Acta Veterinaria Scandinavica 2008, 50:41 http://www.actavetscand.com/content/50/1/41 Staining procedures The sample slides were stained for 40 min with Harris' haematoxylin technique [28], and were permanently mounted before the sperm head was measured. Head measurement The slide was loaded into an IVOS version 12.3 micros- copy system (Hamilton Thorne Research, Beverly, MA, USA) with the aid of a computer-controlled specimen stage. The images were evaluated using commercial mor- phology software (Oval Metrix Version 4.18). Recognition of sperm and the rejection of other cells were performed at an accuracy consistent with the hardware and software specifications. The analysis software settings were mini- mum contrast 15, minimum size 1 μm2, erosion level 7.0, camera gain 50, camera contrast 180, and scale 0.147 μm/ px. The manufacturer-recommended objective magnifica- tion for equine sperm microscopy was 60×. 200 morpho- logically normal sperm heads were acquired in each test, and consequently a total of 800 sperm were analyzed for each animal. The software reported five sperm head fea- tures, namely length (L; μm), width (W; μm), elongation [(width/length) × 100; %], perimeter (P; μm) and head area (A; μm2). In addition, the software calculated four non-dimensional derived parameters, namely ellipticity (e) = (L - W)/(L + W); shape factor 1 (Sf1; rugosity) = 4πA/ P2; shape factor 2 (Sf2) = Sf1 × (L/W) and shape factor 3 (Sf3; regularity) = π L/W/4A [18]. Statistical Analyses Statistical comparisons were made using the SPSS/PC+ statistics package (version 12.0 for Windows, SPSS Inc, Chicago, IL, USA). For each morphometric parameter, the normality and homogeneity of the data's variance distri- bution were assessed using the Kolmogorov-Smirnov and Levene's tests. One-way ANOVA producing significant F- values was followed by an LSD test for comparisons between multiple animals. An independent-samples T test was used for comparisons between groups of animals. All data given were summarized as mean ± standard error of the mean (SE). The coefficient of variation (CV) was cal- culated for both within-animal and between-animal groups [18]. Results The color and aspect of the ejaculates ranged from milky white to opalescent white. For T stallions, the mean ± SE of gel free-volume, motility, progressive motility, living sperm and concentration were 44.0 ± 2.1 ml, 77.8 ± 1.3%, 55.4 ± 1.3%, 75.5 ± 1.3%, 309.0 ± 30.7 × 106sperm, respectively. For the F group, the mean ± SE of gel free-vol- ume, motility, progressive motility, live sperm and con- centration were 47.0 ± 3.2 ml, 73.0 ± 2.0%, 46.8 ± 1.7%, 73.9 ± 1.6%, 374.5 ± 28.4 × 106 sperm, respectively. Sperm Morphology Morphology measurements from the individual ejaculate samples of T and F stallions are presented in Table 1. Sperm morphology varied among stallions with respect to all parameters. On average, the T and F groups were not significantly different (P > 0.05) in respect of percentage of sperm that exhibited normal morphology. However, the percentages of each type of morphologically abnormal sperm were significantly different (P < 0.05). Overall, the most common abnormality in both T and F stallions com- prised sperm that had an abnormal midpiece. Morpho- logically normal and abnormal sperm from the T group are shown in Figure 1 (detected by scanning electron microscopy) and Figure 2 (stained with eosin/nigrosin and detected by light microscopy). The mean of numbers of morphologically normal sperm with progressive motility in each ejaculate ranged from 1.83 ± 0.37 to 4.68 ± 0.47 billion and 3.18 ± 0.53 to 5.41 ± 1.61 billion in T and F stallions, respectively. Sperm Head Morphometry Parameters for the morphometric characteristics of nor- mal sperm heads are summarized in Table 2. There were differences (P < 0.05) between individual stallions in both T and F. Comparisons between the mean values of each characteristic of normal T and F sperm heads showed that the length, elongation, perimeter and area values were higher for T than for F (P < 0.05). Thus, this indicated that the sperm heads of T stallions were rounder and larger than those of F stallions were. The percentage CV values of normal sperm head morpho- metric characteristics were quite low, ranging from 3.3 (shape factor 1 and perimeter) to 8.5 (elongation) and 3.4 (shape factor 1) to 8.8 (elongation) in T and F stallions, respectively (Table 2). Within-stallion group analysis indicated that the CV values in both T and F sperm were also low (Table 3), while analysis of the between-animal groups found that the percentage CVs were quite high for perimeter (19.2), area (19.8), length (28.9), shape factor 1 (25.8) and shape factor 3 (32.0) for T stallions, but were only high in the case of shape factor 1 (26.1) for F sperm. The sperm head parameters with a low within-animal CV and a high between-animal CV were perimeter (2.9, 19.1), shape factor 1 (2.6, 25.8) and shape factor 3 (3.8, 32.0) for T. The latter characteristics were observed only in the shape factor 1 variable (2.9, 26.1) for F sperm. Discussion The percentages of each type of sperm morphology were variable across both T stallions and F stallions. Inter-ani- mal variation was found both within breeds [29] and between breeds [29-31]. The overall percentage of mor- phologically normal sperm was 49.7% and 48.1% for T Page 3 of 9 (page number not for citation purposes)