Effects of Rubber Seed Kernel Fermented with Yeast on Feed Utilization, Rumen Fermentation and Microbial Protein Synthesis in Dairy Heifers

Abstract

Yeast (Saccharomyces cerevisiae) has been used to improve the nutritive value of feedstuffs, especially rubber seed kernel. In the current study, rubber seed kernel was grated and subjected to solid-state fermentation with yeast to enhance the nutritive value. The yeast-fermented rubber seed kernel (YERSEK) was substituted for soybean meal in ruminant diets to evaluate the effect of YERSEK on feed intake, digestibility, rumen fermentation and microbial protein synthesis in dairy heifers. Five Holstein Friesian crossbred heifers with an initial body weight (BW) of 215 ± 20 kg were used in this research. The experimental design was a 5 × 5 Latin squared design and the dietary treatments were five levels of YERSEK at 0, 100, 150, 200 and 250 g/kg dry matter in concentrate at 1% of BW, with rice straw fed ad libitum. The supplementation with YERSEK reduced rice straw and total DM intake linearly (p < 0.05). The intake of neutral detergent fiber and acid detergent fiber decreased linearly (p < 0.05), while ether extract intake increased linearly (p < 0.01) with YERSEK supplementation. The ether extract digestibility tended to be high (p < 0.01) with increasing levels of YERSEK. Supplementation with the YERSEK did not change (p > 0.05) ruminal pH and blood urea nitrogen in this study, but ruminal ammonia nitrogen was increased (p < 0.01) in the heifers receiving YERSEK. Increasing the YERSEK levels did not adversely affect the proportion of volatile fatty acids (VFA), which included acetate, propionate and butyrate and the microbial population (p > 0.05). Microbial protein synthesis was similar among the treatments (p > 0.05). The inclusion of YERSEK at 250 g/kg DM in concentrate feed had no effect on the utilization of feed, rumen fermentation characteristics and microbial protein synthesis. The YERSEK could be used as a protein replacement for up to 86% of the soybean meal in feed concentrate for dairy heifers.

1. Introduction

Soybean meal (SBM) is the most widely used protein source in animal feed, because of its high protein content and beneficial amino acid profile [1]. The rising prices have recently impacted the use of SBM in animal feed production and many nutritionists are looking for more cost-effective alternative sources of supplementary protein.

Rubber seed is a by-product of the rubber tree (Hevea brasiliensis) plantations that are distributed throughout Southeast Asia, and especially in Thailand. The Rubber Authority of Thailand has reported an estimated average of 2.28 million hectares (ha) of rubber plantation in the year 2020. Based on an estimated average of 150 kg seeds/ha/yr. [2], the annual yield of rubber seed was 0.34 million metric tons. The rubber seed kernel (RSK) contains 198 g/kg crude protein (CP), 477 g/kg ether extract (EE), 378 g/kg linoleic acid and 17.6 g/kg linolenic acid [3]. Chanjula et al. [4] reported that the RSK at 200 g/kg in concentrate fed to goats on Briachiaria humidicola hay had no adverse effect on feed intake, nutrient digestibility, rumen fermentation and nitrogen utilization, while increasing levels of the RSK up to 300 g/kg resulted in a slightly lower feed intake and fiber digestibility. Pha-obnga et al. [3] revealed that an RSK level of 136 g/kg in a total mixed ration (TMR) did not affect gas production and digestibility in an in vitro experiment.

Yeast (Saccharomyces cerevisiae), as a source of probiotics in the diets of ruminants, has become a widespread additive for the maintenance of a healthy ruminal environment and to improve the feed efficiency of ruminants [5,6]. It is known to stimulate the growth of fiber-digesting bacteria, stabilize rumen pH by stimulating the population of lactate-utilizing bacteria, support the proliferation of other microorganisms [7,8] and may also increase microbial protein synthesis in the rumen.

Yeast has been widely used to improve crude protein (CP) content [9,10] and has been shown to reduce the anti-nutritional factors of some feedstuffs [11]. Cherdthong and Supapong [12] reported that cassava waste from bioethanol production (CWB) fermented with yeast had a CP content of 251 g/kg, which was two times higher than unfermented CWB. Boonnop et al. [11] reported that adding yeast to fermented cassava root could improve the CP content from 3.2 to 21.1 g/kg.

The use of the RSK fermented with yeast in dairy heifer rations has not yet been studied, so the aim of this study was to evaluate the effects of the YERSEK in concentrate on feed intake, nutrient digestibility, ruminal fermentation and microbial protein synthesis in dairy heifers.

2. Materials and Methods

2.1. Preparation of YERSEK

Fresh rubber seeds were collected from rubber plantations in Sakon Nakhon, Thailand within the harvesting period in August–September. The whole seeds were handpicked from the ground and were stored at room temperature. The seeds were dehulled by a dehulling machine (Incanewlife, Khon Kaen, Thailand). The kernels were sun dried for 3 days, ground to pass a 1 mm sieve and used as an ingredient in the YERSEK production. The YERSEK used in the present work was detailed by Wanapat et al. [13] who, in brief, stimulated S. cerevisiae using a weight of 5 g of baker’s yeast placed into a flask, then adding 20 g sugar and 100 mL distilled water, mixing well and incubating at room temperature for 1 h (A). The preparation of the liquid medium was completed by weighing and mixing well 42 g molasses in 100 mL distilled water followed by 40 g urea and then an adjustment of the pH of the medium solution, using H₂SO₄ to achieve the final pH 3.5–5 (B). The solution A and B were mixed at 1:1 ratio, then flushed with air for 60 h. After 60 h, the yeast medium solution was transferred to mix with the rubber seed kernels at a ratio of 1 mL:2 g, then dried under shade for 72 h, followed by sun-drying for 48 h. The final product was stored in plastic bag and analyzed for its chemical content.

The CP content increase in the YERSEK could be estimated using the following formula: CP (g/kg DM) increase in the YERSEK = ((CP in RSK/(1000 + CP in urea)) ∗ 1000) ∗ (75.3/100). The CP increase of yeast fermented RSK by urea was assumed to be 75.3%, therefore, the CP content (g/kg DM) increase of yeast-fermented RSK = ((212/(1000 + 287.5)) ∗ 1000) ∗ (75.3/100) = 124.0 g/kg DM.

2.2. Animals, Treatments and Experimental Design

Five crossbred dairy heifers (75% Holstein-Friesian × 25% Thai native breed), 215 ± 20 kg of body weight (BW), were randomly assigned according to a 5 × 5 Latin square design. The heifers were fed concentrate at 1% of the BW, and rice straw ad libitum. The YERSEK was included in the concentrate at 0, 100, 150, 200 and 250 g/kg DM. The concentrate and rice straw were offered in two equal meals per day at 08.00 h and 17.00 h. The ingredients and the chemical composition of the dietary treatments are shown in

Table 1. Ingredient and chemical composition of concentrate diet.

Item	Level of YERSEK (g/kg of DM)
	0	100	150	200	250
Ingredient, g/kg of DM
Cassava chip	637	637	637	637	642
Soybean meal	225	154	119	83	32
YERSEK	0	100	150	200	250
Rice bran	90	61	46	32	32
Urea	8	8	8	8	4
Molasses	20	20	20	20	20
Minerals and vitamins 1	10	10	10	10	10
Salt	5	5	5	5	5
Sulfur	5	5	5	5	5
Chemical composition
Dry matter, g/kg	909	903	906	889	889
Organic matter, g/kg DM	941	941	942	949	947
Crude protein, g/kg DM	148	147	149	147	146
Ether extract, g/kg DM	8	28	54	55	72
Neutral detergent fiber, g/kg DM	240	239	231	232	219
Acid detergent fiber, g/kg DM	129	127	126	125	120
Gross energy, MJ/kg DM	16.1	17.0	16.9	17.3	17.3
Price, Thai baht/kg	9.3	8.8	8.6	8.4	8.0

YERSEK, yeast-fermented rubber seed kernel. ¹ Contains per kilogram premix: 10,000,000 IU vitamin A; 70,000 IU vitamin E; 1,600,000 IU vitamin D; 50 g Fe; 40 g Zn; 40 g Mn; 0.1 g Co; 10 g Cu; 0.1 g Se; 0.5 g I.

. The heifers were housed in individual pens with availability of clean fresh water and mineral blocks. The mineral blocks (each kg) contained NaCl, 995.11 g; Na, 390.00 g; Mg, 2.00 g; Zn, 0.81 g; Cu, 0.22 g; I, 0.10 g and Se, 0.01 g (KNZ, Arnhem, Netherlands). The experiment was conducted for five periods and each period consisted of 21 days. The first 14 days were for the feed adaptation period, whereas the last 7 days were for sample collection. There was a switch over period of 7 days between each period.

2.3. Data Collection and Sampling Procedures

The feed that was offered and the refusals were recorded daily in the morning. The BW was measured daily during the sampling period, prior to feeding time. The feeds were sampled daily during the collection period and were composited by period prior to analyses. Fresh fecal samples (about 500 g) were collected twice daily by rectal sampling in the morning (07.00 h) and afternoon (16.00 h). Two successive samples were combined and composited, then stored in the freezer. The composite samples were dried at 60 °C, ground (1-mm screen using Cyclotech Mill; Tecator, Hoganas, Sweden) and analyzed for DM, ash, ether extract (EE) and crude protein (CP) content [14], neutral detergent fiber (NDF) and acid detergent fiber (ADF) [14,15]. The gross energy (GE) was determined in the feeds by bomb calorimetry using an Oxygen Bomb Calorimeter (Parr Instrument Company, Moline, IL, USA) and acid-insoluble ash (AIA). The AIA was used to estimate the digestibility of nutrients [16].

The urine samples (about 100 mL) were collected by spot sampling (morning and afternoon). The urination was induced by manual stimulation of the vulva. The samples were analyzed for allantoin [17] and creatinine [18]. The amount of microbial purines absorbed (X, mmol/d), presumably proportional to the purine derivatives (PD) excreted (Y, mmol/d), was estimated based on the following equation, as described by Chen and Gomes [19]: Y = 0.85X + (0.385W^0.75). The supply of microbial nitrogen (MN) was estimated by the urinary excretion of purine derivatives (PD), according to the predictive equation of Chen and Gomes [19]: MN (g/d) = 70X/(0.116 × 0.83 × 1000) = 0.727X. The N content of purines was 70 mg/mmol, the ratio of purine N to total N in the mixed rumen microbes as 11.6:100 = 0.116 and the digestibility of the microbial purines in the intestines was assumed at 0.83 [19]. The efficiency of the microbial N synthesis (EMNS), to denote the microbial nitrogen (N) supplied to the animal per unit of digestible organic matter apparently fermented in the rumen (DOMR), was calculated using the following formula: EMNS = MN (g/d)/DOMR (assuming that rumen digestion was 65% organic matter of digestion in total tract, DOMR = DOMI × 0.65; DOMI = digestible organic matter intake).

On the last day of each period, approximately 200 mL of rumen fluid was taken using a stomach tube connected with a vacuum pump at 0 and 4 h post-feeding. The ruminal pH was determined using a portable pH meter (FiveGo; Mettler-Toledo GmbH, Greifensee, Switzerland). The samples were then strained through four layers of cheesecloth and divided into two portions. The first portion was comprised of 5 mL of 1 M H₂SO₄ and 50 mL of rumen fluid. It was centrifuged at 16,000× g for 15 min and the supernatant stored at −20 °C. The ruminal NH₃-N concentration was analyzed using a Kjeltech Auto 1030 Analyzer, Tecator, Hoganiis, Sweden [20], and volatile fatty acid (VFA) analysis was performed using HPLC (instruments by controller water model 600E; water model 484 UV detector; column Novapak C18; column size 3.9 mm × 300 mm; mobile phase 10 mM H₂PO₄ (pH 2.5)) [21]. A second portion was fixed with 10% formalin solution in sterilized 0.9% saline solution. The total direct counts of bacteria, protozoa and fungi were made by the methods of Galyean [22], based on the use of a hemocytometer (Boeco, Hamburg, Germany). The methane (CH₄) production was calculated by standard equations according to CH₄ (g/d) = 22.71 × dry matter intake (kg/d) + 8.91 [23].

Blood samples (about 10 mL) were collected from the jugular vein, at the same time as the rumen fluid sampling, into tubes containing 12 mg of ethylene diaminetetraacetic acid, and the plasma was separated by centrifugation at 500× g for 10 min at 4°C and stored at −20 °C until the analysis of blood urea nitrogen (BUN), according to Crocker [24].

2.4. Statistical Analysis

All of the data were subjected to analysis of variance according to a 5 × 5 Latin square design using the general linear models procedures [25]. The data were analyzed using the model Yijk = μ + MiAj + Pk + εijk, where Yijk = observation from treatment I; animal j and period k; μ, the overall mean; Mi = the mean effect of treatments (i = 1 to 5); Aj = the mean effect of animals (j = 1 to 5); Pk = the mean effect of periods (k = 1 to 5) and εijk the residual error. The orthogonal polynomial contrasts (linear and quadratic) were used to estimate the effect of the YERSEK supplementation. Significant effects were identified at p < 0.05.

3. Results

3.1. The Chemical Composition of Diets

The changes in the composition of RSK when it was fermented are important when the feedstuff is new. The RSK contained CP at 212 g/kg DM, while the YERSEK product contained CP at 336 g/kg DM, an increase in CP of 124 g/kg DM. The EE, GE, NDF and ADF content were decreased in the YERSEK (

Table 2. Chemical composition of unfermented- and fermented-rubber seed kernel.

Item	RSK	YERSEK	Rice Straw
Chemical composition
Dry matter, g/kg	913	932	919
Organic matter, g/kg DM	963	958	893
Crude protein, g/kg DM	212	336	33
Ether extract, g/kg DM	343	274	3
Neutral detergent fiber, g/kg DM	215	145	885
Acid detergent fiber, g/kg DM	173	104	608
Gross energy, MJ/kg DM	33.6	26.7	13.7

RSK, rubber seed kernel; YERSEK, yeast-fermented rubber seed kernel.

).

3.2. Feed Intake and Nutrient Digestibility

The increasing levels of YERSEK supplementation meant the DM intake of rice straw and total intake was decreased linearly (p < 0.05), the EE intake was increased linearly (p < 0.01), but the intake of NDF and ADF was decreased linearly (p > 0.05) (

Table 3. Effect of YERSEK on feed intake and nutrient digestibility in dairy heifers.

Item	Level of YERSEK (g/kg of DM)					SEM	Contrast
	0	100	150	200	250		Linear	Quadratic
DM intake, kg/d	3.8	3.6	3.4	3.4	3.2	0.15	0.02	0.88
Rice straw	3.8	3.6	3.4	3.4	3.2	0.15	0.02	0.88
Concentrate	2.5	2.4	2.3	2.5	2.4	0.07	0.45	0.69
Total intake	6.2	6.0	5.8	5.9	5.6	0.19	0.03	0.79
Nutrient intake, kg/d
Organic matter	5.7	5.5	5.3	5.4	5.1	0.18	0.05	0.81
Crude protein	0.490	0.476	0.460	0.474	0.454	0.01	0.09	0.71
Ether extract	0.03	0.07	0.13	0.14	0.18	0.01	<0.001	0.02
Neutral detergent fiber	3.9	3.7	3.6	3.6	3.4	0.13	0.01	0.90
Acid detergent fiber	2.6	2.5	2.4	2.4	2.2	0.09	0.02	0.92
Digestibility coefficients, %
Dry matter	60.5	61.5	60.9	60.5	59.2	1.72	0.60	0.45
Organic matter	62.7	64.3	63.5	63.2	61.7	1.85	0.62	0.41
Crude protein	47.8	45.7	48.4	48.0	47.3	2.02	0.85	0.97
Ether extract	51.7	79.8	88.0	85.6	85.2	1.97	<0.001	<0.001
Neutral detergent fiber	53.4	55.3	53.9	51.6	50.6	2.19	0.22	0.44
Acid detergent fiber	45.0	45.9	45.7	45.6	43.1	1.82	0.51	0.35

YERSEK, yeast-fermented rubber seed kernel.

). Increasing levels of the YERSEK did not change the digestibility of DM, OM, CP, NDF and ADF (p > 0.05), except for the digestibility of EE. The EE digestibility was increased linearly (p < 0.01) with an increase of the YERSEK in the diet, and was highest when inclusion of the YERSEK was at 150 g/kg DM.

3.3. Ruminal Fermentation and Microbial Population

The ruminal pH was similar among groups (p > 0.05) (

Table 4. Effect of YERSEK on ruminal fermentation and BUN in dairy heifers.

Item	Level of YERSEK (g/kg of DM)					SEM	Contrast
	0	100	150	200	250		Linear	Quadratic
Rumen pH
0 h post-feeding	6.9	6.9	6.8	6.9	7.0	0.06	0.21	0.44
4 h post-feeding	6.8	6.5	6.3	6.5	6.6	0.12	0.56	0.05
NH3-N, mg/dl
0 h post-feeding	16.5	16.8	18.1	18.1	19.2	1.05	<0.01	0.83
4 h post-feeding	20.5	22.6	23.1	23.8	24.8	2.08	<0.001	0.29
BUN, mg/dl
0 h post-feeding	5.2	4.0	3.8	3.4	3.5	3.95	<0.01	0.19
4 h post-feeding	6.2	7.2	6.6	6.6	4.5	6.23	0.35	0.26
Total VFA, mmol/d
0 h post-feeding	105.3	106.1	111.1	101.7	87.5	3.24	0.09	0.10
4 h post-feeding	125.6	120.6	118.3	102.1	102.6	2.81	<0.01	0.89
VFA, mol/100 mol
Acetic acid (C2)
0 h post-feeding	63.6	64.1	63.2	64.2	65.1	0.93	0.52	0.91
4 h post-feeding	64.6	64.4	63.4	63.6	63.9	0.99	0.75	0.77
Propionic acid (C3)
0 h post-feeding	28.7	28.0	29.1	28.0	27.1	0.86	0.45	0.91
4 h post-feeding	27.3	27.8	28.7	28.2	28.3	0.95	0.71	0.77
Butyrate (C4)
0 h post-feeding	7.7	7.9	7.7	7.8	7.9	0.11	0.62	0.95
4 h post-feeding	8.1	7.8	7.9	8.1	7.8	0.19	0.81	0.93
C2:C3 ratio
0 h post-feeding	2.2	2.3	2.2	2.3	2.4	0.12	0.52	0.90
4 h post-feeding	2.4	2.3	2.2	2.3	2.3	0.18	0.33	0.47
CH4, g/d	150.3	145.2	139.6	142.8	135.4	6.45	0.44	0.93

YERSEK, yeast-fermented rubber seed kernel; NH₃-N, ammonia nitrogen; VFA, volatile fatty acid; CH₄, methane; BUN, blood urea nitrogen.

). The ruminal NH₃-N at 0 and 4 h post-feeding linearly increased (p < 0.01), whereas BUN and total VFA at 4 h post-feeding linearly decreased (p < 0.01) with increasing YERSEK supplementation. The inclusion of the YERSEK did not affect the acetate (C2), propionate (C3), butyrate (C4), C2:C3 ratio and CH₄ production (p > 0.05). The direct count of the bacterial, protozoal and fungal zoospores’ population was not significantly different among the treatments of increasing the YERSEK supplementation (p > 0.05), as shown in

Table 5. Effect of YERSEK on microbial population in dairy heifers.

Item	Level of YERSEK (%DM)					SEM	Contrast
	0	100	150	200	250		Linear	Quadratic
Microbial population, (cell/mL)
Bacteria, ×109
0 h post-feeding	4.6	6.1	5.1	6.5	4.9	0.71	0.68	0.18
4 h post-feeding	4.4	6.4	4.7	7.3	6.7	0.87	0.07	0.77
Protozoa, ×105
0 h post-feeding	1.3	3.5	2.6	2.4	2.7	0.50	0.34	0.14
4 h post-feeding	3.9	3.8	3.3	3.7	3.0	0.63	0.40	0.91
Fungi, ×104
0 h post-feeding	1.8	1.0	1.2	1.0	2.4	0.87	0.69	0.25
4 h post-feeding	2.4	2.8	2.4	2.4	2.6	1.09	0.99	0.99

YERSEK, yeast-fermented rubber seed kernel.

.

3.4. Microbial Protein Synthesis

The use of YERSEK in concentrate feed for dairy heifers had no effect on the urinary purine derivatives, microbial N supply, microbial CP synthesis and EMNS (p > 0.05) (

Table 6. Effect of YERSEK on microbial protein synthesis in dairy heifers.

Item	Level of YERSEK (%DM)					SEM	Contrast
	0	100	150	200	250		Linear	Quadratic
Urinary purine derivatives, mmol/d
Purine excretion	40.2	38.6	37.9	43.8	25.4	3.52	0.84	0.32
Purine absorption	52.2	51.3	50.1	54.7	40.8	2.66	0.24	0.71
Urine creatinine	7.0	7.6	6.7	6.0	7.6	0.20	0.84	0.32
MN, g/d	29.2	28.1	27.6	31.8	18.5	2.56	0.26	0.70
MCP, g/d	146.7	175.4	172.2	199.0	135.7	16.02	0.26	0.70
EMNS, g/kg OMDR	14.1	13.4	12.3	16.3	11.0	0.07	0.32	0.77

YERSEK, yeast-fermented rubber seed kernel; MN, microbial nitrogen; MCP, microbial protein; EMNS, efficiency of microbial N synthesis.

).

4. Discussion

4.1. The Chemical Composition of Diets

The fermentation process that increased the CP content of the YERSEK may be due to the high production of the yeast cell mass by the addition of 40 g urea and 42 g molasses in solution. The reduction in the NDF and ADF content of the YERSEK could be due to two possible reasons. Firstly, the ingredients, including yeast, sugars, molasses and urea, for the transformation of RSK to YERSEK were reduced in fiber content, resulting in a dilution of the NDF and ADF contents in the YERSEK. Secondly, the secretion of various enzymes by the yeasts and natural cellulase-producing microbes might degrade the hemicellulose and cellulose of the YERSEK. Wanapat et al. [26] reported on an increase in CP and the reduction of fiber content in feed by the supplementation of 20 g yeast and 48 g urea. The fermentation using yeast reduced the EE content of RSK in the present study. The RSK is rich in oleic acid and linoleic acid at 25.1% and 37.8%, respectively. When the plant oils were exposed to light during the feedstuff fermentation with yeast, the fatty acid, or specifically the oleic acid and linoleic acid, content reduced [27]. The oxidation happened because of a hydrogen abstraction reaction between a hydroxy radical and fatty acid [28]. This radical can take a hydrogen atom from the lipid or a hydrogen atom from the lipid hydroperoxides to produce peroxyl radicals [27]. The result of the EE reduction in the YERSEK when compared with RSK indicated that plant oil can be degraded into hydroperoxides under the influence of light and oxygen.

4.2. Feed Intake and Nutrient Digestibility

Chanjula et al. [4] reported that feed intake was reduced when the goats were fed RSK at 200 to 300 g/kg in concentrate. This study also demonstrated that the inclusion of YERSEK in concentrate diets decreased the intake of DM, NDF and ADF. It has often been observed that the negative effect of fat on intake could be due to a low palatability of added fat [29,30,31], so that the levels of unsaturated fatty acids in the YERSEK could be affected by the rice straw intake in dairy heifers. In addition, increasing levels of YERSEK in concentrate increased the EE content from 8 to 72 g/kg DM and GE from 16.1 to 17.3 MJ/kg DM. Therefore, it was expected that the YERSEK would increase the energy supply and nutrient availability and also reduce the fiber intake in the current study. Nutrient digestibility was not affected by the YERSEK inclusion, which suggested that some fatty acids were produced by the hydrogenation of the YERSEK in the rumen. The increased EE intake and digestibility in heifers fed the YERSEK was consistent with the results reported by Chanjula et al. [4], explained by the large amount of EE in the diet and higher EE intake and digestibility.

4.3. Ruminal Fermentation and Microbial Population

The YERSEK had no effect on the rumen pH value of 6.6 to 6.8 in this study and this was consistent with previous research which reported that feeding RSK meal at 100 to 300 g/kg in concentrate had no effect on the rumen pH value of goats fed on a Briachiaria humidicola hay-based diet [32]. The molar proportion of VFA, including acetate, propionate and butyrate was not affected by the dietary treatments in this study. These results indicated that the YERSEK can be used as an alternative feedstuff for components such as soybean meal in dairy heifer diets and does not negatively affect the production of VFA in the rumen. Increasing the YERSEK supplementation did not adversely affect the levels of the bacterial, protozoal and fungal population which ranged from 4.6 to 7.3 × 10⁹, 1.3 to 3.9 × 10⁵ and 1.0 to 2.8 × 10⁴ cells/mL, respectively. In contrast, Wanapat et al. [13] reported that yeast-fermented cassava chips used as a protein source to replace 100% soybean meal in concentrate resulted in a significant increase in the bacterial and fungal population in dairy cows. This could be because fermented cassava chips with added yeast supplied sufficient factors for microbial growth, such as carbon skeletons, amino acids and minerals.

4.4. Microbial Protein Synthesis

The microbial protein synthesis provides 50 to 80% of the total absorbable protein reaching the animals’ small intestines [33,34,35]. Urinary allantoin excretion and the estimated microbial protein synthesis from the rumen were not significantly affected by the YERSEK supplementation.

The protozoal population was not affected by the YERSEK supplementation compared with the control. The results from the present study were similar to those of Promkot and Pornanek [36], who reported that microbial protein synthesis and protozoal population were unaffected by yeast-fermented cassava root in beef cattle, so it seems unlikely that the protozoa substantively contributed to the evaluated increase in the duodenal flow of microbial protein as estimated from the PD excretion in urine [37,38].

The NH₃-N is the major nitrogen source used for protein synthesis by ruminal microbes [39,40]. The NH₃-N concentration was increased from 18.5 to 22.0 mg/dL, despite there being no alteration in the microbial protein synthesis with the increasing levels of YERSEK in concentrate diets. A study by Weakley and Owens [41] resulted in higher ammonia nitrogen, but did not measure the microbial protein synthesis in the rumen. According to Lila et al. [42], the addition of yeast to ruminant diets can not only improve the rumen environment but also enhance the microbial activity, especially cellulolytic activities, which increases their total number, fiber digestion, reduces lactate accumulation and concentration of oxygen in rumen fluid and improves utilization of starch. The S. cerevisiae also stimulates the DM intake and productivity in growing and lactating cattle [43,44] and improved the microbial protein synthesis and milk production in dairy cows [45,46,47].

5. Conclusions

Based on the present study, the inclusion of YERSEK at 250 g/kg DM in concentrate diets had no effect on the feed utilization, rumen fermentation and microbial protein synthesis in dairy heifers. It is concluded that YERSEK could be used as a protein source to replace up to 86% of the soybean meal in concentrate, which would reduce the production costs.