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Expression Profile of Acetyl CoA Carboxylase Beta (ACACB) Gene during the Pre and Post-hatch Period in Chicken
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First Online 19-05-2021|
Methods: The present study was conducted to explore the expression pattern of the ACACB gene in breast muscle tissue during pre-hatch embryonic day (ED) 5th to 18th and post-hatch (18th, 22nd and 40th week of age) periods of White leghorn (IWI line) by using Quantitative real-time PCR (qPCR). Then, fold change of ACACB gene expression was calculated.
Result: Our study showed that the ACACB gene expression was down-regulated during embryonic stages from ED6 to ED18. The gene expression was also down-regulated during adult stages i.e. on 22nd and 40th week of age. This result indicated that the initial expression of the ACACB gene is required for embryo development and during adult periods, low gene expression leads to the less fat deposition in muscle of layer chicken. Finally, it can be concluded that there was a differential expression pattern of the ACACB gene during the pre-hatch embryonic and post-hatch adult periods to mitigate varied requirements of lipids during different physiological stages in layer chicken.
High fat content in the meat exert adverse effect on human health (Milicevic et al., 2014) and its accumulation in excess amount exert a negative impact on reproductive performance of birds (Xing et al., 2009). The expression level of this particular gene at different age groups will form the base for understanding its action and subsequently used for manipulating the gene functionality to reduce fat deposition. Therefore, the present study was conducted to know the expression levels of the ACACB gene during the pre and post-hatch period in the white leghorn IWI line.
MATERIALS AND METHODS
The present study was conducted in IWI chicken line of the White Leghorn breed which is maintained at ICAR-Directorate of Poultry Research, Hyderabad, India during the year 2020. The birds were provided ad lib feeding, watering and standard managemental practices for obtaining good performance with proper welfare (Chatterjee et al., 2008; Rajkumar et al., 2010). This line was undergoing index selection for higher egg production up to 64 weeks of age.
Whole embryos from embryonic days (ED) ED5 to ED8 (n=4 per day) and breast muscle tissues from ED9 to ED18 (n=4 per day) were collected for the study. Also, before (18th week) and after (22nd and 40th week) onset of egg production, 4 birds each were sacrificed and the breast muscle tissues were collected under aseptic conditions following the approved protocol of Institute Animal Ethics Committee (IAEC) of ICAR-Directorate of Poultry Research, Hyderabad. All the samples were collected in 1.5 ml Dnase-RNase-free sterile polypropylene tubes using DEPC (0.1%) treated sterile instruments. They were chilled immediately on ice in order to minimize RNA degradation, transferred to the lab and kept at -80°C until further use.
Total RNA was isolated from embryo and muscle tissues using Trizol (Amresco), according to the standard protocol (Badola et al., 2004; Mishra et al., 2008; Pal et al., 2011). Resulted RNA pellet was re-suspended in 50 µl nuclease-free water and the concentration and quality were determined in Genova plus NanoDrop spectrophotometer and 1.2% formaldehyde agarose gel. The RNA sample showing the OD260:OD280 values in between 1.8 to 2.2 were considered as good quality and were used further.
First strand cDNA synthesis
Each sample of RNA was treated with DNaseI (Fermentas) and converted in to cDNA using Verso cDNA synthesis kit (Thermo Scientific, #00775881) following standard protocol (Bhattacharya et al., 2011a). This reverse transcription was carried out in thermocycler (Himedia) using the components 5X cDNA Synthesis Buffer (4 µl), Anchored Oligo dT (0.25 µl), Random Hexamer (0.75 µl), dNTP Mix (2 µl), RT Enhancer (1 µl), Verso Enzyme Mix (1 µl), RNA template (RNA) (2 µg) and Nuclease-free H2O (make up the volume to 20 µl) under the condition 42°C @ 30 minutes and 95°C @ 2 minutes. The resulted cDNA was stored at -20°C until further use. The ACACB and GAPDH genes were amplified by using cDNA as a template in RT-PCR with gene specific primers and were then confirmed by 1% agarose gel electrophoresis (Fig 1). The GAPDH gene was used as internal control for quantification of ACACB expression in realtime PCR (Bhattacharya et al., 2011b).
Real-time quantitative PCR (qPCR)
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for normalizing different amounts of input RNA. Intron-spanning primers used for qPCR expression were designed using the IDT Primer Quest software (Table 1). The mRNA expression levels of the target (ACACB) and reference (GAPDH) genes were quantified by using thermal cycler Applied Biosystems® Step One Real-time PCR (Life Technologies) with BrightGreen 2x qPCR MasterMix-ROX (abm). All the PCR reactions were performed in three biological replicates with a final volume of 10 µl containing 5 µl of BrightGreen 2x qPCR MasterMix-ROX, 0.5 µl of each forward and reverse primer (10 µM), 1 µl of cDNA and 3 µl of nuclease-free water. Thermal cycling conditions followed were initial denaturation at 95°C for 10 minutes followed by 40 cycles of PCR stage (95°C for 15 seconds and 58°C for 1 minute) and melting stage (95°C for 15 seconds, 60°C for 1 minute and 95°C for 15 seconds) was performed at the end of the qPCR to check the specificity of amplification.
Comparative Ct method was used for estimating the expression levels of gene (Livak and Schmittgen 2001; Bhattacharya and chatterjee 2013). Threshold cycle (Ct) values of ACACB and GAPDH genes were obtained after performing qPCR. These Ct values were used for estimating the “n-fold up/down-regulation of transcription” and ACACB gene expression levels relative to the internal control by using the following formula:
ΔCt= Average Ct of the target gene (ACACB) - Average Ct of the reference gene (GAPDH).
ΔΔCt= Average ΔCt of the target sample - Average ΔCt of the calibrator sample.
RESULTS AND DISCUSSION
Thus, we have chosen IWI line i.e. developed from white leghorn breed that was known for high egg production. These egg laying chickens provides good insight to understand various processes of lipid mobilization, transfer and utilization for yolk precursor synthesis, which is important in deposition of yolk and embryo development (Saarela et al., 2008). Merkin et al. (2012) while studying evolutionary dynamics of gene regulation in mammalian and bird tissues, indicated that ACACB gene expressed at low to medium level in colon, heart, kidney, lung, skeletal muscle tissue, spleen and testis while its expression is below cut off level in brain and liver. Similarly, a study by Barborasa-Morais et al. (2012) also showed that ACACB gene is low to medium in kidney, skeletal muscle and heart and below cut-off in brain and liver. There was no clear-cut idea how the gene is expressed over different developmental stages of chicken. There was no reports are available on expression of the ACACB gene during the pre and post-hatch period in chicken. However, some other genes are studied and reported during pre and post-hatch period in chicken. In domestic chicken, the higher embryonic growth was reported during late embryonic stage (Cogburn et al., 1989). In both control broiler and Aseel, the ACTRIIB gene expressed at ED7, ED11 and ED16 (Vishnu et al., 2017). Similar results were also found in the post-hatch period in layer chicken line, where the highest and lowest expression level of the SCD gene was found on the 2nd (40.75) and 6th week (27.75), respectively (Sagar et al., 2019). In the control layer, the expression of ACACA gene was up-regulated during 2nd, 4th and 6th weeks of age, when compared to the day one post-hatch period (Prasad et al., 2018b). In two indigenous breeds i.e. Ghagus and Aseel, the myoglobin (Mb) gene expression was studied in different organs like muscle, bursa, heart, spleen and gizzard at day old and 4th week, respectively (Prasad et al., 2019). In both Aseel and control broiler, the ACTRIIB gene expression was studied and showed up-regulatedon day one and downward upto 4th week and gradually increased at 6th week, respectively (Vishnu et al., 2017). In Ghagus, the expression of TNNC1 gene was studied in different organs such as muscle, bursa, heart, spleen and gizzard, respectively (Prasad et al., 2018a). In this context, we explore the expression profile of ACACB gene during pre and post-hatch period of IWI line as it no far reported.
Standard curve was plotted to optimize real time PCR, where a regression coefficient (slope) of -2.941 and -4.661 and coefficient of determination (R2) of 0.9543 and 0.8374, respectively, was found for ACACB and GAPDH genes (Fig 2). A significant regression coefficient was found in the target and reference genes representing an optimum standardization of gene expression before carrying out the actual experiments. The amplification plot and dissociation curve of the respective target and reference genes were analyzed by qPCR (Fig 3; Fig 4). The common comparative threshold method of analysis i.e. 2 -ΔΔCt was used to study the relative gene expression during pre and post-hatch period of IWI line. The ED5 and 18th week stage were used as a calibrator to determine the relative expression during pre and post-hatch periods, respectively. A significant (P<0.05) effect of ACACB gene expression was observed in IWI line at different stages during different developmental periods (Fig 5; Fig 6). The expression of the ACACB gene was down-regulated during embryonic period i.e. ED6 to ED18 (on ED6 by 0.40, ED7 by 0.57, ED8 by 0.58, ED9 by 0.38, ED10 by 0.57, ED11 by 0.35, ED12 by 0.26, ED13 by 0.24, ED14 by 0.35, ED15 by 0.62, ED16 by 0.93, ED17 by 0.80 and ED18 by 0.83 folds in this chicken line. During adult stage, the expression was also down-regulated by 0.32 fold on the 22nd week and 0.40 fold on 40th week of age. From this result, we may suggest that due to lower expression of the ACACB gene, the abdominal fat deposition is low in layer chicken. Earlier literature also revealed the same trend in mice where mice lacking ACACB gene showed decreased fat storage in abdomen (Abu-Elheiga et al., 2001). When metabolic fuel in the body is low and sufficient ATP is needed, ACACB pathway is turned off by phosphorylation. Consequently, the level of malonyl-CoA is reduced, which leads to generation of ATP through increased fatty acid oxidation and the decreased consumption of ATP for fatty acid synthesis. Thus, ACACB gene expression is important only during de novo fatty acid synthesis when lipids and triglycerides are synthesized in the body.
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