Donor animals
Four Dorper sheep with body weight of 28 ± 0.54 kg (mean ± standard deviation) and fitted with permanent rumen fistula were used in this study. The animals were fed on roughages and a commercial concentrate diet and kept individually in a cage. The fistulated 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 fistulated Dorper sheep prior to morning feed and strained through four layers of cheesecloth into a thermos flask which was flushed with CO2 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 fiber (NDF), acid detergent fiber (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% H2SO4 were added into one tube which was used for NH3-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 -20oC 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 105oC. Ammonia nitrogen (NH3-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).
CH4 = 0.5 × (A) +0.5(B) - 0.25 × (P)
Where,
CH4= amount (mmol) of methane produced
(A)= concentration (mmol) of acetate
(B) = concentration (mmol) of butyrate
(P) = concentration (mmol) of propionate
Bacteria quantification
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-specific 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 fluid 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 fluid samples.
Apparent biohydrogenation of oleic (C18:1n-9c), linoleic (C18:2n-6c), and linolenic (C18:3n-3) acid was determined based on the difference in the concentration of fatty acids between 0 h and 24 h in vitro incubation using the following formula (Adeyemi et al. 2015):
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 24 h incubation
Statistical analysis
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.