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

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…

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

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 …

Beneficial Effect of Two Culture Systems with Small Groups of Embryos on the Development and Quality of In Vitro-Produced Bovine Embryos

Summary Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro ferti…

Effect of the Bovine Oviductal Fluid onIn VitroFertilization, Development and Gene Expression ofIn Vitro-Produced Bovine Blastocysts

Oviductal microenvironment generally provides better condi-tions for early embryo development than the conventionalin vitro system. In an attempt to simulate the oviductconditions or the main potentially influencing factors, theeffect was studied of a bovine oviductal fluid (bOF) treatmentapplied prior to IVF on (i) IVF parameters, (ii) cleavage rate,(iii) blastocyst yield and (iv) blastocyst quality. Embryo qualitywas assessed by morphological embryo quality and relativetranscript abundance of several developmental genes in bovineblastocysts. Furthermore, to study the effect of bOF withoutthe male effect and zona–sperm interaction, artificially acti-vated metaphase II oocytes were also treated w…

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

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)

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

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

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…

Erratum to “The Trumorph® system: The new universal technique for the observation and analysis of the morphology of living sperm” [Anim. Reprod. Sci. (2015) 1–10]

Sperm Quality Assessment in Honey Bee Drones

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 …

The Trumorph℗® system: The new univ the morphology of living sperm

Abstract Evaluation of sperm morphology is a fundamental component of semen analysis, but its real significance has been obscured by a plethora of techniques that involve fixation and staining procedures that induce artefacts. Here we describe Trumorph℗ ® , a new method for sperm morphology assesment that is based upon examination of wet preparations of living spermatozoa immobilized by a short 60 °C shock using negative phase contrast microscopy. We have observed samples from five animals of the following species: bull, boar, goat and rabbit. In every case, all the components of the sperm head and tail were perfectly defined, including the acrosome and midpiece (in all its length, includin…

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

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

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…

Beneficial Effect of Melatonin on Blastocyst In Vitro Production from Heat-Stressed Bovine Oocytes

Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10(-12) , 10(-9) , 10(-4)  m; Experiment 4: 0, 10(-3)  m), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and …

CASA-Mot in mammals: an update

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…

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

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

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…

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

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 …