243 APOPTOTIC-LIKE CHANGES AND FERTILITY OF TRANSGENIC FOR HUMAN α1,2-FUCOSYLTRANSFERASE GENE AND NONTRANSGENIC BOAR SPERMATOZOA

Abstract

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.