114 Rosiglitazone extends maintenance of frozen-thawed bull sperm for 24 hours at ambient temperature

Cryopreserved bull sperm is utilised almost exclusively for AI in US dairy herds, with increased adoption in beef cattle operations. Maintenance of sperm motility after thawing in preparation for insemination is critical to achieve maximum conception rates. Practical application of AI requires thawing of straws on site, followed by immediate transcervical deposition. Use of thawed sperm is limited to immediate transfer due to rapid decline in sperm motility and thus fertility. Extension of sperm storage time after thawing without altering motility would increase flexibility of AI protocols by allowing for delayed insemination to accommodate unforeseen circumstances associated with animal, technical, or environmental conditions. Furthermore, the ability to store sperm at ambient temperature (∼25°C) would eliminate the need for equipment that is not conducive to working conditions and also allow for short-term transport of sperm between facilities. Using a modified sperm TALP for prolonged storage of sperm, the antidiabetic pharmaceutical rosiglitazone (Ros; 10, 50, or 100 µM), or vehicle control (VC; 0.5% dimethyl sulfoxide (DMSO)) was added to frozen-thawed bull sperm to determine both acute (90 min) and prolonged (24 h) effects on sperm motility kinematics over time. Frozen sperm from three bulls were isolated by single Percoll gradient centrifugation (45/90%), pooled and extended in a modified TALP at 20 × 10⁶ total motile spermatozoa mL⁻¹ followed by the addition of Ros (n = 5 reps). Sperm samples were maintained at 37°C for 90 min and motility kinematics determined by computer-aided sperm analysis every 30 min (CASA, Hamilton-Thorne). Differences between treatments and time × treatment interactions were determined by one-way ANOVA using the Mixed procedure of SAS 9.4 (SAS Institute Inc.) with replicate included as a random effect. Data were analysed for normality and significance was determined (P < 0.05) using Tukey’s post hoc analysis. Addition of Ros did not alter motility parameters over 90 min except for beat-cross frequency (BCF) between 100 and 10 µM treatments (8.2 and 11.6 beats per minute, respectively). To increase physiological relevance and sensitivity of the assay, bull sperm were then maintained in similar conditions but stored at ambient temperature (∼25°C) in the dark and warmed to 37°C for CASA analyses at 24 h (n = 4 reps). Addition of 50 µM Ros maintained both total (TM) and progressive motility (PM) between 0 and 24 h (TM: 63–61%; PM: 45–42%; P > 0.05), whereas VC and 10 µM Ros did not (P < 0.05). Ros (50 µM) was also superior to maintenance of PM compared with VC at 24 h (42 and 28%, respectively). Similarly, average path velocity and curvilinear velocity were maintained for 24 h in 50 and 100 µM Ros treatments (P > 0.05), but not in 10 µM Ros and VC (P < 0.05). These findings demonstrate a potential application for the addition of Ros to frozen-thawed bull sperm to lengthen storage time at room temperature to 24 h, while preserving motility parameters and providing flexibility for AI protocols.