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14 October 2022

Effect of Unsaturated Fatty Acid Ratio In Vitro on Rumen Fermentation, Methane Concentration, and Microbial Profile

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State Key Laboratory of Animal Nutrition, Beijing Engineering Technology Research Center of Raw Milk Quality and Safety Control, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
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Fermentation2022, 8(10), 540;https://doi.org/10.3390/fermentation8100540
This article belongs to the Special Issue Rumen Fermentation
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Abstract

It is well known that dairy cows are fed diets with high fat content, which can adversely affect rumen fermentation. However, whether the effects of high fat content on rumen fermentation are related to the composition of fatty acids (FA) is for further study. We explored the effects of unsaturated fatty acid (UFA) ratios in vitro on rumen, methane concentration and microbial composition under the same fat levels. The experiment included a low-unsaturated group (LU, UFA proportion: 42.8%), a medium-unsaturated group (MU, UFA proportion: 56.9%), and a high-unsaturated group (HU, UFA proportion: 70.9%). The incubation fluid pH and NH3-N levels were not significantly different in the three groups. Total volatile fatty acid (TVFA), acetate, propionate, butyrate, and valerate in the MU group had a decreased trend compared to the LU group (0.05 < p < 0.1), and no difference was found in other volatile fatty acids (VFAs) among the three groups. Furthermore, gas production kinetic parameters among the three groups did not differ significantly. The LU group’s CH4 concentration was significantly higher than the HU group (p < 0.05). The CO2 concentration in the LU group was also significantly higher than the MU and LU groups (p < 0.05). Additionally, 16S rRNA microbial sequencing results showed that the Shannon diversity value significantly increased in the MU group (p < 0.05) compared to the LU group. Other alpha diversity indices (Chao 1, observed species, and ACE) did not differ among the three groups. The increased proportion of UFA significantly decreased the relative abundance of Succinivibrionaceae_UCG_001 and Fibrobacter (p < 0.05). Meanwhile, the multiple Lachnospiraceae bacteria significantly increased in the MU group (p < 0.05). Overall, our findings indicated that the microbial community in the incubation system could be affected by elevating proportions of UFA, affecting the yield of VFA, whereas the CH4 concentration was reduced.

1. Introduction

Dietary fat supplementation has effectively increased dietary energy concentration and milk fat content in dairy cows []. The dietary fat content of high-yield dairy cows is usually about 5–6%; about 3% of the fat derives from forage grass and grains, and fat powder supplements the rest. Generally, the fatty acid (FA) content in 6% fat is >90% []. Fatty acids are the main component of cell membranes and participate in a variety of biological systems and processes, including the immune system [,], coagulation, enzyme activity, cell proliferation, and differentiation [].
Lipids mainly undergo two processes in the rumen of ruminants, fat decomposition and biohydrogenation []. Fat decomposition is mainly fulfilled by lipase released by bacteria in the rumen, while the fatty acids released by fat decomposition can be quickly and completely hydrogenated by bacterial isomerase []. It has been reported [] that when the dietary fat content is >10%, the rumen fermentation of dairy cows reduces by nearly 50%, especially when the dietary unsaturated fatty acid (UFA) is more apparent. The inhibition of FA on rumen fermentation may be due to FA adhering to the feed surface, hindering microorganisms’ decomposition of feed substrate. However, it may also result from the toxic effects of FA on rumen bacteria. Due to the inhibitory effects of fat on rumen fermentation, increasing dietary fat content is recognized as one of the most effective nutritional strategies to reduce rumen methane emissions []. Brask et al. [] increased the dietary fat concentration from 3.5 to 5.5, 6.2, and 6.5%, respectively, by changing the feeding form of rapeseed. The results showed that the daily yield of CH4 decreased by 14.41 and 18.80%, respectively. Sun et al. [] reported that increased UFA in the in vitro substrate also decreased production methane. However, in these experiments, the increased proportion of UFA was also accompanied by an increase in fat concentration. Furthermore, there are different opinions on whether the inhibitory effects of fat on rumen CH4 production are related to FA composition. It has been shown that decreased CH4 production in the rumen by fat is proportional to the unsaturated degree of fatty acids []; however, Grainger C et al. [] believe that decreased CH4 production by fat has nothing to do with FA composition.
Therefore, this study was conducted in vitro to explore the effects of different UFA ratios on CH4 concentration, rumen fermentation, and microbial composition under precursors with consistent dietary fat levels.

2. Materials and Methods

2.1. Animals and Inoculant

Three mid-lactating, rumen-cannulated Holstein dairy cows (26 ± 1.63 kg/d milk yield) from Zhongdi Dairy Holdings Co., Ltd. (Beijing, China) were used as rumen fluid donors. The cows were fed three times per day (07:00, 14:00, 19:00) by a total mixed ratio (TMR) ad libitum, with free access to water, and milked three times daily (06:30, 13:30, 18:30). The ingredients and nutritional composition of the TMR are shown in Table 1. China Agricultural University’s Institutional Animal Care and Use Committee approved all animal procedures (approval number: AW61902202-1-4). On the first day of the batch culture experiment, the rumen fluid was juiced after 2 h of the morning feeding via the rumen cannulas of each cow, filtered through four layers of gauze, and transferred to a thermal flask filled in advance with CO2 at 39 °C in a water bath. The buffer was configured according to a method described by our predecessors []. CO2 was injected continuously for approximately 30 min prior to inoculation.
Table 1. Fermentation substrate composition and nutrients (dry matter basis, %).

2.2. Incubation Substrates

The substrate was obtained from the donor cows’ total mixed ration (TMR) and dried at 65 °C for 48 h in a forced air oven and ground to pass through a 2 mm sieve. We determined the chemical composition using the method described by AOAC []. The experiment was conducted to adjust the proportion of UFA in the whole incubation system by adding different proportions of saturated fatty acids (SFA) and UFA in the fermentation bottle. The experiment included a low-unsaturated group (LU, TMR adding 10 mg palmitic acid, UFA proportion = 42.8%), a medium-unsaturated group (MU, TMR with 5 mg palmitic acid and 5 mg α-linolenic acid, UFA proportion = 56.9%), and a high-unsaturated group (HU, TMR with 10 mg α-linolenic acid, UFA proportion = 70.9%). A palmitic acid with content of >90% was obtained from Yihai Kerry Group (Wuhan, China), and α-linolenic acid with a purity of 99.8% was obtained from McLean Biochemical Technology (Shanghai, China). The FA composition of TMR and palmitic acid was determined as previously described []. The FA composition of the three groups is shown in Table 2.
Table 2. FA composition of the three groups (dry matter basis, %).

2.3. Experimental Design and In Vitro Incubation

The incubation experiments in vitro were conducted in a 120 mL anaerobic glass bottle. To each incubation glass flask, 0.5 g of substrate, 25 mL of filtered rumen fluid, and 50 mL of prewarmed buffer were added. The incubation bottles were injected with N2 quickly after inoculation to maintain the anaerobic condition, rapidly sealed, and immediately connected to the Automated Trace Gas Recording System (AGRS-III, Beijing, China) [] for recording cumulative gas production (GP) in real time. The bottles were fermented at a constant 39 °C; each treatment was performed at 0.5, 1, 2, 3, 6, and 48 h time points; and five replicates were performed for each time point. Meanwhile, airbags were connected to each incubation bottle at 0.5, 1, 2, 3, and 6 h, to be used to collect incubation gas for further composition analysis.

2.4. Sample Collection, Measurement and Calculation

After incubation, all the incubation bottles were separated from the AGRS-III system, and the airbags were removed from the bottles. The Mettler Five Easy Plus series pH meter immediately measured the pH values. The incubation material in the bottles was filtered through dried nylon bags (80 × 150 mm, 42 µm pores) for collecting incubation fluid and dispensing the incubation fluid into five 2 mL sterile tubes for microbial community and fermentation parameters analysis. The 1 mL gas sample was withdrawn from the airbags for gas profile analysis.
As described by Sun et al. [] and Cui et al. [], gas chromatography (Agilent 6890N, Agilent Technologies, Inc., Beijing, China) determined the VFA concentrations of incubation fluid and the gas composition (CO2 and CH4) in gas samples. NH3-N concentrations in incubation fluid were measured by spectrophotometry according to Verdouw et al. [].
The cumulative GP data were recorded by the AGRS-III system and were used to calculate kinetic parameters of gas production according to a nonlinear model [] as follows:
GP48 = A/(1 + (C/48)B)
where GP48 (mL) is the total gas production (ml/g dietary DM) over 48 h; A represents the ideal maximum gas production; B is a parameter reflecting the shape of the curve; C represents the time (h) when the maximum gas production reaches half. The time required to achieve the maximum degradation rate of substrate (TRmaxS) and the maximum gas production rate (TRmaxG) was calculated using B and C. The maximum rate of substrate degradation (RmaxS) was calculated by B, C, and TRmaxS. The maximum gas production rate (RmaxG, mL/h) was calculated by B, C, and TRmaxS [].
TRmaxS = C × (B−1)(1/B)
TRmaxG = C × ((B−1)/(B + 1))(1/B)
RmaxS = (B × TRmax S(B−1))/(CB + TRmaxSB)
RmaxG = (A × CB × B × TRmaxG−B−1)/(1 + CB × TRmaxG−B)2

2.5. DNA Extraction and Determination

As described in the manufacturer’s instructions, total microbial genomic DNA from the total incubation fluid was extracted using FastDNA® SPIN for the soil kit (MP Biomedicals, Solon, OH, USA). DNA concentration and quality were evaluated by a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA) and 1.0% agarose gel electrophoresis. Polymerase chain reaction (PCR) was used to amplify the V3-V4 region of the 16S rRNA gene by primer pairs 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) []. All samples were amplified in triplicate. The PCR reaction mixture system was described by Kong et al. []. The above PCR amplification products were analyzed by electrophoresis, and amplification products were purified from 2% agarose gel using the kit AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified by a real-time quantitative system (Quantus™ Fluorometer, Promega, Madison, WI, USA). The purified amplicon products were mixed equimolarly and paired-end sequenced by using an Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA). The raw sequencing reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: from SAMN30959991-SAMN-30960005 in PRJAN883176).
The sequenced data were saved as raw data in the form of FASTQ files. After the raw data were demultiplexed, the 0.19.6 version of FASTP [] was used for quality control and spliced by FLASH version 1.2.7 [] according to the following criteria: (1) The sliding window method was adopted to filter the bases whose tail quality value was <20 with a set window of 50 bp. If the average quality value of the window was <20, the bases at the tail-end were truncated from the window, and reads <50 bp or containing N bases after quality control were discarded. (2) According to the overlap relationship between PE reads, paired reads with an overlap length of >10 bp were merged into a sequence. The maximum mismatch ratio allowed was 0.2, and non-conforming sequences were eliminated. (3) Samples were distinguished according to the barcodes and primers at the sequence’s beginning and end, and the sequence direction was adjusted. The number of mismatches allowed by the barcode was 0, and the maximum number of primer mismatches was 2. The spliced sequences were performed with operational taxonomic unit (OTU) clustering according to the 97% similarity using UPARSE 7.1 [,] and eliminated chimeras. The confidence threshold was 70% for the RDP Classifier version 2.2 [] compared to the Silva 16S rRNA gene database (v138) for OTU species taxonomy annotation.

2.6. Statistical Methods

The MIXED procedure from SAS version 9.4 software (SAS Institute Inc., Cary, NC, USA) was used to analyze the data. The model for the fermentation kinetics parameters data was Yij = μ + Trti + εij, where Yij is the dependent variable; μ is the overall mean; Trti is the fixed effect of treatment; εij is the random error. The model for pH, VFAs, NH3-N, and gas composition data was Yij = μ + Trti + Timej + Trti × Timej + εij, where Timej is the fixed effect of time; Trti × Timej is the interaction between Trti and Timej, and other parameters were same as the previous model. Data are presented as mean ± standard error, and Tukey’s test was adopted for the multiple comparisons MIXED procedure. Main effects and interaction were considered significant when p < 0.05, and tendencies were considered for 0.05 ≤ p < 0.10.
Sequencing data analysis of the incubation fluid was performed on the Majorbio Cloud platform (https://cloud.majorbio.com, accessed on 27 September 2022). The Mothur v1.30.1 [] software was used to calculate alpha diversity indices (Shannon, ACE, observed species, and Chao 1), and the Wilcoxon rank-sum test was used to analyze the differences between groups. Principal coordinate analysis (PCoA) based on the Bray–Curtis distance algorithm was used to test the similarity between microbial communities in the samples and combined with PERMANOVA non-parametric test to analyze whether the differences in microbial community between sample groups were significant. Incubation fluid genera were compared using the linear discriminant analysis (LDA) effect size (LEfSe) [], and an LDA score checked significant differences >2 and p < 0.05.

3. Results

3.1. pH, Volatile Fatty Acid and NH3-N Concentration

Figure 1 shows the pH variation with incubation time in three groups. The incubation fluid’s pH was not affected by the UFA ratio. The pH of the incubation fluid in all treatment groups significantly decreased as incubation increased (p < 0.05).
Figure 1. Effects of unsaturated fatty acid ratio on in vitro pH. LU: low-unsaturated group (UFA proportion, 42.8%); MU: medium-unsaturated group (UFA proportion, 56.9%); HU: high-unsaturated group (UFA proportion, 70.9%); Treat: Treatment effects; time: incubation time effects; interaction: the interaction effects between treatment and incubation time. Vertical bars represent standard deviation (SD).
Figure 2 shows the effects of LU, MU, and HU on VFAs (Figure 2A–H) and NH3-N (Figure 2I). The concentration of VFAs and NH3-N was not affected by treatment (p > 0.05); however, the concentration of VFAs and NH3-N significantly decreased (p < 0.05) with the increase in incubation time. In the early stages of incubation, the ratio of acetate to propionate (Figure 2C) and NH3-N concentration decreased and reached a minimum concentration at 6 h. Other VFAs accumulated as the incubation time increased, and the concentration was highest at 48 h, significantly higher than other times (p < 0.05).
Figure 2. Effects of unsaturated fatty acid ratio on in vitro volatile fatty acid (A–H) and ammonia concentrations (I). LU: low-unsaturated group (UFA proportion, 42.8%); MU: medium-unsaturated group (UFA proportion, 56.9%); HU: high-unsaturated group (UFA proportion, 70.9%); Treat: treatment effects; Time: incubation time effects; Interaction: the interaction effects between treatment and incubation time. Vertical bars represent standard deviation (SD).

3.2. Gas Production and Fermentation Kinetics Parameter

Table 3 shows the effects of LU, MU, and HU on gas production and fermentation kinetic parameters. All parameters (A, B, C, GP48, RmaxG, RmaxS, TRmaxG, and TRmaxS) did not differ in the three groups (p > 0.05).
Table 3. Effects of unsaturated fatty acid ratio on in vitro gas production and fermentation kinetic parameters.

3.3. CH4 and CO2 Composition

Table 4 shows a significantly higher (p < 0.05) CH4 concentration in the LU group than in the HU group. The CO2 concentration was significantly decreased (p < 0.05) in the MU and HU groups compared with the LU group. The CH4 and CO2 concentrations increased significantly (p < 0.05) with the increasing incubation time.
Table 4. Effects of unsaturated fatty acid ratio on in vitro CH4 and CO2 composition.

3.4. Microbiota Diversity

Figure 3 shows the effects of the LU, MU, and HU groups on the alpha diversity indices of bacterial communities in incubated ruminal fluid. The Shannon index value (Figure 3B) in the MU group was higher than the LU group (p < 0.05), whereas there was no difference between the MU and HU groups (p > 0.05). The value of observed species (Figure 3A), ACE index (Figure 3C), and Chao 1 index (Figure 3D) did not differ across the three groups (p > 0.05). A PCoA plot (Figure 4) based on the Bray–Curtis distances was distinct between the LU and MU groups (PREMANOVA: p < 0.05). There was a close connection between the HU group and other groups.
Figure 3. Alpha diversity indices of bacterial communities in incubated ruminal fluid. (A) Number of observed species; (B) Shannon diversity index; (C) ACE diversity index; (D) Chao 1 diversity index; LU: low-unsaturated group (UFA proportion, 42.8%); MU: medium-unsaturated group (UFA proportion, 56.9%); HU: high-unsaturated group (UFA proportion, 70.9%). Means with different letters imply significant differences (p < 0.05). Different superscript letters indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Vertical bars represent standard deviation (SD).
Figure 4. Beta diversity indices for bacterial communities in incubation fluid. Data are represented by the mean ± SEM. LU: low-unsaturated group (UFA proportion, 42.8%); MU: medium-unsaturated group (UFA proportion, 56.9%); HU: high-unsaturated group (UFA proportion, 70.9%).

3.5. Microbiota Composition

Figure 5 shows the genus composition and significantly different genera of microbiota. Genera with relative abundances >0.5% are shown in Figure 4A, and Figure 4B shows the LEfSe analysis of different genera among the three groups. Overall, 16 genera (including Oscillopira, Allisonela, Lachnospiraceae_ND3007_group, Blautia, Lachnospiraceae_UCG_008, Lachnospiraceae_NK4A136_group, FD2005, Rikenellaceae_RC9_gut_group, Syntrophococcus, Lachnospiraceae_UCG_001, Lachnoclostridium, Eubacterium_hallii_group, and others) were significantly enriched in the MU group (p < 0.05 and LDA > 2). Two genera (Succinivibrionaceae_UCG-001 and Fibrobacter) were significantly enriched in the LU group (p < 0.05 and LDA > 2), and one genus (norank genera) was significantly enriched in the HU group (p < 0.05 and LDA > 2).
Figure 5. (A) Genera composition of the microbiota; (B) significantly different genera of microbiota. Significant differences were tested by linear discriminant analysis effect size (LEfSe) analysis with a linear discriminant analysis (LDA) score of >2 and p value of < 0.05. LU: low-unsaturated group (UFA proportion, 42.8%); MU: medium-unsaturated group (UFA proportion, 56.9%); HU: high-unsaturated group (UFA proportion, 70.9%).

4. Discussion

Previous studies have shown that the addition of fat to dairy cows’ diets produces various changes in rumen fermentation, which are closely related to FA composition [,,]. It has been reported [] that adding excessive fat to the diet of dairy cows inhibits rumen fermentation, especially the addition of UFA, which has a more obvious inhibitory effect on rumen fermentation. This depressive effect might be due to the inhibitory effects of UFAs on various rumen bacteria and the obstructed contact between micro-organisms and feed substrates [].
Rumen fluid pH is a comprehensive index reflecting rumen fermentation and health status. Previous studies have shown that polyunsaturated fatty acids (PUFA) supplementation in dairy cows’ diets did not affect rumen pH [,], and similar results were found in our experiment. In our experiment, rumen pH decreased with incubation time but did not differ among different treatment groups. VFAs are important producers of rumen fermentation and were positively correlated with the digestibility of the substrate. Sun et al.’s [] results showed that the addition of flaxseed tended to increase propionate concentration in the incubation fluid but did not shift the concentration of most VFAs. Meanwhile, a report suggested that flaxseed oil supplementation caused the molar proportion of propionate to increase but did not affect other VFAs []. Our experiment results showed that the VFA concentration in the three treatment groups did not differ significantly with the increase in UFA proportion. However, the TVFA, acetate, propionate, butyrate, and valerate decreased in the MU group compared to the LU group. This result may be due to the significant enrichment of Succinivibrionaceae_UCG_001 and Fibrobacter in the LU group. Succinivibrionaceae_UCG_001 [] ferments various carbohydrates, and the main metabolic end products are acetate, succinate, and lactate. Succinate does not accumulate in the rumen and is rapidly converted to propionate []. The genus Fibrobacter was cellulolytic bacteria originally separated from the rumen []. It can produce cellulase and decompose the cellulose in feed to produce acetate and butyrate. The concentration of NH3-N in incubation fluid depends on the rate at which micro-organisms use NH3-N to synthesize microbial protein, an important index of rumen micro-organisms for nitrogen use in feed []. Some previous studies have found that the effects of rumen plant oil or seeds supplementation on NH3-N are contradictory. For example, the concentration of NH3-N was not affected by plant oil supplementation in Pi et al.’s study []. However, Scollan et al. [] and Broudiscou et al.’s [] studies were positive and negative, respectively. Our experiment found no effect on the NH3-N concentration with the increased UFA proportion. Accordingly, we speculated that under the premise of fat level, the increased UFA ratio would affect the production of rumen TVFA, acetate, propionate, butyrate, and valerate, but not the NH3-N concentration.
The in vitro fermentation gas production is an important index reflecting the rumen microorganisms’ fermentation degree of substrate nutrients. The higher the gas production, the higher the degree of substrate fermentation []. A previous study found that flaxseed supplements increased the maximum gas production of the fermentation substrate and GP48 [], but different results were found in our experiment. We found that with increased linseed oil supplementation, the maximum gas production of the fermentation substrate and GP48 increased numerically, but the difference was not significant. This result may be because we did not have enough flaxseed oil supplements to create a significant impact in our experiment. Ruminants produce a large amount of CH4 rumen fermentation. Studies have shown that adding flaxseed rich in UFA to dairy cows’ diets can significantly decrease the rumens’ CH4 emission, although the extent of the decrease has varied [,,]. There may be two reasons for this: [,] The UFA supplement can reduce the relative abundance of methanogen in the rumen and decrease the activity of methyl coenzyme M reductase. Therefore, the pathway of methane production in the rumen was inhibited, leading to a decrease in CH4 production. Furthermore [], adding UFA would reduce the flow of H to methanogenesis through biological hydrogenation, thus reducing CH4 production. Our results also found that the CH4 and CO2 concentrations decreased significantly with the increased proportion of UFA. However, our microbial results were not annotated with the archaea; thus, the reason for reduced methane is more likely the latter. Hence, elevated UFA ratios reduce the rumens’ CH4 and CO2 production.
Microorganisms in the rumen are important intermediaries for ruminants to digest nutrients in their diets []. The Shannon index of the LU group was the lowest in our experiment, indicating that an increase in the UFA proportion would increase the microbial community’s diversity and abundance. Beta diversity would also have similar results. By contrast, the MU group had the highest Shannon index, which may be due to the more complex fatty acid composition in the MU group. In addition, significant enrichment of multiple Lachnospiraceae bacteria in the MU group, including Lachnospiraceae_ND3007_group, Lachnospiraceae_UCG_001, Lachnospiraceae_UCG_008, Lachnospiraceae_NK4A136_group, Syntrophococcus, Lachnoclostridium, and Blautia. Lachnospiriaceae are reportedly involved in the degradation of carbohydrates and the production of acetate in the rumen []. Although multiple Lachnospiraceae bacteria were significantly enriched in the MU group, the acetate concentration in the MU group was lower than in the LU group. The reason was that the relative abundance of Lachnospiraceae bacteria was much lower than Succinivibrionaceae_UCG_001, but the Succinivibrionaceae_UCG_001 in the MU group was significantly lower than in the LU group. Our results showed a lower relative abundance of Succinivibrionaceae_UCG_001 in the MU group during the in vitro study. However, animal experiments are needed to demonstrate the effects of elevated UFA ratios on rumen fermentation.

5. Conclusions

In conclusion, we showed that an increased UFA proportion in fermentation substrate reduces CH4 and CO2 emissions, affecting rumen fermentation and the microbial community. Additionally, our results indicated that the bacterial richness and diversity in the MU group were higher than in the LU group. However, the relative abundance of Succinivibrionaceae_UCG_001 was significantly decreased, leading to a significant decrease in the main VFAs (TVFA, acetate, propionate, butyrate, and valerate). Meanwhile, our results also showed that increased UFA proportion reduces the emission of CH4 and CO2 through hydrogenation.

Author Contributions

Conceptualization, S.L. (Shengli Li) and W.W.; methodology, W.W. and H.Y.; software, Z.Y.; validation, S.L. (Shengli Li); formal analysis, Z.Y.; investigation, Z.Y., S.L. (Siyuan Liu), T.X., Q.W. and Z.W.; writing—original draft, Z.Y.; writing—review and editing, W.W.; visualization, Z.Y.; supervision, S.L. (Shengli Li) and W.W.; project administration, W.W.; and funding acquisition, W.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Beijing Municipal Science and Technology Project (Z191100004019023) and the 2115 Talent Development Program of China Agricultural University.

Institutional Review Board Statement

All experimental procedures were approved by the Ethics Committee of the College of Animal Science and Technology of China Agriculture University (Beijing China).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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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