\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
\n'}],latestNews:[{slug:"intechopen-partners-with-ehs-for-digital-advertising-representation-20210416",title:"IntechOpen Partners with EHS for Digital Advertising Representation"},{slug:"intechopen-signs-new-contract-with-cepiec-china-for-distribution-of-open-access-books-20210319",title:"IntechOpen Signs New Contract with CEPIEC, China for Distribution of Open Access Books"},{slug:"150-million-downloads-and-counting-20210316",title:"150 Million Downloads and Counting"},{slug:"intechopen-secures-indefinite-content-preservation-with-clockss-20210309",title:"IntechOpen Secures Indefinite Content Preservation with CLOCKSS"},{slug:"intechopen-expands-to-all-global-amazon-channels-with-full-catalog-of-books-20210308",title:"IntechOpen Expands to All Global Amazon Channels with Full Catalog of Books"},{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"}]},book:{item:{type:"book",id:"650",leadTitle:null,fullTitle:"Pathogenesis and Treatment of Periodontitis",title:"Pathogenesis and Treatment of Periodontitis",subtitle:null,reviewType:"peer-reviewed",abstract:"Pathogenesis and Treatment of Periodontitis includes comprehensive reviews on etiopathogenic factors of periodontal tissue destruction related to microbial dental plaque and also host response components. Adjunctive treatment modalities are also addressed in the book. Topics covered range from microbial pathogenic factors of P. gingivalis to the relationship between metabolic syndrome and periodontal disease, and from management of open gingival embrasures to laser application in periodontal treatment.",isbn:null,printIsbn:"978-953-307-924-0",pdfIsbn:"978-953-51-6763-1",doi:"10.5772/1159",price:119,priceEur:129,priceUsd:155,slug:"pathogenesis-and-treatment-of-periodontitis",numberOfPages:214,isOpenForSubmission:!1,isInWos:1,hash:"18d0e34fc1e3a4ee31d85ea586a3f8e1",bookSignature:"Nurcan Buduneli",publishedDate:"January 20th 2012",coverURL:"https://cdn.intechopen.com/books/images_new/650.jpg",numberOfDownloads:48587,numberOfWosCitations:11,numberOfCrossrefCitations:6,numberOfDimensionsCitations:20,hasAltmetrics:0,numberOfTotalCitations:37,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 24th 2011",dateEndSecondStepPublish:"April 21st 2011",dateEndThirdStepPublish:"August 26th 2011",dateEndFourthStepPublish:"September 25th 2011",dateEndFifthStepPublish:"January 23rd 2012",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6",editedByType:"Edited by",kuFlag:!1,editors:[{id:"25979",title:"Prof.",name:"Nurcan",middleName:null,surname:"Buduneli",slug:"nurcan-buduneli",fullName:"Nurcan Buduneli",profilePictureURL:"https://mts.intechopen.com/storage/users/25979/images/system/25979.jpg",biography:"Nurcan BUDUNELİ DDS, PhD, Professor (Ege University, Turkey)\nDr.BUDUNELİ has received her DDS degree in 1990 from Ege University, İzmir, Turkey. She was a visitor in Dental School, Bristol University, UK in 1992. She did her PhD in Periodontology at the same Institute on immune defence systems acting in periodontal diseases, defended her PhD in 1996. She did her post-doc study in 1997 in Copenhagen University, Denmark. She was a guest researcher in Louisville University, KY, USA in 2004. Her main interests are aetiology and mechanisms of interactions between periodontal diseases and systemic diseases/conditions such as diabetes, rheumatoid arthritis, respiratory diseases and pregnancy complications like pre-term low birth weight. Drug-induced gingival overgrowth, experimental periodontitis studies, development of local drug delivery formulations, effects of smoking on periodontal health have also been her research areas. She has published over 116 papers in well-known scientific journals. She has been giving undergraduate and post-graduate courses on epidemiology of periodontal diseases; pathogenesis, diagnosis and prognosis of periodontal diseases; drugs used in periodontal treatment. She is married and has two kids.",institutionString:"Ege University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"Ege University",institutionURL:null,country:{name:"Turkey"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"1000",title:"Periodontology",slug:"periodontology"}],chapters:[{id:"26474",title:"Pathogenic Factors of P. gingivalis and the Host Defense Mechanisms",doi:"10.5772/32150",slug:"pathogenic-factors-of-p-gingivalis-and-the-host-defence-mechanisms",totalDownloads:4034,totalCrossrefCites:0,totalDimensionsCites:2,signatures:"Shigenobu Kimura, Yuko Ohara-Nemoto, Yu Shimoyama, Taichi Ishikawa and Minoru 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Cryopreservation biotechnology has important roles for aquaculture industry and also for conservation of aquatic genetic resources. In this field, sperm cryopreservation has been used for transporting of genetic material between facilities, optimal using of gametes in aquaculture, reducing risk of spreading infections, performing of hybridization studies, conserving of protecting endangered species, and also for conserving of biodiversity [1, 2].
\nCryopreservation technique involves addition of cryoprotectants to the extender and freezing and thawing of sperm samples, which may result in some damage to the spermatozoa and may decrease egg fertilization rate. Therefore, before cryopreservation of spermatozoa, a thorough evaluation of different extender solutions, cryoprotectants, and cooling and thawing rates are essential to develop optimum cryopreservation protocol for various species [3–5].
\nDuring the cryopreservation process, some factors may change the physiological status of sperm. The success of cryopreservation depends not only on preserving the motility of the spermatozoa but also on maintaining their metabolic functions [6]. Extender composition and cryoprotectant concentration are the main factors affecting cryopreservation success [7]. Extenders are required for dilution of fish sperm prior to cryopreservation and are generally designed to be compatible with the physiochemical composition of the fish seminal plasma. Most important function of the extenders is to maintain the spermatozoa in immotile state until required [8].
\nCryoprotectants are added to the extenders to protect the cells against ice crystal formation during freezing and thawing [9]. Although cryoprotectants help to the prevention of cryoinjuries during freezing and thawing, they may become toxic to the cells when exposure time and concentration are increased [10, 11]. Thus, one of the most critical steps in successful cryopreservation of fish semen is the choice of the cryoprotectant and its ratio in the extender during the process.
\nAnother important problem is the handling of sperm produced in small volumes by some fish species such as tilapia. In spite of packaging of sperm in traditional 0.25-mL and 0.5-mL straws has been successfully applied to freeze semen of the most fish species and to fertilize small egg batches [9], there is a lack of information regarding their usage in cryopreservation of Nile tilapia (
The Nile tilapia is one of the most cultivated freshwater fish species in the world aquaculture [12]. This species has great breeding potential due to its hardiness against worse environmental conditions, fast growth rate, adaptation to different environmental conditions (e.g. salinity, temperature), and also good organoleptic characteristics of its flesh [13, 14]. On the other hand, most of studies related with fish sperm cryopreservation have focused on some freshwater species, such as cyprinids [15, 16], salmonids [17, 18], catfishes [19, 20], and loach [21].
\nEven though many successes have been achieved in fish semen cryopreservation, the technique remains as a method that is difficult to standardize and use in all types of fishes. This is due to the fact that cryopreservation of sperm from different fish species required different conditions, where the protocol needs to be established individually [22]. To the best of our knowledge, there is limited information regarding cryopreservation of Nile tilapia sperm. In this concept, the effect of cryoprotectants and packaging methods on freezability and also on post-thaw quality of Nile tilapia sperm still remains unclear. Thus, standardization and simplification of cryopreservation procedure for Nile tilapia sperm are needed for commercial and gene bank applications.
\nThe main aim of this study was to establish an efficient method for cryopreservation of Nile tilapia sperm that can be applied to aquaculture of this species. The present experiment was designed to study the straw volume (0.25 vs 0.5 mL) on Nile tilapia sperm quality after cryopreservation using glucose-Tris–based solution containing 10% dimethylacetamide.
\nThe additives and other chemicals used in this study were obtained from local representative of Sigma-Aldrich Chemicals Company (St. Louis, MO, USA).
\nThe experiments were carried out spawning season of the Nile tilapia. In the pre-spawning period, sexually mature male (n=15) and female (n=5) Nile tilapia were pit-tagged and kept separately in 150 L indoor tanks under constant environmental conditions. The broodstock tanks were provided with freshwater constantly at ratio of 1.5 L/s, while compressed air was provided trough air stones. The water temperature ranged from 27.2°C to 30.5°C, and salinity was maintained at 1.5 ppm. Nile tilapia was fed with floating pellets twice daily (1–5% body weight per day).
\nGametes were collected from healthy mature males and females following immersion anesthetization with 10 ppm quinaldine (Reanal Ltd., Budapest, Hungary) for a few minutes. For sperm collection, 1-mL tuberculin plastic syringe, without needle, was used to aspirate sperm released by gentle abdominal massage to eliminate urine in the ducts. Following, sperm samples were transferred individually into 1.5-mL Eppendorf tubes on ice (0–4°C). A 10-μL pipette tip connected to a mouth pipette was used to extract sperm cells, which were diluted 1:1 in Hanks’ balanced salt solution (HBSS) (280 mOsm/kg, pH 7.0) in 1.5-mL microcentrifuge tubes and placed on ice until analysis. Eggs were also collected by gentle abdominal massaging and stored in HBSS at 25°C and used for fertilization within 30 min following stripping [23].
\nOne microlitre sperm of each sample was placed on a microscope slide and observed under a phase-contrast microscope (Olympus, Japan) at 100× magnification. The motility characteristics of the collected sperm samples were evaluated by adding activation solution (AS) (45 mM NaCl, 5 mM KCl, and 30 mM Tris–HCl, pH 8.2) [24] at a ratio of 1:100. Sperm cells that vibrated in place were considered as immotile. Only samples whose quality parameters ranging between the following values were used for the cryopreservation experiment: osmolarity 50–100 mOsm/kg, pH 7.0–8.0, and progressive motility 80–100% [25]. The quality of ova was determined from their morphological features seen under a dissecting microscope as described in the study of Fauvel et al. [26].
\nSemen samples showing ≥80 motility was pooled into equal aliquots and chosen for cryopreservation experiments. Semen and extenders were kept at 4°C immediately under aerobic conditions prior to dilution. Pooled semen was diluted at a ratio of 1:3 with an extender composing 350 mM glucose and 30 mM Tris [27] containing 10% dimethylacetamide (DMA). The diluted semen were drawn into 0.25-mL or 0.5-mL plastic straws (IMV France) and sealed with polyvinyl alcohol (PVA). Following equilibration of semen for 10 min at 4°C, the straws were placed on a styrofoam rack floating on the surface of liquid nitrogen in a styrofoam box. Samples were frozen 3 cm above of the liquid nitrogen surface and exposed to the liquid nitrogen vapor (≈−140°C) for 10 min [28]. Following, frozen sperm cells were kept into the liquid nitrogen container (−196°C) until analyses for a few days.
\nThe frozen sperm in different volume of straws were thawed in a water bath at 30°C for 20 s (0.25-mL straws) or at 30°C for 30 s (0.5-mL straws). Thawed semen was activated using activation solution (AS) (45 mM NaCl, 5 mM KCl, and 30 mM Tris–HCl, pH 8.2) [24] and observed under a phase-contrast microscope (Olympus, Japan) for progressive motility (%), progressive motility duration (s), and viability (%) evaluations (three replicates). At least five straws were used for each parameter evaluation with three replications.
\nThe percent of motile spermatozoa and motility duration was immediately recorded following activation using a CCD video camera (CMEX-5, Netherland) mounted on a phase-contrast microscope (100×, Olympus BX43, Tokyo, Japan) at room temperature (20°C). Progressive spermatozoa motility and duration of progressive spermatozoa motility were evaluated from sperm with forward movement. Immotile spermatozoa were defined as spermatozoa that did not show forward movement after activation. Percentage of spermatozoa motility was determined within 30 s post-activation. Motility duration was evaluated by counting the time from spermatozoa activation until spermatozoa stopped moving. In order to assess viable sperm percentage, eosin-nigrosin preparations were made according to the method described by Bjorndahl et al. [29] and totally 300 sperm cells were counted on each slide at 1000× magnification. At least five straws were used for each evaluation parameter, and analyses were repeated three times for each treatment.
\nPooled eggs from five mature females were used to assess fertilization rates. In this stage, most of the HBSS was decanted from the eggs, and fertilization was carried out in dry Petri dishes (10 cm diameter). Fresh or thawed sperm was added over the eggs and gently mixed before activation with 20 mL of fertilization solution (3 g urea and 4 g NaCl in 1 L distilled water) [30]. Following fertilization process, 2 mL embryo buffer medium (EBM) (13.7 mM NaCl, 5.40 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.30 mM CaCl2, 1.00 mM MgSO4, 4.20 mM NaHCO3 at 52 mOsm/kg and pH 7.0) was added for activation as described by Westerfield [31]. After 10 min, 100 mL EBM was added again over the eggs and was left undisturbed without movement in a convection type incubator at 27°C (Panasonic MCO-19M-PE, Japan). Unfertilized eggs were removed, and EBM was changed twice daily. After 48 h, the eggs were evaluated for fertilization results. Eggs that developed to stage 11 (embryonic keel and somite formation) were recorded as fertilized eggs, described by Galman and Avtalion [32].
\nFertilization experiments were carried out using 1×105:1 spermatozoa/egg ratio with each straw types (0.25 or 0.5 mL) for the each aliquot of eggs (containing 100 eggs). Three straws were thawed for each fertilization treatment (three replications). For the control, another three aliquot of eggs (containing 100 eggs) were fertilized with fresh semen collected from two other males. Eggs were fertilized with fresh semen samples using the same number of sperm cells (1 × 105 cells) similar to treatments with frozen semen.
\nA two-way analysis of variance (ANOVA) including the straw volumes (0.25 and 0.5 mL) as fixed effects was used. Means were separated by Duncan’s multiple range test and were considered at 5% level of significance. Results are presented as mean ± S.D. All analyses were carried out using SPSS 17 for Windows statistical software package.
\nPost-thaw progressive motility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).
Semen volume was rather variable and ranged from 0.9 to 7.5 mL, with a mean volume of 3.6 ± 0.40 mL. Progressive motility was ranged from 60% to 90%, and mean motility was determined as 80.4 ± 0.15%. In addition, mean progressive spermatozoa motility duration (s), spermatozoa density (× 109/mL), viability (%), and pH values were determined as 64.2 ± 0.45 s, 1.75 × 109/mL, 92.5 ± 4.25%, and 7.2 ± 0.25, respectively. In addition, mean fertilization rate was determined with fresh semen, which was 72.5 ± 0.20%. The findings of the present study indicated that cryopreservation of sperm in glucose-Tris–based extender using 0.5-mL straws increased post-thaw progressive motility (Figure 1), duration of progressive motility (Figure 2), and fertility (Figure 4) (P<0.01). On the other hand, differences in terms of post-thaw cell viability were not significant among the treatments (Figure 3, P>0.01). The fertility of the frozen-thawed sperm showed high positive linear correlation with motility (r2=1.000, Figure 5) and (r2=0.9932, Figure 6) in case of using 0.25-mL and 0.5-mL straws.
\n\nPost-thaw progressive motility duration (s) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).
Post-thaw viability (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).
Post-thaw fertility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).
Relationship between post-thaw spermatozoa motility (%) and fertility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender using 0.25-mL straws.
Relationship between post-thaw spermatozoa motility (%) and fertility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender using 0.50-mL straws.
Tilapias are widely cultured in the tropical and subtropical regions of the world. Several species of tilapia are cultured commercially, but Nile tilapia is the predominant cultured species worldwide that its production reached 3,197,330 mt in 2012 [33].
\nAlthough Nile tilapia is a freshwater fish, it can tolerate a wide range of salinity [34]. Therefore, the expansion of its culture in sea and brackishwater has attracted the attention of fish farmers in recent years. However, limited reports have addressed semen cryopreservation in the Nile tilapia. Therefore, standardization and simplification of the cryopreservation procedure for the Nile tilapia sperm is needed for commercial application. On the other hand, because of limited amounts of data are available, comparison of the methods and results have been mainly made with the cyprinid species in this research. From this point of view, findings of this research significantly contribute improving of the protocols applied for the cryopreservation of the Nile tilapia sperm.
\nSperm cryopreservation is an important biotechnological technique with specific advantages to the aquaculture industry. Improvements in semen cryopreservation techniques require in-depth knowledge of gamete physiology and the biochemical processes occurring during semen collection, processing, freezing, and thawing [35].
\nIn spite of routinely using of cryopreserved semen in aquaculture artificial insemination programs worldwide, there are inconsistencies in experimental results [36]. The success of cryopreservation mainly depends on maintaining the spermatozoal metabolic functions [37]. The major factors affecting the results of insemination with frozen/thawed sperm are the type and properties of extenders and cryoprotectants, the damage caused by the formation of internal ice crystals due to the increase in solute concentration in the extension media and the interaction of these factors [38].
\nIn the present study, glucose-Tris–based extender supplemented with DMA was used to cryopreserve Nile tilapia sperm. In spite of using glucose mainly as energetic substrate, it has been used due to its stabilization effects on the spermatozoa liposomal membrane [39]. It should be noted that, carbohydrate-based solutions such as glucose have also been found effective in some experiments [40, 41], and Tris is the most common buffer solution, not only for cyprinidae but also for other fish species [42].
\nCryoprotectants are added to the extenders in order to protect the spermatozoa from damages during the freezing/thawing process. On the other hand, cryoprotectants can suppress most cryoinjuries when used at higher concentrations but at the same time it can become toxic to the cells. In addition, the amount and type of cryoprotectants used in sperm diluent depend on fish species and can affect the physiological and metabolic structure of the spermatozoa during cryopreservation procedure in different ways [43]. Therefore, selection of suitable type and concentration of the cryoprotectant is needed for the development of an effective cryopreservation protocol. However, comparison of different cryoprotectants and freezing/thawing protocols are difficult when each treatment tested for the ability of fertilization of the eggs by spermatozoa. For this reason, the protective effect of different cryoprotectants varies in different fish species. In this concept, several cryoprotectants have been mainly used for fish sperm cryopreservation, such as dimethyl sulphoxide (DMSO), dimethyl acetamide (DMA), glycerol (Gly), methanol (MeOH), ethylene glycol (EG), and propylene glycol (PG) [44–46].
\nCryoprotectants are essential for preservation, but it is dissimilarly toxic to the cells. The toxicity tolerance level of the cells also depends on cryoprotectant concentration. Also, there are differences in permeability of the cells according to cryoprotectant types. In this study, DMA as penetrating cryoprotectant was used at 10% concentration, and diluted samples were equilibrated for 10 min at 4°C. Some authors recommend having an equilibration period following dilution, allowing cryoprotectants to penetrate the spermatozoa before cryopreservation [9, 47]. However, some authors reported equilibration process did not improve cryopreservation success in fish [48, 49].
\nOn the other hand, the freezing conditions depended on the straw size and were also species specific. Insemination with cryopreserved semen of Arctic charr (
Successful fertilization of eggs using cryopreserved sperm is the final target of cryopreservation process. Fertilization ability of the cryopreserved sperm is a reliable approach to evaluate success of the cryopreservation protocol [52]. According to the results of the present study, Nile tilapia spermatozoa were influenced by cryopreservation process, and depending on this interaction, fertilization ability of frozen/thawed sperm decreased than fresh ones. The reason for the low fertility rate of frozen/thawed spermatozoa may be attributed to the changes in ultrastructural morphology, decrease in progressive motility and motility duration, and also possible toxic effects of the DMA.
\nMotility is one of the most important factors to assess fish sperm quality because it gives important information about the sperm cell’s energy sources. In addition, better knowledge of the characteristics of fresh sperm motility is necessary to evaluate sperm quality in commercial hatcheries before artificial reproduction and also in laboratories before experiments. Spermatozoa motility is induced following releasing of the spermatozoa into the aquatic environment during natural reproduction or after transferring to an activation medium during controlled reproduction [53].
\nThe observed decrease in sperm motility might be due to decrease in the percentage of sperm viability, high damage of sperm cells, or decrease in ATP content following cryopreservation. Similarly, Alavi et al. [54] determined that almost all studies on sturgeon sperm cryopreservation showed significant lower sperm motility and fertilizing ability of frozen/thawed sperm compared to that of the fresh sperm.
\nOn the other hand, when fish spermatozoa are released into water or activation medium, they have a brief spermatozoal activity period [55]. For instance, in fresh sperm, the duration of spermatozoa motility in several cyprinids have been reported to last 120 s [56]. Similarly, in case of silver barb, the maximum motility period was observed until 150 s after water activation [4]. However, in case of frozen/thawed sperm, duration of mean post-thaw spermotozoa motility (32.0 ± 8.16 s) of the Nile tilapia was determined as lower than the results assessed with mirror carp [57] but higher than that of scaly carp [46] when DMSO, DMA, and glycerol were used as cryoprotectant. Similar results for the motility parameters of frozen-thawed spermatozoa were reported in fish in some experiments [48, 58, 59]. On the other hand, it is interesting to note that Godinho et al. [60] reported 241.2 ± 57.3 s post-thaw spermatozoa motility duration in glucose-based cryosolution containing 10% methanol in Nile tilapia.
\nIn the present study, the applied sperm/egg ratio was 1×105:1 for fresh as well as frozen/thawed sperm, which probably resulted in excessive sperm concentrations in all batches. However, according to Lubzens et al. [48], the concentration of frozen/thawed sperm to be used to achieve optimal fertilization and hatching success is approximately 100 times higher than for fresh semen. This may be due to differences in extender compositions, cryoprotectant types, equilibration periods, egg quality, or applied protocols. In the present study, high positive correlation was determined between post-thaw spermatozoa motility and fertilization. This situation was consistent with the results that obtained from turbot (
In conclusion, Nile tilapia sperm can be successfully cryopreserved in a glucose-Tris–based cryosolution containing 10% DMA with 0.5-mL straws. The applied protocol can be used in commercial hatcheries to facilitate artificial reproduction of Nile tilapia due to acceptable post-thaw motility and fertility results obtained. On the other hand, additional researches are needed to examine the growth and survival of the larvae originated from cryopreserved sperm.
\nThis research was funded by the Mustafa Kemal University Scientific Research Fund (Project No: MKU BAP-384).
\nThe development of methods and technologies for the production of glass-ceramic materials and foams, as a particular case of glass-ceramics, has recently revealed an interesting economically grounded field for research in the synthesis of advanced materials with unique properties for application in the civil engineering and in a variety of different industries.
Many industrial streams in the metallurgy provide large amounts of waste raw materials, e.g., in the form of slag, containing the necessary composition making it suitable to be entirely recycled with just minor modifications into new glass-ceramic materials via melting and a subsequent sinter-crystallization treatment [1, 2]. This is especially appropriate when speaking of large-scale production projects.
The most significant producers of iron-rich waste are the iron and steel industries. The different final products (e.g., steel or cast iron) and the different processes used determine the variations in the waste material composition [1]. Blast furnace slag (BF) is easier to be converted to a glass and further to glass-ceramics [3] than the other slags due to the high content of silica and alumina. Other slags like the basic oxygen furnace slag (BOF) and electric arc furnace slag (EAF) are on the contrary more enriched in iron oxides but far more poor in glass formers. They can be used after a certain composition modification [4]. Zinc hydrometallurgy turns out to be another important source of iron-rich waste raw material. Most recent research refers to iron-rich waste from the nonferrous metallurgy [5, 6].
Recent developments in the field of thermal analytical methods and instrumentation result constantly in cutting-edge machines providing faster and far more reliable laboratory measurements. In particular the high-temperature imaging systems like modern optical dilatometers (ODLT) allow in situ contactless observations of the sintering shrinkage during the synthesis of ceramics and glass-ceramics and technology development on a laboratory scale as well. Among the most significant physical parameters governing the technology of sintered glass-ceramic production are the firing temperatures, the thermal treatment rates, the thermal regimes governing the viscosity of the forming material and, as it turns out, the used atmosphere (air or inert).
An innovative part of the presented research is the synthesis in a dual air/argon environment. The authors have initiated pioneering research in this field of glass-ceramic synthesis staring in the year 2000 with some experimentation with air and inert nitrogen atmosphere [7, 8, 9] and a further development recently (started 2017) by N.B. Jordanov and A. Karamanov with argon and dual air/argon atmosphere. This development allows currently the process of synthesis of glass-ceramic samples to be separated and carried out in different stages of controlled environment, thus allowing the effect of redox determined processes to be carefully studied, understood and implemented in the design of new materials. At the same time, the crystallinity of thus obtained materials is considered higher, leading to better properties [1, 10].
The influence of the atmosphere in the particular case of foaming in inert environment of sintered iron-rich pressed powder samples however has not been examined yet and is currently the subject of profound investigations.
The overall theoretical description of effective mechanical properties and structure of cellular solid foam materials was considered and proposed first by Gibson and Ashby in 1982 [11] in terms of their famous original equations.
The theoretic development of Gibson and Ashby on the closed-cell foams however is based generally speaking on the presence of regular shape and size (e.g., cubic or hexagonal) of the closed cells determining the foams. When the case is foam of irregular structure, arbitrary shape and broad cell volume distribution, then the method of 3-D computed tomography is very helpful toward the description and characterization of cellular materials [12, 13].
In the case of cellular solid mechanics, Gibson and Ashby provide equations of the effective Young modulus of foams of closed porosity based on wide experimental data. Here the 3-D computed tomography aid is highly welcome. Actually a certain restriction here is the assumption that all closed cells are filled with a fluid. In the particular case of iron-rich glass-ceramic foams however, the inner cellular space is filled with gas. If one assumes that the pressure inside the cells is comparable to the atmospheric pressure, we can write the following equation of Young modulus [14]:
where
The successful synthesis in the presented investigation of glass-ceramic foams from simultaneously iron-rich and manganese-rich waste slag is crucially determined besides by the thermal treatment, by the redox couple ratio equilibria of Fe(II) ↔ Fe(III) and Mn(III) ↔ Mn(IV) [15]. The state of these redox couples is of greatest importance since it is entirely responsible for the foaming process to proceed successfully. The latter is being realized by the release of gas molecules in the bulk of the sample in the form of oxygen microbubbles determining the porosity. This is possible due to the reversible thermal partial reduction of the manganese and iron oxides of higher oxidation state into lower states, taking place during thermal foam formation. This process is vastly influenced by the environment. Recently it has been shown by the authors and other research teams as well that not only carrying out the thermal synthesis in atmospheric air but also separating the synthesis to subsequent stages in air and in inert gas environment (e.g., argon) can lead to very surprising results. Materials with different structure and properties could be thus obtained. The conditions of synthesis are also technologically favored this way using different environments.
Having already synthesized the sintered glass-ceramic material with appropriately formulated composition, one has obtained a partially crystalline and densified final product with more than satisfactory industrial features obtained at economically favorable conditions (compared to the expensive production of classic ceramic materials). Thus obtained materials from iron-rich industrial slag are ready for implementation in a number of engineering projects as well. They can be (and are actually ready to be) however further processed in terms of just a well-engineered additional thermal exposure scan at temperatures generally speaking higher than the temperature of sintering. This can be realized either isothermally or by a linear thermal scan.
The use of the novel applied method of sinter-crystallization developed experimentally in the last two decades and still being subject to theoretical progress can lead to very promising results mainly due to the relatively moderate treatment temperatures required for simultaneous sintering and crystallization of the samples. Just for the reference of the reader, the foaming process is taking place at higher temperatures after completion of the sintering and the phase formation. Glass-ceramic foams are a particular case of the general division of the so-called cellular glasses possessing high surface area, low density, low specific heat, high thermal and acoustic insulation and high chemical resistance [10]. When most of the cells are closed, the material is referred as foam.
The synthesis of sintered glass-ceramics depends on the relationship between viscous flow sintering and crystallization, while the production of glass-ceramic foams depends on the balance between apparent viscous flow sintering and gas evolution [1].
The gas release usually depends on the oxidation or decomposition reactions with the modifying compounds in glass-ceramic foaming. Oxidation reactions are associated with the release of COx gas from carbon-containing compounds like carbon black, graphite, silicon carbide (SiC) and organics reacting with the oxygen in the atmosphere. Typical decomposition reactions are such with carbonates or sulfates leading to the release of CO2 or SOx [1, 10]. A special case is when the parent glass contains intermediate oxides (called conditional glass formers) either iron oxides or manganese oxides undergoing transition from higher to lower oxidation state, connected to the release of oxygen gas. This is the actual case here which is the subject of investigation in the presented research.
So the production of glass-ceramics from iron-rich metallurgical slag can be terminated at the stage of obtaining just well-sintered glass-ceramics and extended any time further to the stage of glass-ceramic foams (in the particular case of, e.g., iron-rich parent frit).
The sintering of glass-ceramics is a typical example of induced structural densification of a solid sample. The latter is provoked by volume-density variations in the material’s bulk. This is the first step (besides an eventual parallel oxidation process) taking place during new glass-ceramic material formation determined by, e.g., a linear thermal scan. The degree of densification is one of the most important characteristics of sintered glass-ceramics. It is being mainly determined to a great extent by the granulometric composition, the crystallization ability (the crystallization trend) of the parent glass and the rate of heating of the compressed powder sample. At an elevated crystallization trend, the sintering could be blocked because the higher the crystallization trend, the lower the sinter ability and vice versa, respectively. It is well known that the sinterability of a glass-ceramic powder can be significantly improved by using finer glass fractions and/or higher constant linear heating rates [1, 10].
One can thus make the conclusion (and this is actually the case!) that both processes should be carefully balanced in order for a really good material with increased indicators to be successfully produced. This means that both extreme cases of a minimal or a maximal crystallization of the glass-ceramics should be avoided. The crystalline structure determines the stability and durability of the material. On the contrary, a species with higher crystallinity however cannot be sintered.
In the framework of current state of knowledge, it is assumed that both processes of sintering and crystallization are taking place in the same temperature interval. For the sake of a theoretical description however, it is accepted that the sintering stage precedes the crystallization stage. In fact the sintering stops after formation of a critical percentage of crystal phases.
Of crucial importance here is the apparent viscosity of the glass-ceramics. Its value should be maintained in a range, such that the expansion of the structure is possible and the formation and the propagation of open porosity are temporarily unavailable.
The foaming in the studied case is determined by the formation and distribution of a closed porosity population in the bulk of the material due to the release of gas molecules (most often oxygen) during the high-temperature partial reduction of certain oxides from higher to lower oxidation state. These are most often the iron [16] and the manganese [15] oxides. The high-temperature interval of foaming indicates that the mechanism of gas formation is directly related to the oxygen release as a result of the reversible partial Fe(III) and Mn(IV) reduction [15, 16, 17, 18, 19]. Moreover when the oxides of the iron and the manganese are naturally present in a sufficient amount in the parent glass frit, this is the most favorable case because there is no need for the process of foaming to be artificially and additionally catalyzed. An inorganic material is thus being formed by autocatalytic foaming.
For the purpose of current experimentation with sintered materials and foams, a slag from the iron and steel company Helwan, Cairo Governorate, Egypt (with a slag production capacity of 30 kt p.a.), was used for the synthesis of the investigated samples.
This slag is relatively poor in glass formers (SiO2 and Al2O3); that’s why it had to be enriched in silica by mixing 70 wt.% slag and 30 wt.% industrial sand. The parent batch of 150 g was brought to melting in corundum crucibles using an electric furnace. After an exposure for 2 hours at a temperature of 1450°C, the resulting homogenous melt was quenched in water, and a dark brown-colored glass frit was obtained. Thus obtained glass frit was crushed, grinded several times in a planetary mill FRITSCH (Germany) for 10 minutes and sieved below 75 μm with a digitally programmed sieving machine CISA (Spain).
The investigations were performed by a thermal-optical measuring and imaging system with an ESS HSM-1400 MISURA (Italy). This instrument combines two techniques: a high-precision, high-resolution horizontal contactless optical dilatometer and a hot-stage microscope (HSM). This is an established laboratory method in recent years, since it turns out to be reliable and fast and is used already by many research groups worldwide [2, 20].
The sintering behavior of all glass-ceramic samples was investigated by means of ODLT, a method which allows measurements with very high precision. This is mainly due to the absence of a mechanical push rod in the system, as it is the case with classical contact dilatometer devices. A monochrome optical arrangement employing two video cameras providing high magnification and high resolution is used instead. Typical measurements were performed with holding times of 30 minutes at 950°C in air or argon.
To the sintered glass powder, a small amount of 7 wt.% polyvinyl alcohol (PVA) aqueous solution was added to form a granular mass, which then is mechanically homogenized and placed in a matrix of 50 × 5 × 3 mm. Multiple samples of equal forming are prepared by stuffing loosely the material in a pressing matrix. Then by applying uniaxial pressure at 40 MPa in a hydraulic pressing machine NANNETTI (Italy), samples of almost perfectly equal dimensions and densities, with an increased green strength and a decreased porosity, are produced.
Subsequently a burnout step at 270°C is required to be carried out before proceeding further with the heat treatment processes in order to remove the binding agent (e.g., PVA). The burnout can be performed in ODLT separately or as an initial programmed step preceding the oxidation, sintering and foaming steps.
The studied thermal behavior in the range up to 1300°C in air and argon of the sintered samples was studied and analyzed by the optical HSM method. As far as the samples used for ODLT measurements have a standard parallelepiped shape (as described above), the samples used for HSM measurements are not the same but represent upright standard cylinders instead.
In addition the foaming trends of the samples were examined by carrying out measurements both isothermally with holding times of 30 minutes (e.g., at 950°C for simultaneous oxidation and sintering and at 1100°C for foaming initiation) and non-isothermally as well (by using constant linear thermal scans). Typically such thermal scans were used with heating rates of 20°C min−1.
During all measurements the ODLT/HSM instrument was mounted in a closed aluminum box (developed and manufactured at the mechanical workshop of IPC-BAS Sofia), allowing measurements in a controlled environment. The box can be purged with dry argon gas on demand. The latter application allows continuous maintenance of an overpressure of ~10 mbar argon to be kept over the entire synthesis and optical measurement of the thermal variation of the structure of the sample. This is achieved by the use of a fine-graded rotameter, Yokogawa (Japan), for manual control of the gas flux in the sample chamber and a high-precision digital manometer with a ceramic membrane, Profimess (Germany), for overall gas pressure monitoring.
According to the current state of knowledge of the authors, such a laboratory experimental setup—a combined ODLT/HSM mounted in a closed vessel for examinations of the sintering and foaming behavior of iron-rich glass-ceramics in different atmospheres—is unique in Southeastern Europe and even was probably used for the first time in this respect here.
Scanning electron microscopy (SEM) was used to analyze the structure of the sintered glass-ceramics by taking pictures of both fractures and the surfaces of the samples. A JEOL 6390 (Japan) instrument was used. To provide electron conductivity, all samples were metalized with gold by vapor deposition technique.
3-D computed micro-tomography was used for entire bulk scanning of the foam glass-ceramic species. The tomographic measurements were carried out with an X-ray micro-tomograph, Bruker SKYSCAN 1272 (Germany), which uses a white beam with cone geometry. The following setup conditions were applied: X-ray tube voltage 70 kV, current 142 mA and 0.11 mm Cu filter. The voxel (3-D pixel) size was 1 μm and the optical magnification was 7.4. A typical 360° scan took 21 hours and 27 minutes. Reconstruction of the 3-D images was done with the commercial software InstaRecon.
The phase composition of the sintered glass-ceramic foams was determined by X-ray diffraction spectroscopy (XRD) using a Panalytical Empyrean (USA) spectrometer.
As we have already noted, the innovative point of current research is, besides the use of the applied method of sinter-crystallization toward the production of sintered glass-ceramics and/or glass-ceramic foams, the use of a controlled environment during the synthesis of sintered glass-ceramics and in particular extended to the design and production of glass-ceramic foams.
In Figure 1, a comparative graphical representation is presented, together with images at respective temperatures of interest, of the shape alteration during real-time in situ filming of two samples being synthesized in air or in argon atmosphere.
Typical HSM curves of the thermal behavior of pressed iron-rich glass-ceramic samples in air and argon.
Up to temperatures of about 1050°C, the thermal behavior of both species is quite comparable. Both samples undergo similar densification during the interval of low temperature sintering. At higher temperatures however, iron-rich glass-ceramic materials heated up in different (air or inert) atmosphere reveal a completely different thermal behavior compared to each other when they are subject to a subsequent thermal treatment.
In the case of an argon-sintered sample, further heating of the material leads to sample melting (to, e.g., a hemispheric spill at 1155°C). However, the sample sintered in air starts expanding its volume during heat treatment above 1050°C, and at the same reference temperature of 1155°C, it reveals a maximal value of the structural expansion determined by gas evolution—the foaming in the entire bulk of the newly formed material. This difference in the thermal behavior of both samples is mainly due to differences in the respective material’s viscosity values.
The phenomena described above are entirely determined by the state of the redox couple equilibrium Fe(II) ↔ Fe(III) in a sense that an inert environment (e.g., argon) is going to maintain the equilibrium drawn at a maximal extent to the left (oxidation is inhibited [9]), while the air atmosphere is going to keep the ratio Fe(II)/Fe(III) at a minimal value, i.e., the equilibrium is drawn to the right.
In Figure 2a, the sintering curves are presented during an isothermal scan and the respective low temperature behavior of the glass-ceramic samples in air and argon. One can clearly and unambiguously note the effect of the environment on sintering: the onset of the sintering process in argon is shifted to lower temperatures. It starts earlier, and the degree of structural densification is to a certain extent higher than the one in air (14 vs. 12% shrinkage, c.f. again Figure 2a). The shaded area in Figure 2a represents actually the area of temperatures where the glass transition point,
(a) Sintering curves measured by ODLT in air and argon. (b) Sintering curves; zoom-in of the shaded area of (a). The glass transition temperature (Tg) is unambiguously to be recognized in both curves.
In Figure 2a, the shaded area reveals in fact, as it is obvious in Figure 2b, that an ODLT measurement represents certainly a method for truly reliable measurements of the temperature of vitrification,
One can thus summarize that utilizing the ODLT technique for the sake of synthesis of well-sintered glass-ceramic materials is a good approach. It also reveals the possibility for reliable measurements of
XRD spectra of the glass-ceramic foams obtained in air and argon environment are presented in Figure 3. The phase analysis of the samples results in the detection of pyroxene crystal phases. The crystallinity of the samples is between 30 and 40% as referred also by Strnad [21]. The crystallinity in argon however is higher than that in air. A shift of the peak positions in air to lower theta angles has been observed as well.
XRD spectra of glass-ceramic foams synthesized in different environment.
The two features described above confirm that the absence of oxidation in argon environment leads unambiguously to higher crystallinity and to some differences in the chemical composition of the pyroxene phases. These differences can hypothetically be due to the facilitated entering of the Fe(II) and Mn(III) ions in the pyroxene structures. Also a change in the lattice interplanar distance is present.
In order for the microstructure of the sintered glass-ceramic species subject to current investigation to be studied, a series of scanning electron microscope images of two sintered glass-ceramic samples are shown in Figure 4. Images have been taken from the surface and from a fracture of both species. Figure 4a and c represents photos of the surface of the air-sintered sample; Figure 4b and d is from the surface of the argon-sintered sample; Figure 4e, g and h is images from a fracture of the air-sintered sample; and Figure 4f is a picture of a fracture of the argon-sintered sample.
SEM images from a fracture and the surface of two sintered glass-ceramic foam samples in air and argon: 4a and 4c: surface of the air sintered sample; 4e, 4g, 4h: fracture of the air sintered sample. 4b and 4d: surface of the argon sintered sample; 4f: fracture of the argon sintered sample.
Despite the well overall sintering in both atmospheres, it has been found that in argon environment an even better sintering of the sample than that in air is present.
An interesting observation is that on the surface of the argon-sintered glass-ceramics (Figure 4b), the pores reveal a concave morphology. This might be an indication for the formation of these pores in the interval of softening which initiates together with the beginning of the process of high-temperature reduction as well. In both cases of sintering, a large population of small spherical holes on the surface is present, which is almost a certain indication for gas release.
In both cases of sintering, a non-spherical, sharp-edge intergranular porosity is predominant.
The proposed possibility for a selective environment of synthesis of sintered glass-ceramics and glass-ceramic foams provides the option of the synthesis to be carried out and thus to be experimented by using different stages.
Thus after a low temperature sintering stage in air (e.g., see Figure 1), the process of foaming of a pressed glass sample can be carried out in a second stage entirely in argon environment. The process of foaming of this sample traced by a HSM measurement is shown in Figure 5. The snapshots in Figure 5 during the run of a thermal scan represent the evolution of structural alteration at characteristic temperatures.
Measurements of the thermal shape alteration behavior during foaming in argon by HSM; snapshots of the sample at a respective characteristic temperature.
In order for the effect of the atmosphere on the foaming of sintered glass-ceramics to be carefully analyzed, a graphical plot of the structural evolution during foaming in different atmospheres is given in Figure 6. The plot in the expansion-time domain of foaming in air and argon during an isothermal scan at a temperature of 1100°C with 30 minutes holding time (Figure 6) provides a clear and detailed picture on the process under consideration.
Temporal evolution of foaming curves in air and argon of sintered glass-ceramic samples.
The expansion of the structure due to the release of oxygen gas and formation of the closed-cell system during high-temperature reduction initiates in both environments (air and inert) in a similar way and almost at the same time as well. The process of foaming in argon however turns to be more effective. The bloating of the material reaches a maximal value of nearly 200%, considerably earlier than the maximal foaming in air (up to 190%); then after a slight volume shrinkage, a stable material with 180% expanded structure is being formed and maintained in the course of the working isotherm.
The entire foam material formation (i.e., the formation of closed porosity population) proceeds relatively quickly with the programmed isotherm in Figure 6. The foaming in argon takes ~5 minutes to complete followed by a small shrinkage and a structural stabilization. The foaming in air takes longer time to complete than argon (15 minutes) and reaches stabilization again. The foam material formation in air and argon atmospheres results in obtaining a new material characterized in both cases generally with fire resistance properties at temperatures of 1100°C.
For the sake of investigation of the entire bulk structure of the newly formed foam material, 3-D X-ray tomographic analysis has been used. In Figure 7a (left), a false-color 3-D reconstruction of the surface is presented and the volume by tomographic scanning of an iron-rich glass-ceramic foam sample synthesized in air. In Figure 7a (right), a selection of three cross sections of the bulk of the same sample is presented. In Figure 7b (left), similar 3-D false-color surface-volume reconstruction of a glass-ceramic foam sample synthesized in argon is presented. In Figure 7b (right), cross-section slices of the material’s bulk are shown, respectively.
X-ray computed tomography, (a) 3-D reconstruction of the surface and the bulk of a glass-ceramic foam synthesized in air (left); volume cross sections (right), and (b) 3-D reconstruction of the surface and the bulk of a glass-ceramic foam synthesized in argon (left); volume cross sections (right).
From visual observations and the performed analysis, one can clearly note that the porosity in the bulk of both samples is predominantly of closed type and amounts to about 80–85% in both species. It is also evident that the walls of the samples are abundant of pores. Another interesting feature is that the closed cells of the species foamed in air are larger than the cells in the volume of the sample foamed in argon. Moreover the thickness of the walls in the sample synthesized in air is on average lower than the wall thickness in the glass-ceramic foam material obtained in argon environment.
In current investigation the authors have shown the possibility for carrying out successful synthesis depending on the temperature and the applied atmosphere of well-sintered glass-ceramics and/or glass-ceramic foams.
It was shown that synthesis in inert environment leads to the production of materials with higher degree of sintering.
If properly engineered, the practice and experience show that a glassy-crystalline material with low density and high porosity of about 80% can be obtained, which can be considered as more than a satisfactory result [1, 22]. In addition thus obtained foams are characterized by fire resistance features up to 1100°C as well.
By applying a double-stage heat treatment, first stage, low temperature sintering at 950°C, and second stage, foaming at 1100°C, foam materials with improved properties and differences in the structure have been successfully obtained.
The SEM images from the fractures of both glass-ceramic foam species reveal a well-sintered material. They show a good degree of sintering and total porosity below 10–15%.
Inorganic materials obtained by thermal foaming like these subjects of current investigation can be used as modern building materials, as construction materials and as insulation materials in various fields and industries, e.g., as panels or arbitrary shaped.
Inorganic glassy-crystalline foam materials can be considered generally speaking as low-cost thermal insulating, soundproof and fire-resisting low-weight materials.
N.B. Jordanov gratefully acknowledges the financial support of the National Program Young Scientists and Post-docs of the Ministry of Education and Science of Republic of Bulgaria under the project “Sintered self-glazed glass-ceramics and foams from iron-rich industrial wastes.” D. Tatchev and A. Karamanov express their gratitude for the support of Project BG05M2OP001-1.002-0019: “Clean technologies for sustainable environment—waters, waste, energy for circular economy.” All authors are thankful for the financial support for realization of the closed experimental setup for measurements in a dual environment of the Bulgarian Science Fund (FNI) under Project DN 19/7 “Theory and application of sinter-crystallization.”
The authors declare no conflict of interest.
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