Supplements of vitamins B9 and B12 affect hepatic and mammary gland gene expression profiles in lactating dairy cows.

BACKGROUND: A combined supplement of vitamins B9 and B12 was reported to increase milk and milk component yields of dairy cows without effect on feed intake. The present study was undertaken to verify whether this supplementation positively modifies the pathways involved in milk and milk component synthesis. Thus, by studying the transcriptome activity in these tissues, the effect of supplements of both vitamins on the metabolism of both liver and mammary gland, was investigated. For this study, 24 multiparous Holstein dairy cows were assigned to 6 blocks of 4 animals each according to previous 305-day milk production. Within each block, cows were randomly assigned to weekly intramuscular injections of 5 mL of either saline 0.9 % NaCl, 320 mg of vitamin B9, 10 mg of vitamin B12 or a combination of both vitamins (B9 + B12). The experimental period began 3 weeks before the expected calving date and lasted 9 weeks of lactation. Liver and mammary biopsies were performed on lactating dairy cows 64 ± 3 days after calving. Samples from both tissues were analyzed by microarray and qPCR to identify genes differentially expressed in hepatic and mammary tissues.
RESULTS: Microarray analysis identified 47 genes in hepatic tissue and 16 genes in the mammary gland whose expression was modified by the vitamin supplements. Gene ontology (GO) categorizes genes in non-overlapping domains of molecular biology. Panther is one of the online GO resources used for gene function classification. It classifies the 63 genes according to Molecular Function, Biological Process and Protein Class. Most of the biological processes modulated by the vitamin supplements were associated to developmental process, protein metabolic process, transport and response to inflammation. In the liver, most of the genes modulated by the vitamin treatments involved protein metabolic process while developmental process appeared to be more affected by the treatments in mammary gland. Out of 25 genes analysed by qPCR, 7 were validated.
CONCLUSION: The results indicate that several metabolic processes were modulated by the supplementation of vitamins in early-lactating dairy cows. In addition, the results suggest that the vitamin supplements promoted liver regeneration and reduced catabolism of lipids in early lactation.

Background

Bacteria present in rumen synthesize B vitamins in generally sufficient amounts to meet their host’s requirements [1]. Notwithstanding, high-producing dairy cows could benefit from vitamin B9 and B12 supplements, especially during the critical period around calving and in early lactation [2-4]. Vitamin B9 plays a major role in DNA synthesis and in de novo formation of methyl groups required for the methylation cycle. On the other hand, vitamin B12 is involved in two metabolic pathways: the remethylation cycle and as coenzyme of the methylmalonyl-CoA mutase. The former, closely related to folate metabolism, is required for the regeneration of methionine and tetrahydrofolate, whereas the later allows the entry of propionate in the Krebs cycle and gluconeogenesis [5].

Results from two studies suggest that supplementary vitamin B9 might improve efficiency of nutrient utilization, especially for milk protein synthesis [6, 7]. This observation is supported by the observation that in vitro hormonal stimulation of milk protein synthesis by mammary gland explants of dairy cows increased expression of 28 genes; among them, 2 genes related to folate metabolism, FOLR1 and ALDH1L1 [8]. The former is a folate transporter allowing the entry of 5-methyl-tetrahydrofolate into the cells whereas the latter is involved in purine synthesis. As vitamin B9 supplement increases milk protein synthesis, it would be interesting to investigate whether similar transcriptomic process takes place in vivo when vitamin supplements are provided to lactating cows.

A combined supplement of vitamins B9 and B12 given during the peripartum period and in early lactation altered energy partitioning during the first weeks of lactation as compared to control cows [3, 4, 9]. However, the mode of action of this supplement is not fully elucidated. We observed that a combined supplement of vitamins B9 and B12 increases whole-body rate of appearance of glucose which is the sum of glucose from portal absorption, glycogenolysis, and gluconeogenesis [3]. Propionate originating from rumen fermentation is the major precursor of glucose in cows [10, 11] and contributes up to 60 % of glucose flux rate [12, 13]. For ruminants, the glucose, essential for synthesis of milk lactose, is mostly provided by liver (up to 90 %) through gluconeogenesis [14].

It has been shown that at the onset of lactation, the liver as well as the mammary gland undergo numerous adaptations to support milk synthesis [15]. In early lactation, feed intake is not sufficient to meet the nutrient demand for milk production, leading to a negative energy balance [15]. Furthermore, the liver undergoes extensive physiological and biochemical changes mediated by significant alterations in hepatic gene expression in an attempt to re-establish metabolic homeostasis and to counteract the adverse effects of negative energy balance [16]. In addition, during this period, the mammary gland is actively remodeling. In fact, by investigating the transcriptional response of the mammary gland during early lactation, Connor et al. [17] observed changes in mammary expression of genes involved in cell proliferation, cellular remodeling, and nutrient transport.

Therefore, the aim of the present project was to profile the genes for which expression undergoes major changes in hepatic and mammary tissues of lactating dairy cows according to vitamins B9 and B12 supply.

Results

At week 9 of lactation, there was no treatment effect (P ≥ 0.1) on dry matter intake, milk production, milk total solid yields as well as milk component contents. Dry matter intake, milk production, milk total solid yields, milk fat, protein and lactose contents averaged 18.8 (SE 1.9) kg/d, 34.7 (SE 2.9) kg/d, 3.92 (SE 0.33) kg/d, 36.0 (SE 2.0) g/kg, 30.2 (SE 1.4) g/kg, and 46.9 (SE 6.0) g/kg, respectively. During the seven weeks following calving, body condition score losses tended to be reduced (interaction vitamin × time, P = 0.10) in cows receiving the vitamin B9 supplements, alone or combined with vitamin B12. Plasma concentrations of non-esterified fatty acids and β-hydroxybutyrate were also lower (P ≤ 0.06), averaging 175 and 243 μM (SEM 17) and 0.70 and 0.77 mM (SEM 0.03) for cows receiving or not receiving supplementary vitamin B9, respectively.

The microarray analysis revealed that, as compared to control cows receiving no vitamin supplement, the vitamin treatments significantly changed (FDR ≤ 0.05) the expression of some genes by more than 2-fold in hepatic and mammary tissues (Fig. 1 a and b, respectively). The expression of 47 genes in hepatic tissue was modified by at least one of the vitamin treatment compared to control cows (Fig. 1a). From this panel of 47 genes, 41 could be assigned to a biological process using the Panther classification system (Tables 1 and 2). Out of these 47 genes, expression of 14 genes was analyzed by qPCR (Fig. 2). Although nine false positive genes were observed, five genes were confirmed to be differentially expressed in the group of cows supplemented with vitamins.

Fig. 1

Number of genes (and probes) influenced by the vitamin supplementation. Supplements of vitamin B9 (B9), vitamin B12 (B12) or both vitamins (B9 + B12) were given to dairy cows. The Venn diagram shows the number of significant genes. The number of probes is bracketed “()”. (FDR ≤ 0.05) with more than 2-fold change between the control group and each treatment group in (a) hepatic tissue and (b) mammary tissue

Table 1

Gene Ontology annotations of the differentially expressed hepatic genes with the Panther Classification Systema

Treatment	Gene symbol	Gene name	Treatment effect	Molecular function	Biological process	Protein class
B9
DLK1		Delta-like 1 homolog (Drosophila)	Down-regulated	receptor activityextracellular matrix structural constituentreceptor binding	transcription from RNA polymerase II promotercell communicationectoderm developmentnervous system developmentintracellular protein transportreceptor-mediated endocytosisregulation of transcription from RNA polymerase II promoter	membrane-bound signaling moleculereceptorextracellular matrix structural protein
MYOM1		Myomesin-1	Down-regulated	protein kinase activitystructural constituent of cytoskeletonprotein bindingsmall GTPase regulator activityguanyl-nucleotide exchange factor activity	protein phosphorylationcell communicationcell adhesionmuscle contractionmesoderm developmentmuscle organ developmentregulation of catalytic activity	non-receptor serine/threonine protein kinasenon-receptor serine/threonine protein kinaseguanyl-nucleotide exchange factoractin family cytoskeletal proteincell adhesion molecule
B12
HERC6		HECT and RLD domain containing E3 ubiquitin protein ligase family member 6	Up-regulated	ligase activity	catabolic processcellular protein modification processproteolysiscellular process	ubiquitin-protein ligase
SESN2		Sestrin-2	Up-regulated	oxidoreductase activityperoxidase activity	metabolic processcell cycle	Peroxidase
GPNMB		glycoprotein (transmembrane) nmb	Up-regulated	receptor binding	cellular process	membrane-bound signaling moleculecell adhesion molecule
IFI6		Interferon alpha-inducible protein 6	Up-regulated	Unassigned	Unassigned	Unassigned
PPP1R3B		Protein phosphatase 1 regulatory subunit 3B	Up-regulated	phosphatase activityprotein bindingphosphatase regulator activity	glycogen metabolic processregulation of catalytic activity	phosphatase modulator
ID1		ID1 protein	Up-regulated	sequence-specific DNA binding transcription factor activity	transcription from RNA polymerase II promoterregulation of transcription from RNA polymerase II promoter	transcription factor
IFI27		interferon, alpha-inducible protein 27	Up-regulated	Unassigned	Unassigned	Unassigned
MEP1B		meprin A, beta	Up-regulated	oxidoreductase activityserine-type peptidase activitymetallopeptidase activityreceptor activitylipid transporter activitytransmembrane transporter activityreceptor bindingenzyme regulator activity	immune system processproteolysissynaptic transmissioncell-cell adhesionvisual perceptionsensory perceptionectoderm developmentmesoderm developmentskeletal system developmentangiogenesisnervous system developmentheart developmentblood coagulationlipid transportintracellular protein transportendocytosisvitamin transportregulation of catalytic activity	transporterapolipoproteinmembrane-bound signaling moleculereceptormetalloproteaseserine proteaseoxidasemetalloproteaseserine proteaseextracellular matrix proteinenzyme modulatorcell adhesion molecule
FUT5		fucosyltransferase 5 (alpha (1,3) fucosyltransferase	Up-regulated	transferase activity, transferring glycosyl groups	protein glycosylation	glycosyltransferase
G0S2		G0/G1switch 2	Up-regulated	Unassigned	Unassigned	Unassigned
CDK5R1		Cyclin-dependent kinase 5 activator 1	Up-regulated	kinase activityprotein bindingkinase activator activitykinase regulator activity	protein phosphorylationcell cycleregulation of catalytic activity	kinase activator
ISG15		Ubiquitin-like protein ISG15	Up-regulated	structural constituent of ribosomenucleic acid binding	proteolysis	ribosomal protein
LOC515676		NFX1-type zinc finger-containing protein 1-like	Up-regulated	peptidase activityprotein bindingserine-type endopeptidase inhibitor activity	proteolysisregulation of catalytic activity	serine protease inhibitor
SPP1		Osteopontin	Up-regulated	cytokine activity	immune system processcellular processcell adhesioncellular component morphogenesiscellular component organization	cytokineextracellular matrix proteindefense/immunity proteincell adhesion molecule
DLK1		Delta-like 1 homolog (Drosophila)	Down-regulated	receptor activityextracellular matrix structural constituentreceptor binding	transcription from RNA polymerase II promotercell communicationectoderm developmentnervous system developmentintracellular protein transportreceptor-mediated endocytosisregulation of transcription from RNA polymerase II promoter	membrane-bound signaling moleculereceptorextracellular matrix structural protein
MYOM1		Myomesin-1	Down-regulated	protein kinase activitystructural constituent of cytoskeletonprotein bindingsmall GTPase regulator activityguanyl-nucleotide exchange factor activity	protein phosphorylationcell communicationcell adhesionmuscle contractionmesoderm developmentmuscle organ developmentregulation of catalytic activity	non-receptor serine/threonine protein kinasenon-receptor serine/threonine protein kinaseguanyl-nucleotide exchange factoractin family cytoskeletal proteincell adhesion molecule
MT1E		MetallothioneinMT1E	Down-regulated	Unassigned	Unassigned	Unassigned
NEFH		orthologUncharacterized protein (Fragment)NEFHortholog	Down-regulated	Unassigned	Unassigned	Unassigned
SFRP1		Secreted frizzled-related protein 1	Down-regulated	receptor activityprotein binding	reproductioncell communicationsingle-multicellular organism processnervous system developmentresponse to stimulusregulation of biological process	signaling moleculeG-protein coupled receptor
KIAA1324		KIAA1324 ortholog	Down-regulated	Unassigned	Unassigned	Unassigned
MGC126945		Uncharacterized protein	Down-regulated	receptor activity	B cell mediated immunityantigen processing and presentationcellular defense response	immunoglobulin receptor superfamilyimmunoglobulin receptor superfamilymajor histocompatibility complex antigen
MT1A		Metallothionein-1AMT1A	Down-regulated	Unassigned	Unassigned	Unassigned
B9 + B12
SAA3		Serum amyloid A protein	Down-regulated	lipid transporter activitytransmembrane transporter activity	immune system processlipid transport	transporterapolipoproteindefense/immunity protein
LOC100126815		MHC class I-like family A1	Down-regulated	receptor activity	B cell mediated immunityantigen processing and presentationcellular defense response	immunoglobulin receptor superfamilyimmunoglobulin receptor superfamilymajor histocompatibility complex antigen
ACMSD		2-amino-3-carboxymuconate-6-semialdehyde decarboxylase	Down-regulated	Unassigned	Unassigned	Unassigned
THRSP		THRSP protein	Down-regulated	Unassigned	Unassigned	Unassigned
MGC126945		Uncharacterized protein	Down-regulated	receptor activity	B cell mediated immunityantigen processing and presentationcellular defense response	immunoglobulin receptor superfamilyimmunoglobulin receptor superfamilymajor histocompatibility complex antigen
MT1A		Metallothionein-1AMT1A	Down-regulated	Unassigned	Unassigned	Unassigned
C4H7orf57		chromosome 4 open reading frame, human C7orf57	Down-regulated	Unassigned	Unassigned	Unassigned
CACNA2D1		calcium channel, voltage-dependent, alpha 2/delta subunit	Down-regulated	cation transmembrane transporter activity	cation transportprotein targeting	Unassigned
GSTA5		Glutathione S-transferase	Down-regulated	Unassigned	Unassigned	Unassigned
LOC509034		feline leukemia virus subgroup C receptor-related protein 2-like	Down-regulated	transmembrane transporter activity	transport	transporter
NHEDC1		solute carrier family 9, subfamily B (cation proton antiporter 2), member 1	Down-regulated	Unassigned	Unassigned	Unassigned
SH3YL1		SH3 domain-containing YSC84-like protein 1	Down-regulated	structural constituent of cytoskeletonactin binding	cellular process	non-motor actin binding protein
HP		Haptoglobin	Down-regulated	serine-type peptidase activitycalcium ion bindingcalmodulin bindingcalcium-dependent phospholipid binding	gamete generationcomplement activationproteolysiscellular processblood circulationresponse to stressblood coagulation	serine proteaseserine proteasecomplement componentannexincalmodulin
NGEF		neuronal guanine nucleotide exchange factor	Down-regulated	Unassigned	Unassigned	Unassigned
LGALS3		Lectin, galactoside-binding, soluble, 3	Up-regulated	receptor binding	cellular process	signaling moleculecell adhesion molecule
LOC524810		IgM	Up-regulated	Unassigned	Unassigned	Unassigned
LOXL4		Lysyl oxidase homolog 4	Up-regulated	oxidoreductase activityserine-type peptidase activityreceptor activity	macrophage activationapoptotic processproteolysiscell communicationcell-cell adhesionneurological system processcellular defense responseextracellular transportnegative regulation of apoptotic process	receptorserine proteaseoxidaseserine protease
PYCR1		Pyrroline-5-carboxylate reductase 1, mitochondrial	Up-regulated	oxidoreductase activity	cellular amino acid biosynthetic process	reductase
MSMB		Uncharacterized protein	Up-regulated	hormone activity		peptide hormone
IGLL1		immunoglobulin lambda-like polypeptide 1	Up-regulated	antigen binding	B cell mediated immunityhemopoiesisresponse to stimulus	Immunoglobulin
MT1E		MetallothioneinMT1E	Down-regulated	Unassigned	Unassigned	Unassigned
GPNMB		glycoprotein (transmembrane) nmb	Up-regulated	receptor binding	cellular process	membrane-bound signaling moleculecell adhesion molecule

All genes differentially expressed in liver were classified using the Gene Ontology annotations with the Panther Classification System. Some of these genes were not found by Panther genes list analysing system and some could not be classified. Only three ontology categories are presented: Molecular Function, Biological Process and Protein Class

aPanther gene list classification system of the genes differentially expressed in hepatic tissue of cows receiving vitamin supplements (vitamin B9 alone: B9, vitamin B12 alone: B12 or both vitamins: B9 + B12) as compared to no vitamins treatment (Control)

Table 2

Identification of the major biological processes of the genes differentially expressed in hepatic tissuea

Biological process		Genes
Level 1	Level 2/3	Symbol	Fold Change
apoptotic process	negative regulation of apoptotic process	LOXL4	1.6
biological adhesion	cell adhesion	MYOM1 SPP1 MEP1B LOXL4	−2.50.91.31.6
biological regulation	regulation of biological process	DLK1 SFRP1 ID1 LOXL4	−2.5−1.81.11.6
	regulation of molecular function	MYOM1 CDK5R1 PPP1R3B MEP1B LOC515676	−2.50.81.11.31.9
cellular component organization or biogenesis	cellular component organization	SPP1	0.9
cellular process	cell communication	DLK1 MYOM1 SFRP1 MEP1B LOXL4	−2.5−2.5−1.81.31.6
	cell cycle	CDK5R1 SESN2	0.80.9
developmental process	anatomical structure morphogenesis	SPP1	0.9
	death	LOXL4	1.6
	ectoderm development	DLK1 MEP1B	−2.51.3
	mesoderm development	MYOM1 MEP1B	−2.51.3
	system development	DLK1 MYOM1 SFRP1 MEP1B IGLL1	−2.5−2.5−1.81.32.1
immune system process	antigen processing and presentation	LOC100126815 MGC126945	−2.4−1.8
	immune response	LOC100126815 MGC126945 HP IGLL1	−2.4−1.8−1.62.1
	macrophage activation	LOXL4	1.6
localization	transport	DLK1 LOC509034 SAA3 CACNA2D1 MEP1B LOXL4	−2.5−1.5−1.1−1.01.31.6
metabolic process	catabolic process	HERC6	1.8
	carbohydrate metabolic process	PPP1R3B	1.1
	cellular amino acid metabolic process	PYCR1	1.5
	nucleobase-containing compound metabolic process	DLK1 ID1	−2.51.1
	protein metabolic process	MYOM1 HP CDK5R1 MEP1B LOXL4 FUT5 HERC6 LOC515676 ISG15	−2.5−1.60.81.31.61.61.81.92.5
multicellular organismal process	single-multicellular organism process	MYOM1 SFRP1 HP MEP1B LOXL4	−2.5−1.8−1.61.31.6
reproduction	gamete generation	HP	−1.6
response to stimulus	cellular defense response	LOC100126815 MGC126945 LOXL4	−2.4−1.81.6
	immune response	LOC100126815 MGC126945 HP IGLL1	−2.4−1.8−1.62.1
	response to external stimulus	HP MEP1B	−1.61.3
	response to stress	HP	−1.6
Not found		C20H5orf49 LOC789904 SLC22A9 CFH SLC26A10 SAA2 M-SAA3 LOC100847494LOC100335975LOC617654	−1.9−1.8−1.7−1.5−1.4−1.2−1.1−0.91.44.2
Unassigned		C4H7orf57 MT1E MT1A THRSP KIAA1324 NGEF GSTA5 ACMSD NHEDC1 NEFH G0S2 LOC524810 IFI27 IFI6	−4.4−3.4−3.3−1.7−1.6−1.4−1.3−1.2−1.1−1.11.92.63.33.7

All genes modulated in the liver by the vitamins treatment, including those that were not found or classified by the Panther system are listed. The fold change observed by microarray analysis varies from −4.4 to 4.2 and only significantly (FDR ≤ 0.05) expressed genes (compared to control) are shown

aIdentification of the major biological processes of the genes differentially expressed in hepatic tissue of cows receiving vitamin supplements (vitamin B9 alone: B9, vitamin B12 alone: B12 or both vitamins: B9 + B12) as compared to no vitamins treatment (Control)

Fig. 2

Expression of genes measured by quantitative PCR in the liver of the dairy cows that received either no vitamin supplement: Control: no vitamin supplement; vitamin B9 supplement (B9); vitamin B12 supplement (B12) or a combined supplement of vitamins B9 and B12 (B9 + B12). Means different from the control treatment are indicated by *** when P value ≤ 0.01, ** when P values were between 0.01 and 0.05 and * when there was a trend with P values between 0.05 and 0.1

The expression of two genes was significantly modified in liver by the B9 treatment as illustrated in the Venn diagram (Fig. 1a). One gene that also significantly down-regulated by the B12 treatment was confirmed by qPCR for these cows. This repression of DLK1 in liver was down-regulated by the three vitamin treatments (Fig. 2). The B12 treatment had the greatest impact on the liver with 22 genes (totalizing 39 significant probes; Fig. 1a) whose expression level differed by more than 2-fold compared to control (Table 2).

Among the 22 genes affected by the B12 treatment, four genes were also influenced in cows receiving the B9 + B12 treatment: the methallothioneins 1A (MT1A) and 1E (MTIE), the transmembrane glycoprotein (GPNMB), and an uncharacterized protein (MGC126945) (Table 1). Expression of two of these genes (MT1A and GPNMB) was studied by qPCR (Fig. 2). This analysis confirmed the repression of MTIA in liver of cows receiving B12 and B9 + B12 treatments. Results from the microarray analysis indicated an up-regulation of the expression of GPNMB with both B12 and B9 + B12 treatments. The qPCR analysis showed a numerical but not statistically significant increase with these treatments probably due to the large variation in the expression for this gene among the limited number of animals of this study (Fig. 2). Among the other 17 genes affected by the B12 treatment, the expression of seven genes were studied by qPCR but no effect were confirmed for 6 of them (HERC6, IF127, ISG15, MEP1B, PPP1R3B and SFRP1). Only the expression of the G0/G1switch 2 gene (G0S2) was up-regulated in liver of cows receiving the B12 treatment as compared to control (Fig. 2).

In addition to these four genes influenced by B12 treatment, alone and in combination with B9, 24 other genes were affected when both vitamins were administrated simultaneously to the cows; four genes were studied by qPCR. Two of them, namely the haptoprotein (HP) and lysyl oxidase-like 4 (LOXL4), were confirmed as being respectively down and up-regulated by the combined treatment. Nevertheless, the expression of LOXL4 was also up-regulated in liver of B9 cows whereas, the expression of HP was down-regulated by all vitamin treatments (Fig. 2). No difference in expression of GSTA5 and SLC22A9 could be detected.

In mammary gland, the B9 treatment modified the expression of nine genes by 2-fold as compared to cows receiving no vitamin supplement. The B12 treatment affected five genes and the expression of five genes was influenced by the B9 + B12 treatment (Fig. 1b). The B9 + B12 shared one gene with the B12 treatment. Based on results from the microarray analysis, three genes whose expression was modified by the B9 treatment (Fig. 1b), cell death-inducing DFFA-like effector a (CIDEA), the androgen binding protein beta-like (LOC785756), and the periostin (POSTN) genes were studied by qPCR in addition to some candidate genes (Fig. 3). Out of the 11 genes analyzed by qPCR, two genes were confirmed; then, the percentage of false positive in the mammary was 82 %. RAB15 and POSTN were significantly up-regulated respectively by the B12 and B9 as compared to control (Fig. 3). Out of the 16 genes affected in the mammary gland by vitamin supplements, 13 were classified using Panther classification system (Table 3).

Fig. 3

Expression of genes in mammary gland measured by quantitative PCR according to treatments. Control: no vitamin supplement; B9: vitamin B9 supplement; B12: vitamin B12 supplement; B9 + B12: combined supplement of vitamins B9 and B12. Means different from the control treatment are indicated by *** when P value ≤ 0.01, ** when P values were between 0.01 and 0.05 and * when there was a trend with P values between 0.05 and 0.1

Table 3

Panther gene list classification system for the genes differentially expressed in mammary gland tissuea

Treatment	Gene symbol	Gene name	Treatment regulation effect	Molecular Function	Biological Process	Protein Class
B9
POSTN		Periostin	Up-	receptor binding	cell communicationcell-matrix adhesionvisual perceptionsensory perceptionmesoderm developmentskeletal system developmentmuscle organ development	signaling moleculecell adhesion molecule
FUT5		Alpha-(1,3)-Fucosyltransferase	Up-	transferase activity, transferring glycosyl groups	protein glycosylation	glycosyltransferase
ATP6V1G3		V-Type Proton Atpase Subunit G 3	Up-	hydrolase activitycation transmembrane transporter activityproton-transporting ATP synthase activity, rotational mechanism	nucleobase-containing compound metabolic processcation transport	ATP synthasehydrolase
LOC785756		androgen binding protein beta-like	Down-	Unassigned	Unassigned	Unassigned
MGC126945		uncharacterized protein MGC126945	Down-	receptor activity	B cell mediated immunityantigen processing and presentationcellular defense response	immunoglobulin receptor superfamilymajor histocompatibility complex antigen
CIDEA		cell death-inducing DFFA-like effector a	Down-	Unassigned	induction of apoptosis	Unassigned
B12
GPR110		G-Protein Coupled Receptor 110-Related	Up-	G-protein coupled receptor activity	spermatogenesisimmune responsesynaptic transmissionneurotransmitter secretionmesoderm developmentheart developmentresponse to stressintracellular protein transportsynaptic vesicle exocytosis	G-protein coupled receptorantibacterial response protein
IRX6		iroquois homeobox 6	Up-	sequence-specific DNA binding transcription factor activitysequence-specific DNA binding transcription factor activity	transcription from RNA polymerase II promoterectoderm developmentnervous system developmentregulation of transcription from RNA polymerase II promoter	homeobox transcription factornucleic acid binding
RAB15		Ras-related protein Rab-15	Up-	Unassigned	Unassigned	Unassigned
B9 + B12
LOC509034		feline leukemia virus subgroup C receptor-related protein 2-like	Down-	transmembrane	transporter activity transport	Transporter
IDO1		indoleamine 2,3-dioxygenase 1	Down-	Unassigned	Unassigned	Unassigned
SECTM1		SECTM1 protein	Down-	Unassigned	Unassigned	Unassigned

Differentially expressed genes in mammary tissue were assigned to gene ontology pathways using the Panther tool. Some genes were not found in Panther genes list. Furthermore, among the genes ID found by the PANTHER system, some were not classified to a category. Only 3 genes ontology categories are presented: Molecular Function, Biological Process and Protein Class

aClassification with the Panther gene list classification system of the genes differentially expressed in mammary tissue following supplementation with vitamin B9 (B9), vitamin B12 (B12) or both (B9 + B12) as compared to no vitamin treatment (Control)

As shown in Tables 1 and 3, some of the genes identified by microarray in liver and mammary gland were annotated and clustered into three major gene ontology groups: Protein Class, Molecular Function and Biological Process. In Tables 2 and 4, gene expression clusters are categorized within biological processes (levels 1, 2 and 3). This allows looking for statistically over- and under-represented biological process categories among the genes. Most of the genes modulated in the liver by at least one of the vitamin supplements were associated to developmental process, protein metabolic process, transport and immune response (Table 2). However, protein metabolic process was over-represented (with 9 genes: MYOM1, HP, CDK5R1, MEP1B, LOXL4, FUT5, HERC6, LOC515676 and ISG15) in the gene list affected by treatments in the liver (Table 2). In the mammary gland, where very few genes were affected by the vitamin treatments, the over-represented biological process was developmental process involving 4 genes: CIDEA, POSTN, GPR110 and IRX6 (Table 4).

Table 4

Identification of the major biological processes of the genes differentially expressed in mammary gland tissuea

Biological process		Genes
Level 1	Level 2/3	Symbol	Fold change
apoptotic process	induction of apoptosis	CIDEA	−1.9
biological adhesion	cell adhesion	POSTN	1.1
biological regulation	regulation of biological process	IRX6	1.7
cellular process	cell communication	POSTN GPR110	1.11.3
developmental process	death	CIDEA	−1.9
	ectoderm development	IRX6	1.7
	mesoderm development	POSTN GPR110	1.11.3
	system development	POSTN GPR110 IRX6	1.11.31.7
immune system process	antigen processing and presentation	MGC126945	−1.5
	immune response	MGC126945 GPR110	−1.91.3
localization	transport	ATP6V1G3 LOC509034 GPR110	−0.9−0.61.3
metabolic process	nucleobase-containing compound metabolic process	ATP6V1G3 IRX6	−0.91.7
	protein metabolic process	FUT5	3.6
multicellular organismal process	single-multicellular organism process	POSTN GPR110	1.11.3
reproduction	gamete generation	GPR110	1.3
response to stimulus	cellular defense response	MGC126945	−1.9
	immune response	MGC126945 GPR110	−1.91.3
	response to stress	GPR110	1.3
Not found		LOC751574 VAV1 LOC614268 SAA2 C10H14orf53 H1FNT	−1.3−1.2−0.9−0.91.44.9
Unassigned		LOC785756 IDO1 SECTM1 RAB15	−2.1−1.2−0.92.0

All genes modulated in the mammary tissue by the vitamins treatment, including those that were not found or classified by the Panther system are listed. The Fold Change, as using FlexArray microarray analysis, varies from −1.9 to 4.9 and only significantly (FDR ≤ 0.05) expressed genes (compared to control) are shown

aIdentification of the major biological processes of the genes differentially expressed in mammary tissue following supplementation with vitamin B9 (B9), vitamin B12 (B12) or both vitamins (B9 + B12) as compared to no vitamin treatment (Control)

Gene symbols were uploaded to the Panther workspace in order to classify the genes of interest by selecting the Bos taurus reference gene list based on the selected organism [18]. Notwithstanding, some genes in both tissues (liver and mammary gland) could not be assigned to any biological process or molecular function category by Panther classification system (Tables 1, 2, 3 and 4). It is also possible that there is no experimental data to support their biological annotation.

Discussion

Microarray analysis was used to investigate the effects of vitamin B9 and B12 supplements given alone or in combination during the peripartum early lactation period on liver and mammary gland tissues. The expression of only a limited number of genes was modulated by the vitamin treatments in both tissues, which suggests a subtle vitamin effect on the tissue metabolism that would have been better characterized using a larger population. Notwithstanding, the qPCR analyses show that, in liver, expression of keys genes, such as DLK1, LOXL4, G0S2, GSTA5, HP, MT1A, IFI27 and SFRP1 was modulated at different extents (significant effects for five genes and a strong trend for three genes) by the vitamin treatments. Delta-like 1 homolog (DLK1/Pref-1) is a surface marker of hematopoietic progenitor cells (HPCs) associated with less differentiated hepatocellular phenotypes [19] and it has been shown to act in vitro as an inhibitor of Notch signaling [20, 21] to promote liver regeneration [21]. Interestingly, DLK1 is an imprinted gene which is involved in lipid metabolic reprogramming [22]. An increased concentration of this biomarker in blood serum is associated with hepatic cancer [23] whereas downregulation of DLK1 expression through an epigenetic mechanism contributes to attenuate liver disease [24]. Because vitamin B9 plays a major role in de novo formation of methyl groups and vitamin B12 is required for the remethylation cycle, we can speculate that DLK1 expression could also be repressed in the liver of the lactating cows through an epigenetic mechanism. Because DLK1 suppresses glucose production and fatty acid synthesis and oxidation in hepatocytes [25], supplementation of both vitamins B9 and B12 might increase liver metabolism through a genomic imprinting mechanism which negatively impacts the DLK1 pathway.

Lysyl oxidase-like member 4 (LOXL4), a matrix-remodeling enzymes, is extracellularly secreted and significantly contributes to ECM deposition [26]. Activity of lysyl oxidase (LOX) and LOX like proteins are correlated to collagen and elastin deposition and, in adult mammals, are essential to tissue maintenance [27]. Recent studies have provided compelling evidence that G0S2 is abundantly expressed in metabolically active tissues such as liver, and acts as a molecular brake on triglyceride catabolism [28]. Triglyceride hydrolase activity of adipose triglyceride lipase can be selectively inhibited by G0S2 [28]. Hence, increasing the expression of G0S2 decreased lipolysis [29] which is supported by the reduction of plasma concentrations of non-esterified fatty acids in cows receiving vitamin B9 supplements, alone or combined with vitamin B12 in the present study. Although increased IFI27 expression was not significant, the pattern was highly similar to G0S2 thus suggesting a similar B9 supplement effect on the liver for this gene. Expression of the alpha-inducible protein 27 (IFI27) is up-regulated during inflammatory wound repair process [30] and expression of this gene also alters immune response and mitochondrial function [31]. Interestingly, DLK1 locus expression is also associated with a restriction of the mitochondrial metabolism [32]. These gene expression patterns support the hypothesis that both vitamins B9 and B12 improve the hepatic function which might reduce metabolic stress during the transition period and early lactation of dairy cows. This is further supported by the marked reduction of the HP and MT1A genes. The liver is the major site for the synthesis of acute phase proteins including haptoglobin (HP) and metallothionein 1A (MT1A) [30]. During stress response, it is reported that physiological processes aimed on redistribution of energy utilization in specific organs stimulating mobilization of body reserves. In mammary gland, administration of the three vitamin treatments had a very limited effect on gene expression as described above. Interestingly, in the present study, whereas vitamin treatments had no effect on milk total solids yield and dry matter intake, vitamin B9 supplements, given alone or in combination with vitamin B12, decreased body condition score losses during the first weeks of lactation as well as plasma concentrations of non-esterified fatty acids and β-hydroxybutyrate [9] suggesting an improvement in energy balance for these cows.

In the present study, all the genes that have their expression affected by the vitamin treatments in the liver, are involved in tissue repair, resorption of inflammation and lipid metabolism although no mode of action can be clearly identified. During the first weeks of lactation, dairy cows are generally in negative energy balance because nutrient intake increases less rapidly than nutrient demand for initiation of lactation which leads to mobilization of body reserves. Cows are losing body condition score and non-esterified fatty acids are released from adipose tissues and their plasma concentrations increased. During this period, dairy cows are also prone to liver steatosis because hepatic uptake of non-esterified fatty acids is greater than the amounts oxidized or secreted by the liver [33]. Accumulation of lipids in liver affects integrity and function of hepatic cells [33]; in response, liver parenchymal cells produce an acute-phase glycoprotein haptoglobulin [34]. Because a decrease in DLK1 can improved fatty acid oxidation from hepatocytes [25], an improved β-oxidation of non-esterified fatty acids in liver could help to reduce ketone body formation and plasma concentrations of β-hydroxybutyrate. The improvement in energy balance observed in cows receiving supplementary vitamin B9, alone or combined with vitamin B12, likely reduced the liver burden caused by mobilization of body fat reserves which could explain the changes in hepatic gene expression described above. For instance, the increase of LOXL4 and G0S2 strongly support that these treatments protect body fat from catabolism. Prevention of liver damage or improved liver performance is not only important for maintaining liver function but also for general health of high-yielding dairy cows.

Conclusion

In the present study, a supplement of vitamin B9, given alone or in combination with vitamin B12, reduced mobilization of body fat reserves and hepatic lipid catabolism in early lactation. Changes in expression of genes described above support the hypothesis that hepatic tissue integrity in early lactation was improved by these vitamin supplements.

Methods

Animals and treatments

For the purpose of the present study, biopsies of hepatic and mammary tissues were taken from 24 multiparous Holstein cows from the dairy herd at the Agriculture and Agri-Food Canada Research Centre (Sherbrooke, Quebec, Canada) at the end of a larger study [9]. Care of cows followed the guidelines of the National Farm Animal Care Council (2009) [35]. Animals were kept in a tie-stall barn under 18:30 h of light per day (05:00 to 23:30 h) and milked twice daily (07:30 and 19:30 h). The experimental period began 3 weeks before the expected calving date and lasted until 9 weeks of lactation. The cows were fed ad libitum a close-up diet beginning 3 weeks before the expected date of calving until parturition and, then a lactation diet both formulated to meet or exceed the National Research Council (NRC) recommendations [36]. Long hay (0.5 kg) was given at 07:30 h and total mixed ration was served once daily at 08:30 h. Cows had free access to water.

Cows were assigned to 6 blocks of 4 animals each according to their 305-d milk production during the previous lactation. Within each block, cows were randomly assigned to one of the following treatments: weekly intramuscular injections of 5 mL of either saline 0.9 % NaCl (Control group), 320 mg of pteroylmonoglutamic acid (MP Biomedicals, Solon, OH, USA; (Vitamin B9 group), 10 mg of cyanocobalamin (5 000 μg/mL, Vetoquinol, Lavaltrie, Quebec, Canada; (Vitamin B12 group) or 320 mg of pteroylmonoglutamic acid and 10 mg of cyanocobalamin (B9 + B12 group). Thus, there were 6 animals per treatment group.

Biological material collection and tissue handling

Mammary gland and hepatic tissues were obtained from the lactating dairy cows, 64 ± 3 days after calving. Biopsies were performed under local anesthesia. The process of hepatic biopsies uses ultrasound guidance to minimize the hemorrhagic risks [4]. Both procedures were approved by the Institutional Committee on Animal Care of the Sherbrooke Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada according to the guidelines of the Canadian Coucil on Animal Care [37]. Tissues were immediately frozen into liquid nitrogen and stored at −80 °C until use.

Total RNA isolation and purification

Total RNA was extracted from hepatic and mammary tissues by using a QIAzol Lysis Reagent (QIAGEN Inc., Toronto, ON, Canada) following the original manufacturer’s protocol, with slight modifications. Briefly, frozen samples (100 mg of tissue) were homogenized in 2 mL of QIAzol Lysis Reagent on ice using a Tissue-Tearor. A volume of 600 μL QIAzol Lysis Reagent was added to 400 μL of homogenate; the mixture was vigorously vortexed and kept at room temperature for 5 min to promote dissociation of nucleoprotein complexes. A volume of 200 μL of chloroform was added; the mixture was shaken and left at room temperature for 3 min followed by a centrifugation at 12 000 × g for 15 min at 4 °C to remove lipids. After centrifugation, the aqueous fraction (upper layer) was taken and RNA was precipitated by adding an equal volume of 70 % ethanol. RNA was purified according to manufacturer’s procedure using RNeasy Mini Kit (QIAGEN Inc., Toronto, ON, Canada), including on-column DNase digestion. The purity, concentration, and integrity of total RNA intended for qPCR were assessed. Purity of the RNA was evaluated by absorbance (A) readings (ratio of A260/A230 and A260/A280) using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, US). NanoDrop ND-1000 spectrophotometer was also used to measure the concentration. All RNA samples passed the quality control. The RNA Integrity Number calculated by the Bioanalyzer software at McGill University and Génome Québec Innovation Center (Montreal, Quebec, Canada) ranged from 7.3 to 8.7.

Microarray

McGill University and Génome Québec Innovation Center (Montreal, Quebec, Canada) performed the microarray analysis. Cyanine 3-labeled CTP complementary RNA (cRNA) was produced with 50 ng of total RNA using the Low Input Quick Amp Labeling Kit, according to manufacturer’s instructions (Agilent Technologies, Inc). The quality of cRNA was evaluated by capillary electrophoresis on 2100 Electrophoresis Bioanalyzer instrument (Agilent technologies, Santa Clara, CA, USA). A total of 15 525 genes were analyzed via expression levels of 42 789 probes using the Agilent Bovine Genome Oligo Microarrays 4 × 44 K (G2519F-023647) (Agilent technologies, Santa Clara, CA, USA). Labeling, hybridization, and raw data extraction were performed by McGill University and Génome Québec Innovation Center (Montreal, Quebec, Canada) according to the manufacturer’s instructions, as previously described [38]. Hybridizations were performed by batch with samples randomly distributed. The hybridizations of microarrays were compared through a correlation matrix that enables the quick identification of poor and divergent replication (data not shown). Once the slides are scanned, the respective “.tif” image was examined using the Agilent Feature Extraction (FE) software. Scan image information is displayed in the Scan Image Properties for images that were generated using the Agilent Scanner. Then data are extracted with the FE software. A quality control (QC) report is generated for each sample. The FE version 10.7.3.1 with GE1_107_Sep09 protocol and grid associated with the selected type of chip 023647_D_F_20110614 were used. All microarray datasets passed all the quality criteria and were then downloaded into the FlexArray microarray analysis software (http://gqinnovationcenter.com/documents/technicalNotes/technicalNotes_GQ06.pdf). Array data have been submitted to the public databases and assigned Gene Expression Omnibus (GEO) accession number is GSE77421.

FlexArray, a Bioconductor R based software, was developed by Génome Québec to provide researchers with a user-friendly interface for the analysis of microarray experiments. Raw microarray expression intensities were corrected for background using normexp, according to Ritchie et al. [39]. Between-array normalization was performed so that the background corrected intensities have similar distributions across the arrays. Comparison of the vitamin treatments with the control tissue was performed by Cyber-T, a version of the t-test that uses a Bayesian estimate of the within treatment variance [40, 41]. Up- or downregulated genes lists were analyzed on Protein ANalysis THrough Evolutionary Relationships (Panther) classification system [42].

Quantitative real time-PCR

Quantifications by real-time PCR (qPCR) following reverse transcription were performed as previously described [43] with minor modifications. The reverse transcription PCR reactions were performed with the SuperScript II reverse transcriptase (Life Technologies Inc., Burlington ON, Canada) according to the manufacturer’s protocol and using 500 ng of total RNA extracted from each of the 24 animals. An equivalent quantity of cDNA is synthesized in a final reaction volume of 20 μL, giving a concentration of 25 ng/μl of cDNA. A pool of cDNA intended to estimate the efficiencies primers, was made using the cDNA of each animal. Primers were designed for each gene using the Primer Express 3 software package (Applied Biosystems, Life Technologies Corporation, Burlington, ON, Canada) using the reference sequence from the RefSeq database of the National Center for Biotechnology Information depository. Primers for a total of 34 genes for both tissues were designed. Optimizations of primers were performed for each gene by testing different concentrations of both forward and reverse primers, each ranging from 50 to 900 nM. Estimations of primer efficiencies were analyzed using the standard curves made from a serial of seven dilutions (1/7.5, 1/15, 1/30, 1/60, 1/120, 1/240, 1/480) of the pool of the cDNA samples (25 ng/μl of cDNA). As 3 μl of each dilution were used in a final reaction volume of 10 μl for qPCR, the concentrations used per dilution were: 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.015625 (ng/μl). Additional file 1 provides experimental information and PCR amplification efficiency for all genes. The qPCR reactions (10 μL, final volume) were performed on 96 well plates using Fast SYBR Green PCR Master Mix (Applied Biosystems) in a 7500 Fast Real Time-PCR System (Life Technologies, Burlington, ON, Canada) as the manufacturer’s instructions. The PCR thermal cycling conditions comprised an initial 20 s denaturation step at 95 °C followed by 40 cycles at 95 °C for 3 s followed by an annealing/elongation period at 60 °C for 30 s. A dissociation step was included for all amplifications to confirm the presence of single discrete PCR products of the expected size. Twenty-five genes (14 from hepatic tissue data and 11 from mammary gland tissue data) were subject to qPCR validation because they were found differentially expressed by microarray as expressed by log2 of their fold change. In addition, in the mammary gland, four genes (FOLR1, ELF5, B4GALT1 and LALBA) were chosen because of their implication in the metabolic pathway involving vitamins B9 and B12. The expression of 5 putative reference genes, namely actin beta (ACTB), ubiquitously-expressed transcript (UXT), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) was determined for all samples as recommended [44-46]. All the 24 animals were used to perform the qPCR analysis of 34 genes for both tissues. Once the more stable genes were selected, the normalization factor was calculated using a geometrical average as recommended [45]. The combination of ACTB and PPIA was appropriate to normalize the data from hepatic tissue. The appropriated combination was UXT and PPIA to normalize the data from mammary gland.

Data analysis and statistics

Using FlexArray microarray analysis, a significant result at a P value < 0.05 after false discovery rate correction with a minimum of ± two-fold change in gene expression for the respective treatment group compared to control tissues was considered biologically interesting. Gene category over-representation analysis consists in grouping genes into categories by some common biological property and then tested to find categories that are over represented amongst the differentially expressed genes. Gene ontology describes and categorizes gene products in three non-overlapping domains of molecular biology [47]. Panther (http://pantherdb.org) is a visualisation browser of GO [48], using version 10 which included other organisms. Panther ranks proteins (and their genes) according to Family (and subfamily), Molecular function, Biological process and Pathway. The process of classification is extensively explained by Mi and colleagues [42]. Only significant differentially expressed genes were analysed by qPCR. Data from qPCR were analyzed using SAS Institute procedures (2008). Means were assumed to be different at P ≤ 0.05 and tended to differ at 0.05 < P ≥ 0.1. Normfinder indicated the interested combination of reference genes to normalize qPCR data.

Abbreviations

ECM, extracellular matrix components; GO, gene ontology; HPCs, hepatic progenitor cells; Panther, protein analysis through evolutionary relationships; qPCR, quantitative polymerase chain reaction.