Comparative Study of Different Assisted Hatching Techniques and In Vitro Culture Media on Mice Embryo Hatching Rate
Authors:   Department of Animal science, University of Venda, South Africa, NC Negota , ML Mphaphathi ,   Germplasm Conservation and Reproductive Biotechnology, Agricultural Research Council, South Africa, LP Nethenzheni ,   Department of Animal science, University of Venda, South Africa, TL Rammutla ,   Department of Animal science, University of Venda, South Africa, NR Serota ,   Department of Animal science, University of Venda, South Africa, DM Barry ,   Department of Animal science, University of Venda, South Africa, TL Nedambale ,   Department of Animal Science, Tshwane University of Technology, South Africa
Published: April 2021

The study investigated the influence of Assisted Hatching (AH) techniques (mechanical, chemical, enzymatic thinning and laser) and two in vitro culture media (Ham’s F10 and TCM-199) on hatching rate of mice embryos (blastocyststage) following 24 hours of culture. The C57BL/6-black (n=30) and BALB/cwhite (n=30) mouse breeds were raised until maturity and naturally bred to produce a F1 generation. The light in the breeding house was controlled and the mice were fed ad libitum. Female mice (n=30) were injected (peritoneal) with 0.1 ml (5 IU) of eCG into the abdominal cavity with 1 ml syringe and 0.5x16 mm needle to stimulate follicular growth and 46-49 h later was injected with 0.1 ml (5 IU) of hCG to cause ovulation, maintain the corpus luteum and stimulate it to secrete progesterone for maintenance of pregnancy. After the injection, the male and female (1:1) could mate overnight. Female mice with vaginal plugs were observed and kept separately for blastocyst-stage embryos collection on day three following successful mating. They were euthanized, and abdomen sterilized with 70% ethanol. Using a sterile surgical scissor, a fine cut was made, holding the skin firmly above and below the incision, the skin was pulled apart using forceps. The embryos were flushed from the uterus using a 30-gauge needle with culture media. Following the AH techniques, embryos were cultured in TCM-199 or Ham’s F10 for 24 hours and zonal thickness of all hatched embryos were measured. Immediately after assisted hatching, the embryos were cultured into two different in vitro culture media. All embryos hatched were stained and the zonal thickness of embryos were measured. The number of blastomeres were counted and recorded 24 h later. Data collected were subjected to analysis of variance using PROC General Linear Model. The Tukey’s test was used to separate the means. A significant difference was observed between the thickness of Zona Pellucida (ZP) pre and post treatment after 24 hours of culture. However, there was no significance difference among blastocyst hatching rate and the blastomeres nuclei counted after staining. The thickness of the ZP decreased with individual AH techniques. The interaction between AH techniques and in vitro culture was found to be significantly different on blastocyst hatchability. However, laser AH technique had highest hatchability (56.3%) when embryos were cultured in TCM-199 followed by mechanical AH techniques (52.6%). The hatchability rate (33.3%) was recorded in the chemical AH technique group. The blastomeres nuclei counted under interaction of AH techniques and culture media was not differently affected, with the values ranging from 69 to 76%. In conclusion, the use of different AH techniques resulted in varying effect and increase outcomes towards the hatching rate.