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PubMed1999

Purification and cloning of the mitochondrial branched-chain amino acid aminotransferase from sheep placenta.

This paper presents the first purification of the mitochondrial branched-chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82-85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched-chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched-chain amino acid catabolism is specific to each species.

Amino Acid Sequence↗
PubMedDate not supplied

Effects of birth on baroreceptor-mediated changes in heart rate variability in lambs and fetal sheep.

1. In adult unanaesthetized sheep, there is a V-shaped relationship between mean arterial pressure (MAP) and heart rate variability (HRV), measured in both time and frequency domains. In contrast, in fetal sheep, there is only a positive direct relationship between MAP and HRV, which is determined by the cardiac vagus. We postulated that by the time lambs were 8-10 days old, the 'adult like' V-shaped relationship between MAP and HRV would be present and it may appear at or after birth. To test these hypotheses, experiments were performed in six chronically catheterized fetal sheep (aged 132-138 days gestation), 10 newborn sheep (within 10 h of birth) and 10 lambs (aged 8-10 days). The relationships between MAP and HRV (in both time and frequency domains) were studied before and during beta-adrenoreceptor blockade with propranolol and before and during cardiac vagal blockade with atropine. 2. In 8-10-day-old lambs, V-shaped relationships between MAP and HRV (measured in both time and frequency domain) were obtained. The negative limb of this V-shaped relationship between MAP and HRV was present after cardiac vagal blockade. The positive slope of the V was present after beta-adrenoreceptor blockade. 3. In 4-h-old newborn sheep, there was no relationship between MAP and HRV (measured in the time domain), but between 7 and 10 h of age a negative relationship was found during treatment with atropine and a positive relationship was found during beta-adrenoreceptor blockade, when HRV was measured in both time and frequency domains. 4. As described previously, there was only a positive relationship between MAP and HRV in fetal sheep, which was abolished by atropine but not affected by beta-adrenoreceptor blockade. 5. Thus, until relatively late in fetal life, baroreceptor- modulated changes in efferent cardiac sympathetic tone, determined by measuring the effects of autonomic blockade on HRV, could not be elicited, although reflex vagal pathways were active. By 7-10 h after birth, baroreceptor-modulated changes in efferent cardiac sympathetic tone were present. It was possible using measurements of HRV made in the time domain to show that these baroreceptor-modulated cardiac sympathetic effects became stronger over the first 10 days of life (P < 0.01). These studies are the first to show that the influence of baroreceptor-mediated changes in cardiac sympathetic tone on HRV increases in early life. This is probably because maturation of sympathetic innervation of the fetal sheep heart is occurring at this age.

Adrenergic beta-Antagonists↗
PubMed1995

Digestion of a dehydrated lucerne/barley diet (70:30) in defaunated, Isotricha-monoinoculated and mixed-fauna-inoculated rumen in sheep.

Two adult Texel sheep fitted with permanent rumen fistulae were defaunated for 12 weeks in a preliminary experiment. They were then reinoculated with Isotricha sp for 11 weeks, and finally with a mixed fauna (Entodinium, Epidinium, Eudiplodinium and Isotricha) for 10 weeks. They were fed a diet composed of dehydrated lucerne (700 g/d), pelleted barley grain (300 g/d), grass hay (100 g/d) and wheat straw (50 g/d) in one daily meal. Isotricha and the ciliates of the mixed fauna developed rapidly, reaching a maximum concentration 9-17 d after inoculation. Their concentration then fell for 2-3 d and finally stabilized at values close to 10(4)/ml for Isotricha, Eudiplodinium and Epidinium and 2.5 x 10(5)/ml for Entodinium. The estimated ciliate biomass in the rumen was 1.8 g l-1 for a volume of 10.2 ml l-1 in the Isotricha-monoinoculated sheep and 4.7 g l-1 for a volume of 25.0 ml l-1 in mixed-fauna-inoculated sheep. The concentration of Isotricha was unaffected by the inoculation of mixed fauna during the third part of the experiment. No difference in total rumen bacteria counts was observed between defaunated, monoinoculated and completely refaunated animals. The total adenylic nucleotide concentration in filtered rumen juice was 4 times higher in faunated sheep just before feeding and 4-9 times lower after feed intake as compared to defaunated animals; the energy charge was always higher in faunated animals. These results are discussed in relation to the digestive activity and biomass of the protozoa. The in situ degradation of lucerne stems was the highest in the mixed-faunated sheep over a retention time interval of 6-18 h. The values obtained in Isotricha-monoinoculated sheep were intermediate between the defaunated and the mixed-faunated states. The digestibility in the whole digestive tract of dietary dry matter, organic matter, nitrogen and neutral detergent fibre was unaffected by the addition of either Isotricha or the mixed fauna in defaunated rumens. The total volatile fatty acid concentration was higher (p < 0.05) in the defaunated sheep between 6 and 18 h after feeding. The molar proportion of butyrate was significantly (p < 0.05) increased by the presence of mixed fauna in the rumen while that of propionate and valerate was lower. A non-significant decrease in acetate was also observed. The concentration of NH3-N in the rumen was significantly increased (p < 0.05) by the presence of the mixed fauna in the rumen but was unaffected or slightly reduced by Isotricha alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1978

Quantitative aspects of the transformations of sulphur in sheep.

1. [35S]sulphate was used to obtain quantitative estimates of the transfer of sulphur between the blood, rumen and postruminal tract of four sheep given brome grass (Bromus inermis) pellets or lucerne (Medicago sativa) pellets at the rate of 33 or 66 g/h. Sodium sulphate (0-4 g S/d) was infused into the rumen or abomasum of sheep given brome grass during four periods of 19 d and was not infused into the sheep during a subsequent period in which lucerne was given. The flow of sulphide, sulphate, microbial S and non-microbial organic S from the abomasum was estimated using 103Ru and 51Cr. 2. The concentration of inorganic sulphate in serum was increased to maximum values of 35-46 mg S/l by infusion of sulphate into the rumen or abomasum. The rate of irreversible loss of serum sulphate and rumen sulphide was positively related to the amount of sulphate infused. 3. Reabsorption of sulphate by the kidney reached a maximum of 0.69-1.1 mmol sulphate/l glomerular filtrate. 4. The transfer of sulphate from blood to the rumen was related to the concentration of inorganic sulphate in serum, attaining maximum values of 133 (+/- 13) mg S/d for sheep given brome grass plus sulphate, and 127-159 mg S/d for sheep given lucerne. 5. Bacteria derived 0.52-0.67 of organic S from rumen sulphide in sheep given brome grass, and approximately 0.45 of bacterial organic S was derived from sulphide for sheep given lucerne. Protozoa derived approximately 0.90 of organic S from bacteria. 6. It was estimated that endogenous organic S contributed 300-340 mg S/d to the rumen, and that 0.24-0.45 of S digested in the rumen was derived from endogenous sources.

Animals↗
PubMed1981

Acetate supply and utilization by the tissues of sheep in vivo.

1. The supply and utilisation of acetate has been estimated simultaneously in the whole animal and tissues of sheep using a combination is isotope-dilution and arteriovenous-difference techniques. Animals were made alloxani-diabetic and acetate metabolism was compared when stabilized to normal metabolite levels with insulin (ITA sheep) and when food and insulin had been withdrawn for 36 h (fasted, diabetic sheep). 2. Acetate was simultaneously produced and utilized by all tissues. The exogenous (or gut) supply of acetate was the most important determinant of circulating acetate level. Endogenous acetate was produced mainly in the liver; 77 and 94% in fasted, diabetic and ITA sheep respectively. The production of endogenous acetate remained fairly constant and was not related to ketogenesis, which supports the idea that circulating acetate is largely a produce of fermentation. The liver, gut and muscle utilized 17, 25 and 54% respectively (96% total) of the acetate entry rate in ITA sheep; a similar percentage utilization was found in fasted, diabetic sheep. 3. Acetate is largely oxidized to carbon dioxide in the gut and muscles of sheep and may account for 30-40% of their oxidative metabolism. This figure is similar to that for the whole animal. The total acetate taken up by the liver could account for 30% of the oxygen consumption; however, the liver may not directly oxidize all the utilized acetate. 4. The over-all conclusion from this study is that acetate is largely of dietary origin and the major factor determining its rate of utilization is the arterial concentration.

Acetates↗
PubMed1982

Effects of gastrointestinal cannulation and jugular vein catheterization on the metabolism of sheep.

1. Certain digestive and metabolic criteria were examined in mature wethers before and following (1) a simple exploratory laporotomy (two sheep), (2) establishment of a rumen cannula plus simple ('T'-shaped) cannulas in the duodenum and ileum (six sheep), (3) establishment of a rumen cannula plus two cannulas in the caecum (four sheep), (4) establishment of a rumen cannula plus re-entrant cannulas in the duodenum and ileum (four sheep) and (5) insertion of catheters into both external jugular veins (four sheep). 2. Metabolizability of a standard ration and nitrogen balance and rumen retention time of the sheep were not significantly changed by any of the surgical procedures carried out. 3. The amount of heat produced per unit metabolic weight of the sheep prepared with re-entrant cannulas increased significantly (21 +/- 5.6%; P less than 0.001). 4. The use of the different preparations in digestive and metabolic studies is discussed.

Animals↗
PubMed1985

Magnitude of ouabain-sensitive respiration in the liver of growing, lactating and starved sheep.

Oxygen consumption and ouabain-sensitive respiration was measured for liver biopsies from lactating and non-lactating ewes and for hepatocytes isolated from mature, dry ewes. O2 consumption, ouabain-sensitive respiration and 86Rb+ uptake were also measured for hepatocytes isolated from lambs, fed adult sheep and adult sheep starved for 5 d. Ouabain-sensitive respiration in the liver of ewes at peak lactation accounted for 45% of the total liver O2 consumption. This percentage was 24-37% higher (P less than 0.05) than measurements made during late lactation and during the non-lactating period. Total O2 consumption and ouabain-sensitive respiration rates of lamb hepatocytes were greater (P less than 0.05) than similar measurements for hepatocytes isolated from adult sheep. Ouabain-sensitive 86Rb+ uptake by hepatocytes from fed sheep was up to six times greater (P less than 0.05) than that by cells from starved sheep. The magnitude of ouabain-sensitive respiration of hepatocytes from starved sheep was 62% lower (P less than 0.05) than that for hepatocytes from fed sheep.

Animals↗
PubMed1991

The efficiency of chewing during eating and ruminating in goats and sheep.

The total amounts of time spent eating and ruminating per 24 h by goats and sheep were determined. The efficiencies of chewing during eating (mean value of C.EAT) and chewing during ruminating (mean value of C.RUM) on the breakdown of feed particles to below the critical size required to leave the rumen (less than 1.0 mm) were investigated. All studies were done with the animals fed on a chaffed lucerne (Medicago sativa) hay diet. Goats spent more time eating (+3.1 h; P less than 0.01), and less time ruminating (-2.2 h; P less than 0.05) per 24 h, than sheep, when fed hourly at ad lib. intake. The efficiency of chewing during eating (mean value of C.EAT) in breaking down feed particles to less than 1.0 mm was greater in goats (85%; P less than 0.01) than sheep (48%). The process of rumination in sheep served to reduce the feed particles which were greater than 1.0 mm in the rumen to less than 1.0 mm. Sheep tended to be more efficient in this process than goats (59 v. 48%), with the difference not attaining significance (P greater than 0.1). The greater frequency of chews (number of total jaw movements/min) during eating in goats (P less than 0.01), or during ruminating in sheep (P less than 0.001), was the major component explaining differences in efficiency between the two species in both the eating and rumination processes. When corrected for the number of chews/min, the differences in mean value of C.RUM and mean value of C.EAT were not significant between goats and sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Nutritional Physiological Phenomena↗
PubMed2005

Effect of ruminal administration of Escherichia coli wild type or a genetically modified strain with enhanced high nitrite reductase activity on methane emission and nitrate toxicity in nitrate-infused sheep.

The effects of two kinds of Escherichia coli (E. coli) strain, wild-type E. coli W3110 and E. coli nir-Ptac, which has enhanced NO(2) reduction activity, on oral CH(4) emission and NO(3) toxicity in NO(3)-treated sheep were assessed in a respiratory hood system in a 4 x 6 Youden square design. NO(3) (1.3 g NaNO(3)/kg(0.75) body weight) and/or E. coli strains were delivered into the rumen through a fistula as a single dose 30 min after the morning meal. Escherichia coli cells were inoculated for sheep to provide an initial E. coli cell density of optical density at 660 nm of 2, which corresponded to 2 x 10(10) cells/ml. The six treatments consisted of saline, E. coli W3110, E. coli nir-Ptac, NO(3), NO(3) plus E. coli W3110, and NO(3) plus E. coli nir-Ptac. CH(4) emission from sheep was reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac by 6 % and 12 %, respectively. NO(3) markedly inhibited CH(4) emission from sheep. Compared with sheep given NO(3) alone, the inoculation of E. coli W3110 to NO(3)-infused sheep lessened ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production, while keeping ruminal methanogenesis low. Ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production in sheep were unaffected by the inoculation of E. coli nir-Ptac. These results suggest that ruminal methanogenesis may be reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac. The inoculation of E. coli W3110 may abate NO(3) toxicity when NO(3) is used to inhibit CH(4) emission from ruminants.

Animals↗
PubMed2002

The effect of consumption of foods that differ in energy density and/or sodium bicarbonate supplementation on subsequent diet selection in sheep.

The short-term consumption of foods that differed in energy density (ED) and/or NaHCO3 supplementation, on subsequent food intake and diet selection in sheep were measured. Thirty sheep weighing 35.9 (sd 2.89) kg were used. Two foods were formulated: H had 11 and L had 8 MJ metabolizable energy/kg fresh matter. Four further foods were formulated by adding either 40 g NaHCO3/kg or 16.5 g NaCl/kg to foods H and L. NaCl was added to give the same Na concentration as with 40 g NaHCO3/kg to control for any effects of Na per se. In a preliminary test, it was found that a 2 h consumption of food H supplemented with NaHCO3 could buffer potential impact on the rumen environment of subsequent consumption of food H alone (as judged by rumen pH and acid-buffering capacity); however, it was not as effective as the consumption of food L alone in doing so. Each food treatment was offered to one of six groups (n 5) for 2 h following 16 h of food deprivation. Sheep were then offered a choice between H and L for a further 6 h. Supplementing H or L with either NaHCO3 or NaCl had no significant effect on either intake or diet selection. ED significantly (P<0.01) affected intake during the 2 h single feeding period, with sheep offered H or L consuming 540 and 663 (sed 37) g respectively, but had no effect on subsequent intake during the choice period. During the choice period all sheep showed a preference for food H, but sheep previously offered L selected significantly more H (0.873 g/g) than sheep previously offered H (0.544 (sed 0.028) g/g; P<0.001). It is concluded that short-term consumption of foods that differ in ED, and hence in their potential impact on the rumen environment, significantly affects subsequent diet selection. This is in agreement with the hypothesis that ruminant animals select a diet to help maintain the rumen environment within a certain physiological range. Food H with 40 g NaHCO3 added/kg may not have been sufficient to affect subsequent diet selection. It is suggested that larger, rather than smaller, changes in the rumen environment achieved through previous feeding should be expected to alter subsequent diet selection.

Animals↗
PubMed1988

Environmental contamination by heavy metals and fluoride in the Saeftinge salt marsh (The Netherlands) and its effect on sheep.

The Saeftinge salt marsh in the Westerschelde estuary (southwestern part of the Netherlands) represents one of the very few tidal brackish marsh ecosystems in Western Europe. From May 1983 to May 1985 the local pollution of this marsh with metals and fluoride was investigated. Samples from soil and vegetation were analysed monthly for cadmium, lead, copper, zinc, manganese, iron, and fluoride. The amount of these substances in the soil appeared to be related to both the percentage of clay particles and organic matter in the soil and to the frequency of tidal submergences. In vegetation, the pollutants clearly showed a seasonal variation. Adherent clay, deposited on the plants during submergences, contributed considerably to the total amount of these elements. Sheep grazing in the marsh were investigated for renal and faecal excretion of these elements, but no relation between these samples and the seasonal variation in vegetation was found. Presumably sheep consumed vegetation selectively, avoiding the more contaminated plants. Regular clinical inspections of the sheep revealed no signs of acute or chronic intoxication. The organs of sheep that died during the investigation showed increased levels of cadmium in the liver and kidney, and iron in the liver, but not enough to cause alarm. Fluoride found in the rib material, although slightly increased, did not indicate fluorosis. In conclusion, contamination with metals and fluoride, as observed in the salt marsh, apparently does not impair the health of locally grazing sheep. This may be due to selective consumption behaviour of the sheep, their stabling during the winter, limited biological availability of the elements studied, and a sheep management adapted to the local circumstances.

Animals↗
PubMed1975

Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis.

Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important.

Animals↗
PubMed1958

Characterization of the reactant (gamma globulin factor) in the F II precipitin reaction and the F II tanned sheep cell agglutination test.

The material in gamma globulin ("reactant") which reacts with rheumatoid sera in the F II precipitin and F II tanned sheep cell agglutination tests was concentrated by precipitation with sodium sulfate. The concentrated "reactant" appeared to consist of polydispersed molecular aggregates of gamma globulin with s(20) constants as high as 40, as well as the previously described inhomogeneous aggregated material. Aggregated gamma globulin precipitated readily with most rheumatoid sera regardless of the reaction volume, and in low concentration inhibited the F II tanned sheep cell agglutination reaction and sensitized tanned erythrocytes to the agglutinating action of a positive rheumatoid serum. On the other hand, non-aggregated gamma globulin (7S) did not precipitate with rheumatoid sera and in low concentration did not inhibit the F II tanned sheep cell agglutination reaction, or sensitize tanned erythrocytes. Non-aggregated gamma globulin in large excess inhibited the precipitin reaction of aggregated gamma globulin with "rheumatoid factor," and accounted for the characteristic shape of the whole F II precipitin curves and the volume effect described by Vaughan (relation of increased precipitate yield relative to the reaction volume in whole F II precipitin studies). In serological systems other than the F II tanned sheep cell and F II precipitin reactions, the sensitization involves an antigen-antibody combination; i.e., sheep erythrocyte plus hemolysin in the sensitized sheep cell test, and egg albumin plus anti egg albumin in the absorption experiments with specific precipitates. The aggregation of gamma globulin that was essential for sensitization in the F II tanned sheep cell and F II precipitin tests may have simulated the aggregation of antibody gamma globulin that occurs with antigen-antibody union. The present information has been incorporated into a schematic hypothesis for the basis of the rheumatoid serological reactions.

Agglutination Tests↗
PubMed1986

Cyclooxygenase blockade elevates leukotriene E4 production during acute anaphylaxis in sheep.

We examined changes in the levels of eicosanoids in blood and pulmonary lymph of anesthetized sheep undergoing acute anaphylaxis. Within 1-3 min of intravenous antigenic challenge of previously sensitized sheep, there were approximately 7-30-fold elevations in mean arterial plasma levels of thromboxane B2 and 6-ketoprostaglandin F1 alpha, respectively, as measured by RIA. Negligible changes in levels of these cyclooxygenase products were found in both nonsensitized sheep and in sensitized sheep treated with indomethacin before antigenic challenge. In contrast, no changes in levels of sulfidopeptide leukotrienes (SPLT) in pulmonary lymph were detectable by RIA during anaphylaxis in sensitized or nonsensitized sheep, but levels of SPLT in indomethacin-treated sensitized sheep increased more than fivefold above levels in lymph from both other groups of animals. The immunoreactive SPLT in lymph from indomethacin-treated sheep was accounted for as LTE4, as demonstrated by mobility on HPLC and absorbance at 280 nm. These results support the possibility that certain undesirable effects of nonsteroidal antiinflammatory drugs, such as cardiopulmonary reactions in aspirin-sensitive individuals, and impaired renal and cardiac function during therapy with these drugs, may be related in part to augmented synthesis of the 5-lipoxygenase pathway products, especially those of the sulfidopeptide class. Increased LT production could also limit the antiinflammatory effectiveness of these drugs in many disease states.

6-Ketoprostaglandin F1 alpha↗
PubMed1999

Establishment of pregnancy after the transfer of nuclear transfer embryos produced from the fusion of argali (Ovis ammon) nuclei into domestic sheep (Ovis aries) enucleated oocytes.

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.

Animals↗
PubMed2003

Scrapie and experimental BSE in sheep.

Scrapie is a natural disease of sheep, but it can also be successfully transmitted between sheep by experimental inoculation. Although BSE is primarily a disease of cattle, it has also infected humans (causing vCJD) and, in addition, can be transmitted orally to sheep bringing concerns that BSE might naturally have infected the UK sheep population. Because of this, scrapie and BSE are being compared and studied in detail in sheep. PrP genotype controls sheep susceptibility and resistance to scrapie and to BSE, and deposition of the disease-associated PrP(Sc), used as a marker of infection, has the potential to act as a means of identifying BSE-infected animals and describing different pathogenesis mechanisms. Sheep orally dosed with BSE show signs of infection in their blood and this model is of major importance in the study of the safety of blood products for use with human beings.

Animals↗
PubMed1997

Selenium influences tissue levels of selenoprotein W in sheep.

Because selenium increases the levels of other selenoproteins, the influence of this element on selenoprotein W was examined in wether sheep fed either a low selenium diet (0.02 mg/kg) or the same diet supplemented with 3 mg selenium as selenite per kilogram diet. Muscle biopsies were taken initially and at 3.5, 7.0 and 10.5 wk. The sheep were killed after the last muscle biopsy and samples from nine tissues were taken. Selenoprotein W was determined in tissues by Western blots with a polyclonal antibody against a synthetic peptide based on the protein sequence of the homologous rat selenoprotein W. In supplemented sheep, muscle selenoprotein W was significantly increased over initial levels (P < 0.05) at 7 wk and afterwards, whereas in sheep consuming the low selenium diet, muscle selenoprotein W levels declined significantly (P < 0.05) after 10.5 wk. This selenoprotein was found in various amounts in all tissues examined. The highest levels of selenoprotein W were found in skeletal muscles and heart and the lowest was found in liver. Except for selenoprotein W in brain, the concentrations of selenoprotein W, selenium and glutathione peroxidase activity were significantly higher (P < 0.05) in all tissues from supplemented sheep than in those from unsupplemented sheep. The selenoprotein W levels in brains of the two groups were not significantly different. Thus, selenoprotein W levels in all tissues of sheep except the brain are sensitive to selenium status.

Animals↗
PubMed2001

Alanyl-glutamine dipeptide does not affect hemodynamics despite a greater increase in myocardial heat shock protein 72 immunoreactivity in endotoxemic sheep.

The possible beneficial effect of supplemental glutamine (Gln) in critically ill patients has been suggested to be mediated by the induction of the cytoprotective heat shock proteins (HSP)32 and HSP72. There is evidence that HSP72 and HSP32 have opposite effects on the hemodynamic situation during endotoxemia. Therefore, the effect of Gln supplementation on the cardiovascular system is not clear. We investigated the effect of alanyl-Gln (Ala-Gln) dipeptide on cardiovascular function in healthy and endotoxemic sheep. Ten sheep catheterized for chronic studies received Ala-Gln 700 mg/(kg x d) [equal to 470 mg/(kg x d)Gln] on 4 consecutive days, and 10 sheep received NaCl (9 g/L) as the control solution. On d 4, four sheep of each group were killed and myocardial samples were taken for immunohistochemistry. The remaining sheep received a continuous infusion of endotoxin [Salmonella typhosa, 10 ng/(kg x min)]. Hemodynamic parameters were measured before application of Ala-Gln or the control solution, and during endotoxemia. Myocardial HSP72 immunoreactivity was determined by immunohistochemistry. After 24 h of endotoxemia, the sheep exhibited a hyperdynamic circulation. No difference was found in the hemodynamic parameters between treatment and control group. Ala-Gln treated sheep had a greater increase in myocardial HSP72 immunoreactivity compared with controls after (P < 0.05) but not before endotoxemia. In summary, Ala-Gln increased HSP72 immunoreactivity after endotoxemia, but did not alter hemodynamic parameters. Thus, Ala-Gln supplementation does not seem to aggravate the hyperdynamic circulation in endotoxemic shock.

Animals↗