Heat Shock Proteins as Modifying Factors in Physiological Stress Responses in Poultry

Five experiments were conducted to investigate the effects of genetic and neonatal manipulation on physiological stress response and their roles in thermotolerance parameters such as plasma corticosterone concentration (CORT), heterophil to lymphocyte ratio (HLR), heat shock protein 70 (Hsp70) and glucocorticoid receptor (GR) expression, body temperature, performance and Salmonella enteritidis resistance in poultry. In Experiment I, two trials were conducted to compare the thermotolerance of the red jungle fowl (RJF) (Gallus gallus spadiceus), village fowl (VF) and commercial broilers (CB) at (i) a common age (30 days old) and (ii) a common body weight (930±30 g) when exposed to 36 ±1 oC for 3 h. In both trials, irrespective of the stage of heat treatment the RJF had lower HLR, higher CORT concentration and higher Hsp70 expression than the VF and CB. Unlike the RJF, heat exposure significantly elevated the body temperature, HLR, CORT concentration and Hsp70 expression of CB. Experiment II was carried out to further ascertain the functional roles of CORT and Hsp70 in acquisition of thermotolerance through neonatal feed restriction. A total of 180 day-old broiler chicks were subjected to one of the following four feeding regimens: ad libitum (control); 60% feed restriction on days 4, 5 and 6 (FR60); 60% feed restriction on days 4, 5 and 6 +1500 mg/kg metyrapone (FR60M); 60% feed restriction on days 4, 5 and 6 +1500 mg/kg quercetin (FR60Q). To elicit heat stress from day 35 to 42, all chicks were exposed to 37±1°C for 3 h daily. The results suggest that subjecting chicks to FR60, as measured by HLR, FCR and weight gain, can enhance their ability to withstand, high ambient temperature and Hsp70 induction did not appear to play a profound role in the acquisition of thermotolerance. To gain more insights on the roles of Hsp70, the Hsp70 response and S. enteritidis colonization were investigated in neonatally feed restricted broiler chickens subjected to heat stress later in life in Experiment III. Chicks were divided into three feeding regimens: ad libitum (control); 60% feed restriction on days 4, 5 and 6 (FR60); 60% feed restriction on days 4, 5 and 6 +1500 mg/kg quercetin FR60Q). On d 35, all chickens were individually inoculated with 1 mL S. enteritidis (1.5×108 cfu/bird) and exposed to 37 ±1°C for 3 h daily. After heat exposure, the FR60 and FR60Q birds showed a significantly lower S. enteritidis colonization and lower Hsp70 expression than control birds. The least colonization was observed in the FR60Q group (1.38 log10 cfu/g in spleen and 1.96 log10 cfu/g in cecal content) and the highest in the control group (2.1 log10 cfu/g in spleen and 4.42 log10 cfu/g in cecal content). This clearly demonstrated that neonatally feed restricted chicks developed their thermotolerance ability and a greater ability to respond to the S. enteritidis invasion. In Experiment II we emphasised on relationship between CORT and Hsp70 responses under heat stress. To elaborate more on this relationship, Experiment IV was conducted to determine the relationship between Hsp70 expression in the heart and brain and CORT modulated by ascorbic acid and α-tocopherol supplementation in quails subjected to social isolation. Fifty three day old male Japanese quails were randomly assigned to each one of the six feeding regimens for 3 days as follows: (i)Basal diet (control); (ii) Basal diet+1500 mg/kg metyrapone (BM); (iii) Basal diet+30 mg/kg corticosterone (BCO); (iv) Basal diet +250mg/kg ascorbic acid (BC);(v) Basal diet +250mg/kg α-tocopherol (BE); (vi) Basal diet +250mg/kg ascorbic acid and 250mg/kg α-ocopherol (BCE). The birds were subsequently subjected to social isolation stress for 2 hours. Two hours of isolation stress elevated CORT significantly in the control and BE but not in the BC, BCE and BM birds. Isolation stress increased Hsp70 expression of the brain and heart in the control and BM birds. However, Hsp70 expression was not significantly altered after isolation stress by supplementation of ascorbic acid, α-tocopherol or their combination. Although CORT did not increase after isolation stress, Hsp70 expression significantly increased both in the heart and brain. Moreover, exogenous corticosterone supplementation did not result in elevation of Hsp70 expression. The last Experiment (Experiment V) was conducted to investigate the long term impact of neonatal feed restriction on the HPA axis response and hippocampal Hsp70 expression in senescent quail exposed to acute heat challenge. Equal numbers of male Japanese quails (Coturnix coturnix japonica) were subjected to one of the following two feeding regimens: ad libitum (control); 60% feed restriction on days 4,5 and 6 (FR). At the age of 21 (young) and 270 (old) days, four groups of 10 quails each from the control or FR group were randomly selected and blood and hippocampus samples were collected representing the different stages of heat treatment: Basal (no heat treatment), 1 h h at challenge (43°C), 1 h recovery and 2 h recovery. With the use of real-time PCR and EIA, the expression of regulatory genes in the hippocampus and CORT were examined. Aging resulted in a higher CORT,lower Hsp70 and GR expression in control birds, while in senescent FR birds only Hsp70 expression was attenuated following heat challenge. It is interesting to note that regardless of the age, there was a significantly lower basal GR expression in FR than control birds. Moreover, Hsp70 expression was upregulated in senescent FR birds during the recovery period and basal condition compared to control. Together, it can be concluded that selective breeding for phenotypic traits has resulted in tremendous alterations in the physiology of CB and concomitantly the ability to withstand high ambient temperature as compared to the RJF and VF. In other words, selective breeding consciously or unconsciously reduced physiological stress response thresholds in CB, producing more stress susceptible phenotypes rather than stress resistant ones. It is also apparent that genetic differences in body size and age per se may not determine breed or strain variations in response to heat stress. Neonatal modification seems to modify this stress response pattern leading to improved FCR and enhanced weight gain and resistance to S. enteritidis colonization. The Hsp70 and CORT alone are probably insufficient for inducing thermotolerance in chicks during neonatal modification and further studies are needed to investigate the participation of other genes and mechanisms during maturation of the thermoregulatory system. With regard to the relationship between Hsp70 and CORT, it is hypothesized that although Hsp70 expression may have been modulated by the effect of CORT in oxidative stress or glucocorticoid receptor association with heat shock proteins, it may also have been regulated mainly by ACTH functions in the HPA axis. It is also evident that the effect of neonatal modification on HPA axis response may last life-long. Thus, it is revealed to be a functional approach to reduce the allostatic load and restore homeostasis more efficiently in senescent birds leading to the development of adaptive, healthy and resilient phenotypes

Bioavailability of Amino Acids of Soybean Meal in Broilers Subjected to Heat Stress

The effect of high ambient temperature (35 vs 21°C) and acclimation during 5 and 6 week of age on the bioavailability of protein and amino acids was investigated in broilers. Two experiments were conducted to achieve the above objectives. In both experiments, equal numbers of chickens (90 birds: 45 Male, 45 Female) were assigned to three groups namely 2 weeks heat stress (heat acclimated), I week heat stress (non-heat acclimated) and no-heat stress. During the 28-35 days, the 2 weeks heat stress group was challenged to heat stress for 180-min every day. During the 35-43 days, the 1 week heat stress group was challenged to heat stress as well. The no-heat stress group was kept in standard temperature (21 °C) as control. Basal diet which contained soybean meal as a sole source of protein was fed to all birds during day 39-41 as adaptation period. Following this, the birds undergone 24 h fasting. Assay diet was then fed to all birds for one hour before starting the heat challenge. For Experiment I, Assay diet was the same with basal diet without methionine suplimentation and for Experiment 2, it was a nitrogen free diet. Feed conversion ratio (FCR) and deep body temperature (Tb) were measured and distal ileum contents were collected from birds killed by intravenous injection of sodium pentobarbitone through wing vain, immediately after heat challenge. The assay diet for second experiment was a nitrogen-free diet to investigate the endogenous protein and amino acid (AA) output. FCR increased by heat stress (P < 0.05). Heat acclimation resulted in 20% improvement in FCR in 2wk heat stressed birds as compare with non-heat acclimated birds (1 wk heat stress) at 3 5-43 days. Tb of both heat stressed groups increased throughout the heat stress period. 2wk heat stressed birds had significantly lower Tb than l wk heat stressed birds in day 35 and 37 (P < 0.05), but there was no difference at day 39 (P > 0.05). Crude protein and total amino acids apparent and true digestibility was significantly depressed by heat stress (P < 0.05) and this reduction failed to be improved by heat acclimation. Moreover, the reduction was found in all amino acid except for glutamic acid, aspartic acid, methionine, tryptophan, serine, alanine and proline. Acclimation and sex had no effect on total AA availability. Also there was no interaction between sex and heat challenge duration (P > 0.05). The mean AA availability of soybean meal observed in the present study was 82% in no-heat stress group and 78% in heat stressed birds. The relatively low values of AA availability in this study may be attributed to the soybean meal quality which was suspected to be underprocessed. One of the striking findings of this study was the low availability value for tyrosine under heat stress condition (11.9% less than no heat stress) which was the lowest among other AA. Analysis of endogenous excretion from the second experiment revealed that glutamic acid, aspartic acid, serine, threonine, leucine and arginine were the predominant endogenous amino acids present in ileal output. In addition, the lowest ileal endogenous AA losses were tryptophan, methionine and cystine

Phytobiotic Properties of Garlic, Red Ginger, Turmeric and Kencur in Growing Ducks

Abstract. Phytobiotic properties of garlic (Allium sativum), turmeric (Curcuma domestica), red ginger (Zingiber officinale) and kencur (Kaempferia galangal) were studied using standard in vitro antibacterial test and in vivo feeding trial with ducklings. In the in vitro experiment, potency of aqueous extract of these phytobiotic agents were tested against Salmonella pullorum and Escherichia coli. Feeding trial was carried out for 6 week starting at day 28 using ducklings fed diets supplemented with 1% of each of four phytobiotic agents. The highest antibacterial activity against S. pullorum and E. coli was observed with garlic and no additive effect when mixture of phytobiotics was used. Weight gain, fed intake and feed conversion ratio of ducklings were not affected by inclusion of garlic, red ginger and kencur. However, 1% turmeric supplementation significantly reduced growth performance to ducklings. Key words: phytobiotic, antibiotic, duck, medicinal plants Abstrak.  Penelitian karakteristik fitobiotik dari bawang putih (Allium sativum), kunyit (Curcuma domestica), jahe merah (Zingiber officinale) dan kencur (Kaempferia galangal) telah dilakukan secara in vitro melalui uji aktivitas antibakteri dan secara in vivo dengan perlakuan suplemetasi fitobiotik didalam pakan anak itik. Pada percobaan  in vitro, potensi  aktivitas antibakteri dari ektrak  fitobiotik diuji menggunakan Salmonella pullorum and Escherichia coli.  Percobaan suplementasi fitobiotik diberikan masing-masing sebesar 1% didalam pakan anak itik.  Pemberian pakan perlakuan dilakukan selama 6 minggu,  dimulai pada saat anak itik berumur 28 hari.  Hasil penelitian menunjukkan aktivitas antibakteri terhadap S. pullorum dan E. coli paling tinggi adalah ektrak bawang putih dan tidak ada pengaruh yang lebih baik apabila dicampur dengan ektrak fitobiotik lainnya.  Pertambahan bobot badan, konsumsi pakan dan konversi pakan anak itik tidak dipengaruhi oleh penambahan bawang putih, kunyit, jahe merah dan kencur.  Akan tetapi, suplementasi kunyit nyata menurunkan performan pertumbuhan anak itik. Kata kunci: fitobiotik, antibiotik, itik, tanaman obat-obatan

Extrusion enhances metabolizable energy and ileal amino acids digestibility of canola meal for broiler chickens

The aim of the current study was to determine the effect of extrusion process on apparent metabolizable energy (AME), crude protein (CP) and amino acid (AA) digestibility of canola meal (CM) in broiler chickens. A total of 36, 42-day-old broilers were randomly assigned into adaptation diets (no CM or 30% CM) with six replicates. After 4 days of adaptation period, on day 47, birds were allowed to consume the assay diets that contain CM or extruded canola meal (ECM) as the sole source of energy and protein. Following 4 h after feeding, the birds were killed and ileal contents were collected. The results showed that ECM had greater (P<0.001) AME (10.87 vs 9.39 MJ/kg) compared to CM. The extrusion also significantly enhanced apparent ileal digestibility of CP and some of AA such as Asp, Glu, Ser, Thr and Trp. In conclusion, the extrusion treatment appeared to be a practical and effective approach in enhancing the digestibility of AME, CP and some AA of CM in broiler chickens

Inhibition of adrenal steroidogenesis and heat shock protein 70 induction in neonatally feed restricted broiler chickens under heat stress condition.

This study was conducted to determine the roles of plasma corticosterone concentration (CORT) and heat shock protein 70 (Hsp70) expression in the acquisition of thermotolerance in broiler chickens subjected to neonatal feed restriction.Equal numbers of chicks were divided into one of the four feeding regimens: ad libitum (control), 60% feed restriction on days 4, 5 and 6 (FR60), 60% feed restriction on days 4, 5 and 6 + 1500 mg/kg metyrapone (FR60M) and 60% feed restriction on days 4, 5 and 6 + 1500 mg/kg quercetin (FR60Q). From 35 to 42 days of age, all birds were exposed to 37 ± 1°C and 70-80% relative humidity for 3 h/day. The neonatal feed restriction elevated heterophil to lymphocyte ratios (HLR), CORT and Hsp70 expression in FR60 chicks. The FR60M and FR60Q treatments suppressed CORT and Hsp70 expression, respectively. Subjecting birds to FR60 improved feed conversion ratio (FCR) and body weight gain, and reduced HLR when compared to controls in response to heat exposure. Following heat exposure, the HLR and weight gain of FR60M and control birds were not significantly different. Although FR60Q chickens had improved FCR and weight gain, their HLR were similar to controls following heat exposure. The control birds had significantly lower CORT than other groups in response to heat exposure. The Hsp70 expression of FR60 and FR60Q birds was lower than those of control and FR60M groups. It is concluded that Hsp70 expression didnot appear to be a major indicator of thermotolerance acquisitionin neonataly manipulated broiler chickens

Gut microbiota and transportation stress response affected by tryptophan supplementation in broiler chickens

Stimulation of serotonergic activity by tryptophan (TRP) supplementation is known to influence behavioural and physiological processes. One hundred and twenty male broiler chicks were assigned in groups of 5–24 battery cage and fed experimental diets with 0.22, 0.42 and 0.62% of digestible TRP during 21–42 d. On 42 d, birds were challenged with 2 h of transportation stress and samples were collected before or after the transportation. The results revealed that TRP increased feed intake, but had no significant effect on growth performance. Regardless of transportation, heat shock protein 70 (HSP70) and corticosterone (CORT) decreased and serotonin (5-HT) elevated by increasing TRP level in diet. Breast muscle pH and colour were not affected by elevation in dietary TRP, but drip loss decreased and shear force increased. Quantification of gut microflora showed that supplementation of TRP increased Enterococci, and Bifidobacteria populations, while E. coli, Clostridia, Campylobacter and Enterobacteria populations decreased. The effect of diet on Lactobacilli population was not significant. In conclusion, feeding broilers with higher levels of TRP improves their welfare condition both before and after transportation stress, as measured by lower serum CORT and HSP70 and higher 5-HT. Increasing dietary TRP level may shift the balance of pathogenic/non-pathogenic bacteria in gut to a favourable state

Response to withdrawal of vitamin and trace mineral premixes from finisher diet in broiler chickens under the hot and humid tropical conditions

This study was conducted to assess the effects of omitting vitamin (VIT) and trace mineral (TRM) premixes from the finisher diet on growth performance, antibody production against Newcastle disease (ND) vaccination, leg bone strength and incidence of foot pad dermatitis (FPD) in male and female broiler chickens. Birds were raised on floor pens in a naturally ventilated house using wood shavings as litter. Commencing from day 21, equal number of male and female birds were subjected to one of four VIT and TRM premixes withdrawal treatments as follows: (i) withdrawal from 21 to 42 days, (ii) withdrawal from 28 to 42 days, (iii) withdrawal from 35 to 42 days, and (iv) no withdrawal (control). In comparison to the control group, feed intake, body weights, feed conversion ratios and antibody responses to Newcastle disease vaccination were not affected by any of the supplement withdrawal treatment. Male birds had lower antibody production and higher bone-breaking strength as compared to their female counterparts. Irrespective of gender, withdrawal of VIT and TRM premixes from 21 to 42, and 28 to 42 days adversely affected bone-breaking strength. In conclusion, VIT and TRM premixes can be removed from the diet of male and female broiler chickens from 21 to 42 days without any adverse effects on growth performance, mortality, antibody response and incidence of foot pad dermatitis. However, bone-breaking strength was adversely affected by the withdrawal of VIT and TRM premixes for more than seven days

Effects of low-protein diets on acute phase proteins and heat shock protein 70 responses, and growth performance in broiler chickens under heat stress condition

A study with a 4 × 2 factorial arrangement was conducted to investigate the effects of 4 dietary protein levels and 2 environmental conditions on acute phase proteins (APP), brain heat shock protein (HSP) 70 density, and growth performance of broiler chickens. Day-old broiler chicks (Cobb 500) were fed isocaloric diets but with various levels of crude protein (CP), namely, (1) 21.0 and 19.0% CP in starter and finisher diets, respectively (control), (2) 19.5 and 17.5% CP in starter and finisher diets, respectively (Diet A), (3) 18.0 and 16.0% CP in starter and finisher diets, respectively (Diet B), and (4) 16.5 and 14.5% CP in starter and finisher diets, respectively (Diet C). Equal numbers of birds from each diet were subjected to either 23±1°C throughout or 33±1°C for 6 h per d from 22 to 35 d of age. From d 1 to 21, feed intake (FI) and weight gain (WG) decreased linearly (P = 0.021 and P = 0.009, respectively), as CP level was reduced. During the heat treatment period (d 22 to 35), there were significant (P = 0.04) diet × heat treatment interactions for FCR. Diet had no effect on FCR among the unheated birds, but the ratio increased linearly (P = 0.007) as dietary CP level decreased. Irrespective of ambient temperature, there was a significant linear decrease in FI (P = 0.032) and WG (P < 0.001) as dietary CP level decreased. Low-CP diets improved the survivability of heat-stressed broilers when compared to those fed control diets. Low-CP diets linearly decreased (P < 0.01) APP (ovotransferrin and alpha-acid glycoprotein) responses. Both APP and HSP 70 reactions were elevated following heat treatment. In conclusion, feeding broilers with low-CP diets adversely affect the growth performance of broilers under heat stress condition. However, low-CP diets were beneficial in improving the survivability. Because APP are involved in the restoration of homeostasis, the adverse effect of low-CP diet on the synthesis of these proteins could be of concern