In vitro evaluation of sperm quality

This paper highlights selected laboratory analyses that are currently used to evaluate sperm, and describes why results from these assays do not consistently correlate with sperm fertility. Reasons for the disconnect between the two are due in part to the definition and reliability of the fertility data collected, to the complexity of the spermatozoon itself, to imprecision of some measurements, and to uncontrollable factors not associated to either the laboratory analysis or the sperm sample. Each sperm must possess a number of different attributes to fertilize an oocyte, and individual laboratory assays measure only one or a few of these attributes. Current and past data, correlating laboratory assay data with sperm fertility are presented in an effort to determine which types of assays are important to conduct and when to conduct them. Even though laboratory assay results do not allow accurate evaluation of the fertilizing potential of a semen sample, these assays are important to enable culling of poor quality samples.     

New techniques for the assessment of canine semen quality: a review

Until recently, canine semen assessment was routinely performed by conventional light microscopic techniques. The limitations of these methods include subjectivity, variability, the small number of spermatozoa analyzed, and poor correlation with fertilizing potential. The last decade, several new in vitro techniques have been introduced for canine semen assessment that enable a more detailed evaluation of several sperm characteristics. Numerous fluorescent staining techniques have been developed for the evaluation of specific sperm characteristics and functions, including plasma membrane integrity, capacitation status and the acrosome reaction. By combining fluorescent stains, several functional sperm characteristics can be assessed simultaneously. Moreover, by means of flow cytometry, large numbers of fluorescently labelled spermatozoa can be analysed in a short interval. Following thorough standardization and validation, computer-assisted sperm analysis systems provide objective and detailed information on various motility characteristics and morphometric dimensions that cannot be identified by conventional light microscopic semen analysis. In vitro assays, evaluating the capacity of canine spermatozoa to bind to the zona pellucida or oviductal explants, or to penetrate the oocyte, provide additional information on canine gamete interaction that may be useful in predicting the fertilizing potential of spermatozoa. Although substantial improvements have been made in canine semen assessment, surprisingly few parameters were correlated with in vivo fertility. Therefore, further research is required to determine which sperm characteristics are of clinical value for predicting the in vivo fertility in dogs.     

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

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

Comparison of in vitro laboratory analyses with the fertility of cryopreserved stallion spermatozoa

Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability for visual motility, computer assisted motility, and sperm velocity, sperm viability, percent viable-acrosome intact cells and mitochondrial function of cells were all similar, however, intra-assay variability was lower for flow cytometric assays than for motility assays. The reliability of all assays were >0.72, except for sperm velocity (0.32). Although visual motility and the straightness of sperm motility conducted 90 min after thawing were correlated with seasonal fertility (0.56 and 0.55, respectively), data from no single assay were correlated with first-cycle fertility rates (P > 0.05). Best models using data from multiple assays explained 66 to 73, 76 to 89 and 79 to 94% of the variability in fertilizing potential, when two, three and four variables were included, respectively. Caution is required in interpreting these data, as only a few stallions were evaluated and relatively few mares were bred to each stallion, however, they do indicate that using a few rapid and inexpensive sperm assays, we can begin to understand factors important in stallion sperm fertilizing capacity, and we can use these assays to more effectively evaluate new methods for cryopreserving stallion spermatozoa.     

流式细胞术在哺乳动物精液质量检测中的应用

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

Correlations among assays of porcine semen quality following cryopreservation

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

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

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

Prediction of porcine semen fertility by homologous in vitro penetration (hIVP) assay

A field trial was conducted to compare the fertility predicting capacity of different sperm assays applying classical semen analysis, sperm function and the homologous in vitro penetration test (hIVP) to 60 ejaculates from four boars collected over a period of 15 weeks. No differences were found between the groups of fertility (Low Fertility: <20%; Intermediate: 40–60% and High: >80%) for sperm-rich fraction volume collection, sperm concentration, total sperm number, cationic contents in seminal plasma and ATP concentration. Partial differences were found in the parameters of motility, normal morphology, normal apical ridge (NAR), viability with eosin–nigrosin stain, hypo-osmotic swelling test (HOS), osmotic resistance test (ORT) and functional membrane integrity (with carboxyfluorescein diacetate, DCF). These parameters would be useful for detecting sperm with poor fertility, but they are not precise enough to discriminate an ejaculate with higher fertility than the herd median. Only the penetration percentage (10.24±1.45 vs. 55.13±3.35 vs. 84.72±1.73) and sperm number per oocyte (1.29±0.07 vs. 11.29±1.79 vs. 25.86±1.43) in a hIVP system were parameters with a predictive capacity to discriminate between the three fertility groups. Consequently, hIVP was found to be the best seminal assay and it may improve the in vitro assessment of sperm fertilizing ability.     

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.     

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.     

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

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

Fertility evaluation of frozen/thawed semen

In vitro semen analyses have been used for more than half a century to estimate the fertilizing potential of a semen sample. Unfortunately, none of the assays developed provide results that consistently correlate well with fertility. The reasons for this lack of consistency, due in part to the complexity of the spermatozoon itself, the collection of fertility data, and factors beyond control of the semen analyses themselves, are discussed. Different spermatozoal attributes that are necessary for a spermatozoon to fertilize an oocyte are presented and assays used to evaluate each attribute described. Although laboratory assay results do not correlate well with semen fertility, the importance of conducting laboratory assays on every semen sample used for artificial insemination or to attempt to determine causes for infertility, is discussed.     

Factors affecting the plasma membrane function of cooled-stored stallion spermatozoa

The spermatozoon is a highly specified cell that has the abilities of active motility and fertilization of the ovum. Damage to the sperm plasma membrane results in the irreversible loss of its functions. Because of the high content of unsaturated fatty acids in the plasma membrane, mammalian sperm are sensitive to oxidative stress. While mild peroxidation appears to promote capacitation of the sperm cell, excessive peroxidation will damage the plasma membrane and results in loss of motility and fertility. The functional integrity of the sperm plasma membrane can be determined by functional tests (determination of motility, resistance against hypoosmotic media) or different staining methods. Today, fluorescent dyes that allow the evaluation of membrane-intact cells are preferred. Computer-assisted evaluation or the use of flow cytometry has improved the precision of these methods. The use of cooled-shipped semen has become a routine method in the equine industry and semen stored at 5 degrees C for about 24 h maintains fertility close to that of fresh semen. According to the suitability of their ejaculates for cooled-storage, stallions may be classified as "good coolers" or "poor coolers". Semen processing involves a number of factors that may damage the sperm plasma membrane. This includes addition of semen extender, centrifugation, cooling and storage at a temperature of 4-6 degrees C. Extender media and their ingredients protect the sperm plasma membrane against environmental influences, but unsuitable composition of the extender can also promote membrane damage. Recent advantages in extender composition are based on the substitution of undefined factors such as milk or egg yolk by more defined and stable components.     

Current trends in evaluation of sperm function: in vitro selection and manipulation of male gametes for assisted conception

With increasing medical utilization of assisted reproductive technology (ART), scientists and clinicians have been able to study extensively multiple cell functions operating synchronously and flawlessly during the events preceding, before and after fertilization. Critical evaluation of the functional status of spermatozoa for in vitro techniques such as sperm-mucus interaction, acrosome reaction status, sperm-zona pellucida binding and penetration tests, hyaluronic acid binding assay, and computer assisted semen analysis etc. can direct a male partner of an infertile couple to more aggressive forms of treatments. In vitro selection of functionally competent sperm cells is a pre-requisite for successful outcome in in vitro fertilization or in intracytoplasmic sperm injection (ICSI). Direct injections of acrosome-intact spermatozoa into oocyte during ICSI bypassing the normal events of sperm oocyte interaction and fusion events have raised concerns with regard to fertilization abnormalities and genetic issues. The present communication briefly reviews the sperm function tests with emphasis on its correlation with fertility outcome, and the currently employed sperm selection and manipulation procedures which may have implications in assisted conception programs.     

Effects of exposing boars to different artificial light regimens on semen plasma markers and "in vivo" fertilizing capacity

This study was designed to assess the effects of exposing boars to an artificial photoperiod on semen quality in terms of sperm concentration, sperm vitality, sperm motility and acrosome integrity. We also determined biochemical semen plasma variables, such as total protein concentration, phosphorylated tyrosine residues and fructose, glucose and sorbitol contents, along with their effects on the fertility, prolificacy and libido of the boars. Three groups of 10 males were kept for 3 months under experimental conditions of 24, 12 and 0 h of artificial light, and a constant temperature of 21 +/- 1 degrees C and 60-75% humidity. The animals were fed a nutritious diet and subjected to semen collection twice per week. Semen samples were analyzed throughout the entire experimental period. Our results indicate that, while the extreme photoperiods (0 and 24 h of light) affected semen quality in terms of sperm concentration, acrosome integrity and semen volume, its fertilizing capacity was only significantly reduced under conditions of absolute darkness. Sperm motility was found to be a poor indicator of fertilizing ability, while other sperm factors, such as acrosome integrity or other functional variables seemed to behave better. The photoperiod was found to affect the production of accessory sex gland secretions more than their composition. In addition, light effects on fertility, prolificacy and libido seemed to be achieved through independent mechanisms.     

Theoretical aspects of canine cryopreserved semen evaluation

Evaluation of canine cryopreserved semen has the ultimate goal of determining if an individual frozen ejaculate will have acceptable fertility. This is difficult in that there is no accepted normal fertility for the dog. The fertility of the female also plays a crucial role in estimating the fertility of the male. Poor female fertility can make a fertile male appear less fertile. Variability of animals, breeding technique, breeding timing, and number of cells inseminated make comparisons in canine fertility difficult to truly measure. Many more animals are needed to provide meaningful statistical results than are usually used. Several tests, including motility in bright field and phase contrast microscopy, computer analysis of motility, sperm morphology, sperm membrane integrity, capacitation and sperm function tests have been investigated to predict fertility, however few of these tests have actually been correlated with fertility. More work is needed to create one or more tests that accurately predict fertility of cryopreserved canine semen.     

Porcine sperm zona binding ability as an indicator of fertility

The escalated use of artificial insemination in swine has increased the importance of determining fertility of a semen sample before it is used. Multiple laboratory assays have been developed to assess fertilizing potential but they have yielded inconsistent results. This experiment sought to determine the relationship between in vitro competitive zona binding ability and in vivo fertility based on heterospermic inseminations and paternity testing. The zona pellucida binding ability and fertility of sperm from 15 boars was assessed by comparing sperm from one boar with sperm from other individual boars in a pairwise fashion using four ejaculates. The relationship of zona binding ability to the mean number of piglets sired per litter for each boar as well as historic fertility data (litter size and farrowing rate) was assessed. The in vitro competition assay consisted of labeling sperm from each boar of the pair with a different fluorophore and incubating an equal number of sperm from each boar in the same droplet with porcine oocytes. The competitive assay was highly effective in ranking boars by zona binding ability (R2=0.94). Paternity testing using microsatellite markers was used to determine the mean number of piglets sired per litter for each boar during heterospermic inseminations. The pairwise heterospermic insemination assay was effective in ranking boar fertility (R2=0.59). Using historical data from these boars, average litter size and farrowing rate were correlated (r=0.81, p<0.001). However, zona binding ability was not significantly correlated with historic farrowing rate data or historic average litter size. Boar sperm zona binding ability was also not correlated significantly with the mean number of piglets sired per litter following heterospermic insemination. But the number of piglets sired by each boar was related to a combination of zona binding ability, sperm motility, normal morphology, acrosomal integrity, and the presence of distal droplets (R2=0.70). These results suggest that zona binding ability is not an accurate predictor of fertilizing ability when used alone; however, when coupled with other sperm assessments, fertility may be predicted successfully.     

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.     

In Vitro Evaluation of Frozen-Thawed Stallion Semen: A Review

The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h at 37°C and hypoosmotic swelling test after 0 and 3 h of incubation. Significant correlations have been reported between the pregnancy rate and viability of propidium iodide-stained sperm assessed by flow cytometry as well as for glass wool and Sephadex filtration tests. No correlations have been detected between fertility and motility immediately after thawing. In spite of that, motility estimation by light microscope is the most commonly used method to evaluate frozen-thawed stallion sperm. Computer assisted automatic sperm analyzers have replaced light microscopy in research projects, but so far nobody has been able to demonstrate a correlation between fertility of frozen stallion semen and any of the motility parameters obtained by these instruments.     

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

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