- Email: [email protected]
Plasma LH and testosterone in Zebu crossbred bulls after exposure to an estrous cow and injection of synthetic GnRH
THERIOGENOLOGY
PLASMA LH AND TESTOSTERONE IN ZEBU CROSSBRED BULLS AFTER EXPOSURE TO AN ESTROUS COW AND INJECTION OF SYNTHETIC GnRH B.M. Bindon, R.W. Hewetson B T.B. Post. C.S.I.R.O., Animal Genetics, P.O. Box 90, Epping; Wollongbar Research Centre, Lismore; and Cattle Research Laboratory, Rockhampton, AUSTRALIA. (Received for publication, December8, 1975 ABSTRACT Plasma hormone levels were examined in 4 mature Zebu bulls of normal libido (HL) and 4 which were sexually inactive (LL). When used in an artificial insemination programme the 8 bulls had similar fertility. Basal levels of LH and testosterone (T) estimated from 8 sequential blood samples at 30 minute intervals were not different in HL and LL bulls. Exposure of the animals to an estrous cow did not stimulate LH release. Following sexual stimulation plasma T levels actually decreased by an average ('S-E) of 2.9 (c1.9) ng/ml in the HL group and increased by 3.9 (k1.6) ng/ml in the LL group. An injection of 1 mg GnRH (Hoechst) caused LH release of similar magnitude in HL and LL bulls. The elevation of plasma T which followed GnRH injection was significantly larger in HL bulls. Low libido was not associated with a deficiency of basal LH or T, nor with the ability of the pituitary to respond to GnRH. INTRODUCTION The hormonal characteristics of bulls with low sexual drive are not well understood. An investigation designed to study plasma LH and testosterone patterns before and after attempted sexual stimulation and GnRH injection in bulls with normal and abnormal sexual drive is reported here. Synthetic GnRH was generously supplied by Farbwerke Hoechst *g. The work was supported by the Australian Meat Research Committee.
FEBRUARY
1976 VOL. 5 NO. 2
45
TEERIOGENOLOGY METHODS 8, RESULTS The study involved 8 crossbred Bos indicus bulls all aged 2.5 yr, four with normal libido_TRL),oneith low libido and three which were sexually inactive (LL). Semen was routinely collected by artificial vagina (HL bulls) or electroejaculation (LL) bulls. When used in an artificial insemination programme the fertility of the semen from the 8 bulls was similar. Three of the four LL bulls had never served a cow. In April, 1974, jugular blood samples were drawn by venepuncture from each bull at intervals of 30 minutes from 09.00 hr until 12.30 hr. At 12.50 hr the bulls were individually exposed to a restrained, estrous cow until an ejaculatory service was completed or until 4 minutes had Further blood samples were drawn at intervals of elapsed. 10 and 40 minutes after exposure. At this time each bull was injected intramuscularly with 1 mg synthetic GnRH (Hoechst) and blood samples drawn again 60 and 90 minutes later. Plasma samples were assayed for LH by radioimmunassay (1) using anti-ovine LH antibodies and NIH-LH-B3 as Testosterone levels were assessed by radiostandard. immunassay as follows. Testosterone was extracted from 0.1 ml of plasma with An aliquot (0.5 ml) of the 1 ml of toluene:hexane (1:l). extract was evaporated with a stream of nitrogen and the residue incubated overnight at 4oC with 0.5 ml of rabbit antiserum to testosterone (Calbiochem, cospecific for testosterone and dihydrotestosterone). The antiserum, was diluted another 1000 times initially diluted l:lOOO, before use with 0. OSM borate buffer (pH 7.8) containing 0.5% horse serum and 8 nCi 18,26 3H-testosterone (RadioTo separate free from bound chemical Center, Amersham). testosterone 0.5 ml of saturated ammonium sulphate was After centrifugation at added and incubated 10 minutes. 11OOxg for 15 min at 4oC the supernatant (free fraction) was poured into a scintillation vial and extracted into 10 ml of toluene scintillation solution (0.05 gm POPOP and 0.4 gm PPO per liter of toluene) for counting. Testosterone standards (Sigma Chemical Co.) were treated similarly. The effective range of the standard curve was 20 to 400 pg. The results for sexual behaviour and individual testosterone changes are presented in Table 1 and the mean LH and testosterone patterns for the two groups of bulls All bulls behaved exactly as their shown in Text-Fig. 1. ejaculatory services being previous history suggested, completed by HL bulls within seconds of exposure to the
FEBRUARY
1976 VOL.5 NO.2
Sexual drive low or absent ILL)
Normal sexual drive (HL)
Group
Bull No.
to cow
levels
(7.4-19.4) (9.2-17.0) (7.0-19.6) (0.8-6.8)
13.7 13.1 13.0 2.5
-0.2 0.7 0.9
at
+4.9 19.6
14.7
30 min.
+7.4
+3.5
15.0 11.5
10.5
-2.0 13.6
15.6
3.1
-0.2 0.4
-0.8
0.6
1.4
I Change (* ng)
cow
-8.6
,
ng/ml
estrous
40 min after exposure
an
3.2
11.8
2.2
to
Testosterone
exposed
20 min before exposure
in bulls
of values observed in 8 samples estrous cow. to exposure to
(1.0-7.9)
(0.6-2.3)
1.1
2.8
(7.1-17.2)
(2.2-15.0)
11.4
7.4
Basal testosterone* q/ml
and testosterone
2 unsuccessful attempts at intromission
No interest
Immediate ejaculatory service
Response estrous
response
* Mean and range intervals prior
Sexual
TABLE 1
THERIOGENOLOGY
testrous retired;
cow. the
One LL bull made two attempts to mount, others showed complete disinterest.
then
Basal levels of LH were not significantly different for HL and LL bulls. Sequential samples from each animal did not fluctuate greatly from a mean of 1.7 ng/ml although one peak of 10 ng/ml was recorded, The LH pattern in either HL or LL bulls was unaffected by exposure to an estrous cow. After GnRH injection there was a marked increase in plasma LH but this also was similar for HL and LL animals. Although there was no consistent relation between fluctuating patterns of LH and testosterone in the same bull, (the values did not always rise and fall together) there was a significant correlation (r = +0.83) between the mean LH and mean testosterone levels of the 8 bulls during the period up to injection of GnRH. Bulls with highest average LH had highest average testosterone. From Table 1 it can be seen that in the period prior to contact with the estrous cow basal testosterone levels were not consistently different between HL and LL bulls. Some animals from each group had high and some low basal In the 40 minutes after exposure to the cow testoslevels. terone levels actually decreased in the HL group (mean change = -2.9 ng) and increased in the LL group (mean = +3.9 ng). This difference,although not large, is significant (t = 2.73; p < 0.05). It is not possible to conclude that these changes were the direct result of exposure to the estrous cow, although it does confirm that ejaculation is not always followed by an elevation of T levels. In other studies (2) plasma testosterone of bulls was observed to increase after ejaculation. Following GnRH injection the augmentation of plasma LH was accompanied by a rise in testosterone levels (TextThis rise was significantly larger in HL bulls. Fig. 1). Further analysis of the data, however, show that this increase was negatively correlated (r 1-0.88) with the level of testosterone immediately before injection of GnRH (TextSince HL bulls had generally lower testosterone Fig. 2). at this time, it is possible that thelarger response in this group of bulls was due to this factor, rather than any special ability of the testis to respond to LH. Further investigations of the testosterone response to GnRH induced LH release by such animals may resolve this point. DISCUSSION These data leave a rather confused picture of the relation between LH, testosterone and sexual drive in the In the small sample of animals studied it seems bull. fair to conclude that low libido was not associated with a
FEBRUARY
1976
VOL. 5 NO. 2
.5
I
I.5 2 thr)
3
Text-Fig.
Tim-
2.5
1
3.5
4
4.5
5
5.5
6
Patterns of LH (0 = LL, A = HL) and testosterone (A-----= LL, A =HL) before and after exposure of bulls to an oestrous cow (9) and an injection of 1 mg GnRH(+) SE values for testosterone are shown only once since the within-group variation did not change throughout the experiment. SE values for LH are shown twice since within-group variation increased after GnRHinjection.
0
IO
100
120
g .O K cc
0
Text-Fig.
a
2
The correlation (r = -0.88) between the level of testosterone at the time of GnRHinjec0 = I-ILbulls; 0 = LL bulls. tion and change in testosterone level 90 minutes later.
0
THERIOGENOLOGY deficiency of basal LH or testosterone nor with an inability of the pituitary gland to respond to GnRH. The results are in agreement with the fact that injections of testosterone are not consistently successful in reversing the condition of low libido in bulls such as those studied here (R. W. Hewetson, unpublished). Basal LH values reported here are somewhat lower than those for British breed bulls in the same environment and the lack of an LH response to contact with an estrous cow confirms our previous observations on Bos indfcus to confirm bulls (3). Other studies (4,s) have also Eled elevation of LH in bulls after sexual stimulation as previously reported (6).
FEBRUARY
1976 VOL. 5 NO.2
51
THERIOGENOLOGY
REFERENCES (1) Goding, J-R., Katt, K.J., Brown, J.M., Kaltenbach, C.C., Cumming, I.A. and Mole, B.J. Radioimmunoassay for ovine luteinizing hormone. Secretion of luteinizing hormone during estrus and following estrogen administration in the sheep. EndoErinology, 82: 133-142 (1969). (2) Smith, O.W., Mongkonpunya, K., Hafs, H.D., Convey, E.M. and Oxender, W.D. Blood serum testosterone after sexual preparation or ejaculation or after injections of LH or prolactin in bulls. J. Anim. w., 22: 979-984 (1973). (3) Bindon, B.M., Post, T.B. and Hewetson, R.W. Patterns of LH release in mature Zebu crossbred and British breed bulls. J. Reprod. Fert., 36: 450-452 (1974). (4) Convey, E-M., Bretschneider, E., Hafs, H.D. and Oxender, W.D. Serum levels of LH, prolactin and growth hormone after ejaculation in bulls. Biol. Reprod., ,5: 20-24 (1971). (5) Gombe, S., Hall, W.C., McEntee, K., Hansel, W. and Pickett, B.W. Regulation of blood levels of LH in bulls: Influence of age, breed, sexual stimulation and temporal fluctuations. J. Reprod. Fert., 22: 493-503 (1973). (6) Katongole, C.B., Naftolin, F. and Short, R.V. Relationship between blood levels of luteinizing hormone and testosterone in bulls and the effects of sexual stimulation. J. Endocr., 29: 457-466 (1971).
52
FEBRUARY
1976 VOL. 5 NO. 2
PLASMA LH AND TESTOSTERONE IN ZEBU CROSSBRED BULLS AFTER EXPOSURE TO AN ESTROUS COW AND INJECTION OF SYNTHETIC GnRH B.M. Bindon, R.W. Hewetson B T.B. Post. C.S.I.R.O., Animal Genetics, P.O. Box 90, Epping; Wollongbar Research Centre, Lismore; and Cattle Research Laboratory, Rockhampton, AUSTRALIA. (Received for publication, December8, 1975 ABSTRACT Plasma hormone levels were examined in 4 mature Zebu bulls of normal libido (HL) and 4 which were sexually inactive (LL). When used in an artificial insemination programme the 8 bulls had similar fertility. Basal levels of LH and testosterone (T) estimated from 8 sequential blood samples at 30 minute intervals were not different in HL and LL bulls. Exposure of the animals to an estrous cow did not stimulate LH release. Following sexual stimulation plasma T levels actually decreased by an average ('S-E) of 2.9 (c1.9) ng/ml in the HL group and increased by 3.9 (k1.6) ng/ml in the LL group. An injection of 1 mg GnRH (Hoechst) caused LH release of similar magnitude in HL and LL bulls. The elevation of plasma T which followed GnRH injection was significantly larger in HL bulls. Low libido was not associated with a deficiency of basal LH or T, nor with the ability of the pituitary to respond to GnRH. INTRODUCTION The hormonal characteristics of bulls with low sexual drive are not well understood. An investigation designed to study plasma LH and testosterone patterns before and after attempted sexual stimulation and GnRH injection in bulls with normal and abnormal sexual drive is reported here. Synthetic GnRH was generously supplied by Farbwerke Hoechst *g. The work was supported by the Australian Meat Research Committee.
FEBRUARY
1976 VOL. 5 NO. 2
45
TEERIOGENOLOGY METHODS 8, RESULTS The study involved 8 crossbred Bos indicus bulls all aged 2.5 yr, four with normal libido_TRL),oneith low libido and three which were sexually inactive (LL). Semen was routinely collected by artificial vagina (HL bulls) or electroejaculation (LL) bulls. When used in an artificial insemination programme the fertility of the semen from the 8 bulls was similar. Three of the four LL bulls had never served a cow. In April, 1974, jugular blood samples were drawn by venepuncture from each bull at intervals of 30 minutes from 09.00 hr until 12.30 hr. At 12.50 hr the bulls were individually exposed to a restrained, estrous cow until an ejaculatory service was completed or until 4 minutes had Further blood samples were drawn at intervals of elapsed. 10 and 40 minutes after exposure. At this time each bull was injected intramuscularly with 1 mg synthetic GnRH (Hoechst) and blood samples drawn again 60 and 90 minutes later. Plasma samples were assayed for LH by radioimmunassay (1) using anti-ovine LH antibodies and NIH-LH-B3 as Testosterone levels were assessed by radiostandard. immunassay as follows. Testosterone was extracted from 0.1 ml of plasma with An aliquot (0.5 ml) of the 1 ml of toluene:hexane (1:l). extract was evaporated with a stream of nitrogen and the residue incubated overnight at 4oC with 0.5 ml of rabbit antiserum to testosterone (Calbiochem, cospecific for testosterone and dihydrotestosterone). The antiserum, was diluted another 1000 times initially diluted l:lOOO, before use with 0. OSM borate buffer (pH 7.8) containing 0.5% horse serum and 8 nCi 18,26 3H-testosterone (RadioTo separate free from bound chemical Center, Amersham). testosterone 0.5 ml of saturated ammonium sulphate was After centrifugation at added and incubated 10 minutes. 11OOxg for 15 min at 4oC the supernatant (free fraction) was poured into a scintillation vial and extracted into 10 ml of toluene scintillation solution (0.05 gm POPOP and 0.4 gm PPO per liter of toluene) for counting. Testosterone standards (Sigma Chemical Co.) were treated similarly. The effective range of the standard curve was 20 to 400 pg. The results for sexual behaviour and individual testosterone changes are presented in Table 1 and the mean LH and testosterone patterns for the two groups of bulls All bulls behaved exactly as their shown in Text-Fig. 1. ejaculatory services being previous history suggested, completed by HL bulls within seconds of exposure to the
FEBRUARY
1976 VOL.5 NO.2
Sexual drive low or absent ILL)
Normal sexual drive (HL)
Group
Bull No.
to cow
levels
(7.4-19.4) (9.2-17.0) (7.0-19.6) (0.8-6.8)
13.7 13.1 13.0 2.5
-0.2 0.7 0.9
at
+4.9 19.6
14.7
30 min.
+7.4
+3.5
15.0 11.5
10.5
-2.0 13.6
15.6
3.1
-0.2 0.4
-0.8
0.6
1.4
I Change (* ng)
cow
-8.6
,
ng/ml
estrous
40 min after exposure
an
3.2
11.8
2.2
to
Testosterone
exposed
20 min before exposure
in bulls
of values observed in 8 samples estrous cow. to exposure to
(1.0-7.9)
(0.6-2.3)
1.1
2.8
(7.1-17.2)
(2.2-15.0)
11.4
7.4
Basal testosterone* q/ml
and testosterone
2 unsuccessful attempts at intromission
No interest
Immediate ejaculatory service
Response estrous
response
* Mean and range intervals prior
Sexual
TABLE 1
THERIOGENOLOGY
testrous retired;
cow. the
One LL bull made two attempts to mount, others showed complete disinterest.
then
Basal levels of LH were not significantly different for HL and LL bulls. Sequential samples from each animal did not fluctuate greatly from a mean of 1.7 ng/ml although one peak of 10 ng/ml was recorded, The LH pattern in either HL or LL bulls was unaffected by exposure to an estrous cow. After GnRH injection there was a marked increase in plasma LH but this also was similar for HL and LL animals. Although there was no consistent relation between fluctuating patterns of LH and testosterone in the same bull, (the values did not always rise and fall together) there was a significant correlation (r = +0.83) between the mean LH and mean testosterone levels of the 8 bulls during the period up to injection of GnRH. Bulls with highest average LH had highest average testosterone. From Table 1 it can be seen that in the period prior to contact with the estrous cow basal testosterone levels were not consistently different between HL and LL bulls. Some animals from each group had high and some low basal In the 40 minutes after exposure to the cow testoslevels. terone levels actually decreased in the HL group (mean change = -2.9 ng) and increased in the LL group (mean = +3.9 ng). This difference,although not large, is significant (t = 2.73; p < 0.05). It is not possible to conclude that these changes were the direct result of exposure to the estrous cow, although it does confirm that ejaculation is not always followed by an elevation of T levels. In other studies (2) plasma testosterone of bulls was observed to increase after ejaculation. Following GnRH injection the augmentation of plasma LH was accompanied by a rise in testosterone levels (TextThis rise was significantly larger in HL bulls. Fig. 1). Further analysis of the data, however, show that this increase was negatively correlated (r 1-0.88) with the level of testosterone immediately before injection of GnRH (TextSince HL bulls had generally lower testosterone Fig. 2). at this time, it is possible that thelarger response in this group of bulls was due to this factor, rather than any special ability of the testis to respond to LH. Further investigations of the testosterone response to GnRH induced LH release by such animals may resolve this point. DISCUSSION These data leave a rather confused picture of the relation between LH, testosterone and sexual drive in the In the small sample of animals studied it seems bull. fair to conclude that low libido was not associated with a
FEBRUARY
1976
VOL. 5 NO. 2
.5
I
I.5 2 thr)
3
Text-Fig.
Tim-
2.5
1
3.5
4
4.5
5
5.5
6
Patterns of LH (0 = LL, A = HL) and testosterone (A-----= LL, A =HL) before and after exposure of bulls to an oestrous cow (9) and an injection of 1 mg GnRH(+) SE values for testosterone are shown only once since the within-group variation did not change throughout the experiment. SE values for LH are shown twice since within-group variation increased after GnRHinjection.
0
IO
100
120
g .O K cc
0
Text-Fig.
a
2
The correlation (r = -0.88) between the level of testosterone at the time of GnRHinjec0 = I-ILbulls; 0 = LL bulls. tion and change in testosterone level 90 minutes later.
0
THERIOGENOLOGY deficiency of basal LH or testosterone nor with an inability of the pituitary gland to respond to GnRH. The results are in agreement with the fact that injections of testosterone are not consistently successful in reversing the condition of low libido in bulls such as those studied here (R. W. Hewetson, unpublished). Basal LH values reported here are somewhat lower than those for British breed bulls in the same environment and the lack of an LH response to contact with an estrous cow confirms our previous observations on Bos indfcus to confirm bulls (3). Other studies (4,s) have also Eled elevation of LH in bulls after sexual stimulation as previously reported (6).
FEBRUARY
1976 VOL. 5 NO.2
51
THERIOGENOLOGY
REFERENCES (1) Goding, J-R., Katt, K.J., Brown, J.M., Kaltenbach, C.C., Cumming, I.A. and Mole, B.J. Radioimmunoassay for ovine luteinizing hormone. Secretion of luteinizing hormone during estrus and following estrogen administration in the sheep. EndoErinology, 82: 133-142 (1969). (2) Smith, O.W., Mongkonpunya, K., Hafs, H.D., Convey, E.M. and Oxender, W.D. Blood serum testosterone after sexual preparation or ejaculation or after injections of LH or prolactin in bulls. J. Anim. w., 22: 979-984 (1973). (3) Bindon, B.M., Post, T.B. and Hewetson, R.W. Patterns of LH release in mature Zebu crossbred and British breed bulls. J. Reprod. Fert., 36: 450-452 (1974). (4) Convey, E-M., Bretschneider, E., Hafs, H.D. and Oxender, W.D. Serum levels of LH, prolactin and growth hormone after ejaculation in bulls. Biol. Reprod., ,5: 20-24 (1971). (5) Gombe, S., Hall, W.C., McEntee, K., Hansel, W. and Pickett, B.W. Regulation of blood levels of LH in bulls: Influence of age, breed, sexual stimulation and temporal fluctuations. J. Reprod. Fert., 22: 493-503 (1973). (6) Katongole, C.B., Naftolin, F. and Short, R.V. Relationship between blood levels of luteinizing hormone and testosterone in bulls and the effects of sexual stimulation. J. Endocr., 29: 457-466 (1971).
52
FEBRUARY
1976 VOL. 5 NO. 2