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Assessment of sperm damages during different stages of cryopreservation in water buffalo by fluorescent probes

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Abstract

The present study was designed to investigate the sperm damages occurring in acrosome, plasma membrane, mitochondrial activity, and DNA of fresh, equilibrated and frozen–thawed buffalo semen by fluorescent probes. The stability of sperm acrosome and plasma membrane stability, mitochondrial activity and DNA status were assessed by fluorescein conjugated lectin Pisum sativum agglutinin, Annexin–V/propidium iodide, JC-1 and TUNEL assay, respectively, under the fluorescent microscope. The damages percentage of acrosome integrity was significantly increased during equilibration and freezing–thawing process. The stability of sperm plasma membrane is dependent on stability of phosphatidylserine (PS) on the inner leaflet of plasma membrane. The frozen–thawed sperm showed externalization of PS leading to significant increase in apoptotic, early necrotic and necrotic changes and lowered high mitochondrial membrane potential as compared with the fresh sperm but all these parameters were not affected during equilibration. However, the DNA integrity was not affected during equilibration and freezing–thawing procedure. In conclusion, the present study revealed that plasma membrane and mitochondria of buffalo sperm are more susceptible to damage during cryopreservation. Furthermore, the use of fluorescent probes to evaluate integrity of plasma and acrosome membranes, as well as mitochondrial membrane potential and DNA status increased the accuracy of semen analyses.

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Acknowledgments

The authors thank the Director of the institute for provided necessary facilities to conduct this work. Authors thank Dr PP Dubey, Assistant Professor, GADVASU, Ludhiana for assisting in data analysis, and the lab staff of semen freezing lab for helping in semen collection and cryopreservation.

Conflict of interest

The authors have declared that no conflict of interest exists among them.

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Authors and Affiliations

  1. Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Sirsa Road, Hisar, 125001, Haryana, India

    Dharmendra Kumar, Pradeep Kumar & P. S. Yadav

  2. ICAR-National Dairy Research Institute, Karnal, 132001, Haryana, India

    Pawan Singh

  3. Animal Genetics and Breeding Division, ICAR-CIRB, Sirsa Road, Hisar, 125001, Haryana, India

    S. P. Yadav

Authors
  1. Dharmendra Kumar
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  2. Pradeep Kumar
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  3. Pawan Singh
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  4. S. P. Yadav
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  5. P. S. Yadav
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Corresponding author

Correspondence to Dharmendra Kumar.

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Kumar, D., Kumar, P., Singh, P. et al. Assessment of sperm damages during different stages of cryopreservation in water buffalo by fluorescent probes. Cytotechnology 68, 451–458 (2016). https://doi.org/10.1007/s10616-014-9798-9

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  • DOI: https://doi.org/10.1007/s10616-014-9798-9

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