Early post mortem expression of genes related to tenderization in two Italian Simmental young bulls' skeletal muscles differing in contractile type

The early post mortem expression of eight genes potentially involved in meat ageing process and the tenderness of two Italian Simmental young bulls' (Bos taurus) skeletal muscles differing in their contractile type were evaluated. Samples of Longissimus lumborum (LL) and Infraspinatus (IS) muscles were collected from 17 bulls. The messenger RNA (mRNA) abundances of calpain‐1, calpain‐2, calpastatin, caspase 3, caspase 9, heat shock protein 27 (Hsp27), Hsp40 and Hsp70 were detected by quantitative PCR. The myosin heavy chain‐slow and ‐fast isoform content, the pH_48h and the lipid content of the muscles were in line with the contractile and metabolic type. In comparison with the fast LL, the slow IS showed a lower calpain‐1/calpastatin mRNA content ratio after slaughtering and a higher Warner‐Bratzler Initial Yield value after 7 days of ageing. Hsp27 and Hsp70 mRNA abundances were significantly lower in LL than IS, highlighting their potential role in the ageing process of bovine muscles.     

Changes in caspase activity during the postmortem conditioning period and its relationship to shear force in porcine longissimus muscle

The objective of this study was to investigate the protease family caspases in skeletal muscle and their potential contribution to postmortem proteolysis and meat tenderization. Ten Large White gilts were slaughtered, and samples of LM were taken at 0, 2, 4, 8, 16, 32, and 192 h after slaughter and immediately snap frozen in liquid nitrogen. Samples were subsequently analyzed for caspase 3/7 and caspase 9 activity, protein levels of known caspase substrates, alpha II spectrin and poly (ADP-ribose) polymerase (PARP), as well as, at 192 h, shear force. Specific degradation products of alpha II spectrin and PARP, which are known indicators of caspase activity, and apoptosis were detected on immunoblots of muscle samples taken over the postmortem period. The relationships between the changes observed in caspase activities and protein levels of PARP and spectrin across the entire postmortem conditioning period were investigated (n = 70). Protein levels of alpha II spectrin cleavage products across the conditioning period were found to correlate positively to caspase 3/7 activity (r = 0.38, P = 0.003) and caspase 9 activity (r = 0.32, P = 0.012), indicating that caspase-mediated cleavage was occurring in situ. There was a negative relationship between shear force and the 0 to 32 h ratio of caspase 3/7 (r = -0.62, P = 0.053) and caspase 9 activities (r = -0.68, P = 0.044). In addition, there was also a negative relationship between shear force and the level of the caspase-generated alpha II spectrin 120 kDa degradation product (r = -0.75, P = 0.012). The findings of this study indicate that changes in caspase activity and caspase-mediated cleavage take place in muscle during the conditioning period, and this could be associated with the development of tender meat.     

Differences in calpain system,desmin degradation and water holding capacity between commercial Meishan and Duroc × Landrace × Yorkshire crossbred pork

The objective of this study was to examine the differences in calpain system, desmin degradation, pH values and water holding capacity (WHC) between muscles of commercial Meishan and Duroc × Landrace × Yorkshire crossbred pigs. Meishan pork presented better WHC evidenced by lower purge loss at days 1 and 3 and less centrifugation loss at day 1 post mortem (P < 0.05). pH values at 45 min post mortem in Meishan pork were significantly higher compared to crossbred pork (P < 0.05). Calpain‐1 messenger RNA (mRNA) expression was lower in Meishan pork compared to that from crossbred pork (P < 0.05). Additionally, calpain‐1 activity, the ratio of calpain‐1 to calpastatin activity and desmin degradation were lower in Meishan pork compared to those from crossbred pork samples (P < 0.05). The results indicate that the calpain system including mRNA expression and activity were different between commercial Meishan and crossbred pork resulting in difference in the degree of desmin degradation during post mortem aging. pH values at 45 min and 24 h post mortem rather than calpain activity and desmin degradation could explain the higher water holding capacity in commercial Meishan pork.     

Effect of early filleting on the texture of breast muscle of broilers stunned with argon‐induced anoxia

1. Eighty broilers were stunned in batches of 10 with less than 1 % oxygen (air displaced by argon) and their chilled breasts were filleted at 2, 3, 5 or 21 h (argon control) post‐mortem. Twenty broilers were electrically stunned and their breasts filleted at 21 h post‐mortem (electrical control).

2. Meat quality variables studied included pH of the pectoralis major muscle measured at 20 min post‐mortem and at filleting, and texture at 24 h post‐mortem.

3. Argon stunning resulted in a rapid early post‐mortem pH fall, but there was no indication of heat toughening in the breast muscles.

4. Filleting at 2 h post‐mortem, after argon stunning, resulted in 5% of the p. major muscles having greater than 4 kg yield force (slightly tough).

5. Although post‐mortem ageing of argon stunned broilers improved the texture of breast muscle, it is inferred that filleting at 2 h postmortem resulted in breast muscle with acceptable texture.     

MicroRNA‐145 regulates immune cytokines via targeting FSCN1 in Staphylococcus aureus‐induced mastitis in dairy cows

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus‐induced mastitis. In the present study, we overexpressed and suppressed miR‐145 to investigate the function of miR‐145 in Mac‐T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac‐T cytokines and in cell proliferation. We found that overexpression of miR‐145 in Mac‐T cells significantly reduced the secretion of IL‐12 and TNF‐α, but increased the secretion of IFN‐γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real‐time PCR (qRT‐PCR), Western blotting and luciferase multiplex verification techniques, we found that miR‐145 targeted and regulated FSCN1. Knock‐down of FSCN1 significantly increased the secretion of IL‐12, while the secretion of TNF‐α was significantly downregulated in Mac‐T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta‐miR‐145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.     

Severe bilateral physitis with instability and Salter‐Harris type 1 fractures in two foals

This Case Report describes the clinical, radiographic, computed tomographic and post mortem findings in 2 foals with severe bilateral physitis of the distal third metatarsal bones (MtIII). The foals were admitted because of bilateral hindlimb lameness of several weeks' duration. Initial examination revealed that the physeal regions of the distal MtIII were enlarged, warmer than normal and painful on palpation. Case 1 was treated conservatively and discharged but was readmitted 8 weeks later because of clinical deterioration. In both foals, radiographs showed plantar dislocation of both epiphyses through the physis and a Salter‐Harris type 1 fracture. The foals were subjected to euthanasia and a post mortem examination.     

Calpain activation and proteolysis in postmortem goose muscles

Meat tenderness is considered as the most important criterion for meat quality by consumers and can be improved by the actions of endogenous proteases, mainly calpains, during postmortem storage at 0–5°C. The purpose of this study, therefore, was to examine the postmortem calpain activation and proteolysis in breast (BM) and leg and thigh (LM) muscles of White Roman goose. BM and LM were taken from goose carcasses (n = 15) at 0 (10–15 min postmortem), 1, 3, and 7 days of storage at 5°C. The decrease in postmortem pH, calpain‐1 and ‐11 activities, and contents of the calpain‐1 80 kDa subunit and desmin was more rapid (p < .05) in BM than in LM. Our results show that postmortem proteolysis was more extensive in BM than in LM of White Roman goose, not only because the difference in fiber type composition between two muscles, but because the rate and extent of calpain activation were greater in BM as well. These results may provide useful information to optimize meat processing for different muscles in goose industry.     

Changes of microbial spoilage,lipid‐protein oxidation and physicochemical properties during post mortem refrigerated storage of goat meat

Examined was the effect of post mortem refrigerated storage on microbial spoilage, lipid‐protein oxidation and physicochemical traits of goat meat. Seven Boer bucks were slaughtered, eviscerated and aged for 24 h. The Longissimus lumborum (LL) and Semitendinosus (ST) muscles were excised and subjected to 13 days post mortem refrigerated storage. The pH, lipid and protein oxidation, tenderness, color and drip loss were determined in LL while microbiological analysis was performed on ST. Bacterial counts generally increased with increasing aging time and the limit for fresh meat was reached at day 14 post mortem. Significant differences were observed in malondialdehyde (MDA) content at day 7 of storage. The thiol concentration significantly reduced as aging time increased. The band intensities of myosin heavy chain (MHC) and troponin‐T significantly decreased as storage progressed, while actin remained relatively stable. After 14 days of aging, tenderness showed significant improvement while muscle pH and drip loss reduced with increase in storage time. Samples aged for 14 days had higher lightness (P < 0.05) and lower (P < 0.05) yellowness and redness. Post mortem refrigerated storage influenced oxidative and microbial stability and physico‐chemical properties of goat meat.     

Reduced intake of dietary lysine promotes accumulation of intramuscular fat in the Longissimus dorsi muscles of finishing gilts

The present study was conducted to elucidate the effect of dietary lysine levels on the intramuscular fat (IMF) content in the Longissimus dorsi (L. dorsi) muscles of finishing gilts. Eleven gilts in total from two litters of pigs aged 110 days were used. The average initial bodyweight of the pigs was 61.7 kg. Six pigs were assigned to the low lysine (LL) diet group (lysine content: 0.43 or 0.40%) and five pigs were assigned to the control group (lysine content: 0.65 or 0.68%). The diets were iso‐energetic and iso‐protein, and contained all essential amino acids (apart from lysine) in the recommended amounts. The pigs were fed these diets until their live weights reached 110 kg. Live weight gain and feed efficiency tended to be lower in the LL group (P = 0.118 and P = 0.052, respectively). Pigs from the LL group took 5 days longer to reach 110 kg (P < 0.01). The IMF content in the L. dorsi of the LL group was twice as high as that of the control group (6.7 vs 3.5%; P < 0.01). The percentage of oleic acid in the L. dorsi of the LL group tended to be higher than that of the control group (P = 0.052), whereas the percentage of linoleic acid and the total percentage of polyunsaturated fatty acids in the L. dorsi were lower (P < 0.05) in the LL group. Free L‐carnitine content in the L. dorsi was lower (P < 0.05) in the LL group. The average abundance of peroxisome proliferator‐activated receptor gamma mRNA in the L. dorsi of the LL group was threefold higher than that of the control group. The leptin mRNA abundance in the L. dorsi of the LL group was 3.3‐fold higher than that of the control group (P < 0.01). These results suggest that a higher activity of adipogenesis may have been involved in the promoted accumulation of IMF in the L. dorsi muscles of pigs, induced by a dietary LL level.     

Nasal immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis for improving ovarian responses and fertility in cross‐bred buffaloes

This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross‐bred buffaloes. A total of 134 cross‐bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 10¹⁰ CFU/ml of the C501 (pVAX‐asd‐IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF_2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX‐asd‐IS) elicited significant immune responses, and anti‐inhibin IgG antibody titres in group T1 were significantly higher (p < .01) than groups T3 and C. After the second GnRH treatment, the growth speed of the dominant follicles in group T1 was significantly faster (p < .05) than groups T3 and C. The number and diameter of large follicles (≥10 mm) as well as ovulatory follicles in group T1 were the greatest in all groups, resulting in a greater conception rate in buffaloes with positive anti‐inhibin antibodies. These results demonstrate that immunization with the C501 (pVAX‐asd‐IS) vaccine, coupled with the Ovsynch protocol, could be used as an alternative approach to improve reproductive performance in cross‐bred buffaloes.     

Partial autolysis of μ/m‐calpain during post mortem aging of chicken muscle

The objective of this study was to investigate changes occurring in μ/m‐calpain in post mortem chicken muscles and to determine the origin of the unknown bands found in calpain casein zymography. The unknown bands were reported with slightly greater mobility compared to conventional μ/m‐calpain bands in casein zymography. Identification of these bands was accomplished using native polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry and with protein phosphatase treatment. Results showed that the unknown bands were corresponding to μ/m‐calpain, and dephosphorylation by protein phosphatase did not change their appearance. The calpain samples were then incubated with various concentrations of Ca²⁺ to determine the relationship between changes in μ/m‐calpain and the appearance of the unknown bands. The products of μ/m‐calpain partial autolysis were found to be consistent with the appearance of the unknown bands. Therefore, the appearance of these bands did not result from phosphorylation of μ/m‐calpain as previously hypothesized, but from partial autolysis of μ/m‐calpain. Also their presence suggests that μ/m‐calpain undergoes partial autolysis during aging which may play certain roles in meat quality improvement.     

The effect of post‐mortem storage on the water‐soluble proteins of breast muscles of uneviscerated pheasant and chicken carcasses

Electropherograms, obtained by polyacrylamide gel electrophoresis, of water‐soluble proteins extracted from the breast muscles of uneviscerated pheasant and chicken carcasses which had been stored at 10 °C were compared. The results indicated that chicken muscle proteins remained largely unchanged, while marked changes occurred in pheasant muscle proteins especially after 10 d post‐mortem.     

Gene constitution of South-East Asian native chickens, commercial chickens and jungle fowl using polymorphisms of four calpain genes

The gene constitution of polymorphisms of the four calpain genes (µ‐calpain, m‐calpain, p94, and µ/m‐calpain) were analyzed in South‐East Asian native chickens, White Leghorn and Broiler commercial chickens, and Red and Green jungle fowl. Polymorphisms were detected at all loci in chickens and Red jungle fowl, but only for CAPN1 (µ‐calpain gene) in Green jungle fowl. CAPN2 and CAPN1.5 are linked on chicken chromosome 3, and the genotype for these loci were treated as haplotype. Some combinations of calpain loci were tested using principal component analysis, and the best combination (CAPN1, CAPN3, and CAPN1.5) was determined. The proportion of polymorphic loci (P_poly) and heterozygosity (H?) were 1.00 and 0.316–0.465 in domestic chickens and red jungle fowl, and 0.33 and 0.137 in Green jungle fowl, respectively. G_ST values suggested that the degree of subdivision among native chickens was relatively low except for Thailand, which was highest. Pair‐wise F_ST testing, dendrogram and principal component analysis from the results of calpain loci showed that the four South‐East Asian native and commercial chicken populations were close genetically.     

‘Soya Milk Tris‐based Phytoextender Reduces Apoptosis in Cryopreserved Buffalo (Bubalus bubalis) Spermatozoa’

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)‐based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS–PAGE followed by immunoblotting against caspase‐8, caspase‐9, caspase‐3, poly(ADP‐ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC‐1 assay) and apoptotic cells (annexin V‐FITC/PI assay). The results obtained clearly indicate the significant (p < 0.05) reduction in the expression of caspase‐3 (27 kDa), caspase‐8 (53 kDa), caspase‐9 (50 kDa) precursor and cytochrome c (17 kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non‐significant (p > 0.05) reduction in expression of PARP‐DNA‐binding subunit (24 kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p < 0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non‐significant (p > 0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT‐based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non‐animal origin.     

Effect of sperm pretreatment with glutathione and membrane destabilizing agents lysolecithin and Triton X‐100, on the efficiency of bovine intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is an assisted reproduction tool with several applications. Its effectiveness in bovines is lower than that in other species, mainly because of difficulties in the decondensation of the sperm nucleus after injection, and the presence of the acrosome and the plasma membrane which remain intact in this procedure. In this study, we assessed the effect of lysolecithin (LL) and Triton X‐100 (TX), in combination with glutathione (GSH) as sperm pretreatments prior to ICSI. The GSH‐LL and GSH‐TX groups showed 0% of spermatozoa with intact membrane (SYBR 14+/PI), in comparison with the control (63.3%) and GSH (65.7%) groups. The proportions of spermatozoa with damaged acrosome membrane in the GSH‐LL, GSH‐TX, GSH and control groups were 46%, 35.9%, 10.5% and 7.5%, respectively. Sperm chromatin decondensation analysis showed that the groups incubated for 3 hr with GSH presented greater decondensation (p < .05). Although fertilization was improved in all treatment groups evaluated, no differences were observed in the cleavage rate 72 hr after activation in the GSH (73.7%), GSH‐LL (80.2%) and GSH‐TX (77.8%) groups compared to the control (66.3%), neither in the blastocyst rate on day 8 (24.0%, 26.2%, 27.1% and 28.4% for the control, GSH, GSH‐LL and GSH‐TX groups, respectively). No differences were also observed in the total number of cells in all groups. In conclusion, although these sperm treatments promoted nuclear decondensation and induced plasma membrane disruption, these effects were not sufficient to improve bovine embryonic development after ICSI.     

Aortic rupture and aorto‐pulmonary fistulation in the Friesian horse: Characterisation of the clinical and gross post mortem findings in 24 cases

Reasons for performing study: In horses, aortic sinus of Valsalva aneurysms or tears in the aortic root are well‐recognised conditions in breeding stallions, often leading to sudden death. A more uncommon form of aortic rupture, located proximal to the ligamentum arteriosum has been reported in 3 Friesian horses. Objectives: The purpose of this study was to phenotypically characterise aortic rupture and aorto‐pulmonary fistulation in Friesian horses in terms of clinical and post mortem data based on 24 cases. Methods: Friesian horses that were diagnosed with aortic rupture and aorto‐pulmonary fistulation over a period of 13 years (1997–2010) at the Department of Equine Sciences of Utrecht University (n = 15) and Wolvega Equine Hospital (n = 9), were included in this study. Case history, results of clinical examination and gross post mortem findings were screened and analysed. Results: Some cases were found dead without prior symptoms, but in several cases signs such as recurrent colic, peripheral oedema and sustained tachycardia were present for several weeks prior to cardiac failure. Clinical examination during hospitalisation revealed increased rectal temperature, peripheral oedema and increased jugular pulse with a bounding arterial pulse. In the majority of horses an aortic rupture of the aortic arch near the ligamentum arteriosum, concurrent with a circumferential cuff of perivascular haemorrhage and aorto‐pulmonary fistulation, was found at post mortem examination. Conclusions: Aorto‐pulmonary fistulation in conjunction with aortic rupture is more common in Friesians than previously estimated. In some cases findings demonstrate a progressive pathology rather than acute cardiac failure and sudden death. An appropriate approach is necessary during post mortem examination of the heart in order not to overlook the diagnosis. Potential relevance: Equine practitioners should realise that in Friesian horses presented with a history of recurrent false colic, coughing, sustained tachycardia and/or peripheral oedema, aortic rupture and aorto‐pulmonary fistulation should be included in the differential diagnosis.     

Effects of electrical stimulation and pre‐rigor conditioning temperature on aging potential of hot‐boned beef M. longissimus lumborum

The objective of this study was to create various pH/temp decline rates in hot‐boned bull beef M. longissimus lumborum (LL) through a combination of electrical stimulation (ES) and pre‐rigor holding temperature. The relationship between the pre‐rigor interventions, the activities of µ‐calpain and small heat shock proteins (sHSP), and the impacts on meat product quality were determined. Paired LL loins from 13 bulls were hot‐boned within 40 min of slaughter, immediately ES and subjected to various holding temperatures (5, 15, 25, and 35°C) for 3 hr. The rate of muscle pH decline, sarcomere length, shear force, and proteolysis of muscle proteins were measured. ES‐25°C had a longer sarcomere length compared to non‐electrical stimulation samples. ES‐25°C and ES‐35°C samples had lower shear force values, higher µ‐calpain activity and higher desmin, troponin‐T, and sHSP degradation. The above findings suggest that pH/temp decline rates created in hot‐boned muscle impacted muscle protein proteolysis by increasing the activity of proteases and degradation of sHSP.     

FEASIBILITY FOR MAPPING CARTILAGE T1 RELAXATION TIMES IN THE DISTAL METACARPUS3/METATARSUS3 OF THOROUGHBRED RACEHORSES USING DELAYED GADOLINIUM‐ENHANCED MAGNETIC RESONANCE IMAGING OF CARTILAGE (dGEMRIC): NORMAL CADAVER STUDY

Osteoarthritis of the metacarpo/metatarsophalangeal joints is one of the major causes of poor performance in horses. Delayed gadolinium‐enhanced magnetic resonance imaging of cartilage (dGEMRIC) may be a useful technique for noninvasively quantifying articular cartilage damage in horses. The purpose of this study was to describe dGEMRIC characteristics of the distal metacarpus3/metatarsus3 (Mc3/Mt3) articular cartilage in 20 cadaver specimens collected from normal Thoroughbred horses. For each specimen, T1 relaxation time was measured from scans acquired precontrast and at 30, 60, 120, and 180 min post intraarticular injection of Gd‐DTPA^2‐ (dGEMRIC series). For each scan, T1 relaxation times were calculated using five regions of interest (sites 1–5) in the cartilage. For all sites, a significant decrease in T1 relaxation times occurred between precontrast scans and 30, 60, 120, and 180 min scans of the dGEMRIC series (P < 0.0001). A significant increase in T1 relaxation times occurred between 60 and 180 min and between 120 and 180 min post Gd injection for all sites. For sites 1–4, a significant increase in T1 relaxation time occurred between 30 and 180 min postinjection (P < 0.05). Sites 1–5 differed significantly among one another for all times (P < 0.0001). Findings from this cadaver study indicated that dGEMRIC using intraarticular Gd‐DTPA^2‐ is a feasible technique for measuring and mapping changes in T1 relaxation times in equine metacarpo/metatarsophalangeal joint cartilage. Optimal times for postcontrast scans were 60–120 min. Future studies are needed to determine whether these findings are reproducible in live horses.