Metabolomics Reveals Abnormal Citrate Cycle and Phenylalanine Metabolism in Testes from Infertile Hybrid Dzo

This study investigated the metabolomic profiles and molecular basis of hybrid male sterility (HMS) in dzo (the male F1 hybrid offspring of taurine cattle (<i>Bos taurus</i>, ♂) × domestic yak (<i>Bos grunniens</i>, ♀)). In total, 147 co-different metabolites were identified between liver and testis tissues. Metabolomics analysis linked testis-specific abnormal citrate cycle and phenylalanine metabolism to dzo male infertility. Specifically, α-ketoglutaric acid, L-malic acid, and succinic acid were specific elevated in dzo testes, but not significantly different in liver. The testis-specific metabolite phenyllactate was reduced in dzo. Moreover, genes encoding α-ketoglutarate-dependent oxygenases were dysregulated only in dzo testes, including histone demethylations and RNA m⁶A modifications. Reactive oxygen species and m⁶A content were significantly decreased in dzo testes. Multiomics data showed that testis-specific metabolic abnormalities in dzo were linked to upregulated <i>IDH3A</i> and <i>IDH3G</i>, and downregulated testis-specific <i>OGDHL</i> and <i>PDHA2</i>. MiRNA-15b targeting to <i>IDH3A</i> was downregulated in dzo testes. The promoter of <i>PDHA2</i> was hypermethylated and showed lower chromatin accessibility in dzo testes. Notably, testis-specific <i>LDHC</i> downregulation was also associated with lower phenyllactate in dzo testes, which could be an outcome of male infertility. Overall, this study provides comprehensive insights into the citrate cycle as a key pathway associated with dzo sterility, shedding light on the potential mitochondrial-nuclear incompatibility pertinent to addressing this HMS challenge.