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Volatile fatty acids concentration and efficiency of microbial protein synthesis of concentrate cassava peel diet with different levels of protein sources

This study was conducted to evaluate the effects of different levels of protein sources in a concentrate cassava peel diet on volatile fatty acid (VFA) concentration and efficiency of microbial protein synthesis (EMPS) by in vitro method. The five diet treatments in this study consisted of cassava meal (CM), cassava peel (CP), cassava leaves (CL) and moringa leaves (ML) with the proportion applied were T1=CM70% + CP20% + CL5% + ML5%; T2=CM60% + CP20% + CL10% + ML10%; T3=CM50% + CP20% + CL15% + ML15%; T4=CM40% + CP20% + CL20% + ML20%; T5=CM30% + CP20% + CL25% + ML25%. The experiment used a randomized block design with five treatments and three replications. The results showed that increasing the level of protein sources in the ration tended to increase the value of total VFA, acetate, propionate, CO2, and CH4 in which T3 had the highest values. However, the effect of treatments were significant in EMPS value (P<0.01). It was concluded that increasing levels of cassava and moringa leaves in the concentrate cassava peel diet tended to increase the value of total VFA, acetate, propionate, butyrate, CO2, and CH4 which led to a significant increase in EMPS values.


Introduction
Carbohydrates are fermented by rumen microbes to produce volatile fatty acids (VFA) which consist of 60-70% acetic acid, 15-20% propionic acid, and 10-15% butyric acid [1]. The concentration of VFA is influenced by the level of feed ingredient fermentability in the rumen [2]. Apart from their function as a source of energy for the host, VFAs also serve as a source of carbon skeletons as well as ATP for microbial protein synthesis [3]. The microbial protein produced in the rumen and flows into the abomasum provides the majority of protein for further digestion by HCl-pepsin and trypsin to form amino acids in the small intestine for absorption (50-80%). Bach et al. [4] concluded that rumen ammonia is the main precursor, and its efficiency of conversion into microbial protein is determined by the type and amount of feed consumed by ruminants. Ramaiyulis et al. [5] stated that rumen microbial growth, largely determined by the availability of essential nutrients such as soluble carbohydrates and protein.
Retnaningrum et al. [12] reported that the use of cassava meal as an energy source to supplement maize stover basal diet had the highest digestibility and daily weight gain of crossbred Limousine. [13] mentioned that cassava peel can replace corn as an energy source in ration rated from ruminal parameters on small ruminants. Mulyati et al. [14] stated that cassava and moringa leaves potentially as a supplement on low-quality roughages to support rumen microbial growth and activity due to high protein content. This study aimed to evaluate the effects of different levels of protein sources in a concentrate cassava peel diet on VFA concentration and efficiency of microbial protein synthesis (EMPS) by in vitro method.

Location and time
This research was conducted in the Nutrition and Animal Feed Laboratory Faculty of Animal Science Brawijaya University in November.

Materials
Materials used were set of tools for volatile fatty acids (VFA) and efficiency of microbial protein synthesis (EMPS) analysis and feedstuffs consisted of cassava meal (CM), cassava peel (CP), cassava leaves (CL) obtained from Ngajum, Malang, and moringa leaves (ML) took from Kambingan Village, Tumpang, Malang.
The in vitro method used Makkar et al. [15] protocol, in which after the incubation, the residue were centrifuged to separate sediment and supernatant parts. The separated sediment was dried and weighed for measuring fermented organic matter (FOM) value. The separated supernatant was used to determine the VFA total, acetate (C2), propionate (C3), and butyrate (C4) by gas chromatographic techniques. The result of VFA concentration also used to determine CO2 and CH4 by Van Soest [16] equation. For determining of EMPS value, the residues were refluxed using NDS solution and calculated with Bach et al. [4] equation.

Statistical analysis
The data obtained were analyzed by analysis of variance (ANOVA) and followed by Duncan's Multiple Range Test if the treatments gave a significant effect on the variables measured according to Sudarwati et al. [18]. The VFA total in this study was high because the concentration of VFA was generally from 70-150 mMol [19] and this may be due to a high fermentation rate of cassava meal and peels in the rumen. The high starch diet in ration increases acetate and propionate value [20] and increases the amylolytic microbial population [19]. Suharlina et al. and Gumilar et al. [21,22] stated that fibrous feed becomes acetic in the rumen which probably caused acetate and propionate to be higher. In contrast, T3 has a higher concentration of VFA than T4 and T5 which have higher fiber. It also means that the high protein content in the ration without being followed by an adequate energy source cannot be utilized properly by microbes. Retnaningrum et al. [12] reported that the VFA increased with the addition of cassava meal in the ration, but at the 70% proportion of cassava meal, drastically decreased total VFA, acetate, propionate, and butyrate due to inadequate proportion of protein source. Santos et al. [13] reported that cassava peel tended to decrease propionate and increase the acetate concentration in replacement of corn.  [1] mentioned that the organic matter degradation by rumen microbes will produce VFA, CO2, CH4, and other gasses. The higher VFA formed higher the gasses produced. Muchlas et al. [23] stated that the higher CH4 production illustrates more energy wasted, which indicates the more inefficient the feed was.

Results and discussion
Karsi and Russel [24] mentioned that EMPS values generally range from 10-70 g N/kg FOM and the optimal value for EMPS above 30-40g N/kg FOM, thus the EMPS value in this study is low. It may be caused by the low fiber diet and a high concentrate that caused the low rumen pH as stated by Muchlas et al. [23]. The low EMPS in this study may be causing a high value of NH3, in which according to Bach et al. [4], the available energy and amino acid (AA) will be used directly for microbial protein synthesis, while limited energy will cause amino acid (AA) to be deaminated and the carbon skeleton fermented into VFA. The excess of AA will be removed from the cytoplasm into ammonia because some rumen microbes lack of mechanism for emitting AA.

Conclusion
In conclusion, increasing levels of cassava and moringa leaves in the concentrate cassava peel diet tended to increase the value of total VFA, acetate, propionate, butyrate, CO2, and CH4 which led to a significant increase on EMPS values.