INTRODUCTION
Marbling, the visible pattern of fat interspersed between muscle fibers in beef, is a significant indicator of premium beef quality. It presents as white specks or streaks created by adipose tissue between muscle fibers [1]. Intramuscular fat (IMF), the material foundation for marbling, is positively correlated with beef color, flavor, and juiciness [2]. Higher IMF content corresponds with elevated marbling scores [3]. Corbin et al [4] found that consumer preference for beef increased as IMF content rose across 10 quality categories and marbling levels.
Vitamin A is a vital, fat-soluble vitamin. Roels [5] initially suggested a connection between vitamin A and fat metabolism, and subsequent studies indicated that vitamin A could influence marbling quantity. Restricted intake of vitamin A has been shown to notably affect IMF content [6]. Kruk et al [7] found that adding low-dose vitamin A supplements to feed can increase the number and size of marble spots, thus promoting the formation of marble patterns and affecting the IMF. Oka et al [8] noticed that the content of vitamin A in feed was negatively correlated with beef marbling. In addition, Gorocica et al [9] found that limiting the vitamin A dosage in the diet did not significantly affect the carcass weight, meat quality or back fat thickness of Angus hybrid offspring, but it did improve the marbling score of beef. In summary, adding a low dose of vitamin A to fodder can increase IMF deposition and marbling scores. However, the optimal supplemental dosage for specific cattle breeds remains undetermined, and the antioxidant function of vitamin A has not been adequately investigated. Although Palacios found that vitamin A has some antioxidant capacity [10], and subsequent research by Daniel et al [11] implyed that vitamin A restriction might be a management strategy to improve beef quality and extend shelf life. Nevertheless, the impact of restricting vitamin A intake in finishing diets on lipid oxidation and color stability of stored meat remains inconclusive. Compared to other local breeds, Yanbian yellow cattle, one of China’s five outstanding local breeds, has exhibited superior meat quality traits, including marbling patterns [12]. However, the meat quality is characterized by instability, which significantly limits the production of high-quality beef from Yanbian yellow cattle. Currently, the optimal supplementation level of vitamin A during the finishing period for Yanbian yellow cattle has not been established. Moreover, there is a lack of research on the role of vitamin A in delaying beef oxidation and preserving meat quality during storage via antioxidant effects, which severely restricts the industrial development of high-grade beef.
Therefore, the objective of this study was to investigate the effects of vitamin A restriction feeding on the production performance, fat deposition, marbling, meat quality antioxidant capacity, and shelf life in Yanbian yellow cattle, and to determine the appropriate level of its addition and time of restriction feeding in the production, as well as the optimal level of vitamin A added to reduce the oxidation of beef. To provide a reference for the application of vitamin A in beef cattle production and sales, it is of great significance to meet consumers’ demand for high-grade and high-quality beef.
MATERIALS AND METHODS
Animals and treatments
Twenty healthy and disease-free 15-month-old Yanbian Yellow castrated cattle with similar weights (314.13±13.30 kg) were selected from Muluo Livestock, Longjing City, Yanbian Korean Autonomous Prefecture, Jilin Province. The cattle were randomly divided into five groups: one control (CON group) and four experimental groups, each consisting of four animals. The control group (CON) was supplemented with 2,200 IU/kg DM of VA according to the Nutrient Requirements of Beef Cattle (Nutritional Requirements of Beef Cattle 8th Revision). Treatment group 1 (NVA1) and treatment group 2 (NVA2) were not supplemented VA for 180 days and 240 days, respectively, and then recovered to the same level of VA as the CON group. Treatment group 3 (LVA1) and treatment group 4 (LVA2) were supplemented with 1,100 IU/kg DM of VA for 180 days and 240 days, respectively, and then recovered to the same of VA level as the CON group.
A pretrial period of 15 d was followed by a formal experimental period of 15 months. The cattle were raised to the age of 30 months, and standard commercial slaughter practices were applied. The composition and nutritional levels of the diets are presented in Supplement 1.
Feeding, management and growth performance
The experimental cattle were provided with free access to water and ad libitum consumption of roughage. A commercial vitamin A premix, purchased from DSM Company, stored in a cool, dry, and dark place.
Feeding was administered twice daily, at 7:00 AM and 3:00 PM. Before each feeding, the premix was mixed with concentrate feed. Fasting body weights (BWs) were recorded at the start and end of the period to calculate average daily gain (ADG).
Slaughter and carcass determination and sampling
At the end of restriction, jugular blood was collected into sterile tubes, centrifuged (4°C), and serum aliquots were light-protected and stored in liquid nitrogen. After an 18 h fast, cattle were electrically stunned and slaughtered by exsanguination under standard commercial procedures; carcasses were washed, halved, while hide, head, distal limbs, tail, reproductive organs, and surrounding fat were removed. Lean and bone were weighed to calculate dressing yield, lean percentage, and meat:bone ratio. The longissimus dorsi muscle (LD) (12th–13th rib) was sampled. The longissimus muscle area (LMA) was traced on tracing paper and quantified with a transparent grid, and backfat thickness was measured perpendicularly by caliper at the three-quarter point of the LD cross-section near the spine. Marbling score was graded on China’s 5-point scale [13]. LD samples were aseptically collected, and snap-frozen in liquid nitrogen.
Analysis of meat quality traits
Meat quality traits were evaluated as described by Yu et al [14], with slight adjustments. LDs were aged 48 h at 4°C, then evaluated for pH (pH-STAR; MATTHAUS) and color (L*, a*, b*; OPTO-LAB; MATTHAUS) with instruments calibrated before use. Drip loss was determined on standardized strips (5×3×2 cm) suspended in sealed bags at 4°C for 24 h; cooking loss was calculated after heating to a 75°C–80°C core temperature, cooling, blotting, and reweighing. Shear force was measured with a C-LM3B meat tenderness meter by cutting parallel to fiber orientation. Moisture, crude protein, fat, and ash were analyzed as described by Luan et al [15]. For the 4°C storage trial, pH, color, drip loss, cooking loss, and shear force were recorded on days 1, 3, 5, and 7 using the same procedures. All measurements were performed in triplicate.
Measurement of muscle fiber diameter
Muscle fiber diameter was determined using a histological method as described by Zhang et al [16], with slight modifications. Briefly, muscle tissues were fixed in a 10% formaldehyde solution for more than 24 h, rinsed in water for 24 h, and then dehydrated in a graded series of alcohol solutions. Next, xylene and anhydrous ethanol were mixed to decolorize (1:1), and then pure xylene was used for secondary decolorization. The treated tissues were embedded in paraffin, sliced, attached to slides, deparaffinized, and subjected to HE staining and neutral gum sealing. Microscopic images were captured for muscle morphology observation, and ImageJ software was used to measure the muscle fiber diameter in beef.
Biochemical indicators analysis
Serum biochemistry was analyzed by a contract laboratory (Beijing Huaying Biotechnology) using an A6 semi-automatic biochemical analyzer (Beijing Shansheng Technology) and commercial kits (Beijing Huaying Biotechnology). Assays followed manufacturers’ instructions: total protein (TP; HY-50067; biuret, 546 nm), albumin (ALB; HY-50068; bromocresol green, 630 nm), total cholesterol (TC; HY-50061; COD-PAP, 500 nm), triglycerides (TG; HY-50062; GPO-PAP, 500 nm), high-density lipoprotein (HDL; HY-50070; selective detergent method), low-density lipoprotein (LDL; HY-50071; selective detergent method), glucose (GLU; HY-50063; glucose oxidase–peroxidase, 500 nm), urea nitrogen (UREA; HY-N0015; urease/GLDH kinetic, 340 nm), aspartate aminotransferase (AST; HY-50053; rate method with MDH/NADH coupling, 340 nm), and alanine aminotransferase (ALT; HY-50052; rate method with LDH/NADH coupling, 340 nm).
Antioxidant index analysis
In a service company (Beijing Huaying Biotechnology), commercial kits (Beijing Huaying Biotechnology) and A6 semi-automatic biochemical analyzer (Beijing Shansheng Technology) were used to determine the antioxidant indexes in serum and muscle.
Commercial kits for malondialdehyde (MDA; HY-M0003), glutathione peroxidase (GSH-Px; HY-M0004), catalase (CAT; HY-M0018), superoxide dismutase (SOD; HY-M0001), total antioxidant capacity (T-AOC; HY-60021) (Beijing Huaying Biotechnology) were used with an A6 semi-automatic biochemical analyzer (Beijing Shansheng Technology) according to the manufacturers’ instructions.
RNA extraction and quantitative polymerase chain reaction analysis
Total RNA was extracted using the Eastep Super Total RNA Extraction Kit provided by Shanghai Promega Company, and the RNA concentration was assessed using an ultraviolet spectrophotometer. First-strand cDNA synthesis was performed using the FastKing cDNA First-Strand Synthesis Kit (TIANGEN). All procedures were performed in accordance with the protocols provided by the respective kits. These procedures were performed as described by Chen et al [17].
Specific primers were designed using Primer Premier 5.0 software based on GenBank-published cattle gene sequences (Supplement 2; Tianjin GENEWIZ Biology Science and Technology, synthesized all primers).The polymerase chain reaction (PCR) conditions were as follows: an initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 75°C for 25 s. A final extension was performed at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 95°C for 15 s.
The relative gene expression levels were calculated using the 2−ΔΔCT method, where ΔCt target gene = Ct target gene – Ct reference gene, and ΔΔCt = ΔCt experimental group – ΔCt control group. Each sample was run in triplicate.
Statistical analysis
Graphs were generated using Excel. A one-way analysis of variance was conducted using SPSS 26.0. In the case of significant differences, Duncan’s multiple comparison test was used. The results were presented as mean±standard error. A p-value of less than 0.05 was considered statistically significant.
RESULTS
Growth performance and carcass traits
Dietary vitamin A restriction did not significantly affect the growth performance (initial BW, final BW, ADG) (Supplement 3; p>0.05). Similarly, carcass traits, including carcass weight, dressing percentage, net meat percentage, bone weight, meat-bone ratio, weight of high-grade beef parts, and the percentage of high-grade cuts in live weight, were not significantly different from those of the CON group (Supplement 4; p>0.05).
Fat deposition and marbling score of beef
Fat deposition and marbling score in Yanbian yellow cattle were shown in Table 1 and Figure 1. Marbling scores in the LVA1 group were significantly higher than those in the CON, NVA1, and NVA2 groups (p<0.05). The marbling score of the LVA2 group was significantly higher than that of the CON group (p<0.05). Compared with the CON group, the marbling scores of the NVA1, NVA2, LVA1, and LVA2 groups increased by 16.67%, 27.67%, 55.67%, and 39.00%, respectively. The IMF contents of the NVA1, NVA2, LVA1, and LVA2 groups were significantly higher than those of the CON group (p<0.05), with increases of 54.21%, 56.99%, 53.93%, and 50.00%, respectively. No significant differences were observed in LMA and backfat thickness among the groups (p>0.05).
Muscle fiber diameter
As shown in Table 2 and Supplement 5, muscle fiber diameters were significantly smaller in all treatment groups (NVA1, NVA2, LVA1, LVA2) compared to the CON group (p<0.05), indicating that vitamin A restriction effectively reduced muscle fiber size.
Physicochemical characteristics
The effects of vitamin A on the physicochemical characteristics of Yanbian Yellow beef were shown in Table 3. The drip loss rates of the NVA2, LVA1, and LVA2 groups were significantly lower than that of the CON group (p<0.05). Additionally, drip loss rate in the LVA1 group was significantly lower than that in the NVA1 group (p<0.05). The shear forces of the NVA2, LVA1, and LVA2 groups were significantly lower than that of the CON group (p<0.05), and both the LVA1 and LVA2 groups exhibit significantly lower shear forces than that of the NVA1 group (p<0.05). However, no significant differences were observed among the groups in terms of pH, meat color, cooking loss rates, moisture, protein, or ash contents (p>0.05).
Serum biochemical and antioxidant indexes
In Table 4, restricting vitamin A levels in the daily diet did not significantly affect the serum biochemical indexes of Yanbian yellow cattle (p>0.05). There was no significant difference (p>0.05) in SOD concentration between the groups of LVA1 and LVA2 compared with the CON group, but the concentration of SOD in NVA1 and NVA2 groups was significantly lower than that of the CON group (p<0.05), and that in NVA2 were significantly lower than that of LVA1 group (p<0.05). In addition, the concentration of GSH-PX in NVA1 and NVA2 was significantly lower than that in CON, LVA1 and LVA2 groups (p<0.05).
Muscle antioxidant indexes
The effect of vitamin A on muscle antioxidant indexes of Yanbian yellow cattle was shown in Supplement 6. Compared with the CON group, T-AOC, CAT, SOD, GSH-PX and MDA content of the four experimental groups were not significantly different (p>0.05).
Antioxidant gene expression profile in longissimus dorsi muscle
As shown in Figure 2, relative mRNA expression of FOXO1, GSTA1, and SOD in the LD were upregulated in all experimental groups compared with the CON group. For the FOXO1 gene, the relative mRNA expression level in the LVA1 group was significantly higher than that in the CON, NVA1, NVA2, and LVA2 groups (p<0.05). Moreover, the NVA1 group exhibited a significantly higher FOXO1 expression compared to the CON group (p<0.05). For the GSTA1 gene, the mRNA expression in the LVA1 group was significantly higher than that in the NVA1, NVA2, LVA2, and CON groups (p<0.05). The NVA1, NVA2, and LVA2 groups all demonstrated significantly higher GSTA1 expression than the CON group. As for the SOD gene, all experimental groups had significantly higher expression levels compared with the CON group. These findings further confirm that vitamin A restriction enhances the intrinsic antioxidant defense system in the muscle of Yanbian yellow cattle, which is beneficial for maintaining meat quality stability during storage.
Changes in meat quality characteristics during storage
As shown in Supplement 7, the pH value decreased initially and then increased over storage. All groups exhibited similar trends, with no significant differences detected among them (p>0.05).
The evolution of meat color (L*, a*, b*) was shown in Figures 3A–3C. The L* and a* values were not significantly affected by storage time (p>0.05). In contrast, the b* value increased progressively, with the NVA1 and NVA2 groups exhibiting significantly higher values than the CON group by day 7 (p<0.05).
Vitamin A restriction effectively reduced drip loss in Figure 4. The NVA2, LVA1, and LVA2 groups were lower than CON group on days 1, 3, and 5 (p<0.05), and on day 7 these three groups were lower than both CON and NVA1 groups (p<0.05), with the LVA1 group showing the lowest value.
According to Figure 5, it was learned that restricted feeding of vitamin A was able to reduce the cooking loss rate of muscle. Compared with the CON group, the NVA2 group, LVA1 group and LVA2 group decreased significantly on the 7th day of storage (p<0.05).
As shown in Figure 6, shear force exhibited a general decline during storage. On days 1 and 3, the LVA1, LVA2, and NVA2 groups had significantly lower shear force than the CON group, with the LVA1 and LVA2 groups also being significantly lower than the NVA1 group (p<0.05). By day 5, all treatment groups showed significantly lower values than CON, and the LVA groups were significantly lower than the NVA groups (p<0.05). This trend continued on day 7, with the LVA groups being significantly lower than all other groups, and the NVA2 group lower than CON (p<0.05). The LVA1 group consistently demonstrated the lowest shear force throughout the storage period.
DISCUSSION
Regarding beef production, previous studies consistently indicate that restricting vitamin A intake does not significantly affect the growth performance [18–20] or carcass traits [21,22] of steers. Similarly, this study found no significant differences in growth performance, carcass traits, or serum biochemical parameters. This suggests that the VA restriction regimen in this trial had a limited impact on overall metabolic homeostasis, with its effects more likely to be manifested in meat quality-related traits. Therefore, the subsequent experimental analysis were conducted.
Marbling score is a critical indicator for assessing beef quality, and the restricted vitamin A strategy plays a pivotal role in the development of marbling and IMF in beef. Wan [23] observed that reducing dietary vitamin A content significantly increased IMF in Limousin-crossbred steers without affecting fat deposition in other regions. On one hand, higher IMF content is generally associated with improved tenderness. Previous studies have found that vitamin A restriction can reduce beef shear force [22], which aligns with the findings of the present study. On the other hand, tenderness is closely related to muscle fiber diameter: smaller fiber diameters typically correspond to lower shear force and show a negative correlation with IMF [24]. Furthermore, increased IMF is believed to help reduce moisture exudation to some extent, thereby decreasing drip loss and imparting meat juiciness [25]. It had to be emphasized that the LVA1 group had the lowest shear force and drip loss, but the best marbling score.
Vitamin A is involved in regulating redox homeostasis in the body and can help scavenge free radicals [26]. After slaughter, endogenous antioxidants are gradually depleted; once insufficient, ROS-driven damage accelerates and impairs meat quality [27]. Although there was no significant difference in muscle antioxidant level in the results of this experiment, the activities of SOD and GSH-Px in serum were significantly reduced in the group without VA. As SOD and GSH-Px are crucial for clearing reactive oxygen species and protecting against oxidative damage [28]. Our results suggest that low-dose vitamin A restriction has a limited effect on antioxidant capacity, while no vitamin A supplementation may weaken antioxidant defense.
Previous research has reported nonlinear responses of antioxidant parameters to vitamin A in poultry [29]. In our study, we observed a non-linear relationship between dietary vitamin A levels and the expression of antioxidant-related genes. Compared with the CON group, the expression of muscle SOD was significantly up-regulated in all vitamin A-restricted groups. Meanwhile, the expression levels of FOXO1 and GSTA1 peaked in the LVA1 group, while those in the NVA1/NVA2 and LVA2 groups, although relatively lower, still remained higher than the levels observed in the CON group. These results point to changes in the antioxidant network at the transcriptional level, although this does not necessarily mean an immediate improvement in overall antioxidant function. At moderate vitamin A levels, retinoic acid signaling appears to regulate pathways such as Nrf2-ARE and FOXO, which aligns with the increased expression of SOD and GSTA1 observed here [30]. The FOXO family, important transcription factors in oxidative stress response, helps maintain basic antioxidant defense [31]. SOD primarily mediates the superoxide anions, while GSTA1, as a phase II detoxification enzyme, clears lipid peroxides. Under the guidance of FOXO1 and Nrf2 signaling, they form a coordinated antioxidant network [32]. An in vitro study using bovine AD-MSCs suggests that exogenous antioxidant treatment can improve redox status while modulating differentiation-related signaling pathways, thereby promoting adipogenic differentiation [33]. Previous studies have also indicated that vitamin A can promote the transformation of bovine skeletal muscle toward an oxidative muscle fiber phenotype, suggesting its potential to enhance oxidative metabolic capacity [34]. Therefore, these transcriptional changes provide a molecular mechanistic explanation for the improved meat stability observed during storage. As the antioxidant system helps mitigate post-slaughter lipid oxidation damage [35], the significant upregulation of antioxidant genes in the LVA1 group enables it to maintain superior quality throughout storage.
Concurrently, the low-dose vitamin A groups (LVA1 and LVA2) demonstrated the most favorable meat quality attributes during storage, characterized by the highest water retention capacity and optimal tenderness. Notably, in this study, the difference became more pronounced in the later stage of storage. By day 7 of storage, the low-dose vitamin A group exhibited significantly lower drip loss and cooking loss rates, along with a significantly lower shear force, compared to the CON group, a result consistent with previous reports [36]. Given that water loss rate and cooking loss rate are closely related to meat tenderness and juiciness [37], this suggests that low-dose vitamin A restriction may enhance meat quality stability during the later stages of storage. The underlying mechanisms appear to be related to IMF content and alterations in muscle microstructure. Existing research suggests that meat with higher IMF content is generally associated with lower drip loss and improved sensory attributes [38]. During storage, myofibrillar restructuring and protein degradation can influence water distribution in muscle and, consequently, water-holding capacity [39]. In this study, vitamin A restriction significantly reduced cooking loss in Yanbian yellow cattle, with the LVA1 group showing the greatest improvement. This further supports the idea that low-level restriction provides a more stable quality advantage, especially in the later stages of storage.
CONCLUSION
In summary, vitamin A restriction during the finishing period may improve beef quality through two complementary pathways. First, it promote the formation of smaller-diameter muscle fibers, which can help enhance water-holding capacity and tenderness. Second, low-dose restriction induce the upregulation of antioxidant-related genes, potentially extending shelf life. These findings provide useful insight for optimizing vitamin A nutrition strategies during the finishing stage.
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