There are two basic methods for extracting sperm from the epididymis, either by retrograde flushing of the sperm through the epididymis or by utilization of the “float-up” method. In the latter method, the vas deferens and cauda epididymis are placed in petri dish (or embryo search dish), with semen extender ( such as RPMI-1640) or appropriate dilution media (such as saline). The tissue is incised in multiple locations and the dish agitated to encourage release of the sperm from the tissue into the surrounding media.several extenders have been developed to preserve epididymal or semen in liquid form for short--and long-term storage. Does the plasma can be used as extender or dilution media for CASA in rat sperm?
Dear Mehran,
in my opinion, it is in principle possible. But, the blood plasma composition is not well defined. Besides, the negative role may play possible presence of harmfull compounds, presence of anticoagulant etc. Also the viscosity of the plasma differ from the viscosity of the saline and related solutions, what can play a role in the sperm movement evaluation.
Best regards
Pavel Travnik
I will not recommend to use on patients sperm, unless you have done extensive research based on results from different groups.
I am certain it will not work
As Trávník says, plasma (not sure if you refer to seminal plasma or blood plasma) is an undefined medium. It is much more difficult to obtain that a saline solution. Saline and other media are just fine for recovering and storing epididymal spermatozoa. I am attaching a few publications on our experience with epididymal spermatozoa.
Article Comparison of two methods for obtaining spermatozoa from the...
Article Specificity of the extender used for freezing ram sperm depe...
Article Seminal plasma improves cryopreservation of Iberian red deer...
In our laboratory we usually use epididymal sperm for experiments. To obtain it, we separate the cauda epididymis, then place it in a capsule containing Tyrodes medium, we made several cuts in the tissue and then place it in thermostated stove and wait 10 min before starting the study, in order to allow sperm-extruded into the medium. This technique gives very good results.
I enclose a paper where the use of this technique and subsequent analysis of epididymal sperm is described
As was said previously, I do think that the transposition of saline solution by blood plasma need futher research because it composition vary with the intensity of the metabolism and with the physiological state. Even if the pH of blood and seminal plasma seem to be alike, some compounds present in the blood plasma could be negative for sperm mobility.
In my opinion, it is not recommended. Saline or Tyrode have nery nice results.
thanks everybody for your valuable comments, i learned good tricks and hope to utilize it.
I do not think plasma can be used to store epidermal semen considering the fact that composition of plasma depends greatly on the physiological state of that individuate and its metabolic activity,
in addition to good previous answers, you must take into accont the osmotic pressure differences along the epididymal duct. So to know the osmolarity of the extender used is needed
As saying for other colleagues, is more accurate the use of defined media, as well for evaluation and for conservation too.
There is a major difference between plasma and serum. Plasma is fluid portion of blood including fibrin, a protein that makes a network of fibres to stop bleeding. Serum does not contain fibrin. Plasma has never been used in extenders, however, serum is generally added at varying percentages (1-10%) in extenders and in culture media used for embryos. Serum composition varies depending on its source that is why it is called undefined component. Its perfect replacement has not yet been found although a few alternatives have been in use. Any extender for epididymal sperm must have proper, osmolarity, pH, nutrient source, macromolecules and temperature etc., therefore, serum alone is not appropriate rather is useful if supplemented in any other appropriate extender. Serum albumins and/or globulins are now commonly used as replacement of serum which are considered relatively more defined components.
if we are talking about blood plasma, using plasma is not recomended due to its fibrin content and high viscosity as well as lipids content. for my experiance i am using rats' prostate and seminal vesicle fluid extract after hemogenizing with 5ml PBS ph 7,2 and filter the mixutre with very fine mesh.( freshly prepared from health male rat not included in the experiment ) and also you can use Ringer's Solution ph 7.2
Ringer's Solution, per 100 mL:
860 mg NaCl
30mg KCl
35mg CaCl2
Dear chalabi
thank you very much. also, i used vesicle seminal fluid , but in some rats the fluid are very viscid. It should be noted, i extracted the vesicle seminal fluid from each rat that used for epididymal sperm assessment. can you clarify the homogenization steps for me?
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