The cellular retinol-binding proteins (CRBPs) are members of the intracellular lipid-binding protein (iLBP) multigene family. CRBPs are small molecular mass (~15 kDa) proteins that bind specifically to retinol and retinoic acid (Sundelin et al., 1985). CRBPs play important roles in intestinal vitamin A absorption and cellular retinol transport due to the intense hydrophobicity of retinol and also control the metabolism and homeostasis of retinoids through interaction with metabolic enzymes (Ong, 1987, Giguere, 1994, Yin et al., 2014). There are four known CRBP genes, termed CRBP I, CRBP II, CRBP III and CRBP IV and CRBP IV play a role in absorption, transport, metabolism, and homeostasis of retinol and its derivatives. This makes CRBP IV a good candidate gene for enhancing reproductive traits in chickens (Yin et al., 2013). In India, poultry sector has shown a substantial improvement over the years and total egg production reached 82.93 billion with per capita availability of 66 eggs per annum (Singh et al., 2018). Rhode Island Red (RIR) chicken is useful for backyard poultry production and believed to be good egg producer. The basal mRNA expression profile in bird’s different tissues are suggestive of its preparedness and performance ability of egg production. Scanty information available on basal expression level of CRBP IV gene in Rhode Island Red chicken. In view of the above, the present investigation was done to determine the basal mRNA expression level of CRBP IV gene in various tissues of RIR chicken.

Materials and Methods                                           

Experimental Birds                                                                    

A total of 225 straight run chicks were produced by mating of four RIR females each to eleven RIR males through Artificial Insemination. The birds were maintained at the experimental layer farm of this institute by providing ad lib. feed and water and following standard management and vaccination practices (Debnath et al., 2015). Ten pullets were selected randomly at different stages of laying i.e. four birds at pre-laying stage (12 weeks) , three birds at peak laying stage (32 weeks) and three birds at post-laying stage (> 40 weeks)  for mRNA expression studies.

Sample Collection

Three tissues viz. liver, kidney and oviduct, weighing approximately 50-100 mg were aseptically collected from each of the ten experimental birds, in 2.0 ml centrifuge tube containing ~1.0 ml RNAlater^® (Ambion, U.S.A.). Tissues were cut into small pieces to ensure proper infusion of RNAlater^® into it and cryopreserved at -80^oC until used for RNA isolation. Sterilization of lab wares and inactivation of RNase was done by using 0.1% diethyl pyrocarbonate (DEPC) treated water and incubated at 37^oC for overnight.

Isolation of Total RNA

Total RNA from each tissue sample was isolated using TRIzol^® reagent (Invitrogen, U.S.A) following step-wise procedure of Hongbao et al. (2008) and finally dissolved in 50 μl of nuclease- free water. Any possible contamination of genomic DNA was removed by 5 μl of each RNA sample with 5 U of RNase- free DNase (Biogene, USA) at 37^oC for 1 h. The DNase was subsequently inactivated by incubation at 65^oC for 10 min. RNA purity and quantity were determined by NanoDrop® (ND1000- Spectrophotometer) and samples showing absorbance ratio (260/280) of > 1.8-2 were considered to have satisfactory purity and used in subsequent analysis. The concentration of RNA was adjusted to 1000 ng/ μl before proceeding for synthesis of cDNA.

Synthesis of First Strand cDNA and Primers

One μl of total RNA from each sample was taken as template and first strand cDNA was prepared using Thermo Scientific Verso cDNA synthesis kit^® (Thermo Fisher Scientific Inc., U.S.A.). The concentration of cDNA of each sample was equalized to 25 ng/μl for subsequent usage in qRT-PCR. Primer pairs of CRBP IV gene and housekeeping or the reference gene (β-actin) were selected from published literatures (Yin et al., 2013, Higgs et al., 2006) (Table 1). All the primers were synthesized by Xcelris Genomics Labs Ltd., Ahmedabad (India).

Table 1: Details of the primers for qRT-PCR

Reaction Mixture

All PCR reactions were carried out in triplicate in 0.2 ml clear, thin walled nuclease-free 8-tube strips with optically clear flat lid (Axygen Scientific Inc., U.S.A) to avoid pipetting error. A negative control (NTC, no template control) in triplicate containing all the ingredients except the template (cDNA) was also set up to check any contamination.  β-actin gene was used as reference gene. The amplification was performed in 20 μl reaction mixture using DyNAmo ColorFlash SYBR Green qPCR Kit^® (Thermo Fisher Scientific Inc., U.S.A.).

Real Time-PCR Programme, Retrieval Compilation of qRT-PCR Data

Relative mRNA expression of CRBP IV gene in each of the three tissues viz. liver, kidney and oviduct was done by quantitative reverse transcriptase PCR (qRT-PCR) in CFX 96^® in Real Time PCR detection system (Bio-Rad Laboratories Inc., U.S.A.) and Real-time PCR cycling conditions used were as follows: initial denaturation at 95^oC for 7 minutes followed by 40 cycles of denaturation at 95^oC for 10 seconds, annealing at optimized temperature for 20 seconds and extension at 72^oC for 20 seconds; followed by detection of fluorescent signal by the real time detection system to generate amplification curve. After completion of 40 cycles, each sample was subjected to 60-95^oC @ ±0.5^oC increment for 10 seconds to generate dissociation curve or melt curve to identify specific amplification. Afterwards, threshold cycle (C_t) value and melting point temperature of each tube was retrieved and reviewed for its corresponding amplification and dissociation curve to ensure appropriateness of specific amplifications. Subsequently, data were imported into MS-Excel file and saved for further statistical analysis.

Determination of 40-∆C_t and Fold Expression of Genes by 2^(-∆∆Ct) Method

The tissue showing highest ∆C_t value was chosen as the calibrator tissue. For each sample, the ∆C_t value was subtracted from 40 (total cycle number) so as to obtain 40-∆C_t. Higher 40-∆C_t value was considered as higher expression (MacKinnon et al., 2009). The fold change expression was determined by using the formula 2^(-∆∆Ct) method as per Livak and Schmittgen (2001). The standard errors of ∆C_t values were calculated for comparison of the three tissues at different age groups (Yuan et al., 2006).

Statistical Analysis of qRT-PCR Data

Differential expression of target gene in the three tissues at three different ages of RIR pullets was analyzed by least squares analysis of variance (LS ANOVA) using JMP 9.0.0 statistical program package (SAS, 2010). Hatch and age was taken as fixed effect in the model.

Y_ijk = μ + A_i + H_j + e_ijk

Where,

Y_ijk = 40-∆C_t value of mRNA expression of gene under study in i^th age, j^th hatch and k^th tissue of individual pullet

μ    = overall mean

A_i    = fixed effect of i^th age of individual pullet (i = 1, 2 and3)

H_j    = fixed effect of j^th hatch (i = 1, 2)

e_ijk  = random error associated with k^th  individual pullet of i^th age in j^th hatch  (mean ‘0’; variance ‘σ²’)

Results and Discussion

The least squares means revealed that age had significant effect on expression of mRNA of CRBP IV gene in kidney, liver and oviduct of RIR chicken (Table 2).

Table 2: Least squares analysis of variance of relative mRNA expression levels (40-∆C_t values) of CRBP IV gene indifferent tissues at various ages in RIR chicken

MSS: mean sum of squares, ^#p≤0.1, **p≤0.01, df: denotes degrees of freedom

The expression of CRBP IV gene (mean 40-∆C_t values) differed significantly among three age groups in kidney (p≤0.05) and oviduct (p<0.1) in RIR chicken. The 40-∆C_t value at 40 weeks in kidney was 33.96±0.7 which was significantly higher than those at 32 weeks (29.09±0.70) and 12 weeks (27.81±0.60). In oviduct, the highest relative mRNA expression of CRBP-IV gene was seen  at 40 weeks (30.95±0.75).But in liver, the mRNA expression did not differ significantly (p>0.05) among three ages. Analysis also revealed that highest expression (30.29±0.38) was observed in kidney tissue, followed by oviduct (29.36±0.41) and liver (28.85±0.68) tissues (Table 3).

Table 3: Least squares mean ± standard error of 40-∆C_t values of mRNA expression levels of CRBP IV gene in different tissues at various ages in RIR chicken

N: Number of observations; Means with different superscripts in a column differ significantly.

Previously, Yin et al. (2013) studied the mRNA expression in Erlang Mountainous chickens and reported that the CRBP IV mRNA levels changed with age in the various tissues at different ages (12, 24, 32 and 45 weeks) and also reported that high expression was found in all tissues at 32 weeks, except for the heart and low expression was found at 12 weeks and 45 weeks as vitamin A is highly required during laying time and concluded that CRBP IV may play an important role in vitamin A metabolism. In present study, it was seen that expression of CRBP IV gene was tissue- specific and highly expressed at the age of egg production. High expression in kidney might be due to vitamin A metabolism being regulated by kidneys as Yin et al. (2013) reported the same in Erlang Mountainous chickens. In our present investigation CRBP-IV was highly expressed at a later age of 40 weeks. Differences in reports might be due to the differences in the germplasm analyzed. Further, melting curve analysis demonstrated a single predominant peak with a distinct melting temperature for the primer pairs (Fig. 1).

Fig. 1: Melting (A, B & C) curves of CRBP-IV gene mRNA during qRT-PCR in Kidney, Liver and Oviduct tissues, respectively in pure strain of RIR chicken

Fig. 2: Fold expression of CRBP IV gene at various ages in kidney, liver and oviduct of pure strain of RIR chicken

Fold changes of relative mRNA expression of CRBP IV gene in different tissues (kidney, oviduct and liver) of RIR chicken are presented in Fig. 2. It was found that CRBP IV gene expressed 57.87 folds more at 40 weeks of age in kidney than at 12 weeks. The expression at 32 weeks was 2.98 folds more than at 12 weeks. In oviduct, CRBP IV gene expressed 5.5 folds more at 40 weeks than 32 weeks. It was 1.43 folds more expressed at 12 weeks of age than at 32 weeks of age. In liver, CRBP IV gene expressed 9.82 folds more at 12 weeks than at 40 weeks. The expression at 32 weeks was as 2.11 folds more than at 40 weeks of age in liver. However, reports on comparison of basal mRNA expression levels across tissues in RIR and other chicken germplasm were not available in the literature. Thus, it can only be concluded that kidney and oviduct tissues revealed maximum fold expression at the age of 40 weeks of the gene studied.

Conclusion

In view of the above findings, it may be inferred that the basal mRNA expression level of CRBP IV gene in RIR chickens was tissue specific and showed egg laying time- dependent changes. Thus, the results suggest that CRBP IV may play an important role in egg production traits in Rhode Island Red chicken.

Acknowledgement

The authors would like to acknowledge the help rendered by Members of Advisory Committee of first author, Director of ICAR-IVRI for providing IVRI-scholarship to the first author, Director ICAR-CARI for providing all necessary facilities, Director of ARDD, Tripura, for sanctioning leave to the first author and a special thanks to Prof. S. N. Sivaselvam, Head, Department of Animal Genetics & Breeding, C.V.Sc & A.H, R. K. Nagar, Tripura for his guidance to write the paper.

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