| Livestock Research for Rural Development 28 (11) 2016 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
The substrates in an in vitro rumen fermentation were four levels of cassava pulp (0, 20, 40 and 60%, DM basis) replacing Elephant grass. The incubation with rumen fluid was for 48h with measurements of total gas production, content of methane in the gas and pH, ammonia and protozoa in the substrate at the end of the fermentation.
Replacing Elephant grass by cassava pulp: (i) increased the gas production and the concentration of methane in the gas; and (ii) decreased the pH and ammonia in the substrate after 48h; (iii) decreased the protozoa numbers when 20% of the grass was replaced by cassava pulp.
Key words: ammonia, byproducts, protozoa
Cassava pulp is the solid waste produced as a consequence of starch production and contains about 50-60% of starch on dry matter basis (Sriroth et al 2000). During processing of cassava starch from the roots about 10-15% of cassava pulp (as proportion of the root DM) is produced as byproduct. The pulp is high in fermentable carbohydrates and moisture and low in fiber.
The pulp has a low content of crude protein (about 2-3% in DM) and it is necessary to supplement it with protein-richer feeds to make a balanced diet for cattle. One way to improve the protein content of carbohydrate-rich feeds is by combining them with protein-rich forages. The protein content of Elephant grass ranges from 4.4 to 20.4% in DM with the mean around 12% (Rusdy 2016).
The purpose of the present study was to determine effects on methane production in an in vitro rumen incubation when Elephant grass was partially replaced by cassava pulp as the substrate.
The experiment was conducted in the laboratory of the cattle farm in the Research and Technology Transfer Center of Nong Lam University during June 2015.
The experimental design was a completely randomized array of 4 treatments and 4 replications with ratios of cassava pulp: Elephant grass: 0:100, 20:80, 40:60, 60:40 (DM basis).
Cassava pulp was brought from the Wuson starch factory in Binh Phuoc province. Elephant grass was available in the cattle farm. Samples of both substrates were dried in a micro-wave oven and ground (1 mm sieve) before being mixed according to the proportions shown in Table 1. The in vitro system (Photo 1) used recycled PEP water bottles based on the design described by Inthapanya et al (2011). Representative samples of the mixtures (12g DM) were put in the incubation bottle to which was added 960ml of buffer solution (Table 2) and 240ml of rumen fluid. The rumen fluid was taken from a steer immediately after being slaughtered at the local abattoir. It was held in a thermos flask for about 1hr until placed in the incubation bottles. The bottles with substrate were then incubated in a water bath at 39 °C for the different lengths of incubation (6, 12, 24 and 48h).
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Table 1. Ingredients in the treatments (g DM) |
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|
|
CP-0 |
CP-20 |
CP-40 |
CP-60 |
|
Elephant grass |
12 |
9.6 |
7.2 |
4.8 |
|
Cassava pulp |
0 |
2.4 |
4.8 |
7.2 |
|
Crude protein, % in DM |
12.0 |
10.1 |
8.12 |
6.18 |
|
Table 2. Ingredients in the buffer solution (adapted from Tilly and Terry 1964) |
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|
Ingredients |
CaCl2 |
NaHPO4.12H2O |
NaCl |
KCl |
MgSO4.7H2O |
NaHCO3 |
Cysteine |
|
(g/liter) |
0.04 |
9.3 |
0.47 |
0.57 |
0.12 |
9.8 |
0.25 |
|
|
| Photo 1. The in vitro system |
The gas volume was measured at 6, 12, 24 and 48h by water displacement from the receiving bottle which was suspended in water and calibrated at intervals of 50ml. On each occasion, after measuring the volume, the gas was ejected from the receiving bottle though a tube attached to a Crowcom meter (Crowcom Instruments Ltd, UK) fitted with an infra-red sensor to measure methane.
At each stage of the fermentation, samples were taken to measure pH, ammonia and protozoal counts. The pH of the rumen fluid was determined by using an electronic meter (Eco Testr pH2). The concentration of ammonia nitrogen in the rumen fluid was determined by diluting 15 ml of ruminal fluid with 5 drops of concentrated H2SO4 and distilling and titrating the released ammonia by the standard Kjeldahl procedure (AOAC 1990). The protozoa population in the rumen fluid was estimated by diluting 8 ml of ruminal fluid with 16 ml of formaldehyde-saline solution (37 % formaldehyde with saline solution 1:9) and counting the protozoa under light-microscopy (100x magnification) using a 0.2 mm deep Dollfus counting chamber. Four fields in the counting chamber were filled and protozoa counted, according to the method described by Jouany and Senaud (1979) and Dehority (1993).
The data were analysed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2016) Software. Sources of variation in the model were ratio of cassava pulp:Elephant grass and error.
The linear increase in gas production with replacement of Elephant grass by cassava pulp (Table 3; Figure 1).was to be expected in view of the much higher content of fermentable carbohydrate in the pulp. In contrast, the response in methane production was curvilinear reaching a plateau when the cassava pulp represented 60% of the diet DM (Figure 2). The increase in the methane concentration with the length of the incubation has been reported by several authors (eg: Inthapanya et al 2011; Binh Phuong et al 2011).
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Table 3.
Mean values for gas production, percentage methane in the gas and methane produced
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Proportion of cassava pulp, % as DM |
SEM |
p |
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|
0 |
20 |
40 |
60 |
|||
|
Gas production, ml |
||||||
|
0-6h |
370 |
530 |
505 |
478 |
43.0 |
0.084 |
|
6-12h |
293 |
340 |
367.5 |
368 |
18.8 |
0.054 |
|
12-24h |
340 |
357 |
382.5 |
460 |
19.7 |
0.003 |
|
24-48h |
315 |
310 |
320 |
438 |
11.0 |
<0.001 |
|
Total gas |
1318 |
1538 |
1575 |
1742 |
48.3 |
<0.001 |
|
Methane, % |
||||||
|
0-6h |
3.79 |
12.1 |
17.5 |
17.8 |
2.00 |
0.003 |
|
6-12h |
19.8 |
28.8 |
33.0 |
24.8 |
3.40 |
0.083 |
|
12-24h |
29.3 |
28.5 |
26.3 |
27.0 |
1.70 |
0.53 |
|
24-48h |
30.3 |
29.5 |
30.0 |
25.8 |
1.40 |
0.047 |
|
Total methane, ml |
270 |
354 |
406 |
414 |
17.5 |
<0.001 |
|
Methane, ml/g DM substrate |
22.5 |
29.5 |
33.8 |
34.5 |
1.4 |
<0.001 |
 |
 |
 |
| Figure 1. Effect of cassava pulp on gas production in 48h incubation | Figure 2.
Effect of cassava pulp on methane production per unit substrate DM after 48h incubation |
Figure 3. Effect of the length of the incubation on the concentration of methane in the gas with different proportions of cassava pulp added to Elephant grass |
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Table 4.
Mean values for pH and ammonia at the end of the fermentation and for protozoal |
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|
|
Cassava pulp, % in diet DM |
SEM |
p |
|||
|
0 |
20 |
40 |
60 |
|||
|
pH at 48h |
6.08 |
6.03 |
5.83 |
5.63 |
0.025 |
<0.001 |
|
Ammonia, mg/100ml |
5.04 |
4.76 |
4.48 |
3.92 |
||
|
Protozoa 6h, (x 10 -4/ml) |
159 |
144 |
133 |
116 |
||
|
Protozoa 12h, (x 10 -4/ml) |
161 |
133 |
136 |
110 |
||
|
Protozoa 24h, (x 10 -4/ml) |
106 |
109 |
108 |
112 |
||
|
Protozoa 48h, (x 10 -4/ml) |
111 |
88.8 |
87.5 |
101 |
||
The decrease in substrate pH (Figure 4) and in ammonia concentration (Figure 5) reflected the increased DM fermentability and reduced level of crude protein as the cassava pulp replaced Elephant grass. The reduction in protozoa concentrations with increasing proportions of cassava pulp in the substrate (Table 4; Figure 6) was probably due to the decrease in pH as a result of the rapid fermentation of the cassava pulp.
 |
 |
| Figure 4. Effect of adding cassava pulp to Elephant grass on pH after 48h incubation | Figure 5. Effect of adding cassava pulp to Elephant grass on ammonia levels at 48h incubation |
 |
| Figure 6. Effect of cassava pulp on protozoa after 48h fermentation |
Replacing Elephant grass by increasing proportions of cassava pulp (from 0 to 60% in DM) in an in vitro rumen fermentation increased:
This research was done by the senior author as part of the requirements for the MSc degree in Animal Production "Specialized in Response to Climate Change and Depletion of Non-renewable Resources" of Cantho University, Vietnam. The authors acknowledge support for this research from the MEKARN II project financed by Sida. They also acknowledge the Research and Technology Transfer Center, Nong Lam University, Vietnam for providing infrastructure support.
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Received 3 October 2016; Accepted 4 October 2016; Published 1 November 2016