Assessment of the stallion sperm acrosome in two different extenders: Spermac stain in comparison with PNA/PSA/PI triple-staining.

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple-staining detected by flow cytometry with regard to method agreement for detecting non-intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r = .77, p < .001) and fair correlations for Gent (r = .49, p < .001). Interestingly, flow cytometry detected more non-intact acrosomes in EquiPlus than in Gent (p < .001), whereas the Spermac stain showed no differences (p = .902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non-intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results.

Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology.

In brief: The containers used in cell cryopreservation are essential to maintain cell integrity and viability after thawing. This paper reveals the methodology of using biodegradable containers for fish sperm cryopreservation. Cryopreserved sperm in biodegradable containers showed high fertility capability. Biodegradable capsules could be alternative containers to plastic straws for sperm cryopreservation. Abstract: Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from 12South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of the tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

Assessment of semen quality and anti-oxidative enzyme activity between bovine sex-sorted and non-sex-sorted frozen-thawed semen.

In the current study, the difference between the sex-sorted and non-sex-sorted frozen semen of Holstein Friesian breed cattle was investigated. Significant variation (p < .05) was found in the semen quality parameters such as motility; vitality; acrosome integrity rate; the anti-oxidative enzyme activity including GSH (glutathione); SOD (superoxide dismutase); CAT (catalase); GSH-Px (glutathione peroxidase) and the rate of fertilization. The results showed that the sperm acrosome integrity and motility of the non-sorted sperm were higher compared to sex-sorted sperm (p < .05). The linearity index and mean coefficient analysis revealed that the percentage of 'grade a' in sex-sorted sperm were significantly (p < .05) lower than non-sorted sperm. Interestingly, low SOD level and high CAT level was found in the non-sexed semen than in the sexed semen (p < .05). Furthermore, the GSH and GSH-Px activity in the sexed semen was found lower than the non-sexed semen (p < .05). In conclusion, sperm motility characteristics were lower in sex-sorted semen than in non-sex-sorted semen. This might be related to the complex process of sexed semen production, which could reduce sperm motility and movement characteristics, acrosomal integrity, CAT, SOD, GSH and GSH-Px, and finally lead to the decline in the fertilization rate.

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

Quality assessment of cryopreserved Portuguese oyster (Crassostrea angulata) sperm through ultrastructural and flow cytometry analysis.

We investigated the effects of cryopreservation on the quality of Portuguese oyster (Crassostrea angulata) sperm, which were examined before and after freezing; sperm motility, fertilizing capacity, and ultrastructural morphology were analyzed. The motility percentage and fertilizing capacity of the cryopreserved sperm (mean ± standard error) were 16% ± 1% and 17% ± 8%, respectively. In the pre-freezing sperm, these were 58% ± 2% and 76% ± 4%, respectively. The sperm sustained substantial morphological and ultrastructural damage during cryopreservation. The morphological changes varied considerably in nature and extent, ranging from no apparent damage to virtual disintegration. Sperm were stained with fluorescent dyes to assess viability, plasma membrane integrity, mitochondrial activity, acrosomal membrane integrity, oxidation level, and DNA fragmentation and examined through flow cytometry. The methods used for the flow cytometry assays were slightly modified from those used for evaluating the semen quality of livestock. Relative to the pre-freezing sperm, the frozen-thawed sperm exhibited lower acrosomal membrane integrity (acrosomal damage, 59.86 ± 5.29; P < 0.05) and substantially higher oxidation levels (free radicals, 60.06 ± 0.82; P < 0.003). Oxidation level was found to be the most sensitive indicator of cryodamage. Along with ultrastructural analysis, we used flow cytometry to measure the qualitative and quantitative characteristics of Portuguese oyster sperm before and after cryopreservation rapidly, objectively, and accurately. This is the first study to assess the quality of Portuguese oyster sperm through these methods.

A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm.

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.

Preliminary Assessment of the Mucosal Toxicity of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis) Essential Oils on Novel Porcine Uterus Models.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2-0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.

Assessment of a germplasm bank for the autochthonous cattle breed Asturiana de la Montaña: Extender (Biociphos vs. BIOXCell) affected sperm quality but not field fertility.

Semen banking is critical to preserving rare and autochthonous breeds. However, protocols can change with time, leaving heterogeneous semen batches. The objective of this study was to assess differences in sperm quality and field fertility. We report differences between batches frozen with the Biociphos and BIOXCell extenders in the Asturiana de la Montaña cryobank (autochthonous and endangered breed, Northern Spain). Doses from 48 bulls were analysed by CASA and flow cytometry. The 85-days non-return rates from AI records were used to assess the fertility of 23,853 AI. BIOXCell showed higher quality post-thawing. Differences increased after a 5-hr incubation at 37°C, and Biociphos yielded doses with lower resilience. Field fertility did not differ between extenders (Biociphos: 57.4% ± 1.2; BIOXCell: 56.6% ± 3.0), possibly because of AI protocols compensating for differences in quality. However, this needs to be confirmed by controlled intervention studies. In conclusion, batches frozen with Biociphos may require specific strategies for compensating for the lower sperm quality. Regular surveys and evaluation of cryobank procedures may be useful to characterizing stored batches and defining strategies to guaranteeing success in their future use.

Fish Sperm Assessment Using Software and Cooling Devices.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility. Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

Sperm membrane integrity and stability after selection of cryopreserved ovine semen on colloidal solutions.

The aim of this study was to evaluate the effect of four methods of sperm selection, on the integrity and stability of the plasma membrane, integrity of the acrosomal membrane and spermatic morphology in frozen/thawed ovine semen. Two types of colloidal silica: colloidal silica-silane and colloidal silica-polyvinylpyrrolidone (PVP), and two aliquots: 1 and 4 ml, were used for sperm selection. Probes FITC-PSA and PI were used to measure the integrity of the plasma and acrosomal membranes. Plasma membrane stability was measured, using fluorescent probes M540 and YOPRO1. Effective reduction in the incidence of spermatozoa with acrosomal pathologies was only achieved using 1 ml colloidal silica-silane. All methods were efficient in select viable and unreacted spermatozoa. Only methods using 1 ml of silica were efficient in decrease spermatozoa stained by PI (death). Methods using silica colloidal-silane were more efficient to decrease apoptotic cells after selection when compared to silica colloidal-PVP. In conclusion, sperm selection in colloidal silica-silane and colloidal silica-PVP improved sperm quality when compared to the controls. The method using 1 ml of colloidal silica-silane is the preferred method because its effectiveness and lower cost.

Prevalence and Predictors of Sperm Banking in Adolescents Newly Diagnosed With Cancer: Examination of Adolescent, Parent, and Provider Factors Influencing Fertility Preservation Outcomes.

Purpose To estimate the prevalence of sperm banking among adolescent males newly diagnosed with cancer and to identify factors associated with banking outcomes. Patients and Methods A prospective, single-group, observational study design was used to test the contribution of sociodemographic, medical, psychological/health belief, communication, and developmental factors to fertility preservation outcomes. At-risk adolescent males (N = 146; age 13.00 to 21.99 years; Tanner stage ≥ 3), their parents, and medical providers from eight leading pediatric oncology centers across the United States and Canada completed self-report questionnaires within 1 week of treatment initiation. Multivariable logistic regression was used to calculate odds ratios (ORs) and 95% CIs for specified banking outcomes (collection attempt v no attempt and successful completion of banking v no banking). Results Among adolescents (mean age, 16.49 years; standard deviation, 2.02 years), 53.4% (78 of 146) made a collection attempt, with 43.8% (64 of 146) successfully banking sperm (82.1% of attempters). The overall attempt model revealed adolescent consultation with a fertility specialist (OR, 29.96; 95% CI, 2.48 to 361.41; P = .007), parent recommendation to bank (OR, 12.30; 95% CI, 2.01 to 75.94; P = .007), and higher Tanner stage (OR, 5.42; 95% CI, 1.75 to 16.78; P = .003) were associated with an increased likelihood of a collection attempt. Adolescent history of masturbation (OR, 5.99; 95% CI, 1.25 to 28.50; P = .025), banking self-efficacy (OR, 1.23; 95% CI, 1.05 to 1.45; P = .012), and parent (OR, 4.62; 95% CI, 1.46 to 14.73; P = .010) or medical team (OR, 4.26; 95% CI, 1.45 to 12.43; P = .008) recommendation to bank were associated with increased likelihood of sperm banking completion. Conclusion Although findings suggest that banking is underutilized, modifiable adolescent, parent, and provider factors associated with banking outcomes were identified and should be targeted in future intervention efforts.

Cryopreservation of stallion semen: laboratory assessment of sperm injuries after cushioned centrifugation and freezing with conventional and alternative directional freezing methods.

Fresh 36 ejaculates of 13 stallions were split into two volumes, centrifuged with and without cushion and frozen with Conventional and two prototype, Drum and Directional, methods using 0.5 ml straws for the Conventional and Drum, and 2 ml flat straws for both the Drum and Directional. Cushioned centrifugation increased total motility (61.2 ± 18.6% vs. 57.5 ± 18.6%; P < 0.001) and mean velocity (84.3 ± 15.6% vs. 83.2 ± 13.8%; P < 0.05) when compared to not cushioned centrifugation, estimated after cooling the sperm at 4°C for 90 min before freezing. Cushioned centrifugation also increased (P < 0.001) spermatozoa with polarized mitochondrial membranes (46.8 ± 11.4% vs. 43.4 ± 10.6%) and intact plasmatic/acrosomal membranes (41.0 ± 11.2% vs. 38.5 ± 11.3%) of frozen/thawed sperm, with respect to not cushioned centrifugation. However, no effects of the centrifugation were evidenced for classical kinetic parameters. Flat straws had negative effect for almost all the parameters analyzed at thawing (T,) and after 3 hours' incubation at 37°C (T1), while the Drum method with Paillettes did not show appreciable affects. The variability among stallions was relevant (5% to 69% variance for kinetics and membrane status), while the variability among ejaculates was minor (9% to 28%). Factorial analysis identified three relevant, factors with different informational content: Factor 1 represented by membranes status, Factor 2 by kinetics estimated at T0, and Factor 3 by kinetics estimated at T1. Cushioned centrifugation had some beneficial effects for the membrane status of the frozen/thawed sperm, while the use of flat straws needs to be improved.

Cryopreservation of Piau-Breed Wild Boar Sperm: Assessment of Cooling Curves and Centrifugation Regimes.

This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post-thaw viability of Piau-breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800 g for 10 min and 2400 g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour - freezing 1 and automated cooling using a programmed freezing machine - freezing 2) were tested. Therefore, the treatments were divided into M3 - centrifugation at 2400 g for 3 min and freezing 2; M10 - centrifugation at 800 g for 10 min and freezing 2; R3 - centrifugation at 2400 g for 3 min and freezing 1; and R10 - centrifugation at 800 g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post-thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo-osmotic test (HO), sperm-egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post-thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.

Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment.

Due to reduced fertility, cryopreserved semen is seldom used for commercial porcine artificial insemination (AI). Predicting the fertility of individual frozen ejaculates for selection of higher quality semen prior to AI would increase overall success. Our objective was to test novel and traditional laboratory analyses to identify characteristics of cryopreserved spermatozoa that are related to boar fertility. Traditional post-thaw analyses of motility, viability, and acrosome integrity were performed on each ejaculate. In vitro fertilization, cleavage, and blastocyst development were also determined. Finally, spermatozoa-oviduct binding and competitive zona-binding assays were applied to assess sperm adhesion to these two matrices. Fertility of the same ejaculates subjected to laboratory assays was determined for each boar by multi-sire AI and defined as (i) the mean percentage of the litter sired and (ii) the mean number of piglets sired in each litter. Means of each laboratory evaluation were calculated for each boar and those values were applied to multiple linear regression analyses to determine which sperm traits could collectively estimate fertility in the simplest model. The regression model to predict the percent of litter sired by each boar was highly effective (p < 0.001, r(2) = 0.87) and included five traits; acrosome-compromised spermatozoa, percent live spermatozoa (0 and 60 min post-thaw), percent total motility, and the number of zona-bound spermatozoa. A second model to predict the number of piglets sired by boar was also effective (p < 0.05, r(2) = 0.57). These models indicate that the fertility of cryopreserved boar spermatozoa can be predicted effectively by including traditional and novel laboratory assays that consider functions of spermatozoa.

Cationic synthetic peptides: assessment of their antimicrobial potency in liquid preserved boar semen.

Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.

In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen.

Soybean lecithin is a suitable plant-based cryoprotectant for freezing ruminant sperm. Optimum level of lecithin was not clear for goat semen cryopreservation. The objective of this study was to investigate the effects of different levels of soybean lecithin in semen extender on post-thaw sperm quality including CASA-motion parameters, viability, plasma membrane integrity and lipid peroxidation. Semen samples were collected from 4 Mahabadi bucks using an artificial vagina. Different concentrations of soy lecithin (SL, 0.5%, 1%, 1.5%, 2% and 2.5% w/v) were compared to 15% (v/v) egg yolk-based extender (TR-EY). No significant difference was observed for sperm progressive motility, viability or plasma membrane integrity in 1.5% SL media (33.8%, 66%, and 62.7%, respectively) and TR-EY medium (35.4%, 67.2%, and 64.9%, respectively). Sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) and rapid spermatozoa were improved with extender containing 1% and 1.5% SL, compared to TR-EY extender. Furthermore, egg yolk produced significantly higher malondialdehyde (4.02±0.21) than other groups. Results suggest that the optimal lecithin concentration in the semen extender was 1.5% and also soy lecithin can substitute for egg yolk during cryopreservation for caprine sperm.

Comparison of membrane-permeant fluorescent probes for sperm viability assessment in the ram.

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR-14; Hoechst-33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR-14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane-affected sperm (semen treated with three cycles of freezing to -20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma-intact sperm determined by acridine orange and SYBR-14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.

Delayed childbearing: determining responsibilities for prime gamete quality.

Delayed parenting affects fertility in women and in men, and cryopreservation of oocytes and sperm is becoming the latest trend as a solution for those who want or need to postpone procreation, in an attempt to avoid the damage medical conditions or time itself produces in gametes. Although "social freezing" is considered legitimate, its ethical and social aspects are in need of an overdue medical, public and legal debate. Assisted reproduction and cryopreservation, in combination with womb outsourcing, have opened the door to biological ectogenesis and the subsequent question of whether delayed childbearing means we should formally separate procreation from sexual activity. This article briefly summarizes what cryotechniques are capable of presently and in the near future, to separate fact from fiction. It names the implications for and discusses the practically virgin subject of the underlying responsibilities of delayed parenting techniques towards the child-to-be-not only the unborn but also the not-yet-conceived child. Considering the medical, economic, legal and social consequences of these rapidly growing developments in reproduction, several reasons point at the need to formally separate procreation from sexual activity, specifying responsibilities in the first while respecting personal choice in the second.

Long-term preservation of freeze-dried mouse spermatozoa.

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.

Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender.

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.