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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)
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.