The current work evaluated the relative effects of the number of piglets in the litter of origin (high vs. low litter size) and the birthweight (low (LBW) vs. normal (NBW) in large litters) on postnatal performance and quality of carcasses and meat in purebred Iberian pigs. The results indicated that NBW piglets born in large litters had disparities in developmental patterns in weight and size, back-fat deposition, and fatty acid composition of viscerae compared to NBW piglets from small litters, which again were different from those found in LBW piglets when compared to their NBW counterparts. However, both growth patterns were altered and might indicate previous phases of metabolic disorders.Fatty pigs are characterized by a thrifty genotype, adapted to harsh environments based on changes in metabolism and energy saving. Thus, we hypothesized that feto-maternal energy partitioning in large litters might have postnatal effects that might be independent of intrauterine growth restriction (IUGR) processes. Hence, the current work reported the influence of two effects on postnatal performance and carcass and meat quality of purebred Iberian pigs: (a) the effects of the number of piglets in the litter (high vs. low litter size), and (b) the effects of birthweight (low (LBW) vs. normal (NBW)) in large litters. The results confirmed that NBW piglets born in large litters had differences in developmental patterns of weight, back-fat deposition, and fatty acid (FA) composition when compared to NBW piglets from small litters. These results were different from those found in LBW piglets when compared to their NBW counterparts, which showed an initial asymmetrical growth and altered muscle FA composition at slaughtering. The assessment of FA composition indicated better metabolic status in NBW piglets from large litters than in LBW piglets. These data support the concept that the prenatal environment, even when the individual may cope with it, inescapably affects postnatal life. There is currently an increasing demand for high-quality dry-cured products from traditional pig breeds like the Iberian breed. Such demands are transforming the traditional extensive management of this animal into more intensive systems, by adopting management strategies from modern breeds and selecting lines with higher prolificacy to improve farm profitability.Traditionally, the Iberian pig is characterized by lower prolificacy than modern commercial breeds, with around 6–7 piglets per litter ,2 due toThe problems derived from LBW have been widely studied in commercial lean pigs ,7, especthrifty genotype, adapted to harsh environments, which includes changes in genes and pathways driving metabolism and energy saving [In the case of the Iberian pig, there is scarce information about the effects of litter size and birthweight on postnatal development. There have been only a couple of studies using crossbred Iberian × Duroc individuals ,20, whicy saving ,22,23. TLEPR gene (homozygosity for LEPRc.1987T) by pyrosequencing, as previously described [Two experiments, involving 110 purebred Iberian piglets selected from 24 primiparous Iberian sows  from the Retinto strain of this breed, were developed for this study. These sows were genotyped for purity of breed by determining polymorphisms for ®, MSD, Boxmeer, The Netherlands). Sows were allocated to collective outdoor pens with around 7 m2 of surface area per animal until one week before expected parturition, after which these females were housed in single indoor pens of 5.49 m2 until piglets were weaned. The collective pens had individual feeders so, during pregnancy, each sow had her own diet consisting of 2 kg of standard grain-based food diet with mean values of 13.0% crude protein, 2.8% fat, and 3.00 Mcal/kg metabolisable energy. From farrowing to weaning, average daily feed intake was increased to 3.5 kg per sow. Further information on diets is provided in At the beginning of each trial, all of the females weighed around 130 kg and had a mean back-fat depth of around 40 mm. These females were selected, after pregnancy diagnosis on Day 35 of gestation, from a group of animals that were inseminated with purebred Iberian semen after estrus synchronization with altrenogest . From the first month after weaning, the piglets were fed a standard diet with mean values of 18% crude protein, 4.5% fat, and 3.35 Mcal/kg of metabolizable energy; average daily feed intake was 0.5 kg. From 60 to 120 days of age, the piglets were fed a diet containing mean values of 15.1% crude protein, 2.8% fat and 3.08 Mcal/kg of metabolizable energy; the amount of food offered and therefore average daily feed intake were recalculated with age, from 1 to 2.5 kg, to fulfil daily maintenance requirements. Further information on diets is provided in The experimental procedures were assessed and approved by the INIA Committee of Ethics in Animal Research and subsequently by the competent regional authority (report PROEX114/16), according to the Spanish Policy for Animal Protection (RD 53/2013), which meets the European Union Directive 2010/63/UE on the protection of research animals.The selection of the piglets for each experiment was done by prioritizing the LITTER-SIZE experiment  in order to exclude NBW pigs with extreme birthweight and size. Second, LBW and NBW piglets were selected from the same litters for the BIRTH-WEIGHT experiment . The remaining piglets were only maintained until weaning to avoid differences in the productive condition due to litter size during lactation.The first experiment (litter size) aimed to study the effects of the number of piglets in the litter of origin (high vs. low litter size), independently of birthweight, and included 54 piglets from small  and large litters . In order to avoid effects due to birthweight, the 54 piglets selected were to be similar in weight between both groups and representative of the average weight and size of the breed , and, to avoid effects of milk availability during lactation, underwent within-group fostering in order to equalize the number of piglets among sows.The second experiment (birthweight) aimed to study the effects of birthweight and included 18 LBW piglets  and 38 NBW piglets  from large litters (≥8 piglets/litter). The threshold for LBW was a birthweight more than one standard deviation below the average birthweight, adjusted by sex ,26. In aThe remaining 66 piglets were used to determine the effects of litter size and birthweight on juvenile development (patterns of growth and fattening and metabolic traits) and quality of carcass and meat at 180 days old . However, one male from a large litter and one female from a small litter, and two NBW females died due to accidents not related to the experimental design.Body weight and size  were measured at birth and again at 15 and 25 days old (when weaning was performed) to characterize the early postnatal performance. Subsequently, values for body weight and size, subcutaneous back-fat depth, and loin diameter were obtained twice monthly for the first three months after weaning  and monthly after that  to characterize the postnatal performance. Body weights were used to calculate intermediate and total average daily weight gain (ADWG) and fractional growth rates  during ® Systems Europe, Becton Dickinson, Meylan, France) from either the external jugular vein at 120 and 150 days old, or from the orbital sinus at 180 days old for animal welfare, as fattening of the neck made it difficult to get samples from the jugular. Samples were immediately centrifuged at 1500× g for 10 min, and the plasma was separated and stored at −20 °C until it was assayed. Plasma concentrations of parameters indicative of lipids , low-density lipoprotein cholesterol (LDL-c), and triglycerides) and glucose metabolism (glucose and fructosamine) were measured using a clinical chemistry analyzer , according to the manufacturer’s instructions.Indexes of lipids and glucose metabolism were determined for piglets sampled at weaning (25 days old) and then at 120, 150, and 180 days old. Blood samples were drawn between 9:00 and 10:00 a.m. after a fasting period of around 18 hours, as previously described , using hAt 25 and 180 days old, pigs were sequentially euthanized by stunning and exsanguination in compliance with RD53/2013 standard procedures. Immediately, weights of head, carcass, and total and individual viscerae  were determined. The ratios of carcass and viscera weight to body weight were also calculated. Afterwards, samples of brain, liver , muscle (gluteus medius (GM) and longissimus dorsi (LD) at the level of the last rib), and subcutaneous back-fat were obtained and stored at −20 °C to determine fat percentage and fatty acid (FA) composition. A second sample of LD muscle was used on the day of sampling to assess drip-loss capacity. The carcass ratio and data from muscles were used to evaluate carcass and meat quality.Fatty acids in the feed were identified, after extraction and methylation, using a gas chromatograph  with a capillary column . Data aThe lipids from brain, liver, and muscle  were extracted . Dependent variables at 180 and 25 days old  were assessed using two-way ANOVA in a general linear model in each independent trial. The LITTER-SIZE trial included sex  and litter size (<8/≥8 piglets/litter) effects and interactions. The BIRTH-WEIGHT trial included sex and birthweight (LBW/NBW) effects and interactions. Variables with changes over time  were also assessed using a repeated-measures ANOVA with the Greenhouse–Geisser correction in each independent trial, using the same fixed factors described for the previous analyses plus time and its interactions. When the interaction was significant, litter size or birthweight were studied within sex. The pig was the experimental unit, and statistical significance was accepted from p > 0.10). At birth, piglets selected from small litters showed higher body weight, trunk length, and thoracic circumference than piglets from large litters . The ADWG during lactation was greater in piglets from small litters than in their counterparts from large litters , but there were no differences in fractional growth rate ((p > 0.10); further data in p < 0.05 for all). However, piglets from small litters showed less total back-fat .The first trial (LITTER-SIZE) showed a mean of 4.8 ± 1.5 piglets in small litters and 8.8 ± 1.0 piglets in large litters, without significant differences regarding sex distribution . Subsequently, during lactation, NBW piglets had greater ADWG than LBW piglets , but there were no differences in fractional growth rate  than LBW piglets .The litters used for the second trial (BIRTH-WEIGHT) showed a mean of 9.0 ± 0.8 piglets, with 20.5% of them showing a birthweight lower than one standard deviation below the average birthweight . NBW piglets (representative of the breed at 1.3 ± 0.1 kg), as expected due to the study design, showed higher body weight, trunk length, and abdominal and thoracic circumferences than LBW piglets (p < 0.05) and size  from birth to slaughter at 180 days old showed significant differences between pigs from small and large litters, although without a different development in total ADWG or fractional growth rate during the period (p > 0.10). Piglets from large litters showed a greater ADWG than their counterparts from small litters during the post-weaning phase  and a greater fractional growth rate from 25 to 75 days old . The ADWG between 60 and 75 days old was also higher in piglets from large litters . Consequently, body weight and size (p < 0.01). Subsequently (from 75 to 180 days old), pigs from small litters had greater ADWG than pigs from large litters  and greater fractional growth rate than pigs from large litters , and reached higher body weight (p < 0.05) and size at 180 days old (p < 0.01). There were no differences in body weight and thoracic circumferences from 45 to 150 days old (p > 0.10). Interactions with sex were not found either (p > 0.10).In the first trial (LITTER-SIZE), the evaluation of body weight (and size  were simp < 0.01 for both), with significantly higher values in pigs from small litters at 60 and 180 days old , but higher values in pigs from large litters at 75 days old . On the other hand, the assessment of loin diameter also showed higher values in pigs from small litters than in pigs from large litters at 150 days old , without sex-related differences (p > 0.10). There were no differences in the outer layer depth of back-fat (p > 0.10), nor in loin diameter at other time points (p > 0.10). The assessment of subcutaneous back-fat depth evolution showed differences for total depth and inner-layer depth betweenn pigs from small litters and pigs from large litters (p > 0.10). Pigs with LBW only showed greater fractional growth rates from 25 to 75 days old and overall . NBW pigs maintained higher values for body weight and size without different evolution until 150 days old, when no significant differences were found , but without differences in adiposity (p > 0.10) nor at other time points .In the second trial (BIRTH-WEIGHT), NBW and LBW pigs had similar ADWG without differences (p < 0.05) and no significant differences in lipid metabolism  and LDL-c concentrations , without differences in the glucose metabolism (p > 0.10).At weaning, piglets from large litters in the LITTER-SIZE trial showed greater fructosamine levels than piglets from small litters (p > 0.10). Regarding lipid indexes, pigs from large litters showed greater total cholesterol levels at 120 and 150 days old . Pigs from small litters showed higher plasma LDL-c concentrations than pigs from large litters at 150 days old, but pigs from large litters had higher LDL-c levels than pigs from small litters at 180 days old (p < 0.05 for both). On the other hand, there were no differences in metabolic status when comparing NBW and LBW pigs (p > 0.10). Finally, no interactions with sex were found in either of the experiments (p > 0.10).After lactation, the assessment of plasma parameters showed no effects of litter size on glucose metabolism (p < 0.05). There were more profound effects in the birthweight trial because LBW piglets had higher relative weights of head, heart, liver, kidneys, adrenal glands, and pancreas than NBW piglets . No interactions with sex-related effects were found (p > 0.10).In the first trial (LITTER-SIZE), there were scarcely any differences between piglets from small and large litters; only the relative weight of pancreas to body weight was higher in piglets from large litters , but found significant differences in liver and muscles .In the case of LITTER-SIZE, the prolificacy affected the polar lipid fraction of the liver but not the neutral fraction. In this way, piglets from small litters showed higher SFA and MUFA levels and lower total PUFA and ∑n−3 and -6 FA concentrations (p < 0.05). There was a significant interaction with sex because females from large litters showed, in the neutral lipid fraction of both muscles (LD and GM), higher SFA concentrations  and lower desaturation indexes  than males from large litters. Finally, the polar lipid fraction of GM also showed greater MUFA levels and desaturation indexes and lower PUFA concentrations in piglets from small litters than in piglets from large litters . In all muscle lipid fractions, the desaturation index C18:1/C18:0 was higher in piglets from small litters than in piglets from large litters . However, LBW piglets showed higher MUFA concentrations and desaturation indexes than NBW piglets in the neutral lipid fraction, and higher Σn−3 and -6 FA concentrations in the polar lipid fraction of liver .In the case of BIRTH-WEIGHT, there were differences in all lipid fractions of liver and muscle between LBW and NBW piglets. NBW piglets had higher SFA levels than LBW piglets in both lipid fractions of the liver . However, LBW piglets showed a greater Σ-n6/Σ-n3 FA ratio than NBW piglets in the polar lipid fraction of both muscles (p < 0.05 for both). Moreover, in the polar lipid fraction of LD, LBW piglets had higher concentrations of MUFA and lower concentrations of Σn−6 FA than NBW piglets (p < 0.05 for both). LBW piglets also showed higher humidity in the LD muscle than NBW piglets (p > 0.1). Furthermore, there was also a significant interaction with sex, since NBW females showed a greater Σ-n6/Σ-n3 FA ratio than NBW males in the polar lipid fraction of LD .With regards to muscles, NBW piglets had higher ratios of Σ-n6/Σ-n3 FA and C18:1/C18:0 than LBW piglets in the neutral lipid fractions of both muscles (p > 0.1).On the other hand, there were no differences in the carcass weight and ratio between NBW and LBW pigs in the birthweight trial (p > 0.10). However, NBW pigs showed higher relative weights of intestine and kidneys to body weight than LBW pigs .In the LITTER-SIZE trial, there were no significant differences in carcass weight between pigs from small and large litters at 180 days old . Pigs from small litters also showed higher Σn−6 FA levels than pigs from large litters in the neutral lipid fraction of brain (p < 0.05). Moreover, pigs from large litters had greater amounts of Σn−3 FA in both lipid fractions of liver  and a higher desaturation index  in the polar lipid fraction of brain than pigs from small litters. Finally, there were again sex-related effects, since the ratio Σn−6/Σn−3 FA was greater in the polar lipid fraction of liver in males than in females from small litters .There were differences between pigs from small and large litters in the LITTER-SIZE trial when assessing the FA composition of brain and liver. For both viscerae, ratios of Σn−6/Σn−3 FA from neutral and polar lipid fractions were greater in pigs from small litters than in pigs from large litters . Pigs from large litters also had a higher desaturation index (C18:1/C18:0) and lower SFA levels in the outer layer (p < 0.01 for both).The assessment of FA composition in muscle showed that pigs from small litters had greater fat content than pigs from large litters in LD muscle (p < 0.05), without differences in GM muscle (p > 0.10). Pigs from large litters showed lower SFA levels and higher desaturation indexes than pigs from small litters in the neutral lipid fractions of both muscles . Pigs from large litters also had greater MUFA and Σn−3 FA concentrations in the neutral lipid fraction of LD, and a lower Σn−6/Σn−3 ratio in its polar lipid fraction .In the LITTER-SIZE trial, the assessment of FA composition in the subcutaneous fat showed that both back-fat layers had greater levels of PUFA, both Σn−6 and Σn−3 FA, in pigs from large litters than in pigs from small litters (p < 0.01). Assessing the neutral lipid fraction of GM, pigs from large litters showed greater PUFA concentrations, both for Σn−6 and Σn−3 FA, and higher Σn−3 FA levels than pigs from small litters . Finally, pigs from small litters had lower SFA concentrations and higher MUFA concentrations and desaturation indexes in the polar lipid fraction of GM than pigs from large litters .Moreover, the desaturation index C18:1/C18:0 in the neutral lipid fraction of LD was higher in females than in males in the case of large litters ; however, there were no differences when assessing the IMF of LD (p > 0.10). Differences in FA composition were mainly found in the neutral lipid fraction of LD muscle; LBW pigs had greater MUFA concentrations and desaturation indexes and lower SFA levels and Σn−6/Σn−3 FA ratio than NBW pigs (p < 0.05 for both). No interactions with sex were found in main proportions and indexes of FA (p > 0.10).In the BIRTH-WEIGHT trial, the amount of IMF in the case of the GM was higher in LBW pigs than in NBW  and subsequent lowBW (LBW) with its inherent complications. Piglets with normal BW (NBW) but born within large litters (larger than eight piglets), which implies a lower availability of uterine space and therefore of nutrients and oxygen, had differences in developmental patterns, fat deposition, and FA composition when compared to NBW piglets from small litters. These data support the notion that the prenatal environment, even when the individual may cope with it, inescapably affects postnatal life.The current study included two different trials aiming to determine, in animals of the same breed maintained under same environment and management conditions, the relative weight of the effects of either BIRTH-WEIGHT (by comparing NBW and LBW piglets) or the LITTER-SIZE (availability of uterine space and resources) independently of the birthweight .The results of the experiment aiming to determine the effects of BIRTH-WEIGHT showed, as expected, similar results to previous studies on both commercial lean strains ,13 and tAfter weaning, during the juvenile period, NBW pigs remained significantly heavier and larger until 75 days old . Afterwards, differences in body weight and corpulence between NBW and LBW pigs were not statistically significant and the relative sizes of carcass and viscerae were ultimately similar in both groups, except for intestine and kidneys, with lower relative weights in LBW than in NBW at 180 days old, these being two of the systems more affected in case of IUGR ,39. We aThe results of the experiment aiming to determine the influence of LITTER-SIZE showed that piglets from large litters, similarly to LBW piglets in the BIRTH-WEIGHT trial, showed the worst ADWG during lactation and, therefore, the lowest body weight and size at weaning. However, in contrast to the LBW piglets of the BIRTH-WEIGHT trial, piglets from large litters showed higher adiposity than piglets from small litters at weaning,without evidence of asymmetrical growth patterns between groups except in the pancreas. It was not possible to elucidate the metabolic implications of differences in the size of the pancreas under the design of the current study; however, it is known that this organ is one of the most affected in case of prenatal programming, with changes having a prominent role in metabolic disturbances ,46.Subsequently, during the postnatal period, piglets from large and small reached similar values of body weight and size in spite of very different patterns of ADWG. Pigs from large litters had a more precocious weight gain, with higher fractional growth rate, during the post-weaning phase (25–75 days old), and even higher adiposity and larger abdominal circumference at 75 days old . AfterwThe comparison of growth patterns in LBW piglets in the BIRTH-WEIGHT trial and NBW piglets from large litters in the LITTER-SIZE experiment suggested two very different developmental patterns. In brief, LBW piglets were still affected by growth restriction during the post-weaning period (25–75 days old), in spite of developing a higher fractional growth rate than NBW counterparts. They also showed compensatory catch-up growth only during the late-growing period (75–180 days old), as already observed in previous studies in LBW Iberian crossbred pigs . On the The assessment of FA composition at weaning showed, besides similarities in the developmental pattern during lactation, analogous changes in LBW piglets from the BIRTH-WEIGHT trial and piglets from large litters in the LITTER-SIZE experiment when compared to their respective NBW and small-litter counterparts. In brief, there were no differences in the FA profile of the brain among piglets in both trials. On the other hand, the assessment of the polar lipid fraction of the liver showed a decrease in SFA levels with increases in Σn−3 and Σn−6 FA in both LBW and large-litter piglets; piglets from large litters also showed a decrease in MUFA values. The assessment of the neutral fraction of the liver showed no changes in piglets from large litters, but LBW piglets showed increases in MUFA content and desaturation index, which it is a feature commonly related to metabolic disorders such as obesity and insulin resistance in both humans ,49 and pThere were no main differences between LBW and NBW pigs in the BIRTH-WEIGHT trial in the FA profile of brain, liver, and subcutaneous fat—results which are very different from previous studies in Iberian pigs ,20. HoweOn the other hand, there was a plethora of differences in the FA composition of pigs from large and small litters in the LITTER-SIZE experiment. Viscerae (brain and liver) of pigs from large litters showed a decrease in Σn−6/Σn−3 FA ratio in both neutral and polar lipid fractions, which was related to a decrease in Σn−6 FA concentrations in the brain and an increase in Σn−3 levels in the liver. These changes were all protective for the individual. Higher availability of Σn−3 has been found to improve pro-/anti-inflammatory status, insulin function, and physical and mental development during the first years of life ,56,57; hAssessment of the FA content of triglycerides in the subcutaneous fat showed increases of PUFA, Σn−6, and Σn−3 FA values in both outer and inner layers of pigs from large litters. The outer layer also showed a decrease in the SFA content and an increase in the desaturation index. The same changes  were found in the neutral fraction (triglycerides) of the GM muscle. This finding is logical from the physiological point of view, since FAs are mostly distributed in the neutral fraction  and, beHence, the FA profile found in NBW piglets from large litters was mostly protective for their metabolic health (higher levels of PUFA and Σn−6 FA levels with lower ratios Σn−6/Σn−3 FA and lower content of SFA); only the increase in desaturation indexes was a warning. On the other hand, from a productive point of view, increases in the PUFA content and decreases in the SFA content and Σn−6/Σn−3 FA ratio found in the muscle and subcutaneous fat this imply health and sensorial benefits and therefore enhance consumer acceptance ,65,66.In conclusion, piglets with NBW from large litters, in opposition to LBW piglets, are prone to higher adiposity than piglets from small litters as early as during the suckling period. After weaning, these piglets show a very early catch-up growth after exposure to high-energy feed during the post-weaning period (25–75 days old), which may be related to insulin resistance. Afterwards, during the juvenile period (fed with maintenance diets), the catch-up growth slows down and, based on the FA composition assessment, we inferred a better metabolic status in NBW piglets from large litters than in LBW piglets, independently of the litter size. These results, overall, show clear effects of the litter size on the postnatal development in NBW pigs, which are independent of effects due to LBW.


Dental universal adhesives are considered an useful tool in modern dentistry as they can be used in different etching techniques, allow for simplified protocol and provide sufficient bond strength. However, there is still no consensus as to their toxicity towards pulp. Thus, the present study aimed to evaluate the cytotoxicity and genotoxicity of three universal adhesives: OptiBond Universal, Prime&Bond Universal and Adhese in an in vitro experimental model, monocyte/macrophage cell line SC (ATCC CRL-9855). The cytotoxicity was measured by means of XTT assay, whereas the genotoxicity (comet assay) was evaluated based on the percentage of DNA present in the comet tail. Furthermore, the ability of the adhesives to induce apoptosis was analyzed using flow cytometry (FC) with the FITC annexin V/propidium iodide (PI) double staining. The analysis of the cell cycle progression was performed with FC using PI staining. OptiBond Universal presented significant, while Prime&Bond Universal and Adhese Universal had minimal cytotoxicity and genotoxicity towards human SC cells. Moreover, only OptiBond Universal increased the level of apoptosis in SC cell line. None of the adhesives showed significant cell cycle arrest, as revealed by FC analysis. Due to substantial differences in toxicity in in vitro studies of dental adhesives, there is a great need for further research in order to establish more reliable test protocols allowing for standardized methodology. Adhesive dentistry constitutes one of the major branches of the dentistry that is mainly focused on the development of materials that establish an effective bond with tooth tissue. Inasmuch, dental adhesive systems have grown in popularity worldwide and nowadays attract a significant research interest ,2. DentaThe ever-increasing demand for simplified adhesive systems has resulted in the development of a next generation dental adhesives termed universal adhesives. However, the development of universal adhesive systems constituted a great innovation in adhesive dentistry, it is still unknown whether universal adhesive systems may be use in all adhesive procedures, since their detailed characterization is still required . General2 cements [2 cements [Due to the fact that dental adhesives are in direct contact with hard and soft dental tissues, their biocompatibility is extremely important . There a cements ,29,30,31 cements . Scienti cements ,34,35. O cements . The mai cements . Compone cements . Mention cements .In order to precisely define the biologic properties of the investigated material in in vitro experimental models, various research methods should be used including cytotoxicity and genotoxicity assays, apoptosis detection tests as well as evaluation of the cell cycle progression. Both in vitro and in vivo studies give an opportunity for the evaluation of different characteristics of the material ,40,41. SWith regards to all mentioned aspects, the main aim of the present research constitutes a detailed characterization of the properties of dental universal adhesives in a highly standardized, in vitro experimental model.Obtained XTT assay outcomes showed significant differences in the cytotoxic properties of the investigated compound eluates. Monocyte/macrophage peripheral blood SC (ATCC CRL-9855) cells were incubated with the investigated compounds for 24 h. The obtained results showed that only OptiBond Universal significantly decreased their viability. Both in the case of Prime&Bond Universal and Adhese Universal, no significant changes of cell viability were observed .The level of DNA damage was estimated using the alkaline version of the comet assay. The alkaline comet assay enables detection of oxidative DNA damage, single- and double-stranded breaks—as well as presence of alkaline labile sites. The amount of DNA damage was assessed based on the percentage of DNA in the comet tail. The significant increase in DNA damage in SC cell line for OptiBond Universal was observed after 24 h incubation. Both Prime&Bond Universal and Adhese Universal did not induce a significant DNA damage in the tested cell line .In order to assess activation of apoptosis in the SC cell line after exposure to OptiBond Universal, Prime&Bond Universal and Adhese Universal, the FITC annexin V/propidium iodide (PI) double staining and subsequently FC analysis were performed. The 34.96% of SC cells treated with 1-µM staurosporine for 16 h underwent apoptosis than control cells cultured in complete medium for 24 h. After 24 h incubation, only in the case of OptiBond Universal the apoptosis was induced in SC cells (approximately 45% of cells were at the early and late stages of apoptosis) . Prime&BThe cell cycle distribution of PI-stained SC cell line after 24 h exposure to OptiBond Universal, Prime&Bond Universal and Adhese Universal was analyzed by FC. As predicted, cell cycle of SC cells exposed to 1 μM nocodazole was arrested at G2/M phase. The cell cycle progression of SC cells treated with the investigated compounds was similar to SC cells cultured in the complete medium. Thereby, none of the tested compounds induced a significant G2/M cell cycle arrest in SC cells .The dental adhesives have to be examined with highly standardized experimental model with regards to their direct contact with dental tissues. Scientific data concerning the biocompatibility of dental universal adhesives is contradictory. For instance, several research data indicate that the universal adhesives could not be used for direct pulp capping due to the reported inflammation ,34,58. NThe results of the present study regarding the biocompatibility of universal bonding systems showed consistency in all conducted tests. Prime&Bond Universal and Adhese Universal presented minimal cytotoxicity and genotoxicity towards human SC cells in colorimetric XTT assay. Interestingly, OptiBond Universal presented significant cytotoxicity and genotoxicity towards SC cell line. Moreover, only OptiBond Universal showed significant ability to induce apoptosis in SC cell line. On the other hand, none of the tested adhesives showed significant cell cycle arrest in G2/M phase in FC analysis. We suggest that the differences in toxicity of the tested dental bonding systems may resulted from their composition, i.e., acidic monomers and other compounds.The biocompatibility assessment is crucial for the clinical validation of dental materials and to date, only several studies investigated the cytotoxicity and genotoxicity of dental bonding systems ,60. In vThere are different cell types that may be used in in vitro studies on cytotoxicity and genotoxicity. Most studies regarding the toxicity of dental bonding systems were conducted on cells derived from pulp or soft tissues of the oral cavity , whereasHowever, cells cultured in vitro for many generations undergo genomic transformations and/or mutations and thus are not reliable for studies regarding the DNA damage. As far as genotoxicity is concerned, the most preferable studies are those using the diploid cell lines such as human leukocytes . In commIn recent years there were limited studies on dental bonding systems by means of highly sensitive comet assay, which showed insignificant increase of their genotoxicity in human blood cells . TherefoPrevious studies showed that bis-GMA presents the highest cytotoxicity followed by TEGDMA, UDMA and HEMA, which are, on the other hand, moderately cytotoxic ,74,75,76It was demonstrated that typical components of dental bonding systems as well as restorative materials, such as HEMA and TEGDMA, are able to spread through the dentin tubules and thus they reach the pulp tissue at millimolar concentrations. Even negligible levels of mentioned monomers demonstrated an ability to arrest the proliferation of pulp cells ,81. Othein vitro. In other studies, dental adhesives containing MDP showed varied cytotoxic effect, ranging from significant cytotoxicity for Clearfil Liner Bond 2 V, than ED Primer II [Some studies presented that the cytotoxic effect of dental bonding systems and resin monomers are linked to the cell cycle arrest at specific cell cycle phases ,86,87. Trimer II , to the rimer II .The presented study has a specific limitation. The in vitro model shows limited information on the number of residual monomers and other components in the mixture of the adhesives that can penetrate through the dentinal tubules to the dental pulp. There are several variables that affect this leaching including the thickness of dentine, exposed surface area, presence of smear layer ,88. The In the present study, three universal bonding systems were analyzed: OptiBond Universal, Prime&Bond Universal, Adhese Universal .2; 95% humidity), according to the guidelines provided by the vendors. Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 4-mM l-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.05-mM 2-mercaptoethanol, 0.1-mM hypoxanthine and 0.016-mM thymidine (90%); fetal bovine serum (10%).All investigations were performed in in vitro experimental model using a commercially available monocyte/macrophage peripheral blood cell line—SC (ATCC CRL-9855) purchased from the American Type Culture Collection . Cell culture were maintained under standard conditions  according to the manufacturer’s instructions. Then 1 mL of medium was added and incubated for 24 h at 37 °C. The obtained eluate, after centrifugation, was prepared for further experiments.The amount of 50 µL of each investigated dental bonding system was placed in round bottom of Eppendorf tubes and polymerized (LED lamp intensity over 1000 mw/cm3 cells/well) of cell suspension in complete medium, 50 µL of prepared eluate. The positive control constitutes cells suspended in 96% isopropyl alcohol, that high concentrations are highly toxic to cells leading to their lysis, whereas the negative control cells cultured in a complete medium. The analyzed samples were incubated for 24 h. Subsequently, 25 µL of XTT/PMS mixture was added to each well. After 4 h incubation, absorbance was measured at a wavelength of 450 nm using Synergy HT (BioTek) spectrophotometer.The cytotoxicity of the investigated compounds was measured using the XTT colorimetric assay . XTT is used to assess cells’ viability as a function of redox potential. Actively metabolizing cells reduce the tetrazolium salt (XTT) to an orange water-soluble formazan. All of the experiments were performed in triplicate with similar results. Test samples were prepared in 96-well plates by adding up to 50 µL , that high concentrations are highly toxic to cells leading to their lysis, were used as a positive control, whereas cells suspended in 1 mL of complete culture medium as a negative control. All samples were incubated for 24 h. Cell suspension in 0.37% LMP agarose  was placed on microscope slides previously coated with NMP agarose . Preparations were incubated in lysis buffer at pH 10  containing TritonX-100  at a final concentration of 1% at 4 °C for 60 min. After 1 h incubation, the preparations were incubated 20 min in development buffer  at 4 °C, followed by electrophoresis  at 4 °C in electrophoretic buffer . After staining with a DAPI fluorescent dye, preparations were analyzed under a fluorescent microscope. Cell damage was evaluated based on the percentage of DNA in the comet tail.The genotoxicity of analyzed compounds was assessed using a comet assay. Comet assay is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells. Assays were prepared in 12-well plates by adding 5 × 106 cells/well) plated on 12-well plates were incubated with previously prepared compounds filtrates diluted in ratio 1:1 with medium for 24 h. Cells treated with staurosporine  at a concentration of 1 µM for 16 h constituted a positive control, whereas a negative control cells suspended in complete culture medium and incubated for 24 h. Subsequently cells were washed with cold PBS  twice and double stained with annexin V, as a marker of early apoptosis and PI as a marker of cell membrane disintegration, necrosis and late apoptosis. The percentage level of apoptotic cells was analyzed by FC using the Beckman Coulter CytoFLEX. The obtained data were analyzed using the Kaluza analysis 1.5 A software (Beckman Coulter).Apoptotic cell death induced by filtrates of test compounds was assessed using FITC Annexin V Apoptosis Detection Kit I purchased from BD Pharmingen™ . This method is based on high affinity of Annexin V to phosphatidylserine which, as a result of induction of apoptosis, is translocated to the outer parts of the cell membrane as well as on the propidium iodide (PI) that constitutes a marker of cell membrane permeability. SC cells (1 × 106 cells/well) and incubated with the previously prepared compounds eluate diluted in ratio 1:1 with medium for 24 h. Cells treated with 1 µM nocodazole  for 16 h served as a positive control, whereas cells cultured in complete medium for 24 h as a negative control. Cells were washed twice with cold PBS  and fixed with ice-cold 70% ethanol at −20 °C for 20 min. Subsequently, cells were treated with RNase A DNase&Protease-free (10 mg/mL)  and incubated at 37 °C for 1 h before staining with PI solution (10 μg/mL)  staining. Cells were seeded on 12-well plates . Each of the analyzes in individual experiments was based on the results of three independent tests. The differences were statistically significant on the graphs as follows: * It may be concluded that universal bonding systems vary both in cytotoxic and genotoxic effect on human monocyte/macrophage peripheral blood SC cells. Prime&Bond Universal and Adhese Universal presented minimal toxic effect on human SC cells, while OptiBond Universal showed significant cytotoxic and genotoxic effect on SC cell line. Furthermore, only OptiBond Universal showed significant ability to induce apoptosis in SC cell line. None of the compounds showed an ability to arrest the cell cycle in G2/M phase. Studies concerning the cytotoxicity and genotoxicity of dental bonding systems vary in methodology and present different results. Therefore, there is a great need for establishing more reliable test protocols suitable for conducting standardized research on various dental materials including dental bonding systems. According to our results, studies concerning the biologic impact of dental bonding systems should be conducted using various tests including cytotoxicity, genotoxicity, apoptosis detection and cell cycle analysis studies. Colorimetric tests based on the cell metabolism may not provide a reliable result, since dental universal adhesives alter the color of the mixture which may have a significant impact on the obtained results. Thereby, the flow cytometry analysis in dental material research may provide more accurate evaluation and should be recommended by international standards such as ISO. Thus, the present study is the first that evaluate the dental bonding systems by multiple in vitro assays as well as suggest that currently used research techniques should be extended and incorporated into the standardization criteria.


Although AGC, BGC, and SOC showed different partitioning patterns along the altitudinal gradient both in Ecuador and Peru, total carbon stock did not change with altitude in either site. The combination of annual mean temperature and precipitation seasonality explained differences in the observed patterns of carbon stocks across forest compartments between the two sites. This study suggests that the greater precipitation seasonality of colder, higher altitudes may promote faster turnover rates of organic matter and nutrients and, consequently, less accumulation of SOC but greater AGC and BGC, compared to those sites with lesser precipitation seasonality. Our results demonstrate the capacity of TMFs to store substantial amounts of carbon and suggest the existence of a trade-off in carbon stocks among forest compartments, which could be partly driven by differences in precipitation seasonality, especially under the colder temperatures of high altitudes.Tropical montane forests (TMFs) play an important role as a carbon reservoir at a global scale. However, there is a lack of a comprehensive understanding on the variation in carbon storage across TMF compartments  along altitudinal and environmental gradients and their potential trade-offs. This study aims to: 1) understand how carbon stocks vary along altitudinal gradients in Andean TMFs, and; 2) determine the influence of climate, particularly precipitation seasonality, on the distribution of carbon stocks across different forest compartments. The study was conducted in sixty 0.1 ha plots along two altitudinal gradients at the Podocarpus National Park (Ecuador) and Río Abiseo National Park (Peru). At each plot, we calculated the amount of carbon in AGB , BGB , and soil organic matter . The mean total carbon stock was 244.76 ± 80.38 Mg ha HoweverThis study aims to fill some of the existing gaps of knowledge by investigating the role of Andean TMFs as carbon reservoirs. The specific goals were: (1) to understand how carbon stocks from the different forest compartments  and total carbon stock vary along the altitudinal gradient; and (2) to determine the influence of climate—particularly temperature and precipitation seasonality—on carbon stocks. Understanding these carbon trade-offs should be a priority when formulating policies and designing conservation and management plans aimed at mitigating consequences of environmental change .ca. 2000 m) of continuous forest cover, each within a single river basin: the Bombuscaro river in Ecuador and the Montecristo–Abiseo rivers in Peru. Three altitudinal belts were studied in each site: low , middle , and high . Sixty 0.1 ha (50 × 20 m) plots were established between 2015 and 2017: 10 plots within each belt at each site, at least 300 m apart  and Río Abiseo National Park (Peru). These sites extend along wide altitudinal ranges . Annual mean temperature was highly correlated with the rest of temperature-related bioclimatic variables . Precipitation seasonality was selected because it can have an important effect on soil mineralization rates and nutrient availability for plants, even though moist TMFs are not subjected to long periods of water deficit and thus its vegetation does not display adaptations to such conditions, like deciduousness. Precipitation seasonality was calculated as the standard deviation of the monthly precipitation estimates expressed as a percentage of the mean of those estimates . Thus, a higher value of precipitation seasonality means that the total monthly precipitation is more heterogeneously distributed across time. For instance, 50% of precipitation seasonality means that this variability represents 50% of the mean monthly precipitation. Because of the properties of the normal distribution, this implies that differences between the rainiest and driest months could be—with 95% confidence interval—as high as twice (i.e. 200%) the mean monthly precipitation value, whereas if precipitation seasonality were 10% this difference would be only of 40% of the mean monthly precipitation value.We retrieved bioclimatic variables from the CHELSA climatological dataset  for eachIn each plot, all standing woody stems  ≥ 2.5 cm in diameter at breast height  were recorded. For each stem, height was estimated visually since the use of precise instruments proved impractical. For individuals with multiple stems, we used the height of the tallest stem as the individual's height and the square root of the sum of the squares of DBH of each stem as the overall individual's DBH . Field mca. 10 cm long were stripped of cortex, and their volumes calculated as cylinders by measuring their diameter and length fresh. Branch wood density was calculated dividing fresh volume by dry mass after drying at 80°C for 48–72 h. Mean WD and its standard deviation (SD) were calculated for each species using sample values of all conspecifics. For species with just one sample, SD was estimated by multiplying WD by the SD/mean ratio averaged across all the samples in the entire dataset, where SD/mean ratioEcuador = 0.200 and SD/mean ratioPeru = 0.234. Individuals for which WD values were lacking  were assigned the mean WD of their family and their SD estimated as above. The latter included 611 individuals in 57 taxa from Ecuador  and 735 in 59 taxa from Peru (8.5%). Finally, the 63 individuals from Ecuador  and 117 from Peru  with no WD measurement that could not be identified to family or belonged to families with no WD data , were removed from the analyses.Branch wood density (WD) was measured based on We used Chave's three-variable pantropical allometry model  to estimWD wood density (g·cm–3), DBH diameter at breast height (cm), and H is height (m). We then calculated the plot AGB as the sum of the AGB of all trees within each plot. We used the R package “BIOMASS”  was estimated using We assumed that the aboveground carbon stock (AGC) of a plot accounted for the 50% of its AGB, following Mt is the total oven-dry mass of the three cores (g), and Vi the volume of each core i (cm3). Finally, the soil organic carbon stock (SOC) (Mg ha–1) was calculated following Soil samples were collected from the surface (0-15 cm) below the decomposing organic layer  in each plot. Soil samples, consisting of five different subsamples from five different points in the plot, were collected, mixed, air-dried and sifted through a 2-mm sieve . Soil pHC is soil organic carbon concentration (g kg–1) in the soil layer, BD bulk density (g cm–3), LTH layer thickness (cm), and FE content the relative contribution of fine earth fraction  to total soil mass. Despite important variations in BD across sites and altitudinal ranges  was used to assess the goodness of fit of the model:Generalized linear models (GLMs) with a Gamma error distribution were fitted to determine how carbon stocks vary in the different compartments of the forest and along the altitudinal gradient separately for Ecuador and Peru. Quadratic terms of the explanatory variables were included to account for non-linear relationships. To understand the role of climate on the carbon stocks of the different compartments, we fitted another set of GLMs with a Gamma error distribution to relate each of the three carbon stock response variables, as well as total carbon stock, to annual mean temperature (°C) and precipitation seasonality, as well as their interaction, using data from both sites. The significance of predictors was tested using the Chi-squared statistic (α ≤ 0.05), and the explained deviance  than in the Ecuadorian site   and tree height increased in Ecuador from low to mid altitudes and then decreased again at high altitude, whereas in Peru these values did not change much across altitudinal belts , and from 116.73 to 318.52 Mg ha–1 in Peru . The mean total carbon stock found across all study sites was 229.02 ± 68.06 Mg ha–1. All plot-level estimations of AGC, BGC, and SOC are available in AGC ranged from 16.82 to 222.07 Mg haan plots Figure 1–1) and middle altitudes (88.03 ± 51.39 Mg ha–1) from Ecuador, and decreased by ca. 73.2% at high altitude , decreasing by ca. 6% at mid altitude (86.97 ± 117.86 Mg ha–1) and ca. 15.7% at high altitude ; and middle altitudes (70.37 ± 23.85 Mg ha–1) and decreased by 49.7% at high altitude  and middle altitudes  and the lowest mean SOC at low altitude , though the differences across altitudinal belts were not as large as in Ecuador  did not vary with altitude, neither in Ecuador nor in Peru Table 1.Annual mean temperature and precipitation seasonality, but not their interaction, had a statistical significant effect on AGC, BGC, and SOC. There was, however, no effect of either climate variable or their interaction on total carbon stocks Table 2.Our results show that carbon storage at each compartment of the forest  follows a distinct pattern along the altitudinal gradient that differs between sites.ca. 1500 m). This result is similar to the unimodal patterns reported in other studies  are somewhat lower than those reported for other Andean TMFs  was within the low to middle range (87–754 Mg ha–1) reported in a meta-analysis of TMFs by –1; 2 . GB-D-D was funded through a PhD grant by the Spanish Ministry of Education (FPU14/05303).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wooden breast (WB) is a novel myopathy affecting modern broiler chickens, which causes substantial economic losses in the poultry industry. The objective of this study was to evaluate the effect of WB abnormality on meat quality, redox status, as well as the expression of genes of the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway.A total of 80 broilers  raised at Jiujin farm  were used. Twelve unaffected (no detectable hardness of the breast area) and twelve WB-affected (diffuse remarkable hardness in the breast muscle) birds were selected from the commercial broiler farm according to the criteria proposed by previous studies.The results indicated that WB showed histological lesions characterized by fiber degeneration and fibrosis, along with an increase of muscle fiber diameter (p<0.05). Moreover, higher pH value, lightness, yellowness, drip loss and cooking loss were observed in the WB group (p<0.05). Compared with the normal breast (NOR) group, the WB group showed higher formation of reactive oxygen species (p<0.05), increased level of oxidation products and antioxidant activities (p<0.05), accompanied with mitochondrial damages and lower mitochondrial membrane potential (p<0.05). Meanwhile, the relative mRNA expressions of Nrf2 and its downstream antioxidant genes including heme oxygenase-1, NAD(P)H qui none dehydrogenase 1, glutathione peroxidase, superoxide dismutase, and glutamate-cysteine ligase were higher than those of the NOR group (p<0.05).In conclusion, WB myopathy impairs meat quality by causing oxidative damages and mitochondrial dysfunction in broilers, even though the activated Nrf2/antioxidant response element pathway provides protection for the birds. In particular, chicken meat has become one of the most popular muscle foods, owing to its relatively low cost, high nutritive value and suitability for further processing . With thindustry ,4. Althoindustry .pectoralis major muscle [Up till now, several studies carried out to detect differential genes, proteins and metabolites related to the occurrence of WB have evidenced a complex etiology of this defect –8. Howevr muscle . Therefor muscle . Generalr muscle .The excessive production of ROS can stimulate the antioxidant defense system, of which, the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway has been identified as an important endogenous mechanism for suppressing oxidative stress . The NrfYet, the high incidence of WB myopathy leads to an increasing concern for producers and consumers. However, to our knowledge, limited studies have been conducted concerning the redox status in WB abnormality. Therefore, the purpose of this study was to investigate the oxidative status of broilers based on the categorized normal and WB samples, and to evaluate the potential protective roles of Nrf2/ARE pathway in alleviating this myopathy, which can provide a better understanding of the relationship between redox status and meat quality changes in WB abnormality.All experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University , and the serial number of the laboratory animal use certificate issued by Science and Technology department of Jiangsu province is SYXK (Su) 2017–0007.For this study, a total of 80 broilers  raised at Jiujin farm  were used. The broilers were raised on a basal diet formulated according to the NRC (1994) nutrient requirements for 1 to 21 days starter diet and 22 to 42 days grower diet. The composition and nutrition levels of the basal diet are given in pectoralis major muscle.According to the criteria proposed by previous studies, broilers were separated depending on visual observations for posture and manual palpation of the breast muscle in a cranio-caudal direction: unaffected chickens are able to lift wings easily and no detectable hardness of the breast area; WB-affected chickens exhibit bent-forward posture and are unable to lift their wings sufficiently, breast area being markedly firm on palpation ,18. Broipectoralis major muscle was cut to 1 mm×1 mm×1 mm shapes and stored in 2.5% glutaraldehyde phosphate buffer saline  immediately for the ultrastructural observation. At the same location, approximately 0.5 cm wide by 0.5 cm deep by 3 cm long histological samples parallel to the muscle fibers were removed, and fixed in 10% formalin buffer. A piece of pectoralis major muscle tissue was rapidly treated for the detection of the level of ROS and the isolation of mitochondria. Within 20 min postmortem, 10 g of the cranial of the left pectorals major muscle was obtained and frozen in liquid nitrogen, and then stored in −80°C for biochemical tests.After selection, the entire right breast muscle was collected, placed into polyethylene bags and stored at 4°C for the measurement of meat quality. A cranial fraction of the left An examination of the microscopic structure was conducted to ensure the selected samples exhibited typical characteristics of normal and WB. Histological evaluations were performed using the method of Clark and Velleman  with minpectoralis major was evaluated using a HI9125 portable waterproof pH/ORP meter  according to the method of Brambila et al [At 24 h postmortem, the ultimate pH of the la et al  IndividuMeat color (CIE L* a* b*) was measured on the dorsal surface of each sample using a CR410 Chroma Meter . All samples were measured in triplicate, and mean values were calculated.W1), then suspended on a hook and placed in sealed container, stored at 4°C. The samples were reweighed (W2) 24 h later. The drip loss was calculated:Approximately 20 g of each fillet was cut into a regular shape and weighed (W3) and packed in a ziplock bag, then cooked at 80°C in a water bath until the internal temperature reached 70°C. The samples were weighed (W4) after cooking. The cooking loss was calculated:At 24 h post mortem, about 30 g specific-shaped tissue of each fillet was obtained for the cooking-loss determination. The samples were weighed  along the direction of the muscle fibers. Then, each strip was measured three times using a Digital Meat Tenderness Meter , and average values were calculated.The oxidation of 2′7′-dichlorodihydrofluorescein diacetate level was determined as the index of ROS produced by cellular components, using a commercial kit , according to the manufacturer’s instructions.The fresh muscles were treated for the isolation of mitochondria by using a commercial kit . Mitochondrial membrane potential (MMP) assay kit with JC-1  was used to detect the MMP changes of each breast samples, in accordance with the manufacturer’s instructions.Specimens were washed twice with phosphate buffer and then fixed in 1% osmium tetroxide solution (v/v). After dehydrated using acetone and embedded in Epon 812, ultrathin (≤90 nm) sections were cut, and then stained with uranyl acetate and lead citrate. The alterations in mitochondrial morphology were observed with a transmission electron microscope .Muscle tissues were homogenized in chilled phosphate saline buffer, and then centrifuged at a speed of 1,000 rpm for 15 min at 4°C. The supernatant was obtained for the assessment of oxidative parameters. The measurements of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPX), protein carbonyl, malondialdehyde (MDA), and lipid peroxidation (LPO) were performed using commercial kits , in accordance with the manufacturer’s instructions. The 8-hydroxydeoxyguanosine (8-OHdG) was measured using a commercial enzyme-linked immunosorbent assay kit  per the manufacturer’s instructions. The protein concentration was measured using the Coomassie brilliant blue method by a protein assay kit  for standardization.−ΔΔCt method [Total RNA from the breast muscle samples was isolated using RNAiso Plus reagent . After measuring the purity and quality, reverse transcription was performed using PrimeScript RT Master Mix . The real-time polymerase chain reaction (PCR) was performed on an ABI PRISM 7500 Detection System  with the reaction protocol as follows: one cycle at 95°C for 30 s; 40 cycles at 95°C for 5 s, and 60°C for 30 s. Primer sequences used for real-time PCR are shown in t method . Primer The statistical analysis of the data was performed by one-way analysis of variance with software SPSS Statistics 20.0 . The results were presented as mean values and standard error of the means. Significant difference was defined as p<0.05.As shown in The basic indicators of meat quality are shown in The antioxidant capacities are presented in As shown in HMOX1), NAD(P)H qui none dehydrogenase 1 (NQO1), GPX, SOD, glutamate cysteine ligase catalytic subunit (GCLC), glutamate cysteine ligase modifier subunit (GCLM) were significantly higher in the WB group than those of the NOR group (p<0.05).As shown in pectoralis major muscle decreases the available space for vascularization and therefore triggers hypoxia, which might contribute to oxidative stress and, subsequently, affect muscle development and structure.In this study, WB-affected broilers were selected based on the observation of their inability to lift wings and manual palpation. In addition, microscopic observations were further conducted to verify the accuracy of sample selection. All considered WB samples exhibited mild to severe structural abnormalities such as round-shape fibers, muscle fiber hyalinization, nuclear internalization and interstitial thickening with fibrosis as reported by Sihvo et al . MoreoveDamages to the muscle morphological structure ultimately influence meat quality, since meat quality attributes are closely related to the histological structure of muscle tissue . With reThe primary source of ROS in skeletal muscle is the leakage of electrons from the respiratory chain in mitochondria. Meanwhile, mitochondria are vulnerable to attack by ROS owing to the lipid (phospholipid) and protein composition of their membranes. In the current study, a higher level of ROS in WB samples indicated an excessive formation of free radicals, which was in agreement with the findings reported by Salles et al  in whiteIn broiler chickens, excessive production of ROS in skeletal muscle causes cellular damage by leading to protein oxidation, lipid peroxidation and DNA modification . AccordiUnder stressful conditions, excessive free radicals can break down the balance between pro-oxidant and antioxidant systems. Traditionally, it is considered that the excessive ROS can be removed or reduced by antioxidant enzymes such as GPX and SOD. The enhanced ROS generation can lead to oxidative damages and decrease antioxidant enzymes activities . HoweverHMOX-1, NQO1, SOD, GPX and glutamate cysteine ligase, which protect cells from oxidative damages [Nrf2 is a key transcription factor protecting cells against oxidative damage, and is considered as a molecular switch in targeting of ARE-mediated expression of antioxidant genes . In the  damages . In the In conclusion, the occurrence of WB myopathy significantly affects muscle structure and meat quality, which are related to the destroyed cellular redox status. Thus, besides clinical evaluations and histologic examinations, analysis of the cellular redox homeostasis is also important for WB determination.


Laser ablation of the avascular peripheral retina has been the standard method of ROP treatment. Intravitreal anti-VEGF is useful in the management of ROP patients, especially for aggressive posterior ROP. However, ocular and systemic complication after intravitreal bevacizumab was the main concern. This study aimed to investigate the treatment-related ocular and systemic complications of intravitreal bevacizumab (IVB) in patients with retinopathy of prematurity (ROP).This retrospective study included neonates receiving intravitreal injections of bevacizumab (IVB) (0.625 mg) to treat ROP. Medical records of the patients were evaluated about the ocular complications after receiving IVB from 2012 to 2019. Treatment-related complications , and disease-progression signs including retinal fold or stage 4 or 5 detachment were documented. Any reports of systemic events after injections were also recorded.Mean gestational age and birth weight of 441 patients receiving IVB for type-1 ROP were 28 ± 2 (22–34 weeks) and 1121 ± 312 (550–2700 g), respectively. The median follow-up after treatment in all patients and patients with complications was 289.43 ± 257 days (5–1899 days) and 385.89 ± 311.59 (196–1192) days, respectively. Out of 865 eyes, 20 eyes  have been affected by ocular complications. The rates of different complications included progression of retinopathy in 17 eyes (1.96%), cataracts in 2 eyes (0.23%), and vitreous hemorrhage in one eye (0.11%). No cases of endophthalmitis, thromboembolic events, or death occurred in this study. We evaluated the prevalence ratio (PR) on the multiple risk factors to determine the prediction of the complications. The existence of neovascularization of iris has the highest susceptibility to predict the complication  following by the presence of retinopathy in zone 1 of the infant’s retina .The incidence rate of complications related to Intravitreal bevacizumab injection was low, which was compatible with previous studies. Bevacizumab injection seems well tolerated in most cases of ROP. Iris neovascularization and the presence of retinopathy in zone 1 were associated with a higher occurrence of complications than the absence of these risk factors. Retinopathy of prematurity (ROP) is one of the major preventable causes of childhood blindness . ROP is Over the past decades, ablation of the avascular peripheral retina with laser photocoagulation has been the standard method of ROP treatment. It reduces the risk of complications associated with proliferative ROP, but it has disadvantages such as destroying large retina areas and inducing myopia –8. With The vascular endothelial growth factor is essential for the infant's retinal development. Also, growth factors such as VEGF play an important role In normal retinal vascularization, which progresses from the optic nerve to ora serrata .Ophthalmologists, as well as neonatologists, have expressed concerns over the potential ocular and systemic sided effects of anti-VEGF injections in ROP patients, which have been demonstrated to be detectable in serum samples after an intravitreal injection –13.This study aimed to assess the rate of ocular and systemic complications following IVB injection for type-1 ROP patients and evaluate the risk factors associated with occurrence complications.This retrospective case series was performed in Farabi eye hospital, a tertiary referral center. Medical records for patients with type 1 ROP treated with intravitreal anti-VEGF  between 2012 and 2019 were collected from the ROP center and pooled together for data analysis. This study was approved by the Hospital Review Committee and followed the principles of the Declaration of Helsinki. Standard laser treatment, as well as anti-VEGF benefits and complications, were fully explained to parents, and written informed consent was obtained from them as the routine protocol of ROP services of the hospital. Retinal specialists did all the examinations with ROP expertise or by fellowship-trained pediatric ophthalmologists.Subjects were infants born between January 2012 and August 2019, with known gestational age, birth weight, and ROP examination status who had undergone IVB injection. Which included the presence in either eye of Early Treatment of Retinopathy (ETROP) Study type 1  [Infant demographic, medical, and funduscopic data were extracted retrospectively from the patient medical records by a certified data collector (F.B and H.RE) for the infants who had received IVB. Any related ocular complications, including corneal abrasion/ulcer, crystalline lens opacity or cataract, hyphema glaucoma, vitreous hemorrhage, and post-injection endophthalmitis or uveitis, were collected. Further, disease progression was defined as a retinal fold, worsening of the present disease, or stage 4/5 ROP. Any reports of systemic events after injections were also documented.Exclusion criteria were refusal of a parent or guardian, any history of laser therapy, cryotherapy, or surgical procedure before IVB injection, any associated primary ocular or systemic disorders that could confound the interpretation of study results, as well as incomplete medical records.The injection pro0cedure was done in the operation room under topical anesthesia, while the vital signs were monitored throughout the entire procedure. The eyes were prepared in a standard fashion using 5% povidone/iodine. After 5 min, a single intravitreal injection of 0.625 mg (0.025 ml) bevacizumab  was done through pars plicata, 1–1.5 mm posterior to the limbus in all patients. A nurse helped hold the infant during the injection. After the injection, the patients received topical Antibiotics (gentamycin or sulfacetamide 10%) for 3 days. The infants were re-evaluated after 1 day and 7 days, and then every week or biweekly based on the eye's situation of patients to document the progression of the disease and any related complications. Afterward, the examinations continued until complete vascularization of the retina was observed.In the case of inappropriate response (lack of improvement in plus and neovascularization or progression of presence disease) to anti-VEGF treatment, including sustained vascular changes, conventional confluent laser photocoagulation of ROP was performed.Response to IVB was defined as the disappearance or decline in retinal vessel tortuosity and dilation as well as fading of retinal neovascularization, along with the development of vascularization toward the peripheral retina.The primary outcomes of the study were to report the rate of ocular and systemic complications such as corneal opacity, cataract or lens opacity, hyphema, glaucoma, vitreous hemorrhage, endophthalmitis, death, stroke, and cerebrovascular accident. The secondary outcome was to evaluate the rate of disease progression following IVB treatment for ROP.P value less than 0.05 was considered statistically significant. Statistical analysis was performed using SPSS software version 23 .We report the data as Mean ± SD. The rate of complications was reported in prevalence. We analyze the prevalence ratio  to determine ocular and systemic risk factors, to determine the factors to anticipate anti-VEGF associated complications. In this series, we evaluated the gender of patients, the zone of retinal retinopathy involvement in infants, presence of neovascularization in iris prior to treatment, history of intubation in hospitalization period in a neonatal intensive care unit (NICU), case of twin delivery, any history of blood transfusion at the time of hospitalization, history of intraventricular hemorrhage, history of sepsis in the hospitalization period, presence of anemia in lab tests, and history of phototherapy due to neonatal jaundice from the medical records of patients. A This series included 865 eyes from 441 patients who underwent intravitreal bevacizumab injection for type 1 ROP from 2012 to 2019.The mean gestational age of infants was 28 ± 2 weeks (22–34), and the mean birth weight was 1121 ± 312 g (550–2700). The male to female ratio was 252/189 (57.1%/42.9%).We observed 20 complicated eyes from 12 patients  out of 865 eyes  with IVB injection. The complications included a progression of retinopathy in 17 eyes (1.96%), including a case with crunch phenomena  on the gender of patients, the zone of the ROP in infants, presence of neovascularization in iris prior to the treatment, history of intubation in hospitalization period in NICU, case of twin delivery, any history of blood transfusion at the time of hospitalization, history of intraventricular hemorrhage, history of sepsis in the hospitalization period, presence of anemia in lab tests, and history of phototherapy due to neonatal jaundice Table .Table 2TIn our study, 54 eyes (6.2%) had neovascularization of iris before the administration of anti-VEGF, and among them, five eyes (9.3%) had at least one complication from the aforementioned complications. On the other hand, out of 811 eyes (93.8%) without iris neovascularization, 15 eyes (1.8%) showed complications related to anti-VEGF administration. The presence of neovascularization of iris has the highest susceptibility to predict the complication .Retinopathy of prematurity occurred in zone 1 in 187 patients (21.6%) and zone 2 in 678 patients (78.4%). The complication rate in zone 1 ROP and zone 2 ROP following the treatment was 11 eyes (5.9%) and 9 eyes (1.3%), respectively. The presence of retinopathy in zone 1 of the infant's retina had a high chance of causing complications . On the other hand, iris neovascularization and disease in zone 1 were associated with 5 and fourfold greater occurrence of complications as compared with the absence of these risk factors.The current study revealed that among 865 eyes of 441 immature patients treated with intravitreal bevacizumab, the rate of all complications directly attributable to IVB injection for ROP was low (< 2.5%). From different documented complications, the progression of retinopathy was the main complication (2%) observed in our study. We also investigated the risk factors of complications related to IVB injections in ROP patients.As found in this study, the risk of IVB related ocular complications was low. Based on a secondary analysis on the Postnatal Growth and Retinopathy of Prematurity study data (G-ROP), the rate of ocular complications following IVB injection was very low (1 case of vitreous hemorrhage and no case of ROP progression) . AlthougIn the BEAT-ROP study, the retreatment rate due to ROP progression was 4% after IVB injection in infants with zone I or posterior zone II disease. They did not report any cataract, vitreous hemorrhage, or endophthalmitis in their cases .A systematic and meta-analysis was done on the complications of anti-VEGF administration in ROP patients by Pertl et al. . They inWe also revealed the ROP progression in near 2% of our cases. Persistent areas of retinal avascularity are a concern in patients treated with IVB, which may lead to VEGF rise and the ROP progression. We observed that patients with zone 1 of ROP had a higher chance of developing ocular complications, including the ROP progression . Further, we also showed that patients with Iris neovascularization, as an indicator of the severity of ischemia, had a higher chance of developing ROP progression and complications . Thus, ROP progression may be related to baseline ROP severity. An improper patient selection for receiving IVB might be regarded as another reason. We found a crunch phenomenon in one patient. Eyes with any amount of tractions might not be suitable for anti-VEGF therapy.We observed 2 cases with cataract and 1 case with vitreous hemorrhage among 865 the eyes treated with bevacizumab. There was no case with post-IVB corneal opacity, glaucoma, and endophthalmitis. The rate of cataract, vitreous hemorrhage and endophthalmitis was very low in our study, which was consistent with the aforementioned studies. Hwang et al. reported similar rates of vitreous hemorrhage in eyes treated with IVB . The cauAlso, any potential benefit related to lower ocular complications must be weighed against the risk of systemic adverse effects. In an animal study of bevacizumab in rabbit eyes, the detectable level of bevacizumab in blood was shown in 8 days after administration . InfantsIn the BEAT-ROP study, they reported four mortalities due to lung disease in the bevacizumab group compared to one death in the laser group; although non-significant with a very small number of events, it raised concern over pulmonary maturation arrest due to VEGF blockade.  Another Nevertheless, the lack of evidence on long-term safety outcomes of anti-VEGF therapy is still a significant concern and needs further investigation. Importantly, a study that used the data from the Canadian Neonatal Network, after adjusting for main confounders such as gestation, gender, maternal education, Score for Neonatal Acute Physiology II (SNAP-II) score, bronchopulmonary dysplasia, sepsis, and severe brain injury, demonstrated 3.1 times higher odds (95% CI 1.2–8.4) of severe neurodevelopmental disabilities in preterm infants born before 29 weeks of gestation and treated with bevacizumab .There are some case reports on the rare but significant complications of intravitreal anti-VEGF use in ROP patients. Lee et al. reported three patients with fibrous retinal traction band, which occurred after an intravitreal bevacizumab injection for stage 3 ROP with plus disease . ChhablaStrengths of our study included the large number of IVB-treated eyes; All patients were treated at the same center, which used standardized treatment protocols. Our study had some limitations as well. First, the study had a retrospective design. Secondly, some of the infants treated with IVB primarily were subsequently treated with laser due to disease progression; therefore, some late complications of IVB injection may be precluded. Further, some delayed developmental complications of the IVB injections were not evaluated as all patients were not followed until complete vascularization was reached. Moreover, the effect of reduced doses of bevacizumab on the ocular and systemic side effects was not evaluated in this study; though many studies have investigated different doses of anti-VEGF for ROP patients, systemic absorption remains a major concern , 16. Finthe incidence rate of complications related to Intravitreal bevacizumab injection was low (2.31%), which was compatible with previous studies. Disease progression seems to be the most common concern after IVB injection in ROP patients. Infants with Zone 1 of ROP or iris neovascularization before anti-VEGF therapy tended to show a higher ocular complication rate, which can be explained with greater disease severity in these patients. We observed no systemic complications in our series.


Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastating diseases, resulting in significant yield losses in rice. The extensive use of chemical antibacterial agents has led to an increase the environmental toxicity. Nanotechnology products are being developed as a promising alternative to control plant disease with low environmental impact. In the present study, we investigated the antibacterial activity of biosynthesized chitosan nanoparticles (CSNPs) and zinc oxide nanoparticles (ZnONPs) against rice pathogen Xoo. The formation of CSNPs and ZnONPs in the reaction mixture was confirmed by using UV-vis spectroscopy at 300–550 nm. Moreover, CSNPs and ZnONPs with strong antibacterial activity against Xoo were further characterized by scanning and transmission electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction. Compared with the corresponding chitosan and ZnO alone, CSNPs and ZnONPs showed greater inhibition in the growth of Xoo, which may be mainly attributed to the reduction in biofilm formation and swimming, cell membrane damage, reactive oxygen species production, and apoptosis of bacterial cells. Overall, this study revealed that the two biosynthesized nanoparticles, particularly CSNPs, are a promising alternative to control rice bacterial disease.Bacterial leaf blight caused by  Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight in rice, which leads to severe yield losses and consequently alters the food supplies [supplies . The ressupplies ,3,4,5 dusupplies ,7. 2, MgO, and CuO, have strong antibacterial activity against a variety of bacterial pathogen [Xanthomonas pathogenic bacteria isolated from Euphorbia pulcherrima, while the growth of the Xoo pathogen was significantly inhibited in our previous studies by the biosynthesized MgO nanoparticles [Furthermore, a high number of polyphenols, especially flavonoids  in tomatpathogen ,14. For pathogen  reportedarticles , Ag nanoarticles ,18 and Zarticles . Maruyamarticles  developearticles  prepared2 nanocomposite with antibacterial activity against Xoo. The antimicrobial activity of the chitosan polymers and nanoparticles may be mainly attributed to their electric charge and high ability of adsorption as well as chemical reactions that allow them to interact efficiently with the bacterial cell membrane , while tHowever, the concerns are rising on the residue of heavy metal ion in the natural environment, which makes it necessary to exclude the heavy metal ion during the control of Xoo by the combination of chitosan with nanoparticles. Interestingly, chitosan nanoparticles (CSNPs) not only have less toxic impact compared to the chitosan and metal nanoparticles alone ,25,26, bThe purpose of this research was to biosynthesize CSNPs with strong antibacterial activity against the rice pathogen Xoo, while ZnO nanoparticles (ZnONPs) were also biosynthesized as the positive control. Furthermore, the antibacterial activity of CSNPs was compared with that of chitosan, ZnO and ZnO nanoparticles alone. In addition, this study revealed the antibacterial mechanism of the biosynthesized CSNPs and ZnONPs against Xoo.Two grams of dried green tomatoes were placed in a 250 mL flask containing 200 mL deionized water and then boiled in a 60 °C water bath for 4 h. After filtering twice with filter paper Whatman no. 1, the aqueous green tomato extract was directly used in the installation of CSNPs and ZnONPs.6H11NO4)n from shrimp shells, ≥75% (deacetylated) in 200 mL of 2% acetic acid solution and then stirring under continuous magnetic for 30 min. Then, CSNPs were developed by adding 100 mL of the aqueous green tomato extract to 200 mL of a chitosan-TPP mixture dropwise under constant stirring for 30 min. Similarly, ZnONPs were developed by adding 100 mL of the aqueous green tomato extract to 100 mL of 1 M ZnO  and then stirring at 60 °C for 4 h. After discarding the supernatant through centrifugation at 10,000 g for 20 min, the pellets were washed with distilled water and then freeze-dried in ALPHA 1-2/LD-Plus vacuum to obtain the nanoparticles powders.CSNPs and ZnONPs were synthesized according to the method of Dipali et al.  with minThe highest peak absorption levels of CSNPs and ZnONPs were measured according to  by usingThe morphology of two types of the nanoparticles CSNPs and ZnONPs with strong antibacterial activity was observed using transmission electron microscopy (TEM) , which was carried out as described by Ahmed et al.  by fixin−1 regions at a resolution of 4 cm−1.The functional group of two types of the nanoparticles CSNPs and ZnONPs with strong antibacterial activity was determined based on the analysis of Fourier transform infrared spectroscopy (FTIR), which was carried out as described by Hossain et al.  by measuThe quality of two types of the nanoparticles CSNPs and ZnONPs with strong antibacterial activity was determined using Siemens X-ray diffractometer (XRD), which was performed as described by Abdallah et al.  using dr8 CFU/mL) was mixed with five mL of nutrient agar (NA) medium. About 50 µL of dried nanoparticle solutions at different concentrations  were poured at the 6 mm wells and incubated for 24 h at 30 °C. Then the inhibition zone around the well was measured. The antibacterial activity of CSNPs and ZnONPs against Xoo strain GZ 0005 was also examined on liquid broth, which was performed as described by [8 CFU/mL) in total 5 mL nutrient broth  to give different final concentrations of the nanoparticles . The inhibitory effect on bacterial growth was determined by measuring the optical density at OD600 nm using a UV spectrophotometer after 48 h of incubation. The experiment was repeated twice, and each treatment had three replicates.The antibacterial activity of CSNPs and ZnONPs against Xoo strain GZ 0005 was measured on a solid medium by using agar well diffusion assay, which was carried out as described by Perez et al.  with minribed by . The ste570 nm, according to Zhang et al. [Biofilm formation of Xoo strain GZ 0005 was determined in a 96-well plate by measuring the absorption value at ODg et al.  with minw/v) NA media, which was pre-mixed with CSNPs and ZnONPs with a final concentration of 0.0, 4.0, 8.0, and 16.0 μg/mL. After three days of incubation at 30 °C, bacterial migration areas were measured. Each treatment was expressed in triplicates and the experiment was repeated twice.Swimming motility on plates was detected according to the method . In brieBacterial samples for TEM were prepared as described by Sun et al.  with som8 CFU/mL) for 5 min at 5000 g, the bacterial pellets were treated with CSNPs and ZnONPs with the final concentration of 16.0 µg/mL for 4 h, then propidium iodide (PI) was added as described by Cai et al. [CSNPs and ZnONPs induced apoptosis was observed by flow cytometry . After centrifuging the overnight culture of Xoo strain GZ 0005  was centrifuged for 5 min at 5000 g. Then, bacterial pellets were treated with CSNPs and ZnONPs with the final concentration of 16.0 μg/mL for 4 h, respectively, while rotenone was also applied before two types of nanoparticles. The negative control was treated with sterile water. After washing with sterilized water three times, 10 μM DCFH-DA was added and then incubated in the dark at room temperature for 30 min. Eventually, the samples were washed with sterilized water, and fluorescence was detected as described by Applerot et al. [Intracellular ROS was determined using fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA), which interact with ROS to form the fluorescence trapped inside the cell. In brief, one mL overnight culture of Xoo strain GZ 0005 .Data were subjected to analysis of variance (ANOVA) using SAS 2003 software . A general linear model (GLM) procedure was used to check the significant differences among the main treatments. Individual comparisons between mean values were performed using Duncan’s Method , oxygen (40.33%), and phosphorus (5.29%) E. In the−1 , which is a type of fluorochrome that can pass through damaged or dead cells, while the DNA stain was detected using ultraviolet rays. In agreement with the result of TEM observation, result from this study indicated that the two biosynthesized nanoparticles CSNPs and ZnONPs markedly increased the apoptosis of Xoo cells to 99.51% and 74.13%, respectively, compared to 0.82% for the negative control treated with distilled water . CollectEscheria coli following the treatment with nanoparticles and detection by FC analysis [In agreement with the result of this study, cell membrane damage and promoted flowage was observed in analysis . Additioanalysis  reportedGeneration of reactive oxygen species (ROS) in Xoo strain GZ 0005 cells was determined based on CLSM observation of the intensity of the fluorescence after 4 h of incubation. Results from this study indicated that no obvious fluorescence was observed in bacterial cells in the negative control treated with sterile water. Furthermore, a little fluorescence was observed in Xoo strain GZ 0005 cells treated with 16.0 μg/mL of ZnONPs, while a lot of fluorescence was observed in Xoo strain GZ 0005 cells treated with 16.0 μg/mL of CSNPs . HoweverE. coli cells. The formation of ROS promotes the oxidative stress in the cells, which may in turn induce cell damage, causing cell lysis or distortion of bacterial membranes, resulting in the leakage of DNA and protein, even bacterial death [In agreement with the result of this study, Banerjee et al.  found thal death . Indeed,al death ,76. On tal death , this exIn conclusion, this study successfully biosynthesized CSNPs and ZnONPs, which showed a stronger inhibitory effect on the growth of rice pathogenic bacteria Xoo compared to that of bulk chitosan or bulk ZnO, respectively. Furthermore, the two synthesized nanoparticles with strong antibacterial activity were characterized by the analysis of UV–Vis spectroscopy, XRD, and FTIR as well as TEM and SEM observation. In addition, the antibacterial activity of two types of the nanoparticles may be able to, at least partially, be attributed to apoptosis, generation of ROS, reduction in biofilm formation and swimming, destruction or disintegration of the cell walls, and leakage of the intracellular contents, which eventually resulted in cell death. Overall, it could be concluded from this study that the two synthesized nanoparticles particularly CSNPs have a great potential to suppress Xoo infections in rice.