Feces were thawed and diluted to 1:10 in water for pH measurement using a potentiometer (pH and Ion-Meter GLP 22, Crison, Barcelona, Spain). Ammonia-N was determined in 1 g of feces diluted in 200 ml of ultrapure water (18.2 MΩ cm; Arium®, Sartorius, Göttingen, Germany) and subjected to a gas-diffusion microextraction with o-phthalaldehyde labeling for fluorometric determination in a microplate reader (Synergy HT, BioTek Instruments, Bad Friedrichshall, Germany), according to Valente et al. (2017) (link).
For the determination of lactate, 1 g of feces was diluted into 10 ml of ultrapure water and homogenized by vortexing and ultrasound (5 min)-assisted mixing. Samples were then centrifuged for 15 min at 2,415 × g and 4°C and filtered using a 0.45-μm pore size polyethersulfone syringe filter (VWR International, Alfragide, Portugal). The supernatant was recovered and assayed using a commercial kit (D -/L -lactic acid, NZYTech, Lisbon, Portugal) adapted to a microplate format. The UV detection was performed using the above-mentioned microplate reader. Lactic acid is presented as the sum of D - and L -lactic acids.
For VFA analysis, 1 g of feces was diluted in 10 ml of 25% ortho-phosphoric acid solution with internal standard (4 mM of 3-methyl valerate, Sigma Aldrich, St. Louis, MO, United States); the mixture vortexed and centrifuged for 60 min at 5,251 × g, at 4°C. The supernatant was filtered using a 0.45-μm pore size polyethersulfone syringe filter (VWR International) and analyzed by gas chromatography using a Shimadzu GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) equipped with a capillary column (HP-FFAP, 30m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, United States) and a flame ionization detector. Individual VFA was identified by comparison of retention times with a commercial standard and quantified with the internal standard method as described by Maia et al. (2016) (link).
For the determination of lactate, 1 g of feces was diluted into 10 ml of ultrapure water and homogenized by vortexing and ultrasound (5 min)-assisted mixing. Samples were then centrifuged for 15 min at 2,415 × g and 4°C and filtered using a 0.45-μm pore size polyethersulfone syringe filter (VWR International, Alfragide, Portugal). The supernatant was recovered and assayed using a commercial kit (
For VFA analysis, 1 g of feces was diluted in 10 ml of 25% ortho-phosphoric acid solution with internal standard (4 mM of 3-methyl valerate, Sigma Aldrich, St. Louis, MO, United States); the mixture vortexed and centrifuged for 60 min at 5,251 × g, at 4°C. The supernatant was filtered using a 0.45-μm pore size polyethersulfone syringe filter (VWR International) and analyzed by gas chromatography using a Shimadzu GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) equipped with a capillary column (HP-FFAP, 30m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, United States) and a flame ionization detector. Individual VFA was identified by comparison of retention times with a commercial standard and quantified with the internal standard method as described by Maia et al. (2016) (link).
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