Determining the effects of Candida utilis-fermented pea starch vs. unfermented pea starch, alone or in whole diets, on palatability and glycemic response in dogs and cats.

Current research suggests yeast fermentation has the potential to improve palatability of pea-based diets for both cats and dogs. However, to be useful, fermentation should not compromise other healthy attributes of peas such as a low glycemic response. Fermentation of uncooked pea starch with Candida utilis (ATCC 9950) appeared to increase crude protein, crude fiber content, inorganic compounds (phosphorus and iron) and phenols. Whole diets were designed with fermented and unfermented pea starch to assess palatability, food intake, and glycemic responses in unacclimated, mixed sex Beagle dogs and mixed breed cats (n = 8 and n = 7, respectively). For palatability testing, a control diet was formulated with 30% corn starch as well as test diets with 30% inclusion of fermented or unfermented pea starch (all lab-made), then compared to a commercial diet containing pea starch (Legacy/Horizon). Fermentation had little effect on rapidly digestible starch either in uncooked starch form or when incorporated into whole diets, but did decrease resistant starch by 15% and increase slowly digestible starch by 20%. Palatability tests using either two choices or four choices at a time revealed a significant preference for the fermented pea starch diet (p < 0.01) in both species. For the glycemic responses, a total of four different pea products were included: unfermented pea starch, fermented pea starch, and 30% inclusion of unfermented and fermented pea starch in whole formulated diets. There were no significant changes in glycemic responses with the fermented pea diet compared to the unfermented diet, demonstrating that healthful low glycemic properties of pea starch were retained after C. utilis fermentation. Overall, C. utilis-fermentation technique was successfully adapted to pea starch where it resulted in increased palatability and food intake in dogs and cats, with potential to positively contribute to overall health benefits for both species.

Low birth weight and reduced postnatal nutrition lead to cardiac dysfunction in piglets.

Heart disease is the leading cause of death in humans and evidence suggests early life growth-restriction increases heart disease risk in adulthood. Therefore, this study sought to investigate the effects of low birth weight (LBW) and postnatal restricted nutrition (RN) on cardiac function in neonatal pigs. We hypothesized that LBW and RN would reduce cardiac function in pigs but this effect would be reversed with refeeding. To investigate this hypothesis, pigs born weighing <1.5 kg were assigned LBW, and pigs born >1.5 kg were assigned normal birth weight (NBW). Half the LBW and NBW pigs underwent ~25% total nutrient restriction via intermittent suckling (assigned RN) for the first 4 wk post-farrowing. The other half of piglets were allowed unrestricted suckling access to the sow (assigned NN). At 28 d of age (weaning), pigs were weaned and provided ad libitum access to a standard diet. Echocardiographic, vascular ultrasound, and blood pressure (BP) measurements were performed on day 28 and again on day 56 to assess cardiovascular structure and function. A full factorial three-way ANOVA (NN vs. RN, LBW vs. NBW, male vs. female) was performed. Key findings include reduced diastolic BP (P = 0.0401) and passive ventricular filling (P = 0.0062) in RN pigs at 28 d but this was reversed after refeeding. LBW piglets have reduced cardiac output index (P = 0.0037) and diastolic and systolic wall thickness (P = 0.0293 and P = 0.0472) at 56 d. Therefore, cardiac dysfunction from RN is recovered with adequate refeeding while LBW programs irreversible cardiac dysfunction despite proper refeeding in neonatal pigs.

Heart disease is the leading cause of death in humans, and in addition to the known modifiable risk factors, evidence suggests early life undernutrition increases heart disease risk in adulthood. Specifically, low birth weight (LBW) has been linked to poor infant cardiac development which could be made worse by an inadequate postnatal diet. Globally, 160 million children under the age of five experience a poor nutritive environment leading to growth-restriction highlighting the need for continued research. Using a pig model, the present investigation examined the effects of LBW and a restricted diet during postnatal life on cardiac structure and function in preweaning and post-weaning piglets. The most important findings were (1) nutrient-restricted piglets had reduced cardiac function at 28 d old but refeeding reversed cardiac dysfunction at 56 d, indicating that nutrient-induced cardiac dysfunction can be reversed, and (2) LBW pigs presented with cardiac dysfunction at 56 d regardless of feeding level, suggesting potential for an increased risk of heart disease in adulthood with LBW.

Examining the subchronic (28-day) effects of aqueous Cd-BaP co-exposure on detoxification capacity and cardiac function in adult zebrafish (Danio rerio).

The present study aimed to examine the effects of environmentally relevant concentrations of cadmium (Cd) and Benzo[a]Pyrene (BaP) in the adult zebrafish (Danio rerio). To this end, fish were exposed to either 1 or 10 µg/L Cd or 0.1 or 1 µg/L BaP in isolation, or a co-exposure containing a mixture of the two toxicants. Our results showed extensive modulation of the expression of key antioxidant genes (GPx, SOD1, catalase), detoxifying genes (MT1, MT2, CYP1A1) and a stress biomarker (HSP70) differing between control, single toxicant groups and co-exposure groups. We additionally carried out histopathological analysis of the gills, liver, and hearts of exposed animals, noting no differences in tissue necrosis or apoptosis. Finally, we carried out ultrasonographic analysis of cardiac function, noting a significant decrease of E-wave peak velocity and end diastolic volume in exposed fish. This in turn was accompanied by a decrease in stroke volume and ejection fraction, but not cardiac output in co-exposed fish. The present study is the first to demonstrate that a subchronic aqueous exposure to a Cd-BaP mixture can extensively modulate detoxification capacity and cardiac function in adult zebrafish in a tissue-specific manner.

Oligosaccharides and diet-related dilated cardiomyopathy in beagles.

Introduction: In 2018 the US Food and Drug Administration reported a potential link between grain-free, legume-containing dog foods and the development of canine dilated cardiomyopathy in atypical breeds. One hypothesis was that high oligosaccharide content in legumes reduced bioavailability of taurine, an amino acid with some previous links to canine dilated cardiomyopathy. Methods: To address this hypothesis, in the present study, 8 Beagle dogs consumed four diets: a husbandry commercial dental diet, and three test diets formulated with either 30% rice (control), 30% pea (grain-free) or 30% rice with the addition of 1% raffinose (the predominant oligosaccharide found in peas). The study was conducted in a randomized, crossover design with 5 week feeding periods. Measurement of basic health parameters (weight, body condition score, complete blood cell count, chemistry panel), plasma amino acids, cardiac biomarkers (plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) and cardiac-specific troponin I), fecal bile acids and echocardiographic exams were completed pre-study after feeding the husbandry diet as well as after each test feeding period. Results: Echocardiography showed 9-11% reduction in ejection fraction and 17- 20% greater left ventricular end systolic volume with the husbandry diet compared to both grain-containing test diets. Concentrations of plasma NT-proBNP were 1.3-2 times greater after the husbandry diet compared to the grain-based diet, with the oligosaccharide and pea-based diets showing intermediate levels. Plasma taurine levels were unchanged across diets, while plasma methionine levels were highest and cysteine/cystine levels were lowest after dogs ate the husbandry diet. Discussion: Results indicate that raffinose in the diet is sufficient, but not required to see an increase NT-proBNP, but did not induce any changes in cardiac function after 5 weeks of feeding. Whether this could progress to reduction in cardiac function with longer term feeding is uncertain. A reduced cardiac function along with the greatest increase in NT-proBNP was observed after feeding the husbandry diet that contained the highest amount of insoluble fiber but did not contain legumes or oligosaccharide. Further research into the impact of insoluble fiber in the dental diet is needed to support these novel observations.

Effects of a 28-day feeding trial of grain-containing versus pulse-based diets on cardiac function, taurine levels and digestibility in domestic dogs.

In 2018, the US Food and Drug Administration reported a link between canine dilated cardiomyopathy (DCM) and grain-free diets. Evidence to support a link has emerged, but the specific ingredients responsible and the role of taurine or other causative factors remain unclear. We hypothesized dogs fed pulse-based, grain-free diets for 28 days will show decreased macronutrient digestibility, increased fecal bile acid excretion, and reduced plasma cystine, cysteine, methionine and taurine, causing sub-clinical cardiac or blood changes indicative of early DCM. Three diets were formulated using white rice flour (grain), whole lentil (grain-free), or wrinkled pea (grain-free) and compared to the pre-trial phase on a commercial grain-based diet. After 28 days of feeding each diet, the wrinkled pea diet impaired stroke volume and cardiac output, increased end-systolic ventricular diameter and increased plasma N-Terminal Pro-B-type Natriuretic Peptide (NT-ProBNP), albeit in a sub-clinical manner. Digestibility of some macronutrients and sulphur-containing amino acids, excluding taurine, also decreased with pulse-based compared to grain-based diets, likely due to higher fiber levels. Plasma taurine levels were unchanged; however, plasma methionine was significantly lower after feeding all test diets compared to the commercial diet. Overall, DCM-like changes observed with the wrinkled pea diet, but not lentil diet, after only 4 weeks in a breed not known to be susceptible support a link between pea-based diets and canine nutritionally-mediated DCM.

Solidified saturated fats coating subunit vaccines greatly extended vaccine booster release and contributed to a Th1/Th2 mixed immune response in mice.

Delayed release of vaccine coupled with a soluble vaccine acts as a primer and a booster with only a single administration, which would be very beneficial to livestock producers. We developed a subdermal pellet consisting of solid-phase pure stearic acid (SA) or palmitic acid (PA) that was used to encapsulate a small volume liquid vaccine consisting of fluorescently labeled *Ovalbumin (Cy5-*OVA) formulated with Emulsigen-D +/- Poly I:C (EMP) adjuvants. Mice were also immunized via the subcutaneous route with Cy5-*OVA-EMP (soluble liquid). The vaccine leached out of the pellet with very little dissolution of the fat itself resulting in the sustained subdermal delivery of antigens and adjuvants. Cy5-*OVA was still visible 60 days post administration in mice immunized with stearic acid-coated or palmitic acid-coated pellets. In these mice, persistently high IgG1 and IgG2a antibody titres were detected as well as significant IFNγ production at least 60 days post-injection. These responses were significantly higher than those observed after a single subcutaneous injection of the vaccine. A repeat trial with the pellets alone +/- the soluble vaccine showed comparable immune responses after surgical implantation of the pellet, suggesting that pellet alone may be sufficient. The PA-coated vaccines led to dermal inflammation in the mice that would limit usefulness of this vehicle, but this was largely absent when SA was used to coat the pellets. These data suggest that the SA-coated adjuvanted vaccine prolonged the release of the vaccine and triggered a comparable immune response to the mice that received the two liquid injections, and a single pellet vaccine should be tested as a novel immunization method for livestock.

Modulation of Cd and BaP uptake rate during acute aqueous co-exposure in adult zebrafish (Danio rerio).

Cadmium and Benzo[a]pyrene are two toxicants of great environmental importance given their frequency and ability to cause extensive toxicity in aquatic organisms including fish. There is evidence that fish can modulate their respective uptake rate during simultaneous exposures, albeit the mechanism behind this is poorly understood. The present study aimed to examine this interaction by exposing adult zebrafish to either 89.3 nM Cd, 4.25 nM BaP or a combination of the two for 72 hrs prior to examining the uptake rate of either toxicant via short-term exposures (3-6 hrs) to radiotracers (109Cd and 14C-BaP). Our results showed that Cd uptake rate increased significantly in the gills when animals were pre-exposed to both toxicants simultaneously, resulting in an increased maximum uptake rate (Jmax). The increased Cd uptake rate did not correspond to increased expression of gill Cd transporters such as the epithelium calcium channel (ECaC) or the divalent metal transporter 1 (DMT1). Furthermore, BaP uptake rate increased significantly at the whole-body level when animals were exposed to both 5.03 nM 14C-BaP and 89.3 nM Cd concurrently. Additionally, we ran a time-course and observed BaP uptake rate is highest in the 6-12 hrs following the beginning of the exposure. Our results provide evidence that the increased bioaccumulation of Cd and BaP observed during co-exposures is at least in part due to an increase in uptake rate and is driven by separate mechanisms.

Assessing non-protein nitrogen sources in commercial dry dog foods.

Protein is a macronutrient required by dogs for growth and maintenance metabolism. However, a portion of the crude protein listed on pet foods may actually arise from non-digestible organic nitrogen or potentially toxic inorganic non-protein nitrogen sources. Neither non-protein source is retained or used by the animal. However, these compounds may result in adverse effects such as methemoglobin formation and increased oxidative stress or potentially beneficial effects such as improved vascular distensibility and decreased inflammation. To analyze nitrogen retention and screen for non-protein nitrogen, four commercial, dry kibble dog foods and one laboratory-made diet were evaluated and then fed to beagles during two separate feeding trials. During the first trial, dogs were randomly assigned each diet (n = 4 dogs/diet) and fed chromium oxide-coated diets for 48 h, followed by total urine and marked fecal collection, as well as plasma collection for total nitrogen, nitrate, ammonia, and urea determination. The amount of nitrogen retained (93%-96%) did not differ among commercial diets. Protein total tract apparent digestibility (TTAD) ranged from 69% to 84%, with the high protein diets significantly higher than the laboratory-made and mid-ranged diets (1-way ANOVA: P < 0.05). The high protein diet also contained the highest concentration of nitrate with subsequent elevations in plasma nitrotyrosine levels (indicator of oxidative stress). During the second trial, eight dogs (n = 8) were fed the same diets for 6 d, after which echocardiography was completed with blood, urine, and feces collected. For health end-points, methemoblobin, plasma nitrotyrosine, and C-reactive protein (CRP; indicator of inflammation) levels were measured. Methemoglobin levels were significantly lower in the high protein diet (P > 0.05), possible due to the stimulation of methemoglobin reductase while nitrotyrosine was unchanged and CRP was undetectable. Furthermore, there was a positive relationship between crude protein, crude fat (simple linear regression: P = 0.02, r 2 > 0.6), price (P = 0.08, r 2 > 0.6), and caloric density (P = 0.11, r 2 > 0.6). There were no significant cardiovascular differences among any of the diets (P > 0.05). Ultimately, this study shows that in commercial diets, price does reflect protein content but that feeding dogs high protein diets for a long period of time may provide an excess in calories without a change in cardiovascular function or detectable increases in inflammation.

Altered cerebrovascular regulation in low birthweight swine.

Full-term low birthweight (LBW) offspring exhibit peripheral vascular dysfunction in the postnatal period; however, whether such impairments extend to the cerebrovasculature remains to be elucidated. We used a swine model to test the hypothesis that LBW offspring would exhibit cerebrovascular dysfunction at later stages of life. Offspring from 14 sows were identified as normal birthweight (NBW) or LBW and were assessed at 28 (similar to end of infancy) and 56 (similar to childhood) days of age. LBW swine had lower absolute brain mass, but demonstrated evidence of brain sparing (increased brain mass scaled to body mass) at 56 days of age. The cerebral pulsatility index, based on transcranial Doppler, was increased in LBW swine. Moreover, arterial myography of isolated cerebral arteries revealed impaired vasoreactivity to bradykinin and reduced contribution of nitric oxide (NO) to vasorelaxation in the LBW swine. Immunoblotting demonstrated a lower ratio of phosphorylated-to-total endothelial NO synthase in LBW offspring. This impairment in NO signaling was greater at 28 vs. 56 days of age. Vasomotor responses to sodium nitroprusside (NO-donor) were unaltered, while Leu31, Pro34 neuropeptide Y-induced vasoconstriction was enhanced in LBW swine. Increases in total Y1 receptor protein content in the LBW group were not significant. In summary, LBW offspring displayed signs of cerebrovascular dysfunction at 28 and 56 days of age, evidenced by altered cerebral hemodynamics (reflective of increased impedance) coupled with endothelial dysfunction and altered vasomotor control. Overall, the data reveal that normal variance in birthweight of full-term offspring can influence cerebrovascular function later in life.

The Effects of Fermentation of Low or High Tannin Fava Bean-Based Diets on Glucose Response, Cardiovascular Function, and Fecal Bile Acid Excretion during a 28-Day Feeding Period in Dogs: Comparison with Commercial Diets with Normal vs. High Protein.

We have shown that feeding dogs fava bean (FB)-based diets for 7 days is safe and FB flour fermentation with Candida utilis has the potential to decrease FB anti-nutritional factors. In the present study, the effects of 28-day feeding of 4 different FB-based test dog foods containing moderate protein (~27% dry matter (DM)) were compared with two commercial diets with normal protein (NP, grain-containing, ~31% DM protein) or high protein (HP, grain-free, ~41% DM protein). Health parameters were investigated in beagles fed the NP or HP diets or using a randomized, crossover, 2 × 2 Latin square design of the FB diets: unfermented high-tannin (UF-HT), fermented high-tannin (FM-HT), unfermented low-tannin (UF-LT), and fermented low-tannin (FM-LT). The results showed that fermentation increased glucose tolerance, increased red blood cell numbers and increased systolic blood pressure, but decreased flow-mediated vasodilation. Taken together, the overall effect of fermentation appears to be beneficial and improved FB nutritional value. Most interesting, even though the HP diet was grain-free, the diet did contain added taurine, and no adverse effects on cardiac function were observed, while glucose tolerance was impaired compared to NP-fed dogs. In summary, this study did not find evidence of adverse cardiac effects of pulses in 'grain-free' diets, at least not in the relatively resistant beagle breed over a 28-day period. More importantly, fermentation with C. utilis shows promise to enhance health benefits of pulses such as FB in dog food.

Nesfatin-1-like peptide is a negative regulator of cardiovascular functions in zebrafish and goldfish.

Nucleobindins (NUCB1 and NUCB2) were originally identified as calcium and DNA binding proteins. Nesfatin-1 (NEFA/nucleobindin-2-Encoded Satiety and Fat-Influencing proteiN-1) is an 82 amino acid anorexigenic peptide encoded in the N-terminal region of NUCB2. We have shown that nesfatin-1 is a cardiosuppressor in zebrafish. Both NUCB1 and NUCB2 possess a -very highly conserved bioactive core. It was found that a nesfatin-1-like peptide (NLP) encoded in NUCB1 suppresses food intake in fish. In this research, we investigated whether NLP has nesfatin-1-like effects on cardiovascular functions. NUCB1/NLP-like immunoreactivity was found in the atrium and ventricle of the heart and skeletal muscle of zebrafish. Intraperitoneal injection (IP) of either zebrafish NLP or rat NLP suppressed cardiac functions in both zebrafish and goldfish. Irisin and RyR1b mRNA expression was downregulated by NLP in zebrafish cardiac and skeletal muscles. However, cardiac ATP2a2 mRNA expression was elevated after NLP injection. Administration of scrambled NLP did not affect irisin, RyR1b or ATP2a2 mRNA expression in zebrafish. Together, these results implicate NLP as a suppressor of cardiovascular physiology in zebrafish and goldfish.

The sympathetic/beta-adrenergic pathway mediates irisin regulation of cardiac functions in zebrafish.

Irisin is a 23 kDa myokine encoded in its precursor, fibronectin type III domain containing 5 (FNDC5). The exercise-induced increase in the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) promotes FNDC5 mRNA, followed by the proteolytic cleavage of FNDC5 to release irisin from the skeletal or cardiac muscle into the blood. Irisin is abundantly expressed in skeletal and cardiac muscle and plays an important role in feeding, modulates appetite regulatory peptides, and regulates cardiovascular functions in zebrafish. In order to determine the potential mechanisms of acute irisin effects, in this research, we explored whether adrenergic or muscarinic pathways mediate the cardiovascular effects of irisin. Propranolol (100 ng/g B·W) alone modulated cardiac functions, and when injected in combination with irisin (0.1 ng/g B·W) attenuated the effects of irisin in regulating cardiovascular functions in zebrafish at 15 min post-injection. Atropine (100 ng/g B·W) modulated cardiovascular physiology in the absence of irisin, while it was ineffective in influencing irisin-induced effects on cardiovascular functions in zebrafish. At 1 h post-injection, irisin downregulated PGC-1 alpha mRNA, myostatin-a and myostatin-b mRNA expression in zebrafish heart and skeletal muscle. Propranolol alone had no effect on the expression of these mRNAs in zebrafish and did not alter the irisin-induced changes in expression. At 1 h post-injection, irisin siRNA downregulated PGC-1 alpha, troponin C and troponin T2D mRNA expression, while upregulating myostatin a and b mRNA expression in zebrafish heart and skeletal muscle. Atropine alone had no effects on mRNA expression, and was unable to alter effects on mRNA expression of siRNA. Overall, this research identified a role for the sympathetic/beta-adrenergic pathway in regulating irisin effects on cardiovascular physiology and cardiac gene expression in zebrafish.

The Effects of 7 Days of Feeding Pulse-Based Diets on Digestibility, Glycemic Response and Taurine Levels in Domestic Dogs.

Grain-based carbohydrate sources such as rice comprise 30-50% of commercial pet foods. Some pet foods however have removed the use of grains and have instead incorporated pulses, such as peas and lentils, resulting in grain-free diets. The hypothesis was dog diets with higher levels of dietary fiber will produce a low glycemic response due to decreased rates of digestion and lowered bioavailability of all macronutrients and increased fecal bile salt excretion. This in turn was hypothesized to produce lower plasma concentrations of cysteine, methionine and taurine after 7 days of feeding each test diet in dogs. Six diets were formulated at an inclusion level of 20% available carbohydrate, using white rice flour (grain) or whole pulse flours from smooth pea, fava bean, red lentil or 2 different wrinkled pea varieties (CDC 4,140-4 or Amigold) and fed to beagles in a randomized, cross-over, blinded design. After 7 days feeding each diet, fasting blood glucose was the lowest in the lentil (3.5 ± 0.1 mmol/L) and wrinkled pea (4,140-4; 3.6 ± 0.1 mmol/L) diet periods, while peak glucose levels was lowest after feeding the lentil diet (4.4 ± 0.1 mmol/L) compared to the rice diet. Total tract apparent digestibility of all macronutrients as well as taurine differed among diets yet plasma taurine was not outside normal range. Decreased macronutrient and amino acid digestibility was associated with increasing amylose and dietary fiber content but the specific causative agent could not be determined from this study. Surprisingly, digestibility decreases were not due to increased bile salt loss in the feces since increasing dietary fiber content led to decreased fecal bile salt levels. In conclusion, although pulse-based canine diets have beneficial low glycemic properties, after only 7 days, these pulse-based diets decrease macronutrient and amino acid digestibility. This is likely related at least in part to the lower animal protein content, but on a long-term basis could put domestic dogs at risk for low taurine and dilated cardiomyopathy.

The Effect of Fermentation of High- or Low-Tannin Fava Bean on Glucose Tolerance, Body Weight, Cardiovascular Function, and Blood Parameters in Dogs After 7 Days of Feeding: Comparison With Commercial Diets With Normal vs. High Protein.

Fava bean, which is available in high- and low-tannin varieties, is not an approved pet food ingredient and was not included in the "assumed to be safe" category based on its ability to cause favism and hemolytic anemia in susceptible humans. The effects of 7-day feeding of test canine diets containing moderate protein (~27%) were compared with two control commercial diets with normal (NP, grain-containing, ~25% protein) or high protein (HP, grain-free, ~41% protein). Fava bean diets were formulated either with or without Candida utilis fermentation processing to reduce antinutritional factors. Glucose tolerance, body weight, cardiovascular function, and blood parameters were investigated in beagles fed the NP or HP diets or a randomized, crossover, 2 × 2 Latin square design of the fava bean diets: unfermented high-tannin (UF-HT), fermented high-tannin (FM-HT), unfermented low-tannin (UF-LT), and fermented low-tannin (FM-LT). After 7 days, HP decreased red blood cells (RBC) (P < 0.05) compared with NP, while FM increased RBC compared with UF. HP increased blood bicarbonate, calcium, phosphorus, urea, cholesterol, and albumin:globulin ratio while decreasing bilirubin, liver enzymes, and total protein. Sodium:potassium ratio was increased in UF-HT, decreased in FM-HT, and intermediate in LT regardless of fermentation. Blood phosphorus was increased in HT. Blood amylase was increased in FM-HT and decreased in FM-LT, being intermediate in UF regardless of fava bean variety. Blood direct bilirubin was decreased in HT regardless of fermentation. Of note, left ventricular end-systolic volume and cardiac output were increased in NP compared with HP-fed dogs, but were normal and had no significant differences among the fava bean diets. As expected, plasma taurine, cystine, and cysteine levels were increased in HP- compared with NP-fed dogs. Plasma cysteine levels were increased in HT- compared with LT-fed dogs and in FM- compared with UF-fed dogs. Taken together, these results show that fava bean appears to be safe as a dog food ingredient at least in the short term, and its nutritional value appears improved by fermentation. Moreover, blood chemistry parameters and cardiovascular function were impacted by protein content which merits further investigation with longer term feeding trials.

Glycemic, insulinemic and methylglyoxal postprandial responses to starches alone or in whole diets in dogs versus cats: Relating the concept of glycemic index to metabolic responses and gene expression.

Species differences between domestic cats (Felis catus) and dogs (Canis familiaris) has led to differences in their ability to digest, absorb and metabolize carbohydrates through poorly characterized mechanisms. The current study aimed to first examine biopsied small intestine, pancreas, liver and skeletal muscle from laboratory beagles and domestic cats for mRNA expression of key enzymes involved in starch digestion (amylase), glucose transport (sodium-dependent SGLTs and -independent glucose transporters, GLUT) and glucose metabolism (hexokinase and glucokinase). Cats had lower mRNA expression of most genes examined in almost all tissues compared to dogs (p < 0.05). Next, postprandial glucose, insulin, methylglyoxal (a toxic glucose metabolite) and d-lactate (metabolite of methylglyoxal) after single feedings of different starch sources were tested in fasted dogs and cats. After feeding pure glucose, peak postprandial blood glucose and methylglyoxal were surprisingly similar between dogs and cats, except cats had a longer time to peak and a greater area under the curve consistent with lower glycolytic enzyme expression. After feeding starches or whole diets to dogs, postprandial glycemic response, glycemic index, insulin, methylglyoxal and d-lactate followed reported glycemic index trends in humans. In contrast, cats showed very low to negligible postprandial glycemic responses and low insulin after feeding different starch sources, but not whole diets, with no relationship to methylglyoxal or d-lactate. Thus, the concept of glycemic index appears valid in dogs, but not cats. Differences in amylase, glucose transporters, and glycolytic enzymes are consistent with species differences in starch and glucose handling between cats and dogs.

Interactive effects of cadmium and Benzo[a]pyrene in adult zebrafish (Danio rerio) during short-term aqueous co-exposure.

Environmental water quality guidelines often work under the assumption that the toxicity of environmental pollutants is identical when present in isolation or in a complex chemical mixture. Thus, there is a crucial gap in our knowledge regarding how these toxicants interact and alter the toxicological effects in aquatic organisms. The present study examined the effects of acute (72-hr) aqueous exposures of Cadmium (Cd), a highly toxic non-essential trace metal, and Benzo[a]Pyrene (B[a]P), a prototypical polycyclic aromatic hydrocarbon (PAH) in adult zebrafish. Following a range-finding series of individual single-toxicant exposures, a second series was carried out using select concentrations in binary mixture exposures (using 5.8 or 22 µg/L for Cd; 0.44 or 1.07 µg/L for B[a]P). Our results demonstrated that tissue accumulation of both toxicants increased significantly in the presence of the second toxicant relative to single-toxicant exposures. Cd-only and B[a]P-only single toxicant exposures caused a significant downregulation of cytochrome p4501a (CYP1A1) and metallothionein-2 (MT2) mRNA in the gills, respectively, however binary co-exposures using both toxicants resulted in strong up-regulation of CYP1A1 and MT2. Additionally, co-exposures caused a strong induction of SOD1 and CAT mRNA transcript levels in the gill. The observed increase in body burden and transcript modulation did not translate into additive or more-than-additive toxic effects (oxidative stress) in zebrafish.

Comparison of intestinal glucose flux and electrogenic current demonstrates two absorptive pathways in pig and one in Nile tilapia and rainbow trout.

The mucosal-to-serosal flux of 14C 3-O-methyl-d-glucose was compared against the electrogenic transport of d-glucose across ex vivo intestinal segments of Nile tilapia, rainbow trout, and pig in Ussing chambers. The difference in affinities (Km "fingerprints") between pig flux and electrogenic transport of glucose, and the absence of this difference in tilapia and trout, suggest two absorptive pathways in the pig and one in the fish species examined. More specifically, the total mucosal-to-serosal flux revealed a super high-affinity, high-capacity (sHa/Hc) total glucose transport system in tilapia; a super high-affinity, low-capacity (sHa/Lc) total glucose transport system in trout and a low-affinity, low-capacity (La/Lc) total glucose transport system in pig. Comparatively, electrogenic glucose absorption revealed similar Km in both fish species, with a super high-affinity, high capacity (sHa/Hc) system in tilapia; a super high-affinity/super low-capacity (sHa/sLc) system in trout; but a different Km fingerprint in the pig, with a high-affinity, low-capacity (Ha/Lc) system. This was supported by different responses to inhibitors of sodium-dependent glucose transporters (SGLTs) and glucose transporter type 2 (GLUT2) administered on the apical side between species. More specifically, tilapia flux was inhibited by SGLT inhibitors, but not the GLUT2 inhibitor, whereas trout lacked response to inhibitors. In contrast, the pig responded to inhibition by both SGLT and GLUT2 inhibitors with a higher expression of GLUT2. Altogether, it would appear that two pathways are working together in the pig, allowing it to have continued absorption at high glucose concentrations, whereas this is not present in both tilapia and trout.

Echocardiography and electrocardiography reveal differences in cardiac hemodynamics, electrical characteristics, and thermal sensitivity between northern pike, rainbow trout, and white sturgeon.

Doppler and B-mode ultrasonography and electrocardiography (ECG) were used to determine cardiac hemodynamics and electrical characteristics in 12°C-acclimated and metomidate-anesthetized northern pike, rainbow trout and white sturgeon (7-9 per species) at 12°C and 20°C, and at a comparable heart rate (fH , ~60 beats/min). Despite similar relative ventricle masses and cardiac output (Q), interspecific differences were observed at 12°C in fH , ventricular filling and ejection, stroke volume, the duration ECG intervals, and cardiac valve cross-sectional areas. Vis-a-fronte filling of the atrium due to ventricular contraction was observed in all species. However, biphasic ventricular filling (i.e., due to central venous pressure and then atrial contraction) was only observed in rainbow trout and white sturgeon. Changes in atrial and ventricular performance varied between the species as temperature increased from 12°C to 20°C. Rainbow trout had the highest thermal sensitivity for fH (Q10 = 3.73), which doubled Q, and the largest increase in transvalvular blood velocity during ventricular filling. Conversely, northern pike had the lowest Q10 for fH (1.58) and did not increase Q. At ~60 beats/min, the rainbow trout heart had the shortest period of electrical activity, which also resulted in the longest recovery period (TP interval) between successive beats. The QT interval at ~60 beats/min was also longer in the white sturgeon versus the other species. These results suggest that interspecific differences in fish cardiac hemodynamics may be related to cardiac morphology, the duration of electrical impulses through the heart, cardiac thermal sensitivity, and valve dimensions.

Intestinal electrogenic sodium-dependent glucose absorption in tilapia and trout reveal species differences in SLC5A-associated kinetic segmental segregation.

Electrogenic sodium-dependent glucose transport along the length of the intestine was compared between the omnivorous Nile tilapia ( Oreochromis niloticus) and the carnivorous rainbow trout ( Oncorhynchus mykiss) in Ussing chambers. In tilapia, a high-affinity, high-capacity kinetic system accounted for the transport throughout the proximal intestine, midintestine, and hindgut segments. Similar dapagliflozin and phloridzin dihydrate inhibition across all segments support this homogenous high-affinity, high-capacity system throughout the tilapia intestine. Genomic and gene expression analysis supported findings by identifying 10 of the known 12 SLC5A family members, with homogeneous expression throughout the segments with dominant expression of sodium-glucose cotransporter 1 (SGLT1; SLC5A1) and sodium-myoinositol cotransporter 2 (SMIT2; SLC5A11). In contrast, trout's electrogenic sodium-dependent glucose absorption was 20-35 times lower and segregated into three significantly different kinetic systems found in different anatomical segments: a high-affinity, low-capacity system in the pyloric ceca; a super-high-affinity, low-capacity system in the midgut; and a low-affinity, low-capacity system in the hindgut. Genomic and gene expression analysis found 5 of the known 12 SLC5A family members with dominant expression of SGLT1 ( SLC5A1), sodium-glucose cotransporter 2 (SGLT2; SLC5A2), and SMIT2 ( SLC5A11) in the pyloric ceca, and only SGLT1 ( SLC5A1) in the midgut, accounting for differences in kinetics between the two. The hindgut presented a low-affinity, low-capacity system partially attributed to a decrease in SGLT1 ( SLC5A1). Overall, the omnivorous tilapia had a higher electrogenic glucose absorption than the carnivorous trout, represented with different kinetic systems and a greater expression and number of SLC5A orthologs. Fish differ from mammals, having hindgut electrogenic glucose absorption and segment specific transport kinetics.

Cardiometabolic response of juvenile rainbow trout exposed to dietary selenomethionine.

Selenium (Se) is considered an essential trace element, involved in important physiological and metabolic functions for all vertebrate species. Fish require dietary concentrations of 0.1-0.5 µg Se/g dry mass (d.m.) to maintain normal physiological and selenoprotein function, however concentrations exceeding 3 µg/g d.m. have been shown to cause toxicity. As Se is reported to have a narrow margin between essentiality and toxicity, there is growing concern surrounding the adverse effects of elevated Se exposure caused by anthropogenic activities. Previous studies have reported that elevated dietary exposure of fish to selenomethionine (Se-Met) can cause significant cardiotoxicity and alter aerobic metabolic capacity, energy homeostasis and swimming performance. The goal of this study aims to further investigate mechanisms of sublethal Se-Met toxicity, particularly potential underlying cardiovascular and metabolic implications of chronic exposure to environmentally relevant concentrations of dietary Se-Met in juvenile rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were fed either control food (1.3 µg Se/g d.m.) or Se-Met spiked food (6.4, 15.8 or 47.8 µg Se/g d.m.) for 60 d at 3% body weight per day. Following exposure, ultrahigh resolution B-mode and Doppler ultrasound was used to characterize cardiac function in vivo. Chronic dietary exposure to Se-Met significantly increased stroke volume, cardiac output, and ejection fraction. Fish fed with Se-Met spiked food had elevated liver glycogen and triglyceride stores, suggesting impaired energy homeostasis. Exposure to Se-Met significantly decreased mRNA abundance of citrate synthase (CS) in liver and serpin peptidase inhibitor, clad H1 (SERPINH) in heart, and increased mRNA abundance of sarcoplasmic reticulum calcium ATPase (SERCA) and key cardiac remodelling enzyme matrix metalloproteinase 9 (MMP9) in heart. Taken together, these responses are consistent with a compensatory cardiac response to increased susceptibility to oxidative stress, namely a decrease in ventricular stiffness and improved cardiac function. These cardiac alterations in trout hearts were linked to metabolic disruption in other major metabolic tissues (liver and skeletal muscle), impaired glucose tolerance with increased levels of the toxic glucose metabolite, methylglyoxal, increased lipid peroxidation in skeletal muscle, development of cataracts and prolonged feeding behaviour, indicative of visual impairment. Therefore, although juvenile rainbow trout hearts were apparently able to functionally compensate for adverse metabolic and anti-oxidant changes after chronic dietary exposure Se-Met, complications associated with hyperglycemia in mammalian species were evident and would threaten survival of juvenile and adult fish.