Experimental outline
In the first stage of the study, (1) a meta-analysis was performed on the basis of whole-transcriptome microarray data generated in previous experiments10,12 in order to select immune-related genes that were silenced. Bioactive compounds used in those experiments for in ovo delivery were two prebiotics (P1 – GOS; P2 – inulin) and synbiotics ( S1 – Lactococcus lactis subsp. lactis + inulin; S2 – Lactobacillus salivarius + GOS, S3 – Lactococcus lactis subsp. cremoris + GOS in ovo to broiler chickens.
The present experiment follows the same route of the substance delivery and focuses on single synbiotic the synbiotic and its individual components: prebiotic (GOS) and probiotic (Lactococcus lactis subsp. cremoris). These three bioactive substances were in ovo administered into chicken embryos of two contrasting chicken genotypes: Ross 308 and Green-legged partridgelike.
Based on spleen isolated from in ovo stimulated chickens a following analysis were performed: (2) global methylation to verify the epigenetic nature of the changes, and then (3) gene expression analysis (to confirm expression regulation under the influence of various substances) and (4) methylation analysis of individual selected genes.
Meta-analysis of published microarray data for gene selection
Gene selection for methylation analysis was based on two sets of microarray data. These data sets contained broiler chicken transcripts generated from individuals which received prebiotic and synbiotic in ovo on day 12 of egg incubation10,12. Both projects carried out whole-genome microarray analyzes (Affymetrix, Santa Clara, US), based on genetic material isolated from spleen in experiment with in ovo injection of S1, S2 and S3. Meta-analysis aimed to select genes which were silenced at mRNA level in spleen. The analysis was carried out based on gene lists generated by Affymetrix Expression Console software. In silico selection of gene sequences was based on the following criteria: p-value (p<0.05) and fold change (down-regulation; FC< -1.0). Subsequently, selected gene groups were compared with each other based on the Venn diagrams. Connection of the selected genes was analyzed with STRING37. A final list was selected based on gene function and possibility of designing primers for the qMSP reaction.
Experimental setup and tissue collection
600 eggs of Ross 308 (Ross) broiler chicken and 600 eggs of Green-legged Partridgelike (GP) were incubated in standard conditions. On the day 12 of incubation, eggs were randomly distributed into experimental groups (150 eggs per group): (1) probiotic (PRO) – Lactococcus lactis subsp. cremoris, (2) prebiotic (PRE) – galactooligosaccharides (GOS) (3) synbiotic (SYN) – Lactococcus lactis subsp. cremoris with GOS. The set amount of bacteria was 105 bacteria CFU egg-1 and the amount of prebiotic was 3.5 mg egg-1. The control group (C) was mock-injected with 0.2 mM physiological saline (0.9%). Eggs were injected into an air cell with 0.2 mL of aqueous solution of each substance. After hatching, birds were housed in litter pens (4 replicates/group, 8 animals each). Six randomly selected individuals from each group (PRO, PRE, SYN and C) were sacrificed on the day 42 post-hatching and spleen was collected. The experiment was approved by the Local Ethics Committee for Animal Experiments (Bydgoszcz, Poland) (study approval reference number 16/2014). All methods were carried out in accordance with relevant guidelines and regulations.
RNA and DNA isolation
Tissues for RNA isolation were fixed in stabilizing buffer (fix RNA, EURx, Gdansk, Poland). RNA isolation was prepared by using TRI reagent (MRC, Cincinnati, USA) and commercial kit for RNA purification (Universal RNA Purification Kit, EURx, Gdansk, Poland). Spleen was homogenized with the TissueRuptor homogenizer (Qiagen GmbH, Hilden, Germany) in TRI reagent. Isolation of DNA from spleen was carried out by phenol – chloroform method38 as described by Dunislawska et al. 202031. RNA and DNA quality and quantity was checked by electrophoresis and NanoDrop2000 (Scientific Nanodrop Products, Wilmington, USA).
Global methylation analysis
Global DNA methylation analysis was prepared using commercial set for methylated DNA quantification (MDQ1, Imprint Methylated DNA Quantification Kit, Sigma-Aldrich) according to manufacturer's protocol and based on Gryzinska et al. 201339. DNA isolated spleen (n=6) was diluted in binding solution to final concentration 150ng/µl. DNA was intended for estimation of methylated DNA level based on the ELISA principle on 96-well plates. Positive (methylated) and blank controls were analyzed together with DNA samples. The absorbance was measured at 450 nm. For each stimulated group, six samples, each derived from a different individual, were analyzed. The absorbance measurements were performed in two technical repeats, each using the same amount of DNA. Two measurements were averaged, and the mean value was used for the further analyses. Global DNA methylation levels are percentages relative to the methylated control and were calculated using the following formula:
![](https://myfiles.space/user_files/58894_9946feeafa4c1df7/58894_custom_files/img1607051179.JPG)
where A450S is the average absorbance of the sample, A450B is the average absorbance of the blank, A450MC is the average absorbance of the methylated control. Statistical analysis was carried out by using SAS statistical software.
Gene expression analysis – RT-qPCR
Gene expression analysis was performed by quantitative reverse transcription PCR (RT-qPCR). cDNA was synthesized using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific/Fermentas, Vilnius, Lithuania), following the manufacturer's recommendations. The qPCR reaction mixture included Maxima SYBR Green qPCR Master Mix (Thermo Scientific/Fermentas, Vilnius, Lithuania), 1 μM of each primer and diluted cDNA (140 ng). Thermal cycling was performed in a LightCycler II 480 (Roche Diagnostics, Basel, Switzerland). Each RT-qPCR reaction was conducted in two technical replicates. Gene expression analysis was performed for selected genes in meta-analysis step. Sequences of primers were based on the literature or were designed by NCBI Primer BLAST tool40 based on NCBI sequence. Primer sequences are shown in Table 3. Relative gene expression analysis was conducted separately for each experimental group by the ΔΔCt method41 using ACTB42 and G6PDH43 as reference genes. Geometric means of cycle threshold (Ct) values of reference genes were used in the analysis44. Statistical analyses were performed by comparing the Ct value of each experimental group with that of the control group by Student's t-test (P < 0.05).
Table 3. Primer sequences used in the RT-qPCR reaction
Gene
|
Primers sequence
|
Amplicon
size
|
NCBI no.
|
CD72 (chB1)
|
F: AGGAAGGTAGGGCAGCAATG
R: CTGACCTGAGGTTCGCCAAA
|
134
|
395923
|
CXCR5
|
F: GCTCTGACTGTAGGGTGACG
R: TGAAATGATGGGCAGTGGCT
|
145
|
419784
|
NFATC1
|
F: TCGAGTTCAAGCACAGCGAT
R: GAAGGACCCCCTCGGAAGA
|
155
|
420815
|
SYK
|
F: AAGGGACAGCAATGGTTCCT
R: AATTTAACAGACCTGCCAGAGG
|
142
|
427272
|
CYR61
|
F: ATCGCTCGTTCAGACGCATA
R: TGTCTGGGCTCCGCTAAAAG
|
144
|
429089
|
NR4A3
|
F: GGCATCCCCGGAGTTTCTCTG
R: TTTGACGAGGCCGCTCATT
|
237
|
420996
|
SERPING1
|
F: GTCCTCGTGCCACACTTACC
R: TTGACCAATGCTTGCCCACC
|
111
|
423132
|
TNFRSF14
|
F: TGAGCACCATCAGGGGTATC
R: AGGTACGGATGCTTCCCAAG
|
170
|
420403
|
IKZF1
|
F: GCGTGTGAAAGAGCGACTTC
R: GAACACTCCGCACAACACCT
|
149
|
395974
|
KLHL6
|
F: ATGGTTTCTGCGTCAACTCC
R: CATCCTGGCTGGGATGCAATA
|
120
|
424762
|
ANGPTL4
|
F: TCCTCGATTCGCGAGTTCTG
R: CAGGGCACTGGGAGCTG
|
148
|
769087
|
Real-time quantitative methylation-specific polymerase chain reaction (qMSP)
DNA methylation is a specific chemical modification of a nucleic acid, involving the attachment of a methylene group to cytosine or adenine nucleotides. The isolated DNA was subjected to methylation analysis using the qMSP method as described by Dunislawska et al. 202031. The QMSP method is a methyl specific quantitative PCR (qPCR) preceded by DNA conversion using bisulfite. Due to this modification, unmethylated cytosine undergoes deamination and uracil is formed. In contrast, 5-methylcytosine (which is a methylation product) is resistant to this modification, making it possible to make it visible in the qPCR reaction with the help of primers specific for methylated and unmethylated DNA. The mechanism of methylation concerns only cytosines that are part of the cytidine-phosphate-guanosine (CpG) dinucleotide sequence. Therefore the primers for qMSP reactions were designed within CpG islands. Based on the selected gene list in meta-analysis, gene primers in two variants were designed: methylated and unmethylated (Table 4). The conversion was carried out using the EpiJet Bisulfite Conversion Kit (Thermo Fisher Scientific/Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions. In the next stage, the qPCR reaction was performed for the selected genes, where for each gene two primer pairs were designed - specific for methylated and non-methylated DNA using the MethPrimer tool. Primers for qMSP were complementary to the gene promoter region and were designed based on criteria: island size >100, GC% >50.0; obs./exp >0.60. DNA oligonucleotides were synthesized by Sigma-Aldrich. The qPCR analysis was performed in LightCycler 480 (Roche Diagnostics, Risch-Rothreuz, Switzerland) thermal cycler. The reaction mixture contained the Maxima SYBR Green qPCR Master Mix intercalating dye (Thermo Fisher Scientific/Fermentas, Vilnius, Lithuania). The optimized melting temperature was 58°C. After amplification, a melting curve was generated for each product (n=6/group). This was due to a gradual increase in temperature up to 98°C with continuous measurement of fluorescence. The relative level of DNA methylation [%] was calculated based on the results of melting curves (read fluorescence level) for each individual according to the formula45: 100 x (M/M+u), where M - average fluorescence intensity of the methylated product, U - average fluorescence intensity of the unmethylated product. Statistical analysis was performed using the student T-test (n=6; p<0.05) - comparison to control within the genotype and substance between genotypes.
Table 4. Sequences of the primers designed to qMSP reaction by using MethPrimer tool. M – specific for methylated DNA; U – specific for unmethylated DNA.
Gene
|
Primers sequence
|
GC%
|
Amplicon
size
|
NCBI no.
|
CD72
|
M
|
F: AACGGGTTATGTGTCGTTATTAGTC
R: AAACTAAACCCTACTACCTTCTCGC
|
60.00
72.00
|
107
|
395923
|
U
|
F: TGGGTTATGTGTTGTTATTAGTTGT
R: ACTAAACCCTACTACCTTCTCACA
|
64.00
70.83
|
103
|
CXCR5
|
M
|
F: AGAGGTTGGGATTTACGGTAATAAC
R: ACAACTTTCTACCTTTACAAACGCT
|
56.00
56.00
|
156
|
419784
|
U
|
F: AGGTTGGGATTTATGGTAATAATGT
R: ACAACTTTCTACCTTTACAAACACT
|
56.00
56.00
|
154
|
NFATC1
|
M
|
F: CGATTCGGAAATATTAATAAAGC
R: AAAAATAATATAAACCCTACCCGAC
|
52.17
60.00
|
100
|
420815
|
U
|
F: TTTGTATTGATTTGGAAATATTAATAAAGT
R: ACAAAAATAATATAAACCCTACCCAAC
|
46.67
62.96
|
109
|
SYK
|
M
|
F: TATTAGGCGTTTTCGGGAAC
R: AAATTAATACATTTACTCGCCGCT
|
70.00
54.17
|
115
|
427272
|
U
|
F: GTTTATTAGGTGTTTTTGGGAATGA
R: CCAAATTAATACATTTACTCACCACT
|
68.00
57.69
|
120
|
CYR61
|
M
|
F: TTTGGTTTTAGTGTTTAAAGACGT
R: TTATATTTACCTTCAAAAAAACGTA
|
58.33
44.00
|
150
|
429089
|
U
|
F: TTTTGGTTTTAGTGTTTAAAGATGT
R: TATTTATATTTACCTTCAAAAAAACATA
|
56.00
42.86
|
154
|
NR4A3
|
M
|
F: GGGAAAGGATAAAGTTTTTGTAGTC
R: AAACTCAAACGTAACCCTAAACGTA
|
52.00
56.00
|
179
|
420996
|
U
|
F: GGGAAAGGATAAAGTTTTTGTAGTTG
R: AAACTCAAACATAACCCTAAACATA
|
53.85
56.00
|
179
|
SERPING1
|
M
|
F: GGTAACGAGAGTTTGGATTTGTAAC
R: CCTAAATAAACCCTAAAAACTACGC
|
56.00
64.00
|
163
|
423132
|
U
|
F: TGGTAATGAGAGTTTGGATTTGTAAT
R: CTAAATAAACCCTAAAAACTACACC
|
53.85
64.00
|
163
|
TNFRSF14
|
M
|
F: GTTTTAGTTATTTTTGTTTTTACGTTCGT
R: CCGCTATCACTATACAACTTCTCG
|
65.52
62.50
|
298
|
420403
|
U
|
F: AGTTATTTTTGTTTTTATGTTTGT
R: CACTATCACTATACAACTTCTCACC
|
62.50
64.00
|
292
|
IKZF1
|
M
|
F: GTAGTAGTAATTGTTGGAGGAGGC
R: AAAAATAACTTTACGAAACAACGAA
|
62.50
64.00
|
192
|
395974
|
U
|
F: GTAGTAGTAATTGTTGGAGGAGGTG
R: AAAAATAACTTTACAAAACAACAAA
|
64.00
64.00
|
192
|
KLHL6
|
M
|
F: TTTTTTGGATAATGAGTGTTTAACG
R: AAACACCAAAAAAAATCCCGTA
|
52.00
63.64
|
100
|
424762
|
U
|
F: TTTTTGGATAATGAGTGTTTAATGA
R: CTAAAACACCAAAAAAAATCCCATA
|
48.00
64.00
|
102
|
ANGPTL4
|
M
|
F: TAATTTTAACGGGAAGTATTTTCGT
R: CAACTTTAAAACTCTACCTCCAACG
|
56.00
60.00
|
156
|
769087
|
U
|
F: TAATTTTAATGGGAAGTATTTTTGT
R: ACTTTAAAACTCTACCTCCAACACA
|
56.00
60.00
|
154
|