M.A. Silvestre

Intracytoplasmic Sperm Injection in Livestock Species: An Update

Intracytoplasmic sperm injection (ICSI) is a powerful technique in the field of assisted reproduction (ART) and provides exciting opportunities for studying the basic mechanisms of fertilization and early embryo development. Nevertheless, its application in agriculture and conservation biology has been greatly hampered by the low success rate reported for this method in respect of economically important species. Specifically, the rates of blastocyst formation and live newborn are greatly reduced when zygotes are generated by ICSI. Except for humans, ICSI remains a low efficiency technology in comparison with alternatives such as in vitro fertilization (IVF) and its application is less widespread. In this paper, we discuss the present status, applications and factors affecting ICSI in pigs and other species.

Effect of the Bovine Oviductal Fluid on In Vitro Fertilization, Development and Gene Expression of In Vitro-Produced Bovine Blastocysts

Oviductal microenvironment generally provides better conditions for early embryo development than the conventional in vitro system. In an attempt to simulate the oviduct conditions or the main potentially influencing factors, the effect was studied of a bovine oviductal fluid (bOF) treatment applied prior to IVF on (i) IVF parameters, (ii) cleavage rate, (iii) blastocyst yield and (iv) blastocyst quality. Embryo quality was assessed by morphological embryo quality and relative transcript abundance of several developmental genes in bovine blastocysts. Furthermore, to study the effect of bOF without the male effect and zona-sperm interaction, artificially activated metaphase II oocytes were also treated with bOF. In vitro-matured bovine oocytes from abattoir ovaries were treated or untreated with bOF for 30 min and then washed prior to IVF or activation. Subsequently, in vitro-fertilized and parthenogenetic embryos were in vitro cultured for 7 to 8 days. The bOF treatment had no effect on fertilization parameters, cleavage, blastocyst rates both on parthenogenetic and IVF bovine embryos and neither on morphological quality of IVF blastocysts. G6PD and SOD2 genes from IVF blastocysts showed significant changes in their expression after a bOF treatment. Significant differences were found for the expression of SCL2A1, GPX1, BAX, AKR1B1 and PLAC8 genes between excellent or good blastocysts (Grade 1) and fair blastocysts (Grade 2). To our knowledge, this is the first study that evaluates the effect of bOF oocyte treatment on fertilization parameters, development and quality of bovine embryos.

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 procedures using the sperm kinematic and morphometric data resulted in the classification of spermatozoa into three kinematic and three morphometric sperm subpopulations. Logistic regression procedures were used, including fertility at AI as the dependent variable (measured by lambing, 0 or 1) and farm, year, month of AI, female parity, female lambing-treatment interval, ram, AI technician and sperm quality parameters (including sperm subpopulations) as independent factors. Sperm quality variables remaining in the logistic regression model were viability and VCL. Fertility increased for each one-unit increase in viability (by a factor of 1.01) and in VCL (by a factor of 1.02). Multiple linear regression analyses were also performed to analyze the factors possibly influencing ejaculate fertility (N=139). The analysis yielded a significant (P<0.05) relationship between sperm viability and ejaculate fertility. The discriminant ability of the different semen variables to predict field fertility was analyzed using receiver operating characteristic (ROC) curve analysis. Sperm viability and VCL showed significant, albeit limited, predictive capacity on field fertility (0.57 and 0.54 Area Under Curve, respectively). The distribution of spermatozoa in the different subpopulations was not related to fertility.

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 the fluorescence-labeled sperm were recorded with a digital camera, and at least 200 sperm cells per sample were processed using the Image J analysis open software. Air-drying significantly reduced nuclear sperm dimensions in ruminant species, whereas no effect was observed in pigs. For most of the primary morphometric parameters, the relationship between the four species for the sperm nuclear dimensions can be described as follows: bull > ram ≥ boar > goat. However, ram sperm nuclei had greater width than those of the other species studied. For the secondary morphometric parameters, ram sperm nuclei were clearly less elliptical and elongated and showed greater regularity than in the other studied species. In the second experiment, ejaculates from 10 males per species were used to compare the sperm head morphometric results obtained with the computer-assisted sperm morphometry analysis-F system (using the GLUT treatment as reference) to a more conventional CASMA method (semen smears stained with Harriss hematoxylin and processed with the Integrated Sperm Analysis System [ISAS] commercial software [Proiser R&D SL, Buñol, Spain]). Spermatozoa displayed a bigger size when processed with Harriss hematoxylin than with the GLUT method in all primary sperm head morphometric parameters for the four species studied. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters in the four species studied. It was concluded that drying and fixation has little effect on sperm nuclear morphometry, with differences between species, and that there are significant variations in size of the sperm nucleus and in the hydrodynamic properties between the four species studied.

Beneficial effect of two culture systems with small groups of embryos on the development and quality of in vitro-produced bovine embryos.

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 fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF‐ITS (EGF‐ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF‐ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF‐ITS improved the embryo quality when smaller groups of embryos were cultured.

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs.

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca(2+) concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca(2+) concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.

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 resulted in the classification of spermatozoa into three kinematic sperm subpopulations. The distribution of kinematic sperm subpopulations was different between the fluorescent sperm groups (P<0.001), although the correlation between them was low (r=0.113; P<0.01).

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 characteristics. The main fields for CASA-Mot include the study of male fertility and pathologies, evaluation of the effects of physical and chemical agents, improvement of epidemiological survey studies, more precise calculation of seminal doses for farm animals, realisation of basic studies about sperm function, improvement of sperm technologies such as cryopreservation and quality control analysis. Numerous relationships have been established between CASA-Mot and other sperm quality tests, although most of these parameters are complementary. Future CASA-Mot systems will probably be able to integrate several sperm quality parameters with motility.

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- and capillarity-filling chambers respectively, P<0.05). There were no significant differences in total sperm motility between different chamber depths, however, use of the 20μm-chambers resulted in greater sperm progressive motility rate, VSL and LIN, and less VCL and VAP than chambers with a lesser depth. There was less sperm motility and lesser values for kinetic variables as time that elapsed increased between sample loading and sperm evaluation. For sperm motility, use of droplet-loaded chambers resulted in similar values of MOT in all microscopic fields, but sperm motility assessed in capillarity-loaded chambers was less in the central fields than in the outermost microscopic fields. For goats, it is recommended that sperm motility be analysed using the CASA-mot system with a drop-loaded chamber within 2min after filling the chamber.

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 1h at 37 °C with or without genistein) and two sperm concentrations during IVF (1 or 3 × 10(6)sperm/mL) were evaluated to study the influence of genistein on pronuclear formation (PNF). Sperm incubation for 1h before IVF reduced PNF regardless of sperm concentration. However, after sperm incubation and with 3 × 10(6)sperm/mL in IVF, the genistein treatment group had greater fertilization rates than the untreated group. In Experiment 2, six treatments plus the control group were performed to study the effect of genistein (presence or not) and incubation conditions (30 min at 37 °C, 1h at 27 °C or at 37 °C) on PNF using 3 × 10(6)sperm/mL for IVF. When incubation time was reduced to 30 min, PNF rate from the genistein treatment group was no different from either the control group or in the group in which incubation occurred for 1h at 27 °C. In Experiment 3, the effect of several genistein treatments (control; genistein treatment for 30 min of incubation at 37 °C; genistein treatment for 1h of incubation at 27 °C) on sperm motility, viability and DNA fragmentation were evaluated. Genistein did not improve sperm motility and, depending on the experimental group or time, it either reduced or had no effect on sperm motility. Genistein treatment did not improve sperm viability after 5h of incubation. However, genistein treatment for 1h at 27 °C decreased sperm DNA fragmentation compared with the control group after 5h of sperm incubation. In conclusion, the treatment of bull sperm with genistein for 1h at 27 °C could decrease sperm DNA fragmentation, although PNF rate after IVF and sperm motility were reduced.