Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos.
The aim of this study was to compare the fertilising capacity of sperm from 6 transgenic (TG) and 6 non-transgenic (NTG) boars based on analyses of embryos resulting from insemination with sperm from these particular boars. Expanded blastocysts were collected from five groups of synchronised gilts (six gilts per group) inseminated by TG boars bearing a gene construct containing the human alpha1,2-fucosyltransferase gene and by NTG boars. The ejaculates used for insemination were analysed to detect apoptotic changes using two fluorescence methods: an assay to assess early changes in the membrane integrity of the sperm using the YO-PRO-1 fluorophore and an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labelled Annexin-V. Our results, using a combination of YO-PRO-1 and PI fluorophores, revealed no significant differences in the percentage of sperm subpopulations between non-transgenic and transgenic boars (P<0.01). Moreover, the second fluorescent probe also revealed no significant differences between the average values of live (Ann-V(-)/PI(-)), early apoptotic (Ann-V(+)/PI(-)), and late apoptotic/early necrotic sperm (Ann-V(+)/PI(+)) as calculated for TG and NTG boars. Only the percentage of necrotic sperm (Ann-V(-)/PI(+)) was significantly different (P<0.05) between transgenic and non-transgenic boars (3.4%+/-2.7; 7.2%+/-2.1, respectively). The quality of the preimplantation embryos at the blastocyst stage was determined by counting the number of cells, observing a TUNEL-positive reaction and by caspase-3 labelling. We found that expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen showed almost no DNA fragmentation (80%) and 70% caspase-3 activity. The expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen did not differ significantly in their DNA fragmentation, and there were no differences in caspase-3 activity. These results revealed a positive correlation between the percentage of blastocysts with TUNEL-positive nuclei and the percentage of blastocysts with caspase-3 activity (r=0.9787; P<0.0001).
Bryła
M
M
Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, 32-083 Balice, Kraków, Poland. mbryla@izoo.krakow.pl
Trzcińska
M
M
Wieczorek
J
J
Słomski
R
R
Smorag
Z
Z
D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000502:physiology*; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D020287:In Situ Nick-End Labeling; D007315:Insemination, Artificial / Q000662:veterinary; D008297:Male; D010718:Phosphatidylserines / Q000378:metabolism; D011247:Pregnancy; D012661:Semen / Q000166:cytology / Q000502:physiology*; D055101:Semen Analysis / Q000662:veterinary; D013194:Staining and Labeling / Q000379:methods; D013552:Swine / Q000196:embryology / Q000502:physiology*
D010718:Phosphatidylserines