Dietary Supplementation of Fruit from Nitraria tangutorum Improved Immunity and Abundance of Beneficial Ruminal Bacteria in Hu Sheep

Simple Summary The fruit of Nitraria tangutorum (FNT), a medicinal, edible plant, contains a variety of bioactive ingredients with a number of biological functions. However, its effect on sheep is unknown. To fill this gap, we examined the effects of supplementary FNT on serum biochemistry and the rumen bacteria in Hu rams. The results demonstrated that FNT could improve the immunity of sheep and increase the relative abundance of beneficial rumen bacteria. However, the rumen fermentation variables were not altered with supplementary FNT. Abstract The fruit of Nitraria tangutorum (FNT) is reputed to possess medicinal properties; however, its effect on sheep (Ovis aries) is unknown. The aim of this study was to fill this gap. In a 3 × 3 Latin square design, six 12-month-old rumen-fistulated Hu rams (56.2 ± 8.26 kg; mean ± SD) were penned individually and offered one of three levels of FNT, namely, 0 g/d (control; CON), 16 g/d (N16), and 48 g/d (N48). The concentration of serum immunoglobulin G increased linearly (p = 0.03) with an increasing intake of FNT. The serum concentration of β-hydroxybutyrate in the N48 group was lower than in the CON group (p = 0.01) and decreased linearly with increasing FNT (p = 0.001). The concentration of serum lactate dehydrogenase tended to decrease (p = 0.07) linearly with an increase in FNT intake, while the concentration of glucose did not differ among groups (p = 0.14) but displayed a quadratic curve with an increase in FNT (p = 0.05). The rumen concentration of lipase decreased linearly with increasing FNT (p = 0.04). The rumen fermentation variables were not affected by FNT. The FNT intake increased the abundance of beneficial ruminal bacteria, such as Lachnoclostridium, Rhodocyclaceae, and Candidatus Arthromitus. Prevotella, Rikenellaceae_RC9_gut_group, Ruminococcus, Olsenella, Lachnospiraceae_NK3A20_group, and Quinella were the dominant bacterial genera in all treatments. We conclude that FNT can improve immunity and increase the relative abundance of beneficial ruminal bacteria in sheep.


Introduction
Chemicals and antibiotics are used widely as additives in animal feed to improve production. However, some of these additives have been banned in many countries due to negative effects on the environment and on human health [1,2]. Natural herbs are being

Materials and Methods
The experimental procedures, animal handling, and sample collections were approved by the Animal Welfare and Experimental Ethics Committee of the Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences (protocol number: CAS201810082).

Experimental Animals and Design
Sheep feeding trials were carried out at the Gansu Gaolan Field Scientific Observation and Research Station for Agricultural Ecosystems of the Northwest Institute of Eco-Environment and Resources in the Chinese Academy of Sciences (36 • 13 N, 103 • 47 E, 1780 m above sea level) [21].
Six 12-month-old rumen-fistulated Hu rams (56.2 ± 8.26 kg; mean ± SD) were penned (1.2 m × 0.6 m × 1.8 m) individually in a well-ventilated shed. The number of rams was based on the effect size index (d-value), which was calculated using the estimated standard deviations of the means of measured variables from previous similar studies. A sample size of 6 per group resulted in a d-value that was close to 0.5, which, according to Sullivan and Feinn, is a medium and acceptable effect size [22]. The rams were utilized in a 3 × 3 Latin square design, with three treatments, three periods, and 2 sheep in each treatment in each period. Treatments (n = 6 rams/treatment) consisted of three levels of FNT, namely, 0 g/d (control-CON), 16 g/d (N 16 ), and 48 g/d (N 48 ) on a dry matter basis, and each period lasted 23 days, with 14 days for adaptation and 9 days for data collection. The FNT used in this experiment was the residual small fruit, which was purchased from a herbal supplier.
The rams received 1500 g DM per day of a pelleted total mixed ration (Gansu Runmu Biological Engineering Co., Ltd., Jinchang, China): 800 g at 08:00 and 700 g at 18:00. The basal diet was formulated according to the requirements of NRC [23]. Feed ingredients and nutrition levels are presented in Table 1. The FNT was mixed with 100 g of the diet in the morning and was consumed in toto by the rams. Water and mineral salt blocks were freely available.

Sample Collection and Processing
Five ml of jugular-vein blood was collected using evacuated tubes (Shanghai Kehua Bio-Engineering Co., Ltd., Shanghai, China) from each ram before the morning feeding on the last day and centrifuged at 4 • C and 1200× g for 15 min. The serum was stored at −20 • C for analysis of biochemistry variables [24]. In addition, on the last day, at 12:00, approximately 100 mL of rumen content was collected via the fistula using a measuring cup and was strained through four layers of cheesecloth [25]. The pH was measured immediately after collection, using a portable pH meter (PHB-10, Shanghai Hongyi Instrument Limited, Shanghai, China), then the rumen fluid was stored in 10 mL centrifuge tubes at −80 • C for the determination of rumen fermentation variables, rumen enzyme concentrations, and rumen microbiota.

Ruminal Fermentation Variables and Feed Composition Analysis
Five mL of rumen fluid were mixed with 1 mL of 250 g/L of metaphosphoric acid, then the volatile fatty acids (VFAs) were separated and quantified using gas chromatography/mass spectrometry (GC-MC) (7890B GC/5977B MSD, Agilent Technologies, Santa Clara, CA, USA) with a 30 m × 0.25 mm × 0.25 µm capillary column (AT-FFAP, Agilent Technologies, Santa Clara, CA, USA). Feed samples were dried to a constant weight in a forced air oven at 65 • C, air-equilibrated for 12 h, then ground using a 1-millimeter screen. The DM of the feed was determined by drying for 24 h in a forced air oven at 105 • C. The nitrogen (N) content (percentage of dry matter) in feed was determined by the Kjeldahl method, and crude protein content (percentage of dry matter) was calculated as N × 6.25 [26]. The ether extract (percentage of dry matter) was measured via Soxhlet extraction with diethyl ether for six hours.

Extraction of DNA and Polymerase Chain Reaction (PCR) Amplification
The total genomic DNA of the rumen bacteria was extracted using the E.Z.N.A. ® DNA kit (Omega Bio-Tek, Norcross, GA, USA) and following the manufacturer's instructions. The quality of the extracted genomic DNA was tested by 1% agarose gel electrophoresis. The concentration and purity of the DNA were determined with a Nan-oDrop 2000 spectrophotometer (Thermo Scientific Company, Waltham, MA, USA) and stored at −80 • C. With the extracted DNA as the template, the V3-V4 variable region of the 16S rRNA gene of rumen bacteria was amplified by PCR using the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGGACTACHVGGTWTCTAAT-3') [27] carrying the barcode sequence, then the PCR products were recovered and purified.

Sequencing of the 16S rRNA Gene and Bioinformatics Analysis
The purified PCR products were sequenced on the Illumina Miseq PE300 platform (Shanghai Meiji Biomedical Technology Co., Ltd., Shanghai, China). FASTQ version 0.19.6 [28] software was used for quality control, FLASH version 1.2.11 [29] software was used for splicing, and UPARSE version 7.1 [30,31] software was used for operational taxonomic unit (OTU) clustering, according to 97% similarity and eliminated chimera. To minimize the effects of sequencing depth on alpha and beta diversity measurements, the sequence numbers of all samples were rarefied to 20,000 and the average sequence coverage (Good's coverage) of each sample after plateauing was still up to 99.09%. The RDP classifier version 2.11 [32] was used for OTU taxonomic annotation on the Silva 16S rRNA gene database (v138). The confidence threshold was 0.7, and the community composition of each rumen fluid sample was counted at different species classification levels.
Mothur software [33] was used to calculate alpha diversity and Ace and Shannon indices, and the Wilcoxon rank-sum test tested for differences among groups. Constrained principal coordinate analysis (CPCoA), based on the Bray-Curtis distance algorithm, tested the dissimilarity of the microbial community structure between samples, and, combined with permutational multivariate analysis of variance analysis (PERMANOVA), tested whether the bacterial communities were distinct among groups. Linear discriminant analysis effect size (LEfSe) analysis [34] (LDA > 2, p < 0.05) was used to determine the bacterial groups, with significant differences in abundance from phylum to genus level among the different groups. Based on the Spearman correlation |r| > 0.6 and false discovery rate (FDR)-adjusted p < 0.05, species were selected for correlation analysis [35]. The sequencing data are stored in the sequence-reading archive (SRA) of the National Center for Biotechnology Information (NCBI) and can be obtained through the Bio Project number PRJNA880036.

Statistical Analysis
Initially, the Shapiro-Wilk test was used to test the data for normal distribution using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). The data that were distributed normally were analyzed by a one-way analysis of variance (ANOVA) [36], while the Kruskal-Wallis test analyzed data with non-normal distribution. Duncan's test separated means when significance was detected. In addition, coefficients for unequally spaced contrasts were generated by the interactive matrix algebra procedure (IML) of SAS, then the linear and quadratic effects of the increasing FNT levels were assessed using orthogonal polynomial contrasts. The data are represented as means and the standard error of the mean (SEM). A level of p ≤ 0.05 was accepted as significant and 0.05 < p < 0.10 as tending to differ.

Serum Biochemistry
Immunoglobulin G tended to increase (p = 0.08) with an increase in FNT intake and increased linearly (p = 0.03) with an increasing level of FNT (Table 2). Glucose did not differ (p = 0.14) among groups but displayed a quadratic curve (p = 0.05) with an increase in FNT. The FNT did not affect the serum concentrations of total protein, albumin, triglyceride, total cholesterol, and blood urea nitrogen.

Serum Metabolites
The serum concentrations of β-hydroxybutyrate decreased linearly (p = 0.001; Table 4) with increasing FNT and were lower (p = 0.01) in the N 48 group than in the CON group. Lactate dehydrogenase tended to decrease linearly (p = 0.07) with an increase in FNT intake.
There was no difference in the serum concentrations of non-esterified fatty acids (p = 0.51), alkaline phosphatase (p = 0.67), and lactate dehydrogenase (p = 0.19) among the treatments.

Effects of the Fruit of Nitraria tangutorum (FNT) on the Rumen Bacterial Community Composition in Hu Rams
A total of 593,856 16S rRNA sequences were obtained from the sequencing of 18 microbial samples. The coverage rate of all samples was greater than 99%, and 1709 OTUs were obtained via sequence clustering and quality control (similarity > 97%). The curves reflecting the species number and diversity index tended to plateau ( Figure S1 in supplementary materials), so sequencing depth accounted for most of the biomass in the sample. The Ace index, reflecting species richness, was greater (p = 0.045) in N 48 than in CON ( Figure 1B), while the Shannon index, reflecting species diversity, did not differ (p = 0.575) among treatments ( Figure 1A). The rumen microbial community dissimilarity was determined by CPCoA analysis ( Figure 1C). The two groups consuming FNT were separated from the CON group, but the difference was insignificant. The Venn diagram illustrated that there were 918 public core OTUs in the three groups, and 94, 100, and 189 unique OTUs in CON, N 16 , and N 48 , respectively ( Figure 1D). A total of 22 phyla and 334 genera of bacteria were identified in the rumen fluid. At the phylum level (with a relative abundance > 0.1%), Bacteroidota, Firmicutes, Actinobacteriota, Proteobacteria, Patescibacteria, Spirochaetota, and Synergistota were predominant across the 3 treatments (Figure 2A). At the genus level (with a relative abundance > 0.1%), Prevotella, Rikenellaceae_RC9_gut_group, Ruminococcus, Olsenella, Lachnospiraceae_NK3A20_group, Quinella, and Christensenellaceae_R-7_group were the most abundant ( Figure 2B). A total of 22 phyla and 334 genera of bacteria were identified in the rumen fluid. At the phylum level (with a relative abundance > 0.1%), Bacteroidota, Firmicutes, Actinobacteriota, Proteobacteria, Patescibacteria, Spirochaetota, and Synergistota were predominant across the 3 treatments (Figure 2A). At the genus level (with a relative abundance > 0.1%), Prevotella, Rikenellaceae_RC9_gut_group, Ruminococcus, Olsenella, Lachnospiraceae_NK3A20_group, Quinella, and Christensenellaceae_R-7_group were the most abundant ( Figure 2B). Hierarchical cluster analysis on the 50 bacteria genera with the highest relative abundances showed that Prevotella had the highest relative abundance in the three groups. Quinella, Candidatus, Saccharimonas, Ruminococcus, Sharpea, Ruminococcus gauvreauii group, Fretibacterium, and Selenomona were abundant in the CON group. Shuttlewochaeta, Succinivibrionaceae_UCG-001, Succiniclasticum, Selenomonas, Syntrophococcus, and Erysipelotrichaceae_UCG-002 were most abundant in the N16 group. The N48 group was rich in Rikenellaceae_RC9_ gut_ group, Olsenella, Prevotellaceae_UCG-003, NK4A214_group and UCG-004. Sphaerochaeta, Dialister, Christensenellaceae_R-7_group, and Prevotellaceae_UCG-001 increased with an increase in FNT level ( Figure 2C).
LEfSe analysis was used to identify the difference in rumen bacteria among groups. The microbial groups with significant indigenous enrichment and influence on the different groups were counted, among which Oscillospirales and Comamonadaceae were identified in the CON group, genera including Sutterella, Blvii28_wastewater-sludge_group, Lachnoclostridium, and family including Sutterellaceae and Williamwhitmaniaceae were enriched significantly in the N16 group. Candidatus Arthromitus and Rhodocyclaceae, at the level of genus and family, respectively, were enriched significantly in the N48 group ( Figure 2D,E). Hierarchical cluster analysis on the 50 bacteria genera with the highest relative abundances showed that Prevotella had the highest relative abundance in the three groups. Quinella, Candidatus, Saccharimonas, Ruminococcus, Sharpea, Ruminococcus gauvreauii group, Fretibacterium, and Selenomona were abundant in the CON group. Shuttlewochaeta, Succinivibrionaceae_UCG-001, Succiniclasticum, Selenomonas, Syntrophococcus, and Erysipelotrichaceae_UCG-002 were most abundant in the N 16 group. The N 48 group was rich in Rikenellaceae_RC9_ gut_ group, Olsenella, Prevotellaceae_UCG-003, NK4A214_group and UCG-004. Sphaerochaeta, Dialister, Chris-tensenellaceae_R-7_group, and Prevotellaceae_UCG-001 increased with an increase in FNT level ( Figure 2C).
LEfSe analysis was used to identify the difference in rumen bacteria among groups. The microbial groups with significant indigenous enrichment and influence on the different groups were counted, among which Oscillospirales and Comamonadaceae were identified in the CON group, genera including Sutterella, Blvii28_wastewater-sludge_group, Lachnoclostridium, and family including Sutterellaceae and Williamwhitmaniaceae were enriched significantly in the N 16 group. Candidatus Arthromitus and Rhodocyclaceae, at the level of genus and family, respectively, were enriched significantly in the N 48 group (Figure 2D,E).

Discussion
The fruit of Nitraria tangutorum (FNT) is used widely as a food and medicine. This is the first study that has examined the effects of supplementary FNT on serum metabolite variables, rumen fermentation, and the rumen microbiota in sheep.

Effects of the Fruit of Nitraria Tangutorum (FNT) Supplement on Serum Biochemistry Variables in Hu Rams
Serum biochemical variables are used to indicate the condition, metabolism, and health status of the animal [37]. For example, serum concentrations of BUN, TP, ALB, and IgG are key indicators reflecting animal protein metabolism [38]. Different dietary treatments can affect the level of IgG in ruminant serum [39,40]. In the current study, IgG increased with an increase in the level of FNT intake. The IgG affects the growth of lambs [40,41] and their immune functions [39], which may be related to the effective bioactive components, as FNT is rich in polysaccharides [13], flavonoids, alkaloids [20], and anthocyanins [42]. Natural polysaccharides not only enhance immunity, but also regulate it by acting directly on the immune cells and targeting the gut microbiota [43]. The FNT did not affect the serum concentrations of BUN, TP, and ALB levels. The rams in the present study were mature and were not growing. A study on growing lambs is warranted to examine the effect of FNT on protein metabolism and utilization. The above results indicated that the FNT supplementation in this study did not affect the metabolism and utilization of dietary protein, at least in mature Hu rams, but had a tendency to improve immunity.
Blood lipids are essential substances for basal metabolism in the body and can reflect lipid metabolism in animals. TG is affected mainly by dietary intake and nutrient levels and is an important form of energy storage for ruminants. Serum concentrations reflect fat metabolism, and an increase in concentration could indicate malnutrition or stress.

Discussion
The fruit of Nitraria tangutorum (FNT) is used widely as a food and medicine. This is the first study that has examined the effects of supplementary FNT on serum metabolite variables, rumen fermentation, and the rumen microbiota in sheep.

Effects of the Fruit of Nitraria Tangutorum (FNT) Supplement on Serum Biochemistry Variables in Hu Rams
Serum biochemical variables are used to indicate the condition, metabolism, and health status of the animal [37]. For example, serum concentrations of BUN, TP, ALB, and IgG are key indicators reflecting animal protein metabolism [38]. Different dietary treatments can affect the level of IgG in ruminant serum [39,40]. In the current study, IgG increased with an increase in the level of FNT intake. The IgG affects the growth of lambs [40,41] and their immune functions [39], which may be related to the effective bioactive components, as FNT is rich in polysaccharides [13], flavonoids, alkaloids [20], and anthocyanins [42]. Natural polysaccharides not only enhance immunity, but also regulate it by acting directly on the immune cells and targeting the gut microbiota [43]. The FNT did not affect the serum concentrations of BUN, TP, and ALB levels. The rams in the present study were mature and were not growing. A study on growing lambs is warranted to examine the effect of FNT on protein metabolism and utilization. The above results indicated that the FNT supplementation in this study did not affect the metabolism and utilization of dietary protein, at least in mature Hu rams, but had a tendency to improve immunity.
Blood lipids are essential substances for basal metabolism in the body and can reflect lipid metabolism in animals. TG is affected mainly by dietary intake and nutrient levels and is an important form of energy storage for ruminants. Serum concentrations reflect fat metabolism, and an increase in concentration could indicate malnutrition or stress. There was no difference among treatments, probably because the animals were not under nutritional stress. TC is synthesized mainly by the liver and is essential for cell-membrane ACTH, bile acid, vitamin D, and hormones [44,45]. The concentrations for the rams in the present study did not differ among treatments and were similar to the 3.69 to 6.37 U/L reported for dairy cows [46] and the 4.15 mmol/L reported for water buffaloes (Bubalus bubalis) [47].
In this study, FNT did not affect the serum concentrations of MDA, GSH-Px, T-AOC, CAT, and SOD, indicating that dietary FNT supplementation did not affect the antioxidant capacity of the rams. However, FNT did affect the antioxidant capacity in humans [10], while proanthocyanins reduced oxidative stress in rats [16]. This would suggest that sheep respond differently to FNT than humans and rats, or that the dose offered to the sheep in the present study was insufficient. There is some support for the latter, as the serum concentrations of GSH-Px increased numerically with an increase in FNT intake, although the differences were not significant. The increase may be due to the antioxidant effect of FNT [48,49]; however, more evidence is needed to establish the anti-oxidative effect of FNT on sheep.
Serum metabolites are commonly used to assess the nutritional status of livestock [50]. In the current study, BHBA values decreased with increasing FNT levels, but FNT treatment did not affect the serum concentrations of NEFA, GLU, ALP, and LDH. BHBA is commonly used to detect ketosis, and a concentration of 1 mmol/L is considered a subclinical level in cows [51]. Although there was a decrease in the serum concentration of BHBA with an increase in FNT intake, all groups were below the subclinical level. FNT contains abundant polysaccharides [13] that can be used to provide energy for the animal [52]. Therefore, FNT supplementation in the diet can reduce the mobilization of adipose tissue and liver glycogen, and also reduce the serum concentration of BHBA. This could explain the decrease in BHBA concentration with an increase in FNT level. The serum NEFA concentration reflects fat metabolism, and the unchanged concentration may be related to the absence of changes in TC. GLU is regulated mainly by the liver [53], and a serum concentration of less than 2.5 mmol/L, indicates metabolic disorders [54]. The concentration of GLU in this study was between 4.33 and 4.97 mmol/L, which were all above this threshold, indicating that the sheep were well nourished. The serum concentration of LDH decreased linearly with the increasing FNT intake in the current study. LDH is one of the main enzymes involved in gluconeogenesis and is mainly involved in energy metabolism [55]. The concentration of LDH in the present study for all groups was in the normal range of 88-487 U/L commonly reported for sheep [56].

Effects of the Fruit of Nitraria Tangutorum (FNT) Supplement on Rumen pH and Rumen Fermentation in Hu Rams
Rumen pH and VFAs reflect the function and stability of the internal rumen environment. In the present study, the rumen pH ranged between 5.60 and 5.94 and did not differ among treatments. The pH values were slightly lower than the optimal range of 6.2 to 7.2 for fibrolytic bacteria [57]. This may be related to the ratio of concentrate to roughage in the diet for this study, as high-grain diets can lower rumen pH [58,59]. In ruminants, VFAs provide approximately 70% of the energy demands of the ruminant [60]. In dairy cows, the active polysaccharides regulated rumen fermentation and increased the total VFA production [61]. However, in spite of the polysaccharides in FNT, the concentration of ruminal VFAs did not differ among groups in the present study.

Effects of the Fruit of Nitraria Tangutorum (FNT) Supplement on Rumen Microorganisms in Hu Rams
The diversity of the rumen microbial community is affected largely by diet [62]. The Ace index of alpha diversity increased with an increase in FNT, which indicates that the FNT increased the microbial richness in the rumen. Greater microbial diversity in the mammalian gastrointestinal tract usually means stronger metabolic capacity and stability [63]. Its unique community structure and metabolites are essential for regulating host metabolism, growth and development, and immune regulation [64].
In the current study, Firmicutes and Bacteroidetes were the two most abundant bacterial phyla in the rumen, which is consistent with other studies on ruminants [40,[65][66][67].
The relative abundance of Bacteroidetes, which is important in the degradation of polysaccharides such as cellulose, hemicellulose, and pectin [68,69], increased with the addition of FNT to the diet. FNT contains a large number of bioactive components, including polysaccharides, which are beneficial to rumen microorganisms [70,71] that enhance the relative abundance of Bacteroidetes [72].
At the genus level, the relative abundances of Rikenellaceae_RC9_gut_group and Chris-tensenellaceae_R-7_group increased, whereas Quinella and Ruminococcus decreased with supplementary dietary FNT. Rikenellaceae_RC9_gut_group belongs to the Bacteroidetes that promote lipid metabolism [73] and carbohydrate and protein fermentation [74]. Recent studies reported that the Christensenellaceae_R-7_group exists widely in the intestine and mucosa of the host, is essential to health, and is involved in amino acid and lipid metabolism [75]. The relative abundance of the conditional pathogenic bacteria, Quinella [76][77][78], decreased with an increase in FNT, which indicates that FNT has the potential to improve immunity. The active ingredient polysaccharides can reduce pathogenic bacteria, promote the growth and reproduction of beneficial ruminal bacteria, and reduce the consumption of carbohydrates and other nutrients by pathogenic bacteria [7].
We further analyzed the differential bacteria in each group and found that supplementary FNT increased the relative abundance of beneficial bacteria. Oscillospirales and Comamonadaceae were enriched significantly in the CON group, along with Sutterella and Lachnoclostridium in the N 16 group and Candidatus Arthromitus and Rhodocyclaceae in the N 48 group. Sutterella is an anaerobic or microaerobic Gram-negative bacterium [79] that acts as an immunoregulator in the human gastrointestinal tract [80,81]. The increase in the relative abundance of Lachnoclostridium may be due to the utilization of the polysaccharides contained in FNT. Lachnoclostridium is involved in the degradation of polysaccharides and in the synthesis of amino acids in the rumen [82]. It was also reported that Lachnoclostridium can improve the intestinal barrier function of mice [83] and had a positive impact on the colon development of dairy cows [84]. In addition, the intake of FNT increased the relative abundance of Candidatus Arthromitus, commonly referred to as segmented filamentous bacteria (SFB). It is a symbiotic bacterium that regulates the maturation of the host immune system, stimulates the differentiation of Th17 cells, and promotes the secretion of intestinal surface immunoglobulin A (sIgA) [33,85]. Therefore, the increase in Candidatus Arthromitus may be related to the immunomodulation of FNT. Correlation analysis revealed that Candidatus Arthromitus was correlated inversely with lipase, as there was a linear decrease in lipase with an increase in the relative abundance of Candidatus Arthromitus. The denitrifying bacteria, Comamonadaceae, was enriched in the CON group, while Rhodocyclaceae was enriched significantly in the N 48 group. Rhodocyclaceae in the rumen of lambs is correlated positively with rumen development, while Comamonadaceae is correlated negatively with host growth performance and rumen development [86]. In addition, the Spearman correlation results revealed that Rhodocyclaceae was correlated positively with IgG. These results indicate that FNT has beneficial effects on the rumen and immune function in sheep. Other medicinal plants are reputed to possess immunoregulatory effects [87]. Interestingly, the enriched bacteria species differed between the two levels of FNT consumption. The reason for this is unknown and warrants further research.

Conclusions
Supplementary FNT altered the immune-related indicators in the serum and increased the relative abundances of beneficial bacteria, such as Lachnoclostridium, Rhodocyclaceae, and Candidatus Arthromitus in the rumen of Hu rams. In addition, FNT influenced the serum concentrations of BHBA and LDH along with the rumen concentration of lipase, which are all involved in energy metabolism. Further studies are warranted on the effect of supplementing FNT on fattening sheep and in determining the optimal level that should be supplemented in the feed.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12223211/s1, Figure S1: Effect of FNT addition on rarefaction curves in the rumen fluid of Hu rams.

Data Availability Statement:
The data presented in the study are available on request from the corresponding author.