251 CAMELID EMBRYO DEVELOPMENT IN VITRO: EFFECT OF PROTEIN SUPPLEMENT IN MATURATION MEDIUM AND SUBSEQUENT CULTURE IN TWO DIFFERENT MEDIA ON FERTILIZATION AND DEVELOPMENT

Abstract

Successful IVM/IVF can be used to produce large number of embryos cheaply for transfer and for manipulations. The technology has not previously been reported for the dromedary. The objectives of this study were to determine the effect of protein supplement [BSA vs. heat inactivated estrous camel serum (OCS)] in the maturation medium and subsequent culture of in vitro-fertilized zygotes in TCM199 or G1 and G2 medium (Vitrolife, Gothenburg, Sweden) on the rate of cleavage and development of embryos to blastocysts. Cumulus-oocyte complexes (COC) were collected by puncturing the follicles excised from the slaughterhouse ovaries (Nowshari and Wernery, A.E.T.E. 19th Scientific Meeting, September 12–13th 2003, Rostock, Germany). For IVM, TCM199 supplemented with 0.33 mM pyruvate, 10 μg/mL FSH and LH, and 1 μg/mL oestradiol (maturation medium) was used. The maturation medium was supplemented with either 5 mg/mL BSA or 10% OCS. After 30 to 32 h culture, COC were fertilized with epididymal spermatozoa which was stored at 4°C in TRIS-tes-egg yolk diluent for 1 to 8 days and consisted of not less than 50% motile spermatozoa on the day of use. The spermatozoa were swim up in fertilization medium (TALP, Parrish et al. 1985 Theriogenology 24, 537). Oocytes and spermatozoa (2–4 × 10⁶) were incubated in the fertilization medium for 14–16 h at 38°C under 5% CO₂ in air. Intact oocytes were removed from the fertilization medium and washed three times in the respective culture medium. Oocytes from two of the maturation treatments were divided into two subgroups and cultured in either medium TCM199 supplemented with 0.33 mM pyruvate and 10% OCS or medium G1 plus 10% OCS at 38°C under 5% CO₂, 5% O₂, 90% N₂. Zygotes in medium G1 were transferred to medium G2 on Day 3. Zygotes were examined for cleavage on Day 2 and further development on Day 8. The results are presented in Table 1. The results indicate that supplementation of maturation medium with BSA or OCS does not affect the rate of cleavage and development of embryos, however, culture of zygotes in sequential medium (G1-G2) affects the cleavage rate (P < 0.01) but not the further development of in vitro-produced dromedary embryos. Further studies are needed to improve the success of IVF and development during culture in this species.

Table 1.