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In Vitro Production of Goat Embryos in Bangladesh

A. Mondal
Reproductive Biotechnology Laboratory, Department of Animal Breeding and Genetics, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

M. A. M. Y. Khandoker
Reproductive Biotechnology Laboratory, Department of Animal Breeding and Genetics, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

M. A. Mondal
Department of Biotechnology, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

A. H. M. S. Rahman
Reproductive Biotechnology Laboratory, Department of Animal Breeding and Genetics, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

A. S. Apu
Reproductive Biotechnology Laboratory, Department of Animal Breeding and Genetics, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

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

Abstract/ Executive Summary:

The present study was undertaken to collect the quality cumulus-oocyte-complexes (COCs) from ovaries of goat from slaughterhouse by aspiration to establish the suitable culture condition for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Follicular COCs were collected from follicles of 2-6 mm diameter, categorized by microscopic observation and cultured for 22 h in TCM-199 medium supplemented with 5% fetal calf serum (FCS) to determine the success rate of in vitromaturation in a condition of 5% CO2 in air at 38.5oC. The collected ovaries were classified as type-I (corpus luteum absent) and type-II (corpus luteum present). The average numbers of follicles aspirated per ovary were 3.15 and 2.57 in type-I and type-II, respectively. The collected COCs were classified into normal COCs (grade A and B) and abnormal COCs (grade C and D). The number of normal and abnormal COCs collected from two type of ovaries were significantly (P<0.01) differed. Average number of normal COCs per ovary obtained from type-I (1.30) was significantly (P<0.01) higher than that of type-II (0.68). Within the normal COCs significantly (P<0.01) higher maturation was obtained in grade A COCs (71.70%) than that of grade B (51.52%). The matured COCs were cultured for 5 h with fresh buck semen in Brackett and Oliphant (BO) medium and assumed that the COCs were fertilized successfully. In progress, IVC was practiced in TCM-199 supplemented with FCS and bovine serum albumen (BSA) at 38.5oC with 5% CO₂ for 6-7 days. The rate of development to compact morula was found significantly (P<0.01) higher in grade A (25.64%) compared to grade B COCs (6.89%) and similar trend of blastocyst was found in grade A COCs (12.82%) than that of grade of B (3.45%). The results suggested that culture condition for IVM, IVF and IVC was found optimum and grade A COCs might be suitable forin vitroproduction (IVP) of goat embryos.

Keywords: Goat ovaries, Cumulus-oocyte-complexes, In vitroproduction

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Goat

Objectives:

The number of follicles measuring 2-6 mm diameter was found to be higher in ovaries without CL than ovaries with functional and regressed CL. It was also suggested that ovaries having no CL might be the source of quality COCs. With this view in mind, present research work was undertaken to collect the quality COCs by aspiration method.

Methodology:

Ovaries were collected from local slaughterhouse in collection vial containing 0.9% physiological saline kept in a thermos box at 25 to 30ºC and transported to the laboratory within 4 to 5 hours of slaughter. Then, the ovaries were categorized as corpus luteum absent and present group and the number of both types of ovaries were recorded. Collection and grading of cumulus-oocyte-complexes (COCs) After washing 2-3 times in saline solution, the ovaries were placed in a beaker and kept in a water bath at 30^oC. The 10 ml syringe was loaded with PBS (1-1.5ml), and the needle (19G) was put in the ovarian parenchyma near the vesicular follicles (2 to 6 mm diameter) and follicles aspirated near the point at the same time. The aspirated follicular materials were transferred slowly into a 90 mm petridish, avoiding damage to the cumulus cells and the COCs were searched and graded under microscope at low magnification. The COCs was then classified into 4 grades as described 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. : degeneration observed both in oocytes and cumulus cells. Grade A and B were considered as normal COCs and grade C and D will be considered as abnormal. The number of different grades of COCs in each category was recorded. In the meantime another petridish with D-PBS was prepared for pooling COCs and the COCs were picked up with an appropriate glass micropipette. The total work was performed in aseptic condition. The tip diameter of the pipette was checked under the microscope to ensure COCs, which could be easily aspirated without damaging the cumulus cells. Basically the glass micropipettes were prepared slowly stretching the tip of pasteur pipette above burners flame. Then the COCs were washed 2-3 times into D-PBS before initiating the maturation experiment.

The maturation medium, Tissue Culture Medium-199 (TCM-199) supplemented with 5% fetal calf serum (FCS) was prepared and its pH was adjusted at 7.2 and sterilized by passage through a 20µm sartorius Minisart filter. In a culture dish 4 drops of each about 100µl of maturation medium were poured and covered with paraffin oil. Then it was kept in an incubator at 38.5^oC with 5% CO2 in air. Normal COCs (A and B grade) were washed 2-3 times separately in PBS and then transferred into the maturation medium (TCM-199 supplemented with 5% FCS) and washed 2 or 3 times in maturation medium. Droplets containing graded oocytes were kept in a CO₂ incubator at 38.5^oC with 5% carbondioxide in air for 22 hours. After 22 hours of IVM, cumulus expansion was determined according to Rahman et al. (2003) by three levels in same magnification under microscope as i. indicating less expansion of COCs; ii. indicating moderate expansion and iii. indicating marked expansion of cumulus cells with a compact layer. The number of COCs classified on the basis of expansion rate of COCs was recorded.
Semen collection and sperm capacitation The fertilization medium (BO medium) was prepared and its pH was adjusted to 7.8 on the day of use. Finally it was sterilized by filtration. Semen was collected from the buck of departmental Artificial Insemination Center by artificial vagina (AV) method and was brought to the laboratory in icebox within a short period. The concentration of raw semen was calculated by haemocytometer. Fifty micro liter (µl) of raw semen were taken in 10 ml sterilized pipette and 3 ml to 4.2 ml (depending on the sperm concentration) of semen washing solution were added to adjust the sperm concentration to 25x106 per ml. The semen washing with washing solution (BO with 1% BSA) was taken in a centrifuge tube and centrifuged at 800 rpm at room temperature for 5 minutes and then top liquid portion was discarded. After that the same amount of sperm washing solution was added to the centrifuge tube and repeated the procedure twice. Finally the sperm concentration was adjusted at 12.5x106 per ml by adding semen dilution solution (BO with 2% BSA). Four insemination droplets (100 µl) of BO medium were prepared in a 35 mm culture dish, covered with paraffin oil and were kept in the incubator for preincubation The matured oocytes were washed 3 times in the oocyte washing solution (BO with 1% BSA). About 15-20 oocytes with minimum volume of medium were transferred to each of the sperm drops prepared previously and incubated for 5 hours at 38.5oC with 5% of CO₂.
In vitroculture (IVC) and observation After 5 hours incubation, the fertilized ova were taken from the semen drops with cumulus cells by using the appropriate micropipette. After that they were washed three times in pre-incubated medium (TCM-199) and were transferred to other culture drop (600) of TCM-199 with 5% FCS. The dish was then kept in the CO₂ incubator at 38.5^oC with 5% of CO₂ in air. The development was checked every 48 hrs and the culture were continued for 6 to 7 days. The number of compact morula and early blastocysts were recorded on day seven. Data were analyzed using SAS (Statistical Analysis System, 1998) package program in accordance with the principles of CRD (Steel and Torrie, 1980) and Duncun’s Multiple Range Test (DMRT) was also done to identify the significant differences between the mean values (Snedecor and Cochran, 1980).

Source of Information: Bang. J. Anim. Sci. 2008, 37 (1) : 1 -9 ISSN 0003-3588

Article Link (Online Journal):

Funding Source:

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Budget:

Total Budget (Tk.) :

Achievement/Findings:

It was concluded that grade A COCs were important criteria for production of goat embryo in IVP experiment than that of grade B COCs. The results demonstrated to be efficacious for the study of in vitro production of goat embryos and for the application of embryonic manipulation procedure in Bangladesh

Knowledge Management: Journal