A protocol for the study of the rumen biohydrogenation of unsaturated fatty acids of lipid supplements mixed with forages using an in vitro approach

The rumen biohydrogenation of dietary unsaturated fatty acids has an important effect on the nal compositional quality of ruminant meat and milk. Hence, diverse methodological approaches have been developed to understand and modulate this biochemical process that occurs in the rumen. Therefore, in the current method article, a protocol is presented to conduct an in vitro assay for understanding the biohydrogenation of dietary fatty acids in the rumen. The protocol enabled the evaluation of the effects of different relationships between linoleic and alpha-linolenic acids in supplements on the production of fatty acid intermediaries derived from the biohydrogenation of these unsaturated fatty acids in the rumen, with satisfactory results.


Introduction
The understanding of the biohydrogenation process of unsaturated fatty acids (UFA) in the rumen provides valuable insights regarding utilization of lipid supplements by the ruminant (Harvatine and Allen, 2006;Vargas et al., 2012;Vargas et al., 2018;Vargas et al., 2019). For this purpose, several experimental strategies have been proposed using in vivo, in situ, and in vitro approaches (Enjalbert et al., 2003;Baldin et al., 2018;Vargas et al., 2018). Each one possesses advantages and limitations deeply discussed in different reviews (Fievez et al., 2007;Jenkins et al., 2008;Dewanckele et al., 2020); however, the in vitro approach has gained special attention in the recent years due this one is cheap, fast, it does not require specialized operators to conduct the assay, and it provides results potentially applicable under in vivo conditions. These characteristics have enabled the development of multiple in vitro assays exploring the effects of different lipid mixtures on the kinetics of biohydrogenation of UFA in the rumen, whose results have been used for designing nutritional strategies to enhance ruminant production and the quality of derived products (Ferlay et al., 2017). Tilley and Terry (1963) developed an in vitro assay protocol that has been extensively used for evaluating the dry matter and nutrient digestibility of different feedstuffs and forages. This method involves two general steps: the rst one includes a fermentation of the forage or feedstuff in rumen uid inoculum under anaerobic conditions to simulate rumen digestion. The second one involves a digestion of the non-degraded content in the rst step with a mixture of pepsin and HCl to simulate the abomasum digestion. From the aforementioned steps, the rst one is commonly used to understand the biohydrogenation of UFA showing successful results (Ribeiro et al., 2007;Vargas et al., 2012;Vargas et al., 2018). However, to date, this procedure has not been published as a protocol elsewhere. Hence, the objective of this method article is to present the main steps to conduct an in vitro assay based on the Tilley and Terry (1963) method to understand the biohydrogenation of UFA in the rumen. Reagents 1. Carbon dioxide (CO 2 ; gas cylinder). McDougall (McDougall, 1948) preparation (Sigma Aldrich®):

Buffer
a. On the previous day of the analysis: prepare a pre-solution in distilled water, including the following salts: NaHCO 3 (9.80 g/L), Na 2 HPO 4 (2.77 g/L), KCl (0.57 g/L), NaCl (0.47 g/L), and MgSO 4 .7H 2 O (0.12 g/L). Keep the pre-solution in a sealed 1 L volumetric ask under refrigeration.
b. On the day of the analysis: add the pre-solution to a 1 L beaker, and after that, add 0.16 g of CaCl 2 .2H 2 O to it, dissolving this salt under continuous stirring with slow heating when needed. When CaCl 2 .2H 2 O was dissolved and solution gets room temperature, add 1.0 g of urea to the mixture, dissolve it, and transfer the total mixture to the 1 L volumetric ask. Complete the pre-solution to 1 L with distilled water. Finally, transfer the buffer prepared to a plastic or a glass bottle and keep it at 39 ºC in a thermostatic water bath until inoculum preparation.

Equipment
Laboratory equipment -Thermostatic water bath.
-Gasi cation system for CO 2 (composed by CO 2 gas cylinder linked to plastic tube and pipette cap).
-Transfer pipettes with caps.
-Gas chromatograph equipped with autosampler, ame ionization detector, and column.

Materials
-100 mL plastic tubes with one-hole rubber stoppers. The one-hole is essential for the internal gas pressure regulation during in vitro incubation.
-1 L glass or plastic bottle.
-Measuring cylinder, graduated pipette or a burette of at least of 50 mL.
-Vials with glass insert, septum, and plastic caps. Procedure 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).

Ruminal uid collection:
a. From a stulated cattle, sheep or goat, collect at least 50 mL of rumen uid ltering 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, ll it with pre-warmed water at 39 ºC, and empty it at the moment to collect the rumen uid. 2) the rumen uid sample should be composed of rumen uid 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 uid 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 CO 2 gas. Kept the bottle sealed at 39 ºC in a thermostat bat (Figure 2).

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 HgCl 2 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 ask and freeze it at -60 ºC.
3. Activities to be conducted after the day of in vitro assay:

Preparation of incubation systems for fatty acid analysis:
a. Put the ask in a tray of a freezer dryer equipment, covering the ask'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 ask (Figure 4b).
b. Put the ask with lyophilized content in an ultra-freezer at −60 ºC until fatty acid analysis.
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 ux 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. Fatty acid quanti cation: Quanti cation 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).  Tubes with one-hole rubber stoppers used for in vitro incubations into the thermostat bath.

Figure 2
Inoculum prepared for in vitro incubations. See the system tubes to bubble CO2 and serving the inoculum into tubes.
Page 12/14 Lyophilization of incubation contents. a) con guration of ask into the lyophilizer (see that mouth of ask was covered with an absorbent paper tower and adjusted to mouth with a rubber band). b) freeze dried resultant incubation system.