Assessment of plasma membrane and chromatin structure of sperm from transgenic and non-transgenic boars

The aim of this study was to determine the apoptotic changes and chromatin damage in non-transgenic and transgenic boars carrying the human α1,2-fucosyltransferase gene. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars both crossbreds of Polish Landrace and Large White. Two fluorescence methods were employed to measure apoptosis: an assay to assess the early changes in sperm membrane integrity using fluorophore YO-PRO-1 and an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V. The chromatin damage was assessed based on the sperm chromatin structure assay method. No significant differences in the proportion of all detected subpopulations of spermatozoa were found between TG and NTG boars. Similarly, the analysis of the chromatin structure revealed no statistical differences in the sperm chromatin damage between TG and NTG boars. In conclusion, the presence of the human α1,2-fucosyltransferase gene in the genome of TG boars did not cause any spermatogenesis process disturbances leading to the increased production of apoptotic spermatozoa. Moreover, the low level of sperm with damaged chromatin in TG boars confirms the high stability of the spermatogenesis process in the TG boars analyzed.     

Successful low dose insemination of flow cytometrically sorted Sika (Cervus nippon) sperm in Wapiti (Cervus elaphus)

The purpose of this study was to determine a practical method in Wapiti (Cervus elaphus) of using predetermined sexed Sika (Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds (n = 81) were inseminated into the uterus by rectum manipulation with 1 × 10⁶ (X1 and Y1 group, respectively) or 2 × 10⁶ (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 × 10⁷ non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700 IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups (P > 0.05), but higher for control (75%) treatment (P < 0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly (P < 0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250 d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.     

Superovulation and embryo transfer in Holstein cattle using sexed sperm

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 × 10⁶ sexed (X-sorted; n = 5) or unsexed (n = 5) frozen-thawed sperm from one bull at 12 and 24 h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P < 0.0001). In Experiment 2, donors were inseminated twice with 5 × 10⁶ sexed unfrozen sperm (n = 10) or sexed frozen-thawed sperm (n = 9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P < 0.05) inseminated twice with 5 × 10⁶ sperm/dose (10 × 10⁶ total). We concluded that the use of sexed frozen-thawed sperm (≥90% X-sperm biased and 10 × 10⁶ total sperm) may be economically viable for commercial MOET programs in Holstein heifers.     

Heat induced infertility of boars: The inter-relationship between depressed sperm output and fertility and an estimation of the critical air temperature above which sperm output is impaired

Eight groups of Large-White gilts were each inseminated with different numbers of normal motile sperm, in the range 0.28–7.0 × 10⁹. A significant (P < 0.05) relationship between conception rate and the number of motile sperm inseminated was shown. This relationship can be used to equate output of motile sperm with levels of fertility of boars. The optimal number of motile sperm for conception following intra-cervical insemination was near 5 × 10⁹ and the threshold number, below which animals did not conceive, was c. 4 × 10⁸.In a second experiment, three Large-White boars were subjected to graded thermal treatment (air temperature was increased by 1°C per day for 20 days, from a basal level of 20°C to a maximal level of 40°C) and responses of ejaculate and other physiological characteristics were monitored. Scrotal surface temperature, respiration rate and rectal temperature increased (P < 0.05) beyond basal levels at air temperatures of 30°C, 33°C and 35°C, respectively. Motility of sperm in ejaculates decreased when air temperature reached 30°C and this response was presumed to reflect hyperthermia in epididymal tissues, consistent with increasing scrotal surface temperature at this same air temperature. Motility fell below a pre-treatment level of about 93%, to 19% (P < 0.05), 3 weeks after heating. Volumes of seminal plasma and gel in ejaculates were also lower (P < 0.05) following heating. Changes in daily sperm production were minor and, as a result, daily motile sperm production levels paralleled changes in motility. Proportions of abnormal types of sperm increased (P < 0.05) to maximal levels in the last week of heating and all returned to pretreatment values 5 weeks later. High proportions of sperm with kinoplasmic droplets appeared in ejaculates collected after heating (P < 0.05), evidence that epididymal cell types in the boar are sensitive to heat.As a result of heat treatment, normal motile sperm production decreased from control levels (1.28 × 10¹⁰·day^?1) to 0.15 × 10¹⁰·day^?1, 3 weeks after heating ceased. However, the results suggest that normal sperm output by Large-White boars can be maintained at air temperatures as high as 29°C.By relating the results of both experiments, it is concluded that fertility of the boars in the second experiment (if mated once daily) would be depressed for about 5 weeks after heat treatment ceased. The findings support many field reports which indicate a contribution of boars to lower conception rates of sows during and immediately following summer and the results can be used in formulating strategies to circumvent this widespread problem.     

Physiological,morphological and anatomical trait variations between winter and summer leaves of Cistus species

Morphological, anatomical and physiological summer and winter leaf traits of Cistus incanus subsp. incanus, C. salvifolius and C. monspeliensis growing at the Botanical garden of Rome were analyzed. With regard to differences between summer and winter leaves of the considered species, leaf thickness (L) was 21% higher in summer than in winter leaves (mean of the considered species) and this increase was mostly the result of the increased palisade parenchyma thickness over the spongy parenchyma one (24 and 16% higher in summer than in winter leaves, respectively). Leaf mass area (LMA) and leaf tissue density (LTD) were 38% and 17% higher in summer than in winter leaves, respectively (mean of the considered species). The photosynthetic rate (P_N), stomatal conductance (g_s) and chlorophyll content (Chl) of summer leaves were 54%, 17% and 14% lower, respectively, than in winter leaves. C. monspeliensis summer leaves had the highest LMA, LTD, adaxial cuticle thickness (14.6 ± 1.8 mg cm⁻², 1091 ± 94 mg cm⁻³, and 5.8 ± 1.7 μm, respectively) and the lowest mesophyll intercellular spaces (f_ias 38 ± 3%). Moreover, C. monspeliensis had the highest P_N in summer (2.6 ± 0.1 μmol m⁻² s⁻¹) and C. incanus the highest P_N and WUE (84% and 59% higher than the other species) in the favorable period, associated to a higher f_ias (42 ± 2%). C. salvifolius had the highest P_N (54% higher than the other species) in winter. The plasticity index could allow a better interpretation of the habitat preference of the considered species. The physiological plasticity (PI_p = 0.39, mean value of the considered species) was higher than the morphological (PI_m = 0.22, mean value) and anatomical (PI_a = 0.13, mean value) plasticity. Moreover, among the considered species, C. salvifolius and C. incanus are characterized by a larger PI_a (0.14, mean value) which seems to be correlated with their wider ecological distribution and the more favorable conditions of the environments where they naturally occur. The highest PI_m (0.29) of C. monspeliensis indicates that it can play a high adaptive role in highly stressed environments, like fire degraded Mediterranean areas in which it occurs.     

Fixed-time AI pregnancy rate following insemination with frozen-thawed or fresh-extended semen in progesterone supplemented CO-Synch protocol in beef cows

The objective of this study was to compare fixed-time AI pregnancy rate in Angus crossbred beef cows inseminated with frozen-thawed or fresh-extended semen. Two ejaculates from each of two Angus bulls were collected by artificial vagina and pooled for each bull. The pooled semen from each bull was divided into two aliquots; Aliquot 1 was extended using Caprogen^® (LIC, Hamilton, New Zealand) to a concentration of 3 × 10⁶ sperm/straw and Aliquot 2 was extended using egg-yolk-glycerol extender to a concentration of 20 × 10⁶ sperm/straw. Semen extended with Caprogen^® was maintained at ambient temperature and semen extended with egg-yolk-glycerol extender was frozen and maintained at −196 °C until insemination. In each of two breeding seasons (Fall 2007 and Spring 2008), Angus-crossbeef cows (N = 1455) at 12 locations were randomly assigned within location to semen type [Fresh (N = 736) vs. Frozen (N = 719)] and sire [1 (N = 731) vs. 2 (N = 724)]. All cows were synchronized with 100 μg of GnRH im and a progesterone Controlled Internal Drug Release insert (CIDR) on Day 0, and on Day 7, 25 mg of PGF2_α im and CIDR removal. All cows received 100 μg of GnRH im and were inseminated at a fixed-time on Day 10, 66 h after CIDR removal. Timed-AI pregnancy rates were influenced by season (P < 0.05), cows detected in estrus prior to and at AI (P < 0.001), and dam age (P < 0.01). Pregnancy rates were not affected by semen type (Fresh = 51.5% vs. Frozen = 50.4%; P = 0.66) and there were no significant interactions of semen type by estrus expression, semen type by sire, or semen type by season (P > 0.1). In conclusion, commercial beef cows inseminated with fresh-extended semen (3 × 10⁶ sperm/straw) yielded comparable pregnancy rates to conventional frozen-thawed semen in a progesterone supplemented, CO-Synch fixed-time AI synchronization protocol and may provide an alternate to frozen semen for more efficient utilization of superior genetics.     

Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed

The semen of five Majorera breed bucks was collected and processed to reach a final concentration of 200 × 10⁶ spermatozoa/straw in the extender containing 4% of glycerol and 12% of egg yolk. Two freezing techniques were assessed: (LN) straws were frozen and stored in liquid nitrogen, and (ULF) straws were frozen and stored in the ultra-low freezer at −152 °C. Semen quality (sperm motility, acrosome integrity and abnormal sperm cells percentages) was determined for different storage times (1, 30, 90 and 365 days of cryopreservation). Thereafter, 150 Majorera goats were assigned to four experimental groups: for groups LN-1 (n = 40) and LN-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen, respectively, while for groups ULF-1 (n = 40) and ULF-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in an ultra-low freezer at −152 °C, respectively. The pregnancy rate was determined by transabdominal ultrasound scanning; in addition, the kidding rate and prolificacy were recorded at parturition. In vitro results showed that the freezing protocol did not affect sperm quality with similar values for up to 1 year of cryopreservation. The kidding rates were not significantly different between experimental groups (43.6%, 38.5%, 42.8% and 40.0% for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). In all experimental groups, the kidding rate and prolificacy were significantly higher (p < 0.01) in multiparous than in nulliparous goats. Therefore, the in vitro results and fertility trials confirmed the efficiency of the ULF technique for freezing and storage of goat semen.     

A diet supplemented with l-carnitine improves the sperm quality of Piétrain but not of Duroc and Large White boars when photoperiod and temperature increase

It has been reported that a diet supplemented with l-carnitine can improve sperm quality in some mammalian species. Against this background, the current study seeks to determine the effects of feeding l-carnitine (625 mg·day⁻¹) on boar semen characteristics (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) in three different porcine breeds (Sus domesticus) (Piétrain, Duroc, and Large White) exposed to natural environmental changes in temperature and photoperiod over a 20-wk period (February to July 2007). One hundred twenty boars (40 per breed) were randomly separated into two groups (60 boars each): the first (20 boars per breed) was fed a control diet and the second (also 20 males per breed) the same diet supplemented with l-carnitine (625 mg·day⁻¹). Whereas the l-carnitine supplement did not affect ejaculate volume, concentration, motility, viability, or the osmotic resistance of spermatozoa, it did improve sperm morphology in Piétrain boars by reducing the percentage of immature spermatozoa when the temperature and the photoperiod increased. Conversely, no effect on sperm morphology from supplementing feed with l-carnitine was observed in both Duroc and Large White breeds. We can therefore conclude that the addition of l-carnitine to the diet of males may maintain the level of normal sperm morphology in Piétrain boars when a drop in sperm quality occurs (due to increases in photoperiod and temperature), without affecting the other sperm quality parameters.     

Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates

The effects of inorganic nitrogen (N) source (NH₄⁺, NO₃⁻ or both) on growth, biomass allocation, photosynthesis, N uptake rate, nitrate reductase activity and mineral composition of Canna indica were studied in hydroponic culture. The relative growth rates (0.05-0.06 g g⁻¹ d⁻¹), biomass allocation and plant morphology of C. indica were indifferent to N nutrition. However, NH₄⁺ fed plants had higher concentrations of N in the tissues, lower concentrations of mineral cations and higher contents of chlorophylls in the leaves compared to NO₃⁻ fed plants suggesting a slight advantage of NH₄⁺ nutrition. The NO₃⁻ fed plants had lower light-saturated rates of photosynthesis (22.5 μmol m⁻² s⁻¹) than NH₄⁺ and NH₄⁺/NO₃⁻ fed plants (24.4-25.6 μmol m⁻² s⁻¹) when expressed per unit leaf area, but similar rates when expressed on a chlorophyll basis. Maximum uptake rates (V_max) of NO₃⁻ did not differ between treatments (24-35 μmol N g⁻¹ root DW h⁻¹), but V_max for NH₄⁺ was highest in NH₄⁺ fed plants (81 μmol N g⁻¹ root DW h⁻¹), intermediate in the NH₄NO₃ fed plants (52 μmol N g⁻¹ root DW h⁻¹), and lowest in the NO₃⁻ fed plants (28 μmol N g⁻¹ root DW h⁻¹). Nitrate reductase activity (NRA) was highest in leaves and was induced by NO₃⁻ in the culture solutions corresponding to the pattern seen in fast growing terrestrial species. Plants fed with only NO₃⁻ had high NRA (22 and 8 μmol NO₂⁻ g⁻¹ DW h⁻¹ in leaves and roots, respectively) whereas NRA in NH₄⁺ fed plants was close to zero. Plants supplied with both forms of N had intermediate NRA suggesting that C. indica takes up and assimilate NO₃⁻ in the presence of NH₄⁺. Our results show that C. indica is relatively indifferent to inorganic N source, which together with its high growth rate contributes to explain the occurrence of this species in flooded wetland soils as well as on terrestrial soils. Furthermore, it is concluded that C. indica is suitable for use in different types of constructed wetlands.     

Expression of chemokine receptors on natural killer cells in HIV-infected individuals

Chemokine receptors CCR5 and CXCR4 are of major importance in the pathogenesis of HIV-1 infection because they are co-receptors for human immunodeficiency virus (HIV) entry. We examined the frequency of CD3⁻CD56⁺CCR5⁺ and CD3⁻CD56⁺CXCR4⁺ in HIV-infected long-term slow progressors (SPs), HIV typical progressors (TPs) with or without highly active antiretroviral therapy (HAART), and HIV-seronegative controls. The results showed that the frequency of CD3⁻CD56⁺CCR5⁺ was up-regulated, and frequency of CD3⁻CD56⁺CXCR4⁺ was down-regulated in HAART-naïve HIV TPs group compared with HIV SPs group and HIV-seronegative controls (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was down-regulated by HAART therapy (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was lower in HIV SPs compared with controls (P < 0.05). Lower frequency of CD3⁻CD56⁺CXCR4⁺ and higher frequency of CD3⁻CD56⁺CCR5⁺ positively correlated with the level of HIV viral loads and negatively correlated with CD4 T cell counts (P < 0.05). These results indicated that the expression of chemokine receptors on NK cells correlated with HIV disease progression.     

Osmotic tolerance of avian spermatozoa: influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability

Potential factors influencing sperm survival under hypertonic conditions were evaluated in the Sandhill crane (Grus canadensis) and turkey (Meleagridis gallopavo). Sperm osmotolerance (300-3000 mOsm/kg) was evaluated after: (1) equilibration times of 2, 10, 45 and 60 min at 4 °C versus 21 °C; (2) pre-equilibrating with dimethylacetamide (DMA) or dimethylsulfoxide (Me₂SO) at either 4 °C or 21 °C; and (3) inhibition of the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ionic pumps. Sperm viability was assessed using the eosin-nigrosin live/dead stain. Species-specific differences occurred in response to hypertonic conditions with crane sperm remaining viable under extreme hypertonicity (3000 mOsm/kg), whereas turkey sperm viability was compromised with only slightly hypertonic (500 mOsm/kg) conditions. The timing of spermolysis under hypertonic conditions was also species-specific, with a shorter interval for turkey (2 min) than crane (10 min) sperm. Turkey sperm osmotolerance was slightly improved by lowering the incubation temperature from 21 to 4 °C. Pre-equilibrating sperm with DMA reduced the incidence of hypertonic spermolysis only in the crane, at both room and refrigeration temperature. Inhibiting the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ion pumps did not impair resistance of crane and turkey spermatozoa to hypertonic stress; pump inhibition actually increased turkey sperm survival compared to control sperm. Results demonstrate marked species specificity in osmotolerance between crane and turkey sperm, as well as in the way temperature and time of exposure affect sperm survival under hypertonic conditions. Differences are independent of the role of osmotic pumps in these species.     

Pregnancy loss in heifers after artificial insemination with frozen-thawed,sex-sorted,re-frozen-thawed dairy bull sperm

The use of sex-sorted sperm by the dairy industry is often limited by the geographical distance between potential sires and the sex-sorting facility. One method that may be used to overcome this limitation is sex-sorting sperm that have been previously frozen, or transported to the sorting facility as cooled liquid semen. In this study the in vivo fertility of frozen-thawed, sex-sorted, re-frozen-thawed (FSF) and cooled, sex-sorted, frozen-thawed (CSF) bull sperm was determined after artificial insemination (AI) of Holstein heifers. Semen from two bulls was frozen in straws, or transported to the sorting facility in an egg yolk diluent at 5 °C over 24 h. Thawed or re-warmed semen was processed through a PureSperm^® density gradient, and sperm were sorted for sex and frozen (2 or 4 × 10⁶ sperm/straw). Synchronised heifers (n = 183) were inseminated with either non-sorted control sperm (Control; 20 × 10⁶ dose) or with FSF or CSF ‘X’ sperm (2 or 4 × 10⁶/dose). Pregnancy rates (detected at 7–9 weeks) after AI with control sperm were higher than with FSF or CSF sperm (57.4 vs. 4.1 and 7.3% respectively; p < 0.001). There was a significant difference between bulls (Bull 1: Control 63.0%, FSF 8.6%, CSF 10.0%; Bull 2: Control 45.5%, FSF 0%, CSF 4.8%; p = 0.001). Five out of six (83.3%) pregnancies produced with sexed sperm were lost after pregnancy diagnosis. The exception was one heifer inseminated with CSF sperm (2 million sperm dose), which produced a heifer calf. In the non-sorted control group, three pregnancies were lost (8.3%) and three stillbirths occurred (8.3%). The low fertility and high rate of pregnancy loss in the sexed groups, in addition to environmental influences, may be attributed to impaired sperm function caused by sex-sorting and re-freezing, leading to poor embryo quality or altered gene expression. More precise timing of insemination and higher sperm doses might improve the fertility of FSF sperm. Moreover, the in vitro function of double-frozen sexed compared with non-sorted sperm requires further investigation.     

Nitrogen uptake responses of Gracilaria vermiculophylla (Ohmi) Papenfuss under combined and single addition of nitrate and ammonium

The ammonium (NH₄⁺) and nitrate (NO₃⁻) uptake responses of tetrasporophyte cultures from a Portuguese population of Gracilaria vermiculophylla were studied. Thalli were incubated at 5 nitrogen (N) levels, including single (50 μM of NH₄⁺ or NO₃⁻) and combined addition of each of the N sources. For the combined additions, the experimental conditions attempted to simulate 2 environments with high N availability (450 μM NO₃⁻ + 150 μM NH₄⁺; 250 μM NO₃⁻ + 50 μM NH₄⁺) and the mean N concentrations occurring at the estuarine environment of this population (30 μM NO₃⁻ + 5 μM NH₄⁺). The uptake kinetics of NH₄⁺ and NO₃⁻ were determined during a 4 h time-course experiment with N deprived algae. The experiment was continued up to 48 h, with media exchanges every 4 h. The uptake rates and efficiency of the two N sources were calculated for each time interval. For the first 4 h, G. vermiculophylla exhibited non-saturated uptake for both N sources even for the highest concentrations used. The uptake rates and efficiency calculated for that period (V_0-4 h), respectively, increased and decreased with increasing substrate concentration. NO₃⁻ uptake rates were superior, ranging from 1.06 ± 0.1 to 9.65 ± 1.2 μM g(dw)⁻¹ h⁻¹, with efficiencies of 19% to 53%. NH₄⁺ uptake rates were lower (0.32 ± 0.0 to 5.75 ± 0.08 μM g(dw)⁻¹ h⁻¹) but G. vermiculophylla removed 63% of the initial 150 μM and 100% at all other conditions. Uptake performance of both N sources decreased throughout the duration of the experiment and with N tissue accumulation. Both N sources were taken up during dark periods though with better results for NH₄⁺. Gracilaria vermiculophylla was unable to take up NO₃⁻ at the highest concentration but compensated with a constant 27% NH₄⁺ uptake through light and dark periods. N tissue accumulation was maximal at the highest N concentration (3.9 ± 0.25% dw) and superior under NH₄⁺ (3.57 ± 0.2% dw) vs NO3 (3.06 ± 0.1% dw) enrichment. The successful proliferation of G. vermiculophylla in estuarine environments and its potential utilization as the biofilter component of Integrated Multi-Trophic Aquaculture (IMTA) are discussed.     

Nitrogen nutrition of Salvinia natans: Effects of inorganic nitrogen form on growth,morphology, nitrate reductase activity and uptake kinetics of ammonium and nitrate

In this study we assessed the growth, morphological responses, and N uptake kinetics of Salvinia natans when supplied with nitrogen as NO₃⁻, NH₄⁺, or both at equimolar concentrations (500 μM). Plants supplied with only NO₃⁻ had lower growth rates (0.17 ± 0.01 g g⁻¹ d⁻¹), shorter roots, smaller leaves with less chlorophyll than plants supplied with NH₄⁺ alone or in combination with NO₃⁻ (RGR = 0.28 ± 0.01 g g⁻¹ d⁻¹). Ammonium was the preferred form of N taken up. The maximal rate of NH₄⁺ uptake (V_max) was 6–14 times higher than the maximal uptake rate of NO₃⁻ and the minimum concentration for uptake (C_min) was lower for NH₄⁺ than for NO₃⁻. Plants supplied with NO₃⁻ had elevated nitrate reductase activity (NRA) particularly in the roots showing that NO₃⁻ was primarily reduced in the roots, but NRA levels were generally low (<4 μmol NO₂⁻ g⁻¹ DW h⁻¹). Under natural growth conditions NH₄⁺ is probably the main N source for S. natans, but plants probably also exploit NO₃⁻ when NH₄⁺ concentrations are low. This is suggested based on the observation that the plants maintain high NRA in the roots at relatively high NH₄⁺ levels in the water, even though the uptake capacity for NO₃⁻ is reduced under these conditions.     

Oxidation of thiocyanate by iron(V) in alkaline medium

The oxidation of thiocyanate by iron(V) (Fe(V)) was studied as a function of pH in alkaline solutions by a premix pulse radiolysis technique. The rates decrease with an increase in pH. The rate law for the oxidation of SCN⁻ by Fe(V) was obtained as −d[Fe(V)]/dt = k₁₀{[H⁺]²/([H⁺]² + K₂[H⁺] + K₂K₃)}[Fe(V)][SCN⁻], where k₁₀ = 5.72 ± 0.19 × 10⁶ M⁻¹ s⁻¹, pK₂ = 7.2, and pK₃ = 10.1. The reaction precedes via a two-electron oxidation, which converts Fe(V) to Fe(III). Thiocyanate reacts approximately 10³× faster with iron(V) than does with iron(VI).     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

Nitrilotris(methylenephosphonates) in aqueous solution and solid state - dilatometric, potentiometric and NMR investigations

Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H₆L) have been studied by dilatometric, potentiometric and ³¹P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M⁺ + HL⁵⁻ ? MHL⁴⁻ and M⁺ + L⁶⁻ ? ML⁵⁻, respectively, were determined: logK_NaHL=1.08(0.07), logK_KHL=0.86(0.08), logK_NaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pK_a5^app and pK_a6^app. Correction formulae were derived to convert actual dissociation constants pK_a into apparent dissociation constants pK_a^app (or vice versa). The actual dissociation constants were found: pK_a5(H₂L⁴⁻ ? H⁺ + HL⁵⁻)=7.47(0.03) and pK_a6(HL⁵⁻ ? H⁺ + L⁶⁻)=14.1(0.1). The anisotropy of ³¹P chemical shifts of salts M_nH_6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts.     

Kinetic study of base-catalyzed asymmetric nitrogen conversion of cis-folded macrocyclic nickel(II) complex of 1,4,7,11-tetraazacyclotetradecane

In order to examine the effects of coordinated hydroxide ion and free hydroxide ion in configurational conversion of a tetraamine macrocyclic ligand complex, the kinetic of the cis-to-planar interconversion of cis-[Ni(isocyclam)(H₂O)₂]²⁺ (isocyclam = 1,4,7,11-tetraazacyclotetradecane) has been examined spectrophotometrically. All kinetic data have been satisfactorily fitted by the rate law, R = (k₁K_OH[OH⁻]² + k₂[OH⁻])(1 + K_OH[OH⁻])⁻¹(cis-[Ni(isocyclam)(H₂O)₂]²⁺ + [Ni(isocyclam)(OH)]⁺), where k₂ = (3.40 ± 0.12) × 10³ dm³ mol⁻¹ s⁻¹ is almost equal to k_OH determined in buffer solution (lowly basic media), K_OH = 22.7 ± 1.4 dm³ mol⁻¹ at I (ionic strength) = 0.10 mol dm⁻³ (NaClO₄ + NaOH) and 25.0 °C. Rate constants, k₂ and K_OH, are functions of ionic strength, giving a good evidence for an intermolecular pathway. The reaction follows a free-base-catalyzed mechanism where nitrogen inversion, solvation and ring conformational changes are occurred.     

Influence of inflammatory response,infection, and pulmonary function in cystic fibrosis

Aims

Recurrent infections and activation of the inflammatory response affect the prognosis of cystic fibrosis (CF). We investigated the relationship between inflammatory response, infection, and pulmonary function in CF.

Main methods

A clinical-cross-sectional study was conducted with 86 subjects: control group (CG, n = 31, the same age and sex of the CF group), and CF group (CFG, n = 55, age: 1–16 years), further distributed into CFG negative or positive bacteriology (CFGB⁻/CFGB⁺), and CFG negative or positive Pseudomonas aeruginosa (CFGPa⁻/CFGPa⁺). Using the Wald test, multiple linear regression (95% confidence interval) was performed between CG and CFG, and between CG and each of the CF subgroups (CFGB⁻/CFGB⁺ and CFGPa⁻/CFGPa⁺). The inflammatory markers evaluated were myeloperoxidase (MPO), adenosine deaminase (ADA) activities, interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), nitric oxide metabolites (NOx) levels, and total and differential leukocyte counts.

Key findings

After adjusting for sex and age, CFG compared to CG revealed an increase of MPO, IL-1β (P < 0.001 in all subgroups), and CRP: CFG (P = 0.002), CFGB⁻ (P = 0.007), CFGB⁺ (P = 0.009), CFGPa⁻ (P = 0.004) and CFGPa⁺ (P = 0.020). NOx (P = 0.001, P < 0.001), leukocytes (P = 0.002, P = 0.001), and neutrophils (P = 0.003, P < 0.001) were increased in CFGB⁺ and CFGPa⁺, respectively. A negative correlation between FEV₁ and leukocytes (P = 0.008) and FEV₁ and neutrophils (P = 0.031) resulted in CFG.

Significance

The inflammatory response characterized by the increase of MPO, IL-1β, and CRP is determinant for CF. Also leukocytosis due to neutrophilia determines the pulmonary function deficiency in this disease.     

Comparison of efficiency between two artificial insemination methods using frozen–thawed semen in domestic cat (Felis catus): Artificial insemination in domestic cats

The aim of this study was to compare the efficiency of the intravaginal (IVAI) vs. intrauterine artificial insemination (IUAI) using frozen–thawed sperm in the domestic cat. Semen was collected from two tom cats using an artificial vagina and samples were assessed for motility (computer-assisted sperm analysis (CASA)), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP/YOLK (4% of glycerol), sperm samples were loaded into 0.25 mL straws (25 × 10⁶ motile sperm/straw), incubated at 5 °C for 20 min and cryopreserved over liquid nitrogen (LN₂) vapor for 15 min and then immersed in LN₂. For each AI, four straws from the same male were thawed (12 s at 46 °C) and centrifuged at 250 × g for 8 min to pellet the sperm. The supernatant was discarded and sperm pellet resuspended with the remaining liquid, approximately 100 μL, and analyzed as described above. Queens were treated with a single im injection of 100 IU eCG to induce ovarian follicular development. Final oocyte maturation and ovulation was induced with 100 IU hCG given im at 82–84 h after eCG administration. Thirty hours after hCG administration, females were inseminated either intrauterine (n = 8 queens) or intravaginally (n = 8 queens), using thawed sperm from a single male. Although a pronounced decrease in sperm motility, acrosome and plasma membrane integrity was observed in sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method compared with 0% pregnancy when inseminated intravaginally.