extfieldsofstudy: Medicine Chemistry

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Cu-chitosan nano-net improves keeping quality of tomato by modulating physio-biochemical responses

Minimizing the post-harvest losses in fruits and vegetables is one of the challenging tasks in agriculture. To address this issue, we report nano-net of Cu-chitosan nanoparticles (Cu-chitosan NPs) which has the ability to extend the shelf-life of stored tomato. The application of Cu-chitosan NPs (0.01–0.04%) significantly curtailed microbial decay (< 5 versus > 50% in control), physiological loss in weight (14.36 versus 28.13% in control), respiration rate (0.01173 versus 0.01879 g CO2 kg−1 h−1) and maintained fruit firmness (34.0 versus 17.33 N in control) during storage. Further, these NPs significantly retarded loss of titratable acidity, retained total soluble solids, total and reducing sugars, lycopene, ascorbic acid and inhibited polyphenol oxidase. Likewise, NPs effectively preserved L* (lightness), a* (red/green) and b* (blue/yellow) values and maintained organoleptic score. Scanning electron microscopy study confirmed that Cu-chitosan NPs orchestrate into an invisible-intangible nano-net over tomato surface which may plausibly act as a potential barrier at all possible openings (stem scar, cuticle wax, lenticels, and aquaporins) to control microbial infection, moisture loss, gas exchanges and respiration rate. Overall, nano-net extended keeping quality of tomatoes up to 21 days at room temperature (27 ± 2 °C, 55 ± 2% relative humidity).

www.nature.com/scientificreports/ augment valuable functionalities like antimicrobial potential, physical barrier and antioxidant effect to extend the shelf-life of perishables. However, non-degradable highly reactive metal based nanomaterials are major concern for unexpected health hazards due to their residual effect in food. To provide safe solution to mentioned apprehensions, our research group has successfully developed various biodegradable chitosan based nanomaterials functionalized with Cu, Zn, and salicylic acid to prevent crop diseases and improve plant vigor [17][18][19][20] . Among these, Cu-chitosan NPs possess substantial antimicrobial and plant immune booster activities via up-regulating the antioxidant activity and maintaining the cellular homeostasis 17,18 . In these NPs, copper exists in its natural ionic form and is encapsulated into nano-scale chitosan matrix, and considered safer as compared to other zero valance, highly movable metallic nanomaterials. Additionally, the porous network architecture of Cu-chitosan NPs creates an antimicrobial ionic field through its entrapped Cu 17,18 . In spite of this, the composite effect of embedded Cu ions and chitosan is expected to be effective at low dose thus minimizing chemical load on treated fruits and vegetables. In view of aforesaid valuable bioactivities and unique properties of Cu-chitosan NPs, we hypothesized that these NPs would play a crucial role in reducing the post-harvest losses in fruits and vegetables. Therefore, in present study, Cu-chitosan NPs were applied on tomato fruits and concomitantly we report that NPs contrived a nano-net over tomato surface which persuaded intriguing responses over microbial decay, physiological loss in weight, and overall improved the keeping quality of tomatoes. The present study is first report of nano-net of Cu-chitosan NPs to improve the shelf-life of tomato up to 21 days at room temperature.
Effect of Cu-chitosan NPs on quality attributes of tomato. Titratable acidity (TA) of tomato declined throughout storage period in all the stored tomatoes. The decline was lesser in tomatoes treated with Cu-chitosan NPs (0.01-0.16%) as compared with control from 14 to 21d of storage (Fig. 3a). Tomatoes treated with Cu-chitosan NPs considerably abated the increase of total soluble solids (TSS), total and reducing sugars, lycopene, and PPO activity as compared with control ( Fig. 3b-f). Likewise, tomatoes treated with Cu-chitosan NPs significantly retained ascorbic acid (0.16 g kg −1 ) as compared with control (0.090 g kg −1 ) (Fig. 3g).
Scanning electron microscopy (SEM) of tomato surface. At first day, the surface of control tomato exhibited scattered lenticels and at 21 d of storage, tomato got decayed and displayed large fissures on cuticle wax surface (Fig. 4a,b). In contrast, at 1 and 21 d, Cu-chitosan NPs (0.04%) treated tomato showed nano-net structures over the tomato surface ( Fig. 4c,d).
Effect of Cu-chitosan NPs on colour attributes, organoleptic score and Cu content in tomato. The colour values (L * , a * , b * ) decreased during storage period in control tomatoes while the same were notably maintained in Cu-chitosan NPs treated tomatoes during storage (Fig. 5a-c). Organoleptic scores, given on a five-point hedonic scale (9-Excellent, 7-Very good, 5-Good, 3-Fair, 1-Poor), were > 4 in Cu-chitosan NPs treated tomatoes up to 21 days of storage (Fig. 5d). After 21 days of storage, a non-significant change in Cu +2 (0.028-0.035 g kg −1 pulp) was observed in Cu-chitosan NPs (0.01 to 0.08%) treated tomatoes as compared with control (0.027 g kg −1 pulp). However, the maximum accumulation of Cu +2 (0.060 g kg −1 ) was recorded in tomatoes treated with CuSO 4 after 21d of storage (Table 2).

Discussion
chitosan-surfactant nanostructure, alginate + chitosan + ZnO nanoparticles, limonene nano, active packaging of chitosan + TiO 2 NPs, nitric oxide-releasing chitosan nanoparticles, nano-Ca, thymol nanoemulsion + quinoa protein/chitosan edible films and chitosan + nano-silica have been evaluated in tomato, loquat, strawberry, apple, sweet cherry, cherry tomato, black grape, and guavas to study post-harvest losses 6,[9][10][11][12][13][14]16 . The literature survey indicates that chitosan has predominantly been preferred material to minimize post-harvest losses in perishables due to its non-toxic, biodegradable, film-forming, and edible nature 21 . Various chitosan nanomaterials functionalized with Cu, Zn, salicylic acid, etc. have established their role in endorsing plant cellular homeostasis through up-regulation of antioxidant-defense enzymes to protect crops from diseases [17][18][19][20]22 . Amongst these chitosan nanomaterials, Cu-chitosan NPs are known to inhibit various plant pathogenic microbes and incite plant defenseantioxidant system for broad-spectrum disease resistance 17,18,22 . Additionally, We envisage that various favorable physico-chemical characteristics viz. size (368.3 ± 3.2 nm), mono-dispersity (0.2 PDI), positive zeta-potential (+ 22 mV) and porous architecture of Cu-chitosan NPs provided it add-on features to facilitate uniform and firm binding to cuticular wax surfaces of fruits and vegetables (Fig. 1a www.nature.com/scientificreports/ encapsulated Cu ions into nano-scaled chitosan matrix endowed robust antimicrobial activity to Cu-chitosan NPs (see Supplementary Fig. S4). In this realm, we hypothesized that Cu-chitosan NPs would irrefutably be effective to maneuver shelf-life of fruits and vegetables. Therefore, we evaluated Cu-chitosan NPs for the first time to study various factors concomitant to post-harvest losses in tomato.
Curbing microbial infection is of utmost importance as it causes direct rotting and aggravates physiological disorders in stored fruits and vegetables. In the present study, Cu-chitosan NPs significantly precluded microbial infection and notably reduced decay in stored tomatoes (Table 1). Elusive bioactivity of Cu-chitosan NPs on microbial decay was not only due to antimicrobial nature of chitosan and Cu +2 but also by their effect on preventing plant cell death by enhancing plant cell immunity 17 . Moreover, a severe microbial infection on the tomato surface might have created a local acidic environment to induce the quick release of Cu +2 from the chitosan matrix to swiftly inhibit microbial infectivity 18 . Altogether, we assume that reported nano-net of Cuchitosan NPs over the tomato surface acts as a nano-shield and elegantly prevents the microbial infection at low concentrations (0.01-0.04%) as compared to earlier reports (Figs. 4c,d; 6).
Further, Cu-chitosan NPs comprehensively ameliorated various physiological disorders by significantly abating PLW, maintaining fruit firmness, and decreasing respiration rate in stored tomatoes (Fig. 2a-c). Higher www.nature.com/scientificreports/ www.nature.com/scientificreports/ respiration rate in fruit induces metabolic activities, speeds up ripening, and decreases the shelf-life of tomatoes. In our findings, Cu-chitosan NPs significantly controlled respiration rate during storage (Fig. 2c). Earlier, TiO 2 embedded chitosan nanocomposite film was found to retard respiration rate and retain firmness in cherry tomato up to 15 d of storage at 20 °C 11 . In fruits and vegetables, gas exchange and moisture loss and even microbial entry mainly occur through stem scar, cuticular wax, lenticels, and aquaporins [23][24][25] . In present investigation, SEM study confirmed that Cu-chitosan NPs builds a nano-net over tomato surface and we assume that it acts as a potential barrier at all possible openings (stem scar, cuticle wax, lenticels, and aquaporins) to control microbial infection, moisture loss, gas exchanges/ respiration rate (Figs. 4c,d; 6). Contrarily, severe fissures were evident on the cuticular wax surface of control samples after 21 d of storage which were responsible for faster decay of tomatoes (Fig. 4b). These large fissures on tomato surface were caused by severe microbial infection and shrinking of tomato due to unrestricted moisture loss and gas exchange. The sour taste of tomato is largely due to its www.nature.com/scientificreports/ TA which is considered a key quality parameter of tomato. TA in tomato decreased during storage, whereas the application of Cu-chitosan NPs significantly conserved it (Fig. 3a). We foresee that chitosan of Cu-chitosan NPs decreased respiration through nano-net and the low respiration rate eventually averts the consumption of organic acids 26 . During storage, TSS increased from 6.1 to 7.93% from 1 to 21 d in untreated tomatoes, while Cu-chitosan NPs (0.01 to 0.20%) significantly hold it between 7.06 to 6.46% in 21 d of storage as compared to an earlier report by Ali and co-workers (Fig. 3b) 27 . Based on these findings, we further anticipate that chitosan  www.nature.com/scientificreports/ at the nano-scale has higher potential to precisely control tomato TSS during storage. During ripening, starch degrades to yield glucose which contributes to the sweetness of fruit. Therefore, to extend the shelf-life of fruit, contents of total and reducing sugars needs to contain and Cu-chitosan NPs significantly controlled sugars during storage (Fig. 3c,d). The observed results could be attributed to the fact that Cu-chitosan NPs considerably suppress respiration and metabolic activities which in turn control total and reducing sugars. Color change in tomato during storage occurs due to the buildup of lycopene into chromoplasts and this process speeds up with respiration 28 . Cu-chitosan NPs considerably controlled respiration rate thus abated lycopene synthesis during storage (Fig. 3e). Higher activity of PPO is an indicator of post-harvest losses as it is responsible for changes in colour, flavor, and loss of nutritional value. Cu-chitosan NPs remarkably inhibited its activity in tomato during storage as compared with control (Fig. 3f). Ascorbic acid is one of the key antioxidant compounds present in high concentration in green tomato and decreases during ripening 29 . In the present study, ascorbic acid severely decreased from 0.25 to 0.09 g kg −1 in 21 days of storage in control fruits (Fig. 3g). Cu-chitosan NPs, however, significantly maintained the level of ascorbic acid throughout storage period as compared to earlier report on cherry tomato stored in active packaging of chitosan TiO 2 nano-composites 11 . L * (lightness), a* (red/green) and b* (blue/yellow) values and organoleptic score (sensory evaluation) are important parameters that affect the acceptability of fruits in the market. Tomatoes treated with Cu-chitosan NPs significantly conserved L * , a * and b * values and yielded fair to good organoleptic score during storage (Fig. 5a-d).
AAS studies were conducted to assess the Cu +2 content in stored tomatoes. As compared to control, a nonsignificant increase in Cu +2 content (0.028-0.035 g kg −1 pulp) was found in tomatoes treated with Cu-chitosan NPs (0.01-0.08%) which is below the toxic level 30 . However, the maximum accumulation of Cu +2 (0.060 g kg −1 ) was recorded in tomatoes treated with CuSO 4 . These results strongly point out that Cu-chitosan NPs of 368 nm diameter exists as a net structure and hardly infiltrates through the possible openings viz. stem scar, cuticular wax, lenticels and aquaporins into tomato. Majority of nanomaterials used in planta are zero valency metal NPs and below 100 nm size, thus have higher probability of infiltration and translocation into living organisms and raise serious apprehensions [31][32][33][34] . Nano-based materials that are directly or indirectly used in food materials especially for coatings and packaging of fruit and vegetables need to be biodegradable and biocompatible. Herein, we claim that myriad and fecund response of Cu-chitosan NPs to improve the keeping quality of tomato has potential to expend to other fruits and vegetables.

Conclusion
Minimizing post-harvest losses in fruits and vegetables with safe, effective and economical means is a major challenge in agriculture. Demand of chemical free food has unexpectedly risen due to increasing hazardous effects of chemicals on consumers. In this line, Cu-chitosan nanomaterials which are effective at low dose, biodegradable and easy to use, could be the fair alternative of hazardous chemicals. Thus all together, developed nanoformulation has potential to be translated into valuable technology to enhance the shelf-life and quality of fruits and vegetables during prolonged storage.  www.nature.com/scientificreports/ Cu-chitosan NPs. Dry powder of Cu-chitosan NPs was prepared by method described in our earlier study 20 . Before use, NPs were characterized for mean hydrodynamic diameter, zeta-potential, and polydispersity index (PDI) by dynamic light scattering (DLS) using Zetasizer ZS 90 (Malvern, UK) at 25 °C at a scattering angle of 90° in triplicate.

Treatment of Cu-chitosan NPs on tomato.
Stem detached tomatoes of similar size at the orange stage were selected and mechanically damaged with visible decay were abandoned for the study. Selected tomatoes were dipped into different concentrations of Cu-chitosan NPs (0.01, 0.04, 0.08, 0.12, 0.16 and 0.20%, w/v in water), bulk-chitosan (BCH) (0.1%), CuSO 4 (0.01%, w/v) and water (control) for 6 min, and kept in well ventilated paper baskets for storage at room temperature (27 °C ± 2) with the relative humidity 55 ± 2%.
Microbial decay. Before treatments, 1.0 cm long and 0.5 cm deep mechanical cut was placed on tomatoes for induction of microbial infection. Microbial decay was visually inspected in each treatment considering the extent of fungal growth on tomato surface in the scale ranging from 1 to 5, where 1 = normal (no decay on fruit surface), 2 = trace (up to 5% of fruit surface decayed), 3 = slight (5-20% of fruit surface decayed), 4 = moderate (20-50% of fruit surface decayed) and 5 = severe (> 50% of fruit surface decayed) 35 .
Physiological loss in weight (PLW), firmness, and respiration rate. Initial weights of tomatoes were recorded before (0 d) and after treatment (7, 14 and 21 d). Weight loss was recorded as the difference between initial and final weight (taken on a particular day) and expressed as % PLW 36 . The firmness was determined using Texture Analyzer (Model TA-XT Plus, Stable micro System Limited, Godalming, Surrey, UK) equipped with a cylinder probe of 50 kg load cell. Indirect respiration rate (g CO 2 kg −1 h −1 ) in a static system was determined following the method described earlier 37 . In brief, samples (~ 150 g) were placed in polythene bags with silicon septum and CO 2 concentration was measured using HeadSpace Gas Analyzer (WITT-GAS-ETECHNIK GmbH & Co KG, Witten, Germany). After 24 h, CO 2 concentration was again measured and the difference in CO 2 concentration was calculated.
Titratable acidity (TA) and total soluble solids (TSS). TA (%) of tomato juice was determined by titrating a known volume of diluted tomato juice against standard N/10 NaOH solution using phenolphthalein as an indicator until a faint pink color appeared. The results were expressed as percent acidity of the fruit juice 38 . Total soluble solids (%) in the juice were determined using "Pocket Refractometer" bearing the range 0-53% 38 .
Total sugars, reducing sugars, lycopene, ascorbic acid and polyphenol oxidase (PPO). Total sugars (%), reducing sugars (%) and lycopene (g kg −1 ) contents were measured using anthrone, dinitrosalicylic acid and anhydrous sodium sulphate methods, respectively using spectrophotometer 38 . PPO activity (nmol s −1 ) was measured using a spectrophotometer following the method described by Taneja and Sachar 39 . Ascorbic acid was determined by diluting a known volume of filtered juice with 3% metaphosphoric acid and titrating against 2, 6-dichloro phenol indophenol till a stable light pink color appeared. The results were expressed as µmol kg −138 .

Scanning electron microscopy (SEM) of the tomato surface.
To study the tomato surface, SEM analyses were performed in epidermis samples of treated and control tomatoes (at 1 and 21 d of storage). Samples were freeze-dried for 8 h and mounted on double-sided carbon aluminum stubs and sputter-coated with gold-palladium (SC7620, Emitech). SEM images were taken in high vacuum mode using Zeiss EVO MA10 SEM (Carl Zeiss Promenade, Germany) at 20 kV.
Hunter colour values and overall organoleptic score. Changes in L * , a * , b * colour coordinates on the Hunter scale were measured using a colorimeter (Chroma Meter CR 400; Konica Minolta, Tokyo, Japan). The color measurements were expressed in terms of luminosity (lightness) L * (L * = 0 for black and L * = 100 for white) and the chromaticity parameters a (green [-] to red [ +]) and b (blue [-] to yellow [ +]). Overall acceptability of samples was evaluated through standard sensory evaluation techniques. Sensory attributes such as taste, flavor and acceptability were rated on a five point hedonic scale (9-Excellent, 7-Very good, 5-Good, 3-Fair, 1-Poor) by a selected panel of judges (11 members).
Estimation of Cu +2 content. Treated and control tomatoes after 21d of storage were washed properly and pulp samples were taken for estimation of Cu +2 . Samples were dried in oven at 80ºC, burnt in muffle oven and white ash was dissolved in 6 N HCl. Cu +2 content was measured using the atomic absorption spectrophotometer (AAS 4141 model, Electronics Corp. of India Ltd., India) 40 .
Statistical analysis. Statistical analysis was performed using JMP software version 12 41 using Turkey Kramer HSD test. Each experiment was repeated twice wherein each treatment consisted of minimum of three replicates with five tomatoes in each replication.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Effect of environmental factors on the germination of Megathyrsus maximus: an invasive weed in sugarcane in Argentina

Background: Megathyrsus maximus (Jacq.) is a perennial weed that affects many crops. In Argentina, sugarcane is the most affected. Objective: Study effective techniques to break dormancy and the effect of environmental factors on the germination of this weed. Methods: Experiments were carried out in cabinet incubators twice, with five replicates per treatment. The experimental unit was made up for 50 seeds. Results: Seed dormancy was strongly associated with the presence of glumes. Manual extractions of glumes and immersion in sulphuric acid were the most effective techniques for breaking dormancy. Megathyrsus maximus did not depend on light to germinate, and it did so both under a 12 h-light-dark photoperiod and in complete darkness, with maximum mean germination percentages of 73 and 76%, respectively. Mean germination percentage (G) and coefficient of germination (CG: number of germinated seeds per day) showed that this weed responded to a wide range of temperatures, the optimal varied between 25 and 35 °C. Both mean germination percentages and CG decreased as osmotic potential became increasingly negative (0 MPa to -0.6 MPa), and as sodium chloride solution concentrations increased (10 to 130 mmol L-1). No germination was observed at -0.8 MPa and with a 150 mmol L-1 sodium chloride solution. Conclusions: The optimal germination conditions for M. maximus can be found in central sugarcane areas in Argentina, since soils are in ideal conditions and are kept under irrigation. In marginal areas, M. maximus germination would depend on rainfall and certain sodium chloride concentrations in the soil.

INTRODUCTION
Megathyrsus maximus (Jacq.) B.K. Simon & S.W.L. Jacobs var. maximus, formerly known as Panicum maximum, is native to Africa and belongs to the Poaceae family. It is a perennial and robust weed that may be up to 4 m tall and and spreads by seeds. This species is shade-tolerant and frost-susceptible Planta Daninha 2020;38:e020216688 -https://doi.org/10.1590/S0100-83582020380100054 2/8 (Holm et al., 1977;Chaila and Sobrero, 2009). It was introduced in almost every tropical country around the world as forage, then it spread to forest edges, savannahs and crops, thus becoming a pest. The crops most affected by M. maximus worldwide are sugarcane (Saccharum officinarum L.) and cotton (Gossypium hirsutum L.) (Williams and Baruch, 2000).
In Argentina, sugarcane is grown across several agroecological regions with different physiographic, climatic and edaphic characteristics. It is cultivated in areas with 600 mm annual rainfall and salt contents of up to 50 mmol L -1 (Zuccardi and Fadda, 1985). Megathyrsus maximus is regarded as one of the five most important perennial species that affect sugarcane. This is due to the morphological similarities between them, which allow the weed to remain unnoticed, at least in the initial stages (Holm et al., 1977). For several years, the infested areas were restricted to the central sugarcane region. Nowadays, there is a higher number of invaded sugarcane fields as a consequence of important changes in crop management, in particular the switch from intensive soil tillage to reduced tillage. In this scenario, yield losses can be as high as 60% (Chaila et al., 2010).
In general, in perennial grasses multiplication occurs mainly through vegetative reproduction, whereas sexual reproduction is in the background (Benson and Hartnett, 2006). Thanks to their long rhizomes, Panicum repens and Sorghum halepense populations grow and spread rapidly (Hossain et al., 1996;Prostko et al., 1997). By contrast, M. maximus has short rhizomes (Holm et al., 1977), which do not promote significant population growth and dispersion (Cabrera, 2016). Multiplication by basal division is possible (Salariato et al., 2006), but the cuttings exhibit low survival rates when separated from their mother plants (Cabrera, 2016).
Most invasive plants, such as M. maximus (Williams and Baruch, 2000), primarily rely on seed dispersal and seedling recruitment for population establishment and persistence (Ebrahimi and Eslami, 2012). Weed emergence is possible when seeds are non-dormant, and/or when environmental conditions (humidity, temperature and gas exchange) are adequate. Dormancy is the temporary failure of viable seed to germinate under external environmental conditions that later trigger germination, when the restrictive state has ended (Radosevich et al., 2007). In grasses, germination may be inhibited by physical dormancy, that is to say physical factors, as for example seed covering. This rules out the effect of environmental factors on seed germination (Radosevich et al., 2007). Flower structures (lemma and palea) and seed covering were identified as major factors contributing to physical dormancy (Adkins et al., 2002;Richard et al., 2016). Megathyrsus maximus possesses dormant seeds (Smith, 1970;Martins and Silva, 1998). Different techniques were tested to break dormancy, leading to differing results (Smith, 1970;Martins and Silva, 1998). This suggests that differential responses are obtained depending on biotype origin. Whereas in the USA Smith (1970) found that immersion of M. maximus seeds in sulphuric acid was the best treatment, in Brazil (Martins and Silva, 1998) found better responses when seeds were exposed to thermal treatments.
Among the environmental conditions that affect seed germination, temperature is the most important one in the case of non-dormant seeds. Specifically, soil temperature is essential for germination timing and rate (Guillemin et al., 2013). Light can influence the germination of many warm season grasses through the phytochrome system. This environmental factor can explain the stimulating effect of tillage on weed seedling emergence. Many weed seeds that require light for germination normally emerge when they are close to the soil surface (Adkins et al., 2002). Under conventional tillage, a great proportion of seeds remain buried more deeply into the ploughed soil (Ghersa and Martinez-Ghersa, 2000), which keeps them unexposed to light.
Osmotic and salt stress also play an important role, as germination is typically delayed or completely inhibited depending on stress intensity and duration, with seed genetic background also playing a role (Ebrahimi and Eslami, 2012). Ability to germinate under moisture or saline stress conditions is an asset for the species, since these conditions often prevent other species from growing (Javaid and Tanveer, 2014). In Argentina, studying the ability of the seeds of this grass to germinate under water and/or salt stress is important, considering the agroecological distribution of sugarcane in the country (Zuccardi and Fadda, 1985).
There are M. maximus biotypes that have become adapted to different soil and environmental conditions worldwide (Holm et al., 1977). In Argentina, no research has been conducted on the effect of environmental factors on M. maximus germination. Therefore, this study aims to evaluate the effect of different scarification methods on dormant M. maximus seeds, and to determine the influence of environmental factors on the germination of non-dormant seeds: Planta Daninha 2020;38:e020216688 -https://doi.org/10.1590/S0100-83582020380100054 3/8 constant and alternating temperatures, light alternation and continued darkness, and water and salt stress. This information will contribute to the development of new sugarcane management strategies and the improvement of already existing ones.

Seed source and preparation
Megathyrsus maximus seeds -Mature caryopses that were surrounded by two glumes were collected in March 2014, by shaking panicle-type inflorescences gently. The collection of caryopses, hereafter called seeds, came from plants that had grown in sugarcane fields in Tucumán, Argentina (26 o 43'55" and 27 o 46'38"S longitude, and 65 o 15'33" and 65 o 34'13"W latitude). They were air-dried, and then stored in paper bags at 18  2 o C and low relative humidity (15%) until trials began in June 2014. Before starting the study, visibly damaged seeds were excluded and visibly viable seeds were surface sterilized in a 0.5% sodium hypochlorite solution for 10 min, and then rinsed with tap water for 5 min (Ramirez et al., 2014).

Initial seed dormancy test
A test was first carried out to corroborate that freshly harvested M. maximus seeds were dormant (data not shown). Subsequently, the efficiency of chemical, thermal and mechanical treatments in breaking dormancy was tested as follows: Seeds were immersed in concentrated (95%) sulphuric acid for: 1) 3 min; 2) 5 min; 3) 10 min.
In all the cases, the treatment was followed by immersion in tap water for 9, 15 and 30 min, respectively. 4) Seeds were watered with a 0.2% potassium nitrate solution.
5) Glumes were removed by rubbing the seeds with sandpaper blocks (sandpapering).
7) Seeds were first immersed in hot water (70 o C) for 5 min, and then in tap water for 2 min (thermal shock).
8) Seeds were kept at 40 o C for 5 h. 9) Some seeds did not undergo scarification and were used as a control (Martins and Velini, 1997;Martins and Silva, 1998;Ramirez et al., 2014).

General protocol for germination tests
According to the results of the initial seed dormancy test, the main barriers to germination seemed to be the glumes. Therefore, for all germination tests, glumes were removed through immersion in concentrated (95%) sulphuric acid for 5 min, followed by washing with deionized water for 15 min (Figure 1). Then, 50 seeds were placed on 9 cm diameter Petri dishes containing two pieces of Whatman No.1 filter paper, moistened with 2 mL of distilled water, or with solutions having different salt concentrations or osmotic potentials. The Petri dishes were sealed with parafilm (to avoid moisture loss) and kept in cabinet incubators at fluctuating day/night temperatures of 35/15 o C, with a 12 h light photoperiod, except in the study of the effect of light and temperature. Light in the cabinet incubators was provided by cool white fluorescent tubes, with a 35 µmol m 2 s -1 irradiance. Humidity was kept constant by adding distilled water. Germination was recorded every day for a 15 day period, with seeds being considered germinated when their emerged radicle was at least 2 mm long. Germinated seeds were removed after each count, and the remaining seeds, which had not germinated, were tested for viability at the end of each trial, using tetrazolium tests.

Effect of temperature and light
Optimal temperature and light conditions for germination were evaluated in cabinet incubators, with both a 12 h light/12 h dark, and a 24 h dark photoperiod. With both, temperatures were kept constant at 10,15,20,25,30,35,40, and 42 o C, but they were also alternated between the following values: 20/10 o C, 25/15 o C, 30/20 o C, 35/15 o C and 35/25 o C. For germination in complete darkness, dishes were wrapped in an aluminium foil layer and placed in black plastic bags. Germinated seeds were counted at the end of the trial.

Effect of water stress
The effect of water stress on germination was evaluated by incubating M. maximus seeds in solutions with different osmotic potentials: 0, -0.2, -0.4, -0.6, -0.8, and -1.0 MPa. These were prepared by dissolving polyethylene glycol 6000 in distilled water. The solutions and filter papers were renewed every 2 days.

Effects of salt stress
The effect of salt stress on germination was evaluated by incubating M. maximus seeds in 0, 10,30,50,70,90,110,130,150 and 200 mmol L -1 sodium chloride solutions (NaCl). The solutions and filter papers were replaced every 2 days.

Data analysis
For all experiments, mean germination percentage and coefficient of germination (CG) were calculated. CG represents the number of germinated seeds per day and calculates seed germination speed. High CG values mean that a seedling sample is more vigorous than another. CG was calculated with the following mathematical expression (Maguire, 1962): where: CG = coefficient of germination; G = number of germinated seeds in each count; N = number of days elapsing between imbibition and each count.
All experiments were carried out twice, with five replicates per treatment. A completely randomized split-plot design was used in the temperature and light test. The main plots were allotted to constant and alternating temperature tests, and the sub-plots were used for studying the effect of light. The rest of the trials presented a completely randomized design. Mean germination percentage and CG obtained in the dormancy and in the temperature and light tests were analysed using generalized linear models. A binomial distribution was considered for mean germination percentage through the logit linking function, and a normal distribution was considered for CG with an identity linking function. The 0.05% DGC test was Vertical bars represent the standard error of the mean. Different lowercase letters above columns indicate significant differences among treatments, including the control (at P=0.05). used to evaluate differences among treatment averages (Di Rienzo et al., 2013) with P<0.05. This test uses the multivariate technique of cluster analysis. To determine water and salt stress effects on mean germination percentage, non-linear regression models were used. For CG response, linear and quadratic regressions were considered. The best model was chosen on the basis of the lowest AIC (Akaike Information Criterion) value and an appropriate distribution of residues. The data from all the experiments were analysed using Infostat and R software (Di Rienzo et al., 2013;R Core Team, 2015).

Initial seed dormancy test
With regard to effective techniques for breaking dormancy, the best ones were physical and chemical scarification. The highest mean germination percentages were obtained with immersion in sulphuric acid for 3 and 5 min (57% and 62%, respectively), manual extraction of the glumes (53%), and sandpapering (47%). The 10 min immersion in sulphuric acid treatment showed lower intermediate values (45%) ( Figure 1A). CG showed similar results with both 3 and 5 min immersion in sulphuric acid (7.6 and 8.5 seeds day -1 , respectively) and manual extraction of glumes (7.8 seeds day -1 ). These treatments showed the highest values with no significant differences among them. The 10 min immersion in sulphuric acid and sandpapering treatments presented significantly lower values than the other treatments (5.9 and 6.8 seeds day -1 , respectively), and significantly higher values than the control (1.0 seeds day -1 ) ( Figure 1B).
The way in which the seeds responded to the different treatments reveals the importance of seed covering for M. maximus germination. This is in agreement with previous studies that showed that in tropical grasses, like Panicum virgatum or Sporobolus phleoides, seed covering was responsible for germination inhibition (Duclos et al., 2014;Richard et al., 2016). The seed tissue enclosing the embryo plays a primary role in regulating germination and dormancy (Richard et al., 2016). It prevents both gaseous exchange and radicle sprout (Adkins et al., 2002). When sulphuric acid or sandpapering partially removes the seed covering, the seed is then ready for the soaking process that enables germination. This behaviour has also been observed in grasses like Sorghum halepense and P. virgatum (Duclos et al., 2014). In our study, manual extraction of glumes showed similar results to those obtained with the sulphuric acid and sandpapering treatments, suggesting that in M. maximus, dormancy is strongly associated with the presence of glumes.
Regarding the other treatments, seeds of the Argentine biotype watered with a 0.2% potassium nitrate solution, kept at 40 o C for 5 h, or given a thermal shock (5 min in water at 70 o C), presented the lowest mean germination percentages (between 13% and 6%), like the control ( Figure 1A). CG values for these treatments were also similar to those of the control ( Figure 1B). Nitrogenized substances and thermal treatments stimulated mean germination percentages in other populations of this weed (Martins and Silva, 1998). In Brazil, for example, mean germination percentage increased by 7% and 13.5% with nitrogenized substances and thermal treatments, respectively (Martins and Silva, 1998).
All in all, considering that manual extraction of glumes and chemical treatments with sulphuric acid broke dormancy to similar extents, it is very likely that the main barriers to M. maximus seed germination are the glumes.

Effect of temperature and light
Megathyrsus maximus showed a great ability to germinate under a wide range of conditions. Mean germination percentage differed according to the interaction between temperature and light, which was significant (P<0.001) ( Table 1). In the 12 h light photoperiod tests, the treatments with the highest mean germination percentages (between 73% and 61%) were the ones at 35/25 o C, 25 o C, 30/20 o C, 25/15 o C, 20/10 o C, 30 o C, and 35/15 o C. Total darkness did not inhibit M. maximus germination, and the highest percentages were 76%, 65% and 63%, recorded at 35/25 o C, 20/10 o C, and 30/20 o C, respectively. As for CG, the best treatments were the ones at 35 o C, 30 o C, 35/25 o C, 30/20 o C, 25/15 o C and 35/15 o C, giving CG values which ranged between 14.35 and 8.27 seeds day -1 . These results were also found for P. virgatum (Duclos et al., 2014). In contrast, Panicum dichotomiflorum and Panicum capillare did not germinate in total darkness (Taylorson, 1980). Clearly, there are different responses even in the case of closely related species.
Our results suggest that M. maximus could germinate under temperature and light conditions generated by sugarcane canopy or by crop residues in spring and summer. However, in autumn and winter, low temperatures might affect the germination process negatively.

Effect of osmotic stress
Megathyrsus maximus seed germination followed an exponential model. Mean germination percentage decreased from 67% to 4% as water stress intensified between the potentials 0 and -0.6 MPa. Germination was completely inhibited at -0.8 MPa. The osmotic potential required for inhibiting 50% of germination was -0.54 MPa (Figure 2A). A linear regression equation was fitted to the CG data, since values decreased as osmotic potential diminished. The highest value (8.44 seeds day -1 ) was obtained when there was no water stress, and the lowest value (1.04) was reached at -0.6 Mpa ( Figure 2B). It has been reported that once M. maximus germinates and plants are well established, they are tolerant to brief drought spells. Moreover, they are well adapted to soils that are welldrained, fertile and which preferably have a sandy loam texture (Holm et al., 1977).
This behaviour was also recorded in other weed species adapted to sandy soils, such as S. indicus and M. repens (Stokes et al., 2011;Rana et al., 2012). Drought plays an important role not only in determining germination rates, but also in influencing seedling development. If the seeds of these species germinate under a low water potential, seedling establishment might fail if drought continues (Van den Berg and Zeng, 2006). Instead, if the seeds do not germinate until the levels of humidity are appropriate, there is a strong likelihood that the seedlings will survive (Evans and Etherington, 1990). There are associations between rain or water availability and germination (Javaid and Tanveer, 2014). In Argentina, M. maximus germination is successful in irrigated sugarcane fields, whereas in non-irrigated plots it depends on rainfall, which is usually abundant in summer, with average values reaching up to 900 mm per year (Zuccardi and Fadda, 1985).

Effects of salt stress
A decreasing mean germination percentage was observed as NaCl concentration increased from 0 to 200 mmol L -1 . Mean germination percentage in the control treatment was 70%. Germination was higher than 50% at NaCl concentrations between 0 and 45 mmol L -1 , but it diminished by up to 2% at 130 mmol L -1 , and was completely inhibited at 150 mmol L -1 ( Figure 3A).  The relationship between M. maximus CG and salt concentration was described with a quadratic regression model. A decreasing CG was observed as NaCl concentrations increased. Control treatment CG was 7.12 seeds day -1 . At concentrations ranging between 10 and 70 mmol L -1 , CG decreased from 4.86 to 1.67 seeds day -1 , and amounted to less than 1 seed day -1 with 90 to 130 mmol L -1 NaCl ( Figure 3B). Evidence found so far points to M. maximus not being a salt stress tolerant species. In general, maximum salt stress tolerance in grasses occurs with concentrations between 250 and 350 mmol L -1 (Morgan and Myers, 1989). This effect seems to be more noticeable in CG than in mean germination percentage. In marginal sugarcane areas in Argentina, soil salt concentration fluctuates between 5 and 50 mmol L -1 (Zuccardi and Fadda, 1985), hence M. maximus germination would be possible.
It should be taken into account that the effect of salts on germination is conditioned by other factors, such as humidity and soil texture, with the latter having a great influence on ion toxicity through soil cationic exchange capacity (Muscolo et al., 2013). Thus, it is necessary to consider salt stress in combination with these other factors when evaluating if a given area is suitable for M. maximus establishment and growth.
This works provides information about the conditions necessary for M. maximus seed to germinate successfully and exhibit good CG values, while also showing how the dynamics of this process is affected by extreme conditions. The available evidence suggests that glumes are most responsible for seed dormancy. In addition, it can be pointed out that the interaction between light and temperature under crop canopy or underneath its residues in certain sugarcane areas in Argentina would promote M. maximus germination. Lastly, as far as hydric and salinity stress are concerned, although M. maximus has proven not to be tolerant to these types of stress, its ability to germinate under conditions of up to a 130 mmol L -1 concentration and a -0.6 MPa osmotic potential ensures its germination in sugarcane fields.

CONTRIBUTIONS
DCC: data collection and drafting of the article; MP: data analysis and interpretation; MTS: design of the work and critical revision of the article; SC: critical revision of the article and final approval of the version to be published.


extfieldsofstudy: Biology Medicine

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Research Note: Detection of infectious bursal disease virus antibodies in free-living wild birds in Zaria, Nigeria

Infectious bursal disease virus (IBDV) is an immunosuppressive pathogen of poultry causing great economic losses to the poultry industry. In this study, the IBDV antibodies were detected in captured free-living wild birds in Zaria, Nigeria. One hundred and fifty free-living wild birds, comprising 30 birds each of 5 different species, were sampled over a period of 9 months. Blood samples were collected from each bird, and harvested sera were tested for IBDV antibodies using enzyme-linked immunosorbent assay. Results indicated IBDV seroprevalences in speckled pigeon (6.67%) and cattle egret (3.33%). In conclusion, the detection of IBDV antibodies in free-living wild birds in this study is indicative of previous natural exposure of these birds to the virus. These species of wild birds could therefore serve as carriers of these viruses and, consequently, transmit these viruses to chickens.

INTRODUCTION
Infectious bursal disease (IBD) or Gumboro disease is an acute, highly contagious disease of chickens caused by infectious bursal disease virus (IBDV) (Eterradossi and Saif, 2008). The disease is characterized by bursal lesions, bursal atrophy, and immunosuppression in chickens between 3 wk and 3 mo of age (Mahgoub, 2012;Magwod et al. 2014). The virus responsible for IBD is a bisegmented, double-stranded RNA virus, belonging to the genus Avibirnavirus and family Birnaviridae (Baxendale, 2002;Jackwood et al. 2018). It shows selective tropism for lymphoid tissue and has affinity for immature B lymphocytes in the bursa of Fabricius (Lukert and Saif, 2003;Mwenda et al. 2018). It has been reported to also cause lymphoid depletion in the bursa of Fabricius in free-living wild birds, but infection is generally subclinical (AHA, 2009). The natural hosts of IBDV are domestic fowls including chickens and turkeys (Jackwood and Sommer-Wagner, 2007). The mode of transmission of IBDV is primarily through fecal-oral route, with aerosol spread considered to be less important (AHA, 2009). Two serotypes of IBDV (1 and 2) have been recognized with existence of considerable antigenic variation within each serotype (Motohiko et al.1998;Jackwood et al. 2018). Serotype 1 is pathogenic only to chickens and can be further divided into classical virulent, attenuated, antigenic variant and very virulent IBDV based on their virulence and antigenicity (Wang et al. 2007;Mawgod et al. 2014;Jackwood et al. 2018). Serotype 2 viruses are naturally avirulent and do not cause clinical disease in chickens and turkeys (Motohiko et al. 1998). Serological evidence of serotype 1 IBDV infection in wild birds suggests that wild birds may play a role in the epidemiology of IBDV by serving as reservoirs for the virus (Gilchrist, 2005;AHA, 2009). Reports have shown that serotype 2 of IBDV is more prevalent in many species of free-living wild birds, with the natural host considered to be turkeys (Motohiko et al. 1998).
There is dearth of information on the IBDV status of free-living wild birds in Zaria, Nigeria. Therefore, the aim of this study was to detect IBDV antibodies in free-living wild birds in Zaria, Nigeria.

Ethics Statement
The study protocol and all animal studies were approved by the Ahmadu Bello University Committee

Sample Size
The sample size for the study was calculated using the following formula by Mahajan (1997): where N 5 sample, Z 5 the appropriate value from the desired confidence (1.96), p 5 expected prevalence, q 5 1 -Prevalence, d 5 allowable error. Using relative seroprevalence of 6.15% from a previous study by Adamu et al. (2017), To increase the chances of detecting the antibodies, 150 blood samples and 150 sera were collected.

Sampling of Birds
Based on convenience sampling method, a total of 150 birds comprising 30 each of laughing doves (Spilopelia senegalensis), speckled pigeons (Columba guinea), cattle egrets (Bubulcus ibis), village weavers (Ploceus cucullatus), and African silverbill (Euodice cantans) were sampled alive over a period of 9 mo (March-December, 2017). These species of birds were used based on their availability at the particular time of capture. The birds were captured alive and unhurt using wooden traps kept at strategic positions around poultry houses located at different locations within the environ. The 10 poultry farms were selected based on frequency of visit of these free-living wild birds and previous history of IBD outbreaks.
The captured birds were identified by an ornithologist in the Department of Zoology, Faculty of Life Sciences, Ahmadu Bello University Zaria. Thereafter, physical examination was conducted on each bird to be sure there were no lesions, wounds, or ectoparasites.

Blood Sample Collection
Blood sample (0.5-1 ml) was collected from each bird via the wing vein using sterile hypodermic syringes and 23G needles. Sera were harvested from the blood, transferred into labeled sterile plastic containers, and stored at 220 C until used for IBDV antibody detection. After sample collection, each bird was marked to avoid repeated sampling and released into the environment.

Enzyme-Linked Immunosorbent Assay for Detection of IBDV Antibody
The 150 test sera were subjected to indirect ELISA following the manufacturers' instructions (IDEXX IBD-XR Ab Tests Technical Guide). IBD virus antibody test kit (designed for serotype 1 strain) used was obtained from IDEXX IBD-IDEXX Laboratories Inc., Westbrook, ME. The absorbance values were measured and recorded at 650 nm wavelength using an ELISA microtiter plate reader. The relative level of antibody to IBDV in the sample was determined by calculating the sample-to-positive (S/P) ratio. Sera with S/P ratios of less than or equal to 0.20 were interpreted as negative. Sample-to-positive ratios greater than 0.20 were interpreted as positive and indicated vaccination or exposure to IBD virus according to the manufacturers' technical guide.

Data Analyses
The ELISA data were presented as percentages in Table. The prevalence of antibodies to IBDV was calculated for each bird species using the formula outlined by Bennette et al. (1991).

RESULT
The result of this study shows seroprevalence of IBDV antibodies in speckled pigeons (6.67%) and cattle egrets (3.33%) but no serological evidence in laughing doves, village weavers, and the African silverbills in Zaria, North West Nigeria (Table 1).

DISCUSSION
In this region, vaccination of chickens against IBD is frequently carried out. Fagbohun et al. (2000) reported seroprevalence of IBDV antibodies in cattle egrets (20.0%) using ELISA at Ibadan, South West Nigeria. Also, antibodies to IBDV have also been reported in speckled pigeons (6.0%) and laughing doves (13.04%) in Kano using ELISA (Adamu et al. 2017).
From this study, it is believed that these species of free-living wild birds have been previously exposed to IBDV and could play important roles in the natural maintenance and spread of the virus in poultry because of their migratory potentials. The common IBD vaccines used in Nigeria and the surrounding areas are live vaccines from serotype 1 strain, and the wild birds that tested positive did not show signs of having been exposed to these vaccine strains.
The presence of IBDV antibodies in free-living wild birds in this study might have resulted from indirect interaction through frequent visits to commercial poultry farms and feeding around poultry houses in this region (Oladele et al. 2011). Also, these free-living wild birds have been found among the chickens in areas where local chickens are reared on free-range management system and around live bird markets. These interactions allowed for possible ingestion of the viruses by these birds, thus suggestive of the seroprevalence observed in this study. The detection of IBDV antibodies in free-living wild birds is indicative of previous natural exposure of these birds to the viruses at some point in their life. These free-living wild birds therefore may serve as carriers of these viruses after migration to poultry houses and possible dissemination of the virus to chickens.
It was observed that IBDV antibodies exist in free-living wild bird populations. The detection of IBDV (2.0%) antibodies in free-living wild birds in this study indicated previous natural exposure to these viruses. To the best of our knowledge, this is the first report on the serological studies of IBDV in these species of wild birds in Zaria, Nigeria.
Whether these viruses cause clinical diseases and pathology in free-living wild birds require further investigation. It is recommended that further research involving experimental inoculation and more species of birds should be carried out in Zaria, Nigeria, to understand the pathology, pathogenesis, epidemiology, and status of IBDV in wild bird populations.


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Effects of Dietary Fatty Acids from Different Sources on Growth Performance, Meat Quality, Muscle Fatty Acid Deposition, and Antioxidant Capacity in Broilers

Simple Summary The findings in the current study reveal that dietary fish oil or a combination of linseed oil and microalgae could be effective in improving growth performance, carcass traits, muscle fatty acid deposition, and antioxidant capacity in broilers compared with traditional soybean oil in broilers. Abstract This study aimed to investigate the efficiency of dietary fatty acids from various sources on growth performance, meat quality, muscle fatty acid deposition and antioxidant capacity in broilers. 126 Arbor Acres broilers (1 d-old, initial body weight of 45.5 ± 0.72 g) were randomly assigned to three treatments with seven cages per treatment and six broilers per cage. The dietary treatments included: (1) corn–soybean meal basal diet containing 3% soybean oil (control diet, CTL); (2) basal diet + 1% microalgae + 1% linseed oil + 1% soybean oil (ML); (3) basal diet + 2% fish oil + 1% soybean oil (FS). The trial consisted of phase 1 (day 1 to 21) and 2 (day 22 to 42). Compared with CTL, broilers fed ML or FS diet showed improved (p < 0.05) average daily gain in phase 1, 2, and overall (day 1 to 42), as well as a decreased (p < 0.05) feed conversion ratio in phase 1 and overall. On day 42, broilers supplemented with FS diet showed increased (p ≤ 0.05) the relative weights of pancreas and liver, as well as higher (p < 0.05) redness value in breast and thigh muscle compared with CTL. Broilers offered ML or FS diet had lower (p < 0.05) the relative weight of abdominal fat and total serum cholesterol content in phase 1, and increased (p < 0.05) contents of serum glucose, n-3 polyunsaturated fatty acids (PUFA), eicosacagetaenoic acid, docosahexaenoic acid, glutathione peroxidase, superoxide dismutase and total antioxidant capacity, as well as lower (p < 0.05) concentrations of malondialdehyde, n-6 PUFA, and n-6/n-3 PUFA ratio in breast and thigh muscle compared with CTL. This research indicates that diets supplemented with fish oil or a combination of microalgae and linseed oil experience improved performance, antioxidant capacities and n-3 PUFA profile in muscle of broilers compared with traditional soybean oil supplemented diets

Introduction
Meat harvested from animals plays an important role in human diets due to its fatty acids, protein, mineral, and vitamin contents. In recent decades, the consumption of chicken meat has increased and become widely popular around the world. Modern poultry production focuses on improving the performance and health status of broilers, and producing poultry meat that is nutritious for consumers. Studies have shown that high concentrations of n-3 polyunsaturated fatty acids (PUFA), such as

Experimental Animals, Management, and Design
A total of 126 Arbor Acres broilers (1 d-old, initial body weight of 45.5 ± 0.72 g, offered by Beijing Arbor Acres Poultry Breeding Co., Ltd. (Beijing, China)) were randomly assigned to 3 treatments (7 cages in each treatment, 6 broilers per cage). The treatment included a control diet (CTL, corn-soybean meal basal diet + 3% SO, fed in mash form), an ML diet (basal diet + 1% MA + 1% LO + 1% SO), and an FS diet (basal diet + 2% FO + 1% SO). The trial consisted of two phases with phase 1 (day 1 to 21) and phase 2 (day 22 to 42). Nutrient levels in all formulated diets for each phase met or exceeded the nutrient requirements recommended in NRC (1994, Table 2).  All broilers were kept in an environmentally controlled room under continuous illumination (10-20 Lux) and supplied feed and water ad libitum. The room temperature was set and controlled at about 33 • C for the first three days and decreased by 3 • C per week until reaching 24 • C. On day 7 and 28, broilers were vaccinated with Newcastle disease vaccine. On day 14 and 21, all broilers were vaccinated with an inactivated infectious bursal disease vaccine.

Sampling and Processing
All diet samples were ground to pass through a 0.45-mm sieve and samples were prepared for analysis of crude protein (CP, AOAC 2001. , and dry matter (DM, AOAC 2001. ). An automatic isoperibolic oxygen bomb calorimeter (Parr 1281, Automatic Energy Analyzer; Moline, IL, USA) was used to determine the gross energy (GE) content of the diets.
Broilers and feeders were weighed on day 21 and 42 after fasting for 12 h to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) of the broilers.

Serum Parameters Measurement
On day 21 and 42, blood samples (about 8 mL, n = 7) were collected from one broiler close to average body weight in each cage via jugular vein puncture into a 10-mL anticoagulant-free Vacutainer tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). After stewing for about three hours, blood samples were centrifuged at 3000× g for 10 min at 4 • C to recover serum, which was then stored at −80 • C until further analysis. The contents of low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), globulin, albumin, glucose, total protein (TP), and urea nitrogen (UN) in serum were tested (colorimetric method) following the corresponding reagent kit manufacturer instructions (Zhongsheng Biochemical Co., Ltd., Beijing, China) via an automatic biochemical analyzer machine (RA-1000, Bayer Corp., Tarrytown, NY, USA). The concentration of malondialdehyde (MDA) was determined by Thiobarbituric Acid Reactive Substance (TBARS) Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) following the manufacturer's protocols. The activities of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) in serum were determined by spectrophotometric methods using a spectrophotometer (LengGuang SFZ1606017568), according to the SOD, CAT, T-AOC and GSH-Px kits' manufacturer instructions (The Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Meat Quality and Carcass Traits Measurement
On day 21 and 42, one broiler per cage (near the average body weight, n = 7) was chosen to evaluate carcass traits and meat quality. The muscle color (lightness, redness, and yellowness values) in the left breast muscle and thigh muscle were measured from 3 orientations (middle, medial, and lateral) using a Chromameter (CR-410, Konica Minolta, Tokyo, Japan) according to the method by Elokil et al. [13]. The plastic bag method was utilized to determine drip loss in the breast and thigh muscles. On day 42, the relative weights of heart, spleen, liver, abdominal fat, pancreas, and fabricius of broilers were calculated using the following formula: weight of organ/final body weight × 100% = Relative weight of organs (%BW).

Fatty Acid Profile Testing
On day 42, after euthanasia or harvesting, one broiler per cage (near the average body weight, n =7) was chosen to measure the fatty acid profile in the left breast and left thigh muscle. The homogenized skinless freeze-dried breast and thigh muscle along with the experimental milled diets utilized in phase 2 were defrosted. A freeze dryer was used to lyophilize about 10 g diet and 20 g muscle for 60 h. A solvent mixture of chloroform and methanol (2:1, vol/vol) was used to extract total lipids from the homogenized muscle tissue. Gas chromatography (GC, 6890 series, Agilent Technologies, Wilmington, DE, USA) was used to determine the fatty acid profile in diets and muscles following the procedure used by Long et al. [10]. The calculation for n-6 PUFA, n-3 PUFA, the n-6/n-3 PUFA ratio, SFA, the PUFA/SFA ratio, and the monounsaturated fatty acids (MUFA) followed the same procedure outlined in Long et al. [10].

Statistical Analysis
Data were analyzed using the MIXED procedures of SAS (version 9.2, 2008; Inst., Inc., Cary, NC, USA). The dietary treatment served as the fixed effect, and body weight was the random effect. The individual cage was classified as the experimental unit in terms of performance data (body weight gain, ADG, ADFI and FCR). Individual broilers selected in each cage were considered as the experimental unit for all other indices. Student-Neuman-Keul's Multiple Range Test was used to separate the statistical differences among all the treatments. Least squares means and SEM were used to express the results. The significance was defined at p ≤ 0.05, and a trend for significance was determined at 0.05 < p ≤ 0.10.

Fatty Acids Profile in Diets
As presented in Table 3, the, C18:1n-9, C20:0, C16:0, C18:0, and C18:2n-6 were the major fatty acids present in the diets. With the addition of MA and LO, concentrations of n-3 PUFA and DHA increased by approximately 2.3%, whereas the n-6/n-3 PUFA ratio approximately decreased 83% in phase 1 and 94% in phase 2. With the addition of FO, concentrations of n-3 PUFA, EPA and DHA showed an approximate increase of 0.6%; whereas the n-6/n-3 PUFA ratio decreased by approximately 78% in phase 1 and 84% phase 2.

Growth Performance
Compared with CTL, broilers fed the ML and FS diet showed improved (p < 0.05) average daily gain in phase 1 (day 1 to 21), 2 (day 22 to 42), and overall (day 1 to 42), and decreased (p < 0.05) feed conversion ratio during phase 1. Additionally, broilers offered the FS diet tended to have an increased (p = 0.09) ADFI in phase 1 compared with CTL (Table 4).

Meat Quality and Carcass Traits
Compared with CTL, broilers offered the FS diet showed lower (p ≤ 0.05) lightness in thigh muscle on day 21, while expressing higher (p ≤ 0.05) redness on day 42. Broilers fed the FS diet also had greater (p < 0.05) relative weights of the liver and pancreas. Additionally, broilers offered the ML or FS diet showed decreased (p < 0.05) the relative weight of abdominal fat. However, there were no significant effects on the thigh and breast drip loss in broilers supplemented with ML or FS diet compared with CTL (Table 5).

Serum Composition
Compared with CTL, broilers offered the ML or FS diet showed a lower (p < 0.05) content of TC during phase 1. In phase 2, broilers fed the ML or FS diet had higher (p < 0.05) content of glucose and tended to have lower concentrations of TC and UN in serum compared with CTL (Table 6).

Antioxidant Status in Muscle
Compared with CTL, broilers fed the ML or FS diets showed greater (p < 0.05) activities of T-AOC, SOD and GSH-Px, as well as a reduced (p < 0.05) concentration of MDA in the breast and thigh muscles; while broilers fed the ML diet showed an improved (p < 0.05) activity of CAT in the thigh muscles (Table 7).

Fatty Acid Composition in Muscle
In breast muscle, broilers offered the ML or FS diet had improved (p < 0.05) concentrations of EPA, DHA, C14:0, C17:0, and n-3 PUFA, as well as decreased (p < 0.05) contents of n-6 PUFA, C18:2n-6, and n-6/n-3 PUFA ratio compared with CTL. Broilers fed the FS diet showed lower (p < 0.05) concentrations of ALA, PUFA, and PUFA/SFA ratio, as well as a higher (p < 0.05) content of C20:1n-9 compared with CTL. Furthermore, broilers fed the ML diet showed an increased (p < 0.05) content of ALA compared with CTL, and tended to have lower concentrations of C18:1n-9 and MUFA compared with broilers offered the FS diet (Table 8).

Discussion
In the present study, we demonstrated that the ADG was improved in broilers fed ML-supplemented diets compared with a typical soybean meal-corn based diet. This may be partly due to the inclusion of 1% and 2% MA, which could improve ADG and decrease FCR in broilers [7,10]. This result may also be due to the beneficial effects of high level-ALA in LO and high level-DHA in MA on moderating the microflora composition in cecum and improving intestinal morphology in broilers [14]. Moreover, a mixture of MA and LO might slow the feed passage rate in the small intestine and increase nutrient digestibility, thus improving performance [15]. The improved performance of broilers fed a diet containing 2% FO might be due to the high levels of n-3 PUFA (e.g., EPA or DHA) present in FO, that have been shown to improve both the immune status and nutrient digestibility in broilers [6]. Interestingly, low levels of dietary FO (such as 2%) have been demonstrated as being more effective than high levels of FO on improving performance [16]. The improved performance by dietary n-3 PUFA supplementation in broilers is also due to the narrowing dietary n-6/n-3 PUFA ratio (close to 4:1 or 2.5:1) and increasing the n-3 PUFA level that could effectively improve immune responses [17]. Moreover, high levels of n-3 PUFA can improve immunity and anti-inflammatory functions by decreasing the contents of inflammatory biomarkers, consequently leading to better performance in broilers [18]. The performance data in this study provides appropriate proportions as well as a reasonable mixture choice of different sources of n-3 PUFA fed to broilers.
The improved carcass traits in broilers offered the ML diet were partly in agreement with the findings of Baião et al. [19], who reported that dietary oil or fat supplementation can effectively improve the composition and quality of the carcass. The greater liver and pancreas percentages of broilers supplemented with the FS diet reflected that n-3 PUFA in FO could improve the nutrient digestibility and metabolic function [6]. The decreased relative weight of abdominal fat was mainly due to the effect of the high-level n-3 PUFA in MA, LO and FO, rather than SFA or MUFA in SO [20]. Previous studies have also concluded that LO could reduce abdominal fat deposition by promoting fatty acid β-oxidation, rather than suppressing fatty acid biosynthesis [21]. These improved body-mass percentages indicate that dietary n-3 PUFA supplementation could improve the immunity of broilers, as well as provide humans with nutritious chicken meat for consumption while reducing the instances of certain human diseases [22]. Broilers offered the FS-supplemented diet also showed higher redness values in the muscle, which may be due to the ability of FO to minimize the oxidation of myoglobin [23].
The improved concentration of serum glucose in the ML-or FS-fed broilers is due to the fatty acid profile's (particularly PUFA/SFA ratio) ability to regulate glucose metabolism [24] and the effect of LO on lowering the level of serum insulin [25]. Broilers offered high-level n-3 PUFA diets showed lower serum TC, TG and LDL-C, which may be due to high-level n-3 PUFA regulating the contents of triacylglycerols and lipoproteins via suppressing triglycerides and apolipoprotein synthesis B [25,26]. Moreover, n-3 PUFA could also inhibit the activity of ∆9-desaturase, reducing the transformation of hepatic very low-density lipoprotein cholesterol synthesis and deterring triacylglycerol metabolism [10,27]. Furthermore, n-3 PUFA could improve Lipin-1 gene expression, which helps to control DNA-bound transcription factors to regulate gene transcription, thus regulating triglyceride synthesis [28].
Studies have demonstrated that the amount and composition of fatty acids in abdominal fat, animal tissue or muscle, as well as serum composition in broilers, are mainly modified by dietary fatty acid composition [4,26]. Therefore, the improved SFA, EPA, DHA, and n-3 PUFA deposition in the breast and thigh muscles of the FS and ML-supplemented broilers compared to the control group reflect the increased composition of these fatty acids present in the diet [10,19]. Furthermore, previous studies have shown that narrowing the n-6/n-3 PUFA ratio through the addition of FO or LO could improve performance and immune response of broilers, as well as produce n-3 PUFA-enriched chicken meat. Additionally, ALA present in LO could serve as the precursor of EPA and DHA [7]. MA has been demonstrated to improve the transfer of EPA and docosapentaenoic acid into DHA [29]. Therefore, broilers offered the ML-supplemented diet showed higher deposition of DHA. The higher concentration of DHA in broilers fed diets supplemented with FO was due to the high levels of EPA and DHA in FO that could enrich n-3 PUFA concentration in the breast and thigh muscles of poultry [3]. The decreased n-6/n-3 PUFA ratio in the breast and thigh muscles observed in this study was largely related to the dietary n-6/n-3 PUFA ratio. This is because dietary FO, LO and MA supplementation can significantly increase the n-3 PUFA and decrease the n-6 PUFA deposition in chicken muscle [30]. The beneficial nutritional recommendations in human diets of the PUFA/SFA ratio should be above 0.45, whereas the n-6/n-3 PUFA ratio should not exceed 4 and should be nearly 3:1 to 1:1. These ideal ratios could effectively optimize the bioavailability, metabolism, and incorporation of fatty acids into membrane phospholipids [31,32]. Interestingly, the PUFA/SFA ratio and n-6/n-3 PUFA ratio obtained in the breast and thigh muscles of broilers in the current study almost met the recommended guidelines, which indicates that the chicken meat harvested from broilers offered MS-or FS-supplemented diets was more nutritious for human health than broilers fed with traditional SO.
Poultry in traditional production operations may face oxidative stress and lipid oxidation, which may lead to the production of reactive oxygen species. The results in the present study demonstrated that dietary n-3 PUFA supplementation could effectively negate some of these issues, since n-3 PUFA from FO, MA and LO enhanced the antioxidant capacity of thigh and breast muscle in broilers [12]. The decreased concentration of MDA in the muscle reflected in n-3 PUFA could slow the lipid peroxidation in the muscle of broilers, while the enhanced activities of SOD and GSH-Px enzymes could work together to detoxify superoxide anions and hydrogen peroxide in cells of tissues. The increased concentration of T-AOC illustrates the improvement of the non-enzymatic antioxidant defense system in broilers [33].
The improved antioxidant status in the muscle of broilers supplemented with n-3 PUFA enriched diets was mainly due to the ability of n-3 PUFA to scavenge free radicals, H2O2 and lipid peroxides, as well as enhance the hepatic antioxidant enzymes, such as SOD and GSH-Px [34]. Moreover, n-3 PUFA could also suppress the production of pro-inflammatory cytokines (interleukin-6 and interleukin-1β) and tumor necrosis factor-α, resulting in the mitigation of increased oxidative stress and inflammatory insults while modulating lipid metabolism [35]. Furthermore, the improvement of antioxidant capacities in the muscle of broilers offered the ML-supplemented diet may help to prevent n-3 PUFA oxidation [36]. This result might also be due to the oil droplets encapsulated within the cell of MA and LO and their ability to reduce oxidative deterioration [11], as well as the effects of high-level DHA, vitamin A, vitamin E and β-carotene in MA [10,36].

Conclusions
The present research indicates that diets supplemented with 2% FO or a combination of 1% MA and 1% LO could improve the performance, serum glucose, antioxidant capacities and n-3 PUFA profile in the muscle of broilers, as well as reduce the relative weight of abdominal fat and total serum cholesterol content of broilers compared with diets containing traditional soybean oil.
Reducing n-6/n-3 PUFA ratio via the inclusion of 2% fish oil and a mixture of 1% microalgae and 1% linseed oil have been demonstrated as an effective method to improve antioxidant status in broilers and produce n-3 PUFA enriched chicken meat.