263 IN VITRO FERTILIZATION AND EMBRYO DEVELOPMENT OF CUMULUS-OOCYTE COMPLEXES COLLECTED BY ULTRASOUND-GUIDED FOLLICULAR ASPIRATION IN LLAMAS TREATED WITH GONADOTROPIN

Abstract

We have previously documented that both FSH and eCG are equally effective in inducing ovarian superstimulation in llamas, resulting in the recovery of a high number of expanded COC suitable for in vitro fertilization (Ratto et al. 2005 Theriogenology 63, 2445-2457). The objective of the study was to evaluate the ovarian response, morphology, and competence of COC collected by ultrasound-guided follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly into 2 groups (n = 16 per group) and treated for 48 h after follicle ablation with (1)25 mg of FSH (Folltropin, Bioniche Animal Health Canada Inc., Belleville, Canada) i.m. twice daily for 4 d; or (2) 1000IU of eCG (Novormon, Bioniche Animal Health Canada) as a single i.m. dose. The starting of gonadotropin treatment was considered Day 0. Both groups were given an i.m. dose of 5 mg of Armour Standard LH (Lutropin, Bioniche Animal Health Canada) on Day 6, and COC were collected by transvaginal ultrasound follicle aspiration of all follicles ≥7 mm on Day 7. The ovarian response was assessed by transrectal ultrasonography using a 7.5-MHz linear-array transducer (Aloka SSD-500, Clinics, Santiago, Chile) immediately before oocyte collection at 24 to 26 h after LH treatment in both groups. The COC were classified as expanded, compact, denuded, or degenerated. Expanded COC collected from FSH- (n = 147) and eCG-treated llamas (n = 141) were fertilized in vitro using epididymal sperm as previously described (Ratto et al. 2006 Anim. Reprod. Sci. 97, 246-257). Gametes were co-incubated at 38.5°C in air with 5% CO₂ and high humidity for 18 h. After in vitro fertilization, presumptive zygotes were co-culture in SOF medium supplemented with 0.6% of BSA with llama granulosa cells at 39°C, 5% CO₂, 5% O₂, and 90% N² for 7 days. Embryo development was evaluated on Days 2, 5, and 7 of in vitro culture (Day 0 = IVF). Data were analyzed by Student’s t-test or Fisher’s exact test and presented as mean ± SEM. The FSH and eCG treatment groups did not differ with respect to the number of follicles ≥7 mm at the time of COC collection (16.0 ± 2.7 v. 14.0 ± 1.9; P = 0.5), the number of COC collected (11.5 ± 1.9 v. 9.7 ± 1.2; P = 0.4), or the collection rate per follicle aspirated (77.0 v. 71.5%; P = 0.2). No difference was detected between FSH and eCG-treated llamas in the number of expanded COCs (9.8 ± 1.4 v. 9.4 ± 1.2; P = 0.8). The percentage of presumptive zygotes to develop into 2 to 8 cells on Day 2 (65.3 v. 63.1), morulas on Day 5 (46.2 v. 42.5), and blastocyst stage on Day 7 (23.1 v. 20.5) did not differ (P > 0.05) between FSH and eCG-treated llamas, respectively. In conclusion, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. The recovery of a high number of expanded COC can be used directly for in vitro fertilization and their competence is not affected by gonadotropin treatment.

The study was supported by Convenio Desempeño en Investigacion (2007-DGI-CDA-04), Universidad Catolica de Temuco.