G. B. Tactacan

Performance and welfare of laying hens in conventional and enriched cages

Concerns regarding the welfare of laying hens raised in battery cages have led to the development of enriched cages that allow hens to perform natural behaviors including nesting, roosting, and scratching. This study was conducted to compare indices of production and welfare in birds housed in 2 different caging systems. Shaver White hens were housed from 21 to 61 wk in either conventional battery cages (n = 500; 10 cages; 5 hens/cage; floor space = 561.9 cm(2)/hen) or enriched cages (n = 480; 2 cages; 24 hens/cage; floor space = 642.6 cm(2)/hen) and were replicated 10 times. Enriched cages provided hens with a curtained nesting area, scratch pad, and perches. Production parameters and egg quality measures were recorded throughout the experiment. Plumage condition was evaluated at 37 and 61 wk. Bone quality traits and immunological response parameters were measured at 61 wk, and 59 and 61 wk, respectively. Hen-day egg production, feed consumption, egg weight, and percentage of cumulative mortality of laying hens were not affected by the cage designs. Specific gravity and the percentage of cracked and soft-shelled eggs were also similar between the 2 housing systems. The incidence of dirty eggs was, however, significantly higher (P < 0.0001) in enriched cages than in conventional cages. Feather scores were similar between birds except for the wing region, which was higher (P < 0.05) for hens housed in conventional cages. Bone quality measures tended to be higher for hens housed in enriched cages compared with hens in conventional cages. However, the increase was significant only for bone mineral density. Immunological response parameters did not reveal statistically significant differences. Overall, laying performance, exterior egg quality measures, plumage condition, and immunological response parameters appear to be similar for hens housed in the 2 cage systems tested. Enrichment of laying hen cages resulted in better bone quality, which could have resulted from increased activity.

Molecular cloning and tissue distribution of reduced folate carrier and effect of dietary folate supplementation on the expression of reduced folate carrier in laying hens

The reduced folate carrier (RFC) has been postulated to be a major entity for folate transport activity in humans and other mammals. However, there are limited reports of the importance of RFC in an avian system. In the current study, therefore, the molecular cloning and tissue distribution of RFC, as well as the effect of dietary folate supplementation on the expression of this transporter, were investigated in the chicken. Shaver White laying hens (n=8 per diet) received 3 wheat-based diets containing the following: 1) no supplemental folate, 2) folic acid (10.00 mg/kg), or 3) 5-methyltetrahydrofolate (11.30 mg/kg) for 21 d. The mRNA expression levels were analyzed by quantitative real-time PCR. The results showed that the cloned partial RFC cDNA containing the full coding region from duodenum was 99% homologous to the reference gene available in GenBank. A broad expression profile of RFC transcripts was observed, with RFC mRNA detected in the brain, liver, kidney, spleen, lung, intestine, ovary, and testis, as well as other tissues. Real-time PCR analysis revealed that no significant differences (P>0.05) due to diet were found in the mRNA levels of RFC in the duodenum and cecum. However, compared with the basal diet, jejunal mRNA levels of RFC were decreased (P<0.05) in hens fed with the 5-methyltetrahydrofolate diet, but the reduction did not reach significance (P=0.077) in the hens fed the folic acid diet. Overall, the current study demonstrated that the RFC cDNA containing the full coding region was successfully cloned from the duodenum of laying hens. The wide tissue distribution of RFC transcripts is suggestive of an important role of RFC in the process of folate transport in the chicken. Moreover, dietary folate supplementation could downregulate the jejunal mRNA expression of RFC. Such findings will lay the foundation of future work involving the RFC in avian systems, including laying hens.

Performance, serum biochemical responses, and gene expression of intestinal folate transporters of young and older laying hens in response to dietary folic acid supplementation and challenge with Escherichia coli lipopolysaccharide

The present study was conducted to investigate the effects of dietary folic acid (FA) supplementation on performance, serum biochemical indices, and mRNA abundance of intestinal folate transporters in young and older laying hens after acute lipopolysaccharide (LPS) challenge. Two experiments were conducted separately involving 48 Shaver White young laying hens (24 wk of age) in experiment 1 and 48 Shaver White older laying hens (58 wk of age) in experiment 2. Birds were fed 2 diets in a complete randomized design. The diets were wheat-soybean meal based, with or without supplemental 4 mg of FA/kg of diet. Birds were fed for 8 wk, during which time feed consumption and egg production were monitored. At the end of each feeding experiment, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either Escherichia coli LPS or sterile saline. Four hours after injection, blood and intestinal samples were collected for further analysis. Compared with the control, dietary FA supplementation increased egg weight and egg mass and decreased serum glucose levels in the young laying hens, and reduced serum uric acid in the older laying hens (P < 0.05). Relative to saline injection, plasma homocysteine, serum calcium, and phosphorus levels were found to be lower in both young and older laying hens after LPS challenge (P < 0.05). Other serum biochemical variables and the mRNA expression of 2 folate transport genes in the small and large intestine were differentially affected by LPS challenge, and some of those responses varied with the age of the birds. Additionally, interactions between diet and LPS challenge were specifically found in the older laying hens. In summary, in addition to improving production performance, there were effects of dietary FA supplementation and its interaction with LPS challenge on biochemical constituents, and age played a role in the development of responses to diet and bacterial LPS infections.

Characterization of folate-dependent enzymes and indices of folate status in laying hens supplemented with folic acid or 5-methyltetrahydrofolate

The conversion of folic acid (FA) to the biologically active 5-methyltetrahydrofolate (5-MTHF) is necessary for the deposition of folate in the egg. A study was conducted to compare egg folate concentrations, indices of folate status, and activities of folate-dependent enzymes in response to equimolar intake of either FA or 5-MTHF in laying hens. Forty-eight laying hens, 24 wk of age, from 2 different strains (Shaver White and Shaver Brown) were randomly assigned to receive 1 of 3 (n = 8 per strain) dietary treatments: 1) basal diet with no supplemental folate, 2) basal diet + 10 mg/kg of FA, or 3) basal diet + 11.3 mg/kg of 5-MTHF for 3 wk. A completely randomized design with 3 dietary treatments and 2 laying hen strains in a 3 x 2 factorial arrangement was used. Data were subjected to ANOVA, using the PROC GLM procedure of SAS. Plasma homocysteine, serum, and egg folate concentrations; hepatic serine hydroxymethyltransferase; and methionine synthase activity were affected by dietary folate supplementation but not by its form (FA and 5-MTHF). Relative to control hens, plasma homocysteine was decreased (P < 0.05) by 14.2%, whereas serum and egg folate were increased (P < 0.05) by 78.3 and 61.8%, respectively, in hens consuming either folate compound. Hepatic serine hydroxymethyltransferase and methionine synthase activity were increased and decreased (P < 0.05), respectively, in folate-fed birds compared with control-fed birds. Hepatic dihydrofolate reductase was influenced by both the addition and form of dietary folate, being higher (P < 0.05) in FA-fed birds than in 5-MTHF and control-fed birds. Feed efficiency was improved (P < 0.05) in 5-MTHF-fed birds relative to FA-fed birds. Strain of hen influenced serum folate and plasma homocysteine concentrations but not other indices of folate metabolism. Overall, FA and 5-MTHF have equivalent effects in enhancing egg folate concentrations and improving folate status in laying hens. Also, supplementation and form of folate may modulate the activity of folate-dependent enzymes.

Response of older laying hens to an Escherichia coli lipopolysaccharide challenge when fed diets with or without supplemental folic acid

Folic acid plays a key role in nucleic acids and protein synthesis, and has been associated with anti-inflammatory effects in LPS-induced infections. To this end, a study was conducted to investigate the effects of dietary folic acid (FA) supplementation in older laying hens (58 to 66 wk of age) challenged with Escherichia coli lipopolysaccharide (LPS). A total of 24 Shaver White laying hens at 58 wk were fed 2 diets. The diets were wheat-soybean-based, with either 0 or 4 mg of supplemental FA per kg of diet. After 8 wk of feeding and at 66 wk, the hens were injected intravenously with 8 mg of LPS or saline per kg of BW. Four hours after injection, blood was collected and hens were euthanized to obtain spleen and cecal tonsils. The T cell subsets in the blood and the spleen (CD4+ and CD8+), total IgG, and biochemical constituents (total protein, albumin, globulin, and fibrinogen) were not influenced (P > 0.05) by dietary FA supplementation. However, LPS injection decreased (P < 0.05) biochemical constituents, CD4+, and CD8+ cells in the blood, whereas CD4+:CD8+ ratio and total IgG increased (P < 0.05), and fibrinogen was not influenced. Gene expression in the spleen and cecal tonsils was not influenced by dietary FA supplementation except a diet × challenge interaction for interleukin (IL)-8 in the spleen; IL-8 decreased in FA-fed hens that were treated with LPS. Also, FA supplementation decreased the expression of IL-8 in cecal tonsils. Relative to saline-injected hens, expression of IL-1β, interferon-γ, and IL-10 increased in the LPS-injected hens in the spleen and cecal tonsils, IL-8 increased in LPS-injected hens only in the cecal tonsils, whereas Toll-like receptor 4, IL-4, IL-17, and IL-18 increased in the LPS-injected hens only in the spleen; however, LPS decreased expression of IL-13 in the cecal tonsils. In conclusion, FA did not affect inflammatory responses in older laying hens; more studies are required to investigate possible protective effects of FA in laying hens.

Proton-coupled folate transporter (PCFT): molecular cloning, tissue expression patterns and the effects of dietary folate supplementation on mRNA expression in laying hens.

1. The aim was to investigate the molecular characterisation and effects of dietary folate supplementation on mRNA concentrations of the proton-coupled folate transporter (PCFT) in chicken. 2. Twenty-four (n = 8/treatment) laying hens were allocated at random to one of three dietary treatments: a basal diet without supplemental folate, or the same diet with either 10·00 mg/kg folic acid or 11·30 (equimolar) mg/kg 5-methyltetrahydrofolate (5-MTHF) for 21 d. The PCFT cDNA was cloned using the TA cloning system, and mRNA concentrations were determined by real-time polymerase chain reaction (PCR). 3. The PCFT cDNA (1643 bp) was cloned from the duodenum and its transcripts were widely distributed in chicken tissues. Duodenal and caecal PCFT mRNA was not affected by dietary treatments; however, jejunal PCFT mRNA was significantly reduced in hens fed the 5-MTHF diet versus the basal diet. 4. A broad tissue distribution of PCFT transcripts is indicative of a vital role of PCFT in the folate transport in chicken. The expression of jejunal PCFT mRNA was down-regulated by dietary 5-MTHF supplementation. These findings contribute to an understanding of the development of folate transport and the PCFT system in avian systems involving laying hens.

The adaptive transport of folic acid in the intestine of laying hens with increased supplementation of dietary folic acid

Different aspects of folic acid (FA) transport in the intestine of the laying hen have been characterized. Less is known about the adaptive response of this process to a dietary challenge. To this end, a study was conducted to evaluate the effect of increased dietary FA supplementation on the rate of intestinal FA transport and the expression of the intestinal folate transporter genes, the proton-coupled folate transporters (PCFT) and the reduced folate carrier (RFC), in the laying hen. Twenty-four Shaver White hens at 34 wk of age were randomly assigned to receive 1 of 3 dietary treatments: 1) basal diet with no supplemental folate (n = 8), 2) basal diet + 10 mg/kg of crystalline FA (n = 8), and 3) basal diet + 100 mg/kg of crystalline FA (n = 8). A completely randomized design with 3 dietary treatments was used. Production performance was not affected by FA supplementation; however, egg and plasma folate concentrations increased (P < 0.001), whereas plasma homocysteine concentrations decreased (P < 0.011) in birds fed 10 or 100 mg of FA/kg of diet, relative to controls. Mucosal to serosal uptake of FA in the duodenum was decreased (P < 0.002), but the mRNA levels of the duodenal PCFT and RFC genes were not affected when birds were fed 10 or 100 mg of FA/kg of diet. In the jejunum, the mucosal to serosal uptake of FA, as well as the mRNA levels of the PCFT and RFC genes, were not influenced by increased FA supplementation. Overall, increased dietary levels of FA resulted in decreased transport of FA in the duodenum but not in the jejunum of laying hens. This decrease was not associated with decreased mRNA expression of the duodenal PCFT and RFC genes. Therefore, a posttranscriptional or translational adaptation of the intestinal folate transporters may be involved in the much lower transport of FA in the duodenum of laying hens during increased dietary supplementation of FA.

Functional characterization of folic acid transport in the intestine of the laying hen using the everted intestinal sac model

Absorption at the level of the intestine is likely a primary regulatory mechanism for the deposition of dietary supplemented folic acid into the chicken egg. Therefore, factors affecting the intestinal transport of folic acid in the laying hen may influence the level of egg folate concentrations. To this end, a series of experiments using intestinal everted sacs were conducted to characterize intestinal folic acid absorption processes in laying hens. Effects of naturally occurring folate derivatives (5-methyl and 10-formyltetrahydrofolate) as well as heme on folic acid absorption were also investigated. Folic acid absorption was measured based on the rate of uptake of (3)H-labeled folic acid in the everted sac from various segments of the small and large intestines. Folic acid concentration, incubation length, and pH condition were optimized before the performance of uptake experiments. The distribution profile of folic acid transport along the intestine was highest in the upper half of the small intestine. Maximum uptake rate (nmol·100 g tissue(-1)·min(-1)) was observed in the duodenum (20.6 ± 1.9) and jejunum (22.3 ± 2.0) and decreased significantly in the ileum (15.3 ± 1.1) and cecum (9.3 ± 0.9). Transport increased proportionately (P < 0.05) between 0.0001 and 0.1 µM folic acid. Above 0.1 µM, the slope of the regression line was not significantly different from zero (P < 0.137). Folic acid uptake in the jejunum showed a maximum rate of transport at pH 6.0, but was lowest at pH 7.5. The presence of 5-methyl and 10-formyltetrahydrofolate as well as heme impeded folic acid uptake, reducing intestinal folic acid absorption when added at concentrations ranging from 0 to 100 µM. Overall, these data indicated the presence of a folic acid transport system in the entire intestine of the laying hen. Uptake of folic acid in the cecum raises the likelihood of absorption of bacterial-derived folate.

Post-hatching ontogeny of intestinal proton-coupled folate transporter and reduced folate carrier in broiler chickens.

Folate transporters, including the reduced folate carrier and the proton-coupled folate transporter, encoded by Slc19a1 and Slc46a1 genes respectively, play important roles in the transport of folate across biological membranes given the hydrophilic nature of folates. Although a number of studies have demonstrated that these two transporters are regulated ontogenetically in mammals, little data are available on their developmental patterns of expression in poultry. The objective of this study was to investigate the expression patterns of Slc19a1 and Slc46a1 in jejunal and cecal tissue of broiler chickens during post-hatching development. Post-hatch male chicks (Ross × Ross) had free access to water and a soybean/wheat-based diet. Jejunal, cecal and blood samples were collected on day-of-hatch but before feeding (D0), and on D2, D7, D14, D21 and D35 post-hatch (n = 8 at each time point), respectively. Plasma folate concentrations were low on the day of hatch and increased with maturation; by contrast, plasma homocysteine, a marker of folate status, was highest (P < 0.05) in the day-of-hatch birds and decreased thereafter. Increasing age reduced mRNA abundance of Slc19a1 (P < 0.05) in the jejunum and cecum. Abundance of Slc46a1 mRNA (P < 0.05) gradually decreased in the cecum with increasing age and that of Slc46a1 in the jejunum initially decreased and then increased to level similar to that of day-of-hatch. The study provides some initial data on ontogenetic regulation of Slc19a1 and Slc46a1 in the jejunum and cecum of the chicken and lays the ground work for future nutritional studies. Moreover, the expression of Slc19a1 and Slc46a1 transcripts in the cecum provides evidence of the potential for cecally derived folate to contribute to the folate status of the host.