Feeding laying hens a bioavailable soy sterol mixture fails to enrich their eggs with phytosterols or elicit egg yolk compositional changes.

Coronary heart disease (CHD) is the leading cause of death in the United States. Elevated levels of plasma total cholesterol (TC), and particularly plasma low density lipoprotein-cholesterol (LDLC), are primary contributing factors to CHD. Dietary plant sterols (phytosterols) have been shown to significantly reduce plasma TC and LDLC in humans, primarily through inhibition of intestinal cholesterol absorption, and are potentially effective agents for reduction of CHD risk. Although a variety of phytosterol-containing foods are currently available, phytosterol-enriched eggs, which represent a potential value-added product, are conspicuously absent from the marketplace. Therefore, the objectives of this study were 1) to enrich shell eggs with phytosterols; and 2) to determine if feeding phytosterols to hens elicits egg compositional changes, particularly that of yolk cholesterol content. Sixteen 32-wk-old White Leghorn hens were fed a corn-soy-based layer diet without (n = 8) or with (n = 8) 1 g of supplemental soy sterols/100 g of diet for 28 d.. Hen performance was determined on an individual basis, and 1 egg/hen per week was collected, processed, and analyzed for yolk cholesterol, CP, crude fat (CF), and phytosterol content. There was no effect (P > 0.05) of supplemental dietary phytosterols on 28-d weight gain, feed consumption, feed efficiency, plasma TC, hen-day egg production, egg weights, egg component weights, and yolk cholesterol, CP, and CF contents. Small amounts of campesterol were present in most of the eggs (average of 0.29 and 1.02 mg/yolk for control vs. soy sterol-fed hens, respectively; P < or = 0.05), whereas only 3 of the 80 analyzed eggs contained trace amounts of beta-sitosterol and none contained any detectable stigmasterol. It was concluded that phytosterols are either poorly absorbed from the chicken intestine or, if they are absorbed, they are efficiently secreted back into the intestinal lumen, most likely via as yet uncharacterized adenosine triphosphate-binding cassette transporters.

Fatty acid composition of mature human milk of Egyptian and American women.

Fatty acid composition of mature human milk of rural Egyptian and American women was determined by gas-liquid chromatography. Milk of Egyptian women contained significantly higher percentages of capric, lauric, myristic, linoleic and arachidonic acids, saturated fatty acids (SFA), and polyunsaturated fatty acids (PUFA). Conversely, milk of American women contained higher percentages of stearic and oleic acids, total unsaturated fatty acids, and monounsaturated fatty acids. The PUFA:SFA ratio in Egyptian samples was 0.54 +/- 0.18 compared to 0.47 +/- 0.22 in American samples. Increased percentages of medium-chain SFA in Egyptian milk suggested increased mammary gland lipid synthesis. Analysis of Egyptian diets indicated high-carbohydrate and low-fat intakes may have resulted in limited availability and incorporation of dietary fatty acids into milk triglycerides. Thus, increased percentages of medium-chain SFA observed in Egyptian milk may reflect mammary gland synthesis in an attempt to maintain lipid concentrations in milk.

Dietary self-selection and the regulation of protein and energy intake in chicks.

Studies were conducted to determine whether chicks could regulate their protein intake independent of total energy intake in self-selection feeding trials. Day-old White Mountain cockerels were reared in electrically-heated battery brooders and given access to either a 23% protein control ration (no choice) or two diets containing 10% or 60% protein with or without supplemental amino acids. The latter were added to either improve the dietary amino acid balance or to alter plasma and brain levels of free large neutral amino acids (tryptophan, isoleucine, leucine, valine, phenylalanine, and tyrosine) which have been implicated in the neuroregulation of feed intake. Both feed and water were provided ad lib, and the location of the feed troughs within each pen were changed daily. Body weights and feed intakes were measured daily, and total calorie and protein intakes were calculated. Chicks offered 10% and 60% protein diets with no supplemental amino acids exhibited reduced weight gains and markedly higher protein intakes as compared to birds fed either the control ration or those given a choice between 10% and 60% protein diets supplemented with methionine. The higher protein consumption by chicks fed the unsupplemented diets most likely was a result of an attempt to compensate for a dietary methionine deficiency. Chicks fed the 10% and 60% protein diets supplemented with amino acids grew at a slower rate than those fed the 23% protein control diet. In general, plasma and brain data did not support a proposed relationship between certain large neutral amino acid ratios and protein or energy intake.

Proteolysis of Japanese quail and chicken plasma apolipoprotein B and vitellogenin by cathepsin D: similarity of the resulting protein fragments with egg yolk polypeptides.

Plasma very-low density lipoprotein (VLDL) and vitellogenin (VTG) from mature female Japanese quail (Coturnix coturnix japonica) and chickens (Gallus domesticus) were isolated and digested in vitro with cathepsin D (EC3.4.23.5). The incubation mixtures were then reduced and subjected to gradient (4.5-18%) SDS-polyacrylamide gel electrophoresis. Protein fragments were stained with either Coomassie Brilliant Blue R-250 (VLDL digests) or Coomassie Brilliant Blue R-250 containing 20 mM AlCl3 (VTG digests). Fragments resulting from the in vitro enzymatic digestion of quail and chicken plasma VLDL-apolipoprotein B (apo B) and VTG closely resembled those produced in vivo and isolated from egg yolks of each respective species. Phosvitin, a proteolytically derived fragment of VTG, primarily existed as a single band (M(r) approximately 42 kDa) in Japanese quail yolk granules. In contrast, chicken phosvitin mainly consisted of a cluster of phosphoproteins ranging in size from approximately 37 to 45 kDa. In addition to reporting a novel species difference in phosvitin moieties, the present study is the first to examine the role of cathepsin D in the generation of egg yolk proteins from plasma precursors in Japanese quail. Confirmatory evidence also was provided concerning the important role of this aspartic endopeptidase in the proteolytic cleavage of plasma VLDL-apo B and VTG in the chicken.

Substrate specificity of duckling hepatic and renal D-amino acid oxidase.

The substrate specificity of duckling hepatic and renal D-amino acid oxidase (DAAO; D-amino acid: O2 oxidoreductase [deaminating], E.C. 1.4.3.3) was determined using a method based on the combination of coupled enzyme reactions and a colorimetric procedure. When activities were averaged across tissues, D-proline was the most reactive substrate, followed by (in order) D-phenylalanine, D-alanine, D-methionine, D-leucine, D-isoleucine, D-valine, D-tryptophan, D-arginine, and D-lysine. Compared with D-alanine, duckling DAAO had minimal or no reactivity with D-asparagine, D-glutamine, D-histidine, D-threonine, D-cysteine, glycine, or D-serine. These results were in general agreement with data from other vertebrate species.

Visualization of the chicken oocyte lipoprotein receptor by ligand blotting with biotinylated plasma and yolk very low density lipoproteins.

The laying hen 95-kDa oocyte membrane receptor that transports hepatically synthesized very low density lipoprotein (VLDL) and vitellogenin (VTG) from the plasma to growing follicles was visualized by ligand blotting with biotinylated VLDL followed by enhanced chemiluminescence (ECL) detection. Plasma and egg yolk VLDL were isolated by ultracentrifugation and free epsilon-amino groups of lysines of apolipoprotein B (apo B), the protein constituent of VLDL that mediates binding to the 95-kDa oocyte membrane receptor, were biotinylated using D-biotin-N-hydroxysuccinimide ester. An apo B concentration of approximately 223 pM was sufficient to give a signal on 2.5 micrograms of protein from a chicken oocyte membrane detergent extract. Western blotting (immunoblotting) of the laying hen 95-kDa receptor with polyclonal rabbit anti-chicken oocyte VLDL receptor IgG resulted in an ECL signal with the same position of migration as that observed in ligand blots using biotinylated plasma and yolk VLDL. Binding of biotinylated plasma or yolk VLDL to the 95-kDa receptor was abolished by excess unlabeled plasma or yolk VLDL, respectively, as well as by EDTA. Receptor binding activity of biotinylated plasma and yolk VLDL was also demonstrated by a reverse ligand blotting procedure. Compared with conventional techniques involving the use of 125I-labeled ligands or antibodies, the laying hen 95-kDa oocyte membrane lipoprotein receptor can be safely and rapidly visualized with excellent sensitivity using the present nonradioactive method. In addition, it is suggested that ECL detection can be employed to further study the ligand-binding properties and specificity of this protein, which is essential to vitellogenesis in the chicken.

Assessment of reproductive function in mutant restricted ovulator carrier roosters.

Two splice variant forms of the chicken very low density lipoprotein receptor (VLDLR) have recently been shown to exist in both male and female chicken gonads. The longer form, containing an O-linked sugar domain, is produced in the somatic cells of the testes and ovaries, whereas the shorter form, which lacks this domain, is expressed in the male and female germ cells. The latter is essential for female reproduction, because a naturally occurring point mutation in the gene encoding the short form of the VLDLR causes its functional absence, resulting in atherosclerosis and a general failure to lay eggs. Thus, the term restricted ovulator (RO) was originally coined to describe females with the mutant gene. In contrast, the roles of the VLDLR gene products in male reproduction have yet to be defined, because only heterozygous RO roosters, which have a mutant allele of the VLDLR gene (short form) on one of their two Z (sex) chromosomes, can be obtained. However, the question of whether two normal alleles of the VLDLR gene are needed for optimal male reproductive performance can be addressed. Here, the reproductive abilities of heterozygous RO roosters were compared to their wild-type siblings. The RO roosters were found to be phenotypically normal, suggesting that only one normal allele for the VLDLR gene is needed for optimal male reproductive performance. Alternately, it is also possible that the one normal allele compensated for the presence of the mutant one in terms of VLDLR gene product production (i.e., a dosage compensation).

A comparison of methionine sources for broiler chickens fed corn-soybean meal diets under simulated commercial grow-out conditions.

Two experiments, involving 5,760 birds each, were conducted under simulated commercial grow-out conditions to determine the relative response of broiler chicks to two supplemental levels each of DL-methionine (DL-MET), L-methionine (L-MET), and methionine hydroxy analogue-free acid (MHA-FA) fed on an equimolar basis in corn-soybean meal diets. MHA-FA was shown to support 49-day weight gains comparable to those of chicks fed DL-MET, and both sources were superior to L-MET in this regard. However, there were no significant differences due to MET sources in the efficiency of feed utilization. When compared to basal-fed chicks, birds fed supplemental L-MET, DL-MET, or MHA-FA had elevated plasma free MET levels, whereas cyst(e)ine and taurine concentrations were not affected by dietary treatment. There were no effects of diet on either mortality or the incidence of leg abnormalities. The results of these studies suggest that MHA-FA is an acceptable source of MET for broiler chicks fed corn-soybean meal diets from day-old to market weight.

Partial alleviation of phenylalanine toxicity in the chick by supplemental dietary tryptophan.

Studies were conducted to determine the physiological effects of feeding diets containing high levels of phenylalanine to chicks. Marked reductions in weight gain and feed conversion were observed in birds fed a diet containing 2.52% L-phenylalanine and .47% L-tyrosine. Excess dietary phenylalanine caused elevations of phenylalanine and tyrosine in serum and brain, whereas cerebral concentrations of free isoleucine, leucine, and valine were generally decreased. Supraoptimal amounts of glycine, arginine, and tryptophan were fed in an attempt to alleviate the toxic symptoms observed in birds fed high phenylalanine diets. Only tryptophan supplementation proved successful in partially alleviating the growth-depressive effects of phenylalanine toxicity. Both serum and brain levels of tryptophan were increased by tryptophan supplementation. Results of two radiotracer studies with 14C-tryptophan suggested that brain uptake of tryptophan was inhibited by hyperphenylalaninemia.

Comparative effects of dietary tannins in ducks, chicks, and rats.

Studies were conducted to compare the effects of feeding high-tannin sorghum (HTS)- and low-tannin sorghum (LTS)-based diets suboptimal in protein to ducks, chicks, and rats. In the first series of experiments, Savanna (HTS) depressed both growth and feed efficiency of chicks and rats when compared with animals fed RS-610 (LTS). In contrast, ducks fed Savanna-based diets exhibited greater weight gains, but poorer feed efficiency values than birds fed RS-610. In a second series of experiments, Pioneer 8333 (LTS) and DeKalb BR-64 (HTS) sorghum-soybean meal diets were fed to chicks, ducks, and rats. As compared with animals fed 8333, BR-64 depressed both growth and feed efficiency of chicks and rats, but did not significantly affect either parameter in ducks. The lack of effect of HTS on duck growth may have been due to the formation of tannin-protein complexes upon exposure of the ground grain to water. This theory was based on the following observations: 1) ducks consumed water immediately after eating to facilitate swallowing the dry-mash diet; 2) a large amount of feed was deposited on the bottom of each water trough and subsequently consumed; and 3) ground HTS, which had been soaked for 4 h, had virtually no assayable tannin after drying. However, despite the marked reduction in assayable tannin content of treated HTS versus HTS that was dried only, the former material still depressed growth and feed conversion of rats. Thus, the lack of a growth-depressing effect of sorghum tannins in ducks fed dry-mash diets is both unique and unexplained.

Effect of light intensity, litter source, and litter management on the incidence of leg abnormalities and performance of male turkeys.

Two trials were conducted to evaluate the performance of male turkeys reared in either a low or high intensity lighting regimen. Turkeys were also subjected to the following four litter treatments: hardwood shavings intensively managed; hardwood shavings unmanaged; rice hulls intensively managed; and rice hulls unmanaged. Male turkeys reared in high intensity light (20 lx) had significantly heavier body weights, poorer feed to gain ratios, shorter tarsometatarsal bones, earlier growth plate closure, heavier testes, and were less active during the times of behavioral observations than male turkeys reared in low intensity light (2.5 lx). The width of the tarsometatarsal bone, mortality, and the incidence of leg abnormalities and tibial dyschondroplasia were unaffected by lighting. Poor litter conditions or type of litter (hardwood shavings vs. rice hulls) had no effect on the performance of male turkeys.(u3)

Methionine requirement of male white Pekin ducklings.

Two experiments were conducted to determine the methionine (Met) requirement of male White Pekin ducklings. In each study, day-old ducklings were raised in electrically heated battery brooders and fed a corn-peanut meal basal diet (dry mash) supplemented with either 0, .06, .12, .18, .24, or .30% L-Met. Based on the analysis of corn and peanut meal, the basal diet contained 22% crude protein .268% Met, and .284% cystine (Cys). The energy content of the basal diet was calculated to be 2914 kcal ME/kg. Regression analysis of 12- day weight gain data pooled across experiments indicated a quadratic trend. When the quadratic model was solved for 95% of maximum growth, a Met requirement of .422% was predicted (r2 = .49). Analysis of the pooled weight gain data by the broken-line method yielded a requirement estimate of .382% (r2 = .50). Therefore, the duckling's calculated Met requirement most likely lies between .382 and .422% (.666 and .706% total sulfur amino acids, respectively). This .04% discrepancy between models for predicting the duckling's Met requirement is probably of minor practical significance, considering the variability of Met and Cys contents of feedstuffs and the fact that corn-soy diets are usually supplemented with a source of Met activity that includes a margin of safety.

Validation of a modified PCR-based method for identifying mutant restricted ovulator chickens: substantiation of genotypic classification by phenotypic traits.

Upon photostimulation, restricted ovulator (RO) female chickens exhibit endogenous hyperlipidemia, develop atherosclerotic lesions, and generally fail to lay eggs. This phenotype results from a point mutation in the gene specifying the very low density lipoprotein receptor (VLDLR), whose protein product normally mediates the massive oocytic uptake of egg yolk precursors from the circulation. Taking advantage of the single base change in the mutant VLDLR allele, a PCR-based method for the rapid identification of RO chickens was developed at the Biocenter and University of Vienna, Austria. However, this procedure was incompletely validated because phenotypic data were not obtained and conventional progeny testing of sons and grandsons was not performed. Here, the assay validation was completed by providing plasma lipid concentrations, plasma very low density lipoprotein particle sizes, or egg production records of PCR-genotyped females and their brothers and sires to demonstrate that each bird's phenotypic traits substantiated their genotypic classification. Moreover, several methodological modifications resulted in improved chemical safety, speed, and cost of preparing and analyzing genomic DNA from chicken erythrocytes. Because the ovaries of mutant RO females generally contain numerous vitellogenic follicles in the absence of a functional oocyte plasma membrane VLDLR, the existence of an alternate system for the oocytic uptake of plasma very low density lipoprotein and vitellogenin is suggested, whereas a physiological explanation as to why some, but not all, mutant RO hens are able to ovulate and lay eggs is lacking.

Investigations of leg abnormalities in chicks consuming high tannin sorghum grain diets.

Studies were conducted into the etiology of leg abnormalities noted in chicks fed high tannin sorghum grain diets. These anomalies were characterized by a bowing of the legs with a swelling of the hock joints. The incidence of these leg problems was found to be markedly higher when the amino acids in the diet provided by soybean meal were replaced by crystalline amino acids. Supplemental vitamins and minerals had no alleviating effect on the leg problem. Bone mineralization was apparently not influenced by tannins as demonstrated by similar bone ash values for chicks fed high or low tannin sorghums. A possible alteration caused by tannin in the organic matrix of bone is discussed. High tannin sorghum depressed chick growth and feed conversion when compared with low tannin sorghum in both sorghum-soybean meal and sorghum-amino acid rations. This growth depression was overcome by supplementing the high tannin sorghum-soybean meal diet with .15% DL-methionine, but no growth response was observed from a similar supplementation of a high tannin sorghum-amino acid diet.

Effects of high intensity step-up and low intensity step-down lighting programs on the incidence of leg abnormalities in turkeys.

Two trials were conducted to determine if a high intensity (20 lx) step-up lighting program would decrease the incidence of leg abnormalities in turkeys. The step-up lighting regimen was compared to a low intensity (2.5 lx) step-down lighting program. Toms were used in Trial 1 and both sexes were employed in Trial 2. Body weight, feed/gain ratio, and mortality of toms of Trial 1 did not differ significantly between lighting regimens. The incidence of leg deformities and the length of the tarsometatarsal bone were significantly decreased, whereas plasma androgen and testes weights were increased for toms of Trial 1 in the high intensity step-up lighting. The same trends were apparent for toms of Trial 2 with the exceptions that feed/gain ratios were adversely affected and testes weights were significantly lower for toms of the high intensity step-up lighting program. The performance of hens in the high intensity step-up lighting program of Trial 2 was affected adversely; however, incidence of leg abnormalities and length and width of the tarsometatarsus responded in the same manner as with toms. The results of this study indicate that a high intensity step-up lighting program can significantly reduce leg weakness problems in turkeys. Possible explanations for light stimulation reducing the incidence of leg abnormalities are through increased exercise and shortening of long bones.

A rapid, polymerase chain reaction-based procedure for identifying mutant restricted ovulator chickens.

Females of the restricted ovulator (RO) strain of White Leghorn chickens fail to lay eggs upon photostimulation and exhibit endogenous hyperlipidemia and atherosclerotic lesions. A mutation in the gene specifying the oocyte vitellogenesis receptor (OVR), a 95-kDa membrane protein that normally mediates the massive uptake of yolk precursors from the serum, is responsible for this abnormal phenotype. Because a single nucleotide substitution (G-->C) is responsible for the defective OVR, a PCR-based procedure, described herein, was developed in order to provide a rapid and accurate method for identifying chickens possessing the mutant allele. Polymerase chain reaction-amplified fragments of apparently identical size (approximately 400 bp) were obtained from genomic DNA using primer pairs specific for either the wild-type or mutant genes. Through cloning and sequencing of the PCR-amplified products, the fragment sizes were determined to be 413 bp each, which included an intron sequence. Polymerase chain reaction-amplified genomic DNA from wild-type (ovr+/ovr+) males, heterozygous carrier (ovr+/ovr-) males, and wild-type (-/ovr+) females all yielded a 413 bp fragment when a primer pair specific for the wild-type gene was used. Because female chickens are heterogametic (ZW), no PCR product was observed in the case of the mutant (-/ovr-) females. When the primer pair specific for the mutant gene was employed, PCR-amplification of genomic DNA from both heterozygous carrier (ovr+/ ovr-) males and mutant (-/ovr-) females, but not wild-type (ovr+/ovr+) males or (-/ovr+) females, also yielded a 413-bp fragment. Employment of the present rapid and accurate procedure would be expected to obviate the need for conventional progeny testing while reducing the time required to identify RO carrier males and mutant females from approximately 1 yr to several days.

The influence of cecectomy on metabolizable energy and amino acid digestibility of select feedstuffs for white Pekin ducks.

Twenty-four cecectomized and 24 intact White Pekin ducks were used in two experiments to assess the influence of cecectomy on ME and amino acid digestibility of several feed ingredients for ducks. Corn and soybean meal (SBM) were evaluated in Experiment 1, and bakery meal (BM), red dog (RD), and wheat middlings (WM) were evaluated in Experiment 2. Nitrogen-corrected true metabolizable energy and amino acid digestibility of the ingredients were assayed concurrently. In Experiment 1, TME of corn was higher (P < or = 0.05) in cecectomized ducks, and intact ducks demonstrated greater (P < or = 0.05) ability to utilize the energy in SBM. Intact ducks exhibited higher (P < or = 0.05) true digestibilities of lysine and methionine. True digestibility of tryptophan was higher (P < or = 0.05) for cecectomized ducks. True digestibility of indispensable amino acids in SBM did not differ (P > or = 0.05) between cecectomized and intact ducks. In Experiment 2, cecectomized ducks exhibited greater (P < or = 0.05) ability to utilize the energy in RD. Intact ducks exhibited greater (P < or = 0.05) ability to utilize the energy in WM. True digestibility of indispensable amino acids in BM, RD, and WM was variable. Results of the present study suggest that the effect of cecectomy on nutrient digestibility in ducks is dependent on the feedstuff assayed.

Influence of phenylalanine and lysine deficiencies on thyroid morphology in the chick.

A study was conducted to investigate the effects of dietary phenylalanine and lysine deficiencies on thyroid morphology in chicks fed crystalline amino acid diets. Both mean follicle diameter and epithelial cell height were significantly reduced in birds fed either of the deficient diets as compared with controls. These results agree with previous studies on serum T4 and T3 concentrations where the effects of an amino acid deficiency were general and not limited to precursors of thyroxine.

Evaluation of two novel biochemicals on plasma and egg yolk lipid composition and laying hen performance.

PD132301-2, an inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT; EC 2.3.1.26), and 1-stearylboronic acid (SBA), a fatty acid analogue, were orally administered to White Leghorn hens in separate experiments to evaluate their effects on layer performance and plasma and egg yolk lipids. Five 60-wk-old hens each were fed either a corn-soybean meal basal layer ration, or the basal diet supplemented with .0121, .0363, or .1089% PD132301-2. In a second experiment, 12 37-wk-old hens each were fed either a basal layer ration, or the basal diet supplemented with .20 or .40% SBA. The duration of the experiments were 21 and 16 days, respectively. Neither compound significantly affected hen-day production, egg weight, yolk weight, BW gain, feed consumption, feed efficiency, plasma total cholesterol and triglyceride concentrations, or egg yolk cholesterol content. PD132301-2 had no effect on yolk fatty acid profiles, and C22:6n3 was the only fatty acid altered by SBA. Although 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been successful in reducing egg cholesterol, ACAT inhibitors and fatty acid analogues apparently hold little promise in this regard. The results of the present work also support the concept that, in order to pharmacologically alter the cholesterol content of eggs, direct inhibition of key enzymes in the cholesterol biosynthetic pathway is necessary.