Morphologic assessment of stallion sperm (Proceedings)

Sperm quality is the culmination of a variety of factors such as the total sperm number, sperm motility, sperm morphology, and sperm fertilizing ability. Presently in an assessment of the quality of a stallion's ejaculate, it is often based only on the volume, motility, and concentration of the sperm. These are relatively easy parameters to quantify. Morphologic assessment, which an analysis of the sperm shapes, is a more complex task that requires significant expertise. In many cases where the sperm have acceptable motility and the numbers are adequate the underlying problem often lies with the morphology of the sperm. Monitoring sperm morphology along with other parameters such as motility and sperm number allows a manager owner to identify seasonal patterns, or problems in spermatogenesis in breeding stallions.

Reason for morphologic assessment

Morphologic assessment of stallion sperm is commonly performed in a breeding soundness examination. Many stallions are investigated for their fertility potential before entrance into or retirement to stud, as part of a pre-purchase examination for a sire prospect, or prior to semen shipment or cryopreservation. Stallions may also present with a history of low fertility, or declining fertility. Less common are serial monitoring during a breeding season of all semen parameters such as: volume, concentration, motility and morphology. Serial monitoring of semen quality allows management to make adjustments if the quality of the sperm produced declines, or the pregnancy rate suffers. Examples of this are plummeting semen quality measures when the weather becomes very, very hot for a prolonged period of time. In the interpretation of the results of a morphologic assessment of sperm it is essential that all health, fertility and semen characteristics are used in context. If horse breeders in the future wish to achieve the reproductive success of other species, such as beef cattle, or groups of horses such as the mares involved in equine ranching, or the uniform semen quality of warmblood stallions, then a focus on sperm morphologic assessment will be important.

It is customary when working with equine semen to only evaluate the volume, concentration and motility of the sperm and on today's stud farm these characteristics maybe measure using machines such as densimeters for concentration and CASA systems for motility. Progressive motility is correlated with fertility, as is the dose in terms of sperm numbers present in the semen. The variation in the percentage of normal spermatozoa among stallion seasons accounted for 15% of the variation in stallion fertility, and the combination of sperm morphology and motility accounted for 37% of the variation. A fuller picture of a stallion's fertility is achieved through a morphologic assessment of the sperm. A morphologic assessment of the sperm from 2 ejaculates is a requirement for a Breeding Soundness Examination of a stallion. In addition in combination with motility measurements and pregnancy rates, morphologic assessment allows the detection of negative or positive trends in spermatogenesis. The advent of cooled transported and frozen semen has increased the number of semen samples handled by veterinarians and other individuals. There are currently few guidelines available about semen quality, and many samples arrive at farms for mares with no information. A quick assessment of the sample allows for a determination of the viable sperm available for conception. This includes a determination of the number of progressively motile and morphologically normal sperm available in the inseminate. A clinician is then in a position to determine if there are 1 or 2 insemination dosages received, and understands the mare's relative chance of conceiving.

The anatomic features of sperm are simple and easy to assess.

Anatomy of a sperm

Sperm have a head, midpiece, principle piece and end piece. The acrosome covers 2/3rds of head and terminates at the equatorial region. The "tail" includes a midpiece, a principle piece and an end piece. The midpiece contains the mitochondria arranged in a helical fashion which provide energy to the sperm. The principle piece follows and the very end of the sperm has the end piece. Running through the "tail" of the sperm is a structure called the axoneme that stiffens and supports the sperm, and it includes a 9 +2 arrangement of microtubule doublets, which taper off in the end piece.

Historically there were a number of morphologic classification systems for sperm

Author such as Lagerlof, Milovanov, and Blom wrote about methods of classifying sperm in bulls. Blom described 2 systems of classification where sperm defects were either termed as "Primary or Secondary" or "Major or Minor.". Primary defects were those that occurred during spermatogenesis, thus representing a failure of spermatogenesis; and Secondary defects were those that occurred in the excurrent ducts, which represented a failure of maturation. A Primary defect was therefore deemed to be of testicular origin (examples include head defects such as nuclear vacuoles or acrosome defects), and a Secondary defect would be a defect of maturation such as a proximal droplet. Blom also used "Major or Minor" to describe defects. Major defects included problems with heads, midpieces, and proximal droplets, which are defects that are thought to have a greater impact on fertility. Minor defects were deemed those defects which have an unknown role or inconsequential role in fertility, such as distal droplets.

There are problems in using these classification systems, as testis tissue produces sperm with both Major and Minor defects, and sperm with a Primary defect(s) may gain a Secondary defects as they undergo maturation. Therefore both Major and Minor, Primary or Secondary defects may be present on the same sperm so that the most proximal defect or the most severe defect must be chosen. The process of choosing a defect on a sperm, even when multiple defects are present, results in a percentage distribution that will total to 100%. The examiner will not however have a record of the distribution of the sperm defects in the ejaculate. This fact makes it much harder to determine if there are any changes in the types of defects present in sperm over time. This is relevant when we consider the process of spermatogenesis and determine that multiple developmental stages of the sperm may be affected by an injury. More recently a concept of classifying sperm defects as Compensable and Non-compensable System was also proposed. A Compensable defect is a defect where either the sperm are not able to reach the site of fertilization, or the sperm is unable to: bind to the oocyte, induce the cortical reaction, and initiate fertilization. There is no effect on fertility if a numerically adequate population of normal sperm are present for fertilization, because the oocyte may still be fertilized by other fertile sperm. The fertility of the individual is not affected by defects that are effectively overcome by having adequate levels of normal sperm. The normal sperm compensate for the abnormal sperm. Similarly a non-compensable defect is a defect that does not interfere with the sperm reaching the site of fertilization, and the cortical reaction is induced by the sperm as it binds to the oocyte, and may cause syngamy, but the sperm is incapable of supporting embryonic development. These non-compensable sperm defects interfere with fertility. An example of a non-compensable defect is sperm DNA condensation or DNA compaction problems.

Introduction to the differential spermiogram

Another system of morphologic evaluation is called the Differential Spermiogram which has currently gained popularity. The Differential Spermiogram System is performed in an analogous fashion to a differential white count in a leukogram. A total white count and a differential cell count is performed on a blood sample. The differential white blood cell count gives us a variety of information such as the percentage of lymphocytes, neutrophils, monocytes, eosinophils, and basophils in a sample, and changes in the absolute number and percentages of neutrophils and lymphocytes are evaluated. Neutrophils are also scored for signs of activation, such as toxic changes, which indicate impending cell death. Serial daily and weekly samples are used to compare changes in terms of the absolute cell numbers, percentages of cells such as neutrophils, and the condition of the cells in terms of toxic changes in the leukogram. This information helps us to determine if our patient is improving, staying the same or deteriorating. This same sort of logic and principles that apply to a differential white count apply to the use of the Differential Spermiogram in the stallion. The sperm sample is examined for the presence of different cell types in the ejaculate (sperm, neutrophils, lymphocytes, red cells, squames, germinal epithelial cells). The sperm cells are differentially classified by enumerating the defects on the sperm. Using the supravital stain Eosin Nigrosin, live and dead sperm are evaluated in a separate count. Cells which stain pink are considered dead or devitalized, while the cells that stain white are considered alive. The Live / Dead sperm ratios are determined, to indicate the vitality of the sperm, similar to the identification of toxic changes in neutrophils.

Preparation of a sample

In a complete semen analysis the total sperm number is determined (concentration x volume), the progressive motility, and the morphology of the semen is examined. The stallion's sperm sample must be filtered to remove the gel fraction because gel makes the sample impossible to interpret. Sperm are mixed with Eosin Nigrosin for the morphologic assessment using a 60:40 stain to sperm ratio to provide good contrast and density of sperm for evaluation. The stain is hyposmotic to the sperm so the slide should be dried quickly, such as on a warming tray, to prevent any hyposmotic artefacts from forming (usually curved mid and end pieces). Wet mount preparations viewed under phase contrast do not usually allow the same detailed examination of the defects present. The Eosin nigrosin slides are examined using 100x objective (oil immersion) (1000x magnification).

Laboratory errors in preparation

Laboratory errors causing the appearance of sperm defects are sometimes referred to as tertiary defects. There are a few defects that may be produced through errors in sample handling such as failure to filter the semen, excessive stain, cold shock and hyposmotic shock. Gel in the sample makes the background appear white and makes the sperm difficult to see. Excessive stain will create stain cracks along the sperm. Artefacts associated with cold shock of sperm include distal midpiece reflexes, while hyposmotic shock may result in curved midpieces or curled endpieces. In general it is very important to emphasize that the vast majority of defects present in a sample come out of the stallion. Errors or rough handling of the sample will not produce defects such as detached heads, or nuclear vacuoles. Most sperm defects are either developmental or maturational in nature.

Differential spermiogram

A spermiogram, which is a frequency distribution of all defects is performed on a minmum of 100 sperm cells, using a cell counter. The presence of other cell types is noted and additional stained slides may be determined to identify the other cell types. For example a sperm with a macrocephalic head and a broken midpiece are enumerated by simultaneously pressing both keys on the cell counter. This will only advance the counter by one number in the total column. Mathematically the percentages for the categories of defects when added to the percentage of normal sperm will not add to 100% using the Differential Spermiogram System simply because many sperm have more than one defect.Categories in the Differential Spermiogram: The categories used in the Differential Spermiogram such as what might be included in the cell counter spaces are: head, midpiece, detached normal head, detached abnormal head, principle piece, proximal droplet, distal droplet, and acrosomes. All the sperm in a field are counted irrespective of whether they stain white or pink with the Eosin Nigrosin stain. Figure 1 shows a normal sperm. When detached heads are encountered the head but not the corresponding headless midpieces are counted. Certain abnormalities when found together represent more severe disturbances in spermatogenesis and influence the stallion's prognosis [8, 9]. Mentally the relative frequency of subcategories of the head and midpiece defects is noted. Head defects include: Microcephalic (too small), Macrocephalic (too large), Pyriform (pear shaped), Tapered (long and narrow sperm), Teratoid (a coiled sperm which is almost non-recognizable), vacuolated, knobbed acrosome.

There are a number of reports on the head morphometry of sperm in a large number of breeds [10-12]. The range of values for fertile stallions was as follows: length, 4.9-5.7 μm; width, 2.5-3.0 μm. Regarding acrosomes, knobbed, missing, or abnormal acrosomes are noted. Midpiece defects: include: segmental aplasia of the mitochondrial sheath (missing mitochondria), swollen mitochondrial sheath, pseudo-droplet (bunched mitochondria), proximal droplet (cytoplasmic material – which indicates the sperm is immature), fractured midpiece, distal midpiece reflexes. Abaxial insertion of the midpiece is not considered an abnormality in the stallion. Segemental aplasia of the mitochondrial sheath has the appearance of gaps in the mitochondrial shealth, a swollen sheath involves visible thickening of the sheath, a pseudo-droplet is bunching of the mitochondria on the midpiece with a corresponding region on the midpiece of missing mitochondria, fractured midpieces usually have broken fibres extruding at the site of the break, a distal midpiece reflex involves a hairpin turn of the principle piece with or without a distal droplet. Proximal droplets are made of cytoplasmic material and are considered separately and are not a midpiece defect per se. Distal droplets are not considered an abnormality. Comparing the Differential Spermiogram to Other Classification Systems: The percentage of normal sperm is a constant in all of the classification systems. Therefore this measure should be same in all systems.

Brief review of spermatogenesis in the stallion

The sperm in the ejaculate are a reflection of what has happened to the stallion in the last 2 months. In spermatogenesis diploid germinal cells termed spermatogonia, replicate and mature to form haploid, mature sperm. A stallion produces an estimated 16 million sperm per gram of testicular tissue per day. In males defective sperm are eliminated through apoptosis and phagocytosis by the Sertoli cells, and other phagocytic cells in the excurrent ducts, while others are passed into the ejaculate. The seminiferous tubules contain the germinal or spermatogenic epithelium and the supportive Sertoli cells. Cross sections of the seminiferous tubules show that 4 or 5 specific cell types are consistently found together, and these are present in different combinations called cellular associations. Time Frame: The time period from a committed spermatogonium to a mature to a sperm that is released in the lumen of the seminiferous tubule has been shown experimentally to be around 55 - 57 days in the stallion. This is relevant because it allows an examiner a reasonable expectation of when improvement should be seen. This 55 – 57 day period may be divided into 3 phases: spermatocytogenesis where mitosis and differentiation of spematogonia occur (19.4 days); meiosis the period of replication (primary spermatocytes) and then reduction of genetic material into haploid spermatids (19.4 days), and spermiogenesis development and differentiation (18.6 days). Spermiation is the release of spermatids into the lumen of seminiferous tubule. Approximately 9 days are required for transportation of sperm through the excurrent ducts such as the rete testis, and epididymides. A new population of sperm are available for ejaculation every 64 – 66 days. Due to the time it takes sperm cells to divide specific cell types are always present together in what is called cellular associations There is no seasonal influence on the developmental intervals, in other words in winter or summer a sperm takes the same amount of time to produce sperm. There is a seasonal influence and an influence of age on total sperm production, sperm motility and sperm morphology. In the absence of ejaculation sperm are eliminated in the urine.

Stallion sperm morphology

A number of authors have reported that the percentage of morphologically normal sperm in the general population of stallions is around 50%. Therefore in fertile horses there are a relatively large number of morphologically defective sperm. This is important to remember, but pasture mated and feral horses achieve high fertility rates under good nutritional conditions. The question then becomes determining what constitutes a problem when the percentage of morphologically normal sperm is lower than 50%, and / or mares are failing to conceive. A high percentage of defects is associated with lower fertility. Selection for fertility such as in Warmblood breeds does result in higher percentages of normal sperm, or in the case of inbred breeds such as: Clydesdales, Shires, Tennessee Walkers, and Friesians lower fertility and poorer sperm quality is present.

Serial evaluation of the sperm morphology

Serial examinations of semen using the Differential Spermiogram system, is a powerful clinical tool when it is possible to gain insight into the fertility potential of an individual. The problems that arise as a result of the stallion's intrinsic fertility (genetic), a point source problem such as a stress (fever), or a long standing alteration (testicular degeneration) may be diagnosed by serial examinations of ejaculates spaced at least 1 month apart. The clinical interpretation of semen samples is challenging because an examiner must determine which parameters and abnormalities are constitutive and reflect an individual stallion's age and intrinsic genetic ability, and which changes are extrinsic due to a disturbance (nutritional, hormonal, infectious, toxic, degenerative, idiopathic) in spermatogenesis. Age related changes: In general stallions reach puberty at about 18 months of age, but there is wide variability with stallions as young as 8 months achieving puberty. Testicular size is correlated with fertility. Total scrotal width typically is greater than 10cm in fertile horses. Larger mature size breeds tend to mature later. Mature stallions will have an increase in testicular mass until around 4 years of age. Older stallions tend to be more seasonal than younger stallions, but in general fertility does not decline with age in horses.

Puberty – stallion are generally not evaluated before 2 years of age

Young stallions at 2 years of age may still be exhibiting signs of puberty, while others may have poor intrinsic fertility. Until a stallion is producing at least 100 million sperm per ejaculate he is not considered to have reached puberty. Stallions reaching 18 months of age in the winter may have a delayed onset of puberty related to photoperiod. The process of puberty takes approximately 2 months to complete and involves the attainment of patent seminiferous tubules. This explains why germinal epithelial cells appear in the ejaculate at this time. The process of spermatogenesis is not highly efficient when production first begins and there are increased numbers of abnormal sperm. Four types of defects are common in pubertal stallions; germinal cells, head defects, midpiece defects, and proximal droplets. A total scrotal width of 8 cm and soft testes usually indicates that the stallion is immature. A typical ejaculate of a pubertal stallion has a low concentration, low motility, a high percentage of germinal cells (>10%), and other defects such as head, midpiece and proximal droplets. The second ejaculate shows similar parameters except the concentration plummets which reveals a low reserve capacity. Pubertal stallions are classified as Questionable Breeding Prospects, and re-evaluated in 2 – 4 months time or longer depending on the photoperiod conditions

Intrinsic defects

In a population of male animals examined for fertility there will be: below average, average and above average levels of fertility, these attributes are reflected in their testicular size, intrinsic sperm number/concentration and motility / morphology parameters. Intrinsic defects are consistent and there will be only minor seasonal fluctuations, and therefore there is no improvement over time. The ability to produce sperm is largely determined by the stallion's genetics and testicular size. The consistent appearance of a high percentage of morphologically defective sperm in an otherwise healthy young stallion who has no history of illness and who is not receiving exogenous hormone therapy suggests that he has intrinsically low fertility, and he will exhibit only minor seasonal variations in his semen. The presence of intrinsically low fertility is accentuated by inbreeding, and is supported by the practice of not selecting for fertility characteristics in stallions. In some stallions these are manifested by consistent defects present in high percentage of sperm, visible at the light and ultrastructural level, such as Dagg-like sperm (fractured midpieces), severely knobbed acrosomes, large numbers of microcephalic or vacuolated sperm, or missing dyneine arms (immotile live sperm).

Disturbances in spermatogenesis

A disturbance in spermatogenesis is generally manifested by changes in the sperm morphology as quantified using a Differential Spermiogram. Insults to spermatogenesis may be transient and result in temporary fertility impairment, or may result in permanent fertility impairment. Adverse exposure to toxins, radiation, dietary deficiencies or excesses, heat stress, trauma, parasite migrations, tumours etc also influence sperm morphology. A disturbance in spermatogenesis is therefore superimposed on the other factors that influence sperm production.

Permanent fertility disturbances

A variety of problems may result in permanent fertility impairment such as tumours of the testis, testicular torsion/infarction, heavy metal exposure, or unilateral castration. Severe nutritional, neoplastic, vascular, hormonal, traumatic, infectious, toxic, or degenerative insults may permanently impair fertility. These conditions may alter scrotal thermoregulation, cause intense inflammation, may breach the blood testis barrier, interfere with endocrine function, or damage the spermatogonial progenitor cells. More often than not in stallions the cause for the decline in fertility is not identified and is therefore idiopathic. The most common cause of idiopathic sub or infertility in stallions is testicular degeneration. Occasionally testicular degeneration is transient following a dramatic insult, but in most cases it results in permanent alterations. Testicular

Degeneration

The history of the stallion generally includes that previously acceptable (45-55%) per cycle pregnancy rates has declined to a lower per cycle pregnancy rate (<30%). This history should trigger a full breeding soundness examination including an evaluation of the stallion's testicular size and consistency. Testicular degeneration results in a loss of germinal epithelial cells, and will result in small testes, with a soft consistency with prominent connective tissue bands (ribs). A Differential Spermiogram will show low numbers of normal sperm, many germinal epithelial cells (>10%), and high percentages of abnormal sperm. Head and midpiece defects may exceed 25 – 30%.The potential for improvement is low based on testicular consistency and size. The poor motility of the sperm often relate to underlying morphologic problems of the midpiece. An excess number of germinal epithelial cells, high percentages of head and midpiece defects should suggest testicular degeneration. Serial examinations will show persistence of the problem or a continued decline. These stallions would be classified as having an Unsatisfactory Breeding Potential. The stallions with testicular degeneration will tend to stay the same or decline in the percentage of normal sperm over time. True testicular degeneration is not reversible, and improved fertility relies predominately on improving the management of the stallion.

Subfertility

A subfertile stallion may be intrinsically an inefficient breeder, or he may have lost fertility over time. Therefore the history is very important is determining the nature of a stallion's subfertility. Stallions lose commercial viability when their per cycle pregnancy rates drop below 30%. There are a host of reasons why stallions become subfertile, including psychological and medical problems. However the majority of subfertility problems are manifested by high percentages of morphologically abnormal sperm, and the stallion may or may not have low sperm motility. The presence of germinal epithelial cells indicates the stallion may have testicular degeneration, but the absence of germinal epithelial cells indicates the stallion has poor sperm quality. Ultrastructural defects may be present in the sperm. Problems with the head, annulus, midpiece and axoneme have been reported.

Defects

Specific defects for example severely knobbed acrosomes, midpiece defects, DNA condensation defects, and nuclear vacuoles may be present in sperm that are alive and progressively motile in stallions with a testicular size/mass and consistency within normal limits. Spermatogenesis is a complex process, which includes dramatic changes in the chromatin, which refers to DNA and the complex of proteins that associate with it. During maturation in the testes and epididymides there are a number of protein substitutions, often with zinc containing proteins, that neutralize the charges on the DNA, and allowing it to compact or fold up. This process is known as DNA condensation. The proteins need to be substituted in a specific order and in a specific time frame. We know that immature sperm, such as those with proximal droplets, are not able to fertilize because their DNA is not fully condensed. The droplet present on sperm begins is in a proximal location on the midpiece as the sperm enters the epididymis, but is shed from the distal piece just prior to ejaculation. Proximal droplets in a high percentage of sperm indicate that these sperm are immature and have not completed chromatin compaction. The formation of the acrosome occurs concurrently with some stages of chromatin compaction. The presence of acrosomal defects suggests that additional tests should be considered (chromatin assays), as the sperm with knobbed acrosomes have been shown to have more immature chromatin. Sperm with acrosomal defects are a heterogenous population and morphologic problems such as mild knobbing or folding of the acrosome may not influence fertility, but extremely severely knobbed acrosomes or those that contain vesicular material represent a more serious form of the defect, and are more likely to have an underlying problem in chromatin maturation. The knobbed acrosome defect may have a heritable basis, and may be a constitutive problem in some stallions. Therefore one abnormality may signal the presence of another, as acrosome defects are associated with chromatin defects. Serial semen evaluations may be used to determine the persistence of the defect. There is some suggestion that this condition is heritable in many species.

Spermiostasis

Spermiostasis is usually an acquired and persistent idiopathic condition in the stallion. In these stallions one or both ampullae fail to function normally and sperm accumulate. The ampullae may be partially obstructed or completely blocked. In the case of complete obstruction alkaline phosphatase will still usually be elevated in the semen sample indicating the horse is ejaculating. The typical spermiogram shows extremely high numbers of sperm with detached usually pink staining heads, either in every ejaculate or released intermittently. The ampullary plugs are occasionally released and grossly visible within the ejaculate. The detached heads are believed to belong to senescent sperm that are then improperly stored and intermittently released. Cannulation and flushing of the ampullae through a urethral approach has not been successful. The underlying pathogenesis of the condition remains obscure. Rectal massage of the ampulla, and oxytocin before collection have been used along with frequent collection to manage some stallions.

Supplemental tests

When sperm appear to have normal morphology but do not get mares in foal it is time for a different level of examination. Transrectal ultrasound and testicular ultrasound to evaluate testicular echotexture, and Doppler analysis of blood flow may be used. A variety of stains such as coomasie blue, or fluorescent stains may be used to evaluate the acrosome, and tests to induce the acrosome reaction may be performed. Endocrine tests are used to characterize the problem and responsiveness of the hypothalamo-pituitary-testis axis. A Feulgen stain may help identify nuclear vacuoles and chromatin condensation defects. A sperm chromatin structure assay may be used to investigate chromatin stability. An antisperm antibody assay may be indicated if sperm are observed to exhibit head to head agglutination. Scanning or Tranmission electron microscopy may show or reveal the nature of a defect through ultrastructural analysis, such as with immotile sperm that stain alive (primary ciliary dyskinesia)]. Occasionally testicular biopsy is used to determine if the appropriate cell types are being produced and to confirm where the defective sperm are forming, or if a tumour is present.

Treatments to improve sperm morphology

There continues to be interest in improving semen quality through the use of supplements such as docosahexaenoic acid (DHA), and omega 3,6 containing neutriceuticals. Others suggest supplements such as carnitine, and vitamin E selenium may be beneficial. The DHA products are derived from fish meal and are fed for months before a desired effect is reached. Some reports show improvements in sperm morphology, motion characteristics, and ability to survive cooling and freezing. Many times efforts are aimed at enriching the stallion's environment psychologically so he achieves harem status, and are focused on medical management to remove stress, improve health and ensure the stallion is fully ejaculating.

Adjustments to breeding doses,or management routines

Stallions with high percentages of abnormal sperm may still successfully get mares in foals at acceptable rates if they have enough vigorous normal sperm to compensate, especially when breeding at pasture. Planned multiple mating have been reported to increase pregnancy rates in some stallions. Insemination with whole ejaculates in stallions with very low numbers of normal sperm has also been used as a strategy to increase pregnancy rates, as have enriched populations of sperm delivered using deep horn or hysteroscopically guided insemination. Therefore when defects are compensable increasing the number of sperm in an ejaculate will often achieve the desired result. Sperm samples with high percentages of abnormal sperm on the average do not cool as well or freeze very well. Veterinarians may need to adjust the number of sperm in a breeding dose upwards to compensate for poorer morphology. On the average stallions produce around 50% normal sperm, a recommendation is adding at least 100 million more motile sperm for every 10% decrease below 50% in morphologically normal sperm. Stallions generally will begin to leave unacceptably high numbers of mares open when they fall below 100 million morphologically normal and progressively motile sperm per breeding dose. There are some papers that suggest that additional processing steps such as density gradient centrifugation, glass wool sephadex separation, or pelleting sperm followed by swim up may assist in harvesting the best sperm from an ejaculate. The percentage of morphologically normal sperm and sperm with intact chromatin was increased by colloidal centrifugation. The authors reported a decrease in the incidence of proximal cytoplasmic droplets and midpiece defects after centrifugation through colloids. Whether fertility is improved by such processing steps will vary with individual stallions, and likely would only apply to subfertile stallions, and of these only a proportion of the them would realize an increase in fertility.

References available upon request.