P < 0.05); also, broilers that were fed diets containing 0.5 ppm aflatoxin had lower weight gain, carcass yield and breast yield (P < 0.05). The use of 10 000 mg/kg of binder improved (P < 0.05) feed conversion throughout the rearing period. We conclude that aflatoxin negatively affects performance and carcass yield; however, feeding optimum vitamin nutrition improved these performance traits.Vitamins play an essential role in broiler nutrition. They are fundamental for normal metabolic and physiological process, and their requirements for poultry are not fixed and can be affected by multiple factors. In contrast, mycotoxins are a challenging issue because they hinder performance and the immune system. Vitamin supplementation above minimum requirements would permit improvement in productive potential, health, bone and meat quality in a situation of mycotoxin challenge. The objective of this study was to determine the influence of optimum vitamin nutrition in diets contaminated with aflatoxin in broilers from 1 to 44 days of age. A total of 1800 Cobb 500 male chicks were randomized to 15 sets of eight treatment groups, each containing 15 birds using a 2 × 2 × 2 factorial design . The mash diets were corn and soybean meal based, formulated according to commercial practices. Feed intake, weight gain and feed conversion were analyzed for birds from 1 to 44 days of age. To determine carcass characteristics  and black bone syndrome, two birds were slaughtered from each group at 45 days. Other analyses included breast tenderness, water loss by dripping and malonaldehyde concentrations. The results demonstrated that broilers that were fed high levels of vitamins showed better weight gain, feed conversion, carcass yield and breast yield than broilers that were fed diets with commercial vitamin levels ( Dietary vitamin supplementation above the established minimum requirements improves health, performance, bone quality and the welfare of poultry. Optimizing vitamin supplies in broilers minimizes poor bone quality and problems relating to feed contamination with mycotoxin.In recent years, the poultry sector has undergone significant transformations, resulting in better production indexes and quality of products purchased by consumers. Improved nutrition has been one of the factors driving these transformations. There is a large amount of data showing the nutritional requirements for proteins, amino acids and minerals, such as calcium and phosphorus. However, few studies have evaluated broiler vitamin requirements.Vitamins are essential nutrients, involved in over 30 metabolic reactions at the cellular level Marks, . Among oHigher levels of vitamins in broiler diets compensate for changes in intake, bioavailability, issues that compromise the quality of the food and the level of stress. In general, a significant immune response occurs only when vitamins are supplemented at a level 10 times higher than those recommended by the NRC , or two et al.  is a condition that seems to be related to redness of the tissue adjacent to the bone, which can blacken during cooking or storage  in broiler diets from 1 to 44 days of age, with or without aflatoxin, and their impact on performance, carcass yield, meat quality traits and BBS incidence.The aim of this study was to analyze the levels of optimum vitamin nutrition .A total of 1800 one-day-old male Cobb 500 chicks were randomly allocated to 15 sets of eight different treatment groups of 15 birds, using a factorial 2 × 2 × 2 design .The birds were housed in a temperature-controlled 45 × 10 m barn, with negative pressure ventilation and cool-cells with insulated asbestos roofing. There were a total of 120 pens, measuring 1.0 × 1.2 m, and each pen was provided with nipple drinkers, one tube feeder and rice husk litter.ad libitum during the whole trial. The barn was heated using an automatic gas heating system, which was activated depending on the internal temperature. The initial temperature was 32°C, and it was gradually decreased according to the housing recommendations for the strain. The lighting system was set according to the Cobb broiler management guide.Mashed feed and water were provided Aspergillus parasiticus (NRRL 2999) in rice, according to a method developed by Shotweell et al. (The diets were provided in four stages: pre-starter (1 to 7 days), starter 8 to 21 days), grower (22 to 38 days) and finisher (39 to 45 days), formulated according to commercial practices  yield. Breast samples were taken to determine meat quality , and the tibias was used to verify the incidence of BBS.To determine tenderness, the breast muscles were placed in cooking parcels (Cryovac/CN-530) and cooked in a water-bath at 82°C. These were then stored for 24 h at 2°C. After cooling, the samples were cut hexagonally 2 × 1 × 1 cm, according to the method described by Froning and Uijtteenboogaart . The samet al., To determine water loss by dripping, the cuts were placed in polyethylene bags, which were labeled and sealed under atmospheric pressure and refrigerated for 48 h at 4°C. After this period, the cuts were weighed again (Bridi MDA) using the method described by Tarladgis et al.  test. For this purpose, a Minolta 410R colorimeter was placed on the proximal epiphysis of the growth plate. The tibias received a score based on the light parameters, and their appearance was scored as acceptable (no darkening present in the bone – L* > 40), intermediate (presence of slight darkening in the bone – L* between 40 and 35) and unacceptable (severe darkening in the bone – L* < 35) . In cases where the differences were significant, the means were compared using the Tukey test at 5% probability. For performance data, the statistical unit was the pen; and for meat quality, carcass yield and BBS incidence, the statistical unit was the animal.P < 0.05) between vitamin levels and aflatoxin, showing that OVN levels in broiler diets helped to improve weight gain, compared with the control. In addition, the inclusion of a binder in the diet significantly improved feed conversion (Table There was an interaction for weight gain (on Table .P < 0.05). The presence of aflatoxin decreased carcass and breast yield (P < 0.05). There was a relationship between vitamin levels and the presence or absence of aflatoxin with carcass yield (P < 0.05). Broilers that were fed diets without aflatoxin had better carcass yield than diets with aflatoxin, and the supplementation of vitamins at higher than recommended (OVN) levels improved carcass yield in broilers that were fed diets with or without aflatoxin. The analysis of interactions between vitamin levels and the presence or absence of aflatoxins demonstrated that broilers that were fed a control diet with aflatoxin had lower carcass yield. Although the birds that were fed a diet with mycotoxin had lower carcass yield, the use of OVN improved carcass yield compared with the birds that were fed a control diet without aflatoxins.Regarding carcass traits Table , the treP < 0.05). The analysis of breast quality showed that the treatments did not influence water loss by dripping (P > 0.05) (Table OVN treatment decreased the frequency of unacceptable and intermediate levels of BBS Table  and impr5) Table . The addet al., et al., The deficiency of one or more vitamins can cause metabolic disorders, leading to reduced productivity, slow growth and an increased incidence of diseases. Without vitamins, metabolic reactions slow and become ineffective . In addition, broilers that were fed vitamin-free premix diets 1 and 2 weeks prior to slaughter (42 days of age) had lower weight gain, although feed intake and feed conversion were not affected  and aflatoxin-free diets . The percentage of breast yield was higher in birds that were fed aflatoxin-free diets (31.87% with 0 ppm and 31.38% with 0.5 ppm).As expected, this study showed that aflatoxin contamination resulted in a drop in performance and carcass yield; however, higher levels of vitamins in the diet improved broiler performance and carcass yield even when birds were challenged by dietary aflatoxin.et al. . This result contradicts the findings of Giacomini et al. , who ana  even when aflatoxin is present in the diet, and it can effectively minimize the negative effects caused by mycotoxins in birds regardless of the addition of binders.


Subsequently, three blended nonwoven fabrics were selected to assess effects on fluid management properties including topsheet performance indices of rewet, strikethrough, and fluid handling (rate and efficiency of transport to the absorbent core). These materials aligned well with commercial topsheet fluid mechanics. Using the Leeds University Fabric Handle Evaluation System (LUFHES), the nonwovens were tested for total fabric hand. The results of the LUFHES measurements are discussed in light of fiber contributions. Fiber ratios were found to correlate well with improvement in softness, flexibility, and formability. This study provides insights that improves the understanding of the multifunctional properties accessible with greige cotton toward decisions valuable to selecting greige cotton as an environmentally friendly fiber for nonwoven topsheets.Greige cotton (GC) has attracted interest in recent years as an eco-friendly, functional fiber for use in nonwoven topsheet materials. GC imparts favorable fluid management and sensorial properties associated with urinary liquid transport and indices related to comfort in wearable incontinence nonwovens. Nonwoven GC has material surface polarity, an ambient moisture content, and a lipid/polysaccharide matrix that imparts positive fluid mechanic properties applicable to incontinence management topsheet materials. However, a better understanding of the connection between functionality and compositional aspects of molecular, mechanical, and material property relations is still required to employ structure/function relations beyond a priori design. Thus, this study focuses on the relation of key indices of material fluid and sensorial functions to nonwoven topsheet composition. Greige cotton, polypropylene, bleached cotton, and polyester fiber blends were hydroentangled at 60, 80, and 100 bar. Greige cotton polypropylene and bleached cotton were blended at ratios to balance surface polarity, whereas low percentages of polyester were added to confer whiteness properties. Electrokinetic and contact angle measurements were obtained for the hydroentangled nonwovens to assess surface polarity in light of material composition. Notably, materials demonstrated a relation of hydrophobicity to swelling as determined electrokinetically by Δζ, ζ For decades, most incontinence topsheet manufacturers have used polypropylene (PP), which is hydrophobic and less expensive than greige cotton. Petroleum-based PP has been the material of choice in the incontinence topsheet market, with approximately 50 million tons produced annually , compareThe advantageous properties of the cotton may be described in terms of the fiber morphology and molecular composition. The primary cell wall and cuticle of the natural greige cotton fiber contain 1–2% pectins and waxes, as shown in The primary purpose of the absorbent topsheet is to facilitate the inlet of liquid to the acquisition and distribution layers, with rapid strikethrough and minimal rewet, providing efficient uptake of urine to the absorbent core as was first documented for disposable diapers . The oveAnother important goal of topsheet development is fabric comfort at the skin/topsheet interface. Various modelling methods involving both human subjects and instrumentation have examined the fabric–skin interaction in terms of friction (tribology), shear, and pressure for both woven and nonwovens . CottendAn individual’s evaluation of a piece of fabric often employs objective and subjective criteria that involve personal opinion and preference . HoweverHowever, consumer preference for whiteness is accepted by textile manufacturers who tailor processes using oxidizing chemicals, optical brighteners, or bluing agents ,17. As mOver the past several decades, a few methods have been developed to quantify human touch perception. Tactile perception is a complex interplay of rubbing, squeezing, and stretching that involves a multidimensional analysis of the fabric. The conclusions from instrumental evaluation methods, such as the Kawabata Evaluation System for Fabrics (KES-F) and Fabric Assurance by Simple Testing (FAST), are not necessarily objective because of their dependency on statistical data , artificFurthermore, the Kawabata method examines fabric hand in a limited single dimensional approach, and the standard fabrics used are solely based on 100% woven wool fabrics. In a handle assessment method based on extraction ,21, a faAssessment of tactile comfort at the skin–fabric interface strives to improve the objective and subjective evaluations of fabric handle. The criteria for evaluating clothing comfort of woven and nonwoven fabrics has been reviewed , with thPreviously, research identified some of the fluid handling and fabric handle indices that are required for functional incontinence topsheets in hydroentangled greige cotton . Howeverhttp://www.tjbeall.com/natural-fibers-nonwoven/true-cotton). Blue polyester fibers were supplied by Palmetto Synthetics LLC of Kingstree, South Carolina. Bleached cotton fibers were produced from a bale of True Cotton™ that were bleached by Tintoria Piana US in Cartersville, Georgia. The polypropylene fibers were purchased from Fibervisions® of Duluth, Georgia. All fibers were received and used without further modifications.Greige cotton fibers (True Cotton™) were obtained from T. J. Beall of Greenwood, Mississippi. These cotton fibers were precleared using a proprietary process which removed 99.99% of all impurities  in a nonaqueous, mechanical process . The NP fabrics were converted into hydroentangled (HE) nonwoven fabrics on a 1 m wide Fleissner pilot-scale hydroentanglement system  running at a constant production speed of 5 m min−1. The hydroentanglement (HE) system utilized two pressure heads: one low pressure for fabric wet-out maintained at a constant pressure of 30 bar during fabric production; and the second is a high-pressure head maintained at 60, 80, and 100 bar during separate fabric production runs. The final bonding nozzle is suspended over a 24 mesh embossing drum to cast a 24 mesh pattern into the nonwoven material. Each strip on the bonding pressure heads consisted of 50 orifices per inch, with an orifice pore size of 120 µm. The water used for the HE fabric production was ambient temperature, which was approximately 25 °C. Following HE, the fabrics were fed directly through a gas-fired fabric-drying oven (Trützschler Nonwovens GmbH) at ~160–180 °C (depending on fiber melt points) and wound into rolls.The fiber blends were chute fed to a 40 inch-wide textile card fitted with 4 Cardmaster plates (Saco Lowell), followed by feeding the card web into a commercial crosslapper and needlepunch (NP) machine . The NP processing parameters were varied to achieve the target weights  using the 2° observer and the CIE standard illuminant C. L* represents lightness and can be measured independently of color hue. A decrease in lightness is associated with a decrease in fabric reflectance. Normal to the L* axis (lightness) are the +b* to −b* axis and the perpendicular +a* to −a* axis, where b* represents the color yellow (90°), −b* blue (270°), +a* red (360°), and −a* green (180°). Each recorded L*a*b* measurement was the average of three reflectance readings, obtained by rotating the samples after each measurement.The contact angle of a water drop on nonwoven fabrics was measured using a VCA Optima XE . Runs were performed with a 5 µL drop of distilled water syringed onto the fabric. The image of the drop was immediately captured and analyzed to yield a contact angle. Contact angles on twelve different areas were measured, and their average value was recorded.The determination of the ζ-potential was carried out with the Electro Kinetic Analyzer  using the Cylindrical Cell developed for the measurement of fibrous samples. When a fiber absorbs liquid and swells, the surface charges become farther separated, and the absolute value of its ζ-potential decreases. Two kinds of measurements were made on each sample: (1) swell tests to measure the rate and extent of fiber swelling (at a given pH) and (2) a pH titration in which the swelling is measured as a function of pH. All ζ-potential measurements were made in a 1 mM KCl electrolyte.γ and ε0 the dielectric constant and the vacuum permittivity, η the viscosity, and κ is the conductivity of the measuring fluid. Surface conductivity of the fibrous samples was not taken into account. pH titrations were performed over a pH range of 11.0 to 2.5 to ensure recording both the isoelectric point (IEP) and the plateau potential. In the electrokinetic apparatus, the streaming potential is measured, and the zeta potential determined from the Smoluchowski equation ,29:ζ =Fabric handle was evaluated using the Leeds University Fabric Handle Evaluation System (LUFHES). Six nonwoven fabric specimens, three in the cross direction (CD) and three in the machine direction (MD), were tested. Cyclic compression buckling of 30%, cyclic shear deformation of 5° twisting, and fabric–fabric self-friction properties of the six nonwoven fabrics were evaluated during the biaxial deformations of the fabrics. Six fabric handle descriptors and indices are defined based upon the energy consumed either to deform a fabric or to recover the deformation from the deformed state, as defined below.Fabric sponginess (SP): the extent to which a fabric spontaneously recovers from deformation when the external deformation force is withdrawn (elasticity). Fabric crispness (CR): the level of unrecoverable permanent deformation produced under external deformation force. Fabric flexibility (FL): the resistance to recovery of the original dimensions after deformation by an external force. Fabric stiffness (ST): the resistance to deformation by an external force. Fabric softness (SF): the combined effect of the resistance to deformation and the resistance to the recovery subject to an external force. The total fabric handle value (TFHV) is defined asThus, the greater the TFHV value, the greater amount of energy is required to deform the fabric and to recover from the fabric deformation. An additional fabric handle parameter, referred to as fabric smoothness (SN), is defined as the dynamic fabric-to-fabric friction coefficient, or the coefficient of dynamic friction between the fabric and itself. In addition, fabric formability (30b), which is defined to represent a fabric’s ability to be conformed to a 3D shape, could be calculated from the fabric compression buckling force measured in the tests.Liquid acquisition, strikethrough, and rewet tests were performed by Marketing Technology Service, Inc. Kalamazoo, Michigan. According to EDANA/INDA WSP 70.3 and WSP 70.7, the liquid strikethrough time involves determining the time taken for a known volume of test liquid (simulated urine) applied to the surface of a test piece of nonwoven topsheet, which is in contact with underlying standard absorbent pads to pass through the fabric. Rewet testing was performed according to EDANA/INDA WSP 80.10 and WSP 70.8. In this procedure, the wet back of synthetic urine through a prepared sample onto a filter paper is determined. After applying a defined volume of liquid upon the prepared sample (strikethrough time test), a simulated baby weight is applied onto the specimen at a predefined speed and dwell time. Using a specific filter paper and an electronic balance, the amount of liquid that transfers back through the specimen’s surface into the filter paper is determined. The liquid acquisition test evaluates in real time how effectively liquids penetrate into the absorbent core of the absorbent device at any flow rate. A 0.9% saline solution is applied to the specimen at a specific volume and flow rate. The dynamic change of the total volume of fluid acquisition and overflow liquids over the duration of the testing was obtained.At the outset of the study, ten nonwoven fabrics  were desGreater hydroentanglement pressures more effectively removed the yellow surface waxes on greige cotton fibers than lower hydroentanglement pressures. There is also a process-related reduction of L* (lightness), from a control high of 94.29 to a low in sample 10 (97% greige cotton) of 85.41 when hydroentanglement pressure was applied at 100 bar versus 60 bar, respectively as shown in It has been previously reported by Easson et al. that small percentages of blue fiber can give the appearance of increased whiteness using the principles of additive color mixing  without plateau, isoelectric point, Δζ, swell ratios, and rate of swelling (k) have been previously employed to characterize compatible material interfaces of topsheets (TS) and acquisition distribution layer (ADL) materials found in marketed incontinence products [Shown in products .plateau values for the ten blends range from −34 to −25 mv, and the influence of the hydrophobicity of polypropylene is evident, as the blends containing a larger percentage of polypropylene have the more negative zeta potential. This is consistent with the more hydrophobic contact angle values shown in As shown previously for cotton, the swelling of the material, as measured by the Δζ, is a function of the materials’ expansion within the ion shear plane during pH titration ,33,34. TContact angle testing confirmed a finding first reported by Sawhney et al. and identified in GC topsheet ,26, demoThe results of the LUFHES tactile descriptor valuation are shown in −5 J). By contrast, Fabric 7, containing the greatest proportion of greige cotton (90%), is relatively softer (SF = 3.6 × 10−3 J). Fabrics 6 and 8, in the third group, contain the smallest amounts of PP fiber (5% in the fabric 8) or contain a small proportion of blue polyester fibers (2%) and are even softer (SF = 3.1–3.2 × 10−3 J). However, Fabric 9, which contains both the smallest proportion of PP fibers (5%) and a small proportion of blue polyester fibers (0%–2%), is the softest fabric of the six tested (SF = 2.8 × 10−3 J).The two fabrics in the first group containing a greater proportion of PP fibers (about 24%–40%) are the least soft fabrics (SF = 4.0–4.1 × 10−3 J) and the greatest TFHV (TFHV = 6.8 × 10−3 J). On the other hand, fabric 9 (90% greige cotton) has the smallest sponginess (SP= 1.3 × 10−3 J) and TFHV (TFHV = 4.5 x 10-3 J). Fabrics 1, 4, and 7 have relatively great flexibility value (FL = 6.7–7.4 × 10−4 J) and, notably, do not contain bleached cotton. However, fabrics 6, 8, and 9 have relatively smaller flexibility value (FL = 4.7–5.0 × 10−4 J), and 9 has the greatest flexibility .Based on considerations of the relative contributions of greige cotton and polypropylene fabric, 4 has the greatest sponginess (SP = 2.1 × 10−4 J) and, in contrast, fabrics 6 and 9, which have similar values, have smaller crispiness (CR = 3.4–3.7 × 10−4 J). This is consistent with fabrics 6 and 9 containing 2% of the blue polyester fibers.The fabrics 1, 4, 7, and 8 have relatively greater crispiness . This is consistent with fabric 9 being the softest fabric and fabric 4 containing a greater proportion of PP fibers (24%), as they might link to the formation of surface ribs/grooves formed during hydroentanglement process; both of them might have formed a smoother surface during the hydroentanglement process. Fabrics 1 and 6 have medium smoothness  consistent with a greater proportion of PP fibers (24% and 12% respectively) and the presence of a small amount of polyester. Fabric 7 and 8 have the least fabric smoothness (SN = 0.968 and 0.996 respectively), and this is consistent with these fabrics containing the greatest proportion of greige cotton fibers (90%), and polyester fibers that are absent.2, respectively). Both fabrics contain polyester, and are principally composed of greige cotton fibers (86% for the fabric 6 and 93% for the fabric 9). Fabrics 4 and 7 have medium formability (FMR = 0.32 mm2), they contain a lower proportion of cotton fibers  in comparison with fabrics 6 and 9. Fabrics 1 and 8 have the smallest formability , and they are, coincidently, the fabrics containing smallest (60%) and greatest (90%) proportion of cotton fibers, respectively, without the presence of polyester.The fabrics 6 and 9 have the greatest formability , from a control high of 94.29, to a low in sample 10 (97% greige cotton) of 85.41, as hydroentanglement pressures decreased from 100 bar to 60 bar. This trend related to the effect of hydroentanglement pressure on yellowness is especially evident in samples which contain increasing percentages of greige cotton. Similarly, as the percentage of greige cotton increased, the b* measurement also increases.In 2017 Easson et al. reported that small percentages of blue fiber can give the appearance of increased whiteness using the principles of additive color mixing  without Considerations of material surface polarity are central to incontinence topsheet (TS) and acquisition distribution layer design (ADL) . The useplateau is the point where charge equilibrium is attained on the material surface. Zeta plateau (ζplateau) can be either positive or negative, depending on its surface chemistry. Zeta potential analysis is highly applicable to absorbent materials, and provides a tool useful in the analysis of fluid dynamics in aqueous systems based on electrochemical double layer model [The material zeta potential  is characterized through pH titration correlated to change in charge (millivolt increments), and ζer model .The electrochemical double layer model has parallel properties to the fluid transport that occurs at the solid–liquid interface of incontinence material layers, i.e., there is transport of fluid through a material with a certain surface polarity that results in the swelling of a polar, porous material. Thus, zeta potential decrease  is caused by swelling of the fibers and outward movement of the aqueous shear plane where ions are in contact with the outer Helmholtz ion plane on the fiber surface ,29. An i−1, and was more accelerated in materials containing higher levels of bleached cotton (greater than 3%). Rate of swelling is also mediated by the amphiphilic surface polarity of the material, i.e., a combination of bleached cotton, greige cotton, and polypropylene.The indication of a hydrophobic surface on greige cotton nonwovens, shown here, is consistent with incontinence topsheet functionality ,33. In aFabric handle analysis using the Leeds University Fabric Handle Evaluation System (LUFHES) method was limited to six samples representative of a range of surface polarity. As stated earlier, the greater the TFHV value derived in the LUFHES, the more energy that is required to deform the fabric and to recover the deformed fabric. Thus, a lower TFHV value is more desirable for a softer fabric. The results of the LUFHES tactile descriptor valuation are shown in Hydroentangled fabrics containing more greige cotton fibers and a small proportion of blue polyester fibers yield a softer fabric than those with higher PP. It is noticed that fabric 1, containing least greige cotton, is not the least soft fabric, especially in comparison with fabric 4. This is possibly due to the reason that polypropylene fibers have a relatively greater bending rigidity than cotton fibers, and might need greater energy in the hydroentanglement process to form fiber tanglement; thus, fabric 1, which has a higher proportion of PP fibers (40%), might have smaller hydroentanglement intensity (HI) (34b) than fabric 4, which has a smaller proportion of PP fibers (24%) in the level of the water jet pressure applied; smaller hydroentanglement intensity (HI) would lead to a relatively loose and bulky fabric, which should be relatively softer (34b). Previously, it was shown that a 50/50 greige cotton/polypropylene blend had a TFHV that is five times less than a commercial spunbond nonwoven, and this is predominantly due to higher softness and more sponginess, flexibility, and crispiness (18). A similar trend is also found in this study with the fabric stiffness (ST), sponginess (SP), and TFHV. For example, lower amounts of both polypropylene and bleached cotton tend to improve fabric THFV and flexibility when the fabric is predominantly composed of greige cotton.Fabric formability (FMR) represents a fabric’s ability to be conformed to a 3D shape. Thus, a greater FMR value represents the fabric’s greater ability to be conformed to a 3D shape. The relative order of formability observed in this study is consistent with the presence of blue polyester fibers in those fabrics. This suggests the potential to utilize polyester to enable greater formability. Moreover, in the absence of polyester fibers, it is possible that either too small (60%) or too high (90%) amounts of cotton fibers could lead to smaller fabric formability, and fabrics containing a suitable proportion (76–90%) of greige cotton fibers enable a relatively greater formability. This suggests that cuticle waxes from greige cotton may increase formability. This finding also suggests that more malleable hydrophobic materials, such as wax and polyester, may improve fabric formability.Optimal strikethrough, rewet, and fluid acquisition properties in an incontinence topsheet are essential for modulating levels of incontinent episodes in light, moderate, and heavy capacity categories . As showIn contrast with rewet functionality found in the topsheet, designing optimal strikethrough is largely dependent on accelerating both the wicking and retention capacity of the material. Strikethrough properties are, in part, optimized in commercial polypropylene samples by the application of surfactants  to promoOne of the most interesting results of the fluid handling experiments was the improvement in liquid acquisition observed over commercial spunbond topsheet performance in the multi-dose liquid acquisition test. Thus, in contrast with previous studies where 100 percent greige cotton was comparable to spunbond polypropylene topsheets, it is evident, here, that GC/PP blends improve liquid acquisition properties over commercial spunbond polypropylene topsheets.plateau, and contact angles greater than 90°. The influence of the hydrophobicity of polypropylene is evident as the blends containing a larger percentage of polypropylene have the more negative zeta potential. Seven of the ten nonwovens manufactured at 60 bar, with a ratio of polypropylene in the blend greater than 12%, had measurable contact angles greater than 90 degrees and are hydrophobic, which is consistent with incontinence topsheet functionality. The rate of swelling (k) was greater in nonwovens containing a greater amount of bleached cotton and a blend of amphiphilic fibers. Blue polyester fiber did improve fabric whiteness and factored in favorably in handle determination, but whiteness itself was not a factor in handle. On the other hand, improved liquid acquisition may work synergistically with improved formability, as observed with fluid management and fabric handle, respectively. The mechanics of fabric hand and fluid management functioning synergistically could improve incontinence material leakage and promote a more efficient retention of fluid in the absorbent core. The analyses of three nonwoven fabrics for rewet, strikethrough, and fluid handling aligned well with commercial topsheet fluid mechanics. Finally, this research provides valuable insights into nonwoven topsheet material selection, and provides a basis for better understanding of the fluid and hand dynamics that greige cotton imparts for incontinence applications.Greige cotton, polypropylene, bleached cotton, and polyester fibers, were blended in discrete ratios and examined for whiteness, tactile, and fluid handling properties for topsheet application. In Leeds University Fabric Handle Evaluation System (LUFHES) tactile analyses, nonwoven blends containing low percentages of polyester and polypropylene and high percentages of greige cotton and bleached cotton were shown to have the lowest total fabric handle value (TFHV). Fiber ratios demonstrated a relation of hydrophobicity to swelling, as determined electrokinetically by isoelectric point (Δζ, ζ


Progeny born to primiparous sows (gilt progeny) are born lighter, grow slower, and have higher rates of mortality than piglets born to multiparous sows. An understanding of why this might be the case is necessary to implement management strategies that improve the health and welfare of first-litter piglets and increase their production efficiency. Differences in the composition of colostrum and milk between primiparous and multiparous sows might be a contributing factor. Results from this study suggest that primiparous and multiparous sows (parities 3 and 4) have similar levels of immunoglobulin G and energetic components  in colostrum and milk throughout the course of lactation. However, lower lactose levels from day 14 onwards in milk from primiparous sows compared to milk from multiparous sows may indicate that there is a reduction of lactose uptake into the mammary gland in primiparous sows during late lactation, which may reduce milk volume. Lower growth rates and higher rates of mortality in gilt progeny are therefore likely to be as a result of lower colostrum and milk production in primiparous sows and (or) an inability of their piglets to digest these nutritional components effectively.p ≥ 0.05) parity differences in total IgG, fat, protein, lactose, and net energy (NE) concentrations. Primiparous sows had higher lactose levels at day 2  and lower NE at day 3 (p = 0.091), and multiparous sows had higher lactose levels at days 14 and 21. Results suggest that shortcomings of gilt progeny are unlikely due to insufficient nutrient levels in colostrum and milk, and more likely to reduced colostrum and milk intake and their capacity to digest and absorb each component.It is important to understand the biological factors influencing the poorer lifetime performance of gilt progeny in comparison to sow progeny and determine whether this may be partially due to differences in lactation performance between primiparous and multiparous sows. It was hypothesized that primiparous sows would have lower levels of immunoglobulin G (IgG) in colostrum and milk compared to multiparous sows, and lower levels of other energetic components. Differences in colostrum and milk composition between ten primiparous and ten multiparous sows (parities 3 and 4) from a commercial herd were examined throughout lactation . Overall, there were no ( Progeny born to primiparous sows (gilt progeny) are born lighter, grow slower, and have higher rates of mortality than progeny born to multiparous sows ,2,3. As This study investigated differences in total concentrations of immunoglobulin G (IgG), macronutrient levels , and net energy (NE) content in colostrum and milk between primiparous and multiparous sows (parities 2 and 3) from farrowing to day 21 of lactation. It was hypothesized that primiparous sows would have lower concentrations of IgG in colostrum and milk compared to multiparous sows, coupled with lower levels of macronutrients, resulting in lower overall NE content of their lacteal secretions compared to that of older sows.All experimental procedures carried out were approved in May 2016 by both the Rivalea  Animal Care and Ethics Committee (protocol number 16P030) and the Murdoch University Animal Ethics Committee (protocol number N2847/16) in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes .TM, Corowa NSW, Australia) were used. Sows were allocated to the study based on expected farrowing date, parity, and success in lactation. All sows were fed common gestation  and lactation  diets throughout the experiment. Sows were housed in groups of 40 during gestation , with primiparous sows housed separately to multiparous sows. Sows entered the farrowing room at approximately day 110 of gestation and were housed in farrowing crates fitted with drinker nipples for the dam and piglets, and a heat lamp in the creep area. Dams were spread over four separate farrowing rooms according to expected due date and farrowed over a period of 15 days. Piglets were fostered at least 24 h after farrowing according to commercial practices to standardize litters to match the number of productive teats and minimize within-litter weight variation. During lactation, sows were fed according to the commercial feeding regimen and given an allowance of 2.5 kg/d of lactation feed from entry to farrowing, 2.5 kg on day 0, 3 kg on day 1, up to 4 kg on day 2 and ad libitum access thereafter until weaning. Piglets were not given access to creep feed and were weaned at 26.5 (± 0.4) days of age.The experiment was conducted under commercial conditions at a large piggery in New South Wales, Australia , from May to July 2016. Ten F1 primiparous sows (parity 1) and 10 F1 multiparous sows  per parameter by dam parity (primiparous or multiparous) for colostrum and milk composition traits are given in A colostrum sample (approx. 5 mL) was collected from each sow at farrowing , day 1 (approx. 24 h later) and day 2 (approx. 48 h) after farrowing without the use of oxytocin. Milk samples were collected on days 3 (approx. 72 h), 7, 14 and 21 after farrowing following a 1 mL subcutaneous  injection of oxytocin . Colostrum and milk samples were pooled from as many teats as possible, collected throughout the letdown phase, and immediately frozen at −20 °C until further analysis. Several colostrum and milk samples were unable to be collected, or an insufficient amount was obtained for each subsequent laboratory analysis and therefore not all dams were represented at each timepoint in the results for all colostrum and milk composition parameters. Numbers of observations  in a sterile glass tube and briefly mixed. The suspension was then frozen at −20 °C for 1 h and subsequently transferred to small polystyrene tubes and spun at 13,000 × g for 15 min at 4 °C. The supernatant was then transferred to another sterile glass tube and left to reach room temperature. From this solution, 200 μL of each standard and sample was added to a 96 well UV compatible assay plate  in duplicate and read at 208 nm on a plate reader . A standard curve was generated from the absorbance readings for each standard dilution using online software  and totaColostrum and milk samples were spun at 21,000 × g for 40 min at 4 °C to remove fat prior to analysis for IgG, total protein, and total lactose. Concentration of IgG was determined using a commercial pig IgG ELISA kit . Total protein was determined using the Pierce BCA Protein Assay Kit  after dilution of standards and samples in 2% sodium dodecyl sulfate  to remove any interference from remaining lipids as per the procedures used by Geale . The intTotal NE of colostrum on days 0, 1, and 2 were calculated as adapted by Theil et al.  using thFarrowing data and colostrum and milk IgG concentration at each individual timepoint  were analyzed as linear mixed models with dam parity as a fixed factor. Colostrum and milk macronutrient composition data were analyzed as a repeated measures mixed model with dam parity, timepoint, and their interaction as fixed factors, with timepoint defined as a repeated measure with a heterogeneous autoregressive first order covariance structure. Parameter estimates were calculated using the method of restricted maximum likelihood (REML). All data analysis was carried out using the MIXED procedure of SPSS  with dam as the experimental unit. p ≥ 0.10) and the final model for each trait was therefore the simple factorial model. Pairwise comparisons were made between individual treatment means using the least significant difference (LSD) method, and comparisons between interaction means were made when the interaction was significant (p < 0.05) or a trend (p < 0.10) by simple effects analysis in SPSS syntax using the COMPARE function. Estimates stated herein are reported as mean ± standard error of the mean (SEM).Any extreme outliers were carefully considered for accuracy and either removed or kept in the analysis as appropriate , number of stillbirths , mummified fetuses  and number of piglets weaned . Primiparous sows had less piglets born alive (p = 0.031) than multiparous sows .One primiparous sow was removed from the experiment after the day 7 milk sample as her piglets were unthrifty and her udder was drying up; samples from this animal were excluded from the analysis. Primiparous and multiparous sows had similar gestation lengths  in concentration of IgG between primiparous and multiparous sow colostrum or milk at any timepoint (There was no difference (imepoint .p = 0.16). Protein decreased over the course of lactation (p < 0.001), and this trend was similar in both primiparous and multiparous sow lacteal secretions  parity by timepoint interaction effect on lactose content of colostrum and milk, increasing early in lactation for both primiparous and multiparous sows, and the decrease occurring earlier between days 7 and 21 in primiparous sows compared to between days 14 and 21 in multiparous sows (p ≥ 0.10) at days 0, 1, 3, and 7 between primiparous and multiparous sows, tended to be higher (p = 0.063) at day 2 in primiparous sows, and was higher at days 14 and 21  in multiparous sows. There was no main effect of parity (p = 0.21) on colostrum and milk lactose concentration (5.5 ± 0.1% for primiparous sows and 5.3 ± 0.1% for multiparous sows).There was a significant . Overall, fat content increased (p = 0.024) from farrowing until day 14 and decrease again by day 21 prior to weaning. Multiparous sow milk increased in total fat content at day 3 whereas this timepoint represented a drop in total fat in primiparous sow milk (p = 0.11). There was no main effect of parity on total fat concentration in colostrum and milk (p = 0.46) in NE content of colostrum and milk between primiparous and multiparous sows . Net energy content changed over time (p < 0.001) and followed a similar pattern to total fat (p = 0.091), with similar energy levels in primiparous and multiparous sow milk at all timepoints except day 3, where it was lower (p = 0.017) in primiparous sows compared to multiparous sows.There was no difference  compared to older sows (into the third week of lactation). It is, therefore, more likely that the underperformance of gilt progeny compared to sow progeny is attributable to other factors. This is probably a consequence of lower colostrum and milk production in primiparous sows compared to multiparous sows ,6,30,31,n = 20) in the current study. Regardless, the concentration of IgG in colostrum and milk decreased significantly over the course of lactation in the current study, in accordance with others [It has been assumed that primiparous sows may produce colostrum and milk with lower levels of immune factors than multiparous sows due to having had less time for exposure to pathogens over their lifetime and a relatively naïve immune system compared to older sows . Howeverh others ,37,47. Dh others ,47, and Gilt progeny have a lower concentration of serum IgG compared to sow progeny ,41,48,49Values obtained for lactose concentration in colostrum and milk in the current study, and the observation that lactose concentration increased over the course of lactation, are consistent with previous reports ,47. SimiIn support, several studies have found that milk yield increases with increasing parity ,30,31,63Fat is an important source of energy for the newborn piglet. Since these animals have limited body fat reserves at birth, there is little for them to mobilize in periods of fasting and therefore intake of fats through the diet is critical . Fat is Total NE in milk followed a similar pattern to fat content, which is not surprising given the high energy content of fat in the equation used to calculate this value . The curThe results from this study indicate that colostrum and milk composition throughout lactation does not differ considerably between primiparous sows and third and fourth parity sows in terms of IgG, total protein, fat, or total NE content. However, primiparous sows may not be able to meet the additional demands of a prolonged lactation, supported by a drop in lactose content in the late stages of lactation in primiparous sows compared to multiparous sows in the current study, and this requires further investigation. Consequently, it seems that reduced growth and higher mortality in gilt progeny compared to sow progeny may be more likely due to them having a lower intake of colostrum and milk, and (or) a relative inability to digest and absorb nutritional components from colostrum and milk during the pre-weaning period.


Plasmonic gold nanoparticles with sharp tips and vertices, such as gold bipyramids (AuBPs) and gold nanocubes (AuNCs), have been widely used for high-sensitivity localized surface plasmon resonance (LSPR) sensing. However, conventional LSPR sensors based on frequency shifts have a major disadvantage: the asymmetry and broadening of LSPR peaks because of instrumental, environmental, and chemical noises that limit the precise determination of shift positions. Herein, we demonstrated an alternative method to improve the efficiency of the sensors by focusing on homogeneous LSPR scattering inflection points (IFs) of single gold nanoparticles with a single resonant mode. In addition, we investigated the effect of the shape and vertices of AuNCs on the refractive index (RI) sensitivity of homogeneous LSPR IFs by comparing with gold nanospheres (AuNSs) of similar size. The results show that for both AuNCs and AuNSs, tracking homogeneous LSPR IFs allows for higher RI sensitivity than tracking the frequency shifts of the LSPR peaks. Furthermore, single AuNCs with vertices exhibited higher RI sensitivity than single AuNSs of similar size in the homogeneous LSPR IFs. Therefore, we provided a deeper insight into the RI sensitivity of homogeneous LSPR IFs of AuNCs with vertices for their use in LSPR-based biosensors. When gold nanoparticles are irradiated, the conduction electrons on their surfaces are excited and collectively oscillate with the incident electromagnetic field. Furthermore, the strong interaction between gold nanoparticles causes light to be confined into sub-diffraction volumes6.Plasmonic gold nanoparticles (AuNPs) have unique optical properties that depend on their shapes and sizes, and on the refractive index (RI) of the surrounding media. These properties are induced by the localized surface plasmon resonance (LSPR) effect7 and absorption-based photothermal imaging9, without ensemble averaging. It has been reported that the LSPR of AuNPs is strongly dependent on the three-dimensional (3D) structure and size of the nanoparticles10, as well as on the RI of the surrounding medium11. Accordingly, by controlling these parameters, it is possible to tune the characteristic plasmonic properties for specific purposes and applications13. Furthermore, the AuNPs have unique intrinsic properties11, such as biocompatibility14, high chemical stability15, convenient surface modification with organic and biological molecules17, etc. The many advantages of AuNPs has thus led to their use in LSPR-based biosensors19. The conventional LSPR biosensors are based on AuNPs functionalized with receptors that confer specific binding abilities for target molecules, then the LSPR peak is shifted and dampened upon the attachment of the target molecules on the nanoparticle surface20. Thus, the LSPR changes of AuNPs is monitored by the shift of the peak maximum as well as broadening of the peaks21; such changes indicate the presence of target molecules22.For many years, the optical properties of single AuNPs have been intensively investigated by far-field single particle imaging and spectroscopic techniques, such as scattering-based dark-field microscopy23. The accurate determination of LSPR properties is affected by a realistic representation of the wavelength-dependent dielectric function of the nanoparticles10. Therefore, simplistic models negatively impact the fundamental quantities that are necessary for the reliable fabrication of plasmonic devices24. Second, LSPR biosensors are limited by the unsymmetrical broadening of LSPR peaks when measuring the changes in the local environment at the nanoparticle surface25. It should also be noted that alterations in the shape of the LSPR peak can have a negative effect on the sensing efficiency26.Despite the remarkable advantages of LSPR-based biosensors, they still have many fundamental limitations. First, the efficiency of LSPR-based sensor using AuNPs is low in comparison with surface plasmon polariton (SPP) sensors23. Recently, Chen and co-workers reported a different approach that evaluates the changes in LSPR curvature of ensemble samples with respect to RI changes23. They showed that higher RI sensitivity was obtained in the inflection points (IFs) located at the long wavelength side (or low energy side) of the LSPR extinction peak25. However, that report was based on only ensemble samples of Au nanoparticles rather than single nanoparticles. Very recently, a single particle study on homogeneous LSPR IFs of single Au bipyramids was reported, however, our understanding of the effect of the NPs shape on the RI sensitivity at LSPR IFs of single Au nanoparticles is still scarce27.To overcome these limitations, recent studies have, for example, improved the effectiveness by using lithographic methods, but there are some disadvantages such as the high processing cost and low yieldIn this study, we carried out single particle studies to evaluate the shape-dependent RI sensitivity at LSPR IFs of homogeneous scattering spectra experimentally measured for gold nanospheres (AuNSs) and gold nanocubes (AuNCs), to compare structures with and without vertices. We investigated the LSPR sensing effect of single AuNSs and AuNCs deposited on a glass slide with three different surrounding media of known RI values . The results indicate that tracking the homogeneous LSPR IFs of AuNCs with vertices can be effectively used to develop LSPR-based biosensors with high RI sensitivity.The size and shape of AuNSs and AuNCs was characterized by SEM. Figure 28. The experimental setup for single particle DF microscopy and spectroscopy is shown in Fig. Scattering-based DF microscopy and spectroscopy was used to characterize the shape-dependent optical properties of AuNSs and AuNCs with vertices at the single particle level27. Fig. To better understand the shape- and environment-dependent characteristic optical properties, the effect of changing the surrounding medium RI on the LSPR wavelength was further investigated. Therefore, the scattering spectra of single AuNSs and AuNCs were obtained in three different RI environments: air, water, and oil. Figure 27. However, it is necessary to deepen our understanding on the RI sensitivity of homogeneous LSPR IFs of various Au nanoparticles with different shapes, such as multiple sharp branches, vertices, etc. We therefore investigated the shape-dependent RI sensitivity of LSPR IFs in the homogeneous scattering spectra of both AuNSs and AuNCs. The first and second derivatives of the scattering spectra taken from DF experiments were obtained using a convenient method based on the Lorentzian fitting curve function27. The first, second, and third rows in Fig. Homogeneous LSPR inflection points  have been reported to have a higher RI sensitivity than the LSPR wavelength maximum peak in single Au bipyramids with sharp tips25. Furthermore, the zero values of the first order derivatives axis are exactly at the point B (LSPR peak maxima), which is the point of symmetry for the three local RI media. When analyzing the curvatures, it was found that the LSPR scattering curves and second order derivatives are even functions and symmetrical to the axis of intensity, while the first order derivative curves are odd functions and are symmetrical to the axis of photon energy.As shown in Fig. 29, the linearity of the A, B, and C peak energies was examined for air, water, and oil. Figure −1 (R2 = 0.9398) for peak A, 0.133 eV·RIU−1 (R2 = 0.9983) for B, and 0.190 eV·RIU−1 (R2 = 0.9895) for C. It should be noted that the inflection point C exhibited the highest sensitivity with respect to A and the LSPR peak maxima (B), as shown in Fig. 27.As shown in the Supplementary Information Tables –S3, the Next, to better understand the shape-dependent RI sensitivity at the LSPR IFs, DF microscopy and spectroscopy experiments were performed for AuNCs with vertices. The RI sensitivity of LSPR IFs of AuNSs was compared with that of AuNCs with vertices. Both AuNSs and AuNCs of similar size showed a single broad LSPR peak at similar LSPR wavelengths; therefore, this investigation focuses on how the shape of the nanoparticles  affects the RI sensitivity at LSPR IFs at the single particle level.Similar to the analysis method used for AuNSs in Fig. vs. local air, water, and oil RI media. As presented in Fig. −1 (R2 = 0.9685) for peak A, 0.138 eV·RIU−1 (R2 = 0.9868) for B, and 0.206 eV·RIU−1 (R2 = 0.9998) for C. Similar to the experimental result of AuNSs, the inflection point C exhibited the highest sensitivity with respect to the IF A and the LSPR peaks maxima (B) as shown in Fig. Measurement of the LSPR  scattering spectra in multiples of 10 for each local RI index provided the same results, with values of 2.233 (±0.012), 2.194 (±0.017) and 2.153 (±0.013) eV for local air, water, and oil. Similarly, the LSPR IFs, A and C, were 2.136 (±0.011) and 2.328 (±0.017), 2.119 (±0.019) and 2.268 (±0.016), and 2.082 (±0.015) and 2.224 (±0.011) eV, respectively. The peak energy A, B, and C was plotted In summary, we demonstrated the significance of tracking the curvature shapes through homogeneous LSPR IFs near the resonance energy in various local RIs , rather than tracking their counterpart LSPR maximum peak shifts, for both AuNSs and AuNCs of similar size. The homogeneous LSPR scattering IFs of single gold nanoparticles  with a single resonant mode showed an enhanced RI sensitivity in various local RI environments. Furthermore, we found that single AuNCs with sharp vertices and edges showed higher RI sensitivity at homogeneous LSPR IFs than single AuNSs, with no edges, of similar size. Therefore, this study provides a deep insight into shape-dependent RI sensitivity of homogeneous LSPR IFs in single Au nanoparticles having a single resonant mode using DF single particle spectroscopy. Moreover, we showed that tracking the curvature changes in the LSPR scattering spectra of single AuNCs with vertices may be effectively employed in LSPR-based RI sensing studies.Cetyltrimethylammonium bromide (CTAB)-stabilized gold nanospheres (AuNSs) and gold nanocubes (AuNCs) with an average size of 50 nm were purchased from Nanopartz . Immersion oil was purchased from Sigma-Aldrich .The structural characterization of AuNSs and AuNCs was conducted by scanning electron microscopy  to assess the shapes and sizes. Furthermore, the LSPR absorption spectra of the AuNSs and AuNCs dispersed in water were measured using a Varian Carry 300 UV-Vis spectrophotometer .−2 to facilitate the measurement of a single particle without inter-particle LSPR coupling.The preparation of the samples was simple. First, the colloid solution was diluted with distilled water to lower the concentration. The diluted solution was sonicated for 10 min at room temperature and was then dropped on a washed slide glass and covered with a 22 mm × 22 mm No. 1.5 cover glass . To achieve the conditions of air as surrounding medium, the aqueous solution on the slide glass was dried after placing the cover glass. When using the oil as surrounding medium, the same procedure was followed and then, after drying the aqueous solution, the immersion oil was added. The concentration of Au nanoparticles deposited on the glass slide was adjusted to approximately 1 μmWe performed scattering-based dark-field (DF) microscopy using an inverted microscope . In the DF mode, we used a Nikon Plan Fluor oil iris objective (100×) with an adjustable numerical aperture  and a Nikon DF condenser for DF imaging. To obtain DF scattering images with high quality, we used an Andor EMCCD camera . We analyzed the collected DF images with the Image J software. Furthermore, single particle spectra of AuNSs and AuNCs were taken by using an Andor spectrometer  equipped with an Andor CCD camera . We collected the scattered light from AuNPs by an objective lens and sent to the entrance of the spectrometer for taking a spectrum. The scattered light was then dispersed by a grating (300 l/mm) inside the spectrometer, and detected by the Andor CCD camera . We obtained a background spectrum at an area without nanoparticles. Finally, Matlab programs specially designed for this study were used to perform data analysis and to obtain single particle spectra.Supplementary Information


The second population is composed of felids and felid products bearing a predominance of C3 signatures indicative of a more natural dietary regime. As with Copan deer, species-specific δ18O variations within these felid populations further substantiates the postulation that an expansive faunal trade network operated throughout the greater Copan Valley and beyond. Animals routed from sites of capture into the mesh of this network would have been processed into pelts, venison and other secondary goods or delivered alive to centers of state power for ritual usage and display. Our data reveal that at Copan, wild animals were routinely brought into intimate contact with human settlements to be managed and physically manipulated in a variety of ways in order to fulfill ritual and symbolic purposes.From Moctezuma’s zoo to animals kept in captivity at Teotihuacan, there is increasing evidence that Mesoamericans managed wild animals for a myriad of purposes. The present study situates ritualized animal management of highly symbolic fauna in the broader context of Classic Mesoamerica by examining another core site, the Maya center of Copan, Honduras (A.D. 426–822). In this study, we identify two animal populations among the faunal remains from public and private rituals spanning the Copan dynasty. One population, with diets heavily composed of atypically sourced C In way . Other fOdocoileus virginianus) were also interred in the cache. The first individual was represented by a skull identified as that of a male 1.5 to 2.5 years of age. Unfortunately, the deer postcranial bones  were not available for analysis to confirm if they were from the same individual. A pair of antlers inside a ceramic vessel represent a second adult male deer. The remains of crocodile scutes (identified by Randolph Widmer during excavations), also recorded on top of the capstones to the cist, were likewise unavailable for analysis . In addition, several other species of birds and turtles were identified, the majority found within the ceramic vessels. These included two turkeys, one of which was relatively complete. Given the species involved and the intentional placement inside the vessel, some of these remains were likely offered as prepared food .The puma was found complete with no pathological indicators of captivity nor cause of death, and was in relatively good health at the time of death at the age of 2–3 years. An MNI of two white-tailed deer  and wild (owl and deer) diets.th century A.D. in Plaza A of Group 9N-8. The animal accoutrements accompanying this individual identified him as a possible shaman . A recalculation, based mainly on dentition, determined that 16 felids were present, rather than 14 as previously argued . This caA few surface modifications including cutmarks, carnivore tooth marks, and rodent gnawing were recorded in addition to the differential weathering stages which indicate surface exposure . TogethePlatalea ajaja)  were also found. Excavation records also describe the remains of a macaw (Ara macao), although this was not available at time of analysis. The presence of the spoonbill, usually found in coastal habitats, is noteworthy given its non-local distribution. Both the macaw and the spoonbill were highly valued, as their elaborately colored plumage was often incorporated into ritual accoutrements.The remains of a roseate spoonbill  and mineral (carbonate) components. Bone and tooth dentine were analyzed to obtain simultaneous δ13Ccollagen and δ15Ncollagen. Tooth enamel, tooth dentine, and bone were sources for δ13Ccarbonate and δ18Ocarbonate data.Stable isotope analyses of bone and teeth were implemented to examine the diet and geographical origin of the animals. All isotope compositions are given in per mil (‰) units and standard delta notation is utilized for reported isotope values derived from the equation,13C, reflect variations in the relative inputs of different plant resources, distinguished by their photosynthetic pathways. Most trees, shrubs, and temperate grasses are signaled by Calvin cycle (C3) isotope ratios that range around -26.5‰. The Hatch-Slack Cycle (C4) is typically found in arid to sub-arid regions and ranges around -12.5‰. Values for Crassulacean Acid Metabolism (CAM), common to epiphytes and xerophytes living in hot and arid environments, span the C3 and C4 ranges but overlap more substantially with C4 values /590ht, indicate the splitting factor of the phosphate peaks [Degree of diagenesis in bone carbonate samples was assessed utilizing the Fourier-Transform Infrared Spectrometer with the attenuated total reflectance (FTIR-ATR) attachment at the Smithsonian Museum Conservation Institute. Two criteria were utilized: C/P ratios  and IR-SF  values. C/P ratios are constituted by the ratio of the carbonate and phosphate peaks at 1415 cmspectrum ,99. Modespectrum –100. IR-te peaks . Values The three sources of data  are distinguished in the n = 5/20, bone n = 11/34).Descriptive statistics of the entire assemblage are summarized in 13Ccollagen data exhibit a bimodal distribution that cannot be explained by chronological differences  and three felid samples from Altar Q (A.D. 776) have unnaturally high C4-based diets. In fact, their isotope values suggest the felids were consuming more C4 (maize) based resources (average -7.5‰) than the domesticated turkey (-9.0‰) found in the Motmot burial. Although less pronounced than δ15Ncollagen values, δ13Ccollagen do exhibit a roughly 1.2‰ trophic level effect within a single foodweb [Felid δferences . Two fel foodweb , which m13Ccollagen values are significantly lower (p < 0.001) than the rest of the felidae population. The isotope values sourced from the Altar Q and Motmot felids are comparable to those of primary burial felids identified at Teotihucan, with C4 signatures extending as high as -6.2‰ [Motmot and Altar Q felids likely represent instances of primary animal sacrifice. In contrast, the phalanges analyzed from Ruler 12’s burial chamber were most likely secondary by-products of pelts and these two δ15Ncollagen values . The spoonbill and the owl have the expected C3-based diets (δ13Ccollagen = -17.5 and -15.5‰ respectively) with enriched δ15N values (+10.0 and +8.1‰ respectively) correlated with natural resources. The riverine source of the crocodile’s diet could explain both the high δ15Ncollagen values and the presence of a mixed C3 and C4 diet, distinct from the terrestrial habitat, although a more robust sample size is necessary to form any conclusion.As expected, all felid populations have more enriched δn = 3/20, enamel n = 17/19, bone n = 16/34), providing a more robust sample   and δ4 is the assumed end member δ13C value for C4 goods; in this case most likely archaeological maize (-9‰). δb is the measured δ13Ccarbonate value of Copan samples, and Δ is the δ13Ccarbonate-diet spacing (9.7‰) [Out of the 73 samples processed for structural carbonate, 36 passed diagenesis testing accounting for 49% of the total (dentine  sample) . Schwarcg (9.7‰) ,105.4 intake for all carbonate samples was between 3 and 73%. The felid samples extended through this entire range, clustered into two distinct populations. According to a two-tailed t-test, the two specimens from Ruler 12 have a significantly low C4 intake (4), while the rest of the population from Motmot and Altar Q deposits was higher (4) (p < 0.001). Some individuals demonstrate an abnormally high reliance on C4 resources: δ13Ccarbonate values of three felids were more positive than a domestic turkey , suggesting this may be their protein source. These values are as high as some of the felids analyzed from Teotihuacan; a group of animals with clear pathological indicators of captivity  [Applied to the low and high values of the entire assemblage, % C4 resources seem to have played a role among the control deer carbonate samples (4). The owl also exhibits a somewhat elevated C4 intake , which may hint at the degree of landscape alteration caused by the introduction of C4 grasses into the area.Deer naturally congregate around milpa plots, not only to consume agricultural products, but also to lick the salts generated from slash-and-burn agricultural practices. Indeed, garden hunting is documented as an effective strategy among tropical groups . This ma4 intake was overall dietary contribution or protein specific (n = 11). The observations made in collagen and carbonate values are accentuated in this model. There is a strong association of felids from Ruler 12, closely aligned along the C3 protein line, closer to the 100% C3 diet range. Altar Q and Motmot felids, on the other hand, are clearly above the C4/marine protein line, closer to the limit of the 100% C4/marine diet range, at the opposite end of the spectrum. Given that the turkey is found in a very similar location along the C4/marine protein line as the latter, it is likely the felids were fed these domesticated animals. Deer, on the other hand, closely cluster near the two felid values from Ruler 12’s tomb, suggestive of a wild signature. As before, the owl displays an intermediate range.Based on the collagen and carbonate results, a simple carbon model was developed in order to determine whether the source of the high Cspecific . As this18Ocarbonate values  was 2.95‰ more enriched than its bone . While the limited deer sample size is insufficient to accurately document the local signature, this distribution is in agreement with other [While the inter-species differences in oxygen values cannot be used to directly plot movement of animals across the landscape, intra-species variation can signal unusual or abnormal relocation patterns. Deer had the largest variation in δth other ,24 oxyge18Ocarbonate variations observed among jaguars and puma samples are more challenging to interpret. As both jaguars and pumas have extensive home ranges, and are quite intolerant of territorial overlap  [18Ocarbonate variation is not surprising. However, 4‰ variation is high, with some samples clustering above one standard deviation from the mean (+25.2‰). This group consists of two claws from Ruler 12’s tomb (+27.0 and +26.0 ‰) and three specimens from the Motmot tomb and Altar Q felids . This group was statistically distinct from the rest of the felid population . Similarly, two individuals have values just below one standard deviation . Since the claws from Ruler 12 most likely originate from pelt remnants, it is reasonable to interpret that secondary faunal products were traded into Copan, as they exhibit higher δ18Ocarbonate values. Later codices describe such trade networks at play in Mesoamerica, which list important tribute items including jaguar pelts traded from the Maya region to the Aztecs [The δ-300km2) ,107, thee Aztecs .4 intake is indicative of captive behavior. The reservoir effect may explain the elevated δ18O values, caused by giving captive felids stored water in such reservoirs, or they may have been transported from distant regions. More conclusive evidence about where these felids originated would require additional analyses, such as strontium isotopes, to greatly enrich the sources of data available to investigators. Needless to say, the acquisition of these highly territorial and dangerous predators in the large quantities demanded by the commemoration event could have entailed a hazardous and difficult expedition to import them from afar.The three individuals that are above the range from Motmot and Altar Q assemblages must be interpreted more carefully, given that their extremely high C13Ccollagen, δ13Ccarbonate, and simple carbon models identified two distinct population clusters: 1) those fed on heavily C4 based diets, corresponding with the felids interred in Altar Q and Motmot deposits, and 2) felids and felid products—including some interred secondarily into the offering caches—with a predominance of C3 signatures indicative of a wild dietary pattern. Their δ18O values further substantiate the postulation that animals and their by-products were an important part of the expansive trade network established across the greater Copan Valley region and beyond, routing their carcasses from sites of capture to be processed into secondary products like pelts and venison and traded, or delivering them alive and intact to centers of state power for ritual usage and display.The isotope results demonstrate that an integrative approach to investigating and understanding the rich zooarchaeological assemblage at Copan significantly improved interpretive depth and nuance. Stable isotope analyses, in particular, exposed subtle changes in animal diet and mobility that zooarchaeological indicators alone did not detect. Unlike the Teotihuacan case study, there were no pathological indicators of captivity and the age distribution of the assemblage lacked the proportion of young individuals typical of a captive population. Yet the δThe implication of this find is clear: Mesoamerican cultures manipulated (kept in captivity and transported across the landscape) a wide variety of animals ranging from those domesticated for food to highly specialized carnivores used for ritual purposes, and this practice was much more extensive than previously demonstrated. Until recently, academic scholarship concerning animal management practices in Mesoamerica had remained relatively undeveloped. This may be a consequence, in part, of the unusual diagnostic challenges arising from the spectacular and disruptive transformation of Mesoamerican ecology and terrain instigated by the European biotic invasion (e.g. livestock) ,10, espeBased on multiple congruent streams of evidence from the Motmot and Altar Q caches, we propose that felid procurement and captivity were practiced throughout the history of the Copan dynasty (A.D. 426–820). Other fauna, such as deer from both ritual and midden contexts, revealed patterns of variation in their zooarcheological and isotopic markers indicative of animals being traded across the Mesoamerican landscape. The deer-crocodilian collar and related evidence furnished by the shaman burial vividly depict elite usage of animal craft products to construct direct links to creatures from mythological realms usually restricted to royal personages . Some ofThis Classic period Mesoamerican site readily invites comparison with Teotihuacan. As with its mighty northern counterpart, excavation and analysis at Copan continues to yield a profusion of evidence telling the tale of a society deeply and intimately acquainted with the uses, benefits, and behaviors of the animals inhabiting their settlement as well as from beyond their local domain. Animals fulfilled a myriad of roles and functions at all social strata, and were especially significant components of Copan ritual practice and identity construction.Further research into human-animal interactions at Copan, particularly when conducted using an integrated zooarchaeological and isotopic analysis methodology, provides an extraordinarily rich seam to mine for both broadly applicable field datasets as well as for the more focused archaeological insights uniquely expressed by this universal and fundamental feature common to all Mesoamerican lifeways.S1 TableYellow cells indicate samples that were dropped based on the diagenesis tests, d = dentine, e = enamel.(XLSX)Click here for additional data file.


Elaeis guineensis (Eg) is suffering severely from bud rod disease, restricting the potential cultivation areas. Therefore, breeding companies have started to work with interspecific Elaeis oleifera × Eg (Eo × Eg) hybrids which are tolerant to this disease. We performed association studies between candidate gene (CG) single nucleotide polymorphisms (SNP) and six production and 19 oil quality traits in 198 accessions of interspecific oil palm hybrids from five different origins. For this purpose, barcoded amplicons of initially 167 CG were produced from each genotype and sequenced with Ion Torrent. After sequence cleaning 115 SNP remained targeting 62 CG. The influence of the origins on the different traits was analyzed and a genetic diversity study was performed. Two generalized linear models (GLM) with principle component analysis (PCA) or structure (Q) matrixes as covariates and two mixed linear models (MLM) which included in addition a Kinship (K) matrix were applied for association mapping using GAPIT. False discovery rate (FDR) multiple testing corrections were applied in order to avoid Type I errors. However, with FDR adjusted p values no significant associations between SNP and traits were detected. If using unadjusted p values below 0.05, seven of the studied CG showed potential associations with production traits, while 23 CG may influence different quality traits. Under these conditions the current approach and the detected candidate genes could be exploited for selecting genotypes with superior CG alleles in Marker Assisted Selection systems.Oil palm production is gaining importance in Central and South America. However, the main species  East-Asian countries address most of the oil palm production. Actually, Indonesia, Malaysia, and Thailand together produce almost 90% of the palm oil worldwide. Latin-American countries have started climbing positions in production few years ago, since Asian countries suffer lack of space due to increased oil demand and restricted cultivation areas . ColombiElaeis guineensis (Eg) is suffering from bud rot disease “Pudrición de Cogollo” in these countries [Elaeis oleifera and Eg (Eo × Eg) [2 100 g−1, Eg palms showed 52 g I2 100 g–1 and Eo palms an average of 77.4 g I2 100 g−1. However, the main oil palm species ountries ,5 leadinEo × Eg) . These hEo × Eg) ,8. CadenEo × Eg)  reportedElaeis species. Therefore, in the current study a broader collection of Eo × Eg hybrids was analyzed for different traits, divided in two big groups; production and quality traits. Production traits cover agronomic performance in terms of bunch number, bunch weight, and bunch yield and the oil contents in mesocarp and bunch. The analyzed oil quality traits considered different components of lipids and tocols, as well as carotenoids. Even though these last two represent only minor components, they are of nutritional importance [Many breeding and seed companies have started breeding programs to get elite hybrid palms. Marker-assisted selection has emerged as a useful technology for this purpose, particularly for traits controlled by multiple genes, such as those related to oil quality and oil quantity. However, until now only a few studies have been published on this topic. Montoya et al.  identifiportance . The quap < 0.001 as well as 16 quality traits. The SSS triglyceride (SSS), Delta compound (Delta), and Gamma compound (Gamma) traits did not reveal significant differences between origins.Saphiro–Wilk tests revealed 16 traits which were not normally distributed. They are marked with “*” in The results of the Tukey post hoc tests are presented in Three separate amplicon libraries were constructed with a total of 167 candidate genes. The first library was constructed from 56 candidate genes and yielded over 13.9 million raw reads. The second library from 55 CG produced around 9.2 million raw reads and the third library from 56 CG generated around 9.6 million raw reads. This total number of 32.7 million reads was reduced to 9.8 million clean reads after the filtering steps. Approximately 83% of the reads mapped to the Eg reference genome. The Snakemake-capture workflow identified initially 12,200 potential SNP. However, after the mentioned filtering steps, only 115 potential SNP remained for the following analyses. The average observed (Ho) and expected heterozygosity (He) were 0.61 and 0.37, respectively. Bartlett’s test revealed a significant difference between expected and observed heterozygosity. The fixation indices (Fst) values revealed no discriminant differentiation between populations as can be seen in The remaining 115 SNP belong to 62 of the 167 initial CG used in the study and four of them showed multi locus mapping at two loci. SNP numbers for each candidate gene varied between one and four. The remaining CG are shown in p values of each model were drawn as a Quantile-Quantile (QQ) plot for each trait. 2) of the CG data points from the diagonal of the QQ plot was applied for determining the best fitting model for each trait, even though in several cases the differences in the values for alternative models are very small. The results are shown in After running Association Mapping using GAPIT, expected and observed p values < 0.05 between CG SNP and traits are displayed, as well as the genome location of the significant SNP, the applied model, the significance level of the association, the explained variance, and the effect of the marker. The significant SNP which belong to a particular CG were grouped.SNP belonging to a total of seven CG influenced significantly six production traits. Three CG revealed significant effects on two different production traits, while the other four CG influenced only one trait each, leading to a total of 10 significant associations for production traits. The BW trait was influenced by three different CG, OildM and OilB by two CG and BN, BY, and OilfM by only one CG. The explained variances by the model ranged from 8.9% to over 26% for the different CG. p values. Alph3 did not show any association with any of the studied CG SNP. The explained variances by the models ranged from 6.1% to over 28% of the total variance. For nine CG more than one SNP showed associations with different traits. Poly-Un showed associations with six of the studied CG and the Car trait revealed associations with five CG. Four potential associations were observed for the Delta, Delta3 compound (Delta3), Mono-Un, OA, SSS, and Toc traits and three associations for Alpha compound (Alpha), Gamma3 compound (Gamma3), IV, Toc3, Tocph, and UUU. SUU revealed two potential associations and Gamma, Sat and SUS showed only one potential association. It is also worth to notice, that five of the CG—EgNAC, PKP-ALPHA, SEQUI, LIPOIC, and TO1—showed also potential effects on different production traits. However, considering FDR adjusted p values, all detected associations are not significant anymore.For quality traits SNP belonging to a total of 23 CG showed potential significant associations with 18 out of the 19 quality traits using unadjusted Eg had higher CO2 fixation capacities, which are positively correlated with an increase in oil contents [guineensis palms. In our study we also found higher bunch weights (BW) in all origins involving Taisha and a higher bunch yield (BY) in the Taisha × Avros (Oleoflores) accessions. However, Arias et al. [The analyses of production traits revealed larger differences between Coarí × La Mé genotypes and the other four origins where Taisha was involved. The Coarí × La Mé origin presented on average a higher oil to bunch (OilB) percentage and higher oil percentages in fresh and dry mesocarp . Peláez et al.  observedcontents . On the contents  as “Oleis et al.  studied s et al.  are loweConsidering analyses of quality traits, some studies are available from Montoya et al. , Singh eWe performed also a detailed study for tocols contents which are composed of tocotrienols and tocopherols. These components represent different forms of vitamin E and can be found in oil palm as beneficial phytonutrients . Both, tWe used the Ion Torrent Personal Genome Machine (PGM) sequencing platform for convenience, based on previous experiences in other studies and ease of access. Similar studies using the PGM platform were also performed by other authors ,26.Eg var. pisifera genome sequence as reference. The decision to use this genome relied on the fact that actually no reference genome exists for Eo even though Singh et al. [Elaeis species could be used as reference for mapping the sequence reads. Mapping of the sequenced reads were performed using the published h et al.  publisheh et al.  analyzedElaeis species. The high number of SNP was reduced drastically after the filtering steps and only 115 potential markers remained. The 62 targeted CG included two CG with multi-locus characteristics , since they mapped to different chromosomes on the genome. These results suggest that the corresponding CG primers were specific for gene families rather than for individual CG. In our analysis 83% of the reads could be mapped onto the reference genome and 12,200 SNP were identified initially. According to Singh et al.  73% of tRandom seed samples were received descending from multiple crosses made by Oleflores and RGS. However, nothing was known about the population structure a priori. Therefore, we performed some global genetic analyses. The Ho = 0.561 was significantly higher than the He = 0.37 in the accessions of all five origins. This high Ho value is in accordance with Arias et al.  who evalAccording to Johnson and Shaw  the highElaeis species, which are so important in Latin-American regions, have been studied far less so far. Actually, only some QTL studies have been performed in order to improve the crop [Many studies have been published for the important oil palm crop Eg with the objective of crop improvement. However, the hybrids between the the crop ,11,31,32p values was visualized in a QQ plot for each trait. Several authors have used these QQ plots to determine the best fitting models visually [In total four different models were used for association mapping. Two GLM models with population structure (GLM_Q) and principal component analysis (GLM_PCA) as covariates and two MLM models where in addition a K matrix between individuals was included . After the analysis, the coincidence of observed and expected visually ,34,35. 2) of the CG data points from the diagonal of the QQ plot which represents an objective method for determining the best fitting model for each trait. When looking to the example of a QQ plot for carotenes contents in In our study the mixed effects models fitted better for most of our traits, while only a few traits were found to have better associations with GLM models where the K matrix was not taken into account. These findings are in accordance with those of Wang et al. , Nigro ep values from GAPIT is highly stringent, leading to the loss of the detected significant associations using unadjusted p values. A p value of 0.05 was set as threshold for identifying potential CG with potential significant influence on a trait as also in other studies with similar approaches [As pointed out by Gao et al.  the outpproaches ,39,40,41p values, LIPOIC revealed potential associations with one production traits  and six quality traits  It represents a lipoyl synthase gene, responsible for the synthesis of lipoic acid a universal antioxidant under oxidative stress conditions. This gene is required for cell growth, mitochondrial activity, and coordination of fuel metabolism and uses multiple mitochondrial 2-ketoacid dehydrogenase complexes [If not considering FDR adjusted omplexes  for the omplexes . It is komplexes . p values, also TO1 may influence the production traits BN and BW and one quality trait Gamma3. This CG represents a gamma-tocopherol methyltransferase which catalyzes the conversion of gamma-tocopherol into alpha-tocopherol. In Arabidopsis the overexpression of this enzyme resulted in more than 80-fold increase of α-tocopherol at the expense of γ-tocopherol without changing the total tocopherol contents [Using unadjusted contents . C. verbenacea and Cannabis sativa L. [The candidate gene SEQUI showed potential influence with one production trait, BW, and six quality traits; Toc, Toc3, Gamma3, SSS, IV, and Poly-Un. It is an alpha-humulene synthase transcript related to zerumbone biosynthesis. This compound is known as an essential oil of ativa L. ,47 and hativa L.  and antiativa L. . Finally, EgNAC showed that it could be associated with seven quality traits and one production trait. NAC transcription factors have been studied widely in different crops. They are known to regulate different plant functions in plants, such as fruit ripening in tomato , variatiThese findings indicate that many significant candidate genes could be involved in complex biological pathways, but there is still a lot of information missing. Fully understanding these metabolic pathways can help to discover the precise role of these genes influencing particular characters and can be a good starting point to obtain higher yielding oil palm varieties with increased oil contents. Association mapping results could be exploited in potential downstream applications by selecting genotypes with superior alleles of different significant candidate genes in Marker Assisted Selection systems.Production traits are the most interesting characters from a commercial point of view. However, quality traits are becoming more and more important in recent years. Breeding Companies look for high quality oil properties in order to satisfy customer’s preferences. Components such as high levels of unsaturated acids, high carotene contents, or high amount of tocols are becoming more and more important traits for taking into account. Our association mapping approach and whole understanding of the function of these detected candidate genes could help to obtain improved palms with these desired qualities. In our study we only considered partial amplicons from a reduced number of candidate genes, limiting the scope of our approach. Further studies should be conducted in the future to improve the results, considering other molecular tools such as whole genome resequencing, transcriptome sequencing, or bait sequencing in order to increase the number of targets.A broader collection of 198 Eo × Eg F1 genotypes from five different origins were evaluated in the Energy and Palma plantation in San Lorenzo . These consisted of 40 hybrid genotypes from Coari × La Mé origin , 75 accessions from Taisha × Avros , 37 genotypes from Taisha × Avros , 21 genotypes from Taisha × Yangambi , and 25 genotypes from Taisha × Ekona .Partial amplicons from 167 CG related to oil production and oil quality were used for the analysis. These CG were identified randomly by in silico mining using different sources: (i) literature searches related to known genes from oil palm or other species with proven influence on the trait of interest, (ii) relevant patent sequences in oil palm and other species, (iii) exploration of relevant metabolic pathways such as palm oil biosynthesis for potentially useful enzymes, and (iv) analyses of published QTL and co-located transcripts with a relevant biological meaning. Amplicon primers for these CG were designed only in exons, but not in adjacent regulatory regions . The CG Eo × Eg genotypes were planted in 2010 and phenotypic data recording started in 2014. In total, six production traits and 19 quality traits were studied. The phenotypic raw data are shown in The evaluated production traits were bunch number ), bunch weight ), bunch yield ), oil percentage in fresh mesocarp ), oil percentage in dry mesocarp ), and oil percentage in the bunch ). BN and BW data were collected over four years and cumulative data were used for the analysis. OildM data was determined by Soxhlet extractions. OilfM and OilB were calculated according to García and Nañez  as modifiodine/g was measured using the AOCS Official Da 15-48 method [The analyzed oil quality traits considered different components of lipids and tocols, as well as carotenoids. Lipid components included percentages of oleic acid (OA), of saturated acids (Sat), mono-unsaturated acids (Mono-Un), and poly-unsaturated acids (Poly-Un) and were measured using the AOCS Official Ce-1h-05  method. 8 method  and the 8 method . The nom8 method  and are 8 method . Saphiro–Wilk tests were applied in order to check for non-normal distributed data. The traits that showed a significant deviation were normalized by z-score correction and the normalized data were further used for ANOVA analyses. ANOVA analyses of the different traits and origins were performed in order to see how the origin of the different accessions affects oil production and quality. Separation of means for traits with significant differences was performed using a Tukey post hoc test. All analyses were performed using R language. DNA extractions were performed from young leaflet tissue samples using the Analytik JenaLife extraction kit  according to the manufacturer instructions. ® .All PCR primers were designed in exons of the CG by blasting the CG against the oil palm genome sequence from MPOB  and usinThree amplicon libraries were constructed with a total of 167 CG in the mentioned plant materials. First and second libraries were constructed with 55 CG each, while the third had 57 CG. The CG for each library were chosen randomly. The library number in which a particular CG was included is indicated in For the first multiplex PCR reaction fusion primers were used which were composed of a universal part  and a part common to the CG of interest. These primers produced 120–300 bp amplicons. For each library several multiplex reactions were performed. For selecting appropriate primers for these multiplex reactions, each primer pair was tested with all others for Self-Dimers and Cross Primer Dimers formation using Thermo Fisher Multiple Primer Analyzer . Sets ofA total of 20 ng of each genomic DNA, Invitrogen™ Platinum™ SuperFi™ PCR Master Mix , and 0.16 µM primer-mix were used per 25 µL amplification reaction. The PCR conditions were as follows: 98 °C denaturation for 30 s, followed by 30 cycles of 98 °C for 10 s, 58 °C for 30 s, 72 °C for 60 s, and a final elongation step of 72 °C for 5 min. PCR reactions were performed in a Thermal Cycler ABI 2720 . Amplification products were visualized as described and the PCR products were purified using Agencourt AMPure XP .All purified multiplex PCR products of a specific genotype were combined in one pool and used in a second PCR reaction to barcode each genotype. For this purpose, fusion primers were designed which were composed of one part complementary to the universal part of UniA and UniB, a genotype specific MID part, the key part (ACGT) to calibrate the sequencing machine and the specific key sequences A and B used by the sequencing platform. All primers as well as the forward and reverse MID sequences are shown in 2 genotypes can be discriminated. The genotype specific combinations of the MID sequences with UniA and UniB sequences, respectively, allow to identify unambiguously each genotype. By using a combination of forward and reverse MID a large number of genotypes can be barcoded with a relatively small number of primers. With 2n MID primers nFor each barcoding reaction, a 25 µL reaction volume was prepared containing 1 µL of the purified PCR product, 0.2 µM forward and reverse barcoding primer, and Invitrogen™ Platinum™ SuperFi™ PCR Master Mix. PCR reactions and visualization were performed as described before in the first step.PCR products of each barcoded genotype were individually quantified with a Qubit 2.0 device, using the Qubit dsDNA HS assay . Equal concentrations of genotype specific PCR products were mixed in one tube.Each pool was purified with columns using the GeneRead Size Selection Kit . The quality of the libraries was verified on an Agilent 2100 Bioanalyzer using DNA Chips with HS DNA Kit reagents according to the manufacturer’s protocol (Agilent Technologies). The libraries were sent for sequencing to the Center for Applied Medical Research , using the Ion Torrent PGM. Emulsion PCR was performed with Ion PGM™ Template OT2 400 Kit according to the manufacturer’s protocol. All libraries were sequenced using the 318 Chip v2 with the Ion PGM™ Sequencing 400 Kit. Sequencing was performed unidirectionally. http://southgreen.cirad.fr/ [Analyses of the obtained sequences were performed using the South Green Bioinformatics Platform irad.fr/ , which pE. guineensis pisifera genome sequence [The Fastq files of the three libraries were combined and processed together, since all genotypes had the same MID combination in the three libraries. In order to obtain clean amplicon sequences, trimming and demutliplexing steps were performed. First, each genotype was identified by the combination of MIDs in each read. Sequences were separated in genotype specific files. For this purpose the public “demultiplex.py” Python script  was usedsequence  was usedp value < 0.05) was that the population is in equilibrium and pairing occurs randomly. fastStructure software v1.0 [The SNP of the obtained Variant Calling Format (VCF) file were filtered using VCFtools software v4.2 . Markersare v1.0  was applp values also FDR using the method of Benjamini and Hochberg [p values. Association studies were performed on a single marker basis using GAPIT v 3.0  in R envHochberg  adjustedp values of each model were visualized separately for each trait in a QQ plot, in order to get a first impression on the fitting of different alternative models. In addition, an equation was developed to measure the average square distance (d2) of the CG data points from the diagonal of the QQ plot for each model:oP and eP are the expected and observed –log(p) values, respectively and n the number of CG data points. The model with the smallest d2 value was considered as the best fitting model for our data.The resulting observed and expected