Abstract:
This study determined the effects of in vitro culture media (Ham’s F10 and TCM199) and
assisted hatching techniques (laser or mechanical) on mice embryo survival following
cryopreservation. Pure strain C57BL/6 (B6) female (50) and strain BALB /c (C) Male (25) mice
were crossed to produce F1 generation of females which were injected for follicular growth and
super ovulation at 6 weeks of age and from which embryos were produced 21 h later through
in vivo fertilization. Embryos were randomly divided into Petri dishes with different culture
media, and the development of embryos was assessed until the morula stage. At the morula
stage, selected embryos were assisted to hatch using different techniques, and then
cryopreserved in liquid nitrogen using the slow freezing method for a period of 1 week. After
1 week of cryopreservation, the embryos were thawed and cultured in the two different in vitro
culture media for 72 hours. Thereafter, the numbers of embryos hatched or survived were
recorded after 24 h, 48 h and 72 h. Data was analyzed using ANOVA in Minitab Software
Version 16 (2010). Significant difference in embryo quality development was observed
between in vitro culture media and stage of embryo development (P<0.05). In the TCM-199 in
vitro culture medium, embryo quality development yielded 72, 69 and 69% from day 1 to day
3, while in Ham’s F10 embryo quality development yielded 68, 63 and 60% respectively.
Relative to the control (18.1%) assisted hatching improved hatchability significantly (P<0.05)
in the order laser (23.6%)>, mechanical (20.8%). There was significant (P<0.01) interaction
between assisted hatching techniques and evaluation time, whereby laser assisted hatching
was most successful at 48 h (42.0%) while mechanical assisted hatching was most successful
at 72 h (36.8%). Cryopreservation reduced the embryo survival compared to fresh embryos.
In conclusion laser was the best assisted hatching technique, while TCM-199 was the better
medium for in vitro culture of embryo.
