30 Bull sperm kinetics after semen cryopreservation in extender containing propagermanium

Abstract

There is a negative association between a plentiful production of oxygen reactive species and spermatozoa kinetics parameters. Thus, antioxidants have been added to the freezing medium to improve sperm quality due to their protective effect against membrane lipid peroxidation. Propagermanium (GE132) is an organometallic compound that has never been used in freezing medium despite its known antioxidant effect as a free radical scavenger. This study aimed to investigate the effects of different concentrations of GE132 added in a commercial freezing medium (CFM) on frozen-thawed sperm motion. Nine ejaculates of 3 Nellore bulls (3 replicates), collected by an artificial vagina, were evaluated, pooled, and divided into groups D (semen was diluted and kept at 33°C for 30 min before cooling) and C (semen was cooled immediately after dilution). Both groups were submitted to the same experimental treatment, as follows: addition of 0, 500, and 1000 µg mL⁻¹ of GE132 in a CFM resulting in subgroups D0, D500, D1000, C0, C500, and C1000. The sperm samples were diluted to a final concentration of 30 × 10⁶ spermatozoa per straw (0.25 mL) and then cooled at 4°C for 5 h before freezing. Sperm samples were assessed using a computer assisted sperm analyser at 5 and 60 min post-thawing for total motility (TM; %), progressive motility (%), curvilinear velocity (μm s⁻¹), velocity straight line (μm s⁻¹), velocity average path (μm s⁻¹), amplitude of the lateral head displacement (ALH; μm), beat cross frequency (Hz), linearity (LIN; %), and straightness (STR; %). Data were analysed using the R software package version 3.4.4 (2018; https://www.r-project.org/). An ANOVA was applied to assess statistical differences, and Tukey’s test was used to determine differences among subgroups. A significance level of P < 0.05 was adopted. No significant differences (P > 0.05) were observed among subgroups for all sperm parameters except for TM, in which C0 presented higher (P < 0.05) value (68.72 ± 3.36) than D0 (54.67 ± 5.59), D5 (57.10 ± 2.34), and C10 (54.20 ± 2.73), with similar results between D10 (59.5 ± 4.22) and C5 (59.52 ± 4.64). There were significant differences within subgroups when comparing 5 and 60 min post-thawing for TM, ALH, LIN, and STR. Total motility decreased 17.2 and 9.9% in C5 and C10, respectively. Similarly, values of ALH decreased 0.2, 0.4, and 0.2 µm in D0, D5, and C5, respectively. However, the increase in LIN was 11% in D10, whereas the values for STR increased in D10 (10%), C5 (6.3%), and C10 (7.3%). The addition of GE132 to the CFM did not enhance all the sperm parameters after cryopreservation except for a slight improvement in ALH, LIN, and STR over time and TM among groups. The lack of additive effect could be due to the presence of antioxidants in the CFM; therefore, further investigation with fluorescent probes using flow cytometry and free-antioxidants freezing medium could lead to a new approach for bull sperm freezing.

We acknowledge Tairana AI Station, Master Fertility, and Botupharma, Brazil.