243 APOPTOTIC-LIKE CHANGES AND FERTILITY OF TRANSGENIC FOR HUMAN α1,2-FUCOSYLTRANSFERASE GENE AND NONTRANSGENIC BOAR SPERMATOZOA
Authors: Trzcinska M. , Bryla M. , Slomski R. , Smorag Z. 
Published: August 2011

In the current study, we used sperm from transgenic boars with a gene construct containing the human α1,2-fucosyltransferase gene that competes with α1,3-galactosyltransferase for the same substrate, N-acetyl lactose amine. A decreased affinity of the anti-Gal antibodies between the human and the pig can reduce the species-specific immunological difference and reduce the risk of xenographic rejection. The objectives of this research were first to find out if the presence of the human α1,2-fucosyltransferase gene (pCMVFUT gene construct) in boars leads to apoptotic-like changes in the ejaculated spermatozoa, and second, to verify the fertilizing capacity of the sperm from transgenic (TG) boars. Six ejaculates were collected from 5 TG and 5 nontransgenic (NTG) boars, both crossbreds of Polish Landrace and Large White aged 21.3 ± 0.8 months. Only ejaculates with at least 70% motile and 80% morphologically normal sperm were used. Fresh semen were diluted to a final concentration of 2.5 × 109 sperm mL–1 in Biosolwens Plus (Biochefa, Sosnowiec, Poland) extender and used for artificial insemination of gilts (5 gilts per 1 ejaculate). All ejaculates were analysed on the day of insemination using 2 fluorescence methods: an assay to assess the early changes in sperm membrane integrity using the combination of fluorophores YO-PRO-1 and propidium iodide (PI; Molecular Probes Inc., Eugene, OR, USA) and an assay for phosphatidylserine (PS) translocation across the plasma membranes using Annexin-V–fluorescein isothiocyanate (FITC) and PI (Roche, Mannheim, Germany). Fluorescent staining was measured by microscopic observation of at least 200 spermatozoa/sample per slide. Results are expressed as means ± SD. Significance of the differences between means was tested by Student’s t-test. Using the YO-PRO-1/PI assay, we observed 3 groups of sperm: apoptotic sperm (3.1 ± 1.5% v. 3.5 ± 1.6%), necrotic sperm (22.2 ± 5.3% v. 19.7 ± 5.1%), and live sperm (74.7 ± 7.8% v. 76.8 ± 5.6%) in TG and NTG boar semen, respectively. Using the Annexin-V–FITC/PI assay, 4 subpopulations were detectable: early apoptotic sperm (2.3 ± 1.3% v. 2.6 ± 1.5%), late apoptotic/early necrotic (16.9 ± 8.4% v. 15.7 ± 5.6%), necrotic sperm (7.7 ± 3.9% v. 6.2 ± 2.9%), and live sperm (73.1 ± 6.1% v. 75.5 ± 5.8%) in TG and NTG boar semen, respectively. The use of both assays yielded no significant differences (P ≥ 0.05) in all detected subpopulations of sperm of TG boars compared with that of NTG boars. Moreover, no differences in conception rate (87.7 and 83.8%) and in the average number of live-born piglets per litter (10.7 and 11.2) were observed after insemination using the semen from TG and NTG boars, respectively. In conclusion, the presence of human α1,2-fucosyltransferase gene in the genome of TG boars did not increase apoptotic-like changes in sperm and did not affect pregnancy rate or litter size.

This work was supported by Grant NR 12 0036 06 from the National Centre of Research and Development, Poland.