Multi-tissue gene expression profiling of cows with a genetic predisposition for low and high milk urea levels.

Milk urea (MU) concentration is proposed as an indicator trait for breeding toward reduced nitrogen (N) emissions and leaching in dairy. We selected 20 German Holstein cows based on MU breeding values, with 10 cows each having low (LMUg) and high (HMUg) MU genetic predisposition. Using RNA-seq, we characterized these cows to unravel molecular pathways governing post-absorptive body N pools focusing on renal filtration and reabsorption of nitrogenous compounds, hepatic urea formation and mammary gland N excretion. While we observed minor adjustments in cellular energy metabolism in different tissues associated with different MU levels, no transcriptional differences in liver ammonia detoxification were detected, despite significant differences in MU between the groups. Differential expression of AQP3 and SLC38A2 in the kidney provides evidence for higher urea concentration in the collecting duct of LMU cows than HMU cows. The mammary gland exhibited the most significant differences, particularly in tricarboxylic acid (TCA) cycle genes, amino acid transport, tRNA binding, and casein synthesis. These findings suggest that selecting for lower MU could lead to altered urinary urea (UU) handling and changes in milk protein synthesis. However, given the genetic variability in N metabolism components, the long-term effectiveness of MU-based selection in reducing N emissions remains uncertain.

Microbial signature inferred from genomic breeding selection on milk urea concentration and its relation to proxies of nitrogen-utilization efficiency in Holsteins.

Increasing the nitrogen-utilization efficiency (NUE) of dairy cows by breeding selection would offer advantages from nutritional, environmental, and economic perspectives. Because data collection of NUE phenotypes is not feasible in large cow cohorts, the cow individual milk urea concentration (MU) has been suggested as an indicator trait. Considering the symbiotic interplay between dairy cows and their rumen microbiome, individual MU was thought to be influenced by host genetics and by the rumen microbiome, the latter in turn being partly attributed to host genetics. To enhance our knowledge of MU as an indicator trait for NUE, we aimed to identify differential abundant rumen microbial genera between Holstein cows with divergent genomic breeding values for MU (GBVMU; GBVHMU vs. GBVLMU, where H and L indicate high and low MU phenotypes, respectively). The microbial genera identified were further investigated for their correlations with MU and 7 additional NUE-associated traits in urine, milk, and feces in 358 lactating Holsteins. Statistical analysis of microbial 16S rRNA amplicon sequencing data revealed significantly higher abundances of the ureolytic genus Succinivibrionaceae UCG-002 in GBVLMU cows, whereas GBVHMU animals hosted higher abundances of Clostridia unclassified and Desulfovibrio. The entire discriminating ruminal signature of 24 microbial taxa included a further 3 genera of the Lachnospiraceae family that revealed significant correlations to MU values and were therefore proposed as considerable players in the GBVMU-microbiome-MU axis. The significant correlations of Prevotellaceae UCG-003, Anaerovibrio, Blautia, and Butyrivibrio abundances with MU measurements, milk nitrogen, and N content in feces suggested their contribution to genetically determined N-utilization in Holstein cows. The microbial genera identified might be considered for future breeding programs to enhance NUE in dairy herds.

Ruminal background of predisposed milk urea (MU) concentration in Holsteins.

Efforts to reduce nitrogen (N) emissions are currently based on the optimization of dietary- N supply at average herd N requirements. The implementation of the considerable individual differences and predispositions in N- use efficiency and N- excretion in breeding programs is hampered by the difficulty of data collection. Cow individual milk urea (MU) concentration has been proposed as an easy-to-measure surrogate trait, but recent studies questioned its predictive power. Therefore, a deeper understanding of the biological mechanisms underlying predisposed higher (HMUg) or lower (LMUg) MU concentration in dairy cows is needed. Considering the complex N- metabolism in ruminants, the distinction between HMUg and LMUg could be based on differences in (i) the rumen microbial community, (ii) the host-specific transcription processes in the rumen villi, and (iii) the host-microbe interaction in the rumen. Therefore, rumen fluid and rumen epithelial samples from 10 HMUg and 10 LMUg cows were analyzed by 16S sequencing and HiSeq sequencing. In addition, the effect of dietary-N reduction on ruminal shifts was investigated in a second step. In total, 10 differentially abundant genera (DAG) were identified between HMUg and LMUg cows, elucidating greater abundances of ureolytic Succinivibrionaceae_UCG-002 and Ruminococcaceae_unclassified in LMUg animals and enhanced occurrences of Butyvibrio in HMUg cows. Differential expression analysis revealed genes of the bovine Major Histocompatibility Complex (BOLA genes) as well as MX1, ISG15, and PRSS2 displaying candidates of MU predisposition that further attributed to enhanced immune system activities in LMUg cows. A number of significant correlations between microbial genera and host transcript abundances were uncovered, including strikingly positive correlations of BOLA-DRA transcripts with Roseburia and Lachnospiraceae family abundances that might constitute particularly prominent microbial-host interplays of MU predisposition. The reduction of feed-N was followed by 18 DAG in HMUg and 19 DAG in LMUg, depicting pronounced interest on Shuttleworthia, which displayed controversial adaption in HMUg and LMUg cows. Lowering feed-N further elicited massive downregulation of immune response and energy metabolism pathways in LMUg. Considering breeding selection strategies, this study attributed information content to MU about predisposed ruminal N-utilization in Holstein-Friesians.

Identification of Genomic Regions Influencing N-Metabolism and N-Excretion in Lactating Holstein- Friesians.

Excreted nitrogen (N) of dairy cows contribute to environmental eutrophication. The main N-excretory metabolite of dairy cows is urea, which is synthesized as a result of N-metabolization in the liver and is excreted via milk and urine. Genetic variation in milk urea (MU) has been postulated but the complex physiology behind the trait as well as the tremendous diversity of processes regulating the N-metabolism impede the consistent determination of causal regions in the bovine genome. In order to map the genetic determinants affecting N-excretion, MU and eight other N-excretory metabolites in milk and urine were assessed in a genome-wide association study. Therefore phenotypes of 371 Holstein- Friesians were obtained in a trial on a dairy farm under near commercial conditions. Genotype data comprised SNP information of the Bovine 50K MD Genome chip (45,613 SNPs). Significantly associated genomic regions for MU concentration revealed GJA1 (BTA 9), RXFP1, and FRY1 (both BTA 12) as putative candidates. For milk urea yield (MUY) a promising QTL on BTA 17 including SH3D19 emerged, whereas RCAN2, CLIC5, ENPP4, and ENPP5 (BTA 23) are suggested to influence urinary urea concentration. Minor N-fractions in milk (MN) may be regulated by ELF2 and SLC7A11 (BTA 17), whilst ITPR2 and MYBPC1 (BTA 5), STIM2 (BTA 6), SGCD (BTA 7), SLC6A2 (BTA 18), TMCC2 and MFSD4A (BTA 16) are suggested to have an impact on various non-urea-N (NUN) fractions excreted via urine. Our results highlight genomic regions and candidate genes for N-excretory metabolites and provide a deeper insight into the predisposed component to regulate the N-metabolism in dairy cows.