I was doing semen analysis on guinea pig; semen collected from vas deferens. I am pretty sure that tissue around the vas deferens was cleaned properly and had no blood contamination. Equipment used was clean as well.
Medium used was PBS ± 28oC. Why did the sperm adhere to each other in the head? Would you mind suggesting how should I do to fix that in the next semen analysis?
Thanks for your help :)
Yo should include in the PBS, at least, a 0.05% final concentration of BSA (Bovine Serum Albumin) in order to avoid the sperm agglutination. Nevertheless, it depends on the species, there ir a head adhesion when the sperm membrane has any damage or even capacitation, taking into account that the natural responde of sperm in contact with the oocyte is to "breake" its membrane to be able to secret degradative enzymes to enter into the oocyte.
In conclusion, I think that yu could try with the inclusion of BSA.
Regars, Manuel.
Guinea pig sperm form the so-called rouleaux in the epididymis and when you collect them you will see then in stacks. When they are incubated under capacitating conditions, they separate at one point and swim individually. Timing of separation varies according to the conditions used for capacitation.
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Eduardo
I've doing semen analysis with the same method once, and the spermatozoa were separated and move freely. How can i create capacitating condition for inkubation? Thank you :D
hi for capacitation you have use separate media(capacitation TALP, IVF talp, sp-TALP), its also buffer with some ingridients (heparin) which make sperm to capacitate. you have to incubate at 37degree in Co2 incubator. regarding sperm head adhere, if you allow the sperm in room temperature for more time even in PBS, it will adhere. this is due to damage to the sperm membrane when time progress and RT is favorable for capacitation
Hope this helps
Human semen may agglutinate, head to head, tail to tail or mixed.All of them occur in abnormal conditions.
Human semen may agglutinate,Bach all of them occurs in patological conditions
There are two major approaches used to capacitate guinea pig sperm in vitro: (a) One is based on the use of a Minimal Capacitation Medium (MCM) with a 5 hour-incubation (more or less, depending on conditions) and (b) another one based on a modified Tyrode's medium (mT). For this latter one, there are in turn two options: (i) a 17 hour-incubation in a calcium-free medium (Ca2+ deficient actually) followed by addition of Ca2+; under these conditions sperm undergo acrosome reactions quickly (about 10 min) in response to calcium addition; (ii) incubation for 1 hour in the same calcium-deficient medium with addition of lysolecithin (i.e., lysophosphatidylcholine); after addition of calcium, sperm undergo acrosome reactions. The beauty of the guinea pig system is that the acrosomes are large and one can observe motile sperm under the microscope to record the number of acrosome-intact and acrosome-less sperm to quantify proportion of acrosome reactions among motile (live) sperm. The "drawback" of this system is that acrosome rections occur in response to calcium addition but there is no physiological ligand involved. So some people have argued that there is a limit to what one can learn with this system.
The method in which guinea pig sperm are incubated in MCM for about 5 hours and acrosome reactions are induced with progesterone is one in which it is possible to analyze ligand-induced responses at the molecular and cellular levels.
In all cases, the acrosome reaction is taken as a response of capacitated sperm to an inducer (calcium, or any other molecular probe) or a physiological ligand (progesterone). So, if one is interested in capacitation, the proportion of sperm that undergo an acrosome reaction is a reflection of the proportion of capacitated sperm at the end of incubation under capacitating conditions.
To find out more, please see:
Roldan E.R.S., Shibata S., Yanagimachi R. (1986) Effect of Ca2+ channel antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa. Gamete Research 13: 281-292.
Murphy S.J., Roldan E.R.S., Yanagimachi R. (1986) Effects of extracellular cations and energy substrates on the acrosome reaction of precapacitated guinea pig spermatozoa. Gamete Research 14: 1-10.
Roldan E.R.S., Fleming A.D. (1989) Is a Ca2+-ATPase involved in Ca2+ regulation during capacitation and the acrosome reaction of guinea pig spermatozoa ? Journal of Reproduction and Fertility 85: 297-308.
Shi Q.X., Chen W.Y., Yuan Y.Y., Mao L.Z., Yu S.Q., Roldan E.R.S. (2005) Progesterone primes zona pellucida-induced activation of phospholipase A during acrosomal exocytosis in guinea pig spermatozoa Journal of Cellular Physiology 205: 344-354.
Chen W.Y, Ni Y., Pan Y.M., Shi Q.X., Yuan Y.Y., Chen A.J., Mao L.Z., Yu S.Q., Roldan E.R.S. (2005) GABA, progesterone and zona pellucida activation of PLA2 and regulation by MEK-ERK1/2 during acrosomal exocytosis in guinea pig spermatozoa FEBS Letters 579: 4692-4700.
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Eduardo
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