Biohydrogenation Pathway of α-Linolenic Acid in Rumen of Dairy Cow In Vitro

Simple Summary Few studies have investigated a relationship between t9,c12,c15-C18:3 and ALA, an α-Linolenic acid c9,c12,c15-C18:3 in the rumen. These results indicated that t9,c12,c15-C18:3 was an intermediate of the α-linolenic acid shifted rumen biohydrogenation pathway. This study hypothesized a pathway for α-linolenic acid biohydrogenation in rumen. Abstract The t9,c12,c15-C18:3 as an isomer of α-linolenic acid (c9,c12,c15-C18:3; ALA), has been recently detected in milk, but has not been found in the rumen. This study hypothesized that it may be a biohydrogenation product of ALA in rumen and aimed to explore whether it was present in the rumen and help to understand the rumen biohydrogenation mechanisms of ALA. The in vitro experiment included two treatments, a control check (CK group) with 50 µL ethanol added, and ALA group with 50 µL ethanol and 2.6 mg ALA (ALA addition calculated by 1.30% of dry matter base of diet); each sample of fermentation fluid had the composition of C18 fatty acids analyzed at 0, 0.5, 1, 2, 3, 4, 5, and 6 h. The results showed that no t9,c12,c15-C18:3 was detected in the CK group, but ALA addition increased the concentration of t9,c12,c15-C18:3 in fermentation fluid. The content of t9,c12,c15-C18:3 peaked 1 h after fermentation, then declined gradually. At 1 h, no t9c12c15-C18:3 was detected in the fermentation fluid of the CK treatment. The results suggested that ALA converted to the isomer t9,c12,c15-C18:3 through biohydrogenation in the rumen. The addition of ALA can also increase the concentration of t9,c12-C18:2, c9,t11-C18:2, c12-C18:1, t11-C18:1, t9-C18:1, and c6-C18:1 in fermentation fluid. It was concluded using an in vitro experiment that t9,c12,c15-C18:3 was a product of rumen biohydrogenation of ALA.


Introduction
The supplementation of α-linolenic acid (c9,c12,c15-C18:3; ALA) in the ruminant diet can increase the concentration of ALA and longer chain ω-3 polyunsaturated fatty acids (n-3 PUFA) in milk [1], which are essential fatty acids for humans. However, ALA biohydrogenation in the rumen is the main limiting factor influencing the efficiency of dietary ALA transport into milk. Therefore, exploring the pathways of ALA biohydrogenation in rumen could help regulate milk ALA and n-3 PUFA. The first step of ALA biohydrogenation in the rumen is isomerization. Pervious research showed that the cis-trans isomerization of ALA could happen in the ortho-position, and ALA could isomerize to t10,c12,c15-C18:3 Animals 2022, 12, 502 2 of 6 with c9 to t10 [2], c9,t11,c15-C18:3 with c12 to t11 [3], and c9,t13,c15-C18:3 with c12 to t13 [4]. However, few studies have reported that the isomerization of ALA could happen in an in-situ position such as t9,c12,c15-C18:3 with c9 to t9 in the rumen. The in situ isomerization of ALA has been reported in the processing of food. A previous study reported that ALA could isomerize to t9,c12,c15-C18:3 through frying [5]. Recent studies have found that t9,c12,c15-C18:3 was present in milk [6]. Therefore, it can be hypothesized that ALA may also convert to t9,c12,c15-C18:3 through biohydrogenation in rumen. The purpose of this experiment was to explore whether there was t9,c12,c15-C18:3 in rumen fluid using an in vitro fermentation test and to investigate the relationship between t9,c12,c15-C18:3 and ALA. The t9,c12,c15-C18:3 identified in this study may provide a theoretical basis for the exploration of rumen biohydrogenation.

Experimental Design and Animal Management
The ALA pure products were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) and diluted with ethanol. Rumen fluid was collected from three ruminal cannulated Holstein cows fed a diet that met the feeding standards of dairy cattle in China according to the Ministry of Agriculture of China Feeding Standard of Dairy Cattle (NY/T 34-2004, MOA: Beijing, China, 2004). Rumen contents were withdrawn before the first meal in the morning, pooled in equal proportions into a container with CO 2 , transferred to the laboratory, and strained through four layers of cheesecloth to obtain rumen fluid.
Eighty fermentation bottles were separated into two groups as a control (CK group), with 50 µL ethanol added and ALA group with 50 µL ethanol and 2.6 mg ALA (ALA addition calculated by 1.30% of dry matter base of diet). The bottles were prewarmed to 39 • C and flushed by CO 2 , then filled with 200 mg diet (Table S1) and 30 mL oxygen-free buffered rumen fluid, including 10 mL rumen fluid and 20 mL buffer [7]. All the bottles were sealed with rubber stoppers and incubated in the shaking water bath (MRT-60R, shanghai minquan instrument Co., Ltd. Shanghai, China) at 40 r/min and 39 • C for 0, 0.5, 1, 2, 3, 4, 5, and 6 h, respectively. Fermentation fluid was collected in a 5 mL EP tube and stored at −80 • C for further C18 fatty acid analysis.

C18 Fatty Acid Analysis
The extraction of fatty acids and fatty acid methyl esters (FAMEs) from ruminal fluid was according to a previous protocol [6]. A 2 mL aliquot of each sample was transferred to a 15 mL tube and 4 mL of n-hexane/isopropanol (v/v = 3/2) solution was used to extract the fat. The methyl esterification of the fat was then performed using 10% acetyl chlorocarbinol followed by 2% methanolic NaOH. The FAMES were analyzed using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) fitted to a 5975C MS mass spectrometer detector (Agilent Technologies, Santa Clara, CA, USA). A sample of 2 µL FAME mixed with hexane was injected through the split injection port in a 10:1 ratio into a capillary column CP-Sil 88 fused silica 100 m × 0.25 mm × 0.20 µm (Agilent, Palo Alto, CA, USA). The injector temperature was set at 250 • C, and oven temperature was initially 120 • C. After holding for 2 min, the temperature was increased by 3 • C per min to 180 • C and then increased by 1.5 • C per min to 200 • C. This temperature was maintained for three min, then increased by 2 • C per min to 220 • C and held for 20 min. The transfer line and MS ion source temperatures were maintained at 250 and 280 • C, respectively, and the ionizing energy was 70 eV.

Statistical Analysis
The results of C18 fatty acid were analyzed using ANOVA models of SAS (version 9.4, SAS Inst., Inc., Cary, NC, USA). The following statistical model was used: where Yij represents the observed dependent variables, µ was the overall mean, Ti was the effect of treatment, and εij was the residual error. Significant and extremely significant levels were set at p < 0.05 and p < 0.01, respectively.

Statistical Analysis
The results of C18 fatty acid were analyzed using ANOVA models of SAS (version 9.4, SAS Inst., Inc., Cary, NC, USA). The following statistical model was used: where Yij represents the observed dependent variables, µ was the overall mean, Ti was the effect of treatment, and εij was the residual error. Significant and extremely significant levels were set at p < 0.05 and p < 0.01, respectively.

Conclusions
Overall, this study found that t9,c12,c15-C18:3 was the product of ALA biohydrogenation in the rumen, which may provide a new biohydrogenation pathway in the rumen. The cis-trans isomerization of ALA from c9,c12,c15 C18:3 isomerizes to t9,c12,c15-C18:3 could happen in situ and the ortho-position c9,c12,c15-C18:3 isomerized to t10,c12,c15-C18:3. The enzymes and microorganisms involved in isomerization may be different, and any further study should focus on the manipulation of enzymes and microorganisms involved in the biohydrogenation pathway.

Conclusions
Overall, this study found that t9,c12,c15-C18:3 was the product of ALA biohydrogenation in the rumen, which may provide a new biohydrogenation pathway in the rumen. The cis-trans isomerization of ALA from c9,c12,c15 C18:3 isomerizes to t9,c12,c15-C18:3 could happen in situ and the ortho-position c9,c12,c15-C18:3 isomerized to t10,c12,c15-C18:3. The enzymes and microorganisms involved in isomerization may be different, and any further study should focus on the manipulation of enzymes and microorganisms involved in the biohydrogenation pathway.