Gas Production and Rumen Fermentation Characteristics of Buffalo Diets Containing By-Product from Some Sorghum Varieties

Sugoro I, Wiryawan KG, Astuti DA, Wahyono T. 2015. Gas production and rumen fermentation characteristics of buffalo diets containing by-product from some sorghum varieties. Idones J Anim Vet Sci. 20(4): 242-249. DOI: http://dx.doi.org/10.14334/jitv.v20i4.1241 Sorghum is one of potencial fibre sources as buffalo feed. Quality of sorghum could be increased by irradiation mutation breeding. Samurai 1 and Samurai 2 were products of the irradiation mutation breeding of Pahat. This study was conducted to compare buffalo diets containing Samurai 2 sorghum straw and Samurai 1 bagasse sorghum compared with Pahat sorghum straw using in vitro study. Completely randomized design with 6 treatments and 3 replications was applied in this experiment. The treatment diets were P1 (50% Pahat sorghum straw + 50% concentrate), P2 (50% Pahat sorghum straw silage + 50% concentrate), P3 (50% Samurai 2 sorghum straw + 50% concentrate), P4 (50% Samurai 2 sorghum straw silage + 50% concentrate), P5 (50% Samurai 1 sorghum bagasse + 50% concentrate) and P6 (50% Samurai 1 sorghum bagasse silage + 50% concentrate). The 200 mg DM samples of diets were incubated in 30 ml rumen-buffer fluid for 48 hours. Variables measured were total gas production, CH4 production and rumen fermentation characteristics. Results showed that P2 and P4 produce the highest of gas production (P<0.05) with 60.99 and 60.86 ml/200 mg dry matter respectively. Treatments of P1, P2 and P4 produced the lowest CH4 concentration (P<0.05) with 10.57, 10.90 and 9.82% of total gas, respectively. The P4 produced the highest total volatile fatty acids (VFA), dry matter degradability and organic matter degradability with 109.83 mM, 62.93% and 59.97% respectively, meanwhile ammonia (NH3) concentration was not significantly different. The conclusion showed that straw silage of Samurai 2 was comparable to the Pahat sorghum straw and Samurai 1 bagasse sorghum as buffalo diet.


INTRODUCTION
Utilization of sorghum for forage will strongly support livestock management in marginal area.
Sorghum is a multifunctional crop producing food, feed, bioethanol and another industrial material (Sirappa 2003).As a fibre sources, sorghum may be fed in form of fresh straw or silage.Silage processing was Indones J Anim Vet Sci.20(4): 242-249 aimed for preservation and nutrients retaining (Colombo et al. 2007).Combined sorghum straw and concentrate diet had equal quality and may replace combined corn straw and concentrate diet.Fibre of sorghum crop may be also obtained from sorghum bagasse, which is a processing waste of bioethanol made of sorghum.Combined baggase and concentrate (50 : 50) was able to increase milk fat level (7.61%) of Murrah buffalo and to maintain milk yield more than 5 kg/day (Seshaiah et al. 2013).
Several sorghum varieties are suitable to be developed in dry land, ideal for food industrial, suitable for bioethanol and have good palatability.Those varieties are Pahat, Samurai 1 and Samurai 2. Those varieties were breeding result by gamma irradiation conducted by National Nuclear Energy Agency (Human 2013).Advantages of Pahat are high seed productivity (5 t/ha), low tannin (0.012%), semi-short rod (158 cm), and a multifunctional food and feed resources.Advantages of Samurai 1 are high seed productivity (7.5 t/ha), high rod sugar level (12-18%), and high biomass appropriate for bioethanol substance.Meanwhile, advantages of Samurai 2 are high biomass productivity (47 t/ha), leaf rust diseases (Hemileia vastatrix) and rotten midrib (Sihono et al. 2013).Pahat is a sorghum breed producing Samurai 1 and Samurai 2 varieties.
By-products such as straw and bagasse from those varieties may be used as fibre resources for buffalo diet.Those fibre sources fed in form of silage or fresh.Nutrient value of the third varieties needs to be compared considering that the Samurai 1 and samurai 2 are a derivative of Pahat.Comparation of nutrient value of the silage and fresh fibre resources needs to be conducted to determine nutrient value change occurred due to the breeding process.This study was aimed to compare buffalo diet consisting of 50% fibre source from sorghum straw Samurai 2 or bagass Samurai 1(as mutants) with diet consisting of 50% sorghum straw Pahat (as breed).

Materials preparation
Forages used in this study were sorghum straw Pahat, Samurai 1, Samurai 2, silage of sorghum straw Pahat and Samurai 2, bagass Samurai 1, silage of bagass Samurai 1and concentrate.Pahat and Samurai 1 used were the leaves and.Whereras, the bagass Samurai 1 used was squeezed-stems.Pahat and Samurai 2 were harvested in 80 days old.Samurai 1 was harvested in 100 days old as bioethanol raw material.Silage of sorghum straw and bagass were withered and chopped by ±2 cm. the chopped silage was putted in plastic drum silo with capacity 20 kg and incubated anaerobically for 21 days.Additive was not used during the silage processing.After that, study materials were dried in oven 60°C, grilled and filtered by 1 mm filterer.

Experimental design
This study used Complete Randomize Design with 3 replications with following model: where: Yij = the ith treatment observation and jth replication, Μ = general mean, Αi = effect of ith treatment, Εij = random affect of Ith factor and jth replication.Data obtained will be analyzed using analysis of variance (ANOVA) and followed by Duncan further test (Mattjik & Sumertajaya 2006) helped by SPSS 16.0.
Buffalo's rumen collection during diferent incubation played as replication.This study used 6 treatments presented in Table 2. Diet was formulated based on nutrient requirement of lactation buffalo (CP 8%) (Parakkasi 1999).

Hohenheim gas test (Menke et al. 1979)
Two hundred ±10 mg of sample was putted into syringe with vaseline smeared-piston.The sample was incubated according to Menke et al. (1979) for 48 hours into 1241.76ml Mc Dougall fluid (Krishnamoorthy 2001) and 650 ml rumen fluid.Rumen fluid was obtained from fistulated-buffalo routinely fed by grass and concentrate with ratio by 50 : 50 in DM.Rumen collection was carried out in the morning before fed.The liquor was filtered by 4 layer-gauze.Incubation was conducted in waterbath 39 o C. Thirty ml media liquor was putted into each syringe.Piston in the syringe was pressed until no empty cavity.Initial volume before sample incubated was recorded.Variables observed were total gas production, CH4 production, kinetic gas, ammonia (NH3), volatile fatty acid (VFA), dry material (DM) degradation, and organic material (OM) degradation.

Measurement of rumen fermentation variables
Variables observed were total gas production, CH4 production and rumen fermentation products such as NH3, VFA, dry material (DM) degradation, and organic material (OM).
Kinetic gas was measured by exponential method of Ørskov & McDonald (1979) p = a + b (1-e-ct).a and b constants were soluble and insoluble fraction but may be degradable, respectively.The c constant was constantly fraction rate per t unit time.Calculation of a, b, and c used fitcurve Naway® software.
Measurement of CH4 was carried out after determination of CH4 concentration in total gas from fermentation of each syringe.Measurement of CH4 concentration conducted using MRU gas Analyzer®.CH4 concentration measurement was conducted to incubation result of the 48 th hour.Readed value on MRU gas Analyzer® was percentation of CH4 stored in the syringe.Variables observed were CH4 concentration and CH4 production of buffalo diet in every 200 mg digested organic matter.NH3 measurement was carried out by Conway microdifusion method in GLP (1966).VFA measurement was carried out by steam destilation method (Warner 1964).DM and OM measurements were carried out according to measurement of Blümmel et al. (1997).

Total gas production
Measurement result of the total gas production was presented in Table 3.The total gas production between treatments was significantly no different on 2 nd and 4 th hour incubation.Difference began to show in 6 th hour incubation.The highest total gas production was on 8 th -12 th incubation produced by P6.On the 24 th hour incubation, the highest total gas production was produced by P4, P6, and P2 (P<0.05).On the 48 th hour incubation was produced by P4 and P2.Gas production gradualy increased with increasing the incubation time.CO2 and CH4 produced in this method derived from directly and undirectly substrate fermentation through VFA buffering mechanism, that is CO2 which was released from buffer bicarbonate produced during fermentation process (Getachew et al. 1998;Jayanegara et al. 2009).
Gas profile dynamic was related to nutrient content difference in the six diets (Table 2).P1, P3, and P4 treatments had low gas production rate in the early incubation hours due to high crude fibre (CF) content, in the contrary with P2 and P4 that produced the highest gas in the early hour incubation due to the lowest CF content.High fibre in diet might cause decrease on rumen microbial activity on less than 24 hours of diet incubation (Kumar et al. 2007), however gas production rate from fermentation by microbe was higher on 48 hours of incubation.This was caused by rumen microbial started to digest the fiber after 24 hours incubation.
Coefficient of gas production kinetic was calculated by exponential model of Ørskov & McDonald (1979), where maximum gas production (a+b) and gas production rate (c) presented in Table 3.The maximum gas production (a+b) was significantly different (P<0.05) between treatments.Gas production rate was not significantly different in all treatments.The highest maximum gas production was produced by P2 and P4 and has the same trend with CP content of diet (Table 2).P2 and P4 consisted of high CP and tend to produce higher maximum gas production.Different with Luna et al. (2013) who reported that CP content had no correlation with maximum gas production.Protein fermentation would produce aminia affecting buffer bicarbonate balance through ion H + neutralization mechanism without releasing CO2 (Cone & Van Gelder 1999;Salem et al. 2013).This difference was caused by diet substrate difference in the treatment.Combination of sorghum and concentrate in this study might maximize CP quality of diet for microbial fermentation.Non Protein Nitrogen (NPN) obtained from urea in the concentrate was able to be optimized for microbial protein synthesis.Optimal microbial performance affected increase of maximum gas production.

CH4 production
Total gas production increasing was followed by CH4 production.Higher total gas production caused high CH4 production.CH4 variable was used to measure CH4 emition decrease level.CH4 concentration of buffalo diet from 48 hour incubation and CH4 production in every 100 mg organic material were presented in Figure 1.The lowest CH4 concentration was produced by P4 and had no significant difference with P1 and P2.The lowest CH4 production per unit digested OM was produced by P1 and had no significant difference with P4, P2, and P3.The highest CH4 concentration was produced by P5 with higher concentration by 66.60% (P<0.05)compared with P1.CH4 concentration in P6 was also higher by 61.49% (P<0.05)than P1.High CH4 concentration and production per unit OM digested in P5 and P6 were affected by low CP content.Widiawati et al. (2010) reported that higher content and good quality of CP might produce lower CH4.Protein degradation would produce NH4 combined with CO2 to produce lower CH4 (Getachew et al. 1998).Negative effect of fermentative digestion in the rumen was a lot of CH4 wasted.Energy losses in feed become CH4 reached 7% in ruminant (Kamal 1994).Baker (1999) reported that CH4 production indicated a lot of energy losses in form of gas showing low feed efficiency.Low CH4 concentration in P1, P2, and P4 showed the third diets were able to increase efficiency of rumen fermentation.High CH4 concentration in P5 and P6 might also be caused by fibre resources used was bagass sorghum harvested in 100 days old.P1, P2, P3, and P4 used sorghum fibre source harvested in 80 days old, so that CH4 concentration produced was lower.Difference of harvest age affected dissolved and structured carbohydrate content.The dissolved carbohydrate would decrease, whereas structured carbohydrate would increase as well as crop age.Structured carbohydrate would be more difficult to be degraded than dissolved carbohydrate (Van Soest 1994).Widiawati et al. (2010) reported that high cellwall fraction produced high portion of acetat acid and CH4.In Shahbazi (2008), it was reported that cellwall fraction was slowly fermented due to cellulose which was acociated in the lignin matrix.Besides, silage treatment caused the structured carbohydrate getting less due to degradation process carried out by microba during incubation (Yahaya et al. 2002).
It was suspected that there were cellwall fraction in the fibre source in the P5 and P6.It could be seen in low maximum gas production (a+b coefficient) in the P5 and P6 (Table 3).High cellwall fraction produced high CH4.Utilization of sorghum fibre resource Pahat and Samurai 2 in P1, P2, P3, and P4 harvested in 80 days old had low concentration CH4 and CH4 production per unit OM digested compared to the P5 and P6.P2 and P4 diet, which was silage, was able to decrease CH4 and CH4 production per unit digested OM, but not in P6 (bagass silage).This was suspected occurred because bagass consisted of higher structured carbohydrate due to the squeeze processing.Decrease of CH4 production might decrease wasted feed energy losses, so that increasing efficiency of feed utilization (Widiawati et al. 2010).

Characteristic of rumen fermentation
Characteristics of rumen fermentation observed were NH3, VFA, DMD, and OMD produced after 48 hour incubation (Table 4).NH3 was not significantly different between treatments.VFA, DMD, and OMD showed significant different (P<0.05) between treatments.P4 showed highest in those fourth variables.NH3 concentration between treatments showed no significant different, even though P4 tended to produce high NH3 concentration (39.74 mg/100 ml).Not significant different between treatments was caused by percentage of NPN (urea) resources use in identic diet   1).Kang & Wanapat (2013) reported that buffalo rumen microbial had high efficiency in NPN use to synthesize microbial protein compared to another feed stuff.Amount of protein of diet was a factor affecting NH3 production (McDonald et al. 2002).
Different result in this study showed CP content of diet had no affect NH3 concentration.This was suspected due to variated degradation level from different protein concentration feed resources.NH3 showed high concentration around 33.85-39.74mg/100 ml in all treatments.Concentrate content in treatment affected high ammonia production due to increase of sample protein content.This result was different with optimal concentration (5 mg/100 ml) for microbial fermentation in closed culture system and depended on feed fermentability level (Wanapat & Rowlison 2007;Wanapat et al. 2013).Optimal NH3 concentration in the rumen of swamp buffalo was 14 mg/100 ml (Wanapat & Pimpa 1999).
The highest total VFA concentration was produced by P4 compared to another five treatments (P<0.05).Leng and Leonard who were cited by Pamungkas et al. (2006) reported that fermentation rate was correlated with VFA concentration, so that VFA concentration change was a reflection of increase of rumen microbial population.One of factors affecting high production of total VFA in P4 was high concentration of NH3.This was an indicator of increase of microbial protein synthesis to increase rumen microbial population.Gas production rate (c), which tended to high in Table 3, also reflected a high VFA concentration as final product.The P4, P5, and P6 produced high total VFA as well as gas production rate.
Total VFA concentration in rumen-fistulated swamp buffalo fed by sorghum as single diet was 53.5 mM.Buffalo rumen fed by fermented rice straw and concentrate produced total VFA concentration by 44.8, 48.9, and 55.9 mM, respectively (Chanthakhoun & Wanapat 2012).Su-jiang et al. (2016) reported that total VFA from in vitro incubation in sweet sorghum silage and sorghum silage was 35.0 and 27.54 mM, respectively.Result in this study showed total VFA concentration around 86.38-109.83mM, respectively.This higher result was caused by feed substrate difference.Fibre source from sorghum was more optimal to support rumen microbial performance if it was combined with concentrate in the diet.
The highest DMD and OMD was produced by P4, but were no significantly different than P1, P2, and P6.P4 produced higher DMD and OMD than P3 and P5 (P<0.05).Diet with silage as fibre source (P2, P4, and P6) tended to produce higher OMD than P1, P3, and P5.This respon was caused by structured carbohydrate content in the fibre source have been fermented by microb in the silage process.This caused faster degradation of DM.Silage treatment might increase degradation value of OM due to microflora activity during fermentation.The activity was effect of cellulase extra-curricular enzyme cutting cellulose bind in the silage substance (Jancik et al. 2011).OMD value, numericaly, was proportional to NH3 concentration (Table 4) and maximum gas production value (a+b) (Table 3).High maximum gas production reflected organic material degradation level in diet.This proved that optimal fermentation level during 48 hour incubation was produced by P1, P2, and P4.From the production of total VFA, DMD, OMD, and low production of CH4, P4 was better than other treatments.Sorghum silage Samurai 2 might be used to substitute fibre source from sorghum straw and sorghum bagass.

CONCLUSION
Samurai 2 was better than Pahat and Samurai 1 for buffalo diet.This was presented by high production of gas, total VFA, and Dry Matter Degradation.Diet containing of Samurai 2 also produced low CH4 in vitro.It needed further in vitro test to strengthen evaluation study of this buffalo diet.

Figure 1 .
Figure 1.Concentration of CH4 of buffalo diet from 48 hour incubation (left) and CH4 production of the diet in every 100 mg organic matter digested (right)

Table 1 .
Composition of experimental diet based on DM with fibre source and concentrate ratio by 50 : 50

Table 2 .
Composition of experimental diet nutrient basen on DM with fibre source and concentrate ratio by 50 : 50

Table 3 .
Production of total gas and kinetic gas in vitro of buffalo diet containing of sorghum during incubation for 2-48 hours (ml/200mg bk)

Table 4 .
Characteristic of rumen fermentation In Vitro of buffalo diet based on sorghum in 48 hour incubation (Table