Centrifugal Countercurrent Distribution Research Articles

Technologies for Separation of ‘X’ and ‘Y’ Spermatozoa in Bovines: An Overview

Several approaches to sperm sexing such as Albumin gradient, Identification of H-Y antigen, Free-flow electrophoresis, Detection of sex-specific proteins, Centrifugal counter current distribution, Volumetric differences, Percoll density gradient, Flow-cytometry, Laser ablation, etc were put forward by the scientists. However, the alteration in DNA content between X- and Y-sperm remains the key and only discriminating feature for sperm sorting based on which only Flow cytometry and Laser ablation techniques are being used commercially. Flow cytometry is based on precise staining of the DNA of sperm with the nucleic acid-specific dye, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized highspeed sorter. Laser ablation (LA) is a system and method for sorting a mixture of stained particles in a fluid flow path. The system and method includes an electromagnetic radiation source for exciting fluorescence emissions from the stained particles, a photodetector for detecting the fluorescence emissions, a processor for classifying the stained particles, and a photo-damaging laser for damaging selected particles in the flow path.

Centrifugal countercurrent chromatography to elucidate surface differences of adipose tissue-derived stem cells

The current methods of isolation of adipose tissue-derived stem cells result in a heterogeneous population that might interfere with their differentiation potential and makes it difficult to compare the results between different groups. Partition in aqueous two-phase systems is one of the few techniques that separate cells on the basis of surface properties, gentle enough to isolate fragile cell types in isotonic conditions without altering their structure, and can be easily scaled. In this study, stem cells isolated from human adipose tissue seeded and expanded in vitro were fractionated by using centrifugal countercurrent distribution in an aqueous two-phase system. The separated subpopulations revealed the high heterogeneity of adipose tissue-derived stem cell samples. Comparative partition analyses showed that aging induces a loss of heterogeneity, which is not due to a loss of cell viability associated to age. The phosphatidylserine externalization, an apoptotic feature, is the main factor in cell partition that results in a decreased hydrophobicity of the cell surface. This procedure may be suitable for separating adipose tissue-derived stem cell populations enriched in some functional and/or structural surface characteristics. The possibility of a very effective separation of different subpopulations in opposite phases would be an interesting development of the method.

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20°C or cooled down to 5°C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/PI. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V, and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5°C than at 20°C, which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature.

High pre-freezing dilution improves post-thaw function of ram spermatozoa

Despite considerable cryobiology research there is no industry standard for the concentration to which ram spermatozoa should be diluted before freezing. Ram semen is highly concentrated and often frozen at a high sperm concentration, necessitating the use of small laparoscopic insemination doses. The aim of this paper was to ascertain the effect of dilution on the integrity of frozen-thawed ram spermatozoa. In the first experiment, spermatozoa were extended with a Tris-buffered diluent before freezing or after thawing to yield a final sperm concentration of 20 × 10 6/ml, or were not diluted. Motility characteristics, viability and acrosome integrity of spermatozoa were analysed over a 6 h incubation period at 37 °C. In the second experiment, spermatozoa were either diluted before freezing, subjected to sex-sorting or not diluted before freezing. Thawed spermatozoa were separated into sub-populations using centrifugal counter-current distribution (CCCD) and the profile of partition and functional integrity (viability, chlortetracycline status and Annexin-V binding) in the sub-populations assessed. Dilution before freezing significantly improved post-thaw viability, acrosome integrity and total motility whereas dilution post-thaw decreased viability and motility of spermatozoa. Sperm heterogeneity, as assessed by CCCD profile, was not different for control, diluted and sex-sorted spermatozoa. Analysis of CCCD sub-populations showed the proportion of functional cells (displaying the F-Pattern or no PS translocation) was similar for all sperm types. The results show that ram spermatozoa retain normal function at higher pre-freeze dilution rates than are commonly used in the sheep industry. The application of these findings would result in more practicable and functional artificial insemination doses.

Sperm survival and heterogeneity are correlated with fertility after intrauterine insemination in superovulated ewes

Efficient animal production involves accurate estimations of fertilizing ability. One key factor is the plasma membrane of the sperm cell, which is actively involved in the cascade of events before oocyte fusion. Many methods are used to analyze the characteristics of this membrane, including partition in aqueous two-phase systems which is an efficient method to analyze sperm surface changes accounting for loss of viability and different functional states. Centrifugal countercurrent distribution (CCCD) analysis can also be used in an aqueous two-phase system to determine the relationship between sperm parameters and in vivo fertility in ewes. In a previous work, we found a significant correlation between two post-CCCD parameters (heterogeneity and recovered viability) and field fertility when the same sample was used after cervical AI. The present study was intended to find out whether the control of several external factors that affect reproductive efficiency is able to increase the correlation coefficient between post-CCCD parameters and fertility. Thus, 90 Rasa aragonesa ewes were controlled on the same farm and received intrauterine inseminations using the same technical equipment. The fertilizing ability of the raw semen and sperm samples selected by a dextran/swim-up process was compared using a low number of spermatozoa per insemination (7 × 10 7) to enhance possible fertility differences. A new post-CCCD parameter was considered; the loss of viability (LV) occurred during the CCCD process. This variable denotes the sperm surviving ability and corresponds to the difference between the total number of viable cells loaded and recovered after the CCCD run. The mean fertility of eight sperm control samples was 60% (range: 25–76%), and there was no significant correlation between standard parameters and in vivo fertility. LV ranged from 2 to 69% (average 27%) and was negatively correlated with fertility ( r = −0.914, P < 0.01). Ejaculate heterogeneity (H) ranged from 20 to 47% and was positively, but not significantly, correlated with fertility ( r = 0.391). A predictive equation for fertility was deduced by multiple analysis with a very high correlation coefficient ( r = 0.967), and level of significance ( P < 0.005): predictive fertility PF = 52.546 − 0.594 LV + 0.665 H. The mean fertility of 13 swim-up selected samples was 63% (range: 25–86%). Again, only parameters derived from the CCCD analysis were highly correlated with fertility, especially LV and H ( P < 0.05).

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing–thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen–thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen–thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk–yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk–yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.

Prediction of fertility by centrifugal countercurrent distribution (CCCD) analysis: correlation between viability and heterogeneity of ram semen and field fertility

The prediction of the fertilizing ability of a sire or a given insemination dose is a primary aim in the field of artificial insemination. Centrifugal countercurrent distribution analysis (CCCD) was used to determine the relationship between some sperm parameters and the in vivo fertility rate obtained with the same sample after cervical artificial insemination. A total of 522 ewes from 26 different farms was inseminated with 53 ejaculates obtained from 25 mature Rasa aragonesa rams. Semen was diluted to 1.6 x 10(9) cells ml-1 and doses of 0.25 ml were prepared and kept at 15 degrees C until used for insemination. The same ejaculates were used for analysis of standard semen parameters and CCCD analysis. Sperm motility, concentration and viability were determined before and after CCCD. Post-CCCD parameters were derived from the analysis of the profile obtained after CCCD. The recovered viability showed the highest correlation with fertility, especially in the central chambers (V2), r = 0.415, P < 0.005). The ejaculate heterogeneity also showed a positive correlation with field fertility (r = 0.23), with a tendency towards significance (P < 0.1). The mean fertility value of all ejaculates used in this study was 46.75%, ranging from 12.5% to 75.0%. Ejaculates were classified into two categories according to their fertility: higher and lower than the mean value. Only the viability recovered in the central chambers (V2) was a parameter with a predictive capacity to discriminate between the two groups (P < 0.05). A predictive equation for field fertility with a correlation coefficient r = 0.488 and a very high level of significance (P < 0.005) was deduced by multiple analysis: PF = 6.02 + 0.069V2 + 0.315H (where PF is predictive fertility, V2 is the recovered viability in the CCCD profile central chambers and H is heterogeneity).

Prediction of fertility by centrifugal countercurrent distribution (CCCD) analysis: correlation between viability and heterogeneity of ram semen and field fertility

The prediction of the fertilizing ability of a sire or a given insemination dose is a primary aim in the field of artificial insemination. Centrifugal countercurrent distribution analysis (CCCD) was used to determine the relationship between some sperm parameters and the in vivo fertility rate obtained with the same sample after cervical artificial insemination. A total of 522 ewes from 26 different farms was inseminated with 53 ejaculates obtained from 25 mature Rasa aragonesa rams. Semen was diluted to 1.6 x 10(9) cells ml-1 and doses of 0.25 ml were prepared and kept at 15 degrees C until used for insemination. The same ejaculates were used for analysis of standard semen parameters and CCCD analysis. Sperm motility, concentration and viability were determined before and after CCCD. Post-CCCD parameters were derived from the analysis of the profile obtained after CCCD. The recovered viability showed the highest correlation with fertility, especially in the central chambers (V2), r = 0.415, P < 0.005). The ejaculate heterogeneity also showed a positive correlation with field fertility (r = 0.23), with a tendency towards significance (P < 0.1). The mean fertility value of all ejaculates used in this study was 46.75%, ranging from 12.5% to 75.0%. Ejaculates were classified into two categories according to their fertility: higher and lower than the mean value. Only the viability recovered in the central chambers (V2) was a parameter with a predictive capacity to discriminate between the two groups (P < 0.05). A predictive equation for field fertility with a correlation coefficient r = 0.488 and a very high level of significance (P < 0.005) was deduced by multiple analysis: PF = 6.02 + 0.069V2 + 0.315H (where PF is predictive fertility, V2 is the recovered viability in the CCCD profile central chambers and H is heterogeneity).

Sperm washing method alters the ability of seminal plasma proteins to revert the cold‐shock damage on ram sperm membrane

Prolonged exposure of spermatozoa to seminal plasma (SP) has been found to have adverse effects on sperm function. Therefore, the separation of mammalian spermatozoa from SP is a practice routinely used in the laboratory and in assisted reproductive technology application. We have previously shown that adsorption of seminal plasma proteins (SPP) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we have compared the influence of two different semen washing methods, a dextran/swim-up or a filtration procedure, on the SPP adsorption to the sperm surface, and on their ability to recover membrane integrity of cold-shocked sperm. Seminal plasma proteins were added to cold-shocked sperm samples obtained by both washing procedures. Adsorption of proteins to the sperm membrane surface was assessed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system, as well as membrane integrity being determined by fluorescence markers. The percentage of reversion of cold-shock effect in the sample containing plasma proteins with respect to the control sample was determined by assessing the percentage of membrane-intact spermatozoa, i.e. propidium iodide-negative. The addition of 700 microg of SPP to the swim-up damaged samples caused a 32% reversion, whereas the reversal percentage found with the same amount of SPP on filtered damaged samples was 10% (p < 0.05). Likewise, the loss of heterogeneity and the decrease in viability after the cold-shock revealed by CCCD analysis were greatly reversed by the addition of SPP to sperm samples obtained by swim-up. However, this restorative effect was again much lower when SPP were added to the cold-shocked filtered sample. These results strongly suggest that the washing method influences the ability of SPP to recover membrane integrity of cold-shocked sperm. Separation of spermatozoa by swim-up accounts for higher viability recovery than that obtained by filtration washing.

Sperm washing method alters the ability of seminal plasma proteins to revert the cold-shock damage on ram sperm membrane

Prolonged exposure of spermatozoa to seminal plasma (SP) has been found to have adverse effects on sperm function. Therefore, the separation of mammalian spermatozoa from SP is a practice routinely used in the laboratory and in assisted reproductive technology application. We have previously shown that adsorption of seminal plasma proteins (SPP) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we have compared the influence of two different semen washing methods, a dextran/swim-up or a filtration procedure, on the SPP adsorption to the sperm surface, and on their ability to recover membrane integrity of cold-shocked sperm. Seminal plasma proteins were added to cold-shocked sperm samples obtained by both washing procedures. Adsorption of proteins to the sperm membrane surface was assessed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system, as well as membrane integrity being determined by fluorescence markers. The percentage of reversion of cold-shock effect in the sample containing plasma proteins with respect to the control sample was determined by assessing the percentage of membrane-intact spermatozoa, i.e. propidium iodide-negative. The addition of 700 microg of SPP to the swim-up damaged samples caused a 32% reversion, whereas the reversal percentage found with the same amount of SPP on filtered damaged samples was 10% (p < 0.05). Likewise, the loss of heterogeneity and the decrease in viability after the cold-shock revealed by CCCD analysis were greatly reversed by the addition of SPP to sperm samples obtained by swim-up. However, this restorative effect was again much lower when SPP were added to the cold-shocked filtered sample. These results strongly suggest that the washing method influences the ability of SPP to recover membrane integrity of cold-shocked sperm. Separation of spermatozoa by swim-up accounts for higher viability recovery than that obtained by filtration washing.

Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system

Seminal plasma plays an important role in maturation of spermatozoa through hormonal, enzymatic and surface-modifying events. We have previously shown that adsorption of seminal plasma proteins (SPPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm. The effect of seminal plasma proteins, obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season, on cold-shocked ram spermatozoa previously freed from seminal plasma, was analysed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers. Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa. The cold-shocked sperm viability obtained was approximately 20%. The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP, which induced restoration of the surface characteristics of viable-like spermatozoa, as well as an increase in the number of recovered viable sperm. However, this restoring effect was much lower when nbsSPPs were added, even in a sixfold higher concentration than used with bsSPPs. Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods, showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season. The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons. However, the reversion percentages obtained with nbsSPPs were significantly lower ( P<0.05) than those found with bsSPPs in both studied seasonal periods. This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition, as revealed by comparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The bands of 20, 21, 24, 36 and 67 kDa of the bsSP sample profile decreased in winter–spring SP, and were even less intensely stained in summer SP. Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands, and revealed an important decrease in the content of several bands. The 21.5 kDa band showed the highest decrease, lowering to 14% in June–August plasma with respect to the value obtained in September–December plasma.

Separation of ram spermatozoa bearing X and Y chromosome by centrifugal countercurrent distribution in an aqueous two-phase system.

The availability of reliable quantification techniques of X and Y chromosome-bearing spermatozoa in a given insemination dose would allow further approaches to their separation, which is a topic of unquestionable interest in animal production. The aim of the current work was the development of a combined approach of polymerase chain reaction and countercurrent distribution to address both objectives. First, using Sac/polymorphisms for ZFX/ZFYloci in sheep deoxyribonucleic acid, a linear correlation has been established between the densitometric quantification of the restricted fragment length polymorphisms corresponding to the amplified loci ZFX/ZFY by polymerase chain reaction and the theoretical proportions of X and Y chromosomes in standard solutions. The method, subsequently applied to semen samples, estimated an equal proportion of spermatozoa bearing each chromosome. Second, by using centrifugal countercurrent distribution in a sensitive-charge aqueous two-phase system, we achieved the separation of a sperm population enriched in Y chromosome-bearing ram spermatozoa (75%) with a high viability rate (57%).

Assessment of the acrosomal status of ram spermatozoa by RCA lectin-binding and partition in an aqueous two-phase system.

The acrosome reaction is an important marker for sperm function. Because different laboratory techniques may be used to detect this exocytotic process, the objective of this study was to investigate the use of fluoresceinated lectins to assess the acrosomal status of nonpermeabilized ram spermatozoa. In addition, we used centrifugal countercurrent distribution (CCCD) in an aqueous 2-phase system to assess the sperm surface modifications associated with the acrosome reaction by observing changes in their partition behavior. We analyzed the binding of 5-fluorescein isothiocyanate (FITC)-conjugated lectins to ram sperm to select a lectin that bound preferentially to the acrosomal region, which would allow differentiation of acrosome-intact from acrosome-damaged ram spermatozoa. Ricinus communis agglutinin (RCA) bound intensely to the anterior and weakly to the equatorial acrosomal regions. Acrosomal labeling changed when spermatozoa were induced to acrosome-react with calcium ionophore A23187. RCA acrosomal labeling significantly increased (P < .0001) after incubation (84% versus 28% in control samples). To determine if RCA lectin labeling could be used to assess the acrosomal status of fresh ram spermatozoa in suspension, we compared the percentage of acrosome-reacted sperm detected by the carboxyfluorescein diacetate/propidium iodide (CFDA/PI) double-fluorescent staining with the percentage detected by FITC-RCA labeling. The incidence of acrosome-reacted spermatozoa detected by CFDA/PI was not significantly different (P = .704; 13 comparisons in 6 different experiments) from the incidence of spermatozoa detected by FITC-RCA staining. The evaluation of the spontaneous acrosome reaction by RCA labeling (5.83%) was not significantly different (P = .644) from that assessed by CFDA/PI (6.88%). The percentage of induced acrosome reactions detected by CFDA/PI staining (56%) significantly correlated (P < .0001; r = 0.876) with that detected by RCA labeling (56.67%). We simultaneously carried out a comparative CCCD in an aqueous 2-phase system to analyze sperm surface changes associated with the acrosome reaction. Results revealed that sperm surface hydrophobicity decreased in samples that had been incubated with ionophore compared with the untreated-control samples. Likewise, RCA binding after CCCD showed that all acrosome-reacted cells were stained, whereas only 42% of cells were lectin-labeled in the untreated semen sample. This change in lectin reactivity of acrosome-reacted spermatozoa signals the presence of some deep membrane or intracellular residues that would affect partitioning. Therefore, the FITC-RCA-labeling procedure can be used to accurately assess the acrosomal status of ram spermatozoa in suspension.

Sperm-lectin agglutination combined with swim-up leads to an efficient selection of highly motile, viable and heterogeneous ram spermatozoa

Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high-quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56 × 10 9 cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus. With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate.

Improvement of Ram Sperm Cryopreservation Protocols Assessed by Sperm Quality Parameters and Heterogeneity Analysis

Semen cryopreservation processes generally decrease sperm quality and fertility rate, but this is highly dependent on the specific susceptibility of sperm cells to low temperatures. In order to study the effect of cooling, freezing, and thawing on ram spermatozoa, sperm viability (plasma membrane integrity), HOS test (hyposmotic swelling test), and individual motility were assessed in fresh, cooled, and frozen–thawed samples. The cryoprotective ability of four extenders [Triladyl-yolk (20% Triladyl, 20% egg yolk), Salamon (9.9% raffinose, 2% sodium citrate, 15% egg yolk, 5% glycerol), I.N.I.A. (6.31% Tes, 1.51% Tris, 6% egg yolk, 3% glycerol), and Fiser (F1: 3.25% Tris, 9.3% fructose, 1.7% citric acid, 25% egg yolk, 2% glycerol; F2: 0.68% sodium citrate, 0.15% TES, 0.36% glycine, 10.18% lactose, 1.18% raffinose, 0.5% fructose, 3.95% dextran (150,000–200,000 MW); 12% of the obtained solution was replaced by the same volume of glycerol)] was tested, as well as the effect of adding various compounds (bovine lactalbumin and serum albumin, vitamin E, dithiothreitol, bull seminal plasma, glycine betaine, proline, phosphatidylcholine, and cholesterol) to Fiser's medium. Simultaneously, centrifugal counter-current distribution in an aqueous two-phase system was carried out to analyze the effect of the four extenders on sperm surface heterogeneity. The results showed that ram spermatozoa undergo a dramatic loss of heterogeneity, viability, motility, and positive response to the HOS test during freezing and thawing. These changes were partially prevented by diluting samples in Fiser's extender containing egg yolk and glycerol. A considerable increase in sperm quality with respect to that obtained in Fiser's control sample was achieved by the addition of bovine lactalbumin, vitamin E, bovine serum albumin, and bovine and ram seminal plasma.

Improvement of cryopreserved ram sperm heterogeneity and viability by addition of seminal plasma.

The effect of seminal plasma, as well as some seminal plasma fractions, on the heterogeneity and viability of frozen-thawed ram spermatozoa was studied. Fresh and frozen sperm samples were simultaneously assessed by a routine semen-quality assay and centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system analysis. All samples used for this study were obtained from Salz rams by an artificial vagina and frozen according to the Fiser method. Sperm samples, consisting of either raw or washed semen, were frozen in the presence of either whole ram seminal plasma (RSP) or > 10 kDa seminal plasma fraction. Two control samples were considered: one diluted in Fiser's extender and an undiluted sample. Cell-membrane integrity (viability), hypoosmotic swelling (HOS) test, and sperm motility were assessed in all cases after collection, before freezing (at 5 degrees C), after thawing, and after removing the diluent before CCCD analysis. Our results show that sperm cell partition behavior was dependent on the sperm treatment and that the freezing-thawing process accounted for a clear loss of sperm heterogeneity and membrane integrity. These losses were less apparent when seminal plasma had been removed from semen samples before freezing. In addition, washed semen frozen in the presence of either > 10 kDa seminal plasma fraction or whole re-added seminal plasma maintained a higher rate of sperm heterogeneity and viability.

Surface changes of ram spermatozoa by adsorption of homologous and heterologous seminal plasma proteins revealed by partition in an aqueous two-phase system.

Ram spermatozoa freed from seminal plasma by a dextran 'swim-up' procedure were incubated with Tween 20 and fractionated into motile (PEG-rich) and stationary (dextran-rich) fractions by centrifugal countercurrent distribution (CCCD) in an aqueous dextran-Ficoll-polyethylene glycol (PEG) two-phase system. Increasing concentrations of Tween 20 resulted in greater amounts of extracted protein and lower cell viability. Addition of bull seminal plasma increased the proportion of live cells, whereas ram seminal plasma increased the proportion of stationary cells. Proteins isolated from each type of seminal plasma restored the CCCD profile of treated spermatozoa to the right, this effect being reduced when proteins were thermally denatured. Bovine serum albumin induced a slight displacement to the left. No restoration of profile was achieved when ram spermatozoa were exposed to proteins from bull seminal plasma in the presence of protein-free ram seminal plasma. Adsorption of seminal plasma proteins by spermatozoa previously stripped of surface proteins by exposure to detergent reversed the detergent effect on motility. The findings are consistent with the concept that ram seminal plasma contains a factor that interferes with protein adsorption on the cell surface and prevents the protective effect of seminal plasma proteins on maintenance of cell viability.

Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phase systems

Centrifugal counter-current distribution (CCCD) in a dextran, Ficoll, poly(ethylene glycol) two-phase system was used to study the effect of seminal plasma proteins on the partition behaviour of ram spermatozoa exposed to thermal shock. Ram spermatozoa freed from seminal plasma by a ‘swim-up’ procedure were submitted to thermal shock and fractionated by CCCD. Cell viability decreased from 68% to 18% after the treatment, showing a slight displacement of the cells from the right (where a higher enrichment of live cells is found) to the centre of the profile. A change of the distribution profile was shown in the presence of either ram or bull seminal plasma. Bull seminal plasma was able to move the profile to the right, whereas ram seminal plasma increased the proportion of cells with enhanced affinity for the lower dextran-rich phase. Plasma proteins isolated from both seminal plasmas moved the profile to the right. In addition, cell viability rose to 48% after the CCCD run in the presence of ram plasma proteins. This restoring effect was lost when ram plasma proteins were thermally denatured. Bovine serum albumin was not only unable to move the profile to the right but even promoted displacement of the profile to the left. This negative effect was also observed when proteins from bull seminal plasma were in the presence of protein-free ram seminal plasma. However, proteins isolated from ram seminal plasma still restored the profile in the presence of bull seminal plasma freed from proteins. The results presented here strongly suggest that seminal plasma proteins are absorbed by a spermatozoal surface previously exposed to thermic shock. These proteins would exert a highly specific protective effect on ram spermatozoa. In addition, in the ram seminal plasma there must be some factor which avoids this adsorption.

Surface changes associated with ram sperm cryopreservation revealed by counter-current distribution in an aqueous two-phase system effect of different cryoprotectants

Ram sperm was frozen in the presence of the most commonly used cryoprotectants. After thawing, the overall cell surface changes provoked by freezing were assessed by centrifugal counter-current distribution (CCCD). In addition, cell membrane integrity (viability) of all the treated sperm was estimated by fluorescent staining. Fresh and refrigerated sperm were used as controls. Our results show no improvement of the cooling-induced cell surface damage by freezing in the presence of bovine seminal plasma, proline, glycine-betaine and phosphatidylcholine. Better results were obtained with vitamin E and cholesterol. However, the best protective effects were found by employing seroalbumin and lactalbumin. Furthermore, freezing in the presence of bovine lactalbumin resulted in a good maintenance of the cellular viability and of the CCCD heterogeneity in respect to fresh cells.

Short-term inhibition of the energy metabolism affects motility but not surface properties of sperm cells.

Centrifugal countercurrent distribution (CCCD) in aqueous two-phase systems has been proven to be a useful method to study subtle surface properties of spermatozoa. The present work shows that a short-term inhibition of the energy metabolism of sperm cells effected by incubating bovine sperm cells with KCN or ouabain, did not account for changes in the cell surface properties, as assessed either by estimation of the cell viability or by CCCD analysis. However, the short-term inhibition of energy metabolism provoked a clear decrease of cell motility, suggesting that a drop of cellular ATP levels brings about a rapid decrease of motility followed by a very delayed effect on cell surface properties. The relevance of these results on the handling of sperm and on the understanding of the molecular events underlying asthenospermia is discussed.