In sacco probiotic properties of effective microorganisms (EM) in forage degradability

M Syomiti, M Wanyoike*, R G Wahome* and J K N Kuria*

Kenya Agricultural Research Institute (KARI), Muguga South, P.O Box 30148, Nairobi
syomitimargaret@yahoo.com
* University of Nairobi; Animal Production department. P.O Box 29053, Nairobi

Abstract

Effective microorganisms (EM), a composite of selected microorganisms, are used for various purposes that include probiotic for ruminants and non-ruminants but without sufficient scientific evidence of their efficacy. This experiment was designed to determine the efficacy of EM as a microbial feed additive in an in sacco experiment, using a Boran steer of about 350 kg ^0.75 metabolic weight. The EM was included in the drinking water at 0.2% and offered ad libitum. The test samples were incubated for 48, 72, 96 and 120 h before and after introduction of EM into the rumen of the animal. A 10-day adaptation period to the EM was allowed before introduction of the test samples. The test samples were: (i). Maize stover (MS) as control (ii). MS + spent brewers’ grains (iii). MS + Desmodium spp. (iv). cellulose and (v). lignin.

EM addition increased (P<0.01) the degradability values for all the samples, except lignin. N-enriched samples showed (P<0.01) higher degradability values, both with and without EM inoculation than those without protein source. Addition of Desmodium spp. to the maize stover alongside EM inoculation had the greatest impact on DM degradability which increased up to 65% at 120 h (from 44% at 48 h).  Degradation of cellulose increased slowly from 37 % to 40.4% between 48 and 72 hours of incubation, then more rapidly between 72 and 96 h to 54%, and less rapidly to 57% at 120 hours with EM inoculation.

The results suggest that rumen inoculation with EM has beneficial effects on utilization of high fibre roughages. 

Key words: fibrous feeds, microbial feed additives, ruminants’ diet

Introduction

Over the years, significant improvement in forage cell wall digestibility have been achieved through forage breeding programs and agronomic advances. Despite these improvements, low forage digestibility continues to limit the intake of available energy by ruminants (Lewis et al 1999). Correspondingly, this factor contributes to excessive nutrient excretion by livestock. The use of microbial fibrolytic enzymes is one way of increasing forage utilization and improving the productive efficiency of ruminants.

Probiotics are live microbial feed supplements aimed at improving the intestinal microbial balance of the host animal (Maurya 1993, Preston and Leng 1987). The two most commonly used microbial additives are Lactobacillus spp. and Saccharomyces cerevisiae (bacteria and yeast spp. respectively). The results of microbial supplementation are highly variable because a large number of factors regulate their efficiency (Yoon and Stern 1995). According to Agarwal et al 2000, the effects of microbial cultures in the rumen depend on their tolerance to the variable gut environment when they are given orally. This is because they have to survive and work in an environment of pH fluctuations from the mouth and rumen where conditions vary from basic to slightly acidic up to the lower gut where hydrochloric acid digestion is encountered. 

Singh et al (1998) reviewed the effect of directly fed microbes: Aspergillus oryzae, Saccharomyces cerevisiae, Lactobacillus and Streptococcus and concluded that less than 40% of the studies reported a positive response of microbial supplementation. A possible explanation for the positive effects is the loosening and partial breakdown of cell wall structure of forages resulting in greater accessibility of fibre-degrading bacteria and their associated enzymes (Aramble and Kent 1990, Aramble and Tung 1987).

Information and policy guidelines on the use of probiotics is lacking in Kenya. Previous findings (Syomiti unpublished) revealed that a culture of EM available in Kenya (Organic Solutions, Nairobi).contains two microorganisms, namely; yeast and Gram positive non-spore forming bacillus bacterial species and had marked effects on degradability of substrates in vitro. The objective of this study was to investigate the efficacy of EM as a microbial feed additive for ruminants on in sacco degradability of nutrients.         

Material and methods 

Test sample preparation

The test samples were; Maize stovers alone (control), Maize stover supplemented with spent brewers’ grains or Desmodium intortum,each at a ratio of 3:1 (energy:protein ratio) according to Erwin (1993). cellulose and lignin. The chemical composition of some experimental feedstuffs are presented in Table 1.

The spent brewers’ grains (SBG) were collected from East African Breweries Ltd, Ruaraka, Kenya. EM was obtained from Organic Solution Ltd. ( http://www.infonet-biovision.org/res/res/files/481.of7red.pdf). Filter paper discs were used as the cellulose substrate. The filter discs with a diameter of about 7 cm were dried in an oven at 60⁰C for 72 h, chopped into small pieces with a pair of scissors and then milled to form a pulp, using a kitchen fruit juice mixer (hammer mill portion). To get the lignin sample, saw dust was dried in an oven at 60⁰C for 72 h. and milled to pass a 2mm screen. It was then digested using 72% concentrated sulphuric acid for 3 hrs (Goering and Van soest 1970), oven dried at 60⁰C for 48 h and the residue (lignin) crushed by hand in large crucibles. All the samples were stored in plastic sample bottles, pending the degradability studies.

In sacco degradability of nutrients

The test samples were subjected to standard ruminal degradability procedures (Ǿrskov and McDonald 1979) using one ruminally-fistulated boran steer. The steer was fed on Rhodes grass hay as basal diet. In the treatment with EM inoculation, the animal was given water containing EM, at an inclusion rate of 40ml in 20 litres of water, ad libitum. The animal was allowed a 10 day adaptation period to the EM containing water. Approximately 5 g of the test samples were weighed into 3 Dacron bags (7 x 14cm long with 38 μm pore size) per sample and incubated in the rumen for 48, 72, 96 and 120 h, first without and then with EM inoculation. Activated EM (EM-2) was used in the experiment. Activation was done by mixing 1 kg of EM-1 (original EM) with 1 kg of molasses to 18 kg of tap water and stored for 7 days at room temperature and away from sunlight. The bags were then removed at the end of each incubation time and washed in cold running water until the washing ran clear. The bags were then oven-dried at 60⁰C for 48 h. For the control (zero hour) degradability, the test samples were prepared as above and soaked in a water-bath at 39⁰C for 5 minutes, washed, dried and weighed in a similar manner. 

Statistical analysis

Data were subjected to Genstat (Version 10.0  2005) for analysis of variance (ANOVA), and means separated by least significant difference (LSD) with the F-test significant at the 0.05 probability level.

Results and discussion 

In sacco degradability of roughages

Table 2 summarizes the results on the degradability characteristics of test samples with and without EM microbial inoculation.

Higher (P<0.01) degradability values were detected in all the treatments with the addition of EM, except lignin. The degradability of the test samples varied according to the type of roughage and/or nutrient used. The N-enriched samples showed higher values, both with and without EM inoculation, than those without protein source.

In general, degradability values increased with inoculation time with exception of lignin. The lignin degradability remained consistently low and relatively unchanged (P>0.05), both with and without EM addition across all the incubation periods. For this reason, the values for lignin DM degradability are excluded in the calculation of mean values depicted in Figure 1.

These findings can be explained by the fact that enzymes hydrolyze the easily rumen degradable fibre fractions, cellulose and hemi-cellulose, leaving the more recalcitrant fraction, lignin, unaffected (Van Vuuren et al 1989).

According to Preston and Leng (1987), microbes in the rumen degrade lignin slowly, but the feed does not remain in the digestive tract long enough to allow complete degradation.

Degradation of cellulose increased slowly from 37 % between 48 and 72 hours of incubation, then more rapidly between 72 and 96 h, to 57% at 120 hours with EM inoculation (Table 2). A forage degradability of 50% is the recommended minimum value for ruminants (Aramble and Tung 1987). When Desmodium spp. was added to the maize stover alongside rumen EM inoculation, an increase in the degradability of up to 65% at 120 h, from 43% at 48 h was observed.

Desmodium spp. probably served as a source of Nitrogen for microbial cell synthesis leading to the increased degradability in these samples. For similar reasons, addition of spent brewers’ grains with a CP content of 26.4%, increased sample degradability from 44.2 to 60.3% with EM inoculation. Higher DM degradability occurred beyond 72 hours (Figure 1), particularly in EM inoculated rumen. This was expected because of the longer exposure to microbial enzymes. Low substrate degradability is undesirable because it leads to a low rate of passage through gastro-intestinal tract with subsequent lower feed intake and lower productivity of the animal. Therefore, feeds that are fairly degradable in the rumen should be more preferred in feed formulations.   

At 72 hours, the supplementation with Brewers’ grains or Desmodium did not improve the in sacco digestibility in Non-EM treatment. The effect of N supplementation should have been seen there if N was limiting. This observation can not be explained in the current study. However, there are variations in residency time of different feedstuffs in the gastro-intestinal truct (GIT). The rate of passage of fibrous feeds in the GIT is low, unless there is a form of rumen manipulation to enhance degradability of these feeds. Microbial populations (yeasts and bacterial species) previously isolated in the EM (Syomiti 2009), may have had positive effects in feed degradability in the EM treated samples. Yeasts are excellent consumers of oxygen and this could have enhanced the cellulolitic activity of anaerobic bacterial species in the EM treatments. Yeasts have been reported to utilize feeds with high structural components, even pentose sugars (Maurya 1993), and this can explain the higher degradability in the EM-treatments. The inclusion rates of the N-rich feedstuffs such as Brewers’ grain and legume in the Non-EM treatments may not have been enough for microbial population growth at 72h. A study to determine the inclusion levels of these N-rich feedstuffs for maximum roughage degradability is required.      

The positive effect of EM inoculation (Table 2) on apparent degradability of the cell wall constituents (cellulose) was due to the yeasts and bacterial species in this product (EM). These may have stimulated the activity of beneficial microbes, especially the cellulolytic organisms and their associated enzymes in the ruminant (Aramble and Kent 1990; Yoon and Stern 1995). According to Maurya (1993), the yeast cells remain active in the rumen and have a stimulatory effect upon cellulose-degrading bacteria. Yeasts convert available oxygen and sugar into carbon dioxide and usable energy for efficient bacterial cell growth, thereby maintaining the rumen environment anaerobic favorable to rumen cellulolitic bacteria.

Conclusions and recommendations 

• The results of this study suggest that the inclusion of EM (effective microorganisms) in drinking water at 0.2% has beneficial effects on cell wall constituents’ degradability and thus utilization of high fibre diets. Inclusion of a protein-rich feed ingredient in the formulation of ruminant rations enhances feed utilization. However, further investigations are required to screen the tolerance of the isolated microorganisms in the EM against the variable gut environment such as pH fluctuation in the post-ruminal digestive tract. Additional studies are required to screen the specific microbial strains with potential of enhancing fibre degradability.

References 

Agarwal Neeta, Kamra D N, Chaudhary L C, Sahoo A and Pathak N N 2000  Selection of Saccharomyces cerevisiae strains for use as microbial feed additive. Letter in Applied Microbiology  31: 270-273.

Aramble M J and Kent B A 1990 Effect of yeast culture on nutrient digestibility and milk yield responses in early to mid lactation in dairy cows. Journal of dairy science 73: 1560-1563 http://jds.fass.org/cgi/reprint/73/6/1560.pdf

Aramble M J and Tung R S 1987 Evaluation of saccharomyces cerevisiae growth in the rumen ecosystem. In 19^th Biennial Conference on rumen Function, Chicago, Illinois, p. 29.

Erwin K 1993 Integrated smallholder dairy farming manual, Heifer project international.

GenStat Release 10^th Edition 2005 Lawes Agricultural Trust.

Goering H and Van Soest P J 1970 Forage fibre analysis. U. S Department of Agriculture, Hand book No. 379.

Lewis G E, Sanchez W K, Hunt C W, Guy M A, Pritchard G T, Swanson B I and Treacher R I 1999 Effect of direct-fed fibrolytic enzymes on the lactational performance of dairy cows. Journal of Dairy Science 82: 611-617 http://jds.fass.org/cgi/reprint/82/3/611

Maurya M S 1993 Effect of Feeding Live Yeast Culture (Saccharomyces  cerevisiae) on rumen fermentation and nutrient digestibility in goats. PhD Thesis.  Indian Veterinary Research institute, Deemed University, Izotnager. India.   Organic Solution Ltd. http:// www.infonet-biovision.org/res/res/files/481.of7red.pdf.

Ørskov E R and McDonald I 1979 The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. Journal of Agricultural Science 92: 499-503.

Preston T R and Leng R A 1987 Matching Ruminant Production Systems with Available Resources in the Tropics and Sub-Tropics. Pernambul Books, Australia. Pp. 245.

Singh R, Chaudhary L C, Kamra D N and Pathak N N 1998 Effect of dietary supplementation with yeast cell suspension (Saccharomyces cerevisiae) on nutrient utilization and growth response in crossbred calves. Asian-Australasian Journal of Animal Science 11: 268-271.

Syomiti M 2009 Evaluation of Effective Microorganisms (EM) as an Additive to Improve Feed Value of Maize stovers. MSc Thesis. University of Nairobi, Kenya. 105 pp

Van Vuuren A M, Bergsma K, Frol-Kramer F and Van Beers J A C 1989 Effects of addition of cell-wall degrading enzymes on the chemical composition and the in sacco degradation of grass silage. Grass Forage Science Journal 44: 223-230.

Yoon I K and Stern M D 1995 Influence of direct-fed microbials on ruminal microbial fermentation and performance of ruminants: A review. Asian-Australasian Journal of Animal Science 8: 533-555

Received 2 November 2009; Accepted 29 November 2009; Published 1 January 2010

Go to top