Semen Collection and Evaluation

Joni L. Freshman, DVM, MS, DACVIM (Internal Medicine)

Conformation showing and a variety of dog sports are increasingly popular. Propagation of an excellent dog requires normal
fertility. Semen collection and evaluation are essential skills for
small animal practitioners who work with breeders and dog-sport
enthusiasts. Semen collection can be performed on most dogs in
the clinic setting with minimal standard equipment. Preparation
of the collection room and presence of a teaser bitch increase
the success of sample collection. Semen evaluation consists of
several components. First, gross characteristics and sample
volume are recorded. Percentage and quality of motility are
evaluated immediately. The concentration of spermatozoa in the
sample is determined and multiplied by the total sample volume
to determine the total number of sperm in the ejaculate. Morphology of ->100 spermatozoa is evaluated and recorded to
determine the percent normal spermatozoa Total normal sperm
per ejaculate is then calculated. Additional tests are often performed. These include cytology of the second and third fractions,
culture of the semen, and measurement of semen alkaline phosphatase. Knowledge of normal parameters for canine semen
enables the practitioner to evaluate the results of these tests with
confidence.
Copyright 2002, Elsevier Science (USA). All rights reserved.

xhibition of dogs in conformation and obedience competi-

E tion as well as a variety of dog sports (e.g., field, herding,

agility) is an increasingly popular activity in the United States.

The ability to perpetuate a dog's excellent qualities requires his
or her fertility. Veterinarians may be asked to perform prebreeding or prepurchase semen evaluations. In addition, the
use of chilled and frozen semen breeding is increasing. Collection and evaluation of canine semen is essential to these techniques. Finally, the American Kennel Club requires veterinary
certification of fertility for stud dogs younger than the age of 7
months or older than 12 years.
Semen collection and evaluation is readily learned and easily
done within the practice setting. Breeder and dog sport clients
appreciate the availability of these services.

Semen Collection
Precollection Procedures
A thorough history should be obtained before collection. 1-3
This should include breeding history, medical history, and any
medication or supplements administered over at least the previous 6 months. Environmental and genetic or familial infor-

From Canine Consultations, Colorado Springs, CO

Address reprint requests to J. L. Freshman, Canine Consultations,
3060 Woodvlew Court, Colorado Springs, CO 80918.
Copyright 2002, Elsevier Science (USA). All rights reserved.
1096-2867/02/1703-0002535.00/0
doi:l 0.1053/svms.2002.34326

104

mation is also helpful. A pedigree should be examined for

degree of inbreeding. Record the length of time since the last
breeding or collection. 4
Equipment
Essential equipment for canine semen collection and evaluanon is listed in Table 1.3 Many supplies will be available in any
clinical setting. Others may need to be ordered before beginning semen collections.
Concern has been expressed about the effect of latex artificial
vaginas (AV) on motility of canine spermatozoa. Studies that
found decreased motility on contact with latex (gloves or AV)
used a prolonged contact time that will not be an issue if appropriate collection techniques are used. 5-r Disposable AV are
available, but are not as easy to use because they will not adhere
to the engorged penis.
The first collection tube attached to the AV should have a
small hole near the top. This will prevent vacuum formation
that could adversely affect semen collection. 2
The top of the AV should be folded back to form a final length
that will allow the erect penis to be just above, but not contacting the attached collection tube. For toy dogs, the AV can be cut
down in size.
Preparation
The room where collection is to occur should be quiet and
isolated. Interruptions should be prevented. Use of a rubberbacked mat will provide good footing and olfactory cues to the
stud dog. Toy dogs may be more readily collected on a grooming table. Request that the stud owner bring any equipment or
accessories that the dog associates with breeding.
The collector should &vest themselves of any "doctor" paraphernalia, such as white coat or stethoscope. Semen collection
should be performed before physical examination, venipuncture, or any other stressful procedures. ~,7 The dog's comfort and
cooperation are essential for semen collection.
All equipment should be at hand and warmed to body temperature. 8 Attach the AV to the collection tube with the vent
hole. A teaser bitch should be used. Ideally she should be in
proestrus or estrus and near in size to that of the male dog. In
lieu of an estrus teaser, a calm bitch can be used. Estrus scent
can be provided in two ways. Swabs taken from vaginal secretions of estrus bitches can be frozen in plastic bags and thawed
for this purpose. 8 Some have had success using a pheromone
chemical, methyl p-hydroxybenzoate (Aldrich Chemical, Milwaukee, WI) swabbed on the vulvar area and tail head of an
anestrus teaser. 7
Although most dogs are not particular about the teaser bitch,
some are. With dogs that show no interest in the teaser, using a
bitch he knows and likes may be beneficial, even if she has been
ovariohysterectomized. An assistant should be present to restrain the bitch while staying out of the stud dog's way.

Clinical Techniques in Small Animal Practice, Vol 17, No 3 (August), 2002: pp 104-107

TABLE 1. Equipment Needed for Semen Collection

and Evaluation 7,8
Rubber backed mat
Latex artificial vaginas
Plastic graduated centrifuge tubes
Slide warmer
Pipettes
Microscope shdes
Cover slips
Binocular mmroscepe
Unopette system for white blood cell/platelet count
Neubauer hemacytometer
Cell counter
Stains (eosin-nigrosin, modified Giemsa)
Centrifuge

Dog Preparation
The length of sexual abstinence should be recorded before
collection. Ideally, schedule 4 to 5 days of sexual rest before
collection, r-9 Greater than 10 days of sexual rest may result in
increased morphologic abnormalines and decreased motility
due to spermatozoal aging and increased debris, r,9 Semen quality is best if collected no more frequently than every 2 to 5
days/
The dog should be walked before collection to allow opportunity to urinate. After urination, he should be trotted briefly to
help clear the urethra of residual urine.
Presence of the owner at collection depends on the dog. Most
dogs are more comfortable with the owner present, but some
can only be collected in the owner's absence. 2,9
An assistant should restrain the bitch at the end of the rubber-backed mat, with her tail toward the mat. If there is any
concern she may bite, a muzzle should be used. The dog should
be walked in behind the bitch. An inexperienced or nervous
dog can be allowed to sniff and play with the bitch before
collection is attempted.S Lubricating jelly is not recommended
or needed for collection, a,5
While the dog is sniffing the bKch's perineal area, the collector kneels or squats on the left side of the dog (reverse for
left-handed collector). The collector's right hand briskly massages the prepuce over the penis. The collector's left hand holds
the AV with attached collection tube at the tip of the prepuce.
As the dog's penis reaches 40% to 50% erection, the prepuce is
pushed behind the bulbus glandis with the AV. If the bulbus
glandis has enlarged so that the prepuce cannot be moved
behind it, take the dog away from the bitch and allow his
erection to subside before trying again. Complete erection and
ejaculation while the bulbus is within the prepuce can be painful and result in an incomplete collection/ After the AV is
positioned just proximal to the bulbus, circular, firm pressure
should be maintained with the left hand to simulate the copulatory lock or "tie. ''s,>r,s The right hand can continue to massage the prepuce over the proximal portion of the penis, as
needed. The right hand is also used to switch collecuon tubes
between fractions, if desired. For the second and third tubes,
simply fold or lay the tip of the AV within them to continue
collection. Some stud dogs prefer minimal masturbauon. The
technique should be adjusted for each dog's preference.
Dogs ejaculate in three fractions. The first fraction, also
called the presperm fraction, originates from the prostate gland.
Normally it is clear or slightly cloudy and .5 to 20 or more
milliliters. 1,2 The second fraction is the sperm-rich fraction,
normally opaque, milky-white in color and .5 to 2.0 mL. This
SEMEN COLLECTION AND EVALUATION

fraction comes from storage in the tail of the epididymis, as well

as from daily sperm output, u The first and second fractions are
often collected together and are ejaculated during and immediately after the dog's vigorous thrusting movements. 1 For chilled
or frozen semen collection and some diagnostics, these fractions should be separated. Contact of spermatozoa with first or
third fraction fluid may decrease motility after 2 hours. 12
Immediately after the vigorous thrusting, the dog often will
try to step over the collector's arm. The collector should assist
him in doing so, and allow him to reverse 180 degrees so that he
is in the normal position assumed for the tie. 1.r
The third, or prostanc, fraction, is normally clear and may
consist of many milliliters, depending on how long pressure is
maintained proximal to the bulbus glandis. ~1 The dog will often
have a few moments' cessation of ejaculation before releasing
the prostatic f r a c t i o n / C o l l e c t enough of this fraction for desired testing.
After collection, the bitch is removed from the room. Pressure is released from around the dog's penis and the AV may
either be left in place until detumescence or can be gently
peeled off. The dog may continue to ejaculate prostatic fluid for
some time. The dog should be monitored until the pems is back
within the prepuce. Some dogs prefer standing still, whereas
others benefit from slow walking. Sometimes a cool compress
or lubricant is required. With longhmred dogs be sure no hair is
caught inside the prepuce. Lubrication placed at the junction of
the prepuce and the penis minimizes entrapment of the tip of
the penis by the distal preputial skin rolling inward. The dog
should not be kenneled or placed with other dogs until his
penis is back within his prepuce.

Semen Evaluation
Gross Evaluation
The volume of semen collected should be measured and recorded. This measurement should consist of the first and second fractions if collected together, or the second fraction if
collected separately. The amount of prostatic fluid collected is
variable and not used m this calculanon.
The color of the different fractions is also recorded. Yellow
can indicate urine contamination or pus. Green is consistent
with pus. Red or brown indicates fresh or hemolyzed b l o o d /
The most common causes for blood in the semen include prostatic disease or damage to blood vessels on the penis. Blood, in
the amount seen with chnical cases, has no effect on canine
spermatozoal motility until 6 hours of contact/2
pH
pH is measured using pH test-tape with a range of 5.5 to 8 that
measures in units of 0.5, is A drop of the second and the third
fraction is placed on pH tape. The tape should not be dipped
into the collection tube. Seminal plasma has a normal pH range
of 6.3 to 7.0 or 6.3 to 6.7. T M Prostatic fluid has a normal range
of 6.0 to 7.4, with an average of 6.5 or 6.8.1,7,11 The pH is be
helpful for antibiotic selecnon in case of infection.
Motility
Spermatozoal motility should be evaluated immediately after
collection. A drop of semen is placed on a warmed microscope
slide and covered with a warm cover slip. Microscopic evalua-

105

tion of motility should be performed at 20 and 40 with the

condenser lowered. ~.r If the sample is too concentrated to evaluate motility, a drop of semen can be diluted with a drop of
buffered saline solution at the appropriate pH. n
Recent information indicates a warmed slide may not be
required because canine semen is resistant to cold shock, at
least above 70 F.7,13 Other information indicates cold shock
can be a concern in decreasing motility. 9 Subjectively, semen
motility is improved when evaluated on a slide warmer set at
body temperature.
Normal motility is described as rapid, progressive, forward
motion. ~ This is subjectively recorded in percentage of motile
sperm. At least 70% of sperm should show this motility. Spermatozoa that roll or circle are not exhibiting normal motility.
The assessment of slow, medium, and fast speed can also be
made. The percentage of progressive motility increases after 5
days of sexual rest compared to 1 day. r
During the motility evaluation, also evaluate for sperm agglutination. Agglutination is evidenced by sperm sticking to
each other.* Sticking to yolk material in extender, debris or, air
bubbles is not of concern.
Sperm Count
The first step in determining sperm count is to determine concentration of the sample. This procedure is performed using a
Unopette T M (Becton-Dickinson; Rutherford, NJ) system for
white blood cell/platelet count and a Neubauer hemacytometer. 1,2,r,8,1~,~* The sample of first and second fractions, or second-only fraction is gently mixed, then drawn up into the .02
mL Unopette capillary tube. This aliquot is then dispensed into
the 1.98 mL Unopette chamber and mixed. The diluted sample
is dispensed into both sides of the Neubauer chamber and
allowed to settle, which facilitates focusing.
The central square (of nine squares) on the Neubauer chamber is counted on each side and averaged. 2,<r,9 13,s4 The numbers should be within 10% of each other. This number represents the number of spermatozoa (in millions) per milliliter of
semen.
The number of spermatozoa per milliliter is multiplied by the
sample volume to determine the total spermatozoa per ejaculate. This number is dependent on the size of dog and resulting
size of testes. Larger dogs produce larger numbers of spermatozoa. The average size dog should produce at least 250 to 300
million sperm per ejaculate and may produce much more. 3.7,~s~
Toy dogs may produce less and giant breed dogs should produce much more.
The total sperm per ejaculate is dependent on a variety of
factors. Very young and very old dogs produce less sperm. 7 In
Dalmatians, sperm count has been documented to decrease
after 6 years of age. ~ In addition, total sperm number is decreased with inbred dogs.r In normal dogs, total sperm number
decreased in late summer and fall as compared to spring and
early summer, but did not fall below normal values. ~
Frequency of ejaculation also affects the total sperm per
ejaculate. Daily collection for 7 to 10 days exhausts the epididymal storage and results in collection of only the daily sperm
output (DSO). ~5a6 Evaluation of sperm count should never be
based on a single sample. 2.r A poor sample may not be representative for the dog if he is nervous or stressed. An excellent
sample from a dog rarely collected may look quite different by
the third sample within a 7-day period. The time from differen-

106

Table 2. Spermatozoal Abnormalities1,2,8,9

Primary abnormalities
Head abnormalities
Pyriform head
Tapered head
Narrow head
Small head
Giant head
Round head
Deformed head
Double head
Midpiece abnormalities
Double midpiece
Swollen midpiece
Proximal droplet
Tail abnormalities
Tightly coiled tatl
Double tail

Secondary abnormalities
Head abnormahties
Detached head
Midpiece abnormalities
Distal droplet
Tall abnormalities
Bent tail
Reverse tail
Distal coiled tail
Other
Released acrosome

tiation of spermatogonia to release of the resulting sperm is 62

days, so abnormalities in sperm number may be seen much later
than the insult that caused them. 15A normal semen evaluation
does not equate to fertility because sperm function and breeding ability are not evaluated.
Spermatozoal Morphology
Morphology is evaluated by examining a stained semen slide
under 100 (oil immersion) and counting 100 to 200 spermatozoa. Two different techniques may be used to produce a slide
for morphologic examination. In the first, a drop of eosinnigrosin stain (Hancock's stain; Lane Manufacturing, Denver,
CO) is placed on the end of a microscope slide. 2-7,8,1s A drop of
gently mixed semen sample (sperm-rich fraction) is placed next
to it. A second slide is used to gently mix the two drops, and
then draw the mixture along the slide as for a blood smear. The
slide is air-dried.
In the second technique, a drop of semen is smeared as for a
blood smear, then stained with a modified Wright's-Giemsa
stain (Diff-Quick; Baxter Healthcare, Miami, FL). With this
technique, the slide is left in each of the three solutions for 5
minutes before rinsing and air drying, ral
The numbers and types of abnormal morphology are recorded and percentage of normal determined. Count only free
heads, not free tails, s When a spermatozoa has more than one
abnormality, record the most serious one. 8
Abnormalities are considered as primary or secondary (Table
2). Primary abnormalities occur during spermatogenesis and
are therefore more serious. Secondary abnormalities may occur
during passage through the epididymis or during collection and
preparation of the slide, s
Normal semen samples should have <10% primary abnormalities and <20% secondary abnormalities.S, 2,r,9. Total abnormalities should be <20-30%. ~,2,r,9 The total number of normal
sperm in the ejaculate is determined by multiplying the percent
of normal sperm by the total sperm number as calculated
above. 9
Cytology
Cytology of the sperm-rich and prostatic fractions should be
evaluated separately. There are several ways to prepare the
cytology slide. One way is to centrifuge 0.3 to 0.5 mL of sample
at 120g for 7 minutes, r The slide is made from the resulting
pellet and stained with Diffquick as for any cytologic preparaJONI L. FRESHMAN

tion.r Alternatively, a slide can be made from the whole sample;

however, fewer cells will be seen. 9
Normal cytology of the sperm-rich fraction contains spermatozoa, white blood ceils (WBC) (2-4/hpf), epithelial cells, bacteria, and red blood cells, r Increased or degenerate WBC or
intracellular bacteria indicate infection. Prostatic fluid also contains epithelial ceils, bacteria, and WBC (2-4/hpf). r Increased
red blood cells may indicate prostatic disease or bleeding from
penis or prepuce. 8

Semen Culture
Semen is not sterile. A wide variety of normal flora is present, s r
When culture is done, effort should be taken to decrease contamination. Collect part of the sample into a sterile vial. Before
collection, clean the prepuce and tip of penis with sterile saline
moistened gauze.1 Culture of the distal urethra to compare flora
may also be useful. Greater than 10,000 cfu of aerobic bacteria
per mL of semen indicates infection. ~r Anaerobes are not comm o n in canine semen. If they are suspected, an aliquot of semen
should be immediately inoculated into Anatrans media and
taken to the lab. Mycoplasma is a normal finding in the distal
canine reproductive tract. However, it can cause problems if
present in excess amounts. Mycoplasma and ureaplasma can be
cultured by inoculating semen into Amies Transport Media
(Difco Laboratories, Detroit, MI) and shipping it to the laboratory overnight, s No more than 24 hours should elapse before
culture.
Alkaline P h o s p h a t a s e
Alkaline phosphatase (ALP) is produced in the epididymis. 15
This makes it an excellent marker for patency of the ductal
system. In an azoospermic semen sample, measurement of ALP
is essential in determining if the azoospermia is due to problems with libido, testicular failure, or ductal blockage. H,15 In
normal semen samples, the ALP has a range of 5,000 to 40,000
U/L. M With a complete collection, a low ALP indicates ductal
blockage, whereas a normal ALP indicates testicular failure.
An incomplete collection can also result in low ALP.
Serum ALP assays can be used to measure semen ALP. Dilution will be necessary for normal samples. 1~
SllllnIlary
Semen collection and evaluation is a valuable service that can
readily be performed in private practice. Attention to the dog's
comfort and the presence of a teaser bitch maximize collection
quality. Multiple collections may be needed to truly evaluate
the dog. Detailed evaluation of semen, as discussed here, will
enable the veterinarian to provide prebreeding and prepurchase

SEMEN COLLECTION AND EVALUATION

assessment, decide viability for fresh, chilled and frozen artificial insemination, assess infertility cases, and monitor response
to treatment.

References
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Chn North Am (Sm Anim Pract) 31:259-269, 2001
3. Seager SJW. Semen collection and evaluation in the dog. In Morrow
DA (ed): Current Therapy in Theriogenoiogy, ed 2. Philadelphia, W.B.
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4. Olson ON. Collection and evaluation of canine semen. In Kirk RW,
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17. Root Kustritz MV, Johnston SD, Olson PN. Correlation between
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(1987-2000). JAVMA, in press.

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