m-RNA profiling of HSP70 under different tropical stress conditions in various broilers

Aim: The present experiment was conducted to study the effect of different tropical stress conditions on haemato-biological traits in various broiler strains during 3 to 4 weeks of age. Introgressing some important major genes likes Naked neck (Na) and Frizzle (F) into broiler germplasm may substantially improve the heat tolerance. Materials and Methods: The experiment was designed to evaluate three indigenously developed broilers viz. CARIBROTropicana (Naked neck and Frizzle gene bearing), CARIBRO-Mritunjai (Naked neck gene bearing) and CARIBRO-Vishal (Normal plumaged) under different THI (i.e. 72, 85 and 91) for 4 hours daily for 7 days. Total 324 broiler chicks (i.e. 36 chicks in each group) of 3 weeks of age were used in this study. Results: The m-RNA expression of HSP-70 gene was observed highest in frizzle plumaged birds and lowest in normal plumaged birds. The CARIBRO-Vishal showed highest stress as compared to other group. Higher the THI more severe was the effect on the traits. During the 7 day of exposure trial birds of all the genetic group exhibited the phenomenon of acclimatization as reveled by the averages of various traits at different days into the exposure. The mRNA expression analysis of HSP-70 in liver revealed higher expression levels at later days during the exposure trials which indicated the phenomenon of memory (stress memory) and acquired thermotolerance. Conclusion: Among the three genetic groups, CARIBRO-Tropicana exhibited highest means for HSP-70 production as well as tolerated the heat stress in a better way; therefore CARIBROTropicana was adjudged to be the best genetic group for production under tropical climate.


Introduction
size and efficiency of the cardiovascular and respiratory system.However, relatively inferior development The center of poultry industry is shifting to subof such major body systems [3] has led to lower ability tropical countries and this trend is likely to continue.
for balancing energy expenditure and body water Nearly one third of world-wide broiler stock placement content under extreme environment conditions.Thus, is in Asian countries such as India and china and these acute exposure of chickens to extreme conditions results countries are emerging as important locations for the in major economic losses.production and trade of poultry product, whereas, the All living organisms possess surveillance and major international poultry breeders are located in homeostatic mechanisms to adjust the demand of temperate countries.India has distinctly different growth, differentiation, environmental stress and ageing.seasons with variable temperature and humidity.The However, under certain circumstances, these mechanisms high ambient temperature in tropical climate leads to fail to adequately respond to imbalanced and result in heat stress to poultry in general and to broilers in the accumulation of the misfolded proteins inside the particular.Heat stress results in poor performance in cell.growth, feed efficiency and meat yield as well as higher To adapt to these environmental challenges and mortality.Recent decades have seen significant survive different types of injuries, cells have evolved developments in genetic selection of the meat type networks of different responses which detect and fowl, i.e.Broilers [1, 2] and Turkeys [3].The genetic control diverse form of stress.One of these responses, selection has led to rapid growth accompanied by known as the heat shock responses (HSR), has attracted increased feed efficiency and metabolic rate [4], a great deal of attention as a universal fundamental thus providing the poultry industry with heavy domestic mechanism necessary for cell survival under a variety fowls in relatively short growth period.Such developof unfavorable conditions.The heat shock response is ment in body size logically necessitates increase in the transient and lasts only a few hours [5].This phenomenon of HSR is a very well conserved regulatory network across all eukaryotes and is triggered by the synthesis of a group of proteins [6].
The utilization of major genes for broiler hepatocellular injury [28].Open sided House birds had production under tropical conditions exudes much greater HSP-70 expression than the environmentally enthusiasm among the poultry breeders.The important controlled House; the feed restriction chicks showed major genes are Naked neck (Na), Dwarf (dw) and higher HSP-70 density than those of ad libitum feed Frizzle (F).These genes are located on Autosomes and groups [29].The positive signals of HSP-70 mRNA at get inherited in Mendelian fashion.Introgressing some 6 hr heat stress were localized in the liver and lung, important major genes into the poultry germplasm may especially in the walls of vessels.The weak positive substantially improve the heat tolerance ability of signals were seen in the myocardial cells.No significant broilers without reducing their economic performance.
singles were observed in spleen, thymus and bursa of Some major genes results in reduction of feather cover fabricius [30].The liver samples for HSP-70 expression while other exerts their effect by decreasing the concluded that during stress (40 ºC temperature for 2 metabolic heat output [7,8]. hrs) a non-significant (P > 0.05) induction of HSP-70 In prokaryotic and eukaryotic cells, the synthesis expression was observed in turkey embryo [31].The of specific stress proteins increases under a wide variety cells with increased HSPs exhibit tolerance against the of stress conditions.The most extensively investigated additional stress hence they are often called as stress stressors is heat stress in which, a sudden increase in markers [32].Subjected to cold or high incubation temperature induces the synthesis of heat shock temperature, the expression of HSP-70 in younger proteins (HSPs) [9].The role of HSPs in the protection embryos had higher HSP-70 synthesis than older embryos of cells from heat stress is well established.An [17].The moderate heat stress caused significantly important aspect of HSPs is that organisms which have increased HSP-70 levels compared with the control reversed from previous mild stressful conditions groups in 51 and 65 weeks laying hens [33].Resistance expresses elevated level of stress proteins [10].HSPs to acute heat stress and concentration of HSP-70 were work as molecular Chaperones.These stress proteins higher in those birds subjected to more heat stress in can exhibit tolerance against the high levels of the broilers [34].During heat stress (35°C), the increase in stress causing agents, including heat that would both colonic temperature and hepatic HSP-70 concennormally cause developmental abnormalities or death tration were significantly less in high energy fed [10].This phenomenon, referred as "Acquired thermobroilers [35].The oxidative stress has been proposed as tolerance" which is dependent on the increased a key mechanism that mediates HSPs induction [36].expression and accumulation of the HSPs [11] Frizzle plumaged broiler strains developed at Central These proteins also play role in immunity and immuno-Avian Research Institute (CARI), Izatnagar under different pathology [12].Heat shock proteins are induced by a thermal humidity indices using functional genomics variety of agents including stressors.Heat is the most tools in order to throw light on underlying physioloprominent inducer of HSPs.Heat pre-conditioning is gical and genetic mechanism in major genes (Na or F related to the expression of HSPs and antioxidant gene) induced heat tolerance in broiler chicken.The enzymes [21].HSP-70 expression levels could be present study was under taken with the objectives to influenced by the cold stress and the expressions levels undertake mRNA profiling of HSP-70 under different differed in different tissues in different durations of the tropical stress conditions in various broilers strains cold exposure [22].Neonatal feed restriction improved [23].Pituitary and Ethical approval: Authors followed all the national thyroid HSP-70 m RNA transcription level was legislation concerning protection of animal welfare repressed which showed that it was the down regulaand following the guidelines of the Ethics Committee.tion genes of pituitary and thyroid in cold stress reaction This work is approved by the permission of Director, [24].The effect of stresses did not produce any Institutional Animal Ethics Committee, Indian significance effect on mRNA expression of HSP-70 in Veterinary Research Institute (IVRI).ovary and ovarian follicles of Japanese quail at 6 week old age [25].The response of HSP-70 was greater in the Experimental site: The proposed research work was high fear than the low fear group.Both low fear and conducted at Experimental Broiler Farm and Disease high fear showed similar increases in HSP-70

HSP-70 expression in aged birds
Genetics & Biotechnology Lab, Division of Avian expressions after crating [26].Heat stress increases the Genetics and Breeding, CARI, Izatnagar.The trials for level of HSP-70 in brain and ovary [27].Serum levels exposure of chicks under different THIs were carried of HSP-70 in broiler chickens also increased after out in Psychometric chamber, Veterinary Physiology continuous feeding of sodium arsenite in drinking Division, IVRI.water.This particular observation may be attributed Physiology Division, IVRI, Izatnagar, for exposure Hercules, USA), Gel documentation system (Biometra, under particular THI for a period of 7 days.Three UK; Syngene, USA), horizontal submarine electroweeks old chicks (36) of each germplasm were used in phoresis apparatus (Scie-Plas Ltd., Warwickshire, this study in each exposure trial totaling to 108 chicks England), Laminar air-flow apparatus (Tanco, India).
of each genetic group in the three exposure trials and Auto Gamma counter (COBRA III, Packard Bio 324 chicks of all genetic groups in all exposure trials.Science), Double beam UV-VIS spectrophotometer Thermal humidity index (THI) exposures for (ICI, India).
evaluation of broiler stocks: The evaluation of these Glass and plastic wares: Glass wares i.e. beaker, test stocks was carried out at three different THI in three tubes, cylinders, and conical flasks, round bottle flasks separate trials.The birds of each genetic groups were etc. used throughout the experiment were acquired kept in Psychometric chamber at an age of 3 week and from Borosil (India).Plastic wares viz.real time PCR the chamber was set to provide specific temperature tubes and flat caps, microcentrifuge tubes (0.2 ml, 1. 5 and humidity for a particular THI.The desired tempml and 2 ml) and tips (20 µl, 200 µl and 1 ml) were erature and humidity at each THI were provided for a The RNA was pelleted by spinning at 10000 rpm for 10 T Dry-bulb temperature in °F d = min at 4ºC; thereafter the isopropanol supernatant was RH= Relative Humidity in % (In equation, RH is used decanted carefully without disturbing the pellet.The as a decimal; in other words, 50% relative humidity is pellet was washed with 0.5 ml of 70 % ice-cold ethanol indicated as 0.50.and the tube was centrifuged at 10000 rpm for 10 min at Organization of exposure trial: The chicks of each 4ºC.The ethanol was decanted and the RNA pellet was genetic group were simultaneously exposed to air dried in an RNase free environment.The air dried particular THI and different parameters were recorded. RNA pellet was re-suspended in 50 µl of nuclease free The chicks (3 week old) of each genetic group were water.housed in battery brooders inside the Psychometric RNA quantification: The purity and concentration of chamber.The battery brooders were consisted of 4 tiers the total RNA was checked using nanodrop (Nano and two compartments in each tier.Each compartment Drop1000, Thermo Scientific, Singapore).The purity housed 7-8 chicks.Thus, a total of 36 chicks of one of the total RNA was confirmed by considering the genetic group were housed in 5 compartments of ratio of OD values at 260 and 280 nm.The purity was battery brooder.
further checked by Agarose gel electrophoresis.The Molecular analysis: Expression profile analysis of RNA showing any contamination with DNA was th rd th HSP-70 in liver tissue at 0 , 3 and 7 day of experiincubated with RNase free DNase (Biogene, CA USA) ment.
at 37ºC@1U for 1 µl).The DNase was subsequently inactivated by incubation at 65ºC for 10 min.Each Sample size: A sample of 4 birds/ genetic groups/ DNase treated total RNA sample was computed and all experimental day was slaughtered soon after THI the samples were adjusted to equal concentration (5.0 exposure (i.e. 4 hours daily on a particular THI) under µg/µl).each THI exposure and liver tissues were collected for HSP-70 gene expression analysis.
First-strand c DNA synthesis: The first-strand cDNA synthesis was carried out using 200 µl PCR microtubes HSP 70 gene expression studies: Total RNA was TM first in a thermal block using Revert Aid strand cDNA isolated from the liver following standard TRIZOL synthesis kit (MBI, Fermantas).Eleven micro-liters method.The purity of RNA was checked before the (11 µl) diluted total RNA (containing 5.0 µg of RNA) preparation of first-strand cDNA.Prepared cDNA was was taken in RNase free PCR tube (0.2ml).One microstored frozen at -20°C and was used for HSP 70 gene liter (1 µl) of random hexamer primer (0.5 µg/ µl) was expression studies.Expression of HSP 70 gene was added to each RNA samples and spinned down for few quantified by using specific primer pairs for genes of seconds.Each tube containing 12 µl of reaction interest (GOI) in Real-Time PCR.Here β-actin was th mixture was incubated 65 ºC for 5 minutes in thermal used as a reference gene and 0 day sample after cycler.Eight micro liters of master mix containing 5X exposure was taken as control.
reaction buffer (4µl), 10mM dNTP mix (2µl), Ribonu-TM Sterilization and inactivation of RNases: Glasswares clease inhibitor (1µl) and revert Aid reverse transcriused for RNA work were sterilized in hot air oven at ptase (1µl) was added to each tube and mixed thoroughly.180ºc for at least 5 hours to make them RNase free.All The total reaction mixture was incubated in the thermal the plasticwares used for the same purpose were cycler according to the manufacturer's instructions i.e. thoroughly treated with 0.1% Diethyl pyrocarbonate 25ºC for 5min and 42ºC for 60 minutes.Finally, the (DEPC) at 37ºC for about 12 hr in order to destroy the reaction was stopped by heating at 70ºC for 10 min and RNases and then were sterilized by autoclaving.
the resultant first-strand c-DNA was synthesized and stored at -20ºC till further expression studies.Isolation of total RNA: The tissue samples from liver were cut into small pieces and collected in 2 ml RNase HSP-70 mRNA quantification: The relative expression free microcentrifuge tubes containing 600 µl TRIzol of specific gene mRNA was quantified by real-time (M/s Invitrogen, USA) denaturing solution.The tissue PCR detection system (IQ5, Bio-Rad laboratories Inc. sample were homogenized using tissue homogenizer at USA).All reactions were performed in nuclease-free 8 tubes with optically clear flat caps (Axygen Scientific, mean.The data recorded as CT values for gene Inc.USA).
expression analysis of HSP-70 gene was also analysed as per below models.cycling conditions were, initial denaturation at 95ºC variance'σ ' for 20s, annealing at 56ºC or 58ºC for different gene for

20s and extension 72 ºC for 20s. For each sample a dissociation curve (melt curve) was generated after
The mean mRNA expression levels of HSP-70 in th rd th completion of amplification.A negative control liver of birds of three genetic groups at 0 , 3 and 7 containing all the ingredients except cDNA template days of experiment under different THIs have been (Non-template control; NTC) was set up invariably for presented in Table-3 and Fig. 3.The difference among th rd each master mix made for conducting the reactions.βthree genetic groups were significant (P<0.05) at 0 , 3 th actin was used as reference gene.The results were and 7 day of experiment at THI 72, 85 and 91.The expressed in terms of the threshold cycle value (Ct).
higher mRNA expression levels of HSP-70 at all the PCR standard curve of β-actin gene is given in fig.-1     The differences due to THI and genetic groups Poultry.Sci., 82: 1500-1508.
were found significant for HSP-70 traits studied.
Real-time polymerase chain reaction: Twenty micro-Y = µ + G + T + (G x T) + e ijk i j (ij) ijk liter volume of reaction mixture was used to amplify th where, Y = Trait recorded under i genetic group and ijk the gene of interest with final concentration of 1X th th TM j THI on k individual SYBR Green PCR master mix (2x DyNAmo HS, µ = Overall mean Finnzymes, USA) which contain SYBR Green 1dye, th G = Effect of i genetic group (i = 1, 2, 3) Meteor Taq hot start DNA polymerase, dNTPs i th T = Effect of j THI (j = 1, 2, 3) including dUTP and MgCl with 4M final concenj 2 th tration in optimized buffer components, a 0.05 pM (G x T) = Interaction effect between i genetic group (ij) th concentration of gene-specific primer (Table-2) and and j THI 2.5 µl of diluted (1:10) cDNA template.Real-time PCR e = Random error distributed with mean '0' and ijk 2

bA cA 1 )
Figures in parentheses are the number of observations; 2) Figure bearing same superscript (lower case) in a row and (upper case) in a column within day of experiment do not differ significantly (P< 0.05); 3) The figures bearing different superscript (symbol) differ significantly in a particular THI during different days in a column.thermotolerancehas been shown to be dependant on B. T. (1994) Growth, livability and feed conversion of 1991 the increased expression and accumulation of the HSPvs 1957 broilers when fed typical 1957 and 1991 broiler 70 [11].diets.Poultry Sci., 73: 1785-1794.2. Havenstein, G. B., Ferket, P. R. and Qureshi, M. A. (2003 a) Conclusion Growth, livability and feed conversion of 1957 versus 2001 broilers when fed representative 1957 and 2001 broiler diets.
3. Havenstein, G. B., Ferket, P. R. , and Qureshi, M. A. ( 2003b) During the 7 day exposure trial, birds of all the three Carcass composition and yield of 1957 versus 2001 broilers when fed representative 1957 and 2001 broiler diets.genetic groups exhibited the phenomenon of acclimati-Poultry.Sci., 82:1509-1518.zation as reveled by the averages of various traits at 4. Janke, O., Tzschentke, B. and Boerjan, B. (2004) different days into the exposure.Among the three Comparative investigation of heat production and body genetic groups, CARIBRO-Tropicana exhibited highest temperature in modern chicken breeds.Avian Poultry Biol means for HSP-70 production as well as tolerated the Rev 15 : 191-196.

Figure- 3 .
Figure-3.mRNA expression profile of heat shock proteins-70 gene in liver of broiler chickens of different genetic groups at different days during experimental period under different THI (Mean±SE)

Table - 1
. Formula and chemical composition of broiler rations.

Table - 2
. Details of the primers used for real-time PCR analysis of HSP-70 gene in liver tissue of broiler chickens during different stress conditions (THIs).

Table - 3
. mRNA expression profile of heat shock protein-70 gene in liver of broiler chicken of different genetic groups at different days during experimental period under different THI (Mean±SE)