Daniela Alberghina

Analysis of serum proteins in clinically healthy goats (Capra hircus) using agarose gel electrophoresis

BACKGROUND Electrophoretic patterns of serum proteins provide useful information on pathological conditions in ruminants. Their reference values, however, are dissimilar to those of other species. Reference values for goats using agarose gel as the supporting matrix have not been reported. OBJECTIVE The aim of this study was to evaluate serum concentrations of total protein and protein fractions (albumin and globulins) by means of agarose gel electrophoresis (AGE) in goats in order to establish electrophoretic reference intervals and to evaluate potential changes associated with aging. METHODS Blood was collected from 105 clinically healthy Girgentana goats by means of jugular venipuncture. Serum protein concentrations were assessed by AGE. Three age groups were compared: 1-1.5 years, 2-4 years, and 5-12 years. RESULTS Values (mean ± SD) were determined for concentrations of total protein (72.26 ± 6.40 g/L), albumin (31.80 ± 4.00 g/L), α-globulins (6.40 ± 1.23 g/L), β(1) -globulins (10.50 ± 2.58 g/L), β(2) -globulins (5.18 ± 1.60 g/L), and γ-globulins (18.65 ± 5.90 g/L) and for albumin/globulin (A/G) ratio (0.82 ± 0.20). One-way ANOVA showed statistically significant age-related differences for total protein and α-globulin concentrations and A/G ratios. Age influenced protein concentrations with the 5-12-year-old group having higher total protein and α-globulin concentrations and lower albumin concentration and A/G ratios than the 2-4-year-old group. CONCLUSIONS This study provides reference values for total protein concentrations and protein fractions obtained by AGE in goats. Some values vary with age. Age-specific reference intervals are reported in order to provide clinicians with an additional diagnostic aid.

Reference intervals for total protein concentration, serum protein fractions, and albumin/globulin ratios in clinically healthy dairy cows.

The aim of the current study was to evaluate total serum protein concentration measured by the biuret reaction as well as albumin and globulin protein fractions determined by agarose gel electrophoresis. These data were used to establish reference intervals in dairy cows of different ages. Blood was collected from 111 clinically healthy Modicana dairy cows by means of jugular venipuncture. Reference intervals (mean ± standard deviation) were determined for total protein (67.54 ± 11.53 g/l), albumin (31.86 ± 4.60 g/l), α1-globulin (5.77 ± 2.20 g/l), α2-globulin (5.84 ± 1.90 g/l), β-globulin (7.46 ± 1.94 g/l), and γ-globulin (16.73 ± 4.54 g/l) concentrations as well as for albumin/globulin (A/G) ratio (0.88 ± 0.43). Values from 2-, 3-, 4-, 5-, and 6-year-old cows were compared statistically. One-way analysis of variance showed age-related differences for α-globulin and β-globulin fractions only. The results of the current study provide reference intervals for total protein concentration as well as albumin and globulin protein fractions in 2- to 6-year-old dairy cows.

Estrogenic followed by anti-estrogenic effects of PCBs exposure in juvenil fish (Spaurus aurata).

Vitellogenin (Vtg) is a phospho-lipo-glycoprotein produced by oviparous animals in response to estrogen receptor (ER) binding. The presence of Vtg in juvenile and male fish liver and plasma has been used as biomarker to evaluate levels of environmental contaminants as dioxin and PCBs. Interaction of dioxins and PCBs with aryl hydrocarbon receptor (AhR) may affect reproduction by recruitment of estrogen receptor alpha (ERalpha). The aim of this study was to investigate the effects of PCB-126, a co-planar PCB prototypical AhR agonist, and of PCB-153, a non-coplanar PCB lacking dioxine-like activity, on Vtg expression in young fish (Spaurus aurata) after a 12 or 24h exposure to PCBs as well as 48h following PCBs removal. Vtg expression was evaluated by immunohistochemistry and by Western-blot analysis. Our results showed an increased Vtg expression following PCBs administration, with a maximum level after 12h of exposure to either PCB-126, PCB-153 or a mixture of both PCBs. Following this estrogenic activity, an anti-estrogenic activity was detected after 24h of incubation with PCB-126 (alone or mixed with PCB-153), suggested by a decrease in Vtg expression likely through AhR, as a consequence of a hypothetic defence mechanism to endogenous or exogenous ligands.

Pattern of serum protein fractions in dairy cows during different stages of gestation and lactation

In dairy cows the period of transition from late gestation to early lactation is recognized as inducing considerable metabolic adaptation. The aim of this study was to analyse modifications in serum protein values occurring during the dry and the transition period and during lactation in a group of five Holstein cows of high average milk production. For all subjects, selected on the basis of their pregnancy status, blood samples were collected at different physiological phases: dry period (-60, -30 d to calving), transition period (almost 7 d to calving, 7 d after calving), and lactation (weeks 2, 5 and 15 after calving), for a total of eight blood samples for each cow. On each blood sample total proteins and electrophoresis analysis were performed. On the data obtained, normally distributed (P<0·05, Kolmogorov-Smirnovs test), one-way Repeated Measure Analysis of Variance (ANOVA), was applied to evaluate the influence of different stages of gestation and lactation on the considered parameters. Results showed a significant effect on total proteins, α1-globulins, β-globulins, γ-globulins and albumin/globulin ratio. Most of the detected modifications were related to the transition from gestation to lactation, indicating that it is a period of great metabolic stress for cows. On the basis of the obtained results we can affirm that the pattern of serum protein fraction rn could give information about dehydration, plasma volume expansion and hepatic function occurring during the peripartum period in dairy cows.

Electrophoretic Serum Protein Fraction Profile During the Different Physiological Phases in Comisana Ewes

The aim of this study was to evaluate the influence of different physiological phases on serum total proteins and their fractions of ten Comisana ewes housed in Mediterranean area. From each animal, blood samples were collected at different physiological phases: late pregnancy, post-partum, early, mid-, end lactation and dry period. On all samples serum total proteins were determined by the biuret method, and albumin, α-globulins, β(1) -globulins, β(2) -globulins and γ-globulins concentrations were assessed using an automated system. One-way repeated measures analysis of variance was applied to determine the significant effect of different physiological phases on the parameters studied. During the late pregnancy and post-partum, total proteins, β1- and β2-globulins and γ-globulins showed the highest values. Starting from post-partum, α-globulins increased to reach their peaks in mid-lactation. Early lactation was characterized by low γ-globulins values. The increase in serum albumin concentration and the drop in some globulin fractions determined the significant increase in albumin/globulin ratio. The obtained results contributed to improve the knowledge on electrophoretic profile during the different physiological phases in ewes, confirming that pregnancy and lactation periods affect the protein metabolism. Particularly, serum protein fractions pattern could give information about dehydration, plasma volume expansion and hepatic function, which occur during the different physiological phases. Dynamics of the protein profile - from pregnancy to dry period - which are provided by our results, could be considered as guidelines for the management strategies to guarantee the nutritional needs of these animals during the different physiological phases and to avoid a decline of productive performance and consequently an economic loss.

Training‐induced modifications of circadian rhythmicity of peroxidative parameters in horses

The aim of this study was to evaluate the daily rhythms of peroxidative parameters in untrained and trained horses. Blood samples were collected every 4 h for a 48-h period for the determination of reactive oxygen metabolites test (d-ROMs), antioxidant barrier (Oxy-ads), thiol antioxidant barrier (SHp) and lipid peroxidation (LPO). Two-way anova showed a significant effect of time of day on all parameters studied, except on LPO. Higher values of Oxy-ads and SHp were observed in trained horses during both days of monitoring (p < 0.01). All studied parameters, except for LPO, showed robust daily rhythms. Their acrophases occurred during the day in untrained horses and in the evening in trained horses. Our results showed that training increases antioxidative capacity and demonstrated that the daily rhythms of antioxidative parameters can be modified by training.

Peripheral serotoninergic response to physical exercise in athletic horses.

The purpose of this study was to evaluate the influence of exercise on plasma tryptophan (TRP) and free serotonin (f5-HT), whole blood-5-HT (WB-5-HT) and f5-HT/WB-5-HT ratio in Italian Saddle horses. Six clinically healthy Italian Saddle horses were subjected to a 450 meters obstacles course. Blood samples were collected from each horse by jugular venipuncture using vacutainer tubes with K3-EDTA at rest, immediately after exercise, and after 30 min. TRP, f5-HT and WB-5-HT were analyzed by HPLC. Immediately after exercise, statistically significant increases of f5-HT (p<0.001) and WB-5-HT (p<0.001) were observed. After 30 min, f5-HT and WB-5-HT decreased compared to immediately after exercise, but were still significantly higher than rest values (p<0.01 and p<0.05, respectively). A significant linear regression between f5-HT and WB-5-HT was observed during experimental conditions. f5-HT and WB-5-HT modifications after exercise suggest an important role of peripheral serotoninergic markers in response to physical activity. The possible source of extra serotonin detected after show jumping should be clarified by further investigation.

Activation of the Ahr signalling pathway by polychlorobiphenyls causes a marked induction of cytochrome P450 only after depletion of vitellogenin in Sparus aurata

The effects of polychlorinated biphenyl congeners 126 and 153 (PCB-126 and PCB-153) on vitellogenin (Vtg) and cytochrome (CYP1A1) expression were evaluated in 60 juvenile Sparus aurata. Fish were divided into four groups and the control group (Group A) was compared to fish exposed to PCB-126 (10-8M) (Group B), PCB-153 (10-6M) (Group C) singly and also in combination (Group D) for 12, 24 and 72 h. Hepatic expression of Vtg and CYP1A1 were analyzed using histological examinations and by immunochemical (Western blotting and immunohistochemistry) methods. Vtg increased in juvenile fishes of Groups B, C and D after 12h respect to Group A and decreased after 24 and 72 h respect to 12h in each group. CYP1A1 increased after 12 and 24h in all groups vs control group and increased in Group B only at 72 h vs in control group. The results showed that chemical interaction and endocrine disruption in fish might produce deleterious consequences not only for fish but also for human.

Influence of short-term storage on electrophoretic profile of bovine serum proteins

Storage represents a basic step of the pre-analytical phase for the accuracy of screening tests both in human and veterinary diagnostic laboratories. This study aimed to evaluate the effect of short-term storage on electrophoretic profile of serum proteins in dairy cows. Blood was collected from 18 clinically healthy cows and serum was divided into four aliquots. The first aliquot was immediately analysed (time 0). The second and the third aliquots were stored at +4°C for 24 and 48 h, respectively. The fourth aliquot was frozen at −20° C for 48 hours. A statistical analysis was performed on the obtained data. Albumin and α-globulins relative concentration and A/G ratio showed significant changes after 48 h storage. The results obtained in the current study showed that short-term storage influenced electrophoretic pattern of bovine serum proteins and the A/G ratio. Therefore, the refrigeration of bovine sera for 24 h at the most should be suggested to practitioners in order to obtain reliable serum protein concentrations as useful screening test in veterinary diagnostic laboratory.

Modulation of peripheral markers of the serotoninergic system in healthy horses

To evaluate the changes in plasma and platelet serotonin (5-HT) as markers of the serotoninergic system in equines, 5-HT content was measured by high performance liquid chromatography (HPLC) in deproteinized plasma obtained from peripheral blood samples of 12 clinically healthy horses, before and after feeding. 5-HT was measured in platelet rich plasma (PRP) and in platelet poor plasma (PPP). 5-HT in platelets (p5-HT) was obtained by subtracting free 5-HT in PPP (f5-HT) from 5-HT in PRP. After food ingestion, significant increases in p5-HT and f5-HT (p < 0.001), and no changes in the f5-HT/p5-HT ratio were recorded. Increase in the total circulating 5-HT might account both for initiating peristaltic activity and for increasing the f5-HT levels. Augmented 5-HT uptake by platelets could reflect the hypothetical increased activity of the serotoninergic neurons. Besides showing the feasibility to obtain f5-HT and p5-HT through HPLC determination of 5-HT in PRP and PPP equine plasma, these findings are consistent with the postulation that 5-HT is released from enterochromaffin cells following a mechanical and chemical stimulation.