流式细胞术在哺乳动物精液质量检测中的应用

精液检测的首要目标就是快速准确地确定精子的生育力。同时具备多种特性和功能完整的精子才能受精,因而只有同时客观地检测多个指标,才能更好地反映精子的生育力。精子检测的传统方法费时费力,检测精子数量少,指标单一,而且易受操作者的主观影响,不能准确地反映精子功能。流式细胞术(FCM)为精子功能研究提供了一种快速、客观、多指标、大通量的检测手段;利用FCM检测精子的质膜完整性、顶体状态、染色质结构、线粒体功能以及细胞凋亡等,可以得知精子功能的相关情况。随着新的荧光探针、染色方法的不断开发和改进,FCM为精液质量检测提供了一种新的检测平台,应用前景极其广阔。     

Sperm factors related to in vitro and in vivo porcine fertility

The prediction of sperm fertilizing ability has great economic importance for breeding herds when artificial insemination is used. Classical methods of semen evaluation generally measure the sperm concentration, progressive motility, percentage of viable cells, and acrosome morphology. These assays are poor in predicting sperm fertility, because only the samples with markedly poor quality can be detected. The development of new sperm tests that measure certain sperm functions is an attempt to solve this problem. On the other hand, the binding and penetration of the zona pellucida is one of the most important barriers the spermatozoa must overcome in the fertilization process. Also, the interaction with the oocyte plasma membrane appears to explain much of the variability in sperm fertilizing potential among fertile boars. Thus, the study of the relationship between sperm factors and in vitro fertility may be a good strategy and assays that include a study of gamete interaction may lead to a better way to predict male fertility than the routine laboratory evaluation of semen. This review will discuss the relationships between sperm factors and fertility in vitro and in vivo (AI trial) with both diluted and frozen-thawed semen. We will also try to analyze the problems and limitations related to the interpretation of boar sperm tests.     

Assessment of Sperm Viability by Measurement of ATP,Membrane Integrity and Motility in Frozen/Thawed Bull Semen

Control of sperm quality after commercial freezing/thawing of bull semen is still restricted to the subjective assessment of sperm motility, despite its low correlation to fertility (Söderquist et al. 1991, Kjaestad et al. 1993). Although no single in vitro method has yet been designed to predict the fertilizing ability of a given semen sample, the quantitation of viable spermatozoa (with intact plasma and acrosome membranes, and metabolically active) seems to be most promising (Woelders et al. 1991). The present report describes the use of a bioluminiscence technique to determine ATP-levels and a novel supravital stain (using fluorescent dyes) to assess the amount of viable spermatozoa in frozen/thawed semen from 3 A.I. dairy bulls with significantly different motility after thawing.     

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO(2) and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R(2)=63%); percentage of oocytes that interacted with spermatozoa and BCF (R(2)=73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R(2)=64%) and velocity straight line (VSL; R(2)=64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

Correlations among assays of porcine semen quality following cryopreservation

Correlations between in vitro tests of semen quality, used to predict the in vivo fertilizing potential of sperm, indicate that the tests may substitute for each other in predicting fertilizing potential. Lack of correlation between tests suggest that both tests should be used to estimate the fertilizing potential. The purpose of this study was to establish correlations between several in vitro tests of porcine semen quality following freezing. Tests of motility with and without caffeine, spermatozoa with normal apical ridges, sephadex filtration with and without caffeine and acrosin activity were all correlated with each other. Correlations among these tests ranged from 0.45 to 0.83 (P<0.05). Assays for glutamic oxalacetic transaninase (GOT) were not consistently correlated with other tests. None of these tests of semen quality were correlated with the sperm penetration assay except for the test of motility without caffeine, which was correlated with the number of penetrations per hamster oocyte (r = 0.71, P<0.05).     

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14? fluorescent stain), mitochondrial activity (Mitotraker? fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer - CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

Dimethyl-acetamide as a cryoprotectant for rainbow trout spermatozoa

Dimethyl-acetamide was evaluated as a cryoprotectant for rainbow trout (Oncorhynchus mykiss ) semen on the basis of motility, percentage of dead spermatozoa as determined by fluorometry, and fertility of frozen-thawed spermatozoa. Dimethyl-acetamide performed significantly better (P<0.05) than the conventional cryoprotectant, dimethyl sulfoxide, by all evaluation methods. An extender comprised of 0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4) 7H(2)O, 7.5 g/l L-alpha-lecithin and 10 % DMA showed promise for cryopreserving rainbow trout spermatozoa. Future evaluation of new extenders should be carried out by several in vitro techniques and fertility measurements to present a complete assessment of an extender's ability to cryopreserve sperm cells.     

Importance of sperm-binding assays for fertility prognosis of porcine spermatozoa

There has been a considerable effort to establish correlations between the outcome of in vitro sperm-binding assays and the fertility achieved by individual males under conditions of commercial AI. During passage through the oviduct, a fertilizing spermatozoon has to bind to and interact with several targets. Generally, it is assumed that these interactions can be mimicked by in vitro binding assays. However, there is little evidence that assays based on zona binding, zona penetration, or IVF: (a) have been adequately validated; (b) provide data with a high degree of correlation to a boar of average fertility; (c) provide accurate predictions as to pregnancy rate and litter size from a given boar when used for commercial AI. This is due partly to the variability in measurements of pregnancy rate and litter size in a commercial setting and partly to the fact that sperm fertility is multifactorial. A recently developed in vitro test is based on the fact that spermatozoa bind in vivo to oviduct epithelium, creating a functional sperm reservoir, and that fertilization-competent spermatozoa are released in a time-dependent manner from these cells. Mating or insemination occurs usually hours before ovulation thus rendering such temporary sperm binding to the epithelial cells, a prerequisite for successful sperm-oocyte interaction. In vitro binding of porcine spermatozoa to explants derived from fresh oviduct epithelium may provide a useful test system to predict fertility, although detailed validation has not been published. The sperm-oviduct-binding assay tests for multifunctional characteristics of the plasma membrane and may be a valuable in vitro test to identify subfertile boars. We believe that boar subfertility might be indicated in vitro by reduced capacity of his spermatozoa to bind to oviductal cells and that this may provide information as to whether an adequate sperm reservoir will presumably be established in vivo from the sperm population that successfully has passed the barriers of the utero-tubal junction.     

Integration of future biotechnologies into the equine industry

There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on thousands of spermatozoa in a matter of seconds. Development of procedures for insemination of mares with relatively small numbers of spermatozoa has the potential to change how stallions and their semen are managed. This review discusses the use of insemination of fresh, frozen and sex-sorted spermatozoa in relatively small numbers compared with conventional insemination technologies. The recent acceptance of frozen-thawed semen by many of the major breed registries has stimulated an increase in research on frozen semen. Many of the studies have focused on identifying damage during the freezing and thawing process. Numerous studies also have been conducted to modify freezing extenders so that the sperm are protected during the freezing and thawing process. The production of in vitro-produced embryos is extremely limited in the horse due to the failure of in vitro fertilization. However, intracytoplasmic sperm injection (ICSI) has been used for the production of foals from stallions that have less than typical sperm numbers or from stallions that have died and a limited quantity of frozen semen is available. This technique has been used by several laboratories to produce embryos in vitro. The breeder and veterinarian now have access to techniques that allow assessment of semen quality, improvement of procedures for freezing and thawing and insemination of mares with fewer numbers of spermatozoa. It is likely that the next decade will also produce tremendous advances in semen technologies that can be utilized in the horse industry.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

Assessing the quality of raw semen: a review

An analysis of semen characteristics can provide a reasonable basis upon which to develop a strategy for maximizing the fertility of a stallion. However, the repeatability of semen characteristics between ejaculates within stallions is low to moderate. Factors such as season, collection technique, frequency of collection and disturbances in spermatogenesis contribute to this variation. Fertility can, however, be influenced by a host of other factors besides semen characteristics, including management, semen handling procedures and the number of mares being bred. Parameters usually included in a conventional evaluation of raw semen quality are volume, sperm concentration, total number of spermatozoa in the ejaculate, percentages of motile spermatozoa, sperm morphology, seminal pH, longevity of sperm motility and bacteriological status. Although, these evaluations provide a lot of information, their correlations with fertility are somewhat conflicting. However, it seems likely that the prediction of male fertility could be improved if additional parameters based on the functional characteristics of spermatozoa were to be used. Several functional tests have been investigated, such as the use of fluorescent stains as a marker for cell membrane integrity, sperm-oocyte binding tests and the hypoosmotic swelling test. In this study, emphasis is placed on sperm motility and sperm morphology, but in addition, some functional tests are also discussed.     

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

Effect of bovine serum albumin on motility and fecundity of turkey spermatozoa before and after storage.

Motility characteristics of turkey spermatozoa before and after storage for 24 h at 7 degrees C in diluent with and without bovine serum albumin (BSA; 1% final concentration) were measured by computer-assisted semen analysis. BSA significantly increased the percentage of motile spermatozoa and sperm velocity, linearity, lateral head displacement and beat frequency in each treatment, but BSA in fresh or stored semen in diluent did not augment hen fertility over 15 weeks of egg production. Fatty-acid-free BSA, globulin-free BSA and Fraction V BSA all significantly increased each sperm motility characteristic compared with semen in diluent alone. The lack of correlation between sperm motility and fecundity emphasizes the need to develop procedures for semen evaluation that accurately predict the fertilizing capacity of an aliquot of semen.     

Relationship between sperm-zona pellucida binding assays and the 56-day nonreturn rate of cattle inseminated with frozen-thawed bull semen

Assays based on sperm-zona pellucida binding have been developed as diagnostic tests to predict the fertilizing potential of mammalian spermatozoa. Recently, we reported on the development of a sperm-zona pellucida binding assay (SZBA) for bull spermatozoa. The aim of the present study was to develop a hemi-zona assay (HZA) for bull spermatozoa and to investigate the relationship between SZBA and HZA outcomes and in vivo fertility. Frozenthawed semen samples from 8 fertile Swedish Red and White bulls (one ejaculate per bull) designated as the test semen samples and a single ejaculate from a fertile Holstein-Friesian bull designated as the control semen sample were used in this study. In the SZBA, 2 groups of 20 oocytes per semen test sample and in the HZA a minimum of 6 matching pairs of hemizonae were used for comparison of sperm binding with control semen. Sperm binding to matching hemi-zonae of individual semen samples was equal, and clearly demonstrated the feasibility of the HZA for cattle. A significant correlation was found between the SZBA and the HZA indices obtained from the different semen test samples (r = 0.42, P < 0.001; n = 67). There was no significant relation between the SZBA indices and the 56-d nonreturn rate of the test samples. However, the HZA indices of the semen test samples and the 56-d nonreturn rate were significantly correlated (r = 0.46, P < 0.0001; n = 67). It is concluded that HZA can be regarded as a potential assay for predicting the fertilizing ability of bovine semen samples. However, further studies using more semen samples are necessary to confirm this view.     

Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis

The fertility of liquid-preserved boar semen declines during storage at 17°C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17°C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability.     

Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.     

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

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 75mOsm 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 3h 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 3h 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.