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Efficient isolation and long-term viability of bovine small preantral follicles in vitro

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Summary

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.

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  1. Research Institute for the Functional Peptides, 4-3-32 Shimojo, 990-0823, Yamagata, Japan

    Takehiro Itoh & Hiroyoshi Hoshi

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  1. Takehiro Itoh
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  2. Hiroyoshi Hoshi
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Correspondence to Hiroyoshi Hoshi.

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Itoh, T., Hoshi, H. Efficient isolation and long-term viability of bovine small preantral follicles in vitro. In Vitro Cell.Dev.Biol.-Animal 36, 235–240 (2000). https://doi.org/10.1290/1071-2690(2000)036<0235:EIALTV>2.0.CO;2

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  • DOI: https://doi.org/10.1290/1071-2690(2000)036<0235:EIALTV>2.0.CO;2

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