Abstract
This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term.
Highlights
Very little information is available on intracytoplasmic sperm injection (ICSI) in rats, possibly because the large size of rat spermatozoa makes their microinjection into oocytes extremely difficult
The present study has demonstrated for the first time that rat oocytes can be activated following intracytoplasmic injection of sperm heads isolated from heat-dried spermatozoa, and male pronuclei can be formed in the activated oocytes
Injected oocytes were generally difficult to develop to the blastocyst stage when injected oocytes were not induced artificial activation, about 23–29% of oocytes developed to the blastocyst stage when they were induced artificial activation after being injected with heat-dried sperm heads that were stored at 4uC for 1 week in nitrogen gas or in air
Summary
Very little information is available on intracytoplasmic sperm injection (ICSI) in rats, possibly because the large size of rat spermatozoa makes their microinjection into oocytes extremely difficult. This report indicated that removal of the tail from the spermatozoa before injection did not affect development to the blastocyst stage in vitro Using such a technique, it has been reported that freezedried spermatozoa are able to produce normal blastocyst and live young when injected into oocytes in mouse [9,10,11,12], rabbit [13], bovine [14] and porcine [15]. These reports suggest that spermatozoa have lost their motility, acrosome integrity or plasma membrane integrity but have genetic integrity unmarred by drying. Heat-dried sperm are similar to evaporative dried sperm on the technical approach
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