Do X and Y spermatozoa differ in proteins?

This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y-chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins.     

Expression of the G-protein α-subunit gustducin in mammalian spermatozoa

Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit α-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether α-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-α-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the α-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether α-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for α-gustducin. Although a possible function for α-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein α-subunit may process intracellular signals controlling sperm motility. Johanna Fehr and Dorke Meyer contributed equally to this work.     

Can programmed cell death be induced in post-ejaculatory bull and stallion spermatozoa?

Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 °C) or heat shock (40 and 41 °C) for 4 h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 °C. Incubation at 38.5 °C (least-squares mean ± SEM = 4.0 ± 1.4%), 40 °C (6.2 ± 1.4%), and 41 °C (7.0 ± 1.4%) for 24 h did increase the proportion of sperm that were TUNEL positive slightly as compared to non-incubated control sperm (1.0 ± 1.4%). However, the increase in TUNEL labeling was not affected by incubation temperature and occurred even in the presence of the group II caspase inhibitor, z-DEVD-fmk. In addition, exposure of bull sperm to carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which depolarizes mitochondrial membranes, did not increase TUNEL labeling. Stallion sperm were also resistant to increased TUNEL labeling in response to incubation at 41 °C for 4 h or exposure to CCCP. Western blotting was performed to determine whether failure of induction of apoptosis was due to aberrant caspase activation. Procaspase-9 was detected in bull sperm, but cleavage to caspase-9 was not induced by short-term aging at 38.5, 40, or 41 °C, or exposure to CCCP. Procaspase-3 was not detected in bull spermatozoa. In conclusion, post-ejaculatory bull and stallion sperm were resistant to induction of apoptosis; this resistance, at least in bulls, was due to refractoriness of mitochondria to heat shock-induced depolarization, lack of activation of procaspase-9, and an absence of procaspase-3.     

Does long-term storage of spermatozoa occur in the adder (Vipera berus)?

In the adder ( Vipera berus) multiple inseminations during the spring mating season have been demonstrated, with fertilization taking place several weeks after mating activities have ceased. Subsequent sperm competition in the female reproductive tract can result in within-season mixed paternity. Prolonged sperm storage between mating seasons has been suggested as another mechanism of sperm competition in the adder. Competition between new and old spermatozoa would obstruct efforts to reveal reproductive strategies that correlate observed behaviour to paternity in natural populations. In the present study, controlled breeding experiments with adders were performed and DNA fingerprinting was used to determine paternity among all offspring of a total of 15 litters. The offspring of 11 large females, that had most probably also mated in a previous season, were sired exclusively by enclosure males. This result and earlier empirical data support the hypothesis that long-term sperm storage, over one or several reproductive cycles, does not occur in the adder.     

Surface receptors: Are they involved in transformation of spermatozoa of Ascaris?

Exposure of the spheroidal spermatozoa of Ascaris suum to an extract of the male accessory gland causes their transformation into ameboid cells. We have investigated the mechanism of this transformation, also termed activation, by labeling the proteins of accessory gland extracts with fluorescein isothiocyanate (FITC) or [125I], followed by qualitative localization of the sperm activating substances (SAS) and quantitative measurements of [125I]-SAS binding. Fluorescent patches of FITC-conjugated SAS were localized at the spermatozoan surface and were concentrated primarily at the posterior region. Few fluorescent patches were detectable in the region of the newly formed pseudopodia following transformation. Although spermatozoan transformation occurs within 2-5 min after exposure to SAS, the fluorescent patches became more distinct after a minimum of 8 min and reached maximum density at 15-30 min. Spermatozoa activated with [125I]-SAS became radioactively labeled in direct proportion to the amount of available [125I]-SAS until a saturation level was reached. SDS-polyacrylamide gel electrophoresis combined with autoradiography indicated that the cells bind two SAS components, of small (9,000 MW) and large (56,000 MW) sizes. These same two components were also detectable in a membrane fraction, obtained by differential centrifugation, of the spermatozoa after incubation with [125I]-SAS. binding of the two SAS components was not inhibited by preincubation of the spermatozoa with trypsin or Concanavalin A; however, the 56,000 MW component of SAS was not detectable in autoradiograms of spermatozoa incubated with periodic acid (1.6-10 mM) treated SAS. Such cells also failed to transform into ameboid spermatozoa. These results indicate that the two components of SAS that bind to the spermatozoan surface are possibly responsible for inducing the cell transformations associated with activation.     

Sperm-mediated gene transfer–treated spermatozoa maintain good quality parameters and in vitro fertilization ability in swine

A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100 μg/mL) of DNA than usual (5 μg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24 h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24 h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.     

Do human eggs attract spermatozoa?

A key process in human fertilization is bringing the two gametes together, so that the complex molecular events involved in sperm and egg interaction can begin. Does nature allow fertilization to occur only as a consequence of a chance collision, or is there a precontact sperm-egg communication? This review summarizes the bioassays used in testing human spermatozoa for chemotaxis, emphasizing the necessity to distinguish between chemotaxis and other accumulation-causing processes, and the results obtained. It demonstrates that human sperm chemotaxis to a follicular factor(s) does occur, at least in vitro, and that only capacitated spermatozoa are chemotactically responsive. Substances that have been proposed as attractants for human spermatozoa are reassessed. The potential role of sperm chemotaxis in vivo is discussed. Faulty precontact sperm-egg communication may be one of the causes of male infertility, female infertility, or both. On the other hand, interfering with human sperm chemotaxis may represent an exciting new approach to contraception. BioEssays 21:203–210, 1999. © 1999 John Wiley & Sons, Inc.     

Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm(®) gradient

The aims of the present study were to: (1) determine if discrete motile sperm subpopulations exist and their incidence in fresh dog ejaculates, (2) evaluate the effects of cryopreservation on the distribution of spermatozoa within the different subpopulations, and (3) determine the effect of the discontinuous PureSperm(?) gradient on the sperm subpopulation structure of frozen-thawed dog spermatozoa. Semen from 5 dogs were collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(?). Sperm motility (assessed by computerized-assisted semen analysis, CASA) was assessed before freezing, just after thawing and after preparation on the PureSperm(?) gradients. Cryopreservation had a significant (P<0.001) effect on CASA-derived parameters. PureSperm(?) centrifugation yielded sperm suspensions with improved motility (P<0.01). A multivariate clustering procedure separated 19414 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (20.97%), Subpopulation 2 consisting of slow and low-linear spermatozoa (18.24%), Subpopulation 3 consisting of highly active but non-progressive spermatozoa (20.75%), and Subpopulation 4 consisting of high speed and progressive spermatozoa (40.03%). Although, cryopreservation had a significant (P<0.001) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after freezing and thawing. The selected sperm samples was enrich in Subpopulation 4, reaching a proportion of 31.9% of the present spermatozoa, in contrast with the unselected sperm samples, where this sperm subpopulation accounted for 24.9% of the total. From these results, we concluded that four well-defined motile sperm subpopulations were present either in fresh semen, in unselected sperm samples or in selected preparations from dogs. The discontinuous PureSperm(?) gradient is a simple method to improve the quality of canine frozen-thawed semen samples, since Subpopulation 4 (high-speed and progressive spermatozoa) was more frequently observed after preparation on the gradient. Finally, this study also demonstrated that the general motile sperm structure present in dog remains constant despite the effect caused by either cryopreservation or separation on PureSperm(?) gradient.     

The spermatozoa of three species of Xenotrichulidae (Gastrotricha,Chaetonotida): the two “dünne Nebengeisseln” of spermatozoa in Heteroxenotrichula squamosa are peculiar para-acrosomal bodies

The spermatozoa of xenotrichulid gastrotrichs have been studied with the aim of supplying further characters for the phylogenetic analysis of Gastrotricha and to assess the reported biflagellarity of Heteroxenotrichula squamosa. Three species have been examined, belonging to the two hermaphroditic genera of xenotrichulids. The spermatozoa are filiform cells characterized by a scarcely condensed nucleus followed by a single mitochondrion and a flagellum with large accessory fibers. These show an obliquely striated cortex and a core containing some dense material. In Heteroxenotrichula squamosa and Xenotrichula punctata there is also a simple acrosome flanked by two para-acrosomal bodies which are curious long extracellular structures formed by a pile of electron-dense disks connected by thin threads. Xenotrichula intermedia lacks both acrosome and paraacrosomal bodies. The sperm model of xenotrichulids is very different from that of the Macrodasyida and Chaetonotida so far studied, thus supporting an isolated position of the family. The oblique striation of the tail's accessory fibers is similar in to the one period and inclination of the strated cylinder of macrodasyid gastrotrichs, thus being the only spermatological character shared by the two gastrotrich taxa.     

Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super- and single-ovulating cattle?

The collective efficiency of barriers in the female tract against spermatozoa with abnormal heads was studied. In Experiment 1, Day 6 ova/embryos were recovered nonsurgically from superovulated (n = 24) and single-ovulating (n = 44) cows following artificial insemination with semen of bulls selected for normal spermatozoal motility (> or = 50%) and high content (> 30%) of spermatozoa with misshapen heads, random nuclear vacuoles or the diadem defect. To assess characteristics of spermatozoa capable of traversing barriers in the female tract, accessory spermatozoa were classified morphologically (x 1250) and compared with those of the inseminate. Superovulated cows proved inadequate for assessment of accessory spermatozoa due to evidence of poor sperm retention in the zona pellucida; thus, only single-ovulating cows were used. Accessory spermatozoa (n = 479) from 31 ova/embryos recovered from 44 cows were more normal in head shape than those in the inseminate (76 vs 62%; P < 0.05). Spermatozoa with normal head shape, but with nuclear vacuoles appeared as accessory spermatozoa at the same frequency as they were found in the inseminate (20 vs 17%, respectively). Only sperm cells with subtly misshapen heads appeared as accessory spermatozoa. In Experiment 2, semen pooled from 4 bulls having large numbers of spermatozoa exhibiting a gradation from severely asymmetrically misshapen heads to subtly misshapen heads was evaluated. Again, the accessory sperm population (960 sperm cells recovered from 64 ova/embryos) was enriched with spermatozoa of normal head shape relative to the inseminate (53 vs 26%, respectively; P < 0.05). Sperm cells with only nuclear vacuoles and those with subtly misshapen heads were not different between the accessory and inseminate populations (11 vs 8%, and 20 vs 25%, respectively). We conclude that morphologically abnormal spermatozoa are excluded from the accessory sperm population based upon severity of head shape distortion.     

Distribution of endogenous and exogenous 5′-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.     

Superoxide dismutase (SOD) in boar spermatozoa: Purification,biochemical properties and changes in activity during semen storage (16 °C) in different extenders

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16 °C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16 °C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67 kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45 °C. The enzymatic activity of form released after cold shock was inhibited by H₂O₂ and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H₂O₂ and DDC (40%). The molecular form released after urea treatment was a 30 kDa protein with maximum activity at 20 °C and pH 10. Enzymatic activity of this form was inhibited by H₂O₂ by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.     

Capacitation status of frozen–thawed spermatozoa from wild ruminant species

Semen from Barbary sheep (Ammotragus lervia), Bighorn sheep (Ovis canadensis), Mouflon sheep (Ovis musimon), Fallow deer (Dama dama), collected by electroejaculation, and semen from Wildebeest antelope (Connochaetes taurinus), collected post mortem, were frozen using a standardized technique and a commercial freezing medium (Triladyl). Sperm quality was assessed by measuring their capacitation status (chlortetracycline assay) in addition to classic assessment: motility, viability (plasma membrane integrity), and acrosome integrity. Sperm cryosurvival measured in terms of recovery (from the initial, pre-freeze values) of motility, viability, and acrosome integrity was relatively high in most species. However, proportion of premature-capacitated spermatozoa (B + AR patterns) increased many times in relation to that of fresh semen: from 8% to 78% in Barbary sheep; from 27% to 61% in Bighorn sheep; and from 12% to 68% in Mouflon sheep. The use of a standardized technique for freezing of spermatozoa from wild ruminant species produced, in most of the cases, acceptable results. This approach may be extensively used to carry out sperm cryopreservation in field conditions. Chlortetracycline assay allowed identifying the same fluorescent patterns observed in spermatozoa from domestic animals and produced additional information on sperm cryosurvival. That is, it revealed those cells that survive freeze–thawing and are potentially fertile: non-capacitated (F pattern) and capacitated acrosome-intact spermatozoa (B pattern).     

Is a Ca2+ -ATPase involved in Ca2+ regulation during capacitation and the acrosome reaction of guinea-pig spermatozoa?

The Ca2+-ATPase antagonists quercetin and ethacrynic acid accelerated the onset of the acrosome reaction in guinea-pig spermatozoa incubated in the continuous presence of Ca2+, whereas furosemide had no effect, and sodium orthovanadate only affected sperm motility. When spermatozoa were preincubated in a 'Ca2+-free' medium, quercetin and ethacrynic acid shortened capacitation time: spermatozoa incubated for 1 h in 100-200 microM-ethacrynic acid showed 60-80% acrosome reactions when Ca2+ was added. Such spermatozoa were able to fertilize zona-free hamster eggs. Our results therefore point to the possible involvement of a Ca2+-ATPase in the regulation of intracellular Ca2+ in spermatozoa. Cysteine and dithiothreitol, both disulphide reducing agents, prevented the effects of quercetin and ethacrynic acid, suggesting that sulphydryl groups may be important for the expression of Ca2+-ATPase activity. Lysophosphatidylserine (LS) also prevented the stimulatory effect of ethacrynic acid, an effect similar to that shown by LS on lysophosphatidylcholine (LC). It is argued that both LS and LC could exert their action through an effect on the Ca2+-ATPase.     

Localization and expression of a 70 kDa protein in goat spermatozoa having Na+,K+-ATPase inhibitory and arylsulphatase A activities

We have previously isolated and purified a goat sperm protein of 70 kDa molecular weight designated as P70 and characterized it as an inhibitor of Na(+),K(+)-ATPase. Our study reveals that the first 10 amino acid residues from the N-terminal end of P70 has high degree of homology with arylsulphatase A from mice, pig and human. Indirect immunofluorescence study shows the presence of the protein on goat sperm surface. Furthermore, live goat sperm and the extract of peripheral sperm plasma membrane proteins exhibit arylsulphatase A's desulphation activity. The P70 remains on the head surface of in vitro capacitated cauda epididymal sperm as shown by positive immunofluorescence staining of cauda sperm. Immunoblot and flow cytometric studies corroborate the above findings. The presence of P70 on capacitated cauda sperm surface suggest a possible role of this protein in sperm zona pellucida binding. In the present report we demonstrate arylsulphatase A like activity in P70 and describe its localization and expression in goat sperm.     

Enrichment of non–apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting

Cryopreservation increases the rate of apoptotic spermatozoa withdecreased capability to fertilise oocytes. In order to optimise thefertilisation rates, especially in assisted reproduction the use of apoptoticsperms should be avoided. Early events of apoptosis in cryopreservedspermatozoaare not detectable by conventional methods. However, the surface of apoptoticspermatozoa is characterised by externalisation of phosphatidylserine (PS),which has a high affinity to Annexin V. Therefore, colloid paramagneticAnnexin-V-conjugated microbeads (AN-MB) were tested fortheir ability to eliminate apoptotic spermatozoa from a total of 40 fresh andinTEST yolk buffer cryopreserved semen samples which were provided by 15 healthyvolunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) thesperm suspensions were divided into 2 sperm fractions depending on boundmagnetic Annexin V-microbeads (AN-MB) to spermatozoa. Asadditional markers of apoptosis CD95 (Fas, APO-1) on the sperm surfaceand activated caspases in the cytosol were detected in both fractions.Supplementary investigations comprised eosin-supravital staining andcomputer assisted sperm motion analysis. The separation was supervised by flowcytometric analysis of spermatozoa labelled with FITC-conjugated antiAnnexin V-antibodies.Analyses of the magnetic inactive sperm fraction (AN-MB-negative)showed CD95 on 0.6 ± 0.3% (X ± SEM) of spermatozoa andonly3.2 ± 0.5% were stainable with eosin, whereas, 40.6 ±6.7% of the remaining cells in the column appeared to be CD95 positiveand 99.8 ± 0.1% stainable with eosin after cryopreservation.Indeed the overall amount of CD95 positive spermatozoa did not significantlyincrease after cryopreservation (2.5 ± 0.5% vs. 4.3 ±1.2%; p > 0.05). Activated caspases were found in 21.8 ±2.6% of the spermatozoa in fresh and in 47.7 ± 5.8% ofcryopreserved semen samples (p < 0.01). The separation procedure of thecryopreserved spermatozoa reduced significantly the quantity of thosecontainingactivated caspases to 9.3 ± 2.2% within theAN-MB-negative fraction. In contrast 89.1 ± 2.3% ofAN-MB-positive sperms showed activation of these proteolyticenzymes. Flow cytometric analyses using FITC-conjugated anti AnnexinV-antibodies for monitoring of AN-MB-binding to spermatozoashowed 5.2 ± 1.0% labelled spermatozoa in the AN-MBnegative fraction and 72.6 ± 2.7% labelled spermatozoa in theAN-MB positive one. There was no significant influence of the separationcolumn and the magnetic field on the sperm functions. The passage through thecolumn led to a sperm loss of 0.8 ± 1.2%.Conclusion: The binding of paramagnetic AnnexinV-conjugated microbeads is an excellent method to eliminate spermatozoaat early apoptotic stages from cryopreserved semen samples. A deleteriousinfluence of the separation column and the magnetic field on the spermatozoawasnot observed.     

Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen–thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P < 0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P < 0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P < 0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P > 0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P > 0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen–thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.     

Centrifugation on PureSperm(®) density-gradient improved quality of spermatozoa from frozen-thawed dog semen

The main objective of this study was to investigate if centrifugation through PureSperm^® density-gradient can improve the post-thaw semen quality of dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm^®. Assessments of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of Propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, unselected samples and selected preparations. Cryopreservation had a significant (P < 0.001) effect on all studied semen parameters. PureSperm^® centrifugation yielded sperm suspensions with improved motility and viability (P < 0.001). The washing step significantly reduced (P < 0.001) all of the kinematics parameters evaluated as well as reduced the proportion of viable spermatozoa with intact acrosomes (P < 0.05). We concluded that PureSperm^® centrifugation is a successful method for improving the quality of frozen-thawed dog spermatozoa. However, washing after density-gradient centrifugation dramatically reduces the post-thaw semen quality, indicating that the inclusion of such a washing step is unnecessary.