Effects of milk replacer acidification and free-access feeding on early life feeding, oral, and lying behavior of dairy calves.

Acidification is a practical way of preserving the bacteriological quality of milk so that it can be fed to calves under free-access conditions. The objectives of this study were to evaluate how milk replacer acidification and free-access feeding affect dairy calf behavior during the first week of life. Sixteen Holstein male calves were purchased at birth and transported to the University of Guelph Kemptville Campus Dairy Education and Research Centre. Calves were randomly assigned to 1 of 4 milk feeding programs: (1) free-access (ad libitum) feeding of acidified milk replacer (22% crude protein and 17% fat, 150 g/L; FA); (2) restricted (6 L/d, 150 g/L) feeding of acidified milk replacer (RA); (3) free-access feeding of nonacidified milk replacer (FN); and (4) restricted feeding of nonacidified milk replacer (RN). Formic acid was used to acidify milk replacer to a target pH between 4.0 and 4.5. Video recordings of each calf at 1, 2, and 6 d were analyzed continuously over 24 h for all occurrences of each behavior in the ethogram. Feeding behavior observations were organized into sucking bouts, from which feeding behavior outcome variables were calculated. Calves consuming acidified milk replacer demonstrated more fragmented feeding patterns, characterized by more pauses within a sucking bout (FA, FN, RA, and RN calves = 12.4, 4.4, 13.7, and 11.9 pauses/bout, respectively) and longer sucking bout duration (FA, FN, RA, and RN calves = 8.8, 5.2, 9.3, and 8.1 min/bout, respectively), than calves fed nonacidified milk replacer. Restricted-fed calves tended to have longer sucking bouts and performed more within-bout sucks (FA, FN, RA, and RN calves = 10.7, 5.8, 13.5, and 14.1, respectively) and pauses than free-access calves. Acidification and free-access feeding did not affect lying duration. Calves assigned to the acidified feeding treatments tended to perform more grooming behavior than those fed nonacidified milk replacer (FA, FN, RA, and RN calves = 0.9, 0.5, 0.8, and 0.6 h/d, respectively). Free-access feeding did not affect grooming duration. The observed differences in feeding and grooming behavior suggest that acidification to a pH between 4.0 and 4.5 may have altered the palatability of milk replacer. Calves assigned to the acidified milk replacer feeding treatments did not, however, show avoidance toward this feedstuff during the first week of life.

Clinical trial on the effects of a free-access acidified milk replacer feeding program on the health and growth of dairy replacement heifers and veal calves.

The objectives of this study were to evaluate the effects of free-access acidified milk replacer feeding on the pre- and postweaning health of dairy and veal calves. Individually housed calves were systematically assigned at birth to 1 of 2 feeding programs: free-access feeding (ad libitum) of acidified milk replacer (ACD, n=249) or traditional restricted feeding (3L fed twice daily) of milk replacer (RES, n=249). Calves were fed milk replacer containing 24% crude protein and 18% fat. Acidified milk replacer was prepared to a target pH between 4.0 and 4.5 using formic acid. Calves were weaned off milk replacer at approximately 6wk of age. Weaning occurred over 5d, and during this weaning period, ACD calves had access to milk replacer for 12h/d and RES calves were offered only one feeding of milk replacer (3 L) daily. Calves were monitored daily for signs of disease. Fecal consistency scores were assigned each week from birth until weaning. A subset of calves was systematically selected for fecal sampling at 3 time points between 7 and 27d of age. Fecal samples were analyzed for enterotoxigenic Escherichia coli F5, Cryptosporidium parvum, rotavirus, and coronavirus. Hip width, hip height, body length, heart girth, and body weight were measured at birth and weaning. Postweaning body weight measurements were collected from the heifers at approximately 8mo of age. Postweaning body weight and carcass grading information was collected from the veal calves at slaughter once a live weight between 300 and 350kg had been achieved. The odds of ACD calves being treated for a preweaning disease event tended to be lower than that of the RES calves (1.2 vs. 5.2%, respectively). Preweaning mortality, postweaning disease treatment, and postweaning mortality did not differ between feeding treatments. The ACD feeding treatment supported greater preweaning average daily gain (0.59 vs. 0.43kg/d) and structural growth than RES feeding. Postweaning average daily gain and carcass characteristics were similar for ACD and RES calves. These results indicate that free-access acidified milk replacer feeding tended to support improved health, and greater body weight gain and structural growth during the preweaning period; these effects did not persist in the postweaning period. The growth advantage observed before weaning in the ACD calves likely disappeared due to the weaning methods used.

Spectral CT of carotid atherosclerotic plaque: comparison with histology.

OBJECTIVE: To distinguish components of vulnerable atherosclerotic plaque by imaging their energy response using spectral CT and comparing images with histology. METHODS: After spectroscopic calibration using phantoms of plaque surrogates, excised human carotid atherosclerotic plaques were imaged using MARS CT using a photon-processing detector with a silicon sensor layer and microfocus X-ray tube (50 kVp, 0.5 mA) at 38-µm voxel size. The plaques were imaged, sectioned and re-imaged using four threshold energies: 10, 16, 22 and 28 keV; then sequentially stained with modified Von Kossa, Perl's Prussian blue and Oil-Red O, and photographed. Relative Hounsfield units across the energies were entered into a linear algebraic material decomposition model to identify the unknown plaque components. RESULTS: Lipid, calcium, iron and water-like components of plaque have distinguishable energy responses to X-ray, visible on spectral CT images. CT images of the plaque surface correlated very well with histological photographs. Calcium deposits (>1,000 µm) in plaque are larger than iron deposits (<100 µm), but could not be distinguished from each other within the same voxel using the energy range available. CONCLUSIONS: Spectral CT displays energy information in image form at high spatial resolution, enhancing the intrinsic contrast of lipid, calcium and iron within atheroma. KEY POINTS: Spectral computed tomography offers new insights into tissue characterisation. Components of vulnerable atherosclerotic plaque are spectrally distinct with intrinsic contrast. Spectral CT of excised atherosclerotic plaques can display iron, calcium and lipid. Calcium deposits are larger than iron deposits in atheroma. Spectral CT may help in the non-invasive detection of vulnerable plaques.

Spectroscopic (multi-energy) CT distinguishes iodine and barium contrast material in MICE.

OBJECTIVE: Spectral CT differs from dual-energy CT by using a conventional X-ray tube and a photon-counting detector. We wished to produce 3D spectroscopic images of mice that distinguished calcium, iodine and barium. METHODS: We developed a desktop spectral CT, dubbed MARS, based around the Medipix2 photon-counting energy-discriminating detector. The single conventional X-ray tube operated at constant voltage (75 kVp) and constant current (150 microA). We anaesthetised with ketamine six black mice (C57BL/6). We introduced iodinated contrast material and barium sulphate into the vascular system, alimentary tract and respiratory tract as we euthanised them. The mice were preserved in resin and imaged at four detector energy levels from 12 keV to 42 keV to include the K-edges of iodine (33.0 keV) and barium (37.4 keV). Principal component analysis was applied to reconstructed images to identify components with independent energy response, then displayed in 2D and 3D. RESULTS: Iodinated and barium contrast material was spectrally distinct from soft tissue and bone in all six mice. Calcium, iodine and barium were displayed as separate channels on 3D colour images at <55 microm isotropic voxels. CONCLUSION: Spectral CT distinguishes contrast agents with K-edges only 4 keV apart. Multi-contrast imaging and molecular CT are potential future applications.

Zonal centrifuges and other separation systems.

This discussion has included only a partial list of the systems now under development at Oak Ridge as part of the feasibility studies for the Molecular Anatomy Program. It is evident that we are still in the "Robert Goddard" phase of this work. It may not be premature, however, to suggest several conclusions. Biomedical scientists are discouraged on discovering that developmental efforts cost more, by one or two orders of magnitude, than pure research. In part this is because the full cost of development is generally shown, while in pure research some of the costs may be hidden, or the funds supplied by several sources. Regardless of the reason, the fact remains that development is expensive, as is well understood in nuclear physics and space science. The role and mission of the large national laboratories, and the kinds of research that should be done in them, have been discussed by Weinberg (63). The studies described here were in part stimulated by his ideas. We have been unable to find an environment outside a large national laboratory where a program like the Molecular Anatomy Program could be undertaken at the present rate. It appears that programs which attempt to make use of the multidisciplinary approach characteristic of national laboratories should be carefully designed and should evolve experimentally. There is less chance of success when a program is an administrative invention than when it evolves from scientific invention and discovery. It has been pointed out (64) that most program decisions in science are secret decisions in the sense that the scientific community as a whole does not participate in them. If a choice is to be made at some future time between large-scale expenditures for exploring space, for developing new weapons systems, for constructing new accelerators, for designing large reactors, or for systematically developing methods to explore the molecular basis of human disease, then we will need sufficient information to evaluate each alternative fully, and the information should be generally available. It appears desirable, therefore, to allow the Molecular Anatomy Program to proceed to a point where the full range of its contributions and its inherent limitations may be seen. A rational choice may then be made.

Reproducibility of sonographic assessment of fetal liver length in diabetic pregnancies.

OBJECTIVES: Assessing fetal liver size might be useful in monitoring the effectiveness of the treatment of diabetes in pregnancy. We aimed to assess the reproducibility of fetal liver-length measurement in pregnant women with diabetes. METHODS: From 3 April 2006 to 5 December 2006, we assessed intraobserver and interobserver variation of fetal liver-length measurements on 55 sonograms in 44 pregnant women with diabetes, 34 of whom had gestational diabetes. The mean maternal age was 33 years, the mean maternal weight was 92 kg and the mean body mass index (BMI) was 33.7 kg/m(-2). The effect of covariates BMI, gestational age and maternal age on the reproducibility of fetal liver length was assessed by calculating intraobserver SD ratios. We compared liver length with abdominal circumference and gestational age. Nine of 12 sonographers scanned, on average, six women (range, 3-12) as the first sonographer, and all 12 sonographers scanned, on average, four women (range, 1-10) as the second sonographer. The data were analyzed using a hierarchical linear model. RESULTS: Measurement of fetal liver length was reproducible. The intraobserver SD was 3.06 (95% CI, 2.68-3.59) mm; the interobserver SD was 2.17 (95% CI, 0.59-4.83) mm; the intraobserver correlation was 0.77 (95% CI, 0.63-0.87), and the interobserver correlation was 0.84 (95% CI, 0.51-0.99). The covariate effects were minimal, the SD for a 1-unit increase in the covariate was 1.06 for gestational age, 0.98 for BMI, and 0.97 for maternal age. CONCLUSIONS: Measurement of fetal liver length in the diabetic pregnancy is reproducible. It is worthy of further investigation as a parameter for monitoring the effectiveness of treatment of the diabetic pregnancy.

The effect of exposure to dibutyryl cyclic adenylic acid on the membrane antigenicity of SV40 tumor cells.

When SV40 tumor cells (F5-1) in culture were treated with dibutyryl cyclic adenylic acid (DbcAMP),marked alterations occurred in their growth and morphology. Additionally, the incorporation or uptake of labeled thymidine, uridine, phenylalanine, and choline were reduced by this treatment. These modifications with DbcAMP exposure produced conditions simulating those of a contact-inhibited state. The immunization of hamsters with X-irradiated tumor cells previously cultured in the presence of DbcAMP indicated that the tumor-specific transplantation antigens and/or fetal antigens were altered during short-term expsoure to DbcAMP. Further examination of membrane antigens of SV40 tumor cells using the isotopic antiglobulin technique demonstrated a significant reduction in the tumor membrane antigen on cells cultured with DbcAMP.

Agonist-mediated tyrosine phosphorylation of isoforms of the shc adapter protein by the delta opioid receptor.

Maximally effective concentrations of the opioid agonist D-ala2-D-leu5-enkephalin resulted in some 2-3-fold enhancement of tyrosine phosphorylation of the p52 Shc adapter protein in a clone of Rat-1 fibroblasts transfected to express stably the murine delta opioid receptor. More limited modifications of the tyrosine phosphorylation status of the p46 and p66 forms of Shc were observed in parallel. Epidermal growth factor caused some 10-12-fold enhancement of tyrosine phosphorylation of p52 Shc and marked increases in the p46 and p66 forms. The effect of D-ala2-D-leu5-enkephalin was prevented by pretreatment of the cells with pertussis toxin and was not observed in non-transfected parental fibroblasts whereas the effect of epidermal growth factor was still manifest in both these situations. Half-maximal effects of D-ala2-D-leu5-enkephalin on p52 Shc tyrosine phosphorylation were produced with sub-nanomolar concentrations, in agreement with previous results on the tyrosine phosphorylation of p44MAPK (Burt et al., 1996). p52 Shc became tyrosine phosphorylated more rapidly than p44MAPK in response to D-ala2-D-leu5-enkephalin and its enhanced tyrosine phosphorylation was maintained for at least 10 min.

Studies of epidermal growth factor receptor inhibition in breast cancer.

Until recently, there has been little knowledge on the growth control of oestrogen receptor (ER)-negative ductal carcinoma in situ (DCIS) and invasive breast cancer. The recent development of DCIS models, such as transgenic mice, cell-line xenograft models and, importantly, in vivo human DCIS xenograft models has facilitated the investigation and understanding of the control of growth of early pre-invasive breast lesions. Recent studies have shown that ER-negative DCIS, unlike ER-positive DCIS, is hormone independent and does not respond to anti-oestrogen treatment. Moreover, DCIS of the comedo type utilises type I tyrosine kinase growth factors, such as epidermal growth factor receptor (EGFR) and c-erbB-2, in receptor signalling for growth. New data underscore the importance of EGFR as the major modulating growth factor receptor in the control of proliferation in the breast. Pre-clinical studies performed on human DCIS xenografts in nude mice suggest a potential role for EGFR tyrosine kinase inhibitors (EGFR-TKIs). More specifically, ZD1839, a novel orally active and selective EGFR-TKI, has been shown to produce a response in DCIS through a decrease in epithelial proliferation. These findings have enhanced our knowledge of signal transduction pathways in cancer and indicate that tyrosine kinase blockade of EGFR has potential for the treatment and chemoprevention of DCIS. It is hoped that further advances in this area and evaluation of EGFR-TKIs in Phase II/III clinical trials will allow their therapeutic potential as anticancer agents to be appreciated.

C19 steroidal precursors of estrogens.

The pathways of biosynthesis of the estrogens from C19 steroids has been examined by in vitro kinetic experiments using human placental microsomes and two precursors, each labeled with a different radioisotope. The results indicate that under the conditions employed, androstenedione is an obligatory intermediate in the conversion of 3 beta-hydroxy-5-androsten-17-one (dehydroisoandrosterone) or its sulfate, dehydroisoandrosterone sulfate, into estrone and estradiol. The same result was obtained when the microsomal fraction was replaced by either a 1000 x g supernatant preparation from human placenta or a homogenate of placental mitochondria. If alternative pathways involving C19-hydroxylated derivatives of dehydroisoandrosterone or its sulfate exist, they appear to be of minor quantitative significance.

Changes in the expression of guanine nucleotide-binding proteins during differentiation of 3T3-F442A cells in a hormonally defined medium.

Using specific antisera, the expression of the G protein alpha subunits, G8, G(i1), G(i2), G(i3) and G0, were determined in 3T3-F442A cells during their differentiation to adipocytes in a hormonally defined medium. Differentiation caused distinct increases in the expression of two Gs isoforms and decreases in the expression of both G(i2) and G(i3). Differentiation also resulted in a 2- to 4-fold increase in forskolin-stimulated adenylyl cyclase activity and a 15-fold increase in the response of cells to a beta-adrenergic agonist. The increase in Gs expression was also observed, to a lesser degree, in cells maintained at confluence under conditions where morphological conversion was negligible and the decreased expression of G(i2) and G(i3) and the increased beta-adrenergic responsiveness did not occur.

Growth hormone induces the tyrosine phosphorylation and nuclear accumulation of components of the ISGF3 transcription factor complex.

Growth hormone induced the accumulation of the stat91 and p84 subunits of the transcription factor complex ISGF3 in nuclear fractions of 3T3-F442A cells. Nuclear levels of p84 and stat91 peaked 30-60 min after addition of growth hormone. Growth hormone also induced the tyrosine phosphorylation of two proteins, with similar sizes to stat91 and p84, in both nuclear and cytosolic fractions. The time course of growth hormone-induced tyrosine phosphorylation of these proteins paralleled the nuclear accumulation of stat91 and p84. Immunoprecipitation with stat91-specific antibodies confirmed that growth hormone induced the tyrosine phosphorylation of stat91 and an associated protein of M(r) = 120 kDa. These findings suggest a mechanism for the modulation of specific gene transcription by growth hormone.

Human muscle proteins: analysis by two-dimensional electrophoresis.

Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy, denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be caused by generalized muscle-fiber damage.

Synthesis and biological activity of 5-(4-chlorobenzoyl)-4-(hydroxymethyl)-1-methyl-1H-pyrrole-2-acetic acid, a major metabolite of zomepirac sodium.

5-(4-Chlorobenzoyl)-4-(hydroxymethyl)-1-methyl-1H-pyrrole-2-acetic acid (2), the major oxidative metabolite of zomepirac (1), was synthesized starting with ethyl 5-(4-chlorobenzoyl)-1,4-dimethyl-1H-pyrrole-2-acetate (3), the ethyl ester of 1. Compound 3 was oxidized with selenium dioxide to afford the alpha-oxoester, 5. Bromination of 5 with N-bromosuccinimide produced bromomethylpyrrole 7, and reaction of 7 with acetate produced by corresponding acetoxymethylpyrrole 8. Hydrogen sulfide effected the selective reduction of the side-chain carbonyl group if 8 to give 9. Saponification of 9 gave the title compound, 2. Synthetic 2 was identical with the isolated metabolite of zomepirac (1). Biological testing revealed that the metabolite was essentially devoid of the biological activity associated with zomepirac.

Cyclic AMP potentiates growth hormone-dependent differentiation of 3T3-F442A preadipocytes: possible involvement of the transcription factor CREB.

We have examined the effects of cyclic AMP on the differentiation of 3T3-F442A preadipocytes. High concentrations of intracellular cyclic AMP potently inhibited differentiation whereas low concentrations of intracellular cyclic AMP, induced by a number of different agents, promoted differentiation. To analyse these effects of cyclic AMP more closely, we developed a two-phase protocol for the differentiation of 3T3-F442A cells. Growth hormone (GH) was necessary to prime confluent cells during the first phase, following which, the addition of insulin and other adipogenic agents then promoted terminal differentiation. Cyclic AMP potentiated the priming action of GH but exerted an inhibitory effect on terminal differentiation when added to cells which had previously been primed with GH showing that the effects of cyclic AMP on preadipocyte differentiation are stage-dependent. We analysed the stimulatory effects of cyclic AMP during GH priming and found that cyclic AMP induced phosphorylation of the cyclic AMP response element (CRE) binding protein CREB and activated transcription of a CRE-linked reporter gene. Furthermore, GH also stimulated CREB phosphorylation and activation and this effect was potentiated by cyclic AMP. These results suggest a mechanism for the synergistic priming of preadipocytes for terminal differentiation by cyclic AMP and GH via the activation of differentiation genes containing CREs.

Placental compressibility: implications for indirect diagnosis of posterior placenta previa.

A number of problems beset the indirect diagnosis of posterior placenta previa using transabdominal ultrasound. We add a new potential complicating factor. In 128 pregnancies at or beyond 30 weeks' gestation, measurements were taken from the fetal skull to the maternal sacrum before and after compression. Up to 69% compressibility of the placenta was found in vivo and in vitro. Modified fetal skull to maternal sacrum measurement criteria were devised from the results. Placenta previa is highly unlikely if the measurement from the fetal skull to maternal sacrum is less than 10 mm before compression or less than 7 mm after compression. Placenta previa is probable if the measurement is greater than 20 mm before compression or greater than 15 mm after compression. In 40% of the cases, posterior placenta previa could not be excluded. We conclude that placental compressibility is an additional confounding problem for indirect ultrasound assessment of posterior placenta previa and that indirect assessment should be attempted only if maneuvers to image the lower uterine segment directly are unsuccessful.

Placental and luteal steroidogenesis in the pregnant rhesus monkey.

Progesterone, 20alpha-dihydroprogesterone, estrone and estradiol-17beta concentrations were estimated by radioimmunoassay in blood plasma from uterine, uteroovarian and femoral veins of rhesus monkeys (Macaca mulatta) on days 22, 49, 128 and 160 of gestation. Steroids were consistently more concentrated in uterine and uteroovarian that in femoral venous plasma and in many cases levels in the uteroovarian vein were also higher than those in the uterine vein indicating luteal secretion of both progestins and estrogens thoughout gestation. In some animals, however, the corpus luteum appeared quiescent. As reflected in the decline in the uterine venous progesterone/estradiol-17beta concentration ratio, a shift in steroid contribution from the uterus and its contents occurred between days 22 and 49 of gestation with progesterone declining more rapidly than estradiol-17beta. Progesterone/20alpha-dihydroprogesterone was higher in both uterine and uteroovarian than in femoral venous plasma suggesting peripheral metabolism of progesterone to 20alpha-dihydroprogesterone.