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Multiplication of Red Chittagong Cow Using Ovum Pick Up (opu) Approach

Gautam Kumar Deb
Senior Scientific Officer
Bangladesh Livestock Research Institute, Savar, Dhaka

M. R. Islam
Bangladesh Livestock Research Institute, Savar, Dhaka

M. M. Rahman
Principal Scientific Officer
Biotechnolog Division.Bangladesh Livestock Research Institute, Savar, Dhaka

T. N. Nahar
Chief Scientific Officer
Head,Biotechnolog Division.Bangladesh Livestock Research Institute, Savar, Dhaka

M. A. Kabir
Scientific Officer
Bangladesh Livestock Research Institute, Savar, Dhaka

M. Y. A. Khan
Scientific Officer
Animal production Research Division Bangladesh Livestock Research Institute, Savar, Dhaka

Abstract/ Executive Summary:

To achieve above objectives, slaughterhouse bovine ovaries were used during this study. Oocytes were aspirated using a 10-mL disposable syringe attached with 21G needle and searched under stereomicroscope. Oocyte possessing an even cytoplasm and covered with minimum 3 layers of compact cumulus cell were subjected to maturation in vitro (IVM). The matured COC were fertilized in vitro (IVF) using fresh semen capacitated by treating with heparin sodium salt. After IVF, cumulus cells were removed by gentle pipetting and denuded zygotes were subjected to in vitro culture (IVC) for blastocyst development. Cleavage rates were evaluated at day 3 (day 0: day of IVF) and blastocyst rate at day 8 as a proportion of the presumed zygote transferred into IVC-I medium. Mean and standard deviation were calculated using Microsoft Office Excel programme. Results show that duration of treatment of spermatozoa with heparin had no significant effect (P>0.05) on capacitation of spermatozoa as evaluated by cleavage and blastocyst development rates following IVF. The effect of COC density on embryo development efficiency was evaluated in another experiment. Results show that development rate up to cleavage did not affect by COC densities. Single COC did not reach to the blastocyst stage when cultured individually. The development rate to the blastocyst stage was 13.9±9.0%, 10.8±4.9% and 16.6±3.9% for 3, 5, and 10 embryo groups, respectively. Alternatively, the GPS group culture allowed the smaller numbers of COC (1, 2, 3 and 4 COC per chamber) to develop at blastocyst stage. In third experiment, research was carried out to adopt bovine embryo cryopreservation protocol. This experiment was conducted two times but preserved embryos did not survive after post thaw culture. This experiment is continuing for standardization of the protocol. In fourth experiment, the risk of Brucella abortus contamination status of in vitro produced embryos was evaluated. No Brucella abortus organism was detected in blastocyst, laboratory water, semen and different culture media used during IVP. In conclusion, the present experiment developed Brucella abortus free in vitro embryo production system for small number of bovine oocytes.

Keywords: Bovine, Cumulus-oocyte-complexes, Cleavage, Brucella abortus, Blastocyst

Location: BLRI, SAVAR, DHAKA

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Cattle

Objectives:

1. To develop a culture system suitable for culture of smaller number of cumulus-oocyte-complexes (COC) (less than 10 COC)

2. To adopt embryo cryopreservation

3. To evaluate Brucella abortus contamination risk in in vitro produced bovine embryos.

Methodology:

In vitro embryo production - In vitro embryo production (IVP) protocol was described. In summary, the cumulus-oocyte-complexes (COC) were aspirated from 3 to 8 mm diameter follicles of slaughtered cow ovaries using a 10-mL disposable syringe attached with a 21G needle and searched under a stereomicroscope. The COC possessing an even cytoplasm and covered with minimum 3 layers of compact cumulus cells were selected for in vitro maturation (IVM). The selected COC were washed 2-3 times in TL-HEPES followed by 2-3 times washing in IVM medium before placing them into IVM droplet as per experimental design. The matured COC were fertilized in vitro (IVF) using fresh semen capacitated through incubation with heparin sodium salt (20 μg/mL) dissolved in the IVF medium for 15 min. The capacitated sperm were diluted at approximately 1×10⁶ spermatozoa/mL with IVF medium. The matured COC were cocultured with capacitated spermatozoa for 18 to 20 h. After IVF, cumulus cells were removed by gentle pipetting into TL-HEPES. The denuded zygotes were washed in in vitro culture (IVC) medium-I (3 times) and placed them into the culture droplet for 3 days. After 3 days, the 8 to 32 cell embryos were transferred into ICV-II medium for remaining culture period (until Day 8). The incubation conditions during IVM, IVF, and IVC were 5% CO₂ in air at 38.5°C temperature with maximum humidity. Cleavage development rates were evaluated at day 3 (day 0: day of IVF) as a proportion of the presumed zygote transferred into IVC-I medium. Blastocyst development rates were calculated at Day 8 as a proportion of the presumed zygote transferred into IVC-I medium. The data were analyzed using one way ANOVA method by SPSS statistical packages.

Source of Information: BLRI, Paper presented in the annual research review workshop held at Bangladesh Livestock Research Institute, Savar Dhaka in June 2013.

Article Link (Online Journal):

Funding Source:

1.	Budget: What is the budget of the project?

Total Budget (Tk.) :

Achievement/Findings:

The present study developed Brucella abortus contamination free in vitro embryo culture system for small number of COC.

Knowledge Management: Report/Proceedings