Yeasts isolates from bovine rumen can improve the in vitro digestibility of Urochloa decumbens

Leveduras isoladas de rumen bovino podem melhorar a disgestibilidade in vitro de Urochloa decumbens

Abstract

Commercial yeast has been frequently employed to improve the digestibility of ruminant diets, albeit these microorganisms are not indigenous to the rumen microbiome. In this investigation, our aim was to assess the in vitro digestibility of Urochloa decumbens hay (UDH) with the incorporation of two selected yeast strains isolated of ruminal fluid of Zebu cattle. Rumen fluid was collected at the onset, midpoint, and final of the dry period in a semiarid region Brazil. UDH samples were obtained from the same region. Within simulated ruminal chambers, we included McDougall buffer solution, ruminal fluid, Ankon F-57 bags containing UDH samples. Sabouraud broth (control), V62 yeast (Pichia kudriavzevii), V5 yeast (Rhodotorula mucilaginosa), or a combination thereof (mixture) were evaluated as inoculant to the ruminal fluid. The incorporation of V5, the combination of both isolates, and V5 alone resulted in significantly elevated in vitro dry matter digestibility of UDH for ruminal fluid collected at the initial, intermediate, and final stages of the dry season, respectively. Moreover, the yeast isolates demonstrated resilience to the in vitro acid digestion process, indicating their potential utility in the formulation of probiotics or microbial supplements for ruminants raised in tropical pastures during dry season.

Keywords:
yeast interactions; ruminant nutrition; ruminal micobiota; tropical forage; semiarid

Resumo

Leveduras de uso comercial tem sido frequentemente utilizadas para melhorar a digestibilidade das dietas de ruminantes; entretanto esses microrganismos não são nativos do ecossitema ruminal. Nesta investigação, o objetivo foi avaliar a digestibilidade in vitro do feno de Urochloa decumbens (UDH) com a incorporação de duas cepas selecionadas de leveduras isoladas do líquido ruminal de bovinos zebuinos. O líquido ruminal foi coletado no início, no meio e no final do período seco em uma região semiárida do Brasil. Amostras de UDH foram obtidas da mesma região. Em câmara simuladora do rúmen, foram incluídos solução tampão McDougall, fluido ruminal e sacos Ankon F-57 contendo amostras de UDH. Caldo Sabouraud (controle), levedura V62 (Pichia kudriavzevii), levedura V5 (Rhodotorula mucilaginosa) ou uma combinação dessas leveduras (mistura) foram avaliados como inoculantes para o líquido ruminal. A incorporação de V5, a combinação de ambos os isolados e V5 isoladamente promoveram maiores taxas de digestibilidade in vitro matéria seca de UDH para fluido ruminal coletado nos estágios inicial, meio e final da estação seca, respectivamente. Além disso, os isolados de levedura demonstraram resiliência ao processo de digestão ácida in vitro, indicando o potencial para utilidade na formulação de probióticos ou aditivos microbianos para ruminantes criados em pastagens tropicais durante a estação seca.

Palavas-chave: interações de leveduras; nutrição de ruminantes; micobiota ruminal; forragem tropical; semiárido

1. Introduction

In tropical regions experiencing a semi-arid climate, Urochloa genus plants are commonly utilized for establishing pastures (Guamán-Rivera et al., 2024). However, during the dry period, these forages exhibit diminished nutritional value and reduced digestibility due to their elevated content of lignin, cellulose, and hemicellulose, coupled with other indigestible components like cuticle and silica. These factors collectively lead to decreased animal performance (Santos et al., 2008). Enhancing the digestibility of these forages and manipulating the ruminal microbiota are crucial for boosting the productivity of grazing ruminants (Xie et al., 2019).

The incorporation of Saccharomyces cerevisiae into ruminant diets has been shown to augment ruminal bacterial populations and enhance microbial protein production within the rumen (Huebner et al., 2019). However, certain commercial strains of S. cerevisiae exhibit limited growth in the rumen environment (Garcia-Mazcorro et al., 2020; Huebner et al., 2019).

Natural occurrences of yeast in ruminal fluid have been documented in cattle fed various sources of tropical roughage, potentially constituting up to 6.76 log colony-forming units (CFU) per mL of healthy cattle's ruminal fluid across different age groups (Abrão et al., 2014; Marrero et al., 2011; Fernandes et al., 2019).

In preceding study, we selected two yeast isolates from cow rumens that exhibited in vitro potential for Urochloa hay degradation alongside significant biomass production (Duarte et al., 2024). The aims in this study were to assess the in vitro digestibility of dry matter (DMD) and neutral detergent fiber (NDFD) in U. decumbens hay with the addition of these yeast strains to ruminal fluid collected during distinct periods of the dry season.

2. Material and Methods

2.1. Isolates of yeast from the rumen

Two yeast strains (V5 and V62) isolated from the rumen of Nellore cows reared in Urochloa decumbens (UD) lignified pastures in northern Minas Gerais were evaluated. These microorganisms were collected through puncture of the ventral rumen sac and isolated in a previous study (Duarte et al., 2024). These yeast strains were selected due to their high populations in the rumen environment of eight adult beef cows (>6 log CFU per mL of ruminal fluid). Additionally, they demonstrated superior UD hay degradation compared to other yeast strains in our prior investigations. The Isolates V62 and V5 were identified as Pichia kudriavzevii [MN380269] and Rhodotorula mucilaginosa [MN380264], respectively, and their D1/D2 sequences of 28 rRNA genes were deposited in the World Federation for Culture Collections (Duarte et al., 2024).

The strains were cultivated in Sabouraud broth, stored in an ultra-freezer at -80 °C, and deposited in the public Yeast Culture Collection of the Institute of Agricultural Sciences at the Federal University of Minas Gerais. The study of yeast was authorized by the National Management System of Brazil Genetic Heritage and Associated Traditional Knowledge (cadastres AC14923, ABAA22D, and A3B530C). The procedures related to yeast isolation and evaluation were undertaken within the framework of experiments that were duly submitted and approved by the Ethics Committee on Animal Experiments of the UFMG (protocols no. 156/05 and 128/2013), adhering to the regulations stipulated by the National Council for Control of Animal Experimentation of Brazil

2.2. Animal and rumen fluid collection

All procedures performed in this study were approved by the Animal Experiment Ethics Committee of the Federal University of Minas Gerais under CEUA 128/2013 registration. For the in vitro digestibility test, six Nellore heifers aged 1.5 - 2 years, reared on UD pastures, were used as rumen fluid donors. Rumen fluids were collected using an esophageal tube assisted by a vacuum pump, homogenized and stored in thermos containers before being promptly sent for in vitro digestibility testing (Watanabe et al., 2010). The collection occurred in July, August, and September, corresponding to the start, middle, and end of the dry period.

Macroscopic analysis was carried out immediately after collection in a tube containing 5 mL of the fluid sample. Attributes such as color, odor, viscosity, and methylene blue reduction potential (MBRP) at a concentration of 0.03% were evaluated. The pH of the rumen fluid was estimated using a digital potentiometer (De Hoog et al., 2000).

2.3. In vitro digestibility of dry matter and neutral detergent fiber of Urochloa decumbens hay

To prepare the inoculum, the fungi were cultured in Sabouraud broth at 39 °C for 48 hours and standardized to an approximate concentration of 10⁴ colony-forming units CFU/mL for each period under evaluation. Urochloa decumbens hay (UDH) material was collected during the dry season (March to June) from a farm in the Montes Claros region of Northern Minas Gerais, Brazil. The forage species was identified based on its morphological characteristics during flowering periods (Panda, 2014).

UD hay was dried at 40 °C for 72 hours and ground using a Wiley knife mill, resulting in particle sizes of 1-3 mm. Subsamples were then subjected to chemical composition analysis (AOAC, 2010). The composition based on dry matter (g 100 g^-1) was as follows: Dry Matter = 95.38; Crude Protein = 3.06; Protein insoluble in neutral detergent = 21.57; Protein insoluble in acid detergent = 20.59; Neutral detergent fiber = 82.26; Fiber in acid detergent = 53.04; Lignin = 7.50; Ethereal extract = 1.02; Total Sugars = 3.02; Ashes = 5.89; Calcium = 0.14; Phosphorus = 0.10; Potassium = 0.58; Magnesium = 0.20; Sulfur = 0.06; Cellulose = 45.54; Hemicellulose = 29.22; Non-Fibrous Carbohydrate = 8.43.

In vitro dry matter digestibility coefficients (DIVMD) and neutral detergent fiber digestibility (NDFIVD) of UDH were evaluated using the methodology described by Tilley and Terry (1963) with modifications by Holden (1999). These analyses were performed using a ruminal simulator (TE-150, Tecnal Scientific Equipment, São Paulo, Brazil), consisting of four glass chambers (2,500 mL) for incubation at 39 °C with a rotation rate of 5 RPM. F-57 Filter Bags (Ankon®) (5.5 cm × 5.5 cm) that were dried at 55 °C for 24 hours were used. Empty bags were weighed, and 0.5 grams of UD hay standardized to 1mm diameter were placed inside (Nocek and Russell, 1988). Each chamber received 1200 mL of McDougall buffer solution, 400 mL of filtrated ruminal liquid (using CO₂), six Ankon F-57 bags containing UDH samples, and 100 mL of sterile Sabouraud broth (control); or 100 mL of V62 strain (P. kudriavzevii), or 100 mL of V5 strain (R. mucilaginosa), or a mixture containing. The determination of the DMIVD and NDFIVD and was obtained by the formula ANKOM Technology (2014) (Equation 1).

$$\%IVDMDor\%NDFIVD\left(DMbase\right)=100–\left(W3-\left(W1xC1\right)\right)/\left(W2xDM\right)x100$$ (1)

where: W1 = Empty bag weight; W2 = Sample weight; W3 = Bag weight + residue after incubation; C1 = Correction of the white sample (final dry kiln weight / initial weight); DM= Dry matter. NDF after.

2.4. Quantification of yeast population during in vitro digestibility test

Ruminal medium collection within the ruminal simulator was conducted at zero time, 24 hours, and 72 hours, corresponding to the introduction of 6N hydrochloric acid. Using sterile syringes, 10 mL of ruminal fluid was collected. Cultivation was carried out to quantify the colony-forming units of yeast per milliliter of ruminal fluid (CFU/mL). Successive decimal dilutions were prepared in sterile saline solution, and 100 µL aliquots were inoculated onto sterile plates containing Sabouraud Agar. Following inoculation, the plates were incubated at 39 °C and observed for yeast colony growth over a period of up to seven days. Isolated colonies were obtained and subjected to Gram staining for microscopic characterization (Kurtzman and Fell, 1998; Lacaz et al., 2002).

2.5. Statistical analysis

The in vitro digestibility experiment was designed as a 3x4 factorial, comparing the effects of three ruminal fluid collection periods and four types of inoculum, with six repetitions. Normality and homoscedasticity of variances were assessed using the Lilliefors and Bartlett tests, respectively. The data underwent analysis of variance, and means were compared at a 5% significance level using the Scott-Knott test. Statistical analysis was performed using the SAEG statistical package, Version 9.

3. Results

3.1. In vitro dry matter digestibility and the neutral detergent fiber digestibility

A significant interaction was observed between the collection period of ruminal fluid and the evaluated inoculum types for DIVMD (P<0.05). The DIVMD showed significant increases with the inclusion of V5, a mixture of the two isolates, and V5 alone during the initial, intermediate, and late dry periods, respectively (Table 1).

Table 1
In vitro digestibility of dry matter of Urochloa decumbens hay inoculated with V62 yeast (Pichia kudriavzevii), V5 yeast (Rhodotorula mucilaginosa) or the mixture of these yeast from cow rumen.

Dry season	Inoculums
	V5	V62	Mix V5+V62	Control
Beginning	47.64 ± 2.41 Aa	43.33 ± 1.36 Bc	43.66 ± 0.70 Bc	46.89 ± 0.61Ab
Middle	41.28 ± 1.47 Bc	40.02 ± 3.16 Cd	49.13 ± 0.09 Aa	44.76 ± 0.07 Bb
End	49.02 ± 1.19 Aa	48.51 ± 2.30 Ab	42.08 ± 1.78 Cc	43.92 ± 2.04 Cb

• Note: Distinct uppercase letters in the column indicate differences between periods of collect of ruminal fluid. Different Lowercase letters in the lines indicate significant differences between the inoculums by Scott-Knott test at 5% significance.

Furthermore, for NDFIVD, there was a significant interaction between inoculum types and collection periods (p<0.01). The NDFIVD was notably lower when ruminal fluid was collected at the end of the dry period (p<0.001), displaying an average reduction of 7.63% compared to the initial and intermediate collection periods. Conversely, NDFIVD exhibited significant enhancements with the inclusion of the mixture of the two isolates, V62, and the mixture of the two isolates during the initial, intermediate, and final periods of the dry season, respectively (Table 2).

Table 2
In vitro neutral detergent fiber digestibility of Urochloa decumbens hay inoculated with V62 yeast (Pichia kudriavzevii), V5 yeast (Rhodotorula mucilaginosa) or the mixture of these yeast from cow.

Dry	Inoculums
season	V5	V62	Mix V5+V62	Control
Beginning	39.77 ± 1.35 Ab	39.38 ± 0.55 Bd	40.52 ± 0.12 Aa	39.23 ± 0.76 Bc
Middle	40.08 ± 0.79 Ac	40.54 ± 0.83 Aa	38.58 ± 0.66 Bd	40.20 ± 0.69 Ab
End	35.70 ± 0.80 Bd	36.57 ± 0.91 Cc	37.55 ± 3.39 Ca	37.21 ± 0.88 Cb

• Note: Distinct uppercase letters in the column indicate differences between periods of collect of ruminal fluid. Different Lowercase letters in the lines indicate significant differences between the inoculums by Scott-Knott test at 5% significance.

3.2. Quantification of yeasts during in vitro ruminal fermentation

The interaction between the three periods and treatments did not yield significant yeast concentration variations during in vitro ruminal fermentation (Table 3).

Table 3
Yeast population (colony-forming units/mL) in artificial ruminal fluid during in vitro digestibility assay of Urochloa decumbens hay inoculated with autochthonous yeast V5 (Rhodotorula mucilaginosa) and or V62 (Pichia kudriavzevii).

Inoculums	Periods during digestibility assay
	0 hs	24 hs	72 hs
V5	1.2x107	1.1x105	3.9x104
V62	2.6x107	8.4x105	6.6x104
Mix V5 + V62	7.7x107	5.6x105	6x104
Controle	6.5x106	2x105	0.6x103

• Variation coefficient = 25%.

4. Discussion

In this study, the incorporation of these live yeasts effectively contributed to the enhancement of both DMIVD and NDFIVD through direct interaction with active rumen fluid from cattle raised on lignified pasture. These yeasts were chosen from the ruminal environment of mature cows reared in lignified pastures (Abrão et al., 2014), showcasing superior adaptation to function effectively even under the selective pressures exerted by the rumen's native microbiota, which comprises a diverse array of microorganisms and antagonistic compounds (Flint et al., 2008). In this study, the UDH demonstrated limited nutritional quality, a consequence of the collection timing. The forage was gathered during the dry period following seed dispersal, characterized by diminished nutritional value and heightened lignification of plant tissues (Santos et al., 2008).

Ruminal fermentation results from intricate ecological interactions among ruminal microorganisms. The ecosystem's structure can be dynamically influenced by host characteristics and provided food (Weimer et al., 2010; Weimer, 2011; Lukáš et al., 2010). These fluctuations affected the fluid composition in animals during the dry period investigated in this study, thereby influencing the optimal inoculants for each phase of the dry season. Toward the end of the dry season, the ruminal fluid exhibited greater aqueous content and reduced activity, contributing to the lowered digestibility of UDH.

The digestion of plant biomass is facilitated through symbiotic interactions among microorganisms that produce complementary enzymes (Cragg et al., 2015). Fungi that utilize lignified biomass rely on intricate degradation mechanisms that act on the substrate (Cragg et al., 2015). The repertoire of enzymes utilized by these organisms includes a comprehensive set of cellulases and enzymes capable of oxidizing lignin components, encompassing ligninase, peroxidase, and laccase, either individually or in combination (Pollegioni et al., 2015).

The strain Pichia kudriavzevii, assessed by Chan et al. (2012), demonstrated fermentative potential for xylose. The authors identified the expression of genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. Furthermore, the species P. kudriavzevii possesses three genes encoding phytases (Greiner and Konietzny, 2006), which could hold significance in ruminal fermentation by contributing to the availability of substrates for cellulolytic microorganisms.

The species Rhodotorula mucilaginosa can be isolated from diverse natural and extreme environments, including the intestinal microbiota of various animal species (Raggi et al., 2014). Alongside its ability to thrive in challenging conditions, this yeast exhibits notable biotechnological characteristics such as biosurfactant, unsaturated fatty acid, and carotenoid production (Aksu and Eren, 2005; Kawahara et al., 2013; Singh et al., 2013). However, a comprehensive understanding of R. mucilaginosa 's genomic potential is essential, encompassing aspects of cell physiology, biochemistry, and potential biotechnological applications in human and animal health (Deligios et al., 2015).

While numerous microorganisms possess cellulose-degrading capabilities, strategies for lignin removal remain limited. Certain ligninolytic fungi and bacteria function as lignin degraders through the action of oxidative enzymes like peroxidases and laccases (Bugg et al., 2011; Pollegioni et al., 2015). In other studies, yeast supplementation has led to improved in vitro ruminal fermentation of fibrous substrates. For instance, a Candida norvegensis strain stimulated cellulolytic fungal population, resulting in increased volatile fatty acid (VFA) production and DIVMD (Marrero et al., 2015).

The stimulatory impact of R. mucilaginosa as a co-culture for laccase production in Pleurotus ferulae JM301 was demonstrated. The researchers observed elevated total protein concentration due to the yeast, underscoring the biotechnological potential of R. mucilaginosa (Wang et al., 2015). Subsequent investigations should explore the potential advantageous effects of this species on ruminal microorganisms effective in the utilization of lignified forages, including anaerobic rumen fungi (Hess et al., 2020).

In this study, yeast counts were observed even in rumen fluid without the inoculation of the selected strains, indicating the natural presence of yeast in the ruminal fluid of the assessed heifers. This finding aligns with a prior study involving cattle fed the same species of pasture (Abrão et al., 2014). Consequently, forthcoming investigations should explore the impact of yeast inoculation in diets for ruminants with low-fiber diets, assessing its potential benefits in colonizing and stabilizing the ruminal microbiota of feedlot animals, recently weaned animals, or those with gastrointestinal disorders. In this study, yeasts previously selected for better degradation of the UD forage were inoculated, thus the presence of these yeasts and its secreted enzymes in the inoculated ruminal fluid could be contributing to improving in vitro digestibility in relation to the control ruminal fluid.

The reduction of one log in yeast population after 72 hours might be attributed to the addition of hydrochloric acid, which lowered the pH to simulate abomasum conditions. The predominant morphotypes observed with the inoculation of strains V5 and V62 corresponded, respectively, to the genera Rhodotorulla spp. and Pichia spp. This indicates the continued survival and presence of these yeasts in the in vitro fermentation of rumen fluid, even subsequent to simulating acidic conditions akin to those in the abomasum.

5. Conclusion

The inclusion of specific rumen yeast strains, whether combined or singularly, enhances the in vitro digestibility of both dry matter and neutral detergent fiber in U decumbens. Additionally, the efficacy of these yeast strains varies depending on the selection of particular strains tailored to different periods within the dry season in semi-arid regions. Importantly, it is noteworthy that these yeasts exhibit resilience to acidic conditions akin to those found in the abomasum.

Acknowledgements

We are grateful to the Pró reitoria de Pesquisa of Universidade Federal de Minas Gerais and Universidade Estadual do Sudoeste da Bahia, campus Itapetinga. This project was supported by grants from the Pro Reitoria de Pesquisa da Universidade Federal de Minas Gerais, Conselho Nacional de Desenvolvimento Científico e Tecnológico (project 310898/2018-8) and Fundação de Pesquisa do Estudo de Minas Gerais (FAPEMIG projet PPM-00393-17).

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Publication Dates

• Publication in this collection
  11 Feb 2025
• Date of issue
  2024

History

• Received
  29 June 2024
• Accepted
  11 Nov 2024