Growth of rumen papillae in weaned calves is associated with lower expression of insulin‐like growth factor‐binding proteins 2, 3, and 6

Abstract

This study aimed to characterize the relationship between the growth of rumen papillae in calves and the mRNA expression of insulin‐like growth factor‐binding proteins (IGFBPs) in the rumen papillae. The length of rumen papillae, the mRNA expression of IGFBPs in rumen papillae by quantitative real‐time PCR, and the presence of insulin‐like growth factors I and II (IGF‐I and II) by immunohistochemistry (IHC) were analyzed in nine Holstein calves divided into three groups: suckling (2 weeks, n = 3), milk‐continued (8 weeks, n = 3), and weaned (8 weeks, n = 3). The length of rumen papillae was greater (p < 0.01) in weaned calves than in suckling and milk‐continued calves, whereas the expressions of IGFBP2, IGFBP3, and IGFBP6 genes were lower (p < 0.05) in the rumen papillae of weaned calves than in milk‐continued calves. Thus, rumen papillae length and IGFBP2, 3, and 6 expressions were negatively correlated. The IHC analysis showed that IGF‐I and IGF‐II were present in the rumen epithelium of calves. These results suggested that the growth of rumen papillae after weaning is associated with the induction of IGFs by the low levels of IGFBP2, IGFBP3, and IGFBP6.

Full text

Anim Sci J. 2019;90:1287–1292. wileyonlinelibrary.com/journal/asj 
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© 2019 Japanese Society of Animal Science
1 | INTRODUCTION
The rumen is an essential organ in ruminants as it plays major roles
in nutrient metabolism, uptake, and transpor tation (Roh et al., 20 07;
Roh, Suzuki, Gotoh, Tatsumi, & Katoh, 2016; Tamate, McGilliard,
Jacobson, & Getty, 1962). Growth of rumen papillae increases the
surface area of rumen as well as the absorption of volatile fatty
acids (VFA: acetic acid, propionic acid, butyric acid, among others).
Therefore, understanding the mechanism of grow th of rumen papil‐
lae is important for devising strategies to improve ruminant produc‐
tivity. It is known that rumen papillae develop remarkably when feed
is changed from milk to roughage or concentrated diet (Stobo, Roy,
& Gaston, 1966). Growth of rumen papillae is induced by VFA, and
butyric acid is the most potent activator of rumen papillae growth
(Gorka et al., 2011; Kato et al., 2011; Sakata & Tamate, 1978; Shen et
al., 2004). However, the molecular mechanism regulating the growth
of rumen papillae remains unclear.
Six isoforms of insulin‐like growth factor‐binding proteins
(IGFBPs) are known to regulate the proliferation and differenti‐
ation of epithelial cells in some tissues by controlling the activity
of insulin‐like growth factor I (IGF‐I) or insulin‐like growth factor II
(IGF‐II) (Firth & Baxter, 2002). It has been reported that mRNA ex‐
pression of IGFBP3 and IGFBP6 are downregulated while IGFBP5
is upregulated by grain‐induced rumen acidosis, which causes
damage to rumen epithelial cells in non‐lactating Holstein cows
(Steele, Alzahal, Walpole, & McBride, 2012; Steele et al., 2011;
Steele, Dionissopoulos, AlZahal, Doelman, & McBride, 2012). Our
previous study using comparative transcriptome analysis of rumen
Received:22January2019
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Revised:16May2019
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Accepted:4June2019
DOI : 10.1111 /asj .13270
ORIGINAL ARTICLE
Growth of rumen papillae in weaned calves is associated with
lower expression of insulin‐like growth factor‐binding proteins
2, 3, and 6
KokiNishihara1 | Yutaka Suzuki2 |DahyeKim1 |SanggunRoh1,3
1Labor atory of Animal Physiolog y, Graduate
School of Agricultural Science , Tohoku
University, Sendai, Miyagi‐ken, Japan
2Research Faculty of Agriculture, Hokkaido
University, Sapporo Hokkaido, Japan
3Laboratory of Function and Development
Science of Livesto ck Produ ction , Graduate
School of Agricultural Science , Tohoku
University, Sendai, Miyagi‐ken, Japan
Correspondence
Sanggun Roh, Laborato ry of Animal
Physiology, Graduate School of Agricultural
Science, Tohoku University, Sendai, Miyagi‐
ken 980‐0842, Japan.
Email: sanggun.roh@tohoku.ac.jp
Funding information
Rural Development Administration; Japan
Societ y for the Promotio n of Science, Grant /
Award Number: 18H02325 and 19J12823
Abstract
This study aimed to characterize the relationship between the growth of rumen papil‐
lae in calves and the mRNA expression of insulin‐like growth factor‐binding proteins
(IGFBPs) in the rumen papillae. The length of rumen papillae, the mRNA expression
of IGFBPs in rumen papillae by quantitative real‐time PCR, and the presence of in‐
sulin‐like growth factors I and II (IGF‐I and II) by immunohistochemistry (IHC) were
analyzed in nine Holstein calves divided into three groups: suckling (2 weeks, n = 3),
milk‐continued (8 weeks, n = 3), and weaned (8 weeks, n = 3). The length of rumen
papillae was greater (p < 0.01) in weaned calves than in suckling and milk‐continued
calves, whereas the expressions of IGFBP2, IGFBP3, and IGFBP6 genes were lower
(p < 0.05) in the rumen papillae of weaned calves than in milk‐continued calves. Thus,
rumen papillae length and IGFBP2, 3, and 6 expressions were negatively correlated.
The IHC analysis showed that IGF‐I and IGF‐II were present in the rumen epithelium
of calves. These results suggested that the growth of rumen papillae after weaning
is associated with the induction of IGFs by the low levels of IGFBP2, IGFBP3, and
IGFBP6.
KEY WORDS
insulin‐like growth factor‐binding proteins, insulin‐like growth factors, rumen papillae growth

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papillae in suckling and weaned c alves (Nishihara et al., 2018)
showed that IGFBP2, IGFBP3, and IGFBP6 gene expressions were
lower while that of IGFBP5 was higher in the rumen papillae of
weaned calves than in the rumen papillae of suckling calves. There
is a possibility that IGFBPs are involved in the growth of rumen
papillae through controlling the activity of IGF‐I or IGF‐II in rumen
epithelial cells. Therefore, in the present study, we aimed to reveal
the relationship between the growth of rumen papillae and the
expression of IGFBP genes, and to investigate the presence and
localization of IGF‐I and IGF‐II, the targets of IGFBPs, in the rumen
papillae of Holstein calves.
2 | MATERIALSANDMETHODS
2.1 | Ethicsstatement
The experiment on Holstein calves was conducted in accordance
with the Guidelines for the Tohoku University, and the experimen‐
tal procedures were approved by the Animal C are Committee of
Tohoku University (2016AgA‐004).
2.2 | Animals
Nine Holstein calves, bred at the Graduate School of Agricultural
Science, Tohoku University, Sendai, Japan were assigned to three
experimental groups (suckling [n = 3], milk‐continued [n = 3], and
weaned [n = 3]) in a random design. All calves were raised using a
milk replacer in individual teated buckets (Calftop EX, Zenrakuren;
total digestible nutrients (TDN) > 103%, crude protein (CP) > 28%,
crude fat > 15%). All calves were fed at 09:00 and 16:00 hr. Calves in
the suckling group were slaughtered at 2 weeks of age (n = 3), while
calves in the milk‐continued group were slaughtered at 8 weeks of
age (n = 3). Calves in these two groups were raised only by artificial
suckling without feeding starter. Calves in the weaned group (n = 3)
were fed calf star ter (New make star, Zenrakuren; TDN > 72.0%,
CP > 18.0%, crude fat > 2%) at 09:00 hr from 1 week of age, weaned
at 7 weeks of age, and slaughtered at 8 weeks of age. Calves were
allowed ad libitum access to water. The amounts of milk replacer and
starter are described in Table S1.
2.3 | Samplecollection
Rumen tissue samples were collected as previously described (Kato
et al., 2016; Nishihara et al., 2018). After slaughter, rumen papillae
tissues (approximately 1 × 1 cm) were collected from the ventral
cranial sac. The epithelial layer was manually separated from the
muscular layer using surgical scissors, and rinsed with phosphate
buffered saline to remove residual feed particles. All tissue samples
werefrozenimmediatelyinliquidnitrogenandstoredat−80°Cuntil
further analysis. Samples for hematoxylin and eosin (HE) staining
and immunohistochemistr y (IHC) were fixed in Bouin's solution and
embedded in paraffin.
2.4 | Measurementofthelengthofrumenpapillae
Rumen papillae samples were sectioned (3 μm thickness) and then de‐
paraf finized in xyle ne and dehydrated in a s eries of ethanol . Thereafter,
HE staining was per formed, and each tissue sec tion (approximately
1 × 1 cm) was photographed under a Leica S9E stereo microscope
(Leica). The length of all papillae in the rumen tissue of each calf was
determined using cellScan (Olympus) (n = 9–21 from each calf).
2.5 | Quantitativereal‐timePCR
Total RNA was extracted from the rumen papillae tissues using
RNAiso plus (TaKaRa‐Bio), and 500 ng of total RNA was reverse
transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser
Perfect Real Time (TaKaRa‐Bio). Gene expression analysis was
performed by quantitative real‐time PCR (qRT‐PCR) using SYBR®
Premix ExTaq™ II (Tli RNaseH Plus) (TaKaRa‐Bio) in a Thermal Cycler
Dice® Real Time System II ( TaKaRa‐Bio). Primer sequences are de‐
scribed in Table S2; PCR efficiencies were 0.8–1.2 for each primer
pair, as checked by drawing standard curves using serial dilutions of
pooled cDNAs. Beta actin (ACTB), 18S, glyceraldehyde‐3‐phosphate
dehydrogenase, and vacuolar protein sorting protein 4 homolog
A were used as reference genes. Relative transcript expression
was calculated by the modified 2−ΔΔCt method (Suzuki et al., 2016;
Vandesompele et al., 2002) and represented as relative values to
that of the suckling group.
2.6 | Immunohistochemistry
Tissue sections from weaned calves were deparaffinized in xylene, re‐
hydrated withalcohol, autoclaved at 120°C for 10min in 0.01 M cit‐
ric acid buffer (pH 6.0) to retrieve antigens for use in IHC , and finally
cooled to 25°C.After washing in phosphatebuffered saline + Tween
20 (PBST), endogenous peroxidase activity was quenched in 1% hy‐
drogen peroxide (H2O2) in methanol. Tissue sections were blocked
with a buffer containing 5% normal goat serum diluted in 1 × PBST.
Thereaf ter,tissuesectionswereincubated at 4°Covernight withrab‐
bit anti‐IGF‐I antiserum (1:1,000, AFP4892898; National Hormone &
Peptide Program) or rabbit polyclonal anti‐IGF‐II IgG (1:100, ab9574;
Abcam) as the first antibody. After washing in PBST, the tissue sections
were incubated with horseradish peroxidase‐labeled goat anti‐rabbit
IgG(Nic hi re iB io sc ie nc es)at 25°Cf or 1hr.Af terwashingwi th PB ST,th e
immunocomplex was visualized using 3,3‐diaminobenzidine substrate
(Nichirei Biosciences). Hematoxylin was used for counter staining for
10 min. The tissue sections were dehydrated using alcohol and xylene.
Slides were mounted with Softmount (FUJIFILM Wako Pure Chemical
Corporation). To evaluate IGF‐I and II antibodies, liver tissue was used
as a positive control. The negative control comprised rumen tissue
sections without addition of IGF‐I and II antibodies. IHC st aining was
performed with IGF‐I and II antibodies pre‐incubated with its antigen
as an antibody absorption test. Anti IGF‐I and IGF‐II antibody were in‐
cubated with recombinant bovine IGF‐I (RP1330; Kingfisher Biotech,

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NISHIHA RA et A l.
Inc.) and IGF‐II protein (RP1547B; King fisher Biotech, Inc.) for over‐
night, respectively. The ration of the molar concentration of antibody
to recombinant protein was 1–20. Normal rabbit serum was added to
confirm non‐specific binding of rabbit serum or IgG to rumen tissue.
2.7 | Statisticalanalyses
All data are expressed as means ± standard error of the mean (SEM).
Differences in mRNA expression and length of rumen papillae were
analyzed using one‐way analysis of variance and Tukey's multiple
comparisons test. Correlations between the length of rumen papil‐
lae and mRNA expressions of IGFBP2, IGFBP3, and IGFBP6 were
determined using Pearson's product‐moment correlation coef‐
ficient. All statistical analyses were per formed in R version 3.5.1
(https ://www.r‐proje ct.org) considering p < 0.05 as the significance
threshold.
3 | RESULTS
3.1 | Morphologicaldifferencesamongtherumen
papillae of suckling, milk‐continued, and weaned
calves
Figure 1a,b,c show morphological differences in the rumen papillae
of the three different groups. Growth of rumen papillae was ob‐
served in weaned calves (Figure 1c), but not in suckling and milk‐
continued calves (Figure 1a,b). The lengths of rumen papillae were
309.1 ± 36.7, 325.4 ± 65.2, and 1,491.1 ± 243.1 μm in suckling, milk‐
continued, and weaned groups, respectively. The length of rumen
papillae of weaned calves was greater (p < 0.01) than that of suckling
and milk‐continued calves.
3.2 | ChangesintheexpressionofIGFBPgenesand
IGF‐I,IGF‐II,andrespectivereceptorgenesinthe
rumen papillae of suckling, milk‐continued, and
weaned calves
To determine the effect of weaning using solid feed on the expres‐
sions of IGFBP genes, we examined mRNA transcript levels of these
genes in the rumen papillae of suckling, milk‐continued, and weaned
calves. Expressions of the six IGFBP mRNAs in the rumen papillae
from each group are shown in Figure 2. No expression was detected
for IGFBP1 in the rumen papillae of each group, and the expressions
of IGFBP4 and IGFBP5 were not altered in weaned calves compared
to that in suckling claves. The expressions of IGFBP2, IGFBP3, and
IGFBP6 mRNA were lower (p < 0.05, p < 0.05, and p < 0.01, respec‐
tively) in the rumen papillae of weaned calves than in the rumen pa‐
pillae of milk‐continued calves. Gene expression of IGF‐I, IGF‐II, and
their receptors were not different across groups (Figure 3).
3.3 | Correlationsbetweenthelengthofrumen
papillae and IGFBP2, IGFBP3, and IGFBP6 expressions
To characterize the relationship between the length of rumen papil‐
lae and mRNA expression of IGFBPs in the rumen papillae of calves,
we evaluated the correlations between the mean leng th of rumen
FIGURE 1 Morphology of rumen
papillae of (a) suckling, (b) milk‐continued,
and (c) weaned c alves by Hematoxylin
(HE) staining (scale bar = 500 μm)
(a) (c)
(b)
FIGURE 2 Relative mRNA expression of IGFBP1, IGFBP2,
IGFBP3, IGFBP4, IGFBP5, and IGFBP6 in the rumen papillae of
suckling and weaned c alves. Relative mRNA expressions were
analyzed by qRT‐PCR and are shown as fold changes relative to the
expression in suckling calves. N.D., not detected. Different letters
indicate significant differences between each group (p < 0.05).
IGFBP, insulin‐like growth factor‐binding protein; qRT‐PCR,
quantitative real‐time PCR

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papillae and the relative expression of IGFBPs in each c alf (n = 9). The
length of rumen papillae was negatively correlated with the relative
mRNA expression of IGFBP2 (r=−0.78,p = 0.01), IGFBP3 (r=−0.73,
p = 0.02), and IGFBP6 (r =−0.73, p = 0.02) ( Table 1). There was no
correlation between the length of rumen papillae and the relative
mRNA expression of IGFBP4 or IGFBP5 (data not shown).
3.4 | LocalizationofIGF‐IandIGF‐II
Insulin‐like growth factor I and IGF‐II were present in the rumen
epithelium of calves (Figure 4a,b). Liver tissue, where IGF‐I and
II were mainly expressed, was used to validate each antibody.
Figure 4d,e show that IGF‐I and II were present in liver tissue. By
the antibody absorption test, the stainability of IGF‐I and II an‐
tibody was mostly reduced by each antigen (Figure S1a,b,c,d). In
addition, there was no specific reaction by normal rabbit serum in
rumen tissue (Figure S1e).
4 | DISCUSSION
To the best of our knowledge, the present study is the first to char‐
acterize the relationship bet ween the growth of rumen papillae and
the mRNA expression of IGFBPs in the rumen papillae of calves. In
the present study, rumen papillae grew in weaning calves, but not in
suckling and milk‐continued calves indicating that the intake of solid
feed during weaning contributes to the growth of rumen papillae
with changes in IGFBPs mRNA expression.
In a previous study, differences in the expression of IGFBP2,
IGFBP3, IGFBP5, and IGFBP6 genes in rumen papillae were ob‐
served between suckling (5 weeks of age) and weaned (15 weeks
of age) Japanese Black calves (Nishihara et al., 2018). However, in
the present study, there were no dif ferences in the mRNA expres‐
sion of IGFBP5 in rumen papillae among suckling, milk‐continued,
and weaned Holstein calves. In addition, the length of rumen papil‐
lae was correlated with IGFBP2, IGFBP3, and IGFBP6 mRNA ex‐
pression, but not with IGFBP5 mRNA expression. Bec ause IGFBPs
control the release of IGFs to their receptors in several tissues,
overexpression of IGFBP2 inhibited cell proliferation in human
embryonic kidney fibroblast (Hoflich, Lahm, Blum, Kolb, & Wolf,
1998) and of intestinal epithelial cells (Corkins, Vanderhoof, Slentz,
MacDonald, & Park, 1995), and IGFBP3 inhibited the role of IGFs in
pulmonary artery smooth muscle cells proliferation (Cheng, Zhang,
Zhang, He, & Wang, 2017). In addition, IGFBP4 knockout mice had
greater small intestinal growth and deeper crypts than control mice
(Austin, Imam, Pintar, & Brubaker, 2015), and IGFBP6 inhibited
FIGURE 3 Relative mRNA expression of IGF‐I, IGF‐IR, IGF‐II,
and IGF‐IIR in the rumen papillae of suckling and weaned calves.
Relative mRNA expressions were analyzed by qRT‐PCR and are
shown as fold changes relative to the expression in suckling calves.
IGF‐I, insulin‐like grow th factor I; IGF‐II, insulin‐like growth factor
II; qRT‐PCR, quantitative real‐time PCR
TABLE 1 Correlations between the length of rumen papillae and
IGFBP2, IGFBP3, and IGFBP6 expressions (n = 9)
Variable r p‐value
IGFBP2 −0.79 0.01
IGFBP3 −0.73 0.02
IGFBP6 −0.73 0.02
Abbreviation: IGFBP, insulin‐like growth factor‐binding protein.
FIGURE 4 Localization of IGF‐I and
IGF‐II in rumen and liver tissues in weaned
calves by immunohistochemistry. Nuclei
were stained using hematoxylin. (a) IGF‐I
and (b) IGF‐II in rumen papillae. (c) and (f )
IGF‐I (or IGF‐II) immunohistochemistry
in rumen papillae and liver tissue without
the first antibody as the negative control.
(d) IGF‐I and (e) IGF‐II in liver tissues as
positive controls. Scale bar = 100 μm.
IGF‐I, insulin‐like grow th factor I; IGF‐II,
insulin‐like growth factor II
(a)
(d) (e) (f)
(b) (c)

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cell proliferation and/or differentiation of rat myoblast s and colon
cancer cell lines (Bach, Salemi, & Leeding, 1995; Kim, Kang, et al.,
2002; Kim, Schaffer, Kang, Macdonald, & Park, 20 02; Leng, Leeding,
Whitehead, & Bach, 2001). The presence of IGF‐I and II in rumen
epithelial cells was similar to that observed in uterine endometrial
epithelial cells and intestinal epithelial cells (Firth & Baxter, 2002;
Kapur, Tamada, Dey, & Andrews, 1992; Park et al., 1992). Our previ‐
ous repor t using RNA sequencing analysis showed that there were
no differences in the expressions of IGF‐I and IGF‐IR between suck‐
ling (5 weeks of age) and weaned (15 weeks of age) Japanese black
calves (Nishihara et al., 2018). IGF‐I directly regulates cell prolifera‐
tion in cultured rumen epithelial cells of lambs (Baldwin, 1999). Gene
expression of IGF‐I in rumen papillae was lower in lambs fed milk and
starter diet than in lambs fed milk only, while the expression of IGF‐
IR and leng th of rumen papillae were higher in the first than in the
latter (Sun, Mao, Zhu, & Liu, 2018). However, there were no changes
in the expressions of IGF genes and corresponding receptor genes
in the Holstein calves examined in the present study, indicating that
IGFs and their receptors have no direct effects on the growth of
rumen papillae. Therefore, the downregulation of IGFBP2, IGFBP3,
and IGFBP6, which have inhibitory effec ts on IGFs, might potentiate
the growth of rumen papillae by regulating epithelial cell prolifera‐
tion in calves weaned by solid feed.
Our previous study suggested that IGFBP5 was upregulated
by weaning and might be necessary for the proliferation of rumen
epithelial cells of Japanese Black calves (Nishihara et al., 2018).
However, in the present stud y, higher expression of IGFBP5 in rumen
papillae was not obser ved in weaned Holstein calves compared with
suckling and milk‐continued calves. This result indicated that the
expressions of IGFBP5 is not associated with the growth and prolif‐
eration of rumen papillae in Holstein calves. The IGFBP5 present in
rumen papillae was upregulated by subacute ruminal acidosis, dam‐
aging rumen epithelial cells in Holstein cattle (Steele, Alzahal, et al.,
2012; Steele et al., 2011; Steele, Dionissopoulos, et al., 2012). The
decrease in the ratio of dietar y neutral detergent fiber to starch in‐
creased mRNA expression of IGFBP5 in dairy cows (Ma et al., 2017).
Expression of IGFBP5 mRNA was higher in concentrate‐fed sheep
(Jing et al., 2018). These data suggested that higher expression of
IGFBP5 in rumen papillae is associated with the changes of the fer‐
mentation in rumen. Higher expression of IGFBP5 in rumen papillae
might be necessary for adapt ation to highly fermentable diet causing
disruption of rumen epithelial barrier (Greco et al., 2018; Meissner et
al., 2017; Steele et al., 2011). IGFBP5 induced monocyte migration
in lung epithelium in mice (Yasuoka, Yamaguchi, & Feghali‐Bostwick,
2009). IGFBP5 also has a role to enhance the adhesion in cultured
mammar y epithelial cells in mice (Vijayan et al., 2013). IGFBP5 might
be highly expressed by ferment able diet feeding in rumen papillae
to enhance adhesion of epithelial cells or to promote migration of
immune cells for protecting rumen epithelial cells.
Taken together, our findings suggest that the downregulation of
IGFBP2, IGFBP3, and IGFBP6 might induce IGFs in rumen epithe‐
lial cells and promote the growth of rumen papillae in young calves
weaned with solid feed.
ACKNOWLEDGMENTS
This work was partly suppor ted by JSPS K AKENHI (grant number
18H02325 and 19J12823). This work was also carried out with the
support of "Cooperative Research Program for Agriculture Science
and Technology Development (Project No. PJ01439502)" Rural
Development Administration, Republic of Korea.
ORCID
Sanggun Roh https://orcid.org/0000‐0003‐1092‐5691
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