Comparison of Different Glycerol and Egg Yolk Concentrations Added to Tris-based Extender for the Collared Peccaries (Tayassu tajacu) Semen Freezing HM Alves 1 , IRS Oliveira 1 , TS Castelo 1 , GL Lima 1 , ALP Souza 1 , MAP Moreira 2 , VV de Paula 2 and AR Silva 1 1 Laboratory of Animal Germplasm Conservation (LCGA), Universidade Federal Rural do Semi-A ´ rido (UFERSA), Mossoro ´, RN, Brazil; 2 Laboratory of Veterinary Surgical Technique and Anesthesiology, Universidade Federal Rural do Semi-A ´ rido (UFERSA), Mossoro ´, RN, Brazil Contents This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentra- tions of glycerol (3% or 6%) added to a Tris-based extender on the post-thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphol- ogy and functional membrane integrity. The ejaculates were initially extended in Tris-fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris-egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris- based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In con- clusion, the use of Tris-based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries. Introduction The Tayassu tajacu, also known as collared peccary, is an exclusive species from the Americas, which inhabits regions extending from the southern United States to northern Argentina (Bodmer and Sowls 1996). Due to a constant threat to these animals from poaching and deforestation of their habitats, they have been recently classified as vulnerable to extinction in some Brazilian biomes such as the Caatinga and Atlantic Forest (ICMBio 2011). The storage of gametes at low temper- atures allows the long-term availability of genetic resources, which helps the preservation of wild species (De Mercado et al. 2010). Recently, Castelo et al. (2010a,b) reported the first success in cryopreserving the collared peccary semen using Tris-based extenders added of egg yolk (20%) and glycerol (3%). This protocol provided 37% motile sperm after thawing. In spite of the importance of these initial studies, there is a need for improving the cryopreservation protocols for the above-mentioned species. Glycerol is a highly permeable polihidric alcohol, and its cryoprotectant activity was discovered by Polge et al. (1949). Nowadays, glycerol is the most usual agent employed in different species for semen cryopreservation (Curry 2000). Species differences in ability to withstand glycerol exposure therefore interact in a complex fash- ion with the freezing rates used and the degree of cryoprotection conferred (Holt 2000). The ideal cryo- protectant concentration represents a balance between its toxic and protective effects, because extremely high concentrations are associated to diminish the fertilizing capacity of the sperm (England 1993). Since that only 3% glycerol was previously tested for collared peccaries semen (Castelo et al. 2010a,b), the search for other concentrations that provide a better cryoprotection is justified to improve the protocol. Based on the fact that glycerol concentrations as higher as 6% are currently used for the semen freezing in domestic animals (Silva et al. 2006; Bezerra et al. 2011), we hypothesize that this glycerol proportion could also provide a better sperm cryoprotection in collared peccaries. Semen extenders are also composed of chicken egg yolk. The cryoprotectant fraction of egg yolk is found in the low-density lipoproteins (LDLs) (Moussa et al. 2002; Jeyendran et al. 2008), which protects the mem- brane phospholipid integrity during cryopreservation (Amirat et al. 2004). Egg yolk has also buffering properties, and hence, the amount of egg yolk in the media may vary according to the buffering property of the other components in the extender (Farstad 1996). According to Hammerstedt et al. (1990), the addition of egg yolk and glycerol promotes an osmotic increase for values around 1300 mOsm/l in the Tris extender. Therefore, in the search for the ideal media for semen freezing, it is necessary to establish the ideal proportion among the components to avoid an interaction that could negatively affect the semen quality. To determine the ideal concentrations of cryoprotec- tants for the collared peccaries semen freezing, the present study aimed to evaluate the effect of various concentrations of egg yolk (5%, 10% or 20%) in combination with different concentrations of glycerol (3% or 6%) on their post-thawed sperm characteristics. Materials and Methods Animals A total of thirteen sexually mature male collared peccaries (40.7 ± 1.6 months of age and 22.5 ± 2.8 kg of weight) were used. The animals belonged to the Centre of Multiplication of Wild Animals from Univer- sidade Federal Rural do Semi-A ´ rido, located in the northeast of Brazil (Mossoro´, RN, Brazil; 5°10 = S, © 2012 Blackwell Verlag GmbH Reprod Dom Anim 48, 506–511 (2013); doi: 10.1111/rda.12115 ISSN 0936–6768