2.4. In vitro gas and methane production measurement

Rumen fluid was collected from slaughtered sheep following the procedure of Wang et al. [25]. On each experimental day, fresh rumen fluid was collected from two Arsi-Bale sheep slaughtered in abattoir. The donor animals were fed grass hay for 7 days before slaughter. Rumen fluid was collected early in the morning immediately after slaughter and collected into a pre-warmed thermos flask (39 °C) and immediately transported to laboratory. The pooled rumen fluid was strained through 4 layers of gauze then mixed with Menke's buffer (incubation medium) in a 1:2 ratio (v/v). The buffer solution was prepared using the method described by Menke and Steingass [26]. All handling was carried out under continuous flushing with CO₂ to minimize change in microbial population.

In vitro rumen incubation was done using a 100 ml glass syringe. Prior to incubation, 200 mg of dry and milled (1 mm sieve size) maize stover sample was weighed into each glass syringe and 2 ml of the already prepared concentrations of leaf extracts of AN, AZ, CC, LL, MS, RO, and TS, root of EK, seeds of CS, were added to syringes containing maize stover in triplicate in two separate runs/replications [12]. In both runs, each sample was tested with 3 replications of controls (syringes with samples and without plant extracts) and three blanks (syringes incubated with rumen fluid + buffer solution alone). The syringes containing feed samples, pre-warmed at 39 °C overnight and their pistons lubricated with vaseline to ease movement and prevent gas from escaping, then 30 ml of rumen fluid was added (rumen fluid + buffer mixture) under continuous CO₂ flushing. The syringes were incubated at 39 °C in a water bath and shaken manually every hour for an initial 8 h (including 0 h) of incubation [27] and then at each recording time [28]. Gas volume was recorded before incubation (0 h) and after 3, 9, 12, 24, 48, 72 and 96 h of incubation. Total gas volumes were corrected for readings of the blank syringes and initial volume at each time.

Total gas production was determined by reading the position of the piston at each time following the procedure of Menke and Steingass [26]. Net gas production (ml/200 mg) at t hours was calculated as: G_t = [(V_t–V₀–G₀) × 200]/W_S. Where: G_t = gas production value (ml/200 mg) at t hours, G₀ = gas production of blank syringes (ml), V₀ = initial volume in ml, V_t = volume in ml at t hours, W_S = weight of dried sample in mg.

In-vitro organic matter digestibility and metabolizable energy of the samples were calculated using the equations of Menke and Steingass [26] as follows:

Where: OMD = Organic matter digestibility at 24 h.

CP = Crude protein content of feed sample (% DM),

GP = Gas production (corrected for blank at 24 h of incubation), XA = ash content.

ME (MJ/kg DM) = 2.20 + 0.136 GP + 0.057CP

Where: GP= Gas production at 24 h of incubation (ml per 200 mg DM).

CP= Crude protein content of feed sample (% DM).

ME = Metabolizable enrgy.

Short-chain fatty acids (SCFA) were estimated as:

SCFA (mmol/g DM) = (0.0239 GP) - 0.0601 Getachew et al. [29].

Where: GP = Gas Production at 48 h of incubation.

Methane Production: Methane production at 24 h of incubation was measured using the procedure described by Fievez et al. [30]. For measuring methane production at the end of incubations and after recording the final gas volume, the lower end of the syringe was connected to the lower end of another syringe containing 4.0 ml of 10 M sodium hydroxide (NaOH). Sodium hydroxide was then introduced from the latter into the incubated contents, thereby avoiding gas escape. Mixing of the contents with NaOH allowed absorption of CO₂ and the gas volume remaining in the syringe was considered to be CH₄.

Net methane production was calculated by the differences of the methane in the syringe and the equivalent blank. The methane concentration was calculated according to Jayanegara et al. [31]: