Elevated incidence of freemartinism in pigs detected by droplet digital PCR and cytogenetic techniques

Freemartinism is a disorder of sex development (DSD) caused by the formation of placental anastomoses between fetuses of different sex. It leads to development of an abnormal reproductive tract in the female fetus, including the presence of testes or ovotestes. In pig production, such conditions can affect both reproductive performance and meat quality, through undesired boar taint. A common approach to diagnosing freemartinism is to detect leukocyte chimerism (XX/XY). In the study described here, we performed a complex evaluation of 28 DSD pigs with ambiguous external genitalia (enlarged clitoris, presence of scrotum, etc.). Sex chromosomes were cytogenetically identified by Giemsa staining, as well as with fluorescence in situ hybridization (FISH), while Y-linked (ZFY) and X-linked (ZFX) genes were detected by PCR-RFLP. Histological analysis of the gonads was performed on eosin-hematoxylin-stained microscope slides. Chromosome studies revealed that, of 28 DSD animals, 20 were freemartins (38,XX/38,XY), 2 had a female karyotype (38,XX), and 6 had a male karyotype (38,XY). Analysis of breeding data showed that the mean litter size in which DSD animals occurred was about 17. Histology of the gonads could be performed for 8 DSD pigs, including 6 freemartins and 2 XX DSD. In all these cases, testicular tissue without signs of spermatogenesis was seen. In XX DSD pigs, a copy number variation (CNV) polymorphism downstream from SOX9, tentatively associated with this type of DSD, was detected by FISH. On the other hand, the background of the XY DSD remains unknown. Since cytogenetic analysis is laborintensive and time-consuming, we evaluated the usefulness of the droplet digital PCR (ddPCR) approach to diagnosing XX/XY leukocyte chimerism. Estimation of X and Y chromosome copy numbers was based on the quantitative detection of AMELX and AMELY genes. Different sex chromosome sets (XX, XY and XX/XY) were unequivocally identified by ddPCR. In conclusion, we suggest that freemartinism, beside testicular or ovotesticular XX DSD, is an emerging problem in highly prolific pigs and that detection of XX/XY leukocyte chimerism can be performed using a fast and reliable ddPCR approach.