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Research Article

In vitro Dry Matter Digestibility and in vitro Gas Production of Some Acacia Seeds Treated with Sodium Hydroxide and Poly Ethylene Glycol

Balgees, A. Atta Elmnan and Moawia Mohamed Sharaf Ali
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Objectives: The study was carried out to determine the nutritive value and the effect of sodium hydroxide (NaOH) and poly ethylene glycol (PEG) treatments on in-vitro dry matter digestibility (IVDMD) and gas production parameters of some Acacia seeds. Materials and Methods: Seeds of Acacia mellifea (A. mellifea), Acacia senegal (A. senegal), Acacia tortillis (A. tortillis), Acacia seyal (A. seyal ) and Acacia nilotica (A. nilotica) were collected from El-Gadaref State-East Sudan. Samples of each seeds were milled, and divided into 3 groups and assign to one of the three treatments; untreated seeds (control seeds), 0.2% PEG treatment (PEG seeds) and 0.2% NaOH treatment (NaOH seeds). Results: The results revealed that all of acacia seeds under study contained appreciable amount of crude protein (CP), which range from 16.68 to 22.57%. A. mellifea seeds had higher level of CP, lower level of crude fiber (CF) and tannin than the other seeds. Contrary Acacia nilotica seeds contained high level of CF, tannin and low amount of CP. PEG seeds and NaOH seeds had higher IVDMD and gas production than control seeds. The correlation study indicated that the IVDMD of acacia seeds were positively correlated with CP and negatively with tannins and CF content. Conclusion: It is concluded that the PEG and NaOH treatments had a great potential to improve the IVDMD and gas production for acacia seeds with superiority to the PEG treatment.

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  How to cite this article:

Balgees, A. Atta Elmnan and Moawia Mohamed Sharaf Ali, 2020. In vitro Dry Matter Digestibility and in vitro Gas Production of Some Acacia Seeds Treated with Sodium Hydroxide and Poly Ethylene Glycol. Pakistan Journal of Nutrition, 19: 381-387.

DOI: 10.3923/pjn.2020.381.387

Copyright: © 2020. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.


Providing feed throughout the year is a major constraint in animal production of the tropics. Natural pasture especially during the dry season and crop residues have low nutritive value in term of low crude protein, digestible nutrients and high fiber1. Sudan has huge numbers of livestock and browsing trees which are important source of forage for small ruminant and camel. Nowadays, fodder trees and shrubs considered as promising energy and protein sources to be utilized in the different animal production system. They have high potential to fill the gap of the shortage and nutritional deficiencies during dry season on small holder farm2. Tropic areas have a large number of legume fodder trees; their seeds could enhance the consumption of poor quality roughage for animals during dry season. However; the majority of these seeds contain anti-nutritional agents such as tannins which are unpalatable or toxic for animals and rumen microorganisms, thus limiting their use as feeds for animals3,4.

Acacia seeds are promising feed in the tropics due to its capability to thrive in dry zones of the world5 but the presence of tannin could be the major constraints to be used as feedstuff. The basic anti-nutritional factor in Acacia species and many other browse species are condensed tannins (CT)6. Tannins are a polyphenolic compound with high molecular weights and a various complexity7. There are different recommended treatments to eliminate the effect of high content of condensed tannin in browse species. For instance, PEG or NaOH treatment which are effectively bind with condensed tannin have been suggested by many researchers8.

The study was carried out to determine the nutritive value and to assess the effect of PEG or NaOH treatment on in vitro dry matter digestibility and gas production of some acacia seeds collected from El-Gadaref State- East Sudan.


Site of the study: The study was carried out at the laboratory of Department of Animal Nutrition, Faculty of Animal production, University of Khartoum.

Samples collection and preparation: Seeds samples were collected from 10 trees of five Acacias Spp. from EL-Gedaref State-Sudan (Table 1). Seed samples were cleaned by removing foreign particles then about 5000 g of each seeds were weighed, milled by a laboratory miller to 1 mm screen and kept at room temperature for further analysis and treatments. Then seed samples for each spp. were divided into 3 groups and each one was treated with PEG and NaOH at zero level for making control seeds, 0.2% of PEG glycol for making PEG seeds and 0.2 sodium hydroxide for making NaOH seeds. Each treatment had 4 replicates.

Chemical analysis: The chemical composition of the seeds [crude protein (CP), crude fiber (CF), ether extract (EE) and ash] was analyzed according to AOAC9. Nitrogen free extract (NFE) was calculated as follow:

NFE% (DM) = {DM-(EE%+CP%+CF%+ash%)}

Determination of tannin: The total condensed tannin was determined by the butanol-HCl methods10. The reagent was prepared just at need by mixing equal volumes of 1% vanillin methanol and 8% HCl methanol. It was discarded if color appeared. Catechin was used to prepare the standard curve; this was done by adding 600 mg of catechin to 100 mL of 1% HCl methanol. From this stock solution various dilutions were prepared. 5 mL vanillin/HCl reagent was added to 1 mL of each dilution, incubated at 30°C for 20 min. After that the absorbance was read using spectrophotometer (DR3 spectrophotometer) at 500 nm. The absorbance was plotted against catechin concentration.

Weighted of 0.2 g of the sample was placed in a tube, then 10 mL of 1% HCl/methanol was added. The test tube was capped and continuously shaken for 20 min and then centrifuged at 2500 rpm for 5 min. One milliliter of the supernatant was pipetted into each of the tube and then proceeding as was described in the standard curve above. For zero setting prior absorbance was read,1 mL blank solution was mixed with 5 mL (4%) concentrated HCl in methanol and 5 mL of vanillin in a test tube. The absorption was read at 500 nm and the concentration of condensed tannin as catechin equivalent was calculated as follows:

Tannin (%) = W C V 100 V V


C = Concentration corresponding to the optical density

V = Volume of extract (mL)

W = Weight of the sample (mg)

In vitro dry matter digestibility (IVDMD): Dry matter samples from each sample were subjected to 48 h microbial digestion period with Mc Douglass buffer, rumen fluid mixture in sealed plastic bottles, followed by 48 h digestion with pepsin in week acid11.

In vitro gas production: The in vitro gas production was carried out using the method described by Menke and Steingass12. Rumen fluid was collected into pre-warmed insulted bottles from a healthy adult steer weighing 250-300 kg, fed on sorghum straw and commercial concentrate mixture diet. It was strained trough 4 layers of cheesecloth and stored in thermos container saturated with carbon dioxide (CO2) and maintained at 39°C. Then well mixed CO2 flushed rumen fluid was added to the buffer solution (1:2 v/v), which was maintained in a water both at 39°C. Buffered rumen fluid (30 mL) was pipetted into each syringe, containing the feed samples and the syringes were immediately placed into the water bath at 39°C. Two syringes with only buffer rumen fluid were incubated and considered as blanks. The syringes were gently shaken every 2 h and the incubation terminated after recoding the 72 h gas volume. The gas production was recorded after 3, 6, 12, 24, 48 and 72 h of incubation. Total gas values were corrected for the blank incubation and reported gas values are expressed in mL mL1 200 mg of DM.

Statistical analysis: Data obtained was subjected to one way analysis of variance (ANOVA) for a completely randomized design (CRD)13. Where the F test was significant; the treatment means were compared using least significant different (LSD). A p-value used for the determination of statistical significance was 0.05.


Chemical composition: Table 2 shows the results of chemical compositions of seed samples under study. The dry matter of different seeds was rich in CP content, which ranged from 16.68 to 22.57%.

Therefore, these seeds could be used as protein source to improve the consumption of poor quality roughages for animals during dry season. Among the seeds under study, A. mellifera seeds had the highest amount of CP, this result was similar to a previous study conducted by Adewusi et al.14 who stated a range of 15.6-24%.

Analysis of the data indicated that there was a significant (p≤0.05) variation among the seeds on EE level. A. mellifera seeds contained high percent of EE compared with the rest of investigated seeds. The present finding of EE was higher than those reported by Elginaid15 who mentioned a range of 0.84-3.90% for acacia seeds.

Low ash content of acacia seeds was observed in the current study. These results are different from the results obtained by Adewusi et al.14 who reported a range of 6.4-9.6%. This difference between two studies could be attributed to the type of soil16.

Although all seeds under study had high content of NFE, A. seyal recorded the highest value. Similar result was obtained by Adewusi et al.14 who reported that A. seyal contained 54.8% of NFE. The high level of NFE of acacia seeds indicated that it can be ranked and used as a carbohydrate rich feed for animals.

Range of CF content of seeds under study was lower than the results of previous study conducted by Kebede et al.17 who reported a range of 19.54-31.11%. A. nilotica seeds contained higher amount of CF than the other seeds under study. These variations could be attributed to the positive correlation between total condensed tannins and CF content18,19. Therefore, the higher amount of tannin content was noted for A. nilotica seeds than other seeds. This result was near to previous study conducted by Khazaal and Orskov18 who reported a value of 8.2%.

In vitro dry matter digestibility: The results of in vitro dry matter digestibility (IVDMD) for different seeds were given in Table 3. The treated seeds had higher IVDMD than untreated (control) seeds, this result could be due to reaction between PEG or NaOH with tannin, which provide a suitable environment for microbes to ferment feed materials20.

Among the un-treated samples, the highest value of IVDMD was acquired by A. torrtlis, while the lowest value recorded by A. nilotica. This result could be justified by the low CP, high CF and tannin content of A. nilotica seeds. Odenyo et al.21 found that there was a positive correlation between CP content and IVDMD of some browses. Also Adewusi et al.14 reported that the increase in CF content in diet resulted in decreases in DM digestibility.

Gas production parameters: Results of gas production during fermentation period indicated that the cumulative volume of gas production increased with increasing incubation time. Figure 1 shows the gas production (mL/200 mg DM) of control samples at different time of incubation period. For the control samples, at the end of incubation time (72 h) A. mellifera seeds produced more gas compared with other samples. This result could be attributed to relatively low phenols compound and CF in these species22.

The highest gas production for treated seeds was recorded by A. mellifera for NaOH treatment and A. Senegal for PEG treatment (Fig. 2, 3), respectively. A. senegal seeds at different incubation time acquired the highest gas volume among the different species after treated by PEG.

The gas production of all samples slightly increased from 3-12 h, then increased exponentially from 12-72 h for PEG and NaOH treatments. Among the treated seeds A. nilotica seeds recorded low gas volume, this might be explained by the presence of high tannin and CF content in this seeds. Feeds contain high amount of fiber are known to be less degradable than feeds that contain high amount of soluble carbohydrates2,23,24. The adverse effect of the cell wall constituent on gas production may be due to the reduction of the microbial activity through increasing un-favorable environmental conditions25.

The fermentable fractions of gas production of different sample species were presented in Table 4. Among untreated seeds the A. senegal gained the high fermentable fraction (b) and potential gas production (a+b), this result may be due to its high CP content. Atta Elmnan and Alamin26 reported that there is a positive correlation between CP content and gas production.

The fermentable fraction (b) and potential gas production (a+b) were higher for treated seeds than the control seeds. This result could be attributed to the effect of PEG and NaOH on the phenolics compound that complex with protein. Since protein molecule becomes free, the microbe easily will ferment protein which lead to increase the production of gas.