The hypothalamus plays an overarching role that is reflected in the physiological processes observed in the entire organism. The hypothalamus regulates selected metabolic processes and activities of the autonomic nervous system. The avian hypothalamus due to the structural complexity is not well described and has a slightly different function than the mammalian hypothalamus that is the subject of numerous studies. The present study evaluated activities of hypothalamic genes in fast-growing chickens during development (at the 1st day and 3rd and 6th weeks after hatching). The hypothalamic transcriptomes for 3- and 6-week-old cockerels were analysed using an RNA sequencing method in next-generation sequencing (NGS) technology. The differentially expressed gene analysis was conducted using DESeq2 software. In younger 22-day-old cockerels, 389 genes showed higher expression (fold change > 1.5) than that in 45-day-old birds. These genes played a role in several biological processes because they encoded proteins involved in integrin signalling, regulation of hormone levels, camera-type eye development, and blood vessel development. Moreover, surprisingly in the hypothalamus of 3-week-old cockerels, transcripts were identified for proteins involved in both anorexigenic (
The hypothalamus is the part of the brain localized below the thalamus and dorsally to the anterior of the pituitary. The primary role of the hypothalamus is to link the nervous and endocrine systems [
During organismal development, the hypothalamus plays a significant role because the action of growth hormone (GH) is mediated; thus, body composition, fat utilization, somatic growth, physical strength, and agility are affected. Moreover, the hypothalamus controls the intermediary metabolism and secretes important hormones such as GH-releasing hormone (GHRH), somatostatin (SST), and ghrelin (GHRL) that regulate GH action. Additionally, impaired GH secretion results in growth retardation in children [
With the development of molecular biology tools, experimentation is possible in dimensions inaccessible ten years ago. For example, a novel high-throughput RNA sequencing (RNA-seq) method identified the
In birds, the hypothalamic regions cannot be analysed separately for their functionality, whereas in mammals, such analysis is possible thanks to Nissl staining [
For the avian model, 24 fast-growing Ross 308 cockerels were used. The cockerels were hatched on the same day in a commercial poultry hatchery. The eggs were obtained from selected parent stock farms and delivered to the experimental farm of the National Research Institute of Animal Production located in Aleksandrowice (Poland). The chickens were fed ad libitum complete starter (days 1–21), grower (days 22–35), and finisher (days 36–45) diets containing 22, 20.5, and 20.5% completed protein (CP), respectively, and 2990, 3130, and 3130 kcal/kg ME, respectively. The mixtures were composed according to dietary requirements for meat-type chickens [
The chickens were kept in pens on deep litter in the same optimal, electronically controlled environmental conditions (temperature, lighting regime, and air humidity) until 1 (
The isolation of the hypothalamus using the magnifier Benefit 130E Pro7 and minitweezers.
Dissected hypothalamic tissues were quickly collected into tubes with RNAlater® solution (Ambion) and then were left at +4°C for 24 h before being frozen at −20°C. Carcass and growth traits were determined in accordance with the methods described by Połtowicz et al. [
RNA was isolated with PureLink™ RNA Mini Kit (Ambion) according to manufacturer’s recommendations. Hypothalamus samples were homogenized with beads using a Bullet Blender24 homogenizer (Next Advance). RNA was purified by an Agencourt® RNAClean™ XP (Beckman Coulter, Inc.). Quantitative and qualitative evaluation of RNA was performed using a TapeStation 2200 (Agilent). The RIN was in the range 7.3–8.4. The cDNA libraries were prepared for two age groups: 22- and 45-day-old chickens, with a TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the protocol. The libraries were indexed using individual adaptors in the order shown in Table
The FastQC program was used for read quality control, and the Flexbar program was used to remove adapters and poly-A sequences. Reads shorter than 32 and quality scores lower than 20 were removed from the dataset. Basecalls were performed using CASAVA version 1.8.2. The cleaned reads were aligned to Gallus_gallus-5.0 (GCA_000002315.3) with a reference annotation containing 18,346 genes listed in the Ensembl database. Alignment and estimation of gene expression level were made using the RSEM [
DEG analysis was conducted using DESeq2 [
Fifteen transcripts were validated by qPCR using
Several growth and carcass traits of the cockerels were measured during growth and in the dissection. After the sixth week, cockerels were nearly 3-fold heavier than the 3-week-old birds, although the growth rate of birds gradually decreased with age (Table
Traits of investigated cockerels estimated after slaughtering (mean ± SD).
Trait | Group of Ross 308 cockerels | ||
---|---|---|---|
0-week-old |
3-week-old |
6-week-old | |
Body weight (g) | 43 ± 5.3 |
1213 ± 104.7 |
3532 ± 386.9 |
Growth rate (%) | Days 0–22, 185.45% | Days 23–45, 97.3% | |
Average daily gain (g) | 21 days, 83 ± 7.93 |
42 days, 114.3 ± 18.1 | |
Abdominal fat (%) | 0.05 ± 0.04 |
0.73 ± 0.28 |
0.90 ± 0.23 |
Breast muscles (%) | 1.53 ± 0.25 |
17.93 ± 1.17 |
24.70 ± 1.87 |
Leg muscles (%) | 9.90 ± 0.82 |
5.53 ± 0.72 |
16.50 ± 1.30 |
A,B,CValues in rows with different letters show significant results (
The 3- and 6-week-old cockerels presented similar fat content and did not differ regarding the percentage of leg muscles. The share of breast muscle between the 1st and the 45th day after hatching increased more than 16-fold. By contrast, the leg muscle share increased only 1.7-fold during the same period.
The average total number of raw reads per sample was 23,947,897 ± 1,543,653. After the removal of adapters, trimming and filtering feature 90.5% of raw reads remained that were mapped to the chicken reference genome (Galgal 5.0) of which 69.2% and 8.5% matched annotated exonic and intronic regions, respectively (see Table
Age-dependent gene expression analysis of chicken hypothalamus was performed using the DESeq2 method. The comparison of 22 and 45-day-old cockerel transcriptomes indicated 389 genes with elevated expression in younger birds (FC ≥ 1.5, adjusted
Regulation of cellular response to growth factor stimulus (GO:0090287) biological process enriched by genes that showed higher expression level in the hypothalamus of 3-week-old than 6-week-old cockerels. The grey genes are the background (connections) for upregulated genes.
In 3-week-old cockerels, three genes (
Neuropeptide signalling pathway (GO:0007218) enriched by genes that showed higher expression level in the hypothalamus of 3-week-old than 6-week-old cockerels. All presented genes were upregulated in 3-week-old chickens.
CCAAT Enhancer-Binding Protein Delta (CEBPD) induces
The connections of leukocyte cell-derived chemotaxin 2 (LECT2). Colored genes were upregulated in 3-week-old cockerels and grey genes are the background.
In the hypothalamus of 45-day-old cockerels, 246 overexpressed genes were related to neurogenesis (
Fifteen DEGs were validated by the qPCR method and then by Pearson’s correlation. The results are presented in Figure
The validation of RNA-seq results by qPCR and Pearson correlation. The charts were generated with SAS Enterprise v. 7.1.
The relative expression patterns of the most notable hypothalamic transcripts dependent on broiler age (
In vertebrates, the hypothalamus is a brain structure accountable for numerous behavioural, autonomic, and endocrine responses that are involved in regulating metabolism, homeostasis, and reproduction. The avian hypothalamus is not well recognized because of the lack of clear boundaries delineated by Nissl staining [
The Ross 308 broilers are a fast-growing commercial chicken line with growth rates that are 10–15% higher than those of other commercial lines between the ages of 30 and 40 days. The slaughter weight of chickens of the Ross 308 line is obtained between the 35th and 42nd day from hatching, because their abdominal fat content increases significantly after this period, which is undesirable. The producer of this commercial chicken line informs in its instructions [
Numerous notable observations were produced in the comparison of hypothalamic activity at the transcriptome level in broiler chickens between the 3rd and 6th week from hatching. The Ross 308 broiler chickens according to the producer’s instructions show an increase in feed intake and daily gain calculated per BW. Our study also confirmed a significant increase in growth rate in the younger 3-week-old chickens that was associated with an increase in ADG calculated per BW and could be a consequence of increased feed intake. However, in the present study, daily feed intake was not recorded for individuals. Nevertheless, this intensive growth rate was reflected in the hypothalamic activity, because numerous genes associated with hormone status regulation were overexpressed (
Although the cockerels in the present study were fed ad libitum, the 3-week-old chickens that received a feed mixture providing 140 kcal/kg less showed higher daily gain and growth than the 6-week-old chickens. This finding could be associated with the feeding status of younger broilers since increased expression of numerous candidate genes involved in the regulation of appetites and energy balance was observed. However, the activity of hypothalamic proteins involved in the regulation of the satiety centre was unusual, because in 3-week-old cockerels, increase in transcript abundance of genes encoding proteins involved in both anorexigenic and orexigenic pathways was observed. These pathways should exclude one another because they respond to the conflicting signals of satiety and hunger. One of the most notable is the
On the other hand, in 3-week-old chickens,
In older 6-week-old chickens, the activated GO biological processes and enriched pathways were associated primarily with brain structure development. The gene that showed the highest age-dependent change in expression level was
In summary, the feed intake status of 3-week-old chickens was not obvious. Although they were fed ad libitum, their satiety centre did not provide an unambiguous signal of satiety, because many genes involved in the induction of feed intake also showed increased expression levels in the hypothalamus of this age group. However, this situation is also observed in rat foetal brain development in mothers fed a high-fat diet. Both the POMC and NPY mRNA expression increased, although only the anorexigenic pathways should have been activated. In the present study, the RNA-seq method also identified increased NPY expression (1.47-fold change) in the 3-week-old broilers; however, qPCR did not confirm this observation. The activation of genes associated with both anorexigenic and orexigenic pathways in 3-week-old broilers could be a consequence of the intensive growth rate at this age, which could be associated in turn with intensive feed intake; therefore, their anorexigenic pathways were activated to avoid excessive fat deposition. Simultaneously, they also grow intensively, which requires high expenditure of energy; therefore, they induced indirect orexigenic factors. However, further investigation of this focus is required.
The datasets generated during and/or analysed during the current study are available from the corresponding author on request.
The authors declare no conflict of interest regarding the publication of this paper.
The study was supported by the statutory activity of the National Research Institute of Animal Production (no. 01-013.1) and the National Multidisciplinary Laboratory of Functional Nanomaterials NanoFun nr POIG.02.02.00-00-025/09 (Innovative Economy Operational Programme, Priority Axis 2: R&D Infrastructure, Action 2.2: Support of Formation of Common Research Infrastructure of Scientific Units). Adam Mickiewicz University, Faculty of Biology, is a member of KNOW Poznan RNA Centre (no. 01/KNOW2/2014).
Table S1: overall statistics and read annotations obtained for each analysed cockerel sample describing raw and processed data. Table S2: genes used in validation during qPCR analysis and information on primers, probes, and assays purchased from Applied Biosystems. Figure S1: blood vessel development biological process enriched by genes that showed increases in expression level in the hypothalamus of 3-week-old cockerels. The grey genes are the background (connections) for upregulated genes. Figure S2: reactome pathways and GO biological processes generated by PANTHER and enriched by genes that showed increased expression levels in 6-week-old cockerels. Figure S3: the connections of mitogen-activated protein kinase 10 (MAPK10). Colored genes were upregulated in 6-week-old cockerels, and grey genes are the background.