Improving cryopreserved ram sperm quality: a biotechnological application of the recombinant protein SPINK3

INIESTA-CUERDA M; SOLER AJ; MAROTO A; ZALAZAR L; CESARI A

Barcelona

Congreso; 14º CONGRESO INTERNACIONAL DE LA ASOCIACIÓN ESPAÑOLA DE REPRODUCCIÓN ANIMAL; 2017

Asociación Española de Reproducción Animal

Ram sperm is susceptible to the cryopreservation process due to its plasma membrane characteristics, reducing half-life and fertilizing ability. Seminal plasma is known to improve quality of frozen-thawed sperm due to the membrane stabilizing ability of some secretory proteins. We developed a recombinant version of SPINK3, a mouse seminal vesicle secretory protein, which physiologically attaches to the spermatozoa and avoids a premature capacitation. We propose that this peptide would be imitate the protective effect of seminal plasma without its negative aspects. Ejaculates were obtained from 7 rams and each sample was individually cryopreserved. After thawing, samples were incubated with/without 13.65 µM SPINK3 at 37ºC and at different times during incubation (30, 90, 120 and 150 min) sperm quality was assessed by CASA and flow cytometry. SPINK3 significantly enhanced the progressive motility at 30 min whereas velocity parameters (VSL, VAP and VCL) and viability (Yopro-1 and propidium iodide) were improved after 90 and 120 min of incubation, respectively. Moreover, intracellular calcium (Fluo3AM) and lipid peroxidation (Bodipy 581/591), molecular signals of cryocapacitation, were significantly reduced with SPINK3 beyond 90 min of incubation in viable cells. Mixed effect models statistically supported data robustness.The outcomes could result in a biotechnological tool, since SPINK3 attenuates deleterious effects of cryopreservation and would prolong the survival and competence of spermatozoa in the female. Supported by ANPCyT, UCLM and BeCAR