CHAPTER 43Advanced Semen Tests for Stallions

Evaluation of male fertility in horses has been primarily limited to mare cycle and seasonal pregnancy rates, foaling rate, and nonreturn to estrus.¹ In certain countries and on some farms, identification and culling of subfertile males that have been associated with lowered fertility serves as the most economic solution to problems of sire infertility. However, when the value of an individual stallion is high, further investigations into the nature of the subfertility may be warranted. A test for prediction of fertility in unproven sires has been elusive because an array of factors contribute to the interaction between male and female that results in “good fertility.”

In most cases of male subfertility the underlying causes are largely unknown; reduced fertility occurs with a variety of causes and numerous contributory factors. Identifiable factors that can contribute to a male’s decreased fertility include inadequate numbers of morphologically normal, progressively motile sperm; suboptimal management systems; testicular degeneration and genital tract trauma; poor overall health and body condition; and psychologic or behavioral problems. The role of other factors such as environmental toxin exposure; endocrine, autocrine, and paracrine control of spermatogenesis; epididymal dysfunction; and the function of sperm cells is less clearly defined. The objective of this presentation is to discuss several recently developed methods of assessing spermatozoal function and dysfunction as might occur during toxicologic exposure, trauma, or subfertility.

CONVENTIONAL SEMEN EVALUATION

Indirect evaluation of stallion fertility, defined as the ability to cause conception, pregnancy, and the birth of live young, has been performed traditionally by assessment of ejaculated semen. Adjunctive evaluation of testicular parameters such as size, shape, volume, ultrasonographic appearance, and total scrotal width has also been routinely performed. Conventional semen evaluation is not considered to be a direct measure of fertility and consists of measurements of sperm morphologic and movement characteristics and seminal fluid characteristics, including pH, turbidity, color, and presence of immature germ cells or somatic cells. Many of these parameters are relatively well correlated to fertility in most males evaluated; however, few have been able to account fully for decreased fertility in some males.

Clearly, males with extremely low numbers of motile, morphologically normal sperm in their ejaculates are typically incapable of causing conceptions and may be justifiably considered to be infertile. However, some males that have normal to moderately low numbers of morphologically normal, motile sperm in their ejaculates either fail to cause conceptions or cause a decreased conception rate under good breeding management schemes. The subfertility in this latter population is not well defined by conventional semen evaluation. Diagnostic tests, which probe sperm function, are needed to determine the potential causes and explore treatments for this type of subfertility. This would provide diagnostic information about sperm function that would otherwise not be available for males in which conventional semen evaluation fails to identify a cause for diminished fertility. Several laboratories are developing tests for sperm integrity and function based on subcellular mechanisms in differing spermatozoal compartments, including sperm lipid composition, DNA integrity, membrane integrity, resistance to osmotic stress, sperm-zona or sperm-oolemmal binding, signal transduction, and numerous others. It should be mentioned that clinical relevance has not been worked out for many of the following parameters because of small sample populations, but the cell physiologic background should provide rationale to include a number of these assessments on a clinical basis.

EVALUATION OF SPERM FUNCTION

For a single spermatozoon to fertilize an oocyte and develop into an embryo, sufficient numbers of motile sperm must ascend the female genital tract, become capacitated, acquire hyperactivated motility, undergo the acrosome reaction, penetrate the oocyte extracellular investing layers (which include the cumulus oophorus and zona pellucida), and initiate fusion with the oocyte’s plasma membrane. Fertilization also includes sperm nuclear decondensation, male pronucleus formation, and syngamy of male and female pronuclei. Many of these crucial steps in the fertilization process may be artificially isolated as demonstrated by the success of in vitro fertilization in several species. In human medicine the ability to undergo acrosome reactions, to bind and penetrate human ova, and to fuse with zona-free hamster ova have proven to be clinically useful as predictors of in vitro fertilization success and infertility in men. Such function tests have also been performed in livestock species but have not become routinely useful in equine medicine possibly because much subfertility results from conventionally diagnosable sperm deficits such as diminished total numbers of morphologically normal, progressively motile sperm in ejaculates.

Clinical evaluation of fertilization-based sperm function has been applied most widely in human andrology and has included end points such as measurements of total and progressive motility, hypoosmotic swell test, cervical mucus penetration, hyperactivated motility, sperm fusion with zona-free hamster oocytes, or homologous in vitro fertilization. Measures of capacitation and acrosomal physiology have proven to be useful adjuncts to motility and morphologic end points for diagnosis and treatment of human male factor infertility.

In veterinary medicine, bull fertility has been correlated to acrosome reaction inducibility by chondroitin sulfates and dilauroylphosphatidylcholine liposomes.² Other than evaluation of sperm motility, which has not been highly correlated to stallion fertility, there are presently few clinically available indices of sperm function in equine reproduction. For assessment of subfertility, evaluation of sperm function at end points more finitely defined than “fertility” may be appropriate.

CAPACITATION AND ACROSOME REACTION

It has been shown for several species that sperm must undergo the process of capacitation as a prerequisite for most events of fertilization, including cumulus penetration, zona binding and penetration, the acrosome reaction (acrosomal exocytosis), and fusion with the oocyte. Sperm capacitation has been suggested to include increases in intracellular calcium, phosphorylation of protein tyrosine residues, and reversible changes in the sperm plasma membrane, including removal of cholesterol and cholesteryl esters by cholesterol acceptor molecules in the female genital tract or in artificial media. Capacitation in vivo occurs within the female genital tract but can be induced artificially in vitro for sperm of many species, including the horse. The acrosome reaction can occur only following completion of capacitation and can be induced by a variety of chemical and biologic agents. Cytologic techniques for quantitation of acrosome reactions have been applied in several species, including stallions, bulls, and boars. Sperm that are nonviable experience a “false” acrosome reaction, which must be differentiated from the true exocytotic acrosome reaction in order to reflect capacitational changes. Supravital stains, such as Hoechst 33258 or propidium iodide, accomplish this task efficiently. Consequently, the acrosome reaction in live sperm may be considered a rational end point for evaluation of sperm function because it can be used to evaluate a complex set of cell behaviors.

Progesterone has been evaluated for its ability to stimulate acrosome reactions because in some species, including horses, the preovulatory follicle has been reported to secrete progesterone before ovulation and luteinization. Therefore progesterone is likely to be present in oviductal fluids and within the cumulus of ovulated oocytes. Differences have been reported for efficiency of progesterone-induced acrosome reactions in sperm of human fertile and subfertile patients, and this has been attributed to the lack of progesterone receptor on sperm or to a nonfunctional receptor. It is not known whether these subfertile patients had a congenital absence of progesterone receptor, or whether an acquired condition resulted in the loss of functional receptor or receptor subunit. The author and others have reported similar results for fertile and subfertile stallions.^3,⁴ It has been determined that bull and stallion sperm have receptors for progesterone.

Acrosome reaction status in stallion sperm has been evaluated using various staining methods. These include immunofluorescence methods with specific antibodies, both monoclonal and polyclonal,^3,^5,⁶ labeling with fluoresceinated lectins,^6,⁷^–⁹ chlortetracycline labeling (CTC) and transmission electron microscopy¹⁰ (TEM), and nonfluorescence methods.^11,¹² Electron microscopy offers direct imaging of the acrosome-associated membranes and is considered to be the “gold standard” but is the most costly and requires the most skill in comparison to other methods. Few methods offer significant advantages over others; however, the lectin-staining methods generally offer speed and ease of use, although a fluorescence microscope is required. Nonfluorescence methods have also been used, but few staining methods have been validated against TEM. Pratt et al¹³ demonstrated the use of a Coomassie Blue method that was validated for use in canids, but this has not been reported for stallion sperm.