Hello. There are many possibilities why your results are not optimal. But it is very difficult to know where the problem is considering only what you have stated. The problem may be in each of the freezing steps: diluent (quality, pH, type of egg yolk, etc.); temperature drop curve; stabilization period; concentration of semen in the straw; temperature and level of temperature stability of liquid nitrogen vapors; cold stabilization time of the straw; type of defrosting; etc. In your case, I would try to analyze each of the steps in detail to find the problem or to optimize the process. Hope it helps, Do not hesitate in contacting for help. Cheers.

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Humberto O Cisale Professor, it is very informative. After equilibration, the sperm motility was between 40-60%. Egg yolk was separated using whatsman paper with intensive care, not included albumin portion. pH of the tris egg yolk extender was between 6.8 to 7.2 The semen straws were kept in liquid nitrogen vapors above 4 cm for 7 mins. I look forward to probe the problem.

Dear João Diego de Agostini Losano,

Sperm motility drops after vapor freezing in liquid nitrogen. We have used dry ice method to drop temp from 37 to 4oC (it takes 2 hours). Equilibration is done at refrigeration temp for 2 to 3 hours. We are using fresh egg yolk. Styrofoam boxes are used for vapor freezing in liquid nitrogen for 7 to 8 mins.

Dear Tarique Hussain,

I would like to add my opinions.

Firstly I want to summarize your method from what you wrote. You collect ejaculates (1) and determine motility as 80% and high. You are using Tris-Egg Yolk-Glycerol (2) based extender. You apply 3 hours equilibration (3) and freezing with LN vapour for 7 mins (4). And You store in LN.

Firstly (1), goat ejaculate collection method determines success of frezzing. If possible you should use artificial vagina instead of electro ejaculator. Electro ejaculator also increases seminal plasma containing egg yolk cogulant enzyme as known phospholipase A2.

(2) If you use sperm extenders containgin egg yolk or milk. I think you use Tris-Egg Yolk (15 or 20%)-Glycerol (5-7%). Therefore egg yolk detrimentally interacts with phospholipase A2 and detrimentally effect sperm motility. You can see this effect coagulations in microscope fields. It is not occuring everytime however we faced alot and sometimes you don't know why motlity rates are very low. The mayor reason is this in goa semen. So you should wash your goat semen prior to dilution with egg yolk based extender. I advise SP-TALP or simple isotonic solutions with centrifuge not high speed (200-600g) use pelet section of semen and remove supernatant or seminal plasma.

(3) Equilibration phase is an other important issue. Maybe you can not provide enough temperature or you provide very low temperature. And also maybe you can reduce your equilibration time. Please check your refregirator or cooling condition it is proper for this aim.

(4) I think you use strofoam and you arrange the high of level of straws. It is important issue. You have to sure your strofoam has good density provide enough subzero temperature. I mean -110/-130ºC range ideal. And you can increase time 10-12 mins then transfer to LN.

(5) An other important issue is pH changes in extenders when you add antioxidants. Some antioxidants cause to decrease pH. And also you can balance your pH with 1N NaOH. And I advise you provide 7.2-7.4 pH for this aim. Acidity is not good for semen motility. We faced this problem with NAC (N acetly cytein) while we used milk based extender in rams. Some antioxidants may cause toxic effects on semen and decrease motility. Check out your control groups not containing antioxidants.

Lastly check all steps and be sure all solutions are good and suitable for your study.

Good Luck

Umut

I'm sorry, but this is a matter far beyond my expertise

Tarique Hussain as I understand from your answer, you have a lost in motility before freezing. If is so, I guess you have to begin working in the freezing curve between 30º to 5ºC. If you do this in a refrigerator,may be you can put a thermocouple analize how the temperature drop is working. Of course,if yuou have a computer controled freezer is easier, but if not, this is a good step to work on.

Humberto O Cisale, Thank you dear professor.

I want to know, which antioxidant (resveratrol or quercetin) is best for improving post-thaw sperm motility parameters and enhancing in-vivo fertility rate in goats

Dear Tarique Hussain,

I would like to talk about my experience on antioxidants.

I included in Glutathione, Glutathione Peroxidase, Glutathione Reductase (As you see I completed Glutathione Redox Cycle), L-Ergothionine, Thiohistodine , N-acetyl cystein (SH grups) and some others. I mean Vitaimne C, E etc. We carried out research on semen storage and also IVM-IVF-IVC steps. I mean I have enough experiment on antioxidant usage in semen and IVF. I hope other researhers share their experiment and knowledge.

I know you only ask "resveratrol or quercetin". I can say that antioxidant usage is expensive and not needed if your semen or IVF conditions are good. If you carry out your steps smoothtly. I determined that if semen motility is very low and vulnarable to enviromental conditions then maybe antioxidants reveal some beneficial effects on post-thaw semen quality. Otherwise antioxidant usage is expensive and not needed.

I want to share my experience on GSH, GPx, GR these antioxidants have itself antioxidant capacity and also create unique antioxidant chamber/cycle called Glutathione Redox Cycle. Therefore I tried these antioxidants. These cycle creates with NADPH, which is aboundant in biological enviorement and provide "H ion" to reduce GSSG to GSH with GR and also GSH gives its H ions with GPx. GPx also has antioxidant capacity (itself). GPx deals with H2O2 with GSH or itself like catalase enzyme.

Briefly, I determined if semen quality is good. GSH, GPx or GR combinations there is no effect on postthaw semen quality. If pre-freezing semen quality was bad (normal conditions maybe you would reject this kind of quality of semen) post-thaw semen quality was beter than control grup in GPx group. GSH and GPx was behind GPx group. Moreover GSH-GPx-GR group didnot have good results compared with control. This condition was only real in bad semen quality prior to freezing. I determined same effects in L-ergothionine, L-2-thiohistidine...

I may say that if you did not determine any deterimental effect of antioxidants on semen and its cheap. You can use... It has placebo effect on researchers :-)

I want to share my IVF experience about antioxidants in an other answer or question section...

I think there are alot of factor (multifactor) affecting semen and embryo quality and we have test and improve these factors. Otherwise we, as researchers, spent our limited research budget on un-necessary issues like antioxidants...

If you have enough research budget you can try. And share your results with us. I want to know which one is good "resveratrol or quercetin"?

Good luck,

Umut

Dear Dr. Hussain,

Apart from you standardization procedures, you may also revisit your procedure for vapour freezing. Pls check the distance between strws and LN2 level (ideally 4-5cm) and timing (ideally 8-10 minutes)