**7. References**

AbdelHafez, F. F., Desai, N., Abou-Setta, A. M., Falcone, T. & Goldfarb, J. (2010). Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis., *Reproductive biomedicine online* 20(2): 209–22. URL: *http://www.ncbi.nlm.nih.gov/pubmed/20113959*

<sup>7</sup> The Stirling Engine is used in the Asymptote EF600

14 Will-be-set-by-IN-TECH

virus (BIV), that can be considered a model for retrovirus like the human immunodeficiency

Moreover, vitrification requires a greater amount of CPA (CryoProtectant Agent) than Slow

In order to reduce the risk of contamination, *closed supports* for vitrification were developed (*Cryotip* and *Isachenko Method*). However, a lower survival rate is obtained than using an *open*

A lower survival rate after thawing using Slow Freezing instead of Vitrification was claimed by many authors. (Fadini et al., 2009) reports a survival rate of human oocytes of 78.9% using Vitrification, while it is reduced to 57.9 % using Slow Freezing (*p-value* lower than 0.0001); similar results are shown in (Vutyavanich et al., 2010) (where survival rate of human spermatozoa is measured equal to 64.8% using Vitrification and equal to 50.4% using Slow Freezing, *p-value* equal to 0.0036). However, many authors believe that a better understanding of slow freezing principles will improve its performances (Bianchi et al., 2007; De Santis et al.,

Both Vitrification and Programmable Freezers (the most common machines use for Slow Freezing) require a supply of liquid-nitrogen, that is a limiting factor in many situations of inefficient or absent nitrogen distribution network, such as small industries, isolated places and during transport of cells. In order to overcome to this limitation, two alternative systems

Stirling Engine7 and Pulse Tube Cryocooler are closed-cycle machines, reducing risk of contamination and toxicity. A cells freezing system based upon closed-cycle machines is a viable commercial solution, especially for those markets where liquid nitrogen supply is

However, Stirling Engine exhibits high vibration, thus the nucleation process can not be inducted manually. Moreover, vibrations might damage cells. Those problems are avoided

The application of a Pulse Tube Cryocooler for cells cryopreservation is under developing at "Sapienza" - University of Rome Laboratory of Mechanical Engineering. A validation of the proposed system with the assessment of cells survival rate after thawing is envisaged as next step. Future work will also focus on the development of a cost effective control system which

We would really like to thank *MES - Microconsulting Energia & Software S.c.a.r.l.* and *LABOR*

AbdelHafez, F. F., Desai, N., Abou-Setta, A. M., Falcone, T. & Goldfarb, J. (2010). Slow freezing,

meta-analysis., *Reproductive biomedicine online* 20(2): 209–22.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/20113959*

vitrification and ultra-rapid freezing of human embryos: a systematic review and

for Slow Freezing (*Asymptote EF600* and *Pulse Tube Cryocooler*) have been developed.

virus (HIV). Risk of Hepatitis B contamination is analyzed in (Tedder et al., 1995).

Freezing does, increasing the toxicity of the environment.

2007; Edgar, 2009; Fadini et al., 2009; Mcgrath, 2009).

difficult or excessively expensive, or during transport.

allows the operator to set a desired cooling rate.

<sup>7</sup> The Stirling Engine is used in the Asymptote EF600

*S.r.l.* for their contribution to the realization of this work.

using a Pulse Tube cryocooler.

**6. Acknowledgment**

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**20** 

*Poland* 

**Methods of Assessment of** 

*Wroclaw University of Environmental and Life Sciences* 

Agnieszka Partyka, Wojciech Niżański and Małgorzata Ochota

The numerous effects that cryopreservation can induce in spermatozoa, ranging from lethal injuries to those which merely impair their subsequent function. In the last few years, the considerable increase in our understanding of both, the cell physiology of spermatozoa, and the stress of cryopreservation, have contributed to a renewed interest in improving the

Despite the significant progress, the post-thaw viability and fertility of the cryopreserved sperm are still reduced, as a consequence of accumulated cellular injuries that arise throughout the cryopreservation process. Many laboratory tests have already been carried out to verify these detrimental effects and their origin. Their is needed to well understand the whole process of cryopreservation and its influence on sperm function. As a consequence, it would lead to a subsequent improvement of sperm viability by means of reformulated protocols and

Here, we present an overview of the cryopreserved semen assessment methods in the light of sperm physiology, in order to relate these factors to altered functions of cryopreserved

Light microscopy is the most often used to analyze the quality and predict the fertility of the cryopreserved semen in the conventional way. Visual assessment requires such equipment as microscope, heated stage and slides, as well as an experienced evaluator, however the

Motility is one of the most important features of a fertile spermatozoa. It was the first, and continues to be the most widely used indicator of sperm function. Sperm motility is an important attribute, because it is readily identifiable and reflects several structural, and functional competence, as well as essential aspects of spermatozoa metabolism. Sperm motility is expressed as the percentage of total motile or progressively motile spermatozoa. This parameter is usually assessed by the subjective visual examination under a phase contrast microscope at 37°C using low objectives (10 or 20x). Light microscopic evaluation

approaches helping to minimize the detrimental effect of cryopreservation.

sperm and to determine the fertilizing potential of the frozen-thawed semen.

**2. Conventional methods of semen assessment** 

assessment is subjected to the evaluator bias.

**2.1 Sperm motility** 

**1. Introduction** 

performance of cryopreserved semen.

**Cryopreserved Semen** 

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