One-carbon metabolite supplementation increases vitamin B₁₂, folate, and methionine cycle metabolites in beef heifers and fetuses in an energy dependent manner at day 63 of gestation

Abstract

One-carbon metabolites (OCM) are metabolites and cofactors which include folate, vitamin B₁₂, methionine, and choline that support methylation reactions. The objectives of this study were to investigate the effects of moderate changes in maternal body weight gain in combination with OCM supplementation during the first 63 d of gestation in beef cattle on (1) B₁₂ and folate concentrations in maternal serum (2) folate cycle intermediates in maternal and fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF) and (3) metabolites involved in one-carbon metabolism and related metabolic pathways in maternal and fetal liver. Heifers were either intake restricted (RES) and fed to lose 0.23 kg/d, or fed to gain 0.60 kg/d (CON). Supplemented (+ OCM) heifers were given B₁₂ and folate injections weekly and fed rumen-protected methionine and choline daily, while non-supplemented (-OCM) heifers were given weekly saline injections. These two treatments were combined in a 2 × 2 factorial arrangement resulting in 4 treatments: CON-OCM, CON + OCM, RES-OCM, and RES + OCM. Samples of maternal serum, maternal and fetal liver, ALF, and AMF were collected at slaughter on day 63 of gestation. Restricted maternal nutrition most notably increased (./ ≤ 0.05) the concentration of vitamin B₁₂ in maternal serum, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate in maternal liver, and cystathionine in the fetal liver; conversely, maternal restriction decreased (P = 0.05) 5,10-methylenetetrahydrofolate concentration in fetal liver. Supplementing OCM increased (P ≤ 0.05) the concentrations of maternal serum B₁₂, folate, and folate intermediates, ALF and AMF 5-methyltetrahydrofolate concentration, and altered (P ≤ 0.02) other maternal liver intermediates including S-adenosylmethionine, dimethylglycine, cystathionine Glutathione reduced, glutathione oxidized, taurine, serine, sarcosine, and pyridoxine. These data demonstrate that OCM supplementation was effective at increasing maternal OCM status. Furthermore, these data are similar to previously published literature where restricted maternal nutrition also affected maternal OCM status. Altering OCM status in both the dam and fetus could impact fetal developmental outcomes and production efficiencies. Lastly, these data demonstrate that fetal metabolite abundance is highly regulated, although the changes required to maintain homeostasis may program altered metabolism postnatally.

We found moderate changes in body weight gain in beef heifers during the first 63 days of gestation increased maternal serum B₁₂, hepatic folate intermediates, and one-carbon metabolism-related metabolites, and altered fetal hepatic metabolite concentrations. Additionally, supplementation with one-carbon metabolites increased maternal serum and hepatic metabolites, as well as fetal fluid folate intermediates. These changes are likely linked to developmental outcomes.

Introduction

Maternal nutrient restriction, even during the earliest stages of gestation, can have short- and long-term impacts on the fetus and offspring and result in developmental programming (Ford et al., 2007; Long et al., 2009; Caton et al., 2020; Reynolds et al., 2022a). Early gestation is a critical window for fetal growth and development (Winters et al., 1942; Dahlen et al., 2021; Reynolds et al., 2022b). In beef cattle, successful early pregnancy events are essential for maintaining reproductive and whole herd efficiencies. Therefore, there are increasing efforts to identify the mechanisms by which maternal nutrition and supplementation affect fetal metabolism and growth (Paradis et al., 2017; Liu et al., 2021; Crouse et al., 2022c; Menezes et al., 2023). Developmental programming is regulated, in part, by epigenetic modifications, which include DNA methylation and histone modifications (Meyer et al., 2012; Reynolds and Caton, 2012; Reynolds et al., 2017; Crouse et al., 2022a). Epigenetic modifications rely on one-carbon metabolites (OCM), which include methionine, choline, folate, and vitamin B₁₂. A maternal diet deficient in folate, B₁₂, and methionine in pregnant ewes led to altered methylation of CpG sites in the offspring (Sinclair et al., 2007). Additionally, those lambs were heavier and fatter and experienced increased insulin resistance and blood pressure after birth (Sinclair et al., 2007). Supplementing OCM to beef heifers during the first 14 d of the estrous cycle altered serum and hepatic OCM concentrations (Crouse et al., 2022b).

One-carbon metabolism primarily consists of the folate cycle, the methionine cycle, and the transsulfuration pathway, and it plays a major role in the methylation of DNA, RNA, and proteins, among other metabolic processes (Clare et al., 2019). The major function of the methionine cycle is to convert methionine to s-adenosylmethionine (SAM), which acts as universal methyl donor for many biological reactions (Mudd et al., 1995; McFadden et al., 2020). The folate cycle, along with vitamin B₁₂ serves to remethylate homocysteine to methionine as well as being a source of purines and pyrimidines (Finkelstein, 2000; Kalhan and Marczewski, 2012). The transsulfuration pathway is involved in the removal of homocysteine in the absence of remethylation and regulation of redox reactions within cells (Clare et al., 2019). Previous research by our lab group found restricting maternal nutrition altered concentrations of amino acids, including methionine and homocysteine, in maternal serum and fetal fluids (Crouse et al., 2019). Additionally, restricted maternal nutrition results in increased B₁₂ and folate concentrations in allantoic fluid (Syring et al., 2023). Little research exists in the literature investigating the effects of supplementing OCM on beef cattle experiencing moderate nutrient restriction during early gestation.

We hypothesized moderate differences in maternal rate of gain (achieved through nutrient restriction) and supplementation of OCM would alter concentrations of B₁₂ and folate in maternal serum, impact folate metabolic intermediates in maternal and fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF), and alter key aspects of the metabolome associated with the folate cycle, methionine cycle, and the transsulfuration pathway in maternal and fetal liver at day 63 of gestation in beef heifers. Specifically, we hypothesized that OCM supplementation would increase B₁₂, folate, and folate intermediates, saturate the folate and methionine cycle, and shift the metabolome towards secondary pathways (transsulfuration and oxidative phosphorylation) associated with one-carbon metabolism. Additionally, we hypothesized nutrient restriction would increase concentrations of B₁₂ and folate in the maternal serum, maternal and fetal liver, and fetal fluids.

Materials and Methods

Animals, diet, and treatment

All procedures were approved by the North Dakota State University Animal Care and Use Committee. Seventy-two Angus-cross heifers were estrus synchronized using a 7-d Select-Synch and CIDR protocol, and artificially inseminated using female-sexed semen from a single sire (Maternal Made [ST Genetics, Navasota, TX]). All heifers were individually fed in the NDSU Animal Nutrition and Physiology Center at 1 of the 2 levels of gain using Calan gates (American Calan, Northwood, NH): control (CON; targeted: 0.45 kg/d ADG, actual: 0.60 kg/d ADG) or restricted (RES; targeted: −0.23 kg/d ADG, actual: −0.23 kg/d ADG). Diets were a total mixed ration of corn silage, alfalfa hay, corn grain, and alfalfa/grass hay fed to reach targeted gains described above, with a daily top-dressed vitamin/mineral premix (Trouw dairy VTM w/Optimins, Trouw Nutrition USA, Highland, IL) delivered via a fine-ground corn carrier. Further details of the diet composition can be found in Table 1. Heifers were weighed and diet intake was adjusted weekly to reach body weight gain targets. Heifers also either received OCM supplementation (+OCM; 7.4 g/d rumen-protected methionine [Smartamine, Adisseo, China] + 44.4 g/d rumen-protected choline [ReaShure, Balchem Inc., New Hampton, NY] + 20 mg B₁₂ [MWI Animal Health, Boise, ID] + 320 mg folic acid (FA) [Spectrum Chemical Mfg. Corp., New Brunswick, NJ]) or no supplementation (−OCM; corn carrier and saline injections) for a 2 × 2 factorial design with the rate of gain and OCM supplementation serving as the factors. Methionine and choline were fed per the manufacturer’s recommendations and included in the daily top-dressed fine-ground corn carrier. Injections of folate (6 cc), B₁₂ (4 cc), and saline (one 6cc and one 4cc injection) were administered intramuscularly every week per Beef Quality Assurance guidelines for injection. Supplement levels for B₁₂ and folate were determined previously by Crouse et al. (2022a). The 4 treatments (CON − OCM, CON + OCM, RES − OCM, or RES + OCM) were provided for 63 d after breeding. Only heifers pregnant with heifer calves were used, resulting in a final sample size of 31 heifers (CON-OCM, n = 7; CON + OCM, n = 7; RES-OCM, n = 9; RES + OCM, n = 8).

Table 1.

Ingredient and dietary composition (dry matter basis) of heifer diet

Ingredient, % of dietary DM
Ground corn	27.0
Corn silage	9.0
Alfalfa	15.0
Alfalfa/grass hay	45.0
Top-dressed supplement1	4.0
Average estimated dietary composition
Dry matter, %	83.00
Crude protein, %	13.48
Dietary metabolizable energy, Mcal/kg2	2.56
Dietary net energy of maintenance, Mcal/kg2	1.65
Dietary net energy of gain, Mcal/kg2	1.04
Supplement ingredients on a g/d basis
Trouw dairy VTM w/Optimins3	10.0
Rumen protected methionine4	7.4
Rumen protected choline5	44.4
Chemical composition of Trouw VTM premix 6
Ca, %	10.00 to 12.00
Mg, %	5.00
K, %	5.00
Co, mg/kg	180.00
Cu, mg/kg	5,100.00
I, mg/k	375.00
Fe, %	1.20
Mg, %	2.70
Se, mg/kg	132.00
Zn, %	2.70
Vitamin A, IU/kg	2,755,775.00
Vitamin D3, IU/kg	771,617.00
Vitamin E, IU/kg	13,228.00

¹Top-dressed supplement delivered using a fine ground corn carrier. For -OCM heifers, supplement comprised of Trouw dairy VTM w/Optimins mix and fine ground corn carrier. For +OCM heifers, the supplement comprised of Trouw dairy VTM w/Optimins mix, rumen-protected methionine, rumen-protected choline, and fine ground corn carrier.

²Calculated values from the NASEM (2016).

³Trouw dairy VTM w/Optimins, Trouw Nutrition USA, Highland, Illinois.

⁴Smartamine, Adisseo, China.

⁵ReaShure, Balchem Inc., New Hampton, NY.

⁶Trouw VTM premix fed to meet or exceed vitamin and mineral requirements.

Sample collection and preparation

Maternal serum samples were collected via jugular venipuncture on day 62 (the day before slaughter) of gestation using 10 mL serum monoject corvac blood collection tubes (Cardinal Health, Dublin, OH). Blood samples were allowed to clot for 20 min at room temperature before being centrifuged at 2,000 × g for 10 min. The serum was then separated from the rest of the contents and stored at −80 °C. Serum samples (1 mL) were sent to IDEXX BioAnalytics (N. Grafton, MA) and analyzed on a Siemens Immulite 2,000 analyzer by chemiluminescence immunoassay methodology for B₁₂ and folate analysis (Crouse et al., 2022b).

On day 63 of gestation, heifers were slaughtered at the NDSU Meat Lab, which is a USDA Federally inspected facility, using captive bolt and exsanguination. At slaughter, maternal and fetal liver samples were collected, placed on dry ice, and stored at −80 °C. Allantoic fluid (ALF) and amniotic fluid (AMF) samples were collected using sterile hypodermic needles and syringes and stored at −80 °C. Samples were transported to USDA US Meat Animal Research Center in Clay Center, NE, where they were analyzed for folate intermediate concentrations using ultra-high performance liquid chromatography MS/MS (UPLC-MS/MS) as outlined by Crouse et al. (2022a). Approximately 80 mg of each liver sample was weighed out for mass spectrometry analysis. Next, 450 µL HEPES buffer (pH 8.0) and 40 µL charcoal-treated plasma were added to the samples prior to homogenization and incubation. Samples were incubated at 37 °C for 3 h, centrifuged at 14,000 × g for 30 min, and stored at −80 °C until analysis. For ALF and AMF samples, 500 µL of sample was combined with 450 µL HEPES buffer and 40 µL charcoal-treated plasma, vortexed, and incubated at 37 °C for 3 h. After centrifuging for 30 min at 14,000 × g, 100 mL of filtrate was collected and stored at −80 °C for analysis. Additionally, 50 mg of each maternal and fetal liver sample were sent to Metabolon (Metabolon, Inc., Durham, NC) for metabolomic analysis. Briefly, samples were prepared using the automated MicroLab STAR system (Hamilton Company, Reno, NV) and then aliquoted for analysis via reverse phase UPLC-MS/MS and HILIC/UPLC-MS/MS. Compounds were compared with library entries of purified standards or unknown recurrent entities for identification. For a more detailed methodology, see Crouse et al. (2022c). Only results for intermediates involved in one-carbon metabolism and its related pathways will be discussed in this paper.

Statistical analysis

Data for serum B₁₂ and folate concentrations were analyzed using the MIXED procedure of SAS (version 9.4, SAS Institute Inc.). Model effects included fixed effects of heifer gain, OCM supplementation, and their interaction. The LSMEANS with Tukey adjustment were used to separate means. If no significant interactions were found (P > 0.05), main effects were reported. Tendencies were reported at a P-value ≤ 0.10 and >0.05.

The MIXED procedure of SAS was also used to analyze maternal and fetal folate intermediate concentration data. The rate of heifer gain, OCM supplementation, and their interaction were included as fixed effects in the model. The LSMEANS with Tukey adjustment were used to separate means. Values were considered significant at P ≤ 0.05, a tendency at 0.10 ≥ P > 0.05, and main effects were reported if no significant interaction was found.

Raw data from Metabolon were analyzed and quality control was processed by Metabolon’s hardware and software as described by Crouse et al. (2022c). Briefly, one-carbon metabolism-related metabolites were identified by comparison to a library of purified standards or recurrent unknown entities. Two-way ANOVAs were performed with the main effects of the rate of gain and OCM supplementation and their interaction. Contrasts were performed for each of the main effects. The P-values were provided and considered significant if P ≤ 0.05 and a tendency at 0.10 ≥ P > 0.05.

Results

Maternal serum B₁₂ and folate concentrations

Results for maternal serum B₁₂ and folate concentrations can be found in Table 2. There were no gain × OCM supplement interactions (P ≥ 0.49), so only the main effects will be discussed. The B₁₂ and folate concentrations were greater (P < 0.01) in + OCM heifers compared with −OCM heifers. Additionally, gain had an effect on B₁₂ concentration where RES heifers had greater (P = 0.03) concentration than CON heifers.

Table 2.

Effects of gain and one-carbon metabolite supplementation on B₁₂ and folate concentrations in maternal serum

		OCM supplement1				P-value2
Vitamin	Gain3	−OCM	+OCM	Gain	SEM4	Gain	Supp.	Gain × supp.
B12, ng/L	CON	353.0	764.7	558.9	100.7	0.03	<0.01	0.49
	RES	504.0	1,043.0	773.5
	Supp.	428.5	903.9
Folate, µg/L	CON	12.48	17.77	15.13	1.69	0.32	<0.01	0.77
	RES	13.57	19.78	16.67
	Supp.	13.03	18.77

¹OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection.

²Probability values for the effects of gain, OCM supplementation (supp.), and gain × supp. Bold text indicates stastical significance.

³Gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. Supp, the main effect means of −OCM and +OCM treatments.

⁴Greatest SEM for gain × supp interaction.

Maternal and fetal hepatic, allantoic, and amniotic folate intermediate concentrations

Results for maternal and fetal folate intermediate concentrations can be found in Table 3. Only the concentration of the folate cycle intermediate 5-methyl tetrahydrofolate (THF) could be detected in ALF and AMF and it is presented in Table 3 for the fetal fluids. In the maternal liver, there was a heifer gain × OCM supplement interaction for FA (P = 0.04) and dihydrofolate (DHF; P = 0.05), where RES + OCM concentrations were greater than all other treatments. There were also tendencies for an interaction for 5,10-methenyl (P = 0.07) and 5,10-methylene (P = 0.08) concentrations where RES + OCM tended to be greater than all other treatments. There was an OCM supplement effect for THF (P < 0.01), 5,10-methenyl THF (P < 0.01), and 5-methyl THF (P < 0.01), where + OCM heifers had greater intermediate concentrations compared with −OCM heifers. Restricted heifers tended to have greater concentrations of 5,10-methenyl THF (P = 0.06) and 5,10-methylene THF (P = 0.07), while +OCM heifers also tended to have greater concentrations of 5,10-methylene THF (P = 0.10) compared with −OCM heifers. Control fetal livers were found to have a greater (P = 0.05) concentration of 5,10-methylene THF than RES fetal livers. In both ALF and AMF, 5-methyl THF was greater in +OCM (P < 0.01 and P = 0.03, respectively) than in −OCM. There also tended (P = 0.06) to be a gain × OCM supplement interaction for ALF 5-methyl THF where RES + OCM tended (P = 0.06) to be greater than RES − OCM and CON − OCM.

Table 3.

Effects of one-carbon metabolite supplementation and gain on folate cycle intermediate concentrations in maternal liver, fetal liver, allantoic fluid, and amniotic fluid samples at day 63 of gestation

	Intermediate (ng/mL)		OCM supplement1				P-value2
Matrix		Gain3	−OCM	+OCM	Gain	SEM4	Gain	Supp.	Gain × supp.
Maternal liver	Folic acid	CON	3.91a	4.04a	3.98	1.93	0.04	0.03	0.04
		RES	4.04a	12.08b	8.06
		Supp.	3.98	8.06
	DHF	CON	3.87a	4.30a	4.09	1.81	0.04	0.03	0.05
		RES	3.96a	11.56b	7.76
		Supp.	3.91	7.93
	THF	CON	47.68	127.31	87.50	22.33	0.12	<0.01	1.00
		RES	13.69	93.35	53.52
		Supp.	30.68	110.33
	5,10-methylene THF	CON	334.68	323.02	328.85	112.01	0.07	0.10	0.08
		RES	341.81	719.47	530.64
		Supp.	338.24	521.25
	5,10-methenyl THF	CON	3.03	5.83	4.43	2.43	0.06	<0.01	0.07
		RES	3.10	14.76	8.93
		Supp.	3.06	10.30
	5-methyl THF	CON	32.96	148.19	90.58	17.42	0.89	<0.01	0.18
		RES	53.69	123.01	88.35
		Supp.	43.33	135.60
Fetal liver	Folic acid	CON	11.17	7.14	9.15	2.42	0.29	0.19	0.66
		RES	7.77	5.71	6.74
		Supp.	9.47	6.43
	DHF	CON	7.12	5.21	6.17	1.57	0.22	0.27	0.85
		RES	5.02	3.66	4.34
		Supp.	6.07	4.43
	THF	CON	37.61	41.80	39.71	15.26	0.73	0.46	0.65
		RES	36.14	53.13	44.63
		Supp.	36.87	47.47
	5,10-methylene THF	CON	351.13	482.62	416.88	72.67	0.05	0.26	0.43
		RES	265.61	289.03	277.32
		Supp.	308.37	385.83
	5,10-methenyl THF	CON	9.47	8.26	8.86	1.58	0.15	0.91	0.49
		RES	6.27	7.14	6.70
		Supp.	7.87	7.70
	5-methyl THF	CON	12.27	8.46	10.36	2.56	0.17	0.23	0.70
		RES	7.99	6.05	7.02
		Supp.	10.13	7.25
ALF5	5-methyl THF	CON	0.94	1.23	1.09	0.28	0.24	<0.01	0.06
		RES	0.75	2.03	1.39
		Supp.	0.85	1.63
AMF6	5-methyl THF	CON	1.12	1.46	1.29	0.31	0.90	0.03	0.25
		RES	0.80	1.86	1.33
		Supp.	0.96	1.66

¹OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection.

²Probability values for the effects of gain, OCM supplementation (supp.), and gain × supp. Bold text indicates statistical significance.

³Gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. Supp, the main effect means of −OCM and +OCM treatments.

⁴Greatest SEM for gain × supp interaction.

⁵Only 5-methyl THF concentration was high enough to be detected in ALF.

⁶Only 5-methyl THF concentration was high enough to be detected in AMF.

DHF, dihydrofolate; THF, tetrahydrofolate.

^a-bMeans without a common superscript differ (P < 0.05).

Results for maternal liver folate intermediate concentrations were corrected for total maternal liver weights because gain had an effect on maternal liver weight (CON = 4,291.9 ± 230.8 g, RES = 3,071.2 ± 209.8 g; P < 0.01) and can be found in Table 4. No significant (P ≥ 0.10) interactions were found in corrected maternal liver folate intermediates, except for 5,10-methenyl THF where there was a tendency (P = 0.08) for a gain × OCM supplement interaction where RES + OCM tended to have greater concentration than all other treatments. The OCM-supplemented heifers had greater concentrations of FA (P = 0.02), DHF (P = 0.03), THF (P < 0.01), 5,10-methenyl THF (P < 0.01), 5,10-methylene THF (P = 0.05), and 5-methyl THF (P = 0.02) compared with -OCM heifers. Restricted heifers had greater concentrations of 5,10-methenyl THF (P = 0.03) and 5,10-methylene THF (P = 0.05) compared with CON heifers. Restricted heifers also tended to have greater concentrations of FA (P = 0.08) and DHF (P = 0.09). Fetal liver weight was not affected by gain, OCM supplementation, or their interaction as reported by King et al. (2022), and fetal folate intermediate concentrations were not corrected for total fetal liver weight.

Table 4.

Effects of one-carbon metabolite supplementation and gain on folate cycle intermediate concentrations in maternal liver corrected for total liver weight at day 63 of gestation

		OCM supplementation1				P-value2
Intermediate (ng/mg)	Gain3	−OCM	+OCM	Gain	SEM4	Gain	Supp.	Gain × supp.
Folic acid	CON	0.90	1.68	1.29	0.80	0.08	0.02	0.20
	RES	1.36	4.14	2.75
	Supp.	1.13	2.91
DHF	CON	0.90	1.78	1.34	0.79	0.09	0.03	0.26
	RES	1.35	3.97	2.66
	Supp.	1.12	2.88
THF	CON	10.68	36.68	23.68	6.80	0.31	<0.01	0.92
	RES	4.68	29.29	16.99
	Supp.	7.68	32.99
5,10-methylene THF	CON	81.84	115.01	98.43	38.58	0.05	0.05	0.27
	RES	116.96	233.48	175.22
	Supp.	99.40	174.25
5,10-methenyl THF	CON	0.73	1.81	1.27	0.75	0.03	<0.01	0.08
	RES	1.08	4.77	2.92
	Supp.	0.90	3.29
5-methyl THF	CON	7.66	64.02	35.84	15.83	0.65	0.02	0.26
	RES	18.24	39.48	28.86
	Supp.	12.95	51.75

¹OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection.

²Probability values for the effects of gain, OCM supplementation (supp.), and gain × supp. Bold text indicates statistical significance.

³Gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. Supp, the main effect means of −OCM and +OCM treatments.

⁴Greatest SEM for gain × supp interaction.

DHF, dihydrofolate; THF, tetrahydrofolate.

Maternal and fetal hepatic one-carbon metabolism intermediate concentrations

Results for maternal liver one-carbon metabolism-related metabolite concentrations can be found in Table 5. There were gain × OCM supplementation interactions (P ≤ 0.05; Table 5; Figure 1) for betaine, cystathionine, taurine, and sarcosine. For betaine, RES − OCM was greater (P = 0.01; 1.34-fold) than CON − OCM; however, RES + OCM was less than CON − OCM and CON + OCM (0.76 and 0.50-fold, respectively). In the transsulfuration pathway, cystathionine was greater in CON + OCM and RES + OCM compared with CON − OCM and RES − OCM, respectively (1.63- and 2.99-fold). Conversely, RES-OCM was less than CON − OCM (P = 0.05; 0.66-fold). Taurine in CON + OCM was greater than CON − OCM (P = 0.02; 1.98-fold) while RES + OCM was less than compared with CON + OCM (P = 0.02; 0.68-fold). Within the amino acid interconversion pathway, sarcosine was greater (P = 0.02) in RES + OCM vs RES-OCM (1.7-fold), RES-OCM vs CON − OCM (1.66-fold), and RES + OCM vs CON + OCM (2.93-fold).

Table 5.

Metabolites and cofactors involved in one-carbon metabolism and connected pathways including polyamine synthesis and the phosphatidylcholine pathway in day 63 maternal liver

		P-values			Two-way ANOVA contrasts3
Pathway	Metabolite	Gain1	Supp.2	Gain × supp.	CON + OCM CON − OCM	RES + OCM RES − OCM	RES-OCM CON − OCM	RES + OCM CON + OCM
Folate cycle	5-methyltetrahydrofolate	0.21	0.09	0.33	2.05	1.28	1.07	0.67
Methionine cycle	Choline	<0.01	0.11	0.56	1.16	1.10	0.80	0.76
	Betaine	0.76	<0.01	0.01	0.90	0.50	1.34	0.75
	Dimethylglycine	<0.01	<0.01	0.31	1.44	2.24	2.37	3.68
	Methionine	0.51	0.09	0.39	0.94	0.82	1.14	0.99
	s-adenosylmethionine	0.02	0.14	0.29	0.93	0.72	0.81	0.62
	s-adenosylhomocysteine	0.44	0.02	0.50	1.14	1.26	1.01	1.12
	Adenosine	0.02	0.30	0.77	1.12	1.11	0.84	0.84
	Homocysteine	0.89	0.96	0.58	1.14	1.15	1.05	1.07
Transsulfuration pathway	Cystathionine	0.36	<0.01	0.05	1.63	2.99	0.66	1.21
	Homoserine	0.61	0.56	0.10	1.27	0.64	1.26	0.63
	Cysteine	0.18	0.08	0.69	1.16	1.17	0.88	0.89
	Glutathione, reduced	<0.01	0.01	0.53	1.35	2.22	0.40	0.66
	Glutathione, oxidized	0.02	<0.01	0.30	1.87	4.52	0.29	0.71
	Taurine	0.83	<0.01	0.02	1.98	0.99	1.36	0.68
Amino acid interconversion	Glycine	0.01	0.31	0.19	1.02	0.91	1.20	1.07
	Sarcosine	<0.01	0.05	0.02	0.96	1.70	1.66	2.93
	Serine	0.93	<0.01	0.38	0.68	0.50	1.19	0.88
Cofactors	Pyridoxine	0.30	0.02	0.76	0.67	0.79	0.85	1.02
	Pyridoxal phosphate	0.77	0.34	0.52	1.26	0.98	1.19	0.94
	NAD+	<0.01	0.18	0.93	1.09	1.09	0.74	0.75
	NADH	<0.01	0.12	0.56	1.00	1.41	0.35	0.49
	FAD	<0.01	0.39	0.36	1.12	0.99	0.88	0.77
	Riboflavin	0.56	0.42	0.16	1.04	0.86	1.17	0.97
Polyamine synthesis	5-methylthioadenosine	0.06	0.24	0.21	1.03	0.77	0.88	0.65
	Ornithine	<0.01	0.46	0.46	1.00	0.92	0.89	0.82
	Putrescine	<0.01	0.75	0.33	0.89	1.02	1.33	1.52
	Spermidine	<0.01	0.92	0.48	0.90	1.14	1.53	1.93
	Spermine	0.03	0.48	0.60	1.08	1.00	0.88	0.82
Phosphatidylcholine pathway	Phosphatidylcholine	<0.01	0.44	0.94	0.92	0.88	0.48	0.46

¹Gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG.

²OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection.

³Fold change difference between contrasted treatments. Red and green shaded cells indicate P ≤ 0.05 (red, mean values are significantly greater for the comparison; green, mean values are significantly lower for the comparison). Light red and green shaded cells indicate 0.10 > P > 0.05 (light red, mean values tend to be greater for the comparison; light green, mean values tend to be lower for the comparison). Bolded P-values indicate statistical significance.

Figure 1.

Boxplots for the interaction between rate of gain and one-carbon metabolite supplementation in maternal liver at day 63 of gestation on (A) betaine (B) cystathionine (C) taurine and (D) sarcosine concentrations. 1Rate of gain: Control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection. 2P-values for (A) 0.01 (B) 0.05 (C) 0.02 (D) 0.02.

The main effects of OCM supplementation (Table 5; Figure 2) and rate of gain (Table 5; Figures 3 and 4) are discussed below. In the methionine cycle, dimethylglycine and S-adenosylhomocysteine (SAH) were greater (P < 0.02; 1.84 and 1.14-fold, respectively) in +OCM compared with −OCM. Restricted rate of maternal gain decreased (P < 0.03) choline, SAM, and adenosine compared with CON (0.78-, 0.72-, and 0.84-fold, respectively), but dimethylglycine was greater (P = 0.02; 3.03-fold) in RES compared with CON. In the transsulfuration pathway, OCM supplementation had an effect (P ≤ 0.01) on reduced and oxidized glutathione, where +OCM were greater than -OCM for both (1.79- and 3.20-fold, respectively). Additionally, the rate of gain also had an effect (P < 0.02) on reduced and oxidized glutathione, where both were less in RES compared with CON (0.53- and 0.50-fold, respectively). For amino acid interconversions, serine was less in +OCM compared with −OCM (P < 0.01; 0.59-fold) and glycine was greater in RES compared with CON (P = 0.01; 1.14-fold). Cofactors affected by the rate of gain (P < 0.01) included NAD⁺, NADH, and FAD, which were all less in RES compared with CON (0.75, 0.42, and 0.83-fold, respectively). Pyridoxine was less in +OCM compared with −OCM (P = 0.02; 0.73-fold). For polyamine synthesis, ornithine, putrescine, spermidine, and spermine were all affected by a rate of gain (P < 0.03) with ornithine and spermine being less (0.86 and 0.85, respectively) in RES compared with CON, while putrescine and spermidine were greater in RES compared with CON (P < 0.01; 1.425- and 1.73-fold, respectively). Finally, phosphatidylcholine was less in RES compared with CON (P < 0.01; 0.47-fold). Supplementation of OCM tended to increase 5-methyl THF (P = 0.09; 1.67-fold) and cysteine (P = 0.08; 1.17-fold) in maternal liver, but tended to decrease methionine (P = 0.09; 0.88-fold). Additionally, restricting maternal diet tended to decrease (P = 0.06; 0.77-fold) 5-methylthioadensoine.

Figure 2.

Boxplots for the main effect of one-carbon metabolite supplementation in maternal liver at day 63 of gestation on (A) dimethylglycine (B) s-adenosylhomocysteine (C) glutathione, reduced (D) glutathione, oxidized (E) serine (F) pyridoxine. 1Rate of gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection. 2P-values for (A) < 0.01 (B) 0.02 (C) 0.01 (D) < 0.01 (E) < 0.01 (F) 0.02.

Figure 3.

Boxplots for the main effect of rate of gain in maternal liver at day 63 of gestation on (A) choline (B) dimethylglycine (C) s-adenosylmethionine (D) adenosine (E) glutathione, reduced (F) glutathione, oxidized (G) glycine (H) ornithine (I) putrescine. 1Rate of gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline + 20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection. 2P-values for (A) <0.01 (B) <0.01 (C) 0.02 (D) 0.02 (E) <.01 (F) 0.02 (G) 0.01 (H) <0.01 (I) <0.01.

Figure 4.

Boxplots for the main effect of rate of gain in maternal liver at day 63 of gestation on (A) spermidine (B) spermine (C) phosphatidylcholine (D) FAD (E) NAD + (F) NADH. 1Rate of gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection. 2P-values for (A) <0.01 (B) 0.03 (C) <0.01 (D) <0.01 (E) <0.01 (F) <0.01.

Results for fetal liver one-carbon metabolism intermediate concentrations can be found in Table 6 and Figure 5. There were no rate of gain × OCM supplement interactions (P ≥ 0.15) for the fetal liver, so only the main effects will be discussed. Within the transsulfuration pathway cystathionine was greater (P = 0.01; 1.49-fold) in RES compared with controls. Within the folate cycle, 5-methyl THF tended (P = 0.08; 1.87-fold) to be greater in OCM compared with non-OCM-supplemented heifers, whereas betaine tended to decrease (P = 0.10; 0.86-fold).

Table 6.

Metabolites and cofactors involved in one-carbon metabolism and connected pathways including polyamine synthesis and the phosphatidylcholine pathway in day 63 fetal liver³

		P-values			Two-way ANOVA contrasts
Pathway	Metabolite	Gain1	Supp.2	Gain × supp.	CON + OCM CON − OCM	RES + OCM RES − OCM	RES − OCM CON − OCM	RES + OCM CON + OCM
Folate cycle	5-methyltetrahydrofolate	0.39	0.08	0.48	1.57	2.16	0.97	1.34
Methionine cycle	Choline	0.85	0.36	0.15	1.02	0.90	1.05	0.93
	Betaine	0.43	0.10	0.82	0.87	0.85	1.10	1.07
	Dimethylglycine	0.17	0.48	0.56	1.00	0.87	1.29	1.11
	Methionine	0.46	0.43	0.23	0.83	1.03	0.96	1.18
	s-adenosylmethionine	0.15	0.42	0.99	1.09	1.08	1.21	1.20
	s-adenosylhomocysteine	0.23	0.45	0.52	1.10	1.05	1.17	1.12
	Adenosine	0.89	0.38	0.78	1.18	1.08	1.05	0.96
	Homocysteine	0.30	0.63	0.35	1.35	0.90	1.65	1.10
Transsulfuration pathway	Cystathionine	0.01	0.15	0.75	1.22	1.16	1.52	1.45
	Homoserine	0.22	0.62	0.94	0.91	0.99	1.11	1.19
	Cysteine	0.33	0.91	0.14	1.17	0.83	1.39	0.99
	Glutathione, reduced	0.91	0.61	0.93	1.11	1.14	1.16	1.20
	Glutathione, oxidized	0.40	0.24	0.37	1.09	1.21	0.88	0.98
	Taurine	0.27	0.18	0.93	1.10	1.09	0.94	0.93
Other/amino acid interconversion	Glycine	0.56	0.21	0.55	0.96	0.89	1.08	1.01
	Sarcosine	0.19	0.73	0.94	1.04	1.08	1.15	1.19
	Serine	0.36	0.91	0.35	1.04	0.94	0.99	0.90
Cofactors	Pyridoxine	NA	NA	NA	1.00	1.00	1.00	1.00
	NAD+	0.87	0.68	0.65	0.98	1.07	0.95	1.04
	NADH	0.74	0.83	0.22	1.07	0.85	1.25	1.00
	FAD	0.56	0.82	0.20	1.08	0.95	1.03	0.90
	Riboflavin	0.39	0.93	0.52	1.06	0.93	0.99	0.87
Polyamine synthesis	5-methylthioadenosine	0.12	0.26	0.62	1.15	1.06	1.24	1.14
	Ornithine	0.11	0.75	0.55	0.93	1.01	0.86	0.94
	Putrescine	0.28	0.76	0.19	0.69	1.19	0.62	1.06
	Spermidine	0.56	0.28	0.15	0.26	1.16	0.28	1.26
	Spermine	0.91	0.22	0.33	0.47	0.81	0.71	1.20
Phosphatidylcholine pathway	Phosphatidylcholine	0.78	0.29	0.20	1.00	1.17	0.93	1.10

¹Gain: Control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); Restricted (RES) = −0.23 kg/d ADG.

²OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B12 + 320 mg folic acid; −OCM, saline injection.

³Fold change difference between contrasted treatments. Red and green shaded cells indicate P ≤ 0.05 (red, mean values are significantly greater for the comparison; green, mean values are significantly lower for the comparison). Light red and green shaded cells indicate 0.10 > P > 0.05 (light red, mean values tend to be greater for the comparison; light green, mean values tend to be lower for the comparison). Bolded P-values indicate statistical significance.

Figure 5.

Boxplots for the main effect of rate of gain in fetal liver at day 63 of gestation on cystathionine. 1Rate of gain: control (CON) = 0.45 kg/d ADG (actual: 0.60 kg/d ADG); restricted (RES) = −0.23 kg/d ADG. OCM supplementation: +OCM = 7.4 g/d rumen-protected methionine +44.4 g/d rumen-protected choline +20 mg B₁₂ + 320 mg folic acid; −OCM, saline injection. 2P = 0.01.

Discussion

In this study, we observed that OCM supplementation increased B₁₂ and folate concentrations in maternal serum, which is to be expected and was part of the design of the experiment. Additionally, moderately restricted maternal gain increased serum B₁₂ concentration in the dam. Previous work on restricting maternal diet during the first 50 d of gestation reported an increase in B₁₂ and folate concentrations in ALF, but no effect on those concentrations in maternal serum across days 16, 34, and 50 of gestation (Syring et al., 2023). However, an increase in serum B₁₂ concentration due to a roughage-restricted diet in dairy cattle has been observed (Walker and Elliot, 1972). Additionally, increased concentration of B₁₂ in the urine and decreased concentration in the liver were observed in the study by Walker and Elliot (1972) study, leading to the hypothesis that roughage restriction led to an increase in the excretion of B₁₂ and a reduction in its uptake by the liver (Walker and Elliot, 1972). Thus, our observation of an increase in circulating B₁₂ could be due to a decreased need for tissues for B₁₂ resulting from a decrease in metabolism. The average concentration of serum B₁₂ in dairy cattle is approximately 128 to 200 ng/L, and the average concentration of serum folates in dairy cattle is thought to be approximately 10 to 11 µg/L (Grace and Knowles, 2012; McFadden et al., 2020; Duplessis et al., 2023). In an OCM dose titration study, unsupplemented serum B₁₂ concentration ranged from 215 to 228 ng/L, and serum folate concentrations were approximately 11 to 13 µg/L in beef heifers (Crouse et al., 2022b). Over the course of a 2-wk study receiving weekly OCM supplementation, serum B₁₂ concentration ranged from 500 to 800 ng/L, and serum folate concentration ranged from 14 to 18 µg/L in those beef heifers (Crouse et al., 2022b). These concentrations peaked 2 d after treatment and decreased until the next round of treatment was given (Crouse et al., 2022b). The unsupplemented B₁₂ and folate concentrations observed in the current study are similar to those previously reported values in dairy and beef cattle, and our OCM-supplemented values fall within the range of the previously reported values for OCM-supplemented beef heifers. These data are interpreted to indicate that our heifers were normal in their presentation of folate and B₁₂ concentrations and that our dietary treatments were functioning as anticipated.

FA must be converted to DHF and then to the biologically active THF before entering the folate cycle (Clare et al., 2019). Once in the folate cycle, THF is reduced to 5,10-methylene THF and finally to 5,10-methenyl THF or 5-methyl THF, which is the main form of folate found in circulation (Xu et al., 2015; Clare et al., 2019). In maternal and fetal liver, 5,10-methylene THF, which is involved in pyrimidine biosynthesis, was the greatest folate intermediate concentration in this study. In the maternal liver, FA and DHF concentrations were almost 3× greater in RES + OCM heifers compared with other treatments when total liver weight was not considered. For the rest of the intermediates (THF, 5,10-methenyl THF, and 5-methyl THF) in uncorrected maternal liver, concentrations were greater in OCM-supplemented heifers; however, once intermediate concentrations were corrected for total maternal liver weight, all concentrations were increased by OCM supplementation. In ALF and AMF, the only detectable intermediate, 5-methyl THF, was also increased by OCM supplementation. Studies involving folate supplementation to gestating gilts also saw an increase in total folate concentration in ALF (Matte et al., 1993; Guay et al., 2002). Thus, we can conclude that supplementing OCM increases maternal folate intermediate concentrations. Additionally, in the current study, neither diet nor OCM supplementation affected day 63 fetal liver folate intermediates, except when CON fetuses had greater 5,10-methylene THF concentration; however, OCM supplementation can affect fetal fluid folate intermediate concentrations, indicating changes in the dam have the potential to affect the fetal environment.

While one-carbon metabolism may mainly be thought of as a source of methyl groups for epigenetics, it affects many other metabolic pathways. For example, SAM can also donate a carboxyl group to putrescine to synthesize spermidine, or to spermidine to synthesize spermine in the polyamine pathway (Bauchart-Thevret et al., 2009; Clare et al., 2019). Alternatively, ornithine can be decarboxylated to putrescine before being converted to spermidine and then spermine (Bauchart-Thevret et al., 2009; Clare et al., 2019). s-adenosylmethionine can also donate methyl groups to phosphatidylethanolamine to produce phosphatidylcholine, a glycerophospholipid important for lipid transport into animal cells (Clare et al., 2019); however, phosphatidylcholine can also be synthesized from choline (Kent, 2005). If homocysteine does not get remethylated, it can be condensed with serine to become cystathionine. Cystathionine then produces cysteine, a precursor for glutathione and taurine (Clare et al., 2019). In the present study, maternal liver dimethylglycine concentrations were increased, while choline concentrations were decreased, suggesting an increase in the conversion of choline to dimethylglycine in RES heifers. For polyamine synthesis SAM, ornithine, and spermine concentrations were reduced in RES heifers, but putrescine and spermidine concentrations were increased. Polyamines (putrescine, spermidine, and spermine) has a role in DNA and protein synthesis, cell growth, and embryonic development, and thus are important for a healthy pregnancy (Wu et al., 2013). Conversion of ornithine to putrescine increases rapidly after implantation, increasing uterine concentrations of putrescine and spermidine (Fozard et al., 1980). It could be hypothesized that RES heifers were increasing polyamine synthesis to compensate for the limited energy available to the developing embryo. Additionally, RES heifers had decreased glutathione, phosphatidylcholine, and cofactors NAD⁺, NADH, and FAD, and increased glycine concentrations. Glycine is involved in the production of glutathione, which acts as a source of reducing and oxidizing equivalents and may be essential for mitochondrial function (Shi et al., 2000). Restricted heifers may have shifted away from the synthesis of glutathione and phosphatidylcholine and toward glycine production. Finally, OCM supplementation increased concentrations of dimethylglycine, SAH, and glutathione, and decreased serine in heifers. While we can conclude that both moderate nutrient restriction and OCM supplementation shift pathways related to one-carbon metabolism, further investigation of other metabolic pathways should be done to get a more complete understanding of the effects of moderate nutrient restriction and OCM supplementation on the dam and developing fetus.

The goal of this study was to determine the impact of maternal gain on OCM and related metabolite concentrations, and if supplementing OCM would mitigate any effects reported from a reduced maternal diet. We confirmed our hypothesis that a moderately restricted maternal diet, as well as OCM supplementation, would increase B₁₂ and folate concentrations and shift-related pathways in the dam; however, we saw less impact on the fetus. The increases in maternal B₁₂ and folate concentrations help support the assumption that our OCM supplementation was effective in increasing OCM concentrations in the dam. Additionally, the changes in metabolite concentrations due to moderately restricted gain during the early gestational period indicate shifts in the metabolic pathway of the dam. While few changes were observed in the fetus, previous work on nutrient restriction has shown increased folate and B₁₂ concentrations in ALF. In addition, in this study we report increased 5-methyl THF with OCM supplementation and additional data from this study demonstrates altered concentrations of glucose and fructose in ALF and AMF due to maternal gain and supplementation status (Entzie et al., 2022). These findings indicate maternal diet and supplementation can affect, at least in part, metabolite concentrations in fetal fluids. While changes in fetal OCM concentrations were not observed, fetal tissues from this study showed a rate of gain × OCM supplement effect on allometric growth of the heart, left longissimus dorsi, and left and right hemispheres of the brain (King et al., 2022). Thus, our treatments appear to have an effect on the fetus and its development, even if fetal OCM status is not affected at day 63. Further research should be done to determine if fetal changes in response to early gestational treatments occur in later stages of gestation and the effects of those changes on the offspring later in life. These results are yet a further step into understanding the effects of moderate maternal nutrient restriction and OCM supplementation during early gestation on fetal outcomes and development.

Glossary

Abbreviations

ALF

allantoic fluid

AMF

amniotic fluid

CON

control

DHF

dihydrofolate

FA

folic acid

OCM

one-carbon metabolite

RES

low-gain via diet restriction

SAH

S-adenosylhomocysteine

SAM

S-adenosylmethionine

THF

tetrahydrofolate

TMR

total mixed ration

UHPLC-MS/MS

ultra-high-performance liquid chromatography MS/MS

Funding

This project was funded by the USDA National Institute of Food Agriculture, USDA-NIFA-AFRI (2018-07055), and the North Dakota State Board of Agricultural Research Education.

Conflict of interest statement

The authors declare no conflict of interest. Mention of a trade name, proprietary product, or specific agreement does not constitute a guarantee or warranty by the USDA and does not imply approval of the inclusion of other products that may be suitable. USDA is an equal opportunity provider and employer.

Author Contributions

Conception: M.S.C., A.K.W., L.P.R., C.R.D., J.S.C.. Collected data: J.G.S., M.S.C., Y.L.E., L.E.K., M.R.H., A.K.W., L.P.R., P.P.B., C.R.D., and J.S.C.. Performed Analysis: J.G.S., M.R.H., and M.S.C.. Wrote the Paper: J.G.S., M.S.C., and J.S.C..