But there is no published study that

determined the optimal cooling rate of rat sperm. Determination of optimal extender composition for various species has enabled development of better cryopreservation protocols [14], [38] and [53]. An ideal sperm extender should have optimum pH, buffering capacity, suitable osmotic pressure and protect sperm against cold shock [45]. The solutions of Tris-citrate-EY, skim milk-EY, lactose-EY and Tris-TES are the most commonly used sperm extenders [56]. Krebs–Ringer bicarbonate (mKRB) solution containing raffinose, 0.75% Equex STM, 0.05% sodium dodecly sulfate (SDS) and EY greatly enhanced the cryosurvival of rat sperm [57]. The use of EY reduces chilling injury to sperm in many mammalian species [38]. In a recent study [51], we found that the addition of 20% lactose-egg yolk (LEY) into extenders reduced motility loss after chilling. In addition, Y 27632 various SDS-based products improve the effectiveness of EY during sperm freezing for several mammalian species including mouse [40], rat [34], cat [5], dog [39] and pig [8]. Equex Paste (EP) and Orvus ES Paste are the commercial forms of SDS which is a water-soluble anionic detergent. Equex Paste is used for horse and swine sperm cryopreservation. Equex Paste with EY has more

protective effect against freezing damage and cold shock [41] due to increase of protective activity of EY by changing the structure of lipoproteins of egg yolk [4]. Many previous

reports suggest Selleck R428 that sperm from different species respond differently to chilling, CPA, and extenders [2], [17], [31], [44] and [51]. The addition of permeating CPA (e.g. glycerol) and non-permeating sugars (e.g. sucrose, raffinose and trehalose) to extenders has been effective for cryopreservation of sperm from various mammalian species [3] and [4]. Glycerol is the most common CPA used for freezing sperm from various species [42] and [45]. However, addition of glycerol to extenders was found to be detrimental to mouse sperm [26] and not effective for rat sperm freezing [34]. Furthermore, many reports suggested that raffinose is an effective CPA for mouse and rat [25], [26], [32], [33], [37] and [57]. For successful cryopreservation, careful selection of extender as well as an appropriate CPA that works Depsipeptide chemical structure well with the chosen extender to maintain high sperm motility after freezing is necessary [51]. In this study we performed series of experiments to determine appropriate CPA, extender and cooling rate to improve post thaw rat sperm viability. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. 10 to 12 weeks old SD and Fisher 344 (F344) rats were used as sperm donors. The rats were housed in accordance with the policies of the University of Missouri Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals.