Sheep Red Research Articles

Assessment of immune-competence traits among native and cross bred chicken

A 3x3 diallel cross experiment between indigenous breeds [Ankleshwar (AN), Nicobari (NC)] and exotic breed (CARI Red) was carried out to study the immune-competence traits like humoral and cell mediated immune (CMI) response. A total of 540 birds were grouped into nine (three pure and six cross breeds, n=60/group) to conduct this 3 × 3 complete diallel cross experiment. Of which a total of 180 birds (n=20/group) were taken as representative birds as 10 male and 10 female from each genetic group to study the immuno-competence traits. Each bird was injected with 1 ml of 1% suspension of Sheep Red Blood Cells (SRBC) intravenously at 6th week of age to study the humoral immune response. The serum antibody titre against SRBC was measured by Heam agglutination (HA) test. After 10th week of age, the cell mediated immune response was assessed via intra-dermal injection of Phytohemagglutinin type P (PHA-P) on 3rd and 4th toe of the right foot of the chicken. The results of this study showed that there was significant difference (P<0.05) in humoral immune response between mating systems and genetic groups. The crossbreds showed significantly higher humoral and cell mediated immune response than purebreds. However, among purebreds, the CR showed better immune response. Moreover, the crosses of CR×NC showed higher humoral and Cell mediated immune response than others, followed by NC×CR. It is concluded that CARI Red as male and Nicobari as female showed better humoral and cell mediated immune response than others.

Fine-scale population structure analysis of seven local Swiss sheep breeds using genome-wide SNP data

As part of the global sheep Hapmap project, 24 individuals from each of seven indigenous Swiss sheep breeds (Bundner Oberlander sheep (BOS), Engadine Red sheep (ERS), Swiss Black-Brown Mountain sheep (SBS), Swiss Mirror sheep (SMS), Swiss White Alpine (SWA) sheep, Valais Blacknose sheep (VBS) and Valais Red sheep (VRS)), were genotyped using Illumina’s Ovine SNP50 BeadChip. In total, 167 animals were subjected to a detailed analysis for genetic diversity using 45 193 informative single nucleotide polymorphisms. The results of the phylogenetic analyses supported the known proximity between populations such as VBS and VRS or SMS and SWA. Average genomic relatedness within a breed was found to be 12 percent (BOS), 5 percent (ERS), 9 percent (SBS), 10 percent (SMS), 9 percent (SWA), 12 percent (VBS) and 20 percent (VRS). Furthermore, genomic relationships between breeds were found for single individuals from SWA and SMS, VRS and VBS as well as VRS and BOS. In addition, seven out of 40 indicated parent–offspring pairs could not be confirmed. These results were further supported by results from the genome-wide population cluster analysis. This study provides a better understanding of fine-scale population structures within and between Swiss sheep breeds. This relevant information will help to increase the conservation activities of the local Swiss sheep breeds.

Antibody Response of Non-Dewormed and Dewormed Village Chickens to Sheep Red Blood Cells

The economic activities of rural communities rely on small scale enterprises such as raisin g village chickens. However, the impact of many infectious diseases has hampered the activities of these rural communities necessitating the use of vaccines to control and mitigate the negative effects of the diseases. Understanding the effect of helminths on the immune system and ultimately on the outcome of vaccinations is important. In this study, the effect of deworming on the immune response was evaluated by antibody titres to sheep red blood cells using direct hemagglutination in 41 naturally helminth-infested village chickens. We found that the immune response of both the control and dewormed groups of chickens, respectively, to sheep red blood cells were similar at primary (mean titres 726.74 and 819.58; p = 0.78) and secondary (mean titres 1822.24 and 1792.92; p = 0.95) levels. The results show that helminth infestation of clinically healthy chickens may not grossly affect the immune response of village chickens, suggesting that naturally infested chickens can be immunized without deworming.

Effect of Supplementation of Licorice Root (Glycyrrhiza Glabra L.) Extracts on Immune Status in Commercial Broilers

An experimental study was designed with the objective of exploring the effect of supplementation of Glycyrrhiza glabra extracts on immune status in commercial broilers. Fifty four, day old, straight run broiler chicks were randomly allotted to 3 treatment groups (T 1 - unsupplemented; T 2 - supplemented with 1% G. glabra crude extract powder and T 3 - supplemented with 0.1% G. glabra s tandardized extract powder) consisting of three replicates of six chicks each. The birds were reared under standard and uniform management conditions throughout the experimental period. Ranikhet-LaSota strain was used to produce antibodies against Ranikhet disease virus. All the birds were also immunized against Sheep Red Blood Cells (SRBC). HI titre values against Ranikhet (LaSota) and HA titre values against SRBC antigens were estimated to assess the humoral immunity. The Serum Biochemical Parameters viz., serum total protein and albumin were estimated and serum globulin and albumin:globulin ratio were calculated. To assess the cell mediated immunity, total and differential white blood cell counts were performed. The group supplemented with 0.1% licorice standardized extract has shown significantly (p<0.01) higher HI titre value against Ranikhet disease virus, than the other groups, on day 42, whereas, there was no significant differences among the groups on days 7, 14, 21, 28 and 35 in HI titre values. Licorice root standardized extract supplemented group performed better than other groups in increasing HI titre values throughout the study period. Against SRBC antigen, significant differences (p<0.01) were observed among the groups on days 21, 28, 35 and 42 in HA titre values (p<0.01). Licorice root standardized extract supplemented group performed better than other groups in increasing HA titre values throughout the study period. No significant differences were noticed among groups in 2 weeks and 6 weeks except on 4 weeks in which significant (p<0.01) differences were noticed in serum albumin, serum globulin and albumin:globulin ratio. No significant differences were observed in the total WBC and differential cell counts throughout the study period among different treatment groups. This shows that licorice root extracts do not have any direct effect on cell mediated immunity.

Effects of Ground Thyme and Probiotic Supplements in Diets on Broiler Performance, Blood Biochemistry and Immunological Response to Sheep Red Blood Cells

A trial was conducted to study the effects of the aromatic plant thyme, a commercial probiotic (Protexin) and avilamycin on broiler performance, blood biochemical parameters and also the antibody response to sheep red blood cells. A total of 750 broilers were assigned into five replicate groups for each of five dietary treatments, namely; control (C), 2.5 mg/kg avilamycin (AB), 0.1 g/kg commercial probiotic (P), 5 g/kg ground thyme (T1), and 7.5 g/kg ground thyme (T2). In general, body weight, feed consumption and feed conversion ratio were not affected by dietary treatments compared to the control birds (P>0.05). Birds fed the P supplemented treatment had the greatest serum protein levels (P<0.001) and highest albumin levels (P<0.001) when compared with control birds, while the birds fed T2 had the lowest (P<0.001). Dietary supplementation reduced (P<0.001) cholesterol and triglyceride concentration in serum of broilers, with the effect were more noticeable by P supplements. Moreover, blood calcium and phosphorus concentrations were higher (P<0.001) in birds fed the P-supplemented diet compared to the birds fed the control diets.This study suggests that probiotic supplementation in particular, and to an intermediate extent ground thyme supplementation in diets of broiler, resulted in chicks with favorably improved blood biochemical parameters and mineral utilization, compared to the birds fed diets supplemented with avilamycin or without any supplementation.

Correlated Responses to Bi-Directional Selection for Sheep Red Blood Cell Titer in White Leghorns

Correlated Responses to Bi-Directional Selection for Sheep Red Blood Cell Titer in White Leghorns

The Hummoral Immune Response of Heat Stressed Broiler Chicks Fed Different Levels of Energy and Methionine

The present study was conducted to investigate the effect of feeding different levels of energy and methionine on the total antibody, IgG, IgM immunoglobulin titer of broiler chicks. Thirty chicks were reared in an open sided house with temperature range from 29-42°C. The birds were divided into six groups of five replicate with individual bird. Six experimental diets were used with two levels of energy 3050, 2850 kcal/kg and three levels of methionine: control (0%, 0.2% and 0.4%) above the recommended level. At 14 day of age the birds were immunized with 0.2% Sheep Red Blood Cell (SRBC) suspension. The total antibody titer against SRBC was evaluated at 5 and 10 days post immunization using haemagglutination test. 0.1M 2- meracaptoethanol was used to determine IgG, IgM immunoglobulin. The total antibody titer response to SRBC was significantly (p 0.05) effect on total antibody titer. At 5 and 10 days post immunization IgG increased significantly (p<0.05) with 0.2 and 0.4% methionine above the recommended level. However, no change was observed in the titer of lgM. The different levels of energy had no effect on IgG and IgM titer.

Improved Extraction of Saturated Fatty Acids but not Omega‐3 Fatty Acids from Sheep Red Blood Cells Using a One‐Step Extraction Procedure

Several methods are available to extract total lipid and methylate fatty acids from a range of samples including red blood cells (RBC). Fatty acid analysis of human RBC can be undertaken using a two-step extraction and methylation or a combined one-step extraction and methylation procedure. The lipid composition of sheep RBC differs significantly from that of humans and may affect their extraction. The purpose of the current study was to examine the efficiency of extraction of lipid and detection of fatty acids from sheep RBC using a one-step procedure. Fatty acids were analysed using a one-step extraction and methylation procedure using methanol:toluene and acetyl chloride in comparison with a two-step procedure involving extraction of lipid using chloroform:methanol and separate methylation. Concentrations of saturated fatty acids including C16:0 and C18:0 were significantly higher (42.6 and 33.9% respectively) following extraction using the one-step procedure compared with the two-step procedure. However, concentrations of some polyunsaturated fatty acids, including C20:5n-3 and C22:6n-3 were not significantly different between either procedure. The improved detection of fatty acids may be related to the ability of different solvents to extract different lipid fractions. The differential extraction of lipids and detection of fatty acids from sheep RBC may have important implications in studies examining the effect of dietary treatment on the possible health benefits of fatty acids.

Ontogenesis of the Reticulum with Special Reference to Neuroendocrine and Glial Cells: A Comparative Analysis of the Merino Sheep and Iberian Red Deer

The present study was designed to compare the differences in the ontogenesis of the reticulum in sheep (domestic ruminant) and deer (wild ruminant). A total of 50 embryos and foetuses Merino sheep and 50 Iberian deer were used, from the first pre-natal life until birth. The appearance of the reticulum from the primitive gastric tube was earlier in the sheep (22% gestation, 33 days) than in the deer (25% gestation, 66 days). In both cases, it displayed a primitive epithelium of a stratified, cylindrical, non-ciliary type. At around 48% gestation in the sheep (72 days) and 36% (97 days) in the deer, the reticulum was configured of four clearly differentiated layers: mucosa (with epithelial layer and lamina propria), submucosa, tunica muscularis and serosa. The stratification of the epithelial layer was accompanied by modifications in its structure with the appearance of the primitive reticular ribs. The primary ribs began to be formed first in the deer, at 117 days of pre-natal life (40% gestation) and later in the sheep (79 days, 53% gestation). The differentiation of the corneum papillae in the primary ribs coincided with the appearance of secondary reticular ribs. These structures began to be formed first in the deer, at 142 days of pre-natal life (51% gestation) and later in the sheep (83 days, 55% gestation). The presence of neuroendocrine cells (non-neuronal enolase-positive cells) in the reticular mucosa was not detected until 97 days (36% gestation) in deer and 81 days (54% gestation) in sheep. The presence of glial cells (GFAP-positive cells) occurred at around 142 days (51% gestation) in deer and at 112 days (75% gestation) in sheep. In conclusion, the presence of neuroendocrine and glial cells was detected in deer at earlier stages than sheep.

ROLE OF DRIED PURSLANE LEAVES MEAL AND ESSENTIAL PHOSPHOLIPIDS IN LAYING HEN DIETS IN REDUCING CHOLSTEROL BIOSYNTHESIS

The aim of this study was to explain the cholesterol-lowering mechanism of dietary dried purslane leaves meal and essential phospholipids in egg-yolk and hen muscle tissues. Seventy two Inshas laying hens were randomly divided into 4 groups (3 replicate pens of 6 hens each) housed individually in one cage. Birds were fed from 28 to 40 weeks of age, either the control diet (based on corn-soybean meal) or the control diet with 10% purslane leaves meal with or without 450 mg essential phospholipids (EPL) /kg diet. Feed and water were offered ad libitum, feed intake, feed egg mass ratio, egg number and egg weight were recorded weekly. Birds were injected at 11th week of the experiment with Sheep Red Blood Cells (SRBC's) for immune test. At end of the experiment, sensory evaluation of hard boiled eggs was conducted for teste and flavor evaluation, cholesterol levels in each of egg-yolk, liver breast, thigh muscles and serum were determined. The activity of the rate-limiting enzymes in cholesterol biosynthesis was also determined. Four birds/group were slaughtered for sampling analysis. Results obtained showed that feeding laying hens on 10% purslane leaves supplemented diet produced the best values of egg production, egg number, egg mass and feed conversion compared with the other dietary treatment groups. Dried purslane leaves, EPL and purslane + EPL diets decreased (P<0.05) serum total lipids by 3.05, 6.55 and 9.40% ; serum cholesterol by 16.74, 23.25 and 29.30% and serum triglycerides by 6.72, 8.93 and 14.55%, while, serum high density lipoprotein was significantly (P<0.05) increased by 5.35, 6.96 and 8.48%, respectively, and hens fed 10% purslane leaves diet increased (P<0.05) serum low density lipoprotein by 3.06% compared with the control group. Moreover, no significant effects were observed on serum AST and ALT as well as taste and flavor of hard boiled eggs at end of the experimental period. Antibody response to SRBC's and leucocytes (WBC's) and lymphocytes counts were increased (P<0.05) by feeding the purslane diets. Cholsterol level was decreased (P<0.05) by feeding purslane or purslane + ELP diets, in thigh, breast, liver, yolk and serum and this decrease (P<0.05) was more pronounced by feeding the purslane + EPL diet. Supplementation of purslane or EPL to the laying hen diets significantly (P<0.05) decreased the relative weight of liver and oviduct. Abdominal fat percentage of groups while bile volume of gall bladder was significantly (P<0.05) increased compared with the control group. The activity of the rate-limiting enzymes in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl CoA reductase was suppressed (P<0.05) by feeding purslane or EPL and purslane + EPL diets. Fatty acid synthetase activity was not significantly affected by dietary treatment groups. Both purslane and EPL diets reduced cholesterol -7-a-hydroxylase activity. This study indicates that purslane and EPL inhibit cholesterol biosynthesis by a similar mechanism.

Antibody Productivity against Sheep Red Blood Cells and Splenocyte Proliferation Reaction of Tiger Puffer (Takifugu rubripes) Fed with Tetrodotoxin-Containing Diets

Antibody Productivity against Sheep Red Blood Cells and Splenocyte Proliferation Reaction of Tiger Puffer (Takifugu rubripes) Fed with Tetrodotoxin-Containing Diets

Receptors for Sheep Red Blood Cell, Immunoglobulin M and Complement Detected on the Lymphocyte of Eel, Anguilla japonica

Receptors for Sheep Red Blood Cell, Immunoglobulin M and Complement Detected on the Lymphocyte of Eel, Anguilla japonica

Comparison of Four Induced Molting Methods Based on Subsequent Performance and Welfare of Single Comb White Leghorn Hens

A total of 400 Single Comb White Leghorn hens at 80 weeks of age were used in present study. Four treatment groups that were consisted of six replications with 10 hens per each replication were considered. In the first treatment, food and water were removed for the first 2, 4, 6 and 8 days. However, on days 3, 5, 7, 9 and on day 10 until day 28 hens were fed 45 g of food per hen (ON-OFF group). In the second and third groups, food was withdrawn for 10, 14 days, respectively and oyster shell and water were provided for ad libitum until day 28 (California and North Carolina treatments (CAL, NC groups)). The last group (full-food treatments (FF group)) was the hens that were non-food-deprived and were fed corn molt diet for 28 days ad libitum. In all treatment groups at day 29, hens were returned to a full food layer ration and received 16 h of light day(-1). Body weight, egg production, egg weight, relative eggshell weight, internal egg quality, egg specific gravity and mortality were determined. Differential leukocyte count and antibody response were also measured. The results demonstrated that induced molting increased egg production according to pre-molt egg production. There was not a significant difference in the rate of egg production during the peak period of post molt production for treatments. No significant differences were detected in differential leukocyte counts and antibody response against Sheep Red Blood Cell (SRBC) between treatments at pre molt and post molt of the experiment. This study indicated that diets with high corn level (FF groups) are effective and simplified nonfood removal procedure for welfare molting hens.

Effects of Different Forced Molting Methods on Postmolt Production, Corticosterone Level, and Immune Response to Sheep Red Blood Cells in Laying Hens

The objective of this study was to evaluate the effects of different molting methods on postmolt production, plasma corticosterone levels, and antibody production to SRBC for the welfare of laying hens. This experiment was conducted with 120 IGH-type Brown laying hens (70 wk of age), randomly divided into 3 experimental groups. The hens in one group were fed a whole-grain barley diet during the first 10 d (WB diet). On d 11, hens consumed 100 g of layer diet/d until d 28. In the second group, hens were fed a Zn diet containing 10,000 mg/kg of Zn as ZnO for 10 d (Zn diet). Hens were then provided 100 g of a layer diet from d 11 to 28. In the third group, feed was withdrawn for 10 d, and on d 11 hens were fed a cracked corn diet ad libitum until d 28 (California method; CAL diet). Hens in all groups were returned to the layer diet ad libitum on d 29. Egg weight was lower in the Zn treatment than in the other treatments. Feed intake and plasma corticosterone levels were higher and antibody production was lower in the CAL treatment than in the WB and Zn groups. The FCR was better in the WB than in the Zn group. Mortality, egg production, and egg quality were not significantly different among the molting methods. As a result, the WB molting program was the best method for postmolt production among the programs examined.

Use of Whole Sheep Red Blood Cells in ELISA to Assess Immunosuppression In Vivo

An enzyme-linked immunosorbent assay (ELISA) using whole sheep red blood cells (SRBC) has been reported as one of the methods for detecting a T-lymphocyte-dependent antibody response. However, it has not been widely used because of SRBC problems such as the weak attachment to ELISA plates, specificity and short-term stability. The objectives of this study were to address these issues and to validate the SRBC-specific antibody response assay. Male Sprague–Dawley rats were bled after 6 days of SRBC immunization. In our new procedure, glutaraldehyde was added before discarding the supernatant of inoculated SRBC suspension to attach SRBC firmly to the plate, while in the original method it was added after discarding. As a result, the attached SRBC was maintained throughout the ELISA procedures. No interference was observed in the titration curve of IgM and IgG antibodies in rats and IgM-antibody in mice when control sera were analyzed to evaluate specificity of this method. The short-term stability of SRBC was overcome by using the different lots of SRBC. They provided antibody titers, which were consistent with those measured using the same lot for immunization. In addition, cyclophosphamide, cyclosporine, prednisolone and methotrexate, well-known immunosuppressive agents, were tested to confirm the applicability of the improved ELISA method to detect the T-lymphocyte-dependent antibody response. All four compounds inhibited the IgM antibody responses dose-dependently. These results demonstrate that the improved whole SRBC-ELISA method provides reproducible and reliable results in the T-lymphocyte-dependent antibody response assay.

Primary Immune Response to Sheep Red Blood Cells (SRBC) as the Conventional T-Cell Dependent Antibody Response (TDAR) Test

The production of antigen-specific antibodies represents a major defense mechanism of humoral immune responses. Several assays have been developed to assess T-cell-dependent antibody responses (TDAR). Of these assays, the antibody forming cell assay (AFC) or plaque forming cell (PFC) assay and ELISA are the two most often used tests to assess immunotoxicity. Historically, the T-cell-dependent antigen of choice has been sheep red blood cells (SRBC). The SRBC AFC assay is considered the “gold standard” for TDAR based on extensive intra- and inter-laboratory validation in mice and has been utilized for over 35 years. The quantification of the primary AFC response (i.e., the specific IgM antibody-forming cell response) was found to provide one of the best predictors of immunotoxicity in mice. The SRBC-specific ELISA is relatively new, with the first publication of the method appearing in 1993. Data from the application of using both the SRBC specific AFC and ELISA for evaluation of potential immunotoxicity of chemicals in rodents and the pros and cons and associated issues of each method were presented. Specifically, the following was discussed: (1) studies investigating the incorporation of the SRBC-specific IgM ELISA in rats on standard toxicology study; (2) characterization of an approach to developmental immunotoxicology assessment in the rat using SRBC as the antigen; and, (3) data from an inter-laboratory study comparing the AFC assay and ELISA in outbred rodents using both cyclophosphamide and dexamethasone.

Memory Antibody Responses of Broiler and Leghorn Chickens as Influenced by Dietary Vitamin E and route of Sheep Red Blood Cell Administration

Influences of dietary levels of vitamin E fed to hens and their progeny, and routes of SRBC inoculation on antibody responses of diverse populations of chickens were studied. Populations were a commercial broiler sire line (C), 2 commercial broiler dam lines (A and B), and Leghorn lines selected for high (H) or low (L) antibody response to SRBC. Dams from lines A and B were fed diets supplemented with either 10 or 300 IU/kg of vitamin E, whereas dams from lines H and L received only the diet with 10 IU of vitamin E/kg. Progeny from matings of C males with A and B females as well as H and L females mated to males from their respective lines were hatched on the same day and fed diets supplemented with either 10 or 300 IU/kg of vitamin E. Breeders were the same age and eggs were incubated in the same machine. Chicks were inoculated on d 14 intravenously with 0.1 mL of a 0.5% suspension of SRBC or intramuscularly with 0.1 mL of a 25% suspension of SRBC. Antibody response was measured 6 and 14 d later. Chicks received a booster i.m. inoculation of 0.1 mL of 25% SRBC on d 28. Titers were again measured 6 and 14 d later. Level of vitamin E fed to dams did not affect progeny BW or plasma vitamin E levels. Although titers were higher following i.v. than i.m. inoculation, the degree of difference varied among stocks. Dietary vitamin E level interacted with inoculation route with a greater response to the higher than lower level of vitamin E for i.v., but there was no difference for i.m. There were stock × level of vitamin E and stock × route of inoculation interactions for secondary responses to SRBC inoculation. Stock rankings after the first inoculation were not predictive of the rankings after the second inoculation. The 30-fold increase in dietary level of vitamin E resulted in >12-fold differences in plasma levels of vitamin E. Overall, there was a stock-dependent influence of dietary vitamin E on growth and humoral antibody response.

Effects of Dietary Polyunsaturated Fatty Acids on Antibody Production and Lymphocyte Proliferation of Laying Hens

The purpose of present study was to assess the effect of polyunsaturated fatty acids (PUFA) on the immune responses of laying hens. Three hundred and sixty hens at the age of 60 weeks were randomly assigned to ten diets, which contained no oil (CK), 1%, 3%, 5% fish oil (FO); 2%, 4%, 6% linseed oil (LO) and 2%, 4%, 6% corn oil (CO). After 5 weeks of feeding experimental diets, humoral and cellular immune responses were assayed. Laying hens were injected with Sheep Red Blood Cell (SRBC) and Bovine Serum Albumin (BSA) and antibody titers, which were measured on d6, d10, d14 after primary challenge and on d5, d9, d13 after secondary challenge. Concanavalin (ConA) and lipopolysaccharide (LPS) -stimulated proliferation of peripheral blood and spleen lymphocytes were assessed by ( 3 H) thymidine incorporation at the week age of 5 and 10, respectively. The results showed that antibody titers in FO-fed and LO-fed laying hens were higher than that in laying hens fed CO. The proliferation response to ConA was lower in laying hens that fed oils rich in n-3 fatty acids than that in laying hens fed CO. Higher level n-3 fatty acids can improve immune functions of laying hens. In conclusion, dietary fat source and level had a significant impact on immune responses of laying hens. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 9 : 1320-1325)

Role of nitrite, a nitric oxide derivative, in K-Cl cotransport activation of low-potassium sheep red blood cells.

K-Cl cotransport (COT) is the coupled movement of K and Cl, present in most cells, associated with regulatory volume decrease, susceptible to oxidation and functionally overexpressed in sickle cell anemia. The aim of this study was to characterize the effect of the oxidant nitrite (NO2-) on K-Cl COT. NO2- is a stable metabolic end product of the short-lived highly reactive free radical nitric oxide (NO), an oxidant and modulator of ion channels, and a vasodilator. In some systems, the response to NO2- is identical to that of NO. We hypothesized that NO2- activates K-Cl COT. Low potassium (LK) sheep red blood cells (SRBCs) were used as a model. The effect of various concentrations (10(-6) to 10(-1) m) of NaNO2 was studied on K efflux in hypotonic Cl and NO3 media, Cl-dependent K efflux (K-Cl COT), glutathione (GSH), and methemoglobin (MetHb) formation. In support of our hypothesis, K efflux and K-Cl COT were stimulated by increasing concentrations of NaNO2. Stimulation of K efflux was dependent upon external Cl and exhibited a lag phase, consistent with activation of K-Cl COT through a regulatory mechanism. Exposure of LK SRBCs to NaNO2 decreased GSH, an effect characteristic of a thiol-oxidizing agent, and induced MetHb formation. K-Cl COT activity was positively correlated with Methb formation. N-ethyl-maleimide (NEM), a potent activator of K-Cl COT, was used to assess the mechanism of NO2- action. The results suggest that NEM and NO2- utilize at least one common pathway for K-Cl COT activation. Since NaNO2 is also a well known vasodilator, the present findings suggest a role of K-Cl COT in vasodilation.

14C]cyanate labeling of sheep red cells: covalent binding to hemoglobin continues in vivo for a day.

The sheep is a useful model to study fetal and newborn physiology including perinatal erythropoiesis and red cell kinetics. A practical, economical method for measuring red cell survival (RCS) in sheep would be very valuable. However, 51Cr is unsatisfactory, and suitable alternatives have not been published. In the course of investigating [14C]cyanate as a label for sheep red cells, we observed continued covalent labeling over 24 h in vivo that was great enough to introduce a substantial artifact into two commonly used parameters of RCS: posttransfusion recovery (PTR24) and time to 50% decrease (T50) when referenced to time zero. In a simulation of in vivo conditions, the amount of 14C bound to Hb increased 26 +/- 6% (mean +/- 1 SD, n = 11) over 24 h. To investigate the mechanism of the increasing 14C bound, acid-acetone extraction, molecular sieve chromatography, and density gradient separation were used separately or in combination to quantitate intracellular free 14C and 14C covalently bound to intracellular proteins. Free 14C decreased as protein-bound [14C]cyanate increased. These studies provide evidence that covalent binding of [14C]cyanate to intracellular Hb continues in vivo for the first 24 h and that the source of the increase is intracellular free [14C]cyanate. We conclude that 1) PTR24 cannot be accurately determined by [14C]cyanate unless labeled red cells are incubated before infusion to allow the cyanate reaction to approach completion and 2) RCS by [14C]cyanate should be referenced to blood concentrations at 24 h.