Evaluation of In Vitro Capacitation of Buffalo Frozen/Thawed Sperm by Different Techniques
Pages 3-11
A.R. Elkhawagah, V. Longobardi, B. Gasparrini, G.A. Sosa, G. Bifulco, M.E.A. Abouelroos, A.E. Abd el-ghafar and G. Campanile

DOI: http://dx.doi.org/10.6000/1927-520X.2014.03.01.2

Published: 21 March 2014

Abstract: This study aimed to determine the most reliable method to evaluate capacitation of buffalo frozen/thawed sperm. Frozen/thawed sperm cells were incubated in Tyrode albumin lactate pyruvate medium (TALP) in absence of capacitating agents (control) and in presence of 10 µg/ml heparin for 2 and 4 h. Capacitation was assessed by Trypan blue/Giemsa after lysophosphatidylcholine (LPC) exposure, chlortetracycline (CTC) fluorescence assay and immune-localization of tyrosine phosphorylated protein. Furthermore, we evaluated the effect of heparin on penetration, cleavage rates and kinetics of embryo development after heterologous IVF. The percentage of LPC-induced acrosome reacted (AR)-sperm increased (P<0.05) with heparin compared to the control after 2 h (28.2 vs 24.4%, respectively) and 4 h (35.1 vs 32.0 %, respectively). No differences in CTC pattern B (capacitated sperm) were found between groups and incubation times (on average 63%). On the contrary, heparin decreased (P<0.01) the percentage of tyrosine phosphorylation pattern A after 2 and 4 h (34.3 and 35.3%, respectively) compared to the control (54.5 and 51.8%, respectively) and increased (P<0.01) that of pattern EA after 2 and 4 h (59.2 and 54.2 %, respectively) compared to the control group (44.7 and 45.2 %, respectively). Both cleavage and penetration rates, as well as the percentage of fast developing embryos, were higher (P<0.01) in the heparin-treated group (77.2, 80.4 and 74.0 %, respectively) compared to the control (56.6, 58.0 and 55.2 %, respectively). In conclusion, Trypan blue/Giemsa staining to evaluate LPC-induced AR and tyrosine protein phosphorylation assay can be successfully used to evaluate capacitation of buffalo frozen/thawed semen.

Effect of Methyl-B-Cyclodextrin (MBCD) on In Vitro Capacitation of Buffalo Frozen/Thawed Sperm
Pages 12-17
A.R. Elkhawagah, V. Longobardi, B. Gasparrini, G.A. Sosa, A. Salzano, M.E.A. Aboul-roos, A.E. Abd El-Gaffar and L. Zicarelli

DOI: http://dx.doi.org/10.6000/1927-520X.2014.03.01.3

Published: 21 March 2014

Abstract: The aim of this study was to determine the effect of Methyl-B-Cyclodextrin (MBCD) on capacitation of buffalo sperm. Frozen/thawed semen was incubated in the absence of capacitating agents (negative control), in the presence of 10 µg/ml heparin (positive control) and of 1, 2, 4 and 8 mg/ml MBCD for 2 and 4h. At each incubation time, sperm motility was evaluated by phase contrast microscopy. Capacitation was assessed by the sperm ability to undergo acrosome reaction after lysophosphatidylcholine treatment, evaluated with viability by Trypan blue-Giemsa. After 2 h capacitation increased (P<0.01) in MBCD groups (39.2±1.4, 44.5±3.3, 56.7±1.5 and 62.5±3.8, with 1, 2, 4 and 8 mg/ml MBCD, respectively) compared to the negative and positive controls (27.5±1.0 and 28.0±0.8, respectively). Likewise, after 4 h the percentage of live capacitated sperm was higher at increasing concentration of MBCD (31.0±0.7, 34.5±1.7, 42.0±1.9, 49.2±2.8, 62.3±1.5 and 70.8±1.7 in negative control, positive control and with 1, 2, 4 and 8 mg/ml MBCD, respectively; P<0.01). After 2 h sperm motility was lower (P<0.01) in 4 and 8 mg/ml MBCD groups (43.3±2.1 and 25.0±3.2, respectively) than in negative control, positive control, 1 and 2 mg/ml MBCD groups (55.0±1.8, 48.3±2.8, 61.7±2.8, 56.7±1.1, respectively). After 4 h the lowest sperm motility was observed with higher MBCD concentrations (40.0±0.0, 46.7±4.2, 51.7±4.6, 50.0±0.0, 40.0±3.7 and 6.7±1.1, in negative control, positive control, 1, 2, 4 and 8 mg/ml MBCD, respectively; P<0.01). In conclusion, MBCD improved sperm capacitation in a dose-dependent manner while decreasing the sperm motility at higher concentrations.