GNX Males Season influenced (p < 0.01) mean concentration of LH in males from the GNX group. The greatest mean concentration of LH was observed at the spring equinox; lower mean concentrations of LH occurred at the summer solstice and fall equinox (Fig. 1). There was a trend for season of the year to influence (p = 0.09) mean concentration of FSH in GNX males. Mean concentrations of FSH were highest during the spring equinox and lowest during the summer solstice (Fig. ). Mean concentrations of T (overall mean = 70 5 pg/ml) and E (overall mean = pg/ml) were not influenced (p > 0.10) by season in GNX males. Hormone Profiles in INTACT Males There was no influence of season (p > 0.10) on mean concentration of LH (Fig. 1) or FSH (Fig. ) in INTACT males. However, season did influence (p < 0.01) amplitude of LH pulses and both the mean concentration of T (p < 0.01) and the amplitude of T pulses (p < 0.05). The data for variables of LH and T are displayed in Table 1. Amplitude of LH pulses was greatest at the spring equinox (Fig. ). Mean concentration of T was highest at the summer solstice and lowest at the winter solstice (Fig. 4). In addition, amplitude of T pulses was greatest at the summer solstice and lowest at the winter solstice (Fig. ). The -J z // WINTER MEAN CONCENTRATION OF LH INTACT AND GNX FV f\'\\ k\\ SPRING SEASON SEM INTACT t 0.8 (P<0.01) 7-1 GNX + 0. SUMMER FALL FIG. 1. Mean concentrations of luteinizing hormone (LH) in INTACT and gonadectomized GNX) bulls at each season of the year. Significant effect of season is designated in parentheses. Specific treatment mean comparisons are discussed in Results. F r177 N\\

3 SEASON OF THE YEAR AFFECTS LH AND TESTOSTERONE IN BULLS 1091 MEAN CONCENTRATION OF FSH INTACT AND GNX 7- - SEM S INTACT (P<0.10) V GNX z_ WINTER C SPRING SEASON I:- c /;~ SUMMER FALL FIG.. Mean concentrations of follicle-stimulating hormone (FSH) in INTACT and gonadectomized (GNX) bulls at each season of the year. Significant effect of season is designated in parentheses. Specific treatment mean comparisons are discussed in Results. frequency of neither LH nor T pulses was influenced (p > 0.10) by season of the year (Fig. 5). Greater than 90% of the LH pulses were associated with identifiable pulses of T within a bleeding period (data not shown). Season of the year tended (p = 0.09) to influence concentrations of E in circulation (Fig. 4). Mean concentrations of E in males from the INTACT group were highly correlated (r = 0.8; p < 0.01) with concentrations of T and followed the same pattern as mean concentration of T, being greatest at the summer solstice and lowest at the winter solstice. Relationship of LH and T Secretion in INTACT Males The relationship of pulses of LH to pulses of T, as well as the effect of season on profile of T in circulation, is depicted in Figure 6. Greater than 90% of the pulses of LH were associated with identifiable pulses of T within a bleeding period. Discrepancies from this association were due to a pulse of LH observed at the end of a bleeding period TABLE 1. Characteristics of LH and T in INTACT males at each season of the year. FIG.. Amplitude of luteinizing hormone (LH) and testosterone (T) pulses in INTACT bulls at each season of the year. Specific treatment mean comparisons are discussed in Results and presented in Table 1. or a pulse of T observed at the initiation of a bleeding period; thus the corresponding LH or T pulse was not identified because of the blood sampling protocol. In addition, cases were identified in which multiple LH pulses (short interpulse intervals) stimulated a single identifiable testosterone release. Table summarizes the relationship of LH and T pulses at each season of the year in INTACT males. These data were calculated from only those pulses of LH that stimulated a corresponding release of T, in which the entire LH and associated T pulse was characterized. Season of the year tended (p = 0.06) to influence the duration of a pulse of LH. The duration of a pulse of LH was greatest at the spring equinox ( min); duration of pulses of LH was approximately 74 min at the other times of the year. This reflects the increased amplitude of pulses of LH at the time of the spring equinox given a constant half-life of this hormone in circulation. Season of the year did not alter (p > 0.10) duration of pulses of T, time from peak height of an Winter Spring Summer Fall Characteristic solstice equinox solstice equinox SEM LH Mean (ng/ml) LH Pulse amplitude (ng/ml)** b 5.48a LH Pulse frequency (pulses/4 h) T Mean (ng/ml)*.9a bc 6.18c 4.1 b 0.60 T Pulse amplitude (ng/ml)* ' bc 1.14 T Pulse frequency (pulses/4 h) *Main effect of season (p < 0.05). **Main effect of season (p < 0.01). a'cnumbers with different superscripts within rows differ (p < 0.05). Season

4 109 STUMPF ET AL. 8 MEAN CONCENTRATION OF TESTOSTERONE AND E INTACT 7 fr a w : I o WINTER SPRING SUMMER FALL SEASON FIG. 4. Mean concentrations of testosterone (T) and 171-estradiol (E ) in INTACT males at each season of the year. Significant effect of season is designated in parentheses. Specific treatment mean comparisons are discussed in Results and presented in Table 1. LH pulse to initiation of the corresponding T pulse, or time from peak height of an LH pulse to peak height of corresponding T pulse. The average duration of a pulse of T was 194 ± 1 min. The time interval from peak height of a pulse of LH to initiation of the associated T pulse was 15 + min, whereas the time interval from peak height of an LH pulse until the peak height of the corresponding T pulse was 79 ± 5 min. Testicular Measurements and Body Weight There was a trend for season of the year (p = 0.06) to influence scrotal circumference (Fig. 7). Scrotal circumference increased from the time of the winter solstice until the summer solstice and followed the same profile as mean concentration of T and amplitude of T pulses. A trend for scrotal circumference to be related with mean concentration of T was also observed (r = 0.8; p = 0.09) in the INTACT males. Paired testis volume was calculated for each bull at each season of the year [5]. Season of the year did not influence paired testis volume (p > 0.10). Mean volume of paired testes was 579, 65, 715, and 704 cc (SEM = 45 cc) at the winter solstice, spring equinox, summer solstice, and fall equinox, respectively. All males of both treatment groups lost weight from the winter solstice until the spring equinox, then gained weight throughout the remainder of the study. Mean body weights of males of the INTACT group were 461, 450, 519, and 55 kg (SEM = 10 kg) at the winter solstice, spring equinox, summer solstice, and fall equinox, respectively. No relationship between body weight and scrotal circumference was observed in males of the INTACT group, as the correlation of these two variables was not significant (r = 0.8;p > 0.10). Mean body weights FIG. 5. Frequency of luteinizing hormone (LH) and testosterone (T) pulses in INTACT bulls each season of the year. Significant effect of season is designated in parentheses. Specific treatment mean comparisons are discussed in Results and presented in Table 1. of males of the GNX group were 47, 44, 59, and 565 kg (SEM = 10 kg) at the winter solstice, spring equinox, summer solstice, and fall equinox, respectively. DISCUSSION Secretion of LH occurs in a pulsatile manner in bovine males [9, 6, 7], and LH stimulates testicular tissue to produce T [8]. Season of the year influences the pattern of LH in circulation of ovariectomized Holstein heifers [1], with mean concentrations of LH being greatest at the spring equinox in crossbred beef females [1, 14]. This increase in mean concentration of LH at the spring equinox is due to z I.- 1 HOUR FIG. 6. Profile of luteinizing hormone (LH) and testosterone (T) for a representative INTACT male during the summer solstice and winter solstice I 0o

5 SEASON OF THE YEAR AFFECTS LH AND TESTOSTERONE IN BULLS 109 TABLE. Relationship of circulating LH and T in INTACT males.* Winter solstice Spring equinox Summer solstice Fall equinox Duration of LH pulse (min)** 75' 105 b 7 a 74a Duration of T pulse (min) Interval from peak concentration of LH pulse to initiation of corresponding T pulse (min) Interval from peak concentration of LH pulse to peak concentration of corresponding T pulse (min) *Characteristics of hormone secretion calculated only from entire LH and associated T pulses that were identified during the bleeding periods. **Main effect of season (p < 0.10). "bnumbers with different superscripts within rows differ (p < 0.05). Concentration of T has been found not to be influenced by season of the year in Holstein bulls [4]. In other studies, however, mean concentration of T was reported to be higher during April [7] and November [] than at other times of the year in Holstein bulls. In yet another study, mean con- centration of T in Norwegian Red bulls was greatest in June and August, with the lowest T occurring during the winter [1]. The results of the present investigation support these data, as both mean concentrations of T and amplitude of T pulses were greatest at the summer solstice and lowest at the winter solstice. The presently reported findings also indicate that mean concentrations of E follow the seasonal pattern of T in bulls. Discrepancies in the effect of season on secretion of T may have arisen because of the various experimental pro- tocols utilized, as all of the aforementioned studies were conducted in regions of north latitude. In one study, in- dividual blood samples were collected at frequent intervals (0 min) but for only 6-h periods every wk. Season did not influence secretion of T in that study. In another investigation, concentration of T was greatest in April [7]. In that case, samples were collected at hourly intervals for 4 h, but only two bulls were used and samples were obtained an increased amplitude of LH pulse; s [1, 14]. The results of the present study with gonadecton nized bovine males are comparable to those previously rreported for ovariectomized bovine females. Males from the GNX group had the greatest mean concentration of LH at the spring equinox. Mean concentration of FSH in gonaidectomized males was also greatest at the spring equinox. In some previous studies, seasoni of the year did not affect concentration of LH in circulatiion of intact Norwegian Red and Holstein bulls [1, ]; but in another study utilizing Angus bulls [], LH was fountd in greater concentrations during the summer as com: )ared to winter. In the present study, mean concentrationi of LH was not influenced by season in intact males. A'nplitude of LH pulses was greatest at the spring equinox iin intact males; this finding is similar to results for ovariectomized bovine females [1,14]. Mean concentrations of FSHI in intact Holstein bulls have been reported to be greatest during the fall [4]. Resuits of the present study do not stipport this observation, as season of the year did not influerice mean concentration of FSH in intact bulls. 1J I o As, SCROTAL CIRCUN IFERENCE Pooled SEM llrinc rnl'r thrr noanrnc, F tln rl-r Cr,- rtnn nf T nrc -lllt k/lily ail... laktl,... I L Y.-_ - J-t.ll.. l I 11-t reported to be greatest in November in an investigation in which three blood samples were collected from each bull every 6 wk []. When single blood samples were collected at hourly intervals for 1 h [1] and serial blood samples were collected in the current study over a 4-h period, secretion of T was determined to be greatest during June and August with the lowest concentrations of T occurring in the winter. Pulse frequency of LH and T was not influenced by season in either gonadectomized or intact bovine males in our experiments. Similar results regarding LH have been reported previously for intact Holstein bulls [4] and ovariectomized crossbred beef females [1,14]..Jrastiil drerodlo t h -- e U L 1 L- r U- tau- ai _..n_- OCLTtUll tjd[~su LU 11tV: d JUUl~t[ C11CLL U11 nl.aulatt %,- 50LSTICE SEASON cumference in the present investigation. Changes in scrotal circumference followed the seasonal pattern of T and E FIG. 7. Scrotal circumference (SC) of IN' TACT bulls at each season of the year. Significant effect of season is designnated in parentheses. Specific secretion, being lowest in the winter and increasing through treatment mean comparisons are discussed in Results section of text. the summer solstice. This influence of season on the testis 8 11

6 1094 STUMPF ET AL. does not appear to be due to increases in body weight, as all bulls in this study lost weight from the time of the winter solstice to the spring equinox. It is, however, possible that increasing age of the bulls (assumed sexually mature at initiation of study) could be masking distinct seasonal effects on scrotal circumference. A minimal increase (0.5 cm) in scrotal circumference has been reported to occur in Angus bulls from 18 to 6 mo of age [9]. However, these observations are contradictory to a decreased scrotal circumference in Holstein bulls observed during the summer months [4]. Serum prolactin (PRL) concentrations of Holstein, Hereford, and Brahman bulls are elevated [4, 40, 41] during the summer months and depressed during the fall and winter months. This effect of season on secretion of PRL in the bull is thought to be caused by changes in photoperiod and temperature [4]. Interestingly in the bull, the time of year when PRL concentrations are elevated corresponds to the time of year when secretion of T from the testis is greatest. In rodents, PRL has a positive effect on testicular function [4]. Administration of PRL to hypophysectomized mice and rats potentiates the effects of exogenous LH on synthesis of testosterone and spermatogenesis [44, 45]. Testicular PRL receptors have been characterized in rodents [46-48], and a potential mechanism by which PRL increases the sensitivity of Leydig cells to LH is through PRL-induced increases in concentration of LH receptors in the testis [49, 50]. Whether PRL is involved with modulating the increased secretion of testosterone in the bull during the summer months, as observed in the present study, remains to be elucidated. With regard to the working hypothesis of the current study, the effect of season on concentration of LH in the circulation of bovine males could have been directly reflected in a seasonal effect on T in circulation. Alternatively, a seasonal effect on maximal secretion of LH may have preceded a seasonal effect on maximal secretion of T, as occurs in Suffolk and Finnsheep rams [51]. The greatest concentration of LH in bovine males occurred at the spring equinox. We expected the greatest concentration of T to occur at the same time. However, the greatest concentration of T in intact males was at the summer solstice. An increased sensitivity of the testis to LH has been reported to occur during the summer in bulls [5]. Brahman bulls located in Montana displayed the greatest release of T in response to LHRH in the summer, whereas the response was lowest in the winter and intermediate in the spring. The period of increased responsiveness of the testis to LH corresponds to the time of year in which the greatest semen output [0] and greatest sperm concentration in ejaculates occur in Holstein bulls [19, 0]. The mechanisms governing increased sensitivity of the testis to LH are unknown. Possibly season of the year influences Leydig cell number or number of LH receptors within the membrane of Leydig cells, which could then mediate this effect. As discussed previously, the effect may be a result of an increased influence of PRL during the summer. Season of the year has also been shown to influence Sertoli cell numbers within the testis of seasonal-breeding deer [5] and stallions [54]. In the present study, pulse frequency of LH and T was approximately 5 pulses/4 h. Also, the average duration of LH and T pulses was approximately 1.5 and h, respectively, with a 15-min interval between the peak of the pulse of LH that stimulated a T release and the beginning of the T pulse. A possible cause for the variation among seasons in concentration of LH and T in previous studies may be the experimental protocols utilized. Sample collections in previous research varied in frequency from single daily sample collections to serial collections at 0-min intervals for 6 h. Data obtained in the present study indicate that periods of bleeding similar to those used in the present research are needed to quantify the number of pulses of LH and T. Such a protocol in turn allows for a more accurate determination of mean hormone concentrations and secretory profiles of these hormones. In addition, local environment and breed effects may alter the influence of season. Specifically, relocation of Hereford and Brahman bulls caused variations in the effect of season on hormone secretion and semen characteristics [51]. In summary, season of the year influenced the profile of gonadotropins in circulation of gonadectomized and intact bovine males. Generally the greatest secretion of gonadotropins occurred at the spring equinox. Season of the year also influenced secretion of T in intact males. However, more T was released in response to LH during the time of the summer solstice, as both mean concentration of T and amplitude of T pulses were greatest at the summer solstice. ACKNOWLEDGMENTS We thank Laura Rife for her patience in preparation of this manuscript; Karl Moline, Bob Browleit, and Jeff Bergman for management of the experimental animals; Dr. Dan Deaver, Ken Pearson, and Georgette Caddy for technical assistance with hormone analysis; Dr. Jerry Reeves and Dr. Jim Dias for providing LH and FSH antisera and Dr. Leo Reichert, Jr. for the purified LH and FSH. REFERENCES 1. Lincoln GA, Short RV. Seasonal breeding: nature's contraceptive. Recent Prog Horm Res 1980; 6:1-4.. Pelletier J, Almeida G. Short light cycles induce persistent reproductive activity in Ile-de-France rams. 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