At the end of each experimental period, microbial pellets from each fermenter vessel were harvested [38 (link)]. Briefly, total fermenter contents were blended for 1 min and filtered through 4 layers of cheesecloth with 200 mL of saline solution (0.9% NaCl). To remove the remaining feed particles, the filtrate was centrifuged (Allegra X-15R Centrifuge, Beckman Coulter Life Sciences, Indianapolis, IN, USA) at 1000× g for 10 min at 4 °C. The supernatant was collected and centrifuged (Sorvall RC-5B Refrigerated Superspeed Centrifuge, DuPont Instruments) at 11,250× g for 20 min at 4 °C for isolation of the microbial pellet. The microbial pellet was resuspended in 200 mL of McDougall’s solution for pellet purification and centrifuged at 16,250× g for 20 min at 4 °C. The final microbial pellet was resuspended in distilled water and transferred to a new container and kept at −20 °C until further analysis. The microbial pellet was freeze-dried for DM determination and analyzed for ash, total N, and 15N abundance.
We used 15N as a marker for microbial protein synthesis, and it was analyzed on artificial saliva, background digesta (before 15N-enriched saliva was used), digesta, and bacteria samples according to the following procedure. Freeze-dried samples were processed in 2 mL microcentrifuge tubes using 2.0 mm zirconia beads and homogenized (Precellys 24, Bertin, Rockville, MD, USA) at 5500× g for 10 s. Samples were weighed in tin capsules using a microscale (Excellence Plus XP Micro Balance Mettler-Toledo GmbH, Laboratory & Weighing Technologies, Columbus, OH, USA), and 35 µL of K2CO3 solution (10 g/L) were added to alkalinize the samples; the suspension was dried overnight in a forced-air oven at 40 °C to volatilize ammonia [39 (link)]. Analysis of 15N was performed with a mass spectrometer (IsoPrime 100, IsoPrime, Naperville, IL, USA), and the results were obtained as the fractional abundance of isotopic fractions (15N/14N). The equations used for the calculation of ruminal N metabolism are described below:
The percent excess of 15N of NAN (non-NH3-N) effluent was obtained by subtracting % atom 15N in the background from the % atom excess of 15N of NAN effluent [40 (link)].
Flows of NH3-N, NAN, and N metabolism were determined [41 (link)].
Microbial efficiency was determined as follows [40 (link)].
Efficiency of N used was determined as follows [41 (link)].
We used 15N as a marker for microbial protein synthesis, and it was analyzed on artificial saliva, background digesta (before 15N-enriched saliva was used), digesta, and bacteria samples according to the following procedure. Freeze-dried samples were processed in 2 mL microcentrifuge tubes using 2.0 mm zirconia beads and homogenized (Precellys 24, Bertin, Rockville, MD, USA) at 5500× g for 10 s. Samples were weighed in tin capsules using a microscale (Excellence Plus XP Micro Balance Mettler-Toledo GmbH, Laboratory & Weighing Technologies, Columbus, OH, USA), and 35 µL of K2CO3 solution (10 g/L) were added to alkalinize the samples; the suspension was dried overnight in a forced-air oven at 40 °C to volatilize ammonia [39 (link)]. Analysis of 15N was performed with a mass spectrometer (IsoPrime 100, IsoPrime, Naperville, IL, USA), and the results were obtained as the fractional abundance of isotopic fractions (15N/14N). The equations used for the calculation of ruminal N metabolism are described below:
The percent excess of 15N of NAN (non-NH3-N) effluent was obtained by subtracting % atom 15N in the background from the % atom excess of 15N of NAN effluent [40 (link)].
Flows of NH3-N, NAN, and N metabolism were determined [41 (link)].
Microbial efficiency was determined as follows [40 (link)].
Efficiency of N used was determined as follows [41 (link)].
Free full text: Click here
