115 In vivo confocal laser endomicroscopy visualisation of fresh and frozen bull spermatozoa in the genital tract of dairy heifers

Abstract

Fertility in cattle includes the ability of the uterus to provide the appropriate environment for pregnancy success, including the transport of spermatozoa before fertilization. Confocal laser endomicroscopy technology (Cellvizio) has been previously used in small ruminants to visualise labelled spermatozoa in vivo in the uterine horns of ewes (Druart et al. 2009 Reprod. 138, 15-53). Nevertheless, to the best of our knowledge, no in vivo study has reported a live visual analysis of the behaviour of spermatozoa in the bovine uterus upon AI. The aim of this study was to develop an experimental procedure to label bull spermatozoa and to visualise their progression in the uterine horns of dairy cows, using Cellvizio and uterine cervix catheterization. Fresh ejaculated bull spermatozoa were double-labelled with octadecyl rhodamine B chloride and MitoTracker Green FM dyes before dilution and freezing processing. At each step of the process, semen quality was evaluated with a semen quality analyzer (SQA-V b, Medical Electronic System) and compared to nonlabelled bull reference semen. The specificity of the labelling was validated in vitro. We also developed a specific device to insert the Cellvizio fibre probe into the genital tract, in order to image vagina, cervix, and uterine body, as well as the proximal, middle, and distal parts of the uterine horn, including the utero-tubal junction (UTJ), before and after AI with fresh and frozen labelled spermatozoa. Then, 10 heifers were oestrus synchronized. Video sequences were recorded at oestrus (n = 5), just before AI, at the time of AI (7-12h after the onset of oestrus), 30 and 120 min after AI, and 14 days after oestrus (n = 5; luteal phase), to investigate the effect of the steroid-primed uterine environment on the distribution and progression of spermatozoa in the genital tract. Using our approach, labelled spermatozoa were detected (1) at the day of oestrus and 14 days after oestrus, (2) in the uterine body at the time of AI, (3) in various parts of the genital tract 30 min after AI, and (4) in the UTJ, the cervix, and the vagina 120 min after AI. In addition, labelling the spermatozoa did not alter the fertilizing capacity: 4 of the 5 oestrus females were pregnant at Day 35. Additionally, an IVF test showed a blastocyst rate of 45.8% for labelled semen vs. 39.4% for the control group (Carvalho et al. 2017 Reprod. 154, 695-710). In conclusion, our study provides a method to phenotype in situ the transport of spermatozoa in the genital tract of heifers. Further investigations are in progress to optimize video quality, to develop an algorithm for an automatic analysis (spermatozoa count, speed, and path) of the recorded sequences.