الفهرس 
Introduction
Reproductive biotechnology in buffaloOnly 14 pages are availabe for public view
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Abstract
Summary
The current study aimed to compare the quality of harvested spermatozoa separated by Percoll gradient method; swim-up technique and washing of frozen–thawed buffalo semen on the basis of recovered motility, viability, normal morphology and functions of spermatozoa.
Lots of frozen buffalo semen straws were obtained from Veterinary Governmental Authority, Assiut Province and transported in liquid nitrogen tank to the laboratory of Theriogenology Department, Faculty of Veterinary Medicine, Assuit University. Semen samples were pooled after thawing and divided into 4 aliquots, three of them for the different treatments (Percoll, swim-up or washing) and the fourth one was considered as control (without any treatments). The recovered spermatozoa from all treatments and control aliquots were evaluated for progressive motility using a phase contrast microscope set at magnification of 400 and equipped with a warm stage (37°C), sperm membrane integrity using hypo-osmotic swelling (HOST) assay , morphology using phase contrast light microscopy to examine wet smears of unstained buffered formalin solution-fixed spermatozoa , proteolytic activity using fixed gelatin membranes , viability and acrosome reaction using trypan blue/giemsa staining.
The results revealed that density gradient centrifugation (percoll method) using PureSperm ® protocol selected a sperm population with improved quality in regard to motility (86.00%), viability (87.00%), acrosome integrity(80.50%), proteolytic activity (34.80%) and membrane integrity (30.00% HOST positive sperm cells). Moreover, there was a tendency for increasing the percentage of spermatozoa with normal morphology after gradient selection (92.20%) and viable spermatozoa with intact acrosome (72.00%).
In this study, it was found that spermatozoa harvested from swim-up method showed a significant improvement in motility (73.00%), viability (76.00%), proteolytic activity (33.40%), membrane integrity (31.70 % HOST positive sperm cells), and morphologically normal spermatozoa (91.20 %). There were a high percentage of spermatozoa with reacted acrosome separated after swim up (31.80 %). However there was no significant effect on the viable spermatozoa with intact acrosome after swim up preparation (54.30 %).
For washing of frozen thawed buffalo semen, the present data showed a significant increase in progressive motility of recovered spermatozoa (66.50 %), morphologically normal spermatozoa (86.60%), proteolytic activity (22.60 %) and slightly improved membrane integrity (25.80 % HOST positive sperm cells). However, there was a slight reduction on viable spermatozoa (62.40%), while a marked decrease in alive spermatozoa with intact acrosome recovered after washing (47.40 %).
Harvested spermatozoa from frozen–thawed buffalo semen after percoll method were superior in recovered motility, viability, normal morphology, membrane and acrosome integrity when compared with that recovered from swim up and washing