Investigation and response to an outbreak of Neisseria meningitidis serogroup Y ST-1466 urogenital infections, Australia.

Abstract: In 2023, an increased number of urogenital and anorectal infections with Neisseria meningitis serogroup Y (MenY) were reported in New South Wales (NSW). Whole genome sequencing (WGS) found a common sequence type (ST-1466), with limited sequence diversity. Confirmed outbreak cases were NSW residents with a N. meningitidis isolate matching the cluster sequence type; probable cases were NSW residents with MenY isolated from a urogenital or anorectal site from 1 July 2023 without WGS testing. Of the 41 cases, most were men (n = 27), of whom six reported recent contact with a female sex worker. Five cases were men who have sex with men and two were female sex workers. Laboratory alerts regarding the outbreak were sent to all Australian jurisdictions through the laboratories in the National Neisseria Network. Two additional states identified urogenital MenY ST-1466 infections detected in late 2023. Genomic analysis showed all MenY ST-1466 sequences were interspersed, suggestive of an Australia-wide outbreak. The incidence of these infections remains unknown, due to varied testing and reporting practices both within and across jurisdictions. Isolates causing invasive meningococcal disease (IMD) in Australia are typed, and there has been no MenY ST-1466 IMD recorded in Australia to end of March 2024. Concerns remain regarding the risk of IMD, given the similarity of these sequences with a MenY ST-1466 IMD strain causing a concurrent outbreak in the United States of America.

Biomarkers of Recent Cannabis Use in Blood, Oral Fluid and Breath.

Proving driving under the influence of cannabis (DUIC) is difficult. Establishing a biomarker of recent use to supplement behavioral observations may be a useful alternative strategy. We determined whether cannabinoid concentrations in blood, oral fluid (OF) or breath could identify use within the past 3 h-likely the period of the greatest impairment. In a randomized trial, 191 frequent (≥4/week) and occasional (<4/week) cannabis users smoked one cannabis (placebo [0.02%], or 5.9% or 13.4% Δ9-tetrahydrocannabinol [THC]) cigarette ad libitum. Blood, OF and breath samples were collected prior to and up to 6 h after smoking. Samples were analyzed for 10 cannabinoids in OF, 8 in blood and THC in breath. Frequent users had more residual THC in blood and were more likely to be categorized as 'recently used' prior to smoking; this did not occur in OF. Per se limits ranging from undetectable to 5 ng/mL THC in blood offered limited usefulness as biomarkers of recent use. Cannabinol (CBN, cutoff = 1 ng/mL) in blood offered 100% specificity but only 31.4% sensitivity, resulting in 100% positive predictive value (PPV) and 94.0% negative predictive value (NPV) at 4.3% prevalence; however, CBN may vary by cannabis chemovar. A 10 ng/mL THC cutoff in OF exhibited the overall highest performance to detect its use within 3 h (99.7% specificity, 82.4% sensitivity, 92.5% PPV and 99.2% NPV) but was still detectable in 23.2% of participants ∼4.4 h post-smoking, limiting specificity at later time points. OF THC may be a helpful indicator of recent cannabis intake, but this does not equate to impairment. Behavioral assessment of impairment is still required to determine DUIC. This study only involved cannabis inhalation, and additional research evaluating alternative routes of ingestion (i.e., oral) is needed.

Impaired mammary development in tamoxifen-treated prepubertal heifers is associated with altered development and morphology of myoepithelial cells.

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.

Transcriptome analysis of cortical tissue reveals shared sets of downregulated genes in autism and schizophrenia.

Autism (AUT), schizophrenia (SCZ) and bipolar disorder (BPD) are three highly heritable neuropsychiatric conditions. Clinical similarities and genetic overlap between the three disorders have been reported; however, the causes and the downstream effects of this overlap remain elusive. By analyzing transcriptomic RNA-sequencing data generated from post-mortem cortical brain tissues from AUT, SCZ, BPD and control subjects, we have begun to characterize the extent of gene expression overlap between these disorders. We report that the AUT and SCZ transcriptomes are significantly correlated (P<0.001), whereas the other two cross-disorder comparisons (AUT-BPD and SCZ-BPD) are not. Among AUT and SCZ, we find that the genes differentially expressed across disorders are involved in neurotransmission and synapse regulation. Despite the lack of global transcriptomic overlap across all three disorders, we highlight two genes, IQSEC3 and COPS7A, which are significantly downregulated compared with controls across all three disorders, suggesting either shared etiology or compensatory changes across these neuropsychiatric conditions. Finally, we tested for enrichment of genes differentially expressed across disorders in genetic association signals in AUT, SCZ or BPD, reporting lack of signal in any of the previously published genome-wide association study (GWAS). Together, these studies highlight the importance of examining gene expression from the primary tissue involved in neuropsychiatric conditions-the cortical brain. We identify a shared role for altered neurotransmission and synapse regulation in AUT and SCZ, in addition to two genes that may more generally contribute to neurodevelopmental and neuropsychiatric conditions.

Severe refractory hypertension during shoulder arthroscopy.

The use of epinephrine-containing saline irrigating solutions during arthroscopic shoulder surgery gained popularity after it was reported that the addition of epinephrine reduced bleeding and improved visualization without adverse cardiovascular effects. We share a case of a patient undergoing shoulder arthroscopy who received a standard intra-articular infusion of epinephrine-containing normal saline (1 mcg/mL) and experienced severe hemodynamic consequences.

Localization and quantitation of macrophages, mast cells, and eosinophils in the developing bovine mammary gland.

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is highly regulated and in mice is known to be affected by the presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. For the first time, we determined the presence, location (relative to mammary ductal structures), and changes in numbers of eosinophils, mast cells, and macrophages in prepubertal bovine mammary tissue, and evaluated the effects of age, ovariectomy, and exogenous estrogen on numbers of each cell type. Chemical stains and immunofluorescence were used to identify the 3 cell types in formalin-fixed, paraffin-embedded mammary tissue from prepubertal female calves from 3 archived tissue sets. The ontogeny tissue set included samples of mammary tissue from female calves (n=4/wk) from birth to 6 wk of age. The ovary tissue set contained samples from ovary intact and ovariectomized heifers allowing us to investigate the influence of the ovaries on immune cells in the developing mammary gland in prepubertal heifers. Nineteen animals were intact or ovariectomized 30 d before sampling; they were 90, 120, or 150 d old at the time of sampling. A third tissue set, the estrogen set, allowed us to determine the effect of exogenous estrogen on innate immune cells in the gland. Eosinophils were identified via Luna staining, mast cells by May-Grunwald Giemsa staining, and macrophages with immunofluorescence. Key findings were that more eosinophils and mast cells were observed in near versus far stroma in the ontogeny and ovary tissue sets but not estrogen. More macrophages were observed in near versus far stroma in ontogeny animals. Eosinophils were more abundant in the younger animals, and fewer macrophages tended to be observed in ovariectomized heifers as compared with intact heifers and estrogen treatment resulted in a reduction in cell numbers. In summary, we show for the first time that innate immune cells are present in prepubertal bovine mammary tissue, localization varies by immune cell type, and abundance is related to proximity of epithelial structures and physiological state. We suggest a likely role for these cells in control of bovine mammary growth and ductal development.

Ovariectomy in young prepubertal dairy heifers causes complete suppression of mammary progesterone receptors.

Mammary growth and development depends on ovarian steroids and particularly interaction of estrogen and progesterone with their intracellular receptors. The objectives of this study were to determine the effect of ovariectomy on the expression of protein and messenger RNA for estrogen receptor-alpha (ESR1) and progesterone receptor (PGR) and their relation to mammary ductal development and cell proliferation. Prepubertal Holstein heifers 2, 3, or 4 mo of age were randomly assigned to one of 2 treatments, ovariectomized (OVX; n = 8) or sham operated (INT; n = 12). Mammary parenchymal (PAR) tissue samples were harvested 30 d after surgery. Localization and quantitation of ESR1 and PGR in PAR were determined by immunohistochemistry and quantitative multispectral imaging. Relative messenger RNA expression of ESR1 and PGR in PAR was measured by quantitative real time polymerase chain reaction. We observed the complete absence of PGR-positive epithelial cell nuclei and reduced PGR transcript abundance in mammary parenchyma of OVX heifers. The percent of epithelial cells expressing ESR1 did not differ by treatment but was decreased with age. However, average intensity of ESR1 expression per cell was reduced in OVX heifers. The abundance of Ki67 labeled epithelial cells and stromal cells was reduced after ovariectomy. These data suggest that reduced mammary development after ovariectomy may be mediated by loss of PGR expression and reduced ESR1 expression in positive cells. A presumptive relationship with ovarian-derived circulating estradiol remains unresolved, but data suggest other ovarian-derived agents may play a role. Use of specific antagonists to manipulate expression or action of PGR and ESR1 receptors should provide direct evidence for roles of these receptors in prepubertal bovine mammary development.

Transcriptional regulators transforming growth factor-ß1 and estrogen-related receptor-α identified as putative mediators of calf rumen epithelial tissue development and function during weaning.

Molecular mechanisms regulating rumen epithelial development remain largely unknown. To identify gene networks and regulatory factors controlling rumen development, Holstein bull calves (n=18) were fed milk replacer only (MRO) until 42 d of age. Three calves each were euthanized at 14 and 42 d of age for tissue collection to represent preweaning, and the remaining calves were provided diets of either milk replacer + orchard grass hay (MH; n=6) to initiate weaning without development of rumen papillae, or milk replacer + calf starter (MG; n=6) to initiate weaning and development of rumen papillae. At 56 and 70 d of age, 3 calves from the MH and MG groups were euthanized for collection of rumen epithelium. Total RNA and protein were extracted for microarray analysis and to validate detected changes in selected protein expression, respectively. As expected, calves fed MRO had no rumen papillae and development of papillae was greater in MG versus MH calves. Differentially expressed genes between the MRO diet at d 42 (preweaning) versus the MG or MH diets at d 56 (during weaning) were identified using permutation analysis of differential expression. Expression of 345 and 519 transcripts was uniquely responsive to MG and MH feeding, respectively. Ingenuity Pathway Analysis (Qiagen, Redwood City, CA) indicated that the top-ranked biological function affected by the MG diet was the cell cycle, and TFGB1, FBOX01, and PPARA were identified as key transcriptional regulators of genes responsive to the MG diet and associated with development of rumen papillae. Increased expressions of TGFB1 mRNA and protein in response to the MG diet were confirmed by subsequent analyses. The top-ranking biological function affected by the MH diet was energy production. Receptors for IGF-1 and insulin, ESRRA, and PPARD were identified by ingenuity pathway analysis as transcriptional regulators of genes responsive to the MH diet. Further analysis of TGFB1 and ESRRA mRNA expression in rumen epithelium obtained from a separate ontogenic study of Holstein calves (n=26) euthanized every 7d from birth to 42 d of age showed increases in transcript expression with advancing age, supporting their roles in mediating rumen epithelial development and function during weaning. Additional evaluation of gene expression in the rumen epithelium of adult cows ruminally infused with butyrate also suggested that observed changes in ESRRA mRNA expression in developing calf rumen may be mediated by increased butyrate concentration. Our results identify TGFB1 and ESRRA as likely transcriptional regulators of rumen epithelial development and energy metabolism, respectively, and provide targets for modulation of rumen development and function in the growing calf.

Effect of summer forage species grazed during finishing on animal performance, carcass quality, and meat quality.

Angus-cross steers (n = 60) were used to assess the effect of forage species [alfalfa (AL; Medicago sativa L.), bermudagrass (BG; Cynodon dactylon), chicory (CH; Cichorium intybus L.), cowpea (CO; Vigna unguiculata L.), and pearl millet (PM; Pennisetum glaucum (L. R Br.)] in replicated 2-ha paddocks for finishing on cattle performance, carcass quality, and meat quality in a 2-yr study. Steers were blocked by BW and assigned randomly to finishing-forage treatments before the start of the experiment. Steers grazing AL and CH had greater (P < 0.05) ADG than those grazing for BG, CO, and PM, whereas AL produced more (P < 0.05) gain/ha than CH, CO, and PM. Days steers spent grazing were longest (P < 0.05) for PM and shortest (P < 0.05) for CO. Steers grazing BG and CO produced heavier (P < 0.05) HCW than steers grazing BG and PM. Dressing percentage was greatest (P < 0.05) in steers grazing CO, and grazing AL resulted in greater (P < 0.05) dressing percentages than grazing BG, CH, and PM. Grazing AL and CH produced carcasses with more (P < 0.05) fat at the 12th rib than steers grazing warm-season grasses (BG and PM). Marbling scores tended to be greater (P = 0.06) for CO, but carcasses from steers grazing CO received greater (P < 0.05) quality grades than AL and CH. Trans-11 vaccenic (C18:1 trans-11; TVA) acid concentration in the LM was greater (P < 0.05) for BG than CH, CO, and AL. Conjugated linoleic acid, cis-9 trans-11 isomer, concentration was greatest (P < 0.05) for BG and PM than AL, CH, and CO. Grazing CH and PM increased (P < 0.05) the ratio of omega-6 to omega-3 fatty acids in the LM compared with AL, BG, and CO. Grazing legumes (AL and CO) resulted in lower (P < 0.05) Warner-Bratzler shear force values than other forage species. Consumers rated steaks from steers finished on AL and CO pastures greatest (P < 0.05) and steaks from steers finished on BG and CH least (P < 0.05) for overall palatability. Consumer preference was greatest (P < 0.05) for steaks from steers finished on AL and least (P < 0.05) for steaks from steers finished on BG and CH. Finishing steers on AL and CH during summer increased steer performance (> 1 kg/d). Finishing on legumes (AL and CO) increased dressing percentage, reduced Warner-Bratzler shear force values, and increased consumers preference, whereas finishing on grasses (BG and PM) enhanced anticarcinogenic fatty acid concentrations.

Effect of staged ovariectomy on measures of mammary growth and development in prepubertal dairy heifers.

Previous studies in prepubertal heifers suggest that the magnitude of reduction in mammary parenchymal growth in response to ovariectomy varies with the age at which surgery is performed. We hypothesized that ovarian secretions are essential for initiating mammary development but not required to maintain allometric mammary growth in prepubertal dairy heifers. The objectives of this study were to determine the effect of staged ovariectomy during the prepubertal period on mammary growth and tissue composition and the expression of selected genes. Prepubertal Holstein heifers at 2, 3 or 4 months of age were randomly assigned to one of two treatments, ovariectomized (OVX; n = 12) or sham operated (INT; n = 12). Mammary parenchyma (PAR) and fat pad (MFP) were harvested 30 days after surgery. Proximate composition of PAR and MFP (DNA, protein and lipid) as well as expression of the selected estrogen-responsive genes stanniocalcin1 (STC1), tissue factor pathway inhibitor precursor (TFPI) and proliferating cell nuclear antigen (PCNA) were determined in PAR and MFP by quantitative real-time PCR. The relative amount of epithelium and proportion of epithelia cell nuclei expressing the proliferation marker Ki67 were determined by histological and immunohistochemical analyses, respectively. MFP mass was not impacted by treatment but was decreased with age as was lipid content and concentration (P ⩽ 0.01). The mass of mammary PAR was reduced in OVX and increased with age (P ⩽ 0.01). Parenchymal tissue tended to have less total DNA, protein and lipid in OVX heifers. Parenchymal tissue concentrations of protein and DNA were increased with age and there was an age × treatment interaction. Treatment had no effect on either the Ki67 labeling index or percent epithelial area. The relative abundances of STC1, TFPI and PCNA mRNA in PAR were reduced in OVX. We did not find a significant impact of ovariectomy on mRNA expression when surgery was performed at 2 months compared with surgery at 3 or 4 months of age. However, having nearly undetectable PAR in two heifers ovariectomized at the earliest period (2 months of age) suggests that early ovariectomy is especially detrimental to subsequent parenchymal development.

Effect of exogenous somatotropin in Holstein calves on mammary gland composition and proliferation.

Pubertal mammary gland growth and development are hormonally regulated, but the details are poorly understood in calves. Our purpose was to evaluate the effects of exogenous growth hormone (GH) on the biochemical composition of the prepubertal mammary gland, mRNA expression of selected genes, and histological characteristics of the developing parenchyma (PAR). In this experiment, 19 calves (7 ± 4 d of age) were randomly assigned to 1 of 2 treatments: bovine somatotropin (bST, 500 mg; n = 10) or placebo (Sal; 0.9% saline; n = 9). Animals were treated every 3 wk beginning on d 23. Calves were assigned to an early (65 d; tissue harvested after 2 treatment injections) or late collection time (107 d; tissue harvested after 4 treatment injections). Calves were fed milk replacer and calf starter for 8 wk and starter and hay thereafter. Parenchyma and mammary fat pad (MFP) from one udder half were harvested for analysis of protein, lipid, and DNA. Additional tissues were preserved for histological analysis or snap-frozen for quantitative real-time PCR. Somatotropin treatment did not significantly alter the mass of PAR or MFP or the general pattern of development of epithelial structures. Significant increases were observed in protein/100 kg of body weight (BW), total protein, DNA concentration, DNA/100 kg of BW, and total DNA in 107-d calves, and a significant treatment by day interaction was observed for DNA and lipid concentrations in PAR. In MFP, a significant decrease was observed in protein/100 kg of BW in bST-treated calves and in total MFP protein in 65-d calves. A treatment by day interaction was found for total protein, DNA, and protein/100 kg of BW. In PAR, relative expression of ATPase-binding cassette 3 and growth hormone receptor were reduced by bST and both were lower in 107-d-harvest calves. Epithelial cell retention of bromodeoxyuridine (BrdU; possible indicator of stem-like cells) was greatest in 65-d bST-treated calves, and a significant time of sampling response and treatment × time interaction were observed. Expression of the proliferation marker protein Ki67 was numerically higher in bST-treated calves but the difference was nonsignificant. Retention of the BrdU label was reduced in 107-d calves. Exogenous growth hormone given to calves may affect mammary tissue composition and epithelial cell gene expression in subtle ways but exogenous supplementation with bST alone is not likely to alter overall development patterns or affect the mass of mammary parenchymal tissue. Whether such subtle changes have an effect on subsequent development or function is unknown.

Characterization of glucagon-like peptide 2 pathway member expression in bovine gastrointestinal tract.

Glucagon-like peptide 2 (GLP-2), secreted by enteroendocrine cells, has several physiological effects on the intestine of monogastric species, including promotion of growth of intestinal epithelium, reduction of epithelial cell apoptosis, and enhancement of intestinal blood flow, nutrient absorption, and epithelial barrier function. The regulatory functions of GLP-2 in the ruminant gastrointestinal tract (GIT) have not been well studied. The objectives of this investigation were to characterize the mRNA expression of 4 members of the GLP-2 pathway throughout the bovine GIT, including (1) proglucagon (GCG), the parent peptide from which GLP-2 is derived through cleavage by prohormone convertase; (2) prohormone convertase (PCSK1); (3) GLP-2 receptor (GLP2R); and (4) dipeptidyl peptidase IV (DPP4), the enzyme that inactivates GLP-2. Gene expression was evaluated in rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, and rectum collected at slaughter from prepubertal heifers, mature cows in early, mid, and late lactation, and nonlactating cows (n=3 per stage) by a gene expression profiling assay. In addition, mRNA expression of 14 genes involved in nutrient transport, enzyme activity, blood flow, apoptosis, and proliferation were evaluated in the 9 GIT tissues for their association with GCG and GLP2R mRNA expression. Immunohistochemistry was used to localize GLP2R protein in tissues of the lower GIT. Results indicated that mRNA expression of GCG, PCSK1, GLP2R, and DPP4 varies across the 9 GIT tissues, with greatest expression in small and large intestines, and generally nondetectable levels in forestomachs. Expression of DPP4 and GLP2R mRNA varied by developmental stage or lactational state in intestinal tissues. Expression of GCG or GLP2R mRNA was correlated with molecular markers of proliferation, apoptosis, blood flow, enzyme activity, and urea transport, depending on the tissue examined, which suggests a potential for involvement of GLP-2 in these physiological processes in the ruminant GIT. The GLP2R protein was expressed in intestinal crypts of the bovine GIT, which is consistent with the distribution in monogastric species. Our findings support a functional role of the GLP-2 pathway in bovine GIT and the potential for use of GLP-2 as a therapy to improve intestinal function and nutrient absorption in ruminants.

Effects of ovariectomy in prepubertal goats.

The objectives of this study were to determine the effects of ovariectomy on mammary gland development in prepubertal goats and to validate this model to study mammogenesis in young dairy ruminants. In this experiment, 3 months of aged goats were ovariectomized (ovx) while shammed goats played as surgery controls (sham). Thereafter, sham and ovx goats were slaughtered at 7 months of age to provide tissue for the assays. Results demonstrated that proliferation of mammary of mammary epithelial cells was significantly lower in ovariectomized goats compared to control goats. In ovx animal, epithelium structures were completely overstretched and epithelial ducts were undeveloped with limited branching whereas control animals had classical complex arborescent units with multiple round ductules and limited stroma. Concerning ERalpha (estrogen receptor alpha), PR (progesterone receptor) and P450 (aromatase) expression, results showed number of ERalpha, PR and P450 positive cells was higher in shammed goats compared to ovariectomized goats. All this results suggested that goat mammogenesis and ovarian control are similar to prepubertal heifers and that young goats are a good model to study mammary gland development in ruminants. In conclusion, we demonstrated that ovariectomy of prepubertal goats decreased proliferation of mammary epithelial cells with a profound alteration of cell adhesion molecules.

Influence of supramammary lymph node extract on in vitro cell proliferation.

OBJECTIVES: Experiments were conducted to evaluate whether or not bovine supramammary lymph node extract (LNE) could support cell proliferation when it was substituted for bovine growth serum (BGS) in cell culture media. MATERIALS AND METHODS: Two different preparations of LNE were tested. The first yielded protein concentration of 3 mg/mL and the second contained 27 mg/mL protein. Three cell lines (MDA-MB-435, MAC-T and 1C6) were used in serum starvation assays to evaluate LNE. Cell proliferation assays were used to determine growth stimulation in the presence of LNE, and short-term or rapid adaptation cultures were evaluated for LNE effects on cell survival. RESULTS: Heat-inactivated preparation 1 supported cell proliferation as well as or better (12-39%) than BGS following 2 days of serum starvation in culture. The second lymph node preparation provided a stimulatory effect (263-702% greater than BGS across all cell lines) following serum starvation at 2.7 and 5.4 mg/mL protein supplementation. A gradual adaptation process with lymph node supplementation into media maintained cell population growth on a short-term basis. However, once cells were trypsinized or scraped and re-seeded into 2.7 mg/mL LNE protein containing media, cells were unable to re-adhere, leaving them detached, and eventually appearing to be dead. CONCLUSIONS: Substitution of BGS with LNE protein dramatically stimulated cells to proliferate, but did not allow for rapid cell population growth adaptation in vitro.

Mammary response to exogenous prolactin or frequent milking during early lactation in dairy cows.

Frequent milking of dairy cows during early lactation results in a persistent increase in milk yield; however, the mechanism underlying this effect is unknown. We hypothesized that increased exposure of the mammary gland to prolactin (PRL) mediates the milk yield response. Fifteen multiparous Holstein cows were assigned to 3 treatments for the first 3 wk of lactation: twice daily milking with (2x + PRL) or without (2x) supplemental exogenous PRL, or 4 times daily milking (4x). Mammary biopsies were obtained at 7 DIM, and rates of [(3)H]thymidine incorporation into DNA in vitro were determined. Mammary expression of suppressors of cytokine signaling (SOCS)-1, -2, and -3; the long form of PRL-receptor; and alpha-lactalbumin mRNA was measured by real-time reverse-transcription PCR. Incorporation of [(3)H]thymidine into DNA was not affected by frequent milking or PRL treatment; however, analysis of autoradiograms revealed that stromal cell proliferation was greater in 4x cows. Mammary expression of SOCS-1 was not affected by milking frequency or PRL treatment. Expression of SOCS-2 mRNA was increased with frequent milking or PRL treatment, whereas expression of SOCS-3 mRNA was reduced by frequent milking or exogenous PRL. Abundance of PRL-receptor mRNA was reduced, whereas alpha-lactalbumin mRNA was increased with PRL treatment. These results demonstrate that the bovine mammary gland is responsive to exogenous PRL during early lactation. In addition, differences in the response to frequent milking or exogenous PRL during early lactation indicate distinct effects of PRL and milk removal on the mammary function of dairy cows.

Ovarian and IGF-I axis control of mammary development in prepubertal heifers.

Although much is known about the endocrine control of bovine mammary development, most heifer work has focused on periods near the time of puberty or during gestation. However, we have found that ovariectomy in the prepubertal period also markedly impacts mammary development well before the onset of estrus would have normally occurred. Interactions between the pituitary and ovary to control udder development are mediated at least in part via alteration in concentrations of local IGF-I axis molecules within the developing mammary gland. For example, in heifers treated with growth hormone or estrogen, expression of IGF-I binding proteins (IGFBP-3) protein was reduced, thus effecting an increase in free IGF-I. Ovariectomized heifers had reduced rates of epithelial cell proliferation, fewer IGF-I receptors, and less local IGF-I. Mammary tissue expression of fibronectin was increased in ovariectomized heifers, but laminin expression was higher in controls. Thus, alterations in specific extracellular matrix proteins likely impact heifer mammary development. As a result, we have initiated calfhood studies. At 30 days of age, it is difficult to detect parenchymal tissue in the udder. Only a thin cord of parenchymal tissue (150 mg per gland) is discernible. By 75 days of age, a rounded, walnut-like mass of mammary parenchymal tissue becomes very evident and at 90 days of age, this mass of tissue has grown to approximately 10 g, a approximately 60-fold increase. At 2 months of age, most proliferating epithelial cells (>92%) are confined to a population of light and intermediate-staining parenchymal cells. Between 2 and 5 months of age, a dark-staining cell population markedly emerges, but these dark cells were rarely labeled with bromodeoxyuridine (BrdU) and are likely to represent a more differentiated or committed cell lineage. The coordinated change in the proportions of each cell type suggests a progression from light-, to intermediate-, to dark-staining cell phenotypes. We are currently focusing on the importance of the ovary and mammary tissue synthesis of estrogens on emergence of specific populations of putative mammary stem cells.

Exposure to short day photoperiod during the dry period enhances mammary growth in dairy cows.

Exposure to short day photoperiod (SD; 8 h light:16 h dark) during the dry period increased milk yield of cows in the subsequent lactation. We hypothesized that this effect is due to increased growth of mammary cells in response to enhanced prolactin signaling to influence the insulin-like growth factor (IGF) axis. Multiparous Holstein cows were dried off 60 d before parturition and assigned to long day photoperiod (LD; 16 h light:8 h dark) or SD during the dry period. Mammary biopsies were obtained at approximately -40, -20, -10 and +10 d relative to expected calving. Expression of IGF-I, IGF-II, and IGF binding protein-5 mRNA was assessed by real time reverse transcription-polymerase chain reaction. In SD cows, incorporation of [3H]-thymidine in vitro increased from -40 d to -20 d and was greater at -20 d than in LD cows. A later increase in proliferation was observed at -10 d in LD cows. For both groups, cell proliferation decreased during lactation. Analysis by terminal deoxynucleotidyl transferase dUTP nick end labeling revealed that the apoptotic index in mammary epithelial cells was less in SD cows than in LD cows. Expression of IGF-II mRNA increased during the dry period and into lactation and was greater in SD cows. Expression of IGF binding protein-5 mRNA increased during lactation, but was unaffected by day length. Expression of IGF-I did not differ over time or between treatments. We concluded that exposure to SD during the dry period enhanced mammary growth relative to LD, and this may be related to increased expression of IGF-II. Treatment differences in the temporal pattern of proliferation indicated the existence of a critical period wherein photoperiod affects mammary gland development during the dry period.

Mammary epithelial proliferation and estrogen receptor alpha expression in prepubertal heifers: effects of ovariectomy and growth hormone.

The objectives of this study were to determine the effects of ovariectomy and growth hormone on mammary epithelial cell proliferation and estrogen receptor alpha (ER alpha) expression within the bovine mammary gland. Two experiments were performed. In the first experiment, eight Holstein heifer calves aged between 1 and 3 mo were ovariectomized, while six calves served as controls. At 6 mo of age, calves were treated with bromodeoxyuridine (BrdU) to label proliferating cells and sacrificed 2 h later. Coinciding with reduced mammary mass (304 +/- 25 vs. 130 +/- 21 g), proliferation of mammary epithelial cells was significantly lower in ovariectomized heifers compared to control heifers (2.24 vs. 0.25%). ER alpha expression was restricted to mammary epithelial cells and was not observed within intra-lobular stroma of parenchymal tissue. The proportion of ER alpha positive cells was significantly higher in ovariectomized heifers than in controls (36.1% +/- 2.2 vs. 46.7% +/- 2.4). In the second experiment, mammary biopsies were taken from five 6-mo-old heifers, immediately preceding and 7 d following a single injection of bovine growth hormone. Mammary epithelial cell proliferation (assessed by incorporation of 3H-thymidine) was increased by growth hormone. The proportion of ER alpha positive mammary epithelial cells was not increased by growth hormone. In conclusion, reduced mammary epithelial cell proliferation following ovariectomy was associated with an increase in ER alpha expression, whereas increased proliferation caused by bovine growth hormone was not associated with changes in the proportion of ER alpha positive cells.

Lactation persistency: insights from mammary cell proliferation studies.

A persistent lactation is dependent on maintaining the number and activity of milk secreting cells with advancing lactation. When dairy cows are milked twice daily, the increase in milk yield from parturition to peak lactation is due to increased secretory activity per cell rather than to accretion of additional epithelial cells. After peak lactation, declining milk yield is due to loss of mammary epithelial cells by apoptosis. During lactation, only 0.3% of mammary cells proliferate in a 24-h period. Yet this proliferative rate is sufficient to replace most mammary epithelial cells by the end of lactation. Management practices can influence lactation persistency. Administration of bovine somatotropin may enhance persistency by increasing cell proliferation and turnover, or by reducing the rate of apoptosis. Increased photoperiod may also increase persistency of lactation by mechanisms that are as yet undefined. Increased milking frequency during the first weeks of lactation increases milk yield, even after return to less frequent milking, with increases of approximately 8% over the entire lactation. A mammary cell proliferation response to frequent milking during early lactation appears to be involved. Conversely, advanced pregnancy, infrequent milking, and mastitis increase death of epithelial cells by apoptosis. Regulation of mammary cell renewal provides a key to increasing persistency. Investigations to characterize epithelial cells that serve as the proliferative population in the bovine mammary gland have been initiated. Epithelial cells that stain lightly in histological sections are evident through all phases of mammary development and secretion and account for nearly all proliferation in the prepubertal gland. Characterization of these cells may provide a means to regulate mammary cell proliferation and thus to enhance persistency, reduce the effects of mastitis, and decrease the necessity for a dry period.