Development of a Procedure to Determine Standardized Mineral Availabilities in Soybean Meal for Broiler Chicks

The objective of this study was to develop a procedure for estimating the standardized mineral (calcium, Ca; phosphorus, P; copper, Cu; manganese, Mn; zinc, Zn) availabilities (SMA) in soybean meal for broilers. In experiment 1, twelve 24-day-old male chicks were used to determine the length of the pre-experimental fasting period needed to empty the total tract of feed residues. Feed was removed and excreta samples were collected for 12, 24, 36 or 48 h after feed withdrawal, respectively. The result indicated that birds were fasted for at least 24 h after feed withdrawal in order to ensure that no previous feed residues remained in the digestive tract of chicks. In a subsequent experiment, forty-eight 24-day-old male chicks were used to determine SMA in soybean meal. Chicks were fasted for 24 h and then fed mineral-free or soybean meal diet for 4 h, and excreta samples were collected until 24 or 48 h after feed withdrawal. The results showed that the extended excreta collection time of 48 h after feed withdrawal was adequate for estimating SMA. The standardized availability values of Ca, P, Cu, Mn or Zn in soybean meal were 51.14%, 50.22%, 36.28%, 30.79% or 49.66%, respectively. In experiment 3, a similar bioassay was conducted with forty-eight 36-day-old male chicks to measure SMA in the same soybean meal. The standardized availability values of P (51.59%), Cu (36.37%), Mn (31.94%) or Zn (43.37%) were similar (P > 0.12) to the results of experiment 2 except for Ca (41.49%), indicating that the age of birds had no effect on the standardized availabilities of P, Cu, Mn or Zn in soybean meal. The results from this study suggested that this simple and rapid procedure could be used to determine SMA in feedstuffs (e.g., soybean meal) for broiler chicks.     

Immunological cell and serum metabolite response of 60-week-old commercial laying hens to an alfalfa meal molt diet

The practice of induced molting involves the restriction of light, feed removal and optionally water for 5-14 days. However, there is growing concern regarding feed removal and animal welfare issues. With this in mind, alternative diets have been developed to produce similar molting effects as that of feed deprivation. Alfalfa, which largely consists of insoluble fiber, can be used as a molting diet. In this study, heterophil and lymphocyte counts, serum chemistry, and organ weight parameters were evaluated in hens that were deprived of feed or fed alfalfa during a nine day induced molt. Full-fed hens were used as the control. Blood serum parameters assessed included calcium, magnesium, glucose, total protein, ketone bodies, uric acid, and cholesterol. White blood cells were counted and categorized by cell type. On the ninth day of the trial, the hens were euthanized and the liver, spleen, heart, intestine, pancreas, ovary, oviduct, and kidney were collected and weighed. On day 8 birds molted with alfalfa or by feed deprivation had significantly higher (P<0.05) levels of ketone bodies and cholesterol and lower levels of calcium, and magnesium compared to the full-fed hens while birds molted by feed deprivation exhibited significantly lower levels of uric acid. Birds molted by both methods exhibited significant reductions in ovary, oviduct, liver and pancreas weights and increased spleen weights when compared to the non-molted hens. On days 0, 2, and 6 there were no significant differences (P>0.05) in either heterophil or lymphocyte percentages. However, heterophil percentages were higher in feed withdrawal birds than full-fed birds on day 4 but lymphocyte percentages were higher in full-fed birds compared to feed withdrawal birds. On day 8 of the induced molt lymphocyte percentages were higher from full-fed birds when compared to feed withdrawal birds but no significant differences were detectable for heterophil percentages. Based on reproductive organ weight loss and changes in serum and immunological responses of birds during molt, it appears that alfalfa meal can be an effective molt induction alternative.     

Molecular identification of Cryptosporidium parvum from avian hosts

Cryptosporidium species are protozoan parasites that infect humans and a wide variety of animals. This study was aimed at identifying Cryptosporidium species and genotypes isolated from avian hosts. A total of 90 samples from 37 different species of birds were collected throughout a 3-month period from April 2008 to June 2008 in the National Zoo of Kuala Lumpur, Malaysia. Prior to molecular characterization, all samples were screened for Cryptosporidium using a modified Ziehl-Neelsen staining technique. Subsequently samples were analysed with nested-PCR targeting the partial SSU rRNA gene. Amplicons were sequenced in both directions and used for phylogenetic analysis using Neighbour-Joining and Maximum Parsimony methods. Although 9 (10%) samples were positive for Cryptosporidium via microscopy, 8 (8.9%) produced amplicons using nested PCR. Phylogenetic trees identified all the isolates as Cryptosporidium parvum. Although C. parvum has not been reported to cause infection in birds, and the role of birds in this study was postulated mainly as mechanical transporters, these present findings highlight the significant public health risk posed by birds that harbour the zoonotic species of Cryptosporidium.     

Reproductive tissue regression: involvement of caspases, inducible nitric oxide synthase and nitric oxide during moulting in White Leghorn hens

Moulting is a natural physiological process where the reproductive system of birds undergoes complete remodeling in preparation for the next laying cycle. In domestic chickens, moulting is artificially induced by feed withdrawal to recycle the old laying flock for best profit margins. This has received severe criticism from animal welfare organizations, forcing several countries to stop this practice. Several alternative methods to feed withdrawal methods were developed but were found to produce inconsistent results. Understanding the actual mechanism of moulting would help in designing a new animal welfare friendly method. The present investigation attempted to study the molecular mechanism of moulting in White Leghorn hens. Eighty-four layers (75 weeks) were divided into two groups. The birds in the first group were subjected to moulting by feed withdrawal (FW) while the other group received high dietary Zn (ZnF) treatment for 10 days. Six birds from each group were sacrificed on 0, 1-4, 6 and 10 days of moulting and mRNA expression of caspases-1, -2 and iNOS, along with the apoptotic ladder pattern and nitric oxide (NO) in the ovary and oviduct, was investigated. The mRNA expression of iNOS was upregulated with a corresponding increase in NO levels. Caspases-1 and -2 were differentially upregulated in the ovary and oviduct of moulted birds. A constant decline in serum estradiol and progesterone levels was also observed. It can be concluded that the pattern of reproductive regression during moulting by the two methods is different, as the expression of genes studied in the present investigation is different.     

Comparison of morphological and molecular genetic quantification of relative abundance of arbuscular mycorrhizal fungi within roots

Nested PCR amplicons of ribosomal RNA genes have been used to identify individuals within assemblages of arbuscular mycorrhizal (AM) fungi in roots and to estimate their relative abundance. Microscopy has also been used to identify their relative abundance in roots, but only at low resolution, usually the genus level. We evaluated the robustness of using nested PCR amplicons of ribosomal RNA genes to estimate the relative abundance of undefined AM fungi in uniformly aged roots in comparison to visual estimates. The relative abundance of AM fungi was assessed as per cent root length colonised by morphotypes and relative sequence type abundance in clone libraries. Plants were grown in coastal soil to obtain assemblages of unknown AM fungi at two times (spring and autumn). Relative abundance of dominant genera of AM fungi in roots (Archaeospora and Glomus) based on an analysis of ribosomal RNA genes did not consistently correspond with relative abundance of morphotypes. This microscopic vs. molecular genetic comparison supports previous conclusions that there can be limitations in using nested PCR amplicons for quantifying the relative abundance of AM fungi in roots, with a sampling bias likely to be of significance. Both molecular genetic and morphological methods are used to estimate relative abundance of AM fungi as a precursor to understanding mycorrhizal function in field soils, but they are rarely verified using alternative approaches although this may be necessary.     

Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10(8) to 10(9) CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.     

Estrogen induces hyperlipidemia in fasted chicks

To determine whether the estrogen-induced hyperlipidemia is affected by fasting, male growing chicks were administered subcutaneously a single dose of 17 beta-estradiol (25 mg/kg body wt), and the hormone treatment lasted for 2 days with or without feed (Experiment 1). In the second experiment, chicks were initially fasted for 1 or 3 days, and then treated with the same dosage of 17 beta-estradiol as in Experiment 1 for 2 days without feed. Plasma and liver lipids, and the activities of hepatic malic enzyme, glucose-6-phosphate dehydrogenase, and hormone-sensitive lipase in the adipose tissue were determined. Compared with fed control chicks, estrogen treatment in fed birds resulted in a marked elevation of plasma lipids, especially triglyceride during the 2-day period (137 vs 2263 mg/dl). In fasted chicks, the present finding that estrogen also induced a marked hyperlipidemia is noteworthy. Upon estrogen treatment (Experiment 1), the level of plasma triglyceride in fasted birds increased about 16 times over that of the fasted control group (133 vs 2093 mg/dl). Even in chicks fasted for 5 days (Experiment 2), estrogen treatment resulted in a persistent hypertriglyceridemia (75 vs 1369 mg/dl). In fed chicks, estrogen treatment also induced a fatty liver with massive accumulation of triglyceride, but the liver of estrogen-treated/fasted chicks appeared to be normal. In both fed and fasted chicks, malic enzyme was found to be the major NADPH producing enzyme in the liver. Upon fasting, both malic enzyme and glucose-6-phosphate dehydrogenase activities decreased significantly (P less than 0.05). In fed chicks, the total activities of both enzymes increased with estrogen treatment, whereas the effect of hormone on these enzymes was less obvious in fasted chicks. The hormone-sensitive lipase activity in the adipose tissue was much lower in fed chicks compared with that of fasted birds (0.15 vs 0.33 nmol of oleic acid released/min/mg protein). Estrogen treatment in fed chicks had no effect on the hormone-sensitive lipase activity, but its activity was enhanced by the hormone treatment in fasted chicks. The present finding that hyperlipidemia persisted in estrogenized chicks during the fasting seems to indicate the complex nature of this hormonal influence on lipid metabolism.     

Effect of fasting on the structure and function of the gastrointestinal tract of house sparrows (Passer domesticus)

Starvation is a condition that often affects animals in nature. The gastrointestinal tract is the organ system displaying the most rapid and dramatic changes in response to nutrient deprivation. To date, little is known about starvation phases and effects on the organ morphology and digestive function in small passerine birds. In this study, we determined the phases of starvation and examined the effect of final stage of starvation in the organ morphology and, intestinal histology and enzymatic function in the small intestine. Our results show the three phases of the classical model of fasting in a shorter period of time. The mass of heart, pancreas, stomach, small intestine and liver of long-term fasted birds was reduced between 20 and 47%. The mass decrease in small intestine was correlated with reduction in small intestinal histology: perimeter, mucosal thickness, villus height and width. In contrast, the enzyme activity of sucrase-isomaltase and aminopeptidase-N in enterocytes, all expressed per μg of protein, was higher in long-term fasted birds than fed animals. This suggest that, while autophagy of digestive organs is induced by starvation, consistent with phenotypic plasticity, the activity of sucrase-isomaltase and aminopeptidase-N remains high, probably as an anticipatory strategy to optimize digestion at re-feeding time.     

RNA reveals a succession of active fungi during the decay of Norway spruce logs

Current knowledge of the succession of fungi in decaying wood is mostly based on fruit bodies and in vitro culture. Here, we investigated the changing community of metabolically active fungi during the decomposition of fallen Picea abies logs by directly extracting and barcode sequencing precursor rRNA. We also compared rRNA-derived amplicons of the 18S and ITS regions in 21 isolates and discuss the use of RNA as a marker of metabolically active fungi. The richness of active fungi, revealed as separated bands in DGGE, peaked in logs at an advanced stage of decay. Soft-rot fungi were common in the early stages but white- and brown-rot fungi became dominant as decay progressed. Ectomycorrhizal fungi were detected at an early stage, and they became the most abundant group in the late stages of succession. A comparison of rRNA-derived amplicons revealed that although ITS was detected in the form of precursor rRNA, introns within 18S rDNA were already spliced. As such, rRNA- and rDNA-derived amplicons would yield different profiles of active and total communities if profiling method is affected by amplicon length.     

Development of the glycogen body of the Japanese quail, Coturnix japonica. I. Light microscopy of early development

The glycogen body is a functionally enigmatic structure located in lumbosacral region of the spinal cord in birds. This tissue is unique to birds, and, although it is believed to be present in all species, studies on the glycogen body to date have been confined largely to the domestic chicken. The present study is the first to describe the glycogen body of the Japanese quail (Coturnix japonica) during incubation and at hatching. Light microscopy and histochemistry were used to identify the glycogen body in the spinal cord of the developing quail beginning at 7 days of incubation and to ascertain the presence of nerve fibers in that tissue at hatching.     

Species-specific PCR-DGGE markers to distinguish Pyrenophora species associated to cereal seeds

Pyrenophora species, toxigenic cereal pathogens, and causal agents of leaf and kernel diseases, bring about economic and food safety concerns. Traditionally, Pyrenophora taxa have been identified microscopically after a period of incubation on culture media. In this study, a simple nested PCR-denaturing gel electrophoresis (DGGE) method was developed to detect, differentiate and identify six Pyrenophora species in plant tissues. A primer, specific to Pyrenophora species and able to amplify a fragment of the ribosomal RNA (rRNA), following first round amplification with universal ITS primers, was designed by reviewing Pyrenophora ribosomal DNA sequences deposited in GenBank. The specificity of the primer was assessed by submitting its sequence to the GenBank Basic Local Alignment Search Tool (BLAST) algorithm, and was also tested with DNA extracted from several ascomycetous, basidiomycetous, and zygomycetous taxa. No PCR product was obtained from non-Pyrenophora species. PCR amplification of DNA extracted from pure cultures of the different Pyrenophora species generated amplicons of an approximate 350bp. DGGE effectively separated between all six Pyrenophora amplicons. Subsequently, amplicons of known Pyrenophora species were used as molecular markers when Pyrenophora infected wheat seed was analyzed by PCR-DGGE. The molecular-based approach described herein can be used to identify different Pyrenophora species directly from infected plant material.     

Ontogeny and nutritional status influence oxidative kinetics of nutrients and whole-animal bioenergetics in zebra finches, Taeniopygia guttata: new applications for (13)C breath testing

Rapidly growing animals or those that are recovering from nutritional stress may use exogenous nutrients differently from well fed adults. To test this possibility, we compared the rates of exogenous nutrient oxidation among fledgling, fasted adult, and refed adult zebra finches using a technique called breath testing, where animals are fed (13)C-labeled nutrients and (13)C in the exhaled breath is collected and quantified. In order to identify the possible mechanisms responsible for differences in oxidative kinetics of ingested nutrients, we also compared body mass (m(b)), organ mass, core body temperature (T(b)), and metabolic rate (MR). We found that fasted birds had lower T(b), relative liver and intestine masses, MR, and respiratory exchange ratios (RERs) than fed adults. Adult birds recovering from nutritional stress had much lower rates of exogenous nutrient oxidation than fed birds; this difference was particularly evident for fatty acids. Differences in oxidative kinetics were correlated with reduced RER, m(b), and liver mass, suggesting that previously fasted birds were using recently assimilated nutrients to replenish exhausted fuel stores. Rapidly growing fledglings oxidized exogenous nutrients as quickly as fed adults, despite their significantly lower m(b) and T(b). We suggest that fledglings had higher mass-specific rates of exogenous nutrient oxidation because they must compensate for the relatively low conversion efficiency of feather production and other lean tissue growth, which was not taking place in the adults. Although this study demonstrates that ontogeny and nutritional status influence the way that birds oxidize exogenous nutrients, it also underscores the likelihood that environmental and endogenous factors shape how other types of animals spend the nutrients they ingest.     

Pathway analysis of liver metabolism under stressed condition

Pathway analysis is a useful tool which reveals important metabolic network properties. However, the big challenge is to propose an objective function for estimating active pathways, which represent the actual state of network. In order to provide weight values for all possible pathways within the metabolic network, this study presents different approaches, considering the structural and physiological properties of the metabolic network, aiming at a unique decomposition of the flux vector into pathways. These methods were used to analyze the hepatic metabolism considering available data sets obtained from the perfused livers of fasted rats receiving burn injury. Utilizing unique decomposition techniques and different fluxes revealed that higher weights were always attributed to short pathways. Specific pathways, including pyruvate, glutamate and oxaloacetate pools, and urea production from arginine, were found to be important or essential in all methods and experimental conditions. Moreover the pathways, including serine production from glycine and conversion between acetoacetate and B—OH-butyrate, were assigned higher weights. Pathway analysis was also used to identify the main sources for the production of certain products in the hepatic metabolic network to gain a better understanding of the effects of burn injury on liver metabolism.     

Effects of fasting on the control of fatty-acid synthesis in hepatoma 7777 and host liver. Role of long-chain fatty acyl-CoA,, the mitochondrial citrate transporter and pyruvate dehydrogenase activity.

The effects of fasting on the rate of fatty acid synthesis, the properties of the mitochondrial citrate transporter and on pyruvate dehydrogenase activity were investigated in "poorly-differentiated" tmorris hepatoma 7777 and in host liver preparations. The properties of the citrate transporter from hepatoma mitochondria were similar to those of host liver mitochondria, with the exception that the Km for the liver mitochondrial citrate transporter was 248 plus or minus 20 mu M while that in hepatoma mitochondria was less than 75 mu M. The acid-insoluble CoA content was 180 plus or minus 20 pmol/mg protein in the hepatoma and remained essentially unchanged in the fasted state, while the acid-insoluble CoA levels in livers from fed rats was 720 plus or minus 80 pmol/mg protein and were increased to 1050 plus or minus 50 pmol/mg protein during fasting. After a 36-h fast, the rate of lipogenesis and the percentage of pyruvate dehydrogenase present in the active form were each decreased by approximately 80% in host liver preparations. In contrast, the rate of lipogenesis by hepatoma slices did not decrease during fasting, and essentially all pyruvate dehydrogenase present was in the active form of hepatomas obtained from either fed or fasted animals. Implications concerning the identification of possible regulatory sites in the control of lipogenesis were discussed in relation to the above observations.     

Antipredator vigilance in birds: modelling the 'edge' effect

Many animals spend a large proportion of their time either foraging for food or watching out for predators (antipredator vigilance). There have been many theoretical and empirical studies investigating the trade-off between these two activities, especially in birds. Previous models of antipredator vigilance assume that all birds within the group spend the same amount of time feeding. However, many empirical studies have shown that individuals on the edge of flocks are more vigilant. Here we describe a vigilance model which investigates the effect of position on the birds' strategies by dividing the feeding area into an inner and outer region. The model examines how various parameters such as food availability and predation risk affect the strategies of individual birds according to whether they are in the inner or outer region. Our model predicts that birds in the outer group are always more vigilant than those in the inner region. Birds in the centre receive a higher payoff in each of the wide range of scenarios that we have considered, and so our model also indicates why dominant birds would choose to feed in the centre of the group; a prediction in accord with several empirical studies.     

An Efficient RNA Extraction Method for Estimating Gut Microbial Diversity by Polymerase Chain Reaction

An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.