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Influence of Culture Media on in Vitro Fertilization of Ovine Tubal Oocytes^{1 ,2,}

Washington State University³, Pullman 99164

Abstract

The ability of Synthetic Oviductal Fluid (SOF) and Minimum Essential Medium (MEM) to support sperm binding, sperm penetration and subsequent cell division of ovine tubal oocytes in vitro was evaluated. These media contained no protein, 3% (w/w) bovine serum albumin (BSA) or 20% (v/v) lamb serum (LS). Midventral laparotomies were performed on 49 ewes synchronized with progesterone and then superovulated, and tubal oocytes were collected by flushing of the oviducts. Oocytes were pooled and randomly added to 2 x 10⁷ ejaculated spermatozoa in 1 ml of treatment media that had been preincubated in tubes for 3 hr at 37 C under 5% C0₂ in air. After an additional 3 hr of culture in a rotating tissue culture drum, oocytes were further cultured in microdrops of the same medium for 48 hr and observed for sperm penetration, cell division and fragmentation. Oocytes were then stained with acetoorcein and observed for penetration, multiple nuclei and bound sperm. Percentage of oocytes showing penetration in SOF, SOF+ BSA, SOF+LS, MEM, MEM+BSA and MEM+LS was 14, 3.6, 7.8, 11.1, 8.7 and 12.8, respectively; percentage of oocytes cleaving was 8, 3.6, 5.9, 4.4, 4.3 and 4.3 ; percentage of oocytes fragmenting was 40, 60, 60.8, 20, 60.9 and 29.8, and the average number of sperm cells bound per oocyte was 82, 177,117, 41, 56 and 47. No cell division was observed for control oocytes (no sperm added), but 72.7, 80 and 76.9% fragmented in SOF, SOF+BSA and SOF+LS, respectively. No difference was found among the media in their ability to support sperm penetration and subsequent cell division in vitro. All media tested supported a high incidence of sperm binding and oocyte fragmentation.

¹ Scientific paper no. 5437. College of Agr. Res. Center, Washington State Univ., Pullman. Project 0313.

² This investigation was supported in part by funds provided to Washington State Univ. through the NIH Biomedical Research Support Grant.

³ Dept.of Anim. Sci.