Welfare impacts during and after reproductive procedures for in vivo embryo production and transfer in Holstein dairy heifers

Embryo technologies are routinely used in cattle, but the links between embryo technologies and cattle welfare have been poorly investigated. The aim of this study was to describe the behavioural, physiological and clinical responses of Holstein dairy heifers during and after five reproductive procedures: dominant follicle removal (DFR) by transvaginal follicular puncture, superovulation, double artificial insemination (AI1 and AI2), embryo flushing (EF), and embryo transfer (ET). This longitudinal cross-over design used twelve nulliparous pubescent and cycled Holstein heifers, each acting as their own control. Behavioural recordings (video and continuous monitoring sensors), physiological and clinical examinations, and blood sampling were performed at regular intervals on each reproductive procedure from the start of contention up to 24 h after the end of the procedure. The heifers changed their behavioural and physiological responses during and after each of the five reproductive procedures. During the procedures, they displayed more abnormal postures of the body (e.g. arched back during all procedures; P < 0.05), hindlimbs (e.g. base-wide stance during all procedures except EF; P < 0.05), and head (e.g. lowered head during AI2, EF and ET; P < 0.05). They also displayed more signs of agitation, moving their body more (e.g. stepping aside during DFR, AI1 and ET; P < 0.05), their feet more (e.g. hoof lifting during DFR, EF and ET; P < 0.05) and their head more (e.g. neck stretching forward during AI2, EF, and ET; P < 0.05). They also showed increases in both plasma cortisol concentration (during AI2 and EF; P < 0.05) and heart rate (during DFR and AI2; P < 0.05). However, we did not observe any inflammatory response in plasma pro-inflammatory cytokines and haptoglobin or in macroscopic appearance of the vulvae 2 h after the procedures. During the 24 h after the procedures, the heifers spent less time ingesting and standing up after DFR (P < 0.05), less time ruminating after EF (P < 0.05), more time with activity after superovulation and more time without activity after EF (P < 0.05). Each reproductive procedure has specific responses in the heifers. Taken together, our findings suggest that heifer welfare was impacted both during and after the procedures involved in embryo production and in vivo transfer protocol. Acknowledging that some discomfort/pain may be present, it may be welfare-friendly to develop and apply refinement strategies during and after embryo technology procedures.