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Osmotic tolerance of feline epididymal spermatozoa
Animal Reproduction Science xxx (xxxx) xxx–xxx
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Osmotic tolerance of feline epididymal spermatozoa ⁎
Panisara Kunkittib, , Kaywalee Chatdarongc, Junpen Suwimonteerabutrc, Teerawut Nedumpund, Anders Johannissona, Ann-Sofi Bergqvista, Ylva Sjunnessona, Eva Axnéra a
Division of Reproduction, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden Department of Surgery and Theriogenology, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, Thailand c Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand d Inter-department of Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok, Thailand b
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Keywords: Cats Immature sperm Osmotic effect
During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezingthawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10 min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P < 0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P > 0.01) and a difference between caput and corpus after return from 1200 mOsm (P < 0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P < 0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to nonreturning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more
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Corresponding author. E-mail addresses: [email protected] (P. Kunkitti), [email protected] (K. Chatdarong), [email protected] (J. Suwimonteerabutr), [email protected] (T. Nedumpun), [email protected] (A. Johannisson), Ann-Sofi[email protected] (A.-S. Bergqvist), [email protected] (Y. Sjunnesson), [email protected] (E. Axnér). http://dx.doi.org/10.1016/j.anireprosci.2017.08.014 Received 2 March 2017; Received in revised form 28 May 2017 0378-4320/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Kunkitti, P., Animal Reproduction Science (2017), http://dx.doi.org/10.1016/j.anireprosci.2017.08.014
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susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.
1. Introduction Preservation of feline epididymal sperm is currently a subject of interest with the purpose to rescue genetic material from threatened and endangered wild felids species in which the genetic materials could be lost by unexpected death of the animals (Cocchia et al., 2010). Epididymal spermatozoa, similar to ejaculated spermatozoa, can be used by assisted reproductive technologies (ARTs) such as; artificial insemination (AI), in vitro fertilization (IVF) (Tsutsui et al., 2003; Tebet et al., 2006) and intra-cytoplasmic sperm injection (ICSI) to produce offspring (Bogliolo et al., 2001). Currently, spermatozoa from corpus region of epididymis have been proved to have similar abilities as spermatozoa from cauda, capability to undergo capacitation, fertilize oocytes in vitro and to be able to survive cryopreservation (Kunkitti et al., 2015; Kunkitti et al., 2016a; Kunkitti et al., 2016b). However, up to 40–60% of feline epididymal sperm motility is lost in the cryopreservation process (Kunkitti et al., 2016b). In order to facilitate improvement in cryopreservation of feline epididymal sperm, a better understanding of basic cryobiological properties of the cells are needed, including their tolerance limits and the effects of osmotic stress. During the cryopreservation process, spermatozoa are exposed to high concentrations of cryoprotectants (CPAs) which equilibrate the spermatozoa and minimise ice crystal formation within the cells. However, addition and removal of CPAs subjects the spermatozoa to a substantial osmotic stress. Before freezing, high concentrations of CPAs are added to the sperm suspension. Spermatozoa are exposed to a hyperosmotic environment and left frozen in high concentration of CPAs (Guthrie et al., 2002). In the thawing process, the sperm suspensions in hypertonic solution are diluted and returned to near isotonic conditions. The rapid osmolality changes throughout the process can lead to loss of functional integrity, such as sperm plasma membrane integrity, DNA integrity and motility. (Watson, 2000; Ball and Vo, 2001) Spermatozoa from different regions of the epididymis differ in their degree of maturation and may have different susceptibility for osmotic stress depending on the composition of the cell membrane and this will affect their tolerance for the freezing-thawing stress. The objectives of this study were to evaluate the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to determine the effect of osmotic stress on the epididymal spermatozoa of different epididymal regions. The results contributed to a better understanding of basic physiological properties of epididymal spermatozoa from different regions which may be helpful for designing cryopreservation protocols for epididymal sperm.
2. Materials and methods 2.1. Experimental design Epididymal spermatozoa from three different regions (caput, corpus and cauda) were exposed to solutions of various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10 min and then returned to 300 mOsm. Percentage of motile spermatozoa and sperm membrane integrity (SYBR-14/PI) was evaluated after exposure and after returning to 300 mOsm (isotonic condition). The mitochondrial membrane potential (JC1) were evaluated by flow cytometry after the return to isotonic condition. The experiment was performed according to Thailand regulations.
2.2. Animals The study included epididymal spermatozoa from 55 privately owned domestic male cats of various breeds and ages. All cats were subjected to routine castration by closed-technique at the Veterinary Public Health of Bangkok, Thailand. After testes and epididymides were removed from the cats, they were immediately kept in 0.9% (w/v) normal saline solution supplemented with penicillin-streptomycin at room temperature until epididymal spermatozoa were transferred to the laboratory. The experiment was performed within 6 h after the testes were removed from the cats.
2.3. Epididymal sperm recovery The epididymides were dissected free from blood vessels and connective tissues. Each epididymis was divided into three regions; caput, corpus and cauda. The epididymides from 5 cats were randomly pooled as one replicate in order to increase the sperm numbers and also to reduce individual variations. A total of 11 replicates were performed in this study. To collect spermatozoa from epididymides, the tissue segment of each region was transversely cut into small pieces and placed in 2 mL warm (38 °C) phosphate buffered saline (PBS) [137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 280 mOsm] medium for 10 min. After 10 min, the tissues were removed. Sperm concentration and total sperm number were evaluated. 2
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2.4. Single exposure to different osmolality Hypo- and hypertonic solutions were prepared according to Pukazhenthi et al. (2000). Briefly, the hypotonic solution (75 mOsm) was prepared by diluting PBS with double-distilled water, whereas hypertonic solutions were prepared by adding 5 M NaCl solution. Osmolalities of all solutions were determined using osmometer (Micro Osmometer Model 210, Fiske®, MA, USA). To determine the effect of osmotic stress on feline epididymal sperm from different regions, the sperm aliquot (1 × 106 spermatozoa) from each region was pelleted by centrifugation (300g for 6 min) and resuspended in 400 μL of different osmolalities solutions (75, 300, 600, 900, 1200 mOsm). The sperm were left in the solutions for 10 min at room temperature. Sperm motility and membrane integrity (SYBR-14/PI) were evaluated after exposure. 2.5. Return to isotonicity After 10 min of incubation at room temperature, the spermatozoa from each osmolality condition were returned to 300 mOsm by a single step addition of 500 μL of 565 mOsm of PBS, 500 μL of 300 mOsm of PBS or 500 μL of double-distilled water, 1000 μL of double-distilled water and 1500 μL of double-distilled water, respectively. 2.6. Sperm evaluation 2.6.1. Concentration and subjective motility Sperm concentration was determined using a hemocytometer (Boeco, Hamburg, Germany). The proportions of spermatozoa with total motility and progressive motility were subjectively assessed. A 5 μL aliquot of sperm sample was placed on a pre-warmed slide and covered with a pre-warmed cover slip and subjectively assessed on a phase contrast microscope at 200× magnification. 2.6.2. Flow cytometric analysis of plasma membrane integrity Plasma membrane integrity was assessed using the protocol slightly modified from Johannisson et al. (2009) using the dual fluorescence staining SYBR-14 (Molecular Probes, Inc., Eugene, OR, USA) in combination with propidium iodide (PI, Invitrogen, Eugene, OR, USA).The 300 μL sperm samples were stained with 0.6 μL of 20 μM SYBR-14 (final stain concentration 12 μM) and 3 μL of 12 μM PI (final stain concentration 36 μM) (Live-Dead Sperm Viability Kit L-7011; Invitrogen,) and incubated at 37 °C for 10 min. The stained samples were analyzed with flow cytometer (Beckman Coulter FC 500, Beckman Coulter, Pasadena, CA, USA). Excitation was induced by an argon ion 488 nm laser. SYBR-14 fluorescence (green: cells with intact plasma membrane) was detected via channel FL1 (525 nm), PI fluorescence (red: cells with permeable plasma membrane) was detected via channel FL3 (610 nm) A total of 30,000 spermatozoa were evaluated and classified into three populations according to the degree of plasma membrane integrity: living (%) (SYBR14-positive/PI-negative), dead (%) (SYBR14- negative/PI-positive), and dying (%) (SYBR14-positive/PI-positive). 2.6.3. Flow cytometric analysis of mitochondrial membrane potential Mitochondrial membrane potential (MMP) of sperm was analyzed using flow cytometry procedures which was slightly modified from Goodla et al. (2014). Briefly, 300 μL of sperm samples were stained with 1.2 μL of 3 mM JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolcarbocyanine iodide) (final stain concentration 12 μM) and incubated at 37 °C for 40 min. The stained samples were evaluated with a flow cytometer (Beckman Coulter FC500). Excitation was induced by an Argon ion 488 nm laser. Emitted fluorescence was collected using both FL1 (525 nm) and FL2 (575 nm) filters. The green fluorescence was evaluated in FL1 and orange in FL2. A total of 30,000 cells were analyzed and presented in two classifications: spermatozoa with high mitochondrial respiratory activity or high membrane potential (Orange fluorescence) and with low mitochondrial respiratory activity or nomembrane potential (Green fluorescence). 2.7. Statistical analysis The comparisons of sperm quality (motility, membrane integrity and mitochondrial membrane potential) among osmolalities and among epididymal regions as well as the effects of single exposure and return to isotonic conditions were performed using mixed statistical models procedure of the SAS® 9.3 (Proc Mixed, SAS® 9.3, Cary, NC, USA) after checking that the residuals had a normal or close to normal distribution. Pairwise differences were tested by multiple comparisons with Tukey’s range test. The level of significance was set at P ≤ 0.05. 3. Results 3.1. Effect of exposure to different osmolality There was a significant effect of both osmolality and region on sperm motility and an interaction between the two factors (P < 0.001). In the overall comparison with all regions combined the highest sperm motility was observed in the 300 mOsm medium (P < 0.001). Overall motility was significantly higher at 600 mOsm compared to 900 or 1200 mOsm (P < 0.05) but did not differ significantly between 600 and 75 mOsm (P = 0.67). At 300 mOsm spermatozoa from cauda region showed a significantly higher percentage of sperm motility than those from caput region (P < 0.0001) and corpus spermatozoa had significantly higher motility 3
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than spermatozoa from the caput (P < 0.001). Spermatozoa from cauda completely lost their motility at osmolality 900 mOsm and higher, while caput and corpus spermatozoa lost their motility at osmolality 1200 mOsm (Fig. 1A). There was no overall effect of region on the percentage of spermatozoa with intact membranes (P = 0.20) while osmolarity had an effect (P < 0.0001). Spermatozoa exposed to 900 mOsm had a significantly higher percentage of intact sperm membranes compared to 75 and 1200 mOsm (P < 0.05) while other treatments did not differ significantly. Feline epididymal sperm membranes appeared to be highly tolerant to hypo- and hypertonic stress with percentages of spermatozoa with intact membranes ranging from 53% to 69%. (Fig. 1B). 3.2. Effect of returning to isotonic condition after exposure to hypo- and hypertonic solutions Spermatozoa from corpus and cauda which were pre-exposed to isotonic solution (300 mOsm) demonstrated a significant reduction in the percentage of motile spermatozoa after adding 1:1 300 mOsm medium (P < 0.01) (Fig. 1a), while the spermatozoa pre-exposed to 75, 600, 900 and 1200 mOsm solution did not show a significant reduction of motile spermatozoa after being returned to isotonic conditions. In contrast, spermatozoa from the cauda pre-exposed to 75 mOsm showed an increase in motility after being returned to isotonic conditions (P = 0.0015). Returning spermatozoa to isotonic conditions after exposure to 900 or 1200 mOsm resulted in significant membrane damage in spermatozoa from any of the regions while membranes of spermatozoa exposed to 75–600 mOsm where not significantly affected (Fig. 1b). Moreover, comparing between regions of epididymis, spermatozoa from cauda showed significantly more severe membrane damage than caput or corpus spermatozoa after being returned to isotonic conditions after pre-exposure to 900 mOsm (P < 0.01) and significantly more severe than spermatozoa from the caput after being pre-exposed to 1200 mOsm (P < 0.05) (Fig. 1b). There was an effect of both region (P < 0.01) and osmolarity (P < 0.0001) on the percentage of spermaotozoa with hight MMP as well as an interaction between the two factors (p < 0.0001). The corpus and cauda regions contained higher overall percentage of spermatozoa with high MMP compared to the caput region (P < 0.05) (Fig. 2A, B). Spermatozoa from all regions that were preexposed to 900- or 1200 mOsm had lower (P < 0.05) proportion of spermatozoa with high MMP than spermatozoa pre-exposed to 75-, 300- or 600 mOsm (Fig. 2A,B). 4. Discussion This study is the first to investigate the osmotic tolerance of feline epididymal spermatozoa by imitating the conditions that spermatozoa will be exposed during the freezing-thawing process. The experiment was conducted at room temperature and the effect of cryoprotectants was not included in this study. According to the standard protocol for freezing and thawing, firstly, spermatozoa are diluted in tris egg-york extender I which contains 3% (v:v) glycerol (865 mOsm) then diluted in tris egg-york extender II (1495
Fig. 1. Effects of osmotic change on feline epididymal spermatozoa from different regions of epididymis (caput, corpus and cauda): (A) motility after exposure to different osmotic conditions (a) motility after abrupt exposure to different osmotic conditions and return to isotonic condition (B) intact membrane after abrupt exposure to different osmotic conditions (b) intact membrane after abrupt exposure to different osmotic conditions and return to isotonic condition. Results are presented as mean ± standard error of mean (SEM). Different letters indicate significant difference between osmolalities across regions (P < 0.05). * indicate that there is a significant difference between regions within osmolality (P < 0.05).
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Fig. 2. Osmotic effect on mitochondrial membrane potential of feline epididymal spermatozoa from different regions of epididymis (caput, corpus and cauda) after exposure to hypo-and hyperosmotic media and return to isotonic conditions afterwards: (A) percent spermatozoa with high mitochondrial respiratory activity or high membrane potential (Orange fluorescence), (B) percent spermatozoa with low mitochondrial respiratory activity or no-membrane potential (Green fluorescence). Results are presented as mean ± standard error of mean (SEM). Different letters indicate significant difference between osmolalities across regions (P < 0.05). * indicate that there is a significant difference between regions within osmolality (P < 0.05).
mOsm). These will make the final osmolality that sperm will be stored to be around 1180 mOsm (Kunkitti et al., 2016b). In the thawing protocol, it is recommended to dilute with Tris buffered solution (324 mOsm) to reduce toxic effect of cryoprotectant and Equex STM paste (Chatdarong et al., 2010) that made the final osmolality to be around 914 mOsm. As the water freezes during the sperm freezing process electrolytes are concentrated in the fluids surrounding the spermatozoa leading to an increased osmolality in the sperm environment. At thawing this process is reversed. Altogether, feline caudal epididymal spermatozoa are exposed to two levels of hyper-osmotic solutions prior to their return to isotonicity during the freezing and thawing process. Therefore, in the present study the effect of osmotic stress of feline epididymal spermatozoa was investigated by exposure of spermatozoa to different osmolality before returning them to isotonic condition. The current results of sperm motility confirmed that the isotonic osmolality of 300 mOsm promoted the best motility. Spermatozoa from cauda region had a higher percentage of motile spermatozoa compared to corpus and caput regions. This finding consists with an earlier study in feline epididymal sperm (Kunkitti et al., 2015). The decrease in motility after returning spermatozoa from the cauda pre-exposed to 300 mOsm might be a time effect rather than an effect of osmolality as the osmolality in this treatment did not change. The feline epididymal spermatozoa are highly susceptible to osmotic stress as demonstrated by the dramatical decrease when exposed to the solutions at 75 mOsm and 600 mOsm and complete loss of motility at osmolality more than 900 mOsm. This supports the study of Pukazhenthi et al. (2000) in feline and is similar to the previous studies in the mouse (Agca et al., 2005), bovine (Guthrie et al., 2002) and equine (Pommer et al., 2002) in that the sperm motility markedly declined in response to small deviations from isotonicity. The osmotic tolerance of feline epididymal sperm motility might be only a small deviation from isotonicity. More investigation in the response of sperm motility to small deviations in osmolality from 300 mOsm might be needed. Interestingly, from our study an extensive damage of sperm plasma membrane was observed in spermatozoa from all epididymal regions pre-exposed to hypertonic solution of more than 600 mOsm and returned to isotonic condition (300 mOsm). However, the plasma membrane of feline epididymal sperm was more tolerant to the hypo-osmotic media than hypertonic condition demonstrated by the similar percentage of spermatozoa with intact membrane prior to and after exposure to75 mOsm. Comparison between our finding and a previous study on ejaculated feline spermatozoa which exhibited a marked decrease in membrane integrity when spermatozoa were exposed to hypotonic solution (0, 35, 75, and 150 mOsm)(Pukazhenthi et al., 2000), our discovery might indicate biophysical differences in the membrane between epididymal spermatozoa and ejaculated spermatozoa. The exposure of spermatozoa to 900- or 1200 mOsm solution and return to isotonic conditions resulted in at least 30% decline in the percentage of intact membrane. Our findings are consistent with a previous report by Pukazhenthi et al. (2000), the severe disruption of sperm membrane was observed when returning the sperm to isotonic condition in a single step after pre-exposed to osmolality greater than 600 mOsm. The results demonstrated that a rapid return of spermatozoa close to isotonic condition after spermatozoa was exposed to hypo- or hypertonic solution is more harmful for spermatozoa than non-return. Minimizing the effect of osmotic stress during the sperm cryopreservation process might need more investigation. For example, the negative effect might be reduced if spermatozoa are not returned to isotonic conditions or are returned in multiple steps. A previous study in feline ejaculate spermatozoa by Chatdarong et al. (2010) demonstrated the benefit of post-thaw dilution at a rate of 1:2 with Tris buffer contributing to the final osmolarity of more than 300 mOsm and resulted in improved sperm motility, membrane integrity and acrosome integrity. The optimal osmolality postthaw could be another point that might be interesting to investigate. Although, from our results, the osmolality range that feline epididymal sperm plasma membrane could tolerate is between 75 mOsm and 600 mOsm. Sperm mitochondrial membrane potential of spermatozoa has been reported to have a positive relation with fertility. Accordingly, sperm motility and fertility potential were high when mitochondrial membrane potential was high (Kasai et al., 2002). Therefore, assessment of mitochondrial membrane potential is useful to predict sperm fertility potential. The exposure of hypertonic stress affected not only sperm plasma membrane but also the MMP of feline epididymal spermatozoa. A significant reduction in the percentage of spermatozoa with high MMP was detected when spermatozoa were returned to isotonic condition after pre-exposure to 900- and 1200 mOsm and no extensive mitochondrial damage were observed at 75, 300 and 600 mOsm. From this result, the osmolality range that epididymal sperm are capable to preserve their mitochondrial function, as demonstrated by a high MMP, is between 75 mOsm to 600 mOsm. 5
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The osmosensitivity of epididymal spermatozoa has also been studied in the kangaroo (McClean et al., 2006). Comparison between caput and cauda spermatozoa showed that caput spermatozoa were more tolerant to hypotonic solution and hypertonic solution (high glycerol concentration) than cauda spermatozoa. This is similar to our findings as membranes of spermatozoa from the cauda were more sensitive to damage when they were returned to isotonic conditions after being pre-exposed to hypertonic solutions of 900 mOsm or higher compared to caput and corpus spermatozoa. As spermatozoa mature in the epididymis of the ram they acquire susceptibility to cold shock (White, 1993). It is therefore likely that sperm membrane modifications during feline epididymal maturation results in decreased ability to tolerate changes in the sperm environment as has been shown in other species. In conclusion, from the present study each of the sperm parameters (motility, membrane integrity and MMP) showed differences in range of osmotic tolerances. The safety margin of low and high osmolality of feline epididymal sperm might depend on which parameter that you intend to study or use. Our results showed that returning to isotonic condition in a single step after pre-exposure to osmolality greater than 600 mOsm can cause severe damage on sperm membrane and MMP compared to the non-returning group. Spermatozoa from all epididymal regions have a similar tendency in responding to osmotic stress, although cauda epididymis was more sensitive to osmolality changes compared to corpus and caput. Future studies on longevity of the feline epididymal sperm in hypertonic conditions or studies on slow-step returning to isotonic condition after pre-exposure to hypertonic solution might give us more information about the osmotic resistance of feline spermatozoa. Careful consideration and proper protocols for handling of feline spermatozoa and osmotic conditions are required to achieve reliable results and minimise damage. Conflict of interest None of the authors have any conflict of interest to declare. Acknowledgments We would like to sincerely thank The Veterinary Public Health Division of The Bangkok Metropolitan Administration, Bangkok, Thailand for providing cat’s epididymis samples, the Research Unit of Obstetrics and Reproduction and Center of Emerging and Reemerging Infectious Diseases in Animals, Faculty of Veterinary Science, Chulalongkorn University for providing lab facilities and flow cytometry facility. References Agca, Y., Mullen, S., Liu, J., Johnson-Ward, J., Gould, K., Chan, A., Critser, J., 2005. Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa. Cryobiology 51, 1–14. Ball, B.A., Vo, A., 2001. Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. J. Androl. 22, 1061–1069. Bogliolo, L., Leoni, G., Ledda, S., Naitana, S., Zedda, M., Carluccio, A., Pau, S., 2001. Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa. Theriogenology 56, 955–967. Chatdarong, K., Thuwanut, P., Manee-in, S., Lohachit, C., Axner, E., 2010. Effects of thawing temperature and post-thaw dilution on the quality of cat spermatozoa. Reprod. Domest. Anim. 45, 221–227. Cocchia, N., Ciani, F., El-Rass, R., Russo, M., Borzacchiello, G., Esposito, V., Montagnaro, S., Avallone, L., Tortora, G., Lorizio, R., 2010. Cryopreservation of feline epididymal spermatozoa from dead and alive animals and its use in assisted reproduction. Zygote 18, 1–8. Goodla, L., Morrell, J.M., Yusnizar, Y., Stålhammar, H., Johannisson, A., 2014. Quality of bull spermatozoa after preparation by single-layer centrifugation. J. Dairy Sci. 97, 2204–2212. Guthrie, H.D., Liu, J., Critser, J.K., 2002. Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa. Biol. Reprod. 67, 1811–1816. Johannisson, A., Morrell, J.M., Thoren, J., Jonsson, M., Dalin, A.M., Rodriguez-Martinez, H., 2009. Colloidal centrifugation with Androcoll-E prolongs stallion sperm motility, viability and chromatin integrity. Anim. Reprod. Sci. 116, 119–128. Kasai, T., Ogawa, K., Mizuno, K., Nagai, S., Uchida, Y., Ohta, S., Fujie, M., Suzuki, K., Hirata, S., Hoshi, K., 2002. Relationship between sperm mitochondrial membrane potential, sperm motility, and fertility potential. Asian J. Androl. 4, 97–103. Kunkitti, P., Bergqvist, A.S., Sjunnesson, Y., Axner, E., 2015. The ability of feline spermatozoa in different epididymal regions to undergo capacitation and acrosome reaction. Anim. Reprod. Sci. 161, 64–74. Kunkitti, P., Axner, E., Bergqvist, A.S., Sjunnesson, Y., 2016a. In vitro fertilization using frozen-thawed feline epididymal spermatozoa from corpus and cauda regions. Theriogenology. Kunkitti, P., Bergqvist, A.S., Sjunnesson, Y., Johannisson, A., Axner, E., 2016b. The tolerance of feline corpus and cauda spermatozoa to cryostress. Theriogenology 85, 502–508. McClean, R., MacCallum, C., Blyde, D., Holt, W.V., Johnston, S.D., 2006. Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa. Reprod. Fertil. Dev. 18, 469–476. Pommer, A.C., Rutllant, J., Meyers, S.A., 2002. The role of osmotic resistance on equine spermatozoal function. Theriogenology 58, 1373–1384. Pukazhenthi, B., Noiles, E., Pelican, K., Donoghue, A., Wildt, D., Howard, J., 2000. Osmotic effects on feline spermatozoa from normospermic versus teratospermic donors. Cryobiology 40, 139–150. Tebet, J.M., Martins, M.I., Chirinea, V.H., Souza, F.F., Campagnol, D., Lopes, M.D., 2006. Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa. Theriogenology 66, 1629–1632. Tsutsui, T., Wada, M., Anzai, M., Hori, T., 2003. Artificial insemination with frozen epididymal sperm in cats. J. Vet. Med. Sci. Jpn. Soc. Vet. Sci. 65, 397–399. Watson, P.F., 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60 (-61), 481–492. White, I.G., 1993. Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod. Fertil. Dev. 5, 639–658.
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Osmotic tolerance of feline epididymal spermatozoa ⁎
Panisara Kunkittib, , Kaywalee Chatdarongc, Junpen Suwimonteerabutrc, Teerawut Nedumpund, Anders Johannissona, Ann-Sofi Bergqvista, Ylva Sjunnessona, Eva Axnéra a
Division of Reproduction, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden Department of Surgery and Theriogenology, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, Thailand c Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand d Inter-department of Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok, Thailand b
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Keywords: Cats Immature sperm Osmotic effect
During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezingthawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10 min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P < 0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P > 0.01) and a difference between caput and corpus after return from 1200 mOsm (P < 0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P < 0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to nonreturning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more
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Corresponding author. E-mail addresses: [email protected] (P. Kunkitti), [email protected] (K. Chatdarong), [email protected] (J. Suwimonteerabutr), [email protected] (T. Nedumpun), [email protected] (A. Johannisson), Ann-Sofi[email protected] (A.-S. Bergqvist), [email protected] (Y. Sjunnesson), [email protected] (E. Axnér). http://dx.doi.org/10.1016/j.anireprosci.2017.08.014 Received 2 March 2017; Received in revised form 28 May 2017 0378-4320/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Kunkitti, P., Animal Reproduction Science (2017), http://dx.doi.org/10.1016/j.anireprosci.2017.08.014
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susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.
1. Introduction Preservation of feline epididymal sperm is currently a subject of interest with the purpose to rescue genetic material from threatened and endangered wild felids species in which the genetic materials could be lost by unexpected death of the animals (Cocchia et al., 2010). Epididymal spermatozoa, similar to ejaculated spermatozoa, can be used by assisted reproductive technologies (ARTs) such as; artificial insemination (AI), in vitro fertilization (IVF) (Tsutsui et al., 2003; Tebet et al., 2006) and intra-cytoplasmic sperm injection (ICSI) to produce offspring (Bogliolo et al., 2001). Currently, spermatozoa from corpus region of epididymis have been proved to have similar abilities as spermatozoa from cauda, capability to undergo capacitation, fertilize oocytes in vitro and to be able to survive cryopreservation (Kunkitti et al., 2015; Kunkitti et al., 2016a; Kunkitti et al., 2016b). However, up to 40–60% of feline epididymal sperm motility is lost in the cryopreservation process (Kunkitti et al., 2016b). In order to facilitate improvement in cryopreservation of feline epididymal sperm, a better understanding of basic cryobiological properties of the cells are needed, including their tolerance limits and the effects of osmotic stress. During the cryopreservation process, spermatozoa are exposed to high concentrations of cryoprotectants (CPAs) which equilibrate the spermatozoa and minimise ice crystal formation within the cells. However, addition and removal of CPAs subjects the spermatozoa to a substantial osmotic stress. Before freezing, high concentrations of CPAs are added to the sperm suspension. Spermatozoa are exposed to a hyperosmotic environment and left frozen in high concentration of CPAs (Guthrie et al., 2002). In the thawing process, the sperm suspensions in hypertonic solution are diluted and returned to near isotonic conditions. The rapid osmolality changes throughout the process can lead to loss of functional integrity, such as sperm plasma membrane integrity, DNA integrity and motility. (Watson, 2000; Ball and Vo, 2001) Spermatozoa from different regions of the epididymis differ in their degree of maturation and may have different susceptibility for osmotic stress depending on the composition of the cell membrane and this will affect their tolerance for the freezing-thawing stress. The objectives of this study were to evaluate the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to determine the effect of osmotic stress on the epididymal spermatozoa of different epididymal regions. The results contributed to a better understanding of basic physiological properties of epididymal spermatozoa from different regions which may be helpful for designing cryopreservation protocols for epididymal sperm.
2. Materials and methods 2.1. Experimental design Epididymal spermatozoa from three different regions (caput, corpus and cauda) were exposed to solutions of various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10 min and then returned to 300 mOsm. Percentage of motile spermatozoa and sperm membrane integrity (SYBR-14/PI) was evaluated after exposure and after returning to 300 mOsm (isotonic condition). The mitochondrial membrane potential (JC1) were evaluated by flow cytometry after the return to isotonic condition. The experiment was performed according to Thailand regulations.
2.2. Animals The study included epididymal spermatozoa from 55 privately owned domestic male cats of various breeds and ages. All cats were subjected to routine castration by closed-technique at the Veterinary Public Health of Bangkok, Thailand. After testes and epididymides were removed from the cats, they were immediately kept in 0.9% (w/v) normal saline solution supplemented with penicillin-streptomycin at room temperature until epididymal spermatozoa were transferred to the laboratory. The experiment was performed within 6 h after the testes were removed from the cats.
2.3. Epididymal sperm recovery The epididymides were dissected free from blood vessels and connective tissues. Each epididymis was divided into three regions; caput, corpus and cauda. The epididymides from 5 cats were randomly pooled as one replicate in order to increase the sperm numbers and also to reduce individual variations. A total of 11 replicates were performed in this study. To collect spermatozoa from epididymides, the tissue segment of each region was transversely cut into small pieces and placed in 2 mL warm (38 °C) phosphate buffered saline (PBS) [137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 280 mOsm] medium for 10 min. After 10 min, the tissues were removed. Sperm concentration and total sperm number were evaluated. 2
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2.4. Single exposure to different osmolality Hypo- and hypertonic solutions were prepared according to Pukazhenthi et al. (2000). Briefly, the hypotonic solution (75 mOsm) was prepared by diluting PBS with double-distilled water, whereas hypertonic solutions were prepared by adding 5 M NaCl solution. Osmolalities of all solutions were determined using osmometer (Micro Osmometer Model 210, Fiske®, MA, USA). To determine the effect of osmotic stress on feline epididymal sperm from different regions, the sperm aliquot (1 × 106 spermatozoa) from each region was pelleted by centrifugation (300g for 6 min) and resuspended in 400 μL of different osmolalities solutions (75, 300, 600, 900, 1200 mOsm). The sperm were left in the solutions for 10 min at room temperature. Sperm motility and membrane integrity (SYBR-14/PI) were evaluated after exposure. 2.5. Return to isotonicity After 10 min of incubation at room temperature, the spermatozoa from each osmolality condition were returned to 300 mOsm by a single step addition of 500 μL of 565 mOsm of PBS, 500 μL of 300 mOsm of PBS or 500 μL of double-distilled water, 1000 μL of double-distilled water and 1500 μL of double-distilled water, respectively. 2.6. Sperm evaluation 2.6.1. Concentration and subjective motility Sperm concentration was determined using a hemocytometer (Boeco, Hamburg, Germany). The proportions of spermatozoa with total motility and progressive motility were subjectively assessed. A 5 μL aliquot of sperm sample was placed on a pre-warmed slide and covered with a pre-warmed cover slip and subjectively assessed on a phase contrast microscope at 200× magnification. 2.6.2. Flow cytometric analysis of plasma membrane integrity Plasma membrane integrity was assessed using the protocol slightly modified from Johannisson et al. (2009) using the dual fluorescence staining SYBR-14 (Molecular Probes, Inc., Eugene, OR, USA) in combination with propidium iodide (PI, Invitrogen, Eugene, OR, USA).The 300 μL sperm samples were stained with 0.6 μL of 20 μM SYBR-14 (final stain concentration 12 μM) and 3 μL of 12 μM PI (final stain concentration 36 μM) (Live-Dead Sperm Viability Kit L-7011; Invitrogen,) and incubated at 37 °C for 10 min. The stained samples were analyzed with flow cytometer (Beckman Coulter FC 500, Beckman Coulter, Pasadena, CA, USA). Excitation was induced by an argon ion 488 nm laser. SYBR-14 fluorescence (green: cells with intact plasma membrane) was detected via channel FL1 (525 nm), PI fluorescence (red: cells with permeable plasma membrane) was detected via channel FL3 (610 nm) A total of 30,000 spermatozoa were evaluated and classified into three populations according to the degree of plasma membrane integrity: living (%) (SYBR14-positive/PI-negative), dead (%) (SYBR14- negative/PI-positive), and dying (%) (SYBR14-positive/PI-positive). 2.6.3. Flow cytometric analysis of mitochondrial membrane potential Mitochondrial membrane potential (MMP) of sperm was analyzed using flow cytometry procedures which was slightly modified from Goodla et al. (2014). Briefly, 300 μL of sperm samples were stained with 1.2 μL of 3 mM JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolcarbocyanine iodide) (final stain concentration 12 μM) and incubated at 37 °C for 40 min. The stained samples were evaluated with a flow cytometer (Beckman Coulter FC500). Excitation was induced by an Argon ion 488 nm laser. Emitted fluorescence was collected using both FL1 (525 nm) and FL2 (575 nm) filters. The green fluorescence was evaluated in FL1 and orange in FL2. A total of 30,000 cells were analyzed and presented in two classifications: spermatozoa with high mitochondrial respiratory activity or high membrane potential (Orange fluorescence) and with low mitochondrial respiratory activity or nomembrane potential (Green fluorescence). 2.7. Statistical analysis The comparisons of sperm quality (motility, membrane integrity and mitochondrial membrane potential) among osmolalities and among epididymal regions as well as the effects of single exposure and return to isotonic conditions were performed using mixed statistical models procedure of the SAS® 9.3 (Proc Mixed, SAS® 9.3, Cary, NC, USA) after checking that the residuals had a normal or close to normal distribution. Pairwise differences were tested by multiple comparisons with Tukey’s range test. The level of significance was set at P ≤ 0.05. 3. Results 3.1. Effect of exposure to different osmolality There was a significant effect of both osmolality and region on sperm motility and an interaction between the two factors (P < 0.001). In the overall comparison with all regions combined the highest sperm motility was observed in the 300 mOsm medium (P < 0.001). Overall motility was significantly higher at 600 mOsm compared to 900 or 1200 mOsm (P < 0.05) but did not differ significantly between 600 and 75 mOsm (P = 0.67). At 300 mOsm spermatozoa from cauda region showed a significantly higher percentage of sperm motility than those from caput region (P < 0.0001) and corpus spermatozoa had significantly higher motility 3
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than spermatozoa from the caput (P < 0.001). Spermatozoa from cauda completely lost their motility at osmolality 900 mOsm and higher, while caput and corpus spermatozoa lost their motility at osmolality 1200 mOsm (Fig. 1A). There was no overall effect of region on the percentage of spermatozoa with intact membranes (P = 0.20) while osmolarity had an effect (P < 0.0001). Spermatozoa exposed to 900 mOsm had a significantly higher percentage of intact sperm membranes compared to 75 and 1200 mOsm (P < 0.05) while other treatments did not differ significantly. Feline epididymal sperm membranes appeared to be highly tolerant to hypo- and hypertonic stress with percentages of spermatozoa with intact membranes ranging from 53% to 69%. (Fig. 1B). 3.2. Effect of returning to isotonic condition after exposure to hypo- and hypertonic solutions Spermatozoa from corpus and cauda which were pre-exposed to isotonic solution (300 mOsm) demonstrated a significant reduction in the percentage of motile spermatozoa after adding 1:1 300 mOsm medium (P < 0.01) (Fig. 1a), while the spermatozoa pre-exposed to 75, 600, 900 and 1200 mOsm solution did not show a significant reduction of motile spermatozoa after being returned to isotonic conditions. In contrast, spermatozoa from the cauda pre-exposed to 75 mOsm showed an increase in motility after being returned to isotonic conditions (P = 0.0015). Returning spermatozoa to isotonic conditions after exposure to 900 or 1200 mOsm resulted in significant membrane damage in spermatozoa from any of the regions while membranes of spermatozoa exposed to 75–600 mOsm where not significantly affected (Fig. 1b). Moreover, comparing between regions of epididymis, spermatozoa from cauda showed significantly more severe membrane damage than caput or corpus spermatozoa after being returned to isotonic conditions after pre-exposure to 900 mOsm (P < 0.01) and significantly more severe than spermatozoa from the caput after being pre-exposed to 1200 mOsm (P < 0.05) (Fig. 1b). There was an effect of both region (P < 0.01) and osmolarity (P < 0.0001) on the percentage of spermaotozoa with hight MMP as well as an interaction between the two factors (p < 0.0001). The corpus and cauda regions contained higher overall percentage of spermatozoa with high MMP compared to the caput region (P < 0.05) (Fig. 2A, B). Spermatozoa from all regions that were preexposed to 900- or 1200 mOsm had lower (P < 0.05) proportion of spermatozoa with high MMP than spermatozoa pre-exposed to 75-, 300- or 600 mOsm (Fig. 2A,B). 4. Discussion This study is the first to investigate the osmotic tolerance of feline epididymal spermatozoa by imitating the conditions that spermatozoa will be exposed during the freezing-thawing process. The experiment was conducted at room temperature and the effect of cryoprotectants was not included in this study. According to the standard protocol for freezing and thawing, firstly, spermatozoa are diluted in tris egg-york extender I which contains 3% (v:v) glycerol (865 mOsm) then diluted in tris egg-york extender II (1495
Fig. 1. Effects of osmotic change on feline epididymal spermatozoa from different regions of epididymis (caput, corpus and cauda): (A) motility after exposure to different osmotic conditions (a) motility after abrupt exposure to different osmotic conditions and return to isotonic condition (B) intact membrane after abrupt exposure to different osmotic conditions (b) intact membrane after abrupt exposure to different osmotic conditions and return to isotonic condition. Results are presented as mean ± standard error of mean (SEM). Different letters indicate significant difference between osmolalities across regions (P < 0.05). * indicate that there is a significant difference between regions within osmolality (P < 0.05).
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Fig. 2. Osmotic effect on mitochondrial membrane potential of feline epididymal spermatozoa from different regions of epididymis (caput, corpus and cauda) after exposure to hypo-and hyperosmotic media and return to isotonic conditions afterwards: (A) percent spermatozoa with high mitochondrial respiratory activity or high membrane potential (Orange fluorescence), (B) percent spermatozoa with low mitochondrial respiratory activity or no-membrane potential (Green fluorescence). Results are presented as mean ± standard error of mean (SEM). Different letters indicate significant difference between osmolalities across regions (P < 0.05). * indicate that there is a significant difference between regions within osmolality (P < 0.05).
mOsm). These will make the final osmolality that sperm will be stored to be around 1180 mOsm (Kunkitti et al., 2016b). In the thawing protocol, it is recommended to dilute with Tris buffered solution (324 mOsm) to reduce toxic effect of cryoprotectant and Equex STM paste (Chatdarong et al., 2010) that made the final osmolality to be around 914 mOsm. As the water freezes during the sperm freezing process electrolytes are concentrated in the fluids surrounding the spermatozoa leading to an increased osmolality in the sperm environment. At thawing this process is reversed. Altogether, feline caudal epididymal spermatozoa are exposed to two levels of hyper-osmotic solutions prior to their return to isotonicity during the freezing and thawing process. Therefore, in the present study the effect of osmotic stress of feline epididymal spermatozoa was investigated by exposure of spermatozoa to different osmolality before returning them to isotonic condition. The current results of sperm motility confirmed that the isotonic osmolality of 300 mOsm promoted the best motility. Spermatozoa from cauda region had a higher percentage of motile spermatozoa compared to corpus and caput regions. This finding consists with an earlier study in feline epididymal sperm (Kunkitti et al., 2015). The decrease in motility after returning spermatozoa from the cauda pre-exposed to 300 mOsm might be a time effect rather than an effect of osmolality as the osmolality in this treatment did not change. The feline epididymal spermatozoa are highly susceptible to osmotic stress as demonstrated by the dramatical decrease when exposed to the solutions at 75 mOsm and 600 mOsm and complete loss of motility at osmolality more than 900 mOsm. This supports the study of Pukazhenthi et al. (2000) in feline and is similar to the previous studies in the mouse (Agca et al., 2005), bovine (Guthrie et al., 2002) and equine (Pommer et al., 2002) in that the sperm motility markedly declined in response to small deviations from isotonicity. The osmotic tolerance of feline epididymal sperm motility might be only a small deviation from isotonicity. More investigation in the response of sperm motility to small deviations in osmolality from 300 mOsm might be needed. Interestingly, from our study an extensive damage of sperm plasma membrane was observed in spermatozoa from all epididymal regions pre-exposed to hypertonic solution of more than 600 mOsm and returned to isotonic condition (300 mOsm). However, the plasma membrane of feline epididymal sperm was more tolerant to the hypo-osmotic media than hypertonic condition demonstrated by the similar percentage of spermatozoa with intact membrane prior to and after exposure to75 mOsm. Comparison between our finding and a previous study on ejaculated feline spermatozoa which exhibited a marked decrease in membrane integrity when spermatozoa were exposed to hypotonic solution (0, 35, 75, and 150 mOsm)(Pukazhenthi et al., 2000), our discovery might indicate biophysical differences in the membrane between epididymal spermatozoa and ejaculated spermatozoa. The exposure of spermatozoa to 900- or 1200 mOsm solution and return to isotonic conditions resulted in at least 30% decline in the percentage of intact membrane. Our findings are consistent with a previous report by Pukazhenthi et al. (2000), the severe disruption of sperm membrane was observed when returning the sperm to isotonic condition in a single step after pre-exposed to osmolality greater than 600 mOsm. The results demonstrated that a rapid return of spermatozoa close to isotonic condition after spermatozoa was exposed to hypo- or hypertonic solution is more harmful for spermatozoa than non-return. Minimizing the effect of osmotic stress during the sperm cryopreservation process might need more investigation. For example, the negative effect might be reduced if spermatozoa are not returned to isotonic conditions or are returned in multiple steps. A previous study in feline ejaculate spermatozoa by Chatdarong et al. (2010) demonstrated the benefit of post-thaw dilution at a rate of 1:2 with Tris buffer contributing to the final osmolarity of more than 300 mOsm and resulted in improved sperm motility, membrane integrity and acrosome integrity. The optimal osmolality postthaw could be another point that might be interesting to investigate. Although, from our results, the osmolality range that feline epididymal sperm plasma membrane could tolerate is between 75 mOsm and 600 mOsm. Sperm mitochondrial membrane potential of spermatozoa has been reported to have a positive relation with fertility. Accordingly, sperm motility and fertility potential were high when mitochondrial membrane potential was high (Kasai et al., 2002). Therefore, assessment of mitochondrial membrane potential is useful to predict sperm fertility potential. The exposure of hypertonic stress affected not only sperm plasma membrane but also the MMP of feline epididymal spermatozoa. A significant reduction in the percentage of spermatozoa with high MMP was detected when spermatozoa were returned to isotonic condition after pre-exposure to 900- and 1200 mOsm and no extensive mitochondrial damage were observed at 75, 300 and 600 mOsm. From this result, the osmolality range that epididymal sperm are capable to preserve their mitochondrial function, as demonstrated by a high MMP, is between 75 mOsm to 600 mOsm. 5
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The osmosensitivity of epididymal spermatozoa has also been studied in the kangaroo (McClean et al., 2006). Comparison between caput and cauda spermatozoa showed that caput spermatozoa were more tolerant to hypotonic solution and hypertonic solution (high glycerol concentration) than cauda spermatozoa. This is similar to our findings as membranes of spermatozoa from the cauda were more sensitive to damage when they were returned to isotonic conditions after being pre-exposed to hypertonic solutions of 900 mOsm or higher compared to caput and corpus spermatozoa. As spermatozoa mature in the epididymis of the ram they acquire susceptibility to cold shock (White, 1993). It is therefore likely that sperm membrane modifications during feline epididymal maturation results in decreased ability to tolerate changes in the sperm environment as has been shown in other species. In conclusion, from the present study each of the sperm parameters (motility, membrane integrity and MMP) showed differences in range of osmotic tolerances. The safety margin of low and high osmolality of feline epididymal sperm might depend on which parameter that you intend to study or use. Our results showed that returning to isotonic condition in a single step after pre-exposure to osmolality greater than 600 mOsm can cause severe damage on sperm membrane and MMP compared to the non-returning group. Spermatozoa from all epididymal regions have a similar tendency in responding to osmotic stress, although cauda epididymis was more sensitive to osmolality changes compared to corpus and caput. Future studies on longevity of the feline epididymal sperm in hypertonic conditions or studies on slow-step returning to isotonic condition after pre-exposure to hypertonic solution might give us more information about the osmotic resistance of feline spermatozoa. Careful consideration and proper protocols for handling of feline spermatozoa and osmotic conditions are required to achieve reliable results and minimise damage. Conflict of interest None of the authors have any conflict of interest to declare. Acknowledgments We would like to sincerely thank The Veterinary Public Health Division of The Bangkok Metropolitan Administration, Bangkok, Thailand for providing cat’s epididymis samples, the Research Unit of Obstetrics and Reproduction and Center of Emerging and Reemerging Infectious Diseases in Animals, Faculty of Veterinary Science, Chulalongkorn University for providing lab facilities and flow cytometry facility. References Agca, Y., Mullen, S., Liu, J., Johnson-Ward, J., Gould, K., Chan, A., Critser, J., 2005. Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa. Cryobiology 51, 1–14. Ball, B.A., Vo, A., 2001. 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