斑马鱼精子冻存与复苏实验方法与流程

斑马鱼是研究胚胎发育及其遗传机制的重要模式脊椎动物。目前人们已经积累了大量的突变体和转基因鱼系, 如何安全、妥善地长期保存这些品系是每一个研究斑马鱼的实验室都会面临的问题。精子冻存与复苏技术是目前最为简单有效的一种长期保存斑马鱼遗传品系的方法。应用这一技术不仅可以节省大量的鱼房空间与人力、物力, 使鱼系的使用与保存更加灵活和持久, 更重要的是能够防止珍贵鱼系的意外丢失, 为鱼系保种提供额外的保障。这类方法一般是通过体外挤出精子或研磨精巢获取新鲜的精子, 用适量的冻存液混匀后, 分装保存在液氮罐中。需要时可以随时通过体外授精的方法使精子复苏。经过30年的发展, 随着冷冻保护剂的不断改良和冻存与复苏条件的不断优化, 斑马鱼精子冻存与复苏技术已逐渐成熟。文章简单回顾了斑马鱼精子冻存与复苏技术的历史与发展, 并重点介绍本实验室自2005年以来常规使用的斑马鱼精子冻存与复苏的方法及具体流程。
补充资料 ：冻存与复苏实验指南 [视频]     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.     

Improved recovery of post-thaw motility and vitality of human spermatozoa cryopreserved in the presence of dithiothreitol

Semen was collected in the laboratory from nine healthy donors. The concentrations and the percentages of live and motile spermatozoa in all semen samples were within the normal range. Each sample was diluted with citrate-egg yolk-glycerol medium with and without 5 mM dithiothreitol (DTT). Samples were frozen in liquid nitrogen vapor (?70 °C) for 7 min and subsequently stored in liquid nitrogen. The effect of DTT in cryopreservation of sperm was determined by comparing percentage of motile and live spermatozoa between controls and DTT-treated post-thaw samples. Percentage of motile spermatozoa was determined by two techniques, laser Doppler velocimetry (LDV) and light microscopy. The percentage of live spermatozoa was measured by microscopic evaluation after staining with eosin-nigrosin. It was shown that the addition of DTT to the freezing medium significantly improved the recovery of motile and live spermatozoa in the post-thaw samples. The mean motility recovery, as measured by LDV, was 44.9% in the controls as compared to 73.9% in the DTT-treated samples. Similarly the mean recovery of live spermatozoa in the controls and DTT-treated samples was 66.5 and 86.6%, respectively. Based on these results, a new hypothesis implicating lipid peroxidation in cryoinjury is proposed. It is also suggested that the use of DTT in the freezing medium may offer an advantage over the commonly used techniques of human sperm cryopreservation.     

Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose

Glycerol is not an effective cryoprotectant for rabbit spermatozoa; therefore, rabbit spermatozoa were used as a model for developing cryopreservation procedures for other cell types which also freeze poorly when glycerol is used as the cryoprotectant. Experiments were conducted to 1) compare several published protocols for cryopreserving rabbit spermatozoa; 2) determine if removal of seminal granules, required for flow cytometry analysis, affects the motility of rabbit spermatozoa; and 3) determine if using a combination of cell permeating cryoprotectants (acetamide) with cell nonpermeating cryoprotectants (trehalose and methyl cellulose; MC), can increase the recovery of viable rabbit spermatozoa after cryopreservation. Media containing acetamide as a cryoprotectant were found to be most effective for rabbit spermatozoa. The cryoprotectants ethylene glycol, dimethylsulfoxide and glycerol were not effective for cryopreserving rabbit spermatozoa. Second, rabbit spermatozoa could be centrifuged through a Percoll gradient composed of equal volumes of Prcoll and a HEPES-buffered sperm medium. This centrifugation removed all seminal granules without affecting the percentage of motile spermatozoa after initial sperm dilution (85 vs 74%) or after cryopreservation (35 vs 30%), when sperm were either centrifuged or not centrifuged, respectively. The substitution of trehalose in the cryopreservation medium for raffinose did not improve recovery of motile cells following cryopreservation (P > 0.05). However, addition of MC resulted in higher percentages of motile sperm after cryopreservation (43 vs 31%; P < 0.05). In addition, sperm viability and acrosomal integrity were simultaneously evaluated using flow cytometry. The addition of both trehalose and MC to media containing acetamide resulted in higher percentages of live acrosome-intact cells than acetamide alone (53 vs 37%; P < 0.05). These results indicate that a combination of permeating and nonpermeating cryoprotectants (acetamide, trehalose and MC) were more effective in preserving rabbit spermatozoa than acetamide alone and that analyzing multiple sperm characteristics, by flow cytometry, can assess sperm damage not detected by analyzing sperm motion characteristics.     

Development of methods for cryopreservation of rooster sperm from the endangered breed “Gallina Valenciana de Chulilla” using low glycerol concentrations

Glycerol (11%; v:v) is the cryoprotectant most often used for the cryopreservation of rooster sperm. However, chicken breeds differ in the resistance of their sperm to the cryopreservation process and endangered or local breeds usually present low fertilizing ability when conventional sperm cryopreservation protocols are used. The objective of this study was to optimize the protocol for the cryopreservation of the sperm from the endangered breed “Gallina Valenciana de Chulilla”. For this purpose, 10 pools of semen from 43 roosters of this breed were cryopreserved using 8%, 7%, 6%, or 4% glycerol, and the sperm quality was determined immediately after thawing and in the insemination doses. Lohmann Brown Classic laying hens (n = 40) were used for the insemination trials. The sperm quality after cryopreservation progressively decreased as the glycerol concentration was reduced (P < 0.01); samples frozen using 4% glycerol exhibited the lowest quality (38% total motile sperm and 49% live sperm), and samples frozen using 8% glycerol exhibited the highest quality (67% total motile sperm and 66% live sperm). These differences were also observed after the glycerol was removed (P < 0.01). However, the sperm fertilizing ability was similar for all the treatments (23%–30% fertilized eggs), and increased as the glycerol concentration decreased. In conclusion, semen from roosters frozen using 4% glycerol exhibited lower sperm quality but similar fertilizing ability compared with samples processed using higher glycerol concentrations. These results may provide useful information for developing cryopreservation protocols for other breeds.     

A procedure for Poitou jackass sperm cryopreservation

We have tried to establish sperm banking for the endangered Poitou donkeys. No successful cryopreservation technique had been described for spermatozoa of this species; our preliminary work indicated that a particular medium and procedure may be effective for cryopreservation of Poitou jackass spermatozoa as evaluated by sperm motility, membrane integrity and pregnancy rate after AI with frozen-thawed semen. We found that glutamine at 80 mM and 10% (v/v) quail egg yolk in a basal medium containing 4% (v/v) glycerol (T2-94 medium) improved the post-thaw total and progressive motility and velocity assessed with the automated analyzer ATS-M. The T2-94 medium also preserved the sperm nuclear, acrosom, and plasma membrane integrity as assessed with the acridine orange method, fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) lectin procedure, and hypo-osmotic swelling test, respectively. Semen frozen-thawed in T2-94 medium as used to artificially inseminate. 13 Poitou jennies from the beginning of estrus to ovulation during 4 cycles at a rate of one AI per day. Heigh pregnancies and 3 foals were obtained, but only when the glycerol was removed from sperm before AI. We conclude that the cryopreservation of Poitou jackass semen for sperm banking may succeed by using the T2-94 medium and removing the glycerol post-thaw, but before AI.     

CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM

A simple method for preserving live sea urchin embryos and sperm in liquid nitrogen (LN) wasdeveloped through the use of DMSO as a cryoprotective additive. Samples of late embryos in double test tubes were cooled to— I96°C by two-step freezing, first to — 76°C and then by plunging in LN. In the case of fertilized eggs, samples were previously frozen to — 40°C, at which temperature the samples were kept for 15 min; they were then transferred into LN. After preservation in LN for various lengths of time, samples in the double test tubes were thawed in water at 15°C. The post-thaw survival was more than 90% for late embryos, and about 10% for fertilized eggs. Difference in the length of the cryopreservation period did not affect survival. Post-thaw development in cryopreserved embryos often showed abnormalities in structure. Sperm with 1.5 M DMSO was successfully preserved in LN by a similar method to the one used for cryopreservation of late embryos. Fertilizability in cryopreserved sperm was complete, regardless of the length of the preservation period. Nearly all the eggs fertilized by cryopreserved sperm developed to normal plutei.     

Cryoprotective effect of antifreeze polyamino-acid (Carboxylated Poly-l-Lysine) on bovine sperm: A technical note

In this study, investigating Carboxylated Poly-l-Lysine (CPLL) for its effectiveness as a new cryoprotectant for bovine sperm is aimed. CPLL is an ampholytic polymer compound, has cryoprotective properties similar to those of anti-freeze protein. The cryopreservation medium used for control group consisted of 6.5% (v/v) glycerol, the cryopreservation medium used for experimental group consisted of 3.25% (v/v) glycerol + 0.5% (w/v) CPLL. There was no consequential difference in sperm motility parameter after thawing whereas there was huge distinction for sperm membrane integrity rate (control vs experimental; 49.6 vs 60.7%, P < 0.01). Conception rate of artificial insemination of experimental group was significantly higher than that of control group (79.0% vs 53.1%, P < 0.01). These results suggest CPLL has protected sperm membrane and leads to improve fertility. This is the first report using CPLL for bovine sperm cryopreservation, it is also expected CPLL can be applied to other animal species.     

Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18% raffinose and 3% skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.Here we demonstrate a newly developed I•Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (> 60%) and rapid progressive motility (> 45%) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later recovered by IVF.     

Cryopreservation of murine spermatozoa

Cryopreservation of mouse sperm provides an economic option for preserving the large number of mouse strains now being generated by transgenic and targeted mutation methodologies. The ability of a spermatozoan cell to survive cryobiological preservation depends on general biophysical constraints that apply to all cells, such as the avoidance or minimization of the formation of intracellular ice during cooling. This action is typically achieved by use of cryoprotectant substances and by controlled, slow rates of cooling. Superimposed on those general constraints may be special characteristics of mouse spermatozoa, such as more narrow, osmotically driven volume tolerance limits and the fact that relatively successful freezing can be obtained without the use of a permeating cryoprotective agent. The lack of important information regarding sperm cells fundamental cryobiological properties, including their osmotic and membrane permeability characteristics, has hindered progress in developing anything but empirically derived methods. Genetic differences between inbred mouse strains are reflected in motility and fertility characteristics of mouse sperm and contribute to the difficulty of developing successful cryopreservation methods. Recovery of live young from frozen sperm has been much more successful with sperm from hybrid mice than from most inbred strains. There have been no published reports of successful cryopreservation of rat sperm. Nevertheless, in mice, success in deriving live young from intracytoplasmic sperm injection using sperm frozen under suboptimal conditions raises the possibility of using this technique for the ultimate rescue of sperm regardless of the success of cryopreservation. This technique, however, requires additional development and verification of its efficacy before it will be suitable for general laboratory use. Although cryopreservation of mouse sperm is not yet universally successful, it can be used reliably to supplement cryopreservation of embryos and other germline cells or tissues for preserving biomedically important strains of mice for research.     

Workshop report: Cryopreservation of aquatic biomedical models

The genetic resources of aquatic biomedical model organisms are the products of millions of years of evolution, decades of scientific development, and hundreds of millions of dollars of research funding investment. Genetic resources (e.g., specific alleles, transgenes, or combinations) of each model organism can be considered a form of scientific wealth that can be accumulated and exchanged, typically in the form of live animals or germplasm. Large-scale maintenance of live aquatic organisms that carry these genetic resources is inefficient, costly, and risky. In situ maintenance may be substantially enhanced and backed up by combining cryopreserved germplasm repositories and genetic information systems with live animal culture. Unfortunately, cryopreservation has not advanced much beyond the status of an exploratory research for most aquatic species, lacks widespread application, and methods for successful cryopreservation remain poorly defined. For most aquatic species biological materials other than sperm or somatic cells are not comprehensively banked to represent and preserve a broad range of genetic diversity for each species. Therefore, new approaches and standardization are needed for repository-level application to ensure reproducible recovery of cryopreserved materials. Additionally, development of new technologies is needed to address preservation of novel biological materials, such as eggs and embryos of aquatic species. To address these goals, the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health (NIH) hosted the Cryopreservation of Aquatic Biomedical Models Workshop on January 7 to 8, 2017, in conjunction with the 8th Aquatic Animal Models of Human Disease Conference in Birmingham, Alabama. The goals of the workshop were to assess the status of germplasm cryopreservation in various biomedical aquatic models and allow representatives of the scientific community to develop and prioritize a consensus of specific actionable recommendations that will move the field of cryopreservation of aquatic resources forward. This workshop included sessions devoted to new approaches for cryopreservation of aquatic species, discussion of current efforts and approaches in preservation of aquatic model germplasm, consideration of needs for standardization of methods to support reproducibility, and enhancement of repository development by establishment of scalable high-throughput technologies. The following three broad recommendations were forwarded from workshop attendees:1Establish a comprehensive, centralized unit (“hub”) to programmatically develop training for and documentation of cryopreservation methods for aquatic model systems. This would include development of species-specific protocols and approaches, outreach programs, community development and standardization, freezing services and training of the next generation of experts in aquatic cryopreservation.2Provide mechanisms to support innovative technical advancements that will increase the reliability, reproducibility, simplicity, throughput, and efficiency of the cryopreservation process, including vitrification and pipelines for sperm, oocytes, eggs, embryos, larvae, stem cells, and somatic cells of all aquatic species. This recommendation encompasses basic cryopreservation knowledge and engineering technology, such as microfluidics and automated processing technologies.3Implement mechanisms that allow the various aquatic model stock centers to increase their planning, personnel, ability to secure genetic resources and to promote interaction within an integrated, comprehensive repository network for aquatic model species repositories.     

Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold-stored epididymides

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.     

Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status, and future directions

Molecular genetics and developmental biology have created thousands of new strains of laboratory animals, including rodents, Drosophila, and zebrafish. This process will accelerate. A decreasing fraction can be maintained as breeding colonies; hence, the others will be lost irretrievably unless their germplasm can be cryopreserved. Because of the increasingly critical role of cryopreservation, and because of wide differences in the success with which various forms of germplasm can be cryopreserved in various species, the National Institutes of Health National Center for Research Resources held a workshop on April 10-11, 2007, titled "Achieving High-Throughput Repositories for Biomedical Germplasm Preservation." The species of concern were mouse, rat, domestic swine, rhesus monkey, and zebrafish. Our review/commentary has several purposes. The first is to summarize the status of the cryopreservation of germplasm from these species as assessed in the workshop. The second is to discuss the nature of the major underlying problems when survivals are poor or highly variable and possible ways of addressing them. Third is to emphasize the importance of a balance between fundamental and applied research in the process. Finally, we assess and comment on the factors to be considered in transferring from a base of scientific information to maximally cost-effective processes for the preservation of this germplasm in repositories. With respect to the first purpose, we discuss the three methods of preservation in use: slow equilibrium freezing, rapid nonequilibrium vitrification, and the use of intracytoplasmic sperm injection to achieve fertilization with sperm rendered nonviable by other preservation treatments. With respect to the last purpose, we comment on and concur with the workshop's recommendations that cryopreservation largely be conducted by large, centralized repositories, and that both sperm (low front-end but high rederivation costs) and embryos (high front-end but modest rederivation costs) be preserved.     

Elective frozen-thawed embryo transfer (FET) in women at risk for ovarian hyperstimulation syndrome

Elective cryopreservation of cultured embryos has become a treatment option for women at risk for ovarian hyperstimulation syndrome (OHSS). The aim of our study was to investigate the outcome of elective cryopreservation and consecutive frozen-thawed embryo transfer (FET) in a large IVF clinic in Austria. A total of 6104 controlled ovarian hyperstimulation cycles (COH) were performed on 2998 patients including 200 patients (6.7%) who were undergoing elective cryopreservation and FET due to high risk of OHSS. We estimated the cumulative live birth rate using the Kaplan-Meier method and evaluated independent predictors for successful live births with a Cox model. A total of 270 frozen-thawed embryo transfers were performed on 200 patients with up to 4 transfers per patient. The first embryo transfer showed a live birth rate of 42.0%, the second transfer showed a cumulative rate of 58.5%. After a total of 4 FETs from the same COH cycle, a cumulative live birth rate of 61.0% per COH cycle could be achieved. Four cases of OHSS occurred amongst these patients (2.0%), all of them of moderate severity. Multivariate analysis identified maternal age, the use of assisted hatching and the number of embryos transferred at the blastocyst stage as independent predictors for cumulative live birth. Our study clearly suggests that elective FET is safe and shows excellent cumulative live birth rates. This concept can, therefore, be used to avoid the severe adverse events caused by COH and the inefficient use of cultured embryos.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm

Semen cryopreservation is widely used in clinical medicine, agriculture, aquaculture and biomedical research, but it is an inefficient technique that induces extensive cytoplasmic damage and loss of fertilising ability. Whether any genetic damage (i.e. DNA strand breakage or mutation) is also induced is still unclear. However, previous data has indicated that this is likely. The present study was designed to explore this possibility further by using inhibitors of the DNA repair system to block DNA repair in embryos derived from cryopreserved spermatozoa. If cryopreservation causes strand breaks in sperm DNA it might be expected that inhibition of a repair enzyme such as poly(ADP-ribose) polymerase (PARP) would enhance any such negative effect of cryopreservation. To check this hypothesis 3-aminobenzamide (3-AB) was used as an inhibitor of PARP. Weather loach (Misgurnus fossilis) eggs were fertilised using cryopreserved as well as fresh spermatozoa. Embryos derived from cryopreserved spermatozoa were exposed to 10 mM 3-AB for 2 h after fertilisation. The experiments were carried out using 43,544 embryos from 5 females and 10 males. Embryo survival was evaluated at different stages until the hatching stage. Sperm cryopreservation significantly decreased embryo survival (53.6+/-2.79% compared to 76.97+/-2.79% of control; P<0.01). The addition of 3-AB to the medium with embryos derived from cryopreserved sperm further decreased embryo survival from 53.6+/-2.79% to 46.1+/-2.79% (P<0.01) whereas there was no adverse effect of 3-AB exposed embryos derived from fresh sperm (76.97+/-2.79% of control compared to 74.8+/-2.79% of control+3-AB). The effect of 3-AB provides indirect evidence that cryopreservation might induce instability in sperm DNA, and that such damage can be repaired by the oocyte repair system after fertilisation.     

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes’ zona pellucida

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 μM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.     

A comparison of sucrose,saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm

Previous studies on cane toad (Bufo marinus; Bufonidae; Anura) sperm cryopreservation were extended to compare the effects of cryopreservation in established sucrose (non-ionic) diluents with cryopreservation in ionic diluents containing amphibian Ringer solutions (with and without egg-yolk). In addition, methanol was tested as a cryoprotectant for B. marinus sperm for the first time. Twenty-seven cryoprotective solutions were trialled, with each containing one of the three diluents [10% (w/v) sucrose, simplified amphibian Ringer (SAR) or SAR/egg-yolk], with one of the three cryoprotectants (Me(2)SO, glycerol, or methanol) at one of the three concentrations (10%, 15%, or 20% v/v). Sperm were collected by maceration of testes into cryoprotective solutions with post-thaw recovery assessed as the percentage of motile sperm and the degree (vigour) of motility. Percentage motility was the most sensitive measure of post-thaw recovery. The recovery of motility was lowest in Ringer (SAR) diluents and highest in sucrose diluents, with improved motility in SAR diluents when egg-yolk was added. Methanol was the poorest cryoprotectant and Me(2)SO the most effective. Methanol at high concentrations was shown to support recovery in sucrose diluent but not in SAR, although its effectiveness in SAR was improved by egg-yolk. Overall, the efficacy of diluents in supporting a high percentage of sperm recovery was in declining order: sucrose>SAR/egg-yolk>SAR diluents, and with cryoprotectants: Me(2)SO>glycerol>methanol. In conclusion, SAR offers less potential as a diluent than sucrose, presumably due to the presence of inorganic ions.     

Morphometric changes in goat sperm heads induced by cryopreservation

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.