We modeled and explored several hypotheses regarding the influence of H2O2 on the dynamics of propagation of axonal degeneration in LHON. Zonal oxidative stress, corresponding to H2O2 gradients, correlated with the morphology of injury exhibited in the LHON pathology. If the axonal membrane is highly permeable to H2O2 and oxidative stress induces larger production of H2O2, small injuries could trigger cascading failures of neighboring axons. The cellular interdependence created by H2O2 diffusion, and the gradients created by tissue variations in H2O2 production and scavenging, result in injury patterns and surviving axonal loss distributions similar to LHON tissue samples. Specifically, axonal degeneration starts in the temporal optic nerve, where larger groups of small diameter fibers are located and propagates from that region. These findings correlate well with clinical observations of central loss of visual field, visual acuity, and color vision in LHON, and may serve as an in silico platform for modeling the mechanism of action for new therapeutics.Leber hereditary optic neuropathy (LHON) is a rare syndrome that results in vision loss. A necessary but not sufficient condition for its onset is the existence of known mitochondrial DNA mutations that affect complex I biomolecular structure. Cybrids with LHON mutations generate higher rates of reactive oxygen species (ROS). This study models how ROS, particularly H In 1988, in a first for genetically linked disorders, Wallace et al. , is a neMuch progress has been made in unravelling LHON pathophysiology, molecular and environmental triggers, but the causal chain leading to the illness and the roots of its sudden onset and progression are still unelucidated . While nLHON-linked mtDNA mutations affect critical components of the mitochondrial electron transport chain, and, as a result, energetic imbalance and oxidative stress have been used to explain the death of affected RGCs ,19. The Experimental data from rats demonstrates that, in optic nerve axons, mitochondrial volume is directly proportional to the axon section volume in both myelinated and unmyelinated segments . However2O2) as a stress-inducing species, recognizing that H2O2 physical properties make it a more likely candidate as the oxidative stress species. The range of ROS concentrations with beneficial physiological outcomes is relatively narrow. For example, H2O2 concentrations ([H2O2]) between 1 nM to 10 nM benefit neuronal development, while concentrations below 1 nM and over 100 nM led to tissue pathology ) in axonal segments as the element that forces mitochondria to release mediator(s), which induce axonal degeneration  , where ) were tracked at each  voxel position. There were 3 different operating equations for each species: Equations (2)–(4) modeled 2O2 dynamics in the tissue using a single system implementation. However, we focused on H2O2 dynamics because this species is less labile and has a longer range signaling capacity MODEL.Physiological Heustress . Neuronsver 1 µM . HoweverAxonal segments were assigned one of 3 states to account for the concept of oxidative distress: “Healthy” [H], “Oxidative Stress” [S] (which corresponds to oxidative distress), or “Dead” [D]  each of Mitochondrial in glia were placed in the inter-axonal space but without demarcating glial cell boundaries. Each glial mitochondrion could be in one of 3 states , similarAll voxels in an axonal segment were assigned the same state. This assumption limited the 3D axonal mitochondria topologies that could be simulated because mitochondria are dispersed along the axon, and [ROS] can vary substantially along the axon. At the nodes of Ranvier, mitochondria are present next to but not at the node . EnergetThe complex geometry of the optic nerve (ON) makes three-dimensional (3D) modeling of the entire ON difficult. The current work simplified the problem by restricting modeling to axonal sections in the region, starting at the optic nerve head (ONH) and extending to but not including the chiasm. In the model, all axons are parallel, a reasonable abstraction for small optic nerve sections.x = dy = 1/7 µm, dz = 1 µm, and volume V = 1/49 µm3.Samples were mapped to 3D meshes, with equal mesh steps in the X and Y dimensions, while the Z dimension mesh step could be set independently. Thus, each point in the mesh is a voxel corresponding to a rectangular cuboid of the optic nerve. For example, if the XY resolution was 7 pixels/µm and Z resolution was 1 pixel/µm, each pixel represented a physical cuboid of sizes dIn the optic nerve, RGC axon diameter distributions differ between optic nerve areas . To modeONdiam denotes the optic nerve diameter, then an “x”% model of that optic nerve is a model of diameter Healthy young adult optic nerves contain 1.2 million RGCs . Models Axonal mitochondria have a tubular-like appearance with diameters between 50 nm and 300 nm ,50 and laxon)p mitochondria. Mitochondria were placed in the optic nerve volume not occupied by axonal mass with a probability glia),p octant on the left, the nasal (N) octant on the right, the superior (S) octant at the top, and the inferior (I) octant at the bottom. In other words, a right optic nerve viewed from the front was always modeled.The simulation software contained a visualization module which displayed ROS concentrations and axonal states while running simulations. 3D ROS concentrations could be examined through 2D cuts in the 3 anatomical planes: coronal, transverse and sagittal . Coronaltemporal (T), superior-temporal (ST), superior (S), superior-nasal (SN), nasal (N), inferior-nasal (IN), inferior (I) and inferior-temporal (IT) , −101) and measured in dB. The lower limit for loss was selected because the logarithm would be minus infinity when the loss in an octant is complete. A lower limit was needed for display, and the number (−101) chosen is less than the dB value if only one axon in an octant survives.Axons were also grouped into 8 octants for data processing, each covering a 45° angle circle sector. From left to right in the clockwise direction, the octants were ral (IT) . In eachxx, Dyy, Dzz). Diffusion values for the X and Y directions were always equal (Dxx = Dyy), while the diffusion in the Z direction could be set independently. Thus, the model space was anisotropic.Each voxel had a three-dimensional diffusion vector .Under the constant neuronal firing frequency hypothesis for two axons of radii RFCONST.Simulations conducted under the constant firing hypothesis were marked with RPFCONST hypothesis, mitochondria in smaller axons will experience greater oxidative stress.Sadun et al.  proposedH values.S = 0.7 × RPH), the simulation was annotated with NFRP.If the overall ROS generation in the Stress state is decreased (RPS = RPH or RPS = 1.3 × RPH), the simulation was annotated with PFRP.If the overall ROS generation in the Stress state stays the same or increases . We set the −1 nM because reported cellular concentrations of −2 nM range  in the myelinated area.The optic nerve has a narrow geometry, with a diameter less than 3% of its length. In geometries where one dimension is significantly larger than others, diffusion processes proceed faster on the smaller dimensions. Therefore, lengthy areas where scavenging is present without significant ROS production might reduce the influence that high oxidative stress regions can have on their surroundings. We hypothesized that HTo study the hypothesis, simulations were performed with an abridged optic nerve  containing myelinated and unmyelinated regions. Along the Z axis, the top 50 µm modeled the unmyelinated region, followed by 200 µm axonal myelinated region, a 5 µm section comprising a node and another 155 µm region modeling a myelinated axonal shaft. The node’s length was larger than the published data from rats  because In this simulation, the role of scavenging rates along the axon was also examined. For this purpose, either similar scavenging rates across the model  or a 30%2O2] reached a steady state. The unmyelinated regions had significantly higher [H2O2] than the myelinated regions for both uniform scavenging of H2O2  in the unmyelinated region ,52 shoul2O2 produced by axons while varying the mitochondrial H2O2 in the Stress state  and one with decreased production (NFRP). Simulations were performed to test the possibility that mitochondria change their operating points when subject to oxidative stress. For the PFRP cases, we ran simulations where the Stress and Healthy states had either the same production values .To further explore myelinated vs. unmyelinated axons, we again assessed two models for oxidatively stressed axons, one with increased oxidative production in the Stress state , axonal degeneration occurred across a wide range of mito% values (4% to 15%)  . However to 15%) .RP) had greater variability in axonal survival than the NFRP simulations for the same mito%  and for the case when production in the Stress state increases, there were abrupt changes in survival for small variations in mito% around the 10% mito% value , different axonal degeneration patterns occurred depending on ROS production in the Stress state , there was high neighborhood loss, and in most cases, the loss was complete in each octant  and glial mitochondria produced more ROS (PFRP mode) was surprising , although the scale at which local loss occurs was significantly smaller than in the PFRP cases  in different systems, e.g., ROS and calcium, can result in wavelike propagation ,71. Howe2O2 concentrations correlated with axonal degeneration patterns seen in LHON histological sections [2O2 is produced. The degeneration of some axons and the subsequent cessation of ROS production can have a protective effect on the remaining axons. Clinical observations on children under 12 show that despite structural evidence of axonal loss, there is less functional loss and no loss later in life [We found that Hsections . Higher  in life . The pro in life , the inc in life ,24, an i2O2 and relatively impermeability to 2O2 also affects the results. However, it is not known what the RGC axonal permeability to H2O2 is in either the unmyelinated or myelinated regions, and assumptions from non-axonal cell membranes may not be incorrect. Second, although the range of concentrations at which H2O2 causes oxidative stress are known [2O2 concentrations and mechanisms of axonal degeneration are also unknown.This study has several limitations. First, although it is well known that cellular membranes are relatively permeable to Hre known , there iThird, in silico studies are hampered by the balance between computational resources needed, simulation time, and simulation resolution. Our study is no exception. Model sizes and the model resolution were limited by the available GPU with its on-board memory and the need for simulation throughput. The methods presented in this paper can be extended to larger tissue samples. For example, extending the current work to a complete optic nerve and ultimately to an optic nerve-retina system would be worthwhile. Nevertheless, although the model can be extended spatially using more greater computational resources, there are limits to the simulation time that can be afforded. When a second in the biological life of the sample can only be processed in many seconds of computer simulation, longer-term evolving diseases such as LHON cannot be simulated at the time scales of the illness. On the other hand, we do not know how long the pathophysiological process takes, and are limited by patient symptoms, measurements of visual loss, clinical observation of disc edema and other retinal findings, and detection of axon and ganglion cell/inner plexiform layer loss in the retina.Fourth, the timelines of the simulated processes are more rapid than the timeline of visual loss in LHON. Given that the simulation results are based on realistic biological parameters, the difference in timing suggests a possible intermediate mechanism triggered by ROS, which subsequently results in axonal dysfunction and degeneration, and eventually results in somal apoptosis. Such a mechanism could be a delay until the axon becomes dysfunctional, based on a biochemical, inflammatory, or other mechanisms.Finsterer et al.  reviewedNo similar data regarding carriers were found in the literature. An interesting data point would be to see if non-visual diseases occur more or less frequently in carriers vs. controls or vs. LHON cases.In summary, a more comprehensive model of ROS in LHON and the effects on the propagation of axonal degeneration has been developed. The model is more realistic anatomically, biophysically, and biochemically, and has implications for the pathophysiology of the disease and the development of therapeutic approaches. Future studies could address biochemical feedback loops between RGC and glia, the role of other ROS, the specific anatomy of the optic nerve head, and use these refinements to further advance the detection and treatment of LHON in those at risk.


Acid oils, by-products of edible oil refining, are potentially interesting fat sources for farmed fish diets because of their high energy content and usually competitive price. Their use and revaluation may contribute to more efficient and sustainable fish production. They are characterised by presenting a similar fatty acid profile to their respective crude oils, but with a high content of free fatty acids. The present study aimed to investigate the effects of including soybean-sunflower and olive pomace acid oils in European seabass diets, as a preliminary step to determine whether they might be suitable energy sources for fish diets. The results showed that growth was only impaired in animals fed the diet containing olive pomace acid oil, which had the highest moisture, impurities and unsaponifiable matter. They also suggest that dietary free fatty acid content affects digestibility, but not the fatty acid profile of flesh and perivisceral fat. Notwithstanding, further studies assessing the effects of the inclusion of these oils are needed before recommending their use.p < 0.01). In contrast, other diets including acid oils did not impair performance. Acid oil diets did not affect the apparent digestibility of dry matter, crude protein or total fatty acids (p > 0.05), but a lower digestibility of lipids and saturated fatty acids was observed (p < 0.001). Flesh composition and fatty acid profile were not affected by the high dietary free FA content (p > 0.05). Hence the results suggest that the studied acid oils may potentially be used in fish diets although further studies are needed.The effects of dietary inclusion of soybean-sunflower and olive pomace acid oils on growth, digestibility and flesh composition were studied in European seabass. Eight diets were fed for 100 days , differing in the added fat source : S (crude soybean oil), SA (soybean-sunflower acid oil), O (crude olive pomace oil) or OA (olive pomace acid oil); 3 blends: S-O, S-OA, SA-OA at a 1:1 ratio; and a diet containing only fish oil (F) as a control. Animals fed OA showed the worst performance among dietary treatments, with the lowest weight, specific growth ratio, average daily gain and the highest feed conversion ratio ( Dicentrarchus labrax) is one of the most important farmed marine fish species, Turkey, Greece, Spain and Egypt being the countries that account for 88% of total production [The increasing importance of aquaculture, linked to factors such as population growth, the increasing demand for aquatic food products and the bioaccumulation of toxic compounds in wild marine species, has raised the need for safe and efficient production of aquatic species. In Mediterranean aquaculture, European seabass (oduction .Oncorhynchus mykiss) [Sparus aurata) [In farmed fish diets, fish oil (FO) had traditionally been used as the only dietary fat source, partly due to its energy content but mainly to its contribution of eicosapentaenoic EPA; C20:5 n − 3) and docosahexaenoic DHA; C22:6 n − 3) acids. These n − 3 highly unsaturated fatty acids (n − 3 HUFA) are considered essential for marine fish 0:5 n − 3. Aquacul n − 3 ac mykiss) ,13,14,15 aurata)  have beeThe potential use of a new ingredient requires the assessment of its quality and composition. According to Glencross , the cha2O3) was added to the diets as an inert marker for digestibility balance.Eight experimental diets were formulated to be isoproteic and isolipidic using the same ingredient composition except for the added fat source . The added fat consisted of 25% FO and 75% experimental oil. Then, four diets including experimental oils, namely S (crude soybean oil diet), SA (soybean-sunflower acid oil diet), O (crude olive pomace oil diet) and OA (olive pomace acid oil diet); and three blends at a 1:1 ratio  were formulated. A diet was formulated including only commercial fish oil for use as a control (F). Diets were formulated according to the nutritional requirements of the species . Ingrediw/w) were supplied by Bunge Ibérica S.A.U. . Crude olive pomace oil was supplied by General d’Olis i Derivats S.L.  and olive pomace acid oil was supplied by RIOSA S.A. . Comercial FO was obtained from AFAMSA . The experimental diets were manufactured as extruded pellets by Ceimar-University of Almeria  using standard aquafeed procedures. Briefly, feed ingredients were finely ground and mixed in a vertical helix ribbon mixer  before oil and diluted choline chloride were added. All the ingredients were mixed together for 20 min, and then water (350 mL/kg) was added to the mixture to obtain a homogeneous dough. The dough was passed through a single screw laboratory extruder . The extruder barrel consisted of four sections, and the temperature profile in each section (from inlet to outlet) was 95 °C, 98 °C, 100 °C, and 110 °C, respectively. Finally, pellets were dried at 27 °C in a drying chamber  for 24 h and feeds were kept in sealed plastic bags at −20 °C until use.Crude soybean oil and soybean-sunflower acid oil . This system allows for water recirculation of between 1 and 1.5 tank volumes per hour (15 m3/h), and is equipped with an aerobic biofilter for the removal/transformation of ammonia to nitrite and nitrite to nitrate. The supply of fresh water to the system consists of 5–15% of the total volume per day. Each experimental diet was randomly assigned to three tanks and was administered twice a day by automatic feeders  for 100 days. Uneaten feed was collected by filtering effluent water from each tank and collectors were emptied at the end of each meal, so the average feed intake per tank was recorded daily. Water temperature (22.55 ± 0.84 °C), dissolved oxygen levels (7.30 ± 0.66 mg/L), pH (7.9 ± 0.2) and salinity (35.5 ± 0.50‰) were maintained throughout the study. The levels of ammonia (0–0.5 ppm) and nitrites (0–2 ppm) were maintained within the safe levels for the species. During the experimental period (from July to October), the tanks were subjected to natural photoperiod.All the procedures were conducted following the European Union Guidelines for the ethical care and handling of animals under experimental conditions (2010/63/EU) and in accordance with the Animal Protocol Review Committee of the Universitat Autònoma de Barcelona (CEEAH). The trial was carried out at the Aquaculture Center facilities of the Institute of Agrifood Research and Technology . A total of 480 European seabass  were randomly allocated into 24 cylindroconical tanks with a capacity of 500 L (20 fish per tank) in a sea water recirculation system  and at the end of the experimental period (day 100). Each tank had a removable faecal settling system for the collection of faecal samples where feed and faeces are separated on the basis of their different densities. Faecal collection was carried out during the last two weeks of the experimental period and then stored at −20 °C until further analyses. At the end of the experimental period and after 24 h of fasting, six fish from each tank  were euthanized by hypothermia in a mix of water and ice (1:3) and individually gutted. Viscera and abdominal fat pad were removed and weighed. The entire left and right muscles were also removed and weighed. The left muscle was immediately used for fresh colorimetric determination. The right muscle was cut into two different sections  and weighed. All samples were bagged individually and stored at −20 °C until chemical analyses.Prior to chemical analyses, samples of each experimental oil and feed were pooled, homogenized and kept at 5 °C. Faeces, skinned muscle  and perivisceral fat samples were homogenized, freeze-dried  and kept at 5 °C. Fatty acid composition, lipid class composition and MIU  content of experimental oils were analysed in duplicate as described by Varona et al. . AnalytiThe FA content of feed and faeces were analysed following the method described by Sukhija and Palmquist . PerivisLipid class composition and MIU values of experimental oils are shown in The FA composition of experimental oils and diets are shown in L*, a*, b* [L* represents the lightness of the sample, a* defines the position between red/magenta and green and b* defines the position between yellow and blue. Then, C*  and h (hue angle) values were calculated as C* = (a*2 + b*2)1/2 and h = arctan (b*/a*), respectively [Colorimetric determinations were performed on the fresh and thawed left muscle using a Minolta chroma meter  on the Norwegian Quality Cut (NQC) section . Thawed , a*, b* , where Lectively . Three mAll calculations were in accordance with standard formulae ,28. The Growth performance and carcass parameters were calculated according to standard formulae. The average daily gain was calculated from:p < 0.05.The normality of the data and homogeneity of variance were verified using the CAPABILITY procedure of SAS . All data were analysed using the GLM  procedure of SAS. Differences between means were tested using Tukey’s adjust correction for multiple comparisons. For growth performance, digestibility balance, colorimetric and quality assessment of flesh and FA profile of muscle and perivisceral fat, the experimental unit was the tank. For carcass parameters, the experimental unit was the individual. The results in the tables are reported as the least square means, and differences were considered significant at p = 0.002) ADG (p = 0.003) and SGR (p = 0.002) values among dietary treatments. Additionally, they had the highest FCR, which was significantly different to those fed F or SA (p = 0.004). In terms of ADFI, the lowest value was observed in animals fed F (p = 0.005).The effects of added oils on growth performance and carcass parameters are shown in p > 0.05). Mean values of carcass yield and gross and net flesh yields were about 89%, 41% and 46%, respectively.Regarding carcass parameters, no differences among dietary treatments were observed in carcass weight or in the percentages of carcass yield, flesh yield and perivisceral fat (p > 0.05) among dietary treatments. In contrast, differences were observed in the ADC of lipids. Acid oil diets  showed a lower ADC for crude fat than their corresponding crude oil diets . When comparing to F, no differences were observed for diets including crude oils , while lower values of crude fat ADC were obtained for diets composed only of acid oils  (p < 0.001).Feed digestible energy and the ADC of macronutrients and FA are shown in p > 0.05). For SFA, only SA showed lower digestibility than S (p < 0.05).Concerning ADC of FA, significant differences were observed among dietary treatments for total FA (TFA), SFA and MUFA, but not for PUFA, n − 3 or n − 6 FA. No significant differences were obtained for TFA and MUFA digestibility between diets containing acid oils and their corresponding crude oil (p > 0.05). In contrast, higher TFA digestibility was obtained for diets including crude soybean oil  (p < 0.01). Similarly, diets with crude olive pomace oil (O and S-O) showed higher SFA digestibility than F (p < 0.001).When acid oil diets  were compared to F, no significant differences in the ADC of FA were obtained . In thawed muscle, differences were observed for both C* and b* parameters, the flesh from diets O and S-O being those that showed the lowest values among dietary treatments (p < 0.01). When comparing fresh to thawed muscle, L* increased, while h and a* decreased (p < 0.001).The colorimetric assessment, chemical composition and liquid holding capacity of the flesh are shown in p < 0.001). On the other hand, dietary treatments showed no differences in terms of the liquid holding capacity of thawed muscle.No differences were observed in the chemical composition of flesh. Regarding lipid content, no statistical differences were found among dietary treatments either for the dorsal or ventral sections. However, the ventral section of the muscle showed a higher lipid content than the dorsal  content, while those fed olive pomace oil diets (O or OA) did for MUFA (p < 0.001). However, the differences in MUFA and PUFA composition observed between acid oil and their corresponding crude oil diets are more clearly reflected in the FA profile of perivisceral fat than in the two sections of muscle . When compared with animals fed F, higher UFA: SFA and lower n − 3: n − 6 ratios (p < 0.001) were observed in animals fed VO diets. In perivisceral fat, the EPA and DHA contents in treatments including VO were about 30–33% and 43–52%, respectively, of those of F. Higher content (with respect to F) was obtained for the dorsal  and ventral . Moreover, higher DHA content was obtained for the dorsal muscle compared to the ventral muscle (p = 0.046), but no other significant differences were observed in the FA profile of the dorsal and ventral sections.The FA profiles of the dorsal and ventral sections of the muscle and perivisceral fat are presented in In the present study, the level of replacement of FO was formulated according to previous studies in rainbow trout and gilthead seabream ,15,16,29Both carcass and muscle yields are parameters that may be useful for the industry, as the main valuable final product of aquaculture production is the fillet. However, as far as we know, no studies have assessed the effect of different VO or dietary FFA content on carcass parameters or flesh yield. In the present study, the percentage of FO replacement (75%) with VO  had no effect on carcass parameters or flesh yield. In addition, lower gross flesh yields were obtained in the present study (40% to 43% of BW) than those reported by Lanari et al.  and VandThe replacement of FO with crude or soybean-sunflower and olive pomace acid oils or blends had no effect on the digestibility of dry matter and crude protein. These results are in agreement with other studies that found no differences in digestibility of nutrients when FO was replaced with different VO . In contSimilar to the observation for ADC of lipids, a decrease in total FA digestibility was observed for the use of acid oil diets, although it was not significant. Trullàs et al.  reportedL* values in thawed muscles, in agreement with other authors who have confirmed that freezing and storage generally increases flesh brightness [In species with a white flesh colour, such as European seabass, preservation of the expected whiteness is a key attribute for determining sensory quality with regard to consumer acceptance . Acid oiightness ,50,51.When the chemical composition and liquid holding capacity of thawed muscle are considered, a non-significant effect of replacing FO with crude and soybean-sunflower and olive pomace acid oils was obtained. These results are in agreement with those found when replacing FO with crude VO such as soybean, rapeseed, linseed and olive oils ,35,52,53The results of the present study suggest that there is no effect of dietary FFA content on the FA profile of flesh and perivisceral fat, but it is affected by the dietary FA profile. The slight differences between diets containing crude or acid oils were those present in the FA profile of the diets. Animals fed S or SA produced flesh that was richer in PUFA and the flesh of those fed O or OA was richer in MUFA. Hence, the inclusion of acid oils in the diets helped to obtain a final product with a similar FA profile to that obtained with animals fed their respective crude oils.Salmo salar) [Although the FA composition of flesh and perivisceral fat reflected that of the diet, differences in n − 3 HUFA and C18 FA concentrations were less marked in the FA composition of flesh than that of perivisceral fat. The same effect was observed in other studies performed in European seabass ,35, in Ao salar) ,57, in ro salar)  and in go salar) . This coo salar) ,59, whilo salar) ,56. Regao salar) , and couIn conclusion, the substitution of fish oil with different vegetable oils (75%) showed different results depending on the botanical origin and free fatty acid content of the experimental oils. The inclusion of soybean-sunflower acid oil as a replacement for soybean oil does not have a negative effect on performance, feed efficiency or the studied flesh parameters. In contrast, the inclusion of olive pomace acid oil as a replacement for olive pomace oil impaired performance and feed efficiency. However, the negative effects observed for the inclusion of olive pomace acid oil alone disappeared when acid oils were included in a blend with soybean oil or soybean-sunflower acid oil. It is important to note that although similar performance and feed efficiency results could be achieved by including acid oils instead of their respective crude oils, the digestibility of lipids decreased. Hence, the correct evaluation of acid oil quality parameters such as MIU would help to incorporate acid oils in aquaculture diets, since they are highly variable sources in terms of composition.The present results offer a view on the preliminary step for the potential use of acid oils in farmed fish species. However, further studies assessing the effects of the inclusion of these oils on metabolism, immunology, intestinal health and product quality are needed before recommending their use.


Ovarian tissue cryopreservation transplantation (OTCT) is the most flexible option to preserve fertility in women and children with cancer. However, OTCT is associated with follicle loss and an accompanying short lifespan of the grafts. Cryopreservation-induced damage could be due to cryoprotective agent (CPA) toxicity and osmotic shock. Therefore, one way to avoid this damage is to maintain the cell volume within osmotic tolerance limits (OTLs). Here, we aimed to determine, for the first time, the OTLs of ovarian stromal cells (OSCs) and their relationship with reactive oxygen species (ROS) and mitochondrial respiratory chain activity (MRCA) of OSCs. We evaluated the effect of an optimal dose of melatonin on OTLs, viability, MRCA, ROS and total antioxidant capacity (TAC) of both human and bovine OSCs in plated and suspended cells. The OTLs of OSCs were between 200 and 375 mOsm/kg in bovine and between 150 and 500 mOsm/kg in human. Melatonin expands OTLs of OSCs. Furthermore, melatonin significantly reduced ROS and improved TAC, MRCA and viability. Due to the narrow osmotic window of OSCs, it is important to optimize the current protocols of OTCT to maintain enough alive stromal cells, which are necessary for follicle development and graft longevity. The addition of melatonin is a promising strategy for improved cryopreservation media. Advances in cancer diagnosis and treatment have reduced cancer mortality rates and expanded lifespans of cancer survivors ,2. HowevAlthough ovarian tissue cryopreservation has been used at many clinics around the world to preserve female fertility, and approximately 200 live births have been reported using this technique , there aWorldwide, approximately 48.5 million couples (15% of couples) are estimated to be affected by infertility, and oxidative stress has been linked to infertility in both males and females ,25. ReceIn many cell types, including sperm , oocytes+/K+ ATPase, the membrane permeability to water, the lipid phase transition temperature, or ion and water channels, may determine the susceptibility of cells to osmotic stress, which alters their tolerance to CPA equilibration and freezing and thawing stress [Cryopreservation-induced alterations in the volume of the cells in ovarian tissue has been suggested to be a source of cell damage and death, possibly based on the speed and intensity of volume changes . The litg stress ,44. Mostg stress ,46, whicDuring cryopreservation, all cells are exposed to the toxic effect of CPAs and the detrimental effects of freezing and warming steps , but a qWhile the primordial follicles seem to be resistant to the detrimental effects of cryopreservation, and oocytes appear to be preserved after ovarian tissue cryopreservation, various investigations have found that the freezing–thawing method has a deleterious impact on stromal cells, especially when slow freezing methods are used ,57. SlowN-acetyl-5-methoxytryptamine), which is a highly lipophilic molecule, can easily cross the cell membranes and is mainly produced and secreted by the pineal gland [Another candidate antioxidant for protecting ovarian tissue during cryopreservation is melatonin  of OSCs. This novel connection between osmotic stress and ROS lends itself to antioxidant mitigation. Thus, here, we evaluate the effect of melatonin as an antioxidant on osmotic sensitivity, viability, MRCA, ROS production levels and total antioxidant levels of both human and bovine OSCs, plated and suspended, to clarify the influence of cell–cell surface interaction on osmotic tolerance, we divided them in suspended and adherent groups to have better understanding about their reaction in tissue following exposer to osmotic stress. Our results provide fundamental information to improve current approaches for the cryopreservation of ovarian tissues.All chemicals were purchased from the Thermo Fisher Company  unless otherwise stated.n = 24). The samples were then placed in a sample container containing MEM medium supplemented with 25 mM HEPES, 1% streptomycin-penicillin G. Ovaries were subsequently transported to the laboratory on ice at 4 °C within 30 min. Under a laminar flow hood, the exterior of the ovaries was washed once with 75% alcohol and then three times in the fresh medium with supplements as described above.Animal ethics approval was obtained from the University of Saskatchewan University Animal Care Committee . Pairs of whole ovaries were obtained from bovine cows, aged from 14 to 18 months, at a local abattoir . Human ovarian biopsies were collected from three patients (40 to 48 years old) who underwent an oophorectomy. Informed consent was obtained from patients. Ovarian tissues were retrieved by laparoscopy and transported to the laboratory on ice within 20 min in the above-mentioned fresh media.2 for 45 min. The tissue was mechanically dispersed by pipetting digested tissue every 15 min. Following the incubation, an equal volume of cold DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS) was added to inactivate the activity of the enzymes. Then, the digested cell suspension was consecutively filtered through sterilized 80, 40 and 11 µm nylon net filters  and subsequently centrifuged at 500 g for 5 min. The pellet was then re-suspended in 5 mL of DMEM-F12 supplemented with L-glutamine, sodium bicarbonate 1% streptomycin-penicillin G and 10% FBS. Cells were then counted, and 1 × 106 cells were loaded into a T75 culture flask. The cell culture flask was placed in an incubator at 37 °C and 5% CO2. The culture medium was exchanged with fresh culture medium every two days. When the confluency of cultured stromal cells reached approximately 80%, they were trypsinized with Trypsin-EDTA (0.25%)  to disperse the cells into suspension or for use in further experiments. After an initial viability assessment using trypan blue staining, the volume of the cell suspensions was adjusted to equalize the cell density in each well. The cell suspension was divided into two parts and each part was used for the different experiments. One part of the cell suspension was seeded into a 96-well plate at a density of 1 × 104 cells per well and cultured for 2 days, which is when the cells reached an approximate confluency of 80% before experimentation. The second part was used for suspended cell OTL assessments in the concentration of 4 × 104 cells per group.Under the laminar flow hood, following the removal of surface epithelium and medulla with surgical scissors and scalpel, the ovarian cortex was cut into small fragments of 1 × 1.5 × 0.5 mm. OSCs were isolated following the methods described by Soares et al.  with somThe cell count and viability assessments were performed using trypan blue staining. Briefly, 100 µL of the stromal cell suspension was transferred to a microtube and incubated with an equal volume of 0.4% trypan blue solution for approximately 1 min at room temperature (RT). 10 µL of cell suspension was then loaded in a hemocytometer (counting chamber) and observed under a bright-field microscope.To evaluate OTLs, hypertonic and hypotonic solutions were prepared using distilled water, sucrose solution and isotonic culture medium. Hypotonic solutions were prepared by the addition of sterile distilled water to the isotonic culture media to produce solution osmolalities of 25, 50, 75, 100, 150, 200, 250 and 275 mOsm/kg. Hypertonic solutions were made by mixing sucrose solution with culture media to create solution osmolalities of 325, 350, 375, 400, 425, 450, 475, 500, 700 and 1000 mOsm/kg. The osmolalities of test solutions were measured using a freezing point depression osmometer (Fiske 2400 Multi-Sample Osmometer) and then sterilized by filtering through a 0.22 µm pore filter .2, followed by 5 min equilibration with isotonic culture media in the incubator. To measure the cell viability, the cells were washed with an isotonic culture medium and exposed with 2 µL calcein-AM and then returned to the incubator for 45 min at 37 °C and 5% CO2. As a negative control for calcein-AM (dead cells), cells were exposed to 3% formaldehyde for 30 min at 4 °C to induce cell death and subsequently subjected to the same wash procedures using the isotonic culture media. As a positive control for calcein-AM (live cells), cells were subjected to the same washing procedures using the isotonic culture media. To evaluate the background fluorescence of dye, calcein-AM was added to a subset of cell-free wells. All the fluorescence procedures were performed in a dark room or with covered plates or tubes. Calcein fluorescence intensity was then quantified using a plate reader (Varioskan Flash). The excitation and emission wavelengths were 485 nm and 530 nm, respectively. The obtained fluorescence intensity was normalized with the control group to calculate the percentage of viability in all experimental groups.The osmotic sensitivity of stromal cells was evaluated by the percentage of viable cells exposed to a range of anisotonic solutions compared to control groups (300 mOsm/kg) . The via2. After incubation, the formation of fluorescent dichlorofluorescein, as a product of DCFH-DA oxidation in the presence of ROS (primarily hydroperoxide), was measured by using a fluorescence microplate reader (Varioskan Flash) at excitation/emission wavelengths of 500/530 nm. Each sample was tested in duplicate, and results were expressed as the fluorescence intensity. As a negative control, buffer solution without DCFH-DA was added to the cells. As a positive control, cells were exposed to 500 μM H2O2 for 30 min. To assess the background fluorescence of dye, DCFH-DA was added to a subset of cell-free wells. All the fluorescence procedures were performed in a dark room or with covered plates or tubes.To detect intracellular ROS levels, 2′, 7′-dichlorofluorescein diacetate  as a fluorescent oxidative dye was used . Both ad2. After the intracellular reduction in resazurin to resorufin, the fluorescence intensity was measured using a plate reader at 530 nm excitation and 590 nm emission. Two wells of each 96-well plate were allocated for each group. As a negative control, cells were exposed to 3% methanol for 30 min at 4 °C to induce cell death. As a positive control, cells were subjected to the isotonic culture media without the dye. To evaluate the background fluorescence of dye, resazurin was added to the cell-free wells. The resorufin-induced fluorescence intensity was normalized by the control group both in adherent and suspended cells. Then the reduction in MRCA was computed by the difference between the normalized results of the calcein-AM test and resazurin test in each group. Both assays are used to identify cell viability, but with different approaches. Therefore, the difference between normalized results shows the exact amount of reduction in MRCA. Since we seeded the same number of cells in both treatments, we expected to have the same number of viable cells after exposure to a similar osmotic condition [To evaluate MRCA, resazurin  as an indicator of cellular metabolic ability was used. Cells in all groups were subjected to resazurin (6 mM) for 60 min at 37 °C and 5% COondition .2, followed by a 5 min equilibration with isotonic culture media in the incubator. These concentrations of melatonin were selected based on previous studies [After measurement of the osmotic sensitivity of stromal cells, both suspended and adherent bovine OSCs were exposed to selected hypoosmotic (25 mOsm/kg and 150 mOsm/kg) and hyperosmotic (500 mOsm/kg and 1000 mOsm/kg) solutions containing concentrations of melatonin , 0.001 mM, 0.01 mM, 0.1 mM, 1 mM and 10 mM)  and incubated for 15 min at 37 °C and 5% CO studies ,78. To dAfter determination of the optimal dose of melatonin, both human and bovine OSCs were exposed to an isosmotic solution  with or without supplementation with the optimal melatonin dose (0.1 mM). Then, the total antioxidant levels, ROS level, apoptosis, viability, cells proliferation rate and the MRCA were assessed in both species.2+ working solution for 90 min on a shaker at room temperature. The small molecule and protein antioxidants convert Cu2+ ions to Cu+. Absorbance was measured using a microplate reader (Varioskan Flash) at 570 nm. TAC was calculated using a standard curve, and results were expressed as antioxidant concentration (mmol). Each sample was tested in duplicate. Trolox   was used as a standard to determine the Trolox equivalent capacity of the tested samples.The total antioxidant capacity (TAC) of the stromal cells in all groups was evaluated using the colorimetric Total Antioxidant Capacity Assay Kit  according to the manufacturer’s protocol. Briefly, suspended and adherent cells in different groups were collected and washed in cold PBS. Then, cells lysed using ddH2O supplemented with 0.05% Triton and homogenized quickly. TAC was examined by incubating lysates with the Cup < 0.05.SPSS software (version 23) was used to analyze the data. The data were tested for normality by using the Kolmogorov–Smirnov test. One-way analysis of variance was used to compare the mean values across the groups, followed by Turkey’s or Tamhane’s tests. Correlation analysis was conducted using Pearson’s r tests. The results are presented as the mean ± standard error. In the current study, statistical significance was set at 2 = −0.925, r = 0.962) for suspended cells and  for adherent stromal cells. This value showed significant correlation in hyperosmotic conditions, which was  and  for suspended and adherent stromal cells, respectively.The effects of the anisotonic treatments on the viability of bovine OSCs, normalized to the isosmotic group (control group), and their relevant p value is shown in 2 = −0.944, r = −0.972) for suspended stromal cells and  for adherent stromal cells. This correlation was also observed in hyperosmotic conditions, which was  and  for suspended and adherent stromal cells, sequentially (p < 0.05).In humans, a significant correlation was observed between the degree of hypoosmolality and stromal cells viability,  and adherent stromal cells . In hyperosmotic conditions, positive correlation was observed between the degree of hyperosmolality in hyperosmotic conditions and the ROS level production, which was  and  for suspended and adherent stromal cells, respectively. In the human cells, the relation between hypo-osmolality conditions and ROS production were significant with a value of  for suspended cells. These variables showed positive correlation with a rate of  for adherent stromal cells. Our result showed that hyperosmolality is related significantly with ROS levels in suspended stromal cells  and adherent cells  (p < 0.05).ROS level results and their related p value in the bovine adherent and suspended stromal cells exposed to the range of anisotonic conditions are indicated in 2 = −0.970, r = −0.985) and adherent stromal cells , and these results demonstrate a significant correlation between the degree of hyperosmolality and MRC activity, which was  and  for suspended and adherent stromal cells, respectively. In human cells, our results showed a significant reduction in MRC activity in the selected anisotonic solutions compared to the control group  for suspended cells and  for adherent cells. The deviation from isosmolality to the hyperosmotic condition showed significant negative correlation with the MRCA with the value of  for suspended cells and  for adherent cells (p < 0.05).The impacts of the hypo-osmolality or hyperosmolality conditions on MRCA of both adherent and suspended bovine OSCs with their p values are presented in ol group B. Additip values are presented in The impact of the different concentrations of melatonin on OSCs viability is presented in p values are presented in After incubation of ovarian adherent and suspended stromal cells with the above-mentioned chosen anisotonic solutions supplemented with the optimal dose of melatonin (0.1 mM), cell viability was evaluated both in human and bovine cells . Based op values are presented in As mentioned above, when stromal cells were subjected to hypotonic or hypertonic conditions, a significant increase in ROS generation was detected. However, the addition of 0.1 mOsm/kg melatonin to selected anisotonic solutions significantly reduced the osmotic stress-induced ROS levels in treated groups compared to control groups in both human and bovine OSCs. These results and their related p values are presented in Based on our data shown in p value are presented in The result of TAC in all previously mentioned groups and their related In recent years, significant progress has been made toward successful OTCT. However, there are still challenges that include massive follicle loss and short graft longevity after transplantation . TherefoPrevious studies on different cell types indicated the importance of OTLs and the role of osmotic stress in actin filament disruption , membran2+ is increased by osmotic stress and it may cause the activation of the calpain protein  that can accelerate apoptosis via the cleavage of the Bcl-2 family members and then triggers cytochrome c release and caspase-3 activity [An alternative or additional pathway that could be affected is the release of calcium. Caactivity . In hypeactivity .Our results indicated that supplementation of MLT in anisotonic solutions can increase viability and decrease osmotic sensitivity of human and bovine stromal cells. A large body of evidence supports the positive effect of MLT addition to freezing media on cell viability and cell apoptosis during cryopreservation in different cell types ,94. Prev2+ in the cytosol) as well as ROS formation [Another crucial aspect is the role of extra ROS in OSCs viability/apoptosis. Studies in other cell types indicated that an elevated level of ROS may result in mitochondrial impairment and activate the mitochondrial apoptotic pathway ,102. Briormation . Sarcoplormation ,108.2+ into the cytosol and subsequently Ca2+ may flow into mitochondria and induce NOX activity and ROS production. Both Ca2+ and ROS work as secondary messengers in cell signaling and cooperate bidirectionally. Indeed, ROS can control intracellular Ca2+, and enhanced Ca2+ levels can trigger a mitochondrial permeability transition and motivate ROS production. In agreement with previous studies, this study reveals that MLT reduced osmotic stress-induced ROS in human and bovine OSCs. This result is in agreement with previous reports [Based on the above findings, another explanation is that the osmotic stress may trigger the release of Ca reports ,110, sho reports . Another reports . Additio reports .This research reveals that osmotic stress might reduce total antioxidant levels in human and bovine OSCs in isotonic conditions and MLT can ameliorate negative effects of osmotic stress on antioxidant levels, which is in line with previous studies. Li et al. showed that in primary human corneal epithelial cells, hyperosmolality conditions reduced expression of the nuclear transcriptional factor (Nrf2) which is responsible for reduction in ROS levels and increasing the total antioxidant level via regulation of antioxidant gene expression, such as NAD(P)H quinoneoxido reductase 1 (NQO1), glutathione peroxidase (GPx) and glutamate cysteine ligase (GCLC) . SimilarOne of the main sites of ROS production in cells is mitochondria . PreviouAlthough many studies have showed that melatonin is a powerful antioxidant and free radical scavenger, some studies suggest that melatonin might act as a pro-oxidant and promotes the generation of ROS in some cells ,129, mosFinally, our results indicate that OSCs show similar levels of sensitivity to osmotic stress in suspended and adherent groups, and the morphology of the cells related to the cell-cell/surface connection had no significant effect on osmotic tolerance. In agreement with our finding, it is reported that membrane hydraulic conductivity is similar between isolated and adherent cells, suggesting a small impact of the cytoskeletal structure on the water flow rate. In addition, this previous study indicated that the surface area involved with water transport in adherent PC-3 cells was almost the same as one in isolated cells. Therefore, the level and rate of dehydration is similar in PC-3 suspended and adherent cells . On the In conclusion, in this study, the osmotic sensitivity of human and bovine OSCs was defined using cell viability. Our results show that a reasonably safe osmotic range for OSCs is approximately between 200 and 375 mOsm/kg for bovine and approximately between 150 and 500 mOsm/kg for human OSCs. This fundamental information explains why OSCs and, thus, ovarian tissue, are extremely sensitive to cryopreservation-induced damage. Moreover, the current study shows that osmotic stress can cause elevated levels of ROS generation and reduce the MRCA as well as antioxidant level and cell viability. Our results showed conclusively that exogenous melatonin can ameliorate some of the detrimental effects of osmotic stress. Since stromal cells have very narrow OTLs, these data are an important and useful tool for the rational optimization of the current protocol of OTC, allowing the functional preservation of enough stromal cells to enable post-graft follicle development and extended graft lifespan, and the addition of melatonin at the optimal concentration of 0.1 mM to ovarian tissue cryopreservation media is a promising option for clinical application.


Recent trends in ecological agriculture practices are focused on finding optimal solutions for reuse and recycling of pelt waste from tannery industry. In this context, new collagen-based hydrogels with NPK nutrients encapsulated have been functionalized with synthetic and natural additives, including starch and dolomite, to be used as composite fertilizers. Possible interaction mechanisms are presented in case of each synthetic or natural additive, ranging from strong linkages as a result of esterification reactions until hydrogen bonds and ionic valences. Such interactions are responsible for nutrient release towards soil and plants. These fertilizers have been adequately characterized for their physical chemical and biochemical properties, including nutrient content, and tested on three Greek poor soils and one Romanian normal soil samples. A series of agrochemical tests have been developed by evaluation of uptake and leaching of nutrients on mixtures of sand and soils. It was observed that the clay soil exhibits a higher adsorption capacity than the loam soil for most of nutrients leached from the composite fertilizers tested, with this being correlated with a slower control release towards cultivated plants, thus assuring efficiency of these collagen-based composite fertilizers. The most significant effect was obtained in the case of collagen-based fertilizer functionalized with starch. According to the principle of “circular economy”, most of the treatment techniques applied to waste mainly aim at their reuse and recycling while substantially reducing environmental pollution . In thisThese treatments are made in order to ameliorate the biological transformation of organic matter for obtaining a stabilized polymeric bio-composite avoiding any potential risks for plants and soils, while these products are applied as organic amendments. By adding such organic amendments as compost into the soil, major influences are exerted on the physical–chemical properties of the soil, able to modify the acids–bases relationships, with beneficial effects on increasing total cationic exchange and buffering capacities of the soils, as has been intensively studied during the last decades ,10,11.Our interest is focused on bovine leather recycling, which is generally directed towards obtaining protein composites by biochemical treatments using microorganism enzymes and obtaining protein hydrolysates and protein binders with different uses. Such organic biopolymers are an important source of raw materials for agriculture, as the protein waste matrix provides sufficient elements to improve the composition of poor and degraded soils, and many plants can benefit from elements such as nitrogen, calcium, magnesium, sodium, and potassium ,13.A way to valorize the untreated fleshing and trimming, bovine hide waste is the product of a three-dimensional molecular network named hydrogels by cross-linking the proteins hydrolyzed with polymers based on polyacrylamide, polyvinyl alcohol, oligo oxyethylene methacrylate, acrylic acid, maleic acid, cellulose, starch, and gum, which form three-dimensional molecular networks. The hydrogels enriched with nutrients C, N, P, and K can be used as efficient amendments in agriculture for degraded soils ,15.Such an approach meets the stringent demand to counteract the decline of soil fertility and productivity, which is in line with growing interest in the general improvement of the quality of soils by adding organic amendments from different sources ,17.Indeed, for ecological reasons, other solutions for reuse and recycling of pelt waste from the tannery industry have been proposed, one of them being for collagen hydrolysate with micronutrients incorporated to be used as fertilizers for poor soil’s rehabilitation ,19,20.Our previous works ,22,23 reThe current work aims at testing these new biocomposite fertilizers for their nutritive characteristics and nutrient-leaching properties. In this respect, detailed study of the physical–chemical and biochemical characterization of both fertilizers and soils was conducted in order to establish their biocompatibility and possible interactions. For each additive encapsulated, the intercalation mechanisms were analyzed and correlated with the uptake and leaching of nutrients on sand and soils. The beneficial effects in improving soil quality were proven by testing the biological amelioration of soil fertility.Multipolymeric collagen-based agro-hydrogels were prepared, using as raw material the limed hide waste (no haired) from fleshing and trimming bovine hides (lime fleshing), which was provided by SC PIELOREX tannery, Jilava, Ilfov county, Romania. Collagen hydrolysate was obtained according to our previous studies ,25. BrieIt is obvious that during acid hydrolysis for obtaining collagen hydrogel, the calcium content provided by lime is almost completely removed, and its remnant contribution will have not a significant effect on the composite quality, as the fertilizers will be applied for poor soils amendment, and these usually have carbonates (circa 30–50 mg/kg) as calcite and dolomite in their mineralogical composition.For the studied samples, the following codes were allocated:CH—collagen hydrolysate,Ref-CH—collagen hydrolysate with phosphorus and potassium nutrients encapsulated as reference sample,PSSG—Ref-CH functionalized with 5% P(SSNa-co-GMAx) copolymer,POLY—Ref-CH functionalized with 5% poly-acrylamide,AMI—Ref-CH functionalized with 5% starch,DO—Ref-CH functionalized with 5% dolomite.Physical and chemical analyses of soils were performed as follows:pH was determined by potentiometric method in aqueous suspension of soil:water ratio 1:2.5, with glass-calomel combined electrode; the potentiometer was calibrated with buffer solutions of known pH prior to the analysis of samples.Organic matter (humus), OC%, was determined according to STAS 7184/21-82, volumetric method, and by wet oxidation modified Walkley–Black method.2SO4 at 350 °C and potassium sulphate and copper sulphate catalyst—SR ISO 11261:2000.Total nitrogen (TN%) was determined by Kjeldahl method: digestion with HTotal content of soluble salts (extraction ratio 1:10)—STAS 7184/7–87; SR ISO 11265 + A1:1998.Exchangeable  phosphorus (EP%) followed these steps: extraction according to Ègner–Riehm–Domingo procedure and was then determined by spectrophotometry (CINTRA 404 UV–VIS spectrometer) with molybdenum blue after reduction with ascorbic acid .Exchangeable  potassium (EK%) was obtained by extraction after Ègner–Riehm–Domingo procedure and then determination by flame photometry (Flame Photometers S-935).Alkaline soil carbonates of the air-dried soil fraction (particle size < 2 mm) were estimated by calculation of measured carbon dioxide from carbonates decomposed with hydrochloric acid (1:3)-STAS 7184/16–80, total phosphorus by spectrophotometry (CINTRA 404 UV–VIS spectrometer), and total potassium by flame photometry (Flame Photometers S-935) .2O; this means a low-to-reduced fertility state. In contrast, for soil S4, phosphorous was present in the amount of 21 mg/kg P2O5 and potassium 144 mg/kg K2O, with such values corresponding to a high fertility state (>20 mg/kg P2O5 and >80 mg/kg K2O).Soil samples considered as poor soils were collected from three different regions of western Greece: Kernitsa Achaias (S1), Neochori Messolonghiou (S2), and University Patras (S3). For comparison purposes, a fourth sample of normal soil was collected from Aldeni, Buzau, Romania (S4). They belong to the following textural classes: S1—loam soil (S1-L), S2—clay loam soil (S2-CL), S3—silty clay loam (S3-SiCL), and S4—sandy clay loam (S4-SCL). The fertility state of poor and normal soils was assessed by the function of their nutrient content; indeed, for the samples S1–S3, extractible phosphorous was below detection limit, while extractible potassium was below 50 mg/kg K−1 to 10−9 g of soil/mL suspensions. In every following technique described, the proper dilutions within this interval were chosen in order to be applied on the selective media, and the developed microorganisms are expressed as colony-forming units per gram (CFU/g). Each experiment was conducted in triplicate.All samples were collected in sterile containers at a depth of 10 to 30 cm and stored at 4 °C in the dark for no more than 48 h prior to microbial examination. Serial dilutions of soil samples were made, followed by sterile deionized water addition in order to achieve 10The number of CFU of cultivable aerobic mesophilic bacteria was performed by standard plate counting method: 1 mL of the chosen diluted samples, prepared as described above, were added to a Petri dish plate (9 cm) and followed by the addition of 15 mL of Plate Count Agar (PCA) , which was prepared according to manufactures instructions. Plates were then incubated aerobically for 24 to 72 h at 30 ± 1 °C. In order to determine the number of CFU of cultivable proteolytic bacteria present in the soil samples, the proper dilutions were counted onto Plate Count Agar supplemented with 10 g/L of skimmed milk (PCA-SM)  in Petri dishes (9 cm). Plates were incubated aerobically for 24 to 72 h at 30 ± 1 °C. The number of CFU of cultivable soil yeasts and molds (fungi) was determined using the proper dilutions of the soil and spread on Petri dishes (9 cm) containing 20 mL of Rose Bengal Chloramphenicol Agar (PBCA) , which was prepared according to the manufacturer instructions. All plates were incubated aerobically for 3 to 5 days at 20.5 ± 1 °C. To determine the number of CFU of cultivable actinomyces in the soil samples, the proper dilutions of soil were used in order to inoculate Petri dishes (9 cm) containing 20 mL of Sheep Blood Agar , which was prepared according to the manufacturer instructions. All plates were incubated in anaerobic conditions for 3 to 7 days at 35 ± 1 °C.Microbial biomass of Romanian soil S4-SCL amended with fertilizer was determined by indirect methods, such as substrate-induced respiration and fumigation extraction methods , accordiEvaluation of functional bacteria of soil microorganisms was made by CLPP method: Community Level Physiological Profiling .Method. Soil poor in nutritive elements sampled from the western Greece region was placed in an aluminum vessel and mixed with the agro-hydrogel as liquid, dried, or gel in various final proportions: 1%, 5%, 10%, 30%, and 50%. Portions of these mixtures were sampled at different time intervals  and analyzed for ammonium leaching. Changeable ammonium could be evaluated by elution of the soil mixture with a solution 2.0 M KCl, and ammonium nitrogen was determined by a modified Kjeldahl method. The experiments were performed in triplicate. On the graphs are shown the average values. The standard deviation was between ±5%.The agrochemical tests for characterization of fertilizers with slow release and determination of their percolation degree in soils are mainly defined by SR EN 13266/2002—Fertilizers with slow dissolution rate—Determination of nutrients leaching and SR CEN/TR 14405/2009—Waste characterization. Behavioral tests on leaching. Percolation test in ascendant counter-flux.Method. An amount of 5 g of each fertilizer tested was completely dissolved into 5 L distilled water and transferred through a column. A constant elution flux of 225 mL/h was assured for each fertilizer. Volumes of 200 mL of leaching were collected and analyzed for their ammonium, nitrate, and phosphate ions contents. Experiments were performed at 25 °C in triplicate.Physical–chemical characterization of composite agro-hydrogels is given in The fertilizer quality is mainly conferred by the content of NPK nutrients available for plant growth and their leaching in soil solutions. The absolute values in nutrient content are reflected by the elemental analysis data given in 10P6K10, which is significant for agriculture application.According to these data, one may observe that all the fertilizers exhibits quite similar chemical composition, as functionalization with 5% synthetic or natural additive does not result in a major change in proportion of NPK content, with this corresponding to the general chemical formula NAfter the preliminary agrochemical tests, a special attention was directed to the collagen hydrogels functionalized with natural compounds, such as starch (AMI) and dolomite (DO), which fulfilled both requirements referring to fertilization efficiency and those related to economic aspects, as these are available as waste and are therefore cost-effective. In this respect, the agrochemical characteristics of AMI and DO fertilizers were determined by values of nutrient content soluble in water, expressed by total nitrogen, phosphorus, potassium, and total content of soluble salts. The values of pH and electrical conductivity were determined in aqueous suspensions of various concentrations ranging between 0.5% and 10%. The results are collected in The data on the microbial analysis of Greek soils by standard plate counting method are available in the 6, which proves a low microbial activity, with these soils being poor in organic compounds.As can be seen from For a normal soil sample, special microbiological tests were conducted in order to prove the biological amelioration of soil fertility while applying the best-performant collagen hydrogel functionalized with starch. These experiments were performed on a sandy clay loam soil , having as reference sample the moist soil as compared with two samples of soil humectated with aqueous suspensions 0.1% and 0.2% of AMI hydrogels. The main biologic indicators are collected in 9, being a fertile soil rich in biodegradable organic compounds when compared with data from This normal soil sample (S4-SCL) exhibits an initial number of microorganisms at the magnitude order of 10Tests on leaching ammonium and phosphate ions from composite fertilizers were performed on columns filled with gravel and sand in one series of experiments and in another series on columns filled with gravel and a mixture of sand with each of two Greek soils (80 g sand and 20 g soil from samples S1-L and S2-CL). The initial content in nitrogen and phosphorus as water-soluble ions in the fertilizers tested, Ref-CH, AMI, and PSSG, are as follows: 426.5, 363.75, and 588.5 mg N/g and 75, 85, and 83 mg P/g, respectively.The evolution in the time of ions leaching as ammonium and phosphate on columns with gravel and sand is illustrated in From the analysis of In case of the PSSG fertilizer, four zones can be distinguished. One zone of rapid release was in the first 2 days when circa 45% of ammonium nitrogen leached, followed by a slow-release zone of 2% during the next 10 days, with the third zone being once again a rapid release of circa 43% during 20 days, and the remaining nitrogen was slowly released with a rate of 3% per day. The difference between the ammonium-releasing rate of these two fertilizers is in accordance with stronger covalent bonds established in case of synthetic polymer PSSG, when there are some esterification reactions involving various functional groups of the collagen chains, while in the case of starch, the hydrogen bonds are mainly responsible for the affinity of the bio-fertilizer components, as will be discussed in the next section.Reference fertilizer Ref-CH not functionalized has a relative constant rate of ammonium nitrogen leaching after 2–3 days, when 20% of nitrogen was rapidly released; during the next 28 days, the rate of release was 2%/day, while in the last 40 days, the release slowed down to 2%/day.PSSG fertilizers and over 95% in case of AMI. In the next 60 days, the rest of the phosphorus was released, until 98% in case of AMI, 92% for Ref-CH, and 80% for PSSG. This similar behavior of the three fertilizers can be explained by similar structures of these composites, of which the polymers are encapsulated inside the hydrogel matrix between two collagen chains, while the potassium phosphate groups remain outside the capsule, facilitating their easier release towards soil and plants.In contrast to ammonium nitrogen release, in the case of phosphorus, the leaching behavior is quite similar for the three fertilizers tested on sand: with very rapid release during the first five days, when over 90% of phosphorus was released by Ref-CH and The above experiments can be relevant for the sandy soil sampled from Romania (S4-SCL).Similar experiments were carried out for leaching ammonium and phosphate ions of these fertilizers on columns filled with a mixture of sand and two of the tested poor soils (S1-L and S2-CL) in a proportion 4:1 of sand to soil.Aliquots sampled from solutions of each fertilizer were analyzed before (fraction adsorbed) and after passing (fraction leached) through the columns filled with mixtures of sand and soils S1-L and S2-CL, respectively, and the data were graphically represented in PSSG, while for ammonium nitrogen, the adsorption capacity reached its maximum values after 6 h for all three fertilizers tested  (P(SSNa-co-GMAx-PSSG) and two natural compounds, starch and dolomite, are presented in Completely different types of interactions are established during encapsulation of starch and dolomite into hydrogel matrix. The highly saturated structure of natural polymer starch, dominated by α1–4 glycoside linkages along linear chains of amylose and α1–6 glycosidic linkages at branch points in amylopectin, confers a lower reactivity with collagen groups. In this way, higher compatibility between the two biopolymers is assured by means of numerous hydrogen bonds established by glycoside groups of starch with hydroxyl and amino groups of collagen, while only a few carboxyl groups can be involved in esterification reactions. Such rather low- or medium-strong bonds add to this biopolymer composite enough stability when is applied on the soil and at the same time assure an easy release of NPK nutrients together with additional biodegradable organic carbon content.When dolomite is encapsulated within NPK collagen hydrogel, the composite consistency is mainly assured by means of hydrogen bonds between carbonate oxygen and hydrogenated groups of hydrogel, such as carboxyl and hydroxyl, but some ionic interactions caused by calcium and magnesium cations could not be neglected, which could facilitate the ionic exchange toward soil and plants.These interaction mechanisms will provide the theoretical support for further interpretations of the specific behavior of these new composite fertilizers on uptake and leaching toward various types of soils.Improving soils’ fertility by adding various amendments containing organic components is mainly analyzed by means of mineralization of carbon and nitrogen  and theiIn this context, a comprehensive study was performed on these new agro-hydrogels referring to their biodegradability in aerobic conditions in water and in composting environment, completed by the leaching of the ammonium, nitrate, and phosphate ions on columns filled with gravel and sand mixed with each of poor soils tested.The big difference noticed between the leaching rates of the ammonium and phosphorous nutrients can be explained by the special structure of these composite fertilizers, which contain phosphorus and potassium as ionic species incorporated into collagen hydrolysate matrix as a result of encapsulation with dipotassium phosphate, while the nitrogen is available in the covalent form of protein compounds and needs several degradation steps to be transformed from organic into inorganic species as nitrate and ammonium.max) and time to reach the equilibrium on the two tested soils for phosphorus and nitrogen nutrients  and that functionalized with starch (AMI) for phosphorus as a result of its encapsulation in ionic form, as it is a more easily exchangeable species than nitrogen, as explained before. At the same time, the leaching period was quite similar in all the cases, around 8–9 h, which is correlated with nutrient control leaching from soil to the plants. These time intervals in hours seem to be rather short, but we should take into account that these laboratory experiments on columns were rather accelerated, as only a proportion of 4:1 sand to soil was used. It is obvious that in real agriculture conditions in the soils, these time intervals will be much longer, in accordance with vegetation period of plants, as it was also established by Anghel et al. .10P6K10 was calculated using elemental analysis data from Another useful remark after analysis of adsorption capacities values from When comparing the two poor soils analyzed here, one may notice that the clay soil (S2-CL) exhibits a higher adsorption capacity than loam soil (S1-L) for most of the nutrients released from the fertilizers tested, and this higher adsorption capacity on soil could be correlated with a slower control release towards cultivated plants, thus assuring the efficiency of these composite fertilizers.Laboratory tests emphasized the beneficial effects on soil amelioration provided by protein structure of the collagen hydrogel functionalized with starch (AMI) on some biologic indicators, as shown in Bacillus strain by developing some species attesting to the improvement in soil humidity and aeration, such as B. subtilis and B. egaterium. Frequently, some species were isolated from the zymophenic flora belonging to the Pseudomonas strain.For the soil samples treated with AMI fertilizer suspensions, the number of CFU of cultivable mesophilic organotrope bacteria was significantly increased in both concentration variants by 61–81%, indicating the stimulation of development of this taxonomic group. Simultaneously, there was an increase of species diversity belonging to Aspergillus strain, accompanied by Cephalosporium and Penicillium. The Cladosporium herbarum strain, with a role in aggregating soil particles, was isolated in both fertilization variants with protein biopolymers based on collagen hydrolysate and functionalized with starch and dolomite.The number of CFU cultivable mesophilic fungi, with a decreasing tendency, does not significantly differ as a function of treatment. In both variants, there is a number of 7–9 similar species, the most dominant being the 2 released during respiratory processes of soil, were also influenced for a 5% probability by the treatments applied, which showed an increased evolution.Physiological activities of microflora, expressed by the amount of CO2 release. This amendment variant applied for a sugar beet subculture on this sandy clay soil assures an important multiplying effect of microbial mass and soil respiration together with an equilibrated microscopic bacteria/fungi balance.The microbial biomass significantly increased by 19% for the variant with 0.1% hydrogel AMI against reference soil, with this value being mainly supported by the bacterial component of soil microflora, which assures intensification of metabolic activities by means of COOne may conclude that the use of AMI collagen hydrogel with encapsulated starch for poor soil structure amelioration positively influenced the micro-colonies in soil, creating adequate conditions for maintaining strain diversity and their equilibrium balance and thus enhancing their physiologic activities as a result of improving the biophysical properties of soil.A systematic study is presented on reusing hide waste resulting from the leather industry by capitalizing on their valuable protein components to obtain efficient collagen-based hydrolysates as composite fertilizers for poor soils’ amelioration. A series of polymer composite fertilizers was obtained by the functionalization of collagen hydrolysates with some synthetic and natural compounds after encapsulation with P and K nutrients. Two representative organic and mineral amendments, starch and dolomite, were selected, as both of them are available in high amounts as waste to be recycled.Possible interaction mechanisms during encapsulation of nutrients and synthetic or natural polymers/ore are discussed in tight connection with their uptake and leaching on studied soils and the effects on improving soil fertility.10P6K10, which is significant for agriculture applications.All the composite fertilizers tested exhibited a similar composition in atomic ratio of NThe study of nutrient leaching on soils S1-L and S2-CL revealed that the soil adsorbs nitrogen and phosphorus as ammonium and phosphate ions, respectively.6, which proves a low microbial activity, with these soils having a low organic content, while for a normal soil, the level of magnitude order of 1010 proves a high microbial activity in accordance with its high fertility.The initial number of microorganisms in three poor soils was at the level of magnitude order of 10Finally, it was demonstrated that the use of collagen hydrogel with encapsulated starch for poor soil structure amelioration positively influenced the micro-colonies in soil, creating adequate conditions for maintaining strain diversity and their equilibrium balance and thus enhancing their physiologic activities as a result of improving the biophysical properties of soil.


Broilers were injected intraperitoneally with 5'-bromo-2'-deoxyuridine (BrdU) 1 h prior to sampling to label mitotically active cells. Samples were analyzed using cryohistology and immunofluorescence to determine the density of mitotically active cells and macrophages. Mitotically active cell and macrophage densities changed in both the duodenum and ileum over time. Neither dietary protein source nor litter condition affected mitotically active cell or macrophage densities in the duodenum on d 11 and 21 or in the ileum on d 3, 8, 11, and 15. However, on d 3 and 15 in the duodenum (P ≤ 0.0126) and d 21 in the ileum (P ≤ 0.0009), broilers reared on UL had greater mitotically active cell densities than those reared on NL. On d 8 in the duodenum, broilers fed MBM had increased macrophage density compared with those fed PFM and SBM (P ≤ 0.0401). These results indicate dietary protein source and litter condition may impact the physiology of the broiler small intestine, though additional work with this model is necessary to understand the underlying mechanisms.As antibiotic-free (ABF) broiler production continues to increase, understanding the development and local immune response in the intestines of ABF broilers is essential. Mitotically active cells, the majority of which will become enterocytes, help maintain the intestinal epithelial barrier. Macrophages prevent pathogen invasion by their phagocytic activity, functioning as immune response amplifying cells to aid in the recruitment of additional immune cells, and stimulating cytokine production in other adjacent cells. The objective of this experiment was to evaluate commonly used practical production practices on intestinal cell mitotic activity and local intestinal immunological responses. A randomized complete block design experiment with a 3 × 2 factorial treatment structure was conducted. The 3 dietary protein sources were: soybean meal (SBM), a mix of 50% poultry by-product meal and 50% feather meal (PFM), and porcine meat and bone meal (MBM) and broilers were reared on either new litter (NL) or used litter (UL). On d 3, 8, 11, 15, and 21, 6 birds per treatment from 6 blocks (total  The broiler industry is primarily concerned with producing broiler growth via lean muscle accretion; however, several different physiological systems support the complex process of muscle growth and development. Although necessary, these systems can place a burden on broiler metabolism and reduce the efficiency of muscle protein accretion by requiring energy and nutrients that could be utilized for lean muscle growth. It has been estimated that maintenance of the gastrointestinal tract (GIT) alone requires 25% of the total basal metabolic needs of an animal, while this tissue does not contribute as significantly as lean muscle tissue to the profit gained from broiler processing . HoweverIntestinal epithelial cells continuously migrate up the villi and are shed upon reaching the tip. Also, the intestinal epithelium provides some immune protection as its integrity must be maintained to prevent translocation of microorganisms into host tissue. Furthermore, there is a strong presence of immune cells within the GIT, and it contains the largest pool of tissue-resident macrophages found in the body . This heCurrently, the broiler industry is experiencing a shift away from the use of antibiotic growth promoters (AGP). As of 2019, more than 50% of broilers produced in the United States were reared under some type of antibiotic-free (ABF) program . In the Antibiotic-free broiler production has increased the incidence of enteric diseases like coccidiosis and necrotic enteritis (NE), both of which can induce costly production loses –11. CertAll procedures involving the use of live birds in this work were approved by the Auburn University Institutional Animal Care and Use Committee under protocol 2017-3146.n = 50 blocks, each comprised of 6 pens, with each pen representing a different treatment) with a 3 × 2 factorial treatment arrangement. Three diets were fed, differing primarily in the dietary protein source included. The dietary protein sources were soybean meal (SBM), porcine meat and bone meal (MBM), and a 50:50% poultry by-product and feather meal mix (PFM). These diets were formulated based on diets typically fed in the broiler industry, therefore, each of the diets contained some SBM, but the MBM and PFM diets included 4% MBM or PFM, respectively (This experiment was a randomized complete block design (ectively . Two dif2 per bird). On d 8, each pen was culled to 3 birds per pen (0.07 m2 per bird). Each raised floor pen was equipped with 1 feeder and 2 water nipples, providing ad libitum access to feed and water. A crumbled starter diet was supplied throughout the experiment (1–21 d). At placement, ambient temperature was set at 30.5°C and reduced to maintain bird comfort as the birds aged, with a final set point of 24.5°C on d 21. From d 0–7, birds were exposed to 30 lux of light for 23 h, and from d 8 to 21, the lighting was adjusted to 18 h of light at 10 lux with 6 h of darkness. Individual bird body weights were recorded, and pen feeder weights were collected to calculate individual bodyweight gain (BWG) and mortality corrected pen feed intake (MCFI). Mortality was recorded daily, and FCR was mortality corrected  were received from a commercial hatchery. Each bird was tagged with a wing tag for identification, individually weighed to determine d 0 bodyweight, and randomly distributed to 300 pens . Birds were injected intraperitoneally with an aqueous solution (25 mg per mL) of 5'-bromo-2'-deoxyuridine  to label mitotically active cells, as described by Hutton et al.  to remove intestinal contents. The samples were then flash frozen in liquid nitrogen and stored at −80°C. Before freezing, the cylindrical samples were cut lengthwise to better expose the mucosal surface to the liquid nitrogen and promote rapid freezing, rather than slow freezing which would damage the morphology of the tissue.Samples were held at −20°C for 24 h to bring samples to an appropriate temperature for cryosectioning. After being embedded in frozen section compound , 5-μm thick cryosections were collected from the intestinal samples, transverse to the length of the intestine, using a Leica CM 1950 cryomicrotome. The cryosections were mounted on positively charged glass slides  and stored at 4°C for no more than 48 h before immunofluorescence staining.Cryosections were fixed and immunofluorescence stained following previously published methods with some modifications as described below –20. All Primary antibodies were as follows: mouse monoclonal IgG1 anti-chicken monocyte/macrophage  and moulamina propria were counted to calculate mitotically active cell and macrophage density, which are reported as the number of positive cells per square millimeter. All nuclei determined to be positive for BrdU were also labeled with DAPI, a nuclear counterstain. Representative digital images illustrating detection of KUL01+ macrophages, including pro-inflammatory and anti-inflammatory macrophages, and mitotically active cells in duodenal and ileal cryosections of 21-d-old broilers are shown in After immunofluorescence staining, cryosections were imaged at 200X magnification using a semi-automated inverted fluorescence microscope  equipped with a solid-state LED light source (Lumencor SOLA Sm II 365 light engine). Images were captured and analyzed using an Evolve 512 EMCCF camera  and the Basic Research version of Elements software . Two representative images were captured from each slide (1 slide per bird), focusing on the crypt region of the tissue section. The area of mucosa in each image was measured, and BrdU+ mucosal nuclei and macrophages located within the P ≤ 0.05.All data were subjected to 2 and 3-way analysis of variance using the GLIMMIX procedure of SAS version 9.4 , which is suitable for both normally and non-normally distributed datasets. For the 2-way ANOVA dietary protein source and litter condition served as the fixed effects with bird as the experimental unit with 6 replicates per treatment per sampling time point. For the 3-way ANOVA, dietary protein source, litter condition, and time (bird d-of-age) served as the fixed effects to assess their impact on cell populations over time. The Satterthwaite adjustment was used to correct the degrees of freedom and the PDIFF option was used to conduct the pairwise least square means separation analysis. For all variables, the means were separated at P > 0.05). Therefore, the results are presented as fixed effects within a sampling age time point and over time by tissue (duodenum and ileum).The major goal of this work was to establish experimental procedures to assess GIT developmental  and local immune  responses while simultaneously determining how dietary protein source, litter condition, and time (bird age) impact these variables. The ability to successfully evaluate local cellular responses will improve our overall understanding of what is required to successfully feed and manage broilers in ABF production systems. Interestingly, no 2 or 3-way interactions among the fixed effects of dietary protein source, litter condition, and time (broiler d-of-age) were observed for either mitotically active cell density or macrophage density  and followed a pattern of increasing and decreasing with each timepoint thereafter  through Hatch Act funds to the Alabama Agricultural Experiment Station.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.