Effect of different stunning methods on rigor mortis, shape, energetic status and physical characteristics of Salmo carpio fillets.

BACKGROUND: Consumer interest in safeguarding animal welfare and increased demand for fresh aquatic products support the need to understand the effects of stunning methods used in aquaculture on the biochemical process affecting fish fillet quality. The present paper aimed at comparing electrical stunning (ES) and cold shock (ICE) in Salmo carpio, an Italian endemic under-investigated species. Rigor mortis evolution, fillet adenosine 5'-triphosphate (ATP), shape, colour, pH and water holding capacity were assessed by integrating chemical and image analyses. RESULTS: Seventy-two fish (24 fish per treatment) were stunned by ES, ICE or anaesthesia (AN, used as control), then percussively slaughtered. ES and ICE hastened rigor mortis onset and resolution (21 and 28 h post mortem) compared to AN. This was confirmed by the faster ATP degradation in ES and ICE. Fillet shape features varied during rigor mortis, according to the stunning method, with the perimeter showing irreversible variation in ES and ICE groups. Initial circularity was recovered only in AN, while ICE and ES fillets showed significantly different values, between 0 and 192 h. CONCLUSION: ES is a promising stunning technique for S. carpio, but parameters should be optimized, because of the adverse effect on muscle activity which caused a fast pH drop, and the presence of blood spots in the fillets. Further studies are needed to understand whether fillet shape changes can interfere with filleting or fillet processing and consumer appreciation. © 2022 Society of Chemical Industry.

A novel method for post-mortem interval estimation based on tissue nano-mechanics.

Forensic estimation of post-mortem interval relies on different methods, most of which, however, have practical limitations or provide insufficient results, still lacking a gold standard method. In order to better understand the phenomenon of rigor mortis and its applicability to the post-mortem interval estimation, we decided to use atomic force microscopy, a tool often employed to measure mechanical properties of adherent cells. Thus, we surgically removed skeletal muscle samples of three forensic cases from 0 to 120 h post-mortem and quantitatively evaluate two parameters: the Young's modulus (E), which gives information about the sample stiffness, and the hysteresis (H), which estimates the contribution of viscous forces. Despite being a preliminary study, the obtained results show that the temporal behavior of E well correlates with the expected evolution of rigor mortis between 0 and 48 h post-mortem, and then monotonically decreases over time. Unfortunately, it is strongly affected by inter-individual variability. However, we found that H provides measurable data along a time-dependent curve back to the starting point, and these data measured on different subjects collapse onto a single master curve, getting rid of the inter-individual variability. Although a larger sampling should be performed to improve the result reliability, this finding is strongly suggestive that the evaluation of rigor mortis should involve the measure of the nanoscale dissipative behavior of muscular tissues.

Coupling of Rigor Mortis and Intestinal Necrosis during C. elegans Organismal Death.

Organismal death is a process of systemic collapse whose mechanisms are less well understood than those of cell death. We previously reported that death in C. elegans is accompanied by a calcium-propagated wave of intestinal necrosis, marked by a wave of blue autofluorescence (death fluorescence). Here, we describe another feature of organismal death, a wave of body wall muscle contraction, or death contraction (DC). This phenomenon is accompanied by a wave of intramuscular Ca2+ release and, subsequently, of intestinal necrosis. Correlation of directions of the DC and intestinal necrosis waves implies coupling of these death processes. Long-lived insulin/IGF-1-signaling mutants show reduced DC and delayed intestinal necrosis, suggesting possible resistance to organismal death. DC resembles mammalian rigor mortis, a postmortem necrosis-related process in which Ca2+ influx promotes muscle hyper-contraction. In contrast to mammals, DC is an early rather than a late event in C. elegans organismal death. VIDEO ABSTRACT.

Methods for determining time of death.

Medicolegal death time estimation must estimate the time since death reliably. Reliability can only be provided empirically by statistical analysis of errors in field studies. Determining the time since death requires the calculation of measurable data along a time-dependent curve back to the starting point. Various methods are used to estimate the time since death. The current gold standard for death time estimation is a previously established nomogram method based on the two-exponential model of body cooling. Great experimental and practical achievements have been realized using this nomogram method. To reduce the margin of error of the nomogram method, a compound method was developed based on electrical and mechanical excitability of skeletal muscle, pharmacological excitability of the iris, rigor mortis, and postmortem lividity. Further increasing the accuracy of death time estimation involves the development of conditional probability distributions for death time estimation based on the compound method. Although many studies have evaluated chemical methods of death time estimation, such methods play a marginal role in daily forensic practice. However, increased precision of death time estimation has recently been achieved by considering various influencing factors (i.e., preexisting diseases, duration of terminal episode, and ambient temperature). Putrefactive changes may be used for death time estimation in water-immersed bodies. Furthermore, recently developed technologies, such as H magnetic resonance spectroscopy, can be used to quantitatively study decompositional changes. This review addresses the gold standard method of death time estimation in forensic practice and promising technological and scientific developments in the field.

The effect of temperature on the mechanical aspects of rigor mortis in a liquid paraffin model.

Rigor mortis is an important phenomenon to estimate the postmortem interval in forensic medicine. Rigor mortis is affected by temperature. We measured stiffness of rat muscles using a liquid paraffin model to monitor the mechanical aspects of rigor mortis at five temperatures (37, 25, 10, 5 and 0°C). At 37, 25 and 10°C, the progression of stiffness was slower in cooler conditions. At 5 and 0°C, the muscle stiffness increased immediately after the muscles were soaked in cooled liquid paraffin and then muscles gradually became rigid without going through a relaxed state. This phenomenon suggests that it is important to be careful when estimating the postmortem interval in cold seasons.

[The theory of postmortem rigidity: the history and an original concept].

The original theory of postmortem rigidity has been developed and substantiated based on the concept of postmortem muscular contracture. It is postulated that the unrestricted growth of Ca2+ concentration in myoplasm of contractile cells during the immediate postmortal period brings the actin-myosine complex to the force generation state without subsequent relaxation.

Experimental evaluation of rigor mortis IX. The influence of the breaking (mechanical solution) on the development of rigor mortis.

Objective measurements were carried out to study the possible re-establishment of rigor mortis on rats after "breaking" (mechanical solution). Our experiments showed that: *Cadaveric rigidity can re-establish after breaking. *A significant rigidity can reappear if the breaking occurs before the process is complete. *Rigidity will be considerably weaker after the breaking. *The time course of the intensity does not change in comparison to the controls: --the re-establishment begins immediately after the breaking; --maximal values are reached at the same time as in the controls; --the course of the resolution is the same as in the controls.

Immunohistochemical detection of hemoglobin in frost erythema.

Reddish discoloration of exposed skin areas, called frost erythema, is an important criterion for the diagnosis of fatal hypothermia. In the present study, we used immunohistochemistry in a prospective trial to show that on the molecular level, the correlate of frost erythema is hemoglobin without hemorrhage. Furthermore, we compared routine histological and immunohistochemical features of frost erythema, hematoma and livor mortis and established some criteria for their histological differentiation.

Long persistence of rigor mortis at constant low temperature.

We studied the persistence of rigor mortis by using physical manipulation. We tested the mobility of the knee on 146 corpses kept under refrigeration at Torino's city mortuary at a constant temperature of +4 degrees C. We found a persistence of complete rigor lasting for 10 days in all the cadavers we kept under observation; and in one case, rigor lasted for 16 days. Between the 11th and the 17th days, a progressively increasing number of corpses showed a change from complete into partial rigor (characterized by partial bending of the articulation). After the 17th day, all the remaining corpses showed partial rigor and in the two cadavers that were kept under observation "à outrance" we found the absolute resolution of rigor mortis occurred on the 28th day. Our results prove that it is possible to find a persistence of rigor mortis that is much longer than the expected when environmental conditions resemble average outdoor winter temperatures in temperate zones. Therefore, this datum must be considered when a corpse is found in those environmental conditions so that when estimating the time of death, we are not misled by the long persistence of rigor mortis.

Differential rigor development in red and white muscle revealed by simultaneous measurement of tension and stiffness.

Based on the molecular mechanism of rigor mortis, we have proposed that stiffness (elastic modulus evaluated with tension response against minute length perturbations) can be a suitable index of post-mortem rigidity in skeletal muscle. To trace the developmental process of rigor mortis, we measured stiffness and tension in both red and white rat skeletal muscle kept in liquid paraffin at 37 and 25 degrees C. White muscle (in which type IIB fibres predominate) developed stiffness and tension significantly more slowly than red muscle, except for soleus red muscle at 25 degrees C, which showed disproportionately slow rigor development. In each of the examined muscles, stiffness and tension developed more slowly at 25 degrees C than at 37 degrees C. In each specimen, tension always reached its maximum level earlier than stiffness, and then decreased more rapidly and markedly than stiffness. These phenomena may account for the sequential progress of rigor mortis in human cadavers.

The conformation of myosin head domains in rigor muscle determined by X-ray interference.

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T(0)), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T(0)), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T(0), the heads' center of mass moved back by 1.1-1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys(707)-Lys(843) vector and the filament axis increases by approximately 36 degrees between isometric contraction and low-force rigor, and decreases by 7-10 degrees when the rigor fiber is stretched to 0.55 T(0).

Comparison of the histological and histochemical properties of skeletal muscles between carbon dioxide and electrically stunned chickens.

1. Histological and histochemical profiles of Musculus pectoralis (PT, type IIB fibres), M. iliotibialis lateralis (ITL, types IIA + IIB fibres) and M. puboischiofemoralis pars medialis (PIF, type I fibres) were compared in carbon dioxide (37%, 70 s) and electrically (14 V, 5 s) stunned male chickens. 2. Muscle materials were taken at 0, 4 and 24 h from carcases dressed and cooled with ice-water mixture for 30 min. Glycogen and fat contents, and adenosine triphosphatase and reduced nicotinamide adenine dinucleotide dehydrogenase activities of fibres were measured. 3. In PT muscle at 0 h, gas stunned chickens showed many fibres with high glycogen content but those electrically stunned contained few such fibres. Fibres from gas stunned birds had lost almost all their glycogen after 24 h of cold storage. 4. In the ITL muscle of gas stunned chickens at 0 h residual glycogen was observed in type IIB fibres. In contrast, in the electrically stunned birds it was in type IIA, showing the different effects of the stunning methods. During cold storage, glycogen disappeared earlier in type IIB than IIA fibres. 5. In PIF muscle with fibres of low glycogen content, the gas stunned chickens maintained a good fibre structure for 4 h or more, but the electrically stunned had already lost intact fibre structure at 4 h. 6. These results indicated that the carbon dioxide stunning was a better method for chicken welfare and meat quality than electrical stunning.

[Experimental study of restiffening of the rigor mortis].

OBJECTIVE: To observe changes of the length of sarcomere of rat when restiffening. METHODS: We measured the length of sarcomere of quadriceps in 40 rats in different condition by scanning electron microscope. RESULTS: The length of sarcomere of rigor mortis without destroy is obviously shorter than that of restiffening. CONCLUSION: The length of sarcomere is negatively correlative to the intensity of rigor mortis. Measuring the length of sarcomere can determine the intensity of rigor mortis and provide evidence for estimation of time since death.

Experimental study of restiffening of the rigor mortis / 法医学杂志

OBJECTIVE@#To observe changes of the length of sarcomere of rat when restiffening.@*METHODS@#We measured the length of sarcomere of quadriceps in 40 rats in different condition by scanning electron microscope.@*RESULTS@#The length of sarcomere of rigor mortis without destroy is obviously shorter than that of restiffening.@*CONCLUSION@#The length of sarcomere is negatively correlative to the intensity of rigor mortis. Measuring the length of sarcomere can determine the intensity of rigor mortis and provide evidence for estimation of time since death.

Does the sequence of onset of rigor mortis depend on the proportion of muscle fibre types and on intra-muscular glycogen content?

We examined the postmortem changes in the levels of ATP, glycogen and lactic acid in two masticatory muscles and three leg muscles of rats. The proportion of fibre types of the muscles was determined with NIH image software. The ATP levels in the white muscles did not decrease up to 1 h after death, and the ATP levels 1 and 2 h after death in the white muscles were higher than those in the red muscles with a single exception. The glycogen level at death and 1 h after death and the lactic acid level 1 h after death in masticatory muscles were lower than in the leg muscles. It is possible that the differences in the proportion of muscle fibre types and in glycogen level in muscles influences the postmortem change in ATP and lactic acid, which would accelerate or retard rigor mortis of the muscles.

Morphological changes of rats muscles at various postmortem intervals by scanning electron microscopy.

The aim of this study was to observe the morphological changes of muscle in the process of rigor mortis. The quadriceps of 40 rats at various postmortem intervals were observed under the scanning electron microscope (SEM) and the light microscope by phosphotungstic acid-haematoxylin (PTAH) stain. The results showed that the striations of muscle were blurred within 4 h, but they became apparent from 6 h to 24 h after death. The authors suggest that this phenomenon be associated with the increased resistance of muscle against the postmortal changes. The observations by scanning electron microscopy and light microscopy have revealed that the muscles do contract in the process of rigor mortis because the distance between two Z lines shortens and the 1 band narrows, compared with those in anaesthetised animals. The basic biochemical process for the formation of rigor mortis is the same as that of muscle contraction except that the former happens postmortem and the latter antemortem.

Thiophosphorylation of myosin light chain increases rigor stiffness of rabbit smooth muscle.

1. The effect of thiophosphorylation of the regulatory myosin light chain (MLC20) on rigor stiffness was determined in permeabilized rabbit bladder smooth muscle. 2. Rigor stiffness of alpha-toxin-permeabilized smooth muscle was significantly increased by thiophosphorylation of MLC20. This increase may have been due to partial shortening (melting) in the proximal rod region and/or stiffening of the regulatory domain of the myosin head. 3. We suggest that phosphorylation of MLC20, by increasing the stiffness of the S1 lever arm and/or S2 hinge regions of the myosin molecule, favours separation of the two phosphorylated heads and consequent deinhibition of motor domain activity.

[Observations on rat's muscle at various postmortem intervals by scanning electron microscopy].

The aim of this study was to observe the morphological changes of muscle in the process of rigor mortis. The quadriceps of 40 rats at various postmortem intervals were observed under the scanning electron microscope (SEM) and the light microscope by phosphtungstic acid-haematoxylin staining. The results showed that the striations of muscle were blurred within 4 hours, but they became apparent from 6 hours to 24 hours after death. The authors suggest that this phenomenon be associated with the increased resistance of muscle against the postmortal changes. The observations by scanning electron microscopy and light microscopy have revealed that the muscles do contract in the process of rigor mortis because the distance between two Z lines shortens and the I band narrows, compared with those in anaesthetised animals. The basic biochemical process for the formation of rigor mortis is the same as that of muscle contraction except that the former happens postmortem and the latter antemortem.