- Email: [email protected]
Effect of Adrenocorticotropic Hormone and Associated Hormonal Responses on Semen Quality and Sperm Output of Bulls
Effect of Adrenocorticotropic Hormone and Associated Hormonal Responses on Semen Quality and Sperm Output of Bulls M. L. O'CONNOR, 1 F. C. G W A Z D A U S K A S , M. L. M c G I L L I A R D , and R. G. SAACKE ~
Department of Dairy Science Virginia Polytechnic Institute and State University Blacksburg 24061 ABSTRACT
The objective was to determine if semen quality and output could be affected by pharmacological doses of adrenocorticotropin. Three Holstein bulls, one 7 yr old and two yearlings, were treated with 200 IU adrenocorticotropic hormone every 8 h for 6 days. Effects of treatment on semen traits and peripheral concentrations of glucocorticoids, testosterone, and androstenedione were measured. Viability of spermatozoa (percentage motility and percentage intact acrosomes), ejaculate volume, sperm concentration, and weekly sperm o u t p u t were unaffected by adrenocorticotropin treatment up to 8 wk posttreatment. The proportion of spermatozoa with cytoplasmic droplets and head abnormalities was elevated slightly from .8 -+ .1 and 4.2 + .3% to 1.3 -+ .2 and 5.8 -+ .4% during treatment. Total glucocorticoids increased from 11.9 + 2.7 ng/ml before treatment to 73.5 + 4.1 ng/ml during treatment. Testosterone decreased in the yearling bulls from 5.5 + .9 ng/ml plasma before treatment to .5 + .5 ng/ml afterward; the decrease began 8 h following the initial adrenocorticotropin injection and persisted until 24 h following last injection. The mature bull had normal testosterone concentrations for the first 4 days of injection, and decreased concentrations for the last 2 days of injection. Semen viability, concentration, and sperm output are unaffected by a pharmocological administration of adrenocorticotropin
Received February 29, 1984. 1Department of Dairy and Animal Science, The Pennsylvania State University, University Park 16802. 2Reprint requests. 1985 J Dairy Sci 68:151--157
and subsequent marked increase of glucocorticoids and decrease of testosterone. Only a small increase of semen content of immature sperm or sperm with abnormal heads may be associated with these marked endocrine changes. INTRODUCTION
Various stressful conditions alter reproductive functions of mammals and fowl. In studies with mice (16) and chickens (5), increased group size caused a decrease of weights of testes that was accompanied by degeneration of germinal epithelium. Transportation of bulls caused significant decrease of semen quality characterized b y lowered sperm motility and increase of primary abnormalities (10). Willet (24) reported variation of nonreturn rate among certain stud bulls due to transportation; however, he showed that overall, there was no significant effect of transportation on nonreturn rate. Effect of heat stress on the male and on semen characteristics has received considerable attention (for review, 14 and 23). Exposure of bulls to experimental heat stress of 40°C for as little as 12 h resulted in disruption of spermatozoal development in testes and appearance of sperm abnormalities in ejaculates (19). Adrenocorticotropic hormone (ACTH) is the major hormone associated with stress. Administration of synthetic ACTH to men reduced testosterone concentrations (9), and "longlasting" ACTH preparations significantly reduced testosterone concentrations in boars (8, 13). In bulls, dexamethasone treatment lowered both luteinizing hormone and testosterone concentrations (22), and when testosterone was increased by exogenous luteinizing hormone, testosterone response within the next hour was negatively related to glucocorticoid concentrations. Our objective was to determine if semen quality and o u t p u t could be affected by pharmacological doses of ACTH. Concentrations 151
152
O'CONNOR ET AL.
in blood plasma of testosterone, androstenedione, and glucocorticoids were monitored also to judge variations of these hormones that may be compatible with normal sperm production. MATERIALS AND METHODS
One 7-yr-old bull weighing 1,050 kg and two yearling Holstein bulls weighing 500 kg each were given 200 IU of porcine ACTH (i.m.) every 8 h for 6 days. Four blood samples were obtained at 8-h intervals before the first ACTH injection (pretreatment). T w o blood samples were taken 30 and 80 min after initial injection and at 8-h intervals until last injection on day 6. Blood samples were taken just prior to ACTH injections on those 6 days. Five additional blood samples were obtained at 8, 16, 24, 48, and 72 h after the final ACTH injection (posttreatment). Blood for endocrine assays was obtained from the midcoccygeal vein in 10-ml samples with a heparinized syringe. Blood was centrifuged at 12,300 × g at 6°C for 10 min, and plasma was recovered and stored at - 9 0 ° C until assayed. Concentrations in blood plasma of testosterone (11, 20) and androstenedione (1) were determined by radioimmunoassay as validated in our laboratory. Total glucocorticoids were determined by competitive protein binding (15) as modified by (7). Minimum sensitivities for testosterone, androstenedione, and glucocorticoids were .005, .05, and .1 ng/ml. Correlations within and between assays were .97 and .86, .97 and .82, and .92 and .62. Semen was collected from each bull via artificial vagina four times a week (two collections on each of 2 days) for 18 wk. Each collection was preceded by sexual preparation consisting of a false mount, a 2-rain restraint, a second false mount, and a 1-min restraint. For the purpose of statistical analysis, semen data were summarized in 3-wk periods. Periods 1, 2, and 3 were the 9 wk before ACTH treatment (pretreatment). Period 4 included the wk of ACTH treatment and the 2 wk following. Periods 5 and 6 represented wk 3 through 5 and 6 through 8 posttreatment. Seminal volume, sperm concentration, percentage progressive motility, and percentage abnormal cells were determined for each
Journal of Dairy Science Vol. 68, No. 1, 1985
ejaculate. Weekly sperm o u t p u t for each bull was calculated from the four ejaculates collected each wk. Sperm concentration was determined photometrically. Estimations of progressive motility to the nearest 10% were made with a phase contrast microscope equipped with a warm stage; raw semen was diluted into a drop of 20% egg yolk (vol/vol) in 2.9% sodium citrate dihydrate. In addition, the percentage of sperm cells possessing an intact acrosome was determined as another measure of cell viability (17). Sperm abnormalities were determined f r o m eosin-aniline blue semen smears according to the procedure of Shaffer and Almquist (18). Two smears of 100 cells each were evaluated for each ejaculate, and this procedure was replicated. All data (response in semen characteristics, sperm ouput, and plasma hormones) were analyzed by least-squares procedures. Because numerous semen traits and h o r m o n e s were determined for each ejaculate and plasma sample, respectively, multivariate analysis of variance (12) was used. In the model for hormone response, sources of variance were bull (1, 2, and 3), treatment category (pretreatment, treatment, and posttreatment), sampling and interaction of bull × treatment. Sampling was the regression of each characteristic on time and removed any linear time trends from effects of treatment and bulls. Also, linear contrast were used to make specific comparisons of importance. In linear contrast 1, hormone concentrations during treatment were compared with those measured before treatment. In contrast 2, the response of the mature bull was compared with that of the two yearling bulls combined. Sources of variation in the model for semen characteristics were period (1 to 6), bull (1, 2, and 3), and interaction. Periods 1, 2, and 3 were pretreatment periods. Each subsequent period was compared with these three pretreatment periods combined (contrasts 1, 2, and 3). In linear contrast 4, the response of the mature bull was compared with that of the yearlings. Simultaneous confidence intervals (12) were constructed for each semen trait to determine which traits contributed to the significance of each source of variation (P<.05). All percentage data were transformed to arcsin of square root for analysis.
PRODUCTION TECHNICAL NOTE
153
TABLE I. Multivariate analysis of variance for effects of adrenocorticotropic hormone administration on blood plasma concentrations of glucocorticoid, testosterone, and androstenedione. Mean squares Source Treatment Contrast 12 Sampling Bull Contrast 23 Bull × treatment Residual
df 2 1 1 2 1 4 65
Glucocorticoids
Androstenedione
Testosterone
p-Statistic
26,443.8 34,360.9* 3,423.9 81.8 23.5 90.8 556.3
57.5 61.3 87.2 38.2 60.9 14.7 8.3
33.2 44.4 57.3 34.8 57.3 8.9 6.8
.004" .009" .745 * * .787** .831 * .846
z Treatment and Contrast 1 were tested by the bull × treatment interaction; other sources of variation were tested by residual.
2 Contrast 1 = Pretreatment versus treatment. 3 Contrast 2 = Mature bull versus yearlings. *P<.05 as determined by simultaneous confidence intervals. **P<.O1 as determined by simultaneous confidence intervals.
R ESU LTS Hormonal Response to Adrenocorticotropic Hormone
Results o f t h e m u l t i v a r i a t e analysis f o r each h o r m o n e a n d sources o f v a r i a t i o n are in T a b l e 1. T h e r e was significant v a r i a t i o n o f h o r m o n a l r e s p o n s e f r o m t r e a t m e n t a n d buli. C o n t r a s t 1 was p a r t i t i o n e d o u t o f t h e significant t r e a t m e n t effect. In this c o n t r a s t , h o r m o n e c o n c e n t r a t i o n s d u r i n g p r e t r e a t m e n t periods were c o m p a r e d w i t h t h o s e d u r i n g t r e a t m e n t . This c o n t r a s t was significant; h o w e v e r , significance was solely d u e to g l u c o c o r t i c o i d s . G t u c o c o r t i c o i d s were elev a t e d ( P < . 0 5 ) f r o m a m e a n (+ SE) p r e t r e a t m e n t c o n c e n t r a t i o n o f 11.9 + 2.7 t o 73.5 -+ 4.1 n g / m l d u r i n g A C T H a d m i n i s t r a t i o n . I n c o n t r a s t 2, h o r m o n e c o n c e n t r a t i o n s in t h e yearling bulls (Figure 1) were c o m p a r e d w i t h t h o s e in t h e m a t u r e bull (Figure 2). This c o n t r a s t was significant a n d can be seen b y c o m p a r i s o n of Figures 1 a n d 2. B o t h g r a p h s s h o w similar c o r t i c o i d r e s p o n s e f o r all bulls. A l t h o u g h t h e testosterone r e s p o n s e to A C T H administ r a t i o n was n o t statistically d i f f e r e n t in c o n t r a s t 1 or 2 f o r all bulls (Table 1), t e s t o s t e r o n e was decreased b y A C T H a d m i n i s t r a t i o n in t h e yearling bulls, d r o p p i n g f r o m 5.5 -+ .9 t o .5 + .2 n g / m l plasma. This decrease b e g a n at t h e 8-h s a m p l i n g f o l l o w i n g t h e initial A C T H i n j e c t i o n a n d persisted u n t i l 2 4 h f o l l o w i n g t h e last
i n j e c t i o n (Figure 1). In a d d i t i o n , t h e c o r r e l a t i o n between testosterone and glucocorticoids for t h e yearling bulls was - . 4 4 ( P < . 0 1 ) . H o w e v e r , t h e m a t u r e bull m a i n t a i n e d n o r m a l t e s t o s t e r o n e c o n c e n t r a t i o n s f o r t h e first 4 days o f i n j e c t i o n ; t e s t o s t e r o n e t h e n was d e c r e a s e d f r o m 5.8 + 1.1 to .8 + .1 n g / m l f o r t h e n e x t 2 days a n d u n t i l 16 h a f t e r t h e last injection. This a p p e a r e d to
155
,3t
125
12
115
tt
lOS
10
95
~s ~8
s5 ~. r5
~r
~ e
5s ~
~5 o ~4 ~3
45~
25 2
15 I
5
b
i }~---~
i
i
i
ACT H TREATMENT DAYS
i
i
2r
i
i
I
Figure 1. Mean plasma testosterone and total glucocorticoid concentrations in two yearling bulls before, during, and after adrenocorticotropic hormone (ACTH) treatment. ("30 and 80 min" refers to samples obtained at those intervals after the first injection; other bleedings were at 8-h intervals, except for the last two, which were at 24-h intervals.) Journal of Dairy Science Vol. 68, No. 1, 1985
154
O'CONNOR ET AL.
IS 12. II, _ I0 E ~9
/80
Min
A /
©
8
i"' '~
i
i
o
/i/
oN s ~4
•A•I
/ORTICOIDS
;I II
~7
135 125
-115 - I05 95
~'' ~
~ TESTOST/, ERONE
j I
.-"%\
-
75
ss ~ 55
I I:
o _o
4s ~ ~5 8
w I,- 3,
~ 25
2"
15
I-
5
ACTH
TREATMENT DAYS
A l t h o u g h n o t r e p r e s e n t e d graphically, pretreatment androstenedione concentrations for t h e t h r e e bulls were elevated f r o m 1.7 + 1.1 t o 5.4 -+ 1.9 n g / m l d u r i n g A C T H a d m i n i s t r a t i o n . T h i s was n o t a significant change.
i
Figure 2. Mean plasma testosterone and total glucocorticoid concentrations in the mature bull before, during, and after adrenocorticotropic hormone (ACTH) treatment. ("30 and 80 min" refers to samples obtained at those intervals after the first injection; other bleedings were at 8-h intervals, except for the last two, which were at 24-h intervals.)
r e p r e s e n t a d e l a y e d r e s p o n s e t h a t m a y have b e e n r e l a t e d t o t h e l o w e r dosage o f A C T H , o n a b o d y w e i g h t basis, f o r this bull t h a n f o r t h e l i g h t e r yearling bulls (each bull received 2 0 0 IU ACTH).
Semen Characteristics
S e m e n d a t a were g r o u p e d a n d a n a l y z e d in 3-wk periods. M u l t i v a r i a t e analysis o f spermatozoal abnormalities (total cytoplasmic droplets, h e a d a b n o r m a l i t i e s , a n d tail a b n o r m a l i t i e s ) is in T a b l e 2. T h e r e was significant v a r i a t i o n f r o m period. S i m u l t a n e o u s c o n f i d e n c e intervals indicated that total cytoplasmic droplets and h e a d a b n o r m a l i t i e s in p e r i o d 4 were elevated significantly f r o m t h o s e in p e r i o d s 1, 2, a n d 3 c o m b i n e d ; h o w e v e r , in p e r i o d 5 o n l y h e a d a b n o r m a l i t i e s were d i f f e r e n t f r o m t h o s e in p e r i o d s 1, 2, a n d 3 c o m b i n e d (Table 2). T h e n u m b e r o f a b n o r m a l tails was n o t altered significantly b y period. T h e r e were d i f f e r e n c e s ( P < . 0 1 ) d u e to bull a n d i n t e r a c t i o n o f bull x period. H o w e v e r , u n i v a r i a t e analysis o f variance for t o t a l s p e r m a t o z o a l a b n o r m a l i t i e s revealed n o significant effects o f p e r i o d o r individual c o n t r a s t . T h e r e was a highly significant bull e f f e c t a n d i n t e r a c t i o n o f bull × p e r i o d f o r t o t a l abnormalities.
TABLE 2. Multivariate analysis of variance for the effect of adrenocorticotropic hormone administration on total cytoplasmic droplets head and tail abnormalities.a Mean squares (nontransformed) Source Periods Contrast 1 Contrast 2 Contrast 3 Bull 4 Contrast 4 Bull X period Residual
df 5 1 1 1 2 1 10 402
Total droplets
Head
Tail
36.52 137.97* .79 .03 8.79 9.65* 14.85 6.57
4.37 5.42* 10.38" .32 69.87 111.58" 2.01 1.34
18.81 .43 4.19 89.11 58.72 117.33" 29.70 4.37
gt-Statistic2 .086" .287*
.383* .758 .734"* .785** .813"*
I Period and contrast were tested by the bull × period interaction; other sources of variation were tested by residual. ~Dara were transformed to arcsin of the square root for tests of significance. 3Period was partitioned into the following contrasts: Contrast 1 = period 4 versus periods 1, 2, and 3;Contrast 2 = period 5 versus periods 1, 2, and 3; Contrast 3 = period 6 versus periods 1, 2, and 3. 4 Contrast 4 = Mature bull versus yearlings. *P<.05 as determined by simultaneous confidence intervals. **P<.01 as determined by simultaneous confidence intervals. Journal of Dairy Science Vol. 68, No. 1, 1985
PRODUCTION TECHNICAL NOTE
Means for alI spermatozoal abnormalities and other semen characteristics across the six periods are in Table 3. Differences in cytoplasmic droplets and head abnormalities for periods 4 and 5, although significant, were small. Analysis of ejaculate volume, sperm concentration, and percentage progressive motility revealed no significant effects of period or contrast on these semen characteristics• Also, univariate analysis of variance for weekly sperm output and percentage intact acrosomes revealed no significant effect of period•
t~ N
~x o0 ~ - ~
Ox
ox ¢q
¢q
1 ~ . 4 ~
~
155
O~
DISCUSSION ¢q
4
0 tm
i=2
m~
g~ +1 v
~ , ~ o ~
- ~
< <
Through administration of a pharmacological dose of ACTH, an attempt was made to induce endocrine changes that could mimic or exceed those of a stressed animal with anticipation of a subsequent response of semen quality and output. Figures 1 and 2 illustrate endocrine response of glucocorticoids and testosterone to the ACTH. The rapid significant elevation of total glucocorticoid concentrations to 73.5 + 4.1 ng/ml and maintenance of these high concentrations was anticipated from studies with cows (6) and bo~rs (13). Although a depressive effect on testosterone was expected, the magnitude of the drop for yearlings and the delayed testosterone response of the older male were surprising. Nevertheless, testosterone is important in reduction division of spermatocytes and may be involved in early stages of spermiogenesis (21). Therefore, low testosterone concentrations (.5 + .2 ng/ml) in the younger bulls during the 6 days of ACTH administration might be expected to have affected adversely certain aspects of spermatogenesis. However, the large endocrine imbalances that were created did not affect adversely sperm viability, ejaculate volume, sperm concentration, or weekly sperm output. With respect to sperm morphology, there was a statistically significantly increase of total cytoplasmic droplets and head abnormalities during period 4, the period that included the week of ACTH treatment and the 2 wk posttreatment. However, head abnormalities and cytoplasmic droplets were increased only from •8 + .1 and 4.2 -+ .3% pretreatment to 1.3 + .2 and 5.8 + .4% during treatment (period 4). Although it is possible that such a small increase is physiologically significant, longer periods of ACTH administraJournal of Dairy Science Vol. 68, No. 1, 1985
156
O'CONNOR ET AL.
tion w o u l d be necessary to confirm the relatively m i n o r impact on semen of the rather large e n d o c r i n e imbalance created in this study. In a study with bulls, Cupps et al. (4) r e p o r t e d that pharmacological doses of glucocorticoids administered for periods ranging f r o m 2 to 10 wk did not affect percentage live or t o t a l a b n o r m a l cells in ejaculates. H o w e v e r , using differential interference contrast microscopy, Coulter (3) observed an increase in "crater-like" sperm defects associated with a 7-day d e x a m e t h a s o n e treatment. Such defects were e x p e c t e d b u t n o t observed in our study following 6 days of A C T H treatment. A m a n n and Ganjam (2) r e p o r t e d that steroid c o n c e n t r a t i o n s in the testicular vein were considerably higher than peripheral concentrations. Thus, plasma c o n c e n t r a t i o n s in this study do n o t reflect necessarily h o r m o n e c o n t e n t within the testis. Possibly t e s t o s t e r o n e c o n c e n t r a t i o n s within the testicular p a r e n c h y m a and epididymal artery o f the bulls in this s t u d y were high enough to maintain normal spermatogenesis and epididymal f u n c t i o n . In addition, the higher c o n c e n t r a t i o n s of a n d r o s t e n e d i o n e during A C T H t r e a t m e n t m a y have provided enough c o m p e n s a t o r y androgenic activity to maintain normal gametogenesis and epididymal function. CONCLUSION
Results indicate that A C T H t r e a t m e n t for 6 days results in marked increase of glucocorticoids and can cause m a r k e d depression o f testosterone for this t i m e ; however, neither A C T H nor these a c c o m p a n y i n g endocrine changes affect semen viability or sperm o u t p u t in the bull. A slight but significant increase in sperm head abnormalities and i m m a t u r e sperm (possessing cytoplasmic droplets) was observed in response to A C T H ; however, the physiological i m p o r t a n c e of this increase is n o t apparent in view o f the wide variance in testosterone and glucocorticoid caused by the A C T H treatment. ACKNOWLEDGMENTS
We are especially grateful to J. H. Bame for her assistance in quantifying sperm abnormalities and D. A. Aalseth for the endocrine assays. This w o r k was supported partially b y Select Sires, Inc., and a Virginia Agricultural F o u n d a tion Grant. Journal of Dairy Science Vol. 68, No. 1, 1985
REFERENCES
1 Abraham, C. E., M. S. Manlimos, M. Solis, and A. C. Wickman. 1975. Combined radioimmunoassay of four steroids in one ml of plasma: II Androgens. Clin. Biochem. 8: 374. 2 Amann, R. P., and V. K. Ganjam. 1976. Steroid production by the bovine testis and steroid transfer across the pampiniform plexus. Biol. Reprod. 15:695. 3 Coulter, G. H. 1976. Effect of dexamethasone on a sperm defect. J. Anim. Sci. 43:279. 4 Cupps, P. T., R. C. Laben, D. F. Rahlmann, and A. R. Reddon. 1960. Effects of adrenal glucocorticoid and testosterone on the semen. J. Anim. Sci. 19:509. 5 Flickinger, G. L. 1966. Response of the testes to social interaction among grouped chickens. Gen. Comp. Endocrinol. 6:89. 6 Gwazdauskas, F. C., W. W. Thatcher, and C. J. Wilcox. 1972. Adrenoeorticotropin alteration of bovine peripheral plasma concentrations of cortisol, cortieosterone and progesterone. J. Dairy Sci. 55:1165. 7 Gwazdauskas, F. C., W. W. Thatcher, and C. J. Wilcox. 1973. Physiological, environmental and hormonal factors at insemination which may affect conception. J. Dairy Sci. 56:873. 8 Holtz, W., A. Hartig, and A. Konig. 1978. Effect of ACTH on plasma testosterone levels in the male Gottingen miniature pig. Pfluengers Arch. Eur. J. Physiol. 373 Suppl. R56. 9 Irvine, W. J., A. D. Taft, K. S. Wilson, R. Fraser, A. Wilson, J. Young, W. M. Hunter, A.A.A. Ismail, and P. E. Burger. 1974. The effect of synthetic corticotropin analogues on adrenocortical, anterior pituitary and testicular function, J. Clin. Endocrinol. Metab. 39:522. 10 Jaskowski, L., and L. Szule. 1968. Experiments on preventing the decrease of semen quality in bulls caused by transportation. Pages 285 in Proc. Sixth Int. Congr, Anim. Reprod. Artif. lnsem,, Paris. 11 Kattesh,/-I. G., E. T. Kornegay, F. C. Gwazdanskas, J. W. Knight, and H. R. Thomas. 1979. Peripheral plasma testosterone concentration and sexual behavior in young prenatally stressed boars. Theriogenology 12: 289. 12 Kramer, C. Y. 1972. A first course in methods of multivariate analysis. Virginia Polytechnic lnst. State Univ., Blacksburg. 13 Liptrap, A. J., and J. I. Raeside. 1975. Increases in plasma testosterone concentration after injection of ACTH into the boar. J. Endocrinol. 66:123. 14 Lodge, J. R., and G. W. Salisbury. 1970. Seasonal variation and male reproductive efficiency. Pages 739-767 in The testis. Vol. 3. A. D. Johnson, W. R. Gomes, and N. L. Van Demark, ed. Academic Press, New York, NY. 15 Murphy, B. P. 1967. Some studies of the proteinbinding of steroids and their application to the routine micro and ultramicro measurement of various steroids in competitive protein-binding radioassay. J. Clin. Endocrinol. 27:973. 16 Pasley, J. M., and J. J. Christian. 1972. The effect of ACTH, group caging, and adrenalectomy in
PRODUCTION TECHNICAL NOTE
17
18
19
20
peromyscius leucopus with emphasis on suppression of reproductive function. Proc. Soc. Exp. Biol. Med. 139:921. Saacke, R. G., and J. M. White. 1972. Semen quality tests and their relationship to fertility. Page 22 in Proc. Fourth Tech. Conf. Artif. Insem. Reprod., Chicago, IL. Shaffer, H. E., and J. O. Almquist. 1948. Vital staining o f bovine spermatozoa with an eosin-aniline blue staining mixture. J. Dairy Sci. 31:677. Skinner, J. D., and G. N. Louw. 1966. Heat stress and spermatogenesis in Bos indicus and Bos taurus cattle. J. Appl. Physiol. 21:1784. Smith, O. W., K. M o n g k o n p u n y a , H. D. Hafs, E. M. Convey, and W. D. Oxender. 1973. Blood serum
21 22
23
24
15 7
testosterone after sexual preparation or ejaculation, or after injections of LH or prolactin in bulls. J. Anim. Sci. 37:979. Steinberger, E. 1971. Hormonal control of m a m malian spermatogenesis. Physiol. Rev. 51 : 1. Thibier, M., and O. Rolland. 1976. The effect o f d e x a m e t h a z o n e (DXM) on circulating testosterone (T) and luteinizing h o r m o n e (LH) in y o u n g postpubertal bulls. Theriogenology 5:53. Van Demark, N. L., and M. J. Free. 1970. T e m p erature effects. Pages 233-312 in The testis. Vol. 3. A. D. Johnson, W. R. Gomes, and N. L. Van Demark, ed. Academic Press, New York, NY. Willet, E. L. 1957. Effect of transportation u p o n fertility of bulls. J. Dairy Sci. 40:1367.
Journal o f Dairy Science Vol. 68, No. 1, 1985
Department of Dairy Science Virginia Polytechnic Institute and State University Blacksburg 24061 ABSTRACT
The objective was to determine if semen quality and output could be affected by pharmacological doses of adrenocorticotropin. Three Holstein bulls, one 7 yr old and two yearlings, were treated with 200 IU adrenocorticotropic hormone every 8 h for 6 days. Effects of treatment on semen traits and peripheral concentrations of glucocorticoids, testosterone, and androstenedione were measured. Viability of spermatozoa (percentage motility and percentage intact acrosomes), ejaculate volume, sperm concentration, and weekly sperm o u t p u t were unaffected by adrenocorticotropin treatment up to 8 wk posttreatment. The proportion of spermatozoa with cytoplasmic droplets and head abnormalities was elevated slightly from .8 -+ .1 and 4.2 + .3% to 1.3 -+ .2 and 5.8 -+ .4% during treatment. Total glucocorticoids increased from 11.9 + 2.7 ng/ml before treatment to 73.5 + 4.1 ng/ml during treatment. Testosterone decreased in the yearling bulls from 5.5 + .9 ng/ml plasma before treatment to .5 + .5 ng/ml afterward; the decrease began 8 h following the initial adrenocorticotropin injection and persisted until 24 h following last injection. The mature bull had normal testosterone concentrations for the first 4 days of injection, and decreased concentrations for the last 2 days of injection. Semen viability, concentration, and sperm output are unaffected by a pharmocological administration of adrenocorticotropin
Received February 29, 1984. 1Department of Dairy and Animal Science, The Pennsylvania State University, University Park 16802. 2Reprint requests. 1985 J Dairy Sci 68:151--157
and subsequent marked increase of glucocorticoids and decrease of testosterone. Only a small increase of semen content of immature sperm or sperm with abnormal heads may be associated with these marked endocrine changes. INTRODUCTION
Various stressful conditions alter reproductive functions of mammals and fowl. In studies with mice (16) and chickens (5), increased group size caused a decrease of weights of testes that was accompanied by degeneration of germinal epithelium. Transportation of bulls caused significant decrease of semen quality characterized b y lowered sperm motility and increase of primary abnormalities (10). Willet (24) reported variation of nonreturn rate among certain stud bulls due to transportation; however, he showed that overall, there was no significant effect of transportation on nonreturn rate. Effect of heat stress on the male and on semen characteristics has received considerable attention (for review, 14 and 23). Exposure of bulls to experimental heat stress of 40°C for as little as 12 h resulted in disruption of spermatozoal development in testes and appearance of sperm abnormalities in ejaculates (19). Adrenocorticotropic hormone (ACTH) is the major hormone associated with stress. Administration of synthetic ACTH to men reduced testosterone concentrations (9), and "longlasting" ACTH preparations significantly reduced testosterone concentrations in boars (8, 13). In bulls, dexamethasone treatment lowered both luteinizing hormone and testosterone concentrations (22), and when testosterone was increased by exogenous luteinizing hormone, testosterone response within the next hour was negatively related to glucocorticoid concentrations. Our objective was to determine if semen quality and o u t p u t could be affected by pharmacological doses of ACTH. Concentrations 151
152
O'CONNOR ET AL.
in blood plasma of testosterone, androstenedione, and glucocorticoids were monitored also to judge variations of these hormones that may be compatible with normal sperm production. MATERIALS AND METHODS
One 7-yr-old bull weighing 1,050 kg and two yearling Holstein bulls weighing 500 kg each were given 200 IU of porcine ACTH (i.m.) every 8 h for 6 days. Four blood samples were obtained at 8-h intervals before the first ACTH injection (pretreatment). T w o blood samples were taken 30 and 80 min after initial injection and at 8-h intervals until last injection on day 6. Blood samples were taken just prior to ACTH injections on those 6 days. Five additional blood samples were obtained at 8, 16, 24, 48, and 72 h after the final ACTH injection (posttreatment). Blood for endocrine assays was obtained from the midcoccygeal vein in 10-ml samples with a heparinized syringe. Blood was centrifuged at 12,300 × g at 6°C for 10 min, and plasma was recovered and stored at - 9 0 ° C until assayed. Concentrations in blood plasma of testosterone (11, 20) and androstenedione (1) were determined by radioimmunoassay as validated in our laboratory. Total glucocorticoids were determined by competitive protein binding (15) as modified by (7). Minimum sensitivities for testosterone, androstenedione, and glucocorticoids were .005, .05, and .1 ng/ml. Correlations within and between assays were .97 and .86, .97 and .82, and .92 and .62. Semen was collected from each bull via artificial vagina four times a week (two collections on each of 2 days) for 18 wk. Each collection was preceded by sexual preparation consisting of a false mount, a 2-rain restraint, a second false mount, and a 1-min restraint. For the purpose of statistical analysis, semen data were summarized in 3-wk periods. Periods 1, 2, and 3 were the 9 wk before ACTH treatment (pretreatment). Period 4 included the wk of ACTH treatment and the 2 wk following. Periods 5 and 6 represented wk 3 through 5 and 6 through 8 posttreatment. Seminal volume, sperm concentration, percentage progressive motility, and percentage abnormal cells were determined for each
Journal of Dairy Science Vol. 68, No. 1, 1985
ejaculate. Weekly sperm o u t p u t for each bull was calculated from the four ejaculates collected each wk. Sperm concentration was determined photometrically. Estimations of progressive motility to the nearest 10% were made with a phase contrast microscope equipped with a warm stage; raw semen was diluted into a drop of 20% egg yolk (vol/vol) in 2.9% sodium citrate dihydrate. In addition, the percentage of sperm cells possessing an intact acrosome was determined as another measure of cell viability (17). Sperm abnormalities were determined f r o m eosin-aniline blue semen smears according to the procedure of Shaffer and Almquist (18). Two smears of 100 cells each were evaluated for each ejaculate, and this procedure was replicated. All data (response in semen characteristics, sperm ouput, and plasma hormones) were analyzed by least-squares procedures. Because numerous semen traits and h o r m o n e s were determined for each ejaculate and plasma sample, respectively, multivariate analysis of variance (12) was used. In the model for hormone response, sources of variance were bull (1, 2, and 3), treatment category (pretreatment, treatment, and posttreatment), sampling and interaction of bull × treatment. Sampling was the regression of each characteristic on time and removed any linear time trends from effects of treatment and bulls. Also, linear contrast were used to make specific comparisons of importance. In linear contrast 1, hormone concentrations during treatment were compared with those measured before treatment. In contrast 2, the response of the mature bull was compared with that of the two yearling bulls combined. Sources of variation in the model for semen characteristics were period (1 to 6), bull (1, 2, and 3), and interaction. Periods 1, 2, and 3 were pretreatment periods. Each subsequent period was compared with these three pretreatment periods combined (contrasts 1, 2, and 3). In linear contrast 4, the response of the mature bull was compared with that of the yearlings. Simultaneous confidence intervals (12) were constructed for each semen trait to determine which traits contributed to the significance of each source of variation (P<.05). All percentage data were transformed to arcsin of square root for analysis.
PRODUCTION TECHNICAL NOTE
153
TABLE I. Multivariate analysis of variance for effects of adrenocorticotropic hormone administration on blood plasma concentrations of glucocorticoid, testosterone, and androstenedione. Mean squares Source Treatment Contrast 12 Sampling Bull Contrast 23 Bull × treatment Residual
df 2 1 1 2 1 4 65
Glucocorticoids
Androstenedione
Testosterone
p-Statistic
26,443.8 34,360.9* 3,423.9 81.8 23.5 90.8 556.3
57.5 61.3 87.2 38.2 60.9 14.7 8.3
33.2 44.4 57.3 34.8 57.3 8.9 6.8
.004" .009" .745 * * .787** .831 * .846
z Treatment and Contrast 1 were tested by the bull × treatment interaction; other sources of variation were tested by residual.
2 Contrast 1 = Pretreatment versus treatment. 3 Contrast 2 = Mature bull versus yearlings. *P<.05 as determined by simultaneous confidence intervals. **P<.O1 as determined by simultaneous confidence intervals.
R ESU LTS Hormonal Response to Adrenocorticotropic Hormone
Results o f t h e m u l t i v a r i a t e analysis f o r each h o r m o n e a n d sources o f v a r i a t i o n are in T a b l e 1. T h e r e was significant v a r i a t i o n o f h o r m o n a l r e s p o n s e f r o m t r e a t m e n t a n d buli. C o n t r a s t 1 was p a r t i t i o n e d o u t o f t h e significant t r e a t m e n t effect. In this c o n t r a s t , h o r m o n e c o n c e n t r a t i o n s d u r i n g p r e t r e a t m e n t periods were c o m p a r e d w i t h t h o s e d u r i n g t r e a t m e n t . This c o n t r a s t was significant; h o w e v e r , significance was solely d u e to g l u c o c o r t i c o i d s . G t u c o c o r t i c o i d s were elev a t e d ( P < . 0 5 ) f r o m a m e a n (+ SE) p r e t r e a t m e n t c o n c e n t r a t i o n o f 11.9 + 2.7 t o 73.5 -+ 4.1 n g / m l d u r i n g A C T H a d m i n i s t r a t i o n . I n c o n t r a s t 2, h o r m o n e c o n c e n t r a t i o n s in t h e yearling bulls (Figure 1) were c o m p a r e d w i t h t h o s e in t h e m a t u r e bull (Figure 2). This c o n t r a s t was significant a n d can be seen b y c o m p a r i s o n of Figures 1 a n d 2. B o t h g r a p h s s h o w similar c o r t i c o i d r e s p o n s e f o r all bulls. A l t h o u g h t h e testosterone r e s p o n s e to A C T H administ r a t i o n was n o t statistically d i f f e r e n t in c o n t r a s t 1 or 2 f o r all bulls (Table 1), t e s t o s t e r o n e was decreased b y A C T H a d m i n i s t r a t i o n in t h e yearling bulls, d r o p p i n g f r o m 5.5 -+ .9 t o .5 + .2 n g / m l plasma. This decrease b e g a n at t h e 8-h s a m p l i n g f o l l o w i n g t h e initial A C T H i n j e c t i o n a n d persisted u n t i l 2 4 h f o l l o w i n g t h e last
i n j e c t i o n (Figure 1). In a d d i t i o n , t h e c o r r e l a t i o n between testosterone and glucocorticoids for t h e yearling bulls was - . 4 4 ( P < . 0 1 ) . H o w e v e r , t h e m a t u r e bull m a i n t a i n e d n o r m a l t e s t o s t e r o n e c o n c e n t r a t i o n s f o r t h e first 4 days o f i n j e c t i o n ; t e s t o s t e r o n e t h e n was d e c r e a s e d f r o m 5.8 + 1.1 to .8 + .1 n g / m l f o r t h e n e x t 2 days a n d u n t i l 16 h a f t e r t h e last injection. This a p p e a r e d to
155
,3t
125
12
115
tt
lOS
10
95
~s ~8
s5 ~. r5
~r
~ e
5s ~
~5 o ~4 ~3
45~
25 2
15 I
5
b
i }~---~
i
i
i
ACT H TREATMENT DAYS
i
i
2r
i
i
I
Figure 1. Mean plasma testosterone and total glucocorticoid concentrations in two yearling bulls before, during, and after adrenocorticotropic hormone (ACTH) treatment. ("30 and 80 min" refers to samples obtained at those intervals after the first injection; other bleedings were at 8-h intervals, except for the last two, which were at 24-h intervals.) Journal of Dairy Science Vol. 68, No. 1, 1985
154
O'CONNOR ET AL.
IS 12. II, _ I0 E ~9
/80
Min
A /
©
8
i"' '~
i
i
o
/i/
oN s ~4
•A•I
/ORTICOIDS
;I II
~7
135 125
-115 - I05 95
~'' ~
~ TESTOST/, ERONE
j I
.-"%\
-
75
ss ~ 55
I I:
o _o
4s ~ ~5 8
w I,- 3,
~ 25
2"
15
I-
5
ACTH
TREATMENT DAYS
A l t h o u g h n o t r e p r e s e n t e d graphically, pretreatment androstenedione concentrations for t h e t h r e e bulls were elevated f r o m 1.7 + 1.1 t o 5.4 -+ 1.9 n g / m l d u r i n g A C T H a d m i n i s t r a t i o n . T h i s was n o t a significant change.
i
Figure 2. Mean plasma testosterone and total glucocorticoid concentrations in the mature bull before, during, and after adrenocorticotropic hormone (ACTH) treatment. ("30 and 80 min" refers to samples obtained at those intervals after the first injection; other bleedings were at 8-h intervals, except for the last two, which were at 24-h intervals.)
r e p r e s e n t a d e l a y e d r e s p o n s e t h a t m a y have b e e n r e l a t e d t o t h e l o w e r dosage o f A C T H , o n a b o d y w e i g h t basis, f o r this bull t h a n f o r t h e l i g h t e r yearling bulls (each bull received 2 0 0 IU ACTH).
Semen Characteristics
S e m e n d a t a were g r o u p e d a n d a n a l y z e d in 3-wk periods. M u l t i v a r i a t e analysis o f spermatozoal abnormalities (total cytoplasmic droplets, h e a d a b n o r m a l i t i e s , a n d tail a b n o r m a l i t i e s ) is in T a b l e 2. T h e r e was significant v a r i a t i o n f r o m period. S i m u l t a n e o u s c o n f i d e n c e intervals indicated that total cytoplasmic droplets and h e a d a b n o r m a l i t i e s in p e r i o d 4 were elevated significantly f r o m t h o s e in p e r i o d s 1, 2, a n d 3 c o m b i n e d ; h o w e v e r , in p e r i o d 5 o n l y h e a d a b n o r m a l i t i e s were d i f f e r e n t f r o m t h o s e in p e r i o d s 1, 2, a n d 3 c o m b i n e d (Table 2). T h e n u m b e r o f a b n o r m a l tails was n o t altered significantly b y period. T h e r e were d i f f e r e n c e s ( P < . 0 1 ) d u e to bull a n d i n t e r a c t i o n o f bull x period. H o w e v e r , u n i v a r i a t e analysis o f variance for t o t a l s p e r m a t o z o a l a b n o r m a l i t i e s revealed n o significant effects o f p e r i o d o r individual c o n t r a s t . T h e r e was a highly significant bull e f f e c t a n d i n t e r a c t i o n o f bull × p e r i o d f o r t o t a l abnormalities.
TABLE 2. Multivariate analysis of variance for the effect of adrenocorticotropic hormone administration on total cytoplasmic droplets head and tail abnormalities.a Mean squares (nontransformed) Source Periods Contrast 1 Contrast 2 Contrast 3 Bull 4 Contrast 4 Bull X period Residual
df 5 1 1 1 2 1 10 402
Total droplets
Head
Tail
36.52 137.97* .79 .03 8.79 9.65* 14.85 6.57
4.37 5.42* 10.38" .32 69.87 111.58" 2.01 1.34
18.81 .43 4.19 89.11 58.72 117.33" 29.70 4.37
gt-Statistic2 .086" .287*
.383* .758 .734"* .785** .813"*
I Period and contrast were tested by the bull × period interaction; other sources of variation were tested by residual. ~Dara were transformed to arcsin of the square root for tests of significance. 3Period was partitioned into the following contrasts: Contrast 1 = period 4 versus periods 1, 2, and 3;Contrast 2 = period 5 versus periods 1, 2, and 3; Contrast 3 = period 6 versus periods 1, 2, and 3. 4 Contrast 4 = Mature bull versus yearlings. *P<.05 as determined by simultaneous confidence intervals. **P<.01 as determined by simultaneous confidence intervals. Journal of Dairy Science Vol. 68, No. 1, 1985
PRODUCTION TECHNICAL NOTE
Means for alI spermatozoal abnormalities and other semen characteristics across the six periods are in Table 3. Differences in cytoplasmic droplets and head abnormalities for periods 4 and 5, although significant, were small. Analysis of ejaculate volume, sperm concentration, and percentage progressive motility revealed no significant effects of period or contrast on these semen characteristics• Also, univariate analysis of variance for weekly sperm output and percentage intact acrosomes revealed no significant effect of period•
t~ N
~x o0 ~ - ~
Ox
ox ¢q
¢q
1 ~ . 4 ~
~
155
O~
DISCUSSION ¢q
4
0 tm
i=2
m~
g~ +1 v
~ , ~ o ~
- ~
< <
Through administration of a pharmacological dose of ACTH, an attempt was made to induce endocrine changes that could mimic or exceed those of a stressed animal with anticipation of a subsequent response of semen quality and output. Figures 1 and 2 illustrate endocrine response of glucocorticoids and testosterone to the ACTH. The rapid significant elevation of total glucocorticoid concentrations to 73.5 + 4.1 ng/ml and maintenance of these high concentrations was anticipated from studies with cows (6) and bo~rs (13). Although a depressive effect on testosterone was expected, the magnitude of the drop for yearlings and the delayed testosterone response of the older male were surprising. Nevertheless, testosterone is important in reduction division of spermatocytes and may be involved in early stages of spermiogenesis (21). Therefore, low testosterone concentrations (.5 + .2 ng/ml) in the younger bulls during the 6 days of ACTH administration might be expected to have affected adversely certain aspects of spermatogenesis. However, the large endocrine imbalances that were created did not affect adversely sperm viability, ejaculate volume, sperm concentration, or weekly sperm output. With respect to sperm morphology, there was a statistically significantly increase of total cytoplasmic droplets and head abnormalities during period 4, the period that included the week of ACTH treatment and the 2 wk posttreatment. However, head abnormalities and cytoplasmic droplets were increased only from •8 + .1 and 4.2 -+ .3% pretreatment to 1.3 + .2 and 5.8 + .4% during treatment (period 4). Although it is possible that such a small increase is physiologically significant, longer periods of ACTH administraJournal of Dairy Science Vol. 68, No. 1, 1985
156
O'CONNOR ET AL.
tion w o u l d be necessary to confirm the relatively m i n o r impact on semen of the rather large e n d o c r i n e imbalance created in this study. In a study with bulls, Cupps et al. (4) r e p o r t e d that pharmacological doses of glucocorticoids administered for periods ranging f r o m 2 to 10 wk did not affect percentage live or t o t a l a b n o r m a l cells in ejaculates. H o w e v e r , using differential interference contrast microscopy, Coulter (3) observed an increase in "crater-like" sperm defects associated with a 7-day d e x a m e t h a s o n e treatment. Such defects were e x p e c t e d b u t n o t observed in our study following 6 days of A C T H treatment. A m a n n and Ganjam (2) r e p o r t e d that steroid c o n c e n t r a t i o n s in the testicular vein were considerably higher than peripheral concentrations. Thus, plasma c o n c e n t r a t i o n s in this study do n o t reflect necessarily h o r m o n e c o n t e n t within the testis. Possibly t e s t o s t e r o n e c o n c e n t r a t i o n s within the testicular p a r e n c h y m a and epididymal artery o f the bulls in this s t u d y were high enough to maintain normal spermatogenesis and epididymal f u n c t i o n . In addition, the higher c o n c e n t r a t i o n s of a n d r o s t e n e d i o n e during A C T H t r e a t m e n t m a y have provided enough c o m p e n s a t o r y androgenic activity to maintain normal gametogenesis and epididymal function. CONCLUSION
Results indicate that A C T H t r e a t m e n t for 6 days results in marked increase of glucocorticoids and can cause m a r k e d depression o f testosterone for this t i m e ; however, neither A C T H nor these a c c o m p a n y i n g endocrine changes affect semen viability or sperm o u t p u t in the bull. A slight but significant increase in sperm head abnormalities and i m m a t u r e sperm (possessing cytoplasmic droplets) was observed in response to A C T H ; however, the physiological i m p o r t a n c e of this increase is n o t apparent in view o f the wide variance in testosterone and glucocorticoid caused by the A C T H treatment. ACKNOWLEDGMENTS
We are especially grateful to J. H. Bame for her assistance in quantifying sperm abnormalities and D. A. Aalseth for the endocrine assays. This w o r k was supported partially b y Select Sires, Inc., and a Virginia Agricultural F o u n d a tion Grant. Journal of Dairy Science Vol. 68, No. 1, 1985
REFERENCES
1 Abraham, C. E., M. S. Manlimos, M. Solis, and A. C. Wickman. 1975. Combined radioimmunoassay of four steroids in one ml of plasma: II Androgens. Clin. Biochem. 8: 374. 2 Amann, R. P., and V. K. Ganjam. 1976. Steroid production by the bovine testis and steroid transfer across the pampiniform plexus. Biol. Reprod. 15:695. 3 Coulter, G. H. 1976. Effect of dexamethasone on a sperm defect. J. Anim. Sci. 43:279. 4 Cupps, P. T., R. C. Laben, D. F. Rahlmann, and A. R. Reddon. 1960. Effects of adrenal glucocorticoid and testosterone on the semen. J. Anim. Sci. 19:509. 5 Flickinger, G. L. 1966. Response of the testes to social interaction among grouped chickens. Gen. Comp. Endocrinol. 6:89. 6 Gwazdauskas, F. C., W. W. Thatcher, and C. J. Wilcox. 1972. Adrenoeorticotropin alteration of bovine peripheral plasma concentrations of cortisol, cortieosterone and progesterone. J. Dairy Sci. 55:1165. 7 Gwazdauskas, F. C., W. W. Thatcher, and C. J. Wilcox. 1973. Physiological, environmental and hormonal factors at insemination which may affect conception. J. Dairy Sci. 56:873. 8 Holtz, W., A. Hartig, and A. Konig. 1978. Effect of ACTH on plasma testosterone levels in the male Gottingen miniature pig. Pfluengers Arch. Eur. J. Physiol. 373 Suppl. R56. 9 Irvine, W. J., A. D. Taft, K. S. Wilson, R. Fraser, A. Wilson, J. Young, W. M. Hunter, A.A.A. Ismail, and P. E. Burger. 1974. The effect of synthetic corticotropin analogues on adrenocortical, anterior pituitary and testicular function, J. Clin. Endocrinol. Metab. 39:522. 10 Jaskowski, L., and L. Szule. 1968. Experiments on preventing the decrease of semen quality in bulls caused by transportation. Pages 285 in Proc. Sixth Int. Congr, Anim. Reprod. Artif. lnsem,, Paris. 11 Kattesh,/-I. G., E. T. Kornegay, F. C. Gwazdanskas, J. W. Knight, and H. R. Thomas. 1979. Peripheral plasma testosterone concentration and sexual behavior in young prenatally stressed boars. Theriogenology 12: 289. 12 Kramer, C. Y. 1972. A first course in methods of multivariate analysis. Virginia Polytechnic lnst. State Univ., Blacksburg. 13 Liptrap, A. J., and J. I. Raeside. 1975. Increases in plasma testosterone concentration after injection of ACTH into the boar. J. Endocrinol. 66:123. 14 Lodge, J. R., and G. W. Salisbury. 1970. Seasonal variation and male reproductive efficiency. Pages 739-767 in The testis. Vol. 3. A. D. Johnson, W. R. Gomes, and N. L. Van Demark, ed. Academic Press, New York, NY. 15 Murphy, B. P. 1967. Some studies of the proteinbinding of steroids and their application to the routine micro and ultramicro measurement of various steroids in competitive protein-binding radioassay. J. Clin. Endocrinol. 27:973. 16 Pasley, J. M., and J. J. Christian. 1972. The effect of ACTH, group caging, and adrenalectomy in
PRODUCTION TECHNICAL NOTE
17
18
19
20
peromyscius leucopus with emphasis on suppression of reproductive function. Proc. Soc. Exp. Biol. Med. 139:921. Saacke, R. G., and J. M. White. 1972. Semen quality tests and their relationship to fertility. Page 22 in Proc. Fourth Tech. Conf. Artif. Insem. Reprod., Chicago, IL. Shaffer, H. E., and J. O. Almquist. 1948. Vital staining o f bovine spermatozoa with an eosin-aniline blue staining mixture. J. Dairy Sci. 31:677. Skinner, J. D., and G. N. Louw. 1966. Heat stress and spermatogenesis in Bos indicus and Bos taurus cattle. J. Appl. Physiol. 21:1784. Smith, O. W., K. M o n g k o n p u n y a , H. D. Hafs, E. M. Convey, and W. D. Oxender. 1973. Blood serum
21 22
23
24
15 7
testosterone after sexual preparation or ejaculation, or after injections of LH or prolactin in bulls. J. Anim. Sci. 37:979. Steinberger, E. 1971. Hormonal control of m a m malian spermatogenesis. Physiol. Rev. 51 : 1. Thibier, M., and O. Rolland. 1976. The effect o f d e x a m e t h a z o n e (DXM) on circulating testosterone (T) and luteinizing h o r m o n e (LH) in y o u n g postpubertal bulls. Theriogenology 5:53. Van Demark, N. L., and M. J. Free. 1970. T e m p erature effects. Pages 233-312 in The testis. Vol. 3. A. D. Johnson, W. R. Gomes, and N. L. Van Demark, ed. Academic Press, New York, NY. Willet, E. L. 1957. Effect of transportation u p o n fertility of bulls. J. Dairy Sci. 40:1367.
Journal o f Dairy Science Vol. 68, No. 1, 1985