Introduction

The main problem of goat farming in the southern part of Thailand is the lack of adequate and nutritionally valuable roughage resources to meet the demands for goat meat production. This shortage is due to unsuitable land and water conditions for sufficient grass cultivation. Conversely, the southern region is a major economic crop area for oil palm cultivation, with an estimated production area of 6.252 million acres1. Oil palm fronds are often left unused or partially fed to animals, leading to significant waste and underutilization of this agricultural by-product. Current practices in ruminant feeding typically involve unprocessed or minimally treated palm fronds, which are limited by low digestibility and nutritional value. Research has shown that fermenting palm fronds with yeast can enhance their digestibility2and nutritional profile by increasing crude protein content and improving fiber breakdown, ultimately benefiting ruminant productivity and growth performance3. However, farmers have yet to widely adopt yeast fermentation as part of their feeding strategies due to limited awareness and access to efficient methods.

The prioritization of developing techniques to enhance livestock output, decrease production expenses, and mitigate the adverse environmental effects of livestock production was of paramount significance for nutritional scientists, microbiologists, and biochemists. The practice of adding antibiotics, probiotics, or prebiotics to livestock feed has been utilized to attain these aforementioned objectives4. Extensive research has been conducted over the years to investigate the impact of adding microbial feed additives to the diet of cattle on their health and productivity. The predominant yeast supplement utilized in ruminant feed is derived from cultures of Saccharomyces cerevisiae. To optimize the activity of beneficial micro-organisms during fermentation, it is important to minimize energy and nutrient losses. This will ultimately enhance the digestibility of nutrients and boost the production capacity of animals5. S. cerevisiaeis rich in digestible proteins, vitamins (vitamin B6, thiamin, biotin, riboflavin, nicotinic acid and pantothenic acid), magnesium and zinc6.

Yeasts serve as a crucial source for deriving products with probiotic potential, comprising live strains or derivatives of their cell walls. These formulations have exhibited confirmed immunomodulatory effects in livestock, alongside enhancements in gastrointestinal functionality, leading to better production outcomes7. Yeast ranks among the probiotics frequently utilized in studies and practices concerning ruminant nutrition. Incorporating yeast into diets has shown promise in boosting feed consumption and milk yield in dairy cows. Moreover, yeast supplementation holds the potential to optimize fiber digestion within the rumen and foster the proliferation of anaerobic rumen microorganisms, particularly cellulolytic bacteria8.

Therefore, the objectives of this study were to evaluate the effects of high-protein yeast (Pro Bio Feed) combined with oil palm leaves on reducing waste and processing oil palm leaves, as well as enhancing the nutritional value of fermented oil palm leaves on the digestibility of the feed, rumen fermentation and hematological in Thai Native-Boer crossbred goats.

Materials and methods

Animal care

All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Rajamangala University of Technology Srivijaya (Record no. IAC 01–11-2023). The Research and Development Institute, Rajamangala University of Technology Srivijaya, Thailand, authorized all protocols in compliance with relevant guidelines and regulations. Additionally, all methods are reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

Animals, design, treatments, and management

Twelve female Thai Native x Boer crossbred goats were obtained from Experimental Goat Farm of the Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat Province, Thailand. These goats aged 6 ± 1 months with body weight of 18 ± 1.5 kg were randomly distributed in a completely randomized design (CRD) to assess the effect of fresh Napier grass (Treatment 1), fresh oil palm leaves fermented with Pro Bio Feed (Treatment 2) and Napier grass fermented with Pro Bio Feed (Treatment 3). Pro Bio Feed had a content S. cerevisiae of 1.0 × 108 CFU/g and lactic acid bacteria (LAB) of 1.0 × 108 CFU/g was obtained from Bounthavyxay Farm Co., Ltd. The concentrate fed in this experiment (Table 1) at 0.5% BW was formulated9 to meet the nutrient requirements and roughage ad libitum. The goats were fed twice a day, with feedings occurring at 8:00 a.m. and 5:00 p.m. Each feeding session consisted of offering the concentrate first, followed by the roughage. There was a time gap of approximately 9 h between the two feedings, allowing the goats to consume both the concentrate and roughage during the day. Additionally, the goats had free access to water throughout the experimental period, and daily records of both roughage and concentrate intake were maintained to ensure accurate monitoring of their consumption. Vaccinations and other preventative precautions were carried out before the trial’s start in accordance with the Institutional Animal Care and Use Committee of Rajamangala University of Technology Srivijaya. The experiment was carried out over 90-day intervals, with the first 7 days dedicated to adjusting to and measuring feed consumption. Feed and excrement samples were collected once a week during the study for chemical analysis.

Preparation to test diets

Fresh oil palm leaves and Napier grass were selected as the primary roughage sources for the study. The leaves were first cut in half, and then ground using a grinder to reduce their particle size. The chopped roughage, consisting of oil palm leaves and Napier grass, was tightly packed into a fermentation tank in preparation for the fermentation process. To create the fermentation liquid, 10 L of water, 1 L of molasses, 1% urea (based on the total liquid volume), and yeast (Pro Bio Feed) at a ratio of 1 gram per 1 kg of chopped leaves were mixed together. The mixture was stirred thoroughly for approximately 20 min to ensure proper oxygenation, which promotes yeast growth and allows the yeast to feed on the molasses. Once the fermentation liquid was well-mixed, it was added to the packed roughage in the fermentation tank. The tank was then covered with plastic to prevent air from entering and sealed tightly to maintain anaerobic conditions. The tank was stored in a shaded area, away from direct sunlight, to protect the mixture from external environmental factors. The fermentation process lasted for 21 days, during which the mixture was left undisturbed to allow proper microbial fermentation. After the fermentation period, the mixture was ready for use in feeding trials.

Chemical composition of experimental feeds

Based on the study of the nutritional components of the concentrated feed, which includes soybean meal, palm kernel cake, soybean meal, cassava, ground corn, molasses, salt, mineral mix, and dicalcium at 17.7%, 20.0%, 10.0%, 28.3%, 20.0%, 1.0%, 1.0%, 1.0%, and 1.0% respectively, the chemical composition of the concentrated feed and roughage used in the experiment is as follows: Concentrated feed has 84.36% DM, 11.94% CP, 4.88% ash, 49.12% NDF, 12.01% ADF. Fresh Napier grass has 32.89% DM, 2.52% CP, 1.90% ash, 67.41% NDF, 22.92% ADF. Yeast-fermented oil palm fronds has 34.49% DM, 10.84% CP, 2.80% ash, 74.43% NDF, 18.70% ADF. Yeast-fermented Napier grass has 34.73% DM, 6.62% CP, 1.45% ash, 74.63% NDF, 22.71% ADF. These compositions are presented in Table 1.

Table 1 Chemical composition of concentrated feed and roughage used in the experiment.
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Feed and fecal sampling procedures

Daily records of the amount of feed provided and the number of samples were kept throughout the experiment. Using the entire collection method, samples of feces, feed, and refusals were taken from each individual group at the conclusion of experiment. The method of AOAC9 was used to determine, dry matter (DM) and ash. Ankom Fiber Analyzer (Ankom220, Macedon, NY, USA) was used to extract and filter neutral detergent fiber (NDF) and acid detergent fiber (ADF). Analysis of nitrogen in the feeds and faeces samples were determined by the combustion method (Leco FP828 Nitrogen Analyzer, LECO Corporation, Saint Joseph, MI, USA).

Rumen fluid sampling procedures

On the final day of the data collection period, rumen fluid samples (200 mL each) were taken at 0 and 4 h post-feeding using a stomach tube connected to a vacuum pump. The pH and temperature of the rumen fluid were measured right away using a portable temperature and pH meter (HANNA Instruments HI 98153 microcomputer pH meter, Kallang Avenue, Singapore). The rumen fluid samples were then filtered through four layers of cheesecloth. To halt the microbial fermentation process, 45 mL of rumen fluid were collected and immediately mixed with 5 mL of sulfuric acid solution (1 M). Although the addition of sulfuric acid could potentially alter some chemical characteristics, it does not affect the stoichiometry of the fluid for ammonia nitrogen (NH₃-N) and volatile fatty acid (VFA) analysis. Alternatively, rumen fluid samples were preserved by freezing with ice packs to prevent acidification. The mixture was centrifuged for 15 min at 16,000x g. (Table Top Centrifuge PLC-02, FL, USA). The supernatant was analyzed for ammonia nitrogen (NH3-N) using a Kjeltech Auto 1030 analyzer (Tecator, Hoganiis, Sweden) and volatile fatty acids (VFA) were assessed using gas chromatograph (Nexis GC-2030, SHIMADZU, Shimadzu Corp., Kyoto).

About 20 mL of ruminal fluid was collected to analyze for protozoa and one mL was added to the formalin 10% solution produced a 1:10 dilution. Samples were kept chilled after dilution. For protozoa counts, a Sedgewick-Rafter chamber (S.I Scientific Supplies Co., Ltd., Bangkok, Thailand) was used with a cover slip. Finally, to find the average per square, 25 large squares were counted randomly and divided the number of protozoa counted by 25.

Blood sampling analysis

The blood samples were taken from the jugular vein at 0 and 4 h post feeding at jugular vein about 10 mL on the 90 st day of experimented to analyze blood chemistry (Clinical chemistry automatic PKL PPC 125, Salerno, Italy) and routine hematology (Mindray BC-5000, Wanda Center, China).

Statistical analyses

All data were statistically analyzed using one-way analysis of variance (ANOVA) in the General Linear Model (GLM) procedure of SAS. The following equation was used to evaluate the data as a completely randomized design (CRD) using SAS’s (Version 9.4) Proc GLM procedure10 : Yij = µ + αi + βj + ϵij where: Yij = observations; µ = overall mean; αi = effect of treatment i;βj = the effect of time j and ϵij​ is the residual error. Results are presented as mean values with the standard error of the mean. Differences between treatment means were estimated by Duncan’s New Multiple Range Test11 and differences with p < 0.05 were considered to represent statistically significant differences.

Results and discussion

Feed intake in goats

According to a study on the feed intake of crossbred meat goats that were fed with concentrate, fresh Napier grass, yeast-fermented palm fronds, and yeast-fermented Napier grass, it was found that there was no statistically significant difference in total feed intake (p > 0.05). However, when considering roughage intake, Treatment 2 resulted in the highest feed intake (0.67 kg/day), followed by Treatments 3 and 1, respectively (0.64 and 0.62 kg/day). This finding aligns with Musnandar et al.2, who found that replacing grass with fermented palm fronds at levels of 0, 50, and 100% had no effect on digestible nutrients, feed intake (dry matter), and the chemical composition of the carcass (p > 0.05). However, replacing grass with 100% fermented palm fronds significantly increased daily growth rate, carcass weight, and carcass percentage. Cai et al.12 reported that supplementing with S. cerevisiaeimproves production performance by increasing dry matter intake, and supplementing with molasses reduces total fiber content. This is because adding molasses speeds up carbohydrate fermentation and improves the quality of the silage13, thereby allowing goats to consume more yeast-fermented palm fronds, as shown in Table 2.

Table 2 Effect of supplementing Yeast-Fermented forage on feed intake in crossbred goats.
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The effect of yeast-fermented feed plants on digestibility coefficients in goats

Studies have shown that the digestibility coefficients of different yeast-fermented feed plants across three treatments demonstrate significant statistical differences (p < 0.05) in the digestibility coefficients of dry matter (37.29, 34.49, and 34.73), crude protein (2.52, 10.84, and 6.62), NDF fiber (67.41, 74.43, and 74.63), and ADF fiber (22.92, 18.70, and 22.71). However, there is no significant difference (p > 0.05) in the digestibility coefficient of organic matter, as shown in Table 3. The effects of yeast supplementation on nutrient digestibility depending on the ratio of roughage to concentrate feed14. It was found that live yeast supplementation with high amounts of concentrate feed enhances the digestibility of DM and NDF, but has no effect on nutrient digestibility when high amounts of roughage are fed. This outcome could be attributed to the interaction between fiber-degrading bacteria and the yeast Saccharomyces cerevisiae, which promotes the growth of key fiber-degrading bacteria such as R. albus, R. flavefaciens, and F. succinogenes. These bacteria play a significant role in the breakdown of fiber, potentially influencing the digestibility of feed components, especially in concentrate-based diets15. These microorganisms are critical for breaking down complex plant fibers into simpler compounds, which improves fiber digestibility and energy availability. The study further revealed that yeast supplementation modulated the ruminal microbiota by creating a favorable environment for anaerobic fermentation. This resulted in increased production of short-chain fatty acids (SCFAs), especially acetate and propionate, which are vital for energy metabolism in ruminants.

The use of Saccharomyces cerevisiae yeast as a component in animal feed can enhance nutrient digestibility and increase cell counts in the animal’s digestive system. When yeast is broken down, it produces additional protein nutrients. Saccharomyces cerevisiaecontains numerous enzymes that improve the effectiveness of existing digestive enzymes, thereby increasing the digestibility rate and food intake, which in turn leads to weight gain or increased animal productivity16. In addition to yeast, LAB play a significant role in the digestive processes of ruminants. Lactic acid bacteria are known for their ability to ferment carbohydrates, producing lactic acid as a primary end product. The numerical increase in pH might be explained by the balanced microbial activity in the rumen, possibly supported by yeast-fermented forages that promote the growth of beneficial microbes while maintaining a stable rumen environment. Yeast supplementation is known to stabilize ruminal pH by stimulating cellulolytic bacteria and reducing the accumulation of organic acids. As a result, minimal pH fluctuations were observed, even with the presence of LAB. This highlights the potential role of yeast fermentation in enhancing microbial efficiency without significantly altering the ruminal pH.Lactic acid bacteria, such as Lactobacillus, Enterococcus, and Pediococcus species, contribute to the breakdown of complex carbohydrates, enhancing the overall digestibility of the feed. They work synergistically with yeast and other microorganisms in the rumen to improve the fermentation process and nutrient availability. The presence of LAB has been shown to improve the digestibility of fiber and protein, potentially leading to better feed efficiency and animal performance.

Table 3 The effect of Yeast-Fermented feed plants on digestibility coefficients in crossbred goats.
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The effect of yeast-fermented feed plants on digestive processes in the rumen of crossbred goats

Studies have shown that yeast-fermented feed plants affect various aspects of the fermentation process in the rumen of crossbred goats in Table 4. These findings are consistent with Kansagara et al.17, who noted that the rumen maintains a constant temperature around 39 °C, with a dynamic pH range of approximately 6–7. The rumen harbors a dense population of microorganisms, including bacteria, protozoa, and fungi, responsible for 60–70% of total digestion.

The concentration of ammonia-nitrogen at hour 0 was 14.83, 15.13, and 14.86 mg/dL, showing no significant statistical difference. However, at hour 4, the ammonia-nitrogen concentration was 15.90, 18.35, and 16.43 mg/dL, respectively. The optimal ammonia-nitrogen concentration in the rumen is between 15 and 30 mg/dL, influenced by various factors such as feed level, protein degradation, carbohydrate sources, mineral sources, and feeding frequency, which affect microbial digestion and ammonia-nitrogen production. Controlling these factors ensures optimal ammonia-nitrogen levels for microbial growth and digestive efficiency18. Additionally, microbial protein synthesis is related to nitrogen balance, a balance between protein and carbohydrate digestion, and increased nitrogen utilization can improve rumen fermentation processes19.

The number of protozoa at hour 0 was 8.36, 24.38, and 9.63 × 105 cells/mL, and at hour 4, it was 8.50, 26.25, and 7.13 × 105 cells/mL, showing significant statistical differences (p< 0.05). Yeast supplementation, particularly Saccharomyces cerevisiae, has been shown to modulate the protozoa population by enhancing microbial efficiency. Some studies suggest that yeast can have a positive impact on protozoa by promoting a more stable microbial environment, which helps improve fiber digestion and nutrient utilization. Yeast may also enhance the growth of beneficial microbes, including protozoa, by providing a more favorable environment for fermentation. The relationship between yeast and protozoa could be positive, as yeast fermentation products, such as vitamins and growth factors, may support the growth and activity of protozoa. Furthermore, yeast may help reduce the number of harmful protozoa species that can contribute to inefficiencies in digestion20.

In contrast, other studies have suggested that live yeast could suppress protozoa populations under certain conditions, possibly by promoting the growth of competing microbial populations, such as bacteria. This suppression could be beneficial in reducing protozoa-associated inefficiencies, such as excessive degradation of nutrients. Therefore, the effects of yeast on protozoa might depend on the balance of microbial populations and specific feeding conditions21. Thus, yeast supplementation is beneficial for improving the rumen environment, digestion efficiency, and nutrient absorption, benefiting both animals and farmers through improved feed utilization and animal health.

Table 4 The effect of Yeast-Fermented feed plants on fermentation processes in the rumen of crossbred goats.
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The effect of supplementing yeast-fermented feed plants on the concentration of volatile fatty acids in the rumen

The study found that supplementing yeast-fermented feed plants affects the concentration of VFA in the rumen (Table 5).

The study found no statistically significant differences (p > 0.05) except for propionic acid at hour 4, showing a statistically significant difference (p< 0.05). Lactic acid bacteria further enhance the fermentation process by producing lactic acid, which serves as a substrate for other rumen microorganisms to produce VFAs. Lactic acid bacteria can stabilize the rumen environment, reduce pH fluctuations, and inhibit the growth of pathogenic bacteria. Their presence can enhance the production of propionic and butyric acids, contributing to a more efficient fermentation process22,23. The significant increase in propionic acid (C3) concentration observed at Hour 4 in goats fed yeast-fermented feed plants and LAB may reflect enhanced microbial activity stimulated by the yeast and LAB supplementation. Yeast provides a stable rumen environment, promoting the growth of lactate-utilizing bacteria, which convert lactic acid into propionic acid24. LAB, although its direct impact on lactic acid production was not measured, likely contributed to an increased availability of lactate, which serves as a precursor for propionic acid synthesis25. These findings align with previous studies indicating that the synergy between yeast and LAB can enhance rumen fermentation efficiency and the production of specific volatile fatty acids26.

Table 5 Effect of supplementing yeast-fermented feed plants and lactic acid bacteria on the concentration of volatile fatty acids in the rumen of crossbred goats.
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The effect of supplementing yeast-fermented feed plants on blood chemical composition

The study investigated the effects of supplementing yeast-fermented feed plants on the chemical composition of blood, as shown in Table 6. The results revealed statistically significant differences (p < 0.05) in blood urea nitrogen levels, while other blood chemical components did not show significant differences (p> 0.05). The variability in BUN concentrations can be attributed to dietary effects and microbial interactions in the rumen. Yeast-fermented palm oil, with its high protein content, likely contributes to increased nitrogen levels in the blood. BUN is a key indicator of protein and energy utilization in ruminants, and it plays an essential role in growth. Elevated BUN concentrations are closely associated with increased ammonia-nitrogen levels in the rumen, reflecting the amount of protein metabolized and its efficiency in dietary protein utilization27,28.

The results in Table 6indicate significant effects of yeast-fermented feed supplementation on hemoglobin, hematocrit, and red blood cell counts in crossbred goats. These parameters are critical indicators of overall health, oxygen-carrying capacity, and the efficacy of erythropoiesis in animals. The enhanced hemoglobin, hematocrit, and RBC levels observed in this study could result from the improved digestibility of nutrients and potential modulation of the gut microbiota by yeast and LAB supplementation21. These additives likely increased the bioavailability of essential nutrients (e.g., iron, B vitamins), thereby supporting erythropoiesis.

Hemoglobin levels were significantly higher in goats fed with yeast-fermented feeds compared to the control. Hemoglobin plays a crucial role in oxygen transport, and its increase suggests improved nutritional status and possibly enhanced oxygen delivery to tissues, supporting better metabolic activity18. The improvement could be attributed to better nutrient utilization and absorption facilitated by yeast and LAB25.

Hematocrit reflects the proportion of red blood cells in the blood, serving as an indicator of blood viscosity and oxygen-carrying capacity. The significant increase in hematocrit in the supplemented groups suggests an improvement in erythropoiesis, potentially due to enhanced dietary protein and micronutrient availability, which are crucial for red blood cell synthesis26.

Red blood cell counts were significantly higher in goats fed with yeast-fermented feeds, indicating better production of erythrocytes. This aligns with the hypothesis that yeast fermentation improves feed digestibility and nutrient bioavailability, including iron and vitamins required for erythropoiesis29. A high red blood cell count may indicate increased red blood cell production to meet oxygen transport needs or for growth and repair. Conversely, a low red blood cell count may indicate problems with oxygen transport or reduced vascular and respiratory function. Analyzing red blood cell volume can help diagnose medical conditions related to the vascular system and oxygen transport35. Moreover, LAB can enhance the immune response in animals by producing antimicrobial compounds, stimulating the production of antibodies, and modulating the gut microbiota. This can result in improved health and reduced incidence of digestive disorders, further contributing to the productivity of the animals.

Table 6 Effect of supplementing Yeast-Fermented feed plants on blood chemical composition in crossbred goats.
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Conclusions

Based on the study on the effects of yeast (Pro Bio Feed) combined with oil palm fronds, it can be concluded that yeast-fermented oil palm fronds provide a beneficial nutrient profile and enhance feed intake in meat goats. These findings suggest the potential of using yeast-fermented oil palm fronds as an alternative feed to improve the nutritional value and efficiency of feed utilization in goat farming. This study concluded that supplementing yeast with oil palm fronds can significantly enhance the efficiency of nutrient utilization in meat goats. Additionally, incorporating LAB into the diet, either through direct-fed microbials or fermented feed, can complement the effects of yeast and provide a more comprehensive approach to improving feed digestibility and animal health. Future research could focus on the optimal combinations and ratios of yeast and LAB in animal feed to maximize these benefits.