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ABSTRACT: We hypothesized that cryopreservation and incubation in conditions that mimic the female genital tract following insemination increases the susceptibility of ram sperm DNA to denaturation. Ram sperm samples (n = 12) underwent the sperm chromatin structure assay (SCSA) and semen quality tests, including motility parameters, viability, and chlortetracycline fluorescence (CTC) patterns. We also assessed correlations between SCSA variables and semen quality parameters. Analyses were performed for both fresh and cryopreserved samples at 0, 3, and 20 hours of incubation in synthetic oviductal fluid (SOF; 39°C, 5% CO2). The SCSA variables, mean alpha t (Xαt) and standard deviation of alpha t (SDαt), were higher because of cryopreservation (P < .05, P < .001, respectively) after 20 hours in SOF. For both fresh and frozen spermatozoa, SCSA values (Xαt, SDαt, and the percentage of cells outside the main population of αt [%COMPαt]) increased during incubation in SOF. Motility was negatively correlated with both SDαt and %COMPαt, ranging from −0.39 (P < .01) to −0.59 (P < .001) for both fresh and cryopreserved semen; viability also was negatively correlated with Xαt, SDαt, or %COMPαt (−0.36; P < .05, –.40 and -.46; P < .01, respectively) in fresh semen. The %COMPαt was positively correlated to the percentage of CTC pattern AR (P < .001) and negatively correlated to the percentages of patterns F and B (−0.33 to −0.60, P < .05 to P < .001). Variation among ejaculates within ram was observed (P < .01). Cryopreservation clearly facilitates DNA damage in physiological conditions. The low to moderate correlations between SCSA variables and classical semen quality parameters indicate that the SCSA provides additional information to standard tests for evaluating ram sperm quality.
Semen cryopreservation and artificial insemination (AI) offer many advantages to the livestock industry, particularly in conjunction with genetic evaluation and selection programs (Maxwell, 1984). However, the biggest obstacle to exploiting cryopreserved semen of many species is that cooling, freezing, and thawing generally damage sperm membrane structures, leading to fewer viable and motile cells postthaw (Nath, 1972; Hammerstedt et al, 1990). Consequently, fertility following AI is poorer than with fresh semen in most species (Salamon and Maxwell, 1995; Watson, 1995).
Semen quality and its relationship to fertility are of major concern in animal production. Quality tests are routinely used to determine acceptability of processed semen for breeding purposes. Thus accurate measurement is of major importance. Conventionally, the principal laboratory tests for standard semen analysis at most AI centers use light microscopy to estimate sperm survival and the percentage of motile (and progressively motile) spermatozoa (Rowe et al, 1993). Although useful, these tests are not completely reliable or repeatable because of the small numbers of sperm evaluated, lack of objectivity, and human bias (Graham et al, 1980). More objectivity and repeatability in assessing sperm motility can be achieved by computer-assisted sperm analysis (CASA) (Davis and Siemers, 1995). In addition to CASA, flow cytometry (Shapiro, 1988) has emerged as a powerful technique for providing rapid, multiparameter, objective, and nonbiased measurements of parameters on very high numbers of sperm per ejaculate, which might be necessary for predicting fertility potential (Evenson et al, 1994). Flow cytometric measurements of sperm parameters have included mitochondrial function (Evenson et al, 1985b; Graham et al, 1990), viability (Garner et al, 1986), DNA content for sex determination (Johnson et al, 1987, 1989), acrosome integrity (Graham et al, 1990), sperm calcium level (Collin et al, 2000), and chromatin structure integrity, which is defined as the susceptibility of DNA to acid- or heat-induced denaturation in situ (Evenson et al, 1980; Ballachey et al, 1987).
The sperm chromatin structure assay (SCSA) is a technique based on the assumption that structurally abnormal sperm chromatin is more susceptible to denaturation (Evenson et al, 1980). This assay uses the metachromatic properties of acridine orange, which fluoresces green when combined with the intact double DNA helix and red when combined with RNA and denatured DNA (ie, single-stranded helices) (Darzynkiewicz, 1990). Mild acid treatment of spermatozoa denatures structurally abnormal chromatin, thereby intensifying red band fluorescence (Darzynkiewicz, 1990).
The SCSA can determine the importance of DNA structure in assisted reproductive techniques outcomes such as AI. The SCSA is reported to be an unbiased, quantitative assessment of sperm chromatin integrity, and its variables are apparently consistent within individuals over time if they are not exposed to reproductive stressors (Evenson et al, 1991). Furthermore, SCSA parameters are correlated with DNA strand breaks (Sailer et al, 1995b) and fertility in vivo (Evenson et al, 1980, 1999). However, SCSA parameters are only weakly to moderately correlated with classical criteria of sperm quality, including concentration, total number, motility and morphology (Evenson et al, 1991), and viability (Januskauskas et al, 2001). Therefore, SCSA parameters are considered independent descriptors of semen quality and might complement the information derived from the classical andrological assessment. The SCSA has not been used extensively on ram sperm, and associations between traditional andrological endpoints and sperm chromatin structure in this species have not been established.
This study was therefore performed to investigate the effect of commercial semen cryopreservation on DNA structure of ram spermatozoa by evaluating the level of sperm nuclear DNA damage during incubation in medium resembling the natural environment of the female genital tract. Our hypothesis is that the freeze-thaw process reduces sperm fertilizing competence by destabilizing the chromatin, as reflected by increased DNA susceptibility to denaturation in situ. A secondary objective was to determine the relationship between the parameters of SCSA and the classical criteria of ram sperm quality.
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The major objective of this study was to test the hypothesis that cryopreservation disrupts the DNA structure of ram spermatozoa. Although not addressed in this study, it is possible that such DNA damage might account for the poor fertility of frozen-thawed ram semen relative to fresh (Salamon and Maxwell, 1995.)
It is well known that mature mammalian sperm nuclei are stable structures, with highly condensed and organized DNA (Ward and Coffey, 1991). Chromatin stabilization occurs by intermolecular and intramolecular covalent disulfide bonds between the protamines that replace histones during spermatogenesis (Poccia, 1986). This study used the SCSA to evaluate the susceptibility of ram sperm DNA to denaturation resulting from cryopreservation and whether there was any effect of incubation in SOF on sperm DNA damage. The SCSA has been used previously to evaluate sperm DNA in bulls (Ballachey et al, 1988; Karabinus et al, 1990), boars (Evenson et al, 1994), stallions (Love et al, 2002), mice (Evenson et al, 1993), and humans (Evenson et al, 1999; Saleh et al, 2002).
As expected and previously documented (reviewed by Bailey et al, 2000), the classical andrological sperm parameters were reduced following cryopreservation. More importantly, this study demonstrates that SCSA variables (Xαt and SDαt) were different (P < .05 and .001, respectively) between fresh and cryopreserved spermatozoa after 20 hours of incubation in conditions that were intended to mimic the female reproductive tract (in SOF), but not earlier (0 and 3 hours). The elevated SCSA values for cryopreserved spermatozoa after 20 hours in SOF suggest both an increased susceptibility of sperm nuclear DNA to denaturation and heterogeneity of sperm nuclear chromatin in comparison with the fresh spermatozoa. Previous studies (Ballachey et al, 1986, 1987; Karabinus et al, 1990, 1991) demonstrated that elevated heterogeneity of sperm nuclear chromatin is related to spermatogenic disturbances, morphological abnormalities of spermatozoa, and decreased fertility in vivo. In a previous study with boar semen, sperm chromatin structure was not altered by freezing directly on dry ice or in liquid nitrogen in different types of extenders, as determined by the SCSA (Evenson et al, 1994). Similar results were obtained with mouse (Evenson et al, 1989, 1993), bull (Van der Schans et al, 2000), and human spermatozoa (Duru et al, 2001). Furthermore, it was concluded that cryopreservation does not significantly damage sperm DNA immediately or within 30 minutes of thawing, as assessed by an immunochemical assay in bull sperm (Van der Schans et al, 2000) and TUNEL for human sperm (Duru et al, 2001). In contrast, studies on human semen assessed by the SCSA (Spano et al, 1999) or a modified alkaline single-cell gel electrophoresis (comet) assay (Donnelly et al, 2001) showed that sperm DNA integrity deteriorates after cryopreservation. In the current investigation, it is difficult to compare our results with those in the previous studies because of the differences in species, cryopreservation procedure, and time of evaluation postthaw. However, the freeze-thaw processes did not have an immediate or early influence on the degree of DNA damage during incubation in SOF (0 and 3 hours), but damage was apparent later, after 20 hours.
The incubation in SOF, a physiological type of medium, was intended to mimic the ewe genital tract, thereby allowing us to monitor sperm DNA structure within a time frame relevant to the interval between thawing, AI, and fertilization in vivo. In sheep, ovulation usually occurs several hours postinsemination (Walker et al, 1989). Thus, the observation that cryopreserved ram spermatozoa have more susceptible and heterogeneous chromatin than do fresh, suggests that the poorer fertilizing efficiency of frozen ram semen might be at least partly due to abnormal sperm DNA structure, despite having a normal appearance soon after thawing. The increased susceptibility to DNA denaturation in situ during incubation in SOF is in agreement with other reports. Incubation of bull sperm in cryopreservation extenders at temperatures comparable with those of the female genital tract augmented the susceptibility of sperm DNA to denaturation within 30 minutes (Karabinus et al, 1990, 1991). Also, Estop et al (1993) showed that in vitro incubation of mouse spermatozoa induced a sharp increase in abnormal chromatin structure during the first hour (half of the cells were altered), followed by a continual rise to 70% at 12 hours and about 95% at 48 hours. In contrast, undiluted human semen does not show any significant increase of DNA damage when kept at room temperature for up to 4 hours (Alvarez et al, 2002). It seems that the level of sperm DNA damage depends on the composition of the incubation medium, which might contain agents that encourage or inhibit this damage, and this must be considered in any sperm incubation protocol (Estop et al, 1993). Moreover, Karabinus et al (1991) found that frozen-thawed bull spermatozoa incubated at 38.5°C in milk extender were more susceptible to DNA denaturation than in egg yolk extender. The authors suggested that milk components might either possess actively detrimental agents on chromatin quality or lack a protective effect that is present in egg yolk.
Although the mechanism of DNA damage in cryopreserved sperm after 20 hours is unknown, it is tempting to speculate that thawed cells are more susceptible to oxidative stress. Reactive oxygen species can induce sperm DNA damage (Barroso et al, 2000), and cryopreservation is known to reduce sperm antioxidant levels (Bilodeau et al, 2000). This hypothesis implies that the stability of sperm nuclear chromatin might be achieved via the addition of antioxidants to the semen extender. Alternatively, trying to inseminate at a closer time to ovulation might also improve sperm competence.
A secondary objective of this study was to determine whether SCSA parameters and classical criteria of ram sperm quality are correlated. DNA damage and abnormal chromatin structure can occur in bovine spermatozoa classified as normal (Karabinus et al, 1997). However, we show here significant negative correlations, ranging from poor to moderate, among SCSA variables and most classical semen quality parameters from both fresh and cryopreserved semen (Table 4). For example, correlations between either SDαt or %COMPαt and total or progressive motility were similar and varied from −0.39 (P < .01) to −0.59 (P < .001), indicating that as the percentage of ram spermatozoa with good motility declined, susceptibility to chromatin denaturation rose. These results are supported by other studies with bull (Ballachey et al, 1988; Januskauskas et al, 2001) and human spermatozoa (Evenson et al, 1991, 1999; Saleh et al, 2002). In addition, Januskauskas et al (2001) reported negative relationships between SCSA variables and sperm viability parameters and speculated that abnormal chromatin structure might impede the survival of bull spermatozoa during freezing and thawing. On the other hand, no significant correlations were detected between SCSA variables and viability traits also with bovine spermatozoa (Karabinus et al, 1991).
The negative correlations observed in this study were expected because higher values for SCSA variables mean a greater level of DNA denaturation and, consequently, lower semen quality, whereas for the other semen quality parameters, higher values indicate better semen quality. The low and moderate values of the correlation coefficients observed in this study between SCSA and the other semen quality parameters suggest that, together, both types of assays (ie, classical semen quality parameters and SCSA variables) are better predictors of semen quality and male fertility potential than each separately.
The positive correlations between %COMPαt and CTC pattern AR in both fresh and frozen semen indicates that acrosome-reacted spermatozoa are more susceptible to sperm nuclear DNA denaturation. Negative correlations also were observed between SCSA variables and acrosomal integrity of cryopreserved bovine spermatozoa (Karabinus et al, 1990). They added that the mechanism behind this relationship is not clear. Perhaps the chromatin of acrosome-reacted spermatozoa is less tightly coiled, thereby facilitating DNA decondensation that normally occurs after oocyte penetration. Moreover, a direct relationship has been observed between chromatin condensation and the capacity of sperm to fertilize. Human spermatozoa that have incomplete chromatin condensation fertilize a very low percentage of ova in vitro (Hammadeh et al, 1998) or fail to fertilize, even after direct injection of spermatozoa into the ovum (Rosenbusch, 2000).
Of interest in this study was the observation that the variation between the ejaculates within the individual rams was significant (P < .01) for all SCSA variables during the period of the experiment, which was 1 breeding season (October—January). In contrast, SCSA parameters in men (Evenson et al, 1991) and bulls (Ballachey et al, 1987) were constant within individuals. These differences suggest that defects in chromatin structure might be a variable trait (Bochenek et al, 2001). Previous studies reported changes in the frequency of abnormal sperm chromatin structure during extended periods because of disrupted spermatogenesis or external factors such as heat stress (Sailer et al, 1997), ionization radiation (Sailer et al, 1995a), toxic chemicals such as triethlenemelamine and methyl methanesulfonate (Evenson et al, 1989, 1993), and semen extenders (Karabinus et al, 1991). In our study, all rams were apparently in good health, without stress, and no chemical treatments were used during this period. Therefore, other reasons, perhaps genetic or otherwise, must be responsible for the variation between ejaculates within ram.
Semen cryopreservation had little or no effect on the susceptibility of ram sperm DNA to denature in situ when measured immediately at thawing or after 3 hours of incubation, but significant DNA damage appeared later in physiological conditions. This finding supports our hypothesis that freezing and thawing disrupts the stability of ram sperm chromatin, suggesting that the reduced fertilization efficiency of cryopreserved semen in vivo might partly be due to abnormal DNA structure. Furthermore, the low to moderate correlations between the SCSA variables and conventional semen quality parameters corroborate the utility of the SCSA in providing additional information on sperm quality and competence in the AI and andrology laboratories.