CARD FERTIUP® PM 1 mL - CARD MEDIUM® Set

Kyudo

Catalog No.: KYD-004-EX

Refer to the Reproductive Engineering Techniques in Mice Technical Manual for complete instructions of IVF

Product Description

This convenient double medium set comprises CARD FERTIUP® PM and CARD MEDIUM® that when used together synergize to elevate fertilization rates in mouse IVF. The former is a sperm fertility-enhancing preculture medium. The latter is a novel fertilization-promoting culture medium that acts during IVF on the mouse egg zona pellucida.

CARD FERTIUP® Preincubation Medium: PM is a preculture medium that enhances mouse sperm fertility. Used in IVF with thawed C57BL/6 mouse sperm frozen using CARD FERTIUP® Cryoprotectant: CPA, fertilization rates of 30% or more can be expected. CARD FERTIUP® Preincubation Medium: PM also effectively promotes IVF with fresh sperm.

CARD MEDIUM® is a novel culture medium that acts during IVF on the mouse egg zona pellucida to promote high fertilization rates, especially when used in conjunction with CARD FERTIUP® Preincubation Medium: PM. CARD MEDIUM® was developed by The Center for Animal Resources and Development (CARD), Kumamoto University, Japan.

Combined use of FERTIUP® sperm cryoprotectant and CARD FERTIUP® Preincubation Medium: PM greatly improves the fertilization rate of frozen-thawed sperm in C57BL/6 and 129 strains, from less than 10% using conventional methods to more than 30%. Further, when coupled with our new CARD MEDIUM®, fertility rates are boosted to over 80%. Thus, combined usage of the trio of CARD FERTIUP® Cryoprotectant: CPA, CARD FERTIUP® Preincubation Medium: PM and CARD MEDIUM® provides a high and consistent rate of fertilization from frozen-thawed mouse sperm, which will allow you to efficiently recover mouse lines when needed.

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® PM 1 mL - CARD MEDIUM® Set 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.

Can I achieve high IVF rates without using the CARD FERTIUP series together with CARD MEDIUM® (Cat. No. KYD-003-EX)?
The CARD FERTIUP series acts on sperm, while CARD MEDIUM® acts on oocytes to improve fertilization rates. Therefore, using CARD MEDIUM® together with the CARD FERTIUP series can achieve IVF rates of over 80% even in C57BL/6 mice.

Can I achieve high IVF rates in mouse strains other than C57BL/6 using CARD MEDIUM® (Cat. No. KYD-003-EX)?
Yes. High IVF rates have been achieved in a variety of mouse strains when CARD MEDIUM® is used in combination with the CARD FERTIUP series.
Reference:
Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains
Takeo T, Nakagata N. Theriogenology. 2016;86(5):1341-1346. doi:10.1016/j.theriogenology.2016.04.076.

I found that many sperm were attached to oocytes when performing IVF using CARD MEDIUM® (Cat. No. KYD-003-EX) with fresh mouse sperm. Is there a risk of polyspermy?
Mouse oocytes have two mechanisms that prevent multiple sperm from entering the cytoplasm. Even when many sperm attach to the zona pellucida surrounding the oocyte, the zona reaction prevents additional sperm from penetrating the zona pellucida. Furthermore, even if a sperm passes through the zona pellucida, the oocyte membrane provides an additional block to sperm entry. Therefore, it is very rare for more than one sperm to enter the oocyte cytoplasm (polyspermy). In particular, the incidence of polyspermy in C57BL/6 mice is extremely low, approximately 0.2–0.3%, and is not considered a concern.

I performed IVF using CARD MEDIUM® (Cat. No. KYD-003-EX), but embryo development did not progress properly beyond the 2-cell stage. The embryos were cultured in KSOM medium or Whitten medium. What could be the cause?
Several factors may contribute to poor embryo development:
1. The incubator temperature or CO₂ concentration may not be optimal.
2. If a homemade embryo culture medium is being used, the distilled water or medium components may be affecting embryo development. Consider using a commercially available medium such as CARD KSOM (Cat. No. KYD-011-EX).
3. If the mineral oil overlaid on the culture medium is old, embryo development may be impaired.