Short communicationHypoosmotic swelling test in epididymal ram (Ovis aries) spermatozoa
Introduction
The plasma membrane of spermatozoa is susceptible to damage from different factors, like osmotic stress or lipid peroxidation. Physical pressure caused by osmotic stress can end up in plasma membrane damage, but if a certain level of stress is not exceeded, the plasma membrane can respond and the sperm cell will deliver a visual, morphological reaction (Ramalho-Santos et al., 2007). The hypoosmotic swelling (HOS) test capitalizes this susceptibility, exposing sperm samples to hypoosmotical solutions (HSs): if the plasma membrane is functional, endosmosis follows and the cells exhibit volume increase (i.e. swelling) and a notorious curling of the tail, probably due to caudal regions being more malleable compared to the head (Jeyendran et al., 1984). Thus, the HOS test has become a routine evaluation employed to assess the integrity of the plasma membrane and possesses high relevance in assisted reproductive technologies (World Health Organization, 2010).
The HOS test has been routinely used in several species, applying the original conditions described and standardized for human samples (Jeyendran et al., 1984): so far, the HOS test has been reported, among others, in boars (Vazquez et al., 1997), cattle (Rota et al., 2000), dogs (Dobranić et al., 2005), rabbits (Ducci et al., 2002), turkeys (Donoghue et al., 1996), horses (de la Cueva et al., 1997), elephants, emus and baboons (Matson et al., 2009), giant pandas (Pérez-Garnelo et al., 2004), hippopotamuses (Saragusty et al., 2010), red deers (Fernández-Santos et al., 2007), and ram (Kaabi et al., 2003). When analyzing epididymal spermatozoa, a HOS test should not assume identical responses to those reported for ejaculated sperm samples, since they are reported to have different characteristics (Hammond, 1930, Martinez-Pastor et al., 2006, Songsasen and Liebo, 1998, Walton, 1930).
Regarding the incubation time of sperm samples in the HSs, for example, a incubation time of one minute was enough to obtain valid results in dogs (Pinto and Kozink, 2008), meaning that it is possible for exposure time to not have a significant influence on results. The aim of the present study was to determine the appropriate osmolality of the HS and the optimal incubation time for the HOS test in ram epididymal spermatozoa.
Section snippets
Samples
The collection of the biological samples was done in a local abattoir in May and June 2010. Immediately after the animal death, testes (including epididymides) were excised from the body and immersed in a 37 °C tempered 0.9% NaCl solution supplemented with 100 UI/ml penicillin G sodium and 1 mg/ml streptomycin and transported to the laboratory within 1 h at the same temperature (in a vacuum flask). A total of 15 biopsies from the same number of mature rams (at least 3 years old and approximately 40
Results
The epididymal spermatozoa of ram responded differently to the several HSs tested (Fig. 2), for both total and strongly reacted sperm. For both groups, the 75–100 mOsm/kg solutions consistently showed the highest response values (P<0.05), subtly declining from 100 to 200 mOsm/kg and abruptly falling beyond 200 mOsm/kg (Fig. 2). Incubation time showed no significant influence on results (P>0.05). Percentages of sperm motility had a mean of 65.78±4.04% and the vitality parameter had a mean of
Discussion
The HOS assay can be considered a quick, easy to perform, low cost test that has allowed us to assess the functional integrity of the sperm plasma membrane. In this study it was possible to identify an appropriate hypoosmotic solution, i.e. the most sensitive, ranging from 75 to 100 mOsm/kg. Other concentrations reported for mammals (ejaculated sperm only) are, for example, 60mOsm/kg in rabbits (Amorim et al., 2009) and 150 mOsm/kg in bulls and humans (Jeyendran et al., 1984, Revell and Mrode,
Conclusion
In conclusion, adequate conditions for the hypoosmotic swelling test of ram epididymal sperm are a HS at 75–100 mOsm/kg and an incubation time of 15 min at 37 °C.
Conflict of interest statement
The authors declare no conflict of interest related to this study.
Acknowledgment
This research was supported in part by the Peruvian National Council of Sciences, Technology and Innovation (CONCYTEC) through the Grant PROCYT315-2009.
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