Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm.

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.

Lee JW ，Yang X 《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes.

Nakai M ，Kashiwazaki N ，Takizawa A ，Maedomari N ，Ozawa M ，Noguchi J ，Kaneko H ，Shino M ，Kikuchi K ... -  《REPRODUCTION》

Failure of male pronucleus formation is the major cause of lack of fertilization and embryo development in pig oocytes subjected to intracytoplasmic sperm injection.

Lee JW ，Tian XC ，Yang X 《BIOLOGY OF REPRODUCTION》

Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm.

The present study investigated the correlation of sperm movement in the ooplasm, pretreatment of sperm with dithiothreitol (DTT) and sperm freezing with the development of porcine embryos derived from modified intracytoplasmic sperm injection (ICSI). In vitro, matured gilt oocytes without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail-first. In Exp. 1, frozen-thawed sperm were categorized into three groups: impaired, immotile or motile. Oocytes injected with motile sperm (43.6%) showed a higher (P < 0.05) fertilization rate compared to oocytes injected with impaired or immotile sperm (34.5 or 37.2%). The survival rate was significantly higher (P < 0.05) in oocytes injected with impaired sperm (92.9%) than in oocytes injected with immotile or motile sperm (84.8 or 86.7%). No differences were observed in the rates of cleavage or blastocyst formation, and in total cell number of blastocysts among three groups of oocytes. In Exp. 2, motile frozen-thawed sperm were pretreated with DTT before injection and non-treated sperm served as controls. Higher rates (P < 0.05) of fertilization, male pronucleus (MPN) and decondensed sperm head (DSH) formation were observed in oocytes injected with control sperm (41.1, 50.0 and 91.1%, respectively) than in oocytes injected with DTT-treated sperm (22.1, 30.2 and 72.1%, respectively). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In Exp. 3, motile frozen-thawed or fresh sperm without DTT pretreatment were injected into oocytes. The rates of fertilization and MPN formation were significantly higher (P < 0.05) in oocytes injected with fresh sperm (59.8 and 73.5%) than in oocytes injected with frozen-thawed sperm (36.7 and 59.2%). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In conclusion, the present study clearly demonstrated that sperm movement in the ooplasm, use of DTT and fresh spermatozoa did not significantly affect on embryo development in porcine modified ICSI.

Yong HY ，Hong JY ，Kang SK ，Lee BC ，Lee ES ，Hwang WS ... -  《THERIOGENOLOGY》

[Effects of sperm and oocyte quality control on intracytoplasmic sperm injection (ICSI) in goats].

Zhou JB ，Wu YG ，Han D ，Liu LQ ，Tan XW ，Liu N ，Luo MJ ，Chang ZL ，Tan JH ... -  《-》