The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

Cyclic amp-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus: Expression of transformation-associated markers

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by α-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.     

Plasma and luteal concentrations of oxytocin in cyclic and early-pregnant cattle.

Concentrations of oxytocin were measured in corpora lutea obtained from heifers throughout the oestrous cycle and first 30 days of pregnancy. Values were low during the first 3 days of the cycle (less than 250 ng/g tissue), increasing to 1312 ng/g by Day 4. Values then further increased up to a maximum of 2344 ng/g on Day 12. Concentrations were similar in cyclic and pregnant animals throughout the midluteal phase and were maintained at approximately 1500 ng/g until the 18th (cyclic cows) or 19th (pregnant cows) day after oestrus, when they were again low. Values subsequently remained less than 250 ng/g in pregnant cattle. Concentrations of oxytocin in jugular venous plasma of cyclic (n = 5) and pregnant (n = 4) cows were measured in samples collected every 15 min for 8 h on Days 14, 16, 18 and 19 after oestrus. There were no significant differences in mean concentrations (range: 2.5-4.7 pg/ml) or in the number, frequency or area under the curve of episodes between either cyclic and pregnant animals, or between days. Mean basal concentrations were higher on Day 16 than on Day 14 (P less than 0.05), values on Days 18 and 19 being intermediate. These findings suggest that the corpus luteum contains a finite amount of releasable oxytocin, which is exhausted by Day 18-19 after oestrus, whether or not pregnancy occurs, and that there is no further accumulation of oxytocin in the animal during early pregnancy. The contribution of luteal oxytocin to jugular venous concentrations appears to be less than in sheep, in which values in the jugular vein closely parallel those within the corpus luteum.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

The aversion of broiler chickens to concurrent vibrational and thermal stressors

The requirement for assessing the effects of multiple concurrent stressors in improving the welfare of broiler chickens during transport has not been widely recognised. A discrete-choice technique was used to investigate the aversion of broiler chickens to concurrent vibrational and thermal transport stressors. In experiment 1, 12 female broiler chickens, aged 42+/-3 days were studied individually using two choice-chambers. Each chamber had four compartments connected via a central zone and offered four treatments; thermal (T: air temperature; 40 degrees C, 21% RH), vibrational (V: frequency; 2Hz, acceleration; 1ms(-2)), concurrent vibrational and thermal (VT) and no applied stressors (N). Coloured compartment wall panels, allocated at random, assisted chickens' identification of compartments. Birds were fasted overnight and were required to make five consecutive choices on each of four consecutive days. A choice was defined as entering a compartment and feeding (5g pellets), whereupon confinement for 60min was initiated. Choices were totalled over all birds and analysed using a log-linear generalised linear model. The vibration was significantly avoided (V and VT versus N and T; P<0.05) but the thermal stressor was not (T and VT versus N and V; P>0.05) and there was no interaction. In experiment 2, the procedure was repeated with 12 more birds and modifications to increase method sensitivity and maximise bird learning. Choices were more disparate than before with vibration avoided to a greater extent (V and VT versus T and N; P<0.01) but there was still no main effect of the thermal treatment or a significant interaction. Substantial differences between individuals were observed in both experiments. The overall response to vibration supported previous findings for short-term exposure, however, non-avoidance of the thermal treatment was unexpected. Possibly, the birds were unable to associate the delayed heat stress with the compartment. Alternatively, the thermal conditions were not perceived as aversive either initially or throughout the 60min confinement. The preference method provides a useful starting point for assessing combinations of stressors which affect broiler welfare, allowing relative ranking of treatments from an animal-centred perspective.     

Acute and chronic exposure to ammonia and olfactory acuity for n-butanol in the pig

An associative learning method (using a food reward) was developed to measure pigs' olfactory acuity for n-butanol, a standard odourant in human olfactometry. The pig could press two operant paddles but it only received a food reward when it pressed the one over which n-butanol was released. Once each pig had reached a training criterion (10 consecutive roots on the correct paddle on each of two consecutive sessions) this method was used to assess the impact of acute and chronic exposure to an atmosphere containing approximately 40 parts per million (ppm) ammonia gas, compared to fresh air, on its ability to perceive different concentrations of n-butanol. These were presented using a staircase pattern, i.e. if the pig gained or failed to gain a food reward on two consecutive occasions the concentration was reduced or increased, respectively. Acute exposure for approximately 45min to about 40ppm ammonia had no effect (P>0.05) on the lowest detected concentration (LDC) of n-butanol in six pigs. The geometric mean LDC was 1.23 parts per trillion (ppt) in approximately 40ppm ammonia and 2.09ppt in fresh air. The LDC of three pigs increased, i.e. acuity fell, from 5.1 to 175.5ppt over 24 days of exposure to congruent with40ppm ammonia. Ammonia had no effect on one of the other pigs and the high variability in the LDC for the remaining two pigs produced no meaningful assessment of its impact. Subsequent removal to fresh air for a further 24 days led to partial recovery of acuity in one of the three pigs that had shown evidence of olfactory impairment but not in the other two. Collectively our findings suggest that chronic, but not acute, exposure to congruent with40ppm ammonia can interfere with olfactory perception in some pigs (50% of our sample) and that this loss of acuity is not necessarily reversible.     

Investigation of transferability of BovineSNP50 BeadChip from cattle to water buffalo for genome wide association study

Cattle and water buffalo belong to the same subfamily Bovinae and share chromosome banding and gene order homology. In this study, we used genome-wide Illumina BovineSNP50 BeadChip to analyze 91 DNA samples from three breeds of water buffalo (Nili-Ravi, Murrah and their crossbred with local GuangXi buffalos in China), to demonstrate the genetic divergence between cattle and water buffalo through a large single nucleotide polymorphism (SNP) transferability study at the whole genome level, and performed association analysis of functional traits in water buffalo as well. A total of 40,766 (75.5 %) bovine SNPs were found in the water buffalo genome, but 49,936 (92.5 %) were with only one allele, and finally 935 were identified to be polymorphic and useful for association analysis in water buffalo. Therefore, the genome sequences of water buffalo and cattle shared a high level of homology but the polymorphic status of the bovine SNPs varied between these two species. The different patterns of mutations between species may associate with their phenotypic divergence due to genome evolution. Among 935 bovine SNPs, we identified a total of 9 and 7 SNPs significantly associated to fertility and milk production traits in water buffalo, respectively. However, more works in larger sample size are needed in future to verify these candidate SNPs for water buffalo.     

A meta-analysis of the effects of Vitamin E supplementation on the incidence of retained foetal membranes in dairy cows

A meta-analysis was performed to consolidate the results of studies which have evaluated the effects of Vitamin E supplementation during the dry period on the risk of retained foetal membranes (RFM) in the dairy cow. Twenty studies demonstrated a beneficial response to Vitamin E whilst 21 found no benefit and 3 reported an increase in the incidence of RFM in treated cows. The odds ratios (OR) of the available studies exhibited significant heterogeneity, so multivariable logistic regression analysis was performed to enable the identification of factors associated with the response to Vitamin E supplementation. Our multivariable analysis included parity and Vitamin E supplementation (control/treated) in the model, because all other factors were co-linear. Results indicated that Vitamin E supplementation led to a reduction in the incidence of RFM. A second multivariable analysis was undertaken on a subset of the data including only supplemented cows to determine the influence of supplementation factors on the risk of RFM. All factors were co-linear with each other, therefore, only type of Vitamin E supplementation was included in this analysis. The regression model demonstrated that administration of the synthetic Vitamin E alpha-tocopheryl acetate was associated with a lower risk of RFM than treatment with natural Vitamin E (alpha-tocopherol) (P=0.047, OR=0.49), whereas the difference between the synthetic Vitamin E alpha-tocopherol acetate and natural Vitamin E just failed to attain statistical significance (P=0.059, OR=0.53). Overall the analyses indicate that Vitamin E supplementation during the dry period is associated with a reduced risk of RFM, and that the synthetic forms of Vitamin E are more effective than the natural compound.     

Effect of level of dietary n-3 polyunsaturated fatty acid supplementation on systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle

Reproductively normal crossbred beef heifers were individually offered a diet of barley straw and concentrate supplemented with one of four levels of a fish oil (FO) enriched supplement. Following oestrous cycle synchronisation, blood samples were collected at appropriate intervals for the measurement of progesterone (P(4)), oestradiol (E(2)), fatty acids, insulin-like growth factor 1 (IGF-1) and metabolites. On days 15 and 16 of the cycle, oxytocin was administered intravenously and the prostaglandin F(2alpha) (PGF(2alpha)) response was measured as venous concentrations of 13,14-dihydro-15-keto PGF(2alpha) (PGFM). The heifers were slaughtered on days 17 or 18 of the oestrous cycle and endometrial tissue, rumen fluid and follicular fluid were collected for determination of fatty acid concentrations. In general there was no effect (P>0.05) of diet on plasma P(4) or E(2) concentrations. Increasing FO supplementation increased CL diameter on day 7 post-oestrus (P<0.0001) but had no effect on diameter on day of slaughter (P>0.05). On day 15, PGFM concentration was greater on the highest level of FO supplementation compared to controls (P<0.05), however, there were no differences between other diet comparisons (P>0.05). There was no effect of diet on PGFM concentration on day 16 (P>0.05). There was a strong positive relationship between plasma and uterine endometrial concentrations of both EPA (R(2)=0.86; P<0.0001) and total n-3 PUFA (R(2)=0.77; P<0.0001). IGF-1 concentrations increased on all diets and were greatest at the highest level of n-3 PUFA supplementation (P<0.05).