Comparison of two different media and assisted hatching techniques on the embryo hatching rate using the mouse as a model

Abstract

The use of in vitro culture media and assisted hatching techniques remain a challenging obstacle to hatching of blastocyst-stage embryos. Mechanical, chemical, enzymatic thinning and laser assisted techniques have been used previously, but there is still a lack of information on its application and implication in livestock. The aim of this study was to compare the effect of two in vitro culture media ((Ham’s F10 and Tissue Culture Medium 199 (TCM-199)) and four assisted hatching techniques (mechanical, chemical, enzymatic and laser) on blastocyst formation and hatching rate using murine embryos as a model. The C57BL/6 and BALB/c mouse breeds were bred and raised until they reach maturity and then bred naturally to produce a hybrid F1 generation. The light in the breeder house was controlled at 14 hours light and 10 hours darkness. Feed and water were provided ad libitum for the mice. Mature female mice were super-ovulated using equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A total of 400 blastocysts were collected from the F1 generation and these were allocated equally for the four assisted hatching techniques (laser, mechanical, chemical and enzymatic) as well as a non-treated control group. The blastocysts were paired into a group of 10 and replicated 4-four times for each assisted hatching techniques and control group. The embryos were then cultured for 24 hours and the hatching of the embryos were observed. Hatched embryos were stained for blastomere counting. The general linear model (GLM) of statistical analysis software (SAS) version 9.4 was used to analyze the data. Assisted hatching techniques (laser, mechanical, enzymatic and chemical) yielded 46.86±37.12; 51.07±40.19; 39.05±35.83 and 33.32±37.50% of hatching, respectively under in vitro culture in Ham’s F10. There was a significant difference (p<0.05) observed between assisted hatching techniques using Ham’s F10 as culture medium. In the TCM-199, laser, mechanical, enzymatic and chemical assisted hatching techniques yielded 56.25±43.30; 52.55±35.50; 49.16±37.50 and 33.85±35.50%, respectively, with significant differences (p<0.05). However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM-199 compared to those cultured in Ham’s F10, and statistically higher than the control group. In conclusion, laser assisted hatching technique is the best of the techniques to use to assist the hatching of murine embryos and TCM-199 is the best of the two in vitro culture media for the hatching percentage.