The acrosome

The acrosome is a unique sperm organelle that develops from the Golgi apparatus during early stages of spermatogenesis. The acrosome is composed of an inner and outer membrane and a protease-filled matrix. In mammals, the acrosome overlays in a cap-like manner about 60% of the apical region of the sperm nucleus and head. It exhibits species-specific differences in shape, size and precise location on the sperm head (Fawcett, 1975; Mann and Lutwak-Mann, 1981).

The principal piece

The endpiece

The endpiece is the region beyond the distal end of the fibrous sheath. This region contains only the terminal segment of the axoneme, surrounded only by the cell membrane of the sperm tail (Fawcett, 1975; Mortimer, 1997; Garner and Hafez, 2000).

Abnormal Sperm Morphology

It has been well documented that a variety of sperm structural modifications are present in ejaculates of all species. The morphometric shapes and/or size of the sperm cells that are different from the normal structure that is characteristic of the individual species are referred to as abnormal or teratozoic spermatozoa. The distinct morphological deviations from normal structure can be usually identified during routine clinical evaluation of semen quality. The importance of morphologically abnormal spermatozoa to fertility was first brought to the attention of other investigators by Williams (1920). Williams and Savage (1925), and then Lagerlof (1934), reported the production of abnormal spermatozoa by infertile and sterile bulls. For more than 70 years, there have been extensive studies involving the identification of abnormal sperm morphology and its relationship with fertility (Lagerlof, 1934; Salisbury et al., 1942; Cupps and Briggs, 1965; Blom, 1972; Lorton et al., 1983; Barth and Oko, 1989; Söderquist et al., 1991; Foote et al., 1992; Howard et al., 1993; Saacke, 2008).

Abnormal sperm cells have long been associated with sub-fertility or sterility, depending on the type or frequency of the morphological abnormalities. However, the prediction of fertilizing ability is still largely a mystery owing to the fact that abnormal sperm cells coexist along with normal sperm cells, and the presence (i.e. number) of normal cells can be adequate for normal fertility. Still, when a very high frequency of several morphological abnormalities is found in the ejaculate, fertility of the male is expected to decline (Salisbury et al., 1942; Barth and Oko, 1989). Thus, relatively arbitrary thresholds of the percentage of morphological abnormalities have been established by andrologists in the animal breeding industry in order to consider the ejaculate as worthy of use in animal breeding. The economic impact of sub-fertility is of the utmost importance, even in light of superior genetics. Therefore, periodic sperm morphological evaluations during either natural or artificial breeding programmes serve as a valuable tool for maintaining optimal fertility levels.

There are various morphological schemes that are used by andrologists to characterize sperm abnormalities and the importance of each abnormality with respect to fertility (Salisbury et al., 1942; Blom, 1972; Barth and Oko, 1989; Söderquist et al., 1991; Saacke et al., 1994). These schemes include those described below.

The origin of the abnormalities

Earlier researchers classified morphological abnormalities as primary, secondary or tertiary according to their origin (Williams and Savage, 1925; Lagerlof, 1934; Salisbury et al., 1942; Blom, 1950; Garner and Hafez, 2000). The primary abnormalities were considered to be developmental abnormalities occurring within the seminiferous epithelium as a result of abnormal spermatogenesis before spermiation. These abnormalities include sperm head and acrosome abnormalities, midpiece abnormalities, e.g. the Dag defect, proximal cytoplasmic droplets and primordial cells. Secondary abnormalities were considered to be those originating after the sperm cells exit the testis. These abnormalities were attributed to altered epididymal maturation, prolonged retention of the sperm cells in the genital tract and abnormal composition of the seminal plasma introduced during ejaculation (Elmore, 1985; Roberts, 1986; Garner and Hafez, 2000). Secondary abnormalities include distal cytoplasmic droplets, simple bent tails, free normal heads and detached acrosomes. Tertiary abnormalities were characterized as being the result of improper semen handling during or after collection. Examples include fast cooling, high ambient temperature, contamination with urine or water and improper slide preparation for examination (Mitchell et al., 1978; Elmore, 1985; Roberts, 1986; Garner and Hafez, 2000).

Impact on fertility: major and minor sperm abnormalities

Although the classification of sperm abnormalities based on their origin has been widely accepted, it is often difficult to distinguish the anatomical origin of those abnormalities, even with the use of the electron microscope. For example, although the Dag defect was considered to be a primary defect, it also was reported to occur during the caput epididymal transit, although it was normal in the testis (Blom, 1972, 1973). Blom (1972) reported that some sperm head abnormalities were not related to improper testicular function, but occurred after the cells exited the testes. Due to the fact that the origin-based classification was observed to be not consistent, Blom (1972) suggested a new scheme in which a sperm abnormality is either associated with a major adverse impact on male fertility, i.e. ‘major defects’, or is associated with a minor effect on male fertility, i.e. ‘minor defects’. A high incidence of major defects is associated with impaired fertility or sterility and probably results from abnormal conditions in the testis or epididymis, or from hereditary genetic defects. The minor defects are considered to be less important for male fertility, unless they are present in a large percentage within the ejaculate.

Localization of abnormalities

Sperm morphological abnormalities have more simply and traditionally been classified based on where the abnormalities are localized on the spermatozoa. Thus, the abnormalities are categorized as being of the acrosome, the head, the neck or midpiece, or the tail (Söderquist et al., 1991; Al-Makhzoomi et al., 2008).

Compensable and uncompensable sperm defects

The incidence of compensable sperm defects can be overcome by increasing the number of normal spermatozoa in an artificial breeding dose. Therefore, the fertility of the bull will be at the maximum level for that individual. Increasing the number of normal spermatozoa further will not affect (i.e. increase) fertility. It should also be noted that different individuals exhibit different maximum fertility levels (Pace et al., 1981; Saacke et al., 1994; Saacke, 2008).

Increasing the number of spermatozoa does not overcome the negative effects of uncompensable sperm defects, which may be of molecular or genetic origin. Uncompensable sperm defects are likely to be identified as normal or slightly irregular during routine laboratory evaluations. These sperm are often capable of reaching the site of fertilization, and penetrating and activating the oocyte, but fail to support zygotic and embryonic development (Saacke et al., 1998; Evenson, 1999; Saacke, 2008).

Genetic sperm defects

An important component of mitochondria is cytochrome C (cytC), which, in conjunction with Complex IV, participates in establishing the electrochemical gradient of mitochondria and the subsequent production of ATP. When the structural integrity of the mitochondrial inner membrane is compromised, cardiolipin (CL) is released and binds to cytC (Kagan et al., 2009). The resultant cytC/CL complex produces hydroperoxides (Kagan et al., 2006).

Other critical proteins, such as apoptosis inducing factor (AIF), also emerge from the deteriorating mitochondrial membrane (Susin et al., 1999; Candé et al., 2002; Fleury et al., 2002; van Loo et al., 2002a,b; Philchenkov, 2004) and are present in sperm (Martin et al., 2004; Paasch et al., 2004; Grunewald et al., 2005a,b). Mature sperm are terminally differentiated cells that are also transcriptionally silent (Monesi, 1965), unlike somatic cells, which are transcriptionally active during apoptosis. The caspase proteins, which are part of the signalling pathways to programmed cell death, i.e. apotosis, are absent from mature sperm cells. Their presence in sperm then is indicative of immature or damaged cells, for example after the cryopreservation and thawing processes. The presence of activated apotosis signalling, which has been termed ‘apoptotic like’, is indicative of decreased fertilizing ability (Grunewald et al., 2008, 2009).

Not all research groups have noted a clear correlation between increased ROS production and negative fertilization effects or morphological abnormalities (Moilanen et al., 1998). Zan-Bar et al. (2005) found that ram sperm were more susceptible to light-induced ROS generation than were tilapia sperm. It is also possible that the ability to discern differing levels of ROS production could be compromised, as ROS production has been shown to decline over time during the assay period (Kobayashi et al., 2001); this could potentially skew the results.

Since ejaculates are comprised of millions to billions of individual sperm cells, variability in the overall quality of the ejaculate is reflected in the total number of individual defects. Sperm with defects in the nuclear chromatin or the protamine milieu have been demonstrated to cause problems to sustained embryo development (Ward et al., 2000; Seli et al., 2004; Lewis and Aitken, 2005; Nasr-Esfahani et al., 2005; Suganuma et al., 2005). Excess lipid peroxidation has also been shown to be correlated with midpiece abnormalities in human semen (Rao et al., 1989). However, mitochondrial abnormalities cannot be too severe or the resulting pathology will be reflected in the overall midpiece structure, thereby ablating or seriously attenuating the motility and subsequent fertilizing ability. The ability of sperm to progress though cervical mucus is seriously compromised in sperm with midpiece deformities, while sperm of normal morphology are favoured (Jeulin et al., 1985). It would therefore be logical to believe that midpiece deformities prevent sperm from reaching the site of fertilization. Because of the role of mitochondria in creating and regulating ROS during oxidative phosphorylation (Brookes et al., 2004), and because ROS may have a detrimental effect on sperm, significant disturbances in the functional physiology of the midpiece, which are not necessarily reflected in the morphology, are still of great concern.

The Environment and Abnormal Sperm Production

Temperature

Ambient heat is relevant to sperm quality issues not only because it can be reproduced for scientific study, but also because animals are indeed subjected to elevated temperature and humidity indices. Extremes of heat can compromise sperm quality, as evidenced by an increase in the number of abnormal sperm (Austin et al., 1961; Igboeli and Rakha, 1971; Kumi-Diaka et al., 1981; Parkinson, 1987; Mathevon et al., 1998). Spermatogenesis in animals occurs in a progressive, cyclical manner, known as the cycle of the seminiferous epithelium. The time required for these progressive changes is the duration of the cycle, and differs by species. When scrotal insulation is applied under research conditions, specific patterns of sperm cell abnormalities arise in relation to the temporal characteristics of spermatogenesis (Barth and Oko, 1989; Vogler et al., 1993; Karabinus et al., 1997; Zhu and Setchell, 2004; Enwall, 2009; Rahman et al., 2011).

Cryptorchid animals are a ‘natural’ example of the effects of heat on spermatogenesis. The temperature of the cryptorchid testis reflects that of the body temperature. Bilaterally cryptorchid individuals are infertile (Setchell, 1998), but spermatogenesis in cryptorchid pigs (Frankenhuis and Wensing, 1979) and rats (Karpe et al., 1984) has been restored if the testicles are removed from the warmer body cavity and moved into the cooler scrotum. Warming the scrotum in a heated water bath (Sailer et al., 1997), heating the neck of the scrotum (Kastelic et al., 1996), or insulating the entire scrotum (Vogler et al., 1991, 1993; Karabinus et al., 1997; Brito et al., 2003) can also induce changes to normal spermatogenesis via heat stress. When using semen from rams that had been scrotally insulated for 16 h/day on 21 consecutive days, Mieusset et al. (1992) noted that, while pregnancy rates in ewes did not decrease, embryonic mortality was significantly increased.

Spermatogenesis in the bovine bull occurs over a 65 day process whereby the spermatogonial stem cells undergo mitosis and meiosis, and subsequently physiological and morphological alterations, to produce mature sperm (Johnson et al., 1994). When dairy bulls were subjected to 2 or 3 wk long periods of elevated ambient air temperature (in excess of 37°C), sperm concentration, motility and total numbers were decreased and spermatogenesis was almost ablated (Casady et al., 1953). When scrotal insulation is applied for 48 h, specific patterns of sperm cell abnormalities arise in relation to the temporal characteristics of spermatogenesis (Vogler et al., 1993; Brito et al., 2003; Walters et al., 2005a; Enwall, 2009).

Dutt and Hamm (1957) reported that in unshorn rams subjected to an ambient temperature of 32°C for 1 wk, spermatogenesis was negatively affected 5 wk later. In male rabbits exposed to an air temperature of 43°C for 1 h, an effect was seen on fertilization but not on sperm motility (El-Sheikh and Casida, 1955). In roosters, a decrease of sperm numbers in the ejaculates occurred after a 3 h heat stress event, while sperm number recovered within 2 wk following the stress (Boone and Huston, 1963).

Heat stress to the scrotum not only alters the morphology of testicular spermatogenic cells (Kumi-Diaka et al., 1981; Malmgren and Larsson, 1989), but also affects the morphology of ejaculated sperm (Parkinson, 1987; Vogler et al., 1991, 1993; Barth and Bowman, 1994; Kastelic et al., 1996; Sailer et al., 1996; Enwall, 2009). The resulting altered sperm morphology has also been linked to decreased fertility (Skinner and Louw, 1966; Thundathil et al., 1998, 1999; Ostermeier et al., 2000; Jung et al., 2001; Karaca et al., 2002; Brito et al., 2003; Walters et al., 2005a,b, 2006; Enwall, 2009). This is not surprising given that alterations in the nuclear structure of sperm have been shown to correspond to decreased fertility with or without heat stress (Sailer et al.