Formation of the calpain-1/calpastatin complex promotes activation of calpain-1 under oxidizing conditions.

Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.

Protein degradation in skeletal muscle is a key component of protein turnover and maintenance of muscle function. Protein degradation in postmortem muscle is commonly observed and is associated with the accumulation of degradation products and improved meat tenderness. Because there is significant evidence that calpain-1 is involved with proteolysis of muscle proteins in both situations, defining the factors that regulate calpain activity will position scientists to improve calpain-1 activity in both contexts. Calpain-1 is a neutral calcium-dependent proteinase that is inhibited by calpastatin, oxidation, and slightly acidic pH environments. Because oxidation of the calpain/calpastatin complex with hydrogen peroxide appeared to activate calpain-1, we hypothesize that calpastatin binding to calpain may protect the active site cysteine. In the current study, we tested this hypothesis and investigated how n-ethyl maleimide (NEM), an alkylating agent, affects the regulation of calpain in the presence and absence of calpastatin molecules. The results suggest that calpastatin can protect calpain-1 from reacting with maleimide-polyethylene glycol but that exposure of calpain-1/calpastatin complex to NEM or hydrogen peroxide resulted in autolysis and activation of calpain. Under some circumstances, calpastatin appears to protect calpain-1 from inhibition by modification of active site cysteine. These novel observations show a different role for calpastatin and give reason to interpret calpastatin abundance and activity data in a different light.

Secondary Lipid Oxidation Products as Modulators of Calpain-2 Functionality In Vitro.

The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and localization of modification sites. 2-Hexenal and 4-hydroxynonenal incubation significantly decreased calpain-2 activity and slowed the progression of autolysis, while malondialdehyde had minimal impact on calpain-2 activity and autolysis. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde modification sites on the calpain-2 catalytic and regulatory subunits. 2-Hexenal modification sites were observed on the calpain-2 catalytic subunit. Intact protein mass analysis with MALDI-MS revealed that a significant number of modifications on the calpain-2 catalytic and regulatory subunits are likely to exist. These observations confirm that specific lipid oxidation products modify calpain-2 and may affect the calpain-2 functionality. The results of these novel experiments have implications for healthy tissue metabolism, skeletal muscle growth, and post-mortem meat tenderness development.

Distinct myofibrillar sub-proteomic profiles are associated with the instrumental texture of aged pork loin.

Fresh pork tenderness contributes to consumer satisfaction with the eating experience. Postmortem proteolysis of proteins within and between myofibrils has been closely linked with pork tenderness development. A clear understanding of the molecular features associated with pork tenderness development will provide additional targets and open the door to new solutions to improve and make pork tenderness development more consistent. Therefore, the objective was to utilize liquid chromatography and mass spectrometry with tandem mass tag (TMT) multiplexing to evaluate myofibrillar sub-proteome differences between pork chops of different instrumental star probe values. Pork loins (N = 120) were collected from a commercial harvest facility at 24 h postmortem. Quality and sensory attributes were evaluated at 24 h postmortem and after ~2 weeks of postmortem aging. Pork chops were grouped into 4 groups based on instrumental star probe value (group A,x¯â€…= 4.23 kg, 3.43 to 4.55 kg; group B,x¯â€…= 4.79 kg, 4.66 to 5.00 kg; group C,x¯â€…= 5.43 kg, 5.20 to 5.64 kg; group D,x¯â€…= 6.21 kg, 5.70 to 7.41 kg; n = 25 per group). Myofibrillar proteins from the samples aged ~2 wk were fractionated, washed, and solubilized in 8.3 M urea, 2 M thiourea, and 1% dithiothreitol. Proteins were digested with trypsin, labeled with 11-plex isobaric TMT reagents, and identified and quantified using a Q-Exactive Mass Spectrometer. Between groups A and D, 54 protein groups were differentially abundant (adjusted P < 0.05). Group A had a greater abundance of proteins related to the thick and thin filament and a lesser abundance of Z-line-associated proteins and metabolic enzymes than group D chops. These data highlight that distinct myofibrillar sub-proteomes are associated with pork chops of different tenderness values. Future research should evaluate changes immediately and earlier postmortem to further elucidate myofibrillar sub-proteome differences over the postmortem aging period.

A primary goal of meat production is to efficiently produce safe, high-quality products. Competing interests within the goal complicate this seemingly simple aspiration. Consequently, it is necessary to emphasize efforts to enhance our comprehension of biological and molecular factors that influence quality, safety, and efficient meat production. This experiment aimed to define the proteomic profiles of the myofibrillar fraction of fresh pork with differing quality traits. Myofibrils from aged pork chops with a range of tenderness levels were used to achieve this objective. Fifty-four proteins were differentially abundant between the divergent tenderness groups. This was due to the expression profile of proteins in muscle and/or changes in proteins in the myofibrillar fraction during postmortem aging. These results inform and direct the development of antemortem and postmortem applications to ensure success in producing high-quality pork.

Matrix metalloproteinase- 9 may contribute to collagen structure modification during postmortem aging of beef.

Matrix metalloproteinases (MMPs) are responsible for the turnover of intramuscular connective tissue in live animals. We hypothesize that MMPs may play a role in postmortem aging of beef muscles for the degradation of connective tissues. Four different experiments were performed to: 1) characterize MMP activity during postmortem aging of beef; 2) determine if the native beef MMP can contribute to connective tissue degradation in a simulated standard industry postmortem aging condition; 3) explore approaches to improve the native beef MMP activity and 4) characterize MMP activity in beef from cattle supplemented with supranutritional level of Zn. In experiment 1, MMP was active throughout the entire aging periods (3, 21, 42 and 63 d) for beef muscles Longissimus lumborum, Gluteus medius and Gastrocnemius, and the unknown MMP responsible for the collagen degradation was identified as MMP-9 by Western Blot. In experiment 2 and 3, MMP-9 activity was noticeable in the gels after 42 d of storage in the cooler. Moreover, the addition of ZnCl2 in the model system significantly increased MMP-9 activity when compared to the control (P < 0.01). In experiment 4, Longissimus thoracis from animals supplemented with a supranutritional Zn level had increased Zn availability and MMP-9 activity than those from animals fed with a control diet (P < 0.05). Further research is needed better understand MMP-9 mechanism during postmortem aging of meat. With a better understanding of MMP-9 in the aging process, the beef industry can provide better connective tissue management strategies for lower-quality beef cuts.

Lipid Peroxidation Products Influence Calpain-1 Functionality In Vitro by Covalent Binding.

The objective of the current study was to evaluate the effects of lipid peroxidation products, malondialdehyde (MDA), hexenal, and 4-hydroxynonenal (HNE), on calpain-1 function, and liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification of adducts on calpain-1. Calpain-1 activity slightly increased after incubation with 100 µM MDA but not with 500 and 1000 µM MDA. However, calpain-1 activity was lowered by hexenal and HNE at 100, 500, and 1000 µM. No difference in calpain-1 autolysis was observed between the control and 1000 µM MDA. However, 1000 µM hexenal and HNE treatments slowed the calpain-1 autolysis. Adducts of MDA were detected on glutamine, arginine, lysine, histidine, and asparagine residues via Schiff base formation, while HNE adducts were detected on histidine, lysine, glutamine, and asparagine residues via Michael addition. These results are the first to demonstrate that lipid peroxidation products can impact calpain-1 activity in a concentration-dependent manner and may impact the development of meat tenderness postmortem.

Effect of postmortem pH on the physical, biochemical, and sensory characteristics of bovine longissimus thoracis et lumborum muscle.

From a large feeding trial study consisting of 299 bulls and steers, 15 carcasses exhibited stress-related syndromes manifested by atypical color and pH which were then selected for subsequent analysis. Samples of longissimus thoracis et lumborum muscle with postmortem pH in the range of 5.5-6.9 were subjected to a 14-day aging period at 2°C. Sensory panel tenderness, connective tissue, juiciness, and flavor intensity of high pH (6.4-6.9) meat were significantly different (p < .05) from samples of intermediate pH (6.0-6.1) as well as normal pH (5.5). Muscles at pH 6.0-6.1 were the toughest samples. This was confirmed by Warner-Bratzler shear force (WBSF), residual force, and myofibril fragmentation index. Palatability attributes of normal pH (5.5) samples were significantly different (p < .05) from dark-cutting beef in terms of tenderness and flavor and at the high pH extreme. The increase in WBSF at pH 6.0-6.1, lack of extensive degradation of muscle proteins, and the decreased sarcomere length resulted in tougher meat than low or high pH muscles. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis of meat at the high pH extreme (6.7-6.9) revealed that the breakdown of troponin-T to 30 kD was complete while at intermediate pH (6.0-6.1) was incomplete. In addition, the appearance of a 'doublet' on high-molecular-weight resolution gels may also account for the greater tenderness experienced by sensory panelists.

Characterizing the sarcoplasmic proteome of aged pork chops classified by purge loss.

Unpredictable variation in quality, including fresh pork water-holding capacity, remains challenging to pork processors and customers. Defining the diverse factors that influence fresh pork water-holding capacity is necessary to make progress in refining pork quality prediction methods. The objective was to utilize liquid chromatography and mass spectrometry coupled with tandem mass tag (TMT) multiplexing to evaluate the sarcoplasmic proteome of aged pork loins classified by purge loss. Fresh commercial pork loins were collected, aged 12 or 14 d postmortem, and pork quality and sensory attributes were evaluated. Chops were classified into Low (N = 27, average purge = 0.33%), Intermediate (N = 27, average purge = 0.72%), or High (N = 27, average purge = 1.19%) chop purge groups. Proteins soluble in a low-ionic strength buffer were extracted, digested with trypsin, labeled with 11-plex isobaric TMT reagents, and detected using a Q-Exactive Mass Spectrometer. Between the Low and High purge groups, 40 proteins were differentially (P < 0.05) abundant. The Low purge group had a greater abundance of proteins classified as structural and contractile, sarcoplasmic reticulum and calcium regulating, chaperone, and citric acid cycle enzymes than the High purge group. The presence of myofibrillar proteins in the aged sarcoplasmic proteome is likely due to postmortem degradation. These observations support our hypothesis that pork chops with low purge have a greater abundance of structural proteins in the soluble protein fraction. Together, these and other proteins in the aged sarcoplasmic proteome may be biomarkers of pork water-holding capacity. Additional research should establish the utility of these proteins as biomarkers early postmortem and over subsequent aging periods.

Fresh pork can vary in its ability to retain water­commonly termed as its water-holding capacity­where a greater water-holding capacity means it retains more water as it is cut, packaged, and stored. However, commercial pork loins have considerable variability in their water-holding capacity, which can impact the consumer's eating experience. This study aimed to examine water-soluble proteins from aged commercial pork chops and to identify and quantify these proteins with mass spectrometry to confirm the previous observation that the degradation of specific structural proteins is associated with greater water-holding capacity. This analysis identified 40 proteins differentially abundant between pork chops with varying water-holding capacities. Pork chops with greater water-holding capacity had a greater abundance of proteins classified as structural and contractile, calcium regulating, and chaperone. Metabolic proteins were also differentially abundant in aged pork loins with differing water-holding capacity. This study confirmed previous observations that the degradation of key structural proteins is associated with greater water-holding capacity while identifying new proteins that may be biomarkers for water-holding capacity.

Early postmortem muscle proteome and metabolome of beef longissimus thoracis muscle classified by pH at 6 hours postmortem.

The objective was to identify metabolome and proteome differences at 1 h and 1 d postmortem between longissimus thoracis (LT) muscle classified based on 6 h pH values. Twenty beef LT rib sections were sorted based on 6 h postmortem pH values into low (LpH; pH < 5.55; n = 9) and high (HpH; pH > 5.84; n = 8) pH classifications. Warner-Bratzler shear force (WBSF), desmin degradation, and calpain-1 autolysis were measured. Two-dimensional difference in gel electrophoresis (3-10, 4-7, and 6-9 pH range) and Tandem mass tagging (TMT) protein analyses were employed to determine how the sarcoplasmic protein profile varied across pH classification. Non-targeted metabolomic analyses were conducted on extracts prepared at 1 h and 1 d postmortem. The LpH classification had a lower WBSF value at 1 d postmortem, which was explained by greater calpain-1 autolysis and desmin degradation at 1 d postmortem. Proteome and metabolome analysis revealed a phenotype that promotes more rapid energy metabolism in the LpH group. Proteome and metabolome analyses identified energy production, apoptotic, calcium homeostasis, and proteasome systems influencing pH classifications that could explain the observed pH, proteolysis, and beef tenderness differences. SIGNIFICANCE: This study is the first to identify proteomic and metabolomic variations early (1 h and 1 day) postmortem that are linked to differences in early (6 h) postmortem pH values and to tenderness differences at 1 day postmortem. This study integrates postmortem biochemical features (protein degradation, proteome, and metabolome variations) to postmortem pH decline and eating quality of beef steaks. Potential biomarkers of more rapid postmortem metabolism linked to earlier tenderization in beef are suggested. Identification of these biochemical features will assist in predicting the eating quality of beef products.

Nutritional approaches to slow late finishing pig growth: implications on carcass composition and pork quality.

Although pork producers typically aim to optimize growth rates, occasionally it is necessary to slow growth, such as when harvest facility capacity is limited. In finishing pigs, numerous dietary strategies can be used to slow growth so pigs are at optimal slaughter body weights when harvest facility capacity and/or access is restored. However, the impact of these diets on pork carcass quality is largely unknown. Thus, this study aimed to evaluate the efficacy of dietary strategies to slow growth in late finishing pigs and evaluate their effects on carcass composition and pork quality. Mixed-sex pigs (n = 897; 125 ± 2 kg BW) were randomly allotted across 48 pens and assigned to 1 of 6 dietary treatments (n = 8 pens/treatment): (1) Control diet representative of a typical finisher diet (CON); (2) diet containing 3% calcium chloride (CaCl2); (3) diet containing 97% corn and no soybean meal (Corn); (4) diet deficient in isoleucine (LowIle); (5) diet containing 15% neutral detergent fiber (NDF) from soybean hulls (15% NDF); and (6) diet containing 20% NDF from soybean hulls (20% NDF). Over 42 d, pen body weights and feed disappearance were collected. Pigs were harvested in 3 groups (14, 28, and 42 d on feed) and carcass data collected. From the harvest group, 1 loin was collected from 120 randomly selected carcasses (20 loins/treatment) to evaluate pork quality traits. Overall, ADG was reduced in CaCl2, Corn, and 20% NDF pigs compared with CON pigs (P < 0.001). However, ADFI was only reduced in CaCl2 and 20% NDF pigs compared with CON (P < 0.001). Feed efficiency was reduced in CaCl2 and Corn pigs compared with CON (P < 0.001). Hot carcass weights were reduced in CaCl2 pigs at all harvest dates (P < 0.001) and were reduced in Corn and 20% NDF pigs at days 28 and 42 compared with CON pigs (P < 0.001). In general, CaCl2 and 20% NDF diets resulted in leaner carcasses, whereas the Corn diet increased backfat by 42 d on test (P < 0.05). Loin pH was reduced and star probe increased in CaCl2 pigs compared with CON pigs (P < 0.05); no treatments differed from CON pigs regarding drip loss, cook loss, color, firmness, or marbling (P ≥ 0.117). Overall, these data indicate that several dietary strategies can slow finishing pig growth without evidence of behavioral vices. However, changes to carcass composition and quality were also observed, indicating quality should be taken into consideration when choosing diets to slow growth.

Feeding oxidized chicken byproduct meal impacts digestibility more than performance and oxidative status in nursery pigs.

Rendered products from the meat industry provide quality proteins in diets for companion animals. These proteins are exposed to extreme temperatures during processing leading to the potential for decreased diet digestibility and subsequent growth performance. While this would impact production efficiency in livestock species, oxidized ingredients in companion animal diets may impact health and longevity. The objective of this study was to determine the extent to which a feedstuff containing oxidized protein and lipid affect diet digestibility, growth performance, and oxidative stress in nursery pigs. A total of 56 male pigs (21 d of age, initial body weight 5.51 ± 0.65 kg) were randomly assigned to one of the four dietary treatments in a 2 × 2 factorial arrangement with two levels of heat and two levels of antioxidant (AOX). Diets were fed for 35 d and growth performance was measured, while total tract digestibility and nitrogen (N) balance was determined during the trial on day 18-20. Blood plasma was collected on day 34 and jejunum, colon, and liver tissues were collected on day 35 to analyze for markers of oxidative stress. Average daily feed intake (ADFI) was reduced in pigs fed diets without AOXs (P = 0.02). Additionally, pigs consuming diets containing heated chicken byproduct (CBP) meal had decreased gain:feed (GF; P = 0.02). There was an interaction between heat and AOX (P = 0.02) where heating CBP reduced N digestibility in the presence of an AOX but did not have an impact when AOX was not present. The removal of AOX resulted in reduced GE digestibility (P < 0.01). Dry matter (P < 0.01), ash (P < 0.01), and protein (P < 0.01) digestibility were reduced (P < 0.01) as a result of heating. Furthermore, heating (P =0.01) as well as absence of AOX (P =0.01) resulted in reduced digestible energy. No difference was detected in N retention suggesting that oxidation reduces digestibility but has no impact on N utilization. This is supported by the fact that systemic oxidative stress was not consistently affected by heating or AOX inclusion. These results suggest that feeding pigs CBP containing oxidized proteins and lipids did not induce oxidative stress. However, feeding young pigs CBP containing oxidized proteins and lipids did result in reduced energy and nutrient digestibility as well as negatively affected feed efficiency. Because CBP is commonly used in companion animal diets, it is reasonable to revisit their impacts on those species.

Impact of storage conditions on protein oxidation of rendered by-product meals.

Rendered products used in animal feed and pet food undergo extreme temperatures during manufacturing and may be stored up to 2 yr. No information is available on protein oxidation in these products. The objective of this study was to determine the extent to which typical antioxidant inclusion at different storage conditions may limit protein oxidation in typical rendered protein meals. Two experiments were conducted on 14 rendered products stored at either 45 °C for 7 or 14 d, or at 20 °C for 3 or 6 mo to determine the extent to which time, temperature, and antioxidants affect protein oxidation. Results from this study show that fish meal and chicken blood meal are susceptible to protein oxidation during storage at 45 °C (P = 0.05; 0.03) as well as during storage at 20 °C (P = 0.01; 0.04). Natural antioxidants were effective at limiting carbonyl formation in fish meal during short-term storage at 45 °C, whereas ethoxyquin was effective at limiting the extent of protein oxidation in fish meal stored long term at 20 °C.

Impact of dietary oxidized protein on oxidative status and performance in growing pigs.

Rendered products from the meat industry can provide economical quality sources of proteins to the animal and feed industry. Similar to lipids, rendered proteins are susceptible to oxidation, yet the stability of these proteins is unclear. In addition, interest in understanding how oxidative stress can impact efficiency in production animals is increasing. Recent studies show that consumption of oxidized lipids can lead to a change in the oxidative status of the animal as well as decreases in production efficiency. To date, little is known about how consumption of oxidized proteins impacts oxidative status and growth performance. The objectives of this study were to determine if feeding diets high in oxidized protein to growing pigs would: 1) impact growth performance and 2) induce oxidative stress. Thirty pigs (42 d old; initial body weight [BW] 12.49 ± 1.45 kg) were randomly assigned to one of three dietary treatments with increasing levels of oxidized protein. Spray-dried bovine plasma was used as the protein source and was either unheated upon arrival, heated at 45 °C for 4 d, or heated at 100 °C for 3 d. Diets were fed for 19 d and growth performance was measured. Blood plasma (days 0 and 18), jejunum, colon, and liver tissues (day 19) were collected to analyze for markers of oxidative stress (e.g., protein oxidation, lipid oxidation, DNA damage, and glutathione peroxidase activity). Average daily gain (ADG;P < 0.01) and average daily feed intake (ADFI;P < 0.01) had a positive linear relationship to increased protein oxidation, but there was no effect on gain to feed ratio. Furthermore, protein (P = 0.03) and fat (P < 0.01) digestibility were reduced with increased protein oxidation in the diet. Crypt depth showed a positive linear relationship with dietary protein oxidation levels (P = 0.02). A trend was observed in liver samples where pigs fed the plasma heated to 45 °C had increased lipid oxidation compared with pigs fed the plasma either unheated or heated to 100 °C (P = 0.09). DNA damage in the jejunum tended to have a linear relationship with the dietary protein oxidation level (P = 0.07). Even though results suggest dietary oxidized protein did not induce oxidative stress during short-term feeding, differences in performance, gut morphology, and digestibility are likely a result of reduced protein availability.

Effect of a dual enteric and respiratory pathogen challenge on swine growth, efficiency, carcass composition, and pork quality1.

The objective of this study was to determine the influence of a dual respiratory and enteric pathogen challenge on growth performance, carcass composition, and pork quality of high and low feed efficient pigs. Pigs divergently selected for low and high residual feed intake (RFI, ~68 kg) from the 11th generation of Iowa State University RFI project were used to represent high and low feed efficiency. To elicit a dual pathogen challenge, half of the pigs (n = 12/line) were inoculated with Mycoplasma hyopneumoniae (Mh) and Lawsonia intracellularis (MhLI) on days post-inoculation (dpi) 0. Pigs in a separate room of the barn were not inoculated and used as controls (n = 12/RFI line). Pigs were weighed and feed intake was recorded to calculate ADG, ADFI, and G:F for the acclimation period (period 1: dpi -21 to 0), during peak infection (period 2: dpi 0 to 42), and during the remaining growth period to reach market weight (period 3: dpi 42 to harvest). At ~125 kg, pigs were harvested using standard commercial procedures. Carcasses were evaluated for composition (weight, fat free lean, loin eye area, 10th rib fat depth) and meat quality (pH decline, temperature decline, Hunter L, a, and b, subjective color and marbling, star probe, drip loss, cook loss, proximate composition, and desmin degradation). Challenged pigs had lesser ADFI than controls during period 2 (P < 0.05), but had greater ADG and G:F during period 3 (P < 0.05). Selection for feed efficiency did not result in a differential response to MhLI (P > 0.05). Loin chops from the less feed efficient, high RFI pigs, had greater drip loss, greater cook loss, lesser moisture content, greater Hunter L values, and greater Hunter b values (P < 0.05) than loin chops from low RFI pigs. Infection status did not significantly affect carcass composition or pork quality traits (P > 0.05). These results indicate that a MhLI challenge early in growth did not significantly affect ultimate carcass composition or meat quality traits. Selection for greater feed efficiency in pigs did not affect their response to pathogenic challenge.

Association of calpain and calpastatin activity to postmortem myofibrillar protein degradation and sarcoplasmic proteome changes in bovine Longissiumus lumborum and Triceps brachii.

The aim of this study was to determine the extent to which calpastatin (CASN) variants (based on two chromatographic peaks; CASN-P1 and CASN-P2) explain variation in µ-calpain autolysis, protein degradation, and changes in the sarcoplasmic proteome observed during postmortem aging of beef. The Longissimus lumborum (LL) and Triceps brachii (TB) muscles were obtained from six crossbred steers and samples prepared from day 0, 1 and 7 postmortem (pm). The decline of CASN activity during aging was due to decrease of CASN-P2 in both muscles. The CASN-P2:µ-calpain ratio at day 0 was greater for TB, which presented lesser calpain autolysis, myofibrillar protein degradation, and fewer sarcoplasmic proteome changes during aging. Changes in abundance of Heat shock protein 70 family in the sarcoplasmic fraction were positively associated to proteolysis during aging, with greater differences in LL.

Effect of nitric oxide and calpastatin on the inhibition of µ-calpain activity, autolysis and proteolysis of myofibrillar proteins.

The aim of this study was to investigate the dual effect of the nitric oxide donor NOR-3 and calpastatin on µ-calpain activity, autolysis, and proteolytic ability. µ-Calpain and calpastatin were purified and allocated to the following five treatments: µ-calpain, µ-calpain + calpastatin, µ-calpain + NOR-3, µ-calpain + calpastatin + NOR-3, and µ-calpain + NOR-3 + calpastatin. µ-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce µ-calpain S-nitrosylation and effectively block the activity via the inhibition of µ-calpain autolysis. Calpastatin inhibited µ-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on µ-calpain activity, autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin. Our data suggest that S-nitrosylation may play a regulatory role in mediating µ-calpain activity in the presence of calpastatin.

Effect of nitric oxide on myofibrillar proteins and the susceptibility to calpain-1 proteolysis.

This study was designed to investigate the nature of modification of myofibrillar proteins by nitric oxide (NO) and the extent to which S-nitrosylation alters their susceptibility to calpain-1 proteolysis. Isolated myofibrils from porcine semimembranosus muscle were incubated with the NO donor S-nitrosoglutathione (GSNO) at 0, 20, 50, 250, 1000 µM for 30 min at 37 °C and then incubated with purified calpain-1. GSNO treatment decreased the thiol content of myofibrillar proteins and increased their intensity and amount of S-nitrosylation. GSNO caused the formation of proteins cross-linkage through intermolecular disulfide. More desmin and titin (T2, the degraded fragment of original titin) were degraded by calpain-1 when myofibrils were incubated with 1000 µM GSNO. Incubation with 250 and 1000 µM GSNO suppressed calpain-1-catalyzed cleavage of troponin-T. The data suggest that NO could change the redox state of myofibrillar proteins and subsequently affect the extent of proteolysis by calpain-1 in a protein-dependent manner.

Identification of S-nitrosylated proteins in postmortem pork muscle using modified biotin switch method coupled with isobaric tags.

The objective of this study was to identify the S-nitrosylated proteins in aging samples of pork longissimus thoracis muscle (aged 0 and 3 d) and to study the effects of exogenous S-nitrosoglutathione (GSNO, concentration at 10 and 100 µM) treatments of aged 0 d sample. After validating modified biotin switch method, the samples were labeled with tandem mass tags (TMT126-129) for the LC-MS/MS analysis. A total of 366 peptides were identified to be S-nitrosylated corresponding to 339 proteins. Comparison of total intensity and individual S-nitrosylated sites between aging samples revealed that S-nitrosylation did occur in pork muscle during postmortem aging through possible pathways of denitrosylation and transnitrosylation. GSNO treatment groups showed a considerable number of potential cysteines could be modified with high thiol-reactivity. It was deduced that S-nitrosylation could be involved in the postmortem metabolic process possibly through the regulation of activity or function of glycolytic enzymes, calcium release, heat shock proteins, antioxidant enzymes and myofibrillar proteins.

Understanding postmortem biochemical processes and post-harvest aging factors to develop novel smart-aging strategies.

Postmortem aging is a value-adding process and has been extensively practiced by the global meat industry for years. The rate and extent of aging impacts on meat quality characteristics are greatly affected by various biochemical/physiological changes occurring during the pre-rigor phase through post-rigor aging processes. This should also mean that the positive aging impacts on eating quality attributes can be further maximized through establishing specific post-harvest aging strategies. In this review, we propose the smart-aging concept, which is to develop innovative template strategies through identifying optimal aging regimes to maximize positive aging impacts on meat quality and value. The concept requires a good understanding of the physical, biochemical and post-harvest factors that affect the aging of beef. This knowledge coupled with the ability to non-invasively determine muscle composition early postmortem will create opportunities to tailor the process of muscle conversion to meat and the subsequent aging processes to deliver meat with consistent and improved eating qualities and functionality.

Acute Heat Stress and Reduced Nutrient Intake Alter Intestinal Proteomic Profile and Gene Expression in Pigs.

Heat stress and reduced feed intake negatively affect intestinal integrity and barrier function. Our objective was to compare ileum protein profiles of pigs subjected to 12 hours of HS, thermal neutral ad libitum feed intake, or pair-fed to heat stress feed intake under thermal neutral conditions (pair-fed thermal neutral). 2D-Differential In Gel Electrophoresis and gene expression were performed. Relative abundance of 281 and 138 spots differed due to heat stress, compared to thermal neutral and pair-fed thermal neutral pigs, respectively. However, only 20 proteins were different due to feed intake (thermal neutral versus pair-fed thermal neutral). Heat stress increased mRNA expression of heat shock proteins and protein abundance of heat shock proteins 27, 70, 90-α and ß were also increased. Heat stress reduced ileum abundance of several metabolic enzymes, many of which are involved in the glycolytic or TCA pathways, indicating a change in metabolic priorities. Stress response enzymes peroxiredoxin-1 and peptidyl-prolyl cis-trans isomerase A were decreased in pair-fed thermal neutral and thermal neutral pigs compared to heat stress. Heat stress increased mRNA abundance markers of ileum hypoxia. Altogether, these data show that heat stress directly alters intestinal protein and mRNA profiles largely independent of reduced feed intake. These changes may be related to the reduced intestinal integrity associated with heat stress.

Effect of early postmortem enhancement of calcium lactate/phosphate on quality attributes of beef round muscles under different packaging systems.

The objective was to determine the influence of calcium lactate/phosphate enhancement on quality of beef round cuts in high-oxygen modified atmosphere (HiOx-MAP; 80% O2/20% CO2). Mm. semimembranosus (SM), semitendinosus (ST), and adductor (AD) were divided and assigned to water-injected control (CON), 3mM phosphate (STP), or 200mM calcium lactate/3mM phosphate (CAL/STP) treatments at 24h postmortem. Steaks (n=10) were vacuum packaged (VAC) and stored for 9days, then displayed for 7days in VAC or HiOx-MAP. Lipid oxidation, pH, surface color, star probe, and sensory characteristics were evaluated. HiOx-MAP resulted in greater lipid oxidation, more discoloration, and decreased sensory quality of steaks (P<0.05) compared to VAC. However, CAL/STP enhancement significantly reduced lipid oxidation of all steaks, decreased ST and SM star probe values, and improved tenderness of HiOx-MAP packaged AD and SM (P<0.05). Results suggest that CAL/STP enhancement has beneficial effects on lipid stability and sensory attributes of beef round cuts under HiOx-MAP.