All methodologies and experiments adhered to relevant guidelines and regulations. The design of the experiment, the management of animals, and the tissue collection processes were approved by the Institutional Animal Care and Use Committee at North Dakota State University (IACUC #20220059) on October 26, 2022.

Seventy-two Angus heifers were transported from the Central Grasslands Research Extension Center (Streeter, ND, USA) to the Animal Nutrition and Physiology Center at North Dakota State University (Fargo, ND, USA). After a 14-day adaptation to the feeding system, heifers were subjected to a 7-day Select Synch + CIDR estrus synchronization protocol (37) and artificially inseminated 18 to 22 h following the detection of estrus. To explore the effects of maternal nutrition and OCM on fetal development during gestation while removing sex-specific effects (38, 39), only female-sexed semen from a specific bull (Connealy Maternal Made [ST Genetics, Navasota, TX, USA]) was used for insemination. Of these 72 heifers, 29 (~ 14 months of age) successfully conceived [CON − OCM (n = 7), CON + OCM (n = 8), RES − OCM (n = 7), and RES + OCM (n = 7)]. The average initial body weight was 436 ± 42 kg. Upon breeding, the heifers were allocated into four different dietary groups based on a 2 × 2 factorial design that considered the level of nutritional plane (control vs. restricted) and OCM supplementation (with or without OCM). The heifers were individually fed daily in an electronic head gate facility (American Calan; Northwood, NH) at 0800 h daily.

Heifers on the control (CON) diet were fed to achieve 100% of the National Academy of Sciences, Engineering, and Medicine (NASEM) (40) nutritional requirements aiming for an average daily gain (ADG) of 0.45 kg/day, though they actually gained 0.60 kg/day. This regimen intended to bring them to 80% of their mature body weight by calving. Conversely, heifers on the restricted (RES) intake regimen were fed less to achieve a weight loss of 0.23 kg/day, mimicking the natural production responses observed in heifers undergoing dietary and environmental changes in early gestation (40). Their diets consisted of a total mixed ration including corn silage, alfalfa hay, corn grain, mixed alfalfa/grass hay, and a vitamin/mineral premix (Trouw dairy VTM w/Optimins, Trouw Nutrition USA, Highland, IL, USA). Diet compositions are more fully described in (41). Diets were formulated based on initial body weight at breeding to contain 2.25 Mcal/kg ME, 9.75% CP, and 58.6% NDF. Heifers were weighed weekly, and their individual feed intake was adjusted throughout the study to achieve the targeted body weight gains. Heifers supplemented with OCM (+OCM) received daily doses of 7.4 g of rumen-protected methionine (Smartamine, Adisseo, Beijing, China) and 44.4 g of rumen-protected choline (ReaShure, Balchem Inc., New Hampton, NY, USA) in a corn carrier, following dosages from previous studies (42, 43). They also received weekly intramuscular injections of vitamin B₁₂ (50 mg of cyanocobalamin/mL; MWI Animal Health, Boise, ID, USA) and folate (53.33 mg of folic acid/mL; Spectrum Chemical Mfg. Corp., New Brunswick, NJ, USA) supplements, aimed at delivering 320 mg of folic acid and 20 mg of vitamin B₁₂ each week, following previously described methods (43, 44). In contrast, the non-OCM supplemented heifers (−OCM) were given only the corn carrier daily and received weekly saline injections intramuscularly. These treatment protocols continued until day 63 of gestation, after which all heifers were managed on the CON − OCM treatment targeting a daily gain of 0.45 kg for the remainder of the study.

Pregnancy confirmation occurred on day 35 via transrectal ultrasonography by detecting fetal heartbeats, and a subsequent ultrasound on day 63 assessed fetal sex (45), retaining only pregnant heifers with female fetuses for further study stages.

Heifers were slaughtered on day 161 of gestation. The gravid uterus was promptly removed, and the fetus was immediately separated from the placenta, and subsequently dissected. Given that the cranioventral area of the lung is predominantly affected in bovine respiratory disease (46), lung samples from both the fetus and the dam were collected from this region (left cranioventral). Reflecting findings that high levels of AMP mRNA are present in the ileum (47), samples from this region were obtained from a section 10 cm proximal to the ileocecal junction. Additionally, considering the involvement of the mammary gland parenchyma in mastitis (48) and the uniform expression of AMP across different quarters (17), mammary gland samples were taken from the parenchyma of the right forequarters. All samples were wrapped in aluminum foil, snap-frozen in liquid nitrogen, and stored at −80°C until further analysis.

Total RNA was extracted using the RNeasy® Plus Universal Kit (Qiagen, Germantown, MA, USA), following the manufacturer’s protocol and tissues were lysed using the TissueLyser LT (Qiagen Germantown, MA, USA). The RNA concentration was determined using the Qubit® RNA BR Assay Kit (Thermo Fisher Scientific, MA, USA) and measuring it with a Qubit 3.0 Fluorometer (LifeTechnologies, Carlsbad, CA, USA).

The RNA was reverse transcribed into cDNA utilizing the High-Capacity Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA) following the manufacturer’s protocol. The final cDNA concentration was 100 ng/μl in a volume of 20 μl, and samples were stored at −20°C until use.

The target AMP genes were as follows: lingual antimicrobial peptide (LAP), tracheal antimicrobial peptides (TAP), enteric β-defensin (EBD), bovine neutrophil β-defensin4 (BNBD4), bovine neutrophil β-defensin5 (BNBD5), S100 calcium binding protein A7 (S100A7), and cathelicidin5 (CATHL5). For each gene, the primers were designed using NCBI/Primer-BLAST or sourced from previous literature.

Optimization was conducted to determine the optimum cDNA concentration and primer efficiencies in each tissue type (49). The amplification efficiency (E) of target genes was evaluated by plotting the cycle threshold (Ct) versus log concentration. The E was calculated using the equation E % = 10 − 1 / slope − 1 × 100 , with acceptable efficiencies ranging between 90 and 110% (50, 51). Details of the primer sequences used in the qPCR analysis can be found in Table 1. The primer sets for two genes (S100A7 and TAP) fell outside the acceptable efficiency range, likely due to low expression levels, as they exhibited high Ct values at the highest cDNA concentrations. Consequently, the relative mRNA expression of S100A7 and TAP were deemed below our limit of detection and were not further evaluated in this study. All other primers demonstrated efficiency within the acceptable range (90–110%). The results of the amplification efficiency experiment, including slope, R², and efficiency, are summarized in Supplementary Tables S1, S2.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) were used as reference genes. We employed the geNorm tool to assess the stability of their expression across all tissue samples. The lower M-values calculated by geNorm indicate more stable expression (52), and in our study, reference genes were considered stable if their M-values were below 0.5 (53).

Quantitative real-time PCR was performed using a QuantStudio™ 3 System (Thermofisher Scientific, MA, USA), with a 10 μl reaction mixture comprising 2 μl of the diluted cDNA (10 ng), 5 μl of 2× SYBR Green Master Mix (Bio-Rad Laboratories, Hercules, CA), 0.5 μl each of 10 μM forward and reverse primers (final concentration of 500 nM), and 2 μl of nuclease-free water. The amplification protocol included an initial denaturation at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 1 s and annealing/elongation at 60°C for 20 s. A dissociation melt curve was determined at the end of each run, involving a brief denaturation at 95°C for 1 s, annealing at 60°C for 20 s, and a final denaturation at 95°C for 1 s. All runs included a negative control (without cDNA) and were performed in triplicate on a 96-well reaction plate (Thermo Fisher Scientific, MA, USA). Relative gene expression levels were quantified using the 2^-ΔΔCT method (54), employing GAPDH, ACTB, and HPRT1 as reference genes for normalization, and using the CON − OCM group as control (set to 1).

Data were analyzed using the PROC MIXED function of SAS v.9.4 software (SAS Institute Inc., Cary, NC, USA). The normality of the data was assessed both before the analysis and for the resulting residuals after the analysis using PROC UNIVARIATE (Shapiro–Wilk test), followed by the QQ plot statement. In cases where the normality assumption was not met, data were transformed using log 10 x + 1 . Residuals were subsequently re-evaluated for normality using the same methods. The model’s fixed effects included the level of nutrition, OCM treatment, and the interaction between these factors, with each individual heifer serving as the random effect. In the absence of significant interactions, the main effects of maternal nutrition and OCM treatment were presented. Means were separated using the PDIFF function of SAS (Tukey–Kramer adjustment) and all results were reported as least squares means (LSMEANS) and standard error of the mean (SEM). After analysis, the LSMEANS and SEM were back transformed to the original scale. The largest SEM is reported. All plots were generated using ggplot2 v.3.4.1 in R Studio v.4.2.2. Statistical significance was set at a p-value ≤0.05, and tendencies were reported for 0.05 < p ≤ 0.1.

The mRNA expression of BNBD4 in the fetal lung was not influenced by either the nutritional plane × OCM interaction or the main effect of treatment (p > 0.28; Figure 1A; Table 2); however, there was a tendency (p = 0.07) for OCM supplementation to affect the mRNA expression of CATHL5, with expression being 16.5% lower in the +OCM groups (0.81 ± 0.06 relative fold) compared to the −OCM groups (0.97 ± 0.06 relative fold) (Figure 1B; Table 2).

In the fetal mammary gland, the mRNA expression of BNBD5 tended (p = 0.07) to decrease by 35% in the +OCM groups (0.80 ± 0.19 relative fold) compared to the −OCM groups (1.23 ± 0.19 relative fold; Figure 1C; Table 2). No differences were observed in the mRNA expression of CATHL5 among the groups (p > 0.1; Figure 1D; Table 2).

In the maternal lung, the mRNA expression of BNBD4 did not show differences among the groups (p > 0.1; Figure 2A; Table 3). Despite this, there was a tendency (p = 0.06) for an interaction for CATHL5 mRNA expression, with the RES + OCM group having higher expression compared to the CON + OCM (p = 0.07) and RES − OCM (p = 0.08) groups (Figure 2B; Table 3).

The mRNA expression of BNBD5 and LAP was assessed in the maternal mammary gland. No differences (p > 0.1) in the expression of these genes were observed (Figures 2C,D; Table 3).

In the maternal small intestine, the mRNA levels of LAP did not differ among the groups (p > 0.1; Figure 2E; Table 3). However, the expression of EBD was affected by OCM supplementation (p = 0.01), with the +OCM groups (0.77 ± 0.4 relative fold) showing a 67% lower expression compared to the −OCM groups (2.35 ± 0.4 relative fold; Figure 2F; Table 3).

Maternal undernutrition prior to parturition has been shown to interfere with the innate immune system of the ovine fetus and offspring (4, 65). However, to our knowledge, no studies have explored the effects of restricted maternal nutrition during pregnancy on the bovine fetal expression of AMP. Our data indicate that restricted maternal nutrition did not significantly affect the expression of AMP, neither in the fetus nor in the dams.

Several potential reasons may explain why the restricted nutritional plane did not independently result in significant changes in AMP expression. First, the severity and duration of the nutrient restriction imposed in this study may not have been sufficient to elicit significant changes in AMP expression. Although the extent of restricted nutrition was sufficient to assess impacts on gene expression associated with tissue metabolism, accretion, and function in our previous studies (66, 67), it may not have been severe or prolonged enough to affect AMP expression significantly.

Second, epigenetic adaptations might have occurred to maintain baseline AMP expression levels, potentially as a protective measure against nutrient restriction. It has been pointed out that the impact of prenatal events, such as undernutrition, on the immune system is not uniform. These events may lead to differential investments in various subsystems of the immune system, where some aspects might be downregulated or compromised, while others receive increased investment or remain unaffected (68, 69). In this context, it is possible that the body prioritized the maintenance of AMP expression over other immune functions in response to the prenatal nutritional stress experienced in our study. This differential investment could be an adaptive response, as it proposes that the immune system may shift its investment away from more energetically expensive specific immune defenses (like those involving specific antibodies and adaptive immune responses) and towards less costly nonspecific defenses (such as innate immune defenses) (68). This shift could help conserve resources while still providing adequate protection. It has been shown that undernutrition in ewes can upregulate specific genes, such as Cholesterol 25-Hydroxylase and MHC Class I Polypeptide-Related Sequence B, that modulate the innate immune response and inhibit the actions of receptors involved in cytotoxic activities, potentially beneficial for immunotolerance at the embryo-maternal interface (70).

Another factor is the variability in tissue response to stimuli. The AMP are constitutively expressed but can also be induced/increased in response to stimuli such as infection (17). Studies have shown that different energy levels during gestation do not affect the mRNA abundance of innate immune-related genes in weaned piglets under basal conditions; however, when challenged with lipopolysaccharide, the immune response was significantly higher in those born to dams under low-energy diets (71). Therefore, the lack of significant changes in AMP expression in the current study may be due to the absence of an immunological challenge. Hence, it is suggested that evaluating the expression of AMP in both dams and fetal tissues in response to different immunological challenges could provide further insights into the potential effects of maternal nutrition on the inducible expression of these innate immune effectors.

Additionally, maternal organisms may have evolved compensatory mechanisms to mitigate the effects of nutrient restriction on the developing fetus. These mechanisms could include alterations in nutrient partitioning, placental adaptation, or changes in maternal metabolism to ensure the provision of essential nutrients to the fetus, thereby minimizing the impact on fetal development (72, 73), including AMP expression.

Furthermore, sex-specific effects could play a role. Although the effects of sex were not explored in this study, there is strong evidence supporting sexual dimorphism in the innate immune system. Females tend to have a more effective and protective innate immune response, leading to better outcomes in the face of infections and immune challenges (9, 74). The mechanisms behind these observations may include evolutionary adaptations for higher reproductive potential (75) and hormonal differences between males and females (74).

The supplementation of OCM during early gestation influenced the expression of AMP in both fetal and maternal tissues, indicating a modulatory role of these metabolites on the innate immune system.

In the fetal lung and mammary gland, the mRNA expression of CATHL5 and BNBD5, respectively, tended to decrease in the OCM-supplemented groups compared to the non-supplemented groups. Similarly, in the maternal small intestine, the mRNA levels of EBD were significantly lower in the OCM-supplemented groups. These findings demonstrate that the expression of AMP was suppressed by OCM supplementation.

One-carbon metabolism and its metabolites are essential for the proper regulation of immune function through epigenetic modifications and metabolic reprogramming, ensuring that immune cells can respond effectively to infections and stress (13, 14). For instance, a study by Sinclair et al. (9) demonstrated that a maternal diet low in B vitamins and methionine can lead to altered immune responses in ovine offspring, indicating potential negative impacts on the immune system (9). Although our results showed a decrease in the mRNA expression of AMP in response to OCM supplementation, this does not necessarily contradict previous findings. It is possible that the improved overall condition of the OCM-supplemented fetuses reduced the need for immune responses, thereby influencing the expression of these immune-related genes. Further research is warranted to evaluate the activation and function of AMP in response to OCM supplementation.

It is widely acknowledged that the expression of AMP increases during an inflammatory response (76, 77). The nuclear factor-kappa B (NF-κB) through toll-like receptor (TLR)-NF-κB pathway, and the mitogen-activated protein kinase (MAPK) signaling pathway play crucial roles in regulating the expression of AMP (17, 78). On the other hand, a deficiency in one-carbon metabolites has been shown to elevate homocysteine levels, leading to increased inflammation. In agreement, our previous research has shown that nutrient restriction in beef heifers can result in reduced concentrations of methionine in allantoic fluid and increased levels of homocysteine in maternal serum during early gestation (16). Conversely, OCM reduces inflammation in two ways. First, it increases levels of S-adenosylmethionine (SAM) while reducing S-adenosylhomocysteine (SAH). The ratio between these compounds (SAM/SAH) regulates most methyltransferases (79–82). Second, when SAM levels rise, they help decrease inflammation by reducing NF-κB production and suppressing both the NF-κB and MAPK pathways. This leads to lower levels of inflammatory markers (80, 83, 84). Therefore, it is likely that OCM supplementation attenuated AMP expression by exerting its anti-inflammatory properties through the suppression of the NF-κB and MAPK pathways. Future studies should evaluate these pathways in the context of OCM supplementation to further elucidate their role in regulating AMP expression.