Effects of a single trace mineral injection in gestating Brangus cows on growth, physiology, and the liver transcriptomic profile of their offspring

1 Introduction

Trace minerals, such as Zn, Cu, Se, and Mn, are essential for reproduction and conceptus development in livestock (Hostetler et al., 2003). Maternal trace mineral deficiencies are commonly associated with impaired fetal development and reduced growth and health of the offspring after birth (Harvey et al., 2021; Anas et al., 2023). However, the majority of the studies evaluating the role of trace minerals in developmental programming in cattle have relied on dietary supplementation (Harvey et al., 2021; Craner et al., 2025), whereas studies investigating the effects of trace mineral injections (TMIs) remain limited (Stokes et al., 2019).

TMIs are commonly used in grazing beef systems to rapidly improve mineral status during the breeding season, growth phases, or during immune challenges (Vedovatto et al., 2019a; Palomares, 2022; Vedovatto et al., 2024). Commercial TMI formulations typically contain Zn, Cu, Se, and Mn and are administered subcutaneously. Following administration, these minerals are absorbed into the bloodstream and subsequently metabolized by the liver, where they may be stored for weeks or months depending on the physiological and nutritional conditions (Pogge et al., 2012; Vedovatto et al., 2024). Even in well-managed grazing systems with free-choice mineral supplementation, substantial variability in the mineral intake and liver mineral concentrations has been reported among animals within the same herd (McCarthy et al., 2021). Therefore, TMI is an effective strategy for the delivery of precise doses of trace minerals to each animal, potentially reducing variability and ensuring a more homogeneous trace mineral status across the herd.

The majority of research on TMI in cattle has focused on its effects in weaned calves, particularly on the growth performance and immune responses (Palomares, 2022; Vedovatto et al., 2024; McKnight et al., 2026), or in cows with emphasis on reproductive outcomes such as pregnancy rates (Vedovatto et al., 2019a, b). However, the potential role of maternal TMI in fetal programming and its effects on offspring performance remain poorly understood. Furthermore, the administration of a single TMI during mid-to-late gestation can be conveniently incorporated into routine herd management procedures, such as handling cows during weaning of the previous calf crop, making it a practical, low-labor intervention with potential fetal programming benefits. Therefore, we hypothesized that administering TMI to pregnant cows would improve the maternal and fetal trace mineral status and induce lasting programming effects on offspring growth and metabolism. Thus, the objective of this experiment was to evaluate the effects of a TMI at 114 days prepartum in Brangus cows on the growth, physiology, and liver transcriptomic profile of their offspring.

2 Materials and methods

The experiment was conducted at the White Sand Research Unit, Mississippi State University, Poplarville, Mississippi, USA, from September 2024 to August 2025. All animals and experimental procedures were approved by the Mississippi State University Institutional Animal Care and Use Committee (IACUC protocol no. 24-233).

2.1 Animals, treatments, and experimental design

The cows used in the study were confirmed pregnant during the previous breeding season following artificial insemination using conventional semen from a single Brangus sire. At 114 ± 23.6 days prepartum (day −114), at weaning time, 40 spring-calving Brangus beef cows (6 ± 2 years of age) were stratified by body weight (BW; 533 ± 55 kg) and body condition score (BCS; 5.8 ± 0.4) and assigned to one of two treatments: 1) Saline (saline solution, 0.9% NaCl; n = 20) or 2) TMI (solution containing 60, 10, 5, and 15 mg/ml of Zn, Mn, Se, and Cu, respectively; Multimin 90, Axiota Animal Heath, Fort Collins, CO, USA; n = 20). The treatments were administered subcutaneously (1 ml/90 kg BW) on the right side of the neck. Cows were kept in a single group during the entire experiment, consuming ad libitum bahiagrass (Paspalum notatum) hay during prepartum (from day −114 to day 1), grazing perennial ryegrass (Lolium perenne) forage from day 1 to day 80, and then grazing bahiagrass forage until weaning (day 234). The grazing method was rotational, with the animals moved weekly.

During the entire experiment, animals had access to a commercial free-choice mineral mix (12.5%–14.58% Ca, 4.0% P, 18.8% NaCl, 10% Mg, 0.10% K, 3,600 ppm Zn, 4,800 ppm Mn, 1,200 ppm Cu, 18 ppm Co, 60 ppm I, 27 ppm Se, 750 IU/g vitamin A, 75/g IU vitamin D, and 0.75 IU/g vitamin E; Purina Wind & Rain, High Magnesium; Land O’Lakes, Inc., Roseland, LA, USA; target intake of 113g/cow/day). Hay samples were collected on days −114, −60, and 1 and composited into a single sample for nutrient composition analysis. Hand-plucked ryegrass samples were collected on days 0 and 80, whereas hand-plucked bahiagrass samples were collected on days 80, 160, and 234. Samples from each forage type were composed into a single sample before analysis. The hay and forage samples were dried in a forced-air oven at 55°C for 72 h before laboratory analysis (Table 1).

Table 1. Chemical composition (dry matter basis) of the hay and forages available to cows and calves throughout the experimental period.

CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; TDN, total digestible nutrients; NE_l, net energy for lactation; NE_m, net energy for maintenance; NE_g, net energy for growth.

Calculated as described by Weiss et al. (1992).

Calculated using the equations proposed by the NASEM (2016).

2.2 Maternal collections

BW and BCS were collected from all cows on days −114 and 1 (24 h after calving) and on days 80 and 234. The BCS was visually assessed by one trained technician (blinded for treatments) using a 1 to 9-point scale (i.e., 1 = extremely thin, 9 = extremely fat) (Renquist et al., 2006).

Liver biopsies were collected from all cows on days −114 and 1. Liver samples were obtained between the 10th and 11th intercostal spaces using a Tru-Cut biopsy needle (CareFusion, 14-gauge × 15 cm; Becton Dickinson, Vernon Hills, IL, USA), and four core tissue samples were collected from each animal (~50 mg). Samples were placed on ice immediately after collection and stored at −80°C for later analysis of the trace mineral concentration.

Blood samples were collected from all cows on days −114, 1, and 80 via jugular venipuncture using commercial vacuum tubes without anticoagulant for serum collection (BD Vacutainer, 10 ml; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Samples collected on day 1 were obtained individually according to each cow’s calving date. Thereafter, all cows were sampled on common calendar dates; therefore, the sampling days reported represent the average days postpartum for cows and calves at each collection time point. Samples were immediately placed on ice after collection, centrifuged at 2,000 × g for 25 min at 4°C to harvest serum, and stored at −80°C until further analysis.

2.3 Offspring collections

BW and blood samples were collected from all calves on day 1 (24 h after birth), following maternal colostrum consumption, and on days 80 and 234 (weaning). Samples were collected via jugular venipuncture using commercial vacuum tubes without anticoagulant for serum collection (BD Vacutainer, 10 ml; Becton, Dickinson and Company). Samples were immediately placed on ice after collection, centrifuged at 2,000 × g for 25 min at 4°C to harvest serum, and stored at −80°C until further analysis.

Two liver biopsy samples (~50 mg each) were collected from all calves on day 1 using the procedures described above. The first sample was placed on ice immediately after collection and stored at −80°C for later analysis of the trace mineral concentrations. The second sample was wrapped in aluminum foil, snap-frozen in liquid nitrogen, and then stored at −80°C until RNA extraction.

2.4 Laboratory analysis

2.4.1 Hay and forage

The hay and forage samples were sent to a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY, USA) and analyzed using wet procedures according to AOAC (2006) for crude protein (CP; method 976.05) and minerals (method 985.01). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) concentrations were analyzed as described by Van Soest et al. (1991). The total digestible nutrient (TDN) concentrations were calculated according to Weiss et al. (1992), while the net energy for maintenance (NEm), gain (NEg), and lactation (NEl) were estimated using the equations proposed by NASEM (2016).

2.4.2 Liver mineral concentration and serum analysis

The liver trace mineral concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS) at the Veterinary Diagnostic Laboratory from Michigan State University (Lansing, MI, USA). Serum biochemistry analyses were performed using a Beckman Coulter AU480 Chemistry Analyzer (Beckman Coulter Inc., Brea, CA, USA). The concentrations of albumin, total protein, urea nitrogen (serum urea nitrogen, SUN), glucose, and creatinine were determined using commercial colorimetric and enzymatic kits (Beckman Coulter Inc.). The serum insulin and cortisol concentrations were measured using chemiluminescent enzyme immunoassays (Immulite 2000; Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). The intra- and inter-assay coefficients of variation were 4.69% and 4.54% for insulin and were 4.26% and 5.64% for cortisol, respectively.

2.4.3 Liver transcriptome: RNA extraction, library preparation, sequencing, and raw data processing

All laboratory analyses were conducted in samples collected from all animals, except for the liver transcriptomic profile analysis, which was conducted in only nine male offspring per treatment. For this, liver samples were used for total RNA isolation using the Trizol reagent (ThermoFisher Scientific Inc., Carlsberg, CA, USA) following the manufacturer’s protocol, as described in Kertz et al. (2025). RNA purification was performed using DNase I (80 µl; Qiagen, Germantown, MA, USA), followed by a 15-min incubation at room temperature. RNA quality control was performed on a Qubit fluorometer using a Qubit RNA broad-range assay kit (Life Technologies, Thermo Fisher Scientific Inc., Waltham, MA, USA). In addition, the RNA integrity was assessed using the Agilent Bioanalyzer and the Agilent RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA). Directional mRNA libraries were prepared (poly A enrichment) using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA). Sequencing was performed in the Illumina NovaSeqX Plus series platform at Novogene Co. (Sacramento, CA, USA) to generate paired-end 150-bp reads at a depth of 20 million reads/sample.

Raw read quality control included removal of sequencing adapters and filtering of reads with Phred scores below 30. FastQC v. 0.11.9 (Babraham Bioinformatics, Babraham Institute, Cambridge, UK) and MultiQC v. 1.17 (Ewels et al., 2016) were used to evaluate data quality and generate summary statistics. Reads were aligned to the Bos taurus ARS-UCD1.3 reference genome using the STAR aligner v. 2.7.5 (Dobin et al., 2013). Gene-level read counts were generated using the quantMode GeneCounts option in STAR. Post-mapping quality control was conducted using MultiQC and edgeR version 4.0.3 (Robinson et al., 2010).

2.5 Statistical analysis

2.5.1 BW, BCS, liver trace mineral concentration, and serum variables

The animal was considered the experimental unit for all statistical analyses. Data were analyzed using the GLIMMIX procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC, USA) for binary variables (i.e., pregnancy loss and percentage of males and females born) and the MIXED procedure for all continuous variables (i.e., all other variables, except for the transcriptomic data). The Kenward–Roger approximation was used to estimate the degrees of freedom in the denominator for the fixed-effects tests.

The statistical model for non-repeated measurements [i.e., days prepartum at treatment injection, pregnancy loss, percentage of males and females born, cow BW and BCS changes, age at weaning, adjusted 205-day BW, average daily gain (ADG), and the calf’s liver mineral concentration at birth] was tested for the fixed effect of treatment and the random effect of animal (treatment). For the cow variables, BW on day −114 was included as a covariate for BW change, whereas BCS on day −114 was included as a covariate for BCS change. Days prepartum was also included as a covariate for the cow-related variables. For calf ADG, age at weaning was included as a covariate. Only covariates with p ≤ 0.10 were retained in the final model.

All other variables were analyzed as repeated-measures data, tested for the fixed effects of treatment, day, and their interaction, and included the random effect of animal (treatment). The day −114 values for each variable were included as covariates, in addition to the other covariates previously described for the analysis of non-repeated variables. Only covariates with p ≤ 0.10 were retained in the final model. Covariance structures evaluated included autoregressive [AR(1)], compound symmetry, unstructured, and Toeplitz. The AR(1) structure was selected for all repeated-measures variables based on the lowest Akaike information criterion, indicating the best model fit.

After defining the statistical model, the normality of the residuals from all continuous variables was evaluated using studentized residuals from the MIXED procedure. These residuals were evaluated visually through histograms, boxplots, and normal probability (Q–Q) plots and were formally tested using the UNIVARIATE procedure of SAS, which provided the Shapiro–Wilk test. Normality was considered acceptable when residuals exhibited approximate symmetry, no systematic deviations in the Q–Q plots, and p > 0.10 in the Shapiro–Wilk test. Variables that did not meet the normality criteria were log-transformed and then analyzed assuming a normal distribution. Log-transformed data were subsequently back-transformed to the original scale for presentation and interpretation. Means were separated using the protected least significant difference (PDIFF; t-test), and all results were reported as least square means followed by SEM. Significance was defined as p ≤ 0.05 and tendency as p > 0.05 and p ≤ 0.10.

2.5.2 Differential expression and functional overrepresentation analyses

Differential gene expression analysis was performed using DESeq2 v.1.30.1, which models the RNA sequencing (RNA-seq) count data using a negative binomial distribution (Love et al., 2014). Pairwise comparisons between the TMI and Saline groups were conducted to estimate gene dispersion and the log2 fold change (log2FC). Gene annotation was performed using biomaRt v.2.46.3 (Durinck et al., 2009). A combination of significance and fold change was used to determine the differentially expressed genes (DEGs; p ≤ 0.05) and the absolute log2 fold change [(|log2FC|) ≥ 0.5]. The direction of regulation (i.e., up or down) was determined based on the sign of the log2FC in the TMI group.

Functional enrichment analyses for Gene Ontology (GO) biological processes (BPs), molecular functions, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed using WebGestalt (WEB-based Gene SeT AnaLysis Toolkit) (Liao et al., 2019). Expressed genes retained after quality control were used as the background gene set for B. taurus. GO terms for BP were considered significant at false discovery rate (FDR) ≤0.05. In addition, analyses of the immune system processes were conducted using the ClueGO plug-in in Cytoscape (Bindea et al., 2009). Functional terms were grouped using a kappa score of 0.4 and visualized as functionally grouped networks (group p ≤ 0.05).

3 Results

3.1 Maternal

No effects of treatment (p ≥ 0.11) were detected for days prepartum at the time of treatment injection, pregnancy loss, or percentage of males or females born (Table 2). Furthermore, no effects of treatments or treatment × day were detected (p ≥ 0.21) for BW, BW change, BCS, BCS change (Table 3), or liver mineral concentration (Table 4). However, a treatment × day effect was detected (p = 0.05) for serum total protein concentration, with TMI cows having a lower concentration (p = 0.03) on days 1 and 80 than did Saline cows (Table 4). No effects of treatment or treatment × day were detected (p ≥ 0.11) for the other serum biochemistry variables or the serum insulin concentration.

Table 2. Reproductive variables of cows receiving a saline solution (Saline; n = 20) or a trace mineral injection (TMI; n = 20) at approximately 114 days prepartum and their effects on subsequent offspring.

Saline solution consisted of 0.9% NaCl, whereas TMI had 60, 10, 5, and 15 mg/ml of Zn, Mn, Se, and Cu, respectively (Multimin 90, Axiota Animal Heath, Fort Collins, CO, USA). Treatments were administered subcutaneously (1 ml/90 kg BW) on the right side of the neck approximately 114 days prepartum.

Values in parentheses represent the number of positive animals relative to the total number of animals evaluated.

Table 3. Body weight (BW) and body condition score (BCS) of cows receiving a saline solution (Saline; n = 20) or a trace mineral injection (TMI; n = 20) at approximately 114 days prepartum.

Saline solution consisted of 0.9% NaCl, whereas TMI had 60, 10, 5, and 15 mg/ml of Zn, Mn, Se, and Cu, respectively (Multimin 90, Axiota Animal Heath, Fort Collins, CO, USA). Treatments were administered subcutaneously (1 mL/90 kg BW) on the right side of the neck at approximately 114 days prepartum. The BW and BCS were collected on days −114 and 0 (24-h postpartum) and on days 80 and 234 (weaning).

Covariate-adjusted for BW on day −114 (p < 0.01).

Covariate-adjusted for BCS on day −114 (p < 0.01).

Table 4. Liver mineral concentrations, serum insulin concentration, and blood biochemistry variables of cows receiving a saline solution (Saline; n = 20) or a trace mineral injection (TMI; n = 20) at approximately 114 days prepartum.

Values with lowercase letters in the same line differ (p ≤ 0.05).

Saline solution consisted of 0.9% NaCl, whereas TMI had 60, 10, 5, and 15 mg/ml of Zn, Mn, Se, and Cu, respectively (Multimin 90, Axiota Animal Heath, Fort Collins, CO, USA). Treatments were administered subcutaneously (1 ml/90 kg BW) on the right side of the neck at approximately 114 days prepartum. Liver samples were collected on days −114 and 1 (24-h postpartum) and serum samples on days −114, 1, and 80.

Covariate-adjusted for baseline (day −114) concentration (p < 0.01).

Not normally distributed (p < 0.06). The data were therefore log-transformed for statistical analysis and back-transformed to generate the averages presented.

3.2 Offspring BW, serum variables, and liver trace mineral concentration

The age at weaning was similar between treatments (p = 0.16) (Table 2). A treatment × day effect tended to be detected (p = 0.10) for BW, in which TMI calves tended to have greater (p ≤ 0.08) BW on days 80 and 157 compared with Saline calves (Table 5). Furthermore, the ADG tended to be greater (p = 0.10) for TMI vs. Saline calves from day 1 to day 80. The ADG did not differ between treatments (p ≥ 0.53) after day 80, or when the entire period was evaluated (days 0–234).

Table 5. Body weight (BW) and average daily gain (ADG) of calves born to cows receiving a saline solution (Saline; n = 19) or a trace mineral injection (TMI; n = 19) at approximately 114 days prepartum.

Values with lowercase letters in the same line tend to differ (0.05 > p ≤ 0.10).

Saline solution consisted of 0.9% NaCl, whereas TMI had 60, 10, 5, and 15 mg/ml of Zn, Mn, Se, and Cu, respectively (Multimin 90, Axiota Animal Heath, Fort Collins, CO, USA). Treatments were administered subcutaneously (1 ml/90 kg BW) on dams on the right side of the neck at approximately 114 days prepartum.

Covariate-adjusted for age at weaning (p ≤ 0.09).

A treatment × day effect was detected (p ≤ 0.05) for the serum concentrations of insulin, globulin, total protein, and creatinine, with TMI calves having greater (p ≤ 0.05) concentrations of insulin on days 1 and 80, as well as globulin, total protein, and creatinine only on day 0, compared with Saline calves (Table 6). Furthermore, effects of treatment (p ≤ 0.05), but not treatment × day (p = 0.68), were detected for the serum concentrations of albumin and glucose, of which TMI calves had greater concentration compared with Saline calves. No effects of treatment or treatment × day were detected (p ≥ 0.19) for the liver mineral concentrations, the serum cortisol concentration, or SUN.

Table 6. Liver mineral concentration, serum insulin concentration, and biochemistry variables of calves born to cows receiving a saline solution (Saline; n = 19) or a trace mineral injection (TMI; n = 19) at approximately 114 days prepartum.

Values with lowercase letters in the same line differ (p ≤ 0.05).

Saline solution consisted of 0.9% NaCl, whereas TMI had 60, 10, 5, and 15 mg/ml of Zn, Mn, Se, and Cu, respectively (Multimin 90, Axiota Animal Heath, Fort Collins, CO, USA). Treatments were administered subcutaneously (1 ml/90 kg BW) on dams on the right side of the neck at approximately 114 days prepartum. Liver was collected on day 1 (24-h postpartum) and serum on days 1, 80, and 234.

Not normally distributed (p < 0.09). The data were therefore log-transformed for statistical analysis and back-transformed to generate the averages and SEM presented.

3.3 Offspring liver transcriptomic profile

RNA-seq generated an average of 23.8 million clean reads per sample, of which 97.4% were uniquely mapped to the reference genome. Following post-mapping quality control, 15,385 of the 36,075 detected genes were retained across 18 samples for analysis of the DEGs. A total of 283 DEGs were detected, of which 128 and 155 were down- and upregulated in TMI calves, respectively, compared with Saline calves (Supplementary Table 1). The functional classification of the DEGs was as follows: 76.3% protein-coding genes, 10.25% long non-coding RNAs, and 13% others (e.g., pseudogenes, small nucleolar RNAs, and miRNAs). The top 5 annotated upregulated genes, based on log2FC, were ISG15, MX1, MAMLD1, HERC6, and OAS1Y. The downregulated genes were SLCO4A1, AOX1, ATP6V1C2, C11H2orf74, and MYL6B.

We performed a functional overrepresentation analysis to understand the biological and immune process GO terms and pathways affected by the DEGs using WebGestalt and ClueGO tools. Overrepresented terms, such as innate immune response, response to cytokine, and leukocyte migration, were significant (FDR < 0.05) based on the BP (Figure 1; Supplementary Table 2). Overrepresentation analysis of the DEGs in Cluego GO terms for immune system processes (group p ≤ 0.05) highlighted genes underlying the development or function of the immune system (Figure 2; Supplementary Table 3). The terms included response to type I interferon and positive regulation of innate immune response (Figure 2; Supplementary Table 2).

4 Discussion

4.1 Maternal

In our experiment, TMI did not affect the BW and BCS in our mature cows. In growing cattle, TMI also does not appear to increase growth, as observed in our previous experiment (Vedovatto et al., 2024) and in a recent meta-analysis (McKnight et al., 2026). In another experiment, repeated TMI during gestation (at 205, 114, and 44 days prepartum) also did not affect the BW or BCS in heifers (Stokes et al., 2019). However, in thinner Bos indicus mature cows (BCS ≤ 4.7), a TMI at the start of the fixed-time artificial insemination protocol tended to increase the BCS 30 days after insemination (Vedovatto et al., 2019a). As our cows had an adequate BCS at the beginning of the experiment (BCS ≥ 5.78) and had already reached the mature BW, the lack of TMI effects on BW and BCS is expected.

The TMI administered 114 days prepartum did not increase the liver mineral concentrations in cows at calving. Following subcutaneous administration, TMI reached peak plasma concentrations approximately 8–10 h post-injection, with liver uptake occurring within 24 h (Pogge et al., 2012). However, the duration of trace mineral storage in the liver likely depends on several factors, including the animal’s mineral status at the time of injection, the dietary mineral supply and absorption, and physiological requirements. In a previous study conducted by our group, TMI administered at weaning in steers increased the liver Cu and Se concentrations for approximately 14 days post-injection; however, these effects were not sustained through the end of the experiment at day 197 (Vedovatto et al., 2024). In contrast, Stokes et al. (2019) reported that repeated TMI during gestation (with the last injection at 44 days prepartum) increased the liver Cu and Se concentrations at calving. Therefore, in the present study, because the second liver biopsy was collected at calving (i.e., 114 days after TMI administration), the trace mineral concentrations in the liver likely increased transiently following injection but had returned to baseline by the time of the second sampling. Furthermore, free-choice mineral mix supplementation already supplied substantial Zn, Cu, Mn, and Se, which may have limited the incremental effect of the injectable product.

The TMI decreased the serum total protein concentrations at calving and at 80 days postpartum. The biological mechanisms underlying this response are unclear, but may be associated with an improved antioxidant status and a reduced systemic inflammation. In a previous study, it was reported that TMI increased the plasma concentrations of the antioxidant enzymes superoxide dismutase and glutathione peroxidase while decreasing the concentrations of cortisol and haptoglobin; however, these effects were transient and were observed only for a few days following injection (Vedovatto et al., 2024). A reduction in acute-phase proteins, such as haptoglobin, could partially explain the decrease in serum total protein, as these proteins contribute to the circulating protein pool (Gouvêa et al., 2022). Nevertheless, in the present study, this mechanism alone is unlikely to account for the observed response, given that the reduction in total protein persisted for more than 114 days after TMI administration. Therefore, the decrease in the total protein concentration observed herein is unexpected and warrants further investigation.

4.2 Offspring

Maternal TMI increased the offspring BW on days 80 and 157 but did not affect the birth or weaning BW. These findings are in contrast with previous reports. For example, repeated TMI during gestation in Angus heifers decreased the offspring BW at birth but had no effect on the weaning BW (Stokes et al., 2019). On the other hand, TMI administered 4 weeks before calving and again at 4 weeks before the breeding season in British crossbred cows did not affect the birth or the weaning BW (Wilcox et al., 2026). The positive effects of TMI on postnatal growth observed in our study, but not in others, may be partially explained by breed differences. Our experiment included cattle with B. indicus influence, whereas prior studies primarily used B. taurus cattle. These subspecies differ in the mechanisms that regulate Cu and Se metabolism, resulting in distinct trace mineral status in cows and neonates, particularly in liver stores (Ranches et al., 2021). Consequently, the fetal programming responses to maternal TMI may vary by breed, potentially explaining the divergent outcomes across studies.

The trace mineral concentration in the liver of calves at birth was not affected by maternal TMI in the present study. This contrasts with Stokes et al. (2019), who reported increased hepatic Cu and Se concentrations in newborn calves following repeated TMI during gestation, along with greater maternal liver Cu and Zn concentrations at calving. In our experiment, however, the maternal liver trace mineral concentrations were not altered. These results suggest that the effectiveness of TMI in enhancing the fetal trace mineral stores depends not only on supplementation but also on the maternal mineral status near parturition. In our study, TMI was administered only once at 114 days prepartum, likely causing a transient increase in the liver mineral concentrations during mid-gestation, but not during late gestation. Because fetal liver growth accelerates during late gestation (Wathes, 2022) and greater trace mineral accretion in the liver may occur during this period, the lack of TMI administration closer to calving may have allowed the maternal mineral supply to decline before peaking fetal demand, thereby limiting mineral transfer to the fetus.

The increase in the serum insulin and glucose concentrations in calves born to TMI cows is likely explained by in utero programming of metabolic function. Trace minerals such as Zn and Se play key roles in pancreatic development, insulin synthesis, and antioxidant defense, thereby enhancing β-cell function (i.e., cells in the pancreas responsible for producing insulin) and glucose metabolism (Li, 2014; Ojeda et al., 2019; Ferreira et al., 2022). The effects of trace mineral supplementation (especially Se) on fetal programming have been well described in rats (Ojeda et al., 2019), but experiments in livestock are poorly evaluated (Anas et al., 2023). Consequently, maternal TMI may have improved the endocrine and metabolic capacity in the offspring, leading to greater circulating insulin and glucose concentrations and potentially supporting early postnatal growth. Notably, these effects were observed from birth to day 80, which corresponds to the period when TMI calves tended to exhibit greater growth.

The greater serum concentrations of globulin, total protein, and creatinine at birth in calves born from TMI cows suggest improved metabolic and developmental status. Trace minerals such as Zn, Cu, Se, and Mn play essential roles in protein synthesis, enzyme activity, and immune function, thereby potentially enhancing hepatic function and increasing circulating protein concentrations (NASEM, 2016; Palomares, 2022). In addition, these minerals are involved in the regulation of immune responses and antibody production, which can contribute to higher globulin concentrations (Palomares, 2022; Vedovatto et al., 2024).

The liver transcriptomic analysis revealed that maternal TMI markedly altered the expression of the genes associated with innate immune function in the offspring at birth. The upregulation of ISG15, MX1, HERC6, and OAS1Y, together with the enrichment of the pathways related to innate immune response, cytokine signaling, leukocyte migration, and type I interferon response, clearly indicates the activation of the antiviral and early immune defense mechanisms in the liver at birth. This response is biologically plausible given the essential roles of trace minerals such as Zn, Cu, Se, and Mn in regulating cytokine signaling, oxidative balance, and gene expression (Anas et al., 2023; Vedovatto et al., 2024). Although studies evaluating the effects of maternal trace mineral supplementation on offspring immune function remain limited, available evidence suggests that improving the maternal mineral status can enhance the antioxidant capacity and, consequently, the offspring immune function (Palomares, 2022). In this study, these transcriptomic changes are consistent with the observed serum responses. Elevated serum concentrations of globulins, total proteins, and albumin support enhanced hepatic synthesis and immune function, while elevated insulin and glucose concentrations indicate improved metabolic capacity. These transcriptional changes suggest an improved immunological readiness at birth, which may contribute to reduced disease susceptibility early in life in beef calves. Further research evaluating the effects of maternal TMI on vaccination responses and health outcomes in offspring is warranted, as the present results indicate that immune function can be programmed through trace mineral supplementation during gestation.

5 Conclusion

Maternal TMI applied 114 days before calving elicited measurable programming effects in the offspring, although it did not alter the maternal BW and BCS or the liver trace mineral concentrations at calving, indicating that a single administration is insufficient to induce long-term changes in the maternal mineral status. The liver trace mineral concentrations in calves at birth were unaffected, while maternal TMI increased the circulating concentrations of albumin, glucose, and insulin and increased the globulin, total protein, and creatinine at birth, suggesting improved metabolic and developmental status. These physiological responses were accompanied by a tendency for greater BW and ADG during early postnatal life. Liver transcriptomic analysis further demonstrated that maternal TMI modulated the expression of the genes associated with innate immune function, particularly the pathways related to interferon signaling and immune response. These findings indicate that maternal TMI promotes fetal programming of both metabolic and immune functions, resulting in enhanced immunological readiness and metabolic capacity at birth.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fanim.2026.1923158/full#supplementary-material

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Footnotes

Edited by: Nara Regina Brandão Cônsolo, University of São Paulo, Brazil

Reviewed by: Muhammad Anas, Ministry of National Food Security and Research, Pakistan

Gastón Alfaro, National University of Cordoba, Argentina