Are you feeling stressed as your bitch's due date approaches? Worried that you are going to waste the last vial of frozen semen from your champion stud?A successful breeding program does not have to consist of one panic-laden situation to the next, but all too often this occurs. As a veterinarian working in a practice that sees over 900 dogs for artificial insemination and performs over 250 cesarean sections annually, I have learned to rely on and have confidence in the value of accurate and timely progesterone testing in the bitch. Although it seems that some people thrive under stressful conditions, replacing panic with infor­mative scientific data invariably results in larger, healthier litters and happier owners. In today's era of CO-ownership of valuable stud dogs and breeding bitches, it is not uncommon to have four or six differing opinions on the timing of a breeding.

Indeed there are a lot of options. One breeding or two? Should it be a natural, vaginal, transcervical, or surgical breeding? The questions mount and the stress can build, but if you are armed with a basic working knowledge of the bitch's repro­ductive system and accurate progesterone tests, you can make good decisions. The progesterone test is your best friend. First, let's review the basics. Prior to coming in season (estrus), serum progesterone levels in the bitch are low, less than 2ng (ng=nanograms). As the cycle continues, progesterone levels slowly climb to a level of 5ng, upon which ovulation occurs. The ovulation date can be as early as day 7 and as late as day 27 of the estrous cycle, emphasizing the importance of the blood test. Once a bitch hits 5ng and ovulates, subsequent serum progesterone levels can vary greatly. For example, at three days post ovulation we routinely see progester­ones that may range from as low as lOng to as high as 60ng, depending on the bitch. For ovulation purposes we are only interested in the day she exceeds 5ng. I commonly get the question, "Doc, we only ran one progesterone test on my bitch and it was 15ng, when did she ovulate?" The answer is impossible to know based on one test.

I have seen bitches at 15ng one day after ovulation as well as 5, 10, 20, or 40 days after ovulation. You must have one test below 5ng and one above 5ng to determine ovulation day. For most bitches we start testing 6 days after the onset of vulvar swelling and bleeding, and we test every 1-3 days until ovulation, depending on the situation. After ovulation, serum progesterone levels will remain elevated, regardless of whether or not the bitch is pregnant, for approximately two months, at which point they return to near Ong. It is important to note that vaginal bleeding, swelling, "flagging," and receptivity to males is predominantly a re­sponse to estrogen's in the bitch's bloodstream, and not a reliable indi­cator of progesterone and consequently ovulation. She mayor may not be receptive at the proper time. The use of physical indicators alone for breeding may not be a chance worth taking, especially with valu­able semen. So what does knowing the ovulation day do for us? It tells us when the eggs will be fertile, which is important with all breeding, but especially so with fresh chilled extended semen and with frozen semen.

Once ovulated, a bitch's eggs take approximately 48 hours to become fertile. They will then remain fertile for approximately 48 hours. The idea is to maximize contact time between viable semen and fertile eggs. This requires a brief discussion of semen longevity. Fresh se­men can remain fertile in the bitch's uterus for 3-5 days and in some cases even longer, depending on quality. Fresh chilled semen, because of energy lost in the cooling and subsequent warming process, may live only 24-48 hours in the uterus. Frozen semen, due to the stress of the freezing process and energy lost, may live only 12-24 hours in the uterus. Clearly, the shorter the sperm's life span, the more paramount it becomes to know when it should be inseminated to allow for maxi­mum contact time with fertile eggs. In the case of fresh semen, there is more flexibility for two reasons. First, there is usually not a limited quantity, and second, it lives a long time. We routinely recommend breeding days 1 and 3 or days 2 and 4 post ovulation with fresh semen, though one breeding is usually sufficient. With fresh chilled extended semen, because of a 24-48 hour life span, we recommend breeding 2 days after ovulation.

As we discussed earlier, the bitch's eggs are just becoming fertile at this point, so we should have 1-2 days of good con­tact time. In the case of frozen semen, we recommend waiting three days after ovulation to do the breeding. This may make our clients nervous that we are waiting too long. However, it ensures that all the eggs are fertile when we put semen into the uterus that may live only 12 hours. We do not have the luxury of waiting around for eggs to become fertile. We have found the highest levels of success with fro­zen semen following surgical insemination and recommend it exclu­sively. Our levels of success using the above timing recommendations have been excellent; however, the manner in which a bitch is bred natural, vaginal insemination, transcervical insemination with an endoscope, or surgical insemination-must be carefully considered de­pending on factors such as reproductive history, age, physical charac­teristics, and semen quality. Having an accurate ovulation date at the beginning of a pregnancy is also very important at the end of a pregnancy.

A bitch's due date is 63 days from ovulation, regardless of breeding days. This is extremely valuable for planned c-sections, due to such factors as breed confor­mation, litter size, prior medical history, or complications arising dur­ing the pregnancy. It is also very helpful when whelping. Knowing an exact due date not only helps breeders prepare for the arrival of a litter, but, often more importantly, it can alert us to a premature or overdue labor, allowing for prompt medical intervention if indicated. Progesterone monitoring can also be important during gestation. Some bitches have a problem maintaining adequate levels of progest­erone during a pregnancy, usually resulting in resorbed or aborted fe­tuses. Monitoring progesterone levels is critical in bitches with such histories, as supplementation of progesterone at the proper times could mean the difference between live or dead puppies. Often we are confronted with owners who have difficulty "catch­ing" a bitch's heat cycle. This is usually due to minimal outward physi­cal signs such as vulvar swelling and bleeding. We recall that proges­terone levels stay elevated for approximately two months after ovula­tion in all bitches, whether pregnant or not. Obtaining an elevated progesterone level in such an animal at the very least indicates she was in season in the last two months, often helping a breeder know ap­proximately when to expect the next cycle. Conversely, a low proges­terone level, less than 2.5ng, means there have been no eggs ovulated in the past two months.

This may alert a breeder to an upcoming cycle, or, if the level remains chronically low, the veterinarian may be alerted to begin a thorough medical workup. The usefulness of accurate and timely progesterone testing should not be underestimated. It is a valuable tool and in some cases of criti­cal importance in helping a veterinarian and breeder choose the proper course of action. Proper use and interpretation of these tests will re­sult in not only more pregnancies, but larger, healthier litters, fewer complications with your bitch, insight into potential problems, and, as I have experienced, fewer panicked telephone calls at three 0' clock in the morning..

FRESH CHILLED SEMEN

Fresh chilled semen uses energy as it is cooled to 40'F (4'C) and eventually re-warmed to body temperature. The life in-utero of 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'F. (-180'C). This extreme temperature preserves a sperm cell indefinitely but not without stress and energy output. The thawed spermatozoa have a maximum life span of twelve 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 subsequent time of ovulation. When a veterinarian receives a fresh chilled sample, the package should immediately be opened. Attention should be paid to the "impression of coolness". 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 wanns 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 non-motile. 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 fifteen 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 connection 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 remove 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.

FROZEN SEMEN

Frozen semen requires 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 insemination. If no vapor is seen, 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 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 follow the thawing instructions precisely. Most thawing techniques require that the person using the semen have 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.