Department of Animal Science, Texas A&M University, College Station, TX 77843-2471, USA.
Department of Natural Resources Management, Texas Tech University, Lubbock, TX 79409, USA.
J Anim Sci. 2022 Sep 1;100(9). doi: 10.1093/jas/skac259.
The objectives of this multipart study were 1) to assess the efficacy of sampling methods of methane concentration ([CH4]) of headspace gas produced during in vitro gas production (IVGP) fermentation, 2) to verify whether headspace [CH4] sampled from an exetainer has the same [CH4] as the headspace of IVGP bottles, 3) to measure relative humidity (RH) within an IVGP bottle, and 4) to compare [CH4] on a dry-gas (DG) basis when accounting for water vapor pressure (Pw). The original IVGP protocol recommends placing bottles on ice (0 °C) for 30 min to stop fermentation (ICE). A laboratory protocol recommends placing the bottles in the refrigerator (4 to 6 °C) to slow fermentation for 48 h and subsequently allowing the bottles to return to ambient temperature before sampling (FRIDGE). This study evaluated the previous methods against a direct sampling of the headspace gas after incubation (DIRECT). Rumen inoculum from four rumen-cannulated beef steers was combined and homogenized before incubating the fermentable substrate of ground alfalfa hay. After 48 h of IVGP incubation, each bottle was randomly assigned to a treatment protocol. The pressure (P), volume (V), and temperature (T) of headspace gas in each bottle were recorded. Headspace gas was then thoroughly mixed, and 12 mL gas was removed into an evacuated exetainer for [CH4] sampling via gas chromatography (EXET; Objective 1). Eight bottles from ICE and FRIDGE were randomly selected to follow EXET, whereas the remaining bottles had [CH4] directly measured from their headspace (BOTT; Objective 2). Five diets of differing feed composition and nutrient densities were used with a blank to test the RH of the IVGP slurry (Objective 3). Using RH, [CH4] was transformed to a DG basis to account for Pw (Objective 4). Statistical analysis was completed using a random coefficients model. There were no differences between EXET and BOTT (P = 0.28). The RH of the IVGP slurry was 100% (P = 1.00), confirming that IVGP gas is saturated with water vapor. The P, V, and T differed among treatments (P < 0.01). The [CH4] of DIRECT, ICE, and FRIDGE were different (P < 0.01). Dry-gas P, V, and [CH4] differed among treatments (P < 0.01). As the methods differ in their assessment of [CH4], there is no clear recommendation. Instead, to present a more accurate [CH4], P, V, and T should be measured when sampling headspace gas and equations presented should be used to remove volume inflation due to water vapor and present [CH4] on a DG basis.
1）评估体外产气量（IVGP）发酵过程中顶空气体甲烷浓度 ([CH4]) 的采样方法的效果，2）验证从 exetainer 中采集的顶空气体 [CH4] 是否与 IVGP 瓶的顶空气体相同，3）测量 IVGP 瓶内的相对湿度（RH），4）在考虑水蒸气压力（Pw）的情况下比较干气（DG）基础上的 [CH4]。原始的 IVGP 方案建议将瓶子放在冰上（0°C）30 分钟以停止发酵（ICE）。实验室方案建议将瓶子放在冰箱（4 至 6°C）中以减缓发酵 48 小时，然后在采样前让瓶子恢复到环境温度（FRIDGE）。本研究评估了先前的方法与孵育后直接采集顶空气体（DIRECT）的方法。将来自四个瘤胃切开的肉牛的瘤胃液混合并均匀化，然后孵育苜蓿干草的可发酵底物。经过 48 小时的 IVGP 孵育后，每个瓶子随机分配到一个处理方案。记录每个瓶子内顶空气体的压力（P）、体积（V）和温度（T）。然后彻底混合顶空气体，并从每个瓶子中取出 12 毫升气体到抽空的 exetainer 中，通过气相色谱法（EXET；目标 1）进行[CH4]采样。ICE 和 FRIDGE 的 8 个瓶子被随机选择进行 EXET，而其余的瓶子则直接从其顶空测量[CH4]（BOTT；目标 2）。使用不同饲料成分和营养密度的五种饲料与空白一起测试 IVGP 浆液的 RH（目标 3）。使用 RH 将[CH4]转换为 DG 基础以考虑 Pw（目标 4）。使用随机系数模型完成统计分析。EXET 和 BOTT 之间没有差异（P=0.28）。IVGP 浆液的 RH 为 100%（P=1.00），证实 IVGP 气体与水蒸气饱和。处理之间的 P、V 和 T 不同（P<0.01）。DIRECT、ICE 和 FRIDGE 的[CH4]不同（P<0.01）。DG 的 P、V 和[CH4]在处理之间不同（P<0.01）。由于这些方法在[CH4]的评估上存在差异，因此没有明确的建议。相反，在采样顶空气体时，应测量 P、V 和 T，并使用提供的方程去除水蒸气引起的体积膨胀，并以 DG 为基础呈现[CH4]。
