An Efficient Isolation Method for Domestic Hen (Gallus domesticus) Ovarian Primary Follicles

Abstract

Abstract. An enzymatic method of isolating primary follicles in the turkey has been described previously, but no similar work has been done in the hen. In this study, primary follicles from domestic hens (Gallus domesticus) were isolated using an enzymatic method, and the isolated follicular quantity, quality, and development in vitro were assessed. About 400 primary follicles ranging from 60 to 1125 µm in diameter were recovered with trypsin and collagenase from hen ovaries (per ovary).Of these, 76.5% were intact follicles with a complete single layer of granulose cells surrounded by the basement membrane, and their ultrastructures appeared this way in situ. Follicles (351 to 500 µm in diameter) were cultured in vitro, and 46.67% of them survived after 5 days. Ultrastructural examination showed that elongated mitochondria forming a ring were distributed to the periphery of the oocyte, the Golgi was oriented with the maturing face toward the granulosa cell layer, and the oocyte plasma membrane presented a few short microvilli lying on the oocyte surface, which confirmed that the surviving follicles were developmental. These results suggest that a simple, rapid, effective enzymatic method can be used to isolate a great number of intact primary follicles from the hen ovary.

Enzymatic isolation of human primordial and primary ovarian follicles with Liberase DH: protocol for application in a clinical setting

“…Our aim was to replace collagenase and Liberase Blendzyme 3 with an enzyme that could be used for all clinical applications. To evaluate Liberase DH, we compared it with collagenase, considered as the gold standard in ovarian follicle isolation (7,10,11,13,15,(17)(18)(19)(20)(21)(22)(23)(24). Although collagenase allows isolation of a significant number of preantral follicles, their quality and viability may vary according to product lot (29).…”

“…A number of mechanical (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) and enzymatic (3,7,10,11,13,15,(17)(18)(19)(20)(21)(22)(23)(24)(25) methods have been developed to isolate follicles from humans and different animal species. Due to their dense and fibrous ovarian stroma, human preantral follicles are more efficiently isolated using a combination of mechanical and enzymatic procedures, which have been shown to preserve follicle viability (3,(26)(27)(28).…”

MORPHOLOGICAL CHARACTERISTIC OF SPERMATOGONIA AND TESTES DISSOCIATION : A Preliminary Study for the Germ Cell Transplantation in Giant Gouramy (Osphronemus gouramy)

“…Since the enzymes could cause detrimental effect to the cell as reported in some literatures (Nicosia et al, 1975;Bellve et al, 1977;Du et al, 2006) then the dissociation procedure should be arranged carefully including how long the cells should be exposed to the enzymes. Therefore, in this research the period of incubation time to determine the optimal incubation time that yield high number and viable spermatogonia was examined.…”