Determining the relationship between bull sperm kinematic subpopulations and fluorescence groups using an integrated sperm quality analysis technique.

2017

The aim of the present study was to determine whether there is an association between the kinematic sperm subpopulations and fluorescent groups in bulls using a new fluorescent staining method that allows classification of spermatozoa into groups depending on their acrosomal and membrane integrity, as well as functional status, without inhibiting sperm motility. Cryopreserved semen samples from 10 Holstein bulls were used in the study. A multiparametric analysis of results obtained by the ISAS 3Fun kit (Proiser) was performed. The different fluorescent groups were detected and their motility characteristics evaluated using ISAS software. Clustering procedures using the kinematic data resul…

Morphometry and subpopulation structure of Holstein bull spermatozoa: variations in ejaculates and cryopreservation straws

2016

Artículo científico Sperm quality is evaluated for the calculation of sperm dosage in artificial reproductive programs. The most common parameter used is motility, but morphology has a higher potential as a predictor of genetic quality. Morphometry calculations from CASA‑Morph technology improve morphological evaluation and allow mathematical approaches to the problem. Semen from 28 Holstein bulls was collected by artificial vagina, and several ejaculates were studied. After general evaluation, samples were diluted, packaged in 0.25 ml straws, and stored in liquid nitrogen. Two straws per sample were thawed, and slides were processed and stained with Diff‑Quik. Samples were analyzed by a CA…

Effect of sperm concentration and storage temperature on goat spermatozoa during liquid storage

2020

The use of cooled semen is relatively common in goats. There are a number of advantages of cooled semen doses, including easier handling of artificial insemination (AI) doses, transport, more AI doses per ejaculate, and higher fertility rates in comparison with frozen AI doses. However, cooled semen has a short shelf life. The objective of this study was to examine the effect of temperature and sperm concentration on the in vitro sperm quality during liquid storage for 48 h, including sperm motility and kinetics, response to oxidation, mitochondrial membrane potential (MMP) and DNA fragmentation in goats. Three experiments were performed. In the first, the effects of liquid preservation of …

Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men

2016

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 kine…

CASA-Mot technology: how results are affected by the frame rate and counting chamber.

2017

For over 30 years, CASA-Mot technology has been used for kinematic analysis of sperm motility in different mammalian species, but insufficient attention has been paid to the technical limitations of commercial computer-aided sperm analysis (CASA) systems. Counting chamber type and frame rate are two of the most important aspects to be taken into account. Counting chambers can be disposable or reusable, with different depths. In human semen analysis, reusable chambers with a depth of 10 µm are the most frequently used, whereas for most farm animal species it is more common to use disposable chambers with a depth of 20 µm . The frame rate was previously limited by the hardware, although chang…

Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls

2021

The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested

A comparative study of the sperm nuclear morphometry in cattle, goat, sheep, and pigs using a new computer-assisted method (CASMA-F)

2012

This study was designed to compare the sperm nuclear morphometry of four species of domestic artiodactyls (cattle, sheep, goats, and pigs), using the newly developed automatic computer-assisted sperm morphometry analysis-F. The study was divided into two experiments. In the first experiment, samples from 20 males from each species were collected, diluted, and divided into four sample aliquots. The first was labeled directly with Hoechst 33342, and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying, and the other was fixed either with glutaraldehyde (GLUT), or with methanol, and afterward labeled with Hoechst. Digital images of t…

Effect of hypoosmotic swelling test and water test on the distribution of sperm subpopulations in bull

2016

Toward an integrative and predictive sperm quality analysis in Bos taurus

2017

There is a need to develop more integrative sperm quality analysis methods, enabling researchers to evaluate different parameters simultaneously cell by cell. In this work, we present a new multi-parametric fluorescent test able to discriminate different sperm subpopulations based on their labeling pattern and motility characteristics. Cryopreserved semen samples from 20 Holstein bulls were used in the study. Analyses of sperm motility using computer-assisted sperm analysis (CASA-mot), membrane integrity by acridine orange-propidium iodide combination and multi-parametric by the ISAS®3Fun kit, were performed. The new method allows a clear discrimination of sperm subpopulations based on memb…

Effect of different oxidative stress degrees generated by hydrogen peroxide on motility and DNA fragmentation of zebrafish (Danio rerio) spermatozoa.

2018

An increase in reactive oxygen species (ROS) or decrease in antioxidant barriers can provoke lipid peroxidation of the membranes or DNA damage of the spermatozoa. The aim of this work is to study the effect of the different degrees of oxidative stress generated by H2 O2 incubation on total motility, kinetics, and DNA fragmentation of zebrafish (Danio rerio) spermatozoa. For this process, experimental groups were incubated in 50 µM (Low; L) and 200 µM (High; H) H2 O2 , respectively, for 20 min at 4oC. Sperm motility parameters were obtained with a computer-assisted sperm analysis (CASA) system. Sperm DNA fragmentation (SDF) was assessed using the sperm chromatin dispersion test. Both low and…

Role of Antioxidants in Cooled Liquid Storage of Mammal Spermatozoa

2021

Cooled preservation of semen is usually associated with artificial insemination and genetic improvement programs in livestock species. Several studies have reported an increase in reactive oxidative species and a decrease in antioxidant substances and sperm quality parameters during long-term semen storage at refrigerated temperatures. The supplementation of antioxidants in extenders before refrigeration could reduce this detrimental effect. Various antioxidants have been tested, both enzymatic, such as superoxide dismutase and catalase, and non-enzymatic, such as reduced glutathione, vitamins E and C and melatonin. However, the problem of oxidative stress in semen storage has not been full…

Sperm Quality Assessment in Honey Bee Drones

2020

The quality of honey bee drone semen is relevant in different contexts, ranging from colony productivity to pathology, toxicology and biodiversity preservation. Despite its importance, considerably less knowledge is available on this subject for the honey bee when compared to other domestic animal species. A proper assessment of sperm quality requires a multiple testing approach which discriminates between the different aspects of sperm integrity and functionality. Most studies on drone semen quality have only assessed a few parameters, such as sperm volume, sperm concentration and/or sperm plasma membrane integrity. Although more recent studies have focused on a broader variety of aspects …

Study of nuclear and acrosomal sperm morphometry in ram using a computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method.

2014

The aim of this study was to develop a new method that allows morphometric assessment of the sperm nucleus and acrosome in the ram using fluorescence microscopy and free software. The study was divided into three experiments. In the first experiment, semen smears from 20 ejaculates were fixed and labeled with a propidium iodide-pisum sativum agglutinin (PI/PSA) combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed using the ImageJ program. The computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method used allowed the differentiation, capture, and morphometric analysis of most sperm nuclei, acrosomes, and whole heads with…

Comparison of different statistical approaches to evaluate morphometric sperm subpopulations in men.

2016

This study was designed to characterize morphometric sperm subpopulations in normozoospermic men by using different statistical methods and examining their suitability to classify correctly different sperm nuclear morphologies present in human ejaculates. Ejaculates from 21 normozoospermic men were collected for the study. After semen collection and analysis, samples were prepared for morphometric determination. At least 200 spermatozoa per sample were assessed for sperm morphometry by computer-assisted sperm morphometry analysis (CASA-Morph) using fluorescence. Clustering and discriminant procedures were performed to identify sperm subpopulations from the morphometric data obtained. Cluste…

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex

2016

This study was designed to determine the ability of computer-assisted sperm morphometry analysis (CASA-Morph) with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa (SX and SY, respectively). Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average (P < 0.001) although with impo…

Predictive capacity of sperm quality parameters and sperm subpopulations on field fertility after artificial insemination in sheep

2015

This study was designed to evaluate the relevance of several sperm quality parameters and sperm population structure on the reproductive performance after cervical artificial insemination (AI) in sheep. One hundred and thirty-nine ejaculates from 56 adult rams were collected using an artificial vagina, processed for sperm quality assessment and used to perform 1319 AI. Analyses of sperm motility by computer-assisted sperm analysis (CASA), sperm nuclear morphometry by computer-assisted sperm morphometry analysis (CASMA), membrane integrity by acridine orange-propidium iodide combination and sperm DNA fragmentation using the sperm chromatin dispersion test (SCD) were performed. Clustering pro…

CASA-Mot in mammals: an update

2017

Sperm motility is one of the most widely used parameters of sperm quality. Computer-aided sperm motility analysis (CASA-Mot) systems were developed to reduce the subjectivity of sperm motility assessment, and have had broad scientific and practical acceptance. In this review, the sources of variation and current applications of this technology and its relationships with other sperm quality tests are described in detail. Despite remarkable advances in the technique, there is still great need for standardisation in many species, and the numerous factors that affect the results make it difficult to provide universally accepted criteria for classifying semen samples based on sperm motility char…

Computer assisted sperm morphometry in mammals: a review.

2014

Computer-assisted sperm morphometry analysis (CASMA or ASMA) systems were developed to reduce the subjectivity of sperm morphology assessement. This review focuses on a complete description of the CASMA technique, including recent developments, factors of variation, results in the different species and possible applications. Techniques to study sperm morphometry include light microscopy, phase-contrast microscopy and, more recently, fluorescence microscopy. Most published studies on sperm morphometry have been centered on the whole sperm heads, although some of them also measured other parts of the sperm structure, such as the nucleus, acrosome, midpiece or flagellum. The independent study …

Effect of solid storage at 15 degrees C on the subsequent motility and fertility of rabbit semen.

2005

We conducted two studies to improve preservation of rabbit semen. The objective of the first study was determine whether a glucose- and fructose-based extender with two different amounts of gelatin would solidify at 15 degrees C, and to evaluate the influence of gelatin supplementation on sperm motility parameters after storing semen up to 10 days at 15 degrees C. The fertility of rabbit semen diluted in the best gelatin-supplemented extender established in Study 1 and stored for up to 5 days was evaluated in the second study. In Study 1, semen was collected with an artificial vagina from 40 bucks. Each ejaculate was diluted to (80-100) x 10(6) spermatozoa/mL (1:3, semen/extender) at 37 deg…

Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination

2021

The aim of the study was to compare the morphometric features of sperm head sizeand shape from the Pietrain line and the Duroc×Pietrain boar crossbred terminal lines, and toevaluate their relationship with reproductive success after artificial insemination of sows producedfrom crossbreeding the York, Landrace and Pietrain breeds. Semen samples were collected from 11sexually mature boars. Only ejaculates with greater than 70% motility rate and <15% of abnormalsperm were used for artificial inseminations (AI) and included in the study. Samples were analyzedusing an ISAS®v1 computer-assisted sperm analysis system for eight morphometric parameters ofhead shape and size (CASA-Morph). Sub-populat…

Effect of chamber characteristics, loading and analysis time on motility and kinetic variables analysed with the CASA-mot system in goat sperm.

2017

Several factors unrelated to the semen samples could be influencing in the sperm motility analysis. The aim of the present research was to study the effect of four chambers with different characteristics, namely; slide-coverslip, Spermtrack, ISAS D4C10, and ISAS D4C20 on the sperm motility. The filling procedure (drop or capillarity) and analysis time (0, 120 and 240s), depth of chamber (10 or 20μm) and field on motility variables were analysed by use of the CASA-mot system in goat sperm. Use of the drop-filling chambers resulted in greater values than capillarity-filling chambers for all sperm motility and kinetic variables, except for LIN (64.5% compared with 56.3% of motility for drop- a…

Effect of diluent composition on the dynamics of sperm DNA fragmentation and other sperm quality parameters in ram during incubation at 37°C

2015

Abstract This study was conducted to assess the effect of different semen extenders on the dynamics of sperm DNA fragmentation (SDF) and other quality sperm parameters in ram. To this end, second ejaculates from 6 adult males were collected using an artificial vagina and diluted in either INRA-96 ® , milk, MOPS, TRIS, TES, HEPES, citrate or phosphate-based extenders. Semen samples were incubated at 37 °C and the SDF, motility and membrane integrity were assessed after 0, 3, 6 and 24 h intervals. Significantly higher SDF indexes were obtained at 6 and 24 h of incubation in the milk-based than in all the buffer-based extenders. At 24 h, sperm diluted in INRA also reached a higher SDF than MOP…

Use of Relief Contrast®Objective to Improve Sperm Morphometric Analysis by Isas®Casa System in the Ram

2013

The aim of this study was to develop a new method for morphometric assessment of the sperm head and acrosome in the ram. Ejaculates from 10 adult males were collected using an artificial vagina. For each ejaculate, 10 semen smears were prepared, air-dried and divided (in pairs) into the following five treatment groups: (i) washed in distilled water and allowed to dry without further processing (DRY); (ii) fixed in 50% methanol (MET); (iii) fixed in 2% glutaraldehyde (GLUT); (iv) fixed and stained with Hemacolor(®) (HEM) and (v) fixed and stained with SpermBlue(®) (SB). The prepared slides were examined with a 40 × Relief Contrast(®) objective (RC) and processed with ISAS(®) commercial softw…

Effect of genistein added to bull semen after thawing on pronuclear and sperm quality.

2015

The aim of this research was to study the effect of different genistein treatments on bull sperm after thawing on pronuclear formation after in vitro fertilization (IVF) and on different sperm quality variables. Three experiments were performed. In Experiment 1, three treatments (Control, sperm incubation for 1 h at 37 degrees C with or without genistein) and two sperm concentrations during IVF (I or 3 x 10(6) sperm/mL) were evaluated to study the influence of genistein on pronuclear formation (PNF). Sperm incubation for 1 h before IVF reduced PNF regardless of sperm concentration. However, after sperm incubation and with 3 x 10(6) sperm/mL in IVF, the genistein treatment group had greater …