3.1. Chemical Contents in the Diets
Table 1 shows the chemical content of the diets in experiment. Fermented total mixed ration contained CP, NDF, and ADF at 9.6% to 13.1.0% DM, 38.0% to 38.1.0% DM, and 24.1.0% to 24.2.0% DM, respectively. Fermented total mixed ration supplemented with 1.25% urea had a lower pH compared to that supplemented with 2.5% urea. Because ensiling time can provide soluble urea to ammonia-N, it was in the suitable range of 4.91–5.38 in FTMR. The pH affected the extent of preservation allowing nutrient content to be maintained at a high level for long periods of time. The HCN concentrations reduced 99.3% to 99.4% compared with fresh cassava root when FTMR was supplemented with 1.0% and 2.0% sulfur, respectively. Thus, HCN levels in FTMR were not dangerous for feeding animal. These levels of HCN concentration in FTMR are non-toxic for dairy cows. Larson [
4] indicated that HCN concentrations for cattle over 600 ppm are toxic, and at 1000 ppm, death is usually caused. This could be due to ensiling time that can provide high temperature and acidic conditions, thus detoxifying the HCN in FTMR [
25].
In addition, Kimaryo and Massawe [
26] found that during the fermentation process, microorganisms can also interact directly with inactive laminarase enzymes and convert toxins into organic acids. It has been reported that when fresh cassava root was ensiled for 5 days, the HCN decreased from 176.3 to 8.2 ppm. Boonnop et al. [
27] tested different fresh cassava products and showed that in the fermented product HCN reduced by 99.0% after 6 days of fermentation. The mechanism for ensiling microorganism utilizes glucose in organic acids that causes the pH to drop. Laminarase enzymes can break down activity at a lower pH to degrade linamarine to HCN, thereby reducing toxic effects [
28]. In agreement with the present study, Supapong et al. [
12] found that beef cattle fed a diet containing 1.0% to 2.0% sulfur experienced reduced HCN concentrations by 99.3% to 99.5%.
3.2. Nutrient Intake and Digestibility
The DM intake and feed digestion of animals that consumed FTMR are presented in
Table 2. The total FTMR intakes were different ranging from 118.9 to 139.1 g/kg BW
0.75. McSweeney and Denman [
29] reported sulfate-supplemented animals had significantly higher (29.6%) DM intake compared with the unsupplemented group, which might have influenced by increasing in the number of rumen microbes. This is likely due to the relationship of urea and fresh cassava root, which are highly fermentable in the diet. Cows are fed ad libitum, resulting in straight fermentation and sufficient nitrogen to promote fermentation in rumen [
30]. In addition, intake of CP was increased based on urea level addition (
p < 0.05). This could provide sufficient substrate for improved digestibility coefficients and lead essential amino acids to microbial protein synthesis [
7]. There was no difference in estimated energy intake between sulfur levels and urea levels on all parameters. The digestion of DM, OM, and CP when 2.0% sulfur and 2.5% urea was added to the FTMR was significantly higher (
p < 0.05). This might be because increasing concentrations of dietary sulfur and urea may also impact rumen motility and bacterial digestibility of the diet (
Table 3). Promkot et al. [
31] indicated that cows exhibited increased fiber digestibility when fed a diet with 0.4% over those with 0.2% sulfur in their diet. Furthermore, Cherdthong et al. [
2] found that cattle consuming fresh cassava root in the basal diet with feed block at 4.0% sulfur levels showed improved DM and OM digestibility by 13.0% and 12.1%, respectively. An earlier report by Supapong et al. [
12], showed that beef cattle fed a diet supplemented with 2.0% sulfur in the FTMR exhibited increased DM digestibility by 4.2% compared to those fed with a diet supplemented with 1.0% sulfur.
3.3. Rumen Characteristics and Blood Profiles
Rumen parameters, ruminal pH and temperature of the FTMR groups, ranged from 6.5 to 6.6 and 39.0 to 39.4 °C, respectively (
Table 3). It is well recognized to be in the range considered suitable for bacteria breakdown of fiber and activity to ferment the feed in the rumen [
32]. There was no interactions between levels of sulfur and urea on the NH
3-N concentration (
p > 0.05), whereas ruminal NH
3-N concentration was higher in FTMR supplemented with 2.5% urea levels (
p < 0.01). Because urea results in the rapid degradation to ammonia by urease activity, which is excreted from rumen microbes. Ammonia-nitrogen in the rumen can occur at a much faster rate depending greatly on the availability of energy and protein in the rumen. The balance in the overall daily ratio of rumen with available carbohydrate and nitrogen in the diet may improve microbial protein production and utilization [
33]. Inclusion of a high level of urea with the supplement of an extremely fermentable carbohydrate source has improved dairy cows’ milk production [
34].
Blood urea-nitrogen was higher when FTMR was supplemented with 2.5% urea 4 h after morning feeding (
p < 0.05), indicating that after feeding, effective urea may be degraded by bacteria into NH
3-N and absorbed into the blood. In the present experiment, BUN concentration in dairy cows was not affected by sulfur (
p > 0.05) but was always within the normal range for protein utilization. Blood thiocyanate concentration was increased by 21.6% when sulfur was supplemented at 2.0% compared to 1.0% (
p < 0.05). National Research Council (NRC) [
35] reported that the ruminant diets high in HCN level need sulfur supplementation to activate sulfur for expulsion of HCN into the thiocyanate form by rhodanese and β-mercaptopyruvate-sulfur transferase [
25]. This finding was supported by the findings of Uwituze et al. [
36] in sheep that were fed a cassava-based diet and exhibited a blood thiocyanate concentration directly proportional to the level of sulfur supplementation. In addition, Promkot et al. [
31] added 0.5% sulfur to DM with fresh cassava foliage for beef cattle, and the findings suggested improved HCN detoxification.
There was no difference in protozoal concentration, whereas bacterial populations at 4 h after feeding were significantly greater by 6.1% with the FTMR supplemented with 2.0% sulfur and 2.5% urea (
p < 0.01) (
Table 3). In ruminants, ruminal microbes change sulphate to hydrogen sulphite, which is supplied to produce methionine and cysteine to optimize cell synthesis and maintenance for microbial synthesis. Sulfur is mainly needed to maintain maximal rumen microbial growth. Thus, the microbial population might have increased further with consistent availability of nitrogen and sulfur for fermentation in the rumen. This relationship has been investigated in in vitro studies comparing the effect of various sources of sulfur on the bacterial synthesis, the utilization of NH
3-N, and nutrient digestion over a 96-h incubation period [
37]. It can also be noted that sulfur addition increases the performance of microbial protein synthesis to production of MCP and improves the amino acid balance [
38].
3.4. Nitrogen Balance and Purine Derivatives
The data for nitrogen balance and purine derivatives in animals treated with the FTMR are shown in
Table 4. There were differences in nitrogen intake and nitrogen absorption between the two levels of urea in the FTMR (
p < 0.05). The total nitrogen intake was highest in 2.5% urea treatments for all dairy cows. One possible explanation for this is the lack of difference in total DM intake, which showed a difference in total nitrogen intake. Allantoin concentrations, excretion, absorption, and MCP showed significant interactions between sulfur levels and urea levels in cows fed diets supplemented with 2.0% sulfur and 2.5% urea (
p < 0.05). This may be because with urea and sulfur, there has been an apparent improvement in microbial protein synthesis associated with carbohydrate from fresh cassava root, as well as digestibility of the diet. Synchronizing the rate of supply of protein and energy sources to ruminal microbes has been conducted to maximize the capture of rumen degradable protein and to optimize efficiency of microbial protein synthesis. Both energy and protein sources should be continuously used to improve microorganism synthesis and enhance feed efficiency [
39]. The microbes in rumen can benefit inorganic forms of sulfur, cooperating with NH
4-N and carbon skeleton to form amino acids and microbial proteins. Another measure of the effect of sulfur on the activity of ruminant microorganism is the ability to synthesize protein from urea.
3.5. Volatile Fatty Acid (VFA) Concentration in the Rumen
The molar ratios of the VFA profile were affected by dietary FTMR (
p < 0.01) (
Table 5). Mean values of total VFA, acetic acid, propionic acid, and butyric acid were 107.1 to 120.1 m
M, 60.2 to 64.9, 24.5 to 28.1, and 10.5 to 11.7 mol/100 mol, respectively. Furthermore, these values correlated with the nutrient digestibility when 2.0% sulfur and 2.5% urea was supplemented. Volatile fatty acid production interacted with sulfur and urea level (
p < 0.01). Volatile fatty acids produced in the rumen are utilized by the animal as a main source of energy, and their increased production in the rumen is a measure of an increase in the efficiency of feed utilization by the animal. Sulfur in the rumen can be absorbed as sulphide (S
2−) but also by outflow as undegraded protein sulfur or bacterial protein. Portion of degradable sulfur depends on such factors as supply of degradable nitrogen, rate of sulfur degradation by rumen microbes, and the ratio of arrival of readily fermentable energy, which affects ruminal pH and hence S
2− absorption [
40]. Greater degradability might be due to the better activity of microbial stimulate efficiently hydrolyzed to NH
3 by urea. This shows that the NH
3 increased nutrient imbalances for ruminal microorganisms by enhancing the availability of carbohydrate from fresh cassava root and its ability to change into VFA production.
Different microorganisms use elemental sulfur as an electron acceptor; therefore, the reduced forms of sulfur (sulfite and sulfide) are a metabolic end product of fermentation from these microorganisms [
41]. This could be because the non-protein nitrogen utilization is increased by high levels of sulfur in the rumen fluid, indicating microbial growth [
5]. Furthermore, propionic acid increased by 4.6% when diets were supplemented by 2.5% sulfur (
p < 0.01). Animals supplemented with a high amount of sulfur exhibited increased ruminal propionate concentration because propionic acid could be supplied as a sink of hydrogen sulfide when increasing ruminal available sulfur is offered [
42]. Similarly, Supapong and Cherdthong [
37] reported that propionic acid increased by 10.9% after supplementation of 2.0% sulfur compared to an absence of sulfur in TMR containing fresh cassava root. In addition, Promkot et al. [
31] studied 1.0% sulfur supplementation in fresh cassava foliage and noted that the concentration of propionate and microbial protein synthesis were enhanced in the rumen of sulfur-supplemented cattle.
3.6. Milk Production, Composition, Somatic Cells and Thiocyanate Concentration
Improvement in milk yield was not significant when dairy cows were fed fresh cassava root at 40% in an FTMR diet (
p > 0.05) containing sulfur and urea (
Table 6). The milk yield of cows when the level of supplementation with sulfur and urea was not significant ranged from 12.4 to 13.0 kg/day (
p > 0.05). However, these numerical differences could be linked to the numerical feed intake differences shown in
Table 2.
Milk fat and total solids increased when feed was supplemented with 2.0% sulfur and 2.5% urea (
p < 0.05). Efficiency of nutrient digestibility increased when absorbed as glucose rather than when nutrients utilized by rumen microbes and the propionate converted to glucose in the liver [
43]. This enhances the proportion of amino acids and glucose relative to that of acetate and long chain fatty acids in the circulation, resulting in enhanced production of protein, lactose, and to a lesser degree, fat in the mammary gland [
44,
45,
46]. Knika and Zmiev [
45] observed that supplementation of dairy cow rations with 30 g of sodium sulphate per day for 30 days increased cellulose digestibility by 13%. Dageaw et al. [
46] found that as a result of the sulfur treatment, the production of milk solids, fat, protein, and casein increased in dairy cows fed a diet supplemented with 1.5% BW fresh cassava root combined with 4.0% feed block containing high sulfur and increased milk fat by 8.6%. This is consistent with the result that milk fat increases by 3.7% when the diet is supplemented with 2.0% sulfur and 2.5% urea. High propionic acid using the substrate to synthesize glucose in gluconeogenesis by the pentose phosphate pathway produces NADPH, which is a factor in the synthesis of fatty acids [
47,
48,
49]. Feed high in NDF is related to an improved production rate of lipogenic to glucogenic VFA, with the alteration in the proportion of VFA resulting in an enhanced concentration of milk fat [
1,
50,
51,
52].
In this study, the HCN result was critical differentiation in the milk thiocyanate concentration which is related to the levels of sulfur fortified in feed (
Table 6). The diets supplemented with 2.0% sulfur levels resulted in greater concentrations of milk thiocyanate (
p < 0.05); however, the result was not significantly different based on urea levels (
p > 0.05). In the present data, milk thiocyanate ranged from 5.02 to 11.87 ppm. The advantage of a significant dose of HCN in fresh cassava root is to change the thiocyanate through the elimination of feed HCN by changing it to thiocyanate in the liver and kidney of cows by the rhodanese enzyme action and partly through detoxification in the milk [
11], which was observed in milk thiocyanate increases. Srisaikham et al. [
48] reported that effects on milk thiocyanate in the case of dairy cows were also positively correlated in a regression model between milk thiocyanate and HCN in dietary fresh cassava peel results.
The present experiment was conducted in the Research Center of Institute, which controlled external factors such as dirty environment, substrates, or health factors such as pre-clinical mastitis. None of these variables were investigated for in the study; thus, they have not impacted somatic cell count results. Thus, we could be confident that the somatic cell count results were influenced by the treatment study. The decrease in somatic cell count was possibly because the rhodanese enzyme in the liver contains sulfur and transformed HCN into the blood thiocyanate, which is transferred to the milk, saliva, and urine [
11]. The lactoperoxidase system was affected in the antimicrobial function, activated by milk thiocyanate in the milk, to limit the increase in somatic cell count. This effect was due to the restraining the activity of various cytoplasmic enzymes to damage the cell bacterial membranes and reduce growth [
9,
51,
52]. These characteristics enable milk thiocyanate and somatic cell count in the milk and can be activated by lactoperoxidase as an indicator of mastitis, which has been previously elucidated by Isobe et al. [
49]. Thus, milk thiocyanate from HCN can be used to prolong the shelf life of raw milk stored at room temperature by inhibiting microbial growth [
48].