Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success

Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development. We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1+/-2.3 but a high rate of abortions, especially during gastrulation, and only 14.5+/-4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2+/-2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reported. Reproduction (2010) 139 989-997Junta de Castilla y Leon (Spain) [LE007A06]; University of Leoninfo:eu-repo/semantics/publishedVersio

Effect of cryopreservation on fish sperm subpopulations

10 páginas, 5 figuras, 4 tablas.The evaluation of the motility data obtained with a CASA system, applying a Two-Step Cluster analysis, identified in seabream sperm 3 different sperm subpopulations that correlated differently with embryo hatching rates. Hence, we designed an experiment to understand the effect of the application of different cryopreservation protocols in these sperm motility-based subpopulations. We analyzed Sparus aurata frozen/thawed semen motility 15, 30, 45 and 60 s after activation, using CASA software. Different protocols were applied for cryopreservation: three different cryoprotectants (Dimethyl Sulfoxide (Me2SO), Ethylene Glycol (EG) and Propylene Glycol (PG)) each at two different concentrations and two packaging volumes (0.5 ml straws, and 1.8 ml cryovials) were tested. Different freezing rates were evaluated corresponding to 1, 2, 3, 4 and 8 cm above the liquid nitrogen surface for the straws and 1, 2 and 4 cm for the cryovials. Motility parameters rendered by CASA were treated with a Two-Step Cluster analysis. Three different subpopulations were obtained: SP1 – slow non-linear spermatozoa, SP2 – slow linear spermatozoa and SP3 – fast linear spermatozoa. We considered SP3 as the subpopulation of interest and focused further analyses on it. Generally, SP3 was the best represented subpopulation 15 s after activation and was also the one showing a greater decrease in time, being the least represented after 60 s. According to the applied univariate general linear model, samples frozen in straws with 5% Me2SO and in cryovials with 10% Me2SO at 2 and 1 cm from the LN2, respectively, produced the best results (closer to the control). Clustering analysis allowed the detection of fish sperm subpopulations according to their motility pattern and showed that sperm composition in terms of subpopulations was differentially affected by the cryopreservation protocol depending on the cryoprotectant used, freezing rates and packaging systems.This work was supported by a PhD grant (SFRH/BD/31990/2006) and by the project Petri (PTR 95.1026.OP.01).Peer reviewe

Altered gene transcription and telomere length in trout embryo and larvae obtained with DNA cryodamaged sperm

Sperm cryopreservation could entail DNA damage, promoting base oxidization and strand breaks. In a previous work we showed that trout DNA damaged sperm is able to fertilize leading to embryo loss when the repair system of the oocyte is inhibited. Here we have analysed the later effects on embryo and larvae of fertilizing trout oocytes with cryopreserved DNA-damaged spermatozoa. Fish have weak sperm selection mechanisms, are very prolific and have external embryo development, being convenient models for this type of study. We cryopreserved rainbow trout semen using extenders containing egg yolk or their low density lipoprotein fraction to obtain samples with different degrees of DNA damage. DNA fragmentation was evaluated using the Comet assay and telomere length using quantitative-PCR. Fertilization trials were performed and the transcription at different developmental stages of telomerase reverse transcriptase (Tert) and eight genes related with embryo growth and development (Igf1, Igf2, Igfr1a, Igfr1b, Gh1, Gh2, Ins1 and Ins2) were analyzed using quantitative-PCR in surviving embryos and larvae. Results showed an increase in sperm DNA fragmentation after both cryopreservation procedures as well as a decrease in sperm telomere length. Larvae obtained with damaged sperm showed longer telomeres and Tert overexpression. The transcription of the analyzed genes in these embryos and larvae was also modified with respect to the control, most of them as an increase at hatch. We conclude that fertilization with cryopreserved DNA-damaged spermatozoa significantly affects offspring performance, detectable as an increase in telomere length as well as some alterations in gene expression in surviving embryo and larvae. © 2011 Elsevier Inc

Cryopreservation of fish sperm: applications and perspectives

13 páginas, 2 tablas.Cryopreservation is of interest not only for fish farming but also for the conservation and genetic improvement of resources. This technique has been well established in some freshwater fish species mainly, salmonid, sturgeons and carps, however, only in the last decade research was focused in marine fish species. The benefits of sperm cryopreservation include: (i) synchronization of gamete availability of both sexes, (ii) sperm economy; (iii) simplification of broodstock management, (iv) transport of gametes from different fish farms, and (v) germplasm storage for genetic selection programs or conservation of species. These issues would certainly benefit the aquaculture industry. The tremendous impact that biotechnology is having in aquaculture has been particularly obvious in recent years. Several species are being used as research models not only for aquaculture development applications but also for medical research. Sperm cryopreservation can give an important contribution in the germ storage of all transgenic lines. However, in all applications in fish sperm, cryopreservation needs to overcome a lack in standardization of methodologies and procedures, a correct assay of seminal quality and the development of tools to characterize cryoinjury. Many efforts have recently been made in the study of DNA using different approaches such as the comet assay (single cell gel electrophoresis), TUNEL (terminal deoxynucleotidyl transferase-nick-end-labelling), SCSA (sperm chromatin structure assay) and the analysis of specific DNA sequences using RT-PCR, since DNA damage may impair fertility or embryo development. Cryopreservation of gametes would certainly benefit from a higher concern on male improvement, basically through nutrition or selection of resistant stocks (e.g. stress resistant individuals or highly adapted to captivity) producing gametes of higher quality. There is a huge window of opportunities for improve the resistance of cells to cryopreservation through diet supplementation of certain compounds such as amino acids (taurine and hypotaurine), vitamins (Vit. E and C) and lipids or through a direct supplementation of the extender media. An equilibrium of those compounds will improve spermatozoa and seminal plasma composition protecting cells against oxidative stress (lipid peroxidation, protein oxidation, DNA fragmentation, enzyme protection) that is gaining each day more importance in cryodamage research.This work was supported by CRYOSPERM project (FCT, PTDC/MAR/64533/2006) and a ”Ramon y Cajal“ research contract (E. Cabrita).Peer reviewe

The oviduct From sperm selection to the epigenetic landscape of the embryo

The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of preimplantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed

Cryopreservation of canine sperm using egg yolk and soy bean based extenders

Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1℗) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with egg yolk-based extender (CaniPRO) or LP1℗. Freezing the spermatozoa in LP1℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P < 0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P < 0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P < 0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P < 0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1℗, reaching similar values than those cryopreserved in CaniPRO. In conclusion, LP1® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters. © 201

Cultured bovine embryo biopsy conserves methylation marks from original embryo

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. Thiswork analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ~4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cellmass were separated.We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation ismore similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved

Effect of supplementation of valine to chicken extender on sperm cryoresistance and post-thaw fertilization capacity

International audienceRecent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n = 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P = 0.025), curvilinear velocity (P = 0.033), straight-line velocity (P 5 0.040), and average path velocity (P = 0.033), whereas progressive motile sperm (P = 0.019), curvilinear velocity (P = 0.006), straight-line velocity (P = 0.003) and average path velocity (P = 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P = 0.002) in VF (36.3% VF-control vs 31%VF-valine). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% 6 12.1) than VF cryopreserved sperm control (16.1% 1 6.2). Supplementing valine to the freezing extender doubled (P 5 0.026) the fertility rate of VF (32.6% 6 12.2) compared with the control (16.1% 1 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory