Agreement and internal quality assurance of the Neubauer hemocytometer and Makler chamber for human sperm concentration determination.

OBJECTIVE: The Neubauer hemocytometer, as well as the Makler chamber, are devices commonly used in andrology laboratories. The present study aimed to verify if both methods yield comparable results, and whether they can be used interchangeably to determine sperm concentration. METHODS: Sperm and latex beads concentration measurements were performed with the Neubauer hemocytometer and the Makler chamber. Fixed and proportional biases were estimated, and the method agreement was determined by assessing sperm concentration results with the Bland and Altman plot. The Coefficient of Variation (CV) and relative bias were calculated as an index of precision and accuracy, respectively, by measuring latex beads target concentrations in both chambers. RESULTS: The Makler chamber systematically overestimated the Neubauer hemocytometer concentration measurements by a mean of -7.99%, with limits of agreement (LOA) between -41% to 25.61% (p<0.001). The fixed bias was found for concentration values inferior to 40 x 106/ml range (p<0.001), but not higher concentration results (p>0.05). Measurements with the Neubauer hemocytometer showed the greatest consistency in the study with the CV ranging from 3.01% to 6.67%; while the CV with the Makler chamber ranged from 8.46% to 25.64%. The relative bias for the Neubauer hemocytometer determinations varied from 0.12% to 8.40%, while for the Makler chamber varied from 7.6% to an overestimation of 38.0%. CONCLUSIONS: Measurements made with the Makler chamber demonstrated more variability and a higher degree of overestimation. The Makler chamber is a poor substitute to the Neubauer hemocytometer for evaluation of oligozoospermic samples, although both chambers render similar results for highly concentrated samples.

Combined effect of type and capture area of counting chamber and diluent on Holstein bull sperm kinematics.

The evaluation of sperm motion is crucial for processing of seminal doses for artificial insemination. Here, the combined effect of the type and capture area of three counting chambers, together with the type of diluent employed, on sperm motility was analysed. Ejaculates from thirteen Holstein bulls were used for sperm kinematic analysis with the ISAS® v1 CASA-Mot system, using two capillary-loaded counting chambers (Leja® and Cell-Vu® ) and one drop displacement chamber (Makler® ). Nine fixed positions were analysed per chamber type, considering central and lateral and three longitudinal fields. Independent of the diluent used, differences were found between the three chambers. Independent of the extender, no differences in x-axis were observed with Cell-Vu® , while using Leja® , some parameters showed lower values in the centre than in lateral areas. In both counting chambers, the lowest values were observed in the distal area. Results obtained with the two diluents were highly different with a very low correlation between them. In conclusion, the capture area inside the chambers leads to significant changes in sperm kinematic parameters and different dilution media introduce considerable differences in the motility patterns. It is necessary to optimise sampling methods and specific set-ups to be used with CASA-Mot technology.

Eficiência dos diluidores tris e Botu-Crio® sobre os parâmetros seminais de garanhões das raças quarto de milha e mangalarga marchador / Efficiency of tris extenders and Botu-Crio ® on seminal parameters in stallion breeds quarter horse and mangalarga marchador

The limiting factor in the cryopreservation process of equine semen is related to the species, as they present a great variability in the ejaculates characteristics after thawing. The aim of this study was to evaluate sperm viability after thawing, from Quarter Horse and Mangalarga Marchador stallions using two extenders (Botu-crio and Tris) in cryopreservation. To this end, we analyzed the physical characteristics of fresh semen, the sperm membrane functionality by the hypoosmotic swelling test (HOST), total motility and vigor by the Thermoresistance Test (TRT), progressive motility by a computerized system CASA (Computer-Assisted Semen Anlyses) and acrosomal membrane integrity and functionality of the mitochondria by epifluorescence microscopy. After thawing, the extender Botu-crio® better preserved motility, vigor and integrity of the plasma membrane. There was no significant difference between breeds for the thermoresistance test after twaing. Quarter Horse showed higher percentage of bigger defects in the sperm pathology analysis.(AU)

O principal fator a limitar o processo de criopreservação de sêmen equino está relacionado à própria espécie, por apresentar uma grande variabilidade nas características dos ejaculados pós-descongelação. O objetivo desta pesquisa foi avaliar a viabilidade espermática pós-descongelação, do sêmen de garanhões das raças Quarto de Milha e Mangalarga Marchador utilizando-se dois diluentes (Tris e Botu-crio®) na criopreservação. Para tanto, foram analisadas as características físicas do sêmen fresco, funcionalidade da membrana espermática pelo Teste Hiposmótico (HOST), motilidade total e vigor pelo Teste de Termoresistência (TTR), motilidade progressiva pelo sistema computadorizado CASA (Computer Assisted Sêmen Anlyses) e integridade da membrana espermática, do acrossoma e funcionalidade de mitocôndria por microscopia de epifluorescência. O diluente Botu-crio® preservou melhor os valores de motilidade e vigor e a integridade de membrana plasmática, pós-descongelamento. Não houve diferença significativa entre as raças quanto à motilidade total e vigor no sêmen fresco, bem como para a análise desses parâmetros no Teste de Termoresistência pós-descongelamento. Garanhões da raça Quarto de Milha apresentaram percentual maior de defeitos maiores na análise de patologia espermática.(AU)

Eficiência dos diluidores tris e Botu-Crio® sobre os parâmetros seminais de garanhões das raças quarto de milha e mangalarga marchador / Efficiency of tris extenders and Botu-Crio ® on seminal parameters in stallion breeds quarter horse and mangalarga marchador

The limiting factor in the cryopreservation process of equine semen is related to the species, as they present a great variability in the ejaculates characteristics after thawing. The aim of this study was to evaluate sperm viability after thawing, from Quarter Horse and Mangalarga Marchador stallions using two extenders (Botu-crio and Tris) in cryopreservation. To this end, we analyzed the physical characteristics of fresh semen, the sperm membrane functionality by the hypoosmotic swelling test (HOST), total motility and vigor by the Thermoresistance Test (TRT), progressive motility by a computerized system CASA (Computer-Assisted Semen Anlyses) and acrosomal membrane integrity and functionality of the mitochondria by epifluorescence microscopy. After thawing, the extender Botu-crio® better preserved motility, vigor and integrity of the plasma membrane. There was no significant difference between breeds for the thermoresistance test after twaing. Quarter Horse showed higher percentage of bigger defects in the sperm pathology analysis.

O principal fator a limitar o processo de criopreservação de sêmen equino está relacionado à própria espécie, por apresentar uma grande variabilidade nas características dos ejaculados pós-descongelação. O objetivo desta pesquisa foi avaliar a viabilidade espermática pós-descongelação, do sêmen de garanhões das raças Quarto de Milha e Mangalarga Marchador utilizando-se dois diluentes (Tris e Botu-crio®) na criopreservação. Para tanto, foram analisadas as características físicas do sêmen fresco, funcionalidade da membrana espermática pelo Teste Hiposmótico (HOST), motilidade total e vigor pelo Teste de Termoresistência (TTR), motilidade progressiva pelo sistema computadorizado CASA (Computer Assisted Sêmen Anlyses) e integridade da membrana espermática, do acrossoma e funcionalidade de mitocôndria por microscopia de epifluorescência. O diluente Botu-crio® preservou melhor os valores de motilidade e vigor e a integridade de membrana plasmática, pós-descongelamento. Não houve diferença significativa entre as raças quanto à motilidade total e vigor no sêmen fresco, bem como para a análise desses parâmetros no Teste de Termoresistência pós-descongelamento. Garanhões da raça Quarto de Milha apresentaram percentual maior de defeitos maiores na análise de patologia espermática.

Picomolar gradients of progesterone select functional human sperm even in subfertile samples.

More than 1 million infertility treatments are practiced around the world per year, but only 30% of the couples succeed in taking a baby home. Reproductive technology depends in part on sperm quality, which influences not only fertilization but also embryo development and implantation. In order to provide a better quality sperm subpopulation, innovative sperm selection techniques based on physiological sperm features are needed. Spermatozoa at an optimum state may be selected by following an increasing concentration gradient of picomolar progesterone, a steroid secreted by the cumulus cells at the time of ovulation. In this study we developed a method to recruit spermatozoa at the best functional state, based on sperm guidance toward progesterone. The sperm selection assay (SSA) consists of a device with two wells connected by a tube. One well was filled with the sperm suspension and the other with picomolar progesterone, which diffused inside the connecting tube as a gradient. The sperm quality after the SSA was analyzed in normal and subfertile semen samples. Several sperm parameters indicative of sperm physiological state were determined before and after the SSA: capacitation, DNA integrity and oxidative stress. After the SSA, the mean level of capacitated spermatozoa increased three times in normal and in subfertile samples. The level of sperm with intact DNA was significantly increased, while sperm oxidative stress was decreased after sperm selection. Interestingly, the exposure to a progesterone gradient stimulated the completion of capacitation in some spermatozoa that could not do it by themselves. Thus, the SSA supplies a sperm population enriched with spermatozoa at an optimum physiological state that may improve the assisted reproductive technology outcome.

Uso potencial da água de coco na tecnologia de sêmen / Potential use of coconut water in the semen technology

A produção brasileira de coco sempre foi de fundamental importância na vida e economia das populações do Nordeste do país. Sua utilização abrange as mais diversas áreas do conhecimento. O presente estudo teve por objetivo apresentar o potencial da água de coco em suas várias formas de utilização na área de biotecnologia da reprodução, como diluente seminal de diferentes espécies de animais em processos de conservação. Diversos são os trabalhos que demonstram a eficácia da água de coco na manutenção de características in vitro e in vivo da célula espermática de várias espécies, mantendo assim sua capacidade fecundante. Sua utilização como diluente é uma importante alternativa para o transporte de sêmen a curta distância e para a realização da inseminação artificial, além do seu fácil preparo e baixo custo. Com este trabalho, foi possível observar que a água de coco apresenta características bioquímicas necessárias a um diluente seminal e sua utilização pode aumentar a difusão dos programas de inseminação artificial em espécies animais de interesse econômico.

The Brazilian coconut production has a fundamental importance in the life and economy of the Northeast people of Brazil. Your use embraces the most several areas of the knowledge. This review had for objective to present the potential use of coconut water in various forms in the biotechnology of reproduction, as a sperm extender in the conservation process from various species. Several works demonstrate the coconut water effectiveness in the maintenance of the in vitro and in vivo characteristics of the spermatic cell in several species with the maintenance of your fertility capacity. Its utilization as a extender is an important alternative for the semen transport in small distances and your utilization in artificial insemination programs. In addition it is of easy prepare and low cost. With this work we observed that coconut water has the biochemical characteristics needed for a seminal extender and their use may increase the spread of artificial insemination programs in species of economic interest.

Uso potencial da água de coco na tecnologia de sêmen / Potential use of coconut water in the semen technology

A produção brasileira de coco sempre foi de fundamental importância na vida e economia das populações do Nordeste do país. Sua utilização abrange as mais diversas áreas do conhecimento. O presente estudo teve por objetivo apresentar o potencial da água de coco em suas várias formas de utilização na área de biotecnologia da reprodução, como diluente seminal de diferentes espécies de animais em processos de conservação. Diversos são os trabalhos que demonstram a eficácia da água de coco na manutenção de características in vitro e in vivo da célula espermática de várias espécies, mantendo assim sua capacidade fecundante. Sua utilização como diluente é uma importante alternativa para o transporte de sêmen a curta distância e para a realização da inseminação artificial, além do seu fácil preparo e baixo custo. Com este trabalho, foi possível observar que a água de coco apresenta características bioquímicas necessárias a um diluente seminal e sua utilização pode aumentar a difusão dos programas de inseminação artificial em espécies animais de interesse econômico.(AU)

The Brazilian coconut production has a fundamental importance in the life and economy of the Northeast people of Brazil. Your use embraces the most several areas of the knowledge. This review had for objective to present the potential use of coconut water in various forms in the biotechnology of reproduction, as a sperm extender in the conservation process from various species. Several works demonstrate the coconut water effectiveness in the maintenance of the in vitro and in vivo characteristics of the spermatic cell in several species with the maintenance of your fertility capacity. Its utilization as a extender is an important alternative for the semen transport in small distances and your utilization in artificial insemination programs. In addition it is of easy prepare and low cost. With this work we observed that coconut water has the biochemical characteristics needed for a seminal extender and their use may increase the spread of artificial insemination programs in species of economic interest.(AU)

Effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved Gyr bull semen evaluated by CASA and flow cytometry.

The objectives of the present study were to investigate the effects of three equilibration times (0, 2, and 4h) and two extenders (TRIS or Bioxcell) for cryopreservation of bull semen. Semen from 12 Gyr bulls was cryopreserved using an automated freezing machine. There were significant interactions between equilibration times and extenders for sperm motility and membrane integrity. The control treatment (0h equilibration) had the lowest values (P<0.05) for total (MOT) and progressive motilities (PROG), and percentage of sperm with intact plasma and acrosomal membranes (IPIA), with no significant differences between extenders. Extender TRIS had greater cryoprotective action than Bioxcell, with greater MOT, PROG, IPIA at 2 and 4h, as well as the lowest proportion of damaged plasma membrane (DPM, 72.2% vs. 85.8%) for all times. Equilibration for 4h yielded the most desirable (P<0.05) for MOT, PROG, and IPIA, and the least DPM percentage (86.5, 78.0, and 72.6% for 0, 2, and 4h, respectively). Overall, the combination of TRIS and 4h of equilibration was the most desirable semen cryopreservation method, with greatest MOT, PROG, and IPIA (TRIS-T4=26.8%; BIO-T4=18.3%) and the least DPM. In conclusion, based on objective analyses, equilibration during cryopreservation was essential for maintaining motility and integrity of sperm membranes; equilibration for 4h yielded the greatest sperm survival, independent of the extender used.