Assessment of cell viability in oocytes using LIVE/DEAD and MTT assays offers complementary insights, albeit with provisional conclusions. Structural analysis using LIVE/DEAD staining revealed diminished viability during the peak degradation stage (post-rigor) across all cellular developmental stages compared with the alternative assessment criteria (fresh/control, pre-rigor mortis, and rigor mortis). However, the MTT assay failed to corroborate this observation as it yielded no statistically significant differences among the evaluated stages.
The incongruity between the two assays underscores the intricacies associated with the assessment of cell viability amidst rigor mortis progression. While the LIVE/DEAD assay focuses on cell membrane integrity, the MTT assay evaluates metabolic activity. This divergence suggests that, notwithstanding the conspicuous morphological changes evident during the heightened degradation phase, metabolic activity may not be uniformly impacted, thus contributing to discordant outcomes. The absence of significant differences in the MTT results across rigor mortis stages elicits thought-provoking inquiries. It is plausible that oocytes in advanced developmental stages sustain consistent metabolic activity even in the aftermath of rigor mortis. This implies heightened metabolic resilience compared to the less mature growth stages.
In summation, the findings arising from the LIVE/DEAD and MTT assays, delving into cell viability, unveil the intricate cellular dynamics inherent in rigor mortis. These disparities underscore the need to embrace multifaceted evaluation criteria to comprehensively grasp cellular viability within dynamic contexts such as rigor mortis. According to Gryshkov et al. (2015) [27], exclusive assessment of cell membrane viability provides a less comprehensive perspective on the number of viable or non-viable cells. Thus, the concomitant utilization of these analyses with metabolic evaluation assays is imperative, given their demonstrated heightened sensitivity compared to isolated approaches such as the trypan blue staining method [28]. Remarkably, in oocytes, Marques et al. (2018) [29] observed that the MTT assay exhibited enhanced sensitivity for distinguishing between treatments with higher viability than those with lower viability. This phenomenon may be attributed to the elevated mitochondrial concentration inherent to oocytes during specific developmental stages.
It is reasonable to postulate that cellular degradation may induce an increase in metabolic activity, which could potentially be linked to the presence of active bacteria in these environments (e.g., lactic acid-producing bacteria) [30]. These bacterial strains possess oxidoreductase enzymes, which may introduce confounding variables into the MTT assay. However, a comprehensive investigation is necessary to determine whether the bacterial consortia involved in rigor mortis can directly affect the observed outcomes of this assay. Moreover, rigorous exploration is essential to establish whether bacteria participating in rigor mortis can stimulate measurable mitochondrial activity within the confines of this specific analytical framework.
In males, analyses of cell membrane integrity (LIVE/DEAD) and cellular metabolic activity (MTT) revealed no qualitative differences between the various rigor mortis stages and fresh/control semen. However, upon evaluating the outcomes related to sperm kinetics and morphology, discernible qualitative deterioration was observed in these reproductive cells from animals with rigor mortis and post-rigor mortis. In kinetic assessments, across all analyzed variables, spermatozoa obtained from pre-rigor animals did not deviate from samples collected from fresh/control animals. These findings underscore the fact that spermatozoa exhibit kinetic attributes conducive to viability similar to those sourced from live animals. The significance of these findings lies in the prospect of establishing germplasm banks from deceased animals up to the pre-rigor mortis stage, without compromising sperm quality. For the cryopreservation of zebrafish sperm, ensuring that the motility of a fresh sample falls within the range of 80–95% is minimally essential [31, 32]. In this regard, sperm motility analyses yielding results similar to those in the control sample demonstrate the potential utility of this objective.
The assessment of sperm kinetics provided valuable insights into the dynamic aspects of sperm motility in relation to the different stages of rigor mortis. Our findings revealed the noteworthy influence of the rigor mortis phase on various parameters that contribute to sperm motility. Significant variations were observed in key metrics, including VCL, VSL, VAP, WOB, PROG, and BCF. These alterations reflect the complex interplay between the physiological and biomechanical factors that regulate sperm movement.
It is essential to highlight that the effect of rigor mortis on sperm motility was not uniform across all parameters. While certain variables demonstrated substantial declines in post-rigor mortis samples, others exhibited intermediate responses, and a few parameters remained relatively unchanged. Specifically, motility parameters such as VCL and WOB displayed pronounced decreases in rigor mortis and post-rigor mortis samples, indicating compromised sperm propulsion and path trajectory. Conversely, the VSL, VAP, and PROG values exhibited a more nuanced pattern, with the fresh/control and pre-rigor mortis groups displaying higher means than the rigor mortis and post-rigor mortis groups. These variations may reflect alterations in the mechanical properties of sperm cells and their microenvironment during the different stages of rigor mortis.
Furthermore, the elevation of BCF in the rigor mortis group suggests an intriguing connection between beat frequency and the state of rigor mortis. The higher BCF observed in rigor mortis samples could indicate a compensatory mechanism aimed at sustaining motility under challenging conditions. However, the physiological basis underlying this phenomenon requires further investigation.
The observed disparities in sperm kinetics between the rigor mortis stages and fresh/control and pre-rigor mortis groups underscore the multifaceted nature of sperm motility and susceptibility to physiological changes. It is worth noting that the absence of significant differences in STR emphasizes that sperm motility alterations primarily affect curvilinear aspects rather than the overall directional stability of sperm movement.
These findings offer valuable insights into both basic reproductive biology and practical applications of assisted reproductive technologies. Distinct alterations in sperm kinetics during rigor mortis stages may have implications for sperm quality assessment, cryopreservation protocols, and breeding programs. Further research is needed to unravel the precise mechanisms underlying these kinetic changes and explore potential strategies to mitigate their impact on sperm function and fertility outcomes.
The increased percentages of spermatozoa with degenerated heads observed in both the rigor mortis (10.75 ± 3.352%) and post-rigor mortis (8.700 ± 2.288%) groups suggest potential consequences of cellular degeneration due to autolysis [33, 34]. This association between degenerated heads and rigor mortis progression implies a connection between tissue architecture breakdown and membrane integrity disruption, aligning with autolytic processes. Additionally, the higher prevalence of microcephalic spermatozoa in the post-rigor mortis group (8.400 ± 2.331%) compared to the fresh/control group (1.903 ± 1.172%) indicates significant morphological changes, potentially linked to autolytic mechanisms. The decrease in structural integrity and a higher proportion (P < 0.05) of spermatozoa with detached heads in the post-rigor mortis group relative to the pre-rigor mortis group suggested a possible impact of rigor mortis progression on head detachment and structural integrity. These findings highlight the susceptibility of spermatozoa to structural disruptions during the post-mortem period, which affect their functional viability. Tail morphology also showed variations; the rigor mortis group had a higher proportion of spermatozoa with loose tails (11.85 ± 2.625%) compared to the pre-rigor mortis group (6.500 ± 3.512%). Moreover, the pronounced presence of short tails in the post-rigor mortis group (16.00 ± 5.132%) underscores a potential correlation between rigor mortis progression and tail morphology alterations, further implicating autolytic processes. The sperm membrane, which is composed of a lipid layer containing phospholipids and cholesterol, is vital for membrane organization [35–37]. During autolysis, this membrane becomes susceptible to lipid peroxidation by reactive oxygen species, resulting in loss of membrane fluidity and integrity [38].
These morphological deviations have implications for sperm motility and hydrodynamics because morphological abnormalities can negatively affect the mechanical dynamics of sperm propulsion [39, 40]. The interplay between structural disruption and altered motility highlights the complex relationship between form and function, underscoring the need for a comprehensive understanding of morphological intricacies in the context of sperm performance.