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Luteinizing Hormone and Testosterone Concentrations in Plasma of Bull Calves Treated with Gonadotropin Releasing Hormone1
L u t e i n i z i n g H o r m o n e and Testosterone C o n c e n t r a t i o n s in Plasma o f Bull Calves Treated w i t h G o n a d o t r o p i n Releasing H o r m o n e 1 D. J. KESLER and H. A. GARVERICK Department of Dairy Husbandry University of Missouri Columbia 65201 ABSTRACT Twelve bull calves, 2 to 24 days of age, were assigned to two groups of six calves and administered a single 50 pg intramuscular injection of gonadotropin releasing hormone. Bull calves in group 1 were 2 to 5 days of age (mean 3.0 days), and bull calves in group 2 were 10 to 24 days o f age (mean 17.1 days). Blood plasma for radioimmunoassay of luteinizing hormone and testosterone was collected at .5-h intervals for 3-h and at 4and 6-h following treatment. With increasing age, preinjection luteinizing hormone concentrations in plasma decreased while preinjection testosterone concentrations increased. Mean preinjection luteinizing hormone concentrations for group 1 (1.01 -+ .07 ng/ml; mean + standard error) were higher than for group 2 (.61 + .12 ng/ml). Preinjection luteinizing hormone and testosterone concentrations were correlated - . 6 3 . Following treatment, luteinizing hormone increased with mean peak concentrations of 7.28 + .74 and 6.95 +1.79 ng/ml for groups 1 and 2. Peak testosterone response following gonadotropin releasing hormone was higher for group 2 than 1 (1077 -+ 253 versus 436 + 116 pg/ml). I NTRODUCTION Rawlings et al. (8) demonstrated little change in luteinizing hormone (LH) in plasma of bulls from birth to 1 mo of age when samples were collected at monthly intervals. When samples were collected daily, Oxender et
Received November 15, 1976. 1Journal Paper No. 7697, Missouri Agrlcukura| Experiment Station, University of Missouri-Columbia. Approved by the Director.
al. (7) reported that LH in serum did not change from birth to 6 days of age in male and female calves. Testosterone in plasma, however, increased during the 1st mo following birth (8, 9). Pubertal bulls release LH in response to synthetic gonadotropin releasing hormone (GnRH) (2, 11). The earliest age prepubertal bulls have been challenged with GnRH for LH and testosterone response showed bulls at 2, 4, or 6 mo released LH equally (4). Testosterone concentrations of plasma increased in all bulls following treatment with G n R H . There have been no studies demonstrating how early bull calves can release LH and testosterone in response to stimulation by GnRH. Therefore, the objective of this study was to investigate responses of LH and testosterone in plasma to GnRH in bull calves during the first 24 days following birth.
MATERIALS AND METHODS Twelve Holstein and Guernsey bull calves ranging from 2 to 24 days of age were administered a single 50 pg intramuscular injection of GnRH. Heparinized blood samples were collected via jugular venipuncture prior to treatment (time 0) and at .5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 6.0 h following treatment with GnRH. Following collection, the blood was chilled in ice water until centrifugation at 10,000 x g for 10 min at 4 C. The plasma was stored at --20 C until assayed. Plasma LH was quantified by double antib o d y radioimmunoassay as described by Niswender et al. (6) except 12s I was used rather than 1311 as the radioactive label for the LH antigen. Radioimmunoassay procedures reported by Erb et al. (1) were used to measure testosterone in plasma. Plasma testosterone was extracted from duplicate aliquots of 100 pl of samples in one assay. Statistical analysis included correlation and 632
TECHNICAL NOTE analysis of variance (10). For analysis of variance, bull calves were assigned to two groups of six calves. Bull calves in group 1 were 2 to 5 days old (mean 3.0 days), and calves in group 2 were 10 to 24 days of age (mean 17.1 days). Significant mean differences were identified by a multiple range test as described by Kramer et al. (3). RESULTS AND DISCUSSION
Preinjection concentrations of LH in plasma decreased with increasing age up to 24 days of age (r = - . 7 7 ; P<.01). Rawlings et al. (8) reported little change from birth (.70 + .30 ng/ml; X -+ SE) to 1 mo of age (.50 + .10 ng/ml) when samples were collected at monthly intervals. In our study, bull calves 2 to 5 days of age averaged 1.01 -+ 0.7 ng/ml of plasma LH. Bull calves 10 to 24 days of age were lower (P<.02) with .61 +- .12 ng/ml of plasma LH. Oxender et al. (7) reported .36 ng/ml of plasma LH in male and female calves at birth which did not change significantly up to 6 days of age. Similarly in our study, the correlation between days of age and preinjection LH in plasma for bull calves 2 to 5 days of age was n o t significant (r = - . 2 4 ) . This agrees with Oxender et al. (7) with the exception of higher mean LH which difference could be attributed to variation in assay. Preinjection concentrations of testosterone in plasma were low following birth and increased with increasing age (r = .68; P<.05). Bull calves 2 to 5 days of age averaged 152 -+ 35 pg/ml of plasma testosterone, and calves at 10 to 24 days of age averaged 259 + 45 pg/ml. Mongkonpunya et al. (5) reported that male fetuses at 260 days of gestation averaged 210 pg/ml of testosterone in serum. In another study, testosterone in plasma of bull calves increased from undetectable concentration at birth to 720 pg/ml at 1 mo of age (8). Our results agree with those studies. Concentrations of LH and testosterone were correlated - . 6 3 (P<.05) prior to GnRH treatment. Following treatment with GnRH, LH in plasma increased about the same magnitude for all bull calves (Fig. 1). Peak LH was 7.28 -+ .74 and 6.95 -+ 1.79 ng/ml for groups 1 and 2. Differences between groups for peak LH concentrations were not significant nor was peak LH significantly correlated with age (r = - . 3 1 ; P>.05). Similarly, Mongkonpunya et al. (4)
633
10. SYMBOL
AGE
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2-5 DAYS
10-24 DAYS
4
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FIG. i . Concentrations of LH and testosterone in
plasma of bull calves following treatment (0 to 6 h) with GnRH. Each point represents the mean -+ standard error of six observations.
reported no difference in peak LH following GnRH in bull calves at 2, 4, and 6 mo of age. In our study, peak concentrations occurred at .5 h following treatment for all but one calf which had a plasma LH peak at 1.0 h following treatment. Testosterone in plasma increased following GnRH treatment to peak concentrations of 436 + 116 and 1077 + 253 pg/ml for groups 1 and 2. Peak testosterone was correlated with days of age .76 (P<.01), and the values for group 2 were higher than those for group 1 (P<.05). The increase in testosterone from 0 to 6.0 h following treatment was significant ( P < . 0 1 ) f o r group 2 only. By 6 h following GnRH treatment, LH and testosterone concentrations in plasma had returned to preinjection concentrations. Mongkonpunya et al. (4) gave bulls at 2, 4, and 6 mo of age different dosages of GnRH. Serum testosterone increased following treatment at all ages, and the increase was three-fold Journal of Dairy Science Vol. 60, No. 4
634
KESLER AND GARVERICK
in b u l l s 6 m o o f age. In bulls at 2 a n d 4 m o of age, circulating t e s t o s t e r o n e increased f r o m .4 a n d .6 n g / m l t o .6 a n d 1.2 n g / m l a t 2 t o 3 h f o l l o w i n g G n R H t r e a t m e n t . In c o n t r a s t , in 6-too old bulls, s e r u m t e s t o s t e r o n e averaged 1.7 n g / m l p r i o r t o t r e a t m e n t a n d p e a k e d at 5.3 ng/ml. T h e s e results d e m o n s t r a t e t h a t p i t u i t a r y r e s p o n s i v e n e s s to G n R H in bull calves does n o t c h a n g e t h e first 24 days f o l l o w i n g birth. However, t e s t i c u l a r r e s p o n s e to G n R H - i n d u c e d LH release, as e v i d e n c e d b y increased circulating t e s t o s t e r o n e , does increase. Results h a v e b e e n similar in bull calves 2 t o 6 m o of age (4). ACKNOWLEDGMENTS
T h e a u t h o r s are i n d e b t e d to G. D. N i s w e n d e r and S . A . Tillson f o r a n t i b o d i e s for r a d i o i m m u n o a s s a y o f L H a n d t e s t o s t e r o n e , respectively, a n d t o M. D. Brown, A b b o t t L a b o r a t o r i e s , f o r s u p p l y i n g G n R H . T h e a u t h o r s also express a p p r e c i a t i o n t o J a n e t L i e b e r t for assistance w i t h h o r m o n e assays a n d to Phyllis Kuschel for secretarial assistance in t y p i n g o f m a n u s c r i p t .
REFERENCES
1 Erb, R. E., E. L. Monk, T. A. Mollett, P. V. Malven, and C. J. Callahan. 1976. Estrogen, progesterone, prolactin, and other changes associated with bovine lactation induced with estradiol-173 and progesterone. J. Anita. Sci. 42:644. 2 Goiter, T. D., J. J. Reeves, C. C. O'Mary, A.
Journal of Dairy Science Vol. 60, No. 4
Arimura, and A. V. Schally. 1973. Serum LH levels in bulls treated with synthetic luteinizing hormone-releasing hormone/follicle stimulating hormone-releasing hormone (LH-RH/FSH-RH). J. Anita. Sci. 37:123. 3 Kramer, C. Y. 1956. Extension of multiple range test to group means with unequal numbers of replications. Biometrics 12: 307. Mongkonpunya, K., H. D. Hafs, E. M. Convey, H. A. Tucker, and W. D. Oxender. 1975. Serum luteinizing hormone, testosterone and androstenedione in pubertal and prepubertal bulls after gonadotropin releasing hormone. J. Anita. Sci. 40: 682. 5 Mongkonpunya, K., Y. C. Lin, P. A. Noden, W. D. Oxender, and H. D. Hafs. 1975. Androgens in the bovine fetus and dam. Proc. Soc. Exp. Biol. Med. 148:489. 6 Niswender, G. D., L. E. Richert, A. B. Midgley, and A. V. Nalbandov. 1969. Radioimmunoassay of bovine and ovine luteinizing hormone. Endocrinology 84:1166. 7 0 x e n d e r , W. D., H. D. Hafs, and W. G. Ingalls. 1972. Serum growth hormone, LH and prolactin in the bovine fetus and neonate. J. Anita. Sci. 35:56. 8 Rawlings, N. C., H. D. Hafs, and L. V. Swanson. 1972. Testicular and blood plasma androgens in Holstein bulls from birth through puberty. J. Anita. Sci. 34:435. 9 Secchiari, P., F. Martorana, S. Pellegrlni, and M. Luisi. 1976. Variation of plasma testosterone in developing Friesian bulls. J. Anita. Sci. 42:405. 10 Snedecor, G. W., and W. G. Cochran. 1967. Statistical Methods (6th Ed.). The Iowa State College Press, Ames. 11 Zolman, J., E. M. Convey, J. H. Britt, and H. D. Hafs. 1973. Release of bovine luteinizing hormone by purified porcine and synthetic gonadotropin re/easing hormone. Proc. Soc. Exp. Biol. Med. 142:189.
Received November 15, 1976. 1Journal Paper No. 7697, Missouri Agrlcukura| Experiment Station, University of Missouri-Columbia. Approved by the Director.
al. (7) reported that LH in serum did not change from birth to 6 days of age in male and female calves. Testosterone in plasma, however, increased during the 1st mo following birth (8, 9). Pubertal bulls release LH in response to synthetic gonadotropin releasing hormone (GnRH) (2, 11). The earliest age prepubertal bulls have been challenged with GnRH for LH and testosterone response showed bulls at 2, 4, or 6 mo released LH equally (4). Testosterone concentrations of plasma increased in all bulls following treatment with G n R H . There have been no studies demonstrating how early bull calves can release LH and testosterone in response to stimulation by GnRH. Therefore, the objective of this study was to investigate responses of LH and testosterone in plasma to GnRH in bull calves during the first 24 days following birth.
MATERIALS AND METHODS Twelve Holstein and Guernsey bull calves ranging from 2 to 24 days of age were administered a single 50 pg intramuscular injection of GnRH. Heparinized blood samples were collected via jugular venipuncture prior to treatment (time 0) and at .5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 6.0 h following treatment with GnRH. Following collection, the blood was chilled in ice water until centrifugation at 10,000 x g for 10 min at 4 C. The plasma was stored at --20 C until assayed. Plasma LH was quantified by double antib o d y radioimmunoassay as described by Niswender et al. (6) except 12s I was used rather than 1311 as the radioactive label for the LH antigen. Radioimmunoassay procedures reported by Erb et al. (1) were used to measure testosterone in plasma. Plasma testosterone was extracted from duplicate aliquots of 100 pl of samples in one assay. Statistical analysis included correlation and 632
TECHNICAL NOTE analysis of variance (10). For analysis of variance, bull calves were assigned to two groups of six calves. Bull calves in group 1 were 2 to 5 days old (mean 3.0 days), and calves in group 2 were 10 to 24 days of age (mean 17.1 days). Significant mean differences were identified by a multiple range test as described by Kramer et al. (3). RESULTS AND DISCUSSION
Preinjection concentrations of LH in plasma decreased with increasing age up to 24 days of age (r = - . 7 7 ; P<.01). Rawlings et al. (8) reported little change from birth (.70 + .30 ng/ml; X -+ SE) to 1 mo of age (.50 + .10 ng/ml) when samples were collected at monthly intervals. In our study, bull calves 2 to 5 days of age averaged 1.01 -+ 0.7 ng/ml of plasma LH. Bull calves 10 to 24 days of age were lower (P<.02) with .61 +- .12 ng/ml of plasma LH. Oxender et al. (7) reported .36 ng/ml of plasma LH in male and female calves at birth which did not change significantly up to 6 days of age. Similarly in our study, the correlation between days of age and preinjection LH in plasma for bull calves 2 to 5 days of age was n o t significant (r = - . 2 4 ) . This agrees with Oxender et al. (7) with the exception of higher mean LH which difference could be attributed to variation in assay. Preinjection concentrations of testosterone in plasma were low following birth and increased with increasing age (r = .68; P<.05). Bull calves 2 to 5 days of age averaged 152 -+ 35 pg/ml of plasma testosterone, and calves at 10 to 24 days of age averaged 259 + 45 pg/ml. Mongkonpunya et al. (5) reported that male fetuses at 260 days of gestation averaged 210 pg/ml of testosterone in serum. In another study, testosterone in plasma of bull calves increased from undetectable concentration at birth to 720 pg/ml at 1 mo of age (8). Our results agree with those studies. Concentrations of LH and testosterone were correlated - . 6 3 (P<.05) prior to GnRH treatment. Following treatment with GnRH, LH in plasma increased about the same magnitude for all bull calves (Fig. 1). Peak LH was 7.28 -+ .74 and 6.95 -+ 1.79 ng/ml for groups 1 and 2. Differences between groups for peak LH concentrations were not significant nor was peak LH significantly correlated with age (r = - . 3 1 ; P>.05). Similarly, Mongkonpunya et al. (4)
633
10. SYMBOL
AGE
~--o
8
5 =,
~
~
2-5 DAYS
10-24 DAYS
4
:
!I 0
l
$
8110
"7
2OO
0
~
0
1,O
2.0
5.0
~,0
6.0
TIME FOLLOWINGINJECTION (He)
FIG. i . Concentrations of LH and testosterone in
plasma of bull calves following treatment (0 to 6 h) with GnRH. Each point represents the mean -+ standard error of six observations.
reported no difference in peak LH following GnRH in bull calves at 2, 4, and 6 mo of age. In our study, peak concentrations occurred at .5 h following treatment for all but one calf which had a plasma LH peak at 1.0 h following treatment. Testosterone in plasma increased following GnRH treatment to peak concentrations of 436 + 116 and 1077 + 253 pg/ml for groups 1 and 2. Peak testosterone was correlated with days of age .76 (P<.01), and the values for group 2 were higher than those for group 1 (P<.05). The increase in testosterone from 0 to 6.0 h following treatment was significant ( P < . 0 1 ) f o r group 2 only. By 6 h following GnRH treatment, LH and testosterone concentrations in plasma had returned to preinjection concentrations. Mongkonpunya et al. (4) gave bulls at 2, 4, and 6 mo of age different dosages of GnRH. Serum testosterone increased following treatment at all ages, and the increase was three-fold Journal of Dairy Science Vol. 60, No. 4
634
KESLER AND GARVERICK
in b u l l s 6 m o o f age. In bulls at 2 a n d 4 m o of age, circulating t e s t o s t e r o n e increased f r o m .4 a n d .6 n g / m l t o .6 a n d 1.2 n g / m l a t 2 t o 3 h f o l l o w i n g G n R H t r e a t m e n t . In c o n t r a s t , in 6-too old bulls, s e r u m t e s t o s t e r o n e averaged 1.7 n g / m l p r i o r t o t r e a t m e n t a n d p e a k e d at 5.3 ng/ml. T h e s e results d e m o n s t r a t e t h a t p i t u i t a r y r e s p o n s i v e n e s s to G n R H in bull calves does n o t c h a n g e t h e first 24 days f o l l o w i n g birth. However, t e s t i c u l a r r e s p o n s e to G n R H - i n d u c e d LH release, as e v i d e n c e d b y increased circulating t e s t o s t e r o n e , does increase. Results h a v e b e e n similar in bull calves 2 t o 6 m o of age (4). ACKNOWLEDGMENTS
T h e a u t h o r s are i n d e b t e d to G. D. N i s w e n d e r and S . A . Tillson f o r a n t i b o d i e s for r a d i o i m m u n o a s s a y o f L H a n d t e s t o s t e r o n e , respectively, a n d t o M. D. Brown, A b b o t t L a b o r a t o r i e s , f o r s u p p l y i n g G n R H . T h e a u t h o r s also express a p p r e c i a t i o n t o J a n e t L i e b e r t for assistance w i t h h o r m o n e assays a n d to Phyllis Kuschel for secretarial assistance in t y p i n g o f m a n u s c r i p t .
REFERENCES
1 Erb, R. E., E. L. Monk, T. A. Mollett, P. V. Malven, and C. J. Callahan. 1976. Estrogen, progesterone, prolactin, and other changes associated with bovine lactation induced with estradiol-173 and progesterone. J. Anita. Sci. 42:644. 2 Goiter, T. D., J. J. Reeves, C. C. O'Mary, A.
Journal of Dairy Science Vol. 60, No. 4
Arimura, and A. V. Schally. 1973. Serum LH levels in bulls treated with synthetic luteinizing hormone-releasing hormone/follicle stimulating hormone-releasing hormone (LH-RH/FSH-RH). J. Anita. Sci. 37:123. 3 Kramer, C. Y. 1956. Extension of multiple range test to group means with unequal numbers of replications. Biometrics 12: 307. Mongkonpunya, K., H. D. Hafs, E. M. Convey, H. A. Tucker, and W. D. Oxender. 1975. Serum luteinizing hormone, testosterone and androstenedione in pubertal and prepubertal bulls after gonadotropin releasing hormone. J. Anita. Sci. 40: 682. 5 Mongkonpunya, K., Y. C. Lin, P. A. Noden, W. D. Oxender, and H. D. Hafs. 1975. Androgens in the bovine fetus and dam. Proc. Soc. Exp. Biol. Med. 148:489. 6 Niswender, G. D., L. E. Richert, A. B. Midgley, and A. V. Nalbandov. 1969. Radioimmunoassay of bovine and ovine luteinizing hormone. Endocrinology 84:1166. 7 0 x e n d e r , W. D., H. D. Hafs, and W. G. Ingalls. 1972. Serum growth hormone, LH and prolactin in the bovine fetus and neonate. J. Anita. Sci. 35:56. 8 Rawlings, N. C., H. D. Hafs, and L. V. Swanson. 1972. Testicular and blood plasma androgens in Holstein bulls from birth through puberty. J. Anita. Sci. 34:435. 9 Secchiari, P., F. Martorana, S. Pellegrlni, and M. Luisi. 1976. Variation of plasma testosterone in developing Friesian bulls. J. Anita. Sci. 42:405. 10 Snedecor, G. W., and W. G. Cochran. 1967. Statistical Methods (6th Ed.). The Iowa State College Press, Ames. 11 Zolman, J., E. M. Convey, J. H. Britt, and H. D. Hafs. 1973. Release of bovine luteinizing hormone by purified porcine and synthetic gonadotropin re/easing hormone. Proc. Soc. Exp. Biol. Med. 142:189.