Heart Rate and Acceleration Dynamics during Swim-Fitness and Stress Challenge Tests in Yellowtail Kingfish (Seriola lalandi).

The yellowtail kingfish is a highly active and fast-growing marine fish with promising potential for aquaculture. In this study, essential insights were gained into the energy economy of this species by heart rate and acceleration logging during a swim-fitness test and a subsequent stress challenge test. Oxygen consumption values of the 600-800 g fish, when swimming in the range of 0.2 up to 1 m·s-1, were high-between 550 and 800 mg·kg-1·h-1-and the heart rate values-up to 228 bpm-were even among the highest ever measured for fishes. When swimming at these increasing speeds, their heart rate increased from 126 up to 162 bpm, and acceleration increased from 11 up to 26 milli-g. When exposed to four sequential steps of increasing stress load, the decreasing peaks of acceleration (baseline values of 12 to peaks of 26, 19 and 15 milli-g) indicated anticipatory behavior, but the heart rate increases (110 up to 138-144 bpm) remained similar. During the fourth step, when fish were also chased, peaking values of 186 bpm and 44 milli-g were measured. Oxygen consumption and heart rate increased with swimming speed and was well reflected by increases in tail beat and head width frequencies. Only when swimming steadily near the optimal swimming speed were these parameters strongly correlated.

Steroid implants for the induction of vitellogenesis in feminized European silver eels (Anguilla anguilla L.).

Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17ß-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.

Sanitary Conditions on the Farm Alters Fecal Metabolite Profile in Growing Pigs.

The aim of this study was to use fecal metabolite profiling to evaluate the effects of contrasting sanitary conditions and the associated subclinical health status of pigs. We analyzed fecal metabolite profiles by nuclear magnetic resonance (1H NMR) from pigs aged 14 and 22 weeks. Pigs kept under low and high sanitary conditions differed in fecal metabolites related to the degradation of dietary starch, metabolism of the gut microbiome, and degradation of components of animal (host) origin. The metabolites that differed significantly (FDR < 0.1) were from metabolic processes involved in either maintaining nutrient digestive capacity, including purine metabolism, energy metabolism, bile acid breakdown and recycling, or immune system metabolism. The results show that the fecal metabolite profiles reflect the sanitary conditions under which the pigs are kept. The fecal metabolite profiles closely resembled the profiles of metabolites found in the colon of pigs. Fecal valerate and kynurenic acid could potentially be used as "non-invasive" biomarkers of immune or inflammatory status that could form the basis for monitoring subclinical health status in pigs.

Local intestinal microbiota response and systemic effects of feeding black soldier fly larvae to replace soybean meal in growing pigs.

Black soldier fly (Hermetia illucens; BSF) larvae as dietary protein source have the ability to deliver nutrients and could possess functional properties that positively support animal productivity and health. More knowledge, however, is needed to assess the impact of feeding a BSF based diet on gut and animal health. Sixteen post-weaned male pigs were randomly assigned to two groups and fed for three weeks with iso-caloric and iso-proteinaceous experimental diets prepared with either soybean meal (SBM) as reference protein source or with BSF as single source of dietary protein. At the end of the trial, the pigs were sacrificed to collect relevant digesta, gut tissue and blood samples to study changes induced by the dietary treatments using ~ omics based analyses. Inclusion of BSF in the diet supports the development of the intestinal microbiome that could positively influence intestinal health. By amine metabolite analysis, we identified two metabolites i.e. sarcosine and methionine sulfoxide, in plasma that serve as markers for the ingestion of insect based ingredients. BSF seems to possess functional properties indicated by the appearance of alpha-aminobutyric acid and taurine in blood plasma of pigs that are known to induce health beneficial effects.

What Goes Wrong during Early Development of Artificially Reproduced European Eel Anguilla anguilla? Clues from the Larval Transcriptome and Gene Expression Patterns.

In eels, large variations in larval mortality exist, which would impede the viable production of juvenile glass eels in captivity. The transcriptome of European eel larvae was investigated to identify physiological pathways and genes that show differential regulation between non-viable vs. viable larvae. Expression of genes involved in inflammation and host protection was higher, suggesting that non-viable larvae suffered from microbial infection. Expression of genes involved in osmoregulation was also higher, implying that non-viable larvae tried to maintain homeostasis by strong osmoregulatory adaptation. Expression of genes involved in myogenesis, neural, and sensory development was reduced in the non-viable larvae. Expression of the major histocompatibility complex class-I (mhc1) gene, M-protein (myom2), the dopamine 2B receptor (d2br), the melatonin receptor (mtr1), and heat-shock protein beta-1 (hspb1) showed strong differential regulation and was therefore studied in 1, 8, and 15 days post-hatch (dph) larvae by RT-PCR to comprehend the roles of these genes during ontogeny. Expression patterning of these genes indicated the start of active swimming (8 dph) and feed searching behavior (15 dph) and confirmed immunocompetence immediately after hatching. This study revealed useful insights for improving larval survival by microbial control and salinity reduction.

Physiological Effects of Water Flow Induced Swimming Exercise in Seabream Sparus aurata.

A longer on-land rearing period of Gilthead seabream Sparus aurata before transfer to sea-cages would allow the farmer to benefit from exercise-enhanced growth, resilience, and robustness as induced by increasing water flow in the tanks. In this study, the physiological effects of flow-conditioning were investigated by subjecting large groups of experimental fish to minimal flow or to flow regimes inducing swimming exercise at 1 or 2 body length (BL) s-1 for a period of 8 months (February-October) in 1,500 L tanks. Fish representing the three treatment groups were then used for: (1) a stress challenge netting test and plasma cortisol measurement (baseline, peaking, and recovery levels), (2) blood plasma measurements of glucose, triglycerides, lactate, cholesterol, growth hormone (GH), and insulin-like growth factor 1 (IGF1), and (3) heart and muscle gene expression of the GH and IGF1 receptors and the muscle transcriptome by deep RNA sequencing (RNAseq). Fish size after 8 months of flow conditioning was 92 ± 27 g body weight (BW) for fish under minimal flow, 106 ± 24 g BW (+15%) at 1 BL s-1, and 125 ± 27 g BW (+36%) at 2 BL s-1. Flow conditioning at 1 BL s-1 provided optimal conditions for growth and uniformity, but also stress (lowest baseline plasma cortisol), robustness (higher condition factor and larger hearts), and energy mobilization (increased plasma glucose). Although flow enhanced growth linearly with swimming speed, also the percentage of lordotic fish increased with exercise, particularly high for swimming at 2 BL s-1. The absence of important differences in plasma GH and IGF1, and expression levels of their receptors in heart and white skeletal muscle, indicated that other factors may be involved in growth enhancement. RNAseq of the white skeletal muscle showed upregulated expression of genes involved in muscle contraction, muscle development and its molecular regulation, and immune genes that may play a role in the muscle repair mechanism. An exercise regime of swimming at 1 BL s-1 can be considered as optimal for farming robust seabream although the increase of skeletal deformities should be avoided.

Sanitary Conditions Affect the Colonic Microbiome and the Colonic and Systemic Metabolome of Female Pigs.

Differences in sanitary conditions, as model to induce differences in subclinical immune stimulation, affect the growth performance and nutrient metabolism in pigs. The objective of the present study was to evaluate the colonic microbiota and the colonic and systemic metabolome of female pigs differing in health status induced by sanitary conditions. We analyzed blood and colon digesta metabolite profiles using Nuclear Magnetic Resonance (1H NMR) and Triple quadrupole mass spectrometry, as well as colonic microbiota profiles. 1H NMR is a quantitative metabolomics technique applicable to biological samples. Weaned piglets of 4 weeks of age were kept under high or low sanitary conditions for the first 9 weeks of life. The microbiota diversity in colon digesta was higher in pigs subjected to low sanitary conditions (n = 18 per treatment group). The abundance of 34 bacterial genera was higher in colon digesta of low sanitary condition pigs, while colon digesta of high sanitary status pigs showed a higher abundance for four bacterial groups including the Megasphaera genus (p < 0.003) involved in lactate fermentation. Metabolite profiles (n = 18 per treatment group) in blood were different between both groups of pigs. These different profiles suggested changes in general nutrient metabolism, and more specifically in amino acid metabolism. Moreover, differences in compounds related to the immune system and responses to stress were observed. Microbiome-specific metabolites in blood were also affected by sanitary status of the pigs. We conclude that the microbiome composition in colon and the systemic metabolite profiles are affected by sanitary conditions and related to suboptimal health. These data are useful for exploring further relationships between health, metabolic status and performance and for the identification of biomarkers related to health (indices) and performance.

Changes in Plasma Protein Expression Indicative of Early Diet-induced Metabolic Disease in Male Pigs (Sus scrofa).

Recognition of the preclinical stages of metabolic diseases such as diabetes helps to prevent full development of the disease. In our research, we alter the diet composition of pigs to create a model of human metabolic disease. The objective of the current study was to identify plasma proteins and biologic mechanisms that differed in expression between pigs fed a 'cafeteria diet' (considered unhealthy; high in saturated fats) and those fed a 'Mediterranean diet' (considered healthy; high in unsaturated fats). Pigs fed the cafeteria diet showed increased plasma levels of proteins related to LDL ('bad cholesterol'), immune processes, blood clotting, and metal binding. The Mediterranean diet was associated with increased plasma quantities of proteins associated HDL particles ('good cholesterol'), binding of LDL particles, regulation of immune processes, and glycolysis. Pigs fed a cafeteria diet showed molecular signs of diabetes and atherosclerosis-even in the absence of clinical symptoms-which seemed to protect against the development of metabolic disorders. The current results suggest potential biomarkers of the early onset of metabolic syndromes. These biomarkers can help to reveal specific metabolic changes that precede the onset of diabetes, thus enabling the initiation of patient-specific interventions early during pathophysiologic development.

Dietary protein sources differentially affect microbiota, mTOR activity and transcription of mTOR signaling pathways in the small intestine.

Dietary protein sources can have profound effects on host-microbe interactions in the gut that are critically important for immune resilience. However more knowledge is needed to assess the impact of different protein sources on gut and animal health. Thirty-six wildtype male C57BL/6J mice of 35 d age (n = 6/group; mean ± SEM body weight 21.9 ± 0.25 g) were randomly assigned to groups fed for four weeks with semi synthetic diets prepared with one of the following protein sources containing (300 g/kg as fed basis): soybean meal (SBM), casein, partially delactosed whey powder, spray dried plasma protein, wheat gluten meal and yellow meal worm. At the end of the experiment, mice were sacrificed to collect ileal tissue to acquire gene expression data, and mammalian (mechanistic) target of rapamycin (mTOR) activity, ileal digesta to study changes in microbiota and serum to measure cytokines and chemokines. By genome-wide transcriptome analysis, we identified fourteen high level regulatory genes that are strongly affected in SBM-fed mice compared to the other experimental groups. They mostly related to the mTOR pathway. In addition, an increased (P < 0.05) concentration of granulocyte colony-stimulating factor was observed in serum of SBM-fed mice compared to other dietary groups. Moreover, by 16S rRNA sequencing, we observed that SBM-fed mice had higher (P < 0.05) abundances of Bacteroidales family S24-7, compared to the other dietary groups. We showed that measurements of genome-wide expression and microbiota composition in the mouse ileum reveal divergent responses to diets containing different protein sources, in particular for a diet based on SBM.

Amine Metabolism Is Influenced by Dietary Protein Source.

Growth in world population will inevitably leads to increased demand for protein for humans and animals. Protein from insects and blood plasma are being considered as possible alternatives, but more research on their nutritional quality and health effects is needed. Here, we studied the effect of dietary protein source on metabolism and metabolic amine profiles in serum and urine of mice. Groups of mice were fed semi-purified diets containing 300 g/kg of soybean meal, casein, partially delactosed whey powder, spray-dried plasma protein, wheat gluten meal, and yellow mealworm. Feed and water intake as well as body weight gain were measured for 28 days. After 14 and 28 days, serum and urine samples were collected for measurement of a large panel of amine metabolites. MetaboAnalyst 3.0 was used for analysis of the raw metabolic data. Out of 68 targeted amine metabolites, we could detect 54 in urine and 41 in blood serum. Dietary protein sources were found to have profound effects on host metabolism, particularly in systemic amine profiles, considered here as an endophenotype. We recommend serum over urine to screen for the amine metabolic endophenotype based on partial least squares discriminant analysis. We concluded that metabolites like alpha-aminobutyric acid and 1-methylhistidine are sensitive indicators of too much or too little availability of specific amino acids in the different protein diets. Furthermore, we concluded that amine metabolic profiles can be useful for assessing the nutritional quality of different protein sources.

Plasma proteome profiles associated with diet-induced metabolic syndrome and the early onset of metabolic syndrome in a pig model.

Obesity and related diabetes are important health threatening multifactorial metabolic diseases and it has been suggested that 25% of all diabetic patients are unaware of their patho-physiological condition. Biomarkers for monitoring and control are available, but early stage predictive biomarkers enabling prevention of these diseases are still lacking. We used the pig as a model to study metabolic disease because humans and pigs share a multitude of metabolic similarities. Diabetes was chemically induced and control and diabetic pigs were either fed a high unsaturated fat (Mediterranean) diet or a high saturated fat/cholesterol/sugar (cafeteria) diet. Physiological parameters related to fat metabolism and diabetes were measured. Diabetic pigs' plasma proteome profiles differed more between the two diets than control pigs plasma proteome profiles. The expression levels of several proteins correlated well with (patho)physiological parameters related to the fat metabolism (cholesterol, VLDL, LDL, NEFA) and diabetes (Glucose) and to the diet fed to the animals. Studying only the control pigs as a model for metabolic syndrome when fed the two diets showed correlations to the same parameters but now more focused on insulin, glucose and abdominal fat depot parameters. We conclude that proteomic profiles can be used as a biomarker to identify pigs with developing metabolic syndrome (prediabetes) and diabetes when fed a cafeteria diet. It could be developed into a potential biomarkers for the early recognition of metabolic diseases.

Identification of proteomic biomarkers in M. Longissimus dorsi as potential predictors of pork quality.

Meat quality traits have low heritability and large environmental influences. To predict, improve and manage meat quality, proteomic biomarkers are superior to genetic markers. The objectives of this research were (1) to find associations between proteome profiles of longissimus muscle at slaughter and meat quality accuracies of prediction of traits ranged from 20 up to 80%. Differentially expressed proteins related to drip loss and ultimate pH were identified by NanoLC-FTMSMS. The proteins highlight biological mechanisms that may explain how these traits develop biologically and how they are related to each other.

Serum protein profiles as potential biomarkers for infectious disease status in pigs.

BACKGROUND: In veterinary medicine and animal husbandry, there is a need for tools allowing the early warning of diseases. Preferably, tests should be available that warn farmers and veterinarians during the incubation periods of disease and before the onset of clinical signs. The objective of this study was to explore the potential of serum protein profiles as an early biomarker for infectious disease status. Serum samples were obtained from an experimental pig model for porcine circovirus-associated disease (PCVAD), consisting of Porcine Circovirus type 2 (PCV2) infection in combination with either Porcine Parvovirus (PPV) or Porcine Reproductive and Respiratory Syndrome virus (PRRSV). Sera were collected before and after onset of clinical signs at day 0, 5 and 19 post infection. Serum protein profiles were evaluated against sera from non-infected control animals. RESULTS: Protein profiles were generated by SELDI-TOF mass spectrometry in combination with the Proteominer™ technology to enrich for low-abundance proteins. Based on these protein profiles, the experimentally infected pigs could be classified according to their infectious disease status. Before the onset of clinical signs 88% of the infected animals could be classified correctly, after the onset of clinical sigs 93%. The sensitivity of the classification appeared to be high. The protein profiles could distinguish between separate infection models, although specificity was moderate to low. Classification of PCV2/PRRSV infected animals was superior compared to PCV2/PPV infected animals. Limiting the number of proteins in the profiles (ranging from 568 to 10) had only minor effects on the classification performance. CONCLUSIONS: This study shows that serum protein profiles have potential for detection and identification of viral infections in pigs before clinical signs of the disease become visible.

Dietary unsaturated fatty acids affect the mammary gland integrity and health in lactating dairy cows.

BACKGROUND: Information about the effects of unsaturated fatty acids (UFA) supplementation on the health and integrity of the mammary gland in lactating dairy cows is lacking. Therefore, the aim of this study was to determine the effects of unprotected dietary UFA on the global expression pattern of genes in the mammary gland tissue of grazing dairy cows, and to translate this information into relevant biological knowledge. METHODS: Twenty-eight Holstein-Friesian dairy cows were randomly assigned to 4 different concentrated UFA-sources for 23 days after which all cows were switched to a non-UFA-supplemented concentrate for an additional 28 days. On the last day of both periods, mammary gland biopsies were taken to study genome-wide differences in gene expression on Bovine Genome Arrays. RESULTS: Supplementation with UFA reduced the concentration of short chain fatty acids (FA), C16 FA and saturated FA in the milk, whereas that of trans-FA increased. One major finding was that canonical pathways associated with remodelling and immune functions of the mammary gland were predominantly down-regulated during UFA supplementation and negatively correlated with the concentration of milk trans-FA. CONCLUSIONS: Supplementing grazing dairy cows with unprotected dietary UFA can affect the remodelling and immune functions of the mammary gland with potential consequences for its integrity and health, as well as milk quality.

Successive immunoglobulin and cytokine expression in the small intestine of juvenile chicken.

The intestinal mucosa is of major importance for immune development. To further study the ontogeny of avian mucosal immunity, mRNA levels of IgM, IgY and IgA, the polymeric immunoglobulin receptor (pIgR) and a number of cytokines were determined at different ages in jejunum and ileum of non-immunized healthy juvenile layer chickens. Immunoglobulin genes were successively expressed in jejunum and ileum. IgM expression was maximal in week 1, IgY expression peaked in week 5, and IgA expression was most dominant after week 7 post hatch. PIgR gene expression was relatively low in the first 2 weeks post hatch, but increased thereafter. Generally, increased expression levels of IL-1, IL-10, IL-12p40, iNOS and interferon-γ mRNA levels were found between days 14-42 as compared to days 3 and 49-70 post hatch (p<0.05). Correlation was found between IgA and IL-10, TGF-ß and IFN-γ expression levels on days 21, 28 and 35. Cytokine mRNA expression levels decreased to basal levels between 49 and 70 days post hatch, whereas IgA reached its maximum levels in this period. Based on the current results, we hypothesize that chicken sIgA, as mammalian sIgA, may contribute to the maintenance of intestinal homeostasis.

Age, gender and litter-related variation in T-lymphocyte cytokine production in young pigs.

The capacity of farm animals to produce cytokines could be an important determinant of robustness and health. From research in rodents and humans it appears that the production and the balance of T helper 1 (Th1) and T helper 2 (Th2)-type cytokines influences susceptibility to autoimmune and infectious diseases. It is known that pigs show a large variation in many immune response parameters. So far the extent of individual variation in the production of Th1- and Th2-type cytokines in commercial outbred pigs has not been reported. In the current experiment we determined mRNA expression, as well as protein production of cytokines in 32 pigs from eight litters. From each litter two male and two female pigs were tested at 2, 5 and 8 weeks of age. Two Th1-type cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, and two Th2-type cytokines, IL-4 and IL-10, were measured after phytohaemagglutinin (PHA)-stimulation of blood mononuclear cells. Cytokine production and the Th1/Th2-ratio were highly variable. The variation in cytokine protein production was moderately consistent across ages, i.e. pigs that produced high levels of cytokine at 2 weeks of age tended to do so as well at 5 and 8 weeks of age. Cytokine production tended to increase with age, and gilts and boars differed in their IL-2/IL-4 ratio. Unexpectedly, age, gender and litter effects often differed for mRNA and protein production data. We hypothesize that cytokine production is a consistent trait in pigs, especially at the protein production level. Future investigations in more animals and across a wider age range are necessary.