105 Functionality evaluation of two extenders for Leopardus geoffroyi sperm cryopreservation by interspecific IVF with domestic cat oocytes

Abstract

Even though knowledge in sperm cryopreservation of endangered felids advanced in recent years, very little is known about suitable protocols to cryopreserve sperm from Leopardus geoffroyi (LG). In the present study, sperm obtained by electroejaculation from 5 different males were cryopreserved in either a Tes-Tris- or a lactose-based diluent (Gañan et al. 2009 Theriogenology 72, 341-352) with modifications in the freezing process using a one-step method: straws were placed horizontally on a metal rack, 4 cm above the surface of liquid nitrogen in a styrofoam box, and kept for 10 min before plunging them in LN. The objectives were to (1) compare in vitro motility and acrosome status of LG sperm cryopreserved in both extenders and (2) test functionality of LG sperm cryopreserved in both extenders through their ability to fertilize mature domestic cat oocytes. Straws were thawed by exposing them to air for 10 s and then immersing them in a water bath at 37°C for 30 s. The contents of the straws were poured into a sterile 1.5-mL microtube prewarmed to 37°C. The sperm suspension was diluted (1:3 vol/vol) by the slow (drop by drop) addition of a modified Tyrode’s solution. Sperm parameters, percentage of motile spermatozoa, and quality of motility were assessed and sperm motility index (SMI) was calculated as follows: [% motile sperm + (quality × 20)]/2. Acrosome integrity was assessed by staining with Coomassie brilliant blue. For IVF, in vitro-matured domestic cat oocytes (n = 238 Tes-Tris, n = 239 lactose) were co-incubated with 0.5 × 10⁵ motile spermatozoa/mL under 5% CO₂ in air at 38.5°C for 18-20h (Pope et al. 2006 Methods Mol. Biol. 254, 227-244). Presumptive zygotes were cultured in vitro in 50-µL drops of modified Tyrode’s medium at 38.5°C in 5% CO₂, 5% O₂, 90% N₂ atmosphere. Cleavage was assessed 48 h postfertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Data was analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), significant at P < 0.05. Results, mean (±standard error of the means), showed that SMI and acrosome integrity (pre- and post-thawing) were similar for both extenders: prethawed (SMI = 56 ± 3.3 v. 59 ± 5.5; acrosome integrity = 88 ± 3.0% v. 90 ± 2.0%), and post-thawed (SMI = 46 ± 5.0 v. 44 ± 7.0; acrosome integrity = 57 ± 7.5% v. 68 ± 2.4%) Tes-Tris v. lactose, respectively. For IVF, results showed a high cleavage rate in both groups (117/238, 49% v. 117/239, 49%), and a high development to morula (96/238, 40% v. 94/239, 39%) and to the blastocyst stage (61/238, 26% v. 51/239, 21%) for all males Tes-Tris v. lactose, respectively. There were no significant differences between groups at any development stage. In conclusion, we found that both extenders can be used to cryopreserve LG sperm maintaining functional conditions and that fertilizing capacity can be tested using in vitro-matured domestic cat oocytes.