molecules
A icle
T acing Recombinan Bo ine Soma o opin Ab(Use)
Th ough Gene Exp ession in Blood, Hai Follicles,
and Milk Soma ic Cells: A Ma ix Compa ison
Alexand e Lamas * ID , Pa icia Regal * ID , Bea iz Vazquez ID , JoséManuel Mi anda ID ,
Albe o Cepeda and Ca los Manuel F anco ID
Labo a o io de Higiene, Inspección y Con ol de Alimen os, Dp o. de Química Analí ica, Nu ición y
B oma ología, Uni e sidad de San iago de Compos ela, 27002 Lugo, Spain; [email p o ec ed] (B.V.);
[email p o ec ed] (J.M.M.); [email p o ec ed] (A.C.); [email p o ec ed] (C.M.F.)
*
Co espondence: [email p o ec ed] (A.L.); pa icia. [email p o ec ed] (P.R.); Tel.: +34-982-822-455 (A.L. & P.R.)
Recei ed: 27 June 2018; Accep ed: 11 July 2018; Published: 13 July 2018


Abs ac :
The use o ecombinan bo ine soma o opin ( bST) in dai y ca le is o bidden in he
Eu opean Union. Due o he e y low ci cula ing concen a ion o bST in ea ed animals, i s di ec
de ec ion is s ill a challenge. The e o e, he use o indi ec me hods o de ec he ab(use) o bST
in dai y ca le appea s as a good al e na i e. In he pas ew yea s, gene exp ession demons a ed
i s u ili y in sc eening he use o illici subs ances in bo h humans and animals. In his s udy,
a compa ison o h ee ypes o ma ices (milk soma ic cells, blood, and hai ollicles) was ca ied ou
o e alua e hei po en ial use o ou ine con ol o bST using 15 gene-exp ession p o iles. A o al o
six bST- ea ed cows and h ee con ol cows we e included in he s udy. A subcu aneous injec ion
con aining 500 mg o bST was adminis e ed o he ea ed g oup. Samples o he h ee ma ices we e
collec ed be o e bST adminis a ion, and a h ee and nine days a e ea men . The quali y o RNA
ex ac ed was highe in he blood and hai - ollicle samples han in he milk soma ic cells. In he h ee
ma ices, he e we e signi ican di e ences in he exp ession o some genes, wi h milk soma ic cells
and blood p esen ing he he bes ma ices. On his no e, he cyclin D1 (CCND1), in e leukin 1 be a
(IL-1
β
), umo nec osis ac o (TNF), and insulin-like g ow h ac o 1 ecep o (IGF-1R) genes showed
po en ial as bioma ke s o bST ea men . The e o e, blood, soma ic cells, and ollicle hai should
be conside ed as p omising sou ces o RNA, and can be used in gene-exp ession assays o ou inely
con ol he illici use o bST.
Keywo ds: bST; nanoli e PCR; gene exp ession; sc eening; blood; hai ; soma ic cells
1. In oduc ion
The g ow h ho mone (GH) is a single-chain polypep ide sec e ed by he an e io pi ui a y gland o
all e eb a es. This ho mone is in ol ed in a wide ange o biological ac i i ies, such as g ow h, ene gy
me abolism, sexual ma u a ion, and immuni y. These ac ions a e ini ia ed upon he binding o GH
o memb ane-bound ecep o s loca ed in a ious issues, including li e , muscle, bone, o mamma y
issue [
1
]. The GH-media ed ac ions in mamma y issue we e s udied in dai y ca le o many yea s.
Fu he mo e, s udies ca ied ou in Russia and England a he beginning o he 20 h cen u y ound ha
pi ui a y ex ac s om cows inc eased milk yield in dai y ca le [
2
,
3
]. This e ec was due o he p esence
o g ow h ho mone in he ex ac . In he 1980s, ecombinan DNA echnology allowed he p oduc ion
o la ge quan i ies o ecombinan bo ine soma o opin ( bST) in a cos -e ec i e and e icien manne .
The galac opoie ic e ec o he ecombinan a ian s was demons a ed, and he ou ine use o bST
in dai y a ms o inc ease he global milk yield s a ed being e alua ed [
4
]. Thus, in he yea 1993,
he Food and D ug Adminis a ion (FDA) app o ed he use o bST in he Uni ed S a es.
Molecules 2018,23, 1708; doi:10.3390/molecules23071708 www.mdpi.com/jou nal/molecules
Molecules 2018,23, 1708 2 o 15
Despi e i s capaci y o enhance milk p oduc ion in dai y a ms, he use o bST was con o e sial
since he beginning. Some s udies obse ed ha he use o bST could inc ease he incidence o mas i is
and lameness in dai y a ms, wi h a consequen de e io a ion o animal wel a e [
5
]. Mo eo e , i was
sugges ed ha he inc eased p esence o insulin-like g ow h ac o 1 (IGF-1) in milk as a consequence
o bST use could ha e a de imen al e ec in human heal h [
6
]. The e o e, while he use o bST is
allowed in a ious coun ies such as Uni ed S a es, B azil, and Mexico, i s use is banned in o he s such
as Canada, Aus alia, and New Zealand. In 1999, he Eu opean Union (EU) de ini i ely banned he
use o bST in Eu ope, in oking animal wel a e easons and i s impac on Eu opean milk policy and
consume ea s [7].
The p ohibi ion o bST use in he EU canno p e en he illici use o his subs ance in dai y
a ms. Consequen ly, he illegal use o bST in dai y a ms was de ec ed in 2013 in Spain. The pe sons
in ol ed in his scandal in oduced comme cial injec ions om Mexico, whe e he use o his subs ance
is allowed. The e o e, i is clea ha bST should be included in he Eu opean esidue-con ol plans [
8
].
Analy ical chemis y echniques combining ch oma og aphy and mass spec ome y a e commonly
used o de ec he illici use o subs ances in ca le (e.g., g ow h p omo e s). Fu he mo e, some me hods
we e de eloped o de ec bST in bo ine and bu alo se um, using liquid ch oma og aphy/ andem
mass spec ome y [
9
,
10
]. Howe e , due o di e en bST a ian s a ailable wi h a ying e minal
amino-acid composi ions, and he low ci cula ion le els o bST in blood, i is di icul o de ec bST
using hese me hods [
11
]. The e o e, he use o indi ec me hods could be an al e na i e o sc eening
he use o bST in ca le. In he pas ew yea s, ansc ip omics eme ged as a p omising ool o
e alua ing he use o illici subs ances in ca le [
12
]. Mo eo e , some s udies used qPCR o de elop
a sc eening panel ha could elucida e he use o g ow h p omo e s in ca le [
13
,
14
]. Some s udies
e alua ed he gene-exp ession modi ica ions caused by bST in a ious issues o dai y ca le [
15
–
17
].
Howe e , none o hese s udies ocused on he de elopmen o a sc eening panel o be used ou inely
in moni o ing he illici use o bST in dai y a ms.
Rou ine con ols o bST ab(use) should be ca ied ou a di e en ime poin s in dai y a ms o
a oid he ma ke elease o milk p oduced using bST. The e o e, pos -mo em samples om he li e
o muscle a e o ally disca ded in his ype o con ol. F om a p ac ical poin o iew, only ma ices
ha can be collec ed
in i o
a e eally in e es ing o ou ine con ols. The ma ix which is easies o
collec in a dai y a m is ha o milk, and he soma ic cells p esen in milk a e a good al e na i e o
ansc ip omic assays [
18
]. The collec ion o o he ma ices equi es di ec con ac wi h he animal.
A good example o his is blood, which is commonly used o
in i o
ansc ip omics assays [
19
].
Ano he op ion o ca y ou
in i o
ansc ip omics assays is he hai ollicle, as hai is a common
ma ix o de ec he use o illici subs ances in ca le [
20
]. Howe e , un il now, he e a e no s udies
ocusing on he use o hai ollicles o sc eening ansc ip omics assays o con ol he use o banned
subs ances in ca le.
The e o e, he aim o his s udy was o compa e he po en ial o h ee ma ices (milk soma ic cells,
blood, and hai ollicles) o de ec he ab(use) o bST in dai y ca le, using gene exp ession. A o al o
nine dai y cows we e in ol ed in he s udy (six bST- ea ed cows and h ee con ol cows). A o al
o 15 a ge and h ee e e ence genes we e selec ed, and hei exp essions in h ee di e en ma ices
we e de e mined using a high- h oughpu eal- ime pla o m, allowing he simul aneous analysis o
a ious genes in a wide ange o samples.
2. Resul s
2.1. RNA Isola ion and Quali y
The amoun o RNA isola ed om he samples a ied ac oss ma ices (Table 1). The amoun o
RNA isola ed om soma ic cells was signi ican ly mo e abundan (p< 0.001) han ha in blood and
hai ollicles. Fu he mo e, he a io o abso bances a 260 and 280 nm (A260/A280) was signi ican ly
di e en (p< 0.001) ac oss ma ices. The RNA ex ac ed om hai ollicles p esen ed he highes
Molecules 2018,23, 1708 3 o 15
A260/A280 a io, wi h a mean alue o 1.892, while he RNA om blood p esen ed a mean A260/A280
a io o 1.809, and he RNA om milk soma ic cells p esen ed he lowes A260/A280 a io, wi h a mean
alue o 1.710. The RNA in eg i y numbe (RIN) alues showed a simila endency. The RIN alues o
RNA ex ac ed om hai ollicles and blood we e signi ican ly highe han ha o RNA ob ained om
milk soma ic cells.
Table 1.
The concen a ion o RNA, he a io o abso bances a 260 and 280 nm (A260/A280), and he
RNA in eg i y numbe (RIN) alues o RNA isola ed om he h ee ma ices.
Ma ix RNA Concen a ion (ng/µL) A260/A280 Ra io RIN Value
Hai ollicles 53.30 ±11.97 b1.89 ±0.06 a7.76 ±0.41 a
Blood 59.00 ±28.02 b1.81 ±0.04 b7.79 ±0.40 a
Soma ic cells 165.42 ±71.23 a1.71 ±0.08 c6.10 ±0.57 b
Le e s (a–c) in each column e lec signi ican di e ences.
2.2. Re e ence Genes
Th ee di e en genes, namely ubiqui ously exp essed p e oldin-like chape one (UXT), ibosomal
p o ein S9 (RPS9), and mi ochond ial glyce ol 3-phospha e acyl ans e ase (GPAM), we e e alua ed as
in e nal con ols o his gene-exp ession s udy. Thei s abili y was e alua ed using he Bes keepe
®
so wa e. Wi h his so wa e, he s anda d de ia ion (SD) o c ossing-poin alues (CPs) o candida e
e e ence genes was calcula ed, as well as Bes keepe indexes using he SD alues o candida e genes.
The mos s able genes exhibi he lowes a ia ion, and any s udied gene wi h an SD highe han 1 can
be conside ed inconsis en .
The e we e di e ences in he exp ession o e e ence genes. The exp ession o all h ee e e ence
genes included in his s udy was de ec ed in soma ic-cell samples. Howe e , in blood and hai - ollicle
samples, only he exp essions o UXT and RPS9 we e de ec ed. The e o e, he s abili y o he h ee
genes was calcula ed in soma ic cells, while only he s abili y o UXT and RPS9 was calcula ed in
blood and ollicle samples. Table 2shows he SD (
±
CP) o he selec ed genes om each ype o ma ix.
Table 2.
S anda d de ia ion o c ossing-poin (CP) alues ob ained o he h ee e e ence
candida es—ubiqui ously exp essed p e oldin-like chape one (UXT), ibosomal p o ein S9 (RPS9),
and mi ochond ial glyce ol 3-phospha e acyl ans e ase (GPAM). Fo hai ollicles and blood, he
ampli ica ion o GPAM was no de ec ed. Bes keepe indexes we e calcula ed based on he CP alues
o e e ence genes. Fo soma ic cells, he Bes keepe index was calcula ed based on UXT-RPS9.
Ma ix UXT RPS9 GPAM Bes keepe Index Bes keepe Index (UXT-RPS9)
Hai ollicles 0.90 0.91 - 0.96 0.96
Blood 0.89 0.75 - 0.71 0.71
Soma ic cells 0.85 0.79 1.19 0.94 0.89
The GPAM gene was only exp essed in soma ic cells; in he samples analyzed o his s udy, he
SD (
±
CP) was highe han 1, and could be conside ed as inconsis en . The SD (
±
CP) o he o he
wo genes was lowe han 1, and he e o e, could be conside ed as consis en . Fu he mo e, in hai
ollicles, he SD (
±
CP) was simila o bo h genes (UXT and RPS9), while in he blood and in soma ic
cells, RPS9 showed lowe SD (
±
CP) alues. These esul s indica e a be e s abili y o his gene in
compa ison wi h UXT. The blood samples p esen ed he bes Bes keepe index. In he case o soma ic
cells, he use o only UXT and RPS9 allowed us o ob ain a be e Bes keepe index. Hai ollicles
p esen ed he wo s Bes keepe index. In his s udy, only he UXT and RPS9 genes we e used o
no malize he exp ession o a ge genes.
Molecules 2018,23, 1708 4 o 15
2.3. Exp ession Pa e n o Ta ge Genes Analyzed
The exp essions o h ee genes, namely insulin-like g ow h ac o binding p o ein 5 (IGFBP5),
collagen ype III alpha 1 chain (COL3A1), and es ogen ecep o 2 (ESR2), we e no de ec ed in he
ma ices es ed (Figu e 1). In addi ion, he exp ession o IGF-1 was only de ec ed in one cow in he milk
soma ic cells. The numbe o a ge genes in which exp ession was de ec ed in his s udy a ied in each
ma ix. In milk soma ic-cell samples, he exp essions o nine genes we e de ec ed, and he ansc ip ion
o lac o ans e in (LTF) was only de ec ed in his ma ix. In blood samples, he exp essions o eigh
genes we e obse ed, and he ansc ip ion o ca enin alpha-like 1 (CTNNAL1) was only de ec ed in
his ma ix. Finally, in hai - ollicle samples, he ansc ip ions o se en genes we e obse ed, and he
exp ession o insulin-like g ow h ac o binding p o ein 3 (IGFBP3) was de ec ed in his ma ix.
Molecules 2018, 23, x FOR PEER REVIEW 4 o 15
2.3. Exp ession Pa e n o Ta ge Genes Analyzed
The exp essions o h ee genes, namely insulin-like g ow h ac o binding p o ein 5 (IGFBP5),
collagen ype III alpha 1 chain (COL3A1), and es ogen ecep o 2 (ESR2), we e no de ec ed in he
ma ices es ed (Figu e 1). In addi ion, he exp ession o IGF-1 was only de ec ed in one cow in he
milk soma ic cells. The numbe o a ge genes in which exp ession was de ec ed in his s udy a ied
in each ma ix. In milk soma ic-cell samples, he exp essions o nine genes we e de ec ed, and he
ansc ip ion o lac o ans e in (LTF) was only de ec ed in his ma ix. In blood samples, he
exp essions o eigh genes we e obse ed, and he ansc ip ion o ca enin alpha-like 1 (CTNNAL1)
was only de ec ed in his ma ix. Finally, in hai - ollicle samples, he ansc ip ions o se en genes
we e obse ed, and he exp ession o insulin-like g ow h ac o binding p o ein 3 (IGFBP3) was
de ec ed in his ma ix.
Ma ices
Gene Blood Milk Soma ic Cells Hai Follicles
IGF1
IGF1-R
IGFBP3
IGFBP5
IL-1β
TNF
LTF
COL3A1
TPD52L2
ESR2
CTNNAL1
SIRT2
CCND1
MFGE8
EEF1G
Figu e 1. Gene-exp ession pa e ns in blood, milk soma ic cells, and hai - ollicle samples. G een cells
ep esen exp ession o he a ge gene, and g ay cells ep esen no exp ession o he a ge gene, in
he samples o each ma ix. Fo a lis o he gene names e alua ed, see Table 3.
2.3.1. Exp ession o Ta ge Genes in Blood Samples
Gene-exp ession assays we e ca ied ou he week be o e he i s bST adminis a ion, and on
he hi d and nin h days a e bST adminis a ion. Genes di e en ly exp essed be ween bST and
con ol samples we e only de ec ed on he hi d day a e bST adminis a ion (Figu e 2).
Speci ically, euka yo ic ansla ion elonga ion ac o 1 gamma (EEF1G), umo nec osis ac o (TNF),
and in e leukin 1 be a (IL-1β) we e signi ican ly up egula ed in bST samples. Howe e , milk a
globule epide mal g ow h ac o (EGF) 8 (MFGE8) was signi ican ly down egula ed in he bST
g oup on he hi d day.
Figu e 1.
Gene-exp ession pa e ns in blood, milk soma ic cells, and hai - ollicle samples. G een cells
ep esen exp ession o he a ge gene, and g ay cells ep esen no exp ession o he a ge gene, in he
samples o each ma ix. Fo a lis o he gene names e alua ed, see Table 3.
Table 3. Genes included in his s udy o gene-exp ession assays.
Gene Symbol Gene Name NCBI Accession Numbe Assay ID
IGF1 Insulin-like g ow h ac o 1 NM_001077828.1 b 03252281_m1
IGF1-R Insulin-like g ow h ac o 1 ecep o NM_001244612.1 b 03649217_m1
IGFBP3 Insulin-like g ow h ac o binding p o ein 3 NM_174556.1 b 03223809_m1
IGFBP5 Insulin-like g ow h ac o binding p o ein 5 NM_001105327.2 b 03258785-g1
IL-1βIn e leukin 1 be a NM_174093.1 b 03212745_m1
TNF Tumo nec osis ac o NM_173966.3 b 03259156_m1
LTF Lac o ans e in NM_180998.2 b 03217382_m1
COL3A1 Collagen ype III alpha 1 chain NM_001076831.1 b 03249914_m1
TPD52L2 Tumo p o ein D52-like 2 NM_001034615.2 b 03227133_m1
ESR2 Es ogen ecep o 2 NM_174051.3 b 03259198_m1
CTNNAL1 Ca enin alpha-like 1 NM_001191534.1 b 04308229_m1
SIRT2 Si uin 2 NM_001113531.1 b 03258971_m1
CCND1 Cyclin D1 NM_001046273.2 b 03235030_m1
MFGE8
Milk a globule epide mal g ow h ac o (EGF) 8 p o ein
NM_176610.1 b 03216856_m1
EEF1G Euka yo ic ansla ion elonga ion ac o 1 gamma NM_001040487.2 b 03229629_g1
Re e ence genes
UXT Ubiqui ously exp essed p e oldin-like chape one NM_001037471.2 b 03229278_m1
RPS9 Ribosomal p o ein S9 NM_001101152.2 b 03272016_m1
GPAM Glyce ol 3-phospha e acyl ans e ase (mi ochond ial) NM_001012282.1 b 03210379_m1
Molecules 2018,23, 1708 5 o 15
2.3.1. Exp ession o Ta ge Genes in Blood Samples
Gene-exp ession assays we e ca ied ou he week be o e he i s bST adminis a ion, and on
he hi d and nin h days a e bST adminis a ion. Genes di e en ly exp essed be ween bST and
con ol samples we e only de ec ed on he hi d day a e bST adminis a ion (Figu e 2). Speci ically,
euka yo ic ansla ion elonga ion ac o 1 gamma (EEF1G), umo nec osis ac o (TNF), and in e leukin
1 be a (IL-1
β
) we e signi ican ly up egula ed in bST samples. Howe e , milk a globule epide mal
g ow h ac o (EGF) 8 (MFGE8) was signi ican ly down egula ed in he bST g oup on he hi d day.
Molecules 2018, 23, x FOR PEER REVIEW 5 o 15
Figu e 2. Rela i e abundance o a ge genes exp essed in blood samples be o e he adminis a ion o
ecombinan bo ine soma o opin ( bST), and on he hi d and nin h days a e bST adminis a ion.
The ba s ep esen he mean alue o each g oup ( bST and con ol). The alue used o each animal
is he mean o h ee eplica es; * p < 0.05, *** p < 0.001.
2.3.2. Exp ession o Ta ge Genes in Milk Soma ic-Cell Samples
The IL-1β, insulin-like g ow h ac o 1 ecep o (IGF-1R), and TNF genes we e signi ican ly
up egula ed (Figu e 3) in he bST g oup on he hi d and nin h days a e ecombinan -ho mone
adminis a ion. The cyclin D1 (CCND1) gene was signi ican ly up egula ed in he bST g oup on he
nin h day. On he o he hand, LTF and umo p o ein D52-like 2 (TDP52L2) we e signi ican ly
up egula ed on he nin h day in he con ol g oup. The ansc ip ion o EEF1G was signi ican ly
Figu e 2.
Rela i e abundance o a ge genes exp essed in blood samples be o e he adminis a ion o
ecombinan bo ine soma o opin ( bST), and on he hi d and nin h days a e bST adminis a ion.
The ba s ep esen he mean alue o each g oup ( bST and con ol). The alue used o each animal is
he mean o h ee eplica es; * p < 0.05, *** p < 0.001.
2.3.2. Exp ession o Ta ge Genes in Milk Soma ic-Cell Samples
The IL-1
β
, insulin-like g ow h ac o 1 ecep o (IGF-1R), and TNF genes we e signi ican ly
up egula ed (Figu e 3) in he bST g oup on he hi d and nin h days a e ecombinan -ho mone
adminis a ion. The cyclin D1 (CCND1) gene was signi ican ly up egula ed in he bST g oup on

Molecules 2018,23, 1708 6 o 15
he nin h day. On he o he hand, LTF and umo p o ein D52-like 2 (TDP52L2) we e signi ican ly
up egula ed on he nin h day in he con ol g oup. The ansc ip ion o EEF1G was signi ican ly
di e en be ween bo h g oups o all sample poin s, including hose be o e bST adminis a ion, and
MFEG8 exp ession was highe in he bST g oup on he six h day p io o adminis a ion, bu no a e
bST adminis a ion.
Molecules 2018, 23, x FOR PEER REVIEW 6 o 15
di e en be ween bo h g oups o all sample poin s, including hose be o e bST adminis a ion, and
MFEG8 exp ession was highe in he bST g oup on he six h day p io o adminis a ion, bu no
a e bST adminis a ion.
Figu e 3.
Rela i e abundance o a ge genes exp essed in milk soma ic-cell samples be o e bST
adminis a ion, and on he hi d and nin h days a e bST adminis a ion. The ba s ep esen he mean
alue o each g oup ( bST and con ol). The alue used o each animal is he mean o h ee eplica es.
* p < 0.05, ** p < 0.01, *** p < 0.001.
Molecules 2018,23, 1708 7 o 15
2.3.3. Exp ession o Ta ge Genes in Hai -Follicle Samples
In his ma ix, signi ican di e ences be ween he bST and con ol g oups we e only de ec ed in
he exp ession o IGF-1R on he hi d and nin h days, and also in CCND1 on he nin h day (Figu e 4).
The o he genes showed no signi ican di e ences o all ime poin s analyzed.
Molecules 2018, 23, x FOR PEER REVIEW 7 o 15
Figu e 3. Rela i e abundance o a ge genes exp essed in milk soma ic-cell samples be o e bST
adminis a ion, and on he hi d and nin h days a e bST adminis a ion. The ba s ep esen he
mean alue o each g oup ( bST and con ol). The alue used o each animal is he mean o h ee
eplica es. * p < 0.05, ** p < 0.01, *** p < 0.001.
2.3.3. Exp ession o Ta ge Genes in Hai -Follicle Samples
In his ma ix, signi ican di e ences be ween he bST and con ol g oups we e only de ec ed
in he exp ession o IGF-1R on he hi d and nin h days, and also in CCND1 on he nin h day (Figu e 4).
The o he genes showed no signi ican di e ences o all ime poin s analyzed.
Molecules 2018, 23, x FOR PEER REVIEW 8 o 15
Figu e 4. Rela i e abundance o a ge genes exp essed in hai - ollicle samples be o e bST
adminis a ion, and on he hi d and nin h days a e bST adminis a ion. The ba s ep esen he
mean alue o each g oup ( bST and con ol). The alue used o each animal is he mean o h ee
eplica es. * p < 0.05, *** p < 0.001.
3. Discussion
On he basis o he esul s obse ed in his s udy, milk soma ic cells may be highligh ed as he
bes candida e o a a ge ma ix in bST gene-exp ession analysis, as i showed he highes
di e ences in he selec ed genes. T ansc ip ion pa e ns a e expec ed o be issue-speci ic, and, on
his basis, a a ge ma ix should be selec ed ollowing ac ion-based c i e ia. The mamma y gland is
conside ed one o he p incipal a ge issues o bST, and as such, i p o ided he mos in e es ing
ansc ip ion pa e n. Addi ionally, i is wo h men ioning ha he s abili y o he selec ed e e ence
genes a ied acco ding o he ype o ma ix. Hence, i is ele an o explo e new e e ence
candida es o hai ollicles, as his ma ix showed he poo es s abili y o he housekeepe s selec ed
in his s udy. These esul s highligh he impo ance o e alua ing a ious candida es o ind hose wi h
be e s abili y, as e e ence genes a e one o he mos impo an aspec s o RT-qPCR assays [21].
In his s udy, a ious a ge genes we e included wi h he aim o inding a bioma ke signa u e
o bST adminis a ion. Samples o he h ee ma ices e alua ed we e collec ed a h ee di e en ime
poin s (be o e bST adminis a ion, and on he hi d and nin h days a e bST adminis a ion). The
exp ession o insulin-like g ow h ac o 1 (IGF1) was e alua ed in his s udy, and i s exp ession was
only de ec ed in one cow in he soma ic cells. Fo he emainde o he animals, i s exp ession was
no de ec ed in any o he e alua ed ma ices. I is possible ha he ansc ip ion o IGF1 associa ed
wi h bST adminis a ion is ocused in he li e , and IGF1 syn he ized in his o gan ci cula es o he
o he issues, such as he mamma y gland, whe e bST exe s an indi ec unc ion h ough an IGF1
molecule [22]. Fu he mo e, he exp ession o IGF-1R ( he ecep o o IGF1) was de ec ed in all h ee
ma ices. The exp ession o IGF-1R was signi ican ly up egula ed, bo h in soma ic cells and hai
ollicles, in he bST g oup in compa ison wi h he con ol g oup on he hi d and nin h days.
Howe e , in blood, he e we e no di e ences ac oss g oups. Acco dingly, Cas igliego e al. [15]
obse ed no in luence o bST on he exp ession o IGF-1R in he muscle o ea ed cows. These da a
highligh he issue-speci ic e ec o bST, and he need o pe o ming he selec ion o candida e
a ge genes acco ding o he ma ix ha is going o be analyzed. Mo eo e , he exp ession o genes
coding o IGF-1-binding p o eins was analyzed, and only he exp ession o IGFBP3 was de ec ed in
hai ollicles; howe e , no signi ican di e ences we e obse ed ac oss ime poin s. These esul s
disc edi he use o he exp ession o genes coding o IGF-1-binding p o eins as bioma ke s o bST
ea men in he ma ices s udied.
Two genes ela ed o he immune sys em (IL-1β and TNF) we e included in his s udy. The
exp ession o hese genes was de ec ed in milk soma ic cells and in blood, bu no in hai ollicles.
The exp essions o IL-1β and TNF we e clea ly up egula ed in milk soma ic cells on he hi d and
nin h days a e bST adminis a ion. In blood, he di e ences ac oss g oups o hese genes we e
only signi ican on he hi d day, and hese di e ences we e less signi ican han in milk soma ic
cells. A me a-analysis s udy showed ha bST adminis a ion inc eases heal h p oblems in dai y
cows [5]. This exogenous subs ance can al e he immune sys em o cows, and his could explain he
Figu e 4.
Rela i e abundance o a ge genes exp essed in hai - ollicle samples be o e bST
adminis a ion, and on he hi d and nin h days a e bST adminis a ion. The ba s ep esen he mean
alue o each g oup ( bST and con ol). The alue used o each animal is he mean o h ee eplica es.
* p < 0.05, *** p < 0.001.
Molecules 2018,23, 1708 8 o 15
3. Discussion
On he basis o he esul s obse ed in his s udy, milk soma ic cells may be highligh ed as he bes
candida e o a a ge ma ix in bST gene-exp ession analysis, as i showed he highes di e ences in
he selec ed genes. T ansc ip ion pa e ns a e expec ed o be issue-speci ic, and, on his basis, a a ge
ma ix should be selec ed ollowing ac ion-based c i e ia. The mamma y gland is conside ed one o
he p incipal a ge issues o bST, and as such, i p o ided he mos in e es ing ansc ip ion pa e n.
Addi ionally, i is wo h men ioning ha he s abili y o he selec ed e e ence genes a ied acco ding
o he ype o ma ix. Hence, i is ele an o explo e new e e ence candida es o hai ollicles, as his
ma ix showed he poo es s abili y o he housekeepe s selec ed in his s udy. These esul s highligh
he impo ance o e alua ing a ious candida es o ind hose wi h be e s abili y, as e e ence genes
a e one o he mos impo an aspec s o RT-qPCR assays [21].
In his s udy, a ious a ge genes we e included wi h he aim o inding a bioma ke signa u e
o bST adminis a ion. Samples o he h ee ma ices e alua ed we e collec ed a h ee di e en
ime poin s (be o e bST adminis a ion, and on he hi d and nin h days a e bST adminis a ion).
The exp ession o insulin-like g ow h ac o 1 (IGF1) was e alua ed in his s udy, and i s exp ession
was only de ec ed in one cow in he soma ic cells. Fo he emainde o he animals, i s exp ession was
no de ec ed in any o he e alua ed ma ices. I is possible ha he ansc ip ion o IGF1 associa ed
wi h bST adminis a ion is ocused in he li e , and IGF1 syn he ized in his o gan ci cula es o he
o he issues, such as he mamma y gland, whe e bST exe s an indi ec unc ion h ough an IGF1
molecule [
22
]. Fu he mo e, he exp ession o IGF-1R ( he ecep o o IGF1) was de ec ed in all h ee
ma ices. The exp ession o IGF-1R was signi ican ly up egula ed, bo h in soma ic cells and hai
ollicles, in he bST g oup in compa ison wi h he con ol g oup on he hi d and nin h days. Howe e ,
in blood, he e we e no di e ences ac oss g oups. Acco dingly, Cas igliego e al. [
15
] obse ed no
in luence o bST on he exp ession o IGF-1R in he muscle o ea ed cows. These da a highligh
he issue-speci ic e ec o bST, and he need o pe o ming he selec ion o candida e a ge genes
acco ding o he ma ix ha is going o be analyzed. Mo eo e , he exp ession o genes coding o
IGF-1-binding p o eins was analyzed, and only he exp ession o IGFBP3 was de ec ed in hai ollicles;
howe e , no signi ican di e ences we e obse ed ac oss ime poin s. These esul s disc edi he use
o he exp ession o genes coding o IGF-1-binding p o eins as bioma ke s o bST ea men in he
ma ices s udied.
Two genes ela ed o he immune sys em (IL-1
β
and TNF) we e included in his s udy.
The exp ession o hese genes was de ec ed in milk soma ic cells and in blood, bu no in hai ollicles.
The exp essions o IL-1
β
and TNF we e clea ly up egula ed in milk soma ic cells on he hi d and
nin h days a e bST adminis a ion. In blood, he di e ences ac oss g oups o hese genes we e
only signi ican on he hi d day, and hese di e ences we e less signi ican han in milk soma ic cells.
A me a-analysis s udy showed ha bST adminis a ion inc eases heal h p oblems in dai y cows [
5
].
This exogenous subs ance can al e he immune sys em o cows, and his could explain he up egula ion
o he immune-sys em- ela ed genes, TNF and IL-1
β
, in he ea ed g oup. Also, in p e ious sc eening
ansc ip omics s udies, i was obse ed ha anabolic ea men s caused an up egula ion o IL-1
β
in blood and aginal smea cells [
13
,
23
]. In addi ion, o he p ocesses in ol ing in ec ions such
as subclinical mas i is can inc ease he exp essions o IL-1
β
and TNF in mamma y glands [
24
,
25
].
The e o e, hese genes canno indi idually be conside ed as speci ic ma ke s o bST adminis a ion.
The e o e, in sc eening ansc ip omic s udies, i is necessa y o include a ange o genes, as one gene
can be up- o down egula ed on he basis o se e al ac o s. As he numbe o genes included in he
panel inc eases, so does he disc imina ion powe o he designed panel.
O he genes wi h unc ions in ol ing cell cycle, p oli e a ion, di e en ia ion, and adhesion we e
included in his s udy. I is known ha bST inc eases milk syn hesis by inc easing he u no e
(p oli e a ion/apop osis) and ac i i y o mamma y epi helial cells, indica ing ha bST s ongly
in luences me abolic pa hways ha egula e cell u no e /cycle and me abolism [
25
]. Acco dingly,
he CCND1 gene plays an impo an ole in cell physiopa hology because i s dys egula ion is s ongly
Molecules 2018,23, 1708 9 o 15
ela ed o a d i e in inapp op ia e cell di ision, and he gene a ion o genome ins abili y, gene a ing
neoplas ic g ow h [
26
]. This gene was signi ican ly up egula ed on he nin h day in he bST g oup
o milk soma ic cells and hai ollicles. These esul s highligh he close ela ionship be ween bST
and cell-cycle egula ion. The adminis a ion o his exogenous subs ance causes an o e exp ession
o CCND1 ha can esul in he ac i a ion o cells in he G
0
phase. The e o e, he CCND1 gene is
a p omising ma ke o de ec ing he use o bST in dai y ca le, using milk soma ic cells and hai
ollicles. Howe e , in blood, he exp ession o CCND1 was no de ec ed. On he o he hand, he EEF1G
and MFGE8 genes we e signi ican ly up egula ed and down egula ed, espec i ely, in he bST g oup
on he hi d day o blood samples. A p e ious s udy in he mamma y issue o bST- ea ed cows also
obse ed an up egula ion o EEF1G six days a e ho mone adminis a ion [
16
]. EEF1G is in ol ed
in ansla ion elonga ion ia he anspo o aminoacyl ans e RNAs ( RNAs) o he ibosome o
p o ein syn hesis [
27
]. The e o e, he up egula ion o his gene is associa ed wi h a highe le el o
p o ein syn hesis, po en ially ac i a ed by he exogenous adminis a ion o bST. Howe e , in milk
soma ic cells and hai ollicles, i was no possible o es ablish a ela ionship be ween he exp ession o
his gene and bST ea men . The MFGE8 gene is an essen ial ac o o a enua ing in lamma ion and
inhibi ing in lammasome-induced IL-1
β
p oduc ion [
28
]. As such, i is ema kable ha , while TNF and
IL-1
β
exp essions we e up egula ed in blood samples, MFGE8 was down egula ed in ea ed cows on
he hi d day. These esul s highligh he in e se ela ionship be ween he exp essions o hese genes.
Due o i s ole in a enua ing in lamma ion, MFGE8 could be down egula ed as an in lamma o y
esponse o he ex e nal adminis a ion o bST.
In he case o si uin 2 (SIRT2), his gene was up egula ed on he hi d day in he bST g oup o
milk soma ic cells, and no di e ences we e obse ed in he o he wo ma ices. SIRT2 is closely ela ed
o cell ac i i y, and i is in ol ed in he cell cycle. SIRT2 is equi ed o no mal mi o ic p og ession
and he p e en ion o ch omosomal ins abili y. SIRT2 le els a e g ea ly inc eased du ing mi osis, and
i s inhibi ion in e e es wi h cell-cycle p og ession [
29
]. This esul again highligh s he in luence o
exogenous bST on cell-cycle egula ion. Finally, TDP52L2 and LTF we e down egula ed in he bST
g oup on he nin h day, and no bST in luence o exp ession was de ec ed in he o he wo ma ices
es ed. These da a highligh he in luence o ma ices in he di e ences obse ed be ween wo g oups
using gene exp ession, and he impo ance o selec ing genes o he sc eening panel acco ding he
ma ix ha is analyzed.
The ansc ip ional modi ica ions a e bST adminis a ion we e in luenced by bo h ime and
he ma ix used. Blood and milk soma ic cells a e good candida es o be used as ou ine ma ices
o sc eening pu poses. In he case o hai ollicles, IGF-1R and CCND1 we e up egula ed a e bST
adminis a ion. Howe e , wo genes alone a e seemingly no enough o de ec bST adminis a ion.
Howe e , he simul aneous analysis o mul iple ma ices can be combined. Based on he esul s
obse ed ac oss he di e en ma ices, IL-1
β
,TNF,IGF-1R, and CCND1 a e good candida es o be
included in a sc eening panel. Al hough he ansc ip ion o EEF1G and MFGE8 was in luenced by
bST in blood samples, hey we e up egula ed in milk soma ic cells be o e ho mone adminis a ion,
and hei use in a panel should only be conside ed in blood ma ices. I is impo an o no e ha , in
he p esen ed s udy, con ol animals we e no injec ed wi h excipien s o Lac o opina
®
, as i s exac
composi ion is no decla ed by he manu ac u e . On he o he hand, in eal- a m condi ions, con ol
animals a e no ea ed wi h excipien s. None heless, i could be in e es ing o p epa e excipien
injec ions o a known composi ion o con ol animals in any u u e esea ch ocused on bST analysis.
OpenA ay
®
echnology allows he simul aneous analysis o a ious genes in a wide numbe o
samples [
30
]. The e o e, in ou ine con ols, h ee ma ices can be collec ed om he same a m and
can be analyzed simul aneously. The o e exp ession o a ious selec ed genes in di e en ma ices
can be a good indica ion o bST adminis a ion. Realis ically, i is no possible o know when bST
was adminis e ed in a a m. Ideally, e e y day du ing lac a ion should be con olled. Howe e ,
om a p ac ical poin o iew, his sugges ion is un ealis ic. The e o e, a andom collec ion o samples
( o ins ance, once pe week and on al e na ing week days) is p oposed. As bST is injec ed on a egula