340 Ruminal Protozoa Viability Under Diverse Additives Using Trypan Blue Exclusion Method as an Alternative Counting Methodology
Authors: Cuervo Wilmer , Lopez Camila Gomez , Marenchino Ignacio Fernandez , Maderal Araceli B , Vargas Juan , Tarnonsky Federico , Fernandez-Lehman andres , Schulmeister Tessa M , Podversich Federico , DiLorenzo Nicolas 
Published: November 2023

Abstract
Beef cattle are a major contributor to methane (CH4) emissions. Enteric CH4 is produced by archaea, mostly hosted by ruminal protozoa, thus, changes in protozoa counts have been associated with CH4 production. Therefore, reliable ruminal protozoa count (PROT) is essential to evaluate CH4-mitigation strategies. Typically, PROT are done by fixation with methyl-green-formalin-saline solution (MFS), which reveals ciliate numbers but not ruminal protozoa viability (VIAB). The Trypan Blue Exclusion Method (TBEM) involves an intracellular probe that dyes the cytoplasm of apoptotic eukaryotic cells with membrane damage, but few studies report its use for PROT. Therefore, our objectives were to establish culture conditions to use the TBEM for PROT and VIAB under different diets (Exp. 1) and feed additives. In Exp. 1 ruminal fluid was collected from 4 Angus-crossbred ruminally cannulated steers receiving a high-grain diet (NOYE) alone or supplemented with 28 g /d of a product of yeast fermentation (YEAST). Filtered and diluted ruminal fluid was incubated (39ºC, 95% CO2) with (SUB) or without (NOSUB) the substrate diet in three types of containers; 125 mL bottles (BOT), Erlenmeyer (ERL), or 16 × 125 mm Hungate tubes (TUB). After 8, 12, 24, and 48 h PROT and VIAB were determined using the TBEM, gas production, and CH4 concentration were measured. Exp. 2 involved in vitro batch culture incubations with a silage-based substrate (CON), plus a 50:50% mix of condensed and hydrolysable tannins (TAN), cashew nutshell extract (CNSE), or citral (CIT) at 0.3, 0.02, and 0.2% of substrate DM, respectively. At 0 and 48 h PROT, VIAB, volatile fatty acids (VFA), CH4, in vitro organic matter digestibility (IVOMD), and gas were determined. In Exp-1, PROT and VIAB were greater in TUB (P = 0.01) at 24 h and in BOT at 48 h (P &lt; 0.01), particularly with SUB (P &lt; 0.01). Supplementation of YEAST reduced VIAB in BOT and TUB compared with ERL (P &lt; 0.01). In Exp. 2, TAN (P &lt; 0.01) and CIT (p 0.05) reduced PROT and VIAB at 24 and 48 h (P &lt; 0.01). Additionally, TAN reduced gas production (P &lt; 0.01), and IVOMD (P &lt; 0.01) coinciding with previous studies reporting protozoa reduction. Protozoa/mL were similar to previous reports with MFS in cows (1.5 × 104) and steers (3.7 × 103). In both experiments, PROT and VIAB reductions by YEAST, TAN and CIT coincided with a reduction of CH4, gas, and IVOMD. Differences in PROT with some studies using MFS might be associated with losses of small Entodinium during initial filtrations. When cultured in BOT with substrate, TBEM is a viable, simple, rapid, and cost-effective methodology to evaluate PROT and VIAB after a nutritional challenge. The ability to assess membrane disruptions associated with protozoa apoptosis in the TBEM is one its greatest advantages.