Calpastatin Activity Research Articles

Challenges and opportunities of using Bos indicus cattle to meet consumers' demand for quality beef

Beef consumption is expected to increase worldwide, which necessitates the use of Bos indicus cattle that are well-adapted to harsher climates, like the tropics. Yet, beef from these cattle is considered inferior to that of Bos taurus breeds, primarily due to lowered tenderness values and reduced intramuscular fat content. However, the benefits of using Bos indicus genetics are numerous and undeniable. Herein, we explore how decreases in meat quality in these cattle may be offset by increases in livability. Further, we review the knowledge surrounding beef tenderness and explore the processes occurring during the early events of the transformation of muscle to meat that are different in this biological type and may be altered by stress. Growth rate, calpastatin activity and mitochondrial function will be discussed as they relate to tenderness. The opportunities of using Bos indicus cattle are of great interest to the beef industry worldwide, especially given the pressures for enhancing the overall sustainability and carbon footprint of this sector. Delivering a consistently high-quality product for consumers by exploiting Bos indicus genetics in a more sustainable manner will be proposed. Information on novel factors that influence the conversion of muscle to meat is explored to provide insights into opportunities for maximizing beef tenderization and maturation across all cattle. Exploring the use of Bos indicus cattle in modern production schemes, while addressing the mechanisms undergirding meat tenderness should provide the industry with a path forward for building greater demand through producing higher quality beef.

Effect of sex (ram or wether) and short duration, high volt electrical stimulation on tenderisation of Longissimus thoracis et lumborum and Semimembranosus muscles derived from Boer Goat and large frame Indigenous Veld Goat

This study determined whether castration and electrical stimulation (ES) influenced chevon tenderness and related physiological characteristics in Boer Goat (BG; n = 36; 21 bucks and 15 wethers) and large frame Indigenous Veld Goat (IVG; n = 41; 21 bucks and 20 wethers). Half of buck and wether carcasses were ES (20s, 400 Volts peak, 5 ms pulses at 15 pulses/s) 10 min post-mortem. Dressed carcasses were chilled (4 °C within 1-h post-mortem). pH and temperature decline, % drip loss, sarcomere length (SL), myofibril fragmentation length (MFL; 1- and 4-days post-mortem), Calpain-1, −2 and calpastatin activities (1- and 24-h), Warner-Bratzler shear force (WBSF; 1-day post-mortem) and sensory attributes (tenderness and juiciness; 4-days post-mortem) were measured on Longissimus thoracis et lumborum (LTL) and Semimembranosus (SM) muscles. ES carcasses had significantly lower pH irrespective of sex or breed. Buck LTL and SM were less tender (P ≤ 0.05) supported by longer MFL and higher calpastatin activity (P < 0.05), than wether muscles. ES LTL were more tender (WBSF and sensory) (P ≤ 0.001) while ES SM were less affected (P = 0.055). ES caused lower Calpain-1 activity in the LTL. SL do not support cold shortening and calpastatin played a major role in the tenderisation of chevon early post-mortem. A longer ageing period is recommended for goat meat in general to achieve acceptable levels of tenderness.

PSIII-A-1 Acute Stress Effects on Physiological Responses and Meat Quality Changes in Goats

Abstract Duration of stress prior to processing can significantly affect physiological status and meat quality in goats. This experiment was conducted to determine the effects of duration of preslaughter stress on physiological responses and meat quality in goats. In a Randomized Complete Block Design, fifty-four uncastrated male Spanish goats (8-mo old; BW 29.7 ± 2.03 kg) were randomly subjected to one of three treatments (n = 18 goats/treatment): (i) transported for 3 h, (ii) transported for 30 min, or (iii) or held in pens (control) on two different days. Blood samples were collected before and after transport for assessment of stress responses. Goats were slaughtered, using humane procedures, for meat quality analysis. The data were analyzed using MIXED procedures in SAS with treatment and day as fixed variables. Cortisol concentrations were higher (P &amp;lt; 0.01) in both 3 h (117.7 ± 7.96 ng/mL) and 30 min (130.1 ± 8.05 ng/mL) transported groups compared to the control group (27.5 ± 7.59 ng/mL). A similar treatment effect was noticed in plasma non-esterified fatty acid (P &amp;lt; 0.05) concentrations. Glucose concentrations were highest in 180-min, lowest in control, and intermediate in 30-min group (P &amp;lt; 0.01). There were significant positive correlations among muscle desmin concentration after 6 days of aging and 24 h pH (P &amp;lt; 0.05) and calpastatin activity (P &amp;lt; 0.05), suggesting that desmin degradation plays a role in determining the tenderness of aged meat from goats subjected to preslaughter stress. Warner-Bratzler shear force values of loin chops were highest in 180-min, lowest in control, and intermediate in 30-min groups; however, treatment did not influence the color values or cooking loss of loin chops. The results indicate that acute stress prior to processing can affect both physiological responses and meat quality in a duration-dependent manner in goats.

Feed Restriction Improves Lipid Metabolism by Changing the Structure of the Cecal Microbial Community and Enhances the Meat Quality and Flavor of Bearded Chickens.

Simple SummaryWith the development of intensive feeding, the increased cost of feeding and the excessive fat deposition affecting carcass and meat quality in full-fed broiler chickens have attracted more attention from poultry farmers and consumers. Feed restriction (FR) has been adopted to tackle these problems. The present study demonstrated that 80% feed intake showed a significant role in improving lipid metabolism and enhancing the meat quality and flavor of Bearded chickens. Additionally, this study confirms that the major mechanisms are the activation of the calproteinase system and the regulation of the structure of cecal microflora.Excessive fat deposition in full-fed Bearded chickens does not only reduce carcass yield but also causes consumer rejection of meat. Feed restriction (FR) is an effective method to save on feed cost, reduce carcass fat deposition, and improve meat quality. A total of 560 150-d Bearded chickens were randomly divided into seven groups (each with eight replicates of ten birds) for 40 days. The control group was fed with the basal diet ad libitum (CON), and the other six groups were fed with 90% of the feed intake (90% FI), 80% FI, 70% FI, 90% metabolizable energy (90% ME), 80% ME, and 70% ME of the CON, respectively. Compared to the CON group, FR increased meat yield, but the total weight of the Bearded chickens was slighter; 80% FI and 70% ME improved the relative lipid metabolism indices of chickens, especially the levels of triglycerides and total cholesterol in the plasma and liver (p < 0.05), and decreased calpastatin activity in the breast muscle (p < 0.05). Additionally, 16S rRNA sequencing of cecal microbial community indicated that an increase in the abundance of Hydrogenoanaerobacterium and Bacteroides plebeius was observed in the 80% FI group (p < 0.05), and an enrichment in Olsenella, Catabacter, and Lachnospiraceae were observed in the 70% ME group (p < 0.05) compared to the CON group. Moreover, compared to the CON group, the L * value of the breast muscle significantly decreased, and a * value significantly increased in the 80% FI group (p < 0.05). Notably, the concentrations of threonine, lysine, aspartic acid, glutamic acid, proline, and arginine and the activity of calpain in breast muscle increased in the 80% FI group more than in the CON group (p < 0.05), while valine, isoleucine, leucine, phenylalanine, lysine, alanine, tyrosine and proline decreased in ME restriction groups (p < 0.05). Taken together, our results indicated that 80% FI could improve lipid metabolism by changing the structure of the cecal microbial community, and the meat quality and flavor of the Bearded chickens in 80% FI group was improved with a promoted meat color score, flavor substances, and the calproteinase system.

Expressão de transcritos do sistema calpaína responde de forma inversa ao amaciamento após suplementação com vitamina D3 em bovinos Nelore

ABSTRACT The calpain system is the central player for meat tenderization and the calpastatin activity plays an important role in beef tenderness of Bos taurus indicus cattle. This study investigated whether dietary vitamin D3 induced gene expression of μ-calpain (CAPN1), total calpastatin (CAST T), and their variants (CAST I and II). Animals received none or 2 × 106 IU of vitamin D3 for either 2 or 8 days before slaughter and were submitted to different conditions during feedlot: sun exposure or artificial shade. Steaks from Longissimus lumborum were fabricated, aged for 1, 7, and 21 days post-mortem, and later used for the analyses of shear force and the myofibrillar fragmentation index. Vitamin D3 did not influence mRNA abundance; however, it induced a greater CAST II transcript in animals supplemented 8 days before slaughter. There was a negative association between CAST II abundance and the shear force, which revealed an important modulation of the calpain system expression due to vitamin D supplementation. This result is an important tool for strategies to improve beef tenderness.

Feeding strategies and ageing time alter calpain system proteins activities and meat quality of Braford steers.

ObjectiveThe aim of this study was to evaluate the effect of ageing and feeding strategies on the calpain protease system and meat quality traits in Braford steers.MethodsThirty Braford steers were employed; 15 animals were supplemented with corn silage during finishing and 15 were kept only on pasture. Meat quality traits and calpain system protein activity were evaluated in longissimus thoracis et lumborum (LTL) steaks aged for 2, 7, 14, and 21 days.ResultsAged meat showed higher pH and calcium content, while Warner Bratzler shear force (WBSF) decreased to day 21. No interaction between ageing and diet was seen for quality traits. Steers finished with corn silage showed higher values of water holding capacity, WBSF and free calcium, and lower values of pH and cooking loss. Calpain and calpastatin activities decreased with ageing. Finishing steers on pasture produced higher values of calpains and lower values of calpastatin activities. The higher values of calpain 1 activity were observed in muscles aged 2 days from pasture finished animals, and the lower activity of the inhibitor in the 21 days aged samples of the same group.ConclusionThese results suggest a diet by ageing interaction in calpains and calpastatin and this interaction impact in Warner Bratzler Shear Force in Braford LTL muscle.

Calpastatin prevents Angiotensin II–mediated podocyte injury through maintenance of autophagy

The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin II promoting progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy) specifically in the podocyte resulted in accelerated angiotensin II-induced podocytopathy, accentuated albuminuria and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.

Relationship between beta-adrenergic agonists, calpain system activity and beef texture: A systematic review.

The objective was to conduct a systematic review to evaluate the effects of dietary supplementation with beta-adrenergic agonists on calpains and calpastatin activity in bovine muscle and changes in meat tenderness. A survey was conducted in June 2019 on Science Direct, Web of Science, Scopus, PubMed and Capes Periodicals, using four keyword combinations: agonist and calpain and cattle; agonist and calpain and bovine; agonist and calpain and heifers; agonist and calpain and steers. Thirteen studies were selected, 54% concluded that supplementation with beta-adrenergic agonists increases calpastatin activity, 23% observed increase in their gene expression and 23% reported no effect on activity or expression of this enzyme. Nine studies evaluated the influence of beta-adrenergic agonists supplementation on meat texture and all found an increase in shear force values. There is strong evidence that beta-adrenergic agonists may increase calpastatin activity in the muscle, causing damage to meat tenderness.

Expression of calpastatin isoforms in three skeletal muscles of Angus steers and their association with fiber type composition and proteolytic potential

This study aimed to evaluate the expression of calpastatin (CAST) isoforms and their potential associations with fiber type composition (%RA), calpastatin activity (CA) and myofibril fragmentation index (MFI) in three muscles with known differences in tenderness (infraspinatus, triceps brachii and semitendinosus) of Angus steers. Expression of total CAST (CAST-T) and CAST isoforms I, II, III (2–3) and III (2–4) (including or not exon 3) was evaluated by qRT-PCR. CAST expression and CA were significantly higher and MFI was lower in semitendinosus, the muscle with the highest %RA of IIX fibers. Differential expression of isoforms defined the variability in CAST-T among muscles. Semitendinosus had a higher expression of isoforms II and III (2–3), but lower expression of III (2–4) compared to the other two muscles. Relative expression of isoforms II and III that were defined by promoter preference linked to alternative splicing, seem to be the main factors explaining differences in CAST expression and ultimately in MFI among muscles.

TRANSCRIPTOS ALTERNATIVAMENTE POLIADENILADOS DE CALPASTATINA EN MÚSCULOS DE BOVINO

Calpastatin activity has a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditioning. The regulation of calpastatin (CAST) expression is highly complex, the gene has four putative promoters and at least three different polyadenylation sites, and it is also alternatively spliced. We investigated the presence of alternative polyadenylation (APA) isoforms of CAST transcripts in three muscles (infraspinatus, triceps brachii and semitendinosus) of two bovine breeds (Angus and Brahman). The 3´ RACE-PCR was used to specifically amplify the different APA sites. The amplified fragments were cloned and sequenced. Sequencing confirmed the existence of three expected polyadenylation sites corresponding to short, medium and long polyadenylated transcripts. Also, transcripts with a novel APA site were found in the three muscles of both breeds. Because the same APAs isoforms were found between muscles and breeds, we could hypothesize a possible contribution to the relative abundance of different isoforms, probably in coordination with promoter preference and alternative splicing. This knowledge would be useful in the design of future experiments to analyze differential expression of CAST isoforms and their contribution to the definition of beef tenderness. Key words: Beef cattle; Alternative polyadenylation; 3´ RACE-PCR.

Calpain Activity of Jawarandu Does under Four Different Energy Level in the Ration

The aim of the research is to investigate the effect of four different energy level in the ration into the calpain activity of Jawarandu does. The research was done during 5 months in the Experimental Farm of the Faculty of Animal Science, Universitas Jenderal Soedirman. The research material used was 16 heads of the Jawarandu doe with the aged 2.5−3 years. All the animals were randomly assigned to the ration treatment which forms four the different energy levels (82.26% TDN, 85, 87.93, dan 90.74% TDN). The replication of each treatment was four times. Variable measured was a calpain activity on the muscle of Longissimus dorsi. General linear model (GLM) of the SPSS was used to analysis variable measured. Energy content 1.63McalME/heads/day and 1.92McalME/heads/day as well as 1.73McalME/heads/day and 2.06McalME/heads/day were increased of the μ-calpain and m-calpain activities at the Longissimus dorsi muscle, respectively. However, there was decreased of the calpastatin activity at the Longissimus dorsi muscle. Different energy content of the ration increased the μ-calpain and m-calpain activities at the Longissimus dorsi muscle and of those decreased calpastatin activity.

Tough aged meat presents greater expression of calpastatin, which presents postmortem protein profile and tenderization related to Nellore steer temperament

Eighteen Nellore steers were classified as either excitable (high index) or calm (low index) temperament, and high or low WB Shear Force (SF) in Longissimus lumborum at 16 days postmortem, with a subset assigned to four groups: HTHS–high temperament and SF (n = 5); HTLS–high temperament and low SF (n = 4); LTHS–low temperament and high SF (n = 4); LTLS–low temperament and SF (n = 5). Only the second calpastatin peak was detected at 48 h in the all samples, and the calpastatin activity only differed (P < .05) between shear force groups. High WBSF groups had greater expression of CAST gene (P < .05) in the high and low temperament. Western blots showed a calpastatin fragmentation pattern very particular to each group with high molecular weight bands present in the HTHS. Temperament was not a determinant factor in postmortem proteolysis variation. However, greater WBSF aged meat from high temperament animals had greater calpastatin activity and less calpastatin fragments formed during the postmortem period analyzed.

Investigation of the effects of high pressure processing on the process of rigor in pork

This study was conducted to determine whether the application of high pressure processing (HPP) could prevent the process of rigor in fresh pork and to identify the physical and chemical changes occurring in the meat that are responsible for this effect. Pressure (0–400 MPa) and time (10 min) were applied to the pork loin and enzyme samples. The effects of treatment on shear force, myofibrillar fragmentation, enzyme activity and meat texture were measured. The results showed that, compared with untreated samples, HPP treatment inhibited the occurrence of rigor in pork. The myofibrillar fragmentation indices of HPP treated samples were higher than those of control samples and increased linearly with increasing pressure. Application of HPP prevented the process of rigor at 0–300 MPa, reduced calpain activity and markedly decreased calpastatin activity, resulting in an increase of total calpains. Above 300 MPa, the mechanical action of pressure led to continuous prevention of rigor.

The impact of oestradiol only hormone growth promotants (HGPs) on the eating quality of pasture finished steer carcasses

A total of 200 Bos indicus/Bos taurus cross steers were allocated to control (CON) and an oestradiol (OES) implant treatments and pasture finished for 389 days. Longissimus lumborum (LL) and gluteus medius (GM) samples were aged for 5 and 35 days. Live weight, carcass weight and ossification scores (P < 0.05) increased in OES relative to CON. The three-way interaction between treatment, days aged and muscle was significant (P < 0.05) for tenderness, overall liking and meat palatability, whereby the OES had lower scores relative to CON at 5 days in LL (P < 0.05), although the difference halved by 35 days. For the GM, OES scores at 5 days were lower than CON (P < 0.05), apart from like flavour, and differences reduced by 35 days. LL shear force was higher for OES at 5 days (P < 0.05), though not 35 days (P > 0.05), or the GM at 5 or 35 days (P > 0.05). OES samples had a higher calpastatin activity (P < 0.05) in the LL at 19 h post mortem.

The impact of two different hormonal growth promotants (HGPs) on the eating quality of feedlot-finished steer carcasses

The Meat Standards Australia beef-grading model applies a variable adjustment for different cuts of hormonal growth promotant (HGP)-treated carcasses, but does not differentiate between different HGP types. Using 300 non-implanted Bos indicus–Bos taurus composite steers, an experiment was conducted to compare the effects of an oestradiol only (OES) and a combination trenbolone acetate and oestradiol (TBA+OES) implant with non-implanted animals (CON) fed a concentrate ration for 73 days before slaughter, on eating quality of the mm. longissimus lumborum (LL) and gluteus medius (GM) muscles. Sensory and objective LL and GM samples were aged for either 5 or 35 days before freezing at −20°C. Carcass weights from each group were significantly (P &amp;lt; 0.05) different. Corrected for carcass weight, HGP treatment had a significant effect on hump height, ossification score, marble score, P8 fat depth and eye-muscle area. The TBA+OES treatment resulted in significantly (P &amp;lt; 0.05) tougher meat than the OES and CON treatments as assessed by shear force, although this difference was reduced with aging. Sensory scores (tenderness, juiciness, like flavour, overall liking and a composite MQ4 score) confirmed a negative HGP treatment effect, whereby TBA+OES was significantly lower than the CON and OES treatments after 5 days of aging, and these differences were reduced through aging. TBA+OES had a greater impact on sensory scores in the LL when compared to the GM. Both HGP treatments increased calpastatin activity, and the TBA+OES treatment was significantly (P &amp;lt; 0.05) different from the CON and OES treatments. It was concluded that OES and TBA+OES implants have different impacts on meat eating-quality measurements, which could have important implications for the Australian and international beef industry.

Impact of PRRS Challenge on Calpain and Calpastatin Activity in Skeletal Muscle of Young Pigs

ObjectivesPorcine Reproductive and Respiratory Syndrome (PRRS) virus challenges in growing pigs are known to negatively affect growth performance. One hypothesis to explain this observation is that an increase in calpain system initiated protein degradation in muscle contributes to the reduction of muscle protein accretion. Calpain-1 and –2 are calcium dependent cysteine proteinases that are regulated by an endogenous inhibitor, calpastatin. The objective of this experiment was to define the extent to which PRRS challenge influences skeletal muscle calpain and calpastatin activity at 2 stages of infection (viremia and seroconversion).Materials and MethodsThirty-three pigs (11.3 ± 1.54 kg body weight (BW), approximately 6 wk of age) were assigned to a contemporary group (3 pigs per group) based on age and weight. Pigs were housed in individual pens in separate rooms depending on treatment. One pig per group was assigned to 1 of 3 treatments, 1)PRRS [PRRS challenged with ORF5 RFLP1–3-4 isolate (n = 11)], 2)Pair-fed [Non-challenged, daily feed intake matched to challenged pigs (n = 11)], and 3)Ad-lib [Non-challenged, Ad libitum fed (n = 11)]. Weekly BW, feed disappearance, and feed efficiency were assessed over each period. At days post inoculation (dpi) 9–10 (7 groups; viremia period) and 16 to 17 (4 groups; seroconversion period), pigs were euthanized and a 50-g sample of longissimus muscle was collected and homogenized in pre-rigor extraction buffer (100 mM Tris-HCl pH 8.3, 10 mM EDTA, 100 mg/L trypsin inhibitor, 2 μM E-64, and 0.1% 2-mercaptoethanol). Calpains and calpastatins were separated using anion exchange chromatography and quantified using casein as a substrate. Data from each time period were analyzed as a separate experiment. Data were analyzed using the mixed procedure in JMP Pro version 13.1 with fixed effects of treatment and a random effect of contemporary group. Means separations were conducted via Student t test with P < 0.05 considered significantly different.ResultsAt both time points, PRRS pigs had reduced ADG and G:F compared with Ad-lib and Pair-fed pigs (P < 0.01). Additionally, ADFI was reduced in PRRS and Pair-fed pigs compared with Ad-lib pigs (P < 0.001). At dpi 9–10, PRRS challenge resulted in greater calpastatin-2 (1.04) and total calpastatin (1.70) activity compared with the Pair-fed pigs (0.58, 1.06 respectively; P < 0.05). By contrast, at dpi 16 to 17, PRRS challenge resulted in greater calpastatin-1 (1.08) and total calpastatin activity (1.98) compared with Pair-fed controls (0.54, 0.89, respectively; P < 0.05). Calpain-1 and –2 were not significantly altered by treatment at either time point. The ratio of calpain-2 to total calpastatin activity was significantly (P < 0.05) less in muscle from PRRS challenged pigs (1.16) compared with muscle from the Pair-fed (2.47), but not the Ad-lib pigs (1.58).ConclusionThe results demonstrate that a decline in voluntary feed intake was not singularly responsible for the observed response to PRRS challenge. These results are counter to the hypothesis that PRRS challenge would decrease calpastatin activity. Moreover, the results of this study indicate a difference in calpastatin response to PRRS challenge, notably that PRRS challenge increased calpastatin-2 activity at dpi 9 to 10 and calpastatin-1 activity at dpi 16 to 17. The switch indicates a temporal response in skeletal muscle proteolysis due to PRRS virus challenge.

Impact of PRRS Challenge on Calpain and Calpastatin Activity in Skeletal Muscle of Young Pigs

ObjectivesPorcine Reproductive and Respiratory Syndrome (PRRS) virus challenges in growing pigs are known to negatively affect growth performance. One hypothesis to explain this observation is that an increase in calpain system initiated protein degradation in muscle contributes to the reduction of muscle protein accretion. Calpain-1 and –2 are calcium dependent cysteine proteinases that are regulated by an endogenous inhibitor, calpastatin. The objective of this experiment was to define the extent to which PRRS challenge influences skeletal muscle calpain and calpastatin activity at 2 stages of infection (viremia and seroconversion).Materials and MethodsThirty-three pigs (11.3 ± 1.54 kg body weight (BW), approximately 6 wk of age) were assigned to a contemporary group (3 pigs per group) based on age and weight. Pigs were housed in individual pens in separate rooms depending on treatment. One pig per group was assigned to 1 of 3 treatments, 1)PRRS [PRRS challenged with ORF5 RFLP1–3-4 isolate (n = 11)], 2)Pair-fed [Non-challenged, daily feed intake matched to challenged pigs (n = 11)], and 3)Ad-lib [Non-challenged, Ad libitum fed (n = 11)]. Weekly BW, feed disappearance, and feed efficiency were assessed over each period. At days post inoculation (dpi) 9–10 (7 groups; viremia period) and 16 to 17 (4 groups; seroconversion period), pigs were euthanized and a 50-g sample of longissimus muscle was collected and homogenized in pre-rigor extraction buffer (100 mM Tris-HCl pH 8.3, 10 mM EDTA, 100 mg/L trypsin inhibitor, 2 μM E-64, and 0.1% 2-mercaptoethanol). Calpains and calpastatins were separated using anion exchange chromatography and quantified using casein as a substrate. Data from each time period were analyzed as a separate experiment. Data were analyzed using the mixed procedure in JMP Pro version 13.1 with fixed effects of treatment and a random effect of contemporary group. Means separations were conducted via Student t test with P < 0.05 considered significantly different.ResultsAt both time points, PRRS pigs had reduced ADG and G:F compared with Ad-lib and Pair-fed pigs (P < 0.01). Additionally, ADFI was reduced in PRRS and Pair-fed pigs compared with Ad-lib pigs (P < 0.001). At dpi 9–10, PRRS challenge resulted in greater calpastatin-2 (1.04) and total calpastatin (1.70) activity compared with the Pair-fed pigs (0.58, 1.06 respectively; P < 0.05). By contrast, at dpi 16 to 17, PRRS challenge resulted in greater calpastatin-1 (1.08) and total calpastatin activity (1.98) compared with Pair-fed controls (0.54, 0.89, respectively; P < 0.05). Calpain-1 and –2 were not significantly altered by treatment at either time point. The ratio of calpain-2 to total calpastatin activity was significantly (P < 0.05) less in muscle from PRRS challenged pigs (1.16) compared with muscle from the Pair-fed (2.47), but not the Ad-lib pigs (1.58).ConclusionThe results demonstrate that a decline in voluntary feed intake was not singularly responsible for the observed response to PRRS challenge. These results are counter to the hypothesis that PRRS challenge would decrease calpastatin activity. Moreover, the results of this study indicate a difference in calpastatin response to PRRS challenge, notably that PRRS challenge increased calpastatin-2 activity at dpi 9 to 10 and calpastatin-1 activity at dpi 16 to 17. The switch indicates a temporal response in skeletal muscle proteolysis due to PRRS virus challenge.

Effect of postmortem storage on shear force value and calpain/calpastatin activity in longissimus dorsi muscle of rusa deer (cervus timorensis)

The objective of this study was to determine the effects of aging period on shear force values and activities of calpain/calpastatin enzymes of the longissimus dorsi (LD) muscle from rusa deer (Cervus timorensis). The shear force values were measured with instron materials testing machine. Results showed that postmortem storage influenced meat tenderness with a highly significant reduction of shear force value from 9.01±0.83 kg/cm2 at day 1 to 4.34±0.10 kg/cm2 at day 21 (P&lt;0.01). Determination of the enzyme activities indicated that the μ-calpain activity decreased significantly from 1.50±0.42 to 0.19±0.28 units/g of meat (P&lt;0.01), whereas m-calpain activity (22.88±9.64 to 16.95±8.34 units/g of meat) and calpastatin activity (9.93±2.37 to 6.82±2.96 units/g of meat) slightly decreased (P&gt;0.05). Shear force values were significant correlated with higher levels of μ-calpain (r=0.915) and m-calpain (r=0.758), respectively. However, the values were not significant related to the calpastatin activity (r=0.462). Therefore, this study confirmed that in the case of rusa deer, μ-calpain is a key factor controlling postmortem meat tenderness compared to m-calpain and calpastatin. The results suggested that storage of the rusa deer meat for 7 days is enough to achieve aging of the LD muscle, which may help to reduce the cost of meat production.

Biochemical Basis of Calpain and Calpastatin Activity Determination in Blood and Tissue Samples: Potential Role in Post - Mortem Ageing of Guinea Fowl Meat

Biochemical Basis of Calpain and Calpastatin Activity Determination in Blood and Tissue Samples: Potential Role in Post - Mortem Ageing of Guinea Fowl Meat

Differences in skeletal muscle proteolysis in Nellore and Angus cattle might be driven by Calpastatin activity and not the abundance of Calpain/Calpastatin

SUMMARYThe present study aimed to explore the molecular factors underlying differences in Calpain/Calpastatin proteolytic system in Nellore and Angus cattle. Longissimus muscle samples were collected in Nellore (n= 6; body weight (BW) = 373 ± 37·3 kg) and Angus (n= 6; BW = 383 ± 23·9 kg) cattle at slaughter for analysis of gene and protein expression, and Calpastatin enzyme activity. Additionally, the myofibrillar fragmentation index was used to quantify the extension of proteolysis in longissimus muscle samples. A greater myofibrillar fragmentation was observed in skeletal muscle of Angus compared with Nellore cattle. Conversely, no differences were found between breeds for mRNA expression of Calpain 1 (CAPN1) and Calpastatin (CAST). Similarly, no differences were observed for the abundance of Calpain and Calpastatin proteins between skeletal muscles of Nellore and Angus cattle. Despite the lack of differences in mRNA and protein abundance, a greater activity of Calpastatin was observed in skeletal muscle of Nellore compared with Angus cattle. These data indicate that the greater proteolysis in skeletal muscle of Angus compared with Nellore cattle is mainly driven by a greater Calpastatin activity rather than Calpain or Calpastatin mRNA and protein expression.