Ochratoxin A cytotoxicity on Madin-Darby canine kidney cells in the presence of alpha-tocopherol: Effects on cell viability and tight junctions.

Ochratoxin A (OTA) is a potent nephrotoxic fungi metabolite that affects animal and human health. At the cellular level, OTA is able to alter functions and viability by several mechanisms of action. Several strategies to counteract its toxicity have been studied. We investigated the role of α-tocopherol in counteracting OTA oxidative damage in Madin-Darby canine kidney (MDCK) cells by pre-incubating the cells for 3 hr with the antioxidant (1 nm, 10 µm) and then adding OTA (0-1.2 µg/ml) for the following 24 hr. Cell viability, lactate dehydrogenase (LDH) release, TUNEL staining and occludin and Zo1 localization by immunofluorescence were determined. Here, 1 nm α-tocopherol was shown to significantly reduce (p < .05) the cytotoxicity, LDH release and apoptotic rate induced by OTA. The presence of the antioxidant at the same concentration maintained the localization of occludin and Zo1 in the rim of the MDCK cells after the 24-hr OTA exposure. These results indicate that a low concentration of α-tocopherol could block OTA toxicity, supporting its defensive role in the cellular membrane.

Cultured meat in the European Union: Legislative context and food safety issues.

The current food system, which is responsible for about one third of all global gas emissions, is considered one of the main causes of resource depletion. For this reason, scientific research is investigating new alternatives capable of feeding an ever-growing population that is set to reach 9-11 billion by 2050. Among these, cell-based meat, also called cultured meat, is one possible solution. It is part of a larger branch of science called cellular agriculture, whose goal is to produce food from individual cells rather than whole organisms, tracing their molecular profile. To date, however, cultured meat aroused conflicting opinions. For this reason, the aim of this review was to take an in-depth look at the current European legislative framework, which reflects a 'precautionary approach' based on the assumption that these innovative foods require careful risk assessment to safeguard consumer health. In this context, the assessment of possible risks made it possible not only to identify the main critical points during each stage of the production chain (proliferation, differentiation, scaffolding, maturation and marketing), but also to identify solutions in accordance with the recommendations of the European Food Safety Authority (EFSA). Further, the main challenges related to organoleptic and nutritional properties have been reviewed.. Finally, possible future markets were studied, which would complement that of traditional meat, implementing the offer for the consumer, who is still sceptical about the acceptance of this new product. Although further investigation is needed, the growing demand for market diversification and the food security opportunities associated with food shortages, as well as justifying the commercialisation of cultured meat, would present an opportunity to position cultured meat as beneficial.

Review: Nutritional ecology of heavy metals.

The aim of this review is to focus the attention on the nutrition ecology of the heavy metals and on the major criticisms related to the heavy metals content in animal feeds, manure, soil and animal-origin products. Heavy metals are metallic elements that have a high density that have progressively accumulated in the food chain with negative effects for human health. Some metals are essential (Fe, I, Co, Zn, Cu, Mn, Mo, Se) to maintain various physiological functions and are usually added as nutritional additives in animal feed. Other metals (As, Cd, F, Pb, Hg) have no established biological functions and are considered as contaminants/undesirable substances. The European Union adopted several measures in order to control their presence in the environment, as a result of human activities such as: farming, industry or food processing and storage contamination. The control of the animal input could be an effective strategy to reduce human health risks related to the consumption of animal-origin products and the environmental pollution by manure. Different management of raw materials and feed, animal species as well as different legal limits can influence the spread of heavy metals. To set up effective strategies against heavy metals the complex interrelationships in rural processes, the widely variability of farming practices, the soil and climatic conditions must be considered. Innovative and sustainable approaches have discussed for the heavy metal nutrition ecology to control the environmental pollution from livestock-related activities.

The head cartilage of cephalopods. I. Architecture and ultrastructure of the extracellular matrix.

The morphology of head cartilage of the cephalopods Sepia officinalis and Octopus vulgaris has been studied on samples fixed and embedded for light- and electron microscopy and on fresh frozen sections viewed by polarizing microscopy. The organization of extracellular matrix (ECM) varies in different regions of the head cartilage. Superficial zones are made up of densely packed collagenous laminae parallel to the cartilage surface, while radially arranged laminae form a deeper zone where territorial and interterritorial areas are present. A compact arrangement of banded collagen fibrils (10-25 nm in diameter) forms the laminae of the superficial zones and of the interterritorial areas; a loose three-dimensional network of fibrils (10-20 nm) with many proteoglycan aggregates forms the territorial areas. A pericellular matrix surrounds the bodies of extremely branched territorial chondrocytes. Peculiar anchoring devices (ADs) are dispersed with variable orientation within the ECM. A perichondrium is present, but often connectival and muscular bundles are fused with the superficial layers of cartilage. Some vessels were also observed within the superficial inner zone and clusters of hemocyanin molecules were demonstrated both in the ECM and in some cells. The cephalopod head cartilage seems to share some morphological characteristics with both hyaline cartilage and bone tissue of vertebrates.

Effects of vitamin E on urokinase-plasminogen activator receptor expression by bovine neutrophils.

OBJECTIVE: To determine the effect of vitamin E supplementation on urokinase-plasminogen activator (u-PA) receptor (u-PAR) expression by neutrophils of dairy cows. ANIMALS: 16 healthy Holstein dairy cows. PROCEDURE: 16 cows were assigned to 1 of 2 experimental groups: control (no vitamin E supplementation) and vitamin E supplementation. Supplementation of vitamin E started 4 weeks prior to and continued up to 4 weeks after parturition and included oral administration of vitamin E at 3,000 U/cow per day; these cows also received 1 injection of vitamin E (5,000 units), 1 week prior to the expected date of parturition. Blood samples were collected, and neutrophils were isolated weekly throughout the experimental period. The following variables were measured: u-PA (mRNA), total cell-associated u-PA activity, membrane-bound u-PA activity, and free unoccupied u-PA binding sites on the cell membrane of neutrophils. RESULTS: Stimulated neutrophils isolated from cows that received vitamin E supplementation had significantly higher u-PA mRNA and total cell-associated and membrane-bound u-PA activity at postpartum week 1, compared with those of stimulated neutrophils isolated from control cows. There were no differences between groups throughout the whole experimental period in u-PA binding sites of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: The increased total cell-associated and membrane-bound u-PA activity in neutrophils isolated from cows that received vitamin E may facilitate the ability of neutrophils to extravasate and reach the mammary gland at postpartum week 1. Rapid recruitment of neutrophils is critical for proper defense of the gland.

Kinetic behavior of three preparations of alpha-tocopherol after oral administration to postpubertal heifers.

OBJECTIVE: To assess the kinetic behavior of 3 preparations of alpha-tocopherol (vitamin E) after oral administration to heifers. ANIMALS: 8 postpubertal Friesian heifers. PROCEDURE: A single oral bolus of 5,000 U of alpha-tocopherol in oil or encapsulated in liposomes or cyclodextrin was administered to each cow, using a 4 X 4 design with 8 days between treatments. Blood samples for kinetic analyses were obtained at various times for 168 hours after treatment. RESULTS: Mean (+/- SEM) maximal plasma concentrations of alpha-tocopherol were 4.86 +/- 0.49 microg/ml, 5.03 +/- 0.39 microg/ml, and 5.08 +/- 0.56 microg/ml after administration of oil, liposomal, and cyclodextrin preparations, respectively. Plasma concentrations peaked 21 to 34 hours after administration. The disappearance rate constant (Kd) was less after administration of alpha-tocopherol encapsulated in liposomes, compared with the other 2 preparations. Area under the concentration versus time curve was greater after administration of either encapsulated form of alpha-tocopherol, compared with alpha-tocopherol in oil, but these differences were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: The lower Kd determined for alpha-tocopherol encapsulated in liposomes suggests that this formulation may result in longer persistance of the vitamin in plasma than the other 2 preparations. Dietary supplementation with alpha-tocopherol encapsulated in liposomes may enhance plasma availability of this vitamin in cattle and could be useful during periods of increased vitamin E requirements, such as parturition and early stages of life.

Administration of a novel plant extract product via drinking water to post-weaning piglets: effects on performance and gut health.

The present study evaluated the effects of a novel plant extract (PE) product (GrazixTM) on the performance and gut health of weaned piglets challenged with Escherichia coli. The PE was a standardised mixture of green tea leaves (Camellia sinensis) and pomegranate fruit (Punica granatum) obtained by using the LiveXtract™ process. A total of 144 piglets were weaned at 24 days and allocated to 8 for a 35-day experiment with a 2×2×2 factorial design comparing different treatments (water without product (CT) or 8 µl/kg per day PE in drinking water (PE)), feeding regimens (ad libitum (AD) or restricted (RE)) and oral E. coli challenges on day 9 (sham (-) or infected (+)). There were six pens per group with three piglets per pen. On day 35, 24 of the RE feeding piglets were slaughtered. It was found that PE supplementation increased the average daily gain (ADG) from day 28 to day 35 (P=0.03) and increased the gain to feed ratio (G : F) from day 7 to day 14 (P=0.02). RE feeding led to lower feed intake in piglets during the 1st week (P<0.01), 2nd week (P=0.06), 3rd week (P=0.05), and throughout the course of the overall study period (P=0.05). E. coli challenge decreased the ADG and G : F ratio from day 7 to day 14 (P=0.08 and <0.01, respectively) and increased the faecal score (higher values indicate more severe diarrhoea) on days 14, 21, 28 and 35 (P<0.01). PE supplementation decreased the faecal score in the challenged piglets during the 1st week post-challenge (P<0.01). E. coli challenge increased the faecal E. coli level on day 14 (P=0.03) and increased the Enterobacteriaceae level on day 35 (P<0.01). Reduced faecal E. coli was observed on days 14 and 35 (P=0.05 and 0.02, respectively), and reduced Enterobacteriaceae (P<0.01) was found on day 35 in the PE animals. RE feeding increased the faecal Lactobacillus, Enterobacteriaceae and E. coli levels on day 35 (P=0.02, <0.01 and <0.01, respectively). These results suggest that PE supplementation may improve the gut health status of post-weaning piglets and counteract some of the negative effects that occur when piglets are challenged with E. coli.

Computer image analysis: an additional tool for the identification of processed poultry and mammal protein containing bones.

The aims of this study were (1) to evaluate the potential of image analysis measurements, in combination with the official analytical methods for the detection of constituents of animal origin in feedstuffs, to distinguish between poultry versus mammals; and (2) to identify possible markers that can be used in routine analysis. For this purpose, 14 mammal and seven poultry samples and a total of 1081 bone fragment lacunae were analysed by combining the microscopic methods with computer image analysis. The distribution of 30 different measured size and shape bone lacunae variables were studied both within and between the two zoological classes. In all cases a considerable overlap between classes meant that classification of individual lacunae was problematic, though a clear separation in the means did allow successful classification of samples on the basis of averages. The variables most useful for classification were those related to size, lacuna area for example. The approach shows considerable promise but will need further study using a larger number of samples with a wider range.

Nutrition in mammary gland health and lactation: advances over eight Biology of Lactation in Farm Animals meetings.

Over the years, numerous studies have investigated the mechanisms controlling nutrient availability and metabolism in the mammary gland and how dietary interventions can influence these processes. The development of in vivo and in vitro systems made it possible to explore the trafficking and metabolic fate of nutrients and how these are influenced by hormones. To improve the quality and safety of milk products, attention has focused on improving animal health in general and mammary gland health in particular and also on enhancing the milk content of natural bioactive milk components that promote the health of human neonates and adults. In the past, attempts to probe the relationship among nutrition, animal health, and animal products were limited to administering single dietary components and investigating their biochemical and metabolic effects. Today, we have genomics, proteomics, and related technologies that allow us to pursue more holistic investigational strategies. These new technologies are providing new insights into interactions among nutrition, lactation, and product quality. The aim of this paper is to review advances in nutritional support of mammary gland function and health as presented in 14 yr of Biology of Lactation in Farm Animals (BOLFA) meetings.

Rumen-protected choline and vitamin E supplementation in periparturient dairy goats: effects on milk production and folate, vitamin B12 and vitamin E status.

We investigated the effects of rumen-protected choline (RPC) and vitamin E (VITE) administration on milk production and status of folate, vitamin B12 and vitamin E during the periparturient period of dairy goats. Forty-eight Saanen multiparous goats were selected for the 72-day experiment, being moved to a maternity pen 30 days before expected parturition and assigned to one of the four experimental groups: control (CTR), no choline or vitamin E supplementation; choline (RPC), supplemented with 4 g/day choline chloride in rumen-protected form; vitamin E (VITE), supplemented with 200 IU/day vitamin E in rumen-protected form; and choline and vitamin E (RPCE), supplemented with 4 g/day RPC chloride and 200 IU/day vitamin E. Supplements were administered individually before the morning feed to ensure complete consumption, starting 30 days before kidding and continuing for 35 days after. During the experiment, milk yield and 4% fat-corrected milk (FCM) yield were, respectively, 210 and 350 g/day higher in RPC-supplemented goats than in non-supplemented goats. Milk fat concentration and fat yield were also increased by RPC treatment. Milk yield and composition were unaffected by vitamin E supplementation. There were no significant interactions between RPC and VITE for any of the variables measured. Plasma metabolites did not differ between treatments before and after kidding except that plasma folate at parturition was higher in RPC-supplemented goats. Neither choline nor vitamin E affected vitamin B12 plasma concentrations, while a time effect was evident after the second week of lactation, when B12 levels in each treatment group started to increase. Vitamin E administration resulted in plasma α-tocopherol levels that were 2 to 2.5 times higher than in non-supplemented goats. Overall, these results suggest that greater choline availability can improve milk production and methyl group metabolism in transition dairy goats.

Ultrastructural study of the dermis in a case of type IV Ehlers-Danlos syndrome.

Light microscopic and ultrastructural examination of the skin in a case of type IV Ehlers-Danlos syndrome (EDS) showed the presence of morphological changes involving all the dermis components. Collagen fibres were smaller, more irregular and more spaced apart than in normal dermis. Conversely, elastic, microfibrillar and amorphous components were considerably increased. Collagen fibrils presented poorly defined outlines and a loose texture, besides being markedly reduced in size. Fibroblasts were characterized by variable degrees of dilatation of RER cisternae containing flocculent material. In addition to these features, which confirm previous description of dermis structure in a case of type IV EDS (Holbrook and Byers, 1981), we have also observed thickening and delaminations of epidermal, perineurial and perivascular basement membranes and the presence of filamentous banded elements associated to nerve bundles. Moreover Langerhans' cells were found in papillary dermis, probably indicating the existence of an abnormal antigenic stimulation.

The head cartilage of cephalopods. II. Ultrastructure of isolated native collagen fibrils and of polymeric aggregates obtained in vitro: comparison with the cartilage of mammals.

Native collagen fibrils were isolated from cephalopod head cartilage and mammal hyaline cartilage. The analysis with TEM after positive and negative staining demonstrated that the fibrils have a periodic structure similar to that of fibrillar type I collagen of mammals. The banding pattern of polymeric forms (SLS, FLS) obtained in vitro from squid cartilage collagen was remarkably different from the analogous forms of mammal collagen types I and II.

Plasminogen activation system in goat milk and its relation with composition and coagulation properties.

The activity of plasmin (PL), plasminogen (PG), and plasminogen activator (PA) and their correlation with goat milk components and milk clotting parameters were investigated. Seven late-lactating Saanen goats were used to provide milk samples that were analyzed for PL, PG, and PA activity (colorimetric assay) fat, protein, noncasein nitrogen, nonprotein nitrogen, casein content, and somatic cell count (SCC). Milk clotting parameters (rennet coagulating time = coagulation time; K20 = firming rate of curd; A30 = curd firmness) were measured with a formagraph. Average milk yield and composition were similar to those previously observed in other studies. Plasmin, PG, and PA activity, expressed as units/ml, were, respectively, 20.04 +/- 0.94, 3.21 +/- 0.04, and 1154 +/- 57.61. Plasminogen activity was surprisingly low compared with other species (bovine, ovine), but it was consistent with the high activity of PA. A negative significant correlation was observed between PL and milk casein content. The correlation coefficients between PL and casein/protein ratio and PA and casein/protein ratio were negative and significant. A positive significant correlation was observed between PL and rennet clotting time and PA and rennet clotting time. Also positive was the correlation between PL and K20 and PA and K20. The plasmin activity was negatively correlated with A30. High plasmin and plasminogen activator activity in goat milk appeared to be negatively related with coagulating properties in late lactation, most probably via degradation of casein due to plasmin activity.

Effects of vitamin E and different energy sources on vitamin E status, milk quality and reproduction in transition cows.

We investigated whether vitamin E supplementation and supplemental energy sources (fat or starch) influenced plasma and milk levels of vitamin E, and reproductive and other parameters in 28 Italian Friesian multiparous dry cows. From 14 days before expected calving to 7 days after, the animals were assigned to either basal diet (containing 1000 IU/day of vitamin E) or an extra 1000 IU/day of vitamin E (total 2000 IU). In addition they received either 0.5 kg/day of corn or 0.2 kg/day of calcium soaps. Plasma samples were collected 4 days before expected calving and 4 days after calving and analysed for alpha-tocopherol and cholesterol. Milk yield as well as the composition, somatic cell count (SCC) and alpha-tocopherol of milk were determined 7 and 14 days after calving. Milk yield and composition were unaffected by treatments. SCC was significantly lower in (SCC Log 4.62 versus Log 5.1, P < 0.01) 2000 IU/day animals than in the 1000 IU/day group. Milk alpha-tocopherol was higher (P < 0.001) in animals receiving 2000 IU/day (1.11 vs. 0.65 microgram/ml, P < 0.01). Plasma alpha-tocopherol in animals receiving 2000 IU/day was also higher (P < 0.001) than in cows receiving 1000 IU/day (4.85 vs. 3.25 micrograms/ml), but was not affected by dietary energy source. Number of services and days to conception were lower (P < 0.01) in the 2000 IU vitamin E supplemented cows. To conclude, dietary vitamin E supplementation to periparturient dairy cows increased plasma and milk vitamin E, decreased SCC in milk, and improved fertility but different energy sources had no effect on any measured variable.

Synthesis of plasminogen activator inhibitor 1 by bovine mammary epithelial and myoepithelial cell lines.

Conversion of plasminogen to plasmin provides an important source of proteolytic activity in the bovine mammary gland. Plasminogen activator inhibitor 1 (PAI-1) plays a key role in limiting plasminogen activation. The PAI-1 biosynthetic capabilities of various bovine mammary cells were determined. The immortalized epithelial cell lines MAC-T and BME-UV and the myoepithelial cell line BMM-UV were used as model systems. Northern blot analysis indicated that both epithelial and myoepithelial cells contained PAI-1 mRNA. Bovine PAI-1 was encoded by a single mRNA species approximately 3.0 kb long. BME-UV cells contained 2.0-fold (P < 0.01) the PAI-1 mRNA of MAC-T or BMM-UV cells. Reverse zymography indicated that both epithelial and myoepithelial cells synthesized PAI-1 protein with a molecular mass of approximately 50 kDa.

Relative bioavailability of vitamin E in dairy cows following intraruminal administration of three different preparations of DL-alpha-tocopheryl acetate.

DL-alpha-tocopheryl acetate, a synthetic form of vitamin E, is routinely given as a dietary supplement to cattle. In this study we assessed the relative bioavailability of three formulations of DL-alpha-tocopheryl acetate in a kinetic study of plasma alpha-tocopherol in four Italian Friesian dairy cows, following intraruminal administration of a gelatin capsule containing 5,000 IU of DL-alpha-tocopheryl acetate. A Latin square design was used so that each animal received all formulations: (A) adsorbed on silica, (M) microencapsulated and (O) in oil form; 5,000 IU of DL-alpha-tocopheryl acetate was also administered intraperitoneally. The treatments were given following a 2-week period on a diet having no vitamin E supplementation with an interval of 8 days between each administration. Blood samples were collected at 0, 1, 10, 11, 21, 30, 48, 72, 96 and 168 h after each administration. The mean initial plasma alpha-tocopherol concentration (CO) was 2.38 +/- 0.57 micrograms/mL. Maximum plasma concentrations (Cmax) of alpha-tocopherol, adjusted for pretreatment values, were 3.90 +/- 0.13, 3.29 +/- 0.13 and 4.07 +/- 0.19 micrograms/mL, following administration of the A, M and O forms, respectively. The length of time required to obtain the maximum concentration (Tmax) in plasma was 57.5 +/- 7.8, 76.8 +/- 8.9 and 73.1 +/- 14.1 h, and the area under the curve (AUC) was 503.3 +/- 63, 620.25 +/- 108.5 and 465.4 +/- 38.7 micrograms.h/mL for A, M and O forms, respectively. Administration significantly increased the plasma alpha-tocopherol levels in all cases; however the A and M formulations had a lower elimination rate than the O form.

Hormonal and metabolic responses to the stress of transport and slaughterhouse procedures in clenbuterol-fed pigs.

The aim of this study was to evaluate if metabolic and endocrine adaptive responses to stressful stimuli are influenced by clenbuterol treatment in pigs. Two groups of nine Goland barrows of 167 kg liveweight, fed diets containing 0 (control) or 1 ppm of clenbuterol (clenbuterol-treated) for 40 days, were used. Blood samples were collected by jugular venipuncture the day before (day 40) and at slaughter (day 41). On day 40 clenbuterol-treated pigs had lower levels of thyroxin, tri-iodothyronine, insulin, urea and higher levels of lactate and creatine phosphokinase activity. On day 41, thyroxin concentration was lower and creatine phosphokinase activity higher in clenbuterol-treated pigs than in controls. In response to transport and slaughterhouse procedures, most of the parameters considered both in clenbuterol-treated and in control pigs increased.

Effect of body condition score at calving on performance, some blood parameters, and milk fatty acid composition in dairy cows.

Effect of body condition score at calving and of change in body condition score on productive and reproductive performance, on lactation curve parameters, and on postpartum disease occurrence was investigated in 213 Italian Holstein-Friesian cows. Plasma FFA, glucose, and insulin concentrations and milk fatty acid composition were analyzed in 15 cows. The cows calving at highest body condition score lost more subcutaneous fat; condition score change did not exceed 1.05 units. Change in body condition score was positively associated with peak and total milk production. Occurrence of retained placenta was not related to the content of fat stores at calving. Glucose concentrations were relatively constant, FFA concentrations were highest, and insulin concentrations were lower in cows calving at the highest body condition score. Milk fatty acid composition reflected the different utilization of body fat stores.