Aspergillus spp. isolates from bovine rumen can increase the digestibility and degradability of forages Isololados de Aspergillus spp. provenientes do rumen podem melhorar a digestibilidade e degradabilidade de forrragens Aislamientos de Aspergillus spp. del rumen puede mejorar la digestibilidad y degradabilidad del forrajes

The bovine production on pastures is a sustainable activity, responsible to generating income and employments in many tropical regions. However, in this system a major obstacle occurs during long dry periods, when the forages show low nutritional quality. In this research we evaluated the in vitro digestibility and degradability of Urochloa decumbens and Cynodon dactylons var Tiffton 85 inoculated with isolates of Aspergillus spp. from the gastrointestinal tract cattle. Four cows with rumen fistula were used as rumen contents donors to determine the in vitro digestibility of dry matter (IVDMD). The treatments were, as follows: control No fungal inoculum, AT13 – (with Aspergillus terreus isolate), AF69 – (with Asperillus fumigates isolate, MIX (with mix of fungi AT13 and AF69). The analyses were performed in rumen simulator with four replications. The gas production was determined by semiautomatic method for both gas and the mechanics ability to fiber colonization of these fungi isolates were evaluated by scanning electron microscopy. The inclusion of AF69 and MIX promoted significant increase of IVDMD of U. decumbens (41.4 42.1%). The dry matter degradability were not influenced by the inoculums types using the by Research, Society and Development, v. 10, n. 8, e56410817340, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17340 2 semi-automatic gas production method However, they linearly increased with fermentation time. The two isolates of Aspergillus spp. were able to colonize the U. decumbens fiber showing production of mycelium and reproductive structures in electronic microscopic analyses. The addition these Aspergillus isolates promotes significant increase of IVDMD to U. decumbens, indicating promising potential for development of microbial or probiotic additive to cattle raised on lignified tropical pasture.


Introduction
The remarkable capacity of protein production in ruminants is attributed to the pre-stomach system which houses a complex microbial ecosystem (Kamra, 2005;Liu et al. 2020;Russell and Rychlik 2001). However, in semiarid tropical regions, the quantity of available fodder and the pasture quality are compromised during dry periods when the physiological lignification of the vegetal cell wall reduces the forage digestibility (Moore, and Jung 2001).
Alternatively, fungus species produce synergistically-acting lignocellulolytic enzymes which reduce lignocellulosic residues in nature and to favor the cellulose releasing and other carbohydrates for ruminant feed (Dashtban et al. 2001;Russell and Rychlik 20002001).
Anaerobic ruminal fungi shows major importance for tropical forage digestion and degradation lignified fibers (Hartinger et al. 2021;Paul et al. 2004). However theses fungi have showed labors culture conditions in laboratory (Hartinger et al. 2021). Alternatively, facultative anaerobic fungi have notable production of enzymes that facilitate polysaccharide degradation in ruminants. Microbial cultures of Aspergillus oryzae, and the extracts of these fungi may been used as supplements in ruminant diets, improving productivity by 7-8% (Chiou et at., 2002;Sobhy Sallam et al. 2020).
In an earlier study, beef cattle feeding only on lignified tropical pastures contained predominantly Aspergillus spp. in the rumen fluid between fungi isolates ). Isolates of this genus has shown potential for supplementation of animal feeds as well as for industrial purposes (Facchini et al. 2012, Sallam Sobhy et al. 2020Zilio et al. 2019). Aspergillus spp. isolates from the digestive tract of dairy cattle expressed notable capacity for microcrystalline cellulose degradation and were not micotoxin producer of (Almeida et al. 20172014;Abrão et al. 2018). The inclusion of additives containing exogenous fungi and their enzymes to ruminant diets has promoted increase of ruminant production by optimizing digestion (Yang et al. 2000).
We previously selected two isolates of Aspergillus spp. presenting probiotic characteristics and superior ability to degrade vegetal cell wall (Abrão et al. 2017;Abrão et al. 2018). Here we are evaluating the hypothesis that these fungi, isolated from bovine digestive tract, could colonize lignified grass fibers and to improve their digestibility and dry matter degradability when inoculated in a ruminal simulation environment.

Fungal isolates
We evaluated an isolated of Aspergillus terreus (AT13), Acc. Genbank KF781532, from the rumen and one isolate of Aspergillus fumigatus (AF69), Acc. Genbank KF781534,-from the rectum of Nellore calves raised in U. decumbens in the north of Minas Gerais, Brazil, during the dry season ). These isolates were identified after the amplificaction of ITS region of rDNA and the sequencing was performed with DYEnamic (Amersham Biosciences, USA) in a Mega-BACE 1000 automated sequencing system at the Genome Analysis Center and Gene ExpressionThe obtained DNA sequences were analyzed using BLASTn (v.2.215) of BLAST 2.0 at the NCBI website, as reported by (Abrão et al. 2017).
In previous studies, these isolates showed superior production of degradation enzymes of plant cell wall, tolerance to ruminal fermentation fatty acids, did not produce mycotoxins and increased the production of gluconic, butyric and acetic acids in the fermentation of U. decumens (Abrão et al. 2017, Abrão et al. 2018

Evaluated forages
Samples of Urochloa decumbens hay were collected during the dry period in the North of Minas Gerais, a semiarid tropical region. Tifton 85 (Cynodon dactylons) hay was commercially purchased in the region of Belo Horizonte -MG, Brazil.

In vitro digestibility of dry matter of forages
The fungi were cultured in microaerophilia, in Sabouraud broth at 39°C under shaking for 48 h and were standardized to approximately 10 8 spores/ mL. Four cows with ruminal fistulas were used to ruminal fluid donors. These animals were housed in individual stalls for 15 days, with 14 days for adaptation to diets (70% Tifton 85 hay + 30% commercial concentrate + mineral mix) and on the last day the ruminal fluid was collected. The ruminal contents were homogenized, filtered and stored in thermal bottles and immediately sent for analysis. All the procedures were approved by the Animal Experimentation Ethics Committee of the Federal University of Minas Gerais under registration CETEA 128/2013.
In vitro dry matter digestibility coefficients (IVDMD) of U. decumbens and Tifton 85 hay (Cynodon spp.) were evaluated according to the methodology described by Tilley and Terry (1963), following the modifications described by Holden (1999)  The statistical design used in this study was in a randomized block in factorial 4 x 2 (fungi x forage source) with four repetitions. Each block was a period of evaluation. Normality and homoscedasticity of variances were evaluated using the Lilliefors and Bartlett tests, respectively. The data were submitted to analysis of variance using the ASSISTAT 7.7 Beta statistical program and the means compared by the Scott-Knott test at the 5% probability level (P ≤ 0.05).

Evaluation of the degradation and fermentation of the fodders by the semi-automatic gas production method
The inoculations with saline containing the fungus AT13, the AF69 or MIX of both fungi were evaluated. Saline solution without fungal spores was used in the control. Samples of U. decumbens and Tifton 85 hay were ground in a 1 mm sieve mill and incubated in glass vials of 160 mL capacity, prewashed with distilled water and dried. Subsequently the vials were saturated with carbon dioxide (Ankom 2014).
Two flasks (replicates) were used per sample of roughage by gas production time (subplot) incubated with pooled rumen liquid from cows fed as previously described. An Ankom sac was filled with approximately 0.5 g of the bean per fermentation flask along with 12.5 mL of buffer culture medium and 4 mL of the ruminal fluid with 0.5 mL of standardized microbial additive (10 8 spores/mL). In the control the same procedure was adopted replacing the fungal additives with sterile saline solution. To eliminate the residual effect of the sachets, blank bottles containing the empty sachets were evaluated in duplicate for each treatment and control (Ankom 2014).
The dry matter degradability was obtained by the difference between the initial dry weight of the sample bags and the residue obtained at the times of 6, 12, 24, 48, 96 h. The sachets were then kept for 8 h in a 105 ºC oven, after which they were weighed and the weight used to calculate the DM degradability (Ankom 2014).
The pressure data (psi) were used to calculate the volume of gases (mL) produced according to the mathematical equation standardization for tropical forages: mL = -0.085 + 3.582psi + 0.019psi 2 . Subsequently, the accumulated gas productions (mL) were estimated.
Normality and homoscedasticity of variances were evaluated using the Lilliefors and Bartlett tests, respectively. In order to evaluate the degradability of the dry matter and gas production of the two bulks, the design was used in randomized blocks with subdivided plots. In the plots the effects of the inclusion of the fungi and the blocks were studied, while in the subplots. The effects of the incubation time and its interaction with the treatments tested (4 treatments x 16 times x 2 blocks).
The means were compared by the Scott-Knott test (P < 0.05). As the quantitative factor "time" was significant, regression analysis was applied to the degradability variable. The ASSISTAT 7.7 Beta software was used for data analysis. The cumulative gas production was presented in a descriptive way.

Scanning electron microscopy analysis
For inoculation of evaluated fungi isolates, spore suspensions were obtained from colonies cultured for seven days in Cellulose medium at 37 °C. Ten mL of a suspension was prepared in saline plus 0.1% Tween 20® (v/v), containing 10 8 spores/mL.

In vitro dry matter digestibility
As expected, it was observed that of the Tifton 85 of the genus Cynodon presents superior nutritional quality (Table   1). Consequentially, for the four inoculuDM evaluated, U. decumbens IVDMD was significantly lower (35.3 to 42.1%), compared to that observed for Tifton 85 (51.6 to 60.2%). Treatments with A. fumigatus and fungal mix promoted a significant increase in the IVDMD of U. decumbens (Table 2). For Tifton 85 hay the digestibility was higher using the A3 (A. fumigatus) treatment (P < 0.05).
After inclusion of the isolate AF69 and the mix containing both fungi was observed an increase in the U. decumbens digestibility by approximately 7%. For Tifton 85, the increase was observed up to 8.6% with the addition of this isolate. Note: The differences in capital letters in the rows, for the forage types, and low letters in the columns, for the inoculuDM types, indicate different means by the Scott-Knott test at 5% probability. Source: Authors.

Degradation and fermentation by the semi-automatic gas production method
The dry matter degradability was not influenced by the treatments (P > 0.05), as shown in the Table 3. Synergistic effect of the two fungi isolates was not observed to Tifton 85 fermentation (Table 3). However, this effect was verified for the production of gas in the U. decumbens.
Despite the significant interaction between the type of fungal additive and time, the pressure produced by the fermentation (measured in psi) did not differ (P > 0.05) among the treatments evaluated within each time. This result occurred for both forages. Thus, the non-cumulative gas production was not influenced by the fungi addition, but was significant for the fermentation time (Y Urochloa=0.22+0.01X, R 2 =0.99; Y Tifton=0.44+0.07X, R 2 =0.97) (P < 0.05).

Scanning electron microscopy analysis
In the control, no microbial growth was observed, preserving the forage cell wall structure (Figure 1). In the Figure 2 and 3, the colonization of the fungus isolates was observed with 24 and 96 hours of incubation, under a smaller increase.
Development of both fungi isolates on the surface of plant fibers has been confirmed, showing capacity of these organisms to colonize and to degrade tropical forages such as U. decumbens. A distinct development profile was observed among the fungal species evaluated, with the highest mycelial mass produced by AT13 isolate (Figure 2). AF69, while producing less mycelium, there was more evenly distributed throughout the tested forage fragment (Figure 3). Research, Society andDevelopment, v. 10, n. 8, e56410817340, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17340 8 Source: Authors.

Discussion
The increase of the U. decumbens digestibility with the AF69 inclusion could demonstrate the potential of this fungus as a microbial additive in the diet of ruminants fed with tropical forages In a previous study, the analysis of AT13 and AF69 did not show this isolates as micotoxin produces and they were resistant to different proportions of ruminal fatty acids.
Additionally, these fungi presented viability on autochthonous microbiota, showing physiological characteristics to be used with microbial additives or probiotic (Abrão et al. 2018).
In this study, tested fungal additives did not influence the DM degradability, which could have been influenced by the variability of the results obtained in semi-automatic gas production technique. However, the degradability of both forages linearly increased with the fermentation time, indicating that the 96-hour period was more suitable to achieve better rates, allowing better colonization and performance of fibrolytic ruminal microbiota. Future studies should elucidate this effect, as well as the influence of fungal additives on the digestibility of FDN and, crude protein, among others.
As expected, U. decumbens showed lower digestibility and degradability of DM than C. nlenfuensis and were justified by their differences in nutritional compositions. The U. decumbens was collected at the end of the dry season and it presented low nutritional value and high lignification index of the vegetal tissues. Differently, Cynodon sp. presented superior nutritional quality because it was produced in haymaking process, and was collected when presented there was good nutritional characteristics, being removed only the moisture of the food.
The quantity and quality of pastures are drastically reduced during dry periods, in semiarid regions. The forage digestibility is reduced by the physiological lignification process in plant cell wall (Moore and Jung 2001). Fungi produce lignocellulolytic enzymes and contribute synergistically to the decay of lignocellulosic residues in nature (Dashtban et al. 2010). When lignin complex is removed, cellulose and other carbohydrates may be released for ruminal microbiota actuation (Kamra 2005).
In previous study, AT13 and AF69 isolates presented intense production of phenoloxidases, important lignindegrading enzymes (Abrão et al. 2017), suggesting their potential to degrade lignified forage during the dry season.
Additionally, these fungi showed expressive production of avicelases that were higher at 48 h than at 24 h of incubation, probably due to an adjustment period of fungal growth in vegetal fiber (Abrão et al. 2017). Avicelases are import to bioconversion of agricultural wastes to useful products, catalyzing most of the bond-cleavages during saccharification of microcrystalline cellulose (Liu et al. 2011).
Similarly to the observed in this study, anaerobic ruminal fungi promoted major importance for tropical forage digestion and degradation lignified fibers (Lee et al. 2000, Paul et al. 2004) than forages with better quality. In another study, the in vitro analyzes of fiber digestibility and ruminal fermentation revealed that the addition of rumen anaerobic fungal cultures significantly increased acetate production, in vitro dry matter digestibility and microbial biomass synthesis. The inclusion of Orpinomyces spp. showed better plant fiber degrader than Anaeromyces sp. (Sirohi et al. 2012).
Scanning microscopy reinforced the results reported in the other experiments of this study, validating the ability of fungal isolates to colonize tropical forage fiber, demonstrating a distinct growth profile among fungi, which could confirm the higher probiotic capacity of isolate AF69 on the digestibility of U. decumbens. In another study, Curvelo et al. (2010) also demonstrated ability of the fungi Ramularia areola on the development in plant samples, and points to sporulation via stomata in leaf veins because, in direct contact with the xylem vessels.

Conclusion
In this study, the addition of the isolate AF69 (Aspergillus fuminatus), from Nelore calf, promotes a significant increase of the IVDMD for U. decumbens, pointing promising potential for the elaboration of probiotic or microbial additive for cattle raised in pastures containing this forage. The AF69 (A. fumigatus) and AT13 (A. terreus) isolates are able to colonize the surface of the vegetal fiber, which could favor the digestibility of the lignified vegetal fiber and use by other groups of ruminal microorganisms. Future studies should elucidate the best formulations and strategies to use these fungi in diets of cattle fed on lignified forages