Motile sperm subpopulations in frozen–thawed dog semen: Changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress

Animal Reproduction Science 133 (2012) 214–223

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Motile sperm subpopulations in frozen–thawed dog semen: Changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress ˜ ∗ , M. Barrio, J.J. Becerra, L.A. Quintela, P.G. Herradón A.I. Pena Unit of Reproduction and Obstetrics, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Santiago de Compostela (USC), 27002 Lugo, Spain

a r t i c l e

i n f o

Article history: Received 28 March 2012 Received in revised form 18 June 2012 Accepted 21 June 2012 Available online 27 June 2012 Keywords: Dog semen Cryopreservation Motile sperm subpopulations Capacitating conditions Osmotic stress

a b s t r a c t In this study we investigated the changes that in vitro incubation under capacitating conditions could induce on the motile sperm subpopulations present in frozen–thawed dog semen samples. In addition, cryopreserved dog spermatozoa were exposed to CCM (canine capacitating medium) solutions of 300, 150, 100 and 75 mOsm and the proportions of live spermatozoa with swollen tails were recorded (HOST+). Finally, frozen–thawed dog semen samples were submitted to a second cycle of freezing and thawing and the overall sperm motility, as well as the motile sperm subpopulations structure, was determined. Cryopreserved dog semen samples were structured in four sperm subpopulations with different motility characteristics: Subpopulation (Sp) 1 contained moderately rapid and progressive spermatozoa (25.2 ± 8.5%), Sp 2 included poorly motile and non progressive sperm (15.3 ± 8.1%), Sp 3 was represented by moderately slow non progressive sperm (14.9 ± 5.9%), and Sp 4 contained the most rapid and progressive sperm (20.8 ± 14.7%). After 3 h of incubation under capacitating conditions, percentages of spermatozoa assigned to Sp 2 (6.1 ± 3.4%) and 3 (4.9 ± 2.8%) significantly decreased, whereas those assigned to Sp 1 (17.0 ± 11.2%) and 4 (16.2 ± 12.8%) did not significantly change. Significant correlations were found between percentages of HOST+, for the 3 osmolarities tested, and percentages of spermatozoa included in Sp 1 and 4 after 3 h of incubation in capacitating conditions or in Sp 4 after double freezing and thawing. These results indicated that subpopulations with the most rapid and progressive sperm seemed to be highly resistant to in vitro incubation in capacitating conditions and to osmotic stress, suggesting they are likely to be the source of the fertilizing population. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The existence of well-defined sperm subpopulations with specific motility characteristics has been demonstrated in ejaculates from numerous species (Abaigar et al., 1999; Quintero-Moreno et al., 2003, 2004; Rigau ˜ et al., 2008), including the dog et al., 2001; Muino

∗ Corresponding author. Tel.: +34 982 28 59 00; fax: +34 982 82 26 27. ˜ E-mail address: [email protected] (A.I. Pena). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.06.016

˜ (Nunez-Martínez et al., 2006a,b; Mogas et al., 2011; Dorado et al., 2011a,b), and it has been suggested to be a widespread phenomenon among mammalian ejaculates (Quintero-Moreno et al., 2003). As in other species, dog ejaculates have been found to maintain a constant structure of four motile sperm subpopulations during cold storage (Dorado et al., 2011b), after freezing and thawing (Mogas et al., 2011; Dorado et al., 2011a) or after post-thaw centrifugation on PureSperm® gradients (Dorado et al., 2011a). The different subpopulations of motile sperm are thought to represent spermatozoa in different physiological states

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˜ et al., 2008). As individual cells (Abaigar et al., 1999; Muino lose structural and functional integrity and advance toward a late state of cell deterioration, they are believed to shift from subpopulations of high velocity and progressiveness to those with a lower velocity and non progressive trajectories, to finally become immotile. It would seem logical to think, therefore, that the fertilizing capacity of fresh or frozen–thawed semen might preferentially reside in the subpopulations of most rapid and progressive sperm, as slow moving or non progressive spermatozoa would have a reduced capacity to migrate along the genital tract of the female (Gaddum-Rosse, 1981; Shalgi et al., 1992). However, after insemination, the motility patterns of the different sperm subpopulations could be substantially modified by the uterine environment. Moreover, numerous evidences suggest that cryopreservation procedures initiate capacitation-like changes in a proportion of the surviving spermatozoa, and that the extent of these changes determines their ability to survive in the female genital tract (Watson, 1995; Rota et al., 1999; Leahy and Gadella, 2011). Although in vitro incubation in capacitating conditions does not reproduce the same environment found by the sperm in the female genital tract, it is known to induce many of the destabilizing membrane changes in sperm that are ascribed to capacitation in vivo (Harrison, 1996). It is believed that only normal viable uncapacitated sperm are selected by the oviductal epithelium (Yanagimachi, 1994; Pacey et al., 2000); thus, a too rapid or too high hyperactivation response during in vitro incubation in capacitating conditions might indicate an excessive destabilization and a low fertilizing ability of the sample (Petrunkina et al., 2007). Therefore, the identification of individual sperm subpopulations able to survive and maintain forward progressive motility after a given period of incubation under capacitating conditions, more than determining the mean motility changes of the whole population, could be relevant for the fertilizing capacity of the sample. Whether there is a relationship between the incidences of specific motile sperm subpopulations in frozen–thawed dog semen and the proportions of progressively motile vs. hyperactivated sperm (i.e. sperm exhibiting star-spin-like active movement) observed after a given period of incubation in capacitating conditions is not known. On the other hand, the different sperm subpopulations present in frozen–thawed semen should be expected to respond differently to hypotonic stress, as it requires a capacity to regulate cell volume that is related to the functional and structural integrity of the sperm plasma membrane and of the cytoskeleton (Petrunkina et al., 2004a,b). This is an important issue because cryopreservation extenders for dog spermatozoa usually are hypertonic and, after deposition in the genital tract of the bitch, the thawed spermatozoa will have to adapt to the isotonic uterine fluids with new volume changes (Cooper and Yeung, 2003). It would be interesting to investigate if there is any correlation between the outcomes of hypo-osmotic swelling test (HOST) and the proportions of spermatozoa assigned to subpopulations of the highest velocity and progressivity either determined in frozen–thawed semen or after a period of post-thaw incubation in capacitating conditions.

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Moreover, it has long been known that major local osmotic gradients across the sperm membranes are generated during the freeze-thaw cycle (Mazur et al., 1972) and it is believed that cellular damage occurs mainly during the thawing process, when the dehydrated sperm, after undergoing primary hypertonic shock, would be exposed to severely hypo-osmotic conditions (Gao et al., 1993). In this sense, it would be interesting to find out if any of the sperm subpopulations surviving after cryopreservation would be able to survive to a second cycle of freezing and thawing. The ability to control cell volume regulation and maintain membrane integrity in the face of the severe osmotic changes generated during two successive cycles of freezing and thawing could help to identify a subpopulation of sperm cells possessing exceptionally robust membranes. We hypothesize that such subpopulation, if any, might correlate with the proportion of spermatozoa able to maintain a rapid and progressive (vs. hyperactive-like) pattern of movement during post-thaw incubation in capacitating conditions, and with the proportion of spermatozoa able to maintain plasma membrane integrity and functionality after post-thaw incubation in hypotonic solutions. The aims of the present study were to investigate: (1) how the incubation in capacitating conditions could modify the motility patterns of the different sperm subpopulations determined in frozen–thawed dog semen samples; (2) the existence of any correlation between the outcomes of hypo-osmotic swelling test (HOST) and the proportions of spermatozoa assigned to specific subpopulations, either in frozen–thawed semen samples or after post-thaw incubation in capacitating conditions; and (3) the effect of two successive cycles of freezing and thawing on the frequency distribution of motile spermatozoa within the different subpopulations, and its potential relationship to the subpopulations observed after incubation in capacitating conditions and to the HOST test. 2. Materials and methods 2.1. Frozen semen Frozen semen from eight privately owned, clinically healthy dogs (1 Newfoundland, 1 Staffordshire Bull Terrier, 2 Siberian Huskies, 1 Bob tail, 1 Beagle, 1 English bull dog and 1 Labrador retriever, ranging between 3 and 8 years of age) was used for this study. The semen was frozen at the Veterinary Faculty of Lugo (Spain) during the period 2003–2010 and was kept stored for private use. For different reasons, after variable time periods, the owners decided not to keep the frozen semen of their dogs and gave their consent for its use in research. 2.2. Collection and evaluation of ejaculates Ejaculates were collected by digital manipulation into 15 ml Falcon conical tubes (Becton Dickinson, Franklin Lakes, NJ, USA) aided by prewarmed glass funnels. Only the sperm-rich fraction of the ejaculates was collected and was assessed to determine its volume, sperm concentration, sperm motility and the percentages of abnormal spermatozoa and of sperm plasma membrane integrity. The

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percentage of motile spermatozoa was determined by subjective microscopic examination at a magnification of 200× using a phase contrast microscope, and the sperm concentration was determined using a photometer (SpermaCue, Minitüb, Tiefenbach, Germany). Sperm morphology and plasma membrane integrity were determined by eosinnigrosin staining using a phase contrast microscope at 1250× magnification. Ejaculate characteristics varied between dogs, means and standard deviations being as follows: volume 2.4 ± 0.6 ml, total number of spermatozoa (761.2 ± 353.4) × 106 spz, total motility 88.1 ± 4.6%, and percentages of live and abnormal spermatozoa 95.1 ± 5.2% and 13.4 ± 5.6%, respectively. 2.3. Semen freezing All the ejaculates were frozen using the Uppsala method ˜ and Linde-Forsberg, 2000a,b). Immediately after col(Pena lection and evaluation, each ejaculate was centrifuged at 700 × g for 5 min. The seminal plasma obtained after centrifugation was discarded, and the sperm pellet was rediluted in a first extender (Ext-1) at room temperature and at a sperm concentration of 400 × 106 spz/ml. The composition of Ext-1 was as follows: 200 mM Tris, 66 mM citric acid, 44 mM glucose, 17 mM Na-bencylpenicillin, 14 mM streptomycin sulphate, 20% (v/v) egg yolk and 3 mM glycerol. Diluted semen was allowed to equilibrate in a refrigerator at 4 ± 0.5 ◦ C for 1 h. After 1 h of equilibration, the same volume of a second extender (Ext-2) at 4 ◦ C was added to the semen sample. The composition of Ext-2 was the same as that of Ext-1 except that it contained 7 mM glycerol and 1% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA). The addition of the second extender made a final sperm concentration of 200 × 106 spz/ml. Once the Ext-2 had been added, the extended semen was left at 4 ◦ C for 15 min, and then it was packaged in 0.5 ml-straws, which were frozen in a closed styrofoam box on a rack 4 cm above the surface of LN2 for 10 min. The thawing was done by immersing the straws in a water bath at 70 ◦ C for 8 s. The time for straws immersion was controlled with an 8 s-settled timer. 2.4. Experimental design From each ejaculate, three straws were thawed and submitted, respectively, to the following treatments: (1) TRIS (control treatment): the content of one straw (100 × 106 spz) was emptied in 1 ml of Tris buffer (same composition as the buffer fraction of Ext-1 and Ext-2) at 37 ◦ C, and the sperm suspension was incubated for 3 h in a water bath at 37 ◦ C. (2) CCM (canine capacitating medium): contents of a second straw were emptied in 2.5 ml of CCM and centrifuged twice at 300 × g for 5 min to remove the cryopreservation extender. The sperm pellet was rediluted in 1 ml of CCM (initial sperm suspension, 100 × 106 spz/ml).

CCM composition (Mahi and Yanahimachi, 1978): 83.49 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl2 .2H2 O, 1.19 mM KH2 PO4 , 37.61 mM NaHCO3 , 0.25 mM Na pyruvate, 21.55 mM Na lactate 60% syrup, 2.78 mM glucose, 56.4 ␮M phenol red and 0.4% (w/v) BSA; pH 7.8; 305 mOsm. Four CCM solutions of different osmolarity were prepared: - CCM (1:0): 1 ml of CCM (305 mOsm). - CCM (1:1): 500 ␮l of CCM and 500 ␮l of distilled water (approx. 150 mOsm). - CCM (1:2): 333 ␮l of CCM and 666 ␮l of distilled water (approx. 100 mOsm). - CCM (1:3): 250 ␮l of CCM and 750 ␮l of distilled water (approx. 75 mOsm). Aliquots of 100 ␮l (10 × 106 spz) from the initial sperm suspension were respectively added to the four CCM solutions and then incubated at 38.5 ◦ C and 5% CO2 in air. (3) DFT (double freezing and thawing): immediately after thawing, the third straw was not emptied. It was left for 10 min at room temperature, and then was placed at 4 ◦ C in a fridge for 1 h. After 1 h of cooling, the straw was frozen again in a styrofoam box on a rack 4 cm above the surface of LN2 for 10 min, and then stored in LN2 until thawing and evaluation. After the second thawing, the content of the straw was emptied in 1 ml of Tris buffer at 37 ◦ C. In control samples and samples incubated in isosmotic CCM (1:0), motility patterns were analyzed after 0–3 h of incubation by means of a CASA system (Sperm Class Analyzer 5.1; Microptic, Barcelona, Spain). In DFT samples, motility characteristics were only evaluated after thawing but not after post-thaw incubation. Samples incubated in CCM solutions (1:0, 1:1, 1: 2 and 1:3) were assessed after 1 h of incubation by using eosinnigrosin staining and phase contrast microscopy at 1250× magnification, under immersion oil. Two smears were prepared from each sample and 100 cells were counted in each smear. The sperm cells were classified according to four categories: (1) live spermatozoa with normal morphology: excluding eosin, normal acrosomes and normal tails, (2) live spermatozoa with acrosomal damages: excluding eosin, totally or partially reacted acrosomes, independent of tail morphology, (3) live spermatozoa with different degree of tail swelling: excluding eosin, intact acrosomes and bending or coiling tails, (4) dead spermatozoa: eosin stained, independent of acrosomal status or tail morphology. 2.5. Sperm motility evaluation by CASA The CASA system used was based on the analysis of 25 consecutive, digitized photographic images which were taken in a time lapse of 1 s, which implied a velocity of image-capturing of 1 photograph every 40 ms. Images were taken from 5 ␮l semen aliquots, which were placed on a Makler chamber (Israel Electrooptical Industry, Rehovot, Israel). Six microscopic fields were analyzed per sample using a microscope (Eclipse E200, Nikon, Tokyo, Japan)

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supplied with a prewarmed stage at 37 ◦ C and a negative phase-contrast objective at a magnification of 100×. The number of spermatozoa analyzed per sample ranged between 500 and 1000, including the immotile sperm. Objects incorrectly identified as spermatozoa were minimized on the monitor by using the playback function. Total motility was defined as the percentage of spermatozoa with curvilinear velocity (VCL) above 30 ␮M/s. The kinematic parameters recorded for each spermatozoon, as described by Mortimer (Mortimer, 1997, 2000) were: curvilinear velocity (VCL, ␮M/s): the average path velocity of the sperm head along its actual trajectory; straight-line velocity (VSL, ␮M/s): the average path velocity of the sperm head along a straight line from its first to its last position; average path velocity (VAP, ␮M/s): the average velocity of the sperm head along its average trajectory; percentage of linearity (LIN, %): the ratio between VSL and VCL; percentage of straightness (STR, %): the ratio between VSL and VAP; wobble coefficient (WOB, %): the ratio between VAP and VCL; mean amplitude of lateral head displacement (ALH, ␮M): the average value of the extreme side-to-side movement of the sperm head in each beat cycle; and beat cross frequency (BCF, Hz): the frequency with which the actual sperm trajectory crosses the average path trajectory.

3. Results

2.6. Statistical analysis

Four sperm subpopulations were defined after multivariate cluster analysis of 27,952 individual motile spermatozoa. The motility characteristics of those subpopulations are shown in Table 2, and their patterns of movement can be described as follows. Subpopulation 1 represented spermatozoa with relatively high velocity and progressiveness. Values of VCL, VSL and VAP, as well as those of LIN, STR and WOB were the second most elevated when comparing the four subpopulations. This subpopulation included about 31% of the total motile sperm. Subpopulation 2 included about 24% of the spermatozoa, which were poorly motile and non progressive, as indicated by the lowest values observed for all the kinematic parameters. Subpopulation 3 contained spermatozoa with medium velocity and low progressiveness, as indicated by medium values of VCL, ALH and BCF, and relatively low values of VSL, VAP and LIN. This subpopulation contained about 20% of the total motile sperm. Subpopulation 4, containing about 24% of the total motile population, included the most rapid and progressive sperm, as showed by the highest velocity parameters together with the highest LIN, STR, WOB and BCF, and relatively low ALH.

Data from all the motile spermatozoa obtained in all the evaluations were imported into a single data set that represented 27,952 spermatozoa, each one defined by the 8 motility descriptors specified above. A multivariate kmeans cluster analysis was carried out to classify the 27,952 spermatozoa into a reduced number of subpopulations according to their patterns of movement, so that every spermatozoon belonged to one and only one cluster. Spermatozoa that were very close to each other were assigned to the same cluster, whereas spermatozoa that were far apart were put into different clusters. The k-means clustering model used euclidean distances computed from the 8 quantitative variables, after normalization of the data, so that the cluster centres were the means of the observations assigned to each cluster. The specified number of clusters was based on the previous analysis of hierarchical dendograms constructed on individual dogs using the Ward method (Rencher, 2002). For each treatment, contingency tables were used to determine the percentages of spermatozoa assigned to the different clusters for individual dogs. The effect of the treatment on the relative distribution frequency of spermatozoa within subpopulations was analyzed using a general linear model (GLM) procedure. The GLM procedure was also used to evaluate the effects of respective treatments on the mean kinematic parameters defining the different sperm subpopulations, and on the proportions of spermatozoa with acrosomal damages and swollen tails. Differences between means were analyzed by Tukey’s test. Correlation studies were performed by the Pearson analysis. All the statistical analyses were performed using the SPSS 18.0 software (SPSS Inc., Chicago, IL, USA), and differences were considered significant at P < 0.05 level.

3.1. Overall sperm motility The mean sperm motility parameters observed after post-thaw dilution (0 h), after 3 h of incubation in TRIS or CCM, and after double freezing and thawing (DFT) are shown in Table 1. For control samples and samples incubated in CCM, the incubation time had a significant effect (P < 0.001) on total sperm motility, but the incubation medium or the interaction medium*time had no significant effects. Positive correlations were found between total motility in DFT samples and total motility in TRIS (0.662, P = 0.019) or CCM (0.599, P = 0.04) samples after 3 h of incubation, but not after 0 h of incubation. At the onset of sperm incubation in capacitating conditions (0 h), the mean VCL, VSL and VAP, as well as LIN, STR and ALH showed lower (P < 0.05) values than those observed in control samples. However, after 3 h of incubation in CCM both the velocity and progressivity descriptors were higher (P < 0.05) than in control samples. Samples submitted to double freezing and thawing showed lower VCL but higher progressiveness than control samples. 3.2. Motile sperm subpopulations

3.3. Frequency distribution of spermatozoa within subpopulations after incubation in capacitating conditions or after double freezing and thawing Immediately after thawing, centrifugation and redilution in CCM, the proportions of spermatozoa assigned to the four subpopulations did not differ from those observed in control samples diluted in TRIS buffer (Fig. 1). However, after 3 h of incubation, significant differences

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Table 1 Mean motility parameters (±SD) of cryopreserved dog spermatozoa subjected to 3 h of post-thaw incubation in TRIS buffer (37 ◦ C), in CCM (38.5 ◦ C and 5% CO2 in air) or to double freezing and thawing (DFT) (n = 12). TRIS-0 h Total motility (%) Kinematic parameters VCL (␮M/s) VSL (␮M/s) VAP (␮M/s) LIN (%) STR (%) WOB (%) ALH (␮M) BCF (Hz) a–e

CCM-0 h

76.3 ± 14.6 132.8 89.9 105.2 62.6 79.4 75.4 5.3 7.0

± ± ± ± ± ± ± ±

TRIS-3 h

69.4 ± 19.2

a

56.3b 56.8b 54.3b 26.5b 23.2c 17.5a 3.1a 4.0c

125.6 80.9 101.2 58.7 73.0 76.3 3.7 7.1

± ± ± ± ± ± ± ±

CCM-3 h

41.7 ± 25.9

a

68.9c 60.2c 62.6c 27.4c 26.8e 17.7a 1.8e 3.9c

123.3 69.0 83.3 52.5 77.7 65.2 5.0 7.9

± ± ± ± ± ± ± ±

DFT

44.2 ± 26.7

b

44.5 ± 17.0b

b

50.7c,d 45.4d 44.4e 23.4d 22.4d 17.6c 2.0b 4.1b

151.7 102.1 113.0 64.0 84.0 72.6 4.7 10.1

± ± ± ± ± ± ± ±

59.8a 52.4a 50.6a 25.7b 23.1b 19.6b 2.3c 4.5a

120.0 89.3 96.5 68.8 87.1 76.1 4.5 7.2

± ± ± ± ± ± ± ±

52.3d 55.0b 53.0d 24.9a 18.3a 19.0a 2.1d 4.1c

: Different letters indicate significant differences between columns (P < 0.05).

Table 2 Mean values (±SD) of the kinematic parameters defining the four sperm subpopulations identified in frozen–thawed dog semen samples. Sperm subpobpulations

Number of spermatozoa (%) Kinematic parameters VCL (␮M/s) VSL (␮M/s) VAP (␮M/s) LIN (%) STR (%) WOB (%) ALH (␮M) BCF (Hz) a–d

1

2

3

4

8732 (31.2)

6646 (23.8)

5770 (20.6)

6804 (24.3)

160.2 96.5 119.7 62.7 81.5 76.0 6.0 9.0

± ± ± ± ± ± ± ±

25.6b 27.1b 18.7b 21.8b 21.2b 13.2b 2.8a 3.9b

45.2 17.6 26.4 37.8 59.8 58.2 2.7 3.9

± ± ± ± ± ± ± ±

16.1d 12.6d 12.6d 24.2d 27.0d 19.7d 1.2d 2.5d

104.4 56.5 71.7 57.0 78.7 70.6 5.1 6.4

± ± ± ± ± ± ± ±

24.0c 20.6c 15.9c 22.6c 21.4c 15.8c 2.3b 3.2c

190.6 159.7 170.1 84.4 94.0 89.5 4.5 9.7

± ± ± ± ± ± ± ±

22.5a 25.5a 21.4a 12.9a 9.2a 8.1a 1.9c 3.8a

: Different letters indicate significant differences between subpopulations (P < 0.001).

in the sperm subpopulations distribution were found between the two incubation systems. In control samples, the subpopulation of most rapid and progressive sperm (subpopulation 4) significantly (P < 0.05) decreased from 20.8 ± 14.7% at 0 h of incubation to 4.2 ± 5.9% after 3 h at 37 ◦ C, whereas in CCM samples this subpopulation did not significantly change during the same period (18.6 ± 11.3% after 0 h vs. 16.2 ± 12.8% after 3 h at 38.5 ◦ C under 5% CO2 atmosphere). In contrast, subpopulations

2 and 3 did not significantly vary during incubation in TRIS buffer (15.3 ± 8.1% vs. 10.2 ± 6.2% and 14.9 ± 5.9% vs. 12.5 ± 9.1% respectively, for subpopulations 2 and 3, after 0 and 3 h of incubation), but significantly (P < 0.05) declined during incubation in CCM (19.4 ± 7.5% vs. 6.1 ± 3.4% and 12.5 ± 5.5% vs. 4.9 ± 2.8% respectively, for subpopulations 2 and 3, after 0 and 3 h of incubation). In semen samples submitted to double freezing and thawing, the four sperm subpopulations were almost

Fig. 1. Relative frequency distribution of motile spermatozoa (mean percentages ± SD; n = 12) within subpopulations (1, 2, 3, 4) determined after 0–3 h of incubation in Tris buffer (37 ◦ C) or in CCM (38.5 ◦ C and 5% CO2 ), and after double freezing and thawing (DFT). Different letters (a–c) inside or by side columns indicate significant differences within subpopulations between incubation medium-time.

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equally represented among the total motile sperm, and did not significantly differ from those observed after 3 h of incubation in TRIS or CCM media (Fig. 1). A positive correlation (0.720, P = 0.008) was found between the proportion of spermatozoa assigned to subpopulation 4 in DFT samples and that observed in TRIS samples at 0 h, but not after 3 h of incubation. In contrast, there was no correlation between any of the four subpopulations found in DFT samples and those determined in CCM samples at 0 h, but after 3 h of incubation, a correlation was found between the subpopulation 4 obtained in CCM samples and that one in DFT samples (0.632, P = 0.027). Moreover, when subpopulations 1 and 4 in DFT samples were combined and considered as only one population, this one was highly correlated (0.809, P = 0.001) with the subpopulation 4 observed in CCM samples after 3 h of incubation.

showed no correlation with the total motility registered in TRIS samples, after 0 or 3 h of incubation, nor with that in CCM samples at 0 h of incubation, or with total motility in DFT samples. Only after 3 h of incubation, total motility in CCM samples showed to be significantly correlated with HOST(+) (Table 7). When individual subpopulations were considered, the HOST(+) was found to be significantly correlated with the proportion of spermatozoa assigned to subpopulation 4 in: TRIS samples at 0 h, CCM samples at 3 h and DFT samples (Table 7). In addition, subpopulation 1 in CCM samples at 3 h was also significantly correlated with HOST(+). Other sperm subpopulations were not correlated with the HOST test.

3.4. Effect of treatment on the kinematic parameters defining the four sperm subpopulations

In agreement with previous studies (Dorado et al., 2011a,b), the present results confirmed the existence of four distinct sperm subpopulations in frozen–thawed dog semen samples. At the onset of in vitro incubation in capacitating conditions, total sperm motility was slightly lower, though not significantly, than in the respective control samples. This initial effect could have been due to the two centrifugations post-thaw and further re-dilution in CCM, a step not undergone by the control samples. After 3 h of incubation, total sperm motility was not different for the two incu˜ bation systems. In previous studies (Rota et al., 1999; Pena et al., 2003, 2004), cryopreserved dog spermatozoa submitted to in vitro incubation in capacitating conditions showed capacitation-like changes after a 2–4 h incubation period, and therefore, a 3 h incubation period was chosen for this study. The most important change observed after 3 h of incubation in capacitating conditions was that the percentages of spermatozoa assigned to subpopulations 1 and 4, containing sperm cells with the maximal values of both velocity and linearity, were maintained nearly unaltered despite a significant decline in the total motility. Reduction of total motility occurred almost exclusively at the expense of subpopulations 2 and 3, containing sperm cells with low velocity or progressiveness. However, in control samples, the subpopulation 4 was significantly reduced during incubation whereas subpopulations 2 and 3 were unchanged. This indicated that, although total motility most probably declined due to higher mortality rates in subpopulations 2 or 3, there was, in addition, a general impairment of motility causing sperm replacement from subpopulations with rapid and forward motility to those with lower velocity or progressiveness. It seems, therefore, that a simple thermoresistance test, where the thawed semen was diluted with Tris buffer and incubated at 37 ◦ C without removing the cryopreservation extender, is not sensitive enough as a functional test to characterize the response level of the different sperm subpopulations when exposed to a secondary stress, as it is a capacitating environment. Moreover, the different sperm subpopulations reacted differently to the CCM effect. Whereas subpopulations 1 and 4 showed significant increases in VCL as compared

For each one of the four sperm subpopulations (Tables 3–6), the mean kinematic parameters significantly varied depending on the incubation system or whether the spermatozoa had been submitted to double freezing and thawing. Subpopulation 1 (Table 3) responded to incubation in capacitating conditions by increasing VCL, VSL and VAP, as well as LIN, STR, WOB and BCF, as compared with its respective TRIS control. In contrast, spermatozoa included in subpopulation 2 (Table 4), showed a reduction of all the kinematic parameters; spermatozoa assigned to subpopulation 3 (Table 5) responded to incubation in capacitating conditions with a significant reduction of VCL but with higher VSL, VAP and LIN than its respective TRIS controls. Finally, subpopulation 4 (Table 6) showed an increase of VCL and ALH but similar values of VSL, STR and BCF, and a lower LIN, than its respective controls. In samples submitted to double freezing and thawing, the four sperm subpopulations showed lower values of VCL but the highest VSL, LIN, STR and WOB, as compared with TRIS or CCM samples. 3.5. Effect of hypoosmotic CCM on sperm membrane integrity The percentage of total live spermatozoa was not significantly different for the 4 CCM solutions (Fig. 2), ranging from 53% observed in 300 and 75 mOsm solutions to 57% observed in 100 and 150 mOsm CCM solutions. In samples incubated in isosmotic CCM, the proportion of live spermatozoa with normal morphology was 36.0 ± 13.4%, whereas 13.7 ± 11.5% of the live sperm had folded tails. In the samples incubated in hypotonic CCM, for the 3 osmolarities studied, the majority of the live spermatozoa were swollen and had an intact acrosome. Only 3–4% of the live sperm showed a reacted acrosome, and such cells usually had a straight tail. Many of the dead spermatozoa, stained by eosin, showed coiled tails. The percentage of live spermatozoa with intact acrosomes and swollen tails (HOST(+)) in response to incubation in hypotonic CCM, for the 3 osmolarities tested,

4. Discussion

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Table 3 Mean motility parameters (±SD) of spermatozoa assigned to subpopulation 1, after 0 and 3 h of post-thaw incubation in TRIS buffer (37 ◦ C), in CCM (38.5 ◦ C and 5% CO2 in air) or after double freezing and thawing (DFT) (n = 12). Subpobpulation 1

Number of spermatozoa (%) Kinematic parameters VCL (␮M/s) VSL (␮M/s) VAP (␮M/s) LIN (%) STR (%) WOB (%) ALH (␮M) BCF (Hz) a–e

TRIS-0 h

CCM-0 h

TRIS-3 h

CCM-3 h

DFT

3300 (33.4)

1969 (25.7)

1564 (35.2)

716 (37.8)

1183 (29.0)

158.6 96.6 120.8 63.3 80.2 77.4 6.9 7.8

± ± ± ± ± ± ± ±

23.9d 27.5c 16.4b 22.2b 20.9d 12.3b 3.7a 3.8d

165.4 90.3 127.0 57.8 73.0 78.0 5.1 8.9

± ± ± ± ± ± ± ±

29.5b 29.9d 20.3a 24.0c 25.3e 12.1b 1.7d 3.3c

161.5 93.1 110.9 59.2 84.6 69.6 5.9 10.1

± ± ± ± ± ± ± ±

20.9c 24.9d 19.4d 18.5c 18.7c 13.2d 1.8b 3.6b

170.8 103.4 118.3 63.4 87.6 71.5 5.5 11.7

± ± ± ± ± ± ± ±

31.1a 23.4b 17.2c 20.3b 15.6b 16.0c 2.1c 3.6a

147.7 107.0 116.7 73.9 91.7 79.9 5.4 9.2

± ± ± ± ± ± ± ±

18.6e 21.6a 16.0c 17.6a 13.4a 12.5a 2.4c 3.9c

: Different letters indicate significant differences between columns (P < 0.001).

Table 4 Mean motility parameters (±SD) of spermatozoa assigned to subpopulation 2, after 0 and 3 h of post-thaw incubation in TRIS buffer (37 ◦ C), in CCM (38.5 ◦ C and 5% CO2 in air) or after double freezing and thawing (DFT) (n = 12). Subpobpulation 2

Number of spermatozoa (%) Kinematic parameters VCL (␮M/s) VSL (␮M/s) VAP (␮M/s) LIN (%) STR (%) WOB (%) ALH (␮M) BCF (Hz) a–d

TRIS-0 h

CCM-0 h

TRIS-3 h

CCM-3 h

DFT

2020 (20.4)

2239 (29.2)

1102 (24.8)

283 (14.2)

1002 (24.6)

45.9 17.3 26.3 36.1 59.3 56.7 3.0 3.9

± ± ± ± ± ± ± ±

15.6b 12.4b,c 12.3b,c 22.4b 25.5b 17.8b 1.4b 2.4b

39.8 16.0 25.3 38.3 55.0 62.3 2.0 3.6

± ± ± ± ± ± ± ±

15.5c 12.7c 13.1c 26.2b 29.4c 20.5a 0.7c 2.4b

54.3 18.9 29.1 35.2 62.1 53.7 3.1 4.5

± ± ± ± ± ± ± ±

15.2a 11.2b 11.5a 20.5b 23.3b 18.0c 1.1b 2.8a

39.1 12.8 20.5 30.8 50.9 50.8 2.0 3.7

± ± ± ± ± ± ± ±

17.2c 12.9d 13.3d 26.9c 32.1d 22.9d 0.8c 2.8b

47.6 21.6 28.0 45.2 71.8 58.8 3.3 3.8

± ± ± ± ± ± ± ±

13.4b 12.8a 12.0a,b 24.3a 22.1a 20.3b 1.2a 2.5b

: Different letters indicate significant differences between columns (P < 0.001).

with their respective controls, subpopulation 3 showed a reduction in VCL but an increase in LIN, and subpopulation 2 responded with a reduction of both velocity and progressiveness. A similar response of individual motile sperm subpopulations to incubation in capacitating medium has also been observed in boar spermatozoa (Ramió et al., 2008). Whereas subpopulations containing progressive sperm reacted with significant increases in velocity and linearity, those containing non progressive spermatozoa did not significantly modify their motility parameters. Another finding reported in boar sperm (Ramió et al., 2008) also observed in the present study, was that the mean

motility changes detected after incubation in capacitating conditions, determined on the whole sample without considering subpopulations, were of a greater magnitude than those observed on individual subpopulations. Such effect can be explained by the changes in the frequency distribution of spermatozoa within subpopulations observed after incubation. It can be assumed that the ability of the sperm tail to swell in the presence of a hypoosmotic solution is a sign of membrane integrity and normal functional activity (Drevius and Eriksson, 1966; Jeyendran et al., 1984). When the sperm cells lose the ability to swell under

Table 5 Mean motility parameters (±SD) of spermatozoa assigned to subpopulation 3, after 0 and 3 h of post-thaw incubation in TRIS buffer (37 ◦ C), in CCM (38.5 ◦ C and 5% CO2 in air) or after double freezing and thawing (DFT) (n = 12). Subpobpulation 3

Number of spermatozoa (%) Kinematic parameters VCL (␮M/s) VSL (␮M/s) VAP (␮M/s) LIN (%) STR (%) WOB (%) ALH (␮M) BCF (Hz) a–d

TRIS-0 h

CCM-0 h

TRIS-3 h

CCM-3 h

DFT

1931 (19.5)

1408 (18.4)

1292 (29.1)

225 (11.9)

914 (22.4)

104.6 56.0 72.2 56.4 77.5 70.7 5.9 5.7

± ± ± ± ± ± ± ±

23.6b,c 20.7c 15.7b 22.3b 22.1c 14.1b 2.7a 3.1c

98.3 57.6 75.1 61.2 77.1 77.8 3.6 6.6

± ± ± ± ± ± ± ±

21.8d 20.1b,c 15.5a 22.2a 21.5c 13.2a 1.2d 3.0b

:Different letters indicate significant differences between columns (P < 0.001).

112.6 51.6 67.5 47.9 76.2 61.8 5.5 6.7

± ± ± ± ± ± ± ±

24.6a 19.7d 15.8c 20.2c 21.1c 15.7c 2.0b 3.2b

107.3 60.2 71.9 60.9 81.5 71.0 4.0 7.9

± ± ± ± ± ± ± ±

29.4b 24.6a,b 17.4b 27.0a 23.1b 20.9b 2.1c 3.4a

101.1 61.9 70.9 64.0 86.5 72.1 5.3 6.6

± ± ± ± ± ± ± ±

22.2c,d 19.5a 15.6b 21.4a 17.7a 16.0b 2.2b 3.5b

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221

Table 6 Mean motility parameters (±SD) of spermatozoa assigned to subpopulation 4, after 0 and 3 h of post-thaw incubation in TRIS buffer (37 ◦ C), in CCM (38.5 ◦ C and 5% CO2 in air) or after double freezing and thawing (DFT) (n = 12). Subpobpulation 4

Number of spermatozoa (%) Kinematic parameters VCL (␮M/s) VSL (␮M/s) VAP (␮M/s) LIN (%) STR (%) WOB (%) ALH (␮M) BCF (Hz) a–e

TRIS-0 h

CCM-0 h

TRIS-3 h

CCM-3 h

DFT

2636 (26.7)

2037 (26.6)

483 (10.9)

671 (35.4)

977 (24.0)

187.8 162.2 170.1 86.7 95.2 90.7 4.7 9.3

± ± ± ± ± ± ± ±

18.9c 23.4a,b 18.7b 10.8b 7.0b 6.5b 2.3b 4.0b

200.3 159.2 177.6 80.3 89.8 89.0 4.3 9.5

± ± ± ± ± ± ± ±

26.0a 32.7b 26.0a 15.7d 12.9c 8.8c 1.5c 3.4b

185.9 151.9 159.9 82.1 95.0 86.3 4.6 11.8

± ± ± ± ± ± ± ±

17.3c 18.3c 15.2d 10.0c 7.3b 6.4d 1.2b 3.2a

193.7 152.6 160.2 79.7 95.2 83.5 5.2 11.7

± ± ± ± ± ± ± ±

21.9b 19.7c 17.6d 12.8d 5.5b 11.2e 1.9a 3.4a

178.5 162.8 166.2 91.3 97.9 93.2 4.0 8.8

± ± ± ± ± ± ± ±

16.8d 17.9a 16.4c 6.7a 3.5a 4.8a 1.6d 3.7c

: Different letters indicate significant differences between columns (P < 0.001).

Fig. 2. Proportions of live spermatozoa (mean percentages ± SD; n = 12) with normal morphology (normal acrosomes and normal tails), reacted acrosomes (independently of tail morphology) and swollen tails (with normal acrosomes) determined after incubation (38.5 ◦ C and 5% CO2 ) in isoosmotic CCM or in 3 hypotonic CCM solutions.

hypotonic conditions, it may be due to an increased general permeability of the sperm membranes during cell deterioration, and/or to the loss of activity of ionic transport mechanisms, which otherwise would counteract by transferring osmolytes into or out of the cell in order to regulate cell volume and prevent an excessive swelling response (Petrunkina et al., 2004a,b). As capacitation is considered a continuing process of cell destabilization (Harrison, 1996) which, among other phenomena, involves an increasing disorder of plasma membrane phospholipids (Harrison et al., 1996), spermatozoa exposed to capacitating conditions could be expected to lose the ability to swell under hypotonic stress. In fact, such phenomenon has been observed in boar spermatozoa (Petrunkina et al., 2000). In the present study, frozen–thawed dog spermatozoa were

incubated in hypotonic solutions of CCM in an attempt to join the two stress factors, exposure to hypotonicity and to capacitating conditions. Percentages of live spermatozoa with swollen tails after hypotonic stress were found to be only significantly correlated with the sperm subpopulations of high speed and lineal trajectories, either determined immediately after thawing or after incubation in capacitating conditions. The sperm subpopulations with lower velocity and non progressive trajectories, identified after semen thawing, were likely to contain cells with severe sub-lethal damage (Parks and Graham, 1992) or at advanced capacitation-like status (Watson, 1995), as a consequence of cryopreservation. These cells were probably too fragile to withstand the future stress imposed by capacitating conditions or hypotonic media, which would

Table 7 Significant correlations (Pearson correlation coefficient and significance level) observed between percentages of live spermatozoa with swollen tails (HOST(+)) in response to incubation in 3 hypotonic solutions of CCM, and total motility or specific motile sperm subpopulations determined by CASA in samples incubated during 3 h in TRIS medium, in CCM or submitted to double freezing and thawing (DFT) (n = 12).

Subpopulation 4 in TRIS at 0 h Total sperm motility in CCM at 3 h Subpopulation 1 in CCM at 3 h Subpopulation 4 in CCM at 3 h Subpopulation 4 in DFT samples

HOST(+) in 150 mOsm CCM

HOST(+) in 100 mOsm CCM

HOST(+) in 75 mOsm CCM

0.675; P = 0.016 0.764; P = 0.004 0.707, P = 0.010 0.637; P = 0.026 0.601; P = 0.039

0.796; P = 0.002 0.728; P = 0.007 0.701; P = 0.011 0.612; P = 0.035 0.720; P = 0.008

0.749; P = 0.005 0.739; P = 0.006 0.691; P = 0.013 0.640; P = 0.025 0.733; P = 0.007

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explain the high correlations found between the HOST(+) tests and subpopulations of high speed and lineal trajectories. These results supported the hypothesis that the spermatozoa able to maintain rapid and progressive motility patterns during incubation in capacitating conditions were also the most resistant to hypotonic stress, and emphasized the importance of considering the different motile sperm subpopulations instead of the total sperm motility of a semen sample. About 14% of the live spermatozoa incubated in isosmotic CCM also showed swollen tails, suggesting that the abrupt change from the hypertonic cryopreservation extender to the isotonic CCM induced a moderate osmotic stress. The degree of swelling was lower than in hypotonic CCM solutions, as most of the sperm tails showed a simple bending of the distal portion, whereas at the lower osmolarities a spherical shape of the tails was commonly seen. In the present study only a small proportion of about 3–4% of the live spermatozoa showed reacted acrosomes, and these usually had straight tails, indicating inability to control membrane permeability. This subpopulation was believed to have undergone spontaneous acrosome reactions associated to the loss of membrane function. An interesting finding was that about 45% of the total spermatozoa were able to survive to a second freezing and thawing, and that total sperm motility after DFT was positively correlated with total motility after 3 h of incubation in the two media. A somewhat unexpected finding was that after DFT the four sperm subpopulations were still present and almost equally represented among the total motile sperm. Because the DFT process involved such an osmotic challenge to the sperm membranes, one could expect to find only one homogeneous population of poorly motile cells, or at least the disappearance of subpopulation 4, containing the most rapid and progressive sperm. However, after exposure to such extreme conditions, subpopulations 1 and 4, considered as the most competent, still accounted for about 22% of the total sperm. These two subpopulations, when considered together, showed to be highly correlated with the subpopulation 4 observed after incubation in CCM, which supported the hypothesis of a good relationship between resistance to osmotic challenge and to incubation in capacitating conditions. Successful double freezing of spermatozoa from a number of species has been previously reported (Arav et al., 2002; Hollinshead et al., 2004; McCue et al., 2004; Underwood et al., 2006; Maxwell et al., 2007). The purpose of those studies, in general, was to determine the potential feasibility of sperm re-freezing, to be used after procedures such as flow cytometric sex sorting, and the subsequent use of the re-frozen semen for IVF or for AI. It is not possible to compare our results with those previously reported in other species because the processing steps to which the spermatozoa were submitted were totally different. In most of the previous reports the thawed spermatozoa were purified through density gradients, re-diluted with new cryopreservation extenders and finally frozen and thawed. Our purpose, in contrast, was to challenge the sperm membranes with the severe osmotic changes generated during the two successive cycles of freezing and thawing, thus, spermatozoa were not subjected to other processing steps.

The dog semen used in this study was frozen using Equex STM Paste in the freezing extender, which in previous studies was found to improve the post-thaw ˜ and Lindesurvival and longevity of dog spermatozoa (Pena Forsberg, 2000a) and to maintain prolonged sperm viability during post-thaw incubation in capacitating conditions ˜ et al., 2003). Although the exact mechanism for the (Pena protective effect of Equex STM Paste is not known, its inclusion in the extender probably had some stabilizing effect on sperm membranes thus reducing capacitation-like changes during cryopreservation and slowing sperm destabilization during incubation in capacitating conditions. Such increased stability of capacitated sperm was found not to interfere with their responsiveness to calcium ionophore ˜ et al., 2004), or their zona pellucida binding ability (Pena which suggests a normal fertilizing capacity. Although individual variation was observed in this study, in terms of sperm survival after thawing and refreezing or after post-thaw incubation, results concerning the sperm behavior in the face of the diverse conditions imposed on them, were consistent among dogs and within treatments. In conclusion, the present results showed that four sperm subpopulations with specific patterns of movement were present in frozen–thawed dog semen. In vitro capacitation conditions affected different subpopulations differently. Whereas the sperm subpopulations with high velocity and progressivity responded to the capacitating environment with a further increase in velocity, those with the lowest kinematic parameters responded by reducing velocity and/or progressiveness. The two subpopulations of most rapid and progressive sperm seemed to be highly resistant to severe osmotic stress. If the present results could be extrapolated to the in vivo situation (i.e. assuming the same cellular stability after exposure to specific capacitating effectors present in the uterine and oviductal fluids of the bitch, such as glycosaminoglycans (Kawakami et al., 1998, 2000), and other unknown factors), these findings might indicate that only spermatozoa belonging to subpopulations with high speed and forward progressive motility could be able to survive during sperm transport in the female genital tract, and eventually undergo a physiological capacitation in the oviduct. Unfortunately, our results seemed to indicate that from a dog semen sample with an average post-thaw motility of about 75%, only around 30% of the sperm might have the competence to survive during the transport in the female genital tract. Further studies should be aimed to confirm or refute a potential relationship between the incidences of specific motile sperm subpopulations in a dog semen sample and its fertility in vitro and in vivo.

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