ANIMAL SCIENCE

Effects of Aspergillus oryzae on ruminal fermentation of an alfalfa hay:concentrate diet using the rumen simulation technique (Rusitec)

0000-0001-5426-9660Areadne Sosa^{1  *}  , 0000-0002-1887-4529Cristina Saro^{2  3}  , 0000-0002-9764-0404I. Mateos^{2  3}  , 0000-0002-4195-3305A. Díaz^{2  3}  , 0000-0001-8639-4693Juana Galindo¹  , 0000-0002-4221-9057María D. Carro⁴  , 0000-0002-4000-7704María J. Ranilla^{2  3} 

¹Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba

²Departamento de Producción Animal. Facultad de Veterinaria, Universidad de León, 24071 León, Spain

³Instituto de Ganadería de Montaña, CSIC-Universidad de León, Finca Marzanas s/n, 24346 Grulleros, Spain

⁴Departamento de Producción Agraria, Escuela Técnica Superior de Ingeniería Agronómica, Agroalimentaria y de Biosistemas, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040 Madrid, Spain

Abstract

Direct-fed microbial products have been shown to modify ruminal fermentation parameters and to have beneficial effects on ruminant performance. The aim of this study was to evaluate the effect of Aspergillus oryzae on ruminal fermentation of a 65:35 alfalfa hay:concentrate diet in continuous fermentation system Rusitec. Four fermenters corresponded to the control and four received 50 mg of A. oryzae lyophilized culture which was mixed with feed. Treatment with A. oryzae increase (p < 0.001 to p = 0.039) acetate, total short chain fatty acids (SCFA), minor SCFA production and acetate:propionate (Ac:Pr) ratio. A. oryzae addition decreased (p < 0.001) pH values and increased (p < 0.001) ammonia-N concentration. There were no differences among treatments in methane production (p = 0.230). The inclusion of A. oryzae did not affect (p = 0.386 to p = 0.865) the dry matter and fiber disappearance. Rumen fluid from A. oryzae fermenters did not affect (p=0.465 to p=0.938) gas production neither (p = 0.131 to p = 0.942) total SCFA from pure substrates. The increase of acetate, minor SCFA and total SCFA production indicate that A. oryzae strain stimulated rumen fermentation of a 65:35 alfalfa hay:concentrate diet in continuous fermentation system Rusitec.

Key words: continuous fermenters; direct-fed microbial; fiber degradability; volatile fatty acids

The use of direct-fed microbial products as feed additives is a strategy to optimize the production efficiency of ruminants and can be used as alternative to the use of antibiotics as growth promoters (^{Shen et al. 2018}, ^{Mamuad et al. 2019} and ^{Sklyar and Gerun 2020}). Among the microbial species that are used for this purpose is the fungus Aspergillus oryzae. This microorganism enhanced the number of cellulolytic bacteria, total viable bacteria and fungal populations (^{Beharka and Nagaraja 1998} and ^{Sun et al. 2014}). Stimulation of these ruminal populations leads to an increase in the concentration of SCFA and the synthesis of microbial proteins (^{Seo et al. 2010} and ^{Sun et al. 2017}). In dairy cattle, an improvement of productive indicators was demonstrated with the inclusion of A. oryzae in the diet (^{Sucu et al. 2018} and ^{Sallam et al. 2019}).

Most research to evaluate the effect of this microbial additive is made in vivo conditions or using batch cultures of rumen microorganisms, as in vitro system. Few studies evaluate the effect of A. oryzae in continuous fermentation systems (^{Frumholtz et al. 1989} and ^{Newbold et al. 1991}), which is an option that generates short term, useful information to determine the efficiency of the organism under study (^{Martínez et al. 2009} and ^{Wagner et al. 2018}).

In addition, most of these experiments have been conducted with commercial products based on A. oryzae fermentation extract dried on insoluble base of wheat bran (^{Yohe et al. 2015}). Limited data involve the application of live culture of this microorganism and its potential as ruminal fermentation modifier has received much less attention. Furthermore, in tropical countries the systems of animal feeding, specifically for the dairy cattle, are based on grasses and forages of low nutritive value that don’t cover the requirements of these animals. Microbial additive products in the international market have a high price that makes it impossible to import for Latin American developing countries, so it is essential to obtain own additives to respond to the situations generated in animal production.

As strategy to improving the use of available resources to increase the productive efficiency of ruminants, the objective of this study was to evaluate the effect of A. oryzae live culture as activator of rumen fermentation in the continuous fermentation system Rusitec fed with a mixture of alfalfa hay and concentrate.

Materials and Methods

The experimental protocol was approved by the León University Institutional Animal Care and Use Committee (Project AIB2010NZ-00190). Sheep were cared and handled by trained personnel in accordance with the Spanish guidelines for experimental animal protection (Royal Decree 53/2013 of February 1st on the protection of animals used for experimentation or other scientific purposes).

One 10-d incubation trial was carried out using eight Rusitec fermenters with an effective volume of 600 mL each in a completely randomized design. Four fermenters corresponded to the control and four received 50 mg of A. oryzae culture (strain H/6.28.1 from the collection of the Cuban Institute of Research of Sugarcane Derivatives, ICIDCA). The culture was obtained by growth in Malt Extract Broth (Fluka) for seven days. Dose selection was based on the results of ^{Sosa et al. (2010)}.

The general incubation procedure was as described by ^{Czerkawski and Breckenridge (1977)}. On the first day of the experiment, each fermenter was inoculated with 250 mL of strained rumen fluid, 250 mL of artificial saliva (pH 8.4) (^{McDougall 1948}) and 80 g of solid rumen content supplied into a nylon bag (100 µm pore size). Ruminal content was obtained from four rumen-cannulated Merino sheep fed the same diet administered to the fermenters. Ruminal contents of each sheep were obtained immediately before the morning feeding. Solid and liquid fractions were collected in separated flask. The content was mixed and strained through four layers of cheesecloth. Both fractions were transferred to the in vitro system within 30 min as previously described by ^{Carro et al. (1992)}.

Each fermenter received 20 g of substrate dry matter daily, which were placed into nylon bags. The substrate consisted on 13 g of alfalfa hay (65%) and 7 g of concentrate (35%). Ingredients and chemical composition of the substrate are shown in table 1. Alfalfa hay was chopped (approximately 0.5 cm pieces), and the concentrate was ground through a 4 mm sieve. Both feed components were weighed independently and carefully mixed before applying the experimental treatments. The four bags corresponding to the A. oryzae treatment received 50 mg of microorganism which was mixed with the substrate. The flow through fermenters was maintained by continuous infusion of ^{McDougall (1948)} artificial saliva (pH 8.4) at a rate of 610 mL/d (dilution rate of 4.24 %/h).

Table 1 Ingredient composition and chemical analysis of experimental diets fed to donor sheep and used as substrates for the Rusitec fermenters 

Ingredients	g/kg of dry matter
Alfalfa hay	650.0
Barley	75.0
Gluten feed	71.0
Wheat middlings	70.0
Soybean meal	47.0
Palmkern meal	40.0
Wheat	18.0
Corn	11.0
Mineral-vitamin premix^1	18.0
Chemical composition
Organic matter	928.0
Neutral detergent fiber	507.0
Acid detergent fiber	248.0
Crude protein	131.0

¹ Declared composition (g/kg mineral/vitamin premix): Vitamin A, 600,000 IU; Vitamin D3, 120,000 IU; Vitamin E, 1 g; Vitamin Bl, 33 mg; Niacine, 1.5 g; S, 5 g; IK, 300 mg; SO₄Fe, 1 g; ZnO, 4 g; MnO, 2 g; CoSO₄, 60 mg; Na₂SeO₃, 30 mg; Ethoxyquin, 30 mg.

On the second day the nylon bag with solid rumen content was removed, and a new bag with substrate was supplied. On subsequent days and until the end of the experiment, the bag that had spent 2 d in the fermenters was removed, and a new bag with feed was introduced to achieve an incubation time of 48 h.

Sampling, analytical procedures and statistical analyses. In each fermenter the pH was measured daily before the feeding. Every day liquid effluent was collected in a flask containing 20 mL of sulfuric acid solution (20%; vol:vol) to maintain pH values below 2. After 7 d of adaptation, on days 8, 9 and 10, samples for gas, SCFA and ammonia-N determination were collected following the procedures described by ^{Martínez et al. (2009)}. The nylon bag collected from each fermenter was washed twice with 40 mL of fermenter’s fluid, and then washed with cold water for 20 minutes in a washing machine. The dry matter apparent disappearance after 48 h incubation was calculated from the loss in weight after oven drying at 60ºC for 48 h, and the residues were analyzed for neutral detergent fiber and acid detergent fiber content, according to van ^{Soest et al. (1991)}, in an ANKOM220 fiber analyzer (Ankom Technology Corporation, Fairport, NY, USA) to estimate neutral detergent fiber and acid detergent fiber disappearance.

Effluent samples were analyzed for individual SCFA by gas chromatography, as indicated by ^{Carro et al. (1992)} and ammonia-N by the method of ^{Weatherburn (1967)}. Gas production (L/d) was measured with a drum-type gas meter (model TG1; Ritter Apparatebau GmbH, Bochum, Germany). The amount of methane produced (mmol/d) was calculated by multiplying the gas produced by the methane concentration which was determined by gas chromatography as described by ^{Carro et al. (1992)}.

Adaptive changes in the microbial population of fermenters to A. oryzae treatment were studied using the fluid from each fermenter as inoculum for batch cultures and measuring the response in final pH, gas production and SCFA. The fermentative activity of the fluid was tested against three pure substrates (Sigma-Aldrich Química SA, Madrid, Spain): starch (mixture of 40% corn, 40% wheat and 20% potato starch), oat spelt xylan and cellulose. On the last day of Rusitec trial 300 mL of fluid from two pairs of fermenters of each treatment were mixed, obtaining two inocula for control and two for A. oryzae treatment. Six hundred mL of each inoculum were mixed with 150 mL of artificial saliva enriched with N (1 L of artificial saliva containing 472 mg of NH₄Cl and 791 mg of trypticase), and 40 mL of the final mixture were anaerobically (under continuous flushing with CO₂) dispensed into 120-mL serum bottles containing 400 mg of one of the substrates described above. Twelve bottles (three bottles for each substrate and three bottles without substrate) were incubated per each inoculum. The bottles were capped and incubated at 39°C for 9 h for cellulose and for 6 h for the rest of substrates. The amount of gas produced was measured, the bottles were opened the pH was immediately measured and samples of 0.8 mL were added to 0.5 mL of a deproteinizing solution (20 g of metaphosphoric acid and 0.6 g of crotonic acid per liter) for the analysis of SCFA.

Data were analyzed using the PROC MIXED procedure of SAS (version 9.4) (^{SAS, 2013}), for a completely randomized design. Effects included in the model were the treatment, the incubation day, and the interaction between treatments and incubation day, and fermenter as a random effect.

Results and Discussion

Effect of A. oryzae on fermentation parameters in Rusitec fermenters is shown in table 2. In the present study there were observed increases of acetate and total SCFA production (p=0.022 and p=0.039, respectively), with the inclusion of the fungus, which is consistent with that reported by other authors (^{Wiedmeier et al. 1987}, ^{Frumholtz et al. 1989}, ^{Fondevila et al. 1990}, ^{Newbold et al. 1991} and ^{Sun et al. 2017}), as the increase (p < 0.001) in the acetate:propionate ratio (^{Wiedmeier et al. 1987} and ^{Frumholtz et al. 1989}). The SCFA represent the main supply of metabolizable energy for ruminants, and therefore an increase in their production would be nutritionally favorable for the host animal. Increase (p < 0.001 to p = 0.038) in minor SCFA production was also observed after inclusion of the additive. Specifically, the production of the branched SCFA is due to deamination and decarboxylation of branched chain amino acids. A. oryzae is highly proteolytic (^{Kumura et al. 2011} and ^{Su et al. 2011}) and it could be the proteolytic activity of the fungus that contributes to the formation of these compounds. The increase of these isoacids is a relevant aspect because there are growth factors for fibrolytic rumen bacteria as Ruminococcus albus and Fibrobacter succinogenes (^{Hardy 1987}).

Table 2 Effect of A. oryzae on SCFA production (mmol/d), acetate:propionate ratio (Ac:Pr, mol/mol), pH, ammonia-N concentration (mg/d) and methane production (mmol/d) 

SCFA	Control	A. oryzae	SEM	p value
Total SCFA	58.8	65.9	2.23	0.039
Acetate	31.4	35.7	1.21	0.022
Propionate	9.76	10.3	0.390	0.348
Butyrate	9.82	10.5	0.370	0.238
Isobutyrate	0.54	0.69	0.022	<0.001
Isovalerate	1.80	2.09	0.060	0.003
Valerate	3.45	3.87	0.132	0.038
Caproate	2.09	2.78	0.094	<0.001
Ac:Pr	3.22	3.47	0.033	<0.001
pH	6.77	6.69	0.014	<0.001
Ammonia-N	146	193	6.0	<0.001
Methane	20.9	22.3	0.77	0.230

With A. oryzae addition pH values decreased (p<0.001) but remained close to neutrality. This decrease in pH could be attributable to the increase in total SCFA production. The increase (p<0.001) in ammonia-N concentration coincides with the results of ^{Frumholtz et al. (1989)} who observed 30% increases in this issue. Several authors found that A. oryzae inclusion stimulates ammonia-N production by rumen microorganisms (^{Arambel et al. 1987}, ^{Frumholtz et al. 1989} and ^{Martin and Nisbet 1990}), suggesting that microbial additives favor in vitro proteolysis.

With the addition of A. oryzae no difference on methane production (p = 0.230) was observed (table 2). It has been reported that microbial additives are able to reduce rumen methanogenesis (^{Sharma 2005}). ^{Frumholtz et al. (1989)} reported a decrease on methane production by adding an A. oryzae fermentation extract in Rusitec fermenters which was consistent with the increased production of reduced products such as butyrate and valerate. These authors used higher doses of A. oryzae fermentation extract (250 mg in fermenter with effective volume of 850 mL). In the present study increase in the acetate:propionate ratio was observed and consequently the reduction in methane is not expected. The formation of methane in rumen fermentation is thus closely associated to the profile of SCFA formed, propionate competes with methane as a hydrogen sink in rumen fermentation whereas acetate formation release hydrogen that can be utilized by methanogens to reduce CO₂ to methane (^{Ungerfeld 2020}). The difference with other studies could be due to the strain and doses used. ^{Newbold and Rode (2006)} affirm that responses of yeast cultures in Rusitec fermenters were highly variable and depended on the strain used which may be related to differences in metabolic activity of the strains. The influence of strain and dose was also demonstrated by ^{Jiao et al. (2018)} in studies with yeast in batch culture of rumen microorganisms.

Results of A. oryzae effect on diet disappearance are shown in table 3. Fungus inclusion did not affect (p = 0.386 to p = 0.865) the dry matter and fiber disappearance. Among the beneficial effects of this additive is its ability to stimulate fiber degradability, which was demonstrated in in vitro (^{Arambel et al. 1987}) and in vivo (^{Wiedmeier et al. 1987}) research. In previous studies, ^{Newbold et al. (1991)} observed that A. oryzae stimulated dry matter disappearance within 24 hours of fermentation, but not at 48 hours. This probably occurred by an improved rate rather than extent of digestion (^{Fondevila et al. 1990}). In the present study feed bags were removed after 48 hours and this could explain the lack of effect observed in the dry matter and fiber disappearance. On the other hand, the increase in the total SCFA production at 24 hours is consistent with the hypothesis that A. oryzae improved the rate of digestion when added to the feed in Rusitec fermenters.

Table 3 Effect of A. oryzae on diet disappearance 

Diet dissapearance (g/kg)	Control	A. oryzae	SEM	p value
Dry matter	572	570	0.6	0.865
Neutral detergent fiber	275	270	1.2	0.756
Acid detergent fiber	185	171	1.1	0.386

The effect of inoculum from Rusitec fermenters in fermentation of pure substrates is shown in table 4. For all substrates there were no differences (p = 0.824, p = 0.938 and p = 0.465 for starch, xylan and cellulose, respectively) between control fermenters and those with A. oryzae in gas production.

Table 4 Effect of inoculum from fermenters Rusitec on pH, gas (mL) and SCFA production (mmol) and the Ac:Pr ratio (mmol/mmol) in fermentation of pure substrates¹ 

Sustrate and item	Control	A. oryzae	SEM	p value
Starch
Gas production	26.7	26.4	1.09	0.824
pH	6.84	6.92	0.017	0.007
Total SCFA	3.91	3.91	0.059	0.942
Acetate	2.06	2.13	0.033	0.187
Propionate	0.63	0.55	0.013	0.002
Butyrate	0.73	0.68	0.010	0.007
Isobutyrate	0.03	0.04	0.001	<0.001
Isovalerate	0.12	0.13	0.002	0.005
Valerate	0.22	0.21	0.003	0.029
Caproate	0.12	0.17	0.006	<0.001
Ac:Pr	3.28	3.85	0.045	<0.001
Xylan
Gas production	14.4	14.5	0.54	0.938
pH	7.16	7.17	0.023	0.761
Total SCFA	3.75	3.80	0.079	0.682
Acetate	2.08	2.17	0.044	0.190
Propionate	0.62	0.55	0.016	0.018
Butyrate	0.59	0.56	0.015	0.274
Isobutyrate	0.03	0.04	0.001	0.002
Isovalerate	0.11	0.12	0.002	0.010
Valerate	0.21	0.20	0.004	0.163
Caproate	0.12	0.16	0.006	<0.001
Ac:Pr	3.37	3.93	0.066	<0.001
Cellulose
Gas production	15.2	15.8	0.547	0.465
pH	7.26	7.26	0.014	1.000
Total SCFA	3.26	3.43	0.074	0.131
Acetate	1.70	1.86	0.038	0.012
Propionate	0.55	0.51	0.017	0.152
Butyrate	0.56	0.55	0.015	0.546
Isobutyrate	0.03	0.04	0.001	<0.001
Isovalerate	0.10	0.12	0.002	0.003
Valerate	0.20	0.20	0.004	0.783
Caproate	0.11	0.15	0.006	<0.001
Ac:Pr	3.07	3.64	0.060	<0.001

¹ Starch and xylan were incubated for 6 h and cellulose for 9 h

In the case of starch, it was observed that the ruminal fluid containing A. oryzae increased (p=0.007) pH, confirming the stabilizing effect on ruminal pH of this organism in high concentrate diets. Several studies reported that A. oryzae enhances lactate utilization by ruminal bacteria capable of fermenting lactate (Megasphaera elsdenii and Selenomonas ruminantium) (^{Beharka and Nagaraja 1998}). ^{Waldrip and Martin (1993)} demonstrated that the fungus provides growth factors (i.e., sugars, amino acids, vitamins) that are required by these bacteria.

Batch cultures inoculated with ruminal fluid from fermenters fed A. oryzae produced greater (p<0.001 to p=0.010) amounts of branched SCFA and increased (p<0.001) caproate production and the ratio Ac:Pr with all pure substrates than those inoculated with fluid from control fermenters (Table 4). For starch and xylan substrate it was shown a decrease (p=0.002 and p=0.018, respectively) in propionic acid production with rumen fluid corresponding to A. oryzae. In the case of cellulose this treatment caused an increase (p=0.012) of acetic acid.

The lack of effect of inoculum from Rusitec fermenters in fermentation of pure substrates seems to indicate that the strain of A. oryzae or the dose used in the current study are unable to induce the adaptive changes in the microbial population in fermenters with A. oryzae. These results suggest that the microorganism must be supplied daily to maintain its effect on the ruminal ecosystem.

Conclusions

The increase of acetate, minor volatile fatty acids and total volatile fatty acids production indicate that A. oryzae strain stimulated rumen fermentation of a 65:35 alfalfa hay:concentrate diet in continuous fermentation system Rusitec. The results obtained support the development of in vivo further research.

Acknowledgments

The authors wish to acknowledge the financial support received from the MCYT of Spain (project AGL2008-04707-C02-02) and from the MAE-AECID (Project PCI-Iberoamérica A/4951/06). A. Sosa and A. Diaz gratefully acknowledge the receipt of scholarships from the MAEC-AECID.

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Received: May 05, 2020; Accepted: June 04, 2020

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