Allantoin

Growth rate of all the inverted and non-inverted strains was determined using FLUOstar optima microplate reader. Cells were grown to stationary phase in YPD, minimal and allantoin containing medium. Optical density (OD) of the cultures was measured at 595 nm and then diluted to OD_{595 nm} = 0.1 in pre-warmed respective medium. 240 µl of the diluted cultures was transferred to each well of 96 well plate including the media controls. The OD measurement was taken by the microplate reader at 30°C for 40 hours at intervals of 5 minute and with 1 minute linear shaking just before every measurement. Growth curves were plotted using the Optima data analysis programme and standard deviations were calculated in Excel. The growth parameters (growth rate μ and maximum cell biomass A) were measured using R statistic package grofit [29] .

Samples of TMR, orts, and feces were dried at 65°C in a forced-air oven to a constant weight to measure the DM, CP, ether extract, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents by wet chemistry. The DM was determined using a forced-air oven (AOAC method 930.15). The nitrogen content was determined using a Kjeldahl auto-analyzer (VELP DK20, Usmate, Italy), and the CP was calculated as N × 6.25 (AOAC method 990.03). The ether extract content was determined using the AOAC International method 920.39 (AOAC method 920.39). The ADF and NDF contents were measured according to a previously described method (Van Soest et al., 1991 (link)). Milk subsamples preserved in 2-bromo-2-nitropropane-1-3-diol were analyzed for true protein, fat, lactose, and total milk solids using infrared spectroscopy (Foss FT 120, Denmark). The nitrogen content in the milk, fecal, and urine samples was determined using the Kjeldahl method (AOAC method 990.39). The NH₃-N concentration in rumen fluids and urine was measured using a phenol-hypochlorite assay. The rumen AA contents were determined as previously described (Liu et al., 2020 (link)). In the rumen fluid preserved with metaphosphoric acid, the VFA content was analyzed using gas chromatography (Agilent 7890A, USA). The allantoin, xanthine, uric acid, and hypoxanthine contents in the urine samples were measured as previously described (Chen and Gomes, 1992 ).
The total protein, albumin, globulin, creatinine, and β-hydroxybutyric acid content and the activities of alanine and aspartate aminotransferase in the plasma of dairy goats were analyzed using a Beckman AU680 Automatic Biochemical Analyzer (Beckman Coulter Inc., FL, USA). The total AA content, NH₃ concentration, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase, and total antioxidant capacity (T-AOC) in plasma were quantified using total AA (A026-1-1), blood ammonia (A086-1), GSH-Px (A005-1), SOD (A001-3-2), catalase (A007-1-1), and T-AOC (A01502-1) assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), respectively. The levels of growth hormone, prolactin, estradiol, and insulin in plasma were determined using a DFM-96 Gamma Radioimmunoassay Counter (Zhongheng Electromechanical Technology Development Co., Ltd, Hefei, China). Insulin-like growth factor 1 (IGF-1) was determined using a bovine IGF-1 ELISA kit (Shanghai Ketao Biotechnology Co. LTD, Shanghai, China) according to the manufacturer’s instructions.
The total DNA of rumen microorganisms was extracted using the hexadecyltrimethylammonium bromide method and measured using Nanodrop One (Thermo Scientific, MA, USA). The sequencing library was constructed using the MGIEasy kit (BGI, China) and the BGISEQ-500, with read lengths of 2 × 100 base pairs (bp), was used to sequence the libraries. Quality control analysis of the original data was performed using the TrimGalore software to remove low-quality reads with lengths less than 50 bp and average base mass less than 20 (Mehdipour et al., 2020 (link)). The relevant data were then compared with the host (sheep, alfalfa, corn, and soybean) database using BM Tagger to remove the host data (Uritskiy et al., 2018 (link)). The sequence was then annotated with the species using the Kraken 2 software (Kopylova et al., 2012 (link)) and the GTDB-r89 database (Waschulin et al., 2022 (link)). The relative abundance of species was analyzed by principal component analysis and the diversity analysis was performed using MicrobiomeAnalyst (Chong et al., 2020 (link)). The species composition and community structure of the rumen microbiome were tested for significance using linear discriminant analysis effect size analysis.