Journal of Research in Ecology
An International Scientific Research Journal
Association of transferrin gene polymorphism SNPs C14037T and
A14081G with the reproductive performance of Holstein cows
Authors:
Journal of Research in Ecology
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
Web Address:
http://ecologyresearch.info/
documents/EC0590.pdf
Journal of Research
in Ecology
An International
Scientific Research Journal
ISSN No: Print: 2319 –1546; Online: 2319– 1554
Original Research
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 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
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1846-1852| JRE | 2018 | Vol 6 | No 2
www.ecologyresearch.info

INTRODUCTION
Dairy cattle have a crucial role in animal re-
sources sector through supplying different dairy (milk
and cheese), meat, bone and leather products for human
consumers. Therefore, increasing the dairy cattle popu-
lation accompanied with improving their productive and
reproductive performance became the main target for
the animal breeders (McDowell, 1985). The bovine
transferrin (TF) is located on chromosome 1q41-q46 as
reported by Chowdhary et al. (1998). This gene consists
of 17 exons in the 39 kb of genomic DNA. Moreover,
the transferrin is an iron-binding B-globulin synthesized
by the liver and has a molecular weight of about 80 kDa
(Fletcher and Huehns, 1968). Plasma transferrin is an
iron-transport protein with a half-life of 8 to 10 days
that reflects both protein and iron status (Litchford,
2008). Furthermore, Fletcher and Huehns (1968) no-
ticed that the TF has two separate iron-binding sites,
each of them is capable of binding one atom of ferric
iron.
The herd fertility can be defined as the capabil-
ity of reproduction possibility that it has. However, both
males and females contribute the herd fertility efficien-
cy, and the males have a crucial role in this process
(Hafez and Hafez, 2000). The most important reproduc-
tive efficiency parameters in cows are the number of
days open, age at first calving, service per conception,
calving interval and non-return rate (Bayram et al.,
2009; Ulutas and Sezer, 2009 and Sajjad, 2012). The
Table 1. Number and percentage of genotype
distribution of TF gene and allele frequency in
Holstein-Friesian cows
S. No Genotype No. Percentage (%)
1 AA-CC 15 30.61
2 AG-CT 18 36.73
3 GG-TT 16 32.65
4 Total 49 100%
5 Chi-square –χ2 --- 4.091 (P<0.05)
6 Allele frequency --
7 T 0.49
8 G 0.51
1847
Ahmed et al., 2018
genetic markers are tools to identify the specific regions
on the chromosomes, being the candidate genes affect-
ing quantitative trait loci (QTL) (Ge et al. 2001).
The objective of this study was to investigate
the polymorphism transferrin gene (TF) for intron 9,
A14037G, C14081T SNPs and their association with
some reproductive efficiency parameters for Holstein
cows.
MATERIALS AND METHODS
Animals
A study was carried out at the Dairy Cattle Farm
as well as at the Laboratory of Biotechnology pertaining
to the College of Agriculture at the University of Bagh-
dad located at the Abu-Ghraib region (West of Bagh-
dad). Other part of the study was conducted at the La-
boratory of Molecular Genetic Analysis from 1.12.2015
to 12.5.2016. Blood samples (10 ml) were collected
from 49 Holstein cows by jugular venipuncture and
stored under -20°C until the molecular assays of trans-
ferrin gene were conducted.
Genomic DNA extraction
The genomic DNA was harvested from the bo-
vine blood samples according to the procedure de-
scribed by the Promega Company. The DNA concentra-
tion was determined by spectrophotometry and adjusted
to 50 ng /ml. All DNA samples were stored under -20°C
(Burden and Whitney, 1995). Gel electrophoresis was
performed according to Sambrook and Russell (2001) to
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 mix-
ture 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
72°C for 30 s, and a final extension at 72°C for 8 min
(Ju et al., 2011). PCR products were estimated using
agarose gel electrophoresis at 2.5% concentration fol-
lowing staining with ethidium bromide.
DNA products (20 μL) of all concerning cows
were sequenced at the Macrogen Company at USA.
Sequence data were analyzed using peaks by visual
comparison of the colour peaks after alignment with the
base data in the National Centre for Biotechnology In-
formation (NCBI) (Ji et al., 2008).
The statistical analyses were done using SAS
(2012) to study the effect of TF gene polymorphism of
A14037G and C14081T SNPs on some reproductive
characteristics of Holstein-Friesian cows. Least square
means to assess the significant differences among per-
centages (distribution of polymorphism and allele fre-
quency of TF gene based on Hardy and Weinberg low;
Edwards, 2008). The statistical model was as follows:
Yijkl = µ + Gi + Pj + Sk + eijkl
where, Yij : dependent variable (Days open, service per
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


Figure 3. Two SNP of 309 and 354 sequence, Query: Transferrin strand sequence
mean; Gi: Effect of genotype (AA-CC, AG-CT and GG-
TT); Pj: Effect of parity (Second to fourth); Sk: Effect of
calving season (winter, summer, spring and autumn)and
eijkl: Error term.
RESULTS AND DISCUSSION
SNPs detection
Results did reveal that according to the sequence
data, two SNPs were showed. The first one was
A14307G in the intron 8 that previously detected (Ju et
al., 2011) with NCBI accession No. of ss250608651 and
recently reported for Holstein-Friesian cows as 43 pb.
The second detected SNPs were C14081T (Figure 1).
On the other hand, the A14037G and C14081T SNPs
were detected in TF gene of intron 8 as a haplotype
block for Holstein-Friesian cows in Iraq (Figure 2).
Moreover, as explained in Figure 1, two mutations were
existed, first in the 281 sequence (from A to G base as
referred by black line) and second in the 325 sequence
(from C to T base as referred by red line).
The two SNPs occurred in one DNA strand of
Sbjct gene in other cows of heterozygous genotype (AG
-CT). First site is of changing from A to G base at the
Table 2. Genetic polymorphism of TF gene and its relationship with open days and service per conception in
Holstein-Friesian cows
Genetic polymorphism
S. No
No.
1 AA-CC 15
2 AG-CT 18
3 GG-TT 16
4 Level of significance
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Ahmed et al., 2018
sequence 309 and second site is of changing from C to
T base at the sequence 354 (Figure 2). In the other
cows, the SNPs did not occur in the DNA strand; and
the two bases C and T did not change and the genotype
was a wild (AA-CC; Figure 3).
Distribution of TF gene in Holstein-Friesian cattle
studied with sample percentage
Table 1 revealed the percentage of genotype
distribution of TF gene in the sample of Holstein-
Friesian cows as 30.61, 36.73 and 32.65 % for AA-CC,
AG-CT and GG-TT genotypes respectively. On the oth-
er hand, the allele frequencies of T and G alleles were
0.49 and 0.51 respectively (Table 1). Many studies con-
cerning the association of SNPs with the cows perfor-
mance including Ju et al. (2011) deals with the produc-
tive characteristics and Liu et al. (2011) deals with the
reproductive traits of Holstein bulls.
Genetic polymorphisms of TF gene in Holstein-
Friesian cattle
Table 2 noticed the genetic polymorphism of
A14037G and C14081T SNPs in intron 8 of TF gene
and their association with days open and service per
conception of Holstein-Friesian cows. The heterozygous
Mean ± SE
Open days SPC (Service)
132.57 ± 10.04b 2.47 ± 0.42b
108.91 ± 6.95a 1.79 ± 0.26a
139.75 ± 12.82b 2.61 ± 0.39b
P<0.01 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.
Calving interval (day)
1 AA-CC 15
2 AG-CT 18
3 GG-TT 16
4 Level of significance
genotype (AG-CT) recorded the better results (P<0.01)
of open days (108.91±6.95 days) and service per con-
ception (1.79±0.26) as compared with the AA-CC and
GG-TT genotypes. On the other hand, the results were
lesser for AA-CC (132.57±10.04 days and 2.47 ± 0.42)
and moderate for GG-TT (139.75 ± 12.82 days and 2.61
± 0.39; Table 2).
The reasons behind the resultant SNPs in this
segment and in whole TF gene as well as in other genes
related with the productive and reproductive characteris-
tics of Holstein-Friesian cows may return to the expo-
sure of the animals to the harsh environment due to
transportation of the them from their native homelands
to the others differing in temperature, humidity as well
as management conditions. The animals try to be suita-
ble with the new environment in spite of the difficulties.
Accordingly, the heterozygous genotype (AC-GT) rec-
orded a moderate pattern of the reproductive traits, fol-
lowed by wild genotype (GG-TT). These results were
consistent with Senkal et al. (2017) whom found that
the heterozygous genotype (AG-CT) recorded the better
productive performance in Holstein cows.
The occurrence of these SNPs in TF gene and /
or other genes will certainly change the nucleotides se-
quence and gene expression of TF gene consequently.
This will lead to change the TF protein itself and animal
performance positively or negatively according to the
produced protein.
Table 3 revealed the genetic polymorphism of
A14037G and C14081T SNPs in 882 bp segments of TF
gene and their association with the calving interval and
non-return rate of Holstein-Friesian cows. Concomitant
Journal of Research in Ecology (2018) 6(2): 1846–1852
Mean ± SE
Non-return rate (%)
485.39 ± 23.69b 57.83 ± 2.98b
409.61 ± 19.54a 68.04 ± 3.07a
497.25 ± 26.71b 53.68 ± 3.92b
P<0.01 P<0.01
with the Table 2 results, the heterozygous genotype (AG
-CT) recorded the better results (P<0.01) of calving in-
terval and non-return rate being 409.61±19.54 days and
68.04±3.07 % respectively. On the other hand, the re-
sults were lesser for AA-CC namely 485.39± 23.69 days
and 57.83±2.98 % (Table 3).
The superiority of heterozygous genotype in all
reproductive characteristics studied may returns to the
adaptive advantage of harsh environment and climate in
Iraq. The current data were agree with those reported
by Guha et al. (2012) that Gir cows with heterozygous
genotype (TfAE) were superior in some reproductive
traits (age at first calving, age at second calving and
calving interval) as compared with the homozygous
genotypes. Genetic variation is essential to permit im-
provement in cattle productivity. This may be created
from new mutations or from conserving of the existing
genetic variation (Zaabal and Ahmed, 2008). The repro-
ductive efficiency is affected by different factors like
breed, parity, calving season, calving year as well as the
environmental conditions surrounding the animal in-
cluding nutrition and health management (Bearden et
al., 2004). Therefore, the inappropriate environmental
conditions represented by poor grazing land and lesser
healthy and reproductive management of Holstein-
Friesian cows in Iraq led to the lesser productive and
reproductive performance as compared with those in
their native homeland.
CONCLUSION
The reproductive performance of Holstein cows
can be enhanced according to the transferrin gene SNPs
1850
 Ahmed et al., 2018
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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.
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programs in Iraq. Fletcher J and Huehns ER. 1968. Function of trans-
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