Production and purification of melanoma gp100 antigen and polyclonal antibodies.

gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells. Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma. To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides. High yield was achieved in an Escherichia coli expression system. The protein was purified by AKTA Prime using anionic-columns. Polyclonal antibodies developed in chicken against HR-gp100 were efficient at detecting gp100 in melanoma cells, as determined by Western blot analysis and by immunohistochemistry. HR-gp100 can be used to develop a vaccine against melanoma. Antibodies to HR-gp100 may be used to detect tumors of melanocytic origin or to determine the level of gp100 expression in tumors prior to immunotherapy with the protein or one of its peptides.

Non-invasive photoacoustic spectroscopic determination of relative endogenous nitric oxide and ethylene content stoichiometry during the ripening of strawberries Fragaria anannasa (Duch.) and avocados Persea americana (Mill.).

Employing non-invasive photoacoustic spectrometry, emissions of nitric oxide (NO) and ethylene in post-harvest strawberries and avocados were monitored. A clear-cut stoichiometric relationship was found between the two gases: unripe fruit manifesting high NO and low ethylene levels-the converse in ripe fruit. Findings are discussed in the light of putative control of ethylene-promoted fruit senescence by endogenous NO.

Identification of a starting point of breast skin tears during chicken plucking.

A site (STP) was identified on the skin of the chicken, during defeathering in the slaughter house, at which about 90% of breast skin tears started. This site is on the ventral side of the pectoral tract area. There was no difference in location of this site with respect to different commercial lines, sexes, flocks, or time of the day. In order to demonstrate the importance of a small skin tear to ultimate damage, defeathered chickens with a minor tear at a particular site and undamaged defeathered chickens were passed through the defeathering machine a second time. Thirty-six percent of the STP chickens were torn further, but only a small percentage (about 4%) of the undamaged chickens were harmed.

Hypothalamic obese, functionally castrated hens are hypersensitive to estrogenic modulation of lipid metabolism.

Estradiol benzoate (E2) increases plasma lipids in hypothalamic obese, functionally castrated (OFC), obese laying (OL), and control laying hens (CONT). However, E2 reduces fattiness in OFC but not in OL or CONT hens. Antiestrogen, such as tamoxifen (TAM), reduces plasma lipids in OL and CONT, but not in OFC, hens and has no effect on fattiness in any of them. Apolipoprotein VLDL-II (apo-VLDL-II), lipoprotein lipase (LPL), and rate of lipolysis may mediate these estrogenic effects. In the present study, effects of E2 and TAM on fattiness, plasma apo-VLDL-II, in vitro lipolysis, and LPL activity in postheparin plasma and abdominal adipose tissue (AAT) were determined in OFC, OL, and CONT hens. Basomedial hypothalamic lesions were performed in 3-month-old White Leghorn hens. At the static phase, 10 months later, OFC OL, and CONT hens were divided into three subgroups and injected IM on alternate days, with either 2 mg E2/kg b.wt., 10 mg TAM/kg, or vehicle corn oil, for 5 weeks. In OL and OFC hens, body and AAT weights were higher than in CONT poullets. Food intake and ovarian weight were similar in OL and CONT, higher than in OFC hens. Plasma LPL activity was higher, whereas plasma apo-VLDL-II and stimulated lipolysis were lower in OFC than in OL and CONT hens. In OFC hens LPL activity per unit of AAT was half than in OL and CONT. Total LPL activity in AAT was similar in OFC and CONT and higher in OL hens. Levels of basal lipolysis were similar in all experimental hens. TAM did not affect any of the measured parameters in OFC hens. In OL and CONT hens, TAM depressed apo-VLDL-II, increased plasma LPL activity, but had no effects on AAT LPL activity, on stimulated lipolysis, or fattiness. E2 increased apo-VLDL-II to similar levels in all groups and reduced LPL activity in plasma and AAT of obese hens. Only in OFC hens did E2 enhance basal and stimulate lipolysis and reduce FI and fattiness. We conclude that in adult laying hens, unlike in cockerels and juvenile hens, estrogen reduces lipid incorporation in fat depots by enhancing apo-VLDL-II production that reduces plasma and AAT LPL activity. This may increase lipoprotein available for incorporation into developing yolks. The lack of estrogen in OFC hens reduces circulating apo-VLDL-II and thus increases LPL activity and amount of fat depots.

Effects of dietary protein under high ambient temperature on body weight, breast meat yield, and abdominal fat deposition of broiler stocks differing in growth rate and fatness.

The effect of dietary protein on growth, feed intake and efficiency, abdominal fat deposition, and breast meat yield was investigated in broiler males from a commercial stock (WI) and from experimental stocks selected for low (LF) or high (HF) abdominal fat. All birds were kept at constant high ambient temperature (32 C) and were provided with low- (LP) or high-protein (HP) diets from hatch until 8 wk of age (Experiment 1) or from 4 to 8 wk of age (Experiment 2). In both experiments, HP diet significantly increased 4- to 8-wk BW gain in the LF and HF stocks but reduced it in the WI stock as compared with the LP diet. Abdominal fat, as percentage of BW, was almost twofold higher in the HF birds than in the LF ones, with WI mean being intermediate. In contrast to the HF and WI birds, in which abdominal fat decreased with increased protein intake, abdominal fat was not affected by dietary protein in the LF stock. The HP diet substantially increased breast meat yield in LF birds but not in the WI birds, with HF birds exhibiting intermediate increase in breast meat weight. It was concluded that birds of varied inherent growth rate and tendencies toward protein and fat deposition respond differently to dietary protein level under heat stress.

Early transition of the digestive system to exogenous nutrition in domestic post-hatch birds.

The effect of early transition of the digestive system to exogenous nutrition was examined in three experiments with growing birds. A nutrient mixture (0.5 ml) of glucose, starch and oil (1:1:0.5, by vol) was orally administered immediately after hatch to turkey poults (Meleagris gallopavo) having immediate or delayed access to feed (Expt 1). Increasing amounts (0, 0.25 and 0.5 ml) of this mixture were administered immediately after hatch to turkey poults (Expt 2), or to broiler chicks (Gallus domesticus; Expt 3). The relative weights of the gastrointestinal tract (GIT) and its ingesta content, and the amylolytic capability of the pancreas were examined during the immediate post-hatch period (to 30 h). Oral administration of nutrients immediately after hatching only slightly influenced the growth of the pancreas and its amylolytic activity, but significantly increased GIT weight in both species, in a dose-dependent manner. It is suggested that early post-hatching exposure of the digestive system by the forced administration of nutrient mixture induces anatomical and metabolic changes in the digestive system slightly earlier than in birds with late access to feed. This increases GIT content and plasma glucose levels, resulting in enhanced feed consumption and growth promotion.

Hyperglycemia and non-enzymatic glycation of serum and tissue proteins in chickens.

The objectives of these studies were to determine whether elevated plasma glucose concentrations in broiler breeder chickens (200-250 mg/dl) can result in the non-enzymatic attachment of glucose to serum proteins (fructosamine) and eventual cross-linking of tissue proteins (basement membrane thickness), and to investigate the effects of a factor that may influence this cross-linking process. In response to feeding the satiety factor calcium propionate (CaP, 1.7%), plasma glucose and fructosamine concentrations were increased (P < 0.05) from 1 to 9 weeks of age, whereas concentrations of plasma glucose and fructosamine in feed-restricted chicks were reduced for the first 7 weeks after hatch. In a second study, the age-related increase in kidney capillary basement membrane thickness was prevented (P < 0.05) by feeding the cross-linking inhibitor aminoguanidine (AG, 800 ppm) to 30-week-old broiler breeder hens for 34 weeks. The results from these studies suggest that concentrations of plasma glucose in chickens may, in fact, be exerting long-term detrimental effects on tissue proteins, which can be ameliorated by factors that limit the cross-linking reaction.

Broiler chick responses to anorectic agents: dietary acetic and propionic acids and the blood metabolites.

The effects of dietary supplementation with acetic or propionic acid, given at a level of 3% of diets containing 2,500 or 2,750 kcal of metabolizable energy per kg, on levels of blood glucose, and on lipid, protein, and energy metabolisms were studied in female broiler chicks. Voluntary feed and energy intakes, as well as body weight gain, decreased significantly with the inclusion of the acids in either diet, in decreasing order of effect: propionate > acetate > nonsupplemented controls. The relative weight of the abdominal adipose tissue decreased significantly with the inclusion of either acid, but was unaffected by dietary energy level. In contrast, the weight of the liver was higher in birds fed the low-energy diet, but was unaffected by the anorectic agents. Inclusion of the acids increased the concentration of volatile fatty acids in the cecum and blood plasma. The rate of clearance of plasma propionate was greater than that of acetate. Anorectic agents and dietary energy density exhibited a synergistic effect on feed intake. These agents altered blood metabolites involved in lipid and energy metabolisms, but not in that of protein. Plasma metabolites and enzymes, particularly beta-hydroxybutyrate and alkaline phosphatase, reflected the degree of feed restriction, supporting the hypothesis that the hypophagic effect of these acids is due to metabolic intervention.

Broiler chick responses to anorectic agents: 1. Dietary acetic and propionic acids and the digestive system.

The effects of various levels of acetic or propionic acid supplementation on feed intake and the gastrointestinal tract were studied in female broiler chicks. Voluntary feed intake and body weight gain decreased with the inclusion of the acids. Results indicate that acetic and propionic acids exert a similar anorectic effect on chicks fed diets differing in energy content. The inhibitory effect of these acids on voluntary feed intake could not be attributed to alterations in feed utilization, to differences in pancreatic alpha-amylase activity, or to differences in the acid-base balance. The increase in cecal capacity and volatile fatty acid (VFA) content is probably due to increased utilization of nonstarch polysaccharides at that site, the latter compensating for feed inhibition. This reflects a slight adaptive response of the cecum to anorectic agents.

Plasma apolipoprotein VLDL-II and egg production in laying hens: establishment of an ELISA method.

The developmental pattern of apolipoprotein VLDL-II (apo-II) in the plasma with regard to lipid concentration was characterized during the transition of pullet females to the laying phase in commercial laying hens (Gallus domesticus). Apo-II was isolated from the plasma of estrogen-administered roosters, and a rapid ELISA was developed for its quantification in chickens. The sensitivity of the assay was 5 ng/ml, and the inter- and intra-assay coefficients of variation were 4 and 8%, respectively. Whereas no detectable levels of apo-II could be monitored in the blood plasma of immature chickens, the level increased slightly to 23 weeks of age and sharply at the onset of lay. This profile reflects the dramatic changes in lipid metabolism at the onset of lay, and concurs with the concentration of total lipids in the plasma and with egg production rate. Plasma apo-II of 2 laying strains differing in body weights but compared at approximately similar egg production rates, tended to be slightly higher in heavy vs light-weight type hens. Oogenesis was speculated to be the main factor affecting both apo-II and total lipid profiles in chicken plasma, enabling efficient delivery of VLDL to the egg.

Dietary supplementation with xanthophyll as an effective way of identifying low-producing broiler breeder hens.

The relationship between dietary levels of xanthophyll, the degree of pigmentation in the hen, and egg production rate was studied in commercial broiler breeders (Anak 2000). In the first study, the degree of shank and beak coloration, measured with a 15-grade Roche yolk color fan, was determined in broiler breeder pullets until 34 wk of age. Although overall body coloration decreased with age following initiation of egg production, the degree of shank coloration was two- to threefold higher than that of the beak. In the second study, Roche Carophyll-red (canthaxanthin, 10%) was supplemented at levels of 0, 10, and 20 mg/kg in a corn and soybean diet containing xanthophyll, and given to hens for a period of 4 wk beginning at 55 wk of age. Increasing dietary canthaxanthin levels increased pigmentation of beak and egg yolk in a quadratic manner, whereas pigmentation increased linearly in the shank. The production rate was inversely related to the degree of shank and beak pigmentation. In a third study, the relationship between hen pigmentation (using Carophyll-red, 30 mg/kg of diet) and production rate was examined in a commercial flock of broiler breeder hens. Hens were sampled according to shank coloration (Grades 1 to 3) and egg production was monitored. The production rate of hens with high coloration was significantly lower (by approximately 33%) than the flock average. At peak production, shank pigmentation was too low to differentiate visually between degrees of coloration. Dietary supplementation of 5% corn gluten meal increased shank pigmentation and enabled efficient identification of the nonlaying hens.

Effect of dietary polyunsaturated fatty acid concentration on performance, fat deposition, and carcass fatty acid composition in broiler chickens.

The effect of dietary polyunsaturated fatty acid (PUFA) concentration on body fat deposition and composition in broiler chickens was studied. Twenty-day-old birds were fed five isocaloric diets supplemented with different combinations of tallow and vegetable oils. Contents of abdominal adipose tissue (AAT) as well as body fat were determined at 40 days of age. A significant linear effect was observed between dietary PUFA and feed utilization. Fatty acid (FA) profiles in AAT and total body fat were correlated to dietary PUFA content, with the main effect of higher dietary PUFA being a reduction in monoenoic FA (C16:1 and C18:1) and an increase in C18:2. The effect of dietary PUFA on the saturated FA (C16:0 and C18:0) was small and not statistically significant. Increased dietary PUFA modified FA composition such that C18:2, rather than C18:1 became the dominant FA in AAT. In that respect, greater changes were observed in FA composition of AAT, as compared with that on the whole carcass. The deposition of saturated and monoenoic FA in the whole carcass during the experimental period was related to dietary PUFA content in a quadratic manner. However, deposition of C18:2 and C18:3 was not affected by intake and their utilization (deposition:intake ratio) was reduced in a manner parallel to their dietary levels.

The possible prediction of fatness in broiler chickens by biochemical measurements on adipose tissue.

1. The possible prediction of fatness in 6-week-old broiler chickens was examined by measuring lipoprotein lipase (LPL) activity and lipid content in the abdominal adipose tissue. 2. Fat pad weight, as representing the degree of fatness in broiler chickens, was moderately well correlated (r = 0.54) with lipid content (g/100 g pad) and negatively correlated with LPL activity (r = 0.13). 3. It was concluded that the measurement of lipid content in samples from adipose tissue can be indicative of fatness of birds, while enzymic measurements are less valuable.

Relationship between the weight of hatching eggs and subsequent early performance of broiler chicks.

1. The effect of egg weight on the subsequent performance of individual growing chicks was studied. Eggs from commercial broiler breeder hens were collected at 52, 55 and 57 weeks of age and incubated. The chicks were reared for 18 d. 2. Egg weight and hatched chick weight were not affected by the age of the hens during the 52 to 57-week period, but there was a close correlation between egg weight and hatching weight, irrespective of the age of the hens (r = 0.89). Chick:egg weight ratio at hatching was about 0.71 and independent of hen age. One d after hatching, chick weights had decreased by about 1.5 g. 3. The initial high correlation between egg weight and chick weight decreased markedly during post-hatching growth, becoming insignificant 5 d after hatching. At 18 d of age, however, chicks from eggs of older hens tended to be heavier than those from younger birds. The greater 18-d weights appeared to be associated more with higher food intake during that period, than with greater egg weight. 4. It was concluded that, in individual broiler chicks, the advantage of the initially higher weight attributable to a larger egg diminishes rapidly after hatching, and the main factor affecting final body weight is food intake.

Metabolic and anatomical adaptations of heavy-bodied chicks to intermittent feeding. 2. Pancreatic digestive enzymes.

1. Activities of digestive enzymes in meat-type chickens under ad libitum or alternate-day feeding were determined from 14 to 83 d of age. 2. Final body weight of intermittently fed birds attained 75% of that of the ad libitum-fed controls. 3. When compared with the ad libitum-fed counterparts, a marked increase in the relative weight of the pancreas and intestinal contents were found on repletion days. On depletion days the relative weights of the pancreas and of the intestinal contents were about half those found in ad libitum-fed birds. 4. The activity of the digestive enzymes in the pancreas, expressed as U/g pancreas or U/kg body weight, was not affected consistently by the feeding regime. In the small intestine a marked increase in relative activity (U/kg body weight) was observed on repletion days and a marked decrease on depletion days as compared with ad libitum-fed controls. The activities per g intestinal contents following food restoration did not differ significantly from those of ad libitum-fed controls except for trypsin, which was higher in the former. On depletion days the activities per g intestinal contents were lowest, lipase excepted.

Broiler chick response to low protein diets supplemented with synthetic amino acids.

Performance of broiler chicks fed low protein diets supplemented with several essential amino acids (EAA) is generally inferior to that of birds fed a higher protein diet composed primarily of intact protein. Two experiments were conducted to determine whether lowering the minimum requirements of the EAA in proportion to the lowered protein concentration or equalizing the dietary amino nitrogen content by use of glutamic acid would prevent the reduction in performance. Chicks were fed the experimental diets from 7 to 21 days of age, and body weight gain, feed consumption, and feed efficiency were determined. In the first experiment, the chicks were fed diets with 23, 20, or 17% CP with minimum EAA set at 100% of the 1984 National Research Council (NRC) suggested requirements. Other groups were fed the 20% CP diet with EAA set at 93.5% or the 17% CP diet set at either 93.5 or 87.5% of the NRC requirements. In the second experiment, the 23% and 17% CP diets were fed, and glutamic acid was added at 3, 6, or 9% to provide the amino nitrogen equivalent of 23% protein with the minimum EAA set at 100% of 1984 NRC requirements. In the first experiment, body weight gain and feed efficiency, but not feed intake, were depressed by lowering CP content. Reducing the minimum EAA requirements in proportion to the reduction in CP failed to correct the difference in performance between the high and low CP diets. In Experiment 2, body weight gain and feed intake were reduced as intact CP decreased and glutamic acid increased, but feed efficiency was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy requirement of feed-restricted broiler breeder pullets.

The energy requirements for growth and maintenance of broiler pullets were studied during a 17-wk growing period. Four flocks of commercial broiler replacement pullets, housed in conventional poultry houses, received a restricted amount of feed on 4 or 5 days a week, and pullet growth was recorded. The energy needs for maintenance and body weight gain under a feed restriction regimen was calculated using a model based on metabolic body weight and weight gain described by Hurwitz and coworkers. Average daily body weight gain for the entire experimental period was 13.7 g; the daily energy intake increased with age from 114 kcal/day at 3 wk of age to approximately 270 kcal/day at 20 wk. The estimated daily metabolizable energy requirement for maintenance was 1.57 kcal/g of body weight67 and for growth, .71 kcal/g of body weight. It was concluded that the energy requirement for birds under a feed restriction regimen is considerably lower than those under ad libitum feeding.

Muscle growth and composition in heavy and light breed chickens adapted to intermittent feeding.

1. The effect of intermittent feeding in chickens of heavy breed (HB; meat type) and light breed (LB; egg type) on skeletal muscle growth and composition was studied in adapted and non-adapted chickens. 2. Food intake, relative to body-weight, was similar in both breeds but was higher in ad lib.-fed than in intermittently fed birds. 3. On repletion days the relative growth rate was similar in both breeds, while on depletion the LB chickens lost more weight than the HB chickens. In both breeds, the relative growth was higher in the intermittently fed birds during days of food restoration than in those fed ad lib. 4. The relative weight of the breast muscle was higher in HB birds than in LB birds, but deposition rate on the day of food restoration was similar in both breeds. This growth was more pronounced in chickens adapted to alternate feeding than in chickens exposed to this feeding regimen for one cycle. 5. Protein concentration in breast muscle was not affected by age and was slightly higher in LB chickens than in HB chickens. Soluble protein was markedly reduced on days of repletion, and more at 46 d than at 18 d of age. 6. The RNA:DNA ratio was higher in HB than in LB chickens, and lower on days of food deprivation than on days of food restoration. After repletion this ratio returned to the level of the ad lib.-fed chickens. While in LB chickens cell size (as estimated by DNA concentration) remained constant on repletion and depletion days, in the HB chickens it decreased. 7. The rapid growth of breast muscle in HB chickens was attributed to the higher rate of protein synthesis (estimated by RNA:DNA ratio) compared with LB chickens. This may also explain why the breast muscle of LB chickens was less sensitive to intermittent feeding than that of HB chickens.

Glycolic acid and tryptophan effects on feed intake and hypothalamic indoleamines in chicks.

The effect of intraperitoneal injections of glycolic acid, glycine or tryptophan on feed intake and hypothalamic levels of 5-hydroxy-tryptamine (5-HT) and 5-hydroxy indole acetic acid (5-HIAA) was examined in chicks. Glycolic acid significantly depressed feed intake in a dose-dependent manner in comparison to saline or glycine, but only in birds injected with a level higher than 150 mg/kg body weight. The injection of glycolic acid or tryptophan but not glycine inhibited feed intake, and increased hypothalamic 5-HT and 5-HIAA 2 hr post-injection, as compared with saline injected controls. Blood plasma total lipids were elevated by all 3 compounds but plasma glucose was unaffected. The reduced feed intake obtained by injection of glycolic acid appears to result from an effect on the serotonergic system.

The synthesis in vivo of proteins in various tissues in chickens adapted to intermittent feeding.

1. Protein synthesis was estimated in vivo in breast (superficial pectoral) and tibia (gastrocnemius) muscles, liver, kidney, pancreas, crop, duodenum, jejunum and ileum, using L-[U-14C]lysine injection. The effect on incorporation of [14C]lysine 1 and 2 h after injection was examined in five chickens adapted or not adapted to intermittent feeding. 2. Incorporation of [14C]lysine into tissue decreased in magnitude in the following descending order: pancreas greater than jejunum, duodenum greater than ileum, crop, liver greater than kidney greater than tibia, breast muscle and blood plasma. 3. The incorporation of [14C]lysine into muscle protein was higher in chicks after 24 h of refeeding than after 24 h of food deprivation. These differences were higher in adapted than in non-adapted birds. On days of refeeding the rate of incorporation exceeded that found in chickens fed ad lib. 4. Bound 14C from lysine in the intestinal segments was less than in control birds after food deprivation and greater after refeeding in non-adapted chicks only. 5. A negative relation was observed between bound and free 14C in muscles and in other tissues. 6. Short- and long-term adaptations to feeding regimens are discussed.