Elsevier

Livestock Science

Volume 152, Issue 1, March 2013, Pages 94-99
Livestock Science

Short communication
Effects of five cryoprotective agents on quality of sheep epididymal spermatozoa during pre-freezing

https://doi.org/10.1016/j.livsci.2012.12.011Get rights and content

Abstract

The objective of this study was to test five cryoprotective agents during ram epidydimal spermatozoa incubation (at 4 °C), of up to 3 h, typical equilibration time before the freezing step begins, in order to establish a starting point for future freezing and thawing protocols. The parameters analyzed were: progressive motility (PM), vitality, and the plasma membrane functional integrity by the hypoosmotic swelling (HOS) test. Testes and epididymides were collected immediately after death. The tail of both epididymides were isolated and spermatozoa were recovered constituting one sample (n=20). A Tes–Tris–Yolk extender was employed. The extender contained five alternative CPAs: Dimethylacetamide (DMA), Dimethyl sulfoxide (DMSO), Ethylene glycol (EG), Glycerol (GLY) and Propylene glycol (PG) at three final concentrations: 2.5%, 5.0% and 10.0%. Control groups consisted of samples mixed only with the extender, without any CPA. All sample groups were exposed to the CPAs for 1 h or 3 h at 4 °C. EG exposure yielded better responses in both PM and HOS test parameters compared to extender only and also the other CPAs. There was no difference among all the treatments regarding vitality. EG (with best results at 2.5%) is thus proposed as a good CPA (followed by DMA as an explorable alternative) for the implementation of forthcoming ram epididymal spermatozoa freezing protocols.

Introduction

The cryopreservation of gametes and embryos has great relevance in reproductive biotechnologies, both in humans and animals allowing the conservation of valuable genetic material for long periods of time (Leibo and Songsasen, 2002). The retrieval and freezing of viable epididymal spermatozoa, represents an interesting alternative in the preservation of either from wildlife or economically interesting animals that can be lost at anytime by unexpected death; in other cases, as a last resort in animals in which ejaculated semen collection has proven difficult (Gilmore et al., 1998b, McClean et al., 2008). The interest in the preservation of germplasm from some socioeconomically important species has generated attention in studies regarding retrieval, evaluation, commercial storage, conservation and cryopreservation of epididymal sperm from ill or dead animals (Papa et al., 2008, Patrizio, 2000, Thuwanut et al., 2008).

In the particular case of the conservation of ram genetic material, so far, a great number of reports has been published concerning freezing of ejaculated sperm, with several extenders (Dorado et al., 2007, O'Hara et al., 2010, Paulenz et al., 2002) and cryoprotective agents (CPAs) (Molinia et al., 1994, Morrier et al., 2002, Nur et al., 2010.) tested; however, reports regarding the effects of CPAs on epididymal ram spermatozoa are very rare (to our knowledge, only Varisli et al., 2009). It is well known that there are important differences in the physiological characteristics of epididymal versus ejaculated spermatozoa, especially in their membrane properties (Hammond, 1930, Martínez-Pastor et al., 2006, Songsasen and Leibo, 1998, Walton, 1930, Watson and Morris, 1987). Martínez-Pastor et al., 2006 also showed differences between both types in responses to extenders, CPAs, and their concentrations. Regarding the CPAs, a good criterion of election must consider that although they generally confer a good protection against damage induced by ice crystal formation (Mazur, 1970), they also could cause injury to sperm even at relative low concentrations (Blackshaw, 1960). Determination of an appropriate CPA and its optimal addition and dilution is a critical step for a successful cryopreservation (Agca et al., 2002, Gao et al., 1995). CPAs treatments parameters, like exposure time, concentration or temperature could possibly be reduced in order to counteract their inherent toxicity on spermatozoa (Clarke et al., 2004, Katkov et al., 1998, Si et al., 2006, Sztein et al., 2001). An alternative CPA for the conservation and storage of semen is the group of amides; employed in equines, especially for samples in which glycerol (GLY) has not shown good results (Alvarenga et al., 2005, Squires et al., 2004). More recent reports describe the use of amides for cryopreservation of boar semen (Bianchi et al., 2007), gander (Lukaszewicz, 2002), etc. Studies carried out by our group have shown that dimethyl sulfoxide (DMSO) and dimethylacetamide (DMA) give good results for epididymal spermatozoa of alpaca, being DMA the cryoprotective presenting best physiological results after cooling and thawing (Canorio et al., 2006, Valdivia et al., 2005). These precedents led us to include DMA among the CPAs tested for this study. The present study aims to propose an adequate CPA for epididymal ram spermatozoa, by testing its incubation for up to 3 h at 4 °C, typical equilibration time before the freezing step begins, in order to determine the best conditions for the implementation of future freezing and thawing procedures.

Section snippets

Location of the study

The collection of samples was made in a local slaughterhouse (Lima, Peru) in May and June of 2010, and all the analysis were performed in the Animal Reproductive Physiology Laboratory, Biological Sciencies Faculty, UNMSM.

Experimental design

Adult rams, >3 years old, >50 kg of body weight, were initially subjected to postmortem sample collection (necropsy); testes were collected and transported immediately after death and processed within the following first 12 h (Kaabi et al., 2003). 20 individuals were finally

Results

After 1 h of cooling at 4 °C, MP decreased in the DMA, DMSO, GLY, and PG 10% treatments compared to control (P<0.05), but EG preserved its control MP levels (P>0.05) (Fig. 1A). At 5% concentration, DMSO and PG showed significant inferiority (<0.05), and 5% EG (P<0.05) showed superior results. At 2.5%, PG showed inferior results (P<0.05), EG showed again superior results (P<0.05), and the rest of 2.5% CPAs showed no significant differences compared to control. Regarding the plasma membrane

Discussion

It is well known that the concentration and type of CPA influence the success of cryopreservation (Fernández-Santos et al., 2006, Leibo, 1999, Salamon and Maxwell, 2000, Watson and Holt, 2001); other parameters have also been evaluated like temperature of addition and incubation of the CPA (Farshad et al., 2009, O'Hara et al., 2010, Paulenz et al., 2002), with results indicating that species-specific factors are critical and parameters should be accordingly established. These parameters are

Conclusion

2.5% EG treatment shows superior cryoprotectant characteristics in ram epididymal spermatozoa and is a good prospective for future freezing protocols implementation. Despite an increase of physiological insult as pre-freezing exposure times prolongs, EG shows good results up to 3 h.

Conflict of interest statement

I (we) certify that there is no conflict of Interest with any financial organization regarding the material discussed in the manuscript “Effect of five cryoprotective agents on quality of sheep epididymal spermatozoa during pre-freezing”.

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