Canine semen collection and evaluation (Proceedings)

Introduction - a review in brief

The normal male dog attains puberty at approximately 6 – 8 months of age. Sexual maturity is generally attained at 18 – 30 months. Males may successfully breed bitches prior to sexual maturity but they will not achieve maximal fertility or daily sperm output until mature. The normal male can breed once every 2 - 5 days and maintain daily sperm output.

A complete breeding soundness examination in the dog includes history-taking, general physical examination, reproductive system examination, libido determination, semen collection and evaluation, hormonal evaluation, and prostatic examination and testing for disease (eg. Brucellosis).

Semen collection

In addition to artificial insemination breeding, collection of semen is indicated for evaluation in conjunction with a breeding soundness examination, in the diagnostic workup of potentially subfertile or infertile dogs, in the diagnostic workup of reproductive tract disease (infectious, degenerative or neoplastic disease), or for short-term (fresh-chilled extended semen) or long term (frozen semen) storage of gametes to be used in the future. When very young (< 7 months) or aged (>12 years) sires are used for breeding, the American Kennel Club (AKC) requires documentation, including a semen evaluation, of the male's ability to sire a litter.

The collection process - tips for success

     • Ambiance is important. In the typical setting, ejaculation in the dog is a voluntary process and cannot be forced or rushed. The dog needs to be relaxed and comfortable with the place and the people involved.

     • Be prepared. Have all supplies and adequate personnel available. You will need a dog handler, a bitch handler, a estrous teaser bitch (optimally), collection cones, nonspermacidal lubricant, 15-ml conical centrifuge tubes, a microscope, microscope slides, coverslips, bulb pipettes, stain, a means to determine sperm cell concentration (hemacytometer or Spermacue™) and other supplies depending upon the purpose for the semen collection. Some males may not require any external stimulus beyond manual massage to attain erection. If a teaser bitch is not available, many dogs will respond favorable to swab scent from an estrous bitch. Swabs can be collected and saved ahead of time.

     • Perform the collection on a non-slip, dog friendly surface (I have non-slippery epoxy flooring and my "magic carpet").

     • With the bitch adequately restrained, the dog is allowed investigate the bitch's hindquarters. He may sniff or lick her external genetalia or rear limbs. He may or may not mount the bitch. Signs of readiness include salivation and "chomping" of his teeth. During collection, the dog should be observed for ease of achieving an erection, presence of a normal erection, normal thrusting behavior and normal pulsation associated with ejaculation and prostatic fluid emission.

     • It is important to be aware of the onset of penile erection. Some males will achieve erection without any manual manipulation. Other males require brisk massage of the bulbus glandis through the prepuce to elicit erection. As soon as erection begins, the prepuce is moved proximally such that the entire prepuce is proximal to the exposed bulbus glandis. Failure to reposition the prepuce at the correct time may result in an inability to slide the prepuce proximally over the bulbis glandis. The collection process may then become painful to the dog as the skin of the prepuce tightens over the engorged bulbis glandis. It is important to roll over the edge of the cone such that the edge of the cone does not come into direct contact with penis. Failure to do so may result in a "paper cut" type of injury to the penis.

     • Concurrent with prepucial positioning, the collecting cone is introduced over the engorging penis. The collecting cone covers the penis to the level of the bulbis glandis.

     • The collector uses his or her thumb and finger to form a ring proximal to the bulbis glandis and pressure is applied to the penis circumferentially, thus simulating the copulatory lock or "tie."

     • With the onset of ejaculation, dog will thrust vigorously for several minutes. During this period, the first and second seminal fractions are ejaculated. The first or pre-sperm fraction is of prostatic origin and is clear. The pre-sperm fraction arises from the prostate and urethral glands, and is believed to cleanse the urethra of contaminants (urine, bacteria and cellular debris) prior to ejaculation The second or sperm-rich fraction originates in the cauda of the epididymis where spermatozoa are stored.

     • After a brief period of rest, the dog will ejaculate the third seminal fraction. The third fraction is comprised of prostatic fluid and is normally clear. Prostatic secretion provide volume to the ejaculate, assist in propelling the sperm out of the vagina and into the cervix/uterus, and provide nutrients for the sperm while traveling to the site of fertilization in the oviducts. During third fraction collection, the dog may try to step over the collector's arm. If this occurs, the collector should lift the dog's rear limb over his or her arm and redirect the penis caudally to simulate the copulatory lock or "tie." It is important to redirect the penis caudally while maintaining the horizontal plane of the penis. Do not redirect the penis caudally in a vertical "pendulum swing" plane as this may cause injury.

     • During ejaculation of the third fraction, the collector can feel and visualize the rhythmic pulsations in the penile urethral and perineal muscles. There is also an audible spurting of prostatic fluid into the receptacle.

     • For breeding purposes, usually only the second, sperm-rich fraction is collected. If all three fractions of the ejaculate need to be collected, it is advisable to collect the fractions into separate receptacles. Fractionating requires the use of funnels or additional cones, dexterity and quick hands.

     • Once semen collection is completed, the collector releases the penis and removes the collection cone. The dog often will continue to ejaculate pulses of prostatic fluid. Detumescence of the penis may require several minutes and often takes a similar amount of time as would recovery from a natural mating. Care should be taken to ensure that no injury occurs to the penis at this time.

     • Make sure that subsidence has occurred and that the prepuce is positioned normally over the penis before allowing the dog to be discharged. Occasionally the prepuce rolls inward as penile engorgement wains leaving the unprotected glans of the penis vulnerable to dessication and trauma. With the aid of lubricant, the prepuce can readily be manipulated over the tip of the penis.

     • Number of spermatozoa collected is optimized in the presence of an estrus teaser bitch. As much as a four-fold increase in spermatozoa number can be realized when using a teaser bitch. Alternatively, swab the perineum of any tractable female with frozen-thawed swab scent from an estrus bitch.

     • For reticent dogs or when a teaser bitch is not available, semen collection may be facilitated by the administration of prostaglandin F2 alpha (Lutalyse®) at a dosage of 0.1 mg/kg subcutaneously 15 minutes prior to collection. Side effects commonly associated with prostaglandin administration (salivation, emesis, etc.) are minimal at this dosage.

     • For breeding purposes, usually only the sperm-rich second fraction of the ejaculate is collected.

Semen handling

Sperm are motile and vigorous cells, but are also fragile and susceptible to damage and demise by environmental conditions. When collecting and handling semen it is critical to avoid exposing sperm cells to two types of insults - toxic chemicals and thermal insult. Keeping collection equipment clean and disinfected is important, but soap and disinfecting chemicals are quite potent spermicides. For this reason and for their convenience of use, disposable polypropylene collection cones are often preferred over their latex counterparts. When using latex cones, take great care to rinse thoroughly deionized water to remove all soap and disinfectant residue. It is usually best to use new, sterile collection tubes. Finally, be certain to lubricate collection cones with a sterile lubricant known to be non-toxic to sperm. If the tube of lubricant is not clearly labeled as being nonspermacidal then you cannot presume that it is safe to use.

Sperm are sensitive to both heat and cold. Rapid chilling of semen results in a phenomenon called "cold shock" that is often manifest by abnormal sperm motility and morphology. Short periods of exposure to temperatures just a few degrees above body temperature will usually kill large numbers of sperm. To avoid thermal stress, the collection cone should be pre-warmed to body temperature. Additionally, microscope slides, coverslips, stains, extenders and pipets used to handle and examine sperm are best maintained on a warming plate prior to use. If you are performing a large number of analyses, a heated microscope stage is also a valuable piece of equipment.

Semen from most species is not damaged by exposure to room temperature (20-22°C) for an hour or two. If longer periods of storage are required, it is best to dilute the ejaculate in a buffered nutrient solution, called an extender, and cool it slowly to refrigerator temperature (4-5° C). A large number of extenders have been developed and are commercially available for use in short term and long term storage of semen. They are similar in having an energy source (eg. glucose), buffers to maintain pH (eg. Tris or citrate) and a source of protein (eg. egg yolk or skim milk).

Semen evaluation

Putting the evaluation into perspective

Ultimately, the goal of any semen evaluation is to predict the likelihood for a dog to be successful in siring a litter. Routine semen evaluation in the dog includes determination of semen volume, color and pH, spermatozoa motility, velocity, concentration, total number of spermatozoa in the ejaculate, and sperm cell morphology. In humans, The World Health Organization (WHO) has developed strict criteria for semen collection and evaluation. WHO protocols recommend that semen be collected when the man has had sexual rest for at least 2 but no more than 7 days, and that two separate samples, collected 7–21 days apart, be evaluated before any recommendations are made. Similarly with morphologic evaluation, "A normal human sperm has a specified size and shape, with a smooth outline, an acrosome that comprises 40-70% of the sperm head, has no neck, midpiece or tail defects, and has no droplets more than ½ the size of the sperm head." Use of these criteria makes morphologic examination of the semen more standardized and allows for easier comparison of research studies. To date, there is no "WHO equivalent" for the dog. No such criteria have been adapted to any of the domestic species, including the canine, beyond the guideline that a dog should not be condemned on the results of one semen evaluation.

Male factor infertility accounts for up to 50% of failed pregnancy attempts in humans. Compared to human medicine, little is known in canine medicine regarding specific findings on semen evaluation and their correlation with fertility. No estimates have been reported for the canine, but experiential evidence supports the notion that male factor infertility does account for a significant portion of conception failure, early embryonic death and small litter size.

Veterinarians routinely perform semen analysis on dogs. Unfortunately, there is little data associating the parameters measured with the issues practitioners really need to evaluate, i.e. testicular function, fertilizing capability of spermatozoa, and likelihood that puppies will develop normally. With the current methodology, semen analysis may only be reliably predictive of fertility if the semen quality is either very good or very bad.

A big problem is that in vitro analysis ≠ in vivo function. Ejaculated spermatozoa examined in vitro do not exhibit the characteristics they will take on as they traverse the reproductive tract of the female. For a spermatozoon to fulfill its role, it must:

     • develop properly in the testis

     • undergo maturation in the epididymis

     • pass through the cervix and uterus

     • undergo capacitation and the acrosome reaction as it passes from the seminal fluid into secretions from the female reproductive tract

     • bind to the epithelium of the uterus or distal uterine tube

     • detach at the correct time and move into the uterine tube

     • penetrate the cumulus cells surrounding the ovum

     • bind to the zona pellucida

     • and for that lucky "one," penetrate the zona

Another problem is that evaluation techniques are difficult to standardize. Results of tests that may be performed in a semen evaluation are influenced by sample collection technique and timing, concentration of spermatozoa in the sample, amount of time from sample collection to evaluation, temperature at which the sample was held, equipment used, and many other factors, not the least of which is the subjectivity of the evaluator. Another impediment to standardization of semen evaluation in the canine relates to the enormous variability present as a result of breed. Along with the obvious extremes in testicular size, there may be other significant influence of breed on semen parameters.

Acknowledging the problems encountered and limitations inherent with making functional conclusions on the basis of in vitro testing, multiple parameters are used in performing a semen evaluation.

Color and appearance

Color evaluation is subjective but informative. The turbidity or opacity of semen provides a rough indication of concentration. Commonly, the turbidity of a sample is graded on a subjective scale of 0 to 5, with 5 being the most opaque as in the whole milk. A clear sample contains no spermatozoa. Although cloudy or milky samples almost always contain spermatozoa, they must still be checked microscopically because occasionally a dog with azoospermia will shed excessive numbers of fat droplets into the sample, giving the appearance of normal semen. Yellow semen is indicative of urine contamination and is also seen in humans with icterus or after ingestion of certain vitamins. Brown discoloration is indicative of old digested blood and red discoloration is indicative of fresh blood. The most common causes of hemospermia in dogs are prostatic disease and penile trauma. In humans, hemospermia also may be associated with urinary calculi, sexually transmitted diseases such as syphilis and gonorrhea, spermatocele and hydrocele, and treatment with anticoagulant medications.

pH

The value of pH measurement of canine semen is debatable. Values for normal pH in non-fractionated canine semen vary from 6.4 to 6.8. Seminal pH may change in the presence of disease, such as prostatitis, or if the semen sample is contaminated with urine. There are no published data comparing pH analysis techniques for canine semen or specifically describing the clinical value of pH measurement. Alterations in pH may affect sperm longevity and motility. Decreases in prostatic fluid pH are common with prostatic disease. Alterations in pH may occur with use of excessive amounts of lubricant or improper cleaning and disinfection of collection equipment. Standardization in the determination of semen pH with respect to timing and method of measurement (pH meter versus "dipstick" pH paper) would be useful.

Volume

Dog semen is ejaculated in three fractions. The first, pre-sperm fraction is small in volume (less than 5 mls) and contains few to no spermatozoa. The pre-sperm fraction is believed to cleanse the urethra of contaminants (urine, bacteria and cellular debris) prior to ejaculation. The pre-sperm is not usually collected. The second, sperm-rich fraction comes from the epididymes and testes. The sperm rich fraction is cloudy white in color and usually 0.5 – 4 ml in volume. The third fraction consists solely of prostatic fluid and contains few to no spermatozoa. Prostatic secretions lend volume to the ejaculate, assist in pushing the sperm out of the vagina and into the cervix/uterus, and provide nutrients for the sperm during their transit to the oviducts. The volumes of the first and third fractions are variable. In particular, the volume of the third fraction is controlled by the person collecting the sample, as they can choose to collect more or less of the cell-free prostatic fluid. Prostatic fluid is normally clear in color and may range in volume from 3 – 80 ml. Volume is not an indicator of semen quality in dogs. However, the measurement of semen volume is important in the calculation of total number of spermatozoa in the sample.

Motility

Sperm motility is essential for fertilization because it allows or at least facilitates passage of the sperm through the zona pellucida. Without technologic intervention, a non-motile or abnormally-motile sperm is not going to fertilize. Hence, assessing the fraction of a sperm population that is motile is perhaps the most widely-used measure of semen quality.

For canine semen, motility is better maintained if samples are kept at room temperature than at body temperature. Warmer temperatures increase sperm cell metabolism. Delay in evaluation of warmed samples may result in errors due to sperm cell energy depletion. Rapid temperature fluctuations should be avoided. Ambient temperature may affect the motility assessment of a sample if the evaluation room is excessively hot or cold. Percentage of progressively motile spermatozoa from a given dog is not affected by frequency of semen collection.

In evaluating motility, sperm cells are classified as immotile, progressively motile or non-progressively motile. Both total and progressive motility are determined and expressed as a percentage of 100. Sperm cells showing motility of any kind are included in the calculation of total motility. A progressively motile spermatozoan moves forward in an essentially straight line, whereas a non-progressively motile sperm cell moves, but with an abnormal path, such as in tight circles (tail chasing), fish flopping, and when normal movement is prevented by sperm head agglutination. In the dog, the normal percentage of progressively motile spermatozoa is 70% or greater. Speed or quality of motility also may be assessed; a canine spermatozoon with normal motility should traverse the microscopic field of view in 2–3 seconds.

Progressively motility is positively correlated with normal morphology in dogs. Just as is the case with the whole dog "on the hoof," think function follows form. If the sperm cell is put together normally, then it should move normally. Immotile sperm cells that are morphologically normal may have experienced "iatrogenic immotility" as a result of technical errors in semen collection or handling. Exposure to rapid or extreme temperature fluctuations, any kind of residue on collection equipment, or the wrong pH or osmolality of an extender can adversely affect motility. Motility is also affected by periods of sexual inactivity - males that have not ejaculated for prolonged periods often have poor motility on the first ejaculate, but much better motility for a second ejaculate collected soon thereafter. This is the so-called "rusty pipe" condition.

In humans, age is a factor in motility; motility has been documented to decline at rate of 0.27%/year in men > 45 years old. In protocols for humans, two samples of 200 spermatozoa are evaluated separately and graded as A (=rapid progressive motility), B (slow or sluggish motility), C non-progressive motility), and D (complete lack of motility). If results for the two samples don't "jive" (ie., the difference is greater than expected by statistical random variation), two additional slides are evaluated from that semen sample.

Manual motility estimates are easy to perform and require minimal equipment. This commonly-used technique involves placing a sample of semen on a microscope slide, examining it with a microscope and estimating the fraction of the population that is motile. Manual motility estimates are subjective. However, with an experienced evaluator, manual estimates generally provide good estimates of motility. The chief limitation of this technique is its subjective nature.

Computer assisted sperm analysis (CASA)

CASA is a technique employing a computerized system that tracks multiple motility parameters. The computer takes video images of the sperm and stores them for analysis. The system recognizes motile from non-motile sperm and other organic debris by comparing luminosity (gray-scale intensity) and size of the object. There are also preset user-defined thresholds for size and luminosity that help prevent mistaking other cells and debris for non-motile sperm. While some studies have shown that CASA systems are more accurate than subjective assessment of sperm motility, other studies have found that subjective analysis progressively motility is well correlated with computer-based analysis.

Most CASA systems are found in teaching hospitals and research laboratories. Using CASA, many more motility parameters can be evaluated, and be evaluated objectively:

     • Mean percentage of motile sperm

     • Mean percentage of progressively motile sperm

     • Curvilinear velocity

     • Straight-line velocity

     • Amplitude of lateral head displacement

Such parameters can be incorporated into detailed mathematical formulas to produce factors such as a Semen Quality Score or Sperm Motility Index. Normal values have not been established for the dog. Another potential value of these sperm motility factors is recognition of sub-populations of spermatozoa within the sample that may react differently to cryopreservation or other manipulations of spermatozoa.

Concentration and total number

Concentration is important because it is used to calculate total sperm number in the ejaculate. Concentration is inversely related to volume collected. Concentration multiplied by volume is the total number of spermatozoa in the ejaculate; total number of spermatozoa is dependent on testicular size. In dogs, normal total number of spermatozoa is greater than 300 million. A general guideline for total sperm number in an ejaculate is 10 million sperm cells per pound of body weight. Total number of spermatozoa decreases with frequent semen collection, presumably as epididymal reserves are depleted. In humans, concentration and total number decrease with age, declining 2.1%/year after 45 years of age. Normal dogs may ejaculate oligozoospermic or azoospermic samples due to apprehension or pain. In human medicine, apparently azoospermic samples are centrifuged at greater than 3000 g for 15 min and all the fluid systemically examined before that sample is declared completely deficient of spermatozoa.

Concentration can be measured by several methods: CASA systems, hemacytometer, optical density measurement, and spermatocrit. The most common means of determining sperm concentration is to simply count sperm under a microscope with the aid of a hemacytometer. A hemacytometer is a glass slide onto which a precision grid has been etched. They are sold for counting blood cells, but work equally well for counting sperm. Since the dimensions of the grid squares and depth of sample chamber are known, it is a simple matter to calculate cell concentration. The hemacytometer technique has been reported to be accurate and is considered the gold standard.

To determine concentration of canine spermatozoa using a hemacytometer, semen is drawn up into the 20 mL pipette of the Unopette WBC system [Becton- Dickinson, Rutherford, NJ, USA] and dispensed into the diluent chamber. The coverslip specific to that hemacytometer should be used. Do not use disposable coverslips. Concentration measurement with a hemacytometer is time consuming compared when compared to other methods.

The SpermaCue™ [Minitube of America, Verona, WI, USA] is a small, compact and accurate photometer developed for measuring sperm cell concentration. A <10 μl sample of raw semen is loaded into a microcuvette and inserted in to the machine. While it is not the gold standard technique, it is quick, easy and relatively inexpensive. Spermatocrit is determination of concentration by evaluation of percentage solids when semen is centrifuged in a hematocrit tube; this technique is not accurate in dogs.

Total number of spermatozoa in the ejaculate can be calculated by multiplying the volume (in mls) by the concentration (number of sperm cells per ml). A guideline for total sperm number in the ejaculate is 10 million sperm cells per pound of body weight. For example, a 60 pound dog should have 600 million sperm cells in his ejaculate.

Morphology

To evaluate morphology, 100 individual sperm cells are examined for normal shape and structure. The results of a sperm morphology exam are reported as percent normal. Minimally, the size and shape of the head, midpiece and tail are examined. The head contains the DNA and has a cap (the acrosome) which contains the enzymes that allow zona penetration and fertilization to occur. The midpiece contains the motor apparatus that propels the spermatozoum. The tail provides the propulsion to move the sperm cell forward. Regardless of breed, normal canine spermatozoa are 6.8 μm in length, of which the midpiece is 1.1 μm and the tail is 5.0 μm.

Defects may be classified in several different fashions: primary vs. secondary defects (primary occurring in the testicles, and secondary occurring during storage, transport or handling); major vs. minor (major affecting the ability of the sperm to fertilize and minor not affecting the ability to fertilize) or compensable and non-compensable (compensable defects can be overcome by providing access of the sperm to the egg and non-compensable defects cannot be overcome by the sheer presence of providing access to the egg).

The primary vs. secondary abnormality classification scheme is most common. Primary defects are the more severe and occur during the process of spermatogenesis while the sperm cells are still within the semeniferous epithelium of the testis. Secondary defects are less serious and are believed to arise either during passage and storage through the epididymis or by mishandling after collection. A normal ejaculate contains >70% normal sperm.

Examination of sperm cell morphology by phase contrast microscopy can usually be performed on unstained, undiluted semen or when concentration is high, in semen diluted with phosphate-buffered saline. Examination of morphology by light microscopy requires staining of the sperm cells. Morphology artifacts can occur during slide preparation staining technique, possibly the result of temperature shock or alterations in osmolarity or pH of the stain. A variety of stains can be used. As with motility, the vast majority of semen morphology evaluations are manual and subjective but there are computerized systems that can evaluate sperm cell morphology objectively.

Automated sperm morphometric analysis (ASMA)

ASMA provides a more accurate and repeatable evaluation of general sperm morphology. With this automated process, a consistent number of sperm/field may be analyzed. Very subtle abnormalities in the sperm head, midpiece and tail morphology can be detected 95% of the time with these measurements.

In the normal dog, a significant variation in head area, length, width and roundness exists, but the analyzer still provides accurate differentiation of teratozoospermic samples. Ovalness was the least variable factor obtained while length and width had more variation between dogs. Within dogs, there was less variation of any measurement. Similar variability in sperm head shape and size is noted with SCSA analysis of the same dogs, indicating that ASMA may be a valuable tool when assessing sperm for teratozoospermia.

Some of the more common morphology stains are:

     • Eosin-nigrosin (EN) stain - a vital stain which can differentiate live from dead sperm cells.

     • Modified Wright-Giemsa stain [modified Giemsa stain, DiffQuikTM; Baxter Healthcare, Miami FL, USA]. Diff-Quik is a simple, inexpensive, and rapid method of staining sperm. Basic alterations in head size and shape are detectable; acrosomes cannot be differentiated. This stain is able to differentiate round cells from immature germ cells in the ejaculate.

     • India ink is a one-step stain that is simple and inexpensive to use. It provides a black background and a white or clear sperm outline. Size and shape abnormalities can be identified, but it does not allow for good visualization of the acrosome or equatorial region.

     • Spermac is expensive and moderately time-consuming but is technically a simple staining technique. The nucleus of the cell stains dark red while the acrosome stains light green, and the midpiece and tail stain dark green. This is an excellent stain for acrosomal evaluation and also allows for good midpiece and tail piece evaluation.

     • Papanicolaou stain is commonly used in human andrology labs, but is not commonly used in veterinary applications due to the complexity of the procedure.

     • Acrosomal stains: Acrosome function is required in order for the sperm to penetrate the egg during fertilization. In some dogs, adequate sperm may reach the oviduct and surround the egg, but the acrosome reaction may not occur normally resulting in failure of fertilization. In these dogs, all other sperm testing may be normal. To assess acrosome function, a drug called calcium ionophore is added to the semen. This substance will induce the acrosome reaction in normal sperm. Fluorescent dye is then added and those sperm whose acrosomes react will take up the dye, while those that don't react, don't take up any stain. Acrosome testing still being refined for use in the dog, but holds good promise for future use.

          - Giemsa stain makes the acrosome appear dark purple. It provides good detail of the acrosome but needs to be made fresh for each use and does not allow for evaluation of the sperm nucleus, midpiece or tail.

          - Triple stain of Trypan blue stains the spermatozoa blue, Bismarck brown stains the post-acrosome region light brown and Rose Bengal stains the acrosome light red. Sperm can be differentiated into 4 groups with this stain: dead sperm with fully or partially inactivated acrosomes, dead spermatozoa with missing or degenerated acrosomes, live spermatozoa with reacted acrosomes, and live spermatozoa with active or normal acrosomes.

          - Fast green FCF, Rose Bengal and ethyl alcohol has been used to stain the acrosome of cat spermatozoa. This stain allows differentiation of acrosome intact, acrosome reacted or damaged sperm, and acrosome non-intact sperm. The slide is examined using bright field microscopy at 1000x.

          - Coomassie blue stain has been used to assess acrosomal integrity. This staining procedure is relatively simple and results in intense blue staining of intact acrosome and lack of stain uptake in acrosome reacted sperm. The results of Coomassie blue staining correlate well with fluorescent staining, DIC and bright field microscopy techniques.

          -Acridine Orange, a fluorescent stain, can be used to evaluate sperm that are in opaque extender, like skim milk. Acrosomal integrity can be evaluated using fluorescent microscopy, phase-contrast or DIC microscopy.

          - Bis-benzimide dye Hoescht 33 258 and a FITC-Pisum sativum agglutinin (FITC-PSA) uses fluorescent microscopy is used to differentiate acrosome reacted versus non-acrosome reacted sperm. It is a simple, quick technique to assess acrosome status, but it does not allow for morphologic assessment of the cell itself.

          - Trypan blue or Congo red stain can be precipitated by neutral red and then stained with Giemsa to stain bull, boar and rabbit sperm, but not stallion sperm. This is a simple and reliable staining procedure that results in the stained sperm being classified as live or dead with intact acrosomes, loose or damaged acrosomes, detached acrosome and detached acrosome with no post acrosomal ring.

     • Mitochondrial stains

          - Rhodamine 123 (Rh123) is a mitochondrial probe which is combined with the viability stains of PI and carboxydimethylfluorescein diacetate (CDMFDA) to allow for the determination of sperm with intact membranes (CDMFDA+) and functional mitochondria (Rh123+) from dead sperm (PI+). Stallion sperm with functional membranes and mitochondria correlate well with sperm viability and motility.

Miscellaneous tests

Hypo-osmotic swelling test (HOST)

Host involves submersion of spermatozoa into a hypo-osmotic medium. Those spermatozoa that have intact plasma membranes will swell as fluid moves into the sperm cell; this will cause swelling and coiling of the tail. This test is based on the premise that motile sperm have normal membranes and will coil or swell when incubated in a hypo-osmotic solution. Water will cross the membrane and enter the cell if the membrane is intact and the transport mechanisms are functioning normally. Membrane integrity is important in sperm metabolism and changes in membrane properties must occur for capacitation and the acrosome reaction to occur normally. HOST not only assesses the morphologic integrity of the plasmalemma but it also assesses its function and biochemical activity. Use of HOST on rewarmed chilled or post-thaw frozen semen may be predictive of the highest quality samples to be used for insemination by selecting for samples with the highest number of HOS+ cells. The HOST may be a beneficial addition to the semen evaluation in dogs with poor fertility but a normal spermiogram.

Sperm penetration assays

These assays asses the ability of the sperm to undergo capacitation, the acrosome reaction, membrane fusion and chromatin decondensation in the presence of an oocyte. Sperm must be prepared for the assay by incubating overnight in a capacitation medium or storing in a TES-tris buffer with egg yolk for 24 – 48 hours and then applying thermal shock. After this processing step, the sperm are divided into microdrops and zona-free hamster eggs are added. They incubate for 3 hours and the number of eggs penetrated and the number of sperm/egg are counted. The count is performed by looking for swollen heads within the unstained eggs using phase-contrast or phase-interference microscopy or after staining with acridine orange (AO) and using fluorescence microscopy. Use of the TES-tris buffer procedure was more highly correlated with fertility and successful outcomes with IVF in humans in some studies while in other species its predictive power was questionable.

Hemizona assays

This test assesses the availability of the proper molecules on the sperm's head, required for binding of sperm to bind to the zona pellucida and initiate interacting with the oocyte. A bisected zona pellucida from a normal oocyte is used. Each half of the zona is incubated with sperm for 4 hours and then the number of bound cells is counted. Zona pellucida binding assays have been used to evaluate the fertilizing capacity of chilled and frozen-thawed canine spermatozoa]. The test allows an estimation of the damage caused by manipulation of semen on the fertilizing ability of sperm. This test also demonstrates the critical interaction between the zona pellucida and the sperm cell during fertilization and tests multiple sperm functions, including capacitation and ligand-induced acrosome raction. Of the classic sperm parameters, morphology was the best predictor of the ability of sperm to bind to the zona pellucida. In conventional IVF studies in humans, defective sperm-zona binding and zona penetration are common causes of failure of fertilization.

There is a high predictive power of sperm-zona pellucida binding and successful outcome with IVF in humans.

Electron microscopy (EM)

Electron microscopy is a special microscopic technique that allows for very detailed examination of the entire sperm at very high magnifications. Cross sections and full length sections of individual sperm may be examined to see if there are abnormalities of structure that are beyond that seen by the light microscope. There are 2 types of electron microscopy, transmission and scanning. Transmission EM (TEM) provides a two dimensional view of the interior of the sperm cell, while scanning EM (SEM) provides a three dimensional view of the exterior surface of the sperm.

Transmission EM is typically more helpful in the assessment of infertility. TEM may identify lesions of the plasma membrane, acrosome, mitochondria, and nuclear chromatin. TEM may identify defects of the tail in patients with motility issues that are not apparent with light microscopy. TEM may also help identify and characterize other cells in the ejaculate including germ cells, WBC and infectious organisms. DNA fragmentation caused by oxidative stress or exposure to toxins may be identified.

Antisperm antibody assay

In humans, antisperm antibody production is a common cause of male factor infertility. There are two commercial assays [SpermMar®, Conception Technologies, LaJolla CA and Immunobead Test®, Irvine Scientific, Santa Ana, CA] available for human antisperm antibody assay. These tests provide semiquantitative results regarding the degree of antibody binding present and detect the presence of IgA and IgG antisperm antibodies. If ≥ 20% of the sperm bind to the beads, a sample is considered positive for the presence of antisperm antibodies. Serum may be assayed for antibodies using a tray agglutination test. At this time, the importance of antisperm antibodies in domestic animals is unclear but may be a useful test for dogs with autoimmune orchitis/epididymitis.

Flow cytometry for DNA and morphology measurement

Binding of fluorescent dyes to sperm chromatin permits the identification of sperm DNA abnormalities and can be measured using a flow cytometry. Sperm are stained using fluorescent assay and then run through the flow cytometer to differentiate cells with normal DNA integrity from abnormal. Sperm are typically oriented to be in the same plane before they are excited by a laser beam to induce fluorescence and then flow past a fluorescence detector which monitors exactly how much fluorescence each cell has. Sperm that have uniform head size and shape display a uniform degree of fluorescence while cells with abnormal size and shape have amounts of fluorescence outside the normal ranges. In addition to being able to differentiate morphologically normal from abnormal sperm, flow cytometry can also differentiate sperm with normal motility from those with decreased motility. One cause of decreased motility in humans is a result of a break in the DNA strands of the sperm nucleus and their mitochondria. Flow cytometry can confirm the presence of these strand breaks in this population of asthenozoospermic men.

One of the greatest assets of using flow cytometry for sperm evaluation is the sheer number of sperm that can be evaluated in a short period of time. Routine microscopic assessment of sperm involves counting either 100 or preferably 200 cells. But with the use of flow cytometry, thousands of cells can be assessed in a matter of minutes. Another assessment that can be performed using flow cytometry is that of sperm viability.

Cytochemical tests for sperm chromatin integrity

The integrity of nuclear chromatin results from a combination of factors including sperm maturation processes, damage due to oxidative stress and other endogenous factors. Chromatin maturation depends on proper replacement of histone with transition proteins and finally basic protamines. This transition results in compact packaging of the DNA and is enforced by cross-linking with protamine-disulfide bonds. In some abnormal sperm, histones may be partially or completely replaced by protamines resulting in loosely packed chromatin. Staining protocols provide a good estimation of the number of sperm with normal vs loosely packed chromatin structure. If more than 30% of sperm have loosely packed chromatin structure an association with increased infertility is noted.

Sperm chromatin analysis

Sperm morphology is well correlated with semen quality. The head of the sperm consists primarily of nuclear chromatin, so subtle changes in sperm head morphology may be related to abnormalities of DNA content. Measurement of a set of parameters regarding the sperm head (size and shape) and midpiece can align sperm into certain populations of sperm, such that the chromatin content in each sperm head can be determined to be normal or abnormal using automated sperm morphometric analysis (ASMA). Non-compensable defects (those that cannot be overcome by increasing the number of sperm in a breeding dose) are typically related to sperm with abnormal head morphology. Sperm head shape has been correlated with fertility and resistance to cryopreservation. Sperm nuclear DNA fragmentation is positively correlated with lower IVF fertilization rates, impaired implantation, increased risk of abortion, and increased risk of disease in offspring, including pediatric cancer. Fertile sperm have stable DNA which is able to decondense at the appropriate time during the fertilization process such that the oocyte has access to this DNA for combination with its own DNA complement.

Sperm DNA damage may occur on several different levels. Mitochondrial DNA damage can occur and be manifest as deletions, point mutations and polymorphism and is associated with decreased semen quality, asthenozoospermia and male infertility. Nuclear DNA damage may occur as a result of oxidative stress, sperm chromatin packaging and apoptosis. DNA damage may occur as a result of environmental factors, pollutants, infection, inflammation, or the presence of reactive oxygen species (ROS).

Sperm chromatin structure assay (SCSA)

SCSA is a method to assess the DNA content of the sperm head. This assay compares the amount of DNA present in each sperm and how much variation there is between individual sperm cells. Normal dogs have very little variation in the amount of DNA present in each sperm head, while dogs with abnormal sperm may have a wide variation in the DNA content of the sperm.

For SCSA, sperm are treated with an acidic solution (pH1.2) in order to denature their DNA in situ. Sperm with normal chromatin will not denature under these conditions while abnormal DNA will denature. The sperm are then stained with acridine orange (AO). This is a metachromatic DNA stain. Chromatin which has been denatured into single stranded DNA will fluoresce red, while DNA which does not denature (remains double stranded) will fluoresce green. Combining the use of SCSA and ASMA may assist in evaluating dogs with normal spermiograms and poor fertility.

DNA fragmentation

DNA fragmentation may also be evaluated using alkaline single-cell gel electrophoresis testing, TUNEL assay; Comet assay, in situ nick translation, and DNA breakage detection-fluorescent in situ hybridization assay (DBD-FISH). These assays use fluorescence microscopy staining techniques to identify chromatin packaging defects. At this time, few of these assays beyond SCSA are used in clinical practice as it remains to be determined what the clinical relevance of negative outcomes means to fertility. There also still remains significant variability in techniques between labs resulting in disparate results. Certainly in veterinary medicine, data regarding most of these tests in clinical practice is lacking, although they are slowly being introduced.

Fluorescence in situ hybridization (FISH)

This procedure allows for the analysis of chromosome numbers in individual sperm. Individuals with oligoasthenoteratozoospermia are at increased risk of having chromosomal abnormalities such as aneuploidy, double aneuploidy and diploidy. Sperm probes for specific chromosomes each fluoresce in specific color ranges are applied and in situ hybridization is performed. In humans, teratozoospermia in the form of macrocephalic, multi-tailed sperm have an increased incidence of aneuploidy. Other morphologic abnormalities may also be associated with specific chromosomal abnormalities and this area bears the need for further investigation.

In vitro fertilization (IVF)

This is the ultimate test of sperm function. In human medicine, the end point of IVF is what percent of metaphase-II oocytes are fertilized and develop to the 8 cell stage by day 3 post insemination.

Assessment for other cell types

Other cell types that may be present in semen include prostatic or urethral epithelial cells, immature germ cells, red blood cells, and inflammatory cells. Quantification of epithelial cells may be accomplished by determining number of the cells of interest (N) per 100 spermatozoa. Concentration of the cell of interest in millions/mL equals the number of the cells of interest times concentration of spermatozoa in that sample divided by 100. As many as 2000 white blood cells/mL may be present in the first and second fractions of the ejaculate in normal male dogs. Presence of inflammatory cells is not well correlated with lack of significant bacterial growth from semen samples in dogs; 44% of dogs with no inflammatory cells in their semen had significant aerobic bacterial growth from that semen in one study.

Measurement of components of seminal fluid

The components of seminal fluid reported as measured as a component of semen evaluation in dogs include glandular products, proteins, and electrolytes. Components may break down at variable rates after semen collection due to metabolism by spermatozoa and enzyme degradation, and may vary with interval from previous ejaculation, degree of sexual excitement, and health status of accessory sex glands.

Heparin-binding proteins have been identified in semen of several species including bulls and dogs. These proteins may be correlated with fertility, perhaps at the level of the acrosome. Identification of these proteins in canine seminal fluid has been published recently; work regarding correlation with fertility is ongoing.

Filters

Various filters have been described for use in evaluation and improvement of canine semen quality. Filters may be used to assess for normal function of spermatozoa by evaluating motility via determination of extent to which spermatozoa can penetrate the filter, or by binding abnormal spermatozoa. While percentage morphologically normal sperm may be much better in semen after filtration, total number of spermatozoa present in the filtrate may be much lower than that in the original sample. In one study evaluating semen from four dogs before and after passage through a glass-wool filter, average concentration of spermatozoa in the unfiltered samples as 445.7 million/mL and average concentration of spermatozoa in the filtered samples was 53.3 million/mL.

Centrifugation gradients

Centrifugation gradients are media layered in differing concentrations in a test tube over which a layer of semen is laid and the sample centrifuged. Abnormal spermatozoa and other cells are bound while normal spermatozoa move through and accumulate as a pellet in the bottom of the tube. Canine spermatozoa have been demonstrated to be not damaged by centrifugation. These techniques greatly improve the concentration of morphologically normal sperm cells in the centrifuged samples, with type of morphologic defect most improved variable by product. Digested and fresh blood also may be removed using some systems.

Chill or longevity test

When semen will be chilled and shipped for insemination, assessment of sperm longevity may be evaluated (following extension with semen extender), in order to determine the potential success with the use of this type of semen. Semen is collected and extended depending on the semen concentration, the type of extender being used and the type of insemination being performed. Generally, an ejaculate will be extended at a minimum ratio of 2-3:1. Semen should be chilled (slowly) to 4 - 5°C and held for a minimum of 48 hours at this temperature. A small sample of the semen is warmed to 37°C at 24 and 48 hours and total motility, progressive motility and velocity are determined. In some cases, motility may be so good at 48 hours to make evaluation at 72 and 96 hours (or longer) indicated.

Hormonal assays

Hormones that may be evaluated include testosterone, estrogen, prolactin, LH (luteinizing hormone), FSH (follicle stimulating hormone), and thyroid hormones. Dogs with testicular degeneration may have elevated estrogen concentrations and decreased testosterone concentrations. They may also have elevated FSH and LH concentrations. Prolactin concentrations may be increased or decreased depending on normal pituitary (brain) function and feedback from the testes. Thyroid hormones are commonly assessed when faced with infertility problems. There is little direct evidence to substantiate that hypothyroidism directly affects reproductive function. However, it is believed that through indirect mechanisms, chronic thyroid dysfunction may affect the brain's ability to either produce hormones or respond to hormones released by the testicles in feedback loops and may thereby result in reproductive dysfunction and infertility secondarily.

Karyotyping

Males that have decreased fertility without any obvious cause may have genetic defects resulting in testicular hypoplasia or degeneration. Although most of these individuals are sterile, in some cases, one or more litters may have been sired. Evaluation of the DNA of these individuals through karyotyping (chromosome analysis) may indicate a genetic reason for the infertility.

Testicular biopsy

Testicular biopsy can be performed on subfertile or infertile males to help differentiate the cause of the infertility. Biopsies are generally recommended when sperm production is low or steadily decreasing to help elucidate the cause of the problem and help determine if there is a treatment that will improve the dog's fertility. Biopsies are taken under general anesthesia through a small incision in the scrotum.

What does it all mean?

Values determined in semen analysis, no matter how sophisticated, are meaningless unless taken in context. Factors that must be considered include the signalment of the animal; inbred status of the animal; time since semen was last collected; and perhaps season of the year. With this background knowledge, conclusions may be drawn regarding the likelihood of a dog being able to impregnate females. Age was associated with fertility in humans; semen quality declined after 45 years of age. There certainly are reports of men siring children when much older than this, but they are the exception rather than the rule and their success may not have been predicted based on semen analysis alone. Two studies identified 6 years of age as the cut-off after which there was a decline in sperm output, percentage motile spermatozoa, and percentage of morphologically normal sperm in Dalmatians and Rottweilers. Breed also is a factor in canine semen analysis, since total number of spermatozoa in the ejaculate is based on testicular size. There are no breed-specific guidelines for total number of spermatozoa in the ejaculate in dogs. Inbred status of the dog may be responsible for variations in semen quality. In a study evaluating 14 outbred and four inbred male foxhounds bred to 544 outbred and 51 inbred bitches, sperm count, motility, conception rate, and number of pups born alive was decreased in males with inbreeding coefficients of 0.125–0.558.

The owner should be asked how long prior to the evaluation the dog was used for natural service or had semen collected by manual ejaculation. Ejaculation more frequently than every 48 h is associated with a decline in sperm output as epididymal reserves are depleted. However, more frequent semen collection also may be associated with increased normal motility and morphology. Another aspect of time is recognition that semen quality varies over time in dogs; in one study in which semen was collected from six young adult dogs monthly for 6 months, there was considerable monthly variation in total sperm number, motility and morphology. Finally, after prolonged sexual rest, there may be a concern that the ejaculate will contain a greater percentage of morphologically abnormal spermatozoa due to presence of aged spermatozoa from epididymal storage. This has not been rigorously evaluated in dogs and is not supported by research evaluating 6 weeks sexual rest. Season of the year has been documented as a possible factor in several studies. Work done in Japan and England demonstrated that semen quality was best in the spring, and morphologic abnormalities were highest in the summer. Region of the world has not been demonstrated to play a role with semen collection in a tropical country, similar to reports of semen quality from temperate regions.

Males should be evaluated for reproductive function prior to their first attempt at breeding, if it has been several months or years between breedings, or if fertility begins to decline or is questionable. In some cases, a routine reproductive examination will suffice, but in others, advanced diagnostics may be required.

Summary

It is clear that there is much more to be evaluated regarding male fertility than the basic semen evaluation. The concept that simply providing a specified number of normal appearing, motile sperm at the proper time in relation to breeding will result in acceptable pregnancy rates is clearly a misconception. There are many aspects of sperm function that may affect the functional competence of the sperm cell, beyond its basic size, shape and motility. When faced with a dog that has subfertility, the clinician must first rule out the most obvious causes for the problem and then move on to more advanced semen diagnostic testing to exhaust all possible diagnoses. In the process of making the diagnosis, the clinician may discover a method of treating or correcting for the problem. Unfortunately, in some cases, even with exhaustive testing, a diagnosis may remain elusive. Research is needed for all domestic animals including the canine.

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