Urinary excretion patterns of 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophol in various animal species: implications for studies on serotonin metabolism and turnover rate

The concentrations of the serotonin metabolites 5-hydroxyindole-3-acetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL) were determined in spot urine samples of 12 mammalian and one fish species (cat, cow, dog, ferret, golden hamster, guinea pig, horse, monkey, mouse, rabbit, rainbow trout, rat, sheep) and compared with human data. The highest urinary concentrations of 5HTOL were found in the Sprague-Dawley rat (mean 9.5 micromol/L) and NMRI mouse (8.2 micromol/L), and the lowest in rainbow trout, cynomolgus macaque, and human urine (approximately 0.1 micromol/L). The highest 5HIAA concentrations were found in hamster (89.3 micromol/L) and mouse (85.2 micromol/L), and the lowest in rainbow trout, horse and sheep (range 2.0-3.7 micromol/L). Several species showed 5HIAA concentrations similar to that normally observed in human urine (approximately 5-40 micromol/L). This study demonstrated wide inter- and intra-species variations in the urinary concentrations of 5HIAA and 5HTOL, both separately and in the sum of concentrations. The 5HTOL/5HIAA ratio, which is used as an easily accessible index of the relative importance of the reductive and oxidative pathways for serotonin metabolism, also varied considerably between different species. This observation confirms that the much higher urinary 5HTOL/5HIAA ratio in rats (mean 0.35) compared with humans (< 0.01) is due to a higher baseline formation of 5HTOL in the rat. The monkey, ferret, hamster, and rabbit most closely resembled humans in this respect, and at least the two latter species appear to be more suitable than rats as animal models for studying serotonin metabolism and turnover rate, and the metabolic interaction with ethanol.     

Determination of a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist (LY300164) and its N-acetyl metabolite in mouse,rat, dog,and monkey plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor antagonist LY300164 (compound I) and its N-acetyl metabolite (compound II) in plasma was developed. The assay utilized solid-phase extraction on a C₁₈ Bond Elut cartridge followed by reversed-phase HPLC with UV detection at 310 nm. The method exhibited a large linear range from 0.05 μg/ml to 50 μg/ml with an intra-sassay accuracy for compound I and compound II ranging from 89.0% to 114.5% and intra-assay precision ranging from 0.5 to 15.3% in mouse, rat, dog, and monkey plasma. The inter-assay accuracy of compound I and compound II was 93.3% to 101.8% and the inter-assay precision was 1.6% to 11.2% in dog plasma. The lower limit of quantitation was 0.05 μg/ml for compound I in plasma from all species tested. The lower limit of quantitation for compound II was 0.05 μg/ml in dog and monkey plasma and 0.1 μg/ml in mouse and rat plasma. Extracts of compound I and II from dog plasma were shown to be stable for 24 h at room temperature, and both compounds were stable when spiked into rat and monkey plasma frozen at −70°C for 27 days. The method has shown to be useful in the investigation of the pharmacokinetics of the parent compound (I) and metabolite (II) in preclinical studies.     

Use of the polymerase chain reaction for homology probing of butyrylcholinesterase from several vertebrates

Genomic blots from man, monkey, cow, sheep, pig, rabbit, dog, rat, mouse, guinea pig, and chicken DNA were hybridized with probes derived from the four exons of the human butyrylcholinesterase gene (BCHE) (Arpagaus, M., Kott, M., Vatsis, K. P., Bartels, C. F., La Du, B. N., and Lockridge, O. (1990) Biochemistry 29, 124-131). Results showed that the BCHE gene was present in a single copy in the genome of all these vertebrates. The polymerase chain reaction was used to amplify genomic DNA from these animals with oligonucleotides derived from the human BCHE coding sequence. The amplified segment contained 423 bp of BCHE sequence including the active site serine of the enzyme (amino acid 198) and a component of the anionic site, aspartate 70. Amplification was successful for monkey, pig, cow, dog, sheep, and rabbit DNA, but unsuccessful for rat, guinea pig, mouse, and chicken DNA. Amplified segments were cloned in M13 and sequenced. The mouse sequence was obtained by sequencing a genomic clone. The highest identity of the human amino acid sequence was found with monkey (100%) and the lowest with mouse (91.5%). The sequence around the active site serine 198, Phe-Gly-Glu-Ser-Ala-Gly-Ala, was conserved in all eight animals as was the anionic site component, aspartate 70. A phylogenetic tree of mammalian butyrylcholinesterases was constructed using the partial BCHE sequences.     

Population Pharmacokinetics of an Indian F(ab')2 Snake Antivenom in Patients with Russell's Viper (Daboia russelii) Bites

Background

There is limited information on antivenom pharmacokinetics. This study aimed to investigate the pharmacokinetics of an Indian snake antivenom in humans with Russell’s viper bites.

Methods/Principal Findings

Patient data and serial blood samples were collected from patients with Russell’s viper (Daboia russelii) envenoming in Sri Lanka. All patients received Indian F(ab’)₂ snake antivenom manufactured by VINS Bioproducts Ltd. Antivenom concentrations were measured with sandwich enzyme immunoassays. Timed antivenom concentrations were analysed using MONOLIXvs4.2. One, two and three compartment models with zero order input and first order elimination kinetics were assessed. Models were parameterized with clearance(CL), intercompartmental clearance(Q), central compartment volume(V) and peripheral compartment volume(V_P). Between-subject-variability (BSV) on relative bioavailability (F) was included to account for dose variations. Covariates effects (age, sex, weight, antivenom batch, pre-antivenom concentrations) were explored by visual inspection and in model building. There were 75 patients, median age 57 years (40-70y) and 64 (85%) were male. 411 antivenom concentration data points were analysed. A two compartment model with zero order input, linear elimination kinetics and a combined error model best described the data. Inclusion of BSV on F and weight as a covariate on V improved the model. Inclusion of pre-antivenom concentrations or different batches on BSV of F did not. Final model parameter estimates were CL,0.078 Lh^-1, V,2.2L, Q,0.178Lh^-1 and V_P,8.33L. The median half-life of distribution was 4.6h (10-90%iles:2.6-7.1h) and half-life of elimination, 140h (10^th-90^th percentilesx:95-223h).

Conclusion

Indian F(ab’)₂ snake antivenom displayed biexponential disposition pharmacokinetics, with a rapid distribution half-life and more prolonged elimination half-life.     

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')₂ (t_1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')₂, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t_1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t_1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.     

Characteristics of anion transport in cat and dog red blood cells

Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of³⁵SO₄ efflux on the intracellular SO₄ concentration, the pH dependence and the inhibition of³⁵SO₄ efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of³⁶Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV _max andK _1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H₂DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H₂DIDS.     

Infarcted heart uptake and biodistribution of radiolabelled anti-myosin monoclonal antibody in rat and dog myocardial infarct models

A new mouse monoclonal antibody that recognizes α- and β-heavy chains of human atrial and ventricular myosin and β-heavy chain of human slow skeletal muscle myosin was obtained. The ¹²⁵I- and ¹¹¹In-labelled antibody, and its F(ab′)₂ and Fab fragments localize in isoproterenol induced infarcted rat heart, with the F(ab′)₂ fragment showing the highest uptake. Comparison with ^99mTc-pyrophosphate uptake in infarcted dog heart, induced by selective obstruction of a coronary artery, suggest that the ¹¹¹In-labelled F(ab′)₂ localizes specifically in infarcted myocardium only.     

Reactivation of UV and γ-inactivated herpes virus in BHK cells

The DNA-repair capabilities of baby hamster kidney (BHK) cells were investigated by comparing the reactivation of irradiated herpes simplex virus type I (HSV1) in BHK cells with its reactivation in mouse fibroblasts and in normal and repairdeficient human diploid fibroblasts. BHK cells were found to have an intermediate ability to reactive UV-irradiated HSV1 (the viral D_o was 14 J/m²) relative to normal human fibroblasts (viral D_o = 19 J/m²) and xeroderma pigmentosum (XP) group A cells (viral D_o = 4.5 J/m²). With mouse L929 cells as the host, the response of the UV-irradiated virus was biphasic with D_os of 4.6 and 30 J/m² for the low- and high-dose components respectively. In contrast to the response following UV radiation, γ-irradiated HSV1 was similarly reactivated by BHK and normal human cells (the D_os for the irradiated virus in BHK and CRl 1106 were 55 and 51 krad, respectively, whereas xeroderma pigmentosum cells were slightly less efficient in the repair of γ-irradiated virus (D_o = 45 krad). UV irradiation of BHK host cells 0–48 h prior to infection enhanced the reactivation of UV-irradiated HSV.     

Effect of bacteriocinogenic Lactobacillus spp. on the shelf life of fufu, a traditional fermented cassava product

Quantitative determination of antimicrobial compounds produced by the Lactobacillus strains under test was carried out. L. plantarum F1 produced the highest quantity of lactic acid (16.4 g/l) while the lowest amount (0.3 g/l) was produced by L. jensenii F9. All the test organisms produced hydrogen peroxide, with L. brevis OG1 having the highest yield of 0.037 g/l. Diacetyl was also produced by all the organisms, with L. plantarum F1 and L. brevis OG1 having the highest yield of 1.7 g/l, while the lowest producer was L. jensenii F9 (0.86 g/l). Determination of bacteriocin activity was carried out. L. plantarum F1 exhibited 6400 AU/ml bacteriocin activity, while L. brevis OG1 had the lowest activity of 3200 AU/ml using E. coli NCTC10418 as indicator organism. However, L. fermentum F5 and L. jensenii F9 did not produce any detectable bacteriocin. The pH value in the culture supernatant of L. plantarum F1 reached 3.1 within 48 h of incubation, while that of L. jensenii F9 was 5.2. Fufu was prepared using both bacteriocin-producing (BP) L. plantarum F1 and L. brevis OG1, and non-bacteriocin producing L. fermentum F5 and L. jensenii F9. No viable cells of Salmonella typhimurium ATCC13311 and Shigella flexneri AP23498 were detected after 12 h in the cassava products fermented with mixed starter culture of L. plantarum F1 and L. brevis OG1. The rate of survival of enteropathogens in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was much lower when compared to cassava fermented with mixed starter culture of L. fermentum F5 and L. jensenii F9. At 12 h, the viable count of E. coli NCTC10418 in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was 1.1 log₁₀ c.f.u./g whereas in cassava fermented with mixed starter cultures of L. fermentum F5 and L. jensenii F9, 8.5 log₁₀ c.f.u./g was obtained The study revealed that fufu produced with BP mixed starter cultures had a better shelf life and kept for 13 days before spoilage occurred, relative to 5 days observed for fufu produced using non-bacteriocin-producing starter cultures, and 6 days for the traditional fermented fufu.     

Generation,characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (K_D) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.     

Characterization of Nasal Potential Difference in cftr Knockout and F508del-CFTR Mice

Background

Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V_TE) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V_TE in CF mice must be well characterized for correct interpretation.

Methods

We performed V_TE measurements in large-scale studies of two mouse models of CF—B6;129 cftr knockout and FVB F508del-CFTR—and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice.

Results

We determined the typical V_TE values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl^- solution was considered to indicate a normal response.

Conclusions

These data will make it possible to interpret changes in nasal V_TE in mouse models of CF, in future preclinical studies.     

Daily Rhythms in Blood and Milk Lead Toxicokinetics Following Intravenous Administration of Lead Acetate to Dairy Cows in Winter

The objective was to investigate circadian variations of blood and milk lead toxicokinetics in dairy cows in winter. Twenty lactating Holstein animals were randomly assigned to 4 treatments, corresponding to different hours after onset of light (HALO): 2, 8, 14 and 20. Cows received a single intravenous administration of 2.5 mg/kg lead as lead acetate. Blood and milk samples were taken and analyzed by atomic absorption spectrophotometry. For each toxicokinetic parameter, a one-way analysis of variance was performed to outline the existence of daily variations. Significant differences as a function of HALO were detected in blood for the hybrid constant of elimination (β), half-life of elimination (t_1/2β), area under the curve (AUC) and clearance (Cl) (p &lt; 0.01) and volume of distribution at steady state (V_ss) (p < 0.05). Half-life of elimination was highest when lead acetatae was injected at 2 HALO, and lowest following the 14 HALO administration. Milk data showed significant differences for maximum concentration, AUC and Cl (p &lt; 0.001), volume of distribution and V_ss (p < 0.005). The ratio AUC_milk/ABC_blood was utilized to estimate penetration of lead in milk. It differed significantly along the day (p < 0.01). Blood daily variations could be fitted a circadian rhythms. No circadian rhythms were detected in milk parameters or in the ratio AUC_milk/ABC_blood. It was concluded that blood and milk lead toxicokinetics are distinctly affected by the time of the day.     

Loss of alveolar membrane diffusing capacity and pulmonary capillary blood volume in pulmonary arterial hypertension

Background

Reduced gas transfer in patients with pulmonary arterial hypertension (PAH) is traditionally attributed to remodeling and progressive loss of pulmonary arterial vasculature that results in decreased capillary blood volume available for gas exchange.

Methods

We tested this hypothesis by determination of lung diffusing capacity (DL) and its components, the alveolar capillary membrane diffusing capacity (D_m) and lung capillary blood volume (V_c) in 28 individuals with PAH in comparison to 41 healthy individuals, and in 19 PAH patients over time. Using single breath simultaneous measure of diffusion of carbon monoxide (DL_CO) and nitric oxide (DL_NO), DL and D_m were respectively determined, and V_c calculated. D_m and V_c were evaluated over time in relation to standard clinical indicators of disease severity, including brain natriuretic peptide (BNP), 6-minute walk distance (6MWD) and right ventricular systolic pressure (RVSP) by echocardiography.

Results

Both DL_CO and DL_NO were reduced in PAH as compared to controls and the lower DL in PAH was due to loss of both D_m and V_c (all p < 0.01). While DL_CO of PAH patients did not change over time, DL_NO decreased by 24 ml/min/mmHg/year (p = 0.01). Consequently, D_m decreased and V_c tended to increase over time, which led to deterioration of the D_m/V_c ratio, a measure of alveolar-capillary membrane functional efficiency without changes in clinical markers.

Conclusions

The findings indicate that lower than normal gas transfer in PAH is due to loss of both D_m and V_c, but that deterioration of D_m/V_c over time is related to worsening membrane diffusion.     

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human,Monkey, Dog,Rat, and Mouse Liver Microsomes

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH₃-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH₃-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH₃-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH₃-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH₃-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH₃-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH₃-PPD, especially at the C-20 hydroxyl group.     

Differential brain expression of the Alzheimer''s amyloid precursor protein.

The expression of the Alzheimer amyloid protein precursor (AAPP) was examined in human, monkey, dog and rat brains. Two proteins, one identified as AAPP695 and the other as AAPP751, were immunoprecipitated from the in vitro translation of human, dog and rat brain polysomes. The AAPP751 to AAPP695 ratio was highest in human, intermediate in dog and lowest in rat brain polysomes. Human cerebral cortex contained higher levels of the AAPP751 mRNA than either dog or rat cortex. AAPP695 was detected in both cerebral cortex and cerebellum of all species examined. In contrast, AAPP751 was detected predominantly in the cortex of human, monkey and to a lesser extent dog brains while it was not detected in rat brain. These findings indicate that the amyloid precursors are differentially expressed in different mammalian brains and suggest that AAPP751 is mainly expressed in the brain regions involved in plaque formation.     

Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality

The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (V_max/K_m) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Effect of culture density and volume on <Emphasis Type="Italic">Moina micrura</Emphasis> (Kurz, 1874) reproduction,and sex ratio in the progeny

Moina micrura is a cosmopolitan, cyclic parthenogenetic, cladoceran with ample morphological and ecological plasticity that inhabits temperate, tropical, and subtropical regions of the world. Sexual reproduction in cladocerans is a phenomenon induced by environmental factors, mainly associated with adverse conditions, including crowding. In this study we have assessed the effects of population density combined with the volume available for individual development, on the number of newborns and on the sex ratio in the progeny. Three parthenogenetic female densities (D ₁ = 33 ind l⁻¹, D ₂ = 66 ind l⁻¹, D ₃ = 100 ind l⁻¹) were assayed in combination with three culture volumes (V ₁ = 30 ml, V ₂ = 60 ml, V ₃ = 120 ml), at 21°C. Once the reproduction began, progeny was isolated daily and counted, identifying female and male neonates. Culture volume was the only factor that had a significant effect on the total progeny, as well as on the number of males in the progeny; the highest sex ratio was recorded in D ₂ V ₁ (0.58), whereas the lowest was for D ₁ V ₃ (0.06). Female density had no significant effect on the number of sexual M. micrura. We hypothesize that male production (quantity and frequency) was higher in the lowest tested volume because of a reduction in the space available per individual, but not necessarily associated with the physical interaction related with a high encounter rate in more crowded conditions. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera