Assisted Reproductive Technology - Science topic

Please read MRT syndrome in Https://www.hereditics.net. MRT syndrome is a new kind of artificial cytoplasmic hereditary diseases, shortened as Cytohetic diseases.

Your question is bit confusing. From your question, it seems that you want to isolate and denude diplotene arrested immature oocytes from primordial/primary follicles of mouse ovary and to study the gene expression in denuded oocytes cultured in vitro.

If it is so, then remember that due to larger in size as compare to other cell types in ovary, immature oocytes are more susceptible for any minor changes under in vitro culture conditions. These minor changes such as temperature, culture medium, addition of hyaluronidase or trypsin will have negative impact on the oocyte in vitro. Addition of hormones like hCG etc. may also have influence on the gene expression analysis of oocytes due to presence of encircling somatic cells.

You have to be very careful, while isolating and denudation of immature oocytes in vitro in terms of addition of either enzyme or hormones (both exposure time and concentration).

I would suggest you to reduce the diameter of micro-glass pipette, and keeping all physical and biochemical factors into consideration, make repeated pipetting in order to denude the immature oocytes. Yes, it is difficult task but not impossible for a technically skilled scientist.

Hope, you would be able to do it and best of luck

Increased research pertaning to infertility, it physiological and pathological causes as prevelence of infertility in animals is presenting increased trend with time. Similarly, retention of placenta and increase in dystocia, it is all related to chane in physiology.

In your example, you have 3 parameters in your model, so you can pick 3 data points in your data pool and plug these 3 data points into your model to solve the parameter values. Although your model is nonlinear, the equations you need to solve may be linear. In the case that these equations are nonlinear equations, root-finding problem is still easier than your original problem. The solution for these 3 parameters can be used as a reasonable initial guess for nonlinear least squares fitting. More details of this approach (e.g., how to choose 3 data points from the data pool) can be found in the following paper:

dear Omer Ucar

I really appreciate your comprehensive and beneficial explanation.

wish u the best

Yes, We can supply you,

PMSG,pregnant mare serum gonadotropin, Natural glycoprotein, pure natural animal hormone, extracted from serum of pregnant mares , Follicle stimulating and Luteinizing activites,improving fertility rate, inducing superovalutions and estrous synchoronization , used in livestock farming,Pig,prepuberal gilts,Cattle,Dairy,Sheep,Goat and other mammal animals

Full series of PMSG, from Raw mare serum, intermediate, lyophilized powder ,and injection (FDF),

now Scientific research and animal farm are using this products

For more and discussion, please contact us .tommy59@qq.com

Thank you for sharing with me, it means a lots and I really appreciate that! :)

Best wishes!!

Dear Stefan S Du Plessis ,

Thank you so much for your awell articulated suggestions.Now,I got the bsic idea pertaining to the issue.

Regards!!

Osim

Dear Gabor, the following paper may help you:

Mechanical Stresses in Embryonic Tissues: Patterns, Morphogenetic Role, and Involvement in Regulatory Feedback

L.V.Beloussov, S.V.SavelievI, .NaumidiV. V.Novoselov

Our kharkiv`s scientifics stored diluted FSH in liquid nitrogen without losses of activity.

recently I have been in University of Brussel , the Belgian friend’s they depend on BWt , if > 80kg will trigger by 10,000 and of < 80 kg they trigger by 5000 , I think it dose make sense

Hi Maraia

if we spoke about old method coconut fresh was used as preservative for sperms before  And still used it for animals

Dear Meng,

I believe this work will help : Eur Rev Med Pharmacol Sci. 2010 Oct ; 14 ( 10 ) : 891-6 .

Inositol activity in oligoasthenoteratospermia - an in vitro study .

Colone M1 , Marelli G , Unfer V , Bozzuto G , Molinari A, Stringaro A.

If you want I can send you some vials of Myoinositol for your experimentation.

Vittorio

We (my husband, Professor Neal First) and I had a student that studied haploidization in the bovine model.  She did manage to get development to the morula stage but not beyond that.  I will add that a few embryos did look as though they were "trying" to develop into blastocysts but they were far from normal.  With a little work I can dig out her thesis if there is interest.

Well, in my opinion I wouldn't make the solution and store it for so long. According to our experience in laboratory, if you want to use it to obtain a calibration line, for example, I recommend that you make your solution almost immediately, not waiting for more than a week. 

Greetings!

Regarding prevent ovarian hyperstimulation syndrome, think about ORPI (ovarian response prediction index)

Reprod Biol Endocrinol. 2012 Nov 21;10:94. doi: 10.1186/1477-7827-10-94

Hi,

INRA96 is the best extender for cooled stallion semen and INRA freeze is the the best for cryopreservation at -196 C

Magdi

You can try BL6 mice.

I believe there are kits for this.  The American Red Cross  (ARC)Specialty lab does this, as well as NIH. These are micro well plates.  These are serological tests.  I would contact ARC in S. California (formerly Dr. Garratty's lab). Dr. Garraty passed last year. There they do flow cytometry crossmatches.  They would be the first I would talk to. 

Mixed lymphocyte studies is a pretty old method to do this, but it actually works well.  This would be for cellular immunity.

Hope this helps 

Sergio

just because they are close does not make them the same!  The endometrium is a completely different environment and I would say not influenced by what is going on in the cervical region 

Acridine Orange intercalates into nicked or damaged DNA and will foluresce "Red" on 488 blue laser.  Undamaged DNA or sperm cells with correctly packaged DNA will resist intercalation and the compound flouresces "green".  In this way the ratio of red to green can be calculated as the DNA fragmentation index (DFI).  I use AO from sigma aldrich (SIAL) or bioscience also have it. Important to use very high purity and concentration of stock solution should be 1mg/ml.  maintain AO powder at -20.

It depends on what you are injecting & where you anticipate its expression. Check out Xenopus methods here...

sorry I dont know

My co-authored (with Maureen McBrien) book titled Assisted Reproductive Technolgy publishled by the American Bar Association (2011) covers the US. 

Be careful. If you mix the methanol solution with the extender, the oil will probably drop out of the solution.

Pavel

With regards to the acrosome reaction you can either use FITC-PSA or FITC-PNA. Please refer to the WHO Laboratory manual for the Examination and processing of human semen (2010) pages 146-152

Agree with Prof. Tulay Irez:

Use HOS Test to identify sperm with intact normal DNA.

Those sperm exhibiting tail curling can be used for ICSI, we believe.

However, such a study should be reported.

Ref: Stanger et al, 2010. RBM Online 21, 474-484.

Best wishes and do report your experience.

John Yovich

Just take a  sterile disposable pipette tip and pipette the medium from under the oil. I collected in this way medium after human embryo culture,to analyse the glycolitic activity between day 4 and day 5 of in vitro culture.  I worked in drops of 5 micro-liters.  Just keep in mind that if you are overlying cultures with oil that the oil will extract all lipo-soluble substances contained in the medium.  Good luck

thank you very much. I'll read it with joy. I have recently published a book on the subject but there are still topic to include or expand.

If you have blastocysts, they all have survived to that that stage of development. It is understandable that there is a strong desire to be able to assess them and then predict their chance for success. To meet that desire, some have developed protocols.

Yet, it is difficult to assess embryos with confidence.

In cattle, sheep, deer and mice, I have transferred blastocysts and found that. One thing I discovered that was useful was to examine the shape of the zona pellucida. If it was round and the blastocyst looked competent to hatch, we would get better pregnancy rates. However, even when the blastocysts had flattened zona pellucidae, if I cut them out of their zona with a micro-scapula, their pregnancy rates would go way up.

The idea of transferring the most advanced stage ones is interesting . It has been learned that, all other things being equal, the most advance stage mammalian embryos within a female, tend to be males. Other things can influence the relative stages of embryos such as the fact that all follicles are not always ovulated at the same time.

Salamatin, Joint Institute for Nuclear Research. Now the new technology for On-line systems construction is in progress, and some of papers are avaliable, but in russian. Below is abstrfct of one of them. If it is what you need, please write to del@xaker.ru or salam@nf.jinr.ru.

Paper title:"Automation of neutron spectrometry experiments using network technologies". Abstract contents:

A new structure of a distributed software system for experiments automation (SEA) is proposed. The DSEA includes the following interacting parts: 1) a subsystem describing the experimental procedure (SDEP); 2) the experiment control program (ECP); 3) distributed components messaging environment (DICME); 4) sample environment at the instrument; 5) Data Acquisition (DAQ) system; 6) components for auxiliary operations.

SDEP contains data base (DB) and two dialog software: 1) a program producing passports of components which govern conditions of data registration; 2) program of Preparation of a Single Job (PSJ). The first one creates and writes to the database documentation for components, which contains: the name and description of controllers and devices connected to them, globally unique identifiers (GUID), used for addressing components, and parameters descriptions. The second one, PSJ, uses only terminology understandable to the experimenter, and represents the job as a list of ECP states descriptions in JSON format.

ECP program receives from SDEP the list of SEA states, selects the description of the next state of the system (list of the registration data conditions) and sends a description of each condition to the intermediary DiCME. The description of the condition carries information about the component (GUID), sufficient for its search and link to ECP, and it also contains a list of parameters. Communicating through DICME with all necessary components, the ECP serves as a components manager. The choice and procedures procession that implements the conditions of registration data is performed in the components that drive the hardware, based on the interpretation of the referred to them descriptions of the conditions. Each component, to which ECP sent a message, should return to ECP the end of job signal (DONE / ERROR). After receiving signals from all the components listed in the description of the required state, ECP activates the data acquisition subsystem DAQ. The signal of data exposition finishing allows the ECP to switch to processing of the next state description in the job file.

Mediator DiCME, developed using network technology, automatically searches and dynamically binds components. It provides an asynchronous mechanism for remote execution of procedures and access interface to component’s procedures independent on the computer network address. Information transfer and processing of all interactions of components in the SEA is performed by similar means. For dynamic components linking two algorithms are used in the SEA: 1) the "hard" one for the basic operations (conditions formation, registration and archiving of experimental data) , implying a mandatory delivery of the message from a "customer" and of the answer from the "implementer"; 2)the "soft" one: a "subscription" for auxiliary operations (visualization , preprocessing , ...), in which a component consumer declares interest in a particular type of information, and the event manager serves all the "Subscribed" consumers when the information appears. As a result, the components implemented in different experiments and in different SEA can be used without modification.

The presented approach was applied in the development of SEA software for several spectrometers at neutron sources IBR-2 and IRENE in JINR. It can be also applied in other areas, for example, to process automation.

You should be seeing the effect of zona hardening, which is a byproduct of fertilization but might be brought about my the Ca++ transient elicited by activation, depending on the species you are working with.

I have done lot of work on primary cell culture in buffaloes, I have work both on buffaloes and cattle, Their is nothing change between two species, except for buffaloes you need to maintain CO2 incubator temp to 38.5C.

I am using PDMS and lexan.

Follow this link and thank to Prof. Hansen.

Thanks a lot Rohan, I am reading the literature, and I saw there are a lot of genes, which is difficult to decide which one is better. I will read papers that you mentioned and thanks for points.

I`m quite curious & waiting for contributions in this so crucial issue. :

Transvaginal sonogram is helpful before progesterone challenge test , because in atrophic endometrium , no bleeding is occurred with progesterone, if there is bleeding , so there is a thick endometrium.

Hi Loris,

Here is the website of Dr. Evenson's company. You can directly order from them.

Thanks Miriam, please say hello to Shlomi for me. We used glycerol,egg-yolk,citrate (GEYC) cryoprotectant from 1976 until early 2013 with very good results. We have had to switch to a commercially available cryoprotectant because of lab accreditation requirements. Cryosperm has the advantage of not containing any biological products such as egg-yolk or albumin but still giving equivalent post-thaw motility recovery. Cryosperm does contain Raffinose which a previous student of mine (Puti Adla Runisa) found to give good motility recovery in the absence of egg-yolk or HSA (manuscript in preparation). Cryopreservation is a very complex subject and I am convinced that further significant advances in sperm cryopreservation can be made, for example, by developing cryoprotectants which are tailored to be optimal for different categories of semen.

See Ouhibi et al.

Please see article at vrl attached: http://humrep.oxfordjournals.org/content/23/6/1253.full

At our center, the P4 result is just one factor used to determine day of hCG trigger. The ovulation induction protocol matters, as GnRH antagonist IVF cases will evoke a different P4 pattern compared to those receiving standard GnRH agonist downregulation. The value you mention (1.5ng/ml) probably would be a reasonable level, although P4 levels are best interpreted against corresponding E2 levels taken on the same day. Lower E2 responses could be triggered at a lower absolute P4..

There are those days product specially developped for embryology labs. I do not sell those products but could provide you advise visit www.quartec.co.

For basic rules of safety and possible cross contamination pipette tips should not be re-used.

Thanks! For the fusion medium, I use PVA instead of BSA.

We are 100% agree with that ideas the antagonist is not a golden protocol over the agonist but in our (>10 yrs in infertility treatment) experience the outcome of antagonist is better than antagonist protocols. In case of bad responders .. it is the most ,unfortunately, unwanted results for our kind persons. We found that OCP for at lest 3 months improved those bad responders to short antagonist protocols.

Prof. Dr Ahmed K. Allow

Infertility consultant

PhD and M Sc

Director of Allow IVF center

Sana'a Yemen