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MICROSCOPES FOR VAGINAL SMEAR TESTS

Being able to perform and read vaginal smears and analysis sperm quality using a GT Vision Microscope has the potential to greatly improve your success rate in breeding.   The vaginal smear is not perfect but it is certainly better than guessing what day a bitch should be bred on.  One of the best methods available to determine when to breed a bitch is the vaginal smear.  The photos in this article are from a Purina publication.

In medical terms a bitch goes through 4 different stages:

  • Proestrus
  • Estrus
  • Metestrus or diestrus
  • Anestrus

Proestrus begins when the bitch swells and begins to drop blood. It typically can last from 4 to 20 days. During this period a bitch may attract males but will not accept them for breeding.  Estrus is the time a bitch can be bred. This can last from 4 to 13 days. Typical this is the 9th to the 12th day of a bitch's season. But I have seen bitches bred as late as the 26th day of season. Determining estrus is critical in determining when to breed (either naturally or with artificial insemination).  Metestrus is the period after estrus and can last 80 to 90 days. During this period the bitch will not accept a male.  This period represents the quiet phase of the bitch's estrus cycle. It lasts 2 to 3 months after metestrus. During this time period the female is not swollen and will not allow breeding.  Doing vaginal smears requires a good microscope.

PROESTRUS

Determining the first day of a bitch's season (that point where she starts to bleed) is important. If you have not bred a bitch before its good to know that most bitches will blow their coat just before coming into season. This can be anywhere from 3 weeks before she begins to bleed to the week of proestrus. I try and educate the foster parents of my bitches to watch for this sign. When they begin to see the bitches shedding, its time to do a visual inspection each morning before the dogs go out side.  The problem is that many females will clean themselves through licking for several days before the blood is actually noticed.  It's a good idea to establish a base line of slides during proestrus. By starting on the 5th or 6th day and then doing smears every day or every other day you will be able to notice significant changes in the cell structures.  During proestrus the slide has little dark dots and fried eggs. The dark dots are red blood cells. The fried eggs are the epithelial cells. During proestrus there are a lot of the red blood cells and the epithelial cells are nice and round with dark round centers. (See the photo below)

GT Vision ProEstrus Microscope

What we want to watch is the changes to the epithelial cells. As a bitch moves through proestrus there will be a reduced number of red blood cells and the epithelial cells will begin to look distorted. The centers will disappear and the edges of the cells will begin to get rough (the vets call these cells cornified epithelial cells.) This is when the bitch is approaching estrus. When you notice this change make daily slides.  During the early days of proestrus the female will initially not allow the male to mount her, but as she nears the end she will flag and allow the male to mount but she will not allow penetration. When I see this I know I am within a day or so of breeding the female.

GT Vision ProEstrus Microscope

ESTRUS

Estrus normally occurs 8 to 11 days after a bitch starts to discharge. It lasts 4 to 13 days and during this time the female can be bred. During this time a visual exam will indicate swelling of the vagina area and noticeably less blood in the discharge.  When the slides are examined there is a noticeable difference between proestrus and estrus. During estrus there are very few (if any) red blood cells (erythrocytes). There will be a dramatic increase in the cornified epithelial cells. They will often be stuck together in chunks (but this can vary because of the way that you manually roll the cells on to the slide.) You can see what this look like in the photo below.

GT Vision Estrus Microscope

With normal bitches this is the time to start to breed naturally or do an A.I. (artificial insemination). There is a lot of published information that indicated breeding 2 times 2 days apart is all that is necessary.

METESTRUS

During this period the bitch will quit flagging and will not breed anymore. There is a noticeable lack of interest in being friendly to males. This period represents the "leuteral" or regressive stage of a bitch's season.  Once again this period lasts 80 to 90 days. During the first 2 or 3 days there is a noticeable lack of cornified epithelial cells. During the first 3 or 4 weeks there is an increased number of leukocytes. As this period progresses you will begin to see non-cornified epithelial cells again (the fried eggs.)

ANESTRUS

This period lasts 2 or 3 months and during this time there are very few cells noticeable if a slide is taken

Ed Frawley

SPERM ANALYSIS

GT_Vision_Sperm_Count_Microscope

Motility

Sperm motility is essential for fertilization because it allows or at least facilitates passage of the sperm through the zona pellucida. Without technologic intervention, a non-motile or abnormally-motile sperm is not going to fertilize. Hence, assessing the fraction of a sperm population that is motile is perhaps the most widely-used measure of semen quality.

For canine semen, motility is better maintained if samples are kept at room temperature than at body temperature. Warmer temperatures increase sperm cell metabolism. Delay in evaluation of warmed samples may result in errors due to sperm cell energy depletion. Rapid temperature fluctuations should be avoided. Ambient temperature may affect the motility assessment of a sample if the evaluation room is excessively hot or cold. Percentage of progressively motile spermatozoa from a given dog is not affected by frequency of semen collection.

In evaluating motility, sperm cells are classified as immotile, progressively motile or non-progressively motile. Both total and progressive motility are determined and expressed as a percentage of 100. Sperm cells showing motility of any kind are included in the calculation of total motility. A progressively motile spermatozoan moves forward in an essentially straight line, whereas a non-progressively motile sperm cell moves, but with an abnormal path, such as in tight circles (tail chasing), fish flopping, and when normal movement is prevented by sperm head agglutination. In the dog, the normal percentage of progressively motile spermatozoa is 70% or greater. Speed or quality of motility also may be assessed; a canine spermatozoon with normal motility should traverse the microscopic field of view in 2–3 seconds.
Progressively motility is positively correlated with normal morphology in dogs. Just as is the case with the whole dog "on the hoof," think function follows form. If the sperm cell is put together normally, then it should move normally. Immotile sperm cells that are morphologically normal may have experienced "iatrogenic immotility" as a result of technical errors in semen collection or handling. Exposure to rapid or extreme temperature fluctuations, any kind of residue on collection equipment, or the wrong pH or osmolality of an extender can adversely affect motility. Motility is also affected by periods of sexual inactivity - males that have not ejaculated for prolonged periods often have poor motility on the first ejaculate, but much better motility for a second ejaculate collected soon thereafter. This is the so-called "rusty pipe" condition.

In humans, age is a factor in motility; motility has been documented to decline at rate of 0.27%/year in men > 45 years old. In protocols for humans, two samples of 200 spermatozoa are evaluated separately and graded as A (=rapid progressive motility), B (slow or sluggish motility), C non-progressive motility), and D (complete lack of motility). If results for the two samples don't "jive" (ie., the difference is greater than expected by statistical random variation), two additional slides are evaluated from that semen sample.

Manual motility estimates are easy to perform and require minimal equipment. This commonly-used technique involves placing a sample of semen on a microscope slide, examining it with a microscope and estimating the fraction of the population that is motile. Manual motility estimates are subjective. However, with an experienced evaluator, manual estimates generally provide good estimates of motility. The chief limitation of this technique is its subjective nature.

Computer assisted sperm analysis (CASA)

CASA is a technique employing a computerized system that tracks multiple motility parameters. The computer takes video images of the sperm and stores them for analysis. The system recognizes motile from non-motile sperm and other organic debris by comparing luminosity (gray-scale intensity) and size of the object. There are also preset user-defined thresholds for size and luminosity that help prevent mistaking other cells and debris for non-motile sperm. While some studies have shown that CASA systems are more accurate than subjective assessment of sperm motility, other studies have found that subjective analysis progressively motility is well correlated with computer-based analysis.

Most CASA systems are found in teaching hospitals and research laboratories. Using CASA, many more motility parameters can be evaluated, and be evaluated objectively:

     • Mean percentage of motile sperm 
     • Mean percentage of progressively motile sperm 
     • Curvilinear velocity 
     • Straight-line velocity 
     • Amplitude of lateral head displacement

Such parameters can be incorporated into detailed mathematical formulas to produce factors such as a Semen Quality Score or Sperm Motility Index. Normal values have not been established for the dog. Another potential value of these sperm motility factors is recognition of sub-populations of spermatozoa within the sample that may react differently to cryopreservation or other manipulations of spermatozoa.