Aflatoxins in Feed: Types, Metabolism, Health Consequences in Swine and Mitigation Strategies.

Feeding farm animals with aflatoxin-contaminated feed can cause various severe toxic effects, leading to increased susceptibility to infectious diseases and increased mortality, weight loss, poor performance and reduced reproductive capability. Following ingestion of contaminated foodstuffs, aflatoxins are metabolized and biotransformed differently in animals. Swine metabolism is not effective in detoxifying and excreting aflatoxins, meaning the risk of aflatoxicosis is increased. Thus, it is of great importance to elucidate the metabolism and all metabolic pathways associated with this mycotoxin. The damage induced by AFB1 in cells and tissues consists of inhibition of cell proliferation, carcinogenicity, immunosuppression, mutagenicity, oxidative stress, lipid peroxidation and DNA damage, leading to pathological lesions in the liver, spleen, lymph node, kidney, uterus, heart, and lungs of swine. At present, it is a challenging task and of serious concern to completely remove aflatoxins and their metabolites from feedstuff; thus, the aim of this study was a literature review on the deleterious effects of aflatoxins on swine metabolism, as well as alternatives that contribute to the detoxification or amelioration of aflatoxin-induced effects in farm animal feed.

A Multistep DNA-Based Methodology for Accurate Authentication of Sturgeon Species.

The sturgeons (order Acipenseriformes) are caviar producers and some of the most valuable fish species worldwide. Due to different reasons, wild populations are now at the brink of extinction. The high demand for caviar has led to the development of aquaculture for restocking and caviar production. Since the caviar from different species has different prices depending on the quality and attempts of commercial fraud based on species substitution have been found, correct species identification is more than necessary. We report a new multistep methodology for an accurate species identification based on both nuclear and mitochondrial markers. Our test integrates data from the analysis of microsatellites (Afu19, Afu34, Afu39, Afu54, Aox27, AoxD234, AnacC11 and AnacE4), nuclear gene markers (RPS7, vimentin and rhodopsin) and mtDNA barcoding to give a reliable molecular diagnostic for five sturgeon species (Huso huso, Acipenser stellatus, Acipenser ruthenus, Acipenser gueldenstaedtii and Acipenser baerii). In addition to species identification, our methodology allows the identification of bester, sterbe and best beluga hybrids, but also the identification of hybrids of unknown origin. This methodology has a good potential to contribute to the conservation of highly threatened sturgeon populations and also to the traceability of their products.

Liquid Biopsy and Artificial Intelligence as Tools to Detect Signatures of Colorectal Malignancies: A Modern Approach in Patient's Stratification.

Colorectal cancer (CRC) is the second most frequently diagnosed type of cancer and a major worldwide public health concern. Despite the global efforts in the development of modern therapeutic strategies, CRC prognosis is strongly correlated with the stage of the disease at diagnosis. Early detection of CRC has a huge impact in decreasing mortality while pre-lesion detection significantly reduces the incidence of the pathology. Even though the management of CRC patients is based on robust diagnostic methods such as serum tumor markers analysis, colonoscopy, histopathological analysis of tumor tissue, and imaging methods (computer tomography or magnetic resonance), these strategies still have many limitations and do not fully satisfy clinical needs due to their lack of sensitivity and/or specificity. Therefore, improvements of the current practice would substantially impact the management of CRC patients. In this view, liquid biopsy is a promising approach that could help clinicians screen for disease, stratify patients to the best treatment, and monitor treatment response and resistance mechanisms in the tumor in a regular and minimally invasive manner. Liquid biopsies allow the detection and analysis of different tumor-derived circulating markers such as cell-free nucleic acids (cfNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs) in the bloodstream. The major advantage of this approach is its ability to trace and monitor the molecular profile of the patient's tumor and to predict personalized treatment in real-time. On the other hand, the prospective use of artificial intelligence (AI) in medicine holds great promise in oncology, for the diagnosis, treatment, and prognosis prediction of disease. AI has two main branches in the medical field: (i) a virtual branch that includes medical imaging, clinical assisted diagnosis, and treatment, as well as drug research, and (ii) a physical branch that includes surgical robots. This review summarizes findings relevant to liquid biopsy and AI in CRC for better management and stratification of CRC patients.

The Reduction of the Combined Effects of Aflatoxin and Ochratoxin A in Piglet Livers and Kidneys by Dietary Antioxidants.

The purpose of this study was to investigate the combined effects of aflatoxin B1 and ochratoxin A on protein expression and catalytic activities of CYP1A2, CYP2E1, CYP3A29 and GSTA1 and the preventive effect of dietary byproduct antioxidants administration against these mycotoxin damage. Three experimental groups (E1, E2, E3) and one control group (C) of piglets after weaning (TOPIGS-40 hybrid) were fed with experimental diets for 30 days. A basal diet containing normal compound feed for starter piglets was used as a control treatment and free of mycotoxin. The experimental groups were fed as follows: E1-basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal), E2-the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1) and E3-basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform microsomal fraction isolation, followed by protein expression and enzymatic analyses. The protein expressions of CYP2E1 and CYP3A29 were up-regulated in an insignificant manner in liver, whereas in kidney, those of CYP1A2, CYP2E1 and CYP3A29 were down-regulated. The enzymatic activities of CYP1A2, CYP2E1 and CYP3A29 decreased in liver, in a significant manner, whereas in kidney, these increased significantly. The co-presence of the two mycotoxins and the mixture of grape seed and sea buckthorn meal generated a tendency to return to the control values, which suggest that grapeseed and sea buckthorn meal waste represent a promising source in counteracting the harmful effect of ochratoxin A and aflatoxin B.

The Effectiveness of Dietary Byproduct Antioxidants on Induced CYP Genes Expression and Histological Alteration in Piglets Liver and Kidney Fed with Aflatoxin B1 and Ochratoxin A.

The purpose of this study was to investigate the potential of a byproduct mixture derived from grapeseed and sea buckthorn oil industry to mitigate the harmful damage produced by ochratoxin A and aflatoxin B1 at hepatic and renal level in piglets after weaning. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three experimental groups (E1, E2, E3) and one control group (C), and fed with experimental diets for 30 days. The basal diet was served as a control and contained normal compound feed for starter piglets without mycotoxins. The experimental groups were fed as follows: E1-basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal); E2-the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1); and E3-basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform gene expression and histological analysis. The gene expression analysis showed that when weaned piglets were fed with contaminated diet, the expression of most analyzed genes was downregulated. Among the CYP450 family, CYP1A2 was the gene with the highest downregulation. According to these results, in liver, we found that mycotoxins induced histomorphological alterations in liver and kidney and had an effect on the expression level of CYP1A2, CYP2A19, CYP2E1, and CYP3A29, but we did not detect important changes in the expression level of CY4A24, MRP2 and GSTA1 genes.

Oxidative Stress and Antioxidant Defense Mechanisms in Response to Starvation and Refeeding in the Intestine of Stellate Sturgeon (Acipenser stellatus) Juveniles from Aquaculture.

Acipenser stellatus is a critically endangered species due to the anthropic influence. It has been intensively captured for decades because of its high economic value, its roe being used in the caviar industry. Therefore, Acipenser stellatus is intensively raised in fish farms for both conservation and economical purposes. Aquaculture aims to optimize the feeding regime of juveniles in order to improve its profitability. The purpose of this study was to investigate if Acipenser stellatus can adapt to a starvation/refeeding regime by assessing the effects of this regime on oxidative stress biomarkers and antioxidant defense mechanisms in juveniles raised under aquaculture conditions. The juveniles were subjected to two regimes: a 7-day starvation period followed by 21 days of refeeding, respectively a 14-day starvation period followed by 21 days of refeeding. The results showed that both starvation/refeeding regimes induced an enhancement of antioxidant enzymes activities in the intestine of the juveniles. The oxidative damage was counteracted at the protein level. However, lipid peroxidation was significantly induced in the intestine of the juveniles subjected to 14/21-day starvation/refeeding regime. The 7/21-day starvation/refeeding regime proved to be more suitable for Acipenser stellatus and therefore, it could be useful to optimize the feeding practice in aquaculture production.

Effects of Dietary Inclusion of Bilberry and Walnut Leaves Powder on the Digestive Performances and Health of Tetra SL Laying Hens.

The purpose of this study was to examine the effects of dietary inclusion of two additives at the final concentration of 0.5% bilberry (E1) and 1% walnut (E2) leaves powder in the basal diet on digestive health of hens. A total number of 90 Tetra SL hens were divided into two experimental groups (E1 and E2) and one control group (C) consisting of 30 hens each. After four weeks, 10 hens of each group were sacrificed and tissue samples and intestinal content were taken from the duodenum, jejunum, and cecum in order to perform histological, enzymatic, and microbiota analyses. In groups E1 and E2, the histological analysis showed a significant increase of villus height, resulting probably in increased absorption of nutrients in duodenum and jejunum. A decrease in the specific activity of alpha-amylase and trypsin in E1 and E2 for both duodenum and jejunum compared to the control one was also recorded. In addition, the maltase and invertase specific activity in duodenum increased, a tendency that was kept for maltase but not for invertase in jejunum. The cecal microbiota of E1 and E2 individuals was characterized by an increase of Firmicutes and Lactobacilli and a decrease of Enterobacteriaceae. In conclusion, our results indicate that bilberry and walnut leaves additives in feed may improve the health status of the poultry gastrointestinal tract.

Effects of starvation and refeeding on growth performance and stress defense mechanisms of stellate sturgeon Acipenser stellatus juveniles from aquaculture.

Acipenser stellatus represents a species of great economical interest due to its roe used for caviar production. Therefore, it has been intensively captured for decades and nowadays, this species is on the verge of extinction. As a consequence, Acipenser stellatus is intensively raised in fish farms. Aquaculture is focused on optimizing the feeding regime of juveniles. The aim of this study was to investigate if Acipenser stellatus can adapt to a starvation/refeeding regime by assessing the effects of this regime on growth performance, oxidative stress biomarkers and heat shock protein (hsp) gene expression in juveniles raised under aquaculture conditions. The juveniles were subjected to two starvation/refeeding regimes: a 7-day starvation period followed by 21 days of refeeding, and a14-day starvation period followed by 21 days of refeeding. The results had shown that the juveniles subjected to 7/21-day starvation/refeeding regime presented a complete compensatory growth, they were able to counteract the oxidative stress by enhancing activities of the antioxidant enzymes and they presented no significant changes in hsp gene expression. In contrast, 14/21-day starvation/refeeding regime negatively influenced growth performance, it induced a high level of oxidative stress that was impossible to counteract and it determined major changes in the hsp gene expression level in the liver of Acipenser stellatus. Thus, Acipenser stellatus seems to be able to adapt only to the 7/21-day starvation/refeeding regime that does not threaten the growth performance and the welfare of juveniles. Therefore, it could be useful to optimize the feeding practice in aquaculture production.

Expression Characterization of Six Genes Possibly Involved in Gonad Development for Stellate Sturgeon Individuals (Acipenser stellatus, Pallas 1771).

Nowadays, in sturgeon's aquaculture, there is a necessity for sex identification at early stages in order to increase the efficiency of this commercial activity. The basis for a correct identification is studying the different factors that influence the gonad development. The research has been directed towards molecular methods that have been employed with various degrees of success in identifying genes with different expression patterns between male and female sturgeons during their development stages. For the purpose of understanding the sexual development of 4-year-old stellate sturgeon (Acipenser stellatus) individuals, we have selected six genes (foxl2, cyp17a1, ar, dmrt1, sox9, and star). We analysed the gene expression of the selected genes for gonads, anal fin, liver, body kidney, and white muscle. The cyp17a1, ar, dmrt1, and sox9 genes have a significant higher expression in male gonads than in female gonads, while the data shows no significant differences in the expression of the investigated genes in the other organs. We investigate these genes to shed light on aquaculture sturgeon sexual development.

Molecular analysis of phylogeographic subspecies in three Ponto-Caspian sturgeon species.

Sturgeons (Order Acipenseriformes) represent an extremely valuable natural resource that is now facing depletion. In the current study we evaluate if the traditional classification in subspecies of Acipenser gueldenstaedtii, Acipenser stellatus and Huso huso, endemic to Ponto-Caspian region is sustained by molecular analysis and if these represent Evolutionary Significant Units (ESUs) that should be managed separately in conservation programs. To examine the classification of taxonomic entities we sequenced a fragment of the mitochondrial control region in case of three sturgeon species that inhabit the North-western of Black Sea and migrate for reproduction in the Lower Danube. Beside these sequences, we used previously published sequences from sturgeon individuals sampled in the Black Sea, Azov Sea and Caspian Sea. We determined the genetic diversity and genetic differentiation, conducted a Population Aggregation Analysis (PAA) and inferred an intraspecific molecular phylogeny and haplotype network. The results indicated a low level of genetic differentiation between the geographically designated subspecies and did not support a significant divergence or reciprocal monophyly between them. Our results confirm previous genetic studies with smaller samples sizes, but additional analyses including nuclear markers should be conducted for proper recommendations aiming at the development of conservation programs.

Site heteroplasmy in the mitochondrial cytochrome b gene of the sterlet sturgeon Acipenser ruthenus.

Sturgeons are fish species with a complex biology. They are also characterized by complex aspects including polyploidization and easiness of hybridization. As with most of the Ponto-Caspian sturgeons, the populations of Acipenser ruthenus from the Danube have declined drastically during the last decades. This is the first report on mitochondrial point heteroplasmy in the cytochrome b gene of this species. The 1141 bp sequence of the cytb gene in wild sterlet sturgeon individuals from the Lower Danube was determined, and site heteroplasmy evidenced in three of the 30 specimens collected. Two nucleotide sequences were identified in these heteroplasmic individuals. The majority of the heteroplasmic sites are synonymous and do not modify the sequence of amino acids in cytochrome B protein. To date, several cases of point heteroplasmy have been reported in animals, mostly due to paternal leakage of mtDNA. The presence of specific point heteroplasmic sites might be interesting for a possible correlation with genetically distinct groups in the Danube River.

Phylogenetic relationships of the mangalitsa Swine breed inferred from mitochondrial DNA variation.

The Mangalitsa pig, a swine breed belonging to the protected gene fund of original and primitive animal breeds of the FAO (Food and Agriculture Organization), has been known to inhabit Romanian territories since the 19th century. The aim of this study was to compare the Mangalitsa breed with several European and Asiatic swine breeds in order to emphasize its uniqueness and to elucidate its origin. For this purpose, we analyzed a 613 bp mitochondrial DNA D-loop fragment and 1140 bp of the cytochrome b gene in a population of Mangalitsa pigs and the polymorphic sites were compared with sequences from GenBank originating from other swine breeds. Taking into account the total of 24 breeds and 5 different Wild Boar populations analyzed, 86 polymorphic sites representing 32 haplotypes were observed, with an average percentage of polymorphic sites of 4.9%. Three Neighbor-Joining phylogenetic trees were constructed based on Kimura 2-parameter distances, using D-loop, cytochrome b and mitochondrial reunited sequences. For the analyzed Mangalitsa population, four distinct haplotypes were identified, including one that was common to other breeds. Our study suggests that the Mangalitsa swine originate from primitive breeds which might be directly derived from the Wild Boar.

Nuclear markers of Danube Sturgeons hybridization.

Acipenseriformes are composed of 25 sturgeon species and two paddlefish species distributed exclusively in the northern hemisphere. The Danube River and the Black Sea were originally inhabited by six sturgeon species but two are extinct and only four are still reproducing currently in the Lower Danube: Huso huso, Acipenser stellatus, A. gueldenstaedtii and A. ruthenus. Sturgeon species hybridize more easily than other fish and the determination of pure species or hybrid status is important for conservation and for breeding in fish farms. This survey demonstrated that morphological determination of this status is not reliable and a molecular tool, based on eight microsatellites genotypes is proposed. This method, based on three successive statistical analyses including Factorial Correspondence Analysis (FCA), Structure assignation and NewHybrids status determination, showed a high efficiency in discriminating pure species specimens from F1, F2 and two kinds of backcross individuals involving three of the four reproducing Lower Danube sturgeon species.