44 Assessment of quality and fertilising ability of dog epididymal spermatozoa frozen or vitrified with L-carnitine by heterologous IVF

L-carnitine (LC) plays an important role in transporting short-, medium- and long-chain fatty acids into the mitochondria for β-oxidation and increased ATP production. Besides, heterologous IVF has been successfully employed to assess sperm fertilising ability in several species. Hence, we aimed to evaluate the sperm quality and fertilising ability of dog epididymal sperm frozen or vitrified with LC by assessing heterologous IVF using bovine oocytes. 60 epididymides were recovered from 30 orchiectomised adult dogs and the sperm samples were retrieved by retrograde flushing using TCG-EY (tris-citric-acid-glucose + 20% egg yolk) medium, then 20 pools were conformed. In each pool, four groups were conformed according to cryopreservation method (slow freezing [SF] or kinetic vitrification [KV]) and LC supplementation (5 and 0 mM [control-Co]): SF-LC (n = 79), SF-Co (n = 78), KV-LC (n = 20), and KV-Co (n = 20). The SF method used TCG-EY + 5% glycerol and exposed the 0.25 mL straws to liquid nitrogen (LN₂) vapours for 2 min; while the KV method used TCG-EY + 250 mM sucrose and submerged 30-μL drops directly in LN₂. A first analysis evaluated the kinematic parameters and simultaneous integrity of plasmatic and acrosomal membranes (IPIA, %) by using a computer-assisted sperm analysis system (SCA^®, Microptic) and PI/PNA-FICT double fluorescence test, respectively. A second analysis assessed the fertilising ability of cryopreserved sperm from four groups by heterologous IVF using in vitro-matured, zona-intact bovine oocytes: SF-LC (n = 93), SF-Co (n = 91), KV-LC (n = 146), and KV-Co (n = 118). BoviPure^® (Nidacon) selected all sperm samples 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. Two analyses used one-way ANOVA and Tukey tests to compare groups. The results of the first analysis showed that the total (TM, %) and progressive (PSM, %) motilities, curvilinear (VCL, µm/s), and straight-line (VSL, µm/s) velocities with freezing groups (SF-LC and SF-Co) were greater (P < 0.05) than with vitrification groups (KV-LC and KV-Co). However, the IPIA after cryopreservation with both LC groups (SF-LC and KV-LC) was greater (P < 0.05) than with its control groups (SF-Co and KV-Co). In the second analysis, there were no differences (P > 0.05) between groups regarding sperm-bound. However, the SF-LC group yielded a higher (P < 0.05) proportion of pronuclei formation (69.2 ± 2.46%) than the KV-Co group (61.0 ± 1.51%). Additionally, correlations (P < 0.05) between the kinematic parameters (TM, PSM, VCL, and VSL) and pronuclear formation were found in LC groups (SF-LC and KV-LC). In conclusion, the addition of L-carnitine to the freezing and vitrification media produced a positive cryoprotective effect on the plasmatic and acrosomal membranes, even improving the fertilising ability of dog epididymal spermatozoa.