Dynamics of bacteriologic and cytologic changes in the uterus of postpartum dairy cows

The objectives of this study were to characterize clinical, intrauterine, bacteriologic and cytologic changes during the first month after parturition in healthy dairy cows and in cows with subclinical endometritis (SE) or clinical endometritis (CE). Furthermore, risk factors related to clinical bacteriologic and cytologic findings were determined. A total of 170 calvings were enrolled, and intrauterine samples were collected on Days 0, 3, 9, 15, 21, and 28 postpartum using the cytobrush technique. The presence of Escherichia coli and Trueperella pyogenes was determined by Fourier transform infrared spectroscopy. The cows were categorized according to their uterine health status (UHS) on Day 21 as healthy (clear or absent vaginal discharge and <5% polymorphonuclear cells [PMN] in the cytologic sample), SE (clear or absent vaginal discharge and ≥5% PMN), or CE (vaginal mucus containing any signs of pus). The prevalence of SE and CE on Day 21 was 27.9% and 58.4%, respectively. Generally, samples from cows with SE and CE showed a greater bacterial growth density (BGD) than those from healthy cows. The BGD tended to be affected by the interaction of time by UHS (P = 0.057). Differences between healthy, SE, and CE cows were found from Day 3 to the last sampling day. Furthermore, the percentage of PMN differed between healthy, SE, and CE cows and was affected by time in a cubic way (decrease/increase/decrease). Overall, E coli was found in 25.4% of the samples, and T pyogenes was identified in 30.2% of the samples. The risk for CE was increased by BGD and the presence of T pyogenes. Conversely, the presence of E coli had no effect on the risk of CE or the risk of SE. The risk for an infection with T pyogenes was greater in the first-parity cows and in cows with assisted calving. In conclusion, changes in BGD and proportion of PMN varied with the UHS (healthy, SE, and CE), which was affected by the presence of T pyogenes but not E coli.     

Impact of endometriosis on embryo quality and endometrial receptivity in women undergoing assisted reproductive technology

ART is an important treatment method for infertile patients with endometriosis. However, the effects of endometriosis on embryo quality and endometrial receptivity remain unclear. Thus, we aimed to simultaneously investigate the impact of endometriosis and its stage on embryo quality and endometrial receptivity in women undergoing ART. We retrospectively analyzed the data from patients with and without endometriosis who underwent oocyte retrieval and/or high-quality embryos transfer between July 2015 and December 2020, including 1312 IVF cycles and 608 IVF or frozen-thawed embryo transfer (FET) cycles, respectively. The endometriosis group had a lower percentage of good cleavage-stage embryos and fertilization rates than those in the control group (p = 0.038 and 0.008, respectively). The number of retrieved oocytes, MII oocytes, cleavage, blastocysts, and blastulation rates was comparable between two groups. We found no significant difference in clinical pregnancy, implantation, live birth, miscarriage, or multiple pregnancy rates between the two groups among patients who transferred high-quality embryos. Stratification analysis showed that patients with stage III-IV endometriosis had fewer retrieved oocytes than those with stage I-II endometriosis (p = 0.012) and marginally fewer retrieved oocytes than the control group (p = 0.051). The stage I-II group had the lowest percentage of good cleavage-stage embryos, which was significantly lower than that of the control group (p = 0.043). In FET cycles, patients with stage III-IV endometriosis had a higher miscarriage rate than those in the control group (p = 0.023). Our results suggest that endometriosis does not alter endometrial receptivity but affects embryo quality, oocyte fertilization ability, and ovarian response.     

Comparaison de différentes méthodes de segmentation en TEP/TDM pour le ciblage en radiothérapie des carcinomes épidermoïdes des VADS

IntroductionTo assess interobserver variability for biological target volume (BTV) delineation and to compare the reproductibility of different semiautomatic segmentation methods in pretreatment 18-fluorodeoxyglucose positron emission tomography (PET/CT) of head and neck squamous cell carcinoma (HNSCC).Patients and methodsPatients with histologically proved HNSCC referred to the nuclear medicine service in Brest for pretreatment PET/CT were prospectively included from February 2009 to June 2010. Three nuclear medicine physicians (two specialized in oncology) delineated manually and independently BTV on each primary tumor. Four semiautomatic segmentation methods have been studied; three using a fixed threshold and one applying an adaptive threshold based on the signal-to-background ratio (Daisne). The variability between κ observers and/or methods has been assessed. The concordance between the various BTV intersections and unions has been also assessed.ResultsThirty patients (29M; 1F) were included. The primary site location was oropharynx in six patients, oral cavity in 10 patients, hypopharynx in five patients and larynx in nine patients. A statistically significant global interobserver variability (P = 0.01) was showed, but without statistically difference between the two experienced oncologists (P = 0.15). The maximal concordance of the two experienced observers with the semiautomatic methods was found for the Daisne method (CI = 61.5%; κ = 0.68), expressing a good agreement according to the Landis and Koch criteria, better than with the segmentation method using a fixed threshold with 40% of maximal signal intensity (CI = 52.1%; κ = 0.53).ConclusionOur results suggest the feasibility of achieving HNSCC BTV delineation by PET/CT using semiautomatic methods, in particular those which apply an adaptative threshold but under the supervision of an experienced operator.     

Segmentation d’images de lésions cérébrales primitives en TEP FET : influence du volume de travail

Purpose(1) Evaluate the reproducibility of segmentation methods depending on the preselection region for tumour volume determination on ¹⁸F-fluoro-ethyl-tyrosine (FET) PET. (2) Evaluate the intra and inter-operator reproducibility of the manual delineation. (3) Compare this delineation with the segmentation methods.Materials and methodsEighteen FET PET of patients with glioblastoma were analysed. Preselection regions were determined prior to any segmentation. Two physicians delineated the tumour volume manually. The tumour volume was also delineated with a threshold method (40 and 70% of SUV_max), and a random walk based method. Pearson coefficient (r) (P < 0.05 for r > 0.468) and Jaccard indices (JI) were used to compare the volumes.ResultsManual delineation was reproducible with r = 0.97 and IJ = 0.65 for intra-operator, and r = 0.76 and IJ = 0.45 for inter-operator reproducibility. The preselection regions for a given lesion were different and the segmentation varied with the preselection region: r = 0.55 JI = 0.58; r = 0.85 JI = 0.83; r = 0.70 JI = 0.39 respectively for the threshold of 40%, 70% and the random walk. The segmentation differed form de manual delineation with r = 0.37 and JI = 0.16; r = 0.54 and JI = 0.42; r = 0.43 and JI = 0.37 respectively for the threshold of 40%, 70% and the random walk.ConclusionThe reproducibility of the segmentation methods depends extensively on the preselection region. The intra-operator reproducibility of cerebral lesion delineation on FET PET is satisfactory. The inter-operator reproducibility could be improved.     

Évaluation de l’exactitude de latéralisation du foyer épileptogène par la tomographie par émission de positons au 18f-fluorodésoxyglucose dans le bilan préopératoire de l’épilepsie temporale : analyse visuelle simple et par index d’asymétrie inter-hémisphérique par soustraction d’images,analyse quantitative par statistical parametric mapping

IntroductionInter-ictal 18F-2-fluoro-deoxy-D-glucose-positron emission tomography (FDG-PET) plays a key role for the preoperative evaluation of patients with pharmacoresistant temporal lobe epilepsy. PET images are usually analyzed visually, a way that is reported to provide a high diagnostic value but that remains subjective, depending on the expertise and experience of the observer. By contrast, the voxel-based quantitative analyses, such as statistical parametric mapping (SPM), are objective and therefore, observer independent methods of analyses. In this study, the accuracy of the analyses of brain FDG-PET images to lateralize the temporal lobe epileptogenic zone was compared between: (1) a conventional visual method, (2) a quantitative SPM analysis, and (3) a visual analysis of inter-hemispheric asymmetry (IHA) obtained after images substraction.Materials and methodsFDG-PET scans of 31 patients presenting a severe temporal epilepsy and whom the temporal foci had been accurately lateralized (successful subsequent surgical treatment) were retrospectively analysed by (1) a consensual visual analysis from two experienced observers; (2) SPM analysis with voxel-wise comparisons of FDG-PET images of patients with those of age-matched healthy controls, using various statistical threshold (P) and cluster (k) values; and (3) visual assessment by the two same observers of images obtained for assessing the IHA. For this purpose, a flipped image was initially obtained by reversing in the left-right direction the FDG-PET images, which had been previously spacially normalized with the SPM template. Then, flipped and non-flipped images were substracted.ResultsThe temporal hypometabolic area was accurately identified: (1) by the conventional visual analysis in 87 % of patients and with a satisfactory interobserver reproducibility (interobserver Cohen's coefficient = 0.79); (2) by SPM analysis, in 90 % of patients (when using optimal thresholds of 0.01 for P value and of 50 voxels (400 mm³) for k value); and (3) with the visual analysis of IHA in 97 % of patients with an excellent interobserver reproductibility (interobserver Cohen's coefficient = 1).ConclusionIn patients presenting severe temporal epilepsy, visual assessment of FDG-PET images from IHA seems more accurate for lateralizing the epileptogenic temporal areas when compared with either conventional visual or quantitative SPM analyses. Moreover, this method is very easy to use in clinical practice, contrary to the quantitative method using SPM     

Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa

A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)–based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.     

Ki-67 as a prognostic marker in mantle cell lymphoma��consensus guidelines of the pathology panel of the European MCL Network

Mantle cell lymphoma (MCL) has a heterogeneous clinical course and is mainly an aggressive B cell non-Hodgkin lymphoma; however, there are some indolent cases The Ki-67 index, defined by the percentage of Ki-67-positive lymphoma cells on histopathological slides, has been shown to be a very powerful prognostic biomarker. The pathology panel of the European MCL Network evaluated methods to assess the Ki-67 index including stringent counting, digital image analysis, and estimation by eyeballing. Counting of 2 × 500 lymphoma cells is the gold standard to assess the Ki-67 index since this value has been shown to predict survival in prospective randomized trials of the European MCL Network. Estimation by eyeballing and digital image analysis showed a poor concordance with the gold standard (concordance correlation coefficients [CCC] between 0.29 and 0.61 for eyeballing and CCC of 0.24 and 0.37 for two methods of digital image analysis, respectively). Counting a reduced number of lymphoma cells (2 × 100 cells) showed high interobserver agreement (CCC = 0.74). Pitfalls of the Ki-67 index are discussed and guidelines and recommendations for assessing the Ki-67 index in MCL are given.     

Is there any potential link among caspase-8, p-p38 MAPK and bcl-2 in clear cell renal cell carcinomas? A comparative immunohistochemical analysis with clinical connotations

Background

Management of endometrial precancerous lesions has been of much debate due to inconsistencies in their classification, natural history and histologic diagnosis. Endometrial hyperplasia constitutes a wide range of histomorphologic features associated with high intra and interobserver diagnostic variability. Although traditional microscopic diagnosis is by far the most applicable method and the gold standard for histomorphologic diagnosis, digitized image analysis has been used as a powerful adjunct to maximize the histologic data retrieval and to add some detailed objective criteria for correct diagnosis in difficult cases.

Methods

A series of 100 endometrial curettage specimens with diagnosis of endometrial hyperplasia or well differentiated adenocarcinoma were blindly reviewed by 5 pathologists; their intra and interobserver reproducibility determined and further compared to the objective morphometric data i.e. D-score and volume percent of stroma (VPS).

Results

The results were assessed using the weighted kappa statistics. Mean intraobserver kappa value was 0.8690 (99.44% agreement). Mean interobserver kappa values by diagnostic category were: simple hyperplasia without atypia: 0.7441; complex hyperplasia without atypia: 0.3379; atypical hyperplasia: 0.3473, and well-differentiated endometrioid carcinoma: 0.6428; with a kappa value of 0.5372 for all cases combined. Interobserver agreement was in substantial rate for simple hyperplasia (SH) and well differentiated adenocarcinoma (WDA) but was in fair limit for complex hyperplasia (CH) and atypical hyperplasia (AH). Intraobserver agreement was almost perfect. The specimens were divided in two groups according to the computerized morphometric analysis: Endometrial Hyperplasia (EH) ( D Score ≥ 1 or VPS ≥ 55%) and Endometrial Intraepithelial Neoplasia (EIN) (D-Score < 1 or VPS < 55%). Morphometric findings were closely compatible with routine WHO classification made by one expert pathologist; however; diagnosis of (CH) and (AH) made by other pathologists were not concordant with morphometric data.

Conclusion

It may be necessary to make some revisions in WHO classification for endometrial hyperplasia and precancerous lesions.     

High Reproducibility of Histological Characterization by Whole Virtual Slide Quantification; An Example Using Carotid Plaque Specimens

Objective

Tissue biobanks are an important source for discovery and validation studies aiming for new proteins that are causally related with disease development. There is an increasing demand for accurate and reproducible histological characterization, especially for subsequent analysis and interpretation of data in association studies. We assessed reproducibility of one semiquantative and two quantitative methods for histological tissue characterization. We introduce a new automated method for whole digital slide quantification. Carotid atherosclerotic plaques were used to test reproducibility.

Methods

50 atherosclerotic plaques that were obtained during carotid endarterectomy were analysed. For the semiquantitative analysis, 6 different plaque characteristics were scored in categories by two independent observers, and Cohen''s κ was used to test intra- and interobserver reproducibility. The computer-aided method (assessed by two independent observers) and automated method were tested on CD68 (for macrophages) and α smooth muscle actin (for smooth muscle cells) stainings. Agreement for these two methods (done on a continuous scale) was assessed by intraclass correlation coefficients (ICCs).

Results

For the semiquantitative analysis, κ values ranged from 0.55 to 0.69 for interobserver variability, and were slightly higher for intraobserver reproducibility in both observers. The computer-aided method yielded intra- and interobserver ICCs between 0.6 and 0.9. The new automated method performed most optimal regarding reproducibility, with ICCs ranging from 0.92 to 0.97.

Conclusions

The analysis of performance of three methods for histological slide characterization on carotid atherosclerotic plaques showed high precision and agreement in repeated measurements for the automated method for whole digital slide quantification. We suggest that this method can fulfill the need for reproducible histological quantification.     

Comparison between ambulatory measurement of effective thermal conductivity and laser Doppler flowmetry method to assess skin microcirculatory activity

The main objective of this paper was to assess the performance of the ambulatory device μHematron to measure indirectly skin blood flow relative to the well-established Laser Doppler flowmetry method. The μHematron device is dedicated to the non-invasive measurement of effective thermal conductivity of living tissues, based on the thermal clearance method. Its major advantage is its ambulatory functionality, as available methods for evaluation of microcirculatory activity are non-ambulatory methods. An experiment was conducted on ten healthy women exposed for one hour in three different thermal environments (22 °C, 25 °C and 30 °C). Skin microcirculatory activity was analyzed after an acclimatization period of 30 minutes. The time between each exposure was at least one hour. Performances of the μHematron device were assessed and a comparative study with a laser Doppler perfusion monitor (LDPM) was performed. Good correlation coefficients between the two devices (r = 0.71 at T1 = 22 °C, r = 0.77 at T2 = 25 °C and r = 0.83 at T3 = 30 °C) were obtained while the LDPM signal was filtered by a low pass filter (0.1 Hz). These results showed that continuous monitoring of effective thermal conductivity was possible in neutral and warm ambiences. Then, the μHematron device could be considered as a complementary tool to Doppler techniques for the investigation of skin blood flow, when ambulatory conditions are required.     

Effects of carbon source,polyethylene glycol and abscisic acid on secondary embryo induction and maturation in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) microspore-derived embryos

In this study, the effects of carbon sources, abscisic acid (ABA) either alone or in combination with polyethylene glycol (PEG) were evaluated on secondary embryo (SE) induction and maturation in rapeseed microspore-derived embryos (MDE) of cultivars Global, PF₇₀₄ and Option. Among various carbon sources tested (sucrose, glucose, fructose and sorbitol), the use of 0.3 M (300 mOsml⁻¹) glucose and 0.2 M (200 mOsml⁻¹) sorbitol in SE induction medium (for cultivars Global and PF₇₀₄) and sorbitol at 0.2 and 0.3 M (200 and 300 mOsml⁻¹, for cultivar Option), induced the highest secondary embryogenesis percentage (%SE). The highest number of SEs per each MDE (SE/MDE) was observed with 0.2 M (200 mOsml⁻¹) sorbitol in cultivar Global and with 0.3 M (300 mOsml⁻¹) glucose in cultivars PF₇₀₄ and Option. In another part of this study, the effect of different concentrations of ABA (0, 20, 40, 60, 80 and 100 μM) and of a combined use of ABA (0 and 40 μM) and PEG 4000 or PEG 6000 at 15 g l⁻¹ (3.75 and 2.5 mOsml⁻¹, respectively) was examined on induction and maturation of SEs. In the first experiment, the use of ABA in SE induction medium reduced the mean number of SE/MDE in the three studied cultivars, whereas use of 40–80 μM ABA in SE induction medium increased the percentage of mature SEs in each cultivar. The combined use of PEG with or without ABA also reduced the mean number of SE/MDE compared with control, but resulted in significant enhancement of the percentages of mature SEs for the three cultivars.     

Prevalence of bovine subclinical endometritis 4h after insemination and its effects on first service conception rate

The objective of this study was to investigate the prevalence of subclinical endometritis 4 h after AI and its effect on first service conception rate (FSCR) in dairy cows.A total of 201 Holstein-Friesian cows with no signs of clinical endometritis were examined 4 h after first AI for signs of subclinical endometritis. Endometrial samples were collected from the uterus using the cytobrush technique. The proportion of polymorphonuclear cells (PMN) in the cytological sample was used to characterize an inflammation of the endometrium. Cows were categorized into three groups according to the proportion of PMN in the sample. Cows with 0% PMN (n = 115) were assigned to group Zero, cows with >0-15% PMN (n = 59) to group Medium, and cows with >15% PMN (n = 27) to group High. Pregnancy diagnosis was performed between days 38-44 after AI by palpation of the uterus and its contents per rectum.The FSCR was significantly higher in group Medium than in groups Zero and High (57.6% vs. 39.1% and 29.6%). Statistical analysis revealed an interaction between parity and PMN group. Primiparous cows were at higher risk of being classified into group Medium than multiparous cows (OR = 2.27, P = 0.01). Primiparous cows in group Zero had lower odds of pregnancy after first AI than primiparous cows in group Medium (OR = 0.3, P = 0.02). A comparison with cows that were not examined for subclinical endometritis showed that the collection of endometrial samples itself had no effect on FSCR.     

Identification of novel loci influencing refractive error in East Asian populations using an extreme phenotype design

The global “myopia boom” has raised significant international concerns. Despite a higher myopia prevalence in Asia, previous large-scale genome-wide association studies (GWASs) were mostly based on European descendants. Here, we report a GWAS of spherical equivalent (SE) in 1852 Chinese Han individuals with extreme SE from Guangzhou (631 < ?6.00D and 574 > 0.00D) and Wenzhou (593 < ?6.00D and 54 > ?1.75D), followed by a replication study in two independent cohorts with totaling 3538 East Asian individuals. The discovery GWAS and meta-analysis identify three novel loci, which show genome-wide significant associations with SE, including 1q25.2 FAM163A, 10p11.22 NRP1/PRAD3, and 10p11.21 ANKRD30A/MTRNR2L7, together explaining 3.34% of SE variance. 10p11.21 is successfully replicated. The allele frequencies of all three loci show significant differences between major continental groups (P < 0.001). The SE reducing (more myopic) allele of rs10913877 (1q25.2 FAM163A) demonstrates the highest frequency in East Asians and much lower frequencies in Europeans and Africans (EAS = 0.60, EUR = 0.20, and AFR = 0.18). The gene-based analysis additionally identifies three novel genes associated with SE, including EI24, LHX5, and ARPP19. These results provide new insights into myopia pathogenesis and indicate the role of genetic heterogeneity in myopia epidemiology among different ethnicities.     

Interobserver reproducibility of immunohistochemical HER-2/neu evaluation in human breast cancer: the real-world experience

In spite of the large number of studies on technical problems affecting the interlaboratory reproducibility of IHC HER-2/neu determination, only little is known about factors limiting the intra- and interobserver reproducibility in the actual practice of HER-2/neu expression analysis. The aim of the present INQAT study was to evaluate the intra- and inter-observer reproducibility of IHC HER-2 analysis among pathologists routinely working in Italian laboratories. Twenty immunostained slides were distributed to 12 pathologists who had to report, for each slide, the semiquantitative analysis of the percentage of immunopositive cells and the qualitative evaluation of the intensity of membrane staining. The intra- and interobserver reproducibility as well as the reproducibility between each laboratory and the reference values were quantified adopting an approach based on computation of the weighted kappa statistic (Kw). Additionally, in order to evaluate the contribution of each category to the overall agreement, the kappa category-specific statistics (Kcs) were estimated for both classification criteria by jointly considering all the participating laboratories. The intraobserver analyses showed a satisfactory level of reproducibility for both the percentage of positive cells (median Kw, 0.94; range: 0.80-0.96) and membrane staining (median Kw, 0.86; range: 0.78-0.96). Similarly, a fairly good level of reproducibility for the percentage of cells (median Kw, 0.89; range, 0.73-0.96) and the intensity of membrane staining (median Kw, 0.84; range, 0.72-0.92) were observed from comparisons with reference values. When all possible pairwise comparisons were performed, a satisfactory level of interobserver reproducibility was found for most laboratories. Kw values varied between 0.51 and 0.98 (median Kw, 0.80) and between 0.61 and 0.94 (median Kw, 0.81) for semiquantitative and qualitative measurements, respectively. Analysis of the contribution of the extreme categories to the overall agreement showed a substantial or almost perfect agreement for both classification criteria. Conversely, the contribution of intermediate categories appeared to be scarce or slight for the percentage of immunostained cells and slight or fair for the intensity of membrane staining. We conclude that, overall, the interobserver reproducibility in IHC analysis of HER-2/neu expression is satisfactory, although classification of the intermediate categories is problematic, both with regard to the percentage of immunostained cells and the intensity of membrane staining.     

Epigallocatechin-3-gallate (EGCG) addition as an antioxidant in a cryo-diluent media improves microscopic parameters,and fertility potential,and alleviates oxidative stress parameters of buffalo spermatozoa

The disparity between the endogenous antioxidants concentration and free radicals in spermatozoa results in reactive oxygen species (ROS) generation. In this prospect, epigallocatechin-3-gallate (EGCG) preserves vigorous antioxidant features. Current study explored the influence of EGCG in a cryo-diluent media on microscopic parameters, oxidative stress parameters, and fertility potential of buffalo spermatozoa during cryopreservation. Concisely, collected semen from three donor bulls for four times were then evaluated for volume, motility, concentrations and then dilution in a cryo-diluent media with different concentrations of EGCG (EGCG-0 = control; EGCG-50 = 50 μM, EGCG-100 = 100 μM, EGCG-200 = 200 μM, and EGCG-300 = 300 μM) at 37 °C, cooled to 4 °C in 2 h, equilibrated for 4 h at 4 °C, and cryopreserved. At post-thawing, Computer-Assisted Sperm motion Analysis motilities (total and progressive, %) and rapid velocity (%), plasma membrane functionality, supravital plasma membrane integrity, and mitochondrial potential (%) were found higher (P < 0.05) in EGCG-200, and EGCG-300 than control, whereas average-path, straight-line, and curved-linear velocities (μm/sec), and acrosome integrity (%) were recorded higher in EGCG-300 than control. Further, comet length (μm), and tail length (μm), LPO (lipid peroxidation, μM/mL), and apoptosis-like changes (%) in spermatozoa were significantly decreased in EGCG-300 than control. Seminal plasma antioxidant enzymes activities (glutathione peroxidase, U/mL, and superoxide dismutase, U/mL) were increased with EGCG-300 than control. Moreover, EGCG-300 addition in a cryo-diluent media improves the fertility potential (%) of buffalo spermatozoa. In a nutshell, the inclusion of EGCG-300 in a cryo-diluent media enhances post-thaw microscopic parameters, and fertility potential, whereas decreases oxidative stress parameters in buffalo spermatozoa.     

Measuring the sero-prevalence of Leishmania donovani induced cutaneous leishmaniasis: A method comparison study

An in-house enzyme-linked immunosorbent assay (ELISA) based on crude antigen of Leishmania reported a high sero-prevalence (82.0%) in Leishmania donovani induced cutaneous leishmaniasis (CL) in Sri Lanka. ELISA was further compared with established serological tools to identify a suitable point of care diagnostic tool. Sero-prevalence of 100 CL samples were analyzed using in-house ELISA, Indian dipstick test and rK39 strip test. Results obtained were further compared with direct agglutination test (DAT) for 40 CL. Test performance was evaluated using Kappa index value. Clinico-epidemiological characteristics of patients were analyzed using SPSSv25.0. Cost analysis of tests was carried out. ELISA showed a high sero-positivity of 81.0% (n = 81/100) while DAT (57.5%,n = 23/40), Indian dipstick test (22.0%,n = 22/100) and rK39 test (15.0%,n = 15/100) showed a comparatively less sero-positivity. According to Kappa index values, there were no perfect agreement between tests. Among ELISA positive patients (n = 81/100), DAT, Indian dipstick test and rK39 demonstrated sero-positivity rates of 61.3% (n = 19/31), 25.9% (n = 21/81) and 16.0% (n = 13/81) respectively. Among ELISA negative patients (n = 19/100), three assays demonstrated sero-positivity rates of 44.4% (n = 4/9), 5.3% (n = 1/9) and 10.5% (n = 2/19) respectively. DAT can be used as an alternative test when ELISA is not available or negative. Clinico-epidemiological profiles of patients that showed sero-positivity by each assay were different. Cost per patient was approximately 5.5 USD for DAT and 3.0 USD for each other tests. Established serological tests demonstrated different and relatively lower detection rates. Species, strain and antigen heterogeneity, inconsistency in amount of used antigens, sera, antibody expression patterns and testing methodologies could be responsible. This study highlighted the importance of designing an in-house serological assay based on local parasite.     

Rapid quantitation of lipid in microalgae by time-domain nuclear magnetic resonance

A specific strain of Chlorella protothecoides has been studied in heterotrophic fermentation for increasing cell growth rate and lipid content for biodiesel production. For optimizing the process of fermentation to reduce costs of alga-based biodiesel production, rapid determination of lipid content in microalgal cells is critical. Nile Red (NR) staining and time-domain nuclear magnetic resonance (TD-NMR) have been investigated to quantitate the lipid content in C. protothecoides. Both methods were found feasible and simpler than gravimetric methods that are commonly employed. The TD-NMR method showed better agreement (R² = 0.9973) with the measured values from lipid extraction experiments than the NR staining method (R² = 0.9067). Additionally, the smaller standard deviations of the samples (≤ 0.36) analyzed by TD-NMR revealed that the method is accurate and reproducible. The application of TD-NMR for lipid quantitation in C. protothecoides opens up the possibility of determining lipid content in algal fermentation precisely and quickly.     

Risk factors for uterine diseases on small- and medium-sized dairy farms determined by clinical,bacteriological, and cytological examinations

The involution process of the postpartum bovine uterus is usually accompanied by invasion of various bacteria. The objectives of this study were to identify the relationship between the postpartum findings as risk factors for clinical endometritis (CE) and subclinical endometritis (SE). Furthermore, the effects of CE or SE on reproductive performance in small- and medium-sized dairy herds were investigated. A total of 400 cows were examined by vaginoscopy for CE at 20 to 30 days postpartum, and samples were collected for cytological examinations for SE and for bacteriology by cytobrush technique. The vaginoscopic and cytological examinations showed that 27.3% and 21.0% of the cows were found with CE and SE, respectively. The bacterial community analyses revealed a large variety of bacteria. Overall, bacteria from the order Actinomycetales, Lactobacillales, Bacillales, Burkholderiales, Caulobacteriales Enterobacteriales, Pasteurellales, and Pseudomonadales were detected, whereas in 39.5% of the samples no bacterial growth was detectable. The uterine pathogens Escherichia coli and Trueperella pyogenes were found in 16.8% and 13.0% of the samples cultivated under aerobic conditions. Other frequently isolated bacteria were Streptococcus spp. (31.3%), Staphylococcus spp. (20.0%), Corynebacterium spp. (16.5%), and Bacillus spp. (10.5%). The infection with T. pyogenes was the most important bacteriological risk factor for the occurrence of CE (odds ratio (OR) = 5.72; 95% CI = 3.07–10.83) and had a detrimental effect on the hazard of nonpregnancy by 200 days postpartum (hazard ratio = 1.66; 95% CI = 1.12–2.46). Calving assistance (OR = 1.79; 95% CI = 1.16–2.98) and farm (OR = 1.11; 95% CI = 1.02–1.20) were indicated as further risk factors for CE and SE. Effects of CE and SE on reproductive performance parameters could not be demonstrated.     

Impact of sublethal doses of Piper guineense (Schum and Thonn) on the biological parameters of Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) reared on different cowpea varieties

Cowpea varieties show a wide range of characteristics in their composition that can determine Callosobruchus maculatus (Fabricius) response and survival. Consequently, this work investigated the influence of sublethal doses of Piper guineense on C. maculatus raised on four cowpea varieties: ART-98–12, Niger White, Oloyin, and Ife Brown. Our results demonstrated that C. maculatus from different varieties, despite being reared under the same conditions, responded differently to sublethal doses of P. guineense. Cowpea beetles from ART-98–12 were the most susceptible while those reared on Ife Brown were the least susceptible. Across varieties, survival and reproductive parameters of C. maculatus differed. Furthermore, the ranking for the percentage reduction in the number of eggs laid by C. maculatus was ART-98–12 > Niger White > Oloyin > Ife Brown. According to the log-rank statistic test, the difference observed in C. maculatus’ survival can be explained by variety (log-rank statistic: χ2 [df = 3, N = 16] = 15, p = 0.004). Conversely, the survivability of the chrysomelid beetles reared on Niger white was significantly lower (p = 0.012) compared to the other varieties. This study highlights the role of cowpea variety in dictating the efficacy of a given botanical against C. maculatus.