text:
Adaptation of a Grafting Method for Carica papaya Based on Seedling Behavior

. Grafting Carica papaya plants can have several beneﬁts for productive, phytosanitary, and sexing purposes. However, the literature on the subject of papaya grafting is limited. The tongue approach and cleft grafting techniques seem to be the most adequate for C. papaya , but the quality of grafts depends on several factors. With the objective of developing and adapting a grafting method for papaya, experimental assays were carried out in the Valley of Apatzingan, Michoacan, Mexico. The physical condition of the seedlings was assessed, and the most advantageous time for grafting was determined based on the size and thickness of the stems. Three assays were then carried out. The ﬁrst assay was a test of the tongue approach and cleft grafting techniques using two clamping devices. The second assay involved the same techniques with modiﬁcations and the addition of another treatment. In the third assay, the modiﬁed tongue approach grafting method was tested on three containers with papaya plants. Seedling vigor, graft survival, and graft quality were the recorded variables. The results indicated that unwanted tissue should be cut 6 days after grafting.

In Mexico, Carica papaya L. is cultivated in 19 states, with a harvested area of 16,684 ha, concentrated mainly in Veracruz (3196 ha), Colima (2850 ha), Oaxaca (2702 ha), Michoacan (2368 ha), and Guerrero (1124 ha) (SIAP-SAGARPA, 2017). Despite its economic and nutritional importance, the papaya production chain faces several difficulties associated with a low capacity for innovation and development. This could be resolved or diminished through the imple-mentation of technical innovations that increase productivity and resolve existing phytosanitary problems (Feit o and Portal, 2013). Agronomic management and breeding techniques have been the main focus of research associated with the papaya crop. The selection of parents for hybridization and crosses (Mirafuentes and Azpeitia, 2008;Mirafuentes and Santamaria, 2014), genetic engineering (Song et al., 2015), and explant multiplication (Kumar and Kumari, 2013;Singh et al., 2010) are all complex processes in which the time and costs involved must be taken into account. In contrast, micropropagation and production of seedlings from cuttings and grafts are alternatives for improving the commercial production of papaya and have been little studied (Teixeira et al., 2007;Schmildt et al., 2016;Senthilkumar et al., 2014).
Grafting C. papaya plants can have several benefits for productive, phytosanitary, and sexing purposes. Grafting could be used to improve fruiting and yield in papaya plants, as well as to reduce the height of the varieties and hybrids of interest (Allan, 2007;de Lima et al., 2010;Lange, 1969). Thus, grafting could generate improvements throughout the entire production chain of papaya (Senthilkumar et al., 2014). Several grafting techniques have been used in herbaceous species (Alvarez-Hern andez et al., 2009;Davis et al., 2008;Honma, 1977;Kumar and Sanket, 2017;Lee, 1994;Oda, 1995); most of them agree on the following general guidelines: the grafting must be carried out during the first stages of plant development, the seedlings must be kept under controlled conditions of temperature and relative humidity during the period of formation of the graft union callus, and the grafted plants should be acclimated to the prevailing environmental conditions before transplantation. Although few studies have focused on papaya grafting (Allan et al., 2010;Van-Hong and Chung-Ruey, 2018), it is generally agreed that the tongue approach and cleft grafting methods are the most appropriate grafting techniques (Lee and Oda, 2003); however, several factors can influence the quality of the grafts, including the grafting method used; the compatibility, vigor, and health of the plants involved; and postgrafting handling (Kawaguchi et al., 2008). In papaya, a very important factor is bacterial contamination (Allan et al., 2010). In the absence of studies aimed at the development of an effective and efficient grafting method for papaya, the objective of our study was to develop and standardize a grafting method for papaya that can be used in the main papaya-producing region of Michoacan, Mexico.

Materials and Methods
Study area. When the environmental conditions are more favorable for the papaya grafting in the region (Sept.-Dec. 2016 and Jan.-Apr. 2017), different trials were carried out in the nursery of the Valley of Apatzingan Experimental Field, located in Antunez, municipality of Paracuaro, Michoacan, Mexico (Fig. 1). The area's climate is subtype BS 1 [a semidry warm climate (Garcia, 1988], the predominant vegetation of the area consists of species that predominate in low deciduous forests (García and Linares, 2012), and the type of soil corresponds to Pelic Vertisol (clay) (INEGI, 2016).
Seedling development. Papaya seeds from 'Maradol' were used to obtain seedlings for use as rootstock and scion. The seeds were moistened by immersing them in water for 24 h, and only those that stayed on the bottom of the container were seeded in polystyrene trays with either 128 or 200 cavities, and in plastic bags (7.8 · 12.4 cm). The cavities contained wet commercial substrate (Canadian sphagnum moss).
After germination, the development of seedlings was sustained by daily irrigation, and preventive applications of cypermethrin (1 ml · L -1 ) and a fungicide mixture containing Captan (1 g · L -1 ) (Lucaptan Ò , Celaya, Mexico), supplemented with nitrogen fertilizer (1 g · L -1 ) (Urea New Green Ò , Naucalpan, Mexico), every 5 days. This stage lasted 40 d, and the seedlings developed under normal environmental conditions. The nursery was protected by covering it with an antiaphid mesh and a shade mesh.
Description of treatments. The seedlings were evaluated before and after grafting. Before grafting, seedling development was recorded 30 d after emergence. The evaluated seedlings were grown under three different treatments: 1) in trays with 128 cavities (46 mL/cavity), 2) in trays with 200 cavities (22 mL/cavity), and 3) and in plastic bags (7.8 · 12.4 cm). All seedlings were measured for the following parameters: height, using a graduated ruler, from the base to the apical meristem; stem diameter, with a Vernier caliper at the base of the stem; number of leaves, by visual counting; and vigor, using an ordinal scale based on the work of Maiti et al. (1981) and Barchuk and Diaz (2000) ( Table 1).
The grafting procedure was initiated when the seedlings presented adequate physical conditions in terms of size and stem thickness. This occurred 30 d after emergence. To determine the time at which grafting was most likely to succeed using the tongue grafting method with grafting tape laminated as the fastening device, six treatments were evaluated 3 to 8 d after grafting. Tissue fragments were removed from the grafted seedlings every day, so they would function as single plants. The percentage of graft survival and seedling vigor were recorded.
Another three evaluations were developed after grafting (Table 2); however, it is important to note that temperature and humidity were not regulated. The first evaluation involved four treatments: 1) tongue approach grafting with grafting tape as the fastening device, 2) tongue approach grafting with a clip as the fastening device, 3) cleft grafting with grafting tape as the fastening device, and 4) cleft grafting with a clip as the fastening device. In the second evaluation, the tongue grafting method was modified as follows: The meristem of the seedling to be used as rootstock was removed before fixing the tongues. The treatments consisted on the place of formation of the tongue in rootstock seedling, one was formed above the cut site of stem, and other below the cut site of stem. In both cases, the tongues formed of rootstock and scion were combined with the two fastening devices: tape and clip. In the fifth treatment, cleft grafting, transversal cuts were made on both stems, and the tissue of both plants was fastened using a graphite pin. In the third evaluation, a modified tongue approach grafting method (tongues formed below the site cut and fastened with grafting tape) was used, but the grafted seedlings were grown under three treatments: in polystyrene trays with 128 or 200 cavities, or in plastic bags (7.8 · 12.4 cm). The percentage of graft survival was determined 6 d after grafting whereas seedling vigor was assessed 9 and 12 d after grafting, according to the evaluation table (Table 1). The three evaluations are specified in Table 2.
In all treatments, graft quality was assessed based on the physical and visual conditions of the graft site. Four characteristics were evaluated: superficial adherence, compatibility, callus formation, and absence of necrotic tissue. The graft quality was classified as bad regular, or good.
Experimental design. A completely randomized block design was used in all evaluations. Because of the continuity of each experiment and according to the evaluation, the number of repetitions varied. In the pregrafting stage, 10 seedlings were used. After grafting, to evaluate grafting survival, four replications were performed, each with 10 seedlings. To evaluate seedling vigor, seven replications (seedlings) were usedthe plants derived from the graft survival variable. An analysis of variance (ANOVA) was performed on most of the variables, with the exception of the seedling vigor variable, which was analyzed with the Kruskal-Wallis nonparametric test. According to the analysis, means were compared using Tukey's Studentized range test and the Mann-Whitney test (P # 0.05). The statistical software used was SAS (ver. 9; SAS Institute, Cary, NC) and PAST [ver. 3.2 (Hammer, 2018)].

Results and Discussion
ANOVA results showed statistically significant differences only in seedling height and stem diameter (P # 0.01 and P # 0.001, respectively). Treatments 1 and 3, which had statistically similar means, yielded better results than treatment 2. The variables number of leaves and vigor of seedlings did not show differences (Table 3).
Recording seedling development parameters is always important, regardless of whether grafting is intended, because seedlings should always be of the highest quality. The use of quality growth media during sowing had a positive effect on germination, development, and rooting (Meena et al., 2017). There was a report that the addition of gibberellic acid, cow urine, and coconut fiber substrate affected these parameters positively (Desaid et al., 2017). In our study, we followed some general guidelines for the production of seedlings, but the response of the plants was different in different containers. The containers with more substrate (treatments 1 and 3) showed better seedling development (Table 3). Larger containers provided better anchoring and support for the seedlings, in addition to serving as a reservoir of nutrients and water, and facilitating gas exchange (Abad et al., 2002). The space between plants was decisive. When producing seedlings for grafting, the most important parameters were height and stem diameter, so that it was possible to cut and fasten into place the rootstock and the scion (Kumar and Sanket, 2017). The better characteristics, in terms of height and stem diameter, as well as seedling vigor, were shown by the seedlings significantly in treatment 3.
The tongue approach grafting method (Lee and Oda, 2003) was used to determine the best moment to cut off the unwanted parts of the plants to keep only the functional parts of a grafted plant. After the grafting procedure, the percentage of graft survival increased as more days passed, before cutting the unwanted tissue between days 3 and 8 after grafting. But, no grafts survived when the cuts were made on days 3 and 4, which is why the data from those days are not shown in Fig. 2. When the cuts were made between days 5 and 8. All results were positive, with graft survival of 55% to 95% (Fig. 2). Seedling vigor was least favorable when the unwanted tissue was cut off on day 5 after grafting. At days 6 and 8, vigor values were 5 and 4, respectively; from days 8 to 11, the vigor ranking changed to 3, which corresponded to plants with little vigor (Table 1). On day 12, seedling vigor had a value of 2. When the cuts of unwanted tissue were done on days 6, 7, and 8 after grafting, all seedlings showed a vigor value of 3. Nine to 12 d into the observation, with the exception of day 9 d, which corresponded to unwanted tissue cut at a previous day after grafting, the vigor of seedlings improved until reaching a value of 2 (Table 1), which corresponds to plants with good vigor (Fig. 2).
Greater precocity is the desired result in grafted papaya plants. The problems related to the rapid production of grafted seedlings depend on the grafting techniques used, temperature, and the skill with which the grafting procedure is performed. The grafting process should last between 7 and 10 d (Lee and Oda, 2003). In our work, graft survival was evaluated based on the time at which unwanted tissue could be removed without preventing grafted plants from functioning as single plants. When the unwanted tissue was removed 6, 7, or 8 d after the grafting procedure, the percentage of graft survival was more than 90% (Fig. 2). Therefore, the time at which the unwanted tissue was removed was standardized to 6 d after the grafting procedure. This ensured the highest    percentage of graft survival and shortened the time spent taking care of grafted seedlings. Seedling vigor, which improved as time passed, was evaluated 12 d after cutting unwanted tissue (Fig. 2). However, cutting unwanted tissue 6 or 7 d after grafting was also adequate, because seedling vigor improved easily thereafter. Studies of grafting in different species have shown that the time of recovery increases the uniformity and vigor of seedlings, and makes them more likely to resist biotic and abiotic stresses (Leonardi and Romano, 2004), which is in agreement with the results of our study. During the first evaluation, which involved two grafting methods and two fixation devices, ANOVA detected statistical differences in graft survival (P # 0.001). Treatment 1 (T-T) yielded 80% of graft survival, surpassing the other treatments. Treatment 4 [cleft grafting fastened with pin (C-P)] yielded only 50% graft survival, which is considered a low percentage (Table 4). Regarding the vigor of the grafted seedling only had significant differences on day 9 of observation (P # 0.01). Treatment 1 (T-T) statistically was better, because it kept below with value lower to 2, according to the scale of vigor (Table 1), compared with treatments 2 (T-P), 3 (CT), and 4 (C-P), for which the resulting value was higher. The 12-d vigor evaluation, even without differences, all treatments improved due to the time that elapsed favored the vigor (Table 4).
To date, there have been few studies of grafting, although they are not consistent for papaya. However, our study started by evaluating the tongue approach and cleft grafting methods (Khankahdani et al., 2012;Lee et al., 2010) in terms of graft survival and seedling vigor. The results obtained in the first evaluation served as the basis to propose modifications to the grafting methods under study. Of the two grafting methods and the two fastening devices, treatment 1 (T-T) was the most effective because it yielded a percentage of graft survival of 80% in the first evaluation. It should be noted that this method is commonly used in other species (Haghighi et al., 2016). It yielded good results in papaya, but it is worth noting that neither temperature nor humidity were controlled during the growth of the grafted seedlings, as is done for other species and as has been established in the literature (Hassell et al., 2008). With respect to seedling vigor, on days 9 and 12 after grafting, the values were 1.71 and 1.42, respectively. Thus, this method, which was used for treatment 1 (T-T), did not cause any problems in terms of seedling vigor (Table 4). The other treatments-2 [tongue approach grafting fastened with pin (T-P)], 3 (C-T), and 4 (C-P)-which used the cleft grafting method, yielded a low percentage of graft survival and lower seedling vigor after grafting (Table 4), and so they have room for improvement using other strategies ( Van-Hong and Chung-Ruey, 2018).
During the second evaluation using modified grafting methods, there were statistically significant differences in graft survival (P # 0.01). Regarding the percentage of graft survival, treatment 2 (M-T-Bc-T) surpassed the rest of the treatments, with 90% graft survival. Treatments 1 and 4 [modified tongue approach grafting method, tongues formed above the stem-site cut, fastened with tape (M-T-Ac-T) and modified tongue approach grafting method, tongues formed below the stem-site cut, fastened with pin (M-T-Bc-P), respectively] both yielded a graft survival of 65%. Finally, treatments 3 [modified tongue approach grafting method, tongues formed above the stem-site cut, fastened with pin (M-T-Ac-P)] and 5 [modified cleft grafting method, fastened with lace (M-C-L)] yielded a lower percentage of graft survival than the other treatments (Table 5). Regarding the vigor of grafted seedlings, statistical differences were also detected between both observations (9 and 12 d) after grafting (P # 0.01 and P # 0.001, respectively). According to the quality scale, treatment 5 (M-C-L) yielded the lowest quality seedlings for both observation days. The observation to 9 d after grafting, all treatments (M-T-Ac-T, M-T-Bc-T, M-T-Ac-P, and M-T-Bc-P) yielded vigor values ranging from 1.4 to 2.6, but these values improved as time progressed until 12 d after grafting (Table 5).
Although M-C-L works well in tomato grafts (Alvarez, 2012), the results obtained in our study suggest that use should be discarded for papaya as a result of the low percentage of graft survival (only 12.5%) and the low vigor values on the two observation dates (Table 5). Of the modified grafting methods, treatment 2 (M-T-Bc-T) yielded a greater rate of graft survival (90%) and acceptable vigor (Table 5), surpassing our expectations for use in papaya under the conditions evaluated. With this modified method, the meristem at the upper part of the rootstock is removed at the beginning of the grafting procedure. This step allowed us to manipulate more easily both plants during the cutting and joining process, and it had a positive effect on the percentage of graft survival. Thus, grafting methods should be modified based on different influencing factors, and it is necessary to evaluate the effectiveness of each modification (Yassin and Hussen, 2015).
With respect to the evaluation of seedlings grown in different containers, ANOVA results showed statistically significant differences (P # 0.05) for the graft survival variable; the seedling vigor variable (according to the Kruskal-Wallis test), was statistically significant (P # 0.05) for the first observation date. Treatment 3 (M-T-Bc-T-B) and treatment 1 [modified tongue approach grafting method, tongues formed below the stem-site cut, fastened with tape, 128-cavity tray (M-T-Bc-T-128)] yielded higher percentages of graft survival. However, the percentage of graft survival obtained with the three treatments were considered acceptable because of to the spacing between plants. With respect to seedling vigor, the three treatments yielded good results, based on the observations made 9 d after grafting; at day 12, seedling vigor was still improving, although without differences (Table 6).
The grafting method used in this evaluation was the modified tongue approach grafting, which yielded the greatest percentage of graft survival and highest values of seedling vigor. The plants in the container involving the use of plastic bags (M-T-Bc-T-B) were more stable and had a greater percentage of graft survival compared with the tray containers. An advantage of the use of plastic bags was that the root balls were never exposed to the environment during the grafting procedure, which did happen in the treatments involving trays. C. papaya is very sensitive to the exposure of its rootlets during transplantation, which probably explains its response to the treatments involving polystyrene trays, which was independent of the number of cavities in each tray. The favorable effect of plastic bags was also reflected in seedling vigor shown by the seedlings on the two observations dates after grafting (Table 6).
In the last evaluation of graft quality based on the physical and visual conditions of the grafting area, the grafting methods used yielded results between ''regular'' and ''good,'' with the exception of grafting method M-C-L, which yielded ''bad'' results in terms of adherence and compatibility. M-T-Bc-T yielded good results in terms of the four parameters tested and was thus considered the best grafting method for C. papaya (Table 7).
The methods involving the tongue approach, in which the two root systems are preserved during the grafting procedure, yielded the best results. Cleft grafting can be considered an alternative method, but it has the disadvantage that temperature and humidity must be controlled rigorously controlled, which is not necessary for the tongue approach grafting method. In addition, in cleft grafting, the quality of the seedlings is highly influenced by the growing medium used in the nursery (Agbo and Omaliko, 2006) (Table 7).
Plant grafting onto specific rootstocks is used to induce vigor and precocity, improve yield and quality, increase survival rates, reduce the risk of infection by soil pathogens, and increase tolerance against abiotic stress (Kumar and Sanket, 2017). In our study, normal and modified grafting methods were evaluated several times, as the latter was derived from the former. The same was true   for the different containers used for growing grafted papaya seedlings.

Conclusions
Graft survival and seedling vigor were the most important parameters used to determine the quality of grafted seedlings. Under normal environmental conditions in the Valley of Apatzingan, the seedlings were able to function as grafted seedlings 6 d after the grafting procedure. T-T and M-T-Bc-T were the most effective grafting methods, with 80% and 90% graft survival, respectively. With respect to the use of containers, the grafted plants grown in plastic bags had the highest percentage (92.5%) of graft survival. We therefore propose M-T-Bc-T and M-T-Bc-T-B, as reliable grafting methods for papaya that do not require special handling conditions.  G  G  G  T-P  R  G  R  G  C-T  R  G  R  G  C-P  R  G  G  G  M-T-Ac-T  R  G  G  R  M-T-Bc-T  G  G  G  G  M-T


extfieldsofstudy: Medicine Chemistry

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

text:
Effects of Activated Charcoal-Herb Extractum Complex on Antioxidant Status, Lipid Metabolites and Safety of Excess Supplementation in Weaned Piglets

Simple Summary Weaning is the most significant event in the life of pigs and pigs must cope with the sudden disruption of social interaction with sows and litters and pressure to adapt to new environments that may impair the growth performance and health status of piglets. Weaning can induce oxidative stress and disturb the balance of lipid metabolism in pigs. In this study, we innovatively evaluated a complex of activated charcoal and Chinese herb extractum (CHC) in weaning pigs on antioxidant status, serum lipid metabolites and safety supplementation in weaning pigs. This complex combines functions of active charcoal in absorption of toxins and the Chinese medicinal herbs in antioxidant capacity. The results demonstrated a promising additive of this complex CHC in future applications. This complex may be a good nutritional supplement to use in swine production. Abstract This study was aimed at evaluating the effects of activated charcoal-herb extractum complex (CHC) on antioxidant status, serum lipid metabolites and its safety supplement in weaning piglets. In experiment 1, a total of 216 piglets (Duroc × Landrace × Large White) weaned at 28 days of age with initial body weight of 8.55 ± 1.18 kg were assigned randomly to six treatment groups. each treatment group had six pens, with six pigs per pen. Pigs were fed a corn-soybean meal-based diet supplemented with 500, 1000, 1500 or 2000 mg kg−1 of CHC over two 14-d periods. Diets supplemented with 0 and 1000 mg kg−1 of montmorillonite (MMT) were set as the negative and positive controls, respectively. In experiment 2, pigs (n = 108) weaned at 28 days of age with initial body weight of 8.58 ± 0.04 kg were randomly assigned to three treatment groups. Each treatment group had six pens, with six pigs per pen. Pigs were fed a corn-soybean meal-based diet supplemented with 0, 1000 or 10,000 mg kg−1 of CHC over two 14-d periods. In experiment 1, on day 14, supplementation with CHC significantly decreased very low-density lipoprotein (VLDL) concentration while they decreased low-density lipoprotein (LDL) concentration on d 28, CHC at 500, 1000 or 1500 mg kg−1 significantly increase high-density lipoprotein (HDL) concentration. Supplementation with 500 or 1000 mg kg−1 CHC reduced serum malondialdehyde (MDA) concentration during the entire experimental period and increased the concentration of serum total superoxide dismutase (T-SOD) on d 14. CHC at 500 or 1000 mg kg−1 significantly reduced the liver MDA concentration and increased liver T-SOD concentration. In experiment 2, increased ADG was obvious during the first 14 days and the whole period in 1000 mg kg−1 supplemented pigs, similarly F: G was lowest in the first 14 days. There was no difference in growth performance, visceral index, haematological and serum biochemical parameters and visceral organs morphology between pigs fed 10,000 mg kg−1 of CHC and control. Together, 500 to 1000 mg kg−1 CHC was confirmed to improve antioxidant status, and serum lipid metabolites in this study and excess supplementation of CHC is safe in weaning piglets.

. Composition and analyzed nutrient levels of basal diets (%, dry matter basis) 1 .

Items
Phase The purpose of experiment 1 was to evaluate the effects of CHC on antioxidant status and serum lipid metabolites in weaned pigs. Weaned pigs (n =216) with an initial body weight of 8.55 ± 1.18 kg were assigned randomly to 6 treatment groups: a corn-soybean meal basal diet supplemented with 500, 1000, 1500 or 2000 mg kg −1 of CHC. Negative and positive controls were 0 (CON) and 1000 mg kg −1 of MMT (MMT), respectively. Each treatment group had six pens, with six pigs (three barrows and three gilts) per pen.
We selected 36 pigs near the average group body weight in each pen randomly to collect blood in the morning on d 14 and 28 after 12 h of fasting. Serum samples were separated by centrifugation at 3000 g at 4 • C for 10 min and stored at −20 • C. After 12 h of fasting on the d 28, the pigs which collected the blood were euthanized by electric shock and then bled. Samples of liver and kidney were placed in formalin prior to antioxidant status analysis.

Experiment 2
Experiment 2 was conducted to determine the safety of excess CHC supplementation in weaned pigs. Weaned pigs (n = 108) with an initial body weight of 8.58 ± 0.04 kg were assigned randomly to three treatment groups. Each treatment group had six pens (three barrows and three gilts per pen). Pigs were fed a corn-soybean meal-based diet as in Experiment 1 supplemented with 0, 1000 or 10,000 mg kg −1 of CHC.
Each piglet was weighed on days 0, 14, and 28 and recorded feed consumption in pens to determine average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F: G) was calculated. Health data was recorded every day. 18 pigs (1 pig from each pen) were selected randomly to collect blood in the morning on d 14 and 28 after 12 h of fasting. Blood samples were collected from the anterior vena cava and placed in tubes containing ethylene-diaminetetraacetate bi-potassium salt (EDTAK 2 ) and no whole blood and serum anticoagulants. Hematological parameters were measured on whole blood within 1 h after sampling. Serum samples were separated by centrifugation at 3000 g at 4 • C for 10 min and stored at −20 • C until analysis. The pigs from which blood had been sampled were euthanized by electrical stunning followed by exsanguination following a 12-h fasting on d 28. Harvest the heart, liver, spleen, lungs and kidneys and weigh them. The relative weight of the heart, liver, spleen, lungs and kidneys is expressed as the ratio to body weight (g kg −1 ). The heart, liver, spleen, lung, kidney and duodenum were sampled and placed in formalin for morphological examination.

Chemical Analysis
The ingredients and diet were analyzed according to the Association of Official Analytical Chemists' (AOAC) (2012) procedure [13], including crude protein, total phosphorus and calcium. For analysis of most amino acids, the ingredients and diet were hydrolyzed in 6 M HCl for 24 h at 110 • C. Determination of sulfur amino acid content after formic acid oxidation (AOAC, 2012) was undertaken. Amino acid analysis was carried out using liquid chromatograph (Hitachi L-8800 Amino Acid Analyzer, Tokyo, Japan).

Antioxidant Status
Liver and kidney samples were homogenized in 0.1 mol/L Tris (hydroxymethyl) aminomethane buffer at 4 • C, pH 7.4 to make a 10% (w/v) homogenate, using a Polytron homogenizer for 5 minutes. The ultrasonic homogenizer was centrifuged at 3000 g for 5 minutes at 4 • C for 3 minutes, and the supernatant was collected and stored at −20 • C for enzyme analysis.
In the serum from d 14 and d 28, liver and kidney, the total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD) and malondialdehyde (MDA) concentrations were determined. All assays were performed according to the manufacturer's instructions using commercial kits (Jiancheng Biochemical Reagent Co., Nanjing, China) [14].

Serum Lipid Metabolites
Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) concentrations were determined with commercially available kits (Biosino Biotechnology and Science Inc., Beijing, China). The Automatic Biochemical Analyzer was a Hitachi 7160 manufactured by Hitachi High-Tech Corporation (Tokyo, Japan).

Histopathology Analysis
Tissue samples from heart, liver, spleen, lung, kidney and duodenum were fixed in 10% formalin buffer for 48 h, then dehydrated, cleared and paraffin-embedded. 5 µm thick paraffin sections were stained with hematoxylin and eosin. The change of tissue morphology was observed.

Statistical Analysis
The data was analyzed as a completely random design using one-way analysis of variance (ANOVA) according to the general liner model (GLM) procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA), which included terms for treatment. The pen serves as the experimental unit for growth performance, while the individual pig is the experimental unit for all other indicators. Mean differences were tested using Duncan's multi-range test. The coefficients of the unequal contrast are generated by the interactive matrix algebra process (IML) of SAS, and then the linear and quadratic responses of the CHC are evaluated using orthogonal polynomials. Significance was declared at p < 0.05. An unpaired Student's t-test was used when analyzing the differences between MMT and CHC in the effects of lipid metabolites, MDA and T-SOD. The comparison contrasts were: MMT and CHC 500 mg kg −1 , MMT and CHC 1000 mg kg −1 , MMT and CHC 1500 mg kg −1 , MMT and CHC 2000 mg kg −1 . * p < 0.05, ** p < 0.01 were declared as significance. Similarly, the differences between CON and CHC were analyzed with the comparison contrasts of CON and CHC 500 mg kg −1 , CON and CHC 1000 mg kg −1 , CON and CHC 1500 mg kg −1 , CON and CHC 2000 mg kg −1 . # p < 0.05 were declared as significant. controls. Compared with the two control groups, pigs fed CHC showed a significantly higher serum HDL concentration. Serum LDL concentration was significantly lower in the pigs fed 500, 1000 and 1500 mg kg −1 CHC compared with positive control (MMT) and negative control (CON)s. Other serum lipid traits were not influenced by dietary treatments (Table 2). Similarly, the differences between CON and CHC were analyzed with the comparison contrasts of CON and CHC 500 mg kg −1 , CON and CHC 1000 mg kg −1 , CON and CHC 1500 mg kg −1 , CON and CHC 2000 mg kg −1 . Note: 1 Each mean is the average of 6 observations. Significance was declared at p < 0.05. 2 MMT montmorillonite, CHC activated charcoal-herb extractum complex, SEM standard error of mean, TC total cholesterol and TG triglyceride.

Antioxidant Status
We observed pigs fed 500, 1000 and 1500 mg. kg -1 CHC significantly reduced concentration of serum MDA on d 14 while 500 and 1000 mg. kg -1 CHC groups had lower concentration of serum MDA on d 28 when compared with MMT and CON ( Figure 2). In relation to kidney and liver MDA, 500 mg. kg -1 CHC group had significantly reduced concentration of MDA in kidney compared with MMT and in liver compared with CON. Similarly, supplementation of CHC at 500 and 1000 mg. kg -1 significantly increased T-SOD concentration in serum on d 14 and in liver compared with MMT, 500 mg. kg -1 group significantly increase T-SOD concentration in kidney with MMT. There were no differences in serum, kidney or liver T-AOC and GSH-Px activity among all groups (Table 3). An unpaired Student's t test was used when analyzing the differences between MMT and CHC in the effects of lipid metabolites. The comparison contrasts were: MMT and CHC 500 mg kg −1 , MMT and CHC 1000 mg kg −1 , MMT and CHC 1500 mg kg −1 , MMT and CHC 2000 mg kg −1 . * p < 0.05, ** p < 0.01 were declared as significance. Similarly, the differences between CON and CHC were analyzed with the comparison contrasts of CON and CHC 500 mg kg −1 , CON and CHC 1000 mg kg −1 , CON and CHC 1500 mg kg −1 , CON and CHC 2000 mg kg −1 .

Antioxidant Status
We observed pigs fed 500, 1000 and 1500 mg kg −1 CHC significantly reduced concentration of serum MDA on d 14 while 500 and 1000 mg kg −1 CHC groups had lower concentration of serum MDA on d 28 when compared with MMT and CON ( Figure 2). In relation to kidney and liver MDA, 500 mg kg −1 CHC group had significantly reduced concentration of MDA in kidney compared with MMT and in liver compared with CON. Similarly, supplementation of CHC at 500 and 1000 mg kg −1 significantly increased T-SOD concentration in serum on d 14 and in liver compared with MMT, 500 mg kg −1 group significantly increase T-SOD concentration in kidney with MMT. There were no differences in serum, kidney or liver T-AOC and GSH-Px activity among all groups (Table 3). Similarly, the differences between CON and CHC were analyzed with the comparison contrasts of CON and CHC 500 mg kg −1 , CON and CHC 1000 mg kg −1 , CON and CHC 1500 mg kg −1 , CON and CHC 2000 mg kg −1 . # p < 0.05 were declared as significant. Note: 1 Each mean is the average of 6 observations. Significance was declared at p < 0.05. 2 T-AOC total antioxidant capacity and GSH-Px glutathion peroxidase.

Serum Biochemical Parameters
There were no significant differences among all groups in the concentration of serum glucose (GLU), total protein (TP), albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine (CREA) and urea nitrogen (UN) on d 14 and d 28 (Table 4).  Similarly, the differences between CON and CHC were analyzed with the comparison contrasts of CON and CHC 500 mg kg −1 , CON and CHC 1000 mg kg −1 , CON and CHC 1500 mg kg −1 , CON and CHC 2000 mg kg −1 . # p < 0.05 were declared as significant.

Growth Performance and Visceral Index
Compared with the negative control and excess supplementation groups, pigs fed 1000 mg kg −1 CHC displayed significantly increased ADG and reduced F: G during the first phase (Table 5). Over the whole experimental period, CHC at 1000 mg kg −1 significantly increased body weight and ADG and decreased F: G compared with the negative control (0 mg kg −1 ) and excess supplementation group (10,000 mg kg −1 ) s. When it comes to visceral index, there were no differences in heart, liver, spleen, lung and kidney visceral index among the three experimental groups.

Visceral Organ Histopathology
Normal histological structures in the heart, liver, spleen, lung, kidney and duodenum were observed in all pigs fed the different diets for 28 days (Figure 3).
Note: 1, a, b, c Means within the same row without common superscripts differ significantly (p < 0.05). 2 BW body weight, ADG average daily gain, ADFI average daily feed intake, F: G feed to gain ratio.

Visceral Organ Histopathology
Normal histological structures in the heart, liver, spleen, lung, kidney and duodenum were observed in all pigs fed the different diets for 28 days (Figure 3).

Hematological and Serum Biochemical Parameters
On d 28, the high dose of CHC (10000 mg. kg -1 ) caused higher ALP level compared to the negative control and the lower dose of CHC (1000 mg. kg -1 ). No effect (p > 0.05) was observed on hematological parameters (Supplement Table S1) or other serum biochemical parameters (Table 6) among treatments. The results suggest that CHC causes no measurable damage in piglets.

Hematological and Serum Biochemical Parameters
On d 28, the high dose of CHC (10000 mg kg −1 ) caused higher ALP level compared to the negative control and the lower dose of CHC (1000 mg kg −1 ). No effect (p > 0.05) was observed on hematological parameters (Supplement Table S1) or other serum biochemical parameters (Table 6) among treatments. The results suggest that CHC causes no measurable damage in piglets.  Note: 1 a, b Means within the same row without common superscripts differ significantly (p < 0.05). 2 Each mean is the average of 6 observations. Mean differences were tested using Duncan's multi-range test. The coefficients of the unequal contrast are generated by the interactive matrix algebra process (IML) of SAS. Significance was declared at p < 0.05. 3 GLU glucose, TP total protein, ALB albumin, AST aspartate aminotransferase, ALT alanine aminotransferase, ALP alkaline phosphatase, CREA creatinine, UN urea nitrogen, TC total cholesterol, TG total triglycerides and TBILI total bilirubin.

Discussions
Weaning is the most critical period in the life of young piglets. Nutrition, environment and social factors impact weaned piglets in ways that contribute to accumulated oxidative stress [15]. Early weaning stress is associated with oxidative indicators [16] such as elevated plasma MDA and free radicals and impaired cellular antioxidant defense systems in piglets [17].
In a previous study, we found that CHC can promote growth performance, immunological indices, intestinal morphology and microflora in weaned piglets [11]. In the connect study, we further investigated CHC effects on oxidative stress that generated in weaning piglets and its effect on plasma lipid metabolism.
Oxidative stress is known as the imbalance between oxidants and detoxification through antioxidant systems. Oxidative stress is traditionally considered harmful to humans because it damages cellular components including lipids, DNA, proteins and carbohydrates, leading to tissue damage [18,19]. Free radicals can produce reactive oxygen species in cells and can cause damage to living cells and tissues [20]. The ability of cells to maintain functional homeostasis depends on the rapid induction of protective antioxidant enzymes. T-SOD in serum may catalyze the dismutation-reaction of free radicals, GSH-Px can remove lipid peroxide, and T-AOC represents the activity of the oxidase system in serum [21]. These antioxidant enzyme activities can effectively protect the structure and functional integrity of cell membranes, and are the main components of important antioxidant systems in the body [22]. Weaning induces oxidative stress in piglets, resulting in oxidative damage in the gastrointestinal track [23]. Thus, it is important to restore and maintain body antioxidant capacity to combine oxidative stress. The antioxidant system owns complete and complex functions comparable to the immune system. Literature has documented how supplementation of antioxidants such as essential oil blends [24], chitosan oligosaccharide [25] and selenomethionine [14] are closely related to improved growth performance in weaning pigs.
In this study, we found that CHC showed its antioxidative effect by decreasing concentration of MDA in serum at 500 to 1000 mg kg −1 , and kidney and liver MDA at 500 mg kg −1 . A decreased concentration of MDA in the duodenum and jejunum was observed in pigs fed 500 and 1000 mg kg −1 of CHC in previous study [11]. MDA is the end product of lipid peroxidation by free radicals in vivo. A large accumulation of MDA can lead to cross-linking polymerization of proteins, nucleic acids and other living macromolecules. MDA content in vivo can indirectly reflect oxidative stress state of the body [26]. Similarly, we found that CHC increased T-SOD levels in serum. Collectively, these results demonstrated that CHC has beneficial effects on the antioxidant status and protected weaned pigs against oxidative stress.
Compared with CHC diet and the un-supplemented control, MMT did not influence the antioxidative state of pigs. MMT is a widely used adsorbent for improvement of diarrhea in humans and weaned piglets. It possesses a porous structure with positive charges inside tetrahedral and octahedral layers but negative charges on the surface [27]. MMT higher than 1000 mg kg −1 is reported to reduce antioxidant capacity and disrupt liver structure [12]. Lower levels of MMT supplementation are not different from un-supplemented control diets regarding various antioxidant indexes which is in agreement with the current study [12].
CHC is a complex of activated charcoal and extractum of Chinese herb medicines, thus its antioxidative activity can be considered separately. The porous structure of activated charcoal allows it to act as an absorbent in the digestive tract. Previous reports document activated charcoal's chemical efficacy in removing toxins from the gastrointestinal tract of broilers [28][29][30][31] but there are no similar reports with piglets. Toxins are a factor that can generate reactive oxygen species and induce oxidative stress [32]. Absorption of toxins can indirectly reduce the oxidative stress generated by toxins. In this study, we did not examine the effect of activated charcoal independent of the Chinese herb extratum. However, absorption of un favorable components in feedstuffs may have contributed to the improved antioxidative status of CHC-fed pigs.
Four types of Chinese herbs extractum (Pulsatilla chinensis, Portulaca oleracea L., Artemisia argyi Folium and Pteris multifida Poir) are contained in CHC. They are all widely used as antioxidants. For example, Portulaca oleracea L. is linked to strong antioxidant activity and a reduction in blood lipid content [33][34][35] and Pulsatilla chinensis can specifically increase superoxide release and increase SOD activity which minimizes superoxide-mediated toxicity [36]. Artemisia argyi Folium also plays an important role in mitigating oxidative stress [37].
There are limited reports on the effects of dietary absorbents on lipid metabolites of pigs. Dietary MMT has lipid-adsorbing ability which enhances lipid excretion in feces [17]. Kuusisto [38] reported that patients with hypercholesterolaemia were treated for 4 weeks with activated charcoal and plasma TC and LDL decreased and HDL increased. Our results showed that CHC decreased VLDL and LDL and increased HDL concentration in serum. Increasing concentrations of HDL are strongly associated with decreasing accumulation of atherosclerotic plaques within walls of arteries [39]. Conversely, LDL transports cholesterol from the liver to tissues of the body, and can be oxidized into low-density lipoprotein [40]. Herbs such as Portulaca oleracea reduces triglyceridemia, cholesterolemia, and improves reverse cholesterol transport in rats [41], Gatreh-Samani [42] showed that serum TC, TG and LDL decreased and HDL increased in patients who received 50 or 60 g of Portulaca oleracea per day. Their researchers indicated that CHC can balance of lipid metabolism capacity and improve lipid composition to good state through the weaning time.
In comparison to other researches evaluating activated charcoal, only 500 to 1000 mg kg −1 CHC can improve growth performance and maintain healthy functions superior to the reported. This effective dose was lower than that reported in other studies: 0.25% activated charcoal in African catfish juveniles [43] and 0.3% to 1% bamboo charcoal in fattening pigs [44], chicken [45], Nile tilapia, and ducks [46]. Excessive supplementation of MMT as a mycotoxin adsorbent impairs growth performance, liver function, and antioxidant capacity in starter pigs [12]. A previous study reported that the effective dose of CHC for improvement of growth ranged from 500 to 1000 mg kg −1 . The purpose of adding a high level (10,000 mg kg −1 ) of CHC, 10 times greater to the highest effective dose, was to evaluate the safety of CHC supplementation in weaning pigs. On d 28, the high dose of CHC (10000 mg kg −1 ) caused a higher ALP level compared to the negative control and the lower dose of CHC (1000 mg kg −1 ). The serum level of ALP in high dose-treated piglets was within the normal range of piglet serum [47]. Excess supplementation of CHC elicited similar growth performance compared with the negative control. Visceral index, visceral organ histopathology and haematological (except ALP) indices were not different from controls when 10,000 mg kg −1 CHC were included in the diet. It may be due to the combination of activated charcoal and extractum from Chinese herbs.

Conclusions
In conclusion, dietary CHC at an inclusion rate of 500 or 1000 mg kg −1 is recommended for weaning pigs. This level of supplementation improves the antioxidant status and serum lipid metabolism of weaned pigs. Supplementation of CHC at 10,000 mg kg −1 appears to have no detrimental effects on weaned pigs.


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Effects of long-chain fatty acid supplementation on the growth performance of grower and finisher pigs: a meta-analysis

Background Supplementation of feed with long-chain fatty acids (LCFAs) during the grower and finisher phases has long been discussed as a growth promotion strategy in pigs, but its effects are inconsistent. The purpose of our study was to comprehensively evaluate its effects on the growth performance based on the average daily gain (ADG), average daily feed intake (ADFI) and gain: feed (G:F) ratio and to unveil the roles of the basal diet, LCFA concentration and LCFA saturation. Results We searched the PubMed and Web of Science databases (articles published from Jan 1st, 2000, to Sep 30th, 2018; restricted to English) and compared LCFA-supplemented diets with control diets. We retrieved 2346 studies, 18 of which (1314 pigs, 26 records) were eligible for our analysis. We used a random-effects model to calculate the weighted mean differences (WMDs) and 95% confidence intervals (CIs). LCFA supplementation in the grower-finisher phase improved the ADG (WMD = 41.74 g/d, 95% CI: 8.81 to 74.66, P = 0.013) and G:F ratio (WMD = 0.019, 95% CI: 0.006 to 0.032, P = 0.003). For supplementation solely in the finisher phase, we found a similar performance in the ADG (WMD = 39.93 g/d, 95% CI: 26.48 to 53.38, P < 0.001) and G:F ratio (WMD = 0.019, 95% CI: 0.006 to 0.032, P < 0.001) but a reduction in the ADFI (WMD = − 83.863 g/d, 95% CI: − 156.157 to − 11.569, P = 0.023). Specifically, approximately 5% LCFA supplementation in the finisher phase had significant effects on the ADG (WMD = 51.385 g/d, 95% CI: 35.816 to 66.954, P < 0.001), ADFI (WMD = − 102.869 g/d, 95% CI: − 189.236 to − 16.502, P = 0.02) and G:F ratio (WMD = 0.028, 95% CI: 0.018 to 0.039, P < 0.001), whereas a concentration of approximately 1% exhibited no effects. Conclusions Overall, regardless of the basal diet and saturation, LCFA supplementation greatly improves the growth performance of grower and finisher pigs, primarily by increasing the energy density.

Background
Antimicrobial growth promoters (AGPs) have been used in the pig industry for more than 60 years, where they have made impressive contributions in terms of economic benefits and healthy farming [1]. Simultaneously, as a result of extensive use of sub-therapeutic antimicrobials [2], pigs have become an important reservoir of antimicrobial-resistant bacterial strains and genes, which seriously endanger public health [3,4]. In addition, rapidly rising concerns about food safety from consumers are encouraging animal nutritionists to develop reliable alternatives for growth promotion.
Supplementation of long-chain fatty acids (LCFAs), which is the largest category of fatty acids in animal diets [5], or their compounds into daily rations provides a practical method for achieving better growth performance than diets without additional LCFA supplementation. More importantly, few adverse effects of fatty acids have been found, which ensures easier acceptance of LCFAs as a growth promoter by consumers. According to the National Research Council (NRC) [6], supplementation of grower and finisher feed with fatty acids increases the growth speed, reduces the feed intake and improves the gain efficiency. However, in feeding trials, the effects of LCFA supplementation on growth performance are inconsistent. Our review of previous studies reveals that some influential factors, including the basal diet [corn-soybean vs. distillers' dried grains with solubles (DDGS)], LCFA concentration (high concentration vs. low concentration) and LCFA saturation (saturated vs. unsaturated), should be considered when exploring the synergistic effects of basal diet with LCFAs, the dosage-dependent effect of such supplementation and the influence of the physicochemical properties of LCFAs, respectively.
Because separate studies differ in the factors considered and inevitably lack an overall investigation [7], we performed a meta-analysis to reveal the effects of LCFA supplementation on grower and finisher pigs and to elucidate the influential factors based on the average daily gain (ADG), average daily feed intake (ADFI) and gain: feed (G:F) ratio.

Methods
This meta-analysis strictly followed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses [8].

Search strategy
We collected relevant studies published between Jan 1 st , 2000, and Sep 30 th , 2018, in the PubMed (https://www. ncbi.nlm.nih.gov/pubmed; accessed Sep 30 th , 2018) and Web of Science (http://webofknowledge.com; accessed Sep 30 th , 2018) databases. The date range was chosen based on the development of feeding facilities and improvements in growth performance due to breeding [9]. We restricted the language to English. The search principles were as follows: 1) the terms "grower pig" and "finisher pig" were extended to include "pig", "swine", "boar", "piglet", "sow", "gilt" and "barrow"; 2) the terms related to fatty acids were searched in the PubMed database beforehand and shown to be "acids, fatty", "fatty acids, esterified", "acids, esterified fatty", "esterified fatty acids", "fatty acids, saturated", "acids, saturated fatty", "saturated fatty acids", "aliphatic acids" and "acids, aliphatic"; and 3) growth performance was equal to production performance. The detailed search strategy and findings are shown in Table 1. We also extended our search to the articles referenced by the studies identified for the meta-analysis.

Selection criteria and procedure
Studies were eligible for inclusion in our meta-analysis under the conditions that 1) the growth performance parameters (ADG, ADFI and G:F ratio) were reported, 2) LCFAs, LCFA esters, LCFA-rich compounds or LCFA salts were added to the feed throughout the experimental period, 3) the trials were initiated at the growing or finishing phase and terminated at the finishing phase, 4) the ADFI and G:F ratio were calculated by the gross weight of the feed, 5) the genetic background was a commercial breed (e.g., Duroc × Landrace × Yorkshire) and 6) the detailed fatty acid composition and protein density were included, with no difference in the protein level. The exclusion criteria were as follows: 1) the major content of the supplement was not LCFAs (e.g., grape seed cake and rice bran); 2) the study lacked a controlled diet without additional fatty acid supplementation; and 3) the basal diet was not corn-soybean or DDGS (e.g., barley diet). Based on these standards, we selectively screened eligible studies for inclusion in the analysis (Fig. 1a). The information extracted from the included studies was as follows: author information (first author, year); genetic background; sum number of pigs included in the control and treatment groups; mean initial body weight; mean final body weight; initial phase (grower or finisher); supplemental substance; energy difference; basal diet (cornsoybean or DDGS); concentration (low or high); saturation (saturated or unsaturated); and growth performance (ADG, ADFI and G:F ratio) of the treatment and control groups. One study could have more than one record depending on the treatments and growth phases of the pigs.
The study selection procedure was as follows: 1) two investigators (Z. Li and B. Xu) independently screened the titles and abstracts of the acquired studies and identified relevant studies for full-text reading; 2) disagreements during independent study selection were referred to the third investigator (Y. Wang) for an ultimate resolution; and 3) after the eligible studies were verified according to our criteria, one investigator (Z. Li) extracted the data and information from each study, followed by inspection by the other investigator (B. Xu).

Study quality assessment
Two investigators (Z. Li and B. Xu) performed independent study quality assessment according to the criteria provided in the Consolidated Standards of Reporting Trials statement [8] and the Cochrane Collaboration's tool for assessing risk of bias [10]. The assessment aspects included sequence generation, allocation concealment, blinding of participants and personnel, incomplete outcome data, selective reporting and other bias. The divergences were resolved by the third investigator (Y. Wang) (Fig. 1b).

Within-group SD estimate
Within-group standard deviations (SDs) or standard errors (SEs) are required for a meta-analysis. However, in the included articles, these data usually were missing, and the provided SE of the mean could not be used to calculate the within-group SD. In such cases, first we contacted the authors via email to request the withingroup SDs. If raw statistics were not available from the authors, the within-group SDs of growth performance were estimated using 8-15% of the mean value, which was based on the raw statistics of our institute, suggestions from peers in both industry and college settings and relevant data presented by the NRC [6]. Technically, the SD is derived from the random errors in trials and follows a random distribution. Thus, the SD should not Apple et al. [19] Benz et al. [18] Benz et al. [23] 2346 studies identified through database searching Eastwood et al. [20] Engel et al. [15] Jaturasitha et al. [21] Juarez et al. [22] Lee et al. [25] Liu et al. [31] Moran et al. [32] Rickard et al. [24] Stephenson et al. [27] Sun et al. [17] Thiel-Cooper et al. [16] Upadhaya et al. [28] Villela et al. [29] Wang et al. [26] Random sequence generation (selection bias) Allocation concealment (selection bias) Blinding of participants and personnel (performance bias) Blinding of outcome assessment (detection bias) Incomplete outcome data (attrition bias) Selective reporting (reporting bias) Other bias Low risk Unclear High risk be calculated as a single mean ratio. To be prudent, we randomized the SDs of each group and repeated the meta-analysis 10 times to confirm the stability of our results. If we had observed a statistically significant difference, then the meta-analysis would have been regarded as impossible for this topic and ceased. The data used for the subsequent analyses and presentation originated from one of the 10 random processes.

Statistical analysis
The statistical analysis was performed with Stata 12.0 (Stata Corp., USA).

Meta-analysis
We calculated the pooled estimates of the mean differences between the treatment and control groups using a random-effects model [11]. We also used Cochran's Q statistic (significance level of P ≤ 0.1) and the I 2 statistic to quantitatively measure the heterogeneity in our analysis. The grading of heterogeneity was as follows: no heterogeneity, I 2 ≤ 25%; low heterogeneity, 25% < I 2 ≤ 50%; moderate heterogeneity, 50% < I 2 ≤ 75%; and high heterogeneity, I 2 > 75% [12].

Regression analysis
To measure the effects of covariants, which in our study are the basal diet, LCFA concentration and LCFA saturation, on the outcomes (ADG, ADFI and G: F ratio), we performed a regression analysis after the meta-analysis.
To avoid a false positive result, the regression analysis was applied only to groups with more than 10 records.

Subgroup categorization and analysis
We conducted subgroup analyses to elucidate heterogeneity that was significant (P < 0.05) or beyond a moderate level (I 2 > 50%). The included studies were classified into the "corn-soybean diet vs DDGS diet", "high concentration vs low concentration" and "saturated vs unsaturated" subgroups. The low and high concentrations were set to approximately 1% and 5%, respectively, based on the frequency of occurrence in the included studies and, more importantly, the role of fatty acids in the feed. Low-concentration supplementation (approximately 1%) often applied to unsaturated fatty acids (e.g., conjugated linoleic acid) and was more likely to have biological functions, including biomembrane constitution, signal transduction [13,14] and eicosanoid precursor action (e.g., prostaglandins, leukotrienes and thromboxanes) [15]. The saturation classification of fatty acid compounds (e.g., animal fat and vegetable oils) was determined by the dominating ratio (> 50%) in the fatty acid composition [16].

Sensitivity analysis
If the heterogeneity was significant (P < 0.05), a sensitivity analysis was performed to identify the study (or studies) that contributed as the main source of the heterogeneity. Heterogeneity and pooled estimates were recalculated after the study or studies (including all records) was deleted from the outcome.

Publication bias
Publication bias was evaluated using Begg's and Egger's tests, for which the significance level was defined at P < 0.1 [11]. If Begg's and Egger's tests disagreed, Egger's test was used as a reference. Additionally, the trim-and-fill test was used to further test and adjust for publication bias [17].

Results
Of the 2346 studies identified, we included 18 studies (with data for 1314 pigs) and extracted 26 records for our meta-analysis [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. Except for Juarez et al. [25], in which the pigs were from a commercial farm, all of the studies were performed on clearly defined commercial breeds, with 5 studies (6 records) beginning LCFA supplementation at the grower phase and 13 studies (20 records) beginning it at the finisher phase. The mean initial weights of the growing and finishing pigs were 33.98 kg and 67.78 kg, respectively. All studies ended in the finishing phase, with a mean body weight of 115.32 kg. The categorization of growth phases was combined with both the body weights and the experimental design (Table 2). Because of the nonsignificant publication bias (P > 0.1) in the current meta-analysis, the trim-and-fill test was not performed ( Table 3).

Regression analysis
According to the regression analysis of LCFA supplementation in finisher pigs (Table 4), the LCFA concentration T treatment, CT control; a significant difference in the trial is indicated by * P < 0.05 and ** P < 0.01 might play a significant role, especially in determining the ADG (P regression = 0.014) and G:F ratio (P regression = 0.007). In contrast, the basal diet and LCFA saturation were not major causes of heterogeneity, because they exhibited no significant effects (P regression > 0.05) in the regression analyses of the ADG, ADFI, and G:F ratio. Therefore, we focused on the role of the concentration and conducted corresponding subgroup analyses in the subsequent research.
Effects of LCFA supplementation on the G:F ratio of finisher pigs As shown in Fig. 3c, LCFAs had a significant positive effect on the G:F ratio of the finishers (WMD = 0.022, 95% CI: 0.012 to 0.033, P = 0.000), which was especially strong in the case of high-concentration supplementation (WMD = 0.028, 95% CI: 0.018 to 0.039, P = 0.000). In contrast, low-concentration supplementation of finisher feed had no effect on the G:F ratio (WMD = 0.004, 95% CI: − 0.004 to 0.011, P = 0.331). Although high heterogeneity was detected overall (I 2 = 79.3%, P heterogeneity = 0.000), after the subgroup analysis, the majority of the heterogeneity was attributed to the highconcentration subgroup (I 2 = 69.1%, P heterogeneity = 0.000) rather than the low-concentration subgroup (I 2 = 0.0%, P heterogeneity = 0.884).

Discussion
In swine husbandry, AGPs have been shown to be unquestionably stable and to have excellent effects on growth performance and animal health [36]. Therefore, with global banning of AGPs, a long list of alternatives (including antimicrobial peptides, organic acids, enzymes, probiotics, prebiotics, essential oils and metal oxides) has been intensively researched and developed to compensate for the vacancy [37,38]. These alternative substances exhibit similar or even better effects than AGPs against pathogen infections, oxidation and inflammation in animal trials [38]. However, the majority of alternative strategies are focused on health; in terms of promotion of growth performance, they yield mostly inconsistent results that are unequal to the effects of AGPs [37]. In addition, the administration route and economic costs indicate that much work is required for large-scale utilization of the listed alternatives for growth promotion. In this context, we focus on a conventional feedstuff, LCFAs, because of their extensive sources, cost effectiveness, safety, oral administration and potential functional roles [39][40][41]. Based on a systematic, largescale literature search and meta-analysis, we were able to comprehensively and quantitatively confirm the beneficial effects of LCFAs on pig growth performance. Regression analyses of the basal diet, concentration and saturation (Table 4) together with subgroup analysis of the concentration in finisher pigs (Figs. 2 and 3) further suggested that the benefits were concentration-dependent.
As shown in Table 2, the improved changes in growth performance of the growing and finishing pigs fed different LCFAs were mainly associated with the energy level. Of the 18 studies (26 records) included in our meta-analysis, 13 (20 records) revealed an elevated energy density after LCFA supplementation. Thus, even a lower feed intake is able to meet the caloric requirements of pigs. Moreover, intake of additional LCFAs will improve the digestibility of amino acids by lowering the gastric emptying speed and increasing the time of exposition to proteolytic enzymes [42][43][44]. As a consequence, a higher ratio of amino acids in feed will participate in meat production, which in turn increases the weight gain and gain efficiency. Our findings revealed no differences in the effects of saturated and unsaturated LCFAs on growth performance (Table 4). Unsaturated fatty acids (e.g., linoleic acid) are essential in pig feed because of the absence of desaturase enzymes [9]. In pig farming, essential unsaturated fatty acids improve sow fertility and piglet growth [45][46][47]  Stephenson et al. [30] (beef tallow, 4%) Eastwood et al. [23] (flaxseed meal, 5%) Jaturasitha et al. [24] (tuna oil, 1%) Stephenson et al. [30] (soybean oil, 4%) Thiel-Cooper et al. [19] ( (c) Fig. 2 Meta-analysis of differences in the growth performances of grower pigs fed LCFAs. a ADG. b ADFI. c G:F ratio. CI = confidence interval; CLA = conjugated linoleic acid; CWG = choice white grease; WMD = weighted mean difference; P heterogeneity = P value of heterogeneity (significance level P heterogeneity < 0.05). The small solid diamond represents the point estimate for each individual trial, and the horizontal line extending from each solid diamond represents the upper and lower limits of the 95% CI. The size of the shaded square indicates the relative weight of the trial in the meta-analysis. Small solid diamonds located on the positive quadrant of the X-axis favour an increase in the growth parameters (ADG, ADFI and G:F ratio), whereas those on the negative quadrant favour a decrease. The open diamond represents the WMD and 95% CI of the trials their beneficial effects on neural development, immune responses and gut health [39][40][41]. Nevertheless, for growers and finishers, whose body systems are highly mature, the primary role of fatty acids is to be oxidized and to store and supply energy. Additionally, the regression analysis indicated that the addition of DDGS to a cornsoybean diet did not impair the promoting function of LCFAs, which was in accordance with the review of Stein and Shurson [48]. As shown in Fig. 3, the significant heterogeneity in the ADFI and G:F ratio of the finishers was primarily driven by the high-concentration subgroup. In the sensitivity analyses, we found that Villela et al. [32] (2 records) and Liu et al. [34] (4 records) were the major sources of heterogeneity (data not shown). After excluding the 2 studies (6 records), the overall and highconcentration subgroup heterogeneity in the ADFI became nonsignificant. Additionally, exclusion of the 6 records from the ADFI analysis caused the originally negative effects of LCFA supplementation on the overall group and high-concentration subgroup to become nonsignificant. For the G:F ratio, removing the 6 records decreased the heterogeneity but did not influence the significance of the pooled estimates. The differences in heterogeneity and statistical significance of the pooled estimates were primarily due to the variation in energy density. In the trial of Villela et al. [32], the metabolizable energy of the diet with 5% minimally refined cottonseed oil (3537 kcal/kg) in two phases (55-90 kg and 90-120 kg) was 253 kcal/kg higher than that of the control diet (3284 kcal/kg). Because the gossypol concentration (0.001%) was too low to exert any adverse effects on the growth performance [49], Villela et al. [32] concluded that the energy density was the key reason for the changes in growth performance. To clarify the concentration dependency of the results, we initially set the concentrations to approximately 1% and 5% for the low-and high-concentration groups, respectively ( Table 2). The concentration applied in the study of Liu et al. [41] was the highest (6%) among the included studies, and the energy difference between the group with 6% lipid supplementation (3600 kcal/kg) and the control group (3320 kcal/kg) was also considerable (270 kcal/kg). Therefore, a lower ADFI still provided sufficient energy for weight gain and enhanced the gain efficiency, indicating that the promotion of growth performance by LCFAs was related to an increased energy density.
The limitation of our study is the effects of within-group SD estimates on the pooled estimates. Due to the lack of within-group SDs in animal nutrition studies, we had to perform our meta-analysis based on within-group SDs estimated using 8-15% of the mean values. Because SD values impact the 95% CIs and the weight of an individual study, they also affect the pooled estimates and heterogeneity. To improve the validity of our findings, we compared the estimate range with the true values to test its accuracy. With data provided by the Moran group, we found that the true within-group SDs of the ADG and G:F ratio were precisely located within our estimate range. In contrast, the SD for the ADFI was under the lower limit because of the data collection method. Unlike the ADG and G:F ratio, in most cases the ADFI of each commercial pig can be obtained only by dividing the total intake per pen by the number of pigs. This method masks individual variation, causing a lower within-group SD than we would predict. Similar to the SDs of the ADG and G:F ratio, the variation in ADFI should be consistent with the calculated SD range. However, the dominant contributor to the pooled estimates was the mean difference of each study instead of the 95% CI and weight. The random-effects model we used was capable of balancing the differences in individual weights and therefore highlighting the role of mean differences. Taken together, both the accurate SD estimates and the primary role of the mean difference between the treatment and control groups ensure that this meta-analysis conducted on within-group SD estimates is reliable.

High concentration
Liu et al. [34] (palm oil, 6%) Lee et al. [28] (palm kernel oil, 3%) Engel et al. [18] (CWG, 4%) Villela et al. [32] (cotton seed oil, 5%) 90-120kg Apple et al. [22] (beef tallow, 5%) 0.02 (0.01 to 0.03)  Fig. 3 Meta-analysis of differences in the growth performances of finisher pigs fed a high/low LCFA concentration. a ADG. b ADFI. c G:F ratio. CI = confidence interval; CLA = conjugated linoleic acid; CWG = choice white grease; WMD = weighted mean difference; P heterogeneity = P value of heterogeneity (significance level P heterogeneity < 0.05). The small solid diamond represents the point estimate for each individual trial, and the horizontal line extending from each solid diamond represents the upper and lower limits of the 95% CI. The size of the shaded square indicates the relative weight of the trial in the meta-analysis. Small solid diamonds located on the positive quadrant of the X-axis favour an increase in the growth parameters (ADG, ADFI and G:F ratio), whereas those on the negative quadrant favour a decrease. The open diamond represents the WMD and 95% CI of the trials