Low Irradiation Dose for Sorghum Seed Sterilization : Hydroponic Fodder System and In Vitro Study

The purpose of this study was to to evaluate the influence of low gamma irradiation dose on growth performance, in vitro gas production and rumen fermentation product of sorghum hydroponic fodder (SHF) to utilize them in ruminant diets. Three polyethylene packages of Samurai 2 sorghum seeds were exposed at 25oC gamma irradiation in gamma cell (Co-60) at doses of 100, 200 and 300 Gy in the presence of air. Samples were then referigated (< 5oC) before planting. All seeds were planted in nutrient film technique hydroponic system. This study used Completely Randomized Design with four replications The observed parameters were total fresh yield, plant height and coversion ratio from seeds to SHF. In vitro gas test evaluation was done to compare all treatments with sorghum straw. The observed parameters were total gas production, kinetics gas, CH4 concentration, CO2 concentration and rumen fermentation products. Results showed that lower irradiation dose for seeds sterilization decreased plant height and total fresh yield on SHF production. In vitro total gas production of all SHF treatment was higher (p<0.05) than sorghum straw (control) at 2-48 h time of incubation. Gamma irradiation dose of 200 and 300 Gy on SHF seeds sterilization decreased (p<0.05) in vitro CH4 concentration for 19.51 and 15.43% respectively compared to SHF control (hypochlorite sterilization). In the same dose, seeds sterilization with gamma irradiation increased (p<0.05) CO2:CH4 ratio by 23.46 and 20.73% respectively compared to SHF control. The treatment of 100 Gy gamma irradiation for seed sterilization also increased (p<0.05) TVFA by 30.63% compared to sorghum straw. It was concluded that lower irradiation dose for seeds sterilization decreased growth performance of SHF. However, 100 Gy gamma irradiation increased in vitro total gas production.


Introduction
Traditional farmers in Indonesia are a major component to fight against various constraints in the good nutrients supply for livestock.The problems are the limited land for forage and conversion of agricultural land to other purpose.Limited supply of forage land could decrease the supply of green forage.El-Morsy et al. (2013) also reported that the availability of fodder is decreasing due to climate change, higher competition for land and water resources between fodder and cereal crops.According to vulnerability in agriculture system, especially in green fodder production, strategies are needed in order to improved water and land efficiency.Hydroponic fodder is the right solution for the provision of forage in a limited land.
Hydroponic green fodder is produced from forage grains that are germinated and grown in short period time inside controlled growing rooms with appropriate growing conditions (Gebremedhin, 2015).Hydroponic technique can be used for green fodder production in a hygienic environment free of chemical residue (insecticides, herbicides, fungicides and growth promoters).Hydroponic fodder makes seed germinated and sprouted into high quality, highly nutritious and disease free fodder.This green fodder is also extremely high in protein and metabolisable energy (El Morsy et al., 2013;Gebremedhin, 2015).This planting system has enabled the production of green fodder forage from oats, barley, wheat and other grains (Fazaeli et al., 2012).Sorghum is potential cereal crops to be developed in Indonesia as hydroponic fodder.National Nuclear Energy Agency (BATAN) have produced sorghum variety which has the advantage of high biomass production and great nutrient composition (Samurai 2 variety).This varieties have not been optimally utilized as a source for forage, so that it is need to be developed as Sorghum Hydroponic Fodder (SHF).In many case, cereal grains may become contaminated with a range of microorganism especially fungi during their development.
Surface sterilization should be done to eliminate and decontaminated of seed surface affected with fungi.Sodium hypochlorite (NaOCl), mercury chloride (HgCl2) and Methyl bromide are conventional used for surface sterilization and fumigation of seeds (Younesikelaki et al., 2016).Alternative method is gamma irradiation that useful for both sterilization and for preservation of food and seed in nutrition and agriculture.Gamma irradiation also used for long-storage seeds and fumigation (range 1-6 kGy) on Oryza sativaseeds (Maity et al., 2009).Aynehband and Afsharinafar (2012) studied the effect of gamma irradiation (100-250 Gy) on germination characters of amaranth seeds.Surface sterilization effect with gamma irradiation depended on plant species and the dose of irradiation.From the above information, there was no information yet on low gamma irradiation effect for sterilization seed, in growth and degradability of sorghum hydroponic fodder.Therefore the objective of this study was to evaluate the influence of low gamma irradiation dose on growth performance, in vitro gas production and rumen fermentation product of sorghum hydroponic fodder to utilize them in ruminant diets.

Source of seed
Seeds of Samurai 2 sorghum (Sorghum bicolor (L.) Moench) variety were obtained from Agricultural Division, Center of Isotope and Radiation Application, National Nuclear Energy Agency, Indonesia.Samurai 2 variety was sorghum variety from mutation radiation technique breeding.

Gamma Irradiation
Gamma irradiation was carried out in a cobalt-60 irradiator in Center of Isotopes and Radiation Application.Three polyethylene packages of seeds were exposed at 25 o C gamma irradiation in gamma cell (Co-60) at doses of 100, 200 and 300 Gy in the presence of air.Samples were then referigated at < 5 o C before planting.

Hydroponic system
A growing plan was conducted using a steel hydroponic rack in controlled sterilized room equipped with automatic irrigation system with capacity of 10 polyethylene trays sized 60x18 cm 2 each.The hydroponic system was nutrient film technique system (Lee and Lee, 2015).Temperature inside room controlled to get a range of 20-22 o C and the relative humidity adjusted about 60-70% controlled by air circulation.Clean seeds of sorghum were soaked in tap water for 24 h then were spread in the trays.The density obtained was 0.38 g/cm 2 .The irrigation system automated by using digital timer to control water pumping.Trays were irrigated with hydroponic nutrition solution on 1 st day and then tap water on the day of 2-8.Trays were irrigated each two hour periods during daily hours for two minutes each time.

Sample preparation
Sorghum hydroponic fodder was harvested at 8 d after planting, where the fodder biomass was ready for harvest (Figure 1).The following data were recorded per tray: total fresh yield, plant height and coversion ratio from seeds to SHF.Representative fresh plant samples from every tray were taken and oven-dried at 60 o C for 48 h.Samples ground to pass a fine particle size.Organic material (OM), crude protein (CP) and ether extract (EE) (AOAC, 2010) were analyzed.Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were also analyzed using Goering and Van Soest (1970) procedures.
Rumen fluid for in vitro gas study obtained from fistulated male buffalo fed a 30:70 concentrate:field grass.Buffalo was fed twice daily at 8.00 am and 4.00 pm.Rumen fluid was collected from middle part of the rumen.

In vitro gas study
Approximately of 380 mg dry matter (DM) samples were added with 40 ml of rumen fluidbuffer in calibrated 100 ml syringe (Fortuna model, Germany) by following the method of Menke et al. (1979) as modified by Blümmel et al. (1997).The glassware was infused with CO2 and kept at approximately 39 o C before use the rumen strained and filtered through nylon net.The incubation was carried out in waterbath at 39 o C for 48 h.All of measurements were repeated four times.Variables observed were total gas production, kinetics gas, CH4 concentration, CO2 concentration, CH4:CO2 ratio, pH, ammonia (NH3), total volatile fatty acid (TVFA) and protozoa population.

Measurement of rumen fermentation product variables
Gas production was recorded on 2, 4, 6, 8, 10, 12, 24 and 48 h.Gas production reading was quickly conducted to minimize temperature change.Kinetics gas was measured by exponential equation of Ørskov and Mcdonald (1979) as follows: p = a + b (1-e -ct ).Where, p is the gas production at time t, a is the gas production from soluble fraction (ml/380 mg DM), b is the gas production from insoluble fraction (ml/380 mg DM), c is the gas production rate constant (ml/h), (a+b) is the potential gas production (ml/380 mg DM) and t is the incubation time (h).
The CH4 and CO2 concentrations, CH4:CO2 ratio, pH, NH3, TVFA and protozoa population were measured after 48 h incubation.The CH4 and CO2 concentrations were measured using MRU gas Analyzer®.Readed value on MRU gas Analyzer® was percentation of CH4 or CO2 stored in the syringe.The pH value was measured by pH meter Hanna instrument.Measurement of total VFA using AOAC (2010).The NH3 concentration was analyzed using Conway microdifusion methods (Conway, 1950).Protozoa population was measured according to the method of Ogimoto and Imai (1981).

Experimental design and statistical analysis
This study used completely randomized design with four replications from four tray unit with following model: Yij = μ + αi + εij; where: Yij is the i th treatment observation and j th replication, μ is general mean, αiis effect of i th treatment and εij is random affect of i th factor and j th replication.
Data were statistically analyzed using SPSS 20.00.Differences among treatments were compared using Duncan's multiple range test (Steel and Torrie, 1980).

Growth performance
Growth parameters included plant height, total fresh yield and ratio of SHF: seeds conversion are presented in Table 1.All parameters were significantly decreased after gamma irradiation treatments for seed sterilization (p<0.05).Plant height decreased by 16.39, 19.15 and 22.42% after 100, 200 and 300 Gy seeds sterilization treatment, respectively.Total fresh yield also decreased by 18.40, 22.23 and 24.09% respectively.SHF: seeds conversion calculated from comparison of total fresh yield after harvested and seeds yield before planting.SHF:seeds conversion decreased by 15.33, 16.75 and 20.28% after 100, 200 and 300 Gy seeds sterilization treatment respectively.
The higher dose of gamma ray (800 Gy) had negative effect on the morphological characteristics of corn (Emrani et al., 2013).By contrast, Aynehband and Afsharinafar (2012) reported that gamma irradiation were effective in improving germination index.In present study, dose of 100, 200 and 300 Gy for sorghum seed sterilization were not effective in improving plant growth.Gamma irradiation could decrease seed germination (Harding et al., 2012;Aparna et al., 2013).Lower growth performance on treatments of gamma irradiation for sterilization might be caused by DNA structural changes in sorghum seeds.Franco et al. (2015) reported that there is change in lettuce DNA bands after gamma irradiation treatment.These effect might be due to the structural rearrangements in DNA caused by different types of DNA damages.One of proper consideration on gamma irradiation for sterilization is mechanism DNA damage after irradiation process.Maity et al. (2009) also reported that the carbohydrate content and total protein are decrease due to higher metabolic activities and hydrolyzing enzyme activity in germinating seeds (range 3-4 kGy).Change of enzym activity due to induction by stress effect after irradiation process.
The range of SHF:seeds conversion ratio were 3.38 -4.24 (Table 1).Green fodder:seed ratio depended on several factors such as type and quality grain, amount and frequency irrigation, nutritious solution, humidity, lights position and harvested time (Fazaeli et al., 2012).From study Al-Karaki and Al-Hashimi (2012) have shown that sorghum produce lower conversion ratio (fodder:seeds) than cowpea, barley and alfalfa.However, sorghum had conversion ratio higher than wheat.Higher amount of barley green fodder to seed ratio was reported by Fazaeli et al. (2012) who obtained a ratio of 7.21 after harvested at 8 d.

In vitro gas study
Total gas production after in vitro gas study are presented in Table 3. CH4 concentration, CO2 concentration and CO2:CH4 ratio from in vitro total gas measurements are presented in Figure 2. In vitro total gas production of all SHF treatments were higher than sorghum straw (control) at 2-48 h incubation time (p<0.05).However, potential gas production (a+b) in sorghum straw was higher than all SHF treatments (p<0.05).Gamma irradiation dose of 200 and 300 Gy on SHF seeds sterilization decrease in vitro CH4 concentration for 19.51 and 15.43% respectively lower than SHF control (hypochlorite sterilization) (p<0.05).In the same dose, seeds sterilization with gamma irradiation increased CO2:CH4 ratio by 23.46 and 20.73% respectively compared to SHF control (p<0.05).
Total gas production of SHF at 2-48 h incubation was significantly different (p<0.05) when compared to sorghum straw treatment.The increasing of in vitro total gas production on SHF  could be as a result of the higher of soluble carbohydrates, minerals and vitamin of SHF than sorghum straw.Sorghum straw has higest content of NDF and ADF (Table 2).High fibre in sorghum straw might cause decrease on rumen microbial activity on less than 24 h of diet incubation, however gas production rate from fermentation by microbe was higher on 48 hours of incubation.It could be seen on potential gas production (a+b) after kinetics gas calculation.This was caused by rumen microbial started to digest substrate after 24 h incubation.Total gas production from in vitro fermentation represent the extent of feed digestibility and fermentation (Krizsan et al., 2013).Fazaeli et al. (2012) reported that variations in gas production are the result of variations in chemical composition.The increase of total gas production not yet represented the efficiency of feed substrate utility.The feed efficiency should be observed from variable of CH4 and CO2 concentration.The increase of total gas production was followed by CH4 production.Higher total gas production caused high CH4 production.CH4 variable was used to measure CH4 emition decrease level (Wahyono, 2015).SHF 100 treatment had highest total gas production but it had high CH4 concentration.Bodas et al. (2012) reported that CH4 gas production represent energy losses in form of gas reflecting low feed efficiency.The increase of microbial activity followed by increasing in protozoa population (Table 4).Protozoa population could increase due to more easily digested carbohydrate from SHF. Bhatta et al. (2015) reported that CH4 production was related to the protozoa population.CH4 was generated by methanogens bacteria that consumed hydrogen from carbohydrate fermentation.

Rumen fermentation product
In vitro rumen fermentation products of SHF vs sorghum straw were presented in Table 4. pH value in all SHF treatments were higher than sorghum straw (p<0.05).TVFA results showed that SHF control and SHF 100 had higher value than sorghum straw (p<0.05).However, SHF 200 and SHF 300 treatments had lower TVFA than  sorghum straw (p<0.05).CH4 concentration and protozoa population also increased on 100 Gy irradiation for seeds sterilization treatment higher than sorghum straw (p<0.05).There was no significant difference between all treatments in NH3 parameter.
The range of pH from this study at the level > 6.2.The results indicated that all treatments had no negative effect for the growth of cellulolytic bacteria.pH level > 6.2 could inhibits the growth of bacteria.The optimum and suboptimal pH values in the rumen for growth of bacteria were 6.4 and 5.5, respectively (Cardozo et al., 2000).pH balance rumen might effect on cellulose digestibility due to carbohydrate type of forage have a reciprocal relationship with pH value (Wahyono et al., 2014) The lower pH of all SHF treatments due to a higher content of soluble carbohydrate than sorghum straw.Wahyono (2015) reported that the decrease of pH value might be due to higher content of soluble carbohydrate from the substrate.The NH3 value in present study ranged from 36.02 to 41.67 mg/100 ml.These values were higher than normal range value for in vitro culture (5 mg/100 ml) (Wanapat et al., 2013).This is due to incubation technique was carried out in closed system so there was NH3 product accumulated (Wahyono et al., 2014).The higher TVFA value in SHF control and SHF 100 were reflection of the higher total gas production (Table 3).Wang et al. (2016) reported that fermentation rate was correlated with TVFA value.TVFA is the final product of carbohydrate fermentation.It is used for rumen microbial growth (Roza et al., 2013).TVFA also plays a role as a major energy source of ruminant and as the building block for milk synthesis in dairy animals (Jayanegara et al., 2016).High protozoa population is also a reflection of the high concentration of CH4 (Figure 2).

Conclusions
Lower irradiation dose for seeds sterilization decreased growth performance of SHF.Nevertheless, this study showed that SHF was a potential solution for the provision of forage in a limited land.Dose of 100 Gy gamma irradiation could increase in vitro total gas production better than SHF control and sorghum straw.CH4 concentration and protozoa population are increased on 100 Gy irradiation for seeds sterilization treatment, so there is still need more appropiate strategy to overcome this problem.

Table 1 .
Growth parameters of SHF after harvested at 8 th day after planting

Table 2 .
Nutrient composition of sorghum straw and SHF DM (dry matter); SHF (sorghum hydroponic fodder); SHF control (SHF after 10% hypochlorite seeds sterilization); SHF 100 (SHF after dose of 100 Gy seeds sterilization); SHF 200 (SHF after dose of 200 Gy seeds sterilization); SHF 300 (SHF after dose of 300 Gy seeds sterilization).a,b,c Different superscripts at the same coloumn indicate significant differences (p<0.05).Each value is a mean of four samples.SEM: Standard error of the Means.

Table 3 .
Total gas production and gas kinetics of sorghum straw and SHF

Table 4 .
Rumen fermentation products of sorghum straw and SHF after 48 h incubation