An enriched subpopulation of sperm recruited by the Sperm Selection Assay (SSA) allow improve the cleavage rate in the in vitro bovine embryo production

GUIDOBALDI ALEJANDRO; MORENO-IRUSTA, AYELEN; GIOJALAS, LAURA; DOMINGUEZ, ESTEBAN; TRIBULO HUMBERTO

Holderness, NH, US

Congreso; Fertilization and Activation of Development Gordon Research Conference; 2017

Assisted reproduction technology (ART) has been widely used in farm animals, whereas artificial insemination with cryopreserved conventional or sexed semen samples, embryo transfer and in vitro embryo production are the most currently applied. However, the success of some of these technologies is rather low, depending in part on sperm quality, which influences fertilization, embryo development and implantation. In our laboratory, we designed a method to recruit a sperm subpopulation which is at the best physiological state. The Sperm Selection Assay (SSA) is based on the sperm chemotaxis and only the capacitated spermatozoa are able to respond. Originally, this device was developed for human sperm, for this reason the aim of this study was adapt this device for cryopreserved conventional or sexed bovine semen samples. The semen samples were conventionally thawed, removing the seminal plasma and cryoprotectant by migration-sedimentation or centrifugation technique, respectively. Then, spermatozoa were capacitated in Sp-TALP media at 38,5°C under 5% CO2 on air. The SSA consists of a device with two wells connected by a tube. One well was filled with the sperm suspension and the other with the attractant solution, which diffused inside the connecting tube as a gradient. Several features were tested to adapt the SSA in this species and semen samples types: timing, sperm concentration, follicular fluid and progesterone concentration, determining the relative sperm accumulation in the well containing the attractant. The level of sperm capacitation (determined by the pharmacological induction of the acrosome reaction) and sperm DNA fragmentation level (determined by the ?Halo sperm test?) were assessed in the sperm population before and after the SSA. Results show that the optimal SSA conditions were the same either for conventional or sexed semen samples: ten minutes of incubation in the SSA device, 2 million spermatozoa/ml, and 1:10¯³ follicular fluid and 1 pM progesterone concentration. When the SSA was performed under these conditions, the sperm capacitation level was significantly increased and the sperm DNA fragmentation was significantly decreased after the SSA performed either with conventional or sexed semen samples. Furthermore, the cleavage rate were significantly improved when oocytes were fertilized with those selected conventional and sexed spermatozoa. In conclusion, the SSA can be used to recruit a sperm subpopulation at the best functional state in spite of the sample was previously sexed and allow improve the in vitro bovine embryo production, providing the SSA as an assay that might increase the efficiency of current assisted reproduction techniques.