103 Hormonal stimulation and post-breeding sperm induction in the mountain yellow-legged frog, Rana muscosa

The first choice of any captive animal program should be to promote natural breeding through appropriate diet and environmental conditions. However, in cases of poor reproductive performance, the administration of exogenous hormones can induce breeding behaviours and gamete release. Part of the approach to the Mountain yellow-legged frog recovery program is the collection of sperm for cryobanking as a means of preserving genetic diversity in a cost- and space-effective manner. To develop a protocol for induction of sperm release from Mountain yellow-legged frogs, we tested the administration of gonadotropin-releasing hormone agonist (d-Ala6, des-Gly10 ethylamide LHRH derivative) and hCG singly or in combination. Once a month, animals were injected with a single dose of saline amphibian ringers (control) or 1 of 10 hormone treatments. Groups 1-4 received a single intraperitoneal injection of gonadotropin-releasing hormone agonist administered in 4 different doses: (1) 0.3 μg/g body weight (bw); (2) 0.4 μg/g bw; (3) 0.6 μg/g bw; and (4) 1 μg/g bw. Groups 5 and 6 were injected with 5 and 10 IU hCG/g bw, respectively, and groups 7-10 received a combined injection of hCG and GnRH in four different doses: (7) 5 IU/g hCG with 0.3 μg/g GnRH; (8) 10 IU/g hCG with 0.3 μg/g GnRH; (9) 5 IU/g hCG and 0.6 μg/g GnRH; and (10) 10 IU/g hCG and 0.6 μg/g GnRH. Hormone treatments began in July 2015, 2 months after the end of the breeding season to allow males a post-breeding recovery time. Monthly administration continued from July until December 2015, ending before the brumation period. In 2016, treatments resumed again in July, after another breeding period. In 2017, hormone treatments began in April, during the reproductive season, and continued until July. From July 2015 through November 2016, sperm concentration and motility were examined in response to hormones treatment groups 1, 3, 5, 6, and 7-10. In April 2017, based on previous results, experiments were redesigned to include group 9 from 2015-16, and two new groups, 2 and 4. Spermiation was not easily hormonally induced when administered from August-October 2015, August-September 2016, and in July 2017. These low-response periods coincided with post-breeding months when only 3.7% (3/81) of males responded to either 10 IU/g hCG, 0.6 μg/g GnRH, or a combination of 5 IU/g hCG and 0.6 μg/g GnRH. Sperm production was significantly affected (P < 0.05) by the month of hormone administration and hormone treatments during the responsive periods (P < 0.0001) of April-June and October-November. However, hormone doses and treatments had no significant effect on the average or total sperm concentration per male (P < 0.05). Motility and speed of forward progression were not significantly affected by treatment (P < 0.05). This study demonstrates the seasonality of sperm production in this species, which may be attributed to testicular recrudescence during the summer months following breeding. Without hormonal stimulation, spermiation did not occur during the nonreproductive part of the year.