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In Vitro Production of Zygote from Slaughterhouse Driven Buffalo Oocyte

GK Deb
Biotechnology Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341

MFH Miraz
Biotechnology Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341

SMJ Hossain
Biotechnology Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341

MF Afroz
Biotechnology Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341

MA Kabir
Biotechnology Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341

S Akhter
Breed up gradation through progeny test project (3rd phase), Department of Livestock Services

Abstract/ Executive Summary:

Buffalo is a highly potential animal species in terms of milk and meat production but traditionally they are regarded as poor breeder. In vitro embryos production technology has been introduced in many countries to improve reproductive efficiency of buffalo. Considering the above fact, the present study was undertaken aiming to produce in vitro buffalo embryo in the laboratory. Ovaries of slaughtered buffaloes were collected from abattoir and transported to the laboratory within 4 to 5 hr of slaughter. Cumulusoocyte- complexes (COCs) possessing an even cytoplasm and covered with minimum 3 layers of compact cumulus cells was selected for in vitro maturation (IVM) for 24 hr (5% CO₂ in air at 38.5°C with maximum humidity). After IVM, the presumptive matured COCs were co-cultured with capacitated fresh spermatozoa for 18 hr After IVF, the presumptive zygote were denuded, washed and transferred in to in vitro culture medium (IVC 1) for 3 days. After three days cleavage were recorded and 4 cell embryos were transferred in to in vitro culture media II for next 2 days. The development of embryos was evaluated on day 6. A total of 227 buffalo ovaries were collected from the slaughterhouse and categorized into 2 groups based on presence (n=83) or absence (n=144) of corpus luteum (CL). A total of 1464 follicles were counted on the ovarian surface, 1066 being from CL absent and 398 from CL-containing ovaries. A significantly higher (P<0.01) number of follicles, aspirated follicles, normal COCs and total COCs (7.4 ± 0.21, 5 ± 0.00, 1.98 ± 0.77 and 2.98 ± 0.16 respectively) were observed in CL-absent ovaries than those aspirated from CL-containing ovaries (4.80 ± 0.17, 3.92 ± 0.95, 0.88 ± 0.60 and 1.88 ± 0.16 respectively). Total 358 normal COCs were set for in vitro maturation and underwent for IVF and IVC. Results showed that cleavage rates were 56.42%. Among the cleaved embryos, 137 were at 2-cell stage and 65 were at 4-cell stage. Therefore, development rate to 2 cell and 4-cell stage was 38.27% and 18.15% respectively. No embryo developed beyond 4-cell stage. This result indicates that follicle and oocyte numbers and oocyte quality are associated with CL of ovaries and current culture system support in vitro embryo production up to 4-cell stage. The in vitro culture condition may be improved for increasing efficiency of embryo production.

Keywords: Buffalo, oocyte, In vitro maturation, Cumulus oocyte complexes, Cleavage

Location: Kaptan bazar, City Corporation Slaughterhouse, Gulistan, Dhaka

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Buffalo

Objectives:

To produce in vitro buffalo embryo in the laboratory.

Methodology:

Ovaries of slaughtered buffaloes was collected from abattoir located at Kaptan bazar, City Corporation Slaughterhouse, Gulistan, Dhaka in physiological saline (0.9% sodium chloride supplemented with 100 IU/mL penicillin and 0.1 g/mL streptomycin sulfate) at ambient temperature and transported to the laboratory within 4 to 5 hr of slaughter. In the laboratory, extraneous tissue was removed and ovaries were washed with phosphate buffer saline (PBS). The collected ovaries were divided into two groups according to presence and absence of CL. The cumulus-oocyte-complexes (COCs) were aspirated using a 10-mL disposable syringe attached with a 21G needle. The collected cumulus-oocyte-complexes (COCs) were graded as normal and abnormal grade for in vitro maturation (IVM) based on their size, diameter and quality of cumulus cell. The aspirated material were poured onto a 100-mm petridish containing TL-HEPES (114-mM sodium chloride, 3.2-mM potassium chloride, 2-mM sodium bicarbonate, 0.34-mM sodium biphosphate, 10-mM sodium lactate, 0.5-mM magnesium chloride, 2.0-mM calcium chloride, 10-mM hepes, 1 μL/mL phenol red, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin) solution and the cumulus-oocyte-complexes (COCs) were searched under a microscope at low magnification (4x). The ‘normal’ cumulusoocyte-complexes (COC) possessing an even cytoplasm and covered with minimum 3 layers of compact cumulus cells were selected for in vitro maturation (Stojkovic et al., 2001). The selected COCs (50 to 70 per well) were washed 2-3 times in TL-HEPES and 2-3 times in IVM medium (TCM199 + 10% FBS, 1 μg/mL β-estradiol, 10 μg/mL FSH, 0.6-mM cystein, and 0.2-mM sodium pyruvate) before placing them into a well of 4-well dish containing 500 to 700 µL IVM medium for 22 to 24 hr. The matured COCs were fertilized in vitro by fresh semen collected from buffalo bulls of BLRI buffalo farm using artificial vagina method. 10 µL semen was placed in a 15-mL conical tube containing 10 mL D-PBS and pelleted by centrifugation at 750×g for 5 min. The supernatant was removed carefully and 10 mL D-PBS was added in the tube. The sperm was washed for 2-3 times accordingly. Then spermatozoa was capacitated through incubation with 500 μL IVF medium (Tyrode’s lactate solution supplemented with 6 mg/mL BSA, 22 μg/mL sodium pyruvate, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin) containing heparin sodium salt (20 μg/mL) for 15 min. After capacitation, the spermatozoa diluted at approximately 1×106 spermatozoa/mL with IVF medium. The matured COC were co-cultured with capacitated spermatozoa for 18 to 20 h through placing them into a well of 4-well dish (500 to 700 μL). All values relating to follicut or statistics were expressed as Mean±SE. The statistical analysis were done using SPSS IMB 20.0 version software programme.

Source of Information: Bangladesh J. of Livestock Res. 21-25: 127-132, 2018

Article Link (Online Journal): DOI: https://doi.org/10.3329/bjlr.v0i0.45455

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Comparatively higher numbers of ovaries were found without corpus luteum compared to ovaries with corpus luteum confirms that usually noncyclic buffaloes are slaughtered in the slaughterhouse and that is may be due to economic reason. Ovaries without CL contributing larger number of follicles aspirated per ovary compared to ovaries with CL. Furthermore, comparatively higher number of total COCs and superior quality of COCs were possible to obtain from without CL ovaries, suggested to be suitable for collecting COCs for initiating in vitro embryo production experiment in buffaloes. In vitro embryo production experiment concluded that current culture system support in vitro embryo production up to 4-cell stage. Improvement of culture condition may improve the efficiency of embryo production.

Knowledge Management: Journal