Table 1_Physiological and behavioral parameters of pain and stress in mares during and after transvaginal ultrasound-guided follicular aspiration.xlsx

IntroductionThe use of transvaginal ultrasound-guided follicle aspiration (TVA) for oocyte collection has become a widely used procedure in horses for in vitro embryo production (IVEP). The TVA procedure is characterized by various manipulations, which are physical restraint, perineal preparation, transrectal palpation, insertion of an intravaginal device, and needle punctures. These repeated transvaginal and transovarian punctures have raised concerns about their potential effects on mare welfare. Our study aimed to investigate the effects of TVA manipulations, and especially puncturing, on pain and stress in mares in a commercial set-up.
MethodsTherefore, eight mares were subjected to three TVA procedures: two with puncturing (P+) and one without (P−, control). Before, during and after all TVA procedures, blood was collected for serum glucose, lactate and cortisol levels, and facial pain scores were assessed. An electrocardiogram (ECG) was recorded for each mare before and during the procedure to measure heart rate (HR) and heart rate variability (HRV). Parameters in response to manipulations and puncturing were analyzed using linear mixed effect models for each outcome variable at different timepoints during the procedure.
ResultsResults revealed that puncturing during TVA did not significantly influence serum metabolite levels, facial pain scores, HR, or HRV over the complete procedure (p > 0.05). Notably, HR did not increase at the moment of puncturing (p > 0.05), and no significant changes in HRV parameters between P+ and P− were detected (p > 0.05). Both P+ and P− procedures triggered significant increases in cortisol and lactate levels, facial pain scores, and HR during restraint in stocks and perineal preparation compared to the day before and the day after TVA (p < 0.05). Interestingly, even without puncturing, manipulations in the P− procedure were sufficient to induce significant elevations in metabolite levels and facial pain scores compared to the day before and after (p < 0.05).
DiscussionThese results indicate that the TVA procedure induces stress responses in mares, predominantly associated with all manipulations specific to TVA, while the effect of puncturing itself was minimal. All effects were acute, with parameters returning to baseline when measured 24 h later.

引言
经阴道超声引导卵泡抽吸术（transvaginal ultrasound-guided follicle aspiration，TVA）用于卵母细胞采集，现已成为马属动物体外胚胎生产（in vitro embryo production，IVEP）领域广泛应用的操作技术。TVA操作包含多类步骤：躯体保定、阴部备皮、直肠触诊、阴道内器械置入以及穿刺操作。这类反复经阴道及经卵巢穿刺操作，引发了学界对其可能影响母马福利的担忧。本研究旨在探究商业化繁育场景下，TVA操作（尤其是穿刺环节）对母马疼痛与应激状态的影响。
方法
为此，本研究选取8匹母马，开展3次TVA操作：2次为穿刺组（P+），1次为非穿刺对照组（P−）。在所有TVA操作开展前、操作中及操作后，采集血样以检测血清葡萄糖、乳酸及皮质醇水平，并对母马进行面部疼痛评分。在操作前及操作过程中，对每匹母马记录心电图（electrocardiogram，ECG），以检测心率（heart rate，HR）及心率变异性（heart rate variability，HRV）。针对操作过程中不同时间点的各结局变量，采用线性混合效应模型分析操作与穿刺对应的各项参数变化。
结果
结果显示，在整个TVA操作过程中，穿刺环节并未对血清代谢物水平、面部疼痛评分、心率及心率变异性产生显著影响（p＞0.05）。值得注意的是，穿刺瞬间心率并未出现升高（p＞0.05），且穿刺组与对照组的心率变异性参数未检测到显著差异（p＞0.05）。与TVA操作前1日及后1日相比，穿刺组与对照组在保定架固定及阴部备皮环节，皮质醇、乳酸水平、面部疼痛评分及心率均出现显著升高（p＜0.05）。有趣的是，即便未实施穿刺，对照组的操作步骤仍足以使血清代谢物水平及面部疼痛评分较操作前后1日出现显著升高（p＜0.05）。
讨论
上述结果表明，TVA操作会引发母马的应激反应，该反应主要与TVA特有的各类操作相关，而穿刺环节本身的影响极小。所有影响均为急性反应，相关参数在操作24小时后即可恢复至基线水平。