\r\n\tHydrogen gas is the key energy source for hydrogen-based society. Ozone dissolved water is expected as the sterilization and cleaning agent that can comply with the new law enacted by the US Food and Drug Administration (FDA). The law “FDA Food Safety Modernization Act” requires sterilization and washing of foods to prevent food poisoning and has a strict provision that vegetables, meat, and fish must be washed with non-chlorine cleaning agents to make E. coli adhering to food down to “zero”. If ozone dissolved water could be successively applied in this field, electrochemistry would make a significant contribution to society.
\r\n
\r\n\t \r\n\tOxygen-enriched water is said to promote the growth of farmed fish. Hydrogen dissolved water is said to be able to efficiently remove minute dust on the silicon wafer when used in combination with ultrasonic irradiation. \r\n\tAt present researches on direct water electrolysis have shown significant progress. For example, boron-doped diamonds and complex metal oxides are widely used as an electrode, and the interposing polymer electrolyte membrane (PEM) between electrodes has become one of the major processes of water electrolysis.
\r\n
\r\n\t \r\n\tThe purpose of this book is to show the latest water electrolysis technology and the future of society applying it.
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,isSalesforceBook:!1,isNomenclature:!1,hash:"7532579d8c6881554d1812b55d0e8d4d",bookSignature:"Prof. Fumio Okada",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/7702.jpg",keywords:"Hydrogen, Electrode, Polymer Electrolyte Membrane, Ozone Water, Oxygen-enriched Water, Hydrogen-peroxide, Electrolysis for Purification, Pulp drainage, Semiconductor, Food Processing, Medical Treatment, Detoxification",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:1,numberOfTotalCitations:1,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 26th 2019",dateEndSecondStepPublish:"February 14th 2020",dateEndThirdStepPublish:"April 14th 2020",dateEndFourthStepPublish:"July 3rd 2020",dateEndFifthStepPublish:"September 1st 2020",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"2 years",secondStepPassed:!0,areRegistrationsClosed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"147954",title:"Prof.",name:"Fumio",middleName:null,surname:"Okada",slug:"fumio-okada",fullName:"Fumio Okada",profilePictureURL:"https://mts.intechopen.com/storage/users/147954/images/system/147954.jpg",biography:"Dr. Fumio Okada received BE from the University of Tokyo in 1978, MS from the University of California at Irvine in 1989, and Dr. Eng. from the University of Tokyo in 1994. He entered Nippon Mining Co. Ltd in 1978 and worked as an engineer and as a researcher until 2002. After retiring the company, he became a Prof. in the Department of Chemical System Engineering, the University of Tokyo. He moved to the Department of Environmental Chemistry, Kogakuin University in 2012. There he teaches Reaction Engineering, Heat Transfer Engineering, and Physical Chemistry for undergraduate students and Environmental System Engineering for graduate students. His research is now focused on the electrochemical production of functional water, such as ozone dissolved water, hydrogen dissolved water, and advanced oxidation water which comprises of ozone and hydrogen peroxide dissolved in water. He also developed a highly efficient gas-liquid mixer which enables the production of ozone dissolved water without ozone gas emission. In that process, all of the ozone gas formed in water electrolysis is dissolved into water by passing it in the mixer. 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1. Introduction
It has been estimated that about 15–20% of couples worldwide have infertility problems or impaired fecundity. Approximately 40–50% of these cases are due to male infertility, which could be indirectly measured through the assessment of the sperm production and quality. According to WHO’s criteria [1], indeed, men with low sperm concentration (oligospermia), poor sperm motility (asthenospermia), and abnormal sperm morphology (teratospermia) are considered to have male infertility factors. The revised WHO’s parameters and the corresponding lower reference limits for semen analyses are reported in Table 1.
Parameter
Reference value
95% confidence index
Sperm volume
1.5 mL
1.4–1.7
Sperm concentration
15 million sperm/mL
12–16
Total sperm number
39 million sperm per ejaculate
33–46
Morphology
4% normal forms
3–4
Vitality
58% live
55–63
Progressive motility
32%
31–34
Total motility (progressive + nonprogressive)
40%
38–42
Table 1.
WHO’s parameters for semen analysis (2010).
Infertile couple usually resort to assisted reproduction techniques (ART), chosen by the clinician according to the degree (soft, moderate, or severe) and kind (male and/or female) of infertility. For soft/moderate infertility, usually homologous intrauterine insemination (HIUI) or in vitro fertilization (IVF) is carried out. HIUI consists in transferring a small volume of selected and isolated motile and morphologically normal spermatozoa directly into the uterus a few hours before the ovulation. IVF is used after continued failures of HIUI due, for example, to an impervious uterine tube, and the fertilization of the oocytes takes place outside the woman body.
In case of severe male infertility, the intracytoplasmic sperm injection (ICSI) is preferred. In this technique, a single spermatozoon is directly injected into an oocyte, bypassing the physiological selection naturally performed by the female tract. Although ICSI has drastically reduced the number of viable sperm required for fertilization, giving hope of conception to extremely severe cases of oligospermia, the rate of successful pregnancy still remains low (<30%), due to the lack of accurate and reliable methods for selecting the spermatozoon that able to fertilize the oocyte.
The first selection is based on motility and morphology. The sperm cells preparation procedure includes a density gradient centrifugation (morphology-based selection) followed by swim-up method (motility-based selection) in order to mimic the physiological selection of the spermatozoa made by the female genital tract. In most laboratories, the sperm quality assessment is relied on the expertise and the subjective skills of the operator. To increase precision and reproducibility, numerous automated computer-aided sperm analysis (CASA) systems have been introduced, and they can automatically view multiple fields in a shallow specimen chamber to capture images of 500 to >2000 sperm in <2 minutes [2]. By using CASA, it is also possible to retrieve some morphometrical parameters, such as ellipticity and regularity, whereas the measurement of the acrosome area generally requires staining.
Moreover, CASA has difficulty in distinguishing spermatozoa from particulate debris, leading to some extent of inaccuracy. In addition, several studies have shown that the correlation between male fertility and the percentage of morphologically normal or motile sperm cells in semen sample is relatively low [3, 4]. Indeed, in infertile men, a high percentage of spermatozoa with a normal morphology or motility could have a damaged DNA, and therefore, they are, in principle, incapable to fertilize the oocyte [4, 5].
Sperm DNA integrity is generally assessed by biochemical methods such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay [6], comet assay [7], sperm chromatin dispersion (SCD) test [8], or sperm chromatin structural assay (SCSA) [9], which assess the quality of DNA bases, chromatin, and altered protein in sperm nuclei using specific stains. Indeed, all currently employed tests to determine DNA integrity are of limited clinical utility as they are invasive and destructive, rendering the sample unusable for IVF [6, 7, 8, 9, 10, 11].
All these methods mentioned above, only based on assessing morphology or motility, are inadequate to select the fertile spermatozoon as the selection techniques have to be noninvasive, safe, highly discriminative, and relatively easy to perform. Moreover, they should simultaneously assess three conditions: normal morphology, motility, and DNA integrity.
Recently, several techniques are emerging for label-free selection of viable spermatozoa. In this chapter, we focus on two of them: Raman-based spectroscopy and phase contrast imaging. Raman spectroscopy (RS) investigates the biochemical and physiological state of a sample by detecting the inelastic light scattering, without labeling or long preparation procedures [12]. Phase contrast imaging includes basic microscopies (bright-field) [13] and more advanced ones (interferometric microscopies) [14, 15, 16, 17], such as digital holography. These methods exploit the phase delay of light when passing through a sample. Then, these light delays are converted into intensity changes and recorded on a camera as holograms containing the information suited for morphological sperm cell reconstructions.
The physical principles and the technical instrumentation of Raman and holographic techniques will be separately described in Sections 2 and 3, respectively, also highlighting some relevant results in sperm cells analysis. In Section 4, we will report on our recent achievements using the two techniques in a multimodal analysis setting. Indeed, both the techniques are based on intrinsic optical properties of the sample and may offer the unique advantage to simultaneously acquire the holographic and Raman data assessing single spermatozoa on the base of their morphological and biochemical parameters and their motility. The complete analysis is performed by the same optical system, with a strong impact on costs and times for the analysis. This aspect makes the proposed approaches very promising for clinical applications; however, at present, they are only in experimental stages and specific recommendations for routine procedures cannot be made. At the end of this chapter, the difficulty in reaching clinical use will be discussed, as well as the possible solutions offered by new technological improvements.
2. Label-free biochemical sperm analysis
2.1. Basic principles of Raman spectroscopy
Raman spectroscopy is an optical technique based on the scattering of the light interacting with a sample. Light scattering originates from the elastic or inelastic collision between an incident photon and a molecule of the sample. When the collision occurs, the molecule undergoes an excitation to a virtual state followed by a nearly simultaneous de-excitation towards the initial energy level (elastic) or a vibrational level different from the initial one (inelastic) (Figure 1). The virtual state is an energy level created only when photons interact with electrons and its energy is determined by the frequency itself of the incident photons. When molecules return back to their initial energy state, photons are emitted (scattered). In the elastic process, named Rayleigh scattering, the emitted photons have the same energy of the incident ones. Whereas in the inelastic scattering, there are two possibilities (Figure 1): 1. molecules decay from the virtual state to an excited vibrational level emitting photons with a lower energy than the incident ones (Stokes scattering) and 2. when molecules in a vibrational state were excited in a virtual state, they can decay in the ground state, producing photons with energy higher than the incident ones (anti-Stokes scattering). Since the number of molecules in an excited state decreases very fast, anti-Stokes scattering is much less probable than the Stokes scattering (typically by a factor around 1000). For this reason, Raman analysis is usually limited to the observation of the Stokes scattering.
Figure 1.
Jablonski diagram representing quantum energy transitions for Rayleigh, Raman scattering (Stokes and anti-Stokes), and fluorescence emission.
The energy difference between the initial and final vibrational levels (Raman shift) expressed in wavenumbers (cm−1) is given through the relation:
ℏν=ℏ1λinc−1λscatE1
in which λinc and λscat are the wavelengths of the incident and Raman scattered photons, respectively. Therefore, the energy (or frequency) shifts in the scattered radiation provide a direct measure of the vibrational frequencies of the molecule (ν). Since different molecules are characterized by defined vibrational modes, by collecting the photons scattered at different frequencies allows reconstructing a sort of “chemical fingerprint” of the sample.
Therefore, RS turns out to be an extremely powerful tool for interdisciplinary researches, involving physicists, chemists, and biologists, as it allows the characterization of molecules based on properties of chemical bonds. It is a noninvasive and nondestructive technique providing molecular-level information, allowing the investigation of functional groups, bonding types, and molecular conformations.
From an experimental point of view, to observe a Raman spectrum, it is required to suppress the light scattered at the same frequency of the incident radiation (Rayleigh scattering), often more intense than the Raman scattering. Using the notch filter, disperse the spectral components of the light by means of the diffraction grating and image the light onto the detector (high sensitive charge-coupled device (CCD) camera).
In a confocal configuration (micro-RS), a spatial resolution of ~300 nm in the transverse x-y plane and of ~1.2 μm in the axial direction can be achieved. Moreover, for extended samples, it is possible to acquire the Raman spectra on a two- or three-dimensional array of points, scanning the sample with a step comparable with the spatial resolution (Raman imaging). In this way, 2D images (or 3D profiles) are obtained, reporting the spatial variation of a given Raman parameter. This parameter is usually the intensity of a particular Raman band or sometimes it derives from a more complicated analysis of the whole Raman spectrum [18, 19].
2.2. Raman spectroscopy analysis of sperm cells quality
Interestingly, due to the cited characteristics, RS has been successfully employed for the study of several living/fixed cells with subcellular resolution [18]. The first Raman-based single cell analyses were conducted just on individual living salmon sperm cells due to their relative simple structure [20]. Since this pioneer study, no other Raman experiments on sperm cells have been reported until the 2009 when Huser and colleagues [21] examined the spectra obtained from human spermatozoa with different nuclear shapes (box 1 in Figure 2) in order to determine if there was a correlation between DNA-protein complex in sperm chromatin and the morphology of the nucleus. RS results showed that while the DNA packaging of normal and abnormal shaped nuclei was different, the nature and the efficiency of DNA packaging in normal head spermatozoa appeared to vary greatly [21]. Therefore, by selecting sperm cells solely based on morphology, a fraction of the normal considered sperm cells used for in vitro fertilization will contain improperly packaged DNA, thus resulting infertile. In this study, fixed membrane-free cells were used in order to minimize the potentially interfering contributions from membrane proteins.
Figure 2.
Overview of the most representative studies on Raman spectroscopy and imaging for the label-free analysis of sperm cells. Box 1: the highlighted variations in peak intensities in the Raman spectra correspond to different sperm head shapes [21]. Box 2: the peaks at 1095 and 1050 cm−1 represent biomarkers of fragmented DNA in the sperm nucleus [24, 25]. Box 3: panel A shows the chemical Raman reconstruction of distinct sperm regions [23]; panels B–Y represent the biochemical composition of individual immobilized, living human sperm cells [26]; and panel Z shows the efficiency of Raman imaging in revealing small irregularities in the sperm head such as vacuoles (yellow circles) distinguishable based solely on the presence of differing spectra [24].
The results of this study are also relevant from an epigenetic point of view. Indeed, they show the possibility to use RS for detecting epigenetic alterations (DNA packaging and spatial conformation, methylation and histone modification) in those spermatozoa that, if used in assisted reproductive techniques, may increase the incidence of imprinting disorders and have a deleterious impact on embryonic development [21, 22].
In other studies, RS was applied on whole and fixed sperm cells in order to demonstrate the efficiency of the technique in identifying DNA-damaged sperm cells (box 2 and panels A and Z in the box 3 of Figure 2). Usually, the DNA fragmentation as a consequence of oxidative stress is induced by UV radiation [23, 24] or Fenton’s treatment [25]. All the studies found that the PO2 backbone of DNA was significantly affected by the exposure to radiation or treatment, and thus, its corresponding Raman band could represent a significant biomarker of DNA fragmentation [23, 24, 25]. Mallidis et al. previously identified characteristic spectral changes indicative of nuclear DNA damage of single fixed human spermatozoa and using Raman mapping, they localized the most damaged sites [24].
In the following study [25], the same group determines the possibility to use Raman microspectroscopy for identifying different levels of sperm nuclear DNA damage induced by oxidative stress and corroborated the findings using an established assay and an alternative but complementary spectroscopic technique (Fourier-transform infrared (FTIR) spectroscopy). The results of this last work confirm that RS is able to reveal different levels of oxidative DNA fragmentation, especially associated to alterations within the 1050–1095 cm−1 spectral range (Raman spectra in the box 2 of Figure 2), which includes the band associated with the DNA phosphate backbone, changes that were confirmed by similar shifts in the corresponding FTIR peaks (not shown) [25]. Also, the Raman bands associated to protein and lipid content (1400–1600 cm−1) showed some alterations induced by UV radiation, consistent with protein denaturation and lipid peroxidation that are well-known markers of oxidative damage [27]. Raman-based identification of DNA-damaged sperm cells linearly correlated with the findings from the flow cytometric analysis of DNA fragmentation, which represents the most statistically robust, reproducible, and standardized procedure available for the determination of sperm nDNA damage [28].
In 2015, Edengeiser et al. [26] analyzed spermatozoa under near-physiological conditions using confocal Raman microspectroscopy. The spermatozoa are immobilized on pre-treated object slides. More in detail, CaF2 slides were coated with concanavalin A and overlaid with preheated Ringer’s solution just before putting a drop of isolated spermatozoa which, after the analysis, can be easily removed from the substrate. The study demonstrated for the first time the possibility to image and analyze several tens to hundreds individual cells with a rate of one cell per minute with submicrometer resolution (panels B–Y in the box 3 of Figure 2). This opens up possibilities to investigate different physical and biochemical parameters under physiological conditions, leaving the assessed spermatozoa functional.
3. Quantitative phase imaging for sperm analysis
3.1. Basic principles of quantitative phase microscopy
Quantitative phase microscopy (QPM) is a label-free imaging technique, which allows reconstructing both the amplitude and the phase information of an optical field that passes through the sample, and it is particularly interesting in case of transparent biological cells. Respect to differential interference contrast (DIC) microscopy [29] or Nomarski/Zernike’s phase contrast [30], the QPM gives a quantitative measure of the optical path difference (OPD) at each point in the sample. OPD in each position (x, y) of the acquisition plane is defined as the refractive index variation across the cell thickness, t(x, y):
OPDxy=txync−nsE2
where nc and ns are the refractive index of the cell and the surrounding medium (assumed to be homogeneous), respectively. The resulting OPD map of the cell is reconstructed by recording the interference fringes pattern, the so called “hologram,” of two superimposed coherent beams, one that interacts with an object under test and another that does not come in contact with the object and acts as a reference beam, and calculating the phase difference between them [31]. If the hologram is acquired by a digital sensor array, typically a charge-coupled device (CCD) or a complementary metal-oxide semiconductor (CMOS) device, digital holographic microscopy (DHM) technique is implemented. A typical interferometric setup for DHM is reported in Figure 3.
Figure 3.
Typical interferometric setup for digital holography microscopy. BS: beam splitter, M: mirror. Reference and object beams are highlighted.
The acquired hologram is then mathematically analyzed, allowing obtaining the complex field of the object beam that can be reconstructed at different distances, too. Therefore, numerical refocusing of a digital hologram, that is a 2D image, at different object planes, without any z-scan of the optical system, allows to retrieve a 3D quantitative imaging [31]. This makes digital holography a very powerful method for metrology applications, particularly attractive in the field of biology as it is noninvasive, noncontact, and label-free, allowing the characterization of live specimen.
In DHM, in addition to the hologram of the sample under investigation, a second hologram is acquired on a reference region near to the object in order to numerically compensate all the aberrations due to the optical components, comprising the defocusing due to the microscope objective. An “off-axis” configuration is generally adopted to avoid a spatial overlapping of the real and conjugate images due to the holographic reconstruction, leading to the separation of first diffraction order from the entire spatial frequency spectrum. Thus, the spectrum of the sample (object field defined as S(x, y) = |S(x, y)|eiφ(x, y), with |S(x, y)| and φ(x, y) amplitude and phase, respectively) can be retrieved except for a constant [32]. Then, it is possible to propagate the optical wavefront at different distances from the plane of acquisition applying the Fourier formulation of the Fresnel-Kirchhoff diffraction formula [33, 34]. This reconstruction can be obtained by means of the operator algebra proposed by J. Shamir [35], where Fresnel diffraction is described by replacing the Fresnel-Kirchhoff integral, the lens transfer factor, and other operations by operators. The resulting propagated object field Sprop(ξ, η) is expressed as a function of the initial object field S(x, y) and can be written as [36, 37]:
Spropνμ=expikd×I−1exp−ikdλ22p2+q2∙ISxyE3
being Ifx the Fourier transform of the function f(x), k=2πnλ (with n refractive index of the medium), p and q spatial frequencies defined as p=νλd and q=μλd, and d the reconstruction distance. For digital reconstruction, Eq. (3) is applied in a discrete form:
Spropmn=expikdID−1−ikdλ22N2d2U2+V2∙IDShjE4
where N is the number of pixels in both directions and m, n, U, V, h, and j are integer numbers varying from 0 to N − 1.
Intensity and phase distributions can be reconstructed by Sprop(m, n) according to the following equations:
Ipropmn=Spropmn2;E5
φpropmn=arctanImSpropmnReSpropmn.E6
The phase φprop(m, n) includes information about the morphological profile of the object under investigation; in fact, it is related to the OPD:
OPDmn=λ2πφpropmn.E7
The relation between the OPD and the thickness of the cell t is given by Eq. (2).
3.2. Digital holography microscopy for sperm cells assessment
Digital holography (DH) allows retrieving a fully 3D image of the sample, thus offering new prospects for the analysis of sperm cells in a noninvasive, quantitative, and label-free way. Sperm cells were acquired by a digital holographic microscope for the first time in 2008 by Mico et al. [38].
The potential of applying this technique for label-free sperm assessment was recently confirmed by Shaked’s group. Indeed, they demonstrated that DHM allows obtaining equivalent information about key morphological parameters of fixed human spermatozoa to that obtained by bright field microscopy (BFM) imaging of stained sperm cells [39].
Additionally, the opportunity to have information about the third dimension in the sperm analysis can offer a better understanding of this kind of cell and of male infertility [40]. Furthermore, since this technique allows obtaining quantitative information and numerical analysis, estimation area or profiles in a given direction may be carried out. Such kind of analysis can help to study the male infertility and its possible relation with the abnormal morphology [41, 42].
DH has been mainly employed to study the morphology of human sperm cells in order to verify the integrity of their structures and to evaluate their kinematic parameters and concentration. This approach allows to visualize the morphology of abnormal sperm and to analyze in 3D some typical defects such as cytoplasmic droplet along the tail, bent tail, and acrosome broken, as reported in the box 1 of Figure 4 [42].
Figure 4.
Some potentialities of DHM. Box 1: morphological analysis of semen carried out by DHM; top panels: sperm cells with distinct morphological defects; center and bottom panels: difference in height denotes a reduced volume in spermatozoon head with vacuoles respect to the normal spermatozoon [41, 43]. Box 2: examples of some physical parameters obtained by DHM [31, 44]. Box 3: 4D tracking of clinical sperm samples [45].
Additionally, a quantitative study of vacuoles has been performed by DH. In particular, it was demonstrated that the profile of the normal spermatozoon results higher than that of the spermatozoon with vacuoles, whereas their 2D dimensions (such as area and axes length) are similar [43]. The difference in height denotes a reduced volume in spermatozoon with vacuoles respect to the normal spermatozoon; this difference could be ascribed to a modification of the inner structure of the sperm head with loss of material (see box 1 in Figure 4).
In 2013, Merola et al. [44] provided an evaluation of the biovolume of spermatozoa (about 55 μm3). The authors used optical tweezers to trap and rotate the cells; meanwhile, they flow through a microchannel, enabling recording digital holograms of the sperm at different angles and the production of a tomographic 3D model, as showed in the box 2 of Figure 4.
Another important semen parameter, the dry mass of the cell (i.e. the average mass of the proteins, carbohydrates, lipids, and so on within the cell), can be obtained by DHM. Indeed, the OPD of the cell linearly depends on the axially averaged refractive index of the cell relative to the surrounding medium, for a given thickness t [45]. Thus, considering that the human spermatozoa head can be divided in the cell nucleus and the acrosome, which differ in the composition and concentration of proteins, nucleic acids, and other components, Balberg et al. evaluated the dry mass of the cell by starting by the knowledge of the OPD [31]. In particular, the authors measured the dry mass of separate cellular compartments in the OPD maps of unlabeled human spermatozoa, as reported in the box 2 of Figure 4.
Finally, the movements of living spermatozoa have been tracked applying an automatic 4D tracking (movements in the 3D spatial directions over time) of the swimming samples in [46]. The results are showed in the box 3 of Figure 4, where an anomalous spermatozoa behavior, known as “bent tail,” is highlighted. A collection of several holograms at a fixed distance between the sample and the microscope objective was acquired. In order to simultaneously track multiple spermatozoa, a proximity criterion has been included into the algorithm. In particular, by means of this approach, the position in the (n + 1)th frame has been searched in a reasonable neighborhood of the nth frame position.
Therefore, DH could be seen as a breakthrough that can renew the sperm analysis in the spermatology laboratories, encouraging researchers in the field of sperm cell biology to consider using DH as a standard method for their characterization studies.
4. Combined optical approach for the noninvasive analysis of single spermatozoa
As seen in the previous sections, Raman spectroscopy and quantitative phase microscopies have been separately developed for assessing spermatozoa from a biochemical and morphological perspective, respectively. The two photonic techniques, employing intrinsic contrast mechanisms, allow noninvasively selecting the fertile spermatozoon according to its normal morphology as well as its DNA integrity. Kang et al. [47] first proposed a combined system where quantitative phase microscopy and Raman imaging allowed correlating morphological parameters with molecular information, i.e. the red blood cell thickness was correlated to the hemoglobin distribution. Huang and colleagues in 2014 published a study that evaluated the possibility to combine micro-Raman spectroscopy with image analysis for label-free identification of normal spermatozoa [48]. Recently, our group proposed a similar system, which combines Raman spectroscopy/imaging and digital holography microscopy as a potential tool to rapidly and objectively identify the healthy spermatozoa [36, 49, 50].
4.1. The optical setup
The setup used in our works for the simultaneous Raman and holographic analysis essentially consists of a Raman microscope coupled to an interferometer (Figure 5) [36, 49, 50]. We used two different laser sources: a green laser at 532 nm for the Raman excitation and a long coherence (>100 m) red laser at 660 nm for the holographic experiments. The red laser beam was split into two beams: the object beam passing through the sample and the reference beam that directly goes to the detector. Importantly, the reference beam was controllable in intensity and polarization enabling us to improve signal intensity and contrast. The collimated object beam of the holographic pathway is recombined to the reference beam by a beam splitter (BS). The recombined beams are filtered and sent to the CCD camera (CCD1) for the holograms recording.
Figure 5.
Experimental set up of the combined Raman and holographic system used in our works [36, 49, 50].
The green light is focused on the sample by a high numerical aperture objective lens. The high N.A. of the objective and the wavelength chosen for the Raman beam allows to reaching spatial resolutions on the order of less than 0.5 μm, particularly suitable for cell imaging application. The back-scattered light from the sample is collected by the same objective lens (OBJ) and separated from the holographic radiation by a long pass dichroic mirror (DM) reflecting wavelength below 600 nm. The scattered light, consisting of Rayleigh and Raman radiation, is filtered through a dichroic beam splitter (BS45) that rejects the Rayleigh light at 532 nm. The Raman signal is further filtered using a laser-blocking filter (NF0) to eliminate the residual Rayleigh scattering and then focused onto the entrance slit of a monochromator. The Raman signal is finally detected using a cooled CCD camera (CCD2).
4.2. Morphological and biochemical analysis of single sperm cells
In this paragraph, some of the most interesting results we have obtained by applying the Raman/holographic microscope for the sperm cell characterization will be discussed. Our investigations mainly focused on:
correlative analysis of morphological and biochemical alterations [49];
morphological and biochemical analysis of photo-damaged spermatozoa [36];
sex classification of bovine spermatozoa [49, 51].
With the aim of demonstrating the potential applicability of the proposed multimodal imaging approach in identifying the fertile spermatozoa for IVF, we have first performed a qualitative Raman/holographic analysis of single sperm cells [49].
Raman imaging can be performed scanning the laser over a region of interest of the sperm cell or on the entire cells, acquiring in that way a point-by-point spectrum. Similar spectral features correspond to similar molecular structures; consequently, we can identify separate regions of the spermatozoon (acrosome, nucleus, and tail) according to their different chemical composition (Figure 6a and b). Pseudo-color Raman map of the sperm is shown in Figure 6b, in which each color is arbitrarily associated to specific Raman spectral patterns. Therefore, the Raman map delineates not only the distribution of DNA and protein in the nucleus, acrosome and tail but also detects, in a label-free manner, small biochemical discrepancies correlated with the presence of morphological defects, highlighted by digital holography (Figure 6c). We found that the peculiar protuberance in the region of the spermatozoon connecting the head to the tail, the so-called middle piece, well correlated with the biochemical alteration detected in the Raman map. Indeed, by analyzing the typical Raman bands of the spectra acquired in that specific cell region, it was possible to correlate the morphological alteration to an increased amount of proteins in the middle-piece region, where mitochondria are localized [49].
Figure 6.
(a) Raman spectra and (b) false-color Raman map of different regions of the sperm cell head: middle piece (yellow), nucleus (red), acrosome (blue), and membrane (green). (c) 3D digital holographic reconstruction of the sperm cell morphology [49].
Recently, we applied the multimodal imaging tool for the online evaluation of the damages induced by green laser radiation for studying in which dose and how it affects the irradiated sperm cells [36]. Severe spermatozoa variations associated with a topological redistribution of the sample and a gradual decrease in the Raman signal intensity were detected in a label-free configuration (Figure 7a–c). Importantly, at laser fluences (30 MJ/cm2) where no morphological alterations were detected by digital holography, high specific spectral variations were monitored to evaluate the cell photodegradation. More specifically, Raman analysis provided precise information on the most affected biochemical structures, finding that DNA phosphate backbone (900–1100 cm−1) and lipids and proteins (1200–1400 cm−1) are very sensitive to photoxidative denaturation (Figure 7d) [36].
Figure 7.
(Top) Reconstructed phase map of the (a) nonirradiated and (b) irradiated region of interest at the focus plane. The star (*) indicates the cell position where the Raman spectrum is acquired that corresponds to the irradiated area. (c) Raman spectra of the sperm cell at three different selected laser fluences (0, 61 and 107 MJ/cm2). (d) Zoom of four selected Raman spectra acquired at laser fluences of 0, 30, 61 and 107 MJ/cm2 in the spectral region between 700–800 cm−1, 1050–1120 cm−1, 1200–1400 cm−1 and 1550–1720 cm−1 [36].
To test the biochemical/morphological ability of the proposed multimodal approach, we additionally tested bovine sperm cells. The sex preselection of the offspring reveals a high significant impact on animal production management as well as genetic improvement programs. Since a noninvasive method for sex predetermination in animals is still not available, we used our multimodal approach for identifying and separating X and Y-bearing sperm cells. The key parameters estimated in our works [49, 51], on hundreds bovine spermatozoa of different bulls, were the Raman bands correlated to DNA content and head volume.
Indeed, specific peaks related to the vibrational modes of the DNA bases (726 and 785 cm−1) can be used to sort X- and Y-bovine sperm cells (Figure 8a and b). However, additional significant spectral variations can be observed in the Raman bands mainly associated with the presence of lipids and proteins (1400–1600 cm−1, Figure 8a and b), due to the different composition of the sex-associated membrane proteins in X- and Y-bearing sperm cells. In order to quantify the efficiency and the accuracy of the Raman spectroscopy in discriminating the two sperm cell populations, we have analyzed the data using a multivariate statistical method, known as principal component analysis, that allows to visualizing the separation of the two classes of cells into two well distinct clusters (Figure 8c), only on the base of the differences in their Raman fingerprint. The sorting accuracy resulted of 90.6%, which is comparable to the accuracy (90–94%) achievable with standard sorting techniques such as fluorescence-activated cell sorting (FACS).
Figure 8.
(a) Average Raman spectra of 900 X- (purple line) and 900 Y- (blue line) sperm cell spectra acquired in the “fingerprint” spectral region. (b) Measured peak area of the bands at 726 and 785 cm−1, highlighting the different DNA content measured for X and Y sperm cells, ΔA = 4.2 ± 0.9%. (c) PCA score plots of PC2, PC3 and PC4 showing the separation of data corresponding to X- and Y- sperm cells [49, 51].
Then, the spectroscopic results have been correlated with the morphological analysis, where the sperm head volume has been evaluated by applying the Otsu’s method [51] on the holographic phase map, a procedure generally used to indirectly measure morphological properties of the region under investigation (results not reported, see [49, 51]). As the main differences between X and Y chromosomes are the size (X chromosome is bigger than Y one) and the total DNA content, these major differences can be reflected in the head sperm volume, representing therefore a fast and automated way to identify the chromosome type [49, 51].
Therefore, by combining holographic with Raman microscopy provides label-free, quantitative morphological and chemical information from unfixed sperm cells. The combination offers an excellent system for a complete, morphological and physiological, monitoring of the sperm cell quality.
5. Towards clinical applications: a demanding path
Despite the feasibility of Raman spectroscopy and holography microscopies has been successfully demonstrated in many medical and biological applications [14, 16, 30, 52, 53, 54, 55], there is still a significant lack of translation and implementation of such innovative techniques into clinical practice. Indeed, while thanks to the technological advances the capability and information achievable are quickly expanding, there are some concerns to consider [56].
The Raman signals are generally low and often obscured by the presence of the cell autofluorescence. However, the fluorescence-free detection can be achieved using instruments working in the near-IR region of the spectrum, also reducing the cell photodamage. Alternatively, modulating or multiwavelengths approaches can be employed to eliminate the fluorescence background [57, 58, 59].
Another crucial aspect for translational applications is the time required to collect the spectral data and reconstruct the Raman images. Indeed, for reproductive medicine applications, performing experiment in real-time is crucial. Recently, several approaches have been proposed providing a faster imaging modality and allowing investigation of several sperm cells simultaneously such as the Coherent anti-Stokes Raman scattering (CARS) and surface-enhanced Raman scattering (SERS) imaging [60, 61] or the use of structured illumination [62, 63]. Moreover, in our specific case, the sample is moving while measuring. This represents another problem that researchers are trying to overcome using the laser trapping capability [64, 65], slide functionalization procedures [25, 66], or microfluidic devices [67, 68].
A further obstacle to the clinical success of these new methods is the complexity in interpreting the results. Indeed, the newer instruments for Raman/holographic imaging are fast, efficient, and reliable; however, they require specialized operators. An useful system should be easier to use, providing clear and automated answers to biomedical problems instead of spectra or holograms. The ongoing implementation of computer-assisted diagnosis algorithms is helping the interpretation of the holographic images, while further work on the creation of larger Raman database is still required.
The economical aspect has also to be considered. Raman spectroscopy and digital holography use precise equipment. If we consider that a holographic imaging system is sensitive to optical pathway differences on the nanoscale and has to be isolated to any kinds of vibrations for avoiding artifacts, we can well image that the precision required in the construction of such devices is more expansive than that of conventional microscopes currently in clinical use.
The path to clinical implementation of innovative multimodal imaging techniques for sperm cell assessment passes through the following milestones: 1. identification of the medical problem and the need for a new technique; 2. experimental tests of the technique, showing the proof of principle and the feasibility of the specific application; 3. closely collaboration between researchers and clinicians for evaluating the clinical relevance of the information the new technique provides; 4. optimisation of the technique for the specific application in order to improve its sensibility, specificity and robustness; 5. clinical trials; 6. industrial implementation of the system for making it clinician and patient friendly; and 7. Clinical implementation.
The proof of principle of Raman spectroscopy and holographic imaging as sperm selection techniques has been successfully demonstrated. Their use for detecting epigenetic alterations, including DNA packaging and spatial conformation, methylation, and histone modification, that could seriously affect the embryonic development has been showed [21]. Researches in this field are currently focusing on the points 3 and 4, as highlighted in this chapter. Our efforts aim to assess the feasibility and the reliability of the two techniques before initiating the clinical trials, filling in such a way the gap between experimentation and clinical implementation.
\n',keywords:"Raman spectroscopy, digital holography, sperm morphology, label-free analysis, sex-sorting",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/57212.pdf",chapterXML:"https://mts.intechopen.com/source/xml/57212.xml",downloadPdfUrl:"/chapter/pdf-download/57212",previewPdfUrl:"/chapter/pdf-preview/57212",totalDownloads:997,totalViews:145,totalCrossrefCites:0,totalDimensionsCites:1,totalAltmetricsMentions:0,introChapter:null,impactScore:1,impactScorePercentile:66,impactScoreQuartile:3,hasAltmetrics:0,dateSubmitted:"March 6th 2017",dateReviewed:"September 16th 2017",datePrePublished:"December 20th 2017",datePublished:"June 13th 2018",dateFinished:"October 14th 2017",readingETA:"0",abstract:"Current in vitro fertilization (IVF) techniques require a severe selection of sperm, generally based on concentration, morphology, motility, and DNA integrity. Since routinely separation methods may damage the viability of the sperm cell, there is a growing interest in providing a method for noninvasively analyzing spermatozoa taking into account all those parameters. This chapter first reviews the state-of-the-art of label-free sperm cell imaging for IVF, highlighting the limitations of the used techniques. Then, our innovative approach combining Raman spectroscopy and digital holography will be described and its advantages detailed. These include the ability to perform a simultaneous and correlative morphological and biochemical analysis of sperm cells, without labeling, in a fast and reliable way. Finally, the difficulty in reaching clinical use will be discussed, as well as the possible solutions offered by new technological improvements.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/57212",risUrl:"/chapter/ris/57212",book:{id:"6079",slug:"spermatozoa-facts-and-perspectives"},signatures:"Annalisa De Angelis, Maria Antonietta Ferrara, Giuseppe Coppola\nand Anna Chiara De Luca",authors:[{id:"104314",title:"Dr.",name:"Maria Antonietta",middleName:null,surname:"Ferrara",fullName:"Maria Antonietta Ferrara",slug:"maria-antonietta-ferrara",email:"mariaantonietta.ferrara@cnr.it",position:null,profilePictureURL:"https://intech-files.s3.amazonaws.com/a043Y00000f8dcoQAA/Co1_Profile_Picture-1582285984751",institution:{name:"National Research Council",institutionURL:null,country:{name:"Romania"}}},{id:"106792",title:"Dr.",name:"Giuseppe",middleName:null,surname:"Coppola",fullName:"Giuseppe Coppola",slug:"giuseppe-coppola",email:"giuseppe.coppola@cnr.it",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/106792/images/system/106792.jpg",institution:{name:"National Research Council",institutionURL:null,country:{name:"Romania"}}},{id:"206226",title:"Ph.D.",name:"Annalisa",middleName:null,surname:"De Angelis",fullName:"Annalisa De Angelis",slug:"annalisa-de-angelis",email:"a.deangelis@ibp.cnr.it",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"Institute of Protein Biochemistry",institutionURL:null,country:{name:"Italy"}}},{id:"207532",title:"Dr.",name:"Anna Chiara",middleName:null,surname:"De Luca",fullName:"Anna Chiara De Luca",slug:"anna-chiara-de-luca",email:"a.deluca@ibp.cnr.it",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"Institute of Protein Biochemistry",institutionURL:null,country:{name:"Italy"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Label-free biochemical sperm analysis",level:"1"},{id:"sec_2_2",title:"2.1. Basic principles of Raman spectroscopy",level:"2"},{id:"sec_3_2",title:"2.2. Raman spectroscopy analysis of sperm cells quality",level:"2"},{id:"sec_5",title:"3. Quantitative phase imaging for sperm analysis",level:"1"},{id:"sec_5_2",title:"3.1. Basic principles of quantitative phase microscopy",level:"2"},{id:"sec_6_2",title:"3.2. Digital holography microscopy for sperm cells assessment",level:"2"},{id:"sec_8",title:"4. Combined optical approach for the noninvasive analysis of single spermatozoa",level:"1"},{id:"sec_8_2",title:"4.1. The optical setup",level:"2"},{id:"sec_9_2",title:"4.2. Morphological and biochemical analysis of single sperm cells",level:"2"},{id:"sec_11",title:"5. Towards clinical applications: a demanding path",level:"1"}],chapterReferences:[{id:"B1",body:'Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HWG, Behre HM, et al. 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Current Opinion in Biotechnology. 2012;23(1):56-63'},{id:"B55",body:'Managò S, Valente C, Mirabelli P, Circolo D, Basile F, Corda D, et al. A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia. Scientific Reports. 2016;6:24821'},{id:"B56",body:'Webb SJ. Laser-Raman spectroscopy of living cells. Physics Reports. 1980;60(4):201-224'},{id:"B57",body:'Canetta E, Mazilu M, De Luca AC, Carruthers AE, Dholakia K, Neilson S, et al. Modulated Raman spectroscopy for enhanced identification of bladder tumor cells in urine samples. Journal of Biomedical Optics. 2011;16(3):037002-037002'},{id:"B58",body:'De Luca AC, Mazilu M, Riches A, Herrington CS, Dholakia K. Online fluorescence suppression in modulated Raman spectroscopy. Analytical Chemistry. 2009;82(2):738-745'},{id:"B59",body:'De Luca AC, Dholakia K, Mazilu M. Modulated Raman spectroscopy for enhanced cancer diagnosis at the cellular level. Sensors. 2015;15(6):13680-13704'},{id:"B60",body:'Li L, Wang H, Cheng J-X. Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains. Biophysical Journal. 2005;89(5):3480-3490'},{id:"B61",body:'Zito G, Rusciano G, Pesce G, Dochshanov A, Sasso A. Surface-enhanced Raman imaging of cell membrane by a highly homogeneous and isotropic silver nanostructure. Nanoscale. 2015;7(18):8593-8606'},{id:"B62",body:'De Luca AC, Reader-Harris P, Mazilu M, Mariggio S, Corda D, Di Falco A. Reproducible surface-enhanced Raman quantification of biomarkers in multicomponent mixtures. ACS Nano. 2014;8(3):2575-2583'},{id:"B63",body:'Kosmeier S, Zolotovskaya S, De Luca AC, Riches A, Herrington CS, Dholakia K, et al. Nonredundant Raman imaging using optical eigenmodes. Optica. 2014;1(4):257-263'},{id:"B64",body:'De Luca AC, Kosmeier S, Dholakia K, Mazilu M. Optical eigenmode imaging. Physical Review A. 2011 Aug 15;84(2):021803'},{id:"B65",body:'Di Caprio G, Ferrara MA, Miccio L, Merola F, Memmolo P, Ferraro P, et al. Holographic imaging of unlabelled sperm cells for semen analysis: A review. Journal of Biophotonics. 2015 Oct 1;8(10):779-789'},{id:"B66",body:'De Luca AC, Rusciano G, Ciancia R, Martinelli V, Pesce G, Rotoli B, et al. Spectroscopical and mechanical characterization of normal and thalassemic red blood cells by Raman tweezers. Optics Express. 2008;16(11):7943-7957'},{id:"B67",body:'Frimat J-P, Bronkhorst M, de Wagenaar B, Bomer JG, Van der Heijden F, Van den Berg A, et al. Make it spin: Individual trapping of sperm for analysis and recovery using micro-contact printing. Lab on a Chip. 2014;14(15):2635-2641'},{id:"B68",body:'De Wagenaar B, Berendsen JTW, Bomer JG, Olthuis W, van den Berg A, Segerink LI. Microfluidic single sperm entrapment and analysis. Lab on a Chip. 2015;15(5):1294-1301'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Annalisa De Angelis",address:"a.deangelis@ibp.cnr.it",affiliation:'
Institute of Protein Biochemistry - National Research Council, Italy
Institute for Microelectronics and Microsystems - National Research Council, Italy
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1. Introduction
Enteric and respiratory viruses can potentially be transmitted via contaminated environmental surfaces [1, 2]. Infectious viruses present on fomites may be transferred to the fingers and/or hands when touching various surface types under a broad spectrum of environmental conditions [3]. Transfer efficiency is affected by factors including virus species, inoculum size, and skin condition [4]. Subsequent contact with the eyes, nose, or mouth with contaminated fingers and hands may then provide access to susceptible human hosts [5]. Disinfection of environmental surfaces lowers the numbers of infectious microorganisms, thereby reducing the risk for transmission [6, 7]. However, such surfaces are subjected to continuous recontamination events, particularly in high-traffic areas and facilities including hospitals, daycare centers, schools and office buildings where fomites are more likely to serve as reservoirs of pathogens [8, 9, 10].
There are hundreds of liquid-based formulations that are registered as disinfectants with governmental regulatory agencies around the world, and a subset of those also carry label kill claims against non-enveloped and enveloped viruses. The efficacy testing that is required for the issuance of product label claims is performed using internationally-recognized standard test methods such as those produced by the American Standard for Test Materials (ASTM) and the European Standard (EN), among others. Liquid disinfectants can be applied to hard, non-porous surfaces using spray devices, towelettes (wipes), or as bulk liquid volumes to address large, soiled areas. To achieve the antiviral inactivation claims specified on product labels, disinfectants must be used according to the manufacturer’s instructions which may require maintaining a completely wetted surface for up to 10 minutes. However, the habits and practices of product users are contrary to the directions specified on the label. A recent survey of American adults conducted on behalf of the American Cleaning Institute in 2020 revealed that 26% of respondents adhere to label directions during household disinfection routines; however, an equal percentage of those surveyed did profess to wiping surfaces until dry immediately after spraying with no adherence to contact time instructions [11]. An additional 16% of respondents claimed to use a single-pass method for disinfectant wipes rather than the multiple passes that are generally required to maintain surface wetness for several minutes.
The importance of correct disinfection usage has been of increased concern during the COVID-19 pandemic. Alternative disinfecting surface treatments that are capable of inactivating infectious agents, in particular viruses, are under research and development [12, 13]. A number of new and diverse antiviral coatings and films have been synthesized, and fixed or immobilized applications including solids (e.g., antimicrobial plastics), paints, and metals are increasingly of interest for their antiviral capabilities. The factors affecting virus survival and the efficacy of antiviral coatings have been reviewed [2, 14] and include virus structure (i.e. enveloped, non-enveloped), the presence of organic soil (dirt), temperature, relative humidity, coating composition, and contact time (Table 1). The ability of treated surfaces to remain continuously active after repeated cleanings and use of liquid disinfectants is also critical (Figure 1). Unfortunately, there are no generally accepted methods for evaluating anti-viral surface coatings, making it difficult to compare the efficacy of different materials and studies. More research is warranted to better understand breadth of antiviral efficacy of these novel disinfecting technologies, and whether they can exact measurable and meaningful impacts on public health.
Factor
Impact
Type of virus
Non-enveloped viruses are generally more resistant than enveloped viruses
Relative humidity
Drying rates of deposited viruses are affected, impacting viability
Temperature
Protein denaturation results in loss of structural integrity of virus
Soil (dirt) load
Increased demand on antiviral actives, decreasing availability for virus inactivation
Coating composition
Mechanisms of antiviral action differ among viruses and vary according to formulation
Contact Time
Time required for at least a 99.9% (3 log10) reduction in titer may range from minutes to hours
Table 1.
Factors that affect virus survival and efficacy of antiviral coatings [2, 14].
Figure 1.
Continuously active antiviral surface coatings: a) coating applied to hard, nonporous surface demonstrates antiviral activity following virus deposition; b) coated surfaces are cleaned/disinfected with wiping action with passage of time, c) residual coating demonstrates continuous antiviral efficacy following surface cleaning events (Created in BioRender.com).
2. Continuously active disinfectants applied to hard, nonporous surfaces
A number of formulations have been developed and assessed over the past two decades that are capable of antiviral inactivation for extended periods of time following surface application (Table 2) [12, 13, 14, 15, 16]. Such applications have been considered as continuously active disinfectants and impart self-disinfecting properties to treated surfaces. There are many industry-based and third-party contract laboratory studies that have evaluated the antiviral properties of these surface treatments. However, few have been published to-date in peer-reviewed scientific journals [17], with an even smaller subgroup assessing efficacy against infectious viral agents. Continuously active disinfectants are generally evaluated for residual inactivation efficacy using a controlled, standardized wear and abrasion procedure such as that described in United States EPA Protocol #01-1A [18]. Briefly, a product applied to a hard non-porous surface is subjected to alternating dry and moistened wiping procedures over a specified time period (≥ 24 hours) with intermittent reinoculations of the test organism. A minimum of 12 wear cycles is required, and the remaining film of test product is challenged by a final dose of the target organism (≥ 4.8 log10) for up to 5 minutes of contact time. Residual efficacy depends in part on the amount of disinfectant remaining on the surface after the wear and abrasion testing which indicates its durability. Products that are readily removed from surfaces during repeated wet and dry wiping events could require regular reapplication to ensure proper performance against target microbes. As with standard disinfection, residual effectiveness generally follows the hierarchy of susceptibility of viruses to disinfectants, where enveloped viruses are more susceptible to inactivation than non-enveloped viruses [19].
Behaves as a surfactant; disrupts lipid and protein structure
Copper
Influenza A, hepatitis A, feline calicivirus, adenovirus, HCoV- 229E, SARS-CoV2
Reactive oxygen species; protein and nucleic acid denaturization
Silver
Influenza, SARS-CoV2, HCoV-229E, murine norovirus
Reaction with sulfhydryl groups in proteins; prevention of viral attachment to host cells
Zinc
Murine norovirus, SARS-CoV-2, influenza
Inhibiting proteolytic cleavage, preventing synthesis of viral polypeptides
Titanium dioxide
Influenza, adenovirus; SARS-Co-2
Generation of reactive hydroxyl radicals
Table 2.
Common antiviral surface chemistries and mechanisms of action [12, 13, 14, 15, 16].
QAC: quaternary ammonium compound.
HCoV-229E: human coronavirus 229E.
SARS-CoV-2: SARS-related coronavirus 2.
Quaternary ammonium compounds (QAC) have been in general use by industry and consumers for almost 70 years, mostly as rapid-action (≤ 10 minutes contact time) spray disinfectants for contaminated surfaces. They are considered as cationic surfactants or detergents, and are highly effective at disrupting the inner membranes of bacteria and lipid bilayers of enveloped viruses. QAC have undergone formulation changes to enhance effectiveness against non-enveloped viruses [20]. When combined with silane and polymers, they can be applied as a surface coating with antimicrobial properties [21]. Silane-QAC are long-chain molecules comprised of three principal components: 1) a silane base for covalent bonding to surfaces; 2) a centrally-located positively-charged nitrogen component, and 3) a long chain ‘spear’ consisting of a methyl hydrocarbon group. They can be applied to hard surfaces and to fabrics, and their virucidal efficacies may persist from 24 hours to weeks on treated surfaces.
Peer-reviewed studies evaluating the effectiveness of QAC-based surface coating treatments against viruses are currently limited. A quaternary ammonium polymer coating applied to stainless steel coupons demonstrated greater than 99.9% (>3 log10) reduction during 2 hours of contact against SARS-CoV-2 and human coronavirus 229E in the presence of 5% organic soil, although wear testing was not performed to assess residual antiviral activity [22]. Another study evaluating a QAC applied onto acrylic surfaces against subsequent SARS-CoV-2 and human coronavirus 229E contamination events demonstrated rapid inactivation upon contact (>90% [>1 log10] reduction); however, just one cleaning event of the coating using a water-based detergent and microfiber cloth substantially reduced product efficacy [23]. More peer-reviewed research is needed to better understand the breadth of QAC coating efficacy against the spectrum of non-enveloped and enveloped viruses, and under varying soil load and environmental conditions. Additional studies are also warranted to assess the durability of these coatings following simulated touches and cleaning events, and the resulting impacts on antiviral effectiveness.
3. Titanium dioxide
Titanium dioxide (TiO2) is a photocatalytic inorganic chemistry that can be applied to a wide variety of surface types to provide antiviral protection. It does not inactivate viruses directly, but acts as a catalyst in the presence of UVA light (wavelength 315 to 400 nm) to generate reactive oxygen species that cause structural damage to viruses. The presence of moisture (in the air or on the surface) and oxygen are necessary for TiO2 to be an effective antiviral agent. Light intensity is also key in driving the photocatalytic reaction. Residual photocatalytic activity may also occur in the dark after exposure to UV light, but is dependent on the prior exposure intensity.
Most of the studies evaluating the antimicrobial effectiveness of TiO2 have focused on bacteria, and data on viruses remains scant in the literature [16]. TiO2 has demonstrated >3 log10 reduction against influenza A within 4 hours, and > 1 log10 inactivation of feline calicivirus within 8 hours [24]. TiO2 coatings have also been modified with fluorine to increase the production of reactive oxygen species under the low UVA-intensity fluorescent lighting that is typically found within indoor settings. Bacteriophage MS2, feline calicivirus, and murine norovirus infectivity levels were reduced by 2.6, 2.0, and 2.6 log10, respectively, on fluorinated TiO2surfaces [25]. The antiviral action of TiO2 can be further enhanced within indoor environments by the addition of metals [26, 27]. A 1% silver-amended TiO2 formulation yielded >4.00 log10 reduction of influenza A and enterovirus following a 20-minute exposure in the presence of a low intensity (15 W) UVA lamp [28]. More recently, infectious SARS-CoV-2 was reduced to levels below detection on TiO2 and TiO2-Silver (Ag) ceramic-coated tiles within 5 hours of exposure [15].
4. Metals
Metals such as copper, silver, and gold have been recognized since ancient times as having some health benefits, and the antibacterial properties of metals have since been well-studied [29]. In contrast, the mechanisms of metal inactivation of specific viruses remain unclear, although a number have been proposed and evaluated. Certain metals in trace amounts are critical to the function of viral proteins and genetic processes; however, levels in excess cause structural damage and affect viability [14]. The presence of these metals stimulates the generation of reactive oxygen species and damages viral envelopes as well as nucleocapsid proteins [30]. Metals can be incorporated into plastics and fabrics, used as actives in coating formulations, and fashioned directly into surfaces for direct use (e.g., copper sheets for incorporation into high-touch surfaces).
4.1 Copper
The antimicrobial properties of copper have been extensively studied, with efficacy demonstrated over a broad range of temperature and humidity values [1]. The proposed antiviral mechanisms of solid-state copper, copper oxides, and copper alloys against enveloped and non-enveloped viruses have been thoroughly reviewed [31]. Copper (I), (II), (III) ions act directly by denaturing viral surface proteins, and indirectly by the formation of reactive oxygen species that damage viral RNA and DNA. Copper surfaces inactivated infectious influenza A (H1N1) within 6 hours by 3 to 4 log10, relative to virus levels remaining on stainless steel coupons [32]. Although copper has demonstrated broad-spectrum antimicrobial activity, it may be impractical to replace bulk materials within high-traffic areas (e.g., clinical settings) with copper products or components. The recent development of cold- and thermally-applied copper sprays, as well as fixed copper nanoparticle coatings and paints, enables continuously active disinfection measures against a spectrum of viruses [16]. Copper nanoparticles in the oxide form have shown promise against herpes simplex virus, human norovirus, and influenza A (H1N1) [31]. When applied using the cold spray technique, copper nanoparticles reduced infectious influenza A virus particles to levels below detection within 10 minutes [33].
4.2 Silver
The antimicrobial properties of silver have been known for more than a century. Much of the research investigating the antimicrobial properties of silver has examined inactivation in suspension, where lower doses are required to achieve inactivation effects relative to other metals [34]. Silver binds with disulfide (S-S) and sulfhydryl (-SH) groups in proteins, facilitates the production of reactive oxygen species (e.g., free radicals), and is believed to inhibit entry of HIV-1 into CD4+ host cells [35]. Unlike copper, the efficacy of silver decreases markedly at relative humidity levels <20% [1], and solid-state silver appears to be much less effective against bacteriophage Qβ and influenza A than solid-state copper [36]. For surface applications, silver nanoparticles have been extensively researched. Silver nitrate and silver nanoparticles in surface coatings reduced recoverable levels of feline calicivirus and murine norovirus for up to 150 days [37]. Silver has also been incorporated into fabrics (hospital gowns, pillowcases, cotton sheets), textiles, and membranes, demonstrating antiviral properties against feline calicivirus and murine norovirus, as well as enveloped viruses [16, 38].
4.3 Zinc
The antiviral properties of zinc have been researched for the past several decades. Zinc inhibits proteolytic cleavage and the synthesis of viral polypeptides by human rhinovirus [39], and interferes with polymerase function and protein production by herpes simplex virus 1 [16]. For surface applications, pure zinc, itself, does not exhibit high levels of antiviral activity. A 1 log10 reduction of murine norovirus on pure zinc was measured within 2 hours, relative to complete inactivation of the test virus via synergism when exposed to a copper-silver-zinc alloy [40]. On plastic coupons with incorporated silver/copper-zeolites, >1.7 log10 and > 3.8 log10 reductions were achieved for human coronavirus 229E and feline calicivirus, respectively, within 24 hours [41]. More recently, zinc ion-embedded polyamide fibers were found to reduce levels of infectious influenza A and SARS-CoV-2 by approximately 2 log10 within 30 minutes [42].
5. Novel antiviral surface treatments
Research efforts are ongoing for the development of novel and continuously active coatings that are capable of maintaining low levels of bioburden while inactivating pathogenic microorganisms. A thorough review has been published of these coatings and their proposed mechanisms of action [14, 43]. The antiviral actives include biopolymers (e.g., antimicrobial peptides), synthetic polymers (e.g., polyethyleneimines, and graphene [14, 44, 45]. Natural product-based surface coatings and super-hydrophobic surfaces are also under development [46, 47]. Although many of these innovative technologies demonstrate promising antiviral effectiveness, further assessments of efficacy against additional types of viruses under various conditions are required. Reproducibility data generated among different lab groups would also be ideal to ensure product efficacy and reliability. Further, scaling up from the lab bench to assess these technologies under real-world conditions (i.e. placement into high-traffic, high-touch areas) will provide insight as to the consistency of their efficacy.
6. Conclusions and recommendations
From this review, it is clear that promising antiviral continuously active disinfectants are a reality. However, many obstacles exist before their widespread implementation. These include:
Development and validation of standard methods for testing the efficacy of antiviral continuously active disinfectants. Ideally, these methods would indicate appropriate experimental conditions including relative humidity and temperature, organic soil load matrices, and evaluation of virucidal efficacy against enveloped and non-enveloped viruses.
Establishing an acceptable contact time for a 3 log10 (99.9%) decrease in infectious virus. Some continuously active disinfectants can achieve this goal within a few minutes, and others may require 1 to 2 hours.
Demonstration of the reduction in illnesses within facilities in which continuously active disinfectants are used. This is an ideal requirement, but difficult to achieve because of the high cost and multiple routes by which enteric and respiratory viruses can be transmitted. Reductions in hospital-acquired infections have been demonstrated with the use of copper [48, 49] and silane QAC [50] disinfectants, but such studies are not always ideal because of limitations inherent in epidemiological studies, and extracting precision is usually lacking. Further, more information is needed as to the potential human health and environmental impacts of silane QAC usage in these settings.
Application of quantitative microbial risk assessment (QMRA) to quantify the cost/benefits of continuously active disinfectants. QMRA is a lower-cost approach to documenting the probability of disease reduction that can be achieved. It can be used to estimate the difference in benefits from a continuously active disinfectant that inactivates 99.9% of the virus within 1 minute vs. one that achieves this within 2 hours.
Education of regulators, public health officials, and the general public is necessary to ultimately achieve the benefits of continuously active disinfectants. There is concern that their use may provide a false sense of security, causing consumers to clean and disinfect less frequently. Continuously active disinfectants should be looked upon as an additional barrier, and not as a replacement for routine cleaning and disinfection.
Conflict of interest
The authors have no conflict of interest to declare.
\n',keywords:"disinfection, virus, coating, continuously active, fomites",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/79842.pdf",chapterXML:"https://mts.intechopen.com/source/xml/79842.xml",downloadPdfUrl:"/chapter/pdf-download/79842",previewPdfUrl:"/chapter/pdf-preview/79842",totalDownloads:105,totalViews:0,totalCrossrefCites:0,dateSubmitted:"November 19th 2021",dateReviewed:"November 24th 2021",datePrePublished:"December 29th 2021",datePublished:"May 18th 2022",dateFinished:"December 29th 2021",readingETA:"0",abstract:"Antimicrobial surfaces and coatings have been available for many decades and have largely been designed to kill or prevent the growth of bacteria and fungi. Antiviral coatings have become of particular interest more recently during the COVID-19 pandemic as they are designed to act as continuously active disinfectants. The most studied antiviral coatings have been metal-based or are comprised of silane quaternary ammonium formulations. Copper and silver interact directly with proteins and nucleic acids, and influence the production of reactive free radicals. Titanium dioxide acts as a photocatalyst in the presence of water and oxygen to produce free radicals in the presence of UV light or visible light when alloyed with copper or silver. Silane quaternary ammonium formulations can be applied to surfaces using sprays or wipes, and are particularly effective against enveloped viruses. Continuously active disinfectants offer an extra barrier against fomite-mediated transmission of respiratory and enteric viruses to reduce exposure between routine disinfection and cleaning events. To take advantage of this technology, testing methods need to be standardized and the benefits quantified in terms of reduction of virus transmission.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/79842",risUrl:"/chapter/ris/79842",signatures:"Luisa A. Ikner and Charles P. Gerba",book:{id:"11006",type:"book",title:"Disinfection of Viruses",subtitle:null,fullTitle:"Disinfection of Viruses",slug:"disinfection-of-viruses",publishedDate:"May 18th 2022",bookSignature:"Raymond W. Nims and M. Khalid Ijaz",coverURL:"https://cdn.intechopen.com/books/images_new/11006.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",isbn:"978-1-83962-416-2",printIsbn:"978-1-83962-415-5",pdfIsbn:"978-1-83962-417-9",isAvailableForWebshopOrdering:!0,editors:[{id:"104702",title:"Dr.",name:"Raymond W.",middleName:null,surname:"Nims",slug:"raymond-w.-nims",fullName:"Raymond W. Nims"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:[{id:"414979",title:"Dr.",name:"Charles P.",middleName:null,surname:"Gerba",fullName:"Charles P. Gerba",slug:"charles-p.-gerba",email:"gerba@ag.arizona.edu",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"414983",title:"Dr.",name:"Luisa A.",middleName:null,surname:"Ikner",fullName:"Luisa A. Ikner",slug:"luisa-a.-ikner",email:"ikner@arizona.edu",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"University of Arizona",institutionURL:null,country:{name:"United States of America"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Continuously active disinfectants applied to hard, nonporous surfaces",level:"1"},{id:"sec_3",title:"3. Titanium dioxide",level:"1"},{id:"sec_4",title:"4. Metals",level:"1"},{id:"sec_4_2",title:"4.1 Copper",level:"2"},{id:"sec_5_2",title:"4.2 Silver",level:"2"},{id:"sec_6_2",title:"4.3 Zinc",level:"2"},{id:"sec_8",title:"5. Novel antiviral surface treatments",level:"1"},{id:"sec_9",title:"6. Conclusions and recommendations",level:"1"},{id:"sec_13",title:"Conflict of interest",level:"1"}],chapterReferences:[{id:"B1",body:'Castaño N, Cordts S, Kurosu Jalil M, Zhang K, Koppaka S, Bick A, et al. Fomite transmission, physicochemical origin of virus–surface interactions, and disinfection strategies for enveloped viruses with applications to SARS-CoV-2. ACS Omega. 2021;6:6509-6527. DOI: 10.1021/acsomega.0c06335'},{id:"B2",body:'Boone S, Gerba C. Significance of fomites in the spread of respiratory and enteric viral disease. Applied and Environmental Microbiology. 2007;73:1687-1696. DOI: 10.1128/AEM.02051-06'},{id:"B3",body:'Lopez G, Gerba C, Tamimi A, Kitajima M, Maxwell S, Rose J. Transfer efficiency of bacteria and viruses from porous and nonporous fomites to fingers under different relative humidity conditions. Applied and Environmental Microbiology. 2013;79:5728-5734. DOI: 10.1128/AEM.01030-13'},{id:"B4",body:'Julian T, Leckie J, Boehm A. Virus transfer between fingerpads and fomites. Journal of Applied Microbiology. 2010;109:1868-1874. DOI: 10.1111/j.1365-2672.2010.04814.x'},{id:"B5",body:'Nicas M, Best D. A study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection. Journal of Occupational and Environmental Hygiene. 2008;5:347-352. DOI: 10.1080/15459620802003896'},{id:"B6",body:'Rutala W, Kanamori H, Gergen M, Knelson L, Sickbert-Bennett E, Chen L, et al. CDC prevention epicenters program Enhanced disinfection leads to reduction of microbial contamination and a decrease in patient colonization and infection. Infection Control & Hospital Epidemiology. 2018;39:1118-1121. DOI: 10.1017/ice.2018.165'},{id:"B7",body:'Rutala W, Weber D. The benefits of surface disinfection. American Journal of Infection Control. 2004;32:226-231'},{id:"B8",body:'Kraay A, Hayashi M, Berendes D, Sobolik J, Leon J, Loopman B. Risk for fomite-mediated transmission of SARS-CoV-2 in child daycares, schools, and nursing homes, and offices. Emerging Infectious Diseases. 2021;27:1229-1231. DOI: 10.3201/eid2704.203631'},{id:"B9",body:'Weber D, Anderson D, Rutala W. The role of the surface environment in healthcare-associated infections. Current Opinion in Infectious Diseases. 2013;26:338-344. DOI: 10.1097/QCO.0b013e3283630f04'},{id:"B10",body:'Hardy K, Gossain S, Henderson N, Drugan C, Oppenheim B, Gao F, et al. Rapid recontamination with MRSA of the environment of an intensive care unit after decontamination with hydrogen peroxide vapour. Journal of Hospital Infection. 2007;66:360-368. DOI: 10.1016/j.jhin.2007.05.009'},{id:"B11",body:'The American Cleaning Institute. Cleaning and COVID-19: Survey Shows 42% not disinfecting properly [Internet]. 2020. 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DOI: 10.1038/248588a0'},{id:"B40",body:'Warnes S, Summersgill E, Keevil C. Inactivation of murine norovirus on a range of copper surfaces is accomplished by a loss of capsid integrity. Applied and Environmental Microbiology. 2015;81:1085-1091. DOI: 10.1128/AEM.03280-14'},{id:"B41",body:'Bright K, Sicarios-Ruelas E, Gundy P, Gerba C. Assessment of the antiviral properties of zeolites containing metal ions. Food and Environmental Virology. 2008;1:37-41. DOI: 10.1007/s12560-008-9006-1'},{id:"B42",body:'Gopal V, Nilsson-Payant B, French H, Siegers J, Yung W, Hardwick M, et al. Zinc-embedded polyamide fabrics inactivate SARS-CoV-2 and influenza A virus. ACS Applied Materials & Interfaces. 2021;13:30317-30325. DOI: 10.1021/acsami.1c04412'},{id:"B43",body:'Bäumler W, Eckl D, Holzmann T, Schneider-Brachert W. Antimicrobial coatings for environmental surfaces in hospitals: A potential new pillar for prevention strategies in hygiene. Critical Reviews in Microbiology. 2021:1-35. 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DOI: 10.1007/s10999-020-09512-y'},{id:"B48",body:'Michels H, Keevil C, Salgado C, Schmidt M. From laboratory research to a clinical trial: Copper alloy surfaces kill bacteria and reduce hospital-acquired infections. HERD: Health Environments Research & Design Journal. 2015;9:64-79. DOI: 10.1177/1937586715592650'},{id:"B49",body:'Salgado C, Sepkowitz K, John J, Cantey J, Attaway H, Freeman K, et al. Copper surfaces reduce the rate of healthcare-acquired infections in the intensive care unit. Infection Control & Hospital Epidemiology. 2013;34:479-486. DOI: 10.1086/670207'},{id:"B50",body:'Ellingson K, Pogreba-Brown K, Gerba C, Elliott S. Impact of a novel antimicrobial surface coating on health care–associated infections and environmental bioburden at 2 urban hospitals. Clinical Infectious Diseases. 2020;71:1807-1813. DOI: 10.1093/cid/ciz1077'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Luisa A. Ikner",address:"ikner@arizona.edu",affiliation:'
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IntechOpen’s Academic Editors and Authors have received funding for their work through many well-known funders, including: the European Commission, Bill and Melinda Gates Foundation, Wellcome Trust, Chinese Academy of Sciences, Natural Science Foundation of China (NSFC), CGIAR Consortium of International Agricultural Research Centers, National Institute of Health (NIH), National Science Foundation (NSF), National Aeronautics and Space Administration (NASA), National Institute of Standards and Technology (NIST), German Research Foundation (DFG), Research Councils United Kingdom (RCUK), Oswaldo Cruz Foundation, Austrian Science Fund (FWF), Foundation for Science and Technology (FCT), Australian Research Council (ARC).
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If you are associated with any of the institutions in our list below, you can apply to receive OA publication funds by following the instructions provided in the links. Please consult the Open Access policies or grant Terms and Conditions of any institution with which you are linked to explore ways to cover your publication costs (also accessible by clicking on the link in their title).
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Please be aware that you must be a member, or grantee, of the institutions/funders listed in order to apply for their Open Access publication funds.
Open Access publication costs can often be designated directly in the grants or in specific budgets allocated for that purpose. Many of the most important funding organisations encourage, and even request, that the projects they fund are made available at no cost to the wider public. IntechOpen strives to maintain excellent relationships with these funders and ensures compliance with mandates.
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In order to help Authors identify appropriate funding agencies and institutions, we have created a list, based on extensive research on various OA resources (including ROARMAP and SHERPA/JULIET) of organizations that have funds available. Before consulting our list we encourage you to petition your own institution or organization for Open Access funds or check the specifications of your grant with your funder to ascertain if publication costs are included. Where you are in receipt of a grant you should clarify:
\n\n
\n\t
Does your institution already have a budget for covering Open Access publication costs?
\n\t
Does your grant list Open Access publication fees as legitimate direct/indirect costs?
\n
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If you are associated with any of the institutions in our list below, you can apply to receive OA publication funds by following the instructions provided in the links. Please consult the Open Access policies or grant Terms and Conditions of any institution with which you are linked to explore ways to cover your publication costs (also accessible by clicking on the link in their title).
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The prefrontal cortex undergoes maturation during childhood with a reduction of synaptic and neuronal density, a growth of dendrites, and an increase in white matter volume. With these neuroanatomical changes, neural networks construct appropriate for complex cognitive processing. The organization of prefrontal cortical circuitry may have been critical to the occurrence of human-specific executive and social-emotional functions, and developmental pathology in these same systems underlies many psychiatric disorders; therefore, if we understand these developmental process well, we could better analyze the development of psychiatric disorders.",book:{id:"6819",slug:"prefrontal-cortex",title:"Prefrontal Cortex",fullTitle:"Prefrontal Cortex"},signatures:"Merve Cikili Uytun",authors:[{id:"163607",title:"Dr.",name:"Merve",middleName:null,surname:"Cikili",slug:"merve-cikili",fullName:"Merve Cikili"}]},{id:"41594",title:"Amygdala, Childhood Adversity and Psychiatric Disorders",slug:"amygdala-childhood-adversity-and-psychiatric-disorders",totalDownloads:6021,totalCrossrefCites:0,totalDimensionsCites:0,abstract:null,book:{id:"2599",slug:"the-amygdala-a-discrete-multitasking-manager",title:"The Amygdala",fullTitle:"The Amygdala - A Discrete Multitasking Manager"},signatures:"Xiaodan Yan",authors:[{id:"144657",title:"Dr.",name:"X",middleName:null,surname:"Yan",slug:"x-yan",fullName:"X Yan"}]},{id:"58070",title:"MRI Medical Image Denoising by Fundamental Filters",slug:"mri-medical-image-denoising-by-fundamental-filters",totalDownloads:2618,totalCrossrefCites:20,totalDimensionsCites:32,abstract:"Nowadays Medical imaging technique Magnetic Resonance Imaging (MRI) plays an important role in medical setting to form high standard images contained in the human brain. MRI is commonly used once treating brain, prostate cancers, ankle and foot. The Magnetic Resonance Imaging (MRI) images are usually liable to suffer from noises such as Gaussian noise, salt and pepper noise and speckle noise. So getting of brain image with accuracy is very extremely task. An accurate brain image is very necessary for further diagnosis process. During this chapter, a median filter algorithm will be modified. Gaussian noise and Salt and pepper noise will be added to MRI image. A proposed Median filter (MF), Adaptive Median filter (AMF) and Adaptive Wiener filter (AWF) will be implemented. The filters will be used to remove the additive noises present in the MRI images. The noise density will be added gradually to MRI image to compare performance of the filters evaluation. The performance of these filters will be compared exploitation the applied mathematics parameter Peak Signal-to-Noise Ratio (PSNR).",book:{id:"6144",slug:"high-resolution-neuroimaging-basic-physical-principles-and-clinical-applications",title:"High-Resolution Neuroimaging",fullTitle:"High-Resolution Neuroimaging - Basic Physical Principles and Clinical Applications"},signatures:"Hanafy M. Ali",authors:[{id:"213318",title:"Dr.",name:"Hanafy",middleName:"M.",surname:"Ali",slug:"hanafy-ali",fullName:"Hanafy Ali"}]}],onlineFirstChaptersFilter:{topicId:"209",limit:6,offset:0},onlineFirstChaptersCollection:[],onlineFirstChaptersTotal:0},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:8,limit:8,total:0},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:9,numberOfPublishedChapters:90,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:107,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:33,numberOfPublishedChapters:330,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:19,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:14,numberOfPublishedChapters:145,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:9,numberOfPublishedChapters:139,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!0},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:122,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:11,numberOfPublishedChapters:112,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:21,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2753-894X",doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:10,numberOfOpenTopics:1,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!0},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:1,numberOfPublishedChapters:19,numberOfOpenTopics:5,numberOfUpcomingTopics:0,issn:"2753-6580",doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}},{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. The whole process of submitting an article and editing of the submitted article goes extremely smooth and fast, the number of reads and downloads of chapters is high, and the contributions are also frequently cited.",author:{id:"55578",name:"Antonio",surname:"Jurado-Navas",institutionString:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRisIQAS/Profile_Picture_1626166543950",slug:"antonio-jurado-navas",institution:{id:"720",name:"University of Malaga",country:{id:null,name:"Spain"}}}}]},series:{item:{id:"24",title:"Sustainable Development",doi:"10.5772/intechopen.100361",issn:"2753-6580",scope:"
\r\n\tTransforming our World: the 2030 Agenda for Sustainable Development endorsed by United Nations and 193 Member States, came into effect on Jan 1, 2016, to guide decision making and actions to the year 2030 and beyond. Central to this Agenda are 17 Goals, 169 associated targets and over 230 indicators that are reviewed annually. The vision envisaged in the implementation of the SDGs is centered on the five Ps: People, Planet, Prosperity, Peace and Partnership. This call for renewed focused efforts ensure we have a safe and healthy planet for current and future generations.
\r\n
\r\n\t
\r\n
\r\n\tThis Series focuses on covering research and applied research involving the five Ps through the following topics:
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\r\n
\r\n\t1. Sustainable Economy and Fair Society that relates to SDG 1 on No Poverty, SDG 2 on Zero Hunger, SDG 8 on Decent Work and Economic Growth, SDG 10 on Reduced Inequalities, SDG 12 on Responsible Consumption and Production, and SDG 17 Partnership for the Goals
\r\n
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\r\n\t2. Health and Wellbeing focusing on SDG 3 on Good Health and Wellbeing and SDG 6 on Clean Water and Sanitation
\r\n
\r\n\t
\r\n
\r\n\t3. Inclusivity and Social Equality involving SDG 4 on Quality Education, SDG 5 on Gender Equality, and SDG 16 on Peace, Justice and Strong Institutions
\r\n
\r\n\t
\r\n
\r\n\t4. Climate Change and Environmental Sustainability comprising SDG 13 on Climate Action, SDG 14 on Life Below Water, and SDG 15 on Life on Land
\r\n
\r\n\t
\r\n
\r\n\t5. Urban Planning and Environmental Management embracing SDG 7 on Affordable Clean Energy, SDG 9 on Industry, Innovation and Infrastructure, and SDG 11 on Sustainable Cities and Communities.
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\r\n\tThe series also seeks to support the use of cross cutting SDGs, as many of the goals listed above, targets and indicators are all interconnected to impact our lives and the decisions we make on a daily basis, making them impossible to tie to a single topic.
",coverUrl:"https://cdn.intechopen.com/series/covers/24.jpg",latestPublicationDate:"August 2nd, 2022",hasOnlineFirst:!0,numberOfPublishedBooks:1,editor:{id:"262440",title:"Prof.",name:"Usha",middleName:null,surname:"Iyer-Raniga",slug:"usha-iyer-raniga",fullName:"Usha Iyer-Raniga",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRYSXQA4/Profile_Picture_2022-02-28T13:55:36.jpeg",biography:"Usha Iyer-Raniga is a professor in the School of Property and Construction Management at RMIT University. Usha co-leads the One Planet Network’s Sustainable Buildings and Construction Programme (SBC), a United Nations 10 Year Framework of Programmes on Sustainable Consumption and Production (UN 10FYP SCP) aligned with Sustainable Development Goal 12. The work also directly impacts SDG 11 on Sustainable Cities and Communities. She completed her undergraduate degree as an architect before obtaining her Masters degree from Canada and her Doctorate in Australia. Usha has been a keynote speaker as well as an invited speaker at national and international conferences, seminars and workshops. Her teaching experience includes teaching in Asian countries. She has advised Austrade, APEC, national, state and local governments. She serves as a reviewer and a member of the scientific committee for national and international refereed journals and refereed conferences. She is on the editorial board for refereed journals and has worked on Special Issues. Usha has served and continues to serve on the Boards of several not-for-profit organisations and she has also served as panel judge for a number of awards including the Premiers Sustainability Award in Victoria and the International Green Gown Awards. Usha has published over 100 publications, including research and consulting reports. Her publications cover a wide range of scientific and technical research publications that include edited books, book chapters, refereed journals, refereed conference papers and reports for local, state and federal government clients. She has also produced podcasts for various organisations and participated in media interviews. She has received state, national and international funding worth over USD $25 million. Usha has been awarded the Quarterly Franklin Membership by London Journals Press (UK). Her biography has been included in the Marquis Who's Who in the World® 2018, 2016 (33rd Edition), along with approximately 55,000 of the most accomplished men and women from around the world, including luminaries as U.N. Secretary-General Ban Ki-moon. In 2017, Usha was awarded the Marquis Who’s Who Lifetime Achiever Award.",institutionString:null,institution:{name:"RMIT University",institutionURL:null,country:{name:"Australia"}}},editorTwo:null,editorThree:null},subseries:{paginationCount:5,paginationItems:[{id:"91",title:"Sustainable Economy and Fair Society",coverUrl:"https://cdn.intechopen.com/series_topics/covers/91.jpg",isOpenForSubmission:!0,editor:{id:"181603",title:"Dr.",name:"Antonella",middleName:null,surname:"Petrillo",slug:"antonella-petrillo",fullName:"Antonella Petrillo",profilePictureURL:"https://mts.intechopen.com/storage/users/181603/images/system/181603.jpg",biography:"Antonella Petrillo, Ph.D., is a professor in the Department of Engineering, University of Naples “Parthenope,” Italy. She received her Ph.D. in Mechanical Engineering from the University of Cassino and Southern Lazio, Italy. Her research interests include multi-criteria decision analysis, industrial plants, logistics, manufacturing, and safety. She serves as an associate editor for the International Journal of the Analytic Hierarchy Process and is an editorial board member for several other journals. She is also a member of the Analytic Hierarchy Process (AHP) Academy.",institutionString:"Parthenope University of Naples",institution:{name:"Parthenope University of Naples",institutionURL:null,country:{name:"Italy"}}},editorTwo:null,editorThree:null},{id:"92",title:"Health and Wellbeing",coverUrl:"https://cdn.intechopen.com/series_topics/covers/92.jpg",isOpenForSubmission:!0,editor:{id:"348225",title:"Prof.",name:"Ann",middleName:null,surname:"Hemingway",slug:"ann-hemingway",fullName:"Ann Hemingway",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y000035LZFoQAO/Profile_Picture_2022-04-11T14:55:40.jpg",biography:"Professor Hemingway is a public health researcher, Bournemouth University, undertaking international and UK research focused on reducing inequalities in health outcomes for marginalised and excluded populations and more recently focused on equine assisted interventions.",institutionString:null,institution:{name:"Bournemouth University",institutionURL:null,country:{name:"United Kingdom"}}},editorTwo:null,editorThree:null},{id:"93",title:"Inclusivity and Social Equity",coverUrl:"https://cdn.intechopen.com/series_topics/covers/93.jpg",isOpenForSubmission:!0,editor:{id:"210060",title:"Prof. Dr.",name:"Ebba",middleName:null,surname:"Ossiannilsson",slug:"ebba-ossiannilsson",fullName:"Ebba Ossiannilsson",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002g6LkBQAU/Profile_Picture_2022-02-28T13:31:48.png",biography:"Professor Dr. Ebba Ossiannilsson is an independent researcher, expert, consultant, quality auditor and influencer in the fields of open, flexible online and distance learning (OFDL) and the 'new normal'. Her focus is on quality, innovation, leadership, and personalised learning. She works primarily at the strategic and policy levels, both nationally and internationally, and with key international organisations. She is committed to promoting and improving OFDL in the context of SDG4 and the future of education. Ossiannilsson has more than 20 years of experience in her current field, but more than 40 years in the education sector. She works as a reviewer and expert for the European Commission and collaborates with the Joint Research Centre for Quality in Open Education. Ossiannilsson also collaborates with ITCILO and ICoBC (International Council on Badges and Credentials). She is a member of the ICDE Board of Directors and has previously served on the boards of EDEN and EUCEN. Ossiannilsson is a quality expert and reviewer for ICDE, EDEN and the EADTU. She chairs the ICDE OER Advocacy Committee and is a member of the ICDE Quality Network. She is regularly invited as a keynote speaker at conferences. She is a guest editor for several special issues and a member of the editorial board of several scientific journals. She has published more than 200 articles and is currently working on book projects in the field of OFDL. Ossiannilsson is a visiting professor at several international universities and was recently appointed Professor and Research Fellow at Victoria University of Wellington, NZ. Ossiannilsson has been awarded the following fellowships: EDEN Fellows, EDEN Council of Fellows, and Open Education Europe. She is a ICDE OER Ambassador, Open Education Europe Ambassador, GIZ Ambassador for Quality in Digital Learning, and part of the Globe-Community of Digital Learning and Champion of SPARC Europe. On a national level, she is a quality developer at the Swedish Institute for Standards (SIS) and for ISO. She is a member of the Digital Skills and Jobs Coalition Sweden and Vice President of the Swedish Association for Distance Education. She is currently working on a government initiative on quality in distance education at the National Council for Higher Education. She holds a Ph.D. from the University of Oulu, Finland.",institutionString:"Swedish Association for Distance Education, Sweden",institution:null},editorTwo:null,editorThree:null},{id:"94",title:"Climate Change and Environmental Sustainability",coverUrl:"https://cdn.intechopen.com/series_topics/covers/94.jpg",isOpenForSubmission:!0,editor:{id:"61855",title:"Dr.",name:"Yixin",middleName:null,surname:"Zhang",slug:"yixin-zhang",fullName:"Yixin Zhang",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002aYWJgQAO/Profile_Picture_2022-06-09T11:36:35.jpg",biography:"Professor Yixin Zhang is an aquatic ecologist with over 30 years of research and teaching experience in three continents (Asia, Europe, and North America) in Stream Ecology, Riparian Ecology, Urban Ecology, and Ecosystem Restoration and Aquatic Conservation, Human-Nature Interactions and Sustainability, Urbanization Impact on Aquatic Ecosystems. He got his Ph.D. in Animal Ecology at Umeå University in Sweden in 1998. He conducted postdoc research in stream ecology at the University of California at Santa Barbara in the USA. After that, he was a postdoc research fellow at the University of British Columbia in Canada to do research on large-scale stream experimental manipulation and watershed ecological survey in temperate rainforests of BC. He was a faculty member at the University of Hong Kong to run ecological research projects on aquatic insects, fishes, and newts in Tropical Asian streams. He also conducted research in streams, rivers, and caves in Texas, USA, to study the ecology of macroinvertebrates, big-claw river shrimp, fish, turtles, and bats. Current research interests include trophic flows across ecosystems; watershed impacts of land-use change on biodiversity and ecosystem functioning; ecological civilization and water resource management; urban ecology and urban/rural sustainable development.",institutionString:null,institution:{name:"Soochow University",institutionURL:null,country:{name:"China"}}},editorTwo:null,editorThree:null},{id:"95",title:"Urban Planning and Environmental Management",coverUrl:"https://cdn.intechopen.com/series_topics/covers/95.jpg",isOpenForSubmission:!0,editor:{id:"181079",title:"Dr.",name:"Christoph",middleName:null,surname:"Lüthi",slug:"christoph-luthi",fullName:"Christoph Lüthi",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRHSqQAO/Profile_Picture_2022-04-12T15:51:33.png",biography:"Dr. Christoph Lüthi is an urban infrastructure planner with over 25 years of experience in planning and design of urban infrastructure in middle and low-income countries. He holds a Master’s Degree in Urban Development Planning from the University College of London (UCL), and a Ph.D. in Urban Planning & Engineering from TU Berlin. He has conducted applied research on urban planning and infrastructure issues in over 20 countries in Africa and Asia. In 2005 he joined Eawag-Sandec as Leader of the Strategic Environmental Sanitation Planning Group. Since 2015 he heads the research department Sanitation, Water and Solid Waste for Development (Sandec) at the Swiss Federal Institute of Aquatic Research and Technology (Eawag).",institutionString:"Swiss Federal Institute of Aquatic Science and Technology, Switzerland",institution:{name:"Swiss Federal Institute of Aquatic Science and Technology",institutionURL:null,country:{name:"Switzerland"}}},editorTwo:{id:"290571",title:"Dr.",name:"Rui Alexandre",middleName:null,surname:"Castanho",slug:"rui-alexandre-castanho",fullName:"Rui Alexandre Castanho",profilePictureURL:"https://mts.intechopen.com/storage/users/290571/images/system/290571.jpg",biography:"Rui Alexandre Castanho has a master\\'s degree in Planning, Audit, and Control in Urban Green Spaces and an international Ph.D. in Sustainable Planning in Borderlands. Currently, he is a professor at WSB University, Poland, and a visiting professor at the University of Johannesburg, South Africa. Dr. Castanho is a post-doc researcher on the GREAT Project, University of Azores, Ponta Delgada, Portugal. He collaborates with the Environmental Resources Analysis Research Group (ARAM), University of Extremadura (UEx), Spain; VALORIZA - Research Center for the Enhancement of Endogenous Resources, Polytechnic Institute of Portalegre (IPP), Portugal; Centre for Tourism Research, Development and Innovation (CITUR), Madeira, Portugal; and AQUAGEO Research Group, University of Campinas (UNICAMP), Brazil.",institutionString:"University of Johannesburg, South Africa and WSB University, Poland",institution:{name:"University of Johannesburg",institutionURL:null,country:{name:"South Africa"}}},editorThree:null}]},overviewPageOFChapters:{paginationCount:9,paginationItems:[{id:"82936",title:"Soil Degradation Processes Linked to Long-Term Forest-Type Damage",doi:"10.5772/intechopen.106390",signatures:"Pavel Samec, Aleš Kučera and Gabriela Tomášová",slug:"soil-degradation-processes-linked-to-long-term-forest-type-damage",totalDownloads:2,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Forest Degradation Under Global Change",coverURL:"https://cdn.intechopen.com/books/images_new/11457.jpg",subseries:{id:"94",title:"Climate Change and Environmental Sustainability"}}},{id:"82777",title:"Sustainability and Social Investment: Community Microhydropower Systems in the Dominican Republic",doi:"10.5772/intechopen.105995",signatures:"Michela Izzo, Alberto Sánchez and Rafael Fonseca",slug:"sustainability-and-social-investment-community-microhydropower-systems-in-the-dominican-republic",totalDownloads:4,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Globalization and Sustainability - Recent Advances, New Perspectives and Emerging Issues",coverURL:"https://cdn.intechopen.com/books/images_new/11476.jpg",subseries:{id:"91",title:"Sustainable Economy and Fair Society"}}},{id:"82387",title:"Kept Promises? The Evolution of the EU Financial Contribution to Climate Change",doi:"10.5772/intechopen.105541",signatures:"Cecilia Camporeale, Roberto Del Ciello and Mario Jorizzo",slug:"kept-promises-the-evolution-of-the-eu-financial-contribution-to-climate-change",totalDownloads:11,totalCrossrefCites:0,totalDimensionsCites:0,authors:[{name:"Mario",surname:"Jorizzo"},{name:"Cecilia",surname:"Camporeale"},{name:"ROBERTO",surname:"DEL CIELLO"}],book:{title:"Globalization and Sustainability - Recent Advances, New Perspectives and Emerging Issues",coverURL:"https://cdn.intechopen.com/books/images_new/11476.jpg",subseries:{id:"91",title:"Sustainable Economy and Fair Society"}}},{id:"82524",title:"Italy’s Small Exporting Companies: Globalization and Sustainability Issues",doi:"10.5772/intechopen.105542",signatures:"Roberta Pace and Francesca Mandanici",slug:"italy-s-small-exporting-companies-globalization-and-sustainability-issues",totalDownloads:13,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Globalization and Sustainability - Recent Advances, New Perspectives and Emerging Issues",coverURL:"https://cdn.intechopen.com/books/images_new/11476.jpg",subseries:{id:"91",title:"Sustainable Economy and Fair Society"}}}]},overviewPagePublishedBooks:{paginationCount:1,paginationItems:[{type:"book",id:"10897",title:"Food Systems Resilience",subtitle:null,coverURL:"https://cdn.intechopen.com/books/images_new/10897.jpg",slug:"food-systems-resilience",publishedDate:"July 13th 2022",editedByType:"Edited by",bookSignature:"Ana I. 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He has 19 publications in indexed international journals (ISIS), as well as over 60 publications and oral presentations in both Portuguese and international journals and congresses.",institutionString:"University of Trás-os-Montes and Alto Douro",institution:{name:"University of Trás-os-Montes and Alto Douro",country:{name:"Portugal"}}},{id:"38652",title:"Prof.",name:"Rita",middleName:null,surname:"Payan-Carreira",slug:"rita-payan-carreira",fullName:"Rita Payan-Carreira",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRiFPQA0/Profile_Picture_1614601496313",biography:"Rita Payan Carreira earned her Veterinary Degree from the Faculty of Veterinary Medicine in Lisbon, Portugal, in 1985. She obtained her Ph.D. in Veterinary Sciences from the University of Trás-os-Montes e Alto Douro, Portugal. After almost 32 years of teaching at the University of Trás-os-Montes and Alto Douro, she recently moved to the University of Évora, Department of Veterinary Medicine, where she teaches in the field of Animal Reproduction and Clinics. Her primary research areas include the molecular markers of the endometrial cycle and the embryo–maternal interaction, including oxidative stress and the reproductive physiology and disorders of sexual development, besides the molecular determinants of male and female fertility. She often supervises students preparing their master's or doctoral theses. She is also a frequent referee for various journals.",institutionString:null,institution:{name:"University of Évora",country:{name:"Portugal"}}},{id:"283019",title:"Dr.",name:"Oudessa",middleName:null,surname:"Kerro Dego",slug:"oudessa-kerro-dego",fullName:"Oudessa Kerro Dego",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/283019/images/system/283019.png",biography:"Dr. Kerro Dego is a veterinary microbiologist with training in veterinary medicine, microbiology, and anatomic pathology. Dr. Kerro Dego is an assistant professor of dairy health in the department of animal science, the University of Tennessee, Institute of Agriculture, Knoxville, Tennessee. He received his D.V.M. (1997), M.S. (2002), and Ph.D. (2008) degrees in Veterinary Medicine, Animal Pathology and Veterinary Microbiology from College of Veterinary Medicine, Addis Ababa University, Ethiopia; College of Veterinary Medicine, Utrecht University, the Netherlands and Western College of Veterinary Medicine, University of Saskatchewan, Canada respectively. He did his Postdoctoral training in microbial pathogenesis (2009 - 2015) in the Department of Animal Science, the University of Tennessee, Institute of Agriculture, Knoxville, Tennessee. Dr. Kerro Dego’s research focuses on the prevention and control of infectious diseases of farm animals, particularly mastitis, improving dairy food safety, and mitigation of antimicrobial resistance. Dr. Kerro Dego has extensive experience in studying the pathogenesis of bacterial infections, identification of virulence factors, and vaccine development and efficacy testing against major bacterial mastitis pathogens. Dr. Kerro Dego conducted numerous controlled experimental and field vaccine efficacy studies, vaccination, and evaluation of immunological responses in several species of animals, including rodents (mice) and large animals (bovine and ovine).",institutionString:"University of Tennessee at Knoxville",institution:{name:"University of Tennessee at Knoxville",country:{name:"United States of America"}}},{id:"251314",title:"Dr.",name:"Juan Carlos",middleName:null,surname:"Gardón Poggi",slug:"juan-carlos-gardon-poggi",fullName:"Juan Carlos Gardón Poggi",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/251314/images/system/251314.jpeg",biography:"Juan Carlos Gardón Poggi received University degree from the Faculty of Agrarian Science in Argentina, in 1983. Also he received Masters Degree and PhD from Córdoba University, Spain. He is currently a Professor at the Catholic University of Valencia San Vicente Mártir, at the Department of Medicine and Animal Surgery. He teaches diverse courses in the field of Animal Reproduction and he is the Director of the Veterinary Farm. He also participates in academic postgraduate activities at the Veterinary Faculty of Murcia University, Spain. His research areas include animal physiology, physiology and biotechnology of reproduction either in males or females, the study of gametes under in vitro conditions and the use of ultrasound as a complement to physiological studies and development of applied biotechnologies. Routinely, he supervises students preparing their doctoral, master thesis or final degree projects.",institutionString:null,institution:{name:"Valencia Catholic University Saint Vincent Martyr",country:{name:"Spain"}}},{id:"309529",title:"Dr.",name:"Albert",middleName:null,surname:"Rizvanov",slug:"albert-rizvanov",fullName:"Albert Rizvanov",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/309529/images/9189_n.jpg",biography:'Albert A. Rizvanov is a Professor and Director of the Center for Precision and Regenerative Medicine at the Institute of Fundamental Medicine and Biology, Kazan Federal University (KFU), Russia. He is the Head of the Center of Excellence “Regenerative Medicine” and Vice-Director of Strategic Academic Unit \\"Translational 7P Medicine\\". Albert completed his Ph.D. at the University of Nevada, Reno, USA and Dr.Sci. at KFU. He is a corresponding member of the Tatarstan Academy of Sciences, Russian Federation. Albert is an author of more than 300 peer-reviewed journal articles and 22 patents. He has supervised 11 Ph.D. and 2 Dr.Sci. dissertations. Albert is the Head of the Dissertation Committee on Biochemistry, Microbiology, and Genetics at KFU.\nORCID https://orcid.org/0000-0002-9427-5739\nWebsite https://kpfu.ru/Albert.Rizvanov?p_lang=2',institutionString:"Kazan Federal University",institution:{name:"Kazan Federal University",country:{name:"Russia"}}},{id:"210551",title:"Dr.",name:"Arbab",middleName:null,surname:"Sikandar",slug:"arbab-sikandar",fullName:"Arbab Sikandar",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/210551/images/system/210551.jpg",biography:"Dr. Arbab Sikandar, PhD, M. Phil, DVM was born on April 05, 1981. He is currently working at the College of Veterinary & Animal Sciences as an Assistant Professor. He previously worked as a lecturer at the same University. \nHe is a Member/Secretory of Ethics committee (No. CVAS-9377 dated 18-04-18), Member of the QEC committee CVAS, Jhang (Regr/Gen/69/873, dated 26-10-2017), Member, Board of studies of Department of Basic Sciences (No. CVAS. 2851 Dated. 12-04-13, and No. CVAS, 9024 dated 20/11/17), Member of Academic Committee, CVAS, Jhang (No. CVAS/2004, Dated, 25-08-12), Member of the technical committee (No. CVAS/ 4085, dated 20,03, 2010 till 2016).\n\nDr. Arbab Sikandar contributed in five days hands-on-training on Histopathology at the Department of Pathology, UVAS from 12-16 June 2017. He received a Certificate of appreciation for contributions for Popularization of Science and Technology in the Society on 17-11-15. He was the resource person in the lecture series- ‘scientific writing’ at the Department of Anatomy and Histology, UVAS, Lahore on 29th October 2015. He won a full fellowship as a principal candidate for the year 2015 in the field of Agriculture, EICA, Egypt with ref. to the Notification No. 12(11) ACS/Egypt/2014 from 10 July 2015 to 25th September 2015.; he received a grant of Rs. 55000/- as research incentives from Director, Advanced Studies and Research, UVAS, Lahore upon publications of research papers in IF Journals (DR/215, dated 19-5-2014.. He obtained his PhD by winning a HEC Pakistan indigenous Scholarship, ‘Ph.D. fellowship for 5000 scholars – Phase II’ (2av1-147), 17-6/HEC/HRD/IS-II/12, November 15, 2012. \n\nDr. Sikandar is a member of numerous societies: Registered Veterinary Medical Practitioner (life member) and Registered Veterinary Medical Faculty of Pakistan Veterinary Medical Council. The Registration code of PVMC is RVMP/4298 and RVMF/ 0102.; Life member of the University of Veterinary and Animal Sciences, Lahore, Alumni Association with S# 664, dated: 6-4-12. ; Member 'Vets Care Organization Pakistan” with Reference No. VCO-605-149, dated 05-04-06. :Member 'Vet Crescent” (Society of Animal Health and Production), UVAS, Lahore.",institutionString:"University of Veterinary & Animal Science",institution:{name:"University of Veterinary and Animal Sciences",country:{name:"Pakistan"}}},{id:"311663",title:"Dr.",name:"Prasanna",middleName:null,surname:"Pal",slug:"prasanna-pal",fullName:"Prasanna Pal",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/311663/images/13261_n.jpg",biography:null,institutionString:null,institution:{name:"National Dairy Research Institute",country:{name:"India"}}},{id:"202192",title:"Dr.",name:"Catrin",middleName:null,surname:"Rutland",slug:"catrin-rutland",fullName:"Catrin Rutland",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/202192/images/system/202192.png",biography:"Catrin Rutland is an Associate Professor of Anatomy and Developmental Genetics at the University of Nottingham, UK. She obtained a BSc from the University of Derby, England, a master’s degree from Technische Universität München, Germany, and a Ph.D. from the University of Nottingham. She undertook a post-doctoral research fellowship in the School of Medicine before accepting tenure in Veterinary Medicine and Science. Dr. Rutland also obtained an MMedSci (Medical Education) and a Postgraduate Certificate in Higher Education (PGCHE). She is the author of more than sixty peer-reviewed journal articles, twelve books/book chapters, and more than 100 research abstracts in cardiovascular biology and oncology. She is a board member of the European Association of Veterinary Anatomists, Fellow of the Anatomical Society, and Senior Fellow of the Higher Education Academy. Dr. Rutland has also written popular science books for the public. https://orcid.org/0000-0002-2009-4898. www.nottingham.ac.uk/vet/people/catrin.rutland",institutionString:null,institution:{name:"University of Nottingham",country:{name:"United Kingdom"}}},{id:"283315",title:"Prof.",name:"Samir",middleName:null,surname:"El-Gendy",slug:"samir-el-gendy",fullName:"Samir El-Gendy",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRduYQAS/Profile_Picture_1606215849748",biography:"Samir El-Gendy is a Professor of anatomy and embryology at the faculty of veterinary medicine, Alexandria University, Egypt. Samir obtained his PhD in veterinary science in 2007 from the faculty of veterinary medicine, Alexandria University and has been a professor since 2017. Samir is an author on 24 articles at Scopus and 12 articles within local journals and 2 books/book chapters. His research focuses on applied anatomy, imaging techniques and computed tomography. Samir worked as a member of different local projects on E-learning and he is a board member of the African Association of Veterinary Anatomists and of anatomy societies and as an associated author at local and international journals. Orcid: https://orcid.org/0000-0002-6180-389X",institutionString:null,institution:{name:"Alexandria University",country:{name:"Egypt"}}},{id:"246149",title:"Dr.",name:"Valentina",middleName:null,surname:"Kubale",slug:"valentina-kubale",fullName:"Valentina Kubale",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/246149/images/system/246149.jpg",biography:"Valentina Kubale is Associate Professor of Veterinary Medicine at the Veterinary Faculty, University of Ljubljana, Slovenia. Since graduating from the Veterinary faculty she obtained her PhD in 2007, performed collaboration with the Department of Pharmacology, University of Copenhagen, Denmark. She continued as a post-doctoral fellow at the University of Copenhagen with a Lundbeck foundation fellowship. She is the editor of three books and author/coauthor of 23 articles in peer-reviewed scientific journals, 16 book chapters, and 68 communications at scientific congresses. Since 2008 she has been the Editor Assistant for the Slovenian Veterinary Research journal. She is a member of Slovenian Biochemical Society, The Endocrine Society, European Association of Veterinary Anatomists and Society for Laboratory Animals, where she is board member.",institutionString:"University of Ljubljana",institution:{name:"University of Ljubljana",country:{name:"Slovenia"}}},{id:"258334",title:"Dr.",name:"Carlos Eduardo",middleName:null,surname:"Fonseca-Alves",slug:"carlos-eduardo-fonseca-alves",fullName:"Carlos Eduardo Fonseca-Alves",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/258334/images/system/258334.jpg",biography:"Dr. Fonseca-Alves earned his DVM from Federal University of Goias – UFG in 2008. He completed an internship in small animal internal medicine at UPIS university in 2011, earned his MSc in 2013 and PhD in 2015 both in Veterinary Medicine at Sao Paulo State University – UNESP. Dr. Fonseca-Alves currently serves as an Assistant Professor at Paulista University – UNIP teaching small animal internal medicine.",institutionString:null,institution:{name:"Universidade Paulista",country:{name:"Brazil"}}},{id:"245306",title:"Dr.",name:"María Luz",middleName:null,surname:"Garcia Pardo",slug:"maria-luz-garcia-pardo",fullName:"María Luz Garcia Pardo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/245306/images/system/245306.png",biography:"María de la Luz García Pardo is an agricultural engineer from Universitat Politècnica de València, Spain. She has a Ph.D. in Animal Genetics. Currently, she is a lecturer at the Agrofood Technology Department of Miguel Hernández University, Spain. Her research is focused on genetics and reproduction in rabbits. The major goal of her research is the genetics of litter size through novel methods such as selection by the environmental sensibility of litter size, with forays into the field of animal welfare by analysing the impact on the susceptibility to diseases and stress of the does. Details of her publications can be found at https://orcid.org/0000-0001-9504-8290.",institutionString:null,institution:{name:"Miguel Hernandez University",country:{name:"Spain"}}},{id:"350704",title:"M.Sc.",name:"Camila",middleName:"Silva Costa",surname:"Ferreira",slug:"camila-ferreira",fullName:"Camila Ferreira",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/350704/images/17280_n.jpg",biography:"Graduated in Veterinary Medicine at the Fluminense Federal University, specialist in Equine Reproduction at the Brazilian Veterinary Institute (IBVET) and Master in Clinical Veterinary Medicine and Animal Reproduction at the Fluminense Federal University. She has experience in analyzing zootechnical indices in dairy cattle and organizing events related to Veterinary Medicine through extension grants. I have experience in the field of diagnostic imaging and animal reproduction in veterinary medicine through monitoring and scientific initiation scholarships. I worked at the Equus Central Reproduction Equine located in Santo Antônio de Jesus – BA in the 2016/2017 breeding season. I am currently a doctoral student with a scholarship from CAPES of the Postgraduate Program in Veterinary Medicine (Pathology and Clinical Sciences) at the Federal Rural University of Rio de Janeiro (UFRRJ) with a research project with an emphasis on equine endometritis.",institutionString:null,institution:null},{id:"41319",title:"Prof.",name:"Lung-Kwang",middleName:null,surname:"Pan",slug:"lung-kwang-pan",fullName:"Lung-Kwang Pan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/41319/images/84_n.jpg",biography:null,institutionString:null,institution:null},{id:"125292",title:"Dr.",name:"Katy",middleName:null,surname:"Satué Ambrojo",slug:"katy-satue-ambrojo",fullName:"Katy Satué Ambrojo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/125292/images/system/125292.jpeg",biography:"Katy Satué Ambrojo received her Veterinary Medicine degree, Master degree in Equine Technology and doctorate in Veterinary Medicine from the Faculty of Veterinary, CEU-Cardenal Herrera University in Valencia, Spain.Dr. Satué is accredited as a Private University Doctor Professor, Doctor Assistant, and Contracted Doctor by AVAP (Agència Valenciana d'Avaluació i Prospectiva) and currently, as a full professor by ANECA (since January 2022). To date, Katy has taught 22 years in the Department of Animal Medicine and Surgery at the CEU-Cardenal Herrera University in undergraduate courses in Veterinary Medicine (General Pathology, integrated into the Applied Basis of Veterinary Medicine module of the 2nd year, Clinical Equine I of 3rd year, and Equine Clinic II of 4th year). Dr. Satué research activity is in the field of Endocrinology, Hematology, Biochemistry, and Immunology in the Spanish Purebred mare. She has directed 5 Doctoral Theses and 5 Diplomas of Advanced Studies, and participated in 11 research projects as a collaborating researcher. She has written 2 books and 14 book chapters in international publishers related to the area, and 68 scientific publications in international journals. Dr. Satué has attended 63 congresses, participating with 132 communications in international congresses and 19 in national congresses related to the area. Dr. Satué is a scientific reviewer for various prestigious international journals such as Animals, American Journal of Obstetrics and Gynecology, Veterinary Clinical Pathology, Journal of Equine Veterinary Science, Reproduction in Domestic Animals, Research Veterinary Science, Brazilian Journal of Medical and Biological Research, Livestock Production Science and Theriogenology, among others. Since 2014 she has been responsible for the Clinical Analysis Laboratory of the CEU-Cardenal Herrera University Veterinary Clinical Hospital.",institutionString:null,institution:null},{id:"201721",title:"Dr.",name:"Beatrice",middleName:null,surname:"Funiciello",slug:"beatrice-funiciello",fullName:"Beatrice Funiciello",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/201721/images/11089_n.jpg",biography:"Graduated from the University of Milan in 2011, my post-graduate education included CertAVP modules mainly on equines (dermatology and internal medicine) and a few on small animal (dermatology and anaesthesia) at the University of Liverpool. After a general CertAVP (2015) I gained the designated Certificate in Veterinary Dermatology (2017) after taking the synoptic examination and then applied for the RCVS ADvanced Practitioner status. After that, I completed the Postgraduate Diploma in Veterinary Professional Studies at the University of Liverpool (2018). My main area of work is cross-species veterinary dermatology.",institutionString:null,institution:null},{id:"291226",title:"Dr.",name:"Monica",middleName:null,surname:"Cassel",slug:"monica-cassel",fullName:"Monica Cassel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/291226/images/8232_n.jpg",biography:'Degree in Biological Sciences at the Federal University of Mato Grosso with scholarship for Scientific Initiation by FAPEMAT (2008/1) and CNPq (2008/2-2009/2): Project \\"Histological evidence of reproductive activity in lizards of the Manso region, Chapada dos Guimarães, Mato Grosso, Brazil\\". Master\\\'s degree in Ecology and Biodiversity Conservation at Federal University of Mato Grosso with a scholarship by CAPES/REUNI program: Project \\"Reproductive biology of Melanorivulus punctatus\\". PhD\\\'s degree in Science (Cell and Tissue Biology Area) \n at University of Sao Paulo with scholarship granted by FAPESP; Project \\"Development of morphofunctional changes in ovary of Astyanax altiparanae Garutti & Britski, 2000 (Teleostei, Characidae)\\". She has experience in Reproduction of vertebrates and Morphology, with emphasis in Cellular Biology and Histology. She is currently a teacher in the medium / technical level courses at IFMT-Alta Floresta, as well as in the Bachelor\\\'s degree in Animal Science and in the Bachelor\\\'s degree in Business.',institutionString:null,institution:null},{id:"442807",title:"Dr.",name:"Busani",middleName:null,surname:"Moyo",slug:"busani-moyo",fullName:"Busani Moyo",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Gwanda State University",country:{name:"Zimbabwe"}}},{id:"439435",title:"Dr.",name:"Feda S.",middleName:null,surname:"Aljaser",slug:"feda-s.-aljaser",fullName:"Feda S. Aljaser",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"King Saud University",country:{name:"Saudi Arabia"}}},{id:"423023",title:"Dr.",name:"Yosra",middleName:null,surname:"Soltan",slug:"yosra-soltan",fullName:"Yosra Soltan",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Alexandria University",country:{name:"Egypt"}}},{id:"349788",title:"Dr.",name:"Florencia Nery",middleName:null,surname:"Sompie",slug:"florencia-nery-sompie",fullName:"Florencia Nery Sompie",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Sam Ratulangi University",country:{name:"Indonesia"}}},{id:"428600",title:"MSc.",name:"Adriana",middleName:null,surname:"García-Alarcón",slug:"adriana-garcia-alarcon",fullName:"Adriana García-Alarcón",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}},{id:"428599",title:"MSc.",name:"Gabino",middleName:null,surname:"De La Rosa-Cruz",slug:"gabino-de-la-rosa-cruz",fullName:"Gabino De La Rosa-Cruz",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}},{id:"428601",title:"MSc.",name:"Juan Carlos",middleName:null,surname:"Campuzano-Caballero",slug:"juan-carlos-campuzano-caballero",fullName:"Juan Carlos Campuzano-Caballero",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}}]}},subseries:{item:{id:"10",type:"subseries",title:"Animal Physiology",keywords:"Physiology, Comparative, Evolution, Biomolecules, Organ, Homeostasis, Anatomy, Pathology, Medical, Cell Division, Cell Signaling, Cell Growth, Cell Metabolism, Endocrine, Neuroscience, Cardiovascular, Development, Aging, Development",scope:"Physiology, the scientific study of functions and mechanisms of living systems, is an essential area of research in its own right, but also in relation to medicine and health sciences. The scope of this topic will range from molecular, biochemical, cellular, and physiological processes in all animal species. Work pertaining to the whole organism, organ systems, individual organs and tissues, cells, and biomolecules will be included. Medical, animal, cell, and comparative physiology and allied fields such as anatomy, histology, and pathology with physiology links will be covered in this topic. Physiology research may be linked to development, aging, environment, regular and pathological processes, adaptation and evolution, exercise, or several other factors affecting, or involved with, animal physiology.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/10.jpg",hasOnlineFirst:!1,hasPublishedBooks:!1,annualVolume:11406,editor:{id:"202192",title:"Dr.",name:"Catrin",middleName:null,surname:"Rutland",slug:"catrin-rutland",fullName:"Catrin Rutland",profilePictureURL:"https://mts.intechopen.com/storage/users/202192/images/system/202192.png",biography:"Catrin Rutland is an Associate Professor of Anatomy and Developmental Genetics at the University of Nottingham, UK. She obtained a BSc from the University of Derby, England, a master’s degree from Technische Universität München, Germany, and a Ph.D. from the University of Nottingham. She undertook a post-doctoral research fellowship in the School of Medicine before accepting tenure in Veterinary Medicine and Science. Dr. Rutland also obtained an MMedSci (Medical Education) and a Postgraduate Certificate in Higher Education (PGCHE). She is the author of more than sixty peer-reviewed journal articles, twelve books/book chapters, and more than 100 research abstracts in cardiovascular biology and oncology. She is a board member of the European Association of Veterinary Anatomists, Fellow of the Anatomical Society, and Senior Fellow of the Higher Education Academy. Dr. Rutland has also written popular science books for the public. https://orcid.org/0000-0002-2009-4898. www.nottingham.ac.uk/vet/people/catrin.rutland",institutionString:null,institution:{name:"University of Nottingham",institutionURL:null,country:{name:"United Kingdom"}}},editorTwo:null,editorThree:null,series:{id:"10",title:"Physiology",doi:"10.5772/intechopen.72796",issn:"2631-8261"},editorialBoard:[{id:"306970",title:"Mr.",name:"Amin",middleName:null,surname:"Tamadon",slug:"amin-tamadon",fullName:"Amin Tamadon",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002oHR5wQAG/Profile_Picture_1623910304139",institutionString:null,institution:{name:"Bushehr University of Medical Sciences",institutionURL:null,country:{name:"Iran"}}},{id:"251314",title:"Dr.",name:"Juan Carlos",middleName:null,surname:"Gardón Poggi",slug:"juan-carlos-gardon-poggi",fullName:"Juan Carlos Gardón Poggi",profilePictureURL:"https://mts.intechopen.com/storage/users/251314/images/system/251314.jpeg",institutionString:null,institution:{name:"Valencia Catholic University Saint Vincent Martyr",institutionURL:null,country:{name:"Spain"}}},{id:"245306",title:"Dr.",name:"María Luz",middleName:null,surname:"Garcia Pardo",slug:"maria-luz-garcia-pardo",fullName:"María Luz Garcia Pardo",profilePictureURL:"https://mts.intechopen.com/storage/users/245306/images/system/245306.png",institutionString:null,institution:{name:"Miguel Hernandez University",institutionURL:null,country:{name:"Spain"}}},{id:"283315",title:"Prof.",name:"Samir",middleName:null,surname:"El-Gendy",slug:"samir-el-gendy",fullName:"Samir El-Gendy",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRduYQAS/Profile_Picture_1606215849748",institutionString:null,institution:{name:"Alexandria 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