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

ZALAZAR, LUCÍA; INIESTA CUERDA, MARIA; MAROTO, A; CESARI, ANDREINA; SOLER VALLS, ANA JOSEFA

Barcelona

Congreso; 14º Congreso Internacional De La Asociación Española De Reproducción Animal; 2017

European Society for Domestic Animal Reproduction (ESDAR)

Ram sperm is susceptible to the cryopreservation process due to itsplasma membrane characteristics, reducing half-lifeand fertilizingability. Seminal plasma is known to improve quality of frozen-thawedsperm due to the membrane stabilizing ability of some secretory proteins.We developed a recombinant version of SPINK3, a mouse seminalvesicle secretory protein, which physiologically attaches to thespermatozoa and avoids a premature capacitation. We propose thatthis peptide would be imitate the protective effect of seminal plasmawithout its negative aspects.Ejaculates were obtained from 7 rams and each sample was individuallycryopreserved. After thawing, samples were incubated with/without 13.65 μM SPINK3 at 37°C and at different times during incubation(30, 90, 120 and 150 min) sperm quality was assessed by CASAand flow cytometry. SPINK3 significantly enhanced the progressivemotility at 30 min whereas velocity parameters (VSL, VAP and VCL)and viability (Yopro-1and propidium iodide) were improved after 90and 120 min of incubation, respectively. Moreover, intracellular calcium(Fluo3AM) and lipid peroxidation (Bodipy 581/591), molecularsignals of cryocapacitation, were significantly reduced with SPINK3beyond 90 min of incubation in viable cells. Mixed effect models statisticallysupported data robustness. The outcomes could result in abiotechnological tool, since SPINK3 attenuates deleterious effects ofcryopreservation and would prolong the survival and competence ofspermatozoa in the female. Supported by ANPCyT, UCLM and BeCAR