Efeito in vitro e in vivo da adição de cafeína sobre as características do espermatozoide equino pós-descongelamento

Abstract

The cryopreservation of equine semen is a crucial tool in genetic improvement by maximizing the use of sires with high genetic merit. However, the cryopreservation process reduces sperm motility and longevity. Thus, sperm motility activating substances may play an important role for the success of equine artificial insemination (AI) with frozen semen. Caffeine is a stimulant of sperm motility and capacitation, which are important attributes to oocyte fertilization. Consequently, the addition of caffeine prior to the AI with frozen-thawed semen can be an alternative to increase the fertility rate of frozen equine semen. The aim of the present work was to evaluate the in vitro sperm quality and in vivo fertility of frozen-thawed equine semen after addition of different caffeine concentrations. Thus, one ejaculate of 9 stallions (n=9) was frozen with INRA82 frozen extender and after thawing three different concentrations of caffeine were added to the samples performing four treatments: T1) control INRA82 (no caffeine addition), T2) T1+3mM caffeine, T3) T1+5mM caffeine, and T4) T1+7.5mM caffeine. The sperm kinetics and motility were evaluated with a computer assisted sperm analysis (CASA), sperm membrane functionality with a hypoosmotic swelling test, and sperm integrity and spontaneous acrosome reaction rate with flow cytometry. The samples were evaluated in four periods: t0) immediately after thawing, t20) 20 min, t30) 30 min, t40) 40 min, and t50) 50 min after semen thawing. The post-thawed samples were subjected to the swim-up sperm selection method and the number of recovered and morphologically normal sperm were recorded. Such samples were evaluated at four period: t20) 20 min, t40) 40 min, t60) 60 min and t80) 80 min after swim-up and compared to the post-thawing sperm (control). Nitrite and hydrogen peroxide concentrations (μM / μg protein) from the control and treatment with 5mM caffeine addition were measured with spectrophotometry. The in vivo fertility rate was assessed with artificial insemination (AI) of 8 mares/ treatments, control and 5mM caffeine. There was no difference between the rate of sperm with functional and intact membrane, and spontaneous acrosome reaction between the control and 5mM caffeine group (P>0,05). However, the concentration of 5mM of caffeine induced an increase of the total sperm motility (38.9±2.8), reduced the nitrite concentration (11.4±2.1), and also increased the sperm recovery rate (7.9x106/ml) and sperm with normal morphology (79.9±1.0) after swim-up compared to control (32.6±3.4; 12.8±2.9; 3.4x106/ml±0.7; 70.0±2.0, P<0.05), respectively. Moreover, the AI rate of the 5mM caffeine group was significantly higher (62.5%) than the control group (12.5%, P<0.05). In conclusion, 5mM of caffeine play a significant role as antioxidant and motility activator of post-thawed equine sperm. Possibly, these factors were responsible for the higher fertility rate of the inseminated mares with thawed semen with 5mM caffeine compared to the control. Thus, the addition of 5mM of caffeine to the frozen-thawed stallion sperm increased the equine fertility rate, so that it may be an alternative to the use in the mares AI with stallion semen with low post-thawed quality.