Sperm Cryopreservation Techniques Research Articles

Cryopreservation of Yesso scallop (Patinopecten yessoensis) sperm

The germplasm of farmed Yesso scallop Patinopecten yessoensis in China has deteriorated since its introduction more than 40 years ago. The main aim of this study is to develop a sperm cryopreservation technique to assist the newly established program to manage this issue in China. This study investigated the factors important to the development of a non-programmable sperm cryopreservation technique in this species, including cryoprotectant agent [CPA; dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycine, sucrose, glucose and trehalose], equilibration time, rack height and thawing temperature. A low post-thaw sperm fertilization rate of 27.67 ± 2.52% was produced with the parameters optimized with the permeable CPAs (DMSO, PG and EG) only: 6% DMSO, 10 min equilibration time, 5 cm rack height and 30 °C thawing temperature. This rate was further improved to 44.00 ± 2.00% by adding 3% glucose into 6% DMSO. This addition had also improved the integrities of post-thaw sperm DNA, plasma membrane and acrosome, mitochondrial membrane potential and activities of some enzymes. Results from this study also showed that the post-thaw sperm morphology and ultrastructure were intensively compromised. In addition, the sperm agglutination found in the newly spawned sperm might be one of the phenomena resulted from the so-called germplasm deterioration, especially in the stock used in this study. The further exacerbation of agglutination after cryopreservation would also compromise the post-thaw sperm performance. The sperm cryopreservation technique established in this study would provide a better option to assist in managing the genetic diversity of farmed Yesso scallops in China.

Development of a non-programmable sperm cryopreservation technique in dwarf surfclams Mulinia lateralis–a potential model species for bivalve research

Sperm cryopreservation technique has been published in many farmed bivalve species. One of the key factors preventing its application in aquaculture and/or cryobanking is the knowledge gap on the performance of resultant progeny at late developmental stages and subsequent generations. An effective strategy to overcome these challenges is to use a model species with a short generation interval, such as the dwarf surfclam Mulinia lateralis (three months). This study evaluated the parameters key to the development of a non-programmable sperm cryopreservation technique in this species, with a D-stage larval rate similar to control being achieved when the sperm were cryopreserved under the conditions (cryoprotectant agent: 8% dimethyl sulfoxide; equilibration period: 10 min; rack height: 4 cm; thawing temperature: 60°C and sperm to egg ratio: 1100:1) optimized. This technique is the most common method applied in bivalve and the results from this study were all within the ranges published for other bivalve species, indicating this species would be an ideal bivalve model species for addressing cryopreservation evaluation issues that need a long extended time to collect data and/or challenging field experiments.

Post-Thaw Sperm Recovery Rates; A Retrospective Study

Background: Cryopreservation is currently the only fertility preservation option available to patients undergoing gonadotoxic treatment. Much research in the field focusses on techniques, media and equipment employed to cryopreserve cells, and demonstrating cell viability after using such. Comparatively, there is a death of information available on the procedures and equipment used to thaw cryopreserved cells, or estimates for post-thaw recovery. Aim: This is a retrospective study assessing semen parameters in post-thawed specimen from patients who attended the Royal Women’s Hospital Andrology Unit for sperm cryopreservation prior to gonadotoxic or other treatments. The study aims to examine the relationship between sperm cryopreservation technique, media and period of storage with post-thaw sperm parameters. Method: Patients with written consent to discard their stored semen were selected for inclusion in the study (n=59); samples with sperm concentration [Formula: see text]1 million/ml prior to freezing (n=11) were excluded. Semen samples were thawed accordingly: 5 minutes at room temperature followed by incubation at 37[Formula: see text]C for sperm straws; vials were transferred directly to a mixer incubator at 37[Formula: see text]C. All samples were immediately assessed for sperm concentration and motility. Recovery rates were calculated and data were analysed using SPSS (IBM, USA) version 25. Results: Progressive motility recovery was 32.4% ± 20.6. Patients were divided into groups according to period of storage, and showed no significant differences for recovery of motility and concentration (p=0.44, 0.96, respectively). Furthermore, progressive motility recovery rates were not significantly different between freezing techniques (p=0.116) and media (p=0.099). Conclusion: Data from this study are preliminary and further study is warranted to fully extrapolate the relationship between sperm cryopreservation protocols and period of storage on recovery rates. The lack of relationship between these factors exhibited in the data could suggest that research and development in thaw technologies may be more pertinent to improving sperm recovery rates.

Use of Equine Sperm Cryopreservation Techniques as a Conservation Method of Donkey Germplasm

The aim of this study was to test equine semen cryopreservation techniques for the conservation of donkey germplasm. Ejaculates of three male donkeys were used (n = 18; six ejaculates per donkey; six repetitions), collected by the artificial vagina method. To remove the seminal plasma, the ejaculates were split and submitted to filtration or centrifugation methods. To assess the freezing method, each fraction was submitted to the automated system or the conventional system, and groups were formed: automated centrifuge, automated filtrate, conventional centrifuge and conventional filtrate. After thawing (37°C/30 seconds), were analyzed the sperm kinetic parameters, integrity and functionality of the plasma membrane and mitochondrial membrane potential. Highest sperm concentration (P < .05) was observed in the filtrate groups; the conventional filtrate group presented lower (P < .05) progressive motility and curvilinear velocity compared to the other groups; no difference was observed (P > .05) among the groups for the membrane integrity and functionality, and mitochondrial membrane potential. Thus, centrifugation is the most indicated technique to remove donkey seminal plasma and the automated and conventional freezing methods can be used in donkey semen conservation.

A REVIEW ON FACTORS INFLUENCING SUCCESS OF GROUPER SPERM CRYOPRESERVATION

This review was conducted to support information gathering on development of sperm cryopreservation techniques and protocols including factors such as type of cryoprotectants, extenders, cooling rates and dilution ratio and the impacts of those factors towards sperm cryopreservation of 10 different grouper species. Groupers are high-value marine species that contribute significantly to the income of Southeast Asian countries which are important as aquaculture producers. Thus, sperm cryopreservation is essential to support the development of grouper seed production and culture. Giant grouper is the most studied grouper species due to the fast-growing ability which enables to produce high-value interspecies hybrids for commercialization purposes. The most used cryoprotectant for sperm cryopreservation among various grouper species is dimethyl sulphoxide (DMSO). Among the extenders, sodium chloride (NaCl) is reported to be the most commonly used due to easy preparation, relative effectiveness and availability of the solution which enables local farmers to easily extend storage of the grouper sperm. Most grouper species seem to have a wide range of optimal cooling rates. However, some species exhibit the narrow range of optimal cooling rates. Optimal cooling rates were also possibly affected by the type and concentration of cryoprotectant. Dilution ratios used to cryopreserve grouper sperm seemed to vary. Sperm of brown-marbled grouper and seven band grouper to report dilution ratios of 1:49 while other grouper species sperm worked better at dilution ratios of 1:1, 1:2, 1:4 and 1:9. Grouper sperm cryopreservation protocol in general is species-specific and the optimization has to be done species by species to increase overall productivity. Grouper sperm cryopreservation is applicable and can be used to enhance seed production.

Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis discus hannai.

Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p > 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.

Challenges in the development of sperm cryopreservation protocols for snakes.

Snake populations are declining worldwide, but research devoted to the development of sperm cryopreservation techniques for this taxon is very limited. Spermatozoa were collected postmortem from snakes of four squamate families (Elapidae, Colubridae, Viperidae and Pythonidae). Viability assessment was performed before and after cryopreservation. Spermatozoa were extended in TES and Tris (TEST) yolk buffer with 12% dimethylsulfoxide (DMSO) or 12% glycerol and frozen at a rate of 0.3°Cmin-1 . The sperm quality index (SQI), representing three viability parameters (motility, plasma membrane and acrosome integrity), was determined. Despite some species differences, glycerol was a more effective cryoprotectant for Colubridae, whereas DMSO provided greater cryoprotection for spermatozoa of members of the other three families.

Recent updates of sperm cryopreservation technique: a literature review

Background: Nowadays, the decline in male fertility is a significant concern due to the mechanical, systems disturbances, genetic, diet, lifestyle, and environment. Currently, no strategies for medical protection of the germinal epithelium are available. Sperm cryopreservation technique has been established to overcome those issues. Accordingly, this study aims to informed the recent updates of sperm cryopreservation technique from kinds of literature. Methods: A review of relevant literature was performed to elaborate the recent updates in the sperm cryopreservation technique. A total of 45 qualified published literature of all years until 2019 were collected from several electronic database and manual search and included in this review.Results: According to the literature, the first record of sperm cryopreservation was made by Lazzaro Spallanzani in 1776. He used to snow during winter and observed that the motility of human spermatozoa could be preserved after freezing and thawing. Sperm cryopreservation programs provide temporarily or permanently infertile men an opportunity to seek paternity. Sperm cryopreservation is the most effective and the most strongly recommended option for fertility preservation prior to any treatment or occupational gonadotoxic exposures. Sperm cryopreservation should be discussed and offered prior to therapy for malignant diseases. Freezing sperm before the initiation of treatment provides patients with future fertility potential.Conclusion: Sperm cryopreservation could be offered to the patient before the vasectomy procedure in order to prevent reversal vasectomy or surgical sperm retrieval in the future.Â

The effect of cryopreservation technique in a very low number sperm count at Soetomo Hospital, Surabaya, Indonesia

Background: The massive development of Assisted Reproductive Technology (ART) offers new hope for the infertile couple. However, the advanced storage methods to preserve the quality of sperm has been raising by cryopreservation technique. This study aims to analyze the motility rate, recovery rate, and viability in subgroups of cryopreserved spermatozoa from the oligozoospermic patient as the quality parameter.Method: A true-experimental study by pre-test and post-test group design was conducted among 13 oligozoospermic patients with a very low sperm counts at Medical Biology Laboratory Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia. Parameters assessed in this study include motility, viability, and concentration of spermatozoa according to Laboratory Manual for the Examination and Processing of Human Sperm 5th edition. The samples were divided into 4 groups such as Group 1 (71-100 sperm), Group 2 (31-70 sperm), Group 3 (11-30 sperm), and Group 4 (1-10 sperm) based on sperm counts which underwent 30 repetitions of cryopreservation. Data were analyzed using SPSS version 17 for Windows. Results: There was a significant difference in the recovery rate among Group 1 (34.88±1.87), Group 2 (44.77±1.89), Group 3 (61.83±2.09), and Group 4 (70.90±2.71) of cryopreservation technique (P&lt;0.001). However, there was no significantly different in the motility rate of sperm-cryopreservation technique among Group 1 (65.60±14.63), Group 2 (52.50±26.28), Group 3 (39.90±9.95), and Group 4 (44.70±41.77) (p=0.070). Based on the sperm-viability parameter assessed in this study, there was no significant difference among Group 1 (56.40±3.89), Group 2 (58.60±5.99), Group 3 (59.60±9.96), and Group 4 (56.70±11.30) (p=0.504).Conclusions: The sperm-cryopreservation technique significantly affected the recovery rate of very low sperm number count among groups. However, there was no significantly different in the motility rate and viability parameter in this study.Â

Strategies to Minimize Various Stress-Related Freeze-Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa.

The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze-thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze-thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze-thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O2 with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N2 gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.

Description and Outcomes of Current Clinical Techniques for Sperm Cryopreservation

Nowadays, sperm cryopreservation is strongly recommended in cases of malignancy. Moreover, the use of frozen testicular sperm in azoospermic patients prevents the need for repeated sperm retrieval and optimises scheduling between oocyte and sperm obtainment. Even though cryopreservation of human spermatozoa for assisted reproductive purposes is a widely implemented practice, none of the established freezing and vitrification techniques offer optimal cryosurvival results due to the dramatic impact of cryodamage on sperm cells. This comprehensive review describes the most commonly used sperm cryopreservation techniques in order to establish which of them minimise sperm cryodamage and offer better survival rates. Presently, it is not sufficiently demonstrated that sperm vitrification improves survival significantly more than freezing methods. Slow freezing offers the best survival results when compared to other freezing protocols, and owing to its technical advantages, can be considered as one of the preferred protocols to be easily implemented in assisted reproduction laboratories. Moreover, several studies have suggested that sperm preparation prior to cryopreservation can improve thawed sample quality. However, other authors have demonstrated that freezing the fresh sample and performing semen preparation after thawing gives better results in regard to total motile sperm count and motility. Regarding clinical results, it is well established that similar or even better reproductive outcomes are achieved using frozen testicular sperm in cases of azoospermia or anejaculation. Moreover, the use of frozen semen in cancer patients can help to achieve good fertilisation and pregnancy rates. Finally, the use of frozen sperm is not at all associated with worse post-natal development.

The anti-oxidant roles of Taurine and Hypotaurine on acrosome integrity, HBA and HSPA2 of the human sperm during vitrification and post warming in two different temperature

Despite advances in vitrification techniques for sperm cryopreservation, cryo-damages of sperm caused by generation of reactive oxygen species (ROS) continue to impede implementation of this technique. This study analyses the effects of taurine and hypotaurine as anti-oxidants during vitrification of human sperms. The study was performed in two steps. In the first step, 20 normospermic semen samples were vitrified in the presence of varying concentrations of taurine and hypotaurine, and their effects as anti-oxidant agents on classical sperm parameters, hyaluronan-binding assay (HBA), lipid peroxidation (LPO) and acrosome reaction (AR) were studied. Statistical analyses showed that the sperm parameters in all vitrified groups decreased significantly (P < 0.05) compared to the fresh group. However, HBA and acrosome integrity in vitrified groups containing taurine and 50 mM of hypotaurine were better than in the control group (P < 0.05). The morphology of the vitrified group was good only in the group that contained 50 mM of hypotaurine (P < 0.05).Based on the results from the first step, 50 mM of hypotaurine was considered the ideal anti-oxidant formulation and further tests were carried out on 10 normospermic semen samples with this protecting agent. In addition to the mentioned parameters, the expression of heat shock proteinA2 (HSPA2) was studied in the vitrified group with 50 mM hypotaurine, warmed under two different warming temperatures 37 and 42 °C. 50 mM Hypotaurine was found to equally improve motility, morphology, HBA, and AR after warming at 37 °C and 42 °C (P < 0.05). However, at both warming temperatures, the expression of HSPA2 was reduced in all vitrified groups comparing to the fresh group (P < 0.05). In conclusion, taurine and hypotaurine antioxidants, especially 50 mM hypotaurine, are able to reduce deleterious cryo-injuries on morphology, acrosome and HBA and improve sperm recovery at both warming temperatures (37 and 42 °C). However, they do not have any protective action on expression of HSPA2.

Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post-thaw quality optimization.

Sperm cryopreservation facilitates the storage and transport of germplasm for its use in artificial insemination (AI) and other advanced reproductive technologies. The cryopreservation process can damage sperm and compromise functionality. Several cryobiological studies have found that the physical and biological factors that affect sperm survival at low temperatures during the cryopreservation process often involve the integrity of sperm membrane. In this review, the behaviour of the sperm membrane against cooling, cold shock, ice crystal formation, oxidative stress, osmotic changes, reorganization of the lipid bilayer and addition of cryoprotective agents (CPA) is discussed. In addition, the phenomenon of reactive oxygen species (ROS) and its relationship with the cryopreservation process is also described. Semen cryopreservation techniques have progressed slowly in past years, and the current performance, measured as post-thawed survival, is not very different compared to past decades. Recent advances in understanding the structure of the cell membrane, its function and metabolism have driven to new conservation systems, including lyophilization and vitrification. However, none of these technologies is commercially available, although its future appears very promising.

Fish sperm motility assessment as a tool for aquaculture research: a historical approach

AbstractFish sperm motility is nowadays considered the best biomarker for the quality of fish spermatozoa, and sperm motion parameters from more than 300 fish species have been reported in more than 1500 scientific articles covering a wide range of topics, from molecular biology to ecology. The most studied topics have been (i) the sperm storage (involving both the use of chilled‐storage protocols for short‐term periods and sperm cryopreservation techniques for long‐term storage), (ii) the sperm physiology (fathom in the spermatozoa activation process and the whole propulsion machinery of the sperm cells) and (iii) the broodstock management (covering aspects such as rearing conditions, dietary requirements or hormonal induction treatments). In addition, other aquaculture and ecological topics, such as (iv) the knowledge of the breeding cycle of the species, (v) the phenomenon of the sperm competition and (vi) ecotoxicological studies for the evaluation of aquatic environments, have also been approached from the evaluation of sperm motion performance. Therefore, fish sperm motility assessment can serve as a potential tool for aquaculture and ecological purposes, covering key topics of fundamental and applied research. This review gives an overview of the major research areas in which fish sperm motility has been applied successfully.

Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie, Pomoxis annularis, Sperm

AbstractCrappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca2+free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.

Methods of cryopreservation of Tambaqui semen, Colossoma macropomum

This study compared three different techniques for sperm cryopreservation of Tambaqui (Colossoma macropomum). Semen was diluted in Beltsville Thawing Solution with the addition of dimethyl sulfoxide (DMSO) at various concentrations (5%, 10%, 15% and 20%). Cryopreservation was performed using three methods: Box Conditioner Method with straws at a 5cm distance from liquid nitrogen vapor (N2L); Dry Shipper Method placing the straws inside the machine; Vitrification Method placing the straws directly into N2L, amounting to 12 treatments (four DMSO concentrations×three freezing methods). The samples were evaluated for analysis of sperm quality in vivo and in vitro. Use of the Vitrification Method at different concentrations of DMSO provided the least values in the different evaluations. Fertilization, hatching rates and plasma membrane integrity using the Box Conditioner Method with 5% and 10% DMSO did not differ (P>0.05) but use of the concentration of 5% DMSO resulted in greater values than the other treatments (P<0.05) as well as for sperm motility and latency time (P<0.05), although sperm viability was superior using the Dry Shipper Method with 20% of the cryoprotectant. Mitochondrial functionality was impaired by use of the Vitrification Method with all DMSO concentration tested showing the most desirable values when the Box Conditioner Method was used with 5%, 10%, 15% DMSO and the Dry Shipper Method was used with 10% and 15% DMSO. Considering the variables evaluated, the use of the Box Conditioner Method is associated with enhanced Tambaqui semen quality with freeze concentrations of 5% and 10% DMSO.

Sperm cryopreservation in oysters: A review of its current status and potentials for future application in aquaculture

Abstract Cryopreservation has been expected to improve the efficiency of hatchery operation by supplying gametes on demand without live broodstocks and genetic improvement programs by achieving desired mating and establishing across-generation controls. The preservation of genetic materials of improved stocks and the original population is immensely important for oyster aquaculture industry to prepare the potential impacts from epidemic diseases and natural disasters. This review summarizes the research progress of sperm cryopreservation in oysters and discusses the scope of application of preserved sperm in aquaculture. A bulk of about 50 reports have been published on oyster sperm cryopreservation since 1971, nevertheless the application of this technique in aquaculture is limited. These studies primarily focused on the development of protocol for individual species by optimizing a set of interacting variables at different steps from sperm collection to post-thawing and fertilization. A number of approaches such as sperm motility, morphological integrity and fertility are used to evaluate the cryopreserved sperm quality but there are considerable variations in practice. We synthesized the outcomes in the existing literature in an attempt to suggest the standardization of sperm cryopreservation technique and provide directions for future research.

Improvement in non-programmable sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata

This study assessed the effects of the addition of vitamins (l-ascorbic acid), amino acids (glycine and taurine), and monosaccharides (glucose, fructose and galactose) on sperm cryopreservation using a non-programmable freezing technique in farmed greenlip abalone. The results showed that the addition of taurine, glycine or l-ascorbic acid significantly improved the post-thaw sperm motility, whereas the post-thaw sperm fertilization rates were improved by the addition of glycine or l-ascorbic acid. Flow cytometry analysis demonstrated that the addition of glycine significantly enhanced the post-thaw sperm plasma membrane integrity and acrosome integrity. Results from the investigation on monosaccharides demonstrated that glucose, fructose and galactose had a similar cryoprotective effect, resulting in a similar level of the post-thaw sperm fertilization rate, plasma membrane integrity, mitochondrial membrane potential and acrosome integrity. In this study, the highest post-thaw fertilization rate of 96% was achieved by using the cryoprotective mediums containing 6% DMSO, 1% glucose and 0.6% glycine.

Cryopreservation of sperm from wild greenlip abalone (Haliotis laevigata) in Australia

Greenlip abalone (Haliotis laevigata) is one of the most economically valuable marine molluscan species in Australia and its aquaculture production comprises more than 50% of the total production of farmed abalone in this country. The development of a cryopreservation technique for this species will in particular enhance the efficiency of the established genetic improvement programs and thus the long-term development of this aquaculture industry. In this study, a series of experiments have been conducted to investigate factors important for the development of sperm cryopreservation techniques in this species, including the evaluation of (1) cryoprotectant toxicity; (2) pre-freezing temperature; (3) volume of sperm + cryoprotectant mixture in 2-mL cryovial; (4) sperm concentration; (5) cold storage duration; (6) thawing temperature; and (7) sperm to egg volume ratio on post-thaw sperm motility and/or fertility. The results show that 6% DMSO was the best among the cryoprotectants evaluated, resulting in a post-thaw fertilization rate similar to controls. Wild greenlip abalone sperm tolerated a reasonable range of conditions for most parameters crucial to the liquid nitrogen vapor cryopreservation method; pre-freezing temperature from − 60 to − 80 °C, duration of cold storage (for chemical equilibration) up to 1.5 h, and the thawed sperm/fresh egg ratio (by volume) up to 1:3. Although a 50 °C thawing temperature resulted in the highest fertilization rate of 95%, all the other temperatures evaluated (from 40 to 80 °C) produced a fertilization rate of 80% or higher. The use of the cryopreservation protocol established in this study will provide an alternative breeding technique to overcome key obstacles experienced in the existing genetic improvement programs.

Study of enzyme activities and protein content of beluga (Huso huso) semen before and after cryopreservation.

Knowledge gained regarding the biochemical processes that occur during sperm collection, processing and freezing-thawing might improve current sperm cryopreservation techniques. In our present study, we determined the effect of cryopreservation on the total protein concentration (TP) and the activities of certain enzymes in semen samples from the beluga (Huso huso). The TP content of the seminal plasma of fresh semen was 0.47 ± 0.026 g/l, and the TP after cryopreservation was 1.86 ± 0.6 g/l. The activities of acid phosphatase (0.82 ± 0.042 U/l), lactate dehydrogenase (234.4 ± 19.4 U/l), arylsulfatase (143.1 ± 32.5 U/l) and β-N-acetylglucosaminidase (58.39 ± 4.14 U/l) in the seminal plasma of fresh semen were significantly lower than those in the supernatant of frozen-thawed semen samples (7.43 ± 0.64, 3224.6 ± 167.2, 422.6 ± 21.3 and 90.2 ± 5.37 U/l respectively). These parameters may be useful as biomarkers for estimating damage to the cell membrane of spermatozoa caused by freezing-thawing.