Early-life vitamin A supplementation modulates the skeletal muscle transcriptome and intramuscular fat deposition in feedlot-finished beef steers

Abstract

This study evaluated the effects of neonatal vitamin A supplementation on intramuscular fat (IMF) deposition and the molecular mechanisms regulating adipogenesis and lipogenesis in Montana × Nellore crossbred beef steers. We hypothesize that neonatal vitamin A supplementation induces long-term transcriptional changes associated with IMF development. Thirty-four calves were randomly assigned to two groups: a control receiving a placebo and a treatment group receiving a single intramuscular injection of 300,000 IU of vitamin A at birth. Longissimus thoracis (LT) muscle samples were collected via biopsy at 40 days of age (cow-calf phase) and again at 400 ± 10 days of age (finishing phase) immediately after slaughter. At deboning (48 hours postmortem), LT samples were collected between the 12th and 13th ribs for chemical composition assessment. Vitamin A supplementation had no significant effect on weaning weight, average daily gain, final body weight, backfat thickness, or ribeye area (P > 0.05). However, IMF content was significantly higher (P < 0.01) in the vitamin A-supplemented (4.10%) compared to control (2.57%). Transcriptomic analysis revealed 52 and 165 differentially expressed genes (DEGs; FDR < 0.05) during the cow-calf and finishing phases, respectively. Functional enrichment analyses showed that vitamin A supplementation upregulated the actin cytoskeleton, insulin, and calcium signaling pathways during the cow-calf phase, and the PI3K-Akt, AMPK, and PPAR signaling pathways during the finishing phase. Notably, key genes associated with adipogenesis and lipogenesis, such as KRAS, ITGB6, CALML6, CISH, and ATF3 in the cow-calf phase, and LEP, ACACA, FASN, SCD, ELOVL6, and CIDEC in the finishing phase, were upregulated in response to vitamin A, as well as several adipogenic regulators, including PPARG, C/EBPA, and FABP4. Pathway enrichment analysis highlighted the PI3K-Akt signaling pathway as a central mediator of vitamin A’s effects on adipocyte differentiation and lipid metabolism. Thus, neonatal vitamin A supplementation can enhance IMF deposition in beef steers by modulating key molecular pathways, without negatively impacting growth performance or carcass traits.

This study provides new molecular insights into how neonatal vitamin A supplementation influences intramuscular fat deposition in beef cattle, identifying key genes and pathways involved in adipogenesis and lipogenesis. These findings could help optimize nutritional strategies designed to enhance the meat quality of crossbred cattle.

Graphical Abstract

Graphical Abstract.

Introduction

Intramuscular fat (IMF) deposition is an important attribute of beef that directly influences sensory characteristics such as tenderness and juiciness, both of which are strongly associated with consumer acceptance (Roeber et al., 2000; O’Quinn et al., 2015; Choi et al., 2019). In cattle, the development of IMF occurs mainly between late gestation and the first months after birth, a period known as the “marbling window,” which is characterized primarily by an increase in the number of adipose cells (Du et al., 2013). Researchers suggest that specific nutritional interventions beyond simple energy supply can significantly modulate the adipogenic process during this critical period, with a positive impact on the degree of meat marbling (Wang et al., 2016).

The term vitamin A comprises 20 carbon molecules, including retinol and carotenoids, whose biological activity is similar to that of trans-retinol (Carazo et al., 2021). The primary active metabolite of vitamin A, all-trans retinoic acid, directly regulates adipogenesis by stimulating the differentiation of undifferentiated cells and preferentially directing them toward the adipogenic lineage, particularly intramuscular adipocytes (Shaw et al., 2003; Wang et al., 2016; Peng et al., 2021). In a recent study, Dean et al. (2024) reported that vitamin A supplementation during late gestation enhances intramuscular adipogenesis in the offspring by exerting an effect on early adipogenic commitment and increasing pre-adipocytes at birth. Additionally, their findings highlight the impact on IMF deposition throughout postnatal life, from weaning to slaughter. Although the positive role of vitamin A in bovine adipogenesis has been recognized, the results reported in the literature are divergent due to differences in the doses used (Kruk et al., 2008; Harris et al., 2018; Yu et al., 2022) and the physiological stage of animals at the time of administration (Oka et al., 1998; Arnett et al., 2009; Campos et al., 2020; Knutson et al., 2020), and because the studies are limited to a few breeds or specific experimental conditions (Knutson et al., 2020).

Harris et al. (2018) demonstrated that neonatal administration of vitamin A increased marbling score in Aberdeen Angus calves (Bos taurus). Using a similar approach, Maciel et al. (2022) found positive effects on adipogenesis in Montana × Nellore crossbred calves (Bos taurus × Bos indicus), which enhance IMF content. However, these studies evaluated a restricted to a limited number of genes. Given this, we hypothesize that neonatal vitamin A supplementation induces long-term transcriptional changes associated with IMF development in beef cattle. Specifically, we propose that vitamin A administered at birth modulates the expression of genes and pathways related to adipogenesis and lipogenesis, detectable at slaughter.

Therefore, this study aims to identify novel metabolic pathways and genes involved in the physiological mechanisms underlying the effects of neonatal vitamin A supplementation on adipogenesis and lipogenesis. To this end, a comprehensive RNA sequencing-based approach was used to investigate IMF deposition in beef steers supplemented with vitamin A at birth and finished in a feedlot.

Material and Methods

All animal procedures followed the ethical guidelines established by the Animal Use Ethics Committee of the São Paulo State University—UNESP (protocol number 0107, approved on June 21, 2018).

Animals, treatments, and sample collection

Thirty-four male F1 Montana × Nellore calves were used (17 did not receive vitamin A [control] and 17 were supplemented with vitamin A). At birth, vitamin A-supplemented calves received an intramuscular dose of 300,000 IU of Monovin A (Bravet, Rio de Janeiro, Rio de Janeiro, Brazil), while control calves received a saline placebo. The body weight at birth of control and vitamin A-treated animals was 28 ± 1.4 and 26 ± 1.2 kg, respectively. In the creep-feeding system, calves were kept with their mothers in the same paddock but had exclusive and free access to a supplement (ad libitum) corresponding to approximately 1% of their body weight. The amount of supplement provided per animal was adjusted weekly based on performance prediction using a diet evaluation and formulation system (“Ração de Lucro Máximo” [RLM] v. 3.3.57 software) (Lanna et al., 2011; Andrade et al., 2023). The creep-feeding supplement consisted of corn, soybean meal, salt, molasses, and a mineral premix (50, 25, 9, 1, and 5%, respectively).

All animals were immunocastrated with three doses of anti-gonadotropin-releasing factor (GnRF) vaccine (Bopriva, Zoetis, Campinas, São Paulo, Brazil). The first dose was applied at weaning, and the second and third doses 30 and 90 days later, respectively. At 40 days of age, muscle samples from the Longissimus thoracis (LT) were obtained via biopsy from both treatment groups. The procedure was performed at the level of the 13th thoracic vertebra, where the skin over the LT area was previously shaved and disinfected. Lidocaine 2% without epinephrine (Lidovet, Bravet, Rio de Janeiro, RJ, Brazil) was administered subcutaneously at the biopsy site at a dose of 4 mg/kg as a local anesthetic. Following antisepsis, a small incision (~1 cm) was made, and a sterilized Bergström biopsy needle (Ortovet, São Paulo, SP, Brasil) was used to collect approximately 1.0 g of muscle tissue. Samples were immediately immersed in liquid nitrogen and subsequently stored (−80 °C) for further analysis. Following tissue collection, the incision was rinsed with sterile saline and water, then closed using veterinary tissue adhesive. An antibiotic spray was applied to the wound, and all calves were monitored for 48 h post-procedure to ensure proper recovery.

After 210 days of age, the control and vitamin A-supplemented animals were weighted (weaning weight = 237 and 236 ± 11 kg, respectively), transferred to the feedlot, and housed in covered pens with concrete floors that were equipped with individual feeders and drinkers. The two groups of animals were fed a diet formulated using the RLM v. 3.3.57 software (Lanna et al., 2011; Andrade et al., 2023). The finishing diet consisted of 12.7% roughage (sugarcane bagasse) and 87.3% concentrate feeds (ground corn, soybean meal, cottonseed, potassium chloride, and mineral-vitamin supplement). The mineral-vitamin mix contained 1231.10 IU/kg of vitamin A. The diet was supplied ad libitum, twice a day at 8 am and 4 pm (Table 1).

Table 1.

Ingredients and chemical composition of the experimental diet.

Item
Ingredients (% dry matter [DM])
Sugarcane bagasse	12.7
Ground corn	73.8
Soybean meal	4.8
Cottonseed	4.0
Potassium chloride	1.27
Mineral-vitamin supplement1	3.43
Chemical composition (% DM)2
Dry matter	68.00
Crude protein	13.50
Ether extract	3.83
Ash	4.25
Neutral detergent fiber	22.28
Estimated TDN	78.50

¹Mineral-vitamin supplement: sodium: 0.2076%; potassium: 0.87%; magnesium: 0.3585%; sulfur: 0.6%; iron: 75.458 ppm; copper: 14.5 ppm; manganese 38.3 ppm; zinc: 59.4 ppm; cobalt: 0.93 ppm; iodine: 0.59 ppm; selenium: 0.22 ppm; fluorine: 15.3542 ppm; vitamin A: 1231.10 IU/kg; vitamin D3: 576.2042 IU/kg; vitamin E: 46.5 IU/kg; thiamine (B1): 2.9703 ppm; niacin (B3): 22.0183 ppm; biotin: 0.9379 ppm; pyridoxine (B6): 6.3750 ppm; vitamin B12: 0.0767 ppm; folic acid: 1.0875 ppm.

²The composition of the diet was obtained by feed analysis (AOAC, 2005), followed by the determination of dry matter (method 976.05), crude protein (method 976.05, N * 6.25), and ash content (method 942.05). For neutral detergent fiber (NDF) analysis, samples were treated with alpha-amylase at a stable temperature without the addition of sodium sulfite and corrected for ash (aNDFom) (Mertens, 2002). ADFom was measured according to Van Soest et al. (1991)  and ether extract was determined by Soxhlet extraction (method 920.39), following previously described procedures (AOAC, 2005). Total digestible nutrients (TDN) were estimated according to Weiss et al. (1992) .

Performance, slaughter, sample collection, and carcass traits

In the present study, animals were managed in a “super early-maturing” production system, as described by Ferraz and Felício (2010) . For the determination of average daily weight gain (ADG), the animals were weighed at the beginning and end of the experimental period after fasting for 16 hours. After 180 days in the feedlot and reaching approximately 400 kg of final body weight (FBW), all animals were slaughtered at a commercial facility inspected and registered with SISP (Boituva, São Paulo, Brazil).

The animals were slaughtered by cerebral concussion and slitting of the jugular vein, followed by removal of the hide and evisceration. The carcasses did not undergo electrical stimulation. Carcasses were identified, washed, halved, and individually weighed for the determination of hot carcass weight (HCW) and dressing percentage (DP). Individual LT muscle samples from all animals (approximately 10 g; central portion of the LT muscle) were collected immediately after slaughter (hot carcass) from the 12th rib of the left half carcass and snap-frozen in liquid nitrogen. These aliquots were transported to the laboratory and then stored at −80 °C until the time of RNA extraction and analysis. Each half-carcass was chilled at 1 °C for approximately 48 hours. Prior to deboning, backfat thickness (BFT) and ribeye area (REA) were measured according to USDA-AMS Standards for Grades of Carcass Beef (USDA, 1997).

Chemical composition of meat

The chemical composition of meat was assessed using LT samples collected from the section between the 12th and 13th ribs on the left side of each carcass. The meat sample was sliced into 2.54 cm thick steak, vacuum-sealed in low-oxygen-permeability plastic bags, and stored at 2 °C in a biological oxygen demand incubator (model TE-371, TECNAL, Piracicaba, São Paulo, Brazil) for seven days. Subsequently, the steaks were frozen and stored until the time of laboratory analyses. Briefly, the steak was thawed and ground in a multiprocessor for 5 minutes using approximately 180 g of sample. The average levels of moisture, protein, total collagen, and IMF content were determined by infrared spectroscopy in a FoodScan equipment (FOSS, Hillerød, Denmark), as reported (Anderson, 2007). At each reading, the sample was removed from the plate, homogenized again, and returned to the plate for the next reading (three readings in total).

RNA extraction, sequencing, and mapping of sequences to the reference genome

Samples from all 34 animals collected during the cow-calf phase (via biopsy) and immediately at slaughter (at hot carcass) were used for RNA extraction. Approximately 100 mg of LT muscle tissue was utilized for total RNA extraction using the TRIzol reagent (Life Technologies, Carlsbad, California, United States), following the manufacturer’s instructions. A central portion of the LT muscle samples was ground using a mortar and pestle, and approximately 100 mg of tissue was used for RNA extraction. Following RNA extraction, six animals were randomly selected from each experimental group (control vs. vitamin A), totaling 24 samples for RNA-Seq analyses (12 from the cow-calf phase and 12 from the finishing phase). For each sample, three technical replicates were analyzed to assess RNA quality and integrity using the Bioanalyzer 2100 system (Agilent, Santa Clara, California, United States).

Complementary DNA (cDNA) libraries were generated and multiplexed for each sample (in triplicate) using 2 µg of total RNA per replicate, following the protocol provided in the TruSeq RNA Sample Preparation Kit v2 (Illumina, USA). Library size distribution was evaluated with the Bioanalyzer 2100 (Agilent Technologies, USA), and concentrations were determined by quantitative PCR using the KAPA Library Quantification Kit (KAPA Biosystems, United States). Sequencing libraries were clustered and sequenced on a single lane using the HiSeq 2500 system (Illumina, San Diego, California, United States) with the v4 2 × 100 bp chemistry, generating paired-end reads of 100 base pairs. A minimum sequencing depth of 16 million reads per sample was achieved.

After the removal of sequencing adapters and low-quality sequences using the fastp v.0.23.2 program and verification of the results with FastQC and MultiQC v.1.12, the filtered reads were mapped to the bovine reference genome (Bos taurus UMD 3.1.1; http://www.ensembl.org/Bos_taurus/Info/Index/) using the STAR v.2.7.10a program. Mapping was performed independently for each sample, allowing up to two mismatches per read. Sequencing quality was assessed using the QualiMap v.2.2.1 and v.1.12 programs. Finally, a raw count matrix was generated with FeatureCounts v.2.0.

Statistical analysis

Statistical analyses were performed using SAS software, version 9.4 (SAS Institute Inc., Cary, North Carolina, United States). Initial body weight was included as a covariate, and analysis of variance (ANOVA) was conducted to evaluate the effects of treatment (control vs. vitamin A) on performance, carcass characteristics, and chemical composition of meat. The PROC GLM procedure was used, based on the following model:

$${\displaystyle \begin{array}{l}{\displaystyle Y_{ij}=\mu +T_i+{\epsilon }_{ij}}\end{array}}$$

Where:

Y_ij represents the observed value for the experimental unit under treatment i and replicate j; μ denotes the overall mean; t corresponds to the treatment effect (control or vitamin A); and Ɛ_ij is the random error term.

Differentially expressed genes (DEGs) were identified using the edgeR v.3.38.4 package in R. Normalization factors were estimated using the trimmed mean of M-values ( Robinson and Oshlack, 2010) for all pairwise comparisons. The dispersion hyperparameter was estimated using the robust empirical Bayes method; the latter was then applied to obtain quasi-likelihood estimates for the count distribution parameters of each gene (in each group/treatment) based on a generalized linear model, assuming a negative binomial distribution. The likelihood ratio test was applied to evaluate the significance of gene expression comparisons between treatments, assuming a χ² distribution.

Gene expression levels were calculated as the average expression across all samples within each group/treatment and reported as mean log-transformed counts per million (logCPM). The log-transformed fold change (logFC) was computed based on the logCPM ratio between two treatments. The Benjamini and Hochberg (1995)   procedure was applied to control the false discovery rate (FDR), ensuring an expected proportion of falsely rejected null hypotheses. Genes were defined as differentially expressed if they met the significance threshold of an FDR < 0.05 and had logFC values below −0.5 or above 0.5.

Functional inferences regarding DEGs were performed based on genes identified in each treatment. Overrepresentation analysis was performed with the clusterProfiler v.4.2 package ( Yu et al., 2012) in R using a hypergeometric test. Pathways and biological processes were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms.

Results

There were no differences in weaning weight (P = 0.58), ADG (P = 0.70), DMI (P = 0.59) or FBW (P = 0.71) between treatments (Figure 1). Similarly, vitamin A supplementation did not exert any effects (P > 0.05) on the carcass traits (HCW, DP, BFT, and REA). However, differences (P < 0.01) were found in IMF content, which was 2.57% in the control group and 4.10% in the vitamin A group (Figure 1). Besides, moisture, protein, and total collagen levels were similar (P > 0.05).

Figure 1.

Productive performance, carcass traits, and meat chemical composition of feedlot-finished F1 Montana × Nellore beef steers (control vs. vitamin A-supplemented). At birth, calves in the vitamin A group received an intramuscular dose of 300,000 IU, while control calves received a saline placebo. All 34 animals (17 per group) were included as biological replicates throughout all phases of the study. * Statistically significant difference (P < 0.05).

A total of 52 and 165 DEGs (FDR < 0.05) were identified during the cow-calf and finishing phases, respectively. While the majority of genes remained stable comparing control versus vitamin A, gene expression changes were more pronounced during the finishing phase. In the cow-calf phase, 19 genes were downregulated and 33 were upregulated, whereas in the finishing phase, 106 genes were downregulated and 59 were upregulated (Table 2). All DEGs identified during the cow-calf and finishing phases comparing control versus vitamin A were described in Supplementary Tables S1 and S2, respectively.

Table 2.

Absolute and relative number of genes detected in Longissimus thoracis muscle of feedlot-finished F1 Montana × Nellore beef steers (control vs. vitamin A-supplemented).

Treatments1	Total number of DEGs2	Expression	Absolute	Relative (%)
Cow-calf phase 3
Control vs. Vitamin A	52	down	19	0.13
		stable	14.022	99.64
		up	33	0.23
Finishing phase 4
Control vs. Vitamin A	165	down	106	0.75
		stable	13.911	98.83
		up	59	0.42

¹At birth, calves in the vitamin A group received an intramuscular dose of 300,000 IU, while control calves received a saline placebo. Muscle samples from all 34 animals were collected by biopsy (cow-calf phase) and at hot carcass (finishing phase) for RNA extraction. Following RNA extraction, six animals were randomly selected from each experimental group (control vs. vitamin A), totaling 24 samples for RNA-Seq analyses (12 from the cow-calf phase and 12 from the finishing phase).

²DEGs = differentially expressed genes.

³ Longissimus thoracis muscle samples collected via biopsy at 40 days of age.

⁴ Longissimus thoracis muscle samples collected immediately after slaughter (hot carcass).

We identified several key DEGs regulating adipogenic and lipogenic processes during both the cow-calf (Figure 2A) and finishing phases (Figure 2B). Notably, during the cow-calf phase, vitamin A supplementation upregulated the actin cytoskeleton, insulin, and calcium signaling pathways (Figure 3A). In the finishing phase, the PI3K-Akt, AMPK, and PPAR signaling pathways (Figure 3B) were upregulated. Furthermore, we characterized the network of DEGs shared across enriched metabolic KEEG pathways during the cow-calf (Figure 4A) and finishing phases (Figure 4B), revealing differences in the number of DEGs within each pathway between the vitamin A and control groups. Specifically, in the cow-calf phase, KRAS, ITGB6, CALML6, CISH, and ATF3 were significantly upregulated, while in the finishing phase, LEP, PGF, CSF1, and THBS1 were upregulated. Moreover, these genes, along with TNC, CREB5, MYC, EGFR, IL6R, CSF1R, and PPP2R1B, participate in the PI3K-Akt signaling pathway, a central regulator of adipocyte differentiation and adipogenesis.

Figure 2.

Differentially expressed genes (DEGs) in the Longissimus thoracis muscle of feedlot-finished F1 Montana × Nellore beef steers at 40 days of age (A; cow-calf phase) and at slaughter (B; finishing phase), comparing control and vitamin A-supplemented groups. Upregulated genes are indicated in blue, and downregulated genes in red. At birth, calves in the vitamin A group received an intramuscular injection of 300,000 IU of vitamin A, while control calves received a saline solution. Muscle samples from all 34 animals were collected by biopsy (cow-calf phase) and at hot carcass (finishing phase) for RNA extraction. Following RNA extraction, six animals were randomly selected from each experimental group (control vs. vitamin A), totaling 24 samples for RNA-Seq analyses (12 from the cow-calf phase and 12 from the finishing phase).

Figure 3.

Enriched metabolic pathways (Kyoto Encyclopedia of Genes and Genomes—KEEG) in Longissimus thoracis muscle of feedlot-finished F1 Montana × Nellore beef steers at 40 days of age (A; cow-calf phase) and at slaughter (B; finishing phase), comparing control and vitamin A-supplemented groups. At birth, calves in the vitamin A group received an intramuscular dose of 300,000 IU, while control calves received a saline placebo. Muscle samples from all 34 animals were collected by biopsy (cow-calf phase) and at hot carcass (finishing phase) for RNA extraction. Following RNA extraction, six animals were randomly selected from each experimental group (control vs. vitamin A), totaling 24 samples for RNA-Seq analyses (12 from the cow-calf phase and 12 from the finishing phase).

Figure 4.

Network of enriched metabolic pathways (KEGG) and differentially expressed genes (DEGs) shared among pathways in Longissimus thoracis muscle of feedlot-finished F1 Montana × Nellore beef steers at 40 days of age (A; cow-calf phase) and at slaughter (B; finishing phase), comparing control and vitamin A-supplemented groups. Gray dots indicate enriched pathways, while dots in varying shades of red and blue represent the relative expression levels of DEGs based on log-transformed fold change. Different dot sizes indicate the number of DEGs present in each enriched metabolic pathway (vitamin A vs. control). Muscle samples from all 34 animals were collected by biopsy (cow-calf phase) and at hot carcass (finishing phase) for RNA extraction. Following RNA extraction, six animals were randomly selected from each experimental group (control vs. vitamin A), totaling 24 samples for RNA-Seq analyses (12 from the cow-calf phase and 12 from the finishing phase).

Discussion

The formation of IMF cells in cattle occurs from late gestation until approximately 250 days after birth (Du et al., 2013). Researchers have shown that nutrient supplementation of beef calves at pre-weaning increases the number of pre-adipocytes in skeletal muscle, thereby improving lipid biosynthesis during the postweaning period (Santos et al., 2023). This phase (marbling window) represents a critical period for metabolic imprinting, during which nutritional strategies can permanently influence the IMF deposition potential of the animal (Scheffler et al., 2014; Khounsaknalath et al., 2021).

Cow-calf phase

The proto-oncogene encoding a small GTPase (KRAS) plays a central role in cell signaling, proliferation, and differentiation (Yin et al., 2023). It modulates adipogenesis during the differentiation phase by inhibiting key transcription factors, such as PPARγ and C/EBP-β (Yu et al., 2021). In the present study, the upregulation of KRAS at 40 days of age in calves supplemented with vitamin A at birth suggests a mechanism that promotes an increase in the pool of preadipocytes during early development, prior to their differentiation into adipocytes.

Although the mechanisms by which retinoic acid regulates KRAS are not fully understood, retinoids are known to inhibit adipogenic differentiation at early stages, prior to the induction of PPARγ expression, particularly in the absence of C/EBP-α (Malibary, 2023). In this context, early activation of KRAS may support the proliferation of precursor cells, thereby enhancing the potential for intramuscular fat deposition during the finishing phase. Furthermore, KRAS was previously identified as a hub gene enriched in signaling pathways related to integrins, chemokine- and cytokine-mediated inflammation, and in PI3-kinase Nellore beef with greater marbling scores (Muniz et al., 2022). The latter, closely linked to the MAPK pathway, was also enriched in vitamin A-supplemented calves, and this pathway has been implicated in the inhibition of lipid accumulation in other biological models (Yu et al., 2021). Moreover, the MAPK pathway has been associated with the induction of ATF3 expression in various cell types, a well-known adaptive response gene (Lim et al., 2006; Lu et al., 2007), predominantly expressed in the stromal-vascular fraction of adipose tissue (Suganami et al., 2009).

Adipose tissue is highly vascularized, and its vasculature plays a crucial role in adipocyte turnover (Cao, 2013), providing endothelial progenitor cells that can differentiate into adipocytes (Crisan et al., 2008; Tran et al., 2012). Adipogenesis occurs in close association with vascular development (Yu et al., 2022), and retinoic acid has been shown to promote angiogenesis and the emergence of PDGFRα⁺/VEGFA⁺ adipose precursors in rodents (Wang et al., 2017a). Therefore, these findings suggest that, at this early stage, calves treated with vitamin A may exhibit increased vascularization and preadipocyte proliferation, fostering a cellular environment favorable to subsequent adipogenesis and lipogenesis.

Calmodulin and calmodulin-like protein 6 (CALML6) play important roles in calcium signaling, a critical regulator of adipocyte differentiation (Lin et al., 2011). Calmodulin, encoded by the CALM gene family, forms complexes with Ca²⁺ ions, which have high affinity for protein kinases such as CaMKK2 (Lin et al., 2011). Evidence suggests that the presence of CaMKK2 acts as an inhibitory factor in the differentiation of preadipocytes into mature adipocytes (Lin et al., 2011), possibly by suppressing the induction of PPARγ (Shi et al., 2000; Neal and Clipstone, 2002), in a mechanism potentially mediated by Ca²⁺/calmodulin-dependent kinases (Lin et al., 2011). Early work by Colca et al. (1987) already implicated calmodulin in insulin signaling and adipose metabolism. The integration of calmodulin activity with insulin and other kinase networks is therefore central to enhancing IMF development under vitamin A-supplemented conditions.

In addition to calcium-mediated regulation, other proteins are involved in coordinating lipid and glucose metabolism under similar conditions. Cytokine-inducible SH2-containing protein (CISH) is also noteworthy for its role in glucose and lipid metabolism. Our observation of increased CISH expression in vitamin A–supplemented animals align with Fonseca et al. (2020), who also described CISH as a hub gene upregulated in Nellore cattle with higher marbling. Experimental evidence from mice supports this metabolic role; CISH-deficient mice have reduced adiposity and exhibit resistance to diet-induced obesity due to decreased food intake and improved insulin sensitivity under high-fat diets (Naser et al., 2022). Interestingly, despite a lower body weight, CISH knockout mice maintain stable glucose tolerance, emphasizing CISH’s important role in lipid accumulation and insulin signaling rather than systemic glucose homeostasis.

The coordinated upregulation of KRAS, CALML6, and CISH in vitamin A-supplemented calves suggests a shared molecular response that delays preadipocyte differentiation while promoting precursor cell proliferation during early development. KRAS, through MAPK and PI3K signaling, and CALML6, via calcium-dependent inhibition of PPARγ, may expand the progenitor pool before terminal adipogenesis. Likewise, CISH, previously shown to repress PPARγ and adipogenic differentiation-related genes (Chen et al., 2021), possibly by inhibiting the GH–GHR–STAT5 pathway (Karlsson et al., 1999; Landsman and Waxman, 2005), may act similarly. Taken together, these regulatory mechanisms may extend the window of preadipocyte proliferation, establishing a cellular environment conducive to greater IMF deposition at the finishing phase.

Integrin subunit beta 6 (ITGB6) is another gene of interest, given its critical role in modulating mesenchymal cell fate toward either adipogenic or myogenic lineages, which directly impacts IMF content in beef cattle (Park et al., 2018). Besides, ITGB6 is a key molecular hub that connects extracellular cues to cytoskeletal dynamics and intracellular signaling, regulating the formation of myotubes through influencing the myogenic differentiation potential, membrane integrity, and cytoskeletal system stability (Zhang et al., 2022). This is consistent with the enrichment of actin cytoskeleton-associated pathways during the cow-calf phase, suggesting that upregulated ITGB6 expression under vitamin A supplementation may fine-tune the balance between muscle and fat development. Similar to our results, ITGB6 has been reported as a hub gene orchestrating myogenic versus adipogenic fate commitment in skeletal muscle (Sun et al., 2017). Together, these findings highlight ITGB6 as a potentially important regulator of extracellular matrix–cell interactions that facilitate increased IMF formation at the finishing phase.

Finishing phase

Maciel et al. (2022) demonstrated that neonatal vitamin A administration increases IMF content in Montana × Nellore cattle and upregulates lipogenic genes such as FABP4 and SREBF1, enhancing fat accumulation. This activation, evidenced by the upregulation of genes such as PPARG, C/EBPA, and FABP4, highlights the importance of vitamin A for modulation of the PPAR signaling pathway, as demonstrated in the present study.

Peroxisome proliferator–activated receptors (PPARs), particularly PPARG, are essential for adipocyte differentiation and maturation and for the regulation of lipogenesis (Bionaz et al., 2013), acting synergistically with C/EBPA (Rosen and MacDougald, 2006). The results of upregulation of PPARG likely stem from an increase in pre-adipocytes during the early stages of life. A larger number of pre-adipocytes leads to an increase in the proportion of cells maturing into adipocytes in the later stages of development. Additionally, increased intramuscular adipogenesis during the first days of life may have contributed to enhanced pre-adipocyte maturation during the finishing phase as reported in Qinchuan cattle (Wang et al., 2022), suggesting an indirect effect of vitamin A administered at birth, as observed in the current study.

All-trans retinoic acid is an active metabolite that controls cell proliferation and differentiation through the retinoic acid receptor (RAR) and PPARs (Yang et al., 2018). This compound is synthesized in two steps, in which retinol is converted into retinaldehyde, a reaction catalyzed by retinol dehydrogenase (Napoli, 2012). In our study, the RDH14 gene, which belongs to the dehydrogenase/reductase gene family, was found to be downregulated during the finishing phase in animals supplemented with vitamin A at birth. This downregulation seems to be associated with early developmental processes. The exact functions of RDH14 in muscle or adipose tissues are still unknown; however, this gene may play similar roles as RDH10, known for its involvement in postnatal vitamin A metabolism (O’Connor et al., 2022).

Ziouzenkova and Plutzky (2008) indicated a specific regulatory role of retinaldehyde in the transcription of target genes like PPARG, which is consistently reduced in the presence of this enzyme. High neonatal doses of vitamin A (300,000 IU) may result in prolonged hepatic storage, reducing systemic bioavailability (Harris et al., 2018) and leading to compensatory downregulation of enzymes involved in retinoid metabolism, such as RDH14. Another hypothesis is that these early high doses of vitamin A might induce lasting epigenetic effects on retinoid signaling in muscle and adipose tissues, characterizing the phenomenon of neonatal metabolic programming. Besides, COL1A1, COL22A1, COL8A2, and COL13A1 genes were downregulated in cattle treated with vitamin A, suggesting a potential remodeling of the extracellular matrix (ECM) that may favor adipogenic differentiation over fibrogenic activity. These genes encode collagen isoforms that contribute to the structural integrity and stiffness of muscle tissue, and their suppression may indicate reduced fibrotic signaling within the intramuscular environment. Retinoic acid, the active metabolite of vitamin A, is known to exert anti-fibrotic effects by modulating TGF-β signaling and other pathways that regulate ECM deposition. Furthermore, early-life vitamin A exposure may influence the commitment of fibro/adipogenic progenitor cells, promoting their differentiation into adipocytes rather than fibroblasts. This shift has been demonstrated in previous studies as a mechanism supporting increased IMF accumulation. Thus, the downregulation of collagen genes in the present study may reflect a vitamin A-induced muscle microenvironment more permissive to adipogenesis and marbling development, consistent with the programming effects of early-life nutritional intervention as evidenced in multiple studies (Hirano et al., 2004; Hopfer et al., 2005; Adiguzel et al., 2006).

Adipose tissue, recognized as an active endocrine organ, regulates energy metabolism, inflammatory processes, and insulin resistance (Jaganathan et al., 2018). This tissue responds to metabolic challenges through the secretion of adipocytokines such as leptin and adiponectin (Tilg and Moschen, 2006; Jaganathan et al., 2018). In our study, vitamin A injection at birth led to the upregulation of the LEP and ADIPOQ genes in steers at the end of the finishing period, suggesting enrichment of the adipocytokine signaling pathway. This pathway, which in birds is interconnected with the PPAR pathway via the ADIPQ gene in lipid metabolism (Chen et al., 2019), plays a crucial role in controlling lipid and glucose metabolism, potentially improving the insulin sensitivity of adipocytes (Jang et al., 2012).

Studies with 3T3-L1 cells have demonstrated that the ATF3 gene can repress adiponectin expression, potentially contributing to insulin resistance (Kim et al., 2006; Park et al., 2010). The ATF3 gene, a member of the AP1 transcription factor family, is known to respond to oxidative stress. A study using ARPE-19 cells showed that pretreatment with a combination of vitamins, including subtherapeutic doses of vitamin A, decreases ATF3 expression, a marker of oxidative stress (Yin et al., 2015). This stress can also suppress the activity of SREBF1, a transcription factor that is essential for fatty acid biosynthesis (Okuno et al., 2018). Although Maciel et al. (2022) reported early upregulation of SREBF1 in calves treated with vitamin A during the cow-calf and weaning phases, our study did not identify differential expression of this gene. Nonetheless, we observed upregulation of the ACACA gene in muscles of animals treated with vitamin A, highlighting its importance as a target of SREBF1 in de novo fatty acid synthesis (Ladeira et al., 2016, 2018). These findings suggest that vitamin A may have modulated ACACA expression independently of SREBF1, possibly through nuclear receptor-mediated signaling pathways such as RXR:PPARγ or RXR:LXR. These heterodimers have been shown to regulate the expression of genes involved in fatty acid synthesis, including ACACA (Kilu et al., 2021), which encodes a key enzyme in the de novo synthesis of fatty acids (Whitehurst et al., 1981) .

Angiogenesis, a process that plays an essential role in adipose tissue development, facilitates the supply of nutrients and oxygen required for adipogenesis and the transport of crucial growth factors for tissue growth and remodeling (Hausman and Richardson, 2004; Cao, 2013; Wang et al., 2017a). Retinoic acid, a promoter of angiogenesis, stimulates the formation of new adipogenic precursor cells through VEGF signaling (Wang et al., 2017a, 2017b). In the first months of life, upregulation of the VEGFA gene was observed in both groups, which is similar to results reported by Yu et al. (2022) and Maciel et al. (2022). However, although VEGFA expression was not detected during the finishing phase, the present study revealed the upregulation of other genes indicative of ongoing angiogenic activity, including LEP, ACACA, FASN, SCD, ELOVL6, and CIDEC. Additionally, these genes, along with TNC, CREB5, MYC, EGFR, IL6R, CSF1R, and PPP2R1B, are implicated in the PI3K-Akt signaling pathway, which is essential for adipocyte differentiation and adipogenesis.

Leptin serves as a growth signal, connecting adipocytes to blood vessels and facilitating neovascularization (Bouloumié et al., 1998; Park et al., 2001). This role of leptin illustrates the complexity of angiogenesis, a vital process for tissue development and maintenance. This fact highlights the importance of treatments such as vitamin A supplementation that can positively influence this process and other essential biochemical mechanisms. Similarly, the PI3K-Akt pathway, which is crucial for regulating vascular endothelial cell elongation and stability during angiogenesis (Lee et al., 2012; Tsuji-Tamura and Ogawa, 2016), is strongly affected by vitamin A, as demonstrated in the present study. This activation of the PI3K-Akt signaling pathway was accompanied by cell adhesion processes and extracellular matrix organization, significantly contributes to the regulation of angiogenesis in skeletal muscle tissue (Chen et al., 2020).

However, it is important to emphasize that the animals in the present study exhibited minimal marbling. As a result, the contribution of intramuscular adipocytes to the total RNA pool was likely limited. Key lipogenic genes such as ELOVL6, DGAT2, SCD, and FABP4 are expressed not only in adipocytes but also in skeletal muscle fibers. Therefore, the upregulation of these genes observed in our study may reflect transcriptional activity within muscle cells themselves.

Conclusion

This study demonstrates that neonatal vitamin A supplementation in beef steers induces long-lasting transcriptional changes in genes involved in adipogenesis, lipogenesis, and vascular development, ultimately contributing to greater IMF deposition at the finishing phase. The early upregulation of key regulatory genes such as KRAS, ITGB6, CALML6, and CISH suggests a priming effect on mesenchymal precursors, enhancing their adipogenic potential. In the finishing phase, the upregulation of LEP, ADIPOQ, ACACA, FASN, SCD, and ELOVL6, alongside the activation of the PI3K-Akt and PPAR signaling pathways, supports the hypothesis of metabolic programming initiated by neonatal vitamin A exposure.

The increase in IMF content observed in response to vitamin A is further substantiated by the upregulation of canonical adipogenic markers, including PPARG, C/EBPA, and FABP4. Collectively, these findings highlight neonatal vitamin A administration as a viable nutritional strategy to promote marbling and improve beef quality through the early modulation of molecular pathways critical to fat deposition.

Glossary

Abbreviations

ADG

Average daily gain

ADIPOQ

Adiponectin

BFT

Backfat thickness

COL4A1

Collagen type IV alpha 1 chain

COL8A1

Collagen type VIII alpha 1 chain

CREB5

cAMP responsive element binding protein 5

CSF1

Colony stimulating factor 1

CSF1R

Colony stimulating factor 1 receptor

DMI

Dry matter intake

DEGs

Differentially expressed genes

EGFR

Epidermal growth factor receptor

FDR

False discovery rate

FBW

Final body weight

GLUT4

Glucose transporter type 4

HCW

Hot carcass weight

IMF

Intramuscular fat

IL6R

Interleukin 6 receptor

KEEG

Kyoto Encyclopedia of Genes and Genomes

LEP

Leptin

LT

Longissimus thoracis

logFC

Logarithmic fold-change function

logCPM

Logarithmic counts per million function

MYC

MYC proto-oncogene

PGF

Placental growth factor

PI3K-Akt

Phosphoinositide 3-kinase/Akt signaling pathway

PPP2R1B

Protein phosphatase 2 regulatory subunit B

PPAR

Peroxisome proliferator–activated receptor

RAR

Retinoic acid receptor

RDH14

Retinol dehydrogenase 14

REA

Ribeye area

RNA

Ribonucleic acid

SREBP

Sterol regulatory element-binding protein

THBS1

Thrombospondin 1

TNC

Tenascin C

Author Contributions

Welder Baldassini (Conceptualization, Investigation, Resources, Writing - original draft), Germán Ramírez-Zamudio (Data curation, Investigation, Writing - review & editing), Marcio Duarte (Supervision, Validation, Writing - review & editing), Marcio Ladeira (Investigation, Visualization, Writing - review & editing), Guilherme Pereira (Software, Supervision, Writing - review & editing), Otavio Machado Neto (Data curation, Visualization, Writing - review & editing), Rodrigo Torres (Data curation, Software, Writing - review & editing), Murilo Soler de Magistri (Data curation, Investigation, Writing - review & editing), Lucas Camargo (Investigation, Visualization, Writing - review & editing), Rogério Curi (Supervision, Visualization, Writing - review & editing), and Luis Artur Chardulo (Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review & editing)

Funding

FAPESP (Grants 2019/14572-2, 2020/11300-9; 2021/07222-5 and 2023/05002-3) and São Paulo State University (UNESP; PROPe 9/2023).

Conflict of interest statement. The authors declare that there is no conflict of interest regarding this work.

Data Availability

Data will be made available on reasonable request.