The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences ( P < 0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storage.
Twenty-eight soil samples were obtained from open fields and greenhouses used for tomato cultivation in various regions of Colombia. For functional characterization, 99 Bacillus thuringiensis (Bt) strains were isolated and characterized by abundance and morphology of microscopic crystals, SDS-PAGE of protein extracts and M-PCR analyses of genes of the cry1 family, as well as for their insecticidal activity against Tuta absoluta second instar larvae. Native Bt strains had amorphous (5%), bi-pyramidal (27%), square (8%), spherical (38%) and triangular (22%) crystal forms. Based on the presence of 1-4 different crystal forms, 18 different profiles were established. The SDS-PAGE analyses of protein extracts established ten different strain groups based on their protein band weight and potential biological activity. The M-PCR technique identified 35 native Bt strains based on the presence of the 6 genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D, whose frequency of occurrence was 76, 26, 21, 35, 32 and 8.8%, respectively. Thirteen different PCR profiles were found in native Bt strains. Several gene combinations tended to cooccur with elevated frequency, such as the pairs cry1Ac/ cry1C, cry1Ab/cry1Ac and cry1Ab/cry1B, for which Pearson correlation coefficients were 0.69, 0.52 and 0.54, respectively. Native strains ZBUJTL39 and ZCUJTL11 had up to three times higher biological activity against T. absoluta second instar larvae than the reference strain Bt var. kurstaki HD1, with an LD 50 of 2.4 lg/ml (P \ 0.05) for native Bt strain ZCUJTL11. This study suggests a high biodiversity of native Bt strains from tomato growing regions in Colombia, which has important implications for designing biological control strategies for T. absoluta.
Density gradient centrifugation with PureSperm® (PureSperm® 40 + PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400 × 106 sperm mL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200 × 106 sperm mL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4 cm above the surface of liquid nitrogen vapors for 10 min, after which they were directly placed in liquid nitrogen. After 24 to 48 h of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P < 0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65 ± 0.05 v. 83.79 ± 0.13; percentage of progressive motile spermatozoa: 79.38 ± 6.66 v. 54.61 ± 16.11), morphology (86.45 ± 0.01 v. 83.51 ± 0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32 ± 0.04 v. 36.50 ± 0.17; percentage of viable sperm with an acrosome reaction: 2.81 ± 0.01 v. 9.74 ± 0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.
Study question Does the evaluation of oxidation-reduction potential (ORP) levels in blood plasma from subfertile patients and oocyte donors represents the ORP status in follicular fluid? Summary answer ORP levels in blood plasma from oocyte donors and sub fertile patients could be an indicator of oxidative stress in follicular fluid What is known already Oxidation-reduction potential (ORP) measurement of seminal plasma is one method to diagnose oxidative stress in male patients, which can help physician to recommend antioxidants administration when it is elevated. Seminal plasma is easily accessible by masturbation. An empirical analogue for semen could be follicular fluid (FF). Unfortunately, it is very difficult to access to FF, unless a surgical procedure is performed. Hence, it is necessary to find indirect parameters to evaluate the ORP status in FF. Since blood plasma (BP) circulation provides antioxidants to the FF, we studied the BP ORP measurement to indirectly determine ORP levels in FF. Study design, size, duration Prospective study conducted at CITMER, Mexico from December 2022 to January 2023. We included under informed consent 15 oocyte donors (age 26.8 ± 4.06 years old) and 55 patients (age 35.2 ± 4.8 years old) undergoing IVF/ICSI. Participants/materials, setting, methods ORP levels in BP and FF from dominant follicles were measured at the same time of oocyte collection with MiOXSYS system. Oocytes were inseminated and zygotes were cultured until blastocysts stage. We calculated correlation of a) ORP levels in FF vs BP, b) ORP of FF and BP vs number of retrieved oocytes, c) ORP of FF and BP vs number of fertilized oocytes, d) ORP of FF and BP vs number of blastocysts Main results and the role of chance A total of 172 oocytes from oocyte donors were collected. After fertilization check, 135 zygotes (78%) were observed. A total number of 55 blastocysts (31%) were observed at day 5 + day 6. For patients, a total of 550 oocytes were collected, 360 zygotes were observed (65%) and 164 blastocysts at day 5 + day 6 were obtained (45%). The correlation between ORP levels from FF and BP for oocyte donors was 0.90 and 0.51 for patients. The overall ORP levels in FF and BP in patients were 136.2 ± 14.9 mV and 148.3 ± 15.97 mV, respectively. For oocyte donors, ORP levels in FF and BP were 160.3 ± 13.8 mV and 170.28 ± 17.67mV, respectively. There was a mild correlation of ORP levels in BP with numbers of oocytes retrieved (r = 0.25). The correlation of BP ORP with oocyte fertilization was 0.24 in patients with autologous oocyte utilization. The mean ORP levels of FF were 136.2 ± 14.9 mV, versus a mean ORP of 148.3 ± 15.97 mV in BP. The FF ORP levels between 100-150 mV [LRD1] were moderately correlated and predictive of blastocyst development [LRD2]. For donor oocytes there was a weak negative correlation [LRD3] of FF ORP with oocyte fertilization (-0.06) and a weak negative correlation for blastocyst formation was found (-0.18). Limitations, reasons for caution The MiOXSYS System ( Careous Biotechnology, Lituania) is a device calibrated to measure ORP levels in seminal plasma but not to measure ORP levels in FF nor BP. The number of analyzed data should be increased. Wider implications of the findings According to this study, the use of blood plasma to surrogate the measurements of ORP levels in follicular fluid could represent a further strategy to include female patients to be treated by antioxidants before an IVF treatment. Trial registration number None
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