Sperm chromatin integrity of bucks transgenic for the WAP bGH gene.
The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13-18 months old. Semen was collected at 1-week intervals and 3-7 ejaculates from each rabbit were examined in total. Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes. The mean value of COMPalpha(t) was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.alpha(t) for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (-0.68, P<0.05) was found between S.D.alpha(t) of NTG males and percentages of pregnant does. The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene. The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks.
Gogol
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P
Department of Animal Reproduction, National Research Institute of Animal Production, 32-083 Balice/Kraków, Poland. pgogol@izoo.krakow.pl
Bochenek
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M
Smorag
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Z
D000165:Acridine Orange / Q000737:chemistry; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000502:physiology*; D002843:Chromatin / Q000235:genetics* / Q000502:physiology; D005260:Female; D005298:Fertility / Q000502:physiology; D005434:Flow Cytometry / Q000662:veterinary; D005456:Fluorescent Dyes / Q000737:chemistry; D013006:Growth Hormone / Q000235:genetics; D007315:Insemination, Artificial / Q000662:veterinary; D008098:Litter Size; D008297:Male; D011247:Pregnancy; D011817:Rabbits / Q000235:genetics / Q000502:physiology*; D013094:Spermatozoa / Q000002:abnormalities*
D002843:Chromatin; D005456:Fluorescent Dyes; D013006:Growth Hormone; D000165:Acridine Orange