Profiling of in vitro rumen digestibility, fermentation parameters and fatty acid biohydrogenation of palm kernel cake-based diet supplemented with corn


 Corn supplementation can enhance the function of rumen and mitigate methane production. Thus, this study aimed to evaluate in vitro rumen digestibility, fermentation parameters and fatty acid biohydrogenation of palm kernel cake-based (PKC) diet substituted with different levels of corn. Corn was substitution into PKC basal diet at the levels; T1= (0% corn + 75.3% PKC), T2= (5% corn + 70.3% PKC) and T3= (10% corn + 65.3% PKC) of the diet. Rumen liquor was obtained from four fistulated Dorper sheep and incubated with 200 mg of each treatment for 24hrs and 72hrs. Net gas production, fermentation kinetics, in vitro organic matter digestibility (IVOMD), in vitro dry matter digestibility (IVDMD), volatile fatty acids (VFA), rumen microbial population and fatty acid biohydrogenation were determined. The results of the in vitro study showed that production of gas increased from 0 hr until 9 hrs with T2 having the highest gas production during this phase. After 48 hrs, the gas production began to decrease gradually with increase in incubation time. No significant differences were observed in the IVDMD, IVOMD, NH3-N, pH and VFA at 72 hrs. However, higher significant methane gas (CH4) production was observed in T3 when compared with T1 and T2. Microbial population did not differ significantly between treatment groups for total bacteria, F. succinogenes and R. flavefaciens. The rates of biohydrogenation were not affected by corn substitution although a significant difference was observed in that of C18:1n9. In conclusion, corn substitution maintained fermentation characteristics with increasing of unsaturated fatty acids.


Introduction
The high prices of grains have forced farmers to seek alternative sources to feed their livestock. South East Asian countries such Malaysia and Indonesia are blessed with abundant oil palm resources which generated a huge volume of oil palm by-product like palm kernel cake (PKC). The amounts of nutrients in the PKC are considered to have moderate crude protein content and its used as feed for livestock's feeding (Alimon 2004). The protein content of PKC is between 16-18%, which is considered to be

Materials And Methods
Donor animals Four Dorper sheep with body weight of 28 ± 0.54 kg (mean ± standard deviation) and tted with permanent rumen stula were used in this study. The animals were fed on roughages and a commercial concentrate diet and kept individually in a cage. The stulated sheep were fed two times a day, water and mineral blocks were provided ad-libitum. Equal volumes of rumen liquor were obtained from the four stulated Dorper sheep prior to morning feed and strained through four layers of cheesecloth into a thermos ask which was ushed with CO 2 and immediately transported to the laboratory for analysis.

Treatment diets
Three dietary treatments T1: control diet (0% corn + 75.3% PKC), T2 diet containing (5% corn + 70.3% PKC), and T3 diet (10% corn + 65.3% PKC) were formulated. The three diets were approximately isonitrogenous. Minerals premix are excluded from the diets due to the high content of essential minerals in PKC so as to reduce the amount of Cu in the diets. The chemical composition and ingredients of experimental diets are presented in Table 1.

Chemical analysis
The dry matter, crude protein, ether extract, and ash content of the treatments were determined according to (AOAC 1990). Neutral detergent ber (NDF), acid detergent ber (ADF) and acid detergent lignin (ADL) were determined according to the method of Van Soest (1994).

Sampling
The experiment was conducted in three runs. At the end of the trials, the in vitro samples were pooled based on time and treatment (0, 24, and 72 h of incubation). Net gas production was examined at 0, 3, 6, 9, 12, 24, 48, and 72 h. The pH of the rumen buffer were measured at 0 h and after 72 h of incubation using pH meter (Mettler-Toledo, Ltd, Leicester, UK) while substrate was collected from syringes after 0, 24, and 72 h of incubation and divided into two falcon tubes. Two to three drops of 10% H 2 SO 4 were added into one tube which was used for NH 3 -N analysis while the other tube was used for fatty acid and biohydrogenation analysis and microbial population. The substrate in both tubes were kept at -20 o C till analysis.

Kinetic fermentation
The computed gas production was according to the model of Ørskov and McDonald (1979).
V= a+ b (1-e -ct ) using Neway software Where, V = volume of gas formed at time t. a = amount of gas produced from soluble fraction. b = volume of gas produced at an insoluble fraction. c = gas production rate constant for the insoluble fraction. T = incubation time.

Rumen fermentation assessment
In vitro dry matter digestibility was ascertained after 72 h incubation according to (Menke and Steingass 1988). The blank and sample contained in the glass syringes were poured into a pre-weighed sintered glass, the syringes properly rinsed with distilled water to remove all residues, and the water evacuated completely from the sintered glass with the aid of a vacuum pump. Then the sintered glass and its contents were dried for 24 h in an airtight oven at 105 o C. Ammonia nitrogen (NH 3 -N) was determined in accordance to Parsons et al. (1984). The determination of volatile fatty acid (VFA) was done using a gas chromatograph (Hewlett Packard 6890 GC system) according to the procedure of Cottyn and Boucque (1968). Methane generated during in vitro rumen fermentation of the feeds in the syringes was approximated utilizing the equation based on VFA proportion according to (Widiawati and Thalib 2012). The extraction of the total bacterial DNA was done using the QIAamp® DNA Mini stool kit (Qiagen, Hilden, GmbH) in accordance with manufacturer's instruction with some changes. Species-speci c quantitative real-time PCR was carried out using CFX96 Touch Real-Time PCR Detection System (BioRad, USA) with an optical grade plate of SYBR Green mix detection. The microbial populations such as total bacteria, cellulolytic bacteria, methanogenic archaea and total protozoa were determined by quantitative real-time PCR according to (Saeed et al. 2018).

Fatty acid analysis
The fatty acid composition of the rumen substrate was incubated for 0, 24, and 72 h. The rumen uid was removed from the freezer and allowed to thaw at room temperature for about 45 minutes. The total lipid extraction method described by Folch et al. (1957) was used for extraction of fatty acids from the feed and rumen uid samples. The chemical composition and secondary compound metabolism of forage samples were analyzed using a simple mean, while other data were subjected to one-way analysis of variance (ANOVA) of SAS, (9.4). Means were separated using Turkey.

Results
In vitro gas production and fermentations of rumen liquor There were no signi cant differences (P>0.05) in in vitro kinetic fermentation ( Table 2). Figure 1 shows pro le of the cumulated gas production of T1, T2, and T3 following incubation in the buffered rumen uid of sheep. The outcome indicated that gas production increased gradually and reached its peak at an incubation time 9 h, T2 recorded the highest gas production when compared to other treatment groups. However, after 48 h of incubation gas production began to decline gradually with passing of time. No signi cant differences (P>0.05) were observed in IVDMD, IVOMD, and NH 3 -N and pH at 72 h. However, signi cant effect of CH 4 was observed with T3 showing the highest CH 4 concentration when compared with T1 at 2.81, 2.71, and 3.72 (mmol/L of gas) respectively (Table 3). Table 4 showed the quantum of each VFA product (acetate, propionate, butyrate, isopronate and isobutyrate) after in vitro incubation. No signi cant difference was observed on overall VFA at 0 and 72 hrs and mean overall of incubation among the treatment groups. However, at 72 hrs of incubation only propionate, isopropionate and butyrate showed signi cant difference (P<0.05) when corn was used as the energy source. The results showed higher propionate and butyrate with low-level isopropionate recorded in T3 at 72 h of incubation as compared to T1 and T2. Also, no signi cant differences were observed among treatments in isobutyrate, and C2: C3 in the incubation period.

Volatile fatty acid
The Rumen microbial pro le The effect of treatment on total bacteria population in the rumen liquor measured at different hours of in vitro experiment is presented in Table 5. The total bacteria in the rumen liquor concentration was signi cantly higher in the corn substituted group (P<0.01) at 0 hr of the trial. However, no signi cant differences were observed on the total bacteria population after 24 hrs of incubation. The population of cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus avefaciens) was not signi cantly different among the dietary treatments. Furthermore, the F. succinogenes showed a reduction pattern between 0-24 hrs for T2 and T3 than T1 which start to increase gradually as the trial progressed. However, the population of R. albus and R. avefaciens remained unchanged in all treatments during the entire period of the experiment. In this study, the methanogenic archaea population gradually increased in the rumen liquor from 0 h to 24 hrs. At 24 h of incubation, the total methanogenic archaea increased (P<0.001) in the rumen liquor as much as 5.56 and 5.12×10 9 /ml in T1 and T3, respectively while T2 remained unchanged with 4.12×10 9 /ml. Moreover, the overall mean of T1 caused an increase in the average of rumen methanogenic archaea population (P<0.001) compared to T2 and T3 (5.27, 4.02, and 4.43 10 9 /ml respectively). The effect of different levels of corn as substitute into PKC urea-treated rice straw based diet on the rumen protozoa population in the rumen liquor was quanti ed.
The mean concentration of protozoa in ruminal uid was signi cantly (P<0.001) higher at 24 h in T2 than in the other treatments. The average number of protozoa were signi cantly (P<0.001) affected by 3.88, 5.39, and 4.12 10 5 /ml respectively. The protozoa population in the rumen liquor slightly increased at 24 h in all treatments except for T3 where it has recorded the lowest population compared with the rest of the time in the treatments.

Discussion
In vitro gas production and fermentations of rumen liquor Amount of gas produced during in vitro incubation could re ect the degree of degradability and fermentation of a substrate. Substitution of corn had no effect on, gas production kinetics, IVDMD, IVOMD, pH, and NH 3 -N. The present nding is consistent with Chanjula et al. (2010) who reported that an inclusion of up to 30% PKC in ruminants diet did not adversely affects rumen fermentation characteristics and microbial populations. There was no signi cant difference in gas production for the soluble fraction (a), insoluble fraction (b), potential gas production (a + b), and gas production rate constant with the insoluble fraction (c) upon corn substitution for all the three treatments in this study. This was probably due to the inability of a microorganism to utilize the nutrients bound to the structural components of PKC under Maillard reaction that occurred during processing of oil extraction (Sundu and Dingle 2003). Cumulative gas production after 24 h of incubation increased when 5% of corn was included in the diet (T2) and a tendency to a curvilinear pattern is noted when the maximum result was approached. The increase in cumulative gas production is due to the higher initial fermentation rate of the corn treatments. Ruminal NH 3 -N and pH at 0 and 72 h incubation were not altered by diets containing corn with PKC urea-treated rice straw based diets, ranging from 31-36 mg/dL and pH 6.69 -6.79 at 72 h. The level of ruminal NH 3 -N exceeded 5-8 mg/dL in all treatment groups, which is the optimal level of NH 3 - with Satter and Slyter (1974) who reported 5 mg/dL as minimum level of rumen NH 3 -N for optimum microbial protein synthesis. The higher level of NH 3 -N observed in this study may probably be due to the high proportion of non-protein nitrogen (NPN) in the rice straw. The declined of NH 3 -N concentration might be due to more e cient N-utilization by rumen microbes when fermentable energy was available. As shown in the present study, dietary components such as NDF and ADF also contribute to the difference in CH 4 production. High levels of NDF raise CH 4 production by moving the short-chain fatty acid fraction towards acetate which is responsible for producing more hydrogen. The type of carbohydrate present in the diet is thought to dictate CH 4 production via changes in the ruminal microbial (Johnson and Johnson 1995).

Voliatile fatty acid
Fermentation in the rumen produces volatile fatty acids, such as acetate, propionate, and butyrate acids, which can be metabolized by the animal. In this study, the means of total VFA, acetate, propionate, and butyrate concentrations in the rumen were unaffected by dietary treatments but in terms of the numbers and the overall level of VFA was a little lower in T1 when compare with other treatments, most likely because of the low apparent digestibility. In present study, there was a continuous decrease in propionate and butyrate production but no difference in acetate after 72 h which may be due to the present of rice straw that is high in ber in the diet. Treatment groups substituted with 5% and 10% corn (T2 and T3) showed a signi cant increase in propionate at 72 hrs. Generally, in ruminants, rapidly fermentable substrates have a relatively higher propionate acid production. On the other hand, slowly fermentable and cellulose-rich substrates will have high acetate acid-directed fermentation products.
High concentrations of soluble carbohydrate promotes propionate production in the rumen, lower ruminal pH, and inhibits methanogen growth, thereby reducing CH 4

Rumen microbial pro le
Several reports showed that dietary corn substitution affects total bacteria populations in rumen (Saeed et al. 2018). The total bacteria population in T2 and T3 demonstrates that it is signi cantly higher at 0 h but declined at 24 h. This may be due to the increased in the number of protozoa and their engul ng effect on the rumen bacteria. This is contrast to Abubakr et al. (2014) who reported that an increase in rumen bacteria population in goats fed PKC based diet could be due to the rapid multiplication after elimination of protozoa. The reason for this is not clear but it could be due to bacterial predation by rumen protozoa which is mainly reliant on the size and type of protozoa such as the holotrich protozoa which has much lower predatory activity than entodiniomorphids. Moreover, the mean of rumen total bacteria population in this in vitro trials had signi cantly reduced in T1, it is well established that fatty acids composition in substrate could be toxic to total bacteria (Williams and Coleman 1997) while long or short-term defaunation may reduce the rumen bacteria population. Methanogens archaea respond differently to different levels of corn at 24 h. In our experiment, the relative abundance of methanogens archaea slightly increased in response to corn in T3, was reported to be affected slightly (Lu et al. 2020). Methanogenesis frequently makes use of the hydrogen and CO 2 from the fermenting of carbohydrate as VFA are formed. By eliminating hydrogen from the ruminal environment as a last step of carbohydrate fermentation, methanogens permit the microorganisms active in fermentation to function at an optimal rate and to form the complete oxidation of substrates. With regard to this, it could be that increasing methanogens archaea from the higher degradability of feed in T3 at 24 h that releases H 2 subsequently enhances the ability of methanogens archaea to grow. Johnson and Johnson (1995) stated that the digestion of cell wall ber enhances CH 4 production by raising the quantum of acetate production and lowering the propionate produced. The rise in CH 4 emission is because of the fermenting of acetate which creates a methyl group for methanogenesis. The level of methanogens observed in T2 are an indication of low CH 4 emission since the hydrogen ion needed by methanogens to reduce CO 2 to CH 4 Abdullah et al. (1995) stated that the number of protozoa in the rumen uid of sheep was reduced after consuming PKC in the rst two groups of sheep. Regarding, the reduction in the number of protozoa was related to the lowest butyrate production. In addition, a study had reported that unsaturated fatty acids reduce protozoa numbers (Machmüller and Kreuzer 1999) due to the fact that unsaturated C18 fatty acids are toxic to protozoa. Therefore, the use of PKC based diets may have the potential of reducing protozoa numbers thereby changing the ruminal ecosystem, and indirectly increase the bacterial population and activity. However, the protozoa and, hence the production of sulphide, decreases the bioavailability of dietary Cu but our ndings show that protozoa levels declined at the higher levels of corn as energy source which is deemed a motivator for protozoa growth (Saeed et al. 2018).

Fatty acid composition of rumen liquor
The main fatty acids substrate for biohydrogenation in ruminants is linolenic acid (cis-9,cis-12,cis-15-18:3) because it is the most abundant fatty acid present in glycolipids and phospholipids of feed. The current study found that the proportion of fatty acids were not affected by corn dietary intake at 24 and 72 h, this could be due to the composition of fatty acids in these diets. The lower of C18:1n9 in T2 and T3 at 24 h was similar with ndings of Adeyemi et al. (2015) was noted that stearic acid (C18:0) is the primary end product of rumen biohydrogenation. Signi cant reduction of C18:0 could be due to incomplete biohydrogenation of C18:2n6c, C18:1n9c and C18:3n-3 which yielded a higher concentration of biohydrogenation intermediates trans-11 C18:1 and CLAc9t11. C18:1n9 declined signi cantly with increasing levels of corn for this study at 24 and 72 h. This is contrary to what we have expected and may be due to the ability of rumen bacteria to synthesize the biohydrogenation intermediate from C18:2n-6 (Harfoot and Hazlewood 1997). In this study palmitic (C16:0) was the second most abundant ruminal FA at 72 h although its proportion was heavily in uenced by corn diets. A possible explanation for this may be that palm oils and its byproducts are usually considered good source of C16:0 ( In this study, the substitution of different levels of corn in the PCK basal diet increased CH 4 emissions and the biohydrogenation percentage of LA and C18 PUFA under in vitro conditions. The corn substitution improved fermentation characteristics without any adverse effect on total gas production in vitro. It also affected the relative abundance of total bacteria. Short-chain fatty acid absorption seems to help in stabilizing ruminal pH by eliminating the effect of toxic in the rumen.

Declarations
Ethics approval None Author contributions All authors were contributed to the analyzed the data and writing of the present article.

Con icts of interest
The authors declare no con icts of interest.

Consent for publication
Not applicable Availability of data and materials Availability of data and materials used and analyzed during this study is available from the corresponding author on reasonable request.        Figure 1 Cumulative gas production in different incubation time at various level of corn, (♦ =T1: 75.3% PKC + 0% corn), (■ = T2: 70.3% PKC + 5% corn), (▲= T3: 65.3% PKC + 10% corn).