Lacking expression of paternally-expressed gene confirms the failure of syngamy after intracytoplasmic sperm injection in swamp buffalo (Bubalus bubalis).

The objectives were to: (1) examine the efficiency of intracytoplasmic sperm injection (ICSI) technique, with or without chemical activation of in vitro matured buffalo oocytes, on sperm head decondensation; and (2) compare the subsequent development of embryos after activation of ICSI (ICSI (+) activation group) and sham injection (Sham (+) activation group) oocytes (embryos obtained by in vitro fertilization of IVM oocytes served as a control group). Pronuclear formation rates in ICSI (+) activation and Sham (+) activation groups were higher than that of ICSI without activation (P < 0.05). However, because 90.9% of presumptive zygotes in ICSI (+) activation group demonstrated pronuclear formation with an intact sperm head, we inferred that most were parthenotes. Neither developmental competence (morula and blastocyst formation rates) nor mean total cell number of blastocysts was significantly different among ICSI (+) activation, Sham (+) activation, and IVF groups. To clarify whether blastocysts were derived from syngamy or parthenogenesis, expression of Nnat, a paternally expressed gene in blastocysts derived from IVF, ICSI and oocyte activation without sperm or sham injection was additionally examined using reverse transcription polymerase chain reaction (RT-PCR). Expression of Nnat mRNA was not detected in ICSI (+) activation blastocysts, indicating failure of male genome activation. Although blastocyst development after ICSI combined with chemical activation was similar to IVF oocytes, these blastocysts were generated by parthenogenesis, due to failure of male pronucleus formation.

Culture medium and embryo density influence on developmental competence and gene expression of cat embryos.

Morphology and gene expression are the currently preferred methods for assessing the effect of culture medium and density on embryos of several species. To define their effects on domestic cat embryos, groups of 8-10 embryos were cultured in SOF, modified Tyrode's solution or MK-1 medium in a fixed volume (50 µL) and in different volumes (20, 50 and 100 µL). SOF supplemented with different concentrations of glucose (1.5, 3.0 and 6.0 mM) was used to examine the effect of glucose level on embryo development. Real-time reverse transcriptase polymerase chain reaction was used to determine the relative transcripts of BAX, BCL-2 and GLUT-1 genes in blastocysts derived from various concentrations of glucose. SOF and MK-1 supported feline embryo development better than modified Tyrode's solution. Embryos cultured in 20 µL droplets showed decreased development in all three media (P < 0.05). Increasing the glucose concentration in SOF to 6.0 mM adversely affected embryo development and tended to increase the BCL-2 transcript in blastocysts. In conclusion, type of culture medium, embryo density and glucose concentration affected the development of domestic cat embryos. High culture density and glucose concentration negatively affected embryo development. The increase of anti-apoptotic BCL-2 expression found in blastocysts cultured in 6.0 mM glucose may have prevented an increase in the incidence of apoptosis. In the present study, it was clearly demonstrated that differential gene expression occurred in embryos with similar morphology.

The effect of GnRH supplement of FSH and PMSG treatments for prepubertal swamp buffalo calves (Bubalus bubalis).

The objective of the experiment was to improve the multifollicle stimulation technique and the ovarian response examination in prepubertal swamp buffalo calves. Six animals were stimulated by gonadotropin hormone 7 days after a progesterone ear-implant. The first stimulation was done by giving 24 mg FSH + 100 microg GnRH (FSH+GnRH) and the second, one month after by giving 2,000 IU PMSG + 100 microg GnRH (PMSG+GnRH). Twenty-four hours after GnRH, the ovarian responses were checked using rectal palpation and real-time B mode ultrasonography. Five out of six animals (83.3%) responded to both treatments and were selected for oocyte collection. The oocytes were aspirated directly following a caudal midline laparotomy. The results of ovarian responses to FSH+GnRH and PMSG+GnRH averaged 17.6+/-12.1 (L-9.8+/-8.7, R=7.8+/-6.2) and 17.4+/-5.6 (L-9.4+/-2.9, R=8.0+/-3.7), respectively. The average number of recovered oocytes per animal was 9.0+/-6.4 and 8.4+/-1.1, respectively which represented a recovery rate of 56.3 (+/- 9.2)% and 51.9 (+/- 10.3)%. More than eighty percent of the recovered oocytes were in an immature stage with more than 2-3 layers of compact cumulus mass. The present study showed that the oocytes were collected successfully in prepubertal buffalo calves after the FSH+GnRH or PMSG+GnRH stimulation and most of the recovered oocytes were immature, which made them suitable for in vitro maturation and fertilization.