Freely Fed Research Articles

Comparing dietary intake of free-feeding to feeding in restrained imaging chamber environments using seven days post fertilization larvae.

Heart disease and diabetes are both in the top ten for leading causes of death in the United States with both being linked to obesity. Studying human metabolism can prove difficult due to ethical concerns with human subjects but zebrafish larvae (Danio rerio) are optically transparent. Zebrafish larvae at seven days post fertilization also have fully formed digestive tracts and are actively seeking new food due to exhausting their supply of egg yolk. Free feeding environments are typically the standard when introducing new food to the larvae but lack versatility in their imaging. The Walters lab microfluidic chip can image larvae before, during, and after their introduction to the diet. To prove the two types of feeding environments were compared by using fluorescent digestive markers mixed into high fat diets. The free fed larvae were put into a well filled with diet and allowed to feed for three hours while sat on a orbital shaker inside of a 28 degrees Celsius incubator. After three hours, these larvae were rinsed out and imaged. The microfluidic chip larvae were mounted into the chip and fed the diet while inside and under the same Zeiss Discovery V8 microscope the freely fed larvae were imaged under. Future predictions for this data were that the larvae would consume slightly less diet inside of the microfluidic chip than the larvae in free feed. Results for this are still ongoing but seem to lead towards supporting the hypothesis.

EFFECT OF FEED RESTRICTION REGIMEN ON PRODUCTIVE PERFORMANCE AND ECONOMICAL EFFICIENCY OF WEANING NZW RABBITS

This experiment aimed to study the effects of feed restriction (FR) on weaning New Zealand White Rabbit's growth performance. Seventy-five unsexed rabbits (5 weeks of age) were divided to five experimental treatments with five equal replications. Feed restriction program was used in the first two weeks (5-7 weeks old) after weaning as follows; the first treatment (control) was free fed the experimental diet. While other treatments (2 to 5) were fed restricted diets at levels of 10, 20, 30 and 40%, respectively from the free feeding of the control. All rabbits were free fed grower diet from 8 to 12 weeks of age. During this trial rabbit growth performance were evaluated, carcass traits, mortality rate and economic efficiency. Some blood parameters in rabbit serum were also determined. The obtained results illustrated that final weight of rabbit was recovered and feed conversion ratio was improved by FR. FR program did not affect carcass traits. Serum blood parameters were not affected by feed restriction. It could be concluded that feed restriction at levels of 30 or 40% during the first two weeks after weaning had beneficial effects on rabbit growth performance, improved feed conversion and economic efficiency and decreasing mortality rate.

Stress-Induced Reduction of Dorsal Striatal D2 Dopamine Receptors Prevents Retention of a Newly Acquired Adaptive Coping Strategy.

The inability to learn an adaptive coping strategy in a novel stressful condition leads to dysfunctional stress coping, a marker of mental disturbances. This study tested the involvement of dorsal striatal dopamine receptors in the dysfunctional coping with the Forced Swim test fostered by a previous experience of reduced food availability. Adult male mice were submitted to a temporary (12 days) reduction of food availability [food-restricted (FR)] or continuously free-fed (FF). Different groups of FF and FR mice were used to evaluate: (1) dorsal striatal mRNA levels of the two isoforms of the dopamine D2 receptor (D2S, D2L). (2) Forced Swim-induced c-fos expression in the dorsal striatum; (3) acquisition and 24 h retention of passive coping with Forced Swim. Additional groups of FF mice were tested for 24 h retention of passive coping acquired during a first experience with Forced Swim immediately followed by intra-striatal infusion of vehicle or two doses of the dopamine D2/D3 receptors antagonist sulpiride or the D1/D5 receptors antagonist SCH23390. Previous restricted feeding selectively reduced mRNA levels of both D2 isoforms and abolished Forced Swim-induced c-fos expression in the left Dorsolateral Striatum and selectively prevented 24 h retention of the coping strategy acquired in a first experience of Forced Swim. Finally, temporary blockade of left Dorsolateral Striatum D2/D3 receptors immediately following the first Forced Swim experience selectively reproduced the behavioral effect of restricted feeding in FF mice. In conclusion, the present results demonstrate that mice previously exposed to a temporary reduction of food availability show low striatal D2 receptors, a known marker of addiction-associated aberrant neuroplasticity, as well as liability to relapse into maladaptive stress coping strategies. Moreover, they offer strong support to a causal relationship between reduction of D2 receptors in the left Dorsolateral Striatum and impaired consolidation of newly acquired adaptive coping.

Ghrelin, Ghrelin O-Acyltransferase, and Carbohydrate Metabolism During Pregnancy in Calorie-Restricted Mice.

Acylation of ghrelin is mediated by ghrelin O-acyltansferase (GOAT). Exogenous acylated ghrelin (AG) stimulates growth hormone (GH) and food intake. In non-pregnant (NP) animals, the GOAT-ghrelin-GH axis prevents hypoglycemia caused by caloric restriction (CR). In humans, maternal malnutrition challenges glucose metabolism, which is a key determinant of fetal health. To clarify the role of AG and GH, we compared effects of CR on the GOAT-ghrelin-GH axis in pregnant (P) and NP mice. C57BL/6 wild type (WT) and GOAT knock-out (KO) P and NP mice were freely fed (FF) or subjected to 50% CR for one week. CR was started in P mice on Day 10.5 after conception. We measured body composition, blood glucose, plasma ghrelin and GH, stomach, hypothalamus and pituitary GOAT and ghrelin expression, and liver glycogen content and Pck1 expression. GOAT and AG were undetectable in KO. In NP mice, CR did not affect blood glucose (-1.3 mmol/l, p>0.05) in WT but was lowered (-1.8 mmol/l, p<0.0001) in KO. GH and Pck1 mRNA expression increased in WT but not in KO. In P mice, CR markedly lowered glucose (-2.7 mmol/l; p<0.0001) in WT and caused fatal hypoglycemia in KO, despite similarly elevated GH in WT and KO mice. KO animals are more prone to hypoglycemia than WT. GH, which is high in P animals, does not prevent hypoglycemia caused by CR during pregnancy. Our data suggest a specific role of AG in the regulation of gluconeogenesis to maintain euglycemia during pregnancy when energy availability is limited.

Resveratrol Prevents Hypertension and Cardiac Hypertrophy in Hypertensive Rodents

s / Can J Diabetes 37 (2013) S13eS84 S23 and unacylated ghrelin (UAG). Acylation is mediated by ghrelin Oacyltransferase (GOAT). Maintenance of euglycemia during calorie restriction (CR) in pregnancy is key for optimal outcome. Hypothesis: Upregulation of GOAT/ghrelin, increased AG and GH concentrations are part of the hormonal response to hypoglycemia during CR. Methods: Wild-type (WT) and GOAT-KO (KO), pregnant (P) and non-pregnant (NP) mice were freely fed (FF) or submitted to 50% CR for 7 days. In P mice, CR occurred between days 11 to 18 (1⁄4 sacrifice) of pregnancy. Blood glucose (BG), plasma AG, UAG and GH were measured by EIA and GOAT/ghrelin expression by RT-PCR. Results (Table 1): 1. In WT mice, CR did not affect BG in NP mice but caused lower BG in P mice. This was associated with higher AG and GH (from placenta) concentrations and lower stomach GOAT and ghrelin mRNA. 2. In KO mice, where plasma AG is by definition absent, CR caused severe hypoglycemia (causing systematic P termination) in P mice and lower BG in NP mice. The latter was associated with higher UAG and lower stomach ghrelin mRNA. Conclusion: 1. GOAT-KO mice are more prone to hypoglycemia, suggesting a role for AG that is independent from GH secretion; 2. The AG increase during CR not due to increased stomach production but likely decreased degradation. 3. AG is not necessary for GH production (by the placenta) in P mice.

Plasma cortisol and prolactin secretion rhythms in cattle under varying external environments and management techniques

The secretion rhythms of plasma cortisol (CORT) and prolactin (PRL), hormones related to stress responsiveness and biological rhythm and controlled by light and temperature, were investigated under varying external environments and different management techniques. Serial blood samples were collected from female cattle reared in free-stall and freely fed (FF) conditions (n = 4) or in tie-stall and restricted feeding (RF) conditions (hay and concentrate twice daily, n = 4). Plasma CORT and PRL concentrations, eating behavior, and environmental parameters were analyzed. Cyclic patterns for each parameter were examined using spectral analysis, and correlations between CORT, PRL and other parameters were investigated using cross-spectral analysis. Under FF conditions, CORT secretion was not related to the lighting intensity and eating behavior. However, under RF conditions, the CORT secretion rhythm showed a distinct correlation with lighting intensity and eating behavior. Under FF conditions, the PRL secretion rhythm was similar in all seasons. However, under RF conditions, the PRL rhythm oscillated with high frequency in summer and low frequency in winter, indicating a seasonal change in rhythm. The present study demonstrates that hormone secretion rhythms change under different environments and management techniques.

Food-entrained rhythmic expression of apolipoprotein E expression in the hypothalamus of rats

Apolipoprotein E (apoE) is a protein mainly synthesized in the liver and brain. To further understand the role of brain apoE in the management of daily food intake, we have examined the circadian pattern of hypothalamic apoE gene and protein expression in freely-fed (FF) and food-restricted (RF, food provided 4 h daily between 1000 h and 1400 h) rats sacrificed at 3-h intervals throughout the light–dark cycle. In FF rats, hypothalamic apoE mRNA and protein levels fluctuated with peaks occuring during the dark phase and the nadirs occuring during the light phase. This pattern was altered in RF rats, which had a marked increase in hypothalamic apoE mRNA and protein levels during the 4-h feeding period in the light phase. Although corticosterone (CORT) levels temporally coincided with the increasing phase of apoE in the hypothalamus in both FF and RF rats, depletion of CORT by adrenalectomy (ADX) did not significantly influence the hypothalamic apoE levels during either period, implying that the circadian pattern of hypothalamic apoE is regulated by factors other than circulating CORT. The finding that hypothalamic apoE and food intake are positively associated during the normal circadian cycle as well as in the period of restricted feeding suggests that hypothalamic apoE is food-entrained and likely involved in the physiological regulation of daily food intake.

NOS isoenzyme content in brain nuclei as related to food intake in experimental cancer cachexia

Evidence implies that nitric oxide (NO) in the central nervous systems mediates anorexia in tumor-bearing hosts. We have therefore evaluated, by immunohistochemical image analyses, net alterations of nitric oxide synthases (nNOS, eNOS, iNOS) in brain nuclei [paraventricular hypothalamic nucleus (PVN), medial habenular nucleus (MHB), lateral habenular nucleus (LHB), paraventricular thalamic nucleus (PV), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS)] of tumor-bearing mice (TB) with prostanoid-related anorexia. Pair-fed (PF) and freely fed (FF) non-tumor-bearing mice were used as controls. c-fos was analyzed as indicator of neuronal activation. nNOS was significantly increased in VMH and PVN from TB mice, while eNOS was significantly increased in LHB and LHA. iNOS was significantly increased in LHA and PVN nuclei, but decreased in MHB, LHB and VMH from tumor-bearers. However, several of these alterations were similarly observed in brain nuclei from pair-fed controls. Provision of unspecific NOS-antagonists to TB mice increased nNOS, eNOS and iNOS in several brain nuclei (PVN, LHA, VMH), but left tumor-induced anorexia unchanged. c-fos was significantly increased in all brain nuclei in PF mice except for NTS, LHA and PVN compared to controls, while tumor-bearing mice had increased c-fos in LHA and PVN only compared to controls. Our results demonstrate a complex picture of NOS expression in brain areas of relevance for appetite in tumor-bearing hosts, where most changes seemed to be secondary to stress during negative energy balance. By contrast, NOS content in PVN and LHA nuclei remains candidate behind anorexia in tumor disease. However, nitric oxide does not seem to be a primary mediator behind tumor-induced anorexia. NO may rather secondarily support energy intake in conditions with negative energy balance.

Diazoxide effects on hypothalamic and extra-hypothalamic NPY content in Zucker rats

To determine if the anorectic effects of the insulin antagonist diazoxide (DZ) are mediated by reduced central neuropeptide Y (NPY), female Zucker rats, given DZ (150 mg/kg/day) or placebo for about four weeks, were sacrificed following overnight fasting or free feeding. Several hypothalamic and extra-hypothalamic nuclei were extracted for NPY content. DZ reduced weight gain in obese rats and lowered glucose of lean and obese rats without affecting insulin. Contrary to the hypothesis, DZ increased NPY in hypothalamic nuclei of free fed lean and obese rats. DZ elevated hypothalamic NPY levels in fasted obese rats and had more diverse effects in extra-hypothalamic nuclei of lean rats.

Diet restriction modulates lung response and survivability of rats exposed to ozone

Ozone (O 3) is a powerful oxidant component of photochemical smog polluting the air of urban cities. Exposure to low-level O 3 causes lung injury and increased morbidity of the sensitive segment of population, and exposure to high levels can be lethal to experimental animals. Injury from O 3 exposure is generally associated with free radical formation and oxidative stress. Because diet restriction is proposed to enhance antioxidant status, we examined whether it would influence the response to inhaled O 3. Twenty-four Sprague–Dawley rats, 1 month old, weighing 150 g, were divided into two dietary regimens (12 rats/regimen); one was freely-fed (FF), and the second was diet-restricted (DR) to 20% the average daily intake of the FF. After 60 days of dietary conditioning, the body weight of DR rats was reduced to 50% that of FF rats. Then, in one experiment, two groups (six rats/group), one FF and the other DR, were exposed to 0.8±0.1 p.p.m. (1570±196 μg/m 3) O 3, continuously for 3 days. Another two similar groups of rats were exposed to filtered room air and served as matched controls. After exposure, all rats were euthanized and the lungs analyzed for biochemical markers of oxidative stress. In a second experiment, 24 rats were divided into two groups (12 rats/group), one FF and the other DR, then exposed to high-level O 3 for 8 h (4 p.p.m., 7848±981 μg/m 3) and the mortality noted during exposure and for 16 h post-exposure. Following low-level O 3, inhalation, greater alterations were observed in FF rats compared with DR rats. With high-level O 3 exposure, DR rats exhibited a much greater survivability compared with FF rats (90% versus 8%, respectively). These observations suggest that diet restriction leading to significant reduction of body weight is beneficial, and may play a role in the resistance to the adverse effects of O 3.

Administration of opiate receptor antagonist inhibits mucosal atrophy of the gut in fasting rats.

The objective of this study was to determine whether the opiate mu receptor antagonist naloxone would prevent atrophy of the gut in 24-h-fasted rats. Male Sprague-Dawley rats (n = 76, body weight 200-225 g) were catheterized in the jugular vein on Day 0. The rats were fed standard rat chow for 4 days. On Day 4, the diet was changed to the standard liquid diet, and the rats were allowed free access to the liquid diet. On Day 7, the rats were randomized into five groups: (1) free fed, (2) free fed plus naloxone, (3) pair fed, (4) fasting, (5) free fed plus morphine, (6) fasting plus naloxone. Either naloxone (0.16 mg/kg/h) or morphine (0. 21 mg/kg/h) was continuously infused via venous catheter for 24 h. On Day 8, 24 h after fasting or free feeding, the animals were sacrificed. Twenty-four hours of fasting caused atrophy of the jejunum and elevated morphine levels in the brain (free fed, 931. 3 +/- 122.3 fmol/g, vs fasting, 1419.0 +/- 150.0, P < 0.05). Morphine infusion reduced villus height, mucosal weight, and protein content in jejunum as compared with the free fed rats receiving saline. Administration of naloxone caused an increase in villus height (fasting, 587.0 +/- 25.8 microm, vs fasting plus naloxone, 670.0 +/- 17.4, P < 0.05), mucosal weight (fasting, 17.4 +/- 1.8 mg/cm, vs fasting plus naloxone, 22.6 +/- 1.9, P < 0.05), and protein content (fasting, 13.5 +/- 0.7 microg/cm, vs fasting plus naloxone, 16.7 +/- 0.6, P < 0.05) in jejunum. Mucosal atrophy of the jejunum is caused by endogenous opioid in fasting rats.

Effect of diazoxide on brain capillary insulin receptor binding and food intake in hyperphagic obese Zucker rats.

Insulin is believed to act as a central adiposity signal by binding to hypothalamic and other brain insulin receptors. Entry of circulating insulin into the brain is accomplished by a saturable receptor-mediated transendothelial transport system and is believed to be impaired in hyperinsulinemic, insulin-resistant, and hyperphagic obese Zucker rats. Theoretically, if hyperinsulinemia is decreased simultaneously while brain capillary insulin binding is increased, uptake of insulin into the brain can be enhanced leading to reduced food intake. To test this hypothesis, we administered diazoxide (DZ, 150 mg/kg/day), an inhibitor of glucose-mediated insulin secretion, or vehicle (control) to 7-week-old female obese and lean Zucker rats for 4 weeks (n = 24-28/subgroup-strain). Animals were assigned to either fasted (FD) or free-fed (FF) protocol for determination of plasma and cerebrospinal fluid (CSF) insulin and brain capillary insulin binding at the end of 4 weeks. DZ obese consumed fewer calories (P<0.01) and gained less weight than control obese (P<0.01), whereas DZ lean had similar amounts of caloric intake and weight gain compared with lean controls. DZ obese had lower fasting and random plasma glucose than control obese (P<0.05). FD and FF DZ-treated obese and lean rats had lower plasma insulin than their respective obese (P<0.01) and lean (P<0.01) controls. FD and FF DZ-treated obese rats demonstrated higher CSF insulin (P<0.05) and CSF/ plasma insulin ratio (P<0.01) than their controls, while only FF DZ lean animals showed higher CSF/plasma insulin ratio (P<0.01) than their controls (P<0.05). This was associated with enhanced brain capillary insulin binding in FD and FF DZ-treated obese (P<0.01) and lean (P<0.05) animals compared with their respective controls. It was concluded that DZ treatment in obese Zucker rats caused a decrease in insulin secretion and partially reversed impaired insulin binding to brain capillaries, leading to enhanced brain insulin uptake, and resulted in reduced food intake and weight gain observed in these animals.

Effects of Prenatal Cocaine Exposure on Perinatal Morbidity and Postnatal Growth in the Rabbit

Prenatal cocaine (CC) exposure may result in increased fetal loss, growth retardation, altered neurodevelopment, and sudden infant death syndrome (SIDS). We sought to establish an animal model for prenatal cocaine exposure which (1) would allow us to distinguish the direct effects from the indirect and nutritional effects of the drug, and (2) might be used to address questions of cocaine's toxicity, specifically to the developing respiratory control system. The study design included 38 New Zealand White rabbit does among CC, pair-fed (PF), and free-fed (FF) groups. Miniosmotic pumps were implanted in each doe on day 10 of timed gestation providing continuous subcutaneous administration of either 30 mg/kg/day of cocaine HCl in H2O (CC) or sterile H2O alone (PF and FF). Mean (SEM) plasma cocaine concentration was 1.71 +/- 0.21 mumol/l (519.4 +/- 64.4 ng/ml). Pregnancy outcome compared for incidence of stillbirth, maternal death, spontaneous abortion, and gross malformation among 211 pups was significant only for increased stillbirths among CC pups (18%, p less than 0.04) as compared to PF (6%) and FF pups (7%). External and renal malformation and postnatal weight, crown-rump length, and snout-occiput head circumference for pups aged 4 and 5 days of age did not differ among groups. The direct effects of prenatal cocaine evaluated in our model do not reproduce the altered perinatal outcome observed among humans. However, our results do not determine if physiologic function has been altered. Investigation of the physiologic and pathologic abnormalities that are relevant to this human condition, specifically to the developing respiratory control system, should add clarity to the mechanism of action of cocaine during pregnancy.

Increased degradation of albumin in cancer is not due to conformational or chemical modifications in the albumin molecule

This study has evaluated whether increased albumin degradation in a tumor-bearing host is dependent on previously recognized chemical and environmental modifications in the albumin molecule as observed by others and ourselves. For the purpose, adult sarcoma-bearing mice with increased albumin degradation and electrophoretic heterogeneity were used and compared to freely fed (FF) or food restricted control animals. Food restricted control animals such as pair-fed (PF) and pair-weighed (PW) served to match the anorexia and malnutrition observed in tumor-bearing animals. The serum albumin concentration was decreased ( P < 0.05) in tumor-bearing animals (33 ± 5 g/liter) compared to pair-fed (40 ± 3), pair-weighed (41 ± 4), and freely fed control mice (43 ± 3). Isoelectric focusing of plasma between pH 3 and 10 and pH 4 and 6.5 confirmed a different isoelectric point for albumin in tumor-bearing animals compared to control animals. Albumin degradation was 33% higher in tumor-bearing mice compared to freely fed controls ( P < 0.01). Tumor-bearing animals had also significantly increased turnover of albumin compared to all control animals (0.13 ± 0.022 mg/hr/g animal vs 0.05 ± 0.008 mg/hr/g in PW; 0.08 ± 0.009 in PF, and 0.09 ± 0.007 in FF). The acidic fraction of albumin had a more rapid fractional turnover than the more basic components in both tumor-bearing and control animals. However, both the anodal and the cathodal albumin in tumor-bearing mice had a higher turnover compared with corresponding fractions of albumin from control animals. Endogenously or exogenously labeled albumin from tumor-bearing animals did not show any increased disappearance when transfused back into normal animals, while labeled albumin from normal and tumor-bearing animals showed a significantly higher disappearance when transfused into tumor-bearing animals. The results demonstrate that elevated albumin degradation and turnover in cancer disease is dependent on host factors that are not associated with the chemical heterogeneity of the albumin molecule itself generally seen in a tumor-bearing host.