"IT'S ALL IN THE TIMING" - FRESH-CHILLED VERSUS FROZEN
ARTIFICIAL INSEMINATION IN THE BITCH

Jane Barber DVM, MS, DACT

Introduction
Great advances in canine assisted reproduction techniques have been achieved during the
past 20 years. In additional to natural breeding, artificial inseminations with fresh "neat"
(unextended) semen, fresh-chilled extended semen and frozen semen are now viable
breeding options. With each technological advance, so too come more manipulations by
human hands and an increased risk for human error in semen processing and
insemination.
When good quality, fresh semen is used for breeding with either natural mating or "side
by side" artificial inseminations, there is much forgiveness with respect to breeding
timing. This is because fresh semen remains highly viable and "fertilizable" for 5 to 6
days once deposited in utero. Live spermatozoa have been recovered from the uterus up
to 11 days post-breeding. In contrast, survival of fresh, "neat" semen in extrauterine
environments is poor.

Advances in preserving extrauterine longevity of spermatozoa include both reducing
energy consumption and increasing energy sources by and for the sperm cells. Lowering
the temperature of sperm cells slows metabolism and reduces their energy consumption.
Replacing seminal plasma with extender bathes sperm cells in an energy-rich
environment. However, even optimally extended and cooled spermatozoa fail to survive
nearly as long as their fresh, intrauterine counterparts.
Fresh-chilled sperm cells have a relatively short life span. Fresh-chilled semen, once
warmed to body temperature and in utero, lives about 24 to 72 hours. Properly frozen
sperm cells can be stored indefinitely in liquid nitrogen at -322°F (-180°C). Once
thawed, however, sperm cells have an ultra-short life span. Frozen-thawed spermatozoa
live an average of 12 hours, 24 hours maximum! Curtailed sperm cell longevity
necessitates precise ovulation timing and breeding.

Ovulation Timing
Ovulation timing (OVT) is an art as well as a science. Unlike other species in which
ovulation occurs in an estrogen environment, ovulation in the canine occurs only after
progesterone levels have risen to >4ng/ml. Another unique feature is that the bitch
ovulates ova in the primary oocyte stage. These primary oocytes must undergo another
meiotic division and further maturation before they can be fertilized. Unlike most species
in which breeding/insemination is timed to coincide with ovulation, insemination in the
bitch is performed 2 to 4 days after ovulation. It is important to remember that, in the
bitch, the critical timing events occur 4 to 6 days before the optimal breeding dates.

A number of tools are available for ovulation timing. These include LH (luteinizing
hormone) assays, qualitative and quantitative progesterone assays, vaginal epithelial
cytology, vaginoscopy, examination of external genetalia and behavioral assessment.
Success in breeding is optimized when as many tools as possible are utilized to time
ovulation. While ovulation timing constitutes an entire discussion of its own, a brief
description of each tool follows.
Estrogen
Estrogen levels rise, peak and begin to decline during proestrus. Under the influence of
estrogen, the vulva and vagina become edematous, a serosanguinous vulvar discharge (of
uterine origin) is present, the vaginal epithelium becomes thickened and cornified and the
bitch exhibits behavioral signs of estrus. However, blood estrogen levels are highly
variable, do not accurately predict ovulation and are not useful as a tool for timing
ovulvation.
LH (Luteinizing Hormone)
Hypophyseal LH is the biological trigger for the cascade of events that culminate in
ovulation. Following a variable period (1 to 21 days) of estrogen level elevation, the LH
surge signals the transition from proestrus to estrus. LH stimulates follicular granulosa
cells to secrete progesterone. The LH surge is the key timing event dictating days of
peak fertility. Ovulation begins 2 days post-LH surge and continues for another day or
so. Ova mature and are capable of fertilization 2 days later. Mature ova live another 1-3
days. Optimal breeding times are day 4, 5, and 6 post-LH surge.
Pinpointing the day of the LH surge is cumbersome. The LH surge is short-lived,
typically lasting only 12 to 24 hours. Therefore, daily blood testing is required. LH is a
species specific hormone and assays are not currently available from commercial
laboratories in the US. A qualitative (test kit) assay is available for in-hospital use, but it
has a short shelf life (3 months maximum). LH assays are most often used in the timing
for breedings with frozen semen.
Progesterone
Progesterone's initial rise occurs concomitantly with the LH surge. At that time, baseline
progesterone levels (<1.5ng/ml) rise to 1.5-2.0 ng/ml. After the initial rise, progesterone
continues to rise and may reach levels of 10-15 ng/ml by the end of the fertile period.
Ovulation occurs when progesterone levels are between 4 and 10 ng/ml. Plan breedings
4 to 6 days after the initial rise,
and 2 to 4 days after the onset of ovulation. Since
baseline and initial rise levels can vary from individual to individual, it is important to
start testing early enough to define the baseline progesterone level.
The gold standard for determining progesterone levels is quantitative measurement by
radioimmunoassay. Results are reported in ng/ml. Progesterone, like all steroid
hormones, is not species-specific and can be measured commercially by human and
veterinary laboratories alike.
Several semiquantitative ELISA progesterone kits are available, Status-Pro [Synbiotics],
Target [Biometallics], and PreMate [Camelot Farms], for example. Results are
intrepreted from a color change in the test well or membrane. Hemolyzed blood samples
will falsely lower the result. If test kit components are not warmed to room temperature,
then a falsely high result will be interpreted. The general consensus is that test kits are
usually adequate for most natural breedings, but are not accurate enough when planning a
fresh chilled or frozen breeding.
Recommendations for progesterone testing include starting to test around day 5 or 6 from
onset of sign of proestrus, followed by testing every other day until ovulation has been
confirmed. However, some females have very short seasons and require testing at the
first sign of proestrus. Others may not ovulate until after 21 or more days. When in
doubt, start testing early.
Evaluation of External Genetalia
During proestrus, the vulva is swollen and a sersanguinous vulvar diacharge (of uterine
origin) is present. As proestrus transitions to estrus, vulvar edema diminishes and the
vulvar discharge becomes straw colored. Note however, that some normal bitches may
have a hemorrhagic discharge that persists throughout estrus. With the onset of diestrus,
vulvar edema subsides completely.
Behavior
The proestrous bitch attracts males but will not stand to be mounted. During estrus, the
bitch will stand, "flag" and allow the dog the mount and intromit. With the onset of
diestrus, the bitch will refuse to stand and be mounted.
Exfoliative Vaginal Cytology
During early proestrus, estrogen concentrations are low, and cytology specimens show
parabasal and intermediate cells, red blood cells, neutrophils and bacteria. As proestrus
progresses, estrogen levels rises and the vaginal epithelium thickens. The number of
superficial cells increases and the number of red blood cells gradually decreases. By the
end of proestrus, more than 80% of the epithelial cells are cornified red blood cells.
During estrus, vaginal smears contain >90% cornified superficial epithelial cells, red
blood cells are few in number and neutrophils are absent. With the onset of diestrus,
vaginal cytology abruptly changes from that seen during estrus to one showing few
superficial cells, a predominance of parabasal and intermediate cells and many
neutrophils.
Practically, vaginal cytology samples are useful for identifying inflammation in the
reproductive tract. If a large number of neutrophils are seen on an estrus smear, then a
vaginal culture and sensitivity may be indicated. Cytology is also useful for
differentiating proestrus, estrus and diestrus. It is not useful for determining ovulation
date within the estrous period.
Vaginoscopy
Vaginoscopy is a useful tool for estimating the LH surge and optimally fertile period.
During proestrus, the vaginal mucosa becomes edematous and has a billowing pillow
appearance on vaginoscopy. Around the time of the LH surge, vaginal edema decreases
and the vaginal folds become wrinkled or crenulated. Maximal crenulation occurs during
the optimal fertile period several days later. As diestrus approaches, the vaginal mucosa
takes on a blotchy white and pink appearance.
Tips for Success With Fresh Chilled Semen
Planning the Breeding
1. Don't commit to organizing and performing a breeding if you cannot be available
on weekends and holidays.
2. Plan the breeding with young, healthy fertile individuals. Since this is almost
always out of the clinician's control, special measures may need to be taken if
either the bitch or dog is aged or known to be subfertile. For example, fresh
chilled semen of marginal quality may necessitate surgical rather than vaginal
insemination to achieve pregnancy.

3. Recommend that the bitch owner always have a Plan B ready to be implemented
in the event that the scheduled breeding encounters a "hitch".

4. Recommend that the dog have had a negative test result for Brucella canis within
30 days or since his last natural breeding, whichever is the most recent. Be aware
that Brucellosis, often thought to be only a sexually transmitted disease, can also
be transmitted by casual contact.
5. Start ovulation timing early, particularly in females who have not had their cycles
"tracked" before.
6. If the dog has never been collected and shipped before, a trial collection and "chill
check" is advisable at least a week before to the anticipated shipping date. This
ensures that he can be collected and that his semen quality is maintained with
addition of buffer and chilling.
Semen Collection, Processing and Packaging
1. Schedule collections to minimize the collection to insemination time interval.
Shipment usually involves an overnight FedEx service.
2. Use an estrous teaser bitch whenever possible. Spermatozoa number and quality
are maximized when a teaser bitch is available. As much as a four-fold increase
in spermatozoa number can be realized when using a teaser bitch. General
guidelines for adequate semen quality include >70% motility, >70%
morphologically normal spermatozoa, few inflammatory cells, and a normal
sperm count. Sperm count should approximate 10 million spermatozoa per pound
of body weight.
7. If collected sperm numbers are marginal (<250 million sperm), wait 45 minutes
and collect the dog a second time. If the total number of sperm cells remains low,
recommend that the dog owner allow you to call the bitch owner to confirm that
he or she still wants to have the suboptimal collection shipped.
8. When collecting the dog, collect only the sperm-rich second fraction of the
ejaculate. Excess prostatic fluid is deleterious to semen quality. If excess
prostatic fluid is present, centrifuge the ejaculate, decant off the supernatant and
resuspend the sperm pellet in an appropriate extender. Follow semen processing
directions carefully. Many extenders are available, each with its own handling
instructions.
9. When extending semen, it is important to know how the insemination will be
performed. Surgical insemination requires much less total volume than does
vaginal insemination. For routine vaginal insemination, the total volume should
be appropriate for the size of the bitch. Generally, 2 to 3 mls is adequate for small
breeds, 4 mls for medium breeds, 6 mls for large breeds and up to 10 mls for giant
breeds.
10. Send the extended semen sample in a conical shaped centrifuge tube with a screw
cap. Make sure the tube does not leak. It is also a good idea to wrap the cap with
a layer of parafilm. Placing the tube inside a whirl pack or sealable plastic bag
allows for recovery of semen should a leak occur.
11. A number is shipping containers are available commercially, including
Equitainers and Camelot Farms shippers. Styrofoam boxes also work adequately
in most instances. In Europe, insulated Thermos-type containers are popular.
The bagged tube is placed in the box along with 1 or 2 frozen gel pack or Kool-It
brick. The tube should not be placed in direct contact with the frozen coolant.
Make sure that the tube is separated from the brick by at least 4 to 6 layers of
paper towel or newspaper. The tube can be wrapped with an insulating material
or the tube can be placed in a smaller Styrofoam box. More newspaper (sufficient
to fill the dead space) is packed between the brick and the tube. Alternatively,
one refrigerated brick can be placed between the frozen brick and the tube, still
using newspaper to fill dead space.
12. Most fresh chilled semen is shipped via FedEx. UPS can also be used. It is
important to make sure that the package can be shipped overnight with an AM
delivery the next day. When dealing with weekend inseminations, make sure that
package can be delivered on Saturday mornings. There are often special boxes on
the shipping form that need to be checked for AM and Saturday delivery.
13. Prior to the 9/11 terrorist attack, counter to counter shipment of semen offered a
convenient option when weekend inseminations were placed. With a counter to
counter shipment, the dog owner typically picked up the package from the
collecting veterinarian's office and took it to the nearest airport where it was put
on a flight to the airport designated by the bitch owner. Delta Dash and US Air
both offer reliable services. The bitch owner would pick up the package from the
airport and take it to the inseminating veterinarian's hospital. Since 9/11, counter
to counter shipments are problematic because the sender must be a "known
shipper" to be granted shipping privileges by airlines.
14. The collecting veterinarian should call and give the inseminating veterinarian the
FedEx or UPS tracking number of the shipment.
15. The cost of semen collection and shipping is typically the bitch owner's
responsibility. It is helpful to get the bitch owner's credit card information so that
services and FedEx fees can be billed directly to them.
Semen Delivery, Handling and Insemination
1. On the day of scheduled semen collection, call the collecting veterinarian's office
and obtain the tracking number for the shipment.
2. When fresh chilled semen package arrives, it should be opened immediately.
Attention should be paid to the "impression of coldness." The ice packs should be
at least cold, if not still frozen.
3. The tube containing the semen should be removed from the packaging material
(usually newsprint). The tube should contain the extended semen in a liquid state.
Unfortunately, occasional mishandling by the shipping company or by the shipper
placing the semen package in a non-pressurized compartment of the airplane will
cause the sample to arrive frozen. The freezing kills the sperm cells and renders
the sample useless.
4. One drop of the sample should be placed on a warmed microscope slide. The rest
of the sample should be refrigerated. Allowing the chilled sample to warm to
room temperature only allows the sperm cells to speed up, using precious energy
and shortening their life span.
5. Semen arriving looking "DOA" may still be viable. Some extenders render the
sample immotile and additional of an activator buffer is required to restore sperm
cell motility. Warming or transferring apparently lifeless semen into fresh
extender may bring it back to life. Perform test manipulations on only a drop of
semen. As the semen drop warms, side to side motility becomes noted. The
continued warming eventually shows the cells to have achieved a normal forward
progression. As the drop warms on the slide, the accompanying paperwork with
the semen collector's evaluation of the semen quality. Time of collection and postcollection
motility should be studied. The semen drop is then analyzed and
compared to the collector's evaluation.
6. If no motility is noted after 15 minutes, the sample is most likely non-viable. If
this occurs, the collector of the semen should be contacted to determine, if
possible, the cause of the semen's demise. In other cases where only partial semen
recovery is noted, the inseminator must use judgment based on the concentration
of the semen, estimated total spermatozoa numbers and the percent recovered. It
may also be necessary to alter the insemination method to that of an intra-uterine
deposition of the fresh chilled sperm to further reduce sperm cell stress and to aid
its arrival at the fallopian tubes.
7. The refrigerated fresh chilled sample need not be warmed to room temperature or
body temperature before insemination. Having the sample in the uterus as it
warms makes maximum use of the conserved energy. All fresh chilled semen
samples are handled in a similar manner, however, many different commercial
companies sell packaging kits and extenders. One should always read their
instructions for any specific handling recommendations before using the semen.
8. Most often, fresh chilled semen is inseminated vaginally. The pregnancy rate for
vaginal inseminations using good quality semen should be at least 80%. Surgical
insemination or TCI (transcervical insemination) can be performed to increase
pregnancy rate. Deposition of the semen directly into the uterus improves
pregnancy rate when chilled semen quality is marginal. Uterine deposition of
semen also improves pregnancy rate in very small and giant breeds of dogs, which
are reported to have less success with vaginal inseminations.
9. To perform a vaginal insemination, the pipette is introduced through the doral
vulvar comissure, directed dorsocranially through the vagina until it enters the
pelvic vagina. Then the pipette is directed cranially as far as possible, ideally to
the external cervical os. The bitch's hind end is elevated to a 45 degree angle
while the semen is injected through the pipette. The dorsal vaginal wall is gently
"feathered" for a moment or two while the hind end remains elevated for 10
minutes. Care is taken not to place pressure on the caudal abdomen during this
time.
10. Recent reports indicate that elevating the hind end for longer than one minute
offers no advantage over a one minute elevation.
11. Save the semen tube for DNA verification of paternity should that become
necessary.
Tips for Success With Frozen Semen
Planning the Breeding
1. Again, if you agree to plan and perform an insemination with frozen semen, you
must be available to perform the insemination should it need to be performed on a
weekend or holiday.
2. Insemination with frozen semen necessitates precise timing of ovulation. All
available tools should be utilized to pinpoint the period of optimal fertility.
3. In planning a breeding involving international shipment of semen, care must be
taken to ensure that all the receiving country and kennel club requirements are
met. This often involves several forms, health certificates and blood tests. The
addresses and information for a large number of Kennel Clubs world-wide can be
found on the website (www.fci.be) of the Fédération Cynologique International.
4. Dog semen is allowed entry into the United States provided it is accompanied by
a certificate endorsed by the animal health official in the country of origin. The
certificate must certify either that the semen extender does not contain milk
products OR that if milk products were used, they originated from a country
recognized as free of foot-and-mouth disease by the USDA. Requirements for
semen import into the United States can be obtained from the United States Drug
Administration (USDA) P.O. Box 3220, Minneapolis, MN 55403-1503, USA,
website (www.aphis.usda.gov).
5. The AKC requests a prior application to permit AI by imported semen. They also
request a DNA sample, which can be ordered via E-mail:dna@akc.org. It is taken
with the aid of a special cheek-swab kit supplied by the AKC. The AKC can also
be reached at5580 Centerview Drive, Raleigh, NC 27606-3390, USA. Phone:
919-233-9767 or 919-854-0124; Fax: 919-233-3627 or 919-854-0102;
website:www.akc.org.
Semen Collection and Freezing
1. Semen is collected in routine fashion.
2. Semen is frozen. There are many methods of freezing, each with its own set of
buffers, cryoprotectants, and freezing protocol. Some methods are proprietary
and require franchise purchase. Proprietary agencies include Canine Cryobank,
CLONE , International Canine Semen Bank (ICSB), and Synbiotics. Nonproprietary
systems, with published protocols, include the Norwegian Tris
extender, the Uppsala-Equex extender and its modification, the Uppsala-Equex 2
extender.
3. Semen is frozen in either straw or pellet form. The semen is usually frozen so that
the final number of spermatozoa per straw or pellet is between 100 and 200
million. Depending on the semen quality, usually 2-4 straws are used for each AI.
In smaller breeds producing fewer spermatozoa per ejaculate it may be desirable
to freeze a less concentrated semen to obtain more straws.
4. The semen sender must provide the inseminating practitioner with adequate
information about the quality of the semen they send and, in the case of frozen
semen, information about how the semen should be thawed, because the method
of thawing is dependent on how the freezing was done, and the methods vary
among freezing facilities.
5. If, however, the semen is of unsatisfactory quality it should not be shipped unless
the bitch owner or importer is informed about the situation and has given consent.
Semen Shipping and Thawing
1. To ship frozen semen a liquid nitrogen container is required. The container must
maintain the temperature at around -197°C. Today most semen freezing facilities
use dry-shippers, called dewars.which absorb the liquid nitrogen into a porous
material in their walls. These will not spill and therefore need not to be shipped as
dangerous goods, which is more expensive. They should, however, always be sent
as fragile goods, because they are easily broken by rough handling. The tank is
usually shipped in a plastic box for protection.
2. If the inseminating veterinarian has a liquid nitrogen storage tank, then the frozen
semen can be shipped in a dry shipper or dewar well in advance of its anticipated
date of use. This relieves the angst associated with overnight shipments at the last
minute. If a storage tank is not available, most dewars can safely store semen for
up to 4 to 5 days.
3. When removing semen from the storage tank, check the labeling to ensure that the
information on the straw or vial matches that on the accompanying paperwork,
and that the paperwork is as expected (correct name, breed, etc. of dog).
4. Because of the many freezing methods employed today, it is critically important
to follow the accompanying handling and thawing instructions implicitly.
5. Straws are thawed in a water bath. Common thaw methods include immersion at
70°C for 8 seconds, 50°C for 10 seconds or 37°C for 30 - 60 seconds. Thawing is
complete when the bubble rises to the top of the straw. After thawing, any water
remaining on the outside of the straw is carefully wiped off before opening the
straw. Some methods may direct you to empty straws into pre-warmed thaw
medium.
6. Semen frozen in pellets is stored in vials. When removing a vial from the storage
tank, care must be taken to carefully loosen the vial cap and let all liquid nitrogen
vaporize prior to removing the cap. Pellets are then quickly transferred and sealed
in a whirl pack bag which is plunged into a water bath. Pellets are massaged
through the bag to aid in the rapid thaw process.
7. Save the straws or whirl pack bag so that cells are available for DNA verification
should that become necessary.
Insemination of Frozen Semen
1. Normally, frozen semen is inseminated directly into the uterus. At present, there
are three methods for depositing semen directly into the uterus: surgical
insemination, blind transcervical passage of a rigid catheter into the uterus, and
endoscopic transcervical passage of a catheter into the uterus.
2. Surgical insemination is performed with the bitch under general anesthesia. The
uterus is exteriorized through a ventral midline laparotomy approach. The thawed
semen, contained in a sterile syringe, is injected into the uterine horn at a 45°
angle, with a 22 or 23 gauge needle, bevel up. Half of the volume is injected into
each uterine horn. After the needle is withdrawn, a saline-moistened gauze
sponge is held over the injection site until hemostasis is achieved.
3. Both Scandinavian and Norwegian rigid catheters are used for blind transcervical
insemination. To perform the insemination, the outer sheath is passed as far as
possible cranially into the vagina, the internal stainless steal catheter is advanced
into the fornix adjacent to the external cervical os, the cervix is palpated through
the abdomen, grasped and manipulated onto the end to the catheter, then the
catheter is advanced through the cervix. This method requires considerable
training, practice and skill. Anesthesia of the bitch is not required.
4. Endoscopic transcervical insemination is referred to as the New Zealand method.
The equipment needed includes a 5 mm diameter, 36 cm long rigid endoscope
with a 30° viewing angle, a sheath for the catheter, a light source and an optional
viewing monitor. An 8 French polypropylene urinary catheter is inserted into the
channel on the sheath and the endoscope/sheath/catheter combination is passed as
a unit into the cranial vagina. The external cervical os is visualized and passage
of the catheter into the cervix can be confirmed. Anesthesia is not required.
5. With both transcervical techniques, semen should not be thawed until correct
catheter placement is achieved. These can be lengthy procedures and sometimes
catheter passage is not achieved, thereby necessitating a change in plans to
surgical insemination.
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