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CARD FERTIUP® and CARD MEDIUM®

FAQs
General questions regarding our products
Q1.Please tell me the storage method and storage period for CARD FERTIUP® Mouse Sperm Cryoprotectant/ Mouse Sperm Preincubation Medium and CARD MEDIUM®.
A1.

1. CARD FERTIUP® Mouse Sperm Preincubation Medium and CARD MEDIUM® should be stored at a temperature between 2~10℃.
2. CARD FERTIUP® Mouse Cryoprotectant should be stored at a temperature between 18~28℃.
3. For the retention period, please see the documentation sent with the product, or alternatively the notes on the side of the ampoule.

Q2.Once opened, is it possible to divide a product into smaller amounts and cryopreserve it?
A2. No, it is not. We cannot guarantee the quality of our products after cryopreservation,
and so we do not recommend cryopreserving them.
Q3. Are there any substances contained in your products which may be harmful to humans?
A3. No, there are no substances in our products which are classed as harmful to humans by the Material Safety Data Sheet (MSDS).
Q4.How can I purchase reagent products produced by Kyudo Co. Ltd?
A4. 1. For orders within Japan, please contact us directly by e-mail. Our address is fertiup@kyudo.co.jp.
2. For orders from overseas, please contact our overseas supplier, Cosmobio Co.,Ltd.
You can contact them using via e-mail, or alternatively you can visit their website.
 HP : Cosmobio Co.,Ltd.
 E-mail : export@cosmobio.co.jp
Q5. How many days does it take for products to arrive?
A5. This means that your products will arrive two days after we have confirmed your order.
For orders from overseas, please contact Cosmobio Co.,Ltd.
Q6. There is a foreign object in the product. Can I receive a replacement product?
A6. Using our quality control system, we aim for the highest quality products from manufacture to shipping. In the unlikely event that you find glass fragments or other such foreign objects in a product,
please contact us and we will arrange for a replacement product to be sent to you.
Questions regarding the CARD FERTIUP® series of products
Q1. We started that were able to obtain a high fertility rate for the C57BL/6 strain of mouse via in vitro fertilization using cryopreserved spermatozoa treated with CARD FERTIUP® Mouse Cryoprotectant/Mouse Preincubation Medium. Do the media increase the in vitro fertility rate of other strains too?
A1. We are currently investigating this further,
so please wait a little longer until we have compiled the relevant data.
Q2. It was not able to use the entire 0.5mL of the CARD FERTIUP® Mouse Cryoprotectant.
Would it be possible to use the cryoprotectant for a few weeks
if I were to transfer the medium into tubes and store it at refrigerated temperatures?
A2. No, it is not possible. Once open, we recommend using up an ampoule as quickly as you can do the following.
To do so, please purchase an ampoule of reagent which you can use in one occasion.
Please note that the ampoule of cryoprotectant should be stored at room temperature.
This is because the cryoprotectant contain high doses of saccharides and skim milk, which may sink to the bottom of the ampoule after refrigerating or freezing.
Q3. There was precipitant in un-opened CARD FERTIUP® Mouse Cryoprotectant.
A3. In case the precipitant is obtained, please incubate CARD FERTIUP® Mouse Cryoprotectant ample 30 min to 1 hour at 37℃.After dissolving, there is no problem in use.
Q4. Liquid paraffin is used in the usage instructions for the CARD FERTIUP® series; does the paraffin have an adverse effect on the sperm?
A4. Yes, it does. However, as a rule liquid paraffin does not mix in with the sperm suspension (sperm suspended in preservant), so there is no adverse effect on the sperm.
Q5. After preincubating cryopreserved sperm using CARD FERTIUP® Mouse Preincubation Medium, what is the sperm concentration when carrying out in vitro fertilization using an IVF medium?
A5. Normally, the sperm concentration in cryopreserved sperm ranges from 100,000~200,000 sperm/μL. When introducing freeze-thawed sperm suspension (10 μL) into our Mouse Sperm Preincubation Medium (90 μL), the sperm concentration is diluted tenfold.This means that the sperm concentration in the preincubation medium is 10,000~20,000 sperm/μL.Thereafter, the preincubated sperm suspension (10 μL) is further diluted tenfold when it is introduced into IVF medium (90 μL). This results in a sperm concentration in the IVF medium of 1,000~2,000 sperm/μL.Finally, the ratio of motile sperm in after freeze-thawing is around 20%, which means that the motile sperm concentration in the IVF medium is 200~400 sperm/μL.
Q6. I would like to download the instruction manuals for the CARD FERTIUP® series.
Where can I download them from?
A6. They are available on our website,  kyudo Co., Ltd.
Q7. Is there a method or measure to confirm whether the freezing and thawing of sperm has been correctly carried out?
A7. 1. When simply confirming the quality of the sperm after freeze-thawing, pre-incubate the sperm in medium after freezing and judge according to the number of active motile sperm at the edge of the drop.
2. When carrying out final confirmation,in vitro fertilization of unfertilized oocytes and then confirm that you have attained at least a 30% fertility rate. When you check the motility of the sperm before freezing, please dilute the suspension of sperm in the medium, such as CARD mHTF or PB1 medium.
(We carry out IVF during lot checks to check and confirm that all of our products on sale attain an in vitro fertility rate of 30% or above for C57BL/6 mice)
Q8. How about legacy stock of cryopreserved spermatozoa?
A8. Please Check here. Rescue In Vitro Fertilization Method for Legacy Stock of Cryopreserved Spermatozoa
Q9. We does not work in vitro fertilization as well as we thought. Sperm and eggs have no problem but the embryos dead next day. What kind of cause can be considered?
A9. The quality of liquid paraffin is sometimes related as in vitro fertilization rates, or one of the reasons why embryogenesis stops occurring. Moreover, liquid paraffin gradually deteriorates after the bottle is opened. Please use as promptly as possible after open the bottle. We recommend using the following products.
Manufacturer name: Nacalai Tesque
Grade:Specially Prepared Reagent for tissue culture
Name of product: Paraffin liquid
Model number: 26137-85
Questions regarding CARD MEDIUM®
Q1. For how many months after manufacture does the warrantee period for CARD MEDIUM® last?
A1. CARD MEDIUM® is a new product and so at present, the warrantee period is 18 months. This warrantee period may increase in the future, depending on the results of investigations and reviews.
Q2. Is it possible to attain a high in vitro fertility rate without using CARD MEDIUM® in association with the CARD FERTIUP® series?
A2. No, it is not. The CARD FERTIUP® series works on sperm and CARD MEDIUM® works on oocytes in order to raise the fertility rate. As such, using both products simultaneously is the key to attaining a fertility rate of over 80% even for the C57BL/6 strain of mouse.
Q3. Are we able to attain a high in vitro fertility rate for strains apart from the C57BL/6 strain of mouse?
A3. Yes, other mouse strains also show high in vitro fertility levels.
Question regarding CARD HyperOva®
Q1. Please tell me the storage methods and storage period for CARD HyperOva®.
A1. CARD HyperOva® are store at lower -20℃ degrees. You can use them until 36th months from the period of production.
Q2. What do you think about don't use CARD FERTIUP® and CARD MEDIUM® in vitro fertilization, when we stimulate with CARD HyperOva?
A2. May be you should better use CARD FERTIUP® and CARD MEDIUM®. In the case of stimulate with CARD HyperOva®, you can get many oocytes. It's important to use CARD FERTIUP® and CARD MEDIUM® for high performance in vitro fertilization.
Q3. We could get around 100 oocytes from a C57BL/6 mouse of 28-30 days age. In the fertilization dish, there are 200 oocytes, we can usually introduce two mice of Ampulla in one drop. How do you think about our procedure?
A3. There is a problem with many oocytes in one drop such as over 100. It cause low fertilization rates. Please make sure lower 100 oocytes in one drop.
Q4. There are white precipitates in the CARD HyperOva®, when I thawed the media. Is it important active ingredient?
A4. There is not active ingredient of CARD HyperOva®. There is fibrin from goat serum. We can remove fibrin from serum as much as possible.
Q5. We have been using CARD HyperOva® to collect zygotes from female mice. We are successfully with PMSG and hCG injected mice and mating. But If we use CARD HyperOva® instead of PMSG, the number of zygotes recovered is extremely low with the majority of ovulations being unfertilized oocytes.
A5. We think that CARD HyperOva® must not use in vivo mating. Because of the number of sperm were very low in mice oviducts after mating. And then we stimurated ovulation by CARD HyperOva®, there are many oocytes in oviduct. In the case of this procedure, the sperms are short against the oocytes. On the other hand in vitro fertilization, we can adjust the number of sperm from in vitro fertilization medium.