A diet containing Moringa oleifera alters the goat rumen microbiome: an insight into bacteria, ciliates and rumen anaerobic fungi

A diet containing Moringa oleifera alters the goat rumen microbiome: an insight into bacteria, ciliates and rumen anaerobic fungi | 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 A diet containing Moringa oleifera alters the goat rumen microbiome: an insight into bacteria, ciliates and rumen anaerobic fungi Chitra Nehra, Minal Bhure, Tejas Shah, Ramesh Pandit, Niteen V Patil, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8826260/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Background In the present study, we have investigated the impact of Moringa olifera feed on rumen and fecal microbial communities, including bacteria, ciliates, and anaerobic fungi in lactating goats ( Capra hircus ). Lactating goats were divided into three groups based on dietary regimes: masoor straw (MS, n=10), 20% moringa leaf meal (20% MLM, n=8), and 30% moringa leaf meal (30% MLM, n=9). Rumen digesta and fecal samples were collected at the end of the experiment, and amplicon sequencing targeting the 16S rRNA, 18S rRNA, and ITS 1 genes were carried out. Results In the rumen solid and liquid fractions, moringa diet increased the Bacillota:Bacteroidota ratio, which is associated with lower residual feed intake and improved metabolism. An increase in proteolytic bacterial phylum Pseudomonadota was observed with moringa diet . Further, Xylanibacter, Sachharofermentans and Ruminococcus genera, that have active role in volatile fatty acids (VFAs) production during fermentation process ( p -value ≤0.05), were more abundant, while Fibrobacter , Succiniclasticum and Sodliphilus ( p -value ≤0.05), were less abundant in moringa feed groups. Cellulolytic ciliates like Enoplastron and Diploplastron significantly increased ( p -value ≤0.05) while Entodinium reduced in the rumen digesta samples of moringa feed groups. Fiber-degrading fungal genus Neocallimastix was less abundant in the rumen of the goats fed with moringa. Conclusion Overall, these findings suggest that moringa supplementation positively influences rumen microbial communities and provides insights into adjustments in rumen microbiome structure and diversity. These findings are useful in understanding how moringa supplementation influences the rumen fermentation process and further modulating goat feed to improve health and milk production. Biological sciences/Biotechnology Biological sciences/Microbiology Animal nutrition Ciliates Lactating goats Moringa oleifera Rumen anaerobic fungi Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Asia, home to over half of the world's goats with approximately one billion goats, is the cradle of goat domestication. Goats, including dairy breeds, thrive in diverse and harsh environments, providing essential nutrition, food security, and socio-economic benefits across many Asian countries, particularly in India[1]. In India, goats are a crucial source of income for small-scale farmers, providing milk, meat, fiber, hide, and manure[2]. Sustainable livestock production hinges on maximizing feed efficiency, which refers to an animal's ability to convert feed into output and improve nutrient utilization, thereby enhancing the animal's performance. Feed efficiency in small ruminants, such as sheep and goats, is a vital productive trait that significantly impacts farmers’ profitability[3]. Improved feed efficiency is also related to decreased dung and methane production[4]. Consequently, improving feed efficiency can boost livestock industry profitability while simultaneously mitigating environmental impacts. These improvements in feed utilization and environmental impact are mostly mediated by the rumen microbiome, which is vital to digestive and metabolic processes[5]. In ruminant’s complex plant materials are digested to yield volatile fatty acids (VFAs), microbial proteins, and vitamins[6]. The composition and diversity of rumen microbiome, which includes bacteria, archaea, ciliate protozoa and anaerobic fungi, significantly impacts the host's health[7]. Rumen microbes play a key role in various nutrient and carbohydrate metabolism in the gastrointestinal tract of ruminants by producing different enzymes that break down complex carbohydrates and other feed components into simpler forms, allowing their absorption and utilization by the host[8]. Moringa oleifera is a native Indian plant which produces several harvests and grows in hot, dry climates and wet tropical regions[9]. Moringa leaves have high nutritional properties, being a rich source of protein and other necessary elements including vitamins[10]. Previous studies have shown that moringa leaf feed improves rumen fermentation and nutrient digestibility and boosts the quantity and quality of milk in lactating goats [11,12]. Additionally, after moringa feeding, improved growth performance and decreased methane production, without having an adverse effect on animal health, have also been reported[13]. In animals, rumen microbiome and diet interactions are associated with overall health and productivity[14]. Different diets have been reported to improve animal health and performance by affecting the rumen microbiota composition [15]. Although various studies have reported the beneficial effects of the moringa diet on animal health and productivity [13,16], very limited research has been carried out to understand its effect on the rumen bacterial communities[17,18], including our previous studies[19,20]. Moreover, to date, no studies have reported its effects on rumen ciliates or anaerobic fungal populations. Hence, to examine the comprehensive effect of moringa leaf diet on rumen and fecal bacterial, ciliates and anaerobic fungal communities, here, we have analyzed the sequencing data targeting to V3-V4 region of 16S rRNA, ciliates specific18S rRNA, and rumen anaerobic fungal specific internal transcribed spacer 1 ( ITS 1) genes in the lactating goats. In present study, we have analyzed and compared the rumen microbiota (bacteria, ciliates and anaerobic fungus) composition in three groups of lactating goats; first group was fed masoor straw (MS) based diet, second group was fed 20%moringa leaf meal (20%MLM), and third group was given 30% moringa leaf meal (30%MLM) in their regular diet. Simultaneously, fecal samples were also processed to compare bacterial and ciliate diversity among the three groups. We hypothesize that an increasing proportion of moringa in the diet induces a dose-dependent restructuring of the rumen and fecal microbial communities. The current findings help in understanding the rumen and fecal microbial diversity shift in response to a moringa diet in small ruminants and open a new avenue to formulate moringa-fortified diet for improved animal health and productivity 2. Results 2.1 Sequencing Data Analysis The sequencing read counts and the percentage of reads classified at the genus level (after removing samples with low sequencing read counts and outliers identified through PCoA plots) for each sample, are provided in Supplementary Table 2. We generated a total of 12.63, 27.25 and 5.6 million reads to analyze rumen bacterial, protozoal and anaerobic fungal diversity, respectively. Similarly, from fecal samples, a total of 9.2 and 12.50 million reads were generated for bacteria and ciliates, respectively. For rumen anaerobic fungi , we were unable to obtain amplification from fecal samples. 2.2 Rumen and Fecal Bacterial Community Analysis In 16SrRNA data, for rumen solid and liquid fractions as well as fecal samples, we observed that as the number of sequences increased, the rarefaction curve flattened out (Supplementary Fig. 1A-1B, Supplementary Fig. 3A), indicating enough data to represent the microbial diversity present in samples. Beta diversity analysis (Fig. 2A-2D) showed significant differences (PERMNOVA, p -value ≤ 0.05) in bacterial community structure among MS, 20% MLM, and 30% MLM groups. In fecal samples, no significant difference was observed among the groups (Supplementary Fig. 3D-3E). The Shannon diversity and evenness metrics did not reveal significant differences among different dietary groups (Supplementary Fig. 2A-2D, Supplementary Fig. 3B-3C). A total of 46 different phyla were detected both in rumen solid and liquid fractions (Supplementary Table 3A,3B). Cumulative bar charts of different phyla with relative abundance above 1% are provided in Fig. 1A and 1B. The most abundant bacterial phyla in both solid and liquid fractions across all the groups were Bacteroidota and Bacillota , formerly known as Bacteroidetes and Firmicutes , respectively. In addition, phyla such as Verrucomicrobiota , Actinomycota , Cyanobacteriota , Synergistota , Fibrobacterota and Spirochaetota were also present with relative abundance above 1%. Further, to see the effect of moringa, we compared the presence of different phyla among all three dietary groups. In the rumen solid fraction, the proportion of Bacillota and Pseudomonadata (formerly known as Proteobacteria ) increased with the moringa diet, being highest in the 30% MLM group, while the abundance of Synergistota and Fibrobacterotota decreased (Fig. 1A,1C). In the rumen liquid fraction, the proportion of Pseudomonadata was found to increase in the 20% and 30% MLM groups (Fig. 1B, 1D). In both solid and liquid fractions, the ratio of Bacillota to Bacteroidota was higher in the 20% MLM and 30% MLM groups compared to the MS group (Supplementary Table 3C). In fecal samples, a total of 40 phyla were identified with Bacillota (60.09%-64.55%) and Bacteroidota (18.40%-19.63%) being the most abundant taxa across all three dietary groups (Supplementary Table 3D, 3E). At the genus level, in total, 621 and 1,114 different genera were detected in the rumen solid and liquid fraction, respectively (Supplementary Tables 4A, 4B). Segatella had the highest relative abundance in both the solid (12.36-15.18%) and liquid fractions (10.6-11.36%) among all three dietary groups, along with other genera such as Xylanibacter, Subdivision5_genera_incertae_sedis, Sodaliphilus, Hallella , and Succiniclasticum , each with a relative abundance greater than 1% (Supplementary Table 4C). In the rumen solid fraction, Xylanibacter , Saccharofermentans and Ruminococus were significantly higher ( p -value <0.05) in moringa feed groups than MS groups (Fig. 2E-2G). Genera like Leyella, Paraprevetolla Butyrivibrio, Clostridium_sensu_stricto, and Enterocloster also increased in response to moringa feeding (Fig. 2Q). On the other hand, Sodaliphilus, Succiniclassticum and Fibrobacter significantly ( p -value <0.05) reduced in 20%, and 30%MLM feed groups as compared to MS group (Fig. 2H-2J). In the rumen liquid fraction, the abundance of Xylanibacter Saccharofermentans, Lucifera, Butyrivibrio, Enterocloster, remined higher in animals who were fed a moringa comprising diet (Fig. 2K, 2L, 2R). While the abundance of Sodaliphilus, Succiniclasticum, Fibrobacter, Segatella, Hallella, Olsenella, Slackia, Selenomonas, Treponema , Syntrophococus and Pseudobutyrivibrio were higher in the MS group (Fig. 2N-2P, 2R). In fecal samples, 642 different taxa were detected at the genus level (Supplementary Table 4D). In the moringa-fed groups, the relative abundance of Monoglobus, Falsiporphyromonas, Ercella, Luoshenia, and Papillibacter increased, whereas Muriventricula, Ruminococcus , and Saccharofermentans reduced (Supplementary Fig. 3F, Supplementary Table 4E). LEfSe analysis was conducted to identify bacterial taxa that were significantly represented to each feeding group in both the rumen solid and liquid fractions. In the solid fraction of rumen digesta, a total of 12, 5, and 8 taxa were significantly enriched in the MS, 20% MLM and 30% MLM groups, respectively (Supplementary Table 7A). Genera such as Sodaliphilus, Fibrobacter, Olsenella , and Anseongella were enriched in the MS group. Ruminococcus, Xylanibacter, Pseudobutyrivibrio , and Coprococcus were dominant in the 20% MLM group, while Anaeroplasma, Enterocloster, Ruminobacter, Butyrivibrio , and Saccharofermentans were abundant in the 30% MLM group (Fig. 5A). Similarly, in the liquid fraction, 7, 3, and 10 taxa were significantly enriched in the MS, 20% MLM, and 30% MLM diet group, respectively (Supplementary Table 7B). Genera such as Sodaliphilus, Slakia and Olsenella were dominant in the MS group, Duncaniella, Algoriphagus , and Subdivision5_genera_incertae_sedis in the 20% MLM group, and Paraprevotella, Ruminococcus, Succinimonas, Blautia, Acetivibrio , and Ruminococcoides were enriched in the 30% MLM feed group (Fig. 5B). In the fecal samples, we did not observe significant taxa., 2.3 Ciliates in Rumen and Fecal Samples of Three Dietary Groups For ciliates also, we generated sufficient data as shown in Supplementary Fig. 2C and Fig. 4A. Beta diversity analysis showed significant differences (PERMONOVA, p -value ≤ 0.05) in rumen ciliate community structure among the three groups in rumen samples (Fig. 3A-3B). Although, in fecal samples, our analysis revealed no significant difference among the three groups (Supplementary Fig. 4D-4E). Further, alpha diversity analysis using Shannon diversity and evenness did not turn up with any significant difference among different dietary groups in both rumen and fecal samples (Supplementary Fig. 2E-2F, Supplementary Fig. 4B-4C). Here, we identified a total of 73 genera in rumen samples (Supplementary Table 5A, 5B) with Polyplastron (26.65%- 29.97%), Entodinium (3.78%-16.39%) , Enoploplastron (11.89%-20.05%), Trichostomatia_XX (6.25%-8.76%), Diploplastron (7.01%-11.98%) and Epidinium (6.49%-7.92%) were major genera detected in all three dietary groups. Enoploplastron and Diploplastron were significantly higher ( p -value 0.05) in moringa feed groups (Fig. 3E-F). Other genera with differential abundance among feeding groups are provided in Fig. 3G. In fecal samples, Blastocystis was observed as the most abundant (85.86%-97.99%) genus in all three dietary groups (Supplementary Table 5E, 5F).Species-level results for rumen ciliates are provided in Supplementary Fig. 5 andSupplementary Table 5C, 5D. In the LEfSe analysis of rumen samples, we found that 12 genera were significantly enriched in the MS group, while five and eight taxa were significantly enriched in the 20% MLM and 30% MLM groups, respectively (Fig. 5C, Supplementary Table 7C). In the MS group, the genera Entodinium, Entodinium_1, Trichostomatia_XX , and Ophryoscolacidae_1_X had come out as signature genera. Similarly, in the 20% MLM group, Dasytricha, Diplodinium, Eremoplastron , and Isotrichidae_2_X and Diploplastron and Metadinum in the 30% MLM group were identified as key ciliate genera. No significantly enriched taxa were identified in the fecal samples. 2.4 Structure and Composition of Rumen Anaerobic Fungi in Three Dietary Groups As like 16SrRNA and 18SrRNA data, the rarefaction curve of ITS 1 sequences also gradually flattened out, suggesting an adequate amount of sequencing data, which facilitates further analysis (Supplementary Fig. 2D and 2E). Beta diversity analysis couldn’t find any significant differences ( p -value ≥0.05) in anaerobic fungal community structures among the three treatments groups (Fig. 4A-4D). Also, changes in the alpha diversity (Shannon and evenness metrics) remined insignificant among the three different dietary groups (Supplementary Fig. 2G-2J). Phylum Neocallimastigomycota was present with more than 99% relative abundance across all three dietary groups in both the solid and liquid fractions of the rumen (Supplementary Table 6A). At the genus level, 21 genera were identified in the rumen solid fraction, while 19 genera were identified in the rumen liquid fraction samples (Supplementary Table 6B, 6C). Neocallimastix and Pecoramyces were the most abundant genera in all three groups, where Neocallimastix decreased in moringa feed groups compared to the MS group in both solid and liquid fractions (Fig. 4E, 4F, Supplementary Table 6D). Intriguingly, in solid fraction, Piromyces increased with the addition of moringa in the feed, while it was inverse in the liquid fraction. At the species level, the proportion of Pecromyces ruminatium was higher in the moringa feed groups than in the MS group. (Supplementary Fig. 6A, 6B; Supplementary Table 6G). In the LEfSe analysis of both rumen solid fraction samples, we couldn’t find any enriched taxa among the groups. 3. Discussion The rumen microbiome comprises hundreds of species, including bacteria, archaea, ciliates, fungi, and viruses [29], which provide adaptation and resilience to the host against different dietary modifications[30]. A healthy rumen microbiome improves growth performance and production traits of the animal[31]. These microbial communities undergo variations in their composition in response to different dietary regimes[32]. Indeed, diet is considered a main reason for the difference in the taxonomic composition of the rumen microbiome. Various beneficial effects, including enhanced production traits, reduced methane emissions, and improved digestibility, have been reported in goats that consume moringa. Although, only a few studies, including our earlier one[20], have focused on the shifts in the rumen microbiota associated with moringa feed[17,18]. Therefore, the present study provided a comprehensive view of the effects of moringa-added ration on rumen and fecal microbiota in lactating goats. The moringa diet has less effect on the species richness and evenness of goats’ rumen bacteria, ciliates, and rumen anaerobic fungi. However, the proportion of several specific microbial taxa was significantly altered when goats consumed moringa-containing feed. Across the three dietary groups, Bacteroidota and Bacillota were identified as the most abundant bacterial phyla in rumen and fecal samples, which is consistent with findings of previous study[33]. Different genera and species belonging to Bacteroidota and Bacillota have an active role in carbohydrate and protein metabolism[34], indicating their key role in the feed digestion in the rumen. In the present study , we found a shift in the rumen bacterial community in response to moringa feed, which is consistent with previous studies reporting changes in rumen microbiota in response to dietary changes [35]. Notably, the proportion of Bacillota group was higher in moringa feed groups, leading to a higher Bacillota : Bacteroidota ratio compared to control group. A higher Bacillota : Bacteroidota ratio has been linked to lower residual feed intake as well as improved energy absorption and storage in the host [3]. The moringa containing diet also led to significant changes in the rumen bacterial community, with a higher abundance of the Pseudomonadota phylum in moringa feed groups compared to the MS group. Pseudomonadota members have active role in propionate production in the rumen [36] and a lower abundance of Pseudomonadota has been associated with higher methane emissions. In high methane-emitting beef cattle, Pseudomonadota abundance was found four times less than in low-emitters[14]. This suggests that the increased abundance of Pseudomonadota in response to a moringa diet may contribute to lower methane emissions in lactating goats. A similar finding have been reported using metatranscriptome approach where abundance of Pseudomonas and Proteus increased in rumen of sheep fed with 15% moringa along with reduction in methane[37] . However, further investigations are required to confirm this effect. Therefore, we suggest performing moringa feeding experiments that simultaneously measure methane emission along with comprehensive microbiome analysis Further, in this study, the goat rumen harbor Xylanibacter and Segatella as the most dominant bacterial genera. Overall, moringa has higher protein and lower fiber content[38]. In this study, we found relatively more abundance of Xylanibacter in the moringa feed groups compared to the MS group. The higher abundance of Xylanibacter suggests its active role in the digestion of protein-rich moringa feed. Moreover, the fibrinolytic bacterial genera Fibrobacter and Succiniclasticum were less abundant in the moringa feed groups, aligning with previous study describing a lower abundance of these genera in response to low-fiber diets [39]. The elevated abundance of Fibrobacter and Succiniclasticum in the MS group can be attributed to the higher fiber and lignin content of masoor straw compared to moringa. The Saccharofermentans, Butyrivibrio and Ruminococcus genera were higher in moringa feed groups. Ruminococcus are known for producing the majority of cellulase enzymes in the rumen, whereas Saccharofermentans play a role in degrading plant polysaccharides and producing VFAs like acetate and propionate as the final fermentation products[40]. Earlier studies also showed a positive association between crude protein content of forages and the abundance of Butyrivibrio [15]. Species of Butyrivibrio are metabolically diverse, contributing to the production of butyrate, bacteriocins, and vitamins during the fermentation process in the rumen[41]. Additionally, bacterial genera like Clostridium and Butyrivibrio are linked with lower methane emissions, higher digestible dry matter and digestible organic matter intake[41]. In fecal samples, the presence of Monoglobus and Papillibacter was observed more frequently in the moringa feed groups. Papillibacter is considered a beneficial bacterium associated with a healthy gut microbiome, and Monoglobus has been reported to have a positive correlation with enhanced immune responses in goats [34]. Both Papillibacter and Monoglobus are known to produce butyrate, a short-chain fatty acid with significant health benefits[42]. Overall, these findings suggest that moringa feed enriches the bacterial genera which has a beneficial role in lactating goat health and is associated with lower methane emissions. Although, we have not analyzed phytochemical composition of moringa in this study, moringa leaves are rich source of saponins, condensed tannins, flavonoids and other polyphenols [43,44]. These compounds can an modulate rumen microbial ecology and fermentation [45,46]. Tannins and other phenolic compounds could bind with dietary proteins, which can slow down the breakdown of these proteins in the rumen. This process enhances the supply of protein later in the rumen digestion process, leading to a preference for microbial taxa that thrives on higher protein sources. This could explain why we see a greater presence of protein-degrading Xylanibacter and related genera like Butyrivibrio in the groups that include moringa. Additionally, flavonoids and polyphenols have been shown to have antimicrobial and antioxidant properties. They can help keep certain harmful bacteria in check while encouraging the growth of beneficial fermentative bacteria that produce short-chain fatty acids (SCFAs), such as Saccharofermentans, Ruminococcus , Butyrivibrio , Monoglobus , and Papillibacter . In this study, all these genera were enriched in the moringa fed goats might play a crucial role in providing energy and supporting gut health. Rumen ciliates are crucial for feed digestion, significantly enhancing the digestibility of the feed [47]. The genus Entodinium is typically reported as the most abundant one in ruminants. However, in this study, Polyplastron emerged as the most abundant genus in all rumen samples, aligning with some previous studies in goats [48]. It’s also been reported that the abundant ciliate genera in the rumen microbial population can be rehabilitated based on the host species, the diet they consume, and even season [49]. In the present study, we have found a significantly higher abundance of Enoploplastron and Diploplastron in goats fed the moringa diet compared to the masoor straw diet. These genera play an active role in carbohydrate degradation, VFA production during the fermentation process and maintain the bacterial population in the rumen[50]. We also observed a higher abundance of ciliate species such as Enoploplastron triloricatum, Polyplastron multivesiculatum, and Diploplastron affine in moringa-fed groups. These species play a key role in the digestion of cellulose in the rumen [51] . Diploplastron affine can digest starch and murein to meet the host’s energy requirement [52]. These findings highlight the complex interactions between diet and the rumen microbial ecosystem. Moreover, the ciliate species Buxtonella sulcata and Blastocystis ratti, which cause diarrhea in animals [53,54], were found to be reduced in the goats that were fed moringa as compared to the MS group. This could be because of specific activity of different phytochemicals present in the moringa. Saponins found in Moringa have consistently been linked to a decrease in protozoal populations and methanogenic activity. This, in turn, can create a more favorable environment for bacterial groups that enhance microbial protein synthesis and help cut down on methane emissions [55,56]. Overall, these outcomes suggest that moringa feed could positively impact the health of lactating goat. Previous studies on moringa supplementation, conducted both in vivo and in vitro , in different animals, have reported reduced protozoal population[57,58]. However, all these studies are count-based and have not performed comprehensive amplicon sequencing. Therefore, further protozoal challenge studies with moringa feeding, correlating pathogen reduction, will strengthen these findings. Overall, a moringa-based diet has a little less effect on the rumen anaerobic fungal community. This might be because rumen anaerobic fungi are specifically engaged in fiber degradation, and moringa leaves have less fiber as compared to masoor straws in this study. In this study, Neocallimastigomycota is the most abundant phylum across all dietary groups. These results align with earlier studies[59]. Earlier study have shown that the genus Neocallimastix decreases with lower fiber content [60] ; here, in this study, we observed the lower abundance of Neocallimastix in the moringa feed groups compared to the MS group (fiber-rich feed). Pecromyces ruminatium produces xylanase and cellulase enzymes and helps in plant biomass degradation in the rumen[61] . The higher abundance of Pecromyces ruminatium in the moringa feed group suggests potential health benefits to the animal. The lack of previous studies on how a moringa-fortified diet affects the rumen anaerobic fungal population makes the comparison somewhat challenging. Besides, although this study compares a fairly good number of samples (8-10 animals per group), a study recruiting more animals is required for a more solid conclusion on its effect on rumen anaerobic fungi. In the present study, a moringa-based diet has significantly altered the composition of rumen bacteria and ciliates but not the anerobic fungi in the lactating goats, as can be seen by beta diversity analysis. The limited response of protozoa as compared to bacteria agrees with earlier findings showing that these communities remain relatively stable despite nutritional shifts, likely due to their slower turnover and strong host association [62]. Likewise, the unchanged abundance of anaerobic fungi may reflect the lower fiber content of the moringa containing diet compared with masoor stover, since rumen anaerobic fungal populations primarily respond to fiber availability rather than short-term diet variation[63]. Furthermore, unlike bacteria and ciliates, the overall diversity of rumen anaerobic fungi remains very low. For example, only two genera, Neocallimastix and Pecoramyces , represent 60-70 % of the rumen anaerobic fungal population. Therefore, this limited diversity compared to bacteria and ciliates could be a possible reason for their limited responsiveness to dietary treatment. 4. Conclusion In summary, this study highlights the significant variations in the abundance of rumen bacteria, ciliates, and anaerobic fungal taxa in response to the moringa diet in small ruminants, which emphasizes the complex interactions between diet and the rumen microbes. The results show that moringa containing diet significantly improved the abundance of beneficial bacterial and ciliate taxa in rumen and fecal, possibly driven by the complex phytochemical composition of moringa. While the abundance of rumen anaerobic fungal taxa remained largely unaffected. Advancements in deciphering the roles of these microbes are crucial for optimizing rumen performance and improving animal health as well as productivity. Findings of this study are important for developing moringa-fortified animal feed and strategies that enhance production traits. 5. Material and Methods 5.1 Animal Handling and Experiment Design At the Central Arid Zone Research Institute (CAZRI) in Jodhpur, India, all animal studies were conducted in accordance with established standards for animal care and experimentation. Moringa plants (cultivar PKM-1), grown at CAZRI, were cultivated under arid climatic conditions, with an average annual rainfall of 379 mm and potential evapotranspiration of 2002 mm. The experimental field had sandy, shallow soil (50 cm depth) with 0.28% organic carbon, 116.64 kilogram/hectare (kg/ha) available nitrogen, 25.13 kg/ha P₂O₅, 481.81 kg/ha K₂O, and a pH of 7.9. The experiment was conducted in a factorial randomized block design with four levels of irrigation and planting density, and each treatment was replicated three times. Moringa cultivar PKM-1 was planted a year before the experiment and uniformly watered. Plants were headed back to a uniform height of 50 cm from the ground surface, and the five tagged plants were selected for yield attributes. Plants sampled for dry matter estimation were separated into leaves and stems for estimation of respective biomass yields after drying in a hot air oven. The details of the proximate composition of M. olifera are given in Supplementary Table 1A. For this experiment, 30 lactating Marwari goats ( Capra hircus ) were randomly assigned to three groups (n = 10 per group) after weaning of their kids at 90 days post-kidding. The goats were sourced from the institutional livestock farm, CAZRI, Jodhpur, and the experimental feeding continued for six months, and observations on daily intake of feed dry matter and nutrients, milk yield and fortnightly body weight change were recorded. All the lactating goats were allowed to graze for 3-4 hours daily in the open field having Cenchrus setigerus , Cenchrus ciliaris and edible arid weeds. In addition to grazing, the first group of goats were fed Masoor straw (MS, n=10), the second group was fed 20% moringa leaf meal (20%MLM, n=8), and the third group was given 30% moringa leaf meal (30% MLM, n=9) feed. The reduced number of animals in the latter two groups was due to infections observed during the experiment, which led to the exclusion of three animals. The MS group was given a total mixed ration (TMR) with a 70:30 roughage-to-concentrate ratio, consisting of Masoor straw (containing 5% crude protein [CP] and 60% total digestible nutrients [TDN]) and a commercial concentrate mixture (22.7% CP and 70% TDN), resulting in an overall TMR composition of 10.31% CP and 63% TDN. The 20% MLM and 30% MLM groups were fed TMRs with the same 70:30 roughage-to-concentrate ratio, composed of Masoor straw (5% CP and 60% TDN) and their respective concentrate mixtures (each containing 26.98% CP and 70% TDN), resulting in a final TMR composition of 11.59% CP and 63% TDN. The concentrate mixtures for the 20% MLM and 30% MLM groups were formulated by incorporating 20% and 30% moringa leaf meal, respectively, along with other conventional concentrate ingredients. The details of feed ingredients and their nutritive values are provided in Supplementary Table 1B. 5.2 Sample Collection and DNA Extraction At the end of the experiment, rumen digesta samples were collected from each goat using a flexible stomach tube attached to a low-pressure vacuum pump. The whole rumen digesta was then separated into liquid and solid components using sterile four-layered muslin cloth and was transferred into cryovials containing RNAprotect bacteria reagent (Qiagen, Valencia, CA, USA) as described in our earlier studies [21]. Simultaneously, fecal samples were collected by trained veterinarians directly from the rectum wearing sterile gloves and lubricant to ensure hygienic and non-invasive sampling and stored in RNAprotect bacteria reagent (Qiagen, Valencia, CA, USA). All the samples were promptly frozen and then stored at -80 °C until further processing. DNA extraction was carried out using a QIAamp Fast DNA stool mini kit (Qiagen, Valencia, CA, USA). To dislodge the fiber adhered bacteria, solid fractions were treated with Phosphate Buffer Saline (PBS) and 0.1% (v/v) Tween 20 for one hour with incubation at 37ºC and intermittent vortexing. The integrity of the extracted DNA was examined on a 0.8% agarose gel, and the concentrations were determined by Qubit 4.0 fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA). 5.3 Amplicon Libraries Preparation and Sequencing For 16S rRNA and ITS 1 amplicons, we processed rumen solid and liquid fractions separately, while amplification of 18S rRNA was carried out using DNA extracted from whole rumen liquor samples. For the Polymerase Chain Reaction (PCR) process, DNA was diluted to 5 ng/µl for 16S, 10 ng/µl for ciliates (18S), and 20 ng/µl for anaerobic fungi ( ITS 1) using sterile nuclease-free water. For the 16S rRNA gene, specific primers were used to amplify the V3-V4 region with primers 341F- 5′CCTACGGGNGGCWGCAG3′ and 805R- 5′GACTACHVGGGTATCTAATCC3′[22]. For 16S rRNA gene amplification in each reaction, total template DNA input was 12.5ng and primer concentrations for both the forward and reverse were 5 picomoles (pM) each. Thermal cycling conditions were an initial denaturation at 95 °C for 3 minutes; 25 cycles of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, and elongation at 72 °C for 30 seconds; final extension at 72 °C for 5 minutes. For ciliates, a specific primer set F-5′-CGATGGTAGTGTATTGGAC-3′ and R-5′-GGAGCTGGAATTACCGC-3′ targeting the 18S genes, as reported by Tapio et al ., [23]were used. In each reaction, forward and reverse primers were 5 pM, and total template DNA was 25.0 ng. Thermal cycling conditions were an initial denaturation at 95 °C for 3 minutes; 30 cycles of denaturation at 95 °C for 25 seconds, annealing at 60 °C for 30 seconds, and elongation at 72 °C for 40 seconds; final extension at 72°C for 5 minutes. Anaerobic fungi-specific primer F-5′-TACCCTTTGTGAATTTGTT-3′ and R-5′-ATCCATTGTCAAAAGTTGT-3′ targeting the Internal Transcribed Spacer (ITS-1) region were used to study rumen anaerobic fungal diversity[23]. In each reaction, forward and reverse primers were 5 pM, and total template DNA was 45.0 ng. Thermal cycling conditions were an initial denaturation at 95 °C for 3 minutes; 32 cycles of denaturation at 98 °C for 25 seconds, annealing at 55 °C for 30 seconds, and elongation at 72 °C for 40 seconds; final extension at 72°C for 5 minutes. All the amplicon primers were synthesized with a pre-tag of Illumina adapters required for sequencing. In addition, fecal samples were also processed for the amplification of 16S rRNA, 18S rRNA and ITS1genes. For amplicon library preparation, we have followed Illumina’s standard protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). The Quality of all the libraries was evaluated using either LabChip GXII Touch (Revvity) or QIAexcel advanced system (Qiagen). Amplicon sequencing of 16S and 18S libraries was performed on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, United States) at our NGS facility using 2 × 250 chemistry. ITS 1 libraries were sequenced on Illumina MiSeq with 2x150 chemistry. 5.4 Taxonomy Assignment and Statistical Analysis For taxonomy assignments, we used the 16S Metagenomics Labs application available on the BaseSpace sequence hub by Illumina. We used the Ribosomal Database Project (RDP) v.2.14 database[24], PR2 (Protist Ribosomal Reference) v.5.0.0 database, and UNIITE database v.10.0 to classify the bacterial, ciliate, and anaerobic fungus reads, respectively. The taxonomic profiles obtained were further analyzed using various tools. For statistical analysis, the sequence count of all samples was first rarified (normalized) to the minimum library size using Microbiome Analyst[25]. After normalization, we employed Statistical Analysis of Metagenomic Profiles (STAMP v2.0.8) [26] to calculate the relative abundance of each taxon (phylum and genus level) present in the samples. To identify significant differences among groups, we performed ANOVA followed by post-hoc Tukey tests, with multiple testing correction using the Benjamini-Hochberg method. Features with a p -value ≤0.05 were considered statistically significant. Comparative abundance profiles were plotted using GraphPad Prism. We have used the MOCHI online tool to measure alpha diversity and to plot taxonomic profiles[27]. Alpha diversity was calculated using Shannon diversity and Shannon evenness, determining statistical significance at a p -value threshold of ≤0.05 using the Kruskal-Wallis test. For beta diversity, we calculated a Bray-Curtis similarity distance matrix and visualized the results through principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS), with significance set at p -value ≤0.05 by permutational multivariate analysis of variance (PERMANOVA), using the Paleontological Statistical Software (PAST v 4.03) tool[28]. Additionally, we conducted Linear Discriminant Analysis Effect Size (LEfSe) using the LEfSe (v1.1.2) command line tool to identify discriminatory taxa among the groups. The threshold for significance was set at LDA score > 3.0 and a p -value ≤0.05. Abbreviations VFA: Volatile Fatty Acids MS: Masoor Straw MLM: Moringa Leaf Meal ITS1 gene: Internal Transcribed Spacer 1 gene PCoA : Principal Coordinate Analysis NMDS : Non-metric Multidimensional Scaling CAZRI: Central Arid Zone Research Institute TMR: Total Mixed Ration CP: Crude Protein TDN : Total Digestible Nutrients PERMANOVA : Permutational Multivariate Analysis of Variance LEfSe : Linear Discriminant Analysis Effect Size LDA: Linear Discriminant Analysis Declarations Ethics approval All experimental protocols were approved by the Institutional Animal Ethics Committee of the Indian Council of Agricultural Research–Central Arid Zone Research Institute (ICAR–CAZRI), Jodhpur, Rajasthan, India (Approval No. CAZRI/2022/IAEC/01). Consent to participate Not applicable. This manuscript does not contain any individual human data. Consent for publication Not applicable. This manuscript does not contain any individual human data. Availability of data and materials Raw amplicon metagenomic sequence data have been submitted to the Indian Biological Data Center (IBDC) under BioProject number INRP000182 https://www.ibdc.dbtindia.gov.in/inda/completeStudyDetailsById?studyid=INRP000182# ), National Center for Biotechnology Information (NCBI) under BioProject number PRJEB89572 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJEB89572 ) and European Nucleotide Archive (ENA) with Study Number ERP172596 (https://www.ebi.ac.uk/ena/browser/view/ERP172596). The datasets supporting the conclusions of this article are included within the article as tables, figures, and supplementary information. Conflicts of interest The authors declare no competing interests. Funding The project was funded by the Department of Biotechnology (DBT), Government of India. Project Reference No: BT/AQ/1/SP41105/2020. Author contributions C.N.- Methodology, Data curation, Formal analysis, Software, Writing original draft; M.B.- Methodology, Software; T.S.- Supervision, Validation, Writing-review & editing; R.P.-Supervision, Validation, Writing-review & editing; N.V.P. -Resources; A.K.P. -Resources; S. K.– Resources; R. N. K.- Resources; M. J. -Conceptualization, Funding acquisition, Supervision; C.G.J. - Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, validation, Writing-review & editing. Acknowledgements Not applicable. References Liang JB, Paengkoum P. Current status, challenges and the way forward for dairy goat production in Asia - conference summary of dairy goats in Asia. Asian-Australasian journal of animal sciences. 2019;32:1233–43. https://doi.org/10.5713/ajas.19.0272 Modi RJ, Patel NM, Patel YG, Islam MM, Nayak JB. Chapter 4 - Goat farming: A boon for economic upliftment. In: Rana T, editor. 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SupplementryFigure2.tiff Supplementary Fig. S2: Alpha diversity analysis: based on Shannon diversity and Shannon Evenness. (A-B) Rumen solid samples-bacterial taxonomy; (C-D) Rumen liquid samples-bacterial taxonomy. (E-F) Rumen liquor samples-ciliates taxonomy. (E-F) Rumen solid samples-anaerobic fungal taxonomy and (G-H) Rumen liquid samples-anaerobic fungal taxonomy. SupplementryFigure3.tiff Supplementary Fig. S3: The fecal bacterial community composition of MS, 20%MLM and 30%MLM feed groups at genus level. (A) Rarefaction curves; Alpha diversity analysis-(B) Shannon diversity and (C) Shannon Evenness. Beta diversity analysis based on Bray-Curtis similarity matrix (D) PCoA and (E) NMDS plots. (G) Bar plots showing bacterial genera present at over one percent mean relative abundance. SupplementryFigure4.tiff Supplementary Fig. S4: The fecal ciliates community composition of MS, 20%MLM and 30%MLM feed groups at genus level. (A) Rarefaction curves. Alpha diversity analysis-(B) Shannon diversity (C) Shannon Evenness. Beta diversity analysis based on Bray-Curtis similarity matrix (D) PCoA and (E) NMDS plots. (F) Bar plots showing ciliates genera present at over one percent mean relative abundance. SupplementryFigure5.tiff Supplementary Fig. S5: Bar plots showingspecies present at over one percent mean relative abundance. 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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-8826260","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":595164855,"identity":"5b42df27-1afd-4791-bbda-d2ba6b9f16be","order_by":0,"name":"Chitra Nehra","email":"","orcid":"","institution":"Gujarat Biotechnology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Chitra","middleName":"","lastName":"Nehra","suffix":""},{"id":595164856,"identity":"c08531ff-fd13-4450-ba9d-eb894549b63a","order_by":1,"name":"Minal Bhure","email":"","orcid":"","institution":"Gujarat Biotechnology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Minal","middleName":"","lastName":"Bhure","suffix":""},{"id":595164857,"identity":"eb075a34-1856-4ee7-847d-ca177a41a733","order_by":2,"name":"Tejas Shah","email":"","orcid":"","institution":"Gujarat Biotechnology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Tejas","middleName":"","lastName":"Shah","suffix":""},{"id":595164859,"identity":"cd5c554b-5b44-4098-a734-6f166e1c163c","order_by":3,"name":"Ramesh Pandit","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYLACxgYgwczD+ADMAAMD4rQwG5CohYGHTQKhBQ8wbz+d+PDnDgZ7/nbeY5Vfd9yTZ5A+fPgDQ8EdnFpkzuRuNuY9w5A44zBf2m3ZM8WGDXxpaRIMBs9wapFgyN0mzdjGkGDAzGN2W7ItIYGBh8cM6JfDuLXwv93+82cbgz1ISzFEC//nD3i1SORuY+BtY2DcANTC+BFiC9BuvFrebpbmbZMA+SVZmvFMgmEbD5uZRAJeh+Vu/Pizzcaev//swY8/dyTI8/MwP/7w4Q9uLfBQAAFmHiDBBmIlENIAA4w/iFU5CkbBKBgFIwoAAHuaSlEX9DIDAAAAAElFTkSuQmCC","orcid":"","institution":"Gujarat Biotechnology Research Center","correspondingAuthor":true,"prefix":"","firstName":"Ramesh","middleName":"","lastName":"Pandit","suffix":""},{"id":595164860,"identity":"77470ef4-4eeb-4a1f-af0a-3bac11f07876","order_by":4,"name":"Niteen V Patil","email":"","orcid":"","institution":"Central Arid Zone Research Institute (ICAR-CAZRI)","correspondingAuthor":false,"prefix":"","firstName":"Niteen","middleName":"V","lastName":"Patil","suffix":""},{"id":595164861,"identity":"ceae5142-ac8e-44d0-b2ed-3fee6e14277f","order_by":5,"name":"Ashutosh K Patel","email":"","orcid":"","institution":"Central Arid Zone Research Institute (ICAR-CAZRI)","correspondingAuthor":false,"prefix":"","firstName":"Ashutosh","middleName":"K","lastName":"Patel","suffix":""},{"id":595164862,"identity":"16d40796-bf68-4d42-8ca3-84d504ff912b","order_by":6,"name":"Subhash Kachhawaha","email":"","orcid":"","institution":"Central Arid Zone Research Institute (ICAR-CAZRI)","correspondingAuthor":false,"prefix":"","firstName":"Subhash","middleName":"","lastName":"Kachhawaha","suffix":""},{"id":595164863,"identity":"025e6154-7d8b-4a21-9320-cb92b94f0b7d","order_by":7,"name":"Ram N. Kumawat","email":"","orcid":"","institution":"Central Arid Zone Research Institute (ICAR-CAZRI)","correspondingAuthor":false,"prefix":"","firstName":"Ram","middleName":"N.","lastName":"Kumawat","suffix":""},{"id":595164864,"identity":"9dd2544d-650d-473b-aeb6-cc89aaf44c87","order_by":8,"name":"Madhvi Joshi","email":"","orcid":"","institution":"Gujarat Biotechnology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Madhvi","middleName":"","lastName":"Joshi","suffix":""},{"id":595164865,"identity":"af2fbd25-e235-4ddf-911b-52359d8d6060","order_by":9,"name":"Chaitanya G Joshi","email":"","orcid":"","institution":"Gujarat Biotechnology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Chaitanya","middleName":"G","lastName":"Joshi","suffix":""}],"badges":[],"createdAt":"2026-02-09 05:53:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8826260/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8826260/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103265519,"identity":"bc25759e-050e-4ee7-bfc5-5dfdfd9c6d8b","added_by":"auto","created_at":"2026-02-23 19:25:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":163868,"visible":true,"origin":"","legend":"\u003cp\u003eThe rumen bacterial community composition of MS, 20%MLM and 30%MLM feed groups at the phylum level. (A-B) Stacked bar plot showing the bacterial phyla present at over one percent mean relative abundance in rumen solid fraction and rumen liquid fraction; (C-D) Plot showing trend line of mean relative percentage of bacterial phyla present at over one percent, in rumen solid fraction and liquid fractions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/804be14a8457eb5631165a8b.png"},{"id":103265488,"identity":"12f0da01-5ece-4239-abd5-55cbde6dba21","added_by":"auto","created_at":"2026-02-23 19:24:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":297238,"visible":true,"origin":"","legend":"\u003cp\u003eThe rumen bacterial community composition of MS, 20%MLM and 30%MLM feed groups at the genus level. (A) PCoA and (B) NMDS plots of rumen solid fraction; (C) PCoA and (D) NMDS plots of rumen liquid fraction. Box plots showing comparison of mean relative abundance of selected bacterial genera. (E-I) in the rumen solid fraction and (K-P) in the rumen liquid fraction. Bar plots of bacterial genera present at over one percent mean relative abundance, (Q) in rumen solid fraction\u003cstrong\u003e \u003c/strong\u003eand (R) in rumen liquid fraction.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/387532e9a0ee711f8d91d09a.png"},{"id":103265480,"identity":"9e06a8da-a002-4c52-8f1d-690b9ef9e986","added_by":"auto","created_at":"2026-02-23 19:24:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":170020,"visible":true,"origin":"","legend":"\u003cp\u003eThe rumen ciliates taxonomy analysis of MS, 20%MLM and 30%MLM feed groups at the genus level. (A) PCoA and (B) NMDS plots of rumen liquor samples. (C-F) Box plots comparing the mean relative abundance of selected ciliates genera in the rumen liquor. (G) Bar plots showing ciliates genera present at over one percent mean relative abundance in rumen liquor.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/465ba63d07fd805ac100c9ed.png"},{"id":103265484,"identity":"655365e1-59d5-463f-9442-3bed3c266061","added_by":"auto","created_at":"2026-02-23 19:24:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":191981,"visible":true,"origin":"","legend":"\u003cp\u003eThe rumen anaerobic fungi taxonomy analysis of MS, 20%MLM and 30%MLM feed groups at genus level. (A) PCoA and (B) NMDS plots of rumen solid fraction and (C) PCoA and (D) NMDS plots of rumen liquid fraction. Bar plots showing anaerobic fungal genera present at over one percent mean relative abundance. (E) in rumen solid fraction\u003cstrong\u003e \u003c/strong\u003eand (F) in rumen liquid fraction.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/dc91a9952cef5e2b590d6ae8.png"},{"id":103505887,"identity":"d2f55f2f-931d-43eb-8e91-b7de9ca8759e","added_by":"auto","created_at":"2026-02-26 13:33:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174395,"visible":true,"origin":"","legend":"\u003cp\u003eLinear discrimination analysis (LDA) effect size (LEfSe) plots of significantly different bacterial and ciliates genera present across MS, 20%MLM and 30%MLM feed groups. (A) Bacterial genera present in rumen solid fraction; (B) Bacterial genera present in rumen liquid fraction; (C) Ciliates genera present rumen liquid fraction.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/c815a04539db7eda880edb23.png"},{"id":103509684,"identity":"b296c9ad-0c82-4fec-ae58-f093517e5264","added_by":"auto","created_at":"2026-02-26 14:00:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1634995,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/859632db-3d4f-4f9b-9697-4ec5e9bba44e.pdf"},{"id":103265479,"identity":"b03973dd-85f9-4541-9349-5b05cde52630","added_by":"auto","created_at":"2026-02-23 19:24:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/9a2140c56b7f262d15fe1ed0.xlsx"},{"id":103265520,"identity":"0948df74-8a0f-4e5d-96eb-4bdb7a5bc19c","added_by":"auto","created_at":"2026-02-23 19:25:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16686,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/58ac91dbea8b0425cf574941.xlsx"},{"id":103265496,"identity":"a38b4a5e-11a2-4255-b3ba-402bde453c52","added_by":"auto","created_at":"2026-02-23 19:24:54","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":79858,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/fcd1711b5801ac44986288b0.xlsx"},{"id":103265516,"identity":"f35e1cc2-a36a-4a24-870b-ff545252e889","added_by":"auto","created_at":"2026-02-23 19:25:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":164895,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/f585fd18633a6591a778b571.xlsx"},{"id":103265509,"identity":"8ad51fdd-3bc8-4740-ad6f-24d2b1b94bdd","added_by":"auto","created_at":"2026-02-23 19:25:03","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":82580,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/8526d28a3542f2f782137bf5.xlsx"},{"id":103265486,"identity":"624b8202-8930-43f3-8029-e1553827f1ea","added_by":"auto","created_at":"2026-02-23 19:24:50","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16387,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/2a71259ad844cbc77c1faf77.xlsx"},{"id":103265512,"identity":"befccfaf-0fd2-4422-9aea-f71f9fb3c3ce","added_by":"auto","created_at":"2026-02-23 19:25:03","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":971395,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/d6c59f5064672ada18b5bd02.xlsx"},{"id":103265531,"identity":"748c83e6-f408-452b-92f4-14d040ca420f","added_by":"auto","created_at":"2026-02-23 19:25:09","extension":"tiff","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":32495111,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementryFigure6.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/e697037fe922bec28b79220e.tiff"},{"id":103265518,"identity":"25cf8151-e53b-4dde-b686-9552bd25956b","added_by":"auto","created_at":"2026-02-23 19:25:07","extension":"tiff","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":192012782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S1:\u003c/strong\u003e Rarefaction curves of MS, 20%MLM and 30%MLM feed groups. (A) Rumen solid and (B) Rumen liquid samples-bacterial taxonomy. (C) Rumen liquor samples-ciliates taxonomy; (D) Rumen solid and (E) Rumen liquid samples-anaerobic fungal taxonomy.\u003c/p\u003e","description":"","filename":"SupplementryFigure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/90324a2ecb040c974416c8e3.tiff"},{"id":103265532,"identity":"f7b34197-235c-4c08-9b00-8474c8e82036","added_by":"auto","created_at":"2026-02-23 19:25:10","extension":"tiff","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":192012782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S2:\u003c/strong\u003e Alpha diversity analysis: based on Shannon diversity and Shannon Evenness. (A-B) Rumen solid samples-bacterial taxonomy; (C-D) Rumen liquid samples-bacterial taxonomy. (E-F) Rumen liquor samples-ciliates taxonomy. (E-F) Rumen solid samples-anaerobic fungal taxonomy and (G-H) Rumen liquid samples-anaerobic fungal taxonomy.\u003c/p\u003e","description":"","filename":"SupplementryFigure2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/5a0e692d842e725e3797d400.tiff"},{"id":103265530,"identity":"ce21b24b-947d-4ce0-a2ed-edaa79f253e5","added_by":"auto","created_at":"2026-02-23 19:25:09","extension":"tiff","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":192012782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S3:\u003c/strong\u003e The fecal bacterial community composition of MS, 20%MLM and 30%MLM feed groups at genus level. (A) Rarefaction curves; Alpha diversity analysis-(B) Shannon diversity and (C) Shannon Evenness. Beta diversity analysis based on Bray-Curtis similarity matrix (D) PCoA and (E) NMDS plots. (G) Bar plots showing bacterial genera present at over one percent mean relative abundance.\u003c/p\u003e","description":"","filename":"SupplementryFigure3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/eca0e9f1f1e118cf451bf482.tiff"},{"id":103265533,"identity":"37fd0284-2932-47da-806c-0ea3a1018271","added_by":"auto","created_at":"2026-02-23 19:25:14","extension":"tiff","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":192012782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S4:\u003c/strong\u003e The fecal ciliates community composition of MS, 20%MLM and 30%MLM feed groups at genus level. (A) Rarefaction curves. Alpha diversity analysis-(B) Shannon diversity (C) Shannon Evenness. Beta diversity analysis based on Bray-Curtis similarity matrix (D) PCoA and (E) NMDS plots. (F) Bar plots showing ciliates genera present at over one percent mean relative abundance.\u003c/p\u003e","description":"","filename":"SupplementryFigure4.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/6b5d0ef1a54f712cfab43a16.tiff"},{"id":103265489,"identity":"ae873bbc-9552-4c62-80bd-1dac11c3d44d","added_by":"auto","created_at":"2026-02-23 19:24:51","extension":"tiff","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":192012782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S5:\u003c/strong\u003e Bar plots showingspecies present at over one percent mean relative abundance.\u003c/p\u003e","description":"","filename":"SupplementryFigure5.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8826260/v1/456e4df6d9aa0e8cf1b85bcb.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"A diet containing Moringa oleifera alters the goat rumen microbiome: an insight into bacteria, ciliates and rumen anaerobic fungi","fulltext":[{"header":" 1. Background","content":"\u003cp\u003eAsia, home to over half of the world's goats with approximately one billion goats, is the cradle of goat domestication. Goats, including dairy breeds, thrive in diverse and harsh environments, providing essential nutrition, food security, and socio-economic benefits across many Asian countries, particularly in India[1]. In India, goats are a crucial source of income for small-scale farmers, providing milk, meat, fiber, hide, and manure[2]. Sustainable livestock production hinges on maximizing feed efficiency, which refers to an animal's ability to convert feed into output and improve nutrient utilization, thereby enhancing the animal's performance. Feed efficiency in small ruminants, such as sheep and goats, is a vital productive trait that significantly impacts farmers’ profitability[3]. Improved feed efficiency is also related to decreased dung and methane production[4]. Consequently, improving feed efficiency can boost livestock industry profitability while simultaneously mitigating environmental impacts.\u003c/p\u003e\n\u003cp\u003eThese improvements in feed utilization and environmental impact are mostly mediated by the rumen microbiome, which is vital to digestive and metabolic processes[5]. In ruminant’s complex plant materials are digested to yield volatile fatty acids (VFAs), microbial proteins, and vitamins[6]. The composition and diversity of rumen microbiome, which includes bacteria, archaea, ciliate protozoa and anaerobic fungi, significantly impacts the host's health[7]. Rumen microbes play a key role in various nutrient and carbohydrate metabolism in the gastrointestinal tract of ruminants by producing different enzymes that break down complex carbohydrates and other feed components into simpler forms, allowing their absorption and utilization by the host[8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMoringa oleifera\u003c/em\u003e is a native Indian plant which produces several harvests and grows in hot, dry climates and wet tropical regions[9]. Moringa leaves have high nutritional properties, being a rich source of protein and other necessary elements including vitamins[10]. Previous studies have shown that moringa leaf feed improves rumen fermentation and nutrient digestibility and boosts the quantity and quality of milk in lactating goats [11,12]. Additionally, after moringa feeding, improved growth performance and decreased methane production, without having an adverse effect on animal health, have also been reported[13].\u003c/p\u003e\n\u003cp\u003eIn animals, rumen microbiome and diet interactions are associated with overall health and productivity[14]. Different diets have been reported to improve animal health and performance by affecting the rumen microbiota composition [15]. Although various studies have reported the beneficial effects of the moringa diet on animal health and productivity [13,16], very limited research has been carried out to understand its effect on the rumen bacterial communities[17,18], including our previous studies[19,20]. Moreover, to date, no studies have reported its effects on rumen ciliates or anaerobic fungal populations. Hence, to examine the comprehensive effect of moringa leaf diet on rumen and fecal bacterial, ciliates and anaerobic fungal communities, here, we have analyzed the sequencing data targeting to V3-V4 region of 16S rRNA, ciliates specific18S rRNA, and rumen anaerobic fungal specific internal transcribed spacer 1 (\u003cem\u003eITS\u003c/em\u003e1) genes in the lactating goats. In present study, we have analyzed and compared the rumen microbiota (bacteria, ciliates and anaerobic fungus) composition in three groups of lactating goats; first group was fed masoor straw (MS) based diet, second group was fed 20%moringa leaf meal (20%MLM), and third group was given 30% moringa leaf meal (30%MLM) in their regular diet. Simultaneously, fecal samples were also processed to compare bacterial and ciliate diversity among the three groups. We hypothesize that an increasing proportion of moringa in the diet induces a dose-dependent restructuring of the rumen and fecal microbial communities. The current findings help in understanding the rumen and fecal microbial diversity shift in response to a moringa diet in small ruminants and open a new avenue to formulate moringa-fortified diet for improved animal health and productivity\u0026nbsp;\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2.1 Sequencing Data Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing read counts and the percentage of reads classified at the genus level (after removing samples with low sequencing read counts and outliers identified through PCoA plots) \u0026nbsp;for each sample, are provided in Supplementary Table 2. We generated a total of 12.63, 27.25 and \u0026nbsp;5.6 \u0026nbsp; million reads to analyze rumen bacterial, protozoal and anaerobic fungal diversity, respectively. Similarly, from fecal samples, a total of 9.2 and 12.50 million \u0026nbsp;reads were generated for bacteria and ciliates, respectively. For rumen anaerobic fungi , we were unable to obtain amplification from fecal samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Rumen and Fecal Bacterial Community Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 16SrRNA data, for rumen solid and liquid fractions as well as fecal samples, we observed that as the number of sequences increased, the rarefaction curve flattened out (Supplementary Fig. 1A-1B, Supplementary Fig. 3A), indicating enough data to represent the microbial diversity present in samples. Beta diversity analysis (Fig. 2A-2D) showed significant differences (PERMNOVA, \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05) in bacterial community structure among MS, 20% MLM, and 30% MLM groups. In fecal samples, no significant difference was observed among the groups (Supplementary Fig. 3D-3E). The Shannon diversity and evenness metrics did not reveal significant differences among different dietary groups (Supplementary Fig. 2A-2D, Supplementary Fig. 3B-3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 46 different phyla were detected both in rumen solid and liquid fractions (Supplementary Table 3A,3B). Cumulative bar charts of different phyla with relative abundance above 1% are provided in Fig. 1A and 1B. The most abundant bacterial phyla in both solid and liquid fractions across all the groups were \u003cem\u003eBacteroidota\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacillota\u003c/em\u003e, formerly known as \u003cem\u003eBacteroidetes\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFirmicutes\u003c/em\u003e, respectively. In addition, phyla such as \u003cem\u003eVerrucomicrobiota\u003c/em\u003e, \u003cem\u003eActinomycota\u003c/em\u003e, \u003cem\u003eCyanobacteriota\u003c/em\u003e, \u003cem\u003eSynergistota\u003c/em\u003e, \u003cem\u003eFibrobacterota\u0026nbsp;\u003c/em\u003eand \u003cem\u003eSpirochaetota\u0026nbsp;\u003c/em\u003ewere also present with relative abundance above 1%. Further, to see the effect of moringa, we compared the presence of different phyla among all three dietary groups. In the rumen solid fraction, the proportion of \u003cem\u003eBacillota\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePseudomonadata\u0026nbsp;\u003c/em\u003e(formerly known as \u003cem\u003eProteobacteria\u003c/em\u003e) increased with the moringa diet, being highest in the 30% MLM group, while the abundance of \u003cem\u003eSynergistota\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFibrobacterotota\u0026nbsp;\u003c/em\u003edecreased (Fig. 1A,1C). In the rumen liquid fraction, the proportion of \u003cem\u003ePseudomonadata\u0026nbsp;\u003c/em\u003ewas found\u003cem\u003e\u0026nbsp;\u003c/em\u003eto increase in the 20% and 30% MLM groups (Fig. 1B, 1D). In both solid and liquid fractions, the ratio of \u003cem\u003eBacillota\u0026nbsp;\u003c/em\u003eto \u003cem\u003eBacteroidota\u0026nbsp;\u003c/em\u003ewas higher in the 20% MLM and 30% MLM groups compared to the MS group (Supplementary Table 3C). In fecal samples, a total of 40 phyla were identified with \u003cem\u003eBacillota\u0026nbsp;\u003c/em\u003e(60.09%-64.55%) and \u003cem\u003eBacteroidota\u0026nbsp;\u003c/em\u003e(18.40%-19.63%) being the most abundant taxa across all three dietary groups (Supplementary Table 3D, 3E).\u003c/p\u003e\n\u003cp\u003eAt the genus level, in total, 621 and 1,114 different genera were detected in the rumen solid and liquid fraction, respectively (Supplementary Tables 4A, 4B). \u003cem\u003eSegatella\u003c/em\u003e had the highest relative abundance in both the solid (12.36-15.18%) and liquid fractions (10.6-11.36%) among all three dietary groups, along with other genera such as \u003cem\u003eXylanibacter, Subdivision5_genera_incertae_sedis, Sodaliphilus, Hallella\u003c/em\u003e, and \u003cem\u003eSucciniclasticum\u003c/em\u003e, each with a relative abundance greater than 1% (Supplementary Table 4C). In the rumen solid fraction,\u003cem\u003e\u0026nbsp;Xylanibacter\u003c/em\u003e, \u003cem\u003eSaccharofermentans\u0026nbsp;\u003c/em\u003eand \u003cem\u003eRuminococus\u003c/em\u003e were significantly higher (\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05) in moringa feed groups than MS groups (Fig. 2E-2G). Genera like \u003cem\u003eLeyella, Paraprevetolla\u003c/em\u003e \u003cem\u003eButyrivibrio, Clostridium_sensu_stricto,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Enterocloster\u0026nbsp;\u003c/em\u003ealso increased in response to moringa feeding (Fig. 2Q). On the other hand, \u003cem\u003eSodaliphilus, Succiniclassticum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFibrobacter\u0026nbsp;\u003c/em\u003esignificantly (\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05) reduced in 20%, and 30%MLM feed groups as compared to MS group (Fig. 2H-2J). In the rumen liquid fraction, the abundance of \u003cem\u003eXylanibacter\u003c/em\u003e \u003cem\u003eSaccharofermentans, Lucifera, Butyrivibrio, Enterocloster,\u003c/em\u003e remined higher in animals who were fed a moringa comprising diet (Fig. 2K, 2L, 2R). While the abundance of \u003cem\u003eSodaliphilus, Succiniclasticum, Fibrobacter, Segatella, Hallella, Olsenella, Slackia, Selenomonas, Treponema\u003c/em\u003e, \u003cem\u003eSyntrophococus\u003c/em\u003e and \u003cem\u003ePseudobutyrivibrio\u003c/em\u003e were higher in the MS group (Fig. 2N-2P, 2R). In fecal samples, 642 different taxa were detected at the genus level (Supplementary Table 4D). In the moringa-fed groups, the relative abundance of \u003cem\u003eMonoglobus,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFalsiporphyromonas,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eErcella, Luoshenia,\u003c/em\u003e and \u003cem\u003ePapillibacter\u0026nbsp;\u003c/em\u003eincreased, whereas \u003cem\u003eMuriventricula, Ruminococcus\u003c/em\u003e, and \u003cem\u003eSaccharofermentans\u003c/em\u003e reduced (Supplementary Fig. 3F, Supplementary Table 4E).\u003c/p\u003e\n\u003cp\u003eLEfSe analysis was conducted to identify bacterial taxa that were significantly represented to each feeding group in both the rumen solid and liquid fractions. In the solid fraction of rumen digesta, a total of 12, 5, and 8 taxa were significantly enriched in the MS, 20% MLM and 30% MLM groups, respectively (Supplementary Table 7A). Genera such as \u003cem\u003eSodaliphilus, Fibrobacter, Olsenella\u003c/em\u003e, and \u003cem\u003eAnseongella\u003c/em\u003e were enriched in the MS group. \u003cem\u003eRuminococcus, Xylanibacter, Pseudobutyrivibrio\u003c/em\u003e, and \u003cem\u003eCoprococcus\u003c/em\u003e were dominant in the 20% MLM group, while \u003cem\u003eAnaeroplasma, Enterocloster, Ruminobacter, Butyrivibrio\u003c/em\u003e, and \u003cem\u003eSaccharofermentans\u003c/em\u003e were abundant in the 30% MLM group (Fig. 5A). Similarly, in the liquid fraction, 7, 3, and 10 taxa were significantly enriched in the MS, 20% MLM, and 30% MLM diet group, respectively (Supplementary Table 7B). Genera such as \u003cem\u003eSodaliphilus, Slakia\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOlsenella\u003c/em\u003e were dominant in the MS group, \u003cem\u003eDuncaniella, Algoriphagus\u003c/em\u003e, and \u003cem\u003eSubdivision5_genera_incertae_sedis\u003c/em\u003e in the 20% MLM group, and \u003cem\u003eParaprevotella,\u003c/em\u003e \u003cem\u003eRuminococcus, Succinimonas, Blautia, Acetivibrio\u003c/em\u003e, and \u003cem\u003eRuminococcoides\u003c/em\u003e were enriched in the 30% MLM feed group (Fig. 5B). In the fecal samples, we did not observe \u0026nbsp;significant taxa.,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Ciliates in Rumen and Fecal Samples of Three Dietary Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor ciliates also, we generated sufficient data as shown in Supplementary Fig. 2C and Fig. 4A. Beta diversity analysis showed significant differences (PERMONOVA, \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05) in rumen ciliate community structure among the three groups in rumen samples (Fig. 3A-3B). Although, in fecal samples, our analysis revealed no significant difference among the three groups (Supplementary Fig. 4D-4E). Further, alpha diversity analysis using Shannon diversity and evenness did not turn up with any significant difference among different dietary groups in both rumen and fecal samples (Supplementary Fig. 2E-2F, Supplementary Fig. 4B-4C).\u003c/p\u003e\n\u003cp\u003eHere, we identified a total of 73 genera in rumen samples (Supplementary Table 5A, 5B) with \u0026nbsp;\u003cem\u003ePolyplastron\u003c/em\u003e (26.65%- 29.97%), \u003cem\u003eEntodinium\u0026nbsp;\u003c/em\u003e(3.78%-16.39%)\u003cem\u003e, Enoploplastron\u0026nbsp;\u003c/em\u003e(11.89%-20.05%), \u003cem\u003eTrichostomatia_XX\u0026nbsp;\u003c/em\u003e(6.25%-8.76%),\u003cem\u003e\u0026nbsp;Diploplastron\u0026nbsp;\u003c/em\u003e(7.01%-11.98%)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eEpidinium\u0026nbsp;\u003c/em\u003e(6.49%-7.92%) were major genera detected in all three dietary groups.\u003cem\u003eEnoploplastron\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Diploplastron\u0026nbsp;\u003c/em\u003ewere \u0026nbsp;significantly higher (\u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05) in moringa feed groups compared to the MS group, being higher with the addition of moringa in the feed (Fig. 3C, 3D). Conversely, \u003cem\u003eEntodium\u003c/em\u003e and \u003cem\u003eEntodinium_1\u003c/em\u003e were significantly lower (\u003cem\u003ep\u003c/em\u003e-value \u0026gt;0.05) in moringa feed groups (Fig. 3E-F). Other genera with differential abundance among feeding groups are provided in Fig. 3G. In fecal samples, \u003cem\u003eBlastocystis\u0026nbsp;\u003c/em\u003ewas observed as the most abundant (85.86%-97.99%) genus in all three dietary groups (Supplementary Table 5E, 5F).Species-level results for rumen ciliates are provided in Supplementary Fig. 5 andSupplementary Table 5C, 5D.\u003c/p\u003e\n\u003cp\u003eIn the LEfSe analysis of rumen samples, we found that 12 genera were significantly enriched in the MS group, while five and eight taxa were significantly enriched in the 20% MLM and 30% MLM groups, respectively (Fig. 5C, Supplementary Table 7C). In the MS group, the genera \u003cem\u003eEntodinium, Entodinium_1, Trichostomatia_XX\u003c/em\u003e, and \u003cem\u003eOphryoscolacidae_1_X\u003c/em\u003e had come out as signature genera. Similarly, in the 20% MLM group, \u003cem\u003eDasytricha, Diplodinium, Eremoplastron\u003c/em\u003e, and \u003cem\u003eIsotrichidae_2_X\u003c/em\u003e and \u003cem\u003eDiploplastron\u003c/em\u003e and \u003cem\u003eMetadinum\u003c/em\u003e in the 30% MLM group were identified as key ciliate genera. No significantly enriched taxa were identified in the fecal samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Structure and Composition of Rumen Anaerobic Fungi in Three Dietary Groups\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs like 16SrRNA and 18SrRNA data, the rarefaction curve of \u003cem\u003eITS\u003c/em\u003e1 sequences also gradually flattened out, suggesting an adequate amount of sequencing data, which facilitates further analysis (Supplementary Fig. 2D and 2E). Beta diversity analysis couldn’t find any significant differences (\u003cem\u003ep\u003c/em\u003e-value ≥0.05) in anaerobic fungal community structures among the three treatments groups \u0026nbsp; (Fig. 4A-4D). Also, changes in the alpha diversity (Shannon and evenness metrics) remined insignificant \u0026nbsp;among the three different dietary groups (Supplementary Fig. 2G-2J).\u003c/p\u003e\n\u003cp\u003ePhylum \u003cem\u003eNeocallimastigomycota\u0026nbsp;\u003c/em\u003ewas present with more than 99% relative abundance across all three dietary groups in both the solid and liquid fractions of the rumen (Supplementary Table 6A). At the genus level, 21 genera were identified in the rumen solid fraction, while 19 genera were identified in the rumen liquid fraction samples (Supplementary Table 6B, 6C). \u003cem\u003eNeocallimastix\u003c/em\u003e and \u003cem\u003ePecoramyces\u003c/em\u003e were the most abundant genera in all three groups, where \u003cem\u003eNeocallimastix\u0026nbsp;\u003c/em\u003edecreased in moringa feed groups compared to the MS group in both solid and liquid fractions (Fig. 4E, 4F, Supplementary Table 6D). Intriguingly, in solid fraction, \u003cem\u003ePiromyces\u003c/em\u003e increased with the addition of moringa in the feed, while it was inverse in the liquid fraction. At the species level, the proportion of \u003cem\u003ePecromyces ruminatium\u0026nbsp;\u003c/em\u003ewas higher in the moringa feed groups than in the MS group. (Supplementary Fig. 6A, 6B; Supplementary Table 6G). In the LEfSe analysis of both rumen solid fraction samples, we couldn’t \u0026nbsp;find any enriched taxa among the groups.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe rumen microbiome comprises hundreds of species, including bacteria, archaea, ciliates, fungi, and viruses [29], which provide adaptation and resilience to the host against different dietary modifications[30]. A healthy rumen microbiome improves growth performance and production traits of the animal[31]. These microbial communities undergo variations in their composition in response to different dietary regimes[32]. Indeed, diet is considered a main reason for the difference in the taxonomic composition of the rumen microbiome. Various beneficial effects, including enhanced production traits, reduced methane emissions, and improved digestibility, have been reported in goats that consume moringa. Although, only a few studies, including our earlier one[20], have focused on the shifts in the rumen microbiota associated with moringa feed[17,18]. Therefore, the present study provided a comprehensive view of the effects of moringa-added ration on rumen and fecal microbiota in lactating goats.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;The moringa diet has less effect on the species richness and evenness of goats’ rumen bacteria, ciliates, and rumen anaerobic fungi. However, the proportion of several specific microbial taxa was significantly altered when goats consumed moringa-containing feed. Across the three dietary groups, \u003cem\u003eBacteroidota\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacillota\u003c/em\u003e were identified as the most abundant bacterial phyla in rumen and fecal samples, which is consistent with findings of previous study[33]. Different genera and species belonging to \u003cem\u003eBacteroidota\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacillota\u003c/em\u003e have an active role in carbohydrate and protein metabolism[34], indicating their key role in the feed digestion in the rumen. In the present study , we found a shift in the rumen bacterial community in response to moringa feed, which is consistent with previous studies reporting changes in rumen microbiota in response to dietary changes\u0026nbsp;[35].\u0026nbsp;Notably, the proportion of \u003cem\u003eBacillota\u003c/em\u003e group was higher in moringa feed groups, leading to a higher \u003cem\u003eBacillota\u003c/em\u003e:\u003cem\u003eBacteroidota\u003c/em\u003e ratio compared to control group. A higher \u003cem\u003eBacillota\u003c/em\u003e: \u003cem\u003eBacteroidota\u003c/em\u003e ratio has been linked to lower residual feed intake as well as improved energy absorption and storage in the host\u0026nbsp;[3]. The moringa containing diet also led to significant changes in the rumen bacterial community, with a higher abundance of the \u003cem\u003ePseudomonadota\u003c/em\u003e phylum in moringa feed groups compared to the MS group. \u003cem\u003ePseudomonadota\u003c/em\u003e members have active role in propionate production in the rumen\u0026nbsp;[36]\u0026nbsp;and a lower abundance of \u003cem\u003ePseudomonadota\u003c/em\u003e has been associated with higher methane emissions. In high methane-emitting beef cattle, \u003cem\u003ePseudomonadota\u003c/em\u003e abundance was found four times less than in low-emitters[14]. This suggests that the increased abundance of \u003cem\u003ePseudomonadota\u003c/em\u003e in response to a moringa diet may contribute to lower methane emissions in lactating goats. A similar finding have been reported using metatranscriptome approach where abundance of \u003cem\u003ePseudomonas\u0026nbsp;\u003c/em\u003eand \u003cem\u003eProteus\u003c/em\u003e increased in rumen of sheep fed with 15% moringa along with reduction in methane[37]\u0026nbsp;. However, further investigations are required to confirm this effect. Therefore, we suggest performing moringa feeding experiments that simultaneously measure methane emission along with comprehensive microbiome analysis\u003c/p\u003e\n\u003cp\u003eFurther, in this study, the goat rumen harbor \u003cem\u003eXylanibacter\u003c/em\u003e and \u003cem\u003eSegatella\u003c/em\u003e as the most dominant bacterial genera. Overall, moringa has higher protein and lower fiber content[38]. In this study, we found relatively more abundance of \u003cem\u003eXylanibacter\u0026nbsp;\u003c/em\u003ein the moringa feed groups compared to the MS group. The higher abundance of \u003cem\u003eXylanibacter\u003c/em\u003e suggests its active role in the digestion of protein-rich moringa feed. Moreover, the fibrinolytic bacterial genera \u003cem\u003eFibrobacter\u003c/em\u003e and \u003cem\u003eSucciniclasticum\u003c/em\u003e were less abundant in the moringa feed groups, aligning with previous study describing a lower abundance of these genera in response to low-fiber diets [39]. The elevated abundance of \u003cem\u003eFibrobacter\u003c/em\u003e and \u003cem\u003eSucciniclasticum\u003c/em\u003e in the MS group can be attributed to the higher fiber and lignin content of masoor straw compared to moringa. The \u003cem\u003eSaccharofermentans, Butyrivibrio\u003c/em\u003e and \u003cem\u003eRuminococcus\u0026nbsp;\u003c/em\u003egenera were higher in moringa feed groups. \u003cem\u003eRuminococcus\u003c/em\u003e are known for producing the majority of cellulase enzymes in the rumen, whereas \u003cem\u003eSaccharofermentans\u003c/em\u003e play a role in degrading plant polysaccharides and producing VFAs like acetate and propionate as the final fermentation products[40]. Earlier studies also showed a positive association between crude protein content of forages\u003cem\u003e\u0026nbsp;\u003c/em\u003eand the abundance of\u003cem\u003e\u0026nbsp;Butyrivibrio\u003c/em\u003e[15]. Species of \u003cem\u003eButyrivibrio\u0026nbsp;\u003c/em\u003eare metabolically diverse, contributing to the production of butyrate, bacteriocins, and vitamins during the fermentation process in the rumen[41]. Additionally, bacterial genera like \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eButyrivibrio\u003c/em\u003e are linked with lower methane emissions, higher digestible dry matter and digestible organic matter intake[41]. In fecal samples, the presence of \u003cem\u003eMonoglobus\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Papillibacter\u0026nbsp;\u003c/em\u003ewas observed more frequently in the moringa feed groups. \u003cem\u003ePapillibacter\u003c/em\u003e is considered a beneficial bacterium associated with a healthy gut microbiome, and \u003cem\u003eMonoglobus\u003c/em\u003e has been reported to have a positive correlation with enhanced immune responses in goats [34]. Both \u003cem\u003ePapillibacter\u003c/em\u003e and \u003cem\u003eMonoglobus\u003c/em\u003e are known to produce butyrate, a short-chain fatty acid with significant health benefits[42]. Overall, these findings suggest that moringa feed enriches the bacterial genera which has a beneficial role in lactating goat health and is associated with lower methane emissions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough, we have not analyzed phytochemical composition of moringa in this study, moringa leaves are rich source of saponins, condensed tannins, flavonoids and other polyphenols [43,44]. These compounds can an modulate rumen microbial ecology and fermentation\u0026nbsp;[45,46]. Tannins and other phenolic compounds could bind with dietary proteins, which can slow down the breakdown of these proteins in the rumen. This process enhances the supply of protein later in the rumen digestion process, leading to a preference for microbial taxa that thrives on higher protein sources. This could explain why we see a greater presence of protein-degrading \u003cem\u003eXylanibacter\u003c/em\u003e and related genera like \u003cem\u003eButyrivibrio\u003c/em\u003e in the groups that include moringa. Additionally, flavonoids and polyphenols have been shown to have antimicrobial and antioxidant properties. They can help keep certain harmful bacteria in check while encouraging the growth of beneficial fermentative bacteria that produce short-chain fatty acids (SCFAs), such as Saccharofermentans, \u003cem\u003eRuminococcus\u003c/em\u003e, \u003cem\u003eButyrivibrio\u003c/em\u003e, \u003cem\u003eMonoglobus\u003c/em\u003e, and \u003cem\u003ePapillibacter\u003c/em\u003e. In this study, all these genera were enriched in the moringa fed goats might play a crucial role in providing energy and supporting gut health.\u003c/p\u003e\n\u003cp\u003eRumen ciliates are crucial for feed digestion, significantly enhancing the digestibility of the feed [47]. The genus \u003cem\u003eEntodinium\u003c/em\u003e is typically reported as the most abundant one in ruminants. However, in this study, \u003cem\u003ePolyplastron\u003c/em\u003e emerged as the most abundant genus in all rumen samples, aligning with some previous studies in goats\u0026nbsp;[48]. It’s also been reported that the abundant ciliate genera in the rumen microbial population can be rehabilitated based on the host species, the diet they consume, and even season\u0026nbsp;[49]. In the present study, we have found a significantly higher abundance of \u003cem\u003eEnoploplastron\u0026nbsp;\u003c/em\u003eand \u003cem\u003eDiploplastron\u003c/em\u003e in goats fed the moringa diet compared to the masoor straw diet. These genera play an active role in carbohydrate degradation, VFA production during the fermentation process and maintain the bacterial population in the rumen[50]. We also observed a higher abundance of ciliate species such as \u003cem\u003eEnoploplastron triloricatum, Polyplastron multivesiculatum,\u003c/em\u003e and \u003cem\u003eDiploplastron affine\u003c/em\u003e in moringa-fed groups. These species play a key role in the digestion of cellulose in the rumen\u0026nbsp;[51]\u003cstrong\u003e\u0026nbsp;.\u003c/strong\u003e\u003cem\u003eDiploplastron affine\u0026nbsp;\u003c/em\u003ecan digest starch and murein to meet the host’s energy requirement\u0026nbsp;[52].\u0026nbsp;These findings\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehighlight the complex interactions between diet and the rumen microbial ecosystem. Moreover, the ciliate species \u003cem\u003eBuxtonella sulcata\u003c/em\u003e and \u003cem\u003eBlastocystis ratti,\u003c/em\u003e which cause diarrhea in animals\u0026nbsp;[53,54], were found to be reduced in the goats that were fed moringa as compared to the MS group. This could be because of specific activity of different phytochemicals present in the moringa. Saponins found in Moringa have consistently been linked to a decrease in protozoal populations and methanogenic activity. This, in turn, can create a more favorable environment for bacterial groups that enhance microbial protein synthesis and help cut down on methane emissions [55,56].\u0026nbsp;Overall, these outcomes suggest that moringa feed could positively impact the health of lactating goat. Previous studies on moringa supplementation, conducted both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, in different animals, have reported reduced protozoal population[57,58].\u0026nbsp;However, all these studies are count-based and have not performed comprehensive amplicon sequencing. Therefore, further protozoal challenge studies with moringa feeding, correlating pathogen reduction, will strengthen these findings.\u003c/p\u003e\n\u003cp\u003eOverall, a moringa-based diet has a little less effect on the rumen anaerobic fungal community. This might be because rumen anaerobic fungi are specifically engaged in fiber degradation, and moringa leaves have less fiber as compared to masoor straws in this study. In this study, \u003cem\u003eNeocallimastigomycota\u003c/em\u003e is the most abundant phylum across all dietary groups. These results align with earlier studies[59]. Earlier study have shown that the \u003cem\u003egenus\u003c/em\u003e Neocallimastix decreases with lower fiber content\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e[60]\u003cstrong\u003e;\u003c/strong\u003e here, in this study, we observed the lower abundance of \u003cem\u003eNeocallimastix\u003c/em\u003e in the moringa feed groups compared to the MS group (fiber-rich feed). \u003cem\u003ePecromyces ruminatium\u003c/em\u003e produces xylanase and cellulase enzymes and helps in plant biomass degradation in the rumen[61]\u003cstrong\u003e.\u003c/strong\u003e The higher abundance of \u003cem\u003ePecromyces ruminatium\u003c/em\u003e in the moringa feed group suggests potential health benefits to the animal. The lack of previous studies on how a moringa-fortified diet affects the rumen anaerobic fungal population makes the comparison somewhat challenging. Besides, although this study compares a fairly good number of samples (8-10 animals per group), a study recruiting more animals is required for a more solid conclusion on its effect on rumen anaerobic fungi.\u003c/p\u003e\n\u003cp\u003eIn the present study, a moringa-based diet has significantly altered the composition of rumen bacteria and ciliates but not the anerobic fungi in the lactating goats, as can be seen by beta diversity analysis. The limited response of protozoa as compared to bacteria agrees with earlier findings showing that these communities remain relatively stable despite nutritional shifts, likely due to their slower turnover and strong host association [62]. Likewise, the unchanged abundance of anaerobic fungi may reflect the lower fiber content of the moringa containing diet compared with masoor stover, since rumen anaerobic fungal populations primarily respond to fiber availability rather than short-term diet variation[63]. Furthermore, unlike bacteria and ciliates, the overall diversity of rumen anaerobic fungi remains very low. For example, only two genera, \u003cem\u003eNeocallimastix\u003c/em\u003e and \u003cem\u003ePecoramyces\u003c/em\u003e, represent 60-70 % of the rumen anaerobic fungal population. Therefore, this limited diversity compared to bacteria and ciliates could be a possible reason for their limited responsiveness to dietary treatment.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, this study highlights the significant variations in the abundance of rumen bacteria, ciliates, and anaerobic fungal taxa in response to the moringa diet in small ruminants, which emphasizes the complex interactions between diet and the rumen microbes. The results show that moringa containing diet significantly improved the abundance of beneficial bacterial and ciliate taxa in rumen and fecal, possibly driven by the complex phytochemical composition of moringa. While the abundance of rumen anaerobic fungal taxa remained largely unaffected. Advancements in deciphering the roles of these microbes are crucial for optimizing rumen performance and improving animal health as well as productivity. Findings of this study are important for developing moringa-fortified animal feed and strategies that enhance production traits.\u003c/p\u003e"},{"header":"5. Material and Methods","content":"\u003cp\u003e\u003cstrong\u003e5.1 Animal Handling and Experiment Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the Central Arid Zone Research Institute (CAZRI) in Jodhpur, India, all animal studies were conducted in accordance with established standards for animal care and experimentation.\u0026nbsp;Moringa plants (cultivar PKM-1), grown at CAZRI, were cultivated under arid climatic conditions, with an average annual rainfall of 379 mm and potential evapotranspiration of 2002 mm. The experimental field had sandy, shallow soil (50 cm depth) with 0.28% organic carbon, 116.64 kilogram/hectare (kg/ha) available nitrogen, 25.13 kg/ha P₂O₅, 481.81 kg/ha K₂O, and a pH of 7.9. The experiment was conducted in a factorial randomized block design with four levels of irrigation and planting density, and each treatment was replicated three times. Moringa cultivar PKM-1 was planted a year before the experiment and uniformly watered. Plants were headed back to a uniform height of 50 cm from the ground surface, and the five tagged plants were selected for yield attributes. Plants sampled for dry matter estimation were separated into leaves and stems for estimation of respective biomass yields after drying in a hot air oven. The details of the proximate composition of \u003cem\u003eM. olifera\u003c/em\u003e are given in Supplementary Table 1A.\u003c/p\u003e\n\u003cp\u003eFor this experiment, 30 lactating Marwari goats (\u003cem\u003eCapra hircus\u003c/em\u003e) were randomly assigned to three groups (n = 10 per group) after weaning of their kids at 90 days post-kidding. The goats were sourced from the institutional livestock farm, CAZRI, Jodhpur, and the experimental feeding continued for six months, and observations on daily intake of feed dry matter and nutrients, milk yield and fortnightly body weight change were recorded. All the lactating goats were allowed to graze for 3-4 hours daily in the open field having \u003cem\u003eCenchrus setigerus\u003c/em\u003e, \u003cem\u003eCenchrus ciliaris\u0026nbsp;\u003c/em\u003eand edible arid weeds. \u0026nbsp;In addition to grazing, the first group of goats were fed Masoor straw (MS, n=10), the second group was fed 20% moringa leaf meal (20%MLM, n=8), and the third group was given 30% moringa leaf meal (30% MLM, n=9) feed. \u0026nbsp;The reduced number of animals in the latter two groups was due to infections observed during the experiment, which led to the exclusion of three animals. The MS group was given a total mixed ration (TMR) with a 70:30 roughage-to-concentrate ratio, consisting of Masoor straw (containing 5% crude protein [CP] and 60% total digestible nutrients [TDN]) and a commercial concentrate mixture (22.7% CP and 70% TDN), resulting in an overall TMR composition of 10.31% CP and 63% TDN. The 20% MLM and 30% MLM groups were fed TMRs with the same 70:30 roughage-to-concentrate ratio, composed of Masoor straw (5% CP and 60% TDN) and their respective concentrate mixtures (each containing 26.98% CP and 70% TDN), resulting in a final TMR composition of 11.59% CP and 63% TDN. The concentrate mixtures for the 20% MLM and 30% MLM groups were formulated by incorporating 20% and 30% moringa leaf meal, respectively, along with other conventional concentrate ingredients. The details of feed ingredients and their nutritive values are provided in Supplementary Table 1B.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2 Sample Collection and DNA Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the experiment, rumen digesta samples were collected from each goat using a flexible stomach tube attached to a low-pressure vacuum pump. The whole rumen digesta was then separated into liquid and solid components using sterile four-layered muslin cloth and was transferred into cryovials containing RNAprotect bacteria reagent (Qiagen, Valencia, CA, USA) as described in our earlier studies [21]. Simultaneously, fecal samples were collected by trained veterinarians directly from the rectum wearing sterile gloves and lubricant to ensure hygienic and non-invasive sampling and stored in RNAprotect bacteria reagent (Qiagen, Valencia, CA, USA). All the samples were promptly frozen and then stored at -80 °C until further processing.\u003c/p\u003e\n\u003cp\u003eDNA extraction was carried out using a QIAamp Fast DNA stool mini kit (Qiagen, Valencia, CA, USA). To dislodge the fiber adhered bacteria, solid fractions were treated with Phosphate Buffer Saline (PBS) and 0.1% (v/v) Tween 20 for one hour with incubation at 37ºC and intermittent vortexing. The integrity of the extracted DNA was examined on a 0.8% agarose gel, and the concentrations were determined by Qubit 4.0 fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3 Amplicon Libraries Preparation and Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 16S rRNA and \u003cem\u003eITS\u003c/em\u003e1 amplicons, we processed rumen solid and liquid fractions separately, while amplification of 18S rRNA was carried out using DNA extracted from whole rumen liquor samples. For the Polymerase Chain Reaction (PCR) process, DNA was diluted to 5 ng/µl for 16S, 10 ng/µl for ciliates (18S), and 20 ng/µl for anaerobic fungi (\u003cem\u003eITS\u003c/em\u003e1) using sterile nuclease-free water. For the 16S rRNA gene, specific primers were used to amplify the V3-V4 region with primers 341F- 5′CCTACGGGNGGCWGCAG3′ and 805R- 5′GACTACHVGGGTATCTAATCC3′[22]. For 16S rRNA gene amplification in each reaction, total template DNA input was 12.5ng and primer concentrations for both the forward and reverse were 5 picomoles (pM) each. Thermal cycling conditions were an initial denaturation at 95 °C for 3 minutes; 25 cycles of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, and elongation at 72 °C for 30 seconds; final extension at 72 °C for 5 minutes. For ciliates, a specific primer set F-5′-CGATGGTAGTGTATTGGAC-3′ and R-5′-GGAGCTGGAATTACCGC-3′ targeting the 18S genes, as reported by Tapio \u003cem\u003eet al\u003c/em\u003e., [23]were used. In each reaction, forward and reverse primers were 5 pM, and total template DNA was 25.0 ng. Thermal cycling conditions were an initial denaturation at 95 °C for 3 minutes; 30 cycles of denaturation at 95 °C for 25 seconds, annealing at 60 °C for 30 seconds, and elongation at 72 °C for 40 seconds; final extension at 72°C for 5 minutes. Anaerobic fungi-specific primer F-5′-TACCCTTTGTGAATTTGTT-3′ and R-5′-ATCCATTGTCAAAAGTTGT-3′ targeting the Internal Transcribed Spacer (ITS-1) region were used to study rumen anaerobic fungal diversity[23]. In each reaction, forward and reverse primers were 5 pM, and total template DNA was 45.0 ng. Thermal cycling conditions were an initial denaturation at 95 °C for 3 minutes; 32 cycles of denaturation at 98 °C for 25 seconds, annealing at 55 °C for 30 seconds, and elongation at 72 °C for 40 seconds; final extension at 72°C for 5 minutes. All the amplicon primers were synthesized with a pre-tag of Illumina \u0026nbsp;adapters required for sequencing. In addition, fecal samples were also processed for the amplification of 16S rRNA, 18S rRNA and ITS1genes. For amplicon library preparation, we have followed Illumina’s standard protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). The Quality of all the libraries was evaluated using either LabChip GXII Touch (Revvity) or QIAexcel advanced system (Qiagen). Amplicon sequencing of 16S and 18S libraries was performed on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, United States) at our NGS facility using 2 × 250 chemistry. \u003cem\u003eITS\u003c/em\u003e1 libraries were sequenced on Illumina MiSeq with 2x150 chemistry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4 Taxonomy Assignment and Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor taxonomy assignments, we used the 16S Metagenomics Labs application available on the BaseSpace sequence hub by Illumina. We used the Ribosomal Database Project (RDP) v.2.14 database[24], PR2 (Protist Ribosomal Reference) v.5.0.0 database, and UNIITE database v.10.0 to classify the bacterial, ciliate, and anaerobic fungus reads, respectively. The taxonomic profiles obtained were further analyzed using various tools. For statistical analysis, the sequence count of all samples was first rarified (normalized) to the minimum library size using Microbiome Analyst[25]. After normalization, we employed Statistical Analysis of Metagenomic Profiles (STAMP v2.0.8) \u0026nbsp;[26] to calculate the relative abundance of each taxon\u0026nbsp; (phylum and genus level) present in the samples. To identify significant differences among groups, we performed ANOVA followed by post-hoc Tukey tests, with multiple testing correction using the Benjamini-Hochberg method. Features with a\u003cem\u003e\u0026nbsp;p\u003c/em\u003e-value ≤0.05 were considered statistically significant. Comparative abundance profiles were plotted using GraphPad Prism. We have used the MOCHI online tool to measure alpha diversity and to plot taxonomic profiles[27]. Alpha diversity was calculated using Shannon diversity and Shannon evenness, determining statistical significance at a \u003cem\u003ep\u003c/em\u003e-value threshold of ≤0.05 using the Kruskal-Wallis test. For beta diversity, we calculated a Bray-Curtis similarity distance matrix and visualized the results through principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS), with significance set at \u003cem\u003ep\u003c/em\u003e-value ≤0.05 by permutational multivariate analysis of variance (PERMANOVA), using the Paleontological Statistical Software (PAST v 4.03) tool[28]. Additionally, we conducted Linear Discriminant Analysis Effect Size (LEfSe) using the LEfSe (v1.1.2) command line tool to identify discriminatory taxa among the groups. The threshold for significance was set at LDA score \u0026gt; 3.0 and a \u003cem\u003ep\u003c/em\u003e-value ≤0.05.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eVFA: Volatile Fatty Acids\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMS: Masoor Straw\u003c/p\u003e\n\u003cp\u003eMLM: Moringa Leaf Meal\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eITS1\u0026nbsp;\u003c/em\u003egene:\u0026nbsp;Internal Transcribed Spacer 1 gene\u003c/p\u003e\n\u003cp\u003ePCoA : Principal Coordinate Analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNMDS : Non-metric Multidimensional Scaling\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCAZRI: Central Arid Zone Research Institute\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTMR: Total Mixed Ration\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCP: Crude Protein\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTDN : Total Digestible Nutrients\u003c/p\u003e\n\u003cp\u003ePERMANOVA : Permutational Multivariate Analysis of Variance\u003c/p\u003e\n\u003cp\u003eLEfSe : Linear Discriminant Analysis Effect Size\u003c/p\u003e \n\u003cp\u003eLDA: Linear Discriminant Analysis\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were approved by the Institutional Animal Ethics Committee of the Indian Council of Agricultural Research–Central Arid Zone Research Institute (ICAR–CAZRI), Jodhpur, Rajasthan, India (Approval No. CAZRI/2022/IAEC/01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This manuscript does not contain any individual human data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This manuscript does not contain any individual human data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw amplicon metagenomic sequence data have been submitted to the Indian Biological Data Center (IBDC) under BioProject number INRP000182 https://www.ibdc.dbtindia.gov.in/inda/completeStudyDetailsById?studyid=INRP000182# ), National Center for Biotechnology Information (NCBI) under BioProject number PRJEB89572 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJEB89572 ) and European Nucleotide Archive (ENA) with Study Number ERP172596 (https://www.ebi.ac.uk/ena/browser/view/ERP172596). The datasets supporting the conclusions of this article are included within the article as tables, figures, and supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was funded by the Department of Biotechnology (DBT), Government of India. Project Reference No: BT/AQ/1/SP41105/2020.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.N.- Methodology, Data curation, Formal analysis, Software, Writing original draft; M.B.- Methodology, Software; T.S.- Supervision, Validation, Writing-review \u0026amp; editing; R.P.-Supervision, Validation, Writing-review \u0026amp; editing;\u0026nbsp;N.V.P.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e-Resources; A.K.P.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e-Resources; S. K.– Resources; R. N. K.- Resources; M. J.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e-Conceptualization, Funding acquisition, Supervision; C.G.J.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e- Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, validation, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiang JB, Paengkoum P. Current status, challenges and the way forward for dairy goat production in Asia - conference summary of dairy goats in Asia. Asian-Australasian journal of animal sciences. 2019;32:1233\u0026ndash;43. https://doi.org/10.5713/ajas.19.0272\u003c/li\u003e\n\u003cli\u003eModi RJ, Patel NM, Patel YG, Islam MM, Nayak JB. Chapter 4 - Goat farming: A boon for economic upliftment. In: Rana T, editor. Trends in Clinical Diseases, Production and Management of Goats. 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Frontiers in Microbiology. 2017;8:1553. https://doi.org/10.3389/fmicb.2017.01553\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","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":"Animal nutrition, Ciliates, Lactating goats, Moringa oleifera, Rumen anaerobic fungi","lastPublishedDoi":"10.21203/rs.3.rs-8826260/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8826260/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, we have investigated the impact of \u003cem\u003eMoringa olifera\u003c/em\u003e feed on rumen and fecal microbial communities, including bacteria, ciliates, and anaerobic fungi in lactating goats (\u003cem\u003eCapra hircus\u003c/em\u003e). Lactating goats were divided into three groups based on dietary regimes: masoor straw (MS, n=10), 20% moringa leaf meal (20% MLM, n=8), and 30% moringa leaf meal (30% MLM, n=9). Rumen digesta and fecal samples were collected at the end of the experiment, and amplicon sequencing targeting the 16S rRNA, 18S rRNA, and \u003cem\u003eITS\u003c/em\u003e1 genes were carried out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the rumen solid and liquid fractions, moringa diet increased the \u003cem\u003eBacillota:Bacteroidota \u003c/em\u003eratio, which is associated with lower residual feed intake and improved metabolism. An increase in proteolytic bacterial phylum \u003cem\u003ePseudomonadota \u003c/em\u003ewas observed with moringa diet\u003cem\u003e. \u003c/em\u003eFurther,\u003cem\u003e Xylanibacter,\u003c/em\u003e \u003cem\u003eSachharofermentans \u003c/em\u003eand \u003cem\u003eRuminococcus\u003c/em\u003egenera, that have active role in volatile fatty acids (VFAs) production during fermentation process (\u003cem\u003ep\u003c/em\u003e-value ≤0.05), were more abundant, while \u003cem\u003eFibrobacter\u003c/em\u003e, \u003cem\u003eSucciniclasticum\u003c/em\u003e and \u003cem\u003eSodliphilus\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e-value ≤0.05), were less abundant in moringa feed groups. Cellulolytic ciliates like \u003cem\u003eEnoplastron \u003c/em\u003eand \u003cem\u003eDiploplastron \u003c/em\u003esignificantly increased\u003cem\u003e \u003c/em\u003e(\u003cem\u003ep\u003c/em\u003e-value ≤0.05)\u003cem\u003e \u003c/em\u003ewhile \u003cem\u003eEntodinium\u003c/em\u003e reduced in the rumen digesta samples of moringa feed groups. Fiber-degrading fungal genus \u003cem\u003eNeocallimastix \u003c/em\u003ewas less abundant in the rumen of the goats fed with moringa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall, these findings suggest that moringa supplementation positively influences rumen microbial communities and provides insights into adjustments in rumen microbiome structure and diversity. These findings are useful in understanding how moringa supplementation influences the rumen fermentation process and further modulating goat feed to improve health and milk production.\u003c/p\u003e","manuscriptTitle":"A diet containing Moringa oleifera alters the goat rumen microbiome: an insight into bacteria, ciliates and rumen anaerobic fungi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 19:24:17","doi":"10.21203/rs.3.rs-8826260/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-17T06:49:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T14:45:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-31T23:41:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210252112041025204312088860973183448027","date":"2026-03-24T07:06:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191588601270508837989027927747249442754","date":"2026-03-04T05:36:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68198294482604208036581335685269865221","date":"2026-03-02T03:38:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-19T04:12:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T19:04:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-16T15:45:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-13T09:47:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-13T09:39:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"c1542bd1-b2d0-4b7e-b1dd-d2089b7dc66d","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63332835,"name":"Biological sciences/Biotechnology"},{"id":63332836,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-17T12:40:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 19:24:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8826260","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8826260","identity":"rs-8826260","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}