CARD FERTIUP® Cryoprotectant: CPA

Kyudo

Catalog No.: KYD-001-05-EX

Refer to the Reproductive Engineering Techniques in Mice Technical Manual for complete instructions of IVF Product Insight pages:  Achieve Successful Fertilization in IVF-resistant Mouse Strains  Reproductive Engineering Reagents and Equipment for RAT from Kyudo/CARD

Product Description

CARD FERTIUP® Cryoprotectant: CPA is a mouse sperm cryoprotectant that guards against damage to spermatozoa during cryopreservation. This reagent significantly improves in vitro fertilization rates of frozen and thawed sperm even with notoriously IVF-inefficient mouse strains such as C57BL/6 and 129. Use it to preserve and protect your valuable mouse resources.

Sperm cryopreservation is a practical and cost-effective method of archiving laboratory mouse strains. FERTIUP® series products and CARD MEDIUM are high-performance media for mouse sperm cryopreservation, sperm preincubation and IVF. When used in combination, FERTIUP® and CARD MEDIUM provide a high and consistent rate of fertilization from frozen-thawed mouse sperm, which will allow you to efficiently recover mouse lines when you need them.

CARD FERTIUP® Cryoprotectant: CPA improves sperm survival after thawing and subsequent in vitro fertilization. Sperm can be cryopreserved with high survival rate even with the C57BL/6 and 129 lines, which were said to have low survival rates with conventional sperm freezing. Storage of genetic resources with frozen sperm is simpler than the method of storing fertilized eggs and can be reduced to 1/30 or less compared to the cost of live maintenance of stock. It is also ideal as a preparation for system disconnection, natural disasters and unexpected accidents.

For information on techniques for mouse spermatozoa cryopreservation, spermatozoa thawing, in vitro fertilization and obtaining pups through embryo transfer, please refer to the CARD website: https://card.medic.kumamoto-u.ac.jp/card/english/sigen/manual/onlinemanual.html

CARD FERTIUP® Cryoprotectant: CPA FAQs

I observed crystals in unopened CARD FERTIUP® Cryoprotectant: CPA (Cat. No. KYD-001-EX). Is the product still usable?
Occasionally, crystals may form in CARD FERTIUP® Cryoprotectant: CPA due to exposure to low temperatures. The crystals can be dissolved by warming the product in a 37°C incubator for 30 minutes to 1 hour with gentle mixing. The cryopreservation performance of CARD FERTIUP® Cryoprotectant: CPA is not affected after the crystals have dissolved.

I obtained good results with in vitro fertilization using frozen sperm from C57BL/6 mice and CARD FERTIUP® Cryoprotectant: CPA (Cat. No. KYD-001-EX) and CARD FERTIUP® Preincubation Medium: PM (Cat. No. KYD-002-EX). Can I expect improved IVF rates in other mouse strains as well?
The use of CARD FERTIUP® Cryoprotectant: CPA and CARD FERTIUP® Preincubation Medium: PM has been shown to be effective in many commercially available mouse strains.

Can I aliquot and freeze CARD FERTIUP® Cryoprotectant: CPA (Cat. No. KYD-001-EX) or CARD FERTIUP® Preincubation Medium: PM (Cat. No. KYD-002-EX) after opening?
No. We do not recommend freezing CARD FERTIUP® Cryoprotectant: CPA or CARD FERTIUP® Preincubation Medium: PM after opening, as their quality after freezing cannot be guaranteed.

If I cannot use all of CARD FERTIUP® Cryoprotectant: CPA (Cat. No. KYD-001-EX) at one time, can I aliquot it into tubes and store it for several weeks?
We recommend using CARD FERTIUP® Cryoprotectant: CPA as soon as possible after opening the ampoule. Please purchase ampoules in a size that can be used in a single procedure.

The protocol for CARD FERTIUP® Cryoprotectant: CPA (Cat. No. KYD-001-EX) and CARD FERTIUP® Preincubation Medium: PM (Cat. No. KYD-002-EX) uses liquid paraffin. Will it have any negative effects on sperm?
Liquid paraffin does not adversely affect sperm suspended in CARD FERTIUP® Cryoprotectant: CPA or CARD FERTIUP® Preincubation Medium: PM.

Can I cryopreserve mouse sperm in cryotubes instead of straws using CARD FERTIUP® Cryoprotectant: CPA (Cat. No. KYD-001-EX)?
Yes. Although straws are recommended for mouse sperm cryopreservation using CARD FERTIUP® Cryoprotectant: CPA, cryotubes may also be used.
For details, please refer to the online manual of the Center for Animal Resources and Development (CARD), Kumamoto University: CARD Online Manual

What is the expected sperm concentration when frozen sperm are preincubated in CARD FERTIUP® Preincubation Medium: PM (Cat. No. KYD-002-EX) and subsequently used for IVF?
Typically, the sperm concentration in CARD FERTIUP® Cryoprotectant: CPA is 100,000–200,000 sperm/µL. When 10 µL of frozen-thawed sperm suspension is added to 90 µL of CARD FERTIUP® Preincubation Medium: PM, the sperm are diluted tenfold, resulting in a concentration of 10,000–20,000 sperm/µL. Subsequently, adding 10 µL of the preincubated sperm suspension to the IVF medium results in a further tenfold dilution, giving a sperm concentration of 1,000–2,000 sperm/µL in the IVF medium. Assuming that approximately 20% of the sperm remain motile after freezing and thawing, the final concentration of motile sperm in the IVF medium is expected to be 200–400 sperm/µL.

How can I confirm that sperm freezing and thawing were successfully performed using CARD FERTIUP® Cryoprotectant: CPA (Cat. No. KYD-001-EX)?
There are two ways to evaluate the quality of frozen-thawed sperm.
1. For a simple assessment, thaw the sperm and preincubate them in CARD FERTIUP® Preincubation Medium: PM. Sperm quality can then be evaluated based on the number of motile sperm observed around the perimeter of the drop.
2. Alternatively, thaw a portion of the cryopreserved sperm and perform in vitro fertilization (IVF) using unfertilized oocytes. A fertilization rate of approximately 30% should be obtained. To assess sperm motility before cryopreservation, dilute the sperm suspension with CARD mHTF (modified Human Tubal Fluid) (Cat. No. KYD-008-02-EX), PB1, or a similar medium and evaluate sperm motility.