Guanidine acetic acid (GAA) is indispensable for creatine synthesis in vertebrates and plays a pivotal role in the energy metabolism of somatic cells in animals (1, 2). L-arginine glycine amidinotransferase catalyzes the conversion of arginine and glycine into GAA in the kidneys, which is subsequently transported via the bloodstream to the liver. Here, it reacts with n-dimethyltransferase and S-adenosylmethionine to produce creatine (3, 4). Creatine serves as a vital energy reservoir in animal tissues, rapidly converting creatine phosphate into ATP.

In recent years, the reduction in animal protein usage in Chinese livestock feed has heightened concerns over creatine loss during feed processing and heat treatment. This has led to increased interest in exogenous creatine supplementation, despite its costliness, instability, and potential inhibition of endogenous creatine synthesis (5, 6). Conversely, GAA exhibits stability and superior effectiveness compared to creatine in enhancing creatine levels in animals (7), making it a preferred option for creatine supplementation.

Studies have explored the supplementation of GAA in weaned piglets and finishing pigs, revealing that 0.12% GAA enhances average daily gain (ADG) and feed efficiency (Gratio) throughout the growth phase, increases lean meat yield, and reduces backfat thickness (8). He et al. (9) reported improvements in growth performance with 300 mg/kg of GAA, attributed to elevated creatine and ATP levels in tissues. Furthermore, supplementation with 2.4 g/kg of GAA during the nursery stage improves feed conversion efficiency and upregulates mRNA expressions of mTOR and AMPK in skeletal muscle (10). Supplementation with 0.06% GAA during the late fattening stages increases ADG and lean meat percentage by influencing muscle development through modulation of myoblast gene expression and muscle fiber characteristics (11). Additionally, supplementation around 86 kg body weight does not enhance growth performance but increases levels of free amino acids, their metabolites in plasma and tissues, and antioxidant enzymes (12, 13).

However, research on GAA’s impact in sow production remains limited and yields varied results (14, 15). Therefore, this study investigates the effects of GAA supplementation during late gestation and lactation on sow reproductive performance, colostrum composition (including immunoglobulin and amino acid content), routine cord blood parameters, nitric oxide levels in cord blood, inflammatory markers, sow intestinal flora, and growth performance of suckling piglets.

The experiment was conducted at the swine experimental unit of Anyou Biotechnology Group Co., Ltd. (Nanjing, Chian). All experimental procedures were approved by the Committee on Ethics in the Use of Animals (CEUA) of the Anyou Biological Technology Group Co., LTD (No: ANS-CEUA-PJT/ PL/202309/084).

Currently, there is limited research on the application of guanidine acetic acid (GAA) in sows. Pregnant and lactating sows have peak energy requirements, and supplementing with GAA during this period can enhance energy efficiency, thereby increasing sow and offspring productivity.

Research indicates that GAA supplementation during gestation alone can boost the number of live piglets per sow. Starting GAA supplementation in late gestation enhances the average birth weight of piglets, while supplementation during lactation increases protein and amino acid concentrations in sow colostrum (20). Creatine supplementation in the final week of gestation improves the myelin sheath of low birth weight piglets and increases the survival rate of weaker piglets, though it has no impact on sow labor duration, piglet birth intervals, or stillbirth rates (21). Adding 0.1% GAA to gilt diets enhances the number of live piglets produced, with minimal variation in birth weights per litter. Moreover, GAA enhances average daily gain (ADG), weaning weights, and daily milk yield of piglets. During lactation, GAA increases amino acid content in milk by the 7th day, particularly lysine, methionine, arginine, valine, and glutamine (15).

Our results indicate that GAA supplementation significantly increases the number of live and healthy piglets but does not affect sow farrow duration, litter intervals, stillbirth rates, feed intake, or backfat loss. This finding aligns with previous studies, suggesting that GAA’s growth-promoting effects in monogastric animals are primarily related to its role in tissue protein anabolism and improvement of energy metabolism. The synthesis of GAA involves its production in the liver and subsequent methylation in the kidneys. GAA synthesis requires the participation of two amino acids, arginine and glycine. Arginine transfers its amidino group to the amino group of glycine to produce ornithine and GAA, catalyzed by L-arginine: glycine amidinotransferase (AGAT). Guanidinoacetate is then methylated using a methyl group from S-adenosylmethionine (SAM), which is synthesized from methionine. This reaction, catalyzed by guanidinoacetate N-methyltransferase (GAMT), produces creatine and S-adenosylhomocysteine (SAH) (2). Consequently, dietary GAA supplementation reduces the breakdown of arginine and glycine in the body, thereby enhancing their efficacy in protein synthesis and promoting growth (22, 23). Additionally, GAA may be mediated through transport proteins such as creatine transporter (CRT/SLC6A8), taurine transporter (TauT/SLC6A6), and γ-aminobutyric acid transporter (SLC6A13), and is transported into target cells via passive diffusion across the plasma membrane (24), thereby improving reproductive performance in sows. Moreover, GAA’s physiological regulatory functions may include stimulating the secretion of insulin-like growth factor 1 (IGF-1), thereby enhancing growth performance (25). Furthermore, dietary GAA supplementation might boost the secretion of gamma-aminobutyric acid (GABA), which promotes the secretion of growth hormone-releasing hormone by the hypothalamus and subsequently increases growth hormone secretion by the adenohypophysis, fostering animal growth (26). However, our current study on GAA in sows has limitations regarding the mechanisms involved, warranting further investigation and validation.

In conjunction with the nitric oxide findings from our experiment, nitric oxide synthase and nitric oxide levels in sow cord blood increased by 0.7 μmol/L and 0.5 μmol/L in the GAA group, respectively. Optimal nitric oxide levels stimulate blood vessel proliferation in the placenta and umbilical cord, enhancing nutrient flow from mother to piglet and increasing piglet birth weights. This underscores why guanidine acetic acid can increase the number of healthy piglets in sows, resulting in an average weight increase of 130 g for live piglets and 90 g for healthy piglets.

During gestation, arginine deficiency can be effectively mitigated by supplementing with guanidine acetic acid (GAA). Mateo et al. (27) observed that daily supplementation of 1% arginine in lactating sows on the 7th day of lactation increased plasma arginine concentrations and insulin levels, thereby enhancing substance metabolism. This supplementation also elevated nutrient content in milk, with higher concentrations of most amino acids compared to the control group. Additionally, arginine promotes mammary gland development, improves blood circulation, affects nutrient transport, and potentially enhances lactating performance by increasing milk yield and quality (28).

Moreover, our study demonstrated that GAA supplementation increased milk protein, urea nitrogen, and total solids in sow colostrum. This suggests that GAA may influence colostrum composition through its impact on arginine levels. Further investigation into normal milk composition in sows was not conducted in this experiment, highlighting the need for future research.

The intestinal microbiota plays a crucial role in regulating physiological development and health status in pigs, as well as preventing pathogen colonization (29). While research on GAA’s effect on intestinal flora is sparse, studies on arginine have shown that intestinal bacteria utilization can lead to polyamine release in the intestine (30). Arginine also regulates amino acid utilization in intestinal bacteria cultures obtained from pig intestines (31, 32).

In our experiment, the GAA group did not affect the α diversity of sow intestinal flora but did show differences in β-diversity. LEfSe and Simper analyses identified Lactobacillus and Terrisporobacter as the main genera affected. Lactobacillus, a probiotic, inhibits harmful microorganisms (33), alters microbial metabolism (enzyme activity), enhances nutrient absorption (e.g., proteins, monosaccharides) (34), and stimulates immune responses (35). The Terrisporobacter genus includes beneficial bacteria like Bacillus subtilis, Bacillus fungoides, and Bacillus polymyxis, which improve animal production performance. These findings suggest that GAA alters dominant intestinal bacteria, potentially improving nutrient metabolism and absorption, thereby increasing milk protein, total solids, and urea nitrogen in sows.

GAA enters sow milk through the bloodstream and effectively increases creatine and creatine phosphate content in muscle tissue. This reduces carbohydrate, fat, and protein energy supplies while enhancing hydration to inhibit protein breakdown and promote protein and glycogen synthesis, accelerating animal growth (36). Additionally, GAA influences the hypothalamus to secrete growth hormone-releasing hormone, impacting gamma-aminobutyric acid secretion and subsequently stimulating pituitary growth hormone secretion (37). Combined with the increased milk protein levels in sow milk, this contributes to increased average daily gain (ADG) in offspring piglets to a certain extent.

GAA can increase nitric oxide synthase levels in sows’ umbilical cord blood, promoting the development of placental blood vessels and piglet growth. It enhances the number of healthy piglets and their vitality, while also increasing the abundance of beneficial bacteria such as Lactobacillus Parabacteroides, and Pedobacter in the gut. These effects lead to higher protein levels in colostrum and increased total lactation volume, thereby improving the growth performance of offspring piglets (Figure 5).