Validation of Using Multiplex PCR with Sex Markers SSM4 and ALLWSex2 in Long-Term Stored Blood Samples to Determine Sex of the North American Shortnose Sturgeon (Acipenser brevirostrum)

Sex-specific information is crucial for sturgeon culture, conservation, and fisheries management. However, identifying their sex is difficult outside the spawning season. Two recently identified female-specific loci (AllWSex2 and SSM4) are conserved across many Acipenserid species, but they have not been validated for all species within this family. This study aimed to (1) determine whether SSM4 can be used to sex shortnose sturgeon, (2) develop and test a multiplex PCR technique using both ALLWSex2 and SSM4 for sexing shortnose sturgeon, (3) determine if long-term stored blood samples can be used to sex shortnose sturgeon, and (4) test the effect of storage temperature on DNA degradation. DNA was extracted from frozen RBC samples from 36 previously sexed fish. A multiplex PCR was set up using three pairs of primers: AllWSex2 and SSM4, as female-specific loci, and mtDNA, as an internal control. AllWSex2 and SSM4 allowed for perfect discrimination of sex. While long-term storage and storage temperature did cause DNA degradation, the signal was still strong enough after 8 years of cold storage for reliable sex determination. This suggests that researchers now have the ability to re-examine archived/frozen samples to determine the sex of their sturgeon.