Mammary Biopsies Research Articles

Comparing the efficacy of serotonin and EGTA on postpartum hypocalcemia.

Inducing a transient state of hypocalcemia prepartum mobilizes stored calcium (Ca) before the abrupt demand for Ca at parturition thus more tightly regulating postpartum hypocalcemia. Prepartum transient hypocalcemia can be achieved through intravenous infusions of either the precursor to serotonin, 5-hydroxy-tryptophan (5HTP) or a Ca chelating agent, ethylene-glycol-tetraacetic acid (EGTA). This study aimed to compare the ability of 5HTP and EGTA treatments to prevent postpartum hypocalcemia. We hypothesized that the 2 methods would be similarly effective compared with the control. Cows received either 5HTP, EGTA, 5HTP+EGTA, or control saline (n = 6/treatment) beginning 7 d before expected calving date through parturition (range 4-13 d). The 5HTP treatment was administered daily as a single 1 L dose at 1 mg/kg body weight (BW). Saline and EGTA were infused for 6 h/d. Infusion rates of EGTA were adjusted to maintain blood ionized Ca (iCa) between 0.7 and 0.8 mM inducing subclinical hypocalcemia, which occurs when iCa is 0.61-0.9 mM. Mammary biopsies were collected from rear quarters at 6, 30, 54, and 78 h postpartum. Cows in the 5HTP+EGTA group required less EGTA to maintain low iCa during infusions and had the lowest total Ca concentrations during infusions. Therefore, 5HTP and EGTA likely utilize different mechanisms to reduce blood Ca that can occur simultaneously resulting in an additive effect in blood Ca reduction. Control cows were subclinically hypocalcemic through 48 h postpartum, and 2 became clinically hypocalcemic. All other treatments were normocalcemic through 96 h postpartum and had significantly greater iCa than the control from 12 to 24 h postpartum. Administering 5HTP increased blood serotonin concentrations from the start of infusions through 72 h postpartum. Cows receiving EGTA or 5HTP+EGTA had decreased mammary Ca compared with the control at 54 and 78 h after calving. Mammary tissue qPCR and Western blot analysis revealed increased gene and protein expression of Ca release-activated Ca modulator 1 on the day of parturition compared with 30 and 78 h postpartum. Mammary gland gene expression of the calcium sensing receptor was decreased 78 h postpartum compared with 6 and 30 h postpartum. While 5HTP and EGTA both prevented postpartum hypocalcemia, EGTA significantly increased iCa through 48 h post-calving rather than 24 h in the 5HTP group. The 5HTP+EGTA and EGTA treatments were equally effective at regulating periparturient calcemic status indicating that the additive effect of 5HTP and EGTA did not persist beyond parturition.

Systemic and mammary Inflammation and mammary gland development of Holstein dairy cows around dry-off and calving.

This study evaluated systemic and mammary inflammation, and immune cell population and developmental pattern of the mammary gland in Holstein dairy cows transitioning from late lactation to the dry period (LTD, n = 6) and from the dry period to early lactation (DTL, n = 7). All cows were healthy and free of mastitis at enrollment. Mammary biopsies, milk or mammary secretions and blood samples were collected on d -7, 3, 7, and 15 relative to dry-off and on d -8, 3, 7, and 21 relative to calving. After dry-off, LTD cows had increased circulating concentrations of tumor necrosis factor (TNF)-α, haptoglobin and fibrinogen suggesting upregulated inflammation. Protein, SCC, and solid-not-fat contents in milk or mammary secretions increased during the early dry period but decreased after calving. In contrast, lactose concentrations in mammary secretions decreased in the early dry period and increased following calving. Skim milk concentrations of TNF-α, interleukin-10 and haptoglobin increased following dry-off. Consistently, mammary tissue mRNA expression of IL1B and IL10 tended to increase on d 7 and 15 following dry-off, respectively. After calving, mammary alveolar and total cells had higher apoptosis rate. Mammary alveolar, stromal, and total cell proliferation rates peaked on d 7 after dry-off in mammary tissue collected from LTD cows and were greater in the late dry period compared with early lactation. Mammary mRNA expression of HGF increased on d 7 and 15 after dry-off. Mammary tissue collected in the late dry period had greater gene expression of IGF1, IGF2, PRLR compared with early lactation. Mammary tissues collected in the early dry period had greater percentages of CD20+ B lymphocytes, CD172a+ macrophage and neutrophils compared with tissues collected during late lactation. Mammary gland collected from early lactation had greater percentages of CD20+ B lymphocytes, CD3+ T lymphocytes, CD172a+ macrophages than tissue collected from the late dry period. In conclusion, dairy cows experienced upregulated systemic and mammary inflammation during the early dry period which warrants further research to elucidate its impact on mammary development. Our data also suggested that the increased mammary growth during the late gestation and early dry period was mediated by different mechanisms. The concurrent increases in immune cell infiltration and mammary cell proliferation in the mammary gland following dry-off suggest an association between mammary immune responses and growth during the early dry period.

Hiperplasia mamária e cística canina

Mammary hyperplasia is a benign proliferation of the epithelial and mesenchymal stroma of the mammary glands. This condition can be caused by the increase of progesterone in the circulation. This study reports the case of a female Pitbull that presented clinical symptoms of mammary and cystic hyperplasia five months after the weaning of her litter of eight. The cytological test of the dog's breasts via puncture with a fine needle confirmed mastitis. The histopathological test demonstrated mammary hyperplasia with ductal fibrosis and a cystic area in the right breast (M5R). Initially, the homeopathic treatment was established with the prescription of Bryonia alba 6cH, Phytolacca decandra6cH and Arnica montana 30cH. On the fifth day of treatment, the complete resolution of the inflammatory condition of the left mammary chain (ML) was achieved. The right mammary chain (MR) demonstrated the resolution of M1 to M4, with persistence of a focal inflammatory area in the right M5 (M5R). In order to continue treatment of the inflammatory residue in M5R, Calcarea fluorica 9cH was added on the tenth day of treatment, in addition to the other previously mentioned homeopathic medications. The treatment period lasted 45 days, proving effective for the left breast, and resolving 90% of the inflammatory condition in the right breast, from M1 to M4. After this period, the animal underwent mammary biopsy of the M5R in a unilateral mastectomy procedure. It was concluded that the chosen homeopathic medication is an option for the treatment of mammary and cystic hyperplasia in dogs.

PSIII-12 Influence of early gestational heat stress on biomarkers of mammary gland development in replacement gilts genomically selected for thermotolerance or thermosensitivity

Abstract Heat stress (HS) alters physiological and metabolic processes in lactating sows leading to decreased milk production, which is a major factor limiting growth and survivability of piglets. While genomic selection for thermotolerance may be a viable solution to alleviate the negative effects of HS on pig welfare, it may be linked to a reduction in milk production and subsequently, litter growth performance. Therefore, the study objective was to evaluate the effects of genomic selection for thermotolerance and its interaction with early gestational HS on biomarkers of mammary gland development in replacement gilts. We hypothesized that HS exposure as well as genomic selection for thermotolerance would negatively affect mammary epithelial proliferation rate. A total of 36 Landrace (33%) × Large White (67%) crossbred gilts divergently selected for thermotolerance (TOL; n = 18) or thermosensitivity (SEN; n = 18) were balanced by body weight, bred to a single Duroc sire, and then exposed to either thermoneutral (TN; constant 17 to 22°C) or cyclical HS (26 to 36°C) conditions until d 65 of gestation. From d 66 of gestation until farrowing, all pregnant gilts were exposed to TN conditions. Of the 36 total gilts bred, only 28 became pregnant yielding 15 HS gilts (n = 8 SEN and 7 TOL) and 13 TN gilts (n = 7 SEN and 6 TOL). On d 105 of gestation, a mammary biopsy was taken from all gilts, mammary tissue was placed into 10% buffered formalin, and KI67 immunohistochemical staining was performed to identify proliferating mammary epithelial cells (MEC). Proliferating and non-proliferating MEC populations were counted using ImageJ tool. Data were analyzed using PROC GLM in SAS 9.4 with individual gilt as the experimental unit. Overall, early gestational HS decreased (P < 0.01; 16.53 ± 2.12%) the percent of proliferating MEC relative to those kept under TN conditions (26.42 ± 2.39%). However, no effect of genomic line divergence was detected with any comparison (P > 0.05). In conclusion, early gestation HS, but not genomic selection for thermotolerance, had a negative impact on biomarkers of mammary gland development in replacement gilts. These data may suggest that early gestation HS could have a reductive effect on milk production capacity, which may negatively affect litter growth.

Carryover effects of maternal late-gestation heat stress on granddaughters' growth and mammary gland development

Maternal (F0) exposure to late-gestation heat stress reduces their daughter's (F1) mammary gland fat pad mass (FP), parenchyma (PAR) mass, and epithelial cell proliferation when evaluated at birth and weaning, and go on to produce less milk in their first lactation. Herein, we investigated the effect of maternal late-gestation heat stress on whole-body growth and mammary development of their granddaughters (F2). Multiparous F0 cows had access to heat abatement (n = 41, shade, and active cooling via fans and water soakers) or not (n = 41, shade only) for the last 56 d of gestation during a subtropical summer. Consequently, the F1 daughters, born to F0 cows, were heat-stressed (HTF1, n = 36) or cooled (CLF1, n = 37) in utero during the last 2 mo of gestation. All F1 heifers were raised as an identically managed cohort until first calving. The F2 granddaughters, born to HTF1 (HTF2, n = 12) or CLF1 (CLF2, n = 17), were raised as an identically managed cohort until 70 d of age. Dry matter intake (DMI), body weight, hip height, wither height, chest girth, head circumference, mammary gland teat length, and left-right and front-rear teat distances were measured. Average daily gain (ADG) was calculated for the pre-weaned period (0-49 d). Mammary ultrasounds were performed on d 21, 49, and 70 (n = 9/group) on the rear left and right quarters to quantify PAR and FP areas. Mammary biopsies were collected for histological evaluation of epithelial structures (H&E staining), and to quantify cells positive for ERα (estrogen receptor, α subunit), cell proliferation (Ki67), and apoptosis (TUNEL). Heifer growth from birth to d 49 was similar between CLF2 and HTF2 for all parameters evaluated. Distances between teats and teat length were not different between groups. On d 70, CLF2 tended to have a greater average PAR (right and left quarters) relative to HTF2. Although the left FP was smaller in HTF2 relative to CLF2, the average FP was not different. The lumenal and non-lumenal epithelial structures in the PAR of HTF2 were significantly smaller than those of CLF2. In addition, HTF2 had a reduced percentage of proliferating cells in the epithelial and stromal compartments and a greater percentage of apoptotic cells, particularly in the stroma. The percentage of ERα positive cells was significantly reduced in HTF2. In summary, although HTF2 heifer's DMI was similar and they grew at the same rate as CLF2 heifers throughout the pre-weaning phase, their mammary glands had smaller PAR areas with fewer epithelial structures characterized by reduced cell turnover and lower ERα expression. These early changes in the microstructure and cellular turnover of the mammary gland may partly explain the reduction in lactation performance relative to CLF2 counterparts at maturity.

Feed restriction of lactating cows triggers acute downregulation of mammary mammalian target of rapamycin signaling and chronic reduction of mammary epithelial mass

While there is generally no consensus about how nutrients determine milk synthesis in the mammary gland, it is likely that the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) plays a role as a key integrator of nutritional and mitogenic signals that can influence a multitude of catabolic and anabolic pathways. The objectives of this study were to evaluate acute changes (<24 h) in translational signaling, in addition to chronic changes (14 d) in mammary gland structure and composition, in response to a severe feed restriction. Fourteen lactating Holstein dairy cows were assigned to either ad libitum feeding (n = 7) or a restricted feeding program (n = 7). Feed-restricted cows had feed removed after the evening milking on d 0. Mammary biopsies and blood samples were collected 16 h after feed removal, after which cows in the restricted group were fed 60% of their previously observed ad libitum intake for the remainder of the study. On d 14, animals were slaughtered and their mammary glands dissected. In response to feed removal, an acute increase in plasma nonesterified fatty acid concentration was observed, concurrent to a decrease in milk yield. In mammary tissue, we observed downregulation of the mTORC1-S6K1 signaling cascade, in addition to reductions in mRNA expression of markers of protein synthesis, endoplasmic reticulum biogenesis, and cell turnover (i.e., transcripts associated with apoptosis or cell proliferation). During the 14 d of restricted feeding, animals underwent homeorhetic adaptation to 40% lower nutrient intake, achieving a new setpoint of 14% reduced milk yield with 18% and 29% smaller mammary secretory tissue DM and CP masses, respectively. On d 14, no treatment differences were observed in markers of protein synthesis or mammary cell turnover evaluated using gene transcripts and immunohistochemical staining. These findings implicate mTORC1-S6K1 in the early phase of the adaptation of the mammary gland's capacity for milk synthesis in response to changes in nutrient supply. Additionally, changes in rates of mammary cell turnover may be transient in nature, returning to basal levels following brief alterations that have sustained effects.

PSXIV-17 Examining Mammary Gland Transcriptomic Profiles Using Rna-Seq in Slick and Wild-Type Haired Holstein Cattle Under Tropical Conditions

Abstract In lactating dairy cows, heat stress reduces feed intake, alters the metabolism of the animal, and compromises milk production. Holstein cattle carrying the prolactin receptor gene mutation (SLICK) have been shown to have superior performance under tropical conditions by exhibiting increased milk yield compared with wild-type haired Holstein cattle (WT). Recent research indicated that SLICK cattle have greater mammary gland blood flow and lower vaginal temperatures, while maintaining prolonged voluntary exposure to direct solar radiation than their WT counterparts. Nevertheless, differences in molecular mechanisms between genotypes at the mammary gland level have been unexplored. The objective of the current study was to investigate possible differences in the mammary gland transcriptome from SLICK (n = 7) and WT (n = 6) Puerto Rican Holstein cattle. At 160 ± 3 days in milk (DIM), ultrasound guided mammary biopsies were collected from the left rear quarter of each animal and samples were snap frozen. Unstranded RNA-seq libraries were created from the frozen mammary tissue and sequenced using Illumina Novaseq 2x150bp. Quantification of expression was done by salmon [v1.9.0] using the Bos taurus (ARS UCD1.3) transcriptome database. Testing for significant differential expression (DE) was done using a pipeline consisting of the R packages tximport [v1.26.1] and edgeR [v3.40.2]. The fry method from edgeR was used to do gene set enrichment analysis (GSEA) for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms. A single gene (GeneID:513329, major allergen Equ c 1 isoform X2) showed significant (B.H. adjusted P-value ≤ 0.05) differential expression. Aligning the protein sequence for this gene using the BLASTp against the non-redundant (NR) database revealed high similarity to two pheromones, salivary lipocalin 2 and salivary lipocalin-like. Both the KEGG pathway and GO term GSEA showed no statistically significant enrichment at B.H. adjusted P-value ≤ 0.05; however, using a less stringent criteria with a normal P-value ≤ 0.05, multiple relevant pathways were identified. For example, the KEGG pathway analysis revealed that the arachidonic acid metabolism pathway (bta00590) was enriched, and the tight junction function pathway (bta4530) was depleted in the SLICK Holstein samples. Among the GO analysis, genes were found to be downregulated in response to cold (GO:0009409), hair follicle development (GO:0001942), and vasoconstriction (GO:0042310), whereas positive regulation of oxytocin production (GO:0140668), and defense response to Gram-positive bacterium (GO:0050830) were upregulated. In conclusion, differences in gene expression were limited, but after enrichment interesting pathways were revealed and deserve further exploration to aid in elucidating different molecular mechanisms between SLICK and WT cattle.

Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation and metabolism in mammary and liver tissue during an intramammary lipopolysaccharide challenge

The objective of this experiment was to examine the effects of supplementation and dose of rumen-protected choline (RPC) on markers of inflammation and metabolism in liver and mammary tissue during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d of RPC (20.4 g/d of choline ions; CHOL45), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30), or no RPC (CON) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 μg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM (CHOL45, n = 9; CHOL45-LPS, n = 9; CHOL30, n = 11; CHOL30-LPS, n = 10; CON, n = 10; CON-LPS, n = 9). Hepatic and mammary tissues were collected from all cows on d 17 postpartum. Hepatic and mammary tissues were collected at ∼7.5 and 8 h, respectively, after the LPS challenge. An additional mammary biopsy was conducted on LPS-challenged cows (CHOL45-LPS, CHOL30-LPS, and CON-LPS) at 48 h postchallenge. Hepatic and mammary RNA copy numbers were quantified for genes involved in apoptosis, methylation, inflammation, oxidative stress, and mitochondrial function using NanoString technology. Targeted metabolomics was conducted only on mammary tissue samples (both 8 and 48 h biopsies) to quantify 143 metabolites including choline metabolites, amino acids, biogenic amines and derivatives, organic acids, carnitines, and glucose. Hepatic IFNG was greater in CHOL45 as compared with CON in unchallenged cows, suggesting an improvement in type 1 immune responses. Hepatic CASP3 was greater in CHOL45-LPS as compared with CON-LPS, suggesting greater apoptosis. Mammary IL6 was reduced in CHOL30-LPS cows as compared with CHOL45-LPS and CON-LPS (8 and 48 h). Mammary GPX4 and COX5A were reduced in CHOL30-LPS as compared with CON-LPS (8 h), and SDHA was reduced in CHOL30-LPS as compared with CON-LPS (8 and 48 h). Both CHOL30-LPS and CHOL45-LPS cows had lesser mammary ATP5J than CON-LPS, suggesting that dietary RPC supplementation altered mitochondrial function following LPS challenge. Treatment did not affect mammary concentrations of any metabolite in unchallenged cows, and only 4 metabolites were affected by dietary RPC supplementation in LPS-challenged cows. Mammary concentrations of isobutyric acid and 2 acyl-carnitines (C4:1 and C10:2) were reduced in CHOL45-LPS as compared with CHOL30-LPS and CON-LPS. Taken together, reductions in medium- and short-chain carnitines along with an increase in long-chain carnitines in mammary tissue from CHOL45-LPS cows suggests less fatty acid entry into the β oxidation pathway. Although the intramammary LPS challenge profoundly affected markers for inflammation and metabolism in liver and mammary tissue, dietary RPC supplementation had minimal effects on inflammatory markers and the mammary metabolome.

Effects of heat stress abatement on systemic and mammary inflammation in lactating dairy cows

To examine the effects of evaporative cooling on systemic and mammary inflammation of lactating dairy cows, 30 multiparous Holstein cows (parity = 2.4, 156 d in milk) were randomly assigned to 1 of 2 treatments: cooling (CL) with fans and misters or not (NC). The experiment was divided into a 10-d baseline when all cows were cooled, followed by a 36-d environmental challenge when cooling was terminated for NC cows. The onset of environmental challenge was considered as d 1. Temperature-humidity index averaged 78.4 during the environmental challenge. Milk yield and dry matter intake (DMI) were recorded daily. Blood and milk samples were collected from a subset of cows (n = 9/treatment) on d -3, 1, 3, 7, 14, and 28 of the experiment to measure cortisol, interleukin 10 (IL10), tumor necrosis factor-α (TNF-α), haptoglobin, and lipopolysaccharide binding protein (LBP). Mammary biopsies were collected from a second subset of cows (n = 6/treatment) on d -9, 2, 10, and 36 to analyze gene expression of cytokines and haptoglobin. A subset of cows (n = 7/treatment) who were not subjected to mammary biopsy collection received a bolus of lipopolysaccharides (LPS) in the left rear quarter on d 30 of the experiment. Blood was sampled from cows and milk samples from the LPS-infused quarter were collected at -4, 0, 3, 6, 12, 24, 48, and 96 h relative to infusion, for analyses of inflammatory products. Deprivation of cooling decreased milk yield and DMI. Compared with CL cows, plasma cortisol concentration of NC cows was higher on d 1 but lower on d 28 of the experiment (cooling × time). Deprivation of cooling did not affect circulating TNF-α, IL10, haptoglobin, or LBP. Compared with CL cows, NC cows tended to have higher milk IL10 concentrations but did not show effects in TNF-α, haptoglobin, or LBP. No differences were observed in mammary tissue gene expression of TNF-α, IL10, and haptoglobin. Milk yield declined after LPS infusion but was not affected by treatment. Compared with CL cows, NC cows had greater milk somatic cell count following intramammary LPS infusion. Non-cooled cows had lower circulating TNF-α and IL10 concentrations and tended to have lower circulating haptoglobin concentrations than CL cows. Milk IL10 and TNF-⍺ concentrations were higher 3 h after LPS infusion for NC cows compared with CL cows. Additionally, NC cows tended to have higher milk haptoglobin concentration after LPS infusion than CL cows. In conclusion, deprivation of evaporative cooling had minimal effects on lactating cows' basal inflammatory status, but upregulated mammary inflammatory responses after intramammary LPS infusion.

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.

Bacteriological investigation of milk samples and biopsies from the mammary gland of healthy sows.

Milk samples from clinically healthy sows often contain a similar bacterial content as samples from mastitic sows. To verify whether contamination during sample collection is a possible reason for this or bacterial content in the mammary gland postpartum is a regular finding, the aim of the present study was to compare the suitability of milk samples and mammary gland biopsies for assessing the bacteriological status of healthy sows. Twenty-five clinically healthy sows of different parities were selected. The mammary skin and teats were cleaned and disinfected before biopsies and milk samples were taken from the second mammary gland on the left udder side one day postpartum. Needle biopsies were performed after local anaesthesia. Samples were investigated bacteriologically for aerobic bacteria and a semi-quantitative classification of bacterial growth was carried out. Additionally, formalin-fixed paraffin-embedded biopsies from 18 of the healthy sows were stained and scored for inflammatory cells. A low bacterial content could be found in 96 % of milk samples and in 92 % of biopsies from the healthy sows. Both Gram-positive (mostly streptococci and staphylococci) and Gram-negative bacteria (Escherichia coli) were detected. Histopathological examination revealed mild inflammatory cell infiltration with mainly plasma cells and lymphocytes, in rare cases neutrophilic granulocytes. Biopsies of the mammary gland provided similar results with regard to bacteriological investigation compared to milk sampling although these were collected under aseptic conditions. Therefore, it is assumed that ubiquitous bacteria are regularly present in the colostrum and in the mammary gland tissue of clinically healthy sows after parturition. Histopathological findings were not entirely uneventful. Milk samples and biopsies from the thoroughly cleaned and disinfected mammary gland indicate a regular bacterial load of the gland postpartum; biopsies do not provide advantages with regard to hygienic conditions.

Internal mammary lymph node biopsy during delayed free flap breast reconstruction: case series and review of the literature

Internal mammary lymph node biopsy during delayed free flap breast reconstruction: case series and review of the literature

Connecting Metabolism to Mastitis: Hyperketonemia Impaired Mammary Gland Defenses During a Streptococcus uberis Challenge in Dairy Cattle.

β-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1β and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Physiological state and photoperiod exposures differentially influence circadian rhythms of body temperature and prolactin and relate to changes in mammary PER1 expression in late pregnant and early lactation dairy goats

Abstract Short-day photoperiod (SDPP; 8 h light:16 h dark) during the dry period increases milk production compared with long-day photoperiod (LDPP; 16 h light:8 h dark). We hypothesized that the impact of photoperiod on lactation is mediated by alterations in the circadian system. Twelve Saanen goats were blocked at dry off into SDPP (n = 6) and LDPP (n = 6) treatments and mammary biopsies were taken in the middle of light and dark phases at 3 wk prepartum and 5 wk postpartum. Total RNA was isolated, and the expression of clock genes was analyzed by qPCR. SDPP goats produced more milk than LDPP goats (3.15 ± 0.04 vs. 2.7 ± 0.05 kg/d). In the 24 h period, LDPP goats had a greater body temperature than SDPP goats at 3 wk prepartum (39.6 ± 0.06 vs. 39.3 ± 0.1 °C) and 5 wk postpartum (40.1 ± 0.15 vs. 39.7 ± 0.1 °C). Cosinor analysis revealed that physiological state affected body temperature mesor (P

Nutrigenomic analyses reveal miRNAs and mRNAs affected by feed restriction in the mammary gland of midlactation dairy cows.

The objective of this study was to investigate the effects of feed restriction on mammary miRNAs and coding gene expression in midlactation cows. Five Holstein cows and 6 Montbéliarde cows underwent 6 days of feed restriction, during which feed allowance was reduced to meet 50% of their net energy for lactation requirements. Mammary biopsies were performed before and at the end of the restriction period. Mammary miRNA and mRNA analyses were performed using high-throughput sequencing and microarray analyses, respectively. Feed restriction induced a negative energy balance and decreased milk production and fat and protein yields in both breeds. Feed restriction modified the expression of 27 miRNAs and 374 mRNAs in mammary glands from Holstein cows, whereas no significant miRNA change was observed in Montbéliarde cows. Among the 27 differentially expressed miRNAs, 8 miRNAs were associated with dairy QTL. Analysis of target genes indicate that the 8 most abundantly expressed miRNAs control transcripts related to lipid metabolism, mammary remodeling and stress response. A comparison between the mRNAs targeted by the 8 most strongly expressed miRNAs and 374 differentially expressed mRNAs identified 59 mRNAs in common. The bioinformatic analyses of these 59 mRNAs revealed their implication in lipid metabolism and endothelial cell proliferation. These effects of feed restriction on mammary miRNAs and mRNAs observed in Holstein cows suggest a potential role of miRNAs in mammary structure and lipid biosynthesis that could explain changes in milk production and composition.

Elevated serotonin coordinates mammary metabolism in dairy cows.

Serotonin plays a diverse role in maternal and mammary metabolism. Recent research in the dairy cow has shown a relationship between serotonin and calcium, with increased serotonin concentrations improving calcium homeostasis in the peri‐partum dairy cow. Therefore, the objective was to elucidate how administration of 5‐hydroxy‐l‐tryptophan (5‐HTP), the immediate precursor to serotonin, altered serotonin and calcium metabolism in lactating dairy cows. Twelve mid‐late lactation multiparous cows were blocked by parity, production and days in milk and allocated to a daily intravenous infusion of (i) 1.5 mg/kg of 5‐HTP (n = 6) or (ii) saline (n = 6) for 3 consecutive days. Milk samples were collected daily. Blood samples were collected before and after each infusion with mammary biopsies and blood samples collected at 48, 56, and 72 h relative to termination of first infusion. Infusion of 5‐HTP increased (p = 0.001) circulating serotonin concentrations and decreased blood calcium via a transient hypocalcemia immediately after each infusion (p = 0.02). Treatment with 5‐HTP increased milk calcium concentrations (p = 0.02) and calcium release‐activated channel protein 1 (ORAI1) mRNA at 56 h and protein at 48 h relative to termination of first infusion (p = 0.008 and p = 0.09, respectively). Fifty‐six hours from termination of the first infusion mRNA of parathyroid hormone‐related protein and mammary serotonin content were increased relative to control (p = 0.03 and p = 0.05, respectively). These findings demonstrate the ability of 5‐HTP infusion to increase circulating serotonin concentrations and alter endocrine and mammary autocrine/paracrine calcium and serotonin metabolism in the lactating dairy cow.

Effects of evaporative cooling and dietary zinc source on heat shock responses and mammary gland development in lactating dairy cows during summer

The objective of this study was to examine the effects of evaporative cooling and dietary supplemental Zn source on heat shock responses and mammary gland development of lactating dairy cows during summer. Seventy-two multiparous lactating Holstein cows were randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement. Cows were either cooled (CL) or not cooled (NC) and fed diets supplemented with 75 mg of Zn/kg of dry matter (DM) from Zn hydroxychloride (IOZ) or 35 mg of Zn/kg of DM from Zn hydroxychloride plus 40 mg of Zn/kg of DM from Zn-Met complex (ZMC). The 168-d trial included a 12-wk baseline phase when all cows were cooled and fed respective dietary treatments, and a subsequent 12-wk environmental challenge phase when NC cows were deprived of evaporative cooling. Plasma was collected from a subset of cows (n = 24) at 1, 3, 5, 12, 26, 41, 54, 68, 81 d of the environmental challenge to measure heat shock protein (HSP) 70 concentration. Mammary biopsies were collected from another subset of cows (n = 30) at enrollment (baseline samples) and at d 7 and 56 of the environmental challenge to analyze gene expression related to heat shock response, apoptosis and anti-oxidative enzymes, and to examine apoptosis and cell proliferation using immunohistochemistry. Supplemental Zn source did not affect milk yield but NC cows produced less milk than CL cows. Supplemental Zn source had no effect on mammary gene expression of HSP27, 70, and 90 or plasma concentrations of HSP70. The NC cows had greater mammary gene expression of HSP than CL cows. Circulating HSP70 of NC cows gradually increased and was higher at 81 d of environmental challenge compared with CL cows. Relative to IOZ, ZMC cows tended to have lower total mammary cell proliferation but greater mammary apoptosis. There was a tendency of greater TNFRSF1A mRNA expression for ZMC compared with IOZ cows, which may suggest upregulated extrinsic apoptosis. At d 7 of environmental challenge, NC cows had numerically higher mammary apoptosis than CL cows although not statistically significant. The NC cows tended to have greater mRNA expression of CAT and SOD3 regardless of time, and had greater mRNA expression of GPX1 at d 56 and FAS at d 7 of the environmental challenge than CL cows. Relative to CL cows, mammary cell proliferation rate was higher for NC cows at d 56 of the environmental challenge. In conclusion, dietary source of supplemental Zn has substantial effect on mammary cell turnover in lactating dairy cows, and prolonged exposure to heat stress increases mammary cell proliferation.

Bilateral gestational gigantomastia.

Gestational gigantomastia is a rare disease characterized by diffuse, extreme, and incapacitating enlargement of one or both breasts during pregnancy. Although benign, it can lead to a great social, emotional, and physical disability. A good and complete knowledge regarding this rare but distressing clinical situation is a must among all practicing physicians especially obstetricians. We report the case of a 30-year-old woman, admitted for a bilateral massive hypertrophy of the breast occurring on pregnancy and with progressive evolution. She had four pregnancies and two born-infants. Biological exams have shown a hyperprolactinemia. Pathological exam of the mammary biopsy had shown a benign hyperplasia. Medical treatment of our patient by bromocriptin was inefficient. She has had a bilateral mastectomy. Physiological enlargement of the breasts occurs at puberty and during pregnancy. It is known as gestational gigantomastia when enlargement in pregnancy becomes excessive, uncomfortable and embarrassing. Gestational gigantomastia may have far reaching effects for the mother and fetus. This rare condition is associated with considerable morbidity but may be associated with good fetal outcome. Multidisciplinary team effort in the form of obstetrician, plastic surgeon and anesthetist, and pediatrician is required for a successful fetomaternal outcome.

Breast Metastases: Updates on Epidemiology and Radiologic Findings.

PurposeThe aim of this study was to report the prevalence of secondary breast malignancies and analyze their radiological characteristics.Materials and methodsWe collected 42,505 pathological reports of mammary biopsies performed from January 2000 to January 2019 in our hospital database, from which we screened reports of secondary cancer of the breast. We collected and analyzed imaging data from computed tomography (CT), ultrasound (US), and mammography. Mammograms, CT scans, and US images were reviewed by two breast radiologists. Prevalence of secondary breast malignancy among suspicious breast masses and all breast malignancies were calculated.ResultsOut of 42,505 histopathology reports from mammary biopsies, we found 19,354 malignancies. We identified 33 cases of secondary breast cancers (0.08% of suspicious breast lesions, 0.17% of breast malignancies). Most common metastases were from lymphoma (23 cases, 0.05% of suspicious breast lesions, 0.12% of breast malignancies) and melanoma (six cases, 0.01% of suspicious breast lesions, 0.03% of breast malignancies). All secondary lesions were hypoechoic on US and showed high density on mammogram. On CT, 83% of the lesions appeared solid/dense, and 17% were mixed, alternating areas of iso/hyperdensity with areas of hypodensity.ConclusionSecondary breast cancer had a prevalence of 0.17% among all breast malignancies. No specific imaging features, characteristic of secondary breast cancer, were found.

Chronic prepartum light-dark phase shifts in cattle disrupt circadian clocks, decrease insulin sensitivity and mammary development, and are associated with lower milk yield through 60 days postpartum

Circadian and metabolic systems are interlocked and reciprocally regulated. To determine if the circadian system regulates glucose homeostasis and mammary development, the function of the circadian system was disrupted by exposing cattle to chronic light-dark cycle phase shifts from 5 wk before expected calving (BEC) to parturition. Multiparous Holstein cows were exposed to 16 h of light and 8 h of dark (CON, n = 8) or phase shifting (PS, n = 8) the light cycle 6 h every 3 d beginning 35 d BEC. After calving, both treatments were exposed to CON lighting. Mammary biopsies were taken at 21 d BEC and 21 d in milk (DIM), and histological analysis indicated PS treatment decreased the ratio of lumen to alveolar area and percentage of proliferating epithelial cells in the prepartum period. Intravenous glucose tolerance test was performed at 14 d BEC and 7 DIM by administering 50% dextrose. Blood glucose, β-hydroxybutyrate, insulin, and nonesterified fatty acids were consequently measured over 3 h. At 14 d BEC no treatment differences were observed in baseline glucose or insulin. Treatment had no effect on blood glucose or glucose area under the curve at 14 d BEC and 7 DIM. Insulin area under the curve was higher in PS versus CON at 14 d BEC and 7 DIM. The PS cows produced less milk than CON cows through 60 DIM (40.3 vs. 42.6 kg/d). Exposure to chronic light-dark PS in late gestation decreased mammary development and increased insulin resistance in periparturient cows, which may have caused subsequent lower milk yield.