Use Of Animal Proteins Research Articles

Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells.

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach.

Prehistoric animal use on the Great Hungarian Plain: A synthesis of isotope and residue analyses from the Neolithic and Copper Age

The archaeological record of Eastern Hungary indicates that settlement patterns, subsistence strategies, ceramic style, trade patterns and mortuary customs changed from the Late Neolithic to the Copper Age (5000–2700cal BC). Despite a rich archaeological tradition, questions remain regarding the management and use of domesticated animals and the role animal husbandry played in social change during this transition. Some researchers have hypothesized that these changes reflect a shift towards an economy that intensified its focus on primary and perhaps secondary animal products. Here we synthesize isotope data from human and animal remains and residue analysis from pottery sherds from Neolithic and Copper Age assemblages. Results indicate that the consumption and use of animal protein and fat was relatively high for both periods, with an increase in animal fats in ceramic vessels during the Middle Copper Age; however, milk products do not appear to have played an important dietary role. We conclude that livestock management remained small-scale during the Neolithic and Copper Age and that dairy use was minimal. It is proposed that the cultural changes that occurred at this time were associated with the emergence of smaller, independent farmsteads and perhaps the innovative use of secondary products like manure.

PCR-RFLP Identification of Prohibited Animal Derived DNA in Animal Feed

A method for confirming identification of prohibited species tissue in animal feed has been developed on the basis of PCR-RFLP analysis. In Japan, to prevent the spread of BSE through animal feed, the use of animal protein in feed has been regulated. Species-specific PCR detection of prohibited species materials in feed has been used as one of a series of laboratory tests to ensure the proper implementation of the feed regulations. However, since the result of this PCR method is determined only by amplicon length, it is sometimes necessary to confirm whether or not the positive result is due to the effect of a non-specific reaction. For this purpose, DNA sequencing is the best way to confirm the test result but it is not suitable for routine analysis because of the required time and cost. In this study, we developed an easy and rapid method to confirm the species identification (mammals, ruminants and cattle) by using 4 restriction enzymes: SmlI, MboI, BlnI and Hpy188III. This PCR-RFLP method, which ensures identification of prohibited animal species in feed, is useful for enhancing the reliability of feed inspection for BSE prevention. This method will be added to the Official Methods of Feed Analysis.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation. To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue. Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins. This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord. All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation. To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue. Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins. This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord. All materials and procedures will be described.

Use of animal proteins as foaming agent in cementitious concrete composites manufactured with recycled PET aggregates

Manufacturing of lightweight building materials is identified as an alternative procedure for valorising reused materials. With intent to valorise two wastes of food industry at the same time, this paper describes an innovative use of animal protein as foaming agent while consumed plastic bottle wastes are introduced as light aggregates. Relative changes in properties of concrete composites due to introducing proteins are experimentally investigated here. The results presented show that the two wastes clearly improve the flowability of the mortar. Both mechanical properties and bulk density of the composites are affected too, but thermal conductivity is improved. In this way, the obtained composites would appear to be low-cost materials which would contribute to resolving some of the solid waste problems in addition to conserving energy.

Les sources protéiques dans les aliments pour les poissons d'élevage

Fish require diets containing 30 to 55% of crude protein and an amino acid supply precisely adapted to meeting the needs for optimal growth. Fish meal is an ideal protein source for aquafeed because it has a high protein level (65% to 72%) with an ideal proportion of all ten indispensable amino acids that meet the requirements of all fish species. However, finding alternatives to fish meal has become an absolute necessity in order to reduce the reliance of aquaculture on marine ingredients. Animal by-product meals (meat meal, bone meal, blood meal, poultry by-product meal, hydrolysed feather meal) are used as alternative protein sources in aquafeed in extra-European countries. Since the use of animal protein in aquafeed is forbidden in Europe (except non ruminant blood meal), more attention has been given to the use of plant protein sources which are available in large amounts on the market. The limitations as regards their incorporation in diets for fish are related to the level of protein, the amino acid profiles and the presence of anti-nutritional factors (ANF). Protein extracts from oilseeds and cereals are higher in protein than meals and pulses and lower in ANF but currently more expensive, thus limiting their use in aquafeed. Numerous studies undertaken with different fish species have led to improved knowledge on the potential of various plant ingredients. Blends of plant protein sources duly supplemented with the required indispensable amino acids can replace 75 to 95% of fish meal in almost all species, thus reducing the pressure of aquaculture on marine resources. Further research should focus on factors that can facilitate total replacement, on the origin of the adverse effects of ANF and on the dietary supply of micronutrients (minerals and vitamins) in order to improve the ormulation of totally plant-protein-based diets for fish.

Developing PCR Primers Using a New Computer Program for Detection of Multiple Animal-Derived Materials in Feed

To reduce the risk of the bovine spongiform encephalopathy agent being recycled to cattle through animal feed, in October 2001 Japan introduced a feed ban prohibiting the use of animal proteins in feed. PCR identification of feed ingredients is part of the audit program to ensure the proper implementation of the feed ban. For efficient analysis, screening of feed products for materials from multiple species is essential. In our study, we developed a computer program GSPRIMER (http://www.famic.go.jp/ffis/feed/gsprimer/) that facilitates development of PCR primers specific to multiple species. The most important feature of GSPRIMER is its ability to estimate the specificity and homology of a potential primer in incremental steps from the 3′ terminal. We analyzed all regions of mitochondrial DNA from the target and nontarget species using GSPRIMER. We designed species-specific primer sets for three animal species (sheep, goats, and swine) and group-specific primer sets for ruminants and animals susceptible to transmissible spongiform encephalopathy. The primers were efficiently screened by the PCR protocol using a mixture of mitochondrial DNA from nontarget species as a template. As a result, one primer set each for sheep and goats, two for swine, and three for a group of ruminant species were developed. The detection limit of one of the ruminant primer sets ranged from 0.05 to 0.01% bovine meat and bone meal and 0.1pg of bovine DNA. We also successfully applied the primer set to 17 commercial feed samples that were known to be free from ruminant-derived materials. No false-positive results were found.

Detecting and quantifying meat meal or meat and bone meal contamination in fishmeal by visible and near infrared reflectance spectra

The use of animal protein feeds such as meat meal or meat and bone meal (MMBM) play an important role in the feed manufacturing industry, but their safe and healthy use in animal feeds is of public concern in order to prevent the spread of bovine spongiform encephalopathy (BSE). The objective of the present work was to develop a technique using near infrared reflectance spectroscopy (NIRS) that would be suitable for detecting and quantifying contaminating levels of MMBM in fishmeal. To this end, a partial least squares (PLS) discriminant analysis and a modified partial least squares (MPLS) quantitative analysis, using visible and NIRS, were developed using a calibration set of 186 samples including 90 samples of pure fishmeal and 96 samples adulterated with MMBM at levels ranging from 10 to 320 g/kg. An external validation set, comprised of 39 pure samples and 54 adulterated samples, was used to validate the calibration model. A PLS discriminant analysis model developed with mathematic pretreatment 1,4,4,1, successfully detected fishmeal adulterated with MMBM. External validation indicated that all samples were discriminated correctly. A MPLS quantitative model, developed with mathematic pretreatment 1,4,4,1, also successfully predicted the MMBM in fishmeal with standard error of cross-validation (SECV) of 27.89 g/kg and ratio of the standard deviation of the validation set to the standard error of prediction (RPD) of 3.37. The calibration and validation results confirm that NIRS could provide the feed industry and inspection bodies with a rapid, non-destructive and non-invasive technique for the detection and quantification of MMBM in fishmeal.

Secretion of Angiogenic Proteins by Human Multipotent Mesenchymal Stromal Cells (MSC) under Hypoxic Culture Conditions and Clinical Application of MSC in Steroid Induced Osteonecrosis in Children.

Osteonecrosis is a frequent complication after treatment for childhood leukemia.17% of adolescents above the age of 15 years treated in the ALL-BFM 95 trial protocol suffered from avascular necrosis of the bone (AVN). In addition, children receiving steroid-based treatment, e. g. autoimmune diseases, are frequently affected as well. If conservative measures fail, core decompression surgery is required. The success rate of surgery varies depending on the stage from 29% to 84% (Castro et al., 2000). Evidently, improved intervention strategies are warranted. Therefore, we evaluated the safety of implanting autologous MSC during surgical intervention in order to improve bone regeneration. AVN represent an area of absent blood perfusion and consequently low supplies of nutrients and oxygen. Therefore, we cultivated MSC under oxygen tensions of 3% or lower. These cells showed slowed proliferation and differentiation into bone. However, MSC grown under these low-oxygen conditions secreted significant amounts of vascular endothelial derived factor and selected insulin-like growth factors and their binding proteins. These factors might facilitate reperfusion and regeneration of bone at the site of implantation during core decompression surgery. 10 ml of heparinized bone marrow was drawn from the iliac crest of the patient. MSC were expanded under GMP conditions without the use of animal proteins employing platelet lysate and human fresh frozen plasma as source for growth factors (Müller et al., 2006). Recombinant trypsin was used for detachment and harvest of the cells. MSC could be expanded from 5 out of 5 juvenile bone marrow aspiates. Within three weeks of culture, sufficient numbers of cells, i. e. 112·106 ± 67·106, were generated in a certified GMP lab. At each of the three passages diagnostic cultures for aerobic bacteria were established and remained sterile throughout the culture period. MSC grown under these conditions were able to differentiate into osteoblasts. No chromosomal aberrations were detected by matrix-based comparative genomic hybridization using DNA microarrays containing >6,400 genomic DNA fragments. At the day of core decompression surgery, cells were harvested and resuspended to a final volume of 3 ml in saline. As core decompression implies drilling a hole into the necrotic area, the MSC were implanted through the same channel, which was sealed thereafter. Hence, instillation of the MSC did not require additional surgical procedures. Application of MSC was feasible and successful during core decompression surgery in all of the 5 patients. Follow up of the patients is between 14 and 8 months. None of the patients experienced adverse side effects, most importantly, there were no infectious complications. Clinically, no patient deteriorated and all patients reported less or no pain two months after surgery. Although the number of patients in this pilot trial is small, initial data for safety and feasibility of employing MSC in juvenile steroid-induced osteonecrosis is very promising.

Discrimination of Fish Bones from other Animal Bones in the Sedimented Fraction of Compound Feeds by near Infrared Microscopy

Since the bovine spongiform encephalopathy (BSE) crisis, the use of animal proteins in animal feed has been prohibited. From October 2003, the European Union (EU) adopted Regulation (EC) no. 1774/2002 governing animal by-products (ABPs), which seeks to address the possible risks inherent in recycling potential infectivity due to the absence of barriers within species and to exclude the cannibalism which may be induced by intra-species recycling. There is an urgent need to develop fast and reliable methods for identification of low-level ABP origins. In this study, near infrared (NIR) microscopy was used to identify different classes of ABPs. Samples of fish meals ( n = 10) and meals of land-animal origin ( n = 50) were ground, sedimented and analysed using an Auto Image Microscope connected to a Fourier transform near infrared spectrometer (FT-NIR). Sediment fraction particles were spread on a Spectralon plate, presented to the NIR microscope and scanned in the 1112–2500 nm region. The support vector machine (SVM) algorithm was used to construct models to identify class origin. Models correctly classified 100% of the samples in the calibration set and between 95 and 95.5% in the validation set. The results demonstrated the potential of FT-NIR microscopy as a rapid method for distinguishing between fish and land-animal particles.

Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones

Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrations <18 mg/mL on in vitro bovine blastocysts. Increasing concentrations of peptones were then added in the freezing medium. The surviving and hatching rates were not affected by comparison with those observed with BSA. No significant difference was observed between groups either for the total number of cells or for the ratio ICM/Total cell, nor for the rate of apoptosis in surviving embryos. When embryos were cryopreserved in 1.8 mg/mL peptone, the hatching rate and embryo quality as assessed at 48 h post-thawing were not significantly different from those of unfrozen embryos. In a second experiment two additives were added in this animal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 μM β-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

The replacement of fishmeal by plant proteins in piglet production

RiassuntoLa sostituzione della farina di pesce con fonti proteiche vegetali nella produzione del suinetto. 144 suinetti svezzati (età allo svezzamento giorni 28; peso vivo medio kg 7,58), sono stati omogeneamente assegnati a 4 gruppi sperimentali le cui diete prevedevano la sostituzione totale della farina di pesce con fonti proteiche vegetali rappresentate, rispettivamente, da farina di estrazione di girasole parzialmente decorticato, glutine di mais e concentrato proteico di patata. Le diete sono state bilanciate in termini di apporti in aminoacidi ed Energia Netta. Ad eccezione del gruppo alimentato con concentrato proteico di patata, che ha dimostrato un ritmo di accrescimento significativamente migliore, non si sono evidenziate differenze tra le tesi. Lo stato di salute è stato sempre soddisfacente, con una bassa mortalità (4%, circa) e un modesto “punteggio diarrea” (valore massimo punti 1,59). La presente esperienza indica come, nell’alimentazione del suinetto nel post-svezzamento, sia possibile sostituire la farina di pesce con fonti proteiche vegetali, purché vengano correttamente bilanciati gli apporti aminoacidi ed energetici delle diete.

Use of Animal Proteins to Limit Leaching of Active Copper Ions Preservatives from Treated Wood

Summary The concept of animal protein utilization aiming to reduce leaching of treatment salts from wood was tested and verified. Animal proteins in the form of spray-dried animal blood plasma were used as an addition to the water solution of fungicidal salt or included in the solution for soaking pre-treated wood. The wood was subjected to thermal treatment which caused protein denaturation, a process which changes the proteins' spatial conformation and reduces their solubility in water. Scots pine wood (Pinus sylvestris L.) was treated with a water solution of copper sulfate using vacuum, vacuum-soaking and vacuum-pressure methods. The animal proteins were added to the treatment solutions or applied by soaking to samples pre-treated with the salt. After treatment, the samples were heated to 100°C. After suitable conditioning, the wood was subjected to leaching. The content of Cu2+ in the water extracts was determined by Atomic Absorption Spectrophotometry (AAS).

The effects of quebracho tannins on rumen degradation and post-rumen digestion of pea and lupin seeds

The use of alternative protein sources in ruminant diets is of interest because of restrictions on the use of animal proteins, loss of nitrogen (N) compounds to the environment and efforts to reduce costs of systems. A problem of some protein-rich-ingredients is that they degrade rapidly in the rumen. The use of tannins has been proposed as a means of reducing the extensive dietary protein degradation in the rumen. However, condensed tannins have been reported to exert detrimental effects on gut microflora and on the gastrointestinal tract, and they can affect post-rumen digestion. The two experiments reported here aimed to study the influence of quebracho tannins (QTs) on ruminal degradation of peas (Pisum sativum) and sweet lupins (Lupinus luteus), and to investigate the effect of QTs on postrumen digestibility of these feeds using a poultry model.

New perspectives on the use of tropical plants to improve ruminant nutrition.

Inadequate nutrition is the main cause of low productivity by ruminants in sub-Saharan Africa. The primary feed resources in the region include natural pasture and crop residues that have tough texture, poor digestibility and are deficient in nutrients. These deficiencies can be corrected by supplementation with high-density feeds such as oilseed cakes and proteins of animal origin. However, protein sources such as oilseed cakes are beyond the economic reach of most farmers, while the incidence of bovine spongiform encephalopathy in Western intensive animal production may be thought to argue against the use of animal proteins. Local tree legumes have been investigated as potential supplements for ruminants because of their beneficial effect of increasing metabolizable energy intake, N intake and feed efficiency, and improving animal performance. However, our work has suggested that some plant materials may have a nutritional value beyond simply their nutrient content, i.e. as rumen-manipulating agents. The foliage of some tree legumes has been shown to be selectively toxic to rumen protozoa. Rumen protozoa ingest and digest bacteria and fungi, degrading their cellular protein to NH3. Microbial protein turnover due to protozoal predation in the rumen may result in the net microbial protein outflow being less than half the total protein synthesized. Results from in vivo experiments have clearly shown that duodenal flow of both undegraded dietary and bacterial protein is generally increased by defaunation. However, no practical method has been developed to date to eliminate protozoa. Anti-protozoal plants may be promising, safe, natural defaunating agents.

Replacement of fishmeal by rape-seed meal and soya bean meal in concentrates given to pregnant and lactating ewes

Fishmeal has been considered for many years an important component of the diet of pregnant and lactating ewes (Hovell and Ørskov, 1989). Recently concerns have been raised over adverse effects of industrial fishing on fish stocks (House of Lords, 1996) and use of animal protein in ruminant diets (GAFTA, 1997). Alternative sources of protein (vegetable sources) to fishmeal have been proposed but they are either expensive or lacking in supply of essential amino acids. The research reported in this paper examines the performance of an alternative protein source to fishmeal given in late pregnancy to ewes bearing multiple offspring and as a creep feed to lambs.

Replacement of fishmeal by rape-seed meal and soya bean meal in concentrates given to pregnant and lactating ewes

Fishmeal has been considered for many years an important component of the diet of pregnant and lactating ewes (Hovell and Ørskov, 1989). Recently concerns have been raised over adverse effects of industrial fishing on fish stocks (House of Lords, 1996) and use of animal protein in ruminant diets (GAFTA, 1997). Alternative sources of protein (vegetable sources) to fishmeal have been proposed but they are either expensive or lacking in supply of essential amino acids. The research reported in this paper examines the performance of an alternative protein source to fishmeal given in late pregnancy to ewes bearing multiple offspring and as a creep feed to lambs.

Detection and identification of animal-derived protein in feedingstuffs by enzyme-linked immunoassay

In Great Britain the use of animal-derived protein in animal feeds has recently been subject to restrictions (The Bovine Spongiform Encephalopathy Order 1988 and subsequent orders), including a prohibition on the use of animal protein produced from ruminant carcases in feed given to ruminants. More generally, livestock producers are increasingly demanding more detailed information about the ingredients included in the compound feeds they purchase, and may stipulate that meat and bone meal derived from particular animal species is not used in their feed. In order that compliance with these legal and commercial requirements can be monitored and enforced, it is important that feed manufacturers, their customers and the regulatory authorities should have recourse to a means of identifying the species from which animal protein, when present in a feed, is derived.

The Nutritional Contribution to Bovine Spongiform Encephalopathy

Evidence that changes in feeding style alter the membrane fatty acid composition of ruminant tissue is presented here by comparing zoo giraffe with the same species from their natural habitat. The membrane changes seen are similar to those used experimentally to make animals susceptible to basic brain protein and encephalomalacia. Similar membrane responses have been noted in cattle. Use of animal protein and increased nitrogen in cattle feeds would lead to a relative deficiency of essential fatty acids in the cell membranes and hence reduced membrane stability. By analogy with crazy chick disease (nutritional encephalomalacia) and experimental encephalomyelitis in rats, the possibility that the changes in animals feeds would have depleted cattle tissue membranes and made them susceptible to BSE is discussed. The assumption being made is that the principle of a requirement of essential fatty acids for neural integrity and immune system function would apply to cattle as well as to other species.