Glp 21 model

Rumen fluid was collected from three slaughtered Egyptian buffalo heifers (450 ± 50 kg, body weight) in each experiment at an abattoir belonging to the Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt. Collection of ruminal content from slaughtered animals saves money and overcomes the need for surgical cannula in live animals with complete adherence to animal protection law [29 ]. Also, the use of rumen fluid from slaughtered animals has been suggested and documented as an alternative in several earlier in vitro fermentation studies [30 –32 ]. The slaughtered animals were fed on a conventional feed for meat production, which contained 70% of concentrate mixture (16% CP) and 30% of Berseem hay. The rumen was cut open with a knife after 15 minutes of slaughtering, and the contents were taken from various positions within the rumen. The rumen content was immediately strained through four layers of cheesecloth, then placed in pre-warmed thermo-containers to keep its temperature at 39 °C and under anaerobic conditions, and then transported directly to the laboratory. Rumen fluid was again strained through four layers of cheesecloth and mixed before incubation. The initial pH of rumen fluid was measured using a portable pH meter (GLP21 model; CRISON, Barcelona, Spain) in each experiment at the laboratory.

All reagents were of analytical grade and used without further purification. Phosphate buffered saline (PBS, 0.01 M, pH 7.4), TRIS (hydroxymethyl)aminomethane (TRIS, 0.01 M, pH 9.1 and 9.5) and acetate buffer (1 mM, pH 5.1) were used as buffer solutions and prepared with ultrapure water Mili-Q laboratory grade. The pH values were measured with a pH meter (Crison Instruments, GLP 21 model). Graphite powder (fine extra pure, particle size < 50 μm) was purchased from Merck and used as received. Poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles dispersion in H₂O, 8-hydroxy-2-deoxyguanosine (8-OHdG, 98%), uric acid (>99% crystalline), sulphuric acid (H₂SO₄, 95–97%), multi-walled carbon nanotube (MWCNT) and multi-walled carbon nanotube, carboxylic acid functionalized (MWCNT-COOH) were obtained from Sigma Aldrich; poly(vinyl chloride) carboxylated (PVC-COOH) from Fluka; N,N-dimethylformamide (DMF) from Analar Normapur and ascorbic acid from Riedel-de-Haen. All measurements were carried out at ambient temperature.

Rumen pH was determined using a pH meter (GLP 21 model; CRISON, Barcelona, Spain) in all fermentation bottles. Protozoal count was microscopically determined and differentiated by Digital Zoom Video microscope (LCD 3D, GiPPON; Wanchai, Hong Kong) following the procedure described by Dehority et al. [28 (link)].
Individual short-chain fatty acids (SCFAs) concentrations were determined according to Palmquist and Conrad [29 (link)] and adapted to Soltan et al. [22 (link)] using gas chromatography (Thermo fisher scientific, Inc., TRACE1300, Rodano, Milan, Italy) fitted with an AS3800 autosampler and equipped with a capillary column HP-FFAP (19091F-112; 0.320 mm o.d., 0.50 μm i.d., and 25 m length; J & W Agilent Technologies Inc., Palo Alto, CA, USA). A mixture of known concentrations of individual SCFAs was used as an external standard (Sigma Chemie GmbH, Steinheim, Germany) to calibrate the integrator. Concentrations of ruminal NH₃-N were measured colorimetrically using a commercial lab kit (Biodiagnostic kits, Giza, Egypt) [30 (link)].