In ruminants, supplementing guanidinoacetic acid (GAA) and methionine (Met) may improve meat-producing performance. GAA is a natural precursor of creatine, which is essential in muscle mass development through its role in ATP regeneration [1,2]. In previous studies, dietary supplementation of GAA increased average daily gain (ADG) of both lambs (supplemented 0.08% to 0.12% GAA on dry matter [DM] basis [3]) and bulls (0.06% to 0.09% DM [4]; 0.06% DM [5]) compared to controls.

It is well established that Met is one of the most limiting amino acids for growing animals, depending on dietary composition. It is involved in various key physiological processes, e.g., protein production, and is also a methyl donor having a role in transsulfuration and DNA methylation [6]. The impact of rumen-protected methionine (RPM) on daily weight gain has not been clearly established, even though beneficial effects of protected Met on feed efficiency and meat quality were reported in lambs fed from 1.5 to 6.0 g metabolizable Met/d [7] and at the diet inclusion rate of 0.06% to 0.09%, respectively [8].

Beyond productivity, consumers are increasingly concerned about food quality [9]. Whether and how GAA and RPM can interact, and impact meat quality of ruminants is still under investigation. In pigs, dietary creatine or GAA enhanced growth performance and carcass quality, e.g., pH, color, water holding capacity (WHC), shear force, and drip loss [10,11]. However, Jayaraman et al [12] did not observe an effect of GAA supplementation on pork quality, which was inconsistent with previous results. Li et al [13] reported that adding 0.2% GAA in diets improved feed efficiency and beef growth and quality. In an assessment of the impacts of GAA on carcass characteristics of ruminants, Chao et al [3] reported more meat and intramuscular fat, and lower carcass fat content in sheep with GAA supplemented up to 0.12% DM. Regarding RPM, existing results are contradictory. Liu et al [8] reported improved meat quality (higher carcass fat weight and grid reference tissue depth, and lower drip loss) of lambs fed 0.06% metabolizable Met/d respectively. El-tahawy et al [14] reported that adding 3.3 g/kg Met in diet improved performance of Rahmani Lambs, whereas no effect was observed with steers given 8 g metabolizable Met daily [15].

Before being exported to the muscles, creatine is synthesized in the liver from GAA and S-adenosylmethionine originating from Met [1]. Consequently, we hypothesized that feeding ruminants a combination of GAA and RPM may maximize conversion of GAA to creatine, increasing energy supply for the muscle, and promoting growth performance. Moreover, as the uptake of Met for GAA conversion may induce a methyl-group deficiency and a subsequent accumulation of homocysteine, adequate Met supply is required [16].

Therefore, the purpose of this experiment was to study effects of combining GAA with RPM on growth performance, carcass traits and meat quality of Tan lambs, and to provide guidance for their practical application in ruminant production. Two doses of RPM were tested to determine the best dosage of Met required to support creatine synthesis and other metabolic roles of Met. We focused on Tan lambs, as there is currently very limited information on the use of GAA and RPM in this breed.

A lamb from each pen was randomly selected for slaughter at the end of the experiment. Carcasses were cut into halves along the midline, and meat and bone were separated on the right-side before weighing. The left-side of the carcass was weighed for carcass weight. Then, dressing percentage (%) was calculated by dividing the carcass weight by shrunk live BW (BW×0.96).

In this study, Treatment II (0.08% GAA+0.06% RPM) significantly improved growth performance of Tan lambs (higher ADG and final BW) and reduced feed conversion ratio. Using the same breed (Tan sheep) at a close initial BW (20 kg) during the same experimental duration (85 d fattening after 1-wk adaptation), Chao et al [3] also reported a higher ADG of lambs supplemented with 0.08% GAA compared to the control group (158 vs 130 g/d). This range was close to the difference in ADG observed in this study between the control and Treatment II group. This enhanced ADG was supported by variations in serum metabolites. Indeed, the serum content of protein and urea nitrogen respectively increased and decreased with this treatment. The nutritional status of the body can be understood indirectly by measuring serum total protein. Serum urea nitrogen is also an index of nitrogen balance and protein utilization; its concentration is negatively correlated with the rate of utilization amino acids for protein synthesis [24].

The beneficial effects of combining RPM with GAA (interaction) can only be assumed, as unfortunately, the experimental design did not allow us to have an additional treatment with RPM and/or GAA alone. In other words, the positive effects of Treatments II and III may also be attributed to the positive individual effects of RPM and GAA, respectively. As mentioned previously, creatine is synthesized in the liver from GAA and S-adenosylmethionine which originates from Met [1]. Ardalan et al [16] reported a methyl group deficiency after GAA supplementation in cattle without Met supplementation. Indeed, the plasma creatine concentration remained unchanged with GAA supplementation alone, whereas the plasma creatine concentration increased when GAA and RPM were supplemented simultaneously. A lack of Met can limit the conversion of GAA to creatine and limit the beneficial effects of GAA on energy metabolism and protein efficiency. In pigs, a low level of dietary Met and cysteine were a potential cause of the absence of an effect of GAA on growth [25]. Therefore, feeding GAA requires adequate levels of Met, the level of which needs to be determined. In our study, Met may have been used in the conversion of GAA to creatine, as indicated by the increased serum creatinine concentrations in lambs fed Treatments II and III. However, the net meat weight/carcass weight of Treatment III was significantly lower than that of Treatment II, which is contrary to our expectations. Majdeddin et al [27] also reported that when birds received 1.2 g/kg GAA at deficient or excessive Met levels, growth was negatively affected. This may be due to disturbances in methylation homeostasis and/or changes in Arg metabolism. High levels of GAA and RPM indicated greater methylation and s-adenosylmethionine, which may lead an increase of S-adenosylhomocysteine and inhibit most of methyltransferases. Therefore, other methylation reactions could be unbalanced in this case [27]. Moreover, when creatine is sufficient, the activity of glycine amidinotransferase in kidney will decrease, catalyzing the synthesis of GAA from Gly and Arg, thus reduced Arg metabolism to GAA.

Depending on BW, age, sex, and diet, dressing percentage for Tan lambs is usually 44% to 55% [3,7]. Our observations were with this range, with an average dressing percentage of lambs fed the basal diet alone (Treatment I) of 49.3%. This finding may be the consequence of several factors related to GAA, Met, or both. First, creatine produced from GAA can stimulate synthesis of two major contractile proteins, actin and myosin [28]. Second, dietary GAA may spare arginine normally used as a creatine precursor [29], and arginine is the most abundant nitrogen carrier of tissue protein, and it promotes skeletal muscle protein synthesis. A linear positive effect of GAA (from 0.04% to 0.12%) on dressing percentage and proportion of meat in carcass of lambs was also reported [3]. Finally, Met can increase transportation and absorption of amino acids in the small intestine and promote protein synthesis via the mechanistic target of rapamycin complex [30]. Besides, Archibeque et al [31] reported an increase in daily N retention of beef steers supplemented with RPM, even though metabolizable protein requirements were met. Interestingly, Ardalan et al [32] reported that supplementing steers with GAA improved N retention only when Met was supplemented. These studies emphasized the need for further research into the combination of GAA with methyl-donor.

Supplementing lambs with GAA and RPM also increased intramuscular fat content especially for Treatment II, and this can be again related to specific functionalities. The reverse conversion of creatine to phosphocreatine produces ATP and excess energy may be stored in intramuscular fat when phosphocreatine reaches saturation [33]. However, the intramuscular fat content of Treatment II was higher than that of Treatments III, which may be related to our previous speculation that excessive Met or excessive Met combined with GAA can inhibit creatine synthesis and thus suppress the reverse conversion of creatine to phosphocreatine and further reduce intramuscular fat deposition.

Methionine can also affect the metabolism of adipose tissue [30] and a dietary deficiency of Met may reduce the availability of lipoproteins and lipid transport. In the present work, despite the change in intramuscular fat, there were no significant differences among treatments for serum triglyceride and cholesterol concentrations.

Both Treatments II and III also increased glycogen content of muscle meat. As glycogen is the main storage form of glucose in the body, this was consistent with decreased serum glucose concentrations in lambs receiving these treatments. It was reported that creatine can stimulate muscle glycogen storage [34]. Moreover, increased Met supply may stimulate insulin secretion, and though the biological mechanisms remain unclear [35], will favor use of glucose by peripheral tissues.

It is well known that a high drip loss and a low carcass pH are associated with poor WHC which can induce liquid outflow, and loss of soluble nutrients and flavor. In addition, a decreased WHC can lead to increased L* value which is detrimental to meat color. In the present study, dietary GAA and RPM supplementation had a positive effect on the meat quality of lambs, reflected in lower cooking loss and shear force and increased WHC, pH_{45 min} and pH_{48 h}, especially for Treatment II. Meat color was also improved, as indicated by the a* and b* values at 24 h and L* value, which were markedly increased by GAA and RPM. Among other causes, the higher level of protein, fat and glycogen in muscle at slaughter for lambs receiving Treatment II or III must have had a major role in improving meat quality [36,37]. Without being systematic [38], similar improvements in meat quality parameters were observed with GAA or creatine supplementation to non-ruminant species such as pigs [39] and broilers [40]. The differences among studies may result from four sources: supplements in different doses, animal species, analysis on muscle sections, or the availability of methyl-donor in the diet.

This study highlighted the potential of dietary GAA and RPM supplementation to improve growth performance and muscle quality of lambs. When combined with 0.08% GAA, an RPM dose of 0.08% did not bring additional benefits compared to a RPM dose of 0.06% and therefore is not recommended. However further studies are necessary to refine the dosage of these two combined products in order to maximize performance and meat quality of lambs. These findings are an impetus to better understand interactions between RPM and GAA in lambs.