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Article

Biotin and Leucine Alone or in Combination Promoted the Synthesis of Odd- and Branched-Chain Fatty Acids in the Rumen In Vitro

by
Tengfei Zhan
1,2,
Xin Guo
1,
Lu Ma
1,
Shengyong Mao
2,* and
Dengpan Bu
1,*
1
State Key Laboratory of Animal Nutrition, Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100193, China
2
Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
*
Authors to whom correspondence should be addressed.
Agriculture 2023, 13(1), 145; https://doi.org/10.3390/agriculture13010145
Submission received: 22 November 2022 / Revised: 12 December 2022 / Accepted: 30 December 2022 / Published: 5 January 2023
(This article belongs to the Section Farm Animal Production)
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Abstract

:
The odd- and branched-chain fatty acids (OBCFA) accumulated in ruminant products are a class of beneficial fatty acids for human health. Since biotin and leucine are involved in OBCFA synthesis, this study aimed to evaluate their effect on OBCFA synthesis in vitro. There were four treatments: the control group that only provided the basal diet, or the basal diet supplemented with biotin (4 mg/kg dry matter, DM), leucine (4 g/kg DM), or a combination of biotin (4 mg/kg DM) and leucine (4 g/kg DM). The results showed that biotin promoted the degradation of DM (p < 0.10), while leucine significantly increased the concentration of branched-chain volatile fatty acids and valerate (p < 0.05). The concentrations of total odd-chain fatty acids, total iso, total anteiso, total branched-chain fatty acids, total OBCFA, and total fatty acids were significantly increased by the supplementation of biotin or leucine (p < 0.05). Biotin and leucine significantly stimulated the activities of acetyl-CoA carboxylase, fatty acid synthase, and malonyl-CoA, with a significant interaction effect (p < 0.05). In conclusion, the results of this study suggested that biotin and leucine can be used as effective nutrition strategies to promote OBCFA synthesis.

1. Introduction

Odd- and branched-chain fatty acids (OBCFA) usually refer to fatty acids with odd carbon atoms and monomethyl branched-chain, such as C15:0, C17:0, iso-C15:0, iso-C17:0, antiso-C15:0, and antiso-C17:0, which are a kind of bioactive fatty acids [1,2,3,4]. It has been reported that branched-chain fatty acids (BCFA) in the serum of patients with excess weight are significantly lower than those of normal-weight people [5], and odd-chain fatty acids (OCFA) in plasma are negatively correlated with type 2 diabetes [2]. Branched-chain fatty acids have been proven to lower triglyceride levels in a fatty liver model in vitro [6]. In addition, BCFA has been shown to decrease the expression of genes that are associated with inflammation in adipose cells, [7], and also inhibit the viability of MCF-7 human breast cancer cells [8]. In the neonatal rat model, BCFA supplementation can effectively reduce the occurrence of necrotizing enterocolitis [9].
It is generally believed that OBCFA in the diet mainly comes from the intake of ruminant products [4]. The research showed that the concentrations of BCFA in human milk were associated with maternal intakes of beef and dairy [10], and Ran-Resler [11] confirmed that ruminant products were the main source of BCFA in the human diet. Rumen microorganisms are considered to be the main producers of OBCFA in ruminant products [1,2,3,4]. In the rumen, propionate [12], branched-chain volatile fatty acids (BCVFA) [13], and branched-chain amino acids (BCAA) [14,15,16] can be used as precursors for the microbial synthesis of OBCFA. However, malonyl-CoA is also required as a carbon donor for chain elongation [1,17].
Previous studies showed that dietary composition, such as content of fiber, starch and lipids, affected rumen OBCFA synthesis [1,2,3]. High fiber diet is certainly conducive to the synthesis of BCFA, which is related to the increase in cellulolytic bacteria in the rumen [18,19]. Some scholars have studied the effect of dietary lipid types on OBCFA in milk. Unfortunately, most of them showed an adverse impact on OBCFA synthesis, except docosahexaenoic acid (DHA)-enriched lipids (fish oil and algal meal), which can increase the proportion of OBCFA in milk [20,21,22]. Although a high proportion of fiber can increase the proportion of OBCFA, it is impossible for dairy cows to take whole-fiber feeds. Furthermore, it is reported that DHA-enriched lipids may inhibit the intake of dry matter (DM) and the proportion of fat in milk [23,24,25]. It is suggested that the biosynthesis process of OBCFA should be taken into account in the regulation of OBCFA. Notably, biotin, as an important component of acetyl-CoA carboxylase (ACC), participates in the synthesis of malonyl-CoA [17,26,27]. Supplementation of biotin in the diet may be an effective strategy to promote the synthesis of microbial fatty acids. Moreover, leucine can also serve as a precursor to promote the synthesis of microbial BCFA [16,28]. Therefore, this study aimed to explore whether the addition of biotin and leucine alone or in combination could have a promoting effect on the synthesis of OBCFA in vitro, providing an effective nutritional strategy for OBCFA enrichment in milk.

2. Materials and Methods

All animal procedures were approved by the Animal Welfare and Ethics Committee, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing, China (No: IAS2022-91).

2.1. Preparation of Rumen Fluid and Buffer Solution

Three healthy rumen-fistulated Holstein cows feeding at the Changping Animal Experiment Farm of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (Beijing, China), with similar weight and lactation stage were selected as rumen fluid donors. The donor cows were fed twice a day (7:00 and 19:00) and water was available ad libitum. The diet for the donor cows met the nutritional requirements of the United States National Research Council (NRC) of lactating cows [29] producing 30 kg milk. The diet contained 23.98% alfalfa hay, 26.02% corn silage, 21.99% steam flaked corn, 8.19% ground corn grain, 11.38% soybean meal, 2.08% expanded soybean, 4.22% canola meal, 1.19% calcium carbonate, 0.38% salt, and 0.57% premix. On the day of fermentation, the rumen content was collected through the rumen fistula, filtered with four layers of cheesecloth to obtain the filtrate, and one volume of the filtrate was mixed with two volumes of buffer solution to obtain an incubation solution. The buffer solution prepared refers to the formulation of Wang [30], which was established based on the research of Leedle and Hespell [31]. During the preparation process of the incubation solution, CO2 is continuously supplied to remove residual oxygen.

2.2. Treatment and Sample Collection

The workflow and experiment design are summarized in the Figure 1. The total mixed ration (TMR, Table 1) formulated according to NRC [29] met the nutritional requirements of lactating cows producing 30 kg milk, dried at 65 °C for 48 h and passed through a 1 mm screen, was used as an incubation substrate. Finally, 0.5 g substrate and 75 mL incubation solution were transferred into incubation flasks. The CO2 was continuously introduced during the whole inoculation process to ensure an anaerobic environment. There were four treatments in this study: the control group that only provided the basal diet (Con), or the basal diet supplemented with biotin (4 mg/kg DM, Bio), leucine (4 g/kg DM, Leu), or a combination of biotin (4 mg/kg DM) and leucine (4 g/kg DM, BL). Each treatment contained five replicates (flasks), and the experiment was repeated twice. The sealed flasks were incubated in a 39 °C constant-temperature incubator (HWY-211, Zhicheng Inc., Shanghai, China) at 180 rpm shaking for 24 h. After 24 h of fermentation, flasks were taken out and placed in ice water to terminate the fermentation, and the pH values were measured immediately (PB-10, Sartorius, Göttingen, Germany). The liquid and solid in the flasks were filtered by 800 mesh nylon bags, the filtered liquid was divided into cryopreservation tubes and stored at −80 °C for the determination of the fermentation parameters, fatty acids, and the activities of enzymes, and the residues were used to determine the degradation rate of the DM.

2.3. Measurement of Fatty Acids in the Fermentation Liquid

The pretreatment of fatty acids in fermentation liquid was minor modified based on the research of Hara [32], Kramer [33], and Wang [30]. Briefly, 3 mL of the filtered liquid was transferred into a 15 mL centrifuge tube, and 5 mL of hexane/isopropanol (v/v = 3:2) mixture was used to extract the fatty acid in the sample. After that, 2 mL of sodium sulfate solution (66.7 g/L) was used to separate the phase. The mixture was mixed by vortexing for 2 min and centrifuged at 5000× g for 10 min at 4 °C. The upper layer was transferred to the thermostability glass tube, the middle and lower layers were re-extracted, and all the upper layers were combined and dried with N2. Then 0.5 mL n-hexane, 1 mL methanol, and 2 mL of 2% NaOH/methanol solution were used to reconstitute the sample, followed by saponification in the water bath at 50 °C for 30 min. After the saponified sample was cooled to room temperature, 2 mL of 10% CH3COCl/methanol was required for the methyl esterification in a 90 °C water bath for 2 h. Then 3 mL of ultrapure water and 5 mL of n-hexane were added to the esterified sample that had cooled to room temperature; the upper phase was transferred to a 10 mL tube after vortexing and standing, and dried with N2. Finally, 0.5 mL of n-hexane and 0.1 g of anhydrous Na2SO4 were used to resuspend the sample, which was passed through a 0.22 μm filter membrane for fatty acid methyl ester (FAME) detection.
The gas chromatograph (6890N; Agilent Technologies, Santa Clara, CA, USA) equipped with HP-88 capillary column (100 m × 250 μm × 0.20 μm; Agilent Technologies, Santa Clara, CA, USA) and flame-ionization detector (FID) was used to analyze FAME in a constant pressure mode of 190 Kpa, and N2 was used as a carrier gas. The injection volume was 2 μL. The initial oven temperature was held at 120 °C for 10 min, increased by 1.5 °C/min to 230 °C, and held for 30 min. The temperature of the injector and detector was 250 °C and 280 °C, respectively. Individual FAME was identified based on the peak and retention time of fatty acid standards (Larodan AB, Solna, Sweden).

2.4. Analyses of Ruminal Fermentation Parameters and Activities of Enzymes Involved in the Fatty Acid Synthesis

The residue in the flasks was dried at 65 °C for 48 h to calculate the dry matter degradation rate (DMD). Ammonia nitrogen (NH3-N) in the fermentation liquid was determined using the phenolhypochlorite colorimetric method referring to the study of Broderick and Kang [34]. The activity of branched-chain amino acid aminotransferase (BCAT), branched-chain α-keto acid dehydrogenase (BCKD), acetyl-CoA, ACC, malonyl-CoA, and fatty acid synthase (FAS) was measured using ELISA kits purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China).
Analysis of volatile fatty acids (VFA) in fermentation liquid refers to the study of Wang [30]. A total of 5 mL filtered liquid was centrifuged at 4 °C for 10 min at 10,000× g, 1 mL of supernatant was mixed with 0.1 mL of 25% metaphosphoric acid solution, and the samples were placed in the ice bath for 30 min, then centrifuged at 4 °C for 15 min at 10,000× g, and the supernatant was taken for determination. The gas chromatograph (6890N; Agilent Technologies, Santa Clara, CA, USA) equipped with DB-FFAP capillary column (15 m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, USA) and FID was used to analyze VFA in a constant pressure mode of 50 Kpa, and N2 was used as a carrier gas. The injection volume was 1 μL. The initial oven temperature was 70 °C, increased by 3 °C/min to 125 °C, finally increased to 180 °C by 30 °C/min, and held for 5 min. The temperature of the injector and detector was 250 °C and 280 °C, respectively.

2.5. Statistical Analysis

Statistical analyses were performed using the linear mixed model of the nlme package (3.1–157) in R (version 4.2.1). The treatment and their interaction were used as the fixed factor with the incubation batch as the random factor in the model. Tukey method was used for the evaluation of differences between treatments. Data are reported as least square means ± standard error of the mean (SEM). Statistical significance and a tendency of significance were declared at p < 0.05 and 0.05 ≤ p < 0.10, respectively.

3. Results

3.1. Effects of Biotin and Leucine on Fermentation Parameters

Addition of biotin increased DMD slightly (p < 0.10), but the other fermentation parameters were not affected (Table 2). Addition of leucine significantly increased the concentrations of isobutyrate, isovalerate, valerate, total branched-chain volatile fatty acids (TBCVFA), and NH3-N in the fermentation liquid (p < 0.05) and led to an increased tendency in the concentration of butyrate (p < 0.10). The combined supplementation of biotin and leucine had no interaction (p > 0.05) on all the parameters except for the valerate concentration (p < 0.05).

3.2. Supplementation of Biotin and Leucine Alone or in Combination Promoted the Synthesis of Fatty Acids In Vitro

Addition of biotin and leucine alone or in combination significantly affected the synthesis of most fatty acids in vitro (Table 3). The concentrations of C12:0, c9-C14:1, C16:0, C18:0, t9-C18:1, c9-C18:1, c9,c12-C18:2, c6,c9,c12-C18:3, c9,c12,c15-C18:3, c8,c11,c14-C20:3, c11,c14,c17-C20:3, c15-C24:1, TFA1 and TFA2 in fermentation liquid were significantly increased due to the addition of biotin (p < 0.05), while the concentration of t9,t12-C18:2, and c5,c8,c11,c14-C20:4 tended to increase (p < 0.10). Leucine significantly promoted the synthesis of c9-C14:1, C16:0, C18:0, t9-C18:1, c9-C18:1, c9,c12,c15-C18:3, c8,c11,c14-C20:3, TFA1 and TFA2 (p < 0.05), and the concentration of C8:0, c9-C16:1, c9,c12-C18:2, c5,c8,c11,c14-C20:4 and c15-C24:1 in fermentation liquid also showed an increasing trend due to the addition of leucine (p < 0.10), but C6:0 tended to decrease (p < 0.10). The effects of supplementing biotin and leucine alone on the concentration of the individual or total fatty acids were similar in the fermentation liquid, and there was no significant interaction (p > 0.05) between biotin and leucine.
Compared with Con, the addition of biotin and leucine alone or their combination increased the total concentration of OBCFA by 7.1%, 8.5% and 17.1%, respectively (Figure 2). The concentrations of C15:0, C17:0, iso-C15:0, iso-C16:0, iso-C17:0, anteiso-C15:0, anteiso-C17:0, OCFA, ISO, Anteiso, BCFA, and OBCFA in the fermentation liquid were significantly increased due to the addition of biotin or leucine (p < 0.05). C13:0 and iso-C14:0 had an increasing trend with the addition of biotin (p < 0.10), but C11:0 (p = 0.030) and c10-C17:1 (p = 0.055) were decreased with the addition of leucine. In addition, the results showed that there was no significant interaction (p > 0.05) between biotin and leucine except for C21:0 (p < 0.05).

3.3. Effects of Biotin and Leucine Supplementation on Fatty Acid Synthesis-Related Enzymes In Vitro

The effects of biotin and leucine supplementation on fatty acid synthesis-related enzymes activities are summarized in Figure 3. Compared with Con, biotin and leucine supplementation alone or in combination significantly improved the activities of ACC, BCKD, BCAT, FAS, and malonyl-CoA (p < 0.05), while acetyl-CoA only increased significantly when added in combination (p < 0.05). The activities of ACC, BCKD, BACT, and malonyl-CoA in Bio were higher than in Leu (p < 0.05). The activities of FAS and acetyl-CoA were higher in BL than in Bio and Leu (p < 0.05), while ACC and BCKD, BCAT, and malonyl-CoA showed the opposite trend (p < 0.05).

4. Discussion

For a long time, OBCFA, as an important component of microbial membrane lipids and an important factor to ensure the normal growth of microorganisms, mainly came from the synthesis of ruminal microbes [1,2,35]. Typically, through carbon chain elongation of microorganisms, even-chain fatty acids were synthesized with acetate as a substrate [1], OCFA with propionate [12], while BCFA with BCVFA (Isobutyrate,2-methylbutyrate, and Isovalerate) [13] as well as BCAA (leucine, isoleucine, and valine) [14,15,16]. Regardless of the substrate used by the microorganisms, malonyl-CoA is required for the chain elongation as the carbon donor [1,17]. The important source of malonyl-CoA is the formation from acetyl-CoA catalyzed by ACC, whereas biotin is an essential component of ACC [26,27]. Under pure culture conditions, supplementation of biotin and BCAA can promote the synthesis of microbial fatty acids [14]. Therefore, we speculated that the supplementation of biotin in ruminant diets can promote the synthesis of fatty acids, while the addition of BCAA can promote the synthesis of BCFA in rumen by providing more branched-chain precursors.
In this experiment, as expected, the addition of biotin promoted the synthesis of most fatty acids in fermentation liquid, which is consistent with the effect of adding biotin in pure culture conditions [14]. In fact, our previous research already proved that OBCFA increased linearly with the addition of biotin [36]. As an important component of ACC [26,27], biotin can promote the conversion of acetyl-CoA to malonyl-CoA by improving the biological activity of ACC [37], providing more carbon donors for the chain extension in the process of fatty acid synthesis, and thus promoting the synthesis of fatty acid. This has been also confirmed by the increased activity of ACC and malonyl-CoA in this experiment. BCAA catalyzed by BCAT and BCKD can provide suitable substrates for the synthesis of BCFA [1,38,39]. A surprising finding in the current study is that the activities of these two enzymes have been also significantly increased by the addition of biotin. Therefore, the promoting effect of biotin on fatty acids synthesis may be caused by its promoting effect on the overall growth of rumen microorganisms, because the increased DMD and activity of FAS have been also observed in the biotin group in the current experiment.
Leucine, as a BCAA, can promote the synthesis of BCFA by providing substrates for the synthesis of BCFA. Isobutyrate and isovalerate, the precursors for the synthesis of BCFA [13], in the fermentation liquid were significantly increased with the supplementation of leucine, which is consistent with the increase in BCFA. It can be inferred that α-ketoisocaproate, as an important intermediate metabolite for the synthesis of BCFA by leucine [38,39], may be also improved in this experiment, but unfortunately, this indicator has not been analyzed. The metabolites of leucine include not only isovalerate and α-ketoisocaproate, but also produce acetyl-CoA [40], which is an important metabolite in carbohydrate metabolism and a precursor for even-chain fatty acid synthesis [41]. The acetyl-CoA can participate in the synthesis of OBCFA through converting to malonyl-CoA under the ACC catalysis [17,26,27]. The supplementation of leucine can not only provide substrate for the synthesis of fatty acid but also indirectly provide malonyl-CoA as a caron donor for the chain extension. Therefore, both increased OBCFA and even-chain fatty acids concentrations have been observed in the current experiment. The activity of acetyl-CoA has not changed with the leucine supplement alone, but malonyl-CoA has significantly increased compared with the control group, which may be related to the increased activity of ACC promoting the efficient conversion of acetyl-CoA to malonyl-CoA.
Besides participating in fatty acid synthesis, leucine is involved in the synthesis of microbial protein [28] to provide superior conditions for the growth of rumen microorganisms. Therefore, the increased activities of ACC, BCKD, BCAT, and FAS may be related to the growth of microorganisms. Although the addition of leucine to the high-fat diet may inhibit the expression of ACC and FAS in the liver of mice [42], such reports have not been seen in microorganisms. Even if such an inhibition effect exists, the improved activities of ACC and FAS in this experiment have indicated that the growth-promoting effect of leucine on microorganisms was stronger than its inhibition. Notably, the supplementation of leucine has resulted in the increase in the NH3-N concentration in the fermentation liquid, which may be caused by more amino generation. Therefore, more attention should be paid to the efficiency of nitrogen utilization as leucine is supplemented.
Since the single supplementation of either biotin or leucine can significantly promote the synthesis of fatty acids, it is hypothesized that the combined supplementation of the biotin and leucine would further improve the synthesis of fatty acids. Indeed, the content of even-chain fatty acids and OBCFA in the fermentation liquid in the combination group was higher than that in the single supplementation group. However, the activities of ACC, BCKD, BCAT, and malonyl-CoA have been observed to be a bit lower compared to the single supplementation, but still higher compared to the control group. This result might be related to the changes in microbial composition and metabolism caused by these two nutrients. Conversely, the higher activity of FAS in the combination group with the simultaneous improvement of carbon donors and substrates promotes fatty acid synthesis. The accumulation of acetyl-CoA in the combination group may be caused by two factors: the first factor is that leucine metabolism can produce acetyl-CoA [40], and the second factor is the decreased activity of ACC in the combination group relative to the supplementation alone group.

5. Conclusions

Overall, biotin and leucine promoted ruminal OBCFA synthesis under the current experiment, either alone or in combination, and were more effective when supplemented in combination. The regulatory targets of these two nutrients are different. Biotin mainly promotes OBCFA synthesis by elevating the content of malonyl-CoA through ACC, while leucine promotes OBCFA synthesis by providing substrates and carbon donors required in the process of fatty acid metabolism. This study provides a basis for enhancing the content of OBCFA in milk, but changes in enzymes related to fatty acid metabolism independent of biotin and leucine suggest that in-depth research should be conducted on its microbiological mechanism in the future.

Author Contributions

Conceptualization, D.B. and S.M.; formal analysis, T.Z.; funding acquisition, D.B.; investigation, T.Z. and X.G.; project administration, L.M.; writing—original draft preparation, T.Z.; writing—review and editing, T.Z., D.B. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially supported by the Key Research and Development Program of the Ningxia Hui Autonomous Region (2021BEF02018), the Scientific Research Project for Major Achievements of the Agricultural Science and Technology Innovation Program (ASTIP) (ASTIP-IAS07-1) and Beijing Innovation Consortium of Livestock Research System (BAIC05-2022).

Institutional Review Board Statement

The animal study was reviewed and approved by the Animal Welfare and Ethics Committee, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing, China (No: IAS2022-91).

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Agriculture 13 00145 g001 550
Figure 1. The workflow and experiment design of this study. Con, the control group that only provided the basal diet; Bio, basal diet supplemented with biotin (4 mg/kg dry matter); Leu, basal diet supplemented with biotin (4 g/kg dry matter); BL, combination of biotin (4 mg/kg dry matter) and leucine (4 g/kg dry matter); Each treatment contained five replicates (flasks) and the experiment was repeated twice.
Figure 1. The workflow and experiment design of this study. Con, the control group that only provided the basal diet; Bio, basal diet supplemented with biotin (4 mg/kg dry matter); Leu, basal diet supplemented with biotin (4 g/kg dry matter); BL, combination of biotin (4 mg/kg dry matter) and leucine (4 g/kg dry matter); Each treatment contained five replicates (flasks) and the experiment was repeated twice.
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Agriculture 13 00145 g002 550
Figure 2. Effects of biotin and leucine supplementation on OBCFA concentration in vitro. Con, control group; Bio, biotin supplemented group; Leu, leucine supplemented group; BL, biotin and leucine combination group; anteiso-C15:0 and c9-C14:1 could not be separated; OCFA = C11:0 + C13:0 + C15:0 + c10-C15:1 + C17:0 + c10-C17:1 + C21:0 + C23:0; ISO = iso-C13:0 + iso-C14:0 + iso-C15:0 + iso-C16:0 + iso-C17:0; Anteiso = anteiso-C13:0 + anteiso-C15:0 + anteiso-C17:0; BCFA = ISO + Anteiso; OBCFA = OCFA + BCFA. Columns labeled with different letters are significantly different (p < 0.05).
Figure 2. Effects of biotin and leucine supplementation on OBCFA concentration in vitro. Con, control group; Bio, biotin supplemented group; Leu, leucine supplemented group; BL, biotin and leucine combination group; anteiso-C15:0 and c9-C14:1 could not be separated; OCFA = C11:0 + C13:0 + C15:0 + c10-C15:1 + C17:0 + c10-C17:1 + C21:0 + C23:0; ISO = iso-C13:0 + iso-C14:0 + iso-C15:0 + iso-C16:0 + iso-C17:0; Anteiso = anteiso-C13:0 + anteiso-C15:0 + anteiso-C17:0; BCFA = ISO + Anteiso; OBCFA = OCFA + BCFA. Columns labeled with different letters are significantly different (p < 0.05).
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Figure 3. Effects of biotin and leucine supplementation on the activities of fatty acid synthesis related enzymes in vitro. Con, control group; Bio, biotin supplemented group; Leu, leucine supplemented group; BL, biotin and leucine combination group; ACC, acetyl-CoA carboxylase; BCKD, branched-chain α-keto acid dehydrogenase; BCAT, branched-chain amino acid aminotransferase; FAS, fatty acid synthase. Columns labeled with different letters are significantly different (p < 0.05).
Figure 3. Effects of biotin and leucine supplementation on the activities of fatty acid synthesis related enzymes in vitro. Con, control group; Bio, biotin supplemented group; Leu, leucine supplemented group; BL, biotin and leucine combination group; ACC, acetyl-CoA carboxylase; BCKD, branched-chain α-keto acid dehydrogenase; BCAT, branched-chain amino acid aminotransferase; FAS, fatty acid synthase. Columns labeled with different letters are significantly different (p < 0.05).
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Table
Table 1. Ingredients and chemical compositions of TMR diets used for substrate.
Table 1. Ingredients and chemical compositions of TMR diets used for substrate.
Ingredients (% DM 1)Chemical Compositions
Alfalfa Hay9.57NEL 2, Mcal/kg DM1.86
Soybean Hull Powder3.97Crude Protein, % DM17.80
Molasses1.43Neural detergent fiber, % DM26.90
Steam Flaked Corn15.96Acid detergent fiber, % DM17.80
Corn Silage29.12Calcium, % DM0.65
Alfalfa Silage1.76Phosphorus, % DM0.36
Ground Corn Grain (Medium)11.01
Soybean Meal13.93
Corn Gluten Meal2.42
Cottonseed5.96
Fat powder1.31
Yeast Culture0.10
Sodium Bicarbonate0.66
Premix 32.79
Total100.00
1 DM, dry matter; 2 NEL, Net energy lactation, which was calculated by NASEM Dairy 8; 3 The premix provided contained the following per kg: Vitamin A 250,000 IU, Vitamin D 65,000 IU, Ferrum 400 mg, Copper 540 mg, Zinc 2100 mg, Manganese 560 mg, Selenium 135 mg, Cobalt 68 mg, Calcium 99 g.
Table
Table 2. Effects of biotin and leucine supplementation on fermentation parameters in vitro (mmol/mL).
Table 2. Effects of biotin and leucine supplementation on fermentation parameters in vitro (mmol/mL).
ItemsTreatment 1SEM 2p-Value
ConBioLeuBLBioLeuBL
DMD 3 %67.7968.8268.1068.940.6080.0680.6680.847
pH6.776.776.776.770.0070.8930.7880.788
Acetate39.2439.2739.0540.030.8520.3920.6210.417
Propionate10.5010.5710.5810.800.1860.3830.3700.635
Isobutyrate0.72 b0.71 b0.75 ab0.78 a0.0260.4340.0010.151
Butyrate7.167.237.297.550.2780.2070.0850.472
Isovalerate1.30 b1.31 b1.46 a1.52 a0.0780.166<0.0010.301
Valerate0.90 ab0.83 b0.89 ab0.94 a0.0250.6900.0230.021
Acetate/propionate3.743.713.693.710.0270.7260.1000.278
TVFA 459.8259.9160.0261.631.4050.3520.2960.409
TBCVFA 52.02 b2.02 b2.21 a2.30 a0.1030.220<0.0010.231
NH3-N 6 mg/dL28.0728.1729.5930.402.1520.5210.0110.618
1 Con, control group; Bio, biotin supplemented group; Leu, leucine supplemented group; BL, biotin and leucine combination group; 2 SEM, standard error of the mean; 3 DMD, dry matter degradation rate; 4 TVFA = Acetate + Propionate + Isobutyrate + Butyrate + Isovalerate + Valerate; 5 TBCVFA = Isobutyrate + Isovalerate; 6 NH3-N, ammonia nitrogen; a,c Means with different superscripts within the same row differ significantly (p < 0.05).
Table
Table 3. Effects of biotin and leucine supplementation on fatty acid concentrations in vitro (μg/mL).
Table 3. Effects of biotin and leucine supplementation on fatty acid concentrations in vitro (μg/mL).
ItemsTreatment 1SEM 2p-Value
ConBioLeuBLBioLeuBL
C4:00.370.390.380.360.0280.8450.6560.475
C6:00.520.530.470.460.0440.8740.0670.666
C8:00.961.131.271.440.1690.3300.0770.977
C10:00.130.110.120.120.0100.6130.9450.300
C12:00.83 ab0.89 a0.80 b0.90 a0.0340.0010.7180.294
C14:02.973.023.063.260.1350.3240.1890.539
c9-C14:1 32.74 b2.94 ab2.96 ab3.19 a0.1790.0110.0050.840
C16:065.01 b69.89 b71.66 b78.5 a3.2070.002<0.0010.587
c9-C16:10.160.160.170.200.0130.3150.0720.385
C18:0100.01 b107.89 ab110.02 ab119.45 a4.8780.0070.0010.803
t9-C18:110.45 b11.14 ab11.11 ab12.07 a0.3220.0140.0180.687
c9-C18:123.5 b24.27 b24.84 ab27.44 a0.9630.0240.0030.207
t9,t12-C18:20.370.380.350.400.0310.0530.9290.258
c9,c12-C18:23.86 b4.14 ab4.02 ab4.36 a0.1100.0060.0820.741
c6,c9,c12-C18:3 40.580.590.580.630.0300.0370.1780.114
c9,c12,c15-C18:30.48 b0.56 a0.55 a0.59 a0.0770.0010.0020.265
c11-C20:10.600.610.590.640.0380.1370.6400.280
c11,c14-C20:20.550.510.490.510.0240.8430.2400.244
c8,c11,c14-C20:3 50.30 b0.31 ab0.32 ab0.34 a0.0160.0410.0060.563
c11,c14,c17-C20:30.50 b0.55 ab0.51 b0.61 a0.0380.0040.1500.215
c5,c8,c11,c14-C20:4 615.1015.9215.9417.872.7090.0980.0920.497
c13-C22:10.530.490.530.540.0640.5360.2120.139
C24:00.880.900.890.960.0250.1070.2550.267
c15-C24:10.47 b0.52 ab0.50 ab0.54 a0.0660.0010.0580.823
TFA1 7231.89 b247.85 b252.13 b275.39 a10.7510.002<0.0010.542
TFA2 8252.15 b269.53 b274.11 b299.12 a11.9680.002<0.0010.559
1 Con, control group; Bio, biotin supplemented group; Leu, leucine supplemented group; BL, biotin and leucine combination group; 2 SEM, standard error of the mean; 3 anteiso-C15:0 and c9-C14:1 could not be separated; 4 c6,c9,c12-C18:3 and C20:0 could not be separated; 5 c8,c11,c14-C20:3 and C22:0 could not be separated; 6 c5,c8,c11,c14-C20:4 and c13,c16-C22:2 could not be separated; 7 TFA1 refers to all fatty acids except OBCFA in this study; 8 TFA2 refers to all fatty acids detected in this study; a–c Means with different superscripts within the same row differ significantly (p < 0.05).
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Zhan, T.; Guo, X.; Ma, L.; Mao, S.; Bu, D. Biotin and Leucine Alone or in Combination Promoted the Synthesis of Odd- and Branched-Chain Fatty Acids in the Rumen In Vitro. Agriculture 2023, 13, 145. https://doi.org/10.3390/agriculture13010145

AMA Style

Zhan T, Guo X, Ma L, Mao S, Bu D. Biotin and Leucine Alone or in Combination Promoted the Synthesis of Odd- and Branched-Chain Fatty Acids in the Rumen In Vitro. Agriculture. 2023; 13(1):145. https://doi.org/10.3390/agriculture13010145

Chicago/Turabian Style

Zhan, Tengfei, Xin Guo, Lu Ma, Shengyong Mao, and Dengpan Bu. 2023. "Biotin and Leucine Alone or in Combination Promoted the Synthesis of Odd- and Branched-Chain Fatty Acids in the Rumen In Vitro" Agriculture 13, no. 1: 145. https://doi.org/10.3390/agriculture13010145

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