Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size

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doi:10.1071/RD03006

Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size

Muhammad Rizwan Yousaf ^A, Kazim Raza Chohan ^A ^B ^C

^A Department of Theriogenology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan.
^B Andrology and IVF Laboratories, University of Utah Health Sciences Center, 675 Arapeen Drive, Suite 205, Salt Lake City, UT 84108, USA.
^C To whom correspondence should be addressed. email: kazim.chohan@hsc.utah.edu





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Reproduction, Fertility and Development 15(4) 223–229 http://dx.doi.org/10.1071/RD03006
Submitted: 23 January 2003 Accepted: 15 May 2003 Published online: 15 May 2003

Abstract

The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus–oocyte complexes (COCs) were collected from 1–<2, 2–<3, 3–<4, 4–<6 and 6–<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL^-1 hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 μg mL^-1 follicle-stimulating hormone, 10 μg mL^-1 luteinizing hormone, 1.5 μg mL^-1 oestradiol, 75 μg mL^-1 streptomycin, 100 IU mL^-1 penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1–<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1–<2 mm (107.7 ± 1.6 μm), 2–<3 mm (108 ± 1.1 μm) and 3–<4 mm (114.6 ± 1.3 μm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4–<6 mm (124.4 ± 1.3 μm) and 6–<8 mm (131.9 ± 1.4 μm) in diameter. A majority of oocytes (P < 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4–<6- and 6–<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2–<3- and 3–<4-mm follicles, respectively, whereas significantly (P < 0.05) more oocytes from 4–<6- and 6–<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.