Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

In Vitro Culture of Mouse Embryos

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

M. R. Ali
Senior Instructor
Livestock, Youth Training Centre, Khulna, Bangladesh

M. R. Islam
M. S. Student
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh, Bangladesh

M. G. M. Rahman
Senior Instructor
Livestock, Youth Training Centre, Gaibandha, Bangladesh

M. A. S. Talukder
Scientific Officer
AI Laboratory, Dairy and Cattle Improvement Farm, Rajshahi, Bangladesh

Abstract/ Executive Summary:

The present study was undertaken to test the cyclic pattern, to fix the hormonal dose for superovulation and to establish the suitable condition for in vitro culture (IVC) of mice embryos up to blastocyst. The cyclic pattern of mouse was confirmed by checking the vaginal plug. For superovulation the mice of diestrous stage were treated with three different doses (5, 10 and 15 IU) of pregnant mare serum gonadotrophin (PMSG) hormone through intraperitoneal injection and 48h after PMSG, three doses (5, 10 and 15 IU) of human chorionic gonadotrophin (hCG) were injected respectively. Four-cell embryos were collected from mouse by cervical dislocation between 4 and 5 hours on day 3 of pregnancy (60h after hCG injection) and cultured for 48h in CO₂ incubator with 5% CO₂ in the air at 37^oC. Almost 65% of mice were found in normal cyclic pattern. Significantly highest (p <0.01) number of embryos were recovered from dose of 10 IU (11.00 ± 0.50) as compared to the dose of 15 IU (9.56 ± 0.44) and dose of 5 IU (6.00 ± 0.41). The development of mouse embryos in M16 medium were found to be in 88.30% into eight-cell embryos, 83.04% into morula and 71.92% into blastocyst. The results indicate that the culture condition was optimum and expected to provide an important base for the future studies in livestock embryos.

Keywords: Mouse embryo, Culture, hCG, PMSG

Location: Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh, Bangladesh

Start Date: 00-03-2004

End Date: 00-11-2004

Program Area: Risk Management in Agriculture

Commodity: Vertebrate

Objectives:

To fix the hormonal dose for superovulation, and
To establish the suitable condition for in vitro culture (IVC) of mice embryos up to blastocyst.

Methodology:

The experiment was conducted at the Reproductive Biotechnology Laboratory (RBL) of the Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh from March to November 2004. Collection and maintenance of mice: Albino mice of 6 to 8 weeks age were collected from International Center for Diarrhoeal Disease Research, Bangladesh (ICDDRB). The mice were kept under controlled lighting conditions i.e. 12h light: 12h darkness. The mice were allowed to free access of commercial pellet diet and tap water at all time. Observation of reproductive status of mice and preparation for superovulation: The reproductive status of the female mice was confirmed by checking the vaginal plug for a period of two weeks. For this purpose vaginal smear was taken in a slide and stained for 10 minutes with Rose Bengal stain. After washing in tap water the slide was dried in air and observed under microscope for confirming the different stages of estrous cycle. The mice of diestrous stage were then treated with different three doses (5, 10 and 15 IU) of pregnant mare serum gonadotrophin (PMSG) hormone through intraperitoneal injection and after 48 hours, three doses (5, 10 and 15 IU) of human chronic gonadotrophin (hCG) hormone were injected respectively. After hCG injection the females were placed immediately with fertile males of same strain at the ratio of 2: 1. The vaginal plug test was performed in the following morning to confirm whether they were mated or not. Moreover, vaginal smearing was also performed to examine the presence of sperm, which further confirmed copulation. The day of copulation was considered as day-I of pregnancy. Two lots of mice examined for reproductive states were considered two different groups (1 and 2). Media preparation: The established medium for mouse embryo culture is Whittingham's M16. Prior to culture of 4-cell embryos the Whittingham's M16 medium was prepared and the pH of the medium were adjusted at 7.2 to 7.4. Then in a 35-mm culture dish, 4 droplets each of 100µl was prepared and covered with paraffin oil and preincubated with 5% CO₂ in air at 37°C. Each droplet may contain about a number of 10 four-cell embryos and incubated for next 48 hours. Collection of embryos: The mated mice were killed by cervical dislocation between 4 and 5 hours on day-3 of pregnancy (60h after hCG injection). Then the oviduct and uterus of the mice were scratched out in 0.9% NaCl solution through needle under microscope and the 4-cell embryos were collected. Washing and culture of embryos Collected 4-cell embryos were washed 3 times with preincubated M 16 medium and they were then transferred in the 4 droplet of preincubated M 16 medium. The droplets having 4-cell embryos in the 35-mm culture dish were kept in the CO₂ incubator and cultured for 48h with 5% C02 in the air at 37°C. Observation on the in vitro developed embryos Development of embryos were observed under microscope at every 24-hour after culture initiation to obtain the morula and blastocyst. The number of morula and blastocyst developed were recorded. Statistical analyses: The collected data were compiled and tabulated for statistical analysis. Calculation of the mean and standard error (SE) for each of the treatment and students t-test were performed using SPSS computer package (SPSS Ine. 1999, Microsoft Corporation 1998).

Source of Information: Progress. Agric. 16(1): 121-128,2005, ISSN 1017-8139

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The result of the development of mouse embryos in M16 medium was 88.30% into eight-cell embryos, 83.04% into morula and 71.92% into blastocyst. In another experiment the rate of development of mouse embryos up to morula and blastocysts were reported to be 93.3 and 84.4 % (Khandoker et al., 1995) in M16 medium. There has been little deviation in the present result as compared to that of Khandoker et al. (1995), which might be, due to the difference in human and animal error. The results suggest that the culture condition is optimum and will provide a base line information for future studies with embryos of domestic mammals

Knowledge Management: Journal