Stress decreases spermatozoa quality and induces molecular alterations in zebrafish progeny.

BACKGROUND: Chronic stress can produce a severe negative impact on health not only in the exposed individuals but also in their offspring. Indeed, chronic stress may be contributing to the current worldwide scenario of increasing infertility and decreasing gamete quality in human populations. Here, we evaluate the effect of chronic stress on behavior and male reproductive parameters in zebrafish. Our goal is to provide information on the impact that chronic stress has at molecular, histological, and physiological level in a vertebrate model species. RESULTS: We evaluated the effects of a 21-day chronic stress protocol covering around three full waves of spermatogenesis in Danio rerio adult males. The induction of chronic stress produced anxiety-like behavior in stressed males as assessed by a novel tank test. At a molecular level, the induction of chronic stress consistently resulted in the overexpression of two genes related to endoplasmic reticulum (ER) stress in the brain. Gene set enrichment analysis (GSEA) of testes suggested a dysregulation of the nonsense-mediated decay (NMD) pathway, which was also confirmed on qPCR analysis. Histological analysis of the testicle did not show significant differences in terms of the relative proportions of each germ-cell type; however, the quality of sperm from stressed males was compromised in terms of motility. RNA-seq analysis in stress-derived larval progenies revealed molecular alterations, including those predicted to affect translation initiation, DNA repair, cell cycle control, and response to stress. CONCLUSIONS: Induction of chronic stress during a few cycles of spermatogenesis in the vertebrate zebrafish model affects behavior, gonadal gene expression, final gamete quality, and progeny. The NMD surveillance pathway (a key cellular mechanism that regulates the stability of both normal and mutant transcripts) is severely affected in the testes by chronic stress and therefore the control and regulation of RNAs during spermatogenesis may be affected altering the molecular status in the progeny.

Early stress exposure on zebrafish development: effects on survival, malformations and molecular alterations.

The effects of stress during early vertebrate development can be especially harmful. Avoiding stressors in fish larvae is essential to ensure the health of adult fish and their reproductive performance and overall production. We examined the consequences of direct exposure to successive acute stressors during early development, including their effects on miR-29a and its targets, survival, hatching and malformation rates, larval behaviour and cartilage and eye development. Our aim was to shed light on the pleiotropic effects of early-induced stress in this vertebrate model species. Our results showed that direct exposure to successive acute stressors during early development significantly upregulated miR-29a and downregulated essential collagen transcripts col2a1a, col6a2 and col11a1a, decreased survival and increased malformation rates (swim bladder, otoliths, cardiac oedema and ocular malformations), promoting higher rates of immobility in larvae. Our results revealed that stress in early stages can induce different eye tissular architecture and cranioencephalic cartilage development alterations. Our research contributes to the understanding of the impact of stressful conditions during the early stages of zebrafish development, serving as a valuable model for vertebrate research. This holds paramount significance in the fields of developmental biology and aquaculture and also highlights miR-29a as a potential molecular marker for assessing novel larval rearing programmes in teleost species.

miR-29a Is Downregulated in Progenies Derived from Chronically Stressed Males.

Recent research has provided compelling evidence demonstrating that paternal exposure to different stressors can influence their offspring's phenotypes. We hypothesized that paternal stress can negatively impact the progeny, altering different miRs and triggering different physiological alterations that could compromise offspring development. To investigate this, we exposed zebrafish male siblings to a chronic stress protocol for 21 days. We performed RNA-sequencing (RNA-seq) analyses to identify differentially expressed small noncoding RNAs in 7-day postfertilization (dpf) larvae derived from paternally stressed males crossed with control females compared with the control progeny. We found a single miRNA differentially expressed-miR-29a-which was validated in larva and was also tested in the sperm, testicles, and brain of the stressed progenitors. We observed a vertical transmission of chronic stress to the unexposed larvae, reporting novel consequences of paternally inherited chronic stress at a molecular level. The deregulation of mi-R29a in those larvae could affect relevant biological processes affecting development, morphogenesis, or neurogenesis, among others. Additionally, these disruptions were associated with reduced rates of survival and hatching in the affected offspring.

Impact of different levels of handling on Solea senegalensis culture: effects on growth and molecular markers of stress.

Aquaculture routine practices may cause stress induction on the fish and compromise their welfare affecting the production. This experiment aimed to evaluate the potential links between handling during culture with stress responses and growth on Senegalese sole (Solea senegalensis). We worked with two fish cohorts in terms of initial body weight and culture stage: Trial 1 included specimens in the fattening stage (226 ± 4.96 g) and Trial 2 animals in the pre-fattening stage (27.20 ± 0.44 g). The tested culture protocol, which lasted 6 and 4 months for Trial 1 and 2, respectively, mainly reduced handling-derived stressors in the experimental tanks via lowering routine samplings to a minimum. This decrease of the handling-derived stress was reflected in both trials with lower concentration of circulating cortisol in blood plasma from the experimental fish when compared to controls. Moreover, the proposed protocol promoted higher growth in the fish cultured in the less disturbing protocol in Trial 2. Higher specific growth rates and mean body weight and length were reported. In order to further explore the potential beneficial effects of our protocol, we studied the musculoskeletal from Trial 2 gene expression of key genes regulating glucocorticoid signaling pathway and apoptosis: glucocorticoid receptors 1 and 2 (gr1, gr2), heat shock protein 90 AA (hsp90aa), and caspase 6 (casp6). In line with the cortisol reduced level in this trial, gr1, hsp90aa, and casp6 genes showed lower expression in the samples coming from the experimental group. The findings of this study provide valuable information to the aquaculture industry for the management of Solea senegalensis stress and welfare.

Male reproductive dysfunction in Solea senegalensis: new insights into an unsolved question.

Senegalese sole (Solea senegalensis) is a species with a high commercial value that exhibits a reproductive dysfunction in males born and raised in captivity (F1) that hinders their sustainable culture. The present study evaluates the sperm quality and dopaminergic pathway of males born in the wild environment and of F1 males. Traditional sperm analyses were performed, finding only significant differences in curvilinear velocity (VCL) and no significant differences in viability and total motility. No differences in global sperm methylation were observed either in spermatozoa or brain between the two groups (F1 and wild-born males). However, our results point to a different sperm molecular signature between wild fish and fish born in captivity, specifically the differential expression in miR-let7-d and miR-200a-5p between these two groups. miR-let7-d has been correlated with spermatogenesis and sex preferences, whereas the miR-200 family is implied in target innervation of dopaminergic neurons in zebrafish. When we analysed the dopaminergic pathway, no differences were found in terms of different mRNA expression of dopaminergic markers. However, some differences were detected in terms of tyrosine hydroxylase protein expression by western blot analysis, thus suggesting an altered post-transcriptional regulation in F1 males. The results of this study suggest that an altered sperm miRNA signature in F1 males could be one possible mode of transmission of reproductive dysfunction to the progeny.

First study in cryopreserved Crassostrea angulata sperm.

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 extremely important for the oyster aquaculture industry to prevent the potential impacts of epidemic diseases and natural disasters. The Portuguese oyster, Crassostrea angulata, was the most important species commercialized by the shellfish industry. However, inadequate management of this industry and pathology occurrences resulted in a significant decrease in natural populations. For this reason, in this work a successful sperm cryopreservation protocol for this important species has been developed for the first time. Different internal cryoprotectants (DMSO, ethylene glycol, polyethylene glycol and methanol) at several concentrations (5, 10, 20%), containers (straws vs cryovials) and freezing rates (slow and fast rates) were tested. Cryoprotectant toxicity tests corroborated that this assay did not take into account the following steps of cryopreservation protocol as sperm agglutination. A fast freezing rate of cells diluted in10% DMSO and the use of straws as containers were the best cryopreservation conditions for Portuguese oyster sperm. Finally, fertilization assays confirmed the efficiency of the cryopreservation protocol in oyster sperm. These results demonstrated that different susceptibilities have been detected concerning sperm cryopreservation depending on oyster species or genetic material composition.

Molecular basis of spermatogenesis and sperm quality.

Spermatozoan quality can be evaluated in different ways, here we focus on the analysis of DNA, RNA and epigenetic status of germ cells. These characterizations also can be the bases for explaining sperm quality at other levels, so we will see how some of these molecules could affect other sperm quality markers. Moreover, we consider the possibility of using some of these molecules as predictors of sperm quality in terms of the ability to produce healthy offspring. The relevant effect of different types of RNA molecules in germ line specification and spermatogenesis and the importance of germ cell DNA integrity and a proper epigenetic pattern will be also discussed. Although most studies at this level have been performed in mammals, some information is available for fish; these recent discoveries in fish models are included. We provide a general overview on how these molecules could have a deep influence in the final sperm quality.

In vitro generation of zebrafish PGC-like cells.

The possibility of generating primordial germ cells (PGCs) in vitro from noncommitted embryonic cells represents an extremely useful tool in current research. Primordial germ cell 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 date to describe a rapid method for PGC in vitro differentiation in teleosts. Primordial germ cell-like cells were characterized by several lines of evidence, including gene expression, cell complexity, size, and image analysis for the quantification of fluorescence under vasa promoter. Moreover, differentiated cells were able to colonize the genital ridge after transplantation. Differentiation treatments increased the number of PGCs in culture, causing differentiation of cells rather than inducing their proliferation. These results open up the possibility of differentiating genetically modified embryonic cells to PGC-like cells to ensure their transmission to the progeny and could be crucial for an in-depth understanding of germline differentiation in teleosts.

The Direct Effects of Climate Change on Tench (Tinca tinca) Sperm Quality under a Real Heatwave Event Scenario.

Global aquaculture growth will most probably face specific conditions derived from climate change. In fact, the most severe impacts of these changes will be suffered by aquatic populations in restrictive circumstances, such as current aquaculture locations, which represent a perfect model to study global warming effects. Although the impact of temperature on fish reproduction has been characterized in many aspects, this study was focused on recreating more realistic models of global warming, particularly considering heatwave phenomena, in order to decipher its effects on male gametes (spermatozoa). For this purpose, thermal stress via a heatwave simulation (mimicking a natural occurring heatwave, from 24 to 30 °C) was induced in adult tench (Tinca tinca) males and compared with a control group (55.02 ± 16.44 g of average body wet weight). The impact of the thermal stress induced by this climate change event was assessed using cellular and molecular approaches. After the heatwave recreation, a multiparametric analysis of sperm quality, including some traditional parameters (such as sperm motility) and new ones (focus on redox balance and sperm quality biomarkers), was performed. Although sperm concentration and the volume produced were not affected, the results showed a significant deleterious effect on motility parameters (e.g., reduced progressive motility and total motility during the first minute post-activation). Furthermore, the sperm produced under the thermal stress induced by this heatwave simulation exhibited an increased ROS content in spermatic cells, confirming the negative effect that this thermal stress model (heatwave recreation) might have had on sperm quality. More importantly, the expression of some known sperm quality and fertilization markers was decreased in males exposed to thermal stress. This present study not only unveils the potential effects of climate change in contemporary and future fish farming populations (and their underlying mechanisms) but also provides insights on how to mitigate and/or avoid thermal stress due to heatwave events.

The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage.

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.

The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment.

Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial. In this sense, the study of intracellular ROS localization and the relationships between ROS and other conventional parameters could improve the characterization of sperm quality for semen preservation protocols in rams. In this work, a multiparametric study was carried out by analyzing four experimental groups of ram sperm with different initial qualities: fresh semen (from both breeding and nonbreeding seasons), frozen-thawed semen and, a positive control group treated with hydrogen peroxide (300 µM) as a marker of extreme damage. Sperm analyses, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters, were applied to compare several experimental groups with different sperm qualities. After that, the signals from two different ROS probes: CellROX™ Deep Red (CRDR) and Green (CRG), were examined by flow cytometry (percentage of cells that express ROS) and fluorescence microscopy (intracellular ROS location). Comparing conventional parameters, fresh samples from the breeding season showed the highest sperm quality, while the positive control samples showed the worst sperm quality. Concerning the ROS probes, the CRDR levels were higher in fresh samples from the breeding season than in the positive control and cryopreserved samples. Surprisingly, CRG presented its highest level (P < 0.05) in the positive control group treated with peroxide by flow cytometry. CRDR and CRG presented opposite labeling patterns that were corroborated by fluorescence microscopy, which determined that the probes localized in different parts of sperm. CRDR was found in the sperm mitochondrial region, while CRG was observed in the cell nucleus, suggesting that ROS localization is an important factor. Finally, our study indicates that CRDR is correlated with proper viability and sperm motility, and could be associated with high mitochondrial activity, while CRG is associated with sperm damage.

Cryobiology of cephalopod (Illex coindetii) spermatophores.

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 experiments and a cryopreservation test. Five permeating cryoprotectants were tested: Dimethyl sulfoxide (Me2SO), methanol, glycerol, propylene glycol and ethylene glycol. In the first experiment, spermatophores were exposed to the five cryoprotectants at 5% (v/v) and 15% (v/v) at 4 °C for 5 min. In the second experiment, spermatophores were exposed to the cryoprotectants at 15% using different exposure times: 5, 15 and 30 min. In a third experiment, we tested two cryopreservation protocols: LN2 vapor or -80 °C freezer, using a 15% cryoprotectant and 15 or 30 min of exposure. Viability and mitochondrial activity were assessed using Mitotracker deep red, YOPRO1 and Hoechst 33342, by flow cytometry. Spermatozoa in this species remain viable after cryoprotectant exposure but their quality decreased considerably after cryopreservation, only 5-10% of spermatozoa being motile. Flow cytometry demonstrated that Me2SO may be the most appropriate cryoprotectant for I. coindetii spermatozoa, and shows a first approach on cephalopod sperm cryopreservation, opening new possibilities for the research and culture of this group of molluscs.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile.

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

Does Size Matter? Testicular Volume and Its Predictive Ability of Sperm Production in Rams.

Over the years, testicular volume has been used to evaluate the reproductive capacity of rams and the effects of different factors related to reproductive performance. The aim of this study was to determine the most suitable tool and formula to calculate testicular volume under field conditions to guarantee a more accurate determination of sperm production. First, testicles from 25 rams (n = 50) were measured in vivo and postmortem using calipers and ultrasonography during the breeding season (BS). The accurate testicular volume (ATV) was calculated through water displacement. In addition, the sexual status of donor rams was evaluated during a period of four years in a reproduction center, and the three most crucial groups in terms of genetic value and seminal collections were studied in the second part of this experiment: ER-NBS (Elite rams during the non-breeding season), ER-BS-S (Elite rams with a standard frequency of seminal collection), and ER-BS-O (Elite rams with a high frequency of seminal collection). The total testicular volume (TTV), testosterone (T), and total spermatozoa obtained from two consecutive ejaculates in the same day (SPERM) were measured, and the relationship between SPERM and TTV and T was analyzed to predict SPERM. Although all published formulas revealed statistically significant differences (p ≤ 0.05) from the ATV, our proposed formula (ItraULE) (Testicular volume = L × W × D × 0.61) did not show significant differences. In the second part of the study, in the ER as a model donor ram for its high genetic value and high demand from farmers, TTV and T showed strong positive correlations with SPERM (r = 0.587, p = 0.007 NBS; r = 0.684, p = 0.001 BS-S; r = 0.773, p < 0.0001 BS-O). Moreover, formulas were established to predict SPERM in these practical scenarios. In conclusion, the use of ultrasonography and a new formula adapted to rams could improve the prediction of SPERM considering crucial factors such as season and semen collection frequency.

Frequency of Semen Collection Affects Ram Sperm Cryoresistance.

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 in cooled and frozen-thawed semen to evaluate sperm quality after different semen collection frequencies during a month: zero sperm collection (0 CW), four sperm collections per week (4 CW), and ten sperm collections per week (10 CW). Traditional analyses have been applied, in combination with novel technologies related to redox balance. Frozen-thawed semen quality showed a significant decrease (p < 0.05) in 0 CW and 10 CW in comparison to 4 CW, concerning motility and kinetics parameters. However, apoptosis showed a significant increase (p < 0.05) in 10 CW in comparison to 0 CW and 4 CW. The employment methods related to redox balance provided us with the definitive probe to ensure the influence of collection frequency on balance redox after thawing. Specifically, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activity showed a significant decrease (p < 0.05) in 10 CW compared to 0 CW and 4 CW. The characterization of alternative strategies to sperm cryopreservation based on consideration of male sexual regimes, could improve the quality of frozen-thawed sperm.

An optimized centrifugation protocol for ram sperm ensuring high sample yield, quality and fertility.

The optimization and implementation of artificial insemination (AI) in sheep is necessary to increase the livestock productivity through enhanced control of reproductive function. Sperm centrifugation is a common procedure in the ejaculate handling in AI and other assisted reproductive technologies (ART), as part of new methods of sperm analysis, selection or preservation. However, our research group previously established that this simple procedure might cause a large sperm loss and induce deleterious effects on the sperm function of the ovine species when high centrifugation forces are employed. To our knowledge, there are no studies on combined effect of extender and different centrifugal forces on ram sperm yield and quality. Furthermore, evidence of in vivo fertility rate using sperm obtained with various centrifugation forces is also lacking in this species. Thus, the objective of this work was to define the ideal conditions for ram semen centrifugation that will achieve the best quantity and quality sample to ensure unaffected fertilization ability of centrifuged ram sperm. The Experiment 1 evaluated the effect of the centrifugation procedure of two extenders (INRA 96 and Tyrode's) and two cooling protocols (Rapid and Slow Refrigeration -35 °C to 15 °C-) on sperm recovery rate and quality (motility and kinetic parameters, viability, apoptosis and mitochondrial activity). INRA 96 combined with Slow Refrigeration and Tyrode's at room temperature registered the highest sperm recovery and quality values (P ≤ 0.05). In Experiment 2, the influence of three centrifugal forces (600, 1200 and 6000×g for 10 min) was assessed immediately after centrifugation on the technical performance and sperm functionality in diluted samples with INRA 96 and Tyrode's at the conditions set out in Experiment 1. The lowest pellet weight (P ≤ 0.05) without harmful effect on sperm physiological status (P > 0.05) was achieved at 1200×g, since 6000×g induced sperm motility damage (P ≤ 0.05) with both extenders. Finally, to ensure the total safety of the centrifugation protocol, Experiment 3 tested in a combined in vitro and in vivo test the effect of these three centrifugal forces on ram sperm quality after dilution (INRA 96) and liquid storage (6-8 h at 15 °C). The damage produced by 6000×g on sperm motility (P ≤ 0.05) was maintained over time, coinciding with a lower fertility (P ≤ 0.05). In conclusion, ram sperm can be centrifuged in INRA 96 extender up to 1200×g for 10 min at 15 °C as secure values with high recovery rates and without detrimental effects on sperm quality and fertility.

Application of ultrasound technique to evaluate the testicular function and its correlation to the sperm quality after different collection frequency in rams.

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, analyzing the testicular volume, echotexture, and vascular function, was used in three different frequencies of semen collection (abstinence frequency, AF; standard frequency, SF; and intensive frequency, IF). Semen samples were cooled (15°C, 6 h) and evaluated in terms of production, motility, viability, apoptosis, and content of reactive oxygen species. Correlation coefficients were calculated between ultrasonography measurements of echotexture and blood flow and sperm quality parameters. Our results showed an increase in the testicular echotexture when the frequency of semen collection was intensified. Doppler parameters (PSV, RI, PI, TABF) increased (P ≤ 0.05) when the frequency of semen collection was intensified. The sperm motility and functionality decreased in the samples of IF (P ≤ 0.05), evidencing the frequency of semen collection's influence. Moreover, moderate positive correlations were established among echotexture and different Doppler parameters with motility parameters in SF. Furthermore, the influence of abstinence days on AI success was analyzed in a field assay. The highest fertility rates were obtained when males had two to five abstinence days. To conclude, frequency of semen collection could be influenced in terms of semen quantity and sperm quality, showing changes in parenchyma echotexture and testicular vascularization. The standard semen collection frequency was the most adequate option. In addition, ultrasonography may be a predictive tool for estimating variations in the sperm quality of donor rams subjected to different frequencies of semen collection in reproduction centers.

In Vitro Induction of Teleost PGCs.

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 surrogate production. However, it was assumed that the induction of fish PGCs in vitro is not possible because in vivo they inherit germ plasm. In this chapter, we describe a successful differentiation protocol explaining the crucial factors and steps for in vitro PGC generation.

Flow Cytometry and Confocal Microscopy for ROS Evaluation in Fish and Human Spermatozoa.

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 samples. The method can be used with two approaches: (1) flow cytometry, for quantification of DCF+ cells, or (2) confocal microscopy, for the localization of ROS within the cells.

Centrifugal force assessment in ram sperm: identifying species-specific impact.

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 seminal plasma, and basic studies in spermatology is associated with the use of centrifugation. This requires a specific evaluation of the centrifugation protocols considering the species-specific relationship with the potential damage produced by this procedure. No previous studies have determined the effect of different centrifugation forces on ram sperm. Therefore, we aimed to assess the performance of three centrifugal forces (600×g, 3000×g, and 6000×g for 10 min at room temperature) and their effects on ram sperm motility and functionality. RESULTS: Sperm motility and functionality parameters were assessed at 0 h and after 2 h of incubation at 37 °C. As expected, a higher cell packaging degree was obtained at high centrifugation forces (P ≤ 0.0001). Cell packaging was unstable at all centrifugal forces. Thus, there was a high cell resuspension rate after less than 2 min. Regarding sperm quality, there was a change in movement pattern of 3000×g and 6000×g centrifuged sperm after 2 h of incubation at 37 °C, characterized by an increase in rapid progressive motility, linearity, straightness, and beat frequency, and a decrease in medium progressive motility, curvilinear velocity, path velocity, and head lateral amplitude. Non-significant differences were obtained among the different treatments concerning the total viability. However, we observed a significant increase (P ≤ 0.05) in the percentage of viable apoptotic sperm in the samples centrifuged at 6000×g at 0 h. CONCLUSIONS: Centrifugal forces equal to or greater than 3000×g induced some deleterious effects in ram sperm quality, and lower forces did not provide a successful cell packaging degree.