Evaluation of In Vitro Capacitation of Buffalo Frozen/Thawed Sperm by Different Techniques

Authors

  • A. R. Elkhawagah Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt
  • V. Longobardi Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy
  • B. Gasparrini Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy
  • G. A. Sosa Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt
  • G. Bifulco Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy
  • M.E. A. Abouelroos Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt
  • A.E. Abd el-ghafar Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt
  • G. Camapnile Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy

DOI:

https://doi.org/10.6000/1927-520X.2014.03.01.2

Keywords:

Buffalo, Frozen semen, capacitation, Trypan blue/Giemsa, chlortetracycline

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.

Author Biographies

A. R. Elkhawagah, Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt

Theriogenology Department, Faculty of Veterinary Medicine

V. Longobardi, Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy

Department of Veterinary Medicine and Animal Production

B. Gasparrini, Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy

Department of Veterinary Medicine and Animal Production

G. A. Sosa, Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt

Theriogenology Department, Faculty of Veterinary Medicine

G. Bifulco, Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy

Department of Veterinary Medicine and Animal Production

M.E. A. Abouelroos, Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt

Theriogenology Department, Faculty of Veterinary Medicine

A.E. Abd el-ghafar, Theriogenology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor – Toukh, PO Box 13736, Egypt

Theriogenology Department, Faculty of Veterinary Medicine

G. Camapnile, Department of Veterinary Medicine and Animal Production, Federico II University, Via F. Delpino 1, 80137 Naples, Italy

Department of Veterinary Medicine and Animal Production

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2014-03-21

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Elkhawagah, A. R., Longobardi, V., Gasparrini, B., Sosa, G. A., Bifulco, G., Abouelroos, M. A., el-ghafar, A. A., & Camapnile, G. (2014). Evaluation of In Vitro Capacitation of Buffalo Frozen/Thawed Sperm by Different Techniques. Journal of Buffalo Science, 3(1), 3–11. https://doi.org/10.6000/1927-520X.2014.03.01.2

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Vol. 3 No. 1 (2014)

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