CHAPTER 13 Semen Quality

William B. Ley, Wynne A. Digrassie, G. Reed Holyoak, and Steven H. Slusher

The Society for Theriogenology has established standards for a stallion’s breeding “satisfactoriness” or “soundness” (Fig. 13-1).¹ To obtain a satisfactory rating on the examination for breeding soundness, the stallion must (1) be physically capable of mating; (2) possess good libido; (3) have normal external genitalia with scrotal or testicular dimension consistent with good sperm production; (4) test negative for infections and for venereal, bacterial, and viral pathogens; (5) have good semen quality; and (6) have at least 1 × 10⁹ progressively motile, morphologically normal spermatozoa in the second of two ejaculates collected 1 hour apart, preceded by 1 week of sexual rest.²

Fig. 13-1 Stallion Breeding Soundness Evaluation Form.

(Courtesy Society for Theriogenology.)

The best semen samples to evaluate for breeding soundness are those that are representative of the stallion’s daily sperm output, that is, ejaculates collected after the extragonadal sperm reserve has been depleted.³ This usually requires 5 to 7 days of daily semen collection. Because this is both time consuming and labor intensive, however, most practitioners instead collect two samples 1 hour apart to evaluate stallion semen quality.

Conventional Methods

Parameters that determine semen quality are similar for the domestic animal species commonly evaluated. Parameters for the horse have been defined, but their individual correlation to fertility are weak to moderate. A “good” spermatozoon must have (1) an intact acrosome, (2) intact plasma membrane, (3) condensed nuclear chromatin, (4) functional mitochondria, and (5) a flagellum allowing the spermatozoon to reach the site of fertilization, undergo capacitation and the acrosomal reaction, penetrate the zona pellucida of the oocyte, and complete the process of fertilization. These parameters define an individual sperm’s quality.

Semen quality addresses the parameters of the population of spermatozoa present in the ejaculate (or semen sample) as well as the nature of the fluid transport medium (seminal plasma). Minimal parameters of semen quality for the stallion include the (1) gel-free (sperm-rich) volume (milliliters) of the ejaculate, (2) concentration (spermatozoa per milliliter), (3) percentage of progressively motile spermatozoa, and (4) percentage of morphologically normal spermatozoa. Each of these variables can change with the season of the year when the ejaculate was obtained, age of the individual stallion, and frequency of ejaculation for the stallion.

It is critical that semen be collected and handled properly before its laboratory evaluation to prevent artifacts from interfering with interpretation of the sample’s quality. Semen collection and sample handling involve the following basic elements:

• The artificial vagina or other collecting device and all surfaces that will come into direct contact with the ejaculate must be warm (37° C) and free from spermicidal agents.

• Collected semen should be transported to the laboratory as quickly as possible, shielding it from direct sunlight, maintaining it at body temperature (37° C), and placing it into a warming bath or incubator at 37° C.

The gel fraction of the ejaculate can be removed either during the semen collection process by appropriate “in-line” filtration (eg, nylon micromesh filter) or after the sample has been collected in the laboratory. The entire ejaculate can be filtered through a sterile, prewarmed funnel that has one or two glass-wool cotton balls placed in the funnel neck to trap the gel from passing through the funnel. Alternatively, the gel in the ejaculate can be aspirated from the ejaculate using a sterile 60-ml syringe. Ejaculate volume (milliliters, gel-free portion) is usually measured in a prewarmed, sterile graduated cylinder (Fig. 13-2). Color and consistency of the ejaculate should be noted to allow estimation of the sperm number and detection of contaminants (eg, urine, blood, purulent exudate). Stallion semen should be an opaque-white color, and any deviation from this may indicate abnormality. Both urine (yellow color) and blood (red color) will damage spermatozoa if left in contact with the ejaculate for longer than 10 to 15 minutes.⁴

Fig. 13-2 Examination of stallion semen for gel-free volume, color, and opacity.

The gel-free portion of the ejaculate should be split into two parts. One sample is kept as is (“raw,” “neat”) and should be left in the incubator. The other sample should be mixed with an equal volume of prewarmed semen extender and allowed to equilibrate to room temperature. Milk-based semen extenders for the stallion are available as ready-to-use packages (eg, E-Z Mixin, Animal Reproduction Systems; Kenney Skim Milk Extender, Har-Vet; Skim Milk Extender, Lane). Alternatively, the Modified Kenney Extender can be prepared by mixing 24 g of nonfat dry skim milk solids with 26.5 g of glucose and 40 g of sucrose in sufficient deionized water to obtain 1 L. Antibiotics are optional for use in laboratory settings, but if included, 1 million units of potassium penicillin G and/or 1 g of amikacin sulfate may be added to each liter of skim milk extender.

Concentration and pH

Sperm concentration is measured on the “neat” sample using a Neubauer hemacytometer (American Optical) (Box 13-1), a Makler chamber (Sefi Medical), or most often, photometric methods (Fig. 13-3). A photometric test evaluates optical density of the fluid sample, and therefore raw semen must be used to prevent false readings resulting from particles that may exist in common milk-based extenders.^2,⁴ Cells other than mature spermatozoa (eg, red cells, neutrophils, round cells) may also falsely elevate the optical density reading. An accurate measurement (number of sperm per milliliter) is essential, since the evaluation of stallion semen quality and breeding soundness is based on available total sperm numbers in the ejaculate (gel-free volume × sperm concentration). This value is an estimate of total sperm number per ejaculate and is a crucial parameter in defining stallion semen quality and resultant breeding suitability (Table 13-1). Total sperm count can change depending on the season, frequency of ejaculation, testicular volume, incomplete ejaculation, extragonadal sperm reserves, and intrinsic reproductive tract disease or obstruction.

Box 13-1 Hemacytometer Method for Evaluating Concentration of Stallion Spermatozoa

Courtesy Oklahoma State University.