Ruminant nutrition is influenced by various factors, such as protein quality, fiber content, feed type, rumen microorganisms, digestion, animal welfare, species, life stage, climate, and nutrient source (NRC, 2001). Among these, the quality of protein is more crucial than the quantity of protein for the growth, production, and reproduction of ruminants. The inefficient breakdown of high-protein diets in the rumen can lead to excess nitrogen release into the environment, contributing to the production of gases such as nitrous oxide, nitric oxide, nitrate, and ammonia, which pose environmental concerns (Abbasi et al., 2018). In cattle finishing diets, approximately 10% to 20% of the ingested nitrogen is stored in the animal’s tissues, while a significant portion of 30% to 50% is eliminated through feces. The majority of the remaining 40% to 70% of ingested nitrogen is excreted through urine (Cole and Todd, 2009). The Sustainable Development Goals (SDGs), set by the United Nations in 2015, emphasize the need to address poverty, hunger, and environmental preservation. Livestock farming is pivotal for addressing poverty and hunger (SDG 1 and SDG 2), but it must also align with goals focused on environmental preservation and ecosystem protection (SDG 13 and SDG 15) to ensure sustainable growth in the sector (Wanapat et al., 2015). Reducing crude protein (CP) in ruminants’ rations while maintaining animal performance could be a strategy to reduce nitrogen excretion and feed costs. In fact, dietary protein is the most important on-farm variable that can be controlled relatively easily with a significant effect on ammonia emissions (Agle et al., 2010). In dairy cows, Broderick (2003) observed that as dietary CP increased, there were linear increases in milk urea and urinary nitrogen (N) excretion, accompanied by linear decreases in nitrogen efficiency. Specifically, increasing CP from 15.1% to 18.4% resulted in a reduction in milk nitrogen from 31% to 25% of dietary nitrogen, an increase in urinary nitrogen from 23% to 35% of dietary nitrogen, and a decrease in fecal nitrogen from 45% to 41% of dietary nitrogen. In beef cattle, Todd et al. (2006) estimated that there would be an annual reduction in daily ammonia flux of 28% if CP was reduced from 13% to 11.5% in cattle diets.

The efficiency of metabolizable protein (MP) utilization in ruminants depends on the amino acid (AA) profile, as an inadequacy in any specific AA can restrict the use of others, even if they are present in sufficient quantities (Cole and Van Lunen, 1994; Klemesrud et al., 2000a). When certain AAs are lacking or imbalanced in the diet, the ruminant’s capacity to utilize MP is reduced, leading to decreased growth performance and overall productivity (Harper et al., 1964). Although the limitation of methionine (Met) and lysine (Lys) as the primary AAs in dairy cattle is widely acknowledged and documented (Sinclair et al., 2014; Abbasi et al., 2018), limited information is available for beef cattle. In diets with large proportions of corn, Lys is the first limiting AA for growing beef cattle because corn is low in Lys (Klemesrud et al., 2000a) but not in cattle fed diets containing soybean meal (Hussein and Berger, 1995). Therefore, ensuring that ruminant diets contain an optimal balance of AAs is crucial to promote efficient protein synthesis and maximize growth and productivity. This can be achieved through meticulous dietary management and the incorporation of rumen-protected AAs (RP-AAs) to supplement specific AAs as needed. RP-AAs have the advantage of bypassing the rumen and being absorbed directly in the small intestine, preventing degradation by rumen microbes and increasing their availability for absorption and utilization by the animal. However, there have been relatively few studies evaluating the effects of RP-AA supplementation in growing beef cattle, particularly in feeding systems involving cereal concentrates and ad libitum straw, with different results. Recently, Montaño et al. (2019) reported that supplementation with rumen-protected Met and Lys enhanced average daily gain (ADG) and gain efficiency during the early growing phase in Holstein calves. However, Barido et al. (2020) found that supplementation with Met and Lys had no effect on growth performance and carcass weight and yield in Hanwoo steers. Therefore, more research in this area is needed to better understand the benefits and applications of RP-AAs in beef cattle nutrition.

Building on previously published information, this study was conceived with the hypothesis that integrating protected AAs into a diet for beef cattle with reduced protein levels could potentially yield equivalent growth and meat quality to a diet with standard protein levels. The primary objective of this study was to evaluate the impact of incorporating RP-AAs into a diet with a 3% reduction in CP while increasing the levels of digestible Met and Lys. The study aimed to assess the effects on the growth and fattening performance of beef cattle, along with meat composition and quality.

The greater ADG in the 90 days of the trial for animals fed the APR diet could be explained by the better utilization of the N provided in the APR diet in the fattening phase, when less growth occurs, which leads to a lower energetic cost of eliminating the N that is not used by the animal. As cattle increase in body weight and level of fatness, the rate of protein deposition decreases, leading to a reduction in their MP requirements (Teixeira et al., 2019). Logically, future research work is required to explain the better performance in animals fed less CP. Similar findings were reported by Prado et al. (2014), who observed no significant differences in animal performance between those with low- and high-protein diets (14.6% CP vs. 16.8% CP) and different Lys-to-Met ratios (3.0 vs. 3.4). However, the growth response to supplementing diets with protected AAs exhibited variability in previous studies (Klemesrud et al., 2000a; Klemesrud et al., 2000b; Schroeder and Titgemeyer, 2008).

Mazinani et al. (2020) suggested that adding protected AAs may have some benefits for these parameters by enhancing rumen undegradable protein. Furthermore, Montaño et al. (2019) found improved daily gains and feed efficiency in Holstein steers fed a rumen-protected Met and Lys diet. In their study, the consumption of these protected AAs increased the availability of Met and Lys in the intestine, post-ruminal segments, and the entire gastrointestinal tract.

According to Weekes et al. (2006), either a deficiency or imbalance in the AA profiles within MP can inhibit the growth performance and feed conversion efficiency of livestock. Heiderscheit and Hansen (2020) conducted an evaluation of supplemental RP-Lys in Angus crossbred steers. Their hypothesis was that steers fed diets formulated to meet Lys requirements would exhibit better feed efficiency and greater gains than those fed a Lys-deficient diet. However, they found that the ADG throughout the entire trial was higher in the control diet, which was deficient in Lys, than the diet formulated to meet Lys requirements using ingredients other than RP-Lys. They suggest that growth performance responses in RP-Lys-fed steers might have been negatively affected by overconsumption of rumen-bypass Lys during the finishing phase. Nevertheless, these authors concluded that diets formulated to meet Lys requirements resulted in positive metabolizable Lys balances during finishing. The increased metabolizable Lys balance may have impacted performance if other AAs were imbalanced. Ma et al. (2021) observed differences in the ADG and feed-to-gain ratio in yaks in a feedlot with four supplementary levels of RP-Lys and Met in the diets. They concluded that growth performance and meat quality could be improved by supplementing RP-AAs in the diets.

Teixeira et al. (2019) also observed that steers fed a supplemental RP-Lys diet did not experience changes in dressing percentage, but did produce carcasses with reduced fat thickness and increased longissimus muscle area. These findings led them to conclude that the RP-Lys diet improved nitrogen utilization, indicating the enhanced utilization of AAs for anabolic purposes and increased protein accretion and muscle synthesis, as we observed in the APR group, which increased protein content in the meat. Heiderscheit and Hansen (2020) reported no differences in the carcass characteristics of Angus crossbred steers fed supplemental RP-Lys diets. Similarly, Ma et al. (2021), in their study with different levels of protected Lys and Met, found no differences in carcass characteristics. In diets with varying levels of CP and Lys-to-Met ratios, Prado et al. (2014) did not observe significant differences in carcass conformation and fatness score. In addition, Barido et al. (2020) found that meat from cattle supplemented with Met and Lys had higher protein content than meat from non-supplemented cattle. Similarly, Teixeira et al. (2019) conducted a study on Angus × Simmental steers that were fed diets with RP-AAs, and they suggested that Lys supplementation increased protein accretion and muscle synthesis. These observations made them deduce that the RP-Lys regimen boosted nitrogen assimilation, pointing to an improved uptake of AAs for growth activities and heightened protein deposition and muscle development.

The fatty acid composition of cattle meat can be influenced by various factors, including breed, weight, sex, age, and diet (De la Fuente et al., 2009). However, in the context of diet, the fatty acid profile of ruminant meat is relatively unaffected due to ruminal modifications, but it can still reflect differences in the composition of dietary lipids (Díaz et al., 2005). In addition, lipid metabolism can be influenced by the balance of AAs in the diet (Martínez et al., 2017). Research has demonstrated that restricting essential AAs in the diet, such as Met, can result in substantial alterations in lipid metabolism within adipose and hepatic tissues. These changes trigger significant transcriptional and metabolic responses, including a reduction in transcriptional initiation and the expression of the enzyme fatty acid synthase (Anthony et al., 2013), which is responsible for the de novo biosynthesis of fatty acids. Published literature on the effects of RP-AAs on the fatty acid composition of ruminant meat is scarce and presents highly variable results. For instance, Barido et al. (2020) observed only minor effects of dietary Met and Lys supplementation on intramuscular fatty acid composition in Hanwoo cattle, with C14:0 being the only fatty acid affected. The present study found that the effect of diet on the intramuscular fatty acid profile was limited, and, therefore, cattle fed the APR diet, supplemented with Met and Lys, produced meat with slightly lower levels of C18:1n9 and greater C16:0 than the CON meat. For example, Třináctý et al. (2006) observed a reduced proportion of C18:1 in milk fat from cows that were supplemented with RP-AAs (Lys, Met, and histidine) compared with cows that were not supplemented. This finding suggests that Met may influence the de novo synthesis of fatty acids, potentially increasing the production of short- and medium-chain fatty acids within the mammary gland (Pisulewski et al., 1996). In this context, C16:0 emerges as the predominant end product of de novo synthesis by fatty acid synthase (Smith et al., 2003). In our study, the lower proportion of C18:1n9 and greater C16:0 in cattle fed the APR diet could be attributed to Met supplementation, but it might also be influenced by the different composition of the diets provided. Further research needs to be conducted to better understand the mechanism of the AAs’ effect on meat fatty acid composition. Other researchers have ascribed a partial effect of AA lipid coating on the fatty acid composition of milk (Sevi et al., 1998). In our case, the surface coating used for Met protection was palmitic acid, which could partly explain the tendency toward a higher proportion of C16:0 in the APR meat. In the case of growing lambs, Dong et al. (2020) demonstrated that betaine, a choline metabolite closely related to Met, supplementation increased the PUFA content of the gluteus muscles, similar to the results of our study, with near to 1% more PUFAs in APR than in CON meat. In this study, the inclusion of RP-AAs and the reduction of CP levels did not have a significant effect on the AA content of the longissimus muscle. Similarly, Ma et al. (2021) found no significant differences in the AA profile of the longissimus muscle in feedlot yaks with various levels of supplementary RP-AAs in the diet. Similarly, Xue et al. (2010) found no differences in the meat AA profile of Limousin-crossbred calves fed RP-Lys. Teixeira et al. (2019) noticed a tendency for Lys levels in serum to decrease when arginine was added to the diet, suggesting that excessive arginine intake might hinder Lys absorption from the small intestine. However, they reported no notable variations in plasma Lys levels among calves consuming diets containing RP-Lys.

The ultimate pH values were in accordance with those obtained by Thénard et al. (2006) in Montbéliard cattle, and no differences were observed in the meat from cattle fed the CON diet and the APR diet. The pH values were below 5.6, indicating that the animals were not stressed at slaughter (Prado et al., 2014). Similar results were obtained by Barido et al. (2020), who reported no effect of dietary supplementation with rumen-protected Met/Lys on pH at 0 h of longissimus lumborum muscle from Hanwoo steers. In yak meat, Ma et al. (2021) obtained ultimate pH values of 5.44, with no differences due to the supplementary level of RP-AAs. Likewise, the WHC was not affected by diet, with a similar percentage of expelled juice in meat from cattle fed the CON diet and the APR diet supplemented with Met and Lys, in accordance with similar ultimate pH values found in the CON and APR meat. The WHC indicates variations in the charges and structure of muscle proteins, and is closely related to pH (Bouton et al., 1971).

In ruminants, the papers published about the impact of RP-AAs on meat quality are scarce and present variable results. Some authors have observed an effect of Met supplementation on ruminant meat color (Dong et al., 2020; Ma et al., 2021). Ma et al. (2021) noted an impact of dietary supplementation with RP-AAs (Met and Lys) in feedlot yaks, resulting in changes in meat color. They reported increased a* values and decreased L* and b* values in the supplemented groups compared with the control group. In the case of growing lambs, Dong et al. (2020) observed a linear decrease in the b* value of the longissimus muscle with increasing doses of rumen-protected betaine. However, our study did not show any significant effect on meat color, as we observed similar L*, a*, and b* values in both the CON and the APR meat samples. It has been reported that some AAs, including Met, have antioxidant activity (Levine et al., 2000; Lund et al., 2011), improving muscular antioxidant status and enhancing meats’ stability against oxidation. Myoglobin oxidation during storage affects the extent of meat discoloration depend on the length of the storage period, packaging conditions, and storage temperature (Olivera et al., 2013). In the present study, meat samples were stored through vacuum packaging and at congelation temperature. These conditions minimize oxidation (Vieira et al., 2009). This could partially explain the lack of effect of diet on meat color.

Despite the differences in protein content between the CON and APR meat, no significant effect of diet was recorded for texture parameters. According to Koohmaraie et al. (2002), changes in muscle protein synthesis do not affect meat tenderness; however, changes in muscle protein degradation affect meat tenderization/tenderness. Similarly, Prado et al. (2014) did not report differences in texture parameters (shear force) due to diet protein level or Lys-to-Met ratio. However, they reported lower values of shear force (4.63 kg) than in our study. The differences between studies could be due to the different breeds used, which imply differences in the quantity and solubility of collagen, fatness, and calpain and calpastatin activity (Monsón et al., 2005), which affect tenderness, but are also due to differences in aging time (Vieira et al., 2009) between studies.

The findings of this study demonstrate that the inclusion of RP-AAs in cattle fattening diets offers several benefits. It allows for a reduction in CP content without compromising productive yields or the quality of the meat produced. Moreover, it may enhance the efficiency of the utilization of nitrogen from the diet, thereby potentially reducing environmental pollution. In summary, incorporating RP-AAs in cattle diets presents a promising approach for beef producers in southern Europe. It may not only mitigate the challenges associated with volatile raw material prices and deforestation but also offer environmental benefits while maintaining cattle productivity and the quality of the meat produced.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics approval was not necessary, as the study was carried out on production animals, and no invasive procedures were performed on them.

AC: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. JD: Data curation, Formal analysis, Investigation, Writing – review & editing, Validation, Writing – original draft. MD: Data curation, Formal analysis, Methodology, Software, Writing – original draft, Writing – review & editing. RB: Data curation, Formal analysis, Software, Visualization, Writing – original draft, Writing – review & editing. Dd: Funding acquisition, Investigation, Writing – original draft, Writing – review & editing. JM: Funding acquisition, Investigation, Writing – original draft, Writing – review & editing. NL: Data curation, Resources, Writing – original draft, Writing – review & editing. VJ: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing.