A stepwise methodology for developing fish sperm cryopreservation protocols: application to the endangered Pogoniascourbina.

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Elasmobranchs are one of the most endangered vertebrate groups on the planet, but despite this situation the use of reproductive techniques in elasmobranch conservation strategies has been scarce. Among these techniques, sperm preservation is a potential tool forex situconservation and aquaria sustainability. However, there are no widespread preservation protocols for elasmobranch sperm, and shark sperm cryopreservation has never been achieved before. Here we present the establishment of successful cryopreservation protocols for elasmobranch sperm, tested in several species. We have formulated a sperm extender that can be used for different elasmobranch species, capable of maintaining sperm motility for several weeks. Additionally, we achieved the cryopreservation of sperm by previously diluting it in our extender and supplementing it with different combinations of cryoprotectants. The effects of methanol and dimethyl sulfoxide as permeating cryoprotectants were evaluated, as well egg yolk as a non-permeating cryoprotectant. Sperm quality was assessed by studying the motility and membrane integrity post-thawing, demonstrating its effectiveness in the 10 species tested, including two which are considered Critically Endangered. This is the first time that shark sperm cryopreservation has been reported, broadening our knowledge of the reproductive techniques that can be applied to elasmobranchs and laying the foundations for the first cryobanks for shark and ray sperm. Outcomes from this study will be useful forex situconservation efforts developed by public aquaria. A regular supply of frozen sperm will reduce the problems that result from the transport of specimens, inbreeding or lack of synchronized reproductive cycles in captivity.

Cryopreservation of embryos of humpback grouper (Cromileptes altivelis) using combinations of non-permeating cryoprotectants

Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis

The biotechnological potential for manipulating offspring sex in the rhinoceros and the elephant

Commercial-scale out-of-season cryopreservation of Eurasian perch (Perca fluviatilis) sperm and its application for fertilization

To optimize a xeno-free cryopreservation protocol for primary human hepatocytes. The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes. Despite several hepatocyte cryopreservation protocols being available, improvements are urgently needed. We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups. Using the polystyrene box freezing, we compared two xeno-free freezing solutions for freezing of primary human hepatocytes: a new medium (STEM-CELLBANKER, CB), which contains dimethylsulphoxide (DMSO) and anhydrous dextrose, both permeating and non-permeating cryoprotectants, and the frequently used DMSO - University of Wisconsin (DMSO-UW) medium. The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation. The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms (CYPs): CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A7. The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol (P < 0.01). There was no significant difference in viability estimation between both the trypan blue (TB) and the Live-Dead Assay methods. There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols (r(2) = 0.69) using the TB method. However, due to high within-group variability in the activities of the major CYPs, any statistical between-group differences were precluded. Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability. Thus, it may be a better alternative to the standard DMSO-UW protocol. Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples. The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.

A newly developed flow cytometric method for determination of sperm concentration and viability was tested in an insemination trial with cryopreserved bull sperm to establish the relationship between sperm viability and nonreturn rates. Semen for experimental inseminations was produced from 157 young sires (114 Holstein and 43 Jersey), each contributing 4 experimental semen collections. Straws containing approximately 15 x 10(6) motile sperm before freezing were used in 118,680 experimental inseminations performed by 254 artificial insemination technicians in 6352 Danish herds. Statistical analysis based on 44,946 experimental first inseminations showed that the major part (95.4%) of variation in the 56-day nonreturn rate (NRR56) was residual. Only 0.38% of the total variation in NRR56 was due to bulls and differences between ejaculate within bull. However, bulls were preselected, and a relatively high insemination dose was used. Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility. However, flow cytometry makes it possible to achieve an objective and precise determination of sperm viability. It was therefore possible to calculate the effect on NRR56 provided selection of semen is based on the flow cytometric method. Three freezing extenders were used in this experiment, but a significant difference in NRR56 was not observed. Flow cytometric results for 1 extender (Biociphos Plus) indicated poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.

Germplasm banking is a fundamental tool for the preservation of autochthonous breeds. Semen cryopreservation is effective for this task, but protocols are adapted to commercial species, and post-thawing sperm quality could be sensitive to environmental cues. We compared the post-thawing sperm quality in doses from the CBA-SERIDA bank in northern Spain for the Asturiana de la Montaña (AM) and Asturiana de los Valles (AV) autochthonous cattle breeds. Doses from 23 AM and 16 AV bulls (ejaculates from at least three different seasons) were assessed for motility (computer-assisted sperm analysis), physiology and chromatin status (flow cytometry) after thawing and after 5 h at 38°C. Data were analysed using linear mixed-effects and cosinor models for seasonal and breed effects and by correlations with the association of sperm quality with temperature-humidity index (THI), considering the interval of spermatogenesis plus maturation. The breed affected sperm quality, with higher motility for AV and higher apoptotic ratio, mitochondrial activity, reactive oxygen species, DNA fragmentation and chromatin immaturity for AM. However, seasonality effects were minimal, and THI was not associated with sperm quality. In summary, the season seems to be a minor factor in the post-thawing quality of the AM and AV autochthonous breeds, well-adapted to their local environment.

One retrospective and two prospective studies were conducted among 218 couples treated with in-vitro fertilization (IVF) to establish the reproducibility and diagnostic accuracy of computer-assisted sperm analysis (CASA) with swim-up spermatozoa for the prediction of the fertilization rate of oocytes in vitro. Based on the results of a preliminary retrospective analysis in 49 patients, the 'curvilinear velocity' (VCL) was chosen as the most distinctive motion parameter of sperm function and the median was used to represent the entire sperm population. The number of inseminated motile spermatozoa was then adjusted to median VCL during two subsequent prospective studies with clinical IVF. Whereas in the first prospective study (90 couples) the threshold values of VCL with regard to the number of spermatozoa inseminated were based on the results of the preliminary retrospective study (49 couples), in the second prospective study (79 couples) the settings were based on the results of the first prospective study. The reproducibility of CASA was tested by analysing the motion characteristics of spermatozoa at different intervals after termination of swim-up, by repeated analysis of the same video-recording of the incubated spermatozoa by different observers, and by the repeated video-recording of the freshly prepared sperm samples and analysis of both video-recordings by the same observer. Under these conditions the frequency of disagreement between two measurements varied between 2.0 and 8.2%. In both prospective studies the sensitivity of CASA for the prediction of fertilization was high (74.0%), whereas the specificity was low (40.0%). In contrast to successful fertilization, unsuccessful fertilization of oocytes in vitro could not be predicted reliably with CASA. However, the pregnancy rate per cycle among patients with predicted low fertilization rates was significantly lower (5.3%) than in couples with high predicted fertilization rates (24.3%, P < 0.001). Therefore, CASA of washed spermatozoa may still help to identify couples who would benefit more from intracytoplasmic sperm injection (ICSI) than from IVF. A definite threshold level could not be identified for any of the motion parameters to distinguish the motion characteristics of fertilizing and non-fertilizing spermatozoa. Using various algorithms for hyperactivated motility, the percentage of hyperactivated spermatozoa was significantly higher among the successfully fertilizing patients than among the nonfertilizing group. However, the absolute number of hyperactivated spermatozoa added to the oocytes was higher in non-fertilizing couples. Therefore, the lack of fertilization in some patients may be caused by a generalized defect in sperm function rather than by insufficient hyperactivation.

Effect of cryopreservation of individual ejaculates on fertility in genetic resource chicken lines

Improvement of Ram Sperm Cryopreservation Protocols Assessed by Sperm Quality Parameters and Heterogeneity Analysis

Simple SummaryThe use of antioxidant compounds could be a successful tool to improve sperm cryopreservation protocols in ovine species. These molecules have been widely employed in different mammalian species with this purpose. It is important to consider the existence of a species-specific antioxidant effect discarding the extrapolations from other animal species. To corroborate the real effectiveness of these compounds is important to combine two approaches: in vitro sperm quality analyses and in vivo field trials based on fertility. In the first scenario, a multiparametric analyses and novel tests based on spermatozoa redox balance, as the main target of antioxidants, could improve the accuracy on antioxidant effectiveness on sperm quality. Moreover, an extensive field insemination study provides the definitive tool to select the best antioxidant treatment. All these aspects have been applied and extensively discussed throughout this manuscript. Novel approaches have been incorporated, such as RedoxSYS, to provide more accuracy in the integrative studies of Redox status in spermatozoa. The effectiveness of an antioxidant treatment, as trolox in our study, should be demonstrated in an integrative way, from in vivo (fertility trials) to in vitro analyses (sperm quality assays), especially when the final aim is to reach AI implementation.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 redox deregulation. For this reason, the antioxidant supplementation in sperm cryopreservation protocols to decrease reactive oxygen species (ROS) levels and to equilibrate redox status has been widely employed in different species. Despite this, more fertility trials are necessary to provide the definitive tool to ensure the antioxidant effectiveness on sperm quality. For this reason, in this work, we performed a multiparametric analysis of some previously tested antioxidants (crocin, GSH and Trolox) on ram sperm cryopreservation from field trials to sperm quality analyses focused on new strategies to measure redox balance. Attending to fertility trial, Trolox supplementation registered an improvement concerning to fertility (when we considered high fertility males) and multiple lambing frequency and other complementary and descriptive data related to lambing performance such as prolificacy and fecundity. This positive effect was more evident in multiple lambing frequency when we considered low fertility males than in global male analysis. In vitro analyses of sperm quality confirmed in vivo trials registering a positive effect on sperm viability and redox balance. In this study, we provided the definitive evidence that the role of trolox on redox balance maintenance has a direct effect on fertility parameters, such as prolificacy. The effectiveness of antioxidant treatments was tested, for the first time in ovine species, using an integrative and multiparametric approach combining in vivo and in vitro analyses and novel approaches, such as RedoxSYS. These types of strategies should be applied to improve sperm conservation methods and optimize AI technologies upgrading the correlation between in vitro and in vivo analyses.

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre-freezing equilibration times (2-3hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500mm). The highest quality (p<.01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60-85°Cmin-1 ), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100mm) provided the highest (p<.05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified-warmed sperm variables were at their best when the spermatozoa was diluted in TCG-6% egg yolk+100mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p<.05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.

Global declines in seaweed forests are reducing genetic diversity and, consequently, the resilience of these habitats to changing environmental conditions. To conserve genetic diversity and secure these important coastal habitats, new preservation strategies are needed, such as cryopreservation. We aimed to develop a cryopreservation protocol for sperm and germlings of the fucoid seaweed Phyllospora comosa, a species highly vulnerable to anthropogenic stressors. We tested the cytotoxicity of cryoprotectant agents commonly used in seaweed cryopreservation (dimethyl sulfoxide, ethylene glycol, glycerol, methanol and propylene glycol). Non-lethal cytotoxic effects of cryoprotectants on germlings and sperm were observed, including a reduction in length (germlings), motility and concentration (sperm). Freezing trials resulted in post-thaw sperm survival. Best results were observed with 10% glycerol (final, v/v) under a freezing rate of -42.4±5.7°C/min, displaying 18.7% post-thaw motility and 1.5×106 sperm/mL, compared to an initial motility of 44.9% and 6.4×106 sperm/mL in untreated sperm samples. Germlings could not be revived after thawing, despite testing several freezing rates, cryoprotectant concentrations (5-30%), equilibration times (10-60min) and added sugars. Our results demonstrate that live Phyllospora sperm can be recovered following cryopreservation, providing an opportunity to store diverse genotypes for breeding and enhancing genetic diversity in distant populations. However, further work is required to improve the efficiency of cryopreservation protocols and apply them in conservation practices. Our findings provide guidelines for future trials with Phyllospora and other fucoid species to incorporate cryopreservation techniques that help secure genetic diversity in the face of climate change.