Status Of Chickens Research Articles

Immunological status of White Leghorn chicks hatched from eggs inoculated with ochratoxin A (OTA)

This study was designed to evaluate the immunological status of chicks hatched from the ochratoxin A (OTA)-contaminated eggs. For this purpose, 900 fertile White Leghorn (WL) layer breeder eggs were divided into eight groups (A–H). Group A was maintained as untreated control, whereas Group B was kept as sham control (10 µL of 0.1 M NaHCO3). Groups C, D, E, F, G, and H were injected with 0.01, 0.03, 0.05, 0.10, 0.50, and 1.00 µg OTA/egg, respectively. Eggs were incubated at 37.5°C and 65% relative humidity. Hatched chicks from each group were then maintained separately under standard environmental conditions. At Day 18-of-age, chicks (n = 10) from each group were used for lymphoblastogenic response against an intradermal administration of phytohemagglutinin P (PHA-P). At Day 30-of-age, abdominal macrophages, collected from 15 chicks in each group, were utilized for determination of phagocytic potential using sheep red blood cells (SRBC) as particulate antigen and for nitrite production in response to lipopolysaccharide. Antibody (Ab) titers (i.e. total antibodies, IgG, and IgM) against SRBC were determined at 7 and 14 days after primary (at Day 13-of-age) and booster (given 14 days after primary) intravenously administered SRBC doses. The lymphoblastogenic responses of the chicks hatched from OTA-contaminated eggs in response to PHA-P administration were significantly lower at 24, 48, and 72 h after PHA-P injection when compared with responses by control chicks. The percentage of abdominal macrophages displaying phagocytosis of SRBC, the number of SRBC/macrophage, and nitrite production were each significantly lower in cells from chicks in the OTA-administered groups. Total Ab, IgG, and IgM titers against SRBC showed significant reductions in the groups that had been hatched from eggs injected with the higher doses of OTA (as compared with titers associated with chicks in control eggs). These findings suggested that there are substantive immunosuppressive risks in chicks that could be exposed to OTA in ovo.

Zinc Supplementation against Eimeria acervulina-Induced Oxidative Damage in Broiler Chickens.

This study was undertaken to determine the dietary supplements of Zn containing diet on the antioxidant status in chickens experimentally infected with Eimeria acervulina. The antioxidant status was monitored via determination of MDA concentrations and erythrocyte SOD and CAT activities, as well as vitamin E, vitamin C, Cu, and Zn in liver, muscle, and serum. The results showed increased MDA (P < .05), CAT (P < .001), and decreased SOD (P < .001) in the infected birds. Significant changes in Cu and Zn concentrations and dramatically reduction of vitamin C and E concentrations in the infected chickens were found. The observed deviations in the studied enzymes and nonenzymatic parameters evidence the occurrence of oxidative stress following the infection and impaired antioxidant status of chickens, infected with Eimeria acervulina. Our results proved the ameliorating role of CuZn(OH)3Cl (0.170 g per kg food) against Eimeria acervulina-induced oxidative damage in infected chickens.

FAS inhibitor cerulenin reduces food intake and melanocortin receptor gene expression without modulating the other (an)orexigenic neuropeptides in chickens

Cerulenin, a natural fatty acid synthase (FAS) inhibitor, and its synthetic analog C75 are hypothesized to alter the metabolism of neurons in the hypothalamus that regulate ingestive behavior to cause a profound decrease of food intake and an increase in metabolic rate, leading to body weight loss. The bulk of data exclusively originates from mammals (rodents); however, such effects are currently lacking in nonmammalian species. We have, therefore, addressed this issue in broiler chickens because this species is selected for high growth rate and high food intake and is prone to obesity. First, we demonstrate that FAS messenger and protein are expressed in the hypothalamus of chickens. FAS immunoreactivity was detected in a number of brain regions, including the nucleus paraventricularis magnocellularis and the nucleus infundibuli hypothalami, the avian equivalent of the mammalian arcuate nucleus, suggesting that FAS may be involved in the regulation of food intake. Second, we show that hypothalamic FAS gene expression was significantly (P < 0.05) decreased by overnight fasting similar to that in liver, indicating that hypothalamic FAS gene is regulated by energy status in chickens. Finally, to investigate the physiological consequences of in vivo inhibition of fatty acid synthesis on food intake, we administered cerulenin by intravenous injections (15 mg/kg) to 2-wk-old broiler chickens. Cerulenin administration significantly reduced food intake by 23 to 34% (P < 0.05 to P < 0.0001) and downregulated FAS and melanocortin receptors 1, 4, and 5 gene expression (P < 0.05). However, the known orexigenic (neuropeptide Y, agouti gene-related peptide, orexin, and orexin receptor) and anorexigenic (pro-opiomelanocortin and corticotropin-releasing hormone) neuropeptide mRNA levels remained unchanged after cerulenin treatment. These results suggest that the catabolic effect of cerulenin in chickens may be mediated through the melanocortin system rather than the other neuropeptides known to be involved in food intake regulation.

Analysis of Zinc and Magnesium Levels in Pinealectomized Chicks: Roles on Development of Spinal Deformity?

Melatonin is the main product of the pineal gland, and trace metals play a critical role in growth and development. The purpose of this study was to assess the serum zinc (Zn) and magnesium (Mg) levels in pinealectomized chicks and their possible interactions with the development of spinal deformity. Chicks were divided into two equal groups: unoperated controls (group M) and pinealectomized chicks (group N). Pinealectomies were performed at the age of 3 d. After 8 wk, serum Zn and Mg levels of 10 animals from each group were measured by spectrophotometric assay. The results of analyses were compared using the Mann-Whitney U-test. The correlation between serum Zn and Mg levels were assessed by Spearman's correlation. In this study, it was obvious that the serum Zn levels in group N were significantly lower than those in group M (2.8 +/- 0.10 vs 4.2 +/- 0.14 ppm; p < 0.0005). In contrast, Mg levels in group N was high compared with the values in group M, although there was no significant difference (17.8 +/- 0.69 vs 15.7 +/- 0.85 ppm; p > 0.05). In pinealectomized animals, serum Zn levels declined significantly while serum Mg levels increased, albeit insignificantly. Thus, there was a moderately positive but not statistically significant correlation between Mg and Zn levels in unoperated controls (r = 0. 273, p > 0.05), whereas there was a negative but not statistically significant correlation between Zn and Mg levels in pinealectomized chicks (r = -0.115, p > 0.05). In addition, the serum Mg to serum Zn ratio was significantly higher in group N than in the group M control (6. 39 +/- 0.32 vs 3.75 +/- 0.22, respectively; p < 0.001). From the results of the current study, it is clear that surgical pinealectomy in newly hatched Hybro Broiler chicks has a significant effect on serum Zn level. However, the serum Mg did not change significantly. Because serum Mg is not a good indicator of Mg status in chicks, it is speculated that other tissues, such as muscle or spine, might productively be explored as a more sensitive Mg biomarker for this model. The present study provides experimental evidence that serum trace metal levels might be affected in pinealectomized animals because of the lack of its main neurohormone melatonin.

Effects of saturated and unsaturated fats with vitamin E supplementation on the antioxidant status of broiler chicken tissues.

The influence of fish oil (highly unsaturated) and beef tallow (highly saturated) with vitamin E (100 IU/kg) supplementation on the antioxidant status of broiler chicken cockerels was investigated. Chicks were fed a control diet with no added fat, 40 g/kg each of fish oil and beef tallow diets, respectively, from 11 to 42 days of age. Tocopherol concentration and the rate of lipid peroxidation, thiobarbituric acid reactive substance (TBARS) in liver, fatty acid composition of the liver lipids, blood serum total antioxidant status (TAS), and reduced glutathione (GSH) content were determined. Vitamin E supplementation of the diet increased liver alpha-tocopherol content in chicks regardless of the type of dietary fat. Fish oil diet resulted in higher liver TBARS value while beef tallow diet showed lower values compared to the control diet. Vitamin E supplementation reduced liver TBARS as well as serum GSH, and raised serum TAS for all diets. Serum GSH was the same for vitamin E supplemented diets regardless of the fat supplement. Fish oil diets resulted in a significant increase in hepatic lipid n-3 PUFA content. A significant positive correlation was found between liver TBARS and n-3 PUFA content. No relationships were established, however, between liver TBARS and n-6 PUFA or saturated fatty acids. The results suggest that feeding oils rich in n-3 PUFA increases tissue concentration of these fatty acids, consequently increasing tissue lipid peroxidation and reducing the antioxidative status of broiler chickens. Supplementing high levels of vitamin E with such oils may increase tissue oxidative stability. Serum TAS or GSH may be used as a measure of antioxidative status in chickens.

The influence of vitamin c on the antioxidative status of chickens in vivo at slaughter and on the oxidative stability of broiler meat products

Broiler chickens were fed diets containing 0, 210, 420 or 840 mg vitamin C kg−1 feed from 10 to 42 days of age. The diets contained 13.6% fat and 87.7% unsaturated fatty acids. Activity of creatine‐kinase (CK) and glutathione peroxidase (GSH‐Px) decreased with increasing levels of dietary vitamin C. No statistical difference between groups was found with respect to the concentration of vitamin E or lutein in plasma, but chickens fed diets containing 420 or 840 mg vitamin C kg−1 feed had a significantly higher plasma concentration of retinol and ß‐carotene compared with chickens fed diets containing 0 to 210 mg vitamin C kg−1 feed. A considerable erythrocyte haemolysis in vitro was demonstrated in all groups and increased with increasing dietary level of vitamin C. The unsupplemented group had a lower concentration of fatty acids expressed as a percentage in extracted fat compared with the vitamin C supplemented group. No influence of dietary vitamin C on the concentration of antioxidative vitamins in the liver, the breast and the thigh muscle was observed. Nor did vitamin C influence the energy metabolism of breast and thigh muscle. Vitamin C did not affect the oxidative stability of the meat under chill and freezer storage.

Virulent Salmonella typhimurium-induced lymphocyte depletion and immunosuppression in chickens.

The effect of experimental Salmonella infection on chicken lymphoid organs, immune responses, and fecal shedding of salmonellae were assessed following oral inoculation of 1-day-old chicks or intra-air-sac infection of 4-week-old chickens with virulent S. typhimurium wild-type chi 3761 or avirulent S. typhimurium delta cya delta crp vaccine strain chi 3985. Some 4-week-old chickens infected intra-air-sac with chi 3761 or chi 3985 were challenged with Bordetella avium to determine the effect of Salmonella infection on secondary infection by B. avium. S. typhimurium chi 3761 caused lymphocyte depletion, atrophy of lymphoid organs, and immunosuppression 2 days after infection in 1-day-old chicks and 4-week-old chickens. The observed lymphocyte depletion or atrophy of lymphoid organs was transient and dose dependent. Lymphocyte depletion and immunosuppression were associated with prolonged fecal shedding of S. typhimurium chi 3761. No lymphocyte depletion, immunosuppression, or prolonged Salmonella shedding was observed in groups of chickens infected orally or intra-air-sac with chi 3985. Infection of chickens with salmonellae before challenge with B. avium did not suppress the specific antibody response to B. avium. However, B. avium isolation was higher in visceral organs of chickens infected with chi 3761 and challenged with B. avium than in chickens infected with B. avium only. Infection of chickens with chi 3985 reduced B. avium colonization. We report a new factor in Salmonella pathogenesis and reveal a phenomenon which may play a critical role in the development of Salmonella carrier status in chickens. We also showed that 10(8) CFU of chi 3985, which is our established oral vaccination dose for chickens, did not cause immunosuppression or enhance the development of Salmonella carrier status in chickens.

Penicillamine: Pharmacokinetics and Differential Effects on Zinc and Copper Status in Chicks

The pharmacokinetics of D(-)-penicillamine (PA) and the effects of PA on the nutritional status of Cu and Zn in chicks were studied. When administered by subcutaneous or intravenous injection, PA was eliminated from the plasma with biphasic kinetics: i.e., a fast (halftime: 6-7 min) and a slow (halftime: 52-55 min) component. The elimination of PA from the liver or pancreas showed only the slow (halftime: 42-46 min) component. When PA was administered orally, plasma PA concentrations responded over time in bimodal pattern, but the maximal level was only one-fifth that achieved by injection. The subcutaneous injection of PA elicited differential, transient changes in the concentrations of Zn and Cu in the plasma: plasma Zn concentration was decreased, and plasma Cu concentration was increased. Analyses of urine collected from ureter-cannulated chicks showed that PA was rapidly excreted via that route, and that urinary PA excretion was associated with increased urinary excretion of both Zn and Cu. These results show that PA treatment can alter tissue distributions particularly of Zn and, to a lesser extent, of Cu. J. Nutr.

Reduced selenium status in chicks given diets low in Sulphur‐containing amino acids and containing Sodium Nitroprusside as a source of cyanide

1. Two experiments were carried out to study the effect of dietary cyanide, given in the form of sodium nitroprusside (SNP), on the growth and selenium status of chicks fed on diets low in sulphur-containing amino acids. 2. In experiment 1, SNP (0.3 g/kg) depressed growth rate and food intake when included in diets low in total sulphur-containing amino acids. It also reduced plasma and liver glutathione peroxidase activity (GSH-Px) and kidney selenium concentration. These latter variables also showed increases in response to supplements of selenium of 0.1 and 0.2 mg selenium/kg (as sodium selenite) although growth was not affected. 3. In experiment 2, SNP did not affect growth when reduced to 0.1 g/kg in diets low both in methionine and total sulphur-containing amino acids. It did, however, reduce plasma GSH-Px activity, which again increased in response to similar dietary supplements of selenium. 4. It is concluded that dietary cyanide effectively increases the requirement for selenium and could induce deficiency in diets only marginally adequate in selenium, particularly when the supply of sulphur-containing amino acids was marginal or inadequate.

The use of an enzyme-linked immunosorbent assay for estimation of the immune status of chickens artificially immunized against coccidiosis.

Nine thousand commercial breeder chicks (Chankee) reared in a floor pen were exposed to restricted numbers of Eimeria tenella and E. necatrix oocysts to confer immunity. Antibody induction in these chicks was examined by the enzyme-linked immunosorbent assay (ELISA) with antigen prepared from E. tenella oocysts. The oocyst excretion pattern demonstrated recycled infections which continued in these chicks for greater than or equal to 22 days after exposure. Antibody levels in their sera, as determined by the mean absorbence values in ELISA, increased gradually up to 38 days post-inoculation. Mean absorbence values of sera from control chicks remained at a low level. When infected and control chicks were challenged with the two species of coccidia, the test chicks were protected against both species. The antibody level did not change for 8 days in the challenge groups, while in the control chicks, absorbence in ELISA rose significantly and the mean absorbence value was higher than that in immunized chicks. Some factors which influence the results of ELISA are considered and the applicability of this method to measuring immunity against coccidiosis in chickens is discussed.

Relation between some folate-dependent metabolic pathways and dietary folate content in chicks.

Responses of several folate-metabolizing pathways to dietary folic acid were studied in 2-week-old chicks. Oxidation of a histidine load to carbon dioxide was impaired in folate-deficient chicks. There was a curvilinear relation between oxidation and dietary folate, and maximum oxidation occurred with 2 mg supplemental folic acid/kg. Hepatic activities of glutamic acid formiminotransferase (EC 2.1.2.5) and glycine N-methyltransferase (EC 2.1.1.20) were not affected significantly (P greater than 0.05) by dietary folic acid. The activity of dihydrofolate reductase (EC 1.5.1.3) in erythrocytes was elevated in folate-deficient chicks. These studies show that the activities of two folate-dependent pathways can be used as biochemical criteria of folate status in chicks.

Effect of fasting and acute heat stress on body temperature, blood acid-base and electrolyte status in chickens

1. 1. The tolerance of chickens to acute heat stress, evaluated by the time required to reach the critical body temperature ( T r) of 44.5°C, was markedly enhanced as the period of fasting was extended. 2. 2. Fasting reduced the rates of heat-induced changes in blood acid-base and electrolyte status. 3. 3. Changes in T r were correlated with changes in blood pH, pCO 2, [Cl −] and [Pi] but not with changes in [Na +]or [K +]. 4. 4. Blood acid-base and electrolyte status were related to T r rather than time of exposure to heat stress.

Effect of dietary sodium nitroprusside as a source of cyanide on the selenium status of chicks given diets of varying selenium concentration.

1. The interaction between dietary cyanide, given in the form of sodium nitroprusside (SNP), and selenium has been studied in two experiments with growing chicks from 14 to 38 d of age. 2. In experiment 1, dietary selenium at 10 mg Se/kg reduced growth, food intake and efficiency of utilisation, and increased relative liver size and selenium content. All of these effects were eliminated by the addition of 0.1 g SNP/kg except for liver selenium content, which progressively declined towards control values as SNP was increased to 0.4 g/kg in increments of 0.1 g/kg. At 0.3 g SNP/kg, cyanide toxicity, as judged by decreased growth, reached significance. 3. In experiment 2, similar effects were observed with selenium at 10 mg Se/kg and SNP at 0.3 g/kg, but selenium deficiency was not evident from growth indicators when selenium supplementation of the diet was omitted completely, nor did these indicators suggest that deficiency was induced by cyanide. 4. In both experiments, plasma and liver glutathione peroxidase activity reflected the dietary selenium content. There was an interaction with dietary SNP content. With selenium intake at a toxic level, SNP increased enzyme activity, further evidence of alleviation of selenium toxicity, but when selenium intake was low and normal, SNP decreased activity in liver, an indication that cyanide could induce deficiency. 5. A possible mechanism for alleviation of selenium toxicity is proposed.

Mercaptans Decrease Selenium-Dependent Glutathione Peroxidase Activity in the Chick

A variety of mercaptans, especially the β-mercaptocarboxylic acids, inhibited the selenium (Se)-dependent glutathione peroxidase (SeGSHpx) activity of chick liver postmitochondrial supernatant or cytosol and of purified bovine erythrocyte SeGSHpx. The effects of mercaptans and a glutathione analogue on Se utilization were determined by subcutaneous injection of test compounds into vitamin E–deficient chicks fed diets containing 0.1 ppm Se (as Na2SeO3) at times when > 50% of vitamin E– and Se-deficient chicks showed the vitamin E–, Se-deficiency disease exudative diathesis (ED). D-(-)-Penicillamine hydrochloride (the positive control model compound), sodium β-mercaptopyruvate, t-butyl mercaptan and S-methylglutathione (nonmercaptan glutathione analogue) decreased SeGSHpx activity in chick liver postmitchondrial supernatants within 24 h of injection and increased the incidence of ED within 4 d. Other mercaptans tested did not increase ED incidence or affect liver, kidney or plasma SeGSHpx activities. Although mercaptosuccinic acid, N-(2-mercaptopropionyl)glycine and sodium thioglycolate each strongly inhibited SeGSHpx in vitro, each also significantly increased chick mortality in the dosage range tested; therefore, their effects on SeGSHpx in vivo could not be evaluated. It appears that the β-mercaptocarboxylic acids, mercaptans with a high degree of steric hinderance in close proximity to the thiol group and a close structural analogue of glutathione are capable of altering selenium status in chicks.

Effects of dietary selenite, copper, and zinc on tissue trace mineral levels in chicks

Studies were conducted to determine whether nutritional selenium (Se) status affects the nutritional status of the chick with respect to other trace elements, particularly copper (Cu) and Zinc (Zn). Severe Se deficiency was produced in chicks by the use of diets that contained exceedingly low contents (less than 0.010 ppm) of Se, but contained adequate amounts of all other known essential nutrients. This diet was based upon corn and soybean meal produced in areas of China with endemic Se deficiency of geobotanical origin. A level of at least 0.10 ppm Se was found to be required to maintain normal Se status of chicks fed this diet, and Se deficiency resulted in decreased levels of Cu, Zn, and molybdenum in the pancreas (liver and plasma levels were not affected). High dietary supplementation of Zn nor Cu did not affect the short-term utilization of Se, as indicated by the 18-h responses of Se-dependent glutathione peroxidase in plasma and liver.

Interaction of lead toxicity and riboflavin status in chicks ( Gallus domesticus)

1. 1. Chicks ( Gallus domesticus) were fed diets containing 0 or 2000 ppm lead (Pb) and adequate, marginal or deficient levels of riboflavin. 2. 2. In comparison to adequate, marginal riboflavin depressed growth at 2000 ppm Pb but not at 0 ppm Pb. 3. 3. Deficient riboflavin depressed growth at both Pb levels, and Pb depressed growth at all riboflavin levels. 4. 4. Hepatic glutathione reductase activity was reduced by riboflavin deficiency, but Pb was without effect. 5. 5. Hepatic non-protein sulfhydryl concentrations were increased by Pb, and the increases were greater at the lower riboflavin levels. 6. 6. Dietary Pb appears to increase the level of riboflavin required in chick diets.

Changes in serum levels of 1,25-dihydroxyvitamin D3, calcium and phosphorus with age and vitamin D status in chickens.

The effects of vitamin D3 (D3) on serum levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), ionic calcium, total Ca and phosphorus in chicks were studied from the time of hatching until sexual maturity. Chicks fed on a diet low in D3 showed a serum level of 1,25(OH)2D3 higher than that in chicks on a normal-D3 diet, for both sexes and at any given age. A dramatic increase in the serum level of 1,25(OH)2D3 occurred in female birds approaching sexual maturity and in laying hens raised on the low-D3 diet the level was five times that of their counterparts raised on a normal-D3 diet. The serum 1,25(OH)2D3 level in adult males in the low-D3 groups was seven times that of those on the normal-D3 diet. The serum level of 25-hydroxyvitamin D3 remained relatively unchanged at weeks 2 and 15 in birds on a low D3 intake as well as in those fed on a normal-D3 diet. Nevertheless, the levels of 25-hydroxyvitamin D3 were different between the two groups. No significant change was observed in the level of ionized serum Ca in relation to dietary regimen, but there was an increase in total Ca concentration in females with the onset of reproduction. The serum P level decreased gradually with age, reaching a minimum value 3 and 8 weeks before laying commenced in the groups on low- and normal-D3 diets respectively. An increase was observed when the hens began laying.(ABSTRACT TRUNCATED AT 250 WORDS)

Serological Comparison and Antigenic Relationships of Seven Serotypes of Infectious Bronchitis Virus Using the Hemagglutination-Inhibition Test

The antigenic relationships, antigenic spectrum, and immunogenicity of seven isolates of infectious bronchitis virus (IBV) were examined using the hemagglutination-inhibition (HI) test. Because there was a discontinuity of antigenic relationships and a high degree of cross-reactivity among serotypes of IBV in cross-hemagglutination-inhibition tests, the range of antigenic spectrum used to group the serotypes with the HI test should be wider than the limits suggested by the plaque-reduction test. The HI test may provide valuable information in monitoring the immune status of a flock following vaccination when the area has a history of infectious bronchitis infection. It may also be used as a rapid diagnostic test if a flock is experiencing an outbreak of a disease caused by emergence of a new type of IBV. Interpretation of HI titers in evaluating immune status of chickens following infection with IBV depends on further cross-challenge and cross-protection studies of various types of IBV.

Effect of Dietary Fiber Sources on Tissue Mineral Levels in Chicks

Dietary fiber may influence mineral status in chicks. Wheat bran, corn bran, soy bran, oat hulls, rice bran, and cellulose, were formulated into diets and fed to chicks for 4 weeks. Fiber-added diets contained additional 6% dietary fiber from the fiber sources. Body weights and feed intakes were recorded. Livers and tibias were analyzed for copper, zinc, iron, manganese, and magnesium. The rice bran diet resulted in significantly lowered body weights, feed intakes, and tibia concentrations of zinc, iron, and manganese. The other fiber-added diets did not produce significantly different values for the parameters examined. Dietary analyses showed that the rice bran diet did not have extremely high fiber levels as compared to the other diets. Addition of rice bran contributed the highest amount of phytic acid of all the fiber sources at 1.3% of the diet. It was concluded that when included at 6% of the diet, most fibers did not influence growth or tissue mineral levels. However, the addition of rice bran to the diet reduced body growth and deposition of zinc, iron, and manganese in tibias. It is possible that one fiber source may be beneficial while another is detrimental to nutritional status.

The Use of Sentinel Birds to Monitor Coccidial Infection in Litter Pens

Two-week-old chickens susceptible to coccidiosis were introduced into litter pens for periods up to 48 hr in order to estimate the potential coccidial challenge. They were then transferred to cages and the infections acquired were estimated by oocyst output or by body weight gain and lesion scores. Moderate to severe infection occurred even when about 1,000 oocysts/gram were present in the litter. The results suggest that the brief introduction of "sentinel" birds into an infected environment may be a valuable method of assessing the actual coccidial challenge which groups of chickens acquire from exposure to litter. The technique may be used to monitor the effectiveness of anticoccidial drugs or the immune status of chickens kept in litter pens by comparing infections in "sentinels" and "in-pen" birds.