Vailati-Riboni et al.

Journal of Animal Science and Biotechnology (2017) 8:17

DOI 10.1186/s40104-017-0147-7

RESEARCH Open Access

Supplemental Smartamine M in higher-

energy diets during the prepartal period
improves hepatic biomarkers of health
and oxidative status in Holstein cows
Mario Vailati-Riboni1, Johan S. Osorio1,4, Erminio Trevisi2, Daniel Luchini3 and Juan J. Loor1*

Abstract
Background: Feeding higher-energy prepartum is a common practice in the dairy industry. However, recent data
underscore how it could reduce performance, deepen negative energy balance, and augment inflammation and
oxidative stress in fresh cows. We tested the effectiveness of rumen-protected methionine in preventing the
negative effect of feeding a higher-energy prepartum. Multiparous Holstein cows were fed a control lower-energy
diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), or were switched to a higher-energy
(OVE, 1.54 Mcal/kg DM), or OVE plus Smartamine M (OVE + SM; Adisseo NA) during the last 21 d before calving.
Afterwards cows received the same lactation diet (1.75 Mcal/kg DM). Smartamine M was top-dressed on the OVE
diet (0.07% of DM) from -21 through 30 d in milk (DIM). Liver samples were obtained via percutaneous biopsy
at -10, 7 and 21 DIM. Expression of genes associated with energy and lipid metabolism, hepatokines, methionine
cycle, antioxidant capacity and inflammation was measured.
Results: Postpartal dry matter intake, milk yield, and energy-corrected milk were higher in CON and OVE + SM
compared with OVE. Furthermore, milk protein and fat percentages were greater in OVE + SM compared with CON
and OVE. Expression of the gluconeogenic gene PCK1 and the lipid-metabolism transcription regulator PPARA was
again greater with CON and OVE + SM compared with OVE. Expression of the lipoprotein synthesis enzyme MTTP
was lower in OVE + SM than CON or OVE. Similarly, the hepatokine FGF21, which correlates with severity of negative
energy balance, was increased postpartum only in OVE compared to the other two groups. These results indicate
greater liver metabolism and functions to support a greater production in OVE + SM. At 7 DIM, the enzyme GSR
involved in the synthesis of glutathione tended to be upregulated in OVE than CON-fed cows, suggesting a greater
antioxidant demand in overfed cows. Feeding OVE + SM resulted in lower similar expression of GSR compared
with CON. Expression of the methionine cycle enzymes SAHH and MTR, both of which help synthesize methionine
endogenously, was greater prepartum in OVE + SM compared with both CON and OVE, and at 7 DIM for CON and
OVE + SM compared with OVE, suggesting greater Met availability. It is noteworthy that DNMT3A, which utilizes
S-adenosylmethionine generated in the methionine cycle, was greater in OVE and OVE + SM indicating higher-energy
diets might enhance DNA methylation, thus, Met utilization.
(Continued on next page)

* Correspondence: [email protected]
1
Mammalian NutriPhysioGenomics, Department of Animal Sciences and
Division of Nutritional Sciences, University of Illinois, Urbana, IL 61801, USA
Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 2 of 12

(Continued from previous page)

Conclusions: Data indicate that supplemental Smartamine M was able to compensate for the negative effect of
prepartal energy-overfeeding by alleviating the demand for intracellular antioxidants, thus, contributing to the increase
in production. Moreover Smartamine M improved hepatic lipid and glucose metabolism, leading to greater liver
function and better overall health.
Keywords: Energy, Methionine, Nutrigenomics, Transition period

Background targeted hepatic transcriptome analysis from transition

The transition period, defined as last 3 weeks prepartum cows fed prepartum either a control low energy, a
through 3 weeks postpartum, is one of the most import- higher-energy, or a higher-energy diet supplemented
ant stages of lactation in dairy cattle. Years of strong with rumen-protected Met. Production and immune re-
genetic selection and improvement have allowed modern sponses have been published elsewhere [25].
dairy cows to reach high production performance, both
in quantity and quality. However, this has made the tran- Methods
sition between late pregnancy to early lactation a signifi- Experimental design and dietary treatments
cant period of metabolic and immune challenges [1–3]. All procedures were approved by the Institutional
Because failure to adequately meet these challenges can Animal Care and Use Committee (IACUC) of the
compromise production, induce metabolic diseases, and University of Illinois. Complete details of the experimen-
increase rates of culling in early lactation [4], the man- tal design and animal management have been reported
agement of the transition cow remains a focal point for previously [25]. Briefly, 65 multiparous Holstein were
dairy producers. enrolled and completed the trail remaining healthy
Following the “steaming up” concept of RB Boutflour throughout the length of the study. All cows were fed ad
[5], transition cows during the dry period were first trad- libitum the same control lower-energy diet (CON; NEL =
itionally offered a high fiber/low energy density ration, 1.24 Mcal/kg DM; no Met supplementation) during the
to then increase the energy density of the ration with a far-off dry period (i.e., -50 to -21 d relative to parturition).
lower fiber content in the last month of gestation (i.e. Consequently, during the close-up period (i.e. -21 d to
“close-up” period). This early century practice is still em- calving), cows were randomly allocated to either a higher-
bedded in the modern dairy industry. However, multiple energy diet (OVE; NEL = 1.54 Mcal/kg DM), OVE plus
studies have consistently reported negative effects of Smartamine M (OVE + SM; Adisseo, NA) or remained
prepartum energy overfeeding on cow health and prod- on CON. The same basal lactation diet (NEL = 1.75
uctivity. Among these, prepartum hyperglycemia and Mcal/kg DM) was fed to all cows postpartum until d
hyperinsulinemia together with marked postpartum adi- 30 relative to parturition. Smartamine M was top-
pose tissue mobilization (i.e., greater blood NEFA con- dressed during the entire experiment over the OVE
centration) [6–11] have strong negative impact on or lactation diet from -21 through 30 d relative to
postpartal health indices [12–15]. parturition at a rate of 0.07% of offered DM. For the
Our general hypothesis was that supplementation with current study, only a subset of cows were considered for
rumen-protected methionine (Smartamine M, Adisseo blood biomarker (n = 10 per group) and hepatic gene ex-
NA) could ameliorate the transition to lactation and the pression (n = 8 per group) analyses.
health status of the cows, while controlling and reducing
the negative effects of prepartal excess energy. In fact, Blood sampling and biomarker analysis
methionine (Met) itself was able to increase both quan- Blood was sampled at -26, -21, -10, 7, 14 and 21 d rela-
tity and quality of production [16, 17], controlling the tive to parturition by coccygeal venipuncture using evac-
inflammatory and the oxidative stress status that uated tubes (BD Vacutainer; BD and Co., Franklin Lakes,
characterize the transition period [18–20]. These out- NJ) containing either clot activator or lithium heparin
comes are partly due to Met’s ability to enhance liver for serum and plasma, respectively. Blood was used for
function, reducing triacylglycerol accumulation and im- determination of (i) metabolic biomarkers: cholesterol,
proving the metabolic capacity of the liver to orchestrate creatinine, growth hormone (GH), insulin-like growth
the metabolic transition into lactation [16–20]. Further- factor 1 (IGF-1), leptin, urea; (ii) liver health biomarkers:
more, Met itself, and several of its metabolites, display albumin, bilirubin, ceruloplasmin, gamma-glutamyl-
an immunonutritional role both in humans [21–24] and transpeptidase (GGT), glutamic oxaloacetic transamin-
in dairy cows [16]. Therefore, in the present study we ase (GOT), haptoglobin, interleukin 6, serum amyloid A
used serum and plasma biomarkers coupled with (SAA); (iii) and oxidative status biomarkers: β-carotene,
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 3 of 12

glutathione, nitric oxides (NOx, NO2, NO3), paraoxo- microsomal triglyceride transfer protein (MTTP), peroxi-
nase, antioxidant capacity (oxygen radical absorbance some proliferator activated receptor α (PPARA), solute
capacity, ORAC), total reactive oxygen metabolites carrier family 22 member 5 (SLC22A5), trimethyllysine
(ROM), tocopherol. hydroxylase, ε (TMLHE); (iii) hepatokines: angiopoietin like
Concentration of albumin, cholesterol, bilirubin, cre- 4 (ANGPTL4), fibroblast growth factor 21 (FGF21); (iv) the
atinine, urea, GOT, and GGT were assessed using kits methionine cycle: betaine–homocysteine S-methyl
purchased from Instrumentation Laboratory (Lexington, transferase (BHMT), betaine–homocysteine S-methyl
MA) using a clinical auto-analyzer (ILAB 600, Instru- transferase 2 (BHMT2), DNA (cytosine-5-)-methyl-
mentation Laboratory). Concentrations of ROM were transferase 1 (DNMT1), DNA (cytosine-5-)-methyl-
analyzed with the d-ROMs-test, purchased from Diacron transferase 3 α (DNMT3A), methionine adenosy
(Grosseto, Italy). Concentrations of haptoglobin, cerulo- ltransferase 1A (MAT1A), 5-methyltetrahydrofolate-
plasmin, paraoxonase and NOx were analyzed using the homocysteine methyltransferase (MTR), phosphatidyl-
methods previously described [26–28], adapting the pro- ethanolamine N-methyltransferase (PEMT), S-adenosyl
cedures to a clinical auto-analyzer (ILAB 600, Instru- homocysteine hydrolase (SAHH); (v) the antioxidant
mentation Laboratory). SAA and ORAC determinations system: cystathionine-beta-synthase (CBS), cysteine sulfi-
were performed using the Synergy 2 Multi-Detection nic acid decarboxylase (CSAD), cystathionine gamma-
Microplate Reader (BioTek Instruments, Inc., Winooski, lyase (CTH), glutamate-cysteine ligase catalytic subunit
VT). SAA concentration was assessed with a commercial (GCLC), glutathione peroxidase 1 (GPX1), glutathione re-
ELISA immunoassay kit (Tridelta Development Ltd., ductase (GSR), glutathione synthetase (GSS), superoxide
Maynooth, Co. Kildare, Ireland), while ORAC was deter- dismutase 1, soluble (SOD1), superoxide dismutase 2,
mined measuring the fluorescent signal from a probe mitochondrial (SOD2); (vi) and the inflammatory re-
(fluorescein) that decreases in the presence of radical sponse: ceruloplasmin (CP), haptoglobin (HP), nuclear
damage [29]. Quantification of GH, IGF-1, and leptin factor κB subunit 1 (NFKB1), retinoid X receptor α
concentration was as previously described [14]. Bovine (RXRA), serum amyloid A2 (SAA2), suppressor of
IL-6 (Cat. No. ESS0029; Thermo Scientific, Rockford, IL) cytokine signaling 2 (SOCS2), signal transducer and
plasma concentration was determined using commercial activator of transcription 3 (STAT3), signal transducer
ELISA kits, while plasma vitamin A, vitamin E, and β- and activator of transcription 5B (STAT5B). Primer
carotene were extracted with hexane and analyzed by sequences and qPCR performances are reported in
reverse- phase HPLC using an Allsphere ODS-2 col- Additional file 1.
umn (3 μm, 150 × 4.6 mm; Grace Davison Discovery
Sciences, Deerfield, IL), a UV detector set at 325 nm Statistical analysis
(for vitamin A), 290 nm (for vitamin E), or 460 nm After normalization with the geometric mean of the
(for β-carotene), and 80:20 methanol:tetrahydrofurane internal control genes, qPCR data were log2 trans-
as the mobile phase. formed prior to statistical analysis to obtain a normal
distribution. Statistical analysis was performed with
Hepatic gene expression analysis SAS (v9.3). Both datasets (blood and qPCR) were sub-
Liver tissue was harvested via percutaneous biopsy jected to ANOVA and analyzed using repeated mea-
under local anesthesia at -10, 7 and 21 d relative to par- sures ANOVA with PROC MIXED. The statistical
turition. Tissue samples were immediately snap frozen model included diet (D; CON, OVE, and OVE + SM),
in liquid nitrogen and then stored at -80 °C. Complete time (T; d -26, -21, -10, 7, 14, and 21 for blood bio-
information about RNA extraction and qPCR proce- markers, d -10, 7, and 21 for qPCR analysis) and
dures can be found in Additional file 1. Briefly, RNA their interaction (D*T) as fixed effect. Cow, nested
samples were extracted from the frozen tissue and used within treatment, was the random effect. For blood
for cDNA synthesis using established protocols in our data, data pre-treatment at d-26 relative to partur-
laboratory [30]. The qPCR performed was SYBR Green- ition, when available, were used as a covariate. The
based, using a 6-point standard curve. Genes selected Kenward-Roger statement was used for computing
for transcript profiling are associated with (i) energy me- the denominator degrees of freedom, while spatial
tabolism: insulin like growth factor-1 (IGF1), pyruvate power was used as the covariance structure. Data
carboxylase (PC), phosphoenolpyruvate carboxykinase 1 were considered significant at a P ≤ 0.05 using the
(PCK1), pyruvate dehydrogenase kinase 4 (PDK4); (ii) PDIFF statement in SAS. For ease of interpretation,
fatty acid metabolism: acyl-CoA oxidase 1 (ACOX1), expression data reported in Table 1 and Fig. 1 are the
apolipoprotein B (APOB), γ-butyrobetaine hydroxylase 1 log2 back-transformed LSM that resulted from the
(BBOX1), carnitine palmitoyltransferase 1A (CPT1A), 3- statistical analysis. Standard errors were also ad-
hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), equately back-transformed.
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 4 of 12

Table 1 Effect of feeding a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), a
higher-energy (1.54 Mcal/kg DM) diet without (OVE) rumen-protected methionine during the last 21 d before calving, or OVE plus
rumen-protected methionine (Smartamine M; OVE + SM; Adisseo NA) from -21 d before calving through the first 30 d postpartum
on hepatic gene expression (relative mRNA abundance, log2 back-transformed LSM) in Holstein cows
Diet1 P-value3
2
CON OVE OVE + SM SE D T D*T
Energy metabolism
IGF1 1.91 1.97 2.41 0.22 0.19 <.0001 0.17
PC 0.24 0.23 0.20 0.02 0.24 <.0001 0.09
a b a
PCK1 0.33 0.25 0.31 0.02 0.03 0.10 0.59
PDK4 0.34 0.31 0.78 0.37 0.35 0.03 0.98
Fatty acid oxidation, Lipoprotein and Cholesterol synthesis
ACOX1 1.34 1.21 1.27 0.07 0.41 0.75 0.61
APOB 1.93 1.67 1.82 0.11 0.22 0.32 0.42
BBOX1 0.39 0.36 0.38 0.02 0.61 0.04 0.96
CPT1A 0.13 0.13 0.14 0.01 0.89 <.0001 0.49
HMGCS2 1.13 1.00 0.93 0.10 0.32 0.22 0.56
a a b
MTTP 1.48 1.50 1.28 0.08 0.05 0.02 0.84
PPARA 0.43 0.40 0.46 0.02 0.16 <.0001 0.03
SLC22A5 2.89 2.92 3.08 0.29 0.88 <.0001 0.14
TMLHE 0.42 0.40 0.41 0.02 0.68 0.003 0.39
Hepatokines
ANGPTL4 0.01 0.01 0.02 0.002 0.34 <.0001 0.03
FGF21 0.27 0.13 0.22 0.07 0.15 0.005 0.0006
Methionine cycle and methylation
BHMT 1.13 0.96 1.02 0.11 0.54 0.002 0.70
BHMT2 0.38 0.45 0.37 0.04 0.20 0.21 0.57
DNMT1 0.02 0.02 0.02 0.001 0.53 0.03 0.64
DNMT3A 0.99a 1.26b 1.20b 0.09 0.04 0.05 0.91
MAT1A 1.46 1.55 1.52 0.08 0.71 0.09 0.86
MTR 0.05a 0.03b 0.04ab 0.002 0.01 0.31 0.65
PEMT 0.33 0.29 0.30 0.02 0.31 0.07 0.95
SAHH 1.39 1.25 1.39 0.06 0.17 0.0003 0.0009
Antioxidant system
CBS 1.41 1.66 1.51 0.11 0.27 0.08 0.93
CSAD 0.23 0.26 0.22 0.04 0.64 0.0004 0.36
CTH 0.45 0.46 0.45 0.02 0.93 0.24 0.86
GCLC 0.06 0.06 0.05 0.004 0.80 0.01 0.35
GPX1 1.39 1.37 1.39 0.10 0.99 0.04 0.77
GSR 0.28 0.30 0.26 0.02 0.11 <.0001 0.11
GSS 0.45 0.49 0.46 0.02 0.34 0.78 0.95
SOD1 3.56 3.64 3.40 0.12 0.27 0.07 0.52
SOD2 4.06 4.08 4.10 0.21 0.99 0.92 0.53
Inflammatory response
CP 2.48 2.13 2.37 0.22 0.49 0.003 0.85
HP 0.18 0.13 0.14 0.09 0.89 0.01 0.48
NFKB1 2.29 2.14 2.48 0.14 0.25 0.04 0.83
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 5 of 12

Table 1 Effect of feeding a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), a
higher-energy (1.54 Mcal/kg DM) diet without (OVE) rumen-protected methionine during the last 21 d before calving, or OVE plus
rumen-protected methionine (Smartamine M; OVE + SM; Adisseo NA) from -21 d before calving through the first 30 d postpartum
on hepatic gene expression (relative mRNA abundance, log2 back-transformed LSM) in Holstein cows (Continued)
RXRA 0.57 0.56 0.58 0.03 0.86 0.20 0.66
SAA2 0.01 0.01 0.02 0.002 0.68 0.02 0.95
SOCS2 2.09 1.84 2.26 0.24 0.44 0.16 0.60
STAT3 1.38 1.42 1.39 0.11 0.96 0.05 0.43
STAT5B 2.30 2.26 2.45 0.07 0.15 0.002 0.07
1
Prepartum dietary treatment: CON = control energy, OVE = moderate energy, OVE + SM = OVE supplemented with rumen-protected methionine (Smartamine M,
Adisseo Inc.)
2
SE = greatest standard error of the mean
3
D = diet, T = time, D*T = diet by time interaction
a, b
Significant difference among dietary groups (P ≤ 0.05). Differences reported for genes with a significant (P ≤ 0.05) Diet effect

Results 0.05) concentration in OVE + SM cows compared with

Blood biomarkers CON at -21 and -10 d, and to a lower (P < 0.05) con-
Metabolism centration in OVE compared with CON at 14 d relative
Time affected all metabolic biomarkers (cholesterol, cre- to parturition. In the case of retinol, the interaction was
atinine, GH, IGF1, leptin, urea; T, P < 0.001). However, due the increasing (P < 0.05) concentration postpartum
no effect of diet or its interaction with time was detected from 7 to 21 d in OVE + SM cows, while in CON
(D, D*T, P > 0.05) (Fig. 2). and OVE cows the concentration remained constant
(P > 0.05). This led to a greater (P < 0.05) retinol con-
Health status centration in OVE + SM at 21 d postpartum compare
No effects of diet or its interaction with time were sig- with OVE. Diet also affected paraoxonase concentra-
nificant for haptoglobin or IL-6 concentration (D, D*T, tion, with overall greater level (P < 0.05) in CON
P > 0.05). Diet affected albumin concentration (D*T, P < compared with OVE and OVE + SM (D, P < 0.05).
0.05), with greater (P < 0.05) postpartum concentrations This difference was due to greater (P < 0.05) concen-
in OVE + SM compared with both other groups. The tration in CON cows at -21, -10 and 7 d relative to
overall concentration of total bilirubin, ceruloplasmin, parturition (D*T, P < 0.05).
and serum amyloid A tended to be affected by diet (D,
P < 0.10), with greater levels (P < 0.05) in OVE cows Gene expression
compared with CON (total bilirubin), OVE + SM Energy metabolism
(ceruloplasmin), or both other groups (SAA). Diet also Cows fed the CON or OVE + SM diets had greater
affected GGT and GOT concentration, as OVE + SM had PCK1 expression compared with OVE cows (D, P < 0.05).
greater (P < 0.05) overall GGT concentration (D, P < Diet also affected the expression of the fatty acid metabol-
0.10), especially postpartum (d 14 and 21), compared ism related genes MTTP (D, P < 0.05) and PPARA (D*T,
with OVE, and lower (P < 0.05) GOT concentration post- P < 0.05). Expression of MTTP was in fact greater (P < 0.05)
partum (d 7 and 14) (D*T, P < 0.05) compared with in CON and OVE cows, compared with OVE + SM, while
CON cows. Time affected the concentration of all previ- PPARA expression was greater (P < 0.05) prepartum (-10 d)
ous health biomarkers (T, P < 0.01). for OVE + SM compared with CON and OVE, and lower
(P < 0.05) early postpartum (7 d) for OVE compared with
Antioxidant and oxidative status the other two groups.
No effect of diet was detected for ORAC, total ROM
and tocopherol (D, P > 0.05). Total NOx also were not Hepatokines and inflammation
affected by diet, despite the fact that concentrations of Diet alone did not affect genes related to hepatokiens
both NO2 and NO3 had significant interactions or diet and the inflammatory response (D, P > 0.05). However,
effects (NO2, D*T, P < 0.10; NO3, D, P < 0.05, D*T, P < the hepatokines ANGPTL4 and FGF21 had a significant
0.10). Diet had a strong effect on GSH concentration interaction with time (D*T, P < 0.05). For FGF21 this
(D, P < 0.001), with greatest concentration (P < 0.05) in significance was due to a greater (P < 0.05) prepartal
OVE + SM cows compared with both other groups. expression in CON and OVE + SM compared with
When interacting with time, diet tended to affect blood OVE cows, while for ANPTL4 no differences among
concentration of β-carotene and retinol (D*T, P < 0.10). dietary groups were detected across the analyzed time
For the first, the response was due to a greater (P < points (P > 0.05).
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 6 of 12

Fig. 1 Effect of feeding a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), a higher-energy
(1.54 Mcal/kg DM) diet without (OVE) rumen-protected methionine during the last 21 d before calving, or OVE plus rumen-protected methionine
(Smartamine M; OVE + SM; Adisseo NA) from -21 d before calving through the first 30 d postpartum on hepatic gene expression (log2
back-transformed LSM) in Holstein cows

Methionine cycle and antioxidant system cows compared with the other dietary groups; whereas,
No genes concerning the antioxidant system were sig- expression was greater (P < 0.05) early postpartum (7 d)
nificantly affected by diet, or its interaction with time in CON cows compared with OVE and OVE + SM.
(D, D*T, P > 0.05). However, MTR and DNMT3A, genes
of the methionine cycle, had an overall effect of diet (D, Discussion
P < 0.05). Expression of MTR was greater (P < 0.05) in Overfeeding dairy cows in the weeks prior parturition
CON compared with OVE, with OVE + SM having an (e.g. close up period) has been previously linked with a
intermediate level of expression, while DNMT3A expres- more pronounced negative energy balance postpartum,
sion was greater (P < 0.05) in OVE and OVE + SA com- due to bigger drops in voluntary dry matter intake
pared with CON cows. Furthermore, SAHH expression (DMI) along with sustained lipid mobilization and pos-
was greater (D*T, P < 0.05) prepartum in OVE + SM sible accumulation of triacylglycerol (TAG) in the liver
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 7 of 12

SM cows compared with OVE had greater postpartal

DMI and better milk production, matching the perform-
ance of the control-fed group [25]. Despite the fact that
the improved DMI, likely a consequence of the im-
proved health status, could easily explain the improved
production performance, other cellular and physiologic
also likely were contributing factors.
The hepatic transcriptome revealed how Met supple-
mentation restored PCK1 expression (an important glu-
coneogenic gene) to the level of control-fed cows. At
least postpartum this could be explained by the higher
insulin concentration in OVE + SM [25], as hepatic
PCK1 mRNA expression is directly related to insulin
level [32]. The increased insulin concentration also could
explain why circulating glucose was lower in OVE + SM
cows [25] compared with CON, i.e. overfeeding alone does
not affect peripheral insulin resistance [9], and the in-
creased insulin concentration was not followed by changes
in GH or IGF1, hence, the improved milk production with
OVE + SM also might have resulted from an increase in
glucose availability directly channeled to peripheral tissues
and the mammary gland. In the latter case it would have
contributed to greater lactose production. Peripheral
tissues, i.e. adipose and muscle, rely mainly on GLUT4
(an insulin-dependent transporter) for glucose uptake,
while the mammary gland uses mainly GLUT1 (usually
described as insulin-independent) as the preferred glucose
transporter [33]. However, a recent study revealed that
insulin increases GLUT1 expression in bovine mam-
mary explants, thus, providing evidence of a func-
tional link between circulating insulin and mammary
glucose uptake [34].
Supplementing Met also increased both fat and pro-
tein percentage during the first week of lactation [25].
Because biomarkers of muscle catabolism were not
Fig. 2 Effect of feeding a control lower-energy diet (CON, 1.24 Mcal/kg
affected by diet (e.g. urea and creatinine) and DMI
DM; high-straw) during the whole dry period (~50 d), a higher-energy was similar in CON and OVE + SM, we speculate that
(1.54 Mcal/kg DM) diet without (OVE) rumen-protected methionine Met itself, combined with higher circulating insulin,
during the last 21 d before calving, or OVE plus rumen-protected might have been the primary cause of the improved
methionine (Smartamine M; OVE + SM; Adisseo NA) from -21 d before protein percentage. In fact, previous research demon-
calving through the first 30 d postpartum on endocrine profiles in
Holstein cows
strated that an increase in amino acid supply (e.g. ab-
omasal casein infusion) could markedly improve milk
protein yield, especially when the circulating level of
[25]. The present study confirmed the overfeeding- insulin was artificially raised through a clamp [35,
induced depression of DMI postpartum and hepatic 36]. The lower inflammation status and greater liver
TAG accumulation [25]. Furthermore, despite previous function around calving in the OVE + SM cows (lower
studies reporting that overfed cows were always able to concentrations of albumin and greater bilirubin, ceru-
maintain similar levels of milk production as the loplasmin, GGT, GOT, and SAA) would have guaran-
control-fed counterparts [31], these changes led to worse teed higher availability of plasma amino acids [37] to
milk performance including lower milk and energy cor- the mammary gland for protein synthesis. The in-
rected milk yield [25]. crease in fat content, which agrees with several previ-
As hypothesized, supplementation of rumen-protected ous studies [16, 38–41], might have been related to
Met to a moderate energy diet was able to overcome the cellular pathways involving Met and its methylated
detrimental effects of energy overfeeding. In fact, OVE + compounds (e.g. choline [42]), which some data
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 8 of 12

indicate are important for supporting milk fat synthe- liver TAG accumulation [25] despite similar NEFA con-
sis in cows [43]. centration between OVE and OVE + SM [25]. This was
As previously mentioned, overfeeding energy prepar- at least in part due to greater PPARA expression with
tum led to hepatic TAG accumulation [25], a condition Met supplementation.
that, if excessive, could become a potential burden for Among the most important metabolic functions coor-
proper liver function [2]. OVE cows, in fact, had signs of dinated by PPARα are LCFA uptake, intracellular activa-
impaired liver function and inflammatory condition tion, oxidation, and ketogenesis [44]. Thus its greater
postpartum including lower concentrations of albumin expression in OVE + SM cows could have improved
and greater bilirubin, ceruloplasmin, GGT, GOT, and NEFA handling, i.e. through greater oxidation. Further-
SAA (Table 2, Fig. 3). As hypothesized, supplemental more, PCK1 is also involved in glyceroneogenesis, as it
Met was able to correct these effects of the OVE diet. can catalyze the production of glycerol-3-phospate for
Thus, as a primary outcome, OVE + SM cows had less use during fatty acid esterification [45]. Thus the

Table 2 Effect of feeding a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), a
higher-energy (1.54 Mcal/kg DM) diet without (OVE) rumen-protected methionine during the last 21 d before calving, or OVE plus
rumen-protected methionine (Smartamine M; OVE + SM; Adisseo NA) from -21 d before calving through the first 30 d postpartum
on biomarker concentrations of metabolism, liver health, and oxidative status in Holstein cows
Diet1 P-values3
2
Items CON OVE OVE + SM SE D T D*T
Metabolism
Cholesterol, mmol/L 3.24 3.16 3.26 0.11 0.76 <.0001 0.71
Creatinine, μmol/L 97.60 98.88 97.68 1.53 0.77 <.0001 0.17
GH, ng/mL 5.75 4.79 6.95 1.08 0.23 <.0001 0.82
IGF-1, ng/mL 56.65 60.03 59.98 6.64 0.91 <.0001 0.69
Leptin, ng/mL 4.44 5.42 4.40 1.62 0.84 <.0001 0.29
Urea, mmol/L 5.20 5.05 5.05 0.18 0.77 <.0001 0.30
Liver health
Albumin, g/L 35.41 35.54 36.32 0.41 0.24 0.0002 0.05
Bilirubin , μmol/L 2.29a 3.38b 2.57ab 0.41 0.10 <.0001 0.57
Ceruloplasmin, μmol/L 2.77ab 2.91b 2.61a 0.09 0.006 <.0001 0.51
a ab
GGT, U/L 22.96 25.21 26.85b 1.17 0.07 <.0001 0.008
GOT, U/L 84.76 90.30 81.71 5.61 0.48 <.0001 0.04
Haptoglobin, g/L 0.42 0.46 0.41 0.06 0.76 0.003 0.86
IL-6, pg/mL 530.63 586.37 412.76 98.56 0.37 0.001 0.67
SAA, μg/mL 35.55a 54.00b 34.77a 7.79 0.10 0.0005 0.58
Oxidative status
β-carotene, mg/100 mL 0.20 0.19 0.23 0.02 0.14 0.04 0.06
a b c
Liver GSH, mmol/L 953 1281 1693 120 0.0002 0.05 0.14
NO2, μmol/L 6.03 6.66 6.80 0.45 0.44 0.01 0.09
NO3, μmol/L 18.65a 16.90b 16.77b 0.40 0.002 <.0001 0.08
NOx, μmol/L 24.61 23.54 23.67 0.56 0.31 <.0001 0.18
ORAC, TE mol/L 12,731 12,359 12,739 198 0.25 <.0001 0.66
Paraoxonase, U/mL 77.96a 68.41b 66.74b 2.68 0.01 <.0001 0.02
Retinol, μg/100 mL 46.39 41.79 43.42 3.10 0.44 0.0009 0.08
ROM, mg of H2O2/100 mL 14.01 12.99 13.44 0.49 0.31 <.0001 0.79
Tocopherol, μg/mL 3.67 3.68 3.16 0.44 0.46 <.0001 0.31
1
Prepartum dietary treatment: CON = control energy, OVE = moderate energy, OVE + SM = OVE supplemented with rumen-protected methionine (Smartamine M,
Adisseo Inc.)
2
SE = greatest standard error of the mean
3
D = diet, T = time, D*T = diet by time interaction
a, b, c
Significant difference among dietary groups (P ≤ 0.05). Differen reported for biomarkers with a tendency (P ≤ 0.10) or a significan (P ≤ 0.05) Diet effect
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 9 of 12

Fig. 3 Effect of feeding a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), a higher-energy
(1.54 Mcal/kg DM) diet without (OVE) rumen-protected methionine during the last 21 d before calving, or OVE plus rumen-protected methionine
(Smartamine M; OVE + SM; Adisseo NA) from -21 d before calving through the first 30 d postpartum on blood biomarkers of liver function and
antioxidant status in Holstein cows

increase of its expression could have further improved al. [51]. OVE did not cause changes in total ROM and
NEFA handling by the liver. The lower expression of NOx, however, these cows had an impairment of the
MTTP in the OVE + SM cows was lower indicated a po- antioxidant system. Despite similar blood antioxidant
tentially lower capacity of these cows to synthesize and capacity, paraoxonase concentration was in fact lower in
export VLDL. However, the data from Bernabucci et al. OVE cows, a condition that not only indicates liver dys-
[46] indicated that apolipoprotein mRNA transcription function, but one that has been proven to lead to an in-
rather than MTTP might be the limiting step in the re- crease in the inflammatory status (confirmed by higher
packaging of TAG into lipoproteins, hence, explaining ceruloplasmin and SAA), which notoriously causes an
the increase in concentration of plasma VLDL in OVE + increase in oxidative stress, and a reduction of antioxida-
SM cows [25]. As a subsequent outcome, the improved tive protection during the early postpartum period [27,
fatty acid metabolism in liver with Met supplementation 52]. As for paraoxonase, postpartum (d 14) concentra-
reduces the risk of liver dysfunction, an idea supported tion of β-carotene, a precursor of vitamin A, which ex-
by the biomarkers of liver function (e.g. greater albumin erts antioxidant effects [53], also was reduced in OVE
and VLDL, and lower bilirubin) in OVE + SM cows [47]. compared with CON.
Metabolic dysfunction and inflammatory events are Supplementation of rumen-protected Met has been
often linked through oxidative stress, a common out- proven to benefit the oxidative status of periparturient
come to both scenarios [48–50]. The present study cows [19, 20], in large part because it is a precursor for
partly confirmed the possible molecular mechanisms the biosynthesis of glutathione and taurine, two of the
through which prepartum overfeeding could cause an most important cellular antioxidants [54, 55]. In the
increased concentration of oxidants proposed by Loor et present study, Met supplementation to cows fed a higher
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 10 of 12

energy diet prepartum was able to improve their com- [58], but overfeeding cows prepartum never led to its im-
promised antioxidant status. In fact, despite the lack of pairment in our previous experiments [9, 10]. On the other
changes in ROM or paraoxonase compared with OVE, hand, levels of hepatic methylation were associated with
OVE + SM cows had greater glutathione concentrations, fatty liver disease in humans [59, 60]. Because OVE cows
even compared with CON, together with higher retinol had a greater hepatic TAG content [25], DNMT3A expres-
concentrations up to the level of control-fed cows. Con- sion regulation could be explained by the alterations in lipid
cerning retinol, its concentration is also regulated by the metabolism.
hepatic synthesis of its carrier, retinol binding protein
[56]. Thus, a greater plasma retinol concentration, be-
Conclusions
sides suggesting a better antioxidant status, could also
Current results confirm the detrimental outcome (e.g.,
be a response to the better liver functionality detected in
reduced DMI, compromised liver function, and higher
OVE + SM cows. Furthermore, GSR expression was de-
inflammatory status) of a higher-energy diet during the
creased in OVE + SM cows to a similar level than OVE.
close up period in dairy cows, thus, supporting the need
GSR encodes the protein glutathione reductase, a central
for energy restriction in the close-up period. However, if
enzyme of cellular antioxidant defense, which reduces
the practice persists, dairy producers should improve the
oxidized glutathione disulfide to the sulfhydryl form
diet methionine supply. In fact, supplemental rumen-
[57]. This further suggests a lesser oxidative status in
protected methionine was effective in reducing the
cows fed methionine, which despite having a greater
aforementioned effects, by (i) stimulating DMI and milk
glutathione concentration seemed to have less of a need
production, (ii) improving hepatic fatty acid metabolism
to restore the pool of its active form.
and reducing TAG accumulation, (iii) improving general
Other health benefits of methionine supplementation
biomarkers of liver function, and (iv) limiting the post-
could also be noticed in the lower somatic cell count in
partal negative effect of inflammation on the cow anti-
milk. For instance, OVE + SM cows compared with both
oxidant system. Further investigation is needed to assess
CON and OVE had lower milk SCC [25], a result that
the effect of methionine supplementation to a prepartal
further highlights the immunometabolic effects of me-
energy restricted diet during the close-up.
thionine and its metabolites [16, 21–23].
At a molecular level, the greater expression SAHH
prepartum in OVE + SM cows underscores that the in- Additional file
creased Met supply to the liver through supplementation
was directed through the methionine cycle, leading to Additional file 1: Complete gene expression methodology, including
primer sequences and qPCR performance. Figures S1-S5 include the
the higher glutathione concentrations. However, over- two-way interaction graphs not shown in the manuscript. (DOCX 8109 kb)
feeding energy prepartum (e.g. OVE and OVE + SM)
seemed to reduce the overall expression of MTR, as if
regenerating Met was not a hepatic priority. This be- Abbreviations
ACOX1: Acyl-CoA oxidase 1; ANGPTL4: Angiopoietin like 4;
comes relevant in early lactation, because after calving APOB: Apolipoprotein B; BBOX1: γ-butyrobetaine hydroxylase 1;
the decrease in expression of both MTR and SAHH in BHMT: BHMT2, Betaine–homocysteine S-methyltransferase 1, and 2;
all groups indicated that cows might redirect the CBS: Cystathionine-beta-synthase; CP: Ceruloplasmin; CPT1A: Carnitine
palmitoyltransferase 1A; CSAD: Cysteine sulfinic acid decarboxylase;
circulating Met to the mammary gland for milk produc- CTH: Cystathionine gamma-lyase; DMI: Dry matter intake; DNMT1: DNMT3A,
tion. To further complicate this scenario, the greater DNA (cytosine-5-)-methyltransferase 1, and 3 α; FGF21: Fibroblast growth
DNMT3A expression in both OVE and OVE + SM cows factor 21; GCLC: Glutamate-cysteine ligase catalytic subunit; GGT: Gamma-
glutamyl-transpeptidase; GH: Growth hormone; GLUT1: GLUT4, Glucose
indicated a role of overfeeding in its regulation. Its transporter 1, and 4; GOT: Glutamic oxaloacetic transaminase; GPX1: Glutathione
greater expression could indicate a higher need of peroxidase 1; GSR: Glutathione reductase; GSS: Glutathione synthetase;
methyl groups from methionine by the liver, hence, in HMGCS2: 3-hydroxy-3-methylglutaryl-CoA synthase 2; HP: Haptoglobin;
IGF1: Insulin like growth factor-1; IL6: Interleukin 6; MAT1A: Methionine
light of the lower hepatic regeneration (e.g. lower MTR) adenosyltransferase 1A; Met: Methionine; MTR: 5-methyltetrahydrofolate-
but greater utilization (e.g. higher DNMT3A) Met homocysteine methyltransferase; MTTP: Microsomal triglyceride transfer protein;
supplementation (e.g. OVE + SM) favored the mammary NEFA: Non-esterified fatty acids; NEL: Net energy for lactation; NFKB1: Nuclear
factor κB subunit 1; NOx: NO2, NO3, Nitric oxides; ORAC: Oxygen radical
demand. The fact that milk production was restored to absorbance capacity; PC: Pyruvate carboxylase; PCK1: Phosphoenolpyruvate
the level of CON cows in the OVE + SM cows supports carboxykinase 1; PDK4: Pyruvate dehydrogenase kinase 4;
this scenario. PEMT: Phosphatidylethanolamine N-methyltransferase; PPARA: Peroxisome
proliferator activated receptor α; ROM: Reactive oxygen metabolites;
The mechanisms by which prepartal overfeeding RXRA: Retinoid X receptor α; SAA: SAA2, Serum amyloid A, and A2; SAHH:
causes a greater DNMT3A expression, increasing DNA S-adenosylhomocysteine hydrolase; SLC22A5: Solute carrier family 22 member
methylation and leading to greater consumption of 5; SOCS2: Suppressor of cytokine signaling 2; SOD1: SOD2, Superoxide
dismutase 1 (soluble), and 2 (mitochondrial); STAT3: STAT5B, Signal transducer
methyl groups from Met, are not clear. Insulin sensitivity and activator of transcription 3, and 5B; TAG: Triacylglycerol;
was previously associated with increased global methylation TMLHE: Trimethyllysine hydroxylase, ε; VLDL: Very low density lipoprotein
Vailati-Riboni et al. Journal of Animal Science and Biotechnology (2017) 8:17 Page 11 of 12

Acknowledgements 10. Khan MJ, Jacometo CB, Graugnard DE, Correa MN, Schmitt E, Cardoso F,
The authors thank Travis Michels and Mike Katterhenry of the University of et al. Overfeeding Dairy Cattle During Late-Pregnancy Alters Hepatic
Illinois Dairy Research Unit (Urbana) staff for help with animal management. PPARalpha-Regulated Pathways Including Hepatokines: Impact on Metabolism
and Peripheral Insulin Sensitivity. Gene Regul Syst Bio. 2014;8:97–111.
Funding 11. Rukkwamsuk T, Wensing T, Geelen MJ. Effect of overfeeding during the dry
Financial support for the research was provided in part by Adisseo period on the rate of esterification in adipose tissue of dairy cows during
(Commentry, France) and Hatch funds under project ILLU-538–914, the periparturient period. J Dairy Sci. 1999;82(6):1164–9.
National Institute of Food and Agriculture, Washington, DC, USA. 12. Soliman M, Kimura K, Ahmed M, Yamaji D, Matsushita Y, Okamatsu-Ogura Y, et al.
Inverse regulation of leptin mRNA expression by short- and long-chain fatty
Availability of data and materials acids in cultured bovine adipocytes. Domest Anim Endocrinol. 2007;33(4):400–9.
The datasets during and/or analyzed during the current study are available 13. Dann HM, Litherland NB, Underwood JP, Bionaz M, D’Angelo A,
from the corresponding author on reasonable request. McFadden JW, et al. Diets during far-off and close-up dry periods affect
periparturient metabolism and lactation in multiparous cows. J Dairy
Authors’ contributions Sci. 2006;89(9):3563–77.
MVR performed the qPCR statistical analysis and wrote the main draft of the 14. Graugnard DE, Moyes KM, Trevisi E, Khan MJ, Keisler D, Drackley JK, et al.
manuscript, with inputs from DL, ET and JJL. JLL and DL designed the study. Liver lipid content and inflammometabolic indices in peripartal dairy cows
JSO performed the animal study and qPCR. ET and JSO performed the blood are altered in response to prepartal energy intake and postpartal
biomarker analysis. All authors read and approved the final manuscript. intramammary inflammatory challenge. J Dairy Sci. 2013;96(2):918–35.
15. Shahzad K, Bionaz M, Trevisi E, Bertoni G, Rodriguez-Zas SL, Loor JJ.
Competing interests Integrative analyses of hepatic differentially expressed genes and blood
The authors declare that they have no competing interests. biomarkers during the peripartal period between dairy cows overfed or
restricted-fed energy prepartum. PLoS One. 2014;9(6):e99757.
Consent for publication 16. Osorio JS, Ji P, Drackley JK, Luchini D, Loor JJ. Supplemental Smartamine M or
Not applicable. MetaSmart during the transition period benefits postpartal cow performance
and blood neutrophil function. J Dairy Sci. 2013;96(10):6248–63.
Ethics approval 17. Zhou Z, Vailati-Riboni M, Trevisi E, Drackley JK, Luchini DN, Loor JJ. Better
All procedures for this study (protocol no. 09214) were approved by the postpartal performance in dairy cows supplemented with rumen-protected
Institutional Animal Care and Use Committee of the University of Illinois. methionine compared with choline during the peripartal period. J Dairy Sci.
2016;99(11):8716–32.
Author details 18. Zhou Z, Bulgari O, Vailati-Riboni M, Trevisi E, Ballou MA, Cardoso FC, et al.
1
Mammalian NutriPhysioGenomics, Department of Animal Sciences and Rumen-protected methionine compared with rumen-protected choline
Division of Nutritional Sciences, University of Illinois, Urbana, IL 61801, USA. improves immunometabolic status in dairy cows during the peripartal
2
Istituto di Zootecnica Facoltà di Scienze Agrarie, Alimentari e Ambientali, period. J Dairy Sci. 2016;99(11):8956–69.
Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy. 3Adisseo NA, 19. Osorio JS, Ji P, Drackley JK, Luchini D, Loor JJ. Smartamine M and
Alpharetta, GA 30022, USA. 4Dairy and Food Science Department, South MetaSmart supplementation during the peripartal period alter hepatic
Dakota State University, 1111 College Ave, 113H Alfred DairyScience Hall, expression of gene networks in 1-carbon metabolism, inflammation,
Brookings SD 57007, USA. oxidative stress, and the growth hormone-insulin-like growth factor 1 axis
pathways. J Dairy Sci. 2014;97(12):7451–64.
Received: 9 August 2016 Accepted: 18 January 2017 20. Osorio JS, Trevisi E, Ji P, Drackley JK, Luchini D, Bertoni G, et al. Biomarkers
of inflammation, metabolism, and oxidative stress in blood, liver, and milk
reveal a better immunometabolic status in peripartal cows supplemented
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