Effect of fish antifreeze protein type iii supplementation to semen extender on cryopreserved dog spermatozoa

Abstract

Ice binding protein such as antifreeze protein (AFP) type III have been used as a cryoprotectant and proven to improve sperm survival in some species but dog. Addition of Equex STM paste to semen freezing medium provides beneficial effects on cryopreserved dog sperm. Hence, the objectives of this study were to (a) evaluate the effects of AFP (Experiment I) and (b) its combination with Equex STM paste on cryopreserved dog spermatozoa (Experiment II). The semen samples were pooled to allow for a sufficient number of sperm and to reduce an individual variation. Only sperm-rich fractions with total motility more than 70% were used. Pooled semen from three dogs was split in equal portions according to the number of tested extenders in Experiment I or II. Two steps-dilution freezing methods with 4 different tris egg-yolk based extender were used in experiment I, e.g. P0; no AFP added (control), P1; 0.01 µg/mL, P2; 0.1 µg/mL, P3; 1 µg/mL (w/v) and experiment II, e.g. E1; 0.1µl/mL AFP), E2; 0.1µl/ml AFP plus 1% (v/v) Equex STM paste and E3; 1% (v/v) Equex STM paste. Sperm evaluation was done one week after the freezing. Thawing was performed by immerse freeze-straw into 37oC water for 60 second. Sperm motility and velocity were evaluated by Sperm Class Analyzer® (SCA Microptic SL, Barcelona, Spain). Post-thawed samples were stained for sperm viability (SBYR-PI fluorescent staining), acrosome integrity (FITC-PNA and PI) and mitochondrial membrane potential (JC-1), and were evaluated. Plasma membrane functional integrity (PMFI) was evaluated by hypo-osmotic swelling test (HOST). In Experiment I, overall, regardless of time post thawing, addition of AFP type III at a concentration of 1µg/ml (P3) significantly deteriorated total motility, progressive motility and sperm velocity compared to P0 (control), P1 and P2. Semen extenders supplemented with 0.01 or 0.1µg/ml AFP yielded no significantly better quality of frozen-thawed dog sperm than those of the control. Immediately post-thawing, there are no significant differences among 3 treatments and control groups (P>0.05) except for the progressive motility. The progressive motility was significantly lower in P3 (P>0.05). In general, all parameters decreased after 1h incubation at 37°C (P>0.05). However, post-thawed sperm velocity (VCL, VSL and VAP) did not significantly decline at 1h in P2. In Experiment II, overall, Equex-contained extenders (E2, E3) significantly improved sperm quality post-thawing compared to E1 (P<0.0001). A combination of AFP and Equex STM paste provided a beneficial effect on sperm viability immediately after thawing (P<0.05). In conclusion, supplementation of AFP type III at a concentration of 0.01µg/ml provided a beneficial effect on dog sperm velocity. Higher concentration of AFP (1µg/ml) deteriorated post-thawed sperm quality. Addition of 0.01 µg/ml into Equex-contained semen extenders was essential for maintaining sperm viability post-thawing.