BACKGROUND: Environmental heat stress (HS) can have detrimental effects on milk production by compromising the mammary function. Mammary plasma flow (MPF) plays a crucial role in nutrient supply and uptake in the mammary gland. In this experiment, we investigated the physiological and metabolic changes in high-yielding cows exposed to different degrees of HS: no HS with thermal-humidity index (THI) below 68 (No-HS), mild HS (Mild-HS, 68 ≤ THI ≤ 79), and moderate HS (Mod-HS, 79 < THI ≤ 88) in their natural environment. Our study focused on the changes in blood oxygen supply and mammary glucose uptake and utilization.
RESULTS: Compared with No-HS, the MPF of dairy cows was greater (P < 0.01) under Mild-HS, but was lower (P < 0.01) in cows under Mod-HS. Oxygen supply and consumption exhibited similar changes to the MPF under different HS, with no difference in ratio of oxygen consumption to supply (P = 0.46). The mammary arterio-vein differences in glucose concentration were lower (P < 0.05) under Mild- and Mod-HS than under no HS. Glucose supply and flow were significantly increased (P < 0.01) under Mild-HS but significantly decreased (P < 0.01) under Mod-HS compared to No-HS. Glucose uptake (P < 0.01) and clearance rates (P < 0.01) were significantly reduced under Mod-HS compared to those under No-HS and Mild-HS. Under Mild-HS, there was a significant decrease (P < 0.01) in the ratio of lactose yield to mammary glucose supply compared to that under No-HS and Mod-HS, with no difference (P = 0.53) in the ratio of lactose yield to uptaken glucose among different HS situations.
CONCLUSIONS: Degrees of HS exert different influences on mammary metabolism, mainly by altering MPF in dairy cows. The output from this study may help us to develop strategies to mitigate the impact of different degrees of HS on milk production.

For the agroecosystems of the dairy cow industry, dietary carbohydrate (starch, neutral detergent fiber [NDF]) and fat could directly affect rumen methane emissions and host energy utilization. However, the relationships among diet, lactation performance, and methane emissions need to be further determined to assist dairy farms to adjust diet formulations and feeding strategies for environmental and production management. A meta-analysis was conducted in the current study to explore quantitative patterns of dietary fat and carbohydrate at different levels in balancing lactation performance and environment sustainability of dairy cows, and to establish a methane emission prediction model using the artificial neural network (ANN) model. The results showed that the regression relationship between dietary fat, carbohydrate and methane emissions could be shown by the following models: methane = 106.78 + (14.86 × DMI), R2 = 0.80; methane = 443.17 - (46.41 × starch/NDF), R2 = 0.76; and methane = 388.91 + (31.40 × fat) - (5.42 × fat2), R2 = 0.80. The regression relationships between dietary fat, carbohydrate and lactation performance could be shown by the following models: milk fat yield = 1.08 + (0.43 × starch/NDF) - [0.34 × (starch/NDF)2], R2 = 0.79; milk protein yield = 0.68 + (0.15 × fat) - (0.016 × fat2), R2 = 0.82. In the structural equation model, we found that when formulating dietary carbohydrates and fats, it was necessary to balance the relationship between methane emissions and lactation performance. Specifically, dietary starch/NDF was lower than 0.63 (extremum point) and dietary fat was between 2.89% and 4.69% (extremum point), it could ensure that the aim of methane emission reduction (methane emissions decrease with increasing dietary starch/NDF and fat) was achieved without losing lactation performance of dairy cows (lactation performance increase with increasing dietary starch/NDF and fat). Finally, we established the ANN model to predict methane emissions (training set: R2 = 0.62; validation set: R2 = 0.61).

Mastitis affects almost all mammals including humans and dairy cows. In the dairy industry, bovine mastitis is a disease with a persistently high incidence, causing serious losses to the health of cows, the quality of dairy products, and the economy of dairy farms. Although local udder infection caused by the invasion of exogenous pathogens into the mammary gland was considered the main cause of mastitis, evidence has been established and continues to grow, showing that nutrition factors and gastrointestinal microbiome (GM) as well as their metabolites are also involved in the development of mammary inflammatory response. Suboptimal nutrition is recognized as a risk factor for increased susceptibility to mastitis in cattle, in particular the negative energy balance. The majority of data regarding nutrition and bovine mastitis involves micronutrients. In addition, the dysbiotic GM can directly trigger or aggravate mastitis through entero-mammary gland pathway. The decreased beneficial commensal bacteria, lowered bacterial diversity, and increased pathogens as well as proinflammatory metabolites are found in both the milk and gastrointestinal tract of mastitic dairy cows. This review discussed the relationship between the nutrition (energy and micronutrient levels) and mastitis, summarized the role of GM and metabolites in regulating mastitis. Meanwhile, several non-antibiotics strategies were provided for the prevention and alleviation of mastitis, including micronutrients, probiotics, short-chain fatty acids, high-fiber diet, inulin, and aryl hydrocarbon receptor.

Reduced feed intake in early lactation prompts increased fat mobilization to meet dairy cows\' energy needs for milk production. The increased lipolysis in cows presents significant health risks with unclear mechanisms. The objectives of our study were to compare the longitudinal profiles of metabolites and lipids of serum from high and low-lipolysis cows. Forty multiparous Holstein dairy cows were enrolled in the retrospective study. Serum samples were collected on d 7 before expected calving, as well as on d 5, d 7, d 14, and d 21 postpartum. Dairy cows were grouped according to mean serum nonesterified fatty acids on d 5 and 7 after parturition as low (<0.600 mmol/L; n = 8; LFM) and high (>0.750 mmol/L; n = 8; HFM), indicating fat mobilization during early lactation. Lactational performance and serum metabolic parameters related to glucose and lipid metabolism, liver functions, oxidative status, and inflammatory responses were determined. Serum samples were subjected to LC-MS-based metabolomics and lipidomics. Despite differences in postpartum BW change, there were no observed variations in milk yield and composition between 2 groups. Serum BHBA, glucose, leptin, aspartate aminotransferase, IL-6, and TNF-α were greater in cows with HFM than in LFM. Serum adiponectin, revised quantitative insulin sensitivity check index and albumin were lower in cows with HFM than LFM. Intensified fat mobilization in the HFM cows came along with reduced estimated insulin sensitivity, impaired liver functions, and increased oxidative stress and inflammatory responses. Differences in metabolic patterns were observed across the transition period when comparing serum blood matrices (e.g., in different amino acids, acylcarnitines, and sphingolipids). The serum metabolome of the HFM cows was characterized by higher concentrations of glycine, acylcarnitines, carnosine, Cer(d20:0/18:0), Cer(d18:1/16:0), and Cer(t18:0/24:0) compared with LFM. The differential serum metabolites and lipids at different sampling times during the peripartum period were enriched in the sphingolipid metabolism. Differences in serum metabolic status parameters suggest that cows adopt varied metabolic adaptation strategies to cope with energy deficits postpartum. Our investigation found a comprehensive remodeling of the serum metabolic profiles in transition dairy cattle, highlighting the significance of alterations in sphingolipid species, as they play a crucial role in insulin resistance and metabolic disorders.

An established technology to create cloned animals is through the use of somatic cell nuclear transfer (SCNT), in which reprogramming the somatic cell nucleus to a totipotent state by enucleated oocyte cytoplasm is a necessary process, including telomere length reprogramming. The limitation of this technology; however, is that the live birth rate of offspring produced through SCNT is significantly lower than that of IVF. Whether and how telomere length play a role in the development of cloned animals is not well understood. Only a few studies have evaluated this association in cloned mice, and fewer still in cloned cows. In this study, we investigated the difference in telomere length as well as the abundance of some selected molecules between newborn deceased cloned calves and normal cows of different ages either produced by SCNT or via natural conception, in order to evaluate the association between telomere length and abnormal development of cloned cows. The absolute telomere length and relative mitochondrial DNA (mtDNA) copy number were determined by real-time quantitative PCR (qPCR), telomere related gene abundance by reverse-transcription quantitative PCR (RT-qPCR), and senescence-associated β-galactosidase (SA-β-gal) expression by SA-β-gal staining. The results demonstrate that the newborn deceased SCNT calves had significantly shortened telomere lengths compared to newborn naturally conceived calves and newborn normal SCNT calves. Significantly lower mtDNA copy number, and significantly lower relative abundance of LMNB1 and TERT, higher relative abundance of CDKN1A, and aberrant SA-β-gal expression were observed in the newborn deceased SCNT calves, consistent with the change in telomere length. These results demonstrate that abnormal telomere shortening, lower mtDNA copy number and abnormal abundance of related genes were specific to newborn deceased SCNT calves, suggesting that abnormally short telomere length may be associated with abnormal development in the cloned calves.

The placenta plays a crucial role in successful mammalian reproduction. Ruminant animals possess a semi-invasive placenta characterized by a highly vascularized structure formed by maternal endometrial caruncles and fetal placental cotyledons, essential for full-term fetal development. The cow placenta harbors at least two trophoblast cell populations: uninucleate (UNC) and binucleate (BNC) cells. However, the limited capacity to elucidate the transcriptomic dynamics of the placental natural environment has resulted in a poor understanding of both the molecular and cellular interactions between trophoblast cells and niches, and the molecular mechanisms governing trophoblast differentiation and functionalization. To fill this knowledge gap, we employed Stereo-seq to map spatial gene expression patterns at near single-cell resolution in the cow placenta at 90 and 130 days of gestation, attaining high-resolution, spatially resolved gene expression profiles. Based on clustering and cell marker gene expression analyses, key transcription factors, including YBX1 and NPAS2, were shown to regulate the heterogeneity of trophoblast cell subpopulations. Cell communication and trajectory analysis provided a framework for understanding cell-cell interactions and the differentiation of trophoblasts into BNCs in the placental microenvironment. Differential analysis of cell trajectories identified a set of genes involved in regulation of trophoblast differentiation. Additionally, spatial modules and co-variant genes that help shape specific tissue structures were identified. Together, these findings provide foundational insights into important biological pathways critical to the placental development and function in cows.
胎盘是哺乳动物成功繁殖的关键因素之一。反刍动物拥有半侵袭性胎盘，其特征是由母体子宫内膜肉阜和胎儿胎盘子叶形成的高度血管化结构，这对胎儿发育至足月至关重要。牛胎盘含有至少两种滋养层细胞群：单核细胞（uninucleate cell, UNC）和双核细胞（binucleate cell, BNC）。目前，阐明胎盘自然环境转录组动力学的能力有限，导致人们对滋养层细胞和生态位之间的分子和细胞相互作用以及控制滋养层分化和功能化的分子机制了解不足。为了填补这一知识空白，我们使用Stereo-seq以接近单细胞分辨率绘制了妊娠90天和130天奶牛胎盘中的空间基因表达模式。获得了高分辨率和空间信息的基因表达谱。基于聚类和细胞标记基因表达分析，包括YBX1和NPAS2在内的关键转录因子被证明可以调节滋养层细胞亚群的转录异质性。细胞通讯和轨迹分析为理解胎盘微环境中滋养层细胞与细胞的相互作用和向BNCs分化提供了一个框架。细胞轨迹的差异分析确定了一组参与滋养层分化调控的基因。此外，还鉴定了有助于塑造特定组织结构的空间模块和共变异基因。总之，这些发现为了解对奶牛胎盘发育和功能至关重要的重要生物途径提供了基础性见解。.

The inclusion of fibrolytic enzymes in the diet is believed to have positive effects on animal production. Hence, the objective of this study was to investigate the impact of supplementing diets with a commercial fibrolytic enzyme preparation (Vistamax; mixture of xylanase and cellulase) derived from Trichoderma reesei on lactational performance, digestibility, and plasma nutrient levels in high-producing dairy cows. Two dietary energy levels were considered: a normal energy diet (metabolizable energy = 2.68 Mcal/kg) and a slightly lower energy diet (metabolizable energy = 2.55 Mcal/kg). A total of 120 lactating Holstein cows (parity = 2; Days in Milk = 113 ± 23) were randomly assigned to four treatment groups using a 2 * 2 factorial arrangement. The dietary treatments consisted of: (1) normal energy diet without enzyme supplementation (NL); (2) normal energy diet with enzyme supplementation (NLE); (3) slightly lower energy diet without enzyme supplementation (SL); and (4) slightly lower energy diet with enzyme supplementation (SLE). The amount of enzyme added to the diets was determined based on previous in vitro studies and supplier recommendations. The enzyme and premix were mixed prior to the preparation of the total mixed ration, and the trial lasted for a duration of 42 days. The results indicated that the application of the fibrolytic enzyme did not have a significant effect on dry matter intake (DMI), but it did enhance the digestibility of dry matter (DM), neutral detergent fiber (NDF), potentially digestible NDF (pdNDF), organic matter (OM), milk production, milk urea nitrogen (MUN), and blood urea nitrogen (BUN). On the other hand, the slightly lower energy diet resulted in a decrease in DMI, milk production, milk protein yield, plasma free amino acids (FAA), and an increase in plasma B-hydroxybutyrate (BHBA). In conclusion, the inclusion of the fibrolytic enzyme in the diets of dairy cows led to improvements in the digestibility of DM, NDF, pdNDF, OM, milk production, and feed efficiency. Furthermore, the application of the enzyme to the slightly lower energy diet resulted in milk production levels comparable to those observed in cows fed the untreated normal energy diet.

Recurrent mastitis poses a common challenge on dairy farms. While the impact of repeated mastitis within the same lactation has been investigated, the difference from one lactation to the next, particularly concerning the change of milk and blood metabolites, remains unclear. This study aimed to examine the difference in milk yield, milk composition, and metabolic status in the subsequent lactation between healthy and repeated mastitis in the previous lactation. The study population comprised 50 cows chosen from 400 cows, with 25 having no history of mastitis and 25 experiencing mastitis more than three times during the last lactation. Following dry-off and calving, all cows initiated a new lactation, during which no mastitis was diagnosed until the sample collection period. In the group exposed to repeated mastitis, a significant decrease in milk fat levels was observed in the subsequent lactation, while no change was observed in milk somatic cell count (SCC). Milk collected from cows that had experienced repeated mastitis in the previous lactation exhibited significant increases in the levels of free amino acids, namely valine, proline, and alanine. However, no difference in plasma levels of these amino acids was noted. These results indicate that individuals exposed to repeated mastitis have persistent milk quality changes even after dry-off. Biomarker analysis suggested that the milk valine and proline showed a moderate biomarker potential on Kappa coefficients to characterize cows that have experienced repeated mastitis. Furthermore, the results of biomarker combinations for valine and proline provided the highest specificity (100 %), positive likelihood ratio (infinity), and substantial biomarker potential on kappa coefficients (0.68). These findings significantly enhance our understanding of the pathobiology and etiology of recurrent mastitis and provide a biomarker to characterize cows that have experienced repeated mastitis in the past.

This examined the effects of Lonicera japonica extract (LJE) with different chlorogenic acid (CGA) contents on lactation performance, antioxidant status and immune function and rumen fermentation in heat-stressed high-yielding dairy cows. In total, 45 healthy Chinese Holstein high-yielding dairy cows, all with similar milk yield, parity, and days in milk were randomly allocated to 3 groups: (1) the control group (CON) without LJE; (2) the LJE-10% CGA group, receiving 35 g/(d·head) of LJE-10% CGA, and (3) the LJE-20% CGA group, receiving 17.5 g/(d·head) of LJE-20% CGA. The results showed that the addition of LJE significantly reduced RT, and enhanced DMI, milk yield, milk composition, and improved rumen fermentation in high-yielding dairy cows experiencing heat stress. Through the analysis of the serum biochemical, antioxidant, and immune indicators, we observed a reduction in CREA levels and increased antioxidant and immune function. In this study, while maintaining consistent CGA content, the effects of addition from both types of LJE are similar. In conclusion, the addition of LJE at a level of 4.1 g CGA/(d·head) effectively relieved heat stress and improved the lactation performance of dairy cows, with CGA serving as the effective ingredient responsible for its anti-heat stress properties.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1β). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.