Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n = 1,941) and parthenogenetic activated (n = 1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100 ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit β-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.     

Mammalian target of rapamycin signaling and ubiquitin-proteasome–related gene expression in skeletal muscle of dairy cows with high or normal body condition score around calving

The objective of the current study was to investigate the effects of overconditioning around calving on gene expression of key components of the mammalian target of rapamycin (mTOR) pathway and ubiquitin-proteasome system (UPS) in skeletal muscle as well as the AA profiles in both serum and muscle of periparturient cows. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to either a high body condition group (HBCS; n = 19) or a normal body condition group (NBCS; n = 19) and were fed different diets until dry-off (d ?49 relative to calving) to amplify the difference. The groups were also stratified for comparable milk yields (NBCS: 10,361 ± 302 kg; HBCS: 10,315 ± 437 kg). At dry-off, the NBCS cows (parity: 2.42 ± 1.84; body weight: 665 ± 64 kg) had a body condition score (BCS) <3.5 and backfat thickness (BFT) <1.2 cm, whereas the HBCS cows (parity: 3.37 ± 1.67; body weight: 720 ± 57 kg) had a BCS >3.75 and BFT >1.4 cm. During the dry period and the subsequent lactation, both groups were fed identical diets but maintained the BCS and BFT differences. Blood samples and skeletal muscle biopsies (semitendinosus) were repeatedly (d ?49, +3, +21, and +84 relative to calving) collected for assessing the concentrations of free AA and the mRNA abundance of various components of mTOR and UPS. The differences in BCS and BFT were maintained throughout the study. The circulating concentrations of most AA with the exception of Gly, Gln, Met, and Phe increased in early lactation in both groups. The serum concentrations of Ala (d +21 and +84) and Orn (d +84) were lower in HBCS cows than in NBCS cows, but those of Gly, His, Leu, Val, Lys, Met, and Orn on d ?49 and Ile on d +21 were greater in HBCS cows than in NBCS cows. The serum concentrations of 3-methylhistidine, creatinine, and 3-methylhistidine:creatinine ratio increased after calving (d +3) but did not differ between the groups. The muscle concentrations of all AA (except for Cys) remained unchanged over time and did not differ between groups. The muscle concentrations of Cys were greater on d ?49 but tended to be lower on d +21 in HBCS cows than in NBCS cows. On d +21, mTOR and eukaryotic translation initiation factor 4E binding protein 1 mRNA abundance was greater in HBCS cows than in NBCS cows, whereas ribosomal protein S6 kinase 1 was not different between the groups. The mRNA abundance of ubiquitin-activating enzyme 1 (d +21), ubiquitin-conjugating enzyme 1 (d +21), atrogin-1 (d +21), and ring finger protein-1 (d +3) enzymes was greater in HBCS cows than in NBCS cows, whereas ubiquitin-conjugating enzyme 2 was not different between the groups. The increased mRNA abundance of key components of mTOR signaling and of muscle-specific ligases of HBCS cows may indicate a simultaneous activation of anabolic and catabolic processes and thus increased muscle protein turnover, likely as a part of the adaptive response to prevent excessive loss of skeletal muscle mass during early lactation.     

Effects of nutritional level on pork quality and gene expression of μ-calpain and calpastatin in muscle of finishing pigs

The study was designed to investigate the effects of nutritional level (control diet (CD), 14.19% crude protein, 13.81 MJ of DE/kg; low nutritional level diet (LND), 11.08% crude protein, 12.55 MJ of DE/kg) on pork quality and gene expression of μ-calpain and calpastatin in muscle of finishing pigs. The LND treatment increased drip loss (P < 0.05), had a trend to increase intramuscular fat (IMF) content (P = 0.09), decreased Warner–Bratzler shear force (WBSF) of pork (P < 0.05), improved mRNA level of μ-calpain (P < 0.05) in skeletal muscle, but had no effect on gene expression of calpastatin, compared with the CD treatment. These data suggest that a moderately reduced energy and protein diet increased pork tenderness and intramuscular fat. The increase in tenderness by LND treatment may be partly due to increased gene expression of μ-calpain in muscle.     

Degradation of γ- and α-tocopherol and formation of 5-nitro-γ-tocopherol induced by peroxynitrite in liposomes and skeletal muscle

Peroxynitrite, formed from the reaction between nitric oxide and superoxide, can participate in free radical-mediated reactions with cellular components in muscle to (1) initiate lipid oxidation via the production of lipid hydroperoxides, and (2) produce novel nitrated products. 5-Nitro-γ-tocopherol (NGT) is formed by the electrophilic substitution reaction between peroxynitrite and γ-tocopherol. The objective of this research was to examine the utility of NGT as a lipid-phase, peroxynitrite-specific biomarker in muscle foods. NGT was detected when exogenous peroxynitrite was added to liposomes containing γ-tocopherol and homogenates from chicken dark and turkey light muscle with added γ-tocopherol. Detectable levels of NGT were not observed in either minced turkey light muscle stored at -20?°C or chicken dark muscle stored at 4?°C. These results suggest that NGT is not a suitable biomarker to confirm the presence of endogenously produced peroxynitrite in muscle foods.     

Heterologous expression and stability of the Escherichia coli β-galactosidase gene in Streptococcus lactis by translation fusion

An Escherichia coli/Streptococcus lactis shuttle vector for creating translation fusions to the E. coli galactosidase (lacZ) has been constructed. Random S. lactis chromosomal DNA fragments inserted upstream of the lacZ gene and in frame promote the expression of active galactosidase in both E. coli and S. lactis. One fusion pSK216 has been characterized by Western blotting to reveal a fusion protein of 116 kd. S. lactis LM0230, a plasmidless lac⁻ derivative of S. lactis C2 when carrying pSK216, exhibits a Lac⁺ phenotype suggesting the presence of a chromosomally encoded lactose permease. Further evidence for this lactose permease system is provided by assay of lactose uptake by S. lactis LM0230. In the absence of selection pSK216 was less stable in S. lactis LM0230 than its derivative pSK41. The growth rate of S. lactis LM0230 carrying pSK216 is slower in comparison to the plasmid-free strain. Two genetic events were observed, the deletion of the lacZ gene and the loss of the entire plasmid. These results indicate that recombinant plasmids are unstable in S. lactis and imply that there is an obstacle in the genetic engineering of lactic acid bacteria.     

Mammalian target of rapamycin signaling and ubiquitin proteasome–related gene expression in 3 different skeletal muscles of colostrum- versus formula-fed calves

The rates of protein turnover are higher during the neonatal period than at any other time in postnatal life. The mammalian target of rapamycin (mTOR) and the ubiquitin-proteasome system are key pathways regulating cellular protein turnover. The objectives of this study were (1) to elucidate the effect of feeding colostrum versus milk-based formula on the mRNA abundance of key components of the mTOR pathway and of the ubiquitin-proteasome system in skeletal muscle of neonatal calves and (2) to compare different muscles. German Holstein calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) up to 4 d of life. The nutrient content in formula and colostrum was similar, but formula had lower concentrations of free branched-chain AA (BCAA) and free total AA, insulin, and insulin-like growth factor (IGF)-I than colostrum. Blood samples were taken from d 1 to 4 before morning feeding and before and 2 h after the last feeding on d 4. Muscle samples from M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM) were collected after slaughter on d 4 at 2 h after feeding. The preprandial concentrations of free total AA and BCAA, insulin, and IGF-I in plasma changed over time but did not differ between groups. Plasma free total AA and BCAA concentrations decreased in COL, whereas they increased in FOR after feeding, resulting in higher postprandial plasma total AA and BCAA concentrations in FOR than in COL. Plasma insulin concentrations increased after feeding in both groups but were higher in COL than in FOR. Plasma IGF-I concentrations decreased in COL, whereas they remained unchanged in FOR after feeding. The mRNA abundance of mTOR and ribosomal protein S6 kinase 1 (S6K1) in 3 different skeletal muscles was greater in COL than in FOR, whereas that of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) was unaffected by diet. The mRNA abundance of ubiquitin activating enzyme (UBA1) and ubiquitin conjugating enzyme 1 (UBE2G1) enzymes was not affected by diet, whereas that of ubiquitin conjugating enzyme 2 (UBE2G2) was greater (MLD) or tended to be greater (MM) in COL than in FOR. The mRNA abundance of atrogin-1 in MLD and MST was lower in COL than in FOR, whereas that of muscle ring finger protein-1 (MuRF1) was greater (MST) or tended to be greater (MLD). The abundance of MuRF1 mRNA was highest in MST, followed by MLD, and was lowest in MM. The results indicate that colostrum feeding may stimulate protein turnover that may result in a high rate of protein deposition in a muscle type–specific manner. Such effects seem to be mediated by the postprandial increase in plasma insulin.     

In vitro study to evaluate the degradation of bovine muscle proteins post-mortem by proteasome and μ-calpain

The degradation of bovine muscle proteins by proteasome and ubiquitous calpains was explored via 2D gel proteome analysis by inhibition of the physiological level of the proteases by specific inhibitors. The inhibition of the proteasome chymotrypsin- and trypsin-like activity results in the lack of degradation of several fragments of structural proteins such as actin, troponin T, myosin light chain and nebulin. In addition the degradation of several sarcoplasmatic proteins was eliminated when proteasome was inhibited. The inhibition of the ubiquitous calpain only resulted in minor changes in the degradation pattern, which might indicate that p94, which is not inhibited by calpastatin, is involved in the degradation post-mortem. The results of the present study indicate a sequential degradation of the structural proteins post-mortem, where calpain initiates the disruption and destabilisation of the myofibrillar structure, and thereby allows the proteasome to act.     

Is apelin gene expression and concentration affected by dietary intakes? A systematic review

Overproduction of apelin in obesity could be one of the last protective defenses before type 2 diabetes develops. To summarize the existing evidence on the association between dietary intake and apelin gene expression and concentration. We systematically searched MEDLINE, EMBASE, and google scholar and hand-searched bibliographies, including peer-reviewed articles with English abstracts, without restriction in publication date, updated until 21 February 2016 that reported the association between dietary intake and apelin gene expression or concentration. From a total of 1075 articles, we identified 12 relevant studies. There were 6 clinical trials in human and 6 studies in animals. Overall, two of three studies conducted in humans showed that calorie-restriction diet in obese subjects decreases apelin concentration. Five animal studies reported that higher intake of fatty acids and eicosapentaenoic acid (EPA) increased apelin expression and concentration. Given the paucity of data available, the heterogeneity of study designs used, and exposures tested, no quantitative meta-analysis was justified. Based on human studies, hypocaloric diet can reduce apelin concentration in obese individuals. In addition, higher intakes of total fatty acids and EPA may increase apelin gene expression and concentration.     

Effects of dietary digestible energy concentration on growth, meat quality, and PPARγ gene expression in muscle and adipose tissues of Rongchang piglets

This study investigated the effects of dietary digestible energy (DE) concentration (3.20, 3.40, 3.60 & 3.80 Mcal/kg) on growth, meat quality, and PPARγ gene expression in muscle and adipose tissues of Rongchang piglets. There was a quadratic increase in average daily gain and a linear decrease in the ratio of feed intake and gain as dietary DE increased (P < 0.05). Increasing dietary DE resulted in a linear increase of back fat thickness and intramuscular fat content (P < 0.05). A significant linear or quadratic effect (P < 0.01) was detected for shearing force. Increasing dietary DE linearly enhanced the expression of PPARγ in adipose tissues (P < 0.01). These data suggest that dietary DE had an impact on carcass and meat quality of Rongchang piglets. This could be partly due to the increased gene expression of PPARγ in adipose tissues, which may regulate the fat deposition of whole body.     

Application of absolute qPCR as a screening method to detect illicit 17β-oestradiol administration in male cattle

It has been previously demonstrated that the progesterone receptor gene is up-regulated in the sex accessory glands of pre-pubertal and adult male bovines after 17β-oestradiol treatment. In the present study, a qualitative screening method was optimised to detect 17β-oestradiol treatment using absolute quantification by qPCR of the progesterone receptor gene to determine the amount of gene expression in bulbo-urethral glands. An external standard curve was generated and developed with TaqMan® technology. Based on two in vivo experiments, the decision limit CCα, sensitivity and specificity of this screening method were established. Trial 1 consisted of 32 Friesian veal calves divided into two groups: group A (n?=?12), consisting of animals treated with four doses of 17β-oestradiol (5?mg?week^?1 per animal); and group B (n?=?20), consisting of control animals. Trial 2 was performed on 26 Charolaise beef cattle that either received five doses of 17β-oestradiol (group C; 20?mg?week^?1 per animal; n?=?6) or remained untreated (group D; n?=?20). Further, progesterone receptor gene expression was evaluated in beef and veal calves for human consumption. A specific CCα on 20 Piedmontese control beef cattle was calculated to include these animals in a field investigation. Five out of 190 beef cattle and 26 out of 177 calves tested expressed the progesterone receptor gene above their respective CCα and they were classified as being suspected of 17β-oestradiol treatment. Additionally, 58% of veal calves that tested suspect via qPCR exhibited histological lesions of the bulbo-urethral gland tissue, which are typical of oestrogen administration and are consistent with hyperplasia and metaplasia of the glandular epithelium.     

Increased blood-circulating interferon-γ, interleukin-17, and osteopontin levels in bovine paratuberculosis

Paratuberculosis-infected cattle initially develop an effective cell-mediated immune response that declines as the disease progresses. Blood is one of best sources for characterizing the inflammatory status of infected cows and for studying mediators related to chronic diseases. The aim of this study was to evaluate the cow-level association between blood cytokine concentration, the influence of serum on immune cell proliferation, and dairy cows naturally infected with Mycobacterium avium ssp. paratuberculosis (MAP). Positive animals (n = 41) from 19 herds were selected on the basis of 2 positive fecal culture results and divided into 2 groups: single-positive, or serum ELISA-negative cows (n = 32), and double-positive, or cows that gave positive results for both mycobacterial culture and serum ELISA (n = 9). Negative animals (n = 39) were selected from paratuberculosis-negative herds in which at least 80% of the animals had been diagnosed as negative by fecal culture and ELISA and that did not produce positive results during the 2-yr study. Analysis of plasma levels of the cytokines IL-4, IL-10, IL-17, IFN-γ, and osteopontin was performed, revealing distinct patterns. The ELISA-positive cows with MAP shedding had similar plasma concentrations of IL-4 and IL-10 but elevated levels of IFN-γ, IL-17, and osteopontin, which is indicative of inflammatory disease in these subclinical positive cows. In vitro MAP infection of bovine macrophages showed increased gene expression of tumor necrosis factor-α, IL-1β, IL-6, IL-23, and transforming growth factor-β as early as 6 h postinfection for all of the cytokines involved in the establishment of a T-helper type-17 immune response. To determine the systemic influence of serum on immune cell functions, lymphoproliferation assays were also performed in presence of JD serum. The serum from shedding cows showed 15% less proliferation. These results indicate that infected cows have a lower systemic capacity to maintain a protective immune response and that, as the disease progresses, an emerging T-helper type-17 immune response is established.     

β-lactam antibiotics residues analysis in bovine milk by LC-ESI-MS/MS: a simple and fast liquid-liquid extraction method

This study presents the development and validation of a simple method for the detection and quantification of six β-lactam antibiotics residues (ceftiofur, penicillin G, penicillin V, oxacillin, cloxacillin and dicloxacillin) in bovine milk using a fast liquid-liquid extraction (LLE) for sample preparation, followed by liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS). LLE consisted of the addition of acetonitrile to the sample, followed by addition of sodium chloride, centrifugation and direct injection of an aliquot into the LC-MS/MS system. Separation was performed in a C(18) column, using acetonitrile and water, both with 0.1% of formic acid, as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Limits of detection ranged from 0.4 (penicillin G and penicillin V) to 10.0 ng ml(-1) (ceftiofur), and linearity was achieved. The decision limit (CCα), detection capability (CCβ), accuracy, inter- and intra-day repeatability of the method are reported.     

Selective determination of trace 17β-estradiol in dairy and meat samples by molecularly imprinted solid-phase extraction and HPLC

Molecularly imprinted solid-phase extraction (MISPE) combined with HPLC were used to detect trace 17β-estradiol (E2) in different dairy and meat samples. E2 imprinted mono-dispersed microspheres prepared by modified precipitation polymerisation method were used as MISPE sorbents. Under optimum MISPE and HPLC conditions, trace (0.116–0.461 nmol kg⁻¹) E2 in different dairy and meat samples can be detected accurately using UV detector when large amount of sample (500 g) was analysed. Recoveries of E2 from spiked (0.1–5 nmol kg⁻¹) milk, yogurt, beef, pork and chicken were higher than 74.18%, 71.92%, 67.21%, 64.20% and 65.93%, respectively with RSDs less than 8.38%. MISPE can enrich E2 selectively, which is helpful to have better HPLC separation and higher recoveries than C18 SPE. MISPE combined with HPLC-UV are reliable methods to determine trace E2 in dairy and meat samples with high accuracy and repeatability.     

Optimisation and validation of a quantitative and confirmatory LC-MS method for multi-residue analyses of β-lactam and tetracycline antibiotics in bovine muscle

A multi-residue method for the determination of the β-lactam antibiotics ampicillin, cefazolin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V and the tetracyclines chlotetracycline, tetracycline and oxytetracycline was optimised and validated in bovine muscle. The method is based on the extraction of the residues from muscle using water/acetonitrile (2/8, v/v) with subsequent use of dispersive solid-phase C18 and hexane for purification. Extracts were analysed using ultra-performance liquid chromatography (UPLC-MS/MS) coupled with the mass spectrometer in positive electrospray ionisation mode (ESI+) for all analytes. The method was validated according to the requirements of European Commission Decision 2002/657/EC. The validation results were obtained within the MRL range of 0-1.5 of the MRL, with recoveries varying from 90% to 110% and CV < 20% (n = 54), except for cloxacillin, dicloxacillin and nafcillin. However, matrix interference was observed. The decision limit (CCα) ranged from 10% to 15% of the MRL. The uncertainty measurement was estimated based on both bottom-up and top-down strategies and the uncertainty values were found to be lower than 20% of the MRL. The method has a simple extraction procedure whereby analytes are separated with reasonable resolutions in a single 11-min chromatographic run. According to the validation results, this method is suitable for monitoring β-lactams and tetracyclines according to National Program for Residue and Contaminant Control - Brazil (NPRC-Brazil) in bovine muscle.     

Estrone and 17β-estradiol concentrations in pasteurized-homogenized milk and commercial dairy products

Some individuals fear that estrogens in dairy products may stimulate growth of estrogen-sensitive cancers in humans. The presence of estrone (E₁) and 17β-estradiol (E₂) in raw whole cow's milk has been demonstrated. The objectives of this study were to determine if pasteurization-homogenization affects E₂ concentration in milk and to quantify E₁ and E₂ concentrations in commercially available dairy products. The effects of pasteurization-homogenization were tested by collecting fresh raw milk, followed by pasteurization and homogenization at 1 of 2 homogenization pressures. All treated milks were tested for milk fat globule size, percentages of milk fat and solids, and E₂ concentrations. Estrone and E₂ were quantified from organic or conventional skim, 1%, 2%, and whole milks, as well as half-and-half, cream, and butter samples. Estrone and E₂ were quantified by RIA after organic solvent extractions and chromatography. Pasteurization-homogenization reduced fat globule size, but did not significantly affect E₂, milk fat, or milk solids concentrations. Estrone concentrations averaged 2.9, 4.2, 5.7, 7.9, 20.4, 54.1 pg/mL, and 118.9 pg/g in skim, 1%, 2%, and whole milks, half-and-half, cream, and butter samples, respectively. 17β-Estradiol concentrations averaged 0.4, 0.6, 0.9, 1.1, 1.9, 6.0 pg/mL, and 15.8 pg/g in skim, 1%, 2%, whole milks, half-and-half, cream, and butter samples, respectively. The amount of fat in milk significantly affected E₁ and E₂ concentrations in milk. Organic and conventional dairy products did not have substantially different concentrations of E₁ and E₂. Compared with information cited in the literature, concentrations of E₁ and E₂ in bovine milk are small relative to endogenous production rates of E₁ and E₂ in humans.     

Characterization of peptides released from rabbit skeletal muscle troponin-T by μ-calpain under conditions of low temperature and high ionic strength

Rabbit skeletal muscle troponin-T (200 μg ml(-1)) was incubated with μ-calpain (2 μl ml(-1)) under conditions of low temperature and high ionic strength for 180 min at 4°C and the peptides released analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin-T was hydrolyzed rapidly with the simultaneous appearance of eight peptides with masses of less than 14 up to 26 kDa. Two peptides produced by 10 min of incubation were electroblotted and analysis of these peptides by N-terminal sequencing and mass spectrometry showed that the principal cleavage sites of μ-calpain on troponin-T were at Thr(45)-Ala(46), Leu(69)-Met(70), Glu(220)-Lys(221) and Asn(231)-Val(232). The peptides present in insufficient quantity for electroblotting were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Cleavage sites were also identified at Met(151)-Gly(152), Asn(188)-Ile(189), Lys(223)-Arg(224), Arg(233)-Ala(234) and Ala(240)-Lys(241). In general, μ-calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of troponin-T.