Posthatch Thermal Conditioning Reduces Heat Stress In Three Broiler Strains

Heat stress is an increasing challenge to the sustainability of poultry production in the tropics due to global warming. This study determined the effect of posthatch thermal conditioning on heat stress indices, haematological parameters and expression of brain derived neurotrophic factor (BDNF) gene in three meat type chickens; Cobb 500 (C500), Ross 308 (R308) and improved Nigerian indigenous broiler FUNAAB Alpha (FA). The interplay of individual bird’s genetics and thermal treatment at critical periods on thermoregulation was largely unpublished as at the time this study was conducted. Thermal conditioning was carried out on day 6 by exposing 20 chicks from each strain to high temperature of 40±1 °C for 3 hours. Both conditioned and unconditioned chicks were exposed to acute heat challenge of 40±1 °C for 15 minutes on day 10. Blood samples were collected to determine haematological parameters. Tissue samples were collected from which RNA were extracted, synthesized into cDNA and subjected to qPCR. Strain and thermal conditioning interaction was signicant (p<0.05) on haematological parameters with conditioned C500 having the highest means for packed cell volume, haemoglobin and red blood cell counts. Interactive effect was also signicant (p<0.05) on BDNF gene expression, with conditioned FA having the highest. The study concluded that variation in traits due to thermal treatment is strain-specic and thermal conditioning is recommended for commercial broilers in southwestern Nigeria. Gene expression proling of facilitated L-LTP in VP16-CREB mice reveals that BDNF is critical for the


Introduction
Climatic stress has been described as the limiting factor to animal production e ciency and global warming will further accentuate heat stress related problems (Renaudeau et al., 2011). United Nations statistics projects increase in human population by over two billion by 2050 (UN, 2013). Global demand for livestock products is also expected to double (Rojas-Downing et al., 2017). Heat stress is a major challenge against poultry production worldwide, causing decline in growth and meat quality of broilers (Lara and Rostagno, 2013). Rajkumar et al. (2010) and Fathi et al. (2013) reported that genetics contribute substantially to the response of birds to high temperature. Strain differences have been found to play a major role in chicken thermoregulation, which in turn determine how individual birds respond to thermal stress (Altan et al., 2003;Nielsen et al., 2003).
Thermal conditioning, which is the exposure of poultry species to high ambient temperature during critical periods has been found to improve the acquisition of thermo-tolerance (De Basilio et al., 2001;Yahav and Hurwitz, 1996;Yahav and McMurtry, 2001) enabling broilers to cope with extreme environmental conditions.
It has been demonstrated that the pathway leading to thermal stress response set-point establishment is activated by Brain-Derived Neurotrophic Factor and there are transient changes in its expression during both thermal conditioning and re-exposure of conditioned chicks to heat stress, compared to unconditioned chicks of the same age ( Yossifoff et al., 2008). Higher expression of the BDNF gene, which plays a pivotal role in developmental plasticity, has been found to improve memory, adaptation and survival (Johnston et al., 1999;Johnston and Rose, 2001).
However, the interplay of individual bird's genetics and thermal treatment at critical periods on thermoregulation was largely unpublished as at the time this study was conducted. This study was therefore aimed at verifying the interactive effect of strain and thermal conditioning on thermotolerance, to help farmers, especially in the tropics, in breed selection to alleviate the effects of heat stress in raising commercial broilers. Effect of posthatch thermal conditioning on heat stress indices, haematological parameters and expression of brain derived neurotrophic factor (BDNF) gene in three meat type chickens; Cobb 500 (C500), Ross 308 (R308) and improved Nigerian indigenous broiler -FUNAAB Alpha (FA) was determined.

Experimental birds
Fifty chicks each of Cobb 500 (Cobb) obtained from Zartech Farms, Oluyole, Ibadan; Ross 308 (Ross) obtained from Agrited Nigeria Limited, Alomaja Ibadan and improved Nigerian indigenous broiler -FUNAAB ALPHA (FA) obtained from FUNAAB Hatchery was used for the study. The birds were placed into partitioned brooder cages at day old. They were raised in an adequate environmental temperature (32±2 ºC) under continuous arti cial illumination and relative humidity of 70-80% (Gan et al., 2013;) until day six. The experimental birds were fed with conventional starter mash with 23% crude protein and 2100 kcal metabolisable energy throughout the experimental period of ten days. Feed and water were provided ad libitum.
Thermal conditioning and acute thermal challenge On day six, birds from each strain were randomly distributed into two treatment groups of twenty-ve birds each. One group of chicks (thermally conditioned chicks) were exposed to high temperature at 40±1°C for 3 hours while the other group (unconditioned chicks) were left at normal brooding temperature. The age for the thermal conditioning (Day 6) applied in this study was adopted from previous reports (De Basilio et al., 2001Basilio et al., , 2003Tanizawa et al., 2015). On day ten, both groups (conditioned and unconditioned chicks) were challenged to high temperature at 40±1°C for 15 minutes without feed and water (Tanizawa et al., 2015).

Data Collection
Rectal Temperature and Respiratory Rate Rectal temperature of each bird was measured by inserting a digital thermometer into its rectum. The reading was taken when it became stable and thermometer gave an alarm signal (Plyaschenko and Sidorov, 1987). Respiratory rate was measured by counting the movement of the abdominal region or vent per minute using a stopwatch (Plyaschenko and Sidorov, 1987).

Blood Sampling
Immediately after the 15 minutes of acute heat exposure on day ten, blood samples were collected from ten heat challenged birds from each group for all the ve strains by cardiac puncture (Tanizawa, 2015). About 2 ml of blood was collected from the heart of each bird and dispensed into clean Bijou bottle containing ethylene diamine tetra acetic acid (EDTA) as an anticoagulant. The un-coagulated blood was used to determine the Packed Cell Volume (PCV), haemoglobin concentration (Hb), red blood cell (RBC) and white blood cell differentials (heterophils, lymphocytes, basophils and eosinophils) counts.

Tissue Sampling
Ten chicks per breed across the two treatment groups (conditioned and unconditioned) were randomly selected and slaughtered by cervical dislocation. Tissue samples were collected from the anterior hypothalamus and immediately stored in eppendorf tubes containing RNAlater solution in the ratio of 1:5 i.e. 0.5g of tissue to 2.5 µl solution and kept at -40 ºC till needed for further analyses.

Extraction of Total RNA and cDNA Synthesis
Norgen's Animal Tissue RNA puri cation kit was used to isolate Total RNA from the stored samples following the manufacturer's instructions. The First-Strand cDNA Synthesis reaction was carried out using Norgen's TruScript™ First Strand cDNA Synthesis Kit.

Real Time Quantitative Polymerase Chain Reaction (Real Time qPCR)
The cDNA products were subjected to real time PCR performed using the Cepheid SmartCycler. Relative quantitation of the cDNA for BDNF gene was carried out using 5X EvaGreen master mix (manufactured by Solis Biodyne) containing DNA Polymerase, dNTPs, MgCl2 and EvaGreen dye. Total reaction mixture of 20 µl contained 4 µl master mix, 0.3 µl forward and reverse primers ( An initial incubation step of 12 min at 95°C was carried out at the beginning of the qPCR cycle to activate the DNA polymerase in the master mix as recommended by kit manufacturer.  Gomez, 1984) and the result is presented as means ± standard error (S.E).

Heat Stress Parameters
The effect of strain was signi cant on heat stress parameters [rectal temperature before acute heat exposure (RT1), rectal temperature after acute heat challenge (RT2), respiratory rate before acute heat challenge (RR1) and respiratory rate after acute heat challenge (RR2)]. FA had the lowest means in RR2 (114.400). R308 had highest mean values for RT1, RR1 and RR2 (1.900°C, 111.800 counts/min and 131.000 counts/min respectively). There was no difference in RT1 for the three strains but C500 and R308 had signi cantly (p<0.05) higher means for RT2, RR1 and RR2 than FA (Table 2).     (Table 5).

BDNF gene expression
The BDNF gene was most expressed in FA and least expressed in C500 strain (Fig. 1).
The interactive effect of strain and thermal conditioning was also signi cant (p<0.05), with unconditioned C500 having the least and conditioned FA the highest expression of BDNF gene (Fig. 3). However, there was no statistical difference between conditioned and unconditioned FA.

Discussion
Heat stress parameters indicated that FA was less stressed than the two exotic strains. This may be attributed to genetic differences in heat tolerance. Indigenous chickens have greater potential for tropical adaptability and disease resistance than the exotic under the same environmental conditions (Ajayi, 2010;Mahendra, 2016). The reduction in heat stress parameters for conditioned chicks can be attributed to acclimation. There was a sharp increase in heat stress experienced by unconditioned chicks due to acute heat challenge which conditioned chicks were already prepared for. The ability of poultry to resist heat stress is improved if they are exposed to high environmental temperatures during the neonatal period as postulated by Collier et al. (undated). The interactive effect further con rms that thermal conditioning alleviated the effect of the acute heat exposure, especially for the exotic strains (C500 and R308).
Haematological parameters have been studied to understand the relationship of blood characteristics to the environment (Ovuru and Ekweozor, 2004) and have been suggested for use in the selection of animals that are genetically resistant to certain diseases and environmental conditions (Isaac et al., 2013;Mmereole, 2008). Although the three strains used in the study had statistically similar blood pro les, it is only the FA strain mean values that falls within reference ranges (Onyishi et al., 2017) for important erythrocytic and leukocytic indices. A higher level of PCV, Hb, RBC and EOS observed in FA strain could imply it has a healthier status since Packed Cell Volume and Hb are involved in the transport of oxygen and absorbed nutrients (Isaac et al. 2013;Maton et al., 1993).
Effect of the Strain X Thermal Conditioning interaction on haematological parameters in this study (i.e. Genotype X Environment) varied among the strains. Thermal conditioning increased PCV, HB and RBC in the two commercial broiler strains, C500 and R308, whereas it had no effect on FA. This agrees with the ndings of Siegel (1989) who reported that various lines exhibited different responses in the same environmental conditions because of the Genotype X environment interaction.
BDNF gene is essential for synaptic plasticity and maintenance of long-term memory (Barco et al., 2005;Kang and Schuman, 1995); increase in its expression has been found in heat conditioned chicks when compared to unconditioned chicks (Yossifoff et al., 2008). The difference in the expression of the gene in the three strains further a rms strain differences in chicken thermoregulation, which affects how Higher expression of BDNF gene confers greater developmental plasticity which could make the thermally conditioned birds more thermo-tolerant when exposed to heat stress at marketing age (Johnston et al., 1999;Johnston and Rose, 2001). However, the interactive effect of strain and thermal treatment suggests that the effect of posthatch thermal treatment is strain speci c since the improved indigenous strain had the highest expression of the BDNF gene and it showed no statistical difference in the conditioned and unconditioned treatment. This possibly explains why it has lower values for heat stress parameters which are indicators of thermal stress.

Conclusion
Post hatch thermal conditioning is bene cial for alleviating thermal stress in meat type chickens raised in hot climates. Although in this study, the improved indigenous strain had an edge over exotic commercial strains in terms of heat resistance, thermal conditioning enhanced thermo-tolerance across board.

Statements And Declarations
Ethics approval and consent to participate: Not Applicable Consent for publication: Not Applicable Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Declaration of Interests: The authors declare that they have no competing interests.    Effect of Strain X Thermal Conditioning Interaction on Relative Expression of BDNF Gene a,b Means with different superscripts are signi cantly different at 5% (p<0.05) level. C = C500, R = R308, CON = Conditioned, UNC= Unconditioned