Measurement of Concentration and Viability in Stallion Sperm

Sperm concentration and sperm membrane intactness (SMI) or viability are two measures of sperm quality that provide important but different information about a stallion's reproductive capability. Sperm concentration is a measure that, by itself, informs little about the reproductive status of either the stallion or the ejaculate. Nevertheless, it is part of the product, along with semen volume, that determines total sperm number. The correct calculation of total sperm number directly affects the number of mares a stallion can breed and therefore, fertility. If either sperm concentration or semen volume is incorrectly measured, both the number of mares that a stallion can breed and the fertility of those breedings are affected. Although considerable between-stallion variation exists, sperm concentration, semen volume and total sperm number tend to be seasonal and vary with ejaculation frequency.     

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 degrees 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.     

The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility

Pregnancy rates in managed horse populations depend on the innate fertility of the mares and stallions involved and on the quality of breeding management. Of course, because a single stallion usually mates many mares, stallion fertility is a critical factor in the overall success of a breeding program. Unfortunately, accurate evaluation of stallion fertility per se requires a large number of normal mares to be mated and is necessarily retrospective. Rather, the ideal is to predict fertility in advance of the stallion's breeding career, and this is currently attempted by way of a thorough physical examination and a routine analysis of semen quality. However, while such a ‘breeding soundness examination’ identifies stallions that clearly lack the capacity for adequate fertility, it is of limited use for predicting the level of fertility and fails to identify some seriously sub‐fertile animals. Similarly, while various sperm function tests (e.g., sperm head morphometry, the hypoosmotic swelling test, glass wool‐sephadex filtration, progesterone receptor exposure) have been shown to correlate fairly well with fertility in the field, most examine only a single or a narrow range of the attributes that a sperm must possess if it is to fertilize an oocyte in vivo, and are thus more useful for identifying specific causes of sub‐fertility than for predicting the level of fertility. On the other hand, combining the results of the various sperm function tests does improve the reliability of fertility estimation and current research is therefore concentrated on identifying a range of tests that covers as many important sperm attributes as possible but that can be performed rapidly and cheaply. In this respect, flow‐cytometry has proven to be an ideal tool because it allows the objective, rapid and simultaneous analysis of a number of properties in a large number of sperm. Moreover, stains are available for an increasing range of sperm characteristics including viability, capacitation and acrosome status, mitochondrial activity and chromatin integrity. Flow‐cytometric analysis of sperm with appropriate probes thus offers considerable promise for the prediction of stallion fertility.     

Evaluation of stallion semen.

This article outlines a basic method for conducting a stallion semen evaluation. After the removal of the gel fraction of the ejaculate, semen gel-free volume is determined, and any abnormality in appearance is noted. Concentration of sperm cells in semen can be determined with the use of either a hemacytometer or spectrophotometer after appropriate dilution of raw semen. The percentage of progressively motile sperm is evaluated promptly after collection of semen with the use of a phase-contrast microscope. The total numbers of sperm and progressively motile sperm in the ejaculate are calculated. The determination of seminal pH and the classification of sperm morphologic features are additional seminal characteristics evaluated during a semen evaluation. Sperm motion characteristics can be further evaluated with the use of computerized sperm image analysis systems and may add additional information concerning the quality of ejaculated sperm. Unfortunately, no single seminal characteristic has in itself been shown to be highly correlated with fertility, although various seminal characteristics are known to affect fertility. Therefore, to properly interpret the fertility of a semen sample, a complete and thorough semen evaluation must be performed.     

Characterization of Equine Phospholipase C Zeta: A Review and Preliminary Results on Expression Defects in Subfertile Stallions

In all mammalian species studied thus far, fertilization results in a series of intracellular Ca²⁺ ([Ca²⁺]_i) increases, referred to as oscillations, responsible for driving oocyte activation and embryonic development. Current evidence supports the notion that sperm-borne phospholipase C zeta (PLCZ) is responsible for the initiation of these [Ca²⁺]_i oscillations. Although this appears to be a highly conserved mechanism for oocyte activation, differences in PLCZ sequence, activity, and expression do exist among different species. Herein, we summarize the information supporting PLCZ as the oocyte-activating factor in mammals and present our current knowledge regarding the characterization of this protein in the horse. The equine sequence yielded a protein of high relative [Ca²⁺]_i-releasing activity. Equine PLCZ was expressed over the head region overlying the acrosome, equatorial segment, connecting piece between the head and midpiece, and on the principal piece of the flagellum of stallion sperm. Equine PLCZ expressed both over the head and tail sperm regions was catalytically active, with the latter representing a characteristic unique to the horse. We also present preliminary data in subfertile stallions displaying PLCZ expression defects, although further research is required to establish a clear association between these defects and fertility problems in the horse. In summary, the information presented raises the questions of whether equine PLCZ could play diverse roles in sperm physiology and/or become a marker for the evaluation of stallion fertility, both of which are worthy of further investigation.     

The Impact of Reproductive Technologies on Stallion Mitochondrial Function

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen–thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies.     

Current Reproductive Technologies Impacting Equine Embryo Production

Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12–24 hours and transport them to recipient stations. Embryo freezing has become more common using the technique of vitrification of embryos >300 μm or deflating embryos >300 μm before freezing. Oocyte vitrification has resulted in poor pregnancy rates although the technique works well in women. The ability to collect oocytes from mares and fertilize them by sperm injection has revolutionized the veterinarian’s approach to infertility in the mare and/or stallion. A transvaginal approach can be used to collect oocytes from preovulatory follicles and unstimulated follicles 5–25 mm in size. Although traditional in vitro fertilization does not work well in the horse, ICSI can be used to produce blastocysts which, upon nonsurgical transfer into recipients, provide a pregnancy rate similar to fresh embryos collected from donor mares. Sorting sperm by flow cytometry into X- and Y-bearing spermatozoa has been shown to provide about a 50% pregnancy rate with freshly sorted sperm but only 12% with sorted, frozen/thawed stallion sperm. It is likely that more advanced reproductive techniques will be developed in the future. Their acceptance will depend on how well they work, perceived need, cost, and, to some extent, the breed associations.     

Advanced reproductive techniques in goats.

The use of ultrasonography for pregnancy diagnosis and reproductive tract evaluation in the goat has become more common in the past decade. Pregnancy-specific hormone assays are commercially available for pregnancy determination in goats. Hormonal methods of synchronization of estrus for artificial insemination have been refined, but a number of factors still make pregnancy results variable. Caprine embryo transfer is widely used commercially. More advanced reproductive techniques such as in-vitro production of embryos and cloning have been accomplished in goats; success rates with these techniques will likely rapidly improve.     

A fast,low‐cost and efficient method for the diagnosis of sperm DNA fragmentation in several species

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high‐precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue‐stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.     

New Methods for Selecting Stallion Spermatozoa for Assisted Reproduction

Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrode's medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively (P > .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% ± 22% vs. 58% ± 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.     

Review on Effects of Fescue Grass Ergot Alkaloids in the Horse and Preliminary Study on Effect of Fescue Grass Ergot Alkaloid in the Stallion

Feed with ergot alkaloids ingested by horses has a deleterious clinical and economic impact on the industry. The clinical manifestation of the effects in mares is early embryonic mortality, abortions, prolonged gestation, dystocia, thickened (edematous) or retained placental membranes, agalactia, and increased rates of newborn mortality. For the stallion, very little is known, although ergot alkaloids decrease the ejaculate volume. However, a large number of breeding stallions graze endophyte infected (E+) pastures. This is especially true in the southeastern United States, and clinically we do not perceive that any stallions have any defined problems that could be attributed to ergot alkaloids. However, the number of spermatozoa produced by any stallion might mask the effects. The effects on fertility may be more subtle and only evident with sperm cell manipulation such as chilling or freezing. A preliminary study was performed on six breeding stallions fed feed containing infected fescue seed. We were not able to determine any significant (P < .05) detrimental effects on sperm motility, number morphology, and sperm morphology when compared with controls.     

Use of nutraceuticals in the stallion: Effects on semen quality and preservation

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.     

In vitro assessment of semen for the prediction of fertility

The prediction of fertility is a primary goal in the field of reproductive medicine. The aim of the present paper is to describe the value of conventional and modern sperm analysis systems considering the process of fertilization. The classical assessment of motility and morphology enables the rough estimation of semen quality in order to select ejaculates for the use in artificial insemination. Recent methods for sperm diagnosis, such as fluorescent marking for the detection of sperm plasma membrane integrity, the hypoosmotic swelling test, and computer assisted semen analysis allow for the evaluation of a large number of spermatozoa and the assessment of sperm dynamics under in vitro-fertilization conditions. The oocyte penetration test investigates the ability of spermatozoa for capacitation, hyperactivation and acrosome reaction in vitro. The amount of specific seminal plasma proteins is related to fertility and thereby provides an additional semen evaluation method. For the use of a given semen test the specific in vitro condition has to be considered. In addition, the evaluated criteria relevant for the process of fertilization need to be defined. The combination of selected semen tests gives a higher accuracy for the prediction of fertilizing capacity compared with a single test.     

Management Options for the Aged Breeding Stallion with Declining Testicular Function

In our experience, the testicular dysfunction that develops in aged stallions is typically progressive, contributing to a gradual deterioration in sperm output and quality over 2-4 years. As the ability to produce sufficient numbers of normal sperm in ejaculates declines, so do pregnancy rates until the stallion eventually becomes so subfertile that it is no longer commercially feasible to continue breeding. However, more intensive breeding management can sometimes result in pregnancy rates (per cycle and per season) that are sufficient to justify breeding of the aged stallion to a diminishing number of mares during the period of declining fertility.     

Comparison of density gradient and single layer centrifugation of stallion spermatozoa: Yield,motility and survival

Reasons for performing study: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4°C overnight, were also available for testing. Results: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18–20% when cold‐stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. Potential relevance: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.     

Semen Characteristics in a Sub‐Fertile Arabian Stallion with Idiopathic Teratospermia

A 5‐year‐old Arabian stallion was managed for breeding with fresh/extended semen during a period of 8 months with a resulting per cycle pregnancy rate of 26.3%. The stallion was in good health and no abnormalities of the reproductive tract were observed. Evaluation of several ejaculates revealed that sperm production and semen quality were mostly unchanged during the period of evaluation, that sperm production was normal and that semen quality was extremely poor. The most prevalent sperm defects were abnormal heads and mid‐pieces. Most abnormal heads were microcephalic and/or tapered and considerable variation in sperm head dimensions within and among ejaculates was observed. A unique defect characterized by swollen/roughened mid‐piece caused by accumulation of cytoplasmic‐like material and abnormal mitochondrial sheath was observed. Nuclear vacuoles, acrosome defects, and teratoids were also prevalent and most sperm presented multiple abnormalities. The absence of any clear cause or any signs of testicular degeneration, combined with normal sperm production, and constant abnormal sperm production suggest an inherent, congenital disturbance of spermatogenesis as the cause of teratospermia in this case.     

Effects of Repeated Partial Thaw and Refreeze on Post-Thaw Parameters of Stallion Semen Cryopreserved in Cryovials

The current study evaluated post-thaw semen parameters of stallion semen cryopreserved in cryovials and subjected to multiple partial thaw-refreeze cycles. Five fertile stallions were collected twice, and ejaculates were analyzed for concentration, percent membrane integrity, motility, morphology, and sperm chromatin structure (SCSA). Semen processed with freezing extender from each ejaculate was cryopreserved in both 1.2-mL cryovials and 0.5-mL straws. Cryovials were subjected to eight subsequent partial thaw-refreeze cycles. Cryovials were warmed for approximately 30 seconds; then, a sample of cryopreserved semen was removed with a 16-gauge needle, and the cryovial was immediately refrozen in liquid nitrogen. A piece of 0.5-mL straw cut under liquid nitrogen from the same stallion and ejaculate was thawed alongside each cryovial to serve as a control. Thawed samples were analyzed for percent membrane integrity, motility, and SCSA. Post-thaw parameters of motility and membrane integrity were analyzed by one-way or two-way analysis of variance with repeated measures when appropriate. The SCSA data were analyzed using a mixed regression model. Post-thaw motility and percentage of intact sperm were significantly lower when sperm was cryopreserved in cryovials compared to straws. However, these parameters may remain adequate for use in assisted reproductive techniques (ARTs) such as intracytoplasmic sperm injection through all cryovial thaws. Additionally, DNA denaturability was not affected by semen packaging method and was only affected by thaw number, increasing at post-thaws 5 and 6. This technique may offer a unique approach for cryopreservation and utilization of stallion sperm for ARTs in the future.     

Biological markers of boar fertility

The semen evaluation techniques used in most commercial artificial insemination centers, which includes sperm motility and morphology measurements, provides a very conservative estimate of the relative fertility of individual boars. As well, differences in relative boar fertility are masked by the widespread use of pooled semen for commercial artificial insemination (AI) in many countries. Furthermore, the relatively high sperm numbers used in commercial AI practice usually compensate for reduced fertility, as can be seen in some boars when lower numbers of sperm are used for AI. The increased efficiency of pork production should involve enhanced use of boars with strong reproductive efficiency and the highest genetic merit for important production traits. Given that the current measures of semen quality are not always indicative of fertility and reproductive performance in boars, accurate and predictive genetic and protein markers are still needed. Recently, significant efforts have been made to identify reliable markers that allow for the identification and exclusion of sires with reduced reproductive efficiency. This paper reviews the current status of proteomic and genomic markers of fertility in boars in relation to other livestock species.     

Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions

Predicting male fertility on non‐invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer‐assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3–5, 6–10, 11–14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real‐time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations.