One-carbon metabolite supplementation to nutrient-restricted beef heifers affects placental vascularity during early pregnancy

Abstract

We hypothesized that restricted maternal nutrition and supplementation of one-carbon metabolites (OCM; methionine, folate, choline, and vitamin B₁₂) would affect placental vascular development during early pregnancy. A total of 43 cows were bred, and 32 heifers successfully became pregnant with female calves, leading to the formation of four treatment groups: CON − OCM (n = 8), CON + OCM (n = 7), RES − OCM (n = 9), and RES + OCM (n = 8). The experimental design was a 2 × 2 factorial, with main factors of dietary intake affecting average daily gain: control (CON; 0.6 kg/d ADG) and restricted (RES; −0.23 kg/d ADG); and OCM supplementation (+OCM) in which the heifers were supplemented with rumen-protected methionine (7.4 g/d) and choline (44.4 g/d) and received weekly injections of 320 mg of folate and 20 mg of vitamin B₁₂, or received no supplementation (−OCM; corn carrier and saline injections). Heifers were individually fed and randomly assigned to treatment at breeding (day 0). Placentomes were collected on day 63 of gestation (0.225 of gestation). Fluorescent staining with CD31 and CD34 combined with image analysis was used to determine the vascularity of the placenta. Images were analyzed for capillary area density (CAD) and capillary number density (CND). Areas evaluated included fetal placental cotyledon (COT), maternal placental caruncle (CAR), whole placentome (CAR + COT), intercotyledonary fetal membranes (ICOT, or chorioallantois), intercaruncular endometrium (ICAR), and endometrial glands (EG). Data were analyzed with the GLM procedure of SAS, with heifer as the experimental unit and significance at P ≤ 0.05 and a tendency at P > 0.05 and P < 0.10. Though no gain × OCM interactions existed (P ≥ 0.10), OCM supplementation increased (P = 0.01) CAD of EG, whereas nutrient restriction tended (P < 0.10) to increase CAD of ICOT and CND of COT. Additionally, there was a gain × OCM interaction (P < 0.05) for CAD within the placentome and ICAR, such that RES reduced and supplementation of RES with OCM restored CAD. These results indicate that maternal rate of gain and OCM supplementation affected placental vascularization (capillary area and number density), which could affect placental function and thus the efficiency of nutrient transfer to the fetus during early gestation.

Supplementation with one-carbon metabolites affects the vascularity of the placenta during early pregnancy in beef heifers.

Introduction

The placenta, a transient organ formed during pregnancy, acts as a vital conduit for the transfer of nutrients, gases, water, hormones, and waste materials between the maternal and fetal systems. Placental vascular development, or angiogenesis, which is necessary for placentation and placental function to support fetal development, begins early in pregnancy and continues throughout gestation (Reynolds and Redmer, 1995; Reynolds et al., 2010, 2013; Chen and Zheng, 2014). Moreover, in ruminants, the placentomes are composed of cotyledon (COT; fetal placental) and caruncle (CAR; maternal placental) tissues (Reynolds et al., 2006, 2010). Insufficient placental angiogenesis during early pregnancy is believed to cause a reduction in the transport of oxygen and nutrients to the fetus, hindering fetal development (McLean et al., 2017; Caton et al., 2020; Reynolds et al., 2023).

Numerous factors result in inadequate maternal nutrition during pregnancy, such as poor forage quality or limited quantity, often due to periods of drought and high feed costs (Funston et al., 2012; Reynolds et al., 2022). In the production of healthy beef cattle offspring, a large economic burden is the cost of maternal feed throughout pregnancy (Webster, 1989). Moreover, even moderate alterations in maternal diet during the peri-conceptual period and early gestation can affect fetal development, organogenesis, and growth (Ferrell and Jenkins, 1985; Bairagi et al., 2016; Crouse, 2019a; Reynolds et al., 2019; Diniz et al., 2021; Menezes et al. 2022). In ewes, maternal nutrient restriction during pregnancy increased umbilical vascular resistance, which suggests that placental vascular function was compromised (Reynolds et al., 2014). In both ruminants and rodents, placental vascular development and expression of angiogenic factors increase from early to late pregnancy and are reduced by inadequate maternal dietary intake (Reynolds and Redmer 2001; Coan et al., 2010; Grazul-Bilska et al. 2011; McLean et al., 2017; Reynolds et al., 2023).

A previous study from our group demonstrated that a moderate nutrient restriction initiated at breeding in beef heifers resulted in reduced concentrations of methionine in allantoic fluid and greater concentrations of homocysteine in maternal serum at day 50 of gestation compared with the control-fed group (Crouse et al., 2019b). Elevated homocysteine concentrations in maternal plasma may be related to a deficiency in folate or vitamin B₁₂, which could subsequently affect homocysteine methylation and thus methionine levels across the maternal and fetal interface (Girard and Matte, 2005; Castro et al., 2006). Because of the interrelationships among methionine, choline, folate, and vitamin B₁₂ metabolism, a deficiency in any one of these would disturb metabolic pathways related to one-carbon metabolism (Zeisel, 2011). Methionine and folate metabolism via the methionine-folate pathway influences many processes that affect gene expression and cell proliferation, including placental angiogenesis (McMahon et al., 2016; Crouse, 2019a; Xia et al., 2021). However, limited data are available on the effects of OCM supplementation and maternal nutrient restriction on placental vascularity during early pregnancy. We hypothesized that OCM supplemention (methionine, folate, choline, and vitamin B₁₂) to feed-restricted heifers would improve the vascularity of the placenta during early pregnancy.

Materials and Methods

All animal procedures were approved by the North Dakota State University Institutional Animal Care and Use Committee.

Experimental design

Angus-crossbred heifers (BCS 5.0; 60% of mature BW; average initial BW = 402.9 ± 32.6 kg) were housed at the North Dakota State University Animal Nutrition and Physiology Center in Fargo. The experimental design was a 2 (gain) × 2 (OCM supplementation) factorial arrangement. Heifers receiving the control (CON) level of gain were fed 100% of the NASEM (2016) requirements to achieve 0.60 kg of BW gain per heifer per day, which would allow them to reach 80% of mature BW by calving. Heifers receiving the restricted (RES) level of gain were fed to lose 0.23 kg/heifer daily, which mimics the observed production responses in heifers that experience a restricted dietary and environmental change at breeding (Perry et al., 2009). These treatments resulted in four gain × OCM groups: CON—OCM (n = 8), CON + OCM (n = 7), RES—OCM n = 9), and RES + OCM (n = 8). The heifers were adapted to an individual feed intake monitoring system (American Calan, Northwood, NH) and fed per NASEM (2016) guidelines to meet or exceed recommendations for dietary sulfur and cobalt and to achieve targeted gains. Diets were a total mixed ration consisting of grass hay, corn silage, alfalfa haylage, grain, and vitamin/mineral premix (11.5 g/d; Trouw Nutrition USA, LLC, Highland, IL). Diets were formulated based on initial body weight at breeding to contain 2.25 Mcal/kg ME, 9.75% CP, and 58.6% NDF. After a 14-d adaptation to the feeding system, 43 heifers were exposed to the Select Synch + CIDR estrous synchronization protocol (Lamb et al., 2010) and were bred 18 to 22 h after detected estrus with female sexed semen from a single sire (ST Genetics, Navasota, TX). The experiment began on the breeding day by randomly assigned treatment and individual feeding.

The OCM supplemented treatment (+OCM) consisted of daily supplementation (top dressed in a fine-ground corn carrier) of rumen-protected methionine (7.4 g/d; Smartamine M, Adisseo NA, Alpharetta, GA) and choline (44.4 g/d; ReaShure-XC, Balchem, New Hampton, NY), targeting dosages used in previously published work (Jacometo et al., 2017; Crouse et al., 2023). Folate (53.33 mg of folic acid/mL; Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ) and vitamin B₁₂ (50 mg of cyanocobalamin/mL; VetOne, Boise, ID) supplements were administered via weekly intramuscular injections to target 320 mg of folic acid and 20 mg of vitamin B₁₂ per week, as described previously (Gagnon et al., 2015; Crouse et al., 2023) Non-OCM supplemented heifers (−OCM) received fine-ground corn carrier without the addition of rumen-protected methionine and choline supplements, and weekly intramuscular injections of saline. Pregnancy was confirmed with transrectal ultrasonography via visualization of a fetal heartbeat on day 35 of gestation, and fetal sex was determined on day 60 with confirmatory ultrasound revealing that all fetuses were female via transrectal ultrasonography by visualization of the genital tubercle (Lamb et al., 2003). Day 63 was selected for its significance in early pregnancy when embryo implantation and placental formation occur. Our study, focusing on placental vascularization, required a well-developed placenta. This choice also coincides with determining the calf’s gender, confirmed to be female.

Sample collection and analysis

Out of 43 bred cows, 32 heifers became pregnant with female calves were processed for slaughter on day 63 of gestation, resulting in a pregnancy rate of 74.4%. Placental and uterine tissues were collected and processed, as previously described (McLean et al., 2017). Fixed placental tissues were embedded in paraffin via a tissue processor (Leica Biosystems Inc., Buffalo Grove, IL) and blocks were cut on a microtome at 5-µm thickness for immunohistochemistry and 3D image analysis of placental vascularity.

As previously described (McLean et al., 2017; Crouse et al., 2020; Dávila Ruiz et al., 2022), immunofluorescence staining was conducted using rabbit anti-CD31 and anti-CD34 antibodies (Abcam, Cambridge, MA) to stain endothelial cells for vascularity, DAPI (Life Technologies, Grand Island, NY) was used for background nuclear staining, and fluorescein griffonia simplifolia lectin I (BS1 lectin; Enzo Life Sciences, Farmingdale, NY) was used as a marker of fetal cotyledon (COT) tissue. All slide sections were deparaffinized in xylene (3 × 5 min), followed by 100% alcohol (2 × 5 min), 95% alcohol (1 × 5 min), 70% alcohol (1 × 5 min), and distilled water (1 × 5 min). Epitope retrieval was performed with a sodium citrate buffer (10 mM trisodium citrate dihydrate with 0.05% Tween-20, pH 6) for 30 min at 121 °C in a pressure cooker. Antigen blocking was done by treating each slide with 5% normal goat serum (Vector Laboratories, Burlingame, CA) for 1 h at room temperature (20 to 25 °C). Primary CD31 and CD34 rabbit antibodies (Abcam) were diluted 1:500 and 1:50, respectively, in 1% bovine serum albumin (Avantor, Visalia, CA), pipetted onto the prepared slides, and incubated for 1 h at room temperature. Secondary CF 633 goat anti-rabbit antibody (Biotium, Fremont, CA) diluted 1:250 in 1% bovine serum albumin was then incubated with tissue sections for 1 h at room temperature. Fluorescein griffonia simplifolia lectin I (BS1 lectin; Enzo Life Sciences) was diluted 1:250 and incubated with tissue sections for 30 min. Finally, nuclear staining was accomplished via DAPI nuclear stain for 5 min at room temperature. Slides were then mounted using EverBrite hardset mounting medium (Biotium). All of these methods have been described previously (McLean et al., 2017; Crouse et al., 2020; Dávila Ruiz et al., 2022). The negative control utilized in immunofluorescence analysis to correct for potential background staining was the control group (CON − OCM).

Image analysis

Placental vascular images were captured with a Zeiss AxioImager M2 epifluorescence microscope (Carl Zeiss, Thornwood, NY) using a 20×, 0.8 NA objective and an AxioCam HRm camera. All Images were processed with Zeiss AxioVision Rev. 4.8.2 image analysis software. Images were analyzed using ImagePro-Premiere software (Ver.9.0.1 Media Cybernetics, Inc., Silver Spring, MD). Briefly, the areas of interest were imaged, and capillary area density (CAD) was calculated as capillary area divided by tissue area, and capillary number density (CND) was calculated as capillary number divided by tissue area. Areas evaluated included fetal placental cotyledon (COT), maternal placental caruncle (CAR), placentome (CAR + COT), intercotyledonary fetal membrane (ICOT), intercaruncular endometrium (ICAR), and myometrium (MYO). For ICAR, we analyzed the sub-region of endometrial glands (EG). The areas were outlined and then were analyzed by using standardized segmentation values, which allows for objective, quantitative comparison across the samples (McLean et al., 2017; Bairagi et al., 2018; Crouse et al., 2020; Dávila Ruiz et al., 2022).

Statistical analysis

Data were analyzed as a completely randomized design with a 2 × 2 factorial arrangement of treatments using the GLM procedure of SAS, main effects of gain (CON or RES) and OCM supplementation (+OCM or −OCM) and their interactions (SAS version 9.4; SAS Inst. Inc., Cary, NY), with heifer as the experimental unit and significance determined at P ≤ 0.05 and a tendency at P < 0.10.

Results

Representative images of CD31 and CD34 immunostaining are presented in Figure 1. Endothelial cells, representing microvascular elements (capillaries, arterioles, and venules) can be clearly seen as pinkish CD31/CD34 staining. In addition, the greenish BS1 lectin staining clearly identifies the fetal membranes in the placentomes (CAR/COT) and ICOT.

Figure 1.

Representative immunofluorescence images of CD31 and CD34 staining in placenta on day 63 of pregnancy in beef heifers. Treatment factors (along the top of the figure) are maternal rate of body weight gain (control, CON = 0.60 kg of BW gain/heifer/day; restricted, RES = −0.23 kg of BW gain/ heifer/day) and dietary OCM supplementation (with supplement, + OCM = fed 7.4 g/d of rumen-protected methionine and 44.4 g/d of rumen-protected choline in a corn carrier and weekly injections of 320 mg of folic acid and 20 mg of vitamin B₁₂; without supplement, −OCM = fed corn carrier and weekly injections of saline). (A) CON − OCM, (B) CON + OCM, (C) RES − OCM, (D) RES + OCM, and (E) Negative Control. (1) Caruncle and cotyledon (CAR/COT, which represents the whole placentome), (2) itercotyledonary fetal membranes (chorioallantois; ICOT), (3) endometrial glands (EG), (4) ICAR, and (5) MYO. Pinkish color indicates positive staining for CD31 and CD34, greenish color in CAR/COT and ICOT indicates fetal membranes (chorioallantois) stained with BS1 lectin, and bluish color indicates cell nuclei counterstained with DAPI. As indicated, the size bar is 50 µm for all images.

For CAD within placentome (CAR + COT), a gain × OCM interaction was observed (P = 0.02). Specifically, CON − OCM exhibited greater CAD (P = 0.02) than CON + OCM, while RES − OCM and RES + OCM showed intermediate values (CAD: −OCM = 3.88, +OCM = 3.77, CON = 4.12, RES = 3.46; SEM = 1.19). Similarly, within the ICAR, there was a gain × OCM interaction for CAD (P = 0.03). RES + OCM had significantly greater CAD (P = 0.03) compared to RES − OCM, with CON − OCM and CON + OCM displaying intermediate values (CAD: − OCM = 2.44, +OCM = 2.49, CON = 2.42, RES = 2.50; SEM = 1.09).

Moving to main effects, although the initial analysis did not reveal a significant gain × OCM interaction (P ≥ 0.10; Table 1), the subsequent examination identified significant gain × supplement interactions. OCM supplementation increased CAD of EG (P = 0.01: −OCM = 3.59 and +OCM = 5.03, SEM = 1.45) and showed a tendency to increase CAD of MYO (P = 0.06: −OCM = 1.78 and +OCM = 1.23, SEM = 0.81), CND of CAR (P < 0.10: −OCM = 0.22 and +OCM = 0.35, SEM = 0.22), and CND of EG (P = 0.06: −OCM = 0.27 and +OCM = 0.41, SEM = 0.21). Additionally, nutrient restriction exhibited a tendency to increase CAD of ICOT (P = 0.10: CON = 5.91 and RES = 7.67, SEM = 2.85) and CND of COT (P < 0.10: CON = 0.36 and RES = 0.49, SEM = 0.20). No other significant main effects of gain or OCM supplementation were observed in Table 1.

Table 1.

Effects of rate of gain and OCM supplementation on the vascularity of placental regions at day 63 of gestation in beef heifers

Measurement	Region1	Gain2		Supplementation3			P-value4
		CON	RES	−OCM	+OCM	SEM5	Gain	Supplement	Gain × Supplement
Capillary area density, %	Placentome	3.88	3.70	4.12	3.46	1.19	0.67	0.13	0.02
	CAR	5.23	4.97	4.86	5.33	2.13	0.68	0.49	0.29
	COT	4.65	4.85	4.33	5.18	2.14	0.79	0.26	0.50
	ICOT	5.91	7.67	6.28	7.30	2.85	0.10	0.32	0.64
	ICAR	2.44	2.49	2.42	2.50	1.09	0.90	0.83	0.03
	EG	4.35	4.27	3.59	5.03	1.45	0.88	0.01	0.55
	MYO	1.61	1.40	1.78	1.23	0.81	0.46	0.06	0.77
Capillary number density, %	Placentome	0.30	0.34	0.29	0.34	0.17	0.54	0.40	0.98
	CAR	0.25	0.32	0.22	0.35	0.22	0.40	0.10	0.85
	COT	0.36	0.49	0.33	0.48	0.20	0.10	0.16	0.61
	ICOT	0.33	0.49	0.41	0.41	0.29	0.14	0.95	0.98
	ICAR	0.26	0.28	0.23	0.31	0.15	0.82	0.15	0.96
	EG	0.32	0.35	0.27	0.41	0.21	0.63	0.06	0.30
	MYO	0.29	0.22	0.28	0.23	0.16	0.26	0.32	0.42

¹Placentome, unseparated caruncle (maternal portion of the placentome) + cotyledon (fetal portion of the placentome); CAR, caruncle; COT, cotyledon; ICOT, intercotyledonary fetal membranes (chorioallantois); ICAR, intercaruncular endometrium; EG, endometrial glands: MYO, myometrium.

²Maternal rate of body weight gain; CON, control (0.60 kg of BW gain/ heifer/day); RES, restricted (−0.23 kg of BW gain/ heifer/day).

³Maternal one-carbon metabolite supplementation; −OCM, no supplementation; +OCM, supplemented with 7.4 g/d of rumen protected methionine and 44.4 g/d of rumen-protected choline daily, and weekly injections of 320 mg of folic acid and 20 mg of vitamin B₁₂.

⁴ P-values in bold indicate significant differences due to Gain or Supplementation.

⁵Standard error of the mean.

Discussion

We hypothesize the effects of supplementing OCM (methionine, choline, folate, and vitamin B₁₂) during restricted maternal nutrition in early pregnancy on placental vascularity. Our findings indicate that while the effects on placental vascularity are limited and modest in the current dataset, there are nuanced variations, especially in the CAD of the placentome and the stratum compactum of ICAR, influenced by the interaction of gain and OCM supplementation. However, other placentome regions did not show the same interaction effect. Additionally, maternal nutrient restriction significantly affected the placental CAD of ICOT and the CND of COT, both fetal-side placental regions, while OCM supplementation influenced the CAD and CND of EG, indicative of maternal placental effects.

The CAD determines the capacity for placental blood flow, as total capillary area per unit of tissue area has a large effect on vascular resistance and thereby is a major driver of blood flow, whereas CND reflects capillary branching and therefore influences primarily the capillary surface area available for exchange/transport of nutrients, respiratory gases and metabolic wastes (Reynolds and Redmer, 1995; Reynolds et al., 2006). Between days 50 and 150 of gestation in ewes, the CAD of CAR has been shown to increase 3.3-fold and CND 1.5-fold, whereas cotyledonary CAD increased 6.2-fold and CND 12.3-fold, demonstrating that angiogenesis is greater in COT than CAR (Borowicz et al., 2007). As the conceptus grows, its nutrient requirements increase, necessitating a corresponding increase in placental blood flow to supply those nutrients.

This study is interpreted to demonstrate compensatory responses of placental vascularity in the RES group, with RES tending to be greater than CON for both CAD of ICOT and CND of COT, while other regions were not affected by the maternal rate of gain. The placental response may be related to maternal stress which is caused by many factors in addition to maternal nutrient intake, such as maternal age, environment (including temperature and humidity), social stress, and the number of fetuses (Reynolds et al., 2006, 2010, 2014). Experiments of restricted maternal nutrition during early pregnancy in a mouse model suggested physiological mechanisms that affect placental nutrient transport to the fetus (Coan et al., 2010), including compensatory changes in placenta blood flow, and placental vascular development. (Fowden et al. 2006; Borowicz and Reynolds, 2010). Placental transport of glucose and amino acids and the expression of their transporters also suggest early compensatory changes in placental function with maternal nutrient restriction during pregnancy (Crouse et al., 2019b). Despite the compensatory increases in the capacity of the placenta for nutrient transfer to the fetus; however, maternal nutrient restriction still had a negative effect on the placental size and surface area, and the volume of the placental capillary bed (Borowicz and Reynolds, 2010; Coan et al., 2010).

In ewes that were nutrient-restricted, the decrease in uterine arterial resistance that is normally observed during pregnancy did not occur, and umbilical vascular resistance was increased (Reynolds et al., 2013). These observations indicate that placental vascular function, in addition to placental vascular development, also is compromised in response to maternal nutrient restriction (Bairagi et al., 2016). In fact, maternal nutrient restriction may result in maternal, placental, and fetal adaptations to the altered prenatal environment, which allows the survival of the offspring, often at the expense of normal fetal development (Quigley et al., 2008; Nathanielsz, 2021). A compensatory increase in vascularity of the placenta due to maternal nutrient restriction has been shown in many species such as rodents, pigs, sheep, and humans (Hoet and Hanson, 1999; Greenwood et al., 2000; Redmer et al., 2004; Fowden et al., 2006; Reynolds et al. 2023). Similar to the studies in other mammalian models, our results demonstrate a tendency for increased placental vascularity in ICOT and COT of nutrient-restricted heifers.

These data also demonstrated that OCM supplementation increased placental vascular density of EG which is an important source of nutrients via their production of histotroph, which is especially important for embryo growth and development during early gestation (Bazer, 2013). In sheep in which the EG were knocked out by early postnatal estrogen treatment, subsequent embryo development failed during the elongation stage due to the embryo’s lack of ability to respond to interferon-tau for pregnancy recognition which eventually causes abortion to occur (Filant and Spencer, 2013). Our results indicated that perhaps histotroph production was impacted during early pregnancy. Folate supplementation increased the number of blood vessels, angiogenesis, and placental blood flow in humans (Williams et al., 2011), This agrees with our observations of increased placental vascularity with OCM supplementation. Placentation and placental vascularization begin very early after embryo attachment to support fetal development by increasing placental blood flow and thus increasing the transport of nutrients from the mother to the fetus (Reynolds and Redmer, 1995; Ocak et al., 2009; Grazul-Bilska et al., 2011; Bairagi et al., 2016; Reynolds et al., 2023).

Much evidence suggests that maternal nutrient intake is related to the programming of fetal development, particularly during early gestation (Robinson et al., 1997; Igwebuike, 2010; Bairagi et al., 2016; Caton et al., 2020; Reynolds et al., 2022, 2023). Previous research has shown that moderate nutrient restriction in early pregnancy resulted in a high concentration of homocysteine in maternal serum, suggesting that there was a reduction in the ability of the one-carbon cycle to remethylate homocysteine to methionine, resulting in a potential deficiency in methyl donors available to the fetus (Crouse et al., 2019b). As such, when pregnant heifers are in a condition of limited nutrition, there may be a benefit of OCM supplementation on placental and fetal development. Research in sheep has highlighted the significant one-carbon transfer in the fetal liver and placenta, these studies revealed a complex process involving the metabolism of serine to glycine within the uterus/placenta, rather than its direct transfer to the fetus. The fetal circulation was found to have high concentrations of serine and glycine. Additionally, there was a unique exchange of serine and glycine between the fetal liver and placenta, supporting methylation demands and nucleotide synthesis. This research emphasizes the intricate metabolic mechanisms crucial for fetal development (Geddie et al., 1996; Moores et al., 1994). In our study, the OCM supplement included choline, methionine, folate, and vitamin B₁₂.

Folate is an important vitamin throughout pregnancy, as deficiency of folic acid has been associated with intrauterine growth restriction, abortion, and placental abruption (Greenberg et al., 2011). Transfer of folate through the placenta is facilitated by three folate transport proteins: FRα, PCFT, and RFC (Solanky et al., 2010). In humans, culture of placental explants from day 49 of pregnancy with folate increased vascular density as measured by using anti-CD31 immunostaining and image analysis (Williams et al., 2011). In ewes, dietary supplementation of B vitamins (i.e., B₁₂ and folate) and methionine during the periconceptional period affected DNA methylation, insulin resistance, and blood pressure in the offspring (Sinclair et al., 2007).

Vitamin B₁₂ (cobalamin) is a cofactor for methionine synthase, which is responsible for the folate-dependent methylation of homocysteine to regenerate methionine (Kalhan, 2016). Folate deficiency increases the risk of fetal developmental abnormalities and fetal loss during early pregnancy, as well as preeclampsia and premature delivery, which are associated with decreasing weight/size of the placenta, low birthweight, and neonatal morbidity and mortality (Bergen et al., 2012). Choline may not be directly associated with increased placental vascular development, but via its metabolite betaine (trimethyl glycine), and folate via its metabolite methyl-tetrahydrofolate, are involved in the methylation of homocysteine to form methionine. Methionine supplementation during the early stages of pregnancy in dairy cattle can improve placental and fetal development and also has effects on the offspring (Coleman et al., 2021).

Conclusions

Our study explored the intricate interplay of maternal nutrient restriction and one-carbon metabolite (OCM) supplementation during early pregnancy on placental vascularity in beef cattle. First, our findings suggest the possibility of a compensatory increase in vascularity in fetal placental tissues, specifically ICOT and COT, in response to maternal nutrient restriction. However, it is important to acknowledge that these observations, though consistent with previous research, are subtle and not universally significant across all measurements, as evidenced in Table 1. Second, our study highlights that OCM supplementation has a discernible impact on increasing vascularity in maternal placental tissues, notably in EG and CAR. Furthermore, the vascularity was notably greater in RES + OCM compared to RES − OCM in the ICAR and the placentome (CAR and COT). This implies that OCM supplementation might enhance placental vascularity, potentially improving uteroplacental blood flow and, consequently, nutrient transfer to support fetal development, particularly under conditions of nutrient restriction.

However, we acknowledge that examining nutrient transfer and its effects on fetal development from the collected tissue samples would provide a more comprehensive understanding of the mechanisms underlying these changes. The data presented here offer valuable insights into the potential outcomes of maternal nutrient restriction and OCM supplementation on placental vascularity, but additional research is essential to bridge the gap and further clarify the consequences for nutrient transfer and fetal development.

Glossary

Abbreviations

CAD

capillary area density

CAR

maternal caruncle

CAR/COT

entire placentome (maternal caruncle + fetal cotyledon)

CD31

cluster of differentiation 31

CD34

cluster of differentiation 34

CND

capillary number density

CON

control

COT

fetal cotyledon

DAPI

4ʹ,6-diamidino-2-phenylindole

EG

endometrial glands of the intercaruncular endometrium

ICAR

intercaruncular endometrium

ICOT

intercotyledonary fetal membrane

MYO

myomettrium

OCM

one-carbon metabolites

RES

nutrient restricted

Conflict of interest statement. The authors declare no conflicts of interest.