In modern animal husbandry, reproductive biotechnologies such as artificial insemination (AI) play a key role in increasing productivity and improving the genetic potential of farm animals. AI allows not only to use agricultural material issued by producers in a larger amount of the male animal as efficiently as possible, but also to reduce the risk of spreading sexually transmitted diseases. The success of AI is largely determined by the quality of the sperm used, which depends on the methods of processing and storing the ejaculate. Among the existing methods of sperm selection, centrifugation occupies one of the leading places. This method is based on the separation of the power components of the ejaculate under the influence of centrifugal. As an example of centrifugation, two main combinations were found: standard and colloidal centrifugation. Standard centrifugation is a single separation of the ejaculate components, with this centrifugation mode under transformations in accordance with the species characteristics of spermatozoa: 200-400 rpm for 5-12 min for rodents, 720 rpm for 5 min for dogs, 2400 rpm for 5 min for stallions, 5000 rpm for 5 min for bulls, 3000 rpm for 3 minutes for goats, 2400 rpm for 3 minutes for boars. Colloid centrifugation involves the use of special colloidal solutions (Percolltm, Isolate®, Porcicoll®, Bovicoll®, Equipure Bottom Layer®, PureSperm®) for more effective separation of spermatozoa formed with morphological and functional forms. Colloid centrifugation allows you to obtain higher quality ejaculate, since it can separate not only seminal plasma, but also defective, low-motility spermatozoa, as well as cells with damaged DNA.

Inter-comparison of extraction methods for plant water isotope analysis and its indicative significance

Comparative life-cycle assessment of various harvesting strategies for biogas production from microalgae: Energy conversion characteristics and greenhouse gas emissions

IntroductionAdipose tissue (AT) has become a source of mesenchymal stromal/stem cells (MSC) for regenerative medicine applications, in particular skeletal disorders. Several enzymatic or mechanical procedures have been proposed to process AT with the aim to isolate cells that can be locally implanted. How AT is processed may impact its properties. Thus, we compared AT processed by centrifugation (C-AT) to microfragmentation (MF-AT). Focusing on MF-AT, we subsequently assessed the impact of synovial fluid (SF) alone on both MF-AT and isolated AT-MSC to better understand their cartilage repair mechanisms.Materials and methodsMF-AT and C-AT from the same donors were compared by histology and qRT-PCR immediately after isolation or as ex vivo cultures using a micro-tissue pellet system. The in vitro impact of SF on MF-AT and AT-MSC was assessed by histological staining and molecular analysis.ResultsThe main AT histological features (i.e., increased extracellular matrix and cellularity) of the freshly isolated or ex vivo-cultured MF-AT persisted compared to C-AT, which rapidly deteriorated during culture. Based on our previous studies of HOX genes in MSC, we investigated the involvement of Homeobox Protein HOX-B7 (HOXB7) and its target basic Fibroblast Growth Factor (bFGF) in the molecular mechanism underlying the improved performance of MF-AT. Indeed, both these biomarkers were more prominent in freshly isolated MF-AT compared to C-AT. SF alone preserved the AT histological features of MF-AT, together with HOXB7 and bFGF expression. Increased cell performance was also observed in isolated AT-MSC after SF treatment concomitant with enhanced HOXB7 expression, although there was no apparent association with bFGF.ConclusionsOur findings show that MF has a positive effect on the maintenance of AT histology and may trigger the expression of trophic factors that improve tissue repair by processed AT.

Uric acid, as the terminal product of purine metabolism in the body, is an important marker of many diseases. Uric acid is abundant in saliva, offering the possibility of its non-invasive detection. However, it is sensitive to interference in saliva by a variety of factors. A reliable method of processing saliva is centrifugation (CF), but the cost and size of equipment limit its use in everyday life. In this study, a novel portable salivary-sensing system (PSSS) with integrated suction filtration (SF) and temperature insulation was proposed to obtain more accurate salivary uric acid levels through a simple procedure. The PSSS includes a saliva container, a high-sensitive uric acid sensor (UAS), an accompanying printed circuit board (PCB), and a mobile application. The responses produced by the UAS presents excellent linearity (4.6 μA/mM with R2 = 0.9964), selectivity, reproducibility, and stability for the detection of low levels of uric acid. The difference in detection values between the UAS and the commercial sensor is only ~4%. The primary feature of the saliva container is the processing of saliva by SF instead of CF. Samples from CF and SF showed no significant differences regarding uric acid levels, and both exhibited approximately 50% deviation from the untreated samples, while the difference in uric acid levels between the samples after SF and after applying both treatments was ~10%. Besides, insulation of the saliva container can partially eliminate sources of error induced by the environment during uric acid level testing. The PSSS provides a novel strategy for the immediate detection of specific markers in saliva. We believe that the PSSS has promising potential for future application in the rapid saliva testing.

The online version contains supplementary material available at 10.1007/s10068-021-00940-w.

Objective: To compare the pharmacokinetic behavior of abiraterone acetate after oral administration of a lipidbased formulation tablet and a reference preparation, and to study the relative bioavailability of abiraterone acetate released from the lipid matrix-based tablet. Methods: Beagle dogs received a single dose orally. The experimental dosage was 75 mg/tablet, and the reference dosage was 250 mg/tablet. Six beagle dogs in each group were investigated with the method of 3 × 3 cross-administration. Blood plasma was collected and centrifuged before administration and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h after administration, and the plasma samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: The half-life (T1/2) values of lipid-based formulation (LBF) abiraterone acetate tablet samples (75 mg/tablet) and reference tablets (250 mg/tablet) after oral administration were 3.42 and 4.27 h, respectively, while the area under the concentration time curve (AUC0-t) (h·ng/mL) values were 107.71 and 52.83, respectively. The F value of the relative bioavailability was 704.80%. Conclusion: The preparation method based on lipid matrix can significantly improve the bioavailability of abiraterone acetate tablets, which is a feasible method to improve the bioavailability of biopharmaceutical classification system (BCS) class<small>IV</small> drugs.

In this study, a sensitive method for quantitation of the unique small-molecule inhibitor of hepatitis C virus SK7324 in rat plasma has been established using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI/MS/MS). SK7324 and internal standard (tramadol) in plasma sample was extracted using acetonitrile. A centrifuged upper layer was then evaporated and reconstituted with the mobile phase of 0.5% formic acid-acetonitrile (35:65, v/v). The reconstituted samples were injected into a C18 reversed-phase column. Using MS/MS in the multiple reaction monitoring (MRM) mode, SK7324 and tramadol were detected without severe interference from rat plasma matrix. Detection of SK7324 in rat plasma by the UPLC-ESI/MS/MS method was accurate and precise with a quantitation limit of 1.0 ng/mL. The validation, reproducibility, stability, and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of SK7324 in rat plasma. Pharmacokinetic parameters of SK7324 were evaluated after intravenous (i.v.; at doses of 5 mg/kg) and oral (p.o.; at doses of 10 mg/kg) administration of SK7324 in rats. After p.o. administration (10 mg/kg) of SK7324, F (Fraction absorbed) value was approximately 87.1%.

A highly sensitive, simple and speed technique was employed for the determination of brilliant green in fish (Sphyraena jello) and seawater samples by visible spectrophotometry after its extraction and enrichment with chitosan- zinc oxide nanoparticle coupled with dispersive liquid- liquid microextraction. Ultra-trace concentrations of brilliant green were dispersed to organic phase in DLLME method after adding of dispersive solvent and chitosan- zinc oxide nanoparticles. The experimental factors such as amount of chitosan- zinc oxide nanoparticles, concentration of Triton X- 114, type of volume of extraction and dispersive solvents, extraction time, rate and time of centrifugation, volume of sample and pH were investigated to order to enhance of the extraction efficiency. Under optimum extraction condition, volume of chloroform (as extraction solvent) and methanol (as dispersive solvent) were 100.0 µL and 550.0 µL, respectively; amount of chitosan- zinc oxide nanoparticles was 15.0 mg; time of extraction was 4.0 min; rate and time of centrifugation were 3000.0 rpm and 8.0 min, respectively, volume of sample was 8.0 mL, and pH of sample solution was 4.0. After optimizing of the microextraction conditions and instrumental factors, an enrichment factor of 169.0 was achieved. The analytical curve )absorbance vs. concentration( was linear over the range 1.0-200.0 µg/L of brilliant green. The limit of detection and relative standard deviation were 0.3 µg/L and < 6.1 %, respectively. The protocol was successfully employed to the determination of brilliant green in seawater of Chabahar Bay and fish (Sphyraena jello) samples.

Mycobacterium avium Subsp. paratuberculosis (MAP) is a gram-positive, small, acid-fast bacillus with high environmental resistance. In animals, especially ruminants, it leads to Paratuberculosis (PTB) or Johne's disease, which is chronic granulomatous enteritis. This bacterium as the main causative agent of Crohn's disease can be a serious threat to human health. This study aimed to detect MAP in pasteurized milk samples produced in Khorasan Razavi province, Iran, using Direct Nested PCR, PCR, and culture methods. In this study, 544 milk samples from Pasteurized Milk Production Companies were selected randomly during the 3-month period. DNA was extracted from milk fat after centrifugation. In order to identify the bacteria, Direct Nested PCR and PCR tests were applied using IS900 and f57, respectively. Furthermore, to detect viable MAP, positive samples resulted from Direct Nested PCR assays were cultured on Herrold's egg medium. For identification of mycobacterial isolates, all colonies were processed by PCR based on f57. A total of 544 pasteurized milk samples were assayed, and Mycobacterium paratuberculosis was detected in 39% of them by IS900 Nested PCR, and only 4.9% of samples were positive in the culture method. All the colonies were positive for the f57using PCR. The results of this study indirectly indicated a high level of contamination of pasteurized milk to Mycobacterium paratuberculosis which is due to the large number of affected animals in livestock farms in Khorasan Razavi province. However, in comparison with the other researches, the low percentage of viable bacteria in pasteurized milk can be due to changes in temperature and time in pasteurizing systems of milk production companies in Khorasan Razavi province, Northeast of Iran.

Allocation is not enough! A system boundaries expansion approach to account for production and consumption synergies: The environmental footprint of Greek yogurt

Platelet rich plasma (PRP) is a haemoderivative used in clinical practice for the treatment of hard-to-heal wounds. Platelet (PLT) activation is a key factor in the wound healing process leading to the production of extracellular vesicles. We obtained PRP and PRP-derived microvesicles (PLT-MVs) from healthy donors and compared their pro-healing efficacy in an in vitro wound model using human keratinocytes. We evaluated PLT-MVs' direct effect on an in vitro model of wound healing. PRP, PRP activated using calcimycin, and PLT-MVs separated by high speed centrifugation were added to scratched keratinocyte monolayers. Fluorescein diacetate was used in flow cytometry to distinguish PLTs and PLT-MVs from debris, and then, PLT-MVs were quantified on the basis of relative dimensions (Forward Scatter signals). Wound areas were measured at time 0 and after 24 hours and they were healed by 24.80 ± 4.28% in control conditions, while PRP, activated PRP, and PLT-MVs increased closure of 62.94 ± 0.96%, 52.69 ± 17.20% and 52.76 ± 9.44%, respectively. Our results indicate that PRP pro-healing effects were fully replicable by PLT-MVs, suggesting a key role of microvesicles in the healing process and a possible clinical use as an alternative to PRP.

In cattle, sex selection has a significant economic impact when it improves herd capacity of milk or meat production and is commonly used for the production of calves of the desired gender. New separation techniques which present both better accuracy and low costs are necessary. Density gradient centrifugation might be an approach to sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head. The present study was designed to apply the modified percoll discontinuous density gradient for X-sperm enrichment in cross bred bovine semen. We attempted to amplify the level of X-sperm enrichment by using double centrifugation in percoll gradient. In addition, effect of percoll density gradient on fertility of semen was also evaluated at three stages; before centrifugation (stage I), after 1st centrifugation (stage II) and after 2nd centrifugation (stage III) respectively and ultrasonography was carried out to confirm the sex of fetus at 60-90 days post AI. Semen was collected from two cross bred bovine bulls and 34 cows were inseminated with liquid X-sperm enriched semen after double centrifugation in percoll gradient. 22 cows were inseminated with non-sexed fresh liquid semen (control). Results showed that the volume, progressive motility, live sperms, concentration and HOST reactive sperms significantly (P is less than 0.05) decreased at stage II and stage III compared to stage I. However, no significant (P is greater than 0.05) change was observed in pH and mass motility of semen at all three stages. There was non-significant effect on per cent spermatozoa with intact acrosomal membrane and fully damaged acrosomal membrane at all three stages however, partially damaged acrosomal membrane sperms were significantly (P is less than 0.05) decreased at Stage II and Stage III compared to Stage I. The conception rate significantly (P is less than 0.05) decreased with X-enriched semen (41.17%) compared to non-sexed liquid semen (59.09%). The female sex ratio of calves significantly (P is less than 0.05) increased to 66.66% with X- sperm enriched semen compared to the male percentage of 33.33% with sexed semen and female percentage of 46.15% with non-sexed semen.

Luka bakar adalah kerusakan jaringan pada kulit akibat terpajan panas tinggi, bahan kimiawi maupun arus listrik. Salah satu hewan yang disembelih di Rumah Potong Hewan (RPH) adalah sapi. Seekor sapi dapat menghasilkan limbah darah kurang lebih sebanyak 28 liter. Komponen darah terdiri dari plasma, sel darah merah, sel darah putih dan platelet. Platelet mengandung growth factor. Platelet Rich Plasma (PRP) atau plasma yang kaya akan platelet terbukti dapat mempercepat penyembuhan tulang dan jaringan lunak. Pembuatan PRP dari darah sapi dilakukan dengan metode sentrifuge. Hewan coba yang dipakai pada penelitian ini adalah tikus putih sebanyak 10 ekor. Besi panas berbentuk bulat dipanaskan dengan api, lalu ditempelkan pada kulit. Kelompok perlakuan diberi PRP dengan konsentrasi 10%, 20% dan 30% dengan salep vaselin album, kelompok kontrol negatif yang tidak diberi perlakuan dan kelompok kontrol positif yang diberi obat yang terbuat dari ekstrak plasenta sapi 10%+neomycin sulfate 0,5%. Parameter yang dipakai untuk mengukur tingkat kesembuhan luka adalah waktu kesembuhan dan pemeriksaan histopatologi. Pada hasil pengukuran waktu kesembuhan dianalisis dengan metode statistik One-way Analsisis of variance (Anova) tingkat signifikansi 95% dengan Kruskal-wallis, kesembuhan luka terdapat perbedaan signifikan antara PRP konsentrasi 10%, 20%, 30%, dan kontrol positif dengan kontrol negatif. Konsentrasi PRP yang optimal adalah 20%. Analisa tipe jaringan yang terbentuk dilakukan dengan pembuatan preparat histopatologi, hasil menunjukkan bahwa luka pada kelompok perlakuan dan kontrol positif tampak sembuh, sedangkan kelompok kontrol negatif epitelisasi belum tertutup jelas. PRP merupakan obat penyembuh luka bakar yang efektif sebagai pengganti obat komersil yang sudah tersedia.

Natural phospholipid (PL) excipients are native, biocompatible and relatively inexpensive alternatives to synthetic emulsifiers. A well-known PL excipient is lecithin, which primarily contains phosphatidylcholine (PC) and (depending on the purity grade) also contains a well-defined mixture of other PLs with a fatty acid composition, which reflects their natural source. Since all of these lipid species are emulsifiers, natural PLs can be considered as a mixture of (co-) emulsifiers. Many different HLB values for lecithins are given in the literature, which is why this needs to be clarified. To assess this, HLB values of thirteen different plant derived PLs differing in PC content were determined using a centrifugation stress method to determine the relative stability of an emulsion with the respective emulsifier and different oil phases. It could be shown that the studied PLs can be characterized by a broad HLB range, which may be linked to the composition of the PLs and the oil used. In order to emphasize the results of the HLB determination, it could be demonstrated that stable emulsions could be prepared with a wide range of oils using the plant-based PLs and that the preparation method of the emulsions is important in order to obtain stable emulsions. Therefore, assigning a specific exact HLB value to natural PLs instead of a wider range is inappropriate. The broad HLB ranges indicate the suitability of the studied PL emulsifiers for the preparation of oil-in-water emulsions for a wide range of oils: It is recommended to experimentally evaluate the suitability of these natural emulsifiers for the preparation of stable emulsions and to benefit from the wide range of HLB values of these emulsifiers instead of relying on inaccurate and confusing HLB values in the literature.

Preparation of Nano/Submicron Ganoderma Tsugae and Its Stability

The aim of this study was to evaluate the influence of clarification treatments on volatile composition and aromatic attributes of wine samples. ‘Italian Riesling’ icewines from the Hexi Corridor Region of China were clarified by fining agents (bentonite (BT) and soybean protein (SP)), membrane filtration (MF), and centrifugation (CF) methods. The clarity, physicochemical indexes, volatile components, and aromatic attributes of treated wines were investigated. Both the fining agents and mechanical clarification treatments increased the transmittance and decreased the color intensity of icewine samples. Bentonite fining significantly influenced the total sugar content, total acidity and volatile acidity. Total acidity decreased 2–3.5% and volatile acidity 2–12%. MF showed the greatest influence on total phenol content, decreasing the initial content by 12%, while other treatments by less than 8%. Volatile analysis indicated that both the categories and contents of volatile compounds of wine samples decreased. MF treatment showed the most significant influence, while SP fining showed much lower impact. Odor activity values indicated the compound with the highest odor activity in Italian Riesling icewines was β-damascenone. For this compound, BT and SP did not show significant differences, however, in MF and CF it decreased by 20% and 63%, respectively. Furthermore, with high impact on aroma were: ethyl hexanoate which reduced by 20–80% especially in MF; rose oxide which extremely reduced in MF and undetected in BT, SP, and CF; isoamyl acetate which reduced by 3–33% and linalool decreased by 10–20% and undetected for BT. Principle component analysis indicated that icewine clarified by different methods could be distinguished and positively correlated with odor-active compounds. Floral and fruity were the dominant aroma series in icewine samples followed by fatty, earthy, spicy, vegetative and pungent flavor. The total odor active value of these series significantly (p < 0.5) decreased in different clarification treatments. Sensory evaluation showed similar results, but the SP and CF wine samples achieved better sensory quality. This study provides information that could help to optimize the clarification of ice wines.

Introduction . Drug encapsulation efficiency (EE) is the important parameter of the nanoparticle-based drug formulations. Generally, the methods for evaluation of the EE are based on separation of the free and NP-bound fractions of the drug; however, applicability of these methods for a particulate formulation needs careful consideration. Aim. The purpose of the study was to optimize the procedure for evaluation of the EE for a nanoparticle-based drug formulation taking doxorubicin loaded in the PLGA nanoparticles (PLGA-Dox NP) as a model formulation. Materials and methods. The PLGA-Dox NP were prepared by a “double emulsion” method at pH of the external aqueous phase of 7.4 or 6.4. The NP size and size distribution (PDI) were determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM). For the EE evaluation of doxorubicin, the NP were separated by centrifugation (CF), ultrafiltration (UF), or gel filtration. The efficiency of NP separation by the CF method was evaluated by the PLGA content in the supernatant by capillary electrophoresis (CZE). Results and discussion. The average hydrodynamic diameter of the PLGA-Dox/7.4 and PLGA-Dox/6.4 NP (FCS) was 103 ± 10 nm and 141 ± 8 nm, respectively. According to the TEM data, the main fraction of the NP averaged 50 ± 16 nm. The EE of doxorubicin, determined after the NP separation by centrifugation at 48254.g, was 78.9 ± 1.8 % for the PLGA-Dox/6.4 and 91.5 ± 0.9 % for the PLGA-Dox/7.4 NP with the residual PLGA content in the supernatant of ~5 %. At lower acceleration the NP separation was incomplete leading to underestimation of the EE. Also, the ultrafiltration method using the filters with the NMWL of 50 and 100 kDa enabled the reliable NP separation with the minimal doxorubicin adsorption on the filter (<4 %). Separation of the NP by gel filtration led to the underestimation of the EE due to considerable desorption of doxorubicin from the NP surface. Conclusion. The optimal analytical procedures for evaluation of the EE of doxorubicin in the PLGA NP are based on the NP separation by CF at 48254×g and UF using filters with NMWL of 50 and 100 kDa.

Development of Sucrose Density Gradient Centrifugation Purification Process for Rabies Vaccine

Tissues loss due to periodontal disease is typically treated by a variety of regenerative treatment modalities, including bone grafts, guided tissue regeneration (GTR) and growth factors, to reform the supporting tissues of teeth. Concentrated growth factors (CGF) are produced by centrifuging blood samples at alternating and controlled speeds using a special centrifuge. The purpose of this study was to evaluate whether GTR could improve the effect of CGF combined with bone graft in the treatment of classII furcations of mandibular molars. In the present study, thirty-five classII furcation involvements were included and randomly divided into two groups. The experimental group (n=17) accepted GTR combined with CGF and bone graft therapy, and the controlled group (n=18) accepted CGF combined with bone graft therapy. The clinical examinations and cone beam computed tomography (CBCT) were performed at baseline and 1 year post-surgery. Comparisons of clinical and CBCT data before and after operation between the experimental group and the control group were made. The clinical and CBCT data of both groups were not statistically different at baseline (P>0.05). At the end of 1 year post-surgery, the clinical parameters of both groups were significantly improved (P<0.001). The probing depths of the experimental group were (4.81±1.95) mm and (3.56±1.94) mm, respectively, significantly higher than the changes of the control group (P<0.001). The vertical and horizontal attachment gains of the experimental group were (4.11±1.98) mm and (3.84±1.68) mm, respectively, significantly higher than the changes of the control group (P<0.001). At the end of 1 year post-surgery, the experimental group showed significantly higher bone gain at vertical and horizontal directions compared with those of the control group: (3.84±1.68) and (3.88±2.12) mm, respectively (P<0.001). Within the limitation of the present study, GTR showed positive role in the effect of CGF combined with bone graft in the treatment of classII furcation involvements of mandibular molars.