ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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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
Effect of Cryopreservation on Sperm Quality and Fertility 209of bull, stallion and boar semen could be improved by using a programmable freezer(Bianchi et al, 2008; Clulow et al, 2008; Woelders and Chaveiro, 2004). An interactionbetween glycerol concentration and cooling rate has been described for boar semen. Currentcryopreservation methods based on optimal combinations of glycerol and cooling rate hasallowed consistent sperm survival in the frozen semen, with acceptable variation amongindividuals. Another method, termed multi-thermal gradient (MTG), that aims to overcomethe problems of conventional freezing protocol has also been reported (Arav et al, 2002). Thisfreezing technology is based on directional freezing in which the spermatozoa are movedthrough a linear temperature gradient so that, theoretically, the cooling rate and ice frontpropagation are precisely controlled. Thus, the spermatozoa are preserved gently betweenhorizontal columns of ice thereby avoiding the damaging effects of the random ice crystalformation observed in conventional freezing. The technique is also known to allow theincorporation of controlled seeding into the freezing process and prevent the dehydration ofsperm commonly seen in conventional freezing while halving the level of glycerol required(Arav et al, 2002). A slightly different technique, termed unique freezing technology (UFT)which was originally designed to freeze foodstuffs, has been recently tested for semencryopreservation (Goolsby et al, 2004). The UFT involves placing extended samples in a baththat contains an organic fluid with a heat capacity similar to water with a freezing rate of -6.1°C /min. Similar results of post-thaw motilities with sperm frozen in traditional liquidnitrogen procedures have been reported for four UFT treatments (Goolsby et al, 2004).Another front of investigation in the last decade has been the development of methods ofexamining sperm ultrastructural characteristics and alterations. Amongst these are spermkinematics assessed by computer-assisted motility analysis, osmotic resistance tests, plasmamembrane integrity evaluation with fluorescent membrane-impermeable dyes, evaluationof acrosomal status with fluorescein isothiocyanate-conjugated lectins, investigation of DNAintegrity using the SCSA, or assessment of membrane architectural status (Gillan et al, 2004;2005). Most methods require the application of fluorescence microscopy and/or flowcytometric techniques. Because of their highly quantitative, repeatable, and sensitive nature,the techniques are already getting their place in many modern semen laboratories.10. ConclusionCryopreservation continues to be one the most frequently employed technique for use inmodern animal production. Commercial AI will inevitably use this technique to preserveand transport semen over a wider area around the world. However, even with the most upto date procedure cryopreservation still causes detrimental effect on sperm compartmentsand their function. To ensure that semen used for AI are of a relatively uniform, and of highquality, artificial breeding organizations should discard ejaculates based on seminal qualitytests immediately after ejaculation and after freezing and thawing. There are evidencesresulting from different investigation that different sperm compartments are interrelated,the defect in one will invariably affect the other compartment. The success of sperm cell inits ability to fertilize is also affected at different level during its course from its origin until itreaches the ovum. Cryopreservation would be an additional artificial interruption in thisjourney. Therefore, the knowledge of the biochemical basis of the detrimental effects ofcryopreservation and the means to detect these changes easily, cheaply and accuratelyduring semen evaluation would be of great significance. The use of combination of tests,rather than the employment of a single test, would give superior and more complete
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VIContentsChapter 9Evaluation of a
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ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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