Influence of exogenous GnRH on sexual behavior and frozen/thawed semen viability in stallions during the non-breeding season.

Abstract: Twelve fertile stallions were divided into two groups, either receiving gonadotropin-releasing hormone (GnRH) (n = 6) or Placebo (n = 6). Based on the history of frozen/thawed semen characteristics three stallions within each group were assigned as being "good freezers" [GnRH (+); Placebo (+)] and three stallions were assigned as being "poor freezers" [GnRH (-); Placebo (-)]. The study was performed as a "blinded" investigation and stallions were treated twice daily by an intramuscular injection of 1 ml GnRH (Buserelin), 50 microg) or Placebo. The experiment was divided into three time periods. Period A (pre-treatment) was performed between 16 November and 20 December; Period B (treatment) was performed during 6 weeks between 21 December and 31 January; and Period C (post-treatment) was performed between 1 February and 12 February. Semen was collected every Monday, Wednesday, Friday, and analysed for motion characteristics by the use of a computerized semen analyser, and sperm morphology immediately after collection. The spermatozoa were cryopreserved, stored in liquid nitrogen, and evaluated for motility (computer assisted semen analysis), membrane integrity (carboxyfluoresceine diacetate (CFDA) combined with propidium-iodide (PI), CFDA/PI), viability and sperm morphology (Eosine-Nigrosine, EN), and osmotic reactivity (hypo-osmotic swelling test, HOS) following thawing in a water bath. The viability of spermatozoa was expressed as the difference between pre-freeze and post-thaw values. A libido score of 1-4, the number of mounts on the phantom before ejaculation, and ejaculation latency were used to evaluate the stallions sexual behavior. Effect of treatment was analysed by comparing time intervals within groups as well as comparing groups within time intervals using SAS statistics software. GnRH treatment decreased the number of mounts before ejaculation (GnRH (total): 2.5 +/- 1.14 versus 1.8 +/- 1.06, P < 0.05), and shortened ejaculation latency. Cessation of treatment increased ejaculation latency in the GnRH group (4.7 +/- 4.98 min versus 7.2+/-7.88min, P<0.05). With the exception of libido score all parameters of sexual behavior were superior in the GnRH (+) group compared to the Placebo (-) group during the treatment period (P < 0.05). GnRH administration increased progressive motility (GnRH (+): 30.7 +/- 10.74% versus 38.4 +/- 15.1%, P < 0.05; GnRH (total): 24.9 +/- 11.80% versus 31.9 +/- 14.68%, P < 0.05), membrane intact spermatozoa CFDA/PI (GnRH (-): 16.8 +/- 7.17% versus 26.2 +/- 7.02%, P < 0.05; GnRH (total): 23.1 +/- 12.33% versus 29.5 +/- 10.77%, P < 0.05) and HOS positive spermatozoa (GnRH (+): 33.2 +/- 11.29% versus 42.2 +/- 10.36%, P < 0.05; GnRH (total): 32.9 +/- 10.23% versus 40.1 +/- 10.30%, P < 0.05) of frozen/thawed spermatozoa. Following cessation of treatment, the viability of frozen/thawed spermatozoa decreased. GnRH treated stallions had lower losses of live stained spermatozoa (EN) compared to the Placebo group (GnRH (total): 17.6 +/- 4.77 versus Placebo (total): 27.2 +/- 5.44, P < 0.05). This was particularly observed in the "poor freezer" group (GnRH (-): 16.6 +/- 4.35 versus Placebo (-): 31.3 +/- 5.87; P < 0.05). In conclusion, exogenous GnRH was shown to improve sexual behavior and increase the quality of frozen/thawed spermatozoa in fertile stallions during the non-breeding season. Nevertheless, it seems that, although significance was achieved relative to improvement to post-thaw sperm quality, that the "real" change in sperm quality seems negligible in fertile stallions. The mechanism of GnRH effect was not determined but this study may support the possibility of a direct gonadal or epididymal effect of exogenous GnRH in the stallion.