46 Fertilising ability of Arabian stallion sperm frozen with different cryoprotective agents by heterologous IVF

The combination of cryoprotective penetrating agents (CPA) such as glycerol (GLY) and dimethylformamide (DMF) has shown encouraging results on equine sperm cryosurvival. In addition, heterologous in vitro fertilisation (IVF) using zona-intact bovine oocytes has been successfully used to assess the fertilising capacity of stallion sperm. Therefore, this work evaluated the fertilising ability of stallion sperm previously frozen with DMF (5%), GLY (5%), and their combination (DMF+GLY [3% + 3%]) by assessing heterologous IVF using bovine oocytes. Stallion sperm samples from 24 ejaculates collected from four Arabian horses (six ejaculates per stallion) were initially frozen and thawed according to CPA groups: DMF (n = 90 straws), GLY (n = 82 straws), and DMF+GLY (n = 84 straws). A first analysis evaluated the kinematic parameters and the percentage of sperm with intact plasma membrane/intact acrosome (IPIA) by computer-assisted sperm analysis system (SCA®, Microptic) and PI/PNA-FICT double fluorescence test, respectively. Subsequently, a second analysis assessed the fertilising ability of frozen stallion sperm from three CPA groups by heterologous IVF using in vitro-matured, zona-intact bovine oocytes: DMF (n = 258), GLY (n = 214), and DMF+GLY (n = 240) in 15 sessions. In parallel, homologous IVF (control process, n = 200) was performed. All sperm samples were selected with BoviPure® (Nidacon) density gradient centrifugation before IVF. Fertilisation ability was assessed at 24 h post-insemination by sperm-oocyte interaction evaluating the number of bound spermatozoa and the capacity to penetrate the oocyte, fertilise them, and form pronuclei. All oocytes or presumptive zygotes were fixed, stained with Hoechst 33342 (ThermoFisher), and analysed in phase-contrast confocal microscopy. A one-way ANOVA and Tukey tests were used to compare CPAs groups in both analyses. The results of the first analysis showed that the DMF+GLY group yielded higher values (P < 0.05) of total (MT) and progressive (MP) motility, curvilinear velocity (VCL), the amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and IPIA compared with the GLY group. In the second analysis, the DMF+GLY group showed a higher number (P < 0.05) of sperm-bound than those obtained with DMF or GLY groups (4.3 ± 0.45 vs 1.3 ± 0.14 and 0.8 ± 0.11, respectively). However, the percentage of pronuclei formation of stallion sperm frozen with DMF+GLY was similar (P > 0.05) to that obtained in those samples frozen with DMF and GLY (20.0 ± 2.59, 22.4 ± 2.86, and 22.9 ± 2.62, respectively). Moreover, only the DMF+GLY group showed a significant correlation (P < 0.05) between the kinematic variables (e.g. MP, VCL, and straight-line velocity) and the formation of pronuclei. In conclusion, the combination of DMF+GLY is effective to cryopreserve Arabian stallion sperm due to achievements obtained in the kinematic variables and integrity of sperm membranes; however, the in vitro fertilising ability was similar to that obtained after using GLY or DMF. Further studies (e.g. in vivo fertilisation) are needed to validate the effects of those cryoprotective agents.