Nociception in Chicken Embryos, Part III: Analysis of Movements before and after Application of a Noxious Stimulus

Abstract

Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore essential and in the interest of animal welfare to be able to precisely define the point at which a chicken embryo is capable of nociception in ovo. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to a noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied in a randomized order. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the mechanical stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The presented results indicate that noxious mechanical stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15.

Keywords: Gallus gallus domesticus; behavior; chicken embryo; development; movement; nociception; pain.

Figure 1

Flowchart of the experimental procedures. (a) Recordings of the embryo were collected in ovo, and video data were transferred to a computer for editing. The body parts of chicken embryos tracked by DLC are labeled in the schema. (b) The neural network was trained, and the video material was analyzed according to the timeline. (c) The video material was manually analyzed according to the timeline. (Created with BioRender.com, accessed on 6 September 2023).

Figure 2

Beak Distance. This variable was defined as the distance between the upper and lower beak of embryos. It was measured at (a) ED15 (n = 16), (b) ED16 (n = 16), (c) ED17 (n = 16) and (d) ED18 (n = 15) before and after application of a control (Touch) or noxious stimulus (Pinch). The total distance in pixels across 30 s intervals (1500 frames) was evaluated. Plots show the estimated mean ±95% confidence intervals at the following 30 s intervals from Baseline (BL) to post-stimulation, with stimulation occurring at 0 s: −30–0, 0–30, 30–60, 60–90 and 90–120 s. Robust linear mixed effects were applied for all analyses. All contrasts (differences) between particular groups were assessed after model-fitting by the estimated marginal means with Tukey’s p value correction for multiple comparisons. Touch: blue; Pinch: red. * Significant difference between Pinch and Touch; ♦ Significant difference from baseline. p values shown.

Figure 3

Eye Corner Movement. This variable was used to detect head movements of embryos at (a) ED15 (n = 16), (b) ED16 (n = 16), (c) ED17 (n = 16) and (d) ED18 (n = 15) before and after application of two stimuli (Touch and Pinch). The total distance in pixels across 30 s intervals (1500 frames) was evaluated. Plots show the estimated mean ±95% confidence intervals at the following 30 s intervals from Baseline (BL) to post-stimulation, with stimulation occurring at 0 s: −30–0, 0–30, 30–60, 60–90 and 90–120 s. Robust linear mixed effects were applied for all analyses. All contrasts (differences) between particular groups were assessed after model-fitting by the estimated marginal means with Tukey’s p value correction for multiple comparisons. Touch: blue; Pinch: red. * Significant difference between Pinch and Touch; ♦ Significant difference from baseline. p values shown.