Kyudo/Card Reagents for Mouse Sperm Cryoprotection,

Thawing and Mouse IVF

CARD FERTIUP® Cryoprotectant : CPA - Reagent used to freeze sperm for storage.
CARD FERTIUP® Preincubation Medium : PM - Reagent used to pre-incubate sperm after thawing and before IVF.
CARD MEDIUM® Mouse Fertilization Medium - Medium used for IVF
CARD FERTIUP® PM + CARD MEDIUM sets -  Conveniently packaged for convenient ordering

CARD FERTIUP® Cryoprotectant : CPA	Available Sizes / Cat. No. (click here for ordering info)
Fertiup® Cryoprotectant is a cryopreservation solution for mouse sperm that reduces sperm damage during freezing.  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 in 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 maintaining it in vivo. It is also used as a preparation for system disconnection and natural disasters due to unexpected accidents. Learn more...	0.5 ml 5 x 0.5 ml 25 x 0.5ml 50 x 0.5ml 1 x 1.0 ml 5 x 1.0 ml 25 x 1.0ml 50 x 1.0ml	KYD-001-05-EX KYD-001-05-EX-X5 KYD-001-05-EX-X25 KYD-001-05-EX-X50 KYD-001-05-EX KYD-001-EX-X5 KYD-001-EX-X25 KYD-001-EX-X50

For Mouse Sperm Thawing and Pre-IVF Culture

CARD FERTIUP® Preincubation Media : PM	Available Sizes / Cat. No. (click here for ordering info)
Fertiup® Cryoprotectant FERTIUP® Mouse Preincubation Medium : Mouse sperm preculture medium. This is a preculture medium that enhances the acquisition of fertility of mouse sperm. When used for in vitro fertilization with C57BL/6 mouse sperm prepared using CARD FERTIUP ® Mouse Sperm Cryopreservation Solution, an in vitro fertilization rate of 30% or more can be expected and efficient mouse fertilized eggs can be obtained. .. It is effective not only for freeze-thaw sperm but also for in vitro fertilization using fresh sperm  significantly increases the fertilizing ability of spermatozoa. Learn more...	0.5 ml 5 x 0.5 ml 25 x 0.5ml 50 x 0.5ml 1 x 1.0 ml 5 x 1.0 ml 25 x 1.0ml 50 x 1.0ml	KYD-002-05-EX KYD-002-05-EX-X5 KYD-001-05-EX-X25 KYD-001-05-EX-X50 KYD-002-05-EX KYD-002-EX-X5 KYD-002-EX-X25 KYD-002-EX-X50

For MOUSE In Vitro Fertilization Procedure

CARD Medium	Available Sizes / Cat. No. (click here for ordering info)
CARD MEDIUM works on the zona pellucida of oocytes to make them easier to fertilize.	Kit Components Ampoule with medium (A): 3 mL Vial of powdered material (B): 30 mg 1.5mL plastic tube (C) 1.5mL plastic tube (D) 2.5mL disposable syringe Needle Filter unit (pore size: 0.22µm)	KYD-003-EX KYD-003-EX-X5 KYD-003-EX-X10

For Mouse: Sperm Thawing and Pre-IVF culture + IVF Procedure

CARD FERTIUP® : PM + CARD MEDIUM® Sets	Available Sizes / Cat. No. (click here for ordering info)
Fertiup® Cryoprotectant is a cryopreservation solution for mouse sperm that reduces sperm damage during freezing. 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 in 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 maintaining it in vivo. It is also used as a preparation for system disconnection and natural disasters due to unexpected accidents.  High performance in vitro fertilization medium This is an in vitro fertilization medium that acts on the zona pellucida of the egg during in vitro fertilization and improves the fertilization rate. Effective for various strains including C57/BL6 strain mouse. After fertilization, we have confirmed in vitro culture tests that normal development occurs from the pronuclear stage to the blastocyst. For in vitro fertilization between C57BL/6 mice , in vitro fertilization rate of 80% or more can be obtained by using CARD FERTIUP® mouse sperm preculture medium together. The mouse sperm cryopreservation and in vitro fertilization system using CARD FERTIUP ® and CARD MEDIUM ® conserves the valuable genetic resources of mice.	1 ml CARD FERTIUP®:PM  + CARD MEDIUM®   0.5 ml CARD FERTIUP® : PM + CARD MEDIUM®	KYD-004-EX KYD-004-EX-X5 KYD-004-EX-X10   KYD-005-EX KYD-005-EX-X5 KYD-005-EX-X10

About CARD FERTIUP® AND CARD MEDIUM®

Pups obtained via in vitro fertilization using cryopreserved sperm treated with CARD FERTIUP^® and CARD MEDIUM^®

CARD FERTIUP® : CPA is a mouse sperm cryoprotectant. CARD FERTIUP® : PM is a mouse sperm preincubation medium for use prior to IVF. CARD MEDIUM®  is a high-performance fertilization medium for the mouse in vitro fertilization procedure. Sperm cryopreservation carried out using CARD FERTIUP® reagents and CARD MEDIUM® allows for low cost and efficient preservation of mouse strains such as Tg or KO mice. These reagents significantly improve in vitro fertilization rates of frozen and thawed sperm and fresh sperm with notoriously IVF-inefficient mouse strains such as C57BL/6 and 129. Use them to preserve, protect and defend the safety and reliability of your valuable mouse resources.

Benefits of using Card FERTIUP and CARD MEDIUM

• 　High fertilization rate (over 80%)
• 　Improvements in the management of genetically modified mice (Tg/KO)
• 　Reduction in labor, facilities, and breeding cost.
• 　Reduction in time required to expand a mouse colony
• 　Efficient production of strains which are notoriously difficult to breed

 Performance Data

 

Fig. 3: Fertilization rates using CARD FERTIUP® Preincubation Medium and CARD MEDIUM® for fresh and frozen sperm in various strains.

 What They Do

CARD FERTIUP® Mouse Cryoprotectant guards against damage to spermatozoa during cryopreservation.	CARD FERTIUP® Mouse Preincubation Medium significantly increases the fertilizing ability of spermatozoa.	CARD MEDIUM® works on the zona pellucida of oocytes to make them easier to fertilize.

Examples of Use

Even strains difficult to obtain via natural crossbreeding can be produced in a short period of time by treating fresh spermatozoa from a male mouse with CARD FERTIUP^® Mouse Preincubation Medium and CARD MEDIUM Medium^®, using the spermatozoa for in vitro fertilization, then transferring the embryos obtained into recipient parents.

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You can also produce mouse pups without having a male mouse present by using cauda epididymis delivered safely and inexpensively to your lab using the CARD Cold Temperature Transport Kit. In this case cauda, it is recommended to incubate cauda epididymis spermatazoa in CARD FERTIUP® Preincubation Medium before in vitro fertilization with CARD Medium with CARD MEDIUM. Embryos so produced are then be transferred a female host. Obtained embryos can then be transferred into a recipient female. 

 

 High fertilization rates can be obtained even in strains known for their low in vitro fertilization rates such as 129 and BALB/c by preincubating spermatozoa in CARD FERTIUP® Mouse Preincubation Medium and CARD MEDIUM® prior to IVF in CARD MEDIUM®.

 Sperm cryopreservation is a practical and cost-effective method of archiving mouse strains in the laboratory. CARD FERTIUP® and CARD MEDIUM® are high-performance media for mouse sperm cryopreservation, sperm preincubation and in vitro fertilization (IVF).

CARD FERTIUP® (mouse sperm cryoprotectant and mouse sperm preincubation medium) and CARD MEDIUM® (high-performance fertilization medium for in vitro fertilization) are high-performance reagents for mouse sperm cryopreservation, mouse sperm thawing (preincubation) and ithe n vitro fertilization (IVF) procedure .Sperm cryopreservation carried out using CARD FERTIUP® and CARD MEDIUM® allows for the low cost and efficient preservation of mouse strains (Tg or KO mice.).

They also significantly improve the in vitro fertilization rates of frozen-thawed sperm and fresh sperm taken from notoriously inefficient mouse strains such as C57BL/6 and 129. This reagent power trio will help you maintain the safety and reliable preservation of your valuable mouse resources.

 When used in combination, CARD FERTIUP® and CARD MEDIUM® provide a high and stable fertilization rate using frozen-thawed mouse sperm with greatly improved fertilization rates for frozen-thawed sperm in C57BL/6 and 129 Mice strains, from less than 10% (using conventional methods) to more than 30%.

Using all three power trio reagents can enhance fertility rates to over 80%.

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CARD FERTIUP® and CARD MEDIUM® Frequently Asked Questions (FAQ)

Q1	We 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. Does the media increase the in vitro fertility rate of other strains too?
A1	When mouse sperm cryopreservation medium/mouse sperm pre-incubation medium is used together, it is effective in ameliorating the fertility rates in many commercially available mouse strains.
Q2	After opening the product, can it be subdivided and frozen?
A2	We cannot guarantee the quality after freezing, so we do not recommend this method of storage.
Q3	Can I use left over CARD FERTIUP^® Mouse Cryoprotectant if I transfer the medium into tubes and store it at refrigerated temperatures?
A3	Once opened, use up the ampoule as quickly as possible. Please purchase an ampoule size which you can use up in one occasion. Please note that the cryoprotectant ampoule should be stored at room temperature as it contains high doses of saccharides and skim milk, which may sink to the bottom of the ampoule after refrigerating or freezing.
Q4	There are crystals/precipitants in my un-opened CARD FERTIUP^® Mouse Cryoprotectant.
A4	In rare cases, crystals may precipitate due to changes in ambient temperature (low temperature). Incubate at 37°C for 30 minutes to 1 hour, and stir to melt the crystals. There is no problem with cryopreservation performance after thawing.
Q5	Liquid paraffin is used in the usage instructions for the CARD FERTIUP^® series; doesn’t the paraffin have adverse effects on sperm?
A5	Liquid paraffin does not adversely affect sperm suspension (sperm suspended in storage solution).
Q6	After preincubating cryopreserved sperm with CARD FERTIUP® Mouse Preincubation Medium, what is the sperm concentration when carrying out in vitro fertilization using an IVF medium?
A6	Typically, the sperm concentration in cryopreserved sperm ranges from 100,000~200,000 sperm/µL. When introducing freeze-thawed sperm suspension (10 µL) into 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.
Q7	I would like to download the instruction manual for the CARD FERTIUP® series. Where can I download them from?
A7	They are available on our website,  https://www.cosmobiousa.com/content/document/cosmo-bio-ltd/10167_KYD_CARDmanual_english.pdf
Q8	Is there a method to confirm whether the freezing and thawing of sperm has been correctly carried out?
A8	1. To confirm the quality of sperm after freeze-thawing, preincubate the sperm in preincubation medium after freezing and judge according to the number of active motile sperm at the edge of the drop. 2. For final confirmation, carry out in vitro fertilization of unfertilized oocytes and confirm that you have attained at least a 30% fertility rate. When checking the motility of the sperm before freezing, dilute the suspension of sperm in 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)

Q9	How about legacy stock of cryopreserved spermatozoa?
A9	Please Check here. Rescue In Vitro Fertilization Method for Legacy Stock of Cryopreserved Spermatozoa
Q10	In vitro fertilization does not work as well as we thought. Sperm and eggs have no problem but the embryos died the next day. What kind of problems can be considered?
A10	The quality of liquid paraffin could be a problem that effects in vitro fertilization rates, and arresting embryogenesis . Moreover, liquid paraffin gradually degrades after opened. Please use as promptly as possible after opening. 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 the manufacture date is CARD MEDIUM® warranted for?
A1	The warrantee period for CARD MEDIUM® 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 ahigh in vitro fertility rate without using CARD MEDIUM® with CARD FERTIUP® series?
A2	No. The CARD FERTIUP® series works with sperm and CARD MEDIUM® with oocytes  to raise the fertility rate. Use both products simultaneously to attain a fertility rate of 80% or more even with C57BL/6 strain mouse.
Q3	Can high in vitro fertilization rates be obtained with strains other than C57BL/6 mice?
A3	Yes, high in vitro fertilization rates can be obtained in various mouse strains. [Reference link]DOI: 10.1016/j.theriogenology.2016.04.076 (Link: https://www.sciencedirect.com/science/article/abs/pii/S0093691X16301194?via%3Dihub)
Q4	In vitro fertilization was performed with CARD MEDIUM ® , and embryonic development beyond the 2-cell stage did not progress well. The medium used for in vitro culture is KSOM medium or Whitten medium. What problems could have slow down the development of the embryo?
A4	The following problems are possible. 1. The incubator temperature and carbon dioxide gas concentration 2. The in vitro culture medium; If prepared by yourself, there may be problems with the distilled water or ingredients. Please consider using "CARD KSOM". 3. The mineral oil that coats the medium: If old, the embryo development may be inhibited.

 

	Questions regarding the CARD FERTIUP^® 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 preservative), 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, preincubate the sperm in preincubation 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, carry out 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 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 degrades after opening. Please use as promptly as possible after opening. 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.

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Key Literature References

• Cryopreservation of mouse spermatozoa and in vitro fertilization.
  Nakagata N.
  Methods Mol Biol. 2011;693:57-73. PMID: 21080274
• Combination medium of cryoprotective agents containing L-glutamine and methyl-{beta}-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm.
  Takeo T, Nakagata N.
  Lab Anim. 2010 Apr;44(2):132-7. PMID: 20357317
• Short-term storage and transport at cold temperatures of 2-cell mouse embryos produced by cryopreserved sperm.
  Takeo T, Kondo T, Haruguchi Y, Fukumoto K, Nakagawa Y, Takeshita Y, Nakamuta Y, Tsuchiyama S, Shimizu N, Hasegawa T, Goto M, Miyachi H, Anzai M, Fujikawa R, Nomaru K, Kaneko T, Itagaki Y, Nakagata N.
  J Am Assoc Lab Anim Sci. 2010 Jul;49(4):415-9. PMID: 20819385
• Birth of mice from vitrified/warmed 2-cell embryos transported at a cold temperature.
  Takeo T, Kaneko T, Haruguchi Y, Fukumoto K, Machida H, Koga M, Nakagawa Y, Takeshita Y, Matsuguma T, Tsuchiyama S, Shimizu N, Hasegawa T, Goto M, Miyachi H, Anzai M, Nakatsukasa E, Nomaru K, Nakagata N.
  Cryobiology. 2009 Apr;58(2):196-202. PMID: 19168045
• Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells.
  Takeo T, Hoshii T, Kondo Y, Toyodome H, Arima H, Yamamura K, Irie T, Nakagata N.
  Biol Reprod. 2008 Mar;78(3):546-51. PMID: 18046011

ALL PRODUCTS ON THIS PAGE ARE TO BE USED FOR RODENT RESEARCH ONLY, NOT FOR USE IN HUMAN THERAPY OR CLINICAL DIAGNOSIS