Action of leukemia inhibition factor on in vitro maturation, fertilization and blastocyst development of Rabbit oocyte
K Thara
Keywords
blastocyst., in vitro maturation ivm, ivf-in vitro fertilization, leukaemia inhibition factor lif, oocyte
Citation
K Thara. Action of leukemia inhibition factor on in vitro maturation, fertilization and blastocyst development of Rabbit oocyte. The Internet Journal of Veterinary Medicine. 2009 Volume 8 Number 1.
Abstract
Abbreviations: In vitro maturation (IVM), In vitro fertilization (IVF), LIF- Leukaemia inhibitory factor
Introduction
Leukaemia inhibitory factor (LIF) is a polyfunctional glycoprotein cytokine whose inducible production can occur in many, perhaps all, tissues. (Kuriek J, 2000).
Rabbit oviduct epithelium cell monolayers were able to support the development of cleaved bovine embryos to the blastocyst stage as compared with the embryos on cow oviduct epithelium cell monolayers (Prokofieve et al 2005). When added at 120 hours post insemination, glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose (Pavasuthipaisit K et al., 1992).
Stimulation of bovine IVM/IVF embryo development by somatic cell conditioning is due to removal of inhibitory influences from the culture environment (Barry et al., 1992).
In the present investigation effect of LIF in the maturation, fertilization, development of rabbit oocytes were studied. We have investigated the effect of LIF on gametes
Materials and Methods
Chemicals were purchased from M/s. Hi media, Mumbai. LH and FSH were procured form Sigma Aldrich, Bangalore.
Methods
Collection of oocytes and maturation
Oocytes were collected from rabbit ovaries obtained from the slaughter house daily. It was brought to the laboratory within 2-3 hrs. of slaughter in a thermo flasks with warm water at 30-35 0C supplemented with 50µg/ml of gentamycin. Ovaries were crushed and the follicular fluid was collected in a 50ml sterile conical tube and kept at 38 o C for 10-15 minutes to settle down the debris. Then spin at 500 rpm for 5 min. The supernatant was then discarded and the pellet was carefully diluted with TL medium buffered with10 mm HEPES and low bicarbonate TALP (Parish JJ ,1998) which was pre incubated at 38oC in CO2 incubator for 4-5 hrs. It is then screened under microscope for oocytes. The mature oocytes are seen having compact cumulus cells and clear cytoplasm. The number of good quality oocytes collected was counted. The collected oocytes are then thoroughly washed in medium with TCM 199+HEPES and 10% fetal calf serum, 0.2 mM pyruvate and50µg/ml of gentamycin and randomly assigned to experimental groups. Oocytes were kept in maturation drops as 20 oocytes per 50µl drop and layered with paraffin oil which was kept in co 2 incubator for equilibration. Maturation media consists of TCM 199 supplemented with sodium bicarbonate, 10 %FCS, 5g/ml FSH, 50g/ml LH, LIF and 0.2mg/ml gentamicin. These oocytes were again kept in CO2 incubator at 38oC for 18-24 h for maturation.
The experiment was repeated for different concentrations of LIF such as 0, 5 and 10µg/ml in the maturation medium. Total 200 numbers of oocytes were taken for each concentration of LIF and the experiments were done in triplicate.
In vitro fertilization
Frozen thawed semen collected from Kerala Live Stocks department was used for
One straw of semen of a particular male is taken out from liquid nitrogen and transferred to a water bath at 38O C. Dead and live sperm were separated by percoll separation The pellet was then diluted to a required concentration of 2 x 106 sperms /ml with TALP with HEPES (Bavister et al., 1983, Fuki et al 1983, Niwa et al 1986). It was then incubated with 10 oocytes /50 l of the fertilization drops. The fully matured oocytes were collected from maturation media, washed with fertilization media and kept in fertilization drops i.e. 10 oocytes/50l of fertilization drop. Then washed diluted semen is added and kept for fertilization at 380C in a CO2 incubator for 24 to 36 hrs. Fertilization was carried out in TALP-FERT supplemented with heparin, penicillamine, hypotaurine, epinephrine, LIF and BSA. Fertilized oocytes were either used for fixing or transferred to embryo development medium. The experiment was repeated for 0, 5, 10g/ml of LIF concentrations in the fertilization medium.
The oocytes were then taken and the cumulus cells were removed by repeated pip petting with a small bored pipette. Cumulus free oocytes were either mounted or transferred for embryo development. (Totey et al., 1992) .Oocytes were classified as normal if it is fertilized for single sperm otherwise poly spermy if fertilized by multiple sperms. If there is contamination the oocytes get degenerated.
Fertilized oocytes were transferred to CR2 development medium supplemented with 10%FCS(v/v),BSA, gentamicin and LIF as described by Rosekrans and First (1991). It was then observed for blastocyst development at every 24 hours for eight days. Then the blastocysts were observed for embryo hatching stage in the medium with and without LIF. The evaluation of embryos for blastocyst, hatching etc were made according to the International Embryo Transfer Society manual (Stringfellow 1999).In brief the embryos were observed under a phase contrast microscope .The number of cells were counted.
Statistical analysis
The maturation, fertilization rate was observed for different LIF concentration. The same experiment is repeated in replicate and data was analyzed by
Results
Oocytes cultured in the medium with LIF showed an increase in the maturation rate than the medium without LIF
The fertilization rate also increased significantly when LIF at concentration of 10 ng/mL is added in the fertilization medium.(p<0.006
When embryos were transferred to development medium with LIF at a concentration of 50 ng/mL, the inner cell mass increased significantly for the first eight days. After eight days not much differences were observed. The number of embryos reaching the hatching stage was significantly reduced on adding LIF in the development medium.
Figure 1
Figure 2
Figure 3
Discussion
There are different factors which influences the maturation, fertilization and embryo development
I t is reported in sheep that LIF exerted a positive influence on the quality of the blastocysts as revealed by significantly higher number of ICM cells and total number of cells. (.Grazyna Ptak et al.,2006).
Conclusion
In conclusion LIF exerts influence on
Acknowledgement: All staff in the department of Animal reproduction ,cv&asc, KAU , Mannuthy is duly acknowledged.