ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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204Artificial Insemination in Farm Animalsenvironment in vivo as well as in vitro. As a result, fertility from frozen thawed semen ispoorer than that obtained from fresh semen. For this reason, proper evaluation of the postthawquality of spermatozoa is of utmost interest for AI industry to obtain information onthe fertilizing capacity of the cryopreserved semen.Many tests of sperm motility, morphology, acrosomal status, defective sperm organelles andDNA, and metabolism have been correlated with fertility (Evenson et al, 2002; Larsson andRodriquez-Martinez, 2000; Muller, 2000; Saacke et al, 2000; Thomas et al, 1998). All of thesespermatozoal attributes have been shown to be either directly or indirectly affected bycryopreservation or the thawing process. The correlation between fertility and percentage ofmotile sperm in a semen sample has already been demonstrated. In one study, afterinsemination of 55 cows with frozen semen a 30.9% (17 cows) pregnancy rate with anaverage number of services per conception of 2.7 was found. Conception rate to first servicewas only 7.2%. The mean (±SD) alteration of acrosome and positive reaction to HOST forsuccessful (pregnant) and failed insemination (non pregnant) were 47.6 ± 9.9% and 64.7 ±3.0%, and 62.7 ± 7.3% and 42.1 ± 3.9%, respectively with a highly significant (p
Effect of Cryopreservation on Sperm Quality and Fertility 2056. Extenders used in freezing semenThe addition of a cryoprotectant in to the semen sample is needed in order to protectspermatozoa from cold shock. A large variety of extenders combining various components(sugars, electrolytes, buffers, egg yolk, milk and milk products), have been proposed andused for extending sperm. Milk and milk-based extenders are known to be practical andefficient in protecting spermatozoa of various species (Batellier et al, 2001; Varner et al, 1989).Based on the composition and dynamics of the spermatic membranes, some substances suchas lipids, fatty acids and proteins have been incorporated to the semen with the goal ofdecreasing sperm damages related to cryopreservation. Glycerol and egg yolk extenders areamongst the first to be used for freezing semen (Curry, 2000; Garner et al, 1999; Holt, 2000;Medeiros et al., 2002), and today many extenders use glycerol as the major cryoprotectant.Glycerol is used at a relatively high concentration which can be detrimental tospermatozoan viability at higher temperatures hence it is added after the semen has beencooled (Fahy, 1986). The deleterious effects are due to osmotic stress, changes in membraneorganization, fluidity and permeability, as well changes in the lipid composition. Thus, acompromise has to be reached with regards to the concentration of glycerol and the lengthof time that the glycerol is in contact with the spermatozoa prior to freezing, in order tomaximize the beneficial effects of glycerol as a cryoprotectant but minimize its toxic effects.In one study, the inclusion rate of 4% glycerol in an extender containing 20% egg yolk wasfound to be superior to 2% or 6% glycerol as regards to progressive motility (Cochran et al,1984). In addition, the efficiency of glycerol may be affected by the diluents to which it isadded, as well as the method of storage.The cryoprotectant nature of many other substances, including sugars and liposomes hasalso been demonstrated. In equines, it is a common practice today that the preparation ofsemen for cryopreservation involves the use of two extenders: a primary extender for initialdilution, which is aspirated off after centrifugation, prior to the addition of a secondaryextender for freezing. Numerous extenders have been used as primary or secondaryextenders. Examples of extenders for freezing include egg yolk and those based on skimmedmilk with egg yolk (Pickett and Amann, 1993). Good success has been reported with the useof trehalose as a cryoprotectant within a skimmed milk–egg yolk extender. It is suggestedthat trehalose has a stabilizing effect on the spermatozoon plasma membrane (Steinmann,1996).Unlike the bull and the ram in which fructose is the major energy source, most extenders useglucose as the major source of energy for metabolic activity and movement of spermatozoain equines (Katila, et al, 2001). In this regard, three extenders were evaluated for theirefficiency of sustaining the viability of jack sperm as measured by motility characteristics(Ayalew E. and Lemma A., 2010; Unpublished observation). The first extender was a heatedskimmed milk (95°C for 10 minutes and cooled to 37°C before use). The second extenderwas prepared from glucose (4gm), glycerol (4%), and crystalline penicillin (150,000 IU)diluted in heated skimmed milk to make up 100ml extender (represented as SMGLU). Thethird extender contained 4% glycerol in skimmed milk (represented as SMGLY). SM wasused for storage in Equitainer (Agtech Inc, Manhattan, USA) while SMGLU and SMGLYwere used for liquid nitrogen storage after a 2.5hr optimization at +4°C, suspension overliquid nitrogen vapour for 7 minutes and plunging the straws immediately into the liquidnitrogen. SMGLU preserved sperm showed superior results (p
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