Acta Veterinaria Scandinavica 2008, 50:41 http://www.actavetscand.com/content/50/1/41 Background Recent studies have shown that male fertility does not only depend on the absolute number of viable, motile, morphologically normal sperm that can be inseminated in a female. Rather, a more important parameter appears to be the functional competence of sperm cells – since this cannot be evaluated using a single variable, researchers have proposed that semen samples should be subjected to multi-parametric analysis [1-3]. However, gross morpho- logical classification of the sperm in order to assess male fertility can be used as a practical screening tool and is already a part of the breeding soundness examination (BSE) that is used in Thailand for all domestic species, including horses [4]. An ejaculation containing a mini- mum of one billion morphologically normal, progres- sively motile sperm specimens in each of two ejaculates sampled at any time during the year is the guideline for satisfactory stallion BSE as codified by the Society for The- riogenology [4,5]. Under light microscopy, a significant increase in the morphological abnormality of sperm sam- ples was observed in stallions that were either infertile or of dubious fertility [6,7]. The average stallion had approx- imately 50% morphologically normal sperm, but some stallions with less than 40% morphologically normal specimens may achieve acceptable pregnancy rates if a minimum threshold number of normal sperm are present [8]. Sperm head morphometry assessed by Computer-Assisted Semen Analysis (CASA) has been shown to correlate with fertility in various species including horses [9], boar [10,11], Iberian red deer [12], and canines [13]. Substan- tial differences in sperm head shape and size were found within breeds in stallions [14,15], rams [16], bulls [17], alpacas [18], red deer [19], and boar [20]. Between-breed differences were identified in stallions [14], canines [21], bulls [17], boar [22], and buffalo [23]. Such variability could be, in part, due to genotypic effects [24]. There are two main horse groups in Thailand: purebred and Thai native crossbred horses. The country has a total population of 2,327 horses (Statistics of Livestock in Thai- land: 2006, Department of Livestock Development, Min- istry of Agriculture and Cooperatives). Purebred horse strains include Arabians, Standard-bred, Thoroughbred and Warm-blood, all of which were originally introduced to Thailand by importation. The Thai native crossbred horse is a pony horse that may have originated from a Bur- mese breed [25]. However, the scientific origin of the breed remains obscure. Nowadays, this native breed is generally used in religious ceremonies, for recreational activities, and occasionally for transportation in highland areas. Natural breeding with stallions is commonly per- formed to increase horse numbers. However, applications of reproductive technology in Thai native crossbred horses such as chilled semen or frozen semen are not available. Artificial insemination with chilled or frozen- thaw semen may be an important method for increasing population numbers of this horse, and a detailed study of its semen characteristics was therefore considered neces- sary. The study aims to 1) investigate the sperm morphology and normal sperm head morphometry of Thai native crossbred animals, and 2) compare the obtained results with the characteristics of purebred stallion sperm of proven fertility. Materials and methods Chemicals All chemicals in this study were purchased from Sigma Chemical Company (Sigma, St Louis, MO, USA) unless otherwise stated. Animals and Semen Collection The investigation was performed on nine clinically healthy stallions, of which five were Thai native cross- breds (T: T1 – T5) and four were purebred animals of proven fertility (F: F1 was a Standard-bred; F2 was a Warm-blood; F3 and F4 were Thoroughbreds). All were aged between 5 and 12 years. Semen was collected using a Missouri-type artificial vagina on a monthly basis over the period January through June 2007 for a total of six ejacu- lates per stallion. Multiple semen parameters were rou- tinely determined including volume, color, consistency, motility, progressive motility, and concentration. All the ejaculates were analyzed to evaluate sperm morphology. In the case of sperm head morphometry assessment, we used only the final four ejaculates in our analysis. Sperm Morphology Examination Sperm morphology was studied in wet preparations com- prising samples fixed in formal-saline [26] under a phase- contrast microscope (Olympus, Tokyo, Japan) at a magni- fication of 1000×. A total of 200 sperm in each ejaculate were examined for morphological abnormalities accord- ing to the criteria defined by Dowsett et al. [27]. Certain findings of abnormalities in the T sperm group were fur- ther examined under eosin/nigrosin staining or using scanning electron microscopy. Sperm Head Morphometry Measurements A 200 μl semen sample was washed and diluted with Dul- becco's phosphate-buffered saline (DPBS) to a concentra- tion of approximately 100 × 106 sperm/ml. Smears were prepared by taking a 7 μl drop of the diluted sperm, smearing it across a clean glass slide, and air-drying over- night. Page 2 of 9 (page number not for citation purposes)