Marta F. Riesco

The frequency of semen collection is a crucial factor to consider in the rams performance inside breeding centers workout. To evaluate this factor, ram Breeding Soundness Evaluation could include sperm quality evaluation and new predictive and non-invasive tools such as ultrasound technique. In this work, an advanced ultrasonography technology, ana...

The improvement of frozen-thawed sperm quality has been mostly approached from the view of cryopreservation protocol optimization in terms of cryoprotectant solutions, freezing-thawing rates and antioxidant supplementation, while the impact of sperm collection frequency remains unknown in rams. In this work, a multiparametric study was carried out...

To fully understand the histological, morphometrical and heamodynamic variations of different supratesticular artery regions, 20 mature and healthy Assaf rams were examined through ultrasound and morphological studies. The testicular artery images of the spermatic cord as shown by B-mode analysis indicated a tortuous pattern along its course toward...

In ectotherm species, environmental temperature plays a key role in development, growth, and survival. Thus, determining how temperature affects fish populations is of utmost importance to accurately predict the risk of climate change over fisheries and aquaculture, critical to warrant nutrition and food security in the coming years. Here, the pote...

Primordial germ cell (PGC) preservation could allow the conservation of fish diploid genome of genetically valuable individuals, such as endangered species or species with reproductive dysfunctions, in contrast to the haploid genome of their gametes. Cryopreservation is the best technological tool for fish PGC storage. Moreover, after freezing/thaw...

Background Centrifugation is routinely employed in handling the ejaculates of some species, but it is not part of the commonly used protocols in ram. However, the development and implementation of new assisted reproductive technologies, alternative preservation models based on washing sperm from a cellular ageing-accelerating substance such as the...

It is crucial to perform a deep study about the most extensively used antibiotics in sperm extenders. Most of the protocols and concentrations used in ram are direct extrapolations from other species. It is important to establish species-specific antibiotic treatments to optimize their use and if it is possible to reduce the quantity. Previews stud...

Primordial germ cells (PGCs) are unique cells in an embryo. These cells contain all genetic information and therefore represent the best source to store maternal and paternal genomes until embryo cryopreservation is achieved. However, the number of these cells in an embryo is very low limiting their potential application in cryopreservation and sur...

The optimization of sperm cryopreservation protocols in ram is a feasible tool to reinforce artificial insemination technologies considering the desirable application of sperm by vaginal/cervical or transcervical deposition. Cryopreservation provokes different types of damage on spermatozoa and many of these detrimental effects are triggered by red...

Reactive oxygen species (ROS) could have a negative impact on sperm cellular function and viability. This chapter describes a protocol for oxidative stress evaluation using dichlorofluorescein (DCF) which can specifically reveal intracellular reactive oxygen species. The protocol described here has been used in human and teleost species sperm sampl...

L.A.-L.); mneim@unileon.es (M.N.-M.); c.palacin@unileon.es (C.P.-M.); rmong@unileon.es (R.M.-G.); mmalvg@unileon.es (M.A.); ppazc@unileon.es (P.d.P.); laner@unileon.es (L.A.) Abstract: To improve artificial insemination protocols in ovine species it is crucial to optimize sperm quality evaluation after preservation technologies. Emerging technologi...

The aim of this study was to evaluate the effect of long term natural feeding in the subsequent reproductive status of 4 years old cultured Solea senegalensis, and to determine if the potential changes were structural or feeding dependent. To this aim, two different feeding regimes were used from one year after weaning and during the following 3 ye...

There is increasing evidence that gut microbiome could have effects on neurological processes and on behavior. In this study we used the novel tank test (NTT) to analyze zebrafish exploring behavior after four months’ supplementation with probiotics with probed antioxidant and anti-inflammatory properties. Results showed that prolonged ingestion of...

Manipulation is usually required for biomass calculation and food estimation for optimal fish growth in production facilities. However, the advances in computer-based systems have opened a new range of applied possibilities. In this study we used image analysis and a neural network algorithm that allowed us to successfully provide highly accurate b...

The aim of the present experiment is to study the effects of oral ingestion of a mixture of two probiotic bacteria on sperm quality and progenies. Three homogeneous groups of juvenile zebrafish were created. Once having reached adulthood (3 months postfertilization; mpf), each group received different feeding regimens: a standard diet (control), a...

Nowadays a decrease tendency in human sperm quality has been reported mainly in developed countries. Reproductive technologies have been very valuable in achieving successful pregnancies with low quality sperm samples. However, considering that spermatozoa molecular contribution is increasingly important in recent studies, it is crucial to study wh...

The cryopreservation of gametes and embryos is a technique widely used in reproductive biology. This technology helps in the reproductive management of domesticated animals, and it is an important tool for gene banking and for human-assisted reproductive technologies. Antifreeze proteins are naturally present in several organisms exposed to subzero...

Non-coding RNAs (ncRNAs) are crucial regulatory elements in most biological processes and reproduction is also controlled by them. The different types of ncRNAs, as well as the high complexity of these regulatory pathways, present a complex scenario; however, recent studies have shed some light on these questions, discovering the regulatory functio...

Infertility is a medical concern worldwide and could also have economic consequences in farmed animals. Developing an efficient diet supplement with immediate effects on sperm quality is a promising tool for human reproduction and for domesticated animal species. This study aims at elucidating the effect of a short-time probiotic supplementation co...

Senegalese sole (Solea senegalensis) is a species with a high commercial value that exhibits a reproductive dysfunction in males born and raised in captivity (F 1 ) that hinders their sustainable culture. The present study evaluates the sperm quality and dopaminergic pathway of males born in the wild environment and of F 1 males. Traditional sperm...

Background: Vitamin K (VK) is a fat-soluble vitamin known for its essential role in blood coagulation, but also on other biological processes (e.g. reproduction, brain and bone development) have been recently suggested. Nevertheless, the molecular mechanisms behind its particular function on reproduction are not yet fully understood. Methods: Th...

Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as...

Cryopreservation is a well-established technique commonly used in clinical practice. It is used widely for the conservation of gametes and embryos that will be used later for insemination or in vitro fertilization. However, several studies have shown that this technique can produce changes in messenger RNA levels, in the epigenome and induce DNA da...

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under...

The dusky grouper, Epinephelus marginatus, is a potential species for aquaculture production although the limited number of males kept in captivity has been the cause of some constraints in its production. Sperm cryopreservation emerged as a solution for this problem. However, spermatozoa can be severely affected by freezing/thawing processes and p...

The dusky grouper, Epinephelus marginatus, is a potential species for aquaculture production although the limited number of males kept in captivity has been the cause of some constraints in its production. Sperm cryopreservation emerged as a solution for this problem. However, spermatozoa can be severely affected by freezing/thawing processes and p...

Sperm cryopreservation is a widely employed technique that promotes alternative techniques to contribute to broodstock management or restoration programs for species of commercial interest, endangered species or species with an interesting genotype. The preservation of genetic material from improved stocks or from the original population is extreme...

This review provides a general view on teleost germline development using primordial germ cells (PGCs) or spermatogonia (SG), highlighting recent progress in research on these two cell types in teleost fishes. Due to the interest of these cells for gene banking purposes, this chapter also reviews the available protocols for cryopreservation and the...

The preservation of genetic material of both improved stocks and of original population is very important for the oyster aquaculture industry to avoid potential impacts from epidemic diseases and natural disasters. Nowadays, purely wild populations of Crassostrea angulata are rare to find due to multiple factors that have affected this industry. Cr...

The effects of a commercial probiotic diet supplement (Bactocell®), containing lactic acid bacteria Pediococcus acidilactici were evaluated by identifying five zebrafish (Danio rerio) sperm quality markers. P. acidilactici (106 colony-forming units per gram) was supplied to young zebrafish males (age 8 months) as a feed probiotic additive during a...

The possibility of generating Primordial Germ Cells (PGCs) in vitro from non-committed embryonic cells represents an extremely useful tool in current research. PGC in vitro differentiation has been successfully reported in mammals. However, contrary to fish, PGC specification in mammals is an inductive mechanism. This study is the first to describe...

[This corrects the article DOI: 10.1371/journal.pone.0067614.].

Sperm cryopreservation is widely used in clinic for insemination, in vitro fertilization and other procedures such as intracytoplasmic sperm injection. The assessment after freezing/thawing of spermatozoa viability, motility and sometimes DNA integrity (mainly using fragmentation assays) has been considered enough to guarantee the safety and effect...

Cryopreservation is a common procedure for long-term storage of sperm in animal reproduction and is also employed in the aquaculture industry. The freezing–thawing process originates reactive oxygen species (ROS) which cause serious damage at chromatin level. Analyzing and characterizing this type of damage, especially in specific DNA regions, coul...

Cryopreservation is an important tool routinely employed in Assisted Reproduction Technologies (ARTs) and germplasm banking. For several years, the assessment of global DNA fragmentation seemed to be enough to ensure the integrity of genetic material. However, cryopreservation can produce molecular alterations in key genes and transcripts undetecta...

List of primers for DNA damage. Sequences start from 5´ to 3´. Annealing temperature and amplicon size is specified for each pair of primers. (TIF)

List of primers for gene expression and for bisulphite sequencing. Sequences start from 5´ to 3´. Annealing temperature and the species are specified for each pair of primers. (TIF)

actb2 Ct values means in fresh and cryopreserved genital ridges (GRs) from 26 somite zebrafish embryos. Ct values mean ± SE of three independent experiments with three replicates for each. Ct values remained unaffected by cryopreservation. (TIF)

Summary of gene abbreviations, gene complete names and gene functions in zebrafish primordial germ cells (PGCs) and in human sperm. (TIF)

Sequences, amplicon size and number of GpCs islands of the analyzed genes from Figure 4. Underlined CG corresponds with the analyzed CpG islands in the zebrafish genes studied before and after cryopreservation (cxcr4b -240 bp-, pou5f1 -255 bp-, sox 3-274 bp- and vasa -156 bp-). (TIF)

Cephalopod culture is expected to increase in the near future and sperm cryopreservation would be a valuable tool to guarantee sperm availability throughout the year and to improve artificial insemination programs. We have studied the tolerance of spermatophores from the oceanic squid Illex coindetii to several cryoprotectants, in two toxicity expe...

Cryopreservation of Primordial Germ Cells (PGCs) for gene banking in fish is very promising considering the limitations that exist in fish embryo. Via germ line chimerism, these cells allow surrogate production in teleost fish. However, cryopreservation involves a range of extreme factors that might be stressful to cells. Moreover, Reactive Oxygen...

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryopro...