36 Effect of antifreeze protein type I supplementation in the extender for semen cryopreservation in the domestic cat

The supplementation of antifreeze protein type I (AFP I) to cryopreservation media had shown greater freeze–thawed sperm quality in ram and cattle. This study aimed to assess the addition of AFP I in the extender for semen cryopreservation in domestic cats. Sperm were collected from cauda epididymides (n = 86 animals), incubated in Tris buffer (3.025 g TRIS, 1.4% citric acid, 0.8% glucose, 0.1% antibiotic, and antimycotic solution), and pooled in nine replicates. Only sperm samples exhibiting values ≥50% (motility) and 3 (vigour) were used. The pooled fresh semen (69.4 ± 2.3% of motility) was allocated into three experimental groups according to the final concentration of AFP I: 0 (CONT), 0.1, and 0.5 µg mL⁻¹. Semen was centrifuged, diluted in Extender I (same composition as Tris buffer, but with 3% glycerol, 20% egg yolk, and AFP I), and kept at 4°C for 1 h. Subsequently, Extender II (same as Extender I, but containing 7% glycerol and 0.6% sodium dodecyl sulfate (SDS)) was added and the concentration was adjusted to 25 × 10⁶ spermatozoa mL⁻¹. The 0.25-µL straws were filled and equilibrated at 4°C for 1 h. Then, the straws were maintained 7 cm above the liquid nitrogen level for 10 min and immersed. After thawing (37°C for 30 s), samples were evaluated. Normal data were analysed by one-way ANOVA followed by Tukey test and non-normal data by Kruskal–Wallis and Dunn’s test (mean ± s.e.m., in %). There was no difference (P > 0.05) in CONT, 0.1, and 0.5 µg mL⁻¹ groups, respectively, for total motility (14 ± 2.1, 14 ± 2.3, 12 ± 2.6%), supravital test (16 ± 3.8, 14 ± 2.9, 13 ± 2.6%), mitochondrial activity (100%, DAB I (6 ± 1.1, 6 ± 1.0, 8 ± 1.5%); ≥50%, DAB II (55 ± 4.0, 63 ± 2.9, 66 ± 1.4%); <50%, DAB III (30 ± 2.8, 22 ± 2.7, 20 ± 2.1%); 0%, DAB IV (13 ± 2.3, 9 ± 1.1, 7 ± 0.8%)), functional membrane integrity (9 ± 1.6, 8 ± 1.1, 9 ± 1.7%), normal chromatin condensation (67 ± 2.0, 67 ± 0.8, 65 ± 3.1%), and viability and acrosome status (live sperm with intact acrosome (LSIA): 48 ± 6.4, 44 ± 11.3, 20 ± 5.6%; live sperm with acrosome reacted: 21 ± 4.4, 17 ± 4.5, 27 ± 9.2%); dead sperm with intact acrosome: 32 ± 8.8, 46 ± 12.6, 50 ± 10.6%; and dead sperm with acrosome lost: 7 ± 2.3, 6 ± 2.3, 9 ± 2.4%), normal morphology (53 ± 2.1, 57 ± 1.9, 56 ± 2.8%), and sperm binding to egg perivitelline membrane (461 ± 33.5, 416 ± 51.9, 421 ± 59.0 sperm mm⁻²). Percentages of LSIA (P = 0.037) and DAB III class (P = 0.041) were reduced in the 0.5 µg mL⁻¹ group compared with the CONT and 0.1 µg mL⁻¹ groups. In conclusion, the supplementation of 0.1 and 0.5 µg mL⁻¹ AFP I did not promote consistent beneficial effects on overall semen cryotolerance in domestic cats.

This research was supported by FAPERJ, CNPq, and CAPES (Finance Code 001).