Computer assisted semen analyzers in andrology research and veterinary practice.

The evaluation of sperm cell motility and morphology is an essential parameter in the examination of sperm quality and in the establishment of correlations between sperm quality and fertility. Computer-assisted sperm analysis (CASA) allows an objective assessment of different cell characteristics: motion, velocity, and morphology. The development and problems related to this technology are raised in this review, paying particular attention to the biases and standardization requirements absolutely needed to obtain useful results. Although some interesting results, mainly in humans, have already been obtained, many questions remain, which have to be answered to allow for further development of this technology in veterinary medicine, clinical fertility settings, physiological, and toxicology research activities. The main problem is related to the standardization and optimization of the equipment and procedures. The different CASA instruments have all demonstrated high levels of precision and reliability using different sperm classification methodology. Their availability gives us a great tool to objectively compare sperm motility and morphology and to improve our knowledge and ability to manipulate spermatozoa.     

Management of pikeperch Sander lucioperca (Linnaeus, 1758) sperm quality after stripping

The aim of this study was to identify the potential for optimizing management of sperm quality during commercial reproduction of pikeperch Sander lucioperca. Sperm from different males is often pooled prior to fertilization or stored for short periods (hr) until ovulated eggs become available. A novel approach was applied to assess pooling effects by cross‐wise transfusion of sperm and seminal fluid (SF) of males with differing initial sperm quality. In addition, the effects of two different buffers (glucose and KCl) were tested, as well as a supplementation of melatonin and progesterone (1 mmol L^?1) to maintain or improve the quality of freshly stripped and incubated (0.5, 1, 2, 4 and 24 hr) sperm. Sperm motility and curvilinear velocity (VCL) were measured by computer assisted sperm analysis (CASA). The VCL proved to be a more sensitive, reliable parameter compared to motility, since significant differences occurred up to 3.5 hr earlier. Transfusion of SF between low and high quality sperm resulted in a significant decrease in sperm with high initial VCL (seven out of 22 transfusions), whereas VCL of low quality sperm could not be improved. In only one case did a transfusion result in an increased VCL. No treatment prevented a significant quality loss over 24 hr or even enhanced sperm performance. Conclusively, pooling sperm of different qualities as well as short‐term storage has a significant, negative impact on overall sperm quality. Pooling should only be considered when the sperm quality is known.     

Glutathione S-transferase Mu 3 is associated to in vivo fertility,but not sperm quality,in bovine

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.     

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.     

Can external quality control improve pig AI efficiency?

External quality control programmes carried out by central laboratories have been long established in human andrology with the aim of enhancing the accuracy and reproducibility of semen assessment. Compared to human, demands on boar semen assessment in AI stations are more complex, with the need both to identify boars with poor ejaculate quality and to monitor individual boar differences for semen storage. Additionally, appropriate assessment serves as a control instrument to ensure the security and efficiency of semen processing. Despite current limitations regarding the ability of sperm assays to estimate the potential fertility of males, it is evident that boar fertility is related to certain conventional semen tests, e.g. sperm morphology. In central studies carried out on stored semen from 11 AI stations, flow cytometric assessment of plasma and acrosome membrane integrity proved to be more sensitive in detecting sperm damage associated with ageing and temperature stress as compared to light microscopy. Membrane integrity of stored semen differed between AI stations indicating significant influences of semen processing on sperm quality. Thus external control of semen quality in reference laboratories may be useful to monitor the efficiency of internal semen quality control in individual AI stations, to identify males with lower semen quality and/or poor response to semen storage, and to verify the precision of sperm counting. The possibility that central laboratories with sufficient resources may be able to identify functionally different responding sperm subpopulations for better estimation of fertility is discussed. Ideally, external quality control schemes for AI stations would comprise application of validated tests with high relevance for fertility (including bacterial status), analysis of semen processing on the AI station, and training courses for laboratory personnel.     

Accessory sperm: a biomonitor of boar sperm fertilization capacity

The number of accessory sperm found in the zona pellucida of porcine embryos was correlated to their individual quality and to the embryo quality range found within a single sow. Our goal was to determine whether accessory sperm counts provide semen evaluation with additional, useful information. Accessory sperm count was highest when only normal embryos were found in a given sow and diminished if oocytes or degenerated embryos were present (P<0.01). Within a given sow, normal embryos had higher (P<0.05) accessory sperm counts than degenerated embryos, although not when oocytes were also present. Fertilization capacity of sperm is optimal when only normal embryos are found in a given sow; this capacity is indicated by high accessory sperm counts. A decrease in fertilization capacity is reflected in diminishing accessory sperm counts. The boar had a significant effect (P<0.01) on accessory sperm count, but not on the percentage of normal embryos; this suggests that accessory sperm may be more sensitive indicators of the fertilization capacity of sperm than the percentage of normal embryos. We conclude that accessory sperm count can be used for the detection of compensable defects in sperm and is a valid parameter for assessing sperm fertilization capacity.     

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

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

Successive observations on the fertilization of a centric diatomMelosira moniliformis var.octagona

The details of fertilization in the centric diatom,Melosira moniliformis var.octagona, were examined by light microscopy with a video system. The sperm penetrated into the egg cell through a temporary opening of the oogonium. The flagellum of the sperm was brought into the egg cell and it remained active for a few minutes. Three observations on the behavior of the sperm and egg cell during fertilization are shown.     

Quantification and identification of sperm subpopulations using computer-aided sperm analysis and species-specific cut-off values for swimming speed

Abstract

Motility is an essential characteristic of all flagellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computer-aided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer^® (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identified. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

Variations in seminal parameters over a 12-month period in captive bonnet monkeys

Semen samples were collected from adult fertile bonnet monkeys twice a month by penile electroejaculation for twelve consecutive months. Various parameters like semen volume, weight of ejaculate and coagulum, sperm count, sperm motility, sperm morphology, and functional parameters e.g. plasma membrane integrity,in vitro nuclear chromatin decondensation and acrosomal status were evaluated to assess within and between animal variations. Effects of seasonality, if any, on quantity and quality of semen were also studied. Considerable intra- and inter-individual variations in the geometric mean values were observed for semen volume, weights of ejaculate and coagulum, and sperm counts during the study period. On the other hand, sperm motility, morphology, and functional parameters showed less within and between animal variations. Results on motility, morphology, and functional parameters indicated that good semen quality was maintained throughout the year. Various routine and functional parameters did not show any annual variations. The diurnal rhythmicity in circulatory testosterone levels was observed throughout the year. The study shows lack of seasonality in exocrine and endocrine testicular functions and further suggests that motility, morphology, and functional parameters are better indicators of semen quality in captive bonnet monkeys.     

Qualitative aspects of sperm stock in males and females from Eupelmus orientalis and Dinarmus basalis (Hymenoptera: Chalcidoidea) as revealed by dual fluorescence

Abstract The quality of a sperm population can be characterized physiologically and its fecundity predicted by its viable : non-viable sperm ratio. To improve the knowledge of reproductive strategies in two ectoparasitoid hymenopteran species, Eupelmus orientalis Crawford (Hymenoptera: Eupelmidae) and Dinarmus basalis Rondani (Hymenoptera: Pteromalidae) , the assessment of sperm viability using the dual fluorescence staining procedure SYBR-14 : propidium iodide was developed. The aim of the study was to provide a comparative test in vitro applicable to both sexes to study the evolution of sperm quality at various stages of the reproductive processes. The reliability of propidium iodide to detect non-viable sperm (stained in red) was confirmed in both species on the basis of two stress tests (ethanol and Triton X-100) but our study also revealed that propidium iodide concentrations must be adequately adjusted for each single species. This experiment also demonstrated the physiological heterogeneity of sperm populations in E. orientalis and D. basalis males and females. In both species, 40% of the sperm in the seminal vesicles was found to be non-viable. By contrast with E. orientalis, the populations of non-viable sperm estimated from the seminal vesicles of D. basalis were found to be strongly different from those observed in the spermatheca. From the present results, the population of viable sperm detected in the spermatheca of females from both species proved a reliable predictor of fertilization achieved in ovipositing females.     

Evaluation of ram semen quality using polyacrylamide gel instead of cervical mucus in the sperm penetration test

Fertility is a very complex biological function that depends on several properties of the spermatozoa, including sperm motility. Two objectives are analyzed in this study: (1) Replace the cervical mucus by a synthetic medium in a sperm penetration test, and (2) evaluating the results of this test objectively analyzing the sperm number that migrates. In experiment 1, we have tested eight concentrations of acrylamide (1%-2%). Rheological properties of media were analyzed. The plastic straws, loaded with acrylamide, were placed vertically on the semen sample tube for 15 min at 39 °C. After, the acrylamides were placed, by segments of 5 mm, into wells of a 24-well plate, dyed with Hoechst 33342 and the number of spermatozoa were calculated by automated microscopy analysis. The 1.55% and 1.6% acrylamide gel showed a number of spermatozoa emigrating closer to that seen with natural mucus. In experiment 2, we applied the sperm penetration in acrylamide 1.6% and 1.55% using fresh semen and cooled semen at 15 °C and 5 °C. The spermatozoa counts were performed for each segment of 10 mm. Semen chilled at 15 °C presented intermediate values of sperm counts in comparison with fresh semen (higher) and 5 °C chilled semen. The sperm counts do not differ between acrylamides but the rheological properties of acrylamide 1.6% were more similar to those of the natural cervical mucus. In experiment 3, we have observed significant correlations between the number of spermatozoa and several sperm quality parameters (positive: progressive motility and velocity according to the straight path; negative: damaged acrosomes and apoptotic cells) in 1.6% acrylamide media. We conclude that the size of the cell subpopulation, objectively calculated, that migrate beyond 20 mm in 0.5-mL straws filled with acrylamide is a useful parameter in ram sperm quality assessment and further studies are needed to evaluate its relationship with field fertility.     

Beyond Testis Size: Links between Spermatogenesis and Sperm Traits in a Seasonal Breeding Mammal

Spermatogenesis is a costly process that is expected to be under selection to maximise sperm quantity and quality. Testis size is often regarded as a proxy measure of sperm investment, implicitly overlooking the quantitative assessment of spermatogenesis. An enhanced understanding of testicular function, beyond testis size, may reveal further sexual traits involved in sperm quantity and quality. Here, we first estimated the inter-male variation in testicular function and sperm traits in red deer across the breeding and non-breeding seasons. Then, we analysed the relationships between the testis mass, eight parameters of spermatogenic function, and seven parameters of sperm quality. Our findings revealed that the Sertoli cell number and function parameters vary greatly between red deer males, and that spermatogenic activity co-varies with testis mass and sperm quality across the breeding and non-breeding seasons. For the first time in a seasonal breeder, we found that not only is the Sertoli cell number important in determining testis mass (r = 0.619, p = 0.007 and r = 0.248, p = 0.047 for the Sertoli cell number assessed by histology and cytology, respectively), but also sperm function (r = 0.703, p = 0.002 and r = 0.328, p = 0.012 for the Sertoli cell number assessed by histology and cytology, respectively). Testicular histology also revealed that a high Sertoli cell number per tubular cross-section is associated with high sperm production (r = 0.600, p = 0.009). Sperm production and function were also positively correlated (r = 0.384, p = 0.004), suggesting that these traits co-vary to maximise sperm fertilisation ability in red deer. In conclusion, our findings contribute to the understanding of the dynamics of spermatogenesis, and reveal new insights into the role of testicular function and the Sertoli cell number on testis size and sperm quality in red deer.     

Not all sperm are equal: functional mitochondria characterize a subpopulation of human sperm with better fertilization potential

Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.     

Sperm competition, sperm numbers and sperm quality in muroid rodents

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert.     

Characterization of a sperm lysin of Volvox carteri

Summary A cell-wall degrading enzyme has been isolated from mature sperm packets of the green flagellate Volvox carteri (Poona strain). This sperm lysin (S-lysin) is a Ca²⁺-dependent protease of 34 kDa with an essential serine group in its active centre. Neither SH group-blocking reagents nor transition metal chelators inhibit its action. S-lysin degrades the hydroxyproline-rich glycoprotein structures of the cell walls of sheath cells and gonidia (eggs) of vegetative and sexual spheroids in a characteristic manner. In asexual spheroids the somatic envelope is totally disintegrated, whereas in sexual spheroids pores are formed by local lysis at sites of adjacent eggs. Although S-lysin is very similar to the G-lysin of the closely related Chlamydomonads, it is species specific and does not attack the mother or daughter cell walls of Chlamydomonas reinhardtii. S-lysin resembles the aerosin of animal sperm cells in some aspects of its action.Dedicated to Professor Richard C. Starr on the occasion of his 65th birthday. He called the piper and gave the tune     

Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm

Evaluating cryoinjury of canine spermatozoa is crucial to improving the probability of fertilization. Recently, studies on sperm ROS production, phospholipid scrambling, and DNA damage induced by cryopreservation have been reported. However, the consequences of cryopreservation on these crucial factors are lacking with respect to canine semen. Therefore, the current study was designed to investigate the effects of the freezing-thawing procedure on these factors in canine semen. Ejaculates from five dogs were cryopreserved and thawed. Spermatozoa before and after a freezing-thawing process were assessed for phosphatidylserine (PS) translocation (Annexin V [AN]/propidium iodide [PI] assay), intracellular H₂O₂ level (dichlorofluorescein [DCF]/PI assay), DNA integrity (sperm chromatin structure assay), and conventional sperm parameters. The freezing-thawing process decreased motility, viability, normal morphology, and membrane integrity in canine sperm (P < 0.05). The frozen-thawed semen also showed a decrease in AN−/PI− sperm (%) and an increase in the PS translocation index, the intracellular H₂O₂ level in the viable sperm fraction, and the DNA fragmentation compared with that of fresh semen (P < 0.05). In conclusion, the freezing-thawing procedure significantly affects PS translocation, the intracellular H₂O₂ level, and DNA integrity in canine semen, which may explain the lower fertilization rate and in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcome when frozen-thawed spermatozoa are used. It is therefore recommended that these parameters be used as an additional parameter for the assessment of sperm quality after freeze-thawing in canine semen.     

The importance of porcine sperm parameters on fertility in vivo

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

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.