Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 8 (august 2021) : 867-872

Exploration of Neuropeptide Y Gene Variants and its Association with Growth, First Lactation Reproduction and Production Traits in Sahiwal Cattle

Prajwalita Pathak1,*, Anupama Mukherjee1, Shabahat Mumtaz1, Vivek Kumar Nayak1, Suchit Kumar1, Alok Kumar Yadav1
1Division of Animal Genetics and Breeding, ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India.
Cite article:- Pathak Prajwalita, Mukherjee Anupama, Mumtaz Shabahat, Nayak Kumar Vivek, Kumar Suchit, Yadav Kumar Alok (2021). Exploration of Neuropeptide Y Gene Variants and its Association with Growth, First Lactation Reproduction and Production Traits in Sahiwal Cattle . Indian Journal of Animal Research. 55(8): 867-872. doi: 10.18805/IJAR.B-4161.

ABSTRACT

Background: Neuropeptide Y (NPY), a 36-AA neurotransmitter involved in the control of appetite, feeding and energy homeostasis. The bovine NPY gene is located on chromosome 4 and consists of four exons. Earlier studies have reported that genes involved in feed intake regulation, energy production and energy regulation may affect different growth and production traits in cattle. Information associating NPY SNP to reproduction and production traits in dairy cattle is, however, limited. Hence, the present study was planned to explore SNPs in NPY gene and its possible association with reproduction and first lactation production traits in Sahiwal cattle. 

Methodology: DNA samples were isolated from blood that has collected from hundred and three Sahiwal cows. Three regions of NPY were amplified using designed primer pairs. The sequencing of the targeted regions was done in 103 Sahiwal cows. Least  squares analysis of variance was carried out for unequal  and  non-orthogonal data using the technique described by Harvey (1990) to study effect of non-genetic factors. Significance of differences based on genotypes effect of growth, reproduction and  production  traits were tested by general linear models.

Result: Sequence data analysis revealed two transversions in NPY gene. For these identified nucleotide variations, positions were A72073973C (intron 3) and C72068620A (intron 3). This is the first report of the presence of NPY gene polymorphism in purebred Bos indicus of Indian origin. For SNP A72073973C, performances of animals with homozygote AA genotype exhibited significantly higher milk production (P≤0.01) and growth performance (P≤0.05) than heterozygote genotype. Heterozygous genotype exhibited superior performance for the SNP C72068620A which was found to be significantly associated (P≤0.05) with 18M wt, WFS, WFC, FCI and FL305DMY.

INTRODUCTION

The use of polymorphic genes as detectable molecular markers is a promising alternative to the conventional methods of selection, once these genes are proven to be associated with traits of interest in animal. The recent development of Single Nucleotide Polymorphism (SNP) genotyping has opened new selection perspectives with the potential advantages of increased genetic gain and lower costs. The candidate gene approach is a way of identifying genomic region with mutations and an association study of these mutations with economically important traits. Growth, milk production and reproduction traits are typically quantitative characteristics controlled by a number of genes. Since these performance traits are economically important, special attention needs to be focused on studying the genes having important role in growth along with production and reproduction. Till date a large number of candidate genes have been reported among which NPY has been found to be critical for growth, reproduction as well as production traits.
       
Neuropeptide Y, a 36-AA neurotransmitter involved in the control of appetite, feeding and energy homeostasis (Bahar and Sweeney, 2008). Concentrations of NPY are elevated when body fat stores are depleted, causing an increase in appetite (Kalra et al., 1991). Therefore, during negative energy balance, NPY levels are expected to be high. Furthermore, NPY has been identified as a primary regulator of leptin activity in the hypothalamus, influencing the release of LH and somatotropin (Kalra et al., 1991). The bovine NPY gene is located on chromosome 4 (Thue et al., 2004) and consists of four exons (99, 188, 81 and 195 nts). Earlier studies have suggested that genes involved in feed intake regulation, energy production and energy regulation may affect different growth and production traits in cattle. For instance, significant associations of NPY gene variants with different growth and lactation traits have been reported (Nkrumah et al., 2004; Sherman et al., 2008 and Clempson et al., 2011). Information associating NPY SNP to reproduction and production traits in dairy cattle is, however, limited. The objectives of this study were to explore SNPs in NPY gene and its associations with growth, reproduction and first lactation production traits in Sahiwal cattle.

MATERIALS AND METHODS

Samples and traits
 
DNA samples were isolated from blood that has collected from hundred and three standing female animals of Indian origin maintained at Livestock Research Complex, ICAR-National Dairy Research Institute, India under the same management regimen which completed their first lactation. Around 10 ml of venous blood was collected aseptically from the jugular vein of the animals in a 15 ml polypropylene centrifuge tube under sterile condition using 0.5 ml of EDTA as an anticoagulant. The samples were stored at 20°C for further utilization. The different traits considered for association analysis included viz. body weight at different age i.e. Birth, 6 Months (6M), 12 Months (12M), 18 Months (18M), 24 Months (24M), 30 Months (30M), Weight at First Service (WFS), Weight at First Calving (WFC), reproduction i.e. Age at First Service (AFS), Age at First Calving (AFC), First Service Period (FSP), First Calving Interval (FCI) and first lactation production traits i.e. First Lactation 305 Day or less Milk Yield (FL305DMY), First Lactation Total Milk Yield (FLTMY), First Lactation Length (FLL), First Lactation 305 day or less Fat Yield (FL305DFY), First Lactation 305 day or less SNF Yield (FL305DSNFY).
 
DNA extraction and genotyping of animal
 
The DNA was extracted from blood samples by  commercially available  kit  [Genomic  DNA  Purification  Kit  (Cat.#A1620, Promega)]. Three regions i.e. partial intron 1, exon 2 and partial intron 2 (454 bp), partial intron 2, exon 3 and partial intron 3 (437 bp) and partial intron 3 and exon 4 (277 bp) of NPY were amplified. Amplification of the desired NPY gene fragments was performed with designed primer pairs (Table 1).
 

Table 1: Designed primers for amplification of Neuropeptide Y (NPY) gene.


       
PCR was performed in 25-μl reactions with a PCR mix containing 15 mM MgCl2, 200 μM dNTP, 30 pM of each primer, 100 ng of genomic DNA and 1 U of Taq polymerase. Thermal cycling conditions were included an initial denaturation for 5 min at 95°C followed by 32 cycles of 95°C (30 s), annealing temperature 64°C, 59°C and 63°C (30 s) and ending with a final extension for 10 min at 72°C. The PCR products were confirmed by agarose gel electro- phoresis methods using 1.5% agarose gel with ethidium bromide for 20-30 min and visualized under Gel Documentation System.
       
The sequencing of the targeted regions was done to screen the DNA polymorphisms of the NPY gene. Sequence data were analyzed using multiple sequence alignment software, freely available online. The coding regions of NPY gene were subjected to basic local alignment search tool at NCBI (www.ncbi.nlm.org) to identify the sequence homology with the corresponding region of other species available with the data bank. Sequence data from different variants obtained were analyzed to identify the SNP variation present in the Sahiwal cattle maintained at ICAR-NDRI, Karnal.
 
Statistical analysis
 
Genotypic and allelic frequencies
 
Genotypic frequencies for variant genotypes were calculated using the formula:
 
 
Allelic frequencies were calculated as follows:
 
Allelic frequency of A       =  AA + ½ AB
Allelic frequency of B       =  BB + ½ AB
 
Where
AA and BB  =  Genotypic frequency of homozygote
AB              =  Genotypic frequency of heterozygote
A and B    = Allelic frequencies
 
 
Least squares analysis of variance was carried out for unequal and non-orthogonal data using the technique described by Harvey (1990) to study effect of non-genetic factors like season and period. The model was used with assumptions that different components being fitted into the model were linear, independent and additive.
 
Association analysis
 
Association of the animal genotypes with growth, reproduction and milk production traits were determined by analyses of quantitative traits. Statistical analysis was carried out using SAS Enterprise Guide 4.2 software (SAS Institute Inc. 2009) and significance of differences based on genotypes effect of growth, reproduction and production traits were tested by following general linear models:
 
                                      Yij= µ + Gi + eij
 
Where
Yij is the trait value of jth animal of ith genotype, μ is the overall
mean, Gi is the effect of ith genotypes and eij is the residual error NID (0, σ2e).

RESULTS AND DISCUSSION

Genetic variability in NPY gene
 
The primer for partial intron 1, exon 2 and partial intron 2 of NPY gene was designed and the PCR amplification was done for a product size of 454 bp fragment (Fig 1). A total of 103 samples were sequenced. For determining the change in partial intron 1, exon 2 and partial intron 2 of NPY gene of Sahiwal cattle, with the reference sequence in ENSEMBL Accession number (ENSBTAG00000004503) for Bos taurus was compared and aligned with the sequence of Sahiwal cattle by Clustal W multiple sequence alignment and SNP was found in intronic region 2. Two genotypes were identified in the studied population i.e. AA and AC as seen in the chromatogram as shown in (Fig 2). In ClustalW multiple sequence alignment thymine to cytosine change at position 334 (A72073973C) was observed (Fig 2). These two distinguished genotypes had the frequencies of 0.54 and 0.46 in AA and AC respectively. The allelic frequencies for A and C were 0.77 and 0.23 respectively (Table 2). The sequence results of partial intron 2, exon 3 and partial intron 3 of 437 bp fragment of NPY gene revealed no nucleotide variation resulting in monomorphic pattern in all the Sahiwal cows under this study. The PCR amplification was done for another product size of 277 bp fragment (Fig 3) targeting partial intron 3 and complete exon 4 of NPY. For determining the change in partial intron 3 and exon 4 of NPY gene of Sahiwal cattle, with the reference sequence in ENSEMBL Accession number (ENSBTAG00000004503) for Bos taurus was compared and aligned with the sequence of Sahiwal cattle by ClustalW multiple sequence alignment and SNP was found in intronic region. The sequence data analysis revealed two genotypes in the studied population i.e. CC and AC as seen in the chromatogram as shown in (Fig 4). In ClustalW multiple sequence alignment thymine to cytosine change at position 71 (C72068620A) was observed (Fig 4).  These two distinguished genotypes had the frequencies of 0.66 and 0.34 in CC and AC respectively. The allelic frequencies for C and A were 0.84 and 0.16 respectively (Table 2). The present study carried out to evaluate polymorphism in NPY gene in Sahiwal cow and very limited works have been reported on other domestic and exotic dairy cattle. Monomorphic SNP loci in the NPY was detected in the Polish Red and Hereford breeds (Pareek et al., 2017).
 

Fig 1: PCR profile of 454bp fragment of NPY gene.


 

Fig 2: Chromatogram showing A72073973C SNP in Sahiwal cow.


 

Fig 3: PCR profile of 277bp fragment of NPY gene.


 

Fig 4: Chromatogram showing C72068620A SNP in Sahiwal cows.


 

Table 2: Genotype and allelic frequencies of NPY gene using sequencing technique in Sahiwal cattle.


 
Sequence data analysis revealed two transvertions in NPY. For these identified nucleotide variations, positions were A72073973C (intron 3) and C72068620A (intron 3) in NPY gene (Table 3).
 

Table 3: Nucleotide changes in NPY (ENSBTAG00000004503) gene in Sahiwal cattle as compared to Bos taurus.


 
Association of the NPY gene polymorphisms with different economic traits in Sahiwal cattle
Growth trait
 
In this present study NPY SNP A72073973C was found to be significantly associated with weight at 18 months, WFS and WFC (P ≤ 0.05) (Table 4). The SNP A72073973C corresponded to two genotypes among which animals with AA genotype attained significantly higher weight than the AC genotypes at 18 months, first service and first calving. The NPY SNP C72068620A showed a significant association with weight at 18 months, WFS and WFC (P ≤ 0.05). Animals with a heterozygote AC genotype significantly attained higher body weight at 18 months, first service and first calving than the CC genotype. These polymorphisms had no association with the other growth traits at different age viz. birth, 6M, 12M, 24M and 30M. The present study carried out for association of NPY gene with various body weights at different age and no previous study reported association of different growth traits that are considered in this study with NPY gene.
 

Table 4: Association of NPY genotypes with different body weights in Sahiwal cattle.


 
Reproduction traits
 
The association study with various first lactation reproduction traits of NPY gene variants revealed that SNP A72073973C was not found to be significantly associated with various first lactation reproduction traits in Sahiwal cows. For the NPY SNP C72068620A, significant association was observed with first calving interval (P ≤ 0.05) as shown in the Table 5. Animals with heterozygote AC genotype had significantly prolonged first calving interval than the CC genotype. On the contrary non significant association with days to first service and calving interval was reported by Clempson et al., (2011). This genetic variant had no impact on other first lactation reproduction traits viz. AFS, AFC and FSP.
 

Table 5: Association of NPY genotypes with different reproduction traits in Sahiwal cattle.


 
First lactation production traits
 
In present study NPY SNP A72073973C was found to be significantly associated with FL305DMY (P ≤ 0.01), FLTMY (P ≤ 0.01), FL305DFY (P ≤ 0.05) and FL305DSNFYY (P ≤ 0.01) in Sahiwal cows (Table 6).  This SNP corresponded to two genotypes among which animals with homozygote AA genotype had higher FL305DMY and FLTMY whereas lower FL305DFY and FL305DSNFYY than the heterozygote AG genotype. For the NPY SNP C72068620A, significant association was observed with FL305DMY (P ≤ 0.05) as shown in the Table 6. Animals with heterozygote AC genotype had significantly higher FL305DMY than the CC genotype. This genetic variant had no impact on other first lactation production traits viz. FLTMY, FLL, FL305DFY and FL305DSNFYY. Similar result was reported by Clempson et al., (2011) who reported significant association of genetic variant with FL305DMY.
 

Table 6: Association of NPY genotypes with different first lactation production traits in Sahiwal cattle.

CONCLUSION

NPY polymorphisms were found to be associated with different growth, reproduction and first lactation production traits. Heterozygote pertaining to SNP C72068620A of NPY while homozygote for SNP A72073973C of NPY gene tended to have better production performance than the other genotype while showed optimum performance in growth. Therefore, in breeding programme selection of animals with heterozygote genotype may be favourable for improvement in growth, reproduction and production traits in Sahiwal cattle with proper validation in larger population and it could be used in marker-assisted selection to improve reproduction and milk production traits with optimum growth.

ACKNOWLEDGEMENT

The authors are thankful to the Director of ICAR-NDRI and Head of the Division, AGB, NDRI, Karnal for providing the necessary facilities. We wish to acknowledge the hard work and sincerity of the staffs of Livestock Research centre.

COMPLIANCE WITH ETHICAL STANDARDS

The authors declare that they have no conflict of interest.

REFERENCES


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