Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. Although the majority of M. hyorhinis-colonized pigs have no apparent clinical disease, the pathogen can induce diseases such as polyserositis, arthritis, and eustachitis in some cases. To explore the mechanisms for the occurrence of these diseases, we challenged 4 groups of Bama miniature pigs with M. hyorhinis isolated from pigs without clinical symptoms  or with typical clinical symptoms  and investigated the impacts of different strains and inoculation routes  on disease induction. Another group of pigs was set as a negative control. Pigs inoculated with the CO strain through a combined intravenous and intraperitoneal (IV+IP) route showed a significant decrease in average daily weight gain (ADWG), serious joint swelling, and lameness compared with the pigs in the negative-control group. Furthermore, this group developed moderate-to-severe pericarditis, pleuritis, peritonitis, and arthritis, as well as high levels of IgG and IgM antibodies. Pigs inoculated IV+IP with the NCO strain developed less marked clinical, pathological changes and a weaker specific antibody response compared with the pigs inoculated with the CO strain. The challenging results of the NCO strain via different routes  indicated that the combined route (IV+IP) induced the most serious disease compared to the other inoculation routes. Intranasal inoculation induced a smaller decrease in ADWG without obvious polyserositis or arthritis. These data suggest that differences in both strain virulence and inoculation route affect the consequences of M. hyorhinis infection.IMPORTANCEMycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. The mechanisms or conditions that lead to the occurrence of disease in M. hyorhinis-infected pigs are still unknown. The objective of this study was to evaluate the impact of differences in the virulence of strain and the inoculation route on the consequences of M. hyorhinis infection. Mycoplasma hyorhinis is ubiquitous in the pig population, frequently colonizing the tonsils and respiratory epithelium of the nasal cavity and the conducting airways. Understanding of the epidemiology of M. hyorhinis is still very poor due to its relatively recent recognition as an economically significant swine pathogen. Different studies have revealed that M. hyorhinis is often detected in older animals such as fattening pigs , as well as low-virulence and non-virulent strains   , 10. In reported , 12; howM. hyorhinis: one from a pig with no clinical symptoms (asymptomatic) and another from a pig with typical clinical symptoms (symptomatic). These strains were used in challenge experiments to evaluate differences in clinical symptoms and pathological changes in infected pigs. We also explored the influence of infection routes on the disease by using different methods to inoculate pigs with the same strain.In this study, we isolated two strains of M. hyorhinis-induced polyserositis and arthritis, was used for M. hyorhinis isolation. Tissues of the tonsil, lung, heart, and joint (swabs) were sampled. Another clinically healthy pig from a farm with very stable animal health, but which was positive for M. hyorhinis infection, was also sampled for pathogen isolation. None of the cultures with color changes appeared turbid. The pathogens isolated were all identified as M. hyorhinis by specific PCR targeting p37 . The STs of the strains isolated from different tissues of the symptomatic pig were found to be the same, indicating a single strain, which was denoted as a strain of clinical origin (CO). Exact matches for all loci were not available in the PubMLST database; novel sequences were submitted for new allelic profiles and a new ST number (ST144) was assigned to the respective allele combinations. The CO strain isolated from the lung was used for the subsequent experiments.One pig with swollen joints and severe diffuse fibrinous peritonitis, from a farm with a known history of problems with ting p37 . M. hyorP < 0.01, After bacterial challenge, the food intake of the pigs changed dramatically. The pigs infected with the CO strain via the IV+IP route (group 1 [G1]) exhibited depressed appetite from 2 days postinoculation (dpi), which was maintained until the day of necropsy with no obvious increase in body weight. The pigs in G1 had significantly lower body weights than those in the other four groups during the monitoring period (P < 0.01) than that of the pigs in the control group (G5). The lowest ADWG was observed in G1 , although there was no significant difference compared with G2 . Among groups infected with the NCO strain, the ADWG of G2 (IV+IP) was significantly lower than that of G4 (IN) (P < 0.05).As shown in M. hyorhinis infection (P < 0.01). There were significant differences (P < 0.01) between groups infected with different strains (G1 and G2) and between groups infected via different routes .Animals were monitored daily for general health and any clinical signs related to nfection  and B. SM. hyorhinis-induced pleuritis, pericarditis, peritonitis, and arthritis were observed and scored . The total score for G1 (9.6 ± 0.55) was higher than that for G2 (4.8 ± 3.96), although the difference was not statistically significant (P = 0.201). The differences in pleuritis and peritonitis scores between G1 and G2 were significant  analysis showed that all isolates were the same ST as the strains used for infection.Bacteria were re-isolated from tissue samples  of one pig with significant lesions each from G1 to G4, and of one from G5 as a negative control. M. hyorhinis-specific IgG was detected in G1 at 7 dpi, and the titer increased significantly during the monitoring period. The IgG titer of G1 was significantly higher than those of the other groups (P < 0.01). IgG antibody responses in other infected groups were not detected until 21 dpi. The IgM response occurred earlier than the IgG response. The IgM titer of all infected pigs peaked at 7 dpi and decreased slowly thereafter. The IgM titer of the G1 pigs was significantly higher than those of the pigs in other groups (P < 0.01). No IgG or IgM response was detected in G5.IgG and IgM antibody production was measured every 7 days . M. hyorM. hyorhinis is very high in farms all over the world. It has been widely recognized that M. hyorhinis infection can induce systemic inflammation, although most pigs do not develop disease. A key question is what determines the result of the occurrence of apparent diseases or an asymptomatic infection. The objective of this study was to evaluate the impacts of differences in the virulence of strains and of experimental systemic dissemination on the consequences of M. hyorhinis infection. Two M. hyorhinis strains, isolated from pigs with typical clinical symptoms (CO strain) and from clinical healthy asymptomatic pigs (NCO strain), were isolated and used to infect Bama miniature pigs. Consequently, compared with the NCO strain, the CO strain induced more serious body weight loss, joint swelling, lameness, and polyserositis, confirming the existence of differences in virulence between the two strains. Consistent with our results, previous studies have also indicated differences in the virulence of M. hyorhinis strains. Gois et al. (M. hyorhinis infection), while other animals infected with the ATCC strain (ATCC 27717) did not develop gross lung lesions and showed only minimally perivascular infiltration. A difference in tissue-specific tropism between strains were also implied in these studies, which was not observed between the two strains used in our study.The infection rate of s et al.  inoculats et al.  found thM. hyorhinis is very common in pig farms, this result indicates that farms with M. hyorhinis-infected asymptomatic pigs should be alert to the possibility of M. hyorhinis-caused diseases. This also emphasizes the importance of implementing measures to control M. hyorhinis infection in pig farms to achieve long-term benefits. Naturally attenuated M. hyorhinis strains may also exist in the field, and further studies are required to investigate this issue.More importantly, we found that the NCO strain induced clinical and gross pathological changes in infected pigs, suggesting that strains isolated from asymptomatic pigs are not necessarily nonvirulent and may also induce disease under suitable conditions. Because M. hyorhinis infection generally occur 3 to 10 days after exposure to the bacterium or the induction of a systemic reaction. Pigs may exhibit an elevated body temperature, apathy, loss of appetite, swollen joints, lameness, reluctance to move, and labored breathing. The acute clinical signs usually subside after 10 to 14 days. In case of arthritis, lameness and swollen joints may persist for 2 to 3 months  showed typical clinical and pathological signs of diseases. However, those inoculated via the IN route did not show obvious diseases, except for a certain decrease in ADWG (M. hyorhinis preparation harvested from lysed-cell suspension after infection caused few/no signs of disease in cesarean-derived colostrum-deprived (CDCD) pigs. This may partly explain why many pigs do not show clinical symptoms (or maybe they are too slight to be noticed) when M. hyorhinis is widespread. It can be noted that the animals in G2 (IV 4.5 mL + IP 4.5 mL) were more severely affected than those in G3 (IV 3 mL+ IP 3 mL+ IN 3 mL). This difference could be attributed to the lower total amount of M. hyorhinis injected through the IV and IP routes in G3, due to the addition of the IN route. In contrast, IN inoculation of M. hyorhinis has also been reported to induce diseases, including pneumonia, polyarthritis, arthritis, and eustachitis  whether IN or aerosol infection of M. hyorhinis would cause diseases without any other factors; (2) would it finally cause a systemic dissemination; and (3) how does this dissemination happen? In our study, we did not observe differences in tissue tropism among the same strain inoculated through different routes. Pneumonia is not typical in M. hyorhinis infection. However, in some studies, the inoculation of M. hyorhinis induced pneumonia similar to that induced by M. hyopneumoniae, the pathogenesis of which is not yet clear. It seems from previous reports that pneumonia was more common when pigs were infected via IN or intratracheal routes  techniques have been developed for notyping , 26. Gengle farm , 27. Unfgle farm . New gengle farm , 29. WitM. hyorhinis vaccines, therapies, and pathogenic mechanisms.The Bama pigs used in the current study are a minipig breed developed naturally in the Chinese remote mountainous areas of Bama County (Guangxi Zhuang Autonomous Region). Bama minipigs are considered suitable laboratory animals for their genetic stability, high level of inbreeding, and small bodies. Consequently, these animals are inexpensive to feed and easy to handle. The clinical and pathological symptoms of Bama pigs after challenge were very typical, suggesting that this breed is a good animal model for studies of M. hyorhinis showed no apparent clinical disease. One main purpose of this research was to investigate which factor determines the results of M. hyorhinis infection. Therefore, the NCO strain was chosen to compare different routes, which may be more representative of the clinical infections on most farms. These results can help us answer whether there is a potential risk of significant diseases with “harmless” infections of this type of strain. However, a comparison over different inoculation routes with the CO strain was not included in this study, since neither obvious clinical nor pathological changes were observed in pigs which received only IN inoculation of the NCO strain. Further studies could be carried out to explore whether IN infection of a high-virulence strain, such as the CO strain, would induce notable clinical diseases. (ii) In this study, each pig received a total dose of 109 color-changing units (CCU) of M. hyorhinis. In previous studies, doses of 1 × 109 to 5 × 109 CCU per pig were usually used to develop a challenge model of M. hyorhinis  Most of the pigs infected with yorhinis , 30–32. M. hyorhinis strains were isolated from a pig with typical clinical symptoms of M. hyorhinis infection and from an asymptomatic pig. Both strains induced clinical and pathological changes in pigs after inoculation via the combined IP and IV route. The disease induced by the CO strain was more severe than that induced by the NCO strain. Comparison of infection consequences with the NCO strain via different routes revealed that the combined IP and IV route induced the most serious disease. Thus, our findings suggest that both the virulence of strain and systemic dissemination affect the consequences of M. hyorhinis infection.M. hyorhinis infection was confirmed by nested PCR (described below) of nasal swab samples from 55 pigs, which showed a positive ratio of 72.7%. One positive, 6-week-old Duroc-Landrace-Yorkshire pig from this farm, with swollen joints and severe diffuse fibrinous peritonitis, was used for pathogen isolation. Farm 2 had very stable animal health, without any respiratory problems, arthritis, or behavioral disorders based on long-term slaughter examination and clinical history. However, 58.9% of nasal swab samples from 56 pigs tested positive for M. hyorhinis by nested PCR analysis. One positive, 8-week-old Duroc-Landrace-Yorkshire pig from this farm was used for pathogen isolation, and no gross pathological changes were observed at necropsy.Pigs were obtained from two commercial farms. Farm 1 had a known history of problems with polyserositis and arthritis, which were determined based on necropsy results. M. hyorhinis isolation, palatine tonsil (hereinafter referred to as “tonsil”), lung, heart, and joint tissues (swabs) were sampled from each pig. Tissues were rinsed three times with sterile phosphate-buffered saline (PBS) and then soaked in 10 mL KM2 medium (a modified Friis medium). The tissues were then cut into small pieces (approximately 1 to 3 mm2). The suspensions were shaken for 1 min before the supernatant was carefully aspirated and filtered (0.45-μm pore). Finally, the filtrates were diluted (10×) with KM2 medium and cultured at 37°C. Cultures were checked daily and subcultured when the red color of the medium changed to yellow. Mycoplasma species were identified by PCR. After three passages, the cultures were plated on solid KM2 medium. A single colony was picked, cultured with liquid medium, and confirmed again by PCR.For p37 gene of M. hyorhinis. The isolated pathogens were identified by regular PCR. The PCR mix  contained 10 pmol of each primer, 2 × Taq Master Mix , and 2 μL DNA template. Amplification reactions were performed in a thermocycler under the following conditions: denaturation at 95°C for 5 min followed by 30 cycles of 95°C for 1 min, 52°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min. To improve detection sensitivity, a nested PCR strategy was used to detect M. hyorhinis in nasal swab samples. After the amplification described above, inner primers were added and the product of the first amplification reaction was used as the template for the second-round PCR. The second-round amplification was performed under the following conditions: denaturation at 95°C for 5 min followed by 30 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. Primer sequences are listed in Total DNA was extracted from the mycoplasma isolates and nasal swab samples using the Bacterial DNA Kit  according to the manufacturers’ instructions. All samples were identified by PCR targeting the M. hyorhinis strains were cultured in KM2 medium until the medium color changed from red to yellow. Cells were collected by centrifugation at 15,000 × g for 20 min, resuspended in 70 μL double-distilled water (ddH2O), and boiled for 15 min. The crude lysate samples were stored at −20°C prior to testing. Six M. hyorhinis housekeeping genes  were selected for multilocus sequence typing using previously described primers and procedures (www.pubmlst.org) to assign allele and ST numbers. Sequences that did not match any of the existing ST types were registered as novel.The isolated ocedures . The PCRM. hyopneumoniae infection, and M. hyorhinis infection based on the routine serological tests and PCR tests performed every 6 months. Twenty-five pigs were picked and confirmed to be free from M. hyorhinis colonization by nested PCR of nasal swab samples. Animals were randomly separated into five groups (n = 5 per group) , including intravenous, intraperitoneal, and intranasal. Details of the M. hyorhinis strains, inoculation volumes, and routes are shown in Bama miniature pigs (aged 6 weeks) were obtained from a pig farm originally developed from cesarean-derived colostrum-deprived pigs. The pigs from this farm were negative for PRRSV, pseudorabies virus, PCV2, classical swine fever virus, african swine fever virus, r group) . AnimalsAfter infection, pigs were monitored daily for clinical signs, including elevated body temperature, apathy, loss of appetite, swollen joints, lameness, and labored breathing. Mean body weights were measured on days 0, 7, 14, and 21. Average daily weight gain during the entire experimental period was calculated and lameness and arthritis were scored as previously described : 0 = no The piglets were euthanized at 21 days postinfection, and a gross pathological examination was performed. The thoracic and abdominal cavities were exposed and examined for signs of polyserositis . The tarsal and carpal joints were exposed and examined for arthritis. Pleuritis, pericarditis, peritonitis, and arthritis were scored separately to reflect relative severity according to previously defined criteria , and a tM. hyorhinis strains that occurred prior to the experiment or unexpected infection from the environment during the experimental period. PCR and MLST analysis were carried out to confirm the species and strains.For one pig in each group, bacteria were re-isolated from tonsil, lung, heart, and joint tissues (swabs) to exclude undetected infection with other mycoplasmas or other M. hyorhinis-specific serum IgG and IgM antibodies were evaluated every 7 days postinoculation using an in-house indirect enzyme-linked immunosorbent assay (ELISA). Briefly, M. hyorhinis cells were collected by centrifugation at 15,000 × g for 20 min at 4°C, washed, and resuspended in PBS. After lysis by ultrasonication, the supernatant was collected, and the protein concentration was determined using a bicinchoninic acid (BCA) assay kit  according to the manufacturer’s instructions. The supernatant was diluted to 5 μg/mL with carbonate-bicarbonate buffer (pH 9.6) and used to coat 96-well ELISA plates overnight at 4°C. After blocking with 5% bovine serum albumin (BSA), each well was incubated with 100 μL of serum sample (1:100 in PBS containing 5% BSA) for 30 min at 37°C, followed by 100 μL horseradish peroxidase (HRP)-conjugated goat anti-swine IgG /IgM  . After washing, the reaction was visualized by incubation with tetramethylbenzidine-hydrogen peroxide  substrate for 10 min at room temperature. The optical density (OD) of the solution was measured at 450 nm using a BioTek uQuant microplate reader .post hoc Bonferroni comparison for selected pairs. For pathological evaluation at necropsy, data were analyzed using the Kruskal–Wallis test with a Dunn’s post hoc test for selected pairs. Repeated measures ANOVA were used to analyze changes in body weight, clinical symptoms, and antibody production during the experimental period. P < 0.05 was set as the threshold for statistical significance.Data were expressed as the mean ± standard deviation (SD). ADWG data were analyzed using a one-way analysis of variance (ANOVA) with The animal experimental procedures conformed to the guidelines of Jiangsu Province Animal Regulations (Government Decree No. 45). The animals in this study were under ethical approval by the Committee on the Ethics of Animal Experiments in Jiangsu Academy of Agricultural Sciences (Protocolnumber PDC 2021001). All efforts were made to minimize animal suffering in experiments.The data sets supporting the conclusions of this article are included within the article and its additional files.


Salix gordejevii saplings growing on inter-dune land (no erosion) and on the windward slope of the dune (20cm wind erosion) in Hunshandake Sandy Land were chosen and their morphology, biomass and physiological traits were investigated, respectively. Wind erosion significantly reduced plant growth, biomass accumulation, gas exchange and chlorophyll fluorescence, and obviously disrupted osmotic regulation function and antioxidant enzyme system in both sexes, especially in males. Under wind erosion condition, females exhibited higher sapling height (SH), basal diameter (BD), leaf dry mass (LDM), root dry mass (RDM), total dry mass (TDM), root percentage in total dry mass, net photosynthesis rate (Pn), maximum efficiency of photosystem II (Fv/Fm), effective quantum yield of PSII (ΦPSII), relative water content (RWC) of leaves, superoxide dismutase (SOD) and peroxidase (POD) activities, but lower malondialdehyde (MDA), proline as well as soluble sugar content than did males. However, no significant sexual differences in most of these traits were observed under no erosion condition. Our results demonstrated that females possess a greater resistance to wind erosion than do males, with females having a better photosynthetic capacity, stronger water retention capacity and more efficient antioxidant system to alleviate negative effects caused by aeolian environment.Effects of wind erosion on growth and adaptability have been widely reported in many plants, but little attention has been paid to dioecious plants. Recent studies have shown that sex-specific responses to environmental changes in many plants exist. To explore sexual differences in response to wind erosion, female and male  Aeolian environment is regarded as one of the extreme environments that seriously affect plant growth and reproduction in the dune ecosystem of arid and semi-arid areas . AffectePopulus cathayana, P. yunnanensis and Morus alba, males are better adapted to the majority of the stress conditions than females, exhibiting less damage, better growth, higher photosynthetic capacity and antioxidant activity  determine the effects of wind erosion on morphological and physiological characteristics in S. gordejevii; (b) evaluate whether females exhibit more resistance against wind erosion than males; (c) analyze the possible reasons for inconsistent sexual performance related to species in Salicaceae.id areas . Despite studies , no sex--1, with the main wind direction being northwest wind and frequent sandstorms in spring and autumn. The soil type in the area is chestnut soil, which is distributed with aeolian sandy soil. Dominant plant species include xylophyta such as Ulmus pumila var. sabulosa, S. gordejevii, S. microstachya var. bordensis, Artemisia halodendron, and perennial herbage such as Potentilla acaulis, Leymus chinensis, Cleistogenes squarrosa, etc. , which is located in Zhenglan banner of Xilin Gol league of Inner Mongolia Autonomous Region, China. The climate type is a mid-temperate continental monsoon climate, with a long cold winter and a short warm summer. The mean annual temperature, precipitation and evaporation are 1.7°C, 355 mm and 1931.4 mm, respectively. The mean annual evaporation is much larger than the mean annual precipitation. The frost-free season lasts approximately 110 days. The annual mean wind speed is about 4 m·ssa, etc. .S. gordejevii saplings that were artificially planted by the local forestry bureau in 2015. When planting, the cuttings (about 50 cm long) were inserted 45 cm into the soil with a 5-cm height above the soil surface. In April 2018 (the flowering season of S. gordejevii), we identified and labeled the sex of saplings growing on inter-dune land and on the windward slope of the dune, respectively. In August, we selected experimental materials for control group and wind erosion group in each habitat, according to the exposure of cuttings that were used for planting in 2015. Among them, the exposed degree of cuttings in control group was still 5-cm height above the soil surface (no erosion), while that in wind erosion group was about 25-cm height (20 cm wind erosion). 20 male and 20 female saplings in each condition were selected and used for growth and physiological index analysis. The experimental layout was completely randomized with two factors (sex and wind erosion). There were finally four conditions: (1) females with no erosion (control); (2) males with no erosion (control); (3) females with 20 cm erosion (wind erosion); and (4) males with 20 cm erosion (wind erosion).The experiment was carried out in an area of semi-fixed dune of Hunshandake Sandy Land, with vegetation dominated by Sapling height (SH), basal diameter (BD) and crown size (CS) of each sapling were measured on 22 August, 2018. Then all saplings were harvested and divided into leaves, stems and roots after washing out of soil. After oven-dry to constant weight at 70°C for 48 h, leaf dry mass (LDM), stem dry mass (SDM) and root dry mass (RDM) were weighed and recorded. The total dry mass (TDM) was calculated as the sum of leaf, stem and root dry mass. The percentage in total dry mass was calculated for leaf, stem and root, respectively. All biomass did not include the weight of cuttings that were used for planting in 2015.Pn), stomatal conductance (Gs), intercellular CO2 concentration (iC) and transpiration rate (rT) were measured between 09:00 a.m. and 11:30 a.m. with a LI-6400 portable photosynthesis system  under the following conditions: leaf temperature, 26°C; photosynthetic photon flux (PPF), 1500 µmol m-2 s-1; relative air humidity, 30%; and ambient CO2 concentration, 380 ± 10 µmol mol -1. Data were recorded once steady-state gas exchange rates were observed.Three male and female plants of each condition were randomly selected, and the fourth fully expanded leaf from apex of each plant was selected for gas exchange and chlorophyll fluorescence measurements on August 21. Net photosynthetic rate  and ΦPSII, effective quantum yield of PSII) were measured with a LI-6400 chlorophyll fluorescence system . The leaf samples were placed in dark treatment for 30 min using aluminum foils cover, and the minimum fluorescence (F0) and the maximum fluorescence (Fm) were measured by low modulated light and a saturating pulse, respectively. The Fv/Fm was then calculated using the equation: Fv/Fm = (Fm − F0)/Fm (-2 s-1 for 15 min to measure the maximum light-adapted fluorescence (Fm′) and the steady-state fluorescence (Fs). The ΦPSII was then calculated using the equation: ΦPSII = (Fm′ − Fs)/Fm′ (Chlorophyll fluorescence kinetics parameters (F0)/Fm . Subsequs)/Fm′ .fW), then were immersed in distilled water to saturation and weighed saturated weight (sW). Finally, the saturated leaves were dried to a constant weight (dW) at 70°C for 48 h. The formula for calculating RWC of leaves was the following:Three fully expanded and exposed leaves were randomly selected from each condition for relative water content (RWC) and malondialdehyde (MDA) content determination. RWC was determined by the method of Malondialdehyde (MDA) content was determined by the method of Proline assay was measured according to the method of Soluble sugar content was measured as described by 0.5 g fresh leaves of each sample were ground with a pre-cooled mortar and pestle with 50 mM potassium phosphate buffer (pH 7.8) to extract enzyme for the measurements of superoxide dismutase (SOD) and peroxidase (POD) activity . The sam2 and 20 μM riboflavin. After mixing enough, the mixture was carried out for 30 min under irradiance of 170 µmol photons m-2 s-1 provided by a white fluorescent lamp. The absorbance was measured at 560 nm using a non-illuminated identical tube as a blank. The SOD activity was expressed as unit g-1 FW.The SOD activity was determined by measuring the ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), as described by -1 FW min-1.The POD activity was determined by the method of P < 0.05.SPSS 20.0 statistical software  was used for statistical analyses. The data were checked for normality and homogeneity of variances, and log-transformed to correct deviations from these assumptions when needed. Two-way ANOVA was used to evaluate the effects of sex, wind erosion and their interaction. Individual differences among means of different conditions were determined by Duncan’s multiple range test. Differences were considered significant at P < 0.001) but CS only in males compared with control. There were significant differences in SH and BD between sexes in the control condition, with females having higher SH and lower BD than males. Under wind erosion condition, females had significantly higher SH and BD than males, but there was no significant difference in CS between male and female saplings. Similarly, wind erosion significantly inhibited biomass accumulation of all saplings (P < 0.001), and induced females to exhibit higher LDM, RDM and TDM than males. While there were no significant differences in these traits (except for SDM) between the sexes in the control condition . Except females always had significantly higher Pn than males, there were no significant sexual differences in Gs, Ci and Tr both under control and wind erosion condition . When comparing the sexes, females exhibited significantly higher RWC but lower MDA content than did males under wind erosion conditions, whereas there were no significant sexual differences under the control conditions (P < 0.001) but proline content only in males. Females had lower the content of proline and soluble sugar, but higher the activities of SOD and POD than males under wind erosion conditions. However, no significant sexual differences were detected in these traits under the control conditions  was greater than that on females (42.25%) in Pn, which is in agreement with previous studies that Pn decreased more in weaker sex under the same stress intensity  and XIX (Populus) of their sex determining genes, indicating that the sex determination loci in the willow and the poplar most likely do not share a common origin and has thus evolved separately , the Science and Technology Research Project of Colleges and Universities in Inner Mongolia Autonomous Region (NJZZ22589), and the Scientific Research Funding Project for Introduced High-level Talents of IMNU (2019YJRC018).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Medicago sativa L.) in salinity are not clear. For a wider and more detail evaluation, alfalfa were planted in saline sandy loam soil in fall, the effects of two plant systems  on soil moisture, root zone salinity, soil microbial community structure, seedling emergence number in the early stage of the growth period and soil nutrient contents, alfalfa production characteristics in the second growth year were determined in a 2-year field experiment. The result showed that, compared with FL, FU resulted in increased soil moisture content and seedling emergence, and significantly reduced relative abundance of Actinobacteria and Choroflexi in soil, but it did not affect root zone salinity at the seedling stage. In April of second growth year, the soil salinity was lower, and the soil available phosphorus, potassium, nitrogen, and soil organic matter contents of the root zone were higher in FU than in FL. Compared with FL, FU resulted in increased yield (by 37.5%), protein content (by 3.6%), and potassium concentration (by 33.2%), and decreased ash content (by 7.7%), and sodium concentration (by 19.0%) in alfalfa plants. Pearson’s correlation analysis indicated that the increased yield was positively correlated with seedling emergence, soil available potassium, total nitrogen, and organic matter contents, and shoot potassium content and negatively correlated with shoot sodium content. The relative abundance of Actinobacteria was negatively correlated with alfalfa ash, calcium, and sodium concentrations, and positively correlated with shoot potassium content. Taken together, the results indicate that Furrow-bed seeding in early fall alleviated salt stress of alfalfa and have the potential to enhance the yield and quality of alfalfa cultivated in saline soils by improving the soil environment and regulating the growth and physiology of alfalfa.Salt stress account for large decreases in crop yield all over the world. Furrow-bed system is an efficient practice to promote plant growth in saline soil. However, the effects of Furrow-bed system on the soil environment and the growth of alfalfa ( 1. Furrow-bed seeding system increased soil moisture content and alfalfa seedling emergence number in the seedling stage.2. Seeding pattern derived soil microbial community change in seedling stage in saline soil.3. Furrow-bed seeding system decreased root zone salinity in spring.+ concentration was decreased and yield was enhanced by furrow-bed seeding system.4. Alfalfa shoot Na Soil salinization is a growing problem for agriculture worldwide , more th+) concentration . This leads to sparse germination, stunted plants, and/or reduced crop yield and quality  is a well-known soil–water conservation practice used in crop production , and it In the soil microbial community, bacteria are one of the richest and most diverse microbial groups, and they play important roles in many soil processes. They participate in soil nutrient cycling, the decomposition of organic matter and waste, the degradation of pesticides and contaminants, and soil aggregation and humus formation. In addition, they affect the soil structure and the growth and health of plants . Soil miSoil ridging with plastic film has been shown to improve rainwater utilization and improve alfalfa yield in semiarid areas where alfalfa production is largely rainfed , HoweverA field experiment was conducted in an area with saline sandy loam soil in the Hohhot district  on the Tumochuan plain, China, during two consecutive alfalfa-growing seasons: 2019 and 2020. The site has a typical continental climate with mean annual air temperature of 7.6°C and mean annual maximum and minimum air temperatures of 23.3°C (July) and −11.0°C (January), respectively. The mean annual precipitation is 392.6 mm , and around 74.8% of the precipitation occurs between July and September. The potential annual evaporation is about 1757.1 mm. Mean annual sunshine exceeds 2829.8 h, and the frost-free period is about 137 days . The distribution of precipitation in 2019 and 2020 is shown in −1 soil organic matter (SOM), 930 mg kg−1 total N, 14 mg kg−1 available phosphorus (P), and 225 mg kg−1 available potassium (K), with an ECe  of 15.8 dSm−1 in spring and with an ECp  of 1.1dSm−1 in early fall. Alfalfa seeds were sown in early August  in 2019.The properties of the background surface soil (0–15 cm) were as follows: 15.9 g kgM. sativa L. cv. “Zhongcao NO.3,” bred by Linqing Yu, Chinese Academy of Agricultural Sciences, was selected for this study, as it is widely cultivated on the Inner Mongolia plateau in China. The experiment comprised two cultivation systems: furrow-bed seeding  and flat-bed seeding . The experiment had a completely randomized block design with four replicates. Each plot was 20.0-m long and 3.2-m wide. Alfalfa seeds were planted on 8th August 2019 using the drill planting method with a hand-pushed vegetable planter. For the FU treatment, the width of the ridges and the furrows was 35 and 5 cm, respectively, and the ridge height was 15 cm. The furrows were leveled as planting belts  concentration, shoot potassium (K+) concentration, shoot calcium (Ca2+) concentration, and plant nutritional components.Data were collected for rainfall, soil temperature, soil salinity, soil moisture content, seedling emergence number, soil bacterial diversity, soil properties, plant height, plant yield, shoot sodium . The number of emerged seedlings  was counted in five 100-cm-long seed row sections and the average number per 100-cm-long seed row in each plot at 4, 6, and 9 days after sowing (DAS) was calculated. The soil moisture content, salinity, and temperature during the seedling stage at 4, 6, and 9 DAS were determined by analyzing 10 surface (0–10 cm) soil samples with a HH2 Moisture Meter and a WET-2 sensor  to determine soil salinity. Soil chemical characteristics were tested following the method described by 3 in vials for 48 h. Then, the diluted extracts were analyzed to determine their Na+, K+, and Ca2+ contents using an M410 flame photometer .Plant height was measured five times from 17 October 2019 to 3 July 2020. At each measurement time, 20 plants were selected from each plot and the height was measured and the average height of alfalfa were used. In 2020, the yield of the first cut of alfalfa was measured. Alfalfa plants were cut in a 1-m long part of the row, with three replicates per plot. The average yield from plants in 1 m was calculated and used to estimate the yield per hectare. The harvested material was dried at 65°C for 48 h and then weighed to determine dry matter content. Then, the dried samples were ground to pass through 1-mm screen using a laboratory knife mill  for later analysis. Neutral detergent fiber NDF;  and acid®Soil DNA Kit  according to the manufacturer’s instructions. The reagents in this kit were designed to isolate DNA from trace amounts of sample, and are effective for isolating DNA from most bacteria. Nucleic acid-free water was used as the blank. The total DNA was eluted in 50-μl elution buffer and stored at −80°C until PCR analyses by the LC-Bio Technology Co., Ltd. .DNA was extracted from 0.25-g soil using an E.Z.N.A.The V3–V4 region of the prokaryotic  small-subunit 16S rDNA gene was amplified with slightly modified versions of the primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The 5′ ends of the primers were tagged with specific barcodes for each sample and universal sequencing primers .Each PCR amplification reaction mixture contained 25-ng template DNA, 12.5-μl PCR premix, 2.5 μl each primer, and PCR-grade water to complete the volume to 25 μl. The thermal cycling conditions to amplify the prokaryotic 16S fragments were as follows: initial denaturation at 98°C for 30 s; 35 cycles of denaturation at 98°C for 10 s, annealing at 54°C/52°C for 30 s, and extension at 72°C for 45 s; and then final extension at 72°C for 10 min. The PCR products were confirmed by 2% agarose gel electrophoresis. Throughout the DNA extraction process, ultrapure water, instead of a sample solution, was used to exclude the possibility of false-positive PCR results as a negative control. The PCR products were purified using AMPure XT beads  and quantified by Qubit . The amplicons were prepared for sequencing using a Library Quantification Kit for Illumina , and the size and quantity of the amplicon library were assessed using an Agilent 2100 Bioanalyzer . The PhiX Control library  was combined with the amplicon library (expected at 30%). The libraries were sequenced with 300PE MiSeq runs. One library was sequenced with both protocols using standard Illumina sequencing primers, eliminating the need for a third (or fourth) index read. The sequencing data were submitted to the NCBI Sequence Read Archive database (accession number: PRJNA826619).Samples were sequenced on the Illumina MiSeq platform according to the manufacturer’s recommendations (LC-Bio). Paired-end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequences. Paired-end reads were merged using FLASH. Quality filtering of the raw tags was performed under specific filtering conditions to obtain high-quality clean tags according to FastQC (V 0.10.1). Chimeric sequences were filtered using Verseach software (v 2.2.4). Sequences with ≥97% similarity were assigned to each representative sequence using the RDP  classifier. Differences in dominant species among different groups were detected and multiple sequence alignments were conducted using PyNAST software, which revealed the phylogenetic relationships among different operational taxonomic units (OTUs). Abundance information for OTUs was normalized using a standard sequence number corresponding to the sample with the least sequences. Alpha diversity was determined by calculating four indices  using QIIME (V 1.8.0). Differences in beta diversity (species complexity) among samples were detected by a principle co-ordinates analysis (PCoA) conducted using QIIME (V 1.8.0).n = 4) in two crop systems using Systat version 12.0 . The relationships between the soil bacterial community composition, at both phylum and class level, with soil and plant properties were analyzed with redundancy analysis (RDA) using the package vegan of the software R version 3.6.3.One-way analysis of variance (ANOVA) was used to evaluate statistical significance of the effects of the two seeding patterns on soil properties, plant properties, relative abundance of dominant bacterial phyla, classes, and genera; bacterial community richness, diversity indices, and OTUs. T-test were conducted to ascertain any significant difference between two seeding patterns at the 0.05 probability level. Proc GlM procedure was used for analysis of variance. Levene’s test were used for homogeneity of variance. All above data analysis was performed using SAS version 8.02 . SigmaPlot was used to generate bar graphs. Spearman correlation analysis was performed to detect relationships among soil and plant properties and soil fungal abundance, diversity, and relative abundance of dominant bacteria phyla and classes using the package stats of the software R version 3.6.3. Pearson’s correlation analysis was performed to detect relationships between alfalfa yield and soil and plant properties , and diversity (Shannon’s index). However, the values of OTU richness, Chao1’s, and Shannon’s indices were higher in FU than in FL .A PCoA was used to analyze beta diversity, to identify the differences in soil bacterial community composition between FU and FL . At the Proteobacteria , Actinobacteria (22.76%–31.60%), Acidobacteria (16.92%–18.06%), Gemmatimonadetes (6.54%–8.57%), and Chloroflexi (3.77%–5.38%), were the main bacteria phyla . LatesciThe main bacterial classes were Actinobacteria , Alphaproteobacteria (14.07%–14.34%), Acidobacteria (15.90%–16.73%), Betaproteobacteria (4.58%–6.06%), Gemmatimonadetes (4.57%–5.59%), and Deltaproteobacteria  and unclassified Actinomycetales  were lower in FU soil than in FL soil, while that of unclassified Betaproteobacteria (Betaproteobacteria_unclassified) was higher in FU than in FL , and decreased contents of ash and Na+ .The seeding pattern significantly affected alfalfa yield, and the contents of CP, NDF, ADF, ash, and ions in alfalfa plants . Compare+ concentration   . In our us study . During Notably, FU improved alfalfa quality compared with FL. For the first cut of alfalfa, the CP content was higher and NDF, ADF, and ash contents in alfalfa plants were lower in FU than in FL in the second year. The results of the Spearman correlation analyses revealed that the alfalfa CP content was positively correlated with soil total N and SOM . Thus, t+ and reduced Na+ accumulation in shoots may be mechanisms to avoid salinity stress. Overall, our results show that specific cultural practices, such as FU, with sowing in early fall have immense potential for alleviating salt stress and improving alfalfa productivity and quality in saline fields. Additionally, FU with sowing in early fall with no need for mulching with plastic, it is inexpensive and do not pollute the soil. Therefore, FU is the effective way of sustaining the alfalfa productivity in saline soil.In conclusion, compared with FL, FU increased the soil moisture content and seedling emergence and altered the soil bacterial community during the seedling stage. The FU system also increased the available soil nutrient contents and decreased soil salinity at the returning green stage in the second growth year. Compared with FL, the FU system resulted in significant improvements in alfalfa plant height, yield, and quality, and decreased salt injury. The improved yield was due to higher soil moisture content, higher seedling emergence, higher contents of soil available nutrients and SOM, and lower salt content in the plant rows in spring of the second growth year. Enhanced Khttps://www.ncbi.nlm.nih.gov/, PRJNA826619.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: KJ and JS conceived and designed the research. JS, JZ, LY, and TZ conducted the experiment and collected the field data. JS and JZ analyzed the data. KJ and JS wrote the manuscript. All authors reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Key Projects in Science and Technology of Inner Mongolia (2021ZD0031), Natural Science Foundation of Inner Mongolia (2018MS03028), Agricultural Science and Technology Innovation Program of CAAS (27-GRI-01), and Science and Technology Project from Inner Mongolia (2021GG0068 and 2019GG260).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Eimeria oocyst counts in feces, blood metabolites, and the meat quality of growing rabbits. A total of 160 hybrid rabbits  of 30 days of age and 643 ± 8.0 g body weight (BW) were assigned to four treatments with 10 replicates each (four rabbits/replicate). The treatments were as follows: (1) CON: rabbits fed basal diet and not supplemented with BP or PRO; (2) BP500: CON + BP (500 mg/kg BW); (3) PRO50: CON + PRO (50 µL/kg BW); and (4) BP + PRO: CON + BP (500 mg/kg BW) + PRO (50 µL/kg BW). Higher daily weight gain (p = 0.04) and lower feed conversion rate (p = 0.03) were observed in rabbits supplemented with PRO50. In addition, supplementation with PRO50 and BP + PRO reduced the amount of Eimeria oocysts per gram of feces (p < 0.05). Most hematological and serum biochemical parameters were similar in rabbits of all treatments. Protein content, collagen, and meat color were similar between treatments. In conclusion, propolis supplementation (50 µL/kg BW) can prevent coccidiosis and act as a natural growth promoter in rabbits without affecting animal health and meat quality.The objective of this study was to evaluate the effect of supplementation with bee pollen (BP) and propolis (PRO) on productive performance,  Apis mellifera) products, such as bee pollen (BP) and propolis (PRO), could be used for this purpose because they contain various nutritional compounds and some bioactive metabolites with pharmaceutical properties [It has been discussed that, due to its short production cycle, rabbit production could be used as an alternative to solve the growing meat shortage in developing countries . Howeveroperties ,6.BP is a mixture of flower pollen grains collected by bees with nectar and secretions from the hypopharyngeal glands of bees ,7. AccorEscherichia coli and Salmonella spp. in the cecum of growing rabbits [Pasteurella multocida [On the other hand, PRO is a complex mixture of resinous, gummy, and balsamic substances collected by bees from plant buds, flowers, and exudates ,13,14,15 rabbits . Likewisultocida . The effultocida  or by miultocida . Howeverultocida . FurtherEimeria oocyst count per gram of feces, blood biometry, blood biochemistry, and meat quality in growing rabbits.Due to the positive effects of BP and PRO, this work hypothesizes that supplementation with BP and PRO will benefit the productive performance of rabbits without affecting health and meat quality. Furthermore, the combination of BP and PRO could act synergistically due to their chemical composition and mechanisms of action. Therefore, the objective of this study was to evaluate the effects of supplementation with bee pollen, propolis, and the combination of both products on productive performance, The experiment was conducted during April and May 2022 at the “Conejos” Centro de Investigación Científica del Estado de México A.C. (COCICEMAC). This site is located in San Miguel Coatlinchán, State of Mexico, Mexico (latitude 19°26′56″ N and longitude 98°52′20″ W). San Miguel Coatlinchán is 2240 m above sea level, the mean annual temperature is 15 °C, and the mean annual precipitation is 645 mm [BP was collected in hives located in the municipality of Contla de Juan Cuamatzi, State of Tlaxcala, Mexico, located at 2320 masl (latitude 19°20’00″ N and longitude 98°10’00″ W). The BP collection was carried out from September to November 2021 using intermediate pollen traps placed in the hives’ inner part .The PRO was collected in the village Charco de Pantoja, in the municipality of Valle de Santiago, Guanajuato State, Mexico, located at 1709 masl (latitude 20°23’34″ N and longitude 101°11’29″ W). PRO collection was carried out from October 2020 to February 2021 using the plastic net mesh technique .BP and PRO samples were analyzed in the laboratory to determine the content of dry matter (method 967.03), crude protein (method 981.10), ether extract (method 920.29), and ash (method 942.05), following the procedures previously described by AOAC . The nutTM, Waltham, MA, USA). Similarly, the total flavonoid content of the PRO extract was obtained following the colorimetric method using aluminum chloride, as previously described by Chang et al. [TM, Waltham, MA, USA). The total content of phenolic compounds of the PRO was 23.67 mg gallic acid equivalent per g dry weight of the extract. Likewise, the total flavonoid content of the PRO was 9.21 mg quercetin equivalent per g dry weight of the extract.The total phenolic compound content of the PRO extract was determined using the colorimetric method described by Folin–Ciocalteu . For thig et al. . In thisAll rabbits used in the present study were clinically healthy at the start of the experiment. One hundred sixty freshly weaned California × New Zealand hybrid rabbits , 30 days of age and with an average body weight (BW) of 643 ± 8.0 g, were used. The rabbits were weighed to form four homogeneous groups and were entirely randomized for one of four treatments. Each treatment had 10 replicates (cages), and in each replicate there were four rabbits with similar initial weights and of both sexes . The treatments used were as follows: (1) rabbits fed a basal diet and without BP or PRO in the drinking water (CON); (2) BP500: CON + BP (500 mg/kg BW); (3) PRO50: CON + PRO (50 µL/kg BW); and (4) BP + PRO: CON + BP (500 mg/kg BW) + PRO (50 µL/kg BW). The BP dose (500 mg/kg BW) was chosen based on a previous report in which supplementation with this dose improved the digestibility of ingested nutrients in growing rabbits . The PROad libitum access to drinking water without treatment. The animals of the CON treatment had the same conditions as the experimental groups but without applying BP or PRO to the drinking water. Instead, drinking water was provided through nipple drinkers. The BP500, PRO50, or BP + PRO treatments were adjusted each week based on the body weights obtained. Oral administration of the treatments was performed in the morning (09:00 h).Treatments were administered five times per week (Monday through Friday) throughout the experimental period. Based on a previous experiment carried out by our work team (unpublished data), we verified that the doses of BP and PRO were well dissolved in the drinking water and that the rabbits consumed this water adequately. Therefore, the BP500, PRO50, or BP + PRO treatments were first applied to 200 mL of drinking water in each of the cages using automatic drinkers, as other authors previously used ,31,32,33Environmental, hygienic, and handling conditions were the same for all rabbits. Also, the health status of the rabbits was monitored throughout the experimental phase. The rabbits were housed in a module with natural ventilation system and thermal insulation on the walls but not on the ceiling. The rabbits were kept in galvanized metal cages 78 cm long, 56 cm wide, and 30 cm high (four rabbits/cage) arranged in a flat-deck system at 45 cm above the floor level. Each cage had a galvanized English-type hopper feeder  with a capacity of 1.5 kg and a nipple-type automatic drinker. During the entire experimental period (April and May), the average daylight was 13 h.®, Texcoco, Mexico), which came from the same production lot. According to the labeled feed package, antibiotics are not included, but 1 g of Diclazuril per ton as coccidiostat is added to prevent Eimeria spp. Each week, feed samples were taken and analyzed to determine the chemical composition of the feed following the procedures described by AOAC [ad libitum during the 42 days of the experimental phase. All rabbits were fed a commercial pelleted basal diet  for growing rabbits  located inside the rabbit facility. With the temperature and relative humidity data, the temperature and humidity index (THI) was estimated using the following equation [Ambient temperature and relative humidity were recorded daily using a Traceableequation : THI = T® brand digital scale , with an accuracy of ± 0.5 g. Subsequently, the rabbits were weighed weekly on days 7, 14, 21, 28, 35, and 42 of the experimental periods. The feed intake of the rabbits was recorded each week and divided by the number of days of the period to estimate daily feed intake . Likewise, average daily gain  was calculated using the weekly BW data. In addition, the rabbits’ feed conversion ratio (FCR) was calculated using the equation: FCR = DFI/ADG.The body weight (BW) of the rabbits was recorded before morning feeding at the beginning (day 0) of the experimental phase, using a TORREYEimeria spp. in rabbits, freshly voided feces samples were collected from eight replicates (cages) of each treatment each week . The feces were collected at the same time (9:00 h) on each sampling and were used to perform oocyst counts using the McMaster counting technique [To evaluate the parasite load of echnique : OPG = oechnique .® K2 EDTA anticoagulant  were used to collect the first sample, which was stored at 4 °C, for subsequent determination of the complete blood count and differential leukocyte count using an EasyVet® hematology analyzer , as previously reported by several authors [®  anticoagulant-free tubes were used to collect the second blood sample. These blood samples were centrifuged at 3500 rpm for 20 min to obtain blood serum using a refrigerated centrifuge . The blood serum was stored in Eppendorf tubes and frozen at −20 °C. Finally, for the analysis of the blood serum samples, an EasyVet® autoanalyzer  and Spinreact kits  were used to determine the contents of glucose (kit 41011), cholesterol (kit 41021), albumin (kit 1001020), globulin (kit 1001032), total protein (kit 1001291), urea (kit 41041), uric acid (kit 41001), bilirubin (kit 1001046), creatinine (kit 1001113), liver enzymes , lactate dehydrogenase (kit MX41274), and aspartate aminotransferase (kit MX41264)), calcium (kit 1001060), and phosphorus (kit 1001155), as described by other authors [To determine hematological and biochemical parameters, at the end of the experimental phase (day 42), blood samples were randomly taken from the ear vein of 10 rabbits  from each treatment. Two 2.5 mL blood samples were taken from each rabbit. Tubes with BD Vacutainer authors ,39,40. B authors ,41.Longissimus dorsi muscles (LDM) were removed from the hot carcass, following procedures similar to those described and recommended by Blasco and Ouhayoun [After obtaining the final body weight (day 42 of the experiment), all rabbits were kept fasting for 12 h. Subsequently, 20 rabbits  from each treatment were sacrificed following the procedures of the Mexican Official Standard (NOM-033 SAG/ZOO-2014). After completing the slaughter and bleeding of the rabbits, all internal organs, skin, feet, and heads were separated from the carcass, and the hot carcass weight (HCW) was recorded. The hot carcass yield (HCY) was estimated by the equation: HCY =  × 100. Subsequently, the leg muscles (HLM) and ® brand pH meter .Before meat quality analyses, samples were thawed at 4 °C for 24 h. Then, meat pH was determined using the procedures previously described by Orzuna-Orzuna et al. . For thi® brand thermometer , and when it reached 70 °C, the steaks were removed from the grill and allowed to cool for 1 h at room temperature (20–25 °C). The percentage of CL was estimated with the equation [Meat color parameters were measured in triplicate in LDM samples from each rabbit using the procedures described by Miltenburg . For thiequation : CL, % =HLM samples from each rabbit were separately ground and homogenized for 5 min using a Ship to Shore brand meat grinder  . SubsequAll data were analyzed by analysis of variance (ANOVA) using SAS statistical software . Before ijk represents the value observed in treatment i in week j for rabbit k; µ represents the overall mean; Ti represents the fixed effect of the i-th treatment (i = 1 (CON), 2 (BP500), 3 (PRO50), 4 (BP + PRO)); Wj represents the fixed effect of the j-th week of the experimental period ; (T × W)ij represents the fixed effect of the interaction between the j-th week and the i-th treatment; Rk represents the random effect of a cage (repetition) within treatment ; and eijk represents the random error.In this model, YThe variables of blood biometry, blood biochemistry, meat quality, and HCY were analyzed using a completely randomized design. For this, the GLM procedure of SAS  was usedij represents the observations, µ represents the overall mean, Ti represents the fixed effect of the i-th treatment, and eij is the random error. For all variables analyzed, treatment means were compared using Tukey’s test. The mortality rate was analyzed as a percentage using chi-square analysis [p ≤ 0.05. In addition, p > 0.05 and ≤ 0.10 was considered as tending to be significant.In this model, Yanalysis . A statip < 0.05) than rabbits assigned to the CON treatment. ADG was 8.8% greater (p = 0.04) in the PRO50-group rabbits than in the CON-treatment rabbits. FCR was 7.1% lower (p = 0.03) in PRO50-treatment rabbits than in CON-treatment rabbits. Compared to the CON treatment, rabbits in the PRO50 and BP + PRO treatments had 50 and 55% lower OPG (p < 0.05), respectively. On the other hand, mortality was higher (p = 0.05) in the BP500-treatment rabbits than in the rabbits assigned to the other treatments (eatments .p = 0.02) percent hematocrit was observed in BP500-supplemented rabbits than in CON rabbits (p = 0.009). Mean corpuscular volume was lower (p = 0.02) in PRO50-treatment rabbits than in CON rabbits. Lower (p = 0.001) blood concentration of monocytes was observed in BP + PRO-treatment rabbits than in rabbits assigned to the other treatments. Rabbits in the PRO50 treatment had higher (p < 0.05) blood concentrations of band neutrophils and eosinophils than in the CON, BP500, and BP + PRO treatments.Higher ( rabbits . Comparep = 0.001) serum urea concentration compared to rabbits in the CON, BP500, and BP + PRO treatments. In addition, rabbits supplemented with BP500 had a higher albumin/globulin ratio (p = 0.04) and lower serum total protein and globulin concentration (p < 0.05) than CON-treatment rabbits. Serum aspartate aminotransferase concentration tended to be lower in BP500-supplemented rabbits than in CON-treatment rabbits  HCY was observed in rabbits supplemented with BP500 and PRO50 than in rabbits assigned to the CON and BP + PRO treatments (p = 0.001) in meat from rabbits supplemented with BP + PRO than in meat from rabbits in the other treatments. PRO50 supplementation reduced (p < 0.05) the CL of meat compared to the other treatments. However, lower fat content (p = 0.05) and higher moisture content (p = 0.03) were observed in meat from rabbits supplemented with BP500 than in meat from CON rabbits  between BW and Eimeria spp. OPG count [Eubacteriaceae, Peptococcaceae, and Natranaerobiaceae) that are positively correlated with ADG. Likewise, the addition of quercetin in diets for rabbits increases up to 7.6% the serum concentration of growth hormone [Zeedan et al.  observedPG count . FurtherPG count . PreviouPG count ,18 have PG count  reported hormone . SimilarFCR was not affected by supplementation with BP500 and BP + PR, suggesting that the doses of BP and BP + PR used in the present study do not improve feed efficiency in growing rabbits. In similar studies, Attia et al. ,10 also Erysipelotrichaceae and Haloplasmataceae) that are negatively correlated with FCR. Consequently, similar PRO and flavonoid consumption effects observed in our study could partially explain the lower FCR in rabbits on PRO50 treatment. The lower FCR observed in rabbits supplemented with PRO50 suggests that PRO could improve the profitability of rabbit production systems since FCR is a crucial indicator for judging an animal production system [On the other hand, FCR was lower in rabbits supplemented with PRO50. PRO supplementation has been reported to improve rabbits’ digestibility of organic matter and ingested crude protein . North en system .Eimeria protozoa, which in rabbits causes growth retardation and high mortality [Eimeria decreased in rabbits supplemented with PRO50 and BP + PRO. This result suggests that these products could be used as natural coccidiostats for growing rabbits. This hypothesis is supported by the average OPG values observed in rabbits supplemented with PRO50 and BP + PRO, which were lower than the range (4000–5000 OPG) at which the application of prophylactic treatment against Eimeria protozoa is required [Coccidiosis is an infection caused by ortality . In the required .On the other hand, although mortality was higher in rabbits supplemented with BP500, the mortality observed in the present study was high (between 17.5 and 40%) for all treatments. In the present study, a seasonal effect and climatic conditions might have an essential role in the high mortality rates observed , particuWhen evaluating a new feed additive for domestic animals, it is essential to analyze the effects of the consumption of the additive on animal health . Accordi3/mL) [Eimeria parasites [The blood concentration of leukocytes was similar between treatments. However, the blood concentration of leukocytes in CON treatment rabbits was higher than the normal range reported for healthy rabbits . In addi3/mL) . These rarasites . Furtherarasites . On the arasites . ConsequSerum glucose concentration and lipid metabolites are indicators of rabbits’ energy status . In the Supplementation with BP500 reduced serum globulin and total protein levels. However, rabbits from all treatments had serum urea, albumin, globulin, and total protein concentrations within the reference range considered normal for healthy rabbits . This reIf analyzed at appropriate reference intervals, serum uric acid and creatinine concentrations can be used as biomarkers of renal function status in rabbits . For exaSerum levels of hepatic enzymes are often used as markers of liver disease . In addiIt has been mentioned that serum calcium and phosphorus levels can be used as good indicators of nutritional status in domestic animals because their variability is low ,73. In tIn the present study, supplementation with BP500 and PRO50 increased HCY. In a similar study, Zeedan et al.  also obsThe pH is considered one of the most critical parameters determining rabbit meat quality because it is related to its color, flavor, water-holding capacity (WHC), tenderness, and shelf life . For exaPrevious studies have reported that meat color can be modified by changes in fat content and pH ,77. In tIn the present study, PRO50 supplementation reduced the CL of meat. This result suggests that PRO supplementation can improve the WHC of rabbit meat because CL and WHC are negatively correlated . LimitedThe chemical composition of rabbit meat from all treatments had values within the normal range . This reThe results of this study indicate that propolis supplementation (50 µL/kg BW) can be used to prevent coccidiosis and as a natural growth promoter in rabbits without affecting animal health and meat quality. However, more research is needed to understand and explain the impact of bee pollen and propolis supplementation on productivity, parasite load, health status, and the meat quality of growing rabbits.


To report a novel 11-cis retinol dehydrogenase gene (RDH5) variant discovered in a 57-year-old male with fundus albipunctatus (FA) complicated by severe macular atrophy.  The patient was evaluated with a complete ophthalmic examination, optical coherence tomography (OCT), color fundus photography, green wavelength fundus autofluorescence, visual field testing, full-field ERG (ffERG), and multifocal ERG (mfERG). Genetic analysis investigating gene variants involved in inherited retinal disorders was performed. RDH5 gene were identified, including a novel missense variant, c.814_815del (p.Leu272Aspfs∗63), and a known pathogenic nonsense variant, c.160C > T (p.Arg54∗). Fundus autofluorescence demonstrated bull's eye maculopathy and hyperautofluorescent perifoveal rings bilaterally. OCT showed foveal atrophy of the outer retina and scattered hyper-reflective lesions in the peripheral macula. The ffERG results showed a severely diminished scotopic and photopic response. The mfERG results demonstrated minimal response in the central macula.  The patient presented with a rapid decline in visual acuity and a history of poor night vision. On fundoscopy, he exhibited a phenotype characteristic of FA accompanied by severe macular atrophy bilaterally. Heterozygous variants in the RDH5 gene. This patient's clinical presentation, diagnostic studies, and genetic testing confirmed the diagnosis of FA. Additionally, he exhibited severe macular atrophy, not typically found in FA. Two RDH5 gene variants were identified, one of which is the novel variant, c.814_815del (p.Leu272Aspfs∗63). We suggest that this RDH5 genotype may be associated with a more progressive phenotype of FA contributing to macular atrophy. Fundus albipunctatus is a rare, congenital form of stationary night blindness caused almost exclusively by the  Fundus albipunctatus  is a rare, congenital form of stationary night blindness that is characterized by the appearance of numerous small, round, yellow-white lesions throughout the retina, typically found in the retinal periphery and perimacular area while sparing the fovea . PatientRDH5-related FA further demonstrated high frequencies of macular involvement and cone dysfunction in older patients. In this study, 17 of 23 patients (73.9%) examined with full-field ERG demonstrated diminished cone responses in addition to severely decreased rod responses. Macular involvement was noted in 12 patients (48%), 2 of which (8%) had diffuse macular disruption and decreased visual acuity [However, in recent decades, there have been increasing reports of a subset of FA patients who progress to develop macular atrophy and cone dystrophy, particularly in the older population, with a review of the literature by Skorczyk-Werner et al. reporting an incidence of up to 30% , 9–11. Acis retinol dehydrogenase gene (RDH5) which produces an enzyme predominantly expressed in the retinal pigment epithelium (RPE) of the eye [RDH5 gene have been identified to date worldwide, with variant types consisting of 46 missense/nonsense variants, 8 small deletions, 4 small insertions/duplications, 2 small indels, and 1 splicing substitution. Of these variants, 49 have been reported in association with a disease phenotype consistent with FA [The pathogenesis of FA at the molecular level can be attributed almost exclusively to a variation in the 11- the eye , 13. Cur gene RDH which pr with FA , 14–27.RDH5 genetic variants and cone dystrophy or macular degeneration in addition to nyctalopia [RDH5 variants would be helpful in identifying and counseling the subset of patients that are genetically predisposed to developing early-onset macular atrophy or cone dystrophy.Reported cases in the literature demonstrate that different genetic variants can result in a spectrum of phenotypic presentations of FA, as recent studies have suggested a causal relationship between certain ctalopia . FurtherRDH5 gene variants, one of which is a previously unreported, novel missense variant of presumed pathogenicity, c.814_815del (p.Leu272Aspfs∗63), as well as a previously described pathogenic nonsense variant, c.160C > T (p.Arg54∗) also known as c.343C > T in the literature [In this report, we present a patient with FA complicated by severe macular atrophy who was heterozygous for two identified terature . We alsoA 57-year-old Hispanic male was referred for retinal evaluation due to progressive decline of central vision bilaterally, more prominent in the right eye than the left, that occurred over the course of 7 months. He complained of difficulty reading and watching TV as well as bilateral light sensitivity. He denied new distortion in vision, new floaters, flashing lights, head or eye trauma, prior eye surgery, and a history of strabismus or amblyopia. His ophthalmic history was unremarkable aside from poor night vision since childhood. His family history was negative for retinal diseases, and his two children and seven grandchildren denied night vision disturbance. His general medical condition was unremarkable aside from a history of hypertension, well-controlled on lisinopril.On presentation, visual acuity was measured by counting fingers at 4 feet OD and counting fingers at 3 feet OS. Anterior segment exam, pupillary reflexes, and extraocular movements were normal. Dilated fundus exam as well as ultra-wide-field confocal scanning laser ophthalmoscopy showed significant macular atrophy in both eyes, with 3+ diffuse, fine, yellow flecks Figures . OpticalRDH5 gene: a known pathogenic variant, c.160C > T (p.Arg54∗), and a novel variant of presumed pathogenicity, c.814_815del (p.Leu272Aspfs∗63), which previously have not been reported in association with an RDH5-related condition. Genetic analysis of the patient's 35-year-old, asymptomatic son revealed heterozygosity for one pathogenic RDH5 variant, c.160C > T (p.Arg54∗), demonstrating in-trans segregation of the variant.Following detailed informed consent, a blood sample was sent to the Invitae Genetic Testing Laboratory  where the inherited retinal disorders panel was performed, consisting of sequence analysis and deletion/duplication testing of 248 genes for variants associated with genetic disorders. Our patient was heterozygous for two variants in the RDH5 gene variant c.814_815del (p.Leu272Aspfs∗63), while not reported in the literature in individuals with FA, was presumed to be pathogenic by the Invitae Genetic Testing Laboratory, given that this variant disrupts the C-terminus of the RDH5 protein—a region disrupted by other variants reported in association with FA. We confirmed that this variant is present in population databases (rs765714290). Per the Exome Aggregation Consortium database, the reported allele frequency is 8.24e−6. This variant is also listed in the ClinVar database under the variation ID 281407, however, without literature evidence for a direct pathogenic association to FA. The reference article listed in ClinVar for this variant describes patients with FA with other RDH5 variants that disrupt the same C-terminal region of the RDH5 protein as c.814_815del, however, without report of this specific variant [RDH5 AND c.814_815del,” “RDH5 AND p.Leu272Aspfs ∗63,” “c.814_815del,” and “p.Leu272Aspfs ∗63.” The Human Gene Mutation Database was also searched without a matching result under small deletions and gross deletions in the RDH5 gene.The novel  variant . To furtRDH5 gene. In most patients, it classically presents as night blindness from early childhood with an otherwise stationary clinical course with normal visual acuity, visual field, and color perception and no further progression to vision loss. However, since the initial description of the stationary clinical course of FA in the early 1920s, there has been an increasing number of reports indicating a more progressive form of the disorder accompanied by macular degeneration or cone dystrophy particularly in the elderly population, as demonstrated in our patient [Fundus albipunctatus is an autosomal recessive form of stationary night blindness caused primarily by variants of the  patient , 14, 16. patient , 9–11. F patient .RDH5 gene [cis retinol dehydrogenase catalyzes the final step in the synthesis of 11-cis retinaldehyde, the universal chromophore of visual pigments [RDH5 gene variants has facilitated the accurate diagnosis of FA in patients whose phenotypic presentation can be clinically confused with other flecked retina syndromes such as retinitis punctata albescens.The pathogenesis of FA at the molecular level can be attributed almost exclusively to variants in the DH5 gene , 13. TheRDH5 gene have been identified with reports in the literature which suggest that different variants are associated with specific phenotypic presentations of FA. For example, Pras et al. in their case series reported an 18-year-old patient with a missense c.565G > A transition variant who developed cone dysfunction at adolescence, while older middle-aged patients affected by a different variant had not developed functional photoreceptor cell dysfunction at the time of the study [Several genetic variants in the he study . The varRDH5 gene variants were identified: c.160C > T (p.Arg54∗) and c.814_815del (p.Leu272Aspfs ∗63). The first variant, c.160C > T (p.Arg54∗), is a nonsense variant resulting in an absent or disrupted protein product, which was previously reported in 2012 by Pras et al. in 10 patients with FA from ages 19 to 55 [In our patient, two ∗63), which results in a frameshift mutation in the RDH5 gene that disrupts the last 47 amino acids of the RDH5 protein and extends the protein by an additional 15 amino acids. This exact variant has not been previously reported in the literature in individuals with RDH5-related conditions such as FA. However, a different pathogenic genetic variant that affects the same region of the RDH5 protein (C-terminus) has been reported in a patient with FA by Liu et al. [∗), in a 6-year-old male patient with FA with early-onset cone dysfunction, suggesting not only the involvement of this variant in the development of FA but that a disruption in this region may also result in a greater negative effect on the cone system compared to other RDH5 gene variants.The second gene variant is the novel missense variant c.814_815del , in an individual with FA, which may exist in a subset of RDH5 variants associated with macular atrophy or cone dystrophy. To further elucidate the correlation of genotype and phenotype among the various RDH5 variants, additional data and further work in this area is needed.While the specific roles of the various


Tuberolachnus salignus with a length of 16,868 bp, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, a control region located between rrnS and trnI, and a repeat region located between trnS2 and trnF. All protein-coding genes are initiated with ATN and terminated with TAA, except the cox1, nad3 and nad4 with termination of T. The length of the repeat region is 1,407 bp, which repeats 4.04 times with a 305-bp unit. Our phylogenetic analysis showed T. salignus is sister to Stomaphis sinisalicis.We sequenced the complete mitochondrial genome of  Tuberolachnus salignus  has an almost cosmopolitan distribution, which mainly feeds on stems and branches of Salix as well as occasionally recorded from Populus .The aphid T. salignus is 16,868 bp in size (GenBank accession number OK642815), containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), a control region, and a repeat region. The genes are arranged in a different order from the inferred ancestral insect mitochondrial genome  arm. The rrnL and rrnS genes are 1316 and 772 bp long, with 85.2% and 84.7% A + T content, respectively. The control region is 737 bp in length located between rrnS and trnI, showing high A + T content (86.2%). The repeat region between trnS2 and trnF is 1407 bp long with an A + T content of 87.0% in the mitogenome of T. salignus. A 305-bp repeat unit repeats 4.04 times in this region.All PCGs initiated with typical ATN codon and used typical TAG or TAA as the stop codons, except Tuberolachnus salignus and 30 other aphids including one outgroup species Adelges tsugae using RAxML v8.2.10 (Stamatakis T. salignus was presented as a sister to S. sinisalicis with well-support.We reconstructed a maximum-likelihood phylogenetic tree with 13 protein-coding genes of Gexia Qiao, Jing Chen and Shuang Xu contributed to the conception and design of the study. Shuang Xu and Xiaolu Zhang conducted the analysis and interpretation of the data. Gexia Qiao, Liyun Jiang and Shuang Xu determined the final published version. All authors agree to be accountable for all aspects of the work.Tuberolachnus salignus is a common pest on Salix in China as well as not recorded in the species list-of-ethics committees for research involving animals of the Institute of Zoology Chinese Academy of Sciences. Therefore, no ethical approval or other relevant permission can be provided for the study.


The treatment recommended for stage IVa Adrenocortical carcinoma (ACC) not amenable to radical resection is the mitotane plus loco-regional treatment (LR) strategy, which has not yet been validated. Moreover, prognosis factors for this strategy are not yet established. This study aimed to determine which stage IVa ACC patient population would benefit the most from this association. For this purpose, we reviewed all stage IVa patients  treated with mitotane and LR from 2008 to 2021 in our institution. This study included 60 patients and 109 LR were performed. The primary endpoint was disease control (DC). We found that DC was associated with longer Time to second line Treatments (TTC). Moreover, DC rate was higher in patients that had ≤5 metastases or a maximum metastasis diameter below 3 cm. Based on those results we propose the first definition of oligometastatic ACC: stage IVa patients with ≤5 metastases or a maximum metastasis diameter below 3 cm. It is vitally important that scientists are able to describe their work simply and concisely to the public, especially in an open-access on-line journal.p < 0.001) and OS . Patients with less than or equal to 5 metastases (HR: 6.15 (95% CI: 1.88–20.0), p = 0.002) or a maximum metastasis diameter below 3 cm had higher rates of DC (HR: 3.78 (95% CI: 1.09–13.14), p = 0.035). Conclusion: stage IVa ACC patients with ≤5 metastases or a maximum metastasis diameter below 3 cm had favorable responses to LR. We propose the name oligometastatic ACC for this subgroup of patients.Objective: The recommended first-line treatment for low-tumor-burden ACC (stage IVa ACC) not amenable to radical resection is mitotane in association with loco-regional treatments (LRs). The aim of this study was to determine the patient population that would benefit the most from LR. Materials and methods: This retrospective monocentric expert center chart review study was performed from 2008 to 2021 and included stage IVa patients  treated with LR . The primary endpoint was disease control (DC). Correlations between DC, time to systemic chemotherapy (TTC), overall survival (OS), and tumor characteristics were analyzed using Kaplan–Meier survival analysis and Cox’s proportional hazards regression model for multivariate analysis. Results: Thirty-four women (57%) and 26 men with a median age of 48.1 years (IQR: 38.3–59.8) were included. One hundred and nine LRs were performed, with a median of 2 (IQR: 1–3) per patient. DC was achieved in 40 out of 60 patients (66.7%). Patients with DC had a significantly longer TTC (HR: 0.27,  Adrenocortical carcinoma (ACC) is a rare and aggressive malignant tumor with an incidence of 0.7–2.0 new cases per million population per year and less than a 15% 5-year overall survival in the metastatic stage . PatientThe new European Network for the Study of Adrenal Tumor Stage classification (mENSAT) allows a better prognostic stratification of patients with metastatic ACC ,5, and sThe treatment recommended for stage IVa ACC  not amenThe objective of this retrospective monocentric study was to identify prognostic factors of LR in stage IVa ACC patients in order to better specify the population that would benefit the most from this strategy.This retrospective single-center study from a tertiary referral cancer center was approved by the Gustave Roussy Institutional Ethics Committee (n°: GR 2021-14). Written informed consent was obtained from each patient in accordance with the policy of our institution regarding chart reviews and the Declaration of Helsinki.Between July 2003 and May 2018, all consecutive patients treated with mitotane for mENSAT stage IVa ACC  were identified and followed until January 2021. The inclusion criteria were a confirmed diagnosis of ACC by expert pathologists, stage IVa ACC , tumor bThe following relevant data were collected from patient charts by two on-site investigators, AB and CR: age, sex, Weiss score, Ki67 percentage, mitotic count, primary surgery status , and GRAS prognostic factors . The disease-free interval (DFI) was defined as the duration between ACC diagnosis and stage IVa diagnosis, with DFI < 1 year representing synchronous metastasis and DFI  studies –19 [16,1 (16–19) , the metLR was decided by the tumor board by consensus among neuroendocrine oncologists, surgeons, radiotherapists, and interventional radiologists. LR characteristics and locations were collected; procedures included either surgery, stereotactic body radiation therapy (RT), chemoembolization (CHE), or percutaneous ablation (PA). The median number of procedures per patient was reported. Because of the retrospective nature of the study, only grade 3–5 adverse events according to Common Terminology Criteria for Adverse Events (CTCAE V.4)  were repThe best response to the LR strategy during the follow-up was defined as follows: regardless of the number of LRs needed to reach it, complete response (CR) indicated the disappearance of all lesions; a partial response (PR) indicated at least a 30% decrease in the largest diameters of the lesions; and progressive disease (PD) indicated at least a 20% increase; otherwise, the tumor was classified as stable disease (SD). The patients whose best responses to LR during follow-up were complete response, partial response, or stable disease were defined as patients with controlled disease (DC). The time to progression after best response to LR was collected. The time to best response from LR initiation was collected, as well as the median number of LRs needed to reach the best response during patient follow-up.Patients were followed by thoracic and abdomen–pelvic computer tomography (CT) scans plus or minus fluorodeoxyglucose (FDG) positron emission tomography (PET) scans every 3 months. They were reviewed by a single investigator (CR) to assess the best response to LR during follow-up according to the RECIST 1.1 criteria .Mitotane initiation was defined as the time of initiation of mitotane in synchronous metastatic patients or as the time of stage IVa diagnosis in cases of metachronous metastatic patients already receiving mitotane as adjuvant treatment. Time to second-line chemotherapy initiation (TTC) was defined as the time from mitotane initiation until systemic polychemotherapy initiation or clinical trial inclusion. Overall survival (OS) was defined as the time from mitotane initiation to death or censored at the time of last news.The primary endpoint of this study was DC, as defined by a best response obtained at any point during follow-up of either CR, PR, or SD. Secondary endpoints were correlations between DC and median TTC and median overall survival as well as DC and tumor characteristics. Categorical variables were analyzed using a Fischer exact test, and continuous variables were analyzed using the Mann–Whitney U test.p < 0.05.TTC and OS were calculated by Kaplan–Meier analysis. The correlations between tumor characteristics and DC and between DC and TTC or OS were analyzed. If significantly associated with DC in the univariate analysis (log-rank test), they were further tested in multivariate analyses. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were estimated using Cox’s proportional hazards regression model, with the lowest risk group as the reference group. All tests were two-sided. The statistical analysis was conducted using JMP software version 16. Results were statistically significant if Between July 2003 and May 2018, 79 patients with stage IVa ACC were diagnosed and discussed by our adrenal tumor board. Thirty-four female and twenty-six male  ACC patients or 75.9% (60/79) of stage IVa ACC patients were considered eligible for LR, with a median age of 48.1 years old (IQR: 38.3–59.8) . The chaAll patients received mitotane  and 41  achieved mitotane >14 mg/L at the time of loco-regional treatment initiation.One-hundred and nine LRs were performed with a median of two (range: 1–12) per patient. Thirty-four  patients had surgeries, fourteen  had adrenal lodge RT, three  had organ radiotherapy, twenty  had CHE, and thirty (50%) had PA. The locations of the metastasis according to loco-regional treatments are detailed in The complication rate per procedure was 6.4% (7/109). Two post-procedure bleedings with spontaneous hemostasis were reported (CTCAE grade 3). One gastrointestinal fistula and one mild pancreatitis occurred after LR (CTCAE grade 3). CTCAE grade 4 complications were two post-procedural adrenal insufficiencies and one pleural hemorrhage requiring embolization and two units of packed red blood cells.Overall DC rate and progressive Disease (PD) rate was 66.7% (40/60). The best response to LR during follow-up was stable disease in 10% (6/60), partial response in 8.3% (5/60), and CR in 29 patients .DC  was achieved after a median of 1 LR (min: 1–max: 10) and at a median time of 16 months (IQR: 19–3) from mitotane and LR initiation.The median durations from SD, PR, and CR to systemic therapy were 44 (95% CI: 8–not reached), 70 (95% CI: 13–not reached), and 97 (95% CI: 16–not reached) months, respectively; at the time of progression after LR, 18 were still stage IVa .The median OS and TTC were 68 (95% CI: 43–117) and 42 (95% CI: 15–100) months, respectively .p < 0.001) and OS (HR: 0.22 (95% CI: 0.10–0.47), log-rank p < 0.001) (Patients with DC had significantly longer TTC (HR: 0.27 (95% CI: 0.13–0.54), log-rank < 0.001) . The onep = 0.002), Dmax < 3 cm (HR: 3.78 (95% CI: 1.09–13.14), p = 0.035). Both factors were independently associated with DC  and during overall follow-up (p = 0.025).Finally, patients with DC had less cytotoxic chemotherapy within 6 months of mitotane initiation than patients with PD , with the majority of best responses to LR strategy being complete response (48.3%). Importantly, LR in our study signifies the potential use, repetition, and combination of all available LR techniques as best determined during the tumor board meeting. This strategy is based on different profiles of indications and contraindications of each LR tool. In brief, surgery can access large lesions; IR is minimally invasive and can be repeated over time; and RT is able to treat infiltrative lesions. The varying number of LRs and time needed to reach DC can be explained by both the delay to achieve a therapeutic plasma mitotane level as well as the time to finalize the LR procedures and obtain the best possible response .The median durations from SD, DC, and CR to untreatable progression with M + LR were 44, 70, and 97 months, respectively, emphasizing that a prolonged response can be achieved and systemic chemotherapy delayed. Interestingly, the best duration of DC was achieved with PR or even CR in 56.6% of patients. Such rates of PR or CR cannot be achieved by any systemic option, including mitotane alone, currently.We looked for prognostic factors for DC with LR treatments in this homogenous population of metastatic ACC patients and identified two simple radiological parameters: five metastases at most or a maximum diameter below 3 cm are associated with higher rates of DC . These results confirm the results of previous reports obtained in ACC populations and in other cancers, showing the importance of the maximum diameter of each metastasis and the number of metastases as a prognostic factor of response to several interventional radiology techniques. Surgery may be the ideal tool in cases of large but limited-number metastases. In case of an infiltrative lesion or a location at risk, radiotherapy could be the technique of choice. Overall, a combination of such techniques can be proposed and repeated over a patient’s treatment course . These rThere is a common effort among all oncologic specialties to provide specific definitions of oligometastatic disease for each cancer type. Hellman et al.  first deLibé et al.  demonstrOn the basis of our results, one may distinguish several possibilities in ACC patients: first, the targeting of all tumor sites at first treatment initiation, if achievable, and repeating LR until the best response is achieved; second, as has been previously suggested in other settings ,33, in cThere are some limitations to this study, which include its retrospective and monocentric nature, the focus on a very selected subgroup of patients, the use of LR based on institution experience , and the low number of patients with bone metastases.We identified a subgroup of stage IVa ACC patients according to modified ENSAT classification with specific behavior and favorable response to mitotane plus LR: patients with five metastasis at most or maximum diameter below 3 cm. On the basis of these simple radiological prognosis factors, we propose the name oligometastatic ACC for this subgroup of patients.