Determining Canine Estrus Stage via Progesterone Testing & Vaginal Cytology for Good Canine Breeding Management Practices
When it comes to reproduction “timing” is everything to make sure that the sperm and eggs meet on time! We use lots of different indicators and tools besides just animal behavior to determine when is the “best” time. The measurement and charting of hormones is fast becoming one of our most reliable tools. There are multiple hormones that help to regulate the estrus (heat) cycle and pregnancy in dogs. These include:
- Estrogen:Stimulates the ovaries to produce eggs.
- Luteinizing Hormone (LH):Stimulates the ovaries to release the eggs.
- Progesterone:Maintains a pregnancy.
We need to fully understanding how the hormone levels change so they can help us in determining the best time to breed naturally or with artificial insemination or surgical implantation and also when to anticipate whelping or to do an elective C-section.
It seems that usually most mammals ovulate when the estrogen level in the blood is still increasing. Dogs, however, ovulate when the estrogen level is already declining and the progesterone level is increasing. Estrogen levels can give us a general idea of when a dog will come into “standing” heat, but are not sufficient to determine when breeding should actually take place for optimal conception rates.
Vaginal cytology can also provide some general information. Vaginal cytology is performed on female dogs during their estrus cycle to help determine the optimum time to breed and enhance the success of pregnancy. The types of cells present in the vagina vary depending on the stage of estrus. A swab is taken of the cells lining the vagina and analyzed under a microscope. Usually more than one swab is required to determine the optimum time to breed.
Progesterone levels and luteinizing hormone (LH) levels are the best indicators of when ovulation will take place and when is the best time to breed. They are also useful in determining whelping dates, allowing an owner to reserve the appropriate days on the calendar and even to schedule a C-section (Cesarean) weeks in advance.
The progesterone test is not species specific so the test can be run in in clinic, in human labs or veterinary labs. Results should be available in less than 24 hours to keep things on time. Different labs have different tests and report result in different measurement units. To switch between the American units and the International units multiply by 3.18.
These are the International numbers that we use from our IDEXX laboratory: The normal progesterone ranges are as follows:
Anestrus (not in heat) is less than 3 nmol/L
Late Proestrus (early heat) is 6-8 nmol/L
LH surge is 8-12 nmol/L
Ovulation is 12-24 nmol/L
Past Ovulation is greater than 24 nmol/L
The progesterone test can be done every 2-3 days starting about 3-5 days into the heat. Timing of the test can be more certain if the lengths of the dog’s previous heat cycles are known. The beginning progesterone levels are typically less than 1.0 ng/ml until the day before the LH surge. The day of the LH spike, serum progesterone concentrations are 2-3 ng/ml; the day following the LH surge, the serum progesterone concentration is 3-4 ng/ml. Ovulation occurs at a progesterone level of 5 ng/ml.
Vaginal Cytology
A vaginal cytology is an evaluation commonly done. For many years, this was our only practical tool with which to time breedings.
The cells seen at the ideal time for breeding are a high percentage of cornified epithelial cells, with few red blood cells (RBC’s), white blood cells (WBC’s) or debris. The vaginal smear may also indicate the presence of other problems such as vaginitis. The test is simple, safe, inexpensive, well tolerated by the bitch approaching estrus, and very helpful in timing breeds.
Examination of a single smear can provide useful information, but can also be misleading. For example, it is often difficult to differentiate proesterus and disestrus from an isolated smear. It is most useful to evaluate multiple smears taken from the same bitch. These slides can be labeled with the date collected and stored for evaluation sequentially as she moves through her estrus cycle to monitor trends in cellular cornification.
Techniques in vaginal smears
The preferred method to obtain a good vaginal cytology, is the application of a cotton swab in the cranial vagina of a bitch . The lip of the vulva are gently separated with the fingers and a cotton swab is passed into the dorsal aspect of the vulva. We must avoid clitoris fossa and advance in a craniodorsal direction, toward vertebral column. At this point we rotate the cotton swab and withdraw it. We must observe the color, and type of secretions and rotate gently the cotton tip in a slide. Usually three or four slides are fixed every time, with the name and date. A lot of stains has been described, but usually Diff-Quick, new methylene blue and Schor’s stain modification (with criteria for keratinization) are used.
In the queen the cotton swab must be of adequate size (urethral human culturette tube) and introduced only 1 to 1,5 cm in the vagina. The main differences between queen and bitch is the relatively straight horizontal anatomy of the vestibule of the vagina, the vagina, and the cervix. Usually when we introduce the cotton swab we are inducing the ovulation of queen (cervico-hypothalamus reflex) with the subsequent external behaviour aspect (vocalization and others).
Type of cells that we can see in a vaginal cytology
We can classify the cells in keratinized versus nonkeratinized or cornified versus noncornified. The cornification or keratinization is a reflex of circulating estrogens. The basal cells (near blood supply) are small, round and with a clear nucleus, but in the cornified cells (with no blood supply) the nucleus become smaller, pyknotic and finally is disintegrated leaving an anuclear cell.
We will describe the principal characteristics of different types of cells:
Parabasal cells: small, round, slightly oval, large vesiculated nucleus and small cytoplasm. Good staining.
Intermediate cells: slightly larger than parabasal cells to twice that size. Smooth, oval to rounded irregular borders, nucleus smaller than in parabasal cells. More cytoplasm than parabasal cells.
Superficial cells: Are dead cells, are the largest in vaginal cytology with a sharp, flat, angular cytoplasmic borders and a small pyknotic, fading nuclei or without nuclei.
Anuclear squames: irregular vaginal cells, no nucleus, smaller than superficial. These are the cells that have also been called “fully cornified.”
Metestrus cells: Large intermediate vaginal cells that appear to have one or more neutrophils in their cytoplasm. Are typical of bitch diestrus.
Foam cells: parabasal and intermediate cells with obvious cytoplasmic vacuoles. May be associated with diestrus and anestrus.
Neutrophils: Inflammatory cells that can be normal or abnormal in vaginal cytology in function of the estrus period
Red blood cells: Blood can be normal in the bitch but always is abnormal in the queen.
Correlation between ovarian cycle and vaginal cytology
In the bitch
Dogs has two ovarian cycles twice yearly, in spring and again in autumn, however some bitches experience more or less than two cycles a year. These are in function of breed, climate, and others.
The bitch ovarian cycle is divided in four phases: proestrus, estrus, diestrus and anestrus. We can correlate this phases with vaginal cytology.
Proestrus: Period of high follicular activity. The female attracts the males but is not receptive to the copula, the vagina has a bloody discharge and is edematous. The duration of this phase is usually from 6 to 11 days, but the average can be from 2-3 days to 25 days. The vaginal cytology is typical of estrogens secreted by developing ovarian follicles with RBC, numerous parabasal, small and large intermediate vaginal cells, neutrophils are common, bacteria, and a dirty background in the early proestrus and with no neutrophils, less blood cells, more than 80% of superficial cells and a clear background in the late proestrus. This late proestrus is difficult to distinguish from early estrus.
Estrus: In this phase the bitch allows the mating. Estrus starts with this allowing and ends when she no longer accepts the male. Another criterion to define the finish of estrus is the vaginal cytology start of diestrus. Estrogen concentrations reach a peak 1 or 2 days prior to the onset of estrus, and during estrus starts the decline in circulating estrogens and the start in progesterone secretion by luteinized follicular cells first and corpus luteum after. The vaginal cytology is relatively constant with superficial cells and anuclear squames accounting more than 80% of the total vaginal cells and often reaching 100%. No neutrophils must be present and red blood cells are not usually presents.
Diestrus: Is the phase with predominance of progesterone secretion with or without pregnancy. Vaginal cytology is characteristic in this phase with a dramatic “shift” form a estrus with a 80-100% superficial cells to one of 80-100% of parabasal and intermediate cells plus neutrophils and metestrus cells. This change is produced in about 24-48 hours.
Anestrus: Is defined as the period of sexual arrest. The uterus involution is produced and start with whelping and ends with a new proestrus. In non pregnant bitches the beginning of anestrus is not readily easy to distinguish clinically with no obvious demarcation between diestrus and anestrus.
In the queen
Queen reach the sexual maturity after she attains at least 80% of adult body weight and if the photoperiod is appropriate (usually between 6 and 9 months of age). By definition the cat is seasonally polyestrous. She cycles repeatedly throughout a breeding season unless the cycle is interrupted by pregnancy, ovulation, illness or pseudogestation. This is a important difference with the bitch, the influence of photoperiod and hours of daylight.
The phases of the queen sexual cycle are the same that in the bitch, but with some differences. Usually we aggregate proestrus and estrus, because they are difficulty distinguished. Another characteristic of the queen is that she has a interestrual period, between estrus, in the adequate photoperiod season with a typical changes in the vaginal cytology. A typical cycle of a queen is: anestrus-follicular phase (proestrus + estrus)-interestrus-follicular phase ……. – anestrus
Another important characteristic in the physiology of the queen, is that is a ovulation induced female. When the tom introduce the penis in the queen vagina, he induce the ovulation reflex. In some occasions a female can ovulate without copula.
Proestrus: Is the period of follicular function after anestrus or interestrus period, estrogen synthesis, secretion and changes in behaviour and vaginal cytology. The behavioral changes are rubbing, vocalizing, lordosis, and rolling.
Estrus: Is associated with follicular estrogen synthesis and secretion. Usually is associated with proestrus as a “follicular phase.”
Vaginal cytology in “follicular phase”: clear background, without cellular debris and easy visualization of vaginal cells. The squames increase to 10% of the total on the firsts days reaching 40% at seven days, and then slowing again to 10%. In this phase the intermediate cells decrease from 40-50% in the interestrus period to 10% during follicular phase. No parabasal cells, no RBC, and no PMNN are typical of this phase.
Interestrus: follows cessation of follicular activity and the suppression of estrogens secretion. In this phase the behaviour is normal for the queen without rubbing or vocalization. The vaginal cytology is really curious and has a lot of typical characteristics: we can observe all types of vaginal cytology cells, intermediate, squames, parabasal and superficial and again we can see debris in the background.
Diestrus: As in the bitch is the phase with progesterone dominance. The corpus luteum produce progesterone after ovulation (induced ovulation in the queen).
Anestrus: Again, anestrus is defined as a period of clinical reproductive quiescence. The female do not attract males and not display sexual behaviour. In the vaginal cytology we have low cellularity with all possible types of cells and a dirty background with debris.
Clinical usefulness of vaginal cytology
We will see with practical cases the normal and possible applications of vaginal cytology as:
Management of normal breeding
Shipping or receiving a bitch
Study of unusual cycles
Predicting whelping dates
Infertility problems
Diagnosis of vaginal problems, follicular problems and uterine diseases
Application of reproductive drugs: abortifacients
Limitations of vaginal cytology
Vaginal cytology doesn’t permit gestation diagnosis, doesn’t permit identify ovulation day, and is just a complement of a good clinical examination, anamnesis and hormone assays.
Progesterone Testing
A quantitative progesterone blood test is the single most accurate method for timing breedings. The test is reported as a numeric result, usually ng/dl. Combined with observing the behavor of the dog and bitch, vaginal cytology, ovulation can be pinpointed with great accuracy in most bitches.
An exact progesterone level is also needed when the dog or bitch will be traveling a long distance for breeding, when fresh chilled or fresh semen is to be used, or when breeding dogs or bitches that have a history of being difficult to breed. In addition, an exact progesterone level for timing ovulation is necessary when a C-section is anticipated or when the bitch is near term and fetal survivablility needs to be assessed.
Progesterone Interpretation
The canine’s reproductive system is unlike any other species. This is probably the most confusing part of canine reproduction. Many breeders and many veterinarians do not understand the cycle well until they see it graphed. Once you understand that progesterone rises slowly and stays high throughout the cycle.
At the start of the strous cycle, proestrus, the estrogen levels are rising. The hormone of interest, progesterone, of the bitch is at avery low level, less than 2ng/ml. This is often reffered to as “baseline”. During proestrus in a normal bitch that has started estrus spontaneously (without drug or hormone intervention), the first progesterone level should be run on day 5 to day 6 of her cycle.
As the bitch enters estrus, she approaches her fertile period and her progesterone level will rise above 2ng/ml. She will become increasingly attractive to the male and receptive to his advances. This slight rise is often called the initial rise. Its only significance is that you will want to monitor her progesterone levels more closely now as she is about to ovulate. Bitches are only fertile for a few short days during estrus, not the entire time they are in estrus.
As the progesterone rises above 2 ng/ml, continue to draw serial serum samples for testing, usually every 1 to 3 days.
Ovulation is though to occur when the progesterone level reaches 5 ng/ml. This may be the single most important concert regarding breeding in this book. The bitch’s ovary responds to the LH from the pituitary, which allows the release of one egg from each of many multiple mature follicles.
But stop: it is too early to breed if you are using fresh chilled or frozen semen. When the bitch ovulates, the eggs are not yet mature and ready to fertilize. Unlike in other species, the eggs mature over the next 48 hours before they are ready to fertilize. The timing of the breeding must occur when the eggs are mature and ready to fertilize and viable semen is in the oviduct, estimating how long the semen is anticipated to survive in the reproductive tract.
Breeding with fresh semen, by either natural breeding or vaginal AI with fresh semen, can be done on the day of ovulation, when the progesterone level reaches 5 ng/ml. Although the eggs are not mature yet, most fresh semen is viable enough to survive in the bitch’s reproductive tract until the eggs are ready.
Breeding with fresh chilled semen should be delayed until approximately 48 hours after ovulation. Fresh chilled semen usually will not survive as long in the reproductive tract as fresh semen. If the fresh semen is deposited vaginally or directly into the uterus 2 days post-ovulation, the eggs should be fertile when the semen appears in the oviduct.
Breeding with frozen semen should be delayed even longer, 60 to 80 hours after ovulation (Ovulation occurs when the progesterone is at a 5 ng/ml). It is thought most frozen semen will only live 12 to 24 hours (or less) in the reproductive tract. For this reason, the semen should arrive in the oviduct when the eggs are mature and ready to fertilize. If necessary to choose, it is better to breed slightly too late than slightly too early. most breedings with frozen semen are done by depositing the semen directly into the uterus, usually by surgical insemination but in some cases by transcervical insemination.
Once the progesterone level is 5 ng/ml we are still not done testing. Although it is believed that ovulation occurs around the time the bitch’s progesterone reaches 5 ng/ml, we still need to keep our eye on one more number; that number is a progesterone level of a 20 ng/ml. To assure that ovulation is complete and the progesterone level is high enough to maintain a pregnancy, delay either the final or the surgical insemination until the progesterone level has reached or exceeded 20 ng/ml.
After ovulation and breeding are completed, the progesterone level will continue to rise. The level typically rises to 40 to 50 ng/ml (the normal range can be 10 to 90) whether the bitch is bred, pregnant, or not. An elevated progesterone level only indicates that the corpus lutea in the ovaries can support a pregnancy, not that the bitch is pregnant. Bitches maintain this level unless that have ovarian or uterine pathology.
Timing of breeding
The aim is to identify when the progesterone level reaches 2.5 ng/ml so the mating schedule can be set up, or the veterinarian and owner of the male dog can be notified that they should be prepared to collect and ship a semen sample. Depending upon the type of semen used, optimal times for natural or artificial insemination are:
- Natural breedingshould occur 3 days after the 2.5 ng/ml mark. Sperm in fresh semen survive 5-7 days after insemination.
- Artificial insemination using fresh chilled semenshould be used for a 1-time breeding. Insemination should take place 4 days after the progesterone reaches the 2.5 ng/ml mark or 48 hours after the 5 ng/ml mark. Sperm in chilled semen survive 48-72 hours after insemination. With artificial insemination, the semen should be deposited into the cervix to increase the chance of it being drawn into the uterus.
- Artificial insemination using frozen semenshould be performed 5 days following the 2.5 ng/ml mark or 72 hours after the 5 ng/ml mark. Sperm in frozen semen survives less than 24 hours after insemination. Frozen semen is ideally deposited directly into the uterus through surgery to increase the chance of pregnancy.
Breeding Management of the Bitch
The canine estrous cycle consists of 4 phases: proestrus, estrus, diestrus and anestrus. Proestrus and estrus are commonly called “heat” or “season”. During proestrus, the start of the estrous cycle, the bitch attracts male dogs, but is still not receptive to breeding. She may become more playful and passive as proestrus continues. A blood tinged vaginal discharge (of uterine origin) is present and the vulva is moderately enlarged and turgid. The cells from vaginal cytology smears change over a period of 4 to 7 days from non-cornified (small “parabasal” cells, small and large “intermediate” cells) to cornified cells (“superficial-intermediate” cells, and “anuclear” cells). These changes in the vaginal cytology reflect the increasing estrogen from the ovarian follicles. Red blood cells are usually, but not invariably, present. Proestrus can last from 3 days to 3 weeks, with 9 days being the average. Proestrus progresses to estrus.
During estrus, the normal bitch displays receptive or passive behavior, enabling breeding. Vaginal discharge normally diminishes. Vulvar edema tends to be maximal and the vulva is flaccid. Vaginal cytology during estrus consists of 80 to 100 percent cornified (“superficial” and “anuclear”) cells. Red blood cells tend to diminish, but sometimes persist throughout estrus. Estrus can last 3 days to 3 weeks, with 9 days being the average. Receptive behavior begins when estrogen concentrations decline and progesterone concentrations increase. The duration of receptivity to male dogs is variable, and may not coincide precisely with the fertile period, which occurs during estrus. Ovulation is triggered by a surge in luteinizing hormone (LH) from the pituitary gland in the brain. Ovulation of immature, infertile primary oocytes (eggs) begins approximately 2 days after the LH surge and oocyte maturation occurs over the following 1 to 3 days. The life span of the secondary (fertile) oocytes is 2 to 3 days. Thus, the bitch’s actual fertile period extends from 3 through 6 to 7 days after the LH surge. The LH surge occurs at the same time as an initial increase in progesterone concentration, enabling ovulation timing by measurement of either hormone.
Ovulation timing should be performed using a combination of serial vaginal cytologic exams and ideally, serum progesterone concentrations. Testing for LH can be used for some cases (infertility, frozen breedings). Vaginal cytology exams are started during the first few days of proestrus and performed every 2 to 3 days. When >70% of the epithelial cells are cornified (“superficial” cells), serum progesterone concentrations are obtained every 48 hours to detect the day of the initial progesterone rise (usually between 2-3 ng/ml), which correlates with the LH surge triggering ovulation. That day is called “day zero”. The bitch is most fertile, and can be bred with good conception rates, between 2 and 7 days after “day zero”. The number of breedings and specific day(s) of breedings depends on the type of semen (fresh, chilled/extended, or frozen). If LH testing is used to provide the most precise ovulation timing, daily serum samples must be acquired for testing once the vaginal cytology contains >70% “superficial” cells. The initial rise in progesterone or the occurrence of the LH surge is confirmed 48 hours later by running an additional progesterone test. To economize ovulation timing, daily serum samples can be saved (refrigerated or frozen) and selected for later LH testing based on the estimated initial rise in progesterone.
A Dog’s Heat Cycle
There are four stages to a dog’s heat cycle.
Stage 1: Proestrus
This is the first stage of your dog’s heat cycle. It will usually last around nine days, but there can be a lot of variation between dogs. Typical signs are vulva swelling, bloody discharge, behaviour changes, and potential changes in eating habits.
Stage 2: Estrus
This is the second stage of your dog’s heat cycle, which typically will happen from day 10 up to 28 days within her cycle.
Usually, the girl’s blood will start to thin down at this stage, it becomes more of a pinky straw discharge type. Her vulva may start to swell down a little bit.
This is her optimum fertility window and the ideal time to mate.
Stage 3: Diestrus
The third stage in your dog’s heat cycle is when she is beginning to decline in her fertility.
Stage 4: Anestrus
Your dog is now in a rest phase before gearing up for her next season. This stage lasts approx four months, depending on your individual dog.
IMAGE-CREDIT-GOOGLE
REFERENCE-ON REQUEST
DR UDAY KUMAR, CANINE SPECIALIST, HAZIPUR,BIHAR
