**5.1.7 Cobalt**

Cobalt is a parametric-based computer-aided design (CAD) and 3D modeling software for both the Macintosh and Microsoft Windows. It integrates wireframe, freeform surfacing, feature-based solid modeling, photo-realistic rendering, and animation (http://www.ashlar.com/3d-modeling/3d-modeling-cobalt.html).

#### **5.1.8 Electric Image Animation System (EIAS)**

EIAS is an eminent 3D animation and rendering package available on both Mac OS X and Windows. Mostly known for its rendering quality and rendering speed it does not include a built-in modeler. EIAS is extensively used in Hollywood, and by professional studios all over the globe to create world-class animations. The popular film *Pirates of the Caribbean* and the television series *Lost* used the software (http://www.eias3d.com/products/eiasoverview).

## **5.1.9 Houdini**

This is used for visual effects and character animation. It was used in Disney's feature film *The Wild*. Houdini uses a non-standard interface that it refers to as a "NODE system". It has a hybrid micropolygon-raytracer renderer, Mantra, but it also has built-in support for commercial renderers like Pixar's Renderman and mental ray (http://www.sidefx.com).

#### **5.1.10 LightWave 3D**

This software was first developed for the Amiga. It was originally bundled as part of the Video Toaster package and entered the market as a low cost way for TV production companies to create quality CGI for their programming. It first gained public attention with its use in the TV series *Babylon 5* and it is used in several contemporary TV series. Lightwave is also used in a variety of modern film productions. It is available for both Windows and Mac OS X. LightWave's tools are designed for fast productivity and easy, intuitive use (http://www.newtek.com/lightwave.html).

A Survey of Some Virtual Reality Tools and Resources 37

This simulation software incorporates 3D displays and is designed to be used where first class presentation of a key proposal is required. This is an established general purpose simulation tool used by thousands of organizations to support continuous process improvement activities. WITNESS VR comes complete with a world building and editing package called mantra4D. This enables objects to be created from simple primitives (spheres, cylinders, boxes, etc) or to be imported from a variety of CAD and solid modeling packages (3DS, WRL formats included). Complex CAD models can be simplified using tools in mantra4D to render them suitable for real time fly-around VR. Backdrops can be created easily using simple surfaces and applied digital images which can be taken with any typical

Flexsim DS incorporates the latest advances in 3D simulation software-from realistic VR environments to network collaboration. Flexsim DS can create custom installations and

OpenSimulator is a 3D application server. It can be used to create a virtual environment (world) which can be accessed through a variety of clients, on multiple protocols (Mousa, n.d). OpenSimulator allows you to develop your environment using technologies you feel work best (Onyesolu & Eze, 2011). OpenSimulator has numerous advantages which among

a. OpenSimulator is released under BSD license, making it both open source, and

b. It has many tools for developers to build various applications (chat application,

c. OpenSimulator can be extended via modules to build completely custom configuration.

e. Supports many programming languages for application development such as Linden Scripting Language / OpenSimulator Scripting Language (LSL/OSSL), C#, and/or

Croquet is an open source 3D graphical platform that is used by experienced software developers to create and deploy deeply collaborative multi-user online virtual world applications on and across multipl e o p e r a t i n g s y s t e m s a n d d e v i c e s (http://www.opencroquet.org/index.php/Main\_Page). Croquet is a next generation virtual operating system (OS) written in Squeak. Squeak is a modern variant of Smalltalk. Squeak runs mathematically identical on all machines. Croquet system features a peer-based messaging protocol that dramatically reduces the need for server infrastructures to support virtual world deployment and makes it easy for software developers to create deeply

d. It is a world building tools for creating content real time in the environment.

f. It incorporates rich and handy documentations and tutorials.

build distributed modeling environment (http://www.flexsim.com/products/ds/).

digital camera (http://www.laner.com/en/media/witness/vr.cmf).

**5.3.1 WITNESS VR**

**5.3.2 Flexsim DS**

other things are:

**5.3.4 Croquet**

**5.3.3 OpenSimulator (OpenSim)**

Jscript and VB.NET

commercially friendly to embed in products.

buildings, and avatars among others).

#### **5.1.11 MASSIVE**

This is a 3D animation system for generating crowd-related visual effects, targeted for use in film and television. It was originally developed for controlling the large-scale CGI battles in *The Lord of the Rings*. Massive has become an industry standard for digital crowd control in high end animation and has been used on several other big-budget films. It is available for various Unix and Linux platforms as well as Windows (http://en.wikipedia.org/ wiki/MASSIVE\_(software)).

#### **5.1.12 Maya**

Autodesk Maya 3D animation software delivers an end-to-end creative workflow with comprehensive tools for animation, modeling, simulation, visual effects, rendering, match moving, and compositing on a highly extensible production platform. This software is currently used in the film, television, and gaming industry. Maya has developed over the years into an application platform in and of itself through extendibility via its MEL programming language. It is available for Windows, Linux and Mac OS X (http://usa.autodesk.com/maya)

#### **5.1.13 Poser**

Poser is a 3D CGI rendering and animation software program optimized for models that depict the human and animal figures in three-dimensional form. Poser has a basic library of human, animal, robotic, and cartoon figures. Poser software package also include poses, hair pieces, props, textures, hand gestures and facial expression (http://en.wikipedia.org/ wiki/Poser).

#### **5.2 2D graphics software**

2D graphics software is used to manipulate texture to be applied to the objects which enhance their visual details. 2D graphics software (also known 2D graphics editor or drawing program) is an application-level software for the creation of images, diagrams and illustrations by direct manipulation (through the mouse, graphics tablet, or similar device) of 2D computer graphics primitives. These editors generally provide geometric primitives as well as digital images; and some even support procedural models. The illustration is usually represented internally as a layered model, often with a hierarchical structure to make editing more convenient. These editors generally output graphics files where the layers and primitives are separately preserved in their original form. MacDraw, introduced in 1984 with the Macintosh line of computers, was an early example of this class; recent examples are the commercial products Adobe Illustrator and CorelDRAW (http://en.wikipedia.org/ wiki/CorelDRAW), and the free editors such as **xfig** or **Inkscape**. There are also many 2D graphics editors specialized for certain types of drawings such as electrical, electronic and VLSI diagrams, topographic maps, computer fonts, etc.

#### **5.3 VR simulation software**

Simulation software brings the components of VR together. It is used to program how these objects behave and set the rules that the virtual world follows. Some examples are presented:

#### **5.3.1 WITNESS VR**

36 Virtual Reality and Environments

This is a 3D animation system for generating crowd-related visual effects, targeted for use in film and television. It was originally developed for controlling the large-scale CGI battles in *The Lord of the Rings*. Massive has become an industry standard for digital crowd control in high end animation and has been used on several other big-budget films. It is available for various Unix and Linux platforms as well as Windows (http://en.wikipedia.org/

Autodesk Maya 3D animation software delivers an end-to-end creative workflow with comprehensive tools for animation, modeling, simulation, visual effects, rendering, match moving, and compositing on a highly extensible production platform. This software is currently used in the film, television, and gaming industry. Maya has developed over the years into an application platform in and of itself through extendibility via its MEL programming language. It is available for Windows, Linux and Mac OS X

Poser is a 3D CGI rendering and animation software program optimized for models that depict the human and animal figures in three-dimensional form. Poser has a basic library of human, animal, robotic, and cartoon figures. Poser software package also include poses, hair pieces, props, textures, hand gestures and facial expression (http://en.wikipedia.org/

2D graphics software is used to manipulate texture to be applied to the objects which enhance their visual details. 2D graphics software (also known 2D graphics editor or drawing program) is an application-level software for the creation of images, diagrams and illustrations by direct manipulation (through the mouse, graphics tablet, or similar device) of 2D computer graphics primitives. These editors generally provide geometric primitives as well as digital images; and some even support procedural models. The illustration is usually represented internally as a layered model, often with a hierarchical structure to make editing more convenient. These editors generally output graphics files where the layers and primitives are separately preserved in their original form. MacDraw, introduced in 1984 with the Macintosh line of computers, was an early example of this class; recent examples are the commercial products Adobe Illustrator and CorelDRAW (http://en.wikipedia.org/ wiki/CorelDRAW), and the free editors such as **xfig** or **Inkscape**. There are also many 2D graphics editors specialized for certain types of drawings such as electrical, electronic and

Simulation software brings the components of VR together. It is used to program how these objects behave and set the rules that the virtual world follows. Some examples are

**5.1.11 MASSIVE**

**5.1.12 Maya**

**5.1.13 Poser**

wiki/Poser).

**5.2 2D graphics software**

**5.3 VR simulation software**

presented:

VLSI diagrams, topographic maps, computer fonts, etc.

wiki/MASSIVE\_(software)).

(http://usa.autodesk.com/maya)

This simulation software incorporates 3D displays and is designed to be used where first class presentation of a key proposal is required. This is an established general purpose simulation tool used by thousands of organizations to support continuous process improvement activities. WITNESS VR comes complete with a world building and editing package called mantra4D. This enables objects to be created from simple primitives (spheres, cylinders, boxes, etc) or to be imported from a variety of CAD and solid modeling packages (3DS, WRL formats included). Complex CAD models can be simplified using tools in mantra4D to render them suitable for real time fly-around VR. Backdrops can be created easily using simple surfaces and applied digital images which can be taken with any typical digital camera (http://www.laner.com/en/media/witness/vr.cmf).

#### **5.3.2 Flexsim DS**

Flexsim DS incorporates the latest advances in 3D simulation software-from realistic VR environments to network collaboration. Flexsim DS can create custom installations and build distributed modeling environment (http://www.flexsim.com/products/ds/).

#### **5.3.3 OpenSimulator (OpenSim)**

OpenSimulator is a 3D application server. It can be used to create a virtual environment (world) which can be accessed through a variety of clients, on multiple protocols (Mousa, n.d). OpenSimulator allows you to develop your environment using technologies you feel work best (Onyesolu & Eze, 2011). OpenSimulator has numerous advantages which among other things are:

#### **5.3.4 Croquet**

Croquet is an open source 3D graphical platform that is used by experienced software developers to create and deploy deeply collaborative multi-user online virtual world applications on and across multipl e o p e r a t i n g s y s t e m s a n d d e v i c e s (http://www.opencroquet.org/index.php/Main\_Page). Croquet is a next generation virtual operating system (OS) written in Squeak. Squeak is a modern variant of Smalltalk. Squeak runs mathematically identical on all machines. Croquet system features a peer-based messaging protocol that dramatically reduces the need for server infrastructures to support virtual world deployment and makes it easy for software developers to create deeply

A Survey of Some Virtual Reality Tools and Resources 39

collaborative applications. Croquet provides rich tutorials, resources and videos as

Ogoglio is an open source 3D graphical platform like Croquet. The main goal of the Ogoglio is to build an online urban style space for creative collaboration. Ogoglio platform is built from the languages and protocols of the web. Therefore, it's scripting language is javascript; it's main data transfer protocol is hypertext transfer protocol (HTTP), it's 2D layout is hypertext markup language (HTML) and cascading style sheet (CSS), and it has lightwave object geometry format for its 3D (http://foo.secondlifeherald.com/slh/2007/01/ interview\_with\_.html). Ogoglio is very different from the other virtual reality world development platforms because it uses Windows, Linux, Solaris operating system platforms

This is a cross-platform, distributed operating system for PC-cluster-based VR. Syzygy is a programming toolkit for writing virtual reality or other graphical applications. Syzygy applications can run on a single computer, but Syzygy is especially designed for the creation of applications to run on clusters of networked computers. Programs or instances of the same program running on different computers in the cluster communicate with one another and share data. The cluster as a whole effectively becomes a single computer with multiple screens and input devices. The rendering of your virtual world is synchronized on all of the computers (to within a few milliseconds), so different screens in the cluster give you different simultaneous views on the virtual world. Included utilities handle communication with 6DOF trackers and other input devices. Spatially-localized sound is supported using the FMOD libraries and non-localized text-to-speech on Windows

Digital sound editing software is used to mix and edit sounds that objects make within the virtual environment. There are so many digital sound editors. Some are presented in table 1.

There are so many VR resources and tools from industry providers. Some companies specialize in providing hardware components for use in VR system such as accelerator cards, pointing and manipulation devices, helmets, shutter glasses, motion platforms, projection system and spatial position and detection systems. There some others companies that specialize in software tools such as 2D software tools, 3D software tools and VR simulation tools. There are a quite handful of companies and institutions that specialize in providing VR applications for manufacturing, education, simulation, design evaluation (virtual prototyping), architectural walk-through, ergonomic studies, simulation of assembly sequences and maintenance tasks, assistance for the handicapped, study and

treatment of phobias, entertainment, rapid prototyping and much more.

educational materials for developers (Onyesolu & Eze, 2011).

and runs on web browsers such as Internet Explorer, Firefox, and Safari.

**5.3.5 Ogoglio**

**5.3.6 Syzygy**

(http://www.isl.uiuc.edu/syzygy.htm).

**5.4 Digital sound editing software**

**6. Conclusion**

Table 1. Some digital sound editing software and compatible operating systems. Source: http://en.wikipedia.org/wiki/Digital\_audio\_editor

collaborative applications. Croquet provides rich tutorials, resources and videos as educational materials for developers (Onyesolu & Eze, 2011).

#### **5.3.5 Ogoglio**

38 Virtual Reality and Environments

Acoustica Acon Digital Media No No No Yes No Amadeus Pro HairerSoft No Yes No No No Ardour Paul Davis Yes Yes Yes No Yes Audacity Dominic Mazzoni Yes Yes Yes Yes Yes

Dementia Holladay Audio No No No Yes No

BIAS Peak BIAS No Yes No No No

Wavestudio Creative Technology No No No Yes No

Ecasound Kai Vehmanen Yes Yes Yes No Yes Eko Peter Semiletov Yes Yes Yes No Yes Freecycle Yes No No Yes Fast Edit Minnetonka audio No No No Yes No Fission Rogue Amoeba No Yes No No No

Wave Editor FlexiMusic No No No Yes No Goldwave Goldwave Inc. No No No Yes No Jokosher Jokosher community Yes No Yes Yes Yes LMMS Tobias Doerffel Yes Yes Yes Yes Yes Logic Pro Apple No Yes No No No

Music Maker MAGIX No No No Yes No

Digitalizer Digitope No No No Yes No

Editor 3delite No No No Yes No MusE Yes No No Yes

Studio FAsoft No No No Yes No NU-Tech Leaff Engineering No No No Yes No Pro Tools Avid No Yes No Yes No Table 1. Some digital sound editing software and compatible operating systems. Source:

No No No Yes Yes

yes (in beta)

Inc. No No No Yes No

(WINE) No Yes Yes

**OS X Unix Windows Free**

No Yes No

**Software**

**Name Creator Linux Mac**

Audition Adobe Systems No

mp3DirectCut Yes

http://en.wikipedia.org/wiki/Digital\_audio\_editor

Enhancedaudio.com,

Audiobook Cutter Free Edition

Audio

Creative

Diamond Cut DC7

FlexiMusic

MAGIX

Media

MP3 Stream

n-Track

Ogoglio is an open source 3D graphical platform like Croquet. The main goal of the Ogoglio is to build an online urban style space for creative collaboration. Ogoglio platform is built from the languages and protocols of the web. Therefore, it's scripting language is javascript; it's main data transfer protocol is hypertext transfer protocol (HTTP), it's 2D layout is hypertext markup language (HTML) and cascading style sheet (CSS), and it has lightwave object geometry format for its 3D (http://foo.secondlifeherald.com/slh/2007/01/ interview\_with\_.html). Ogoglio is very different from the other virtual reality world development platforms because it uses Windows, Linux, Solaris operating system platforms and runs on web browsers such as Internet Explorer, Firefox, and Safari.

#### **5.3.6 Syzygy**

This is a cross-platform, distributed operating system for PC-cluster-based VR. Syzygy is a programming toolkit for writing virtual reality or other graphical applications. Syzygy applications can run on a single computer, but Syzygy is especially designed for the creation of applications to run on clusters of networked computers. Programs or instances of the same program running on different computers in the cluster communicate with one another and share data. The cluster as a whole effectively becomes a single computer with multiple screens and input devices. The rendering of your virtual world is synchronized on all of the computers (to within a few milliseconds), so different screens in the cluster give you different simultaneous views on the virtual world. Included utilities handle communication with 6DOF trackers and other input devices. Spatially-localized sound is supported using the FMOD libraries and non-localized text-to-speech on Windows (http://www.isl.uiuc.edu/syzygy.htm).

#### **5.4 Digital sound editing software**

Digital sound editing software is used to mix and edit sounds that objects make within the virtual environment. There are so many digital sound editors. Some are presented in table 1.

#### **6. Conclusion**

There are so many VR resources and tools from industry providers. Some companies specialize in providing hardware components for use in VR system such as accelerator cards, pointing and manipulation devices, helmets, shutter glasses, motion platforms, projection system and spatial position and detection systems. There some others companies that specialize in software tools such as 2D software tools, 3D software tools and VR simulation tools. There are a quite handful of companies and institutions that specialize in providing VR applications for manufacturing, education, simulation, design evaluation (virtual prototyping), architectural walk-through, ergonomic studies, simulation of assembly sequences and maintenance tasks, assistance for the handicapped, study and treatment of phobias, entertainment, rapid prototyping and much more.

A Survey of Some Virtual Reality Tools and Resources 41

Lane, C. (1993). Display Technologies. *Encyclopedia of Virtual Environments*. Retrieved June

Louka, M.N. & Balducelli, C. (2001). Virtual reality tools for emergency operation support

Onyesolu, M.O. & Eze, F.U. (2011). Understanding Virtual Reality Technology: Advances

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Onyesolu, M.O. (2009b). Virtual reality laboratories: An ideal solution to the problems

Onyesolu, M.O. & Akpado, K.A. (2009). Virtual reality simulations in computer engineering

Onyesolu, M.O. (August, 2006). Virtual Reality: An Emerging Computer Technology of the

Perry, L.D.S., Smith, C. M., & Yang, S. (2004). An investigation of current virtual reality

Sowizral, H. (1995). Tutorial: An Introduction to Virtual Reality. *Virtual Reality Annual*

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Francisco, USA, October 2009, Newswood Limited, Hong Kong.

Vol. 3, No.3, (July 2009) 56-61, ISSN 0795-2260.

http://www.acm.org/crossroads/xrds3-3/vrhci.html

*Computer Graphics and Applications*, March: 79-89.

http://en.wikipedia.org/wiki/3D\_computer\_graphics\_software http://en.wikipedia.org/wiki/Cave\_Automatic\_Virtual\_Environment

http://company.mmosite.com/forterra /index.shtml

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html

and-engineering

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*International Symposium*.

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The exciting field of VR has the potential to change our lives in many ways. There are many applications of VR presently and there will be many more in the future. Many VR applications have been developed. VR technology is now widely recognized as a major breakthrough in the technological advance of science. In Japan today, with the help of VR technology, a customer can walk into a department store, done a VR helmet (HMD) and glove, and proceed to design his own kitchen. The customer can walk around inside the virtual kitchen, open and close drawers; turn on the water, etc. Once the customer is satisfied, the plans are produced and the kitchen is made to order. The Japanese are further developing this system to include various aspects of lighting, noises, humidity, and drafts.

#### **7. References**

http://www.hitl.washington.edu/scivw/EVE/I.D.1.a.ActiveInteraction.html

The exciting field of VR has the potential to change our lives in many ways. There are many applications of VR presently and there will be many more in the future. Many VR applications have been developed. VR technology is now widely recognized as a major breakthrough in the technological advance of science. In Japan today, with the help of VR technology, a customer can walk into a department store, done a VR helmet (HMD) and glove, and proceed to design his own kitchen. The customer can walk around inside the virtual kitchen, open and close drawers; turn on the water, etc. Once the customer is satisfied, the plans are produced and the kitchen is made to order. The Japanese are further developing this system to include various aspects of lighting, noises, humidity, and drafts.

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ejournals/JITE/v41n4/ausburn.html

I.D.1.b. TrackingDevices.html

*Graphics and Applications* January: 55-59.

vrl.umich.edu/intro.html/

pe=pdf

Source:

*the Oklahoma Association of Teacher Educators, 7*, 1-20.

**7. References**

http://en.wikipedia.org/wiki/Cave\_Automatic\_Virtual\_Environment

http://en.wikipedia.org/wiki/CorelDRAW

**1. Introduction**

<sup>1</sup> IST FP6 ICT-IP-035005-2006

practice through repeating exposures to the task.

A high percentage of the human activities are based on *procedural tasks*, for example cooking a cake, driving a car, fixing a machine, etc. Reading an instruction book, watching a video or listening the explanation from an expert have been the *traditional methods* to learn procedural tasks. But, most researchers agree that procedural tasks are learnt gradually as a result of

**Training of Procedural Tasks Through the Use**

**of Virtual Reality and Direct Aids**

Sara Casado2 and Iker Aguinaga1

*2Tecnalia Spain*

**3**

Jorge Rodríguez1, Teresa Gutiérrez2, Emilio J. Sánchez1,

*1CEIT, Centro de Estudios e Investigaciones Técnicas de Gipuzkoa*

Nowadays, *Virtual Reality (VR) technologies* can be used to improve the learning of a procedural task and moreover to evaluate the performance of the trainee. For example, a Virtual Environment (VE) can allow the trainees to interact physically with the virtual scenario, integrating a haptic device with the computer vision system, so that trainees can interact and manipulate the virtual objects, feeling the collisions among them and, the most important, they can practice the task under the approach of *learning by doing*. In this way, trainees can

This chapter introduces a **new Multimodal Training System (MTS)** that provides a **new interactive tool** for assisting the trainees during the **learning of a procedural task** in the assembly and disassembly operations. This MTS uses the haptic feedback to simulate the real behaviour of the task and special visual aids to provide information and help trainees to undertake the task. One of the main advantages of this platform is that trainees can learn and practice different procedural tasks without the necessity of having physical access to real tools, components and machines. For example, trainees can assemble/disassemble the different components from a virtual machine as it would be done in the real life. This system

During the *learning of a task*, most of the times, **trainees need to receive aids with information about how to proceed** with the task. From the point of view of the authors, these aids can be divided into two groups according to the type of information that they provide and how this information is provided: *direct aids and indirect aids***.** The difference between both types of aids is in the cognitive load requested by the trainees to interpret and understand the information provided by the aid. In this way, *indirect aids* require that trainees are active in

practice in order to improve their abilities until they are proficient with the task.

was designed and implemented as an activity in the context of the SKILLS project1.

http://en.wikipedia.org/wiki/Poser

http://en.wikipedia.org/wiki/Video\_card

http://en.wikipedia.org/wiki/Workstation

http://usa.autodesk.com/maya

http://vresources.org/company\_link\_list

http://www.3dace.com

http://www.3dwebtech.co.uk

http://www.5dt.com

http://www.acroneng.com/3D%20Modeller.htm

http://www.adacel.com

http://www.alibre.com

http://www.ashlar.com/3d-modeling/3d-modeling-cobalt.html

http://www.cae.ca/

http://www.edgedsign.com

http://www.eias3d.com/products/eias-overview

http://www.flexsim.com/products/ds/

http://www.icarusstudios.com/

http://www.isl.uiuc.edu/syzygy.htm

http://www.laner.com/en/media/witness/vr.cmf

http://www.maxon.net/products/cinema-4d-studio/who-should-use-it.html

http://www.newtek.com/lightwave.html

http://www.sidefx.com

http://www.simulation.com/products/glstudio/glstudio.html

http://www.thegamecreators.com/?m=view\_products&id=2092

http://www.virtualheroes.com/about.asp

http://www.vxsim.com


**2.2 Biological aspects of freezing**

Living cells have an isotonic condition with a melting point of their intracellular water of approximately -0.6°C. When cells are cooled below this standard freezing point, supercooling takes place and remains in a metastable state up to -5°C (Katkov et al., 2006; Mazur et al., 1972). Water crystallization and ice formation begin between -5 and -15°C, beginning with the formation of an ice nucleus (seed crystal) in the extracellular water. This 'nucleation' can be induced at a higher temperature by the planned external facilitation of ice formation, often referred to as "seeding". Prior to that stage, water remains unfrozen inside the cell as the membrane prevents ice crystals from intracellular penetration (Woods et al., 2004). Solutes are excluded from ice formation which results in rising concentrations

methodology and reagents. While freezing aims to preserve cells it can also easily destroy them if certain precautionary steps are not taken into consideration. During cryopreservation cells and tissue undergo dramatic transformation in chemical and physical characteristics as the temperature drops from +37 to -196°C. The cells can lose up to 95% of their intracellular water. The concentration of solutes increases considerably, triggering the possibility of osmotic shock. Moreover, potential intracellular ice crystallization and mechanical deformation by extracellular ice may cause significant injury leading to cell death. Furthermore, if cells survive freezing, they might sustain additional damage during the thawing process due to osmotic shock, uncontrollable swelling and ice re-crystallization

Recently scientists have begun to re-investigate the utility of ultra rapid freezing in the search for alternative methods of sperm cryopreservation. Slow freezing of sperm utilizes cooling rates of 1–10°C/min, while the rapid freezing, or vitrification, technique allows for cooling rates to reach more than 40-1000°C/min in order to avoid intracellular ice formation. As new techniques are perfected, there is a potential for sperm cryopreservation to greatly

Remarkably, the first reference of empirical sperm freezing dates as far back as the late 16th century, but it was only with the discovery in 1937 by Bernstein and Petropavlovski that glycerol can aid spermatozoa in surviving long term freezing, that sperm cryopreservation became practical. Expansion of artificial insemination for the dairy industry led to further important research in the field of cryobiology (E. Isachenko, 2003, as sited in Bernstein & Petropavlovski, Polge et al., 1949). Shortly after these practices were initiated with animals, the first pregnancies were reported in humans after insemination with frozen spermatozoa. The next milestone was the discovery of the possibility to store human spermatozoa in LN2 at -196°C, resulting in superior recovery rates compared to storage at higher temperatures between -20 and -75°C. After the era of empirical freezing; cryobiology matured to its fundamental stage, focusing on the biophysical and biochemical principals of cryopreservation, further advancing the field (Mazur et al., 1972. A comprehensive review of the historical background of sperm freezing was recently published and is recommended

Living cells have an isotonic condition with a melting point of their intracellular water of approximately -0.6°C. When cells are cooled below this standard freezing point, supercooling takes place and remains in a metastable state up to -5°C (Katkov et al., 2006; Mazur et al., 1972). Water crystallization and ice formation begin between -5 and -15°C, beginning with the formation of an ice nucleus (seed crystal) in the extracellular water. This 'nucleation' can be induced at a higher temperature by the planned external facilitation of ice formation, often referred to as "seeding". Prior to that stage, water remains unfrozen inside the cell as the membrane prevents ice crystals from intracellular penetration (Woods et al., 2004). Solutes are excluded from ice formation which results in rising concentrations

(Woods, et al., 2004).

improve in the future.

**2. Cryopreservation of human spermatozoa 2.1 History of human spermatozoa cryopreservation**

for readers looking for more details (Katkov et al., 2006).

**2.2 Biological aspects of freezing**

of solutes within extracellular water. Due to the permeability of the plasma membrane, this chemical imbalance sets up the diffusion of solutes into the cell, forcing water out of the cell. Cells thus undergo excessive dehydration, losing up to 95% of their intracellular water content. This increases the intracellular concentration of solutes, resulting in denaturation of proteins, pH shifts and potential cell death.

Since velocity of cooling is crucial, inaccurate cooling rates can negatively affect sperm survival, motility, plasma membrane integrity and mitochondrial function (Henry et al., 1993). When cooling is slow enough, there is sufficient time for intracellular water efflux and balanced dehydration. If cooling is too slow, damage may occur due to exposure of cells to high concentrations of intracellular solutes. Extreme cellular dehydration leads to shrinkage of cells below the minimum cell volume necessary to maintain its cytoskeleton, genomerelated structures, and ultimately cellular viability (Mazur, 1984). On the other hand, if cooling rates are too fast, external ice can induce intracellular ice formation and potential rupture of the plasma membrane and damage intracellular organelles. In addition, mechanical damage of cells is possible due to of extracellular ice compression and close proximity of frozen cells can result in cellular deformation and membrane damage (Fujikawa & Miura, 1986). In contrast, with ultra rapid cooling, the amount of ice formation is insignificant and the entire cell suspension undergoes vitrification. At this stage water transitions, ice formation slows, molecular diffusion and aging stops, and liquids turn into a glass-like condition (Katkov et al., 2006).

Despite the relative insensitivity of human sperm to freezing, optimal cooling rates are needed to ensure appropriate sperm recovery. Currently, there are two types of slow freezing, either static vapour phase freezing to a certain temperature, or the multistep approach using nonlinear controlled-rate freezers, followed by plunging into LN2. Most laboratories and sperm banks adapt simple static vapour phase cooling in order to avoid induction of ice nucleation by seeding. For this technique samples are lowered into a vapour phase just above the LN2 level, allowing them to cool for 15-20 minutes before being plunged into LN2. Alternately, controlled rate freezers can be used to cryopreserve human semen. Most of these protocols utilize a "no seeding" option where samples are cooled from room temperature to -4°C at the rate of 2°C/min, followed by an increase of the cooling rate to 10°C/min until -100°C is reached, and finally plunging into LN2 (Morris et al., 1999). In contrast to these slow freezing techniques, single step ultra rapid cooling is used for the vitrification technique.


**6. Future Recommendation**

Such coverage in the localization of QTLs controlling different quantitative traits suggested a close genotypic correlation among these traits or a pleiotropic effect of a single gene. It remains to be tested whether these common genomic regions have pleiotropic effects or there are clusters of tightly linked genes for some related traits in these regions. A more numerous mapping population and more closely spaced markers in the map are needed to determine whether the QTLs correspond to a gene with pleiotropic effects or to several separate but closely linked genes, each controlling a single character.

**Author Contributions:** Conceptualization, S.R., M.B. and S.A.M.; writing and draft preparation, S.R., M.B., T.N. and S.A.M.; writing, review, and editing, S.R., M.B., T.N. and S.A.M.; supervision, M.B. and S.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** Authors are grateful to the Higher Education Commission (HEC) Pakistan, for providing funds under PIN 074-0747-Bm4-151, Indigenous 5000 Fellowship Program for this study.

**Acknowledgments:** The presented study is part of research proposal Mapping QTL's associated with heat tolerance in cotton using microsatellite markers. Author is grateful to Allah almighty, supervisors, Peng Chee (Associate University of Georgia, USA), Edward Lubbers (Research Associate at cotton molecular breeding lab, university of Georgia, Tifton campus, USA) Bahauddin Zakariya University Multan and Higher Education Commission (HEC) Pakistan.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.


**Author details**

Lavanya Suneetha\*, Prasanna Marsakatla, Rachel Supriya Suneetha and Sujai Suneetha

\*Address all correspondence to<EMAIL_ADDRESS>
Infectious Disease Research Laboratory, Nireekshana ACET, Hyderabad, India


One of the most common materials that is used for soil stabilization is PVA. When the PVA solution is absorbed in the soil, the polymers enveloping the soil particles that remain aggregated after the evaporation processes. PVA is especially efficient in soils with granular texture and with small specific surface area, in which it creates a stable crust with strong tensile strength. However, the environmental impacts of this synthetic polymer and its efficiency in fine-grained soils with high content of dust are still unclear. This raises the need to develop an effective material from the perspectives of dust control, soil stabilization, and environmental footprint. Geopolymers are a group of mineral-based materials that commonly used in the building industry [9]. Geopolymer can be synthesized by mixing calcined kaolin and strongly alkaline solutions (such as NaOH or KOH) and then curing at a room temperature [10]. Several studies have examined the use of various geopolymers for loess soil stabilization, including fly ash, lime, and cement, etc., [11–16]. However, the use of the anhydrous calcined form of the clay mineral kaolinite (metakaolin) was given less attention in loess stabilization. Loess is a widespread surface deposit in many parts of the world. There is a strong need for loess stabilization in construction requirements and geohazard mitigation due to its propensity to collapse and subsidence after loading and wetting [11,12]. Moreover, unpaved roads in loess are associated with strong erosion by wind and water, which leads to soil disintegration [7]. This study examined the use of a metakaolin-based geopolymer with different compounds and concentrations for the reinforcement of the topsoil of a semi-arid loess soil.

#### **2. Materials and Methods**

The experiments conducted on a typical loess soil of the northern Negev, Israel. The semi-arid region of the northern Negev is characterized by annual average rainfall of ~200 mm. Most of the loess soils in northern Negev are a secondary material that reworked under pedogenic and geomorphological processes after investment of aeolian and/or pluvial materials [17]. Currently, the northern Negev loess is subjected to intense human activities and related soil erosion and dust emission, e.g., [6,18].

The texture of the soil is silt-loam with a relatively high content of silt and clay particles. Particle size distribution was obtained by the ANALYSETTE 22 MicroTec Plus (Fritsch, Idar-Oberstein, Germany) laser diffraction, which measures particles in the size range of 0.08–2000 μm [19,20]. Replicas (100 mg) were dispersed in Na-hexamvetaphosphate solution (0.5%) by sonication (38 kHz). The data was calculated using the Fraunhofer diffraction model with a size resolution of 1 μm using MasControl software (Figure 1a). Mineralogical analysis, which was obtained by the X-ray power diffraction (XRPD) method, shows typical loess composition with high content of quartz, clays (illite, kaolinite), as well as Ca-based components that are found in arid loess (Figure 1b).

For the dust control experiment, the soil was placed in trays that fit the wind tunnel dimensions (0.5 × 1.0 m, with height of 0.02 m). Three trays (replicas) were prepared for each soil treatment/product. Table 1 presents the products used for the laboratory experiment and their application on the soil. The composition of the solution of each substance and the concentration of the solution to be applied by spraying on the soil surface were provided by the suppliers of the products and are typical in dust control. The soil samples were left outside for a drying process of several days before the experiment to avoid the impact of the amount of the solution (water content) on wind erosion and dust emission. The geopolymer for the dust control experiment was composed of metakaolin (MK) with an activation solution containing NaOH and sodium silicate in a constant ratio. The activation solution was added to the soil mixture in over-saturated until reaching a shed state of the mixture. The MK amount added to the soil sample was 30% of the soil sample weight. The geopolymer was mixed in the soil sample and tested after a drying process of 28 days.

The soils were tested in a wind tunnel for dust emission—overall 30 runs were performed by the wind tunnel (5 samples, 3 replicates, 2 wind velocities). These experiments were performed in the portable boundary-layer wind tunnel of the Aeolian Simulation Laboratory at BGU [8]. Boundary-layer wind tunnels enable simulations under standardized quasi-natural wind conditions and provide quantitative information on dust emission rates from the soils. The wind tunnel has a cross sectional area in the order of 0.5 × 0.5 m with open-floored working sections of up to 10 m length. Air push or air suction flow in the tunnel is generated by an axial fan up to a maximum velocity of 18 m s−1. Instruments installed in the test section of the tunnel enable quantification of wind characteristics and dust (PM) concentrations.

**Figure 1.** Characteristics of the Loess sample used for the experiments: (**A**) particle size distribution obtained by the laser diffractometer technique with the statistical parameters (right side). (**B**) Mineralogical composition of the loess by the X-ray power diffraction (XRPD).

**Table 1.** List of products for dust control in the loess soil used in the laboratory experiment. The composition of the solution of each substance and the concentrations of the solution to be applied by spraying on the soil surface were provided by the suppliers and are typical in dust control. The geopolymer was prepared and applied as described in the text.

The tunnel was operated under two wind velocities, 6.5, and 9.5 m s−1, which represent medium and strong natural winds that are associated with dust emission in semi-arid loess soils [13]. Each run of specific wind velocity lasted 30 s to record the trend of dust emission. Dust concentrations (μg m−3) PM10 were recorded by a light-scattering device, DustTrak DRX 8534 (TSI, Shoreview, MN, USA), in the range of 0.001–150 μg m−3 (±0.1% of reading) at 1-s intervals, which was placed at 25 cm above the tunnel bed. The recorded PM10 concentrations were converted into mass flux emitted from the soil surface (mg m−2 s−1) based on the wind tunnel dimensions, the area of the soil bed, and the wind

velocity (for in depth description refer to Katra et al. 2016 [21]). The PM10 flux from the soil surface can be used in parameterization of dust emission and transport models [7,21].

The soil sample mixed with the geopolymer was examined also for soil stabilization by the tensile strength test. A 300 g quantity of soil was mixed with MK (with the activation solution) to form test sample in three replicates. The mixtures were then poured into a 3.5 cm diameter cylinder on a length of 7.5 cm. Each sample series was dried for 28 days until the bonding material stabilized before testing the strength of the stabilized soil sample. The tensile strength of the sample was measured using the Universal Testing Systems for Tensile, Compression, and Flexure Testing of Instron 5900 Series (Instron, Norwood, MA, USA) at a crosshead speed of 10 mm min−1. This device is commonly used to measure the strength of samples in cement engineering and other materials. The soil sample was placed at the center of the lower surface of the pressure clamps for pressing small pulses until the graph stabilized after the collapse of the sample. The strength tests were conducted for two groups of geopolymer compositions (Table 2). (1) Geopolymer with different MK percentage (0–30%) in the soil sample. (2) Metakaolin (MK) based geopolymer with an alternative activation solution, replacing the NaOH with lime (CaO). Different concentrations of CaO were tested, where the measured solubility level of lime at 20 ◦C (1.79 g L) was taken as a starting point. The amount of lime suspension (in water) used in the activation solution was the same as the NaOH in the origin activation solution, and up to 20-times. The content of the MK was 30% in this group.

**Table 2.** The compositions of the geopolymer as a loess soil stabilizer used for the tensile strength test. Group 1: different MK content (%) with a constant activation solution of sodium silicate and NaOH. Group 2: different concentrations of CaO as a replacement to the NaOH in the activation solution with MK content of 30%.

#### **3. Results and Discussion**

Figure 2 presents the results of the dust emission experiment. The recorded PM10 concentrations are a result of dust emission from the surface of the loess soil samples under two wind velocities. It is clearly shown that no dust emission and PM10 concertation was recorded in the application of the clay-based geolopyler. The "straight" line that was similarly recorded for the two wind velocities in the test of the geopolymer application is a representation of the background level (~20 μg m−3) measured in the wind tunnel before the test. In all the other applications, bitumen, brine and PVA, the averaged PM10 concentrations were significantly (*p* < 0.05) higher than the background level. In general, the PM10 concentrations received for the brine, PVA, and bitumen, were higher under 9ms−1 compared with 6.5 m s−1. The wind velocity OF 9.5 m s−1 was run promptly after the prior velocity (6.5 m s−1), and since the amount of dust particles in the soil samples is limited, the dust emission in 9.5 m s−1 could have been an underestimation for non-limited samples. The PM10 values of the control sample were significantly higher than those of all the other samples in both wind velocities, suggesting that all the applied products are efficient for dust suppression at a certain level. It is interesting to see that the brine, which was found to be very efficient by reducing > 90% of the dust emission in calcareous soils under the same experimental conditions [8], is less effective in loess soils. The results of the brine are at the range of the PVA. However, the brine is not used for the purpose of soil stabilization as the PVA and the geopoylmers.

**Figure 2.** PM10 concentrations recorded during the laboratory wind tunnel experiment on the diverse dust control products under wind velocities 6.5 (**right** side) and 9.5 m s−1 (**left** side). Note, the "control" is the Loess soil with no treatment. The background level measured in the wind tunnel before the test was ~0.020 mg m−3.

Tensile tests were conducted for the geopolymer as a soil stabilizer. The first test was to examine the effect of the MK amount at the range of 0–30% in the geopolymer composition (Group 1, Table 2). The activation solution (sodium silicate and NaOH) remained in a constant ratio. The results in Figure 3a show that the soil sample without MK (0%) resulted in resistant of less than 1000 N. Application of MK content at the range of 2.5–10% improved the soil resistant up to 6000 N. A significant change was obtained for samples with MK content at the range 20–30%. These samples resulted in a consistent increase in resistance to pressure when the peaks ranges from ~11,000 N (20% MK) to ~25,000 N (30% MK). Just for comparison, we tested also the PVA as soil stabilizer, which has been used extensively in soil stabilization [22]. The PVA results after a curing time of 28 days were up to 4200 N. The drying time until significant strength is reached is an important factor when there is a need to stabilize an unpaved road. External factors, such as solar radiation and water regime, can have a great impact on the nature and duration of the soil stabilization. As such, this study examined the hardness of the geopolymer during a short seven-day cure. The geopolymer composition of 30% MK with the activation solution (sodium silicate and NaOH) yielded a result of 15,510 N.

**Figure 3.** (**A**) Resistance to load of stabilized Loess soil with different contents of Metakaolin (MK, 0–30%) in an activation solution containing sodium silicate and NaOH (Group 1, Table 2). (**B**) Resistance to load of stabilized Loess soil with calcium oxide (CaO) as a substitute for NaOH in the activation solution (Group 2, Table 2). The results are CaO at the same amount as the NaOH (X1), which was used in the geopolymer presented Figure 3A, and with enlarged amount of CaO up to 20-times (X5-X20). The content of the Metakaolin was 30% for all samples in Figure 3B.

A second tensile test was conducted for geopolymer with replacement of the NaOH with the base of lime (CaO) that is more "natural" soil material (Group 2, Table 2). The addition of lime is used in applications of soil loess stabilization, e.g., [23]. The lime improves the cation exchange of the soil and absorbs water, which lowers the pH. Adding lime also affects the structure of the soil through the soil ability to absorb water. The results of the test for soil samples stabilized by the geopolymer with different amounts of CaO in the activation solution are presented in Figure 3b. The MK content of 30% was constant for all the samples. The resistance increased as the concentration of the lime was higher. This was true up to lime concentration that is 15 (X15) times that of the original concentration (X1). At the highest concentration (X20), the resistance was lower (peak at 6450 N) than in X15 (peak at 7400 N). This can be explained by a rapid and unsteady solidification of the material, which does not allow us to pour the soil mixture into the molds. However, the results of the NaOH in the activation solution (Figure 3a) are significantly higher than those of the replacement base (CaO) with 30% MK (Figure 3a).

The results in Figures 2 and 3 show the potential of the clay-based geopolymer examined in this study to suppress dust emission and to stabilized loess soil. The application of the geopolymer for dust control in loess soil in comparison with common products (brine, bitumen, PVA) resulted in no dust emission. As a soil stabilizer, the geopolymer tested in this study provides remarkably good results in the tensile test. The most successful composition of the geopolymer, which is activation solution of sodium silicate and NaOH together with an addition of 30% MK, obtained soil strength of 23,900 N after 28 days. The attempt to replace NaOH with CaO in the activation solution was far inferior to the original composition. The factor of relatively rapid firming of the soil after seven days (strength of 15,510 N) is very important in stabilizing unpaved roads.

#### **4. Conclusions**

The clay-based geopolymer tested in this study showed high potential for application as a dust control product as well as soil stabilized in loess soils. There are important factors that should be considered in the development of such a geopolymer. (1) First, some elements of the activation solution are substances that require production by chemical or physical processes such as MK and sodium silicate, which involve certain costs not taken into account in this study. Second, this study did not take into account the effects of the final product on the environment over long time after the application in the soil. Nevertheless, there is a strong potential to develop natural soil stabilizers from a mineral base that even surpass their capabilities over existing synthetic stabilizers. The ability of these materials to generate reactions with the soil components will allow us to reach high levels of strength of the upper crust of the stabilized soil.

**Author Contributions:** Conceptualization, I.K.; methodology, I.K. and N.H.; formal analysis, N.H.; writing —original draft preparation, N.H.; writing—review and editing, I.K. and N.H.; funding acquisition, I.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by KKL-JNF, grant number 03-02-040-14.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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