33 Caffeine improves equine sperm motility after thawing

Abstract

The pattern of sperm transport and survival in the mare’s reproductive tract is different between fresh and frozen-thawed semen. A probable reason for this difference is the biophysiological changes in sperm during cryopreservation of equine semen. These changes can impair motility of stallion sperm after thawing. The aim of the present work was to test the effect of different caffeine concentrations on stallion sperm motility after thawing. One ejaculate of 9 stallions was frozen with the INRA82 frozen extender, and after thawing, different caffeine concentrations were added to the semen samples according to the treatments: control INRA82 without caffeine addition (T1), T1 + 1 mM caffeine (T2), T1 + 2 mM caffeine (T3), T1 + 3 mM caffeine (T4), T1 + 5 mM caffeine (T5), T1 + 7.5 mM caffeine (T6), and T1 + 10 mM caffeine (T7). The analysis of sperm motility parameters was performed with a computer-assisted semen analyser in 4 time periods: immediately after semen samples thawing (t0) and 15 min (t15), 30 min (t30), and 40 min (t40) after semen sample thawing. One semen sample of each treatment was thawed, and an aliquot was analysed for the following computer-assisted semen analysis characteristics: velocity curvilinear (VCL; µm s⁻¹), velocity straight line (µm s⁻¹), velocity average path (µm s⁻¹), linearity (%), straightness (%), wobble (%), amplitude of lateral head displacement (µm), beat cross frequency (BCF; Hz), and percentage of total sperm motility (TM) and progressive sperm motility. The statistical analysis was performed with ANOVA and Duncan’s test. The sperm parameters progressive sperm motility, linearity, wobble, and amplitude of lateral head displacement did not differ among the treatments (P > 0.05). Immediately after addition (t0) of 5, 7.5, and 10 mM caffeine concentrations, an increase of TM was observed (T5: 53.1%; T6: 45.9%; and T7: 47.4%) compared with the other treatments (T1: 37.5%; T2: 36.0%; T3: 36.6%; and T4: 32.3%; P < 0.05). Although after 15 min of incubation (t15) the TM decreased compared with t0 in T5, T6, and T7 treatments, the percentage was comparable with the other treatments at t15, t30, and t40. The mean value for TM was higher with 5 mM caffeine compared with the control group (38.6% v. 34.7%; P < 0.05), whereas for the 10 mM caffeine treatment velocity straight line (19.9 v. 17.1 µm s⁻¹), velocity average path (25.6 v. 22.9 µm s⁻¹), and straightness (75.4 v. 72.3%) were higher than the control (P < 0.05). For the 5, 7.5, and 10 mM caffeine treatments, VCL and BCF were higher than the control (VCL: 33.9, 34.5, 36.8, and 31.5 µm s⁻¹, respectively; BCF: 8.1, 8.6, 9.0, and 7.2 Hz, respectively). The remaining motility parameters did not differ until 40 min after the treatment (P < 0.05). In conclusion, the addition of 5, 7.5, and 10 mM caffeine concentrations after semen thawing increased TM and most of the sperm motility characteristics. However, given the complexities of sperm transport, capacitation, and so on, further experiments are needed to test whether caffeine treatments could be used to improve the fertilization rate of frozen-thawed equine semen.