Evaluation of Dietary Supplementation of Ammonium Sulphate on in vitro Gas Production and Rumen Fermentation Characteristics of WAD Ewes

carried out in collaboration between all authors. Author TOO designed the study and the protocol. However, author SAS anchored the field study, gathered the initial data and performed preliminary data analysis. Authors TOO and UAI managed the literature searches and produced the initial draft. All authors read and approved the final manuscript. ABSTRACT Ammonium sulphate, a chemical compound was utilized to evaluate its potential through in vitro gas production technique and rumen fermentation characteristics as a rumen stimulator and mitigates methane gas. Four experimental diets were formulated with ammonium sulphate (AS) included at varying levels: T1 (control/0% AS), T2 (control diet + 0.25% AS), T3 (control diet + 0.50% AS) and T4 (control diet + 0.75% AS). Rumen fluid was collected from the ewes, sixteen in number and weighing 24 kg averagely, using suction tube method to evaluate in vitro gas production, determine microbial population and rumen fermentation characteristics. Gas production was determined over a 96 hour period. Other data collected included organic matter digestibility (OMD%), short Chain Fatty Acids (SCFA µml), Metabolisable Energy (ME MJ/KJ DM), Degradability (D%), Volatile Fatty Acids (VFA), rumen pH, temperature and ammonia nitrogen (AN). Results showed no significant differences (p>0.05) for in vitro gas production profiles (i.e. total gas, OMD, ME, SCFA). However, as inclusion increased it stimulated the parameters measured to be numerically higher than control. Methane gas was highest for control diet (4 ml/200 mg DM) while T2 recorded the least (2 ml/200 mg DM). Degradability increased as the inclusion level of AS increased. Control diet was the lowest (32%) while T4 had the highest (45%). The rumen fermentation characteristics showed significant (p<0.05) differences for all the parameters measured except rumen temperature. Animals on T4 diet recorded the highest (p<0.05) pH value (6.85) while those on T2 diet had the least (6.53). Animals on Control diet recorded the highest values (p<0.05) for AN and acetic acid (100.40 mg/l and 8.98 mol/100L) while the least values were observed in animals on T2 diet (AN) and those on T4 (acetic acid). Increase inclusion of AS caused a decrease in acetic acid. Rumen bacteria was highest (p<0.05) in animals on T4 (1.40 x 10 6 cfu/mm 3 ) and lowest in those on T3 (0.65 x 10 6 cfu/mm 3 ). For fungi and protozoa, animals on T4 and T2 diets were higher (p<0.05) respectively than those on Control diet (1.15 and 1.09 x 10 6 cfu/mm 3 compared to 1.10 and 0.85 x 10 6 cfu/mm 3 respectively). Supplementation of AS has been validated by this study, with a 0.75% inclusion level resulting in lowest methane gas, and higher ME, SCFA, pH, bacteria and fungi and may be recommended for ruminant feeding.

measured to be numerically higher than control. Methane gas was highest for control diet (4 ml/200 mg DM) while T2 recorded the least (2 ml/200 mg DM). Degradability increased as the inclusion level of AS increased. Control diet was the lowest (32%) while T4 had the highest (45%). The rumen fermentation characteristics showed significant (p<0.05) differences for all the parameters measured except rumen temperature. Animals on T4 diet recorded the highest (p<0.05) pH value (6.85) while those on T2 diet had the least (6.53). Animals on Control diet recorded the highest values (p<0.05) for AN and acetic acid (100.40 mg/l and 8.98 mol/100L) while the least values were observed in animals on T2 diet (AN) and those on T4 (acetic acid). Increase inclusion of AS caused a decrease in acetic acid. Rumen bacteria was highest (p<0.05) in animals on T4 (1.40 x 10 6 cfu/mm 3 ) and lowest in those on T3 (0.65 x 10 6 cfu/mm 3 ). For fungi and protozoa, animals on T4 and T2 diets were higher (p<0.05) respectively than those on Control diet (1.15 and 1.09 x 10

INTRODUCTION
Apart from volatile fatty acids and microbial proteins, methane gas and carbon (IV) oxide (CO 2 ) are also released into the atmosphere as a result of rumen microbial fermentation. Enteric fermentation from livestock is a large source of methane, which has a global warming potential 23 times than that of CO 2 [1]. Ruminant livestock estimate for enteric methane is estimated to be 17-37% [2]. Hence, a need to mitigate methane is of utmost importance due to its effect on the environment and man. Several feed additives have been utilized to suppress this gas such as probiotics, antibiotics, plant extract and essential oils. Methanogenesis can be reversed by using sulphate as the terminal electron acceptor [3]. This submission had earlier been reported that methane gas is decreased immediately following the addition of sulphate [4,5]. It had been suggested the more energetically favourable nitrate and sulphate as having potential for high affinity electron acceptors competing with CO 2 for hydrogen and thus maintaining the availability of oxidized cofactors generated in fermentative degradation of carbohydrates, which at the same time lowers methane production [6]. The product of nitrate and sulphate reduction is the ammonium ion and sulphate ion respectively. Hence, this study was carried out to evaluate the effect of ammonium sulphate on in vitro gas production, degradation of diets and rumen fermentation characteristics of WAD sheep.

Experimental Site
The study was carried out at the sheep unit of Teaching and Research Farm and in the laboratories of the Department of Animal Science, University of Ibadan, Oyo State, Nigeria.

Collection and Preparation of Experimental Diets
The feed ingredients as presented in Table 1 were sourced from the locality dried and ground before mixing to make a total mix ration (TMR) with 50:50 concentrate to roughage (Panicum maximum) ratio without supplementation (T1) and TMR with 0.25% (T2), 0.5% (T3) and 0.75% (T4) ammonium sulphate respectively. Ammonium sulphate was gotten from a chemical store. Sixteen dry ewes aged 18 months with initial average weight of 24.00±0.13 kg were randomly allocated so as to ensure uniformity in the four treatments.

Rumen Fermentative Characteristics and
In vitro Gas Production Procedure About 100ml of rumen liquour was collected before morning feeding from each ewe with the aid of suction tube as described by [7]. One portion was used for determination of temperature and pH immediately after collection using a portable thermometer and digital pH meter respectively. The liquor was further processed by straining and the filtrate was analyzed for ammonia-nitrogen using micro kjedahl method [8], volatile fatty acid was determined on HPLC system (Shimadzu IOAD, Japan) according to procedure described by [9]. Carbon dioxide (CO 2 ), Hydrogen sulphide (H 2 S) and methane (CH 4 ) were analyzed for. Total viable bacteria, protozoa and fungi were enumerated by the method of [10,11].
For the in vitro gas production, incubation procedure was as reported by [12] and carried out at 39±1°c and the volume of gas production was measured at three hourly intervals up to 96 hours. Rumen liquor was obtained with the help of a stomach tube, transferred into pre heated thermos flask, strained through a sieve cloth and flushed with CO 2 . The buffer containing 9.8 g NaHCO 3 + 2.77 g Na 2 HPO 4 + 0.57 g KCl + NaCl + 0.12 g MgSO 4 .7H 2 O + 0.16 g CaCl 2 .2H 2 O was used and kept in the incubator for warming prior to being mixed with rumen fluid (1:4) as inoculums, all under continuous flushing with streams of CO 2 . About 200 mg of the substrate (sealed within porous bags) was measured and introduced into the syringe after removing the plunger. At post incubation period, 4 ml of NaOH (10m) was introduced to estimate methane production as reported by [13]. Data for gas production were fitted to an exponential equation as proposed by [14]: GP = a + b (1 -exp -ct ). Where: GP = Gas production (ML) at time t; a + b = Gas Potential Production; C = Rate of gas production (ML/h). The organic matter digestibility (OMD%), short chain fatty acids (SCFA mmol/200 mg), metabolisable energy (ME; MJ/Kg DM) and degradability were calculated using formula -OMD% = 14.88 + (0.889*GV) + (0.45*CP) + (0.651*XA); SCFA= -0.00425 + (0.0222*GV ml); ME= 2.20 + 0.136*GV + 0.057*CP + 0.0029*CP2 and Degradability= (Initial weight of substrate -Final weight after incubation)/Initial weight*100%.

Chemical and Statistical Analysis
Dried and ground samples of the experimental diets were used for chemical analysis. Crude protein, crude fibre, ether extract and ash were determined according to methods of [8]. Fibre fractions were determined using the methods of [15]. The values are as presented in Table 2. Data collected were subjected to one-way analysis of variance according to the procedure of [16] to determine the significance of treatment effects on the various parameters measured and where differences were observed, the means were separated using Duncan Multiple Range Test of the same package.   Table 3 shows the in vitro fermentation characteristics of ammonium sulphate diets at 96 hr. The potential of gas production 'b' was highest in T2 (10.50 ml) while the lowest was recorded for T4 (6.38 ml). The advantage of 'b' is for predicting feed intake and can account for 88% of variance intake [17]. It is well known that gas production is basically the result of fermentation of carbohydrate to volatile fatty acids [18]. Low (a+b) recorded in T1 (7.50 ml) was probably due to absence of ammonium compound. The highest inclusion level of ammonium sulphate did not produce the highest volume of gas and this supports the finding of [19] who reported that protein fermentation does not lead to much gas.

Methane Gas Production and Degradation
Methane (ml/200 mg DM) production for 96 hr as shown in Fig. 1 ranged from 2 ml -4 ml / 200 mg DM, with the least being from T2 and the highest from T1. Increased inclusion of ammonium sulphate in the diet resulted in increase in methane gas that was still lower than control (T1). It had been reported that methane increased with increase in ammonium sulphate application rate, caused by competition for substrates between sulphate -reducing bacteria and methanogens [20] and this agrees with our finding. Fig. 2 was the result of dry matter degradability (%) of ammonium sulphate supplemented diets (TMR). Ammonium Sulphate (AS) supplementation was effective in stimulating degradability as Increased inclusion of AS  indicated that it stimulated the rumen bacterial proliferation thereby resulting in a greater rate of degradation. The present result was in agreement with those of [21,22]. Bacteria can use carbohydrates as carbon skeletons for proteins in combination with ammonia [23] and also utilize sulphur (organic and inorganic) to synthesize sulphur containing amino acids [24] to produce microbial protein. Table 4 shows the rumen fermentation and microbial count of WAD Sheep fed ammonium sulphate supplemented diets. Rumen temperature did not differ significantly (p > 0.05) across the treatments and was close to the range for sheep in the tropics (39 -40°C). However, significant (p<0.05) difference was observed in rumen liquor pH amongst supplemented diets.

Rumen Fermentation and Microbial Count
Ewes on T4 recorded the highest (6.85) while those on T2 had the lowest (6.53), but the values recorded were within the level for optimum ruminal fermentation. The ammonia-nitrogen was highest (p < 0.05) for animals on the control diet (100.40 mg/L) while those supplemented with AS were lower compared to control. The reason may be attributed to higher ammonia-nitrogen uptake by microbes in the AS supplemented animals. This observation is accordance with the submission of [25] of reduced ammonia nitrogen in sulphate supplemented diets.
The volatile fatty acids were significantly (p < 0.05) different across the treatment diets with animals on T2 recording the highest (14.19 mol/100ml) and the least was observed for those on T4 (13.11 mol/100 ml). Increased inclusion of AS elicited a decrease in VFA values and this was also seen in acetic acid values. The propionic values were higher for AS  Ewes fed diet T4 had the highest (p<0.05) bacterial population (1.4x10 6 cfu/ml) while those on T1 recorded the least (1.15x10 6 cfu/ml). The animals on AS supplemented diets showed an increase in bacterial population as AS inclusion increased over those on control (T1) diet except for those on T3. This is suggestive of AS stimulating the proliferation of bacterial population.

CONCLUSION
This study has validated the potential of ammonium sulphate as a feed additive in ruminant nutrition. Supplementation of ammonium sulphate led to increase in rumen fermentation, efficiency of rumen microbes and stimulating them which occasionally higher in vitro gas production, and fermentation characteristics. The animals were able to tolerate 0.75% inclusion level of ammonium sulphate, hence it is recommended for supplementation in ruminant feeding. oxidation of methane mechanisms, bioenergetics and the ecology of associated micro organisms.