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In Vitro Maturation and Fertilization of Buffalo Oocytes Cultured in Media Supplemented with Bovine Serum Albumin

A.N.M.I. Rahman
Department of Animal Nutrition, Genetics and Breeding, Sher- e - Bangla Agricultural University, Dhaka, Bangladesh.

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

L. Asad
Department of Animal Nutrition, Genetics and Breeding, Sher- e - Bangla Agricultural University, Dhaka, Bangladesh.

S. Saha
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

R.C. Paul
Department of Genetics and Animal Breeding, Patuakhali Science and Technology University, Babugong, Barisal, Bangladesh.

S. Debnath
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Abstract/ Executive Summary:

The aim of this study was to determine the quality of cumulus-oocyte-complexes (COCs) and the effects of bovine serum albumin (BSA) supplementation on in vitro maturation and fertilization rate of buffalo oocytes. COCs were collected from slaughterhouse buffalo ovaries by aspiration method. Only normal grades COCs were matured for 48 hours in TCM-199 media. Two groups were created: one for the maturation medium supplemented with 5% of BSA, the other without supplementation (control). Matured oocyte fertilized with capacitated frozen-thawed semen in Brackett and Oliphant (BO) medium for 5 hours, in an incubator at 38.5 ° C with 5% CO₂ under humidified air. A significantly higher number of normal quality COCs per ovary (P<0.05) were obtained from ovaries devoid of corpus luteum (CL) compared to ovaries having CL (1.84 vs. 0.81) respectively. The percentage of oocytes reaching Metaphase-II (M-II) stages was 58.07±2.08 and 68.10±0.75% for control and 5% level of BSA respectively. The fertility level was assessed by pronuclei formation: the normal fertilization rate (2PN) obtained was 19.63±3.11 and 29.52±1.98% for control and BSA supplementation respectively. Significant differences (P<0.05) were observed in maturation (M-II) and fertilization (2PN) rate of buffalo ooc yte by adding 5% level of BSA supplementation in culture media. Thus, data gathered in this study showed that 5% BSA supplementation in both maturation and fertilization media can be used for enhance the maturation and fertilization rate of buffalo oocytes, as well as to improve the grade of collected buffalo COCs.

Keywords: BSA, Buffalo, Cumulus, oocyte, Complexes, IVF, IVM

Location: Bangladesh agricultural university dairy farm, Mymensingh.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Buffalo

Objectives:

1. To collect and evaluate buffalo ovaries, follicles and COCs obtained at the slaught erhouse.

2. To analyze the effects of BSA supplementation in the maturation and fertilization media in the in vitro maturation and the fertilization rate of buffalo oocytes.

Methodology:

Buffalo ovaries were obtained from a slaughterhouse, were placed in normal saline (0.9% Na Cl) and transported to the laboratory in a thermo flask maintained at 25 °C to 30 °C within 5 to 6 hours. The ovaries were rinsed thoroughly, twice, in physiological saline solution at 25 °C. In the laboratory, each ovary was isolated from the surrounding tissues and overlying bursa, and washed three times in D-PBS, and then twice in oocyte harvesting medium (D-PBS+4 mg/mL BSA+1.50 IU/mL Penicillin) as described. After collection and trimming, ovaries were evaluated on the basis of presence and absence of CL. Buffalo oocytes were aspirated using a 18 G needle coupled to a 10 mL syringe filled with D-PBS (1.0-1.5 mL); all 2-6 mm diameter follicles were aspirated. The follicular content was then transferred to a 90 mm Petri dish, slowly to avoid damaging the cumulus cells. The total number of oocytes harvested was counted under a stereomicroscope. COCs were classified into 4 grades as described, according to the morphology of the cumulus cells and nucleus. Briefly, in Grade A the oocytes completely sur- rounded by cumulus cells; in Grade B the oocytes partially surrounded by cumulus cells; Grade C oocytes are not sur- rounded by cumulus cells; and in Grade D, degeneration was observed in both the oocytes and the cumulus cells. Grades A and B were considered as normal and grade C and D as abnormal COCs. For in vitro maturation of COCs, two different culture media were used: one using 5% BSA supplementation and the other without it. Unless otherwise mentioned, all chemicals and media were purchased from Sigma Chemical co. The IVM of oocytes was performed using a medium consisting of bicarbonate-buffered tissue culture medium (TCM-199), 0.22 m M sodium pyruvate, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. To study the effects of bovine serum albumin on the maturation rate of buffalo oocytes, to this medium 5% BSA was added in the tested BSA supplemented medium. The pH was adjusted to 7.3-7.4 in all the media and they were filtrated through a 20 μm sartorius Minisart filter. From each group of maturation media, about 1-4 drops of 100 μL were prepared into each of two 35 mm culture dishes. Batches of 13-15 oocytes were then transferred into these, cove red with paraffin oil and incubated at 38.5 °C in a humidified atmosphere of 5% CO₂, for 48 hours. The level of nuclear maturation was then assessed in each dish. For this purpose, 25% of cultured COCs were randomly sampled from each droplet. COCs were then denuded and mounted on glass slide and fixed with ethanol:acetic acid (3:1). Semen was collected by artificial vagina (AV) from buffalo in the Bangladesh agricultural university dairy farm, Mymensingh. After semen assessment, semen was diluted in the Triladyl based cryodiluent (diluter+cryoprotectant) in a final concentration of 2×10 6 spermatozoa per mL. The motility of the equilibrated sperm was checked and only samples with more than 60-70% motility were used for freezing, as described. The frozen semen straws were transferred into the liquid nitrogen container, at -196 °C, until use. Data generated in this experiment were entered in Micro- soft Excel worksheet, organized and processed for further analysis. The analysis of variance (ANOVA) was used to compare the oocyte recovery rates, while the effects of CL presence on oocyte recovery were analyzed using Student’s t-test. For in vitro maturation and fertilization, the values analyzed by ANOVA and followed by a least square difference (LSD) test, using SPSS software version 16.0.

Source of Information: Iranian Journal of Applied Animal Science (2015) 5(3), 545-551

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The present study focused on the influence of the presence or absence of CL on quantity, quality and developmental ability of oocyte from buffalo ovaries, as well as on the BSA supplementation in IVM and IVF of buffalo oocytes. After the above discussion, we could conclude that the oocyte yield per ovary was higher in ovaries without corpus luteum and that normal grade COCs (Grade A and Grade B) were suitable for in vitro production (IVP) of buffalo embryos. Considering the effects of BSA on the in vitro maturation and fertilization of buffalo oocyte, 5% BSA can be advantageous as a supplement of maturation and fertilization media to increase the developmental rate of buffalo oocyte. Moreover, this result creates a great opportunity of conducting further research on buffalo embryo production in Bangladesh.

Knowledge Management: Journal