Cow Mammary Gland Research Articles

The mammary glands of cows abundantly display receptors for circulating avian H5 viruses.

Influenza A viruses (IAVs) from the H5N1 2.3.4.4b clade are circulating in dairy farms in the USA.; ruminants were presumed not to be hosts for IAVs. Previously, IAV-positive mammalian species were hunters and scavengers, possibly getting infected while feeding on infected birds. It is now recognized that H5N1 viruses that circulate in US dairy cattle transmit through a mammary gland route, in contrast to transmission by aerosols via the respiratory tract. The sialome in the cow mammary and respiratory tract is so far solely defined using plant lectins. Here, we used recombinant HA proteins representing current circulating and classical H5 viruses to determine the distribution of IAV receptors in the respiratory and mammary tract tissues of cows. We complemented our study by mapping the glycan distribution of the upper and lower respiratory tracts of horses and pigs. Most of the sialome of the cow respiratory tract is lined with sialic acid modifications, such as N-glycolyl and O-acetyl, which are not bound by IAV. Interestingly, the H5 protein representing the cow isolates is bound significantly in the mammary gland, whereas classical H5 proteins failed to do so. Furthermore, whereas the 9-O-acetyl modification is prominent in all tissues tested, the 5-N-glycolyl modification is not, resulting in the display of receptors for avian IAV hemagglutinins. This could explain the high levels of virus found in these tissues and milk, adding supporting data to this virus transmission route.IMPORTANCEH5N1 influenza viruses, which usually affect birds, have been found on dairy farms in the USA. Surprisingly, these viruses are spreading among dairy cows, and there is a possibility that they do not spread through the air but through their milk glands. To understand this better, we studied how the virus attaches to tissues in the cow's respiratory tract and mammary glands using specific viral proteins. We found that the cow-associated virus binds strongly to the mammary glands, unlike older versions infecting birds. This might explain why the virus is found in cow's milk, suggesting a new way the virus could be spreading.

Detection of Listeria monocytogenes in cheese and in cow mastitis – a case report

Listeria monocytogenes is ubiquitous and causes listeriosis, which manifests in animals in the form of septicaemia, encephalitis, abortions, and, rarely, mastitis. In humans, food is the main source of infection, which is the reason food business operators take samples and test for Listeria. We report a case of L. monocytogenesdetection in traditional cheese made from raw milk, and subsequent investigation revealed that L. monocytogenes was excreted from a cow mammary gland. Although treatment was carried out according to the results of the antibiogram, there was no improvement in the subclinical mastitis and the excretion of L. monocytogenes after treatment was 8.8x102cfu/mL, and therefore the cow was removed from the herd.

Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle

The highly pathogenic avian influenza (HPAI) H5N1 virus clade 2.3.4.4b has caused the death of millions of domestic birds and thousands of wild birds in the USA since January 2022 (refs. 1–4). Throughout this outbreak, spillovers to mammals have been frequently documented5–12. Here we report spillover of the HPAI H5N1 virus to dairy cattle across several states in the USA. The affected cows displayed clinical signs encompassing decreased feed intake, altered faecal consistency, respiratory distress and decreased milk production with abnormal milk. Infectious virus and viral RNA were consistently detected in milk from affected cows. Viral distribution in tissues via immunohistochemistry and in situ hybridization revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Whole viral genome sequences recovered from dairy cows, birds, domestic cats and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of the HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the ability of the virus to cross species barriers.

Activation of ARP2/3 and HSP70 Expression by Lipoteichoic Acid: Potential Bidirectional Regulation of Apoptosis in a Mastitis Inflammation Model.

Mastitis typically arises from bacterial invasion, where host cell apoptosis significantly contributes to the inflammatory response. Gram-positive bacteria predominantly utilize the virulence factor lipoteichoic acid (LTA), which frequently leads to chronic breast infections, thereby impacting dairy production and animal husbandry adversely. This study employed LTA to develop models of mastitis in cow mammary gland cells and mice. Transcriptomic analysis identified 120 mRNAs associated with endocytosis and apoptosis pathways that were enriched in the LTA-induced inflammation of the Mammary Alveolar Cells-large T antigen (MAC-T), with numerous differential proteins also concentrated in the endocytosis pathway. Notably, actin-related protein 2/3 complex subunit 3 (ARPC3), actin-related protein 2/3 complex subunit 4 (ARPC4), and the heat shock protein 70 (HSP70) are closely related. STRING analysis revealed interactions among ARPC3, ARPC4, and HSP70 with components of the apoptosis pathway. Histological and molecular biological assessments confirmed that ARPC3, ARPC4, and HSP70 were mainly localized to the cell membrane of mammary epithelial cells. ARPC3 and ARPC4 are implicated in the mechanisms of bacterial invasion and the initiation of inflammation. Compared to the control group, the expression levels of these proteins were markedly increased, alongside the significant upregulation of apoptosis-related factors. While HSP70 appears to inhibit apoptosis and alleviate inflammation, its upregulation presents novel research opportunities. In conclusion, we deduced the development mechanism of ARPC3, ARPC4, and HSP70 in breast inflammation, laying the foundation for further exploring the interaction mechanism between the actin-related protein 2/3 (ARP2/3) complex and HSP70.

Prevalence and sensitivity of contagious and environmental cow mastitis-causing pathogens to antibiotics in Ukrainian farms

Mastitis is considered the most common and problematic disease, resulting in significant economic losses due to reduced milk yields, reduced quantity and quality of milk, treatment costs, and premature culling of animals. One of the traditional methods of treating mastitis in cows is using antibiotics, which leads to the emergence of polyresistant strains of microorganisms, the so-called Superbugs. The emergence of Superbugs, which are not sensitive to most existing antibiotics, is a major concern in veterinary and humane medicine. This study aimed to identify pathogens isolated from the secretion of the mammary gland of cows with mastitis to determine their spread and sensitivity to antibiotics. The samples of secretion from the udder were examined by bacteriological method. The isolates were identified by conventional methods and by the modern method – mass spectrometry (MALDI-TOF MS). The sensitivity of the bacterial isolates to antibiotics was determined by the disc diffusion method (Kirby-Bauer). The results of studies of cow mammary gland secretion samples indicate that 49.2% of the isolates are contagious, and 50.8% are environmental ones. The most common among the isolates of mastitis-causing pathogens were Streptoccocus аgalactiae – 16.9%, Streptococcus uberis – 10.9%, Staphylococcus aureus – 10.7%, E. Coli – 9.6%, Corynebacterium bovis – 7.3%, Staphylococcus haemolyticus – 4.8%, Staphylococcus chromogenes – 3.6%, Streptococcus dysgalactiae – 3.4%. Mastitis is caused by algae and yeast – 1% of all detected pathogens, respectively. According to the results of the determination of the sensitivity of isolates of mastitis-causing pathogens to antibiotics, it was found that most isolates were sensitive to Amoxicillin, Ceftiofur, and Rifampicin, and least of them – to Neomycin, Tylosin, Tilmicosin, Bacitracin.

Different Immune Control of Gram-Positive and Gram-Negative Mammary Infections in Dairy Cows.

In the dairy industry, bovine mastitis represents a major concern due to substantial production losses and costs related to therapies and early culling. The mechanisms of susceptibility and effective response to intra-mammary infections are still poorly understood. Therefore, we investigated innate immunity in acellular bovine skim milk through cytofluorimetric analyses of bacterial killing activity against both Gram-positive and Gram-negative pathogens. Freshly cultured E. coli and S. aureus strains were incubated with colostrum and milk samples at different lactation time points from two groups of cows, purportedly representing mastitis-resistant and mastitis-susceptible breeds; bacterial cells were analyzed for vitality by flow cytometry following incorporation of vital dyes. N-acetyl-β-D-glucosaminidase (NAGase) activity was also investigated in milk and colostrum samples. Our findings revealed that colostrum and milk bacterial killing activity was greater against S. aureus compared to E. coli., with this activity correlated with milk NAGase levels. Furthermore, both killing of S. aureus and NAGase activity were negatively correlated to the elapsed time of lactation. Interestingly, samples from the allegedly mastitis-resistant breed displayed higher bacterial killing and NAGase activities. Our study suggests that diverse control mechanisms are exerted against Gram-positive and Gram-negative pathogens in the mammary glands of cows, probably beyond those already described in the literature.

Using infrared thermography to determine changes in teat skin surface temperature after machine milking in dairy cows

Mastitis is among the costliest diseases affecting dairy cows, partly due to the resulting permanent reduction in the quantity and quality of milk produced. Most mastitis cases involve pathogenic organisms entering the cow's mammary gland through the teat canal. The teat has natural defenses against these pathogens that can be disrupted during milk harvesting. These disruptions of the teat's circulatory system and tissue integrity can predispose them to mastitis. Traditionally, machine milking–induced changes in teat blood circulation and tissue integrity have been assessed by means of manual evaluation and ultrasonography. Infrared thermography has previously been shown to produce precise and consistent measurements of skin surface temperatures (SST) on cows' hind teats. Our objective was to describe the variability in the teat SST following machine milking. Describing the variability in teat SST before and after milking could be useful to guide further studies to elucidate the physiology of the effects of milking on teat defense mechanisms. In this observational study, thermographic images of both hind teats from 140 cows immediately pre- and postmachine milking were analyzed. The average SST were subsequently determined at the proximal, middle, and distal aspects of each hind teat using image analysis software. The LSM (95% CI) from general linear mixed models of the pre- and postmilking SST, respectively, were 33.6 (33.5–33.8)°C and 35.4 (35.3–35.5)°C at the proximal aspect, 33.2 (33.1–33.4)°C and 35.2 (35.1–35.3)°C at the middle aspect, and 32.3 (32.1–32.5)°C and 34.0 (33.9–34.1)°C at the distal aspect. The observed increase in SST from pre- to postmilking SST at all 3 aspects of the teat suggest that some of the variability in the SST can be attributed to the milking event. Future research is warranted to investigate the biological relevance of SST changes during machine milking and any potential change in teat defense mechanisms, risk of mastitis, or other pathologies.

Morphological and biochemical blood parameters of newborn calves

The purpose of these studies was to determine the possibility of the effect of Polyoxidonium on the accumulation of immunoglobulins and other immunogenic factors in the mammary gland of cows before calving, their isolation in colostrum, as well as the influence of these factors on the state of colostral immunity and the formation of nonspecific resistance in calves after drinking such colostrum. An increase in the number of erythrocytes, leukocytes, due to lymphocytes was noted; the level of total protein, especially the fractions of albumins and γ-globulins, was higher; as well as the hemoglobin level 6 hours after birth in experienced calves compared to control ones.

Microbiota Characterization of the Cow Mammary Gland Microenvironment and Its Association with Somatic Cell Count.

Subclinical mastitis is a common disease that threatens the welfare and health of dairy cows and causes huge economic losses. Somatic cell count (SCC) is the most suitable indirect index used to evaluate the degree of mastitis. To explore the relationship between SCC, diversity in the microbiome, and subclinical mastitis, we performed next-generation sequencing of the 16S rRNA gene of cow's milk with different SCC ranges. The data obtained showed that the microbiota was rich and coordinated with SCC below 2 × 105. SCC above 2 × 105 showed a decrease in the diversity of microbial genera. When SCC was below 2 × 105, the phylum Actinobacteriota accounted for the most. When SCC was between 2 × 105 and 5 × 105, Firmicutes accounted for the most, and when SCC exceeded 5 × 105, Firmicutes and Proteobacteria accounted for the most. Pathogenic genera such as Streptococcus spp. were absent, while SCC above 2 × 105 showed a decrease in the diversity of microbial genera. SCC was positively correlated with the percentage of Romboutsia, Turicibacter, and Paeniclostridium and negatively correlated with the percentage of Staphylococcus, Psychrobacter, Aerococcus, and Streptococcus. Romboutsia decreased 6.19 times after the SCC exceeded 2 × 105; the SCC increased exponentially from 2 × 105 to 5 × 105 and above 1 × 106 in Psychrobacter. Analysis of the microbiota of the different SCC ranges suggests that the development of mastitis may not only be a primary infection but may also be the result of dysbiosis in the mammary gland.

Bacterial count and somatic cell count in milk from community tanks in Alagoas

Milk is a highly nutritious liquid produced by the mammary glands of cows, serving as a crucial food source for a significant portion of the population. Therefore, ensuring its quality and safety is essential. The purpose of this study is to evaluate the Plate Count and Somatic Cell Count of raw milk from community expansion tanks in the state of Alagoas. For the research, 160 milk samples were collected from mammary quarters in the months of January, February, May, and August 2013, across 23 municipalities covering the three mesoregions of the state of Alagoas. The samples were sent in appropriate containers to the Milk Quality Network Laboratory for Somatic Cell Count (SCC) and Plate Count (PC) analysis. After analysis, it was found that 79.4% (127/160) of the samples met the criteria established by legislation for SCC, while 20.6% (33/160) did not. For PC, 100% (160/160) of the samples were non-compliant with the limits set by legislation. The study highlighted a higher percentage of samples outside the SCC standard in August and the PC with the highest percentage of non-compliance occurring in May. The average SCC and PC for the studied regions showed a significant difference in SCC mean (748,235.3 cells/mL) in the East region, with no significant difference in PC, having the lowest mean (1,853,029.4 CFU/mL) compared to other regions. It was noted that 12.5% of the samples had a fat measurement outside the allowed range, total proteins ranged from 2.4 to 3.86 (g/100g), and the amount of lactose ranged from 3.42 to 5.42 (g/100g). Thus, it can be concluded that all samples were non-compliant with legislation for PC, particularly in the eastern region of the state, indicating issues with the milk extraction, preservation, or cooling process. Implementation of corrective and educational measures is suggested to improve the quality of the product.

A Study on Differential Biomarkers in the Milk of Holstein Cows with Different Somatic Cells Count Levels.

Dairy cow mastitis is one of the common diseases of dairy cows, which will not only endanger the health of dairy cows but also affect the quality of milk. Dairy cow mastitis is an inflammatory reaction caused by pathogenic microorganisms and physical and chemical factors in dairy cow mammary glands. The number of SCC in the milk of dairy cows with different degrees of mastitis will increase in varying degrees. The rapid diagnosis of dairy cow mastitis is of great significance for dairy cow health and farm economy. Based on the results of many studies on the relationship between mastitis and somatic cell count in dairy cows, microflora, and metabolites in the milk of Holstein cows with low somatic cell level (SCC less than 200,000), medium somatic cell level (SCC up to 200,000 but less than 500,000) and high somatic cell level (SCC up to 5000,00) were analyzed by microbiome and metabolic group techniques. The results showed that there were significant differences in milk microbiota and metabolites among the three groups (p < 0.05), and there was a significant correlation between microbiota and metabolites. Meanwhile, in this experiment, 75 differential metabolites were identified in the H group and L group, 40 differential metabolites were identified in the M group and L group, and six differential microorganisms with LDA scores more than four were found in the H group and L group. These differential metabolites and differential microorganisms may become new biomarkers for the diagnosis, prevention, and treatment of cow mastitis in the future.

CircRNA-02191 regulating unsaturated fatty acid synthesis by adsorbing miR-145 to enhance CD36 expression in bovine mammary gland

CD36 functions as a receptor for long-chain fatty acids, promoting the absorption and transport of long-chain unsaturated fatty acids. However, the regulatory influence of upstream circRNAs or miRNAs on its expression in cow mammary gland remains unclear. Herein, we performed high-throughput sequencing to screen for differentially expressed miRNAs and mRNAs in bovine mammary tissue during the late-lactation and the dry period to screen and conducted bioinformatics analysis to identify 420 miRNA/mRNA pairs, including miR-145/CD36. Experimental results indicate that miR-145 can directly target CD36 and inhibit its expression. Additionally, the circRNA-02191 sequence is predicted to contain a miR-145 binding site. As shown by dual luciferase reporter system detection, circRNA-02191 bound to miR-145 and its overexpression significantly reduced the expression of miR-145. Furthermore, the overexpression of miR-145 inhibited triglyceride accumulation, while circRNA-02191 enhanced the expression of the miR-145 target gene CD36. The above results indicate that circRNA-02191 can regulate triglyceride and fatty acid components by binding miR-145 and subsequently alleviating the inhibitory effect of miR-145 on the expression of CD36. Taken together, these findings present a novel approach to improve milk quality by analyzing the regulatory effect and mechanism regulating the circ02191/miR-145/CD36 pathway on fatty acid synthesis in the mammary gland of dairy cows.

Influence of liposomal thiosulfonate drug on the blood parameters of cows suffering catarrhal mastitis

Diseases of the mammary gland in cows are of multi-factor nature, which significantly complicates their control and treatment. One of the factors influencing development of mastitis is bacterial microflora. Because of limited use of antibiotics, development of novel alternative antmicrobial drugs is of great relevance. Therefore, the objective of the study was identifying the effect of an ethyl-thiosulfanilate-based liposomal drug on the hematological and biochemical parameters of blood of the cows suffering catarrhal mastitis. The animals were three times intramuscularly injected with the liposomal drug in the dose of 0.04 mL/kg of body weight. Blood from the cows was taken from the jugular vein on the day prior to administration of the drug, and also on the 3rd and 7th days after its application. During the bacterial study of the secretion of udder of sick cows, we collected 51 bacterial isolates, in which dominated Staphylococcus aureus (27.5%) and Streptococcus spp. (21.6%). In blood of the sick cows, we found higher number of leukocytes, increases in the concentrations of circulating immune complexes, lipid hydroperoxides, and ketone derivatives of oxidative modification of proteins. At the same time, we observed decreases in the number of lymphocytes and concentration of protein and the bactericidal activity of blood serum. Administration of the complex liposomal drug had a normalizing effect on the analyzed parameters of the cows. This was evidenced by 9.4% decrease in the total number of leukocytes, 18.5% decrease in the content of circulating immune complexes, 9.3% increase in lymphocytes, and 13.6% increase in the level of total protein. In the sick cows, the drug enhanced the bactericidal (by 35.4%) and lysozymic (by 36.3%) activities of blood serum, glutathione peroxidase activity, and increased the content of reduced glutathione. On the seventh day after injecting the drug into blood of the cows, we saw decrease in the level of intermediate and end products of lipid peroxidation and derivatives of oxidative modification of proteins. Therefore, the complex ethyl-thiosulfanilate-based liposomal drug promoted reduction of metabolic homeostasis of the organism, had positive effect on the activity of natural defense mechanisms in the organism and can be recommended for the treatment of cows with catarrhal mastitis.

РОЛЬ УСЛОВНО-ПАТОГЕННОЙ МИКРОФЛОРЫ В ЭТИОЛОГИИ МАСТИТА У КОРОВ

During the bacteriological study of 412 samples of udder secretion from cows with mastitis, 121 cultures of microorganisms of 15 species were isolated, which were represented in 54.9% of cases by gram–positive cocci, the remaining 45.1% by representatives of the Enterobacteriaceae and Ps. aegidinosa families, staphylococci prevailed – 28.6%, streptococci – 22.6%, enterococci – 19.2%. At the same time, in some farms, the mammary gland of cows is infected to a greater extent with staphylococci, in others with streptococci, in some mixed microflora prevails. With the incidence of mastitis in cows exceeding 30%, microflora was isolated from 81.8% of udder secretion samples, including pathogenic microflora (Staph. aureus, E. soli) – in 32.7% of cases, conditionally pathogenic (C. diversus, Ent. faecalis) – 23.6% and 20.0% – saprophytic microflora (Staph. saprophyticus, Staph. epidermidis, Ent. aegodes, Ps. aeruginosa) and fungi (Candida) – 5.5%. Pathogens were absent in 18.2% of udder secretion samples from cows with mastitis. At the same time, it should be noted that gross violations in the technology of machine milking of cows were noted in these farms. The results of the study showed that in clinically healthy lactating cows, the coefficient of bactericidal activity of the udder skin varies depending on the functional state of the breast and has a maximum value of 91.8±2.0% in the first week after delivery, decreasing to 87.8±2.4% by the middle of lactation and becomes minimal during the launch period – 79.7± 2.3%. The change in the bactericidal coefficient of the udder nipple skin is associated with the state of other non-specific local protection factors, in particular, the lysozyme M titer and the level of somatic cells. The above indicates that the violations that took place led to a decrease in their general and local resistance and infection not only with pathogenic and conditionally pathogenic (incidence 10-30%), but also with saprophytic microflora (incidence more than 30%), and the absence of microflora in 18.2% of cases indicates that mastitis initially begins to develop as aseptic, and then when infected with microflora − as an infectious process of varying severity.

CircRNA screening and ceRNA network construction for milk fat metabolism in dairy cows.

Milk fat is one of the main reference elements for evaluating milk quality and is a primary objective trait in dairy cattle breeding. In recent years, circular RNAs (circRNAs) have been found to play crucial roles in many biological processes. However, the function and expression profiles of circRNAs in milk fat synthesis in cows are not completely understood. We performed RNA sequencing to analyze the genome-wide expression of circRNA transcripts in bovine mammary epithelial cells (BMECs) from cows with extreme differences in milk fat percentage. We identified candidate differential circRNAs associated with milk fat metabolism using functional enrichment analysis and constructed a lipid metabolism-related competing endogenous RNA (ceRNA) interactive regulatory network. A total of 290 circRNAs were significantly differentially expressed (DE-circRNAs) in high milk fat percentage (HMF) cows compared to that in low milk fat percentage (LMF) cows. Of the 290 circRNAs, 142 were significantly upregulated and 148 were significantly downregulated. Enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) identified four DE-circRNAs (circ_0001122, circ_0007367, circ_0018269, and circ_0015179) that potentially regulate milk fat metabolism. Among them, circ_0001122, circ_0007367, and circ_0015179 had relatively high expression levels in cow mammary gland tissue compared to other tissues (heart, liver, kidney, uterus, ovaries, and small intestine) of cows. The regulatory networks circ_0001122:miR-12043:LIPG, circ_0007367:miR-331-3p:CIDEA/PML, and circ_0018269:miR-11989:RORC/HPX are potential networks to explore the mechanism of milk fat regulation. These results reveal the possible role of circRNAs in milk fat metabolism in dairy cows. Several important circRNAs and ceRNAs affecting milk fat synthesis were identified, providing insights into the complex biology of milk fat synthesis as well as a novel theoretical perspective for future research on lactation, milk quality, and breed improvement in dairy cows.

Mammary Fibrosis Tendency and Mitochondrial Adaptability in Dairy Cows with Mastitis.

Dairy cow mammary gland fibrosis causes huge economic losses to livestock production, however, research on dairy cow mammary gland fibrosis is in its infancy and it lacks effective treatments. Therefore, the purpose of this experiment was to explore the correlation between mastitis and fibrosis and mitochondrial damage, and to further explore its pathogenesis. In vivo, mammary tissue and milk samples were collected from healthy cows (n = 10) and mastitis cows (n = 10). The results of the study showed that compared with the control group, the mastitis tissue showed tissue damage, accumulation of collagen fibers, and the content of TGF-β1 in mammary tissue and milk was significantly increased; the level of inflammatory mediators was significantly increased; the fibrotic phenotype, collagen 1, α-SMA, vimentin gene, and protein levels were significantly increased, while the E-cadherin gene and protein levels were significantly decreased. In vitro, based on TGF-β1-induced bMECs, the above experimental results were further confirmed, and TGF-β1 significantly promoted the fibrotic phenotype of bMECs. On the other hand, in vivo results showed that fibrotic mammary tissue had a significantly stronger mitochondrial damage phenotype and significantly higher ROS than the control group. In vitro, the results also found that TGF-β1 induced a significant increase in the mitochondrial damage phenotype of bMECs, accompanied by a large amount of ROS production. Furthermore, in a TGF-β1-induced bMEC model, inhibiting the accumulation of ROS effectively alleviated the elevated fibrotic phenotype of TGF-β1-induced bMECs. In conclusion, the fibrotic phenotype of mammary gland tissue in dairy cows with mastitis was significantly increased, and mastitis disease was positively correlated with mammary fibrotic lesions. In an in vitro and in vivo model of cow mammary fibrosis, bMECs have impaired mitochondrial structure and dysfunction. Inhibiting the accumulation of ROS effectively alleviates the elevated fibrotic phenotype, which may be a potential therapeutic approach to alleviate mammary fibrosis.

LncRNA MPFAST Promotes Proliferation and Fatty Acid Synthesis of Bovine Mammary Epithelial Cell by Sponging miR-103 Regulating PI3K-AKT Pathway.

Long noncoding RNAs (lncRNAs) have an essential role in mammary gland development and lactation. Our earlier study showed that the lncRNA mammary proliferation and fatty acid synthesis-associated transcript (MPFAST) is highly expressed in the Holstein cow mammary gland during the middle lactation period compared to the dry period, which indicates its potential role in lactation. Therefore, gain- and loss-of-function experiments were performed on bovine mammary epithelial cells (BMECs) by cell counting kit 8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), real-time quantitative polymerase chain reaction (RT-qPCR), and western blot. The results indicated that MPFAST promoted the viability and proliferation of BMECs. The oil red O staining and cellular triglyceride assay further showed that MPFAST promoted the number of lipid droplets and cellular triglyceride synthesis in BMECs. Bioinformatics analysis showed that MPFAST could act as a molecular sponge for miR-103, and PIK3R1 was a potential target of miR-103, which was further confirmed by the dual-luciferase reporter assay, RT-qPCR, and western blot. The overexpression of MPFAST promoted the expression of PIK3R1 at mRNA and protein levels. It also significantly increased the mRNA relative expression levels of AKT, mTOR, and SREBP1, and the protein relative expression levels of AKT and p-AKT in the PI3K-AKT signaling pathway. In contrast, the inhibition of MPFAST resulted in the downregulation of the PI3K-AKT signaling pathway genes. These results indicated that MPFAST regulates the expression of the genes in the PI3K-AKT signaling pathway through sponging miR-103 and promotes the proliferation and synthesis of fatty acids of BMECs. Our results would provide a new direction for further exploring the regulatory mechanism of lncRNA in the mammary gland.

Molecular mechanisms in the miR-33a/LPPR4 pathway regulating unsaturated fatty acid synthesis in bovine mammary epithelial cells

The regulatory mechanisms governing metabolism of fatty acids in cow mammary gland are crucial for establishing relationships between milk quality and fatty acid content. Both, microRNAs (miRNAs) and protein-coding genes are important factors involved in the regulation of milk fat synthesis. In this study, high-throughput sequencing of miRNAs and mRNAs in bovine mammary gland tissue was performed during peak lactation (3 samples) and late lactation (3 samples) periods to characterize expression profiles. Differential expression (DE) analyses of miRNA and mRNA and miRNA–mRNA regulatory pathway screening were performed. Two-hundred eighty regulatory miRNA–mRNA pairs were identified, including the miR-33a-lipid phosphate phosphatase-related protein type 4 (LPPR4) pathway. Bioinformatics prediction, dual-luciferase reporter system detection, qRT-PCR, and Western blotting revealed that miR-33a can directly target LPPR4 and inhibit its expression. Experiments also revealed that miR-33a promotes the synthesis of triglycerides and increases the content of unsaturated fatty acids (UFAs) in bovine mammary epithelial cells (BMECs). These results indicate that miR-33a via LPPR4 plays an important role in the regulation of milk fat synthesis and UFA levels.

Functional anatomy of cow mammary glands with special reference to its defence mechanism

Functional anatomy of cow mammary glands with special reference to its defence mechanism - IJCS- Print ISSN No: - 0973-2241 Online ISSN No:- 0974-441X Article DOI No:- 10.18231/j.ijcs.2022.005, International Journal of Cow Science-Int J Cow Sci

Identification of Key Genes and Potential Pathways Associated with Mastitis Induced by E. coli.

Mastitis is one of the most important diseases of dairy cattle. It is an infectious disease leading to an inflammatory reaction in the cow's mammary gland. Escherichia coli is one of the common bacteria which induce mastitis in cows. The aim of this study was to identify key genes and potential pathways associated with mastitis induced by E. coli in dairy cattle using bioinformatics analysis. The gene expression profile of ten samples including five adjacent tissues from a quarter infected with Escherichia coli and five tissues from a healthy quarter of dairy cattle was assessed using GEO2R. Gene ontology and pathway analysis were performed using bioinformatics tools. A total of 156 differentially expressed genes were detected which 95 genes were upregulated and 61 genes were downregulated in adjacent tissue of quarter infected compared with healthy tissue. Cellular oxidant detoxification and oxidation–reduction process were the most significant biological process terms in gene ontology analysis. The most important pathways of DEGs were the biosynthesis of amino acids, p53 signaling pathway, and Metabolic pathways. Three important modules were identified and their path enrichment analysis was performed. There are 10 core genes, among which SOD2, COL1A2, COL3A1, POSTN, ALDH18A1, and CBS may be the main genes associated with mastitis, which can be considered as candidate genes in the prevention and carly diagnosis program of mastitis.