Xinjiang Brown cattle are a unique and widely distributed breed of dual-purpose cattle in the Xinjiang area of China, whose milk production performance differs from Holstein cattle. It has been known that variations in bacterial species of the gastrointestinal tract influence milk protein, fat, and lactose synthesis. However, the microbiota differences between Xinjiang Brown and Holstein cattle are less known. This study aims to compare the bacterial community composition of the rumen and feces of these two cattle breeds under the same dietary and management conditions. The 16s rRNA sequencing data and milk production of 18 Xinjiang Brown cows and 20 Holstein cows on the same farm were obtained for analysis. The results confirmed differences in milk production between Xinjiang Brown and Holstein cattle. Microbiota with different relative abundance between these two cattle breeds were identified, and their biological functions might be related to milk synthesis. This study increases the understanding of the differences in microbiota between Xinjiang Brown and Holstein cattle and might provide helpful information for microbiota composition optimization of these dairy cattle.

Introducing Holstein cows on Qinghai-Tibetan Plateau is a potential solution to enhance local milk production. However, the relationship between milk quality and altitude in China remains unknown. Therefore, the components and plasmin (PL) system of raw milk from different altitudes (sea level, 1600, 2700, and 3800 m) were investigated. The daily milk production of Holstein cows and PL activity decreased as the altitude increased. However, the components content of raw milk, plasminogen (PLG)/PL ratio, activities of PLG and plasmin activator (PA) increased with altitude. The pasteurization resulted a significant decrease in PA activity of all milk and a significant increase in PL activity in milk collected at higher altitudes (2700 and 3800 m), suggesting the pasteurization was unsuitable for preserving milk at higher altitudes. This study offered references for the production and storage of milk after introducing Holstein cows on Qinghai-Tibetan Plateau.

Milk production traits as the most important economic traits of dairy cows, they directly reflect the benefits of breeding and the economic benefits of pasture. In this study, A disintegrin and metalloproteinase-12 (ADAM12), Parkinson\'s disease gene 2 (PRKN) and dipeptidyl peptidase-like protein subtype 6 (DPP6) polymorphism in 384 Chinese Holstein cows were detected by time-of-flight mass spectrometry and through statistical analysis using software such as Popgene 32, SAS 9.4 and Origin 2022, the relationship between single nucleotide polymorphisms (SNPs) of three genes with four milk production traits such as daily milk yield (DMY), milk fat percentage (MFP), milk protein percentage (MPP) and somatic cell score (SCS) was verified at molecular level. The results showed that four polymorphic loci (116,467,133, 116,604,487, 116,618,268 and 116,835,111) of DPP6 gene, two polymorphic loci (97,665,052 and 97,159,837) of PRKN gene and two polymorphic loci (45,542,714 and 45,553,888) of ADAM12 gene were detected. PRKN-97665052, DPP6-116467133, ADAM12-45553888, DPP6-116604487 and DPP6-116835111 were all in Hardy-Weinberg equilibrium state (p > .05). ADAM12-45542714, PRKN-97159837 and PRKN-97665052 were moderately polymorphic (0.25 ≤ PIC <0.50) in Holstein. It is evident that the selection potential and genetic variation of these five loci are relatively large, and the genetic richness is relatively high. The correlation analysis of different genotypes between these eight loci and milk production traits of Holstein showed that ADAM12-45542714 and DPP6-116835111 (p < .01) had an extremely significant effects on the DMY of Chinese Holstein in Ningxia, while PRKN-97665052 had an extremely significant effect on MFP (p < .01). The effect of PRKN-97665052 and DPP6-116467133 on MPP of Holstein were extremely significant (p < .01). DPP6-116618268 had an extremely significant effect on the SCS of Holstein in Ningxia (p < .01), and AA genotype individuals showed a higher SCS than GG genotype individuals; the other two loci (ADAM12-45553888 and DPP6-116604487) had no significant effects on milk production traits of Holstein (p > .05). In addition, through the joint analysis of DPP6, PRKN and ADAM12 gene loci, it was found that the interaction effect between the three gene loci could significantly affect the DMY, SCS (p < .01) and MPP (p < .05). In conclusion, several different loci of DPP6, PRKN and ADAM12 genes can affect the milk production traits of Holstein to different degrees. PRKN, DPP6 and ADAM12 genes can be used as potential candidate genes for milk production traits of Holstein for marker-assisted selection, providing theoretical basis for breeding of Holstein.

The aim of this study was to evaluate the effect of the supplementation of rumen-protected (RP) methionine and lysine on milk yield, solids, and body weight over time on fall-calving grazing multiparous Holstein cows from Chile. Four treatment groups were studied and compared for the outcomes over time. The treatments were as follows: (i) CON: control (n = 26); (ii) RP lysine group (LYS; 20 g per cow per day; n = 26); (iii) RP methionine group (MET; 20 g per cow per day; n = 26); and (iv) LYS × MET (RP lysine and RP methionine 20 g of each amino acid per cow per day; n = 26). Data were analyzed with general linear mixed model ANOVAs for repeated measures to primarily test the main effects of each amino acid and their interactions. The supplementation of the amino acids was conducted from 2 to 70 days postpartum. Overall, milk production tended to be higher in the MET and in the LYS × MET group when compared to the control group. Furthermore, CON produced significantly less milk protein (kg) and milk fat (kg) than the LYS, MET, and LYS × MET groups. Milk urea tended to be lower during the entire study in the CON group than the rest of the groups. There was a trend for a reduction in the losses of postpartum body weight in the LYS × MET than the CON. In conclusion, RP methionine and lysine improved milk fat and protein yield in grazing multiparous cows with fall parturitions; consequently, both RP amino acids can be used as a strategy for improving grazing cows\' production performance.

Holstein cattle are well known for their high average milk yield but are more susceptible to disease and have lower fecundity than other breeds of cattle. The purpose of this study was to explore the relationship between ruminal metabolites and both milk performance and ruminal microbiota composition as a means of assessing the benefits of crossbreeding Montbéliarde and Holstein cattle. This experiment crossbred Holstein with Montbéliarde cattle, aimed to act as a reference for producing high-quality dairy products and improving the overall efficiency of dairy cattle breeding. Based on similar age, parity and lactation time, 46 cows were selected and divided into two groups (n  =  23 per group) for comparison experiment and fed the same formula: Montbéliarde×Holstein (MH, DIM  =  33.23  ±  5.61 d), Holstein (H, DIM  =  29.27  ±  4.23 d). Dairy herd improvement (DHI) data is an important basis for evaluating the genetic quality of bulls, understanding the quality level of milk, and improving feeding management. We collected the DHI data of these cows in the early lactation, middle lactation and late lactation period of 10  months. The results showed that the average milk production and protein content in Montbéliarde×Holstein were 1.76  kg (34.41  kg to 32.65  kg, p  >  0.05) and 0.1% (3.54 to 3.44%, p  <  0.05) higher than in Holstein cattle. Moreover, milk from Montbéliarde×Holstein cattle had lesser somatic cell score (1.66 to 2.02) than Holstein cattle (p  <  0.01). A total of 10 experimental cattle in early lactation were randomly selected in the two groups (Lactation time  =  92.70  ±  6.81), and ruminal fluid were collected by oral gastric tube. Using 16S rRNA microbial sequencing, we compared the ruminal microbiota composition and found that Montbéliarde×Holstein cattle had a lower abundance of Alphaproteobacteria (p  <  0.05) and higher abundance of Selenomonas than Holstein cattle (p  <  0.05). These bacteria play roles in protein degradation, nitrogen fixation and lactic acid degradation. The abundance of Succiniclasticum was also greater in Montbéliarde×Holstein cattle (p  =  0.053). Through ruminal metabolome analysis, we found that the levels of trans-ferulic acid, pyrrole-2-carboxylic acid, and quinaldic acid were significantly increased in Montbéliarde×Holstein cattle, while that of lathosterol was significantly decreased. The changes in the levels of these metabolites could confer improved antioxidant, anti-inflammatory, and antibacterial activities.

The quality of colostrum is a key factor contributing to healthy calf growth, and pasteurization of colostrum can effectively reduce the counts of pathogenic microorganisms present in the colostrum. Physiological changes in calves fed with pasteurized colostrum have been well characterized, but little is known about the underlying molecular mechanisms. In this study, key genes and functional pathways through which pasteurized colostrum affects calf growth were identified through whole blood RNA sequencing. Our results showed that calves in the pasteurized group (n = 16) had higher body height and daily weight gain than those in the unpasteurized group (n = 16) in all months tested. Importantly, significant differences in body height were observed at 3 and 4 months of age (p < 0.05), and in daily weight gain at 2, 3, and 6 months of age (p < 0.05) between the two groups. Based on whole blood transcriptome data from 6-months old calves, 630 differentially expressed genes (DEGs), of which 235 were upregulated and 395 downregulated, were identified in the pasteurized compared to the unpasteurized colostrum groups. Most of the DEGs have functions in the immune response (e.g., CCL3, CXCL3, and IL1A) and metabolism (e.g., PTX3 and EXTL1). Protein-protein interaction analyses of DEGs revealed three key subnetworks and fifteen core genes, including UBA52 and RPS28, that have roles in protein synthesis, oxidative phosphorylation, and inflammatory responses. Twelve co-expression modules were identified through weighted gene co-expression network analysis. Among them, 17 genes in the two modules that significantly associated with pasteurization were mainly involved in the tricarboxylic acid cycle, NF-kappa B signaling, and NOD-like receptor signaling pathways. Finally, DEGs that underwent alternative splicing in calves fed pasteurized colostrum have roles in the immune response (SLCO4A1, AKR1C4, and MED13L), indicative of potential roles in immune regulation. Results from multiple analytical methods used suggest that differences in calf growth between the pasteurized and unpasteurized groups may be due to differential immune activity. Our data provide new insights into the impact of pasteurization on calf immune and metabolic-related pathways through its effects on gene expression.

Low-temperature conditions influence cattle productivity and survivability. Understanding the metabolic regulations of specific cattle breeds and identifying potential biomarkers related to cold challenges are important for cattle management and optimization of genetic improvement programs. In this study, 28 Inner-Mongolia Sanhe and 22 Holstein heifers were exposed to -25°C for 1 h to evaluate the differences in metabolic mechanisms of thermoregulation. In response to this acute cold challenge, altered rectal temperature was only observed in Holstein cattle. Further metabolome analyses showed a greater baseline of glycolytic activity and mobilization of AA in Sanhe cattle during normal conditions. Both breeds responded to the acute cold challenge by altering their metabolism of volatile fatty acids and AA for gluconeogenesis, which resulted in increased glucose levels. Furthermore, Sanhe cattle mobilized the citric acid cycle activity, and creatine and creatine phosphate metabolism to supply energy, whereas Holstein cattle used greater AA metabolism for this purpose. Altogether, we found that propionate and methanol are potential biomarkers of acute cold challenge response in cattle. Our findings provide novel insights into the biological mechanisms of acute cold response and climatic resilience, and will be used as the basis when developing breeding tools for genetically selecting for improved cold adaptation in cattle.

In our previous GWAS of Chinese and Nordic dairy cattle, genes CACNB2, SLC39A12, and ZEB1 locating on BTA 13 were suggested as candidate genes for reproduction. In this study, validation of these associations was performed in an independent population with records of nine reproductive traits. More importantly, functions of these genes in the reproductive process were verified by employing the expression data of ovarian follicles. The potential variants within the three genes were firstly detected in 68 Chinese Holstein bulls, and then screened in 1,588 Chinese Holstein cows using the KASP (Kompetitive allele-specific PCR) method. There were nine variants with polymorphisms in CACNB2, five in SLC39A12, and four in ZEB1, respectively, of which one SNP was in the upstream regulatory region, two in exon region, four in downstream regulatory region, and 11 SNPs in intronic regions. Amongst the 18 variants, g.33267056T/G in CACNB2 explained the largest phenotypic variance for age at first calving (0.011%), interval from first to last insemination (0.004%), and calving ease (0.002%), while g.32751518G/A in SLC39A12 contributed the most to stillbirth in heifers (0.038%). Two haplotype blocks were constructed for CACNB2 while one each for SLC39A12 and ZEB1, which were significantly associated with five reproductive traits, including age at the first service, age at the first calving, calving ease in heifers and cows, and the interval from calving to the first insemination. We then studied the profile of gene expression in granulosa cells isolated from four developmental stages of ovarian follicles from eight dairy cows. All three genes were differentially expressed between ovarian follicles with different sizes (p < 0.05), indicating their potential roles in the reproductive process of dairy cows. This study successfully demonstrated the associations of three BTA 13 genes CACNB2, SLC39A12, and ZEB1 with reproduction and further examined their expression levels in ovarian follicles directly. These findings can be beneficial for the ongoing genomic selection program for reproductive traits which have long been considered as traits that are difficult to achieve genetic improvement due to the lack of efficient genetic markers.

Heat stress influences rumen fermentative processes with effects on the physiology and production of dairy cows. However, the underlying relationship between rumen microbiota and its associated metabolism with heat tolerance in cows have not been extensively described yet. Therefore, the main objective of this study was to investigate differential heat resistance in Holstein cows using rumen bacterial and metabolome analyses. We performed both principal component analysis and membership function analysis to select seven heat-tolerant (HT) and seven heat-sensitive (HS) cows. Under heat stress conditions, the HT cows had a significantly (P < 0.05) higher propionic acid content than the HS cows; while measures of the respiratory rate, acetic, and butyric acid in the HT cows were significantly (P < 0.05) lower compared with the HS cows. Also, the HT cows showed lower (P < 0.01) rectal temperature and acetic acid to propionic acid ratio than the HS group of cows. Omics sequencing revealed that the relative abundances of Muribaculaceae, Rikenellaceae, Acidaminococcaceae, Christensenellaceae, Rikenellaceae_RC9_gut_group, Succiniclasticum, Ruminococcaceae_NK4A214_group and Christensenellaceae_R-7_group were significantly (P < 0.01) higher in the HT cows; whereas Prevotellaceae, Prevotella_1, Ruminococcaceae_UCG-014, and Shuttleworthia were significantly (P < 0.01) lower in HT cows compared to HS cows. Substances mainly involved in carbohydrate metabolism, including glycerol, mannitol, and maltose, showed significantly higher content in the HT cows (P < 0.05) compared to that in the HS cows. Simultaneously, distinct metabolites were significantly correlated with differential bacteria, suggesting that glycerol, mannitol, and maltose could serve as potential biomarkers for determining heat resistance that require further study. Overall, distinct changes in the rumen microbiota and metabolomics in the HT cows may be associated with a better adaptability to heat stress. These findings suggest their use as diagnostic tools of heat tolerance in dairy cattle breeding schemes.

Milk protein is one of the most important economic traits in the dairy industry. Yet, the regulatory network of miRNAs for the synthesis of milk protein in mammary is poorly understood. Samples from 12 Chinese Holstein cows with three high ( ≥ 3.5%) and three low ( ≤ 3.0%) phenotypic values for milk protein percentage in lactation and non-lactation were examined through deep small RNA sequencing. We characterized 388 known and 212 novel miRNAs in the mammary gland. Differentially expressed analysis detected 28 miRNAs in lactation and 52 miRNAs in the non-lactating period with a highly significant correlation with milk protein concentration. Target prediction and correlation analysis identified some key miRNAs and their targets potentially involved in the synthesis of milk protein. We analyzed for enrichments of GWAS signals in miRNAs and their correlated targets. Our results demonstrated that genomic regions harboring DE miRNA genes in lactation were significantly enriched with GWAS signals for milk protein percentage traits and that enrichments within DE miRNA targets were significantly higher than in random gene sets for the majority of milk production traits. This integrated study on the transcriptome and posttranscriptional regulatory profiles between significantly differential phenotypes of milk protein concentration provides new insights into the mechanism of milk protein synthesis, which should reveal the regulatory mechanisms of milk secretion.