The Prolactin Receptor Gene Expression Variance in Marshes and Riverine Buffalos in Iraq
Dhia HusseinJassim AL-Delemi*, AlaaKamil Abdulla Al-Gewary and Basim Hameed Abd Ali
Department of Surgery and Obstetrics, University of AL-Qadissiya, Iraq
Submission: July 16, 2016; Published: August 10, 2016
*Corresponding author: Dhia Hussain Jassim Al-Delemi, Department of Surgery and Obstetrics, College of Veterinary Medicine, University of AL-Qadissiya, AL-Qadissiya, Dewaniya, Iraq.
How to cite this article: Dhia HJ AL-D, AlaaKamil A Al-G, Basim H A A. The Prolactin Receptor Gene Expression Variance in Marshes and Riverine Buffalos in Iraq. Agri Res & Tech: Open Access J. 2016; 2(1): 555580. DOI: 10.19080/ARTOAJ.2016.02.555580
The study area eight stations in southern Iraqi marshes in Missan governorate, and six stations were in Al-Qadisiya and Al-Najaf governorates, the southern Iraqi marsh was proper environment to culture of the riverine buffalo breed, yet the water buffalo which preferring the middle area , because the molecular information of the local buffalo in Iraq is very rare , the presence study selection is the improvement of yield of milk with the advances in molecular biology, the identification of the genes underlying by quantitative (rt-PCR) technique to possible efficient the prolactin (PRL-R) gene expression and to better understand the actions of mammary gland gene expression on milk production in two buffalo breeds in Iraq by determined levels of the PRL-R gene expression in somatic cells in the mammary gland by randomly milk samples of swamp and riverine buffalos during first medal and last lactation period, the results appeared showed high expression of PRL-Rgene in last lactation period of riverine buffalo than swamp buffalo breed, this gene may be have potential direct or indirect effect on milk production.
The transcription levels of PRL-R gene in milk of swamp buffalo were found to be significantly down-regulated in first stage of lactation period, but its where up-regulated in second stage of this period, and significantly decreased this regulation in last period, while very down regulation in first stage of lactation of riverine buffalo and curved to a highly induced regulation in second and late lactation period.
The Buffaloes is considered one of the animals that has wide spread in the marshes area in the south of Iraq [1,3] it’s an important dairy animal, because of the opportunities of milk production despite feeding with low quality roughage [4-6]. The domestic buffalo belongs to the family Bovidae, sub family Bovinae, genus bubalis [7,8]. The buffalo population in iraq exist as two main types, swamp and river buffalo [9,10].
In the present study, we evaluated the PRL-R gene expression in milk somatic cells of lactating period, by used the quantitative real-time PCR (qPCR) is considered the gold standard for gene expression analyses because of its high sensitivity, specificity, and reproducibility . The prolactin hormone and other environmental factors which stimulates growth of the mammary gland [12,13], this Prolactin hormone binding to specific receptor (PRL-R) which responsible on this hormonal action [14-16]. In cattle, two isoforms of PRL-R have been found, resulting from alternative splicing:a long form, with a length of 557 amino acids, and a short one, with a length of 272 amino acids. The PRL-R gene is mapped on the bovine chromosome 20 and is originally described as having 10 exons [17-19], this polymorphic sites in the bovine PRL-R gene were discovered in 2006 , also the first polymorphism in the bovine prolactin receptor gene, identified in 2005, in the region involved in the alternative splicing of the transcript [21-23], this gene coding for bovine PRL-R was mapped to chromosome 20 in cattle and chromosome 19 in buffalo .
Most transcripts of the PRL-R gene are processed for the synthesis of PRL-R mRNA in the epithelial cells of mammary ducts and alveoli [25-27], and has nine exons that code for a polypeptide of 581 amino acids , yet the PRL-R belongs to type I transmembrane receptor family and structurally resembles the class I cytokine receptor super family [17,28].
The aim of this study was to examine the changes of mRNA expression using quantitative real-time polymerase chain reaction because its technique considered the gold standard for gene expression analyses because of its high sensitivity, specificity, and reproducibility , with (GAPDH) as a reference gene which have been found to vary with tissue type, developmental stage, and environmental stimuli . In this study we trying a
compartment between of the prolactin receptor gene expression
level in milk somatic cells or lactating mammary gland of cows
during early, mid and last lactation period in both swamp and
riverine buffalos breeds, due to milk collection is routinely
performed and is less expensive and more easily accomplished
than blood collection according to study of .
Twenty adult lactating buffalo were selected from local
breed swamp (n=10) from the middle (Diwaniya and Al-Najaf),
and riverine buffalos (n=10) were collected from south of
Iraq (Missan and ThiQar), the milk samples were determined
to be free of mastitis defect according to routine testing by
CMT (california mastitis test), they volume of samples which
collected from each animal were(50 ml) during three stages of
the lactation period which are classification on physiochemical
characteristic of milk samples according to stage of lactation
period in swamp buffalo where, first stage (S1) 10-100 days,
second stage (S2) 101-180 days and third stage (S3) --> 181 days, while in riverine buffalo were first stage (R1) 10-100 days,
second stage (R2) 101-180 days and third stage(R3) --> 181 days
 with minor modification, this samples placed in ice box for
3- 5hrs, later Centrifugation 12000 rpm for 10 min at 4oC, and
the supernatant containing the hard fat layer was aspirated and
discarded, leaving a 5ml residual fluid at the bottom of tube PBS
(phosphate buffer slain) (5ml) was added to the fluid and was resuspended
and centrifuged again at 1200 rpm for 5 min at 4OC.
The supernatant was discarded and 1ml of the residual fluid at
the bottom was transferred by the pipette into 1.5ml free RNase
eppendorf tube and centrifuged at 1200 rpm for 5min at 4OC. The
pellet obtained was washed with PBS three times, and stored at
-70 to -80OC in deep freeze system until total RNA extraction,
according to method of .
Two primer pairs were designed using the Primer Premier 5.0
software (http://www.premierbiosoft.com); the Housekeeping
genes are GAPDH gene primer and other primer used for PRL-R
gene as a target gene (Table 1). The specificity of the primer sets
designed was confirmed by sequencing analysis of amplicon
The total RNA was extracted from somatic cells using
Trizol® Reagent by, AMBION/RNA (LIFE TECHNOLOGIES
Ltd.)CALIFORNIA, according to the manufacturer’s protocol,
the integrity of total RNA extracted was verified by agarose
gel electrophoresis by, NANOPAC-300 (CLEAVER SCIENTIFIC Ltd.) UK, the RNA was quantitated using SP-3000 Nano (UV/
Vis Spectrophotometer, OPTIMA Tokyo, JAPAN), also the purity
and quantity of the extracted total RNA were assessed, with the
optical density (OD) ratio of OD260/OD280 being 1.8 to 2.0 for
all samples (Table 2). To removal of the contamination from
genomic DNA, we used the DNase I (Ta- KaRa) Kit.
The First-strand cDNA was synthesized from 1500 ng of
RNA using the Cloned AMV (INVETROGEN®) First-Strand cDNA
Synthesis Kit (USA), The Reverse Transcriptase PCR is performed
according to manufacturer’s protocol, under the following
conditions, by using PCR system (Multi Gene Opti Max Thermal
Cycler TC9610 /TC9610-230), MIDSCI®, USA (in biotechnology
college of Al-Qadissiya univ.) finally the samples were stored at
-20OC until performed q (rt- PCR).
The q (rt-PCR) was carried out as described by , for
relative expression of target genes in somatic cells of swamp
and buffaloes in comparison with riverine buffaloes breed,
the ΔCT USING A REFERENCE GENE METHOD can be used by
normalizing gene expression of target (PRL-R) genes with gene
expression of housekeeping (GAPDH) gene as a reference gene,
by using a following formula:-
Expression value (Fold yield) = 2^CT (reference) – CT (target)
The mean of Ct numbers for target genes were normalized
with reference (GAPDH) gene expression were determined by
using the Microsoft excel according to , and evaluation of this
results according of recommended by .
The SYBER Green I based The two-step reaction of q(rt-
¬PCR) which performed by manufacturer’s protocol of Power
SYBR® Green PCR Master Mix kit (applied biosystem ) California
USA and Exicycler™ 96 Real-Time Quantitative Thermal Block
instrument(Bioneer, Korea), in veterinary hospital laboratory of
Al-najaf, according to method described by Chen , therefore
preparing two q(rt-PCR) master mixes, for the PRL, and GAPDH
genes, as the following: (20 μL) of total volume cDNA template for
target genesfrom cDNA template(10 μL), forward (2 μL) & revers (2 μL) primers and DEPC water (6 μL), this master mixes were
added into Power SYBR® Green PCR Master Mix kit q(rt-PCR)
PreMix tube, then start Exicycler™ 96 Real-Time Quantitative,
thermal Block instrument to relative quantification, according to
In our study, the value of total RNA concentration (Table 2)
was highly significant different (94.374 ±3.07 ng/μl) in somatic
cells of the mammary gland, its total RNA samples were used in
cDNA synthesis step by using First-Strand cDNA Synthesis Kit.
Data analysis of SYBRgreen I based rt-PCR assay were divided
into primer efficiency estimation and relative quantification of
PRL-R gene expression level which normalized by housekeeping
gene expression (GAPDH).The Ct value of GAPDH are 23.2676
in swamp breed, and 23.4184 in riverine breed, there for the
result of normalizing the PRL-R gene expression by The delta CT
method Using a Reference Gene .
In swamp buffalo the expression value of PRL-R gene during
lactation period which declines in first stage (0.6145), second
stage up-regulation (1.653) and decreased this regulation
(1.1272) in last period, while in riverine buffalo this expression
are very down-regulation (0.4045) in first stage of lactation and
curved to a highly induced regulation in second (2.1312) and late
lactation period (4.4201) in mammary gland. (Table 3), (Figure
In mammals, the hormone prolactin (PRL) is best known for
its role in the regulation of lactation [39-41], also it has important
functions like the development of mammary gland affecting milk
yield and composition [42-45]. Also according to  this is
hormone stimulant for growth of mammary ductal and alveolar
cells by binding to PRL-R. yet the physiological functions of the
PRL hormone to induce lactation by this acting through the
PRI-R . The prolactin receptor (PRL-R) gene was reportedly
associated with milk protein and milk fat yields in the swamp
buffalo  also the statistically significant of study the 
were confirmed the associations between PRL-R milk production
in livestock, therefore the results of the previous study were
conferment that up-regulation of PRL-R gene expression in last
lactation period of riverine buffalo (4.4201) in more than swamp
buffalo which are down-regulated (1.1272) in this period, this a
results agreement with Previous studies like [46,49].
[50,51] as well as these data support the concept that the
PRL-R were higher sensitivity to PRL during lactation period
may be associated with an increase in subsequent milk yield
in riverine buffalo more than swamp buffalo, consequently the
previous studies like  were observed the swamp buffalo
produce relatively small quantities of milk, yet  recorded
that the high milk yield is about six to seven liters per day in
riverine breeds Indian buffalo.
In the mammary gland of lactating mice, the PRL-R is highly
expressed both at the end of pregnancy and during lactation .
PRL-R1 mRNA expression is highly induced in the mammary
gland during late pregnancy and abruptly declines on the first day
of lactation for the HT rats , but this observe disagreement
with study of , that were recorded a short day photoperiod
was associated with reduced PRL, whereas milk yield and
expression of PRL-R mRNA in lymphocytes and mammary tissue
were increased. The swamp buffalo produce low amount of milk
(1.0 - 1.5) litres per day, so they are not heavily used in milk
production , but the milk yield of Indian riverine breeds,
there is about (6 - 7) liters per day . The PRL-R numbers
begin to decrease in early pregnancy and are maintained at a low
level until late pregnancy .
The data normalization of the reference gene (GAPDH)
with PRL-R gene expression in milk somatic cells of lactating
two local buffalo breeds. In the present study, we compared the
transcription levels of PRL-R gene in milk of swamp buffalo were
found to be significantly down regulated in first stage of lactation
period, but its where up regulated in second stage of this period,
and significantly decreased this regulation in last period, while
in riverine buffalo this expression are very down regulation in
first stage of lactation and curved to a highly induced regulation
in second and late lactation period.