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Canine semen collection and evaluation (Proceedings)
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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 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).
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.
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).
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
• 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 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.
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.
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 indic
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