5.1 Ruminal fluid collection and sample preparation:
1. From a fistulated cattle, sheep or goat, collect at least 50 mL of rumen fluid filtering it through three cheese-cloth layers. Make the collection in a 39 ºC pre-warmed thermos and transport it to the laboratory as soon as possible.
Notes: 1) to thermos pre-warming, fill it with pre-warmed water at 39 ºC, and empty it at the moment to collect the rumen fluid. 2) the rumen fluid sample must be composed of rumen fluid from three rumen regions: front and half of the ventral sac, and from cranial sac (Zijderveld et al., 2011).
2. In the laboratory, add 2 mL of the previously filtered rumen fluid to a 5 mL centrifuge tube.
3. Add 2 mL of extraction solution (see section 3.1) to the centrifuge tube plus rumen fluid and stir it in vortex using medium speed for 30 s.
4. Freeze the sample at -20 ºC until analysis.
Note: if the sample is immediately analyzed after collection, refrigerate the tube at 4 ºC for 30 min to allow rumen fluid protein precipitation.
5.2 Extraction of volatile fatty acids from rumen fluid:
1. On the day of analysis, thaw samples at room temperature (20 ºC).
2. Centrifugate samples at 4,000 rpm for 10 min at room temperature (20 ºC).
3. Recover the liquid phase after centrifugation using a plastic syringe and filter the liquid phase through a 0.45 μm PES membrane filter. Collect the volume in a plastic tube of 4 mL.
5.3 Chromatographic analysis:
5.3.1. Chromatographic analysis of the calibration curve:
1. Prepare the calibration curve as described in 3.3.2 item of reagents section..
2. Analyze each point of the curve by RP HPLC-DAD.
3. Identify and integrate the resulting chromatographic peaks.
5.3.2 Chromatographic analysis of the ruminal fluid samples:
1. Place 500 μL of the final extract in a chromatographic vial.
2. Add 500 μL of Milli-Q water.
3. Analyze by RP HPLC-DAD.
4. Identify and integrate the resulting chromatographic peaks.
5.3.3 Chromatographic conditions:
- Column temperature: 30 ºC.
- Injection volume: 20 μL.
- Stationary phase: Hypersil GOLD C18 column (dimensions: 150 x 4.6 mm) equipped with Hypersil GOLD C18 guard column (dimensions: 10 x 4 mm).
- Working wavelength: 210 nm.
- Mode: gradient, constant flow: 0.750 mL/min (see Table 2 in supplementary files).
Note: According to the literature findings, the most suitable column for VFA chromatographic separation by HPLC-DAD is an ion-exchange column. However, the Hypersil GOLD C18 column allows an efficient VFA separation, except for butyric and iso-butyric acids. This condition may constitute a limitation of the Hypersil GOLD C18 column use for VFA determination. However, considering that the concentration of iso-butyric acid is significantly lower than that of butyric acid, a good estimation of butyric acid can be obtained.