{"paper_id":"3dc0874a-c047-4961-b419-d8040595da89","body_text":"Characteristics of Gut Microbiota in Captive Asian Elephants (Elephas maximus) from Infant to Elderly | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characteristics of Gut Microbiota in Captive Asian Elephants (Elephas maximus) from Infant to Elderly Sarisa Klinhom, Sirawit Sriwichaiin, Sasiwan Kerdphoo, Jaruwan Khonmee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3241323/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Dec, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Gut microbiota play an important role in the health and disease in captive Asian elephants, however, its characteristics at each stage of life have not been thoroughly investigated. This study, therefore, aimed to characterize the profiles of the gut microbiota of captive Asian elephants from infants to the elderly. The diversity of the gut microbiota was lowest in infants, stable during adulthood, and slightly decreased in the geriatric period. Gut microbiota were identified by 16S rRNA sequencing from the feces of captive Asian elephants with varying age groups, including infant calves, suckling calves, weaned calves, subadult and adult elephants, and geriatric elephants. The gut microbiota of the infant elephants was dominated by milk-fermenting taxa. The fiber-fermenting taxa emerged in suckling elephants. The stabilization of gut microbiota profiles has been observed after weaning until the adult period. However, the composition of the gut microbiota was found to change again in geriatric elephants. Understanding of the composition of the gut microbiota of captive Asian elephants at each stage of life could be beneficial to support good health during each period of the elephant's lifespan. Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Zoology gut microbiome elephants captive age nutrition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Asian elephants (Elephas maximus) , originally from the wild, have been captured for use and domesticated under human care for thousands of years. Being both hindgut fermenters and megaherbivores, the colon, or specifically, the caecum, serves as the primary fermentation chamber crucial to the good health status of the elephant. Seventy percent of the energy intake of the elephant is from the polysaccharide breakdown, which is mainly processed by the large community of microorganisms residing in the colon or caecum, widely known as the ‘gut microbiota’ 1 . The gut microbiota contributes to gastrointestinal (GI) physiology by maintaining the structural integrity of the mucosal barrier, protecting against pathogens, and developing immune functions all of which have impact on host health 2 , 3 . The gut microbiota has been shown to facilitate the fermentation of a hitherto indigestible plant diet to short-chain fatty acids (SCFAs) that provide the body with maximum energy and essential nutrients from the dietary fiber, which are crucial for herbivores 4 . The composition of the gut microbiota is influenced by several factors, including diet and age 5 . The initial colonization of the mammalian gut by the microbes occurs shortly after birth 6 , 7 . Subsequently, the diversity and composition of gut microbiota in herbivores are subject to change depending on the developmental period and environmental factors. During the growth and development of the animal, each stage of growth requires different types of nutrients/food consumption, for example during progression from breastfeeding to roughage in elephants, resulting in changes in the diversity of gut microbiota in each growth period 8 . However, information about the transition of gut microbiota profiles of elephants in different periods of growth and development is still limited. The deviation of the composition of gut microbiota from normal conditions or ‘gut dysbiosis’ might impair the normal GI physiology and might greatly influent the health status of elephants which mainly relies on the functioning of the gut microbiota. Gut dysbiosis in animals is associated with GI disorders, including GI stasis, indigestion, constipation, enteritis, colic, and diarrhea 9 – 11 . Notably, GI disorders are also one of the most common disorders found in captive elephants, resulting in the illness or even death of the elephants 12 . Therefore, a study into the imbalance of gut microbiota in elephants might increase our understanding of these conditions. However, this is difficult as the characteristics of the gut microbiota of healthy elephants as a reference for what purports as normal composition have not been thoroughly investigated. A few studies about gut microbiota had been performed in captive 13 – 15 and wild Asian elephants 16 and also in African species 17 , 18 . However, those previous studies were mainly conducted with limited samples in zoos. Interestingly, the majority of captive elephants in Thailand are held in semi-free-ranging conditions, which differ in nutrition, water, and environmental factors from the conditions in a zoo. Therefore, the present study aims to characterize the profiles of gut microbiota of different ages of Asian captive elephants in Northern Thailand by using the next-generation sequencing (NGS) technique. In addition, the associations between gut microbiota profiles in healthy adult elephants with several blood parameters, including hematological parameters, liver and kidney functions, and lipid profile, were also investigated. Understanding the normal profiles of gut microbiota at each stage of life would help to determine the factors associated with health issues in Asian elephants in the future. Results General characteristics and details of food and water consumption of elephants in each age group All elephants (n = 134) in this study were categorized into 5 groups based on nutrition and growth pattern, including: 1) infant calves, 2) suckling calves, 3) weaned calves, 4) subadult and adult elephants, and 5) geriatric elephants. The demographic data are summarized in Table 1 . The body condition scores in all groups were within the normal range without any significant differences between the groups. The information regarding food and water consumption in each group is presented in Table 1 . For the first four months of their lives, maternal milk was the major source of nutrition for infant calves. Suckling calves were still consuming breast milk but also began to consume solid food. Weaned calves and subadult and adult elephants were fed with Napier grass ( Pennisetum purpureum ) and corn stalks ( Zea Mays L. ) as their main roughage. Geriatric elephants were fed with roughage that had been chopped into small pieces and mixed with pellets. Elephant concentrate pellets (Erawan®, CPF, Thailand) were given as a daily supplement to the geriatric elephants and some suckling and weaned calves. Seasonal fruit supplements including banana, sugar cane, mango, tamarind, and watermelon were provided to all elephants except newborn calves. Elephants could access the water sources ad libitum or as given by the mahout, which was 3–4 times a day. Water was sourced from mountain streams, rivers, or ponds (Table 1 ). Table 1 General characteristics and details of food and water consumption of healthy captive elephants in Northern Thailand categorized by age group N = 134 Infant calves Suckling calves Weaned calves Subadult and adult elephant Geriatric elephants General Information N 6 12 27 78 11 Age (years) (mean ± sd) 0.20 ± 0.00 b, c, d, e 1.58 ± 0.50 a, c, d, e 5.70 ± 1.80 a, b, d, e 31.53 ± 11.70 a, b, c, e 58.90 ± 3.70 a, b, c, d Male : Female 4:2 6:6 12:15 14:64 0:11 Body condition score (mean ± sd) 3.00 ± 0.00 3.08 ± 0.30 3.33 ± 0.60 3.62 ± 0.70 3.18 ± 0.40 Diet Breast milk (%) 100 100 0 0 0 Roughage (%) 0 100 100 100 100 Napier grass (%) 0 33.33 96.30 87.18 45.45 Corn stalk (%) 0 16.67 48.15 67.95 100 Total amount per day (kg) (mean ± sd) - 85.00 ± 25.05 157.96 ± 32.56 163.91 ± 32.87 180.00 ± 21.21 Feeding frequency (time/day) (mean ± sd) - 3.67 ± 0.49 3.85 ± 0.82 4.12 ± 0.77 4.36 ± 0.50 Food pellets (%) 0 41.67 9.52 0 63.64 Amount per day (kg) 0 0.5 1 0 1 Feeding frequency (time/day) 0 1 1 0 1 Supplementary (%) 0 100 88.89 92.31 100 Banana (%) 0 100 66.67 79.49 90.91 Sugar cane (%) 0 100 66.67 79.49 90.91 Mango (%) 0 33.33 33.33 16.67 0 Tamarind (%) 0 100 51.85 51.28 54.55 Watermelon (%) 0 25.00 3.70 12.82 54.55 Amount per week (kg) (mean ± sd) 0 35.00 ± 30.90 26.67 ± 33.28 78.46 ± 62.15 63.64 ± 60.54 Feeding frequency (time/week) (mean ± sd) 0 3.50 ± 3.09 1.5 ± 1.69 4.25 ± 3.01 3.18 ± 3.027 Water consumption Source of water River (%) 0 58.33 70.37 50 63.64 Pond (%) 0 33.33 59.26 57.69 36.36 Mountain water supply (%) 100 50 88.89 80.78 63.63 Tap water (%) 0 0 11.11 19.23 36.36 Frequency (time/day) (mean ± sd) 3 3 3.11 ± 0.32 3.19 ± 0.40 3.36 ± 0.50 The data are presented as mean ± standard deviation or percentage as appropriate. a p<0.05 vs infant calves, b p<0.05 vs suckling calves, c p<0.05 vs weaned calves, d p<0.05 vs subadult and adult elephants, e p<0.05 vs geriatric elephants for Bonferroni multiple-comparison correction test Alpha diversity and beta diversity were significantly different among age groups. The diversity of gut microbiota in each age group were determined and are presented in terms of alpha diversity in Fig. 1 . Infant calves exhibited the lowest fecal microbial alpha diversity when compared to other groups (Fig. 1 A-D). In addition, geriatric elephants showed a significant decrease in alpha diversity when compared to subadult and adult elephants, as evidenced by Pielou’s evenness (Fig. 1 A) and Shannon’s index (Fig. 1 D). Notably, the alpha diversity of weaned calves was significantly higher than those of subadult and adult elephants and geriatric elephants as shown in observed feature (Fig. 1 B) and by Shannon’s index (Fig. 1 D), The similarities of the composition of gut microbiota between groups were calculated based on beta diversity and are presented as Principle coordinate analysis (PCoA) plots (Fig. 1 .). PCoA plots based on Bray-Curtis, Jaccard, unweighted and weighted UniFrac revealed that the gut microbiome of the elephants showed distinct gut microbiotal composition across the different age ranges ( p-value of pseudo-F in pairwise PERMANOVA test < 0.05 in pairwise comparison of all groups ) ( Fig. 1 E-H ) . The PCoA plots showed the patterns associated with the age group of elephants. The groups of elephants with adjacent ages had a closed distance between the groups in the PCoA plot, suggesting that there was a similar composition of the gut microbiota of the elephants across the adjacent ages. These patterns were observed in PCoA plots following analysis using Bray-Curtis, Jaccard, and unweighted UniFrac distances ( Fig. 1 E-G ) . Taxonomic composition of bacterial populations in different age groups of elephants The taxonomy of gut microbiota was identified based on the data of hypervariable region V3-V4 of the 16s rRNA gene. Forty-three phyla and 1,134 genera of gut microflora were identified within total elephant fecal samples. The relative abundance at the phylum level of gut microbiota in all age groups are shown in Fig. 2 . The dominant bacterial phyla in fecal samples of all groups of elephants were Firmicutes followed by Bacteroidetes and Actinobacteria. In the infant calves, Bacteroidetes and Spirochete showed less relative abundance than those in other age groups. On the other hand, greater relative abundance of Actinobacteria and Euryarchaeota were detected in the infant calves. The composition of the elephant's gut microbiota changed markedly from infants to suckling calves, and from subadult and adult to geriatric elephants. The relative abundance of fecal microbiota at the phylum level was shown to be only slightly altered from suckling calves to weaned calves and subadult and adult elephants. According to the information from human guts, the microbiota is practically stable in healthy adults 19 . This phenomenon was similar to Asian elephants, which reach maturity between 10–14 years of age 20 . Therefore, in this study, we used subadult and adult elephants as reference for the composition of gut microbiota in healthy elephants for further analyses. Infant elephants showed a distinctly different composition of gut microbiota, when compared with subadult and adult elephants The analysis of differential abundance was conducted by using ANCOM-BC. The results of significantly different taxa of fecal microbiota among age groups with top 10 of log fold changes are presented in Fig. 3 . All significantly different taxa are shown in Supplementary Table 1. We used subadult and adult elephants as a reference for each comparison. In the infant calves, the abundance of the phyla Euryarchaeota, Actinobacteriota, Verrucomicrobiota, Proteobacteria, and Desulfobacterota were higher than that of subadult and adult elephants while the phyla Cyanobacteria, SAR324_clade (Marine_group_B), Spirochaetota, Armatimonadota, and Elusimicrobiota were found to be lower (Fig. 3 ). At the family and genus levels, the taxa from the families Bifidobacteriaceae, Akkermansiaceae, Villonellaceae, Bacteroidaceae , and Butyricioccaceae , together with the genus Bifidobacterium spp., UCG-008 spp., Olsenella spp., Akkermansia spp ., and Bacteroides spp. in infant calves were higher than those of subadult and adult elephants (Fig. 3 ). On the other hand, the families Spirochaetaceae , p-251-o5 , Paludibacteraceae , Planococcaceae , and Gastranaerophilales , and the genus Solibacillus , Agathobacter , XPB1014 group in the families Lachnospiraceae , and Treponema , and an uncultured genus in Paludibacteraceae were lower in infant calves when compared to subadult and adult elephants (Fig. 3 ). Suckling and weaned elephants showed slightly different gut microbiota composition when compared with adult elephants When compared to the differences between the infant calves and subadult and adult elephants, both the number of statistically distinct taxa and their magnitude were much smaller between the suckling and weaned calves and subadult and adult elephants. At the phylum level, only Euryarchaeota was higher in suckling calves and Planctomycetota, Euryarchaeota, and Desulfobacterota were higher in weaned calves when compared to subadult and adult elephants (Fig. 3 ). On the other hand, Synergistota and Fibrobacterota were lower in weaned calves while Cyanobacteria and SAR324_clade (Marine_group_B) were decreased in both suckling and weaned calves (Fig. 3 ). At the family level, Bifidobacteriaceae , Veillonellaceae , and Bacterroidaceae were higher in both suckling and weaned calves whereas Tannerellaceae and Butyricicoccaceae were higher only in sucking calves, and Atopobiaceae and Micrococcaceae were higher only in weaned calves (Fig. 3 ). However, the families Paracaedibacteriaceae , Clostridiaceae , COB_P4-1_termite_group , Endomicrobiaceae , and Leuconostocaceae in suckling calves, and Nocardiaceae , Clostridiaceae , Leuconostocaceae , Desulfotomaculales , and MVP-15 in weaned calves were lower than in those subadult and adult elephants (Fig. 3 ). At the genus level, Bifidobacterium , Olsenella , and Lachnospiraceae_NK3A20_group were higher in sucking and weaned calves while Bacteroides and UCG-008 were higher only in suckling calves, and the [Ruminococcus]_gauvreauii_group and Syntrophococcus were higher only in weaned calves (Fig. 3 ). Meanwhile, the genera Enterobacter , Endomicrobium , Weissella , Clostridium_sensu_stricto_13 , and Sarcina in suckling calves, and [Eubacterium]_oxidoreducens_group , Terrisporobacter , Enterobacter , Weissella , and MVP-15 in weaned calves were lower those in subadult and adult elephants (Fig. 3 ). Geriatric elephants showed distinct gut microbiota, when compared with adult elephants. Geriatric elephants showed a distinct pattern of abundance within the gut microbiota when compared to infant, suckling, and weaned calves. At the phylum level, Chloroflexi, and Euryarchaeota were increased while Cyanobateria, Desulfobacteria, Fibrobacterota, and Bacteroidota were decreased when compared to subadult and adult elephants (Fig. 3 ). At the family level, Lactobacillaceae, Xanthobacteraceae, Pseudomonadaceae, Caloramatoraceae , and Micrococcaceae were more abundant than in subadult and adult elephants. Meanwhile, the taxa from families Proteobacteria_Rickettsiales_uncultured , Desulfovibrionaceae, Bacteroidetes_BD2-2, Paludibacteraceae , and Planococcaceae were lower in composition when compared to those of subadult and adult elephants (Fig. 3 ). At the genus level, Lactococcus , Lactobacillus , Garicola , Succinivibrio , and UBA1819 were higher in geriatric elephants while Schwartzia , Acinetobacter , Lysinibacillus , Solibacillus , and Bacteroidetes_BD2-2 were lower in geriatric elephants when compared to subadult and adult elephants (Fig. 3 ). Major gut microbiota composition in subadult and adult elephants The relative abundance of the taxa in the family and genus level of each elephant in the subadult and adult age group are shown in Fig. 4 . At the family level of subadult and adult elephants, Lachnospiraceae was the most dominant family, followed by Oscillospiraceae, Clostridiaceae, Christrensenellacease, Anaerovoraceae , and Rikenellaceae (Fig. 4 A). The beneficial bacteria were present in fecal samples of subadult and adult elephants, including the fiber-digesting taxa at Phylum level of Firmicutes, Bacteroides, Spirochaetota, and Actobacteriota, also including Lachnospiraceae; NA, Christrensenellacease_R-7_group, NK4A214_group, Sarcina, Lachnospiraceae_XPB1014_group, [Eubacterium]_coprostanoligunes_group, UCG-005, Planococcaceae; NA, Family_XIII_AD3011_group, Lachnospiraceae_AC2044_group, Ruminococcus, Solibacillus, and Saccharofermentans , Rikenellaceae_RC9_gut_group, p-251-o5, F082, and Prevotellaceae_UCG-003 , Treponema , and Olsenella. In addition, Archaea Methanobrevibacter of class Methanobacteria were found to be present in subadult and adult elephants (Fig. 4 B). Blood parameters of subadult and adult elephant To further understand the association between gut microbiota and the health of elephants, the associations between gut microbiota composition and blood parameters were determined. All blood parameters are presented in Table 2 . The hematological and biochemical parameters of subadult and adult elephants were within the normal range. The serum lipid profiles including triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) were all determined to be within the normal range, when compared to the previous study by Norkaew, et al. 21 . In these analyses, the blood parameters were used as the numerical outcome in the ANCOM-BC analysis, and the results with a log-fold change of gut microbiota greater than 0.5 are shown in Fig. 5 . The results showed that several gut microbiota of subadult and adult elephants showed a correlation with blood parameters including RBC count, total protein, and serum albumin. Table 2 Characteristics of blood parameters in subadult and adult elephants. Parameters Mean ± standard deviation Pack cell volume (%) 35 ± 4 Hemoglobin (g/dl) 12.69 ± 1.58 RBC count (×10 6 cells/µl) 2.88 ± 0.4 MCV (fl) 123 ± 6 MCHC (g/dl) 35.9 ± 0.75 WBC count (cells/µl) 12005 ± 2372 Segmented neutrophil (cells/µl) 2690 ± 898 Lymphocyte (cells/µl) 6193 ± 1805 Monocyte (cells/µl) 2725 ± 1333 Eosinophil (cells/µl) 339 ± 218 Basophil (cells/µl) 202 ± 95 Platelet count (×10 3 cells/µl) 353 ± 72 BUN (mg/dl) 10 ± 3 Creatinine (mg/dl) 1.47 ± 0.28 AST (U/L) 17 ± 6 ALT (U/L) 2 ± 1 ALP (U/L) 97 ± 49 Total serum protein (g/dl) 8.55 ± 0.63 Albumin (g/dl) 3.24 ± 0.35 CK(U/L) 166 ± 69 TC (mg/dl) 45 ± 10 TG (mg/dl) 23 ± 14 HDL (mg/dl) 12 ± 2 LDL (mg/dl) 29 ± 8 Abbreviation s : ALT, Alanine transaminase; AST, Aspartate transaminase; BUN, Blood urea nitrogen; CK, Creatine kinase; HDL, High density lipoprotein; LDL, Low density lipoprotein; MCHC, Mean corpuscular hemoglobin concentration; MCV, Mean corpuscular volume; RBC, Red blood cell; TC, Total cholesterol; TG, Triglyceride; WBC, White blood cell. The number of RBC showed a positive association with orders Pedosphaerales and Victivallales, families Pedosphaeraceae and Muribaculaceae , and genera DEV114 from the family Pedosphaeraceae and UCG-004 from the family Erysipelatoclostridiaceae (Fig. 5 ). However, the phyla SAR324_clade(Marine_group_B) and Euryarchaeota, classes Methanobacteria, Desulfotomaculla, and SAR324_clade(Marine_group_B), and orders Methanobacteriales and Desulfotomaculales showed a negative association with RBC count (Fig. 5 ). The total protein content showed a negative association with the genus Lactobacillus and the family Lactobacillaceae (Fig. 5 ). The genera Mycoplasma with corresponding family Mycoplasmataceae and order Mycoplasmatales, Mailhella with corresponding family Desulfovibrionaceae , order Desulfovibrionales, and class Desulfovibrionia, DEV114 with corresponding family Pedosphaeraceae , order Pedosphaerales, and class Verrucomicrobiae, Muribaculaceae with corresponding family Muribaculaceae , and Prevotella were negatively associated with the level of serum albumin (Fig. 5 ). The family p-251-o5 , Atopobiaceae , Paludibacteraceae , and Methanomethylophilaceae from order Methanomassiliicoccales, class Thermoplasmata also showed a negative association with plasma albumin level (Fig. 5 ). However, at the phylum level, Chloroflexi showed a positive association with serum albumin (Fig. 5 ). The other blood parameters are listed in Table 2 . The relationships of less significant association with the composition of gut microbiota and the details are shown in Supplementary Table 2. Discussion This study is the first study to characterize the gut microbial community in Asian captive elephants of different ages. In addition, the association between the gut microbiota and blood parameters was analyzed. The major findings of this study include: 1) the diversity and the composition of gut microbiota were different between age groups and significant differences were observed between the infant calves and suckling calves, and between adult and geriatric elephants; 2) the composition of the gut microbiota of subadult and adult elephants was stable with a high abundance of Firmicutes, followed by Bacteroidetes, and Actinobacteria, and 3) several gut microbiota in adult elephants were associated with blood parameters such as RBC count, total protein, and albumin. Our findings suggest that gut microbial composition in elephants might be dependent on the types of food consumed as has been observed in other animals. Infancy is a critical time for the gut microbiome to grow since it shapes the host immune system and stabilizes the metabolic condition 22 , 23 . Infant calves consume milk as their main source of energy, and the beta diversity analysis showed that the samples in infant calves were separately clustered from the rest of the older elephant groups in PCoA plots and the ANCOM-BC analysis also revealed that the composition of the gut microbiota composition was distinct. Milk fermenting bacteria including the Bifidobacteriaceae family together with Bifidobacterium spp were significantly higher as regards abundance in infant calves. This finding suggested that maternal milk was the key component determining the microbial composition of the gut in the infants enabling the newborn's intestines to adapt to the environment. Oligosaccharides and milk glycans are some of the major solid bioactive components in maternal milk; however, newborns were unable to digest and absorb them. When they passed through the gut lumen, these compounds have been served as prebiotics by shaping and stabilizing the microbiota in early life 24 . In the milk of Asian elephants, 40% of milk carbohydrate content was found to be oligosaccharides, a higher concentration than in other animal milk 25 . Milk oligosaccharides were mainly utilized by glycan-degrading enzymes, encoded by the genome of an infant-associated Bifidobacterium as well as other bacterial species within Bacteroides spp . found to be in higher abundance in both infant and suckling elephants; and Akkermansia spp . 26 found in higher abundance only in infant elephants. It is worth noting that the milk utilizing bacteria such as Bifidobacteriaceae were still higher in suckling and weaned calves when compared to adult elephants. However, during the pre-weaning period, the composition of the gut microbiota is very dynamic and unstable 27 , 28 . As elephant calves are completely weaned at age 3 years old, the abundance of milk utilizer bacteria declined relative to earlier infantile and suckling stages. This finding indicated that the milk from the mother elephants had a significant influence on microbial composition throughout early life. Changes in the fecal microbial composition after the infantile life of elephants were observed in suckling calves or at the age of around four months due to the beginning of plant-based diet consumption. These elephants gradually switched from consuming milk to a variety of high-fiber diets, until completely weaned. Consequently, the gut microbiota of suckling and weaned calves were approaching the adult-like pattern as demonstrated by the overlapping of the composition of the gut microbiota of sucking and weaned calves with adult elephants as shown in the beta diversity analysis. As expected, the fiber-fermenting bacterial families including Spirochaetaceae, p-251-o5, Paludibacteraceae, Planococcaceae , and Gastranaerophilales which were low in abundance in the infant elephants, became no significantly different between suckling and weaned calves and adult elephants. Furthermore, there was a higher abundance of SCFA-producing bacteria including genera Lachnospiraceae_NK3A20_group in both suckling and weaned calves, and Ruminococcus_gauvreauii_group and Syntrophococcus in weaned calves. One study described evidence of a correlation between the genera Lachnospiraceae_NK3A20_ and Ruminococcus_gauvreauii_groups with the concentration of volatile fatty acids and levels of microbial crude protein in the rumen of pre-weaning lambs 29 . In addition to the introduction of forage feeding in herbivores, coprophagia events had an impact on the foal microbiome which promoted the establishment of suitable gut microbial flora in young animals optimizing gut health during the pre-weaning period 8 , 30 . Coprophagic behavior was normally observed in juvenile elephants in the first 4–6 months of life 31 . Conceivably, the introduction of a high-fiber diet and coprophagia might shape the composition of the gut microbiota instigating an adult-like profile even though young elephants were still suckling. In this study, the adult elephant’s main roughage consisted of Napier grass and corn stalks, which were reported to contain 42.6% 32 and 30.5–36.2% of dry weight 33 , respectively. As expected, a significant amount of fibrolytic bacteria were observed in adult elephants. The presence of Lachnospiraceae was necessary for the breakdown of complex carbohydrates in grass 34 , 35 . In addition, it has been shown that Lachnospiraceae are the main producers of SCFAs, especially butyrate which serves as a source of nutrients and growth factors for healthy gut epithelia and also contribute to the prevention of inflammation 36 . The family Oscillospiraceae together with the genera NK4A214_group, [Eubacterium]_coprostanoligenes_group , and UCG-055 were likely to be able to utilize intestinal host glycans and produce the important SCFAs such as butyrate and their metabolites 37 . The genus Sarcina of the family Clostridiaceae , a cellulose-producing bacteria with carbohydrate fermentative metabolism, was also dominant in adult elephant feces. Christensenellaceae R-7_ group , a genus in the family Christrensenellacease which presented as a prominent bacterium genus in adult elephant feces, was reported to be associated with the host’s body mass index (BMI) and leanness in humans 38 . Other genera of microorganisms that were found predominantly in the fecal matter of adult elephants, including Rikenellaceae_RC9_gut_group, Treponema, Ruminococcus, Saccharofermentans , and Prevotella were known to play an important role in fiber degradation. The subadult and adult elephants in this study were healthy with normal body condition scores. No illnesses had been reported 6 months before the investigation. In the mature period of life, several gut bacteria were found to show significant correlations with RBC count, total protein, and albumin. Information regarding these correlations was scarce in elephant research and could be found only in previous studies of other species, such as mice, pigs, and humans. Albumin is the most abundant circulating protein found in plasma. Low albumin levels have usually been found to be as a result of liver disease, kidney disease, heart failure, malnutrition, or vitamin deficiency. We found genera Mycoplasma and Prevotella had negative associations with the level of serum albumin. According to a previous study, Mycoplasma presumably plays an important role in the etiology and pathology of primary biliary cirrhosis in humans 39 . Prevotella is believed to benefit host health as it can produce a significant amount of SCFA in pig model 40 , and sufficient to have anti-inflammatory effects in mouse model 41 . Nonetheless, Prevotella was reported to be associated with chronic inflammatory conditions and was found to have an increase in abundance in patients with chronic liver disease with advanced fibrosis 42 , 43 . We also found the bacterial family Lactobacillaceae , in particular the genus Lactobacillus , was negatively associated with total protein in subadult and adult elephants. Lactobacillus is recognized as a beneficial microbe in humans and animals due to its involvement in immunity, metabolism, and maintaining the gut microbiota ecosystem 44 . In this study, we are unable to determine whether changes in the blood parameters were directly caused by changes in the gut microbiome or as a consequence of pathological changes. Future mechanistic studies into associations between bacterial taxa and total protein, albumin, and RBC count are warranted to delineate their significance and implications in elephant health issues. The geriatric elephants showed a distinct gut microbiota profile as demonstrated by decreasing alpha diversity and the separated cluster of the microbes in the geriatric elephants from the other age groups in beta diversity analysis. The differential abundance analysis also revealed a higher abundance of the family Lactobacillaceae and the genus Lactobacillus in geriatric elephants when compared to adult elephants. Lactobacillus has been identified as one of the major bacterial taxa in the healthy horse 45 . Interestingly, overconsumption of high-starch diets and oligofructose has been associated with the overgrowth of Lactobacillus and has been observed in horses with colic frequently leading to the development of laminitis 45 , 46 . Consumption of elephant concentrate pellets by most geriatric elephants, which contained crude protein 11.6% and nitrogen-free extract 46.4% of dry matter 32 , might be associated with the higher abundance of Lactobacillus than in the subadult and adult group. Furthermore, the archaea class Methanobacteria, after having dipped in the middle age group, shifted to an increase again in geriatric elephants. This was in accordance with elderly macaques, which indicated a positive association of these methanogens with host aging 47 , and a high abundance of centenarian human gut microbiota 48 . The decline in gut microbiota diversity in geriatric elephants might be due to the loss of the last molar teeth (M6) at the age of 50 leading to poor mechanical and hence chemical digestion 49 and resulting in rough feces. The resulting age-related gut dysbiosis, as indicated by decreased bacterial community richness, could affect the host's health and lifespan 50 . Gut dysbiosis can trigger the innate immune response and induce chronic low-grade inflammation, leading to many age-related degenerative pathologies and unhealthy aging processes 22 . Therefore, further investigation into gut dysbiosis and potential variation in diet in geriatric elephants might be one of the pivotal points to improving the quality of life of old elephants. This study demonstrated the composition of gut microbiota in captive Asian elephants by using the 16s rRNA data from a number of elephants which could provide important information on the composition and diversity of these gut microbiota. To increase the strength of the findings of this study, some limitations need to be addressed in further research. Despite a large number of subadult and adult elephants in the sample, the other age groups especially the infant group contained low numbers. Extending the range of data acquisition or collaborating with other elephant camps might increase the number of young elephants. The definition of age group in this study was mainly based on age range and other factors might be more suitable when categorizing the groups based on the biology of elephants and should be included in further study. In addition, this study includes only healthy elephants in all age groups, further research that includes both normal elephants and elephants with certain diseases might reveal more about the roles of gut microbiota in the health and biology of elephants. Conclusion Our findings indicate that the diversity and relative abundance of gut microbiota in captive elephants were affected by age and the diet consumed. This knowledge will inform more appropriate food management to facilitate the development of more beneficial bacteria for each elephant at the different stages of their life. The elephants in this study, all lived in similar environments and were raised and handled similarly. In the future, a comparison of zoo husbandry conditions and semi free-ranging environments, as well as differences in food management, should be further investigated to promote the most effective management and nutrition for a healthy gut in captive elephants. Methods This study was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand (FVM-ACUC; R3/2563) and all experiments were performed in accordance with relevant guidelines and regulations. All methods used in this study are reported in accordance with ARRIVE guidelines. Fecal samples of approximately 50 grams, were single collected from 134 healthy captive elephants aged between two months and 67 years old working in tourist camps in Chiang Mai and Lampang provinces, during January – December 2020. All elephants in this study were divided into 5 groups based on nutrition and growth pattern including infant calves (1 day – 4 months old, food mainly milk based), suckling calves (4 months – 3 years old, food based on milk and grass), weaned calves (3–10 years old, food based on grass), subadult and adult elephants (10–55 years old, food based on grass), and geriatric elephants (more than 55 years old, food based on chopped grass mixed with pellets). The group sizes in each category were determined based on the existing elephant population in the Northern region. Furthermore, a significant number of samples of subadult and adult elephant groups were collected to ensure representation across a broad range of ages. Individual body condition score was estimated scored Morfeld, et al. which ranges from 1 to 5 (where 1 signifies the thinnest and 5 signifies the fattest), with an optimal score of 3 (Table 1 ) 51 . All the elephants had been housed and had worked in the camp for over 6 months, with elephant calves born and raised in the original camp without relocation. Calves and juveniles were housed together with their mother without interacting with the tourists. The adult elephants routinely worked with tourists, trekking, participating in elephant shows, or for observation 52 between 8:00 and 15:00 for no more than 5 hours per day. The geriatric elephants were not involved in any physical activities with tourists. The information on types of work, habitat use, food intake, and foraging behavior was collected individually. The subjects were verified as being clinically healthy by experienced elephant veterinarians, based on history and clinical examination, with no reports of GI issues or antibiotic or drug administration for at least six months before the beginning of the study. Blood collection 10 ml blood samples were collected from each elephant in the subadult and adult groups from an ear vein. Blood samples were submitted to the Veterinary Diagnostic Laboratory of Faculty of Veterinary Medicine, Chiang Mai University, Thailand within 24 h of collection. Blood samples were analyzed using the Auto Haematology Analyzer (Mindray BC5300, Mindray Medical, Thailand) and using the Biochemical Analyzer Vitalab Flexor XL (Vital Scientific NV, Netherlands). Hematology parameters i.e. packed cell volume (PCV), hemoglobin, RBC count, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), White Blood Cell count (WBC count), and platelet count, and biochemical parameters i.e. Blood Urea Nitrogen (BUN), creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), creatine kinase (CK), total serum protein and albumin were analyzed. Serum lipids were quantified using a Mindray BS Series analyzer (Mindray BS-380, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.), total cholesterol was measured using a cholesterol oxidase-peroxidase (CHOD-POD) method, and triglycerides were measured using a glycerokinase peroxidase-peroxidase (GPO-POD) method. Fecal samples collection and analysis Fresh fecal samples (approximately 50 grams) were collected directly from the rectum or immediately after defecation and stored at -20 o C within 3 hours of collection until DNA extraction. After the samples were thawed, indigestible roughage such as grass, leaves, fruit seeds, and peel were separated from the stool contents. Up to 250 mg of prepared stool was used for DNA extraction. Bacterial genomic DNA was extracted from elephant fecal pellets using a commercial genomic DNA isolation kit (QIAamp PowerFecal Pro DNA Kit (QIAGEN), Germany). The extracted bacterial genomic DNA were exposed to an amplification process of hypervariable region V3-V4 of 16s rRNA and then underwent NGS methods. DNA amplification, data quality control methods, and sequencing were conducted by Novogene Inc (Singapore City, Singapore). A Double-blind study with samples categorization was used to avoid bias. Sequencing data analysis DNA sequence data from NGS were processed using the Quantitative Insights Into Microbial Ecology 2 (QIIME2-2021.4) open-source software 53 . The raw datasets containing pair-ended reads and quality scores were merged, denoised, trimmed according to quality scores, and assigned to amplicon sequence variants (ASV) by using q2-dada2 plugin 54 . A feature table including the number of each ASV per sample was also generated 55 . The ASVs were aligned with mafft and used to generate a phylogenetic tree for further analysis 56 , 57 . The diversity analyses were conducted with rarefication at the sequencing depth of 56,700 as this is the maximum number to retain all samples in this study 58 . The alpha diversity including observed species, Pielou’s evenness 59 , Faith’s phylogenetic diversity 60 , and Shannon’s index 61 were calculated. The beta diversity including Bray Curtis, Jaccard, unweighted UniFrac 62 , and weighted UniFrac 63 distance matrices were analyzed and were illustrated as Principal Coordinate Analysis (PCoA). The taxonomy was assigned to ASVs by using q2‐feature‐classifier classify‐sklearn naïve Bayes taxonomy classifier 64 by using the data reference from SILVA database version 138 65 . For visualization of population structure and relative abundance, taxonomical bar plots indicating relative abundance at phylum, class, and genus levels of each sample from the different age groups were generated. The differences in taxa abundance between categories and the association between taxa and blood parameters were estimated with a statistical framework: analysis of composition of microbiomes with Bias Correction (ANCOM-BC) 66 . The data visualization was conducted through R version 4.1.1 by using package qiime2R. Data availability The datasets generated and/or analyzed in this study are available in the NCBI sequence read archive under the Accession Number PRJNA1005601. Declarations Data availability The datasets generated and/or analyzed in this study are available in the NCBI sequence read archive under the Accession Number PRJNA1005601. Acknowledgments We would like to thank the Center of Excellence in Cardiac Electrophysiology Research, Faculty of Medicine, the Center of Elephant and Wildlife Health Animal Hospital, Faculty of Veterinary Medicine, and the Erawan HPC Project, Information Technology Service Center (ITSC), Chiang Mai University, Chiang Mai, Thailand for all their support. We also wish to register our appreciation to the owners of the elephant camps, their managers, and the mahouts for providing samples and data. Author contributions S.Kl., N.C., S.C.C, and C.T conceived the idea and designed the research. S.Kl. and S.Ke. carried out the preparation of material and measurements. S.Kl. and S.S. analyzed and investigated the data. S.Kl and S.S. wrote the original draft menuscript. S.Kl., S.S., N.C., S.C.C., and C.T. reviewed and edited the menuscript. N.C., S.C.C., and C.T. were responsible for the funding acquisition. J.K., N.C., S.C.C, and C.T. supervised the research. Fundings This study was supported by the Thailand Research Fund (C.T); the CMU Presidential Scholarship and Chiang Mai University grant number 59/2565 (S.KI.); a Senior Research Scholar Grant from the National Research Council of Thailand (S.C.C.); the NSTDA Research Chair Grant from the National Science and Technology Development Agency Thailand (N.C.); and a Chiang Mai University Center of Excellence Award (N.C., C.T.) Competing interests The authors have no conflicts of interest to declare that are relevant to the content of this article. Supplementary Information The supplementary materials will be submitted with the manuscript. Gut microbiota sequences of elephants in this study are on requested to the co-responding authors. References Flint, H. J., Bayer, E. A., Rincon, M. 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Supplementary Files KlinhomSupplementaryTable1.xlsx KlinhomSupplementaryTable2.xlsx Cite Share Download PDF Status: Published Journal Publication published 27 Dec, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 13 Oct, 2023 Reviews received at journal 15 Sep, 2023 Reviewers agreed at journal 08 Sep, 2023 Reviewers invited by journal 08 Sep, 2023 Editor assigned by journal 08 Sep, 2023 Editor invited by journal 17 Aug, 2023 Submission checks completed at journal 17 Aug, 2023 First submitted to journal 07 Aug, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3241323\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":227073174,\"identity\":\"f8860d11-0fc6-4248-9bbd-2c28b49911e6\",\"order_by\":0,\"name\":\"Sarisa Klinhom\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chiang Mai University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sarisa\",\"middleName\":\"\",\"lastName\":\"Klinhom\",\"suffix\":\"\"},{\"id\":227073176,\"identity\":\"7e6c2634-fc1e-4538-9a1a-973b14bd66e0\",\"order_by\":1,\"name\":\"Sirawit Sriwichaiin\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chiang Mai University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sirawit\",\"middleName\":\"\",\"lastName\":\"Sriwichaiin\",\"suffix\":\"\"},{\"id\":227073179,\"identity\":\"7a7a7637-dee0-45ff-bab0-f9c2bda378b1\",\"order_by\":2,\"name\":\"Sasiwan Kerdphoo\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chiang Mai University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sasiwan\",\"middleName\":\"\",\"lastName\":\"Kerdphoo\",\"suffix\":\"\"},{\"id\":227073181,\"identity\":\"713b252d-4e1d-4e30-99b3-341b28ab288e\",\"order_by\":3,\"name\":\"Jaruwan Khonmee\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chiang Mai University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jaruwan\",\"middleName\":\"\",\"lastName\":\"Khonmee\",\"suffix\":\"\"},{\"id\":227073183,\"identity\":\"5354e5b9-80a9-4268-9555-5c5eafb1dc88\",\"order_by\":4,\"name\":\"Nipon Chattipakorn\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chiang Mai University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nipon\",\"middleName\":\"\",\"lastName\":\"Chattipakorn\",\"suffix\":\"\"},{\"id\":227073185,\"identity\":\"0ca311c6-fd4f-463d-9001-1849823bbbb8\",\"order_by\":5,\"name\":\"Siriporn C Chattipakorn\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chiang Mai University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Siriporn\",\"middleName\":\"C\",\"lastName\":\"Chattipakorn\",\"suffix\":\"\"},{\"id\":227073186,\"identity\":\"a121f72f-b6b3-4929-a319-b8272bc6bc06\",\"order_by\":6,\"name\":\"Chatchote Thitaram\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYDACCSBmbAAz2RgSwDTzAZgkGw4tjA1ALUCdzEAtYD1sCVA5ZiK0QKzhMcCrhX928/MHP3fY1PEz8B978PCHXT7/tDMfP91sY5DnbwCKYLPkzjHDxt4zaRKSDczsBgkJyZYzbuduls5tYzCccQAogkWLgUSCYTNj22EJgwPMbBIJCcwGDLdztzEDtTBuADpMAquW9I9gLfYQLfUG8rdznoG02OPWkgO1hQGs5bCBwe0cNpCWRFxaJG7kFM7sbUuTnHGY2UwiIe24geHtNGPpnHMSyWARbCE2I33Dh59tNvz87Y3PJH/YVBvI3U5++DmnzMa2HyiCTQsCMKNZjyEyCkbBKBgFo4B4AADUvVo2FSrgwAAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Chiang Mai University\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chatchote\",\"middleName\":\"\",\"lastName\":\"Thitaram\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-08-07 08:59:27\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3241323/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3241323/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41598-023-50429-1\",\"type\":\"published\",\"date\":\"2023-12-27T15:00:53+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":42031586,\"identity\":\"951437cb-10f0-4f24-bbbb-49b6301622b8\",\"added_by\":\"auto\",\"created_at\":\"2023-08-23 17:52:34\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":58799,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAlpha diversity and beta diversity of gut microbiota of healthy captive elephants in Northern Thailand categorized by age group (Group 1 - infant calves; Group 2 - suckling calves; Group 3 - weaned calves; Group 4 - subadult and adult elephants; Group 5 - geriatric elephants). (A-D) alpha diversity including (A) Pielou’s evenness, (B) Observed feature, (C) Phylogenetic diversity, and (D) Shannon’s index. (E-H) beta diversity including (E) Bray Curtis, (F) Jaccard, (G) unweighted UniFrac, and (H) weighted UniFrac. *p \\u0026lt; 0.05, **p\\u0026lt;0.01, ***p\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/a055fa6120f1c16bfe1adeb5.png\"},{\"id\":42031587,\"identity\":\"bc5c6db8-dfcf-46f3-afbc-744a91b91486\",\"added_by\":\"auto\",\"created_at\":\"2023-08-23 17:52:34\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":108995,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRelative abundance of predominant bacteria at the phylum level of gut microbiota of healthy captive elephants in Northern Thailand categorized by age group (Group 1 - infant calves; Group 2 - suckling calves; Group 3 - weaned calves; Group 4 - subadult and adult elephants; Group 5 - geriatric elephants).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/0ebe0fcf652b5cebae93c4c3.png\"},{\"id\":42031591,\"identity\":\"0640002a-05aa-4ca3-ac4d-2d28ea1e06cb\",\"added_by\":\"auto\",\"created_at\":\"2023-08-23 17:52:34\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":141585,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe differential abundance of gut microbiota of healthy captive elephants in Northern Thailand between age groups (Group 1 - infant calves; Group 2 - suckling calves; Group 3 - weaned calves; Group 4 - subadult and adult elephants; Group 5 - geriatric elephants). The data are presented as Log fold change by using Group 4 as the reference group. Full lists of significant differential abundances are provided in Supplementary Table 1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/c14e15e74f64240eafff3498.png\"},{\"id\":42031589,\"identity\":\"809f242a-17a7-43d2-aac2-d1405b74c7cc\",\"added_by\":\"auto\",\"created_at\":\"2023-08-23 17:52:34\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":161369,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRelative abundance of gut microbiota of subadult and adult healthy captive elephants in Northern Thailand at Family (A) and Genus (B) level. The top 20 taxa are presented as distinct colors.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/44178cc90efeb3177d36dade.png\"},{\"id\":42031588,\"identity\":\"e6e4ec51-4bfa-4f79-8874-92de98171858\",\"added_by\":\"auto\",\"created_at\":\"2023-08-23 17:52:34\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":66514,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eLog fold change of taxa in association with blood parameters in subadult and adult healthy captive elephants in Northern Thailand. Only Log fold changes greater than 0.5 are shown in this figure. Full lists of significant associations are provided in Supplementary Table 2.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/73de8c545c5f65a8a08c5fef.png\"},{\"id\":49028245,\"identity\":\"1e0056f7-1059-4bf5-b515-09ea8651e6ec\",\"added_by\":\"auto\",\"created_at\":\"2024-01-01 15:05:04\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1787608,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/448b63b7-794c-49e5-9a6b-bc97eff0d18d.pdf\"},{\"id\":42031592,\"identity\":\"ec8d15cb-3aa1-4774-be98-c54b136ae658\",\"added_by\":\"auto\",\"created_at\":\"2023-08-23 17:52:34\",\"extension\":\"xlsx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":73890,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"KlinhomSupplementaryTable1.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/b31f7ed2971bfc0ff43a589f.xlsx\"},{\"id\":42031590,\"identity\":\"7d0ab6b4-4b64-4648-bcf9-6e068fabfbbb\",\"added_by\":\"auto\",\"created_at\":\"2023-08-23 17:52:34\",\"extension\":\"xlsx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":87510,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"KlinhomSupplementaryTable2.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3241323/v1/0323c6b75a05d9eadbfbe25e.xlsx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Characteristics of Gut Microbiota in Captive Asian Elephants (Elephas maximus) from Infant to Elderly\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eAsian elephants \\u003cem\\u003e(Elephas maximus)\\u003c/em\\u003e, originally from the wild, have been captured for use and domesticated under human care for thousands of years. Being both hindgut fermenters and megaherbivores, the colon, or specifically, the caecum, serves as the primary fermentation chamber crucial to the good health status of the elephant. Seventy percent of the energy intake of the elephant is from the polysaccharide breakdown, which is mainly processed by the large community of microorganisms residing in the colon or caecum, widely known as the \\u0026lsquo;gut microbiota\\u0026rsquo; \\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe gut microbiota contributes to gastrointestinal (GI) physiology by maintaining the structural integrity of the mucosal barrier, protecting against pathogens, and developing immune functions all of which have impact on host health \\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. The gut microbiota has been shown to facilitate the fermentation of a hitherto indigestible plant diet to short-chain fatty acids (SCFAs) that provide the body with maximum energy and essential nutrients from the dietary fiber, which are crucial for herbivores\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. The composition of the gut microbiota is influenced by several factors, including diet and age\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. The initial colonization of the mammalian gut by the microbes occurs shortly after birth \\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. Subsequently, the diversity and composition of gut microbiota in herbivores are subject to change depending on the developmental period and environmental factors. During the growth and development of the animal, each stage of growth requires different types of nutrients/food consumption, for example during progression from breastfeeding to roughage in elephants, resulting in changes in the diversity of gut microbiota in each growth period \\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. However, information about the transition of gut microbiota profiles of elephants in different periods of growth and development is still limited.\\u003c/p\\u003e \\u003cp\\u003eThe deviation of the composition of gut microbiota from normal conditions or \\u0026lsquo;gut dysbiosis\\u0026rsquo; might impair the normal GI physiology and might greatly influent the health status of elephants which mainly relies on the functioning of the gut microbiota. Gut dysbiosis in animals is associated with GI disorders, including GI stasis, indigestion, constipation, enteritis, colic, and diarrhea \\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR10\\\" citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Notably, GI disorders are also one of the most common disorders found in captive elephants, resulting in the illness or even death of the elephants\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, a study into the imbalance of gut microbiota in elephants might increase our understanding of these conditions. However, this is difficult as the characteristics of the gut microbiota of healthy elephants as a reference for what purports as normal composition have not been thoroughly investigated.\\u003c/p\\u003e \\u003cp\\u003eA few studies about gut microbiota had been performed in captive \\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR14\\\" citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e and wild Asian elephants \\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e and also in African species \\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. However, those previous studies were mainly conducted with limited samples in zoos. Interestingly, the majority of captive elephants in Thailand are held in semi-free-ranging conditions, which differ in nutrition, water, and environmental factors from the conditions in a zoo. Therefore, the present study aims to characterize the profiles of gut microbiota of different ages of Asian captive elephants in Northern Thailand by using the next-generation sequencing (NGS) technique. In addition, the associations between gut microbiota profiles in healthy adult elephants with several blood parameters, including hematological parameters, liver and kidney functions, and lipid profile, were also investigated. Understanding the normal profiles of gut microbiota at each stage of life would help to determine the factors associated with health issues in Asian elephants in the future.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGeneral characteristics and details of food and water consumption of elephants in each age group\\u003c/h2\\u003e \\u003cp\\u003eAll elephants (n\\u0026thinsp;=\\u0026thinsp;134) in this study were categorized into 5 groups based on nutrition and growth pattern, including: 1) infant calves, 2) suckling calves, 3) weaned calves, 4) subadult and adult elephants, and 5) geriatric elephants. The demographic data are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. The body condition scores in all groups were within the normal range without any significant differences between the groups. The information regarding food and water consumption in each group is presented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. For the first four months of their lives, maternal milk was the major source of nutrition for infant calves. Suckling calves were still consuming breast milk but also began to consume solid food. Weaned calves and subadult and adult elephants were fed with Napier grass (\\u003cem\\u003ePennisetum purpureum\\u003c/em\\u003e) and corn stalks (\\u003cem\\u003eZea Mays L.\\u003c/em\\u003e) as their main roughage. Geriatric elephants were fed with roughage that had been chopped into small pieces and mixed with pellets. Elephant concentrate pellets (Erawan\\u0026reg;, CPF, Thailand) were given as a daily supplement to the geriatric elephants and some suckling and weaned calves. Seasonal fruit supplements including banana, sugar cane, mango, tamarind, and watermelon were provided to all elephants except newborn calves. Elephants could access the water sources \\u003cem\\u003ead libitum\\u003c/em\\u003e or as given by the mahout, which was 3\\u0026ndash;4 times a day. Water was sourced from mountain streams, rivers, or ponds (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eGeneral characteristics and details of food and water consumption of healthy captive elephants in Northern Thailand categorized by age group\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eN\\u0026thinsp;=\\u0026thinsp;134\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eInfant calves\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSuckling calves\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eWeaned calves\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eSubadult and adult elephant\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eGeriatric elephants\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGeneral Information\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eN\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e12\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e27\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e78\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e11\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAge (years) (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;sd)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.00\\u003csup\\u003eb, c, d, e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.50\\u003csup\\u003ea, c, d, e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e5.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.80\\u003csup\\u003ea, b, d, e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e31.53\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11.70\\u003csup\\u003ea, b, c, e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e58.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.70\\u003csup\\u003ea, b, c, d\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMale : Female\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4:2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e6:6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e12:15\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e14:64\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0:11\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBody condition score (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;sd)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.00\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.00\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.08\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.60\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.62\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e3.18\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eDiet\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBreast milk \\u003cb\\u003e(%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRoughage \\u003cb\\u003e(%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eNapier grass (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e33.33\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e96.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e87.18\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e45.45\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCorn stalk (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e16.67\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e48.15\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e67.95\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTotal amount per day (kg) (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;sd)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e85.00\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;25.05\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e157.96\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;32.56\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e163.91\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;32.87\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e180.00\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;21.21\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFeeding frequency (time/day) (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;sd)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.67\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.49\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.85\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.82\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e4.12\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.77\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e4.36\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eFood pellets (%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e41.67\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e9.52\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e63.64\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAmount per day (kg)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFeeding frequency (time/day)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eSupplementary (%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e88.89\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e92.31\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBanana (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e66.67\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e79.49\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e90.91\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSugar cane (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e66.67\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e79.49\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e90.91\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMango (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e33.33\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e33.33\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e16.67\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTamarind (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e51.85\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e51.28\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e54.55\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eWatermelon (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e25.00\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e12.82\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e54.55\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAmount per week (kg) (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;sd)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e35.00\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;30.90\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e26.67\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;33.28\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e78.46\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;62.15\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e63.64\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;60.54\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFeeding frequency (time/week) (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;sd)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.09\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.69\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e4.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.01\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e3.18\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.027\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eWater consumption\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSource of water\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRiver (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e58.33\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e70.37\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e63.64\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePond (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e33.33\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e59.26\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e57.69\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e36.36\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMountain water supply (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e88.89\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e80.78\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e63.63\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTap water (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e11.11\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e19.23\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e36.36\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFrequency (time/day) (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;sd)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.11\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.32\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.19\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e3.36\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003ctfoot\\u003e \\u003ctr\\u003e\\u003ctd colspan=\\\"6\\\"\\u003eThe data are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation or percentage as appropriate. \\u003csup\\u003ea\\u003c/sup\\u003ep\\u0026lt;0.05 vs infant calves, \\u003csup\\u003eb\\u003c/sup\\u003ep\\u0026lt;0.05 vs suckling calves, \\u003csup\\u003ec\\u003c/sup\\u003ep\\u0026lt;0.05 vs weaned calves, \\u003csup\\u003ed\\u003c/sup\\u003ep\\u0026lt;0.05 vs subadult and adult elephants, \\u003csup\\u003ee\\u003c/sup\\u003ep\\u0026lt;0.05 vs geriatric elephants for Bonferroni multiple-comparison correction test\\u003c/td\\u003e\\u003c/tr\\u003e \\u003c/tfoot\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAlpha diversity and beta diversity were significantly different among age groups.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe diversity of gut microbiota in each age group were determined and are presented in terms of alpha diversity in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. Infant calves exhibited the lowest fecal microbial alpha diversity when compared to other groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA-D). In addition, geriatric elephants showed a significant decrease in alpha diversity when compared to subadult and adult elephants, as evidenced by Pielou\\u0026rsquo;s evenness (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA) and Shannon\\u0026rsquo;s index (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). Notably, the alpha diversity of weaned calves was significantly higher than those of subadult and adult elephants and geriatric elephants as shown in observed feature (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB) and by Shannon\\u0026rsquo;s index (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD),\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe similarities of the composition of gut microbiota between groups were calculated based on beta diversity and are presented as Principle coordinate analysis (PCoA) plots (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.). PCoA plots based on Bray-Curtis, Jaccard, unweighted and weighted UniFrac revealed that the gut microbiome of the elephants showed distinct gut microbiotal composition across the different age ranges \\u003cem\\u003e(\\u003c/em\\u003ep-value of pseudo-F in pairwise PERMANOVA test\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 in pairwise comparison of all groups\\u003cem\\u003e) (\\u003c/em\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE-H\\u003cem\\u003e)\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe PCoA plots showed the patterns associated with the age group of elephants. The groups of elephants with adjacent ages had a closed distance between the groups in the PCoA plot, suggesting that there was a similar composition of the gut microbiota of the elephants across the adjacent ages. These patterns were observed in PCoA plots following analysis using Bray-Curtis, Jaccard, and unweighted UniFrac distances \\u003cem\\u003e(\\u003c/em\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE-G\\u003cem\\u003e)\\u003c/em\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTaxonomic composition of bacterial populations in different age groups of elephants\\u003c/h2\\u003e \\u003cp\\u003eThe taxonomy of gut microbiota was identified based on the data of hypervariable region V3-V4 of the 16s rRNA gene. Forty-three phyla and 1,134 genera of gut microflora were identified within total elephant fecal samples. The relative abundance at the phylum level of gut microbiota in all age groups are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. The dominant bacterial phyla in fecal samples of all groups of elephants were Firmicutes followed by Bacteroidetes and Actinobacteria. In the infant calves, Bacteroidetes and Spirochete showed less relative abundance than those in other age groups. On the other hand, greater relative abundance of Actinobacteria and Euryarchaeota were detected in the infant calves. The composition of the elephant's gut microbiota changed markedly from infants to suckling calves, and from subadult and adult to geriatric elephants. The relative abundance of fecal microbiota at the phylum level was shown to be only slightly altered from suckling calves to weaned calves and subadult and adult elephants. According to the information from human guts, the microbiota is practically stable in healthy adults \\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. This phenomenon was similar to Asian elephants, which reach maturity between 10\\u0026ndash;14 years of age \\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, in this study, we used subadult and adult elephants as reference for the composition of gut microbiota in healthy elephants for further analyses.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eInfant elephants showed a distinctly different composition of gut microbiota, when compared with subadult and adult elephants\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe analysis of differential abundance was conducted by using ANCOM-BC. The results of significantly different taxa of fecal microbiota among age groups with top 10 of log fold changes are presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e. All significantly different taxa are shown in Supplementary Table\\u0026nbsp;1. We used subadult and adult elephants as a reference for each comparison.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the infant calves, the abundance of the phyla Euryarchaeota, Actinobacteriota, Verrucomicrobiota, Proteobacteria, and Desulfobacterota were higher than that of subadult and adult elephants while the phyla Cyanobacteria, SAR324_clade (Marine_group_B), Spirochaetota, Armatimonadota, and Elusimicrobiota were found to be lower (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). At the family and genus levels, the taxa from the families \\u003cem\\u003eBifidobacteriaceae, Akkermansiaceae, Villonellaceae, Bacteroidaceae\\u003c/em\\u003e, and \\u003cem\\u003eButyricioccaceae\\u003c/em\\u003e, together with the genus \\u003cem\\u003eBifidobacterium spp., UCG-008 spp., Olsenella spp., Akkermansia spp\\u003c/em\\u003e., and \\u003cem\\u003eBacteroides spp.\\u003c/em\\u003e in infant calves were higher than those of subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). On the other hand, the families \\u003cem\\u003eSpirochaetaceae\\u003c/em\\u003e, \\u003cem\\u003ep-251-o5\\u003c/em\\u003e, \\u003cem\\u003ePaludibacteraceae\\u003c/em\\u003e, \\u003cem\\u003ePlanococcaceae\\u003c/em\\u003e, and \\u003cem\\u003eGastranaerophilales\\u003c/em\\u003e, and the genus \\u003cem\\u003eSolibacillus\\u003c/em\\u003e, \\u003cem\\u003eAgathobacter\\u003c/em\\u003e, XPB1014 group in the families \\u003cem\\u003eLachnospiraceae\\u003c/em\\u003e, and \\u003cem\\u003eTreponema\\u003c/em\\u003e, and an uncultured genus in \\u003cem\\u003ePaludibacteraceae\\u003c/em\\u003e were lower in infant calves when compared to subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eSuckling and weaned elephants showed slightly different gut microbiota composition when compared with adult elephants\\u003c/h3\\u003e\\n\\u003cp\\u003eWhen compared to the differences between the infant calves and subadult and adult elephants, both the number of statistically distinct taxa and their magnitude were much smaller between the suckling and weaned calves and subadult and adult elephants. At the phylum level, only Euryarchaeota was higher in suckling calves and Planctomycetota, Euryarchaeota, and Desulfobacterota were higher in weaned calves when compared to subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). On the other hand, Synergistota and Fibrobacterota were lower in weaned calves while Cyanobacteria and SAR324_clade (Marine_group_B) were decreased in both suckling and weaned calves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAt the family level, \\u003cem\\u003eBifidobacteriaceae\\u003c/em\\u003e, \\u003cem\\u003eVeillonellaceae\\u003c/em\\u003e, and \\u003cem\\u003eBacterroidaceae\\u003c/em\\u003e were higher in both suckling and weaned calves whereas \\u003cem\\u003eTannerellaceae\\u003c/em\\u003e and \\u003cem\\u003eButyricicoccaceae\\u003c/em\\u003e were higher only in sucking calves, and \\u003cem\\u003eAtopobiaceae\\u003c/em\\u003e and \\u003cem\\u003eMicrococcaceae\\u003c/em\\u003e were higher only in weaned calves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). However, the families \\u003cem\\u003eParacaedibacteriaceae\\u003c/em\\u003e, \\u003cem\\u003eClostridiaceae\\u003c/em\\u003e, \\u003cem\\u003eCOB_P4-1_termite_group\\u003c/em\\u003e, \\u003cem\\u003eEndomicrobiaceae\\u003c/em\\u003e, and \\u003cem\\u003eLeuconostocaceae\\u003c/em\\u003e in suckling calves, and \\u003cem\\u003eNocardiaceae\\u003c/em\\u003e, \\u003cem\\u003eClostridiaceae\\u003c/em\\u003e, \\u003cem\\u003eLeuconostocaceae\\u003c/em\\u003e, \\u003cem\\u003eDesulfotomaculales\\u003c/em\\u003e, and \\u003cem\\u003eMVP-15\\u003c/em\\u003e in weaned calves were lower than in those subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAt the genus level, \\u003cem\\u003eBifidobacterium\\u003c/em\\u003e, \\u003cem\\u003eOlsenella\\u003c/em\\u003e, and \\u003cem\\u003eLachnospiraceae_NK3A20_group\\u003c/em\\u003e were higher in sucking and weaned calves while \\u003cem\\u003eBacteroides\\u003c/em\\u003e and \\u003cem\\u003eUCG-008\\u003c/em\\u003e were higher only in suckling calves, and the \\u003cem\\u003e[Ruminococcus]_gauvreauii_group\\u003c/em\\u003e and \\u003cem\\u003eSyntrophococcus\\u003c/em\\u003e were higher only in weaned calves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Meanwhile, the genera \\u003cem\\u003eEnterobacter\\u003c/em\\u003e, \\u003cem\\u003eEndomicrobium\\u003c/em\\u003e, \\u003cem\\u003eWeissella\\u003c/em\\u003e, \\u003cem\\u003eClostridium_sensu_stricto_13\\u003c/em\\u003e, and \\u003cem\\u003eSarcina\\u003c/em\\u003e in suckling calves, and \\u003cem\\u003e[Eubacterium]_oxidoreducens_group\\u003c/em\\u003e, \\u003cem\\u003eTerrisporobacter\\u003c/em\\u003e, \\u003cem\\u003eEnterobacter\\u003c/em\\u003e, \\u003cem\\u003eWeissella\\u003c/em\\u003e, and \\u003cem\\u003eMVP-15\\u003c/em\\u003e in weaned calves were lower those in subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGeriatric elephants showed distinct gut microbiota, when compared with adult elephants.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eGeriatric elephants showed a distinct pattern of abundance within the gut microbiota when compared to infant, suckling, and weaned calves. At the phylum level, Chloroflexi, and Euryarchaeota were increased while Cyanobateria, Desulfobacteria, Fibrobacterota, and Bacteroidota were decreased when compared to subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAt the family level, \\u003cem\\u003eLactobacillaceae, Xanthobacteraceae, Pseudomonadaceae, Caloramatoraceae\\u003c/em\\u003e, and \\u003cem\\u003eMicrococcaceae\\u003c/em\\u003e were more abundant than in subadult and adult elephants. Meanwhile, the taxa from families \\u003cem\\u003eProteobacteria_Rickettsiales_uncultured\\u003c/em\\u003e, \\u003cem\\u003eDesulfovibrionaceae, Bacteroidetes_BD2-2, Paludibacteraceae\\u003c/em\\u003e, and \\u003cem\\u003ePlanococcaceae\\u003c/em\\u003e were lower in composition when compared to those of subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAt the genus level, \\u003cem\\u003eLactococcus\\u003c/em\\u003e, \\u003cem\\u003eLactobacillus\\u003c/em\\u003e, \\u003cem\\u003eGaricola\\u003c/em\\u003e, \\u003cem\\u003eSuccinivibrio\\u003c/em\\u003e, and \\u003cem\\u003eUBA1819\\u003c/em\\u003e were higher in geriatric elephants while \\u003cem\\u003eSchwartzia\\u003c/em\\u003e, \\u003cem\\u003eAcinetobacter\\u003c/em\\u003e, \\u003cem\\u003eLysinibacillus\\u003c/em\\u003e, \\u003cem\\u003eSolibacillus\\u003c/em\\u003e, and \\u003cem\\u003eBacteroidetes_BD2-2\\u003c/em\\u003e were lower in geriatric elephants when compared to subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMajor gut microbiota composition in subadult and adult elephants\\u003c/h2\\u003e \\u003cp\\u003eThe relative abundance of the taxa in the family and genus level of each elephant in the subadult and adult age group are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. At the family level of subadult and adult elephants, \\u003cem\\u003eLachnospiraceae\\u003c/em\\u003e was the most dominant family, followed by \\u003cem\\u003eOscillospiraceae, Clostridiaceae, Christrensenellacease, Anaerovoraceae\\u003c/em\\u003e, and \\u003cem\\u003eRikenellaceae\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The beneficial bacteria were present in fecal samples of subadult and adult elephants, including the fiber-digesting taxa at Phylum level of Firmicutes, Bacteroides, Spirochaetota, and Actobacteriota, also including \\u003cem\\u003eLachnospiraceae; NA, Christrensenellacease_R-7_group, NK4A214_group, Sarcina, Lachnospiraceae_XPB1014_group, [Eubacterium]_coprostanoligunes_group, UCG-005, Planococcaceae; NA, Family_XIII_AD3011_group, Lachnospiraceae_AC2044_group, Ruminococcus, Solibacillus, and Saccharofermentans\\u003c/em\\u003e, \\u003cem\\u003eRikenellaceae_RC9_gut_group, p-251-o5, F082, and Prevotellaceae_UCG-003\\u003c/em\\u003e, \\u003cem\\u003eTreponema\\u003c/em\\u003e, and \\u003cem\\u003eOlsenella.\\u003c/em\\u003e In addition, Archaea \\u003cem\\u003eMethanobrevibacter\\u003c/em\\u003e of class Methanobacteria were found to be present in subadult and adult elephants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBlood parameters of subadult and adult elephant\\u003c/h2\\u003e \\u003cp\\u003eTo further understand the association between gut microbiota and the health of elephants, the associations between gut microbiota composition and blood parameters were determined. All blood parameters are presented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. The hematological and biochemical parameters of subadult and adult elephants were within the normal range. The serum lipid profiles including triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) were all determined to be within the normal range, when compared to the previous study by Norkaew, et al. \\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. In these analyses, the blood parameters were used as the numerical outcome in the ANCOM-BC analysis, and the results with a log-fold change of gut microbiota greater than 0.5 are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e. The results showed that several gut microbiota of subadult and adult elephants showed a correlation with blood parameters including RBC count, total protein, and serum albumin.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eCharacteristics of blood parameters in subadult and adult elephants.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"2\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eParameters\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eMean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePack cell volume (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e35\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHemoglobin (g/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e12.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.58\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRBC count (\\u0026times;10\\u003csup\\u003e6\\u003c/sup\\u003e cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.88\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMCV (fl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e123\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMCHC (g/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e35.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.75\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eWBC count (cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e12005\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2372\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSegmented neutrophil (cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2690\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;898\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLymphocyte (cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6193\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1805\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMonocyte (cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2725\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1333\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEosinophil (cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e339\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;218\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBasophil (cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e202\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;95\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePlatelet count (\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e cells/\\u0026micro;l)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e353\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;72\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBUN (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e10\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCreatinine (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.28\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAST (U/L)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e17\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eALT (U/L)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eALP (U/L)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e97\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;49\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTotal serum protein (g/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8.55\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.63\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAlbumin (g/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.35\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCK(U/L)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e166\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;69\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTC (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e45\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;10\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTG (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;14\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHDL (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e12\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLDL (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e29\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;8\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e\\u003cstrong\\u003eAbbreviation\\u003c/strong\\u003e\\u003cstrong\\u003es\\u003c/strong\\u003e: ALT, Alanine transaminase; AST, Aspartate transaminase; BUN, Blood urea nitrogen; CK, Creatine kinase; HDL, High density lipoprotein; LDL, Low density lipoprotein; MCHC, Mean corpuscular hemoglobin concentration; MCV, Mean corpuscular volume; RBC, Red blood cell; TC, Total cholesterol; TG, Triglyceride; WBC, White blood cell.\\u0026nbsp;\\u003c/p\\u003e \\u003cp\\u003eThe number of RBC showed a positive association with orders Pedosphaerales and Victivallales, families \\u003cem\\u003ePedosphaeraceae\\u003c/em\\u003e and \\u003cem\\u003eMuribaculaceae\\u003c/em\\u003e, and genera \\u003cem\\u003eDEV114\\u003c/em\\u003e from the family \\u003cem\\u003ePedosphaeraceae\\u003c/em\\u003e and \\u003cem\\u003eUCG-004\\u003c/em\\u003e from the family \\u003cem\\u003eErysipelatoclostridiaceae\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). However, the phyla SAR324_clade(Marine_group_B) and Euryarchaeota, classes Methanobacteria, Desulfotomaculla, and SAR324_clade(Marine_group_B), and orders Methanobacteriales and Desulfotomaculales showed a negative association with RBC count (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe total protein content showed a negative association with the genus \\u003cem\\u003eLactobacillus\\u003c/em\\u003e and the family \\u003cem\\u003eLactobacillaceae\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The genera \\u003cem\\u003eMycoplasma\\u003c/em\\u003e with corresponding family \\u003cem\\u003eMycoplasmataceae\\u003c/em\\u003e and order Mycoplasmatales, \\u003cem\\u003eMailhella\\u003c/em\\u003e with corresponding family \\u003cem\\u003eDesulfovibrionaceae\\u003c/em\\u003e, order Desulfovibrionales, and class Desulfovibrionia, \\u003cem\\u003eDEV114\\u003c/em\\u003e with corresponding family \\u003cem\\u003ePedosphaeraceae\\u003c/em\\u003e, order Pedosphaerales, and class Verrucomicrobiae, \\u003cem\\u003eMuribaculaceae\\u003c/em\\u003e with corresponding family \\u003cem\\u003eMuribaculaceae\\u003c/em\\u003e, and \\u003cem\\u003ePrevotella\\u003c/em\\u003e were negatively associated with the level of serum albumin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The family \\u003cem\\u003ep-251-o5\\u003c/em\\u003e, \\u003cem\\u003eAtopobiaceae\\u003c/em\\u003e, \\u003cem\\u003ePaludibacteraceae\\u003c/em\\u003e, and \\u003cem\\u003eMethanomethylophilaceae\\u003c/em\\u003e from order Methanomassiliicoccales, class Thermoplasmata also showed a negative association with plasma albumin level (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). However, at the phylum level, Chloroflexi showed a positive association with serum albumin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The other blood parameters are listed in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. The relationships of less significant association with the composition of gut microbiota and the details are shown in Supplementary Table\\u0026nbsp;2.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThis study is the first study to characterize the gut microbial community in Asian captive elephants of different ages. In addition, the association between the gut microbiota and blood parameters was analyzed. The major findings of this study include: 1) the diversity and the composition of gut microbiota were different between age groups and significant differences were observed between the infant calves and suckling calves, and between adult and geriatric elephants; 2) the composition of the gut microbiota of subadult and adult elephants was stable with a high abundance of Firmicutes, followed by Bacteroidetes, and Actinobacteria, and 3) several gut microbiota in adult elephants were associated with blood parameters such as RBC count, total protein, and albumin. Our findings suggest that gut microbial composition in elephants might be dependent on the types of food consumed as has been observed in other animals.\\u003c/p\\u003e \\u003cp\\u003eInfancy is a critical time for the gut microbiome to grow since it shapes the host immune system and stabilizes the metabolic condition \\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. Infant calves consume milk as their main source of energy, and the beta diversity analysis showed that the samples in infant calves were separately clustered from the rest of the older elephant groups in PCoA plots and the ANCOM-BC analysis also revealed that the composition of the gut microbiota composition was distinct. Milk fermenting bacteria including the \\u003cem\\u003eBifidobacteriaceae\\u003c/em\\u003e family together with \\u003cem\\u003eBifidobacterium spp\\u003c/em\\u003e were significantly higher as regards abundance in infant calves. This finding suggested that maternal milk was the key component determining the microbial composition of the gut in the infants enabling the newborn's intestines to adapt to the environment. Oligosaccharides and milk glycans are some of the major solid bioactive components in maternal milk; however, newborns were unable to digest and absorb them. When they passed through the gut lumen, these compounds have been served as prebiotics by shaping and stabilizing the microbiota in early life \\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. In the milk of Asian elephants, 40% of milk carbohydrate content was found to be oligosaccharides, a higher concentration than in other animal milk \\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. Milk oligosaccharides were mainly utilized by glycan-degrading enzymes, encoded by the genome of an infant-associated \\u003cem\\u003eBifidobacterium\\u003c/em\\u003e as well as other bacterial species within \\u003cem\\u003eBacteroides spp\\u003c/em\\u003e. found to be in higher abundance in both infant and suckling elephants; and \\u003cem\\u003eAkkermansia spp\\u003c/em\\u003e. \\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e found in higher abundance only in infant elephants. It is worth noting that the milk utilizing bacteria such as \\u003cem\\u003eBifidobacteriaceae\\u003c/em\\u003e were still higher in suckling and weaned calves when compared to adult elephants. However, during the pre-weaning period, the composition of the gut microbiota is very dynamic and unstable \\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. As elephant calves are completely weaned at age 3 years old, the abundance of milk utilizer bacteria declined relative to earlier infantile and suckling stages. This finding indicated that the milk from the mother elephants had a significant influence on microbial composition throughout early life.\\u003c/p\\u003e \\u003cp\\u003eChanges in the fecal microbial composition after the infantile life of elephants were observed in suckling calves or at the age of around four months due to the beginning of plant-based diet consumption. These elephants gradually switched from consuming milk to a variety of high-fiber diets, until completely weaned. Consequently, the gut microbiota of suckling and weaned calves were approaching the adult-like pattern as demonstrated by the overlapping of the composition of the gut microbiota of sucking and weaned calves with adult elephants as shown in the beta diversity analysis. As expected, the fiber-fermenting bacterial families including \\u003cem\\u003eSpirochaetaceae, p-251-o5, Paludibacteraceae, Planococcaceae\\u003c/em\\u003e, and \\u003cem\\u003eGastranaerophilales\\u003c/em\\u003e which were low in abundance in the infant elephants, became no significantly different between suckling and weaned calves and adult elephants. Furthermore, there was a higher abundance of SCFA-producing bacteria including genera \\u003cem\\u003eLachnospiraceae_NK3A20_group\\u003c/em\\u003e in both suckling and weaned calves, and \\u003cem\\u003eRuminococcus_gauvreauii_group\\u003c/em\\u003e and \\u003cem\\u003eSyntrophococcus\\u003c/em\\u003e in weaned calves. One study described evidence of a correlation between the genera \\u003cem\\u003eLachnospiraceae_NK3A20_\\u003c/em\\u003eand \\u003cem\\u003eRuminococcus_gauvreauii_groups\\u003c/em\\u003e with the concentration of volatile fatty acids and levels of microbial crude protein in the rumen of pre-weaning lambs \\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. In addition to the introduction of forage feeding in herbivores, coprophagia events had an impact on the foal microbiome which promoted the establishment of suitable gut microbial flora in young animals optimizing gut health during the pre-weaning period \\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. Coprophagic behavior was normally observed in juvenile elephants in the first 4\\u0026ndash;6 months of life \\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. Conceivably, the introduction of a high-fiber diet and coprophagia might shape the composition of the gut microbiota instigating an adult-like profile even though young elephants were still suckling.\\u003c/p\\u003e \\u003cp\\u003eIn this study, the adult elephant\\u0026rsquo;s main roughage consisted of Napier grass and corn stalks, which were reported to contain 42.6% \\u003csup\\u003e32\\u003c/sup\\u003e and 30.5\\u0026ndash;36.2% of dry weight \\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, respectively. As expected, a significant amount of fibrolytic bacteria were observed in adult elephants. The presence of \\u003cem\\u003eLachnospiraceae\\u003c/em\\u003e was necessary for the breakdown of complex carbohydrates in grass \\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. In addition, it has been shown that \\u003cem\\u003eLachnospiraceae\\u003c/em\\u003e are the main producers of SCFAs, especially butyrate which serves as a source of nutrients and growth factors for healthy gut epithelia and also contribute to the prevention of inflammation \\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e. The family \\u003cem\\u003eOscillospiraceae\\u003c/em\\u003e together with the genera \\u003cem\\u003eNK4A214_group, [Eubacterium]_coprostanoligenes_group\\u003c/em\\u003e, and \\u003cem\\u003eUCG-055\\u003c/em\\u003e were likely to be able to utilize intestinal host glycans and produce the important SCFAs such as butyrate and their metabolites \\u003csup\\u003e\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e. The genus \\u003cem\\u003eSarcina\\u003c/em\\u003e of the family \\u003cem\\u003eClostridiaceae\\u003c/em\\u003e, a cellulose-producing bacteria with carbohydrate fermentative metabolism, was also dominant in adult elephant feces. \\u003cem\\u003eChristensenellaceae R-7_ group\\u003c/em\\u003e, a genus in the family \\u003cem\\u003eChristrensenellacease\\u003c/em\\u003e which presented as a prominent bacterium genus in adult elephant feces, was reported to be associated with the host\\u0026rsquo;s body mass index (BMI) and leanness in humans \\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. Other genera of microorganisms that were found predominantly in the fecal matter of adult elephants, including \\u003cem\\u003eRikenellaceae_RC9_gut_group, Treponema, Ruminococcus, Saccharofermentans\\u003c/em\\u003e, and \\u003cem\\u003ePrevotella\\u003c/em\\u003e were known to play an important role in fiber degradation.\\u003c/p\\u003e \\u003cp\\u003eThe subadult and adult elephants in this study were healthy with normal body condition scores. No illnesses had been reported 6 months before the investigation. In the mature period of life, several gut bacteria were found to show significant correlations with RBC count, total protein, and albumin. Information regarding these correlations was scarce in elephant research and could be found only in previous studies of other species, such as mice, pigs, and humans. Albumin is the most abundant circulating protein found in plasma. Low albumin levels have usually been found to be as a result of liver disease, kidney disease, heart failure, malnutrition, or vitamin deficiency. We found genera \\u003cem\\u003eMycoplasma\\u003c/em\\u003e and \\u003cem\\u003ePrevotella\\u003c/em\\u003e had negative associations with the level of serum albumin. According to a previous study, \\u003cem\\u003eMycoplasma\\u003c/em\\u003e presumably plays an important role in the etiology and pathology of primary biliary cirrhosis in humans \\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003ePrevotella\\u003c/em\\u003e is believed to benefit host health as it can produce a significant amount of SCFA in pig model \\u003csup\\u003e\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e, and sufficient to have anti-inflammatory effects in mouse model \\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. Nonetheless, \\u003cem\\u003ePrevotella\\u003c/em\\u003e was reported to be associated with chronic inflammatory conditions and was found to have an increase in abundance in patients with chronic liver disease with advanced fibrosis \\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e. We also found the bacterial family \\u003cem\\u003eLactobacillaceae\\u003c/em\\u003e, in particular the genus \\u003cem\\u003eLactobacillus\\u003c/em\\u003e, was negatively associated with total protein in subadult and adult elephants. \\u003cem\\u003eLactobacillus\\u003c/em\\u003e is recognized as a beneficial microbe in humans and animals due to its involvement in immunity, metabolism, and maintaining the gut microbiota ecosystem \\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. In this study, we are unable to determine whether changes in the blood parameters were directly caused by changes in the gut microbiome or as a consequence of pathological changes. Future mechanistic studies into associations between bacterial taxa and total protein, albumin, and RBC count are warranted to delineate their significance and implications in elephant health issues.\\u003c/p\\u003e \\u003cp\\u003eThe geriatric elephants showed a distinct gut microbiota profile as demonstrated by decreasing alpha diversity and the separated cluster of the microbes in the geriatric elephants from the other age groups in beta diversity analysis. The differential abundance analysis also revealed a higher abundance of the family \\u003cem\\u003eLactobacillaceae\\u003c/em\\u003e and the genus \\u003cem\\u003eLactobacillus\\u003c/em\\u003e in geriatric elephants when compared to adult elephants. \\u003cem\\u003eLactobacillus\\u003c/em\\u003e has been identified as one of the major bacterial taxa in the healthy horse \\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e. Interestingly, overconsumption of high-starch diets and oligofructose has been associated with the overgrowth of \\u003cem\\u003eLactobacillus\\u003c/em\\u003e and has been observed in horses with colic frequently leading to the development of laminitis \\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e. Consumption of elephant concentrate pellets by most geriatric elephants, which contained crude protein 11.6% and nitrogen-free extract 46.4% of dry matter \\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e, might be associated with the higher abundance of \\u003cem\\u003eLactobacillus\\u003c/em\\u003e than in the subadult and adult group. Furthermore, the archaea class Methanobacteria, after having dipped in the middle age group, shifted to an increase again in geriatric elephants. This was in accordance with elderly macaques, which indicated a positive association of these methanogens with host aging \\u003csup\\u003e\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u003c/sup\\u003e, and a high abundance of centenarian human gut microbiota \\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e. The decline in gut microbiota diversity in geriatric elephants might be due to the loss of the last molar teeth (M6) at the age of 50 leading to poor mechanical and hence chemical digestion \\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e and resulting in rough feces. The resulting age-related gut dysbiosis, as indicated by decreased bacterial community richness, could affect the host's health and lifespan \\u003csup\\u003e\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e. Gut dysbiosis can trigger the innate immune response and induce chronic low-grade inflammation, leading to many age-related degenerative pathologies and unhealthy aging processes \\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, further investigation into gut dysbiosis and potential variation in diet in geriatric elephants might be one of the pivotal points to improving the quality of life of old elephants.\\u003c/p\\u003e \\u003cp\\u003eThis study demonstrated the composition of gut microbiota in captive Asian elephants by using the 16s rRNA data from a number of elephants which could provide important information on the composition and diversity of these gut microbiota. To increase the strength of the findings of this study, some limitations need to be addressed in further research. Despite a large number of subadult and adult elephants in the sample, the other age groups especially the infant group contained low numbers. Extending the range of data acquisition or collaborating with other elephant camps might increase the number of young elephants. The definition of age group in this study was mainly based on age range and other factors might be more suitable when categorizing the groups based on the biology of elephants and should be included in further study. In addition, this study includes only healthy elephants in all age groups, further research that includes both normal elephants and elephants with certain diseases might reveal more about the roles of gut microbiota in the health and biology of elephants.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eOur findings indicate that the diversity and relative abundance of gut microbiota in captive elephants were affected by age and the diet consumed. This knowledge will inform more appropriate food management to facilitate the development of more beneficial bacteria for each elephant at the different stages of their life. The elephants in this study, all lived in similar environments and were raised and handled similarly. In the future, a comparison of zoo husbandry conditions and semi free-ranging environments, as well as differences in food management, should be further investigated to promote the most effective management and nutrition for a healthy gut in captive elephants.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003e This study was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand (FVM-ACUC; R3/2563) and all experiments were performed in accordance with relevant guidelines and regulations. All methods used in this study are reported in accordance with ARRIVE guidelines. Fecal samples of approximately 50 grams, were single collected from 134 healthy captive elephants aged between two months and 67 years old working in tourist camps in Chiang Mai and Lampang provinces, during January – December 2020. All elephants in this study were divided into 5 groups based on nutrition and growth pattern including infant calves (1 day – 4 months old, food mainly milk based), suckling calves (4 months – 3 years old, food based on milk and grass), weaned calves (3–10 years old, food based on grass), subadult and adult elephants (10–55 years old, food based on grass), and geriatric elephants (more than 55 years old, food based on chopped grass mixed with pellets). The group sizes in each category were determined based on the existing elephant population in the Northern region. Furthermore, a significant number of samples of subadult and adult elephant groups were collected to ensure representation across a broad range of ages. Individual body condition score was estimated scored Morfeld, et al. which ranges from 1 to 5 (where 1 signifies the thinnest and 5 signifies the fattest), with an optimal score of 3 (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) \\u003csup\\u003e\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eAll the elephants had been housed and had worked in the camp for over 6 months, with elephant calves born and raised in the original camp without relocation. Calves and juveniles were housed together with their mother without interacting with the tourists. The adult elephants routinely worked with tourists, trekking, participating in elephant shows, or for observation\\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e between 8:00 and 15:00 for no more than 5 hours per day. The geriatric elephants were not involved in any physical activities with tourists. The information on types of work, habitat use, food intake, and foraging behavior was collected individually.\\u003c/p\\u003e \\u003cp\\u003eThe subjects were verified as being clinically healthy by experienced elephant veterinarians, based on history and clinical examination, with no reports of GI issues or antibiotic or drug administration for at least six months before the beginning of the study.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBlood collection\\u003c/h2\\u003e \\u003cp\\u003e10 ml blood samples were collected from each elephant in the subadult and adult groups from an ear vein. Blood samples were submitted to the Veterinary Diagnostic Laboratory of Faculty of Veterinary Medicine, Chiang Mai University, Thailand within 24 h of collection. Blood samples were analyzed using the Auto Haematology Analyzer (Mindray BC5300, Mindray Medical, Thailand) and using the Biochemical Analyzer Vitalab Flexor XL (Vital Scientific NV, Netherlands). Hematology parameters i.e. packed cell volume (PCV), hemoglobin, RBC count, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), White Blood Cell count (WBC count), and platelet count, and biochemical parameters i.e. Blood Urea Nitrogen (BUN), creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), creatine kinase (CK), total serum protein and albumin were analyzed.\\u003c/p\\u003e \\u003cp\\u003eSerum lipids were quantified using a Mindray BS Series analyzer (Mindray BS-380, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.), total cholesterol was measured using a cholesterol oxidase-peroxidase (CHOD-POD) method, and triglycerides were measured using a glycerokinase peroxidase-peroxidase (GPO-POD) method.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFecal samples collection and analysis\\u003c/h2\\u003e \\u003cp\\u003eFresh fecal samples (approximately 50 grams) were collected directly from the rectum or immediately after defecation and stored at -20 \\u003csup\\u003eo\\u003c/sup\\u003eC within 3 hours of collection until DNA extraction. After the samples were thawed, indigestible roughage such as grass, leaves, fruit seeds, and peel were separated from the stool contents. Up to 250 mg of prepared stool was used for DNA extraction. Bacterial genomic DNA was extracted from elephant fecal pellets using a commercial genomic DNA isolation kit (QIAamp PowerFecal Pro DNA Kit (QIAGEN), Germany). The extracted bacterial genomic DNA were exposed to an amplification process of hypervariable region V3-V4 of 16s rRNA and then underwent NGS methods. DNA amplification, data quality control methods, and sequencing were conducted by Novogene Inc (Singapore City, Singapore). A Double-blind study with samples categorization was used to avoid bias.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSequencing data analysis\\u003c/h2\\u003e \\u003cp\\u003eDNA sequence data from NGS were processed using the Quantitative Insights Into Microbial Ecology 2 (QIIME2-2021.4) open-source software \\u003csup\\u003e\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e. The raw datasets containing pair-ended reads and quality scores were merged, denoised, trimmed according to quality scores, and assigned to amplicon sequence variants (ASV) by using q2-dada2 plugin \\u003csup\\u003e\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e\\u003c/sup\\u003e. A feature table including the number of each ASV per sample was also generated \\u003csup\\u003e\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e\\u003c/sup\\u003e. The ASVs were aligned with mafft and used to generate a phylogenetic tree for further analysis \\u003csup\\u003e\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e\\u003c/sup\\u003e. The diversity analyses were conducted with rarefication at the sequencing depth of 56,700 as this is the maximum number to retain all samples in this study \\u003csup\\u003e\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e\\u003c/sup\\u003e. The alpha diversity including observed species, Pielou’s evenness \\u003csup\\u003e\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e\\u003c/sup\\u003e, Faith’s phylogenetic diversity \\u003csup\\u003e\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e\\u003c/sup\\u003e, and Shannon’s index \\u003csup\\u003e\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e\\u003c/sup\\u003e were calculated. The beta diversity including Bray Curtis, Jaccard, unweighted UniFrac \\u003csup\\u003e\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e\\u003c/sup\\u003e, and weighted UniFrac \\u003csup\\u003e\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e\\u003c/sup\\u003e distance matrices were analyzed and were illustrated as Principal Coordinate Analysis (PCoA). The taxonomy was assigned to ASVs by using q2‐feature‐classifier classify‐sklearn naïve Bayes taxonomy classifier \\u003csup\\u003e\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e\\u003c/sup\\u003e by using the data reference from SILVA database version 138 \\u003csup\\u003e65\\u003c/sup\\u003e. For visualization of population structure and relative abundance, taxonomical bar plots indicating relative abundance at phylum, class, and genus levels of each sample from the different age groups were generated. The differences in taxa abundance between categories and the association between taxa and blood parameters were estimated with a statistical framework: analysis of composition of microbiomes with Bias Correction (ANCOM-BC) \\u003csup\\u003e\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e\\u003c/sup\\u003e. The data visualization was conducted through R version 4.1.1 by using package qiime2R.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2 name=\\\"removable\\\"\\u003eData availability\\u003c/h2\\u003e \\u003cp name=\\\"removable\\\"\\u003eThe datasets generated and/or analyzed in this study are available in the NCBI sequence read archive under the Accession Number PRJNA1005601.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated and/or analyzed in this study are available in the NCBI sequence read archive under the Accession Number PRJNA1005601.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe would like to thank the Center of Excellence in Cardiac Electrophysiology Research, Faculty of Medicine, the Center of Elephant and Wildlife Health Animal Hospital, Faculty of Veterinary Medicine, and the Erawan HPC Project, Information Technology Service Center (ITSC), Chiang Mai University, Chiang Mai, Thailand for all their support. \\u0026nbsp;We also wish to register our appreciation to the owners of the elephant camps, their managers, and the mahouts for providing samples and data.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eS.Kl., N.C., S.C.C, and C.T conceived the idea and designed the research. \\u0026nbsp;S.Kl. and S.Ke. carried out the preparation of material and measurements. \\u0026nbsp;S.Kl. and S.S. analyzed and investigated the data. \\u0026nbsp;S.Kl and S.S. wrote the original draft menuscript. \\u0026nbsp;S.Kl., S.S., N.C., S.C.C., and C.T. reviewed and edited the menuscript. \\u0026nbsp;N.C., S.C.C., and C.T. were responsible for the funding acquisition. \\u0026nbsp;J.K., N.C., S.C.C, and C.T. supervised the research.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFundings\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was supported by the Thailand Research Fund (C.T); the CMU Presidential Scholarship and Chiang Mai University grant number 59/2565 \\u0026nbsp; (S.KI.); \\u0026nbsp;a Senior Research Scholar Grant from the National Research Council of Thailand (S.C.C.); the NSTDA Research Chair Grant from the National Science and Technology Development Agency Thailand (N.C.); and \\u0026nbsp;a Chiang Mai University Center of Excellence Award (N.C., C.T.)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe supplementary materials\\u0026nbsp;will be submitted with the manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eGut microbiota sequences of elephants in this study are on requested to the co-responding authors.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eFlint, H. 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Analysis of compositions of microbiomes with bias correction. \\u003cem\\u003eNat\\u003c/em\\u003e\\u003cem\\u003e. \\u003c/em\\u003e\\u003cem\\u003eCommun\\u003c/em\\u003e\\u003cem\\u003e. \\u003c/em\\u003e\\u003cstrong\\u003e11\\u003c/strong\\u003e, 1-11 (2020). \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"gut microbiome, elephants, captive, age, nutrition\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3241323/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3241323/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eGut microbiota play an important role in the health and disease in captive Asian elephants, however, its characteristics at each stage of life have not been thoroughly investigated. This study, therefore, aimed to characterize the profiles of the gut microbiota of captive Asian elephants from infants to the elderly. The diversity of the gut microbiota was lowest in infants, stable during adulthood, and slightly decreased in the geriatric period. Gut microbiota were identified by 16S rRNA sequencing from the feces of captive Asian elephants with varying age groups, including infant calves, suckling calves, weaned calves, subadult and adult elephants, and geriatric elephants. The gut microbiota of the infant elephants was dominated by milk-fermenting taxa. The fiber-fermenting taxa emerged in suckling elephants. The stabilization of gut microbiota profiles has been observed after weaning until the adult period. However, the composition of the gut microbiota was found to change again in geriatric elephants. Understanding of the composition of the gut microbiota of captive Asian elephants at each stage of life could be beneficial to support good health during each period of the elephant's lifespan.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Characteristics of Gut Microbiota in Captive Asian Elephants (Elephas maximus) from Infant to Elderly\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-08-23 17:52:29\",\"doi\":\"10.21203/rs.3.rs-3241323/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2023-10-13T07:21:41+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2023-09-15T19:30:08+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"c3c15e86-0095-43be-acb4-712572532fcf\",\"date\":\"2023-09-08T13:37:58+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-09-08T13:28:56+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2023-09-08T13:18:20+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2023-08-17T12:27:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2023-08-17T12:24:08+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2023-08-07T08:57:30+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"9566fb6f-389d-44bb-a25c-085ba439e71e\",\"owner\":[],\"postedDate\":\"August 23rd, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":24109689,\"name\":\"Biological sciences/Microbiology\"},{\"id\":24109690,\"name\":\"Biological sciences/Molecular biology\"},{\"id\":24109691,\"name\":\"Biological sciences/Zoology\"}],\"tags\":[],\"updatedAt\":\"2024-01-01T15:03:23+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-3241323\",\"link\":\"https://doi.org/10.1038/s41598-023-50429-1\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2023-12-27 15:00:53\",\"publishedOnDateReadable\":\"December 27th, 2023\"},\"versionCreatedAt\":\"2023-08-23 17:52:29\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-023-50429-1\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-023-50429-1\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3241323\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3241323\",\"identity\":\"rs-3241323\",\"version\":[\"v1\"]},\"buildId\":\"WrCJVZZCHTDjtuVLN7oU0\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}