Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media

Abstract

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.

A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects

“…Following microinjection, the percentage of oocytes with 1 pronucleus and no clear signs of spermatozoa decondensation was between 9% and 11% for the 4 bulls and was similar to the rate reported earlier (Keefer et al, 1990;Yang et al, 1990). It is uncertain whether these oocytes with 1 pronucleus and no clear signs of spermatozoa1 decondensation are due to parthenogenetic activation, due to failure to place a spermatozoon in the cytoplasm, or due to expulsion of the spermatozoon from the oocyte.…”

“…For microinjection, spermatozoa were further diluted with TALP, suspended in 0.9% NaCl containing 10% polyvinyl pyrrolidone (PVP), and transferred into a droplet of PVP solution near a drop-let containing the oocytes. The procedure for microsurgical injection of spermatozoa has been described previously (Yang and Foote, 1987;Yang et al, 1990;Heuwieser et al, 1992).…”

Hypertonic Medium Treatment for Localization of Nuclear Material in Bovine Metaphase II Oocytes1

“…4, C and D). This is not surprising since sucrose treatment has been widely used in cryopreservation of gametes and in many types of manipulations in which enlargement of the subzonal space is desirable, such as sperm injection and blastomere insertion into oocytes [52][53][54].…”

Freeze-Dried Sperm Fertilization Leads to Full-Term Development in Rabbits1

“…The ovulation of the recipient does had been synchronized with that of the oocyte donors by i.m. injection of 1.2 g of GnRH analogue (Cystorelin; Fort Dodge Laboratory, Fort Dodge, IA) 16 h before embryo transfer [22].…”