Abstract
During the conservation of feline semen, the freeze–thaw procedure in particular is responsible for inducing severe spermatozoal damage, which diminishes fertilizing ability. Therefore, cold-induced damage represents a limiting factor for the conservation of semen, particularly semen from felids, which are often affected by teratospermia. In this article, feline sperm characteristics are reported, with special reference to motility and morphology, which are more likely to be affected by conservation protocols; and moreover, the causes of cold-induced damages are described.
Attention has been focused on methods to evaluate functional integrity of spermatozoa, and those applied to cat semen are reviewed. Among these, a rather recently developed technique involves fluorescent staining methods, and in particular chlortetracycline. The chlortetracycline assay applied to cryopreserved cat epididymal sperm shows that it is suitable to evaluate the functional status of cat sperm.
Feline semen characteristics
Once semen is obtained, and before its use for assisted reproduction techniques (ART), the evaluation of sperm quality is a necessary step in order to know whether the sample of semen is suitable for conservation treatment. It is important to note that cats, as well as non-domestic felids, are very often affected by teratospermia, a condition in which more than 60% of the spermatozoa show aberrant forms (in normal laboratory cats this is less than 30%) (Pukazhenthi et al 2001). Predominant anomalies include a bent mid-piece with or without a cytoplasmic droplet, a bent flagellum and a tightly coiled flagellum. Moreover, high proportions of spermatozoa show acrosomal defects, such as large vacuoles, protrusion of acrosomal matrix and folding of the acrosome back on to itself(Pukazhenthi et al 2001).
The etiology of teratospermia in the domestic cat is unknown, but structurally defective spermatozoa observed in wild felids, such as the cheetah (Wildt et al 1983, 1984, 1987b, 1988) and geographically isolated lion populations (Wildtet al 1987a) have been related to decreased genetic variation and low circulating testosterone concentrations. Howard et al (1990) demonstrated that testosterone concentrations in teratospermic cats are 33% lower than in normospermic males.
Motility of ejaculated spermatozoa can be affected by morphological anomalies. Percent motility and association of morphological anomalies in domestic cat and wild felids have been reported and are described in Table 1.
Total motility and morphological anomalies of ejaculated spermatozoa in felids
00030-5/unzip/10.1016_S1098-612X(03)00030-5-table1.png)
Experiments conducted using cat epididymal spermatozoa show that they are more frequently affected by morphological abnormalities, with values ranging from 36 to 54% (Goodrowe and Hay 1993, Hay and Goodrowe 1993, Lengwinat and Blottner 1994). This is partially due to the fact that spermatozoa continue the maturation process in epididymides and vasa deferentia, and samples obtained from these structures often contain immature forms.
Motility of epididymal sperm is reported to be 71–79% (Goodrowe and Hay 1993, Hay and Goodrowe 1993, Stachecki et al 1993, Lengwinat and Blottner 1994, Stachecki et al 1994) and 57% (Stachecki et al 1993) in normospermic andteratospermic cats, respectively.
Cold-induced damage
In felids, fertility from artificial insemination (AI) with stored semen is poorer than fertility with fresh semen, a fact that can only be partially compensated by inseminating greater numbers of live spermatozoa close to the site of fertilization by means of an intrauterine insemination (Tsutsuiet al 2000). The cryopreservation process includes several steps, from sperm preparation and dilution to the post-thawing maintenance of functional capability: at each of these steps, spermatozoa can lose their ability to function normally.
As a result of cooling, some workers have reported a decreased motility in ejaculatedspermatozoa compared with fresh semen (43.5 vs 52.2%; Glover and Watson 1985), decreased progressive motility and altered morphology with evidence of acrosomal damage. Pukazhenthi et al (1999) reported a marked decline in intact acrosomes (65.6 vs 81.5% in the fresh sample). Motility and acrosomal integrity are also severely affected by the freezing process. In fact, Wood et al (1993) reported for ejaculated cat sperm a marked decrease in intact acrosomes (28 vs 90% in the fresh sample), and similar results were obtained by Swanson et al (1996b) in the jaguar andcheetah.
Acrosomal damage has been reported even in epididymal frozen–thawed semen. Hay and Goodrowe (1993) observed 30–50% loss of acrosomal integrity. Lengwinat and Blottner (1994) reported a decreased motility (75.8 vs 53%) and decreased acrosomal integrity (69.5 vs 22.5%) after thawing. Therefore, cooled andcryopreserved sperms undergo severe damages that alter their motility and morphology, thus affecting their fertilizing potential. This is explained by the fact that some stages of the process can be very stressful, such as the change in temperature (cold shock), as well as the formation and dissolution of ice crystals. The mechanism by which cold shock acts on the sperm cell is not entirely clear, but it is probably related to the phase transitions of membrane lipids, resulting in phase separations and loss of the selective permeability characteristics of living biological membranes (Watson 1995). Ice crystals cause rupture of cellular membranes when the cells are exposed to a rapid rate of cooling (4°C/min) from body temperature to +5°C, while decreasing the cooling rate to 0.5°C/min minimizes the structural damage (Pukazhenthi et al 1999). As the temperature is reduced below 0°C, the water content in the extracellular medium undergoes crystallization. The solute is concentrated in the remaining fluid portion, and the cell is subjected to osmotic dehydration and shrinkage due to efflux of intracellular water (Watson 1995).
The cryoprotectant agent usually present in the freezing diluent (glycerol) provides protection to the cells from the consequences of ice formation by increasing the unfrozen water fraction, but the osmotic effect of molar concentrations of this compound may result in membrane damage. Moreover, the toxicity of glycerol for cat spermatozoa has been reported (Nelson et al 1999). The decline in motility could be due to changes in osmolality (Pukazhenthi et al 2002) and in active transport and permeability of the plasma membrane in the tail region, combined with an alteration of energy availability or damage to theaxonema elements (Watson 1995). In the cell, several of the organelles are enveloped by a membrane, which is particularly vulnerable during the cryopreservation cycle, and many of the cytoskeleton proteins exhibit a temperature-dependent depolymerization and repolymerization, which could have severe implications for sperm cell viability (Watson 1995). Thus, frozen–thawed spermatozoa demonstrate a loss of internal mitochondrial structure (Watson 1979). Even the nucleus can be altered during cryopreservation, and the degree of denaturation of DNA is influenced by diluents (Karabinus et al 1991).
It is important that spermatozoa from teratospermic males are more susceptible to cold and osmotic stress, which induce membrane disruption, than those from normospermic males (Pukazhenthi et al 1999). These observationsindicate that there may be membrane differences between spermatozoa from normospermic vs teratospermic donors that, in turn, may influence the kinetics of water and solute movement across membranes.
One of the consequences of destabilization of membranes by cooling or cryopreservation is a premature acrosome reaction that shortens the life span of the spermatozoa and reduces fertility. In fact, cryopreserved spermatozoa may be considered to be in a state resembling partially capacitated spermatozoa. Their membranes have undergone similar fluidity changes as those seen during capacitation; they are permeable to calcium ions that promote both capacitation and the acrosome reaction. Their viability is thus limited because capacitated spermatozoa do not have a prolonged survival: they are usually activated close to the time of meeting the oocyte. Moreover, a false acrosome reaction has also been described in thawed spermatozoa, in which acrosomal changes are associated with irreversible membrane damages (Bedford 1970, Meizel 1978). It has been reported that a significant higher proportion of cat epididymal spermatozoa after thawing showed the pattern of acrosome reaction compared with fresh or diluted semen before freezing. The proportion of acrosome-reacted spermatozoa in frozen–thawed samples indicates that many cells were damaged during freezing and thawing and, therefore, are functionally compromised (Marinoni 2001). Spermatozoa must undergocapacitation and the acrosomal reaction in order to penetrate the oocyte, and this is possible only if sperm acrosomal integrity has not been altered during storage.
Assessment of motility and morphology
Motility may be evaluated as percent motility and progressive motility, and the latter is classified according to a scale ranging from 0 (no movement) to 5 (steady, rapid forward progression). From these two parameters, a spermatozoamotility index (SMI) can be derived: SMI=0.5×[(progressive motility×20)+(%) motility] (Howard 1993). Another method for determining motion characteristics of cat spermatozoa is Computer Assisted Semen Analysis (CASA), which makes it possible to measure different kinematic parameters, such as curvilinear velocity, linearity, straight-line velocity and amplitude of lateral head displacement. The CASA technique can evaluate various motility parameters, and it could be useful for identifying a hyperactivated status in spermatozoa, which could suggest their capacitated condition(Stachecki et al 1993).
Morphology of cat spermatozoa can be evaluated fixing a semen sample in glutaraldehyde 1% and evaluating spermatozoa with a phase contrast microscopy, or with all the usual stains used in semen analysis, like eosin–nigrosin, eosin–fast green FCF etc (Byers et al 1989, Hay and Goodrowe, 1993). A staining technique (1% rose bengal, 1% fast green FCF and 40% ethanol in citric acid–disodium phosphate buffer) for evaluating acrosomal morphology of feline spermatozoa was described by Pope et al (1991). There are several classifications of spermatozoal morphological abnormalities in fresh semen, and the one most commonly used differentiates them into: primary, when they occur during spermatogenesis in the testicles, such as a coiled flagellum, and microcephalic or macrocephalic defect and secondary, due to damages during their maturation and transport along the epididymes, such as a bent mid-piece, a bent flagellum, protoplasmic droplets and acrosomal anomalies. Primary defects are normally considered more detrimental to fertility than secondary deformities (Wildt et al 1983, Howard et al 1986). With regard to preserved semen, the evaluation of abnormalities focuses on the acrosome, since this structure is vulnerable and often altered by cooling and freezing processes.
Accurate methods for evaluation of cold-induced damage on acrosome membrane as indicator of fertilization ability have been developed and are described subsequently.
Assessment of spermatozoal fertilizing ability
Methods of evaluating capacitation status and acrosomal reaction of cat spermatozoa can be classified into biological assays and fluorescent staining techniques.
Biological assays include different types of in vitro assays, such as
Homologous zona pellucida (ZP) adhesion: this assay is possible only for capacitated sperm. Some authors have reported that cooled and frozen epididymal spermatozoa bind to homologous ZP in a larger number than fresh epididymal sperms, and this seems to be caused by loss of acrosomal integrity, which on the other hand reduces the fertilizingpotential (Goodrowe and Hay 1993, Hay and Goodrowe 1993).
Homologous or heterologous (intraspecific) ZP penetration: it is a better indicator of capacitation, as sperms not only attach to ZP surface, but they also penetrate it. This assay may be performed with salt-stored oocytes, as such storage prevents oocytes from undergoing cortical reaction, thus allowing more than one spermatozoa to penetrate the ZP. This provides information on percentage of capacitated spermatozoa, and cat oocytes can be employed even for testing wild felid semen (Howard and Wildt 1990, Andrews et al 1992, Donoghue et al 1992, Wood et al 1993, Swanson et al 1996b).
Hamster ‘zona-free’ egg penetration: in this assay, oocytes are treated with a 0.1% hyaluronidase solution to remove cumulus cells and with a 0.2% trypsin solution in order to remove the ZP, that otherwise would prevent heterologous (interspecific) spermatozoa from penetrating the eggs. As homologous eggs of endangered species may not always be available, the heterologous gametes may turn out to be very useful and may allow sparing such valuable oocytes (Byers et al 1989, Nelson et al 1999, Da Paz et al 2002).
Homologous oocytes fertilization: even though cold storage techniques reduce quality of spermatozoa and their fertilizing potential, both cooled and cryopreserved felid spermatozoa employed for in vitro fertilization have proven capable of producing embryos, even though the results are still poor (Pope et al 1989, Donoghue et al 1992, Lengwinat and Blottner 1994, Leoni 1999, Nelson et al 1999, Bartels et al 2000).
Fluorescent staining techniques include
Arachis hypogea (peanut): sperm cells are stained using fluorescein isothiocyanate conjugated A hypogea agglutinin (FITC-PNA). This stain is specific to the outer acrosomal membrane: acrosomes exhibiting uniform bright staining are classified as acrosome-intact and those exhibiting a fragmented appearance or bright staining only in the equatorial segment are classified as acrosome-damaged (Pukazhenthi et al 1999, 2001). Chlortetracycline (CTC): this is an antibiotic with a fluorescent component that can be used to visualize the course of spermatozoal capacitation and acrosome reaction. Using CTC, it appears possible to discriminate between uncapacitated and capacitated acrosome-intact spermatozoa, a feature not offered by other staining methods, which can only differentiate between presence and absence of acrosome. The CTC assay is based on transfer of neutral and uncomplexed CTC across sperm membranes. CTC enters intercellular compartments containing high levels of free calcium, ionizes to an anion and binds calcium, becoming more fluorescent as a result (Tsien 1989). The CTC–Ca2+ complex preferentially binds to the hydrophobic regions of the cell membrane, resulting in a pattern of membrane staining characteristic of the various transitional phases, which spermatozoa display (Saling and Storey 1979). Recently, it has been showed that CTC assay is a suitable method for evaluating functional integrity of cat epididymal spermatozoa (Marinoni 2001). The fluorescent patterns of cat epididymal spermatozoa stained with CTC are shown in Fig 1.
00030-5/unzip/10.1016_S1098-612X(03)00030-5-fig1.png)
Cat epididymal sperm cell stained with CTC assay (×2250). Pattern F1/F2: there is uniform fluorescence over the whole head with about half the sperm population showing a brighter line of fluorescence across the equatorial segment. It is characteristic of uncapacitated, acrosome-intact cells. Pattern B: with a fluorescence-free band in the post-acrosomal region, is characteristic of capacitated, acrosome-intact cells. Pattern AR: with dull or absent fluorescence, is characteristic of acrosome-reacted cells. Bright fluorescence in the mid-piece is seen in all cells.
Conclusions
The final target of semen conservation is the preservation of spermatozoal fertilizing potential in order to develop ART and to increase felid survival chances. Studies conducted on conservation protocols have produced important results in domestic cat as well as in wild felids, but have also elicited many problems, most of which are still unsolved. Cooled and cryopreserved sperms undergo several types of damage, which alter their motility and morphology and are responsible for low pregnancy rate after AI. For these reasons, there is the need to define the ideal diluent components and to optimize cooling and freezing protocols to prevent membrane modification. Moreover, it is necessary to study these damages and to develop new methods to evaluate them. Among currently applied methods, biological assays and fluorescent staining techniques not only allow a morphological evaluation of the spermatozoa, but also provide information on its functional integrity. The CTC, in particular, allows monitoring of various transitional phases that spermatozoa display from capacitation to acrosome reaction resulting from cold-induced damages.