219 EFFECTS OF GROWTH HORMONE AND GNRH ANTAGONISTS ON FOLLICULAR AND OOCYTE DEVELOPMENT IN SHEEP

Abstract

Embryo output in sheep is increased when superovulatory FSH treatments are started in the presence of a high number of small follicles (2–3 mm in size) and in absence of large follicles (>6 mm, Gonzalez-Bulnes et al., 2002. Theriogenology, 57, 1263–1272). Administration of GnRH antagonists (GnRHa) suppresses large follicles (Cognie et al., 2003. Theriogenology, 59, 171–188), whereas the use of growth hormone (GH) would increase the number of small follicles (Campbell et al., 1995. J Reprod Fertil Suppl, 49, 335–350). Our aim was to evaluate the usefulness of pre-treatments with GH or GH plus GnRH antagonists for sheep embryo production. First, we studied the effects on follicular population by serial ultrasonographies. Thereafter, we determined whether such treatments can affect oocyte developmental competence. In a first trial, a total of 18 Manchega ewes were treated with intravaginal FGA sponges (Chronogest^®, Intervet Int., H) during breeding season (beginning of April). Six animals received daily i.m. doses of 15 mg of ovine GH (Tuenre, GA) for 6 days, while six females received GH plus two s.c. doses of 1.5 mg of GnRHa (Antarelix™, Zentaris, G) on Days 0 and 3 of GH treatment, and six ewes acted as controls receiving saline. Number of follicles >2 mm, determined by daily transrectal ultrasonography, increased to reach significant differences on Day 4 in sheep treated with GH/GnRHa (22.7 ± 0.8 v. 16.7 ± 0.5, P < 0.001) and on Day 5 in ewes injected with GH (20.3 ± 0.4 v. 17.0 ± 0.6, P < 0.05). The second trial involved 18 Manchega ewes treated with progestagen sponges on Day 0 and distributed in three groups at the beginning of breeding season (end of July). In the first group (n = 7), sheep were treated with two doses of GnRHa on Days 0 and 3 after sponge insertion and with three doses of 15 mg of GH on Days 3, 4, and 5. Thereafter, ewes from this group and from a second experimental group (n = 7) were treated with 3 doses of 1.5 mL of FSH (Ovagen™, ICP, NZ) every 12 h, starting on the afternoon of Day 5. A third group of sheep (n = 4) did not receive GH/GnRHa or FSH, acting as controls. On Day 7, follicles were aspirated and the cumulus-oocyte complexes (COC) were cultured for 24 h at 38.5°C, 5% CO₂, in TCM-199 supplemented with ovine FSH (Ovagen), LH, FCS, 17-βoestradiol, cysteamine, and sodium pyruvate (Sigma Chemical Co., MO, USA). Nuclear maturation was measured by Hoechst 33342 fluorescence. Mean number of COC was higher in GH/GnRHa + FSH group (8.7 ± 0.9 v. 6.8 ± 1.3 in FSH group, NS and 4.5 ± 0.8 in control, P < 0.05) due to higher number of follicles with similar recovery rates (45.0 ± 4.5, 40.3 ± 1.4, and 39.1 ± 7.1%, respectively). There were no significant differences on the ability of COC to resume meiosis, although this was higher in FSH group (63.1 ± 9.5% for GH/GnRHa + FSH, 79.5 ± 6.3% for FSH and 60.0 ± 8.8% for control group), which can indicate the necessity of a higher FSH supply to induce final development in follicles/oocytes from ewes treated with GH and GnRHa. In conclusion, the use of GH and GnRHa would help to increase the number of gonadotrophin-responsive follicles prior to gonadotrophin injections;; also, adjustment of FSH treatments improved embryo yields in superovulatory protocols.