These are poorly understood and often unprotected, despite their prevalence within the Mediterranean region and their importance. Qualitative and quantitative characterisation of small lagoons, in terms of functioning and sensitivity to global and local changes, are needed to develop appropriate management strategies. For this purpose, this work provides the first inventory of all Corsican lagoons and has investigated three of them of small size , characterised by contrasting anthropogenic contexts . At the regional level, 91 of the 95 lagoons identified are < 0.5 km2, making Corsica a good example for the study of small Mediterranean lagoons. The three case studies showed differences in their seasonal biogeochemical cycles and phytoplankton communities . Arasu and Santa Giulia lagoons showed an increase in watershed urbanisation (+ 12% and + 6% in 30 years), high phytoplankton biomass, low diversity and blooms of potentially harmful dinoflagellates. Conversely, Balistra lagoon showed a good status overall, but some anthropogenic pollution sources within its watershed. This study demonstrates the importance of small lagoons at regional and Mediterranean scale, and provides knowledge on studied local sites but also potential applications elsewhere. The importance of an integrated approach considering lagoons within their adjacent connected systems (watershed and sea) and anthropogenic contexts is highlighted.Mediterranean coastal lagoons are affected by multiple threats  expected to increase in the future and impact the ecosystem services provided. Conservation norms and scientific studies usually focus on large lagoons (> 0.5 kmThe online version contains supplementary material available at 10.1007/s12237-023-01182-1. Coastal lagoons are water bodies, generally parallel to the coastline, separated from the sea by a barrier but connected at least intermittently to the marine environment through one or more restricted inlets Kjerfve . They arPhytoplankton is considered a relevant indicator of ecological status and ecosystem functioning and it is often a central element in management strategies and norms E.C., . In coas3−) concentrations in waters are unequivocal markers of anthropogenic pressures, mostly deriving from synthetic fertilizers and from septic tanks . Since smaller coastal lagoons are not included in the regulation, they remain neglected, poorly understood and ultimately poorly or even not at all managed. Among Mediterranean coastal countries, at least four  have lagoons smaller than 0.5 km2  2) but present three different anthropogenic contexts and histories and contrasting current human pressures. We have considered their connection to adjacent systems, in order to better characterise the ecosystems in their hydrogeological and anthropogenic contexts. To do so, anthropogenic tracers from the watersheds of the three case studies were detected to describe sources of human pressure. Then, seasonal samplings over two consecutive years were performed in the lagoons to analyse environmental characteristics and phytoplankton variations, to assess seasonal patterns and to detect relationships between abiotic and biotic factors.An excellent example with regard to this major challenging goal of the conservation of coastal lagoons, especially in the Mediterranean region, is Corsica Island (France), located in the north-western Mediterranean Sea. The island has more than 200 coastal wetlands, mostly brackish coastal lagoons, highly diverse and is a good example for the study of small-sized coastal lagoon environments Ghilardi . Only thThe main aim of the study was to raise awareness of these little known small-surface systems. To do that, firstly, we contextualised the importance of small-sized lagoons at regional and Mediterranean scales. Secondly, for the three case studies two main questions are addressed: (i) how do these three small lagoons, of comparable size but with different levels of anthropogenic impact in their watersheds, differ in terms of their physical and ecological functioning? (ii) what implications do these differences have for the lagoons’ vulnerability towards climate change and other environmental stressors? Ultimately, our aim is to provide new knowledge to stimulate the development of management strategies dedicated to small-sized coastal lagoons at regional and global scale.Pôle Relais Lagunes Méditerranéennes – Tour du Valat  on the number, surface areas, perimeter, ownership and management of all Corsican lagoons. The water masses considered in this study are classed as “Mediterranean coastal lagoons,” according to the definition of the habitat 1150*-2, provided by the European Directive 92/43/CEE (Habitats Directive) applied in France , over a selected hydrological period (2018–2020) using the ArcGIS 10.3 software.A qualitative description of the large-scale distribution of lagoons in the Mediterranean basin was performed on the basis of available data  website , which is predominantly constituted of densely fractured and intensively weathered granite (72%). In the southern-lower part of the Arasu lagoon watershed, the geology is characterised by clay-sand deposits, favourable to the presence of a restricted alluvial aquifer, formed by Quaternary sediment deposits and hydraulically connected to the lagoon in this area. The main freshwater sources are three streams in the western part of the basin, while exchanges with the sea take place in the eastern part only, through a small channel, with a bifurcation . It has been designated under the Special Areas of Conservation (SAC) protection status as a Natura 2000 (N2000) site (Habitats Directive) and it is under the management of the Collectivité de Corse (CdC). Major threats for this basin are the growing urbanisation of the north-eastern side and tourism during the summer period (e.g. beach restaurant).Arasu lagoon . The geology in the area of Santa Giulia lagoon is very densely fractured (79% granite), with many major clear regional lineaments. The lower watershed is covered by a thick sedimentary layer made of clayey sands that favours the infiltration of water and the rapid saturation of soils as well as the emergence of visibly flowing groundwater springs. The 15.5 km2 watershed provides freshwater inputs mainly at the southern end of the basin, near to the outlet of the lagoon, where small temporary creeks flow and are associated with a restricted alluvial aquifer, pumped through several wells and boreholes by local residents for agriculture and gardening purposes. The sea channel is also located in the southern part . The Balistra lagoon watershed is mostly granitic (93%) and very densely fractured and weathered, favouring the creation, in the immediate vicinity of the mouth of the Francolu river, of a restricted alluvial aquifer made of coarse sand. This configuration shows a similar hydrogeological structure to that of Santa Giulia, with the emergence of multiple isolated groundwater springs and high porosity, favouring continuous groundwater flow contribution to the rivers and thus to the downstream lagoons.The Balistra lagoon  and Cap Pertusato  weather stations were chosen , based on their proximity to the study sites  for various municipalities, depending on the lagoon considered. The municipalities taken into account are the following: Zonza and Lecci for the surroundings of Arasu lagoon, Porto-Vecchio and Bonifacio for the surroundings of Santa Giulia and Balistra lagoons, respectively. Data on principal and secondary residential homes were available from 1968 to 2018, with a 7-year frequency until 2006 and every 2 years afterwards. The CORINE land-cover map of catchments for 2018 and available previous years  were then defined  at the Hydrogeology Department of the University of Corsica .Surface water and groundwater were sampled to get an overview of the entire hydrological functioning of the three watersheds, with a focus on the interactions between surface water, groundwater and lagoons. Groundwater was sampled see Fig.  in five 4+) concentrations were determined through the fluorescence method , nitrate (NO3−), dissolved inorganic phosphorus (DIP) and silicates concentrations by the colorimetric method , May (spring), August (summer) and November (autumn). Seasonal subsurface (−0.2 m) water samples were hand-collected in the water basins at three sampling stations for Arasu lagoon and at two sampling stations for Santa Giulia and Balistra, between 7:30 and 10:30 a.m.  was used as the proxy of phytoplankton biomass. To determine its concentration, water subsamples (250 mL) were filtered on Whatman GF/F filters  and further stored at −20°C until spectrofluorometric analyses (Neveux and Lantoine ® CKX41). At least 400 cells were counted (estimation error within ± 10% limits) . In order to characterise community functional diversity, the smallest fraction of phytoplankton communities was also taken into account and analysed through flow cytometry. A total of 1.8 mL of water samples were fixed with glutaraldehyde  and with addition of Pluronic (Poloxamer 188) at a final concentration of 0.01%, as described in Marie et al.  and F0 is the intrinsic initial fluorescence under non-actinic light when all PSII centres of reaction are opened and potentially available for electron transport  on samples dark-adapted for at least 30 minutes. Samples were kept in a cooler between 10 and 15 °C and analysed between 2 and 6 h from field collection in order to respect recommended storage conditions for Phyto-PAM fluorimetric analyses  transformed to normalise data, reduce effects of extreme values and meet ANOVA conditions.All data were previously logp < 0.05), differences were further investigated through post-hoc Tukey’s test. For some variables, since normality was not achieved despite transformation, a Kruskal-Wallis (K-W) non-parametric test was applied and eventual significant differences were further tested through Dunn’s post-hoc test. Then, a global analysis was performed, with visualisation through Non-metric Multidimensional Scaling (NMDS) of abiotic parameters (water temperature (°C), salinity, turbidity (FNU), dissolved oxygen concentration (%), silicates (µM), DIP (µM), NO3− (µM), NO2− (µM) and NH4+ (µM)) and biotic variables (Chl a (µg L−1), Fv/Fm, Diatoms (cell L−1), Dinoflagellates (cell L−1), Cyanophyceae (cell L−1), Cryptophyceae (cell L−1), Chlorophyceae (cell L−1), Others (cell L−1), PE-cyan (cell L−1), PC-cyan (cell L−1), PEUK (cell L−1), NANO (cell L−1), species richness and Shannon’s Diversity Index) separately. To test potential effects of season, lagoon and stations, datasets were investigated through the permutational multivariate analysis of variance (PERMANOVA) test and significant differences (p < 0.05) were afterwards detailed through the pairwise post-hoc test.Spatial and seasonal differences within lagoons were assessed through analysis of variance (ANOVA) tests for each variable of interest. When significant  of the total lagoon surface area on the island, and among them, 68 cover less than 0.05 km2 . The Balistra lagoon watershed contains no urbanisation and only very little agricultural development. However, it showed the highest values of NO3− in groundwater, with occasional peaks of 837.1 and 1269.4 µM at two sampling sites upstream of the basin, far from the lagoon (3 and 7 km respectively), in hamlets without public sanitation .For all three lagoons, water temperatures followed the seasonal norms and ANOVA detected a strong effect of season .Salinity ranged between 2.8  and 49.9  in Arasu lagoon with lowest values systematically registered at St2 or St3, except for autumn 2021 , while highest mean values for NO2− and NO3− concentrations were registered in Balistra lagoon at 0.2 and 5.6 µM, respectively. At Arasu lagoon, ANOVA detected a moderate effect of the season for NH4+ , resulting in highest values in winter, compared to other seasons according to the Tukey test. In addition, effects of season and station were found for NO3−  and H = 0.236, n = 24, p = 0.031 (station)), indicating significant differences of winter from spring and summer seasons and highest values at St1 compared to the other stations . A similar result was found for silicate concentrations, except in Balistra lagoon  where they seemed to follow a seasonal decreasing gradient from winter to summer  and DIP  revealed significant differences in N inputs and consequent nutrients imbalance. Results showed an effect of season factor for Arasu and Balistra lagoons , but different trends for the two cases: for Arasu the ratio was higher in winter than other seasons, while for Balistra summer showed a lower value. To characterise the lagoons’ trophic status, nutrients ratios were compared to Redfield et al. ANOVA tests on the ratio between total dissolved inorganic nitrogen  and lagoon . All seasons were different from each other according to the pairwise test, while Santa Giulia was different from Arasu  and Balistra  lagoons, which did not differ from each other .In order to obtain an overall characterisation of the three ecosystems, a comprehensive analysis was performed on abiotic parameters datasets to detect potential differences between lagoons and eventually their seasonal and spatial patterns. The analysis highlighted significant effects of both the season and the lagoon factors. From NMDS visualisation, seasons resulted clustered Fig. a. Lagoona content, with a mean of 7.1 µg L−1 reaching a maximum of 17.2 µg L−1 in autumn 2020 at St2 , with highest contributions in summer and autumn compared to spring , indicating St1 differed from the others according to the Tukey test. In particular, St1 repeatedly showed higher diversity but lower photosynthetic efficiency of the community , signifying a difference of summer season  and lagoon  emerged from PERMANOVA, and according to the pairwise test all lagoons differed from each other. Conversely, no clear distinction could be highlighted for seasons, except summer, which differed from winter  and autumn  according to the pairwise test.Overall seasonal and spatial patterns between and within lagoons were investigated through a comprehensive analysis on overall biotic variables. Significant effects of both the season and the lagoon factors were detected. In the NMDS visualisation of biotic variables, season clusters were superposed Fig. b. Lagoon6 cell L−1 , Prorocentrum micans in spring and autumn 2021  and Protoperidinium sp. occurring occasionally  and Santa Giulia  lagoons. They were also positively correlated with Fv/Fm in Arasu  and Santa Giulia  lagoons together with Chl a . In Santa Giulia lagoon Shannon’s Diversity Index was also negatively correlated with Chl a , while Fv/Fm was negatively correlated with salinity , water temperature  and PC-cyan . Balistra lagoon showed a completely opposite mode of functioning, with a positive correlation between Chl a and Shannon’s Diversity Index , and between Fv/Fm and salinity , water temperature , PC-cyan  and PE-cyan .At lagoon scale, some interesting significant correlations emerged. Overall, Diatoms were not correlated with indexes of richness, diversity or photosynthetic activity, but Dinoflagellates were negatively correlated with Shannon’s Diversity Index both in Arasu , during the 1999–2021 period the resident population increased linearly by a 1.3% annual rate, with the year 2021 reaching an increase of 33% compared to 1999. Furthermore, over the same time span, the number of summer travellers increased by a 0.8% annual rate, with a peak in 2018 at 23% more travellers than the average over the entire period . This suggests that during summer the lagoons are probably subjected to higher nutrient inputs and hydric stress, due to increased water demand for usages  Below is the link to the electronic supplementary material.


Cicer reticulatum. The F5 interspecific lines were tested for agronomic and seed quality traits including reaction to ascochyta blight disease under field conditions at two locations in 2018. A subset of 195 lines were selected based on resistance to ascochyta blight and acceptable seed quality. These lines were evaluated for their performance under suboptimal conditions at Lucky Lake (2019 and 2020) and Moose Jaw (2019), Saskatchewan, Canada, and Yuma, Arizona, United States (2019–2020). The lines were grown and evaluated at two seeding dates, normal (SD1) and late (SD2) seeding dates, at each location and year. The same lines were genotyped using Cicer60K Axiom® SNP chip. The population structure was determined based on 35,431 informative SNPs using fastStructure, and the interspecific lines were clustered at a k-value of 15. Significant marker-trait associations were identified for seed yield from SD1 and SD2 seeding dates, and stress tolerance indices  using phenotypic values both from individual locations and combined analyses based on BLUP values. SNP marker Ca2_34600347 was significantly associated with yield from both the seeding dates. This and other SNP markers identified in this study may be useful for marker-assisted introgression of abiotic stress tolerance in chickpea.Chickpea is a cool season crop that is highly vulnerable to abiotic stresses such as heat and drought. High temperature during early flowering and pod development stages significantly reduces the crop yield. The wild relatives of chickpeas can be potential donors for the introgression of heat and drought tolerance into cultivated chickpeas for crop improvement. Initially, 600 interspecific lines were derived from crosses between two elite cultivars, CDC Leader (kabuli chickpea) and CDC Consul (desi chickpea), and 20 accessions of  Cicer arietinum L.) is the world’s second most important pulse crop after the common bean  and bread wheat (Triticum aestivum L.) cultivars by the reintroduction of alleles from wild emmer wheat (Crop wild relatives (CWR) preserve higher levels of genetic diversity as they have been challenged in natural environments for many years in comparison to domesticated cultivars. Hence, these wild relatives are crucial genetic resources used by plant breeders for crop improvement . CWR hasatizocoi . Anotherer wheat . More ster wheat  and wheaer wheat .Cicer reticulatum, which survives under suboptimal environment, is an important resource to improve stress tolerance in current chickpea cultivars  provides a higher resolution of marker-trait association than classical QTL analysis using bi-parental populations . Both GWultivars . LinkageC. arietinum) and 20 accessions of Cicer reticulatum were developed. The elite cultivars were CDC Leader (kabuli chickpea) and CDC Consul (desi chickpea). The design of this population is like a nested association mapping (NAM) design. Each of the 20 C. reticulatum accessions (tester lines) was crossed with CDC Leader and CDC Consul (founder lines). A single F1 seed from each cross was grown in the greenhouse in 2014 and selfed to produce the F2 seed. The F2 plants of each cross were advanced to F5 as single seed descents (SSDs). The seeds of each F5 line were bulked and used for the evaluation of their agronomic performance including their reaction to ascochyta blight disease and seed visual quality at two locations, Limerick and Lucky Lake, SK during the 2018 growing season. One hundred and ninety-five F7 interspecific lines with improved resistance to ascochyta blight and acceptable visual seed quality were selected and used in the current study. All the selected lines were derived from crosses with CDC Leader as a common parent.A chickpea interspecific population consisting of 600 lines derived from crosses between elite cultivars (7 interspecific lines were grown at Lucky Lake (2019 and 2020) and Moose Jaw (2019), Saskatchewan, Canada, and Yuma, Arizona, United States (2019–2020). The population was planted at two different seeding dates [normal (SD1) and late seeding (SD2)] at each location and year. The purpose of late seeding was to expose the plants to higher temperatures during flowering. The normal seeding date in Saskatchewan was in the second week of May  while the late seeding was planted around 2 weeks after the normal seeding  with three replications was used at each location and seeding date. Daily maximum and minimum temperatures and other meteorological data were recorded at each location. There were multiple occurrences of temperatures above 26°C–27°C in all the locations during flowering on late seeding dates (The 195 Fng dates .1STI). These have been reported as reproducible indices under severe stress conditions in chickpeas (Days to flowering (DTF) were recorded for each line on the plot basis when 50% of the plants had flowered. Days to maturity (DTM) was also documented for each line when 50% of the plants in each plot were matured. Plant height was measured from the ground level to the tip of the plants when the pods reached physiological maturity. Flower color was recorded based on visual observation during flowering. Seed weight was measured by weighing 1,000 seeds per line after harvest. Reaction to ascochyta blight of each line was recorded on a plot basis, using a mixed quantitative and qualitative 0–9 score scale as described by hickpeas . The folTOL = Yp–Ys .MP = (Yp + Ys)/2 .ATI = [(Yp–Ys)/(Y̅p/Y̅s)] × 100 .SSPI = [(Yp–Ys)/(2Y̅p)] × 100 .1STI = (Yp2/Y̅p2) × [(Yp + Ys)/Y̅p2)] (K)/Y̅p2)] .In the above formulas, Ys, Yp, Y̅s, and Y̅p represent seed yield in stress and non-stress conditions for each genotype, and mean seed yield in stress and non-stress conditions for all genotypes, respectively.2), variance components were calculated using the SAS PROC VARCOMP procedure  for each seeding date were conducted. ANOVA was done using PROC MIXED in which genotypes were considered as a fixed factor and years as a random factor. The LSMEANS statement was used to compute the average phenotypic score for each line. For a separate analysis of each year, ANOVA was done using the PROC MIXED procedure, in which the lines were considered as a fixed factor and replication was considered as a random factor. To estimate the broad sense heritability  at Eurofins, WI, United States. Individual plants from each line were grown under controlled conditions in phytotron chambers. Young leaf tissue was harvested from 2 to 3 weeks old plants. These leaf tissues were freeze dried using Labconco FreeZone 6-L Console Freeze Dry System and sent to Eurofins BioDiagnostics, WI, United States, for DNA extraction and genotyping.K) into which the interspecific population can be grouped, and their degree of admixtures, based on genotypic data of 35,431 SNPs. The value of K was determined based on the lowest prediction error, and the smallest number of iterations for convergence. For each line, the value of Q, which is the probability of belonging to one of the clusters was derived from the matrix of contributions. A shared allele index derived from the dissimilarity matrix estimated from the SNP genotypic data were used to construct an unweighted neighbor-joining tree  of each chromosome was calculated as the correlation between marker-pairs among the SNP markers of each chromosome. The calculated Pearson correlation coefficient [r] was used to calculate LD decay by Quantile regression  for all site years, indicating a change in the ranking of the genotypes for seed yield production due to stress.Late seeding (stress condition) lowered the yield in comparison to normal seeding (non-stress condition) in all site years, except Lucky Lake in 2020 where the difference in seed yield was marginal but significant  was positively correlated with MP and K1STI, but negatively correlated with TOL, ATI, and SSPI in all site years.Positive significant relationships were observed between the seed yield under non-stress conditions (Yp) and TOL, MP, ATI, SSPI, and KA total of 35,429 SNPs were used to determine the population structure. Of these SNPs, 32,228 were located on the eight chromosomes of chickpea and 3,101 SNPs were on scaffolds and are not located on any of the chromosomes. After filtering for a minimum allele frequency (MAF) of 0.05, 20,679 SNPs were used for association analysis.2) between marker pairs of each chromosome. The LD decay differed among the eight chromosomes. The r2 max,90, which is calculated as the maximum r2 in the 90th percentile of each chromosome, for chromosomes 1 to 8 is between 0.24 and 0.26. The physical distance in Mb at which LD of each chromosome has decayed to half of r2max,90 is 0.51, 0.15, 0.25, 0.21, 0.38, 0.23, 0.24, and 0.43 Mb, for chromosomes 1 to 8, respectively  was tested from 2 to 20. A k-value of 15 is best suited to describe the genetic structure of 195 chickpea interspecific lines along with the two parents CDC Consul and CDC Leader, and six CDC cultivars used as checks, CDC Orion, CDC Cory, ILC 533, ICC 4958, and ILC 3279. The probability of membership of individual lines in each cluster was estimated by admixture analysis  and generally produces fertile progeny because of good chromosome pairing (C. reticulatum to introgress stress tolerance in cultivated kabuli and desi chickpea (C. arietinum) germplasm. In this study, we developed progeny from interspecific crosses between adapted elite cultivars, CDC Leader and CDC Consul, and 20 C. reticulatum accessions. Further lines with improved response to ascochyta blight and acceptable seed quality were selected and used in this study. Interestingly, all these selected lines were derived from CDC Leader (kabuli chickpea) and 17 wild accessions. The design of our population is the same as the nested association mapping (NAM) design. Generally, the bi-parental population has a lack of mapping precision and low genetic diversity. Therefore, using NAM helps to capture additional genetic diversity and increase genetic recombination.The wild relatives of chickpea have been an invaluable source for improving elite chickpea germplasm through resistance to biotic stress . Moreoveolerance . C. retichickpea . It is k pairing . TherefoChickpea is a dry season food legume that is mostly grown on residual moisture after the rainy season. Toward the end of the growing season chickpeas often experience terminal drought stress. Moreover, if the sowing is delayed, the crop may deal with heat stress during the reproductive phase . Low moi1STI which shows that selection of the genotypes based on these indices will improve the yield under non-stress conditions. Some studies have also found a positive correlation between non-stress conditions and MP, K1STI  was used to understand the genetic basis of complex traits in chickpea  and heatIn the current study, we used the measurement of seed yield in heat stress conditions as an important criterion to identify heat stress tolerance in chickpea interspecific lines. Understanding the genetic basis of yield in heat stress conditions is important to develop heat stress tolerant high yielding chickpea varieties. Previous studies have identified quantitative trait loci (QTLs) associated with yield , outliniC. reticulatum accessions. The late seeding and exposure of this population to suboptimal growth conditions has significantly reduced the seed yield. The study characterized the variation of the interspecific population for its yield performance and yield-related indices under suboptimal conditions. Individual lines that were identified for superior performance in suboptimal conditions can be used as trait donors in breeding for stress tolerance. The trait-associated SNP markers identified can be used for marker-assisted selection in the breeding pipeline. Future detailed analysis of these SNPs would allow us to identify the genes underlying tolerance to abiotic stress, providing new alleles and molecular markers to use in chickpea crop improvement.Wild relatives are a source of novel alleles to improve chickpea adaptation to suboptimal environments. In the current study, we used an interspecific population derived from The original contributions presented in the study are included in the article/SK conducted all the experiments, analyzed phenotypic data, and wrote the manuscript. KG analyzed genotypic data, conducted association analysis, and wrote the manuscript. BT conceived, designed, and supervised the research and reviewed the manuscript. All authors contributed to the article and approved the submitted version.We acknowledge the financial support from the Saskatchewan Ministry of Agriculture through the Agricultural Development Fund.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.


To fully understand the histological, morphometrical and heamodynamic variations of different supratesticular artery regions, 20 mature and healthy Assaf rams were examined through ultrasound and morphological studies. The testicular artery images of the spermatic cord as shown by B-mode analysis indicated a tortuous pattern along its course toward the testis, although it tends to be less tortuous close to the inguinal ring. Doppler velocimetric values showed a progressive decline in flow velocity, in addition to pulsatility and vessel resistivity when entering the testis, where there were significant differences in the Doppler indices and velocities among the different regions. The peak systolic velocity, pulsatility index and resistive index were higher in the proximal supratesticular artery region, followed by middle and distal ones, while the end diastolic velocity was higher in the distal supratesticular region. The total arterial blood flow and total arterial blood flow rate reported a progressive and significant increase along the testicular cord until entering the testis. Histological examination revealed presence of vasa vasorum in the tunica adventitia, with their diameter is higher in the proximal supratesticular zone than middle and distal ones. Morphometrically, the thickness of the supratesticular artery wall showed a significant decline downward toward the testis; meanwhile, the outer arterial diameter and inner luminal diameter displayed a significant increase distally. The expression of alpha smooth muscle actin and vimentin was higher in the tunica media of the proximal supratesticular artery zone than in middle and distal ones. The testis consists of a high metabolic tissue rate and any reduction in the testicular blood flow will affect negatively in spermatogenesis and testicular morphology2.Sperm production and semen quality are affected mainly by testicular hemodynamics, which is the main route for nutrients, hormones, and oxygen supply3. Previous study was performed on the testicular artery of the Egyptian buffalo bull, and reported that the artery includes four parts during its course; abdominal, funicular, marginal, and parenchymal. The abdominal part extends from the origin of the testicular artery at its exit from the abdominal aorta till the internal opening of the inguinal canal, while the funicular part occupies the spermatic cord, which starts from the external opening of the inguinal canal till the proximal extremity of the testis. The marginal part of the testicular artery is the normal continuation of the funicular part, and is located on the epididymal margin of the testis and extends between proximal and distal extremities of the testis. The parenchymal part is formed of group of branches run through the texture of the testicular parenchyma towards the mediastinum testis4.Testicular artery is considered the main route for testicular blood supply. It originates directly from the ventral surface of the abdominal aorta, and its diameter is wider in the ram than other medium and small-sized animals5. Doppler is widely used in human medicine to evaluate testicular artery blood flow and pathological conditions, which are associated with blood flow alternation6. Recently, it has been used for assessment of testicular blood flow in domestic species as camels7, stallions8, bulls9, rams10, dogs11 and goats12. The abovementioned clarity of testicular artery is not used in most studies during characterizing the arterial regions, and it is believed that absence of anatomical description consistency and recent improvements in ultrasound equipment quality may clarify conflicting results detected between studies; specially regional differences in testicular artery hemodynamic parameters13.Color pulsed-wave Doppler and B-mode ultrasonography is a non-invasive tool, which was developed to allow the study of anatomical data of blood vessels and to evaluate functional data regarding vascular hemodynamics; including its presence or absence, direction and speed. It is the method of choice for assessing the vascularity of different organs, including the testes15. The supratesticular region is the most important region of the testicular artery, and it is frequently used in the evaluation of testicular blood flow in rams16.Recently, three regions in the testicular artery have been used frequently for evaluation of blood flow to the testis in routine clinical procedures and hemodynamic differences among these regions have been reported. These regions are defined as supratesticular (in the spermatic cord), marginal , and intratesticular regions17.Histologically, testicular artery is considered a muscular artery, where its wall consists of three layers or tunics. The tunica intima is formed of endothelium, thin layer of loose connective tissue and prominent internal elastic lamina. The tunica media is consisted mainly of smooth muscle cells, with elastic fibers, elastic lamellae and reticular fibers. The outermost tunica adventitia is constituted of collagen and elastic fibers, and contains a network of unmyelinated autonomic vasomotor nerve fibers18.Because of significant importance of the supratesticular region in evaluation of the testicular vasculature and its extension in the spermatic cord, this study aimed to investigate histological, ultrasonographic, and morphometrical characteristics of different parts of this region  in Assaf rams for further standardization of the measurement site of the routine clinical procedure for evaluation of testicular blood flow, since a few centimeters can affect greatly in the hemodynamic parametersA total of 20 mature and healthy Assaf rams (2–7 years old) with no history of any previous reproductive disorders were used during the breeding season. The rams were kept at Centro de Selección y Reproducción Animal (CENSYRA), León, Spain. The current study was performed in compliance with the recommendations in the ARRIVE guidelines according to the Guidelines of the European Union Council , following Spanish regulations  for the use of laboratory animals. All experimental protocols and procedures were approved by the institutional animal care and use committee at the University of León (Spain) (ETICA-ULE-034-2020). Scrotal circumference was measured just before ultrasonographic assessment. After slaughtering, testes and spermatic cords were directly taken by inguino scrotal incision and were washed by normal saline to remove blood. The testicular artery in the supratesticular region was dissected, and was subdivided into three parts; 1) proximal of testicular artery near the inguinal ring, 2) middle of testicular artery in the middle way of the spermatic cord, 3) distal of testicular artery at the distal end of the spermatic cord, proximal to the testis.The ultrasound equipment used in this study is an EXApad®  with two different probes: 5–10 MHz linear transducer (LR760V®) and 7.5–15 MHz linear transducer (LC1038V®). All ultrasound examinations were performed using the same operator to evade variation. The rams were restrained with an injection of tranquilization with Xylazine 20 mg . To avoid the presence of air spaces, the wool of the scrotum was shaved and washed with water, followed by application of alcohol. Finally, the transducer was covered with a plenteous amount of gel to favor ultrasonographic imaging.19, where Total Testicular Volume (TTV) is the sum of the both testicles volume. At least three separated images of supratesticular region of testicular artery were described in three locations; (1) near the inguinal ring , (2) in the mid-way of the spermatic cord (middle part), (3) At the distal part of the spermatic cord, proximal to the testis . The testicular artery diameter at each part was analyzed.This imaging modality was carried out to identify different anatomical structures of the testis and also to diagnose probable pathologic conditions. At least three separated longitudinal and transverse testicular images were recorded, and the length, width and thickness of the testis were measured using electronic calipers. The testis volume (TV) was calculated through the formula for measuring an oblate ellipsoid volume: 4/3π × A × B × CDoppler analysis was performed firstly by the B-mode and CFD modality to recognize the arterial vessel. Therefore, PWD was carried out to quantify the blood flow velocity inside the vessel. For distinguishing the artery from the vein using Doppler evaluation, the artery has a wave form on the spectral graph parallel with the arterial pulse, while the venous flow is almost stable, without a pulse. The used angle between the long longitudinal vessel axis and Doppler beam was between 30 and 40 in the blood flow direction. The gate of the Doppler was kept steady at 1 mm.20. Total arterial blood flow  and total arterial blood flow rate (TABF rate: TABF/TTV × 100) were also recorded for all studied animals as testicular perfusion indices. Three to five measurements were determined for each parameter in various locations over the testicular artery. The wave forms of the testicular artery are characterized as either resistive or non-resistive. Resistive wave forms are characterized by high variation degree between systolic and diastolic velocity of blood with high values of resistive index, in contrast to non-resistive wave forms. All ultrasound settings including brightness, gains, focus, and contrast were fixed, and used equally for all examinations in order to reduce recording variations.After spectral pattern appearance of the testicular artery, the Doppler velocites (cm/s) including End Diastolic Velocity (EDV), Peak Systolic Velocity (PSV), and the Time Average Maximum Velocity (TAMV), as well as Doppler indices including Pulsatility Index (PI): PSV-EDV/TAMV and Resistive Index (RI): PSV-EDV/PSV were studied21. Sections were then examined using an OPTIKA B-293 microscope, and digital images were acquired by OPTIKA C-B10 camera and OPTIKA PRO View software.For histological analysis, samples from different supratesticular zones of the supratesticular region of the testicular artery were washed with normal saline, and were fixed in Bouin's solution for 24 h. Fixed samples were then dehydrated through passing in an ethanol ascending series, and were cleaned in methyl benzoate and were embedded in paraffin wax. Sections of 5–8 µm thickness were cut and were stained with Harris hematoxylin and Eosin for general structure, Crossmon's trichrome for collagen and muscle fibers, and Wigert's Elastica for identification of elastic fibersParaffin sections from different supratesticular zones of the testicular artery in the supratesticular region were prepared for further immunohistochemical studies. After deparaffinization using xylene, sections were rehydrated in ethanol and were washed in distilled water. Specimens were heated in sodium citrate buffer  for 15 min in a microwave oven in order to increase epitope exposure. Following that, samples were cooled, and were washed with phosphate buffered saline before being blocked using 10% bovine serum albumin (BSA) at room temperature for 1 h. Sections were then incubated overnight at 4 °C using mouse monoclonal anti-human α-smooth muscle actin (α-SMA)  and mouse monoclonal anti-vimentin . Primary antibodies were visualized using an SABC Kit Elite and 0.05% 3, 3-diaminobenzidine tetrachloride (Sigma) in PBS. The slides were finally counterstained with hematoxylin and were mounted and covered with cover slips. 1% BSA was used instead of primary antibody to examine antibody specificity.Various morphometrical parameters of the testicular artery including thickness of the arterial wall and its different layers, outer arterial diameter, inner luminal diameter, number of smooth muscle cell rows of tunica media, and diameter of vasa vasorum were measured on images of light microscopy using ImageJ version 1.53 processing software.P < 0.05. Data were presented as mean ± SEM.Statistical analyses of the obtained data were performed using GraphPad Prism software version 8.4.0 . Moreover, one-way ANOVA with a Tukey’s post hoc test was used for comparing significance between studied parts. Differences were considered significant when B-mode analysis for testicular measurements was conducted in all studied animals. The summary of the descriptive statistics for both testicular and scrotal measurements are shown in Table . B-mode,The spermatic cord appeared as a non-echoic circular zone with a black colour, surrounded by whiter hyperechoic regions, and the colour Doppler of the pampiniform plexus was detected as large spots with varying degree of orange and blue colouration during scanning Fig. A–C.FigurThe diameter of the testicular artery revealed an increase from proximal to distal zones of the supratesticular region, where there was a significant raise in the diameter of distal and middle zones compared with proximal one, meanwhile the diameter values between middle and distal zones were not significant. The diameter of the proximal, middle and distal zones of supratesticular region of the testicular artery was 2.1 ± 0.1 mm, 2.5 ± 0.1 mm, and 2.6 ± 0.1 µm, respectively  was 0.9 ± 0.02, 0.6 ± 0.02, and 0.4 ± 0.03, in proximal, middle, and distal zones of supratesticular region of testicular artery, respectively. Furthermore, PI was 2.1 ± 0.1 in the proximal supratesticular zone, 1 ± 0.1 cm/s in the middle supratesticular zone, and 0.5 ± 0.04 cm/s in the distal supratesticular zone ; however, the ultrasound equipment couldn’t interpret the given Doppler waves because of the small-sized vessel and decreased blood flow velocity in this area. In this concern, the hemodynamic measurements of the testicular artery of the dog at the marginal zone were evaluated using Doppler ultrasonographyp > 0.05); and subsequently, the means of both testes were used for other performed analyses. This finding is in agreement with recent studies, where testes develop in a highly symmetrical manner in ram lambs19. Similar results were obtained in Barki ram25, Dorper ram19 and Egyptian fat-tailed rams10. Furthermore, the Doppler testicular artery indices were not significantly affected by body weight, age, and pulse rate25.As shown here, right and left testes of Assaf rams revealed no significant difference  are the main cell type of the tunica media. These cells regulate vascular tone and blood flow through dynamic cell contraction and relaxation. α-SMA is considered the first SMC marker to be expressed during embryogenesis, and at the latest is the most considerable protein in SMCs39. Furthermore, vimentin is also expressed in endothelial cells of blood vessels, and that make it a good marker of microvascular distribution, since endothelial cells of blood vessels of all sizes, from capillaries to arteries, are strongly reactive40. Noticeably, vimentin showed immunoreactivity in the endothelial layer in all studied supratesticular zones. At proximal supratesticular zone, tunica media showed higher vimentin expression than in middle and distal supratesticular zones.Vimentin is a master intermediate filament protein in smooth muscle cells and tissues. It regulates the actin cytoskeleton in the smooth muscleThe obtained morphological and hemodynamic data showed to a great extent that the middle and distal supratesticular zones are nearly similar, where there were no significant differences between the two zones in about 60% of the obtained data. However, there were huge significant differences between the proximal and distal supratesticular zones in all obtained data. According to these findings, it is proposed that the middle supratesticular zone can be used as the best one to assess the hemodynamic of testicular artery in regular clinical diagnosis.The findings of this study can be summarized as follows: (1) The arterial diameter as shown by B-mode ultrasonography and histological analyses was widest in the distal supratesticular zone of testicular artery, followed by the middle and proximal zones; (2) Doppler indices RI and PI recorded high values in proximal supratesticular zone of testicular artery; however, in middle and distal ones, these values were lower. PSV showed the highest value at the proximal supratesticular zone of testicular artery, meanwhile, EDV showed a progressive increase distally toward the testis; (3) Thicknesses of tunica intima, media and adventitia were highest in the proximal supratesticular zone and decreased distally; (4) Vasa vasorum diameter was highest in the proximal supratesticular zone of testicular artery and decreased downward toward the testis; (5) Middle supratesticular zone of testicular artery was recommended as a best site to assess the hemodynamics of testicular artery in regular clinical diagnosis; (6) Alpha smooth muscle actin (α-SMA) and vimentin showed higher expression in the tunica media of proximal supratesticular zone than in the middle and distal ones. Findings reported here are considered baseline information for differentiation with pathological affections of the supratesticular region of testicular artery in this species. Future electron microscopic studies on different zones of the testicular artery within the supratesticular region should be done.


Enterococcus faecium as a replacement for soybean meal on growth performance, apparent total tract digestibility, blood profile and gut microbiota of weaned pigs. Eighty piglets (weaned at 21 days) [(Landrace × Yorkshire) × Duroc] with average body weight of 6.52 ± 0.59 kg) were selected and assigned to 4 treatments/4 replicate pens (3 barrows and 2 gilts). The four diets  were formulated using fermented soybean meal to replace 0, 3, 6 and 9% of soybean meal, respectively. The trial lasted for 42 days phase 1, 2 and 3. Result showed that supplemental FSBM increased (P < 0.05) the body weight gain (BWG) of piglets at day 7, 21 and 42 and ADG at days 1–7, 8–21, 22–42 and 1–42, and ADFI at days 8–21, 22–42 and 1–42 and G: F at days 1–7, 8–21 and 1–42, and crude protein, dry matter, and gross energy digestibility at day 42, and lowered (P < 0.05) diarrhea at days 1–21 and 22–42. The concentration of glucose levels, WBC, RBC, and lymphocytes were increased while, concentration of BUN level in the serum was lowered in the FSBM treatment compared to the SBM group (P < 0.05). Microbiota sequencing found that FSBM supplementation increased the microbial Shannon, Simpsons and Chao indexs, (P < 0.05) and the abundances of the phylum Firmicutes, and genera prevotella, Lactobacillus, Lachnospiraceae and Lachnoclostridium (P < 0.05), lower in the abundances of the phylum bacteroidetes, Proteobacteria, genera Escherichia-Shigella, Clostridium sensu stricto1, Bacteroides and Parabacteroides (P < 0.05). Overall, FSBM replacing SBM improved the growth performance, apparent total tract digestibility, and blood profiles; perhaps via altering the faecal microbiota and its metabolites in weaned pigs. The present study provides theoretical support for applying FSBM at 6–9% to promote immune characteristics and regulate intestinal health in weaning piglets.This study investigates the effects of soybean meal fermented by  Weaning pigs are immediately required to undergo a change from high digestible sow’s milk to solid diets as well as complex protein4. Therefore, it is important to explore potential protein sources that should be added to diets to alleviate the weaning stress of piglets. The high cost, finite supply, and unstable variation of sources animal protein had become major reasons for limiting its supplement in diets on weaning pigs. Soybean meal (SBM) is an important protein feed ingredient in livestock diet, but variety of antinutritional factors (ANF) as well as β-conglycinin, glycinin and trypsin inhibitor, which would lead to immune responses, digestive disorders and negative effects on animal health5.In the swine husbandry, weaning produce gut system dysfunctions and results from inconstant impairment of the gut barrier function, oxidative stress and absorption as a result of poor growth, diarrhoea and other diseases6. Enterococcus faecium is a gram-positive gamma-hemolytic or non-hemolytic bacterium in the genus Enterococcus is a kind of facultative anaerobic lactobacillus that can colonize the gastrointestinal tract of humans and animals7. Enterococcus faecium bacterium feeds have shown positive effects in piglets intestinal microbiota, increasing immunoreactivity while reducing diarrhea8. The administration of living microbial preparations is one part of probiotic treatments. While, the another effective pathway is microbial fermentation of feed. Cheng et al.9 and Li et al.10 reported that bacterial fermentation could reduce content by ANFs and improving on nutritional quality and nutrient bioavailability. Fermented soybean meal (FSBM), a manufactured product mixed with solid SBM, liquid phases and the vaccinating the mixture with E. faecium11 could improve protein quality and reduce on ANFs levels of SBM with solid state fermentation12. It has been reported that FSBM has partial or total replacement of SBM to improve the growth performance and apparent tract total digestibility (ATTD) of crude protein and gross energy immune and antioxidant capacity in weaning pigs2. Jeong et al.13 found that compared with SBM, FSBM had improve the growth performance and the apparent ileal digestibility of crude protein, dry matter, and gross energy and amino acids in weaning piglets. Moreover, compared with SBM, FSBM showed greater concentration of crude protein and amino acid and reduce trypsin inhibitor and ANFs14.Probiotics are alternatives to in-feed antibiotics because they are successful in improving livestock production, efficiency and welfareEnterococcus faecium (E. faecium) toproduce a fermented soybean meal (FSBM) in weaning pigs has not been stated earlier and a probiotic-fermented SBM might show some different effects on piglets. The objective of the current study was to compare the effects of dietary SBM and FSBM on growth performance, ATTD, blood profile and gut microbiota of weaning pigs.The ideal situation would be to select feed probiotics that would improve soybean meal quality and enhance resistance to weaning stress by boosting the gut microbiota. Using https://arriveguidelines.org).The animal care and experimental procedures described in this experiment were conducted according to the Animal Welfare Committee guidelines and had the approval of the Ethics committee of Animal Resource and Science College of Dankook University . And the experiments were performed in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines   medium at 37 °C. Fermentation was initiated by soaking SBM in distilled water to a achieve 30% moisture content. Water-soaked SBM and inoculated with a 10% of Enterococcus faecium SLB130 to achieve 10Eighty [(Landrace × Yorkshire) × Duroc] crossed weaned piglets  were randomly selected and allocated into 4 diets with 4 pens replicates according to the average initial body weight and sex, and each pen has 5 piglets (3 barrows and 2 gilts). Four diets were formulated using FSBM to replace 0, 3, 6 and 9% of SBM, respectively. The trial lasted for 42 days (phase 1 (days 1–7), phase 2 (8–21) and phase 3 (days 22–42). Basal diets were formulated to meet the NRC (2012) requirements , average daily feed intake (ADFI) and gain to feed ratio (G:F = ADG/ADFI).17. The number of piglets with diarrhea per treatment was counted at the days 1–21 and 22–42 and the diarrhea rate was calculated with the following formulation:The piglets’ anuses were checked one by one at 09:00 and 17:00 daily during the experiment to observe and recorded any fecal contamination and redness method by Ma et al.18 procedures.To determine dry matter, crude protein, and gross energy digestibility, chromium oxide was added to the diet as an indigestible marker at 2 g/kg of the diet for7 day prior to fecal collection. Fecal samples were collected from 8 pigs randomly selected per treatment via rectal massage, and the sample was stored in a freezer at − 20 °C and was dried in a 65 °C for 72 h and the feed and fecal samples were grounded to passed through 1-mm sieve for the measurement of dry matter, crude protein, and gross energy of FSBM, SBM, diets and feces samples were determined following to the Association of Official Analytical Chemists19 The concentrations of lymphocyte counts, Red blood cells (RBC), White blood cell (WBC), Blood urea nitrogen (BUN), and glucose in serum were measured with an automatic biochemical analyzer . The serum creatinine concentration was determined using anAstra-8 analyzer .At 06:00 on days 21 and 42, two pigs for each replicate was randomly selected to collect blood from the jugular vein and subsequently centrifuged at 3,000 × g for 15 min at 4 °C to obtain the serum sample and was kept at − 80 °C until analysis by Muniyappan et al.20. Then the samples were grouped into microbiome taxonomic profile sets for further analyses. Relative abundance cut-offs at the phylum and genus levels were set to 0.1%. Charts depicting the results from the alpha-diversity and taxonomic analyses were generated using GraphPad Prism software version 9.0 .On day 42, fresh fecal samples were collected from six pigs for microbiota analysis. Genomic DNA of fecal samples was extracted by using a DNA Kit , according to manufacturer’s instructions. The quantity and quality of extracted genomic DNA were checked using a UV spectrophotometer . Amplification and sequencing of the V5–V6 hypervariable region of the 16S rRNA gene was performed using an Illumina MiSeq platform . We performed alpha-diversity and taxonomic analyses of the raw paired-end sequences using EZBioCloud pipelineP < 0.05 was considered as significant.All results are presented as mean ± standard deviation (SD). GraphPad 9.0 was used for figures. Statistical analyses were performed using IBM SPSS 20.0  and the differences between treatments were compared with one-way ANOVA followed by Dunnett’s multiple comparison procedure. For all tests, P < 0.05) during days 7, 21 and 42, 9% FSBM (P < 0.05) during days 7 and 22, (P < 0.01) during day 42 all showed significant increases. Compared with SBM treatments, piglets fed 3, 6 and 9% FSBM (P < 0.05) on the higher ADG, ADFI and G:F during days 1–7, 8–21, 22–42 and 1–42. Importantly, the diarrhea rate lower in FSBM treatments . (P < 0.05).As shown the Fig. P < 0.05) the diarrhea score on days 1–21 and 22–42  the ATTD of dry matter, crude protein, and gross energy compared to the SBM  dry matter was found in the piglets dietary supplemented with 6% FSBM than 3 and 9% FSBM. However, 9% FSBM diet showed elevated crude protein levels than piglets fed 3 and 6% FSBM.Dietary supplementation with 3, 6 and 9% FSBM increased (P < 0.05) the glucose levels, WBC, RBC, and lymphocytes and lowered (P < 0.05) the BUN level on days day 21 and 42. High (P < 0.05) glucose, RBC, WBC, Lymphocyte, and lower BUN compared with SBM was observed  on FSBM compared to the SBM  and lower the abundance of Bacteroidetes and Proteobacteria (P < 0.05). At the genus level, FSBM group increased Lactobacillus, prevotella, Lachnospiraceae and Lachnoclostridium (P < 0.05) in fecal microbiota (P < 0.05). Compared with SBM treatment, FSBM treatments lower in the abundance of Escherichia-Shigella, Clostridium sensu stricto1, Bacteroides and Parabacteroides (P < 0.05)  Fig. .Figure 64. Earlier studies, illustrated that dietary FSBM supplementation increased the growth performance on weaning pigs23 and broilers9. Zhu et al.24 reported a significantly in the increased growth performance in weaning piglets due to dietary FSBM supplementation. In current study, the BW, ADG, and ADFI were improved in weaning pigs supplemented with FSBM treatments. Trypsin inhibitor content and antigenic protein content was reduced to 0.33 and 20%, respectively. Stachyose and raffinose were completely absent in FSBM. Pigs fed with FSBM could improve nutrient digestibility that may explain an enhanced ADG in weaning pigs.Sources of plant protein like FSBM have been extensively used in the diets of weaning pigs to enhance growth and immune status26. These proteins are partially digested by proteases secreted by microorganisms responsible for the fermentation process28. Similarly, Jeong et al.11 showed that degradation of most antigenic proteins (glycinin and β-conglycinin) and protease inhibitors in SBM fermented by Enterococcus Faecium improved intestinal morphology and digestive enzyme activities in weaned pigs. Fermentation of soybeans also degrades its proteins and carbohydrates to the extent of low molecular weight and water-soluble compounds that facilitate their digestibility29. The combination of a better nutritional status and a reduced immunological challenge when pigs are fed FSBM, or FSB may help to prevent diarrhea after weaning. Previous study, we demonstrated that dietary FSBM inclusion reduce the diarrhea in weaning pigs30. In the current study, piglets fed FSBM decreased the diarrhea in weaning pigs. In addition, it has been proposed that some microorganisms present in fermented products can inhibit intestinal colonization of pathogens that causes diarrhea in pigs31. The reduction of diarrhea is of great importance in pig production as it decreases the predisposition of these animals to Escherichia coli infection and improves feed efficiency, especially in weaning pigs32.The positive effect of SBM substitution by FSBM on diarrhea reduction may be attributed to the degradation of the main antigenic soybean proteins such as α and α′ subunits of β-conglycinin and acidic subunits of glycinin and some antinutritional factors such as trypsin inhibitors33 and Muniyappan et al.15 who reported that FSBM inclusion increased apparent total tract of crude protein, dry matter, and gross energy in weaning pigs. The beneficial effects of FSBM on nutrient digestibility may be attributed to the effects of FSBM on gastrointestinal development and the concomitant increase in digestive enzyme secretion4. Similarly, Min et al.34 found that dietary FSBM did not have significant effect on the digestibility of dry matter, crude protein, and gross energy in weaning pigs. In contrast, the negative effects of crude protein, dry matter, and gross energy on nutrient digestibility were also observed in previous studies35. Similarly, Hossain et al.36 and Lan and Kim37 found that dietary inclusion of FSMB did not have a significant effect on the digestibility of crude protein, dry matter, and gross energy in weaning pigs. The inconsistent results may be related to many factors such as FSBM source, additional levels, and composition of the basal diet, with effects being more pronounced when moderate amounts of FSBM are added to SBM basal diets34.In the current study, supplementation of FSBM significantly increased digestibility of crude protein, dry matter, and gross energy which was consistent with Yan and Kim38. Creatinine exists a natural waste result arises from the muscles and is eliminated from the body through kidney. Pigs fed FSBM showed decreased diarrhea score and the glucose levels improved which was in accordance with earlier studies39. Intriguingly, serum blood urea nitrogen reduced in FSBM treatments compared with the control. This shown that the fermenting procedure change nitrogen distribution inside the feed40. The levels of white blood cells, lymphocytes, were also increased, in the current studies. Lymphocyte growth exists as a major phase during the immune reaction in an animal and a proliferative reaction is a specific antigen41. Gizzarelli et al.42 and Wang et al.43 reported that weaning pig immunity is lower on β-conglycinin is not adequate deactivated during fermentation. Our results found a higher immune resistance on piglets this directly corresponded with a decrease in glycinin (80.29%) and β-conglycinin (69.43%) on FSBM. Therefore, overall growth performance and blood profiles were in accordance with each other.The BUN concentration and producing are affected by way of protein catabolism, and its concentrating is negatively associated with digestibility of proteins and amino acids2. Generally, dietary nutrients are absorbed and digested in the foregut, later undigested food residues and endogenous are fermented by gut microbiota in the hindgut45. Fermentation of carbohydrates is enhanced to colonic cells due to the production of SCFA45, during fermentation of indigestible protein manufacture potentially metabolite toxicity inclusive of Ammonia, amines, phenols, and indoles that have harmful effects on intestinal health44. Alpha diversity can be utilized as an indicator of the functional resilience of ecological diversity of the intestinal microbiota, including species richness indices , and species diversity (Shannon and Simpson)46. In the current study, dietary inclusion with FSBM significantly increased the Shannon, Simpson, and Chao1 richness, which was in accordance with our previous findings in pigs that dietary inclusion of FSBM significant difference the Shannon, Simpson47. Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Tenericutes were the most pre-dominant bacteria phylum in the piglets48. In the current study, FSBM group shapes gut microbiota in weaning pigs, including lower in the abundances of the phylum Bacteroidetes, and Proteobacteria and the genera Escherichia-Shigella, Clostridium sensu stricto1, Bacteroides and Parabacteroides, and a higher in the abundances of the phylum Firmicutes and the genera Lactobacillus, prevotella, Lachnospiraceae and Lachnoclostridium. Bacteroidetes, Proteobacteria and Firmicutes as three major communities, are essential to growth performance and energy metabolism homeostasis50. Shin et al.51 found a lower in the abundance of the phylum Proteobacteria in the gut of healthy humans. Therefore, Litvak et al.52 reported that increase in Proteobacteria abundance have been associated with in humans with gut inflammation, colorectal cancer, irritable bowel syndrome and metabolic syndrome and could be bacterial signature of gut dysbiosis51. The abundance of the phylum Proteobacteria contains many possible opportunistic pathogens, as well as Campylobacter spp, Klebsiella spp,Escherichia, and Salmonella and its rise could be shown as a potential indicator of gut diseases. The Firmicutes abundance have been evidence to be positive relationship with energy and active transport, facilitated diffusion, endocytosis and passive diffusion, whereas improve in fecal Proteobacteria and Bacteroidetes is associated with inferior nutrient digestibility2. Therefore, higher abundances of the phylum Firmicutes along with lower abundances of the phylum Proteobacteria and Bacteroidetes might promote nutrient digestibility in weaning pigs. Eren et al.53 reported that the herbivores have an increase abundance of Lachnospiraceae than carnivores in animal. Vacca et al.54 reported that reduce abundance of Lachnospiraceae, while multiple sclerosis and ulcerative colitis patients. All participants of Lachnospiraceae are anaerobic, hemoorganotrophic and fermentative and could degrade non-starch polysaccharides and butyrate and acetic acid. Butyrate provide the major energy source for intestinal epithelial cell growth, increased intestinal protection mediated epithelial cells and favoring the suppression of inhibits inflammatory responses56. Stanley et al.57 showed that Lachnospiraceae is associated with improve growth performance in animals. The abundances of the genera Lachnoclostridium—butyric-acid-producing microbes that have been associated in the mitigation on intestinal inflammation- were better in the FSBM groups10. The Lactobacillus as a possibility probiotic, possess the opposition to pathogen, anti– inflammatory, antioxidant capacity, and capability to higher of fecal microbiota59. Zhu et al.48 reported an improved Lactobacilli and totality anaerobic bacteria counts in the gut microbiota weaning piglets due to dietary FSBM supplementation. In the current study, inclusion of FSBM treatments an increased in the abundances of the genera Lactobacillus were significantly increased RBC, WBC, Lymphocyte, glucose levels and decreased BUN. Lactobacillus is familiar to have a positive influence on the GIT, growth performance, and nutrient digestibility in pigs and regularly used as probiotics in livestock production61. Yan et al.62 reported that Lactobacillus could commonly increase growth performance and the GIT of animals by defense the intestine from pathogens and encourage efficient nutrient and energy extraction by the host. The genus Prevotella is saccharolytic and produce succinic and acetic acids as ending fermentation products63. Prevotella specialized in degrading fiber diet, which had also been correspondent with could improve intestinal immune and decrease diarrhea64. Wu et al.65 reported evidence indicated a close relationship among Prevotella and long period of time carbohydrates diets or carbohydrates from fiber-rich diets. It was also reported that a higher abundance of Prevotellaceae dominated in fecal microbiota of healthy piglets when compared to post-weaning diarrheic piglets 64. Feng et al.47 and Forsyth et al.66 reported a higher Prevotella abundance, the higher the mucin composite content, but bacterial toxins would reduce gut penetrability and the sensitivity of the region-specific gut to systemic exposure. Accordingly, higher in the Prevotella abundances in FSBM it could be helpful in improve on growth performance in weaning piglets. The relative abundance of Clostridium sensu stricto1 and bacteoides showed a positively correlated with the frequency of diarrhea. Clostridium is the main cause of diarrhoea in humans and is responsible for community-acquired out breaks67. Harlow et al.68 reported that Clostridium perfringens, Clostridium difficile and Salmonella spp. are the most common microbes associated with diarrhea. In our study, lower in the abundances of the genera Clostridium sensu stricto1 in the FSBM group, and was positively correlated with decreased diarrhoea. Another result of FSBM groups were a decrease Escherichia-Shigella, and lower Escherichia-Shigella was main participant of the decreased in Proteobacteria abundance compared to the SBM treatments. The genera Escherichia–Shigella comprises an opportunistically pathogenic bacterium. Sousa et al.69 and Gong et al.70 reported the genera Escherichia–Shigella could destroy the gut structure and must be pro-inflammatory actions through multiple pathways and like the secretion of virulence factors, consequent in the improved danger of infection and diarrhea in the host. Parabacteroides and Bacteroides, which occur at the initial phase of the lifetime, have been informed to generate gamma amino butyric acid, closely related to growth72. A higher abundance of the genera Bacteroides is commonly used in the events of colorectal cancer, functional gastrointestinal disorders and ulcerative colitis75. Therefore, the biological results found that appropriate inclusion of FSBM in diets can inhibit the gut pathogens  and enhance beneficial bacteria (such as Prevotella and Lactobacillus) and further enhance the immunity and health status of weaning piglets.Diets offer available substrates inclusive of protein carbohydrates for the gut microbiota and affect microbial structure and metabolism might encourage the performance and gut health of weaned pigsIn conclusion, FSBM replacing SBM improved the growth performance, apparent total tract digestibility, blood profiles, possibly via altering gut microbiota profile in weaned piglets. The present study provides theoretical support for applying FSBM at 6 to 9% promote blood profiles and regulate intestinal health in weaning piglets.


Negaprion acutidens (sicklefin lemon shark) is distributed in the Indo-Pacific and in close association with coral reefs. Under the protection of the Dongsha Atoll National Park, a small but well-established juvenile population of N. acutidens inhabiting coastal areas of Dongsha Island was recently observed to display site fidelity by using acoustic telemetry. This study was designed to reveal the fine scale genetic structure and relatedness within and among 5 juvenile shark cohorts inhabiting 3 sampling sites at Dongsha Island. A total 188 juveniles were caught and sampled between 2016 and 2017, and genotyped with twelve loci. They were assigned to 5 year cohorts (2013–2017) based on the body length and date they were caught, also assigned to 3 sites based on where they were caught. Among five cohorts, the percentage of unrelated pairs within a cohort is more than 62% in average, suggesting a potential high mortality during their early life stage. The results of Fst and assignment testing showed that there was no significant genetic structure between sites and cohorts indicating that there was no fine scale genetic structure, even though the juveniles possessed strong site fidelity. A small effective population size (Ne) was detected (Ne = 86.7) which indicates the presence of a potentially isolated and vulnerable population at Dongsha. These results provide the genetic diversity as a baseline for future management and conservation of N. acutidens in the South China Sea.  Information on abundance and distribution of most of the shark species is relatively scarce, with deficient data occurring in 45% of the species, according to red list assessments of the International Union for Conservation of Nature (IUCN)3. According to the report of Food and Agriculture Organization of the United Nations (FAO), global landing of chondrichthyans increased since 1950s, reaching a maximum in 2000  before declining. However, global landings data are likely to represent a gross underestimation of actual landings due to illegal fishing. The high value of shark fins has led to an increase in illegal shark finning, resulting in an increase in the number of discarded bodies which often go unreported4. In Southeast Asia, the shark catch records are often inadequate and incomplete5. Lam and Sadovy6 suggested all known shark fisheries in the South China Sea collapsed between the 1970s and the 1990s and of the 109 species historically present in the South China Sea, only 18 species were recorded in their fish market surveys between 2006 and 2008. Additionally, sharks observed in the markets are almost all subadults, which is a sign of overfishing and inappropriate management. Therefore, there is an urgent need to enhance conservation efforts and implement management plans for these rapidly declining species on either a regional or global scale.Sharks are the most endangered group of marine animals due to their vulnerability to fishing pressureNegaprion is comprised of two species, including Negaprion brevirostris (lemon shark) an inhabitant of shallow, inshore waters throughout the western Atlantic also occurring in eastern Pacific and eastern Atlantic and Negaprion acutidens (sicklefin lemon shark), which is found in similar habitats across the Indo-Pacific region. Both species are associated with reefs. The former species has been overexploited7 and the latter species is locally extinct in India and Thailand, endangered in Southeast Asia, and considered vulnerable throughout its range . Both species have shown strong site fidelity9. The lemon shark, N. brevirostris, is one of the most well-studied elasmobranch species in the world. The juvenile phase has been found to utilize shallow coastal environments (lagoons) as nursery habitat. Strong site fidelity has been revealed by genetic and acoustic telemetry in different regions of the western Atlantic11. Additionally, female lemon sharks exhibit natal philopatry to one of these sites 12. This understanding of the connectivity, behavior, and spatial ecology of elasmobranch specie is crucial for the development of effective conservation strategy and management plans.The genus N. acutidens), largely because of its overall scarcity due to recent population decline . This species has been extirpated in India, Thailand, and Bali (Indonesia) due to overexploitation, but it remains relatively abundant in Indian Ocean and coastal areas of Australia13. Schultz et al.14 conducted the first genetic study on sicklefin lemon shark in the region between western Australia to central Pacific showing a minor genetic structure which may be due to the isolation by oceanic distance in between, the juveniles exhibit site fidelity by a narrow home range, similar to the closely related lemon shark16. There are only a few records of the occurrence of sicklefin lemon shark in the South China Sea18. The Dongsha Island (20° 40′ 34.5″N 116° 49′ 31.7″ E) is a remote island, part of Dongsha Atoll which is located at the northern boundary of the South China Sea approximately 450 km from Taiwan. The island is 2.8-km long and 0.865-km wide with an embedded lagoon (64 ha). A recent survey of the fish composition in the embedded lagoon showed that juvenile N. acutidens are among the three most abundant fish taxa found in the lagoon18. Like other coastal sharks, sicklefin lemon sharks, use shallow coastal habitats such as seagrass beds and lagoons as nursery grounds, then gradually expand their territory as they grow older9. In addition, site fidelity has been observed in not only juveniles but also resident adults of N. acutidens19. Based on the results of long-term acoustic telemetry data for studying the movement of juveniles inhabit around Dongsha Island, juveniles inhabit different sites around the island  exhibited strong site fidelity (Chen unpublished data). These behaviors related to fidelity for specific breeding and nursery areas may result in genetic structure despite the high mobility of adults on a relatively large scale (100 s–1000 s km) 21. This aggregation of juvenile sharks indicate Dongsha Island may represent as an essential habitat for sicklefin lemon shark to reproduce or as nursing ground like other sharks23 and have disproportionate management implications for shark conservation24. In the present study, in order to reveal the spatial and temporal genetic structure of juvenile N. acutidens at Dongsha Island, we developed eight novel microsatellite loci in combination with four previously published microsatellite loci to determine the genotype of 188 juveniles collected during 2016 and 2017. This information was used to determine whether the site fidelity affects spatial genetic structure around the Island.In contrast, little is known about the movements, behavior, and reproductive biology of the sicklefin lemon shark  while we observed the caught juveniles with open or recently closed umbilical scar between April and May  from 2013 to 2017 to estimate the average total length of newborn pup  of juveniles Table . Additioup Table . Using tTL = Total length of captured juvenile, TL = Average total length of newborn pup, GR = Average growth rate of juvenile (cm/month).25. In total, eight novel microsatellite loci (Table O), expected (HE) heterozygosities and Fst were estimated using Microsatellite Analyzer (MSA) 26. An exact test on genotype counts was done to test for significant deviations from Hardy–Weinberg equilibrium by GENEPOP. MICRO-CHECKER, version 2.2.3 27 was used to check for the possible occurrence of null alleles and allelic dropout. STRUCTURE, version 2.2 29 was used to infer population structure and assign individuals to clusters based on microsatellite genotype. The software was run using the admixture model that assumes that all individuals are potentially of mixed ancestry, and assigns each individual to a designated population  with a partial probability. Ten independent runs, incorporating a burn-in of 1 000 000 Markov chain-Monte Carlo iterations followed by 1 000 000 replicates of data collection. For data set of location, we set K = 1 to 3 because we assigned 188 juveniles to 3 populations according to where they were caught . For cohort data sets, we set K = 1 to 5 because we assigned 188 juveniles  to 5 cohorts . Structure Harvester 0.6.9430 was used based on the ΔK obtained through each run to determine the most likely number of clusters for each data set. CLUMPP31 was used to summarize the results of replicates under the best K-value. To further understand the clustering patterns, genetic distance-based (Pairwise Nei genetic distance) principal coordinate analysis (PCoA) was carried out using GENALEX 6.532. Analysis of molecular variance (AMOVA) was further performed by separating 188 individuals into two groups based on the PCoA result with ‘poppr’ package33 in R.Genomic DNA was extracted from muscle tissue using commercial DNA extraction kits . Through the raw sequences from a paired-end Illumina Miseq sequencing run, 41,546 contiqs (> 500 bp) were obtained after QC and assembly. A total of 84 sequences with different microsatellite motifs were isolated and primers were designed by Primer 3 (version 0.4.0) including 78 dinucleotide, 2 complex-nucleotide, 2 tetra-nucleotide and 2 hexa-nucleotide repeats. all quality filtered and assembled reads were used for screening repeated motifs following the methods described in 34, computing 5000 iterations for each year cohort. The program calculates the maximum likelihood relationship between individual pairs. It determines which of the following yield the greatest likelihood: parent offspring (PO), full-sibling (FS), half sibling (HS) and unrelated (U) categories. Meanwhile, COLONY version 2.0.6.6 was also used with marker type error rate as 0.001 for each locus, both male and female were polygamy to determine whether the relationship of a pair of juveniles belongs to one of three possible candidate relationships, including full-sibling, half-sibling and non-sibling. In order to quantify the effective population size of the N. acutidens inhabiting the waters of Dongsha, we used NeEstimator v2.135 based on linkage disequilibrium (LD) for two allele frequency thresholds (0.02 and 0.05) and one without any constraints.Maximum likelihood estimates of pairwise relatedness coefficients and genealogical relationships were calculated with the software ML-RELATEAll field work was carried on in accordance with the relevant guidelines and regulations under Marine National Park Headquarters permit No. 1051000471 and No. 106000672. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at National Sun Yat-sen University and conducted the experiments following the IACUC guidelines. All methods are reported in accordance with ARRIVE guidelines.The observed heterozygosity and expected heterozygosity of each locus in each population were given in the Table The average juvenile growth rate was 0.868 cm/month, and the average total length of newborn pup was 67.47 cm. Ten juveniles were assigned to the 2013 cohort, 24 juveniles were assigned to 2014, 85 juveniles were assigned to 2015, 45 juveniles were assigned to 2016, and remaining 24 juveniles were assigned to 2017 Table .P > 0.05).Among them, we developed, characterized and applied 8 novel polymorphic microsatellite loci including seven dinucleotide loci and one tetra-nucleotide loci, with another 4 published loci Table  to genotP < 0.01, 13.4% of variance). The estimated effective population size (Ne) by the linkage disequilibrium method under the allele frequency thresholds of 0.02, 0.05 and no constraint were 86.7 (95% CI = 75.3–100.6), 87.9 (95% CI = 71.7–109.5) and 199.9 (95% CI = 170.2–239.1), respectively.The results of STRUCTURE showed that K = 2 has the highest delta K for both location and year cohort data sets. The bar charts represent the results of the assignment test while K = 2 Fig. . Both baP < 0.05, unpaired t-test). Among these cohorts, an average of 91% of the pairwise relationships were unrelated  relationship in each cohort. The first order genetic relationships  accounted for 2%, 10%, 13%, 11% and 10% of all pairwise relationships in year 2013, 2014, 2015, 2016 and 2017, respectively Fig. . In contN. acutidens has a relatively restricted home range at St. Joseph Atoll in the Indian Ocean9, northern Australia (< 1.9 km2) 36 and even smaller in western Australia16 (0.6 ± 0.04 km2). And fine spatial side fidelity was also detected between three sites  where juveniles were collected in the present study. The results of pairwise Fst test showed no genetic structure among these three sites which suggest that this behavior may not affect the fine scale genetic structure of juvenile N. acutidens inhabit in the water of Dongsha Island. Surprisingly, we observed a clear genetic partition based on the PCoA plot . Collection of tissue samples from adults could allow us to verify our findings by reconstructing the pedigree between adults and juveniles inhabiting the Dongsha. Additionally, further study using popup satellite tagging is needed to better understand adult movement in the Dongsha Atoll.Even previous studies showed that the juvenile N. acutidens is placentally viviparous, producing 1–13 (mean = 9.3) pups per litter39. With an extremely low first order genetic relationship found in each year cohort and high sampling intensity of the capture-recapture experiment (monthly), we suggest that the natural mortality rate of newborn sicklefin lemon sharks may be very high in Dongsha population, resulting in survival of only a few pups per litter to reach the juvenile stage instead of causing by incomplete sampling.Our results of relationship test also showed that an average of 91% and 62% of the pairwise relationships were unrelated by ML-RELATE and COLONY, respectively. This evidence may also suggest that we observed an outcross population in Dongsha Island. Previous study documented in French Polynesia showed two times higher first-order genetic relationship in Moorea than in Dongsha. Although the Moorea study focused on only 40 mature sharks and our study focused on juveniles only , we suggest the genetic network of different geographic populations may vary. Additionally, N. acutidens in the South China Sea with high juvenile abundance in the shallow habitats18, (Chen unpublished data) and it is located far from other potential habitats such as West Luzon coast (500 km), Spratly Islands (1160 km) and Paracel Islands (670 km). Although previous study has showed that N. acutidens could potentially travel up 300 km between Moorea and Bora Bora15. Unlike French Polynesia which contains many islands adjacent to each other to serve as stepping-stones to support their seasonal movement, Dongsha Island is relatively isolated. The migratory path to West Luzon coast and Spratly Islands crosses more than 500 km deep ocean basin without any island in between, therefore, the only possible migratory path may follow the continental shelf toward west to reach Hanan Island or Paracel Islands and the travel distance is more than 650 km. Under the protection of the Dongsha Atoll National Park, the nursery ground at Dongsha Island may have acted as the population source for other local populations and for the whole northern South China Sea area since 2013. It is important to improve understanding of the effective population size (Ne) of this putative source population for purposes of assessing extinction risk and improving conservation management of this threatened species42. Frankham et al.43 proposed threshold requirements of Ne ≥ 100 to avoid inbreeding depression and Ne ≥ 1000 to retained evolutionary potential. Considering these recommendations, the estimated Ne of N. acutidens  is insufficient for short-term and long-term health of this species, perhaps representing an isolated population at Dongsha Island44. However, the Ne estimated by microsatellite may lack accuracy and precision when derived from insufficient numbers of samples and loci45. Dudgeon and Ovenden46 provided guidelines for establishing the minimum numbers of samples (91 samples or 20% of the census population) at least 10 loci in order to obtain adequate precision and finite values for estimating Ne of the zebra shark, Stegostoma fasciatum. In the present study, we used 188 samples with 9 microsatellite loci, which should be enough to obtain a fairly robust Ne estimation under the threshold provided by46. Therefore, the Ne value of the present study suggest that the Dongsha Island population is relatively isolated with potential to suffer inbreeding depression in the future.The Dongsha Island is the only known nursery ground for N. acutidens in Dongsha. In present study, we have provided robust evidence that a well-established population of N. acutidens inhabits Dongsha waters, using the Dongsha Island as nursery ground. We found that the site fidelity observed in juveniles is not affecting the fine scale genetic structure, and no genetic structure was found among cohorts. Surprisingly, the principal components analysis showed that the population in Dongsha might be comprised of individuals derived from two breeding families. Additionally, the small Ne value that we obtained indicates the juvenile N. acutidens found in Dongsha may represent a small, isolated and outcross population. These results provide useful insights for future management and conservation of the sicklefin lemon shark in the South China Sea.In conclusion, the coupling of genetic analyses with long-term demographic experiments is crucial to improving our understanding of the movement patterns of the Supplementary Information.