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1 April 2000 EFFICIENT ISOLATION AND LONG-TERM VIABILITY OF BOVINE SMALL PREANTRAL FOLLICLES IN VITRO
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Abstract

A comparison of isolation techniques for small preantral follicles (30–70 μm) from bovine ovaries using a mechanical method with a grating device or collagenase treatment was performed. The mean number (157.0) of intact follicles per ovary isolated by the mechanical method was significantly greater (P < 0.05) than that (26.0) of follicles isolated by the enzymatic method. Isolated morphologically normal follicles (MNF) were cultured for up to 30 d either in control cultures (non-coculture) or in coculture with bovine ovary mesenchymal cells (BOM), fetal bovine skin fibroblasts (FBF), and/or bovine granulosa cells (BGC). In control cultures, most of the follicles degenerated and only a few MNF (1.2%) were present after 30 d in culture. In contrast, the cocultures with BOM, FBF, and BGC resulted in 50.7, 46.6, and 21.4% viable MNF, respectively. Trypan blue and Hoechst 33258 staining were used for a quick and sensitive assessment of oocyte and granulosa cell viability during follicle isolation and culture in vitro. After 30 d, percentages of viable follicles in coculture with BOM (18.6%) and FBF (17.1%) were significantly greater than those of follicles in the control cultures (0%) or in coculture with BGC (10.0%). There was a gradual increase in the average diameter of the MNF during culture. The mean diameter of the follicles increased by 15.4 and 30.0% in coculture with BOM and FBF, respectively, by day 30. In conclusion, small bovine preantral follicles were efficiently isolated using a mechanical method that utilizes a grating device, and could be maintained for up to 30 d in the presence of mesenchymal cell cocultures such as BOM and FBF. This in vitro culture system that supports long-term survival of bovine preantral follicles should be beneficial for studying follicle growth and development.

TAKEHIRO ITOH and HIROYOSHI HOSHI "EFFICIENT ISOLATION AND LONG-TERM VIABILITY OF BOVINE SMALL PREANTRAL FOLLICLES IN VITRO," In Vitro Cellular & Developmental Biology - Animal 36(4), 235-240, (1 April 2000). https://doi.org/10.1290/1071-2690(2000)036<0235:EIALTV>2.0.CO;2
Received: 21 September 1999; Accepted: 1 November 1999; Published: 1 April 2000
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