142 RELATIONSHIP BETWEEN APOPTOSIS IN BOAR SEMEN AND DNA FRAGMENTATION IN PORCINE EMBRYOS

Abstract

Although apoptosis in somatic cells and in spermatocytes and spermatids in vivo is well established, the presence and significance of apoptosis in ejaculated animal sperm and its correlation with developmental competence of preimplantation embryos is still unresolved. The aim of this experiment was to study the relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos. Two ejaculates from the same boar were used in the experiment. Both fresh ejaculates were analyzed using Vybrant Apoptosis Assay Kit #4 (Molecular Probes, Inc., Eugene, OR, USA). Then one of them was diluted in Biosolwens plus extender, stored for 5 days at 15°C and analyzed using YO-PRO-1/PI assay, which detect changes in the membrane of boar spermatozoa, based on the slight increase of membrane permeability. Both fresh and stored semen were used for insemination, of superovulated gilts (8 per group). After 5.5 days of insemination embryos were flushed out of the uterus and DNA integrity of obtained embryos were analyzed. DNA fragmentation and caspase-3 activity were detected in embryos using kits, Roche Diagnostics (Mannheim, Germany) and PhiPhiLux G2D2 (Calbiochem, San Diego, CA, USA), respectively. For both the fresh and stored semen, 3 groups of sperm were observed under a fluorescence microscope. In the fresh semen, 3 and 2% of apoptotic sperm, 13 and 9% of necrotic sperm, 84 and 89% of live sperm in first and second ejaculated, respectively, were observed. In stored semen, 14% of apoptotic sperm, 27% of necrotic, and 59% of live sperm were noted. In total, 141 expanded blastocysts for DNA fragmentation were analyzed. The results are summarized in Table 1. In conclusion, apoptosis in fresh boar semen was lower than in stored semen and was correlated with the TUNEL nucleus index in blastocysts. The expression of caspase-3 was positively correlated with cells positive for TUNEL. Table 1. Relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos This study was supported by Polish Research Committee, grant no. 2 P06D 024 30.