Adipose-derived mesenchymal stromal cells (ASC) transplant to recover the optimal tissue structure/function relationship is a promising strategy to regenerate tissue lesions. Because filling local tissue defects by injection alone is often challenging, designing adequate cell carriers with suitable characteristics is critical for in situ ASC delivery. The aim of this study was to optimize the generation phase of a platelet–lysate-based fibrin hydrogel (PLFH) as a proper carrier for in situ ASC implantation and (1) to investigate in vitro PLFH biomechanical properties, cell viability, proliferation and migration sustainability, and (2) to comprehensively assess the local in vivo PLFH/ASC safety profile . We first defined the experimental conditions to enhance physicochemical properties and microscopic features of PLFH as an adequate ASC vehicle. When ASC were mixed with PLFH, in vitro assays exhibited hydrogel supporting cell migration, viability and proliferation. In vivo local subcutaneous and subgingival PLFH/ASC administration in nude mice allowed us to generate biosafety data, including biodegradability, tolerance, ASC fate and engraftment, and the absence of biodistribution and toxicity to non-target tissues. Our data strongly suggest that this novel combined ATMP for in situ administration is safe with an efficient local ASC engraftment, supporting the further development for human clinical cell therapy. Regenerative therapies using natural or synthetic materials to promote long-lasting tissue structuration and reconstruction remain a challenge, as they still provide very unpredictable and only partial regeneration outcomes ,2,3,4. WAmong the cellular carriers proposed in regenerative medicine, platelet-derived products and especially carriers based on platelet–lysate (PL) have recently received particular attention. PL refers to the content of the platelet cytoplasm released after membrane disruption and that can be obtained from plasma platelet concentrates. Indeed, benefiting from both plasma and platelet content, such products are strongly enriched in cytokines/growth factors , PDGF (Platelet-Derived Growth Factor), EGF  or TGF-and ions ,18. Humaand ions ,20,21. Iand ions . These sand ions . This tyand ions . Howeverand ions . In addiand ions .Such an association of a carrier hydrogel and ASC are classified by the European Medicines Agency  as a coThus, the aim of this study was to optimize the generation step of the PLFH/ASC ATMP for the purpose of medical use and (1) to investigate in vitro PLFH biomechanical properties, cell viability, proliferation and migration sustain, and (2) to assess in vivo PLFH/ASC biosafety: tolerance after the local grafting, biodegradability of the PLFH carrier, ASC fate  and finally biodistribution and toxicity of the cells (study of the non-target tissues).The process for obtaining PLFH is summarized in Briefly, a pro-polymerizing solution containing  process A. In ord process B. CaCl2 bability C, leavinbability C and an bability D, showinbability C. The 0.bability C,D, is tbability F were seThe rheometer frequency sweep indicated that for the four conditions tested, the G’ modulus was greater than the G” modulus, confirming the rheological solid state of the structure and therefore the possibility of qualifying it as a gel G. The coThe Cryo-MEB investigation on PLFH demonstrated that the fibrin network organization depended on mix type. Gel mix with the greatest PL amount displayed the highest fibrin network meshes density A, Gel 1, dextran B, confir 3 and 4 C. Averag 3 and 4 D.Whatever the PLFH component proportions, ASC displayed “fibroblastic” star-like appearance with spreading and adhesion 48 h after seeding within the gel A. For eaIn vitro experiments demonstrated than Gel 2, with 69% PL and 3% PLFH ergonomics (consistency and elasticity) for surgical handling was suitable for both subcutaneous and subgingival implantation site, malleable enough to fill a tissue defect  and strong enough to retain its shape over time despite progressive degradation (subcutaneous implantation). Macroscopic examination of flank subcutis grafted sites at necropsy demonstrated the local successful engraftment, the supportive angiogenesis development and the progressive shrinking and disappearance of the smooth whitish mass over time .Similar changes were observed after subcutaneous  or subgit day 30 D,E. Thert day 30 E.Immediately after implantation (day 1), ASC were observed as scattered  non-cohesive large round cells entrapped in PLFH matrix B. Death PLFH mouse subcutaneous and subgingival PLFH delivery procedures (with or without ASC) did not result in any mortality nor local or systemic adverse effect. At necropsy, there was no recorded macroscopic changes. HE histological examination of the various sampled organs  did not reveal any findings related to the ASC on this set of distant organs, ruling out a general toxicity of the locally implanted combined PLFH/ASC ATMP. The biodistribution analysis  focused To overcome the current limits associated with therapies based on cell suspension injection, the aim of engineered biomaterial is conversely to allow a proper tissue integration by recapitulating the physiological microenvironment, thus providing essential biophysiochemical signals that may preserve stemness, direct cell fate and differentiation, promote reprogramming by acting on genomic and epigenomic features, and drive cells towards the appropriated functional phenotypes. It is assumed that the encapsulation of MSC with hydrogels enhance their therapeutic efficacy. Such 3D scaffolding of MSC before administration seems to improve local targeted delivery, increase the retention at the injury site, and provide tunable elastic materials with many tissue-like properties essential to enhance the function of MSC, including the continuous release of the MSC secretome ,30. In aIn order to develop a 3D hydrogel to enhance the ASC transplantation outcome, this study reported the development of a PL-based biomaterial for tissue regeneration from in vitro mechanical and biological characterization to in vivo preclinical testing. We were able to determine the combined ATMP safety profile and tolerance and the PLFH properties to support cells viability and migration into the host environment. It is assumed that carriers need to mimic the microenvironment of endogenous cells and may contribute to their cell fate by enhancing cell-extracellular matrix interactions.We have demonstrated that an accurate control of PL and tituents . This cotituents , whose ptituents . SEM obstituents ,34. Incr reports , impairiper se, paracrine activity of ASC and host growth factors) [The use of PL as a biomaterial provides many advantages for MSC therapy. In a coagulated form, PL organizes as a hydrogel of natural fibrin, combining ergonomic handling, adequate wound filling, functional support for grafted MSC and microenvironment conditioning close to the endogenous context. Indeed, the three-dimensional fibrin network mimics an extracellular matrix , providifactors) . PL contfactors)  that canfactors) , includifactors) ,38,39. Gfactors) ,40. HoweDuring polymerization, a dense fibrin network trapped the cells. From 72 h, ASC showed spreading and adhesion within the matrix network, without migration capacity failure. The embedding process did not alter cell viability except for gel 3 (lowest amount of PL), where preliminary observations by optical microscopy displayed a higher cell mortality compared to the other hydrogels. These results support the importance of the cell–matrix link to support cell survival . Indeed,Use of PL is compatible with the addition of other biomaterials, which could open up other fields of application and additional properties, such as collagen , hyaluroThe purpose of the current study was also to evaluate the in vivo PLFH implantation feasibility, local evolution of both the carrier and the cells and to provide biodistribution and safety data in two different local delivery sites .Only a few ATMP have received a European Union market authorization, and this rate of new product authorization is considered low compared to the authorization rates of other types of medicinal products . For celWell designed Good Laboratory Practice (GLP) compliant safety studies with sufficient large group sizes and long observation times are essential before clinical trial. However, this non-GLP preclinical study was conducted with regards to the regulatory recommendations in order to give critical preliminary results of the biosafety profile that could support the design of further studies for the translation to human . In thisThe optimal cell dose for a given delivery route is also a question to be addressed ,79. In tThe precise identification and tissue tracking of post-delivery cell products is a technological critical issue that cannot be circumvented to access to a better understanding of biology and the fate of transplanted cells and that must meet the requirements of the medical agencies and the ISSCR recommendations (reliable biomarker for long term follow-up and high sensitivity method) . We usedIn vivo implantation of PHFL with and without ASC provides data on the biodegradability and tolerance of the combined ATMP. As expected, the fibrin content of the carrier exhibited tissue biocompatibility and controlled biodegradation  The biodOverall, we were able to demonstrate the long-term survival, engraftment and tissue integration as well as collagen matrix secretion of PLFH/ASC, suggesting that this ATMP provides an efficient physico-chemical support for the survival of ASC. Most grafted cells in the carrier certainly survived and developed a long fusiform stromal phenotype, suggesting cell–cell interaction and oriented bundles’ organisation along with collagenous extracellular matrix secretion, which also constitutes a favourable niche for persistence. Regarding the biodistribution, such a local carrier-based site is less detrimental than the general blood circulation injection followed by secondary tissue entrapment of the therapeutic cells. Local injection of ASC in suspension can lead to the mechanical drainage of cells that can passively migrate along the anatomical route to reach the drainage lymph node or the blood vessels and further the lungs and other organs, such as the liver and spleen ,87. CompHuman PL have been obtained from batches prepared by the French Blood Establishment . PL were then mixed with an activating solution containing calcium chloride  was used to measure the viscoelastic properties of PLFH (the temperature was set at 20 °C for these experiments). The 4 mL hydrogels used in this experiment were formulated as described above with a 0.45 µm-filtered platelet lysate. Strain sweeps from 0.1 to 100% strain at 1 Hz were performed to define the linear regime and then frequency sweeps from 1 Hz to 0.1 Hz at 0.5% strain were performed to determine the frequency-dependence of both the storage modulus (G’) and the loss modulus (G”). The average moduli over the frequency range were calculated for the four PLFH conditions.The surface behavior of 1 mL hydrogel  was studied using a texturometer  set in compression with a 4 mm diameter cylinder stainless probe at a crosshead speed of 2 mm per minute. The recording was stopped after the rupture of the gel’s surface layer, and the maximum stress applied was thus noted.PLFH was recovered from the wells and its central part was cut with a blade as a 1 mmPorosity was assessed by monitoring the diffusion of high molecular weight dextran through the fibrin network . A 250 µPLFH swelling corresponds to its hydrous imbibition. The gels were collected and weighed on a precision balance. They were then dried on a compress and reweighed. The swelling corresponds to the difference between the weight of the wet gel and its dry weight, expressed in absolute values. PLFH pH was assessed with an electronic pH meter  at room temperature immediately after their constitution.g for 8 min to remove debris and mature adipocytes. Red blood cells were lysed in hypotonic buffer containing 140 mM g for 5 min. After counting, cells were seeded for expansion at 4000 cells/cm² in PL ASC were isolated from human subcutaneous adipose tissue samples following a previously described protocol . BrieflyPLFH were performed with 200,000 ASC per layer, then kept at 37 °C in g, 4 °C). The Quant-iT PicoGreen dsDNA Assay kit  was used for quantification, according to the manufacturer’s recommendations. The results were adjusted to the basal DNA level of the cell-free hydrogel. Cells left adherent on plastic were used as control.For DNA assessment over time, 20,000 ASC per layer were seeded in PLFH, kept at 37 °C in For the Ki-67 immunofluorescence assay, 100,000 ASC were seeded in PLFH in 8-wells Labtek (Sigma-Aldrich) then kept in A total of 200,000 ASC per layer were seeded in PLFH and kept at 37 °C in All animal experiments were carried out by specialized researchers, engineers and technicians, meeting the requirements of the protection of animals used for scientific purposes, as defined in the European regulation (directive 2010/63/UE) and the applicable French welfare legislations. All animal experimentation procedures were approved by the competent authorities (MESR- APAFIS n°16348-2018073110473357v3 and 22399-2019100916269812v3). Mice were bred in the CREFRE facility  and maintained in microisolators and ventilated racks to reduce potential exposure to pathogens. Mice were housed in a controlled environment , fed ad libitum with complete diet and had free access to water. At the initiation of treatment, the animals were 7–8 weeks old. For ethical considerations, we performed a combined study of biosafety and biodistribution of the local surgical graft of the PLFH.in-life phase on survival, welfare, clinical signs, and by the macroscopic changes recorded at necropsy. At different time points , animals (6 to 9 mice at each timepoint) were euthanized.PLFH with or without ASC were grafted in immunocompromised mice  to overcome the host immune response to human cell transplantation. Using 200,000 ASC per PLFH layer, the cell concentration was therefore set to s et al. . PLFH wiSubcutaneous PLFH/ASC implanted on the left side of the animal was used for histological study, while the right-side one was recovered using an 8 mm biopsy punch for RT-qPCR transcriptional analysis. To assess the systemic biodistribution and toxicity, axillary, inguinal, and iliac lymph nodes as well as kidneys, livers, spleens and lungs were sampled. Samples in the oral position were collected 24 h, 72 h and 7 days after implantation (3 mice at each time). Similarly, the left hemi-mandible was devoted for histology and the right for RT-qPCR . For biodistribution, cervical lymph nodes and lungs were sampled. Samples for histology were fixed in 10% buffered formalin for 2 days. Samples for RT-qPCR were stored at 4 °C for 24 h in RNA-later and then frozen at −80 °C until processing.Hemi-mandible specimens were decalcified in 10% EDTA at 37 °C for 7 days before routine processing. Fixed tissues and organs were embedded in paraffin wax, sectioned in 3 µm-thick slices and processed for hematoxylin and eosin and Masson trichrome stainings. Anti-human-vimentin immunohistochemistry (IHC) was used as a specific, accurate and sensitive method to track the transplanted human ASC , and antg, 4 °C), the aqueous phase containing nucleic acids was collected. The RNeasy Micro Kit  was used for RNA extraction according to the manufacturer’s protocol. The assay of these RNA was performed with Nanodrop 2000c  at 260 nm and then the synthesis of complementary DNA (cDNA) was performed from 1000ng of RNA using the high-capacity cDNA reverse-transcriptase kit according to the manufacturer’s recommendations . RT-qPCR was performed on StepOne  using the Fast SYBR Green MasterMix  to evaluate the gene transcription at different times encoding the proteins IL-1Skin or gingival samples were mechanically disrupted with a steel ball (“tissue lyser”). After the addition of chloroform and centrifugation  at the 5% significance level, taking into account multiple comparisons (Bonferroni correction). Stata v13®, R® and GraphPad Prism 5® software were used for data processing. For the mixture experiment, the R packages “mixexp” and “AlgDesign” were used to define the mixture values to be tested and to model the probabilities and the polymerization times. Each experiment was replicated at least 5 times. Data were expressed as mean ± the standard error of the mean unless specified.Taken together, our data strongly support the safety of our ATMP for a human clinical purpose. Indeed, the PLFH accurate design development, biomechanical property determination, cell activity and safety demonstration provide robust prerequisite to go further in the translational research and clinical trials in humans. These encouraging results are the basis for regulatory preclinical testing for the Investigational Medicinal Product Dossier.


CAV1) signaling participate in the development of pulmonary vascular disorders, limited reports of CAV1‐associated heritable pulmonary arterial hypertension (HPAH) exist. This is a case report of a 2‐year‐old female with late recognition of HPAH due to a CAV1 pathogenic variant: c.474del, (p.Leu159Serfs*22)(NM_001753.5). The pedigree demonstrates autosomal dominant transmission with reduced penetrance of PAH, suggestive that additional genetic or environmental factors modify PAH development. Genetic testing and the discovery of rare genetic alterations in PAH during infancy and childhood may aid in identifying disease etiologies, guide therapeutic decisions, and ultimately identify novel therapeutic targets. Moreover, CAV1 genetics implicate variable expressivity and incomplete penetrance for HPAH and underscores the utility of predictive genetic testing for unaffected family members no matter their age.There has been a growing interest in the role that genetic factors influence pediatric pulmonary vascular disease. In fact, data suggests that genetic factors contribute to ~42% of pediatric‐onset pulmonary hypertension. Although animal and human studies suggest that aberrations in Caveolin1 ( CAV‐1‐related PAH has been reported.CAV1 signaling participate in the development of pulmonary vascular disorders, limited reports of CAV1‐associated HPAH exist. This is a case report of a 2‐year‐old female with late recognition of HPAH due to a CAV1 pathogenic variant: c.474del, (p.Leu159Serfs*22)(NM_001753.5).The molecular genetic basis of heritable pulmonary arterial hypertension (HPAH) is evolving and heterogenous, with at least 26 genes currently displaying varying levels of evidence for disease causality. In fact, current data suggests that genetic factors contribute to >40% of pediatric‐onset PAH.CAV1:c.474del, (p.Leu159Serfs*22). A pedigree of the child's affected family members is presented  pressure, severely dilated RV, and severe RV dysfunction. She was started on inhaled nitric oxide (iNO) and transferred to UCSF Benioff Children's Hospital in San Francisco. Despite maximal medical treatment with iNO, inhaled iloprost and inotropes, she had marginal hemodynamics . Neurologic evaluation was concerning for extensive hypoxic‐ischemic injury. Given her poor neurological prognosis, all resuscitative efforts were ceased, and she died in her parent's arms. Postmortem genetic testing was sent for Blueprint Genetics Pulmonary Arterial Hypertension panel consisting of sequencing/deletion/duplication analysis of 23 genes. Testing revealed that she carried a rare variant in CAV1 signaling participate in the development of pulmonary vascular disorders, limited reports of CAV1‐associated HPAH exist. The CAV1 c.474del, p.(Leu159Serfs*22) variant deletes one base pair in the last exon of CAV1 and generates a frameshift, leading to a premature stop codon at position 22 in a new reading frame. Of note, as this frameshift variant occurs in the last exon of CAV1, the truncated transcript is expected to escape nonsense mediated decay, suggesting that the variant may act as a dominant negative mutation. If so, the persistence of the variant transcript results in a mutated protein product which ultimately promotes pathogenic processes that cause or contribute to the PAH phenotype. There is precedent for this situation in PAH, as we previously reported that mutations which are presumably dominant negative have a more deleterious phenotype in subjects with PAH associated with BMPR2 mutations.CAV1 c.474del, (p.Leu159Serfs*22) variant in 6 individuals within a three generation family who were diagnosed with PAH between the ages of 4 and 67.CAV1 expression compared to control samples.CAV1 variations experimentally, Gairhe et al. demonstrated the important role that CAV1 signaling plays in protecting human pulmonary arterial endothelial cells from many of the pathologic hallmarks of PAH, including exuberant proliferation, impaired apoptosis, dysregulated cellular migration, and perturbed inflammatory response to insult.Although animal and human studies suggest that aberrations in CAV1 variant, and of the eight family members tested, only one of four individuals testing positive for the variant expressing the PAH phenotype. Her disease was acute and severe and resulted in her untimely death at 2 years of age after a series of inflammatory insults associated with infection. The correct identification of her CAV1 variant has allowed identification of those who are genotype positive in the family to seek appropriate cardiac surveillance to monitor for signs of PAH. In general, the penetrance of the majority of disease‐causing PAH genes is not well known. For example, penetrance for BMPR2 variants has been estimated at ~20% overall with sex‐dependent penetrance due to higher penetrance observed in female (42%) versus male (14%) carriers.ACVRL1, KCNK3, CAV1, SMAD9, orBMPR1B is not yet known. Pathogenic variants are identified in about 20%‐30% of patients with idiopathic PAH, and in about 75% of patients with familial PAH, although these percentages are likely higher in those diagnosed during childhood.De novo genetic variants contribute to a significant proportion of pediatric PAH cases, approximately 15%, suggesting the presence of other genetic, epigenetic and environmental factors interact with one's risk for PAH.CAV1 variant carriers. Given the incomplete penetrance of PAH genes, including CAV1, predictive testing can illuminate who may be truly at risk. However, given the incomplete clinical sensitivity for genetic testing for familial PAH, a negative result does not rule out a genetic cause or contribution to a patient's PAH.In our particular case, three generations of family members have been tested for the CAV1 variant in childhood presents with severe disease and poor survival, as a prior study reported.CAV1 genetics implicate variable expressivity and incomplete penetrance for HPAH and underscores the utility of predictive genetic testing for unaffected family members no matter their age.This case report demonstrates HPAH associated with All authors have made substantial contributions to the conception of the work; revising the work critically for important intellectual content; have approved the final version to be published; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The author declares no conflict of interest.This study was performed under an Institutional IRB approval.


The degree of ageing was assessed using three different indexes known to be related to changes in MPs: Carbonyl Index (CI), Hydroxyl Index (HI), and Carbon-Oxygen Index (COI). The overall results showed that the regions reflecting changes  appeared significantly modified in artificial and natural weathered particles compared to the pristine materials. The indexes calculated for polymers degraded under the artificial photo and thermo ageing conditions displayed a general tendency to increase with the time in contact with irradiation time. Particular enhancements of CI of PS fragment and PE pellet, HI of PE and PS fragments and PE pellet, and COI of PS fragment were observed. Otherwise, the following incubation of the same particles at a constant temperature of 45 °C did not further affect the chemical composition of the particles. Moreover, new unique peaks were also observed in the freshwater particles, almost all in the fingerprint region (1500–500 cm−1). Differences in CI, HI, and COI were evidenced among the different morphological MP shapes. On the one hand, the CI calculated for the environmental PE pellets showed values ranging from 0.05 to 0.26 with a mean value of 0.17 ± 0.10. Most samples (57%) presented a CI with values between 0.16 and 0.30. On the other hand, fragments presented slicer modifications in the carbonyl region with CI values lower than pellets (0.05 ± 0.05). This index helps evaluate the degradation of PE MPs by UV light, increasing with enhancing residence time in the environment. Conversely, fragments showed greater values of HI (5.90 ± 2.57) and COI (1.04 ± 0.48) than pellets, as well as lines, which presented the maximum value of HI (11.51). HI is attributed to the bond vibrations of hydroxyl, carboxyl, or phenol groups. In contrast, COI is frequently attributed to the vibrations of C_O bonds found in carbohydrates, alkanes, secondary alcohols, and ketones. In conclusion, our results showed characteristics spectra acquired from environmental particles compared to pristine and artificial aged ones. The interpretation of our main results emphasizes the need to conduct ecotoxicological experimental studies using naturally weathered particles due to the unicity of their properties, which are more helpful for understanding microplastic pollution effects.Fourier transform infrared (FTIR) is a spectroscopy technique widely used to identify organic materials. It has recently gained popularity in microplastic (MP) pollution research to determine the chemical composition of unknown plastic fragments. However, it could also be used to evaluate the degree of ageing of MPs collected from the environment. In this context, the principal aim of our research has been to qualitatively evaluate the natural weathering of environmental MPs collected in an Italian freshwater body (the Ofanto River) using ATR-FTIR technology. Furthermore, we compared environmental particles to weathered artificial MPs under controlled light and temperature conditions and to unaltered pristine materials to assess the results. FTIR spectra were acquired using a Nicolet Summit FTIR (ThermoFisher Scientific) equipped with an Everest ATR with a diamond Crystal plate and a DTGS KBr detector (wavenumber range 4000–500 cm Fourier transform infrared (FTIR) is a spectroscopy analytical technique widely used to identify organic materials . It has The chemical structures of unknown polymers can mainly be recognized through their specific IR absorption frequency. However, their exact chemical structures can only be deduced from IR spectra if compared with known data or their monomers’ IR spectra.One of the most critical issues of plastic and microplastic research is their degradation potential, one of the most important factors that can be used to calculate their persistence in the environment in certain conditions . AlthougThe photodegradation of plastic by UV radiation is considered the most important weathering process for the MPs generation, which induces the splitting of chemical bonds, branching, and formation of oxygen-containing groups such as carboxylic, aldehyde, ketone, and hydroxyl ,8. The fWeathering has been reported to significantly change the physicochemical properties of MPs, critically affecting the sorption behaviour of pollutants  onto MPs . These cMoreover, ageing processes potentially affect the MPs ingestion by organisms, including humans, due to the increasing size reduction in weathered particles, resulting in more easily ingested and more complex egested particles by biota. Indirect risk to humans and biota can be induced concerning the leaching of toxic additives, which has also been observed in previous studies ,15. Grea−1 [In this regard, FTIR is a valuable tool for measuring the changes in chemical bonds typical of MPs ageing  ,17. The −1 . Carbony−1 .Previous research ,21 simulEven polypropylene showed an increase in the absorption of hydroxyl and carbonylic groups under the influence of both artificial light (UV-B) and natural sunlight for six months.Another study  simulateHowever, the complexity of the natural processes requires the simulataneous study of numerous variables, and the exposition times set up in laboratory studies are logistically lower compared to what really occurs in the environment.If simulations of MPs degradation in the laboratory provide a reasonable basis for understanding the process, the results obtained are often only partially comparable to natural environmental systems.Most plastic degradation studies were conducted under experimental laboratory conditions to simulate various environmental factors ,25,26,27To fill in gaps of information between artificial and actual weathering processes, our study’s objective has been to investigate the influence of UV radiation and heating on microplastic weathering, evaluating differences among polymers and morphologies. In addition, the natural ageing of environmental MPs collected in an Italian freshwater body (the Ofanto river) was assessed compared to pristine, artificial, and natural weathered particles. The approach adopted provided the use of ATR-FTIR technology through the calculation of weathering indexes used as a standard approach for estimating the ageing stage of environmental particles. To the best of our knowledge, no studies assessed the ageing of naturally weathered particles collected from a freshwater environment.Polymers composed of polyethylene, polypropylene, and polystyrene were selected as pristine materials due to their overall production and dispersion into the environment ,32.A red polypropylene plastic cup, a black polyethylene mulch sheet, and a white polystyrene box were cut in the laboratory to obtain fragments of virgin MPs . ParticlArtificial ageing of pristine MPs was simulated by photo and thermo degradation. Particles were transferred into Petri dishes and placed in a climate room equipped with UVA lamps, set to specific temperatures, irradiance, and humidity . The phoRiver surface microplastic samples were collected from the Ofanto river, the most important river in the Apulia Region , during Spring of 2018.Three surface plankton nets (2.5 × 0.55 m) of 333-μm mesh size fixed in the middle of the river were used to filter water. Material captured by trawl was separated through stainless steel sieves. Material retained on the 0.3 mm sieve was placed in a 40 °C drying oven for 24 h until sample dryness. The biggest plastic particles (5000–2000 µm) were picked manually using forceps and categorized according to colour  and morphology as fragments, pellets, and lines.−1, 32 scans per spectrum, spectral resolution of 4 cm−1). The background was measured with the same settings against air. Ten different PE pellets, fragments, and lines extracted from the Ofanto river were acquired one by one. Comparisons among virgin MPs pellets purchased from the local industry and weathered MPs collected in the river were also made to identify and characterize any changes in the spectra.FTIR spectra were acquired using a Nicolet Summit FTIR (ThermoFisher Scientific) equipped with an Everest ATR with a diamond Crystal plate and a DTGS KBr detector , Hydroxyl Index (HI), and Carbon-Oxygen Index (COI).−1, and at 1495–1423, 1460, and 1452 cm−1 for reference peak of PE /(1495–1423)CI➢PP = Abs (1715–1735)/(1460)CI➢PS = Abs (1635–1650)/(1452)CI➢PE = Abs (3353–3021)/(1504–1467)HI➢PP = Abs (3300–3400)/(986–952)HI➢PE = Abs (924–1197)/(2987–2866)COI➢PP = Abs (1000–1200)/(2885–2940)COIIn the same way, Hydroxyl Index was calculated as the absorbance ratio of hydroxyl groups at 3353–3021, 3300–3400 cmThe artificial ageing process of pristine MPs induced surface alterations  and changes in the FTIR spectra acquired before and after the artificial weathering process of particles.−1), with new absorption peaks appearing in all the spectra polymers after 20 days of UVA exposition in the climate room , are also evident.In the spectra related to the PE fragments and pellets, new weathering bands from 1510 to 1770 cm−1 showing a higher intensity for the PE fragments than the pellets , hydroxyl (HI), and carbon-oxygen (COI) indexes representative of the degree of degradation of particles.The PE particles showed values of CI increased already after 20 days of UVA exposure, as well as the HI and COI indexes. A further enhancement of values is possible following the incubation of PE particles at 45 °C, where an increase in especially the HI from 7.00 − 7.89 ± 0.23 − 3.79 to 8.15 − 8.21 ± 2.11 − 1.95 is notable.In the PP particles, the indexes’ remarkable values are notable, particularly for the HI already after 15 days of photo-exposition and for the PS fragments. The CI increases after 20 days of incubation in the climate room, reaching a mean value of 0.71 ± 0.51.Otherwise, the following incubation of particles at 45 °C further slightly increased the values of indexes; the increase is particularly notable from the value of the HI index of PE particles (both pellets and fragments) and PP fragments  which shIn the present work, changes in the surface composition and the level of degradation of particles of various colours and shapes, subjected to different weathering conditions , were compared to pristine pre-production standard materials.Unfortunately, it was impossible to compare each particle directly for polymer, morphology, and level of ageing due to the scarcity of natural particles detected. Indeed, the vast majority of environmental particles detected in the Ofanto river were composed of PE . Therefo−1, alkenes or carbon double bonds, 1600 and 1680 cm−1, and carbonyl groups, 1690 and 1810 cm−1) appeared significantly modified in the PE environmental particles . Differences in CI, HI, and COI were evidenced among the different morphological MP shapes.Moreover, new peaks are present almost in the fingerprint region  presented a CI with values between 0.16 and 0.30, defined as medium by . On the Comparing the degradation indexes results of PE environmental and artificial aged pellets , the valThis study compares the surface degradation process of pristine particles, artificially weathered materials of microscopic size and naturally environmentally aged MPs through spectroscopy investigations.−1 (–CH2 rocking deformation), 2847 and 2915 cm−1 (–CH2 symmetric and asymmetric stretching), 1462 and 1472 cm−1 (–CH=CH– stretching) [−1 (–CH3 oscillating vibrations), 1375 cm −1 (–CH3 symmetric bending vibrations), 2952 cm −1 (–CH3 asymmetric stretching vibrations), 1455, 2838, and 2917 cm−1  [The pristine reference materials had characteristic absorption bands . In partetching) . The PP Finally, the PS was characterized by absorption bands at 3060, 3026 (–CH aromatic stretching vibrations), 1600, 1492, and 1452 (–C=C aromatic stretching), indicating the existence of benzene rings. Otherwise, peaks at 756 and 698 due to C–H out-of-plane bending vibration indicate that only one substituent in the benzene ring is present .−1), hydroxyl (3300–3400 cm−1), vinyl (1600–1680 cm−1), peroxides (1100–1300 cm−1), and unsaturated groups (880–920 cm−1) [In agreement with previous works, the degradation of the pristine materials under different conditions  and the environmentally weathered particles resulted in the formation of new functional groups such as carbonyls (1700–1760 cm20 cm−1) ,40,41.The calculation of degradation indexes lets us quantify the presence of hydroxyl (HO), carbonyl (C–O), and carbon-oxygen bonds (C–O).−1 region [Indeed, the CI helps evaluate the degradation of MPs by UV light and their surface oxidation level; it increases with enhancing residence time in the environment . All ket1 region .HI is attributed to the bond vibrations of hydroxyl, carboxyl, or phenol groups. In contrast, COI is frequently attributed to the vibrations of C–O bonds found in carbohydrates and alkanes, secondary alcohols, and ketones ,34.The indexes calculated for polymers degraded under the artificial photo and thermo-ageing conditions displayed a general tendency of the indexes to increase with the time in contact with irradiation time. Indeed, after 20 days of exposition to UVA, particular enhancements of CI of PS fragment and PE pellet, HI of PE and PS fragments and PE pellet, and COI of PS fragment were observed. When UV radiation meets with plastic, the polymer’s photo-oxidation occurs, resulting in the scission of C-H bonds and free radicals production .Otherwise, the following incubation of the same particles for 25 days at a constant temperature of 45 °C does not seem to have further altered the chemical composition of the particles; with just some increase in the HI index  observed. Indeed, as also observed by other authors .Considering the comparison between PE artificial and naturally weathered particles of different colours and morphologies, we can observe the highest value of CI calculated for the white fragments spectra (0.34). A CI value above 0.30 is considered high by . This reA similar study  conducteMoreover, the CI values were higher in the coastal sediments close to the high-tide line due to the exposition of the wave action over the solar and microorganisms exposition. Indeed, as also mentioned by other authors , plasticAnother study  also evaEven the colour of these particles confirms what has been detected; in fact, the white colour of these particles is an indication of a loss of colour, the same as which led to a general yellowing probably caused by fragmentation of macro and mesoplastics present in the environment, which occurred over a considerable time. Otherwise, coloured environmental weathered fragments showed considerably lower CI values than the white ones, suggesting younger particles. However, a medium value of CI  equal toWe can explain these deviations of CI based on differences in crystallinity or density of the PE particles. It is known that the more amorphous the polymer material, the easier the diffusion of oxygen and the higher the CI.Indeed, several types of polyethylene polymer are synthetized from chemists  exhibiting a random orientation of side chains for LDPE, linear chain for HDPE and short side chains for LLDPE. The difference in the PE chains’ linearity translates into different crystallizing properties ,41,48.−1) of artificially aged polymers and all PE samples collected from the Ofanto river , indicating the spectral range of carbon-oxygen bonds, particularly relevant just in environmental weathered particles (− 1 is attributed to the stretching vibration absorptions of the C-O (around 1160 cm−1), indicating different C-O-containing degradation products but may also indicate phosphate ions, bonds of the glycosidic linkages [−1) in previous work [Intense peaks appeared in our samples, between 1000–1200 cmarticles . The reglinkages , or alipous work  that, co−1, usually called the fingerprint region, appeared considerably different compared to artificially aged and pristine particles; this region is indeed unique for various compounds [Finally, the region 700–400 cmompounds .These peaks can be attributed to fouling by sand, debris, and organic material (biofilm) absorbed on the polymer’s surface  as well Detecting characteristics spectra acquired from environmental particles indicates that artificial weathered polymers do not account for all the observed functional groups and possibly microplastic pollution effects. These differences represent the heterogeneity of the surrounding environment in terms of nutrients, organic matter, conductibility, and microbial environment, indicating the necessity of more studies representing environmental conditions to fully understand microplastics’ ageing processes.Given the high intensity of fingerprint signals, it is assumed that a considerable amount of material has adhered to the particles sampled from the environment. Indeed, freshwater microplastics most likely originate from terrestrial environments where they are subjected to the mechanical actions of the soil for a long time, resulting in the adhesion of foreign substances to their surface .The major microplastic drivers identified in the terrestrial environment are irrigation water, urban and agricultural runoff, fertilizers, and soil amendments, including sewage sludge, which have to be considered in the counter-measuring policies and institutionalization process ,55.Necessary policies and governance measures must support the creation of appropriate barriers to MPs entering humans through food chains.Indeed, present plastic waste management regulations mainly focus on waste disposal on land. In contrast, the policies regarding MPs have primarily focused on preventing just marine aquatic MP pollution .The analysis of the spectroscopic investigations carried out by ATR-FTIR, allowed us to assess the level of ageing of particles weathered under controlled conditions of light and temperature and of microplastics collected in a natural freshwater environment such as the Ofanto river.The spectra analysis of particles aged under controlled conditions let us conclude that UVA radiation (315–400 nm) exposition was the most critical condition influencing and starting the degradation processes of PE, PP, and PS polymers. The calculation of degradation indexes indicated suggested that PE pellets resulted in the most resistant microplastic category to photo and thermo ageing. Otherwise, PS fragments showed the most altered spectra.By calculating degradation indexes and observing new peaks detected in both artificial and natural aged particles, we can likely conclude a permanence time in the environment of freshwater particles above the total ageing period considered in the laboratory for the weathering (45 days).Real freshwater microplastic samples showed further alteration of all spectra with the appearance of new unique absorption peaks respected to artificial weathered particles demonstrating that several variables interact together in the weathering process of polymers.In light of our results, conducting field studies is paramount to thoroughly implementing and revising plastic policies, considering all the environmental variables influencing weathering plastic processes. It is, in fact, known that aged plastic has a greater susceptibility to pollutants adsorption and microbial adhesion. Indeed, one of the primary concerns related to microplastics stems from their capacity to act as carriers of dangerous substances. .Compared to previous studies, based on experiments that accelerated plastic particle ageing in the laboratory or simulated environmental conditions, our study is the first experiment representing a realistic assessment of the degradation process that occurs on freshwater microplastics.


Citrus macrophylla and Citrus aurantium/Citrus sinensis rootstocks showed a significantly higher production than the Citrus aurantium. All the fruits presented a predominantly yellow color appropriate for the market (0 < CI < +5). Nutritional and chemical parameters were consistent with data reported for the ‘Verna’ clones. All the obtained lemons were suitable for marketing and consumption both in fresh and processed forms. The results indicated the limited influence that the studied variables have on the quality parameters of lemon fruits, but they also could confirm the potential of marine sediment as a culture substrate.Due to its high content of bioactive compounds, the lemon is considered one of the most relevant species around the world. Its great economic importance is motivated, in addition to its fresh consumption, by its applications in the medical, pharmaceutical, and food industries, etc. However, the chemical and nutritional composition of lemon is not constant and can be influenced by external factors such as variety, weather conditions, crop management, etc. Determining the compositional variations of the fruit, essential to defining its potential use, was the main objective of this study. The physicochemical characteristics of the ‘Verna’ lemon were studied as a function of two controlled variables, the growing substrate and the rootstock. For this, 90 lemon trees were cultivated in three rootstocks and three different culture media. Lemon trees cultivated with 50% sediment/peat mix substrate presented a higher total production (590 lemons and 90.53 kg) while this production was 80% lower on trees cultivated with 75% marine sediment.  Citrus limon (L.) Burm f.) is considered the third most important citrus species in the world, only behind orange and tangerine, and is socially known for its valuable nutritional, medical, pharmacological, industrial, and cosmetic properties and uses [The lemon , Greece (5%), and Portugal 1.6%) among other minority countries . Due to .6% amongAmong all the varieties of lemons, the ‘Verna’ variety is the second most relevant in the Valencian Community behind the ‘Fino’ variety. The main characteristic of this lemon variety is that, in general, they present an abundant harvest with medium/big sized fruits, a good acidity of its juice, and few seeds. Another advantage is that the fruits do not present a loss of quality over time, which allows for a staggered harvest ,12.Citrus macrophylla and Citrus aurantium are the most common rootstocks used in southeast Spain for lemon cultivation. Citrus macrophylla is the most demanded rootstock used for lemon trees, mainly due to its vigor or speed of growth; that is, the trees with Citrus macrophylla rootstocks reach a larger size in less time, which can translate into great productivity. However, when grafted with ‘Verna’, it can produce oversized lemons. Citrus aurantium is already the traditional lemon rootstock, and although its use gives the tree great resistance to hydro edaphic environments, its affinity with the ‘Verna’ variety is limited and therefore its commercial and productive life are also limited [Citrus aurantium/Citrus sinensis is commonly used both to avoid possible incompatibilities between rootstock and variety, as well as to improve the quality of the fruits.As the variety choice based on productive and fruit quality criteria is a relevant decision for lemon cultivation, the rootstock selection also is fundamental due to its impact on crop management, since the rootstock can present greater resistance to diseases and/or different adaptation capacities to edaphoclimatic conditions. In this sense,  limited . IntermeOn the other hand, different studies confirm the influence of the culture medium on the quality of the fruit , mainly due to the adaptation/resistance of the trees to the different parameters of the substrate, such as pH (which determines the mobility and availability of microelements), conductivity , and porosity (oxygen availability), among others ,14,15,16In this context, the objectives of this work were: (a) evaluate the potential use of phytoremediated marine sediment as an agricultural substrate for lemon cultivation; (b) define the influence of the cultivation substrate on the morphological and quality characteristics of lemons, with emphasis on their biocomposites; and (c) identify the potential variations/adaptations of the physicochemical composition of lemons related to the most common commercial rootstocks used in southeastern Spain.Citrus limon (L.) Burm var. ‘Verna’) was studied by using nine different treatments (substrate/rootstock combinations) as shown in n = 3) and the culture media (n = 3). The most common rootstocks used in commercial lemon cultivation were: (i) Citrus macrophylla; (ii) Citrus aurantium; and (iii) a combination between Citrus aurantium and Citrus sinensis, usually called ‘sweet orange intermediate wood’. Related to the grown media, three substrates composed of peat and phytoremediated marine sediment in different proportions were evaluated: (i) 25% sediment + 75% peat; (ii) 50% sediment + 50% peat; and (iii) 75% sediment + 25% peat. The marine sediment used in this work came from Livorno port , it was previously phytoremediated for three years and has already been used successfully in other ornamental and food crops [In this work, the rootstock/substrate impact on lemon fruit characteristics , a total of 10 trees were evaluated with a random distribution experimental design by blocks (n = 5) and two trees per block (n = 2). In total, 2-year-old 90 lemon trees (3 substrates × 3 rootstocks × 5 blocks × 2 trees per block), grown in polypropylene pots, were evaluated in an experimental plot of Miguel Hernandez University  located in the Southeast of Spain . The average annual temperature was 19 °C, with moderate winters (minimum of 11 °C in January) and warm summers (maximum of 28 °C in August). The mean annual precipitation was 300 mm, and most of this precipitation was recorded in the spring and autumn seasons. The area presented high insolation, with approximately 3000 h of sunshine per year. The highest sunshine percentage was registered during the summer months (more than 352 h in July) and the lowest in December and January [As highlighted above, the present study considered 9 different treatments (1 lemon variety × 3 rootstocks × 3 substrates). For each treatment ( January .−1). In total, during the entire two-year experiment, an average of 3.12 m3 of water per tree was applied. Both the cultivation conditions and the crop management were kept homogeneous throughout the trial to minimize external influences and to study the morphological and nutritional lemon variations/differences objectively.Fertigation water was supplied through a drip irrigation system, with one pipeline for each differentiated treatment based on the content of the marine substrate , with one drip per tree (4 L hrodrejo’ harvest (between August and October), depending on weather conditions. In this study, the lemons evaluated correspond to the main bloom.Related to plant material, the ‘Verna’ variety of lemon fruits are considered spring/summer lemons, with the main harvest between March and September. The main bloom of the ‘Verna’ variety begins in April–May, but it shows several continuous blooms during the year. In general, up to five or six blooms can be distinguished during the year, from which three commercial harvests are obtained, these being: (i) the main harvest (March to June); (ii) the second lemon harvest (June), and (iii) the ‘In all cases, the lemon fruits were harvested manually once the fruit reached commercial maturity  in accordance with the provisions of the marketing standard for citrus fruits . The pron = 9). The fruit weight  was determined with an analytical balance with a precision of 0.02 g . Measurements of equatorial diameter , fruit length , mamelon length , and peel thickness  were evaluated using a digital caliper . Additionally, the number of seeds and carpels was also monitored by simple visual and manual counting. All measurements were made at constant room temperature (22 ± 2 °C). The results presented are the mean values (91 ≤ n ≤ 590) with the standard deviation in parentheses.The morphological characterization was carried out on all the fruits collected for each treatment  was used. For the external color, four equidistant peel measurements were made in the equatorial zone of the fruit, while the internal color was studied through the juice color obtained as specified in the chemical and nutritional characterization section of this work. All measurements were performed at constant room temperature (22 ± 2 °C). The color was evaluated according to the Commission Internationale de l’Éclairage (CIE)  and exprectively . Addition = 9). Lemon juice was carefully obtained using a commercial manual juicer  at constant room temperature (22 ± 2 °C). Immediately after being squeezed, the juice was characterized for color, as specified in the previous section, and pH, titratable acidity  and total soluble solids  parameters following the methodology described by Aguilar-Hernández et al. [Chemical analyses were performed using five juice samples for each treatment n = . Lemon juirement . The res+•); (ii) radical method 2.2′-Diphenyl-1-Picrylhydrazyl (DPPH•); and (iii) reduction of the ferric ion (FRAP) by means of a UV–visible spectrophotometer . The results correspond to the mean values (n = 5) with its standard deviation and are expressed in mmol Trolox per liter of juice. The total phenolic content (TPC) was also determined using the Folin–Ciocalteu colorimetric reagent as described by Singleton et al. [n = 5).The antioxidant activity (AA) of lemon juice was studied using five juice samples per treatment  followinn et al. . The mea2 ≥ 0.999. The results obtained correspond to the mean values (=5) and are expressed in g 100 mL−1.The quantification of sugars and organic acids was performed by Agilent 1100 high-performance liquid chromatography (HPLC) with ChemStation software by installing a Supelcogel C610H column, 30 cm × 7.8 mm; and a pre-column Supelguard, 5 cm × 4.6 mm . For the organic acids measurement, a diode array detector (DAD) (Diode Aray DAD G1315A) set at 210 nm was used, while a refractive index detector (RID) (G-1362-A) was employed for sugar content determination. Reference standards were used for organic acids  and sugars  supplied by Sigma Aldrich  with calibration curves with Rp < 0.05. In addition, principal component analysis (PCA) and cluster analysis (CA) were also performed. Cluster analysis was applied to the standardized data for hierarchical associations using Ward’s method for clustering and squared Euclidean distance as a measure of dissimilarity. Statistical analyses were performed using Statgraphics software .The experimental data obtained were evaluated by means of a one-way analysis of variance (ANOVA) followed by a mean separation contrast using the Tukey HSD test for n = 3), rootstock (n = 3) and, additionally, by treatment (n = 9) aiming to identify and define the impact, differences, and/or similarities of the parameters evaluated.Due to the large number of experimental data and the possibility of multiple combinations of the variables studied, the results are presented differently by the substrate , equatorial diameter (64.07 mm), and fruit length (98.05 mm), configuring the most characteristic morphology of ‘Verna’ lemons [Citrus macrophylla lemons presented a shorter mamelon length (15.01 mm). Again, the results for peel thickness, number of carpels, and juice yield parameters did not present differences between the rootstocks.Related to the rootstock type used, the ’ lemons . In addiCitrus aurantium/Citrus sinensis rootstock and with 75% marine; since ‘Verna’ lemons tend to present excessive fattening, mainly in rainy springs, exceeding their commercial size and, therefore, losing their economic value.In addition, considering the fruit size as a fundamental parameter for its market valorization, it should be noted that—in all cases—the lemon fruits obtained presented a medium/large size, so they would be suitable for fresh consumption and, therefore, its acceptance in the market. This decrease in the weight of cultivated plants/fruits with high contents of marine sediments was already observed and confirmed for lettuce and strawberry ,26. ThusCitrus volkamerina) presented both smaller equatorial diameter and fruit weight, and in some cases, bigger peel thickness [The lemon fruits reported in the bibliography for different varieties (‘Eureka’ and ‘Fino’) and grafted on other rootstocks  were identified for the fruits obtained with the substrate with 25% sediment (70.12) and for the Citrus macrophylla lemons (70.80); the other treatments did not present significant differences for luminosity values (<71). All the fruits, regardless of the substrate and/or rootstock variable, showed greenish-yellow colorations as indicated by the positive values of a* and b*, respectively. The parameters C* and H° were not affected by the rootstock, but presented a significant difference between the substrates used, where the lemon fruits obtained with the substrate with 25% sediment presented the lowest values of C* (53.32) and higher values of H° (84.92). In general, all the fruits presented a predominantly yellow color appropriate for market (0 < CI < +5) [Related to the peel color, the less luminous lemons (iment 70. and for CI < +5) .L*) was obtained with the lemons grown with the substrate with 75% sediment (41.21). Only the C. aurantium lemon juice presented a reddish coloration  compared to the other treatments with greenish chlorination . All the juices presented yellowish coloration  being: (i) by substrate 75% > 50% > 25%; and (ii) by rootstock C. aurantium > C. macrophylla > Citrus aurantium/Citrus sinensis. In relation to the CI, all the juices presented yellowish green tones with the most intense values for the fruits with 25% sediment (−8.85) and C. aurantium as standard (7.49).Like the external color of the fruits, the juice color results were quite homogeneous. Thus, in this case, the least luminous juice , being much more influenced by other factors—such as variety, temperature, solar incidence and humidity, among others ,43,44,45In order to determine the potential of the remediated marine sediment as an agricultural substrate applied to lemon cultivation and its interaction with the rootstock, it was necessary to determine the quality parameters of the juice obtained since the variations in the biocompounds content could be a relevant factor for decision making. The results are shown in Citrus aurantium/Citrus sinensis rootstock; while for the other treatments the values remained between 2.49 and 2.54 without significant differences. The lemons obtained with 25% sediment presented the lowest values of total soluble solids (8.96 °Brix), the other treatments did not present significant differences and their values were around 9 °Brix. These results are higher than the data reported on the bibliography for ‘Verna’ variety , but similar to those determined for ‘Fino’ variety [The highest pH values (3.04) were identified for lemons cultivated with 75% sediment and those obtained with  variety ,46.−1) followed by 50% sediment and 25% sediment, with 62.79 and 59.71 g citric acid L−1, respectively. Depending on the rootstock, Citrus aurantium/Citrus sinensis lemons presented the highest values (63.88 g citric acid L−1) followed by C. macrophylla (62.33 g citric acid L−1) > C. aurantium (59.75 g citric acid L−1). The results, although a bit higher than those reported in the bibliography, could be considered within the expected range for lemon juice [On the other hand, TA showed a similar trend for the two studied variables, with significant differences both as a function of the substrate and the rootstock. In this sense, the lemon cultivated with 75% sediment showed the highest TA values  and antioxidant activity—evaluated by DPPH−1) while with 25% sediment barely reached 1.08 mmol Trolox L−1. The results agree with those reported for ‘Verna’ lemon, but are different from those defined for other ‘Verna’ clones [Citrus aurantium/Citrus sinensis lemons showed the lowest result for DPPH (2.91 mmol Trolox L−1) but the highest iron oxidation capacity FRAP (1.25 mmol Trolox L−1). Czech et al. [−1 determined with ABTS and DPPH, respectively).When analyzing the results according to the substrate, significant differences were only observed for FRAP method, where lemons grown with 75% sediment presented the highest values , which could indicate a lower degree of maturity compared to the fruits of the other rootstocks [The highest TPC, significantly different from the other rootstocks, was determined for otstocks . Xi et aotstocks  determinAs indispensable contributors to fruit quality, organic acids and soluble sugars can affect fruit flavor and indirectly exert beneficial and disadvantageous effects on marketable fruit quality . In this−1) followed by fructose (≥0.51 g 100 mL−1), and no significant differences between the variables analyzed (rootstock or substrate) were observed. The predominance of glucose against fructose confirms what was established for “acidic” citrus varieties, in contrast to “non-acidic” varieties where fructose predominates against glucose [−1 and 2.7–4.0 g 100 mL−1 for glucose and fructose, respectively, for ‘Fino’ and ‘Verna’ clones but cultivated soil directly.For all the lemon fruits, glucose was the majority sugar  and grapefruit , but with higher values of malic and citric acid than those determined for the sweet orange, tangerine, and sour orange cultivated in Turkey [The main organic acid quantified in the juices was citric acid followed by succinic acid > malic acid > ascorbic acid, which coincides with what was previously reported ,9,55. Inn Turkey ,57,58,59More specifically, the results were analyzed individually for each of the nine treatments (one variety × three substrates × three rootstocks) in order to identify the differences and/or similarities between them in more detail. The results indicated differences mainly related to production, number of seeds, total phenolic content, and malic acid, while the rest of the morphological parameters presented homogeneity of the results between the samples . Since tCitrus macrophylla, Citrus aurantium, and Citrus aurantium/Citrus sinensis).To achieve a better understanding of the trends and relationships between the parameters studied (37) for the variables used (2), principal component analysis (PCA) was applied, considering the analysis based on the type of substrate used  and rootstock  and juice color parameters , citric acid, malic ascorbic acid, succinic acid, glucose, and fructose  explained 100% of the total variation. The first component (PC1), which represents 71.96% of the total variance, was related to the variables analyzed: mamelon length, equatorial diameter, peel weight (PW), TA, MI, ABTS, DPPH, FRAP, the fruit color parameters  and PC2 discriminates the substrate with 50% sediment from the other two (25% and 75%). The cluster analysis indicates that 50% and 75% are close to each other and far from 25% a.L*, b*, C*, and H° for the peel and L*, a*, b*, C*, H° for the juice color; citric acid; malic acid; ascorbic acid; succinic acid; and fructose. These variables are related directly with the production, shape of the fruit, fruit and juice color, organic acids, and fructose. PC2 represented 24.06% of the total variance and was correlated with mamelon length; peel thickness; number of carpels; juice content; peel weight (PW); TSS; FRAP; DPPH; and the color parameters L*, a*, C*, and H° from the fruit. It basically describes the production, shape, and color of the fruit and antioxidant activity.On the other hand, the statistical analysis based on the rootstock used again indicates that the first two main components (PC) explained 100% of the total variation b. In thiCitrus aurantium to be discriminated from Citrus macrophylla and Citrus aurantium/Citrus sinensis; and PC2 discriminates between the three combinations . The cluster analysis indicates that the rootstocks Citrus macrophylla and Citrus aurantium/Citrus sinensis are close to each other and far from Citrus aurantium , while the The impact of marine sediment on the reduction of the fruit size/weight could be used as a tool to control excessive fruit development in some lemon varieties such as ‘Verna’.The lemons obtained in the nine substrate/rootstock combinations studied presented appropriate characteristics for their categorization as commercial. The nutritional value of the ‘Verna’ lemon juices was very homogeneous among the treatments studied; that is, the limited influence of both the rootstock and the culture substrate on the chemical characteristics of the fruits would be confirmed.


Saccharomyces cerevisiae) and microalgae (Spirulina platensis and Chlorella vulgaris) as feed supplementation on feed intake, rumen disappearance of barley straw, bacteria, and fermentation, blood parameters of camels and sheep. Three fistulated camels and three fistulated rams were fed a concentrates mixture and ad libitum barley straw as a basal diet alone or supplemented with YA mixture. The dietary supplementation improved the feed intake, rumen disappearance of barley straw nutrients, and the blood immunity parameters. The YA supplementation affected rumen fermentation as well as the composition and diversity of rumen bacteria; however, the response to the supplementation varied according to animal species. Principle Coordinate Analysis (PCoA) separated bacterial communities based on animal species and feeding treatment. Phylum Bacteroidetes and Firmicutes dominated the bacterial community; and the dominant genera were Prevotella, RC9_gut_group, Butyrivibrio, Ruminococcus, Saccharofermentans, Christensenellaceae_R-7_group, and Succiniclasticum. Our results suggest positive impacts of YA supplementation in rumen fermentation and animal performance.The combination of live yeast and microalgae as feed supplementation could improve rumen fermentation and animal productivity. This study aimed to investigate the impact of a mixture of (YA) yeast ( A huge amount of crop straw is generated globally every year2. Barley is grown in a wide range of adverse environmental conditions, including drought and soil salinity; thus, it is considered the main crop in desert regions3. Barley provides large quantities of lignocellulosic straw that could be used in the feeding of desert ruminants5. However, higher lignocellulose represents a barrier towards the efficient microbial fermentation of barley in the animal rumen6.The low productivity of animals in the arid regions is attributed mainly to feed deficiency, wherever animal feeding relies on the low-protein and high-fiber roughages7. Rumen microbiota convert the components of animal diets such as cellulose, hemicellulose, starch, and protein to microbial proteins and volatile fatty acids that provide the host animal with about 70% of daily protein and energy requirements8. Therefore, any modulations in the rumen microbiota and fermentation could have a significant impact on the physiology and efficiency of the host-animal. Rumen bacteria represent the majority of the microbial communities in the rumen, approximately 88% of microbial populations, with more than 1010cells /ml9. The composition of the animal diet and animal species are the main drivers of the composition of rumen microbial groups9. Understanding the alterations of the rumen microbial ecosystem in different animal species and under different dietary supplementation offers the possibility to design suitable feeding strategies to optimize the rumen microbial fermentation, including fiber digestion of desert ruminants9. Desert dwelling ruminants such as camels and Barki sheep contribute significantly to food security in arid countries by providing milk and meat under harsh conditions10. These animals have been adapted to live in adverse desert conditions, including heat stress, scarcity of water, and availability of poor-quality feedstuff11. Camel is multipurpose animal that is adapted to desert harsh conditions by the unique feeding behavior, physiological mechanisms, and the functional structure of the digestive tract12. Barki sheep is the main sheep breed in the arid regions in North Africa and the Middle East, as this breed is well adapted to desert conditions13. The differences in the anatomy and physiology of the digestive tract as well as feeding behavior between camels and other ruminants resulted in differences in the feeding requirements, rumen microbial fermentation, and metabolism14. Gihad et al.15 indicated that camels have high efficiency than sheep in dry matter and crude fiber digestion, and nitrogen utilization. Moreover, a comparative study on camels and sheep16 showed some discrepancies in the composition of the rumen bacterial community; for instance, camels showed higher Ruminococcus and Butyrivibrio, and Prevotella, while sheep have higher RC9_gut_group. Therefore, the response of animal species to dietary intervention could be varied17.The fermentation in the rumen depends on the interactions between the microbial groups, consisting of bacteria, archaea, protozoa, and fungi19. Probiotic and prebiotic feed additives or their combinations have gained interest to manipulating rumen fermentation21. However, feed additives need to be studied continuously to examine their efficacy, which depends on the nature of compounds, the composition of animal diet, and the response of the rumen microbiome22. Supplementation of the animal diet with probiotics such as live yeast (Saccharomyces cerevisiae) alone or with phytogenic substrates has been shown to affect the rumen microbiome22. Previous studies24 indicated that the supplementation of the cattle and camel with live yeast improved the total VFA production and stabilized rumen pH, and affected the abundance of some bacterial groups, including fibrolytic bacteria. Moreover, in recent years, algae or their extracts have gained interest as feed additives in livestock diets19.Furthermore, intensification of animal production and restriction of antibiotics use in animal production are the main drivers of the transition to the safe and novel feed additivesChlorella and Spirulina are well-known microalgae worldwide; they are rich sources of proteins, essential amino acids, vitamins, pigments, fatty acids, minerals, and other natural bioactive compounds26. Therefore, microalgae supplementation can boost the rumen microbiota to enhance diet fermentation. Previous studies indicated that microalgae feed additives improved animal health, growth, and fertility27. Tsiplakou et al.28 reported that the inclusion of Chlorella in goats’ diet affected the abundance of rumen bacteria and milk fatty acids profile. The availability of polysaccharides and other bioactive compounds is the main reason to use of microalgae or their derivatives as a prebiotic to promote gut health and modulate gut microbiota composition29. Therefore, it was hypothesized that using mixtures of prebiotics such as microalgae, and probiotics such as live yeast, as feed additives is an effective strategy to improve animal health and productivity by promoting the activities of rumen microbiota to enhance rumen fermentation and stimulate the immunity31. Grimm et al.21 supplemented the horses with a combination of live yeast and microalgae (Aurantiochytrium limacinum) and found that the supplementation affected the abundance of some fibrolytic gut bacteria.S. cerevisiae and microalgae mixture (Spirulina platensis and Chlorella vulgaris) on feed intake, rumen fermentation and bacteria, blood parameters, and ruminal disappearance of dry matter (DM), crude protein (CP), and neutral detergent fiber (NDF) of barley straw in two different animal species, camels and sheep.Very limited information is available regarding the supplementation of the combination of live yeast and microalgae to animal diet. This study aimed to determine the effect of a mixture of Camelus dromedaries, Maghraby breed; age, 6 years; average body weight, 450 ± 5.5 kg) and three fistulated adult rams  were assigned to receive two dietary treatments successively. This study was conducted under guidelines set by the Department of Animal and Poultry Production, Desert Research Center, Egypt. Moreover, the project was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, University of Sadat City, Egypt (Project Reference: VUSC00008). All methods were performed in compliance with the ARRIVE guidelines. In addition, the project does not include euthanasia of the experimental animals. The sample size was decided based on the availability of similar animals with similar physical and physiological status.The present experiment was conducted at Maryoout Research Station, Desert Research Center, Alexandria, Egypt. Three fistulated adult camels . The YA combination was offered gradually and was supplied to the camels at 48 g/head and for sheep at 12 g/head, which represented 1% of daily total dry matter intake. The experiment was conducted for two periods, in the first period, the animals were offered the control diet for 21 days before the sampling period. Subsequently, all animals were switched to the second period with the supplemented concentrate mixture for 21 days before the sampling period. Thus, the experimental groups were (1) camels fed the control diet without supplementation (CC); (2) Rams fed the control diet (CR); (3) camels supplemented with YA mixture (TC); and (4) rams supplemented by YA mixture (TR). All animals were housed individually in well-ventilated pens and offered free drinking water and received the experimental diet as per treatment twice daily at 08:00 and 14:00 h. All refused feed was quantified to determine the feed intake. Concentrate feed mixture consisted of corn 57.5%, soybean meal 23%, wheat bran 19%, limestone 2.5%, salt 1.5%, sodium bicarbonate 0.5%, premix 0.4%, and antitoxins 0.1%. The chemical analysis of barley straw and concentrates feed mixtures are presented in Table Throughout the experiment, all animals were offered ad libitum barley straw. Moreover, concentrates mixture was offered to camels at 1400 g/head and rams at 450 g/head. The concentrates mixture was either unsupplemented (control) or supplemented with yeast and algae combination (YA) . One nylon bags was prepared for each of four times for each animal . A total of four bags were placed into the rumen of each camel or ram before morning feeding. The bags were retrieved from the animal rumen after 3, 6, 12, 48 h. After the removal from the rumen, the bags were rinsed with water until the water ran clear; then squeezed and dried at 60 °C for 48 h to determine the disappearance of dry matter (DMD), neutral detergent fiber (NDFD), and crude protein (CPD) of barley straw when animal fed the control diet (C) and treatment diet (YA).At the end of every experimental period, rumen and blood samples were collected before morning feeding. Rumen contents were collected from the animal’s fistula before feeding and strained by two layers of cheesecloth. The rumen pH was recorded using a digital pH meter (WPA CD70). The rumen liquid was used in the analysis of rumen fermentation parameters and DNA isolation. Blood samples were collected from the jugular vein, and serum was separated by centrifuging at 10,000× g for 5 min and then was frozen for further analysis.S. cerevisiae at the concentration of 2 × 107 colony-forming units (CFU)/g. All the Chlorella and Spirulina that were used in the experiment were obtained in the form of green dried powder from Algal Biotechnology Unit, National Research Centre, Cairo, Egypt, and their chemical composition was previously reported in El-Sayed and El-Sheekh; and El-Feky et al.33. The contents of dry matter (DM), neutral detergent fiber (NDF), and crude protein (CP) were determined in experimental diets and barley straw before and after incubation in the rumen. DM was measured by drying the material for 48 h at 60 °C. NDF was determined by the method of Van Soest et al.34 without sodium sulfite. CP was determined according to AOAC35. The rumen ammonia (NH3-N) and total VFA concentrations were determined by steam distillation in Kjeldahl distillation equipment36. In addition, individual VFAs were measured using high-performance liquid chromatography (HPLC) and the VFA were separated using C18 column with a mobile phase containing 0.3% phosphoric acid37. Serum metabolites were analyzed after thawing using a spectrophotometer with commercial reagents  according to manufacturer’s recommendations and immunoglobulin IgA, IgG, and IgM were determined using Enzyme-Linked Immunosorbent Assay (ELISA).The live yeast was a commercial product containing 38. PCR amplification was conducted under the following conditions: 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s; and 72 °C for 10 min. PCR products purification, preparation for sequencing using Illumina MiSeq system were conducted according to the protocol described by Comeau et al.39 in Integrated Microbiome Resource . Briefly, PCR-amplicons were cleaned up and normalized using the high-throughput Invitrogen SequalPrep 96-well plate kit. Then, the samples were finally pooled to make one library for the sequencing.One milliliter of every rumen sample was centrifuged at 13,000 rpm, and the remained precipitate was used for DNA extraction by i-genomic Stool DNA Extraction Mini Kit  according to the manufacturer’s instructions. DNA was eluted in 50µL elution buffer, and DNA quality and quantity were checked by agarose gel electrophoresis and Nanodrop spectrophotometer, respectively. The V4 region of the bacterial 16S rDNA gene was amplified using 515F and 926R primersPrevotella sp, Ruminococcus albus, Butyrivibrio hungatei purchased from DSMZ . Serial dilutions of the standards ranging from 101 to 106 copies of the 16S rDNA gene were used. The qPCR was performed using the Applied Biosystems StepOne system . Bacterial primers F (5′-CGGCAACGAGCGCAACCC-3′) and R (5′-CCATTGTAGCACGTGTGTAGCC-3′)40 were applied. The 10-μL reaction consisted of 1 μL genomic DNA, 1 μL of each primer, and 7 μL SYBER Green qPCR- master mix . The PCR conditions were as follows 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The linear relationship between the threshold amplification (Ct) and the logarithm of 16S rDNA copy numbers of the standards was used to calculate the copy numbers of rumen bacteria per μL of DNA.Quantitative real-time PCR (qPCR) was used to determine the total bacterial 16S rDNA copy number in the rumen samples. Standards were generated using serial dilutions of DNA isolated from 41. Paired-end fastq files were demultiplexed and quality checks of forward and reverse reads were conducted based on the quality scores. Then sequence reads were quality filtered, trimmed, and dereplicated followed by merging the forward and reverse reads together to obtain the full denoised sequences. The reads were inspected for chimeras that were removed, and Amplicon Sequence Variants (ASVs) were generated. Taxonomic assignment of sequence variants was performed using a combination of the functions assign Taxonomy and assignSpecies and was compared using the SILVA reference database. Various alpha diversity indices, Chao1, Shannon, and InvSimpson were obtained. Beta diversity was assessed as the principal coordinate analysis (PCoA) based on bray–curtis dissimilarity.All the paired-end (PE) Illumina raw sequences were processed in R (version 3.5.2) using the DADA2 (version 1.11.3) pipeline as described by Callahan et al.42. The between-subjects factor was the animal species, and the within-subjects factor was feeding treatment. The Duncan test through One-Way ANOVA was carried out for mean separation between all experimental groups. For all statistical tests, a P < 0.05 was used as a threshold of statistical significance. Pearson correlation analysis was used to identify correlation relationships between feed intake, rumen fermentation parameters, blood parameters, nutrients disappearance, and relative abundances of bacterial genera. The correlation scores were visualized as a heatmap using PAST software43. All the sequences were deposited to the sequence read archive (SRA) under the accession number: PRJNA767400.The results of the relative abundance of bacteria were tested for normality using the Shapiro–Wilk test, and non-normal values were then arcsine transformed. The effect of animal species (A), feeding treatment (T), and A × T interactions on the differences in feed intake, rumen fermentation parameters, bacterial copy numbers, blood biochemical and immunological parameters, microbial diversity, and relative abundances of bacterial groups were studied by Mixed ANOVA model using Repeated Measures function in IBM SPSS software v. 20.00.75 (Kilogram metabolic body weight) are described in Table P < 0.05). Camel group TC showed higher dry matter intake (DMI), organic matter intake (OMI), crude protein intake (CPI) from roughage, and neutral detergent fiber feed intake (NDFI). In contrast, sheep group TR showed a higher total CPI ; and animal species did not affect the disappearance. Supplemented camel group (TC) showed the highest DMD at 48 h. Moreover, supplemented sheep group (TR) showed higher NDFD at all incubation times except for 48 h, whenever TC group showed the highest NDFD. Also, sheep group TR showed greater CPD at 48 h.The disappearances of barley nutrients  at 3, 6, 12, 48 h are described in Table P < 0.05) (Table P < 0.05). For example, the molar proportions of propionic was increased in supplemented sheep (TR) and decreased in the supplemented camel (TC). The molar proportion of isobutyric was increased in camels and sheep by YA supplementation. Additionally, the bacterial population was affected by YA supplementation and animal species (P < 0.05) as it was increased in supplemented camels (TC).The effect of the inclusion of YA supplementation on rumen fermentation parameters is shown in Table 5) Table . The supP < 0.05). Higher IgA was observed in camel group CC; and camel group TC showed higher glucose, triglycerides, IgG, and IgM. Moreover, sheep group TR showed higher total protein.The effect of YA supplementation and animal species on serum biochemical parameters and serum immunoglobulins IgA, IgG, and IgM are presented in Table P < 0.05). Chao1 index was increased in camel group TC compared to CC and was declined in sheep group TR compared to CR. Shannon and InvSimpson indices were increased by YA supplementation. Principle Coordinate Analysis (PCoA) based on Bray Curtis dissimilarity metrics showed that bacterial communities of CC, CR, TC, and TR groups were separated distinctly by feeding treatment and animal species   Table a.Table 7Prevotella that was affected significantly (P < 0.05) by YA supplementation, whenever it was declined in camel group TC compared to CC and was increased in sheep group TR compared to CR. Family Rikenellaceae was dominated by genus RC9_gut_group  and total protein were enhanced in supplemented sheep (TR) Table  by supplDFD Fig. . The dec62. The concentration of acetic was declined and propionate was increased in sheep, which was also indicated in previous studies53. Song et al.63 included the yeast culture in the diet of growing lambs and observed that rumen pH and total VFA were not affected; rumen ammonia and butyric acid were increased, and acetic acid was decreased. Previous studies57 revealed that fiber degradation stimulates acetic acid production, which could demonstrate higher acetic acid in supplemented camel group (TC). The increase in the propionic acid production and the decline in the acetic acid could be a positive point for the YA supplementation to reduce the methane production in the rumen as the propionic acid is an alternative hydrogen sink that depresses the methanogensis64. The higher bacterial population in supplemented camels (TC) could be attributed to the availability of growth substrates in animal diets56.It was observed that discrepancies in total VFA production and VFA profile between camel and sheep, which might be attributed to discrepancies in microbial diversity and the relative abundance of bacterial groups65 and yeast/ microalgae combination supplementation (YA) in the horse21. Grimm et al.21 attributed the reduction in total VFA production after the YA supplementation to the changes in the taxonomy of hemicellulolytic and pectinolytic bacteria such as genus Prevotella. This speculation is supported by the decline in the relative abundance of Prevotella in supplemented camels (TC). However, investigating the bacterial functions and enzymes is recommended to get a better understanding of the supplementation impact21.The reduction in total VFA production in supplemented camels (TC) was previously reported by studies that evaluated the microalgae supplementation8. YA supplementation and animal species affected the diversity and relative abundances of bacterial communities in the rumen of camels and sheep (Tables 63 who investigated the effect of yeast supplementation in the diet of lambs on rumen microbiota. Limited information is available about the effect of the microalgae Spirulina and Chlorella on rumen microbiota, and few studies quantified some of rumen bacterial genera using qPCR and concluded that algae supplementation affected the copy number of those genera, which supports our results67. The majority of phylum Bacteriodetes was assigned to genus Prevotella and RC9_gut_group, reflecting the importance of these genera in rumen fermentation63. Genus Prevotella is involved in the degradation of different substrates in the rumen, including protein, xylan, pectin, and starch, and can produce propionate7, which might demonstrate the positive correlation between genus Prevotella, propionic acid, and CPD (Fig. 7.The chemical composition of the animal diet and animal species are the main determiner of rumen microbial community structurep Tables , 2. Simi7. This phylum was dominated by Christensenellaceae_R-7_group, Butyrivibrio, Ruminococcus, Saccharofermentans, Anaerovorax, Succiniclasticum (Table 71. Christensenellaceae_R-7_group was also found in the rumen of goats and Yak and has a potential role in cellulose degradation72. Genus Anaerovorax is involved in butyrate production and has a role in ruminal biohydrogenation73. Additionally, Butyrivibrio has proteolytic and fibrolytic activities and is involved in the production of butyrate and ruminal biohydrogenation74, which could explain the positive correlation between genus Butyrivibrio and butyric acid. Genus Anaerovibrio was not detected in the rumen of the camels after YA supplementation; also, the relative abundance of genus Anaerovorax and Butyrivibrio were affected by YA supplementation; these genera are involved in the ruminal biohydrogenation75, which indicate that the biohydrogenation pathway could be regulated by yeast and algae supplementation.Members of phylum Firmicutes have an important role in fiber degradation in the rumenum Table ; Fig. 2,Papillibacter genus was previously found in a high proportion in cattle- fed corn stover76, indicating that this genus could be involved in fiber digestion77. Moreover, genus Succiniclasticum has a potential role in fiber degradation78 and converts succinate to propionate79, which could illustrate the increase of this genus and propionic acid in the TR group. Genus Saccharofermentans is involved in polysaccharides degradation and produces acetate and propionate71, which could support the positive correlation between this genus and NDFD. Unclassified Ruminococcaceae represented a higher proportion of the bacterial community and some of which were increased by YA supplementation; these bacteria might have a role in fiber degradation76. Phylum Spirochaetes was dominated by genus Treponema and Sphaerochaeta, which were increased by the supplementation. Previous studies80 indicated that Treponema is fiber-associated bacteria, with potential fibrolytic activities. Sphaerochaeta is involved in pectin degradation and produces acetate, lactate, and ethanol81.82. Phylum Verrucomicrobia was found only in camel and increased by YA supplementation, and has potential role the digestion of plant polysaccharides7. Planctomycetes were previously observed in the rumen of the dromedary camel83 and have a potential role in cellulose and hemicellulose degradation84. Phylum Cyanobacteria that was increased by supplementation are aerobic bacteria capable of scavenging oxygen in the rumen and fermenting carbohydrate during the deficiency of nitrogen85. Therefore, this phylum ensures the anaerobic environment to facilitate cellulose degradation by anaerobic microorganisms86. Overall, the results showed that the bacterial community was dominated by polysaccharides degrading bacteria, such as, Prevotella, RC9_gut_group, Butyrivibrio, Ruminococcus, Saccharofermentans, Christensenellaceae_R-7_group, and Treponema, Fibrobacter78. These findings indicate the importance of these genera in the utilization of forage in the rumen. In addition, the relative abundances of some of these genera were affected by YA supplementation, which is in agreement with Ghazanfar et al., and Jiang et al.87, who reported that yeast promotes the colonization of cellulolytic bacteria to dietary fiber as well as stabilize the rumen pH, which could explain the increase in NDFD at 48 h by YA supplementation.Members of phylum Lentisphaerae are involved in cellobiose fermentation63. Previous studies61 mentioned that the improvement in animal immunity could be attributed to the increase in the activity of some cellulolytic bacteria that represent a barrier against pathogens. This trend was observed in the current study, wherever positive correlations between immunity parameters and some cellulolytic bacteria such as Butyrivibrio, Unclassified _Ruminococcaceae, Saccharofermentans, Treponema, and Fibrobacteres (Fig. 88 explained that combining the yeast with prebiotics such as carbohydrates stabilized the rumen pH, modulated the immune response, and improved fiber digestion; besides it inhibited pathogenic bacteria in the gut and alleviated the heat stress. Previous studies90 that linked animal efficiency with rumen microbiota, showed that efficient cows showed lower methane emission; higher feed intake, milk solid, higher relative abundance of Prevotella, Succinclasticum; Treponema, Fibrobacter, Ruminococcus; except for milk quality and methane emission that were not studied in this study, all the previous trends were observed in the supplemented animals. These findings highlight the yeast and algae combination as potential promising feed additives. Previous studies showed that live yeast and algae provide a diverse of chemical compounds that are required for the growth of rumen microbiota, such as carotenoids, phycobiliproteins, polysaccharides, lipids, essential fatty acids, n-3 fatty acids peptides, amino acids, and vitamins64, which encourage the use of yeast and algae combination to supplement the animal diet.Rumen microbiota plays a pivotal role in animal immunity52 who noted an individual variation in animals in response to microalgae supplementation. Ghazanfar et al.61 concluded that the impact of live yeast on animal performance relies on animal breed, physiological stage, type of animal diet, feeding system, type of yeast, and supplementation dose. It is well known that the anatomy and motility of the camel rumen is different from other domestic ruminant animals14, and the retention time of ingested feed is longer in the rumen of the camels than in other ruminants, which affects the response to dietary intervention, diet fermentation, the composition of rumen microbiota, metabolism, and blood biochemical parameters92.Our results indicated that feed intake, the rumen disappearance of DM, CP, and NDF, and blood immunity were improved in both camels and sheep by YA supplementation. Furthermore, the response of rumen bacteria, rumen fermentation, some of the blood parameters to YA supplementation varied according to animal species. A similar pattern was observed by Lamminen et al.The study provides insights into the effect of YA supplementation on feed intake and the rumen ecosystem. The results suggest that YA supplementation modulated the rumen microbial community towards increasing degradation of dietary fiber and crude protein, which improved feed intake and affected rumen fermentation parameters. Moreover, blood immunological parameters were improved. The response to YA supplementation varied according to animal species. Combining the microalgae with yeast might be beneficial to animals’ performance.