Mammary Health Research Articles

Carry-over effects of dry period heat stress on the mammary gland proteome and phosphoproteome in the subsequent lactation of dairy cows

Exposure to heat stress during a cow’s dry period disrupts mammary gland remodeling, impairing mammary function and milk production during the subsequent lactation. Yet, proteomic changes in the mammary gland underlying these effects are not yet known. We investigated alterations in the mammary proteome and phosphoproteome during lactation as a result of dry period heat stress using an isobaric tag for relative and absolute quantitation (iTRAQ)-based approach. Cows were cooled (CL; n = 12) with fans and water soakers in a free stall setting or were heat stressed through lack of access to cooling devices (HT; n = 12) during the entire dry period (approximately 46 days). All cows were cooled postpartum. Mammary biopsies were harvested from a subset of cows (n = 4 per treatment) at 14, 42, and 84 days in milk. Overall, 251 proteins and 224 phosphorylated proteins were differentially abundant in the lactating mammary gland of HT compared to CL cows. Top functions of differentially abundant proteins and phosphoproteins affected were related to immune function and inflammation, amino acid metabolism, reactive oxygen species production and metabolism, tissue remodeling, and cell stress response. Patterns of protein expression and phosphorylation are indicative of increased oxidative stress, mammary gland restructuring, and immune dysregulation due to prior exposure to dry period heat stress. This study provides insights into the molecular underpinnings of disrupted mammary function and health during lactation arising from prior exposure to dry period heat stress, which might have led to lower milk yields.

Stem Cells in Mammary Health and Milk Production.

Adult stem cells like mammary and mesenchymal stem cells have received significant attention because these stem cells possess therapeutic potential in treating many animal diseases. These cells can be administered in an autologous or allogenic fashion, either freshly isolated from the donor tissue or previously cultured and expanded in vitro. The expansion of adult stem cells is a prerequisite before therapeutic application because sufficient numbers are required in dosage calculation. Stem cells directly and indirectly (by secreting various growth factors and angiogenic factors called secretome) act to repair and regenerate injured tissues. Recent studies on mammary stem cells showed in vivo and in vitro expansion ability by removing the blockage of asymmetrical cell division. Compounds like purine analogs (xanthosine, xanthine, and inosine) or hormones (progesterone and bST) help increase stem cell population by promoting cell division. Such methodology of enhancing stem cell number, either in vivo or in vitro, may help in preclinical studies for translational research like treating diseases such as mastitis. The application of mesenchymal stem cells has also been shown to benefit mammary gland health due to the 'homing' property of stem cells. In addition to that, the multiple positive effects of stem cell secretome are on mammary tissue; healing and killing bacteria is novel in the production of quality milk. This systematic review discusses some of the studies on stem cells that have been useful in increasing the stem cell population and increasing mammary stem/progenitor cells. Finally, we provide insights into how enhancing mammary stem cell population could potentially increase terminally differentiated cells, ultimately leading to more milk production.

Interactions between human milk oligosaccharides, microbiota and immune factors in milk of women with and without mastitis

Lactational mastitis is an excellent target to study possible interactions between HMOs, immune factors and milk microbiota due to the infectious and inflammatory nature of this condition. In this work, microbiological, immunological and HMO profiles of milk samples from women with (MW) or without (HW) mastitis were compared. Secretor status in women (based on HMO profile) was not associated to mastitis. DFLNH, LNFP II and LSTb concentrations in milk were higher in samples from HW than from MW among Secretor women. Milk from HW was characterized by a low bacterial load (dominated by Staphylococcus epidermidis and streptococci), high prevalence of IL10 and IL13, and low sialylated HMO concentration. In contrast, high levels of staphylococci, streptococci, IFNγ and IL12 characterized milk from MW. A comparison between subacute (SAM) and acute (AM) mastitis cases revealed differences related to the etiological agent (S. epidermidis in SAM; Staphylococcus aureus in AM), milk immunological profile (high content of IL10 and IL13 in SAM and IL2 in AM) and milk HMOs profile (high content of 3FL in SAM and of LNT, LNnT, and LSTc in AM). These results suggest that microbiological, immunological and HMOs profiles of milk are related to mammary health of women.

Relationships among Indicators of Metabolism, Mammary Health and the Microbiomes of Periparturient Holstein Cows.

Simple SummaryParturition is the most important physiological event in the lifecycle of dairy cows; it mediates changes in the microbiota composition. However, the complete picture of the dynamics of these phenomena and how they affect health and metabolism is unknown. This study documents the composition of the microbiota in the mammary gland, on reproductive surfaces and those associated with the rectum immediately after parturition. The microbiomes of different maternal niches were different, as predicted by their different functional roles in cows. Based on the results of this research, the conclusion that the microorganisms that colonize different mucosal tissues of cows were linked to the state of systemic energy metabolism and had an impact on the health of the mammary gland cows following calving was drawn.During the period called “transition”, from the ceasing of milk production to the reestablishment of full milk production, it is postulated that the microbiota of cows undergo changes in composition driven by the fluxes in systemic energetics and that these changes appear to impact the health of cows. The primary objective of this study was to document the make-up of the microbiota in the mammary gland compared with those in the vagina and in feces in an attempt to determine any correlations between the composition of the microbiota, the impact of blood indicators of energetic metabolites and the health of the mammary gland at the time of calving. Samples were collected from 20 Holstein dairy cows immediately following calving to assess their general health and measure the microbiomes associated with each cow using 16S rRNA sequencing. The results indicated that the microbiomes found within each maternal niche were different. A set of significant negative associations between the blood energetic biomarkers (NEFAs, BHB, triglycerides and cholesterol) and the taxa Pseudomonas, Christensenellaceae and Methanobrevibacter were observed in this study. In contrast, Escherichia and Romboutsia were positively correlated with the same energetic metabolites. Therefore, it was concluded that there appears to be a set of relationships between the microorganisms that colonize several niches of cows and the sufficiency of systemic energy metabolism. Furthermore, both the microbiome and energy dynamics impact the health of the mammary gland of the host.

Study on the Regulation Mechanism of Lipopolysaccharide on Oxidative Stress and Lipid Metabolism of Bovine Mammary Epithelial Cells

The long-term feeding of a high-concentrate diet (the concentrate ratio is greater than 60 %) leads to mammary gland inflammatory response in ruminants and decreased quality in dairy cows and affects the robust development of the dairy industry. The main reason is closely related to elevated lipopolysaccharide (LPS) in the body. In this experiment, a bovine mammary epithelial cell line (MAC-T) was used as a model, and LPS at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, 10000 ng/ml) was added to the cells. The cell survival rate, oxidative stress indicators, total lipid droplet area, triglyceride content and key genes regulating lipid metabolism were detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), assay kit, microscope observation and RT-PCR methods to explore the regulatory mechanism of mammary health and milk fat synthesis. The results showed that compared with those of the control group, the survival rates of cells were significantly decreased after 9 h of stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.01). The contents of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in cells were significantly decreased (P<0.05). Compared with that of the control group, the content of malondialdehyde (MDA) in cells was significantly increased (P<0.05) after stimulation with 10000 ng/ml LPS for 9 h. After 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS, the total lipid drop area and triglyceride (TG) content of MAC-T cells were significantly decreased (P<0.05). The expression levels of fatty acid synthesis-related genes Acetyl-CoA carboxylase (ACC) and Stearoyl-CoA desaturase 1 (SCD-1) were significantly decreased after 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS (P<0.05), while the expression levels of Fatty Acid synthetase (FAS) were significantly decreased after stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.05). TG synthesis by the related gene Diacylglycerol acyltransferase-1 (DGAT1) was significantly lower than that of the control group after stimulation with 1000 ng/ml and 10000 ng/ml LPS for 9 h (P<0.05), and Diacylglycerol acyltransferase-2 (DGAT2) also showed a significant decrease after 10000 ng/ml LPS stimulation (P<0.05). In conclusion, adding different concentrations of LPS to MAC-T cells not only led to a decrease in cell activity, resulting in oxidative damage, but also affected fatty acid and TG synthesis, which may ultimately be closely related to the decrease in milk fat synthesis.

Study on the regulation mechanism of lipopolysaccharide on oxidative stress and lipid metabolism of bovine mammary epithelial cells.

The long-term feeding of a high-concentrate diet (the concentrate ratio is greater than 60 %) leads to mammary gland inflammatory response in ruminants and decreased quality in dairy cows and affects the robust development of the dairy industry. The main reason is closely related to elevated lipopolysaccharide (LPS) in the body. In this experiment, a bovine mammary epithelial cell line (MAC-T) was used as a model, and LPS at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, 10000 ng/ml) was added to the cells. The cell survival rate, oxidative stress indicators, total lipid droplet area, triglyceride content and key genes regulating lipid metabolism were detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), assay kit, microscope observation and RT-PCR methods to explore the regulatory mechanism of mammary health and milk fat synthesis. The results showed that compared with those of the control group, the survival rates of cells were significantly decreased after 9 h of stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.01). The contents of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in cells were significantly decreased (P<0.05). Compared with that of the control group, the content of malondialdehyde (MDA) in cells was significantly increased (P<0.05) after stimulation with 10000 ng/ml LPS for 9 h. After 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS, the total lipid drop area and triglyceride (TG) content of MAC-T cells were significantly decreased (P<0.05). The expression levels of fatty acid synthesis-related genes Acetyl-CoA carboxylase (ACC) and Stearoyl-CoA desaturase 1 (SCD-1) were significantly decreased after 9 h of stimulation with 100 ng/ml, 1000 ng/ml and 10000 ng/ml LPS (P<0.05), while the expression levels of Fatty Acid synthetase (FAS) were significantly decreased after stimulation with 1000 ng/ml and 10000 ng/ml LPS (P<0.05). TG synthesis by the related gene Diacylglycerol acyltransferase-1 (DGAT1) was significantly lower than that of the control group after stimulation with 1000 ng/ml and 10000 ng/ml LPS for 9 h (P<0.05), and Diacylglycerol acyltransferase-2 (DGAT2) also showed a significant decrease after 10000 ng/ml LPS stimulation (P<0.05). In conclusion, adding different concentrations of LPS to MAC-T cells not only led to a decrease in cell activity, resulting in oxidative damage, but also affected fatty acid and TG synthesis, which may ultimately be closely related to the decrease in milk fat synthesis.

Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response

BackgroundBovine milk contains extracellular vesicles (EVs) that play a role in cellular communication, acting in either an autocrine, paracrine, or an exocrine manner. The unique properties of the EVs protect the cargo against degradation. We profiled the ncRNAs (non-coding RNA) present in the EVs from seven dairy products - raw whole milk, heat-treated skim milk, homogenized heat-treated skim milk, pasteurized homogenized skim milk, pasteurized heavy whipping cream, sweet cream buttermilk and cultured buttermilk with four replicates each, obtained at different processing steps from a commercial dairy plant. EVs and their cargo were extracted by using a validated commercial kit that has been shown to be efficient and specific for EVs. Further, to find the annotation of ncRNAs, we probed bovine and other organism repositories(such as miRBase, miRTarBase, Ensemble) to find homolog ncRNA annotation in case the annotations of ncRNA are not available in Bos Taurus database.ResultsSpecifically, 30 microRNAs (miRNAs), were isolated throughout all the seven milk samples, which later when annotated with their corresponding 1546 putative gene targets have functions associated with immune response and growth and development. This indicates the potential for these ncRNAs to beneficially support mammary health and growth for the cow as well as neonatal gut maturation. The most abundant miRNAs were bta-miR-125a and human homolog miR-718 based on the abundance values of read count obtained from the milk samples.bta-miR-125a is involved in host bacterial and viral immune response, and human homolog miR-718 is involved in the regulation of p53, VEGF, and IGF signaling pathways, respectively.Sixty-two miRNAs were up-regulated and 121 miRNAs were down-regulated throughout all the milk samples when compared to raw whole milk. In addition, our study explored the putative roles of other ncRNAs which included 88 piRNAs (piwi-interacting RNA), 64 antisense RNAs, and 105 lincRNAs (long-intergenic ncRNAs) contained in the bovine exosomes.ConclusionTogether, the results indicate that bovine milk contains significant numbers of ncRNAs with putative regulatory targets associated with immune- and developmental-functions important for neonatal bovine health, and that processing significantly affects the ncRNA expression values; but statistical testing of overall abundance(read counts) of all miRNA samples suggests abundance values aren’t much affected. This can be attributed to the breakage of exosomal vesicles during the processing stages. It is worth noting, however, that these gene regulatory targets are putative, and further evidence could be generated through experimental validation.

Clinical Mastitis Incidence in Dairy Cows Housed on Recycled Manure Solids Bedding: A Canadian Cohort Study.

Bedding can affect mammary health of dairy cows. The objectives of this study were to evaluate clinical mastitis incidence in cows housed on recycled manure solids bedding and, more specifically, to determine which pathogens were involved. We followed 26 recycled manure solids farms and 60 straw-bedded farms as a comparative group during 1 year (2018–2019). For each episode of clinical mastitis, defined as a visual alteration of the milk, with or without local or systemic signs of infection, producers sampled aseptically the affected quarter, provided some details about the animal, and sent the sample to the research team at the Université de Montréal. We received and analyzed 1,144 milk samples. The samples were cultured according to the National Mastitis Council guidelines and the different colony phenotypes were subsequently identified with mass spectrometry. In 54.6% of CM cases, a single phenotype of bacteria was cultured (pure culture), while two different phenotypes were found in 16.8% of the samples (mixed culture), and no growth was observed in 14.4% of the samples. Samples with three or more phenotypes were considered contaminated and were not included in the pathogen-specific analyses (14.3% of the submitted samples). The most frequently identified bacterial species in pure and mixed culture in farms using recycled manure solids were Streptococcus uberis (16.0%), Escherichia coli (13.8%), Klebsiella pneumoniae (13.2%), Streptococcus dysgalactiae (6.2%), and Staphylococcus aureus (3.4%). In straw farms, the most frequent species were S. aureus (16.6%), S. uberis (11.0%), E. coli (9.1%), S. dysgalactiae (8.0%), and K. pneumoniae (1.1%). The incidence of clinical mastitis (all cases together) was not higher in recycled manure solids farms (14.0 cases/100 cow-year; 95% CI: 8.3–23.7) compared with straw-bedded farms (16.3 cases/100 cow-year; 95% CI: 9.0–29.6). However, K. pneumoniae clinical mastitis episodes were 7.0 (95% CI: 2.0–24.6) times more frequent in recycled manure solids farms than in straw farms. Adjusted least square means estimates were 1.6 K. pneumoniae clinical mastitis cases/100 cow-year (95% CI: 0.8–3.4) in recycled manure solids farms vs. 0.2 cases/100 cow-year (95% CI: 0.1–0.6) in straw-bedded farms. Klebsiella pneumoniae clinical mastitis is in general severe. Producers interested in this bedding alternative need to be aware of this risk.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription–polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

Impact of High-Fat Diet in Early-Life on Mammary Metabolic and Inflammatory Status in Later-Life in Mice

Abstract Objectives Emerging evidence indicates a potentially important role for early-life events and exposures in cancer development later in life. Moreover, accumulating evidence suggests that the incidence of cancers has reached a plateau in elders, whereas it continuously rises in young to middle adult. The present study aimed to investigate the potential impacts of high-fat diet in early-life, mimicking childhood/adolescent in humans, on mammary health in later-life of mice, equivalent to the young to middle age in human. Methods Female C57BL/8 mice (4 weeks of age) were fed a low-fat diet (LF: 10% kcal from fat) or a high-fat diet (HF: 60% kcal from fat) for 8 weeks, which is equivalent to child/adolescent age in humans. Mice in early-life groups were sacrificed after 8 weeks feeding, whereas mice in later-life groups were switched to standard chow diet (Lab Diet#5P76) and fed for additional 12 weeks before sacrifice. A panel of metabolic parameters, inflammatory cytokines, as well as gene expression related to tumorigenic Wnt-signaling were assessed by qPCR and immunoblotting analysis. Results Compared with LF group, the body weight in HF group was significantly elevated after 8-wk HF diet feeding (P &amp;lt; 0.05). After switching to the standard chow diet for 12 weeks, the significance remained until 24 weeks of age although with a reduced degree of magnitude (P &amp;lt; 0.05). For the metabolic factors, HFD reduced the expression levels of both Pparγ (P = 0.08) and adiponectin (P &amp;lt; 0.05) at 12 weeks and the reductions remains at 24 weeks (P &amp;lt; 0.01). Meanwhile, expressions of aromatase, estrogen receptor α and Tnf-α, Il-6, Il-10 as well as Cox2 among examined inflammatory mediators (Tnf-α, Il-6, Il-10, Il-2, Il-1β, Ifn-γ, Cox2) were significantly higher in HF than in LF group at 24 weeks (P &amp;lt; 0.05). For Wnt-signaling target genes (Cyclin D1, C-Myc, and Axin 2), a significant increase for C-Myc was observed in HF group at 12 weeks (P &amp;lt; 0.01). Conclusions Our results suggested that HF diet in early-life enhances adiposity and alters mammary metabolic and inflammatory status, creating a microenvironment in favor of breast tumorigenesis in later-life. Funding Sources This project was supported by USDA/Hatch (#1013548).

Effects of Pre-Parturient Iodine Teat Dip Applications on Modulating Aversive Behaviors and Mastitis in Primiparous Cows.

Simple SummaryThe first few days after calving can be stressful for young dairy cows since they must acclimate to many unfamiliar situations that are required for milking, including novel noises and sensations to their udders. Furthermore, the human–animal relationship is simultaneously jeopardized during this time since cows may display undesirable or dangerous behaviors during milking, such as kicking and stomping. These cows are also at risk for bacterial mammary infections that can be painful and damage tissue. In a controlled experiment, young cows that investigated the milking area and had their teats sanitized weekly for 3 weeks prior to calving were more comfortable during post-calving milking procedures as indicated by reduced kicking and restlessness behaviors. Furthermore, they had fewer mammary infections caused by the bacteria Staphylococcus aureus after calving. Results from this experiment indicate that sanitizing teats and providing opportunities to acclimate to the milking area prior to calving may improve the wellbeing and mammary health of young cows and promote a positive human–animal relationship.Heifers and their human handlers are at risk for decreased welfare during the early lactation period. This experiment investigated pre-parturient teat dipping and parlor acclimation to reduce mastitis and aversive behaviors in early lactation heifers. Three weeks prior to calving, heifers were randomly assigned to receive either: (1) a weekly 1.0% iodine-based teat dip in the parlor (trained; n = 37) or (2) no treatment (control; n = 30). For the first 3 days of lactation, heifers were milked twice daily, and treatment-blinded handlers assessed behaviors and clinical mastitis. Aseptic quarter milk samples were collected within 36 h of calving and analyzed for pathogens. Control heifers had (OR ± SE) 2.2 ± 0.6 times greater (p < 0.01) odds of kicking during milking. Trained heifers had (OR ± SE) 1.7 ± 0.4 times greater (p = 0.02) odds of being very calm during milking, while control heifers had 2.2 ± 0.8 and 3.8 ± 2.1 times greater (p < 0.04) odds of being restless and very restless or hostile during milking, respectively. Quarters of control heifers had (OR ± SE) 5.4 ± 3.4 greater (p < 0.01) odds of intramammary Staphylococcus aureus infection, yet clinical mastitis was similar among treatments. The results indicate that teat dipping in the parlor weekly for 3 weeks before calving may alleviate some aversive milking behaviors and protect against early lactation S. aureus intramammary infections.

Effect of Chlorophytum borivilianum supplementation on milk production, composition and fatty acid profile in crossbred cows during hot-humid season.

The present investigation was carried out to test the efficacy of Chlorophytum borivilianum (CB) supplementation on milk production, composition, and fatty acid profile in crossbred (KF) cows. Eighteen crossbred cows were selected randomly from the institute herd and divided as control (n = 6), treatment 1 (T1, n = 6) and treatment 2 (T2, n = 6) based on body weight (av. 410.45, 424.47, 414.45 kg) and milk yield (av. 15.17, 15.80, 15.95 kg/d), respectively. The CB was supplemented at low dose at a dose rate of 40 mg/kg BW/day (T1) and high dose at a dose rate of 80 mg/kg BW/day (T2) during hot-humid (HH) season. The parameters like milk yield, dry matter intake (DMI), and body weight were recorded and milk composition viz., milk fat, solid not fat (SNF), protein, and fatty acid profile, somatic cell count (SCC), and plasminogen were estimated. Temperature humidity index (THI) was calculated to assess the level of heat stress on animals during hot-humid (HH) season. Body weight of crossbred cows did not vary between the groups during HH season. C. borivilianum supplementation in higher dose (80 mg/kg BW/day) increased DMI (per 100 kg BW) (p < 0.01) in T2 group KF cows in comparison to T1 and control group. The supplementation of CB in high dose increased milk yield, milk fat, protein, plasminogen and decreased SCC in comparison to low dose and control. Further, ratio of saturated to unsaturated fatty acids (SFAs:UFAs) was significantly reduced (p < 0.01) due to CB supplementation at higher dose in comparison to lower dose. It was concluded that supplementation of C. borivilianum at a dose rate of 80 mg/kg BW/day was found effective to augment milk production. The reduced saturated fatty acid, milk SCC, and the increase in unsaturated fatty acid content of milk, milk plasminogen suggest that its supplementation could be used in improving the mammary health and quality of milk production in dairy animals.

Breast Milk Virome and Bacterial Microbiome Resilience in Kenyan Women Living with HIV

ABSTRACTBreast milk is nutritionally and immunologically beneficial in early life but is also a potential source of infection. Little is known about breast milk microbiota of women living with HIV (WLHIV), the impact of severe immunosuppression, and the contribution to mortality of HIV-exposed infants. Here, we performed metagenomic sequencing to characterize the bacterial microbiome and DNA virome of breast milk samples at 1 month postpartum from Kenyan WLHIV who were not receiving combination antiretroviral therapy (cART), 23 women with CD4 counts of <250 and 30 women with CD4 of >500; and additionally, 19 WLHIV with infants that lived and 26 WLHIV with infants that died during the first 2 years of life were included. We found that breast milk bacterial microbiomes in this study population were highly diverse but shared a core community composed of the Streptococcaceae, Staphylococcaceae, Moraxellaceae, and Eubacteriaceae families. The breast milk virome was dominated by human cytomegalovirus (CMV) and included the bacteriophage families Myoviridae, Siphoviridae, and Podoviridae. Bacterial microbiome and virome profiles and diversity were not significantly altered by HIV immunosuppression, as defined by a CD4 of <250. CMV viral load was not associated with maternal CD4 counts or infant mortality. In conclusion, we show that the core bacterial and viral communities are resilient in breast milk despite immunosuppression in WLHIV.IMPORTANCE Breastfeeding plays an important role in seeding the infant gut microbiome and mammary health. Although most studies focus on the diverse breast milk bacterial communities, little is known about the viral communities harbored in breast milk. We performed the first breast milk virome study of an HIV population. In this study cohort of Kenyan women living with HIV from the pre-antiretroviral therapy era, we found that breast milk harbors a core bacterial microbiome and a virome dominated by human cytomegalovirus. The virome and bacterial microbiome were not substantially altered by immunosuppression or associated with infant mortality. Together, these findings indicate resilience of the microbial community in breast milk compartmentalization. These findings advance out fundamental understanding of the breast milk core microbiome and virome interactions in the context of HIV disease.

Intergenerational cycle of disease: Maternal mastitis is associated with poorer daughter performance in dairy cattle

Adverse prenatal environments, such as maternal stress and infections, can influence the health and performance of offspring. Mastitis is the most common disease in dairy cattle, yet the intergenerational effects have not been specifically investigated. Therefore, we examined the associations between the dam's mammary gland health and daughter performance using somatic cell score (SCS) as a proxy for mammary health. Using data obtained from Dairy Records Management Systems (Raleigh, NC), we linked daughter records with their dam's records for the lactation in which the daughter was conceived. Linear and quadratic relationships of dam mean SCS with the daughter's age at first calving (AFC; n = 15,992 daughters, 4,366 herds), first- (n = 15,119 daughters, 4,213 herds) and second-lactation SCS (n = 3,570 daughters, 1,554 herds), first- and second-lactation mature-equivalent 305-d milk yield, and milk component yields were assessed using mixed linear regression models. We uncovered a phenomenon similar to those found in human and mouse models examining prenatal inflammation effects, whereby daughters born from dams with elevated SCS had poorer performance. Dam mean SCS was positively associated with daughter's AFC and first- and second-lactation mean SCS. Furthermore, for every 1-unit increase in dam mean SCS, daughter's first- and second-lactation mature-equivalent fat yield declined by 0.34% and 0.91% (-1.6 ± 0.49 kg, -4.0 ± 1.0 kg, respectively), although no effect was found on first- or second-lactation milk or milk protein yield. When accounting for genetics, daughter SCS, and AFC (first lactation only), dam mean SCS was associated with reduced second-lactation milk fat yield (-3.5 ± 1.8 kg/unit SCS), and a tendency was found for first-lactation milk fat yield (-1.9 ± 1.0 kg/unit SCS). Taken together, the association of greater dam mean SCS with lesser daughter milk fat yield is likely due to a few underlying mechanisms, in particular, a predisposition for mastitis and alterations in the epigenome controlling milk fat synthesis. As such, future studies should examine epigenetic mechanisms as a potential underpinning of this phenomenon.

289 Effects of dietary Zn on ewe milk minerals and somatic cell count

Abstract The objectives of the current study were to evaluate the effects of dietary Zn fed at approximately 3 times NRC recommendations on milk Zn concentrations and mammary health. Within Rambouillet (WF) and Hampshire (BF) breeds, ewes were ranked by BW and randomly assigned down the rank into 2 treatment groups: Control (n = 45, 37 mg Zn/kg DM) and Zn treatment (n = 44, 113 mg Zn/kg DM). Treatments were delivered via a ZnSO4-fortified alfalfa pellet fed at a rate of 0.45 kg/d DM from a RFID-activated automated feeder from approximately 6 wk before to 4 wk after lambing. Ewe milk was collected twice weekly, and analyzed for mineral content (d 0, 10, and 30 of lactation) and somatic cell count (SCC; d 3–5, 6–9, 10–12, 13–16, 17–19, 20–23, 24–26, 27–29, or 30–32). Single-bearing ewes had greater Ca, Mg, and P (P ≤ 0.04) than multiple-bearing ewes. Day of lactation influenced milk Mg, P, and Zn (P &amp;lt; 0.01), and values generally decreased as lactation progressed. Milk Zn was 1.7-fold greater (P &amp;lt; 0.01) for Zn treatment than Control ewes. Milk Ca, Mg, and P were greater for Control than Zn treatment (P ≤ 0.02) ewes. A breed × litter size effect was detected for LogSCC (P = 0.02). Single-bearing WF ewes had lower LogSCC than multiple-bearing WF ewes (5.36 ± 0.09 vs 5.74 ± 0.07; P &amp;lt; 0.01) but litter size did not affect BF ewe LogSCC (5.80 ± 0.08 vs 5.79 ± 0.09; P = 0.92). Day of lactation impacted ewe SCC (P &amp;lt; 0.01), with peak SCC between d 6 and 9, which began to decline as lactation progressed. In conclusion, dietary Zn above NRC recommendations increased milk Zn.

The Microbiota of the Human Mammary Ecosystem.

Human milk contains a dynamic and complex site-specific microbiome, which is not assembled in an aleatory way, formed by organized microbial consortia and networks. Presence of some genera, such as Staphylococcus, Streptococcus, Corynebacterium, Cutibacterium (formerly known as Propionibacterium), Lactobacillus, Lactococcus and Bifidobacterium, has been detected by both culture-dependent and culture-independent approaches. DNA from some gut-associated strict anaerobes has also been repeatedly found and some studies have revealed the presence of cells and/or nucleic acids from viruses, archaea, fungi and protozoa in human milk. Colostrum and milk microbes are transmitted to the infant and, therefore, they are among the first colonizers of the human gut. Still, the significance of human milk microbes in infant gut colonization remains an open question. Clinical studies trying to elucidate the question are confounded by the profound impact of non-microbial human milk components to intestinal microecology. Modifications in the microbiota of human milk may have biological consequences for infant colonization, metabolism, immune and neuroendocrine development, and for mammary health. However, the factors driving differences in the composition of the human milk microbiome remain poorly known. In addition to colostrum and milk, breast tissue in lactating and non-lactating women may also contain a microbiota, with implications in the pathogenesis of breast cancer and in some of the adverse outcomes associated with breast implants. This and other open issues, such as the origin of the human milk microbiome, and the current limitations and future prospects are addressed in this review.

Effects of Using an Alternative Bedding Composition on the Levels of Indicator Microorganisms and Mammary Health in Dairy Farm Conditions

The aim of this study was to compare an improved bedding composition with conventional straw bedding under farm conditions, regarding its effects on the influence of indicator microorganisms on the hygiene levels of cubicle floors and the occurrence of mastitis in dairy cows. Dairy cows were housed in newly built stalls divided into two parts, each with four subsections, and bedded cubicles arranged in three rows. Five stall subsections from each 9-bedded cubicle were selected for study, and 30 dairy cows were monitored according to the time intervals of bedding treatment for cubicles. In the first subsection (control), the cows were housed in bedded cubicles layered with straw up to a height of 20 cm. Sections 2–5 had alternative bedding (AB) as follows: fresh AB, AB 1 month old, AB 2 months old, and AB 3 months old, which were bedded one day before (fresh) and 1–3 months before the actual observation period, respectively. The alternative bedding per one cubicle consisted of ground limestone (100 kg), water (80 L), recycled manure solids (RMS; 15 kg), and straw (25 kg). After laying, the bedding was treated with a concrete selector to provide strength and sufficient resistance. A total of 180 bedding and 600 quarter milk samples were taken simultaneously from all five monitored subsections for microbiological determination. Comparing classical straw bedding with the alternate bedding showed a stabilizing effect by keeping the bedding thickness up to the floor barrier level, which had a beneficial effect by reducing the level of fecal contamination in the rear of the cubicle. Fecal coliforms and fecal streptococci were found to be reduced in one-day-old bedding as well as after the first, second, and third months. By evaluating the health status of the mammary glands, a positive effect was noted in reducing the occurrence of subclinical mastitis, which was reflected in a reduced number of infected quarters in the group of cows housed in cubicles for three months after use of improved bedding.

Evaluation of the Udders Health Status through Somatic Cell Count and the Californian Mastitis Test in Algerian Dairy Farms

Mastitis is the most common infectious disease in dairy cattle farming. It is responsible for the quantitative and qualitative milk deterioration. The objective of this study is to evaluate the mammary health status, based on the individual cell count (ICC) of milk explained by the Californian Mastitis Test (CMT) score. A total of 280 milk samples from the central region of Algeria were examined. The mammary health diagnosis has divided the milk samples in two groups: the first has 55% of samples from the healthy udders. The second group includes 45% of samples from infected udders, about 5% of them with clinical mastitis which showed cell levels greater than 5 million / ml indicating a very alarming epidemiological situation. The results obtained vary from one farm to another and within the same farm, depending on the hygienic practices diversity (P less than 0.05). The work carried out shows that good farming practices are necessary to maintain the safety of the product.

Characterization of bovine mammary gland dry secretions and their proteome from the end of lactation through day 21 of the dry period

The dry period in dairy cows is critical to mammary health. We used label-free mass spectroscopy to quantify whey proteins from milk/dry secretions collected on days 0, 3,10, and 21 of the dry period. The number of proteins identified was 776. The number of proteins upregulated at one or more times in the dry period was 109. The most significant enriched Gene Ontology (GO) terms for the upregulated proteins were immune function and stress-related. The number of proteins that are downregulated in the dry period was 68. The most significant enriched GO terms for the down-regulated proteins were stress and immune function related. In other assays, total protein and lactoferrin concentrations rose significantly by days 10 and 21 of the dry period. We then examined day 21 dry secretions for their ability to inhibit bacterial growth. Bacterial growth inhibition did not correlate with lactoferrin for 4 coliforms tested nor for all 7 mastitis pathogens tested. Eleven proteins out of the total number of proteins identified were significantly associated with positive or negative coliform growth in day 21 dry secretions. The dry secretion protein composition changes we found may provide new information for understanding the dry period and indirectly involution. Biological significanceThe dry period in a cow is critical to the success of a cow's subsequent lactation for many reasons. Of particular importance is the rate of establishment of new intramammary infections (IMI), which is significant in the dry period. IMI susceptibility is most significant in the first few weeks of the dry period. There are both physical and mechanical reasons for increased IMI susceptibility in the first few weeks of the dry period. We also know that dry secretions have high levels of antimicrobial proteins and bacterial growth is diminished in dry secretions compared to milk. However, we found that by day 21 of the dry period, antimicrobial proteins, such as lactoferrin, are no longer correlated with reduced bacterial growth in dry secretions. We established a dry secretion proteome for the first 3 weeks of the dry period. The library of proteins in dry secretions presented provides a better understanding of the critical dry period in dairy cow management.

Characterization of the colostrum and transition milk proteomes from primiparous and multiparous Holstein dairy cows

Colostrum plays a vital role in the nutrition, development, and immunity of a newborn calf. This study aimed to characterize the protein profile of colostrum and to identify changes in the colostrum proteome across parity during the transition to mature milk. Colostrum and transition milk samples were collected at milkings 1, 2, 4, and 14 after calving from multiparous (n = 10) and primiparous cows (n = 10). Samples were skimmed, fractionated, and enriched before analysis for low-abundance proteins by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Changes in protein abundances were analyzed using PROC MIXED in SAS (SAS Institute Inc., Cary, NC) with determination of the adaptive false discovery rate adjustment using a MULTTEST procedure to identify effects of parity (P), milking number (MN), and their interaction (MN×P). We identified 86 proteins through LC-MS/MS, including 3 low-abundance proteins that were affected by P, 78 that were affected by MN, and 36 affected by MN×P. Prominent ontological groupings of proteins affected by MN included defense or immunity proteins, such as immunoglobulins. Proteins involved in the plasminogen activating cascade and more broadly, blood coagulation, were affected by MN×P. The results of this study add to increasing knowledge of the colostrum and transition milk proteomes, and this is the first study to find evidence of different abundances of these proteins when examined across P, MN, and MN×P. These findings aid in the identification of potential milk protein biomarkers for mammary health during the early postpartum period.