ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

208Artificial Insemination in Farm Animalsidentification of samples and considerably reduces the risk of cross-contamination duringcryopreservation.As different methods of storage have been used, the question of whether the means ofstorage has any effect on the success rate of cryopreservation has been raised. In this regard,different authors have compared spermatozoa stored in different packages (Heitland et al,1996; Kneissl 1993; Park et al, 1995). Their results showed an effect on spermatozoa qualitymanifested through reduced motility and conception rate. The reports further stressed theroles of different extenders used, the interaction between extender, and means of packaging.However, the reasons for these discrepancies was not fully explained, and it was also notclear in all work how the dimensions of the straws change with volume, in addition towhich different extenders and concentration of spermatozoa were used. On the other hand,a more recent work in stallion demonstrated that stallion spermatozoa can be frozen at aconcentration as low as 40×106 mL −1 in 0.25mL straws without a negative effect on spermmotility, morphology or acrosome integrity (Clulow et al, 2008).9. Recent advances in cryopreservationThe application of frozen-thawed semen technology is currently increasing worldwide.Several studies have focused on identifying damages during freezing and thawing, tests toscreen sperm quality post-thaw, evaluation of alternative cryoprotectants and otheradditives, and freezing procedures to improve sperm viability and fertility (Clulow et al,2008; Goolsby et al, 2004; Medeiros et al, 2002; Squires et al, 2004;). Most of the progress inimproving survival of frozen-thawed spermatozoa centers on minimizing the oxidativedamage and decreasing the osmotic stress on spermatozoa. Equine sperm are particularlyknown to be susceptible to oxidative stress, relative to other species, because of their highcontent of unsaturated fatty acids. In addition to membrane effects, lipid peroxidation canalso damage DNA. The addition of antioxidants to extenders has been used as a method todecrease lipid peroxidation and oxidative stress associated with cryopreservation (Bilodaeuet al, 2001; Pe˜na et al, 2003; Roca et al, 2004). Different amides, compounds with lowermolecular weight than glycerol and penetrate the sperm plasma membrane more readily,have been evaluated as alternative cryoprotectants to glycerol in different animals (Bianchiet al, 2008; Medeiros et al, 2002; Squires et al, 2004;). These compounds include methylformamide (MF), dimethyl formamide (DMF) or ethylene glycol (EG) and dimethylacetamide. They were known to provide greater post-thaw motility when used at differentconcentrations. Particularly, MF and DMF or EG have been used as alternativecryoprotectants for individual males whose sperm has lesser post-thaw motility whenfrozen in glycerol (Bianchi et al, 2008; Squires et al, 2004). The use of low-density lipoproteins(LDLs), most often isolated from egg-yolk from different species, as additive has provenbeneficial for sperm function post-thaw, particularly for DNA-integrity(Rodriguez-Martinezand Wallgren, 2011). Attempts to minimize osmotic stress during cryopreservation haveincluded step-wise dilution of cryoprotectants, by incorporating cholesterol-loadedcyclodextrins (CLC) in freezing diluents (Wessel and Ball, 2004). As an alternative to addingCLC to extenders provision of polyunsaturated fatty acids in the feed as a means of alteringthe sperm-lipid membrane profile has been tried with some success in boars and stallions(Brinsko et al, 2005; Purdy and Graham, 2004).Different kinds of freezing procedures have also been reported in the last several years in anattempt to controlling the rates of cooling. Recent results indicate that the cryopreservation