What makes geese special is their ability to store energy through overfeeding in the liver to form fatty liver served as a delicious food [20, 21]. In this study, we built a good model of goose fatty liver, with the body weight and liver weight significantly increased after overfeeding for 7 and 25 days. In particular, the liver weight of geese overfed for 25 days increased by approximately 800 g, accounting for about 12% of the body weight. This is in accord with previous results which showed the liver weight increased up to 10-fold after two weeks of overfeeding and accounted for up to 10% of the body weight . Indeed, these results all confirmed the excellent characteristic of fatty liver production in Landes geese.
In clinical livers with disease such as liver cirrhosis and liver steatosis, the detection of serum enzymatic activities is the most direct method to assess liver damage [23, 24]. Serum enzymes mainly come from the liver as the liver steatosis can lead to the hepatocellular inflammation and enzyme synthesis intensification, which will promote the increase of serum enzyme concentration . In the present study, overfeeding for 25 days increased the amounts of serum ALT, AST and GGT, indicating abnormal in liver function of the overfed geese. Similarly, data from published article showed that long-term overfeeding induced liver cell inflammation, accompanied with higher serum enzyme activities [2, 25]. On the other hand, the formation of fatty liver is essentially a disorder of lipid synthesis and secretion, resulting in the accumulation of serum lipid . Our results showed overfeeding for 25 days caused the significant higher levels of TC and HDL, and a numerical increased TG concentration. These variations in serum lipidemic parameters are consistent with previous studies reporting that overfeeding can induce elevated concentrations of serum lipids [21, 26]. Additionally, we noticed that overfeeding for 7 days did not cause significant variation in serum biological parameters, suggesting that short-term fattening may change the apparent performance, while the body’s metabolism is still in relatively normal operation.
Different aspects of the development of liver steatosis in Landes geese have been studied under experimental conditions [5, 9, 27, 28]. So far, however, the serum metabolic mechanism in this process has not been clarified. Therefore, metabolic profiling with the aid of GC-TOF/MS combined with multivariate statistical analysis was implemented in our study to explore the serum metabolic patterns, and to identify potential contributors to the formation of fatty liver and the correlated metabolic pathways. In the early stage of overfeeding (0 day to 7 day), 34 differentially expressed endogenous serum metabolites were identified, which were enriched in 22 pathways including arginine and proline metabolism, nicotinate and nicotinamide metabolism, pyruvate metabolism, glycolysis or gluconeogenesis, and glutathione metabolism. In the late stage of overfeeding (7 days to 25 days), the levels of 51 metabolites were significantly changed, which were involved in valine, leucine and isoleucine biosynthesis, glycine, serine and threonine metabolism, taurine and hypotaurine metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism, glycerolipid metabolism, glutathione metabolism, etc. During the whole period of overfeeding, the most differential metabolites were observed, up to 55. The distinct reprogrammed metabolism was mainly involved valine, leucine and isoleucine biosynthesis, propanoate metabolism, TCA cycle, alanine, aspartate and glutamate metabolism, taurine and hypotaurine metabolism, glycerolipid metabolism, glyoxylate and dicarboxylate metabolism, as well as pyruvate metabolism. Just as Ma et al.  said in the report, the metabolic pathways identified through the significantly different metabolites represent the typical characteristics response of living systems to pathophysiological stimuli or genetic modification . Comprehensive consideration of our research results, metabolites changed at different stages, such as pyruvic acid, alanine, proline, beta-glycerophosphoric acid, 3-hydroxypropionic acid, canavanine and lactic acid, were selected as potential biomarkers for the early diagnosis of fatty liver disease. In addition, it is noteworthy that several metabolic pathways occur repeatedly at different stages of overfeeding, such as TCA cycle in both the late and the whole stages of overfeeding. The disturbed metabolic pathways identified in the current are consistent with classic metabolism and represent the typical features of the dietary or medical intervention on organisms [31, 32].
In order to systematically demonstrate the metabolic response to overfeeding, the significantly different metabolites combined with corresponding metabolic pathways are shown Table 1. The metabolite with the largest difference between the D0 group and the D7 group was glutathione (8.101 fold higher in the D7 group than that in the D0 group), which is an amino acid and a tripeptide, as well as a well-known antioxidative factor . This finding is in line with a previous study in human, in which γ-glutamyl dipeptide could serve as biomarkers for discrimination among different forms of liver disease, and had a positive correlation with glutathione, providing specific information for different liver diseases . The elevated concentrations of citrulline, proline and creatine, which are products of arginine and proline metabolism, implying the increase of amino acid utilization. Relevant metabolic articles indicated that disturbed metabolism of arginine and proline might play an important role in the obesity progression [33, 34], in complete agreement with the increase in body weight and liver weight of Landes geese.
From the dynamic perspective, the changes of serum metabolism in the late stage of overfeeding were more similar to that in the whole period of overfeeding. The same metabolic pathways screened for these two periods are as follows: valine, leucine and isoleucine biosynthesis; taurine and hypotaurine metabolism; TCA cycle; alanine, aspartate and glutamate metabolism; glycerolipid metabolism. The significantly different metabolites enriched in these pathways were pyruvic acid, succinic acid, fumaric acid, 2-keto-isovaleric acid, glyceric acid, threonine, leucine, l-cysteine, taurine, glycerol, which were all up-regulated in the serum of overfed geese. Notably, pyruvic acid could be found in three metabolic pathways, and it also appeared as a key product in pyruvate metabolism, which can be converted into carbohydrates via gluconeogenesis, to fatty acids or energy through acetyl-CoA, and to amino acids, and ethanol . The elevated level of pyruvic acid in the serum of overfed geese might be related to the intensification of energy conversion. TCA cycle, glycerolipid metabolism and pyruvate metabolism are essential metabolic pathways involved in energy supply. As the center of three major nutrients metabolism (carbohydrates, lipids, and amino acids), TCA cycle is the conversion site among sugar, lipid and amino acid metabolism, and the main way to obtain energy for the body . The concentrations of pyruvic acid, succinic acid and fumaric acid enriched in TCA cycle were increased, indicating that overfeeding exerted an important influence on energy metabolism of Landes geese. Other representative metabolites, such as 2-keto-isovaleric acid, threonine, l-cysteine, leucine, are crucial components involved in protein synthesis. The enhanced of protein synthesis may be the main reason for the improvement of apparent growth performance of overfed geese.