Male castration alters the gut microbiota, leading to increased serum levels of branched-chain amino acids and adiposity

Background : Testosterone deficiency is positively correlated with fat accumulation and obesity-related comorbidities, such as metabolic syndrome. Castration of young males is widely used in the cattle industry to improve meat quality. However, the mechanism linking hypogonadism and host metabolism is not clear. We aimed to evaluate the effect of male hypogonadism on the gut microbiota and serum metabolites, and the contribution of the altered microbiota to the host metabolic phenotype during hypogonadism. Results : We used metataxonomic and metabolomic approaches to evaluate the intestinal microbiota and host metabolism in male, castrated male (CtM), and female cattle. We then used a male mouse castration model to evaluate the causative factor(s) that underpin the alteration of the intestinal microbiota and host metabolic phenotype in response to hypogonadism. After pubescence, the CtM cattle harbored distinct ileal microbiota dominated by the family Peptostreptococcaceae , and exhibited distinct serum and muscle amino acid profiles (i.e., highly abundant branched-chain amino acids), with increased extra- and intramuscular fat storage. Castration of male mice phenocopied both the intestinal microbial alterations and obese-prone metabolism observed in cattle. Antibiotic treatment and fecal microbiota transplantation experiments in a mouse model further revealed that the intestinal microbial alterations associated with hypogonadism are a key contributor to the obese phenotype in the CtM animals. Conclusions : We demonstrated altered gut microbial profiles in the hypogonadal animals, with a negative feedback between the serum testosterone levels and the ileal abundance of Peptostreptococcaceae , and a distinct metabolic phenotype, with an enhanced amino acid metabolism. These findings suggest targeting the gut microbiota as a potential therapeutic strategy for the treatment of both hypogonadism and obesity. reaction (PCR) using bacterial universal primers 8F GAT CMT GGC TCA G-3′) and 338R (5′-TGC TGC CTC CCG TAG GAG T-3′); 10 b sample-specific barcodes (designated as X) were added to the 5′ primer terminus to tag the PCR products. In preparation for the Illumina MiSeq sequencing of the gastrointestinal samples from the adult Hanwoo and mice, a fragment of the 16S rRNA gene spanning the hypervariable V3–V4 regions was amplified by PCR using the forward primer 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3′ and the reverse primer 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′. The PCR was performed in a C 1000 thermal cycler (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: initial denaturation at 96 °C for 6 min; followed by repeated cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 30 s, and extension at 72 °C for 90 s; and a final extension step at 72 °C for 10 min. For the PCR of DNA extracted from the rectum, rumen, cecum, and colon contents, 18 repeated cycles were used; for the PCR of DNA extracted from the ileum content, 25 repeated cycles were used. Three PCR reactions with the same template were pooled and the products were purified using the QIAquick PCR purification kit (Qiagen). Equimolar amounts of the purified amplicons were then combined in a single tube. For the GS-FLX sequencing, the amplicons were pyrosequenced by Macrogen using the Genome Sequencer FLX For Illumina statistical analyses Comparisons between two samples were made using the non-parametric Mann-Whitney U test and the unpaired Student’s t -test (one-tailed). Comparisons between multiple samples were conducted by the analysis of variance (ANOVA), followed by Tukey’s post-hoc test (*p<0.05, **p<0.01, and ***p<0.001). The statistical significance for the observed variations was assessed using the PERMANOVA function with 999 permutations. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively. The values and scattered dots in the bar graphs represent the means ± SEM and the individual replicates, respectively.


Background
Beyond its contribution as the principal male sex hormone, testosterone plays a key role in metabolism and body composition. Elderly men and patients with Klinefelter's syndrome who usually suffer from testosterone deficiency, termed hypogonadism, often show an unfavorable body composition with increased fat mass and reduced muscle mass [1,2].
Conversely, the incidence of hypogonadism is increased in obese men compared to healthy men [3,4]. This bidirectional relationship between obesity and male hypogonadism (meaning that primary hypogonadism promotes fat accumulation while obesity attenuates endogenous testosterone production) has been highlighted in the current obesity epidemic [5]. Several animal model studies also support this reciprocal parallelism. For example, genetically obese (leptin-deficient ob/ob) male mice exhibit hypogonadism-mediated impaired reproductive function [6, 7] and androgen receptor knock-out mice or castration models predispose to development of obesity [8,9]. It has been proposed that the enzymatic conversion of testosterone to estradiol by adipocytederived aromatase can cause hypogonadism in obese individuals [10,11] and that testosterone inhibits differentiation of preadipocyte to adipocyte in vitro [12]. However, the mechanisms linking hypogonadism with fat accumulation are still not fully elucidated.
( Fig. 1a). At the postpubescent stage, but not at the juvenile stage, the serum testosterone levels in the male Hanwoo were significantly higher than those in CtM and females ( Fig. 1b and Additional file 1: Supplementary Fig. S1a; ANOVA, p < 0.001). The serum 17β-estradiol levels were not significantly different between groups at the postpubescent and juvenile stage ( Fig. 1c and Additional file 1: Supplementary Fig. S1b). To evaluate the effects of changes in the gut microbiota induced by male castration on the host metabolomic profile, we next analyzed the serum metabolome profiles of postpubescent cattle by using gas chromatography time-of-flight mass spectrometry (GC-stage (Fig. 2a). PCoA of the microbiota in the rumen, ileum, and colon revealed that the bacterial communities in the different segments were significantly different in the male and CtM Hanwoo (PERMANOVA, p < 0.005 for each segment; comparison of the male and CtM animals in the rumen, ileum, and colon) (Fig. 2b). We next evaluated the gastrointestinal segment in which the bacterial communities were mostly affected by male castration. When all gastrointestinal samples were plotted together, only the ileal microbiota in the male and CtM groups were separated on both the abundance-weighted (weighted UniFrac, Fig. 2c and Additional file 1: Supplementary Fig. S4a) and -unweighted PCoA (unweighted UniFrac, Additional file 1: Supplementary Fig. S4b). Similar, analysis of the weighted UniFrac distance between the male and CtM groups revealed the largest dissimilarity value for the ileal microbiota (Fig. 2d).
We then compared the relative abundances of major taxa (> 0.5% of the mean abundance) at the family level. The difference of the ileal microbiota in the male and CtM groups was apparent, with a marked increase in the family Peptostreptococcaceae in the CtM Hanwoo ( Fig. 2e and Additional file 1: Supplementary Fig. S4c). Phylogenetic analysis of the operational taxonomic units (OTUs) assigned to the family Peptostreptococcaceae further revealed that the abundant OTUs mostly belonged to the genera Romboutsia and Paeniclostridium (Additional file 1: Supplementary Fig. S5).
To investigate the relationship between the intestinal microbiota and the resultant microbial metabolites (especially BCAAs) in detail, we subsequently analyzed metabolomic profiles of the luminal contents of the ileum, cecum, and colon of the CtM Hanwoo (Additional file 3: Supplementary Result). In the ileum, a strong positive correlation (Spearman's rank correlation analysis, p < 0.001) was detected between the BCAAs and two unclassified genera belonging to the family Peptostreptococcaceae, and the genera Butyrivibrio, Acetitomaculum, and Atopobium (Additional file 1: Supplementary Fig. S6a). Furthermore, the ileal levels of intestinal BCAAs were significantly higher (Mann-Whitney U test, p < 0.001) in the CtM Hanwoo than those in the male controls; the BCAA levels in the cecum and colon were much lower than those in the ileum, and no quantitative difference in the BCAA levels was observed between the male and CtM groups (Additional file 1: Supplementary Fig. S6b).
Male castration results in increased intramuscular fat accumulation with high BCAA levels in the adult cattle We next evaluated the consequence of the distinct metabolomic profile (i.e., high levels of BCAAs in the serum and ileum) of the CtM cattle. After the bodies were dressed (n = 10 in each group), we observed clear differences in the body composition of the male and CtM Hanwoo (Fig. 3a). The thickness of the dorsal subcutaneous fat, total fat weight, and serum BCAA levels in the CtM group were significantly higher than those of the male controls ( Fig. 3b; Mann-Whitney U test, p < 0.001). We then analyzed the degree of intramuscular fat accumulation in the fresh striploin muscle in the male and CtM Hanwoo carcasses (n = 5 in each group). We observed a significantly higher (Mann-Whitney U test, p < 0.01) intramuscular fat accumulation in the CtM muscle than in the male muscle ( Fig. 3c,d). We next evaluated the intramuscular metabolites profiles. Similar to the serum metabolite profiles described above, the intramuscular metabolomes of the male and CtM carcasses clustered significantly separately by PCoA ( Fig. 3e; PERMANOVA, p = 0.008; comparison of the male and CtM Hanwoo). The levels of BCAAs (i.e., isoleucine and leucine), as well as phenylalanine, tryptophan, and tyrosine, were significantly higher (Mann-Whitney U test, p < 0.05) in the CtM muscle than in the male muscle (Fig. 3f).
Among the amino acids whose levels were increased in the CtM muscle, leucine, phenylalanine, tryptophan, and tyrosine can be degraded into acetyl-CoA, a precursor of ketone bodies. We subsequently measured the intramuscular levels of β-hydroxybutyrate (3-HB), widely used to diagnose ketosis in dairy cow [21], in the male and CtM muscles.
The 3-HB levels were significantly higher (Mann-Whitney U test, p < 0.01) in the CtM muscle than in the male muscle (Fig. 3g). Interestingly, Jang et al. [19] reported that 3-HB secreted by the muscle cell activates endothelial fatty acid transport and promotes lipid accumulation in the muscle. This supports the data presented herein, bridging the high levels of serum BCAAs and the elevated intramuscular fat accumulation in the CtM cattle.
Male hypogonadism leads to an ileal microbial alteration, adiposity, and increased serum BCAA levels in mice The correlation-based analysis in cattle presented above suggested an obesogenic effect(s) of the castration of young male on the subsequent host metabolic phenotype in association with altered intestinal microbiota and systemic metabolite levels. We next evaluated the causative role of the alteration of the gut microbiota following hypogonadism in the obese metabolic phenotype in a mouse model. Male mice underwent prepubertal castration by orchiectomy and were fed a high-fat diet (HFD) to promote the obese phenotype. Age-matched male mice that underwent sham-operation and fed low-fat diet (LFD) were included as the castration and diet controls, respectively (n = 6 in each group; Fig. 4a). At the end of the experiment, the serum testosterone levels in the CtM groups were significantly lower than those in the Sham groups (unpaired Student's t-test, groups). However, the difference between the ileal microbiota was more obvious than that between the colonic microbiota in HFD-fed mice ( Fig. 4c and Additional file 1: Supplementary Fig. S7a,b). In both LFD-and HFD-fed mice, male hypogonadism amplified the expansion of the family Peptostreptococcaceae in the ileum ( Fig. 4d and Additional file 1: Supplementary Fig. S8a,b), and resulted in significantly increased levels of serum BCAAs ( Fig. 4e; unpaired Student's t-test, p < 0.05).
To reveal the effect(s) of gut microbiota on the host metabolism, the HFD-fed groups To determine whether the gut microbiota was responsible for the obese phenotype in castrated mice, we performed FMT from the HFD-fed Sham or CtM donor mice to agematched surgery-naïve eugonadal male recipient mice (Sham-R and CtM-R, respectively; n = 5 in each group) (Fig. 5a). Intriguingly, the difference in serum testosterone levels in Collectively, these observations suggest that the hypogonadism-mediated gut microbial changes contribute to the obese-prone metabolic phenotype per se. In terms of the gutbrain axis, the microbial shift in CtM-R mice toward that in CtM mice might decrease serum testosterone levels by regulating a negative feedback loop between gonadotropinreleasing hormone in the hypothalamus, luteinizing hormone/follicle-stimulating hormone in the pituitary, and testosterone in the testis.

Feeding BCAA-enriched diet elicits an obese phenotype in male mice
To evaluate whether the surplus BCAAs are the key factor contributing to the obese phenotype of the castrated animal, we fed non-castrated male mice with HFD (0% BCAA group) or customized isocaloric BCAA-HFDs (3% and 5% BCAA groups) for 8 weeks (Fig. 6a). For the BCAA-HFD, casein in HFD was substituted by BCAAs (Additional file 2: Supplementary Table S2). Feeding BCAA-HFD increased the body weight gain to a greater extent than feeding HFD, with a meaningful difference after 3 weeks of feeding ( Fig. 6b; unpaired Student's t-test, p < 0.05). At the end of the experiment, we observed a significantly greater adiposity (characterized by the increased percentage to body weight ratio of the posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the BCAA-HFD-fed groups compared with the HFD-fed group (Fig. 6c,

Hypogonadal male animals exhibit a high intestinal microbial urease activity
Testosterone acts not only as the major androgenic hormone but also as a metabolic hormone, affecting the hepatic urea cycle to regulate the whole-body protein catabolism [22,23]. Because the metataxonomic analyses of the ileal microbiota of castrated animals revealed a high abundance of the family Peptostreptococcaceae, we hypothesized that a positive feedback loop might exist between the hepatic urea cycle and ureolysis undertaken by the abundant gut microbiota. Accordingly, we assessed the systemic urea/ammonia metabolism activities in cattle and mice in response to male hypogonadism.
In adult Hanwoo, the microbial urease activity in both the rumen and ileum in the CtM group was significantly higher (Additional file 1: Supplementary Fig. S12a; Mann-Whitney U test, p < 0.01) than that in the male group. The intestinal microbial ureases hydrolyze urea to ammonia [24]. Hence, we subsequently measured the intestinal ammonia levels.
The ileal ammonia levels in the CtM group were significantly higher (Additional file 1: Supplementary Fig. S12b; Mann-Whitney U test, p < 0.05) than those in the male group. In the gut, ammonia produced by ureolytic bacteria is absorbed into the bloodstream and used to fuel hepatic ureagenesis [25]. Accordingly, we determined ammonia levels in the mouse serum. For the HFD-fed mice, the serum ammonia levels in the CtM group were significantly higher than those in the Sham control (Additional file 1: Supplementary Fig.   S12d; unpaired Student's t-test, p < 0.05). In both cattle and mice, however, no differences in the serum urea levels in the male and CtM groups were apparent (Additional file 1: Supplementary Fig. S12c,e). This was probably because of a simple diffusion of the systemic urea into the gastrointestinal tract [26]. Collectively, the above observations suggested that testosterone deficiency in the castrated animals might positively regulate the hepatic ureagenesis, and that urea (and its hydrolysis products, i.e., ammonia and carbon dioxide) potentially affects the ileal microbial profile in the CtM animals.

Discussion
Although reciprocal parallelism between obesity and hypogonadism in men has been reported by a number of cross-sectional studies, the mechanisms linking testosterone and the obese phenotype are not fully understood. Considering the obesity epidemic, the prevalence of hypogonadism is expected to increase in the coming decades, highlighting the importance of understanding these mechanisms. For the livestock, however, animal welfare, and economical feeding and management must be considered in equal measure.
In this context, understanding the metabolic features that could potentially influence the metabolism of beef cattle is important in breeding animals representing the "metabolically healthy obese" phenotype [27] in terms of the production of high-quality animal products.
Keeping these bimodal aspects in mind, we investigated the effect of male castration on the intestinal microbiota and host metabolism in cattle and mice. We demonstrated (i) altered gut microbial profiles in the CtM animals, with a negative feedback between the serum testosterone levels and the ileal abundance of Peptostreptococcaceae, and (ii) a distinct metabolomic signature of the CtM animals with an enhanced amino acid metabolism. Furthermore, (iii) the obese phenotype associated with hypogonadism was transferrable via FMT.
We used a mouse model to examine whether alterations in the gut microbiota following hypogonadism play a causative role in the obese metabolic phenotype. Generally speaking, the mouse is a monogastric animal; as such, the digestive processes and subsequent energy metabolism are different from those in ruminant animals. Importantly, however, we found a marked increase in the ileal abundance of Peptostreptococcaceae in  Supplementary Fig. S6). Amino acids that are abundant in the ileum might be processed in several ways, including absorption by the small intestinal epithelial cells to produce amino acid metabolites, and/or digestion by the colonic microbiota [31].
Relatively high levels of amino acids were also detected in the serum and striploin muscle (but not in the large intestine) of the CtM cattle, suggesting that the fate of the ileal amino acids in the CtM cattle may mostly be weighted towards the small intestinal absorption. Particularly, in the current study, we demonstrated high serum BCAA levels in the castrated cattle and mice ( Fig. 3b and 4e). Furthermore, disruption of the gut microbiota abolished the effect of castration on BCAA levels in mice, indicating that the increased circulating BCAA levels can be attributed to altered gut microbial activity rather than hypogonadism itself. Furthermore, chronic feeding of the BCAA-enriched HFD to male mice significantly increased its body weight and adiposity, and exacerbated glucose intolerance and insulin resistance ( Fig. 6d and Additional file 1: Supplementary Fig. 11a The high levels of global BCAAs in cattle and mice might be attributed to high production of BCAAs by gut microbiota. However, we were not able to obtain clear evidence of causation to bridge the gap between high BCAA levels and the marked increase in microbial taxa (i.e., Peptostreptococcaceae) in CtM animals. Rather, a high rate of protein breakdown may contribute to increased levels of circulating amino acids, including BCAAs.
Testosterone, as a metabolic hormone, affects whole-body anabolism by negatively regulating the hepatic urea cycle [23,35]. The opposite is also true, as testosterone deficiency may lead to increased hepatic ureagenesis, resulting in whole-body nitrogen loss. Indeed, several lines of evidence (i.e., increased serum ammonia levels and intestinal ammonia and urease activity) support the notion of increased hepatic ureagenesis in CtM animals (Additional file 1: Supplementary Fig. S12). Mammals cannot hydrolyze urea but urease-producing bacteria cleave urea into ammonia in the gut [36].
From the perspective of the hologenome theory (namely, that the host and all of its associated microbiota form a unit of selection during the evolutionary change) [37], the ileal microbial alteration and enhanced microbial urease activity can both conceivably be used to cope with nitrogen loss. Wiebler et al. [38] described the role of an enhanced gut microbial urea hydrolysis in the urea-nitrogen recycling in a hibernating frog.
Metataxonomic analysis presented in the current study revealed the presence of abundant sequences assigned to the genera Romboutsia and Paeniclostridium in the ileal samples of the CtM Hanwoo (Fig. 2e and Additional file 1: Supplementary Fig. S5). Interestingly, recent genomic analyses of species belonging to the genera Romboutsia (e.g., R. ilealis CRIB T ) and Paeniclostridium (e.g., P. sordellii ATCC 9714 T ) revealed the presence of a highly conserved urease gene cluster in the respective genomes [39,40]; this suggests that these two bacterial taxa can utilize urea as a nitrogen source. The metataxonomic data reveal the gut microbial communities in cattle and mice at the genus level of taxonomic resolution. Given that different microbial species within a genus have different metabolic functions, isolation of key species from the gut of CtM animals to evaluate the effects of microbial signatures for host metabolism will be of interest. In addition, several sex steroid hormones regulate the secretion of digestive enzymes by the pancreas to the small intestine [41]. In a rodent, an impaired pancreatic secretion (i.e., antimicrobial peptide secretion to the small intestine) affects the intestinal microbiota and, consequently, the fitness of the host, e.g., the weight and survival rate [42]. The results of the current study, together with those of recently published studies, warrant further investigation to identify the mechanism linking hypogonadism and alteration of the intestinal microbiota.

Conclusions
Taken together, the observations presented herein indicate that primary hypogonadism as an outcome of castration leads to alterations in the ileal microbiota (i.e., dominance of the family Peptostreptococcaceae); changes of the systemic, muscular, and intestinal metabolome profiles (i.e., high abundance of BCAAs); and increased fat storage in the cattle and mice. We also uncovered bidirectional regulation of the circulating testosterone levels by the gut microbiota as revealed by the FMT experiment. These findings suggest that hypogonadism may integrate microbiota-derived cues to modulate the host metabolism, suggesting that targeting the gut microbiota could constitute a potential therapeutic approach for the treatment of hypogonadism, as well as the associated metabolic disorders.

Cattle studies
To obtain the CtM animals, male cattle were orchiectomized by a veterinarian (both testicles were removed). Prior to sample collection, the cattle were examined for symptoms of diarrhea, and antibiotic and other medication treatment within 1 month of sampling. Rectal material and the blood were sampled from 45 healthy animals: juvenile (male, CtM, and female; mean age: 11.9 ± 1.1 months; n = 5 of each) and postpubescent (male, CtM, and female; mean age: 19.3 ± 1.5 months; n = 5 of each) Hanwoo, and postpubescent (male, CtM, and female; mean age: 12.1 ± 1.6 months; n = 5 of each) Holsteins. The rectal luminal content was collected by rectal enema using clean disposable latex gloves. The blood samples (5 ml) were collected from the jugular vein by a veterinarian and immediately centrifuged in BD Microtainer chemistry tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for serum collection. The blood and muscle samples, and the luminal content of the rumen, ileum, cecum, and colon of the adult male (mean age: 31.2 ± 5.9 months) and CtM Hanwoo (mean age: 33.9 ± 1.4 months, n = 10 in each group) were collected from a local slaughterhouse (Gunwi-Gun, South Korea) under the supervision of the official veterinarian; the serum was collected from whole-blood samples as described above. The collected samples were transported to the laboratory on dry ice and stored at − 80 °C until use.

Castrated male mouse studies
To obtain the CtM animals, 4-week-old male mice were castrated by orchiectomy. For the Sham groups, age-matched male mice underwent a sham operation (i.e., the muscle layer and skin incision). After the surgery, both groups were given antibiotics (enrofloxacin) and an analgesic (Repellent). To reverse the effect of enrofloxacin on the gut microbiota, the Sham and CtM mice underwent FMT (the fecal samples were collected from the strain-, age-, and sex-matched non-operated donors, n = 10) once a day for 2 continuous days prior to the dietary intervention. Subsequently, the mice were provided either a LFD (10% fat; D12450H, Research Diets, New Brunswick, NJ, USA) or a HFD (45% fat; D12451, Research Diet). For the antibiotic-treated groups, the HFD-fed mice were given a combination of ampicillin (1 g/l), neomycin (1 g/l), metronidazole (1 g/l), and vancomycin (0.5 g/l) in drinking water for 5 weeks (n = 6 per group; Fig. 4a). The antibiotic cocktail was renewed every 4 d.

Mouse FMT studies
Non-castrated male mice (6-week-old) were maintained on LFD for 10 weeks. Prior to FMT, the mice received an antibiotic cocktail in drinking water. After a 12 h washout period, the mice were subjected to FMT (once a day for 3 continuous days) and fed HFD for 4 weeks. Fecal pellets from the Sham-HFD and CtM-HFD mouse donors (n = 6 in each group) were freshly collected and transferred immediately to an anaerobic chamber (Bactron II-2, Sheldon Manufacturing) filled with an atmosphere of 5% H 2 , 5% CO 2 , and 90% N 2 . The pellets were resuspended in sterile and reduced PBS (100 mg feces/ml PBS), vortex-mixed for 3 min, and sieved through a 70 ∝m cell strainer. The fecal suspension (inoculum) was kept in Hungate anaerobic culture tubes and administered to the recipient mice (Sham-R and CtM-R, n = 5 in each group; Fig. 5a).

Evaluation of contaminating DNA
Possible DNA contamination of all reagents used for DNA extraction and sequencing was investigated. PCR analysis for the presence of contaminating bacterial 16S rRNA genes (in a 30 cycle reaction) revealed no apparent contamination of any reagent used.

Sequence analysis
To generate the GS-FLX sequencing datasets, the raw sequences were preprocessed using the QIIME software package 1.9.0 [44] to exclude poor-quality sequences and/or sequencing errors. Briefly, raw sequences containing more than one ambiguous base call, with errors in the barcode or primer regions, with average quality scores < 25, or shorter than 200 bp were removed. To generate the Illumina MiSeq sequencing datasets, pairedend reads (forward and reverse fastq files) were joined and quality-filtered using the QIIME software. Chimeric sequences were excluded from the quality-filtered sequence sets

Metabolite extraction
Both extracellular and intracellular extracts of intestinal luminal contents, serum, and muscle were prepared for metabolite profiling as follows: the luminal contents (250-500 mg) of the ileum, cecum, and colon were extracted into 1 ml water using the MM400 mixer mill (Retsch®, Haan, Germany) at a frequency of 30 s − 1 for 5 min. After sonication (10 min) and centrifugation (12,578 ⋅ g, 4 °C, 10 min), the supernatant was filtered through a 0.2 ∝m polytetrafluoroethylene (PTFE) filter and concentrated using a speedvacuum concentrator. For the serum extractions, 1 ml methanol was added to 200 ∝l of the serum, and then extracted by sonication and shaking for 10 min. After centrifugation (12,000 rpm, 4 °C, 10 min), the supernatant was filtered through a 0.2 ∝m PTFE filter and dried using a speed-vacuum concentrator. The muscle samples (5 g) were finely chopped before extraction, following which 10 ml methanol was added; the samples were then sonicated (10 min) and agitated (1 h). After centrifugation (12,000 rpm, 4 °C, 10 min), the supernatant was filtered through a 0.2 ∝m PTFE filter and dried using a speed-vacuum concentrator.

Metabolite profiling and data processing
The

Hormone measurements
Serum samples from cattle and mice were prepared as described above. The concentrations of serum testosterone and 17β-estradiol were determined by enzymelinked immunosorbent assay (ELISA) using a testosterone ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) and a 17β-estradiol high sensitivity ELISA kit (Enzo Life Sciences), respectively. Concentration of the serum insulin was determined by using a mouse ultrasensitive insulin ELISA kit (ALPCO, Salem, NH, USA).

Analysis of gastrointestinal urease activity
Urease activity was assayed in the ruminal and ileal luminal contents of the adult Hanwoo.
The luminal contents were suspended in 10 volumes of PBS. The samples were homogenized in an assay buffer using a bead-beating method. After centrifugation, urease activity in the clear lysate solutions was determined by using a urease activity assay kit (MAK120, Sigma). A standard curve was constructed for each experiment using a series of ammonium chloride standards (MAK120C, Sigma). The amount of urease enzymatic activity in the samples was calculated according to the manufacturer's instructions.

Ammonia and urea measurements
The ammonia levels in the rumen and ileum of the adult Hanwoo were determined using the urease activity assay kit as described above. Ammonia levels in the mouse serum were determined using an ammonia assay kit (AA0100, Sigma), according to the manufacturer's instructions. A standard curve was constructed for each experiment using    The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.
The data were analyzed by using the unpaired Student's t-test (b, d, and e).

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. Additional_file_3_Supple_results.pdf Additional_file_1_Supple_Figs.pdf Additional_file_2_Supple_Tables.pdf