1. Activities to be conducted on the previous day of in vitro assay:
a. In 100 mL plastic tubes as replications you want to prepare, carefully weight 500 mg of a dried and milled forage (e.g., kikuyu grass).
b. Seal the tubes with one-hole rubber stoppers and keep them in a test tube rack (Figure 1).
c. Prepare the mixtures of lipids you want to test (treatments) using automatic viscous liquid transfer pipettes and keep them in glass recipients under refrigeration.
2. Activities to be conducted on the day of in vitro assay:
2.1 Pre-warm the buffer McDougall at 39 °C in a thermostatic water bath, as stated in section 2, item b of the reagents section.
2.2 Add lipid mixtures to the 100 mL plastic tubes prepared on the previous day of the analysis. For lipid addition, use the automatic viscous liquid transfer pipettes (see section 1a).
2.3 Ruminal fluid collection:
a. 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 should be composed of rumen fluid from three rumen regions: front and half of the ventral sac, and from cranial sac (Zijderveld et al., 2011).
2.4 Inoculum preparation: in a plastic or glass bottle kept at 39 ºC in a thermostat bath, mix the rumen fluid collected in the step 2.3 with the buffer McDougall in a ratio 1:4 to constitute the inoculum to be used during in vitro incubation. After that, adjust the pH of inoculum to 6.8 bubbling CO2 gas. Kept the bottle sealed at 39 ºC in a thermostat bat (Figure 2).
2.5 Preparation of incubation systems and in vitro assay.
a. Put the 100 mL plastic tubes with forage and lipid mixture in the 39°C thermostat bath, using appropriate support rack. Assure that these tubs are equilibrated at 39 ºC keeping it at this temperature at least 15 min before the in vitro assay.
b. Take a 100 mL tube from incubation bath and add 50 mL of 39 °C inoculum previously prepared using a 50 mL measuring cylinder (graduated pipette or burette can be used as well) equilibrated at 39 °C. Seal the tube with one-hole rubber stopper and kept it back to 39 °C thermostat bath. Repeat this process with the other tubes. The tubes must be shaken manually every 2 hours.
c. To evaluate the changes of fatty acid concentrations across time (e.g., 0, 2, 4, 6, 8, and 16 h), stop the UFA biohydrogenation at different times. For this purpose, remove the plastic tube from the thermostat bath, add 500-uL of a 2% w/v HgCl2 solution to it, and place the tube in an ice bath (Figure 3).
d. Transfer the content of each tube to a 100 mL glass flask and freeze it at – 60 ºC.
3. Activities to be conducted after the day of in vitro assay:
3.1. Preparation of incubation systems for fatty acid analysis:
a. Put the flask in a tray of a freezer dryer equipment, covering the flask’s mouth with an absorbent paper tower and adjusting it with a rubber band (Figure 4a). The lyophilization time can be superior to 48h, and it could vary according to the sample type and number. However, the process can be stopped when all water was removed from the flask (Figure 4b).
b. Put the flask with lyophilized content in an ultra-freezer at −60 ºC until fatty acid analysis.
3.2. Extraction, methylation, and quantification of fatty acids from lyophilized incubation systems (Figure 5; Garcés and Mancha; 1993; Yamasaki et al., 1999):
a. Weight 50 mg of lyophilized incubation system in a 16 x 160 mm screw cap tube.
b. Add to the tube the following reagent quantities: 2148 μL methanol, 990 μL toluene, 66 μL of 99.9% sulfuric acid, 1000 μL dimethylsulfoxide (DMSO), and 2-mL hexane.
c. Seal the tube and heat it at 80 ºC during 2 hours in a water bath.
d. Let the tube equilibrate at room temperature and recover the hexane layer (top layer in the mixture) in an Eppendorf ® 2 mL Safe-Lock tube.
e. Evaporate the hexane under a nitrogen flux and re-dissolve the resultant residue in 500 uL of dichloromethane.
f. Transfer 250 uL of dichloromethane solution to a chromatographic vial with an appropriate glass insert, septum, and plastic cap.
g. Inject in a GC-FID in accordance with the following conditions:
Equipment: Shimadzu GC-2014 gas chromatograph (Shimadzu Manufacturing, Inc., Canby, OR, USA) with autosampler.
Column: fused silica capillary (Rt-2560, 100 m x 0.25 mm i.d. x 0.2 μm film thickness; Restec®, Inc, Belefonte, PA, USA).
Carrier gas: Helium 5.0.
Flux: 1.12 mL/min.
Detector and injector temperatures: 260 and 270 °C, respectively.
Split ratio: 30:1.
Injection volume: 1.0 uL.
Temperature program: The oven temperature is programmed at an initial value of 140 °C remaining for 5 min, which was increased at 4 °C/min for 5 min more, up to a temperature of 220 °C. After that, it was subsequently increased at 2.0 °C/min for 10 min, up to a final temperature of 240 °C.
Fatty acid identification: The fatty acids in samples can be identified by comparison of their retention times with those observed in commercial standards as Nu-Check® Prep (Elysian, MN, USA; GLC-603).
Fatty acid quantification: Quantification can be made by direct comparison of the peak areas for obtaining the relative proportions of fatty acids. The fatty acid concentration or absolute quantities can be obtained using appropriate calibration curves prepared from commercial standards (external standard method) or using cis-10-17:1, 19:0, or 23:0 fatty acids as an internal standard (internal standard method). For this method, an appropriate amount of fatty acids needs to be added at the beginning of fatty acid extraction and methylation procedure (Section 3.2a).
3.3. Fatty acid kinetics analysis:
Finally, fatty acid concentrations can be computed in function of time to define potential intermediary changes across time, as well as treatment effects on that relationship. The association between fatty acid concentration and the time can be further analyzed under different statistical approaches. Take as examples, the approaches used in Ribeiro et al. (2007), Jenkins et al. (2008), and Vargas et al. (2018).