Low-pressure,microwave-induced plasma spectrometry method for the determination of subnanogram quantities of mercury: Application to mercury-carboxypeptidase analysis

A simple, sensitive, and rapid method for the determination of subnanogram quantities of mercury has been devised using a tantalum-filament vaporization system and low-pressure, microwave-induced emission spectrometry. This method is applied to the analysis of mercury-substituted carboxypeptidase with a sample volume of 5 μl and an analysis time of 3 min. The coefficient of variation for 2 × 10⁻¹⁰ g of mercury in mercury-carboxypeptidase is 6.0%, and the detection limit is 3 × 10⁻¹² g.     

The effects of transmitters on the affinity of the insoluble fraction of Ox brain for Na+ and K+

The insoluble fraction of ox-brain, which had previously been shown to have a non-linear affinity for Na⁺ and K⁺, was prepared. Acetylcholine (1×10^–8 mol/l and 1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero, while at 1×10^–6 mol/l, the affinity for the cations was almost as high as in the absence of the transmitter; the affinities for Na⁺ and K⁺ were particularly high, when the supernatant concentrations of these ions exceeded 80–100 mM. Addition of eserine (3×10^–5 mol/l) considerably modified the response of the fraction to acetylcholine (1×10^–5 mol/l). Atropine (1×10^–8 mol/l) in the absence or presence of acetylcholine (1×10^–5, or 1×10^–4 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Epinephrine (3×10^–10 mol/l) lowered the affinity for Na⁺ and K⁺, while ergotamine itself (1×10^–5 mol/l) reduced it to zero. The addition of both epinephrine and ergotamine at the latter concentrations restored the affinity of the fractions for Na⁺ and K⁺ to what it had been in the absence of the transmitter or antagonist, previously reported. Norepinephrine (3×10^–10 mol/l), or ouabain (1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Thus, the transmitters and antagonists altered the affinity of the insoluble fraction for Na⁺ and K⁺ nonlinearity, dependent upon their concentrations, the concentrations of the cations, and the interaction of transmitter and antagonist.     

Coronary vasoconstrictor effect of ghrelin is not mediated by growth hormone secretagogue receptor 1a type in dogs

Ghrelin (GHR) is a recently discovered endocrine regulatory peptide of gastrointestinal origin with multiple functions including cardiovascular effects. However, contradictory data are available on the vascular actions of GHR in different organs and species. The aim of this study was to characterize the direct effect of the peptide on the canine coronary bed and to evaluate the role of the growth hormone secretagogue receptor (GHS-R) in the effect of GHR on coronary arterioles. The presence of GHS-R1a and 1b subtypes in canine coronary arterioles was investigated using Western blotting and immunohistochemistry. Responses of coronary arterioles with spontaneous and elevated vascular tone (the latter evoked by the thromboxane mimetic agent U46619, 10⁻⁷-10⁻⁶ mol/l) to GHR (10⁻⁹-3 × 10⁻⁷ nmol/l) were recorded by video-microscopy as changes of vessel diameter. Positive immunostaining for both GHS-R subtypes was found in the wall of intramural arterioles. The microarteriographic study results showed that GHR alone could not elicit any significant effect on vessel diameter of arterioles with spontaneous tone. However, when vascular smooth muscle was preconstricted by the thromboxane mimetic agent U46619, administration of GHR induced further constriction (+31 ± 9% increase in contraction p < 0.01). This was not abolished by the specific blockade of GHS-R1a by d-Lys³-GHRP-6 (5 × 10⁻⁶ mol/l). The results suggest that GHR induces tone-dependent constriction of canine coronary arterioles which is mediated by a receptor other than GHS-R1a.     

A new interference-free lysine biosensor using a non-conducting polymer film

An electrochemical biosensor for the determination of lysine to be used for rapid evaluation of food quality has been developed. Platinum electrodes have been coated by electropolymerisation with 1,2-diaminobenzene (1.2-DAB) using cyclic voltammetry. The reduction in the oxidation of interferents compared with the bare platinum electrode was 100% for ascorbic acid, 99% for acetaminophen and 99% for cysteine. The enzyme L-lysine--oxidase was then immobilised onto the polymer layer by passive adsorption and a calibration curve for lysine constructed. This gave a linear range of 1×10⁻⁵ mol/l to 1×10⁻³ mol/l and a limit of detection of 2×10⁻⁷ mol/l.     

Deletion of the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> lysine biosynthesis gene <Emphasis Type="Italic">lysF</Emphasis> encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and -aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5×10³ conidia per mouse. In this murine model, the lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.     

Physical-chemical parameters and validation of a colorimetric method for deoxycholic and ursodeoxycholic acids: kit reagent and optical sensor

The simple and low cost β-cyclodextrin (β-CD)–phenolphthalein (PHP) inclusion complex was used for both the study of physical–chemical parameters and validation of analytical procedures for deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) determinations in different formulations. The usefulness of this inclusion complex is proposed either in the form of kit reagent and as an original optical sensor for DCA and UDCA. The results showed that temperature had a negative effect on the equilibrium constant resulting in high negative values of enthalpy and positive values of entropy. The half-life values for DCA and UDCA measurements were 68.71 and 294.71 days, respectively. The method was validated showing limits of detection and quantification of 4.92 × 10⁻⁵ mol L⁻¹ and 1.64 × 10⁻⁴ mol L⁻¹ for DCA, 1.14 × 10⁻⁵ mol L⁻¹ and 3.79 × 10⁻⁵ mol L⁻¹ for UDCA, respectively. The developed optical sensor also showed response linearity, ease of implementation and potential application in fast screening tasks even out of the laboratory.     

Voltammetric behavior of complexation of salbutamol with calf thymus DNA and its analytical application

The interaction of salbutamol (Sal), an animal growth promoter, with DNA was investigated by differential pulse voltammetry (DPV), cyclic voltammetry (CV), and fluorescence spectroscopy. An irreversible reduction was observed from the cyclic voltammograms, and the reaction mechanism involved a one-electron change irreversible oxidation. In the presence of DNA, the DPV peak current decreased and the Sal peak shifted to higher potentials, indicating that Sal interacted with DNA to form an intercalation Sal–DNA complex. In addition, reaction binding parameters were extracted from the DPV data with the use of the multivariate curve resolution–alternating least squares (MCR–ALS) method; the binding constant and ratio were found to be (2.0 ± 0.5) × 10⁵ M⁻¹ and 1:1, respectively. Quantitative voltammetric analysis of Sal was performed in the concentration range of 3.02 × 10⁻⁶ to 1.23 × 10⁻⁴ mol L⁻¹, and it was found that the detection limit was 5.11 × 10⁻⁷ mol L⁻¹ in the presence of 1.00 × 10⁻⁶ mol L⁻¹ DNA. The method was applied for the determination of Sal in spiked urine and human serum samples, and the calibration was successfully verified.     

Electrochemical determination of antihypertensive drug irbesartan in pharmaceuticals

A sensitive voltammetric method has been developed for the determination of irbesartan in a Britton–Robinson buffer medium. Irbesartan exhibited a well-defined cathodic peak over the entire pH range from 2.0 to 12.0. The mechanism of reduction was postulated on the basis of controlled potential electrolysis, coulometry, and spectral analysis. Under optimal conditions, a linear response of irbesartan was obtained in the range from 3.0 × 10⁻⁵ to 5.7 × 10⁻³ mol L⁻¹ and with a limit of detection of 5.33 × 10⁻⁷ mol L⁻¹. The effect of cationic surfactant on the voltammetric reduction peak of irbesartan in Britton–Robinson buffer is also described.     

Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts

Recent studies suggest that both osteopontin and urotensin II (UII) play critical roles in vascular remodeling. We previously showed that UII could stimulate the migration of aortic adventitial fibroblasts. In this study, we examined whether osteopontin is involved in UII-induced migration of rat aortic adventitial fibroblasts and examined the effects and mechanisms of UII on osteopontin expression in adventitial fibroblasts. Migration of adventitial fibroblasts induced by UII could be inhibited significantly by osteopontin antisense oligonucleotide (P < 0.01) but not sense or mismatch oligonucleotides (P > 0.05). Moreover, UII dose- and time-dependently promoted osteopontin mRNA expression and protein secretion in the cells, with maximal effect at 10⁻⁸ mol/l at 3 h for mRNA expression or at 12 h for protein secretion (both P < 0.01). Furthermore, the UII effects were significantly inhibited by its receptor antagonist SB710411 (10⁻⁶ mol/l), and Ca²⁺ channel blocker nicardipine (10⁻⁵ mol/l), protein kinase C (PKC) inhibitor H7 (10⁻⁵ mol/l), calcineurin inhibitor cyclosporine A (10⁻⁵ mol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10⁻⁵ mol/l) and Rho kinase inhibitor Y-27632 (10⁻⁵ mol/l). Thus, osteopontin is involved in the UII-induced migration of adventitial fibroblasts, and UII could upregulate osteopontin gene expression and protein synthesis in rat aortic adventitial fibroblasts by activating its receptor and the Ca²⁺ channel, PKC, calcineurin, MAPK and Rho kinase signal transduction pathways.     

Selective control of Microcystis using an amino acid – a laboratory assay

An amino acid (L-lysine) was screened against eighteen strains ofCyanophyceae, Bacillariophyceae and Chlorophyceae. OnlyMicrocystis strains (Cyanophyceae) were sensitive tolysine; other strains were not affected. Cells of sevenMicrocystis strains (10⁶ cellsmL^–1) were completely killed within 48h by lysine at concentrations between 0.6 and 5.0 mgL^–1. Two Microcystis strains wereinhibited by 92 and 98 %. Similar results were obtained when lysine malonateandlysine copper were used as algicides. Microcystis specieswere killed by lysine malonate at concentrations between 0.6 and 5 mgL^–1, and by lysine copper at concentrations between 0.5and 20 mg L^–1.     

Square-wave adsorptive voltammetry of dexamethasone: redox mechanism, kinetic properties, and electroanalytical determinations in multicomponent formulations

The electrochemical reduction behavior of dexamethasone at a hanging mercury drop electrode was investigated by cyclic and square-wave adsorptive voltammetries in a Britton–Robinson buffer at pH 2.0. The optimized experimental conditions consisted of a pulse potential frequency of 100 s⁻¹, a pulse amplitude of 15 mV, and a potential step height of 2 mV, with E_acc = −0.60 V and t_acc = 15 s. From these parameters, it was also possible to develop a detailed study about the kinetic and mechanistic events involved in the reduction process. Two well-defined peaks were observed in the cathodic scan, and peak 2 was used to obtain analytical curves. A linear range between 4.98 × 10⁻⁸ and 6.10 × 10⁻⁷ mol L⁻¹, with a detection limit of 2.54 × 10⁻⁹ mol L⁻¹ and a quantification limit of 8.47 × 10⁻⁹ mol L⁻¹, was observed. Moreover, it was possible to achieve a simple, selective, and versatile methodology adaptable to the quantification of dexamethasone because common excipients used in multicomponent commercial formulations caused no interference. The satisfactory recoveries and the low relative standard deviation data reflected the high accuracy and precision of the proposed method for the determination of dexamethasone in injectable eye drops and elixir samples.     

Lysine side-chain dynamics derived from 13C-multiplet NMR relaxation studies on di- and tripeptides

Summary ¹³C NMR relaxation data have been used to determine dipolar auto- and cross-correlation times for the di- and tripeptides GK, KG and GKG, primarily to analyze lysine side-chain motional dynamics. In general, correlation times are largest for backbone positions and decrease on going through the lysine side chain, consistent with the idea of increased mobility at C and C methylenes. Correlation times, however, vary with the peptide ionization state. In the zwitterionic state of GK, for example, both auto-and cross-correlation times are at their maximum values, indicating reduced internal motions probably resulting from intramolecular electrostatic interactions. Modifying the charge state increases motional fluctuations. Activation energies determined from the temperature dependence of CH rotational autocorrelation times at neutral pH are approximately equal for glycine and lysine C and lysine C and C positions (4.1±0.2 to 4.5±0.2 kcal/mol) and tend to decrease slightly for lysine C and C (3.8±0.2 to 4.3±0.2 kcal/mol). The sign of lysine side-chain cross-correlations could not be explained by using any available rotational model, including one parameterized for multiple internally restricted rotations and anisotropic overall tumbling. Molecular and stochastic dynamics calculations were performed to obtain insight into correlated internal rotations and coupled overall tumbling and internal motions. Relatively strong correlations were found for i,i+1 backbone and lysine side-chain internal bond rotations. Stochastic dynamics calculations were more successful at explaining experimentally observed correlation times. In the fully charged state, a preferred conformation was detected with an all-trans lysine side chain.Abbreviations rf radio frequency - GK dipeptide glycine-lysine - KG dipeptide lysine-glycine - GKG tripeptide glycine-lysine-glycine     

One-step "green" preparation of graphene nanosheets and carbon nanospheres mixture by electrolyzing graphite rob and its application for glucose biosensing

The graphene nanosheets and carbon nanospheres mixture (GNS–CNS) was prepared by electrolyzing graphite rob in KNO₃ solution under constant current, which was characterized by TEM, AFM, SEM, FT-IR, XRD, XPS, TGA and UV–vis. The nano-mixture can keep stable in water for more than one month. Based on this kind of mixture material, a novel electrochemical biosensing platform for glucose determination was developed. Cyclic voltammetry of glucose oxidase (GOD) immobilized on GNS–CNS/GCE exhibited a pair of well-defined quasi-reversible redox peaks at −0.488 V (E_pa) and −0.509 V (E_pc) by direct electron transfer between the protein and the electrode. The charge-transfer coefficient (α) was 0.51, the electron transfer rate constant was 2.64 s⁻¹ and the surface coverage of HRP was 3.18 × 10⁻¹⁰ mol cm⁻². The immobilized GOD could retain its bioactivity and catalyze the reduction of dissolved oxygen. The glucose biosensor has a linear range from 0.4 to 20 mM with detection limit of 0.1 mM. Moreover, the biosensor exhibits acceptable reproducibility and storage stability. The fabricated biosensor was further used to determine glucose in human plasma sample with the recoveries from 96.83% to 105.52%. Therefore, GOD/GNS–CNS/GCE could be promisingly applied to determine blood sugar concentration in the practical clinical analysis.     

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 ± 0.33 × 10⁻¹⁵ mol s⁻¹, 1.38 ± 0.58 × 10⁻¹⁵ mol s⁻¹, and 3.44 ± 2.07 × 10⁻¹⁵ mol s⁻¹, respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).     

Effect of antagonists of ethylene action on binding of ethylene in cut carnations

Five hours after cut carnations had been treated with a pulse of 1 or 4 mM silver thiosulfate (STS), in vivo ethylene binding in petals was inhibited by 22 and 29%, respectively. When binding was measured 4 days after the 4-mM STS treatment, binding was inhibited by 81%. 2,5-Norbornadiene, which substantially delays carnation senescence, inhibited ethylene binding by 41% at a concentration of 1000 l/l. The Kd for ethylene binding in carnations was estimated to be 0.1 l/l in petals and 0.09 l/l in leaves. The concentration of binding sites was estimated to be 6.0×10^–9 mol/kg of petals and 2.0×10^–9 mol/kg of leaves     

Rationalizing 5000-fold differences in receptor-binding rate constants of four cytokines

The four cytokines erythropoietin (EPO), interleukin-4 (IL4), human growth hormone (hGH), and prolactin (PRL) all form four-helix bundles and bind to type I cytokine receptors. However, their receptor-binding rate constants span a 5000-fold range. Here, we quantitatively rationalize these vast differences in rate constants by our transient-complex theory for protein-protein association. In the transient complex, the two proteins have near-native separation and relative orientation, but have yet to form the short-range specific interactions of the native complex. The theory predicts the association rate constant as where k_a0 is the basal rate constant for reaching the transient complex by random diffusion, and the Boltzmann factor captures the rate enhancement due to electrostatic attraction. We found that the vast differences in receptor-binding rate constants of the four cytokines arise mostly from the differences in charge complementarity among the four cytokine-receptor complexes. The basal rate constants (k_a0) of EPO, IL4, hGH, and PRL were similar (5.2 × 10⁵ M⁻¹s⁻¹, 2.4 × 10⁵ M⁻¹s⁻¹, 1.7 × 10⁵ M⁻¹s⁻¹, and 1.7 × 10⁵ M⁻¹s⁻¹, respectively). However, the average electrostatic free energies () were very different (−4.2 kcal/mol, −2.4 kcal/mol, −0.1 kcal/mol, and −0.5 kcal/mol, respectively, at ionic strength = 160 mM). The receptor-binding rate constants predicted without adjusting any parameters, 6.2 × 10⁸ M⁻¹s⁻¹, 1.3 × 10⁷ M⁻¹s⁻¹, 2.0 × 10⁵ M⁻¹s⁻¹, and 7.6 × 10⁴ M⁻¹s⁻¹, respectively, for EPO, IL4, hGH, and PRL agree well with experimental results. We uncover that these diverse rate constants are anticorrelated with the circulation concentrations of the cytokines, with the resulting cytokine-receptor binding rates very close to the limits set by the half-lives of the receptors, suggesting that these binding rates are functionally relevant and perhaps evolutionarily tuned. Our calculations also reproduced well-observed effects of mutations and ionic strength on the rate constants and produced a set of mutations on the complex of hGH with its receptor that putatively enhances the rate constant by nearly 100-fold through increasing charge complementarity. To quantify charge complementarity, we propose a simple index based on the charge distribution within the binding interface, which shows good correlation with . Together these results suggest that protein charges can be manipulated to tune k_a and control biological function.     

Inositol-1,4,5-trisphosphate mass content in isolated perfused rat heart during alpha-1-adrenoceptor stimulation

Inositol-1,4,5-trisphosphate (IP₃) has been proposed to be a second messenger in response to alpha-1-adrenoceptor stimulation also in myocardial cells. We studied the effect of alpha-1-adrenoceptor stimulation (5 × 10^–5 mol/l phenylephrine or 5 × 10^–5 mol/l noradrenaline both in the presence of 10^–6 mol/l timolol) on IP₃ mass content in isolated perfused rat hearts. IP₃ content was determined by a specific receptor-binding assay-kit (TRK 1000, Amersham) after validating the method. For comparison also the effect of muscarinic stimulation (10^–4 mol/l carbachol in the presence of 10^–6 mol/l timolol) on IP₃ content was measured in corresponding preparations. A basal IP₃ level of about 75 pmol/mg protein was found. There were no prominent effects of alpha-1-adrenoceptor stimulation on total IP₃ content in isolated perfused rat hearts. Phenylephrine gave a statistically significant increase of about 40% at 1/4 min and a statistically significant decrease of about 25% at 4 min after start of exposure. Noradrenaline, however, gave no statistically significant change of IP₃ at the time-points studied. Muscarinic stimulation caused a slight, statistically insignificant, increase of IP₃ at 1/4 min. The results are compatible with an assumption that agonist stimulation evokes a localized increase of IP3 which may be masked by a relatively high total IP₃ mass content. The IP₃ peak after phenylephrine coincided with the early positive inotropic phase of the response reported earlier in perfused rat hearts for alpha-1-adrenoceptor stimulation by phenylephrine. Although this might be compatible with a role for IP₃ in this early and transient phase, a mediator function of IP₃ in the inotropic response is not established.     

Transport of neutral, cationic and anionic amino acids by systems B, b, XAG, and ASC in swine small intestine

Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b^o,+ were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b^o,+ is present and accessible also to -glutamine. -aspartate (K_1/2 0.3 mM) and -glutamate (K_i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 μmol cm⁻² h⁻¹, while only -glutamate with a K_1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 μmol cm⁻² h⁻¹, system ASC, against which serine has a K_i of 1.6 mM. In the presence of 100 mM lysine, -glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of -glutamate but also of glutamine (K_i 0.5 mM), and -glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.     

Sperm concentration at freezing affects post-thaw quality and fertility of ram semen

We have investigated the effect of sperm concentration in the freezing doses 200, 400, 800, and 1600 × 10⁶ mL⁻¹ on the post-thaw quality and fertility of ram semen. Semen was collected from seven adult Churra rams by artificial vagina during the breeding season. The semen was diluted in an extender (TES-Tris-fructose, 20% egg yolk, and 4% glycerol), to a final concentration of 200, 400, 800, or 1600 × 10⁶ mL⁻¹ and frozen. Doses were analyzed post-thawing for motility (computer-assisted sperm analysis system [CASA]), viability, and acrosomal status (fluorescence probes propidium iodide [PI]/peanut agglutinin conjugated with fluorescein thiocyanate (PNA-FITC), SYBR-14/PI [Invitrogen; Barcelona, Spain] and YO-PRO-1/PI [Invitrogen; Barcelona, Spain]). Total motility and velocity were lower for 1600 × 10⁶ mL⁻¹ doses, while progressive motility and viability were lower both for 800 and 1600 × 10⁶ mL⁻¹. The proportion of viable spermatozoa showing increased membrane permeability (YO-PRO-1+) rose in 800 and 1200 × 10⁶ mL⁻¹. Intrauterine inseminations were performed with the 200, 400, and 800 × 10⁶ mL⁻¹ doses at a fixed sperm number (25 × 10⁶ per uterine horn) in synchronized ewes. Fertility (lambing rate) was similar for semen frozen at 200 (57.5%) or 400 × 10⁶ mL⁻¹ (54.4%), whereas it was significantly lower for 800 × 10⁶ mL⁻¹ (45.5%). In conclusion, increasing sperm concentration in cryopreserved semen, at least at 800 × 10⁶ mL⁻¹ and more, adversely affects the postthawing quality and fertility of ram semen.