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This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982). We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity. First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes. The rates of sperm freezing and thawing (δ°C/time) are probably the two most critical variables to control in this procedure. For this reason, do not substitute different tubes for those specified. Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs. Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.
NOTE: ORDER OF ADDITION IS IMPORTANT TO PREVENT PRECIPITATION
In 450 ml sterile ddH2O dissolve:
Bring final volume to 500 ml with sterile ddH2O and store at 4°C.
Materials needed for sperm freezing
1 A simple method for making finely crushed dry ice is to wrap it in a towel and pulverize it with a hammer. A more efficient method is to use a dry ice grinder, e.g. Clawson model RE-2.
We find that this works best with two people. One person squeezes eggs from females while the other person thaws the sperm.
We used this crypreservation methodology to make a library of 8600 frozen sperm samples for TILLING between the years 2001 and 2003. We have thawed 106 of these samples and determined their fertility. Fertility is the ratio of viable embryos produced in an in vitro fertilization divided by the total number of eggs that were fertilized with the frozen sperm. Fresh (non cryporeserved) sperm typically has >95% fertility; cryopreserved sperm has much lower fertility: an average of 29% (n=106 vials). The vial-to-vial variability in fertility is large, ranging from less than 1% to as high as 62%. This variability is represented by the large error bars representing standard deviation in Fig. 2. However in 106 sperm samples thawed to date we have only once experienced 0% fertility in both sperm samples and failed to recover the corresponding TILLING mutation.
By thawing sperm vials from our library over the course of several years (2001-2008) we have found that the average fertility of sperm samples frozen by this method remains stable over time (Fig. 2). The average fertility of frozen sperm samples thawed in 2001 was 23.5% (based on 11 sperm samples) and in 2008 was 25% (based on 21 sperm samples).
A number of other investigators are now using this sperm freezing protocol in their labs. Some have reported good success with fertility rates and sample-to-sample variability similar to what we have observed. Others have reportedly failed to make this protocol work in their hands. We believe that likely sources of problems are:
Figure 1. Schematic of sperm freezing methodology.
Figure 2. Cryopreserved sperm fertility remains stable over time. Percent fertility is the number of viable embryos produced in an in vitro fertilization divided by the total number of eggs that were fertilized with the frozen sperm. "n" refers to the number of cryopreserved sperm vials whose fertility was tested in the corresponding year. Error bars represent standard deviation. Since each sperm vial is from a different male, vial-to-vial variability is observed.
A number of other investigators are now using this sperm freezing protocol in their labs. Some have reported similar fertility and the same sample-to-sample variability that we have observed. Others have reportedly failed to make this protocol work in their hands. We believe that likely sources of problems are:
This work was supported by NIH R01 HG002995 "TILLING the Zebrafish Genome: a Reverse Genetics Approach" to CBM.