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Methods for Cryopreservation of Guinea Fowl Sperm  [PDF]
éva Váradi, Barbara Végi, Krisztina Liptói, Judit Barna
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062759
Abstract: Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet) and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide). The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining). Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols (based on in vitro assessment of post-thaw semen quality) were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively). During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.
Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART  [PDF]
Marlea Di Santo,Nicoletta Tarozzi,Marco Nadalini,Andrea Borini
Advances in Urology , 2012, DOI: 10.1155/2012/854837
Abstract: Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates. 1. Introduction The procedure that makes it possible to stabilize the cells at cryogenic temperatures is called cryopreservation, also known as an applied aspect of cryobiology or the study of life at low temperatures. Many advances in the cryopreservation technology have led to the development of methods that allow for low-temperature maintenance of a variety of cell types including male and female gametes, small multicellular organisms, and even more complex organisms such as embryos. Cryopreservation of human spermatozoa—introduced in the 1960’s [1]—has overcome many space and time limitations and now forms integral part of assisted reproduction technologies (ARTs). This technique becomes particularly important in cases of preservation of male fertility before radiotherapy or chemotherapy [2] which may lead to testicular failure or ejaculatory dysfunction. In fact, semen cryostorage seems to be the only proven method that may offer these couples a chance of having children in the future: cancer therapy could in fact lead to damage, resulting in subfertility or sterility due to gonad removal or permanent damage to germ cells caused by adjuvant therapy. In particular, the risk associated to therapy depends on several factors: the age of the patient at the time of treatment, the dose, site, and type of treatment [3]. Also some nonmalignant diseases, such as diabetes and
Effect of Trolox addition to cryopreservation media on human sperm motility
Mohammad Baqer Minaei,Mohammad Barbarestani,Saeid Nekoonam,Mir Abbas Abdolvahabi
Iranian Journal of Reproductive Medicine , 2012,
Abstract: Background: Sperm parameters and motion kinetics are affected by cryopreservation. Objective: The main purpose of the current study was to determine the effect of different concentrations of Trolox as an antioxidant to freezing-thawing procedure on human sperm kinematic parameter. Materials and Methods: Semen was collected from 20 normal donors and divided into five aliquots prior to cryopreservation. The first aliquot was analyzed by computer-assisted sperm analysis (CASA). Other aliquots were mixed with cryo-protective agent containing 0, 20, 40, and 80 μmol Trolox and treated samples were cryopreserved in liquid nitrogen. After two weeks samples were thawed and sperm motion kinematics was measured by CASA. Percent motility (Mot), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH) were compared before and after freeze. Results: Addition of 40μmol Trolox resulted in significantly higher (p<0.05) post thaw VCL, VSL and VAP compared to other groups. Therefore the percentage of post thaw motile spermatozoa were significantly higher (p<0.01). Conclusion: The supplementation of Trolox significantly improved the post-thawed human semen quality, especially progressive motility and average path velocity.
Sperm Cryopreservation in Cancer Patients  [PDF]
Skrzypek J,Krause W
Journal für Reproduktionsmedizin und Endokrinologie , 2007,
Abstract: Cancer patients are treated multiprofessionally by medical specialists. We predominantly see testicular tumors and haematological malignancies. From 2002 to 2006, 82 cancer patients presented for cryopreservation of semen in our department. Age distribution and various oncological diseases are consistent with epidemiological data. In all malignancies, semen quality was reduced, with a pronounced reduction in testicular tumors. In cancer treatment, a variety of therapeutic strategies such as surgery, radiotherapy, and chemotherapy are well established. Especially radiotherapy and chemotherapy are potentially hazardous for spermatogenesis, the individual risk, however, cannot be predicted. Therefore, cryopreservation of spermatozoa should be offered to all patients prior to cancer treatment.
Role of Membrane Lipid Fatty Acids in Sperm Cryopreservation  [PDF]
Rajes Mandal,Damodar Badyakar,Jitamanyu Chakrabarty
Advances in Andrology , 2014, DOI: 10.1155/2014/190542
Abstract: Lipid is an important constituent of cell membrane. Membrane lipid composition of spermatozoa has been correlated to different function. Many researchers have related membrane lipid with survival success after cryopreservation or cold shock. Sperm maturation and acrosome reactions are natural phenomenon, but cryopreservation or cold shock is not. Therefore, sperm cells are not programmed for such change and undergo stress. So the change in membrane lipid composition due to cold shock or cryopreservation may be looked upon as response of spermatozoa to a certain stressed condition. A significant body of research worked on the relationship between membrane lipid and fatty acid composition and ability of cell to tolerate adverse change in temperature. However, as the approach of different research groups was different, it is very difficult to compare the changes. Studies have been done with different species, ejaculated/seminal or epididymal sperm. Lipid analyses have been done with whole cell membrane isolated by different methods. Fatty acids estimated were from whole cell, plasma membrane, head membrane, or phospholipids. The cryopreservation condition, media composition, and diluents/cryoprotectants were also different. At this onset a comprehensive review is needed to cover changes of sperm membrane lipid composition of different species under different cryopreservation conditions. 1. Introduction Sperm cell is unique in many respects including structure and function. It is capable of fertilizing egg; it functions in a body different from its origin and gender. Its plasma membrane is also different from most other cell membranes in lipid composition. It contains high amount of polyunsaturated fatty acids (PUFA), especially diPUFA (phospholipids esterified with two PUFA), which is found only in sperm, retina, and certain brain areas [1, 2]. In particular, PUFA are known to contribute to membrane fluidity and flexibility [3–5]. Membrane lipid composition has been related to their specific functions, because it promotes the creation of microdomains with different fluidity, fusogenicity, and permeability characteristics [2], required for reaching and fusing with the oocyte. Phospholipids are the most representative lipid fraction of the sperm cell membranes, of which phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin are the major components [6]. Lipid and fatty acid composition of sperm cells differ not only for different animals [7, 8] but also for different species [9–12], even for fertile and subfertile population of same species
Computer assisted semen analysis for quantification of motion characteristics of bull sperm during cryopreservation cycle  [cached]
M. N. Sundararaman,J Kalatharan,K Thilak Pon Jawahar
Veterinary World , 2012, DOI: 10.5455/vetworld.2012.723-726
Abstract: Aim: The study was undertaken to quantify and to analyze the changes in the motion characteristics of bull spermatozoa during various stages of cryopreservation cycle. Materials and Methods: Using computer assisted semen analysis (CASA) technique, 26 ejaculates, collected from two Jersey bulls were analyzed for motility, head behaviour and swimming pattern of spermatozoa on dilution, pre-freeze and post-thaw stages of cryopreservation. French straw technique was employed for deep-freezing of semen using liquid nitrogen. Results: Equilibration of diluted semen at 5 C has significantly (P< 0.01) reduced sperm motility, progressive motility, path velocity, and progressive velocity. Beat cross frequency was also affected significantly (P<0.05) by equilibration. Freezing and thawing processes drastically affected all the motility, velocity and head behaviour characteristics (P< 0.01). Conclusion: CASA facilitate objective evaluation sperm motion characteristics. Adoption of CASA technique has the potential for improvements in evaluation of semen thereby the quality of frozen semen for fertility can be enhanced. [Vet World 2012; 5(12.000): 723-726]
Zebrafish Reproduction: Revisiting In Vitro Fertilization to Increase Sperm Cryopreservation Success  [PDF]
Mary Hagedorn,Virginia L. Carter
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0021059
Abstract: Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 106 cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 106 motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization.
Effects of Dimethyl Sulfoxide, Ethylene Glycol, Propylene Glycol and Glycerol on Cryopreservation of Wild Tree Shrew (Tupaia belangeri Chinese) Cauda Epididymal Sperm
Shu-Huang Ping,Feng Yue,Cai-Yun Wang,Ying Luo,Wei Si,Shi-Hua Yang
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2012.3568.3574
Abstract: The objective was to examine the effect of Permeable Cryoprotectant Agents (CPAs) on sperm cryopreservation of tree shrew. Epididymal sperm were surgically harvested from captured wild male tree shrews and cryopreserved with Tes-Tris-Egg yolk based cryodiluent (TTE) containing either of the four CPAs, Dimethyl Sulfoxide (DMSO), Ethylene Glycol (EG), Propylene Glycol (PG) and Glycerol (Gly) at concentrations of 1, 3, 6 and 10%, respectively. Sperm motility, acrosome integrity and fertility were assessed. In Experiment 1, sperm equilibrated at 4°C in TTE containing 1, 3 and 6% DMSO, respectively showed similar motility to that in TTE without CPA (p>0.05). Following the increase of concentration of CPAs and equilibration time (30-90 min), the other CPAs reduced sperm motility (p<0.05). In Experiment 2, sperm frozen in TTE containing 3% DMSO showed the highest post-thaw motility (p<0.05) and recovery rate of motility (p<0.05) among groups. In Experiment 3, there were no differences in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes inseminated with fresh and thawed sperm frozen in TTE containing 3% DMSO (p>0.05). In conclusion, among the permeable CPAs tested, DMSO provided the best cryoprotective ability for captured wild tree shrew epididymal sperm.
Sperm Cell Population Dynamics in Ram Semen during the Cryopreservation Process  [PDF]
Manuel Ramón, M. Dolores Pérez-Guzmán, Pilar Jiménez-Rabadán, Milagros C. Esteso, Olga García-álvarez, Alejandro Maroto-Morales, Luis Anel-López, Ana J. Soler, M. Rocío Fernández-Santos, J. Julián Garde
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0059189
Abstract: Background Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. Methodology/Principal Findings We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. Conclusions/Significance Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.
Effects of different antioxidants on cryopreservation of Jinhua boar sperm

WANG Zheng-guang,HUA Jian-qing,CHEN Xiao-feng,JIN Gen-lu,YU Song-dong,

浙江大学学报(农业与生命科学版) , 2010,
Abstract: To compare the effects of different antioxidant concentrations of glutathione (GSH), green tea polyphenols (GTPs), vitamin E (V_E) and superoxide dismutase (SOD) on cryopreservation of Jinhua boar sperm, freezing extenders were supplemented with the GSH (0.125, 0.25 and 0.5 mmol·L~(-1)), GTPs (10, 15 and 20 mmol·L~(-1)), V_E (1, 2 and 4 mg·mL~(-1)) and SOD (1000, 2000 and 4000 IU), respectively. The boar sperms were collected and frozen with extenders containing above antioxidants. Sperm motility after 4 ℃cooling, sperm motility, membrane integrity and acrosome integrity after thawing were examined in four treatment groups. The results show that sperm motility after 4 ℃cooling, sperm motility and membrane integrity after thawing were increased significantly by supplemented boar freezing extenders with 15 mmol·L~(-1) GTPs, 2 mg·mL~(-1) V_E or 4000 IU SOD, respectively (P< 0.05).
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