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Collection Grading and Evaluation of Cumulus-oocytecomplexes for in Vitro Maturation in Sheep

M. N. S. Talukder
Department of Genetic Engineering and Biotecnology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

A. Iqbal
Department of Genetic Engineering and Biotecnology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

M. A. M. Y. Khandoker
Department of Animal Breeding and Genetics, Faculty of Animal Husbandry, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Z. Alam
Department of Genetic Engineering and Biotecnology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

Abstract/ Executive Summary:

An alternative to superovulation is in-vitro production (IVP) of embryos where the efficient collection and grading of oocytes is important. Ovaries from an abattoir were collected and categorized as type I with no corpus luteum (CL), and type II with CL. The length, width and weight of type I and type II ovary were 1.4 ± 0.03 and 1.5 ± 0.08 cm; 0.8 ± 0.04 and 1.0 ± 0.07 cm; 0.6 ± 0.07 and 0.7 ± 0.04 gm, respectively, each significantly (P<0.05) higher in type II ovaries. A total of 80 and 78 follicles were observed and 60 and 61 follicles aspirated from left and right ovaries, respectively, from each of 25 ovaries. Out of 133 follicles 100 were aspirated from 40 type-I ovaries, and 21 aspirated from 10 type-II ovaries. The differences in the number of normal, abnormal and total cumulus-oocytecomplex (COCs) per ovary between left and right ovaries were not significant (P>0.05). The number of normal (1.9 ± 0.11) and total (2.5 ± 0.14) COCs per ovary were significantly (P<0.05) higher in ovaries without than in those with CL (1.2 ± 0.36 and 2.0 ± 0.30, respectively). But the number (0.80 ± 0.13) of abnormal COCs per ovary was significantly (P<0.05) higher in ovaries with CL than in those without (0.7 ± 0.09). Significantly (P<0.05) higher percentage of COCs expansion was grade A (6.9 ± 2.05) than grade B (53.1 ± 1.27) COCs. It is suggested that type I (without CL) ovaries and follicles of 2-6 mm diameter are suitable to collect good quality COCs for in-vitro maturation (IVM) of oocytes and the culture condition for IVM of sheep COCs are reported.

Keywords: Grading , Evaluation of cumulus-oocytecomplexes , In vitro maturation in sheep

Location: Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Sheep

Objectives:

To compare collection and grading procedures of cumulus-oocyte-complexes (COCs) in sheep obtained from ovaries at slaughter
To establish the relationship between ovarian conditions and the quality of collected COCs
To establish suitable culture conditions for in vitro maturation (IVM) of sheep oocytes.

Methodology:

Physiological saline (0.9% NaCl) was prepared for washing. Dulbecco’s Phosphate Buffered Saline (D-PBS) was prepared by adding one pack of PBS salt (Sigma, USA) in one litre of distilled water. It was autoclaved and refrigerated for further use. The ovaries were kept in collection vial containing 0.9% physiological saline in a vacuum flask at 25 - 30^oC and transported to the laboratory within 4 -5 hours of slaughter. Sheep ovaries were collected from Municipal slaughterhouse, Mymensingh and numbered. The ovaries were classified into type I, without CL, and type II with CL. After collection and trimming of ovaries, each ovary was weighed and measured with callipers. The visible follicles were counted. Syringe (10 ml) loaded with PBS (1.0 - 1.5 ml), with 19G needle was used for the aspiration of follicles of 2 to 6 mm diameter. The aspirated material was transferred slowly into a 90-mm Petri dish, and the COCs were graded under microscope at low magnification of x10. The COCs were classified as described by Khandoker et al. (2001). Briefly, grade A: oocytes completely surrounded by cumulus cells; grade B: oocytes partially surrounded by cumulus cells; grade C: oocytes not surrounded by cumulus cells; and grade D: degenerated oocytes and cumulus cells. Grade A and B were considered normal COCs and grade C and D abnormal. Maturation medium, TCM-199 supplemented with 5% Fetal Calf Serum (FCS) was prepared and its pH adjusted to 7.4 on the day of aspiration and sterilized by filtration (22 μ Millipore filter). Normal graded COCs were washed 2-3 times in D-PBS, transferred into the maturation medium (TCM-199 + 5% FCS) and washed 2-3 times with the help of glass micropipette. About 2.5 - 3.5 ml of the medium was poured into each of two 35 mm culture dishes. In another culture dish 3 drops of 100 μl of maturation medium were poured and covered with paraffin oil. Droplets containing normal graded COCs were kept in a carbon dioxide incubator at 38.5oC with 5% carbon dioxide in air for 24 hours. After 24 hour of IVM, cumulus expansion was determined by three levels in same magnification of x 10; 1: indicating less expansion of COCs; 2: indicates moderate expansion; and 3: indicating marked expansion of cumulus cells with a compact layer or corona radiate.

Source of Information: The Bangladesh Veterinarian (2011) 28(1) : 31-38

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The number of normal (1.9 ± 0.11) and total (2.5 ± 0.14) COCs per ovary were significantly (P<0.05) higher in ovaries without than in those with CL (1.2 ± 0.36 and 2.0 ± 0.30, respectively). But the number (0.80 ± 0.13) of abnormal COCs per ovary was significantly (P<0.05) higher in ovaries with CL than in those without (0.7 ± 0.09). Significantly (P<0.05) higher percentage of COCs expansion was grade A (6.9 ± 2.05) than grade B (53.1 ± 1.27) COCs. It is suggested that type I (without CL) ovaries and follicles of 2-6 mm diameter are suitable to collect good quality COCs for in-vitro maturation (IVM) of oocytes and the culture condition for IVM of sheep COCs are reported.

Knowledge Management: Journal