Environmental enrichment improves growth and fillet quality in rainbow trout.

BACKGROUND: Some environmental enrichment methods, such as occupational enrichment (OE), can improve fish growth, but little is known about its effects on fillet quality. In this study, we evaluated the effects of OE using underwater currents on different aspects of fillet quality and muscle metabolism in rainbow trout (Oncorhynchus mykiss), before and after a handling procedure (fasting). The trout were placed in groups of 30 in separate tanks in three treatments for 30 days: no artificial currents (CON), randomly fired underwater currents (RFC), and continuous underwater currents (CUC). Additionally, half of the individuals in each treatment were fasted (5 days, 45.2 °C days). RESULTS: Slaughter weight, condition factor, and relative growth were lower in CON fish, indicating a positive effect of OE on growth. Rigor mortis, muscle pH, and muscle glycogen levels were similar among treatments, indicating no effect of OE on classical measures of fillet quality. However, significant differences were found regarding fillet colour and muscle enzymes. The fillets of RFC fish were more salmon-pink in colour, which is favoured by consumers. Also, activity levels of pyruvate kinase and glycogen phosphorylase in muscle were significantly higher in CUC fish, probably due to increased energy demands, as pumps were on continually in that treatment. CONCLUSION: Overall, RFC fish seemed to have received enough stimulation to improve growth while not being excessive in terms of exhausting the animals (avoiding negative effects on muscle metabolism), whereas OE may have provided a hormetic effect, allowing fish to better adjust to fasting. © 2023 Society of Chemical Industry.

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.

Ultrasound Elastographic Measurement of Rigor Mortis in an Animal Model: A Feasibility Study for Improved Time-of-Death Estimates in Forensic Investigations.

OBJECTIVE. The purpose of our study was to assess the feasibility of 2D shear wave ultrasound elastography to quantitatively measure changes of rigor mortis. SUBJECTS AND METHODS. Muscle stiffness of two live pigs and nine sacrificed pigs was measured in kilopascals using ultrasound elastography. The nine sacrificed pigs were divided into three groups of three pigs each and placed in one of three environments at 90°F (32°C), 70°F (21°C), or 34°F (1°C). Ultrasound elastography of five muscles was performed at 1- to 2-hour intervals for up to 50 hours postmortem. For each pig and muscle location, the time to start, peak intensity, duration of peak, and time to decline of rigor mortis were identified from the graphs of muscle stiffness values over time. These outcome variables were then compared across ambient temperature, body weight, and age groups using the Wilcoxon rank sum test. RESULTS. Postmortem measurements show a rise, peak, and decline of muscle stiffness after death. Rigor mortis was highly significantly affected by ambient temperature (p < .001), was significantly affected by body weight (p = .04), and was not significantly affected by animal age or muscle location (facial vs truncal vs limb) (p > .50). Peak intensity of rigor mortis developed more quickly but attained lower levels of muscle stiffness at 90°F (80-100 kPa) compared with 70°F and 34°F (280-300 kPa) (p < .001). The duration of peak rigor mortis and the time to decline of rigor mortis were significantly longer for the lower temperatures (p < .001). CONCLUSION. Two-dimensional shear wave ultrasound elastography can quantifi-ably measure the trajectory of rigor mortis in an animal model. This new approach may have direct implications for human forensic investigations.

An evaluation of the objectivity and reproducibility of shear wave elastography in estimating the post-mortem interval: a tissue biomechanical perspective.

Cadaveric rigidity-also referred to as rigor mortis-is a valuable source of information for estimating the time of death, which is a fundamental and challenging task in forensic sciences. Despite its relevance, assessing the level of cadaveric rigidity still relies on qualitative and often subjective observations, and the development of a more quantitative approach is highly demanded. In this context, ultrasound shear wave elastography (US SWE) appears to be a particularly well-suited technique for grading cadaveric rigidity, as it allows non-invasive quantification of muscle stiffness in terms of Young's modulus (E), which is a widely used parameter in tissue biomechanics. In this pilot study, we measured, for the first time in the literature, changes in the mechanical response of muscular tissues from 0 to 60 h post-mortem (hpm) using SWE, with the aim of investigating its applicability to forensic practice. For this purpose, 26 corpses were included in the study, and the muscle mechanical response was measured at random times in the 0-60 hpm range. Despite the preliminary nature of this study, our data indicate a promising role of SWE in the quantitative determination of cadaveric rigidity, which is still currently based on qualitative and semiquantitative methods. A more in-depth study is required to confirm SWE applicability in this field in order to overcome some of the inherent limitations of the present work, such as the rather low number of cases and the non-systematic approach of the measurements.

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.

Survival After Cardiac Arrest With Instantaneous Rigorlike Stiffness: A Case Report.

Instantaneous rigor is the immediate appearance of rigor mortis after cardiac arrest. To our knowledge, no previous reports exist on resuscitation of such patients. A young athlete suddenly collapsed with cardiac arrest during a marathon; his legs stiffened with instantaneous rigorlike stiffness. This stiffening provoked hyperkalemia, rhabdomyolysis, and multiple organ failure. We decided to amputate both legs, with venoarterial extracorporeal membrane oxygenation support. The patient recovered and was discharged without neurologic impairment. This rare case highlights the potentially significant effect of instantaneous rigor.

Effects of three different stunning/slaughtering methods on physical, chemical, and sensory changes in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Electrical stunning (E) and asphyxia in the air (A) are two common stunning/killing methods for fish. However, they can stress the fish and affect flesh quality, so an alternative should be found. The present study aimed to evaluate the effects of carbon monoxide asphyxia (CO) compared to E and A on the evolution of post rigor mortis changes in rainbow trout (Oncorhynchus mykiss) fillets related to chemical, textural, and sensorial properties during storage time. RESULTS: Fish fillets from the E group showed a higher pH (P < 0.05) than the A ones, both at time of rigor resolution (TRR 0) and from TRR 4 until TRR 7 (7 days after rigor resolution, which corresponded to 10 days post mortem), with the CO group being always intermediate. The CO treatment ensured that the fillets showed the lowest yellowness index (b*), even if only at TRR 2 and TRR 5. Lipid oxidation and texture profile analyses were unaffected. Sensory analysis revealed that the CO fillets had the lowest odor intensity and the highest juiciness scores (P < 0.05). The sensory descriptors resulted the most discriminant variables of the fillets obtained from the differently stunned animals, as resulted by the canonical discriminant analysis. CONCLUSION: The carbon monoxide stunning/slaughtering method could be applied to rainbow trout without detrimental effects on physical, chemical, or sensory characteristics of the fish flesh. © 2018 Society of Chemical Industry.

Relationship between the characteristics of rigor-mortis-related actomyosin and muscle fiber types in the ordinary muscle of various fishes.

BACKGROUND: Rigor mortis occurs when muscle extension vanishes through the irresistible coupling of actin and myosin by the consumption of adenosine triphosphate as energy. To clarify the cause of the differences in the progression of rigor mortis, seven fish species were used as samples. The superprecipitation reaction and Mg2+ -ATPase activity of actomyosin in dorsal ordinary muscle were measured, and the slope of the regression line between these two variables was calculated for each fish specimen. The fiber types of the dorsal ordinary muscle in each sample fish were discriminated by the stability of actomyosin ATPase at acid and alkaline preincubations. RESULT: Positive correlations were found between Mg2+ -ATPase activity and the superprecipitation reaction of actomyosin in all 27 fish specimens. The slopes of the regression lines were different not only between fish species but also in fish specimens within the same species. The area ratios of pink muscle fibers and the IIa and/or IIb subtypes of white muscle fibers in the dorsal ordinary muscle were also different between fish species, as well as in specimens within the same fish species. A positive correlation was found between the area ratios of pink muscle fibers in dorsal ordinary muscle and the slopes of the regression line. CONCLUSION: It was suggested that the differences in characteristics of rigor-mortis-related actomyosin of fish might have been caused by the differences in the interposition ratio of muscle fiber types, especially of the pink muscle fiber type, in the dorsal ordinary muscle. © 2019 Society of Chemical Industry.

Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths.

At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the 'roll and lock' mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin.

Re-establishment of rigor mortis: evidence for a considerably longer post-mortem time span.

Re-establishment of rigor mortis following mechanical loosening is used as part of the complex method for the forensic estimation of the time since death in human bodies and has formerly been reported to occur up to 8-12 h post-mortem (hpm). We recently described our observation of the phenomenon in up to 19 hpm in cases with in-hospital death. Due to the case selection (preceding illness, immobilisation), transfer of these results to forensic cases might be limited. We therefore examined 67 out-of-hospital cases of sudden death with known time points of death. Re-establishment of rigor mortis was positive in 52.2% of cases and was observed up to 20 hpm. In contrast to the current doctrine that a recurrence of rigor mortis is always of a lesser degree than its first manifestation in a given patient, muscular rigidity at re-establishment equalled or even exceeded the degree observed before dissolving in 21 joints. Furthermore, this is the first study to describe that the phenomenon appears to be independent of body or ambient temperature.

Carbon monoxide stunning of Atlantic salmon (Salmo salar L.) modifies rigor mortis and sensory traits as revealed by NIRS and other instruments.

BACKGROUND: Methods of stunning used in salmon slaughter are still the subject of research. Fish quality can be influenced by pre-, ante- and post-mortem conditions, including handling before slaughter, slaughter methods and storage conditions. Carbon monoxide (CO) is known to improve colour stability in red muscle and to reduce microbial growth and lipid oxidation in live fish exposed to CO. Quality differences in Atlantic salmon, Salmo salar L., stunned by CO or percussion, were evaluated and compared by different techniques [near infrared reflectance spectroscopy (NIRS), electronic nose (EN), electronic tongue (ET)] and sensory analysis. RESULTS: Thawed samples, freeze-dried preparates and NIRS devices proved to be the most efficient combinations for discriminating the treatments applied to salmon, i.e. first the stunning methods adopted, then the back-prediction of the maximum time to reach rigor mortis and finally to correlate some sensory attributes. A trained panel found significant differences between control and CO-stunned salmon: reduced tactile crumbliness, reduced odour and aroma intensities, and reduced tenderness of CO-treated fillets. CO stunning reduced radiation absorbance in spectra of thawed and freeze-dried fillets, but not fillet samples stored in ethanol, where it may have interacted with myoglobin and myosin. CONCLUSIONS: The good results in a rapid discrimination of thawed samples detected by NIRS suggest suitable applications in the fish industry. CO treatment could mitigate sensory perception, but consumer tests are needed to confirm our findings. © 2015 Society of Chemical Industry.

Post mortem rigor development in the Egyptian goose (Alopochen aegyptiacus) breast muscle (pectoralis): factors which may affect the tenderness.

BACKGROUND: Baseline research on the toughness of Egyptian goose meat is required. This study therefore investigates the post mortem pH and temperature decline (15 min-4 h 15 min post mortem) in the pectoralis muscle (breast portion) of this gamebird species. It also explores the enzyme activity of the Ca(2+)-dependent protease (calpain system) and the lysosomal cathepsins during the rigor mortis period. RESULTS: No differences were found for any of the variables between genders. The pH decline in the pectoralis muscle occurs quite rapidly (c = -0.806; ultimate pH ∼ 5.86) compared with other species and it is speculated that the high rigor temperature (>20 °C) may contribute to the increased toughness. No calpain I was found in Egyptian goose meat and the µ/m-calpain activity remained constant during the rigor period, while a decrease in calpastatin activity was observed. The cathepsin B, B & L and H activity increased over the rigor period. CONCLUSION: Further research into the connective tissue content and myofibrillar breakdown during aging is required in order to know if the proteolytic enzymes do in actual fact contribute to tenderisation.

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.

Myosin S2 origins track evolution of strong binding on actin by azimuthal rolling of motor domain.

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosin's α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location. However, previous studies did not adequately resolve the S2 domain. Here we used electron tomography of rigor muscle swollen by low ionic strength to pull S2 clear of the thick filament backbone, thereby revealing the azimuth of its point of origin. The results show that the azimuth of S2 origins of those rigor myosin heads, bound to the actin target zone of actively contracting muscle, originate from a restricted region of the thick filament. This requires an azimuthal reorientation of the motor domain on actin during the weak to strong transition.

The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation.

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 µM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.

Estimation of the post-mortem interval; added value of mechanical excitability of human skeletal muscle.

Estimation of the post-mortem interval (PMI) is mainly based on the state of body cooling, post-mortem lividity (livor mortis) and post-mortem muscle stiffness (rigor mortis). However, the time span of development of these post-mortem phenomena are influenced by a variety of factors concerning the body of the deceased and the environment in which the body is found. Subsequently, this leads to a substantial spread in upper and lower limits of PMI based on determination of the state of these phenomena. Moreover, interpretation of post-mortem phenomena like lividity, rigor and interpretation of the correction factor for Henssge's nomogram is subjective. For this reason, PMI estimations are often broad, possibly too broad to be helpful for answering questions which are relevant for the criminal investigation. Therefore, combining the outcome of different methods for estimating the PMI, the so-called compound method, is recommended. Supravital muscle reaction by mechanical stimulation of skeletal muscle is a less known aspect of the compound method. Here we present a series of cases series in which supravital muscle reaction contributed to a more precise estimation of the PMI.

A case of instantaneous rigor?

The question of whether instantaneous rigor mortis (IR), the hypothetic sudden occurrence of stiffening of the muscles upon death, actually exists has been controversially debated over the last 150 years. While modern German forensic literature rejects this concept, the contemporary British literature is more willing to embrace it. We present the case of a young woman who suffered from diabetes and who was found dead in an upright standing position with back and shoulders leaned against a punchbag and a cupboard. Rigor mortis was fully established, livor mortis was strong and according to the position the body was found in. After autopsy and toxicological analysis, it was stated that death most probably occurred due to a ketoacidotic coma with markedly increased values of glucose and lactate in the cerebrospinal fluid as well as acetone in blood and urine. Whereas the position of the body is most unusual, a detailed analysis revealed that it is a stable position even without rigor mortis. Therefore, this case does not further support the controversial concept of IR.

Slaughter of Atlantic salmon (Salmo salar L.) in the presence of carbon monoxide.

The different stunning methods for Atlantic salmon can still be improved with regard to animal welfare. Salmon exposed to carbon monoxide expressed no aversive reactions towards CO as such. CO exposed fish showed an earlier onset of rigour mortis and a faster decrease in muscle pH due to depletion of oxygen during the treatment. Exposure to CO did increase the level of cortisol compared to undisturbed control fish, but the increase was less than in the water only control group. Neuroglobin, a CO binding globin, was found in salmon brain and Saccus vasculosus, a richly vascularized sac connected to the fish brain. Binding of CO to neuroglobin during sedation might possibly improve animal welfare.

A comprehensive review of the spoilage of shrimp and advances in various indicators/sensors for shrimp spoilage monitoring.

Shrimp is a popular internationally traded shellfish due to its unique taste, texture, and nutritional value. Shrimp is highly perishable because it has enough free amino acids, high moisture levels, non-nitrogenous compounds used for microbial growth, and melanosis. Shrimp spoilage after death is caused by various reasons, like autolysis (endogenous proteinases actions during shrimp storage), growth of spoilage microorganisms, ATP degradation, melanin formation, and lipid peroxidation. A microbial byproduct, total volatile basic nitrogen, is one of the major reasons for the generation of foul odors from shrimp spoilage. Shrimp freshness monitoring is crucial for market sellers and exporters. Traditional methods for estimating shrimp freshness are expensive and inaccessible to the general public. Sensors are rapid, sensitive, selective, and portable food toxins' detection tools, devoid of expensive instruments, skilled people, sample pretreatment, and a long detection time. This review addresses shrimp spoilage causes. The mechanisms of different stages of shrimp spoilage after death, like rigor mortis, dissolution of rigor mortis, autolysis, and microbial spoilage mechanisms, are discussed. This review highlights the last five years' advances in shrimp freshness detection sensors and indicators like colorimetric pH indicators, fluorescence sensors, electronic noses, and biosensors, their working principles, and their sensitivities. Commercially available indicators and sensors for shrimp spoilage monitoring are also discussed. A review highlighting the applications of the different sensors and indicators for monitoring shrimp freshness is unavailable to date. Challenges and future perspectives in this field are explained at the end.