1. Introduction
Rumen fermentation plays an important role in feed degradation and health condition of ruminants [
1]. Rumen microorganisms convert low-quality protein and nonprotein nitrogen into high-quality microbial protein (MCP), and fibers into volatile fatty acids (VFAs) [
2], thereby providing 50–80% of the total absorbable protein [
3] and approximately 75% of the total metabolic energy [
4] required for ruminants. Abnormal rumen function can lead to multiple disorders in ruminants such as acidosis and feed accumulation [
1]. Targeting improvements in rumen fermentation is an efficient way to enhance feed digestion and health of ruminants.
During rumen fermentation, microbes in the rumen can also convert nutrients into CH
4 and skatole, which are a concern in ruminant agriculture [
5]. CH
4 emissions account for about 44% of all greenhouse gases emissions from livestock agriculture [
6], previous studies demonstrated that the average yield of CH
4 for dairy cattle under dietary patterns is 304.44 g/d [
7] and that for sheep is 9.10 g/d [
8]; therefore, reducing methane emissions is an important goal in ruminant agriculture [
9]. Skatole, also known as 3-methylindole, is produced by the degradation of tryptophan by rumen bacteria; tryptophan has moderate toxicity and can cause intestinal diseases, pulmonary edema and emphysema in ruminants [
10]. However, skatole is one of the major contributors to the odor of feces and easily deposits in adipose tissue, thus, has a negative role in environmental and meat quality [
11,
12].
Fruit oligosaccharides (FOS) are a green and safe plant additive purified mainly from Jerusalem artichoke. They accounted for 23.93% of the global total production of prebiotics in 2019. Because of its benefits for animals, low cost and convenient use [
13], there is increasing interest in the use of FOS as a feed additive [
14,
15]. Previous studies demonstrated that FOS could improve rumen fermentation performance, food intake [
16], feed utilization efficiency [
17], absorption and utilization of protein [
18], production performance [
19] and antioxidant capacity of ruminants [
20]. It also has the ability to regulate the hormone neuroendocrine axis related to fat metabolism by affecting beneficial microorganisms and their metabolites in the intestine [
21]. Some studies have shown that FOS supplementation in ruminant diets can also reduce CH
4 production [
22] and fecal odor [
23]. However, the dose effect of FOS on rumen fermentation on feed digestibility, CH
4 and skatole production is still unclear.
Therefore, this study adopts the method of in vitro rumen fermentation with Hu sheep to investigate the dose-response of FOS on nutrient degradation and CH4 and skatole production by adding different FOS doses to the diet. The findings in the current experiment might provide a guide for FOS usage in ruminants by demonstrating the impact of FOS on rumen fermentation and environmental issues.
2. Materials and Methods
This study was conducted between December 2021 and August 2022 at the animal center of the College of Animal Science and Technology of Hebei Agricultural University, and the experimental protocol was approved by the Ethical Committee of Hebei Agricultural University (ID: 2021006).
2.1. Experimental Design and Experimental Diet
The Hu sheep (ewe,
n = 6) in the current experiment had similar body conditions (2.80 ± 0.35 in body condition score, 49.74 ± 2.51 kg in body weight) and age (1.5 years old), and all were raised at the animal center of the College of Animal Science and Technology of Hebei Agricultural University. All sheep were prepared with a rumen fistula 3 months before the experiment, provided a formula diet following NRC 2007 (body weight 50 kg, daily gain 200 g) at 9:00 a.m. and 5:00 p.m. each day and given free access to fresh water. The composition and nutritional content of the diet was shown in
Table 1.
2.2. In Vitro Rumen Fermentation
Rumen contents from six Hu sheep were collected through the fistula before morning feeding, and filtered using four layers of sterile cheesecloth. The fresh fluid was immediately transferred to the laboratory in heated vacuum flasks (39 °C) under anaerobic conditions. The composition of the basal diet for in vitro fermentation is presented in
Table 1. The experimental diet was supplemented with 0%, 0.2%, 0.8%, 1.2%, 1.8% and 2.4% FOS in the basal diet.
The rumen fermentation experiment in vitro was performed following the method of Menke [
24] and our previous study [
25]. Briefly, rumen fluid from six sheep was mixed together, and the rumen fluid was then mixed with the buffer solution (pH = 6.86) at a ratio of 1:2 to achieve artificial rumen fluid. The artificial rumen fluid was then preheated to 39 °C and CO
2 was used to deoxygenate. Then, 1.5 g of feed was accurately weighed, and put into fiber packages with a pore size of 25 μm. Two fiber packages and 300 mL of artificial rumen fluid were added to each plastic incubation bag with a 500 mL capacity (Anscitech Company, Hangzhou, China). All incubation bags (6 treatment × 6 replicates) were deoxygenated and sealed using a bag vacuum packer (Aodeju Company, Hu-zhou, China) to create anaerobic conditions. Then, the incubation bags were placed in a 39 °C thermostatic water bath (Jerriel Company, Changzhou, China) with a speed of 45 r/min for incubation. The gas production (GP) readings (mL) were measured and recorded by a graduated syringe, and CH
4 production was measured by a CH
4 detector (JQ-AZ-2(T), JingQi Company, Shanghai, China) at 2, 4, 6, 8, 10, 12, 24, 36, and 48 h after incubation. Additionally, three blank incubation bags with rumen fluid and buffer (without feed substrate) were used to correct the GP readings. After 48 h fermentation, all fermentation bags were immediately moved into ice water (0 °C) for 30 min to stop fermentation.
2.3. Sampling
After fermentation, the fiber package was taken out, washed with distilled water, and dried at 65 °C for 48 h for further analysis. The artificial rumen fluid of each incubation bag was collected into eight 2 mL sterile tubes after fermentation. Two tubes were centrifuged by SIGMA 3K15 Centrifuge (4000× g, 15 min, 4 °C, SIGMA, Osterode am Harz, Germany) to obtain a supernatant, and mixed with a meta-phosphoric acid solution (0.2 mL, 250 g/L, 30 min, 4 °C). The mixtures were then centrifuged by SIGMA 3K15 Centrifuge (10,000× g, 10 min, 4 °C, SIGMA, Osterode am Harz, Germany) for VFA determination. The other six tubes were centrifuged by SIGMA 3K15 Centrifuge (3000 r·min−1, 10 min, 4 °C, SIGMA, Osterode am Harz, Germany), and the supernatant was collected and stored at −20 °C for NH3-N (two tubes), MCP (two tubes) and skatole (two tubes) analysis.
2.4. Chemical Analysis
For the diet substrate and undegraded residuals, DM content was determined by drying at 105 °C for 24 h; GE content was determined by an oxygen bomb calorimeter, model No.XRY-1A; CP content was determined by the Kjeldahl nitrogen determination method and NDF and ADF content were determined using a Ringbio fiber analyzer following the AOAC (2016) method.
For the fermented rumen fluid, the pH was measured with pH EL-20 acidometer (Lecurn Fllid Controls Company, Shanghai, China), NH
3-N was measured by phenol-hypochlorite colorimetry [
26] and MCP was measured by Coomassie brilliant blue method [
22].
VFAs in rumen fluid were measured by gas chromatography [
27] (7890A, Agilent, Milton Keynes, UK). Briefly, H
2 was used as the carrier gas with a 30 m × 320 µm × 0.5 µm capillary column (AT-FFAP). The column temperature was set at 1-min hold (60 °C), increased 5 °C·per minute to 120 °C (not held) and then increased 10 °C·per minute to 180 °C. The detector temperature was set at 250 °C, and the injection port temperature was set at 220 °C.
Skatole content in rumen fluid was measured by the 4-Dimethylaminobenzaldehyde (DMAB) colorimetry method [
28]. First, the standard curve of skatole solution was prepared: acetone and tris buffer were mixed in a 3:1 volume ratio for 3L as the mixed solution, 3-methylindole was added into the mixed solution with concentrations at 0.2 mg/L, 0.4 mg/L, 0.6 mg/L, 0.8 mg/L and 1 mg/L to achieve standard solutions. To achieve the color reagent, 480 mL 99.9% ethanol, 8 g DMAB, 240 mL H
2SO
4 (75%) and 80 mL distilled water were mixed. After adding 2.84 mL of the color reagent into 2 mL of the standard solution and incubation for 3–5 min with 3 repetitions, absorbance was measured by PowerWaveXS2 (580 nm, Biotek, Winooski, VT, USA). Finally, the skatole content in the rumen fluid was measured by mixing the rumen fluid with the color reagent solution with the proportion of 0.7:1, and incubated for 3–5 min until the absorbance measured by PowerWaveXS2 (580 nm, Biotek, Vermont, USA).
2.5. Data Processing and Analysis
Cumulative GP (or CH
4 production) was calculated using the GP (or CH
4 production) measurements at each time point. The gas production parameters were calculated by using the exponential model with discrete lag time [
29] as below: GP (or CH
4 production) = v × (1 – exp(−k × (t – LAG)), where t is the time of fermentation (h), GP is the cumulative gas production (mL), v is the theoretical maximum GP (mL), k is the rate of GP (%/h) and LAG is the discrete lag time. The digestibility of nutrients is calculated as: degradation rate of certain nutrients (%) = [(certain nutrient content before fermentation – certain nutrient content after fermentation)/certain nutrient content before fermentation] × 100%.
All data were processed by Excel 2016, and ANOVA analysis was performed by IBM SPSS Statistics 21.0 (SPSS, Chicago, IL, USA). Multiple comparisons were performed using Duncan’s multiple range test, and a significant difference was considered at p < 0.05.
5. Conclusions
With a huge range of FOS dosages added to basal diet from 0% to 2.4%, we observed the effect of FOS on rumen fermentation parameters, CH4 production and skatole production in vitro was dose-dependent. For improving the digestibility of nutrients, MCP and VFA production, a higher FOS dosage might be helpful, while considering CH4 and skatole production, a dose of FOS at 1.2% was recommended.