Responsiveness of prepubertal crossbred bull calves to exogenous GnRH and its impact on reproductive hormones under tropical conditions
© Bharath Kumar et al. 2016
Received: 1 December 2015
Accepted: 1 March 2016
Published: 8 March 2016
This study investigated the age related variations in luteinizing hormone (LH), androstenedione, testosterone, and total estrogens response to exogenous gonadotropin-releasing hormone (GnRH) in Holstein–Friesian (HF) × Tharparkar bull calves. Fifteen bull calves were selected and, based on their age, were divided into Group I (14–16 months, n = 5), Group II (9–12 months, n = 5), and Group III (6–8 months, n = 5). All bull calves were administered with 10 µg of GnRH intramuscularly. Blood samples were collected at an interval of 30 min commencing 1 h prior to GnRH treatment until 4 h post-GnRH treatment and thereafter, at an interval of 1 h for the next 3 h. Endocrine response in terms of pretreatment values, peak values, area under curve, and time taken to attain peak values for LH, androstenedione, testosterone, and total estrogens was evaluated in all the bull calves. Significant differences were observed in pretreatment values, peak concentrations, and area under curve for androstenedione and testosterone between the groups; with response being higher in Group I bull calves. The results indicated that the HF × Tharparkar bull calves of 14 months age and above respond to exogenous GnRH by secreting significant amounts of testosterone.
The Holstein–Friesian (HF) × Tharparkar has evolved as a potent milk producer among various cross breeds in Tropical India. However, male offspring’s of this cross breed are inferior to Bos indicus and Bos taurus in semen production. Impaired semen production capacity, poor libido, and low freezability are the major reasons for rejection of these crossbreds in semen stations (Sethi et al. 1989; Bhavsar 1993; Sahni and Mohan 1998). From the data collected over a period of 15 years, Mukhopadhyay et al. (2010) observed that the mean ± SE age at first semen collection (AFSC) in HF × Tharparkar bulls was 872 ± 19.1 days (~27 months). Testosterone and gonadotropins are essential to initiate and support the process of spermatogenesis (Kerr et al. 1993). Follicle stimulating hormone acts synergistically with testosterone to influence the efficiency of spermatogenesis and fertility (Sharpe 1994; Mc Lachlan et al. 1994). The trend line for testosterone profiles in growing Sahiwal (indigenous breed) males indicated an exponential increase in testosterone with age when compared to an almost linear increase in HF × Tharparkar males (Gulia et al. 2010), indicating low testosterone production with age as probable cause for poor libido and poor semen production in these crossbred males.
Administration of GnRH analogue, on a weekly basis, to Egyptian puberal buffalo bulls of 15–18 months of age significantly improved the libido and semen quality (El-Khawaga et al. 2011). Bulls provided with additional energy in the diet combined with weekly administration of GnRH significantly increased the testosterone levels and scrotal circumference in comparison to bulls that were fed only with the additional energy in the diet (Ali et al. 2012). The combined strategy of providing additional energy in the diet with weekly administration of GnRH to pre-pubertal HF × Tharparkar bull calves might augment testosterone levels and decrease the AFSC. For these reasons, the age at which the pre-pubertal bull calves respond to exogenous GnRH by secreting significant amounts of testosterone has to be investigated. Hence, this study was designed with an objective to determine the appropriate age at which pre-pubertal HF × Tharparkar bull calves are responsive to exogenous GnRH.
Mean ± SE pre-treatment concentrations of LH, androstenedione, testosterone, and total estrogens in Group I (14–16 months), Group II (9–12 months), and Group III (6–8 months) Holstein × Tharparkar bull calves
2.90 ± 0.84a
2.35 ± 0.67a
2.46 ± 0.35a
2.10 ± 0.17a
0.79 ± 0.06b
0.05 ± 0.01c
0.97 ± 0.08a
0.32 ± 0.06b
0.19 ± 0.03b
Total estrogens (pg/ml)
43.1 ± 23.6a
47.3 ± 26.0a
20.93 ± 5.48a
Mean ± SE peak concentrations of LH, androstenedione, testosterone, and total estrogens in Group I (14–16 months), Group II (9–12 months), and Group III (6–8 months) Holstein × Tharparkar bull calves
29.5 ± 8.49a
23.4 ± 4.41a
11.6 ± 9.91a
3.01 ± 0.72a
2.59 ± 0.71a
0.31 ± 0.22b
3.04 ± 0.29a
0.93 ± 0.27b
0.52 ± 0.17b
Total estrogens (pg/ml)
21.8 ± 4.7a
29.9 ± 14.4a
26.0 ± 11.0a
Mean ± SE area under response curve of LH, androstenedione, testosterone, and total estrogens in Group I (14–16 months), Group II (9–12 months), and Group III (6–8 months) Holstein × Tharparkar bull calves
LH (ng/ml h)
67.7 ± 16.4a
69.4 ± 15.9a
39.7 ± 19.4a
Androstenedione (ng/ml h)
18.2 ± 4.32a
8.32 ± 2.93a, b
0.91 ± 0.35b, c
Testosterone (ng/ml h)
11.4 ± 2.22a
3.63 ± 0.53b
2.8 ± 0.59b, c
Total estrogens (pg/ml h)
115 ± 29.8a
169 ± 22.5a
128 ± 44.9a
To the best of our knowledge, this is the first report that has evaluated the appropriate age at which the pre-pubertal HF × Tharparkar bull calves are responsive to the exogenous GnRH under tropical environmental conditions. The non-significance in the LH levels among the different age groups after GnRH administration in the present study is similar to the results obtained by Mongkonpunya et al. (1975). The LH concentrations after GnRH administration in Hereford bulls as observed by Schanbacher and Echternkamp (1978) are in accord with our results. The mean peak concentration of testosterone in Group I bull calves was observed as early as 2 h, whereas in both Group II and Group III bull calves, the peak was observed at 4 h after GnRH administration (Fig. 3). The pre-treatment testosterone concentrations and their increment with age are in accord with the study conducted by Gulia et al. (2010) on HF × Tharparkar bulls and bull calves. The difference between pre-treatment and peak levels of androstenedione in Group II bull calves were significant (p < 0.05) while the difference between pre-treatment and peak testosterone levels did not differ (p > 0.05). Among the three groups, only Group I bull calves showed a significant (p < 0.0001) difference between the pre-treatment levels and the peak concentration of testosterone after GnRH administration, indicating the responsiveness to GnRH. Since testosterone is essential for initiation and maintenance of spermatogenesis (Kerr et al. 1993), the administered GnRH seemed to have a functional role only among Group I bull calves, i.e. bull calves of 14 months of age and above. The results suggest that the weekly administration of GnRH (Ali et al. 2012) in order to augment testosterone levels and decrease AFSC might be successful only in pre-pubertal HF × Tharparkar bull calves of 14 months of age and above.
Since the differentiation of Leydig cells is completed by 6 months of age in bull calves (Amann et al. 1986), steroidogenesis has to take place in presence of LH. In the present study, LH levels were increased in the bull calves of all the three age groups but significant (p < 0.0001) amounts of testosterone was produced only in Group I (14–16 months of age) bull calves whereas Mongkonpunya et al. (1975) observed testosterone levels as high as 5.30 ng/ml in 6 months old HF bull calves after administration of exogenous GnRH. Growth hormone, insulin like growth factor-1 (Lin et al. 1986; Horikawa et al. 1989), insulin-like peptide 3 (Pathirana et al. 2012) and other growth factors are required, in adequate amounts, for steroidogenesis and production of testosterone. Holstein–Friesian × Tharparkar crossbreds have not adapted to the tropical Indian environmental conditions (Gulia et al. 2010) and their body weight gain is poorer than the HF and Sahiwal breeds. The concentration of the metabolic hormones and growth factors vary with the body weight and growth (Verde and Trenkle 1987); this may be the reason for significant differences in the pre-treatment and post-treatment concentrations of testosterone between the bull calves of three different age groups in the present study and for lower magnitudes than the HF bull calves as observed by Mongkonpunya et al. (1975). The total estrogen levels did not differ after administration of exogenous GnRH in all the groups, which agreed with a similar study carried out on adult HF × Tharparkar bulls (Bharath Kumar et al. 2015). We did not find any significant difference in the peak concentrations and area under response curve of total estrogens and time taken to attain peak levels between the groups.
From the present study, it was concluded that the HF × Tharparkar bull calves of 14 months age and above respond to exogenous GnRH by secreting significant (p < 0.0001) amounts of testosterone and the studies pertaining to decreasing the AFSC by frequent exogenous GnRH administration could be conducted on HF × Tharparkar bull calves of ≥14 months of age.
Selection, feeding, and management of bull calves
The experiment was conducted at National Dairy Research Institute (NDRI), Karnal, Haryana, India. Fifteen HF × Tharparkar bull calves were selected, and based on their age, were divided into group I (14–16 months, n = 5), group II (9–12 months, n = 5), and group III (6–8 months, n = 5). The mean ± SE body weights of group I, II, and III bull calves were 186.6 ± 5.16, 141.4 ± 3.31, and 104.6 ± 1.57 kg, respectively. The trial was conducted during the month of September. All the animals were housed in individual pens and were fed with maintenance ration as per kearl (1982) standards to gain @ 500 g/day. The concentrate offered consisted of 22 % CP and 70 % TDN and the concentrate mixture composed of 32 % groundnut cake, 33 % maize, 26 % wheat bran, 6 % de-oiled rice bran, 2 % mineral mixture, and 1 % salt. Concentrate was offered in the forenoon whereas adlib green fodder was offered in the afternoon. Water was offered twice daily. Approval for animal experimentation was obtained from the Institutional Animal Ethical Committee of National Dairy Research Institute (1705/GO/ac/13/CPCSEA Dt. 3/7/2013).
Animal treatment, blood sampling, and hormone analysis
All the bull calves were treated with 10 µg of GnRH [Receptal® (Buserelin acetate)] intramuscularly. The blood samples were collected at an interval of 30 min commencing 1 h prior to GnRH treatment until 4 h post-GnRH treatment and thereafter, at an interval of 1 h for the next 3 h. In all the bull calves, blood sampling and GnRH administration were carried out between 0700 and 1500 h. Immediately after collection, the blood samples were centrifuged at 1077×g for 15 min, and the plasma samples were stored at −20 °C until they were analyzed for testosterone (Gulia et al. 2010), luteinizing hormone (LH; Prakash et al. 2002), androstenedione (Mallick et al. 2015), and total estrogens (Mondal et al. 2006) by enzyme immunoassay procedures previously developed in the laboratory. The intra-assay coefficients of variation for testosterone, androstenedione, LH, and total estrogens were 3.12, 8.1, 6.48, and 2.97 %, respectively. The inter-assay coefficients of variation for testosterone, androstenedione, LH, and total estrogens were 7.43, 9.34, 14.21, and 9.68 %, respectively.
Definition of pre-treatment concentration and statistical analysis
The average concentrations from the samples collected before and during the GnRH administration constituted the pre-treatment levels of the corresponding hormones. Repeated measures ANOVA was used to determine the significant increase in peak levels from the pre-treatment concentrations in each group for all hormones. Tukey’s multiple comparison tests (one way ANOVA) was used to compare the pre-treatment levels, peak concentration of hormones, and area under curve between Group I, Group II, and Group III bull calves. Student’s t test was employed to compare the pre-treatment concentrations with the peak levels of the hormones. The total area formed on graph between ½ h after GnRH administration till 7 h was considered for calculating area under curve for all the hormones. Graphpad prism (version 5) and SPSS (version 16) softwares were used for statistical analysis.
BSBK, SM, and BM collected the samples, performed the assays, and analysed the data. BSP, SP, and BSBK planned the investigation, designed the protocols, and drafted the paper. All authors read and approved the final manuscript.
Financial support provided by the Director, ICAR-NDRI, Karnal, India and technical assistance by Mr. Dheeraj Kumar are greatly acknowledged.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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