SUCCESS WITH FROZEN SEMEN: BROOD BITCH MANAGEMENT

Dr John F Newell B.V.Sc.
Rocky Ridge Frozen Semen Facility
PO Box 1166 Warners Bay, NSW Australia

• Results - What can be achieved?
• Procedures - How can this be achieved?
• Vaginal Flora considerations
• Progesterone Testing
• Implantation considerations
• References
  

The Greyhound Industry has played a major role in the development of Frozen Semen technology worldwide. The necessity for integral practices, monitoring and accountability has seen the emergence of accurate reporting systems to gauge validity of claimed successes of operators and implant facilities.

With no requirement to register breedings, the compilation of accurate conception rate statistics is not possible in the Show Dog world and the accountability of different freezing methods is difficult to gauge without such accurate recording and reporting practices.

Over the past two decades potential profits have attracted many opportunists to establish as providers of frozen semen services, however many have a less altruistic agenda, rather than embracing the proven efficiencies of frozen semen for the improvement of various breeds worldwide. Other operators do have specific breed improvement at heart but utilize technology and systems that will not achieve maximal conception rates.

Results - What can be achieved?

(1) National Greyhound association – USA Statistics for year ended 2004

• Natural Breedings are 17% of total Breedings, 14% of total litters & 13% of puppies
• AI’s of Fresh Semen are 17% of total Breedings, 15% of total litters & 15% of puppies
• Frozen Semen is 66% of total Breedings, 70% of total litters & 72% of Puppies

(2) Dr John Katakasi (Australia)
Dr John Katakasi from the Adelaide Plains Veterinary Hospital – South Australia – has achieved a 99% conception rate using the Camelot SPS.

(3) Dr Kent Law (USA)
Dr Kent Law has achieved a 94% conception rate over 11,000 frozen semen inseminations over the past 12 years.

(4) Dr John Newell (Australia)
Dr John Newell has also achieved a 94% conception rate with the last 1000 frozen semen inseminations.

The Camelot Farms frozen semen technology has been employed by the veterinarians listed above to achieve such levels of success. 98% of frozen semen breedings registered with the NGA were by the Camelot Farms system as superior results have simply established it as the system of choice.

We are often presented with bitches that have missed once or twice and an owner prepared to spend any amount of money to find out why? Sometimes in retrospect there is no answer. Rather the focus of energy and expense should be on taking control of the breeding, eliminating or minimizing all the causes of a missed conception and facilitating fertility – not trying to diagnose suspected infertility! – The bitch, the Dog or You?

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Procedures - How can this be achieved?

Such results are achieved based on “The Four Steps to Breeding Success” developed by Richard & Sharyn Conole with the Camelot Farms Semen Preservation System.
The main considerations with Brood Bitch Management are

Critical to achieving such results is Correct Sire Management, Semen quality and Extender technology and these aspects are discussed in the companion lecture – Sire Management and constitute the third step to breeding success - Checkmate

The fourth step is the insemination itself and many aspects need to be considered for success with this step

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Vaginal Flora considerations

The debate over the relevance and significance of pre-mating vaginal culture and sensitivity early in the pro-oestral phase presents many aspects that need to be considered. It is however generally agreed that antibiotics are contra-indicated as a routine prior to insemination or breeding. A vaginal culture and sensitivity may be recommended but unfortunately the interpretation of resultant expected flora growth is not easy and many vets place the bitch on antibiotics anyway – often unnecessary and inappropriate antibiotic therapy. In our facility less than 15% of pre-mating culture and sensitivies would be placed on antibiotics.

Some adopt the attitude that without clinical signs of infection, odour and irritation or previous history of breeding complications attributable to infection then culture and sensitivity is not indicated. One study of vaginal cultures obtained from normal fertile breeding bitches found 98% +ve Pasteurella multocida, 89% +ve _-Haemolytic streptococci, 84% +ve Eschericia coli, 59% +ve Mycoplasma species, 33% +ve Staphylococcus intermedius and 25% +ve Proteus mirabilis. Simply by isolating bacteria from the vagina is no basis of diagnosing disease. These bacteria are normal vaginal flora.

Additionally using antibiotics in healthy bitches promoted the growth of opportunistic pathogens such as Eschericia coli and Mycoplasma. Mycoplasma in particular exists in a carrier state with no clinical effect until changes occur in the bitch’s defense system – immunosuppression or well meaning antibiotics, and the clinical disease or embryonic, foetal death may be the likely sequellae.

Antibiotic sensitivity testing and appropriate therapy has proven effective in eradicating bacteria in one to two days, however bacteria re-colonised within one to four days after the course of treatment finished. This study involved the use of trimethoprimsulfamethoxazole and ampicillin selected as appropriate antibiotics for cultured Pasteurella multocida, _-Haemolytic streptococci and Eschericia coli. Highly significant however was the emergence of mycoplasma both during and after the treatment periods! (1)

Brucella canis is known to be a specific bacterium causing infertility in the bitch. Although not in Australia it is mentioned for the sake of completeness as screening for B canis is an integral part of the export preparation testing of sires prior to semen import to Australia. Routine screening of sires and brood bitches is recommended to maintain a high level of fertility in the kennel.

Considerations from Research -So what do we do?

1. Determine past breeding history – Healthy litter? Foetal death? Neonatal losses? Protracted post whelp discharge? – Review prior culture and sensitivity isolates.
2. Current season – Assess discharge – smear – White blood cell % - Malodorous Remember white blood cells tend to disappear during oestrus – the thickened vaginal lumen block neutrophil movement and a finding of many neutrophils at oestrus is significant for infection considerations. Neutrophils return in large numbers in dioestrus.
3. Culture and sensitivity with an open mind – Don’t use antibiotics based on a positive culture – 95% of cultures will be positive! Antibiotics justified by a positive culture have the potential to create worse problems once the flora balance is upset and pathogenic opportunists invade. Having a positive culture result presented with a bitch is no reason to refuse that bitch for mating. Frozen semen implants remove any risk considerations for the bitch and sire.
4. Remember if we do a culture and sensitivity on day two of season and complete a six day course of antibiotics, the original cultured flora will usually have returned within four days of the antibiotics finishing along with the increased likelihood of the nastys – Pathogenic E coli and Mycoplasma.
5. I tend to be more guided by vaginal smears and checking the bitch every two days rather than completing a C & S on day two with the expectation that antibiotics to save the day – Only use when clearly necessary and ignore the easy sale of antibiotics based on a “positive” culture to the lay person.

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Progesterone Testing

The main reason for a missed conception is the improper timing of mating. Accurate progesterone testing has enabled a precise monitoring of the timing of the implant but even so, much theory can cloud this determination and procedural variables and sampling techniques significantly add to the variance which may mean the difference between a success and a missed conception.

Ovulation timing is an art as well as a science. The art lies in the interpretation of rates of progesterone rise, the recognition of environmental effects and a “split season”, regular monitoring with vaginal cytology as indicated and the realization that strict adherence to defined theory will not work in every situation. Given these variables we must look closely at the validity of a number (progesterone level) returned from a laboratory and consider if we are confident to make a decision to breed based on that number. We must also be aware that the correlation between the behavioural and physical changes during oestrus and the time of ovulation is poor. Additionally some dominant bitches do not exhibit standing heat whilst submissive bitches often allow mating when not in oestrus.

The more one observes the oestrous cycle it is easy to come to the conclusion that there are no abnormal cycles – rather extensive variances of the “normal” cycle. Some bitches may show a silent heat before overt signs of oestrus whilst others commence their season with a distinct definable beginning. Consecutive seasons vary in their character and assumptions cannot be made regarding ovulation timing based on the pattern of the previous season. Some bitches loose their “scent” at ovulation and the male may be misled with such confusing pheromone signals. Some bitches do not exhibit standing oestrus even though ovulation has occurred.

The fallacy of “counting days” and the results obtainable by doing so correlate well with the six year average 62% conception rate demonstrated in the National Greyhound Association figures for 1988 to 1993. If we consider 10 bitches, 5 of which ovulate on day 8 and 5 of which ovulate on day 18, then the average day of ovulation is 13. But if you breed them all on day 13 you may miss them all!

As every bitch is different, let us consider they ovulate on days 8,9,10,11,12,13,14,15,16 & 17 respectively. As it takes 48 hours for maturation of the oocytes, if you breed them on day 13 (assuming sperm will live in the bitch for 4 days) conception should occur in those which ovulated on days 11 thru 16 or a 60% conception rate!

Reliance on vaginal cytology is most useful to define the end point of oestrus and the commencement of metoestrus. Cytologically oestrus is defined as complete cornification with greater than 50% of cells with no discernable nuclei. There is poor correlation between the onset of cytologic oestrus and ovulation. (3)

The events leading to ovulation commence with a rise in oestradiol levels from the ovaries commencing the overt signs of oestrus or heat. Oestradiol levels peak approximately 24 -48 hours before the luteinizing hormone (LH) surge then decrease rapidly. It is interesting to note that the decrease in oestradiol varies greatly from one bitch to another. At times oestradiol remains elevated to the end of oestrus or even later resulting in the clinical presentation of bitches that continue to bleed until almost the start of metoestrus. High oestrogen levels are also often present with follicular ovarian cysts and partial anovulation.

A Luteinizing hormone surge from the hypothalamus stimulates granulosa cells to secrete progesterone. Ovulation occurs two days after the LH surge and continues for 24 hours. The bitch ovulates ova as primary oocytes which must undergo further division and maturation before fertilization can take place. In the dog maturation is completed at least 48 hours after ovulation. Breeding then should not be timed to coincide with ovulation as occurs in many other species. Insemination, we are told, should occur two to three days after ovulation or four to six days after the initial progesterone rise. Another theory is to inseminate 3.25 days after progesterone reaches 6.5ng/ml. But no one has necessarily told the bitch this and to make the assumption to inseminate on the basis of this predictive theory will lead to a higher level of missed conceptions.

It is imperative to track the season with sequential progesterone tests to establish an inexorable rise in the progesterone curve to the desired point of insemination, which may vary from facility to facility! At Rocky Ridge Frozen Semen Facility we implant generally between 15ng/ml to 18ng/ml (47.7nmol/L to 57.2nmol/L)
This is not a simple number then implant – rather it is considered in the light of the rate of progesterone rise and vaginal cytology where indicated. For example if we have a steady rise from 8ng/ml to 15ng/ml over six days then we may not breed her until she is closer to 18ng/ml. A bitch rising from 8ng/ml to 15ng/ml in 1.5 days will be bred at the 15ng/ml level. The window of opportunity is narrower.

This is an art – there is no set number on which success is based an implant but rather consider the progression of each bitch’s season armed with knowledge of oestral endocrinology. If we are presented with a bitch that tests at 25ng/ml (79.5nmol/L) – she may only be on day 9 – The owner is cautioned about the risks of such a one off implant with no serial curve history. We assess discharge – still blood?, moist or very dry?, odour clean or rotten oysters?, Vaginoscopy – cobble stoned & mucoid or blotchy white / pink metoestral, cytology – squamous or plump metoestrus cells? We then decide the chances of conception for that particular individual. The last ten bitches presenting like this – three had progressed into metoestrus, two were elected by the owners to return on day five of their next season for a sequential work-up and five bitches were mated resulting in five pregnancies and forty nine pups.

Remember counting days bears no relevance to successful breeding program. The way we used to breed was to count days from the start of the season and mate on day fourteen – As we have seen this resulted in a 62% conception rate.

As progesterone technology progressed it was suggested that we observe the first progesterone rise then count 5.5 days then inseminate. At this time the bitch may have a split season, a false season or have ovulated three days ago if she is a giant breed, seasonal in summer, or if you’re lucky she is right for mating.
Therefore by default this theory implies that it doesn’t matter what the progesterone level is on the day of mating as we are calculating from a defined event and ignoring subsequent variances. Such theory is based on determining the first rise in progesterone, with ovulation occurring 24 to 72 hours after the LH surge and insemination to occur 5.5 days after peak LH surge – counting more days to allow for follicle maturation.

It is however essential with a predictive theory to define variances to maximize conception rates and the progesterone level at the time of implant IS important.

False seasons occur commonly and need defining with a progesterone and implant protocol to avoid implanting unnecessarily and wasting valuable semen. Pro-oestrus develops normally but no LH surge follows peak oestrogen levels. Clinically the bitch is well on season, with normal vaginal turgid swelling and bloody discharge. She may even tail twitch and so receptiveness to the male. As there is no ovulation or rise in progesterone, the partially developed follicles shrink up and subside into the body of the ovary.
Progesterone levels need to be monitored to ensure a continued rise reflective of ovulation. Bitches exhibiting a false oestrus will generally return to an ovulatory season within 4 to 10 weeks.

Split seasons also occur relatively commonly and will be defined with regular progesterone tracking. It is likely that these are a direct result of waxing and waning hormone surges but may be overlapped by temperature variances which may delay ovulation if very cold conditions occur. Clinically as progesterone rises behavioural signalments indicate standing oestrus. The male is keen and the bitch may be receptive. Progesterone levels tracked the next day may reveal a reduced value, and this may stay low for 4 – 7 days before a final surge. If mating is occurs without identifying the “split” oestrus a successful conception is highly unlikely.

In a split season the second hormone surge occurs rapidly. Regular (sometimes twice daily) progesterone is essential to define this second surge as it affords a reduced window of opportunity in which to time an insemination. Bitches that have split a cycle one season may not necessarily split their cycle next season.

The 15 – 18 protocol works for many large implant facilities around the world. These implant at least fifty bitches per month and include Rocky Ridge Farm – Australia, John Tuohy – Trade Kennels – Ireland and Dr Paul Boland – Manchester – United Kingdom. Success is based of course on the Camelot frozen semen system and a repeatable progesterone routine that has proven conceptions many times over. For the average practitioner however there are many factors which may superimpose on this module to provide variance in the progesterone value determined by the laboratory. The extent and implications of these variances needs to be considered as technique and circumstance for this one parameter may mean the difference between a successful conception or implant failure.

Laboratory Variance
Samples drawn at one time, centrifuged and dispatched to three different laboratories at the same time will likely yield three different results to a high degree of statistical variance significance .Laboratory results do vary and it is the question to answer – which number will we believe and why? Variation may stem from the analysis method used, equipment age and maintenance, calibration and adjustment, frequency of running this test, cross contamination from other batch tests and temperature time considerations superimposed as discussed below.

Variance starts with the Sample
Large dedicated surgical implant facilities with in house chemiluminesence or radioimmunoassay analysers provide results within one hour and accurate progesterone tracing is possible with morning & night testing critical in some giant breeds.

Samples are drawn at our facility to a gel clot tube and centrifuged after five minutes, then processed immediately. It is our contention that the value we get at that point in time may be different to the value we would receive if we had drawn blood, awaited a courier pickup, maybe refrigerated immediately or were too busy to centrifuge the sample immediately and faced a weekend where the blood would not be processed for another 24 hours! To this end we carried out a series of experiments to define and assess variability with sample handling, time and temperature effects. These experiments were based on the designs of Volkmann (1) and the results generally parallel his findings. Additionally the effect of the gel clot tube was investigated with blood added to achieve a different mix proportion with the gel. In 5ml ml gel tubes 1.5ml, 3ml & 5ml of blood was added and compared to 3ml of blood added to a plain tube.

The results in summary are

• Serum Progesterone Concentration (SPC) declines within the first two hours after sampling & did not change significantly thereafter
• Decline in SPC only happens if samples are refrigerated after collection
• Blood samples that cannot be centrifuged immediately after collection should be held at room temperature for at least 2 hours prior to any refrigeration to allow clot maturation. Refrigeration subsequently after this time does not significantly affect SPC.
• There was no significant variance in the gel clot tubes incompletely filled compared to serum tubes or a filled 5ml SST gel clot tube.

These results generally parallel those determined by Volkmann (1)
Given this level of variance of up to 50% in certain permutations of sample handling, when considered with laboratory variance, it is not surprising that reliable in house testing provides such excellent results as the majority of these variables are eliminated. Those practices with only a small case load of breeding management or frozen semen may find qualitative in house testing a more accurate option to consider. The methodology that the Camelot Farms system is based on is the ELISA “Date to Mate” kit which has stood the test of time. Prior to establishing the Rocky Ridge Farm facility we routinely utilized Date to Mate for not only fresh artificial inseminations but also for frozen semen implants achieving conception rates in excess of 90%.

“Date to Mate” procedural methodology is well defined on the instructions but of concern is iatrogenic tactile contamination during placement of the pipette tubes into the dropper. One must be extremely careful not to touch the distal end of the pipette that has been placed in either Control A or Control B as dilution contamination can occur to both Sample 1 onward as well as temporal contamination, when the kit is next used, as contamination from previous testing will be cumulative and affect clear colour definition development.

This is particularly important when considering the sensitivity of the Date to Mate ELISA test. With a sensitivity of 1,000 millionth of 1 gram.

Breed Variance
It has been our experience that some giant breeds require special monitoring for a satisfactory outcome. This is especially true with the South African Boerbel and Newfoundland.

We have observed this several times now and routinely monitor giant breeds 12 hourly once they have reached 8ng/ml. This vertical progesterone surge may account in part for the belief that “frozen semen doesn’t work” with these breeds.

Climatic Variance
Monitoring the frozen semen program at a large facility with thirteen stud dogs and up to forty five oestrus bitches in house at peak breeding time it provides the opportunity to observe trends over and above the usual expected progression of the oestrous cycle.

We have observed that there is a direct correlation between heat wave conditions and a rapid rise in progesterone levels. This is not a general effect on the Immulyte anaylser as it operates on a narrow temperature range for incubation and will alarm if outside this designated range. It is however an individual bitch response affording elevated results by way of a larger than expected diurnal jump of up to 16ng/ml!

Conversely with low ambient temperatures (15°C) it is not unusual to see many bitches “hang” and not show the expected rate of progesterone elevation, or even set back for a few days.

Transport Variance
It is well known that extended transport of the brood bitch may have profound effects on the progression of her season. Consequently “Don’t ship the bitch – ship the semen!” Stress, confinement, anxiety and hyperthermia all initiate a prolactin rise and inhibition of normal hypothalamic cascade. The season may be delayed or may not progress to ovulation. False seasons are common in such circumstances. With such delayed seasons we see a high incidence of bitches with malodorous discharges that may have swabbed clear on day one. Many of these fail to ovulate – examine incoming bitches thoroughly for this transport stress syndrome and be aware of the likelihood of progesterone curve variances.

Diurnal Variance
We have followed the progesterone levels of several peri-ovulatory oestral bitches to assess daily variation in progesterone levels. Levels are usually higher with the morning sample and may drop by 15% overall by noon. Such a finding is statistically significant and does not simply reflect any variances or inaccuracies with the analyzer itself. Moreover there is no curvi-linear trend but rather samples oscillate up and down when taken hourly with an overall trend depending on the cycle type that is occurring.

Ovarian Functional Variance
As a clinical report I have observed on two occasions now regarding the over-riding effects of functional cysts and their effects on the progesterone levels on which we base all our decisions.

Vaginal cytology was still cornified and I have never observed such a sudden jump. Analyser function was considered normal as no such extreme values were evident with eighteen additional samples processed that day. I decide to do an exploratory to assess the ovaries with the view to a possible surgical implant. Both ovaries contained large 4.5cm cysts which were drained and ruptured at the time of surgery. Some evidence of ovulation was present on the surface of the ovary and on that basis a surgical implant was completed with frozen semen from a locally available sire.

A natural mating with the same sire was undertaken and the bitch stood solidly with tail arching. Five weeks later at least 8 pups were scanned and she finally whelped 9 healthy puppies.

Such an observation has widespread implications on the progesterone values we receive and base all our decisions. The reduction in progesterone after surgery and rupturing the ovarian cysts would suggest that in this case these cysts were functional and giving a false reading relative to the true underlying progesterone values on which so many implant decisions are made. Although surgical implants are often criticized as being overly invasive and unnecessary, I would suggest that it is good medicine to have the ability to examine the ovaries at the time of surgical implantation to assess ovarian integrity +/- the presence of cysts. This is discussed in more detail below.

We must ask ourselves in how many cases are cysts present and functional giving a progesterone value higher than it would have been if no such cysts were present. By examining the ovaries at the time of implant is the only empirical way to assess any complicating physiology stemming from the ovaries that may reduce the chances of a successful mating by acting as another variance to a true progesterone reading.

Ovarian Bursa – Venous Cascade
Of particular interest is the small level of variance in progesterone that we base our implant decisions on. Research at Monash University (Melbourne) has found progesterone levels in pre-ovulatory follicles of 7,850ng/ml (Dr S Metcalfe pers. comm.), Ovulatory progesterone levels are only 0.15% of this and it the slight change from 10ng/ml to 15ng/ml that we base our decision to implant is only .064% of the progesterone level bathing the ovarian follicle.

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Implantation considerations

The surgical implant is a safe and short procedure with rapid recovery after a gaseous anaesthetic. Propofol is used for induction following sedation with Xylazine & Atropine. Gaseous anaesthesia is then maintained after intubation, with Isoflurane (2%). We prefer the midline approach as it affords greater comfort for the patient than a flank incision.

The incision is made just behind the umbilicus allowing easier access to the ovaries for identification and assessment of SES or follicular cysts. Care must me taken to access the linea alba as fibrosis reactions are reduced should implants at subsequent seasons be required.

Pelleted semen is thawed in a WhirlPac® bag suspended in a 37.5°C waterbath for 5 minutes. It is checked for motility and introduced into the uterine body by two 22G x 1.25” catheters. A triple mattress closure completes the procedure and recovery to sternal recumbency occurs within 15 minutes.

The literature often reports that frozen semen survives for 24 hours once thawed. Thawed pelleted semen processed by the Camelot system has been regularly observed in our laboratory to have motility for 2 to 3 days when held at room temperature in a test tube. Its effective survival within the bitch would be expected to be at least this and as such the window of opportunity is increased for a successful conception.

With natural mating sperm may survive in the tract for up to 5 or 6 days. Fertilization may occur up to 7 or 8 days after semen deposition. Some sires with good semen motility but compromised quality may have survival times of only 1 or 2 days in the female tract and if insemination occurs before the LH surge - conception rates will be reduced due to compromised longevity.

Ovarian Cystic Structures – Significance
Bitches that fail to cycle or have one or two missed conceptions are usually treated with a surgical procedure wherein the ovarian bursa is opened and recent ovarian activity is viewed and ovarian parenchyma stimulated with needle fenestration. Additionally retrograde flushing with Lincomix® - Lincomycin 300mg/ml – 1.5ml each side – determines oviduct patency. There is no need to suture the ovarian bursa. At the time of examination any ovarian cystic structures are identified and removed.

It has become routine at this facility during a surgical implant to extend the incision anteriorally and examine the ovaries on each occasion. We find a high incidence of ovarian cysts, particularly in our greyhound surgical implants, and many of these are “problem” bitches that may have failed to conceive once or twice already. Some of these individuals may have undergone an “Ovary flush” procedure and the ovarian cyst has regrown.

The results of draining and removing these cysts at the time of implant are remarkable and subsequent conception rates for these difficult breeders are in the mid 90’s. Although the general literature says these structures are of little or no significance, our results subsequent to employing this procedure at the time of implant have been encouraging to employ it as a “must do” for every implant. We are in the process of defining the cystic fluid with oestrogen and progesterone assays and will have a better picture of trends within the next six months.

As this is more commonly seen in greyhounds we have to consider if anabolic steroid administration may have played a role in their formation. Apart from ovarian tumours with cystic components, ovarian cysts are defined in three main groups.

• Cystic Subepithelial Structures (SES) – infoldings of the epithelial cells that cover the surface of the ovary. These cells proliferate and are often associated with cystic distention. Clinically seen as grape like clusters on the surface of the ovary with transparent walls and containing a clear fluid.
• Cystic Rete Ovarii – within the ovary stroma near the base of the ovary.
• Follicular cysts – are grossly larger than normal follicles (normal follicle 8mm in the bitch) and contain clear fluid. These cysts are lined with tan / orange tissue and are usually grouped in multiples.

Both Follicular cysts and SES have been treated at the time of implantation and resultant conception rates have been excellent even in difficult bitches who had prior multiple missed conceptions. The clinical effect is direct and repeatable yet the precise endocrine basis of success is not empirically defined.

After the Implant?
Success with frozen semen doesn’t end with the implant itself. There is a need to monitor bitches regularly to identify potential re-absorption individuals often indicated by past reproduction failures. The bitch ovulates many more eggs than are ultimately implanted. Blastocysts enter the uterus at about days 9 – 11 with trophoblast attachment occurring about day 18 and implantation approximately day 20 after the LH surge.

Progesterone is essential for implantation and maintaining pregnancy. It is secreted from the corpus luteum which is in effect an ephemeral endocrine gland arising from the rapid proliferation and differentiation of both granulosa and theca cells within the ovary. Serum progesterone levels rise with some variance after ovulation, peaking at 15 to 90ng/ml by 10 to 25 days after the LH peak and gradually reducing over the term of pregnancy. Progesterone falls abruptly to less than 1ng/ml on the day preceeding or the day of whelping. (2)

Clinically, with the use of ultrasound, we have observed many cases of viable and active foetal units at 3.5 weeks, even as late as 6 weeks gestation only to find no pups present at full term (63 days). There is no correlation difference between natural mating, AI, frozen implant or chilled semen and infectious causes of foetal loss are usually not evident in these cases. We were concerned that these cases of pregnancy failure may be due to hypoluteoidism – the premature failure of progesterone production by the corpus lutea with resultant decrease in serum progesterone and pregnancy failure.

Several syndromes seem to exist with hypoluteoidism. A familial tendency may exist. We have observed a bull terrier breed line, prone to such re-absorption, with the drop in progesterone occurring early in the pregnancy or rather never achieving a very high level. One breeding with a bitch from this line revealed a progesterone of 22.4ng/ml 14 days after mating. This dropped to 12.3ng/ml 25 days after mating and five viable fetuses were present. Therapy with hydroxyprogesterone hexonate (depo progesterone 100mg/ml) every two weeks at a dose of 8mg/kg IM was effective in maintaining pregnancy to full term in this individual. The last dose was given 10 days before whelping and progesterone levels were monitored every three days after this last injection.

Another scenario observed relates to the maintenance of a normal progesterone curve throughout pregnancy followed by a precipitous drop in progesterone earlier than expected. This occurs in affected individuals typically at about day 52 – 54, compromising foetal viability. This may account for many still born pups or litters and the need for progesterone monitoring to maximize the chances of a successful conception is becoming a viable option in those valuable litters that all care must be exercised to ensure a successful outcome.

As a guide the circumstances where progesterone investigation or supplementation is considered include

• Previous history of reproduction failure – no pups, high stillborn % or dead litter
• Simply good practice to identify a problem before it occurs
• In the first 8 weeks of pregnancy draw samples weekly
• Between days 7 to 56 post ovulation progesterone should ideally be above 15ng/ml
• Between days 57 to 60 post ovulation progesterone should be above 5ng/ml
• If outside these ranges samples may be drawn every 2 – 3 days to define a falling trend

It is good practice to recommend to clients that progesterone checks be carried out throughout pregnancy to identify potential problems and institute appropriate therapy.

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References

1. Bjurnstrom L, Linde-Forsberg C, Effects of Ampicillin and Trimethoprimsulfamethoxazole on the vaginal bacterial flora of bitches Am J Vet Res 54; Aug 1993.
2. Smith MS, McDonald LE, Serum levels of luteinizing hormone and progesterone during the oestrus cycle, pseudopregnancy and pregnancy in the dog. Endocrinology 94; 404 – 412 1974
3. Root Kustritz MV, Use of Commercial Luteinizing Hormone and Progesterone Assay Kits in Canine Breeding Management. Recent Advances in Small Animal Reproduction, Recent Advances in Small Animal Reproduction; Concannon PW, England G, Verstegen J (Eds.) Pub. IVIS, Ithaca, New York, USA.

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Dr. John F. Newell B.V.Sc	Phone: 02 4375 1001
Tony Wiseman Crt. Camelot.F.S.	Fax: 02 4375 1010