Reproductive Engineering Reagents and Equipment

for RAT from Kyudo/CARD

CARD FERTIUP® Rat Sperm Cryopreservation Agent - high performance reagent for rat sperm cryopreservation.
CARD Rat Sperm Freezing Kit - hardware set to promote proper sperm freezing. CARD Rat IVF Medium - high performance reagent for rat IVF.
 

As a result of joint research carried out by Kyudo Co., Ltd. and Kumamoto University, new high performance reagents for use in RAT reproductive engineering are now available worldwide.

Rats are widely used in medical and life science research. In recent years, various strains of genetically modified rat have been produced worldwide thanks to the development of genome editing technologies. Moreover, it is predicted that even more genetically modified rats will be produced in the future. Therefore, there is a pressing need for effective preservation techniques for these genetically modified rat strains.

Through sperm freezing, it is possible to quickly cryopreserve a large number of sperm taken from the cauda epididymis of a male rat. For this reason, sperm cryopreservation is expected to be an easier, more effective and most economically viable technique for the preservation of genetically modified rat strains than embryo cryopreservation. However, rat sperm is vulnerable to the effects of physical changes to pH, osmotic pressure, temperature and other similar factors. Moreover, it is difficult to maintain the motility of frozen-thawed sperm via conventional sperm cryopreservation methods, which means that it is not viable to use these sperm in in vitro fertilization (IVF).

Therefore, the establishment of rat sperm cryopreservation and rat IVF techniques was required to solve these issues. A research team led by Dr. Naomi Nakagata and Dr. Toru Takeo at the Institute of Resource Development and Analysis, Kumamoto University have developed a practical rat sperm cryopreservation technique and an IVF method using freeze-thawed rat sperm. And now, as a result of joint research and development carried out by Kyudo Co., Ltd. and Kumamoto University, these new reagents for use in rat reproductive engineering are available to laboratories around the world. 

Together, the reagents:

• Enables IVF to be carried out using frozen-thawed rat sperm.
• Contribute to a reduction in breeding costs through improvements in rat strain preservation efficiency.
• Facilitates the timely acquisition of live rats when they are required for experiments through application to genetically modified rats. 

Performance Characteristics

Rat sperm cryopreservation using CARD FERTIUP® Rat Sperm Cryopreservation Agent and rat IVF carried out using frozen-thawed sperm are revolutionary techniques which have enabled the production of high quality frozen rat sperm and the acquisition of high IVF efficiency rates using frozen-thawed rat sperm at a practical level for the very first time. The fertilized oocytes obtained via IVF develop well, and after transferring embryos to a female rat, one can efficiently acquire genetically modified rat pups (see Reference Data).

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Reference Data 

Table 1 and Figure 1. IVF fertilization rates using fresh and frozen sperm.

Sperm	No.	No. inseminated oocytes	No. fertilized blastocysts	Fertilization Rate %
Fresh	1	198	185	93.4
	2	183	170	92.9
	3	195	181	92.8
	4	182	174	95.6
	Total	758	710	93.7±1.34
Frozen	1	161	135	83.9
	2	191	160	83.8
	3	201	161	80.1
	4	196	159	81.1
	Total	749	615	82.1±1.9

Table 2. Rate of development of fertilized oocytes derived from frozen-thawed rat sperm to the blastocyst stage, and birth rate of said fertilized oocytes after oocyte transfer.

Sperm	No.	No. of 2-cell embryos	No. of blastocysts	Developmental rate(%)	Fertilized Oocytes Transferred	Number of Pups	Birth Rate %
Fresh	1	100	58	58	20	13	65.0
	2	100	63	63	20	12	60.0
	3	100	65	65	20	13	65.0
	4	100	71	71	20	10	50.0
	Total	400	257	64.3±5.4	80	48	60±7.1
Frozen	1	100	72	72	20	12	60.0
	2	100	57	57	20	12	60.0
	3	100	64	64	20	10	50.0
	4	100	53	53	20	12	60.0
	Total	400	246	61.5±5.4	80	46	57.5±5.0

Data from: Naomi Nakagata, Nobuyuki Mikoda, Satohiro Nakao, Ena Nakatsukasa & Toru Takeo, Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats. SCIENTIFIC REPORTS, 10:93, 2020 

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Ordering Information  

For Rat Sperm Vitrification

Product Name Information	Catalog Number	Size
CARD FERTIUP® Rat Sperm Cryopreservation Agent  is a newly-developed agent which enables researchers to efficiently carry out cryopreservation of rat sperm, which until now has been notoriously difficult to cryopreserve. Conventional rat sperm cryopreservation methods are unable to effectively maintain the motility of frozen-thawed sperm. On the other hand, rat sperm cryopreservation carried out using CARD FERTIUP® Rat Sperm Cryopreservation Agent yields superior motility even in frozen-thawed rat sperm, allowing for the production of many rat pups via IVF and embryo transfer.	KYD-FR-001	1 x 1ml
	KYD-FR-001-X5	5 ml
	KYD-FR-001-X10	5 x 1ml

Product Name Information	Catalog Number	Size
CARD Rat IVF Medium is a new IVF medium developed specifically for use in rat IVF.	KYD-FR-002	5 ml
	KYD-FR-002-X5	10 x 1ml
	KYD-FR-002-X10	5 ml

Product Name Information	Catalog Number	Size
CARD Rat Freezing Kit The CARD Rat Sperm Freezing Kit is hardware accessory that facilitates the production of high-quality frozen rat sperm during cryopreservation with CARD FERTIUP® Rat Sperm Cryopreservation Agent. The kit contains a sperm straw holding plate and a foam float for the holding plate. Together, they permit sperm-filled sperm straws to be floated just above the liquid in a container of liquid nitrogen, in order to control the freezing rate prior to full immersion in a liquid nitrogen tank. Similarly, the tray and float can also be used to float sperm straws above liquid nitrogen during the rat sperm thawing procedure.	KYD-GAR-001	1 x 1 ml

For MOUSE and RAT In Vitro Fertilization Procedure

Product Name Information	Catalog Number	Size
CARD mHTF Medium Improved in vitro fertilization rate and development rate in mouse and rat reproductive engineering. National University Corporation Kumamoto University Life Resources Research and Support Center Recommended Resources Development Area	KYD-008-02-EX	1 x 2 ml
	KYD-008-02-EX-X5	5 x 2 ml
	KYD-008-02-EX-X25	25 x 2 ml
	KYD-008-02-EX-X50	50 x 2 ml
	KYD-008-05-EX	1 x 5 ml
	KYD-008-05-EX-X3	3 x 5 ml
	KYD-008-05-EX-X15	15 x 5 ml
	KYD-008-05-EX-X30	30 x 5 ml

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CARD FERTIUP® Rat Sperm Cryopreservation Agent and CARD Rat IVF MEDIUM Frequently Asked Questions (FAQ) 

	General Questions regarding CARD FERTIUP^® Rat Sperm Cryopreservation Agent
Q1	Q1. How should I store CARD FERTIUP® Rat Sperm Cryopreservation Agent? By when should I use it?
A1	1. Please store CARD FERTIUP® Rat Sperm Cryopreservation Agent in a freezer at -20°C or below. 2. The best before end of CARD FERTIUP® Rat Sperm Cryopreservation Agent is two years (24 months) from the date of manufacture. Please use it before the best before end date.
Q2	After opening a vial of CARD FERTIUP® Rat Sperm Cryopreservation Agent, can I separate out the contents into smaller portions and use it for cryopreservation?
A2	No. CARD FERTIUP® Rat Sperm Cryopreservation Agent is sold in 1.5mL measures, and one vial (1.5mL) is to be used per rat cauda epididymis. Please follow the usage instructions and use one vial (1.5mL) of CARD FERTIUP® Rat Sperm Cryopreservation Agent per rat cauda epididymis.
Q3	Does CARD FERTIUP® Rat Sperm Cryopreservation Agent contain any ingredients which are harmful to humans?
A3	No. It contains no ingredients which are harmful to humans.
Q4	There appears to be a foreign object in my vial of CARD FERTIUP® Rat Sperm Cryopreservation Agent. Can you replace the vial with a new one?
A4	Kyudo carries out thorough quality control from manufacture through to shipment; however, if a foreign object has contaminated your vial of CARD FERTIUP® Rat Sperm Cryopreservation Agent, we would be happy to replace your vial with a new one.
	General Questions regarding CARD Rat IVF Medium
Q1	Q1. How should I storeCARD Rat IVF medium? By when should I use it?
A1	1. Please store CARD Rat IVF medium in a refrigerator at 4°C. 2. The best before end of CARD Rat IVF medium is 1.5 years (18 months) from the date of manufacture. Please use it before the best before end date.
Q2	After opening a vial of CARD Rat IVF medium, can I separate out the contents into smaller portions and use it for cryopreservation?
A2	No. Kyudo cannot guarantee the quality of the sperm after freezing, so we do not recommend this method.
Q3	Does CARD Rat IVF medium contain any ingredients which are harmful to humans?
A3	No. It contains no ingredients which are harmful to humans.
Q4	There appears to be a foreign object in my vial of CARD Rat IVF medium. Can you replace the vial with a new one?
A4	Kyudo carries out thorough quality control from manufacture through to shipment; however, if a foreign object has contaminated your vial of CARD Rat IVF medium, we would be happy to replace your vial with a new one.