Pathophysiological studies on Dicrocoelium dendriticum infection in sheep

Red cell kinetics and plasma protein metabolism were investigated in two experiments using 20 adult sheep naturally infected with Dicrocoelium dendriticum, but free of other liver and gastrointestinal helminths. In the first experiment, where groups of animals with low to high Dicrocoelium burdens were injected with 51Cr-labelled red cells and 125I-labelled albumin, the results indicated that there were no significant differences in the turnover rate of labelled red cells or albumin between any of the groups. In the second experiment, two groups of sheep with low and high worm burdens were studied using the same radioisotope tracers; in addition, 59Fe-citrate was used to assess red cell iron incorporation rates in the two groups. Although the red cell half-lives of the infected sheep were just significantly longer, both were within normal limits and the difference was attributed to random variation within the two small groups of sheep. No significant differences were found in the other parameters. It was concluded that burdens of up to 4000 D. dendriticum do not cause significant blood or plasma protein loss in sheep.     

Pathophysiology of ovine trypanosomiasis: ferrokinetics and erythrocyte survival studies.

The haematological changes, erythrokinetics and ferrokinetics of sheep were investigated after infection with Trypanosoma congolense. Following the detection of parasites in blood, the infected sheep developed macrocytic hypochromic anaemia. Studies with 51Cr-red cells, 125I-albumin and 59Fe as ferric citrate 11 weeks after infection revealed that infected sheep had significantly lower mean circulating red cell volumes but higher plasma and blood volumes than control sheep. The infected sheep also had enhanced erythropoietic activity as judged by significantly higher plasma iron turnover rates, faster disappearance of radio-labelled iron and higher iron incorporation rates than control sheep. The rate of disappearance of 51Cr-labelled red cells was greater in infected than in control sheep. It was concluded that the anaemia observed at this stage of infection was due to an increased rate of removal of red cells from the circulation coupled with haemodilution, with no evidence of dyshaemopoiesis.     

Influence of dietary protein on the pathophysiology of ovine haemonchosis in Finn Dorset and Scottish Blackface lambs given a single moderate infection

Two experiments were conducted to examine the influence of dietary protein on the pathophysiology of Haemonchus contortus in lambs of two breeds, namely Finn Dorset and Scottish Blackface. Erythrokinetic and metabolic studies using 51Cr-red cells, 59Fe-transferrin and 125I-albumin, and digestive efficiency and nitrogen balance measurements were conducted between four and six weeks after infection. Identical radioisotopic and nutritional studies were also conducted on pair-fed control lambs of both breeds. The results demonstrated that in normal control animals, there were no significant effects of diet or breed on any of the erythrokinetic, metabolic or nutritional parameters, with the possible exception of red cell survival which was considerably longer in the Finn Dorset lambs. However, in the infected animals diet and breed did influence those parameters. The severest effects were seen in the infected Finn Dorset lambs given the low protein diet. These animals had the highest level of abomasal blood loss and this was associated with low red cell volumes and high levels of both albumin catabolism and plasma iron turnover. These animals were also in negative nitrogen balance which was partly accounted for by a significantly greater urinary nitrogen loss compared with their pair-fed controls and partly due to the poorer digestibility of the crude protein fraction of the diet. In contrast, although the infected Scottish Blackface lambs had high levels of blood loss and associated pathophysiological changes, these did not differ between dietary treatments and were similar to the changes in the infected Finn Dorset lambs given the high protein diet.     

Pathophysiology of Haemonchus placei infection in calves.

This study was conducted to investigate the pathophysiology of Haemonchus placei infection in Friesian calves. Seven calves were divided into two groups, three uninfected calves (control group) and four infected animals. The latter group were infected orally with 500 H. placei larvae kg-1 body weight. Five weeks after infection they were all housed in metabolic crates and injected with 125I-bovine albumin. 51Cr-red cells and 59Fe-transferrin, to study albumin metabolism, erythrokinetics and ferrokinetics. The results showed that there was a significant reduction in the mean haematocrit values and reduced weight gains in the infected calves compared with the controls. There was also a change in the distribution of albumin from the extravascular to the intravascular pool and a significant increase in the plasma and blood volumes of infected calves although the blood and albumin loss via the gastrointestinal tract recorded in this study was similar in both groups.     

Intestinal plasma and blood loss in piglets infected with Strongyloides ransomi

Five piglets were infected subcutaneously with 10 000 third-stage larvae of Strongyloides ransomi per kg body weight; another five animals served as controls.Simultaneous measurement of plasma and red cell loss into the intestinal tract in infected and control animals have been carried out using porcine albumin labelled with ⁹⁵Nb and red cells labelled with ⁵¹Cr. Between the 10th and 20th days the daily average “clearance” of plasma was significantly greater in the infected animals (111.9 ml) than in the controls (18.8 ml). Daily average “clearance” of red cells was 4.1 ml in the infected group and 0.83 ml in the control group. By comparing the faecal “clearance” of plasma and red cells between the infected and control animals, it would appear that there is a leakage of plasma into the gut which is greater than can be accounted for in terms of whole blood loss.     

Studies on vitamin E and selenium deficiency in young pigs. III. Effect on kinetics of erythrocyte production and destruction.

The effect of vitamin E and/or selenium deficiency on the kinetics of erythrocyte production and destruction has been investigated in swine. The plasma iron turnover rate, 59Fe incorporation into newly formed red cells as well as the 51Cr apparent red cell half-life, were not found to be significantly affected by either vitamin E deficiency, selenium deficiency or deficiency of both, as compared to replete animals. The results of this study suggest that vitamin E is not a limiting factor for normal erythropoiesis in young growing pigs. Erythropoiesis appeared, however, to be slightly decreased in selenium deficient pigs and will need to be further investigated.     

The influence of dietary protein on live bodyweight, degree of anaemia and erythropoietic responses of Scottish Blackface sheep infected experimentally with Trypanosoma congolense

The present study investigated the influence of dietary protein on the intensity of parasitaentia, degree of anaemia and erythropoietic responses, in sheep experimentally infected with Trypanosoma congolense and given either a high protein diet (116 g digestible crude protein [dcp] per day) or a low protein diet (51·5 g dcp per day). It was observed that infected and control animals on the high protein diet grew at similar rates while infected animals on the low protein diet experienced marked retardation of growth compared with their uninfected controls. Dietary protein had no influence on the degree of anaemia that followed infection. Measurement of blood volumes revealed that low protein infected group had significantly lower mean circulating red cell volume than their controls. Ferrokinetic measurements indicated that plasma iron turnover rates (pitr) and ⁵⁹Fe incorporation rates were higher in the high protein infected group than in the low protein infected group, although these differences were not significant. These observations indicate that infected animals on a high protein tended to show greater enhancement of erythropoietic activity that infected animals on low protein diet.     

Evaluation of Canine Red Blood Cells Stored in a Saline, Adenine, and Glucose Solution for 35 Days

An additive solution for the storage of red blood cells was evaluated for use in dogs. Blood collected from 6 dogs was processed into packed red blood cells and stored for 35 days in the additive solution Nutricel (Miles, Inc, Pharmaceutical Division, West Haven, CT). Packed red blood cells stored in citrate-phosphate-dextrose-adenine (CPDA-1; Fenwal Laboratories, Baxter Health Care Corp, Deerfield, IL) also were evaluated for comparison. Red blood cell 2,3-diphosphoglycerate (2,3-DPG) concentration, adenosine triphosphate (ATP) concentration, percentage hemolysis, and pH were determined. The red blood cell post-transfusion viability (PTV) after 35 days of storage was assessed with both single-labeled chromium 51 (⁵¹Cr) and double-labeled technetium 99m/chromium 51 (^99mTc/⁵¹Cr) techniques. Mean ATP concentration and percentage hemolysis of the cells stored in Nutricel were 1.1 μmol/g hemoglobin (Hb) and 0.28% respectively and did not differ significantly (P < .05) from the values of 1.0 μmol/g Hb and 0.33% from the CPDA-1-stored red blood cells. The mean pH of red blood cells stored in Nutricel was 6.19, which was significantly lower than the pH of 6.47 for cells stored in CPDA-1. The mean 2,3-DPG concentration of red blood cells stored in Nutricel was significantly higher at 10.1 μmol/g Hb than the 2,3-DPG concentration of 3.4μmol/g Hb for cells stored in CPDA-1. The mean PTV of canine red blood cells stored in Nutricel for 35 days was 85% with ⁵¹Cr and 90% with ^99mTc/⁵¹Cr. This was significantly higher than the mean PTVs of 38% and 36% for the CPDA-1 stored cells as assessed with ⁵¹Cr and ^99mTc/⁵¹Cr techniques, respectively. It was concluded that 35-day-old canine red blood cells stored in Nutricel are of acceptable quality for transfusion purposes.     

Changes in the blood biochemical and haematological profile of neonatal calves with age

Fourteen calves were used to investigate the changes from birth to 83 days of age in the concentrations of serum albumin, alkaline phosphatase, beta-hydroxybutyrate, plasma cortisol, serum creatine kinase, creatinine, iron, plasma fibrinogen, serum gamma-glutamyl transferase, plasma glucose, haptoglobin, serum non-esterified fatty acids, total protein, transferrin, triglycerides, urea and gamma globulin; the haematological variables measured were: basophils, eosinophils, haematocrit, haemoglobin, lymphocytes, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, monocytes, band neutrophils, neutrophils, platelets, red blood cells and white blood cells. The changes are presented as a series of graphs and the values are discussed in relation to the published reference ranges for adult cattle. Two populations of calves were identified which gave rise to a bimodal distribution for some of the variables. Differences in haematocrit, haemoglobin and red blood cell counts were apparent at birth, with raised values for these measurements being associated with an increased white blood cell and neutrophil count between three and 27 days of age.     

Linognathus vituli (Anoplura: Linognathidae): population growth,dispersal and development of humoral immune responses in nai;ve calves following induced infestations

Growth of Linognathus vituli populations following establishment at a point source was followed for 16 weeks on eight louse-na?ve Holstein calves. Dispersal of lice from the point of infestation (withers) was monitored by examination of known louse predilection sights. Mean louse indices increased during the initial weeks reaching maximum mean values at 8 weeks post-infestation (p.i.). Subsequently, mean indices decreased, reaching a plateau that persisted through weeks 14 and 15 p.i. There was little movement away from the withers until 5 weeks p.i. Subsequently, numbers of lice on the withers declined, while increasing at sites on the head. A few lice were noted on the dewlap and topline sites. Host antibody responses to crude nymph antigens, measured by enzyme-linked immunosorbent assay (ELISA), were generally evident within 2-4 weeks of infestation. Examination of individual host data showed louse populations could be categorized relative to the mean population size for the group. Two animals were considered 'chronic', having louse populations that persisted throughout the study and whose louse indices were >150% of the group mean. Two animals were classified as 'resistant' having lice present for only a portion of the study and whose louse indices <40% of the group mean. The remaining animals had louse indices between 62 and 137% of the group mean. Antibody profiles of the individuals were not as clearly categorized, although the 'resistant' animals were only positive for short periods of time, roughly corresponding to the time when lice were observed.     

Haematological studies on frizzled and naked neck genotypes of Nigerian native chickens

Variation in haematological parameters of Nigerian native chickens was studied using 60 clinically normal frizzle-feathered, naked-neck, and normal-feathered native chickens. These included red blood cell count, haemoglobin, packed cell volume, white blood cell count, mean corpuscular volume, mean corpuscular haemoglobin concentration, serum glucose, urea, cholesterol, albumin, globulin and creatinine. Normal-feathered birds had higher (p < 0.05) mean values compared to frizzled and native neck genotypes except for albumin, red blood and white blood cells, and mean cell haemoglobin concentration. Males generally had higher mean values than their female counterparts across all genotypes. Correlation coefficients among the parameters were significant (p < 0.001) with r values ranging from 0.26 between red blood cell and mean corpuscular haemoglobin to 0.92 between red blood cell and cholesterol. Sufficient genetic variation therefore exists for haematological parameters among Nigerian native chickens that may represent indicator traits for further study. However, the application of molecular tools will provide better understanding and application of these differences.     

Mucosal uptake,mucosal transfer and retention of iron in veal calves

A method for studying iron absorption in humans was adapted to veal calves. Three 10-week-old calves with moderate (calves 1 and 2) or severe (calf 3) iron deficiency were given an abomasal injection of⁵⁹Fe and⁵¹Cr and all their faeces were collected over 15 days in order to measure mucosal uptake, mucosal transfer and retention of iron. The mucosal uptake was 62.2, 53.4 and 71.8% in calves 1, 2 and 3, respectively. The iron retention measured 14 days after administration of the test dose was 57.4, 52.3 and 56.4% in calves 1, 2 and 3, respectively. Maximal plasma activity was found in all three calves between 1 1/2 and 2 h after injection of the test dose. The plasma activity decreased rapidly, with a slight increase between the 5th and the 10th hour. After 21 h, less than 0.25% of the injected dose was still present in 1 litre of plasma. Not all the⁵¹Cr was recovered in the faeces. No⁵⁹Fe was found in the urine but some⁵¹Cr could be detected. The results of this study show that the method described is useful for measuring the different steps of iron absorption in iron-deficient veal calves.Abbreviations Hb haemoglobin concentration - MCV mean corpuscular volume - PCV packed cell volume - PI plasma iron - RBC red blood cell count - R iron retention - TIBC total iron-binding capacity - TIBC-SAT saturation of the total iron-binding capacity - U mucosal uptake     

Evaluation Of An Additive Solution For Preservation Of Canine Red Blood Cells

The effect of an additive preservative solution on canine red blood cell posttransfusion viability (PTV) and on selected canine red blood cell biochemical parameters was studied. One unit (450 mL) of blood was collected from 6 clinically normal dogs into the anticoagulant citrate phosphate dextrose, centrifuged, and the plasma removed. The red blood cells were then suspended in 100 mL of a saline, adenine, dextrose, and mannitol solution and stored at 4°C. Aliquots were removed for study at 1, 10, 20, 30, 37, and 44 days. The 24-hour PTV of autologous red blood cells was determined using a sodium chromate (⁶¹Cr) label. Red blood cell concentrations of 2,3-diphosphoglycerate (2,3-DPG), adenosine-5'-triphosphate (ATP), and pH were also determined. Canine red blood cell PTV, pH, ATP, and 2,3-DPG concentrations decreased during storage ( P < .05). The PTV decreased from 94% using day 1 red blood cells to 80% and 75% using day 37 and day 44 red blood cells, respectively ( P < .05). Although the mean PTV of the day 44 stored units equaled the Food and Drug Administration (FDA) minimum standard for human red blood cells, the PTV was substandard in 75% of the day 44 units. The FDA standard was exceeded in 83% of the day 37 units. It was concluded that 37-day-old canine red blood cells preserved with a saline, adenine, dextrose, and mannitol solution are of acceptable quality for transfusion.     

Factitious Hyperkalemia in Dogs With Thrombocytosis

To determine the effect of platelet count on the accurate assessment of serum electrolyte concentrations, simultaneous platelet counts and electrolyte determinations were performed on serum and plasma from 40 dogs. Dogs were grouped according to platelet count as follows: thrombocytopenic (less than 150,000/microliters), normal (150,000 to 600,000/microliters), or thrombocytotic (greater than 600,000/microliters). Serum potassium concentration was significantly higher than plasma potassium concentration in normal dogs (mean difference, 0.63 +/- 0.17 mEq/l) and in dogs with thrombocytosis (mean difference, 1.55 +/- 0.73 mEq/l). This difference in potassium concentration between serum and plasma was positively correlated with platelet count (r2 = 0.86). In the blood of dogs with thrombocytosis, the serum-plasma potassium difference was further increased when the time period between blood collection and separation of serum or plasma from cells was lengthened. Differences between serum and plasma concentrations of sodium or chloride were not seen in any platelet group. These results suggest that a portion of the measured serum potassium concentration is released from platelets during the clotting process. In fact, profound elevations in serum potassium concentrations can occur factitiously in dogs with thrombocytosis. Therefore, the actual concentration of potassium in blood is determined more accurately by measuring the plasma concentration rather than the serum concentration of this electrolyte.     

Effect of the method of capture on the haematology and blood chemistry of red deer (Cervus elaphus)

Haematological and plasma biochemical constituents can be affected during capture and handling operations in wildlife. The present study compares the blood constituents of 40 red deer (Cervus elaphus) captured using two different methods: 20 were captured by physical means, using long nets and box traps, and 20 by chemical means, using a mixture of xylazine and ketamine. Significant differences were found in the haemogram: red blood cell count, packed cell volume, haemoglobin concentration, and segmented neutrophil, lymphocyte, monocyte and total leukocyte counts were higher in animals captured by physical means. Conversely, the eosinophil count was higher in deer captured using anaesthetic. The plasma activity of alanine amino transferase, and the concentrations of cholesterol, triglycerides, albumin, alpha-2 globulins, total proteins, sodium and chloride, were significantly higher in the group captured by physical means. With the exception of the eosinophil count, all the blood constituents which presented higher values were in the group of physically captured deer, and thus it is necessary to take into account the method of capture when interpretation or establishment of reference values is performed.     

Effects of ochratoxin A on some production traits, lipid peroxide and glutathione redox status of weaned piglets

The effect of feeding ochratoxin A (OTA) contaminated diet (379.6 and 338.1 microg/kg in starter and grower diets) on production traits, lipid peroxidation and some parameters of the glutathione redox system were investigated in weaned piglets over a seven-week period. Feed intake and feed conversion ratio (FCR) did not differ significantly, but in the first phase (0-28 days) the daily weight gain was significantly lower in the piglets fed the OTA-contaminated diet. Lipid peroxidation, as measured by the amount of malondialdehyde, glutathione content and glutathione peroxidase activity, did not change significantly in the blood plasma and red blood cell haemolysate in the OTA-loaded group, while malondialdehyde content increased significantly in the liver and markedly but not significantly in the kidney of piglets fed OTA-contaminated feed. Glutathione content did not differ significantly in the studied organs of the two groups while glutathione peroxidase activity of the OTA-loaded animals was significantly lower both in the liver and in the kidney. The results suggest that the use of feed-stuffs contaminated with low levels of OTA for seven weeks did not cause marked differences in the production traits or in lipid peroxidation and amount or activity of the glutathione redox system in the blood plasma, red blood cells and kidney, while significant changes occurred in the liver homogenate.     

Assessment of histamine, bradykinin, prostaglandins E1 and E2 and carrageenin as vascular permeability agents in the horse

The vascular leakage induced by histamine, bradykinin, serotonin and prostaglandin E1 and E2 was assessed. The test agents were injected intradermally into the shaved thoracic skin of horses and the vascular leakage estimated either semi-quantitatively by recording the diameter of the lesions or by measuring the actual volume of extravasated plasma in microliters using iodine-125-labelled human serum albumin (125I-HSA) as a marker in the blood plasma. Using the latter method, the vascular leakage induced by carrageenin and the effect of coadministered prostaglandins E1 and E2 upon the vascular leakage of both histamine and bradykinin were also investigated. No obvious lesions resulted when serotonin (10(-2) mol/l) was injected but histamine and bradykinin produced circular lesions which increased in diameter for approximately 30 min. The size of the lesions and volume of extravasated plasma was dose dependent. On a molar basis, bradykinin (10(-6) mol/l, 10(-5) mol/l) was more potent than histamine but they were equipotent at 10(-4) mol/l. The size of the lesions induced by carrageenin were independent of their anatomical location on the thorax. Except for the second hour, the hourly volume of vascular leakage increased until the fifth hour when the experiment was concluded. The maximum vascular leakage resulting from the injection of prostaglandin E1 or E2 (1, 10, 100 or 1000 ng) was 7 microliters but when co-administered with bradykinin (10(-6) mol/l), the volume of leaked plasma increased from 29 to 78 microliters. No synergy was observed when either prostaglandin was co-administered with histamine (10(-5) mol/l).     

Serum proteins,blood and plasma volumes in experimentalTrypanosoma vivax infections of sheep and goats

Summary Measurements of blood and plasma volumes using¹³¹I-albumin, and of red cell volume using⁵¹Cr in goats and sheep infected withTrypanosoma vivax for 1 to 2 months (at which time the anaemia was severe) showed statistically significant increases in blood volume (29 per cent and 57 per cent) and plasma volume (44 per cent and 59 per cent), and decreases in red cell volume per kilogram body weight (49 per cent and 50 per cent) in goats and sheep respectively. Total serum proteins and gamma globulins increased, while serum albumin decreased,inT. vivax infected sheep and goats, These findings indicate that the anaemia manifested, with mean packed cell volume decreased by 60 per cent and 47 per cent in goats and sheep respectively at the time of red cell volume measurements, is attributble partly to haemodilution and partly to an actual decrease in total volume of circulating red blood cells.

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Resumen Mediciones del volumen plasmático y sanguíneo utilizando¹³¹I-albúmina y del volúmen eritrocítico utilizando⁵¹Cr en cabras y ovejas infectadas conTrypanosoma vivax por 1 a 2 meses (cuando la anemia era severa), mostraron incrementos estadísticos significativos en volúmen sanguíneo (29% y 57%) y disminución del volúmen eritrocítico por kilogramo de peso corporal (49% y 50%) respectivamente. El total de proteínas séricas y de gama globulinas aumentaron mientras que las sero-albúminas disminuyeron. Estos cambios indican que la anemia observada cuando el hematocrito decrece en un 60 y 47 por ciento en cabras y ovejas respectivamente cuando se efectúan las mediciones del volúmen eritrocítico, es atribuíble parte a la hemodilución y parte a la disminución del volúmen total de eritrocitos circulantes.

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Résumé L'étude des volumes de sang et de plasma à l'aide de l'albumine I¹³¹ et cell du volume des globules rouges par le Cr⁵¹ chez des chèvres et des brebis infestées parTrypanosoma vivax depuis un à deux mois (à ce moment l'anémie est sèvère) montre des différences statistiquement significatives dans l'accroissement du volume sanguin (29 p. 100 et 57 p. 100) et du volume plasmatique (44 p. 100 et 59 p. 100) et dans la diminution du volume des globules rougespar kg de poids vif (49 p. 100 et 50 p. 100) respectivement chez les chèvres et les moutons. Le total des protéines sériques et des gamma globulines a augmenté alors que les albumines sériques diminuaient chez les animaux infestés parT. vivax. Ces recherches montrent que l'anémie constatée, avec dimunition de 60 p. 100 chez les chèvres et de 47 p. 100 chez les moutons due volume des globules rouges, est attribuable pour partie à l'hémolyse et pour partie à une diminution certaine du volume total des globules rouges en circulation dans le sang.

     

Electrolyte composition of mink (Mustela vison) erythrocytes and active cation transporters of the cell membrane

Red blood cells from mink (Mustela vison) were characterized with respect to their electrolyte content and their cell membranes with respect to enzymatic activity for cation transport. The intra- and extracellular concentrations of Na+, K+, Cl-, Ca2+ and Mg2+ were determined in erythrocytes and plasma, respectively. Plasma and red cell water content was determined, and molal electrolyte concentrations were calculated. Red cells from male adult mink appeared to be of the low-K+, high-Na+ type as seen in other carnivorous species. The intracellular K+ concentration is slightly higher than the extracellular one and the plasma-to-cell chemical gradient for Na+ is weak, though even the molal concentrations may differ significantly. Consistent with the high intracellular Na+ and low K+ concentrations, a very low or no ouabain-sensitive Na+, K(+)-ATPase activity and no K(+)-activated pNPPase activity were found in the plasma membrane fraction from red cells. The Cl- and Mg2+ concentrations expressed per liter cell water were significantly higher in red cells than in plasma whereas the opposite was the case with Ca2+. The distribution of Cl- thus does not seem compatible with an inside-negative membrane potential in mink erythrocytes. In spite of a steep calcium gradient across the red cell membrane, neither a calmodulin-activated Ca(2+)-ATPase activity nor an ATP-activated Ca(2+)-pNPPase activity were detectable in the plasma membrane fraction. The origin of a supposed primary Ca2+ gradient for sustaining of osmotic balance thus seems uncertain.     

Potassium concentration in equine red blood cells: Normal values and correlation with potassium levels in plasma

The concentration of potassium in plasma and in red blood cells was determined in 948 horses. The coefficient of correlation between the two parameters was low. In 436 of these horses, which were clinically healthy, the red blood cell potassium (RBCK⁺) levels did not fit within a normal distribution curve, but a bimodal distribution was observed with a section point at 90 mmol/litre. In 90 per cent of these normal horses, mean RBCK⁺ content was 97.5 mmol/litre. In the remaining 10 per cent, mean RBCK⁺ concentration was 93.8 mmol/litre. A subdivision into a ‘low potassium group’ and a ‘high potassium group’ was made. In 10 out of 15 horses in the ‘low potassium group’, bimonthly sampling over a period of one year showed that RBCK⁺ content remained low. In the remaining five horses an increase was observed.