1. Introduction
The lack of roughage and protein feed sources limits the production of beef cattle in tropical countries [
1,
2]. Utilizing alternative feed resources is crucial for cost reduction and diversification of the ruminant diet [
3]. The indigo plant, known as
Indigofera tinctoria L., has become established in Africa and tropical Asia [
4]. In Thailand, a variety of small-scale industries use indigo and traditional methods to make natural indigo dye for cotton fabrics [
5]. Following the dye production process, the stem and leaves consist of the remaining indigo waste, which is about 237–261 kg/ha [
6]. The indigo waste contains 19.5% crude protein (CP), 72.4% NDF [
6], and 3488 kcal/kg DM gross energy (GE) [
7]. Our previous studies showed that adding up to 20% of dried indigo waste in the diets did not impact the feed availability, rumen fermentation patterns, hematology, or immune functions of beef cattle [
7]. A concentrate diet suitable for cattle production in the tropics incorporates 10% of dried indigo waste. Ruminants can effectively utilize indigo by-products as a source of protein and fiber while also contributing to environmental sustainability and reducing feed cost. However, Thailand harvests the majority of its indigo plants for indigo dye production during the rainy season. Hence, indigo waste is extremely difficult to sun-dry during this season; hence, ensiling is considered the optimal preservation method [
6].
Silage is a suitable method of preserving forage for later use as ruminant feed [
8]. Silage additives can be incorporated into the forage to enhance the efficiency of silage preservation at different stages, from fermentation to ruminant feeding [
9]. Cellulase is widely used to treat silage due to its ability to breakdown structural carbohydrates into soluble sugars that serve as substrates for the growth of lactic acid bacteria (LAB) under anaerobic fermentation [
10,
11]. Molasses can provide easily fermentable sugars for LAB growth, which results in the production of lactic acid [
12,
13]. The alkaline agents, particularly calcium hydroxide, facilitated the cleavage of ester bonds between lignin and hemicellulose, leading to a decrease in the fiber content of silage and an improvement in feed utilization in ruminants [
14,
15]. Our earlier study found that treating indigo waste with CH, M, or C has potential to improve the quality of silage, in vitro degradability, and rumen fermentation characteristics [
6]. Accordingly, in vivo confirmation is required for the data obtained from in vitro trials. However, there has never been any research on the various additives used for ensiling indigo waste in cattle. Consequently, the objective of this study is to examine the influence of CH-, M-, or C-ensiled indigo waste on the nutritive value, feed utilization, rumen fermentation patterns, blood chemistry, and hematological indices of beef cattle.
4. Discussion
After 30 days of ensiling, the DM content of the indigo waste silage in group M was reduced. The moisture content of liquid molasses is 24.4–35.8% [
23,
24]. Adding M may increase the moisture content, which in turn reduces the DM content in an indigo waste silage. The addition of CH to indigo waste silage increased the content of ash and led to a lower OM content. Adding lime to silage produced a high concentration of alkaline chemicals, which led to an increase in pH. Furthermore, the indigo waste silage in this study had a CP content range of 23.0–26.1%, which was greater than our earlier study’s CP content range of 19.5–21.5% [
6]. This result could be attributed to the collection of indigo waste from different areas, the harvest period, or the proportion of stems and leaves, all of which can affect the CP content in the silage. Our recent study found that CH-treated indigo waste silage increased the CP content [
6]. However, adding CH to indigo waste silage lowers the CP content when compared with the CON in the current study. This study used small-scale silos (plastic drums) with a silage capacity of approximately 80 kg, whereas previous studies used vacuum bags containing 0.5 kg. This difference could potentially impact the nutritive value of the ruminant diet, particularly the CP content. For other reasons, adding lime to silage does not inhibit the growth of
Clostridium. Instead, it increases the breakdown of proteins and amino acids into other substances, including NH
3-N and volatile organic acids. Furthermore, previous research indicated that adding C groups reduces the NDF content in silage [
25,
26]. This agrees with the present study, with the lower NDF content by C-treated indigo waste silage. Cellulase hydrolyzed cellulose and hemicellulose to release soluble sugars [
27], thereby reducing the NDF content in silage. The additives had no impact on the content of ADF. The results were similar to those of Gunun et al. [
6], who reported that adding CH, M, or C, or a mix of them, had no effect on the amount of ADF in indigo waste silage. This may be attributable to the time required to induce the effects of any additive on plant cell walls.
The additives to silage had no effect on the DM intake in cattle. This is consistent with recent studies that found the M- and C-treated silage did not change the feed intake [
28,
29]. In contrast, adding CH and M to silage increased the DM intake in cows and cattle [
14,
30]. It is plausible that recent studies used ruminant-fed silages ad libitum and supplemented with concentrate diets, whereas this study included indigo waste silage in the FTMR, which might not impact the DM intake of cattle. The M-treated silage in the FTMR increased CP digestibility compared to the other treatments. Previous studies have confirmed that adding MO to Napier grass silage enhances CP digestibility in cows [
30]. These findings have two plausible explanations. First, the addition of M may stimulate fermentation, resulting in lactic acid production and a drop in pH, which in turn inhibits the hydrolyzed CP in the silage. Therefore, feeding silage to cattle may increase their intake of CP and improve their digestibility. Second, molasses may provide energy that synchronizes with non-protein nitrogen (NPN) for microbial protein synthesis in the rumen, thereby improving nitrogen utilization and rumen fermentation efficiency [
31,
32]. In addition, previous investigations reported that the addition of M, C, and CH in grass and sugarcane bagasse silage increased the NDF and ADF digestibility in cows and cattle [
14,
30,
33]. So et al. [
28] found that adding 40% of C- and M-treated sugarcane bagasse to the total mixed ration (TMR) of dairy cows increased the fiber digestibility. However, the current study found that the additives to indigo waste silage had no effect on the NDF and ADF digestibility. It is plausible that previous studies used ruminant-fed silages ad libitum, while this study’s inclusion of indigo waste silage with additives at low levels (20%) in the FTMR may not have had an impact on the fiber digestibility in cattle. Another explanation is that different levels of additives in silage, types of forage, fecal collection methods, etc., might influence the NDF digestibility of animals.
Ensiling indigo waste with additives did not alter the pH of the rumen. This agreed with earlier studies that found that adding M, C, and calcium oxide to silage did not alter rumen pH [
29,
30,
34]. After feeding, the rumen’s concentration of NH
3-N increased [
35,
36]. Rumen microbes convert the majority of dietary CP to NH
3-N, the primary source of nitrogen for microbial protein synthesis [
37,
38]. The amount of NH
3-N at 0 and 4 h after feeding in all treatments ranged from 22.2 to 23.4 mg/dL and from 25.7 to 26.9 mg/dL, respectively. This is the optimal concentration (8.5–30 mg/dL) for the growth of rumen microbes [
39,
40]. The additives in fermented indigo waste silage did not influence the levels of NH
3-N. In contrast, our previous research found a lower NH
3-N when adding CH and C to indigo waste silage in an in vitro study [
6]. This different result could be attributed to the use of different techniques (in vitro and in vivo), which could potentially affect the rumen fermentation patterns.
The rumen’s VFAs are considered to be a major energy source for ruminants, contributing mostly 70–80% of their requirement for energy [
41,
42]. The concentrations of VFAs are greatly influenced by the sampling time [
43]. Before feeding, the levels of total VFA in the cattle were lower than after feeding in our study. In addition, the content of total VFA was not affected by silage additives. In contrast, the C- and M-treated silage increased the total VFA concentration in an in vitro study [
44,
45]. Our prior in vitro research found that the amount of rumen total VFA was lowest in group C, while it was highest in group M of indigo waste silage [
6]. In a recent in vitro study, forage was ensiled with additives, whereas the present study used indigo waste silage with additives at 20% in the FTMR fed to cattle, which may have a different effect on the total production of VFA in the rumen. The proportion of VFA in the rumen is impacted by additives, or types of forage [
46,
47]. Previous investigations reported that adding M with C and CH to silage reduced the proportion of C2, while increasing C3 in the rumen of cattle [
14,
29]. Gunun et al. [
6] reported that the use of M and C to ensile indigo waste led to higher levels of C3 and C4, but lower levels of C2 and C2:C3 in an in vitro study. Contrary to this study, the VFA production in the rumen did not change in cattle fed all additive-treated indigo waste silage. However, the addition of M at 4 h after feeding resulted in an increase in C2:C3 levels. These could be caused by the addition of M to silage, which raised the proportion of C2 while decreasing the C3; nevertheless, the change was not statistically significant, leading to greater levels of C2:C3 in the rumen of the cattle.
The rumen’s C3 has a correlation with the ruminant’s glucose levels. Rumen microbes produce C3, a fermentation substrate of carbohydrate, which is a major substrate for glucose synthesis in the liver [
48]. The additions to the indigo waste silage had no effect on C3, which in turn did not alter the glucose concentration of the cattle. The addition of C-treated silage increased the total protein in the cattle’s blood. This result is probably due to ensiled C groups increasing protein utilization in cattle, which led to increased protein in the blood. However, this mechanism is unclear because the addition of C groups did not affect the chemical composition of silage, digestibility, or NH
3-N concentration. The concentration of total protein in all treatments ranged from 6.4 to 6.6 g/dL, which was within the normal range (5.7 to 8.1 g/dL) for cattle [
49].
Hematological analysis is commonly used to examine the health and nutrition as well as the source of abnormalities or malfunctions in cattle [
50]. When silage was fermented with additives, the hematological parameters (hemoglobin, hematocrit, WBCs, neutrophils, monocytes, and eosinophils) remained unchanged. The amounts of hemoglobin, WBCs [
50], hematocrit [
51], neutrophils, monocytes [
52], and eosinophils [
53] in ruminant blood are within the usual range, according to previous research. Lymphocytes are responsible for the incredible specificity of adaptive immune responses [
54,
55]. In the present study, using M- or C-treated silage resulted in lower lymphocyte concentrations. This result is possible because M and C improved the quality of the silage by increasing the lactic acid bacteria, leading to higher levels of lactic acid and a lower pH [
6], which in turn inhibited the growth of pathogens. This may cause a decrease in lymphocytes in the cattle’s blood, indicating improved health. Moreover, beef cattle had a normal lymphocyte concentration (43.5–57.8%) in comparison to prior studies (43.2–66.3%) [
52,
56].