Supplementation of 5,6-Dimethylbenzimidazole and Cobalt in High-Concentrate Diet Improves the Ruminal Vitamin B₁₂ Synthesis and Fermentation of Sheep

Abstract

The objective of this experiment was to investigate the effects of 5,6-dimethylbenzimidazole (5,6-DMB) and cobalt (Co) on the ruminal vitamin B₁₂ synthesis and fermentation parameters of sheep under high concentrate conditions. Twenty-four Kazakh rams (body weight = 39.23 ± 2.61 kg and 8 months old) fitted with permanent ruminal fistulas were randomly divided into four groups with six rams in each group. The control (CON) group was fed a basal ration with a concentrate-to-roughage ratio of 70:30, and the experimental groups (T60, T75 and T90) were fed a basal diet with 60 mg 5,6-DMB+ 0.25 mg Co, 75 mg 5,6-DMB+ 0.5 mg Co and 90 mg 5,6-DMB+ 0.75 mg Co supplied to each kilogram of basal diet, respectively. The experiment lasted for 26 days, with the first 14 days being an adaptation period to allow the sheep to adapt to the diet type and environment, and the second 12 days being a sample period. On 0, 7 and 12 d of the sample period, all sheep were weighed before the morning feed. Rumen fluid samples were collected from all sheep on the last 4 days of the sample period. The results showed that the ruminal vitamin B₁₂ content was higher in trial groups than that of the CON group at 3 h after feeding (p < 0.05). Rumen pH was higher in trial groups than in the CON group at 1, 3 and 5 h after feeding (p < 0.05). The concentration of ruminal ammonia-N was significantly increased in trial groups when compared to the CON group (p < 0.05) at 1 and 3 h after feeding. At 1 and 3 h after feeding, the concentration of propionate in trial groups was higher than that in the CON group (p < 0.05). The rumen microbial protein content reached the highest value at 3 h after feeding, and all trial groups were higher than the CON group (p < 0.05). Thus, the supplementation of 5,6-DMB and Co increased vitamin B₁₂, propionate, ammonia-N and microbial protein contents and pH in the rumen of sheep, and the best results were obtained by the amounts of 75 mg/kg 5,6-DMB and 0.5 mg/kg Co, respectively.

1. Introduction

Vitamin B₁₂ is a peculiar octahedral-like natural compound containing cobalt ions, with the central 4 pyrroles attached to the central metal cobalt ion with 4 N atoms to form a planar coacervate ring. The 5,6-dimethylbenzimidazole (5,6-DMB) attached to the cobalt ion with N-7 atoms to be the lower ligand of the VB₁₂ molecule, the adenosine or methyl group is linked to the cobalt ion to form the upper ligand of the VB₁₂ molecule, with nine asymmetric carbon atoms in the parent nucleus [1], cobalt (Co) is an essential trace element for ruminants and a necessary active ingredient in the structure of vitamin B₁₂ [2].

Vitamin B₁₂ plays an important role in energy metabolism of ruminants [3]. Carbohydrates in the ration are degraded into short-chain volatile fatty acids (VFA) such as acetate, propionate and butyrate by rumen microorganisms, and VFA is the main energy source for ruminants, which can satisfy 70–80% of the total energy requirement of organism [4]. Microbial fermentation of carbohydrates in the rumen is dominated by the production of propionate under high concentrate ration conditions. In the ruminal epithelial cells, approximately 3% of propionate that is produced by the organism is converted to lactate, most of which is transported via the portal vein to the liver for gluconeogenesis or into the tricarboxylic acid cycle for oxidative energy supply. Vitamin B₁₂ is a coenzyme of methylmalonyl coenzyme A isomerase in propionate during its entry into the tricarboxylic acid cycle for energy supply via propionyl coenzyme A, methylmalonyl coenzyme A, and succinyl coenzyme A. Generally, ruminants are able to synthesize vitamin B₁₂ through rumen microorganisms to satisfy their own growth and metabolic needs [5], but the amount of vitamin B₁₂ synthesized in the rumen under high-concentrate conditions cannot meet the requirements of high-yielding dairy cows’ and fattening goats’ organisms. Therefore, exogenous supplementation is required to improve the rumen metabolism of ruminants [6]. Stemme et al. [7] results showed that the addition of Co to the ration increased the vitamin B₁₂ synthesis in the rumen of dairy cows. Wang et al. [8] found that the synthesis of vitamin B₁₂ in the rumen of sheep increased with elevated Co levels, and the increase in vitamin B₁₂ synthesis slowed down at Co supplementation levels above 0.50 mg/kg. In addition, 5,6-dimethylbenzimidazole (5,6-DMB) is an important precursor substance for the synthesis of vitamin B₁₂, which is also important for the synthesis of vitamin B₁₂ to satisfy the body’s needs, and a deficiency of 5,6-DMB leads to impairment of vitamin B₁₂ synthesis [9]. Vitamin B₁₂ synthesis in the rumen increased by 7.4 mg/d following the supplementation of 1.5 g/d of 5,6-DMB to the diet of dairy cows [10]. A previous study has shown that supplementation of 5,6-DMB and Co in the high concentrate rations increased dry matter intake (DMI) and average daily weight gain of sheep, and promoted growth performance and digestive metabolism of the animals [11].

In order to achieve the purpose of rapid growth and fattening of sheep, high-concentrate rations (concentrate supplements at 60% or more, with a composition dominated by energy and protein feeds) are often used in practical production under modern large-scale sheep farming. In this experiment, it is proposed to regulate the synthesis of vitamin B₁₂ by rumen microorganisms through dietary supplementation of 5,6-DMB and Co from the point of view of synthesizing vitamin B₁₂ precursor substances 5,6-DMB and Co, so as to improve the energy utilization of sheep organisms, and to further increase the degree of energy supply of the tricarboxylic acid cycle. The aim of this experiment was to study the effects of supplementing 5,6-DMB and Co on rumen fermentation and vitamin B₁₂ synthesis in sheep under the condition of basal ration with concentrate-to-roughage ratio of 70:30, so as to provide a theoretical basis for the application of a 5,6-DMB and Co in the actual production of animals.

2. Materials and Methods

2.1. Experimental Design

This study was conducted at Hui Kang livestock breeding farm (Changji, Xinjiang, China). Twenty-four Kazakh rams (body weight = 39.23 ± 2.61 kg and 8 months old) fitted with permanent rumen fistulas were randomly divided into four groups with six rams in each group. The control (CON) group with no Co (cobalt supplemented as cobalt chloride, zeolite powder as a carrier, Yuanda Zhongzheng Biotechnology Co., Ltd., Shijiazhuang, Hebei, China; with cobalt (Co) ≥ 1%) and 5,6-DMB (Yuanye Biotechnology Co., Ltd., Shanghai, China; analytically pure ≥ 99%). CON group was fed a basal ration with a concentrate-to-roughage ratio of 70:30, and the experimental groups (T60, T75 and T90) were fed a basal diet with 60 mg 5,6-DMB+ 0.25 mg Co, 75 mg 5,6-DMB+ 0.5 mg Co, and 90 mg 5,6-DMB+ 0.75 mg Co supplied to each kilogram of basal diet, respectively. The experiment lasted for 26 days, with the first 14 days being an adaptation period to allow the sheep to adapt to the diet type and environment, and the second 12 days being a sample period. On d 0, 7 and 12 of the sample period, all trial sheep were weighed before morning feeding (07:30). Rumen fluid samples were collected from all trial sheep on the last 4 days of the sample period.

The base ration for the experiment was formulated according to the meat-type sheep feeding standard (NY/T-816,2021) and its compositions and nutritional levels are shown in

Table 1. Compositions and nutrient levels of basal diets (DM basis) %.

Items	Content	Nutrient Levels 2	Content
Ingredients		DM	91.33
Corn stalk	21.00	CP	17.13
Alfalfa	9.00	EE	1.91
Corn	35.00	CEL	13.59
Wheat bran	8.40	HC	9.71
Soybean meal	14.00	Lignin	3.31
Cottonseed meal	9.10	Ca	1.02
Premix 1	3.50	P	0.55
Total	100.00	ME (MJ/kg)	14.12

ME= metabolic energy; DM = dry matter; CP = crude protein; EE = ether extract; CEL = cellulose; HC = hemicellulose. ¹ The premix provided the following per kg of the diet: VA 7000 IU; VD₃ 1785 IU; VE 14 IU; nicotinic acid 14 mg; biotin 0.04 mg; Cu (as copper sulfate) 8.8 mg; Fe (as ferrous sulfate) 26.32 mg; Mn (as manganese sulfate) 29.14 mg; Zn (as zinc sulfate) 35.53 mg; I (as potassium iodide) 0.57 mg; Se (as sodium selenite) 0.23 mg. ² ME was a calculated value, while the others were measured values.

.

2.2. Feeding and Management

The experimental animals were housed in a single pen with separate feed and water buckets for each sheep and were fed twice daily at 07:30 and 19:30, respectively. The daily feed was weighed in advance, divided equally into two portions and bagged separately. The supplementation was mixed with 25 g of concentrate supplement feed before feeding to ensure that all the trial sheep were fed, and then the concentrate was fed before the roughage. During the adaptation period, concentrate and roughage were mixed tighter twice a day and a high surplus of concentrate appeared, then the feeding method was changed to roughage followed by concentrate, which gave better results. Daily feeds were increased or decreased according to the previous day’s intake in order to keep daily leftovers to 2–4% of the feed. Feed intake and leftovers were weighed daily, and feed intake was calculated from feed offered and leftovers. During the adaptation and sample period, experiment sheep were ensured to feed and water ad libitum.

2.3. Sample Collection

The sampling period was 4 days and rumen fluid samples were collected from 6 sheep per day (2 sheep per group), with a total of 24 sheep in 4 days. The samples were collected at 0 h before feeding and 1, 3, 5 and 7 h after feeding. The 60–80 mL of rumen fluid was collected at each time point with a 100 mL plastic beaker and filtered through 4 layers of cheesecloth. The pH was measured immediately and then divided into numbered 10 mL lyophilized tubes and stored frozen at −20 °C in the refrigerator.

2.4. Mesurement of Index

2.4.1. Measurement of Growth Performance

The sampling period was 12 days and all trial sheep were weighed in the morning (7:30) before feeding on days 0, 7 and 12 d of the sampling period.

2.4.2. Mesurement of Vitamin B₁₂ Content

The content of vitamin B₁₂ in rumen fluid was determined by an enzyme-linked immunoassay kit, which was purchased from Jiangsu Jingmei Biotechnology Co. (Yancheng, China).

2.4.3. Measurement of Rumen Fermentation Parameters

Ruminal fluid pH was determined using a pH meter (Anscitech Co., Ltd., Wuhan, Hubei, China) immediately after each collection; ammonia-N (NH₃-N) concentration was determined using the indophenol blue colorimetric method [12]; and VFA were determined using a Shimadzu GC 2010 gas chromatograph in Japan with the following indicators: acetate, propionate, butyrate, isobutyrate, valerate and isovalerate, with 4-methylvaleric acid as an internal standard [13]. The microbial protein (MCP) content in rumen fluid was determined by referring to the determination method of Gao [14]. The concentrations of L-lactate and D-lactate in rumen fluid were detected by spectrophotometry and enzyme-linked immunosorbent assay, respectively, with the purchase of kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.5. Statistical Analysis

Data were initially collated using Excel 2020, and statistical analysis was performed using one-way ANOVA in SPSS 26.0 software (Version 20.0 for Windows; SPSS, Chicago, IL, USA). Multiple comparisons were performed using Duncan’s method, and the results were expressed as means, and the degree of variation of each group was expressed as standard error of variation (SEM). p < 0.05 indicates a significant difference, and p > 0.05 indicates the difference was not significant. The correlation analysis of growth performance, rumen fermentation parameters and vitamin B₁₂ was performed using Spearman’s correlation test (r = 0.6) implemented in Origin 2021.

3. Results

3.1. Effect of the Supplementation of 5,6-Dimethylbenzimidazole and Cobalt on Growth Performance of Sheep

As shown in Figure 1, DMI and ADG were significantly higher in trial groups than the CON group (p < 0.05), among which the T75 was higher than that of T60 and T90 (p < 0.05). The feed/gain (F/G) was higher in the CON group (p < 0.05), and that of the T60 and T90 groups was higher than that of the T75 group (p < 0.05).

3.2. Effect of the Supplementation of 5,6-Dimethylbenzimidazole and Cobalt on the Vitamin B₁₂ Content of Sheep Rumen

As shown in

Table 2. Effect of the supplementation of 5,6-dimethylbenzimidazole and cobalt on the vitamin B₁₂ content of sheep rumen (n = 6) (nmol/L).

Sampling Time	Control Group	T60 Group	T75 Group	T90 Group	SEM	p-Value
0 h before feeding	40.72	41.32	40.73	41.23	0.51	0.967
1 h after feeding	40.11	41.77	41.95	41.48	0.58	0.697
3 h after feeding	39.68 b	43.40 a	44.19 a	43.46 a	0.50	0.001
5 h after feeding	39.33	42.29	43.31	42.58	0.59	0.073
7 h after feeding	39.17	40.79	42.17	42.07	0.48	0.087

T60 group = trial 60 mg/kg 5,6-DMB+ 0.25 mg/kg Co; T75 group = trial 75 mg/kg 5,6-DMB+ 0.5 mg/kg Co; T90 group= trial 90 mg/kg 5,6-DMB+ 0.75 mg/kg Co; SEM = standard error of mean. Mean values with different letters were significantly different (p < 0.05) according to Duncan’s multiple range test.

, the vitamin B₁₂ content in the rumen was higher in the trial groups at 3 h after feeding (p < 0.05), and the difference between the trial groups was not significant (p > 0.05).

3.3. Effect of the Supplementation of 5,6-Dimethylbenzimidazole and Cobalt on Ruminal pH in Sheep

As shown in

Table 3. Effect of the supplementation of 5,6-DMB and Co on ruminal pH in sheep (n = 6).

Sampling Time	Control Group	T60 Group	T75 Group	T90 Group	SEM	p-Value
0 h before feeding	6.45	6.43	6.49	6.44	0.02	0.852
1 h after feeding	5.81 b	5.98 a	6.03 a	6.01 a	0.03	0.001
3 h after feeding	5.55 b	5.79 a	5.80 a	5.75 a	0.03	0.001
5 h after feeding	5.77 c	5.91 b	6.06 a	5.92 b	0.03	0.002
7 h after feeding	5.96 c	6.09 b	6.20 a	6.08 b	0.02	<0.001

T60 group = trial 60 mg/kg 5,6-DMB+ 0.25 mg/kg Co; T75 group = trial 75 mg/kg 5,6-DMB+ 0.5 mg/kg Co; T90 group = trial 90 mg/kg 5,6-DMB+ 0.75 mg/kg Co; SEM = standard error of mean. Mean values with different letters were significantly different (p < 0.05) according to Duncan’s multiple range test.

, at 1 and 3 h after feeding, the pH was greater in the trial groups (p < 0.05), while no significant difference was found among the trial groups (p > 0.05). At 5 and 7 h after feeding, the pH was higher in the T75 group (p < 0.05), and higher than that of the T60 and T90 groups (p < 0.05), and trial T60 and T90 groups were higher than the CON group (p < 0.05).

3.4. Effect of the Supplementation of 5,6-Dimethylbenzimidazole and Cobalt on Ruminal NH₃-N Concentration in Sheep

As shown in

Table 4. Effect of the supplementation of 5,6-DMB and Co on ruminal NH₃-N concentrations in sheep (n = 6) (mg/100 mL).

Sampling Time	Control Group	T60 Group	T75 Group	T90 Group	SEM	p-Value
0 h before feeding	18.81	19.94	19.50	18.93	0.70	0.943
1 h after feeding	22.26 c	26.14 b	29.84 a	26.07 b	0.74	<0.001
3 h after feeding	16.59 c	19.74 b	23.70 a	19.15 b	0.59	<0.001
5 h after feeding	14.14 b	16.70 ab	17.67 a	16.26 ab	0.34	0.052
7 h after feeding	12.56 b	13.74 ab	16.31 a	13.35 ab	0.60	0.135

T60 group = trial 60 mg/kg 5,6-DMB+ 0.25 mg/kg Co; T75 group = trial 75 mg/kg 5,6-DMB+ 0.5 mg/kg Co; T90 group = trial 90 mg/kg 5,6-DMB+ 0.75 mg/kg Co; SEM = standard error of mean. Mean values with different letters were significantly different (p < 0.05) according to Duncan’s multiple range test.

, the concentration of NH₃-N in the trial groups was greater than that in the CON group at 1 and 3 h after feeding (p < 0.05), with the NH₃-N concentration in the T75 group being higher than in the T60 and T90 groups (p < 0.05). At 5 and 7 h after feeding, the NH₃-N concentration in the T75 group was higher than that in the CON group (p < 0.05).

3.5. Effect of the Supplementation of 5,6-Dimethylbenzimidazole and Cobalt on Ruminal VFA Concentrations in Sheep

As shown in

Table 5. Effect of the supplementation of 5,6-DMB and Co on ruminal VFA concentrations in sheep (n = 6) (mmol/L).

Items	Sampling Time	Control Group	T60 Group	T75 Group	T90 Group	SEM	p-Value
Acetate	0 h before feeding	59.14	59.43	58.55	57.04	0.85	0.784
	1 h after feeding	66.09	68.71	70.43	69.59	1.25	0.663
	3 h after feeding	74.40	76.82	81.12	75.91	1.43	0.407
	5 h after feeding	67.70	72.80	74.54	69.55	1.77	0.542
	7 h after feeding	63.70	66.84	68.20	66.16	1.15	0.597
Propionate	0 h before feeding	15.89	16.48	16.50	16.83	0.17	0.264
	1 h after feeding	19.15 c	21.74 b	25.48 a	22.16 b	0.52	<0.001
	3 h after feeding	24.37 c	27.09 b	30.48 a	27.56 b	0.49	<0.001
	5 h after feeding	22.41 b	23.45 ab	24.56 a	23.68 a	0.24	0.007
	7 h after feeding	19.77	19.95	20.59	20.66	0.27	0.589
Isobutyrate	0 h before feeding	1.39	1.36	1.45	1.24	0.08	0.861
	1 h after feeding	1.40	1.31	1.27	1.34	0.08	0.960
	3 h after feeding	1.48	1.31	1.36	1.46	0.07	0.825
	5 h after feeding	1.68	1.49	1.32	1.36	0.06	0.169
	7 h after feeding	1.71	1.53	1.45	1.42	0.05	0.203
Butyrate	0 h before feeding	15.95	18.11	16.79	15.04	0.57	0.285
	1 h after feeding	10.89	11.48	9.99	10.19	0.37	0.500
	3 h after feeding	9.13	8.25	9.06	9.66	0.29	0.413
	5 h after feeding	9.31	9.21	8.25	8.64	0.33	0.656
	7 h after feeding	12.43	11.25	10.59	10.70	0.27	0.054
Isovalerate	0 h before feeding	2.44	3.00	2.72	2.32	0.14	0.301
	1 h after feeding	1.49	1.42	1.30	1.40	0.05	0.684
	3 h after feeding	1.24	1.24	1.24	1.42	0.05	0.419
	5 h after feeding	1.39	1.39	1.31	1.28	0.06	0.859
	7 h after feeding	1.94	1.51	1.62	1.48	0.08	0.177
Valerate	0 h before feeding	2.14	2.36	2.08	1.77	0.16	0.658
	1 h after feeding	1.70	1.78	1.54	1.66	0.05	0.442
	3 h after feeding	1.59	1.67	1.41	1.59	0.05	0.400
	5 h after feeding	1.65	1.52	1.25	1.35	0.07	0.119
	7 h after feeding	1.69	1.53	1.40	1.40	0.09	0.469
Acetate/propionate	0 h before feeding	3.73	3.61	3.55	3.39	0.07	0.353
	1 h after feeding	3.45 a	3.16 ab	2.76 b	3.16 ab	0.08	0.013
	3 h after feeding	3.05	2.84	2.66	2.75	0.06	0.077
	5 h after feeding	3.02	3.10	3.04	2.94	0.07	0.894
	7 h after feeding	3.23	3.35	3.31	3.20	0.06	0.872
T-VFA	0 h before feeding	96.95	100.74	98.09	94.24	1.26	0.347
	1 h after feeding	100.72	106.44	110.01	106.34	1.55	0.207
	3 h after feeding	112.21	116.38	124.67	117.60	1.77	0.081
	5 h after feeding	104.14	109.86	111.23	105.86	1.92	0.551
	7 h after feeding	101.24	102.61	103.85	101.82	1.29	0.911

T60 group = trial 60 mg/kg 5,6-DMB+ 0.25 mg/kg Co; T75 group = trial 75 mg/kg 5,6-DMB+ 0.5 mg/kg Co; T90 group = trial 90 mg/kg 5,6-DMB+ 0.75 mg/kg Co; SEM = standard error of mean; T-VFA = total volatile fatty acids. Mean values with different letters were significantly different (p < 0.05) according to Duncan’s multiple range test.

, the concentration of propionate in the trial groups was higher than that in the CON group at 1 and 3 h after feeding (p < 0.05), and that in the T75 group was higher than that in the T60 and T90 groups (p < 0.05). At 5 h after feeding, the propionate concentration in the T75 and T90 groups was higher than that in the CON group (p < 0.05). At 1 h after feeding, acetate/propionate was higher in the CON group than in the T75 group (p < 0.05).

3.6. Effect of Supplementation with 5,6-Dimethylbenzimidazole and Cobalt on Ruminal L- and D-Lactate in Sheep

As shown in

Table 6. Effect of the supplementation of 5,6-DMB and Co on ruminal L-lactate, D-lactate concentrations in sheep (n = 6) (μg/mL).

Items	Sampling Time	Control Group	T60 Group	T75 Group	T90 Group	SEM	p-Value
L-lactate	0 h before feeding	31.11	32.20	26.36	28.13	2.08	0.766
	1 h after feeding	45.22	46.04	30.96	31.76	3.13	0.154
	3 h after feeding	62.64	51.97	48.34	43.71	2.68	0.068
	5 h after feeding	75.76	55.38	54.75	56.81	3.50	0.091
D-lactate	0 h before feeding	1.74	1.69	1.32	1.49	0.07	0.155
	1 h after feeding	1.54	1.44	1.03	1.44	0.07	0.106
	3 h after feeding	1.25	1.14	0.96	1.02	0.06	0.272
	5 h after feeding	1.09	0.83	0.95	0.96	0.05	0.255

T60 group = trial 60 mg/kg 5,6-DMB+ 0.25 mg/kg Co; T75 group = trial 75 mg/kg 5,6-DMB+ 0.5 mg/kg Co; T90 group = trial 90 mg/kg 5,6-DMB+ 0.75 mg/kg Co; SEM = standard error of mean.

, at 3 and 5 h after feeding, the concentrations of L-lactate in the trial groups showed a tendency to decrease. There were no significant differences in ruminal D-lactate concentration among all groups at 0 h before feeding, 1, 3 and 5 h after feeding (p > 0.05).

3.7. Effect of the Supplementation of 5,6-Dimethylbenzimidazole and Cobalt on Ruminal MCP Content in Sheep

As shown in

Table 7. Effect of 5,6-DMB and Co supplementation on ruminal MCP content in sheep (n = 6) (μg/mL).

Sampling Time	Control Group	T60 Group	T75 Group	T90 Group	SEM	p-Value
0 h before feeding	116.85	119.97	125.92	124.15	4.51	0.906
1 h after feeding	123.09	137.51	147.79	151.41	7.47	0.567
3 h after feeding	126.78 b	149.07 a	164.54 a	164.26 a	4.01	<0.001
5 h after feeding	109.01	130.88	142.37	146.15	5.37	0.051
7 h after feeding	106.03	120.66	134.13	128.09	4.23	0.094

T60 group = trial 60 mg/kg 5,6-DMB+ 0.25 mg/kg Co; T75 group = trial 75 mg/kg 5,6-DMB+ 0.5 mg/kg Co; T90 group = trial 90 mg/kg 5,6-DMB+ 0.75 mg/kg Co; SEM = standard error of mean. Mean values with different letters were significantly different (p < 0.05) according to Duncan’s multiple range test.

, at 3 h after feeding, the MCP content of the trial groups was higher than that of the control group (p < 0.05). No significant difference was observed among the trial groups (p > 0.05).

3.8. Correlation Analysis of Growth Performance, Vitamin B₁₂ Content and Ruminal Fermentation

As shown in Figure 2, DMI was highly positively correlated with ADG, vitamin B₁₂, pH, NH₃-N, propionate and T-VFA concentration (p < 0.01), and positively correlated with acetate (p < 0.05) and highly negatively correlated with F/G, D-lactate (p < 0.01) and L-lactate (p < 0.05); ADG was highly positively correlated with DMI, vitamin B₁₂, pH, NH₃-N, propionate and T-VFA concentration (p < 0.01), significantly positively correlated with acetate (p < 0.05) and highly negatively correlated with F/G, L-lactate, D-lactate and A/P (p < 0.01); vitamin B₁₂ were highly positively correlated with DMI, ADG, pH and propionate concentration (p < 0.01), whereas with NH₃-N, acetate and T-VFA was positively correlated (p < 0.05), and negatively correlated with F/G, D-lactate (p < 0.01); pH were highly positively correlated with DMI, ADG, vitamin B₁₂, NH₃-N, acetate, propionate and T-VFA concentrations (p < 0.01), and highly negatively correlated with F/G, D-lactate (p < 0.01); NH₃-N concentration were highly positively correlated with DMI, ADG, pH, propionate and T-VFA concentration (p < 0.01), positively correlated with acetate, vitamin B₁₂ (p < 0.05) and highly negatively correlated with F/G, D-lactate (p < 0.01); acetate concentration was highly and positively correlated with pH, A/P and T-VFA (p < 0.01) and with DMI, ADG, vitamin B₁₂ and NH₃-N, propionate concentration (p < 0.05), with D-lactate was negatively correlated (p < 0.05); propionate concentration were highly positively correlated with DMI, ADG, vitamin B₁₂, pH, NH₃-N, T-VFA concentration (p < 0.01) and acetate concentration (p < 0.05), whereas they were negatively correlated with F/G, A/P, L-lactate and D-lactate (p < 0.01); A/P was highly positively correlated with F/G, acetate (p < 0.01) and T-VFA (p < 0.05), with ADG and propionate concentration were negatively correlated (p < 0.01); T-VFA concentrations were highly positively correlated with DMI, ADG, pH, NH₃-N, acetate and propionate concentrations (p < 0.01) and with A/P, vitamin B₁₂ (p < 0.05), whereas they were negatively correlated with D-lactate (p < 0.01), F/G (p < 0.05); L-lactate was highly positively correlated with F/G (p < 0.01), D-lactate (p < 0.05), with ADG, propionate concentration (p < 0.01) and DMI (p < 0.05); D-lactate was highly positively correlated with F/G (p < 0.01) and L-lactate concentration (p < 0.05), whereas they were negatively correlated with DMI, ADG, vitamin B₁₂, pH, NH₃-N, propionate, T-VFA concentration (p < 0.01) and acetate (p < 0.05).

4. Discussion

4.1. Effects of Supplemental 5,6-Dimethylbenzimidazole and Cobalt on Growth Performance of Sheep

Animals consume nutrients by feeding on rations, and feed intake is a parameter of the amount of nutrients consumed. The digestive tract, liver and nervous system are the main endogenous regulatory systems that regulate feed intake. Environmental factors (temperature and humidity, noxious gases, light, sound, etc.), feed type (ratio of concentrate to roughage, grain size, etc.), dietary nutrient levels (carbohydrates, proteins, functional amino acids, fats, etc.) and micronutrient and vitamin content all have an impact on intake. When ruminants are fed high-concentrate rations, a large amount of VFA are produced by rumen microbial fermentation, in which the amount of propionate increases accordingly. In addition to providing energy for the body through the oxidation of the tricarboxylic acid cycle, propionate is also an important precursor of gluconeogenesis, thus propionate is an important factor in the regulation of intake of ruminants. It was found that the supplementation of B vitamins to rations has an important role in the regulation of rumen fermentation parameters, feed intake and performance of ruminants [15]. 5,6-DMB and Co are precursors for the biosynthesis of vitamin B₁₂, and vitamin B₁₂, as a coenzyme in the propionate metabolism pathway in the body of the animal, can effectively promote the growth and development of ruminants and improve the performance of the animals. The supplementation of 5,6-DMB and Co to the diet can increase the synthesis of vitamin B₁₂, which in turn can promote the digestive metabolism of the body [16]. In this experiment, the DMI of sheep increased with the supplementation of 5,6-DMB and Co, with the T75 group showing the highest increase in DMI and a highly significant difference as compared with the control group. This may be related to the low content of 5,6-DMB and Co, precursors of vitamin B₁₂ synthesis in the basal ration, which could not meet the needs of rumen microorganisms to synthesize vitamin B₁₂, and thus affect the metabolism of propionate in the body. The reason leading to the decrease in the animal’s feed intake, and the significant difference in feed intake of the T60 group as compared with the control group, were mainly caused by the synergistic reason of 5,6-DMB and Co. The relevant study has reported that a cobalt content of 0.30 mg/kg or more in the ration enhances microbial synthesis of vitamin B₁₂ [17]. The simultaneous supplementation of 5,6-DMB may have enhanced the synthesis of vitamin B₁₂ and, consequently, feed intake in sheep. Singh et al. [18] found that within a certain range, the sheep Vitamin B₁₂ synthesis increased with increasing cobalt content in the diet. However, high doses of cobalt hindered the growth of rumen bacteria and protozoa in sheep, which may be the reason why feed intake was significantly lower in the T90 group than in the T75 group. It was shown that the supplementation of 75 mg/kg 5,6-DMB to the ration increased vitamin B₁₂ synthesis in the rumen of sheep by 1817 μg/d [19]. In the study of Wang [20], the appropriate level of cobalt supplementation was considered to be 0.50 mg/kg, which was confirmed by the results of the present experiment, where the most significant increase in feed intake and daily weight gain was observed at the supplementation levels of 75 mg/kg 5,6-DMB and 0.50 mg/kg Co.

4.2. Effects of Supplementation 5,6-Dimethylbenzimidazole and Cobalt on Vitamin B₁₂ in Sheep

Vitamin B₁₂ is the main form of cobalt in ruminants to fulfill its biological function and can participate in the body’s energy metabolism [21]. Long-term feeding of concentrate-based rations to ruminants results in metabolic disorders of propionate [22], and the body synthesizes much less vitamin B₁₂ than it would if it were fed a roughage-based ration. A large amount of carbohydrates enter the animal’s body under high concentrate ration conditions, and the body’s own synthesis of vitamin B₁₂ is deficient. Vitamin B₁₂ is the coenzyme of methylmalonyl coenzyme A, and vitamin B₁₂ deficiency leads to the body’s propionate metabolism disorder [23]. Therefore, exogenous supplementation of Co and 5,6-DMB accelerates the production of vitamin B₁₂ and improves the metabolic efficiency of propionate, which in turn provides further energy to the organism. Tiffany et al. [24] studied the effect of different cobalt sources and concentrations on beef cattle and results showed that vitamin B₁₂ content was not affected by cobalt source, but increased with elevation of cobalt supplementation concentration. In a previous study of lactating cows, Sui et al. [25] found that the content of vitamin B₁₂ in rumen fluid was positively correlated with the supplementation level of cobalt. Stemme et al. [7] supplemented 0.17, 0.29 and 2.5 mg/kg cobalt to dairy cows’ diets and found that the microorganisms in the rumen could only use a small amount of cobalt to synthesize VB₁₂, and the synthesis efficiency was very low. When the cobalt content in the body was insufficient, the efficiency of synthesizing VB₁₂ was 7.1%, when the cobalt content in the body was sufficient, the efficiency of synthesizing VB₁₂ was only 9.5%, and when excess the cobalt content in the body, the linear growth was no longer valid, and its synthesis of VB₁₂ efficiency was only 4.4%. This is consistent with the results of the present study, in which ruminal vitamin B₁₂ content gradually increased with increasing levels of 5,6-DMB and Co supplementations, and when Co supplementations reached a certain level, the efficiency of vitamin B₁₂ synthesis began to decrease, and high doses of cobalt began to produce vitamin B₁₂ analogues and derivatives [26].

4.3. Effect of Supplementation 5,6-Dimethylbenzimidazole and Cobalt on Rumen Fermentation Parameters in Sheep

Nutrients in the ration are degraded and absorbed by microorganisms in the rumen, and the intra-rumen environment directly or indirectly influences microbial activity [27]. Rumen pH reflects the acidity and alkalinity within the rumen of ruminants, with a normal range of 5.50–6.80 [28]. The increase in the proportion of ration concentrates and the shift in the feeding pattern have led to a higher enrichment of VFA and lactate in the rumen of ruminants, resulting in a decrease in rumen pH [29]. Bao et al. [30] study showed that rumen pH tended to decrease gradually with the increasing proportion of concentrate. Carbohydrates in the ration enter the rumen and are catalyzed by a series of enzymes to generate glucose, which further generates pyruvate. On the one hand, the pyruvate is converted into lactate, which is more acidic than VFA, and the generated lactate cannot be decomposed quickly, and then the pH in the rumen decreases rapidly. On the other hand, pyruvate is generated into VFA through different metabolic pathways, which accelerates the decrease in the pH in the rumen. In this experiment, under the condition of a high concentration, the pH was increased in the trial group, and the change range was from 5.55 to 6.49. The supplementation of 5,6-DMB and Co increased the synthesis of vitamin B₁₂ in the organism. Vitamin B₁₂, as a coenzyme of methylmalonyl-CoA translocase, can catalyze the conversion of methylmalonyl-CoA to succinyl-CoA and promote the conversion of propionate to glucose through gluconeogenesis in the organism to glucose, which reduces the production of rumen lactate and thus increases rumen pH.

The rumen NH₃-N concentration reflects the efficiency of rumen microorganisms in degrading nitrogen-containing substances and utilizing ammonia, and the microorganisms in the rumen produce NH₃-N during the digestion and absorption of nutrients, and they also utilize NH₃-N for protein synthesis [31]. Rumen NH₃-N concentration was correlated with dietary protein level, and rumen microbial viability increased with dietary protein level in the effective NH₃-N range of 5.00–30.00 mg/100 mL, at which time the NH₃-N concentration likewise showed an increasing trend. In the present study, NH₃-N concentration varied from 12.56–29.84 mg/100 mL, which was within the normal range. The NH₃-N concentration was higher in the 5,6-DMB+ Co group. This has been associated with the supplementation of 5,6-DMB and Co increasing the synthesis of vitamin B₁₂ in the organism, which in turn increases the abundance of rumen flora [32], as well as increasing the nitrogen intake and retention in ruminants [11].

Ruminal VFA is the main product of rumen fermentation of carbohydrates in ruminants, and its content and composition are important indicators of rumen’s digestive and metabolic activities. Acetate and butyrate are the main precursors of fat synthesis, while propionate is the main precursor of gluconeogenesis in ruminants and is involved in various metabolic activities of the organism [33]. Dezfoulian et al.’s [34] findings showed that the rumen propionate and isobutyrate concentrations were higher in lambs than in the control group when cobalt glucuronate was supplemented to the lamb ration at 0.50 mg/kg DM. In this study, at the 5,6-DMB and Co supplementation levels of 100 mg/d and 0.50 mg/kg, the production of total volatile fatty acids could be increased, of which the proportion of propionate increased, which may be rumen microorganisms use 5,6-DMB and Co to synthesize vitamin B₁₂. This promotes rumen propionate production and metabolism, and, with the increase in rumen propionate concentration leading the total volatile fatty acid content of the rumen to increase, causing the overall VFA ratio to change.

Lactate is an intermediate metabolite of the carbohydrate metabolism process in the rumen of ruminants [35], and the level of lactate concentration in rumen fluid is an important indicator of normal rumen function. Carbohydrates in ruminant diets are degraded to glucose by rumen microbial fermentation, and glucose further produces pyruvate. On the one hand, pyruvate is generated under the action of lactate dehydrogenase through the reaction of “pyruvate + reduced nicotinamide adenine dinucleotide (NADH) + hydrogen ion (H⁺) ↔ lactate + oxidized nicotinamide adenine dinucleotide (NAD⁺)” [36,37]; on the other hand, pyruvate is further oxaloacetate generated through the action of pyruvate carboxylase, then malate dehydrogenase and succinate dehydrogenase, then malate dehydrogenase and succinate dehydrogenase generate oxaloacetiate, then generates the corresponding malate and succinate under the action of malate dehydrogenase and succinate dehydrogenase, and, finally, generates propionate, which provides energy for the body [38,39]. In the present study, with increasing levels of 5,6-dimethylbenzimidazole and cobalt, the content of vitamin B₁₂ was increased, while accelerating the conversion of lactate to pyruvate and depleting the concentration of lactate in the rumen, which suggests that sufficient amounts of vitamin B₁₂ accelerated the glycoheterotrophic reaction and increased the amount of propionate synthesized, which corresponds to the results of the propionate mentioned above in the present study.

Rumen MCP content is an important indicator of the ability of rumen microorganisms to utilize ammonia nitrogen and the population and number of rumen microorganisms. The 60–65% of the protein content of the ruminant organism is supplied by rumen MCP [40]. In the present study, the supplementation of 5,6-DMB and Co resulted in a higher MCP content in the 5,6-DMB+ Co group than in the control group. This is consistent with the findings of Li [41], who found that supplementation of cobalt and vitamin B₁₂ increased the MCP content, mainly because vitamin B₁₂ can increase the activity of different ammonia enzymes in the rumen such as glutamine synthetase and asparagine synthetase. This promotes the synthesis of glutamic acid and aspartic acid, etc., and therefore effectively enhances MCP synthesis, and the increase in the concentration of NH₃-N after the supplementation of 5,6-DMB and Co also provides favorable conditions for the synthesis of MCP.

Correlation analysis showed that vitamin B₁₂ content was positively correlated with pH, NH₃-N and propionate concentrations, and vitamin B₁₂ content was strongly correlated with cobalt addition levels, which corroborated Sui’s findings [25]. From this, it was inferred that the addition of 5,6-dimethylbenzenepropylimidazole and cobalt enhanced the promotion of vitamin B₁₂ synthesis. On the one hand, the vitamin B₁₂ content was correlated with the concentration of propionate. On the other hand, vitamin B₁₂, as a co-enzyme could increase the relative abundance of propionate-producing bacteria under the phylum Thickylobacteria in the rumen, which plays an important role in energy conversion [42]. In addition, vitamin B₁₂ was negatively correlated with starch concentration [23,43], and a high concentration of vitamin B₁₂ may have inhibited the production of starch-degrading bacteria (Prevotella) to some extent, thereby increasing rumen pH. This is consistent with Zhang’s [44] findings, which found that Prevotella is negatively correlated with pH. On the other hand, vitamin B₁₂ is not only related to carbohydrate metabolism, but also to protein metabolism [45]. As a nitrogen source for rumen microorganisms, NH₃-N is mainly related to protozoan and bacterial populations and affects protein utilization. In disagreement with the results of the Brito study [10], the supplementation of 5,6-dimethylbenzoimidazole had no effect on the number of protozoa and it is possible that the addition of 5,6-dimethylbenzoimidazole or cobalt alone was not sufficient to improve the distribution of protozoa and bacteria. Vitamin B₁₂ ensures the diversity of microorganisms in the rumen by improving pH, NH₃-N and propionate concentrations, which is beneficial for healthy ruminant growth. In addition, from the figure, it can be seen that D-lactate concentration was negatively correlated with pH, acetate, and propionate, and when pH continued to decrease, the lactate concentration showed a continuous increase, which in turn also affected the concentration of SCFA (acetate and propionate).

5. Conclusions

Under the conditions of this experiment, the supplementation of 5,6-DMB and Co increased vitamin B₁₂, propionate, NH₃-N and MCP contents in the rumen and rumen pH of sheep. The appropriate recommended amounts of 5,6-DMB and Co were 75 mg/kg and 0.50 mg/kg, respectively.