Effect of FOUR Assisted Hatching Techniques and Two in Vitro Culture MEDIA on Embryo Hatching RATE

The objective of the study were to determine the effect of four assisted hatching (AH) techniques (mechanical, chemical, enzymatic and laser ) and use of two in vitro culture media (Hams F10 and TCM-199) on hatching rate of blastocyst-stage embryos following 24 hour in vitro culture. The C57BL/6-black and BALB/c-white breeds were raised until maturity and naturally bred to produce F1 generation. The light in the breeding house was controlled and the mice were fed without restriction. Superovulation was done using both equine chorionic gonadotropins and human chorionic gonadotropins and the F1 generation was used for collection of the blastocyst-stage embryos at six weeks of age. There was a significant difference (P<0.05) observed between the diameter of ZP diameter before AH and after 24 hours of culture, while no significance difference (P>0.05) among hatching rates and the blastomeres counted after staining. The diameter of the ZP decreased with assisted hatching techniques. Also, the interaction between AH techniques and in vitro culture was found to be significantly different (P<0.05) on hatchability, but with highest hatchability in laser AH techniques 85.4±13.30% with TCM-199 culture medium followed by mechanical AH techniques at 79.13±19.50% in the same in vitro culture medium. The lowest hatchability (69.36±30.50%) was recorded in the chemical AH technique group The number of blastomeres counted under interaction of AH techniques and culture media was not significantly different (P˃0.05), with the values ranging from 69±6.35 to 76±10.92 blastomeres for hatched embryos. Data collected were subjected to analysis of variance using PROC General Linear Model of SAS version 9.4, where samples were randomly collected. Where significant differences between groups was detected and the Tukey’s test was used to separate the means. In conclusion, the results of the study indicated that the use of different AH techniques has got varying and increase outcomes towards hatching rate, commonly when they are subjected to different in vitro culture.