Physical properties of springs.
\r\n\t
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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"2270",title:"Fourier Transform",subtitle:"Materials Analysis",isOpenForSubmission:!1,hash:"5e094b066da527193e878e160b4772af",slug:"fourier-transform-materials-analysis",bookSignature:"Salih Mohammed Salih",coverURL:"https://cdn.intechopen.com/books/images_new/2270.jpg",editedByType:"Edited by",editors:[{id:"111691",title:"Dr.Ing.",name:"Salih",surname:"Salih",slug:"salih-salih",fullName:"Salih Salih"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"117",title:"Artificial Neural Networks",subtitle:"Methodological Advances and Biomedical Applications",isOpenForSubmission:!1,hash:null,slug:"artificial-neural-networks-methodological-advances-and-biomedical-applications",bookSignature:"Kenji Suzuki",coverURL:"https://cdn.intechopen.com/books/images_new/117.jpg",editedByType:"Edited by",editors:[{id:"3095",title:"Prof.",name:"Kenji",surname:"Suzuki",slug:"kenji-suzuki",fullName:"Kenji Suzuki"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"57587",title:"Sensory Feedback Device for Myoelectric Prosthetic Hand",doi:"10.5772/intechopen.71690",slug:"sensory-feedback-device-for-myoelectric-prosthetic-hand",body:'\nA myoelectric prosthetic hand is an electrically driven artificial hand that is controlled based on biosignals generated by muscle movement. Therefore, the myoelectric prosthetic hand can be moved freely as intended by the user. However, the user cannot feel sensations when he/she touches an object using the prosthetic hand. Healthy person uses the tactile sense and temperature sense to check the state of the object that he/she touched. A myoelectric prosthetic hand cannot produce any sensations. Therefore, the user has to operate the prosthetic hand only based on visual information. Thus, the user needs to watch the object constantly. This is a burden to the user. To solve this problem, sensory feedback which provides sensation to the user has been studied. Sensory feedback systems for an upper limb prosthesis in the initial stage have been reported in [1]. Summary of studies involving sensory feedback in upper limb prosthetics is listed in [2].
\nA method of providing a sense of touch realized by vibration stimulation was proposed in [3], and a method of sensory feedback realized by the combination of vibration stimulation and electric stimulation was proposed in [4], respectively. On the other hand, Otsuka et al. [5] developed a device that perceives the temperature when an object was touched by a myoelectric prosthetic hand using a hot and cold pad. Morimitsu and Katsura [6] examined transfer of temperature sense using the Peltier element.
\nIn this study, two types of sensory feedback device were developed to enhance the quality of life (QOL) of myoelectric prosthetic hand users. First, a compact feedback device of force sense (hereinafter referred to as FFB device) with a safety mechanism was developed. The FFB device is mounted on the user’s upper arm, and when a prosthetic hand holds an object, a belt in the device is winded by a motor to present the holding force to the user’s arm. Besides, the winding speed of the belt is changed according to the hardness of the object held by the prosthetic hand. In the control system of the FFB device, a reference input creation model creates reference input signals according to the hardness. A self-tuning proportional-integral-derivative (PID) control method proposed by [7] was employed to adjust the gain of the PID controller based on the state of a target object and control the belt-winding speed of the FFB device following the reference input. For the verification of the effectiveness of the control system for the FFB device, a myoelectric prosthetic hand made by [8] was combined with a control method proposed by [9], and experiments to distinguish among five kinds of springs of different hardness were conducted.
\nSecond, a feedback device of temperature sense (hereinafter referred to as TFB device) was developed by using a Peltier element to present an object temperature when the user touches the object by a prosthetic hand. A temperature prediction algorithm was proposed to shorten the temperature measurement. Besides, the temperature sense differs at each body site. Therefore, the TFB device developed in this study transfers temperature sense felt by the fingertip to the upper arm based on the result of the experiment on temperature sense investigation. Furthermore, experiments to distinguish among five different temperatures were performed to verify the effectiveness of the TFB device. Finally, two-sensory feedback devices were united, and a two-sensory feedback device was built.
\nIn this study, the myoelectric prosthetic hand made by [8] shown in Figure 1(a) is used. The prosthetic hand consists of motors and wires, and the fingers are bent by winding the wires. A pressure sensor is attached to a finger cushion on the prosthetic hand’s index finger, and a temperature sensor is attached at the fingertip of the prosthetic hand’s middle finger.
\n(a) Myoelectric prosthetic hand. (b) positions of electrodes.
The myoelectric prosthetic hand used in this study only has three fingers, namely, the thumb, index finger, and middle finger. Therefore, the prosthetic hand grasps an object by bending the index finger for contact force feedback case and bending the middle finger for temperature sense feedback case. The motion of the index finger and the middle finger is identified by measuring the surface electromyogram (SEMG) of the flexor digitorum superficialis (ch 1) and extensor carpi radialis longus (ch 2) shown in Figure 1(b) to control the proximal interphalangeal (PIP) joint of the prosthetic hand’s index finger and middle finger.
\nIn this study, amputees who lost the lower half of a single forearm were chosen as subjects, and electrodes were placed on the upper half of the forearm to operate a prosthetic hand. To let prosthetic hand users recognize the sense more intuitively, the sense of pressure was given to the prosthetic hand users when they grasp an object. The sense of pressure was presented by the tightening force of a belt on the upper arm of the users.
\nThe difference of the contact force according to the hardness of the object is expressed as the winding speed of the belt. The pressure value added to the finger and displacement of the finger are measured, and they are used to estimate the object hardness. Then, the winding speed of the belt is changed to present the estimated hardness. Namely, the high winding speed is for a hard object, and the low winding speed is for a soft object.
\nFigure 2 shows the developed FFB device and its attached state on the user’s upper arm. The working principle is described as follows. Contact force between an object and a finger is measured by the pressure sensor attached to the finger cushion on the prosthetic hand’s index finger. The main shaft for winding the belt and the motor are connected through two gears. When the prosthetic hand grasps the object, namely, when the contact force is sensed, the motor rotates. Therefore, the main shaft is also rotated by the rotation of the motor. Thus, the belt is winded and tightens the upper arm.
\nFFB device and its attached state.
The device is small with the dimensions 97 mm (width), 117 mm (depth), and 39 mm (height).
\nA safety measure against the motor’s failure or other emergent case needs to be taken. In the case of emergency, the belt is released by simply opening the cover, and then the device is released from the arm.
\nIn this study, a control strategy proposed in [9] is used for the operation method of the myoelectric prosthetic hand. It is well known that an integrated electromyogram (IEMG) reflects muscle activity. Hence, the IEMG is employed to identify the input motion for the operation of the prosthetic hand, and a support vector machine (SVM), which is one of the techniques of the machine learning, is used as an identifier. For the control of the prosthetic hand, a target angle of the finger of the prosthetic hand is set based on how long the user keeps the muscle force. This allows the user to arbitrarily control the finger angle.
\nA pressure sensor “FSR402 Short Tail” made by Interlink Electronics was attached on the fingertip to measure the reaction force 𝐹 [N] from the grasped object. The displacement of the fingertip [m] is measured from the encoder attached on the driving motor of the finger. Then, the spring constant 𝐾 [N/m], hereinafter referred to as the hardness parameter, can be calculated from Hooke’s law:
\nA preliminary experiment was conducted in [10] to derive a conversion formula from the pressure (voltage
With Eq. (2), a reaction force is calculated from the measurement value obtained with the pressure sensor. Thus, one can calculate the hardness parameter from Eq. (1).
\nFor the purpose of this study, the winding speed of the belt in the FFB device needs to be adjustable according to the hardness of the grasped object. A reference input creation model is defined as shown in Figure 3, in which a reference input
Reference input creation model.
The following relation is obtained from Figure 3:
\nA small time constant of the primary delay filter produces a rapidly rising reference input, and a large time constant produces a gradually rising reference input. The time constant
For safety reason, the FFB device is configured so that up to 10 mm of the belt is wound up.
\nA function of the time constant
Numerical simulation was performed to verify the reference input creation model. In the verification, the hardness parameter was increased from 1000 to 6000 for each 1000, and the reference input derived from the reference input creation model was checked. The result is shown in Figure 4.
\nReference inputs.
Figure 4 shows that the rise of the curve becomes rapid as the hardness parameter increases. This result indicates that the reference input could be efficiently created so that the user can feel a difference of the hardness.
\nThe FFB device gives feedback of the force sense by pressing the upper arm of the user. The amount of fat and muscle of the human arm differs from person to person, and the arm hardness could also change depending on how much the user strains the arm. Thus, the arm hardness has a nonlinear characteristic. Therefore, an adaptive control is used for the control of the belt-winding motor of the FFB device. To this end, the self-tuning PID control proposed by [7] is used. Since the self-tuning PID control scheme is based on a discrete time control, Eq. (3) is discretized by a bilinear transformation to design a controller of the FFB device.
\nThe control aim is to determine the control input
Here, 𝑘 is the number of steps. Then, the control input is given by the following equation:
\nFor the verification of the functions of the whole system, the prosthetic hand was operated based on the SEMG of a subject, and the hardness of an object that was grasped by the hand was estimated. Then, how the FFB device controlled by the self-tuning PID could follow the reference input created from the estimated hardness was examined.
\nFive kinds of springs with different hardness were chosen as objects to grasp. Table 1 shows the physical properties of the springs.
\nNo. | \nSpring constant | \nDiameter [m] | \nLength [m] | \n
---|---|---|---|
S1 | \n990 | \n14 × 10−3 | \n25 × 10−3 | \n
S2 | \n1800 | \n8 × 10−3 | \n25 × 10−3 | \n
S3 | \n2980 | \n10 × 10−3 | \n30 × 10−3 | \n
S4 | \n4390 | \n13 × 10−3 | \n25 × 10−3 | \n
S5 | \n5340 | \n7 × 10−3 | \n25 × 10−3 | \n
Physical properties of springs.
The subject was an adult male in 20s. The results in which each of the springs was grasped once are shown in the following figures and tables. Figure 5 shows how the FFB device followed the reference input. Table 2 shows the results of the hardness estimation. Table 3 shows self-tuning PID gains and the time constant calculated from the estimated hardness.
\nReference input and output of the FFB device.
Spring | \nForce [N] | \nDisplacement [m] | \nEstimated hardness parameter [N/m] | \nReal hardness parameter [N/m] | \n
---|---|---|---|---|
S1 | \n5.23 | \n3.76 × 10−3 | \n1390 | \n990 | \n
S2 | \n5.27 | \n2.56 × 10−3 | \n2060 | \n1800 | \n
S3 | \n5.66 | \n1.80 × 10−3 | \n3150 | \n2980 | \n
S4 | \n5.15 | \n1.27 × 10−3 | \n4060 | \n4390 | \n
S5 | \n4.96 | \n0.99 × 10−3 | \n5030 | \n5340 | \n
Estimated and real hardness parameter.
Spring | \nTime constant | \nPID gain | \n||
---|---|---|---|---|
\n\n | \n\n\n | \n\n\n | \n||
S1 | \n1.78 | \n0.22706 | \n0.0125 | \n0.01154 | \n
S2 | \n1.58 | \n0.22707 | \n0.0125 | \n0.01154 | \n
S3 | \n1.06 | \n0.22713 | \n0.0125 | \n0.01150 | \n
S4 | \n0.69 | \n0.22710 | \n0.0125 | \n0.01151 | \n
S5 | \n0.49 | \n0.22717 | \n0.0125 | \n0.01147 | \n
Time constant and self-tuned PID gains.
Figure 5(a) and (b) shows a time delay of about 0.2 s in the response of the FFB device for any spring. However, the device clearly followed the reference input. Although a small error between the estimated value and the real value of the hardness parameter is seen in Table 2, the time constant was successfully calculated for all of the springs as shown in Table 3. Therefore, it was confirmed that the reference input could be created according to the estimated hardness of the grasped object. In addition, the belt-winding action of the FFB device under the control of the self-tuning PID controller followed the reference input obtained from the estimated hardness. Thus, it was verified that the system worked properly.
\nThe usefulness of the FFB device with the proposed system implemented is objectively verified with a psychophysics experiment method. Five kinds of springs, shown in Table 1 in the previous section, were used as the target of the hardness identification.
\nIn the experiment, the myoelectric prosthetic hand was not used, but the spring constant of each spring was input directly to a computer, and then the hardness identification experiment using the FFB device was conducted.
\nThe experiment was conducted by following the procedure of a constant method. The hardness of a brass spring (
FFB device is attached on the upper arm of the subject.
The subject is trained so that he can recognize the behavior of the FFB device.
The standard stimulation is given to the subject.
A 4-second interval is taken.
The comparative stimulation is given to the subject.
The subject answers which stimulation is harder or whether the two are the same.
25 sets of operations [(3)–(6)] are conducted. In the operations, all the comparative stimulations are used five times in random orders.
The operations [(3) and (5)] were replaced, and 25 sets of the operations [(3)–(6)] are conducted again.
The experiments were performed for five healthy subjects in their 20s. In the operations, a 1-min break was taken every five sets to prevent the subject from getting tired.
\nTable 4 shows the results of experiments. The bold numbers show the ratio of correct identification of the stimulations.
\nStimulation | \nRate of the subject’s answer [%] | \n||
---|---|---|---|
Hard | \nEqual | \nSoft | \n|
S1 | \n4 | \n12 | \n|
S2 | \n16 | \n16 | \n|
S3 | \n24 | \n30 | \n|
S4 | \n22 | \n0 | \n|
S5 | \n12 | \n0 | \n
Identification results of hardness.
From the results on the hardness identification experiment, one can see that the proposed method of presenting different varying hardness levels by using different belt-winding speeds was effective to identify the hardness of the five kinds of objects.
\nA combination of cold and warm sensations is called a temperature sense. The temperature sense differs at each body site even when an object of the same temperature is touched. In addition, when the skin is exposed to extreme heat or cold, the pain along with the risk of burns arises. Therefore, it is necessary to regulate the temperature of the TFB device.
\nThe Peltier element is an electronic device that enables both cooling and heating by applying a voltage on the basis of the Peltier effect, and temperature is adjustable by regulating the voltage. The TFB device has a Peltier element; thus, the temperature sense is transferred to the user. The Peltier element used in the TFB device is “TEC1-12708” made by HB Electronic Components.
\nA thermocouple is a temperature sensor on the basis of the Seebeck effect. The thermocouple is attached to the silicone finger mounted on the fingertip of the myoelectric prosthetic hand to measure temperature of an object when the fingertip touches the object. A K-type thermocouple “AD-1214” made by T&D Corporation is used in this study.
\nFigure 6 shows the developed TFB device and its attached state on the user’s upper arm. The Peltier element is attached to the inside of an aluminum board, and in order to raise a heat dissipation efficiency, a radiation sheet and a heat sink are attached to the other side of the aluminum board. The TFB device is attached on the upper arm of the user in contact with the user’s skin, and the temperature sense that is corresponding to the temperature detected by the temperature sensor at the fingertip is transferred in the upper arm of the prosthetic hand user.
\nTFB device and its attached state.
The device is small enough with the dimensions 50 mm (width), 60 mm (depth), and 17 mm (height).
\nWhen the TFB device is used for a long period, the accumulated heat causes a high temperature of the TFB device. Therefore, the continuous operating time of the TFB device is limited to 5 s.
\nWhen the TFB device decreases the temperature, it enables refrigeration of surface temperature to a minimum of 15°C for 5 s. On the contrary, when the TFB device increases the temperature, it enables heating of surface temperature exceeding 50°C. Hence, the temperature of the TFB device is limited to 40°C for safety.
\nAfter the myoelectric prosthetic hand contacts with the object, the temperature sensor needs a long time for measuring the temperature. Therefore, a temperature prediction is performed to shorten the measurement time.
\nLet relationship between a sensor output
To determine the transfer function
Let
where ∆
Let
The predicted temperature is updated to reduce the error when the sensor detected a temperature variation; thus, the temperature when reached to the equilibrium state is given by Eq. (10). As a result, the sensor can detect the temperature of the object in a short time.
\nThe verification experiment was performed to verify an effectiveness of the proposed temperature prediction algorithm. In the experiment, under the room temperature of 26°C, the sensor touched an object of 40°C. Then, the temperature was predicted by the proposed algorithm. Figure 7 shows the result.
\nResult of the temperature prediction for the object of 40°C.
The result showed that prediction of the temperature in a short time is possible by using the proposed temperature prediction method.
\nThe temperature sense of an individual differs at each body site. For example, the temperature senses when an object with the same temperature is touched with a finger and with the upper arm are different. Therefore, in order to investigate the difference in the temperature sense between the fingertip and the upper arm, a temperature sense investigation was performed. Thus, the temperature experienced by the upper arm which is equivalent to the temperature experienced by the fingertip was determined as feedback temperature to the upper arm.
\nThe temperature which should be presented at the upper arm and the voltage which should be applied to the TFB device were determined in [11] as follows. The relationship between the temperatures experienced by the fingertip and by the upper arm was interpolated. Then, the relationship between the predicted temperature at the fingertip
In the previous section, the developed TFB device was controlled in an open-loop system, in which the constant voltage computed from Eq. (12) was applied to the TFB device. In the case where the voltage is continuously provided to the TFB device, the temperature keeps increasing or decreasing. Hence, it is impossible to maintain the temperature using this control system. Therefore, the continuous operating time of the TFB device was limited to 5 s.
\nTo use the TFB device continuously, a closed-loop control system was constructed. For this purpose, another temperature sensor was additionally attached on the surface of the Peltier element of the TFB device. The target temperature in the upper arm
In order to verify the effectiveness of the closed-loop control system with PID controller, experiment was performed. In the experiment, the target temperature is suddenly decreased from 40 to 15°C. The transition of the temperature of the TFB device and the input voltage to the TFB device are shown in Figure 8.
\nTemperature of the TFB device and input voltage to the TFB device.
As shown in Figure 8, the results showed that the constructed closed-loop system enabled the adjustment of the temperature of the TFB device according to the temperature change and also enabled a long-time continuous operation of the TFB device.
\nIn order to verify the performance of the TFB device controlled in the closed-loop control system, a temperature identification experiment was performed. The usefulness of the TFB device controlled by the closed-loop system is objectively verified with a psychophysics experiment method.
\nIn this experiment, the myoelectric prosthetic hand was not used, but each temperature was input directly to a computer, and then the temperature identification experiment using the TFB device was conducted.
\nThe temperature of 30°C was used as standard stimulation, and five kinds of temperature, 28, 29, 30, 31, and 32°C, were used as comparative stimulation. The following describes the experimental procedure:
\nTFB device is attached on the upper arm of the subject.
An experimenter inputs the standard stimulation (30°C) to the computer, and standard stimulation is presented to the subject by the TFB device.
An experimenter inputs the comparative stimulation that is randomly selected from the five kinds of temperatures to the computer, and comparative stimulation is presented to the subject by the TFB device.
The subject answers which temperature is higher or whether the two are almost same.
25 sets of the operations [(2)–(4)] are performed.
The operations [(2) and (3)] were replaced, and 25 sets of the operations [(2)–(4)] are performed.
In the operations, all the comparative stimulations were used 10 times in random orders. The experiments were performed for five healthy subjects in their 20s.
\nTable 5 shows the results of experiments. The bold numbers show the ratio of correct identification of the stimulations.
\nTemperature of comparison stimulus [°C] | \nRate of the subject’s answer [%] | \n||
---|---|---|---|
Hot | \nEqual | \nCold | \n|
28 | \n0 | \n14 | \n|
29 | \n4 | \n36 | \n|
30 | \n6 | \n12 | \n|
31 | \n34 | \n6 | \n|
32 | \n10 | \n4 | \n
Identification results of temperature.
From the results on the temperature identification experiment, one can see that the proposed method of presenting temperature by the TFB device controlled in the closed-loop control system was effective to identify the five kinds of temperatures.
\nTo improve the operability of the myoelectric prosthetic hand, a new myoelectric prosthetic hand was designed and built by imitating the commercial prosthetic hand, which is shown in Figure 9. The pressure sensor and the temperature sensor were attached to the fingertip of the thumb and index finger of the prosthetic hand, respectively. Thus, this myoelectric prosthetic hand makes it possible to detect the force and temperature when the prosthetic hand holds an object.
\nNew myoelectric prosthetic hand.
Finally, the FFB device and the TFB device were united, and a two-sensory feedback device was built, which is shown in Figure 10. The dimensions of the device are 75 mm (width), 82 mm (depth), and 34 mm (height).
\nTwo-sensory feedback device and its attached state.
In this study, force feedback device (FFB device) and temperature feedback device (TFB device) were proposed and built. When a user of a myoelectric prosthetic hand grasps an object, the FFB device provides pressure to the user’s upper arm by winding a belt using a motor, and the TFB device presents the temperature sense to the user’s upper arm using the Peltier element.
\nIn the FFB device, the hardness of the object was estimated by a pressure sensor attached on the fingertip of the myoelectric prosthetic hand, and a reference input was produced by a reference input creation model according to the hardness. In addition, a self-tuning PID controller was employed to control the FFB device so as to make the motor’s output angle follow the reference input. Furthermore, the hardness of the grasped object was presented by the winding speed of the belt. Hardness identification experiment to distinguish among the five kinds of springs of different hardness was carried out. The experimental results on the hardness identification experiment showed that the proposed method was effective to identify the hardness of the five kinds of objects.
\nIn the TFB device, a temperature prediction algorithm was proposed for short-time temperature detection. Then, based on the results of the temperature sense investigation, the corresponding temperature sense when the object was touched by a fingertip was transferred to the user’s upper arm by the TFB device. However, it was difficult to operate the TFB device continuously because this device was controlled in an open-loop control system. To solve this problem, a closed-loop control system was constructed for the TFB device and was tested for sudden change of the temperature. Temperature identification experiment to distinguish among five different temperatures was carried out to verify the effectiveness of the TFB device controlled in the closed-loop control system. The experimental results on the temperature identification experiment showed the sufficient capability of the TFB device controlled in the closed-loop control system.
\nIn addition, a new myoelectric prosthetic hand was built to improve the operability of the myoelectric prosthetic hand. Finally, two-sensory feedback devices were united, and a two-sensory feedback device was built.
\nThe author thanks Mr. T. Morita, Y. Ueda, and M. Isobe for their assistance in experimental works.
\nEver since the human evolution and civilization, human has been exploiting animals either for food, transport and or as companions. The use of animals in biomedical and behavioral research has greatly increased scientific knowledge and has benefitted human health enormously. Tremendous advancement took place in the field of medical sciences with the usability of animals for experimental research. Currently, around 75–100 million vertebrates are used annually in research and testing [1]. The most frequently used animals are mice and rats that constitute approximately 95% of experimental animals; mouse being the most commonly used animal in biomedical research [1]. Animal models are used in research for wider understanding of vital physiological processes in human and animals. Animal models are also useful in investigating various diseases including metabolic disorders such as diabetes, cardiovascular disease (CVD), disorders in reproductive endocrinology, infertility, cancer and infectious diseases [2, 3, 4, 5, 6, 7]. Human lives endangered due to organ failure were restored after successful organ transplantation accomplished in animal models. Optimization of cryopreservation protocols will significantly facilitate organ transplantation and/or replacement. In addition, animals like dogs, pigs, cats, sheep, non-human primates (NHP) and fish are widely used for genetic and physiological studies in human health and disease [8]. The chapter is focused on the fundamentals of cryobiology and strategies in male (sperm) and female fertility (oocyte and ovarian tissue) cryopreservation. The last section is focused on the frontiers in cryopreservation of most widely used animal models like rodents and higher animals used in biomedical research and toxicological studies.
Large numbers of animal breeds worldwide are either extinct or endangered with few at the verge of extinction. Hence, it is crucial to develop and apply rescue strategies to ensure survival of these species for the future. One way is to preserve the genetic resources or the germplasm of these species for their maintenance and future development. Genetic diversity is another threat resulting from animal husbandry errors that can result in genetic drift of existing colonies and genetic contamination of lines. Furthermore, weather related natural disaster is a major threat to animal husbandry and vivarium, especially in case of experimental animals that require special care in breeding and rearing colonies. Cattle and breeding industries are modified for the large-scale production of the animal species. There is an urgent need to improve the efficiency and sustainability of producing animals for food in the face of the ever-increasing world population. Improved understanding of mechanisms and challenges of reproductive technologies are vital for improving the viability of the livestock industry [9]. Hence, one solution to preserve animal species is by freezing. Cryopreservation has emerged as the most efficient and compatible method for freezing human and animal genetic resources [10]. Cryobiology is an integrated study of various biological and physical sciences. Semen, embryos, oocytes, somatic cells, nuclear DNA, and other types of biological material such as blood, serum, and tissue, can be stored using cryopreservation, in order to preserve genetic materials or for other applications [8, 11]. The primary benefit of cryopreservation is the ability to save germplasms for extended periods of time, and thus maintaining the genetic diversity of a species or a breed [12]. Also, germplasm of genetically engineered animals (GEA) and cell lines of various species can be preserved by cryopreservation method. Gene banks/cryobanks are established and contain repository of cryopreserved genetic resources to regenerate a particular population in the future [13, 14, 15]. Sperm cryopreservation has been successfully applied in various fields to benefit the mankind and animals. Of prime significance, assisted reproduction technology (ART), the forefront in infertility treatment today might be inconceivable without the efficient cryopreservation techniques.
Method of cryopreservation and recovery involves following steps. Initially, cells are treated with cryoprotective agents (CPAs) to prevent cryoinjury/damage. Later, cells are cooled in a controlled manner at subzero temperatures, at which the metabolic processes of the cell stops. The recovery of the cells follows a reversed procedure; cells are rapidly thawed and then the CPAs are gradually removed. The viability of the cryopreserved sample is enhanced with the use of appropriate CPA type and concentration and the appropriate rate of freezing [10].
The two basic methods of cryopreservation include the conventional slow freezing (SF) and the rapid freezing method, known as vitrification (Vit). These protocols require different concentrations of CPAs and apply different cooling rates. Figure 1 illustrates the two methods of cryopreservation.
Comparison of slow freezing and vitrification method.
Slow freezing involves progressive cooling of sample over a period of 2–4 h either manually or automatically using a semi programmable freezer. This method was developed by Behrman and Sawada [20]. In SF, a phase transition occurs from liquid to solid on temperatures below freezing point. Slow-cooling protocols involve the use of <1.0 M of cryoprotective agents (CPAs), such as glycerol or dimethyl sulphoxide (DMSO), which have minimal toxicity at lower temperatures with the use of a high-cost controlled rate freezer or a benchtop portable freezing container [21].
Main advantage of SF is the reduced risk of contamination during the procedure, without the need of highly skilled professionals. However, SF has many disadvantages. It is time consuming and expensive. There is a high risk of cryoinjury due to the formation of extracellular ice. Although, slow cooling considered a successful method, the success rate is considerably low and might not be suitable for all kinds of cells and tissues [22]. SF is a commonly used method for preservation of animal germplasm for majority of farm animals like sheep, cow, zebrafish etc. [23, 24]. Though with certain drawbacks, nonetheless SF was found more efficient method to cryopreserve ovine embryos in comparison to vitrification [25].
To circumvent the process of ice crystallization, cost and duration for cryopreservation in SF, an alternative technique called rapid freezing or vitrification (Vit) was developed. Vit is an ultrarapid cooling method with high cooling rate, which enables putting cells at cryogenic temperatures, forming what is known as ice-front status, while avoiding ice crystallization, hence also termed ice-free cryopreservation. Vitrification is now the recommended protocol for embryos and cells freezing. The process includes cooling the cells or tissue to cryogenic temperatures using liquid nitrogen, after their exposure to high concentrations of CPAs, with subsequent rapid cooling to avoid ice nucleation [11]. The cell suspensions are transformed directly from the aqueous phase to a glass state upon exposure to LN2. High CPA concentration and higher cooling and warming rates eliminates ice formation [26]. Several factors like viscosity or thickness of the sample, cooling and warming rates, sample volume, CPA type and concentration have considerable effect on the process. Thus, a balance of all the important factors is required for successful vitrification. Vitrification is of two types—equilibrium and nonequilibrium. Formulation of multimolar CPA mixtures and their injection into the cell suspensions is termed as Equilibrium vitrification. Non-equilibrium vitrification includes the use of carriers like plastic straws or cryoloops for obtaining minimum drop volume and carrier-free systems that employs higher freezing rate and lower CPA mix concentration. In comparison to slow freezing, vitrification is more advantageous as is associated with decreased risk of cryoinjury, thereby ensuring sufficiently higher cell survival rate [11]. To note, there is no universal protocol followed for the animal species. However, germ cells from species like mouse ovary [27], fish embryos [28], ovarian sheep [29] and many other species has been cryopreserved by vitrification [24].
Liquidus tracking (LT) is a slow and controlled vitrification protocol with gradual increase in the cryoprotectant concentration simultaneously with continuous decrease of the temperature at subzero ranges in a specified rate. With LT, recovery and restoration of chondrocytes was achieved successfully from cryopreserved articular cartilage [30] and promising results has been reported in case of ovarian tissue cryopreservation [31]. The principle of LT is the dynamic control of CPA concentrations, throughout the cooling process, in order to maintain the cell just above its freezing point at all times, without the formation of ice [11]. An example, Ovarian Tissue Cryopreservation (OTC) by LT has been reported successful in restoration of ovarian function in sheep model [29].
Fowler and Toner proposed the applicability of laser light in cryopreservation process. A successful recovery of rapidly frozen red blood cells by vitrification was achieved without the use of CPAs. Principally, laser targets only the intracellular ice causing it to melt and resolidify into glassy state. After thawing and use of laser light, around 80% of cells treated remained viable [32]. In an attempt, Jin
New approach in the cryopreservation method is freezing under pressure. Previous method discussed above employed constant-standard pressure (isobaric) conditions near 1 atm of pressure. Isochoric (constant-volume) cooling provides means to significantly lower nonfrozen storage temperatures without any or with only minimum requirements for CPAs, achieving greater metabolic reduction without injury associated with freezing, CPA toxicity, or increased amounts of osmotic solutes. The isochoric cryopreservation is a two-phase equilibrium process, in which ice and liquid exist simultaneously at equilibrium under constant temperature and volume, while hyperbaric cryopreservation the solution is maintained in a single phase as liquid, the survival rate is however low in this method [34]. RBCs were successfully cryopreserved by this method [35]. Despite multiple attempts, scientists have not been able to cryopreserve and restore normal functions of complex bio-samples, such as mammalian tissues and organs.
Prior to the understanding of the role of cryoprotective agents (CPAs), the impact of subzero temperatures on healthy tissues and basic principles in cryobiology must be knowledged. As known, water is one of the most essential elements present in every cell, tissue, and organ of the living organisms on earth. It constitutes around 80% of tissue mass [36]. On lowering temperature, water undergoes phase transition (liquid to solid) and results in ice crystallization. The formation of intracellular ice cause damage to cellular structure and its function consequently leads to cryoinjury. Freezing can cause two types of harmful effects on cells. The formation of ice crystals damages the cell membrane and thus regain of structurally intact cells on thawing would be difficult. Further, ice formation increases the solute concentration leading to osmotic imbalance and cellular damage. To minimize or to mitigate these effects, two protective actions viz. selection of effective cryoprotectant, and appropriate cooling and thawing rates must be undertaken.
The discovery of CPA and its role in reducing cryoinjury was a significant step in cryopreservation success. Biological acceptability, cell penetration, low toxicity, are some of the properties, a CPA should possess. As mentioned in previous section, best survival rate of cells and tissues depends on the optimization of factors like cooling rate, warming rate, sample volume. CPA concentration is a major factor influencing the success of the cryopreservation [37]. Based on their penetrating capabilities through cell membrane, CPAs are classified into two categories- membrane permeable/permeating and membrane impermeable/non permeating.
Permeating CPAs are smaller sized (typically less than 100 daltons), and amphiphilic in nature [38]. Owing to these properties these molecules can penetrate through the cell membrane easily, tend to equilibrate within the cytoplasm and replace the intracellular water in order to avoid excessive dehydration. Henceforth, they protect the cell from intracellular ice formation (IIF) and salt accumulation. Examples of CPAs in this category are: glycerol (the first agent discovered), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propanediol (propylene glycol) [39]. The protective role of CPAs is due to hydrogen bonding with water molecules, and lowering the freezing point of water. As a result, less water molecules are available to interact with themselves to form critical nucleation sites required for crystal formation [40]. To minimize toxicity, vitrification mixtures are often added in a stepwise fashion at temperatures near 0°C. Addition of permeating agents prevent the formation of ice and permit cell storage at supercool temperatures. Besides vitrifying, few CPAs, example DMSO have properties to enhance the cell permeability in a dose dependent manner. DMSO of about 5% is reported to increase permeability by reducing the thickness of the cell membrane. DMSO at 10% concentration is more effective and commonly used as it induces water pore formation in biological membranes. Intracellular water can thus easily be replaced by CPAs to promote vitrification. At higher, toxic concentrations (40%) however, lipid bilayers begin to disintegrate [41]. Thus selection of appropriate CPA concentration is vital for maintaining structural integrity and viability after freezing.
Unlike permeating, non-permeating agents are covalently linked dimers, trimers or polymers with a larger size. They cannot pass through the cell membrane and exert their protective effect extracellularly. Most commonly used non-penetrating CPAs are polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), raffinose, sucrose, and trehalose [42, 43]. The mode of action of non-permeating agents is similar to permeating agents by controlling osmolarity but works extracellularly and at a lower degree.
List of different permeating and non-permeating cryoprotective agents used in gamete and embryo cryopreservation in different species is listed in Table 1. Besides these commonly used CPAs, protein like sericin from silkworm and small antifreeze proteins derived from marine teleosts or fishes have also garnered attention as CPAs in cryobiology [11].
Dimethyl sulphoxide | Oocyte, ovarian tissue, embryo, sperm | Mouse, cow, human |
Formamide | Sperm | Mouse, horse, dog, goose |
Acetamide | Oocyte, embryo | Mouse, rat, rabbit, pig |
Propionamide | Sperm | Rabbit |
Lactamide | Sperm | Rabbit |
Butyramide | Sperm | Rabbit |
Malonamide | Sperm | Rabbit |
Methanol | Sperm | Horse |
Ethylene glycol | Oocyte, embryo, sperm | Mouse, cow, human |
Glycerol | Oocyte, embryo, sperm | Mouse, cow, human |
Xylitol | Embryo | Rat |
Arabitol | Embryo | Rat |
Erythritol | Embryo | Rat |
Glucose | Sperm | Cat |
Galactose | Sperm | Horse |
Sucrose | Oocyte, embryo, sperm | Mouse, cow, human |
Trehalose | Oocyte, embryo, sperm | Mouse, cow, human |
Lactose | Sperm | Mouse, cow |
Maltose | Sperm | Mouse, rabbit |
Raffinose | Oocyte, sperm | Mouse, horse |
Dextran | Embryo | Mouse, cat |
Polyethylene glycol | Oocyte, embryo | Mouse, human, cow |
Ficoll | Oocyte, embryo | Mouse, human, cow |
Polyvinyl alcohol | Oocyte, embryo | Mouse, sheep, cow |
Hyaluronan | Embryo, sperm | Mouse, sheep, cow |
List of different permeating and non-permeating cryoprotectants used in assisted reproductive technologies.
Choice of appropriate cooling and thawing rates is another vital step for a successful cryopreservation. Mazur [44] has previously demonstrated the significant correlation between cooling rates and survivability of various cells [44]. The cooling rate was directly proportional to intracellular ice formation and inversely proportional to survivability among various cells. Figure 2 illustrates the survival of several cell types after cryopreservation in relation to the cooling rate. All cells exhibited a characteristic inverted U-shaped curve indicating that the survival rate increases with an increase in cooling rate upto a point after which the survival gradually decreases. Larger cells dehydrate slowly compared to smaller cells. Hence, in the light of this, rates of cooling and thawing should be adjusted. With an exception to larger cells, a cooling rate of approximately 1°C/min is often recommended. Controlled rate freezers that modulate chamber temperatures are used for this purpose. Following cryopreservation, cells are stored for future thawing and appropriate use.
An inverted U shape curve demonstrating relationship between cooling rate and cell survival (adapted from article by Cipri
Unlike cooling rate, thawing rate has been given inadequate attention. Nevertheless, it is advisable to warm or thaw cells rapidly to prevent recrystallization of ice [47]. This can be explained thermodynamically as vitrified state is quasi-stable and can change into a lower energy crystal structure on thawing. Currently, to achieve maximum viability it is suggested to transport cryovials on vapor LN2 and warmed rapidly in a 37°C water bath for 90–120 s [39]. Decrease in viability after post thaw recovery is inevitable, no matter how well the cells were stored and thawed. Care should be taken to remove dead cells timely using density media like Ficol or other methods from the recovered cells to increase the viability of recovered cell prior to use. Density gradients can be utilized to increase viable cell density although this method often involves exposing cells to additional, potentially-harmful centrifugation. Current strategies for identifying cells that remain viable after preservation utilize organic fluorophores, and dyes.
The complete cryopreservation procedure is associated with great efforts to maintain cell viability and function. However, some of the cryopreserved cells often demonstrate decreased viability following thawing. Therefore, it is essential to remove dead cells and increase the viability of live cells. Currently, organic fluorophores are employed for viable cells selection. Viable cell selection using this method is commonly practiced in ART in animal reproduction example semen/spermatozoa of bull, buffalo, rabbit, alpaca, stallion/horse and many other species [48, 49, 50]. The earlier practice to achieve this, involved the use of density gradient. Although a little expensive, advanced technologies like magnetic affinity cell separation (MACS), and fluorescence activated cell sorting (FACS) appeared beneficial [51, 52]. Cryosurvival and recovery of sperm in species like stallion, bovine etc. using FACS has been investigated [53, 54]. New strategies like the introduction of nano-science had revolutionized the field of cryobiology and forwarded it to more higher stages as nanoparticle mediated cell sorting is non-destructive and more beneficial than FACS [55].
After the successful live birth with 21 years cryopreserved sperm sample, sperm viability in cryopreserved sample was evident [56]. Currently, sperm cryopreservation as a method of fertility preservation had gained tremendous significance and applicability in human and animals [57, 58]. Sperm survival rate was found to increase with glycerol as CPAs and increasing concentration of glycerol added at constant cooling rate for long term storages [59]. Sperm freezing can be done by one of the main two established methods; slow freezing or ultra-rapid freezing [60, 61]. Owing to the deleterious effects of SF on sperm physiochemical activity and motility, the rapid freezing approach was suggested to be the potential solution to preserve cells without allowing the nucleation of ice crystals. Currently, ultra-rapid freezing adopted ensures both intra and extracellular vitrification thus improving sperm survival [62].
Like sperm, female germ cells can be frozen by cryopreservation. Fertility preservation in female cancer survivors is a major concern in oncology and assisted conception. Fertility in females can be preserved by freezing egg either embryo, oocytes and oocytes within ovarian tissue. The results with oocyte cryopreservation were unsatisfactory. Ovarian tissue cryopreservation on the other side have attracted the interest of the medical and scientific communities. Cryopreservation protocols for oocyte and ovarian tissue are discussed briefly below.
For oocyte cryopreservation, currently acknowledged methods are SF using equilibrium freezing and Vit/non-equilibrium protocols. In cryopreserving oocytes by SF method, the protocol involves gradual cooling of the specimen to lower temperature (−150°C)with controlled slow cooling rates in presence of low concentration of DMSO (1.5 M) plus non-permeating sugars like sucrose or trehalose at 0.3 M concentration. Specimens are stored at −196°C in LN2. The survival rate achieved by SF remains relatively unsatisfactory. Evidence from earlier investigations indicates survival rates plateau around 70–80% with this method [63]. Hence, the recommended method for cryopreservation of oocytes is vitrification. Initially, oocytes are equilibrated with a solution containing PEG and DMSO at 7.5% v/v for 5–15 min. Prior to storing the cells in LN2, the cells are exposed to vitrification media (PEG and DMSO-15% v/v, -and sucrose (0.5 M) for a minute and stored. For thawing, CPAs are gradually removed to avoid ice crystal formation. Later, the cells are revived following incubation in culture medium [64]. Intriguing results were obtained in systematic analysis conducted by Arav and Natan. Vitrified oocytes were reported to have higher oocyte survival and fertility rates compared to slow-cooled oocytes. Furthermore, no differences were observed in pregnancy rate, formation of top quality embryo and fertilization between vitrified and fresh oocytes thus strongly signifying vitrification as the superior procedure for the oocytes cryopreservation [65]. During the past few years, a significant progress has been made in the cryopreservation of oocytes of different species using new vitrification methods. High rates of survival and development after solid-surface vitrification have been reported for
Parrot’s study, which resulted in first mice offspring was developed following ovarian tissue cryopreservation (at −79°C) and isografting was a breakthrough in the early 1960s [68, 69]. Later, with further discoveries of CPAs like DMSO, propanediol, and ethylene glycol, cryopreservation methods gradually improved. Like oocyte, ovarian tissues are also preserved by SF and Vit method, specially ovarian cortical tissue from mammals [70]. After the advent of Vit for oocyte cryopreservation and in comparison, to SF, ovarian tissue vitrification is now considered promising for ovarian cortical tissue cryopreservation [71, 72]. Ovarian tissue cryopreservation is successfully accomplished in many animal species like mice [68], ewe [73], cow including human [74] and other species.
Table 2 shows the comparison between SF and vitrification protocol for ovarian tissue cryopreservation. Vitrification was suggested to have several advantages over the conventional SF in characteristics reviewed. However, SF remains the standard clinical method until further reports show improved success rate for Vit to be applied in human clinical use over SF. Currently, the main purpose for OTC is fertility preservation known so far, especially in young women diagnosed with cancer or some genetic disease that destroys ovarian reserve. Access to immature oocytes from antral follicles and restoration of organ function have evoked new perspectives in utility of OTC for social reasons besides medical use.
Features | ||
---|---|---|
Period of freezing process | Slow usually 3–4 h | Fast <10 min |
Direct contact with liquid N2 | No | Yes |
Ice formation | Yes | No |
CPA concentration | Low | High |
Cooling rates (°C/min) | 0.15–0.3 | 15,000-30,000 |
Cost | Expensive | Inexpensive |
Special equipment | Yes | No |
Techical expertise | Simple | Risky |
Sample volume | 100–200 μl | 1–2 μl |
Mechanical damage | More | Less |
Chemical change | Less | More |
Comparison between SF and vitrification protocol.
Germ cell cryopreservation certainly had emerged as effective method for long term fertility preservation in the field of reproductive medicine in both human and animals. Germ cells cryopreservation would be applicable in restoring fertility in animals and humans and preservation of endangered animal species. Over a decade there has been increase in production of GEA models from cryopreserved animal genetic resources for disease investigations. The practice of genetic engineering has increased the number of mouse and rat lines to tenfold the actual number. Currently, sperm cryopreservation is a fundamental technique in assisted reproduction technology (ART) like artificial insemination (AI),
The birth of mouse from 50 years old cryopreserved embryo had revolutionized the field of animal reproduction. Later, to achieve greater reproductive outcomes, cryopreservation protocols have been continuously refined over the years. In the given section, few of the important animal models has been discussed.
Mouse is one of the most commonly used animal model for research. In early 1990s, the first attempt in freezing mouse was a grand success. Later, cryopreservation of mouse spermatozoa resulted in production of a large number of mouse inbred and hybrid strains [75, 76]. There is a growing demand for the production of genetically modified mouse strains since mouse has become the most profound model system to investigate the genetics and pathogenetics of human diseases. Moreover, knock-out projects started in Europe and USA over a decade. As life maintenance of the growing number of mice is difficult and uneconomical in animal laboratories, germplasm cryopreservation provides a valuable means of maintaining transgenic mouse strains used in biomedical research [77]. Moreover, animal gametes- sperm, eggs, and embryos preservation are now successfully preserved and maintained in cryobanks owing to the cryopreservation technique. Historically, embryo cryopreservation served as the gold standard for maintaining transgenic mice strains with single, multiple mutations, or complex genetic background [78, 79]. However, it is often more expensive due to costly and time-consuming superovulation procedures and subsequent cryopreservation. However, mouse sperm cryopreservation for long-term storage is simple and inexpensive, and it only requires few donor animals for protecting those commonly used inbred strains (e.g., C57BL/6, FVB, and 129/Sv) with single mutations [80]. Thus, sperm cryopreservation provides an efficient management of these genetic resources by reducing maintenance space and cost and by safeguarding them against, for example, disease, breeding failure, and genetic drift.
Although current sperm cryopreservation protocols showed relatively high success, there has been variation in the sperm motility and IVF outcomes post thawing. Many researchers investigated and worked to develop the gold standard protocol for mouse sperm freezing. Nakagata’s protocol became the most widely used by many research laboratories and clinical and scientific facilities around the world [80]. The initial freezing solution simply contains 18% dehydrated skim milk and 3% raffinose in water, and cooling is achieved in LN2 vapor phase for 5 min followed by plunging the samples into LN2 at −196°C. Since the introduction of this initial protocol, there have been few changes in an effort to improve post-thaw fertilization potential of mouse sperm.
For sperm cryopreservation in mouse, epididymal sperm collected from the cauda epididymis of matured male mice are suspended in sugar-based cryoprotectant, which is loaded in freezing straws and preserved at −196°C [81]. The cryopreserved sperm can be used for efficient fertilization using improved IVF systems featuring methyl-β-cyclodextrin (MBCD) and reduced glutathione (GSH) [82, 83].
Several scientist across the globe had investigated the role of varied antioxidants like monothioglycerol [84], methyl-β-cyclodextrin (MBCD) [82] and the latest refinement was the introduction of reduced glutathione (GSH) to protect spermatozoa against oxidative stress during IVF treatment [83] and increase the fertilization rates. C57BL/6 is a major inbred strain used for the production of transgenic mice, and also as a back-cross for targeted mutant mice [85]. Therefore, it is necessary to establish cryopreservation method for C57BL/6 mouse spermatozoa that could maintain a high fertilizing ability after thawing. Recently, a preincubation medium containing methyl-beta-cyclodextrin used demonstrated increased fertility [82]. Further, it is augmented that sperm freezed in a cryomedia containing 18% raffinose and 3% skim milk, increased fertilization rate [86]. Sperm banking can be used for mice, but in some instances it is also important to bank the female genome [87].
Further, ovarian gamete cryopreservation, as harvested oocytes or contained within the primordial follicles in the cortical patch tissue, can be used for long-term storage of the female germline [88]. Cryopreservation protocol for ovarian tissue based on the slow-cooling procedures was initially developed and used for mouse eggs and embryos in 1970s [10]. This process requires a biological controlled rate freezer or equivalent equipment. It is now documented that in the mouse both fresh and frozen thawed grafts have the potential to restore long-term fertility (i.e., for 1 year) to the graft recipient [89]. A more recent advancement in duration of graft function is in case of ovarian tissue transplanted with transplanted graft functional for 5 years, or more persisting for more than 9 years [90]. Slow freezing is overshadowed with the development of more advanced closed vitrification system, proven more beneficial in ovarian cryopreservation.
Animal model that is also commonly involved in the scientific work is rat, mainly in basic biology, physiology, brain science, and medicine. Over 40 year ago, the success of IVF was reported in rats. Characteristics, such as a short gestation and a relatively short life span, docile behavior, and ready availability of animals with well-defined health and genetic backgrounds make rat an ideal experimental model. The rat is a standard species for toxicological, teratological, and carcinogenesis testing by the pharmaceutical industry and governmental regulatory agencies [91, 92]. Rats are still continued to be used for nutritional, neurological and endocrinology studies. Cryopreservation has not been performed in rats as often as it has in mice, but the technique is becoming more widespread, for the same reasons that it is used widely in mice. Cryopreservation can be an efficient method of maintaining the potential of raising live mice of the thousands of genetically modified genotypes currently available [93, 94]. Sperm cryopreservation has been similarly successful in rats. Offspring are obtained from thawed sperm using intrauterine insemination or via IVF. Seita
A schematic representation of Assisted Reproduction techniques and In vitro fertilization; IVF in rats from cryopreserved sperm and oocytes.
AI in rabbit using cooled semen stored for short time is a commonest ART practice in country like Europe, where rabbit rearing is common [96, 97]. High fertility rates and proliferation are obtained in this process. However, with limiting factors like low productivity and issues with cryopreserved rabbit sperm, AI is used for experimental or genetic resource bank purposes. Although cryopreserved sperm is not used for commercial purposes at the present, there is a need for reliable methods of rabbit sperm resource banking, especially as this species is a valuable animal in therapeutics(for production of vaccines, antibodies, hormones) [98, 99]. The extent of cryoinjury varies with the species due to differences in the gamete plasma membrane composition among them and cell size as well [100]. Hence, there is no universal protocol followed for all the animal species. It is obligatory to standardize cryopreservation protocol, extender and CPAs levels and composition, for each single species or even breeds.
Researchers observed significantly varying outcomes with the type of CPAs used in cryopreserving rabbit sperm. The use of CPAs like DMSO or ethylene glycol is recommended to enhance the storage of rabbit sperm [101]. Few other studies observed ethylene glycol to be less toxic than glycerol in cryofreezing rabbit sperm. Despite lower toxicity, ethylene glycol failed to provide protection to sperm cells when compared to other DMSO. Importantly, CPAs concentration need to be optimized for sufficient protection. To note, addition of CPAs at 5°C instead of 37°C was reported effective in cryopreserving rabbit sperm as permeability increases with increased temperatures [102]. Data on the CPA type and concentration for optimal cryopreservation is contradictory. However, it is well documented that a balance between all the involved parameters is a key for successful freezing. Sperm quality varies with the type of extenders used. The extenders used for rabbit sperm cryopreservation include a mixture of permeable and non-permeable CPAs. The use of two permeable CPAs is a common practice [103]. Sperm frozen in acetamide extender was found to be of superior quality than sperm DMSO and glycerol or other mixtured extenders [104]. The mixture of CPAs with egg yolk has differential effect on the sperm quality. Sperm frozen in extender containing egg yolk and DMSO demonstrated better sperm quality than for sperm frozen in the extender that contained high DMSO and lacking egg yolk [102]. Hence, it is obvious that a balance of all the essential factors is a key for successful freezing.
Recently, Domingo
From rodents to larger animals, cryopreservation is proved to be beneficial in fertility preservation, transplantation and breeding livestock. Advances in cryopreservation pioneered with transplantation of cryopreserved mouse primordial follicles in 1993 by J. Carroll and R. Gosden [106] followed by recognition of sheep as a larger model to study ovarian function. Gosden, in collaboration with D. Baird [107], developed techniques with vascular anastomosis that formed the basis of transplantation of larger organs such as kidney in human. The development of ART techniques has gained significance in the production of commercially important farm animal breeds and a few exotic or endangered species. Livestock industry especially cattle had benefitted to a major extent from the application of cryopreserved semen or embryos over the past decades. This was also the case in experimenting gamete cryopreservation. Larger animal like sheep is greatly emphasized to study human diseases particularly respiratory diseases and lung cancer, since the anatomy and physiology of the sheep respiratory system is more similar to that of humans than rodents. Sheep has been proposed as a good model for vaccines, asthma pathogenesis and inhalation treatments [108]. The gradual decline of genetic diversity in domestic breeds imposes a major threat to livestock, hence, international community strives harder to conserve the livestock genetics. Semen from most of the mammalian species has been successfully frozen [109].
With regards to temperature tolerance, sperm from different species exhibits varied responses. Bovine sperm shows higher tolerance to low temperatures, while porcine and ovine sperm are more sensitive and at risk of cold shock when exposed to temperature between 5° and 22°C leading to rapid loss of vitality. Animal sperm is highly vulnerable to oxidative damage owing to the loss of antioxidase enzyme and increased fatty acid oxidation on freezing [110]. The stability and viability of sperm is enhanced by adding semen extenders during freezing. The first semen extender for bovine sperm preservation used was egg yolk-sodium citrate diluent (EYC) and was gradually replaced with tris-buffered egg yolk (TRISEY)or tris-fructose yolk-glycerol [111]. Most of the industries use tris and citrate as active components in bovine sperm extenders. Addition of compounds like vitamin E to semen extenders is found to increase the structural integrity of acrosome, and thereby preventing sperm from oxidative damage via its antioxidant properties [24].
Vitrification of embryos was invented in 1985 [112]. Later vitrification emerged as one of the powerful methods for cryopreserving embryo from farm animals including cattle, goat, sheep and pig [28, 29, 113, 114]. The birth of the first calf was achieved from frozen/thawed oocyte was reported in 1992 [115]. Vitrification showed high success in bovine oocyte cryopreservation in 1998 [116]. Applicability of macromolecules with lesser toxicity as CPAs, the use of cryotop and solid surface virtification emerged gradually over time to overcome cryoinjury. Treatment with docetaxel improved cryopreservation of bovine oocyte as its protective against cytoskeleton injury thus can potentially enhance survival rate of post thawed oocytes [117]. High rates of survival and development after solid-surface vitrification have been reported for
OTC has been reported successful in restoration of ovarian function in sheep model. Complete restoration of acute ovarian function and high rates of natural fertility with multiple live births, were obtained following whole ovary cryopreservation and autotransplantation of adult sheep ovaries [29]. Although ovarian tissue cryopreservation has developed from experiments in sheep in early 1990s, it is now becoming recognized as relatively successful procedure for OTC in human, particularly to preserve the fertility of cancer patients to avoid gonadotoxic damage resulting from the therapy [118]. While there has been considerable success with cryopreservation of oocytes, embryos and semen in farm animals, this technology still requires refinement and further optimizing studies.
Based on the utility and need of the animals for varied purposes, fertility preservation is a prerequisite to be practiced in animal husbandry and animal house unit for production of animal models. Understanding of the fundamentals of cryopreservation allows the development of more efficient procedures for cryopreservation of germ cells and further expand their clinical applications and utility in livestock, which can also be transferred to human application. Although, the field of cryobiology has advanced over the years, further research remains required to optimize cryoprotectant concentration, cooling and thawing rates to aim high success in animal reproduction.
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",metaTitle:"Waiver Policy",metaDescription:"We feel that financial barriers should never prevent researchers from publishing their research. With the need to make scientific research more publically available and support the benefits of Open Access, more institutions and funders have dedicated funds to assist their faculty members and researchers cover the APCs associated with publishing in Open Access. Below we have outlined several options available to secure financing for your Open Access publication.",metaKeywords:null,canonicalURL:"/page/waiver-policy",contentRaw:'[{"type":"htmlEditorComponent","content":"At IntechOpen, the majority of OAPFs are paid by an Author’s institution or funding agency - Institutions (73%) vs. Authors (23%).
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\\n\\nThe application process is open after your submitted manuscript has been accepted for publication. To apply, please fill out a Waiver Request Form and send it to your Author Service Manager. If you have an official letter from your university or institution showing that funds for your OA publication are unavailable, please attach that as well. The Waiver Request will normally be addressed within one week from the application date. All chapters that receive waivers or partial waivers will be designated as such online.
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\\n"}]'},components:[{type:"htmlEditorComponent",content:'At IntechOpen, the majority of OAPFs are paid by an Author’s institution or funding agency - Institutions (73%) vs. Authors (23%).
\n\nThe first step in obtaining funds for your Open Access publication begins with your institution or library. IntechOpen’s publishing standards align with most institutional funding programs. Our advice is to petition your institution for help in financing your Open Access publication.
\n\nHowever, as Open Access becomes a more commonly used publishing option for the dissemination of scientific and scholarly content, in addition to institutions, there are a growing number of funders who allow the use of grants for covering OA publication costs, or have established separate funds for the same purpose.
\n\nPlease consult our Open Access Funding page to explore some of these funding opportunities and learn more about how you could finance your IntechOpen publication. Keep in mind that this list is not definitive, and while we are constantly updating and informing our Authors of new funding opportunities, we recommend that you always check with your institution first.
\n\nFor Authors who are unable to obtain funding from their institution or research funding bodies and still need help in covering publication costs, IntechOpen offers the possibility of applying for a Waiver.
\n\nOur mission is to support Authors in publishing their research and making an impact within the scientific community. Currently, 14% of Authors receive full waivers and 6% receive partial waivers.
\n\nWhile providing support and advice to all our international Authors, waiver priority will be given to those Authors who reside in countries that are classified by the World Bank as low-income economies. In this way, we can help ensure that the scientific work being carried out can make an impact within the worldwide scientific community, no matter where an Author might live.
\n\nThe application process is open after your submitted manuscript has been accepted for publication. To apply, please fill out a Waiver Request Form and send it to your Author Service Manager. If you have an official letter from your university or institution showing that funds for your OA publication are unavailable, please attach that as well. The Waiver Request will normally be addressed within one week from the application date. All chapters that receive waivers or partial waivers will be designated as such online.
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