Molecular characterization of IGF-1 gene expression in crossbred calves exposed to acute heat stress in a climate-controlled environment

Introduction

Heat stress in livestock causes tremendous impacts on their overall production, productivity and health. Cattle are homeotherms, maintains their thermal homeostasis by initiating various thermo-regulatory and adaptive mechanisms which promote their welfare and survival under stressful conditions (Indu and Pareek, 2015) [8] . Thermo-neutral zone (TNZ) or comfort zone of cattle is the zone where there is no energy expenditure for thermoregulation. If the temperature exceeds its upper critical limit, the animal experiences HS (Bagath et al., 2019) [2] . Molecular adaptations are considered as dominant mechanism in thermoregulation. There are changes in the expression patterns of certain genes that are fundamental for thermotolerance at the cellular level in cattle (Gupta et al., 2021) [7] . Insulin-like Growth Factor-1 (IGF-1) also called Somatomedin, is a hormone similar in structure to insulin, has its significant role in regulating growth, metabolism, and thermoregulation in cattle. The insulin-like growth factors (IGF) which includes IGF-1 and IGF-2 are single chain, ubiquitously expressed polypeptides plays a pivotal role in the regulation of cell growth, cell differentiation and maintenance of differentiated cell function (Jones and Clemmons, 1995; Cohick, 1998) [10,4] . The IGF-1 gene in cattle is located on chromosome five and is expressed in various tissues including the liver, muscle and mammary gland, influencing growth traits and milk production. Heat stress can reduce IGF-1 expression in cattle, which can subsequently affect their growth. The expression of the IGF-1 gene is influenced by multiple factors, mainly temperature-humidity challenges during HS, which poses a significant challenge to livestock productivity, especially in tropical and subtropical regions (Lima et al., 2017; Barrera et al., 2023) [13,3] . Heat stress in cattle disrupts the homeostatic balance, leading to alterations in molecular and biochemical pathways to mitigate its effects (Collier et al., 2008) [5] .

International Journal of Advanced Biochemistry Research 2024 ; SP-8(12): 660-666 IGF-1 is primarily produced in the liver under the influence of growth hormone, acts as a key mediator of anabolic processes, supporting growth and tissue repair. During HS, changes in IGF-1 gene expression act as an adaptive response aimed at conserving energy while mitigating physiological stress (Gupta et al., 2013) [7] . The down regulation of IGF-1 gene expression during HS is considered as broader shift of gene expression towards thermoregulation rather than towards metabolic functions (Rhoads et al., 2009) [16] . Studies have shown that IGF-1 expression is often downregulated during acute HS due to reduced feed intake, altered growth hormone signaling and metabolic shifts prioritizing thermoregulation over anabolic functions (Pragna et al., 2018) [15] . These changes can impair growth rates, reproductive performance, and overall health. Additionally, IGF-1 interacts with other regulatory pathways involving heat shock proteins (HSPs) and oxidative stress markers, highlighting its role in the complex network of heat stress adaptation (Fabris et al., 2020) [6] . Understanding IGF-1 gene expression during HS is crucial for developing strategies to enhance cattle resilience, such as genetic selection for heat tolerance and nutritional interventions. Measuring these bio-markers played a significant role in evaluating the impact of high temperature-humidity-index (THI) on growth as well as metabolism in heat-stressed cattle. In view of this, the present study was conducted with the objective of exploring the molecular adaptation exhibited by crossbred (Holstein Friesian × Sahiwal) calves exposed to acute heat stress in a climate-controlled environment, by characterizing IGF-1 gene expression using GAPDH as a reference gene.

Materials and Methods

Ethical approval of experiment

The research was conducted at the climatic chamber present at the Climate Controlled Research Complex (CCRC) of Centre for Animal Adaptation to Environment and Climate Change Studies (CAADECCS), Kerala Veterinary and Animal Sciences University. The experimental protocol was approved by the Committee for the Control and Supervision of Experimentation on Animals (CCSEA), New Delhi.

Experimental layout

Six healthy crossbred (Holstein Friesian × Sahiwal) female calves of eight to twelve months age were selected randomly from University Livestock Farm, Mannuthy and the same animals were distributed in control group or thermo-neutral zone (TNZ) and acute heat stress or treatment group. The selected animals were given an acclimatisation period of 10 days each initially in the animal holding facility and in the environmentally controlled chamber (Fig. 1) in TNZ for three hours a day for adaptation of the animals to novel environmental conditions of the chamber. This was followed by experimental period of 10 days each under TNZ and in climatic chamber for acute HS study. In TNZ, the comfort chamber was maintained at a temperature of 27ºC and relative humidity of 45-55 per cent (THI < 72). For acute HS study, maximum temperature of 40ºC and relative humidity of 55-65 per cent (Maximum THI of 95.04) was simulated in the climatic chamber for three hours a day (from 11 a.m. to 1 p.m.) for 10 days to assess the impact of HS on IGF-1 expression in crossbred calves. The animals were re-shifted for rehabilitation and reuse to the respective sheds of the cattle farm after the experiment, as per the recommendations of CCSEA.

Collection of blood samples and procedure of RNA isolation

Isolation of RNA from blood sample was done using commercial RNA isolation kit supplied by Origin Diagnostics and Laboratory, Kerala (ORIonX Total RNA Isolation Kit, Origin). Fifteen mL of venous blood sample was collected on day 10 of the experiment at 2 p.m. from each of the experimental animals in both the control and treatment groups in a 15 mL centrifuge tube using EDTA (1mg/mL of blood) as anticoagulant.

The collected blood samples were centrifuged at 3000 rpm for 8 min and plasma is discarded. Two to three volumes of "ice-cold RBC lysis buffer" were then added to the remaining erythrocytes and leukocytes in centrifuge tube and incubated the mixture in ice with occasional shaking for 10 min for the complete lysis of erythrocytes. After ten min, it was centrifuged at 6000 rpm for eight min to pellet the contents. Pellet of leukocytes was recovered after decanting the red coloured supernatant, containing the lysed erythrocytes and haemoglobin. This procedure was repeated until pellet is clear without any unlysed erythrocytes. After centrifugation one mL of TRI reagent was added and mixed well by pipetting. This was then transferred into a two mL RNase free Eppendorf tube and again mixed by pipetting and vortexing. Vortexed sample was then incubated for five min at room temperature to permit complete dissociation of nucleoprotein complex. After incubation 200 µL of chloroform per mL of TRI Reagent was added for homogenization and vortexed vigorously for five min and incubated again for 10 min at room temperature. The sample was then centrifuged at 12000 rpm for 15 min at 4 °C. The mixture separates into a lower red phenol chloroform phase, an interphase and a colourless upper aqueous phase in which RNA is present. Aqueous phase was removed carefully by angling the tube at 45° and pipetting the solution out into a new RNAse free spin column provided in the kit. 500µL of Buffer RD is added to the RNase-Free spin column and centrifuged at 12,000 rpm for 1 min at 4°C. Flow-through was discarded and 700 µL Buffer RW was added to the RNase-Free spin column and centrifuged again at 12,000 rpm for one min at 4 °C. Flow-through was discarded and 500 µL Buffer RW was added to the RNase-Free spin column and centrifuged at 12,000 rpm for one min at 4 °C. Flow-through was discarded and dry spinning was done at 12,000 rpm for 2 min at 4 °C. After dry spinning RNase-Free spin column was placed in a new 1.5mL RNase-Free Eppendorf tube and 35µL of RNase-Free water directly to the spin column membrane. It was then incubated in room temperature for two min and centrifuged at 12,000 rpm for two min at 4 °C to elute the RNA. Concentration and purity of RNA was determined immediately for complementary DNA (cDNA) synthesis.

Determination of concentration and purity of total RNA

The quantity and purity of total RNA were estimated by Nanodrop (Thermo-Scientific NanoDrop™2000 Spectrophotometer) method spectrophotometrically. The concentration of total RNA per microliter of the sample was quantified by placing 1 µL of the sample in the Nano-Drop spectrophotometer after calibrating it with the blank solution, nuclease-free water. The purity of total RNA was checked by A260/A280 and A260/A230 ratio using Nano-Drop spectrophotometer. A ratio of 1.8 or above for both A260/A280 and A260/A230 was preferred for cDNA synthesis.

Checking the quality of RNA by agarose gel electrophoresis

The quality and integrity of total RNA were analysed electrophoretically using agarose gel (0.8%) electrophoresis. Agarose gel (0.8%) was prepared in 1X Tris Acetate EDTA buffer (TAE), which was prepared using diethyl pyrocarbonate (DEPC) treated water. Ethidium bromide (to a final concentration of 0.5g/mL) was added into the molten agarose, mixed thoroughly and poured into gel casting tray to polymerise. After solidification, the comb and the seal were removed. The gel with the tray was immersed in 1X TAE buffer in the electrophoresis tank. RNA sample (5 µL) was mixed with 1uL of RNA gel loading dye in a 0.25mL microcentrifuge tube and denatured at 70°C for 10 min, snap chilled in ice and loaded into the wells along with a molecular size ladder. Horizontal electrophoresis unit was set at 70 V for 50 min and the gel was visualized under gel documentation system (BioRad, Gel Doc 2000TM). After analysing the quantity and integrity, the RNA samples were taken for cDNA synthesis.

Preparation of complementary DNA (cDNA)

Complementary DNA is the DNA copy of RNA molecules, synthesized by the enzyme RNA dependent DNA polymerase (Reverse transcriptase). The total RNA was reverse transcribed into DNA using Origin cDNA synthesis kit with RNase inhibitor (Origin Diagnostics and Laboratory, Kerala) as per manufacturer's protocol using oligo (dT)18 and random hexamer primers. For cDNA synthesis, 500 ng of RNA was reverse transcribed. The RNA samples were set up as per the standard protocol of the cDNA synthesis kit. The product of first strand cDNA synthesis was stored at -80 °C until use. The 1µL of cDNA was used for further qRT-PCR experiments.

Protocol

Designing and selection of primers

Exon-spanning primers (Table 2) were designed using online NCBI primer design software (Primer3, http:/bioinfo.ut.ee/primer3/) and specificity of the primers was checked using BLAST. Designed primers were assessed for quality using PCR primer stats (Bioinformatics.org). The primers purchased in the lyophilized form was reconstituted with nuclease-free water to make a concentration of 100 pM/µL for the stock and then stored at -20 °C. Working solution of each primer was prepared by diluting the stock to 10 pM/µL using nuclease free water.

Standardization for quantitative real-time PCR (qRT-PCR) conditions by gradient PCR using Thermal cycler:

Complementary DNA samples with good purity and concentration determined by Nano drop spectrophotometer (Fig. 3) were used as templates for PCR. The polymerase chain reaction (PCR) was carried out to amplify IGF-1 (target gene) and GAPDH (house-keeping or reference gene) genes. The conditions were optimized by setting different temperatures for annealing processes that was calculated by using thermal melting temperatures (Tm°) of forward and reverse primers. The optimum annealing temperature for the primers was determined by gradient PCR (Biorad, Thermal cycler, Fig. 2) and in quantitative real-time PCR system using SYBR green chemistry. The gradient PCR reaction mix and cyclic conditions used are given in Table 3. The tubes were spun briefly and placed in a thermal cycler.

The following Table 4 describes the details of PCR conditions used for the reaction. The PCR amplified products were stored at -20 °C till further processing. The amplified products were used in 2% agarose gel [1X Tris-Borate-EDTA buffer (TBE)] electrophoresis. Five microlitre of the product was mixed with one microliter of 6 X gel loading buffer and loaded into the wells carefully. As a molecular size standard, 1 µL of 100 bp DNA ladder (Thermo scientific, USA) was also loaded into one of the wells. Electrophoresis was then carried out at 70 V for 50 min or until bromophenol blue dye migrated more than 4 cm length of gel in 1X TBE buffer. The gel was documented in a gel documentation system (BioRad, Gel Doc 2000TM). After detecting the bands by electrophoresis, the product size of IGF-1 was confirmed. Annealing temperature at good dark bands for each gene were standardised for real-time PCR reaction. 2.9 Quantitative real-time PCR amplification of genes After validation of primer annealing temperature, the cDNA of target gene along with the reference gene was studied for mRNA expression by Real Time PCR (Biorad, USA) and analysed using CFX Maestro software.The expression of IGF-1 and GAPDH was studied using iTaq™ Universal SYBR Green Supermix, Bio-Rad Laboratories, USA (Table.5). GAPDH was used as an internal control. The CT of the target gene and CT of control gene were determined for each sample and calibrated (ΔΔCT method) (Livak and Schmittgen, 2001) [14] . Statistically analyzed the data using SPSS version 24.0 software. ΔCT = CT (target gene) -CT (reference gene)

ΔΔCT = ΔCT (test sample) -ΔCT (reference sample) RQ = 2-ΔΔCT

Results

The mean values of CT, ΔCT, ΔΔCT and fold change (2 -ΔΔC T)

in IGF-1 gene expression of crossbred calves in thermoneutral zone (control) and acute heat stressed conditions (treatment) are presented in Table 7. The fold change of IGF-1 gene expression is presented pictorially in Fig. 5 and the amplification plot and melt peak obtained by qRT-PCR is depicted in Fig. 6. Relative quantification of IGF-1 gene expression in peripheral blood leukocytes shown a nonsignificant (p>0.05) 0.71 fold down regulation in crossbred calves exposed to acute heat stress when compared to those under TNZ.

Discussion

The present molecular study of relative quantification of IGF-1 gene expression in peripheral blood leukocytes showed a non-significant (p>0.05) 0.71-fold down regulation in crossbred calves exposed to acute HS when compared to TNZ which is in accordance with the results reported by Pragna et al. (2018) [15] and Shashank et al. (2023) [18] . Pragna et al. (2018) [15] found a significantly lower expression of IGF-1 mRNA at high THI during hotter periods of summer stress which validates the current findings. Fabris et al. (2020) [6] evidenced that Holstein cows exposed to HS had down regulation of IGF-1 (p≤0.10) when compared to cows provided with cooling further supported the present findings. Kim (2023) [12] found that extreme heat stress of 41°C upregulated (p<0.01) the expression of IGF-1 in bovine myocytes when compared to the control group (38°C) which is contradictory to the present findings. The down regulation of IGF-1 in the present study might be involved in energy conservation for thermoregulation. The down regulation of IGF-1 and upregulation of HSP-70 expressions during HS could also be considered as a broader shift in gene expression towards thermo-regulatory adaptation. During HS, there might be decline in growth hormone (GH) levels which could down regulate IGF-1 expression (Aggarwal and Upadhyay, 2013; Pragna et al., 2018) [1,15] . It might also be due to low GH during HS that might not be sufficient to induce hepatic GH-IGF axis thereby reducing GH-dependent hepatic IGF-I hormone production which could down regulate its mRNA expression in heat-stressed calves.

Conclusion

It was evident from the current findings that down regulation of IGF-1 gene expression in peripheral blood leukocytes of crossbred calves (Holstein Friesian × Sahiwal) exposed to acute HS under climate-controlled environment thrown light on molecular adaptation mechanisms exhibited by them when exposed to a maximum temperature of 40ºC and relative humidity of 55-65 per cent, contributing in energy conservation for heat stress adaptation due to temperature-humidity challenges.

Acknowledgement

Conflict of InterestThe authors declared no conflict of interest.

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