Reprod Dom Anim 50, 378–385 (2015); doi: 10.1111/rda.12496 ISSN 0936–6768
Determination of Appropriate Cryopreservation Protocols for Epididymal Cat Spermatozoa K Buranaamnuay Reproductive Biology Research Group, Institute of Molecular Biosciences (MB), Mahidol University, Nakhon Pathom, Thailand
Contents Effects of Equex and glycerol additions and sample dilution step on frozen–thawed epididymal cat spermatozoa were investigated. The epididymal sperm pellets were resuspended in extenders using one- (groups III and IV) or two- (groups I, II, V and VI) step dilution. For one-step dilution, the pellets were resuspended in plain egg yolk-Tris medium (EYT) + 5% glycerol with (IV)/without (III) 0.5% Equex and cooled (4°C, 1 h). For two-step dilution, the pellets were resuspended in EYT (I and V) and in EYT + 3% glycerol (II and VI), cooled and further diluted with EYT + 10% glycerol with (I)/without (V) 1% Equex and with EYT + 7% glycerol with (II)/without (VI) 1% Equex. Immediately after freeze–thawing, no differences (p > 0.05) were found in the motility, viability and membrane integrity (HOST) among the groups except the lowest HOST in IV (p = 0.005 to p = 0.04). The acrosome integrity (FITC) in group I was comparable to that in group II (p > 0.05) and was higher than the rest (p < 0.001 to p = 0.02). At 2 h after thawing, the motility, viability and HOST were comparable among the groups (p > 0.05) except the lower percentages of viability in III (p = 0.008 to p = 0.3) and of HOST in IV (p = 0.005 to p = 0.2). Two-step dilutions with Equex (I, II) were more beneficial for the FITC at 2 h than without Equex (V) (p = 0.005 and p = 0.02) and than one-step dilutions (III, IV) (p < 0.001 to p = 0.02). In conclusion, epididymal cat sperm quality after freeze–thawing could be improved when Equex was added and two-step dilution was performed during freezing. The extenders prepared for the first step of dilution could be with (3%) or without (0%) glycerol.
Introduction In general, spermatozoa can be collected from live male domestic cats (herein called ‘cats’) using artificial vagina (AV) (Tsutsui et al. 2000), electroejaculator under general anaesthesia (EE) (Baran et al. 2004) or urinary catheter after pharmacological sedation (urethral catheterization) (Zambelli et al. 2008). However, in case of sudden death of a sperm donor, it is impossible to conduct the above mentioned methods. Consequently, collection of spermatozoa from the epididymides becomes ‘a method of choice’. Nowadays, study on cryopreservation of epididymal cat spermatozoa is of interest, in part because researchers want to use domestic cats as a model of wildcats and other animals that are in the same biological family (Felidae) such as tigers, lions and leopards which are close to extinction (Martins et al. 2009). According to previous reports, there have been a number of sperm cryopreservation protocols applied in cats, with different outcomes
demonstrated after thawing (Tsutsui et al. 2000; Axner et al. 2004; Baran et al. 2004; Martins et al. 2009; Jimenez et al. 2013; Villaverde et al. 2013). In many of these protocols, a water-soluble anionic detergent sodium dodecyl sulphate (SDS), which is an active compound of commercial preparations named Equex STM (Equex; Nova Chemical Sales Inc., Scituate, MA, USA) and Orvus ES Paste (OEP), was added to spermfreezing extenders; the sample dilution during freezing process was performed in two steps, and glycerol was used as a permeating cryoprotectant (CPA). Equex was incorporated in freezing extenders used for both epididymal and ejaculated cat sperm cryopreservation, and after thawing, the improved in vitro sperm quality was verified (Axner et al. 2004; Zambelli et al. 2010). The inclusion of Equex helps to disperse the egg yolk contained in freezing extenders and thus enhances cryoprotective effect of lipoproteins in egg yolk on the sperm membranes (Ponglowhapan and Chatdarong 2008). Although the advantages of Equex have already been recognized, there may be some other cofactors influencing its action and efficacy which should not be overlooked. Therefore, this study was conducted partly to determine the cryopreservation conditions under which effect of Equex could be optimized. The two-step dilution with freezing extenders is ordinarily conducted in cat sperm cryopreservation. The first step of dilution is performed at room temperature (i.e. 20–30°C depended on a place of doing an experiment), and the second step is undertaken at a lower temperature (i.e. 4–5°C) (Chatdarong et al. 2010; Jimenez et al. 2013). The second dilution with the extenders containing glycerol is usually conducted at a very short time before freezing, to minimize a detrimental effect of high glycerol concentrations on the spermatozoa during the equilibration period. On the contrary, sperm cryopreservation protocols for some other mammalian species such as bulls especially in Europe (Chaveiro et al. 2006), stallions (Papa et al. 2008) and dogs (Szasz et al. 2000) are generally accomplished with one-step dilution at room temperature, and the acceptable post-thawed sperm quality is demonstrated. This thereby raises the question of whether it is possible for cat spermatozoa to be frozen with the onestep dilution protocols, which in common sense are more convenient than two-step dilution. Although glycerol is known to be toxic to sperm cells particularly at high levels, this substance is still being the © 2015 Blackwell Verlag GmbH
Freezing Protocols for Epididymal Cat Sperm
most frequently used CPA for sperm cryopreservation in many species including cat spermatozoa (Barbas and Mascarenhas 2009). In cat sperm cryopreservation, glycerol has been incorporated in freezing extenders at final concentrations between 3 and 8% (Hay and Goodrowe 1993; Nelson et al. 1999), being 4–5% glycerol the most commonly used (Thuwanut et al. 2010; Villaverde et al. 2013). To achieve 5% in final concentration, according to previous reports, some researchers included glycerol in both steps of dilution (i.e. 3% and 7% glycerol, respectively) (Axner et al. 2004; Chatdarong et al. 2010), whereas another group of researchers added this CPA to freezing extenders used for the second step of dilution only (i.e. extenders for the first and second steps containing 0% and 10% glycerol, respectively) (Villaverde et al. 2013). This study was undertaken to clarify whether there are any differences between these two freezing protocols (3% and 7% glycerol vs 0% and 10% glycerol) in the aspect of in vitro quality of frozen–thawed epididymal cat spermatozoa. Altogether, the objectives of conducting this experiment were to appraise effects of a supplement of Equex (Equex-added vs Equex-free extenders), the number of sample dilution steps during freezing process (one- vs two-step dilutions) and inclusion of glycerol in freezing extenders (3% and 7% glycerol vs 0% and 10% glycerol) on the quality of frozen–thawed epididymal cat spermatozoa, so as to find the appropriate sperm cryopreservation protocol for this species.
Materials and Methods Unless otherwise stated, all media components used in this study were obtained from Sigma-Aldrich (St Louis, MO, USA). Animals A total of 126 mature male cats of mixed breed (1– 5 years old and 2–5 kg live weight) brought to the Veterinary Public Health Division of the Bangkok Metropolitan Administration, Bangkok, Thailand, for orchiectomy during August to October 2013 were included in the study. During surgical process, the removed testicles and epididymides of many cats were pooled and placed in a warm (37°C) sterile 0.9% NaCl solution. The samples were sent to the laboratory within 2 h after surgery. The number of cats in each replicate was between 7 and 24, and 10 total replicates were conducted. Epididymal sperm collection In the laboratory, the testicles and epididymides were further washed (2–3 times) with a warm sterile 0.9% NaCl solution and transferred to a Petri dish (100 9 15 mm; SCHOTT GLAS, Mainz, Czech Republic). Three to five millilitres of warm Tris-glucose-citrate © 2015 Blackwell Verlag GmbH
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solution [a Tris buffer; containing 3.025 g Tris(hydroxymethyl)aminomethane, 1.4% (w/v) citric acid, 0.8% (w/v) glucose, 0.06% Na-benzylpenicillin and 0.1% (w/ v) streptomycin sulphate in distilled water] was added to the dish. The cauda epididymides were carefully separated from the testicles; the blood vessels and connective tissue were removed. The remaining epididymides were cut into small pieces using scissors. The medium containing cut epididymides was transferred to a beaker (Duranâ; DURAN Group GmbH, Wertheim, Germany), and approximately 30 ml of warm Tris buffer was additionally added. The suspension was incubated at 37°C in water bath for 15 min to allow epididymal spermatozoa swimming up and epididymal tissues precipitating. The upper fluid containing epididymal spermatozoa was transferred to another warm beaker, gently and thoroughly mixed and allocated to 7 aliquots: 6 equal aliquots in 15-ml centrifuge tubes (Corningâ; Corning Incorporated Life Sciences, Tewksbury, MA, USA) to be frozen (groups I to VI) and the 7th aliquot (2 ml) to be evaluated for the fresh sperm quality, which this process was undertaken after sperm isolation and addition with trace amount of Tris buffer. In all 7 aliquots, epididymal spermatozoa were isolated from the diluents by centrifuging at 500 9 g for 10 min. Sperm cryopreservation The sperm pellets remaining after centrifugation were resuspended at room temperature (25°C) in egg yolkTris-based extenders (Rota et al. 1997; Table 1), using one- (in groups III and IV) or two- (in groups I, II, V and VI) step dilutions. In groups I and V, the sperm pellets were resuspended in plain egg yolk-Tris extender [EYT; Tris buffer plus 20% (v/v) egg yolk]. In groups II and VI, EYT plus 3% glycerol was added to the pellets. The sperm concentration after first-step resuspension in groups I, II, V and VI was 50 9 106 spermatozoa/ml. In group III, the pellet remaining after centrifugation was resuspended in EYT containing 5% glycerol; instead, in group IV, EYT with 5% glycerol and 0.5% Equex was added. The final concentration of processed spermatozoa in groups III and IV was 25 9 106/ml and no further dilution was conducted in these two groups. The thoroughly mixed samples in every group were gradually cooled down from room temperature to 4°C. Following 1 h of cool incubation, the sample in groups I, II, V and VI was further diluted 1 : 1 (v/v) with EYT containing 10% glycerol and 1% Equex (for group I), EYT plus 7% glycerol and 1% Equex (for group II), EYT and 10% glycerol (for group V), and EYT plus 7% glycerol (for group VI). At this step, the final concentrations of spermatozoa (25 9 106/ml) and glycerol (5%) were comparable among the six groups, and only in the Equex-added samples, final concentration of Equex (0.5%) was also the same. The processed samples were maintained at 4°C for 10 min before being loaded in a 0.25 ml plastic straw (Minitub
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Table 1. Composition of egg yolk-Tris-based extenders prepared for cryopreservation of epididymal cat spermatozoa in groups I–VI Egg yolk-Tris-based extenders
Tris (g) Citric acid (g) Glucose (g) Glycerol (%, v/v) Egg yolk (%, v/v) Penicillin (g) Streptomycin (g) Equex (%, v/v) Distilled water added to (ml) Used in group(s)
(Gly, Equex)*
EYT+3% Gly
EYT+5% Gly
EYT+5% Gly, +0.5% Equex
3.025 1.4 0.8 – 20 0.06 0.1 – 100
3.025 1.4 0.8 3 20 0.06 0.1 – 100
3.025 1.4 0.8 5 20 0.06 0.1 – 100
3.025 1.4 0.8 5 20 0.06 0.1 0.5 100
I, V (1st step)
II, VI (1st step)
III
IV
EYT+7% Gly 3.025 1.4 0.8 7 20 0.06 0.1 – 100 VI (2nd step)
EYT+7% Gly, +1% Equex
EYT+10% Gly
EYT+10% Gly, +1% Equex
3.025 1.4 0.8 7 20 0.06 0.1 1 100
3.025 1.4 0.8 10 20 0.06 0.1 – 100
3.025 1.4 0.8 10 20 0.06 0.1 1 100
II (2nd step)
V (2nd step)
I (2nd step)
Gly, glycerol; Equex, Equex STM. * This extender was herein called ‘plain egg yolk-Tris extender (EYT)’.
GmbH, Tiefenbach, Germany) and frozen. The freezing procedure was performed in a Styrofoam box by placing the filled straws horizontally 6 cm over liquid nitrogen (LN2) vapours for 20 min and subsequently immersed into LN2 and stored (Martins et al. 2009). After thawing (70°C for 6 s), the samples were transferred to a 15-ml centrifuge tube and diluted 1 : 1 (v/v) with a Tris buffer. The extended thawed samples were incubated in a warm water bath during 0- (15 min) and 2-h evaluation. The sperm cryopreservation protocols are summarized in a flow chart and shown in Fig. 1. Sperm quality evaluations The quality of spermatozoa was evaluated before and after freeze–thawing. The sperm concentration was evaluated with a haemocytometer (Hausser Scientific, Horsham, PA, USA) after extending aliquot of the sample in 3% NaCl (1 : 200, v/v). The percentage of total sperm motility was evaluated subjectively by only one experienced scientist throughout the study. An aliquot (5 ll) of the sample was dropped onto a pre-warmed (37°C) glass slide, covered with a pre-warmed cover slip and thereafter assessed (4– 5 fields/slide) with a light microscope (Helmut Hund GmbH, Wetzlar-Nauborn, Germany) at 4009 magnification. The sperm viability was studied in smears stained with an eosin–nigrosin dye (Way et al. 1995). Briefly, 10 ll of the sample placed on a glass slide was mixed with a drop of the dye. Few amount of the stained sample was taken to another clean slide and smeared. The dried smear was evaluated under a bright-field microscope (10009); a total of 200 spermatozoa per smear were observed. Unstained bright-head spermatozoa were counted as viable, whereas spermatozoa with red/pink head were classified as dead cells.
The integrity of the sperm plasma membrane was appraised through the hypo-osmotic swelling test (HOST). The HOST predicts membrane integrity by determining the ability of the sperm membrane to maintain osmotic equilibrium between intracellular and extracellular fluids. Influx of the fluid due to hypoosmotic stress causes the sperm tail to swell and coil. The method of Perez-Llano et al. (2001) was partially modified and described as follows: 50 ml of the sample was mixed with 200 ll of hypo- (75 mOsm/kg) or iso- (300 mOsm/kg) osmotic solutions, in a 1.5-ml microtube (Eppendorfâ, Sigma-Aldrich). The osmotic solutions were composed of fructose and Na-citrate in distilled water; their final osmolalities were checked with freezing point depression (Osmomat 030-3P, Gonotec GmbH, Berlin, Germany). Following 15 min of 37°C incubation, the reactions were stopped by adding 50 ll of the hypo/iso-osmotic solution plus 5% formaldehyde (Merck, Darmstadt, Germany) to the mixtures. Upon evaluation, an aliquot of well-mixed sample was dropped on a slide and covered with a slip. Two hundred total spermatozoa per slide were observed under a light microscope (4009), and the number of spermatozoa with coiled tail was recorded. The proportion of coiled-tail spermatozoa in a hypo-osmotic sample was subtracted by the value of iso-osmotic condition to determine the percentage of spermatozoa having intact plasma membranes. The acrosomal status of spermatozoa was assessed with a dual fluorescent staining technique (Axner et al. 2004). Briefly, a 10 ll aliquot of the sperm sample was smeared on a microscope slide, air-dried and membrane-permeabilized with 95% ethyl alcohol for 30 s. Thereafter, 100 ll of fluorescein isothiocyanate conjugated with peanut agglutinin (FITC-PNA) (100 lg/ml in phosphate-buffered saline; PBS) was mixed with 5 ll of propidium iodide (PI) (340 lM in PBS; Molecular Probes Inc., Eugene, OR, USA). Twenty microlitres of © 2015 Blackwell Verlag GmbH
Freezing Protocols for Epididymal Cat Sperm
381 Testicles from a total of 126 cats (n = 10) Cauda epididymides + Tris buffer Cut into small pieces, incubated at 37°C for 15 min Remaining fluid Allocated to 6 equal aliquots (I-VI), centrifuged at 500Хg for 10 min
I) pellets
+ EYT
II) pellets
+(EYT+3%Gly)
(Gr. I and II; 50x106 spz/mL)
III) pellets
IV) pellets
V) pellets
+(EYT+5%Gly) +(EYT+5%Gly, 0.5%Equex) (Gr. III and IV; 25x106 spz/mL)
+ EYT
VI) pellets
+(EYT+3%Gly)
(Gr. V and VI; 50x106 spz/mL)
Cooled to 4°C, 1 h
+(EYT+10%Gly, + (EYT+7%Gly, 1%Equex) 1%Equex) (Gr. I and II; 25x106 spz/mL)
-
-
+(EYT+10%Gly) +(EYT+7%Gly) (Gr. V and VI; 25x106 spz/mL)
Kept at 4°C, 10 min Loaded in 0.25 mL straws Frozen in a Styrofoam box Thawed at 70°C, 6 s Fig. 1. A summary of the cryopreservation protocols for epididymal cat spermatozoa conducted in the present study (EYT; plain egg yolk-Tris extender, Gly; glycerol, spz; spermatozoa)
the solution was spread over the dried smeared slide. The slide was kept in a dark humidified chamber at 4°C for 30 min. Subsequently, the slide was gently rinsed with cold distilled water. The stained slide was always kept in the dark at 4°C until evaluation was undertaken. At the time of evaluation, 200 spermatozoa per slide were observed using epifluorescent microscopy (Axioskop, Carl Zeiss Jena GmbH, Jena, Germany). Two categories of spermatozoa could be classified according to their staining patterns. Spermatozoa with acrosome intact were judged when the spermatozoa exhibiting bright green fluorescence over the entire acrosomal region, and acrosome-reacting/reacted spermatozoa were considered when the cells performing disrupted green or red fluorescence over the acrosomal area. Only the percentage of acrosome-intact spermatozoa was included in the results. © 2015 Blackwell Verlag GmbH
Diluted 1:1 (v/v) with a Tris buffer Incubated at 37°C Sperm quality evaluations
Statistical analysis A pool of sperm sample (from 7 to 24 cats) was used for each replicate, and ten replicates were performed. Analysis of variance (ANOVA) of a software package named SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. When significant differences were shown, the least significant difference (LSD) test was applied to compare statistical differences among groups (I–VI) and time (0 and 2 h). Percentages of motility, viability, plasma membrane integrity and acrosome integrity are presented as mean SEM, and p < 0.05 was considered statistically significant.
Results Based on the data of 10 replicates, cat spermatozoa retrieved from the cauda epididymides had motility of
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61.5 3.9%, viability of 79.2 2.5%, plasma membrane integrity of 82.3 2.2% and acrosome integrity of 71.7 3.0%. Almost all in vitro characteristics of epididymal cat spermatozoa appraised in the present experiment were significantly affected by the sperm cryopreservation protocol (I–VI) and time of post-thawed incubation (0 and 2 h) (p < 0.001 to p < 0.01). At 15 min after thawing (0 h incubation), the motility and viability of spermatozoa frozen with protocols I–VI were not significantly different from one another (p > 0.05). The integrity of sperm plasma membrane detected by HOST was also not significantly different among the groups (p > 0.05) excluding the sample frozen with protocol IV in which the lower percentage (59.9 2.5%) was shown (p = 0.005 to p = 0.04). The proportion of acrosomeintact spermatozoa demonstrated in group I was similar to that in group II (p > 0.05) and was significantly higher than those in the rest four groups (p < 0.001 to p = 0.02). Among the rest four groups, the percentage of spermatozoa with acrosome intact was the lowest in group III (25.1 2.2%); this value was significantly lower compared to those in groups IV (p = 0.004) and VI (p = 0.01). After incubation for 2 h, the percentages of motile, viable and membrane-intact (HOST) spermatozoa were declined to comparable levels among the groups (p > 0.05). These were exceptions for the viability of spermatozoa in group III and membrane integrity of spermatozoa in group IV, where the poorest percentages were found (Table 2). The less viable spermatozoa in group III (41.1 5.5%) differed significantly from the viability of spermatozoa in groups I, II and V (p = 0.008 to p = 0.02). The membrane-intact spermatozoa in group IV (55.6 2.2%) was inferior significantly to
the values in groups III, V and VI (p = 0.005 to p = 0.03). There was no significant difference in the percentage of acrosome-intact spermatozoa frozen with protocols I and II during the 2 h incubation (p > 0.05). These two values were significantly greater than those demonstrated in groups III, IV and V (p < 0.001 to p = 0.02). An equal percentage of acrosome-intact spermatozoa in groups IV and V (i.e. 23.3 1.6%) did not significantly differ from the value in group VI (25.1 1.4%, p > 0.05), but it was significantly higher compared with 17.5 1.4% reported in group III (p = 0.02). Comparing between incubation periods within each cryopreservation protocols, the quality of frozen– thawed spermatozoa in all six groups decreased after incubation for 2 h. Nevertheless, the significant reductions were only observed in the motility of spermatozoa in group I (p = 0.007), viability of spermatozoa in groups III (p < 0.001) and IV (p = 0.017) and acrosome integrity of spermatozoa in every group (p < 0.001 to p = 0.01).
Discussion To determine the appropriate cryopreservation protocol for epididymal cat spermatozoa, effects of adding Equex, diluting the samples in one or two steps and including glycerol in the freezing extenders on the sperm quality were investigated. It was found that egg yolkTris-based extenders containing Equex added to the processed spermatozoa just for 10 min before freezing (groups I and II) were beneficial for the percentage of acrosome-intact spermatozoa both immediately and 2 h after thawing, compared to the extenders without Equex (groups V and VI). These results are in agreement with
Table 2. The in vitro quality of post-thawed epididymal cat spermatozoa, at 0 and 2 h of 37°C incubation, frozen in egg yolk-Tris-based extenders using six different freezing protocols (n = 10) Sperm parameters (%) Incubation time 0 h (15 min)
2h
Experimental groupa I II III IV V VI I II III IV V VI
Motility 20.0 17.0 15.5 15.5 16.5 12.5 1.5 2.0 4.0 7.5 2.5 3.5
3.11 3.5 3.5 4.4 3.2 3.7 0.82 1.1 1.5 2.3 1.3 1.3
Viability 62.6 61.0 57.4 58.8 58.9 56.3 56.4 54.0 41.1 47.2 53.9 50.0
2.2 2.8 3.21 2.21 3.0 3.0 3.2a 3.3a 5.5b,2 4.4a,b,2 3.2a 3.5a,b
HOSTb 67.7 70.0 67.7 59.9 70.9 69.3 60.8 63.1 64.6 55.6 67.2 65.9
3.1a 1.7a 2.8a 2.5b 3.3a 2.3a 3.1a,b 2.7a,b 2.6a 2.2b 3.4a 2.8a
FITCc 43.6 42.0 25.1 35.5 32.0 33.9 30.3 29.1 17.5 23.3 23.3 25.1
3.0a,1 1.5a,b,1 2.2d,1 3.6b,c,1 2.0c,d,1 1.5c,1 1.6a,2 2.4a,b,2 1.4d,2 1.6c,2 1.6c,2 1.4b,c,2
Values with different superscripts indicate significant difference within columns [a, b, c, d comparing among groups (I–VI) within each incubation times; 1, 2 comparing between incubation times (0 and 2 h) within each groups, p < 0.05]. Values without any superscript mean no significant difference within columns. a I = 2-step dilution with 0% and 10% glycerol plus 1% Equex; II = 2-step dilution with 3% and 7% glycerol plus 1% Equex; III = 1-step dilution with 5% glycerol; IV = 1-step dilution with 5% glycerol plus 0.5% Equex; V = 2-step dilution with 0% and 10% glycerol; VI = 2-step dilution with 3% and 7% glycerol. b The plasma membrane integrity of spermatozoa detected by the hypo-osmotic swelling test (HOST). c The acrosome integrity of spermatozoa assessed with FITC-PNA/PI staining (FITC).
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Freezing Protocols for Epididymal Cat Sperm
previous studies conducted in the spermatozoa of many species such as boars (Buranaamnuay et al. 2009), dogs (Rota et al. 1997; Pena and Linde-Forsberg 2000) and even of cats (Axner et al. 2004). In boars, it has been found that the motility, viability and acrosome morphology of thawed spermatozoa were improved when Equex was included in a freezing extender (Buranaamnuay et al. 2009). Similarly, addition of Equex to the freezing medium significantly increased the membrane integrity, motility and longevity of the frozen–thawed dog spermatozoa (Rota et al. 1997; Pena and LindeForsberg 2000). In cats, Axner et al. (2004) found that Equex had a positive effect on the percentage of intact acrosomes immediately after thawing but did not have any effect on the percentage of intact sperm membranes and the motility evaluated at the same time. In addition, the authors reported that reductions of the motility and membrane integrity of cat spermatozoa during the 6-h post-thawed incubation were more accelerated in Equex-added samples than in Equex-free counterparts (Axner et al. 2004). The present findings confirm the results of Axner et al. (2004) that Equex improved the percentage of acrosome-intact spermatozoa but did not improve the percentages of plasma membrane-intact and motile spermatozoa, as well as it exacerbated a decrease in motility during 2 h of post-thawed incubation, as observed in group I. According to the results of the present and Axner et al. (2004) studies, it thereby seems that Equex has a cryoprotective effect on the acrosomal membranes but has an adverse effect on the motility of epididymal cat spermatozoa during warm incubation in vitro. The present study additionally found that among Equex-added samples (groups I, II and IV), positive effects of this compound on the thawed spermatozoa were obvious in the samples processed with two-step dilution (i.e. shorter exposure to Equex before freezing); this is similar to what has been observed in dog spermatozoa (Pena and Linde-Forsberg 2000). These findings led us to believe that the longer the exposure to Equex before freezing, the more the toxic to spermatozoa after thawing. It has been suggested that prolonged exposure to Equex or Equex-treated egg yolk lipoproteins rendered excessive fluidity to the sperm membranes, which would indicate that the beneficial effect of Equex is partly depended on an exposure period (Pena and Linde-Forsberg 2000). Even though the effect of Equex on in vitro sperm quality has been well recognized, the influence of this substance on the life span of spermatozoa in the female genital tract after artificial insemination (i.e. in vivo sperm quality) has not been extensively studied and thus remains to be investigated further. However, it is supposed that spermatozoa will leave the freezing extender after being deposited in the female tract; consequently, they may no longer be positively/negatively affected by the presence of Equex (Axner et al. 2004). Comparing between one- and two-step dilutions, it was investigated that when Equex was present, the membrane integrity (HOST) and the acrosome integrity © 2015 Blackwell Verlag GmbH
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(FITC) of spermatozoa evaluated immediately after thawing (0 h) were likely to be (i.e. FITC between groups II and IV) or significantly higher in the samples diluted twice, compared to those diluted once. The advantages of two-step dilution on the frozen–thawed sperm quality were still found, at least in one parameter assessed, even after incubation for 2 h. Similar phenomena were also observed in the Equex-free samples that two-step dilution resulted in comparable (i.e. FITC between groups III and V at 0 h) or significantly higher percentages of intact acrosomes compared with one-step dilution both at 0 and 2 h of post-thawed incubation. Based on the concordant data between the Equex-added and Equex-free samples, it is likely that glycerol per se had some toxic effect on epididymal cat spermatozoa only in condition that relatively high concentration (5% glycerol) together with long exposure period (one-step dilution) was applied in the cryopreservation protocol. The present observations, however, did not totally agree with a previous report in cat spermatozoa conducted by Vick et al. (2012), where the percentage of intact acrosomes evaluated after thawing was not significantly affected by temperature (i.e. room temperature vs 5°C) of glycerol addition during the cryopreservation process. The discrepancy between this and previous findings might be partly related to differences in the source of spermatozoa (i.e. epididymal spermatozoa are different from ejaculated spermatozoa in the susceptibility to cold shock) and/or in the sperm cryopreservation protocol. With regard to the cryopreservation protocol, Vick et al. (2012) used the TEST yolk medium as a freezing extender and included 4% final glycerol concentration in the medium, whereas the present study selected the egg yolk-Tris-based extender supplemented with 5% glycerol as a freezing medium. It has been recognized that there are many factors influencing achievement of sperm cryopreservation, for example, extender composition, freezing and thawing methods, freezing and thawing rates, as well as size and volume of straw (Almlid and Johnson 1988); therefore, a comparison among different protocols is very complicated and offers less reliability (Baran et al. 2004). Effects of glycerol addition on post-thawed sperm quality have been investigated in several species including cats. Concerning concentration of glycerol, it has been demonstrated that in the range of 0–7% glycerol, the percentage of acrosome integrity of boar spermatozoa was negatively affected by increased glycerol concentrations (Almlid and Johnson 1988). Likewise, in cats, Villaverde et al. (2013) compared between 3, 5 and 7% glycerol and found that the percentage of dead spermatozoa presenting intact acrosomes was increased as glycerol concentration decreased. From the results of Villaverde et al. (2013) and the present study, it seems that a detrimental effect of high glycerol concentration on acrosome integrity presents in cat spermatozoa; this finding is consistent with the results reported in other species (Almlid and Johnson 1988; Fernandez-Santos et al. 2006). The negative effect of such compound on
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the acrosome could be explained by the fact that increased glycerol concentrations result in increased enzyme leakage from the acrosome of spermatozoa (Schmidt et al. 1974), which can be detected indirectly through reduced proportions of intact acrosomes after thawing. With regard to time of glycerol exposure before freezing (i.e. 10 min vs 1 h for the present study), this sole factor was not a reason for differences in the frozen–thawed sperm quality in this study. This suggestion was supported by earlier observations on boars’ (Almlid and Johnson 1988), bulls’ (Berndtson and Foote 1972) and dogs’ (Pena and Linde-Forsberg 2000) spermatozoa. In boars, it was found that glycerol exposure times of 0.5, 2, 5, 15 and 75 min before freezing did not significantly influence the sperm quality after thawing, indicating that glycerol is able to penetrate quickly into the spermatozoa and that equilibrium is reached within a very short time (
Determination of Appropriate Cryopreservation Protocols for Epididymal Cat Spermatozoa K Buranaamnuay Reproductive Biology Research Group, Institute of Molecular Biosciences (MB), Mahidol University, Nakhon Pathom, Thailand
Contents Effects of Equex and glycerol additions and sample dilution step on frozen–thawed epididymal cat spermatozoa were investigated. The epididymal sperm pellets were resuspended in extenders using one- (groups III and IV) or two- (groups I, II, V and VI) step dilution. For one-step dilution, the pellets were resuspended in plain egg yolk-Tris medium (EYT) + 5% glycerol with (IV)/without (III) 0.5% Equex and cooled (4°C, 1 h). For two-step dilution, the pellets were resuspended in EYT (I and V) and in EYT + 3% glycerol (II and VI), cooled and further diluted with EYT + 10% glycerol with (I)/without (V) 1% Equex and with EYT + 7% glycerol with (II)/without (VI) 1% Equex. Immediately after freeze–thawing, no differences (p > 0.05) were found in the motility, viability and membrane integrity (HOST) among the groups except the lowest HOST in IV (p = 0.005 to p = 0.04). The acrosome integrity (FITC) in group I was comparable to that in group II (p > 0.05) and was higher than the rest (p < 0.001 to p = 0.02). At 2 h after thawing, the motility, viability and HOST were comparable among the groups (p > 0.05) except the lower percentages of viability in III (p = 0.008 to p = 0.3) and of HOST in IV (p = 0.005 to p = 0.2). Two-step dilutions with Equex (I, II) were more beneficial for the FITC at 2 h than without Equex (V) (p = 0.005 and p = 0.02) and than one-step dilutions (III, IV) (p < 0.001 to p = 0.02). In conclusion, epididymal cat sperm quality after freeze–thawing could be improved when Equex was added and two-step dilution was performed during freezing. The extenders prepared for the first step of dilution could be with (3%) or without (0%) glycerol.
Introduction In general, spermatozoa can be collected from live male domestic cats (herein called ‘cats’) using artificial vagina (AV) (Tsutsui et al. 2000), electroejaculator under general anaesthesia (EE) (Baran et al. 2004) or urinary catheter after pharmacological sedation (urethral catheterization) (Zambelli et al. 2008). However, in case of sudden death of a sperm donor, it is impossible to conduct the above mentioned methods. Consequently, collection of spermatozoa from the epididymides becomes ‘a method of choice’. Nowadays, study on cryopreservation of epididymal cat spermatozoa is of interest, in part because researchers want to use domestic cats as a model of wildcats and other animals that are in the same biological family (Felidae) such as tigers, lions and leopards which are close to extinction (Martins et al. 2009). According to previous reports, there have been a number of sperm cryopreservation protocols applied in cats, with different outcomes
demonstrated after thawing (Tsutsui et al. 2000; Axner et al. 2004; Baran et al. 2004; Martins et al. 2009; Jimenez et al. 2013; Villaverde et al. 2013). In many of these protocols, a water-soluble anionic detergent sodium dodecyl sulphate (SDS), which is an active compound of commercial preparations named Equex STM (Equex; Nova Chemical Sales Inc., Scituate, MA, USA) and Orvus ES Paste (OEP), was added to spermfreezing extenders; the sample dilution during freezing process was performed in two steps, and glycerol was used as a permeating cryoprotectant (CPA). Equex was incorporated in freezing extenders used for both epididymal and ejaculated cat sperm cryopreservation, and after thawing, the improved in vitro sperm quality was verified (Axner et al. 2004; Zambelli et al. 2010). The inclusion of Equex helps to disperse the egg yolk contained in freezing extenders and thus enhances cryoprotective effect of lipoproteins in egg yolk on the sperm membranes (Ponglowhapan and Chatdarong 2008). Although the advantages of Equex have already been recognized, there may be some other cofactors influencing its action and efficacy which should not be overlooked. Therefore, this study was conducted partly to determine the cryopreservation conditions under which effect of Equex could be optimized. The two-step dilution with freezing extenders is ordinarily conducted in cat sperm cryopreservation. The first step of dilution is performed at room temperature (i.e. 20–30°C depended on a place of doing an experiment), and the second step is undertaken at a lower temperature (i.e. 4–5°C) (Chatdarong et al. 2010; Jimenez et al. 2013). The second dilution with the extenders containing glycerol is usually conducted at a very short time before freezing, to minimize a detrimental effect of high glycerol concentrations on the spermatozoa during the equilibration period. On the contrary, sperm cryopreservation protocols for some other mammalian species such as bulls especially in Europe (Chaveiro et al. 2006), stallions (Papa et al. 2008) and dogs (Szasz et al. 2000) are generally accomplished with one-step dilution at room temperature, and the acceptable post-thawed sperm quality is demonstrated. This thereby raises the question of whether it is possible for cat spermatozoa to be frozen with the onestep dilution protocols, which in common sense are more convenient than two-step dilution. Although glycerol is known to be toxic to sperm cells particularly at high levels, this substance is still being the © 2015 Blackwell Verlag GmbH
Freezing Protocols for Epididymal Cat Sperm
most frequently used CPA for sperm cryopreservation in many species including cat spermatozoa (Barbas and Mascarenhas 2009). In cat sperm cryopreservation, glycerol has been incorporated in freezing extenders at final concentrations between 3 and 8% (Hay and Goodrowe 1993; Nelson et al. 1999), being 4–5% glycerol the most commonly used (Thuwanut et al. 2010; Villaverde et al. 2013). To achieve 5% in final concentration, according to previous reports, some researchers included glycerol in both steps of dilution (i.e. 3% and 7% glycerol, respectively) (Axner et al. 2004; Chatdarong et al. 2010), whereas another group of researchers added this CPA to freezing extenders used for the second step of dilution only (i.e. extenders for the first and second steps containing 0% and 10% glycerol, respectively) (Villaverde et al. 2013). This study was undertaken to clarify whether there are any differences between these two freezing protocols (3% and 7% glycerol vs 0% and 10% glycerol) in the aspect of in vitro quality of frozen–thawed epididymal cat spermatozoa. Altogether, the objectives of conducting this experiment were to appraise effects of a supplement of Equex (Equex-added vs Equex-free extenders), the number of sample dilution steps during freezing process (one- vs two-step dilutions) and inclusion of glycerol in freezing extenders (3% and 7% glycerol vs 0% and 10% glycerol) on the quality of frozen–thawed epididymal cat spermatozoa, so as to find the appropriate sperm cryopreservation protocol for this species.
Materials and Methods Unless otherwise stated, all media components used in this study were obtained from Sigma-Aldrich (St Louis, MO, USA). Animals A total of 126 mature male cats of mixed breed (1– 5 years old and 2–5 kg live weight) brought to the Veterinary Public Health Division of the Bangkok Metropolitan Administration, Bangkok, Thailand, for orchiectomy during August to October 2013 were included in the study. During surgical process, the removed testicles and epididymides of many cats were pooled and placed in a warm (37°C) sterile 0.9% NaCl solution. The samples were sent to the laboratory within 2 h after surgery. The number of cats in each replicate was between 7 and 24, and 10 total replicates were conducted. Epididymal sperm collection In the laboratory, the testicles and epididymides were further washed (2–3 times) with a warm sterile 0.9% NaCl solution and transferred to a Petri dish (100 9 15 mm; SCHOTT GLAS, Mainz, Czech Republic). Three to five millilitres of warm Tris-glucose-citrate © 2015 Blackwell Verlag GmbH
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solution [a Tris buffer; containing 3.025 g Tris(hydroxymethyl)aminomethane, 1.4% (w/v) citric acid, 0.8% (w/v) glucose, 0.06% Na-benzylpenicillin and 0.1% (w/ v) streptomycin sulphate in distilled water] was added to the dish. The cauda epididymides were carefully separated from the testicles; the blood vessels and connective tissue were removed. The remaining epididymides were cut into small pieces using scissors. The medium containing cut epididymides was transferred to a beaker (Duranâ; DURAN Group GmbH, Wertheim, Germany), and approximately 30 ml of warm Tris buffer was additionally added. The suspension was incubated at 37°C in water bath for 15 min to allow epididymal spermatozoa swimming up and epididymal tissues precipitating. The upper fluid containing epididymal spermatozoa was transferred to another warm beaker, gently and thoroughly mixed and allocated to 7 aliquots: 6 equal aliquots in 15-ml centrifuge tubes (Corningâ; Corning Incorporated Life Sciences, Tewksbury, MA, USA) to be frozen (groups I to VI) and the 7th aliquot (2 ml) to be evaluated for the fresh sperm quality, which this process was undertaken after sperm isolation and addition with trace amount of Tris buffer. In all 7 aliquots, epididymal spermatozoa were isolated from the diluents by centrifuging at 500 9 g for 10 min. Sperm cryopreservation The sperm pellets remaining after centrifugation were resuspended at room temperature (25°C) in egg yolkTris-based extenders (Rota et al. 1997; Table 1), using one- (in groups III and IV) or two- (in groups I, II, V and VI) step dilutions. In groups I and V, the sperm pellets were resuspended in plain egg yolk-Tris extender [EYT; Tris buffer plus 20% (v/v) egg yolk]. In groups II and VI, EYT plus 3% glycerol was added to the pellets. The sperm concentration after first-step resuspension in groups I, II, V and VI was 50 9 106 spermatozoa/ml. In group III, the pellet remaining after centrifugation was resuspended in EYT containing 5% glycerol; instead, in group IV, EYT with 5% glycerol and 0.5% Equex was added. The final concentration of processed spermatozoa in groups III and IV was 25 9 106/ml and no further dilution was conducted in these two groups. The thoroughly mixed samples in every group were gradually cooled down from room temperature to 4°C. Following 1 h of cool incubation, the sample in groups I, II, V and VI was further diluted 1 : 1 (v/v) with EYT containing 10% glycerol and 1% Equex (for group I), EYT plus 7% glycerol and 1% Equex (for group II), EYT and 10% glycerol (for group V), and EYT plus 7% glycerol (for group VI). At this step, the final concentrations of spermatozoa (25 9 106/ml) and glycerol (5%) were comparable among the six groups, and only in the Equex-added samples, final concentration of Equex (0.5%) was also the same. The processed samples were maintained at 4°C for 10 min before being loaded in a 0.25 ml plastic straw (Minitub
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Table 1. Composition of egg yolk-Tris-based extenders prepared for cryopreservation of epididymal cat spermatozoa in groups I–VI Egg yolk-Tris-based extenders
Tris (g) Citric acid (g) Glucose (g) Glycerol (%, v/v) Egg yolk (%, v/v) Penicillin (g) Streptomycin (g) Equex (%, v/v) Distilled water added to (ml) Used in group(s)
(Gly, Equex)*
EYT+3% Gly
EYT+5% Gly
EYT+5% Gly, +0.5% Equex
3.025 1.4 0.8 – 20 0.06 0.1 – 100
3.025 1.4 0.8 3 20 0.06 0.1 – 100
3.025 1.4 0.8 5 20 0.06 0.1 – 100
3.025 1.4 0.8 5 20 0.06 0.1 0.5 100
I, V (1st step)
II, VI (1st step)
III
IV
EYT+7% Gly 3.025 1.4 0.8 7 20 0.06 0.1 – 100 VI (2nd step)
EYT+7% Gly, +1% Equex
EYT+10% Gly
EYT+10% Gly, +1% Equex
3.025 1.4 0.8 7 20 0.06 0.1 1 100
3.025 1.4 0.8 10 20 0.06 0.1 – 100
3.025 1.4 0.8 10 20 0.06 0.1 1 100
II (2nd step)
V (2nd step)
I (2nd step)
Gly, glycerol; Equex, Equex STM. * This extender was herein called ‘plain egg yolk-Tris extender (EYT)’.
GmbH, Tiefenbach, Germany) and frozen. The freezing procedure was performed in a Styrofoam box by placing the filled straws horizontally 6 cm over liquid nitrogen (LN2) vapours for 20 min and subsequently immersed into LN2 and stored (Martins et al. 2009). After thawing (70°C for 6 s), the samples were transferred to a 15-ml centrifuge tube and diluted 1 : 1 (v/v) with a Tris buffer. The extended thawed samples were incubated in a warm water bath during 0- (15 min) and 2-h evaluation. The sperm cryopreservation protocols are summarized in a flow chart and shown in Fig. 1. Sperm quality evaluations The quality of spermatozoa was evaluated before and after freeze–thawing. The sperm concentration was evaluated with a haemocytometer (Hausser Scientific, Horsham, PA, USA) after extending aliquot of the sample in 3% NaCl (1 : 200, v/v). The percentage of total sperm motility was evaluated subjectively by only one experienced scientist throughout the study. An aliquot (5 ll) of the sample was dropped onto a pre-warmed (37°C) glass slide, covered with a pre-warmed cover slip and thereafter assessed (4– 5 fields/slide) with a light microscope (Helmut Hund GmbH, Wetzlar-Nauborn, Germany) at 4009 magnification. The sperm viability was studied in smears stained with an eosin–nigrosin dye (Way et al. 1995). Briefly, 10 ll of the sample placed on a glass slide was mixed with a drop of the dye. Few amount of the stained sample was taken to another clean slide and smeared. The dried smear was evaluated under a bright-field microscope (10009); a total of 200 spermatozoa per smear were observed. Unstained bright-head spermatozoa were counted as viable, whereas spermatozoa with red/pink head were classified as dead cells.
The integrity of the sperm plasma membrane was appraised through the hypo-osmotic swelling test (HOST). The HOST predicts membrane integrity by determining the ability of the sperm membrane to maintain osmotic equilibrium between intracellular and extracellular fluids. Influx of the fluid due to hypoosmotic stress causes the sperm tail to swell and coil. The method of Perez-Llano et al. (2001) was partially modified and described as follows: 50 ml of the sample was mixed with 200 ll of hypo- (75 mOsm/kg) or iso- (300 mOsm/kg) osmotic solutions, in a 1.5-ml microtube (Eppendorfâ, Sigma-Aldrich). The osmotic solutions were composed of fructose and Na-citrate in distilled water; their final osmolalities were checked with freezing point depression (Osmomat 030-3P, Gonotec GmbH, Berlin, Germany). Following 15 min of 37°C incubation, the reactions were stopped by adding 50 ll of the hypo/iso-osmotic solution plus 5% formaldehyde (Merck, Darmstadt, Germany) to the mixtures. Upon evaluation, an aliquot of well-mixed sample was dropped on a slide and covered with a slip. Two hundred total spermatozoa per slide were observed under a light microscope (4009), and the number of spermatozoa with coiled tail was recorded. The proportion of coiled-tail spermatozoa in a hypo-osmotic sample was subtracted by the value of iso-osmotic condition to determine the percentage of spermatozoa having intact plasma membranes. The acrosomal status of spermatozoa was assessed with a dual fluorescent staining technique (Axner et al. 2004). Briefly, a 10 ll aliquot of the sperm sample was smeared on a microscope slide, air-dried and membrane-permeabilized with 95% ethyl alcohol for 30 s. Thereafter, 100 ll of fluorescein isothiocyanate conjugated with peanut agglutinin (FITC-PNA) (100 lg/ml in phosphate-buffered saline; PBS) was mixed with 5 ll of propidium iodide (PI) (340 lM in PBS; Molecular Probes Inc., Eugene, OR, USA). Twenty microlitres of © 2015 Blackwell Verlag GmbH
Freezing Protocols for Epididymal Cat Sperm
381 Testicles from a total of 126 cats (n = 10) Cauda epididymides + Tris buffer Cut into small pieces, incubated at 37°C for 15 min Remaining fluid Allocated to 6 equal aliquots (I-VI), centrifuged at 500Хg for 10 min
I) pellets
+ EYT
II) pellets
+(EYT+3%Gly)
(Gr. I and II; 50x106 spz/mL)
III) pellets
IV) pellets
V) pellets
+(EYT+5%Gly) +(EYT+5%Gly, 0.5%Equex) (Gr. III and IV; 25x106 spz/mL)
+ EYT
VI) pellets
+(EYT+3%Gly)
(Gr. V and VI; 50x106 spz/mL)
Cooled to 4°C, 1 h
+(EYT+10%Gly, + (EYT+7%Gly, 1%Equex) 1%Equex) (Gr. I and II; 25x106 spz/mL)
-
-
+(EYT+10%Gly) +(EYT+7%Gly) (Gr. V and VI; 25x106 spz/mL)
Kept at 4°C, 10 min Loaded in 0.25 mL straws Frozen in a Styrofoam box Thawed at 70°C, 6 s Fig. 1. A summary of the cryopreservation protocols for epididymal cat spermatozoa conducted in the present study (EYT; plain egg yolk-Tris extender, Gly; glycerol, spz; spermatozoa)
the solution was spread over the dried smeared slide. The slide was kept in a dark humidified chamber at 4°C for 30 min. Subsequently, the slide was gently rinsed with cold distilled water. The stained slide was always kept in the dark at 4°C until evaluation was undertaken. At the time of evaluation, 200 spermatozoa per slide were observed using epifluorescent microscopy (Axioskop, Carl Zeiss Jena GmbH, Jena, Germany). Two categories of spermatozoa could be classified according to their staining patterns. Spermatozoa with acrosome intact were judged when the spermatozoa exhibiting bright green fluorescence over the entire acrosomal region, and acrosome-reacting/reacted spermatozoa were considered when the cells performing disrupted green or red fluorescence over the acrosomal area. Only the percentage of acrosome-intact spermatozoa was included in the results. © 2015 Blackwell Verlag GmbH
Diluted 1:1 (v/v) with a Tris buffer Incubated at 37°C Sperm quality evaluations
Statistical analysis A pool of sperm sample (from 7 to 24 cats) was used for each replicate, and ten replicates were performed. Analysis of variance (ANOVA) of a software package named SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. When significant differences were shown, the least significant difference (LSD) test was applied to compare statistical differences among groups (I–VI) and time (0 and 2 h). Percentages of motility, viability, plasma membrane integrity and acrosome integrity are presented as mean SEM, and p < 0.05 was considered statistically significant.
Results Based on the data of 10 replicates, cat spermatozoa retrieved from the cauda epididymides had motility of
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61.5 3.9%, viability of 79.2 2.5%, plasma membrane integrity of 82.3 2.2% and acrosome integrity of 71.7 3.0%. Almost all in vitro characteristics of epididymal cat spermatozoa appraised in the present experiment were significantly affected by the sperm cryopreservation protocol (I–VI) and time of post-thawed incubation (0 and 2 h) (p < 0.001 to p < 0.01). At 15 min after thawing (0 h incubation), the motility and viability of spermatozoa frozen with protocols I–VI were not significantly different from one another (p > 0.05). The integrity of sperm plasma membrane detected by HOST was also not significantly different among the groups (p > 0.05) excluding the sample frozen with protocol IV in which the lower percentage (59.9 2.5%) was shown (p = 0.005 to p = 0.04). The proportion of acrosomeintact spermatozoa demonstrated in group I was similar to that in group II (p > 0.05) and was significantly higher than those in the rest four groups (p < 0.001 to p = 0.02). Among the rest four groups, the percentage of spermatozoa with acrosome intact was the lowest in group III (25.1 2.2%); this value was significantly lower compared to those in groups IV (p = 0.004) and VI (p = 0.01). After incubation for 2 h, the percentages of motile, viable and membrane-intact (HOST) spermatozoa were declined to comparable levels among the groups (p > 0.05). These were exceptions for the viability of spermatozoa in group III and membrane integrity of spermatozoa in group IV, where the poorest percentages were found (Table 2). The less viable spermatozoa in group III (41.1 5.5%) differed significantly from the viability of spermatozoa in groups I, II and V (p = 0.008 to p = 0.02). The membrane-intact spermatozoa in group IV (55.6 2.2%) was inferior significantly to
the values in groups III, V and VI (p = 0.005 to p = 0.03). There was no significant difference in the percentage of acrosome-intact spermatozoa frozen with protocols I and II during the 2 h incubation (p > 0.05). These two values were significantly greater than those demonstrated in groups III, IV and V (p < 0.001 to p = 0.02). An equal percentage of acrosome-intact spermatozoa in groups IV and V (i.e. 23.3 1.6%) did not significantly differ from the value in group VI (25.1 1.4%, p > 0.05), but it was significantly higher compared with 17.5 1.4% reported in group III (p = 0.02). Comparing between incubation periods within each cryopreservation protocols, the quality of frozen– thawed spermatozoa in all six groups decreased after incubation for 2 h. Nevertheless, the significant reductions were only observed in the motility of spermatozoa in group I (p = 0.007), viability of spermatozoa in groups III (p < 0.001) and IV (p = 0.017) and acrosome integrity of spermatozoa in every group (p < 0.001 to p = 0.01).
Discussion To determine the appropriate cryopreservation protocol for epididymal cat spermatozoa, effects of adding Equex, diluting the samples in one or two steps and including glycerol in the freezing extenders on the sperm quality were investigated. It was found that egg yolkTris-based extenders containing Equex added to the processed spermatozoa just for 10 min before freezing (groups I and II) were beneficial for the percentage of acrosome-intact spermatozoa both immediately and 2 h after thawing, compared to the extenders without Equex (groups V and VI). These results are in agreement with
Table 2. The in vitro quality of post-thawed epididymal cat spermatozoa, at 0 and 2 h of 37°C incubation, frozen in egg yolk-Tris-based extenders using six different freezing protocols (n = 10) Sperm parameters (%) Incubation time 0 h (15 min)
2h
Experimental groupa I II III IV V VI I II III IV V VI
Motility 20.0 17.0 15.5 15.5 16.5 12.5 1.5 2.0 4.0 7.5 2.5 3.5
3.11 3.5 3.5 4.4 3.2 3.7 0.82 1.1 1.5 2.3 1.3 1.3
Viability 62.6 61.0 57.4 58.8 58.9 56.3 56.4 54.0 41.1 47.2 53.9 50.0
2.2 2.8 3.21 2.21 3.0 3.0 3.2a 3.3a 5.5b,2 4.4a,b,2 3.2a 3.5a,b
HOSTb 67.7 70.0 67.7 59.9 70.9 69.3 60.8 63.1 64.6 55.6 67.2 65.9
3.1a 1.7a 2.8a 2.5b 3.3a 2.3a 3.1a,b 2.7a,b 2.6a 2.2b 3.4a 2.8a
FITCc 43.6 42.0 25.1 35.5 32.0 33.9 30.3 29.1 17.5 23.3 23.3 25.1
3.0a,1 1.5a,b,1 2.2d,1 3.6b,c,1 2.0c,d,1 1.5c,1 1.6a,2 2.4a,b,2 1.4d,2 1.6c,2 1.6c,2 1.4b,c,2
Values with different superscripts indicate significant difference within columns [a, b, c, d comparing among groups (I–VI) within each incubation times; 1, 2 comparing between incubation times (0 and 2 h) within each groups, p < 0.05]. Values without any superscript mean no significant difference within columns. a I = 2-step dilution with 0% and 10% glycerol plus 1% Equex; II = 2-step dilution with 3% and 7% glycerol plus 1% Equex; III = 1-step dilution with 5% glycerol; IV = 1-step dilution with 5% glycerol plus 0.5% Equex; V = 2-step dilution with 0% and 10% glycerol; VI = 2-step dilution with 3% and 7% glycerol. b The plasma membrane integrity of spermatozoa detected by the hypo-osmotic swelling test (HOST). c The acrosome integrity of spermatozoa assessed with FITC-PNA/PI staining (FITC).
© 2015 Blackwell Verlag GmbH
Freezing Protocols for Epididymal Cat Sperm
previous studies conducted in the spermatozoa of many species such as boars (Buranaamnuay et al. 2009), dogs (Rota et al. 1997; Pena and Linde-Forsberg 2000) and even of cats (Axner et al. 2004). In boars, it has been found that the motility, viability and acrosome morphology of thawed spermatozoa were improved when Equex was included in a freezing extender (Buranaamnuay et al. 2009). Similarly, addition of Equex to the freezing medium significantly increased the membrane integrity, motility and longevity of the frozen–thawed dog spermatozoa (Rota et al. 1997; Pena and LindeForsberg 2000). In cats, Axner et al. (2004) found that Equex had a positive effect on the percentage of intact acrosomes immediately after thawing but did not have any effect on the percentage of intact sperm membranes and the motility evaluated at the same time. In addition, the authors reported that reductions of the motility and membrane integrity of cat spermatozoa during the 6-h post-thawed incubation were more accelerated in Equex-added samples than in Equex-free counterparts (Axner et al. 2004). The present findings confirm the results of Axner et al. (2004) that Equex improved the percentage of acrosome-intact spermatozoa but did not improve the percentages of plasma membrane-intact and motile spermatozoa, as well as it exacerbated a decrease in motility during 2 h of post-thawed incubation, as observed in group I. According to the results of the present and Axner et al. (2004) studies, it thereby seems that Equex has a cryoprotective effect on the acrosomal membranes but has an adverse effect on the motility of epididymal cat spermatozoa during warm incubation in vitro. The present study additionally found that among Equex-added samples (groups I, II and IV), positive effects of this compound on the thawed spermatozoa were obvious in the samples processed with two-step dilution (i.e. shorter exposure to Equex before freezing); this is similar to what has been observed in dog spermatozoa (Pena and Linde-Forsberg 2000). These findings led us to believe that the longer the exposure to Equex before freezing, the more the toxic to spermatozoa after thawing. It has been suggested that prolonged exposure to Equex or Equex-treated egg yolk lipoproteins rendered excessive fluidity to the sperm membranes, which would indicate that the beneficial effect of Equex is partly depended on an exposure period (Pena and Linde-Forsberg 2000). Even though the effect of Equex on in vitro sperm quality has been well recognized, the influence of this substance on the life span of spermatozoa in the female genital tract after artificial insemination (i.e. in vivo sperm quality) has not been extensively studied and thus remains to be investigated further. However, it is supposed that spermatozoa will leave the freezing extender after being deposited in the female tract; consequently, they may no longer be positively/negatively affected by the presence of Equex (Axner et al. 2004). Comparing between one- and two-step dilutions, it was investigated that when Equex was present, the membrane integrity (HOST) and the acrosome integrity © 2015 Blackwell Verlag GmbH
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(FITC) of spermatozoa evaluated immediately after thawing (0 h) were likely to be (i.e. FITC between groups II and IV) or significantly higher in the samples diluted twice, compared to those diluted once. The advantages of two-step dilution on the frozen–thawed sperm quality were still found, at least in one parameter assessed, even after incubation for 2 h. Similar phenomena were also observed in the Equex-free samples that two-step dilution resulted in comparable (i.e. FITC between groups III and V at 0 h) or significantly higher percentages of intact acrosomes compared with one-step dilution both at 0 and 2 h of post-thawed incubation. Based on the concordant data between the Equex-added and Equex-free samples, it is likely that glycerol per se had some toxic effect on epididymal cat spermatozoa only in condition that relatively high concentration (5% glycerol) together with long exposure period (one-step dilution) was applied in the cryopreservation protocol. The present observations, however, did not totally agree with a previous report in cat spermatozoa conducted by Vick et al. (2012), where the percentage of intact acrosomes evaluated after thawing was not significantly affected by temperature (i.e. room temperature vs 5°C) of glycerol addition during the cryopreservation process. The discrepancy between this and previous findings might be partly related to differences in the source of spermatozoa (i.e. epididymal spermatozoa are different from ejaculated spermatozoa in the susceptibility to cold shock) and/or in the sperm cryopreservation protocol. With regard to the cryopreservation protocol, Vick et al. (2012) used the TEST yolk medium as a freezing extender and included 4% final glycerol concentration in the medium, whereas the present study selected the egg yolk-Tris-based extender supplemented with 5% glycerol as a freezing medium. It has been recognized that there are many factors influencing achievement of sperm cryopreservation, for example, extender composition, freezing and thawing methods, freezing and thawing rates, as well as size and volume of straw (Almlid and Johnson 1988); therefore, a comparison among different protocols is very complicated and offers less reliability (Baran et al. 2004). Effects of glycerol addition on post-thawed sperm quality have been investigated in several species including cats. Concerning concentration of glycerol, it has been demonstrated that in the range of 0–7% glycerol, the percentage of acrosome integrity of boar spermatozoa was negatively affected by increased glycerol concentrations (Almlid and Johnson 1988). Likewise, in cats, Villaverde et al. (2013) compared between 3, 5 and 7% glycerol and found that the percentage of dead spermatozoa presenting intact acrosomes was increased as glycerol concentration decreased. From the results of Villaverde et al. (2013) and the present study, it seems that a detrimental effect of high glycerol concentration on acrosome integrity presents in cat spermatozoa; this finding is consistent with the results reported in other species (Almlid and Johnson 1988; Fernandez-Santos et al. 2006). The negative effect of such compound on
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the acrosome could be explained by the fact that increased glycerol concentrations result in increased enzyme leakage from the acrosome of spermatozoa (Schmidt et al. 1974), which can be detected indirectly through reduced proportions of intact acrosomes after thawing. With regard to time of glycerol exposure before freezing (i.e. 10 min vs 1 h for the present study), this sole factor was not a reason for differences in the frozen–thawed sperm quality in this study. This suggestion was supported by earlier observations on boars’ (Almlid and Johnson 1988), bulls’ (Berndtson and Foote 1972) and dogs’ (Pena and Linde-Forsberg 2000) spermatozoa. In boars, it was found that glycerol exposure times of 0.5, 2, 5, 15 and 75 min before freezing did not significantly influence the sperm quality after thawing, indicating that glycerol is able to penetrate quickly into the spermatozoa and that equilibrium is reached within a very short time (
