Identifying and solving semen quality problems in stallions

Introduction

There are numerous steps between development of spermatozoa within the testicle to the sperm penetrating the oocyte. If any of these steps go awry, pregnancy will not result. After spermatogenesis occurs in the testicle, the sperm undergo maturation in the epididymis. When ejaculated, they mix with accessory sex gland fluids and are deposited within the uterus. The sperm must move through the inflammatory environment of the uterus to the uterotubal junction. A select few sperm enter the oviduct where they are bound to the oviductal epithelium until ovulation when the cells are hyperactivated and released. The sperm then seek out and bind to the oocyte. The zona pellucida must be penetrated for fertilization to occur.

To successfully make this journey, good semen quality is necessary. What is good semen quality? One definition of good semen quality is an ejaculate that contains a high population of normal sperm cells that provides a normal rate of fertilization. A normal rate of fertilization would be 80–90%. Fertilization rate is difficult to measure because the rate of early embryonic death is high.

At the most basic level, semen with acceptable quality will get a majority of mares pregnant. At the most extensive level, quality would be assessed by a battery of tests that evaluate motility, morphology, sperm numbers, ratio of live/dead, membrane integrity, acrosome integrity, quality of acrosome reaction, quality of DNA, mitochondrial function, ability to bind and penetrate an oocyte and possibly fertility.

In the horse, as compared to the other domestic farm species, we do not select our sires on fertility but rather on performance and athletic ability. This puts the equine industry at a disadvantage from a reproductive standpoint. The frequency of evaluating semen quality and type of examination performed varies within the equine industry. Many stallions have semen evaluated before the imposed breeding season, whereas others are only examined if a decrease in fertility is noted. In the classic breeding soundness examination, semen is evaluated for motility, morphology, concentration, volume, total sperm and microbial contamination. The stallion’s testicles and external genitalia are examined and evaluated. In the thoroughbred industry it is common to perform a breeding soundness examination before the stallion is first mated in February. This exam evaluates the seminal parameters, but not the genitalia. It is usually not until a horse exhibits fertility problems that a more in-depth analysis of semen quality is performed. Due to the limited number of facilities capable of performing the tests and the expense of such tests, in-depth evaluations are performed on only a small number of subfertile horses. Additionally, the true relationship of fertility to many of the tests is not known, as they have not been evaluated in a large number of fertile horses.


Identifying semen quality

The tests available for evaluating semen quality range from basic to highly involved. The following battery of tests is critically assessed when equine semen is evaluated, as there appears to be some
relationship between the test results and fertility.


MOTILITY

Motility is examined in both the raw and extended semen. The semen is most typically extended in glucose/skim milk-based extender. Motility is described and recorded as total and progressive. The motility can be evaluated visually with use of light microscopy or with computer-assisted semen analysis (CASA). While trained operators can reliably assess motility visually, CASA has the ability to repeatably provide more information if semen is properly prepared.

Although the relationship between motility and fertility is poor (Voss, 1981; Jasko, 1991), it is still one of the most common traits evaluated. Longevity of motility of both raw and extended samples can be evaluated. Longevity of motility has been suggested as an indicator of fertility (Bielanski, 1975). The system described by Bielanski graded motility at 18°C and survival times were classified as <18, 18 to 20 and >20 hrs. Semen that maintained motility for greater than 20 hrs was considered to be capable of producing a normal pregnancy rate (>75%).

Motility is a reliable indicator of viability of fresh semen, but not frozen or shipped-cooled semen (Merkies et al., 2000). Intuitively, one would expect that low motility is indicative of low viability, and therefore, low fertility. However, frozen and shipped-cooled semen will contain a percentage of cells that are viable but not motile. It is unknown if these cells regain their motility.


MORPHOLOGY

Morphology can be evaluated using several techniques. Semen can be analyzed using bright field, phase contrast, differential interference contrast (DIC) or electron microscopy. The sample may be prepared as a wet mount in buffered formal saline or stained with a variety of stains such as eosin-nigrosin, hematoxalin-eosin, Giemsa, or new methylene blue. Abnormalities can be classified as the absolute numbers of the abnormality or classified as primary or secondary. Primary abnormalities are considered to have occurred during spermatogenesis while secondary abnormalities are considered to have occurred after spermiogenesis. There is an inverse correlation in stallions between the percent of sperm showing primary abnormalities and the fertility of the stallion (Bielanski, 1975; Jasko, 1990). Dott (1975) has shown that when a high proportion of sperm have abnormal morphology, the potential fertility is low, while others show no meaningful relationships between fertility and morphology parameters (Dowsett, 1984; Dowsett, 1982; Voss, 1981). Much of this work has been performed in the natural breeding situation, where the mare is bred with the entire ejaculate. This relationship may differ in semen programs using shipped-cooled semen and frozen-thawed semen where the number of sperm is limited as each breeding dose is a portion of the total ejaculate.


CONCENTRATION (TOTAL SPERM NUMBERS)

The number of spermatozoa produced per ejaculate is a function of the amount of normal testicular parenchyma present. Concentration of raw semen is determined by the use of a modified spectrophotometer (Densimeter®) or by dilution and a manual count using a hemocytometer. Total sperm number is a product of gel free volume of the ejaculate and the concentration. Daily sperm output
(DSO) is the number of sperm produced in 24 hrs by the horse. This number is used to estimate the number of mares that the stallion should be able to breed if used in a natural service or artificial insemination program. Daily sperm output can be estimated using testicular volume (Love et al., 1991). Testicular volume can be calculated by measuring the length, width and height of each testicle using calipers or ultrasonography. If actual DSO is below estimated DSO, it may be indicative of disease conditions of the testes, epididymides or accessory glands.

The Society for Theriogenology has developed guidelines for evaluating the potential fertility of the horse (Kenney, 1983). The examination includes evaluation of mating ability, physical well-being and semen quality. The semen parameters motility, morphology and total sperm numbers are measured and the values obtained are multiplied together to estimate the DSO. Candidates are defined as suitable, unsuitable or questionable breeding prospects. A suitable breeding prospect is a horse that should have a reasonable pregnancy rate (>75% seasonal pregnancy rate) when bred to 40 mares by natural service or 120 mares by artificial insemination.


ADVANCED TESTING PROCEDURES

More in depth and involved analysis of the various compartments of the sperm can be performed to evaluate semen quality. Compartments evaluated include plasma membrane, acrosome, DNA and mitochondria.


Plasma membrane

Sperm cells with intact plasma membranes are considered viable and thus potentially capable of fertilization. The plasma membrane can be evaluated by several techniques (Magistrini, 2000) including staining spermatozoa with exclusion or supravital stains such as eosin-nigrosin, Casarett’s, Giemsa and the Spermac® stain which typically do not penetrate an intact plasma membrane. They can be used with light microscopy. Interpretation of the viability of the plasma membrane varies with these stains likely because of differences in staining methodology between laboratories and personnel. Fluorescent probes can be used to evaluate plasma membranes but require a fluorescent microscope or a flow cytometer. Flow cytometry allows evaluation of large numbers of sperm and there is less variation in interpretation, but the procedures are expensive. Two stain combinations currently used to evaluate the plasma membrane are carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) and SYBR-14 (a membrane permanent nucleic acid stain)/PI (Live/Dead Stain, Molecular Probes). Cells with damaged plasma membranes will stain red while live cells will stain green. Using this technique, Magistrini et al. (1997) found a positive correlation between percentage of live sperm and percentage of motile sperm. The plasma membrane may also be evaluated by the zona or hemizona pellucida-binding assay (Pantke, 1995; Fazeli, 1995). Fertile stallions have a higher binding rate to the zona than subfertile stallions. Functionality of the plasma membrane can be determined by the hypo-osmotic swelling test (Nield, 1999; Nie and Wenzel, 2001). In this test, sperm are incubated in a hypo-osmotic solution and swelling of the cell is observed as evidenced by coiling of the tail. A functional membrane responds to the hypo-osmotic environment by drawing solution into the cell and exhibiting swelling of the tail. In humans, the hypo-osmotic swelling test has been positively correlated with in vitro and in vivo fertilization as well as sperm penetration assays (Jeyendran, 1992). The relationship to in vivo fertility has not been closely examined in the horse.


Acrosome

An intact acrosome is needed for the sperm to bind to and penetrate the zona pellucida. It can be evaluated with a variety of stains. A triple stain used in humans (Talbot and Chacon, 1981) has been assessed by Magistrini and Palmer (1991) to differentiate intact from damaged acrosomes. This technique was shown by electron microscopy to reliably detect dead acrosome- reacted sperm and live acrosome-intact sperm. In a second technique, lectin, either Pisum sativum agglutinin (Varner, 1987) or Arachis hypogaea agglutinin (Graham, 1996), is combined with FITC and the acrosome evaluated with fluorescence microscopy. Sperm stain in one of four patterns. Acrosome-intact cells will fluoresce green over the acrosomal cap; sperm undergoing the acrosome reaction will display patchy fluorescence. If the cell has already acrosome-reacted it will not take up stain or will stain on the equatorial segment. Transmission and scanning electron microscopy have been used to evaluate the acrosome (Varner et al. 2000). Images must be interpreted with caution as vesicle formation has been associated with certain sample preparation techniques (Abraham-Peskir, 2000).


Capacitation

Capacitation of equine spermatozoa can only be evaluated indirectly. The ability of sperm to acrosome-react after being exposed to capacitating conditions is evaluated. Varner and coworkers (1992) induced capacitation with heparin sulfate. They reported that the percentage of viable sperm that is acrosome-reacted is comparable to that reported for other species (20-40%). Meyers et al. (1993) evaluated capacitation by incubating sperm in a TALP-TEST buffer and assessing the ability of sperm to acrosome-react in the presence of progesterone.


Filtration assays

Filtration of a semen sample is used to remove acrosome-reacted, acrosome-damaged sperm or sperm with capacitation-like changes. Two common filtration techniques are the use of glass wool columns or Sephadex filtration. Glass wool columns trap sperm with acrosomal changes while Sephadex filtration traps sperm with capacitation-like changes. Pregnancy rate per cycle was correlated directly with the percentage of sperm that passed through the Sephadex column. Frozen-thawed semen from nine stallions was filtered and used to inseminate 177 mares (Samper, 1991).


Mitochondria

Mitochondrial activity of the sperm can be evaluated by staining with either Rhodamine 123 or JC-1, fluorescent dyes. Rhodamine is used to label a negative potential. Since the inside of the mitochondria is negative, only coupled respiring mitochondria will take up the dye (Papaioannou et al., 1997). The JC-1 evaluates changes in mitochondrial membrane potential that are indicative of mitochondrial function (Gravance, 2000). Mitochondria that have high membrane potential are motile and stain orange. Cells with low potential are non-motile and stain green.


Penetration assays

Penetration of the oocyte by sperm requires that sperm are motile with functional receptor proteins to bind the zona pellucida. Sperm must be capable of undergoing an acrosome reaction and binding to the plasma membrane of the oocyte. In vitro penetration tests have been developed to evaluate these attributes (Graham, 1997). The zona pellucida penetration assay evaluates motility, zona binding and penetration, sperm capacitation and the acrosome reaction. Sperm are incubated in a medium to induce capacitation and the acrosome reaction. These sperm are then incubated with oocytes for a variable period of time. The oocytes are fixed and then examined to determine the number of sperm bound to the egg. This test has been performed with frozen-thawed oocytes and oocytes stored in salt at 4°C. Semen from fertile stallions bound more oocytes than that from subfertile stallions (Meyers et al., 1996; Pantke et al., 1995). To decrease variability in binding of sperm to oocytes due to oocyte age, hemizonas have been created by bisecting an oocyte. Using this technique, a significant difference (P<0.0001) was found between mean number of spermatozoa bound and fertility indices of the stallion (Fazeli et al., 1995). The zona-free hamster oocyte test evaluates the motility of sperm and the ability of acrosome-reacted sperm to bind to and penetrate the oocyte. This test evaluates the fertilizing ability of more sperm in a sample than do homologous oocytes as multiple sperm can penetrate a single egg. Unfortunately, correlations with sire fertility have not been established in the horse (Graham et al., 1987).

In vitro fertilization has the potential to be a good test of sperm function. It evaluates motility, acrosome integrity, ability to undergo capacitation, ability to acrosome-react and ability to bind to and fertilize the oocyte. It has been used sparingly in the human (Yavetz et al., 1995) as it is invasive and expensive. In the horse, in vitro fertilization has a low success rate, so it is not used as a screening tool to assess fertility of semen.


Solving semen quality problems

The ability to solve fertility problems in the horse varies greatly. In the thoroughbred industry little canbe done to improve semen quality because only natural breeding is allowed. The entire ejaculate is
deposited in the mare so limited manipulations are possible. If the problem is secondary to illness, disease or injury, the primary problem can be addressed and sometimes corrected. If the cause of the problem is testicular in origin, little can be done. Semen collected for artificial insemination, for cooling and shipping or for freezing may have quality improved by modifying the environment in which the spermatozoa are stored.

Decreased motility is due to many factors. Stallions that produce a dilute ejaculate may have decreased motility due to contact of the sperm with excessive seminal plasma (Maxwell and Johnson, 1999). Horses used in cooled or frozen semen programs can have the ejaculate centrifuged to remove the excessive seminal plasma in an attempt to improve motility. Motility and longevity of the sperm are typically evaluated in a variety of extender/antibiotic combinations. Motility can be affected by the composition of extender used. This demonstrates the importance of evaluating the motility in both the raw and extended sample. If motility is lower in a properly extended sample, the type of extender may be the problem. Improper handling of the semen can adversely affect motility. Extenders that will be added to sperm need to be maintained at 37°C and have an osmotic pressure of 300-370 mOsmole. Semen rapidly exposed to cold temperatures will exhibit a circular pattern. After semen is added to an extender, the resulting solution must be cooled slowly to avoid cold shock of the sperm.

Morphological abnormalities can occur during spermatogenesis, sperm transit, disease, trauma or can be created artifactually. For example, cold shock can cause the tails to bend back upon themselves, resulting in a morphologic abnormality (Amann and Graham, 1993). Testicular degeneration, various toxins, illness, high temperatures and trauma are associated with morphological abnormalities. Increased scrotal temperature due to trauma or longstanding fevers can cause morphological changes in spermatozoa. Within four days of insult, an increased incidence of detached heads will be seen. Sperm numbers will decrease in one to two months as spermiogenesis decreases (Amann, 1993). If the cause of the increased scrotal temperature is identified and treated, sperm morphology will eventually return to normal provided the duration of the insult did not affect the testicular parenchyma.

Decrease in concentration and total sperm numbers is associated with testicular degeneration, exogenous steroid use and use of progesterone in young stallions (Koskinen et al., 1997; Brady et al., 1997). As mentioned previously, increased scrotal temperature is associated with decreased sperm numbers 1 to 2 months after the insult.

Changes to the plasma membrane or acrosome are typically noted when the semen is being used in a cooled or frozen semen program. During cryopreservation, plasma membranes are damaged as the membranes go through extreme temperature and physical state changes. These effects can be decreased by controlled cooling rates and by adding lipids (egg yolk) or lipoproteins to the cryopreservation media (Graham, 1996). Addition of cholesterol to cryopreservation media increased the percentage of intact membranes post-thaw and decreased the percentage of acrosome-reacted sperm post-thaw. Fertility, however, was significantly (P<0.05) lower in frozen-thawed sperm treated with cholesterol (Zahn et al., 2002). Addition of Concanavalin A improves motility and the percentage of cells with intact acrosomes (Blanc et al., 1991). Cryoprotectants are added to cryopreservation media to decrease cell and membrane damage. Penetrating cryoprotectants act intra- and extracellularly to protect the cell. Glycerol is the most commonly used penetrating cryo-protectant. Ethylene glycol, dimethylsulfoxide and dimethyl formamide have also been used (Henry et al., 2002; Vidament et al., 2002; Pickett and Amann, 1993). As these alternative cryoprotectants do not result in marked improvements in motility, membrane integrity and fertility, glycerol is still the most commonly used cryoprotectant. Extracellular cryoprotectants include egg yolk, skim milk and various sugars. Modifying the composition of cryopreservation media can improve the post-thaw motility and plasma membrane integrity of frozenthawed semen (Braun et al., 1995).

Unlike other domestic species where cryopreservation techniques are fairly standardized, the horse requires an individualized technique. The semen from individual stallions responds differently to specific freezing protocols. Therefore, the centrifugation media and cryopreservation media must be altered to maximize post-thaw motility and membrane integrity.
 
In all species, oxygen free radicals form as part of aerobic respiration in the mitochondria. These free radicals cause membrane lipid peroxidation that results in structural alteration of the plasma membranes. A variety of antioxidants have been examined in the horse (Denniston et al., 2000; Ijaz and Ducharme, 1995; Aurich et al., 1997). Structural alteration of the plasma membrane will decrease the ability of spermatozoa to withstand cooling and freezing. Although experiments showed improvements in motility and membrane integrity, the effect on fertility has not been examined.

A few studies have been performed in an attempt to improve quality of ejaculated semen. Exogenous hormones have been given to stallions, however, they are not beneficial. Treatment with gonadotropin
releasing hormone, human chorionic gonadotropin or follicle stimulating hormone, which function at the hypothalamic-pituitary level, has had limited success (Roser, 2000). This may be because idiopathic infertility is thought to originate at the testicular level.

No definitive work has been performed to evaluate nutritional supplements and their effect on semen quality. Changing ration composition as well as addition of vitamins and minerals to the feed has not been shown to be beneficial or detrimental to semen quality. Increasing feed intake to result in an obese horse will negatively affect semen quality, as excess fat insulates the testes. Many oral supplements and nutriceuticals have been touted based on testimonial evidence to improve semen quality but no studies have been performed to evaluate these claims (Hintz, 1993).


Conclusions

The ultimate test of semen quality is fertility. To measure fertility of a stallion requires breeding the horse to a large number of mares. This is not commonly performed in the equine industry due to time and cost. Therefore laboratory tests are used to evaluate semen quality, but no one test consistently measures semen quality. Combining three or more tests increases the likelihood of identifying the cause of infertility. However, how to determine which of the tests are the most appropriate is a dilemma.


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