Ontogeny of erythropoietin receptor mRNA expression in various tissues of the foetal and the neonatal pig.

Erythropoietin receptor (EPOR) mRNA expression in liver, spleen, bone marrow and testes of foetal and neonatal pigs was analysed using a real-time RT-PCR assay. The results showed that early in the foetal life, EPOR expression is greatest in the liver. Later in foetal life, the spleen has the greatest expression of EPOR, whereas at 2 weeks after birth, the main expression of EPOR is found in the bone marrow. These findings contradict our earlier hypothesis that erythropoietin (EPO) acting in a paracrine fashion can account for an extensive erythropoiesis at birth, a point of time when plasma EPO concentrations are low. Results presented in the present paper suggest that the spleen or, alternatively, the bone marrow is able to respond to very low concentrations of circulating EPO around the time of birth. The testes were found to express significant amounts of EPOR. Since EPO mRNA has previously been found in the testes, a paracrine function of EPO may exist in this organ.

Responses of plasma Epo and kidney and liver Epo mRNA to hemorrhage in perinatal pigs.

Despite the fact that pig fetuses in late gestation have extensive erythropoiesis, low blood pO(2) and low hemoglobin concentrations, piglets are born without detectable concentrations of plasma erythropoietin (Epo). In the present study, we have examined the hypothesis that long-term hypoxic stimuli are less efficient than short-term stimuli in stimulating Epo production in perinatal pigs. From fetuses collected by hysterectomy 5 days before term, new-born piglets and piglets 2 and 5 weeks old, blood in amounts corresponding to 2% of body weight was withdrawn from the jugular vein. Twenty-four hours later the animals were killed and their kidney and liver Epo mRNA analysed by a competitive RT-PCR assay. Plasma Epo concentration was estimated by a solid-phase, two-site sequential chemiluminescent enzyme immunometric assay. We found that in nearly fully developed fetuses and in new-born piglets, the concentration of Epo mRNA did not increase upon bleeding. This is in contrast to earlier findings in sheep. In 2- and 5-week-old piglets, bleeding was associated with a 12-15-fold increase in kidney Epo mRNA. In the 2- and 5-week-old piglets, bleeding evoked increased translation of Epo mRNA into the protein hormone. Also in new-born piglets, increased plasma levels of Epo accompanied bleeding, whereas significant changes in gene Epo expression were not observed.

Ontogeny of erythropoietin mRNA expression in liver, kidneys and testes of the foetal and the neonatal pig.

Erythropoietin (EPO) mRNA expression in kidneys, liver and testes of foetal and neonatal pigs was analysed using a competitive RT-PCR assay. The results indicate that in the foetal pig, erythropoietin expression is greatest in the liver, at birth; hepatic and renal expression are nearly identical, and by 5 weeks of age there is mainly renal expression. The dynamics of the renal expression of EPO mRNA in the perinatal period provide a correlate for observations made earlier of plasma EPO concentrations. Early in foetal life (30 days after artificial insemination), the mesonephroi contained large amounts of EPO mRNA. As in the rat, the testes produced EPO mRNA in amounts comparable to the liver on a per gram tissue basis, though much less on a per organ basis.

The porcine erythropoietin gene: cDNA sequence, genomic sequence and expression analyses in piglets.

The porcine erythropoietin (EPO) gene and its cDNA have been cloned and characterized. The cDNA encodes a protein of 194 amino acids. The gene structure and sequence show a high degree of homology to the corresponding human and murine gene. Steroid hormone receptor binding sites are present both in the promoter and in the 3' flanking region of the gene, which also contains an oxygen-sensing sequence. The promoter lacks classical promoter elements such as TATA and CAAT boxes. Expression analyses using a competitive RT-PCR assay showed that the kidneys contain about ten times more erythropoietin mRNA than the liver in five-week-old piglets, thus indicating that the shift from mainly hepatic to mainly renal EPO production has taken place at this age. The testes showed a higher ratio of EPO mRNA to total RNA than the liver. Spleen showed very low levels of expression, while no expression of erythropoietin mRNA was detected in brain tissue, bone marrow, lung, lymph nodes, and ovaries.

Polyamines in the gastro-intestinal tract of goat kids and in the regenerating ruminal epithelium of sheep.

The aim of the present study was to investigate whether temporal changes in polyamine concentration and synthesis could be found in the luminal content and wall tissue of the rumen and abomasum, two organs which have entirely different growth patterns during the first month of life. In the abomasal mucosa there was a marked gradual decrease in the ornithine decarboxylase (ODC) activity during the first month of life, while the ODC activity in the ruminal mucosa was low during the whole experimental period. However, injury of the rumen wall was followed by increased ODC activity. The ODC activity in duodenal mucosa was about 10 times higher than in the ileal mucosa and the ruminal epithelium. In ruminal liquid a clear peak in ODC activity was observed during the period 51-70 days after birth. The polyamine concentration did not parallel the ODC activity, in either the ruminal epithelium or the ruminal liquid. Of the polyamines, the spermine concentration was always highest, and with the exception of duodenal mucosa, the putrescine concentration was lowest. In liver a clear decrease in spermidine concentration from day 1 to about day 60 after birth was observed. Otherwise no marked temporal changes in tissue polyamine concentrations were observed. Two and a half hours after oral administration of 14C-labelled spermine, nearly all of the radioactivity was found in the lumen of the gastrointestinal tract. On the other hand, 1 h after intravenous injection of polyamines the walls of the gastrointestinal tract were strongly labelled. In conclusion, the polyamines needed for ruminal epithelial development seem to come from sources other than the ruminal epithelium itself or the ruminal lumen.

Histamine in lambs with abomasal bloat, haemorrhage and ulcers.

The median concentration of histamine in abomasal fluid of lambs with abomasal haemorrhage and/or ulcers (group 2) was significantly (P < 0.05) higher than the concentrations in lambs presenting abomasal bloat (group 1) and in the healthy and the diseased controls. In group 2, there was also a strong correlation (R2 = 0.81) between the histamine concentrations in abomasal tissue and abomasal fluid, although the median value of histamine in the abomasal tissue was not statistically higher in this group than in the others. The urine of lambs in group 2 also had numerically higher median concentration of histamine than the other groups. Five out of eight tested strains of Lactobacillus spp. and one out of two strains of Clostridium sordellii, isolated from abomasal contents of lambs with abomasal disease, were strong producers of histamine. Bacterial production is one possible source for the increased histamine concentrations in lambs suffering from abomasal haemorrhage and/or ulcers.

Plasma erythropoietin in newborn lambs.

In the present study early postnatal changes in erythropoietin (Epo) level and hemoglobin concentration in 8 lambs were examined. Plasma Epo was estimated by a monoclonal enzyme-immunoassay (ELISA), developed for human Epo. In all the lambs, except one, Epo was low, or undetectable, immediately after birth. Within 6-12 h after birth, a marked increase in plasma Epo was found in 4 of the lambs. Within 3-7 days after birth, Epo was back to low levels. In the lamb with high Epo levels at birth, drastic decreases were observed during the next 6 h. There was no obvious correlation between the hemoglobin concentrations and the changes in plasma Epo.

Minor redistribution of ventilation and perfusion within the lung during exercise in sheep.

Considerable heterogeneity unrelated to the effect of gravity has been demonstrated for both local ventilation (V) and perfusion (Q) in the lung. Local ventilation and perfusion are well matched, so that the heterogeneity of the V/Q ratio is less than for ventilation or perfusion alone (Melsom et aL 1997). We are searching for the mechanisms responsible for the coordinate heterogeneity of ventilation and perfusion. Here, we ask how and to what extent physical exercise induces changes in the distribution of ventilation and perfusion. We measured local (approximately 1.5 cm3 tissue volume) pulmonary ventilation and perfusion simultaneously in six sheep before, during and after running on a treadmill. Local ventilation was determined from the deposition of labelled aerosol particles and local perfusion from trapping of radioactive microspheres. Cardiac output increased approximately 2.5-fold during exercise. V/Q-ratios were not normally distributed and we therefore present the heterogeneity as the interquartile range. At rest, the average interquartile ranges for local ventilation, perfusion and V/Q-ratio were 0.48, 0.51 and 0.39, respectively. During exercise, the corresponding values were 0.44, 0.40 and 0.32. Thus, the distribution of local V/Q-ratio was narrower than for ventilation and perfusion also during exercise. We found a moderate redistribution of relative flow towards the dorsal parts of the lungs when perfusion increased, but the increase in total perfusion and ventilation was for the most part throughout the lung. The results indicate that the coupling between local ventilation and perfusion is at least as potent during exercise as at rest. The correlation (r) between paired values in the two resting periods was 0.93 for ventilation and 0.91 for perfusion and thus indicates time stability for the two variables.

Apparent ruminal degradation and rumen escape of lysine, methionine, and threonine administered intraruminally in mixtures to high-yielding cows.

We studied the kinetics of lysine, methionine, and threonine in six high-yielding dairy cows at peak lactation (stage 1) and 6 mo later (stage 2). The cows were fitted with cannulas in the rumen and duodenum and were automatically fed every 4th h. The three amino acids (AA) were administered intraruminally in mixtures at dosages of 100, 200, 300, and 400 mmol of each, together with polyethylene glycol (PEG) as a liquid marker. Mean rumen liquid pools at stages 1 and 2 were not significantly different. The mean liquid outflow decreased from 13.6 to 9.5 L/h, and there was a significant linear increase in the liquid outflow with increasing dosages of AA. No significant interaction was found between feeding levels and AA dosages on rates of apparent degradation and rumen escape values. Expressed as percentages of the dosage, all three AA studied showed a significant linear decrease in degradation and a significant increase in rumen escape values with increasing dosage. At the feeding levels in stages 1 and 2, the highest relative degradation rates (percentage of dosage) were observed for threonine. The relative degradation rate of methionine was significantly lower than of lysine at the high feeding level but significantly higher at the low feeding level. The mean rumen escape values of threonine, methionine, and lysine across dosages and feeding levels were 16.7, 22.1, and 20.5%, respectively. The flow of the administered AA into the duodenum during an 8-h period after administration increased with increasing dosages, with peak concentrations after 1 h. Thus, the amounts of rumen escape of the three AA were considerable at all dosages, even when the AA were administered in unprotected form.

Ruminal escape and apparent degradation of amino acids administered intraruminally in mixtures to cows.

The apparent ruminal degradation and escape of amino acids (AA) administered in 9 different mixtures of essential AA and 8 different mixtures of nonessential AA were studied using two cows fitted with ruminal cannulas. The 600-mmol AA mixtures, which were administered intraruminally using polyethylene glycol as a liquid marker, contained equal amounts of two, four, or eight AA. The amounts of each of the AA in the mixtures were 300, 150, and 75 mmol, respectively. Ruminal degradation and escape were compared with values previously reported for AA administered individually. Across doses, the mean rate of initial degradation (degradation during the 1st h after administration) of essential AA was 26% when the AA were administered in mixtures and 45% when the AA were administered individually. For nonessential AA, the corresponding values were 34 and 54%. Across doses, mean ruminal escape during the first 8 h after essential AA administration was 22% when the AA were administered in mixtures and 16% when the AA were administered individually. For nonessential AA, the corresponding values were 13 and 11%. After intraruminal administration of AA, both individually and in mixtures, significant negative correlations were found between rates of degradation during the 1st h and ruminal escape during an 8-h period. Some AA mixtures caused a net increase in the concentration of other AA in ruminal fluid 1 h after administration. Twelve of the mixtures that did not contain Ala caused a considerable net increase in the concentration of this AA, and 3 AA mixtures containing Arg and Ala caused a marked net increase in the concentration of Trp.

Dietary polyamines promote the growth of azoxymethane-induced aberrant crypt foci in rat colon.

We have examined whether dietary polyamines influence the formation and initial growth of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rat colon. Effects of a combination of dietary polyamines at three dose levels (putrescine: 50, 280, 740 nmol/g; spermidine: 10, 261, 763 nmol/g; spermine: 1, 31, 91 nmol/g) in the polyamine-poor AIN-76A diet were studied in animals in two different experimental situations: animals treated with AOM alone and animals treated with AOM + difluoromethylornithine (DFMO), a specific inhibitor of endogenous polyamine synthesis. In both experimental situations, dietary polyamines enhanced the growth of ACF, expressed as the number of large ACF (foci with three or more aberrant crypts, ACF > or = 3), whereas the formation of ACF, expressed as the number of ACF, was apparently not altered. In animals treated with AOM alone, maximal growth enhancing effect on ACF was nearly obtained with the median level of dietary polyamine. In rats fed a low polyamine diet, basic AIN-76A, DFMO reduced the growth of AOM-induced ACF by 83%. This inhibitory effect of DFMO was counteracted by dietary polyamines in a dose-dependent manner, and it was abolished at the highest level of polyamines. In conclusion, it was demonstrated that dietary polyamines are able to enhance the growth of AOM-induced ACF. Further, dietary polyamines reversed the DFMO-caused inhibition of ACF growth, probably by compensating for the DFMO-reduced endogenous polyamine synthesis.

Rumen escape and apparent degradation of amino acids after individual intraruminal administration to cows.

Apparent rumen degradation and escape of 18 amino acids (AA) after individual administration intraruminally were studied using two nonpregnant, nonlactating, rumen-cannulated cows fed a ration containing hay and concentrate twice daily. The pulse-dosages of AA were 75, 150, 300, and 600 mmol. Polyethylene glycol was used as the liquid marker. Rates of apparent degradation during the first 60 min (initial degradation) varied markedly among the AA; rates were highest for Ser, Asn, Glu, and Gln and lowest for Tyr, Val, Arg, and His. At the 75-mmol dosage, which corresponded to an initial substrate concentration of about 1 mM of rumen fluid, initial degradation varied between 95 and 40% of the dosage, depending on the AA. Rates, expressed as percentages of the dosage, decreased as dosages increased. During the first 8 h after administration, escape of the AA increased from a mean of 9% at the lowest dosage to a mean of 21% at the highest dosage. At the highest dosage, Met caused a net increase in the concentration of 11 of the other AA in rumen fluid. Twelve of the AA administered caused a net increase in the concentration of Ala in rumen fluid. Methionine and Lys are among the AA considered to be limiting to milk yield. These AA showed relatively high rumen escape values and may be useful as feed supplements even in an unprotected form.

Effect of iron on erythropoietin production in anaemic piglets.

In the present study the response of plasma erythropoietin to iron injection in anaemic piglets was examined. At the age of 16 days, 4 piglets from the same litter were given 180 mg iron subcutaneously. After iron injection, blood samples for estimation of erythropoietin activity in plasma, haemoglobin concentration, and reticulocyte counts were taken every 6 or 12 h for 3 1/2 days. Plasma erythropoietin activity was estimated by a monoclonal enzyme-immunoassay (ELISA), developed for human erythropoietin. On the day of iron injection, haemoglobin concentration ranged between 41 and 48 g/l, reticulocyte counts from 9 to 17 percent, and plasma erythropoietin between 22 and 144 mU/ml. In 3 of the 4 piglets, iron injection resulted in a 2-6 fold increase in erythropoietin activity. Maximal erythropoietin activities were observed 24-42 h after injection, and after 66 h, the activities were close to the pretreatment values. It is concluded that in our experiment, iron, per se, has stimulated erythropoietin production.

Both gravity and non-gravity dependent factors determine regional blood flow within the goat lung.

Distribution of pulmonary blood flow has traditionally been regarded as determined by gravity. This view has been challenged recently by reports describing marked gravity-independent distribution of flow. These reports were based on experiments in which local blood flow was measured by methods that have not been thoroughly evaluated. In the present study, we showed that in the goat lung regional trapping of i.v. infused microspheres (O = 15 microns) correlated to endothelial uptake of a simultaneously i.v. infused diamine (r = 0.99, region size approximately 1.5 cm3, dry weight approximately 40 mg). This indicates that the deposition of microspheres reflects true regional pulmonary blood flow. Using the microsphere method, we found a marked gravity-independent heterogeneity in blood flow (coefficient of variation approximately 40%) in the awake goat. We could find no pattern related to anatomy that could account for this variability. We re-examined the influence of gravity by analysing the distribution of pulmonary blood flow in anaesthetized goats both in prone and supine positions. The dorsal to sternal distribution of flow appeared to be inverted when the animals were turned from prone to supine recumbency, indicating that gravity influenced the distribution of pulmonary blood flow along this axis. However, along the gravitational axis, distribution of blood flow varied considerably from lung to lung. It appears that in awake goats the distribution of pulmonary blood flow is the result of several different determinants.

Etiology of acetonemia in Norwegian cattle. 1. Effect of ketogenic silage, season, energy level, and genetic factors.

Plasma acetoacetate concentration in the 1st mo of lactation and its relation to BW change, milk yield, DMI, and BW postpartum were studied in 361 first lactation cows during 6 yr. The cows were fed concentrate at 6 and 3 kg/d. Calvings took place from August to December. Single observations for all cows were fitted by a multitrait animal model that accounted for all genetic relationships. Heritability for acetoacetate was .11 with a genetic correlation of .87 for milk yield, -.65 for weight change, and -.13 for BW postpartum. Acetoacetate was higher at 3 kg/d of concentrate than at 6 kg/d, and calving after 3 to 4 mo of indoor feeding was related to higher acetoacetate than was calving shortly after the pasture season. Acetoacetate was related to weight loss postpartum, but at a different degree in different years. In some years, compounds of the silage caused strongly elevated plasma concentrations of acetoacetate after feeding. Experiments were performed to compare hay with silages of different qualities. Rumen concentration of different amines 3 h postfeeding was taken as an index of the amine load of the cow. The concentration of several amines in rumen fluid were high after feeding ketogenic silage.

Effect of iron treatment on erythrocyte parameters in postnatal anemia of the pig.

The development of postnatal anemia and the preventive and curative effect of iron supplementation were examined in 34 piglets from three litters of Norwegian Landrace pigs. A prostaglandin analog was given on day 111 or 112 of pregnancy, and the piglets were removed by caesarean section. Seventeen piglets were given 180 mg iron as colloidal ferridextran subcutaneously at birth (0 = day group); the remaining 17 were given the same amount on day 13 (13-day group). The piglets had access to a milk substitute from day 1 to day 7 and pelleted food for piglets after day 13. From about 4 weeks of age the piglets ate considerable amounts of pellets. The red blood cell count (RBC) and hemoglobin concentration (Hb) at birth were 3.2 +/- 0.4 (SD) x 10(12)/L and 80.4 +/- 8.1 x 10(12) g/L, respectively. In both groups Hb, RBC, mean corpuscular volume (MCV), and particularly packed cell volume (PCV) decreased markedly the first day after birth. In the 13-day group there was a further decrease until treatment with iron on day 13. Injection with iron on day 13 led to a rapid increase in the above mentioned parameters, with statistically significant increases for Hb, PCV, and MCV four days after treatment. The calculated mass of hemoglobin was fairly constant until treatment in the 13-day group. In the group given iron at birth the data obtained indicate that the amount of iron given is insufficient to sustain a production of normal-sized erythrocytes with a normal mean corpuscular hemoglobin concentration for more than approximately 21 days. Furthermore, the present study also indicates that MCV is a sensitive indicator of iron availability in piglets.

Adenine nucleotides, serotonin, and aggregation properties of platelets of blue foxes (Alopex lagopus) with the Chediak-Higashi syndrome.

Bleeding times, concentrations of serotonin in whole blood, and concentrations of adenine nucleotides as well as aggregation properties of platelets were examined in 18 blue foxes with Chediak-Higashi-like syndrome (CHS) and 16 controls. A claw of each ketamine-sedated fox was cut until bleeding started and the bleeding time was recorded as the time from the first to the last drop. The bleeding time was greatly increased in CHS foxes. Platelet counts of CHS foxes were normal, but aggregation induced by adenosine diphosphate (ADP), serotonin, collagen, and arachidonate was impaired. Adrenaline and serotonin was impaired. Adrenaline and serotonin potentiated the aggregatory effect of ADP on control as well as on CHS platelets. The mean concentration of ADP in CHS platelets was about one-third that in controls, whereas adenosine triphosphate (ATP) was approximately one-half that in controls. Serotonin could not, in most cases, be detected in blood of CHS foxes. These findings suggest that the prolonged bleeding time in the CHS foxes is, at least partly, due to a storage pool deficiency. The drastically reduced, and in some cases absent, aggregation of CHS platelets in response to arachidonate suggests that defective arachidonate metabolism contributes to the impaired hemostasis.

Plasma progesterone in reindeer in relation to ovariectomy and hysterectomy.

Four semi-domesticated reindeer were ovariectomized and 3 both ovariectomized and hysterectomized, 30-70 days before expected parturition. Anaesthesia with etorphine/propyonylphenthiazine was followed by an increase in the plasma concentration of progesterone of peripheral blood. The concentration of progesterone returned to pre-anaesthetic values before ovariectomy. Ovariectomy did not result in any substantial change in the plasma progesterone concentration within an observation period of 35-120 min. Hysterectomy was followed by a marked decrease in peripheral plasma progesterone within 60 min. The progesterone concentration in ovarian venous blood was slightly higher than the jugular venous blood in 1 animal and 150 and 400 times higher in the 2 other animals examined. Thus, despite lack of marked changes of progesterone in jugular venous blood upon ovariectomy, it is concluded that the ovaries are a major production site of progesterone in pregnant reindeer. The progesterone in uterine venous blood was close to that in the jugular vein in 1 animal, and slightly higher in 2 others. This observation, and the maintenance of pregnancy in spite of ovariectomy in 2 out of 4 animals, point to the foeto-placental unit as an additional source of progesterone production.