Parthenogenesis is a phenomenon in which the development of oocyte oocur without fusion of male gamete. During fertilization spermatozoa trigger intracellular Ca+2 oscllation in M-II stage oocyte which initiates the embryonic development. The rises of intracellular calcium (Ca2+) ions is the basic step for the parthenogenesis. During parthenogenetic activation calcium channel open from endoplasmic reticulnum or depletion of calcium store and facilitate the calcium (Ca2+) from extracellular environment. Parthenogenetic technique is applied in cloning and production of embryonic stem cell lines for used to treat different diseases. Many scientists used different chemicals agents for artificial activation such as strontium, Ionomycin and Ethanol. Strontium chloride has been used widely for parthenogenetic activation of mouse oocyte, but its result to blastocyst development is poor. The objective of present study is to improve parthenogenetic activation and embryo development by combination of Ionomycin with strontium. Hypothesis of my study was Addition of Ionomycin in Strontium based activation protocol improves embryonic development.
The present study was conducted in embryology lab of department theriogenology, university of veterinary and animal sciences, Lahore.Six to eigth week old female mice (n=100) were super ovulated with intra-peritoneal injections of eCG (5iu) followed by hCG injection (5iu) at 48 hrs interval. 14 hrs post hCG, the cumulus oocyte complexes were collected from oviduct of the mice. In experiment 1, the oocytes were activated by using Ionomycin with concentration of 5, 10 and 15 µmol/l for 5 and 10 followed by this activation with strontium chloride (10mmol/l). In experiment: 2, The oocytes were activated by activation medium having strontium (10 mM/l) and Ionomycin (5, 10 or 15 µmol/l) in combination. CZB medium were used for oocyte cultured in CO2 incubator of 5% CO2 at 37°C. Number of activated oocytes were analyzed by cleavage rate to blastocyst stage. In-vitro developmental potential of the activated oocytes were assessed by blastocyst. In experiment: 3, Zygotes were collected 18 h post-hCG and treated with the optimum concentration to check the toxicity effects on embryo development.
In experiment 1, There were insignificant results observed on the bases of cleavage rate in each groups and time of activation as compared to control group. The tendency of morula and blastocysts formation rate was higher (p<0.05) in the 15µM for 10 min activation time as compared to other treatment groups and control group. In experiment 2, The tendency of cleavage rate was significantly higher in the 10 µM and 15µM groups as compared to other treatment group. The blastocyst formation rate was no statistically difference in all treatment and control group. While the toxicity experiment, there was no toxic effect of Ionomycin with Strontium Chloride.
In conclusion, there was higher cleavage rate, 4 cells, morula and blastocyst formation rate in 15µM concentration of Ionomycin for 10 min with Strontium Chloride, there was no toxic effect of Ionomycin with Strontium Chloride on embryos and Ionomycin improved the activation rate and embryo development in combination with strontium chloride.