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In vitro culture of oocytes with surrounding cumulus complexes and granulosa cells (COCGs) from bovine early antral follicles

Published online by Cambridge University Press:  18 August 2016

S. Saha ,
M. Shimizu ,
M. Geshi  and
Y. Izaike
S. Saha*
Affiliation:
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norindanchi, PO Box 5, Ibaraki 305-0901, Japan
M. Shimizu
Affiliation:
National Agricultural Research Centre for Tohoku Region, Morioka, Iwate, 020-0198, Japan
M. Geshi
Affiliation:
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norindanchi, PO Box 5, Ibaraki 305-0901, Japan
Y. Izaike*
Affiliation:
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norindanchi, PO Box 5, Ibaraki 305-0901, Japan
*
†Present address: Agricultural and Forestry Research Centre, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. E-mail:[email protected]
‡ Present address: National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
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Abstract

Cumulus-oocyte complexes with surrounding granulosa cells (COCGs) in early antral follicles (0·5 to 0·7 mm in diameter) were surgically collected from sections of bovine ovarian cortex under a dissection microscope and subsequently cultured in vitro using follicle stimulating hormone (FSH), epidermal growth factor (EGF), insulin-transferrin-selenium (ITS) and hypoxanthine, singly or in combination, to obtain fully grown matured oocytes. Oocytes cultured in the presence of FSH + hypoxanthine increased (P < 0·05) in diameter from 93 µm on the day of commencement of culture to 106·37±0·34 µm on day 5. Oocytes cultured in the presence of FSH, hypoxanthine or hypoxanthine + ITS + FSH increased (P < 0·05) to mean diameters of 105·40 (s.e. 0·47) µm, 105·50 (s.e. 0·39) µm and 105·35 (s.e. 0·55) µm, respectively. By day 11 of culture, oocyte diameters 110·50 (s.e. 0·35) µm, 110·13 (s.e. 0·39) µm, 109·49 (s.e. 0·46) µm, 109·53 (s.e. 0·58) µm and 109·16 (s.e. 0·43) µm were recorded for treatments FSH + hypoxanthine, hypoxanthine + ITS + FSH, FSH, hypoxanthine and FSH + EGF + hypoxanthine + ITS, respectively. The proportions with COCGs which formed an antrum while cultured in vitro; were categorized as morphologically normal following recovery from the gel; matured in vitro; showed germinal vesicle break down and reached metaphase II were highest (P < 0·05) for the FSH + hypoxanthine treatment (49/60 (81·7%), 48/60 (80·0%), 47/60 (78·3%), 45/60 (75·0%) and 15/60 (25·0%), respectively, followed by hypoxanthine + ITS + FSH (47/60 (78·3%), 44/60 (73·3%), 41/60 (68·3%), 41/60 (68·3%) and 12/60 (20%), respectively), FSH (43/60 (71·7%), 42/60 (70%), 40/60 (66·7%), 39/60 (65·0%) and 9/60 (15%), respectively) and hypoxanthine (41/60 (68·3%), 38/60 (63·3%), 36/60 (60%), 35/60 (58·3%) and 8/60 (13·3%), respectively). In experiment II, the in vitro fertilization and cleavage rates of COCGs were highest (P < 0·05) for FSH + hypoxanthine treatment (17/60; 28·3%) followed by hypoxanthine + ITS + FSH (13/60; 21·6%), FSH (12/60; 20%) and hypoxanthine (11/60; 18·3%) treatments. The results of this study show that COCGs from early antral follicles can be isolated, cultured and grown in vitro. Furthermore, supplements like FSH and hypoxanthine can be used singly or in combination(s) in culture medium to enhance the growth of COCGs.

Keywords

cowsgraafian folliclesin vitro cultureoocytes
Type
Reproduction
Information
Animal Science , Volume 77 , Issue 1 , August 2003 , pp. 141 - 147
Copyright
Copyright © British Society of Animal Science 2003

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