Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts

The effects of bovine cumulus cell-conditioned medium on cloned bovine embryonic development and subsequent chromosome complement were examined using an air-dry procedure. Conditioned media were prepared using CR1aa supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). Nuclear-transferred embryos were reconstructed with nuclei from cumulus cells. Similar cleavage, morula, and blastocyst development was observed in conditioned media groups compared with the co-culture group. No differences (P > 0.05) were observed in the composition of blastocyst chromosomes after co-culture in different media, either with or without starvation of donor cells. The overall diploid blastocyst rate ranged from 75% to 84%. Chromosomal complement of blastocysts, however, was very different between conditioned medium and co-culture treatments. Overall incidence of chromosomal anomalies was 40% in conditioned medium, which was significantly higher (P < 0.001) than the co-culture group (20%). Moreover, a higher incidence (P < 0.05) of chromosomally abnormal blastocysts (41.5%) was observed after culture with FBS-containing conditioned medium than those cultured in BSA-containing conditioned medium (31.4%). No diploid improvement was observed after exchange of the culture system from conditioned medium to co-culture, or from co-culture to conditioned medium after the first 72 h of culture. The results of this study also indicated that the overall cell number was much lower (P < 0.01) in blastocysts with chromosomal abnormalities than those with a normal diploid state. We have concluded that medium conditioned with bovine cumulus cells increases the incidence of chromosomal anomalies in nuclear reconstructed embryos.     

Flavonoids and cell membrane fluidity

The membrane fluidity characteristics of multilamellar (MLV) and extruded liposomes prepared with kaempferol (K), kaempferol-3-glucoside (KG), (−)-epigallocatechin (EGC) or (−)-epigallocatechin-3-gallate (EGCG) are presented. Kaempferol caused the highest increase in fluorescence polarisation of DPH in both liposomes (other compounds had not) indicating that K with n_K/n_Lip below 0.2 or 0.1 decreased the membrane fluidity, while at higher molar ratios the membrane fluidity increased. EPR measurements with MLV and spin probes MeFASL(10,3) and MeFASL(2,11) showed a significant decrease in fluidity in the upper part of the membrane for all flavonoids measured, and in the core of the membrane an increase in fluidity for EGCG and EGC. Computer simulation of the EPR spectra showed that the membrane of the MLV used was composed of at least three coexisting domain types with different fluidity and that the order parameter of the most ordered domains is responsible for membrane fluidity alterations.     

Isolates of cell types from sorghum stems: Digestion,cell wall and anatomical characteristics

Cell types were separated from internode 5 of sorghum stems to study the interrelationship between digestion characteristics and cell wall composition. Isolates of epidermis (EPI), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) were freeze-dried and ground for analysis. The cell fractions were digested in rumen fluid for times between 0 and 96 h, and wall composition measured using detergent extraction procedures. In-vitro dry matter digestibility (g kg^?1 after 48 h) of cell fractions was in the order of PITH (849-906) > IVB (794-816) > SCL (692-701) > VBZ (641-679) > EPI (608-628). Total cell wall content (CWC), indigestible CWC, and lignin content followed the inverse order. Lignin concentration on a dry matter or cell wall basis was highly correlated with indigestible wall residue after 96 h. The proportion of cell wall digested after 96 h was higher for SCL and VBZ cells (61·8-68·2%) than for PITH cells (48·4-56·1 %), despite the former having lignin content three to five times higher than that of PITH cells. Clearly, there were differences between the cell types in wall composition or chemical linkages between wall components that lead to the observed differences in wall digestion.     

Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos

Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases, NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity and POU5F1, SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may lose NANOG. Cystic embryoid body-like structures expressing POU5F1, CDX2 and EOMES were produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressing POU5F1 and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.     

Composition of cell walls isolated from cell types of grain sorghum stems

Cell types were isolated from sorghum stems at two stages of development, anthesis and grain maturity, to study cell wall characteristics. Cell walls were isolated from epidermis (EPID), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) and analysed for total carbohydrate, acid insoluble lignin, total uronosyls, neutral sugars and hydroxycinnamic acids. In addition, walls from SCL, VBZ, IVB and PITH were subjected to chemical fractionation to separate wall carbohydrate into polysaccharide groups. Although wall characteristics were similar at both plant maturities, there were differences in lignin concentration, hydroxycinnamic acids, and carbohydrate composition among the cell wall types. Lignin was lowest in the PITH walls (169 g kg⁻¹) and highest in SCL and EPID (c 211 g kg⁻¹). Cellulose was most abundant in VBZ and SCL walls with greater secondary wall formation. Pectic materials were most abundant in PITH walls. Xylans were similar among wall types except for EPID that contained higher amounts of xylose. Releasable hydroxycinnamates were not as consistent among the cell wall types. Total ferulates, including ester linked and releasable ether linked, tended to increase from PITH to SCL (8 to 15 g kg⁻¹ CW) with an increase in the proportion etherified within the wall matrices (PITH 51%; SCL 66%). Total p‐coumarates showed opposite trends with PITH walls having significantly more (35 g kg⁻¹ CW) than VBZ or SCL (19 and 13 g kg⁻¹ CW). EPID walls contained the least pCA (6.5 g kg⁻¹ CW). Except for the hydroxycinnamates, compositional trends for the different wall types would reflect changes from primary walls to increased amounts of secondary wall. Neutral sugar analysis of indigestible residues indicated similar carbohydrate compositions among the cell wall types, with xylose being less degradable than all other wall sugars. © 1999 Society of Chemical Industry     

Practical cell labeling with magnetite cationic liposomes for cell manipulation

Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10³–10⁵ cells for personalized cell processing, we determined that 10 μg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method.     

Chitosan kills bacteria through cell membrane damage

The bactericidal activity of chitosan (CS) acetate solution against Escherichia coli and Staphylococcus aureus was evaluated by the enumeration of viable organisms at different incubation times. Morphologies of bacteria treated with CS were observed by transmission electron microscopy (TEM). The integrity of the cell membranes of both species and the permeabilities of the outer membrane (OM) and inner membrane (IM) of E. coli were investigated by determining the release from cells of materials that absorb at 260 nm, changes in the fluorescence of cells treated with the fluorescent probe 1-N-phenylnaphthylamine (NPN) and release of cytoplasmic beta-galactosidase activity. In addition, the interaction of CS with synthetic phospholipid membranes was studied using gel permeation chromatography (GPC), UV-VIS spectrophotometery, Fourier-transform infrared spectroscopy (FT-IR) and thermal analysis. Results showed that CS increased the permeability of the OM and IM and ultimately disrupted bacterial cell membranes, with the release of cellular contents. This damage was likely caused by the electrostatic interaction between NH(3)(+) groups of CS acetate and phosphoryl groups of phospholipid components of cell membranes.     

Evaluation of the overall accuracy of the DeLaval cell counter for somatic cell counts in ovine milk

The DeLaval cell counter (DCC) is a portable device designed for on-farm somatic cell count (SCC) analysis in bovine milk. This study evaluated the performance of the DCC when analyzing ovine milk. A total of 29 composite ovine milk samples, ranging between 20 × 10³ and 2,200 × 10³ cells/mL, were divided into 15 aliquots/milk sample corresponding to 5 SCC methods using 3 types of preservation (unpreserved, azidiol, and bronopol). The SCC methods were the Fossomatic (FSCC), the DCC in undiluted samples, and the DCC in samples diluted 1:1 in 3 different types of diluents (PBS + Triton X-100, PBS + ethidium bromide + Triton X-100, and PBS + propidium iodide + Triton X-100). All analyses were carried out in duplicate. In addition, each sample was analyzed in quadruplicate by the direct microscopic method (DMSCC) using Pyronin Y-methyl green as a stain. Comparison of methods was based on overall accuracy studies (means comparison, repeatability, and regression studies vs. DMSCC and FSCC as reference methods). The DCC methods used to analyze milk samples diluted in staining solution (with ethidium bromide or propidium iodide) showed large coefficients of regression (b = 0.91 to 1.01) and correlation (r > 0.99) when compared with the DMSCC and FSCC methods. In these samples the DCC gave repeatability values (s_r = 33 to 48 × 10³ cells/mL) similar to the DMSCC (s_r = 36 × 10³ cells/mL), and their log SCC means (5.52 to 5.54) did not differ from the reference value (5.54). However, undiluted samples analyzed by the DCC method showed large standard deviations of repeatability and SCC values lower than those by the DMSCC or FSCC methods, probably because of the high solids content in ovine milk. The type of preservation did not affect the outcomes. Consequently, the DCC was determined to be accurate when analyzing diluted ovine milk based on comparison with the SCC reference methods.     

Flow cytometry and cell sorting for yeast viability assessment and cell selection

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P⩽0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.     

Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.     

Impact of early lactation somatic cell count in heifers on somatic cell counts over the first lactation

The objective of this study was to estimate the impact of somatic cell count in early lactation (SCCel) from Belgian dairy heifers on test-day somatic cell count (SCC) in first lactation. Geometric mean SCCel [5 to 14 d in milk (DIM)] of the 14,766 available samples was 104,000 cells/mL, and decreased from 178,000 at 5 DIM to 74,000 cells/mL at 14 DIM. Proportion of SCCel >200,000 cells/mL was 27.5. Heifers calving in the period April-June had highest SCCel.In total, 117,496 monthly SCC were measured. A multilevel regression analysis revealed that an increase of the natural log-transformed SCCel (LnSCCel) by one unit on average resulted in an increase of test-day natural log-transformed SCC (LnSCC) by 0.22 unit. The impact of LnSCCel on LnSCC depended on when LnSCCel was measured; an elevated LnSCCel at 14 DIM was more consequential than an equally elevated LnSCCel at 5 DIM. The probability of having a test-day SCC >200,000 cells/mL during the first lactation, also increased with an increasing LnSCCel. The negative effect of an elevated LnSCCel was still present, although to a lesser extent, in heifers with a second test-day SCC 相似文献   

Viable cell yield from active dry yeast products and effects of storage temperature and diluent on yeast cell viability

Active dry yeast (ADY) products are commonly fed in the dairy industry, but research regarding quality control for such products is limited. The objectives of this study were to determine yeast viability in field samples relative to manufacturers’ guarantees (experiment 1), measure the effects of high-temperature storage on yeast viability (experiment 1), and determine the effect of vitamin-trace mineral (VTM) premix on yeast viability (experiment 2). Commercially available ADY products were acquired in triplicate through normal distribution channels and stored at 4°C upon receipt. Initial samples were evaluated for colony-forming units and compared with product label guarantees. Only 1 of the 6 products sampled in experiment 1 met product guarantees for all 3 samples. To determine effects of storage temperature and duration on viability, ADY samples were stored in an incubator at 40°C with ambient humidity for 1, 2, and 3 mo. High-temperature storage significantly decreased viability over the 3-mo period; approximately 90% of viable cells were lost each month. Three of the 5 products sampled in experiment 2 met product guarantees. Fresh samples of 4 of these 5 ADY products were mixed in duplicate with ground corn (GC) or a VTM premix to achieve a target concentration of 2.2 × 10⁸ cfu/g. For each product, GC and VTM samples were stored at ambient temperature (22°C) and at an elevated temperature (40°C) for 2 wk. No differences in viable yeast count were observed between GC and VTM samples immediately after mixing or after storage at ambient temperature. Yeast viability in GC and VTM samples decreased during storage at an elevated temperature. There also was a significant interaction of diluent and storage temperature; VTM samples had higher cell viability than GC samples when subjected to high-temperature storage. Results suggest that (1) ADY products failed to consistently meet product guarantees; (2) viability of ADY products was greatly diminished during storage at 40°C for 2 wk; and (3) the loss in viability at elevated temperatures may be attenuated when ADY products are diluted with a premix containing VTM.     

Impact of somatic cell count combined with differential somatic cell count on milk protein fractions in Holstein cattle

Udder health in dairy herds is a very important issue given its implications for animal welfare and the production of high-quality milk. Somatic cell count (SCC) is the most widely used means of assessing udder health status. However, differential somatic cell count (DSCC) has recently been proposed as a new and more effective means of evaluating intramammary infection dynamics. Differential SCC represents the combined percentage of polymorphonuclear neutrophils and lymphocytes (PMN-LYM) in the total SCC, with macrophages (MAC) accounting for the remaining proportion. The aim of this study was to evaluate the association between SCC and DSCC and the detailed milk protein profile in a population of 1,482 Holstein cows. A validated reversed-phase HPLC method was used to quantify 4 caseins (CN), namely α_S1-CN, α_S2-CN, κ-CN, and β-CN, and 3 whey protein fractions, namely β-lactoglobulin, α-lactalbumin, and lactoferrin, which were expressed both quantitatively (g/L) and qualitatively (as a percentage of the total milk nitrogen content, %N). A linear mixed model was fitted to explore the associations between somatic cell score (SCS) combined with DSCC and the protein fractions expressed quantitatively and qualitatively. We ran an additional model that included DSCC expressed as PMN-LYM and MAC counts, obtained by multiplying the percentages of PMN-LYM and MAC by SCC for each cow in the data set. When the protein fractions were expressed as grams per liter, SCS was significantly negatively associated with almost all the casein fractions and positively associated with the whey protein α-lactalbumin, while DSCC was significantly associated with α_S1-CN, β-CN, and α-lactalbumin, but in the opposite direction to SCS. We observed the same pattern with the qualitative data (i.e., %N), confirming opposite effects of SCS and DSCC on milk protein fractions. The PMN-LYM count was only slightly associated with the traits of concern, although the pattern observed was the same as when both SCS and DSCC were included in the model. The MAC count, however, generally had a greater impact on many casein fractions, in particular decreasing both β-CN content (g/L) and proportion (%N), and exhibited the opposite pattern to the PMN-LYM count. Our results show that information obtained from both SCS and DSCC may be useful in assessing milk quality and protein fractions. They also demonstrate the potential of MAC count as a novel udder health trait.     

Biphasic modulation of cell proliferation by sulforaphane at physiologically relevant exposure times in a human colon cancer cell line

Sulforaphane (SFN), a cancer chemopreventive compound derived from broccoli, is able to induce cell cycle arrest and apoptosis in various tumor cell lines. Here we show that cell growth inhibition by SFN follows a biphasic pattern: Transient exposure of 40-16 human colon carcinoma cells for up to 6 h resulted in reversible G(2)/M cell cycle arrest and cytostatic growth inhibition even at elevated concentrations, whereas a minimum continuous exposure time of 12 h was necessary for SFN to irreversibly arrest cells in G(2)/M phase and subsequently induce apoptosis. IC(50) values after 12 h of exposure followed by drug-free recovery up to 72 h (6.4-8.1 microM) were indistinguishable from those of chronic exposure for 24 to 72 h (5.4-6.6 microM). Low concentrations of SFN caused a transient decrease in glutathione (GSH) levels followed by GSH induction, which may be related to reversible G(2)/M arrest and cytostatic effects. Depletion of GSH does not seem to play a role in SFN-mediated apoptosis induction. Our data clearly contribute to a better understanding of the kinetics of antiproliferative activity of SFN.     

Modulation of oxidative cell damage by reconstituted mixtures of phenolic apple juice extracts in human colon cell lines

Diets rich in fruits and vegetables are associated with a lower risk of tumour induction in the intestine and other sites. Apple juice with high amounts of antioxidative phenolics might protect the intestine against reactive oxygen species-mediated cell damage. We investigated to which extent the preventive effectiveness of polyphenolic juice extracts is governed by the amounts of five major constituents (rutin, phloridzin, chlorogenic acid, caffeic acid and epicatechin). In human colon cell lines (Caco-2, HT29), reconstituted mixtures of these phenolics were investigated in comparison to the original juice extracts, originating from cider and table apples. Parameters studied were (oxidative) DNA damage (Comet assay), cellular redox status (dichlorofluorescein assay) and Trolox equivalent antioxidant capacity (TEAC). The TEAC of the reconstituted mixtures was higher compared to the respective original extracts (4.7-7.3 mM vs. 3.6-4.2 mM Trolox). After 24 h cell incubation, menadione-induced (oxidative) DNA damage was more effectively reduced by the reconstituted mixtures (1-100 microg/mL, 24 h), as compared to the original extracts. In contrast, the cellular ROS level was reduced to a rather similar extent by original extracts and reconstituted mixtures. The results lead to the conclusion that the selected constituents in their authentic proportions substantially account for the antioxidative effectiveness of phenolic apple juice extracts.     

Antibiotic resistance profiles and cell surface components of Salmonellae

Salmonellae were isolated from raw chilled retail poultry meats (n=100) using the procedures outlined in the Bacteriological Analytical Manual and Microbiology Laboratory Guidebook. These isolates and 36 Salmonella strains from our laboratory culture collection were tested for their resistance to 12 different antibiotics and for their ability to produce thin aggregative fimbriae and/or cellulose, two of the most important surface components influencing the ability of cells to attach to surfaces and form biofilms. The sensitivity of the salmonellae to the antibiotics was determined with a disc diffusion assay. Of 52 Salmonella isolates, 25 (48.0%) were resistant to one antibiotic, 5 (9.6%) were resistant to two, 4 (7.7%) were resistant to three, 6 (11.5%) were resistant to four, and 5 (9.6%) were resistant to five antibiotics. Two (3.8%) of the isolates were resistant to up to nine of the antibiotics tested. Fifty-one (98%) of the isolates were resistant to novobiocin, 18 (34.6%) were resistant to streptomycin, 14 (26.9%) were resistant to tetracycline, and 14 (26.9%) were resistant to oxytetracycline. In separate experiments, the isolates were grown on Luria-Bertani no-salt agar supplemented with Congo red (40 microg/ml) and Coomassie brilliant blue (20 microg/ml) or Calcofluor (200 microg/ml) to determine whether they produced thin aggregative fimbriae and/or cellulose. Of the total 52 Salmonella isolates, 25 expressed only thin aggregative fimbriae, and 1 synthesized only cellulose. Ten isolates produced both thin aggregative fimbriae and cellulose, and the remaining 16 isolates expressed neither surface structure. The findings of this study reveal a prevalence of Salmonella on raw retail poultry products in central Georgia and suggest that salmonellae have the ability to develop resistance to multiple antibiotics and to synthesize cell surface components that help them survive in hostile or suboptimal environments.     

Characterization of distributions of somatic cell counts

There is more useful information in distributions of somatic cell count (SCC) than is currently used in practice. Analysis of SCC of individual quarters (n = 450,834 quarter records of 133,102 cows) showed that the presence of pathogens did not change the peak of the SCC distribution. Instead, the percentages of observations in the tail changed. Probability density functions of specified sets of up to 5 standard distributions were then fitted on the number of records per class, using a maximum likelihood procedure. Analysis of cow SCC (2 data sets: n = 335,135 test-day records of 41,567 cows on 407 farms and n = 1,665,431 test-day records) showed that a mixture of a normal, a log-normal and an exponential density function (N+LN+E) best described the distribution of SCC. A mixture of 4 normal and an exponential distribution (4N+E) was also a good approximation. For this last mixture, each distribution could be associated with presence or absence of pathogens. The first 2 normal distributions appear to consist of uninfected cows and cows recovering from an infection, the third normal distribution may be associated with minor pathogens, and the fourth normal and the exponential distribution with major pathogens and persistent infections. Estimated percentages of records in each underlying distribution differed between parities, between stages of lactation, and between records with previous records being above or below 100,000 cells/mL. The categorical nature of cow-SCC can be utilized by deriving new traits such as the fraction of cow-SCC records in a lactation that are associated with an infection with a major pathogen.     

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.     

Somatic cell count during and between milkings

The objectives of the study were to determine 1) how sampling time between milkings affects the sensitivity and specificity of somatic cell count (SCC) as an indicator for intramammary infection (IMI) status, and 2) which cells are responsible for the diurnal variation in SCC. Six Prince Edward Island, Canada, dairy herds were selected. Quarter samples for SCC were collected immediately before the a.m. milking (pre-a.m.), halfway through the a.m. milking, immediately after the a.m. milking, every 60 min after detachment of the milking unit, and immediately before the p.m. milking (pre-p.m.). Compared with the geometric mean SCC at the pre-a.m. milking, SCC of quarters with no IMI between milkings was higher up to 7 h after milking. The pre-p.m. SCC was significantly lower than the pre-a.m. SCC in quarters with no IMI. Specificity of SCC at a cutoff of 200,000 or 500,000 cells/mL as an indicator for IMI status declined substantially after the a.m. milking. In quarters with elevated SCC, the proportion of polymorphonuclear leukocytes was larger immediately after milking. For accurate interpretations of SCC tests—whether by a laboratory, portable SCC device, or the California Mastitis Test—veterinarians, researchers, and udder health advisors should take milk samples immediately before milking.