Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.     

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.     

Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis

Background: Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and α‐, β‐, and γ‐globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. Objectives: The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm‐blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Methods: Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi‐automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within‐run and within‐assay precision. Data from warm‐blooded and draught horses were compared using the Mann–Whitney U test. Results: Within‐run and within‐assay CVs were <5% for all protein fractions. No significant difference was found between warm‐blooded and heavy draught horses and so combined reference intervals (2.5–97.5%) were calculated for total protein (51.0–72.0 g/L), albumin (29.6–38.5 g/L), α₁‐globulin (1.9–3.1 g/L), α₂‐globulin (5.3–8.7 g/L), β₁‐globulin (2.8–7.3g/L), β₂‐globulin (2.2–6.0 g/L), and γ‐globulin (5.8–12.7 g/L) concentrations, and albumin/globulin ratio (0.93–1.65). Conclusion: Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.     

Effects of blood contamination of cerebrospinal fluid on western blot analysis for detection of antibodies against Sarcocystis neurona and on albumin quotient and immunoglobulin G index in horses.

OBJECTIVE: To determine effects of blood contamination on western blot (WB) analysis of CSF samples for detection of anti-Sarcocystis neurona antibodies, and on CSF albumin and IgG concentrations, albumin quotient (AQ), and IgG index in horses. DESIGN: Prospective in vitro study. SAMPLES: Blood with various degrees of immunoreactivity against S neurona was collected from 12 healthy horses. Cerebrospinal fluid without immunoreactivity against S neurona was harvested from 4 recently euthanatized horses. PROCEDURE: Blood was serially diluted with pooled nonimmunoreactive CSF so that final dilutions corresponded to 10(-3) to 100 microliters of blood/ml CSF, and WB analysis was performed on contaminated CSF samples. Number of RBC, albumin and IgG concentrations, AQ, and IgG index were also determined. RESULTS: Antibodies against S neurona were detected in CSF contaminated with 10(-3) microliters of strongly immunoreactive blood/ml. In CSF samples contaminated with 10 microliters of blood/ml, AQ remained within reference range. Volume of blood required to increase IgG index varied among blood samples and was primarily influenced by serum IgG concentrations. Number of RBC in contaminated samples was correlated with volume of blood added, but not with degree of immunoreactivity detected in contaminated CSF samples. CONCLUSIONS AND CLINICAL RELEVANCE: During collection of CSF from horses, contamination with blood may introduce serum antibodies against S neurona at concentrations sufficient for detection by WB analysis, thus yielding false-positive results. When blood is moderately or strongly immunoreactive, the amount of contaminating albumin may be small enough as to not increase AQ above reference range. In these cases, AQ and IgG index should be interpreted with caution.     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.     

Evaluation of Equine Albumin Solution in Fluid Therapy in Horses with Colic

Equine albumin solution can be a good therapeutic option in fluid replacement for treatment of horses with colic. The purpose of this study was to evaluate the effects of initial fluid therapy with equine albumin solution in horses presenting with colic and mild-to-moderate dehydration, and to compare this therapy with fluid therapy based on crystalloids alone. Nineteen horses of both genders presenting with colic and mild-to-moderate dehydration were used. Animals were randomly assigned to one of two groups (control: fluid therapy based on crystalloid solutions; experimental: fluid therapy based on equine albumin and crystalloid solutions). Physical examination, hematocrit determination, blood gas analysis, serum biochemistry, blood and peritoneal lactate assessment, and measurement of colloid osmotic and arterial pressure were performed at predetermined times. Good results were obtained with equine albumin solution. More fluid is attracted into and maintained in the intravascular compartment, despite infusion of small volumes, as indicated by higher arterial pressure, lower capillary refill time, lower hematocrit and serum protein concentrations, lower colloid osmotic pressure, and better skin turgor. Equine albumin solution has good oncotic action and is a safe fluid therapy option for horses with colic and mild-to-moderate dehydration. Our results suggest it can be a good choice of fluid for correction of severe dehydration, although further research is necessary to determine the adequate dose in such cases.     

Serum concentration of surfactant protein D in horses with lower airway inflammation

Reasons for performing study: Surfactant protein D (SP‐D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP‐D are routinely used as biomarkers for pulmonary injury. To date, serum SP‐D levels have only been investigated in horses in an experimental model of bacterial airway infection. Objectives: To compare serum SP‐D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). Methods: Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD‐affected and 20 classified as controls. Serum SP‐D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. Results: Serum concentrations of SP‐D in IAD‐affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD‐affected group, no significant correlation was found between serum SP‐D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP‐D levels. Conclusions: This is the first study determining serum SP‐D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP‐D levels. Potential relevance: Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.     

The virus neutralizing activity of antibodies specific to the envelope and nucleocapsid of equine herpesvirus type 1.

Antibodies specific to the envelope or nucleocapsid of the Kentucky-D strain of equine herpesvirus type 1 were isolated from convalescent horse serum by immunoadsorption on cyanogen bromide-activated Sepharose conjugated with equine herpesvirus type 1 envelopes or nucleocapsids, with subsequent elution by glycine buffer. In double immunodiffusion and immunolectrophoresis reactions, the eluted proteins appeared to belong to the IgG fraction of horse serum. Antibodies directed against the viral envelope neutralized equine herpesvirus type 1 in a plaque neutralization test, while antibodies against the nucleocapsid showed no virus neutralizing activity.     

A comparison of equine and bovine sera as sources of lipopolysaccharide-binding protein activity in equine monocytes incubated with lipopolysaccharide

Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using LPS-induced expression of procoagulant activity (PCA) by equine monocytes as a readout and (2) evaluate the use of commercial equine serum as a source of LBP activity using LPS concentration response and time course studies to validate the response. Monocytes were isolated from eight horses and incubated with five different serum preparations in the presence or absence of Escherichia coli LPS. The sera tested were heat-inactivated fetal bovine serum (HI-FBS), pooled commercial equine serum (CES), heat-inactivated pooled commercial equine serum (HI-CES), autologous equine serum (AES), and heat-inactivated autologous equine serum (HI-AES). In the absence of LPS, monocytes from half of the horses in the study had increased expression of PCA when incubated with HI-FBS alone; PCA was unaffected by incubation with the other sera. There was a four-fold increase in PCA when monocytes were incubated with LPS in the presence of CES, HI-CES or AES compared to LPS without serum. The combination of HI-FBS and LPS increased PCA 20-fold compared to LPS without serum. The HI-AES serum lacked significant LBP activity. Whereas maximal expression of PCA was induced by 1ng/ml of LPS in the absence of serum, inclusion of 1% CES reduced the LPS concentration required for maximal PCA to 30pg/ml. Monocytes incubated with LPS in the presence of CES had increased PCA at 3h and peaked at 6h. In conclusion, monocytes from many horses are directly stimulated by HI-FBS, suggesting that HI-FBS is not an optimal source of LBP for in vitro studies of LPS with equine monocytes. In contrast, CES and AES are effective sources of LBP activity for such studies, as they do not directly induce activation. Although the heat inactivation process did not affect the LBP activity in CES, it ablated LBP activity in AES. Consequently, investigators are advised to utilize either CES or AES in future studies, but not heat-inactivated AES.     

Sarcocystis neurona-specific immunoglobulin G in the serum and cerebrospinal fluid of horses administered S neurona vaccine

A vaccine against Sarcocystis neurona, which induces equine protozoal myeloencephalitis (EPM), has received conditional licensure in the United States. A major concern is whether the immunoglobulin G (IgG) response elicited by the vaccine will compromise the use of Western blotting (WB) as a diagnostic tool in vaccinated horses with neurologic disease. Our goals were to determine if vaccination (1) causes seroconversion: (2) causes at least a transient increase in S neurona-specific IgG in the cerebrospinal fluid (CSF); and (3) induces an IgG response that can be differentiated from that induced by natural exposure. Horses included in the study (n = 29) were older than 6 months with no evidence of neurologic disease. The presence or absence of anti-S neurona antibodies in the serum of each horse was determined by WB analysis. Seropositive horses had CSF collected and submitted for cytology, CSF index, and WB analysis. The vaccine was administered to all the horses and boostered 3-4 weeks later. On day 14 after the 2nd administration, serum and CSF were collected and analyzed. Eighty-nine percent (8 of 9) of the initial seronegative horses seroconverted after vaccination, of which 57% (4 of 7) had anti-S neurona IgG in their CSE Eighty percent (16 of 20) of the seropositive horses had an increase in serum S neurona IgG after vaccination. Of the 6 of 20 horses that were initially seropositive/CSF negative, 2 were borderline positive for anti-S neurona IgG in the CSF, 2 tested positive, and 2 were excluded because the CSF sample had been contaminated by blood. There were no WB banding patterns that distinguished samples from horses that seroconverted due to vaccination versus natural exposure. Caution must be used in interpreting WB analysis from neurologic horses that have been recently vaccinated for EPM.     

Identification and isolation of calcium-dependent and -independent bovine immunoglobulins reactive with sepharose 4B

Calcium-dependent and -independent Sepharose 4B-binding bovine serum proteins were isolated by affinity chromatography on unsubstituted Sepharose 4B using 2 mM ethylenediamine tetraacetic acid followed by 0.1 M galactose. They appeared in two different protein peaks by gel filtration on Sephacryl S-200. The earlier eluted protein was demonstrated to be immunologically IgM, whereas the retarded one IgG. From these findings, Sepharose 4B-binding bovine serum proteins are suggested to be calcium-dependent and -independent immunoglobulins IgM and IgG.     

Validation of a radioimmunoassay for measurement of gastrin in equine serum

A commercial radioimmunoassay kit designed for measuring gastrin in human serum was validated for use with equine serum. This nonextraction, double-antibody procedure uses an antiserum with broad specificity for molecular forms of gastrin. Synthetic human gastrin (G17-I) was added to pooled equine serum, and the observed assay values were compared with the mass added. Recovery was 99 to 115% in the gastrin concentration range of 40 to 640 pg/ml. Dilutions of postprandial serum with serum from fasted horses were assayed, and the inhibition curves were compared with those of the human gastrin kit standards, using a log-logit transformation. The slopes of the sample dilution plots were not significantly different from the slopes of the standard curves. Ethylenediamine tetraacetate and heparin adversely affected the assay, resulting in lower assayed gastrin concentration values. The intra-assay coefficient of variation (n = 10) was 3.8%, and the interassay coefficient of variation (n = 6) was 11.2%. The assay sensitivity, as reported by the manufacturer, is 8 pg/ml. Gastrin concentrations in serum from fasted horses ranged from undetectable values (less than 8 pg/ml) to 17.5 pg/ml, and peaked at a mean value (n = 6) of 70 pg/ml 3 hours after feeding. Serum cortisol values monitored during the postprandial blood collection period were in the normal range for horses.     

Albumin and immunoglobulins G, A and M in aqueous humour from clinically normal equine eyes

The levels of albumin, immunoglobulin G (including IgG [T]), IgA and IgM in the serum and aqueous humour of 47 horses and ponies with no ophthalmic abnormalities were determined by single radial immunodiffusion. There was a linear relationship between serum and aqueous albumin levels. No relationship of serum and aqueous IgG levels was observed. IgA was detected in only one aqueous sample. IgM was not detected in aqueous humour from any eye in this study. The factors which may influence albumin and immunoglobulin levels in normal aqueous humour are discussed.     

Temporal effects of plasmapheresis on serum proteins in horses.

The temporal response of blood and serum proteins to chronic plasmapheresis was determined in 8 horses used in a commercial antibody enterprise. Plasmapheresis of between 4 and 11 L induced significant decreases in total protein, albumin, and IgG values. With the exception of a high hematocrit value for the first postplasmapheresis blood sample, there were no changes in erythrocyte or leukocyte measurements, and no changes in the proportions of serum protein in an electrophoretic profile. Regression equations generated for recovery of proteins after plasmapheresis indicated a return to preplasmapheresis values of total protein and albumin at approximately 1 month. Complications of repeated plasmapheresis were not detected when plasma extractions were done between 7 and 19 times at 30-day intervals.     

Immunoglobulin M-capture enzyme-linked immunosorbent assay testing of cerebrospinal fluid and serum from horses exposed to west nile virus by vaccination or natural infection

The West Nile (WN) virus, present in the United States since 1999, is a cause of encephalomyelitis in birds, alligators, humans, and horses. No data exist regarding detection of anti-WN virus immunoglobins in equine cerebrospinal fluid (CSF). The aims of this study were to evaluate the blood-brain barrier (BBB) in WN virus-infected (WNE) horses, to compare diagnostic testing in serum and CSF, and to describe the immunoglobulin M (IgM) response in serum and CSF of vaccinated horses. CSF was collected from the lumbosacral (LS) space (n = 13) or the allanto-occipital (AO) space (n = 14) of WNE horses. The albumin quotient (AQ) and IgG index were calculated, and the IgM-capture-enzyme-linked immunosorbent assay (MAC-ELISA) was used to detect anti-WN virus IgM in serum and CSF. CSF collected from the LS site had a higher (P < .02) IgG index compared to the AO site (0.34 +/- 0.04 versus 0.22 +/- 0.04 [mean +/- SE], respectively). The mean AQ, irrespective of collection site, did not exceed reference values. There was 100% agreement between CSF and serum testing for IgM by MAC-ELISA testing. However, the positive to negative antigen ratios were higher (P < .001) in CSF (34.5) versus serum (8.5), indicating lower nonspecific reactivity in CSF samples. Horses vaccinated against WN virus did not develop an IgM response at 1:400 mg/dL in serum; however, a few horses developed a weak IgM response in serum but not in CSF. In conclusion, MAC-ELISA testing of serum and CSF were equivocal. Also, examination of CSF data from WNE horses suggests a normal BBB integrity and increased intrathecal production of antibodies.     

Physiological Concentrations of Acute-Phase Proteins and Immunoglobulins in Equine Synovial Fluid

Synovial fluid (SF) is capable of reflecting infectious, immunological, or inflammatory joint conditions in horses by altering its composition and appearance. Although plasma and SF compositions are quantitatively different, this latter compartment reflects changes in plasma macromolecules. Therefore, changes in serum immunoglobulin protein concentrations tend also to alter intracapsular levels. Therefore, it is necessary to know the physiological concentrations of proteins present in SF. The aim of this study was to determine the levels of total protein, albumin, transferrin, haptoglobin, α1-acid glycoprotein, ceruloplasmin, and immunoglobulins A and G in SF of six healthy horses. The synovial proteinogram was obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The SF proteins reached a maximum of 25% of serum concentrations, varying inversely with molecular weight of the protein, except for the ceruloplasmin.     

基于Label-free技术比较马和驴血清蛋白质组分差异

旨在探究马和驴血清蛋白质生物学特征,为马属动物种间生理特征比较和健康保障提供理论依据。本研究选取辽宁省大连市某集约化养殖场的9匹蒙古马和9头辽西驴,雌性,年龄4~10岁,均处于正常发情周期的间情期,健康无病、精神状态及采食状况良好,分别提供相同的饲养条件。采集马和驴各9份血清样品分别随机分成3组,每组内的3份血清样品均匀混合成1个生物学重复,各获得3个生物学重复的血清样品。利用Label-free蛋白质组学技术及生物信息学方法对马和驴的血清蛋白质组分进行比较研究,提取血清蛋白质,再对蛋白质进行酶解,液相色谱-串联质谱（LC-MS/MS）进行蛋白质组分分析,数据库检索分析马和驴血清样品中蛋白质表达情况,并应用生物信息学方法筛选马和驴血清差异关键调控蛋白质。结果显示,本研究共鉴定出361种蛋白质,其中,马血清中表达288种蛋白质,分子量范围为1.52~511.24 ku;驴血清中表达244种蛋白质,分子量范围为1.53~611.47 ku;共获得231种显著差异表达蛋白质（差异倍数≥1.5,P≤0.05）。差异表达蛋白质主要参与蛋白质激活、补体激活、免疫应答、凝血和脂蛋白氧化调控等生物学过程。显著富集的KEGG通路为补体和凝血级联,吞噬体,内质网蛋白质加工,抗原加工递呈,甘氨酸、丝氨酸和苏氨酸的代谢,癌症蛋白多糖等。通过差异蛋白互作分析显示,差异表达蛋白质紧密相连富集最为显著的功能模块主要为代谢途径、补体和凝血级联、吞噬体,HSP90AA1、HSPA8、APOD、APOM、SERPING1、MASP1、CALR、TUBA1B、TUBB等处在关系互作网的重要节点。马和驴在生理条件下的血清蛋白质组成特征存在一定差异,该研究为进一步揭示马属动物种间生理特征差异和有效保障健康提供数据支撑。     

Serum proteins in guinea-pigs and horses infected with Trypanosoma evansi (Steel, 1885)

The serum protein pattern in guinea-pigs infected with T. evansi was analysed and compared with those found in horses with either a natural or experimental infection. In both species, a highly significant decrease in albumin levels and an increase in gamma-globulins were seen, leading to a very low albumin/globulin ratio. No significant differences in total protein levels between healthy and infected animals were registered. Likewise, alpha-globulins were not significantly affected. A decrease in beta-globulins was observed in one horse and in guinea-pigs with experimental infection, while in horses with natural infections this decrease was not constant. The serum protein patterns in guinea-pigs infected with T. evansi appeared similar to those occurring in horses infected with this parasite. Guinea-pigs, therefore may be useful laboratory models for the study of equine trypanosomosis caused by T. evansi.     

Induction of a Th-1-biased IgG subclass response against equine herpesvirus type 1 in horses previously infected with type 4 virus

An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were na?ve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses na?ve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.     

HAEMATOLOGY OF THE RACEHORSE: A Recent Study of Thoroughbreds in Victoria

ab>The study was based on 216 blood samples collected from 136 Thoroughbred racehorses in 17 stables. Lower variability in the population studied and lower means than in previous studies were attributed in part to measures taken to reduce excitement of the horses during blood collection. The demeanour of the animals during collection was assessed subjectively as placid, timid, apprehensive or excited. The five horses which became excited had significantly higher RBC, Hb, PCV, WBC, total eosinophil count, serum total protein and probably lower ESR. Timid and apprehensive horses did not show these responses. Because of the location of eight stables, samples from them were stored overnight at 4°C before analysis. These samples had higher mean MCV, PCV, MCH, and serum total protein and lower mean MCHC. The likelihood of this arising from swelling and perhaps rupture of some erythrocytes during transportation and storage was discussed. Serum total protein estimates were within the range determined from other reports from horses of various breeds. However albumin and the A/G ratio determined by electrophoresis were higher, but comparable with two other reports from Thoroughbreds. In comparative studies on the estimation of A/G ratios it appeared that equine serum albumin determination was higher using the bromcresol green reagent than by electrophoresis. After eliminating 14 cases with abnormalities of the total and/or differential leucocyte count, parameters were calculated from 202 samples for the definition of normal ranges. The absence of skewness and kurtosis indicated that the individual estimates of RBC, Hb, PCV, WBC, MCV and MCH followed the normal distribution and that the means and standard deviations were appropriate for defining normal ranges. With only ESR and TEC were the coefficients of variation, skewness and kurtosis so great as to cast doubt on the practical use of these estimates.