Sperm Cryopreservation

To initiate a project, visit the appropriate option link below and follow the instructions. Services for each option are detailed under the Quality Control of Cryopreserved Material section. 

• Option 1
• Option 2
• Option 3 

Please read carefully the instructions below. Due to the volume of requests and the related communications we experienced recently, our instructions here and on NUcore have been updated and edited to include current and very detailed steps. TTML staff will not respond to emails. If they do, you will be directed to this website or NUcore.

For all other questions, please call 312-503-0773. We will respond as our work allows.

Steps:

1. As soon as the order is placed, TTML will review and approve the order.
2. Upon approval, TTML staff will contact you to schedule cryopreservation. You will be required to confirm the name of the line(s) and the time/date of the scheduled cryopreservation. Please refrain from emailing the core staff.
3. Two males, ideally between 3-5 months of age, although older males are acceptable, for each line should be identified for cryopreservation. Please refrain from submitting orders until your mice are at the correct age. TTML will cancel your order without notice if the mice are younger than 12 weeks.
4. On the day of the cryopreservation, submit an AOPS request to transfer the males to the TTML animal protocol (we will walk you through this).
5. Immediately after AOPS, bring the cage of males to our lab in Simpson Querrey (B-700) in a fresh cage with food and a water bottle at the scheduled time.
6. Failure to bring the cages on time without notice (15-minute grace period), will result in the cancellation of the appointment and charge of the full fee.
7. Sperm is then isolated from both males, pooled and frozen into 20 straws.
8. A test straw of cryopreserved sperm is thawed from each frozen line and QC performed to determine the quality of the sperm.
9. Upon confirming the successful process, TTML will contact you to confirm that your colony can be culled.

A minimum of 17-20 straws of frozen sperm is generated for each line. A test straw from each freeze is thawed to determine the success of the procedure.

As of September 1, 2024, our core offers three options for sperm cryopreservation. The recommended method is Option 1, unless there are specific reasons for caution that should be discussed with the core prior to freezing.

Regardless of the option selected, it is critical that you are confident in the genotype of the provided males.

Option 1: 

This service includes the thawing of a straw of sperm and assessing its viability, motility and mobility. These parameters are generally highly correlative with the ability of the cryopreserved sperm to generate pups should recovery of the line be requested in the future. This option is a commonly used method followed by many nationally top ranked cores and it allows projects to move more quickly through the facility so that investigators can cull colonies sooner.

Option 2:

This service includes testing the fertilization capacity of the sperm after freezing. In vitro Fertilization (IVF) is performed with thawed sperm using WT B6 oocytes, and the percent fertilization is used to determine the fertilization capacity of the sperm. Percent fertilization (the percentage of fertilized two-cell stage embryos generated during IVF) and development of IVF-generated embryos to blastocyst stage are higher-level indicators that the line can be recovered successfully, however, as above, they don’t guarantee a successful recovery.

Option 3:

This service includes IVF as in Option 2 and the surgical transplantation of blastocyst stage embryos into the uteri of pseudopregnant recipients. Tails from the resulting offspring are provided to the lab for genotyping to confirm that the desired mutation can be obtained after cryopreservation. This test provides an even higher level of confidence, however as above the core doesn’t provide any guarantee. This is the most time-consuming and costly option and the need for it must be discussed and approved by the core director.

Cryopreserved sperm from each line is stored in two separate liquid nitrogen dewars in two different buildings.

There is a modest annual fee for each line maintained in our dewars.

Sperm can be cryorecovered at any time simply by contacting our lab.

Sperm can also be shipped worldwide from our lab.