Effective postoperative analgesia may enhance early rehabilitation after total knee arthroplasty (TKA). The purpose of this study is to perform a randomized controlled trial to compare the efficiency of adductor canal block (ACB) with periarticular infiltration (PAI) versus PAI alone for early postoperative pain treatment after TKA.After institutional review board approval, written informed consent was obtained from patients undergoing elective TKA. Subjects were randomized into 2 groups as follows: adductor canal blockade with 30 mL of 0.5% ropivacaine and 100 mcg of clonidine. All patients received a periarticular infiltration mixture intraoperatively with scheduled and patient requested oral and IV analgesics postoperatively for breakthrough pain. The primary outcome was morphine consumption in the first 24 hours. Secondary outcomes included pain scores, morphine consumption at 48 hours, opioid-related side effects , functional outcomes, quadriceps strength, and length of hospital stay.For the present trial, we hypothesized that patients receiving adductor canal block + PAI would have significantly lower morphine consumption and pain scores after surgery.researchregistry5490 The goal of achieving an ideal analgesia modality which facilitates early rehabilitation, prevents knee stiffness, reduces hospital stay due to pain, and promotes good functional outcomes continues to be elusive. Multimodal analgesia has been devised to control postoperative pain and reduce opioid consumption including periarticular injection (PAI) and peripheral nerve blocks.,5 The adductor canal block (ACB) has recently gained popularity as an alternative to femoral nerve block due to reduced incidence of quadriceps muscle weakness. It provides a more distal nerve blockade, at the mid-thigh, ideally providing sensory blockade in the distribution of the saphenous nerve, posterior branch of the obturator nerve and vastus medialis nerve while sparing quadriceps function. Literature has supported improved motor function with similar pain control when comparing ACB to femoral nerve block.891089Femoral nerve block has been one of the most commonly used peripheral nerve blocks following TKA. However, because the femoral nerve is comprised of both sensory and motor branches, this block can lead to quadriceps weakness thus delaying mobilization and presenting an increased risk of falls.,12 and non-inferior compared to femoral nerve block.1415161415 PAI during TKA has thus been recommended for routine application considering effective pain relief and smoother rehabilitation.The PAI is a surgeon-controlled analgesic technique that used to reduce the pain in the early postoperative period with no influence on quadriceps strength. It is a mixture of medications that typically includes, but is not limited to, a local anesthetic, morphine, and Toradol. It has been shown to have superior analgesic effects compared to placebo1819201819 We, therefore, further designed a randomized controlled study to compare ACB + PAI with PAI in the treatment of TKA. For the present trial, we hypothesized that the addition of an ACB to PAI, compared with PAI alone, would reduce the time to meet discharge criteria after total knee replacement. Additionally, we hypothesized that patients receiving ACB + PAI would have significantly lower pain scores after surgery.Despite the evidence above, concerns exist regarding the incomplete analgesia of PAI. Previous studies have been published to explore the efficacy between ACB + PAI versus PAI alone in reducing pain after TKA, but with different conclusions.22.1This prospective, blinded randomized controlled trial was conducted at our single university hospital. The study was approved by the institutional review board of the hospital (CQ001023). The trial was registered with Research Registry (researchregistry5490) prior to the enrollment start. Patients were enrolled after signed written consent was obtained. All surgeons, recovery room and floor nurses, research assistants, statisticians, and patients were blinded to group allocation. Only the anesthesiologists performing the blocks and operating room nurses were not blinded.2 and an American Society of Anesthesiologists functional status of I-III were included. Exclusion criteria included a knee flexion deformity of ≥30°, varus-valgus deformity of ≥30°, a diagnosis of nonosteoarthritis , known allergy to the drugs used in this study, or a past history of opioid consumption, excessive alcohol consumption, cognitive impairment, psychiatric illness, narcotic dependency, recognized neuromuscular disorder, knee surgery (including arthroscopy and open surgery), knee infection, or thrombolytic events . Patients who had an inability to communicate verbally or who were unwilling to give informed consent were also excluded.Patients aged 50-80 years with a body mass index of 18-36 kg/m2.2www.randomizer.org) in a 1:1 ratio with 20 numbers in each block. Every participant received a consecutive study number from 1 to 69 and received the treatment assigned according to the randomization list. The list was stored and only 2 nurses, who prepared the study medications, had access to it. They had no interactions with the patients. All other clinical personnel, participants and outcome assessors were blinded to the intervention. The randomization key was first broken when all enrolled patients had completed the study. After discharge, the participant's personal information was eliminated from the study number and was therefore not traceable back to the patient  and extended-release oxycodone (10 mg). Both groups received a spinal anesthetic with 0.5% bupivacaine (10 or 12.5 mg). Patients received 4 mg of ondansetron and 20 mg of famotidine. Patients were not given intravenous opioids or ketamine. The surgeons administered a deep periarticular injection prior to cementation and a second superficial injection prior to closure. The deep injection consisted of 30 mL of 0.5% bupivacaine with epinephrine, 1 mL of 8 mg/mL morphine, 1 mL of 40 mg/mL methylprednisolone, 500 mg of cefazolin in 10 mL, and 22 mL of normal saline solution. The superficial injection was 20 mL of 0.25% bupivacaine.For ACB, an ultrasound transducer was used to identify the adductor canal. The transducer located the adductor canal at mid-thigh, halfway between the inguinal crease and patella. Superficial femoral artery, sartorius muscle, adductor longus muscle, and adductor magnus muscle were identified. The hyper echoic structure located anterolateral to the artery  was identified as the target injection site. A 22-guage, 100 mm needle was introduced lateral to medial under ultrasound guidance using linear probe of a sonosite machine. Solution containing 30 mL of 0.5% ropivacaine and 100 mcg of clonidine was injected after ensuring correct placement of the needle.2.4Patients in both groups received 1 session of physical therapy on the day of the surgical procedure and 3 sessions at 24 and 48 hours until they achieved discharge criteria. Patients defaulted to 2 physical therapy sessions if they did not meet discharge criteria by 72 hours. Each rehabilitation session assessed patients for discharge. The criteria for discharge were the ability to independently transfer in and out of a bed, chair, or toilet seat; to walk approximately 50 m with or without an assistive device; to negotiate stairs with or without a rail or cane; and to perform a home exercise plan. All subjects underwent a standard postoperative multimodal pain management regimen. Postoperative medications included acetaminophen, ketorolac followed by celecoxib (for 3 months), gabapentin (standing order for 10 days), oral opioids (as needed), and intravenous hydromorphone for breakthrough pain.2.5The primary end point was Morphine consumption in the first 24 hours (including Morphine administered intra-operatively and in recovery). Secondary outcomes included pain scores, Morphine consumption at 48 hours, opioid-related side effects , functional outcomes, quadriceps strength, and length of hospital stay. Pain scores were recorded at 1, 6, 12, 24, and 48 hours post-operatively, using a Numerical Rating Scale (0–10) at rest and during 45° passive flexion of knee.Nausea was defined as the unpleasant sensation associated with awareness of the urge to vomit; vomiting was defined as the forceful expulsion of gastric contents from the mouth. The presence/absence of nausea and the number of episodes of vomiting (more than 10 mL) were recorded. The usage of antiemetics was also recorded. The level of sedation was assessed on a 4-point scale .Functional outcome measures used were the 30 Seconds Chair Stand Test (30-CST) and the Timed Up and Go (TUG) test. 30-CST tested the number of times each subject was able to stand up from a seated position on a chair in 30s. The TUG test assessed the time taken for each subject to get up from a seated position on the chair, walk 3 meters away from the chair before returning to it. Both 30-CST and TUG test had been shown to be reliable and validated tools for rehabilitation of orthopedic patients. Quadriceps strength was assessed as a percentage of the baseline measurements at 24 and 48 hours post-operatively, using the method previously described. The 30-CST and TUG tests were performed at 24 and 48 hours. We also assessed for early complications such as hematoma, infection and neurological deficits at 48 hours. Any other early complications, including falls within the first 48 hours were recorded. The length of hospital stay was also noted.2.6The sample size calculation was based on a pilot study that we conducted on 18 patients (whose data were not included in the present study). In this prior study, the mean difference and standard deviation of the Numerical Rating Scale scores 24 hours after the operation between the ACB + PAI and PAI groups were 0.40 and 0.19, respectively. From this, it was determined that 42 subjects would be required to reach an α value of 0.05 and a power of 85%. It was estimated that the attrition rate due to canceled surgery or reasons of late patient ineligibility could be up to 20% and, therefore, to account for this, the final sample size selected was n = 100 (50 per group).2.7U variance analysis was used for discrete numerical variables that did not show normal distribution. Relationships between the categorical variables were determined by preparing crosstabs and using the Chi-squared (χ2) test. P < .05 was accepted as statistically significant.The statistical analyses in this study were performed using the Statistical Package for the Social Sciences ) 20.0 software. Continuous variables were presented in the form of mean ± standard deviation or error. The Kolmogorov–Smirnov normality test was used to assess continuous variables. Group comparisons on the variables that showed normal distribution were performed using one-way analysis of variance. Mann–Whitney 334534 Use of systemic opioids can cause adverse effects that may affect functional rehabilitation, such as nausea, vomiting, pruritus, sedation, and respiratory depression. Hypotension, urinary retention, and pruritus are more common in patients with epidural analgesia.TKA is a common surgical procedure that can cause severe postoperative pain. Various methods for postoperative analgesia management are available, such as systemic opioids, epidural local anesthetic, peripheral nerve block, and local anesthetic infiltration analgesia.,21 Moreover, PAI has been shown to improve pain management and to preserve motor function. With PAI, the nerves, muscle, and tissue in the posterior, lateral, and medial aspects of the knee are infiltrated intraoperatively with local anesthetic, morphine, and ketorolac to provide analgesia.The introduction of ACB to manage pain after TKA is relatively new. The adductor canal is an aponeurotic space in the middle third of the thigh. It contains nerve branches that supply sensory innervation to the knee, including the posterior branch of the obturator nerve and the saphenous nerve. Blocking these nerves provides analgesia to the medial aspect but not to the lateral or posterior aspects, of the knee.The main limitation of the current study was the inability to blind both the participants and the physicians to comparisons between peripheral nerve blockade and periarticular injection. This lack of blindness may have introduced some risk of bias from both the patients and the physicians. The outcome assessments from the adjudicators and all the statistical analyses were conducted in a blinded manner. In addition, the impossibility of measuring quadriceps muscle power before and after the operation using special instruments was another limitation. If this had been possible, the evaluation between the ACB + PAI and the PAI groups could have been more objective.Conceptualization: Yongcheng Ren.Data curation: Yongcheng Ren.Formal analysis: Yongcheng Ren.Funding acquisition: Jianming Yang.Investigation: Xiaoyan Qin, Jiacai Liao.Methodology: Xiaoyan Qin, Jiacai Liao.Resources: Jianming Yang.Software: Xiaoyan Qin.Supervision: Jianming Yang.Validation: Xiaoyan Qin.Visualization: Jianming Yang.Writing – original draft: Yongcheng Ren.Writing – review & editing: Yongcheng Ren, Jianming Yang.


Quyu Shengji Formula (QSF), a Chinese medicine formula widely used in the clinic, has proven therapeutic effects on diabetic ulcers. Nevertheless, the potential mechanism of how QSF cures diabetic ulcer remains elusive.  To assess the mechanism of QSF against wound healing defects in diabetes. 2 (PGE2), and angiogenesis factor vascular endothelial growth factor (VEGF) were evaluated by immunostaining (IHC) analysis, ELISA assay, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot analysis in vivo. Human dermal microvascular endothelial cells (HDMECs) and the shRNA interference technique were used to explore the effects of QSF on cell migration, PGT, PGE2, and angiogenesis factor VEGF in vitro.  Db/db mice were adopted to determine the therapeutic potential of QSF. Further histology analysis was performed by hematoxylin and eosin (H&E) staining. Moreover, the expression patterns of prostaglandin transporter (PGT), prostaglandin E2 and increased angiogenesis factor VEGF levels were shown after QSF treatment in vivo and in vitro. Furthermore, QSF promoted HDMEC migration. Inhibition of the expression of PGT by shRNA reversed phenotypes of QSF treatment in vitro.  Applied QSF on the wound of db/db mice significantly accelerated wound closure. Reductions of PGT and elevations of PGE Taken together, our findings reveal that QSF ameliorates diabetes-associated wound healing defects by abolishing the expression of PGT. A diabetic ulcer is a serious complication of diabetes mellitus. Ulcers are often difficult to heal for a long time and easily lead to amputation and eventual disability. It has been reported that approximately 6.3% of the world's population suffers from diabetes ; moreoveDebridement and dressing change are often ineffective in treating diabetic ulcers and always cost a lot, which imposes a heavy body-heart burden on the patient, family, and even the whole society. However, some newly developed biological agents and new excipients are expensive or have poor efficacy, and some even pose an oncogenic risk . Traditi2 (PGE2) has a wide range of physiological functions, especially in the functions of dilating blood vessels and increasing blood flow. Recent studies showed that it has a considerable effect on the healing of gastric ulcers [2 level is decreased in chronic diabetic ulcer, which may be an essential reason why diabetic ulcer is difficult to heal [2 is synthesized by cyclooxygenase (COX) and transported by prostaglandin transporter (PGT) to intracellular degradation. Recent research showed that COX increases in diabetic ulcer wounds [2 can only be analyzed from the perspective of metabolism.Prostaglandin Ec ulcers . PGE2 le to heal . Howeverr wounds . Notably2 showed an obvious negative correlation, and reducing PGT could increase the content of PGE [2, leading to a decrease in local PGE2 content, which is a crucial reason for delayed wound healing. Inhibition of PGT can facilitate wound healing of diabetic ulcers [PGT was found relatively late, and its research is still at a relatively preliminary stage. PGT and PGEt of PGE , and vict of PGE –15. Studc ulcers .2, leading to local occlusion of blood vessels similar to what TCM calls “Yu” (blood stasis). We adopted db/db mice and human microvascular endothelial cells (HDMECs) as the research object to evaluate QSF regulating the expression of PGT during diabetic healing time and its effects on the metabolism of PGE2 and VEGF.Here, we created QSF based on the theory of “Quyu Shengji.” “Quyu” means “removing blood stasis.” In diabetic chronic skin ulcers, the increase in PGT causes a decrease in PGEDrug Preparation. QSF was comprised of 8 Chinese herbs, as shown in The original medicinal materials were decocted twice in water according to the conventional method of decocting traditional Chinese medicine, then filtered and concentrated into 10 g/ml medicinal solution, added with 70% ethanol for precipitation, and left for 24 hours. After the ethanol is recovered, it is diluted with an appropriate amount of water to make a mixture and finally made into a crude drug-containing 2 g/ml . All theFemale clean-grade db/db mice and C57 mice, weighing 20 ± 4 g, were provided by the Experimental Animal Center of the Chinese Academy of Sciences, Shanghai. Animals were maintained under specific pathogen-free conditions. All experiments were performed according to the guidelines of the Committee on Protection, Welfare and Ethics of Experimental Animals in Yueyang Hospital of Integrated Traditional Chinese and Western Medicine affiliated to Shanghai University of Traditional Chinese Medicine (No. YYLAC-2019-043).All mice were anesthetized by intraperitoneal injection of 0.1% ketamine hydrochloride injection (100 mg/kg). The modeling area was marked with a stamp with a diameter of 8 mm, and both sides of the back spine were shaved with a mechanical razor. To form an open wound with a full-thickness skin defect with a diameter of 8 mm under strict aseptic operation, the left and right sides of the mouse spine were cut to the subcutaneous fascia layer. There are 4 mouse wound models with skin ulcers on the left and right sides .μg/g. An equal volume of distilled water was administered by gavage in the blank and control groups. The intragastric volume was calculated by 2 g/ml once a day.After adaptive feeding for 1 week, 12 mice were randomly divided into 3 groups as follows: blank group, control group, and QSF group. The adult dose of QSF was 130 g per day according to the adult standard weight of 70 kg, the mouse coefficient was 9, and the daily dose of rats was 16.7 We repeated the experiment three times, sacrificing animals on day 1, day 5, and day 9, respectively.The measurement of the wound area was performed as follows: the quick wound healing adhesive plaster was pressed to the ulceration surface, and a marking pen was used to draw the outline of the wound on the adhesive plaster. A photo of the plaster was taken using a digital camera against a white background. Wound area measurement: then the photo was evaluated by calculating the wound area with image analysis software Image J v1.42q . The wound area on day 1, day 3, day 5, and day 9 after modeling was compared.2 and the tissues of the skin wounds were taken with an 8 mm diameter metallic punch. The tissue was put into liquid nitrogen immediately and kept at −80°C.On day 1, day 5, and day 9 after the wounding, animals were euthanized by COHE and immunohistochemical staining were carried out according to the relevant protocols. For general histological analyses, tissue samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned from the midline of wounds, and stained with H&E.The expression of PGT in wound tissues was determined with immunohistochemical analysis and the following antibodies: rabbit anti-PGT , and the secondary antibodies goat anti-rabbit IgG HRP (diluted: 1 : 2500). Under the same experimental conditions as the negative control, each sample was diluted and incubated with the same type of antibody. Images were acquired using a light microscope  and analyzed with the image processing software (Image-Pro Plus 6.0).IHC average optical density value analysis method: each slice in each group was randomly selected with at least 3 200x fields of view for photography. The same brown color was selected as the unified criterion for judging the positivity of all photos by using Image-Pro Plus 6.0. The cumulative optical density value (IOD) and the pixel area (AREA) of each photo were obtained by analyzing each photo. Calculate the average optical density value ; AO = IOD/AREA; the higher the AO value, the higher the positive expression level.The sampling procedure is the same as above. Extracting total RNA: the homogenate was taken and total RNA was extracted according to the Trizol reagent kit method and steps . The concentration of total RNA was measured by ultraviolet spectrophotometer. The purity of RNA was measured with agarose gel electrophoresis. Reverse transcribed to synthesize cDNA, primers used were as μl. Real-time PCR amplification: SYBR Green kit  was used to make real-time PCR amplification of GAPDH, PGT, and VEGF. Real-time fluorescent PCR was used to make a real-time fluorescent quantitative PCR reaction . After the reaction was finished, fluorescent quantitative data were collected and analyzed. The data included the amplification curve, working curve, dissociation curve, and corresponding CT value. Light Cycler Software (Version 3.5) was used for data analysis.Reverse Transcription System First Strand cDNA Synthesis Kit was used, with a total reaction volume of 20.0 2 in wound tissue or cell supernatant were determined using ELISA  according to the manufacturer's instructions. Set the blank to zero and measure the absorbance  of each hole in sequence at 450nm wavelength.The levels of PGEThe tissues were lysed and the supernatant was used for protein quantification. GAPDH was used as a protein loading control. The following primary antibodies were used in subsequent experiments: antibody PGT  and VEGF antibody . The secondary antibodies used are goat anti-rabbit HRP and goat anti-mouse HRP . A Tanon-5200 imaging system  was used to check the target protein and was observed with autoradiography film.2. Blood samples were taken from the heart aseptically. Samples were left at room temperature for 3-4 hours. They were centrifuged for 10 min at 3000 revolutions/min. Then, we took the supernatant, and it was inactivated in a water bath at 56°C for 30 minutes. We filtered the serum through a 22 μm filter membrane in an ultraclean table to remove bacteria. Serum samples were dispensed into autoclaved centrifuge tubes. The drug serum was stored at −20°C [Male clean-grade SD rats, weighing 250 ± 20 g, were provided by the Experimental Animal Center of the Chinese Academy of Sciences, Shanghai. Animals were maintained under specific pathogen-free conditions. When QSF was given by gavage for 7 days continuously, the rats were killed by COat −20°C .Low glucose (blank) group: 5 mmol/L glucose + 10% blank serumHigh glucose (control) group: 25 mmol/L glucose + 10% blank serumQSF prescription medicine serum group: 25 mmol/L glucose + 10% QSF drug serumHuman dermal microvascular endothelial cells (HDMECs) were purchased from Kilton Biotechnology (Shanghai) Co., Ltd. The experiment is grouped as follows:HDMECs were cultured in endothelial cell medium (ECM) containing 5% fetal bovine serum, 1% endothelial cell growth supplement (ScienCell Research Laboratories), 1% penicillin-streptomycin, and 5 or 25 mmol/L glucose.Each group had 4 wells; each group was seeded in the corresponding glucose environment at 37°C for 3 days. Medicated serum was added and cultured for 12 hours and then digested with trypsin. Cells were collected for further experiment.We completed the design of the target gene interference target and then sent it to the gene synthesis company  to synthesize the interference target shRNA as Briefly, we infiltrate the chamber before inoculation. We put the Transwell chamber into the culture plate, prepared the cell suspension, and inoculated the cells. Cells were fixed at room temperature and observed after staining.x ± SD, and the count data was expressed by the rate. If multiple groups of measurement data conform to the normal distribution  and the variances were uniform, the analysis of variance and Tukey's method for pairwise comparison would be applied. The nonparametric test was used for those who do not meet the conditions of the variance test and Mann–Whitney U method. The Kruskal-Wallis method was applied for the comparison of multiple groups, and the Dunnett method was used for the comparison between groups.GraphPad Prism 8  software was used for statistical analysis. The measurement data was expressed by in vivo. From Figures To test if QSF can overcome wound healing defects seen in db/db mice, we examined the topical application of QSF 2 and VEGF. We detected the expression of the molecules mentioned above. As shown in 2 content in the control group was significantly decreased, while that in the QSF group increased PGE2 content. Such an effect even appeared in the middle stage of wound healing.Next, we examined whether the observed accelerated wound closure of QSF wounds could, at least in part, contribute to the inhibition of PGT and the upregulation of PGEFrom Figures Due to HDMEC's ability to improve local angiogenesis of the wound and enhancing blood supply, we examined whether QSF affects the migration ability of HDMEC. Through Figures Three shRNA sequences were used for the validation test. It was found in 2 increased significantly, and the QSF group also achieved similar results. However, if shPGT inhibited the expression of PGT, QSF did not play a role as before, indicating that the effectiveness of QSF depended on PGT to a certain extent. Interestingly, similar results were obtained in the Transwell test in the same situation, which also indicates that the effect QSF group is to some extent dependent on the expression of PGT.By the ELISA test, we found that after shPGT inhibited the expression of PGT, the content of PGE2. It is difficult for PGs to pass through the cell membrane in a freely diffused manner [2, while decreasing the glucose concentration will increase PGE2 [Prostaglandin transporter (PGT) is the main protein that mediates the transmembrane transport of PGs such as PGEd manner , 19. Thed manner . Previouase PGE2 .2 content, leading to a series of pathological changes such as local tissue capillary occlusion and local tissue hypoxia [In recent years, studies on diabetic ulcer wounds have shown that increased tissue glucose content will cause PGT-mediated reduction of PGE hypoxia , 23.2 is transferred into the cell through the intercellular PG transporter and is rapidly inactivated by 15-ketoprostaglandin (15-PGDH). In diabetic animals, PGE2 synthase did not decrease, but the expression of PGE2 decreased, which made the wound difficult to heal. High glucose levels will increase PGT and accelerate PGE2 metabolism. PGT inhibitors can accelerate wound healing and increase VEGF expression [PGEpression , 25.β-catenin signaling pathways [The project that the author participated in the early stage partly clarified the mechanism of TCM on wound healing from the perspectives of the “stasis-removing and tissue-generating” method regulating growth factors, collagen metabolism, MAPK, and Wnt/pathways –8, 17. IWe created QSF based on the theory of “Quyu Shengji.” “Quyu” means “removing blood stasis.” In diabetic chronic skin ulcers, the increase in PGT causes a decrease in PGE2, leading to local occlusion of blood vessels similar to what TCM calls “Yu” (blood stasis). The theory of “Quyu” (removing blood stasis) is one of the main basic theories of TCM. In particular, great progress has been made in the past 200 years, making certain new treatment methods available for diseases that were difficult to treat in the past –29.2 and VEGF expression, and increased PGT expression. The traditional Chinese medicine QSF Decoction improves the series of pathological changes by inhibiting the expression of PGT and increasing PGE2 and VEGF in vivo and in vitro.In the current study, db/db mouse skin ulcer model was used as the research object. It was found that, compared with the blank group, the control group had difficulty in wound healing, impaired local blood supply, decreased tissue PGEIn summary, QSF attenuated PGT expression which promoted diabetic wound healing. Further studies on the effects of PGT knockout might reveal more molecular roles for PGT in diabetic wound healing. Our future direction is to further investigate the effect of PGT on nonhealing wounds.


Felis catus) and wild cats. The results of our research showed that ovaries obtained from prepubertal felids may be a rich source of good quality oocytes that are competent for in vitro maturation and able to reach blastocyst stage after in vitro fertilization. The results are important in the context of the possibility of using oocytes from prepubertal felids threatened with extinction in conservation programs based on assisted reproductive techniques (ART).This study was conducted with the aim of determining the morphological similarities and developmental potential of oocytes obtained from adult and prepubertal domestic cats (Felis catus) and wild cats . The average number of oocytes obtained from an adult domestic cat was 23 ± 11, which was significantly lower than from kittens (43 ± 29). A similar number of oocytes was derived from adult Pallas’s cats (28 ± 8), and serval (30). The lowest number of oocytes was collected from the lynx (5 ± 3). No oocytes were obtained from newborn Amur tiger while in the case of older domestic and Pallas’s cat and lynx kittens (1–3 months) 43, 48 and 41 oocytes were collected, respectively. Significant differences (p < 0.001) were observed between the number of oocytes with dark cytoplasm from adult and prepubertal animals of all analyzed species. The diameter of oocytes from adult and prepubertal animals was similar in all species, and was on average 161 ± 4 µm for oocytes with dark cytoplasm and 150 ± 18 µm for oocytes with light cytoplasm. In all species, oocytes with light cytoplasm were significantly smaller (p < 0.05) than dark ones, and their population was more diverse. Results of in vitro maturation of the domestic and wild cat′s oocytes obtained from adult and prepubertal females were similar (47–52%). The cleavage rate after in vitro fertilization (IVF) was lower for prepubertal than adult domestic cats . Moreover, we observed differences in the quantity  and quality of blastocysts and even greater problems with hatching blastocysts from prepubertal kittens . More blastomeres were detected in blastocysts of adult cats. They also demonstrated significantly higher number of inner cell mass (ICM) (p < 0.001) and higher number of trophoblast cells (TE) (p < 0.05).The aim of the study was to compare the morphology and developmental potential of oocytes obtained from adult and prepubertal domestic cats ( Wild felidae are among the most endangered species in the world. Endangered wild cat species conservation programs implemented all over the world are being supported by the latest developments in the assisted reproductive technologies (ART) .Felis catus) are frequently used for research as a model for felid species that are threatened with extinction. Actually, many assisted reproduction methods are tested using the domestic cat model (Felis catus) due to their widespread availability of this research material [Domestic cats , ICSI assisted fertilization or somatic cloning, depends largely on the quality of the oocytes, their selection and appropriate in vitro maturation (IVM) conditions. To adapt ART programs for the protection of wild cats and minimize the risk of failure of these procedures, it is necessary to identify similarities and differences between oocytes from wild feline species and the domestic cat, including discrepancies in their morphology. Moreover, an in-depth understanding of the importance of the morphological features is necessary to ensure that an adequate number of oocytes from the most valuable species, maintained through cryopreservation and storage in cell banks, is available in the future for cloning or in IVF programs .Felis catus) and wild felids represented by the northern lynx (Lynx lynx), serval  and Pallas’s cat (Felis manul) at reproductive age, as well as prepubertal domestic cat (Felis catus), Pallas’s cat (Felis manul), northern lynx (Lynx lynx), and Amur tiger . The study included comparison of the IVM capacity of oocytes retrieved from adult and kittens of domestic cat, Pallas’s cat (Felis manul) and lynx (Lynx lynx). Additionally, developmental competence of the oocytes after IVF and quality of blastocyst in prepubertal and adult domestic cats (Felis catus) were analyzed.The aim of the study was to compare the morphology of oocytes obtained from adult domestic cats .Media used for IVF, such as “Sperm AirOther chemicals and reagents used in this study were purchased from Sigma-Aldrich .Lynx lynx, Leptailurus serval, Felis manul) and prepubertal (up to 3 months old) wild cats  were collected post mortem in emergency situations at the Zoological gardens in Poland. All adult wild cat females were of reproductive age (4–8 years), fertile, and produced offspring regularly. The causes of death or euthanasia were: femur fracture, complications during sedation, severe diarrhea, acute pneumonia, rejection by the mother and hypothermia, or poisoning. Oocytes were obtained immediately after death or euthanasia.Ovaries from 20 healthy adult (cycling > 1 year of age) domestic cats were obtained by ovariohysterectomy conducted at local veterinary clinics. Ovaries from prepubertal domestic cats  were collected post mortem, after euthanasia from veterinary clinics. Ovaries from adult  supplemented with 100 μg/mL streptomycin and 100 IU/mL penicillin at 4 °C in a thermal box.oocytes tightly surrounded by at least three layers of cumulus cells,oocytes partially surrounded by cumulus cells,oocytes surrounded only by the corona radiata,degenerated oocytes with morphological defects, with damaged zona pellucida, distorted shape or fragmented cytoplasm.Cumulus-oocyte complexes (COCs) were obtained by scarification in a washing medium . Oocytes were then assessed using a stereoscopic microscope and divided into four classes based on the quality and quantity of the cumulus cells:A-oocytes with very dark cytoplasm,B-oocytes with dark, mosaic cytoplasm,C-oocytes with pale cytoplasm,The oocytes were also classified according to the color of their cytoplasm :A-oocyteThe numbers of oocytes with dark  and pale cytoplasm (C) were counted and the oocyte diameter was measured.2 in air. About 20–40 COCs were cultured per well except for the lynx, from which only 5 oocytes were obtained. After IVM, oocytes of wild felids were denuded of cumulus cells by mechanical pipetting in hyaluronidase (80 IU/mL) for 2 min, and only those with a visible first polar body were cryopreserved and stored in a cell bank. Some of the domestic cat oocytes were completely denuded of granulosa cells in order to visualize the polar body and determine the maturation rate. The remaining oocytes were partially denuded and subjected to in vitro fertilization.Only oocytes with dark cytoplasm , surrounded by compact layers of cumulus cells , were selected for IVM. Selected COCs were placed in 4-well dishes 20–40 COCs per 400 µL TCM 199 with Earle’s salts, supplemented with 0.02 IU FSH/mL and 0.02 IU LH/mL 0.25 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, and 0.15 mg/mL L-glutamine, and cultured for 24 h at 38.5 °C and 5% CO5 motile spermatozoa/mL in 400 μL IVC-CULT  medium at 37 °C in 5% CO2 in air.Thawed spermatozoa of domestic cats, isolated from cauda epididymis and frozen according to the procedure described by Niżański et al.  were useAt 16 h after IVF, presumptive zygotes were transferred to 20 µL droplets (5 embryos/drop) of pre-equilibrated CULT medium  under mineral oil and cultured up to 8 days. Every second day, half the volume of culture medium was exchanged for fresh medium.Blastocysts on day 7 after IVF were incubated for 15 s in PBS containing 0.5% Triton-X and 100 µg/mL propidium iodide to stain trophoblast cells (TE) in 37 °C. Blastocysts were then immediately transferred into 100% ethanol with 15 mg/mL Hoechst 33,342 and incubated for at least 40 min in 4 °C to stain inner cell mass (ICM). Cells were mounted onto a glass microscope sidle with a drop of glycerol, flattened with a coverslip, and evaluated using epifluorescence inverted microscope (Nicon Eclipse 200) with UV excitation of 345 and 535 nm resulting in bisbenzimide-stained nuclei fluorescing blue (ICM), whereas propidium iodide-stained cells fluorescing red (TE). The ICM ratio was counted as the proportion of the total cell number of blastomeres (TCN).p < 0.001.Non-parametric data, such as differences in the mean percentage values between groups, were assessed by the chi-squared test. Parametric data, such as oocyte dimension and number of blastomeres in blastocysts, were expressed as means, SD and were compared by two-way ANOVA. Significant statistical difference was noted at p < 0.001) were observed between the number of oocytes with dark cytoplasm from adult and prepubertal animals of all analyzed species  than dark ones, and their population was more diverse with an average diameter of 150 ± 18 µm, but similar for all individuals from a species.In all species oocytes with light cytoplasm were significantly smaller (p > 0.05) were obtained from the adult and prepubertal females, regardless of species (47–52%).p < 0.05%). Moreover, we observed differences in the quantity  and quality of blastocysts (p < 0.001). More blastomeres were detected in blastocysts from adult cats (p < 0.001) and higher number of trofoblast cells (TE) (p < 0.05).stocysts  and evenult cats . They alImplementation of reproductive biotechnologies to breed wild felids is extremely difficult. It is seriously hindered by limited knowledge on the physiology of reproduction of particular species of wild cats, poor gamete quality, and a lack of optimized ART treatments that have been developed mostly by using the domestic cat as a model animal ,24. WildPanthera lion), puma (Felis concolor), jaguar (Panthera onca), and golden cat (Felis temmincki) obtaining oocytes from individuals over 14 years old is impossible [Panthera tigris), leopard (Panthera pardus), cheetah (Acinonyx jubatus) and clouded leopard (Neofelis rufus), oocytes were obtained only from individuals in very good health, but the number of oocytes retrieved was lower than from younger females [In this study, we obtained an average of 23 oocytes from adult domestic cats, 30 from serval, 28 from Pallas’s cats and 5 from lynx. The available bibliographic resources fail to provide any information on the characteristics of oocytes collected from the lynx and Pallas’s cat. Johnson et al.  reportedpossible  and is apossible . In othe females .There are no references on the collection of oocytes from very young cats. Due to the high mortality rate in newborn and very young wild cat kittens, both living in the wild and kept in zoos, we attempted to determine whether collection of oocytes from young individuals is possible and to compare their quality with oocytes derived from mature females.p < 0.001) than from adults. In the case of the tiger, no oocytes were collected from a newborn cub, while oocytes retrieved from other young cats were morphologically normal and were subjected to IVM.In the analyzed species, except for the tiger, the number of oocytes collected from young individuals was significantly higher .


Cyprinus rubrofuscus). Analyses of video data shows that overall, koi spent more time than expected in close proximity to the human and even sought out physical contact. Moreover, individual fish displayed unique interaction patterns, with some frequently engaging in tactile interaction and others only periodically or rarely doing so. By demonstrating that koi will voluntarily interact with humans and that individual differences play an important role in interaction style, this study provides the first evidence that individuated human–fish relationships may be possible, which has powerful implications for how we think about, treat, protect, and provide care for fish.To assess the possibility of forming stable and trusting relationships between humans and fishes, we documented the interactions between a familiar human experimenter and seven koi (Carassius auratus) and seven adult and two juvenile koi (Cyprinus rubrofuscus). After a habituation period (July–September 2019) during which time the fish were regularly provided with the opportunity to engage with the researcher’s submerged hand, but were not forced to interact with the researcher, we collected video data on 10 non-consecutive study days during the month of October. This procedure produced 18~20-min interaction sessions, 10 during T1 (when the experimenter first arrived and the fish had not been fed) and eight during T2 . Interactions between the researcher and the seven adult koi were coded from video based on location  and instances of physical, tactile interaction. Analyses revealed that overall, koi spent more time than expected within reach of the researcher during both T1 (p < 0.02) and T2 (p < 0.03). There were also substantial differences between individuals’ overall propensity for being within-reach and engaging in physical interaction. These results show that koi will voluntarily interact with humans and that individual koi display unique and consistent patterns of interaction. By providing quantitative data to support anecdotal claims that such relationships exist around the world, this research contributes to the ongoing discoveries highlighting the profound dissonance between how humans think about and treat fish and who fish actually are, thereby emphasizing the necessity of stronger moral and legal protections for fishes.The study of human–animal interactions has provided insights into the welfare of many species. To date, however, research has largely focused on human relationships with captive mammals, with relatively little exploration of interactions between humans and other vertebrates, despite non-mammals constituting the vast majority of animals currently living under human management. With this study, we aimed to address this gap in knowledge by investigating human–fish interactions at a community garden/aquaponics learning-center that is home to approximately 150 goldfish ( Human–animal interaction (HAI) is an umbrella term used to describe any form of interaction, relationship or bond between a human and nonhuman . HAI resDanio rerio) are popular model organisms increasingly used in studying vertebrate development and gene function in laboratories around the world  = 0.07) higher for within-reach than periphery (t(310.28) = 5.06, p < 0.007) and 1.71 (SE = 0.31) times more than expected during T2 (t(7) = 2.26, p < 0.06). Gabriel spent the second-most amount of time within-reach: 2.22 (SE = 0.37) times more than expected during T1 (t(9) = 3.31, p < 0.01) and 1.67 (SE = 0.32) times more than expected during T2 (t(7) = 2.07, p < 0.08). The individual who spent the least amount of time within-reach, Tigerlily, spent an average of 0.95 (SE = 0.22) times less than expected during T1 (t(9) = 0.24, p > 0.8) and 0.95 (SE = 0.10) times less than expected in T2 (t(7) = 0.48, p > 0.6, Fish showed strong individual differences within these patterns, however. Margaret spent the most amount of time within-reach: 2.38 (SE = 0.40) times more than expected during T1 (t(9) = 3.49, Fish also showed strong individual differences in their tendency to seek out physical, tactile interaction with the human experimenter. Gabriel’s rate of physical interaction was highest, seeking physical contact on average 0.63 (SE = 0.16) times per minute during T1 and 0.29 (SE = 0.09) times per minute during T2, whereas other fish rarely engaged in this behavior  seconds in physical contact with the human during T1 and a median of 18  seconds during T2. While all study fish initiated in at least some physical interaction, several had median physical interaction durations of 0  serves as a source of tactile and/or sensory enrichment. Sensory stimulation is an important aspect of welfare for many animals, including humans, for whom massage therapy reduces stress  by stimuCtenochaetus striatus), a tropical reef species that regularly visits cleaner wrasses (Labroides dimidiatus) to have ectoparasites removed. Previous studies showed that cleanerfish are able to influence client fish decisions by physically touching the surgeonfish with its pectoral and pelvic fins [Experiencing pleasure as the result of physical interaction with a receptive conspecific partner is believed to be a fundamental and highly conserved element of both human and nonhuman welfare. While touch in fishes has not yet been explored from an explicitly welfare angle, there is biological and behavioral evidence to suggest that teleost fishes possess the physiological prerequisites necessary for experiencing pleasurable sensation . Furthervic fins . Soares vic fins . These sA complimentary hypothesis as to why the fish spent more time than expected within reach during both T1 and T2 is that their interest in interaction may be motivated by curiosity, suggesting that fish-directed human interaction may serve as a means of cognitive enrichment by presenting an opportunity to explore and exercise agency. Research on the effects of boredom on captive animal welfare suggest that boredom is likely experienced by a wide range of captive species and is generally perceived as aversive, particularly by those living in barren conditions and lacking the agency necessary to remedy the situation, and thereby leading to frustration and anxiety ,30. CognAnalyses of location and behavior data see  reveal tThese results provide preliminary insight into the personalities of the seven fish who participated in the study, particularly in their interest in seeking interaction and willingness to take the risk of interacting. Interest in interacting, as defined by the relative amount of time spent within reach and interacting physically, ranged substantially between individuals. For example, the comparatively low rates of physical interaction and time spent within reach of the two least interactive fishes (Bessie and Tigerlily) signify a general lack of interest in interaction, but also did not show a substantial avoidance of the researcher. On the other hand, Gingko, whose rate and duration of physical interaction was similar to those of Bessie and Tigerlily, spent substantially more time within reach than the other two least interactive fishes, a behavior profile indicating attentiveness and interest but less interest in initiating physical interaction. Dominick showed stable interaction patterns across the board, with very little variation between T1 and T2 and a relatively low propensity to linger within reach of the researcher when not interacting.Investigating these individual behavior profiles can benefit fish welfare in several ways. First, provided that the patterns are found to be relatively stable over time, they can serve as a baseline by which to evaluate individual welfare. Recognizing and tracking patterns of individual behavior is a useful tool in measuring welfare in zoo housed animals ,102,103,As discussed briefly in the introduction, it has been well-documented that interactions with non-human animals, when perceived as positive by the human participant, can have a positive effect on humans’ perception of the species in question . Human pIn this study, we investigated whether or not koi would voluntarily choose to interact with a familiar human and, if so, whether individual behavior patterns could be identified. We found that koi not only engaged with the human both before and after feeding but also that the fish remained within reaching distance of the human more often than expected based on a random motion model during all sessions. These results suggest that koi are not solely motivated to interact with a familiar human in anticipation of a food reward and that the baseline interest in interaction may be motivated by a desire to interact for its own sake. Additionally, we found substantial, individual patterns of interaction behavior and proclivity for interaction between the seven koi participating in the study, suggesting that voluntary HAI may be used to investigate animal personality in conjunction with traditional personality assessment paradigms. Overall, this study provides important preliminary evidence that human–fish interaction research will be a fruitful area of future inquiry. More work is needed in examining human-mediated enrichment opportunities for fish, determining the qualitative nature of the human–fish interactions, and exploring the implications of carp sociality and personality for their welfare in captivity.


Scientific Reports 10.1038/srep23041, published online 09 March 2016Correction to: This Article contains errors. Figure 2A, which is an illustration of the surgical procedure, was inadvertently duplicated from a different publication provided below as Reference In addition, the bar graph in Figure 4A was inadvertently copied from the preliminary pilot study. The correct bar graph and raw data appear below as Figure


Diabetes mellitus (DM) is one of the fastest growing chronic diseases in the world and one of the main causes of vision loss. Whether or not diabetic choroidopathy (DC) is involved in the initiation and progression of diabetic ocular complications needs to be explored. We included 54 diabetic eyes from 36 diabetic patients, and 54 healthy eyes from 32 control subjects after propensity scores matching. All of the subjects were given pupil light and dark adaptation examination and optical coherence tomography angiography (OCTA). Scotopic pupil diameter (SPD), pupil contraction amplitude, and velocity of pupil contraction of the diabetic group were significantly lower than that of the healthy control group (P < 0.05).Choroidal thickness at temporal quadrant (at 750 μm) and superior quadrant (at 1500 μm and 2250 μm) increased in diabetic group compared to control group(P < 0.05).In the diabetic group, choriocapillaris blood flow signal density (CCBFSD) in the macular area (diameter = 2000 μm) were significantly decreased compared with the healthy control group (P < 0.05). Apparent changes in pupil and choroidal blood flow were observed in the diabetic patients. According to the World Health Organization (WHO), the total number of people with DM will grow from 171 million in 2000 to 366 million in 20302. At present, the pathological mechanisms of diabetic retinopathy are mainly explained by the effects of vascular leakage, vascular abnormalities, neuronal cell dysfunction, and inflammatory mechanisms3.Diabetes mellitus (DM) is one of the fastest growing chronic diseases in the world and one of the leading causes of vision loss4. Histopathological studies revealed a variety of choroidal changes secondary to DM7. The choroid accounts for more than 85% of the retinal blood supply and supplies all the nutrients for the retinal pigment epithelium and photoreceptors9. It has been reported that photoreceptors die rapidly when the choroidal blood flow decline drastically10. The regulation of choroidal blood flow includes neuromodulation, autoregulation, and local choroidal ganglion regulation13.The concept of diabetic choroidopathy was proposed by Saracco et al. as early as 198214. Studies have shown that nerve modulation of the uvea affects blood flow in the iris, ciliary body, and choroid16.Diabetic autonomic neuropathy (DAN) refers to a disorder of the autonomic nerves caused by DM, which involves diseases related to multiple systems including orthostatic hypotension, tachycardia, gastroparesis, diarrhea, constipation, etc. Its ocular complications include a reduction of scotopic pupil diameter and an Argyll-Robertson-like pupil21, little research on diabetic choroidal blood flow and its relation to pupil lesions is currently available. Previous studies have shown that the blood flow of choroidal capillaries in diabetic patients is significantly lower than that of healthy people23. Some studies have shown that their blood flow further decreases as the severity of the disease increases24.Both the choroid and the iris are parts of the uvea, and they have an inseparable relationship in anatomy, structure, and development of each other. Although there is a lot of evidence confirming the presence of pupillary lesions and changes in choroidal thickness caused by diabetic autonomic nerve damage25. OCT angiography (OCTA) is a recently developed technique that allows noninvasive visualization of vascular structures28. Moreover, the higher speed of the new OCT equipment has facilitated the optical reconstruction of complex vascular structures25. This feature of OCTA makes it an extremely valuable tool for studying choroidal blood flow29.In recent years, advances in optical coherence tomography (OCT) technology has revolutionized retinal research and clinical practiceA total of 108 eyes  were included for final analysis after PSM. The demographic and clinical characteristic details can be identified in Table 2, 1 cd/m2, 10 cd/m2, and 100 cd/m2, pupil contraction amplitude (PCA), velocity of pupil contraction (PCV) and velocity of pupil dilation (PDV) are shown in Table 2, 10 cd/m2, 100 cd/m2, and PDV were not significantly different from those in the healthy control group (P > 0.05).The details of the diabetic and healthy control groups for pupil diameters at light intensities of 0 cd/mChoroidal thickness (CT) at different locations of the fundus of the diabetic and the control groups is shown in Fig. CCBFSD in the macular area in the diabetic group were significantly lower than that in the healthy control group (P < 0.05). The details are shown in Fig. 21. We consider that this difference may result from pupillary autonomic nerve disorders in diabetic patients, suggesting that hyperglycemia damages the autonomic nerves of the iris.Our study showed that SPD, PCA, and PCV in the diabetic group were all lower than those in the healthy control group, which is consistent with results of previous studies31; however, some conflicting results suggest early thinning, late thickening19, or completely contrast choroidal thickening34. Some of these studies have their own characteristics that may explain the heterogeneity of the results. Querques et al. only compared CT in the central region18, and we know that choroidal changes in diabetes not only affect the central region but, more importantly, affect the circumference. The Vujosevic’s study included all stages of DR, including some patients with PDR17. In Esmaeelpour’s study, some patients had a history of hypertension and were taking medications30, which could affect choroidal circulation.We found that the CT at temporal quadrant (at 750 μm) and superior quadrant (at 1500 μm and 2250 μm) in the diabetic group significantly increased compared with the healthy control group. For this point, previous studies have had different results. Some clinical findings suggest choroidal thinning of early diabetic retinopathy (DR)OCTA, as a method for non-invasive visualization technique of fundus blood vessels, can provide a more comprehensive assessment of choroidal circulation. Compared with OCT, it provides a richer amount of blood flow information. Although OCTA has been frequently used clinically, it is rarely used as a tool for the study of DC. This is an innovative aspect of this research.Compared with previous studies, choriocapillaris blood flow signal density assessment method has been further updated. We use the self-programmed code to position and analyze each image. First of all, we perform batch analysis of pictures based on code, which can reduce manual errors and have good repeatability. Secondly, the code can accurately calculate the blood flow signal of each picture, thus forming 512 B-scan pictures into a "map" representing the choriocapillaris blood flow volume in the macular area, and finally calculating the percentage of choriocapillaris capillary blood signal density in this area.35. We speculate that there are two reasons for decreased CCBFSD in the macular area with diabetes. Firstly, decreased CCBFSD in diabetic patients is caused by vascular damage and blood flow decreasing due to long-term hyperglycemia. Secondly, although it is not realistic to completely exclude the effect of bleeding and exudation on signal acquisition, the patients we included were all patients with no DR, and mild or moderate DR whose bleeding and exudation were not severe. It should be noted that there is no evidence that slight retinal hemorrhage could significantly affect the collection of choroidal blood flow signals.Our study showed a lower CC perfusion in diabetic eyes compared with healthy eyes. Previous study has illustrated that eyes with NPDR are affected by macular hypoperfusion and photoreceptor damage and a lower CC perfusion may be associated with a greater damage of photoreceptorAt the same time, the increase of choroidal thickness and the decrease of SPD occurred in the diabetic group, and both were regulated by autonomic nerves, suggesting that this may be related to diabetic autonomic nerve damage. As we know, the decline of SPD is related to autonomic nerve damage. Furthermore, the middle vascular layer and large vascular layer of choroid also have autonomic and trigeminal innervation. The mechanism and their correlation deserve further research.36. It might suggest that an increased choroidal thickness may be associated with a lower CC perfusion, which is not surprising. Increased choroidal thickness and decreased choriocapillaris perfusion in patients with central serous chorioretinopathy, similar to the results in our study of patients with diabetes38. This reminds us that there may be some similar mechanisms in these two diseases. Firstly, thicker choroid may be caused by increased choroidal vessel wall permeability in an inflammatory state in both diseases. Secondly, choriocapillaris hypoperfusion may be caused by mechanical stress resulting from compression based on focal or diffusely enlarged choroidal vessels37.A previous report by Borrelli et al. demonstrated that a further dilation of the area occupied by medium-sized and larger-sized choroidal vessels and/or choroidal stroma appears to be associated with a progressive reduction in CC perfusion in healthy eyesCompared with previous studies, we better matched certain factors, such as age, gender, intraocular pressure, axial length and refractive status, to minimize the effect of these confounding factors on CT. At the same time, the OCT examination is performed at a constant time period of a day, which reduces the influence of the daily rhythm on the choroidal measurement.At the same time, we also need to consider the following points. First, differences in choroid assessment methods may lead to conflicting results. Diabetic choroidal vascular abnormalities are most common in the mid-circumference. However, OCT examinations focus on the macular/foveal area, so the results of these studies may not accurately reflect the actual situation. Second, OCTA has limitations as well: that is, only the velocity of blood flow within a certain range can be detected by the equipment. The velocity of choroidal capillaries may occasionally be too low to generate a disassociation signal, preventing OCTA from recording blood flow signals in capillaries. Thirdly, compared with the choroidal capillary layer, it is difficult to accurately evaluate blood flow of medium and large choroidal vessels because of the occlusion of retinal vessels and choroidal capillaries, and high blood flow velocity in these vessels.In short, with the advent of the latest OCTA technology, DC has become one of the most compelling diseases to study for ophthalmological researchers. Nonetheless, the relationship between DC and DR, and the role of DC in diabetic ocular diseases remain unknown. This important field requires further research.This study was performed according to the Declaration of Helsinki and was approved by the Ethics Committee of Tongji Hospital. Informed consent was obtained from all study participants before examination.We included 47 diabetic patients (84 eyes) and 48 healthy people (89 eyes) from the Department of Ophthalmology and Department of Endocrinology of Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology. Because the baselines of age, intraocular pressure, sex and spherical equivalent (SE) in two groups were not well matched, propensity scores matching was used. 54 diabetic eyes from 36 diabetic patients and 54 healthy eyes from 32 control subjects were included for final analysis after propensity scores matching, which were used to balance the two groups according to several baseline variables, including age, sex, intraocular pressure, SE. DM was diagnosed according to the 1999 WHO diagnostic criteria.Diabetic patients were included who had no signs of DR, or those diagnosed with mild or moderate non-proliferative diabetic retinopathy (NPDR).These included: (1) hypertension, kidney disorders, and other systemic diseases that may affect fundus circulation; (2) glaucoma, cataract, and history of ocular surgery; (3) vitreous opacity; (4) high myopia; (5) age-related macular degeneration, choroidal neovascularization, central serous retinal choroidopathy, and other eye diseases that affect the fundus circulation; (6) DM patients treated with laser and intraocular anti-vascular endothelial growth factor (VEGF), or macular edema; (7) patients with a history of ocular trauma.2 OCTA scanning, and foveal OCT line scan. Subjects with B-scan OCT image quality lower than 30 and OCTA quality lower than 35 were excluded.All subjects underwent visual acuity (VA), intraocular pressure (IOP), slit lamp examination, ophthalmoscopy, optometry, fundus photography, examination of pupil light and dark adaptation, parafoveal 3 × 3 mm2, 1 cd/m2, 10 cd/m2, and 100 cd/m2), pupil contraction amplitude (PCA), velocity of pupil contraction (PCV), velocity of pupil dilation (PDV).Pupil function tests were performed using a comprehensive pupillometer . Examinations were performed by the same ophthalmologist, including pupil diameter (PD) at different light intensities  was used to analyze the choriocapillaris blood flow signal in this region. The OCT B-scan was performed horizontally and vertically through the fovea. CT at the nasal, superior, temporal, and inferior choroid quadrants  and subfoveal choroidal thickness (SFCT) were recorded. Mean CT is their average. Choroidal thickness was measured as the perpendicular distance between the hyperreflective outer border of the retinal pigment epithelial/Bruch’s membrane layer and the sclero-choroidal interface, manually drawn blindly by the examiner. OCTA and OCT examinations were performed using the Heidelberg SPECTRALIS OCT equipment in 8:30 a.m.–13:00 p.m. for reducing choroidal changes with daily rhythm.The 3 × 3 mmWe used SPECTRALIS Software Version 6.9a to layer the acquired images. We would make manual adjustments if the automatic segmentation was incorrect, and then used the self-written code to calculate choriocapillaris blood flow signal density (CCBFSD) in the macular area , which is the percentage of the yellow signal to the total area. Choriocapillaris was segmented with an inner boundary Bruch membrane and an outer boundary 49 μm below the Bruch membrane. The positioning method of the choroidal capillary area for calculation in the 256th image of 512 pictures is described detailly in Fig. All the data in this study were analyzed using IBM SPSS Statistics ver.26.0 . The propensity score was estimated using a logistic regression model with 1:1 nearest neighbour matching without replacement, based on an acceptable caliper width of 0.25 times. Kolmogorov–Smirnov test was used for the normality test. Independent sample t-test was used to compare the data between these two groups that satisfied the normal distribution; if it did not, non-parametric test was used. Chi-square test was used for gender comparison between the two groups. Measurement data were expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.


Sparus aurata, containing low inclusion of two microalgae-based products . Fish (12–13 g) were fed for 13 weeks a control diet or one of the four diets supplemented with both products at 0.5% or 1%. LBGb and LBGh did not affect specific growth rate or survival, but increased feed efficiency by decreasing feed intake and enlarging the intestines. LBGb increased hepatosomatic index and reduced cortisol levels in plasma, while both products lowered plasma lactate. Extensive metabolite and metabolic enzyme profiling revealed that microalgae supplementations, especially 1% LBGh: (i) decrease plasma lactate and increase hepatic glycogen, (ii) reduce hepatic gluconeogenesis, (iii) enhance hepatic lipogenic activity and lipid secretion, (iv) led fish to double triglyceride content in muscle and to stimulate its lipid oxidative capacity, and (v) increase the content of monounsaturated fatty acids and the omega-3 alpha-linolenic acid in muscle. This study demonstrates that both microalgae-based products are suited to improve feed efficiency and orchestrate significant changes in the intermediary metabolism in gilthead seabream juveniles.The aim of this work was to evaluate two functional feeds for the gilthead seabream,  One major issue related with both economic and environmental sustainability is the replacement of fish meal (FM) and fish oil (FO) in aquafeeds. Significant achievements have been made during the last decade, and the fish aquaculture industry has significantly reduced the dependence on these marine ingredients by incorporating balanced mixtures of plant meals and oils in feed formulations5, and higher replacement rates are expected through the use of novel alternatives such as genetically engineered oilseed crops and the use of microalgae6.World aquaculture production is continuously growing at a high rate, but the European industry is stagnant and needs to advance to a more competitive and high-performance industry, while ensuring high level of sustainability7 and lipids with high amount of highly unsaturated fatty acids8, thus they have been evaluated in different fish species as alternatives for FM and FO. Several studies indicated that microalgae inclusion in freshwater fish diets does not affect growth rate10, or can induce positive effects on their growth, metabolism, and meat quality11. Results are more variable in salmonids and marine fish, depending on the microalgae species employed and the level of inclusion in diet . Although the inclusion of high level of microalgae in most fish feeds is still limited by high production costs, different companies have announced algae-based products to be used in high-valued commercial fish  as alternatives to FO6, and different products (e.g. AlgaPrimeDHA) are already being produced at large-scale through fermentation-based technologies18.Microalgae and microalgae-derived products are rich in proteins21, two features that have been traditionally improved through the optimization of feed formulations and feeding practices. Feed efficiency, in particular, is difficult to increase using phenotypic trait selection. Other aspects that can be improved by the use of functional feeds are wellbeing, health, stress resistance, or product quality, which are also of a great interest today for both producers and consumers. Functional feeds  trigger, by definition, beneficial effects upon physiological functions22. Bioactive compounds from microalgae such as protein, polyunsaturated fatty acids, polysaccharides, carotenoids, vitamins and minerals, phenolic compounds, volatile compounds, and sterols play important roles in functional feeds for both humans and livestock24. The most widely microalgae-derived product used by the aquaculture industry is astaxanthin, mainly to improve the coloration, but also other important physiological processes25. However, different microalgae have immunostimulating and health promoting effects properties in fish26, or improve growth and fillet quality27. For instance, the addition of moderate levels (~ 10%) of a Chlorella-derived product in feed is able to ameliorate plat-induced enteritis in salmonids28, and ~ 4.5–5.0% inclusion of Isochrysis galbana biomass in diets for the marine fish Trachinotus ovatus, improves growth performance, lipid deposition and content of muscular n-3 fatty acids, DHA, and EPA27.Yet, studies on microalgae-derived products used at lower inclusion levels in diets for farmed fish are scarce, despite this would have a positive impact on feed cost. In other livestock such as broiler chicks, feed supplementation with only 0.1–0.2% of microalgae products, considered as a very-low inclusion level, produces positive effects such as the improvement of fatty acid composition of meat without affecting the growth performance29.While the use of microalgae-derived products as alternative raw material in fish feeds has received most of the attention, there is also an increasing interest in their use as functional feed additives. In this sense, the use of microalgae products at relatively low level (< 10%) in fish functional feeds has the potential to improve aquaculture production by enhancing growth and/or feed efficiencyGb) and LB-GUThealth (LBGh), developed by LifeBioencapsulation S.L., a spin-off from the University of Almería (Spain). The gilthead seabream is one of the main fish species farmed in Europe, especially in the Mediterranean region. In particular, the effects of low dietary inclusion (0.5% and 1%) of these products on growth performance, nutrient utilization, and intermediary metabolism were studied after a 13-week feeding trial. This study shows that the products evaluated are suited for improving some important indicators of culture performance and physiological condition of gilthead seabream juveniles, thus revealing the potential for their inclusion in new functional feeds for this, or even others cultured species.The aim of this work was to evaluate the potential benefits of two functional feeds for the gilthead seabream, which contain low level of two microalgae-based products, LB-GREENboost  and European Union (2010/63/UE) legislation. The Ethical Committee from the Autonomous Andalusian Government approved the experiments .https://www.ual.es/stecnicos_spe). FM and FO were included at 20% and 9.2%, respectively, in all experimental diets. This formulation constituted the control diet (CTRL). In addition, two commercial compounds extracted from microalgae, (i) LB-GREENboost (LBGb) and (ii) LB-GUThealth (LBGh) developed by LifeBioencapsulation S.L. , were added at 0.5% and at 1%, constituting the four supplemented diets  and LBGh  are concentrated mixtures containing 800 g kg−1 and 200 g kg−1, respectively, of a blend of microalgae extracts. The rest of compounds used in those commercial products are lipotropic substances (choline and betaine) and calcium carbonate used as excipients. The ingredient composition and fatty acid profile of experimental diets are shown in Tables Five isoproteic, isolipidic and isoenergetic diets were formulated with a composition that is close to that of commercial feeds for the gilthead seabream, and produced at the University of Almería facilities . Experimental diets were offered to visual satiety three times per day and 6 days per week from February to May . Fish were counted and group-weighed every 3 weeks, and feed intake was recorded for each experimental replicate to calculate growth performance parameters. No mortalities were registered in any experimental group.After an initial acclimation period (10 days) to the experimental facility , gilthead sea bream juveniles with 12–13 g of initial mean body weight were randomly distributed in fifteen 100-L tanks  coupled to a recirculation aquaculture system (RAS), equipped with physical and biological filters, and programmable temperature and Og for 20 min at 4 ºC, and plasma samples were snap-frozen in liquid nitrogen and stored at − 80 ºC until biochemical and hormonal analysis. Prior to tissue collection, fish were killed by cervical section, and livers were extracted and weighed. Intestine was taken for length measurements. Samples of liver and white skeletal muscle were rapidly taken, snap-frozen in liquid nitrogen, and stored at − 80 ºC until biochemical analyses.At the end of the trial (day 87), overnight fasted fish  were randomly selected, deeply anaesthetised with clove oil, and then sampled for blood and tissue collection. Blood was drawn from caudal vessels with heparinised syringes, centrifuged at 3000×i)/Lb, where Li and Lb are the intestine and fork body length, respectively.The following growth parameters were evaluated: (i) specific growth rate (SGR) = /days; (ii) weight gain (WG) = (100 × (body weigh increase)/initial body weight; (iii) feed efficiency (FE) = weight gain/total feed intake; and (iv) condition factor = (100 × body weight)/fork length. Biometric indices were estimated in accordance with the following equations: (i) Hepatosomatic index (HSI) = (100 × liver weight)/fish weight; and (ii) Intestine length index (ILI) = . Glucose, lactate and triglycerides levels in plasma were measured using commercial kits from SPINREACT  adapted to 96-well microplates. Plasma total protein concentration was determined with a BCA Protein AssayKit  using BSA as the standard. All assays were performed using a POWERWAVE 340 microplate spectrophotometer  using the KCJUNIOR data analysis software for MICROSOFT.3, centrifuged , and then supernatants isolated to determine tissue metabolites. Tissue triglycerides and lactate levels were determined spectrophotometrically with commercial kits . Tissue glycogen concentration was quantified using the method described from30, where glucose obtained after glycogen breakdown with amyloglucosidase (SIGMA-ALDRICH A7420) was determined with a commercial kit (SPINREACT) as described before.Frozen tissues used for the assay of metabolites were homogenized by ultrasonic disruption in 7.5 volumes ice-cold 0.6 N perchloric acid, neutralized using 1 M KCOg and 4 ºC, and supernatants stored at − 80 ºC for further analysis. The assays of several enzymes involved in glycogenolysis , glycolysis , gluconeogenesis , Krebs Cycle , pentose phosphate pathway , and lipid metabolism  were performed as previously described for S. aurata tissues34. Enzyme activities were determined using a POWERWAVE 340 microplate spectrophotometer using the KCJUNIOR data analysis software for MICROSOFT. Activities were expressed as specific activities per mg of protein in the homogenate (U mg prot−1). Proteins were assayed in duplicate, as described above for plasma samples.Frozen tissues for enzyme activity assays were homogenized by ultrasonic disruption in 10 volumes of ice-cold homogenization buffer  and 250 mM sucrose; pH 7.5). Homogenates were centrifuged for 30 min at 3220×35 procedures. Lipids were extracted following the Folch method36 using chloroform/methanol (2:1 v/v) as solvent, and total lipid content was calculated gravimetrically. Fatty acid (FA) profile was determined by gas chromatography following the method described in37, by means of a gas chromatograph , using a modification of the direct transesterification method described by38 that involves no prior separation of the lipid fraction.Proximate analysis  of feed and muscle samples were determined according toResults are shown as mean ± standard error of the mean (mean ± SEM). After assessing homogeneity of variance and normality, statistical analysis of the data was carried out using one-way analysis of variance followed by the Tukey test. A comparison of triplicate tanks for all parameters was also performed with one-way analysis of variance. The level of significance was set at p < 0.05. All tests were performed using GRAPHPAD PRISM (v.5.0b) software for Macintosh.Gb and LBGh) and levels of inclusion (0.5 and 1%)  increased in a dose-dependent manner in fish grown-up with both products  of ~ 200% and specific growth rates (SGR) of 1.26–1.30%  (Table Gb and 1% LBGh) (Table Gh diet experienced a significant (twofold) increase in triglyceride accumulation, whereas the content of glucose, glycogen, and lactate was not modified by dietary supplementation (Table Plasma cortisol levels decreased significantly in fish fed both doses of LBps Table . Dietaryup Table . Moreoveh) Table . In the h) Table . Howeverh) Table . In the on Table .Table 4BGh diet. An overall opposite pattern was detected for the gluconeogenic enzyme FBP, with the lowest activity found in livers of the 1% LBGh group. Also, we observed a dose-dependent increase in LDH enzyme with both compounds. In contrast to the observations made on HK activity, the glycolytic enzyme G3PDH shown a significant reduction in its hepatic activity in fish fed both 1% LBGb and 0.5% LBGh supplemented diets. No effects were found on the hepatic activity of PK , MDH  and HOAD  enzymes. In the white skeletal muscle  were able to increase HOAD activity. Moreover, a dose-dependent increase in muscle MDH activity was detected when both compounds where used in feed. No dietary effects were found on the muscular activity of GPase total (F = 1.840 p = 0.343) and active (F = 0.669 p = 0.618), HK (F = 0.049 p = 0.995), FBP (F = 0.271 p = 0.895) and G6PDH (F = 1.969 p = 0.121) enzymes, whereas PK activity was found to be at undetectable (ND) levels in this tissue.The effect of dietary supplementation was also evaluated, both in the liver and the white skeletal muscle, on the activity of several metabolic enzymes related to glycogenolysis, glycolysis, gluconeogenesis, Krebs cycle, pentose phosphate pathway, and lipid metabolism. In the liver Fig. , GPase dGb fillets , followed by monounsaturated fatty acids , and then saturated fatty acids  Table . No majo1% Table . The maint Table .Artemia), or directly in larval culture tanks for keeping the nutritional quality of the zooplankton used for feeding fish40. Also, because of their high content of proteins and lipids8, microalgae have been evaluated in different fish species as alternatives for FM and FO. High production cost of microalgal biomass remains the major drawback of this strategy20, although different products , produced at large-scale through fermentation-based technologies, are already being used commercially in salmon feeds18. However, there is also an increasing interest in the use of microalgae as nutraceuticals in functional feeds23, which effects can be exerted at low dietary inclusion level. The products evaluated in this work were included into aquafeeds at levels that can be considered low (1%) or very low (0.5%). Results showed that even at the lowest inclusion level, these products produce positive changes in feed efficiency, welfare and metabolism of gilthead seabream juveniles.Most widely application of microalgae in fish aquaculture is their use, alive or freeze-dried, to enrich zooplankton  indicates that the incorporation of these products in functional feeds for the gilthead seabream would reduce in up to 148 kg the amount of feed needed to produce one ton of farmed fish, and in saving up to 222 € per ton of fish produced, with a different balance in terms of economic issues depending on the inclusion level , and deposition of hepatic glycogen and lipid in the muscle of fish52. These effects may be exerted by supplementation with microalgae-based products in our study, as suggested by the higher feed efficiency, higher hepatic glycogen content, and higher triglyceride levels in muscle of 1% LBGb and 1% LBGh fed fish. It is known that dietary fatty acids play important roles in the regulation of cortisol release in fish. For instance, in gilthead seabream, dietary deficiencies on n-3 HUFA raised the basal plasma cortisol levels and altered the pattern of cortisol release after stress53, and high concentration of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) reduced cortisol secretion in this species55. However, given the low inclusion level of LBGb in this study, it may exert its effects on cortisol levels through a different mechanism, that remains unknown, although it can be suggested that the lower doses of aquafeeds needed to accomplish apparent satiety produces a decrease in anticipatory activity that leads to improve the welfare status of the fish, which is clearly reflected in lower cortisol levels. Even so, whether lower levels of baseline cortisol in plasma also affect the response of fish to stressful condition such as high stocking density or handling remains unknown and deserves further investigation.Under stressful conditions, cortisol plays a key regulatory role in skeletal muscle metabolism, inhibiting glycogen synthesisGb and LBGh, we measured the level of different metabolites, and the activity of several metabolic enzymes, both in the liver and white skeletal muscle. We observed that both products lowered plasma lactate levels, although LBGb promoted this effect at a lower inclusion (0.5%) than LBGh (1%). This result suggests that both products may favor oxidative over anaerobic metabolism in the white skeletal muscle, or that lactate uptake and clearance by the liver or other tissues is stimulated. We found no differences among treatments in glycogen, free glucose, and lactate in the white skeletal muscle, and the activity of LDH in muscle of all supplemented fed fish was not different to that of control fish, except for slightly higher values in 1% LBGh. Thus, our observations do not sustain a lower production of lactate from muscle anaerobic metabolism in fish fed diets supplemented with the microalgae-based products. Interestingly, the higher hepatic storage of glycogen in fish fed diets supplemented with LBGh and LBGb at 1% and a trend for hepatic LDH activity to increase suggests that both products may promote the hepatic uptake of lactate. However, the conversion of lactate to pyruvate by LDH in the liver (i.e. Cori cycle), and its further conversion to glycogen, is not clear in fish. Indeed, an early study analyzing lactate metabolism in 36 fish species suggested that little blood lactate is taken up by the liver in fish56, and more recent studies also suggested that using lactate as a precursor for liver glycogen is unlikely in fish57. Other tissues of gilthead seabream using lactate as energy source59 should also contribute to the clearance of lactate60 and part of the lactate could return to the muscle60. Our observations on plasma lactate would be also related with a decrease in the level of fish activity when fed the micro-algae supplemented diets, and this may in turn be associated with decreased cortisol levels and improved feed efficiency as discussed above.To further study the biochemical basis of the observed phenotypic outputs of microalgae-based product supplementation, and to better differentiate the metabolic effects of LBGh increased hepatic HK activity, most significantly at 1% inclusion in feed, while PK activity was unaltered by dietary supplementation. HK is the first step in glycolysis, phosphorylating glucose to be used by cells, while PK catalyzes the last step of glycolysis producing pyruvate and ATP. Together, these results support that 1% LBGh supplementation enhanced the liver capacity for glucose uptake, which seems to be stored as glycogen instead of being oxidized for energy. This inferred scenario agrees with reduced hepatic gluconeogenic enzyme (FBP) activity in 1% LBGh fed fish. A non-significant trend for FBP to decrease activity in 1% LBGb fed fish may explain why this fish also exhibited more hepatic glycogen than control fish. However, it remains unknown the metabolic significance of higher activities of GP in 1% LBGh and 1% LBGb groups. It would be related with the turnover of liver glycogen for glucose to be used in other metabolic pathways such as the synthesis of fatty acids. Increased glucose uptake by the liver, or production of glucose from glycogen, is known to have a stimulatory effect on the lipogenic enzymes G6PDH and MDH, which provide NADPH for the biosynthesis of fatty acids, and that this leads to a higher lipid storage or export form the liver62. Indeed, we found for hepatic G6PDH a trend to increase its activity with microalgae supplementation, with higher activity for 1% LBGh, while MDH exhibited higher values  in both LBGh and LBGb at 1% inclusion. The absence of differences in hepatic triglycerides stored in our study supports the export as the most likely fate of synthesized triglycerides, in agreement with a higher triglyceride level in plasma in 1% LBGh and 1% LBGb fed fish. This would be also the cause of increased triglyceride content in muscle of 1% LBGh groups, doubling the triglyceride content of muscles of control fish. It is difficult to fully explain the effect of 1% LBGh on muscle triglycerides, as fat deposition depends upon balance between various metabolic pathways and trade-offs among different organs. Some of our observations suggest that de novo lipogenesis (DNL) in the muscle might contribute to higher triglyceride content in 1% LBGh fed fish. For instance, we observed higher activity for the lipogenic enzyme MDH in muscle in 1% LBGh and 1% LBGb, with the highest mean value induced by the former. MDH activity has been related to intramuscular fat content in other meat-producing animals63. However, the contribution of DNL in muscle to fat accumulation is debatable in fish as it mostly takes place in the liver64. Indeed, we found no evidence of differences in muscle DNL among treatments by using the DNL index (16:0/18:2n6 ratio = 0.7). Higher activity of G3PDH in 1% LBGh fed fish is also in agreement with higher triglyceride content of muscle, as it is a marker of lipid synthesis in fish65 and other animals63. This enzyme produces glycerol-3-phosphate to which fatty acids are esterified, thus it is involved in the synthesis of triglycerides from imported fatty acids (e.g. from liver) rather than in muscle DNL. Finally, we recognize that changes in the triglyceride content of muscle of 1% LBGh fed fish may also result from modifications in the metabolic fate of other nutrients induced by this microalgal product. For instance, in gilthead seabream, between 22 and 30% of the total lipid deposited may come from dietary proteins66.We also found that the inclusion of LBGh fed fish). It is known that in higher vertebrates, lipid availability per se increases mitochondrial fatty acid oxidative capacity in muscle67. In fish (rainbow trout), an experimental high muscle fat line is known to exhibit an enhanced fatty acid oxidation potential68. Increased HOAD activity in our study may be a compensatory mechanism to control excessive fat accumulation in fish muscles supplemented with 1% LBGh, or might be involved in lipid remodeling within the muscle.One interestingly finding of this study is the higher muscular HOAD activity, the third step of beta oxidation, in fish with increased muscle triglycerides . This result is also in accordance with the boost in muscle triglycerides in 1% LBGh fed fish, as triglycerides are rich in MUFA. Therefore, it seems that storage lipids became slightly enriched in MUFA after feeding the microalgae products. Enhanced MUFA in fish muscle is thought to be positive, as long as SFA and n-6 PUFA do not increase, and the n-3 to n-6 ratio is not altered, as occurred in our study. Given that our assessment was performed in juveniles, major effects of increased MUFA and the ALA (n-3 PUFA) in muscle should be related with physiological processes (e.g. permeability and fluidity of membranes) and overall health status. In rats, dietary ALA supplementation increased the movement of lipids across the sarcolemmal membrane, a rate-limiting step in fatty acid oxidation, and led to higher triglyceride content and rates of fat oxidation71, while it is known to have antioxidant and anti-inflammatory effects in both rats and humans72. However, the use of the products evaluated in this study in finishing diets for adult gilthead sea bream farmed on diets with high substitution of FM and FO is worthy to be also explored. Information on the use of dietary supplements to increase the efficiency of finishing protocols is scarce, and their effects on muscle fatty acid composition would be different in fast growing juveniles (this study) and in commercial size (near harvest) fish. Moreover, given that in this and other fish species the content of lipids in fillet is correlated with the levels of different LC-PUFA73, and that 1% LBGh increases the triglyceride content of muscle, it would result in adults with more MUFAs, and maybe PUFAs, per gram of fillet. Although dietary FA profiles are generally reflected in fish muscle76, the significant increase in muscle MUFAs observed in the present study could be attributed to slightly differences in dietary fatty acids as a result of additive inclusion. The marginal higher content of 18:1n-9 and 18:3n-3 in the dietary treatments supplemented with the microalgae products might contribute to the observed increase of those fatty acids in the muscle of fish compared to the results obtained for the control diet  content in all supplemented groups. We have no explanation for this increase in ALA content, but its effects might be related with the observed high protein content in muscle of microalgae fed fish, as ALA is known to prevent muscle wasting in higher vertebratesGb and LBGh additives in gilthead seabream diets does not affect growth and improve feed efficiency. Different positive effects of microalgae products in feeds have been achieved at low level of supplementation such as 5% in salmon77, 5–7% in pigs78, and only 0.1–0.2% in broiler chicks29. In our study, it was found that LBGb and LBGh exerted their positive, although different, effects at 0.5–1% in gilthead seabream juveniles. Given this low inclusion level in diet (e.g. 1%) and their reasonable cost (about 20€ per kg), the use of these products would result in saving from 85 to 148 kg of feeds per ton of farmed fish, which might reduce feed cost up to 33.4 € per ton of fish in the case of LBGh (Table In general, this study demonstrated that the use of LBGh Table . Moreove