Obesity has been declared as a major global health problem in both human beings and domesticated animals, and it has been most commonly observed in companion animals (Chandler et al., 2017). Previous studies have showed that approximately 59% of dogs are overweight or obese (Courcier et al., 2010). Obese dogs have been demonstrated to be more prone to suffering from a subclinical inflammatory state than their lean counterparts, characterized by the higher levels of certain inflammatory markers, which are associated with a higher risk of cardiovascular illnesses and cancer (Vecchiato et al., 2023). Thus, accurate diagnosis is crucial for early obesity treatment in dogs, thereby enhancing their welfare and quality of life. The most common tool used by veterinarians to assess the nutritional status of dogs is the body condition score (BCS) system (Broome et al., 2023), which has relatively high accuracy and reproducibility, but it still has a certain degree of subjectivity in determining canine obesity because the evaluation primarily relies on visual examination and palpation (Tesi et al., 2020; Miller et al., 2018). Even among experienced veterinarians, discrepancies in evaluating BCS for the same dog may occur. Therefore, the additional biomarker indicators are needed to identify for providing supplementary references in the diagnosis of canine obesity by the veterinarians using BCS system.

Serum biochemistry parameters are commonly used to detect the metabolic health of humans and animals. Alanine aminotransferase (ALT) and total bilirubin (TB) serve as significant potential biomarkers in the clinical assessment of liver inflammation-related diseases not only in humans, but also in feline and canine populations (Dirksen et al., 2017; Kozat and Sepehrizadeh, 2017). Furthermore, recent research has demonstrated a positive correlation between ALT levels and body weight in humans, highlighting its potential utility in evaluating metabolic health (Bekkelund and Jorde, 2019).

Although obesity is highly prevalent in dogs, there are higher frequencies of overweight conditions in certain breeds of dogs including Golden Retrievers, Pug, Beagle and English Springer Spanie than in others (Reddy et al., 2019; Pegram et al., 2021). The breed susceptibility of Golden Retriever to obesity is 49.53% according to the clinical study (Mounika et al., 2020), implying that Golden Retrievers should be paid more attentions on their obese status (Pegram et al., 2021; O’Neill et al., 2021). In recent years, increasing evidence has shown that gut microbiota dysbiosis played a causal role in the early onset of obesity and lipid metabolism dysfunction (Yu et al., 2019; Zhang and Gérard, 2022). On the other hand, obese individuals have harbored a distinct gut microbiome from those lean individuals, indicated by reduced microbial diversity and altered taxonomic abundances in the overweight cohort (Lippert et al., 2017). The higher Firmicutes and Bacteroidetes ratio has been related to obesity in some studies, but these observations are inconsistent across studies, and it is unknown which specific microbial taxon is directly related to the development of obesity (Xie et al., 2012). Likewise, overweight Beagle dogs also have exhibited a significantly different gut microbiome from the normal ones, characterized by higher abundances of genera Faecalibacterium, Phascolarctobacterium, Megamonas, Bacteroides, Mucispirillum (Kim et al., 2023). These evidences further support the contention that gut microbiota may be involved in the metabolic health of the host. Owing to the close relationship between gut microbiota and host obesity, several attempts have been made to reveal the potential microbial biomarkers for detecting dietary responsiveness in obese individuals with impaired metabolic health. Indeed, some microbial biomarkers have been identified to hold the potential to predict obesity, and interventions based on these microbial biomarkers might be beneficial to weight loss and metabolic risk improvement (Barengolts et al., 2019). It remains unknown the differences in gut microbiota composition between non-obese and obese Golden Retriever dogs, and thus it is necessary to identify microbial biomarkers of obesity in Golden Retrievers.

Gut microbiota dysbiosis in obese individuals has been shown to affect host physiology possibly via modulating the metabolism and excretion of microbial-derived metabolites including lipids and lipid-like metabolites, amino acids, bile acids derivatives, and catabolites of plant bioactive components, which were also proposed to be predictors of overweight or obesity (Machate et al., 2020; Lippert et al., 2017). Metabolomics has been enlightened as a useful tool to evaluate changes in metabolites due to overweight and obesity at the cellular level and body fluid level (Xie et al., 2012). Thus, metabolite profiling with metabolomics might represent an opportunity to methodically establish biomarkers for obesity diagnosis and control that are relatively simple to measure in comparison to traditional approaches like the BCS system (Rauschert et al., 2014).

Therefore, the alterations in composition of the gut microbiota and the plasma metabolites profile may be informative in predicting the obese status of Golden Retrievers. The present study adopted microbiome and metabolomics techniques to test the differences in gut microbiota and plasma metabolites profile between non-obese and obese Golden Retrievers to reveal the potential obesity biomarkers from microbiota and/or metabolites.

The experimental protocols related to animal treatment in the present study were approved by the Institutional Animal Care and Use Committee of Southwest University of Science and Technology (No. L2023029).

Obesity has reached pandemic proportions in dogs (Courcier et al., 2010; Butterwick, 2000), in which has significant implications for canine physiology, health and welfare, especially causing gut microbiota dysbiosis and metabolism dysfunction. Previous research has identified potential microbial and metabolomic biomarkers associated with obesity in humans and rodents (Metwaly et al., 2022; Wang et al., 2021; Giudetti, 2023). However, the similar kinds of biomarkers for diagnosis of canine obesity remain unclear. Accordingly, the present study compares the serum biochemical indices, fecal microbiota and plasma metabolites profile of non-obese and obese Golden Retrievers to preliminarily reveal the potential microbial or metabolic biomarkers for obesity in Golden Retrievers.

Studies in both humans and animals have showed that obesity causes the disruption of the homeostasis of lipid metabolism and increases fat accumulation not only in adipose tissue but also in the liver, resulting in adipose inflammation and hepatic damage (Giudetti, 2023; Richard et al., 2000). Obesity-associated fatty liver has been shown to induce a substantial increase in the activity of ALT, which suggests a potential connection between ALT and obesity. A previous study has demonstrated that obese dogs exhibited higher activities of ALT and gamma-glutamyl transferase (Tribuddharatana et al., 2011). Consistently, a tendency toward higher activity of ALT was found in the obese Golden Retrievers compared to the control dogs. Besides, previous study found that serum TB was associated with an elevated risk of obesity-associated metabolic syndrome (Wei et al., 2021). TB including direct and indirect bilirubin, the degradation products of the heme moiety of hemoglobin and other hemoproteins, is potentially toxic to the liver where it is detoxified (Roy-Chowdhury et al., 2020). Bilirubin has been proposed as a biomarker to monitor hepatic function and of which the high level in blood was the consequences of liver dysfunction according to the previous study (Guerra Ruiz et al., 2021). In the present study, a significantly higher level of bilirubin was found in the Obe group, indicating the potential liver impairment in overweight dogs.

Gut microbiota play a crucial role in host metabolism and physiology, but the pathophysiological mechanisms and clinical outcomes were still unclear. On one hand, the presence of obesity has been associated with the imbalance of gut microbiota, which has been demonstrated to play a causal role in the induction of metabolic dysfunction (Le Chatelier et al., 2013; Stanislawski et al., 2019). On the other hand, several studies observed significant differences in the abundance and biodiversity of gut microbiota between non-obese and obese individual (Pinart et al., 2021; Yun et al., 2017). The Firmicutes/Bacteroidota ratio was considered as a microbial indicator for obesity by some studies, as the higher abundance of phylum Firmicutes and increased ratio of Firmicutes/Bacteroidota were observed in obese individuals or animals than their lean counterparts. However, others hold the different opinion that there is a weak connection between obesity and the Firmicutes/ Bacteroidota ratio (Schwiertz et al., 2010). Previous studies showed that the most predominant taxa at the phylum level in dogs were Firmicutes (You and Kim, 2021), which is also the most abundant phylum in the feces of Golden Retrievers. In addition, phylum Firmicutes has been found more abundant in non-obese dogs than the obese ones (Macedo et al., 2022; Park et al., 2015; Kim et al., 2023). Consistent with these findings, non-obese Golden Retrievers exhibited the higher level of phylum Firmicutes in feces than the Obe group, implying that phylum Firmicutes might be negatively correlated with the presence of obesity in dogs, which needs to be further verified in the future study. Genus Fusobacterium was found related to the whole-body inflammation state and has been more abundant in the fecal microbiota of children with obesity as well as in the feces of type 2 diabetes subjects (Sedighi et al., 2017; Yuan et al., 2021; Chen et al., 2020; Lee et al., 2018). In agreement with these findings, the increased abundance of genus Fusobacteria was observed in the Obe group in the present study. A previous study showed that fat pigs had significantly higher proportions of Prevotella. copri (Chen et al., 2021). However, a higher abundance of genus Prevotella has been associated with enhanced insulin sensitivity in rodents. These contradictory results might stem from the inter-study difference in the genetic background of studying animals, which has been proved as a determinant factor for the microbiota composition. A previous study on dogs showed that the abundance of Prevotella. copri in fecal microbiota elevated with increasing BCS (You and Kim, 2021). Consistently, in the present study, obese Golden Retrievers exhibited higher levels of genus Prevotella in feces. Likewise, another study also demonstrated that high carbohydrate diet-fed obese dogs exhibited higher abundance of Prevotella. copri in feces than dogs with normal weight (Li et al., 2017). Previous study compared the microbiota differences between dogs with differed degree of overweight have found that highly overweighed dogs had higher abundance of Prevotella in feces compared to their counterparts (Forster et al., 2018). Therefore, the abundance of Prevotella might be used as a potential biomarker for the detection of obesity in Golden Retrievers, but its effects on and reasons for its abundance in obese dogs should be examined in the future study. A higher level of Peptoclostridium was found in dogs with normal BCS compared to those with high BCS in a previous study on Beagle dogs (Kim et al., 2023). Consistently, in this study, the obese Golden Retrievers exhibited a lower abundance of Peptoclostridium in feces. Genus Blautia has been reported to exhibit beneficial effects on intestinal health and metabolic disorders (Liu et al., 2021). A previous study showed that oral administration of Blautia can ameliorates obesity and type 2 diabetes by increasing the production of short chain fatty acids (SCFAs) and activating SCFAs-related signaling pathways (Patel and Preedy, 2017). In the present study, non-obese Golden Retrievers had higher levels of genus Blautia in feces, implying that increasing the colonization of genus Blautia might be helpful for weight management in dogs, which needs to be studied in future research. The alterations in abundances of genus Ruminococcus_gnavus_group might the unique response of Golden Retrievers to obesity, because overweight had no effects on the abundances of these genera in other dog breeds like Beagle dogs (Kim et al., 2023). Therefore, the verification of the application of the genus as a biomarker indicator of obesity should be further studied.

The metabolite profiling via metabolomics is frequently used to reveal metabolic biomarkers for obesity and associated metabolic syndrome. In the present study, the plasma metabolite profile in obese Golden Retrievers differed from the non-obese dogs, which was consistent with previous findings that the presence of obesity significantly altered the whole-body metabolism in humans and rodents (Lai et al., 2014). Citrulline was produced by the hydrolysis of peptidyl arginine catalyzed by protein arginine deiminases, of which high activity was observed in a host of human diseases including metabolic syndrome and inflammation (Allerton et al., 2018). The conversion process of peptidyl arginine into citrulline also called protein citrullination, has been demonstrated to be an inflammation-dependent process since it is upregulated in various inflammatory states (Makrygiannakis et al., 2006). In this study, the plasma level of citrulline was higher in obese Golden Retrievers than in the control ones, suggesting that the presence of obesity might activate this posttranslational process, namely protein citrullination, in tissues of Golden Retrievers dogs. Besides, a previous study found that chronic administration of 11-dehydrocorticosterone to mice increased the circulating glucocorticoid level and downregulated the expression of genes related to the hypothalamic–pituitary–adrenal axis, leading to insulin resistance, adiposity and an elevation of body weight (Marzolla et al., 2014). In the current study, a higher concentration of 11-dehydrocorticosterone in plasma was observed in obese Golden Retrievers, indicating that the elevated cortisol secretion might contribute to the pathogenesis of obesity in Golden Retrievers (Abraham et al., 2013).

The catabolites of prostaglandin E2 (PGE2), including 15-keto-PGE2 and 8-iso-15-keto-PGE2, have been shown to alleviate hepatic inflammation in diet-induced obesity mouse model, indicative of lower activities of ALT and aspartate transferase and inhibited macrophage infiltration (Hee et al., 2023). Consistently, lower plasma levels of 8-iso-15-keto-PGE2 in obese dogs and the negative correlation between 8-iso-15-keto-PGE2 and aminotransferase activity were observed in the current study, suggesting that the approaches increasing the levels of catabolites of PGE2 might be effective for the treatment of obesity in Golden Retrievers. N-hydroxy-L-isoleucine is a derivative of L-isoleucine which has been shown to enhance muscle growth, inhibit fat deposition, and exhibit anti-inflammation activity (Patil et al., 2012). Given the beneficial effects of L-isoleucine on the metabolism and inflammation of the host, it can be speculated that N-hydroxy-L-isoleucine might exhibit similar effects as well. In the present study, obese dogs exhibited lower concentrations of N-hydroxy-L-isoleucine, implying that the intake of L-isoleucine and its derivatives might be useful for controlling overweight in dogs.

Overall, obesity significantly altered the concentrations of certain serum biochemistry parameters, which might be associated with the changes in gut microbiota and plasma metabolites profile. The verification of these possible biomarkers for diagnosing obesity in Golden Retrievers and the dietary intervention approaches targeting the microbiome and metabolome for weight management in dogs need to be further studied and explored.

In summary, obesity notably modified the levels of specific serum biochemical markers, which may correlate with alterations in gut microbiota and plasma metabolite profiles. Further investigation and exploration are necessary to validate these potential biomarkers for diagnosing obesity in Golden Retrievers, alongside dietary intervention strategies aimed at the microbiome and metabolome to manage weight in dogs.