Since the American Kennel Club's recognition of litters conceived from frozen semen in 1981 and the subsequent acceptance and fresh chilled semen, practioners are being asked more frequently to assist clients with maximizing conception rates using these breeding techniques. The gratification one feels when successful is one of the great rewards in veterinary medicine.

The basis of fresh chilling and freezing semen is energy conservation within the sperm cell so that the semen can be shipped or used at a later date. The drawback to these methods is that even though some energy is conserved, enough energy is used to shorten the sperm cell's life.

Fresh semen is thought to easily live 4-6 days in utero. Some papers have reported live sperm found in the uterus 11 days post-breeding. The natural sperm length of life allows for successful breeding when the bitch first exhibits standing and acceptance of the male, even though in many cases the timing is 4-6 days before the prime fertilization period of the mature ova.

Fresh chilled semen uses energy as it is cooled to 40 degrees F (4 degrees C) and eventually re-warmed to body temperature. The life in utero of a spermatozoa having experienced the chilling and subsequent warming process is 24 to 72 hours, necessitating a more precise manner of ovulation timing and breeding.

When sperm is frozen for future use, it is eventually stored at -322 degrees F (-180 degrees C). This extreme temperature preserves a sperm cell indefinitely, but not without stress and energy output. The thawed spermatozoa has a maximum life span of 12 to 24 hours after insemination into the bitch. The ultra-short life span makes success with frozen semen only possible with precise mapping of the bitch's estrous cycle with definitive detection of the lh release and the exact time of ovulation.

When an individual receives a fresh chilled sample, the package should immediately be opened. Attention should be paid to the "impression of coldness." The ice packs should be at least cold, if not still frozen.

The package 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 cell and renders the sample useless.

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.

As the drop warms on the slide, the accompanying paperwork with the semen collector's evaluation of the semen quality. Time of collection and post-collection motility should be studied. The semen drop is then analyzed and compared to the collector's evaluation.

In many cases, the semen will appear to be normal. However, as the semen drop warms gradually, side to side motility becomes noted. The continued warming eventually shows the cells to have achieved a normal forward progression. 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.

It is recommended that the refrigerated fresh chilled sample 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.

FROZEN SEMEN

Frozen semen does require even more precise handling than fresh cooled semen. Frozen semen will usually be shipped in a dewer with nitrogen vapor keeping the sample frozen rather than with actual liquid nitrogen in the container.

When the shipper arrives, the tank will be in a protective shell. The shell should be opened and the plug pulled (not twisted) from the nitrogen tank. Visible vapor should be seen, assuring the recipient that the tank is charged properly and that the sample is still frozen. If vapor is noted, the plug should be replaced until the time of the insemination. If no vapor is noted, an attempt should be made to ascertain that the sample is still in a proper frozen state. This can be accomplished by grasping the cane that holds the semen and raising it to the mouth of the dewer. If the sample is thawed, the semen will not be viable and a replacement sample should be obtained, if possible.

If the sample is still frozen, the person that shipped the sample should immediately be contacted to discuss the tank status and determine if some liquid nitrogen should be added to the dewer to assure that the sample will remain frozen until time for the insemination.

Canine semen is frozen in two different forms, 1) pellets and 2) straws. Both are extremely successful in achieving conception, however, it is essential that the doctor doing the insemination follows the thawing instructions precisely. Most thawing techniques require that the person using the semen has a form of warm water bath available. A form describing the semen's quality, both pre and post thaw, should have accompanied the shipment. These values can be used to judge the quality of the thaw before insemination. Each individual that freezes canine semen has a specific manner of handling and thawing their product. There is no room for error !!

TIMING OF INSEMINATION

Historically, methods of timing the estrous-cycle of the bitch such as color of discharge, swelling of the vulva and flagging of the tail allowed breeders to get bitches bred. But using these signs, in many cases, created more confusion than answers. Vaginal cytology, a rough gauge of estrogen level, has been used by veterinarians and breeders to "pinpoint" the time of ovulation. Vaginal smearing, at best, gives a retrospective view of the cycle with estrus, day 1, occurring six days post-ovulation. However, vaginal smearing does not allow prospective timing, other than giving information as to when other testing needs to begin. Vaginal cytology is not useful by itself in timing fresh cooled or frozen semen. The same can be said of vaginoscopy, a visual method of evaluating vaginal fold swelling and crenulation that equivocates with estrogen rise and fall.

The serial testing of serum samples from the bitch for either progesterone testing or luteinizing hormone (LH) testing will give the veterinarian a prospective view of the ovulation, ova maturation and the prime fertile period for using the fresh cooled or frozen semen.

PROGESTERONE

The release of progesterone from the ovary and its subsequent rise to an average of 2 to 3 ngm corresponds with release and signifies the start of the ovulatory process. The rise of progesterone to greater than 5 ngms (5 to * ngm) indicates that ovulation has occurred. Once the ovulation day has been confirmed, fresh cooled semen insemination is 2 to 3 days or frozen semen insemination in 3 to 4 days, can be planned.

Progesterone not being species specific, can be tested for by many methods. Numerous ELISA kits for in office use is currently available. Progesterone can also be evaluated by radio-immune assay (RIA) by many human and veterinary laboratories.

Progesterone rises and continues to stay elevated for approximately two months in the bitch, whether she is pregnant or non-pregnant. The bitch needs to be tested every 48 hours to anticipate the prime breeding time.

When using the progesterone ELISA kits, negative features include that hemolyzed blood samples will give lower than normal results. If the ELISA kits are not handled properly or warmed to room temperature, and artificially high result will be recorded. Ideal testing for progesterone level would include screening with the ELISA kits, but obtaining RIA progesterone numbers, will define the day of ovulation and the corresponding breeding time.

Cortisol release needs to be a concern when doing progesterone testing as stressed bitches may have a delayed period between a rise of 2 to 3 ngm/ml and the time that the progesterone rises greater than 5 ngm/ml. Some bitches under stress have been shown to not ovulate even after a rise of progesterone above 2 ngm. It is essential, if using progesterone testing, to confirm that the bitch has risen above 5 ngm/ml and has indeed ovulated.

LUTEINIZING HORMONE

Luteinizing hormone is a species specific (describe type of hormone) hormone arising from the pituitary gland. The release of the hormone spikes over a 12 to 24 hour period. The release of the LH triggers ovulation in 48 hours, allowing for a final merotic division of the ova and shedding of the polar body (approximately another 48 hours). An accurate evaluation of the LH spike can give the veterinarian and breeder 4 to 5 days anticipation of the prime insemination dates and reasonable confidence as to the maximum chance of conception.

Unfortunately, LH assays are not easily available for canines at the time of this writing. As LH is species specific, human laboratories cannot run this assay.

An immuno chromatinographic testing kit is currently available for in office use. The test, status LH, (International Canine Genetics, Inc., Malvern, PA. 19355 USA) uses a wicking paper with a reaction zone of test and is run with 4 drops of serum.

The test develops a distinct colored line (red) when the serum lh rises above 1 ngm/ml. When the line appears, one would anticipate prime breeding time to be in 4 to 5 days with fresh cooled semen and 5 to 6 days with frozen semen (the variation based on anticipated sperm life in uter).

Negatives to the LH test are that daily samples must be drawn (even greater than 24 hours between tests may miss the LH rise). The cost of daily testing, owner compliance and veterinarian availability for testing every day have created some problems for this testing method. Serum samples that are hemolyzed or lipemic also affect the testing results. LH testing is especially useful in bitches previously tested by progesterone assay that did not conceive to that showed irregular progesterone levels.

ARTIFICIAL INSEMINATION

Artificial insemination has been a commonly used procedure in small animal reproduction. However, the advent and common usage of canine frozen and fresh cooled extended semen has refocused clinicians attention to more precise methods of insemination. The shortness of spermatozoa life, loss of sperm cell energy in fresh cooled extended semen and especially in frozen semen has necessitated the development of new methods of semen delivery in the bitch. Frozen semen especially benefits with techniques by-passing the cervix and the cervical mucous and being deposited directly into the uterine lumen.

Two techniques of artificial insemination are routinely performed and are amenable to the veterinary practioner dealing with canine reproduction. The two techniques to be described are vaginal insemination and the surgical intra-uterine deposition of semen.

VAGINAL INSEMINATION

When proper timing of the estrous cycle is performed on the bitch and proper semen handling and delivery is accomplished, conception rates should rival those of natural breedings. Delivery technique problems such as improper placement of the insemination rod, improper semen placement, or semen damage due to mishandling, have convinced many dog breeders and veterinarians that artificial inseminations are only used as a last ditch desperate measure.

PROCEDURE

The estrous cycle for the bitch should be monitored using serum progesterone assaying or some other reliable manner to ascertain that ovulation has occurred. Semen motility is evaluated as previously described.

The bitch is positioned with her rear elevated either manually or on a breeding ramp. Care should be taken to avoid pressure on the bitch's abdomen. The semen is drawn from the collection tube through the insemination rod of proper length is used. It is important for the semen to be deposited at the entrance to the cervix sot hat the semen can be drawn into the uterus. With gloved hands, the veterinarian gently inserts the insemination rod through the lips of the vulva at an upward 45 degree angle. The rod is gently passed over the pubis and along the dorsal median fold until it is parallel with the lumbar spine and localized in the area of the cervix. If resistance is encountered, the rod should be gently twisted or withdrawn a short distance, then readvanced.

When the insemination rod is properly positioned, the semen should be gently inseminated. The syringe is then removed from the rod. It is not necessary to push large amounts of air into the rod nor normal to get semen backflow if the rod is properly positioned and the bitch in the estrus stage of estrous. Excess air "bubbling" through the semen is detrimental to the fragile plasma membrane of the head of the spermatozoa.

The bitch is then "feathered" digitally for 1 minute, the rear of the bitch is maintained in an elevated position for 6 minutes to allow gravitational feeding of semen to the anterior vagina. The bitch owner is instructed to confine the bitch or restrict her activity for 1 to 2 hours post-insemination.

SURGICAL INTRA-UTERINE DEPOSITION OF SEMEN

A technique for surgical insemination in the bitch was first described in 1974. The intra-uterine deposition was initially used to increase poor conception rates in the use of canine frozen semen. Since that time, surgical deposition of semen into the uterus has become a routine technique used in numerous situations encountered in canine reproductive medicine.

A) Frozen semen--due to the lack of spermatozoa energy, buffer chemical makeup, or cervical resistance, the conception rates from cryo-preserved canine semen have been historically low when used with a vaginal insemination. Surgical deposition into the uterine lumen has resulted in conception rates equal to those from natural breeding.

B) Fresh cooled extended semen--shipment of semen rather than shipping bitches has become common place. Due to shipment time and spermatozoa energy depletion, numerous clients have chosen surgical semen insemination for their bitches in hopes of increasing conception chances.

C) Bitches with suspected uterine or ovarian disease--the ability to access uterine and ovarian health at breeding time is advantageous to clinicians presented with bitches with histories and reproductive failure or problems.

D) Giant and toy breeds-- individuals on the extreme ends of size have historically been recognized for conception difficulties. The ability to overcome anatomical barriers and inseminate directly into the uterus has increased litter numbers and relieved client frustration.

E) Males with lowered or compromised spermatozoa numbers--little work has been done in the canine to definitively define minimum semen parameters necessary for conception. By directly depositing the semen into the uterus, bypassing the cervix and vaginal vault, conception can possibly be achieved with lesser sperm numbers and lesser overall semen quality.

TECHNIQUE

A pre-surgical evaluation is performed on the bitch. Anesthetic induction consists of coning smaller individuals with isoflurane. Larger bitches may require an intravenous injection of a standard short acting anesthetic to achieve relaxation for intubation and maintenance with isoflurane (i.e., propofol).

The bitch is prepared in the same manner as a bitch undergoing an ovarian-hysterectomy. The ventral abdomen is clipped and the bitch is placed in the surgical theater in dorsal recumbency. Prepping of the surgical site is done in a routine manner. The abdomen is draped in preparation for the surgery.

A 4 to 6 centimeter incision is made midway between the pubis and the umbilicus. The incision is made in the skin, subcutaneous fat and through the linea alba. The uterus is identified and elevated to the surface through the incision. The uterus is draped with a saline moistened laparotomy pad if the semen is not ready for the injection procedure.

A volume of semen varying between .5 ml and 4 ml is prepared for insemination. If the volume is greater than 4ml, the semen should be centrifuged for five minutes. The supernatant is decanted and disposed. The semen pellet is gently re-suspended with the remaining supernate or with a semen extender.

The semen to be injected is gently drawn into a 6ml syringe through an insemination rod. The rod is removed and a 22 gauge 3/4 inch needle is attached to the syringe.

The surgeon inserts the needle into the lumen of the uterine body at a 45 degree angle with the bevel of the needle up. The semen is slowly injected into the uterus. The semen should flow easily with obvious distention of the uterine horns. If the injection cannot be achieved or is difficult, the needle should be repositioned.

A saline moistened gauze is held over the injection site after the needle is withdrawn. After one minute, the gauze is removed and the uterus is replaced into the abdomen. Closure of the fascia, muscle, subcutaneous tissue and skin is routine.

The bitch's rear is elevated as she recovers from the anesthesia. The intra-uterine pressure during the surgical deposition of the semen may cause mild backflow through the cervix into the vaginal cavity. This appears to be of no concern. If the surgeon has doubt as to the semen being placed in the uterine lumen, a post-operative vaginal smear will confirm spermatozoa, if the technique has been performed properly.