ABSTRACT
This study evaluated the influence of coastcross hay substrate hydration and equine fecal inoculum dilution on the parameters of fermentation and microbiology in in vitro essays. A 2 × 2 factorial block design was used. The first factor was hydration of the coastcross hay substrate 12 h before incubation or at the time of incubation, and the second factor was the dilution of fecal inoculum with a nutrient solution in a weight: weight ratio of 1:1 or 1:3. Degradation of the dry matter (DM), organic matter (OM), and neutral detergent fiber (NDF) were evaluated at 24, 48, and 72 h. Microorganisms were counted 24 h after inoculation. The ammoniacal nitrogen concentration (NH3-N), pH, and cumulative gas production were measured up to 72 h and adjusted by the non-linear Gompertz regression model. Hydration of substrate and time of incubation increased nutrient degradation of coastcross hay, as well as the final volume of gases and the concentration of Streptococcus spp. The 1:3 dilution increased the final pH and Streptococcus spp. concentration. The hydration of substrate did not have any effect on NH3-N, Lactobacillus spp., cellulolytic, and total anaerobic bacteria concentrations. In addition, no effect of hydration was observed on the fermentation rate and the maximum fermentation time on the model used. The fermentation profile of the grass substrate is not affected by dilution, and, therefore, horse feces can be used as a source of inoculum in in vitro fermentation trials. Hydration increases the gas volumes and the nutrient degradation of grass hay, renders the lag phase time insignificant and, therefore, can be irrelevant in terms of fermentation model settings.
Key Words:
degradation rate; gas production; microbiology; modeling; roughage
Introduction
The in vitro gas production technique is important to better understand horse nutrition, in which the diet effects on the activity of intestinal microbiota can be evaluated, and the nutrient degradation can be kinetically quantified without the need of fistulated animals (Murray et al., 2014Murray, J. M. D.; McMullin, P.; Handel, I. and Hastie, P. M. 2014. Comparison of intestinal contents from different regions of the equine gastrointestinal tract as inocula for use in an in vitro gas production technique. Animal Feed Science and Technology 187:98-103. https://doi.org/10.1016/j.anifeedsci.2013.10.005
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), making it an important alternative to in vivo studies. Equine feces are easy to obtain and are mildly invasive.
However, some studies indicate that feces may present less fermentative capacity than other equine inoculum sources (Murray et al., 2014Murray, J. M. D.; McMullin, P.; Handel, I. and Hastie, P. M. 2014. Comparison of intestinal contents from different regions of the equine gastrointestinal tract as inocula for use in an in vitro gas production technique. Animal Feed Science and Technology 187:98-103. https://doi.org/10.1016/j.anifeedsci.2013.10.005
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). There are differences in fecal inoculation procedures in the in vitro tests: feces dilution (Elghandour et al., 2014Elghandour, M. M. Y.; Chagoyán, J. C. V.; Salem, A. Z. M.; Kholif, A. E.; Castañeda, J. S. M.; Camacho, L. M. and Buendía, G. 2014. In vitro fermentative capacity of equine faecal inocula of 9 fibrous forages in the presence of different doses of Saccharomyces cerevisiae. Journal of Equine Veterinary Science 34:619-625.) and substrate hydration (Rymer et al., 1999Rymer, C.; Huntington, J. A. and Givens, D. I. 1999. Effects of inoculum preparation method and concentration, method of inoculation and pre-soaking the substrate on the gas production profile of high temperature dried grass. Animal Feed Science and Technology 78:199-213.). The lack of standardization of fecal inoculum preparation generates differences in results and makes comparisons difficult, impairing the technique.
Mathematical modelling assists in the interpretation of the in vitro fermentation kinetics studies, which provides a dynamic approach applied to the model parameters (Mould et al., 2005Mould, F. L.; Kliem, K. E.; Morgan, R. and Mauricio, R. M. 2005. In vitro microbial inoculum: a review of its function and properties. Animal Feed Science and Technology 123:31-50. https://doi.org/10.1016/j.anifeedsci.2005.04.028
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). The mathematical models for gas production applied to equine studies were originally from research on ruminants (Groot et al., 1996Groot, J. C.; Cone, J. W.; Williams, B. A.; Debersaques, F. M. and Lantinga, E. A. 1996. Multiphasic analysis of gas production kinetics for in vitro fermentation of ruminant feeds. Animal Feed Science and Technology 64:77-89. https://doi.org/10.1016/S0377-8401(96)01012-7
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).
There are anatomical differences between ruminant and equine species; therefore, some essential characteristics of the substrate that will be fermented must be considered (Sunvold et al., 1995Sunvold, G. D.; Hussein, H. S.; Fahey, G. C.; Merchen, N. R. and Reinhart, G. A. 1995. In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using fecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle. Journal of Animal Science 73:3639-3648. https://doi.org/10.2527/1995.73123639x
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), such as the substrate already predigested and hydrated when the first contact with the microorganisms occurs in the cecum. Such conditions play an important role in the substrate recognition time, consequently, on the estimates of fermentation model parameters that will be influenced. There is no standardized protocol for the use of equine feces as a source of inoculum in gas production trials, and there is also a lack of fermentative studies with microbiological evaluation using fecal inoculum.
The objective of this study was to evaluate the influence of hay hydration and the equine feces dilution on in vitro fermentation parameters and microbial population.
Material and Methods
The experiment was carried out in Seropédica, Rio de Janeiro, Brazil (22°45′52.7” S, 43°41′22.4” W, and 33 m altitude). Research on animals was conducted according to the institutional committee on animal use registered under case no. 2015/002590.
The feces from three stallions of Mangalarga Marchador breed were used to provide the fecal samples, and the coastcross hay provided for feeding the animals was used as a substrate in the in vitro fermentation essays throughout the experimental period.
A 2 × 2 factorial block design was used. The first factor was hydration of the substrate 12 h before incubation and at the time of incubation. The second factor was dilution of the feces (inoculum) with a nutrient solution in a weight:weight ratio of 1:1 or 1:3. The cumulative gas production and nutrient degradation were analyzed in a randomized block design with repeated measures over time. The test was repeated for five consecutive weeks, each week being an experimental block.
The horses with an average body weight of 400 kg, which had been previously dewormed with ivermectin (Eqvalan Golden®, Merial), were kept in individual stalls with a feeder and water ad libitum and exercised three times a week for 30 min. The diet of the horses was composed of coastcross hay (Cynodon spp. cv coastcross), mineral salt (Essencefós®, Presence), water ad libitum, and concentrate meal (Table 1) provided at 07:00 and 16:00 h. The average dry matter intake of the diet was 26 g kg−1 of liveweight in the ratio of 66:34 of roughage:concentrate.
The coastcross hay and concentrate meal samples were milled to 1-mm sieve in a Willey mill. The analysis performed were of dry matter, crude protein, and ash (AOAC, 1995AOAC - Association of Official Analytical Chemistry. 1995. Official methods of analysis. 16th ed. AOAC International, Arlington, VA.), and ether extract and crude energy (Silva and Queiroz, 2006Silva, D. J. and Queiroz, A. C. 2006. Análise de alimentos: métodos químicos e biológicos. 3.ed. UFV, Imprensa Universitária, Viçosa, MG.). The neutral detergent fiber (NDF) (Van Soest et al., 1991Van Soest, P. J.; Robertson, J. B. and Lewis, B. A. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74:3583-3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2
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) and lignin were analyzed by oxidation of lignin with permanganate (Van Soest and Robertson, 1980Van Soest, P. J. and Robertson, J. B. 1980. Systems of analysis for evaluating fibrous feeds. p.49-60. In: Standardization of analytical methodology for feeds. Pigden, W. J.; Balch, C. C.; Graham, M., eds. IDRC, Ottawa, Canada.).
The gas production incubations were perfomed as follows: feces were collected simultaneously from the rectum of horses at 09:00 h, two hours after the meal. The fecal samples were kept closed in preheated thermal bottles until processing in the laboratory. The time elapsed between the collection of feces and preparation of the inoculum in the laboratory was 20 min.
The fermentation test protocol required the insertion of 1 g of hay milled to 2 mm into 160 mL flasks, plus 90 mL of nutrient solution (Theodorou et al., 1994Theodorou, M. K.; Williams, B. A.; Dhanoa, M. S.; McAllan, A. B. and France, J. 1994. A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Animal Feed Science and Technology 48:185-197.). The nutrient solution was added 12 h before and at the moment of inoculation, constituting, therefore, the procedures adopted to perform the hydration treatment. The flasks that had been hydrated for 12 h were kept under refrigeration at 4 °C and, 2 h before inoculation, they were heated in a water bath at 39 °C.
The procedude adopted to prepare the fecal inoculum were: the fecal samples from three animals were proportionally mixed, then divided into two fractions - the first was diluted with nutrient solution at a ratio of 1:1 (w:w) and the second diluted with nutrient solution at the ratio of 1:3 (w:w). The diluted feces were then shaken for 1 min in a blender, after which they were placed in a water bath at 39 °C under constant spraying with CO2 (Desrousseaux et al., 2012Desrousseaux, G.; Santos, A. S.; Pellikaan, W. F.; Van der Poel, A. F. B.; Cone, J. W. Guedes, C. M. V.; Ferreira, L. M. M. and Rodrigues, M. A. M. 2012. Effect of collection time on the fermentative activity of microbes in equine faeces. Animal Feed Science and Technology 178:183-189. https://doi.org/10.1016/j.anifeedsci.2012.09.016
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). After 1 h, the feces diluted with nutritive solution of each treatment were filtered through a 45-μm nylon cloth to obtain two different inoculums (1:1 and 1:3). Finally, 10 mL of inoculum were added to bottles already prepared, and correspondent to the treatment described above, and sealed with rubber stoppers.
Fourteen flasks (replicates) were incubated for each treatment. After inoculation, the flasks were kept in a water bath at 39 °C with constant stirring. The gas production was measured manually by means of a pressure transducer (LOGGER AG100, Universal Datalogger) at times: 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 21, 24, 27, 30, 36, 42, 48, 54, 60, 66, and 72 h, making a total of 23 readings. After each reading, accumulated gases were released and flasks were shaken. Pressure in psi was converted to volume per gram of dry matter using the specific equation for experimental conditions of gas production:
in which each psi corresponds to 3.80 mL (Martins, 2012Martins, J. A. 2012. Suplementação dietética de equinos com eletrólitos. Dissertação (M.Sc). Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ.).
The dry matter degradation (DMD), organic matter degradation (OMD), and neutral detergent fiber degradation expressed exclusive of residual ash (NDFD) were determined by the difference in nutrient weight present in the substrate before incubation and after 24, 48, and 72 h of fermentation. After 72 h of incubation, pH (pH 300; Analyzer®) was recorded and NH3-N ammonia nitrogen (mg/dL) was evaluated after centrifugation at 10,000 g for 10 min at 4 °C. Thereafter, 5 mL of the supernatant were pipetted and subjected to distillation and titration according to the micro-Kjeldahl method (Fenner, 1965Fenner, H. 1965. Method for determining total volatile bases in rumen fluid by steam distillation. Journal of Dairy Science 48:249-251. https://doi.org/10.3168/jds.S0022-0302(65)88206-6
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; Souza et al., 2013Souza, N. K. P.; Detmann, E.; Valadares Filho, S. C.; Costa, V. A. C.; Pina, D. S.; Gomes, D. I.; Queiroz, A. C. and Mantovani, H. C. 2013. Accuracy of the estimates of ammonia concentration in rumen fluid using different analytical methods. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 65:1752-1758. https://doi.org/10.1590/S0102-09352013000600024
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).
Microorganisms were quantified 24 h after inoculation. The concentrations of total viable anaerobic bacteria were determined with complete modified agar medium (Leedle and Hespell, 1980Leedle, J. A. Z. and Hespell, R. B. 1980. Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations. Applied and Environmental Microbiology 39:709-719.) after 48 h of incubation at 39 °C by the most-probable-number at 10−6, 10−7, and 10−8 dilutions. The concentrations of cellulolytic bacteria were determined by the most-probable-number at 10−3, 10−4, and 10−5 dilutions with modified (Halliwell and Bryant, 1963Halliwell, G. and Bryant, M. 1963. The cellulolytic activity of pure culture strains of bacteria from the rumen of cattle. Journal of Genetic Microbiology 32:441-448.) liquid culture medium (Julliand et al., 1999Julliand, V.; Vaux, A.; Millet, L. and Fonty, G. 1999. Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum. Applied and Environmental Microbiology 65:3738-3741.) with readings taken after 15 days incubation at 39 °C. The concentrations of Lactobacillus spp. and Streptococcus spp. were determined by counting colony forming units in Petri dishes prepared with MRS Lactobacillus base agar medium (200019, Biolog, Italy) and Azide Bile Esculine agar (06105-500g, Sigma-Aldrich, Buchs, Switzerland), respectively, in dilutions of 10−4, 10−5, and 10−6 after 48 h of incubation at 39 °C in anaerobic jars.
The cumulative production of gases was adjusted using the nonlinear Gompertz equation (Winsor, 1932Winsor, C. P. 1932. The Gompertz curve as a growth curve. Proceedings of the National Academy of Sciences 18:1-8.):
in which Vf = final volume of gases (mL/g DM), estimated as a function of Vm; Vm = volume of gases at time t (mL/g DM); b = fermentation rate (mL/hour); t* = time (hours) when fermentation rate is maximum; and t = time (hours). The estimated equation was used to calculate the fermentation rates (mL/g DM) as a function of time (t) by derivation of the Gompertz equation:
The NLIN procedure of the Statistical Analysis System, version 9.2 was used for parameter estimation. The parameter estimates were obtained by a modified iterative Gauss-Newton method developed by Hartley (1961)Hartley, H. O. 1961. The modified Gauss Newton method for the fitting of non linear regression functions by least squares. Thechnometrics 3:269-280. for nonlinear models. The distribution of the studentized residuals, used to detect discrepant points, in general, is considered as a marginally discrepant observation if |Rti*|>3 (Lemonte, 2008Lemonte, A. J. 2008. Diagnostic in normal linear regression: principle and applications. Biometric Brazilian Journal 26:7-26.). The parameters of the models were compared using the likelihood ratio test (Regazzi, 2003Regazzi, A. J. 2003. Teste para verificar a igualdade de parâmetros e a identidade de modelos de regressão não-linear. Revista Ceres 50:9-26.).
The results from DMD, OMD, NDFD, pH, NH3-N, and the bacteriological count (logarithmic basis) were evaluated for the homoscedasticity of variances and normality. When the assumptions were met, they were subjected to ANOVA (α = 0.05) and compared by the Fisher test (α = 0.05). The analyses were carried out with the SISVAR statistical package (Ferreira, 2011Ferreira, D. F. 2011. Sisvar: a computer statistical analysis system. Ciência e Agrotecnologia 35:1039-1042. https://doi.org/10.1590/S1413-70542011000600001
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).
Results
There was no interaction between the inoculum dilution and substrate hydration in nutrient degradation (P>0.05), but there was an effect of substrate hydration on DMD (P<0.05), OMD (P<0.05), and NDFD (P<0.05), and time (P<0.001) (Table 2).
Nutrient degradation (mg g−1) of coastcross hay hydrated for 12 h or immediately before inoculation with feces diluted in the ratios 1:1 and 1:3 up to 72 h (n = 60)
After adjusting the model, no effect of inoculum dilution on the parameters of the model was observed (P>0.05). However, the substrate hydration produced a significant effect on the final gas volume (P<0.001), but not on fermentation rate and time of maximum fermentation between both treatments of 12 h hydration before incubation or at the time of incubation (Table 3).
Parameters of the fermentation model with coastcross hay hydrated for 12 and 0 h before inoculation (mean±SEM)
There was an effect of dilution factor on pH at the end of the fermentation (P<0.001), but no effects of hydration or interaction on pH were observed (P>0.05) (Table 4). However, there was no effect of treatments on NH3-N concentration of the fermentation residue after 72 h of incubation.
Mean values of pH from the residual solution and ammoniacal nitrogen concentration (mg dL−1) after 72 h of coastcross hay fermentation under different hydration and dilution (n = 20)
There was no effect of substrate hydration or inoculum dilution (P>0.05) on cellulolytic and total anaerobic Lactobacillus spp. bacteria concentration after 24 h of fermentation (Table 5). However, there was an effect of substrate hydration (P<0.05) and inoculum dilution (P<0.05) on the concentration of Streptococcus spp., but without significant interaction effect, with greater concentrations of microorganisms observed with the hydration starting at the moment of inoculation and with dilution 1:3.
Concentration of Lactobacillus spp., Streptococcus spp., cellulolytic bacteria, and total anaerobes (log10 cfu mL−1) of the residual solution after 24 h fermentation of coastcross hay subjected to different hydration and dilution (n = 20)
Discussion
No correction for soluble fraction on degradation of DM, NDF, and OM were performed. Pre-hydration of the substrate increased DMD, and, even disregarding the mineral fraction, this procedure increased OMD by approximately 10.2%. Considering the total degradable substrate until 72 h, the degradation extend reached 62.5 and 56.7% for DMD and OMD, respectively, after 24 h, showing that there was greater activity of fecal microorganisms at the beginning of the in vitro tests with the grass substrate.
In addition, there was an increase of NDFD when the substrate was pre-hydrated; consequently, different degradation profiles were observed for the treatment 0 h compared to 12 h of hydration. The relative difference observed between treatments at 24 h of degradation was approximately 30%, reducing the observed difference a long time; as observed at the end of 72 h, this difference was reduced to 5.2%. According to Julliand et al. (2001)Julliand, V.; De Fombelle, A.; Drogoul, C. and Jacotot, E. 2001. Feeding and microbial disorders in horses: Part 3 - Effects of three hay: grain ratios on microbial profile and activities. Journal of Equine Veterinary Science 21:543-546. https://doi.org/10.1016/S0737-0806(01)70159-1
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, microorganisms that degrade the plant cell wall are considered when quantifying cellulolytic bacteria. Although no difference was observed in the populations at 24 h, maximum fermentation occurred between 7 and 8 h after incubation, indicating that this period is the best moment to observe differences in the cellulolytic community. At this time, gas production includes methane and CO2, which are derived from the substrate fermentation (Coles et al., 2005Coles, L. T.; Moughan, P. J. and Darragh, A. J. 2005. In vitro digestion and fermentation methods, including gas production techniques, as applied to nutritive evaluation of foods in the hindgut of humans and other simple-stomached animals. Animal Feed Science and Technology 123:421-444. https://doi.org/10.1016/j.anifeedsci.2005.04.021
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).
Hydration of the substrate speeds up the fermentation process at the initial incubation times; however, at this stage, only the gas production variable had been recorded. Even the fermentation prolonged up to 72h, nutrient degradation and final volume of gases were not compensated when equine feces were used as inoculum. According to Coles et al. (2005)Coles, L. T.; Moughan, P. J. and Darragh, A. J. 2005. In vitro digestion and fermentation methods, including gas production techniques, as applied to nutritive evaluation of foods in the hindgut of humans and other simple-stomached animals. Animal Feed Science and Technology 123:421-444. https://doi.org/10.1016/j.anifeedsci.2005.04.021
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, the goal of an in vitro study is to represent in vivo events, adapting the physiological conditions of the species under study, such as pH, temperature, incubation time, and so on.
Several studies with equine species were performed using in vivo or in situ methodologies to better understand the digestion process, which invariably involves kinetics of passage and nutrient degradation. Therefore, we have used the kinetic of passage (Van Weyenberg et al., 2006Van Weyenberg, S.; Sales, J. and Janssens, G. P. J. 2006. Passage rate of digesta through the equine gastrointestinal tract: a review. Livestock Science 99:3-12. https://doi.org/10.1016/j.livprodsci.2005.04.008
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; Silva et al., 2014Silva, V. P.; Almeida, F. Q.; Pimentel, R. R. M.; Godoi, F. N.; Santos, T. M. D. and Pires, M. S. 2014. Passage kinetics of digesta in horses fed with coastcross hay ground to different degrees. Ciência e Agrotecnologia 38:506-514. https://doi.org/10.1590/S1413-70542014000500010
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) and nutrient degradation information (Hyslop, 2006Hyslop, J. J. 2006. In situ and mobile bag methodology to measure the degradation profile of processed feeds in different segments of the equine digestive tract. Livestock Production Science 100:18-32. https://doi.org/10.1016/j.livprodsci.2005.11.007
https://doi.org/10.1016/j.livprodsci.200...
; Silva et al., 2010Silva, V. P.; Almeida, F. Q.; Morgado, E. S.; Rodrigues, L. M.; Santos, T. M. D. and Ventura, H. T. 2010. In situ caecal degradation of roughages in horses. Revista Brasileira de Zootecnia 39:349-355. https://doi.org/10.1590/S1516-35982010000200018
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) to support the interpretations of the present study. Additionally, due to limitation to obtain fistulated animals for in vitro fermentation studies, a limiting factor nowadays, we had to use the information currently existing in the literature.
Therefore, Van Weyenberg et al. (2006)Van Weyenberg, S.; Sales, J. and Janssens, G. P. J. 2006. Passage rate of digesta through the equine gastrointestinal tract: a review. Livestock Science 99:3-12. https://doi.org/10.1016/j.livprodsci.2005.04.008
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described values of mean retention time (MRT) in horses fed different feedstuffs no longer than 48 h. Verifying the in situ degradation of nutrients of fibrous feed, Hyslop (2006)Hyslop, J. J. 2006. In situ and mobile bag methodology to measure the degradation profile of processed feeds in different segments of the equine digestive tract. Livestock Production Science 100:18-32. https://doi.org/10.1016/j.livprodsci.2005.11.007
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used the incubation up to 48 h as the maximum time for cecal incubation, and recently, Silva et al. (2014)Silva, V. P.; Almeida, F. Q.; Pimentel, R. R. M.; Godoi, F. N.; Santos, T. M. D. and Pires, M. S. 2014. Passage kinetics of digesta in horses fed with coastcross hay ground to different degrees. Ciência e Agrotecnologia 38:506-514. https://doi.org/10.1590/S1413-70542014000500010
https://doi.org/10.1590/S1413-7054201400...
fed fistulated horses coastcross hay with different particle size and observed values of 36.7 h of MRT. Consequently, the incubation time in in vitro fermentation essays with equine fecal inoculum should not exceed 48 h.
According to Silva et al. (2010)Silva, V. P.; Almeida, F. Q.; Morgado, E. S.; Rodrigues, L. M.; Santos, T. M. D. and Ventura, H. T. 2010. In situ caecal degradation of roughages in horses. Revista Brasileira de Zootecnia 39:349-355. https://doi.org/10.1590/S1516-35982010000200018
https://doi.org/10.1590/S1516-3598201000...
, who evaluated the in situ cecal degradation of nutrients in fistulated horses, a value of 34.3% NDFD of coastcross hay (Cynodon spp. cv coastcross) was observed after 48 h of incubation, which was greater than that observed in vitro in this study. The nutrient degradation depends on the fermentative activity and rate of digesta passage, which provides a constant supply of nutrients to microorganisms, maintaining the degradation activity of the bacteria on the fibrous substrate; this is a limiting element in in vitro techniques.
Total gas production is the sum of H2, CO2, and CH4 production along with in vitro incubation, and these gases are used as fermentative activity indicators in the hindgut of horses (Elghandour et al., 2016Elghandour, M. M. Y.; Kholif, A. E.; López, S.; Mendoza, G. D.; Odongo, N. E. and Salem, A. Z. M. 2016. In vitro gas, methane, and carbon dioxide productions of high fibrous diet incubated with fecal inocula from horses in response to the supplementation with different live yeast additives. Journal of Equine Veterinary Science 38:64-71. https://doi.org/10.1016/jjevs.2015.12.010
https://doi.org/10.1016/jjevs.2015.12.01...
; 2018Elghandour, M. M. Y.; Khusro, A.; Greiner, R.; Salem, A. Z. M.; de la Fuente, J. L.; Márquez-Molina, O.; Barbabosa-Pliego, A. and Jiménez, R. M. D. O. 2018. Horse fecal methane and carbon dioxide production and fermentation kinetics influenced by Lactobacillus farciminis: supplemented diet. Journal of Equine Veterinary Science 62:98-101. https://doi.org/10.1016/jjevs.2017.12.006
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). Additionally, by quantifying the total gas production, it is possible to estimate the metabolizable energy and in vitro digestibility of organic matter (Menke et al., 1979Menke, K. H.; Raab, L.; Salewski, A.; Steingass, H.; Fritz, D. and Schneider, W. 1979. The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. The Journal of Agricultural Science 93:217-222. https://doi.org/10.1017/S0021859600086305
https://doi.org/10.1017/S002185960008630...
) as well as the short-chain fatty acid concentration (Getachew et al., 2002Getachew, G.; Makkar, H. P. S. and Becker, K. 2002. Tropical browses: contents of phenolic compounds, in vitro gas production and stoichiometric relationship between short chain fatty acid and in vitro gas production. The Journal of Agricultural Science 139:341-352. https://doi.org/10.1017/S0021859602002393
https://doi.org/10.1017/S002185960200239...
). However, the procedures described above were developed with ruminal fluid studies but have been recently used in in vitro studies in equine nutrition (Elghandour et al., 2016Elghandour, M. M. Y.; Kholif, A. E.; López, S.; Mendoza, G. D.; Odongo, N. E. and Salem, A. Z. M. 2016. In vitro gas, methane, and carbon dioxide productions of high fibrous diet incubated with fecal inocula from horses in response to the supplementation with different live yeast additives. Journal of Equine Veterinary Science 38:64-71. https://doi.org/10.1016/jjevs.2015.12.010
https://doi.org/10.1016/jjevs.2015.12.01...
; 2017Elghandour, M. M. Y.; Cardenas-Chantres, J. C.; Esquivel-Velázquez, A.; Barbabosa-Pliego, A.; Cipriano, M. and Salem, A. Z. M. 2017. In vitro cecal gas and methane production of soybean hulls-containing diets in the presence of Salix babylonica extract as a fermentation modulator in horses. Journal of Equine Veterinary Science 53:45-54. https://doi.org/10.1016/jjevs.2016.12.004
https://doi.org/10.1016/jjevs.2016.12.00...
and 2018Elghandour, M. M. Y.; Khusro, A.; Greiner, R.; Salem, A. Z. M.; de la Fuente, J. L.; Márquez-Molina, O.; Barbabosa-Pliego, A. and Jiménez, R. M. D. O. 2018. Horse fecal methane and carbon dioxide production and fermentation kinetics influenced by Lactobacillus farciminis: supplemented diet. Journal of Equine Veterinary Science 62:98-101. https://doi.org/10.1016/jjevs.2017.12.006
https://doi.org/10.1016/jjevs.2017.12.00...
).
Rymer et al. (1999)Rymer, C.; Huntington, J. A. and Givens, D. I. 1999. Effects of inoculum preparation method and concentration, method of inoculation and pre-soaking the substrate on the gas production profile of high temperature dried grass. Animal Feed Science and Technology 78:199-213. identified that the gas production of the fibrous substrate increased after 6 h of hydration; however, 12 h were used in the present study to facilitate the semi-automatic experimental procedure.
The kinetics of gas production model developed for ruminant nutrition presents a sigmoid profile characterized by a lag phase followed by the exponential growth phase that slows down and reaches the asymptotic phase (Rymer et al., 2005Rymer, C.; Huntington, J. A.; Williams, B. A. and Givens, D. I. 2005. In vitro cumulative gas production techniques: history, methodological considerations and challenges. Animal Feed Science and Technology 123:9-30. https://doi.org/10.1016/j.anifeedsci.2005.04.055
https://doi.org/10.1016/j.anifeedsci.200...
; Vieira et al., 2008Vieira, R. A. M.; Tedeschi, L. O. and Cannas, A. 2008. A generalized compartmental model to estimate the fibre mass in the ruminoreticulum: 1. Estimating parameters of digestion. Journal of Theoretical Biology 255:345-356. https://doi.org/10.1016/jjtbi.2008.08.014
https://doi.org/10.1016/jjtbi.2008.08.01...
). As the fermentation profile of coastcross hay hydrated for 12 h presented a short lag phase and was not detectable by the model, the authors suggested that an exponential model that disregards the lag phase would be more adequate for equine studies, even using equine feces as inoculum.
According to Rymer et al. (1999)Rymer, C.; Huntington, J. A. and Givens, D. I. 1999. Effects of inoculum preparation method and concentration, method of inoculation and pre-soaking the substrate on the gas production profile of high temperature dried grass. Animal Feed Science and Technology 78:199-213., 6 h of hydration were sufficient to provide the soluble fraction from substrate into the medium, which does not change along the in vitro study period prior inoculation. Immediately after inoculation, the soluble nutrients are rapidly consumed; after that, the insoluble and potentially fermentable fraction of substrate will predominate. However, it needs to have been previously hydrated for the colonization of microorganisms (Groot et al., 1996Groot, J. C.; Cone, J. W.; Williams, B. A.; Debersaques, F. M. and Lantinga, E. A. 1996. Multiphasic analysis of gas production kinetics for in vitro fermentation of ruminant feeds. Animal Feed Science and Technology 64:77-89. https://doi.org/10.1016/S0377-8401(96)01012-7
https://doi.org/10.1016/S0377-8401(96)01...
). The initial development of the microorganisms present in the feces and the adhesion to the insoluble particles are time-dependent. Therefore, the hydration of coastcross hay facilitated adhesion to the substrate, optimizing the fermentation process. Murray et al. (2006)Murray, J. M. D.; Longland, A. C.; Moore-Colyer, M. and Dunnett, C. 2006. The effect of feeding a low- or high-starch diet on the in vitro fermentative capacity of equine faecal inocula. Animal Science 82:627-635. observed that lag phase time differences would occur depending on the chemical characteristics of the substrate, so that fibrous feedstuffs will present lag phases greater than the soluble substrates. However, they did not mention the hydration procedure on their protocol.
Coastcross is a C4 grass widely used in the tropics to feed horses and has a high lignification of the plant cell wall (Van Soest, 1994Van Soest, P. J. 1994. Nutritional ecology of the ruminant. Cornell University Press, Ithaca, NY.). Therefore, the 12-h hydration was fundamental to reduce the lag phase to 0.28 h, allowing rapid access to the substrate by the fecal microbial community. Although we did not use the lag phase information for statistical comparisons, it was determinant in the choice and adjustment of the model. In addition, it made the in vitro procedures similar to what occurs naturally in vivo in the equine species.
The inoculum from equine feces may present a fermentation pattern similar to cecal inoculum, but it differs when compared to that of the colon (Murray et al., 2014Murray, J. M. D.; McMullin, P.; Handel, I. and Hastie, P. M. 2014. Comparison of intestinal contents from different regions of the equine gastrointestinal tract as inocula for use in an in vitro gas production technique. Animal Feed Science and Technology 187:98-103. https://doi.org/10.1016/j.anifeedsci.2013.10.005
https://doi.org/10.1016/j.anifeedsci.201...
). However, Julliand and Grimm (2016)Julliand, V. and Grimm, P. 2016. The microbiome of the horse hindgut: History and current knowledge. Journal of Animal Science 94:2262-2274. https://doi.org/10.2527/jas.2015-0198
https://doi.org/10.2527/jas.2015-0198...
reported that the fecal bacterial population was similar to that seen in the dorsal colon and lower colon. Thus, the production of gases may be more associated with the ability of the inoculum microorganisms to ferment a particular substrate than its concentration. In the present study, the same composite sample of feces diluted in 1:1 or 1:3 (weight:weight) was used, and the gas production appeared to be more influenced by the physical characteristics of the substrate than by the concentration of the fecal microorganisms. In addition, there was no difference in the concentration of microorganisms from the fecal inoculum of horses when collected at different times throughout the day; however, the gas production differed according to the substrates used (Desrousseaux et al., 2012Desrousseaux, G.; Santos, A. S.; Pellikaan, W. F.; Van der Poel, A. F. B.; Cone, J. W. Guedes, C. M. V.; Ferreira, L. M. M. and Rodrigues, M. A. M. 2012. Effect of collection time on the fermentative activity of microbes in equine faeces. Animal Feed Science and Technology 178:183-189. https://doi.org/10.1016/j.anifeedsci.2012.09.016
https://doi.org/10.1016/j.anifeedsci.201...
). Therefore, the dilution of feces in the proportions used in the present in vitro fermentation assay can be used without affecting fermentation parameters of hay grasses and some microorganisms. In addition, the use of feces for the inoculum avoids the use of surgically modified or euthanized animals for collection of inoculum (Murray et al., 2014Murray, J. M. D.; McMullin, P.; Handel, I. and Hastie, P. M. 2014. Comparison of intestinal contents from different regions of the equine gastrointestinal tract as inocula for use in an in vitro gas production technique. Animal Feed Science and Technology 187:98-103. https://doi.org/10.1016/j.anifeedsci.2013.10.005
https://doi.org/10.1016/j.anifeedsci.201...
).
Cone et al. (1997)Cone, J. W.; Gelder, A. H. and Driehuis, F. 1997. Description of gas production profiles with a three-phasic model. Animal Feed Science and Technology 66:31-45. https://doi.org/10.1016/S0377-8401(96)01147-9
https://doi.org/10.1016/S0377-8401(96)01...
incubated glucose and observed an alteration in the fermentation profile after 45 h, suggesting microbial turnover on in vitro fermentation systems. Additionally, these authors reported that the microbial protein was greater between 5 and 10 h of incubation, which coincided with the total intake of glucose, and the microbial protein was reduced after a prolonged period of fermentation with the formation of NH3. This suggests that the microbial nutrients were recycled, i.e., the development of new cells using dead cells as a nutrient source.
The microbial population present in the cecum uses non-protein N to generate volatile fatty acids (VFA) in detriment of population growth as a metabolic strategy (Santos et al., 2012Santos, A. S.; Ferreira, L. M. M.; Martin-Rosset, W.; Cotovio, M.; Silva, F.; Bennett, R. N.; Cone, J. W.; Bessa, R. J. B. and Rodrigues, M. A. M. 2012. The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation. Animal 6:1096-1102. https://doi.org/10.1017/S1751731111002527
https://doi.org/10.1017/S175173111100252...
). However, the quantification and identification of groups that would benefit from this nitrogen source were not carried out (Santos et al., 2012Santos, A. S.; Ferreira, L. M. M.; Martin-Rosset, W.; Cotovio, M.; Silva, F.; Bennett, R. N.; Cone, J. W.; Bessa, R. J. B. and Rodrigues, M. A. M. 2012. The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation. Animal 6:1096-1102. https://doi.org/10.1017/S1751731111002527
https://doi.org/10.1017/S175173111100252...
). The concentration of NH3-N in in vitro fermentation assays is inversely proportional to the concentration of microbial protein (Cone et al., 1997Cone, J. W.; Gelder, A. H. and Driehuis, F. 1997. Description of gas production profiles with a three-phasic model. Animal Feed Science and Technology 66:31-45. https://doi.org/10.1016/S0377-8401(96)01147-9
https://doi.org/10.1016/S0377-8401(96)01...
). In the present study, the NH3-N values observed after 72 h were greater than those observed by Santos et al. (2012)Santos, A. S.; Ferreira, L. M. M.; Martin-Rosset, W.; Cotovio, M.; Silva, F.; Bennett, R. N.; Cone, J. W.; Bessa, R. J. B. and Rodrigues, M. A. M. 2012. The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation. Animal 6:1096-1102. https://doi.org/10.1017/S1751731111002527
https://doi.org/10.1017/S175173111100252...
, possibly due to the greater true protein fermentation present in the substrate.
The reduction of pH in in vitro studies is an indication of the accumulation of final fermentation products, such as VFA and lactate (De Fombelle et al., 2001De Fombelle, A.; Julliand, V.; Drogoul, C. and Jacotot, E. 2001. Feeding and microbial disorders in horses: 1-Effects of an abrupt incorporation of two levels of barley in a hay diet on microbial profile and activities. Journal of Equine Veterinary Science 21:439-445. https://doi.org/10.1016/S0737-0806(01)70018-4
https://doi.org/10.1016/S0737-0806(01)70...
; Coles et al., 2005Coles, L. T.; Moughan, P. J. and Darragh, A. J. 2005. In vitro digestion and fermentation methods, including gas production techniques, as applied to nutritive evaluation of foods in the hindgut of humans and other simple-stomached animals. Animal Feed Science and Technology 123:421-444. https://doi.org/10.1016/j.anifeedsci.2005.04.021
https://doi.org/10.1016/j.anifeedsci.200...
) or the saturation of buffering agents in the medium. At the 1:3 dilution, there was a greater proportion of buffering agents from the nutrient solution in relation to the total number of microorganisms incubated. Therefore, for the fermentation products, the buffer was more effective as it left the solution less acidic compared to the 1:1 dilution. However, pH remained within the normal fermentation range (from 7.2 to 6.2) of large intestine for healthy horses (Clarke et al., 1990Clarke, L. L.; Roberts, M. C. and Argenzio, R. A. 1990. Feeding and digestive problems in horses: Physiologic responses to a concentrated meal. The Veterinary Clinics of North America, Equine Practice 6:433-450.). The 1:3 dilution may be a strategy for in vitro studies in which there is an limited amount of feces to prepare the inoculum, such as in studies with new-born foals (Silva, 2017)1
1
Silva, V P. 2017. Personal communication. Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brasil.
.
Although the pH was within clinical normality, the concentration of the genus Streptococcus spp., which is related to metabolism of rapidly fermenting carbohydrates, may have interfered with acidification (Julliand et al., 2001Julliand, V.; De Fombelle, A.; Drogoul, C. and Jacotot, E. 2001. Feeding and microbial disorders in horses: Part 3 - Effects of three hay: grain ratios on microbial profile and activities. Journal of Equine Veterinary Science 21:543-546. https://doi.org/10.1016/S0737-0806(01)70159-1
https://doi.org/10.1016/S0737-0806(01)70...
). The reduction of these nutrients caused by greater concentrations of microorganisms of the diluted 1:1 inoculum may have affected their growth. Besides, the hydration of the substrate also had an inhibitory effect on the growth of these microorganisms. Although the genus Streptococcus spp. is associated with the development of laminitis, they have a proteolytic and degradative activity of mono-, di-, and oligosaccharides of plant origin providing rapid access to these nutrient sources without relying on other microorganisms (Jans et al., 2015). However, a high concentration of Streptococcus leads to a reduction in the medium pH, which may compromise the development of other important microorganisms in the fermentation of fibrous carbohydrates. Therefore, high concentrations of this microorganism are not desirable. Thus, treatments with lower dilution and greater hydration could offer a more balanced environment among the important bacterial populations.
The concentrations of Streptococcus observed in the present study were similar to those observed by Julliand et al. (2001)Julliand, V.; De Fombelle, A.; Drogoul, C. and Jacotot, E. 2001. Feeding and microbial disorders in horses: Part 3 - Effects of three hay: grain ratios on microbial profile and activities. Journal of Equine Veterinary Science 21:543-546. https://doi.org/10.1016/S0737-0806(01)70159-1
https://doi.org/10.1016/S0737-0806(01)70...
of 7.64 log10 cfu/mL in the colon of fistulated horses fed a diet composed of 50% hay and 50% barley. When the diet ratio was changed to 70% hay and 30% barley, similar to the ratio of 66:34 (roughage:concentrate) used in the present study, the authors observed concentrations of 6.84 log10 cfu/mL in the colon. The concentrations of cellulolytic bacteria observed in the present study resemble that quantified by Julliand et al. (2001)Julliand, V.; De Fombelle, A.; Drogoul, C. and Jacotot, E. 2001. Feeding and microbial disorders in horses: Part 3 - Effects of three hay: grain ratios on microbial profile and activities. Journal of Equine Veterinary Science 21:543-546. https://doi.org/10.1016/S0737-0806(01)70159-1
https://doi.org/10.1016/S0737-0806(01)70...
of 4.78 log10 cfu/mL in the cecum and colon of equines fed different proportions of barley, and this community is correlated with fiber utilization efficiency (Drogoul et al., 2001Drogoul, C.; De Fombelle, A. and Julliand, V. 2001. Feeding and microbial disorders in horses: 2: effect of three hay: grain ratios on digesta passage rate and digestibility in ponies. Journal of Equine Veterinary Science 21:487-491. https://doi.org/10.1016/S0737-0806(01)70211-0
https://doi.org/10.1016/S0737-0806(01)70...
). In vitro studies have shown the concentration of cellulolytic bacteria of 5.11 log10 cfu/mL fecal inoculum from animals fed meadow hay and commercial concentrate (60:40) (Desrousseaux et al., 2012Desrousseaux, G.; Santos, A. S.; Pellikaan, W. F.; Van der Poel, A. F. B.; Cone, J. W. Guedes, C. M. V.; Ferreira, L. M. M. and Rodrigues, M. A. M. 2012. Effect of collection time on the fermentative activity of microbes in equine faeces. Animal Feed Science and Technology 178:183-189. https://doi.org/10.1016/j.anifeedsci.2012.09.016
https://doi.org/10.1016/j.anifeedsci.201...
).
Assuming that the fecal inoculum was diluted three times as much in 1:3, consequently, there was an initial incubation of smaller amounts of microorganisms when compared with the population present in the 1:1 dilution, which suggests that there was a slower development of the bacterial population until 24 h when the 1:1 dilution was used. As a result, the microorganisms of the 1:3 inoculum developed rapidly due to the greater amount of nutrients available from the nutrient solution (Santos et al., 2011Santos, A. S.; Rodrigues, M. A. M.; Bessa, R. J. B.; Ferreira, L. M. and Martin-Rosset, W. 2011. Understanding the equine cecum-colon ecosystem: current knowledge and future perspectives. Animal 5:48-56. https://doi.org/10.1017/S1751731110001588
https://doi.org/10.1017/S175173111000158...
).
According to Bueno et al. (2005)Bueno, I. C.; Cabral Filho, S. L.; Gobbo, S. P.; Louvandini, H.; Vitti, D. M. and Abdalla, A. L. 2005. Influence of inoculum source in a gas production method. Animal Feed Science and Technology 123-124:95-105. https://doi.org/10.1016/j.anifeedsci.2005.05.003
https://doi.org/10.1016/j.anifeedsci.200...
, the cellulolytic bacteria adhere to the solid particles of the inoculum and, therefore, with an increased solid phase in the incubation, are able to reduce the lag phase period of the fibrous substrates. In the present study, adjustments were made to the inoculum preparation protocol: feces were kept diluted in nutrient solution for 1 h in a water bath at 39 °C, under constant agitation and sprayed with CO2. This process aimed to increase the activity of cellulolytic bacteria present in the fecal inoculum.
Total anaerobes and Lactobacillus spp. values observed in the present study showed the same dilution factor as the concentrations of microorganisms observed by Julliand et al. (2001)Julliand, V.; De Fombelle, A.; Drogoul, C. and Jacotot, E. 2001. Feeding and microbial disorders in horses: Part 3 - Effects of three hay: grain ratios on microbial profile and activities. Journal of Equine Veterinary Science 21:543-546. https://doi.org/10.1016/S0737-0806(01)70159-1
https://doi.org/10.1016/S0737-0806(01)70...
and De Fombelle et al. (2001)De Fombelle, A.; Julliand, V.; Drogoul, C. and Jacotot, E. 2001. Feeding and microbial disorders in horses: 1-Effects of an abrupt incorporation of two levels of barley in a hay diet on microbial profile and activities. Journal of Equine Veterinary Science 21:439-445. https://doi.org/10.1016/S0737-0806(01)70018-4
https://doi.org/10.1016/S0737-0806(01)70...
in the colon of horses fed a diet with the same concentrate: roughage ratios as the horses of the present study.
The peak microbial growth was observed at approximately 10 h after incubation, when the cumulative gas production and nutrient degradation data were associated. However, the quantification of the microorganisms occurred at 24 h, which was after the point of maximum bacterial development. Thus, in in vitro studies with fecal inoculum, the microbial community should be quantified in the exponential growth phase.
Conclusions
Dilution does not alter the fermentation profile of the roughage substrate. Therefore, this procedure does not limit the comparison between studies and allows the use of equine feces as source of inoculum for in vitro fermentation tests. The hydration of the substrate increases the fermentation process; thus, the lag phase time becomes insignificant and, therefore, can be irrelevant in terms of fermentation model settings. In addition, it increases the extent of substrate fermentation and nutrient degradation. Hydration of the substrate should be considered in in vitro fermentation test protocols for dehydrated roughage feedstuffs.
-
1
Silva, V P. 2017. Personal communication. Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brasil.
Acknowledgments
The manuscript is part of the dissertation of the first author presented to the Graduate Program in Animal Science of the Universidade Federal Rural do Rio de Janeiro and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.
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Publication Dates
-
Publication in this collection
22 Oct 2018 -
Date of issue
2018
History
-
Received
12 Jan 2018 -
Accepted
08 Apr 2018