To investigate the changes in the volatile flavor compounds and the characteristic components responsible for flavor deterioration in the muscle of Litopenaeus vannamei under cold storage, the odor profiles of shrimp stored at 4 ℃ for 0–4 days were quantitatively and qualitatively analyzed using an electronic nose and headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). Key flavor compounds were selected based on their relative odor activity values (ROAVs), and differential compounds were analyzed using a partial least squares discriminant analysis (PLS-DA) model. The results showed that the electronic nose sensors W1W, W6S, and W3C exhibited the strongest responses and HS-GC-IMS identified 14 volatile flavor compounds, with aldehydes, ketones, pyrazines, and sulfur-containing compounds markedly accumulating in the late storage period. PLS-DA highlighted abhexone, safranal, acetone, and pentan-1-ol as key volatile compounds. These findings reveal key chemical components contributing to flavor deterioration in cold-stored shrimp, providing a scientific basis for understanding the mechanism of flavor deterioration and developing targeted control strategies.

In the present study, in order to improve the reliability of Western blotting results, the effects of extraction buffers, grinding methods, inhibitor type and concentration, and extraction time on protein extraction from the hepatopancreas of Litopenaeus vannamei were explored by considering protein dissolution rate and the completeness of protein bands in electrophoretic gels. Next, the expression levels of apoptosis-related proteins in the hepatopancreas of Litopenaeus vannamei were detected with Western blotting. The results showed that the protein dissolution rate was higher using RIPA lysis buffer compared with phosphate buffer saline (PBS) or water as the extraction solvent. Electric homogenization and liquid nitrogen grinding provided more complete protein bands. The degradation of proteins by endogenous proteases was obviously suppressed by addition of 4% protease and phosphatase inhibitor cocktail. In addition, Western blotting results revealed that in addition to inducing Litopenaeus vannamei into dormancy, low temperatures induced slight apoptosis in the hepatopancreas tissue. Furthermore, the apoptosis level increased with waterless duration and this effect was alleviated after removal of environmental stress.

In order to explore the mechanism of oxidative and immune injury by which combined stress of acute cold exposure and air exposure during waterless live transport induces the death of Litopenaeus vannamei, oxidant stress injury and changes in related immune system indicators in the hepatopancreas in response to the combined stress were analyzed under conditions simulating actual transportation. The results showed that compared to the control group, the survival rate declined after acute cold + 9 h waterless stress. The level of reactive oxygen species (ROS) significantly increased in response to acute cold stress (P < 0.05), but declined in response to subsequent waterless stress. Meanwhile, the content of malondialdehyde (MDA) in the cold stress and combined stress groups was significantly higher than that in the control group (P < 0.05), and the maximum value was observed in the acute cold + 3 h waterless stress group. In addition, the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) increased with air exposure duration, reaching 2.82, 2.12 and 5.26 U/mg at 9 h, respectively. After 2 h resuscitation, they nearly returned to normal. In the combined stress group, the activities of the non-specific immune enzymes phenol oxidase (PO), peroxidase (POD) showed a downward trend, while the activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) increased incessantly with air exposure duration, reaching their maximum at 9 h and nearly returning to normal after resuscitation. Furthermore, histopathological examination showed that slight decomposition of epithelial tubules and partial exfoliation of cells in the inner wall appeared in the acute cold + 9 h waterless stress group, which indicated that the defense system was damaged. As L. vannamei can increase its antioxidant enzyme activities and non-specific immune activities in response to environmental stress, intolerable stress can cause irreversible damage to the hepatopancreas and ultimately cause the death of L. vannamei.

To elucidate the physiological regulation mechanism of Penaeus vannamei in response to combined stress of acute cold exposure and waterless duration, and provide a solid foundation for waterless live transport management and improvement in the survival rate, the effects of acute cold exposure combined with waterless duration on the metabolic homeostasis and hepatopancreas histopathology of Penaeus vannamei were explored under conditions simulating waterless live transport. The results showed that the level of lactic acid (LD) in the hemolymph and the activity of lactate dehydrogenase (LDH) in the hepatopancreas and muscle increased, the activities of LDH, hexokinase (HK), succinate dehydrogenase (SDH), phosphofructokinase (PFK) and Na⁺/K⁺-ATPase in the hemolymph increased initially and then decreased with increasing waterless duration, while the contents of adenosine triphosphate (ATP) and glycogen decreased. After resuscitation, the concentrations of glucose (Glu) and LD in the hemolymph were (23.92 ± 0.59) and (6.27 ± 0.32) mmol/L, respectively, and were still significantly higher than those in the normal control group (P < 0.05); the contents of ATP and glycogen in muscle tissues were (4.88 ± 0.31) μmol/g and (1.74 ± 0.10) mg/g, respectively, and were significantly lower than those in the normal control group (P < 0.05). The other indices tested returned to the normal levels. The glycolysis reaction of Penaeus vannamei accelerated with increasing waterless duration from 0 to 3 h, and aerobic respiration was the major respiration pattern. After 3 h, the gluconeogenesis reaction was strengthened, and the anaerobic metabolism was dominant. A waterless duration of 9 h resulted in metabolic disorder, but it could be restored after resuscitation. These findings show that the combined stress of acute cold exposure and waterless duration results in compensatory adjustments in Penaeus vannamei and causes damage to the structure of hepatopancreas, which will provide a theoretical basis for targeted control of metabolism imbalance.

To clarify the key material basis for the rapid softening of shrimp muscle during refrigeration, this study established a specific histological detection method for cytoskeletal proteins and analyzed the correlation between the degradation of cytoskeletal proteins and texture deterioration. By comparing the effects of different fixatives, dehydration modes, clarification times, and staining parameters on the staining of cytoskeletal proteins, it was found that the suitable fixative solution for shrimp muscle tissue was Carnoy’s fixative; the specific staining process encompassed five steps: 1) 15 h fixation at room temperature, 2) dehydration with a series of gradient ethanol concentrations, 3) sequential clarification with a mixture of xylene and ethanol followed by xylene, 4) paraffin embedding for sectioning, 5) and Masson’s trichrome staining, clearly showing the morphology of nuclei, myofibrils, and collagen fibers. The pathological results showed that the gap between muscle fiber bundles increased with refrigeration time, and the structure of myofibrillar bundles was partially destroyed. The color of stained collagen fibers became gradually lighter indicating a decrease in its amount. Quantitative analysis showed that refrigeration led to a significant decrease in the volume fraction of collagen fibers and myofibrils in muscle tissue (P < 0.05). The volume fraction of myofibrils in cross-section and longitudinal sections decreased from (91.03 ± 1.37)% and (93.39 ± 0.91)% to (41.14 ± 3.78)% and (24.36 ± 3.67)%, respectively, over a 5-day refrigeration period. The cross-sectional and longitudinal areas of shrimp myofibrils and collagen fibers were significantly positively correlated with muscle hardness and elasticity (P < 0.05), indicating that the reduction in myofibril and collagen contents during refrigeration was closely related to texture deterioration. The significant correlation between the structural degradation of cytoskeletal proteins and texture deterioration revealed their key role in texture preservation and was an important target for early monitoring and intervention of muscle softening.