Dynamic genetic architecture and integrative association analysis of milk nitrogen traits in primiparous and multiparous Holstein cows

Milk nitrogen traits are useful proxy phenotypes for characterizing nitrogen metabolism and partitioning in dairy cows. This study analyzed milk urea nitrogen (MUN), milk urea nitrogen yield (MUNY), and estimated milk true protein nitrogen after urea correction (MTPN) in primiparous and multiparous Holstein cows. Phenotypic and genetic analyses included 759,955 test-day records from 59,122 cows, of which 6,461 had genotype data. Linear mixed models were used to compare adjusted phenotypic levels between parity groups, and random regression test-day models were used to estimate dynamic genetic parameters throughout lactation. Subsequently, adjusted phenotypes derived from test-day records were used for single-trait and bivariate genome-wide association studies (GWAS) based on imputed sequence-level variants. Candidate genes within ± 200 kb of significant single nucleotide polymorphisms (SNPs) was subjected to functional enrichment analysis. Furthermore, transcriptome-wide association analyses (TWAS) were conducted for MUN, MUNY, and MTPN using tissue-specific gene-expression prediction models for mammary gland, liver, rumen, and milk cells. The results showed that adjusted MUN was higher in primiparous cows, whereas MUNY and MTPN were higher in multiparous cows. Daily heritability estimates varied across lactation stages. MUN heritability ranged from approximately 0.06 to 0.13, whereas MUNY heritability ranged from 0.08 to 0.15 in primiparous cows and from 0.09 to 0.13 in multiparous cows. MTPN heritability generally increased during mid-to-late lactation, reaching maxima of approximately 0.16 and 0.22 in primiparous and multiparous cows, respectively. Genetic and permanent environmental correlations were generally highest between adjacent test days and declined as the test-day interval increased. Both single-trait and bivariate GWAS identified BTA14 as a major associated region for MUN and MTPN in the two parity-group analyses, whereas no genome-wide significant SNPs were detected for MUNY. Functional enrichment highlighted different biological categories in the two parity groups. The TWAS identified associations involving DGAT1 , EXOSC4 , FBXL6 , CCDC166 , and LYNX1 in multiple tissues, traits, or parity-group analyses. These results indicate that milk nitrogen traits exhibit phenotypic differences between parity groups and show lactation stage-dependent genetic architectures, thereby providing a basis for further validation of the genomic regions and candidate genes associated with milk nitrogen traits.