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Evaluation of Bovine Cumulus-oocyte-complexes in View of in Vitro Nuclear Maturation and Fertilization

S. Pervage
Scientific Officer
Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh .

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

M. J. Uddin
Scientific Officer
Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh.

A. B. M. R. Bostami
Scientific Officer
Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh.

A. Mondal
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

The present study was undertaken to evaluate the slaughterhouse cow ovaries and cumulus-oocyte-complexes (COCs) and to establish the suitable method of COCs collection in view of in vitro nuclear maturation and fertilization. Cow ovaries were categorized as right, left, corpus luteum (CL) absent and present group. Ovaries were then evaluated on the basis of weight (g), length (cm), width (cm), total number of follicle on the surface of each ovaries, number of follicle aspirated and collected, total number of COCs, normal and abnormal COCs. It was observed that comparatively higher number of normal COCs per ovary was obtained by intact follicle collection method (3.35) than aspiration method (1.1 0). In case of abnormal COCs, the reverse results were found. The significantly (p<0.01) higher number of normal COCs (2.56) was found in CL-absent group than that of CL-present group. Similarly, an increase of total number of follicles (5.68), number of follicle aspirated and collected (4.02) and total number of COCs (3.87) but decrease in weight (3.19), length (2.22), width (1.67) and abnormal COCs (1.22). The higher morphological maturation of COCs was observed in CL-absent group (75.74%) than CL-present group (70.83%). The cellular maturation rate up to metaphase-Il of grade A and B COCs was recorded at 54.75% and 40%, respectively. The fertilization and further development was tested. The highest fertilization rate was observed in grade A (35.16%) than that of grade B (29.85%) but no further development was recorded after 24 hours of in vitro culture (IVC).

Keywords: Bovine, Grading, Maturation, COCs, Fertilization

Location: Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh.

Start Date: 00-07-2005

End Date: 00-09-2006

Program Area: Variety and Species

Commodity: Cow

Objectives:

To the present study are collection and evaluation of slaughterhouse cow ovaries, follicles and COCs; establishing the suitable method of oocyte collection; the relationship between ovarian condition and morphological quality of COCs and the procedure of in vitro nuclear maturation of oocytes.

Methodology:

The experiment was conducted at Reproductive Biotechnology Laboratory from July 2005 to September 2006 under the Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh. Chemicals and reagents required: i. Phosphate buffer saline (PBS) stock solution, ii. Fetal calf serum (FCS), iii. Bovine serum albumin (BSA), iv. Paraffin oil, v. Tissue culture medium (TCM-199) for maturation, vi. Brackett and Oliphant (BO) medium for sperm capacitation and fertilization, vii. Semen washing solution, viii. COCs washing solution, ix. Semen dilution solution, x. Distilled water. Ovaries were collected from local slaughterhouse. The ovaries were kept in collection vial containing 0.9% physiological saline in a thermos box at 25 to 30°C and transported to the laboratory within 4 to 5 hours of slaughter. The ovaries were recorded as right and left and the presence or absence of corpus luteum was also recorded. Individually right, left, CL-present and CL-absent ovaries were weighed and were recorded in tabular form. Blunt dissection and aspiration methods were practiced to collect the COCs from the ovaries. In case of aspiration method, 10 ml syringe was loaded with PBS (1.0-1.5 ml) and the needle (19G) was put into the ovarian parenchyma near the vesicular follicles (2-6 mm diameter) by right hand and all 2-6 mm diameter follicles were aspirated near the point at the same time. After aspiration, the follicular materials slowly transferred into a 90 mm petridish for avoiding damage of the cumulus cells. In dissection method, each ovary was incised with forceps and scissors to collect all the visible follicles. From each follicle the follicular materials were harvested with the help of forceps and needle by blunt dissection on a sterilized culture dish. The COCs collected both by blunt dissection and aspiration methods were searched and graded under microscope at low magnification. The oocytes in culture dishes with evenly distributed medium and dish with droplet were kept in an incubator at 38.5°C with 5% CO₂ in air. After 22 hours of IVM, both the morphological and nuclear level maturation was checked in representative sample. Two 35 mm culture dishes filled with oocyte washing solution were prepared and the oocytes were washed 3 times in the oocyte washing solution. About 20-25 oocytes with minimum volume of medium were transferred to each of the sperm drops prepared previously. Then the 35-mm culture dish with inseminated oocytes was incubated for 5 hours in incubator at 38.5°C with 5% of CO₂, At 5 hours after initiation of IVF the representative COCs from each group were denuded from cumulus cells by repeated pipetting. Then the oocytes were observed under microscope with emersion oil to observe second polar body formation and finally the rate of fertilization were calculated. The remaining COCs were cultured in embryo culture medium for further development and checked regularly. All values were expressed as mean±SE. Statistical significance of differences between different parameters, maturation and rate of development was evaluated by using students unpaired 't' test.

Source of Information: Bang. J. Anim. Sci., Vo- 36 (1 & 2), Pag: 16-23 , 2007, (ISSN 0003-3588)

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Total Budget (Tk.) :

Achievement/Findings:

Comparatively higher number of normal COCs per ovary was obtained by intact follicle collection method than that of aspiration method. The left ovaries contain comparatively higher number of normal COCs per ovary than that of right ovaries with an increase of total number of follicles and number of follicle aspirated and collected. On the other hand in case of abnormal COCs the reverse results were found in right ovaries than that of left ovaries. An increasing weight, length and width were recorded in right ovaries although the total number of COCs per ovary was found higher in left ovaries. The higher number of normal COCs were found in CL-absent group than that of CL-present group with an increase of total number of follicles, number of follicle aspirated and collected and total number of COCs but decrease in weight, length, width and abnormal COCs. The highest maturation rate was obtained in the COCs recovered from CL-absent group than CL-present group. The maturation rate up to metaphase-II of grade A and B COCs was recorded higher. Although the highest fertilization rate was obtained in grade-A, but no noticeable development was found after 24h of the initiation of Iv C. Finally it can be concluded that the intact follicle collection method and CL-absent group ovaries are suitable for oocyte collection in view of in vitro nuclear maturation and fertilization of bovine oocytes.

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