Profiling of Fatty Acids and Rumen Ecosystem of Sheep Fed on a Palm Kernel Cake-Based Diet Substituted with Corn

Abstract

The purpose of this study was to analyze the effects of corn substitution on the rumen functions and fatty acid profile of Dorper lambs fed a diet based on palm kernel cake (PKC). Corn was replaced with PKC basal diet at the following levels: C0% = (0% corn + 75.3% PKC), C1% = (5% corn + 70.3% PKC) and C2% = (10% corn + 65.3% PKC) of diet. The rumen fermentation was carried out in vitro, and feeding trials were in vivo. Twenty-seven lambs were used to determine gastrointestinal tract content and rumen fluid fatty acid. Rumen liquor was obtained from four fistulae Dorper sheep and incubated with 200 mg of each treatment for 24 and 72 h. In vitro organic matter digestibility (IVOMD), in vitro dry matter digestibility (IVDMD), volatile fatty acids (VFA), methane estimation (CH₄), rumen microbial population and fatty acid biohydrogenation were determined. The results of the in vitro study showed there were no significant differences in IVDMD, IVOMD, NH₃-N, pH and VFA at 72 h. Higher significant CH₄ production was observed in C0% when compared with C1% and C2%. Microbial population did not differ significantly between treatment groups. The rates of biohydrogenation were not affected by corn substitution, although a significant difference was observed in C18:1n9 (in vitro) and C18:1 t-11 (in vivo). In conclusion, the present study indicated that the corn substitution in the PKC diets maintained fermentation characteristics with an increase in unsaturated fatty acids in the rumen.

1. Introduction

The high cost of grains has forced farmers to seek an alternative source to feed for their livestock. South-East Asian countries such as Malaysia and Indonesia have abundant oil palm resources, resulting in a large volume of oil palm byproducts such as palm kernel cake (PKC). Palm kernel cake is now recognized as an important and cost-effective source of high-quality ruminant feed [1]. It is also a rich source of protein in the diet that could be a cost-effective feed and fattening strategy in comparison with imported concentrate feed [2]. Palm kernel cake was reported to have between 16–18% crude protein, which is considered to be moderately digestible and suitable for livestock’s feeding [3]. Chanjula, et al. [4] reported an inclusion level of 35% PKC in the diets of goats on a digestibility trial that lasted 21 days with no adverse effects on rumen fermentation characteristics, nutrient utilization, or microbial populations. However, corn has been a good source of energy in ruminants’ diets which can influence growth performance. Recently, corn supplementation has become a common practice in order to increase the energy density of diets for high-production ruminants [5,6,7]. The inclusion of corn in diets in the current study could compensate for the lack of energy that may occur when feeding on high levels of PKC. We hypothesized that the partial inclusion of corn in a PKC-based diet could increase the rate of fermentation [8]. Furthermore, there is a lack of information regarding the utilization of PKC as a primary constituent of high-concentrate diets that also include corn for in vitro fermentation and in vivo trials. Therefore, the purpose of this investigation was to evaluate the rumen digestibility and fermentation parameters of a PKC-based diet with varying amounts of corn as a substitute.

2. Materials and Methods

2.1. Experimental Design of Study

Two experiments were conducted, involving in vitro rumen fermentation and in vivo feeding trials. The in vivo experiment was carried out at the small ruminant field of Universiti Putra Malaysia. The in vivo experiment used a total of twenty-seven Dorper crossbred lambs, each about six months old and weighing about 15 ± 0.59 kg, which were randomly assigned to one of three dietary treatments (nine lambs per treatment). Individual pens with feeders and water bowls were provided for the lambs. The specific objective of the in vivo study was to determine gastrointestinal tract content and rumen fluid fatty acids. The in vitro study used four Dorper sheep with body weights of 28 ± 0.54 kg (mean ± standard deviation) fitted with permanent rumen fistula as rumen content donors. The aim of the in vitro trial was to study the parameters of rumen fermentation, the fatty acids in rumen fluid, and microbial population in the rumen. The animals were fed roughages and a commercial concentrate diet and kept individually in cages. The fistulae sheep were fed twice a day, water and mineral blocks were provided ad libitum. The fistulae sheep were fed a 50:50 rice straw and concentrate diet. Equal volumes of rumen liquor were obtained from the four fistulae Dorper sheep prior to morning feeding and strained through four layers of cheesecloth into a thermos flask, which was flushed with CO₂ and immediately transported to the laboratory for incubation. Three dietary treatments, a control diet (0% corn + 75.3% PKC), a C1% diet containing (5% corn + 70.3% PKC), and a C2% diet (10% corn + 65.3% PKC), were formulated. The chemical composition and ingredients of experimental diets are presented in

Table 1. Ingredients and chemical compositions of the treatment diets (DM basis).

Item	Corn Concentration (%)
	C0%	C1%	C2%
Rice straw urea treated	20	20	20
PKC	75.3	70.3	65.3
Protected fat (Megalac)	3	3	3
Corn	0	5	10
CaCO3	1	1	1
NaCl	0.5	0.5	0.5
Vitamin premix	0.2	0.2	0.2
Total	100	100	100
Chemical composition:
DM	91.78	91.66	91.55
Ash	13.80	12.72	12.74
OM	86.19	87.27	87.26
CP	15.42	14.88	14.09
EE	5.3	5.1	4.33
CF	26.6	24.50	20.83
NDF	62.36	60.06	55.66
ADF	45.60	40.96	37.30
ADL	6.56	6.10	5.43
Gross energy (MJ/kg DM)	16.89	17.29	17.65
Calculation:
Hemicellulose	16.76	19.10	18.36
Cellulose	39.03	34.86	31.86
NFE	40.44	41.11	48.39
ME (MJ/kg DM)	7.36	8.23	8.92
TDN (%)	43.91	49.24	53.46

The ratios of concentrate to straw were given on 80:20 dry matter basis, vitamin premix A: 10,000,000 IU; Vitamin E: 70,000 IU; Vitamin D: 1,600,000 IU; DM: dry matter; OM: organic matter; CP: crude protein; EE: ether extract; CF: crude fiber; NDF: neutral detergent fiber; ADF: acid detergent fiber; ADL: acid detergent lignin; NFE: nitrogen-free extract; ME: metabolizable energy; TDN: total digestible nutrients.

. Corn starch incorporated into the diets as a source of energy was derived from corn.

The fatty acids of experimental diets are presented in

Table 2. Chemical and fatty acid composition of treatments.

Diets
Fatty Acids	C0%	C1%	C2%
C12:0	8.11	4.74	5.57
C14:0	5.88	6.32	3.94
C15:0	3.67	2.43	2.68
C15:1	0.63	0.26	0.73
C16:0	31.97	35.43	39.97
C16:1 n-9	0.33	0.39	0.37
C18:0	14.14	14.74	7.59
C18:1n9	31.44	31.71	36.01
C18:2n6	3.16	3.10	2.55
C18:3n-3	1.27	1.09	1.29
ΣSFA	63.78	63.68	59.77
ΣUFA	36.21	36.31	40.22
ΣMUFA	32.41	32.37	37.12
Σn-3 PUFA	1.27	1.09	1.29
Σ n-6 PUFA	3.16	3.10	2.55
Σ PUFA	4.43	4.20	3.83
n-6:n-3 Ratio	2.71	2.85	2.18
UFA:SFA	0.56	0.57	0.67
Poly:Sat Ratio	0.07	0.06	0.07

MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; UFA, unsaturated fatty acids.

. The diets included a combination of roughage and concentrate. The lambs were fed at 8:00 a.m. and 6:00 p.m. daily with a 20:80 ratio of roughage to concentrate. After 120 days, five lambs from each treatment were slaughtered in the Meat Science Laboratory, Department of Animal Science, Agriculture Faculty, Universiti Putra Malaysia, following the standard protocol outlined in MS 1500:2004. Gentle emptying and weighing were performed on the rumen, reticulum, omasum, and abomasum after the gastrointestinal tract had been carefully dissected.

2.2. Sampling and Incubations Procedures of In Vitro Experiment

The in vitro experiment was conducted in three runs, glass syringes with internal diameters of 32 mm, lengths of 200 mm, and measured volumes of 100 mL were used for incubations. A rumen fluid-buffer medium (30 mL) was poured into a 100-mL glass syringe containing about 200 mg of dry treatment samples and fitted with a silicon tube. A blank containing only the rumen fluid-buffer mixture, not samples as well as standard hay and standard concentrate, was also incubated in syringes. Samples, blank, standard hay, and concentrate were incubated in triplicate. Two standards (hay and concentrate samples) were provided by the University of Hohenheim (Stuttgart, Germany) and were those proposed by Menke and Steingass [9]. At the end of the trials, the in vitro samples were pooled based on time and treatment (0, 24, and 72 h of incubation). Two to three drops of 10% H₂SO₄ were added to one tube which was used for NH₃-N analysis while the other tube was used for fatty acid and biohydrogenation analysis (0 and 72 h of incubation) and microbial population (24 h of incubation). The substrate in both tubes was kept at −20 °C until analysis.

2.3. Rumen Fermentation Assessment

In this study, seven samples were used for each assessment per treatment after the samples were pooled together. The in vitro dry matter digestibility (IVDMD) was ascertained at 72 h of incubation according to Menke and Steingass [9]. The in vitro organic matter digestibility (IVOMD) was determined as follows [10].

The blank and sample contained in the glass syringes were poured into a pre-weighed sintered glass, the syringes were properly rinsed with distilled water to remove all residues, and the water was 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 °C. Ammonia nitrogen (NH₃-N) was determined in accordance with Parsons et al. [11]. A standard curve was made to determine whether a linear relationship existed between the varying concentrations of ammonium sulphate standard solution and the intensity of color produced. The intensity of color thus developed was measured at a wavelength of 420 nm using a spectrophotometer (Secomam, Domont, France) within 5–10 min after setting it at 0 absorbance with the blank. The determination of volatile fatty acid (VFA) was performed using a gas chromatograph (Hewlett Packard 6890 GC system) according to the procedure of Cottyn and Boucque [12]. The rumen fluid sample from the freezer was kept at room temperature for about 2 h to defrost and 1 mL was pipetted into a falcon tube and 200 μL of 25% (w/v) meta-phosphoric solution added and allowed to remain at room temperature for about half an hour. Then it was centrifuged at 3000 g for 10 min at 24 °C. About 500 μL of supernatant was pipetted into a pre-labeled GC vial and 500 μL of 20 Mm×4-methyl N-valeric acids were added as an internal standard. Ruminal VFA was separated and quantified by gas chromatography (Agilent 6890, Mississauga, ON, Canada). Methane generated during in vitro rumen fermentation of the feeds in the syringes was approximated utilizing the equation based on VFA proportion according to Moss, et al. [13].

CH₄ = 0.45 × (A) − 0.275 × (P) + 0.4 × (B)

where,

• CH₄ = amount (mmol) of methane produced
• (A) = concentration (mmol) of acetate
• (B) = concentration (mmol) of butyrate
• (P) = concentration (mmol) of propionate

2.4. Microbial Populations and Quantification

The microbial populations were quantified after 24 h. The extraction of the microbial populations’ DNA was carried out using the QIAamp^® DNA Mini Stool Kit (Qiagen, Hilden, GmbH) in accordance with the manufacturer’s instructions with some little modifications. The strength of the DNA was measured using an ND-1000 NanoDrop (NanoDrop Technologies, Silverside, Wilmington, DE, USA) spectrophotometer. Species-specific quantitative real-time PCR was carried out using the CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA) with an optical grade-plate for 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. [14]. The PCR reaction was carried out in a total volume of 25 µL using the QuantiFast SYBR^® Green RT-PCR Kit. Every sample’s reaction contained 12.5 µL of SYBR Green Supermix, 1 µL of Forward Primer, 1 µL of Reverse Primer, 2 µL of DNA samples, 9.5 µL of DNA template, and RNAase-free water. Axygen scientific 0.2, thin wall, transparent Real-Time PCR strips with flat clear caps were used to hold the mixed samples. Every well received the following PCR reaction conditions: a 5 min initial incubation at 94 °C, followed by 40 cycles of denaturation at 94 °C for 20 s, annealing temperature for 30 s, and extension to 72 °C for 20 s.

2.5. Fatty Acid Analysis

The rumen liquor was collected immediately after slaughtering the animal in falcon tubes for fatty acid determination. The incubated rumen substrates for 0 and 72 h were removed from the freezer and allowed to thaw at room temperature for about 45 min. The total lipid extraction method described by Folch et al. [15] was used for the extraction of fatty acids from the rumen fluid samples. The total fatty acids were derived from 10 mL rumen liquor, and fatty acids were detected using a gas chromatograph (Agilent 7890A). The rumen liquor column was made of fused silica capillaries (Supelco SP-2560, 100 m, 0.25 mm ID, 0.20 mm film thickness).

Apparent biohydrogenation of oleic acid (C18:1n-9c), linoleic acid (C18:2n-6c), and α-linolenic acid (C18:3n-3) were determined based on the difference in the concentration of fatty acids between 0 and 72 h in vitro incubation using the following formula [10]:

Apparent biohydrogenation (%) = [100 × [(CFA)_I − (CFA)_f/(CFA)_i]

where,

• (CFA) _i = % concentration of unsaturated fatty acid at 0 h incubation
• (CFA) _f = % concentration of unsaturated fatty acid at 72 h incubation

2.6. Statistical Analysis

The chemical composition and secondary compound metabolism of samples were analyzed using the simple mean, while other data were subjected to a one-way analysis of variance (ANOVA) of SAS, (9.4). Mean differences were accepted as significant at (p < 0.05) and separated by Tukey’s test. The plots of residuals against expected values were used to assess the homogeneity of variances. The data were subjected to an analysis of variance by using the statistical model:

Yij = μ + si + eij

in which Yij = dependent variable; μ = overall mean; si = the fixed effect for supplementation of the level of corn (5 and 10%); and eij = experimental error that was assumed in the segments.

3. Results

3.1. Evacuation and Fermentations of Rumen

Major gastrointestinal tract (GIT) weight differences were also recorded among the three treatment groups (

Table 3. Effect of different levels of corn on weight of component of full and empty gastrointestinal tract.

Parameters	C0%	C1%	C2%	SEM	p-Value
Rumen full (kg)	5.30	4.96	5.16	0.15	0.18
Rumen empty (kg)	0.66	0.80	0.87	0.05	0.43
Omasum & Reticulum full (g)	100.00	116.67	113.33	13.54	0.89
Omasum & Reticulum empty (g)	100.0	100.0	100.0	0.01	0.93
Abomasum full (g)	180.00 b	293.33 a	230.00 b	18.02	0.001
Abomasum empty (g)	170.00 b	200.00 ab	210.67 a	8.83	0.05
Intestine full (kg)	1.30 a	1.18 b	1.33 ab	0.03	0.05
Intestine empty (kg)	0.86 b	1.11 ab	1.21 a	0.09	0.05

C0%: (75.3% PKC + 0% corn), C1%: (70.3% PKC + 5% corn), C2%: (65.3% PKC + 10% corn). ^a,b Means in the same row with different superscripts are significantly different.

). Lambs fed on 5% corn (C1%) had heavier abomasum weights compared to the C0% corn group (base diet) (p < 0.001) while those fed on C1% and C2% had the lowest intestine weight compared with those on base diet, which had a high value (p < 0.05). No significant changes in the rumen, omasum, and reticulum were observed in lambs provided with the dietary corn at any of the administered levels.

No significant differences (p > 0.05) were observed in IVDMD, IVOMD, and NH₃-N and pH at 72 h. However, a significant effect of CH₄ was observed, with C0% showing the highest CH₄ concentration when compared with C1% and C2% (

Table 4. Effect of variations among treatment diets on some fermentation characteristics.

Parameters	C0%	C1%	C2%	SEM	p-Value
IVDMD	51.78	44.37	49.91	1.25	0.46
IVOMD	80.54	85.04	82.97	1.82	0.61
CH4 (mmol/L)	3.36 a	2.95 b	2.81 b	0.16	0.01
NH3-N (mg/dL)
0 h	23.76	23.86	25.46	1.98	0.67
72 h	36.79	35.69	32.79	1.78	0.95
pH
0 h	6.80	6.69	6.75	0.02	0.34
72 h	6.83	6.79	6.81	0.01	0.67

C0%: (75.3% PKC + 0% corn), C1%: (70.3% PKC + 5% corn), C2%: (65.3% PKC + 10% corn), ^a,b Means in the same row with different superscripts are significantly different.

).

3.2. Volatile Fatty Acid

Table 5. Concentration of volatile fatty acids (VFA, mmol/ml) after 72 h in vitro fermentation of different levels of corn substitution.

Parameter	C0%	C1%	C2%	SEM	p-Value
Total VFA
0 h	12.77	12.87	12.80	0.38	0.42
72 h	13.61	14.67	16.86	0.51	0.51
Acetate
0 h	6.11	6.19	6.50	0.16	0.46
72 h	7.20	6.48	7.99	0.04	0.51
Propionate
0 h	3.56	4.05	4.23	0.67	0.59
72 h	2.94 b	3.12 ab	4.53 a	0.31	0.05
Butyrate
0 h	3.36	3.62	3.47	0.68	0.93
72 h	2.22 b	2.62 ab	3.52 a	0.22	0.05
C2: C3
0 h	1.68	1.74	1.78	0.10	0.54
72 h	2.06	1.75	2.06	0.07	0.20

C0%: (75.3% PKC + 0% corn), C1%: (70.3% PKC + 5% corn), C2%: (65.3% PKC + 10% corn), ^a,b Means in the same row with different superscripts are significantly different.

showed the quantum of each VFA product (acetate, propionate and butyrate) before (0 h) and after (72 h) in vitro incubation. No significant difference was observed in overall VFA at 0 and 72 h. However, at 72 h of incubation only butyrate showed a significant difference (p < 0.05) when corn was used as the energy source. The results showed higher propionate and butyrate recorded in C2% at 72 h of incubation as compared to C0% and C1%. Furthermore, no significant differences were observed among treatments in acetate: propionate (C2: C3) in the incubation period.

3.3. The Rumen Microbial Profile

The effect of treatment on total bacteria population in the rumen liquor measured at different hours of in vitro experiment was presented in

Table 6. The effect of dietary treatments of sampling on microbial population (copies/ mL) in vitro trial.

Item	Diets
Species	C0%	C1%	C2%	SEM	p-Value
Total bacteria (×1010)
24 h	11.58	11.60	11.29	0.06	0.08
F. succinogenes (×109)
24 h	7.75	7.01	7.50	0.16	0.46
R. albus (×106)
24 h	7.86	6.77	7.78	0.30	0.27
R. flavefaciens (×107)
24 h	6.84	7.46	6.91	0.14	0.17
Methanogenic archaea (×109)
24 h	5.56 a	4.12 b	5.12 a	0.18	0.001
Total protozoa (×105)
24 h	4.37 b	5.72 a	5.87 a	0.24	0.001

C0%: (75.3% PKC + 0% corn), C1%: (70.3% PKC + 5% corn), C2%: (65.3% PKC + 10% corn), ^a,b Means in the same row with different superscripts are significantly different.

. However, no significant differences were observed in the total bacteria population after 24 h of incubation. The population of cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) was not significantly different among the dietary treatments. Furthermore, the F. succinogenes showed a reduction pattern at 24 h for C1% and C2% than C0% as the trial progressed. However, the population of R. albus and R. flavefaciens remained unchanged in all treatments during the entire period of the experiment. In this study, the methanogenic archaea population gradually reduced in the rumen liquor. At 24 h of incubation, the total methanogenic archaea increased (p < 0.001) in the rumen liquor by as much as 5.56 ×10⁹/mL in C0% while C1% remained unchanged with 4.12 × 10⁹/mL. The results showed that the mean concentration of protozoa in ruminal fluid was significantly (p < 0.001) higher at 24 h in C1% than in the other treatments. The average number of protozoa was significantly (p < 0.001) affected by the treatments (4.37, 5.72 and 5.87 × 10⁵/mL respectively for C0%, C1% and C2%) for 24 h of incubation. The protozoa population in the rumen liquor slightly increased at 24 h in all treatments except for C0% where it recorded the lowest population compared with the rest of the time in the treatments.

3.4. Fatty Acid Composition of Rumen Liquor

The effects of corn on the fatty acid profile in the rumen fluid of slaughtered lambs are presented in

Table 7. Fatty acid composition (g/100 g total fatty acids) of rumen fluid in vivo fermentation of different levels of corn substitution.

Fatty Acids	C0%	C1%	C2%	SEM	p-Value
C12:0	3.79	3.97	3.80	0.16	0.91
C14:0	3.94	3.97	4.15	0.15	0.86
C15:0	0.36 b	0.52 ab	0.64 a	0.05	0.05
C15:1	0.44	0.59	0.58	0.04	0.27
C16:0	40.72	37.34	35.63	1.02	0.10
C16:1n-7	0.35	0.39	0.23	0.03	0.12
C16:1n-9	0.20	0.19	0.38	0.04	0.15
C18:0	33.10	32.21	36.70	0.93	0.09
C18:1 c 9	10.01	11.54	10.04	0.59	0.54
C18:1 t-11	1.33 b	3.17 a	2.09 ab	0.28	0.05
C18:2n-6	2.01	1.78	2.39	0.19	0.50
CLAc9 t-11	0.85	0.74	0.94	0.06	0.52
CLAc12 t-10	0.56	0.79	0.68	0.07	0.53
C18:3n-3	1.54	1.28	1.20	0.09	0.37
C20:4n-6	0.32	0.29	0.31	0.01	0.86
C20:5n-3	0.09	0.52	0.12	0.09	0.09
C22:5n-3	0.53	0.45	0.51	0.02	0.30
C22:6n-3	0.24	0.23	0.23	0.01	0.93
ΣSFA	81.93	78.02	80.94	0.96	0.25
ΣUFA	18.07	21.97	19.05	0.96	0.25
ΣMUFA	12.33	13.32	16.45	0.85	0.10
ΣPUFA n-3	2.42	2.49	2.07	0.12	0.37
ΣPUFA n-6	2.33	2.08	2.60	0.20	0.65
ΣPUFA	4.75	4.57	4.67	0.27	0.97
Σ Trans FA	1.33 c	3.17 a	2.09 b	0.28	0.05
ΣCLA	1.41	1.54	1.63	0.12	0.80
n-6:n-3	0.98	0.81	1.24	0.09	0.14
UFA:SFA	0.22	0.28	0.23	0.01	0.22
PUFA:SFA Ratio	0.06	0.05	0.05	0.004	0.95

MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; UFA, unsaturated fatty acids. ^a,b Means in the same row with different superscripts are significantly different.

. The corn diet affected (p < 0.05) the level of C15:0, C18:1 t-11, and Σ Trans FA. The corn substitution increased the concentration of C15:0, C18:1 t-11, and Σ Trans FA at levels C1% and C2% but decreased that of C0%. Further, the diets did not influence (p > 0.05) the other fatty acids detected. From the in vivo results of fatty acid composition, palmitic acid (C16:0) was the most abundant ruminal FA due to ruminal biohydrogenation, and its proportion reduced as the level of corn in the diet increased. On the other hand, the reduction of C16:0 levels may be due to a higher concentration of C16:1n-9 in the diet.

Table 8. Fatty acid composition (g/100 g total fatty acids) of rumen fluid and rate of biohydrogenation at 72 h incubation.

Fatty Acids	C0%	C1%	C2%	SEM	p-Value
C12:0	3.53 a	2.24 b	3.05 a	0.24	0.05
C14:0	2.62	2.74	2.97	0.07	0.12
C15:0	2.51	2.81	2.94	0.11	0.34
C15:1	0.91 b	1.24 a	1.19 a	0.06	0.05
C16:0	16.96 b	24.34 a	23.56 a	1.49	0.001
C16:1n-7	1.17	1.44	0.79	0.18	0.46
C16:1n-9	1.16	0.60	1.14	0.16	0.34
C18:0	49.64 a	39.68 b	40.20 b	2.19	0.05
C18:1 c 9	3.32 b	4.91 a	4.73 a	0.32	0.001
C18:1 t-11	1.89 b	3.05 a	2.77 a	0.23	0.05
C18:2n-6	10.27	12.56	10.91	0.66	0.43
CLAc9 t-11	1.39	1.12	1.39	0.18	0.85
CLAc12 t-10	0.81	1.11	0.91	0.10	0.59
C18:3n-3	1.23	0.93	1.53	0.23	0.69
C20:4n-6	0.60 b	0.90 a	0.59 b	0.07	0.07
C20:5n-3	1.40	0.55	1.21	0.21	0.27
C22:5n-3	0.55 a	0.30 b	0.28 b	0.05	0.05
C22:6n-3	0.91	0.69	0.99	0.08	0.44
ΣSFA	75.26	71.83	72.72	0.93	0.37
ΣUFA	24.73	28.16	27.27	0.93	0.37
ΣMUFA	8.46 b	11.24 a	10.64 a	0.54	0.01
ΣPUFA n-3	4.10	2.48	4.02	0.37	0.10
ΣPUFA n-6	10.87	13.46	11.50	0.72	0.39
ΣPUFA	14.97	15.94	15.52	0.60	0.87
Σ Trans FA	1.89 b	3.05 a	2.77 a	0.23	0.05
ΣCLA	2.20	2.23	2.31	0.20	0.98
n-6:n-3	2.63 b	5.43 a	2.94 b	0.59	0.05
UFA:SFA	0.33	0.39	0.37	0.01	0.35
PUFA: SFA Ratio	0.20	0.22	0.21	0.009	0.69
Apparent biohydrogenation (%)
C18:1n9	87.01 a	80.79 b	81.50 b	1.25	0.001
C18:2n-6c	47.73	36.05	44.44	3.36	0.43
C18:3n-3	63.65	72.63	54.82	4.17	0.23

MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; UFA, unsaturated fatty acids. ^a,b Means in the same row with different superscripts are significantly different.

shows the fatty acid profile of rumen liquor after 72 h of incubation. The addition of corn significantly (p < 0.05) increased the proportion of medium chain FA (C12:0, C15:1, C16:0). It also significantly (p < 0.01) increased the percentage of C18:1trans-11, C18:1n-9 but reduced the percentages (p < 0.05) of C18:0, C20:4n-6 and C22:5n-3. The ability of corn to grow Σ Trans FA was more (p < 0.05) noticeable in high concentrate substrates. However, small substitutions of substrates had significantly (p < 0.05) higher biohydrogenation intermediates at C0%. Apparent biohydrogenation of C18:2n-6 and C18:3n-3 was not as affected as the level of corn substituted in the substrate. Corn reduced (p < 0.001) the apparent biohydrogenation of C18:1n-9 in C1% and C2% compared with C0%.

4. Discussion

4.1. Evacuation and Fermentations of Rumen

This is while noting that the inclusion of PKC in the diet caused a certain degree of feed restriction, thereby lowering the weight of the primary metabolic organs such as the liver, heart, and small and large intestines [16]. The full intestine was lighter in lambs fed on the C1% and C2% diets than in other treatments (Table 3). An increase in rumen and intestine capacity is associated with a higher grass and roughage intake, and consumption enhances together with GIT weight increase [17]. Furthermore, the larger organ size could result in higher caloric requirements [18]. The results of GIT in the present study are in agreement with Abdelrahman et al. [19] in sheep and Buranakarl et al. [20] in goats fed a PKC diet. However, no difference was observed for the weights of the rumen and omasum-reticulum, possibly due to the chemical composition of PKC, which was used as a replacement for concentrates, perhaps, PKC indigestibility resulted in a rumen fill effect [16]. The present study found that lambs fed on a low level of corn diet were lighter in empty body weight and attributed this to the effect of gut fill caused by such a diet. In the previous study, the use of sugarcane stalks in the diet of cattle conducted by Kawashima et al. [21] revealed that harder fiber of the stalk might remain in the rumen for a relatively extended period, which would dampen consumption.

The amount of gas produced during in vitro incubation could reflect the degree of degradability and fermentation of a substrate. Substitution of PKC by corn had no effect on IVDMD, IVOMD, pH, and NH₃-N. The present finding is consistent with Chanjula, Mesang and Pongprayoon [4] who reported that an inclusion of up to 30% PKC in ruminants’ diets did not adversely affect rumen fermentation characteristics or microbial populations. This was probably due to the inability of a microorganism to utilize the nutrients bound to the structural components of PKC under the Maillard reaction that occurred during processing of oil extraction [22]. Ruminal NH₃-N and pH at 0 and 72 h of incubation were not altered by diets containing corn with PKC diets, ranging from 31–36 mg/dL and pH 6.69–6.79 at 72 h. The level of ruminal NH₃-N exceeded 5–8 mg/dL in all treatment groups, which is the optimal level of NH₃-N for microbial protein synthesis [23,24], while all treatment pH means were also within the normal range, with the values showing relative stability at 6.22–6.53, which is the optimal level for microbial digestion of fiber and protein at 6.0–7.0 [4,25,26]. The rumen NH₃-N concentration has not differed, but the observed value of NH₃-N was slightly higher in C0% and C1%. This was a relatively greater concentration of rumen NH₃-N and agreed with Satter and Slyter [23], who reported 5 mg/dL as minimum level of rumen NH₃-N for optimum microbial protein synthesis. The higher level of NH₃-N observed in this study may probably be due to the high proportion of non-protein nitrogen (NPN) in the rice straw. The decline in NH₃-N concentration might be due to more efficient 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 CH₄ production. High levels of NDF raise CH₄ 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 CH₄ production via changes in the ruminal microbial [27,28,29].

4.2. Volatile 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 the present 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, they were a little lower in C0% when compared with other treatments, most likely because of the low apparent digestibility. In the present study, there was a continuous decrease in propionate and butyrate production, but no difference in acetate after 72 h, which may be due in the presence of rice straw, which is high in dietary fiber. Treatment groups substituted with 5% and 10% corn (C1% and C2%) showed a significant increase in propionate at 72 h. Generally, in ruminants, rapidly fermentable substrates have a relatively higher production of propionate acid. On the other hand, slowly fermentable and cellulose-rich substrates will have high acetate acid-directed fermentation products. High concentrations of soluble carbohydrate promote propionate production in the rumen, and a lower ruminal pH inhibits methanogen growth, thereby reducing CH₄ production per unit of fermented organic matter. This is in agreement with Atasoy et al. [30] who reported that VFA produced in the rumen is affected by several factors such as pH, feed composition, and microbial species. However, Dias [31] reported that higher levels of PKC in the diet of goats may probably reduce the digestibility of energy from PKC, mainly because of the low content of starch. Butyrate production was significantly higher in C2% than the control at 72 h of incubation and this could be as a result of ruminal protozoa dominance that produces butyrate acid as their end product of carbohydrate fermentation [14,32]. The acetate-propionate ratio was the same across dietary treatments in the present study, as reported by Zhang et al. [33].

4.3. Rumen Microbial Profile

In recent years, there has been an increasing amount of literature on dietary corn substitution effects on total bacteria populations in the rumen [14]. The total bacteria population in C1% and C2% declined after 24 h. This may be due to the increase in the protozoa and their engulfing effect on the rumen bacteria [34]. This is in contrast to the findings of Abubakr et al. [35], who attributed the increase in rumen bacteria population in goats fed a PKC-based diet to the rapid multiplication after elimination of protozoa. The reason for this was 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 [36]. Moreover, the mean of rumen total bacteria population in these in vitro trials had significantly reduced in C0%; it is well established that fatty acids composition in substrate could be toxic to total bacteria [32], 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 C2%, was reported to be affected slightly [37]. Methanogenesis often utilizes the resultant hydrogen and CO₂ formed during the fermentation of carbohydrates as VFA. Therefore, eliminating hydrogen from the ruminal environment during carbohydrate fermentation could allow methanogens to permit the active fermentation microorganisms to function at an optimal rate with the resultant formation of complete oxidation of substrates [38]. With this, therefore, increasing methanogens archaea from the higher degradability of feed in C2% at 24 h with the releases of H₂ subsequently may enhance the ability of methanogens archaea to grow. Johnson and Johnson [27] reported that the digestion of cell wall fiber enhances CH₄ production thereby raising the quantum of acetate production and lowering the propionate produced. The rise in CH₄ emissions is due to the fermentation of acetate, which creates a methyl group for methanogenesis. The level of methanogens observed at C1% is an indication of low CH₄ emission, since the hydrogen ions needed by methanogens to reduce CO₂ to CH₄ were drastically reduced [39]. As a component of medium-chain fatty acids, it can potentially hamper rumen methanogenesis and methanogens because of its toxic effect. Rumen protozoa are in control of the symbiotic transfers of H₂ to methanogens, which are utilized to increase CH₄ and produce over 25% of CH₄ in the rumen [40]. Meanwhile, Machmüller et al. [41] reported that the relationship between protozoa and methanogens does not play a significant role in rumen methanogenesis. Changes in the proportion of the different methanogen species are due to a lower O₂ pressure and greater H₂ availability within the protozoal cells [32]. Rumen protozoa were affected by treatments at 24 h incubation, and overall protozoal populations were increased by the substitution of PKC with 5% and 10% of corn as sources of energy in the treatment (C1% and C2%), perhaps as a result of a higher level of corn in the diets which enhanced the dominance of protozoa over the rest of the rumen microbial population. This finding was in agreement with Abdullah and Hutagalung [42], who reported a negative influence of PKC on rumen protozoa of cattle fed a PKC-based diet. Some dietary factors may decrease or even remove ruminal protozoa. In addition, Abdullah et al. [43] reported that the number of protozoa in the rumen fluid of sheep was reduced after consuming PKC in the first two groups of sheep. The reduction in rumen protozoa population numbers may be attributed to lower butyrate production. It has also been reported that unsaturated fatty acids reduce protozoa numbers [44] due to the fact that unsaturated C18 fatty acids are toxic to protozoa. Therefore, the use of PKC-based diets may have the potential to reduce protozoa numbers, thereby changing the ruminal ecosystem, and indirectly increasing the bacterial population and activity. However, because the protozoa and, hence, the production of sulfide, decrease the bioavailability of dietary Cu, our findings show that protozoa levels are raised at higher levels of corn as an energy source, which is deemed a motivator for protozoa growth [14].

4.4. Fatty Acid Profile of Rumen

The presence of C15:0 in the diet contrasts with previous studies conducted by Wu et al. (1991) and Adeyemi et al. (2015b) who reported that, although C15:0 was absent from the diets, the abundance of C15:0 detected in the ruminal digesta suggested that it was created by the microbial population. Bauman et al. (2003), however, report that CLAc9t11 is only a transitory intermediate, and it is the trans-11 C18:1 that accumulates.

The corn substitute group had a higher proportion of C18:1trans-11 than the control group. This could be due to the ability of rumen bacteria to synthesize the biohydrogenation intermediate from C18:2n-6 [45] which is abundant in the C1% and C2% diets. Therefore, a greater proportion of C18:1trans-11 is expected in diets containing a higher proportion of C18:2n-6. The total trans FA increased with increasing levels of corn. This could be due to biohydrogenation, which yielded more C16:0 and C18:0 in the C1% and C2% compared with the control diet. The total FA increased linearly with the increase in energy, reflecting the level of dietary fat.

The main fatty acid substrate for biohydrogenation in ruminants was linolenic acid (cis-9,cis-12,cis-15-18:3) because it was the most abundant fatty acid present in glycolipids and phospholipids of feed [10,46]. The current study found that the proportion of fatty acids were not affected by corn dietary intake at 72 h; this could be due to the composition of fatty acids in these diets. A significant 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 C18:1 t-11 (72 h) C0%. The C18:1n9 declined significantly with increasing levels of corn for this study at 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 [47]. In the present study, palmitic (C16:0) was the second most abundant ruminal FA at 72 h although its proportion was heavily influenced by corn diets. A possible explanation for this may be that palm oils and their byproducts are considered good sources of C16:0 [48]. The control diet had a lower proportion of C18:1trans-11 at 72 h than other treatments. It was established that these unsaturated fatty acids include CLA and vaccenic acid [49], further increasing the possibility of significance of protozoa in the delivery of health-promoting FA from the rumen. Beam, et al. [50] reported that the extent of biohydrogenation increases in tandem with the degree of unsaturation of fatty acids. The increasing levels of substituted corn could probably be responsible for the significantly lower concentrations of C18:1n9c. It has been established that the incomplete biohydrogenation of linoleic acid [51,52], linolenic acid, and oleic acid yielded t-11-18:1, CLAc9 t11, CLAc12 t10 and other conjugated isomers. Protozoa play a significant role in biohydrogenation of the rumen microbial FA and contain proportionally more unsaturated fatty acids and CLA [53]. It is well-known that protozoal lipids contain proportionally more unsaturated FA than the bacterial fraction [14,53]. However, Serrapica et al. [54] indicated that due to differences in the source material, such as the presence of gelatinous compounds and the absence of surface area for bacteria to adhere to, bacterial adhesion is inhibited.

5. Conclusions

In conclusion, the inclusion of corn in the PCK basic diet at varying levels did not have an effect on the rumen’s fatty acid content. However, the biohydrogenation percentage of C18:1n9 was decreased under in vitro conditions. The substitution of corn improved fermentation characteristics in vitro with no adverse effects. In addition, it had a quantitative impact on the total number of protozoa. Short-chain fatty acid absorption seems to help stabilize ruminal pH by eliminating the effect of toxic substances in the rumen.