Culture of Porcine Follicles

Systems to support development of porcine oocytes are more advanced than those for other large animal species. The greatest success has been achieved by using preantral follicles from prepubertal pigs. Oocytes from pig preantral follicles grown within a collagen gel matrix have been found to be meiotically competent if they reach a diameter of at least 100 mm (37), but fertilization of in vitro-grown oocytes has not been achieved using this system (37). The follicles isolated in this study consisted of an oocyte of 70-90 mm and granulosa cells with no theca, so that these structures were granu-losa-oocyte complexes. Although 30% of the cultured complexes reach the preovulatory size after 16 days of culture, only 3.5% developed an oocyte with a diameter > 110 mm. After removal of the oocyte from its granulosa layers and maturation for 2 days, only 1.3% of oocytes from the initially cultured follicular structures were capable of reaching metaphase II stage of meiosis. This low rate of success to resume and progress in meiotic maturation will be the result of many factors, but the main ones will be the selection and stage of follicles that start the culture and the culture conditions.

The Hirao study (37) grew follicles within a collagen gel matrix, and we have found that this is suitable for porcine preantral follicles < 100 mm in diameter, but once follicles reach a size of 150-200 mm in diameter, transfer to the surface of a collagen matrix improves oocyte growth. It may be that a three-dimensional support and closer contact with other follicles is required at an early stage of development but that as development proceeds this environment becomes inhibitory.

A recent study reported in vitro development of porcine oocytes from preantral follicles that were capable of maturation and fertilization after 4 days of development in vitro (38). This study used North Carolina State University 23 medium (NCSU23) supplemented with 3 mg/ml BSA (Sigma, Poole, Dorset, UK; fraction V), 3.5 mg/ml insulin, 10 mg/ml transferrin; 100 mg/ml ascorbic acid, and 10% porcine serum. Optimum conditions were found if three follicles were cultured per well. The study (38) reported a remarkable growth rate of the oocyte during a short culture period, although this laboratory has been unable to replicate these results.

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