The objective of this study was to evaluate the effect of fecal bacterial community and metabolomics in goats when consuming a lower-protein diet with different energy levels. Eight healthy Leizhou goats, with 11 ± 0.78 kg of body weight, were selected and housed individually in cages. The animals were randomly allocated to a lower-protein diet that varied with four metabolites energy levels (7.01, 8.33, 9.66, and 10.98 MJ/kg DM) in a replicated 4 × 4 Latin square design. Notably, energy-dependent microbial restructuring was observed at both phylum and genus levels. At the phylum level, the relative abundances of Firmicutes and Spirochaetote increased linearly, whereas the Bacteroidota and Patescibacteria decreased linearly with increasing dietary energy levels (p < 0.05). The relative abundances of Verrucomicrobiota increased quadratically, whereas others decreased quadratically with increasing dietary energy levels (p < 0.05). At the genus level, a total of 316 bacteria were identified in the 32 fecal samples. The relative abundances of Christensenellaceae_R-7_group, unclassified_f__Lachnospiraceae, Ruminococcus, norank_o__Clostridia_UCG-014, Treponema, [Eubacterium]_siraeum_group, and [Eubacterium]_ruminantium_group increased linearly, whereas the Oscillospiraceae_UCG-005, norank_f__[Eubacterium]_coprostanoligenes_group, Prevotellaceae_UCG-004, unclassified_c__Clostridia, norank_f__Ruminococcaceae, unclassified_f__ Oscillospiraceae, and others decreased linearly with an increasing dietary energy levels (p < 0.05). In addition, the metabolomic analysis of feces showed that there are many differential metabolites in goats when consuming a lower-protein diet with different energy levels; for example, lipid metabolism and amino acid metabolic pathways were increased in MLE, MHE, and HE groups compared to the LE group. In conclusion, this study provides further information regarding the effects on fecal bacterial community composition and metabolites in goats when consuming a lower-protein diet with different energy levels.

Macadamia integrifolia husk has great potential for use as a feedstuff in ruminants. However, little information regarding its effects on ruminal fermentation traits is available at present. Hence, this in vitro study aims to investigate the effects of Macadamia integrifolia husk as a substrate, supplemented with enzymes at different levels (0 g/kg cellulases + pectinases, CON; 0.5 cellulases + 0.5 g/kg pectinases, TRE1; 1.0 g/kg cellulases + 0.5 g/kg pectinases, TRE2; 1.5 cellulases + 0.5 g/kg pectinases, TRE3), on gas production, fermentation traits, and bacterial communities in goats. The results demonstrate that gas production was increased in TRE1 and TRE3 groups compared to the CON group at 6, 9, 12, 24, and 48 h (p < 0.05); DMD was the highest in the TRE3 group and the lowest in the CON group at 6, 12, 24, and 48 h (p < 0.05); and NDFD was the highest in the TRE1 group and the lowest in the CON group at 6 h, then the highest in the TRE3 group and the lowest in the CON group at 12, 24, and 48 h (p < 0.05). The concentrations of MCP, TVFAs, acetate, and propionate were the highest in the TRE3 group and the lowest in the CON group (p< 0.05). The abundance of Bacillota was the highest in the TRE1 group and the lowest in the TRE2 group (p < 0.05), whereas that of Synergistota and Actinomycetota was the lowest in the CON group and the highest in the TRE2 group (p < 0.05). Acidobacteriota was the most abundant in the TRE3 group and the least abundant in the TRE1 group (p < 0.05). The most abundant genus was norank_p_Bacteroidota, comprising approximately 29.3%, 30.6%, 30.4%, and 31.7% of the total bacteria in the CON, TRE1, TRE2, and TRE3 groups, respectively; this was followed by Succiniclasticum, comprising approximately 6.3%, 5.8%, 6.1%, and 7.0% of the total bacteria in the CON, TRE1, TRE2, and TRE3 groups, respectively. This study provides new insights regarding the use of Macadamia integrifolia husk as a feedstuff in goats; in particular, supplementation with cellulases and pectinases could effectively improve its utilization. The results suggest that the optimal supplementary levels of cellulases and pectinases are 1.5 g/kg and 0.5 g/kg, respectively.

This study investigated the effects of Bacillus subtilis (B. subtilis) supplementation on microbiota and metabolites in the feces of Leizhou goats. Eight Leizhou goats were used in a replicated 4 × 4 Latin square design according to their gender (nanny goats and billy goats) with a 4 × 2 factorial arrangement of treatments that included four B. subtilis additive doses (control [0 g/d; NC, BC], low [2.5 g/d, NL, BL], medium [5 g/d, NM, BM], and high [7.5 g/d, NH, BH]) and 28 d periods (n = 4 per group), each consisting of 27 d adaption and 1 d sample collection. After collecting 32 fecal samples, 16S rRNA gene sequencing and LC-MS were performed to analyze microbial composition and metabolites, respectively. At the genus level, the relative abundance of Rikenellaceae_RC9_gut_group was significantly higher (p < 0.05) in the NM group than in the NC group. The relative abundance of Treponema sp. was significantly lower (p < 0.05) in the NM group than in the NC group. In billy goats, the relative abundances of UCG-005 and Rikenellaceae_RC9_gut_group were significantly higher (p < 0.05) in the BH group than in the BC group. The relative abundance of Treponema sp. was significantly lower (p < 0.05) in the BL, BM, and BH groups than in the BC group. Furthermore, metabolomic analysis revealed that B. subtilis significantly altered the concentrations of glucose metabolism modulators (1-deoxynojirimycin, 1-DNJ) and certain bioactive peptides. Many amino acid metabolic pathways were also enriched. Correlation analysis demonstrated close connections between differential metabolites and the top 10 bacterial genera in fecal samples. These results provide new insights into the impact of B. subtilis on the microbial community and metabolic profile of the feces of Leizhou goats. In this experiment, the appropriate doses of B. subtilis for nanny goats and billy goats were 5 g/d and 7.5 g/d, respectively, but the optimal doses still need to be verified based on performance-based feeding tests in the next study.

The gut microbiota plays a crucial role in maintaining the host’s metabolism and can influence the host’s productivity. Both dietary composition and gender have distinct effects on the composition of the gut microbiota. Therefore, to investigate the differences in the structure and function of the gut microbiota between female and male goats, we analyzed their fecal microbiota and metabolites when fed a 10% crude protein diet at four different energy levels—7.01, 8.33, 9.66, and 10.98 MJ/kg DM. Four non-pregnant female and four male Leizhou goats (all 8 months of age) were used in the experiment, with an average body weight of 10.3 ± 0.8 kg for females and 13.6 ± 1.1 kg for males (mean ± SD). The animals were assigned to two separate 4 × 4 Latin square designs according to their gender, each consisting of four treatments and four 28-day periods, including 25 days of a dietary adaptation period and 3 days of fecal sample collection per period. The data were analyzed using the SAS statistical package and Pearson’s correlation analysis. The dominant phyla for all samples were Firmicutes and Bacteroidota, regardless of dietary energy levels or gender. Among fecal bacteria, unclassified_f_Lachnospiraceae was the dominant genus in the female goats, and Oscillospiraceae_UCG-005 was the dominant genus in the male goats. The relative abundance of unclassified_f_Lachnospiraceae (p < 0.001), Bacteroides (p = 0.007), norank_f_Ruminococcaceae (p = 0.024), Mediterraneibacter (p = 0.001), and norank_f_Muribaculaceae (p = 0.008) was greater in the female goats than in the male goats. In contrast, the relative abundance of Oscillospiraceae_UCG-005 (p < 0.001), Ruminococcus (p = 0.035), Monoglobus (p = 0.006), Oscillospiraceae-NK4A214_group (p = 0.008), norank_f_F082 (p < 0.001), and Prevotellaceae_UCG-003 (p < 0.001) was lower in the female goats than in the male goats. The volcano plot showed that there were 153, 171, 171, and 183 differential metabolites between the female and male goats at dietary energy levels of 7.01, 8.33, 9.66, and 10.98 MJ/kg DM, respectively. Numerous correlations were observed between differential metabolites and microflora genera. We concluded that the non-pregnant female and male goats exhibited distinct metabolic abilities when consuming a 10% crude protein diet at four different energy levels. Interestingly, in the female and male goats, the fecal microbiota also showed some differing responses to the energy levels. These results provide a gender-based reference for formulating low-protein dietary strategies for 8-month-old Leizhou goats.

This study investigates the effects of rumen-protected methionine and lysine (RPML) on the fecal microbiota of Leizhou goats, focusing on growth performance and fecal microbial community composition. A total of 10 three-month female Leizhou goats (9.90 ± 0.08 kg) were randomly assigned to one of two dietary treatments: a CON group fed a basal diet and an RPML group receiving the basal diet supplemented with 1.5 g/d/head of rumen-protected methionine and 4.5 g/d/head of rumen-protected lysine. Results indicated that RPML significantly enhanced average daily gain (ADG) and final body weight (FBW), as well as significantly decreased the ratio of dry matter intake (DMI) to ADG (p < 0.001). Fecal microbiota composition showed a decrease in abundance of UCG-005, Phascolarctobacterium, and norank_f__Bacteroidales_RF16_group and an increase in others like Christensenellaceae R-7 and unclassified_c__Clostridia (p < 0.05). Moreover, the correlations between the abundance of certain bacterial genera and the concentrations of short-chain fatty acids (SCFAs) suggest that the modulation of the gut microbiota is associated with improved growth performance and feed efficiency in Leizhou goats, indicating that RPML supplementation can modulate the gut microbiota to improve growth performance and feed efficiency in Leizhou goats.

Macadamia integrifolia husk (MIH) is a major byproduct of Macadamia integrifolia processing; however, there is limited information available on its rumen fermentation characteristics and associated bacterial communities when used as a feed for ruminants. This study evaluated the effects of different Bacillus subtilis (BS; ≥10¹³ CFU/g) inclusion levels on MIH using an in vitro technique. A total of six healthy goats (female, 7-month-old) with an average body weight of 15.20 ± 0.20 kg were selected as rumen fluid donors, and the rumen fluid was collected before morning feeding. Four BS inclusion levels were studied: a control group with 0 g/kg (CON), and three treatment groups supplemented with 1.5 g/kg (low dose, BSL), 3.0 g/kg (medium dose, BSM), and 4.5 g/kg (high dose, BSH) of BS. MIH supplemented with BS improved the gas production at all the observed incubation times (p < 0.05). The in vitro dry matter degradability was increased by 3.5%, 2.0%, and 3.1% in the BSL, BSM, and BSH groups, respectively, compared to the CON group at a 48 h incubation time (p = 0.049). The in vitro neutral detergent fiber degradability was increased by 6.0% and 7.8% in the BSL and BSM groups, respectively, compared to the CON group at a 48 h incubation time (p = 0.027). The in vitro acid detergent fiber degradability was increased by 11.7%, 16.8%, and 3.2% in the BSL, BSM, and BSH groups, respectively, compared with the CON group at the 48 h incubation time (p = 0.014). The concentrations of ammonia-N, microbial protein, total VFAs, acetate, propionate, butyrate, iso-butyrate, valerate, and iso-valerate were quadratically changed with increasing BS supplementation levels (p < 0.05). The relative abundances of Bacteroidota and Pseudomonadota were highest in the BSL group and lowest in the BSM group (p < 0.05), whereas Bacillota and Kiritimatiellota were highest in the BSH group and lowest in the BSL group (p < 0.05). Synergistota was highest in the BSM group and lowest in the BSH group (p < 0.05). The relative abundances of norank_p_Bacteroidota and norank_o_Bacteroidoles were highest in the BSL group, whereas they were lowest in the BSM group (p < 0.05). The relative abundances Hoyesella and Succinivibrio were highest in the BSL group, whereas they were lowest in the BSH group (p < 0.05). The relative abundances of Prevotella, Succiniclasticum, and Selenomonas were highest in the BSM group, whereas they were lowest in the BSL group (p < 0.05). These in vitro results indicate that supplementation with Bacillus subtilis could improve the utilization of MIH in goats, which is possibly associated with the altered rumen bacteria communities. The supplementation of 1.5 to 3.0 g/kg Bacillus subtilis (≥10¹³ CFU/g) on Macadamia integrifolia husk could improve its utilization as a feasible alternative feed for ruminants using an in vitro technique.