1. Introduction
Proteins are an indispensable nutrient source for animal growth and development. The quality of protein feed affects the health and productivity of animals. Soybean meal is a high-quality protein feed, but its high price has seriously affected its use in the domestic-animal-breeding industry, especially in China.
In China, the annual output of mixed-meal feed, such as cottonseed meal, is more than 30 million tons. However, animal nutritionists have increasingly focused on plant protein substitutions for soybean meal (SBM). Due to their lower prices and high crude CP content, cottonseed meal (CSM) and rapeseed meal (RSM) are of interest. CSM, a by-product of cottonseed oil extraction, has a CP content of 34–40%, a crude fiber (CF) content of 11%, a neutral detergent fiber content of 25–30%, and comparatively high levels of organic phosphorus and vitamin B [
1]. Rapeseed is crushed to produce RSM after the oil has been extracted. It has a high protein content (34–38%), an amino acid composition that is well balanced, and an NDF content of 25–30% [
2]. The RSM protein contains more sulfur amino acids than other plant proteins and has a nutritional value comparable to that of the SBM protein. However, because they include anti-nutritional components such as erucic acid and glucosinolate in RSM and FG in CSM, their use in animal diets is still limited [
3].
Numerous detoxification strategies have been developed for CSM and RSM, including biological [
4], chemical [
5], and physical [
6,
7] treatments. Yeasts and
Aspergillus oryzae are the most commonly used microorganisms of solid fermentation [
8]. Physical detoxification methods include heating the rapeseed cake by using dry heat and steam. This method of operation is simple and inexpensive, but the detoxification rate is low and irregular [
9]. Chemical detoxification involves the addition of chemicals to the rapeseed. Under certain conditions, glucosinolates in rapeseed undergo a glycosidase reaction and develop poisons. The method is simple and highly targeted; however, owing to the effect of a single seedling, blending makes it difficult to achieve the effect [
10].
Our previous study confirmed that the fermentation with 1.0 × 10
9 and 5.0 × 10
9 CFU/kg DM for
B. clausii and
S. cariocanus to TMR with CSM and 1.0 × 10
10 and 5.0 × 10
9 CFU/kg DM for
B. clausii and
S. cariocanus to TMR with RSM improved the nutritional value and decreased the content of anti-nutritional factors [
11]. In addition, an increased CP content and decreased NDF content, as well as a reduction in the content of anti-nutritional factors in both TMRs containing CSM/RSM, were observed after fermentation. Therefore, the following were the ideal fermentation conditions for fermented TMR (FTMR) with CSM: mixed microbial strains (1.0 × 10
9 and 5.0 × 10
9 CFU/kg DM for
B. clausii and
S. cariocanus, respectively) incubated aerobically for 60 h at 32 °C and 50% humidity. The following were the ideal fermentation conditions for FTMR incorporating RSM: 60 h incubation, 28 °C temperature, 50% moisture, and mixed microbial strains (1.0 × 10
10 and 5.0 × 10
9 CFU/kg DM for
B. clausii and
S. cariocanus, respectively) [
12]. These findings served as the basis for this study’s fermentation conditions.
Fermented TMR (FTMR) refers to a type of ruminant feeding technology in which the finished TMR is inoculated by a particular compound strain [
13] for anaerobic fermentation so that it can be stored for a predetermined amount of time. In comparison to TMR, FTMR preparation is more efficient and less time-consuming for daily feeding management, so it may significantly save labor and resources [
14]. In the lack of TMR mechanical equipment, FTMR can be purchased and used by small-scale farmers, and it can be transported over long distances [
14]. Moreover, FTMR has the potential to enhance feeding levels while also promoting the economic development of animal husbandry [
15]. A TMR optimization method using
L. casei TH14 and fermented sugarcane bagasse significantly changed the intake, digestibility, rumen ecology, and milk production of mid-lactation Holstein cows [
15].
The profitability of farms is severely affected by the aerobic decomposition of silage. In addition to being less palatable, spoiled silage also has a negative impact on livestock productivity. FTMR results in highly enhanced aerobic stability [
16,
17]. This makes it possible to transport and use FTMR in various ways for a considerable amount of time, without experiencing aerobic deterioration [
16]. Numerous studies have investigated the aerobic stability of FTMR. Yeast counts can decrease below the limit of detection (10
2 colony forming units CFU/g) when anaerobic fermentation is sustained for a month or more, substantially enhancing aerobic stability [
18]. Silage with a high yeast population (>10
5 CFU/g) has been observed to spoil upon contact with oxygen [
19]. Even with more than 10
6 CFU/g of yeast measured at silo opening, aerobic stability in FTMR was attained [
13]. This shows that the loss of aerobic stability may be associated with the dominant yeast species found in FTMR; however, there is no discernible relationship between yeast counts and the loss of aerobic stability. However, the replacement of SBM by CSM and RSM in TMR by fermentation has an effect on the degradation of anti-nutritional factors. To explore the effects of fermentation and aerobic exposure to FTMR on nutrients and anti-nutritional factors, this study aimed to ascertain the impact of
B. clausii and
S. cariocanus inoculations on TMR fermentation quality, aerobic stability, anti-nutritional factors, and in situ rumen-degradation characteristics’ variables.
2. Materials and Methods
2.1. Preparation of a Total Mixed Ration and Anaerobic Fermentation
The entire corn plant after cob harvest at the wax-ripe stage (Sanbei 21) was cut immediately when part of the leaves remained green after ear picking on 30 August 2021, at the wax-ripe stage. The following ingredients were used to manufacture TMR: whole-plant corn silage (lactic acid at 67.63 g/kg DM, acetic acid at 15.88 g/kg DM, and propionic acid at 1.72 g/kg DM), corn stalk, SBM, CSM, RSM, wheat bran and fat powder, urea, and premix. The dietary feed was formulated according to the guidelines for meeting the nutrient requirements of sheep and allowing them to gain 300 g per day (NRC, 2007). The diet’s components and chemical composition are listed in
Table 1.
A mixture of the microbial strains at a ratio of 1:5 (1.0 × 109 CFU/kg DM B. clausii: 5.0 × 109 CFU/kg DM S. cariocanus) was inoculated into the FTMR with CSM (F-CSM) at 50% moisture content. A moisture content of 50% and a microbial strain combination ratio of 1:5 (1.0 × 1010 CFU/kg DM B. clausii: 5.0 × 109 CFU/kg DM S. cariocanus) were used for the FTMR with RSM experiment. The combination was fermented for 60 h at 32 °C for F-CSM or 28 °C for F-RSM in a fermenter machine (Model SSJX-WH-3.0, Shengshun Machinery Manufacturing Co., Ltd., Shenyang, China) with a capacity of 500 kg volume. The mixture was combined with silage after fermentation and mixed before being stored in a plastic bag (using the V5 Reelanx vacuum sealer). The TMR in the control group was mixed when the other two groups finished fermentation. Except for the non-fermented TMR, F-CSM, and F-RSM groups during 60 h anaerobic fermentation, when they were finished going through the fermentation process, fermented groups were tested for aerobic stability. Three TMRs were used in this study.
2.2. Aerobic Stability Tests
Three 6 L polyethylene barrels were set up in triplicate for each TMR treatment, and 10 kg of the substance was introduced to each barrel, without compacting, and fully mixed for the aerobic stability test. The remaining contents were loosened, returned to the barrel, and left uncovered for the duration of the air-exposure period. Half of the contents (weight) were discarded. After the aerobic stability test began, sub-samples of non-fermented and FTMRs were collected at 0, 12, 24, 48, 72, 96, 120, 144, and 168 h. The temperatures of the ambience and the TMRs were measured with a mercury thermometer, and aerobic deterioration was deemed to have occurred when the temperature difference between the TMRs and the atmosphere exceeded 2 °C [
13].
2.3. Fermentation Profiles and Microbial Counts
About 20 g of sample was blended with 180 mL distilled water and macerated for 24 h at 4 °C. The extract was filtered through 4 layers of a gauze. The filtrate was used for pH, volatile fatty acid (VFA), and ammonia nitrogen (NH
3-N) determinations. The pH was measured with a pH meter (Testo-206-pH, Testo Co., Berlin, Germany). VFA profiles were determined using a gas chromatography instrument (GC-6800; Beijing Beifen Tianpu Instrument Technology Co., Ltd., Beijing, China) [
20]. The NH
3-N was determined using the phenol-hypochlorite reaction method [
21].
The microorganism populations were enumerated using the technique described by Xu et al. [
22]. Samples (10 g) were serially diluted from 10
−1 to 10
−9 in a mixture of 90 mL of sterilized distilled water. At appropriate dilutions, colonies were enumerated from the plates, and the CFU was expressed per gram of dry matter (DM). After 48 h of anaerobic incubation at 37 °C, the presence of lactic acid bacteria (LAB) was determined by plate counting on deMan, Rogosa, and Sharpe agar (Difco Laboratories, Detroit, MI, USA). On nutritional agar medium (Nissui-Seiyaku Ltd., Tokyo, Japan) incubated for 48 h at 30 °C under aerobic conditions, aerobic bacteria were enumerated. After incubation for 24 h at 30 °C, yeasts were counted on potato dextrose agar (Nissui-Seiyaku Ltd., Japan) that was acidified with a sterilized tartaric acid solution to pH 3.5. Based on the appearance of the colony and the shape of the cells, yeast and aerobic bacteria were separated. By streaking each yeast colony on peptone–dextrose agar, each colony was purified. The purified strains were then stored at −80 °C with 10 g/L glycerine for investigations (Difco Laboratories, USA).
2.4. Chemical Composition and DM Loss
To determine the DM content, 800 g of the sample was dried at 65 °C for 72 h. The DM loss in the samples was calculated from the difference in weight before and after fermentation. Samples were ground using a Wiley Mill after drying (1 mm screen, Arthur H. Thomas, Philadelphia, PA, USA). We tested OM use GB/T 6435-2014 [
23] and GB/T 6438-2007 [
24], ether extract (EE) (Horwitz et al., 1970), and true protein (TP) by using Sniffen et al.’s (1992) method; and NDF (add 0.5 mL/sample Alpha Amylase, Ankom A2000 and A200 instrument, Macedon, OH, USA) and ADF by using an Ankom 200 system (Ankom Technology Corporation, Macedon, NY, USA) according to the manufacturer’s instructions. Starch was determined by the perchloric acid hydrolysis method [
25]. Using the N data collected with a Foss KJELTEC 8400 analyzer, the CP was estimated as the multiplication of the total N by 6.25. (FOSS; Nil Foss, Hilleroed, Denmark). GE used a Parr-6400 oxygen and nitrogen calorimeter (Parr-6400, Beijing Oriental Shenglongda Technology Co., Ltd., Beijing, China) to determine the oxygen and nitrogen levels.
2.5. Anti-Nutritional Factor Analyze
The method approved by the American Oil Chemists Society [
26] was used to calculate the free gossypol concentration. Accordingly, the palladium chloride method was used to determine the F-RSM glucosinolate concentration [
27].
2.6. In Situ Disappearance of Various TMR
A nylon cloth with a diameter of 300 mesh or 35~50 µm was cut into a rectangle measuring 17 × 13 cm; the rectangle was then folded in half, and we used nylon thread to sew it twice. A 12 × 8 cm nylon bag was prepared, with the edge of the bag sealed using a soldering iron or on the alcohol lamp to avoid silk. Then, the bag was numbered, rinsed with tap water, soaked for 50 min, dried at 65 °C to constant weight, and set aside.
To test the rumen’s capacity for degrading feed, three Hu sheep with fistulas were used. We started the experiment by placing a nylon bag into the sheep’s rumen abdominal sac. The bags were incubated for 0, 6, 12, 24, 36, and 48 h in the rumen (0 h was used to determine the soluble fraction), using different methods of simultaneously putting in and taking out. A total of 1.5 kg DM of grass-based 40% and 60% sheep-fattening pellets was provided to the Hu sheep. The bags were withdrawn from the rumen after incubation, washed under running water for 20 to 25 min until the effluent water was clear, and then dried for 48 h in a 65 °C oven. The contents of conventional nutrients in the original samples and those at different time points were measured, and the soluble fraction was estimated to correct the weight of feed samples and calculate the effective degradation.
2.7. Statistical Analysis
One-way analysis of variance (ANOVA) was used to analyze the data in the general linear model (GLM) function of the SAS software (version 9.4, SAS Institute, Cary, NC, USA). One-way analysis of variance was used to assess the data on chemical composition, fermentation quality, and anti-nutritional factors (ANOVA). A two-way ANOVA with a mixed model was also performed on the data for aerobic stability. Tukey’s multiple comparisons test was used to calculate the statistical difference between means, and the differences between means were deemed significant at p < 0.05. The exponential equation of Orskov and McDonald (1979), Y(t) = a + b (1 − e−ct), was used to determine the fraction of the incubated material that was degraded at time t (h of incubation); “a” stands for the water soluble and instantly degradable fraction, “b” stands for the potentially degradable fraction, and “c” stands for the fractional rate of degradation of fraction b (/h). The fractional rate of passage (k) was assumed to be 0.05/h for calculating the effective degradability (ED) of dry matter (DM) and starch, which was calculated as ED = a + (bc)/(c + k). The SAS NLIN procedure was used to calculate estimates for variables “a”, “b”, and “c” (version 9.4, SAS Institute, Cary, NC, USA).