Agarose gel electrophoretic fractionation of serum proteins in adult cattle. II. A study of cows with different diseases.

In 283 dairy cows suffering from internal disorders the serum proteins were studied by agarose gel electrophoresis supplemented by total protein and albumin determinations. The clinical diagnoses could be grouped according to protein pattern. Group 1 (abomasal displacement and traumatic muscle injury) did not appreciably affect the serum protein concentrations and represented primarily non-inflammatory diseases or diseases of non-infectious origin. Group 2 (leukosis) occupied an exceptional position, with heavy lowering of albumin unaccompanied by a corresponding rise of total globulin. The γ-globulin concentration was significantly lowered. Group 3 (acute traumatic peritonitis) represented an acute inflammatory process with increase chiefly of the α-globulins, while the γ-globulin concentration was normal. Group 4 (chronic traumatic peritonitis, summer mastitis, chronic subclinical mastitis, chronic laminitis and polyarthritis, urinary tract infection, abscess, and sub-acute-chronic pneumonia) comprised diseases chiefly of chronic inflammatory character and with infectious origin. Especially characteristic was the heavy rise of globulins in general and of γ-globulin in particular. In most cases there was a large increase also of the α- and p-globulin fractions.     

Some effects of protein deficiency in young growing pigs. I. The serum protein system

No significant differences between the serum protein concentrations of the totally starved pigs and control animals given a milk protein supplemented diet were found. Serum lipid levels rose in the totally starved group. During the first 42 days of the experimental period sharp falls in serum protein concentrations were noted in the protein deprived pigs. The losses were greatest in the albumin and two β-globulin fractions, γ-globulin levels rose but at a much slower rate than in the control group. In the pigs which were refed a protein containing diet during the second 42 day period, the concentrations of all the serum protein fractions except for γ-globulin had reached control levels by the end of the investigation. The pigs which continued with the protein free diet showed further losses of albumin, a reduction in some α-globulin fractions and a cessation of net γ-globulin synthesis. The trends observed by measurements of protein bound carbohydrate were similar to those obtained from polypeptide determinations indicating quantitative rather than qualitative changes in the serum proteins. The reduction in the serum lipid concentrations of the pigs undergoing protein starvation was largely accounted for by losses of α- and β-lipoproteins. The protein deprived pigs maintained their initial body weight, while a continuous fall in weight was found in the totally starved group and a continuous increase in the control pigs.     

Agarose gel electrophoretic separation of blood serum proteins in cattle.

The agarose gel electrophoresis described by Johansson (1972) was modified so that a buffer of pH 7.9 was used in the gel, whereas the buffer in the electrode vessels had a pH of 8.6.The cattle blood serum protein picture is described in detail. The β₁-globulin zone shows a very distinct picture of the genetically polymorphic bovine transferrins. The region between the α- and β-globulins shows a number of faint and often very distinct bands. A faint background staining over the whole electrophoretogram may partly be caused by a rather strong lipoprotein in the α₁-region, lipids thus having migrated all over the electrophoretogram.The modified method described is well suited as a “screen electrophoresis” for cattle serum and is also useful e.g. in studying bovine transferrin polymorphism.     

Changes in serum protein electrophoresis profiles and acute phase proteins in calves with diarrhea

Calf diarrhea leads to substantial economic losses in the livestock industry worldwide due to medical treatment costs, retarded growth performance, and even death. The objective of this study was to investigate changes in serum protein profiles and acute phase proteins in calves with diarrhea and identify the association between these changes and diarrhea. A total of 185 Korean beef calves were used and divided into 3 groups by age: 1 to 10 days (n = 46), 11 to 20 days (n = 65), and 21 to 30 days (n = 74). Blood and fecal samples were collected from each calf. Serum concentrations of total protein, protein fractions (albumin, α1-globulin, α2-globulin, β-globulin, and γ-globulin), haptoglobin (Hp), and serum amyloid A (SAA) were analyzed. Compared to calves without diarrhea, calves with diarrhea had significantly lower albumin concentrations at 11 to 20 days and 21 to 30 days of age (P = 0.017 and P = 0.000, respectively) and significantly higher α1-globulin fractions at 21 to 30 days of age (P = 0.01). Interestingly, α2-globulin fractions were significantly higher in diarrheic calves in all age groups, whereas γ-globulin fractions were significantly lower in calves with diarrhea aged 1 to 10 days, compared with normal animals. In calves with diarrhea, the concentration of Hp was significantly higher, whereas SAA levels were not different between normal and diarrheic calves. In addition, a positive correlation was found between α2-globulin and Hp (P = 0.0004). Taken together, these results provide useful information about the use of serum protein profiles and Hp as prognostic and diagnostic markers for animal health status.     

Effect of storage time and temperature on the total protein concentration and electrophoretic fractions in equine serum

Serum protein electrophoresis (SPE) is a technique that could be considered one of the most useful diagnostic aids available to the clinician. The effect of storage time and temperature on the total proteins and electrophoretic fractions (albumin, α₁-, α₂-, β₁-, β₂-, and γ-globulins) was assessed in 24 healthy horses. All samples, collected by jugular vein puncture, were centrifuged and divided into 4 aliquots. The 1st aliquot was analyzed within 3 h from collection (time 0), the 2nd was refrigerated at +4°C for 24 h, the 3rd was refrigerated at +4°C for 48 h, and the last was frozen at −20°C for 48 h. One-way repeated-measures analysis of variance (ANOVA) showed a significant effect (P < 0.05) of the different storage conditions on the concentrations of all the parameters studied and significant variations in the percentages of albumin, α₁-globulins, α₂-globulins, and γ-globulins. Compared with time 0 the total protein concentration increased significantly after 48 h at −20°C, the albumin percentage decreased after 48 h at −20°C, the α₁-globulin percentage increased after 24 h at +4°C, the α₂-globulin percentage increased after 48 h at +4°C and at −20°C, and the γ-globulin percentage increased after 48 h at −20°C. The results should help veterinary practitioners handle and store equine serum samples appropriately. Further investigations at different storage times and temperatures could be useful.     

Vitamin E status of healthy Swedish cattle

Using high pressure liquid chromatography the serum concentration of vitamin E was measured in dairy cows fed either hay or silage as their main roughage, in calves fed milk-replacer, and in young intensively fed bulls. The concentrates fed to the cows, calves and bulls were supplemented with 5–10, 25 and 5–10 mg DL-α-tocopheryl acetate per kg, respectively, and the milk-replacer for the calves was supplemented with 50 mg DL-α-tooopheryl acetate per kg powder. Cows fed silage as their main roughage had higher serum vitamin E concentrations (: 3.8–5.2 mg/l) than cows fed only hay (: 2.5–4.1 mg/1). Lactating cows had higher vitamin E concentrations than dry cows (: 4.1–5.2 and 2.5–3.8 mg/l, respectively) and calves and bulls had much lower vitamin E concentrations (: 1.4 and 1.2 mg/l, respectively) than cows. Thirty per cent of the calves and 41 % of the bulls had serum vitamin E concentrations less than 1.0 mg/l, suggesting that in these animals the conventional level of supplementation of feeds with DL-α-tocopheryl acetate in Sweden is probably inadequate for the prevention of nutritional muscular degeneration and other negative effects.     

Pregnancy and peripheral plasma progesterone levels in cows inseminated after synchronization of estrus with prostaglandin F2 alpha

Fifteen Holstein cows were treated with two doses of 25 mg of a prostaglandin F₂α (PGF₂α as dinoprost tromethamine) administered intramuscularly 11 days apart. The cows were then divided into three groups and inseminated either at 72, 80 or 72 and 96 hours after the second dose of PGF₂α. Thirteen cows ovulated after the second prostaglandin treatment. Eight cows were diagnosed pregnant by rectal palpation 42 days after insemination but only five calved. PGF₂α induced luteolysis in cows with active corpora lutea as evidenced by the dramatic decreases in the plasma progesterone concentrations after treatment. In contrast, following PGF₂α administration to cows in follicular or late luteal phase the concentrations of plasma progesterone either increased gradually or remained low for several days before increasing to maximal levels. The ovulatory rate after the two doses of PGF₂α11 days apart was high. However, the pregnancy rate after this treatment was influenced by other factors including abnormal ovarian function.     

Exogenous prostaglandin F2 alpha promotes uterine involution in the cow

Three newly delivered dairy cows were given prostaglandin F_2α during the immediate postpartum period. PGF_2α was administered from day 3–13 post partum in doses of 25 mg twice daily. Endogenous release of PGF_2α and progesterone was studied in blood plasma during the experimental period. Rectal examination of the uterus was performed every second day in order to establish the end of uterine involution. Uterine involution in the three cows was completed days 16, 23 and 20, respectively. These figures are to be compared with earlier investigations of uterine involution times, which show about 27 days. It was concluded that PGF_2α had a positive effect on the uterine muscular tone.Key words: uterine involution, prostaglandin F_2α, bovine     

Studies of Enteric Pathogens and γ-Globulin Levels of Neonatal Calves in Sweden

Viring, S., S.-O. Olsson, S. Alenius, U. Emanuelsson, S.-O. Jacobsson, B. Larsson, N. Linde and A. Uggla: Studies of enteric pathogens and γ-globulin levels of neonatal calves in Sweden. Acta vet. scand. 1993, 34, 271-279.– Faecal and blood samples were taken from 10-30% of calves, 36 hours to 14 days old, in 47 dairy herds in different regions of Sweden from September 1987 to October 1988 (Olsson et al 1993). Faecal samples from 279 calves were analysed for the presence of Escherichia coli (K99⁺), rotavirus and Cryptosporidium sp. Twenty (7.2%) of these samples were from diarrhoeic calves. An ELISA was developed and used for the rotavirus analysis. E. coli K99⁺ was detected in 11.5%, Cryptosporidium sp. in 6.1% and rotavirus in 5.4% of the faecal samples. The presence of rotavirus alone and the combination rotavirus and E. coli (K99⁺) was found to be associated with diarrhoea (p<0.001 and p<0.01 respectively).Blood samples from 327 calves were analysed for the level of total protein and γ-globulin. In 43 of these samples (13%) γ-globulin did not separate from the ß₂-region by electrophoresis. The mean total protein concentration was 53.6 g/1 in calves free from diarrhoea. The mean γ-globulin concentration, adjusted to 7 days age was 5.9 g/1. The 20 diarrhoeic calves had lower levels of both total protein and γ-globulin, compared with calves without diarrhoea, but the difference was not significant. One litre more of colostrum at the first feed increased the level of total protein of the calves’ sera by 1.4 g/1 (p = 0.05). Calves born between May and September had a 2.0 g/1 higher (p<0.001) serum concentration of γ-globulin than calves born between October and April.     

Studies of electrophoresis on cellulose acetate membrane of serum proteins from normal horses, sheep and pigs

A method for the rapid electrophoresis on a cellulose acetate membrane of serum proteins from horses, sheep and pigs is discussed.The various main globulin fractions in the serum of these animals were experimentally identified.Normal values for the percentage composition of serum from normal horses, sheep and pigs were calculated.In the horse there was great individual variation in the shape of the β-fraction, assumed to be due to different transferrin types. The mean value for β-globulin of 19.5 % in the horse was higher than for the other two species.The albumin percentage was highest in the sheep and lowest in the pig, 48.5 % and 43.2 % respectively. The sheep had the highest γ-globulin percentage, 22.8 %, while the horse had the lowest with 19.0 %.Finally the values were compared with corresponding figures reported by other authors and the results discussed.     

Disposition kinetics and urinary excretion of ciprofloxacin in goats following single intravenous administration

We evaluated the pharmacokinetics of ciprofloxacin in serum (n = 6) and urine (n = 4) in goats following a single intravenous administration of 4 mg/kg body weight. The serum concentration-time curves of ciprofloxacin were best fitted by a two-compartment open model. The drug was detected in goat serum up to 12 h. The elimination rate constant (β) and elimination half-life (t_1/2β) were 0.446 ± 0.04 h^-1 and 1.630 ± 0.17 h, respectively. The apparent volume of distribution at steady state (Vd_ss) was 2.012 ± 0.37 l/kg and the total body clearance (Cl_B) was 16.27 ± 1.87 ml/min/kg. Urinary recovery of ciprofloxacin was 29.70% ± 10.34% of the administered dose within 36 h post administration. In vitro serum protein binding was 41% ± 13.10%. Thus, a single daily intravenous dose of 4 mg/kg is sufficient to maintain effective levels in serum and for 36 h in urine, allowing treatment of systemic, Gram-negative bacterial infections and urinary tract infections by most pathogens.     

Sequential Hormonal Changes in the Postpartum Dairy Cow

Peripheral plasma levels of 15-keto-13,14-dihydro-PGF_2α, progesterone, Cortisol, LH and prolactin were studied in 6 primiparous postpartum dairy cows. The cows were followed by hormone measurements and clinical examinations from parturition until pregnancy was established. Blood was collected 3 times per day. The cervix, uterus and ovaries were examined by rectal palpation at 6–10 days intervals. The cows were observed for signs of oestrus twice daily and were additionally teased with a bull to provoke standing heat.Four cows had a normal parturition and dropped their fetal membranes shortly afterwards. (NR group). The remaining 2 retained their fetal membranes for more than 24 h following parturition (RFM group). One out of 6 cows showed standing oestrus at the first ovulation, 4 animals were in oestrus at the second ovulation and all cows showed signs of oestrus at the third ovulation. Although the length of the first luteal phase varied from 9 to 22 days a corpus luteum was in all cases palpated. The secretion of progesterone during the first luteal phase was terminated by a PGF_2α release.A significant difference in 15-keto-13,14-dihydro-PGF_2α levels between the 2 groups was found on days 0–4 (2.39 vs 6.87 nmol/1 at Ρ < 0.06). Postpartum prostaglandin F_2α release as reflected by the level of 15-keto-13,14-dihydro-PGF_2α lasted shorter in the NR group than in the RFM group (15–17 vs 21 days). Significant positive correlations between 15-keto-13,14-dihydro-PGF_2α and Cortisol as well as between prolactin and Cortisol during the first 24 days postpartum were noted only in cows having normal parturition. The most pronounced daily prolactin variations occurred during the second luteal phase (NR group), when a significant difference between the times 8.00, 12.00 and 15.00 was recorded (14.7, 31.5 and 19.7 μg/l, respectively). Moreover, a partial negative correlation between log value of prolactin and arithmetical value of LH was found in these cows only during the first luteal phase after parturition.     

Electrophoretic patterns of serum proteins in blue foxes with special reference to changes associated with nosematosis.

The serum proteins in 1 group of healthy breeders, 1 group of healthy pups and 4 groups of foxes suffering from various diseases were separated electrophoretically on cellulose acetate membranes. In most of the sera, the proteins were separated into 1 albumin fraction and 5 globulin fractions designated alpha₁, alpha₂, alpha₃, beta and gamma. The mean concentrations of the total proteins and the various serum fractions in the disease groups were compared statistically with the mean values of the normal groups.A nosematosis group was characterized by a distinct hypergammaglobulinaemia together with an increase in the total protein — and a decrease in the albumin concentrations. However, in 2 cubs recovering from nosematosis the hypergammaglobulinaemia was shown to be reversible.In a feed intoxication group the concentration of albumin was found to be lower and the alpha₃-globulin higher than the corresponding values in the healthy group. A virus hepatitis group was characterized by a decrease in the concentrations of albumin and beta-globulin and an increase in the alpha₁- and alpha₃-globulins. In the toxoplasmosis group the total protein and alpha₁- and alpha₃-globulins showed concentrations below the normal values.     

Effects of prostaglandin f(2alpha) on corpora lutea formation and function in mated bitches

Fifteen mated bitches were used to study the effects of prostaglandin F_2α on ovarian endocrine function during the early and midluteal phase. Five dogs were kept as controls, five were given 250 μg/kg prostaglandin F_2α twice daily between the first and fifth day of metestrus, and five were similarly treated with prostaglandin F_2α between 31 and 35 days of metestrus. Function of corpora lutea was monitored by measuring serum progesterone concentrations during the first 45 days of gestation.

Dogs treated with prostaglandin F_2α during the early luteal phase had progesterone concentrations similar to controls and pregnancies were undisturbed in both groups. A dramatic decrease in serum progesterone concentration and abortion resulted after prostaglandin F_2α administration at midpregnancy.

These results indicate that prostaglandin F_2α was not luteolytic during the early luteal phase and was therefore ineffective for preventing pregnancy at that time. However, at the dosage and frequency used in this study, prostaglandin F_2α was luteolytic and abortifacient at midgestation.

     

Serum proteins in mink with endotoxin-induced amyloidosis and infectious plasmacytosis.

The serum proteins in Sapphire mink from the experimental and control groups in 2 endotoxin experiments and in a group of normal mink of the Standard type, were separated electrophoretically on cellulose acetate membranes.In experiment No. 1, in which the experimental mink were given repeated injections of endotoxin, and the controls were untreated, significantly increased total protein and significantly decreased albumin concentrations in the experimental group compared to the normal group were demonstrated. The concentration of the alpha₁-globulin was significantly elevated and the alpha₂-globulin significantly reduced in the experimental and the control groups compared to the normal group. Significant differences between the gamma-globulin concentrations in the various groups were not found.In experiment No. 2, all the animals were inoculated intraperitoneally with a crude tissue suspension containing the plasmacytosis agent 10 days before the experimental animals received the first of a series of injections with endotoxin. Significantly increased concentrations of total protein and gamma-globulin and significantly decreased albumin concentrations were, compared with the normal group, demonstrated in sera collected from the experimental group on the 94th, 201st and 254th day after the first injection of endotoxin. In the control group, compared with the normal group, significantly elevated concentrations of total protein, alpha₂-, beta- and gamma-globulins and significantly reduced albumin- and alpha₁-globulin were found but only in the second set of samples, while significantly decreased albumin- and significantly increased gamma-globulin concentrations were found in the third set.The results showed no significant differences between the gammaglobulin concentrations or between the other serum fractions in the experimental and the control groups in the 2 experiments. A possible explanation may be that there is no direct interrelationship between hypergammaglobulinaemia and amyloidosis, and that a common basic mechanism may stimulate related stem cells which thereafter differentiate in different ways.     

Serum Biochemical and Hematological Parameters of Sinclair(S-1) Miniature Sows During Gestation and Lactation

Venous blood samples were collected from 29 Sinclair(S-1) miniature sows at 14, ten, six and two weeks prior to parturition and two, four and six weeks postpartum to determine the effect of pregnancy and lactation upon 19 serum biochemical and 12 hematological parameters. During gestation, the levels of serum cholesterol, blood urea nitrogen and alpha₁-globulin, as well as packed cell volume and hemoglobin concentration, decreased; whereas, the level of serum beta-globulin increased. During lactation, the concentrations of serum glucose, total protein, albumin, beta-globulin, calcium, sodium and hemoglobin, as well as packed cell volume, decreased; whereas, the concentration of serum cholesterol and the activity of serum alkaline phosphatase increased.     

Peptococcus (S. Micrococcus) indolicus. The demonstration of two varieties of hemolysin forming strains.

On the basis of biochemical and serological criteria 2 hemolysin forming varieties of peptococci were identified as Peptococcus indolicus.Of a total of 16 hemolytic strains examined 9 originated from the vagina of clinically healthy cows, 4 from mastitis secretions from dry cows, 2 from the interdigital skin of clinically healthy sows, and 1 from a subcutaneous abscess in a pig.Two strains were designated a-hemolytic and 14 β-hemolytic.On blood agar plates colonies of the α-hemolytic variety were surrounded by narrow zones of almost complete hemolysis, while colonies of the β-hemolytic variety were surrounded by broad zones of incomplete hemolysis. The hemolysins were termed α- and β-hemolysin, respectively.The β-hemolysin, but not the α-hemolysin, could be demonstrated in cultures grown in liquid media. The β-hemolysin was found to be filtrable, relatively thermoresistant, and non-dermonecrotic.By gel diffusion analyses the 2 α-hemolytic strains were referred to Serotype C. Ten of the β-hemolytic strains belonged to Serotype C, 2 to Type B, 1 to Type D, and 1 to Type E.     

Oral 1,25-dihydroxyvitamin D3 in prevention of milk fever

The effect of daily oral doses of 1,25-dihydroxy vitamin D₃ (1,25-(OH)₂D₃) on plasma mineral concentrations and incidence of milk fever was tested in 39 aged cows. Three dose levels of 1,25-(OH)₂D₃ (0, 100 and 200 µg/d) were compared in cows supplemented with 100 g Ca/d (250 g CaCO₃) and in cows receiving no Ca supplement. 1,25-(OH)₂D₃ treatments were given from day 5 before expected calving (day –5) until the day after calving (Calving = day 0).Expected minima in Ca concentrations in plasma were seen on day 1 in placebo treated cows, while Ca concentrations in 1,25-(OH)₂D₃ treated cows were normal or increased in the period from start of treatment until day 3–5. In the parturient period (day –1 to +2) 1 of 21 treated cows developed milk fever, as compared to 4 of 18 untreated. A pronounced hypocalcaemia developed, however, in the 1,25-(OH)₂D₃ treated cows from day 3 to 5 onwards, culminating day 8–11 with 7 cases of milk fever. Ca supplements reduced the development of hypocalcaemia in the 1,25-(OH)₂D₃ treated cows, but could not completely prevent the occurrence of milk fever.     

Presence of multiple corpora lutea affects the luteolytic response to prostaglandin F2α in lactating dairy cows

Since the 1970s, luteolytic doses used for synchronizing estrus in dairy cattle have remained unchanged. This study aimed to evaluate the dose-response effect of prostaglandin F_2α (PGF_2α), which is used for synchronizing estrus, and subsequent fertility in cows with two or more corpora lutea (CL). The study population consisted of 1,683 cows with a single CL (1CL), 501 cows with multiple CL receiving a single dose of PGF_2α (2CL1), and 252 cows with multiple CL receiving a 1.5 × PGF_2α dose (2CL1.5). Cows with a single CL (n = 1,245) showed estrus significantly (P < 0.01) earlier (3.01 ± 1.23 days; mean ± SD) than cows with multiple CL (n = 287; 3.33 ± 1.69 days). Using 1CL cows as reference, the odds ratio (OR) for the estrus response in 2CL1 cows was 0.13 (P < 0.0001), whereas the ORs for estrus response and pregnancy of 2CL1.5 cows were 1.8 (P = 0.0001) and 1.7 (P = 0.001), respectively. Based on the results for only the 2CL1 cows, the OR for the estrus response was 0.7 (P = 0.01) for cows producing ≥ 45 kg of milk at treatment, compared to the remaining cows producing < 45 kg of milk. Our results showed that the presence of multiple CL reduced the estrus response to that induced by a single PGF_2α dose and milk production was inversely associated with this response, whereas an increased PGF_2α dose improved the estrus response. Therefore, an increase in the standard PGF_2α dose is recommended.     

Calcium potentiates the effect of estradiol on PGF2α production in the bovine endometrium

Background

Estradiol (E₂) is required for luteolysis in cows and its injection stimulates prostaglandin F2α (PGF2α) release. The main goal of our study was to investigate the ability of endometrial explants and cells treated with E₂ and the calcium ionophore (CI) A23187 to synthesize PGF2α.

Results

Treatment with E₂in vivo resulted in a 48.4% increase of PGF2α production by endometrial explants treated in vitro with A23187. Production of PGF2α was better stimulated with A23187 at concentrations of 10^-6 and 10^-5 mol/L compared with other concentrations used. The concentration of PGF2α for untreated bovine endometrial cell cultures was 33.1 pg/mL, while for cultures treated with E₂, A23187, or a combination of E₂ and A23187, the PGF2α concentration was 32.5, 92.4 and 145.6 pg/mL, respectively.

Conclusions

Treatment with A23187 tended to stimulate PGF2α production. In the presence of E2, A23187 significantly stimulated PGF2α synthesis. It appears that A23187 potentiates the effects of E₂ with respect to synthesis of endometrial PGF2α in cattle.