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Biology of Reproduction Jan 2022Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm...
Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences.
Topics: Animals; Cryopreservation; Male; Metabolome; Phenotype; Semen; Semen Analysis; Semen Preservation; Spermatozoa; Sus scrofa; Temperature; Time Factors
PubMed: 34725678
DOI: 10.1093/biolre/ioab200 -
European Journal of Pediatrics Mar 2023Manually performed double-volume exchange transfusion (DVET) is tedious, error-prone, and may incur the risk of embolism. We aimed to develop a device that automates the...
UNLABELLED
Manually performed double-volume exchange transfusion (DVET) is tedious, error-prone, and may incur the risk of embolism. We aimed to develop a device that automates the DVET procedure performed through the umbilical venous route. We evaluated changes in blood passing through the device during DVET. We developed an electro-mechanical device with accessories (tubing and valve assembly) to perform a complete DVET. It comprises two syringes driven by a common pump that moves back and forth to withdraw aliquots of the patient's blood and infuse equal volumes of donor blood. In tandem, it draws donor blood from a blood bank bag and pushes the patient blood drawn from the previous cycle into a waste bag, respectively. One-way duckbill valves and a two-way pinch valve ensure the separation of the donor and patient blood. A sensor detects bubbles and clots. A dashboard displays set and measured parameters. We tested the accuracy of the delivered flow rate and volume, electrical safety, embolus detection, and changes in hematological and biochemical values. The delivered flow and volume were within 5% of the set parameters. All electrical safety parameters were within normal limits. The sensor consistently detected microbubbles and clots. There were no clinically significant differences in laboratory parameters between samples drawn directly from the blood bank bag and drawn from the exit port at 80, 100, 120, and 160 s with a fixed aliquot volume.
CONCLUSIONS
Our prototype of a novel device can safely automate a DVET. Further trials of this device are warranted.
WHAT IS KNOWN
• Double volume exchange transfusion is often performed manually, but this is time-consuming and error-prone. • Previous attempts at automation were not widely adopted because they involved inserting two catheters and did not have mechanisms to prevent embolism.
WHAT IS NEW
• This novel device fully automates double volume exchange transfusions through a single-lumen umbilical venous catheter. • It prevents air and clot embolism and has a screen for input and output parameters and alarms.
Topics: Humans; Infant, Newborn; Blood Transfusion; Umbilical Cord; Embolism
PubMed: 36625935
DOI: 10.1007/s00431-022-04791-3 -
Pediatric Pulmonology Nov 2022A bronchoscopy is an essential tool in pediatric pulmonology. However, the practices involved in the procedure are variable.
BACKGROUND
A bronchoscopy is an essential tool in pediatric pulmonology. However, the practices involved in the procedure are variable.
OBJECTIVE
To evaluate prevalent practices and variations in pediatric flexible bronchoscopy in India.
METHODS
An online survey was conducted via Google forms between September 2018 and March 2019. We circulated the survey among members of various respiratory societies and personal contacts. Physicians performing pediatric flexible bronchoscopy were requested to respond. The survey had 95 questions in seven domains: demographics, patient preparation, sedation, procedural aspects, monitoring, bronchoscope cleaning, and complications.
RESULTS
The survey received 24 complete responses; the respondents were from 14 cities. Pediatric bronchoscopy was done mainly for diagnostic purposes. Most (19, 79%) respondents reported using conscious sedation for the procedure. The preferred regimen for sedation was midazolam plus fentanyl [9 (37.5%)]. Atropine was used routinely by 4 (16%). For topical anesthesia, nebulized lignocaine only, both nebulized and spray as go lignocaine, and spray as go lignocaine only were used by 1 (4.2%), 6 (25%), and 17 (71%) respondents, respectively. The methods of providing oxygen during bronchoscopy were free flow (9, 37.5%), nasal prongs (8, 33.3%), mask (6, 25%), and laryngeal mask airway (1, 4.2%). The common therapeutic procedures included removal of mucus plugs (17, 71%), bronchoscopic intubation (11, 45%), and foreign body removal (10, 41%). The number of aliquots used by respondents for bronchoalveolar lavage varied from 2 to 6, and the volume for each aliquot was also varied (1-2 ml/kg or 5-10 ml). Almost all the respondents reported complication rates of less than 5%.
CONCLUSION
There is a considerable variation in pediatric flexible bronchoscopy practices across the country, highlighting the need to develop a uniform guideline.
Topics: Atropine Derivatives; Bronchoscopy; Child; Fentanyl; Humans; Laryngeal Masks; Lidocaine; Midazolam; Oxygen; Surveys and Questionnaires
PubMed: 35869591
DOI: 10.1002/ppul.26081 -
New Zealand Veterinary Journal Jul 2017AIMS To examine the agreement between spore counts of Pithomyces chartarum measured in a single aliquot of wash water with counts from multiple aliquots from the same... (Comparative Study)
Comparative Study
AIMS To examine the agreement between spore counts of Pithomyces chartarum measured in a single aliquot of wash water with counts from multiple aliquots from the same 60 g pasture sample, and between spore counts measured in an individual 60 g pasture sample with counts from three 60 g pasture samples selected from the same 200 g paddock sample. MATERIALS AND METHODS Four Waikato dairy farms were visited once weekly from early January to late May 2013. One paddock, with 40 sampling sites, was selected per farm. At each visit, ∼200 g of pasture was collected per site. Spore counting was undertaken using a standard method, except that three separate 60 g pasture samples per 200 g paddock sample was counted; and for each 60 g pasture sample, spores were counted in 10 aliquots of wash water. The relationship between the results of a single aliquot and 6-10 aliquots of wash water from the same 60 g grass sample were assessed by calculating 95% prediction intervals. Limits of agreement analysis was used to assess the agreement between counts from one, two or three aliquots per 60 g pasture sample compared with 10 aliquots, and between counts from one and three 60 g pasture samples from the same 200 g paddock sample. RESULTS Comparing spore counts from individual aliquots with multiple aliquots resulted in large prediction intervals and 95% limits of agreement, which increased with increasing spore count. For an individual aliquot count of 2 spores, the 95% prediction interval for the count from 10 aliquots was 3-49 spores, and for an individual count of 10 spores the 95% prediction interval was 28-222 spores. Increasing the number of aliquots counted improved agreement. For a total count of 10 spores measured in 10 aliquots, the 95% limits of agreement, based on a single aliquot, were 2-50 spores, and for three aliquots were 5-20 spores. The agreement in spore counts measured in one compared with three 60 g pasture samples was moderate and also decreased with increasing spore count; the 95% limits of agreement were 4-14.5 for a mean spore count of 10. CONCLUSIONS AND CLINICAL RELEVANCE Measuring the spore counts of three aliquots of wash water per 60 g grass sample improved repeatability, and should be used as the standard technique, particularly when determining whether to start or finish facial eczema control programmes.
Topics: Animals; Cattle; Cattle Diseases; Colony Count, Microbial; Dairying; Eczema; Mitosporic Fungi; New Zealand; Poaceae; Spores, Fungal; Sporidesmins
PubMed: 28273429
DOI: 10.1080/00480169.2017.1303794 -
Journal of Dairy Science Jul 2023The objectives of this study were to evaluate the abundance and viability of leukocytes, the abundance of microRNA, and the activity of the complement pathway in (1)...
The objectives of this study were to evaluate the abundance and viability of leukocytes, the abundance of microRNA, and the activity of the complement pathway in (1) colostrum following heat-treatment or freezing, and (2) colostrum, transition milk, and mature milk. In experiment 1, composite colostrum samples were harvested from individual cows (n = 14) on a commercial dairy farm in NY and split into 3 aliquots using single-use colostrum bags. One aliquot was immediately cooled on ice following harvest (RAW) and stored at 4°C overnight, one was heat-treated for 60 min at 60°C (HT) before being cooled on ice and stored at 4°C overnight, and one was frozen at -20°C overnight (FR). The following morning, all samples were warmed to 40°C before further processing. In experiment 2, cows were sampled in a longitudinal study where composite samples were collected from colostrum (first milking, n = 23), transition milk (3 to 4 d postpartum, n = 13), and mature milk (6 to 7 d postpartum, n = 13). In both experiments colostrum was harvested from the first milking within 8 h of calving and samples were processed within 14 h of collection. Colostral leukocytes were isolated before viability was determined by trypan blue exclusion and manual differential cell counts were performed. Extracellular vesicles were isolated from whey by ultracentrifugation to isolate and quantify microRNA. Activity of the alternative complement pathway was determined in casein-depleted whey by semi-solid phase hemolysis assay. Somatic cell counts were determined for all raw samples. Macrophages and neutrophils made up the greatest proportion of leukocytes in colostrum followed by lymphocytes. Lymphocyte proportion increased as colostrum transitioned to mature milk, but overall somatic cell numbers declined concurrently. Viable cells were not isolated from HT or FR samples. Abundance of microRNA isolated from transition and mature milk was decreased compared with colostrum, did not differ between HT and RAW, but was increased in FR compared with RAW. Alternative complement pathway activity was decreased in HT, but not FR compared with RAW, and was not measurable in transition or mature milk. Postharvest heat-treatment and freezing of colostrum eliminated viable colostral leukocytes and affected microRNA abundance and complement activity. Leukocyte proportions, microRNA abundance, and complement activity changed as colostrum transitioned to mature milk. Although there were clear changes in the colostral components under study in relation to treatment and transition to mature milk, the biological significance of the described treatment effects and temporal changes were not investigated here.
Topics: Pregnancy; Female; Cattle; Animals; Milk; Colostrum; Hot Temperature; MicroRNAs; Freezing; Ice; Longitudinal Studies; Leukocytes; Lactation
PubMed: 37164855
DOI: 10.3168/jds.2022-22876 -
Biopreservation and Biobanking Aug 2023The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the...
The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the extender before cryopreservation. In a preliminary experiment, different levels of BSP were supplemented (1, 3, 5, 7, and 9% v/v) in egg yolk (7.5% egg yolk)-tris (EYT) extender and used for cryopreservation of Pantja buck semen. Results in terms of motility, viability, plasma membrane integrity, acrosome integrity, and lipid peroxidation showed that 5% BSP was suitable for maintaining Pantja buck semen quality during cryopreservation. In the final experiment, pooled semen from four Pantja bucks was split into three aliquots (I, II, and III). Aliquot I was directly diluted in EYT extender and grouped as the control (C); aliquot II and III were washed separately with TALP solution and diluted as D1 (Washed semen with EYT extender) and D2 (Washed semen with EYT extender containing 5% BSP), respectively. Seminal attributes (sperm individual motility, viability, plasma membrane integrity, acrosome integrity, and total morphological abnormalities) were assessed at the postdilution, postequilibration, and post-thawing stages. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) concentration, and glutathione peroxidase (GSH-Px) activity were measured at post-thaw. Washed semen significantly improved ( < 0.05) seminal parameters at post-thaw compared with unwashed semen (control). A significant difference ( < 0.05) was observed in seminal attributes between freezing stages and between dilution groups. Significantly higher ( < 0.05) post-thaw sperm motility, viability, plasma membrane integrity, acrosome integrity, and GSH-Px activity, and significantly lower ( < 0.05) MDA concentration and extracellular release of enzymes (ALT, AST) were observed in group D2 compared with control and D1. The results of the present study demonstrated that cryopreservation of washed Pantja buck semen diluted with 5% BSP-supplemented EYT extender can improve post-thaw semen quality.
Topics: Male; Animals; Cattle; Semen; Spermatozoa; Semen Analysis; Egg Yolk; Sperm Motility; Cryoprotective Agents; Semen Preservation; Acrosome; Cryopreservation; Antioxidants; Dietary Supplements
PubMed: 35856825
DOI: 10.1089/bio.2022.0013 -
Applied Radiation and Isotopes :... Mar 2022Thermoluminescence (TL) dating is one of the most significant chronological tools used in Quaternary research. However, for changes in the characteristics of quartz, the... (Review)
Review
Thermoluminescence (TL) dating is one of the most significant chronological tools used in Quaternary research. However, for changes in the characteristics of quartz, the larger deviation is still a problem in TL dating, especially with the single-aliquot regeneration-does (SAR) procedure. In the SAR-TL protocol, changes in the characteristics of quartz inevitably cause a shift in the TL peak position and a reduction in the sensitivity of the TL peak during repetitive thermal treatment. In this paper, we studied the optimal TL parameters to minimize the effect of the above problems for TL dating. Based on the optimization experiment combining OSL and TL measurements, the optimal preheat temperature was found to be 300 °C for both silt-sized grains and sand-sized grains, which eliminates the remainder of the 325 °C TL signals and inhibits the 375 °C TL peak position shift. Referring to the test does in SAR-OSL dating protocol, the optimal test doses, 200 Gy and 250 Gy for the silt-sized grains and sand-sized grains respectively, were determined to correct the reduction in TL sensitivity, and they were added to improve the SAR-TL protocol. The improved SAR-TL protocol with the optimal measurement parameters, which we called the accurate-parametric SAR-TL protocol, improves the accuracy of quartz TL dating and expands the range of accurate TL dating. For the experimental doses of 400 Gy and 700 Gy, the relative error of D obtained by the accurate-parametric SAR-TL protocol was less than ±5.5% for both silt-sized grains and sand-sized grains. In addition, we discussed the application conditions of the accurate-parametric SAR-TL protocol and the method that obtains the same level of thermal lag for different luminescence measurement equipment.
PubMed: 34968882
DOI: 10.1016/j.apradiso.2021.110072 -
Journal of Oral and Maxillofacial... 2022The micro-flora of oral cavity is a myriad of micro-organism. Any infection of oral cavity leads to diseased condition which is a transitional transformation of the...
Detection and comparison of prevalence of through culture and Real Time-polymerase chain reaction in subgingival plaque samples of chronic periodontitis and healthy individuals.
INTRODUCTION
The micro-flora of oral cavity is a myriad of micro-organism. Any infection of oral cavity leads to diseased condition which is a transitional transformation of the micro-organism in a specific paradigm depending upon the diseased condition. Periodontitis is one of the predominant chronic diseases which is a multifactorial infection. is a key etiological agent in causing periodontitis. To study the predominance of these bacteria in the diseased condition is important to detect, quantify and to find its efficacy by comparing different methods for identification.
AIM AND OBJECTIVES
The aim of the study is to determine the prevalence of by anerobic culture and by real-time polymerase chain reaction (PCR) from subgingival plaque samples of chronic periodontitis and healthy individual and to compare efficacy of two methods.
MATERIALS AND METHODS
A total of 400 subjects were considered, and subgingival plaque was collected using paper points. Individual were equally divided into two groups: chronic periodontitis (200) and healthy individuals (200). Each plaque sample collected was divided into two aliquots of which the first aliquot was subjected for anerobic culture to isolate . Phenotypical identification was done morphologically and biochemically further quantification of was done by colony-forming unit. The second aliquot was subjected for DNA extraction and real-time PCR was conducted to detect and quantify using specific primer.
RESULTS
Out of 400 samples, 73% showed detection of by culture method and through reverse transcription-PCR (RT-PCR), the detection was 75%. Individual detection of by culture in chronic periodontitis was 89.5% and 54.4% in healthy individuals, while detection by RT-PCR was found to be 91.5% in chronic periodontitis and 58% in healthy individuals. However, comparison between two techniques in detection of was statistically insignificant.
CONCLUSION
When we compared RT-PCR with culture RT-PCR showed higher positivity. RT-PCR is more sensitive and requires less time to detect. However, in the present study, culture also showed good positivity, suggesting proper dilution and with extended incubation, the specificity of culture can be improved to a great extent.
PubMed: 35968159
DOI: 10.4103/jomfp.jomfp_163_21 -
Transfusion Aug 2022Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to...
BACKGROUND
Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to indicate that both the preparation and storage of small-dose RCCs does not alter in vitro red cell quality. The present study seeks to provide data to support this practice.
MATERIALS AND METHODS
To evaluate quality of stored small aliquots, six ABO/Rh matched leukoreduced citrate phosphate-dextrose/saline-adenine-glucose-mannitol (LR CPD/SAGM) RCCs were pooled and split into 30 ml aliquots, 80 ml aliquots, and a standard 290 ml unit, with testing performed for up to 43 days post-collection. To evaluate the impact of irradiation on small-dose RCC preparation, a total of 48 independent LR CPD/SAGM RCCs were used (non-irradiated: n = 24; irradiated: n = 24). Aliquoting with/without irradiation was performed within 7 days of collection and baseline testing was performed within 24 h of aliquot production.
RESULTS
Limited variability in hemolysis, mean cell volume, and extracellular potassium concentrations were seen between the different aliquot sizes throughout the 43-day storage period. Aliquot production did not accentuate damage based on any of these tested parameters in both the non-irradiated and irradiated subsets. A significant increase was seen in the potassium concentrations in the irradiated parent and aliquot samples relative to their non-irradiated counterparts.
CONCLUSIONS
Non-irradiated small-aliquot dose RCCs meet in vitro quality criteria required for safe transfusion throughout the 42-day storage period. The same can be said for aliquots derived from irradiated units and tested within 24 h of aliquot production.
Topics: Blood Preservation; Carcinoma, Renal Cell; Child; Erythrocytes; Gamma Rays; Hemolysis; Humans; Infant, Newborn; Kidney Neoplasms; Potassium; Time Factors
PubMed: 35869790
DOI: 10.1111/trf.17027 -
Journal of the Formosan Medical... Feb 2019Although great interest has been displayed by researchers in the contribution of gut microbiota to human health, there is still no standard protocol with consensus to... (Review)
Review
Although great interest has been displayed by researchers in the contribution of gut microbiota to human health, there is still no standard protocol with consensus to guarantee the sample quality of metagenomic analysis. Here we reviewed existing methodology studies and present suggestions for optimizing research pipeline from fecal sample collection to DNA extraction. First, we discuss strategies of clinical metadata collection as common confounders for microbiome research. Second, we propose general principles for freshly collected fecal sample and its storage and share a DIY stool collection kit protocol based on the manual procedure of Human Microbiome Project (HMP). Third, we provide a useful information of collection kit with DNA stabilization buffers and compare their pros and cons for multi-omic study. Fourth, we offer technical strategies as well as information of novel tools for sample aliquoting before long-term storage. Fifth, we discuss the substantial impact of different DNA extraction protocols on technical variations of metagenomic analysis. And lastly, we point out the limitation of current methods and the unmet needs for better quality control of metagenomic analysis. We hope the information provided here will help investigators in this exciting field to advance their studies while avoiding experimental artifacts.
Topics: DNA; Feces; Gastrointestinal Microbiome; Humans; Metagenomics; Reagent Kits, Diagnostic; Sequence Analysis, DNA; Specimen Handling
PubMed: 29490879
DOI: 10.1016/j.jfma.2018.02.005