Spermatozoa Concentration,Seminal Plasma Composition and Their Physiological Relationship in the Endangered Stellate Sturgeon (Acipenser stellatus) and Russian Sturgeon (Acipenser gueldenstaedtii )

Comparative studies of ionic composition, osmolality, protein concentration and pH of seminal plasma along with spermatozoa concentrations were carried out in stellate sturgeon, Acipenser stellatus, and Russian sturgeon, Acipenser gueldenstaedtii. Analysis of A. gueldenstaedtii sperm showed significantly higher concentrations of Na⁺ (34.58 ± 4.61 mm ), Ca²⁺ (0.35 ± 0.12 mm ), Mg²⁺ (0.70 ± 0.25 mm ), Cl^? (13.50 ± 4.04 mm ) and proteins (0.60 ± 0.29 mg/ml) in the seminal plasma than did seminal plasma of A. stellatus: Na⁺ (20.08 ± 10.75 mm ), Ca²⁺ (0.28 ± 0.06 mm ), Mg²⁺ (0.29 ± 0.05 mm ), Cl^? (7.50 ± 3.00 mm ) and 0.30 ± 0.11 mg/ml proteins. Significantly higher concentration of K⁺ (5.42 ± 1.06 mm ) was observed in A. stellatus compared to A. gueldenstaedtii K⁺ (2.29 ± 0.50 mm ). Concentration of Na⁺ was positively correlated with osmolality (r = 0.819), levels of Cl^? (r = 0.922) and Mg²⁺ (r = 0.727) and pH (r = 0.848). The concentration of Mg²⁺ was positively correlated with protein concentration (r = 0.774), Na⁺ (r = 0.727), Cl^? (r = 0.872) and Ca²⁺ (r = 0.801). A positive relationship was also found between concentration of K⁺ and spermatozoa concentration (r = 0.709). Results revealed strong inter‐species differences in several parameters. The data should be useful for artificial fertilization and for cryopreservation of sturgeon sperm.     

Retracted: Determination of some blood and seminal plasma ions in the beluga,Huso huso (Linnaeus, 1758)

Blood and seminal plasma ionic parameters are essential for monitoring health status, detecting illnesses, fish stock conservation and development of artificial propagation methods via extender improvement. In this study, comparison of blood and seminal plasma ionic parameters in beluga, Huso huso (30–45 kg, 1–2 m, n = 10), was made. The results obtained show that Na⁺ (82.54 ± 5.46), Cl^‐ (15.95 ± 0.72) and K⁺ (3.57 ± 0.15) were predominant ions in the seminal plasma (as mm ). Blood ionic values (as mm ) were determined for Na⁺ (110.2 ± 1.26), K⁺ (3.77 ± 0.081), Cl^? (60.12 ± 1.5), Ca²⁺ (2.05 ± 0.35) and Mg²⁺ (1.9 ± 0.16). Results of the comparison between ionic parameters of seminal and blood plasma indicated that the concentrations of all parameters of blood plasma with the exception of K⁺ were significantly (p < 0.05) higher than those of seminal plasma.     

Branchial Ionic Flux Responses in Rainbow Trout to Chloramine-T after Acclimation to Different Levels of Water Hardness

Abstract

Whole-body unidirectional and net Na⁺ and Cl^– fluxes were determined in rainbow trout Oncorhynchus mykiss exposed to chloramine-T (9 mg/L) alone or in combination with 17 mmol NaCl/L (0.1 % weight per volume, w/v) after acclimation to either city of Ottawa tap water (TW: Na⁺ 0.121 ± 0.004 mmol/L; K⁺ 0.015 ± <0.001 mmol/L; Ca²⁺ 0.351 ± 0.004 mmol/L; Cl^– 0.141 ± 0.002 mmol/L) or artificial soft water (ASW: Na⁺ 0.043 ± 0.003 mmol/L; K⁺ 0.005 ± <0.001 mmol/L; Ca²⁺ 0.063 ± 0.002 mmol/L; Cl^– 0.026 ± 0.002 mmol/L). Preexposure fluxes revealed that ASW-acclimated fish had reduced rates of Na⁺ uptake and efflux compared with TW-acclimated fish. However, rates of Cl^– uptake among ASW-acclimated fish were comparable with those of TW-acclimated fish. Exposure to chloramine-T in TW resulted in significant increases in the Cl^– efflux, giving rise to a significant and negative net flux. Exposure of ASW-acclimated fish to chloramine-T caused a significant negative increase in the Na⁺ efflux and net flux but only a negative increase in the Cl^– net flux. The addition of 17 mmol NaCl/L (0.1% w/v) eliminated the negative net ionic fluxes, resulting in significant increases in influx. The combined use of chloramine-T with NaCl is a potential method of remediating ionic disturbances that may be caused by treating trout with chloramine-T.     

Study of Sperm Concentration,Seminal Plasma Composition and their Physiological Correlation in the Persian Sturgeon,Acipenser persicus

The objectives of the present study were to determine ionic and organic composition of seminal plasma, sperm concentration and their relationships in the Persian sturgeon (Acipenser persicus). In this regard, ionic content (Na⁺, K⁺, Cl^?, Ca²⁺ and Mg²⁺) and organic content (total protein, glucose, cholesterol and triglyceride) along with sperm concentration were measured in 17 specimens of the Persian sturgeon. The seminal plasma contained 59.53 ± 2.56 mm /l sodium, 9.1 ± 1.42 mm chloride, 4.72 ± 0.3 mm potassium, 1.45 ± 0.075 mm calcium and 0.7 ± 0.072 mm magnesium. The following organic contents were found: total protein 0.11 ± 0.02 g/dl, glucose 22.18 ± 4.16 mg/dl, cholesterol 6.67 ± 1.04 mg/dl and triglyceride 15.2 ± 0.65 mg/dl. The mean sperm concentration was estimated to be 1.6 ± 0.12 (×10⁹ sperm/ml). A significant relationship was found between sperm concentration and K⁺ of seminal plasma (r = 0.533, p < 0.05). Significant correlations were observed between ionic contents: Na⁺ vs Cl^? (r = ?0.854, p < 0.01) and Mg²⁺ vs K⁺ (?0.583, p < 0.05). Also, level of triglyceride was negatively correlated with Mg²⁺ (r = ?0.503, p < 0.05). Presented data could be considered as a complementary study for developing special extenders and protectant solutions for improving artificial fertilization in this valuable species.     

Composition of seminal plasma and ovarian fluid in Ide Leuciscus idus and Northern pike Esox lucius

Seminal plasma (SP) and ovarian fluid (OF) plays an important role as storage media to prevent the activation of gametes both in vivo and under artificial conditions. The objectives of this study were to quantify gamete biochemistry and explore correlations among quantitative characteristics of SP, OF and sperm performance traits of Ide Leuciscus idus and Northern pike Esox lucius. Generally, Na⁺, K⁺ and Cl^? were found to be the most dominating ions, although concentrations of K⁺ were higher in SP, while Na⁺ and Cl^? concentrations were higher in OF for both species. Several significant correlations among the biochemical properties such as total protein, glucose, osmolality, cholesterol, K⁺, Ca²⁺, Cl^? and Mg²⁺ were observed for SP and OF. Total protein content of Ide SP was positively correlated with sperm activity traits (r ≥ .89, p ≤ .05), while K⁺ concentration was negatively correlated with sperm traits (r ≥ ?.89, p ≤ .05). Moreover, Ca²⁺ concentration in Northern pike SP was positively correlated with the percentage of sperm motility (r = . 98, p < .01). In conclusion, these results can be used to better understand the biochemistry of SP and OF, improve methods for short‐ and long‐term storage of gametes and standardize fertilization protocols.     

Influence of copper sulphate on the water and electrolyte balance of the freshwater snail Bulinus (Bulinus) tropicus

The water and electrolyte balance of Bulinus (Bulinus) tropicus, a freshwater pulmonate, was determined when subjected to different sublethal concentrations of copper sulphate. It was found that the lethal dose (LD₅₀), which is the dose lethal to 50% of the snails, is 1,0 ppm in water of pH 8,5. Increasing dosages disturb the ionic and water balance. Haemolymph concentrations of Na ⁺ , Ca²⁺ and Cl^- decrease markedly. The rates of ion influx and efflux for Ca²⁺ , Na ⁺ , Cl^- and ³H₂0, measured With their corresponding radioisotopes, were drastically changed. The results indicate that the physiological effects of copper are not limited to particular organs or tissues but probably occur throughout the snail body. The possible effect of copper sulphate on the in- tegumental exchange mechanisms for Ca²⁺, HCO3^-, Na⁺ , K⁺ and Cl^- are discussed.     

酒精阳性乳患牛血清及乳清离子和微量元素分析

通过检测不同酒精阳性乳血清和乳清中Ca²⁺、Mg²⁺、Na⁺、K⁺浓度及Cu、Zn、Fe、Mn含量,探讨其与酒精阳性乳的关系,结果显示,酒精阳性乳患牛体内的微量元素Zn、Cu和Fe代谢紊乱,会导致抗氧化酶的活性降低、毒性产物含量升高而使自由基代谢平衡紊乱,且Zn、Cu含量的缺乏会降低机体的免疫力;患牛乳中过多的Ca²⁺、Mg²⁺中和酪蛋白微胶粒表面的电荷,导致酪蛋白稳定性下降,致使乳胶体聚集能力增强;这些都易导致酒精阳性乳的形成。     

Salt-Lick–Induced Soil Disturbance in the Teton Wilderness,USA

Manmade salt licks on public lands throughout the Rocky Mountain West have been created to attract large game for hunting purposes. This practice is both illegal and controversial, but is of particular importance in otherwise pristine wilderness landscapes. The impact of widespread saltlicks on public lands has never been quantified. This study was undertaken to examine the degree of change in soil physical and chemical properties caused by approximately 10–60 years of salt application in the Teton Wilderness of Wyoming, USA. A total of 27 sites were identified, surveyed, and paired with non–salt-affected control areas. Three replicate sampling points were located within each salt site and in each of the paired control areas. Soil samples from each site were analyzed for soil bulk density, soil salinity as electrical conductance (EC), pH, organic matter content, sodium absorption ratio (SAR), and exchangeable concentrations of sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), and magnesium (Mg²⁺). Salt-treated site centers were found to have elevated EC, bulk density, pH, SAR, and Na⁺ concentration compared to the no-salt controls. Salt-affected sites also contained decreased organic matter contents and decreased concentrations of Ca²⁺ and Mg²⁺. Observed differences were due to the addition of Na⁺ to the soil solum as well as direct effects of ungulates. Soil compaction appears to have a greater impact on plant establishment than the actual presence of NaCl. Salt licks established in wilderness areas habituate animals to localized zones causing extensive soil trampling and consumption of surface soils by grazing ungulates.     

Effects of dietary cation-anion balance on blood PH, acid base parameters, serum and urine mineral levels, and parathyroidhormone (PTH) in weanling horses.

This study demonstrated that the feeding of treatment diets with calculated dietary cation-anion balances (DCAB) of +370.43 (H) and -25.69 (L) did not have significant effects on blood pH, pCO₂, and HCO₃-. Serum Ca²⁺, P, Na⁺, and Cl^- as well as plasma PTH did not differ (P > .05) between the two treatment groups. Serum K⁺ was higher (P< .05) in horses fed diet H rather than diet L. The DCAB of the diet significantly affected urinary Ca²⁺, P, Na⁺, K⁺, and Cl^- excretion in the young growing horse. Urine Ca²⁺ and Cl- levels were higher (P < .01) in horses fed diet H versus diet L. Furthermore, levels of P, Na⁺, and K⁺ in the urine were higher (P < .01) in horses on diet H as opposed to diet L. Results of this study indicate that horses were able to maintain acid-base status regardless of diet. However, these data imply that growing horses consuming diets low in DCAB may be predisposed to abnormal bone mineralization due to the increase in calcium excretion which could lead to a weakening of the skeletal system.     

The concentration of ionized magnesium in serum during the periparturient period of non-paretic dairy cows

Ion-selective electrodes have recently been designed for determining the ionized concentration of magnesium (Mg²⁺) in serum. This development may allow new insights into some metabolic diseases of cattle. For this report, the concentrations of Mg²⁺, total magnesium (Mg_tot), ionized calcium (Ca²⁺), total calcium (Ca_tot), and inorganic phosphate (P_i) were determined in sera from seventeen 3-to 16-year-old Brown Swiss and crossed Simmental/Red Holstein cows during the periparturient period. In each animal, a transient increase of Mg²⁺ and Mg_tot serum concentrations was observed in association with the transient decrease in serum concentrations of Ca²⁺, Ca_tot and P_i after parturition. On average, throughout the study, the serum Mg²⁺ concentrations were 68.5% of those of Mg_tot, whereas the serum Ca²⁺ concentrations were 52% of those of Ca_tot. The possible mechanisms involved in the transient increase of Mg²⁺ and Mg_tot serum concentrations are discussed.Abbreviations Ca²⁺ ionized calcium - Ca_tot total calcium - Mg²⁺ ionized magnesium - Mg_tot total magnesium - P_i inorganic phosphate - PTH parathyroid hormone - PTHrP parathyroid homrone related protein     

Effects of aerobic and anaerobic fluid collection on biochemical analysis of peritoneal fluid in healthy horses and horses with colic

Objective: To determine whether in healthy horses and those with colic, exposure of peritoneal fluid to room air affects values obtained on biochemical analysis. Study Design: Prospective study. Animals: Adult horses with a primary complaint of acute abdominal pain (n=29) and 12 healthy horses. Methods: Peritoneal fluid was aseptically collected under aerobic and anaerobic conditions. After collection, pH, PCO₂, PO₂, HCO₃^?, Na⁺, ionized Ca²⁺, K⁺, lactate, and glucose were immediately measured using a commercial blood gas analyzer. Biochemical variables were compared between aerobically and anaerobically obtained samples using a paired t‐test. Results: In healthy horses, peritoneal fluid samples collected under anaerobic conditions had higher PCO₂ and ionized Ca²⁺ and lower PO₂, HCO₃^?, and pH compared with samples exposed to air. No differences were observed for K⁺, Na⁺, glucose, and lactate. In horses with colic, samples collected anaerobically had higher PCO₂, ionized Ca²⁺, Na⁺, and glucose and lower PO₂, HCO₃^?, and pH value compared with samples exposed to air. No differences were observed for K⁺ and lactate. Conclusion: Exposure of peritoneal fluid to room air had a significant effect on pH, PCO₂, PO₂, and variables associated or dependent on changes in pH such as HCO₃^? and ionized Ca²⁺. Interpretation of biochemical analysis of peritoneal fluid may be influenced by sample collection method.     

Preliminary Studies on the Concentration of Na+,K+-ATPase in Skeletal Muscle of Draught Cattle in Mozambique: Effect of Sex,Age and Training

The effect of training on the potential for work in draught cattle was assessed by measuring the Na⁺,K⁺-ATPase in the muscle cell membrane and the elevation in the concentration of K⁺ in plasma during exercise. Biopsies of the semitendinosus muscle and venous blood samples were taken from the cattle used for draught work in Mozambique. No differences were found in the plasma ion or Na⁺,K⁺-ATPase concentrations in samples taken from Nguni, Africander and Angoni breeds. There were no significant differences in plasma ions (Na⁺, K⁺ and Cl^–) or muscle Na⁺,K⁺-ATPase concentrations between the Angoni males and females, although the males showed an increase in Na⁺,K⁺-ATPase with age, while the females showed a decrease. The increase in males might be attributed to their higher level of activity in the herds than that of females. After a training period of 15 days, a significant increase in Na⁺,K⁺-ATPase concentration in semitendinosus muscle was found in Angoni cattle. In females, this was significant after 8 days of training (about 30%); in males after 15 days of training (about 16%). On day 15, there was a reduction in the elevation of plasma K⁺ during the 2 h of draught work, indicating an increased capacity of the Na⁺,K⁺ pumps to maintain the extracellular K⁺ concentration in working muscles and a possible delay in the moment of fatigue.     

Altered adenosine triphosphatase activities in pigs with naturally occurring hypertrophic cardiomyopathy

The purpose of this study was to determine whether myocardial adenosine triphosphatase (ATPase) activities were reduced in pigs with naturally occurring hypertrophic cardiomyopathy (HCM). The selection of hearts for the HCM and the normal control groups depended on histological examination. Specific ATPase activity and 5-nucleotidase activity were measured in left ventricular myocardium obtained from HCM (n=7) and normal control (n=7) animals. The histological features of HCM included marked disorientation of muscle cells, thickening of the intramural coronary arterial wall with a narrowed lumen, endocardial fibrosis and myocardial fibrosis. The HCM group showed significant increases in both heart weight (32%) and heart weight to body weight ratio (46%). The total ATPase activity in crude homogenates from the HCM group was significantly decreased by 16%. Azide-sensitive ATPase (mitochondrial ATPase) activity, ouabain-sensitive ATPase (Na⁺,K⁺-ATPase) activity, basal Mg²⁺-ATPase activity and Ca²⁺-ATPase activity were all significantly decreased by 18%, 30%, 20% and 50%, respectively. In contrast, no significant decrease was found in the mean values for 5-nucleotidase activity. These results suggest that myocardial ATPase activities are suppressed in pigs with naturally occurring HCMAbbreviations ATP adenosine triphosphate - gww grams wet weight - HCM hypertrophic cardiomyopathy - Pi inorganic phosphate     

pH-dependent modulation of intracellular free magnesium ions with ion-selective electrodes in papillary muscle of guinea pig

A change in pH can alter the intracellular concentration of electrolytes such as intracellular Ca²⁺ and Na⁺ ([Na⁺]_i) that are important for the cardiac function. For the determination of the role of pH in the cardiac magnesium homeostasis, the intracellular Mg²⁺ concentration ([Mg²⁺]_i), membrane potential and contraction in the papillary muscle of guinea pigs using ion-selective electrodes changing extracellular pH ([pH]_o) or intracellular pH ([pH]_i) were measured in this study. A high CO₂-induced low [pH]_o causes a significant increase in the [Mg²⁺]_i and [Na⁺]_i, which was accompanied by a decrease in the membrane potential and twitch force. The high [pH]_o had the opposite effect. These effects were reversible in both the beating and quiescent muscles. The low [pH]_o-induced increase in [Mg²⁺]_i occurred in the absence of [Mg²⁺]_o. The [Mg²⁺]_i was increased by the low [pH]_i induced by propionate. The [Mg²⁺]_i was increased by the low [pH]_i induced by NH₄Cl-prepulse and decreased by the recovery of [pH]_i induced by the removal of NH₄Cl. These results suggest that the pH can modulate [Mg²⁺]_i with a reverse relationship in heart, probably by affecting the intracellular Mg²⁺ homeostasis, but not by Mg²⁺ transport across the sarcolemma.     

日粮添加棉酚对哈萨克羊水代谢影响的研究

试验选用5只2岁、体重约40 kg的健康雄性哈萨克羊,单笼饲养。试验共4期(每期18 d,共72 d),第1期不添加棉酚,第2、3、4期在混合精料中分别添加984、1353、1722 mg/d棉酚。结果表明,随着棉酚添加量的增加,试验羊的饮水量、排尿量、麦秸采食量、排粪量及粪中含水量无明显变化(P>0.05);抗利尿激素、肾素、皮质酮水平无显著变化(P>0.05),促肾上腺皮质激素、醛固酮水平在前3期呈下降的趋势,第4期与第3期相比显著上升(P<0.05);血清中Na⁺水平在2、3期明显下降(P<0.05),第4期恢复到第1期水平,血清中Cl^-水平呈下降趋势,Ca²⁺水平也呈下降趋势,在4期试验中K⁺、无机磷、Mg²⁺含量无明显变化(P>0.05);尿中Na⁺、无机磷含量无变化(P>0.05),Cl^-含量前3期上升,第4期恢复到第1期水平,K⁺、Mg²⁺、Ca²⁺含量第4期较前3期均显著下降(P<0.05)。     

Effects of dietary protein and energy levels on digestive enzyme activities and electrolyte composition in the small intestinal fluid of geese

The present study was conducted to evaluate the effects of dietary protein and energy levels on digestive enzymes and electrolyte composition in jejunum of geese. A 3×3 factorial and completely randomized design was adopted with three protein levels and three energy levels. The experiment included four replicates for each treatment, and three geese for each replicate. Isovolumetric supernate from centrifugal jejuna fluid were mixed in each replicate. Activities of digestive enzymes and ions were analyzed. The results showed trypsin and chymotrypsin activities were significantly increased with increasing of dietary protein and energy levels (P<0.05). The concentrations of Ca²⁺ and pH value were significantly decreased by increased dietary protein and energy levels. However, no significant differences were found for the activities of amylase and cellulase, as well as the concentration of Na⁺ among groups with different protein and energy levels. In conclusion, digesta enzymes and electrolytes in the small intestine adapted to the protein and energy levels. The activities of protease, rather than amylase and cellulase were induced with increasing of protein and energy levels. The imbalance of positive and negative ions was possibly adjusted by the fluctuant concentrations of K⁺, Cl^‐ and Ca²⁺ for maintaining normal physiological function.     

Blood Chemistry Values for Shovelnose and Lake Sturgeon

Abstract

Blood chemistry panels are commonly used for assessing the general health of vertebrate animals. Here, we present novel blood chemistry data for two North American sturgeon species, shovelnose sturgeon Scaphirhynchus platorynchus and lake sturgeon Acipenser fulvescens. Measurements were done using a portable chemistry analyzer (VetScan Analyzer; Abaxis). Among the plasma values measured (mean ± SD for shovelnose and lake sturgeon, respectively) were total proteins (3.7 ± 0.9 and 2.8 ± 0.4 g/dL), albumin (2.0 ± 0.5 and 1.1 ± 0.2 g/dL), globulin (1.7 ± 0.7 and 1.7 ± 0.3 g/dL), glucose (107 ± 46 and 62 ± 9.7 mg/dL), sodium (Na⁺; 132 ± 3.6 and 150 ± 14 mEq/L), potassium (K⁺; 3.5 ± 0.2 and 2.8 ± 1.7 mEq/L), phosphorus (10.4 ± 1.9 and 11.6 ± 3.6 mg/dL), and aspartate aminotransferase (AST; 676 ± 433 and 634 ± 234 IU/L). Higher values for total proteins, albumin, glucose, and Na⁺ in shovelnose sturgeon than in lake sturgeon probably are the result of handling stress. In addition, the plasma of male shovelnose sturgeon had higher concentrations of AST, glucose, and globulin than did that of females, whereas the plasma of females had higher concentrations of albumin and K⁺ than that of males. This study is the first to report blood chemistry data for shovelnose sturgeon. Robust blood chemistry databases can be used by aquaculturists and fish managers for monitoring sturgeon health.

Received July 8, 2011; accepted February 2, 2012     

Ouabain-sensitive respiration and protein synthesis in duodenal mucosa and liver in rats fed increasing levels of pea fibre or protein and housed in 18 or 28°C environments

Seventy‐two Wistar rats were used in two studies to investigate the effect of environmental temperature (18 or 28°C), and increasing levels of dietary fibre (low, 68 g/kg dry matter (DM); medium 110 g/kg DM; high, 157 g/kg DM) and protein (low, 91 g/kg DM; medium, 171 g/kg DM; high, 262 g/kg DM) on respiration attributable to Na⁺,K⁺‐ATPase activity and protein synthesis in duodenal mucosa and liver of rats. In vitro O₂ consumption in tissues was measured polarographically using a Clark‐style YSI biological O₂ monitor. Whole‐body O₂ consumption was measured with two open‐circuit respiration chambers. Whole‐body O₂ consumption was higher (p < 0.05) at 18°C than at 28°C. Rats fed the low protein diet had significantly higher (p < 0.05) whole‐body O₂ consumption than those fed the medium or high protein diet. Compared with 28°C, the environmental temperature of 18°C caused an increase (p < 0.05) in total O₂ consumption and O₂ consumption attributable to Na⁺,K⁺‐ATPase activity in duodenal mucosa. There was no effect (p > 0.05) of environmental temperature on total O₂ consumption, Na⁺,K⁺‐ATPase activity attributable to protein synthesis dependent on O₂ consumption in the liver. Total O₂ consumption and O₂ consumption attributable to Na⁺,K⁺‐ATPase activity increased (p < 0.05) in duodenal mucosa in rats fed the low level of dietary fibre compared with rats fed the medium level of dietary fibre. In vitro O₂ consumption determined in duodenal mucosa and in liver did not always correspond to whole‐body O₂ consumption. This may indicate that respiration in the duodenum and liver adapts differently and may not reflect changes in whole‐body respiration in response to dietary modification and changes in thermal environment.     

Haemolytic activity of sheep complement for two assay systems

Sheep complement (C) is haemolytic for sheep erythrocytes sensitized with rabbit antibody (sheep E-rabbit A) provided serum is used as soon as possible after collection. If left at 4 °C to separate from the clot, serum C activity for sheep E-rabbit A is markedly reduced. Heparinized plasma retains its haemolytic titre for at least 24 h at 4 °C. Plasma from Mg²⁺-ethyleneglycoltetraacetic acid (EGTA) blood is non-haemolytic, but addition of Ca²⁺ partially restores the titre. A high concentration of rabbit A is necessary to sensitize sheep E.Sheep C is haemolytic for human erythrocytes sensitized with sheep antibody (human E-sheep A) in the presence of Mg²⁺-EGTA. This C activity is stable at 4 °C for 24 h in serum, Mg²⁺-EGTA plasma and heparinized plasma. Haemolytic activity of serum heated at 50 °C for 30 min was restored by a factor B containing CM-cellulose fraction of foetal lamb serum in the presence of Mg²⁺-EGTA for human E-sheep A but not sheep E-rabbit A.These findings show that sheep C haemolysis of sheep E-rabbit A requires a Ca²⁺-Mg²⁺-dependent pathway that is labile in vitro for 24 h at 4 °C.     

Haemolysis of human erythrocytes heavily sensitized with sheep amboceptor by sheep complement chelated with EGTA or Mg2+-EGTA

Human erythrocytes sensitized with an agglutinating dose of either sheep anti-human erythrocyte antiserum, IgM, slow or fast γ-globulin antibody are haemolysed by foetal lamb or sheep serum complement (C) in the presence of ethyleneglycoltetraacetic acid (EGTA) or Mg²⁺-EGTA but not ethylenediaminetetraacetic acid (EDTA) or triethylenetetraminehexaacetic acid (TTHA). Dialysis of the C source against phosphate buffered saline rendered it inactive, but addition of Mg²⁺ or Mg²⁺-EGTA restored haemolytic activity. Haemolysis of sensitized erythrocytes took place more rapidly in buffer containing Ca²⁺ and Mg²⁺ than in EGTA or Mg²⁺-EGTA. Heating the C source at 50 °C for 30 min or 56 °C for 3 min abolished haemolytic activity. In the presence of EGTA or Mg²⁺-EGTA, haemolytic activity was restored to 50 °C-heated serum by a CM-cellulose fraction that contained sheep factor B.These results show that sheep C, in the presence of EGTA or Mg²⁺-EGTA will haemolyse human erythrocytes heavily sensitized with sheep amboceptor. The alternative pathway of sheep C is thought to be involved.