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Detection of Brucella Abortus During in Vitro Bovine Embryo Production

A Akter
Beximco Pharmaceutical ltd, Dhaka

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

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

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

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

MR Islam
Bangladesh Agricultural Research Council, Farmgate, Dhaka 1342, Bangladesh.

SK Dey
Department of Microbiology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh.

Abstract/ Executive Summary:

The in vitro embryo production (IVP) technology has emerged as a potential biotechnological approach to multiply genetically high-yielding dairy cows. Its commercial application is increasing in many developed and developing countries over the years. Bangladesh Livestock Research Institute (BLRI) adopted in vitro embryo production protocol from the bovine ovaries of slaughterhouse. However, the risks of transmission of contagious diseases like Brucella abortus with embryos are not evaluated so far. Considering these facts, the present experiments were conducted to evaluate the efficiency of in vitro embryo production protocol with slaughterhouse ovaries as well as the risk of contamination of produced embryos with Brucella abortus. To identify sources of contamination of embryos with Brucella abortus (if any), the laboratory water, different media used in the IVP process, semen, and follicular fluids were evaluated for confirmation of the organisms. In addition, vaginal swabs were collected from 2 buffaloes aborted due to suspected Brucella abortus infection. Molecular tests were used to detect Brucella abortus contamination. Brucella abortus-specific PCR product was not detected on agarose gel electrophoresis. The efficiency of IVP measured by cleavage and blastocyst development rates were 75.5±2.7% and 16.6±3.9%, respectively. The present study inferred that the in vitro produced embryos are free from Brucella abortus infection.

Keywords: In vitro embryo production, Brucella abortus, Contamination, PCR

Location: Biotechnology Division and Bacteriology laboratory of Animal Health Research Division, Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka-1341.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Livestock

Objectives:

To identify the presence of Brucella abortus to assess the sanitary status of the in-vitro embryo production system in Bangladesh condition.

Methodology:

The research was conducted in the Reproductive Biotechnology laboratory of Biotechnology Division and Bacteriology laboratory of Animal Health Research Division, Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka-1341. Ovaries of slaughtered cows were collected from an abattoir located at Mohammodpur, Dhanmondi, Dhaka for IVP embryo production. The IVP protocol was described in a previous report. In brief, 10 cumulus-oocyte-complexes (COC) aspirated from 3 to 8 mm diameter follicles of slaughtered cow ovaries 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 120 µL droplet of IVM medium. 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 approximately1×106 spermatozoa/mL with IVF medium. The matured COC was co-cultured 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 into the culture droplet for 3 days. After 3 days, the 8 to 32 cell embryos were transferred into ICV-II medium for the 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. IVP was conducted in six batches. For detection of Brucella abortus infection during bovine IVP, samples were collected from six independent batches of the IVP experiment. Laboratory produced blastocyst, the laboratory water, TL-HEPES, Dulbecco’s phosphate buffer saline, in vitro maturation medium, in vitro fertilization medium, in vitro culture medium I and II, follicular fluid, and semen used for IVP were simultaneously analyzed for detection of Brucella abortus. Vaginal swabs collected from two aborted buffaloes (abortion was taken place during 6-7 months of pregnancy) were used as a susceptive positive control for Brucella abortus. The clinical symptoms of aborted buffaloes resembled Brucella infected buffalo symptoms. Moreover, extracted genomic DNA was collected from Bangladesh Agricultural University, Mymensingh as a known positive control for molecular confirmation of Brucella abortus. The vaginal swabs were diluted in a 200µl phosphate buffer saline solution. Initially, all samples (50 µL/plate) were plated into bacteriological nutrient agar media for 48 hours at 370 C in a humidified condition. If bacterial colonies were grown into the nutrient agar plate, then the colonies were streaked onto the Kuzdas and Morse selective agar media (Oxoid, UK) and incubated for 72 hours at 37° C. The media was sterilized in an autoclave at 121° C with 15 psi pressure for 15 min before streaking of colonies. Colonies developed into Kuzdas and Morse selective agar medium were sub-cultured on nutrient agar plate overnight and samples were taken for molecular characterization. Genomic DNA was isolated from bacterial colonies using the Wizard Genomic DNA Purification Kit according to the manufacturer’s instructions. Isolated DNA was quantified by Nanodrop 2000c. The concentration of extracted DNA was 22.2 to 35.6 nanogram/µl. The PCR was conducted using two sets of previously designed primers. PCR reactions were performed using a thermal cycler. The final volume for amplification was 15 µL, containing 1.5 µL of 10X reaction buffer, 0.45 µL of dNTPs, 0.15 µL of Taq polymerase, 2 µL of DNA, 1 µL of 10 pmol forward primer, 1 µL of 10 pmol reverse primers, and 7.7 µL of PCR water (DNase, RNase, and Protease free water). The reaction mixture was first denatured by heating at 94°C for 1 minute, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 54°C/60°C for 1 minute, and extension at 72°C for1 minutes. At the end of the cycles, the reaction mixture was maintained at 72°C for 5 minutes. The PCR products were loaded into a 1.5% agarose gel containing 1X TAE (Tris- Acetate-EDTA) buffer at 90 volts for 60 minutes. The gel was stained with ethidium bromide before being photographed using ultraviolet illumination.

Source of Information: Bang. J. Livs. Res. Vol. 27 (1&2), 2020: P. 105-112

Article Link (Online Journal): DOI: https://doi.org/10.3329/bjlr.v27i1.55174

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The present findings inferred that the in vitro embryo production protocol of BLRI support normal cleavage and blastocyst development and developed blastocysts are free from Brucella abortus.

Knowledge Management: Journal