129 In vitro embryo production using a sperm separation device: effects of culture system and sperm concentration

Centrifugation in colloid density gradients, such as Percoll, has been broadly used for the preparation of sperm for IVF in cattle. However, there is a growing body of evidence in the literature that this procedure can damage sperm acrosome and DNA and thus negatively affect blastocyst rates. In the current study, we evaluated the use of a sperm separation device (VetMotl, VetMotl Inc.) as an alternative method to prepare sperm for IVF. We used frozen-thawed sperm from Nelore bulls (n = 10) with records of blastocyst rates ranging from 13.3% to 27.6% after IVF of a significant number of cumulus–oocyte complexes (COCs, average 4053 per bull). Sperm from each bull was prepared by conventional centrifugation in a Percoll gradient (45%–90%, group DGC) or diluted 1:1 in FERT-TALP medium, and 750 µL was loaded into the separation chamber. The upper part of the chamber was then filled with 750 µL of FERT-TALP medium. After incubation for 30 min at 5% CO₂ and 38.5°C, the supernatant (750 µL) was collected with a pipette and gently centrifuged (260g for 2 min) to concentrate sperm, if necessary. Sperm processed by both methods (~1 × 10⁶ sperm mL⁻¹) were used to fertilize COCs (n = 1985) collected by ovum pickup from Nelore donors. These COCs were previously pooled and submitted to IVM in TCM-199 at 5% CO₂, 5% O₂, and 38.5°C. The sperm were added to IVF drops containing 70 µL of FERT-TALP medium and 10 COCs. After 20 h, the presumptive zygotes from four bulls were transferred to 50-µL drops of SOF medium supplemented with 1.0% FCS, whereas the zygotes from the remaining bulls were cultivated in SOF medium with 2.5% FCS, both under the same culture conditions of IVM. Cleavage and blastocyst rates were compared by the Chi-squared method. Associations were evaluated using the Pearson correlation method. Both cleavage and blastocyst rates were greater when the sperm separation chamber was used compared with DGC, regardless of the concentration of FCS in the culture medium (1.0% FCS: 62.7% vs. 42.3% cleavage and 17.2% vs. 7.5% blastocyst rates, respectively; P < 0.0001; and 2.5% FCS: 55.0% vs. 32.9% cleavage and 21.2% vs. 14.8% blastocyst rates, respectively; P = 0.0033). Only one out of the 10 bulls evaluated had lower cleavage and blastocyst rates when the chamber was used compared with DGC (22.2% vs. 44.4% and 3.3% vs. 16.2%, respectively; P < 0.05). The semen from this bull was characterized by low sperm concentration (30 × 10⁶ sperm mL⁻¹) and poor motility (16.1%). We observed a significant positive correlation between blastocyst rates and straw sperm concentration when the chamber was used (R = 0.95; P = 0.0451), but not with DGC (R = 0.33; P = 0.6691), even after adjusting for the concentrations for IVF. In summary, the use of a sperm separation chamber is an alternative means to improve IVEP outcomes in cattle, but the results may be affected by the inital sperm parameters.

This study was supported by VetMotl Inc. and CNPq INCT 406866/2022-8.