Sperm Subpopulations Research Articles

Sperm subpopulations in ejaculated sperm and spermatozoa recovered from ovine epididymides up to 48 h after death

The objectives of this study were threefold: to identify subpopulations of sperm based on the kinetics of frozen/thawed sheep epididymal spermatozoa or semen collected with an artificial vagina; to evaluate the effects on sperm subpopulations in the thawed samples of post mortem storage at room temperature and the addition of 20% of seminal plasma to the freezing extender and to correlate the percentage of subpopulations with gestation rate following artificial intrauterine insemination. The categorization of the subpopulations was based on sperm kinetic data from Computer Assisted Sperm Analysis (CASA). A hundred ewes were inseminated with thawed spermatozoa and gestation rate was correlated with the proportions of each subpopulation using Pearson correlation matrix and linear regression. Three distinct subpopulations were identified in the thawed samples of either ovine ejaculate collected in artificial vaginas (AV) or ovine spermatozoa retrieved from the cauda epididymis. Subpopulation 1 (SP1) was characterized by spermatozoa with slow and non-linear motion, subpopulation 2 (SP2) was classified as hyperactived spermatozoa and subpopulation 3 (SP3) was composed of spermatozoa with fast, linear motion. The largest subpopulation in all groups was SP1. The semen collected in an artificial vagina had a higher (P<0.05) percentage of SP2 and lower (P<0.05) percentage of SP1 when compared to spermatozoa recovered after death. Increasing time of storage after death had a detrimental effect on sperm samples, increasing (P<0.05) the percentage of SP1 and decreasing (P<0.05) SP2. Length of storage after death was the only variable that influenced, with an inversely proportional relationship, SP3. In samples stored for 48h after death no SP3 spermatozoa were present. The addition of seminal plasma to the cryopreservative decreased (P<0.05) the subpopulation of hyperactived spermatozoa (SP2). We conclude that, after thawing there are three sperm subpopulations in the spermatozoa obtained from the cauda epididymides and the semen collected in AVs and that the relative proportions of these subpopulations varies with the time of storage post mortem and the presence of 20% of seminal plasma in the extender. However, we conclude that these subpopulations do not correlate with fertility after intrauterine artificial insemination.

Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na.

In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+ -K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p<0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p<0.05) in intracellular Na+ . These changes occurred in relation to activation of caspase 3 (p<0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+ -K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+ -K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.

Effect of sexual steroids on boar kinematic sperm subpopulations.

Here, we show the effects of sexual steroids, progesterone, testosterone, or estradiol on motility parameters of boar sperm. Sixteen commercial seminal doses, four each of four adult boars, were analyzed using computer assisted sperm analysis (CASA). Mean values of motility parameters were analyzed by bivariate and multivariate statistics. Principal component analysis (PCA), followed by hierarchical clustering, was applied on data of motility parameters, provided automatically as intervals by the CASA system. Effects of sexual steroids were described in the kinematic subpopulations identified from multivariate statistics. Mean values of motility parameters were not significantly changed after addition of sexual steroids. Multivariate graphics showed that sperm subpopulations were not sensitive to the addition of either testosterone or estradiol, but sperm subpopulations responsive to progesterone were found. Distribution of motility parameters were wide in controls but sharpened at distinct concentrations of progesterone. We conclude that kinematic sperm subpopulations responsive to progesterone are present in boar semen, and these subpopulations are masked in evaluations of mean values of motility parameters. © 2017 International Society for Advancement of Cytometry.

Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis.

Does dynamin regulate human sperm acrosomal exocytosis? Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. N/A. This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.

Use of Assisted Reproduction Techniques in Male Pathologies: Molecule Based Sperm Selection Methods

Infertility is a widespread problem that is found in over 15% of couples. In 50 % of the cases, the male factor is the key. In order to improve pregnancy rates in assisted reproductive techniques (ART), new methods to select sperm subpopulations are required. These methods are under constant evaluation, and their possible genetic and epigenetic effects on the offspring must be considered. New concepts such as DNA fragmentation and sperm apoptosis are being introduced in andrology and are considered to develop new techniques. Recently, molecule-based methods have been developed for the purpose of overcoming deficiencies in classical selection methods that involve sperm morphology and movement. One of these methods is physiologic intracytoplasmic sperm injection (PICSI), which selects spermatozoa through their property to bind to hyaluronic acid, a component of the cell-oocyte complexes that acts as a natural selector of spermatozoa. But the top rated method is based on the property of the protein annex in A5 (ANXA V) to bind to phosphatidylserine (PS), a marker of apoptosis. Magnetic-Activated Cell Sorting (MACS) conjugated with ANXA V (MACS-ANXA V) is used to remove apoptotic sperm with exposed PS from a semen sample. The use of MACS-ANX V seems to produce good quality sperm when it is evaluated by in vitro assays. But when MACS-ANXA V is used previous to ART, controversies persist regarding the benefits of sperm selection in reproductive outcomes. In this review, we consider the use of MACS-ANXA V, particularly in cases of male pathologies.

The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm

Adding cryopreservation media with reduced glutathione (GSH) has previously been shown to maintain the motility, membrane integrity and fertilizing ability of frozen-thawed boar sperm, although the effects of GSH on good (GFE) and poor freezability (PFE) ejaculates rely upon the intrinsic ejaculate freezability. The resilience to withstand freeze-thawing procedures has previously been related to the existence of a specific distribution of motile sperm subpopulations, which differs between GFE and PFE. Thus, the main aim of this study was to determine whether the addition of GSH to freezing media has any impact on the distribution of motile sperm subpopulations in GFE and PFE. With this purpose, 18 GFE and 13 PFE were cryopreserved with or without 2 mM GSH. Sperm quality and motile subpopulations were evaluated at 30 min and 4 h post-thawing. Three subpopulations were identified and the percentages of spermatozoa belonging to the fastest and most linear subpopulation, which was referred as ‘SP1’, decreased over post-thawing time. Good freezability ejaculates that were cryopreserved in the presence of 2 mM exhibited a significantly higher percentage of spermatozoa belonging to SP1 than the other combinations of treatment and freezability both at 30 min (mean ± SEM: GFE-C: 16.6 ± 0.4; GFE-GSH 27.7 ± 0.6) and 4 h post-thawing (GFE-C: 7.8 ± 0.2 vs. GFE-GSH: 16.7 ± 0.4). In conclusion, the positive effect of GSH on the motility of frozen-thawed sperm is related to a specific sperm subpopulation (SP1), which could coincide with the fertile sperm one.

Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility

Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as “hyperactivated” (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables.

Effect of two freezing extenders on characteristic of fresh and frozen-thawed semen in endangered Old Kladruber stallions - A pilot study

The aim of the study was to evaluate the effect of two different extenders on sperm characteristic before equilibration and post-thaw in the endangered Old Kladruber stallions. Also, the response of individual stallions to the extenders used was tested. Semen was collected from six stallions every other day within one week. After centrifugation of the collected sperm-rich fraction, the supernatant was removed and sperm pellets were divided to two aliquots; these were diluted either with Gent (Minitube, Germany) or privately manufactured lactose-EDTA-egg yolk extender (Lact). Three cryopreserved insemination doses (IDs) from each extender (Gent and Lact) were prepared for each stallion from one collection (108 samples from six stallions in total). As a parameter of quality, the motility (computer assisted sperm analysis), viability (fluorescence staining), and morphology (eosin/nigrosine staining) were evaluated after dilution with freezing extenders (fresh) and after thawing (frozen-thawed). The different effects of chosen extenders on the quality of fresh semen were only manifested in higher kinematic parameters of sperm when the Lact extender was used. However, in frozen-thawed samples, the Gent extender yielded significantly better results in all of the evaluated parameters. The representation of sperm subpopulation was significantly influenced by extender in fresh as well as frozen-thawed samples; moreover, we found a significant effect of freezing on the distribution of these subpopulations. The response of individual stallions to chosen extenders was evident in the different quality of fresh as well as frozen-thawed IDs; Gent extender yielded better frozen-thawed IDs. Based on our results, among others describing quality parameters of ejaculate in endangered Old Kladruber stallions, we can recommend using Gent extender for the production of frozen-thawed IDs.

Immature and mature sperm morphometry in fresh and frozen-thawed falcon ejaculates

Sperm morphometry is one characteristic which may be useful in prediction of fertility and sperm freezability in a species. Knowledge of the sperm characteristics of the ejaculate and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. The aim of the current study was to provide a general description of the sperm from two falcon species (Peregrine falcon Falco peregrinus peregrinus/brookei and Gyrfalcon Falco rusticolus) including immature sperm, sperm head morphometric descriptors, and the existence of mature sperm subpopulations.Semen samples were collected by massage and voluntary false copulation and diluted with Lake and Ravie medium. Smears were prepared of the diluted samples, stained with Hemacolor®, and subjected to: 1) morphological analysis (bright field optical microscopy), and 2) computerised morphometric analysis; each sperm head was measured for length, width, area and perimeter. In addition, in the Gyrfalcon, pooled semen was frozen in pellets using DMA as a cryoprotectant and the analyses repeated after thawing. The mean percentage of immature sperm (spermatocytes and spermatids) was similarly high in all species/subspecies: Brookei Peregrine falcon (F. p. brookei) 55.5%, European Peregrine falcon (F. p. peregrinus) 65.5% and Gyrfalcon 64.7%. Clustering analyses identified four subpopulations of mature spermatozoa with different morphometric characteristics (P < 0.001). The relative proportions of these subpopulations were similar in all three species. The mean values recorded for the morphometric variables of the four subpopulations were, however, lower (P < 0.001) in the thawed Gyrfalcon samples than in fresh samples. The results support the idea of pleiomorphy as a characteristic of raptor mature sperm. This finding, plus that of the existence of four sperm subpopulations with different morphometric characteristics, may be important in the future development of cryopreservation protocols for falcon sperm.

Motility of sea urchin Paracentrotus lividus spermatozoa in the post-activation phase

The characterization of sperm motility patterns, particularly post-activation changes, is the first step in setting up species-specific protocols involving gamete management and embryo production, for both aquaculture and laboratory research purposes. This study is aimed at the characterization of the sperm motility pattern of the purple sea urchin Paracentrotus lividus. Semen samples were individually diluted in artificial sea water for sperm motility activation. They were then incubated at 18°C for up to 24 hr. Motility was evaluated on dilution, and 1 hr, 3 hr and 24 hr after activation, by computerized analyser. The semen fertilization capacity was also evaluated. Under our experimental conditions (dilution 1:1,000 in artificial sea water plus 0.05% BSA, 18°C, in the dark), P. lividus semen remained viable for up to 24 hr, as the total motile sperm and the fertilization percentages did not change significantly during the incubation time. In contrast, the mean curvilinear velocity and the subpopulation of rapid sperm (those having a curvilinear velocity > 100 µm/s) slightly but significantly decreased after 3 hr, thereafter remaining unchanged for up to 24 hr after activation. In conclusion, our results show that diluted P. lividus semen can be used for a longer period than that of most fish species, with no need for motility inhibition procedures, supporting its wider use in laboratory research. In addition, the development of artificial fertilization protocols for aquaculture production is simplified by long-lasting sperm motility.

Measuring Male-to-Male Differences in Fertility or Effects of Semen Treatments.

Fertility is a convenient but meaningless term unless the outcome measure is stipulated and accounts for dependence of male fertility on the female population. We describe outcome measures and detail the impacts of the physiological status of each female and her external environment, as well as management imposed by humans. We explain the dominant role of the female reproductive tract as a series of hurdles for sperm seeking an ovum. Each spermatozoon in an ejaculate is unique, although usually most are morphologically similar. Semen seemingly contains three subpopulations of sperm, based on fate within a female and role in hampering the success of the ultimate winning spermatozoon; we define these subpopulations. The numerical size of each subpopulation placed into a female determines the shape of the dose-response curve leading to possible live young. Heterospermic artificial insemination provides far greater sensitivity to detect differences, partly because the female environment is identical for each comparison.

Toward an integrative and predictive sperm quality analysis in Bos taurus

There is a need to develop more integrative sperm quality analysis methods, enabling researchers to evaluate different parameters simultaneously cell by cell. In this work, we present a new multi-parametric fluorescent test able to discriminate different sperm subpopulations based on their labeling pattern and motility characteristics. Cryopreserved semen samples from 20 Holstein bulls were used in the study. Analyses of sperm motility using computer-assisted sperm analysis (CASA-mot), membrane integrity by acridine orange-propidium iodide combination and multi-parametric by the ISAS®3Fun kit, were performed. The new method allows a clear discrimination of sperm subpopulations based on membrane and acrosomal integrity, motility and morphology. It was also possible to observe live spermatozoa showing signs of capacitation such as hyperactivated motility and changes in acrosomal structure. Sperm subpopulation with intact plasma membrane and acrosome showed a higher proportion of motile sperm than those with damaged acrosome or increased fluorescence intensity. Spermatozoa with intact plasmalemma and damaged acrosome were static or exhibit weak movement. Significant correlations among the different sperm quality parameters evaluated were also described. We concluded that the ISAS®3Fun is an integrated method that represents an advance in sperm quality analysis with the potential to improve fertility predictions.

Sperm subpopulations, buffalo semen, sperm kinematics, cryopreservation, computer assisted sperm analyzer.

Sperm subpopulations, buffalo semen, sperm kinematics, cryopreservation, computer assisted sperm analyzer.

Identification of sperm morphometric subpopulations in cooled-stored canine sperm and its relation with sperm DNA integrity.

The aims of this study were to (i) identify different morphometric subpopulations in cooled-stored canine sperm and their patterns of distribution during cool-storage for up to 240hr and (ii) determine whether or not morphometric sperm subpopulations (sP) are related to sperm DNA integrity. For that purpose, morphometric parameters were analysed by computer-assisted sperm analysis (CASA) and sperm DNA fragmentation (sDFi) using the sperm Halomax test. Four morphometric sperm heads subpopulations were identified: sP1 (large and rounded), sP2 (large and elongated), sP3 (small and rounded) and sP4 (small and elongated). sP1 was the most predominant subpopulation for up to 72hr and thereafter sP3 increased progressively. sDFi increased after 48hr of cool-storage. Although sP3 showed a positive correlation with sDFi, and both increased over time, it could not be ensured that only the sperm with fragmented DNA are accumulated in sP3. In conclusion, sP3 and DNA fragmentation increased progressively during cool-storage, becoming possible indicators of sperm damage. However, it cannot be concluded that sP3 only contains sperm with fragmented DNA.

Comparison of sperm subpopulation structures in first and second ejaculated semen from Japanese black bulls by a cluster analysis of sperm motility evaluated by a CASA system

In the present study, bull sperm in the first and second ejaculates were divided into subpopulations based on their motility characteristics using a cluster analysis of data from computer-assisted sperm motility analysis (CASA). Semen samples were collected from 4 Japanese black bulls. Data from 9,228 motile sperm were classified into 4 clusters; 1) very rapid and progressively motile sperm, 2) rapid and circularly motile sperm with widely moving heads, 3) moderately motile sperm with heads moving frequently in a short length, and 4) poorly motile sperm. The percentage of cluster 1 varied between bulls. The first ejaculates had a higher proportion of cluster 2 and lower proportion of cluster 3 than the second ejaculates.

Use of Annexin V based Sperm Selection in Assisted Reproduction

Innovative methods to select sperm subpopulations with the best fertilizing ability are needed in assisted reproductive techniques (ART) in order to improve fertilization and pregnancy rates, while also considering possible epigenetic effects on the offspring. Molecular based selection methods are searched for, under the premise that they could be an improvement over classical selection by morphology and movement. One of these methods sustains the elimination of sperm that can bind to annexin A5 (ANX V), coupled to paramagnetic beads, through the phosphatidyl-serine exposed on their membranes upon apoptosis. Although reports accumulate about the use of this method, controversy persists as to the benefits of ANX V based sperm selection in ART. In this review we consider the arguments in favour and against this method and conclude that to the moment the evidence does not support MACS regular use in ART.

Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes.

The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.

Sperm kinematic, head morphometric and kinetic-morphometric subpopulations in the blue fox (Alopex lagopus).

This work provides information on the blue fox ejaculated sperm quality needed for seminal dose calculations. Twenty semen samples, obtained by masturbation, were analyzed for kinematic and morphometric parameters by using CASA-Mot and CASA-Morph system and principal component (PC) analysis. For motility, eight kinematic parameters were evaluated, which were reduced to PC1, related to linear variables, and PC2, related to oscillatory movement. The whole population was divided into three independent subpopulations: SP1, fast cells with linear movement; SP2, slow cells and nonoscillatory motility; and SP3, medium speed cells and oscillatory movement. In almost all cases, the subpopulation distribution by animal was significantly different. Head morphology analysis generated four size and four shape parameters, which were reduced to PC1, related to size, and PC2, related to shape of the cells. Three morphometric subpopulations existed: SP1: large oval cells; SP2: medium size elongated cells; and SP3: small and short cells. The subpopulation distribution differed between animals. Combining the kinematic and morphometric datasets produced PC1, related to morphometric parameters, and PC2, related to kinematics, which generated four sperm subpopulations – SP1: high oscillatory motility, large and short heads; SP2: medium velocity with small and short heads; SP3: slow motion small and elongated cells; and SP4: high linear speed and large elongated cells. Subpopulation distribution was different in all animals. The establishment of sperm subpopulations from kinematic, morphometric, and combined variables not only improves the well-defined fox semen characteristics and offers a good conceptual basis for fertility and sperm preservation techniques in this species, but also opens the door to use this approach in other species, included humans.

Thawing boar semen in the presence of seminal plasma improves motility, modifies subpopulation patterns and reduces chromatin alterations.

Seminal plasma could have positive effects on boar semen after thawing. In the present study we investigated changes in the motility and chromatin structure in spermatozoa over 4h incubation (37°C) of boar semen thawed in the presence of 0%, 10% or 50% seminal plasma from good-fertility boars. Cryopreserved doses were used from seven males, three of which were identified as susceptible to post-thawing chromatin alterations. Motility was analysed by computer-aided sperm analysis every hour, and data were used in a two-step clustering, yielding three subpopulations of spermatozoa (slow non-linear, fast non-linear, fast linear). Chromatin structure was analysed using a sperm chromatin structure assay and flow cytometry to determine the DNA fragmentation index (%DFI) as a percentage, the standard deviation of the DFI (SD-DFI) and the percentage of high DNA stainability (%HDS), indicating chromatin compaction. Thawing without seminal plasma resulted in a rapid loss of motility, whereas seminal plasma helped maintain motility throughout the incubation period and preserved the subpopulation comprising fast and linear spermatozoa. The incidence of chromatin alterations was very low in samples from non-susceptible males, whereas samples from males susceptible to post-thawing chromatin alterations exhibited marked alterations in%DFI and%HDS. Seminal plasma partly prevented these alterations in samples from susceptible males. Overall, 50% seminal plasma was the most efficient concentration to protect motility and chromatin. Some changes were concomitant with physiological events reported previously (e.g., semen thawed with 50% seminal plasma increased the production of reactive oxygen species and yielded higher fertility after AI). Thawing in the presence of seminal plasma could be particularly useful in the case of samples susceptible to post-thawing chromatin damage.

Fish sperm subpopulations: Changes after cryopreservation process and relationship with fertilization success in tambaqui (Colossoma macropomum)

Fish tambaqui (Colossoma macropomum) is the native Brazilian fish with the highest agricultural production under intensive aquaculture in South America. However, the decrease in the genetic variability in fish farms has become necessary the improvement of cryopreservation process through new statistical studies of spermatozoa (like subpopulation studies). The evaluation of the kinetic data obtained with a computer-assisted sperm analysis system, applying a two-step cluster analysis, yielded in tambaqui three different subpopulations in fresh sperm: SP1, considered as a slow nonlinear subpopulation; SP2, considered as a fast nonlinear subpopulation, and finally; SP3, considered as a fast linear subpopulation. For cryopreserved sperm, the cluster analysis yielded only two sperm subpopulations: SP1′, considered as a slow nonlinear subpopulation and SP2′, which seemed to be an intermediate subpopulation (showing medium motility and velocity values) merged from SP2 and SP3 obtained from fresh sperm. Coefficients of correlation (r) and determination (r2) between the sperm subpopulations from fresh sperm and the fertilization rates were calculated, and SP2 and SP3 (the fast-spermatozoa subpopulations) showed a high-positive correlation with the fertilization rates (r = 0.93 and 0.79, respectively). In addition, the positive significant correlations found in curvilinear velocity (r = 0.78), straight line velocity (r = 0.57), and average velocity (r = 0.75) indicate that sperm kinetic features seem to be a key factor in the fertilization process in tambaqui, as occur in other fish species.