Spermatozoa concentration influences cryopreservation success in sea trout (Salmo trutta m. trutta L.)

Variation among individuals is substantial for spermatozoa concentration in fresh milt in sea trout (Salmo trutta m. trutta L.). The objective of the present study was to examine effects of spermatozoa concentration in this species on subsequent cryopreservation success. Milt with high spermatozoa concentration was diluted with seminal plasma to obtain concentrations ranging between 6 and 24 × 10⁹ mL⁻¹ with steps of 2 × 10⁹ mL⁻¹. Diluted milts were cryopreserved in 0.25-mL straws with extender (0.3 M glucose) containing 10% methanol and 10 % (vol/vol) supplement of hen egg yolk. The dilution ratio was 1:3 (milt:cryomedium). Cryopreservation efficacies were assessed according to evaluation of motility of frozen/thawed spermatozoa and quantification of fertilizing ability. Percentage of motility of frozen/thawed spermatozoa was influenced by spermatozoa concentration in the cryomedium (P < 0.05). The highest motility was observed in samples with 3.0 to 4.0 × 10⁹ spermatozoa per mL of cryomedium, which corresponds to 12 to 16 × 10⁹ spermatozoa per mL in fresh milt. Higher sperm concentrations and lower sperm concentrations in cryomedium reduced the effectiveness of cryopreservation when compared with the optimum. Cryopreservation success measured according to fertilization rate was in agreement with results for motility of frozen/thawed spermatozoa, but the optimum could not be determined with statistical precision because of differences in fertilization rate among individual donor males. However, a significant positive correlation was found between postthaw motility and fertilization rate and between cryopreserved spermatozoa velocity and fertilization rate (P < 0.05). In sea trout, cryopreservation efficiency is influenced by spermatozoa concentration in cryomedium. Individual adjustment of the dilution ratio, based on initial spermatozoa density, is recommended in the freezing protocol. Maximum cryoresistance of the cell was obtained when spermatozoa concentration in cryomedium ranged from 3.0 to 4.0 × 10⁹ mL⁻¹.     

Field fertility of sex-sorted and non-sorted frozen-thawed stallion spermatozoa

In the 2004/2005 breeding season, the fertility of sex-sorted (SS) and non-sorted (NS) frozen stallion spermatozoa from two Hannovarian stallions was compared. A hysteroscopic insemination technique [Morris, L.H., Tiplady, C., Allen, W.R., 2003a. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen–thawed spermatozoa onto the uterotubal junction. Equine Vet. J. 35, 197–201] was used to deposit low doses (6, 13 or 25 × 10⁶ frozen–thawed SS or NS spermatozoa) onto the utero-tubal junction at 32 or 38 h after the administration of Chorulon (2500 IU, Intervet). Fertility was low, with one pregnancy (13 × 10⁶ spermatozoa, 500 μL) obtained after artificial insemination with frozen SS spermatozoa (n = 29 cycles) which resulted in the birth of a filly. Two pregnancies were obtained in mares inseminated with 6 × 10⁶ NS spermatozoa in 250 μL (n = 31 cycles). Mares failing to conceive on two experimental cycles were allocated to the conventional insemination group. Insemination with >500 × 10⁶ motile NS frozen–thawed spermatozoa, yielded satisfactory per cycle conception rates (35.5%, 22/62) for both stallions combined and was within the values of their normal fertility as quoted by the stud's records. This suggests that the quality of the frozen semen was acceptable and that the freezing processes yielded viable spermatozoa capable of fertilisation. The poor fertility after hysteroscopic insemination with low doses of sex-sorted or non-sorted spermatozoa from the same stallions may be directly attributable to the low dose insemination conditions with frozen–thawed rather than sex-sorted spermatozoa.     

Cryopreservation of human adipose tissues

Scientific studies on cryopreservation of adipose tissues have seldom been performed. The purpose of our present study is conducted both in vitro and in vivo to develop a novel cryopreservation method that can be used successfully for long-term preservation of human adipose tissues for possible future clinical application. In this study, samples of adipose aspirates were obtained from 36 adult white female patients after liposuction and collected from the middle layer after centrifugation. In the in vitro study, suitable cryoprotectant agents (CPAs) and their concentrations and possible combinations were selected from our preliminary experiment. A combination of dimethyl sulfoxide (Me₂SO) and trehalose as CPA with the optimal concentration (0.5 M Me₂SO and 0.2 M trehalose) was chosen and then used throughout the study. In addition, maximal recovery of adipose tissues was achieved after cryopreservation using slow cooling without seeding (1–2 °C/min to −30 °C, followed by plunging to −196 °C for storage) and fast warming (in 40 °C water bath, averaging 35 °C/min). Fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were evaluated by integrated adipocyte counts and histology. In the in vivo study, fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were injected into a nude mouse. The retained adipose aspirates (fat grafts) were harvested in each animal at 4 months and their weight, volume, and histology was assessed. In the in vitro study, significantly higher integrated viable adipocyte count (2.06 ± 0.54 × 10⁶ mL⁻¹ vs. 1.07 ± 0.41 × 10⁶ mL⁻¹, p < 0.0011) of adipose aspirates was found in Group 3 compared with Group 2. Group 3 had only a marginally lower integrated viable adipocyte count compared with Group 1 (2.06 ± 0.54 ×10⁶ mL⁻¹ vs. 2.57 ± 0.56 × 10⁶ mL⁻¹, p = 0.083). Histologically, more tissue shrinkage was evident in Group 2 compared with Group 3. In the in vivo study, various degrees of absorption of injected fat grafts were seen in all 3 groups. However, Group 3 had significantly more retained weight and volume of the injected fat grafts than Group 2 (both p < 0.0001) but had significantly less retained weight and volume than Group 3 (weight, p = 0.009178; volume, p = 0.007836). Histologically, a large amount of tissue fibrosis was seen in Group 2, and reasonably well maintained fatty tissue with only a small amount of tissue fibrosis was seen in Group 3. The results from the present in vitro and in vivo studies, for the first time, demonstrate that our preferred cryopreservation method, the combination of 0.5M Me₂SO and 0.2 M trehalose as CPA in addition to the controlled slow cooling and fast rewarming protocol, appears to provide the maximum recovered results in cryopreservation of human adipose tissues and may become a real option after further refinements for cryopreservation of human adipose aspirates in a clinical setting.     

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III under laboratory culture

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III, two species of causative bloom dinoflagellates in China, were investigated using bi-algal cultures under controlled laboratory conditions. The growth of P. donghaiense and S. trochoidea were significantly suppressed when the initial cell densities were set at 1.9 × 10⁴ cells mL⁻¹ or 1.9 × 10⁵ cells mL⁻¹ for P. donghaiense and 1.0 × 10⁴ cells mL⁻¹ for S. trochoidea when the initial size/density ratio was 1:1 or 10:1, respectively, but no out-competement was observed in either bi-algal culture by the end. The simultaneous assay on the culture filtrate showed that P. donghaiense filtrate prepared at a lower initial density (1.9 × 10⁴ cells mL⁻¹) stimulated the co-cultured S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹, but filtrate at a higher density (1.9 × 10⁵ cells mL⁻¹) depressed its growth. Differently, the filtrate of S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹ significantly suppressed the growth of P. donghaiense at a density of 1.9 × 10⁴ cells mL⁻¹, but had little stimulatory effect on P. donghaiense at a density of 1.9 × 10⁵ cells mL⁻¹compared to the control (P > 0.05). It is likely that these two species of microalgae interact with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. We then quantify their interactions in the bi-algal culture by using a mathematical model. The estimated parameters from the model showed that the inhibition exerted by S. trochoidea on P. donghaiense was about 43 and 24 times stronger than the inhibitory effect that P. donghaiense exerted on S. trochoidea when the initial size/density were 1:1 and 10:1, respectively. S. trochoidea seemed to have a survival strategy that was superior to P. donghaiense in the bi-algal culture under controlled laboratory conditions. We also observed a closely positive relationship between the initial cell density and its effect on the co-cultured microalga by measuring the fluorenscence: filtrate prepared from higher initial cell density had stronger interference on the co-cultured microalga. Moreover, pre-treated under different temperature conditions (30 °C, 60 °C and 100 °C) would significantly changed the effect of culture filtrate on the co-cultured microalga. Result inferred that P. donghaiense or S. trochoidea would release allelochemicals into the bi-algal culture medium and the allelochemicals might be a mixture with temperature-sensitive components in it.     

Pre-freezing equilibration for 22 h improves post-thaw sperm functions in cryopreserved ram semen by reducing cholesterol efflux

Failure of cervical insemination with cryopreserved semen is hindering implementation of AI in sheep in field condition. Here the effect of equilibration time and catalase on post-thaw qualities of ram semen was investigated. Pooled semen was diluted (800 × 10⁶ sperm mL⁻¹) with a TES-Tris-fructose extender with 6% glycerol, 15% egg yolk and supplemented with 0, 50, 100 and 200 U mL⁻¹ catalase and packaged into 0.25 mL straws. In experiment 1, straws were equilibrated at 5 °C either for 3 h in a cold cabinet (E3) or for 10 (E10) and 22 h (E22) inside a refrigerator. In experiment 2, all straws were equilibrated for 22 h inside refrigerator. Straws were frozen at −25 °C min⁻¹ up to −125 °C using a cell freezer and finally plunged into liquid nitrogen. The post-thaw total and rapid motility were higher (P < 0.05) in E22 compared to E3 and E10. Sperm kinetics was comparable between E3 and E22, but lower in E10. Similarly, acrosome integrity, functional membrane integrity, percent high cholesterol (mCHO) and live-high mitochondrial membrane potential (MMP) were higher (P < 0.05) while live-high intracellular calcium and acrosome-reacted sperm were lower in E22 compared to E3 and E10. The percent rapid motile, high mCHO and live-high MMP were significantly (P < 0.05) lower in catalase-treated samples compared to the control, while the membrane integrity was comparable within the groups. In conclusion, pre-freezing equilibration for 22 h compared to 3 or 10 h resulted in higher post-thaw sperm functions while catalase had negative impact on cryopreservation of ram semen.     

Pine nut peroxidase immobilized on chitosan crosslinked with citrate and ionic liquid used in the construction of a biosensor

A biosensor based on the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI·Tf₂N) and a novel source of peroxidase (tissue from the pine nuts of Araucaria angustifolia) was constructed. This enzyme was immobilized on chitosan crosslinked with citrate and the biosensor used for the determination of rosmarinic acid by square-wave voltammetry. The peroxidase in the presence of hydrogen peroxide catalyzes the oxidation of rosmarinic acid to quinone and the electrochemical reduction of the product was obtained at a potential of +0.15 V vs. Ag/AgCl. Different analytical parameters influencing the biosensor response, that is, peroxidase units, pH, hydrogen peroxide concentration and parameters for the square-wave voltammetry (frequency, pulse amplitude and scan increment), were investigated. The best performance was observed for the biosensor under the following conditions: 1000 units mL⁻¹ peroxidase, pH 7.0 and 8.3 × 10⁻⁴ mol L⁻¹ hydrogen peroxide with a frequency of 30 Hz, pulse amplitude of 100 mV and scan increment of 5.0 mV. The biosensor gave a linear response to rosmarinic acid over the concentration range of 9.07 × 10⁻⁷ to 4.46 × 10⁻⁶ mol L⁻¹ with a detection limit of 7.25 × 10⁻⁸ mol L⁻¹. The recovery of rosmarinic acid in plant extracts ranged from 97.0% to 109.6% and the determination of this substance in these samples using the biosensor compared favorably with that using the capillary electrophoresis method.     

Graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose and study of its swelling, metal ion sorption and flocculation behaviour

The present paper reports the graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose by free radical polymerization using potassium peroxymonosulphate/thiourea redox system in an inert atmosphere. The reaction conditions for maximum grafting have been optimized by varying the reaction variables, including the concentration of N-vinylformamide (12.0 × 10⁻²–28.0 × 10⁻² mol dm⁻³), potassium peroxymonosulphate (4.0 × 10⁻³–12.0 × 10⁻³ mol dm⁻³), thiourea (1.2 × 10⁻³–4.4 × 10⁻³ mol dm⁻³), sulphuric acid (2.0 × 10⁻³–10.0 × 10⁻³ mol dm⁻³), sodium carboxymethylcellulose (0.2–1.8 g dm⁻³) along with time duration (60–180 min) and temperature (25–45° C). Water swelling capacity, metal ion sorption and flocculation studies of synthesized graft copolymer have been performed with respect to the parent polymer. The graft copolymer has been characterized by FTIR spectroscopy and thermogravimetric analysis.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 °C over 1.5 h (−0.16 °C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration–equilibration, after freezing and thawing, and 2 h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.     

Short-term variations of virus-like particles in a tropical lake: relationship with microbial communities (bacteria, ciliates and flagellates)

The short-term dynamics of virus-like particles (VLPs) abundance, bacterioplankton, ciliates and flagellates were analyzed in a small tropical lake, during a rainy day (June 9–10, 2003) and a dry day (February 18–19, 2004), with intervals of 3 h between the samplings. Frequent sampling in intervals of 15 min were conducted. During the rainy day, the VLP mean abundance was 7.0×108 mL⁻¹ and bacterial density was 5.75×107 mL⁻¹. During the dry day, VLP and bacterial mean were 5.78×108 and 4.1×107 mL⁻¹, respectively. The virus/bacterium rate (VBR) varied from 11 to 18 on the rainy day and from 4 to 22 on the dry day. The density of VLP was higher during the night, especially on the dry day, suggesting a virucidal action of the solar radiation on them. When registered in intervals of 15 min, the densities were not associated with the fluctuations of bacteria or chlorophyll a (Chl a), but a strong negative correlation between VLP and protozooplankton was observed (Spearman: R=−0.71; p=0.04), possibly associated with the occurrence of viral lyses on these organisms. The variations of VBR in the system, indicate that the elevated densities and fluctuations of VLP is suggestive of an active and important participation of these biological entities in the dynamics of the microbial communities in the studied environment.     

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.     

Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

Effects influencing boar semen

The aim of this study was to analyze the main influences on the quality and quantity of boar semen. A total of 230,705 records of semen collections were utilised to estimate statistics of semen traits of 2712 boars belonging to the following breeds: Czech Meat Pig, Duroc, Hampshire, Landrace, Large White, Czech Large White and Pietrain, and various crosses of these breeds. The evaluation was based on semen volume (VO), concentration of spermatozoa (CO), progressive motion of spermatozoa (MO), abnormal spermatozoa (AB), total number of spermatozoa (NO_T) and corrected number of spermatozoa (NO_C). The breeds differed significantly for all examined traits. The maximal differences between the breeds were 95 ml for VO, 109 × 10³ mm⁻³ for CO, 9% for MO, 1.6% for AB, 24 × 10⁹ for NO_T and 19 × 10⁹ for NO_C. The maximal heterosis effect reached 12% for VO, 17% for CO, 4% for MO, −14% for AB, and 8% for NO_T and NO_C. The results demonstrate that the year-season effect has a clear effect on semen quality. The lowest values of semen traits were observed in summer while the highest values were found in autumn and winter. Age of boar was found to have a strong impact on sperm output. Sperm output tended to increase up to a boars’ age of 3.5 years. An acceptable level of semen volume occurred after a sexual pause of 3 days and the pool of spermatozoa was restoring after 5–7 days and fully after 10–11 days.     

A sugar-aza-crown ether-based fluorescent sensor for Hg and Cu

A fluorescent sensor, 5, based upon the sugar-aza-crown ether structure with two anthracenetriazolymethyl groups was prepared and its fluoroionophoric properties toward transition metal ions were investigated. In methanol, the sensor exhibits highly selective recognition of Cu²⁺ and Hg²⁺ ions among a series of tested metal ions. The association constant for 5·Cu²⁺ and 5·Hg²⁺ in methanol was calculated to be 4.0 × 10⁵ M⁻¹ and 1.1 × 10⁵ M⁻¹, respectively. The detection limits for the sensing of Cu²⁺ and Hg²⁺ ions were 1.39 × 10⁻⁶ M and 1.39 × 10⁻⁵ M, respectively.     

Xanthine oxidase/laponite nanoparticles immobilized on glassy carbon electrode: Direct electron transfer and multielectrocatalysis

In this work, colloidal laponite nanoparticles were further expanded into the design of the third-generation biosensor. Direct electrochemistry of the complex molybdoenzyme xanthine oxidase (XnOx) immobilized on glassy carbon electrode (GCE) by laponite nanoparticles was investigated for the first time. XnOx/laponite thin film modified electrode showed only one pair of well defined and reversible cyclic voltammetric peaks attributed to XnOx–FAD cofactor at about −0.370 V vs. SCE (pH 5). The formal potential of XnOx–FAD/FADH₂ couple varied linearly with the increase of pH in the range of 4.0–8.0 with a slope of −54.3 mV pH⁻¹, which indicated that two-proton transfer was accompanied with two-electron transfer in the electrochemical reaction. More interestingly, the immobilized XnOx retained its biological activity well and displayed an excellent electrocatalytic performance to both the oxidation of xanthine and the reduction of nitrate. The electrocatalytic response showed a linear dependence on the xanthine concentration ranging from 3.9 × 10⁻⁸ to 2.1 × 10⁻⁵ M with a detection limit of 1.0 × 10⁻⁸ M based on S/N = 3.     

Characteristics and seasonal variation in the semen of Markhoz bucks in western Iran

The aims of the present study were to evaluate if seasonality in semen characteristics and plasma testosterone concentrations exist in Markhoz male goats. Ten Markhoz (Angora) bucks were housed and fed according to standard recognized practices. During the observation period, semen was collected monthly with the aid of an electro-ejaculator and examined microscopically immediately after collection. Physical parameters of semen and the semen index were recorded. Blood samples were also taken monthly throughout the observation period and the plasma testosterone concentration monitored. Bucks demonstrated a higher semen quality (P < 0.05) in autumn and summer (semen index of 965 × 10⁶ and 752 × 10⁶ ml⁻¹, respectively), compared to spring and winter (semen index of 606 × 10⁶ and 512 × 10⁶, respectively). This coincided with a higher (P < 0.05) plasma testosterone concentration in autumn and summer (8.1 and 10.1 ng ml⁻¹, respectively), compared to that obtained in spring (3.0 ng ml⁻¹) and winter (2.5 ng ml⁻¹). During autumn and summer, the ejaculate volume (average of 1.2 and 1.0 ml), sperm output (1159 × 10⁶ and 1005 × 10⁶ sperm ml⁻¹), sperm mass motility (4.2 and 4.3), sperm progressive motility (83.9 and 82.0%) and percentage live sperm (90.7 and 88.2%, respectively) of the bucks were higher (P < 0.05) than in the spring (0.6 ml, 880 × 10⁶ sperm ml⁻¹, 3.3, 71.5% and 80.2%) and winter (0.7 ml, 863 × 10⁶ sperm ml⁻¹, 4.0, 71.5% and 84.9%, respectively). During autumn and summer, the percentage of sperm abnormalities (5.0 and 9.2%) was significantly lower than that in spring (12.9%) and winter (11.2%). The semen pH was slightly alkaline being significantly (P < 0.05) lower in the autumn (7.1) than in spring (7.3). Data showed season of the year to influence all semen parameters evaluated—indicating that optimal buck performance may be obtained in late summer and autumn. It can thus be said that Markhoz bucks have distinct seasonal spermatogenic activity, with poorer semen characteristics being recorded during winter and spring. This may be a critical obstacle when implementing an intensive breeding system of three kidding seasons in 2 years, with natural mating being implemented.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis–Menten kinetics; the apparent efficiency (k_cat/K_T) of this process increases over 2-fold (2.1 × 10⁶–4.5 × 10⁶ s⁻¹ M⁻¹) upon adipocyte differentiation. The V_max values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 × 10⁶ s⁻¹ M⁻¹ versus 1.5 × 10⁶ s⁻¹ M⁻¹). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V_max values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 × 10⁶ s⁻¹ M⁻¹ to 1.35 × 10⁶ s⁻¹ M⁻¹).     

Zinc oxide/redox mediator composite films-based sensor for electrochemical detection of important biomolecules

Electrochemical oxidation of serotonin (SN) onto zinc oxide (ZnO)-coated glassy carbon electrode (GCE) results in the generation of redox mediators (RMs) that are strongly adsorbed on electrode surface. The electrochemical properties of zinc oxide-electrogenerated redox mediator (ZnO/RM) (inorganic/organic) hybrid film-coated electrode has been studied using cyclic voltammetry (CV). The scanning electron microscope (SEM), atomic force microscope (AFM), and electrochemical techniques proved the immobilization of ZnO/RM core/shell microparticles on the electrode surface. The GCE modified with ZnO/RM hybrid film showed two reversible redox peaks in acidic solution, and the redox peaks were found to be pH dependent with slopes of −62 and −60 mV/pH, which are very close to the Nernst behavior. The GCE/ZnO/RM-modified electrode exhibited excellent electrocatalytic activity toward the oxidations of ascorbic acid (AA), dopamine (DA), and uric acid (UA) in 0.1 M phosphate buffer solution (PBS, pH 7.0). Indeed, ZnO/RM-coated GCE separated the anodic oxidation waves of DA, AA, and UA with well-defined peak separations in their mixture solution. Consequently, the GCE/ZnO/RMs were used for simultaneous detection of DA, AA, and UA in their mixture solution. Using CV, calibration curves for DA, AA, and UA were obtained over the range of 6.0 × 10⁻⁶ to 9.6 × 10⁻⁴ M, 1.5 × 10⁻⁵ to 2.4 × 10⁻⁴ M, and 5.0 × 10⁻⁵ to 8 × 10⁻⁴ M with correlation coefficients of 0.992, 0.991, and 0.989, respectively. Moreover, ZnO/RM-modified GCE had good stability and antifouling properties.     

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species—the Spanish ibex—and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w v⁻¹), 2.2% citric acid (w v⁻¹), 0.6% glucose (w v⁻¹), 5% glycerol (v v⁻¹), and 6% egg yolk (v v⁻¹). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility (P < 0.001), membrane integrity (P < 0.001), and viability (P < 0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P = 0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.