Structure of equine spermatozoa

The spermatozoon consists of the head, neck, and tail and is entirely covered by the plasma membrane. The tail is the longest part of the spermatozoon and consists of midpiece, principal piece, and end piece (Figs 1/A, 1/B, Amann and Graham 1993, Brito 2007). The length of equine spermatozoon is approximately 60 µm (Pesch and Bergman 2006, Brito 2007). Every part of the spermatozoa plays important role in the fertilization process. The plasma membrane surrounds the spermatozoon in total and is characterised by a regional specific glycoprotein and lipid, mainly phospholipids and cholesterol constitution. These so-called surface domains are important for the function of the membrane areas. For example, the part of the membrane at the equatorial segment is responsible for the contact to the oocyte membrane in fertilization (Busch and Holzmann, 2001, Pesch and Bergman 2006). Lipids are arranged as a bilayer. Proteins are intermingled with the lipids as integral or peripheral proteins. The ratio of cholesterol to phospolipids and the nature of phospholipids determine the flexibility of the membrane, which is “fluid” at body temperature (Hammerstedt et al. 1990, Juhász et al. 2000). Plasma membrane of stallion sperm contains approximately 57% phospholipids, 37% cholesterol and 6% glycolipids, such that the stallion sperm differs primarily with regard to its relatively high cholesterol content. Phospholipids are the major lipid components and they are largely composed of polyunsaturated fatty acids (PUFA) (Scott 1973) in particular, docosahexaenoic acid (DHA; 22:6 n-3, an omega-3 fatty acid) and docosapentaenoic acid (DPA; 22:5 n-6, an omega-6 fatty acid). Stallion sperm differ from those of the other mammalian species by having a surprisingly very high content of 22:5 fatty acids in their phosphocholineglycerides and phosphoethanolamineglycerides fractions of phospholipids and relatively few 22:6 fatty acids (Yanagimachi 1994, cited: Gadella et al. 2001).

The spermatozoon head is formed by the acrosome, the postacrosomal lamina, and the nucleus (Fig. 1/A). The anterior two-thirds of the nucleus is overlaid by the acrosome, which is a specialized vesicle formed from a double-layered membrane which contains hydrolytic enzymes essential for spermatozoon penetration of the zona pellucida of the oocyte. The acrosome is covered by the inner and outer acrosomal

membrane and can be divided into apical, principal and equatorial segments. The equatorial segment does not contain enzymes and is not involved in the acrosomal reaction, but the plasma membrane in this area fuses with the plasma membrane of the oocyte. The postacrosomal lamina may have a role in attachment of the spermatozoa to the oocyte. The nucleus compromises most of the spermatozoon head and contains the genetic material in the form of highly condensed DNA. The nucleus is contained by a double-layered nuclear envelope. The base of the nucleus terminates with the implantation fossa, where the outer layer of the double-layered nuclear envelope thickens to form the basal plate, which provides the attachment of the head to the capitulum of the neck. Position of the implantation fossa is often abaxial in the stallion, therefore abaxial position of the tail is considered normal in equine spermatozoa. This results in curvilinear movement of the sperm. The border between the head and neck is clearly defined by a posterior ring (Amann and Graham 1993, Juhász et al. 2000, Brito 2007).

The head of the equine spermatozoon is elliptical shape, slightly thicker at the posterior part. Reported means for dimensions of the stallion spermatozoon head include: 5.33 µm to 6.62 µm for length, 2.79 µm to 3.26 µm for maximum width, 0.43 to 0.52 for length/width ratio, 13.76 µm to 15.64 µm for perimeter and 11.43 µm² to 16.28 µm² for area. All reports indicate a significant stallion effect (Dott 1975, Bielanski and Kaczmarski 1979, Ball and Mohammed 1995, Gravance et al. 1996, 1997, Casey et al. 1997, Pesch and Bergman 2006, Brito 2007). The variation in the shape of normal stallion sperm heads is considerable, ranging from somewhat thinner and elongated to shorter and broader forms. The correct classification of sperm with extreme head shape morphology may be difficult, and the distinction of tapered and microcephalic sperm heads requires comparison among several sperm to establish what the “normal” sperm head shape for an individual stallion is (Brito 2007).

Substantial differences in sperm head shape and size were found between breeds and within breeds in stallions. However preparation of sperm for morphometry analyses was also important, sperm head size as determined from Feulgen-stained spermatozoa was smaller than that determined from live, unfixed spermatozoa (Ball and Mohammed 1995). Differences in sperm head size within breed have been reported in both Warmblood (Ball and Mohammed 1995) and Spanish Thoroughbred stallions (Hidalgo et al. 2008). Similarly, differences between breeds have been observed in Arabian, Warm-blood, Thoroughbred and Morgan stallions (Ball and Mohammed 1995). The results of study Phetudomsinsuk et al. (2008) confirmed the previous observations; morphometric characters of normal sperm heads were significantly different among individual Thai native croosbred (T) or control warmblood (purebred)

stallions, and between T and Purebred stallions. The heads of stallion spermatozoa were analysed by computer-assisted sperm head morphometry (ASMA) in several studies. The mean values for length, width, area and perimeter in the major cluster of sperm head dimensions of fertile stallions (>60% per cycle conception rate) were significantly different from those of the subfertile stallions (<40% per cycle conception rate). The range of values of the major cluster of fertile stallions was length: 4.9-5.7 µm, width: 2.5-3.0 µm, width/length ratio: 0.45-0.59, area: 10.3-12.1 µm², and perimeter: 12.9-14.2 µm. On the basis of these values, a significantly higher percentage of normal sperm heads were found in the fertile group than in the subfertile group of stallions (52% versus 19%). The results suggest that a value of < 30% of spermatozoa with normal head morphometry may indicate impaired fertility in stallions, while a value > 40% would be indicative of a fertile stallion. The mean measurements for length, area and perimeter were significantly larger in the subfertile than the fertile group (5.77 vs 5.33 µm, 12.66 vs 11.37 µm² and 14.59 vs 13.64 µm respectively). Sperm in subfertile stallions also tended to be more tapered than in fertile stallions (Gravance et al. 1996, Casey et al. 1997). The larger sperm heads found in subfertile stallions may reflect disturbances in spermatogenesis, particularly involving altered chromatin structure. However, it is important to note that subfertile stallions also had lower total sperm number and percentages of motile and normal sperm in the ejaculate than fertile stallions, which likely also influenced fertility (Brito 2007). Révay et al. (2004) measured the head area of bull spermatozoa after viability and acrosome staining using trypan blue and Giemsa stains, followed by X- and Y-chromosome-specific fluorescence in situ hybridisation (FISH). In all bulls, morphologically normal, viable cells with intact acrosomes were significantly smaller than dead cells with damaged acrosomes. No significant difference in the head area between X- and Y-chromosome-bearing viable, acrosome-intact spermatozoa was found in individual bulls. It seems that live/dead status of the sperm also influences head-morphometry and partly can be the reason for higher values of spermatozoa of subfertile males. Arruda et al. (2002) studied the effects of extenders and cryoprotectants on stallion sperm head mophometry using ASMA. The morphometric parameters such as length, perimeter and area were significantly smaller in cryopreserved sperm than in fresh-extended sperm. Changes in dimensions might be due to acrosomal damage or alteration in chromatin condensation associated with cryopreservation, or extender’s osmolarity.

The spermatozoon neck is a short linking segment between the head and the tail contains the connecting piece, the proximal centriole, which are the base of the dense fibers and the axoneme (Fig. 1/B). This region is the site, where beat of the tail is

initiated. The tail of the spermatozoon includes the middle piece, the principal piece and the end piece. The length of the tail is in average 54 µm (midpiece: 10 µm, principal piece: 40 µm, end piece: 4 µm). Diameter of the midpiece: 0.9 µm and of the principal piece: ≤ 0,6 µm. The midpiece is the widest part of the tail formed by the axoneme surrounded by the nine outer dense fibers and the mitochondrial sheath, extends from the caudal end of the neck to the annulus (or Jensen’s ring).

Mitochondrial sheath is the helically arranged mitochondria, which contains enzymes and cofactors for production of ATP. The axoneme and dense fibers of the midpiece continue through the principal piece, but the dense fibers become narrower and terminate at different levels in the distal principal piece. The principal piece is the longest part of the spermatozoon. It contains the axoneme, the dense fibers and a fibrous sheath, which is characteristic of this part. The axoneme consists of a central pair of microtubules surrounded by nine microtubular doublets which are the elements that contract to produce sperm tail movement. Axoneme microtubules extend from the neck region through the midpiece and principal piece into the end piece, where they terminate at slightly different sites. The dense fibers and fibrous sheath do not contract; however, they provide rigidity and flexibility at the same time for the flagellar movement. The end piece is the short terminal segment of the tail containing only the axoneme or single microtubules which are surrounded by the plasma membrane (Bielanski and Kaczmarski 1979, Amann and Graham 1993, Juhász et al.

2000, Brito 2007).

Figure 1/A. Structure of stallion spermatozoon (Source: Amann and Graham 1993, Brito 2007)

Figure 1/B. Structure of stallion spermatozoon (Source: Amann and Graham 1993, Brito 2007)