Intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes with stored epididymal spermatozoa in camel (Camelus dromedarius): Effect of exogenous activation on in vitro embryo development

Abstract

Experiments were conducted to investigate the development of in vitro matured camel oocytes after their intra-cytoplasmic sperm injection (ICSI) with epididymal sperm collected from slaughtered male camels. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 °C and on ice (0–1 °C), respectively. Cumulus-oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20–25 COCs/well) containing 500 μL of maturation medium and cultured at 38.5 °C in an atmosphere of 5% CO2 in air for about 30 h. Spermatozoa were collected from the cauda epididymites in syringes containing 2–3 mL of tris-tes egg yolk extender. They were cooled down slowly and stored at refrigeration (4 °C) temperature. On the day of use, spermatozoa were prepared by the swim up technique before use in ICSI. The injected oocytes were either activated by ionomycin and roscovitine or put into the culture without any chemical activation. In Experiment 1, presumptive zygotes were fixed and stained with Hoechst 33342 for evaluation of fertilization after 18 h of culture, while, in Experiment 2, they were cultured in 500 μL of the culture medium at 38.5 °C in an atmosphere of 5% CO2, 5% O2 and 90% N2 in air for 7 days to evaluate their development. The proportion of oocytes activated when ICSI was followed by chemical activation was significantly higher (P < 0.05) when compared with non-activated ones. In experiment 2, a higher number of oocytes cleaved (59 vs. 35%) and developed to blastocysts (20 vs. 7%) in the group with post-ICSI activation when compared with the group without chemical activation, respectively.In conclusion, to the best of our knowledge, this is the first report where embryos were produced by ICSI in camels. Chemical activation of oocytes by ionomycin and roscovitine, post -ICSI, enhanced their cleavage and development to blastocyst stage.