Artificial Insemination With Fresh Semen...
Ideally, several days of sexual rest should be allowed prior to
semen collection and evaluation.
Canine semen is most easily collected using masturbation with a
gloved or bare hand, although some operators find the process
distasteful and prefer to use an artificial vagina (AV). However,
most artificial vaginas are made of latex and most latex is
spermicidal. In addition, these artificial vaginas cannot be gas
sterilized because the residues of gas sterilization are also toxic
to spermatozoa. If an AV is used, it should be cleaned by washing
and chemical disinfection then rinsed several times with distilled
water and air dried.
The artificial vagina and attached plastic tube are warmed to body
temperature and lubricated with a small amount of sterile aqueous
It is much easier to obtain an ejaculate when an estrual bitch is
present; therefore owners should be asked to bring in teasers when
appointment are made. Alternatively, a non estrous bitch of the same
breed or size may be used. A commercially available pheromone
(methyl paraben "Eau d'estrus, Synbiotics corporation phone
18584513771, fax at 18584515719 ) may be used to stimulate the male
but we have no experience in its use. It is sometimes very difficult
to collect semen if no bitch is available at all can it can be done.
Optimally, the male and female are brought together on leashes in a
quiet room with nonslip flooring. As the dog sniffs at the bitch's
vulva or mounts her, the collector quickly moves the prepuce back,
behind the bulbus glandis and directs the tip of the penis into the
AV, held in the left hand. Once the artificial vagina is slipped
onto the penis, the right hand is used to hold the artificial vagina
onto the penis while exerting firm pressure around the back of the
bulbus glandis. Once this occurs, the dog will usually show pelvic
thrusting and normal ejaculation. An AV is certainly not essential
to collect semen from dogs. Excellent ejaculates can be obtained by
hand collection alone.
If a gloved or bare hand is used instead of an AV, the dog is
masturbated rapidly for a few seconds until he gains a full
erection. In the process, the prepuce is slipped behind the bulbus
glandis. Masturbation ceases and the hand held behind the bulbus
glandis using very firm pressure, until ejaculation is complete. The
other hand is used to hold a plastic bag over the end of the penis.
Almost any warm receptacle can be used to collect the semen but most
commonly sterile "Whirlpak" bags are used. Syringe casings and other
hard objects should be avoided as the penis is very easily
traumatised during collection and substantial bleeding may occur
into the ejaculate. This does not seem to decrease fertility in dogs
(Cf horses) but it interferes with semen evaluation and of course,
Semen can be collected when the bulbus glandis expands within the
prepuce but some dogs object to this. Therefore, it is usually best
to be sure that the bulbus glandis is out of the prepuce before it
The reader can see therefore than the term "masturbation" is
somewhat misleading. Most of the contact time consists of pressure
exertion behind the bulbus glandis; a process identical to that used
with an artificial vagina!
Ejaculation occurs intermittently over a variable period, perhaps
five to 15 minutes, usually just long enough to deprive the
collector (squatting on the floor) of all blood flow and feeling to
If pressure is maintained firmly around the bulbus glandis,
pulsations can be palpated in the urethra. The anus will also be
observed to contract in a rhythmic fashion. The dog may stop
ejaculating for several minutes then pulsations will resume.
Initially, a few drops (one to 3 ml) of clear to slightly cloudy pre
sperm fraction are ejaculated, followed by a whitish spermrich
fraction (0.1 to 6.0 ml) but most often these fractions are mixed
and only a homogeneous light greyopalescent ejaculate is obtained.
The collector should try keep one hand around the collection vessel
to keep it near body temperature. This is easiest when a plastic bag
is used as a collection vessel.
Soon after the dog begins to ejaculate, he will often lift his hind
limb as though attempting to step into the rumptorump position that
occurs during natural breeding. If this is observed, the collector
should allow the dog to step over his/her arm so that the penis then
extends out caudally from the dog. Soon the clear, third fraction of
the ejaculate (mostly prostatic fluid) is ejaculated increasing the
volume to as much as 60 ml. If the semen is being collected for
artificial insemination as well as evaluation, enough prostatic
fraction is collected to bring the total volume to three to 10 ml so
large numbers of sperm are not lost in the insemination process and
the insemination volume is comfortable to work with. Frequently,
only a few ml of semen are collected but total sperm numbers, not
semen volume, is what is important in A.I.
After collection is complete, the male is observed until his
erection subsides. Paraphimosis may occur following collection, so
the dog must never be kennelled or sent home until the penis is
completely inside the prepuce. To prevent paraphimosis, one should
lubricate the preputial opening liberally after semen collection.
Semen should be kept at a 35 to 370C until progressive motility has
been determined, after which it can be allowed to cool to room
temperature. Semen volume is not important, but it is necessary to
record the volume of the spermcontaining portion so that the total
number of sperm per ejaculate can be calculated. Sperm cell
motility, morphology, and concentration are determined in the
Normal prostatic fluid makes a good diluent if one is required for
motility estimates. It can also be pooled and frozen for future use
as a diluent for per vagina insemination of frozenthawed semen (a
relatively new approach to the use of frozen semen).
A normal canine ejaculate:
Colour: Opalescent to milky white with a clear prostatic
supernatant or homogeneous greyish white.
Volume: Pre sperm fraction: 0.1 to 3 ml, Spermrich fraction: 0.1 to
6 ml Prostatic fraction: one to 50 ml Total volume: one to 60
Progressively Motile Sperm: 60 to 90%
Number of Sperm per Ejaculate: 200 to 3000 X 106 (the population of
the United States)
Morphologically Normal Sperm: 70 to 90%
Bacteria: Many; usually more than10,000/ ml. However, only
the presence of many white blood cells is an indication for
bacterial culture of the semen.
The presence of epithelial cells, red blood cells, inflammatory
cells, and germinal epithelial cells are noted under low
magnification. All cells other than sperm (COTS) are easy to see if
a smear is stained with WrightsGiemsa or DiffQuick but difficult to
differentiate using common sperm morphology stains.
Semen from males that have not ejaculated recently may contain more
epithelial cells and debris than semen from a male that is used
frequently but if large amounts of debris or dead sperm are present,
a second sample should be collected 24 hours later.
Artificial insemination is performed when behavioural problems
prevent mating (especially female dominance) when mounting is
difficult or unlikely due to orthopedic problems or inexperience, or
when transported or frozen semen is used.
Semen is collected from the dog using masturbation or an artificial
vagina and only the spermrich fraction and a small amount of
prostatic fluid is used (total volume three to 10 ml).
Semen collection should not be more frequent than once every two
days. Daily ejaculation results in very low concentrations of
ejaculated sperm after five to seven days.
Artificial insemination of the bitch is most easily performed by
depositing the semen in the cranial vagina with a Cassou sheath
shortened to about 25 cm. These sheaths are normally used to cover
the rigid A.I. Cassou rods used for inseminating cattle. They are
available from any veterinary supplier or A.I. cooperative. They are
soft and flexible and therefore, far superior to the rigid plastic
cattle inseminating pipettes sometimes used. In addition, they fit
directly on a syringe and do not require adapters like the rigid
pipettes. The Osiris apparatus from France is recommended by some
operators but its superiority to other methods has not been
In all cases, the vulva is washed, rinsed and dried. The pipette is
then inserted into the vagina, first dorsally for several
centimetres, then cranially until the cervix is reached. If
resistance is felt, the pipette is withdrawn one or two centimetres,
then reinserted at a slightly different angle. The vagina of the
bitch is long and an insemination pipette may have to be inserted to
a depth of more than 20 cm in large bitches. The hindquarters of the
bitch are then elevated so that the spinal column is at an angle of
45 60 degrees and held there for as long as possible, up to 10
One excellent study demonstrated that this was advantageous for
sperm transport. Some inseminators also stroke the dorsal wall of
the vagina with a gloved finger (æfeathering') or massage the
clitoris for about one minute when the hind quarters are elevated.
This may promote semen transport within the uterus but it has not
been objectively studied. Along the same line of thought, after
insemination, the bitch should not be allowed to squat or jump for
another 10 to 15 minutes.
It is usual to inseminate the total volume of undiluted ejaculate
and prudent to use the ejaculate as soon as possible after
collection. Insemination doses should contain at least 200 million
motile sperm because fertility decreases with ejaculates containing
less than 50 million live cells. Single estrus pregnancy rates up to
90% can be achieved with A.I. using fresh semen in fertile dogs.
Optimal extension rates for dogs have not been studied but
experience with horses suggests that it should be extended at a rate
of between 1:1 to 1: 6 (semen: extender) for transport or if it is
to be used more than an hour or two after collection. In most cases,
a dilution rate of one part semen to two parts extender works well.
It is also packed and transported in cooling containers like those
used for equine semen transport. The "Equitainer" (Hamilton Thorne;
firstname.lastname@example.org) cools the semen at 0.3oC/minute
maintaining better motility than other cooling rates. Perhaps more
important, this container has the best insulation on the market, a
consideration when semen is to be transported through various
Various semen extenders can be used for canine A.I. with
satisfactory results. However a simple and effective extender can be
made for shippedcooled semen by heating skim milk to about 95 o C
for ten minutes in a double boiler. It is then cooled to 37 o C for
use. The heating step denatures a spermicidal albumin component in
milk. It can be frozen in 50 ml aliquots for several weeks but its
exact shelflife is unknown. A specific canine extender called "Fresh
express" is available from the Synbiotics corporation (phone
18584513771 or fax at 18584515719). Commercial extenders used for
equine semen transport can also be used; for example Kenney's or "EZ
mixin" extenders (email@example.com or firstname.lastname@example.org)
but no data exist to show that any of these extenders are superior
to the others.
The extender is warmed to exactly the same temperature as the semen
and slowly added to the semen until the final dilution rate is
obtained. The semen is then packaged according to the instructions
that come with the shipping system. Often this involves just placing
syringes loaded with semen into a styrofoam shipper. The method used
for the Equitainer is more involved.
Some operators recommend warming the chilled semen to room
temperature just before insemination but this recommendation is not
universal. It is a good idea to evaluate the motility of the semen
when it is inseminated just so its status at the time of receipt is
known. Occasionally all the sperm are dead! Motility should be
evaluated in a drop of semen warmed to 370C.
It is recommended that a longevity trial be conducted with the semen
of any male animal (any species) before it is shipped over long
distances in the chilled state.
Pregnancy rates of 50 to 60% higher have been reported with the use
of chilled semen