213 EFFECTS OF TIME OF MATURATION AND SHEEP SERUM ON IN VITRO FERTILIZATION IN THE ENDANGERED ELD’S DEER (CERVUS ELDI THAMIN) OOCYTES

Abstract

The Eld’s deer, native to Southeast Asia, is threatened with extinction. Although artificial insemination is effective for offspring production, in vitro fertilization (IVF) would be more useful for rapidly disseminating genetic material from valuable founders. The objectives of this study were to: 1) determine if oocytes recovered from exogenous gonadotropin-treated hinds require additional in vitro maturation;; and 2) assess if fertilization is enhanced by supplementing Deer Synthetic Oviduct Fluid (DSOF;; Berg DK et al., 2003 Theriogenology 59, 189–205) with 1-day postestrus sheep serum (SS). Estrous cycles in Eld’s deer hinds (n = 10) were synchronized with PGF2α analog (Lutalyse^™, 500 mg), followed by a 14-day intravaginal CIDR-G insertion;; ovine FSH (Ovagen^™; 0.05 unit × 8 injections) was administered at 12-h intervals beginning 84 h before CIDR-removal. COCs (n = 160) were retrieved laparoscopically 40–46 h post-CIDR-removal and either fixed or matured in vitro (for 12 h v. 24 h) in TCM-199 (Earle’s salt) supplemented with 0.33 mM pyruvate, 2 mM glutamine, 100 IU mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin, 10% fetal calf serum, 5 μg mL⁻¹ FSH and LH and 1 μg mL⁻¹ E₂ (5% CO₂, 38.5°C). After 12- or 24-h IVM, cumulus cells were partially removed and oocytes (n = 110) fertilized in DSOF with pooled frozen-thawed sperm (3 males;; 2 × 10⁶ motile sperm mL⁻¹), in the absence or presence of SS (20%, v/v). Additional oocytes (n = 18) were used for parthenogenetic control. At 20-h postinsemination, presumptive zygotes were fixed and stained (Hoechst 33342) to assess fertilization success (presence of two pronuclei). Data were analyzed by ANOVA. Overall, 16.0 ± 2.6 (mean ± SEM) COCs were recovered/female. The majority of COCs were of excellent quality (grade I; 67.7 ± 3.8%). At time of aspiration, 85% of the oocytes (n = 11/13) were in metaphase I stage, 7.5% in telophase and 7.5% degenerate. No parthenogenic activation was observed. Likewise, no polyspermy was observed in any treatment. Fertilization was higher (P < 0.05) in oocytes matured for 24 h and fertilized in the absence (64.4 ± 3.1%) compared to presence (26.9 ± 11.2%) of SS. In the absence of SS, a higher (P < 0.05) proportion of oocytes were fertilized after 24 h (64.4 ± 3.1%) compared to 12 h (27.1 ± 9.0%) IVM. There was no effect (P > 0.05) of SS on fertilization among oocytes subjected to 12-h IVM (27.1 ± 9.0% v. 12.5 ± 9.5%). When SS was present during fertilization, no difference (P > 0.05) was observed among oocytes matured for 12 or 24 h. Results demonstrate that: 1) Eld’s deer oocytes require an additional 24-h IVM to complete maturation;; 2) DSOF supports sperm-oocyte interaction;; and 3) SS is not essential for successful fertilization. (Supported by Morris Animal Foundation.)