Padronização do Sperm Class Analyzer® (SCA®) para avaliação de espermatozoides equinos criopreservados / [Standardization of the Sperm Class Analyzer® (SCA®) for the evaluation of cryopreserved equine sperm]

Objetivou-se, no primeiro experimento, avaliar o efeito da velocidade de captura de imagens de 25Hz, 30Hz e 50Hz na cinética dos espermatozoides equinos criopreservados. Todas as velocidades mostraram-se adequadas para capturar o movimento espermático (P>0,05). No segundo experimento, objetivou-se avaliar o efeito da deposição de sêmen em lâmina sob lamínula, Leja®10 e 20, na cinética espermática. O uso de lâmina e lamínula foi superior às lejas para manter a LIN e o WOB (P<0,05). No terceiro experimento, objetivou-se avaliar o efeito das concentrações de 25, 50 e 100x106 na cinética espermática. As concentrações de 25 e 50 x106 foram superiores a 100x106 para preservar a LIN, a STR e a BCF e não afetar negativamente a motilidade (P<0,05). No quarto experimento, objetivou-se avaliar o efeito dos diluidores BotuCrio®, BotuSêmen®, TALP sperm e da solução fisiológica na cinética espermática. O BotuCrio® foi superior a todos os diluidores em preservar a BCF e os hiperativos (P<0,05). Conclui-se que o emprego da velocidade de captura entre 25 e 50Hz, a deposição do sêmen entre lâmina e lamínula e a rediluição em diluidor de congelação para atingir 25 a 50x106 de espermatozoides/mL são ideais para o SCA® avaliar, de forma fidedigna, o sêmen equino criopreservado.(AU)

The objective of the first experiment was to evaluate the effect of 25, 30 and 50Hz frame acquisition rate on equine cryopreserved sperm. All frame acquisition rates tested were adequate to capture the sperm movement (P>0.05). The aim of the second experiment was to evaluate the effect of chambers, slide-coverslip, Leja®10 and 20 on sperm movement. The use of slide-coverslip was superior to maintain LIN and WOB (P<0.05). The aim of the third experiment was to evaluate the effect of 25, 50 and 100x106 sperm/mL concentration on sperm movement. Concentrations of 25 and 50x106 sperm/mL were greater than 100x106 to preserve LIN, STR and BCF and did not adversely affect motility (P<0.05). The aim of the fourth experiment was to evaluate the effect of BotuCrio®, BotuSêmen®, TALP sperm and physiological solution on sperm movement. BotuCrio® was superior among other extenders in preserving BCF and hyperactive (P<0.05). It is concluded that the use of the frame acquisition rate between 25 and 50 Hz; the deposition of semen between slide and coverslip and new dilution in the freezing extender to 25-50x106 of sperm/mL is ideal to reliably evaluate cryopreserved equine semen by SCA®.(AU)

Internal quality control products for computer-assisted sperm analysis: Research and application / 中华男科学杂志

Objective@#To investigate the application of the self-made semen quality control (QC) product in internal QC of computer-assisted sperm analysis (CASA).@*METHODS@#CASA was calibrated with high- and low-concentration commercially available semen QC product and meanwhile 15 samples of self-made mixed semen QC product were placed in 75 cryotubes containing liquid nitrogen, followed by CASA of the concentration, motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), wobble (WOB) and straightness (STR) of the sperm using standard procedures and 50 days of continuous monitoring. The Makler counting plate was used to measure the concentration and motility of the self-made sperm.@*RESULTS@#The coefficients of variation (CV) of the commercially available semen QC product at high and low concentrations were 6.18% and 7.85%, respectively. CASA showed that the concentration of the self-made QC product was (25.97 ± 1.41) ×10⁶/ml, with a CV of 5.42%, and the sperm motility, VCL, VSL, VAP, LIN, WOB and STR were (22.15 ± 1.75)% (CV = 7.9%), (59.18 ± 2.05) μm/s (CV = 3.46%), (26.79 ± 1.2) μm/s (CV = 4.48%), (34.98 ± 1.4) μm/s (CV = 4.01%), 46.81 ± 1.55 (CV = 3.3%), 60.52 ± 1.3 (CV = 2.15%) and 76.46 ± 1.98 (CV = 2.59%), respectively. The concentration and motility of the self-made sperm detected with the Makler counting plate were (34.39 ± 2.37) ×10⁶/ml (CV = 6.89%) and (38.04 ± 1.69)% (CV = 4.44%), respectively. Levey-Jennings QC charts were plotted for the indicators using the means and standard deviation.@*CONCLUSIONS@#The self-made internal QC product by liquid nitrogen cryopreservation is feasible and effective for monitoring the accuracy and precision of CASA-derived sperm concentration and motion parameters, and it has a smaller CV than the commercially available QC product in measuring sperm concentration.

Hamilton-Thorne Integrated Visual Optical System Ⅰ (IVOSⅠ) versus IVOS Ⅱ for human sperm analysis / 中华男科学杂志

Objective@#To compare the computer-assisted sperm analysis (CASA) systems Hamilton-Thorne Integrated Visual Optical System Ⅰ (IVOSⅠ) and IVOS Ⅱ after verifying the performance of the latter so as to ensure the accuracy of the results of analysis.@*METHODS@#Based on the criteria established in the 5th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen (WHO 5th Ed), we compared the main semen parameters obtained from IVOSⅠ with those generated by IVOS Ⅱ, and examined the consistency between the results of the two sperm analyzers.@*RESULTS@#The linear relationship of the outlier test, bias estimation and scatter plot and the results of the outlier test of the two systems all met the requirements of comparison analysis and showed an obvious correlativity. The application scope of the results obtained from the apparatus indicated a reasonable value range, with r = 0.988 for sperm concentration, r = 0.975 for sperm progressive motility (PR), and r = 0.981 for total sperm motility. Evaluation of the acceptability of the predicted bias showed that the allowable total error (TEa) to be 6.67% with sperm concentration at 12 × 106 /ml and 2.34% with PR < 31%, their upper limit of the allowable error < 1/2. The results of IVOS Ⅱ conformed to the requirements of the WHO 5th Ed.@*CONCLUSIONS@#The main parameters derived from IVOSⅠ and IVOS Ⅱ are comparable and consistent, indicating that both can be used for the examination of semen samples.

Transcutaneous electrical acupoint stimulation for asthenozoospermia / 中华男科学杂志

Objective@#To study the effect of transcutaneous electrical acupoint stimulation (TEAS) in the treatment of asthenozoospermia.@*METHODS@#We randomly divided 72 asthenozoospermia patients into a 2 Hz TEAS (n = 29), a 100 Hz TEAS (n = 20), and a blank control group (n = 23), those in the former two groups treated by 30 minutes of TEAS at 2 Hz and 100 Hz respectively, applied to the acupoints of bilateral Shenshu, left Zusanli, and Guanyuan, once a day for 60 days, while those in the blank control group left untreated. Using computer-assisted sperm analysis (CASA), we examined sperm concentration and motility as well as the percentages of grade a and grade a+b sperm in different groups of the patients.@*RESULTS@#Compared with the baseline, 2 Hz TEAS significantly increased sperm motility (［12.76 ± 1.39］ vs ［18.89 ± 2.46］%, P<0.05) and the percentage of grade a+b sperm ( ［10.68 ± 1.22］ vs ［16.32 ± 2.10］%, P<0.05) in the asthenozoospermic patients, while 100 Hz TEAS improved not only sperm motility (［12.32 ± 2.21］ vs ［23.81 ± 3.42］%, P<0.01) and the percentage of grade a+b sperm (［10.45 ± 1.98］ vs ［20.25 ± 2.82 ］%, P<0.01), but also the percentage of grade a sperm (［6.44 ± 1.16］ vs ［13.31 ± 2.30］%, P<0.05). Moreover, in comparison with the blank control group, 2 Hz TEAS also remarkably increased sperm motility (［9.57 ± 1.60］ vs ［18.89 ± 2.46］%, P<0.05) and the percentage of grade a+b sperm (［7.81 ± 1.31］ vs ［16.32 ± 2.10］%, P<0.05) in the asthenozoosperma patients, while 100 Hz TEAS improved not only sperm motility (［9.57 ± 1.60］ vs ［23.81 ± 3.42］%, P<0.01) and the percentage of grade a+b sperm (［7.81 ± 1.31］ vs ［20.25 ± 2.82］%, P<0.01) but also the percentage of grade a sperm (［4.87 ± 1.01］ vs ［13.31 ± 2.30］%, P<0.01). Meanwhile, the rate of clinical effectiveness was significantly higher in the 100 Hz TEASthan in the blank control group either in intention-to-treat (ITT) analysis (100% vs 18.18%) orper-protocol (PP) analysis (90% vs 0%), and so was it than in the 2 Hz TEAS group based on the data of ITT (100% vs 33.33%).@*CONCLUSIONS@#Both 2 Hz and 100 Hz TEAS are effective for the treatment of asthenozoospermia by improving sperm motility and vitality.

Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men / 亚洲男科学杂志(英文版)

This study was designed to analyze the sperm kinematic and morphometric subpopulations in the different fractions of the ejaculate in normozoospermic men. Ejaculates from eight normozoospermic men were collected by masturbation in three fractions after 3-5 days of sexual abstinence. Analyses of sperm motility by computer-assisted sperm analysis (CASA-Mot), and of sperm morphometry by computer-assisted sperm morphometry analysis (CASA-Morph) using fluorescence were performed. Clustering and discriminant procedures were performed to identify sperm subpopulations in the kinematic and morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three kinematic subpopulations (slow with low ALH [35.6% of all motile spermatozoa], with circular trajectories [32.0%], and rapid with high ALH [32.4%]), and three morphometric subpopulations (large-round [33.9% of all spermatozoa], elongated [32.0%], and small [34.10%]). The distribution of kinematic sperm subpopulations was different among ejaculate fractions (P < 0.001), with higher percentages of spermatozoa exhibiting slow movements with low ALH in the second and third portions, and with a more homogeneous distribution of kinematic sperm subpopulations in the first portion. The distribution of morphometric sperm subpopulations was also different among ejaculate fractions (P < 0.001), with more elongated spermatozoa in the first, and of small spermatozoa in the third, portion. It is concluded that important variations in the distribution of kinematic and morphometric sperm subpopulations exist between ejaculate fractions, with possible functional implications.

Dois diluentes comerciais na criopreservação de espermatozoides do epidídimo de touros / Two commercial extenders for cryopreservation of epididymal bull sperm

A recuperação e a criopreservação de espermatozoides do epidídimo constituem alternativas viáveis para a preservação de material genético de animais valiosos. O objetivo deste estudo foi comparar o desempenho de dois diluentes comerciais Botu-Bov(r) (BB) e Bovimix(r) (BV), sobre a viabilidade pós-descongelação de espermatozoides do epidídimo de touros Tabapuã (Bos taurus indicus) pós-castração. Os espermatozoides foram colhidos da cauda de 20 epidídimos utilizando a técnica de fluxo retrógrado, centrifugados e diluídos com BB ou BV para posterior criopreservação a -196°C. Após a descongelação, as amostras foram avaliadas utilizando a análise computadorizada (CASA) e por análises microscópicas para a determinação da integridade de membranas plasmáticas, acrossomal e morfologia espermática. A avaliação estatística dos dados foi realizada pela análise de variância (ANOVA) com o pós-teste de comparações múltiplas de Tukey-Kramer, com nível de significância (P<0,05). Os resultados do movimento espermático avaliado pelo CASA, não diferiram para o diluente BB e BV. Também não foi observada diferença significativa entre os grupos no percentual de espermatozoides morfologicamente deformados, defeitos de acrossoma e espermatozoides com membrana plasmática íntegra após o descongelamento. Conclui-se que ambos os diluentes (BB e BV) são eficientes e podem ser utilizados na tecnologia do congelamento de espermatozoides colhidos da cauda do epidídimo de touros, não apresentando diferença na viabilidade espermática para os parâmetros estudados.

Recovery and cryopreservation of epididymal sperm is a viable alternative for preservation of genetically valuable animals. The aim of this study was to verify and to compare the effect of two commercial extenders for conventional semen on post-thawing viability of bovine epididymal sperm. For this purpose, the spermatozoa was recovered from the tail of 20 epididymis of Tabapuã bulls (Bos Taurus indicus) using retrograde flow method. After sperm recovery, the cells were centrifuged and divided for dilution with the diluents Botu-Bov(r) (BB) or Bovimix(r) (BV) for cryopreservation at -196°C. After thawing, all samples were evaluated using computer assisted sperm analysis (CASA), and by microscopic analysis for determination of integrity of plasma and acrossomal membrane and morphology. Statistical evaluation was performed by analysis of variance (ANOVA) with post-test for multiple comparisons, the Tukey-Kramer test, with significance level (P<0.05). The results of the sperm movement for diluent BB and BV evaluated with CASA, showed no difference for both (P>0.05). There was also no difference between the percentage of deformed sperm, acrosome defects and the sperm with intact plasma membrane after thawing with BB or BV. We conclude that both extenders (BB and BV) are efficient and can be used for freezing sperm collected from the epididymis of bulls, showing no difference for all the parameters studied.