Regucalcin Expression in Bovine Tissues and Its Regulation by Sex Steroid Hormones in Accessory Sex Glands

Regucalcin (RGN) is a mammalian Ca²⁺-binding protein that plays an important role in intracellular Ca²⁺ homeostasis. Recently, RGN has been identified as a target gene for sex steroid hormones in the prostate glands and testis of rats and humans, but no studies have focused on RGN expression in bovine tissues. Thus, in the present study, we examined RGN mRNA and protein expression in the different tissues and organs of veal calves and beef cattle. Moreover, we investigated whether RGN expression is controlled through sex steroid hormones in bovine target tissues, namely the bulbo-urethral and prostate glands and the testis. Sex steroid hormones are still illegally used in bovine husbandry to increase muscle mass. The screening of the regulation and function of anabolic sex steroids via modified gene expression levels in various tissues represents a new approach for the detection of illicit drug treatments. Herein, we used quantitative PCR, western blot and immunohistochemistry analyses to demonstrate RGN mRNA and protein expression in bovine tissues. In addition, estrogen administration down-regulated RGN gene expression in the accessory sex glands of veal calves and beef cattle, while androgen treatment reduced RGN gene expression only in the testis. The confirmation of the regulation of RGN gene expression through sex steroid hormones might facilitate the potential detection of hormone abuse in bovine husbandry. Particularly, the specific response in the testis suggests that this tissue is ideal for the detection of illicit androgen administration in veal calves and beef cattle.     

We are what we eat: food safety and proteomics

In this review, we lead the reader through the evolution of proteomics application to the study of quality control in production processes of foods (including food of plant origin and transgenic plants in particular, but also meat, wine and beer, and milk) and food safety (screening for foodborne pathogens). These topics are attracting a great deal of attention, especially in recent years, when the international community has become increasingly aware of the central role of food quality and safety and their influence on the health of end-consumers. Early proteomics studies in the field of food research were mainly aimed at performing exploratory analyses of food (bovine, swine, chicken, or lamb meat, but also transgenic food such as genetically modified maize, for example) and beverages (wine), with the goal of improving the quality of the end-products. Recently, developments in the field of proteomics have also allowed the study of safety issues, as the technical advantages of sensitive techniques such as mass spectrometry have guaranteed a faster and improved individuation of food contaminating pathogens with unprecedented sensitivity and specificity.     

Thymus and meat physicochemical measurements to discriminate calves treated with anabolic and therapeutic doses of dexamethasone

To preserve the Europe consumers' health, the use of glucocorticoids as growth promoters is prohibited in cattle fattening. In 2008, the Italian Ministry of Health associated to the official control a national monitoring plan based on the histological thymus analysis to identify animals illegally treated with corticosteroids. However, since corticosteroids are authorized and widely used for therapeutic purposes, it is necessary to verify whether the thymus histological test and some physicochemical traits in meat are able to discriminate doped calves from dexamethasone therapeutic treated ones. The aims of this study were (i) to establish whether the therapeutic and illicit corticosteroid treatments of calves could be differentiated through histological evaluation of thymus and by physicochemical meat traits; (ii) to identify a restricted number of physicochemical traits that could differentiate dexamethasone treated from untreated calves. Three groups of 15 calves each were included in this study: group dexamethasone therapeutic treatment treated with dexamethasone 21-phosphate disodium salt at a therapeutic dose (2 mg/kg of live weight for three consecutive days); group dexamethasone anabolic treatment orally treated with dexamethasone 21-phosphate disodium salt according to a presumed anabolic protocol (0.4 mg/day per animal for 20 days); group placebo control treated with a placebo served as control. Results demonstrated that groups could be easily discriminated by thymus microscopy as well as by two meat markers, namely, cooking loss and shear firmness or Warner-Bratzler shear force. The combination of thymus microscopic features and meat physicochemical traits could be used as a practical, economic and accurate screening strategy to discriminate between meat from illegally and therapeutically treated calves. This new reliable and simple tool could contribute to identify animals treated with dexamethasone in those countries where glucocorticoids are illegally used as growth promoters. More in general, this system could be included in the framework of official controls, and applied to verify suppliers' reliability by the meat industry.     

Proteomic investigation in the detection of the illicit treatment of calves with growth-promoting agents

The use of beta-agonists, sexual steroids, and corticosteroids as growth-promoting agents (GPAs) in veal calves is forbidden in the European Union (EU) and subjected to restrictions in the US because it may be potentially noxious for both treated animals and the consumer. Although official controls performed in the EU have revealed a limited number of positive samples, the analysis of seized preparations indicate that the use of illegal GPAs is far from being abandoned. The presence of these compounds in matrixes of biological origin often goes unnoticed because of the use of very low dosages and/or of molecules of unknown chemical structure. It is therefore necessary to develop screening methods based on the biological effects of these substances that allow the simultaneous screening of many components, as proteome analysis. When hepatic cytosols and microsomes from calves treated with a combination of GPAs were analyzed by 2-DE, we found changes in the expression of two proteins, which we identified as adenosine kinase and reticulocalbin. Our aim was not to speculate about molecular mechanisms, but to show the ability of the proteomic approach to find biomarkers of illicit treatments and to use it as a basis to develop large-scale screening methods.     

Separation and properties of enterovirus and reovirus recovered from a fecal sample of calf with diarrhea.

In April, 1971, a disease with pyrexia and diarrhea as main symptoms broke out collectively among calves. Fecal samples were collected from calves involved and inoculated into bovine kidney (BK) cell cultures. As a result, the diarrheal feces of one calf were suspected to contain two agents simultaneously. One agent (C-121 E strain) was isolated from the primary infected BK cell culture fluid by terminal dilution passages. It had been predominant in replication and shown a cytopathic effect which gave rise to a granular appearance in the early stage of culture. The other agent (C-121 R strain) was isolated from the primary infected BK cell culture fluid by neutralizing the C-121 E strain contained in this fluid with antiserum against this strain. It caused cytoplasmic inclusion bodies to form. On the basis of their physico-chemical properties, the C-121 E strain was identified as bovine enterovirus and the C-121 R strain as reovirus. Serological tests indicated that some of the affected calves had been infected not only with the two strains isolated, but also with bovine viral diarrhea virus, bovine adenovirus type 7, and bovine parvovirus.     

Molecular traceability of beef from synthetic Mexican bovine breeds

Traceability ensures a link between carcass, quarters or cuts of beef and the individual animal or the group of animals from which they are derived. Meat traceability is an essential tool for successful identification and recall of contaminated products from the market during a food crisis. Meat traceability is also extremely important for protection and value enhancement of good-quality brands. Molecular meat traceability would allow verification of conventional methods used for beef tracing in synthetic Mexican bovine breeds. We evaluated a set of 11 microsatellites for their ability to identify animals belonging to these synthetic breeds, Brangus and Charolais/Brahman (78 animals). Seven microsatellite markers allowed sample discrimination with a match probability, defined as the probability of finding two individuals sharing by chance the same genotypic profile, of 10(-8). The practical application of the marker set was evaluated by testing eight samples from carcasses and pieces of meat at the slaughterhouse and at the point of sale. The DNA profiles of the two samples obtained at these two different points in the production-commercialization chain always proved that they came from the same animal.     

Proteomics for routine identification of microorganisms

The invention of MALDI-TOF-MS enormously contributed to the understanding of protein chemistry and cell biology. Without this technique proteomics would most likely not be the important discipline it is today. Besides 'true' proteomics, MALDI-TOF-MS was applied for the analysis of microorganisms for their taxonomic characterization from its beginning. This approach has since been developed as a diagnostic tool readily available for routine, high-throughput analysis of microbial isolates from clinical specimens by intact-cell mass spectrometry (ICMS), the direct analysis of whole bacterial cell without a preceding fractionation or separation by chromatography or electrophoresis. ICMS exploits the reproducibility of mass fingerprints for individual bacterial and fungal strains as well as the high similarity of mass fingerprints within a species. Comparison of mass spectral data to genomic sequences emphasized the validity of peak patterns as taxonomic markers. Supported by comprehensive databases, MALDI-TOF-MS-based identification has been widely accepted in clinical laboratories within only a few years.     

Effect of ionizing radiation on rat tissue: proteomic and biochemical analysis

Reactive oxygen species (ROS), generated by ionizing radiation, has been implicated in its effect on living tissues. We confirmed the changes in the oxidative stress markers upon irradiation. We characterized the changes in the proteome profile in rat liver after administering irradiation, and the affected proteins were identified by MALDI-TOF-MS and ESI-MS/MS. The identified proteins represent diverse sets of proteins participating in the cellular metabolism. Our results demonstrated that proteomics analysis is a useful method for characterization of a global proteome change caused by ionizing radiation to unravel the molecular mechanisms involved in the cellular responses to ionizing radiation.     

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.     

A Longitudinal Study of Escherichia coli in Cows and Calves with Special Reference to the Distribution of O-antigen Types and Antibiotic Resistance

The faecal excretion of E. coli from 6 adult cows was followed over a period of 105 days. E. coli were excreted in only 23·4% of specimens and the patterns of excretion varied between animals. All isolates of E. coli were antibiotic sensitive and fell into 6 O-antigen types; very few were non-typeable. The O-types excreted by each animal, compared with those from other cows in the same house, were traced with time. A similar but less intense study was undertaken in 8 calves. In contrast E. coli was excreted from all samples and included both antibiotic sensitive and resistant strains; 37 O-types were represented. The persistence of specific O-types within each calf and their spread to other calves in the same house were studied. A comparison of the O-types in the bovine species with those isolated from man has been made.     

Species Identification of Archaeological Skin Objects from Danish Bogs: Comparison between Mass Spectrometry-Based Peptide Sequencing and Microscopy-Based Methods

Denmark has an extraordinarily large and well-preserved collection of archaeological skin garments found in peat bogs, dated to approximately 920 BC – AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the production of skin garments. Until recently, species identification of archaeological skin was primarily performed by light and scanning electron microscopy or the analysis of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphological and molecular degradation within the samples. We compared species assignment results of twelve archaeological skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and scanning electron microscopy. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extracted ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine foetal haemoglobin, indicating the production of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029.     

Chromosome studies in bovine quintuplets

Chromosome analysis has been carried out on the four surviving calves of a bovine quintuplet set. Blood samples from all calves exhibited lymphocyte chimerism with greater percentages of male cells. Chromatid breaks and the presence of a hypodiploid cell carrying a metacentric marker were noted in varying proportions in all four calves. The possible relationship between chromosome abnormalities and immunological diversity between the calves have been discussed.     

Mapping of alkaline proteins in bovine skeletal muscle

In agricultural sciences, proteomics has become the new hope for analyzing the meat quality traits that are closely related to the skeletal muscle traits. 2-DE muscle maps of many species have been recently reported and used to find molecular markers of meat quality traits. However, one limitation of 2-DE based analyses is due to the limited alkaline protein separation. Considering this problem, there is a need to use recent advances that have markedly improved the 2-DE based analysis of alkaline proteins. Hence, the present study provides additional information concerning the alkaline proteome of bovine skeletal muscle by using an appropriate protocol to characterize proteins over the entire range of pH 7-11. A total of 32 distinct gene products corresponding to 60 protein spots were identified by PMF and grouped in seven categories according to their main function. This 2-D map will contribute to muscle proteome studies since a significant portion of proteins is in the alkaline pH range.     

Using gamma irradiation and low temperature on microbial decontamination of red meat in Iran

Gamma irradiation can be used as one of the most efficient methods to reduce microorganisms in food. The irradiation of food is used for a number of purposes, including microbiological control, insects control and inhibition of sprouting and delay of senescence of living food. The aim of this study was to study effects of gamma irradiation, refrigeration and frozen storage as the combination process for improvement of red meat shelf-life. The bovine meat samples were treated with 0, 0.5, 1, 2 and 3 kGy of gamma irradiation and kept in refrigerator for 3 weeks and in freezer for 8 months. The control and irradiated samples were stored at 4–7°C and at −18°C for refrigeration and frozen storage, respectively; and microbial and chemical analyze was done at 1 week and 2 months intervals. In this study the optimum dose of gamma radiation in order to decrease the total count of Mesophilic bacteria, Coliforms, Staphylococcus aureus and especially for elimination of Salmonella was obtained at 3 kGy. Microbial analysis indicated that irradiation and storage at low temperature had a significant effect on the reduction of microbial loads. There was no significant difference in chemical characteristics during freezing storage in bovine meat. Also, irradiated meat samples (3 kGy) were stored in 4–7°C for 14 days, compared to 3 days for non irradiated samples.     

Specific discrimination of chicken DNA from other poultry DNA in processed foods using the polymerase chain reaction

In the present study, specific discrimination of chicken DNA contamination in processed foods using the polymerase chain reaction was investigated. The primer pair was designed to amplify a 102-bp fragment of the chicken mitochondrial 16S ribosomal RNA gene. While the DNA from chicken meat was amplified, the DNA from other poultry meat, mammalian meat, fish, shellfish, and cereals was not amplified. The primer amplified DNA fragments derived from model processed and nonprocessed food samples containing 0.001, 0.01, 0.1, 1, 10, and 100% chicken.     

System expansion and allocation in life cycle assessment of milk and beef production

Background, Goal and Scope  System expansion is a method used to avoid co-product allocation. Up to this point in time it has seldom been used in LCA studies of food products, although food production systems often are characterised by closely interlinked sub-systems. One of the most important allocation problems that occurs in LCAs of agricultural products is the question of how to handle the co-product beef from milk production since almost half of the beef production in the EU is derived from co-products from the dairy sector. The purpose of this paper is to compare different methods of handling co-products when dividing the environmental burden of the milk production system between milk and the co-products meat and surplus calves. Main Features  This article presents results from an LCA of organic milk production in which different methods of handling the co-products are examined. The comparison of different methods of co-product handling is based on a Swedish LCA case study of milk production where economic allocation between milk and meat was initially used. Allocation of the co-products meat and surplus calves was avoided by expanding the milk system. LCA data were collected from another case study where the alternative way of producing meat was analysed, i.e. using a beef cow that produces one calf per annum to be raised for one and a half year. The LCA of beef production was included in the milk system. A discussion is conducted focussing on the importance of modelling and analysing milk and beef production in an integrated way when foreseeing and planning the environmental consequences of manipulating milk and beef production systems. Results  This study shows that economic allocation between milk and beef favours the product beef. When system expansion is performed, the environmental benefits of milk production due to its co-products of surplus calves and meat become obvious. This is especially connected to the impact categories that describe the potential environmental burden of biogenic emissions such as methane and ammonia and nitrogen losses due to land use and its fertilising. The reason for this is that beef production in combination with milk can be carried out with fewer animals than in sole beef production systems. Conclusion, Recommendation and Perspective  Milk and beef production systems are closely connected. Changes in milk production systems will cause alterations in beef production systems. It is concluded that in prospective LCA studies, system expansion should be performed to obtain adequate information of the environmental consequences of manipulating production systems that are interlinked to each other.     

Total Iron and Heme Iron Content and their Distribution in Beef Meat and Viscera

To determine the content of total iron (TFe) and heme iron (HeFe) in major cuts of meat and principal viscera of bovine origin. ⁵⁵Fe (30 mCi) was injected into two 4-month-old calves. Triplicate samples of the 12 basic American cuts of meat and major viscera were obtained from each specimen. Samples were acid digested and their iron content was read by atomic absorption spectrophotometry. Duplicate samples of the basic cuts of meat and major viscera were analyzed to determine the concentration of ⁵⁵Fe using a double isotopic technique. The mean and standard deviation of TFe for all cuts was 1.4?±?0.3 mg/100 g of meat. The mean TFe for organs was (per mg/100 g): 0.9?±?0.1 brain, 3.0?±?0.05 kidney, 3.2?±?0.04 heart, 5.7?±?0.2 lung, 6.0?±?0.1 liver, and 31.2?±?0.4 spleen. HeFe was 64% of TFe in meat and 72.8% in spleen, 53.8% in lung, 35.7% in brain, 35.0% in kidney, 27.3% in heart, and only 13.6% in liver. Blood contained 85.5% of the radioisotope and only 1.4% was found in muscle and 1.6% was found in viscera. Results suggest that bovine cuts of meat have a low variation in TFe and that HeFe comprises more than 60% of TFe.     

Specific Discrimination of Chicken DNA from Other Poultry DNA in Processed Foods Using the Polymerase Chain Reaction

In the present study, specific discrimination of chicken DNA contamination in processed foods using the polymerase chain reaction was investigated. The primer pair was designed to amplify a 102-bp fragment of the chicken mitochondrial 16S ribosomal RNA gene. While the DNA from chicken meat was amplified, the DNA from other poultry meat, mammalian meat, fish, shellfish, and cereals was not amplified. The primer amplified DNA fragments derived from model processed and nonprocessed food samples containing 0.001, 0.01, 0.1, 1, 10, and 100% chicken.     

Rapidly maturing field of proteomics: A gateway to studying diseases

The proteomics work reported by Smith et al. represents a giant step forward in characterizing the cerebrospinal fluid (CSF) proteome in mouse models of human diseases. Whereas prior studies were limited to analysis of CSF pools, Smith et al. (Proteomics 2014, 14, 1102–1106) base their conclusions on data derived from individual mice, thereby capturing a fuller range of the biological diversity present. These results underscore how far proteomics has come in the past few years, developing into a modern tool with the capacity to remove bottlenecks in the study of neuropsychiatric diseases. Past efforts with mass spectrometry (MS) have been hampered by limitations in access to CSF samples, and small volumes when available. These barriers have been overcome with newer MS platforms and advances in sample preparation. We are far closer than before to producing the production of clinically useful proteomic data for biomarker discovery and for deriving insights into pathogenesis that can lead to more effective treatments for many diseases.     

Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review

Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.