Preweaning nutrient supply alters mammary gland transcriptome expression relating to morphology, lipid accumulation, DNA synthesis, and RNA expression in Holstein heifer calves.

The objective of this study was to analyze the mammary gland transcriptome to determine how preweaning nutrient supply alters the molecular mechanisms that regulate preweaning mammary development. Holstein heifers were fed via milk replacer (MR) either an elevated level of nutrient intake (ELE; on average, 5.9 ± 0.2 Mcal of ME in 8.4 L of MR/d, n = 6) or a restricted amount of nutrients (RES; 2.8 ± 0.2 Mcal of ME in 4 L of MR/d, n = 5) for 54 d after birth, at which point they were slaughtered and samples of mammary parenchyma tissue were obtained. Parenchymal mRNA was analyzed, and the fold change (FC) of 18,111 genes (ELE relative to RES) was uploaded to Ingenuity Pathway Analysis (IPA) software (Qiagen Bioinformatics, Redwood City, CA) for transcriptomic analysis. Using a threshold of P < 0.05, IPA identified that the FC of 1,931 of 18,811 differentially expressed genes (DEG) could be used for the analysis. A total of 18 molecular and cellular functions were relevant to DEG arising from the treatments; the 5 functions most associated with DEG were cell death and survival, cellular movement, cellular development, cellular growth and proliferation, and lipid metabolism. Based on the directional FC of DEG, the mammary gland of ELE heifers was predicted to have increased epithelial-mesenchymal transition (Z = 2.685) and accumulation of lipid (Z = 2.322), whereas the synthesis of DNA (Z = -2.137), transactivation of RNA (Z = -2.254), expression of RNA (Z = -2.405), transcription (Z = -2.482), and transactivation (Z = -2.611) were all predicted to be decreased. Additionally, IPA predicted the activation status of 13 upstream regulators with direct influence on DEG as affected by ELE feeding that were ligand-dependent nuclear receptors (n = 2), enzymes (n = 1), or transcription regulators (n = 10). Of these, 6 were activated (Z > 2) and 7 were inhibited (Z < -2). In summary, feeding ELE preweaning altered the mammary transcriptome of Holstein heifers, affecting cell functions involved in the morphological and physiological development of the mammary gland.

Symposium review: Integration of postweaning nutrient requirements and supply with composition of growth and mammary development in modern dairy heifers.

To optimize first lactation and lifetime milk yield, growth benchmarks were established to help meet the appropriate growth objectives of breeding weight and age at an economically viable time and to achieve the optimum body size and composition at first calving. These guidelines provide a framework that helps to minimize overfeeding and, thus, potential overconditioning of heifers, which can lead to postpartum metabolic issues and reduced milk yield. Concerns still exist that mammary development is impaired when body weight gain exceeds a certain threshold, which would negatively affects milk yield. The objective of this review was to integrate concepts of nutrient requirements, body growth and composition, mammary development, and milk yield to provide a systems-based perspective on first-lactation milk differences that have been associated with mammary development. Work in the early 1980s described the effect of high energy intake on mammary development and the relationship with circulating growth hormone linked the relationship between prepubertal growth, mammary development, and future milk yield. The primary outcome of that research was to provide an intuitive mechanism to explain why rapid growth during the prepubertal phase resulted in reduced milk yield. The observation of reduced mammary development could be repeated in almost every experiment, leading to the conclusion that high energy intake and increased average daily gain reduced mammary development through altered hormone status or some signaling processes. However, further work that looked at mammary development over the entire prepubertal growth phase recognized that mammary development was not reduced by high energy intake, and instead accumulated at a constant rate; thus, overall mammary parenchymal growth was a function of the time to reach puberty and the associated signals to change from allometric mammary growth. The mammary gland, similar to most reproductive organs, grows in proportion to the size of the body and not in proportion to nutrient intake during the postweaning, prepubertal phase. First-lactation milk yield, mammary development, and body composition will be further discussed in the context of mechanisms and opportunities.

Abrupt weaning reduces postweaning growth and is associated with alterations in gastrointestinal markers of development in dairy calves fed an elevated plane of nutrition during the preweaning period.

The benefits of feeding elevated quantities of milk to dairy calves have been well established. However, there is a reluctance to adopt this method of feeding in commercial dairy production because of concerns around growth, health, and ruminal development during weaning. The objective of this study was to characterize the effect of an abrupt (0 d step-down) or gradual (12 d step-down) feeding scheme when calves are fed an elevated plane of nutrition (offered 1.35 kg of milk replacer/d). For this experiment, a total of 54 calves were randomly assigned to an abrupt or a gradual weaning protocol before weaning at 48 d of life. Calves were housed and sampled in individual pens for the duration of the experiment, and milk, starter, and straw intake were measured on a daily basis. Body weight was measured every 6 d, whereas blood, rumen fluid, and fecal samples were collected on d 36 (pre-step-down), 48 (preweaning), and 54 (postweaning) of the experiment. Although the growth rates of the step-down calves were lower from d 37 to weaning (0.62 ± 0.04 vs. 1.01 ± 0.04 kg/d), the postweaning average daily gain was greater compared with the group that was abruptly weaned (0.83 ± 0.06 vs. 0.22 ± 0.06 kg/d). Total ruminal volatile fatty acid was greater in the step-down group on the day of weaning (d 48; 59.80 ± 2.25 vs. 45.01 ± 2.25 mmol), whereas the fecal starch percentage was lower during postweaning compared with the abruptly weaned calves (d 54; 3.31 ± 0.76 vs. 6.34 ± 0.76%). Analysis of the digestive tract of bull calves on d 55 revealed minimal differences between gross anatomy measurements of gut compartments as well as no morphological differences in rumen papillae development, yet the total mass of rumen when full of contents was larger in the step-down calves (7.83 ± 0.78 vs. 6.02 ± 0.78 kg). Under the conditions of this study, the results showcase the benefits of a step-down feeding strategy from an overall energy balance standpoint, due to increased adaptation of the gastrointestinal tract preweaning.

Effects of preweaning nutrient intake in the developing mammary parenchymal tissue.

Historically, mammary gland growth has been considered isometric the first 2 mo of life and then allometric until peripuberty. However, recent work indicated that the mammary gland might be responsive to nutrient intake preweaning. The objectives of this study were to describe the effects of nutrient intake preweaning on mammary gland development and to investigate cell specific proliferation during this phase of development. Twelve dairy heifer calves were fed either a fixed amount of milk replacer (MR; control, n = 6) or an amount of MR adjusted for BW (enhanced, n = 6). Control calves received a constant amount of a 28% crude protein, 15% fat milk MR per day that was equivalent to 2.8 Mcal of metabolizable energy intake per day; enhanced calves received 0.3 Mcal of metabolizable energy intake per kilogram of metabolic body weight (from 4.2 to 8.4 Mcal of metabolizable energy intake per day). All calves had constant access to water and a 22% crude protein commercial calf starter. Calves were killed at 54 ± 2 d. Control calves consumed 32.6 ± 2.4 kg of MR and 6.7 ± 0.5 kg of calf starter per calf, whereas the enhanced calves consumed 69.5 ± 2.4 kg of MR and 1.9 ± 0.5 kg of calf starter per calf over the 54-d period. Further, to evaluate putative stem cell proliferation, BrdU (5-bromo-2'-deoxyuridine; 5 mg/kg) was injected intramuscularly once per day between 12 to 15 d and again once per day between 24 to 27 d of life. Initial and final body weight for the control and enhanced treatments were 39.2, 61.0, 39.7, and 83.2 kg, respectively. At euthanasia, weights of liver, kidneys, pancreas, whole skinned mammary gland, and mammary parenchyma were measured. The growth rate of each organ was calculated using the concept of allometry as the difference in the change in organ weight as a percentage of body weight. The mammary glands of calves fed the enhanced diet were significantly heavier at euthanasia; when mammary parenchymal weight was analyzed, enhanced calves had 5.9 times greater mammary parenchymal mass, indicating the mammary gland was responsive to nutrient intake before weaning. Allometric growth of the mammary gland was initiated preweaning in the calves fed the enhanced treatment. Further characterization of mammary cells that retained BrdU label revealed no significant differences among the tissue slices analyzed between treatments; however, as calves fed the enhanced diet had more mammary parenchymal mass, if the number of label-retaining cells per counted slide were similar between treatments then the enhanced calves had a larger total population of putative mammary stem cells present in the mammary gland.

Fabrication and characterisation of spin coated oxidised PMMA to provide a robust surface for on-chip assays.

A method to prepare a highly stable carboxylic acid functional surface on various substrates for use in bioassays is reported. A thin layer of a poly(methylmethacrylate) (PMMA) is achieved by spin coating dissolved PMMA onto a variety of underlying substrates at various thicknesses in a range of c. 5-27 nm. Varying the PMMA concentration, time and spinning speed controls the thickness of the spin coated layer. The root-mean-squared roughness values of the spin coated PMMA are less than 1.5 nm, resulting in smooth and uniform layer. Substrate functionalisation is carried out by either ultraviolet/ozone (UV/O3) or oxygen plasma oxidation. Both techniques result in initially stable, highly functional films as demonstrated by the covalent attachment of amino-modified oligonucleotides, however longevity studies comparing the stability of films attached following oxidative activation show better stability for UV/O3 activated substrates when compared to oxygen plasma activated substrates. PMMA films activated by UV/O3 yield highly stable (for up to 24 days) functional surfaces that retain immobilised biomolecules after several extended wash steps. In contrast, films attached to surfaces pre-treated with oxygen plasma discharge lose their functionality within 5 days of oxidation. Direct DNA and sandwich antibody assays were successfully demonstrated on the UV/O3 functionalised surfaces, showing a low level of non-specific binding. Furthermore, the quenching of fluorescently labelled biomolecules bound to PMMA-coated gold-coated slides is shown to be dependent on the PMMA thickness, indicating potential usage in surface-plasmon resonance-based assays.

Lactation Biology Symposium: The effect of nutrient intake from milk or milk replacer of preweaned dairy calves on lactation milk yield as adults: a meta-analysis of current data.

Data from calf studies conducted over the past 20 yr has shown that preweaning nutrient intake, from milk or milk replacer, can have profound effects on development of the calf that enhance first lactation and lifetime productivity. Many of the studies show positive but not significant effects of preweaning nutrition on long-term productivity, primarily due to a lack of power, usually due to inadequate animal numbers per treatment. Meta-analyses were conducted using Comprehensive Meta-Analysis (CMA) software (version 2.2.064; Biostat, Englewood, NJ; Borenstein et al., 2005) to evaluate the effects of preweaning nutrient intake and preweaning ADG on first lactation milk production from studies where milk yield and preweaning treatment data were adequately described. Currently, 12 studies have been reported, describing milk yield of calves that had been fed various levels of preweaning nutrients, from both milk and milk replacer, and 11 evaluated the effect of preweaning ADG on long-term productivity. The estimated effect size for treatment (level of milk or milk replacer intake) and ADG were calculated. Using a random effects model, the overall milk yield response based on treatment was 435 ± 117 kg/lactation (P < 0.001), demonstrating that among the data sets milk yield in the first lactation was increased by increasing nutrient intake from milk or milk replacer in preweaned calves. Meta-regression of the effect of ADG resulted in the following equation: milk yield = -106 kg + 1,551.4 kg × ADG (kg/d; P = 0.01) indicating that for every kilogram of preweaning ADG, first lactation milk yield increased by 1,550 kg. Furthermore, the meta-analysis yielded an odds ratio of 2.09 (P = 0.001) indicating that calves fed for greater preweaning ADG were 2 times more likely to have greater milk yield in the first lactation. Finally, the 2001 Dairy NRC calf model was used to estimate the difference, within study, of intake over maintenance from milk replacer, using either the actual data or the published ADG; the difference in intake over maintenance was used as a predictor in a meta-regression and resulted in the following equation: milk yield = -60 kg + 1,100 kg × estimated difference in intake over maintenance (P = 0.02). The data demonstrate that nutrient intake from milk or milk replacer during the preweaning period positively impacted long-term productivity of dairy calves and provides new management opportunities to improve milk yield of dairy cattle. These data appear to indicate that there are significant developmental functions being programmed in the neonatal calf that require further investigation.

Preweaning milk replacer intake and effects on long-term productivity of dairy calves.

The preweaning management of dairy calves over the last 30 yr has focused on mortality, early weaning, and rumen development. Recent studies suggest that nutrient intake from milk or milk replacer during the preweaning period alters the phenotypic expression for milk yield. The objective of this study was to investigate the relationship between nutrient intake from milk replacer and pre- and postweaning growth rate with lactation performance in the Cornell dairy herd and a commercial dairy farm. The analysis was conducted using traditional 305-d first-lactation milk yield and residual lactation yield estimates from a test-day model (TDM) to analyze the lactation records over multiple lactations. The overall objective of the calf nutrition program in both herds was to double the birth weight of calves by weaning through increased milk replacer and starter intake. First-lactation 305-d milk yield and residuals from the TDM were generated from 1,244 and 624 heifers from the Cornell herd and from the commercial farm, respectively. The TDM was used to generate lactation residuals after accounting for the effects of test day, calving season, days in milk, days pregnant, lactation number, and year. In addition, lactation residuals were generated for cattle with multiple lactations to determine if the effect of preweaning nutrition could be associated with lifetime milk yield. Factors such as preweaning average daily gain (ADG), energy intake from milk replacer as a multiple of maintenance, and other growth outcomes and management variables were regressed on TDM milk yield data. In the Cornell herd, preweaning ADG, ranged from 0.10 to 1.58 kg, and was significantly correlated with first-lactation yield; for every 1 kg of preweaning ADG, heifers, on average, produced 850 kg more milk during their first lactation and 235 kg more milk for every Mcal of metabolizable energy intake above maintenance. In the commercial herd, for every 1 kg of preweaning ADG, milk yield increased by 1,113 kg in the first lactation and further, every 1 kg of prepubertal ADG was associated with a 3,281 kg increase in first-lactation milk yield. Among the 2 herds, preweaning ADG accounted for 22% of the variation in first-lactation milk yield as analyzed with the TDM. These results indicate that increased growth rate before weaning results in some form of epigenetic programming that is yet to be understood, but has positive effects on lactation milk yield. This analysis identifies nutrition and management of the preweaned calf as major environmental factors influencing the expression of the genetic capacity of the animal for milk yield.

The effects of increased milking frequency during early lactation on milk yield and milk composition on commercial dairy farms.

Increased milking frequency (IMF) during early lactation has the potential for carryover responses following the return to normal herd milking frequency. The objective was to determine the consistency of response of cows in commercial dairy farms to IMF during early lactation. Cows (n=398) were assigned randomly at calving within each of the 4 participating farms to 1 of 2 treatments. The control group was milked twice-daily (2×) during the entire lactation. The IMF group was milked 4-times daily (4×) starting on d 1 to 7, depending on farm, until d 21 postcalving and 2× thereafter. Cows in the IMF group were milked at the beginning and again at the end of the normal milking routine. Milking intervals differed across the farms for the 4× cows with a minimum interval of 3.5, 4.0, 5.0, and 6h for each of the 4 farms, respectively. The milk yield of cows subjected to IMF increased by 2.2±0.4 kg/d during the first 7 mo of lactation. Interactions of treatment with lactation group (primiparous vs. multiparous) were not significant. Although percentages of fat and protein in milk were decreased by early lactation IMF (3.69%±0.03 fat and 3.05%±0.02 true protein for control vs. 3.57%±0.03 fat and 2.99% ± 0.02 true protein for IMF), overall yields of protein were increased by IMF (1.02±0.01 vs. 0.98±0.01 kg/d). Early lactation IMF did not affect udder health as assessed by somatic cell count linear score. Cows subjected to IMF were 1.4 times more likely classified as subclinically ketotic than the control cows. Early lactation IMF has the potential to increase milk yield on commercial dairy farms. Although the direction of response was the same on all farms, the magnitude of the response was different among farms and appears influenced by management practices specific to each farm, which included, but were not limited to, housing system, stocking density, nutrition, genetics, and other covariates differing among farms.

Disinfection of meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a remote non-thermal gas plasma.

The effective disinfection of hospital surfaces is recognised as an important factor in preventing hospital-acquired infections. The purpose of this study was to quantify the disinfection rate of a novel gas plasma system on clinically relevant biofilms. Clinical isolates of Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus (MRSA) were grown as biofilms on glass surfaces and tested in a disinfection container remote from the plasma source. The strains used in this study were known to produce substantial quantities of biofilm and average log10 counts were 9.0 and 9.1 cfu/cm(2) for S. epidermidis and MRSA respectively. Counts were reduced by between 4 and 4.5 log10 after 1h of exposure for MRSA and S. epidermidis respectively. More prolonged treatment in the case of MRSA biofilms resulted in a 5.5 log10 reduction after 90 min. Biofilm samples were also placed in medical device packaging bags and similar rates of disinfection were observed.

Effects of increased milking frequency on metabolism and mammary cell proliferation in Holstein dairy cows.

Results of previous studies have shown that increased milking frequency (IMF) during early lactation results in increased milk yield not only during the period of IMF but also after cows have returned to a decreased milking frequency. The cellular mechanisms underpinning this increased milk yield and the overall effects of IMF on metabolism have not been well characterized. The objective of this study was to determine the effects of IMF on metabolism and mammary epithelial cell proliferation in dairy cows. Thirty primiparous and 30 multiparous Holstein cows were assigned randomly at calving to 1 of 2 treatments. The control group was milked twice daily (2x) for 119 d, whereas the IMF group was milked 4 times daily (4x) from d 2 postcalving until d 21 and then 2x from d 22 until d 119. Overall milk yield did not differ between treatments throughout the 119 d monitored; however, the interaction of treatment by week was significant in that IMF cows yielded 4.8kg/d more milk than control cows during wk 2 and 3 and had similar levels of milk yield during the remainder of the study period. Reanalysis of data excluding data from cows subjected to mammary biopsy suggested that the mammary biopsy procedure contributed to the lack of overall responses of milk yield, but that responses overall to IMF were greater in primiparous cows compared with multiparous cows. Plasma nonesterified fatty acid concentrations were elevated in multiparous cows subjected to IMF during the period of IMF, but were not influenced by treatment in primiparous cows. Plasma beta-hydroxybutyrate concentrations were not affected by treatment. Mammary tissue was collected by biopsy in a subset of cows (n=8 cows per parity and treatment) at calving and at d 21 and 75 postpartum and used for immunohistochemical localization of the cell proliferation antigen, Ki67. Effects of treatment on mammary epithelial cell proliferation were not significant, suggesting that other mechanisms must be responsible for carryover effects of IMF on lactational performance.

Reliability of intraobserver and interobserver sonographic endometrial stripe thickness measurements.

OBJECTIVE: To determine the intraobserver and interobserver reliability of endometrial stripe thickness measurements in women undergoing controlled ovarian hyperstimulation. DESIGN: Prospective blinded study. SETTING: Tertiary care, university hospital. PATIENT(S): Sixty-three patients undergoing controlled ovarian hyperstimulation and being monitored with transvaginal ultrasound were studied. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Intraobserver and interobserver variability of endometrial stripe thickness measurements between 2 blinded observers, with 2 observations made by each observer. RESULT(S): A statistically significant correlation was detected between the 2 measurements of each observer. The mean (+/-SD) interobserver difference between the average of the 2 measurements performed by both observers was 1.02 +/- 0.82 mm. A statistically significant correlation between the measurements of the 2 observers was detected. For both observers A and B, who used < or =6 mm as an abnormal endometrial thickness, an excellent level of agreement was detected between the 2 measurements made on each patient. When comparing the average values obtained by the 2 observers for each of the patients, an excellent level of agreement was detected. CONCLUSION(S): There is an excellent correlation between intraobserver and interobserver measurements of endometrial stripe thickness.

[Observations on pe-eclampsia-eclampsia and the advances in the evolution of some laboratory tests]. / Algunos conceptos sobre preeclampsia-eclampsia y los avances en su evalución por algunas pruebas de laboratorio.

The preeclampsia-eclampsia syndrome is a vasospastic disorder and probably has a placental origin. Once the hypertensive syndrome is established the uteroplacental blood flow is reduced as well as the intervillous blood flow. Since 18-24 weeks of gestation and before the symptoms of preeclampsia become overt, changes in placental flow velocity can be detected with Doppler technics. The placental theories for the etiology of preeclampsia are focused on the hypoxic effect in the trophoblastic tissue of second trimester. The placental ischemic changes are evident and seen in the uteroplacental bed. They are interrelated with the stages of trophoblastic invasion of the spiral arteries during the 14 and 20 weeks. When the trophoblastic invasion is over, the spiral arteries become a high resistance system. The defect observed in preeclampsia is the lack of invasion of the trophoblast to the maternal arteries. The diminished placental perfusion probably creates endothelial damage. This damage has several effects: decreased prostaglandin production, activated coagulation cascade, stimulated fibrin aggregation, and increased vascular permeability. The ideal laboratory test for preeclampsia shall predict the onset of this entity. Recent findings seem promising. The fibronectin concentration increases 2-3 wks. prior to the clinical manifestation of preeclampsia. Severe hypertension shows an abnormal decrease in fibronectin levels. Hypocalciuria has been described as an early predictor in the development of preeclampsia. Other agents undergoing extensive evaluation as predictors are: uric acid, b-thromboglobin, prolactin and atrial natriuretic peptide. Recently high levels of b-HCG (human corionic gonadotrophin) have been linked to a lack of trophoblastic invasion during the second trimester, therefore this is a potential marker for those patients that will eventually develop preeclampsia.

[Uterine arteriovenous malformations, Review of the literature]. / Malformaciones arteriovenosas del útero. Revisión de la literatura.

Uterine arteriovenous malformation (AVMs) are fairly rare. Seventy four cases have been reported in the medical literature. The clinical presentation of this entity is quite diverse. The approach to uterine AVM requires clinical alertness and to make the diagnosis a high index of suspicion is required. Prompt treatment is often essential. This article reviews the entire literature on uterine AVM emphasizing the various clinica presentations and treatment modalities available for the gynecologist.

Markedly elevated cytokines in pleural effusion during the ovarian hyperstimulation syndrome: transudate or ascites?

OBJECTIVE: To study levels of proinflammatory cytokines in pleural fluid during the severe ovarian hyperstimulation syndrome (OHSS). DESIGN: Case report. SETTING: Tertiary academic medical center. PATIENT(S): A 35-year-old female with a 6-year history of unexplained infertility on menotropin therapy and 28 healthy normal controls. INTERVENTION(S): Thoracentesis for severe pleural effusion and venipunctures. MAIN OUTCOME MEASURE(S): Interleukin-1 beta (IL-beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) levels were measured by ELISA and compared between pleural effusion and serum from normal controls. RESULT(S): Pleural effusion IL-1 beta and IL-6 levels were higher than serum. Interleukin-6 levels were elevated particularly in pleural effusion (1,961.89 pg/mL) compared with serum (3.9 +/- 0.41 pg/mL). CONCLUSION(S): Our results confirm the high cytokine levels observed in OHSS. Cytokines have been implicated in capillary permeability, extravasation of fluid, oliguria, and shock. We have postulated that these mediators are released from the corpora lutea into the peritoneum and systemic circulation. Alternatively, the presence of high cytokine levels in pleural fluid maybe the result of diaphragmatic defects, which allow for the migration of ascites into the pleural space.

Uterine arteriovenous malformation in a patient with recurrent pregnancy loss and a bicornuate uterus. A case report.

BACKGROUND: Arteriovenous malformations (AVM) of the uterus are a rare but potential cause of recurrent pregnancy loss. Only four cases of uterine AVM have resulted in a live birth after conservative management. There is no previous report in which a combination of a müllerian anomaly and an AVM existed concomitantly. CASE: A 33-year-old woman with a history of recurrent pregnancy loss was found to have a coexistent uterine AVM and a bicornuate uterus. The patient underwent arterial embolization and Strassman metroplasty and subsequently had a term pregnancy with a live birth. CONCLUSION: Management of uterine AVM should be individualized, taking into account the patient's desire for future fertility and the stability of her health at presentation.

[Placenta accreta during the first trimester of pregnancy. A case report]. / Placenta increta en el primer trimestre de embarazo. Presentación de un caso.

Placenta accreta is defined as the abnormal adherence of the placenta, totally or in part, to the underlying uterine surface. It is uncommon to find this abnormality of the attachment of the placenta in the first half or pregnancy. Only 10 cases have been reported. We present a case of placenta accreta in the first trimester in a patient with three previous curettages as a risk factor, diagnosed during curettage for a fourth missed abortion that required total abdominal hysterectomy. This case follows the pattern of those previously reported.