Short Communication: The effect of in ovo creatine monohydrate on early pen use activity in chicks.

First-week survival and egg hatchability are lower in chicks from younger broiler breeder hen flocks. Creatine is a naturally occurring compound synthesised from the amino acid arginine or obtained from the diet and is important in the storage and transport of energy. Previous research found an improvement in the hatch rate but no posthatch performance improvements when fertile eggs from young breeder hens were injected with creatine monohydrate (CrM) on embryonic day 14. This pilot study aimed to further investigate the possibility of early posthatch improvements by examining the activity of chicks during the 1st week posthatch. Behaviours were broadly classified as active or inactive, the pen was split into three areas, and the amount of time spent in the heat lamp, feed hopper, or drinker line areas was recorded. Chicks given in ovo CrM spent less time in the heat lamp area over the whole 7 days compared to saline (t = 2.352, P = 0.021) and control groups (t = 3.336, P = 0.003) and more time in the feed hopper area during the first 4 days compared to the control group (t = 2.174, P = 0.033). This finding suggests that creatine may improve energy reserves in young chicks allowing them to spend more time away from the heat lamp.

Reduced fasting periods increase intestinal permeability in chickens.

Fasting of up to 24 hr has been shown to increase intestinal permeability (IP) in chickens. The aim of this study was to determine whether fasting duration of 4.5 and 9 hr increased IP and whether l-glutamine (a non-essential amino acid) supplementation before fasting provided some protection of barrier function as shown in other species. Ross 308 male broilers (n = 96) were fed either a control diet or the same diet supplemented with 1% glutamine from d0 to d38 post-hatch. On d37, the birds were assigned to single-bird metabolism cages and were fasted for either 0, 4.5, 9 or 19.5 hr. This study design was 2 × 4 factorial with two levels of glutamine and four levels of fasting. Birds in the 0-hr fasting group had free access to feed. All birds had ad libitum access to water. To measure IP on day 38, following their respective fasting periods, birds were administered two separate oral gavages of fluorescein isothiocyanate dextran (FITC-d) followed by lactulose, mannitol and rhamnose (LMR) sugars, 60 min apart. Whole blood was collected from the jugular vein 90 min post-LMR sugar gavage. FITC-d and L/M/R ratios were measured by spectrophotometry and high-performance ionic chromatography respectively. Lipopolysaccharide (LPS) endotoxins in plasma of the birds fed the control diet were also measured using chicken-specific LPS antibody ELISA. Serum FITC-d and plasma L/M and L/R ratios for 4.5, 9 and 19.5 hr were significantly (p < .05) higher compared to the non-fasting group. However, IP was not different in the glutamine-supplemented group (p > .05) compared to the control group. LPS concentrations measured by the ELISA were below the detectable range. We conclude that fasting periods of 4.5 and 9 hr increased IP compared to non-fasted birds and dietary glutamine supplementation did not ameliorate changes in IP.

Vitamin A and marbling attributes: Intramuscular fat hyperplasia effects in cattle.

Twenty Angus steers were fed a diet low in ß-carotene and vitamin A for 10months. Ten steers were supplemented with vitamin A weekly, while the other ten steers did not receive any additional vitamin A. The results demonstrated that the restriction of vitamin A intake increased intramuscular fat (IMF) by 46%. This was a function of the total number of marbling flecks increasing by 22% and the average marbling fleck size increasing by 14%. Vitamin A restriction resulted in marbling flecks that were less branched (22%) and slightly more round (4%) with an increased minor axis length (7%). However, restricting vitamin A did not affect the size of the intramuscular or subcutaneous adipocyte cells or the subcutaneous fat depth. The results suggest that vitamin A affects the amount of marbling and other attributes of the marbling flecks due to hyperplasia rather than hypertrophy. This may explain why vitamin A restriction specifically affects IMF rather than subcutaneous fat deposition.

Gene expression and morphological changes in the intestinal mucosa associated with increased permeability induced by short-term fasting in chickens.

Short-term fasting for 4.5 and 9 hr has been demonstrated to increase intestinal permeability (IP) in chickens. This study aimed to investigate the effects of 0, 4.5, 9 and 19.5 hr fasting on intestinal gene expression and villus-crypt architecture of enterocytes in jejunal and ileal samples. On day 38, Ross-308 male birds were fasted according to their group and then euthanised. Two separate intestinal sections (each 2 cm long, jejunum and ileum) were collected. One section was utilised for villus height and crypt depth measurements. The second section was snap-frozen in liquid nitrogen for quantitative polymerase chain reaction (qPCR) analysis of tight junction proteins (TJP) including claudin-1, claudin-3, occludin, zonula occludens (ZO-1, ZO-2), junctional adhesion molecules (JAM) and E-cadherin. Additionally genes involved in enterocyte protection including glucagon-like peptide (GLP-2), heat-shock protein (HSP-70), intestinal alkaline phosphatase (IAP), mammalian target of rapamycin (mTOR), toll-like receptors (TLR-4), mucin (MUC-2), cluster differentiation (CD-36) and fatty acid-binding protein (FABP-6) were also analysed. Normally distributed data were analysed using one-way analysis of variance ANOVA. Other data were analysed by non-parametric one-way ANOVA. Villus height and crypt depth were increased (p < .05) only in the ileum after fasting for 4.5 and 9 hr compared with non-fasting group. mRNA expression of claudin-3 was significantly reduced in the ileum of birds fasted for 9 and 19.5 hr, suggesting a role in IP modulation. However, all other TJP genes examined were not statistically different from control. Nevertheless, ileal FABP-6 of all fasted groups was significantly reduced, which could possibly be due to reduced bile acid production during fasting.

New biomarkers for increased intestinal permeability induced by dextran sodium sulphate and fasting in chickens.

Increased intestinal permeability (IP) can lead to compromised health in chickens. As there is limited literature on in vivo biomarkers to assess increased IP in chickens, the objective of this study was to identify a reliable biomarker of IP using DSS ingestion and fasting models. Male Ross chickens (n = 48) were reared until day 14 on the floor pen in an animal care facility, randomized into the following groups: control, DSS and fasting (each with n = 16), and then placed in metabolism cages. DSS was administered in drinking water at 0.75% from days 16 to 21, while controls and fasted groups received water. All birds had free access to feed and water except the birds in the fasting group that were denied feed for 19.5 h on day 20. On day 21, all chickens were given two separate oral gavages comprising fluorescein isothiocyanate dextran (FITC-d, 2.2 mg in 1 ml/bird) at time zero and lactulose, mannitol and rhamnose (LMR) sugars (0.25 g L, 0.05 g M and 0.05 g R in 2 ml/bird) at 60 min. Whole blood was collected from the brachial vein in a syringe 90 min post-LMR sugar gavage. Serum FITC-d and plasma LMR sugar concentrations were measured by spectrophotometry and high-performance ion chromatography respectively. Plasma concentrations of intestinal fatty acid binding protein, diamine oxidase, tight junction protein (TJP), d-lactate and faecal α-antitrypsin inhibitor concentration were also analysed by ELISA. FITC-d increased significantly (p < 0.05) after fasting compared with control. L/M and L/R ratios for fasting and L/M ratio for DSS increased compared with control chickens (p < 0.05). TJP in plasma was significantly increased due to fasting but not DSS treatment, compared with controls. Other tests did not indicate changes in IP (p > 0.05). We concluded that FITC-d and LMR sugar tests can be used in chickens to assess changes in IP.

Intestinal permeability induced by lipopolysaccharide and measured by lactulose, rhamnose and mannitol sugars in chickens.

Increased intestinal permeability (IP) can lead to compromised health. Limited in vivo IP research has been conducted in chickens. The objectives of the current study were to develop a model of increased IP utilizing lipopolysaccharide (LPS Escherichia coli O55:B5) and to evaluate IP changes using the lactulose, mannitol and rhamnose (LMR) sugar permeability test. In addition, fluorescein isothiocyanate dextran (FITC-d), d-lactate, zonula occludens (ZO-1) and diamine oxidase (DAO) permeability tests were employed. Male Ross chickens were reared until day 14 on the floor in an animal care facility and then transferred to individual cages in three separate experiments. In each of experiments 1 and 2, 36 chicks were randomly allocated to receive either saline (control) or LPS (n=18/group). Lactulose, mannitol and rhamnose sugar concentration in blood was measured at 0, 30, 60, 90, 120 and 180 min in experiment 1, at 60, 90 and 120 min in experiment 2 and at 90 min in experiment 3 (n=16/group). Lipopolysaccharide was injected intraperitoneally at doses of 0.5, 1 and 1 mg/kg BW in experiments 1, 2 and 3, respectively, on days 16, 18 and 20, whereas control received sterile saline. On day 21, only birds in experiments 1 and 2 were fasted for 19.5 h. Chicks were orally gavaged with the LMR sugars (0.25 gL, 0.05 gM, 0.05 gR/bird) followed by blood collection (from the brachial vein) as per time point for each experiment. Only in experiment 3, were birds given an additional oral gavage of FITC-d (2.2 mg/ml per bird) 60 min after the first gavage. Plasma d-lactate, ZO-1 and DAO concentrations were also determined by ELISA in experiment 3 (n=10). Administration of LPS did not affect IP as measured by the LMR sugar test compared with control. This was also confirmed by FITC-d and DAO levels in experiment 3 (P>0.05). The plasma levels of d-lactate were decreased (P<0.05). Plasma levels of ZO-1 were increased in the third experiment only and did not change in the first two experiments. Lipopolysaccharide at doses of 0.5 and 1 mg/kg did not increase IP in this model system. In conclusion, the LMR sugar can be detected in blood 90 min after the oral gavage. Further studies are needed for the applicability of LMR sugars tests.

Effect of oral polyamine supplementation pre-weaning on piglet growth and intestinal characteristics.

A high proportion of piglets fail to adapt to the changing composition of their diet at weaning, resulting in weight loss and increased susceptibility to pathogens. Polyamines are present in sow milk and promote neonatal maturation of the gut. We hypothesised that oral spermine and spermidine supplementation before weaning would increase piglet growth and promote gastrointestinal development at weaning. In Experiment One, one pair of liveweight (LW)-matched piglets per litter from first and third lactation sows received 2 ml of a 0 (Control) or 463 nmol/ml spermine solution at 14, 16, 18, 20 and 22 days of age (n=6 piglets/treatment per parity). Villus height and crypt depth in the duodenum and jejunum were measured at weaning (day 23 postpartum). In Experiment Two, piglets suckling 18 first and 18 third lactation sows were used. Within each litter, piglets received 2 ml of either water (Control), 463 nmol/ml spermine solution or 2013 nmol/ml spermidine solution at 14, 16, 18, 22 and 24 days of age (n=54 piglets/treatment per sow parity). Piglets were weighed individually at 14, 18, 24 (weaning) and 61 days of age. In Experiment One, oral spermine supplementation resulted in a 41% increase in villus height, a 21% decrease in crypt depth and 79% decrease in the villus height : crypt depth ratio compared with control piglets (P<0.01). In Experiment Two, spermine and spermidine-supplemented piglets suckling first lactation sows grew faster (P<0.05) between days 14 and 18 postpartum than control piglets: 0.230±0.011 and 0.227±0.012 v. 0.183±0.012 kg/day, respectively. Spermine supplementation tended (P<0.1) to increase piglet LW gain from weaning to day 37 post-weaning compared with control piglets (0.373±0.009 v. 0.341±0.010 kg/day). In conclusion, spermine supplementation increased villus height at weaning, and appears to have the potential to improve the pre- and post-weaning growth of conventionally weaned piglets.

Quantitative analyses of genes associated with mucin synthesis of broiler chickens with induced necrotic enteritis.

Clostridial infection of the intestine can result in necrotic enteritis (NE), compromising production and health of poultry. Mucins play a major role in protecting the intestinal epithelium from infection. The relative roles of different mucins in gut pathology following bacterial challenge are unclear. This study was designed to quantify the expression of mucin and mucin-related genes, using intestinal samples from an NE challenge trial where birds were fed diets with or without in-feed antimicrobials. A method for quantifying mucin gene expression was established using a suite of reference genes to normalize expression data. This method was then used to quantify the expression of 11 candidate genes involved in mucin, inflammatory cytokine, or growth factor biosynthesis (IL-18, KGF, TLR4, TFF2, TNF-α, MUC2, MUC4, MUC5ac, MUC5b, MUC13, and MUC16). The only genes that were differentially expressed in the intestine among treatment groups were MUC2, MUC13, and MUC5ac. Expression of MUC2 and MUC13 was depressed by co-challenge with Eimeria spp. and Clostridium perfringens. Antimicrobial treatment prevented an NE-induced decrease in MUC2 expression but did not affect MUC13. The expression of MUC5ac was elevated in birds challenged with Eimeria spp./C. perfringens compared with unchallenged controls and antimicrobial treatment. Changes to MUC gene expression in challenged birds is most likely a consequence of severe necrosis of the jejunal mucosa.

Effects of necrotic enteritis challenge on intestinal micro-architecture and mucin profile.

1. This study investigated the effect of Eimeria spp./Clostridium perfringens induced necrotic enteritis and traditional antibiotic preventatives on intestinal micro-architecture and mucin profile. 2. A total of 600 Cobb 500 broiler chickens were randomly assigned to the following three groups: (i) unchallenged, (ii) challenged, and (iii) zinc bacitracin/monensin (ZnB/monensin) (n = 25 chickens/pen, 8 pens/group). The challenged and ZnB/monensin chickens were individually inoculated with Eimeria acervulina, E. maxima and E. tenella and C. perfringens type A (EHE-NE18) at 9 and 15 d post-hatch respectively, to induce necrotic enteritis. 3. The challenge procedure significantly decreased villus height, increased villus width and increased crypt depth in the challenged compared to the unchallenged chickens. Zinc bacitracin and monensin maintained villus-crypt structure similar to that of the unchallenged chickens. 4. Mucin profile was not affected by Eimeria spp./C. perfringens challenge as demonstrated by periodic acid-Schiff and high iron diamine-alcian blue pH 2 x 5 staining. Zinc bacitracin and monensin decreased the number of intestinal mucin-containing goblet cells. 5. Lectin histochemistry showed a trend towards greater Arachis hypogea (PNA) reactivity in unchallenged chickens. 6. In summary, Eimeria spp./C. perfringens challenge disrupted intestinal micro-architecture; however, challenge did not appear to affect intestinal mucin profile. Traditional antibiotics, zinc bacitracin and monensin maintained micro-architecture.

Bacterial modulation of small intestinal goblet cells and mucin composition during early posthatch development of poultry.

Mucins possess potential binding sites for both commensal and pathogenic organisms and may perform a defensive role during establishment of the intestinal barrier. To observe the effects of bacteria on intestinal goblet cell mucin production during posthatch development, differences in the small intestine of conventionally reared (CR) and low bacterial load (LBL) broiler chicks were examined. Jejunal and ileal goblet cells were stained with either periodic acid-Schiff stain or high iron diaminealcian blue pH 2.5 to discriminate among neutral, sulfated, and sialylated acidic mucins. Total goblet cell numbers and morphology of goblet cells containing neutral and acidic mucins did not differ significantly between CR and LBL birds. However, significant differences in acidic mucin composition from primarily sulfated to an increase in sialylated sugars at d 4 posthatch were observed in CR chicks, with greater numbers of jejunal and ileal goblet cells displaying this mucin type (CR, 0.5 +/- 0.1 x 10(3) cells/mm(2); LBL, 0.04 +/- 0.02 x10(3) cells/mm(2)). This change in mucin profile in response to bacterial colonization suggests a potential role as a protective mechanism against pathogenic invasion of the intestinal mucosa during early development.