Determination of gastrointestinal tract colonization sites from feedlot cattle transiently shedding or super-shedding Escherichia coli O157:H7 at harvest.

AIMS: The objective of the study was to determine levels of Escherichia coli O157:H7 colonization in the gastrointestinal tract (GIT) of naturally shedding cattle shedding the pathogen at low- or super-shedder levels. METHODS AND RESULTS: Over 2 years, feedlot cattle were sampled multiple times for faecal shedding of E. coli O157:H7. Just prior to harvest (1-2 days), animals that were super-shedders (≥104  CFU per gram of faeces) were specifically identified, and based on the longer term screening data, pen cohorts that were low-shedders (years 1 and 2) or chronic-shedders (year 1) were also identified. At harvest, samples were collected from throughout the GIT, including the rectoanal junction (RAJ) for enumeration and enrichment of E. coli O157:H7. The mouth samples exhibited the greatest prevalence for the pathogen, and the abomasum and rumen exhibited the lowest prevalence (P < 0·05). Super-shedders had significantly greater prevalence for all GIT locations except the mouth and abomasum compared to low-shedders, but the super-shedders were the only animals with positive abomasum samples. Samples from the super-shedders were enumerable for most GIT locations, and the rectum and RAJ locations were the only locations that were significantly greater than other locations (P < 0·05). CONCLUSIONS: Across all animals naturally exposed to E. coli O157:H7, the risk of ingestion is high, but rumen and abomasum are potential barriers to passage. In super-shedders, the passage through the GIT was greater, allowing colonization in the rectum and at the RAJ. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 low-shedding cattle had lower pathogen levels throughout the GIT, indicating intrinsic GIT factors to these cattle may reduce pathogen passage through the GIT, including the abomasum, and minimize risk of RAJ colonization.

Inhibitory effects of grape seed extract on growth, quorum sensing, and virulence factors of CDC "top-six" non-O157 Shiga toxin producing E. coli.

Non-O157 Shiga toxin producing Escherichia coli (STECs) have become a growing concern to the food industry. Grape seed extract (GSE), a byproduct of wine industry, is abundant in polyphenols that are known to be beneficial to health. The objective of this study was to evaluate the effect of GSE on the growth, quorum sensing, and virulence factors of Centers for Disease Control and Prevention (CDC) "top-six" non-O157 STECs. Minimal inhibitory concentration (MIC) of GSE was 2mg/ml against E. coli O26:H11, and 4mg/ml against the other non-O157 STECs tested. Minimal bactericidal concentration (MBC) was the same as MIC for all six non-O157 STECs tested. At 5×10(5)CFU/ml inoculation level, 4mg/ml GSE effectively inhibited the growth of all tested strains, while 0.25-2mg/ml GSE delayed bacterial growth. At a higher inoculation level (1×10(7)CFU/ml), GSE had less efficacy against the growth of the selected six non-O157 STECs. Its impact on bacterial virulence was then assessed at this inoculation level. Autoinducer-2 (AI-2) is a universal signal molecule mediating quorum sensing (QS). GSE at concentration as low as 0.5mg/ml dramatically reduced AI-2 production of all non-O157 STECs tested, with the inhibitory effect proportional to GSE levels. Consistent with diminished QS, GSE at concentration of 0.125mg/ml caused marked reduction of swimming motility of all motile non-O157 STECs tested. In agreement, GSE treatment reduced the production of flagella protein FliC and its regulator FliA in E. coli O103:H2 and E. coli O111:H2. Furthermore, 4mg/ml GSE inhibited the production of Shiga toxin, a major virulence factor, in E. coli O103:H2 and E. coli O111:H2. In summary, GSE inhibits the growth of "top-six" non-O157 STECs at the population level relevant to food contamination. At higher initial population, GSE suppresses QS with concomitant decrease in motility, flagella protein expression and Shiga toxin production. Thus, GSE has the potential to be used in food industry to control non-O157 STEC.

Effects of early- to mid-gestational undernutrition with or without protein supplementation on offspring growth, carcass characteristics, and adipocyte size in beef cattle.

Angus × Gelbvieh cows with 2 to 3 previous pregnancies were used to evaluate effects of maternal nutrient restriction on offspring adipose tissue morphology at standard production endpoints. At 45 d after AI to a single sire, pregnancy was confirmed and cows randomly allotted into groups and fed a control (Con, 100% of NRC recommendations), nutrient-restricted (NR, 70% of Con diet), or nutrient-restricted + protein-supplemented (NRP, 70% of Con + essential AA supply to the small intestine equal to Con) diet. At d 185 of gestation, cows were commingled and received the Con diet thereafter. Bull calves were castrated at 2 mo of age. Calves were weaned at 210 d, backgrounded for 28 d, and then placed in the feedlot for 195 d. Steers and heifers were slaughtered at an average 12th-rib fat thickness of 7.6 mm. Adipose tissue from selected depots was collected for adipocyte size analysis. There was no significant difference in BW or BCS between Con, NRP, and NR cows at d 45 of gestation, which averaged 489.7 ± 17.7 kg and 5.35 ± 0.13, respectively. At d 185 of gestation, Con and NRP groups had similar BW (566.1 ± 14.8 and 550.2 ± 14.8 kg) and BCS (6.34 ± 0.27 and 5.59 ± 0.27), but NR cows exhibited reduced (P < 0.05) BW (517.9 ± 14.8 kg) and BCS (4.81 ± 0.27). Among offspring (steers and heifers) at slaughter, there were no significant differences in BW or organ weights among treatment groups. Yield grade was reduced (P < 0.05) and semitendinosus weight/HCW tended (P = 0.09) to be reduced in NR offspring compared with Con and NRP offspring. Average adipocyte diameter was increased (P < 0.05) in subcutaneous, mesenteric, and omental adipose tissue and tended (P = 0.09) to increase in perirenal adipose tissue in NR compared with Con offspring with NRP offspring adipocyte diameter being either intermediate or similar to Con calves. The adipocyte size alterations observed in NR offspring were confirmed by DNA concentration of the adipose tissue depots. There also was an increased mRNA expression (P < 0.05) of fatty acid transporter 1 in subcutaneous adipose tissue from NR offspring compared with Con and NRP offspring. Nutritional restriction during early and mid gestation increased or tended to increase (P < 0.09) adipocyte diameter in all adipose tissue depots in finished steer and heifer calves.

Nutrition during mid to late gestation affects growth, adipose tissue deposition, and tenderness in cross-bred beef steers.

The objective of this study was to examine whether the plane of nutrition of cows at a critical time for fetal skeletal muscle and adipose tissue development would affect meat quality and carcass composition of offspring. To alter maternal nutrition, beef cows were placed on improved pasture (IP) or native range (NR) pasture from 120 to 150 through 180 to 210days of gestation. Esophageal extrusa samples collected from cows grazing IP varied from 11.1% crude protein of organic matter early in the test period to 6.0% crude protein of organic matter at the end of the grazing period; whereas, extrusa samples of cows grazing NR ranged from 6.5% crude protein of organic matter during early grazing to 5.4% crude protein of organic matter at the end of the grazing period. Steers were slaughtered and carcass characteristics were collected. Warner-Bratzler shear force was performed on longissumus steaks, western blotting was used to measure proteolysis, and myosin isoform typing was performed. Improved pasture steers had heavier live and hot carcass weights. Tenderness was greater in IP compared to NR steers. No difference in calpastatin content and troponin-T degradation was observed between treatments. The 12th rib fat thickness was greater for IP than for NR steers. Subcutaneous adipose tissue of IP steers tended to have a greater number of cells per field of view than NR steers. Data show improving nutritional status of cows during mid to late gestation affects tenderness, adipose tissue deposition and growth in steers.

Mouse AMP-activated protein kinase gamma3 subunit R225Q mutation affecting mouse growth performance when fed a high-energy diet.

The Rendement Napole (RN) genotype widely exists in Hampshire pigs. Recently, RN gene was identified as a R200Q mutation in AMP-activated protein kinase (AMPK) gamma3 subunit. The effect of RN genotype on the growth performance of animals and the underlying mechanisms remain controversial. Using transgenic mice carrying an analogous R225Q mutation, the objective of this study was to study the role of RN gene in the growth performance of animals at different energy levels. Wild-type (WT) mice and those with the RN mutation were assigned to 4 groups: 1) WT plus normal diet, 2) RN plus normal diet, 3) WT plus high-energy diet, and 4) RN plus high-energy diet. Mice were weaned at 21 d old and fed the trial diets for 1 mo and then killed for carcass measurements. The pH of postmortem muscle from RN mice was less (P < 0.01) than that from WT mice. No difference in growth performance was observed when mice were fed a normal diet. When fed a high-energy diet, RN mice showed a greater fat accumulation (WT vs. RN, 1.11 vs. 1.63 g for gonadal fat and 1.40 vs. 1.84 g for subcutaneous fat; P < 0.05). Muscle weight was also increased (WT vs. RN, 0.27 vs. 0.30 g for gastrocnemius muscle; P < 0.05). The food consumption was greater in RN compared with WT mice (2.95 vs. 2.49 g; P < 0.05). The AMPK content and its downstream target, acetyl-CoA carboxylase (ACC), content were greater in RN mice (P < 0.05). The phosphorylation of ACC at Ser 79, a site exclusively phosphorylated by AMPK, was increased (P < 0.05), showing greater AMPK activity in RN mouse muscle. No difference in muscle fiber composition and mitochondrial content was observed between WT and RN mice. High fat diet downregulates protein kinase B but upregulates extracellular signal-regulated kinase signaling. In conclusion, the R225Q mutation has no major effect on the growth performance of animals fed a normal diet; a high-energy diet increased fatness in RN mice, likely due to their greater consumption of feed compared with WT mice.

Caspase 3 is not likely involved in the postmortem tenderization of beef muscle.

Postmortem proteolysis is an important determinant of beef tenderness. Caspase 3 is a protease that functions in apoptosis and has been shown to degrade myofibrillar proteins. Our objective was to evaluate whether caspase 3 activity is related to beef tenderness and muscle growth, and whether caspase 3 is activated in postmortem beef muscle. In experiment 1, longissimus thoracis (LT) and sternomandibularis muscle samples were obtained at 0, 0.25, 1, 3, 24, 72, and 240 h postmortem from 5 steers. In experiment 2, a group of 40 beef cattle was slaughtered at the University of Wyoming Meat Lab with 10 steers of different tenderness and growth characteristics chosen for the analysis of caspase 3 activity in the LT. In experiment 3, 10 steers with different tenderness but matched growth characteristics were chosen for analyses. In experiment 1, no significant activation (P = 0.70) of caspase 3 activity was detected; only a decreased activity at 72 (P = 0.05) and 240 h (P = 0.02) postmortem was observed. Western blot analysis of both muscle samples showed only the pro-caspase 3 form and failed to detect the activated enzyme. In experiment 2, caspase 3 activity in the LT immediately postmortem was greater (P = 0.05) for the cattle with increased Warner-Bratzler shear force values. No difference in caspase 3 activity was detected for experiment 3. Our results demonstrate that caspase 3 activity is not activated, with its activity decreasing with time postmortem, and caspase 3 activity is not associated with Warner-Bratzler shear force at slaughter. Therefore, caspase 3 is not anticipated to be involved in postmortem tenderization of beef.

AMP-activated protein kinase is negatively associated with intramuscular fat content in longissimus dorsi muscle of beef cattle.

Marbling, or intramuscular fat, is an important factor in meat quality. As a key regulator of lipid metabolism, AMP activated protein kinase (AMPK) may be associated with intramuscular fat accumulation. Our objective was to evaluate the relationship among AMPK and its associated signaling mediators, with marbling and lean growth in beef cattle. Steers with high intramuscular fat content (High IMF, 5.71±0.36%, n=5) and low intramuscular fat content (Low IMF, 2.09±0.19%, n=5) were selected. High IMF was associated with increased tenderness (P<0.05) and backfat thickness (P<0.01). Muscle weights were higher in Low compared to High IMF (P<0.05). High IMF steers had a reduced AMPK activity (P<0.01), reduced acetyl-CoA carboxylase phosphorylation (P<0.05), and reduced total mTOR (P=0.02) content. Data provide evidence that AMPK is involved in IMF deposition in beef cattle.

Effect of early gestational undernutrition on angiogenic factor expression and vascularity in the bovine placentome.

The effect of early gestation maternal undernutrition followed by realimentation on placentomal vascular growth and angiogenic factor expression was determined in multiparous beef cows bred to the same bull. Cows gestating only female fetuses (n = 30) were fed in equal numbers to meet the NRC requirements (control) or were fed below the NRC requirements to lose BW (nutrient restricted; NR) from d 30 to 125 of gestation. After slaughter on d 125 of gestation, 10 control and 10 NR cows were necropsied. The remaining NR cows (n = 5) were then fed to achieve a BCS equal to their control group contemporaries (n = 5) by d 220 of gestation. All cows were fed the control diet from d 220 until 250 of gestation, when the remaining control and NR cows were slaughtered and necropsied. At necropsy, placentomes were fixed via perfusion of the caruncular and cotyledonary arteries to determine capillary vascular density. Cotyledonary (fetal placental) and caruncular (maternal placental) tissues also were snap-frozen in liquid nitrogen, and mRNA concentrations of vascular endothelial growth factor and its 2 specific receptors, fms-like tyrosine kinase and kinase insert domain containing receptor, as well as placental growth factor, were determined. There was no effect of diet or day of gestation on the percentage of proliferating caruncular cells. Although diet did not impact cotyledonary cellular proliferation, there was an increase (P < 0.05) in the percentage of proliferating cells on d 250 compared with d 125 of gestation. Nutrient restriction from d 30 to 125 increased (P < or = 0.10) placental mRNA concentrations of placental growth factor and fms-like tyrosine kinase; however, there was no alteration in vascularity. By d 250 of gestation, NR cows had increased (P < 0.05) caruncular capillary surface density and decreased (P < 0.05) cotyledonary capillary area density, capillary number density, and capillary surface density compared with control cows. Although nutrient restriction had little effect on placental vascularity by d 125, upon realimentation, alterations in vascularity became apparent by d 250 of gestation, suggesting a placental programming effect.

Maternal undernutrition during early to mid-gestation in the ewe results in altered growth, adiposity, and glucose tolerance in male offspring.

This study utilized maternal undernutrition from early to midgestation in the ewe to determine the impact(s) of intrauterine growth restriction on postpartum growth of male offspring and the potential mechanisms involved. Multiparous ewes were fed 50% (nutrient-restricted) or 100% (control-fed) of their nutrient requirements (NRC, 1985) between d 28 and 78 of gestation, and then all ewes were fed 100% of the NRC requirements from d 79 through lambing. Male lambs born to nutrient-restricted (n = 9) and control-fed (n = 9) ewes exhibited similar BW (5.8 vs. 6.0 +/- 0.3 kg) and crown-rump lengths (53.8 vs. 55.4 +/- 1.0 cm) at birth. At 63 and 250 d of postnatal age, wether lambs were subjected to a glucose tolerance test, in which a bolus of glucose was administered i.v. to evaluate changes in glucose and insulin concentrations. After i.v. glucose administration at 63 d of age, lambs from nutrient-restricted ewes exhibited a greater area under the curve for glucose (AUCg; 6,281 vs. 5,242 +/- 429; P < 0.05) and insulin (AUCi; 21.0 vs. 8.6 +/- 1.9; P < 0.001) than lambs from control-fed ewes. After glucose administration at 250 d of age, lambs from nutrient-restricted ewes had greater AUCg (7,147 vs. 5,823 +/- 361; P < 0.01) but a lower AUCi (6.4 vs. 10.2 +/- 1.9; P = 0.05) than lambs from control-fed ewes. Lambs from nutrient-restricted ewes were heavier (26.6 vs. 21.8 +/- 2.3 kg; P < 0.05) and had more backfat (0.30 vs. 0.21 +/- 0.03 cm, P < 0.05) by 4 mo of age than the lambs from control-fed ewes. At slaughter at 280 d of age, lambs from nutrient-restricted ewes remained heavier than lambs from control-fed ewes, had greater (P < 0.05) amounts of kidney and pelvic-area adipose tissue, and tended (P < 0.10) to have reduced LM and semitendinosus muscle weights as a percentage of HCW. These data demonstrate that a bout of maternal undernutrition during early to midgestation in sheep increased BW and fat deposition during adolescence and dysregulated glucose uptake in the absence of any change in birth weight.

The halothane gene, energy metabolism, adenosine monophosphate-activated protein kinase, and glycolysis in postmortem pig longissimus dorsi muscle.

The presence of the halothane gene results in PSE meat. However, the exact mechanisms linking the halothane gene and the incidence of PSE meat remain unclear. We hypothesize that the presence of the halothane gene accelerates energy consumption in postmortem muscle, which activates adenosine monophosphate-activated protein kinase (AMPK), leading to enhanced glycolysis and PSE meat. To test our hypothesis, energy status, AMPK activity, and glycolysis in the postmortem LM of the halothane gene carrier and halothane-negative pigs were compared. The results showed that the presence of the halothane gene accelerated energy depletion in postmortem muscle immediately after exsanguination, leading to rapid and early depletion of ATP, as shown by an increase in the (adenosine monophosphate + inosine monophosphate):ATP ratio in postmortem LM. In addition, an early AMPK activation was observed in LM from halothane carriers. The fructose-2,6-diphosphate concentration in postmortem LM was well correlated with AMPK activation. To be a potent stimulator of phosphofructose kinase, the increase in fructose-2,6-diphosphate is expected to activate phosphofructose kinase, a key enzyme controlling glycolysis, leading to enhanced glycolysis and early accumulation of lactic acid. In summary, this study showed that the presence of the halothane gene induced early energy depletion, which could be a primary reason causing AMPK activation, leading to accelerated glycolysis and an increased incidence of PSE meat. However, AMPK might also be activated by other mechanisms besides energy depletion, which warrants further studies.

Maternal nutrient restriction upregulates growth signaling pathways in the cotyledonary artery of cow placentomes.

This study evaluated the role of MAPK/ERK1/2 and/or PI3-K/Akt signaling pathways in modulating bovine placentomal vascularity in response to maternal nutrient restriction. Beef cows were randomly assigned to control fed (Control, n=15, 100% of requirements) or nutrient restricted (NR, n=15, 50% requirements) diets from day 30 to day 125 of gestation. Ten cows from each dietary group were necropsied on day 125 (approximately 45% gestation), and the remaining cows in each diet group were then fed control diets and necropsied on day 250 (approximately 90% gestation). At day 125 of gestation, NR cows exhibited increased (P=0.06) COT vascularity, improved (P<0.05) placentome efficiency (fetal weight/placentomal weight), and increased (P<0.05) phosphorylated Akt and ERK1/2 in COT arteries compared to Control cows. By day 250, however, treatment differences in COT vascularity and phosphorylated Akt and ERK1/2 in COT arteries were lost. On both gestational days, no treatment difference was observed in the levels of phosphorylated Akt or ERK1/2 in CAR arteries. CAR vascularity was similar across treatment on day 125, but tended to be greater (P<0.10) in NR than Control cows on day 250. These data suggest that conceptuses react to an early gestational nutrient restriction by up-regulating COT growth signaling pathways associated with angiogenesis, and that these compensations do not persist to term.

Pre-slaughter transport, AMP-activated protein kinase, glycolysis, and quality of pork loin.

Numerous studies have revealed that pre-slaughter stress, like transport, increases the occurrence of pale, soft, and exudative (PSE) pork meat. The molecular mechanism underlying this phenomenon, however, is poorly defined. In this study, we investigated the effects of pre-slaughter transport and subsequent rest on energy metabolism, AMP-activated protein kinase (AMPK) activation, and glycolysis in postmortem pork loin. Results indicated that pre-slaughter transport accelerated ATP depletion, which led to lower energy status in postmortem muscle immediately post-exsanguination when compared with control. The lower energy status led to AMPK activation within 1h postmortem, subsequently increasing glycolysis, leading to rapid glycolysis and high incidence of PSE meat. Allowing pigs to rest after transport restored energy status in muscle ante-mortem. Higher energy status then prevented premature and rapid AMPK activation in postmortem muscle and lessened the negative effects of pre-slaughter transport on meat quality. AMPK regulated glycolysis in postmortem muscle, at least partially, through phosphorylation and activation of phosphofructose kinase-2, since fructose-2,6-diphosphate content, an allosteric activator of phosphofructose kinase-1, was well correlated with AMPK activation and glycolytic rate. This suggests that AMPK is a potential molecular target for the control of PSE incidence in pork.

Nutrient restriction differentially modulates the mammalian target of rapamycin signaling and the ubiquitin-proteasome system in skeletal muscle of cows and their fetuses.

The mammalian target of rapamycin (mTOR) signaling controls nutrient-stimulated protein synthesis in skeletal muscle, whereas ubiquitin-proteasome systems control the degradation of myofibrillar proteins. The objective of this study was to elucidate the effect of nutrient restriction on the mTOR signaling and ubiquitin-proteasome system in the skeletal muscle of cows and their fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet that provided 100% nutrient requirements or a nutrient-restricted diet at 68.1% of NE(m) and 86.7% of metabolizable protein requirement. Cows were slaughtered on 125 d of gestation, and the LM of both cows and fetuses was sampled for the measurement of mTOR, ribosomal protein S6, adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein ubiquitylation. When comparing the muscle samples from nutrient-restricted and control cows and their fetuses, no difference was observed for the content of mTOR and ribosomal protein S6, but the phosphorylation of mTOR at Ser(2448) and ribosomal protein S6 at Ser(235/336) were greater (P < 0.05) in control muscle than in muscle from nutrient-restricted animals. Because the phosphorylation of mTOR and ribosomal protein S6 upregulates translation, these results showed that nutrient restriction inhibits protein synthesis in muscle. The activity of AMPK in the muscle of nutrient-restricted cows was significantly lower (P = 0.05) than that of control cows. The protein ubiquitylation, however, was greater (P < 0.05) in the muscle from nutrient-restricted cows, showing accelerated protein degradation. No difference in the protein ubiquitylation was detected for fetal muscle. Data suggested that the decreased protein synthesis and promoted protein degradation resulted in muscle atrophy of pregnant cows, but not in fetal muscle. Results of this study show that in response to nutrient restriction, protein degradation was differentially regulated between cow and fetal muscle. The atrophy of cow muscle during nutrient deficiency may involve the enhanced degradation of muscle proteins.

Effect of nutrient restriction on calpain and calpastatin content of skeletal muscle from cows and fetuses.

Calpains are crucial for the degradation of myofibrillar proteins in muscle. Calpastatin is a specific inhibitor of calpains. The objective of this study was to elucidate the effect of nutrient restriction on the activity of calpains and calpastatin in the skeletal muscle of both cows and fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet consisting of native grass hay fortified with vitamins and minerals at recommendations for a mature cow to gain 0.72 kg/d or half the vitamins and minerals and millet straw at 68.1% of NEm requirements. Cows were slaughtered on d 125 of gestation, and the LM was sampled at the 12th rib for calpain and calpastatin measurement. When comparing the muscle samples from nutrient-restricted and control cows, no difference in the activity of calpain I and II was observed; however, there was a significant difference (P < 0.05) in calpastatin activity. Muscle samples from control cows had greater calpastatin content than those of nutrient-restricted cows (P < 0.05); in contrast, the calpastatin content of fetal muscle was greater in fetuses gestated by nutrient-restricted cows than those of control cows (P < 0.05). Further, there were three calpastatin isoforms of 125, 110, and 70 kD detected in fetal muscle, whereas only the110-kD isoform was detected for cow muscle. These results indicate that the activity of the calpain system in skeletal muscle is mainly controlled through the expression of calpastatin. Alternating the calpastatin content in muscle and thereby modulating calpain activity may provide a mechanism for the maintenance of fetal muscle growth during nutrient restriction, whereas skeletal muscle loss in cows is upregulated.

Bovine metalloprotease characterization and in vitro connective tissue degradation.

Metalloproteases that selectively hydrolyze connective tissue proteins may tenderize meat without creating texture problems associated with myofibrillar protein degradation. Our objective was to characterize the activity of bovine placental proteases to determine whether they can improve meat tenderness through disruption of the connective tissue matrix. Enzymes were extracted, crudely purified, and proteolytic activity was assessed against gelatin and collagen under varying pH and temperature conditions using both SDS-PAGE and zymography. Gelatin zymography revealed proteolysis between 57 and 63 kDa, with decreased activity as buffer pH decreased from pH 7.4 to 5.4 (37 degrees C). Proteolytic activity was pronounced at 37 degrees C, moderate at 25 degrees C, and absent at 4 degrees C following 48-h incubation (pH 7.4). Placental enzymes were metalloproteases inhibited by excess EDTA. Maximum proteolysis was achieved in the presence of Ca2+, with or without Mg2+ and Zn2+. Absence of Ca2+ decreased proteolytic activity. Complete degradation of both the 125- and 120-kDa proteins of the alpha-chains of gelatin was achieved following enzyme incubation for 6 h at 37 degrees C or 24 h at 25 degrees C. No degradation was observed following enzyme incubation with native Type I collagen. Given the marked decrease in enzyme activity at pH 5.4 and 4 degrees C (standard industry conditions), bovine placental metalloproteases would not be expected to contribute to connective tissue degradation or improve meat tenderness.

Feeding lambs high-oleate or high-linoleate safflower seeds differentially influences carcass fatty acid composition.

Our objective was to determine effects of dietary high-oleate (Oleate; 76% 18:1) or high-linoleate (Linoleate; 78% 18:2) safflower seeds on fatty acids in muscle and adipose tissue of feedlot lambs. White-faced ewe lambs (n = 36) were fed a beet pulp, oat hay, and soybean meal basal diet (Control), blocked by BW, and allotted randomly to dietary treatments. Cracked safflower seeds were used in isocaloric and isonitrogenous replacement of beet pulp, oat hay, and soybean meal so that Oleate and Linoleate diets contained 5.0% additional fat. Fatty acids were determined in semitendinosus, longissimus dorsi (longissimus), and adipose tissue from the tail head (tailhead adipose tissue), adjacent to the 12th rib (s.c. adipose tissue), and kidney and pelvic fat (KPH adipose tissue) depots. Fatty acid data were analyzed within muscle and adipose tissue as a split-block design. Single degree of freedom orthogonal contrasts were used to compare treatment effects. Average daily gain, feed efficiency, and carcass characteristics did not differ (P = 0.15 to 0.96) across dietary treatments. Adipose tissue saturated fatty acids were greater (P = 0.04) for Controls but were not different (P = 0.36) in muscle. Trans-vaccenic acid (18:1(trans-11)) increased (P < 0.0001) with safflower supplementation and was greater (P < 0.0001) in Linoleate than in Oleate for both tissue types. Linoleate lamb had greater (P < 0.0001) PUFA than Oleate lamb in muscle and adipose tissue. Conjugated linoleic acids (CLA; cis-9, trans-11 and trans-10, cis-12) were greater (P < 0.0001) in muscle and adipose tissue of lambs fed safflower seeds. Lambs fed Linoleate had greater (P < 0.0001) CLA in adipose tissue and muscle than lambs fed Oleate. Saturated fatty acids were greater (P < 0.0001) in s.c. adipose tissue than in tailhead adipose tissue. Mono- and polyunsaturated fatty acids were greater (P < 0.0001) in tailhead adipose tissue than in s.c. adipose tissue. Weight percentages of 18:1(trans-11) ranked tailhead adipose tissue = KPH adipose tissue > s.c. adipose tissue and semitendinosus > longissimus, whereas CLA ranked tailhead adipose tissue > s.c. adipose tissue > KPH adipose tissue and semitendinosus > longissimus. Feeding mono- and polyunsaturated fatty acids increased tissue 18:1(trans-11) and CLA, which is a favorable change in regard to current human dietary guidelines.

Bovine placental protease specificity toward muscle connective tissue proteins.

Enzymes currently used to tenderize meat are not substrate-specific, resulting in extensive myofibrillar protein degradation that often produces an undesirable texture. Bovine placental metalloproteases, which selectively hydrolyze connective tissue proteins while leaving myofibrillar proteins intact, may tenderize meat without causing texture problems. Therefore, our objective was to extract and crudely purify bovine metalloproteases from bovine placenta for possible use as tenderizers in meat systems. Enzymes were extracted from homogenized tissue and purified by ammonium sulfate precipitation. Samples were collected before (crude enzyme) and after gel filtration on a Sephadex G-100 column. Spectrophotometric analysis identified one major peak (filtered enzyme). Gelatin, casein, and type I acid-soluble collagen zymography were used to determine substrate specificity. Beef myofibrillar proteins were incubated with crude and filtered enzyme fractions, enzymes quenched, and substrate degradation visualized using SDS-PAGE. Active gelatinases and collagenases exhibiting molecular weights of 57 to 65 kDa were detected on zymograms. Banding patterns from crude enzyme indicated two enzymes with both gelatinase and collagenase activity and a third enzyme with gelatinase activity only. Banding patterns from filtered enzyme indicated two enzymes with both gelatinase and collagenase activity. Proteolytic activity was not detected with casein, actin, or myosin heavy-chain substrates. Due to specificity for collagen and gelatin, these enzymes may be capable of improving the tenderness of certain cuts relatively high in connective tissue, while avoiding myofibrillar protein hydrolysis.

Antioxidant activity of mechanically separated pork extracts.

Utilization of synthetic carnosine as a food additive is limited by both regulatory and economic hurdles. Therefore, the potential of producing carnosine-containing antioxidant extracts from an underutilized skeletal muscle source, mechanically separated pork (MSP), was investigated. Carnosine-containing MSP extracts were capable of inhibiting lipid oxidation both in vitro and in salted ground pork. Heating (60-80°C) the MSP extract removed iron and increased in vitro antioxidant activity. Isolation of a low molecular weight fraction of the MSP extract by ultrafiltration was effective at decreasing iron but did not substantially increase in vitro antioxidant activity. Freeze dried extracts (untreated, 80°C, ultrafiltration permeate) were capable of inhibiting both TBARS and lipid peroxide formation in ground, salted pork stored at -15°C. While MSP extracts were capable of inhibiting lipid oxidation both in vitro and in salted, ground pork, their antioxidant activity was low suggesting that their use as a food additive would be impractical.

Derivation of an equation to estimate marrow content of bovine cervical vertebrae.

Marrow content of bovine cervical vertebrae from Choice- and Select-grade carcasses weighing 294 to 343 kg was determined so that a method to monitor the amount of marrow in meat from advanced meat/bone separation machinery and recovery (AMR) systems could be developed. The marrow determination requires cleaning and then ashing bones. Because a large difference in ash content of bone and bone marrow exists and because cartilage content of cervical vertebrae in Choice and Select beef is relatively constant, it was possible to derive the following equation: Weight of marrow = [weight of cartilage (% ash in cartilage - % ash in bone) + % ash in bone (total weight) - (total ash)]/[(% ash in bone - % ash in marrow)]. Constants for ash in fresh bone, marrow, and cartilage were 58.51, .57, and 2.14% with SD of 2.23, .15, and .30%, respectively. A cartilage content of 9.5% along with cervical vertebrae weight and total ash weight were also used to calculate 33.9% marrow in cervical vertebrae. Means for marrow pressed or centrifuged from bovine cervical vertebrae were lower than those obtained from the equation. Therefore, pressing and centrifuging left some marrow in spongy bone. Our ashing method for determining the amount of marrow in whole cervical vertebrae should be useful for determining marrow remaining in cervical vertebrae of bone cakes from AMR systems. Percentage ash in pressed bones is higher and the calculated marrow content is lower when pressed bones are compared to cervical vertebrae that are not pressed. The amount of marrow in whole cervical vertebrae minus the amount left in cervical vertebrae from bone cakes equals the amount in meat from AMR systems.