Association of transferrin gene polymorphism SNPs C14037T and A14081G with the reproductive performa

Journal of Research in Ecology

Journal of Research in Ecology

ISSN No: Print: 2319 –1546; Online: 2319– 1554

An International Scientific Research Journal

Original Research

Association of transferrin gene polymorphism SNPs C14037T and A14081G with the reproductive performance of Holstein cows Authors: Jaafar Ahmed R, Talal Abdulkareem A and Nasr Al-Anbari N Institution: Department of Animal Production, College of Agriculture, University of Baghdad, Iraq.

Corresponding author: Jaafar Ahmed R

ABSTRACT: This study was undertaken to investigate the association of transferrin gene polymorphism SNPs (intron 8) with the reproductive performance (service per conception; SPC, number of open days, calving interval; CI and non-return rate; NRR) of fifty Holstein cows in Iraq. This study was conducted at the Dairy Cattle Farm and Physiology Laboratory pertaining to the College of Agriculture, University and Baghdad and also at the Laboratory dealing with the analysis of molecular genetic during the period from 1.12.2015 to 12.5.2016. Significant (P≤0.05) differences were found among genotypes distribution of the Holstein cow’s transferrin gene, namely 30.61, 36.73 and 32.65% for AA-CC, AG-CT and GG-TT respectively. The cows with heterozygous genotype (AG-CT) exhibited better (P≤0.01) results concerning with the number of open days (108.91±6.95 days) and SPC (1.79±0.26) as compared with the other genotypes. On the other hand, the wild genotype (AA-CC) recorded undesirable reproductive traits represented by long open days and lesser SPC in comparison with the other genotypes. Shorter CI (409.61±19.54 days) and lesser NRR percentage (68.04±3.07%) were noticed for heterozygous genotype (AG-CT) as compared with the other two genotypes. In conclusion, the reproductive performance of Holstein cows can be improved based on the transferrin gene SNPs (C14037T and A14081G) at the intron 8. Investigating the other SNPs of this gene using larger number of cows and follow those through generations may give more accurate results for the selection programs.

Keywords: Transferrin gene, SNPs, Intron 8, Reproduction, Holstein cows.

Article Citation: Jaafar Ahmed R, Talal Abdulkareem A and Nasr Al-Anbari N Association of transferrin gene polymorphism SNPs C14037T and A14081G with the reproductive performance of Holstein cows Journal of Research in Ecology (2018) 6(2): 1846-1852 Dates: Received: 01 July 2018 Web Address: http://ecologyresearch.info/ documents/EC0590.pdf Journal of Research in Ecology An International Scientific Research Journal

Accepted: 27 July 2018

Published: 29 August 2018

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

1846-1852| JRE | 2018 | Vol 6 | No 2

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Ahmed et al., 2018 genetic markers are tools to identify the specific regions

INTRODUCTION Dairy cattle have a crucial role in animal resources sector through supplying different dairy (milk

on the chromosomes, being the candidate genes affecting quantitative trait loci (QTL) (Ge et al. 2001).

and cheese), meat, bone and leather products for human

The objective of this study was to investigate

consumers. Therefore, increasing the dairy cattle popu-

the polymorphism transferrin gene (TF) for intron 9,

lation accompanied with improving their productive and

A14037G, C14081T SNPs and their association with

reproductive performance became the main target for

some reproductive efficiency parameters for Holstein

the animal breeders (McDowell, 1985). The bovine

cows.

transferrin (TF) is located on chromosome 1q41-q46 as reported by Chowdhary et al. (1998). This gene consists

MATERIALS AND METHODS

of 17 exons in the 39 kb of genomic DNA. Moreover,

Animals

the transferrin is an iron-binding B-globulin synthesized

A study was carried out at the Dairy Cattle Farm

by the liver and has a molecular weight of about 80 kDa

as well as at the Laboratory of Biotechnology pertaining

(Fletcher and Huehns, 1968). Plasma transferrin is an

to the College of Agriculture at the University of Bagh-

iron-transport protein with a half-life of 8 to 10 days

dad located at the Abu-Ghraib region (West of Bagh-

that reflects both protein and iron status (Litchford,

dad). Other part of the study was conducted at the La-

2008). Furthermore, Fletcher and Huehns (1968) no-

boratory of Molecular Genetic Analysis from 1.12.2015

ticed that the TF has two separate iron-binding sites,

to 12.5.2016. Blood samples (10 ml) were collected

each of them is capable of binding one atom of ferric

from 49 Holstein cows by jugular venipuncture and

iron.

stored under -20°C until the molecular assays of transThe herd fertility can be defined as the capabil-

ity of reproduction possibility that it has. However, both

ferrin gene were conducted.

Genomic DNA extraction

males and females contribute the herd fertility efficien-

The genomic DNA was harvested from the bo-

cy, and the males have a crucial role in this process

vine blood samples according to the procedure de-

(Hafez and Hafez, 2000). The most important reproduc-

scribed by the Promega Company. The DNA concentra-

tive efficiency parameters in cows are the number of

tion was determined by spectrophotometry and adjusted

days open, age at first calving, service per conception,

to 50 ng /ml. All DNA samples were stored under -20°C

calving interval and non-return rate (Bayram et al.,

(Burden and Whitney, 1995). Gel electrophoresis was

2009; Ulutas and Sezer, 2009 and Sajjad, 2012). The

performed according to Sambrook and Russell (2001) to

Table 1. Number and percentage of genotype distribution of TF gene and allele frequency in Holstein-Friesian cows S. No 1 2 3 4 5

Genotype AA-CC AG-CT GG-TT Total Chi-square –χ2

6 7 8

Allele frequency T G

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No. 15 18 16 49 ---

Percentage (%) 30.61 36.73 32.65 100% 4.091 (P<0.05) -0.49 0.51

affirm the presence of DNA in the extracted samples.

PCR amplification and sequencing In order to amplify the bovine TF gene (NW_001493777) including intron 8 and the primers which were designed by Ju et al. (2011) [F: 5'GGTCTGA CTGCCCTCTCTC -3' and R: 5' – GTTCAAACACACC TCTAATG -3']. The PCR mixture with a final volume of 25 μL consisted of 12.5 μL ProFi Taq PCR PreMix Kit 2.5, 1μL PF, 1μL PR, 21μL template DNA, 8 μL H2O. PCR was done according the Journal of Research in Ecology (2018) 6(2): 1846–1852

Ahmed et al., 2018

Figure 1. DNA sequencing pattern explaining the A14037G and C14081T SNPs following schedule: 94°C for 5 min, followed by 30

Statistical analysis

cycles of 94°C for 30 s, annealing 65°C for 30 s, and

The statistical analyses were done using SAS

72°C for 30 s, and a final extension at 72°C for 8 min

(2012) to study the effect of TF gene polymorphism of

(Ju et al., 2011). PCR products were estimated using

A14037G and C14081T SNPs on some reproductive

agarose gel electrophoresis at 2.5% concentration fol-

characteristics of Holstein-Friesian cows. Least square

lowing staining with ethidium bromide.

means to assess the significant differences among per-

DNA products (20 μL) of all concerning cows

centages (distribution of polymorphism and allele fre-

were sequenced at the Macrogen Company at USA.

quency of TF gene based on Hardy and Weinberg low;

Sequence data were analyzed using peaks by visual

Edwards, 2008). The statistical model was as follows:

comparison of the colour peaks after alignment with the

Yijkl = µ + Gi + Pj + Sk + eijkl

base data in the National Centre for Biotechnology In-

where, Yij : dependent variable (Days open, service per

formation (NCBI) (Ji et al., 2008).

conception, calving rate and non-return rate); μ: overall

Figure 2. Two SNP sequences in one DNA strand of Sbjct gene, Query: transferrin strand sequence Journal of Research in Ecology (2018) 6(2): 1846–1852

1848

Ahmed et al., 2018

Figure 3. Two SNP of 309 and 354 sequence, Query: Transferrin strand sequence mean; Gi: Effect of genotype (AA-CC, AG-CT and GG-

sequence 309 and second site is of changing from C to

TT); Pj: Effect of parity (Second to fourth); Sk: Effect of

T base at the sequence 354 (Figure 2). In the other

calving season (winter, summer, spring and autumn)and

cows, the SNPs did not occur in the DNA strand; and

eijkl: Error term.

the two bases C and T did not change and the genotype was a wild (AA-CC; Figure 3).

RESULTS AND DISCUSSION

Distribution of TF gene in Holstein-Friesian cattle

SNPs detection

studied with sample percentage

Results did reveal that according to the sequence

Table 1 revealed the percentage of genotype

data, two SNPs were showed. The first one was

distribution of TF gene in the sample of Holstein-

A14307G in the intron 8 that previously detected (Ju et

Friesian cows as 30.61, 36.73 and 32.65 % for AA-CC,

al., 2011) with NCBI accession No. of ss250608651 and

AG-CT and GG-TT genotypes respectively. On the oth-

recently reported for Holstein-Friesian cows as 43 pb.

er hand, the allele frequencies of T and G alleles were

The second detected SNPs were C14081T (Figure 1).

0.49 and 0.51 respectively (Table 1). Many studies con-

On the other hand, the A14037G and C14081T SNPs

cerning the association of SNPs with the cows perfor-

were detected in TF gene of intron 8 as a haplotype

mance including Ju et al. (2011) deals with the produc-

block for Holstein-Friesian cows in Iraq (Figure 2).

tive characteristics and Liu et al. (2011) deals with the

Moreover, as explained in Figure 1, two mutations were

reproductive traits of Holstein bulls.

existed, first in the 281 sequence (from A to G base as

Genetic polymorphisms of TF gene in Holstein-

referred by black line) and second in the 325 sequence

Friesian cattle

(from C to T base as referred by red line).

Table 2 noticed the genetic polymorphism of

The two SNPs occurred in one DNA strand of

A14037G and C14081T SNPs in intron 8 of TF gene

Sbjct gene in other cows of heterozygous genotype (AG

and their association with days open and service per

-CT). First site is of changing from A to G base at the

conception of Holstein-Friesian cows. The heterozygous

Table 2. Genetic polymorphism of TF gene and its relationship with open days and service per conception in Holstein-Friesian cows S. No

Genetic polymorphism

1 2 3 4

AA-CC AG-CT GG-TT Level of significance

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Mean ± SE No. 15 18 16

Open days 132.57 ± 10.04b 108.91 ± 6.95a 139.75 ± 12.82b P<0.01

SPC (Service) 2.47 ± 0.42b 1.79 ± 0.26a 2.61 ± 0.39b P<0.01

Journal of Research in Ecology (2018) 6(2): 1846–1852

Ahmed et al., 2018 Table 3. Genetic polymorphism of TF gene and its relationship with calving interval and non-return rate in Holstein-Friesian cows S. No

Genetic polymorphism

No.

1 2 3 4

AA-CC AG-CT GG-TT Level of significance

15 18 16

Mean ± SE Calving interval (day) 485.39 ± 23.69b 409.61 ± 19.54a 497.25 ± 26.71b P<0.01

Non-return rate (%) 57.83 ± 2.98b 68.04 ± 3.07a 53.68 ± 3.92b P<0.01

genotype (AG-CT) recorded the better results (P<0.01)

with the Table 2 results, the heterozygous genotype (AG

of open days (108.91±6.95 days) and service per con-

-CT) recorded the better results (P<0.01) of calving in-

ception (1.79±0.26) as compared with the AA-CC and

terval and non-return rate being 409.61±19.54 days and

GG-TT genotypes. On the other hand, the results were

68.04±3.07 % respectively. On the other hand, the re-

lesser for AA-CC (132.57±10.04 days and 2.47 ± 0.42)

sults were lesser for AA-CC namely 485.39± 23.69 days

and moderate for GG-TT (139.75 ± 12.82 days and 2.61

and 57.83±2.98 % (Table 3).

± 0.39; Table 2).

The superiority of heterozygous genotype in all

The reasons behind the resultant SNPs in this

reproductive characteristics studied may returns to the

segment and in whole TF gene as well as in other genes

adaptive advantage of harsh environment and climate in

related with the productive and reproductive characteris-

Iraq. The current data were agree with those reported

tics of Holstein-Friesian cows may return to the expo-

by Guha et al. (2012) that Gir cows with heterozygous

sure of the animals to the harsh environment due to

genotype (TfAE) were superior in some reproductive

transportation of the them from their native homelands

traits (age at first calving, age at second calving and

to the others differing in temperature, humidity as well

calving interval) as compared with the homozygous

as management conditions. The animals try to be suita-

genotypes. Genetic variation is essential to permit im-

ble with the new environment in spite of the difficulties.

provement in cattle productivity. This may be created

Accordingly, the heterozygous genotype (AC-GT) rec-

from new mutations or from conserving of the existing

orded a moderate pattern of the reproductive traits, fol-

genetic variation (Zaabal and Ahmed, 2008). The repro-

lowed by wild genotype (GG-TT). These results were

ductive efficiency is affected by different factors like

consistent with Senkal et al. (2017) whom found that

breed, parity, calving season, calving year as well as the

the heterozygous genotype (AG-CT) recorded the better

environmental conditions surrounding the animal in-

productive performance in Holstein cows.

cluding nutrition and health management (Bearden et

The occurrence of these SNPs in TF gene and /

al., 2004). Therefore, the inappropriate environmental

or other genes will certainly change the nucleotides se-

conditions represented by poor grazing land and lesser

quence and gene expression of TF gene consequently.

healthy and reproductive management of Holstein-

This will lead to change the TF protein itself and animal

Friesian cows in Iraq led to the lesser productive and

performance positively or negatively according to the

reproductive performance as compared with those in

produced protein.

their native homeland.

Table 3 revealed the genetic polymorphism of A14037G and C14081T SNPs in 882 bp segments of TF

CONCLUSION

gene and their association with the calving interval and

The reproductive performance of Holstein cows

non-return rate of Holstein-Friesian cows. Concomitant

can be enhanced according to the transferrin gene SNPs

Journal of Research in Ecology (2018) 6(2): 1846–1852

1850

Ahmed et al., 2018 (C14037T and A14081G) at the intron 8. Exploring the

Association of transferrin polymorphism with reproduc-

other SNPs of this gene using larger number of cows

tive traits of Gir cattle. Exploratory Animal and Medical

may give more accurate results for the future selection

Research, 1(2): 141-144.

programs. The polymorphic nature of transferrin in Holstein-Friesian cows in Iraq explaining the greatest amount of heterozygosity may improve the intra breed variability status and could be useful in cattle breeding programs in Iraq.

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Fletcher J and Huehns ER. 1968. Function of transferrin. Nature, 218: 1211-1214.

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