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Full Research Article

Comparative Sequencing and Detection of Polymorphism in Targeted Regions of Ryanodine Receptor 2 Gene in Fast and Slow Growing Chickens

K
K.F. Olayiwola1,*
0009-0000-2119-2265
A
A.A. Toye1
0009-0008-4105-8790
M
M.K. Sulaiman2
0000-0002-8295-7855
1Department of Animal Production and Health, Faculty of Agriculture, University of Ilorin, Ilorin, Nigeria.
2University of Ilorin Teaching Hospital Molecular and Diagnostic Research Laboratory, Ilorin, Nigeria.

ABSTRACT

Background: Sudden death syndrome (SDS) is a metabolic and cardiovascular disorder of fast-growing broilers, associated with rapid growth, physiological stress and cardiac dysfunction. Ryanodine receptor 2 (RYR2) regulates calcium release during cardiac contraction and is therefore a relevant candidate gene for SDS-related cardiovascular risk. This study detected sequence variation in selected RYR2 regions and examined its association with growth and cardiovascular risk indicators in chickens raised in Nigeria.

Methods: One hundred and fifty chickens comprising ross 308, arbor acres and noiler were evaluated for growth traits, body mass index and selected blood-based cardiovascular indicators. Fifteen representative samples, five per breed, were used for DNA extraction, PCR amplification and Sanger sequencing of selected RYR2 exons and flanking regions. Sequences were aligned with the reference chicken RYR2 sequence to identify polymorphisms.

Result: Sequence analysis identified an A/G single nucleotide polymorphism in the flanking region of RYR2 exon 40 at chicken genome position 3:36968262. Arbor Acres and Noiler chickens carried the G allele, while ross 308 chickens carried the A allele. No polymorphism was detected in exon 23, while exon 44 was not successfully sequenced. Ross 308 chickens showed higher body mass index from weeks 1 to 6, whereas most blood-based cardiovascular risk indicators were not significantly different between breed-associated haplotype groups, except high-density lipoprotein. The detected RYR2 flanking-region SNP may serve as a preliminary breed-associated molecular marker, but further validation using larger populations and individual genotyping is required.

 

INTRODUCTION

Broiler chickens are important to Nigeria’s animal protein supply because they produce meat within a short production period of about 6-10 weeks and support employment, income and food security (Oloso et al., 2020). Their commercial value depends largely on rapid growth, high feed conversion efficiency and profitability (Riber and Wurtz, 2024). Nigeria has over 200 million commercially raised chickens, making poultry production a major contributor to national food supply (Oloso et al., 2020).

However, genetic selection for rapid growth, muscle deposition and feed efficiency has increased the occurrence of metabolic and cardiovascular disorders, including sudden death syndrome (SDS) (Sosnowka-Czajka and Skomorucha, 2022; Riber and Wurtz, 2024). Sudden death syndrome is a metabolic and cardiovascular disorder of fast-growing broiler chickens, characterized by the sudden death of apparently healthy birds, often without obvious external clinical signs (Sosnowka-Czajka and Skomorucha, 2022).

Although the exact aetiology of sudden death syndrome is not fully understood, nutrition, genetics, environmental stress and cardiovascular dysfunction are key contributing factors (Sosnowka-Czajka and Skomorucha, 2022). Intensive selection for rapid growth and feed efficiency has increased cardiovascular workload in commercial broilers, predisposing them to metabolic imbalance, increased oxygen demand, excessive heat production, cardiac arrhythmias and sudden death, particularly under tropical conditions where high temperature and humidity increase physiological stress (Egbuniwe and Ayo, 2024; Apalowo et al., 2024; Huang et al., 2017; Riber and Wurtz, 2024).

Fast-growing broilers have high metabolic activity because they rapidly convert feed into body mass, especially muscle tissue, generating substantial internal heat that must be dissipated to maintain physiological balance (Hridoy et al., 2021; Bora et al., 2024; Apalowo et al., 2024; Riber and Wurtz, 2024; Monika et al., 2025). However, heat loss is limited because chickens lack sweat glands, are feather covered and rely mainly on respiratory and behavioural thermoregulation, while subcutaneous fat and a smaller surface area-to-volume ratio in heavy broilers may further increase thermal burden (Egbuniwe and Ayo, 2024; Rokade et al., 2018; Riber and Wurtz, 2024). Thus, internal metabolic heat and external heat stress may intensify cardiovascular strain, oxidative stress and metabolic imbalance, making sudden death syndrome a multifactorial condition involving genetic, metabolic, cardiovascular, nutritional and environmental factors (Egbuniwe and Ayo, 2024; Apalowo et al., 2024; Sosnowka-Czajka and Skomorucha, 2022).

In contrast, slow-growing and tropically adapted chickens generally have lower metabolic pressure, reduced cardiovascular load and better tolerance to local conditions than fast-growing commercial broilers (Ajayi et al., 2020; Riber and Wurtz, 2024). The Noiler chicken, a Nigerian dual-purpose breed developed from improved and locally adapted genetic backgrounds, is therefore useful for comparison because of its slower growth, local adaptation and relevance to Nigerian poultry production (Ajayi et al., 2020; Animashahun et al., 2022).

The contrast between fast-growing broilers such as Ross 308 and Arbor Acres and slower-growing adapted chickens such as Noiler provides a useful basis for investigating genetic factors linked to growth, cardiovascular risk and susceptibility to sudden death (Ajayi et al., 2020; Riber and Wurtz, 2024).

Because sudden death syndrome is influenced by genetic, metabolic, cardiovascular and environmental factors, molecular approaches can help explain its biological mechanisms and may provide greater precision than phenotypic selection alone (Khalil et al., 2021; Sosnowka-Czajka and Skomorucha, 2022). Candidate gene studies are particularly useful because they focus on genes with known biological roles related to the traits or disorders of interest (Basaki et al., 2019; Khalil et al., 2021).

For sudden death syndrome and related cardiac disorders, polymorphisms have been reported in genes involved in calcium regulation and cardiac muscle function, including calsequestrin 2 and ryanodine receptor 2 (Basaki et al., 2016; Basaki et al., 2019). The ryanodine receptor 2 gene is a strong candidate for cardiovascular risk studies because it regulates calcium release and excitation-contraction coupling in cardiac muscle, processes essential for normal cardiac rhythm and contraction (Peng et al., 2016; Basaki et al., 2019; Roston et al., 2022). Therefore, sequence variation in selected regions of this gene may help explain breed-associated differences in susceptibility to sudden death syndrome and related cardiovascular traits (Basaki et al., 2019; Khalil et al., 2021).

This study assessed sequence variation in selected regions of the ryanodine receptor 2 gene and its association with body mass index and selected cardiovascular risk indicators in ross 308, arbor acres and noiler chickens.

MATERIALS AND METHODS

Experimental site
 
The study was conducted from July 2023 to July 2024 at the University of Ilorin, Ilorin, Nigeria. The animal phase, including rearing and data collection, was carried out from July to October 2023 at the University of Ilorin Teaching and Research Farm (8°27'09.5"N, 4°39'44.5"E) under the Department of Animal Production and Health. DNA extraction, PCR amplification and sequencing were performed at the University of Ilorin Central Research Laboratories, University of Ilorin Teaching Hospital Molecular Diagnostic and Research Laboratories and Inqaba Biotechnology Limited, South Africa, respectively.
 
Experimental birds and management
 
A total of 150 day-old chicks, comprising 50 ross 308, 50 arbor acres and 50 noiler chickens, were used in a completely randomized design. Ross 308, arbor acres and noiler chicks were sourced from agrited hatchery, lagos, Nigeria; valentine hatchery, Ilorin, Nigeria and amo sieberer hatchery, ibadan, Nigeria, respectively. Birds from each breed were distributed into three environmentally homogenous pens as replicates to reduce micro-environmental effects.

On arrival, chicks received commercial multivitamins and electrolytes, were wing-tagged for identification and reared on deep litter with ad libitum access to feed and water. Birds were fed commercial starter mash from day-old to week 4 and finisher mash from weeks 5 to 6, with nutrient composition shown in Table 1. Infectious bursal disease vaccine (Gumboro, intermediate plus strain) was administered on days 7 and 21 via drinking water, while newcastle disease vaccine (LaSota strain) was given on days 14 and 28 via drinking water. Ross 308 and arbor acres represented fast-growing commercial broilers, while Noiler served as a slower-growing, tropically adapted genetic contrast with lower metabolic load and greater resistance to metabolic stress.

Table 1: Nutrient composition of commercial broiler starter and finisher diets (Ultima feeds).


 
Body mass index data collection
 
Growth and morphometric traits were recorded weekly from weeks 1 to 6. Body weight was measured in grams using a top-loading scale, while body length was measured in centimeters using a measuring tape, following Oludoyi and Toye (2012). Body mass index was calculated as:
 
 
 
Blood collection and DNA extraction
 
Blood samples were collected from the brachial vein of selected birds into EDTA bottles using sterile 2 ml syringes and needles for hematological analysis, serum biochemistry and DNA extraction. Genomic DNA was extracted following Udeze et al., (2020) and DNA concentration and purity were assessed using a NanoDrop spectrophotometer.

Primer design
 
The Gallus gallus domesticus RYR2 gene sequence was obtained from ensembl using transcript ENSGALT000 10021632.1 on chromosome 3. RYR2 was selected because of its role in calcium regulation, cardiac function and sudden death syndrome in broilers. Primers targeting exons 23, 40 and 44, including flanking intronic regions, were designed using Primer3web version 4.0.0 (Table 2) and synthesized by Inqaba Biotechnical Industries, South Africa.

​

Table 2: Primer sequencing results for RYR2 exons in ross 308, arbor acres and noiler chickens.


 
PCR amplification
 
Fifteen DNA samples, five per breed, were amplified using a MyGene™ Series Peltier Thermal Cycler at the Molecular Diagnostic and Research Laboratory, University of Ilorin, Nigeria. Each 25 μl PCR reaction contained 2X PCR master mix, genomic DNA, forward and reverse primers and nuclease-free water. Cycling involved denaturation at 95°C, primer-specific annealing at 55°C, 58°C or 62°C, extension at 72°C and final extension at 72°C. PCR products were resolved on 2% ethidium bromide-stained agarose gel at 145 V for 45 minutes and visualized under ultraviolet light using a 100 bp DNA ladder.
 
DNA sequencing
 
PCR products with clear bands were purified and sent to Inqaba Biotechnology Limited, South Africa, for sanger sequencing using an ABI 3500XL genetic analyzer with POP7™ polymer. Chromatograms were viewed and checked using FinchTV version 1.4.0 (Patterson et al., 2006).
 
Sequence alignment and polymorphism detection
 
Obtained sequences were aligned against the reference chicken RYR2 sequence using MultAlin to confirm target amplification and identify nucleotide differences among ross 308, arbor acres and noiler chickens. Sequence variation was assessed in both exon and flanking intronic regions (Corpet, 1988).
 
Phylogenetic and comparative sequence analysis
 
Comparative sequence analysis was performed to assess conservation around the amplified RYR2 regions. Sequences from the three breeds were compared with the reference chicken sequence and selected orthologs, while phylogenetic analysis was conducted using MultAlin and Phylogeny.fr to evaluate relationships and conservation among breeds and related species (Dereeper et al., 2008).
 
Statistical analysis
 
Quantitative performance traits, including body weight, BMI and growth velocities, together with biochemical and hematological indicators, were analyzed using a general linear model in IBM SPSS statistics version 27.0. Because sequencing was performed on a representative subset per breed, the identified polymorphism was treated as a breed-associated RYR2 haplotype rather than an individually independent genotype. One-way ANOVA was used to assess the fixed effect of haplotype group, using the following model:
Yij = μ + Hi + eij
 
Where,
Yij = The dependent trait.
μ = The overall mean.
Hi = The fixed effect of the breed-associated RYR2 haplotype (A or G).
eij = The random residual error.

Homogeneity of variance was tested using levene’s test, mean differences were compared using duncan’s multiple range test and significance was set at P<0.05.

RESULTS AND DISCUSSION

All three targeted RYR2 exons were amplified from the 15 DNA templates, with clear bands obtained in 66.7%, 66.0% and 60.0% of samples for exons 40, 23 and 44, respectively (Fig 1 and 2). The clearest bands from each breed were selected for sequencing, but exon 44 sequencing was unsuccessful despite repeated attempts.

Fig 1: Gel image of RYR2 exon 40 and 23 PCR products showing lane M ladder and fragment sizes.



​

Fig 2: Gel image of RYR2 exon 44 PCR products showing lane M ladder and fragment size.


 
Nucleotide sequence polymorphism in RYR2
 
RYR2 gene exon 23
 
Multiple sequence alignment of RYR2 exon 23 and its flanking intronic regions from Ross 308, Arbor Acres and Noiler against the reference chicken RYR2 sequence (ENSGALT00010021632.1) revealed no sequence polymorphism (deletion, insertion, inversion or substitution) in either the coding or non-coding regions (Fig 3).

​

Fig 3: Multiple sequence alignment of RYR2 exon 23 and flanking intronic sequences from ross 308, arbor acres and noiler chickens against the reference chicken RYR2 sequence, showing no polymorphic site.



RYR2 gene exon 40
 
Multiple sequence alignment of RYR2 exon 40 and its flanking intronic region showed no coding-region polymorphism, but an A/G SNP was detected in the 32  non-coding flanking region at position 3:36968262 of the RYR2 primary transcript ENSGALT00010021632.1. Arbor acres and noiler carried the G allele, while Ross 308 carried the A allele (Fig 4-5).

​

Fig 4: Multiple sequence alignment of RYR2 exon 40 and flanking intronic sequences from ross 308, arbor acres and noiler chickens against the reference chicken RYR2 sequence, showing the polymorphic site.




​

Fig 5: Chromatograms of the 3¢ flanking intronic region of RYR2 exon 40 showing the polymorphic site corresponding to 36968262insA in the reference RYR2 primary transcript ENSGALT00010021632.1.


 
Phylogenetic analysis
 
RYR2 gene exon 23
 
Phylogenetic analysis of RYR2 exon 23 and its flanking intronic regions in noiler, arbor acres and ross 308 showed sequence conservation when compared with red jungle fowl and other avian orthologs. Conserved motifs included GTACCTTC near the 32 splice donor site, TCTCTTT near the 52  splice acceptor site and conserved exon sequences, indicating conservation of this region among the examined avian sequences (Fig 6).

​

Fig 6: Phylogenetic alignment of RYR2 exon 23 and flanking intronic regions, 100 bp upstream and downstream, from arbor acres, ross 308 and noiler chickens compared with red jungle fowl and other avian orthologs, showing conserved regions.


 
RYR2 gene exon 40
 
Phylogenetic analysis of RYR2 exon 40 and its flanking intronic regions in noiler, arbor acres and ross 308, compared with red jungle fowl and other avian orthologs, revealed several conserved regions, except in duck and turkey orthologs. These included a conserved TTCAG motif at the 52  splice acceptor site corresponding to position 36968349 of the primary transcript ENSGALT00010021632.1 (Fig 7).

​

Fig 7: Phylogenetic alignment of RYR2 exon 40 and flanking intronic regions, 100 bp upstream and downstream, from arbor acres, ross 308 and noiler chickens compared with red jungle fowl and other avian orthologs, showing highly conserved regions.


 
SNP association with trait related to cardiovascular risks and physiological stress
 
Birds were classified by SNP allele, with ross 308 carrying the adenine (A) allele and Arbor Acres and Noiler carrying the guanine (G) allele. This classification was used to assess haplotype effects on cardiovascular risk indicators, including BMI, blood glucose, inflammation, lipidemia and stress level measured by the heterophil/lymphocyte ratio.
 
Effect of genotype on body mass index
 
BMI differed significantly between haplotype groups from weeks 1 to 6, with ross 308 birds carrying the A allele consistently showing higher BMI than Arbor Acres and Noiler birds carrying the G allele (P<0.05; Table 3).

Table 3: Body mass index of chickens according to breed-associated RYR2 haplotype group from week 1 to week 6.


 
Effect of genotype on blood glucose
 
Glucose levels did not differ significantly between the A and G haplotype groups (Table 4).

​

Table 4: Blood biochemical and hematological indicators of chickens according to breed-associated RYR2 haplotype group.


 
Effect of genotype on inflammation (White blood count, heterophil, lymphocyte)
 
White blood cell counts, heterophil percentages and lymphocyte percentages did not differ significantly between the A and G haplotype groups (Table 4).
 
Effect of genotype on lipidemia (TC, LDL, HDL)
 
Total cholesterol and LDL did not differ significantly between the A and G haplotype groups, but HDL was significantly lower in the A group than in the G group (P<0.05; Table 4).
 
Effect of genotype on physiological stress (Heterophil/lymphocyte ratio)
 
The heterophil/lymphocyte ratio was not significantly affected by haplotype group, although the near-significant P value suggests a weak trend that may require greater replication and lower environmental variation to confirm (Table 4).

This study assessed structural variation in selected regions of the avian ryanodine receptor 2 (RYR2) gene and its association with growth, physiological stress and cardiovascular risk indicators. RYR2 encodes a cardiac calcium release channel essential for excitation-contraction coupling and polymorphisms in this gene have been linked to calcium leakage, arrhythmias and sudden death syndrome in avian and mammalian species. Sequence alignment showed complete conservation of exon 23 among Ross 308, Arbor Acres and Noiler chickens, suggesting strong evolutionary constraint in this region. However, an A/G SNP was detected in the 32  non-coding flanking region of exon 40 at position 3:36968262, with Ross 308 carrying the A allele and arbor acres and noiler carrying the G allele.

The marked BMI divergence from week 1 (P<0.003) reflects rapid tissue accretion in ross 308 compared with the slower-growing noiler. Such accelerated growth increases visceral and cardiovascular burden because cardiac development may not match skeletal muscle demand, requiring greater cardiac output and predisposing fast-growing broilers to pulmonary hypertension, right ventricular hypertrophy, arrhythmias and sudden death.

Biochemical analysis showed significantly lower HDL cholesterol in birds carrying the Ross 308-associated A allele (6.5±0.52 mg/dL) than in G-allele birds (8.4±0.36 mg/dL; P = 0.008). Since HDL supports reverse cholesterol transport and reduces vascular inflammation, its reduction alongside high BMI suggests greater cardiovascular vulnerability in ross 308 birds. However, total cholesterol, LDL and glucose remained similar between groups, indicating that some baseline blood lipid traits may remain stable despite underlying cardiovascular risk.

The heterophil-to-lymphocyte (H/L) ratio is a useful indicator of chronic physiological and environmental stress in birds. Although the H/L ratio was not statistically significant, its near-significant value (P = 0.063) suggests a possible biological trend, with the slightly higher ratio in G-allele carriers likely influenced by the Noiler cohort and its greater alertness compared with more sedentary commercial broilers.

Similar to the present study, Basaki et al., (2019) examined RYR2 in relation to sudden death syndrome in broilers and reported increased RYR2 mRNA expression and point mutations in SDS-affected birds. In contrast, this study detected an A/G SNP in the flanking region of RYR2 exon 40, while exon 23 showed no polymorphism. Because this SNP is intronic, it does not directly alter the RYR2 amino acid sequence, but it may influence gene function through splicing, regulatory activity, non-coding RNA binding or linkage with another functional variant. Therefore, it should not yet be considered a causal mutation, but rather a preliminary breed-associated marker for further investigation of RYR2-related cardiovascular dysfunction and sudden death syndrome in chickens.

CONCLUSION

This study detected sequence variation in selected regions of the ryanodine receptor 2 (RYR2) gene among Ross 308, Arbor Acres and Noiler chickens. Exon 23 and its flanking region were conserved, while an A/G SNP was identified in the 32 flanking intronic region of exon 40 at position 3:36968262, with Ross 308 carrying the A allele and Arbor Acres and Noiler carrying the G allele. Ross 308 chickens also had significantly higher BMI from weeks 1 to 6, reflecting faster growth and greater body mass accumulation. Most cardiovascular risk and physiological stress indicators did not differ between RYR2 haplotype groups, except HDL, which was lower in Ross 308, suggesting that the A allele group may be linked to a growth and lipid profile relevant to cardiovascular risk assessment. However, the exon 40 flanking-region SNP should not be considered causal at this stage because of the small sequenced sample size, targeted gene coverage and breed-associated haplotype classification. It should instead be regarded as a preliminary marker for breed differentiation and further investigation using larger populations, individual genotyping, broader RYR2 sequencing, successful exon 44 analysis and functional validation.

ACKNOWLEDGEMENT

The present study was supported by the Department of Animal Production and Health, Faculty of Agriculture, University of Ilorin, Ilorin, Nigeria, through institutional and academic supervision. The authors also acknowledge the University of Ilorin Central Research Laboratories and the University of Ilorin Teaching Hospital Molecular and Diagnostic Research Laboratory for technical support during molecular analysis.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal handling and experimental procedures were carried out in accordance with the ethical guidelines of the Department of Animal Production and Health, Faculty of Agriculture, University of Ilorin, Ilorin, Nigeria. The study was approved by the departmental animal ethics committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Comparative Sequencing and Detection of Polymorphism in Targeted Regions of Ryanodine Receptor 2 Gene in Fast and Slow Growing Chickens

    K
    K.F. Olayiwola1,*
    0009-0000-2119-2265
    A
    A.A. Toye1
    0009-0008-4105-8790
    M
    M.K. Sulaiman2
    0000-0002-8295-7855
    1Department of Animal Production and Health, Faculty of Agriculture, University of Ilorin, Ilorin, Nigeria.
    2University of Ilorin Teaching Hospital Molecular and Diagnostic Research Laboratory, Ilorin, Nigeria.

    ABSTRACT

    Background: Sudden death syndrome (SDS) is a metabolic and cardiovascular disorder of fast-growing broilers, associated with rapid growth, physiological stress and cardiac dysfunction. Ryanodine receptor 2 (RYR2) regulates calcium release during cardiac contraction and is therefore a relevant candidate gene for SDS-related cardiovascular risk. This study detected sequence variation in selected RYR2 regions and examined its association with growth and cardiovascular risk indicators in chickens raised in Nigeria.

    Methods: One hundred and fifty chickens comprising ross 308, arbor acres and noiler were evaluated for growth traits, body mass index and selected blood-based cardiovascular indicators. Fifteen representative samples, five per breed, were used for DNA extraction, PCR amplification and Sanger sequencing of selected RYR2 exons and flanking regions. Sequences were aligned with the reference chicken RYR2 sequence to identify polymorphisms.

    Result: Sequence analysis identified an A/G single nucleotide polymorphism in the flanking region of RYR2 exon 40 at chicken genome position 3:36968262. Arbor Acres and Noiler chickens carried the G allele, while ross 308 chickens carried the A allele. No polymorphism was detected in exon 23, while exon 44 was not successfully sequenced. Ross 308 chickens showed higher body mass index from weeks 1 to 6, whereas most blood-based cardiovascular risk indicators were not significantly different between breed-associated haplotype groups, except high-density lipoprotein. The detected RYR2 flanking-region SNP may serve as a preliminary breed-associated molecular marker, but further validation using larger populations and individual genotyping is required.

     

    INTRODUCTION

    Broiler chickens are important to Nigeria’s animal protein supply because they produce meat within a short production period of about 6-10 weeks and support employment, income and food security (Oloso et al., 2020). Their commercial value depends largely on rapid growth, high feed conversion efficiency and profitability (Riber and Wurtz, 2024). Nigeria has over 200 million commercially raised chickens, making poultry production a major contributor to national food supply (Oloso et al., 2020).

    However, genetic selection for rapid growth, muscle deposition and feed efficiency has increased the occurrence of metabolic and cardiovascular disorders, including sudden death syndrome (SDS) (Sosnowka-Czajka and Skomorucha, 2022; Riber and Wurtz, 2024). Sudden death syndrome is a metabolic and cardiovascular disorder of fast-growing broiler chickens, characterized by the sudden death of apparently healthy birds, often without obvious external clinical signs (Sosnowka-Czajka and Skomorucha, 2022).

    Although the exact aetiology of sudden death syndrome is not fully understood, nutrition, genetics, environmental stress and cardiovascular dysfunction are key contributing factors (Sosnowka-Czajka and Skomorucha, 2022). Intensive selection for rapid growth and feed efficiency has increased cardiovascular workload in commercial broilers, predisposing them to metabolic imbalance, increased oxygen demand, excessive heat production, cardiac arrhythmias and sudden death, particularly under tropical conditions where high temperature and humidity increase physiological stress (Egbuniwe and Ayo, 2024; Apalowo et al., 2024; Huang et al., 2017; Riber and Wurtz, 2024).

    Fast-growing broilers have high metabolic activity because they rapidly convert feed into body mass, especially muscle tissue, generating substantial internal heat that must be dissipated to maintain physiological balance (Hridoy et al., 2021; Bora et al., 2024; Apalowo et al., 2024; Riber and Wurtz, 2024; Monika et al., 2025). However, heat loss is limited because chickens lack sweat glands, are feather covered and rely mainly on respiratory and behavioural thermoregulation, while subcutaneous fat and a smaller surface area-to-volume ratio in heavy broilers may further increase thermal burden (Egbuniwe and Ayo, 2024; Rokade et al., 2018; Riber and Wurtz, 2024). Thus, internal metabolic heat and external heat stress may intensify cardiovascular strain, oxidative stress and metabolic imbalance, making sudden death syndrome a multifactorial condition involving genetic, metabolic, cardiovascular, nutritional and environmental factors (Egbuniwe and Ayo, 2024; Apalowo et al., 2024; Sosnowka-Czajka and Skomorucha, 2022).

    In contrast, slow-growing and tropically adapted chickens generally have lower metabolic pressure, reduced cardiovascular load and better tolerance to local conditions than fast-growing commercial broilers (Ajayi et al., 2020; Riber and Wurtz, 2024). The Noiler chicken, a Nigerian dual-purpose breed developed from improved and locally adapted genetic backgrounds, is therefore useful for comparison because of its slower growth, local adaptation and relevance to Nigerian poultry production (Ajayi et al., 2020; Animashahun et al., 2022).

    The contrast between fast-growing broilers such as Ross 308 and Arbor Acres and slower-growing adapted chickens such as Noiler provides a useful basis for investigating genetic factors linked to growth, cardiovascular risk and susceptibility to sudden death (Ajayi et al., 2020; Riber and Wurtz, 2024).

    Because sudden death syndrome is influenced by genetic, metabolic, cardiovascular and environmental factors, molecular approaches can help explain its biological mechanisms and may provide greater precision than phenotypic selection alone (Khalil et al., 2021; Sosnowka-Czajka and Skomorucha, 2022). Candidate gene studies are particularly useful because they focus on genes with known biological roles related to the traits or disorders of interest (Basaki et al., 2019; Khalil et al., 2021).

    For sudden death syndrome and related cardiac disorders, polymorphisms have been reported in genes involved in calcium regulation and cardiac muscle function, including calsequestrin 2 and ryanodine receptor 2 (Basaki et al., 2016; Basaki et al., 2019). The ryanodine receptor 2 gene is a strong candidate for cardiovascular risk studies because it regulates calcium release and excitation-contraction coupling in cardiac muscle, processes essential for normal cardiac rhythm and contraction (Peng et al., 2016; Basaki et al., 2019; Roston et al., 2022). Therefore, sequence variation in selected regions of this gene may help explain breed-associated differences in susceptibility to sudden death syndrome and related cardiovascular traits (Basaki et al., 2019; Khalil et al., 2021).

    This study assessed sequence variation in selected regions of the ryanodine receptor 2 gene and its association with body mass index and selected cardiovascular risk indicators in ross 308, arbor acres and noiler chickens.

    MATERIALS AND METHODS

    Experimental site
     
    The study was conducted from July 2023 to July 2024 at the University of Ilorin, Ilorin, Nigeria. The animal phase, including rearing and data collection, was carried out from July to October 2023 at the University of Ilorin Teaching and Research Farm (8°27'09.5"N, 4°39'44.5"E) under the Department of Animal Production and Health. DNA extraction, PCR amplification and sequencing were performed at the University of Ilorin Central Research Laboratories, University of Ilorin Teaching Hospital Molecular Diagnostic and Research Laboratories and Inqaba Biotechnology Limited, South Africa, respectively.
     
    Experimental birds and management
     
    A total of 150 day-old chicks, comprising 50 ross 308, 50 arbor acres and 50 noiler chickens, were used in a completely randomized design. Ross 308, arbor acres and noiler chicks were sourced from agrited hatchery, lagos, Nigeria; valentine hatchery, Ilorin, Nigeria and amo sieberer hatchery, ibadan, Nigeria, respectively. Birds from each breed were distributed into three environmentally homogenous pens as replicates to reduce micro-environmental effects.

    On arrival, chicks received commercial multivitamins and electrolytes, were wing-tagged for identification and reared on deep litter with ad libitum access to feed and water. Birds were fed commercial starter mash from day-old to week 4 and finisher mash from weeks 5 to 6, with nutrient composition shown in Table 1. Infectious bursal disease vaccine (Gumboro, intermediate plus strain) was administered on days 7 and 21 via drinking water, while newcastle disease vaccine (LaSota strain) was given on days 14 and 28 via drinking water. Ross 308 and arbor acres represented fast-growing commercial broilers, while Noiler served as a slower-growing, tropically adapted genetic contrast with lower metabolic load and greater resistance to metabolic stress.

    Table 1: Nutrient composition of commercial broiler starter and finisher diets (Ultima feeds).


     
    Body mass index data collection
     
    Growth and morphometric traits were recorded weekly from weeks 1 to 6. Body weight was measured in grams using a top-loading scale, while body length was measured in centimeters using a measuring tape, following Oludoyi and Toye (2012). Body mass index was calculated as:
     
     
     
    Blood collection and DNA extraction
     
    Blood samples were collected from the brachial vein of selected birds into EDTA bottles using sterile 2 ml syringes and needles for hematological analysis, serum biochemistry and DNA extraction. Genomic DNA was extracted following Udeze et al., (2020) and DNA concentration and purity were assessed using a NanoDrop spectrophotometer.

    Primer design
     
    The Gallus gallus domesticus RYR2 gene sequence was obtained from ensembl using transcript ENSGALT000 10021632.1 on chromosome 3. RYR2 was selected because of its role in calcium regulation, cardiac function and sudden death syndrome in broilers. Primers targeting exons 23, 40 and 44, including flanking intronic regions, were designed using Primer3web version 4.0.0 (Table 2) and synthesized by Inqaba Biotechnical Industries, South Africa.

    ​

    Table 2: Primer sequencing results for RYR2 exons in ross 308, arbor acres and noiler chickens.


     
    PCR amplification
     
    Fifteen DNA samples, five per breed, were amplified using a MyGene™ Series Peltier Thermal Cycler at the Molecular Diagnostic and Research Laboratory, University of Ilorin, Nigeria. Each 25 μl PCR reaction contained 2X PCR master mix, genomic DNA, forward and reverse primers and nuclease-free water. Cycling involved denaturation at 95°C, primer-specific annealing at 55°C, 58°C or 62°C, extension at 72°C and final extension at 72°C. PCR products were resolved on 2% ethidium bromide-stained agarose gel at 145 V for 45 minutes and visualized under ultraviolet light using a 100 bp DNA ladder.
     
    DNA sequencing
     
    PCR products with clear bands were purified and sent to Inqaba Biotechnology Limited, South Africa, for sanger sequencing using an ABI 3500XL genetic analyzer with POP7™ polymer. Chromatograms were viewed and checked using FinchTV version 1.4.0 (Patterson et al., 2006).
     
    Sequence alignment and polymorphism detection
     
    Obtained sequences were aligned against the reference chicken RYR2 sequence using MultAlin to confirm target amplification and identify nucleotide differences among ross 308, arbor acres and noiler chickens. Sequence variation was assessed in both exon and flanking intronic regions (Corpet, 1988).
     
    Phylogenetic and comparative sequence analysis
     
    Comparative sequence analysis was performed to assess conservation around the amplified RYR2 regions. Sequences from the three breeds were compared with the reference chicken sequence and selected orthologs, while phylogenetic analysis was conducted using MultAlin and Phylogeny.fr to evaluate relationships and conservation among breeds and related species (Dereeper et al., 2008).
     
    Statistical analysis
     
    Quantitative performance traits, including body weight, BMI and growth velocities, together with biochemical and hematological indicators, were analyzed using a general linear model in IBM SPSS statistics version 27.0. Because sequencing was performed on a representative subset per breed, the identified polymorphism was treated as a breed-associated RYR2 haplotype rather than an individually independent genotype. One-way ANOVA was used to assess the fixed effect of haplotype group, using the following model:
    Yij = μ + Hi + eij
     
    Where,
    Yij = The dependent trait.
    μ = The overall mean.
    Hi = The fixed effect of the breed-associated RYR2 haplotype (A or G).
    eij = The random residual error.

    Homogeneity of variance was tested using levene’s test, mean differences were compared using duncan’s multiple range test and significance was set at P<0.05.

    RESULTS AND DISCUSSION

    All three targeted RYR2 exons were amplified from the 15 DNA templates, with clear bands obtained in 66.7%, 66.0% and 60.0% of samples for exons 40, 23 and 44, respectively (Fig 1 and 2). The clearest bands from each breed were selected for sequencing, but exon 44 sequencing was unsuccessful despite repeated attempts.

    Fig 1: Gel image of RYR2 exon 40 and 23 PCR products showing lane M ladder and fragment sizes.



    ​

    Fig 2: Gel image of RYR2 exon 44 PCR products showing lane M ladder and fragment size.


     
    Nucleotide sequence polymorphism in RYR2
     
    RYR2 gene exon 23
     
    Multiple sequence alignment of RYR2 exon 23 and its flanking intronic regions from Ross 308, Arbor Acres and Noiler against the reference chicken RYR2 sequence (ENSGALT00010021632.1) revealed no sequence polymorphism (deletion, insertion, inversion or substitution) in either the coding or non-coding regions (Fig 3).

    ​

    Fig 3: Multiple sequence alignment of RYR2 exon 23 and flanking intronic sequences from ross 308, arbor acres and noiler chickens against the reference chicken RYR2 sequence, showing no polymorphic site.



    RYR2 gene exon 40
     
    Multiple sequence alignment of RYR2 exon 40 and its flanking intronic region showed no coding-region polymorphism, but an A/G SNP was detected in the 32  non-coding flanking region at position 3:36968262 of the RYR2 primary transcript ENSGALT00010021632.1. Arbor acres and noiler carried the G allele, while Ross 308 carried the A allele (Fig 4-5).

    ​

    Fig 4: Multiple sequence alignment of RYR2 exon 40 and flanking intronic sequences from ross 308, arbor acres and noiler chickens against the reference chicken RYR2 sequence, showing the polymorphic site.




    ​

    Fig 5: Chromatograms of the 3¢ flanking intronic region of RYR2 exon 40 showing the polymorphic site corresponding to 36968262insA in the reference RYR2 primary transcript ENSGALT00010021632.1.


     
    Phylogenetic analysis
     
    RYR2 gene exon 23
     
    Phylogenetic analysis of RYR2 exon 23 and its flanking intronic regions in noiler, arbor acres and ross 308 showed sequence conservation when compared with red jungle fowl and other avian orthologs. Conserved motifs included GTACCTTC near the 32 splice donor site, TCTCTTT near the 52  splice acceptor site and conserved exon sequences, indicating conservation of this region among the examined avian sequences (Fig 6).

    ​

    Fig 6: Phylogenetic alignment of RYR2 exon 23 and flanking intronic regions, 100 bp upstream and downstream, from arbor acres, ross 308 and noiler chickens compared with red jungle fowl and other avian orthologs, showing conserved regions.


     
    RYR2 gene exon 40
     
    Phylogenetic analysis of RYR2 exon 40 and its flanking intronic regions in noiler, arbor acres and ross 308, compared with red jungle fowl and other avian orthologs, revealed several conserved regions, except in duck and turkey orthologs. These included a conserved TTCAG motif at the 52  splice acceptor site corresponding to position 36968349 of the primary transcript ENSGALT00010021632.1 (Fig 7).

    ​

    Fig 7: Phylogenetic alignment of RYR2 exon 40 and flanking intronic regions, 100 bp upstream and downstream, from arbor acres, ross 308 and noiler chickens compared with red jungle fowl and other avian orthologs, showing highly conserved regions.


     
    SNP association with trait related to cardiovascular risks and physiological stress
     
    Birds were classified by SNP allele, with ross 308 carrying the adenine (A) allele and Arbor Acres and Noiler carrying the guanine (G) allele. This classification was used to assess haplotype effects on cardiovascular risk indicators, including BMI, blood glucose, inflammation, lipidemia and stress level measured by the heterophil/lymphocyte ratio.
     
    Effect of genotype on body mass index
     
    BMI differed significantly between haplotype groups from weeks 1 to 6, with ross 308 birds carrying the A allele consistently showing higher BMI than Arbor Acres and Noiler birds carrying the G allele (P<0.05; Table 3).

    Table 3: Body mass index of chickens according to breed-associated RYR2 haplotype group from week 1 to week 6.


     
    Effect of genotype on blood glucose
     
    Glucose levels did not differ significantly between the A and G haplotype groups (Table 4).

    ​

    Table 4: Blood biochemical and hematological indicators of chickens according to breed-associated RYR2 haplotype group.


     
    Effect of genotype on inflammation (White blood count, heterophil, lymphocyte)
     
    White blood cell counts, heterophil percentages and lymphocyte percentages did not differ significantly between the A and G haplotype groups (Table 4).
     
    Effect of genotype on lipidemia (TC, LDL, HDL)
     
    Total cholesterol and LDL did not differ significantly between the A and G haplotype groups, but HDL was significantly lower in the A group than in the G group (P<0.05; Table 4).
     
    Effect of genotype on physiological stress (Heterophil/lymphocyte ratio)
     
    The heterophil/lymphocyte ratio was not significantly affected by haplotype group, although the near-significant P value suggests a weak trend that may require greater replication and lower environmental variation to confirm (Table 4).

    This study assessed structural variation in selected regions of the avian ryanodine receptor 2 (RYR2) gene and its association with growth, physiological stress and cardiovascular risk indicators. RYR2 encodes a cardiac calcium release channel essential for excitation-contraction coupling and polymorphisms in this gene have been linked to calcium leakage, arrhythmias and sudden death syndrome in avian and mammalian species. Sequence alignment showed complete conservation of exon 23 among Ross 308, Arbor Acres and Noiler chickens, suggesting strong evolutionary constraint in this region. However, an A/G SNP was detected in the 32  non-coding flanking region of exon 40 at position 3:36968262, with Ross 308 carrying the A allele and arbor acres and noiler carrying the G allele.

    The marked BMI divergence from week 1 (P<0.003) reflects rapid tissue accretion in ross 308 compared with the slower-growing noiler. Such accelerated growth increases visceral and cardiovascular burden because cardiac development may not match skeletal muscle demand, requiring greater cardiac output and predisposing fast-growing broilers to pulmonary hypertension, right ventricular hypertrophy, arrhythmias and sudden death.

    Biochemical analysis showed significantly lower HDL cholesterol in birds carrying the Ross 308-associated A allele (6.5±0.52 mg/dL) than in G-allele birds (8.4±0.36 mg/dL; P = 0.008). Since HDL supports reverse cholesterol transport and reduces vascular inflammation, its reduction alongside high BMI suggests greater cardiovascular vulnerability in ross 308 birds. However, total cholesterol, LDL and glucose remained similar between groups, indicating that some baseline blood lipid traits may remain stable despite underlying cardiovascular risk.

    The heterophil-to-lymphocyte (H/L) ratio is a useful indicator of chronic physiological and environmental stress in birds. Although the H/L ratio was not statistically significant, its near-significant value (P = 0.063) suggests a possible biological trend, with the slightly higher ratio in G-allele carriers likely influenced by the Noiler cohort and its greater alertness compared with more sedentary commercial broilers.

    Similar to the present study, Basaki et al., (2019) examined RYR2 in relation to sudden death syndrome in broilers and reported increased RYR2 mRNA expression and point mutations in SDS-affected birds. In contrast, this study detected an A/G SNP in the flanking region of RYR2 exon 40, while exon 23 showed no polymorphism. Because this SNP is intronic, it does not directly alter the RYR2 amino acid sequence, but it may influence gene function through splicing, regulatory activity, non-coding RNA binding or linkage with another functional variant. Therefore, it should not yet be considered a causal mutation, but rather a preliminary breed-associated marker for further investigation of RYR2-related cardiovascular dysfunction and sudden death syndrome in chickens.

    CONCLUSION

    This study detected sequence variation in selected regions of the ryanodine receptor 2 (RYR2) gene among Ross 308, Arbor Acres and Noiler chickens. Exon 23 and its flanking region were conserved, while an A/G SNP was identified in the 32 flanking intronic region of exon 40 at position 3:36968262, with Ross 308 carrying the A allele and Arbor Acres and Noiler carrying the G allele. Ross 308 chickens also had significantly higher BMI from weeks 1 to 6, reflecting faster growth and greater body mass accumulation. Most cardiovascular risk and physiological stress indicators did not differ between RYR2 haplotype groups, except HDL, which was lower in Ross 308, suggesting that the A allele group may be linked to a growth and lipid profile relevant to cardiovascular risk assessment. However, the exon 40 flanking-region SNP should not be considered causal at this stage because of the small sequenced sample size, targeted gene coverage and breed-associated haplotype classification. It should instead be regarded as a preliminary marker for breed differentiation and further investigation using larger populations, individual genotyping, broader RYR2 sequencing, successful exon 44 analysis and functional validation.

    ACKNOWLEDGEMENT

    The present study was supported by the Department of Animal Production and Health, Faculty of Agriculture, University of Ilorin, Ilorin, Nigeria, through institutional and academic supervision. The authors also acknowledge the University of Ilorin Central Research Laboratories and the University of Ilorin Teaching Hospital Molecular and Diagnostic Research Laboratory for technical support during molecular analysis.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal handling and experimental procedures were carried out in accordance with the ethical guidelines of the Department of Animal Production and Health, Faculty of Agriculture, University of Ilorin, Ilorin, Nigeria. The study was approved by the departmental animal ethics committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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