Post-thawing quality and incubation resilience of cryopreserved ram spermatozoa are affected by antioxidant supplementation and choice of extender

The performance of cryopreserved semen in ovine artificial insemination still needs improvement. Some antioxidants have been tested, with variable success. We cryopreserved semen from Churra rams using TES-Tris-fructose with 4% glycerol and 10% egg yolk (EY) or 3.5% soybean lecithin (SL), with 1 mM of reduced glutathione (GSH), Trolox, crocin, or cysteamine. Samples were analyzed after thawing and incubation (6 hours, 38 °C) for motility (computer-assisted sperm analysis [CASA]), viability, acrosomal integrity, apoptosis, mitochondrial activity, chromatin status, and lipoperoxidation (malondialdehyde production). Interactions (antioxidant/extender/incubation) were significant for most variables. Extenders yielded similar results, although SL depressed mitochondrial activity and linearity (P < 0.001), it improved motility (P < 0.05), DNA fragmentation (P < 0.05), and acrosomal damage (P < 0.001). The control, GSH, and Trolox showed greater viability with SL (P < 0.01). Cysteamine depressed motility (0 hours: 51.6 ± 2.0% vs. 32.3 ± 4.3%; 6 hours: no motility vs. 32.5 ± 1.9%; P < 0.001), but improved viability when using EY (P = 0.004). Crocin increased acrosomal damage (P = 0.022) but improved linearity-related parameters after thawing (P = 0.014). Trolox considerably reduced malondialdehyde production in both extenders (8.6 ± 0.4 nmol per 108 cells vs. 14.2 ± 0.3 in EY and 20 ± 0.6 in SL; P < 0.001). Interestingly, thiol antioxidants (cysteamine and GSH) increased DNA fragmentation (percentage of DNA fragmentation index), whereas crocin reduced it (P < 0.05). Interactions between extender and antioxidant must be taken into account for improving sperm cryopreservation. Soybean lecithin seems to be a suitable replacement for EY, but its effect on mitochondria must be investigated. Trolox and crocin might be useful for ram semen freezing.