**6. General conclusion**

Data presented in this review shown that the technique of cryopreservation by direct plunging into liquid nitrogen (vitrification) in absence of permeable cryoprotectants has a great perspective. This technique allows significantly protect the important physiological parameters of mammalian and fish spermatozoa against cryo-injures.

## **7. References**

68 Current Frontiers in Cryobiology

inclusion of sucrose in the vitrification solution was ineffective for rainbow trout spermatozoa. According to Lahnsteiner (2007), lipoproteins in the seminal plasma of rainbow trout likely maintain the lipid composition of the plasma and may increase the cryostability of spermatozoa. Our results support this point of view and we suggest that the method of sperm vitrification described here could also be applied to other species. As a rule, carbohydrates are used for sperm cryopreservation to compensate for the decrease in osmotic pressure caused by the permeable cryoprotectant glycerol, which works as an additional dissolvent and has the ability to decrease the medium's osmotic pressure. Based on this evidence, we investigated whether sucrose had a similar cryoprotective effect on fish spermatozoa during freeze–thaw. We found that its inclusion in vitrification medium has no visible protective effect on mitochondrial membrane integrity nor does it provide significant protection for spermatozoa when compared to other vitrification mediums containing BSA or BSA + seminal plasma. Indeed, the addition of these non-permeable cryoprotectants did not increase either the motility or plasma membrane integrity of rainbow trout spermatozoa. However, described here technology of cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen has big disadvantage because did not protect the biological material against direct contact with liquid nitrogen. In this connection in the future investigation it would be necessary to find a synthetic substitute for seminal plasma to avoid the possible microbial contamination. In fact, any technology in reproductive biology, and especially in a therapeutic medical approach, must guarantee the full protection of cells from microorganisms that might survive in liquid nitrogen temperatures (Gardner, 1998; Bielanski et al., 2003), and it has been suggested that liquid nitrogen can be contaminated by microorganisms (Tedder et al., 1995). The problem of potential microbial contamination of spermatozoa during cryopreservation, especially by the virus of Infectious Salmon Anemia is significant in the fish industry, especially in Latin America (Ellis, 2007; Fortt and Buschmann, 2007; Sommer, 2009). In spite of that the results of our experiments conformed that for fish spermatozoa the developed method of cryopreservation by direct plunging into liquid nitrogen (vitrification) without permeable cryoprotectants is potentially significant for this industry, but the development of "aseptic" methods, in which the spermatozoa suspension is enclosed in capillaries or straws to prevent direct contact of sperm with liquid nitrogen, will need to be considered. Filtration or ultraviolet treatment of liquid nitrogen cannot guarantee the absence of contamination of biological material by viruses. For example, Tedder et al. (1995) reported the contamination of blood probes by hepatitis virus during the storage of probes in liquid nitrogen. Different types of viruses, such as hepatitis virus, papova virus, vesicular stomatitis virus and herpes virus, which are simple and very cryostable structures, may increase their virulence after direct plunging and storage in liquid nitrogen (Hawkins et al., 1996; Charles and Sire, 1971; Schaffer et al., 1976; Jones and

Data presented in this review shown that the technique of cryopreservation by direct plunging into liquid nitrogen (vitrification) in absence of permeable cryoprotectants has a great perspective. This technique allows significantly protect the important physiological

parameters of mammalian and fish spermatozoa against cryo-injures.

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**3** 

*Canada* 

**Cryopreservation of Human Spermatozoa by** 

*Department of Obstetrics & Gynaecology, Sunnybrook Health Sciences Centre and* 

Many advances in reproductive medicine in the past five decades have made cryopreservation of human spermatozoa an invaluable tool for the clinical management of infertility and sperm banking. The advent of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with microsurgical sperm handling techniques along with advances in female gamete acquisition have resulted in an increased demand for the cryopreservation of semen and tissue samples, often containing a very limited number of spermatozoa. Sperm cryopreservation also makes it possible for cancer patients to preserve their fertility prior to gonadotoxic chemotherapy or radiation. Applications of sperm banking are not limited to cancer patients but extend to patients undergoing certain types of pelvic or testicular surgeries; those who suffer from degenerative illnesses such as diabetes or multiple sclerosis; spinal cord disease or injury; and persons in occupations where a significant risk of gonadotoxicity prevails. Sperm cryopreservation is also available to men undergoing surgical sterilization such as vasectomy, in the event that children may be desired in the future. Another use for semen cryopreservation is to allow donor semen samples to be quarantined while appropriate screening is performed to prevent the transmission of

Although major improvements have been made in sperm cryopreservation, there are many unresolved technical issues. Since freezing protocols differ between types of cells, the ideal conditions for human sperm freezing and thawing need to be perfected. To add more complexity, samples with abnormal semen parameters, such as severe oligospermia or high seminal fluid viscosity, often require unique cryopreservation conditions. For example, the particular cryoprotectants can affect cooling rates. In addition, storage temperature can significantly influence cryopreservation outcome. Liquid nitrogen (LN2) can offer long-term survival of spermatozoa due to essentially absent metabolic activity, such as chemical reactions, genetic modification or aging of cells (Mazur, 1984). A conventional slow freezing protocol has been in use for many years and very little has changed in terms of

infectious pathogens during therapeutic donor insemination (TDI).

**1. Introduction** 

**Vitrification** *vs.* **Slow Freezing:** 

*3Division of Reproductive Endocrinology and Infertility,* 

S.I. Moskovtsev1,2, A.G-M. Lulat1 and C.L. Librach1,2,3

*2Department of Obstetrics & Gynaecology, University of Toronto* 

 **Canadian Experience** 

*Women's College Hospital, Toronto, Ontario* 

*1CReATe Fertility Centre* 

