Ascorbic Acid Effect on Frozen and Thawed on Sperm Motility, Plasma Membrane Integrity, Livability and Acrosome Integrity of Ring-Necked Pheasant (Phasianus colchicus) Semen <strong> </strong>
Authors: Ali Umer , Bilal Asif , Iqbal Aasma , Ansari Muhammad Sajjad , Rakha Bushra Allah , Akhter Shamim 
Published: May 2022

Ring necked-pheasant (Phasianus colchicus) is bird of order Galliformes. Ring necked-pheasant is also known as gallinaceous bird and game bird. It looks like chicken bird. It is national bird of South Dakota. It is mostly found in wild areas but in Pakistan it exists as Domestic bird. It has high proteins and low fat in its meat. The present study was conducted to check the effect of ascorbic acid on sperm Motility, sperm cytoplasmic membrane integrity, sperm livability and acrosome integrity of Ring-necked pheasant at different concentrations of ascorbic acid (0mM, 1mM, 2mM, 3mM and 4mM) were used. Semen was collected by abdominal massage technique. Motility of sperm was greater than 70%.Then it was processed further. The cryopreservation of semen was checked at various stages like Post Dilution, Post Cooling, Post Equilibration and Post Thawing. Semen was cooled from post-dilution stage (370 C) to post-cooling (200C).Then it further cooled gradually to get post-equilibration stage (40 C) within 24 hours. After that 10% glycerol was added to the sample. Then it was transferred to liquid nitrogen (LN2) cylinder for 24 hours. After thawing stage sample was removed from LN2 cylinders and placed in water bath for 4 hours to achieve post-thawing. Then performed sperm quality assays at each stage of cryopreservation. Sperm motility was assessed by Neubauer Chamber Hemocytometer. Plasma Membrane Integrity was checked by using Hypo Osmotic Swelling Test with the help to 2% Eosin. Sperm livability was assessed by Lake&rsquo;s glutamate solution and sperm Acrosome Integrity was checked by dual staining technique by using Formal citrate solution and Giemsa stain. The better results were seen at 3mM treatment of ascorbic acid. Sperm motility percentage was significantly different (P &lt; 0.05) on 3mM treatment on all the stages of cryopreservation rather than other treatments. Plasma membrane integrity assay also showed good results at same concentration (P &lt; 0.05) on all the stages of cryopreservation rather than other treatments. At the sperm livability stage 3mM treatment showed significantly difference (P &lt; 0.05) on all the stages of cryopreservation rather than other treatments. The sperm acrosome integrity showed highest percentage on 3mM treatment (P &lt; 0.05) on all stages of cryopreservation rather than other treatments. Effect of ascorbic acid on all the quality parameters (sperm motility, plasma membrane integrity, livability and acrosome integrity) was showed significant difference (P &lt; 0.05) at 3mM treatment of ascorbic acid as compare to control as well as remaining treatments. It was seen that by increasing the concentration of ascorbic acid from 3mM concentration to onward it shows negative results on its cryopreservation. Now this cryopreserved semen can be transfer from one place to another place to obtain good varieties as well as better genetic characteristics of this species.