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.

Macadamia is an emerging woody nut with high economic value. Although genome assemblies are now available for all four species, a comprehensive evolutionary analysis integrating divergence time estimation, lineage-specific gene family dynamics, and whole-genome duplication (WGD) history has not yet been undertaken. Here, we performed a comparative genomic analysis of four macadamia species (M. integrifolia, M. tetraphylla, M. jansenii, and M. ternifolia) alongside 11 other plant species. A total of 72,992 gene families were identified, with species-specific gene families numbering 277 in M. integrifolia, 234 in M. tetraphylla, 236 in M. ternifolia, and 183 in M. jansenii. KEGG enrichment analysis revealed that M. integrifolia-unique genes were enriched in terpenoid biosynthesis and ABC transporter pathways, whereas the unique genes of the other three species were predominantly associated with phenylpropanoid biosynthesis and amino acid/carbohydrate metabolism. Phylogenetic analysis revealed a well-supported monophyletic macadamia clade, with M. integrifolia and M. tetraphylla forming one subclade, and M. ternifolia and M. jansenii forming another. Divergence time estimation indicated that the genus diverged approximately 10.3 million years ago (Mya), with species-level divergences occurring at 7.8–8.4 Mya. Genomic synteny analysis revealed a conserved genomic architecture, with 57.36%–59.68% of genes being shared in syntenic blocks among species. Gene family dynamics revealed lineage-enriched patterns in the terpenoid biosynthesis pathways: expansion in M. integrifolia but contraction in the other three species. WGD analysis detected two WGD events. On a relative Ks scale, the more recent WGD event overlapped with the species-level divergence, whereas the older WGD event was shared by the common ancestor of the genus. These findings provide a comprehensive genomic foundation for understanding macadamia evolution and may facilitate breeding and conservation efforts.

This study was conducted to measure Macadamia integrifolia husk (MIH) as a feed ingredient for goats using an in vitro rumen fermentation system. Four isonitrogenous and isoenergetic substrates were prepared by using MIH to replace 0, 5%, 10%, or 15% of corn as a dry matter basis, namely MIH0, MIH5, MIH10, and MIH15 groups, respectively. Gas production decreased linearly as corn was replaced by increasing levels of MIH. The DMD decreased linearly at the 24 h and 48 h incubation times as corn was replaced by increasing levels of MIH (p < 0.01). The NDFD decreased linearly at the 6 h, 12 h, 24 h, and 48 h incubation times as corn was replaced by increasing levels of MIH. At 48 h, pH differed extremely significantly (p < 0.001) and followed a quadratic trend (p < 0.05). The MIH0 group had the highest NH₃–N, butyric, and isovaleric acids concentration (p < 0.05); the MIH5 group had the highest MCP, acetic acid, and propionic acid concentration (p < 0.05). Bacteroidota and Pseudomonadota were the dominant phyla in the fermentation fluid at the 48 h incubation timepoint among the four groups. The relative abundance of Kiritimatiellota and Synergistota was highest in the MIH15 group. The relative abundance of Pseudomonadota was highest in the MIH5 group. At the genus level, norank_p_Bacteroidota and Ruminobacter were the dominant genera, and the bacterial community structure differed significantly among the four treatments (p < 0.05). In conclusion, we suggest that the MIH replacing 5% of corn did not affect fermentation, VFA profile, or microbial protein synthesis and it could alter the bacterial communities. Moreover, it is necessary to conduct an in vivo study to investigate the growth performance of goats in a further study.