Combined Supplementation of Probiotics and Enzymes Improves Performance and Regulates Rumen Microbiota in Fattening Goats

Background: The aim of this study was to explore the effects of growth performance, slaughter performance, serum biochemical, immune and antioxidant indexes and rumen microora fed with a basal diet (CON group), added B. subtilis and B. licheniformis (PRO group), supplemented with B. subtilis, B. licheniformis and enzyme preparations (COM group) on fattening goats. Methods: 39 male goats were randomly divided into 4 groups with 13 individuals in each group for feed period of 80 d. Goats were fed as follows: CON diet, PRO diet with B. subtilis and B. licheniformis, and COM diet with B. subtilis, B. licheniformis and compound enzymes. Results: ADFI of COM group signicantly increased compared with CON group and PRO group (P < 0.01), as well as COM group and PRO group dramatically promoted ADG versus with CON group (P < 0.05). As a consequence, the body weight of fattening goats in the COM group was predominantly higher than that in the CON group (P < 0.01). In addition, the PRO group and COM group enhanced the TNF-α (P < 0.05) and IL-10 content (P < 0.01) in the serum. No differences were observed in serum biochemical and antioxidant indexes of three groups (P > 0.05). Likewise, the GR values of PRO group and COM group were noteworthy improved in comparison with CON group (P < 0.01). The VFA contents in rumen uid were insignicantly different (P > 0.05). COM group also enriched the relative abundance of Proteobacteria compared with CON group and PRO group (P < 0.05). Nevertheless, the relative abundance of Actinobacteria decreased of PRO group and COM group in rumen uid microorganisms (P < 0.05). Apparently, COM group signicantly enriched nitrogen metabolism, glycolysis and TCA cycle (P < 0.05), whereas nucleotides biosynthesis was notably reduced (P < 0.05). Conclusion: The combined feed of probiotics and enzymes had more profound effects than probiotics Effects of probiotics combined with enzymes on alpha diversity of rumen uid microorganisms in fattening goats.

The compound biological preparations of Bacillus licheniformis, Saccharomyces cerevisiae and protease fed fattening lambs, which improved the growth performance, antioxidant competence and immunocompetence, also increased the rumen microbial diversity [14]. Supplementation with probiotics and cellulose was con rmed of having the impact on improving growth performance and promoting the expression of functional genes in rumen. The combined feed of probiotics and cellulose has an immense importance for the health of sheep [15]. Nevertheless, the functions of probiotics (B. subtilis, B. licheniformis) and enzymes (xylanase, protease, amylase, β-mannanase, cellulase, β-glucanase) on fattening goats are still unknown. Herein, this research investigated the effects of B. subtilis, B. licheniformis and enzymes on growth performance, slaughter performance, antioxidant capacity, immune ability and rumen microorganisms of fattening goats.

Experimental design
The procedures were approved by the Animal Care and Use Committee of Nanjing Agricultural University and the experiments were followed by the National Research Council Guide. 39 male goats (Yantse River Delta White) with near weights and healthy state were randomly divided into 3 groups and 13 individuals in each group: CON group, fed a basal diet; PRO group, fed a basal diet with B. subtilis (600 mg/kg) and B. licheniformis (600 mg/kg); COM group, fed a basal diet with B. subtilis (600 mg/kg), B. licheniformis (600 mg/kg) and compound enzymes (200 g/t). For PRO group and COM group, B. subtilis, B. licheniformis and compound enzymes and other ingredients were made to granule then fed to goats as experimental diets. The basal diets were purchased from Jiangsu Agriportal Co., Ltd. The composition and nutritional levels are shown in Table 1. The goats were raised in house and fed with basal diets twice a day at 08:00 and 16:00, goats had fed and water ad libitum.

Sample collection
The ruminal contents were collected by a gastric catheter on day 20, day 40, day 60 and day 80 of the experimental process, after the ltration of four layers of gauze, the rumen uid was transferred to 50 mL tube, then quickly measured the pH of rumen uid using a pH meter (Wuhan, China) and frozen in liquid nitrogen and stored at -80 ℃.
Blood samples of four goats in each group were collected on day 80 and centrifugated at 3000 rpm for 10 min to obtain the serum samples then stored at -20 ℃ before detection. Serum biochemical, immune and antioxidant indexes were analyzed using kits (Nanjing Jiancheng, Biotechnology, Jiangsu, China).
The content of NH 3 -N was detected according to the means [16]. The VFA contents were measured according to the following method: 1 mL rumen uid samples were blended with 0.2 mL mixed solution of metaphosphoric acid and crotonic acid (metaphosphoric acid 25 g/100 mL, crotonic acid 0.6464 g/100 mL), and placed at -20 ℃ for 12 h. After thawing, subsequently, quickly injecting 0.6 mL samples to gas chromatography (Agilent 7890A GC, Japan).

Growth performance
The goats were weighted without being fed on the 1st and 80th days of the experiment to determine the initial weight and nal weight, then calculated ADG of each goat. ADG = ( nal weight -initial weight) / experimental days. The fed intake of goats was recorded each day during the experimental period, ADFI = total feed intake / experimental days, thus F/G was determined: F/G = ADFI/ADG.

Slaughter performance
Five goats in each group were randomly selected to slaughter and fasted 12 h before slaughter. Live weight before slaughter and carcass weight were recorded. Carcass weight = live weight before slaughter -head weight -skin weight -hoof weight -tail weight -reproductive organs weight -internal weight (remain kidney and perirenal lipid). Then dressing percentage was determined: Dressing percentage = carcass weight / live weight before slaughter. Loin eye area was calculated using a planimeter (KP-90 N) followed a previous study [17]. GR  Statistics and analysis SPSS 23.0 was performed to analyze data through one-way ANOVA and Duncan's test was conducted to compare the differences. The results were shown as the Mean ± Standard Error (M ± SE). The differences among the three groups were described as signi cant differences at P < 0.05 and trends at P < 0.10. GraphPad Prism 8.0 was used to make graphs.

Results
As Table 2 shows, the nal weight and ADG of goats signi cantly increased in the PRO and COM group compared with that in the CON group (P < 0.05). COM treatment dramatically enriched ADFI in comparison with that in CON and PRO. There were no signi cant differences in F/G among the three groups (P > 0.05). The values were expressed as "mean ± standard deviation".

Slaughter performance
As shown in Table 3, probiotics feed and combined feed had no notable effects on live weight before slaughter, carcass weight, dressing percentage and loin eye area of fattening goats compared with those in CON (P > 0.05). Nevertheless, supplementing with probiotics or combined with enzymes pronouncedly enhanced the GR value than that in CON (P < 0.05). Serum biochemical indexes Figure 1 shows the effects of biochemical indexes in fattening goats fed probiotics and combined with enzymes. There were no differences in ALT, AST, ALP, TP, ALB, GLB, TG, TC, UN, Cr and HDL contents of three groups (P > 0.05). The results illustrated that supplementation of probiotics and combined with enzymes had no dramatical function of changing the physiological state of goats (P < 0.05).

Serum immune and antioxidant indexes
No differences were observed in SOD, MDA, GSH-Px and T-AOC contents among CON, PRO and COM groups (P > 0.05, Fig. 2). Furthermore, administrating probiotics and combined with enzymes predominantly increased the TNF-α and IL-10 concentrations of goat serum (P < 0.05), while the content of IL-6 had no remarkable changes in the PRO and COM group in comparison with that in the CON group (P > 0.05).
Rumen fermentation parameters Figure 3 shows the rumen fermentation parameters of fattening goats administration of probiotics or combined with enzymes. Feeding PRO or COM unaffected the pH, NH 3 -N and VFA contents compared with the CON, while the isobutyrate content signi cantly increased in the PRO group (P = 0.040) and tended to enrich in the COM group (P = 0.064) than that in the CON group.

Rumen microbiota and interactions
As shown in Table 4, no dramatical differences were detected in the Chao 1, Goods_coverage, Observed_species, Shannon and Simpson indexes in CON, PRO and COM group, which indicated that supplementing probiotics and combined with enzymes had no central effects on alpha diversity of rumen uid microorganisms in fattening goats. Principal Coordinate Analysis (PCoA) demonstrated 30.47% variation of total rumen microbiota based on unweighted_unifrac. Besides, the stacked bar chart exhibited that Bacteroides and Firmicutes were the dominant phylum of rumen microbiota. Compared with CON group, the relative abundance of Proteobacteria pronouncedly enriched in COM group, while the relative abundance of Actinobacteria signi cantly reduced in PRO and COM (Fig. 4B). Additionally, heatmap manifested remarkable bacteria at the genus level of three groups (Fig. 4C). Circos showed Prevotella_1, Prevotella_7 and Prevotellaceae_unclassi ed accounted for a large proportion in three groups (Fig. 4D). Fig. 3E shows sixteen predominant in three groups. Speci cally, adding probiotics signi cantly advanced the relative abundance of Butyrivibrio and Clostridiales_unclassi ed (P < 0.05), the relative abundance of Bilophila and Succiniclasticum dramatically enhanced in COM group (P < 0.05). Figure 4F re ected the evolutionary branching cladogram of rumen micro ora in three groups. The PRO group had no biomarker of rumen micro ora. The dominant biomarkers were Butyrivibrio, Shuttleworthia and Syntrophococcus in the COM group. LEFSe analysis found that Aeromonas_sp_18III_A01_071 was a biomarker in PRO group (LDA SCORE > 3), and there were seventeen biomarkers in COM, in which the number of biomarkers was the largest among three groups. Speci cally, the relative abundance of Firmicutes, Lachnospiraceae, Ruminococcus_1_unclassi ed and Ruminococcus_1(LDA SCORE >4)were signi cantly improved in COM group. Sparcc analyzed the most abundant microbial species in the top 30 to get the correlation between two bacteria (Fig. 4H). The results indicated that Bacteroidaceae_unclassi ed and Prevotellaceae_ unclassi ed, Bacteroidetes_unclassi ed and F082_ unclassi ed had the strongest positive correlations, a negative correlation existed in Prevotella_1 and Lachnospiraceae_NK3A20_group (Fig. 4I). PICRUST functional predictions PICRUST (Phylogenetic investigation of communities by reconstruction of unobserved states) were applied to predict the functional abundance of micro ora. The COM group signi cantly upregulated nitrogen metabolism, ATPase and GMP reductase compared with the CON group (Fig. 5A). The results found that the contents of DNA synthesis, some proteins and transcriptional regulators were signi cantly enriched in COM based on the KO database (Fig. 5B). Furthermore, glyoxylate cycle, glycolysis and glyoxylate were dramatically upregulated in the COM group compared with that in the CON group, COM group signi cantly upregulated nucleotide synthesis and TCA cycle in functional proteins in the COM group compared with that in the CON group (Fig. 5D).
Correlation analysis goat performance, serum indexes, rumen fermentation parameters and rumen microbiota Pearson's correlation was emphasized to analyze the relevance of goat performance, basic parameters and rumen microbiota (Fig. 6).

Discussion
These results observed that feeding probiotics combined with enzymes enhanced the growth performance and slaughter performance of fattening goats. Besides, COM had more advanced effects than PRO. In addition, the immunity was enhanced and the rumen bacterial structure was changed in the PRO group and COM group, which might be the reasons to improve the performance of fattening goats.
A number of studies have shown the profound effects on pigs supplemented with B. subtilis and B. licheniformis [20][21][22][23][24]. Nevertheless, there were little researches into which the in uence of B. subtilis and B. licheniformis were conducted on goats. Moreover, various researches have studied the effects of compound enzymes on weaned pigs [25,26]. In addition, combined feed probiotics and enzymes had predominant effects on growth performance and rumen bacteria of sheep [15]. Based on these reports, we investigated the functions of B. subtilis, B. licheniformis and multiple enzymes on fattening goats, individually and in combination. Importantly, the effects of combined feed were better than single feed which was explored in this study.
The results observed that ADG signi cantly increased of fattening goats in the PRO group. A previous study reported that the basal diet administration of B. subtilis and B. licheniformis signi cantly improved the growth performance of piglets [20]. The growth performance of nisher pigs was noteworthily improved supplemented with B. subtilis and B. licheniformis [23]. Adding B. subtilis, B. licheniformis, B. coagulans and C. butyricum ameliorated the weaning pig performance [27]. Some ndings above indicated that feeding B. subtilis and B. licheniformis played a central role in improving the growth performance of pigs. In our study, supplementing B. subtilis and B. licheniformis also accelerated the improvement of goat performance. Furthermore, COM group raised the nal weight, ADG and ADFI in goats in comparison with CON group. There was a report that supplementing cellulose increased the growth performance of Hu sheep [28]. Previous research found that treatment of compound probiotics and cellulase enabled the increase of the total weight gain and ADG in sheep [15]. It may be explained that supplementation with enzymes could promote the absorption of nutrients, enhance the feed e ciency, keep the healthy condition [29][30][31]. Additionally, more combinations of probiotics and enzymes should be investigated to put to good use in the feed industry.
Slaughter performance re ects the applicability in animals, which decides the economic pro t in the practical application [32]. Adding cellulase and xylanase to steers failed to improve the carcass and slaughter percentage, which were similar to our research results [33]. Pigs fed probiotics included Bacillus subtilis and Clostridium butyricum had no effect on loin eye area [34]. A previous study reported that supplementation with cellulose did not signi cantly change the dressing percentage and loin eye area of Hu sheep [28]. Consistently, PRO and COM feed unaffected the loin eye area of fattening goats.
Compared with the CON group, the GR value dramatically in PRO group and COM group, while there was no signi cant difference between PRO group and COM group, which indicated that probiotics administration helped to deposit the muscle fat, besides, combined feed cannot enhance the deposition effect. The results showed that probiotics alone or combined with enzymes could improve the slaughter performance of fattening goats.
We elucidated that PRO and COM feed enriched the contents of IL-10 and TNF-α compared with CON feed. IL-10 and TNF-α play vital roles in in ammation [35]. TNF-α affects the carbohydrate and immune response of animals, which is a kind of proin ammatory cytokines [36,37], and the content of TNF-α would enrich if the tissues were in ammatory [38]. IL-10 performs an important effect on in ammatory reaction as an anti-in ammatory factor, importantly, inducing the function of immunity [39,40].
Supplementing probiotics could in uence the immune function, inducing the improvement of immune cytokines contents [41][42][43]. Studies reported that administration of B. subtilis lead to the bene t of the host immune system [44,45]. CD4+ cells are part of the T lymphocytes, whose subpopulations are Th cells that include Th1 and Th2 cells [46]. Th2 secretes different cytokines, such as IL-2, IL-4, IL-6 and IL-10, which play a major role in immune regulation and in ammatory responses [46,47]. Importantly, Th2 accelerates the multiplication and differentiation of B lymphocytes, resulting in the production of antibody and intense responses of immune systems [46]. Apparently, B. subtilis secretes the antiin ammatory cytokines IL-10 to regulate the immune response [48]. Consequently, supplementation with B. subtilis, B. licheniformis and in combination with compound enzymes had an intense importance for enhancing the immunity of fattening goats.
Rumen pH, NH 3 -N and VFA contents are vital parameters in ruminants which illustrate the function and steady state of the rumen [14], and VFA are principal products of rumen fermentation, which relate the balance of energy in ruminants [49]. Probiotics could improve the function of rumen via maintaining the pH and enriching the VFA concentrations [14]. A previous study found that Lactobacillus plantarum and Bacillus subtilis feed unaffected the pH, NH 3 -N and VFA concentrations, which is consistent with our results [50]. Administration of B. licheniformis enriched the total VFA contents and reduced the NH 3 -N content [51]. There were no remarkable changes in the rumen fermentation parameters of cows fed with B. subtilis [51]. Nevertheless, the in uence of rumen fermentation of fattening goats fed with probiotics or combined with enzymes still remains unclear. In our study, we found that adding probiotics (B. subtilis and B. licheniformis) or blended enzymes insigni cantly in uenced the rumen pH, NH 3 -N and VFA concentrations of fattening goats in basal diet. Moreover, PRO and COM had potential tendencies to increase the isobutyrate contents. Isobutyrate predominantly produced from the oxidative-deamination and decarboxylation of ruminal isoleucine, which illustrated that PRO feed and COM feed had the potential to enhance the metabolism of isoleucine [52].
The microbiota has profound effects on the health and performance of animals. Hence, it is necessary to regulate diversity based on the composition and structure of bacteria in the host [53]. Interestingly, the alpha diversity of rumen microorganisms (Chao 1 index, Goods_coverage index, Observed_species index, Shannon index and Simpson index) showed no signi cant differences (P > 0.05). Administration of probiotics combined with enzymes had no impact on the bacterial diversity of goat rumen. At the phylum level of rumen microorganisms, COM group remarkably upregulated the relative abundance of Proteobacteria in contrast with CON group and PRO group (P < 0.05). Besides, the relative abundance of Actinobacteria in both PRO and COM signi cantly decreased compared with that in CON (P < 0.05). The combined feed showed notable upregulation of Ruminococcaceae, Lachnospiraceae and Succinaceae in ruminal microorganisms (P < 0.05). Prevobacteriaceae, Clostridium and Succinaceae are strict anaerobes, and Ruminococcus can produce a large amount of VFA in the hindgut of cows [54][55], which may lead to the enrichment of isobutyrate in goat rumen. A previous study found that supplementation with compound enzymes (xylanase, α-amylase, β-glucanase and protease) to pigs resulted in the increase of the abundance of Firmicutes in treatment group, which is in coherence with our ndings [56]. The colonization of anaerobic microbes contributes to the creation of an anaerobic environment in rumen, which is conducive to the maintenance of rumen health and effective digestion in rumen, therefore, promoting the health of fattening goats. Butyrivibrio was one of the distinct bacteria in the COM group (P < 0.05), which was in agreement with that previously reported [57]. Prevotella is a kind of potential pathogenic bacterium [58]. The relative abundance of Prevotella_1 signi cantly reduced in COM in comparison with that in PRO, indicating that combined feed of probiotics and enzymes could decrease the number of pathogenic bacteria to keep the health of fattening goats. Clostridiales bene cially maintained the balance of animal intestines and optimized the structure of intestinal microenvironments, thus inhibiting the reproduction of harmful bacteria in the intestine [59]. The relative abundance of Clostridiales_unclassi ed dramatically enriched in COM compared with that in PRO, which plays a central role in keeping the balance of goat rumen.
This study rst revealed the metabolic functions added to probiotics or blended enzymes of fattening goats. COM group signi cantly upregulated nitrogen metabolism, DNA synthesis, glyoxylate cycle, nucleotide synthesis and TCA cycle. COM greatly regulated the rumen microbes possibly be a reason to the differences of functional metabolism [60].
The correlation analysis con rmed there were relevance among goat performance, serum biochemical, immune and antioxidant indexes, rumen fermentation parameters and rumen microorganisms, which indicated that feeding probiotics or combined with enzymes regulated the rumen microbiota to ameliorate the performance and immunity of fattening goats. Particularly, Saccharofermentans and Olsenella positively related to VFA and improved the performance and serum indexes of goats. Consequently, these results above emphasized that PRO feed and COM feed had bene cial effects on the rumen micro ora to facilitate the performance of goats. The performance and healthy state of lactating sows were promoted supplementation with B. subtilis and B. licheniformis [24], which was aligned to our research results. Accordingly, it elucidated an original method to enhance the performance and immunity of fattening goats by attenuation of the relative abundance of Saccharofermentans and Olsenella in rumen.

Conclusion
In conclusion, this research reveals the profound effects on the growth performance, slaughter performance, serum biochemical, immune and antioxidant indexes, rumen fermentation parameters and rumen microbiota of fattening goats administration of B. subtilis, B. licheniformis and combined with enzymes, which explains the potential mechanism to improve the performance and immunity of PRO feed and COM feed. Nonetheless, further research is still worthy of being investigated in regulating the performance and immunity of fattening goats added PRO and COM feed.

Availability of data and materials
The datasets analyzed in the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The procedures were approved by the Animal Care and Use Committee of Nanjing Agricultural University and the experiments were followed by the National Research Council Guide.

Consent for publication
Not applicable.     Relationships of goat performance, serum indexes, rumen fermentation parameters and rumen microorganisms. A. At the phylum level. B. At the genus level. Red represents a positive correlation and blue represents a negative correlation. The darker the color, the stronger the correlation. The signi cance is shown as ** 0.001 < P < 0.01, and * 0.01 < P < 0.05. Values included nine duplicates.