Schmoll Proc B ESM R Code.txt from Sperm velocity in a promiscuous bird across experimental media of different viscosities

In species with internal fertilization, the female genital tract appears challenging to sperm, possibly resulting from selection on for example ovarian fluid to control sperm behaviour and, ultimately, fertilization. Few studies, however, have examined the effects of swimming media viscosities on sperm performance. We quantified effects of media viscosities on sperm velocity in promiscuous willow warblers Phylloscopus trochilus. We used both a reaction norm and a character-state approach to model phenotypic plasticity of sperm behaviour across three experimental media of different viscosities. Compared to a standard medium (Dulbecco's Modified Eagle Medium, DMEM), media enriched with 1% or 2% w/v methyl cellulose decreased sperm velocity by up to about 50%. Spermatozoa from experimental ejaculates of different males responded similarly to different viscosities, and a lack of covariance between elevations and slopes of individual velocity-by-viscosity reaction norms indicated that spermatozoa from high- and low-velocity ejaculates were slowed down by a similar degree when confronted with high-viscosity environments. Positive cross-environment (1% versus 2% cellulose) covariances of sperm velocity under the character-state approach suggested that sperm performance represents a transitive trait, with rank order of individual ejaculates maintained when expressed against different environmental backgrounds. Importantly, however, a lack of significant covariances in sperm velocity involving a cellulose concentration of 0% indicated that pure DMEM represented a qualitatively different environment, questioning the validity of this widely used standard medium for assaying sperm performance. Enriching sperm environments along ecologically relevant gradients prior to assessing sperm performance will strengthen explanatory power of in vitro studies of sperm behaviour.

在体内受精的物种中，雌性生殖道对精子似乎具有筛选作用，这可能源于诸如卵巢液为调控精子行为、最终调控受精过程而产生的选择压力。然而，鲜有研究探讨精子运动介质黏度对精子运动性能的影响。我们以混交繁殖的欧柳莺（Phylloscopus trochilus）为研究对象，定量分析了介质黏度对精子运动速度的影响。我们同时采用反应规范（reaction norm）与性状状态分析法，针对三种不同黏度的实验介质，构建了精子行为的表型可塑性（phenotypic plasticity）模型。相较于标准介质达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM），添加1%或2%重量体积比（w/v）甲基纤维素的介质可使精子运动速度降低最多约50%。来自不同雄性实验精液样本的精子对不同黏度介质的响应模式相似；且个体精子速度-黏度反应规范的截距与斜率间不存在显著协方差，这表明：当暴露于高黏度环境时，高活力精液与低活力精液中的精子均会出现相近程度的运动速度下降。通过性状状态分析法计算得到的、1%与2%纤维素介质间的精子速度跨环境正协方差表明，精子性能属于可递推性状，即不同环境背景下，各精液样本的性能排名保持稳定。但重要的是，涉及0%纤维素浓度（即纯DMEM）的精子速度协方差未达到显著水平，这说明纯DMEM属于性质截然不同的实验环境，进而对这一广泛用于精子性能检测的标准介质的有效性提出了质疑。在开展精子性能检测前，按照生态相关的黏度梯度调整精子所处环境，将有助于提升精子行为体外（in vitro）研究的解释效力。