O-12: Studies on Sequestration of PDC-109 Protein on Cryodamage and In Vitro Fertility of Crossbred Bull Spermatozoa

Background: Plasma membrane of the spermatozoa interacts with and is altered by abundant seminal PDC-109 present in their immediate milieu in vivo with damaging effect in a time- and concentration- dependent manner. Therefore we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial for cryopreservation of sperm cells. To this aim we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY+Ab in minimizing cryoinjury to bull spermatozoa. Materials and Methods: Seminal PDC-109 protein was purified and injected in rabbits to raise antibodies. Immuno-diffusion assay followed by western blot to ascertain cross-reactivity of other HBPs was carried out. Ab were quantitated and added to ejaculates, either alone or in addition to EY in Tris-Glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY+TG1 (Group I), EY+TG2 (direct collection of ejaculates in EY, Group II), EY+Ab+TG (Group III) or Ab+TG (Group IV). Semen quality parameters (SQP) viz. viability, individual motility, biochemical integrity of membrane, cholesterol content of spermatozoa, acrosome integrity (Giemsa and fluorescent stain FITC-PSA), cryoinjury to spermatozoa (Chlortetracycline, CTC assay) and in vitro fertility parameters were evaluated. Results: A significant (p<0.05) improvement in post-thaw SQP observed at pre-freeze and post-thaw stages of cryopreservation in Group III compared with other groups indicated improvement in freezability. Study revealed highly significant (p<0.01) increase in non-capacitated spermatozoa at pre-freeze as well as post-thaw stages in AB+EY treated group signifying reduced cryounjury. Conclusion: We report for the first time novel method of sequestration of PDC-109 protein by Ab+EY synergistically to improve freezability and minimize cryoinjury to bull spermatozoa. Further this protocol will open new opportunities for understanding and diagnosis of infertility and /or subfertility not only in bulls but in other species thereby eliminating low fertile sires, before semen is processed for cryopreservation.