Spermatocrit as a measure of the concentration of spermatozoa in canine semen.

HISTORICALLY, practitioners have determined the concentration of spermatozoa in canine ejaculates using the Unopette microcollection system (Becton Dickinson) and a haemocytometer (Root and Johnston 1994). Other techniques described include the use of computer automated semen analysis (CASA) equipment, automated cell counters and spectophotometry. These techniques are not used commonly by practitioners to evaluate canine semen, which is naturally more dilute than is the semen of other species. In addition, CASA systems have been shown to be less accurate for determination of the total number of spermatozoa in the ejaculate than assessment using a haemocytometer (Kuster 2005). Many practitioners no longer use Unopettes and haemocytometers for blood testing, and so do not have this equipment to hand should they be required to evaluate semen. Spermatocrit, in which the percentage of solids in semen is determined by centrifugation of a semen sample drawn up into a microhaematocrit tube, is a technique that has been used for experimental work and clinical management of farmed fish (Winnicki and Tomasik 1976, Clearwater and Crim 1998, Rakitin and others 1999, Tvedt and others 2001, Park and others 2002). In fish, the relationship between sperm density and spermatocrit is linear, described by the equation Y = 3·68X – 2·718, where Y is spermatocrit and X is sperm density (Park and others 2002). Spermatocrit also has been described for evaluation of the concentration of spermatozoa or sperm density in poultry and mammals (Jones 1980, Pholpramool and others 1982, Wen and Wong 1988, Stewart and others 1998, Haenlein and others 1999, Siopes 1999, Lagares and others 2000, Al-Daraji 2001, Arabi 2004). This short communication describes the use of spermatocrit to evaluate samples of semen from dogs. Attempts were made to collect semen from 44 dogs by manual ejaculation in the absence of a teaser bitch. The dogs were all client-owned. Their average age was 2·9 years (range seven months to seven years). Twenty-four breeds were represented, of which the two most common were Samoyeds (n=11) and Nova Scotia duck tolling retrievers (n=6). The total number of spermatozoa in one sample was assessed using a standard haemocytometer technique (Root and Johnston 1994). The sample was split into aliquots containing concentrations of 0, 52 x 106, 104 x 106, 209 x 106 and 417 x 106 spermatozoa/ml, reflecting the normal range of concentration of spermatozoa in canine semen, reported by Johnston and others (2001) to be from 4 x 106 to 400 x 106 spermatozoa/ml (Johnston and others 2001). Five microhaem atocrit tubes in each of two sizes (internal diameter 0·53 mm and 1·1 to 1·2 mm) were filled with the samples and centrifuged for one minute. The percentage solids was read using the automated reader on the microhaematocrit centrifuge (HemataStat-II; Separation Technologies). The five percentages measured for each concentration were averaged. A standard spermatocrit scale was created by plotting the average percentage solids at each known concentration, determining the line of best fit for the data, and calculating the formula for that line for each size of haematocrit tube. From the remaining ejaculates, two microhaematocrit tubes of each diameter were filled, centrifuged for one minute, and the percentage solids were read using the automated reader as described above. The average of the values for each tube size was determined and the concentration in that sample was determined using the formula developed in the first phase of the study. The concentration of spermatozoa in each of the samples was also determined using the standard haemocytometer technique, and the two values were compared by analysis of variance (ANOVA). Ten of the samples contained no spermatozoa or were too small in volume for the appropriate number of haematocrit tubes to be filled, and so were not included in the study. The quality of the remaining semen samples was variable, with the total number of spermatozoa in the ejaculate averaging 547·4 x 106, with a range from 14 x 106 to 2·3 x 109. The percentage of morphologically normal spermatozoa averaged 72 per cent (range 21 to 96 per cent). The standard scale for the tubes with an internal diameter of 0·53 mm was described by the equation (concentration [x 106 ml] = [51·69][% solids] – 47·43), and that for tubes with an internal diameter of 1·1 to 1·2 mm was described by the equation (concentration [x 106/ml] = [113·1][% solids] – 262·6). The values for the remaining 33 samples determined by spermatocrit were compared with those determined with the haemocytometer by ANOVA and determined to be significantly different (P<0·001) for both sizes of haematocrit tube. In dogs, even semen samples that contain a relatively high concentration of spermatozoa will yield a low percentage total solids. For example, based on the spermatocrit equation generated for tubes with an internal diameter of 0·53 mm, if a semen sample contained 400 x 106 spermatozoa/ml, among the highest concentrations likely to be encountered in practice, the percentage solids in a haematocrit tube of this size would be only approximately 9 per cent. Difficulty in reading the percentage solids in the haematocrit tube at such low percentages precludes sufficient accuracy and repeatability to make spermatocrit a clinically meaningful measurement in dogs. While steps could be taken to try to enhance the accuracy of this technique, they would negate the original intent, which was to establish a simple way to evaluate the concentration of spermatozoa in canine ejaculates. On the basis of these results the authors conclude that determination of the concentration of spermatozoa with a haemocytometer is still the preferred technique for semen analysis in dogs.