Effects of different proportions of stevia stalk on nutrient utilization and rumen fermentation in ruminal fluid derived from sheep

Test materials

Stevia stalk was collected from Wulan Town, Jingyuan County, Gansu Province, in November 2020. After drying at 65 °C for 48 h, initial moisture was determined, and plant material was crushed through a 40-mesh sieve for further use. Stevia samples were placed into nylon bags (9 cm × 5 cm, 400-mesh), which was placed into a 100 mL trachea with a chitin plug and plastic screw top. The basal diet was ground with a plant crusher and then prepared into dry matter substrate. The main nutrients in stevia stalk were shown in Table 1.

Rumen fluid collection

All experimental protocols were approved by the Livestock Care Committee of Gansu Agricultural University (GAU-LC-2022-0555). Rumen fluid was obtained from animals at Zhonghua Sheep Farm in Lanzhou, and the experiment was conducted with the consent of the farmer. It was collected from three sheep (Small Tailed Han Sheep, 3 months old, male) at different sites in rumen, then mixed with CO₂ gas in an insulated bottle preheated to 39 °C. After the rumen fluid was extracted, the experimental sheep were in good health and had no adverse reaction. The bottle mouth was immediately covered, then transported to the laboratory quickly. All of the mixed contents were filtered through four layers of gauze and stored in containers in a 39 °C water bath. CO₂ gas was continuously injected, all of which were completed as soon as possible.

Diet composition and nutrition

The trial sheep diet was made by Lanzhou Zhengda Co., Ltd. and formulated according to nutritional requirements of rams (body weight 45 kg, daily gain 50 g, fine to coarse ratio 4: 6) established by Agricultural Industry Standard of People’s Republic of China (NY/T816-2004). Dietary composition and nutritional information was shown in Table 2.

Test design

A single-factor experiment was conducted in seven treatments, these were, 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, and 1.5% (dry matter basis) proportions of stevia stalk, added to total mixed basal diet and mixed evenly for fermenting substrate. The specific test design was shown in Table 3.

In vitro fermentation method

Stevia stalk with concentrations of 0, 2, 4, 6, 8,10 and 15 g/kg was accurately weighed and placed in a homemade nylon bag (9 cm ×5 cm, 400-mesh) with 0.5000 g basal diet for substrate, numbered, sealed and then placed with forcep in the bottom of 100 mL tracheas, which were preheated to 39 °C for 30 min, in order to prevent gas from escaping, an appropriate amount of petroleum jelly was applied to the plunger of the syringe and then the filtered rumen fluid and artificial culture medium were mixed evenly at a volume ratio of 1: 2. 30 mL microbial culture mixture saturated with CO₂ was accurately measured and placed in each trachea, sealed with rubber tube and clips, and the initial volume (mL) of each trachea was recorded. Each treatment contained 17 replicates. In order to ensure the representativeness of the test sample, three blank samples were made when the sample was incubated to eliminate test errors. After reading the initial volume, the tracheas were immediately transferred to water bath for culture preheated to 39  °C (the water surface height of the bath should be higher than liquid surface height of trachea culture solution) for 48 h. All data of the gas production at 2 h, 6 h, 12 h, 24 h, 36 h and 48 h were recorded. After 2 h, 6 h, 12 h, 24 h and 48 h of fermentation, the fermentation tube was placed into an ice water bath to stop fermentation. Three replicates were removed after 2 h, 6 h, 12 h and 24 h fermenting respectively, keeping the last five replicates for 48 h fermenting were placed into an ice water bath to stop fermentation, collected fermentation broth and nylon bags for index measurements.

Indexes and measurement methods

Fermentation indexes

pH value of rumen fluid was determined by pH meter (P611 type). Determination of ammonia nitrogen concentration (NH₃-N), 10 mL of rumen fluid was centrifuged at 3,500 r/min for 10 min, 2 mL of supernatant was taken and placed in 15 mL test tube, then 8 mL of 0.2 mol/L hydrochloric acid was added to 10 mL, shaking, and the content of NH₃-N was determined by colorimetry (Feng & Gao, 2010).

For volatile fatty acids (VFAs), sample of each treatment at each time was centrifuged at 5,400 r/min for 10 min at 4 °C; 1 mL of supernatant was added to a 1.5 mL centrifuge tube containing 0.2 mL of 25% phosphonic acid solution containing internal standard 2-ethyl butyric acid. The samples were mixed well, placed in an ice water bath for more than 30 min, centrifuged at 10,000 r/min for 10 min, filtered through a 0.22 µL filter membrane, and detected by gas chromatography (Li, 2021).

Rumen degradation rates

After 2 h, 6 h, 12 h, 24 h and 48 h of fermentation, nylon bag was washed with distilled water until clear. After natural drying, the nylon bag was transferred to an oven at 65  °C for 48 h and dried to a constant weight. The weight of the residue was weighed and used to determine DMD, CPD, ADFD and NDFD of different treatments at different times. ADF and NDF contents were determined by fiber washing method.

Every index of rumen degradation rate of feed was calculated as: (Mass of certain components in feed-the mass of associated components in residue)/the mass of associated components in feed ×100% (Liu et al., 2021).

Data processing and statistical methods

Microsoft Excel 2010 was used for preliminary data collation. SPSS 23.0 software was used for single factor analysis of variance, duncan method was used for multiple comparisons. The results were expressed as means ± standard deviations, and P < 0.05 was considered significant. The data standardization of 48 h fermentation was transformed by membership function method.

The positive indexes were transformed according to the formula “Uij = (Xij-Ximin)/(Ximax-Ximin)”; the negative indexes were transformed according to the formula “Uij = 1 - (Xij-Ximin)/(Ximax-Ximin)”. In the formula Uij was the index (j) membership function value of different stevia stalk treatments (i); Xij was the measured value, Ximax and Ximin were the maximum and minimum measured values, respectively. The membership value of each index was obtained by the membership function method, and the average membership value of the measured index was calculated and used as the comprehensive evaluation of different stevia stalk treatments (Liao et al., 2022).

Effects of stevia stalk on gas production, DMD, CPD, NDFD and ADFD of sheep diet

Effects of different stevia stalk proportions on gas production

The change trend of fermented gas production remained the same under different stevia stalk treatments. It tended to be slight at 2 h-6 h and increased at 6 h-24 h in each treatment, but they were significantly higher in 0.8%, 1.0% and 1.5% treatment at 2 h-6 h than that in the control (P < 0.05). All of treatment reached to flat at 36 h-48 h among treatments. The maximum value was obtained in 0.8% treatment at 36 h (P < 0.05) while got the highest value in 1.0% treatment at 48 h (P <  0.05) (Fig. 1; Table 4).

Table 4:

Significance among treatments.

Item	Fermentation time
	2 h	6 h	12 h	24 h	36 h	48 h
0%	c	d	a	a	ab	c
0.2%	c	c	b	ab	b	c
0.4%	ab	c	ab	b	b	c
0.6%	bc	c	ab	ab	b	c
0.8%	a	a	ab	ab	a	ab
1.0%	a	b	a	ab	ab	a
1.5%	a	b	a	ab	ab	bc
P- value	0.000	0.000	0.148	0.218	0.072	0.004

DOI: 10.7717/peerj.14689/table-4

Notes:

Different lowercase letters in the same column indicate significant differences (P < 0.05) between different stevia stalk concentrations at the same time.

Effects of stevia stalk on DMD, CPD, NDFD and ADFD

The DMD of sheep diet increased with fermentation time extention. It was lower in each treatment fermented at 2 h, 6 h and 12 h than that in the control. After 48 h of fermentation, there were no significant differences among treatments (P > 0.536). But the DMD_48h in 0.2% and 1.0% treatments were higher than that in the control (P > 0.05). CPD_12 h, CPD_24h and CPD_48h in 1.0% and 1.5% treatments were higher than that in the control (P < 0.05). And the ADFD_48h and NDFD_48h of 0.8%, 1.0% and 1.5% treatments were significantly higher than those of other treatments (P < 0.05) (Table 5).

Effects of stevia stalk on in vitro fermentation characteristics

As shown in Table 6, the pH value of rumen fluid derived from sheep treated with different stevia concentrations fluctuated between 6.21 and 7.25. It increased first and then decreased in 0%, 0.2%, 0.4% and 0.6% treatments while decreased in 0.8%, 1.0% and 1.5% treatments during fermentation. In short, with the extension of fermentation, the pH of rumen fluid declined. At 48 h, the pH was significantly lower in the 0.8% and 1.0% treatments than in the control (P < 0.05).

The concentration of NH₃-N in sheep rumen fluid decreased with the extension of fermentation time. 0.4% treatment had significantly lower NH₃-N_48h than other treatments (P < 0.05), while there had no significant different NH₃-N_48h between other treatments and control (P > 0.05).

During period of 2 h-48 h, the concentrations of acetic acid and propionic acid varied significantly among treatments, while the concentrations of isobutyric acid, butyric acid, isovalerate and valerate changed only slightly and remained relatively stable. At 6 h and 12 h, the total acid content of 0.8% and 1.0% treatments were significantly higher than that of other treatments (P < 0.05), while at 48 h, it had no difference in 0.8%, 1.0% , and 1.5% treatment, compared to the control. But showed the higest value in 1.0% treatment.

Comprehensive analysis

It was insufficient to evaluate the fermentation characteristics with single index. Using the membership function method to analyze the relevant indicators of rumen fermentation characteristics comprehensively could reflect the overall performance of rumen fermentation accurately. In this study, the membership function method was used to comprehensively analyze pH, NH₃-N, VFAs, DMD, CPD, NDFD and ADFD (Table 7). The higher the comprehensive analysis value, the better the fermentation of rumen nutrients. The results showed that order of the treatments was 1.0% >0.8% >1.5% >0.4% >0.6% >0.2%.

Substrate degradation rate

Dry matter degradation rate, gas production and in vivo digestibility are highly correlated (Blümmel, Makkar & Becker, 1997). In this experiment, the trend of gas production with different concentrations was consistent, and they were stable at 2 h-6 h, it was because the rumen microbial activity was weak and a small amount of gas produced in early fermentation stage, while to 6 h-36 h, rumen microbe proliferated, acting on substrate completely to be digested enough, thus producing large amounts of gas rapidly. The gas production decreased slowly with the nutrients being consumed. Menke et al. (1979) showed that the rate of forage organic matter degradation was positively correlated with gas production during in vitro incubation, i.e., higher gas production indicated higher forage degradation in rumen and higher microbial activity. The results of this study showed that the CPD_48h, NDFD_48h and ADFD_48h of 0.6%, 0.8%, 1.0% and 1.5% stevia stalk treatments were higher than those without stevia stalk, and 1.0% treatment has the highest gas production at 48 h, which was consistent with the result of DMD_48h, CPD_48h and NDFD_48h. It showed that appropriate concentration of stevia stalk could improve sheep rumen digestive function and promote crude fiber digestion and absorption in diet, adding 1.0% stevia stalk could promote the microbe fermentation activities in rumen more effectively, thus improved feed digestibility. Studies showed that the active ingredients such as stevia or flavonoids in stevia rebaudiana could reduce rumen protozoa and increase cellulolytic bacteria amount to facilitate fiber decomposition and change rumen micro ecological environment (He et al., 2017). This was consistent with the results of the Sarnataro’s study (2020). Studies reported that sea buckthorn flavonoids (Bai et al., 2020) and sea onion flavonoids (Bao et al., 2015) significantly increased rumen gas production, which was consistent with the results of the present experimental study. Its specific mechanism needs further research in feeding experiments.

Rumen fermentation characteristics

The pH value is a comprehensive index reflecting rumen fermentation level, it not only reflects the strength of rumen microbial activity but also serves as an important indicator to evaluate nutrients decomposition status and assess rumen internal environment stability. The normal rumen pH is 5.5 ∼7.5, and pH values higher or lower than this range will affect normal fermentation in rumen. When pH value is lower than 6.0, the number of fiber-decomposing bacteria and protozoa will decrease, and the cellulose and protein degradation rates in diet will also decrease (Liu et al., 2012; Krause & Oetzel, 2005). In this experiment, the pH range was 6.21 to 7.25, and high stevia stalk concentration (0.8%, 1.0%, 1.5%) could decrease the pH of the rumen culture medium, but the final pH still remained within the normal physiological range. This was consistent with the results of He’s study (2017). It indicated that the artificial rumen was in a normal fermentation state and had not adverse effects on normal metabolism of rumen microbe, ensuring rumen microbe normal growth and reproduction. An explanation was that rumen microbe decomposed starch and structural carbohydrates in feed, producing a large number of VFAs. Moreover, with the extension of fermentation, VFAs was accumulated in fermentation tubes, resulting in a further decline in pH value. Consistent with the result of a negative correlation between VFAs and rumen pH by Stritzler et al. (1998).

The NH₃-N concentration in rumen is an important indicator of nitrogen metabolism, microbial protein synthesis and protein degradation. Maintaining an appropriate NH₃-N concentration is a prerequisite for microbial protein synthesis in rumen (Feng et al., 2017). A suitable rumen NH₃-N concentration is beneficial to growth of microbe, and higher NH₃-N concentration is beneficial to fibrolytic bacteria growth and reproduction and DM degradation. The most suitable NH₃-N concentration range for microbial activity was 0.35 ∼29 mg/dL (Galdi et al., 2002). In this experiment, the NH₃-N concentration was 12.17 ∼45.92 mg/dL, and NH₃-N concentrations were relatively high in all treatments for 48 h. He et al. (2017) found that stevia residue reduced ruminal NH₃-N concentration, contrary to the results of the present study. This might be related to factors such as feed type, feeding form and in vitro fermentation. In this experiment, after 12 h of rumen fermentation, NH₃-N concentration in each treatment increased significantly, which might be due to the longer retention time of chyme in rumen and the longer interaction time between rumen microbe and feed particles (He et al., 2020). This might also be associated with lower microbial utilization. With the extension of fermentation, the pH of rumen fluid decreased, and the decreased pH environment was not conducive to the growth of microbe that decomposed structural carbohydrates. The utilization efficiency of NH₃-N by microbe that decompose structural carbohydrates decreased, resulting in increasing of NH₃-N concentration in rumen. This was also consistent with the decreasing trends of the pH value.

VFAs in the rumen are important indicators of rumen fermentation in sheep and can reflect the environmental conditions in the rumen, the fermentation degree and the type of feed in rumen (Liu et al., 2012). In this experiment, at 48 h of fermentation, the content of acetic acid and propionic acid increased in all treatments, and acetic acid contents were higher than propionic acid contents, and the total acid content of 1.0% treatment was the highest. It could be seen that adding 1.0% stevia stalk could increase the concentration of VFA in vitro fermentation, better improve rumen fermentation capacity, maintain the normal growth, development and reproduction of rumen microbe and ensure the normal function of rumen, which was consistent with the results of pH value decreased to the lowest and NH₃-N concentration increased. However, Jiang (2019) found that the addition of stevia pellets to lactating cows did not affect rumen fermentation. Studies had shown that in vitro rumen fermentation of stevia residue was a typical acetic acid type fermentation, which could promote rumen carbohydrate fermentation, improve energy utilization and VFAs production (He et al., 2017). This indicated that stevia stalk also had good feeding value as a by-product. Stevia extract could reduce the number of rumen protozoa, thereby affecting rumen metabolism (Chiara & Mauro, 2020). It was also reported that stevia could help digest and prevent constipation. Stevia could promote the proliferation of bifidobacteria and lactobacillus in human body, improve human immunity, prevent and treat constipation and diarrhea (Xu, 2013). Stevia rebaudiana residue could significantly inhibit the growth of harmful bacteria and promote the proliferation of beneficial bacteria (Zhong et al., 2022). Therefore, it was speculated that the glycosides in stevia stalk also had an effect on sheep gastrointestinal fermentation. However, whether the increase of VFAs content in this experiment was related to steviosides in stevia rebaudiana, its mechanism needs further study.