Dietary sialylated oligosaccharides in early-life may promote cognitive flexibility during development in context of obesogenic dietary intake.

Introduction: Oligosaccharides found in mammalian milk have shown the potential to alter brain development across multiple species. The diversity and concentration of these oligosaccharides is species-specific and varies greatly between individuals, thus understanding their role in cognitive development is warranted. We investigated the impact of early life dietary fucosylated/neutral or sialylated human milk oligosaccharides (HMO) on behaviours in tasks assessing anxiety, motivation, appetite, learning, and memory.Methods: Sixty-four female Göttingen minipigs were artificially reared from 2 weeks postnatal and provided milk replacers. The study used four groups: no additional oligosaccharides (Con), fucosylated and neutral oligosaccharides (FN, 4 g/L), sialylated oligosaccharides (SL, 0.68 g/L), or both FN and SL (FN + SL, 4 g/L) from 2 to 11 weeks postnatal. One reference group was sow-reared. Weaning occurred between 10 and 11 weeks postnatal, and thereafter an obesogenic diet was provided. Behavioral tasks were conducted over three periods: 1) 0-11 weeks; 2) 16-29 weeks; 3) 39-45 weeks. Tasks included a spatial holeboard task, open field task, exposure to a novel object, runway task, single-feed task, and home pen behaviour observation.Results: In the holeboard, the SL group demonstrated improved reference memory during reversal trials between 16-29 weeks. All groups demonstrated equivalent behavior in open field, novel object, runway, and single-feed tasks, as well as in their home pens (Ps > 0.05).Discussion: These results suggest that early life dietary intake of sialylated oligosaccharides may provide an improvement to cognition during the equivalent developmental stage of adolescence.

Targeted vaccination against the bevacizumab binding site on VEGF using 3D-structured peptides elicits efficient antitumor activity.

Therapeutic targeting of the VEGF signaling axis by the VEGF-neutralizing monoclonal antibody bevacizumab has clearly demonstrated clinical benefit in cancer patients. To improve this strategy using a polyclonal approach, we developed a vaccine targeting VEGF using 3D-structured peptides that mimic the bevacizumab binding site. An in-depth study on peptide optimization showed that the antigen's 3D structure is essential to achieve neutralizing antibody responses. Peptide 1 adopts a clear secondary, native-like structure, including the typical cysteine-knot fold, as evidenced by CD spectroscopy. Binding and competition studies with bevacizumab in ELISA and surface plasmon resonance analysis revealed that peptide 1 represents the complete bevacizumab binding site, including the hairpin loop (ß5-turn-ß6) and the structure-supporting ß2-α2-ß3 loop. Vaccination with peptide 1 elicited high titers of cross-reactive antibodies to VEGF, with potent neutralizing activity. Moreover, vaccination-induced antisera displayed strong angiostatic and tumor-growth-inhibiting properties in a preclinical mouse model for colorectal carcinoma, whereas antibodies raised with peptides exclusively encompassing the ß5-turn-ß6 loop (peptides 15 and 20) did not. Immunization with peptide 1 or 7 (murine analog of 1) in combination with the potent adjuvant raffinose fatty acid sulfate ester (RFASE) showed significant inhibition of tumor growth in the B16F10 murine melanoma model. Based on these data, we conclude that this vaccination technology, which is currently being investigated in a phase I clinical trial (NCT02237638), can potentially outperform currently applied anti-VEGF therapeutics.

Critical review evaluating the pig as a model for human nutritional physiology.

The present review examines the pig as a model for physiological studies in human subjects related to nutrient sensing, appetite regulation, gut barrier function, intestinal microbiota and nutritional neuroscience. The nutrient-sensing mechanisms regarding acids (sour), carbohydrates (sweet), glutamic acid (umami) and fatty acids are conserved between humans and pigs. In contrast, pigs show limited perception of high-intensity sweeteners and NaCl and sense a wider array of amino acids than humans. Differences on bitter taste may reflect the adaptation to ecosystems. In relation to appetite regulation, plasma concentrations of cholecystokinin and glucagon-like peptide-1 are similar in pigs and humans, while peptide YY in pigs is ten to twenty times higher and ghrelin two to five times lower than in humans. Pigs are an excellent model for human studies for vagal nerve function related to the hormonal regulation of food intake. Similarly, the study of gut barrier functions reveals conserved defence mechanisms between the two species particularly in functional permeability. However, human data are scant for some of the defence systems and nutritional programming. The pig model has been valuable for studying the changes in human microbiota following nutritional interventions. In particular, the use of human flora-associated pigs is a useful model for infants, but the long-term stability of the implanted human microbiota in pigs remains to be investigated. The similarity of the pig and human brain anatomy and development is paradigmatic. Brain explorations and therapies described in pig, when compared with available human data, highlight their value in nutritional neuroscience, particularly regarding functional neuroimaging techniques.

Considerations on pig models for appetite, metabolic syndrome and obese type 2 diabetes: From food intake to metabolic disease.

(Mini)pigs have proven to be a valuable animal model in nutritional, metabolic and cardiovascular research and in some other biomedical research areas (toxicology, neurobiology). The large resemblance of (neuro)anatomy, the gastro-intestinal tract, body size, body composition, and the omnivorous food choice and appetite of the pig are additional reasons to select this large animal species for (preclinical) nutritional and pharmacological studies. Both humans and pigs are prone to the development of obesity and related cardiovascular diseases such as hypertension and atherosclerosis. Bad cholesterol (LDL) is high and good cholesterol (HDL) is low in pigs, like in humans. Disease-relevant pig models fill the gap between rodent models and primate species including humans. Diet-induced obese pigs show a phenotype related to the metabolic syndrome including high amounts of visceral fat, fatty organs, insulin resistance and high blood pressure. However, overt hyperglycaemia does not develop within 6 months after initiation of high sugar-fat feeding. Therefore, to accelerate the induction of obese type 2 diabetes, obese pigs can be titrated with streptozotocin, a chemical agent which selectively damages the insulin-producing pancreatic beta-cells. However, insulin is required to maintain obesity. With proper titration of streptozotocin, insulin secretion can be restrained at such a level that hyperglycaemia will be induced but lipolysis is still inhibited due to the fact that inhibition of lipolysis is more sensitive to insulin compared to stimulation of glucose uptake. This strategy may lead to a stable hyperglycaemic, non-ketotic obese pig model which remains anabolic with time without the necessity of exogenous insulin treatment.

Physiological and behavioral reactions elicited by simulated and real-life visual and acoustic helicopter stimuli in dairy goats.

BACKGROUND: Anecdotal reports and a few scientific publications suggest that flyovers of helicopters at low altitude may elicit fear- or anxiety-related behavioral reactions in grazing feral and farm animals. We investigated the behavioral and physiological stress reactions of five individually housed dairy goats to different acoustic and visual stimuli from helicopters and to combinations of these stimuli under controlled environmental (indoor) conditions. The visual stimuli were helicopter animations projected on a large screen in front of the enclosures of the goats. Acoustic and visual stimuli of a tractor were also presented. On the final day of the study the goats were exposed to two flyovers (altitude 50 m and 75 m) of a Chinook helicopter while grazing in a pasture. Salivary cortisol, behavior, and heart rate of the goats were registered before, during and after stimulus presentations. RESULTS: The goats reacted alert to the visual and/or acoustic stimuli that were presented in their room. They raised their heads and turned their ears forward in the direction of the stimuli. There was no statistically reliable rise of the average velocity of moving of the goats in their enclosure and no increase of the duration of moving during presentation of the stimuli. Also there was no increase in heart rate or salivary cortisol concentration during the indoor test sessions. Surprisingly, no physiological and behavioral stress responses were observed during the flyover of a Chinook at 50 m, which produced a peak noise of 110 dB. CONCLUSIONS: We conclude that the behavior and physiology of goats are unaffected by brief episodes of intense, adverse visual and acoustic stimulation such as the sight and noise of overflying helicopters. The absence of a physiological stress response and of elevated emotional reactivity of goats subjected to helicopter stimuli is discussed in relation to the design and testing schedule of this study.

The pig as a model animal for studying cognition and neurobehavioral disorders.

In experimental animal research, a short phylogenetic distance, i.e., high resemblance between the model species and the species to be modeled is expected to increase the relevance and generalizability of results obtained in the model species. The (mini)pig shows multiple advantageous characteristics that have led to an increase in the use of this species in studies modeling human medical issues, including neurobehavioral (dys)functions. For example, the cerebral cortex of pigs, unlike that of mice or rats, has cerebral convolutions (gyri and sulci) similar to the human neocortex. We expect that appropriately chosen pig models will yield results of high translational value. However, this claim still needs to be substantiated by research, and the area of pig research is still in its infancy. This chapter provides an overview of the pig as a model species for studying cognitive dysfunctions and neurobehavioral disorders and their treatment, along with a discussion of the pros and cons of various tests, as an aid to researchers considering the use of pigs as model animal species in biomedical research.

Welfare of the minipig with special reference to use in regulatory toxicology studies.

This paper reviews the animal welfare challenges associated with the use of minipigs in toxicology testing, and compares these to published knowledge on the other widely used non-rodent species (dogs and non-human primates). Welfare challenges arise from housing and management of populations under laboratory conditions, and from the procedures carried out for product evaluation. Welfare assessment requires a multidisciplinary approach: cardiovascular parameters, adrenocortical hormones and behaviour are well known parameters. However, reliable non-invasive methods to assess welfare and species-specific benchmarks need further development in minipigs. Husbandry of minipigs (housing, diet, and socialisation needs) to promote good welfare is described in the revised Appendix A of the European Convention (ETS 123). This has been supplemented by knowledge of species biology and expert opinion from experienced minipig users. Challenges when using minipigs in toxicity testing have been reviewed in detail. Although deeper location of the peripheral blood vessels makes blood sampling more challenging, samples can be taken with minimal distress when staff members are well trained. Temporary and chronic vascular catheters can also be used for frequent sampling, and are likely to improve the welfare of the animals. Available training courses with a focus on stress free handling and dosing, as well as surgical placement of temporary and chronic vascular catheters, should be utilised to improve welfare during these procedures. Humane endpoints have been described, mainly based on current industry practices, but further scientific investigations are required. From an animal welfare perspective there are no basic restrictions to using minipigs in toxicity testing that are unique to this species. We conclude that it is easier to keep minipigs to a good standard of welfare under laboratory conditions than it is for dogs or non-human primates, since minipigs are not athletic (like dogs) or arboreal (like non-human primates).

Effects of chronic stress: a comparison between tethered and loose sows.

The present study aimed to investigate whether long-lasting, recurrent tethering of sows leads to enduring effects on measures that may be indicative of chronic stress. Sows that had experienced tethering for about 1.5 or 4.5years and age-matched sows kept in a social housing system (loose sows) were compared. Immediately after slaughter, blood samples were taken to measure plasma cortisol levels, and the brain, spleen, and adrenals were dissected and weighed. Gene expression in the frontal cortex and hippocampus was analyzed. Plasma cortisol levels were higher in the tethered sows than in the loose sows. The older, but not the younger, tethered sows had heavier adrenal glands than their loose counterparts. The weight of the spleen was not affected by the housing conditions, but the pituitary gland was lighter in tethered sows than in loose sows. Microarray analyses revealed an increased expression of beta-globin mRNA in the hippocampus and to a lesser extent in the frontal cortex of the older tethered sows, compared with the older loose sows. Taken together, the findings indicate that chronically stressed pigs develop depression-like symptoms. However, it can be questioned whether the pig subjected to repeated, long-term stress can be regarded an animal model of major depression.

Emotional reactivity and cognitive performance in aversively motivated tasks: a comparison between four rat strains.

BACKGROUND: Cognitive function might be affected by the subjects' emotional reactivity. We assessed whether behavior in different tests of emotional reactivity is correlated with performance in aversively motivated learning tasks, using four strains of rats generally considered to have a different emotional reactivity. METHODS: The performance of male Brown Norway, Lewis, Fischer 344, and Wistar Kyoto rats in open field (OF), elevated plus-maze (EPM), and circular light-dark preference box (cLDB) tasks, which are believed to provide measures of emotional reactivity, was evaluated. Spatial working and reference memory were assessed in two aversively motivated learning and memory tasks: the standard and the "repeated acquisition" versions of the Morris water maze escape task, respectively. All rats were also tested in a passive avoidance task. At the end of the study, levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid, and 5-HT turnover in the hippocampus and frontal cortex were determined. RESULTS: Strain differences showed a complex pattern across behavioral tests and serotonergic measures. Fischer 344 rats had the poorest performance in both versions of the Morris water escape task, whereas Brown Norway rats performed these tasks very well but the passive avoidance task poorly. Neither correlation analysis nor principal component analysis provided convincing support for the notion that OF, EPM, and cLDB tasks measure the same underlying trait. CONCLUSIONS: Our findings do not support the hypothesis that the level of emotional reactivity modulates cognitive performance in aversively motivated tasks. Concepts such as "emotional reactivity" and "learning and memory" cannot adequately be tapped with only one behavioral test. Our results emphasize the need for multiple testing.

The d-amphetamine-treated Göttingen miniature pig: an animal model for assessing behavioral effects of antipsychotics.

RATIONALE: Rodents are usually used to assess the ability of antipsychotic drugs to antagonize hyperlocomotion induced by dopamine agonists, such as the psychostimulant d-amphetamine. However, the substantial differences between rodents and humans may hinder extrapolation of experimental results to humans. For this reason, we speculated that Göttingen miniature pigs, which show strong physiological and genetic homology with humans, might be a better model for investigating the effects of antipsychotics. To investigate this, we determined whether d-amphetamine induced hyperlocomotion in miniature pigs and whether this effect was reversible by antipsychotics. MATERIALS AND METHODS: d-amphetamine was tested in the dose range of 0.2 to 2.0 mg kg(-1) for its ability to induce hyperactivity in the open field, and the effects of two antipsychotics, haloperidol and risperidone, on amphetamine-induced hyperactivity were examined. RESULTS: d-amphetamine increased open-field activity at 0.2, 0.4, and 0.7 mg kg(-1) s.c. but not at higher doses. The stimulation of open-field activity induced by 0.4 mg kg(-1) s.c. d-amphetamine was antagonized by haloperidol and risperidone (0.01 and 0.04 mg kg(-1) s.c.). CONCLUSION: d-amphetamine-induced hyperlocomotion in miniature pigs may be a useful model for studying the effect of putative antipsychotics.