Effect of increasing levels of fat-soluble and water-soluble vitamins on improving performance and plasma vitamin concentration in modern hybrids pig’s growth and finishing phase

ABSTRACT The objective was to evaluate the supplementation of fat-soluble and water-soluble vitamins on the performance and plasma concentrations of vitamins in modern hybrids pigs. A total of 144 commercial hybrid castrated male, 43.531 ± 1.099kg, were used in a randomized block design with six treatments, eight replicates and three animals per pen. The experimental treatments were different vitamin supplementation levels - 0, 25, 50, 75, 100, and 125% of the recommended by Rostagno et al. (2011) for male pigs in growth I (40 to 50kg), growth II (50 to 70kg), and finishing I (70 to 90kg) phases. For growth phases I and II, a linear effect (P<0.05) due to increase in vitamin supplementation was observed on performance. For finishing phase I and total phase, a linear effect (P<0.05) was observed with increased final average weight (FAW) and improved feed conversion ratio (FCR). For average daily weight gain (ADWG) and average daily feed intake (ADFI) a quadratic effect was observed (P<0.05). A linear (P<0.05) increase in plasmatic α-tocopherol and B12 was observed with the 125%. Thus, it is concluded that the 125% vitamin supplementation improved performance of modern hybrids pigs (40 to 90kg).


INTRODUCTION
Vitamins are organic compounds required in small amounts in the diet and participate in numerous metabolic reactions essential to promote performance, health and, consequently, the productive efficiency of pigs.Vitamins are basically classified according to their solubility, being fat-soluble (A, D 3 , E and K) when soluble in lipophilic substances and water-soluble (complex B and C vitamins) when soluble in water.Because most of them are not synthesized efficiently by the animal, it is imperative to provide them in the diet.
In general, vitamin A contributes to maintain the epithelial tissue integrity, vision, and immunity while vitamin D 3 , considered a hormone, acts in the regulation of calcium and phosphate ion homeostasis, bone mineralization and in increasing macrophage activity (Combs Jr, 2008;Isabel et al., 2012).Vitamin E is a natural biological antioxidant and acts at the cell membrane level with effects on the reproductive and immune systems, showing synergism with selenium (McDowell, 2006).Vitamin K, on the other hand, contributes to the blood clotting process (Combs Jr, 2008).Regarding the watersoluble vitamins (B complex), it is possible to state that they act as enzyme cofactors in the energy (mitochondrial and lipidic) and amino acid metabolism, for example, in interconversion processes of compounds, methylation, acetylation, remethylating, electron transport chain, DNA formation and in biosynthesis of intermediary compounds essential for health and performance improvement (McDowell, 2006;Combs Jr, 2008;Isabel et al., 2012).
The scientific literature available on the recommendation of vitamin levels in the diet of growing and finishing pigs is very variable.While the average recommendation of vitamins (fat-soluble and water-soluble) for growing and finishing pigs increased by 1.51% according to the National Research Council (Nutrient…, 1988(Nutrient…, , 1998(Nutrient…, , 2012)), Rostagno et al. (2005Rostagno et al. ( , 2011) ) increased by an average of 7.07%.However, when comparing the levels recommended by Rostagno et al. (2017), there is an average increase of up to 2.9 times in relation to the levels recommended by Rostagno et al. (2011) for some specific vitamins and at certain rearing stages.
Based on this, Dalto and Silva (2020) reviewed the vitamin levels recommended by nutritionists in the Brazilian swine industry.These authors noted that the average supplementation values for fat-soluble and water-soluble vitamins can reach up to 1.4 and 2.2 times when compared to the levels recommended by Rostagno et al. (2017) for the growing and finishing phases, respectively.The difference between the vitamin recommendations can be explained by the fact that companies with high productivity rates are adopting the concept of diet formulation with higher vitamin inclusions.Thus, the objective is not only to provide the minimum vitamin requirements to avoid deficiency symptoms in pigs.There is also an interest in maximizing performance associated with increased lean meat content in the carcass, reduced lipid peroxidation of the meat, improved immune status against stressors in the production of confined pigs, among other factors that are related mainly to the genetic advances of the adopted lines, which are constantly evolving.
Given the above, the objective was to evaluate the effect of fat-soluble and water-soluble vitamin supplementation on the performance and plasma concentrations of α-tocopherol and B 12 in growing and finishing modern pigs.

MATERIAL AND METHODS
All methods involving the handling of the animals were performed in accordance with the regulations approved by the Ethics Committee on Animal Use (CEUA) of the Federal University of Viçosa (UFV) under protocol number 119/2014.The study was carried out in the Swine Production Sector of the Animal Science Department of the Agricultural Sciences Center at the UFV, in Viçosa, Minas Gerais, Brazil.
A total of 144 castrated male pigs, commercial hybrids of high genetic potential for meat deposition (AGPIC 426 x Camborough), with initial average weight of 43.531±1.099kgwere used, distributed in a randomized block design with six treatments, eight repetitions, and three animals per experimental unit.
The treatments were 0, 25, 50, 75, 100, and 125% supplementation of fat-soluble and watersoluble vitamins.The treatment with 100% vitamin supplementation was based on the Brazilian Tables for Poultry and Swine (Rostagno et al. 2011) recommendations for castrated male pigs in the phases from 30 to 50kg, from 50 to 70kg and from 70 to 100 kg live weight, the inclusion levels being 1.000, 0.880 and 0.750kg/ton of feed, respectively (Table 1).The experimental diets for these production phases (Growth I, Growth II, and Finishing I) were formulated based on corn and soybean meal according to Rostagno et al. (2011), being isoprotein and isoenergetic (Table 2).
The animals were housed in masonry stalls (2 x 2m) with concrete feeders (60 x 30 cm) and nipple drinkers, located in a masonry shed with a concrete floor and covered with clay roof tiles.The facilities were cleaned daily during the experimental period.
Experimental feed and water were provided ad libitum throughout the experimental period.Feed, leftovers, and animals were weighed at the beginning and end of each production phase and during the total experimental period to verify the final average weight (FAW), average daily feed intake (ADFI), average daily weight gain (ADWG), and feed conversion ratio (FCR) of the pigs.
Blood samples were collected from the pigs on the final experimental period to measure vitamin E and vitamin B 12 in the plasma of one pig per pen.The pigs were transferred to a cage and 20mL of blood were collected through the jugular vein.Vitamin E concentration (αtocopherol) were determined with HPLC (Van Kempen et al., 2016) and Vitamin B 12 were determined according to Yang et al. (2020).
The experimental data were submitted to normality tests (Shapiro-Wilk test) and analysis of variance (ANOVA) and subsequent regression, at 5% level of significance, using the SAS program (SAS, 2002).To assess the fit of the models, values of the root mean square of the residuals (RMSE) and the coefficients of determination (R 2 ) were considered.

RESULTS
Table 3 shows the performance data of pigs in growth phases I and II.The duration of growth phase I, II, and total growth (I and II) were 11, 15, and 26 days, respectively.A linear effect (P<0.05) was observed for growth phase I (40-50 kg), with higher FAW and ADWG and improved FCR because of increasing vitamin supplementation levels.However, no effect (P>0.05) was observed on ADFI.
A linear effect (P<0.05) was observed for growth phase II (50-70 kg), with higher FAW and improved FCR.However, no effect (P>0.05) was observed on ADFI and ADWG.When analyzing the total growth phase (I and II, 40-70 kg), a linear effect (P<0.05) was observed with higher ADWG and improved FCR.Thus, an improvement in performance was found because of the 125% vitamin supplementation in the diet of pigs in growth phase I and II. 1 FAW = final average weight; 2 ADFI = average daily food intake; 3 ADWG = average daily weight gain; 4 FCR = feed conversion ratio; CV (%) = coefficient of variation; L = linear effect; Q = quadratic effect.
Table 4 shows the performance data of the pigs in finishing phase I (70-90kg) and total experimental period (40-90kg).The duration of finishing phase I and total experimental period were 21 and 47 days, respectively.For finishing phase I and total experimental period (growth I, II and finishing I) a linear effect (P<0.05) was also observed with increase in FAW, ADWG, ADFI and improvement in FCR due to increasing the vitamin supplementation levels in the pigs' diet.A quadratic effect (P<0.05) was also observed on ADFI and ADWG for the mentioned experimental periods. 1 FAW = final average weight; 2 ADFI = average daily food intake; 3 ADWG = average daily weight gain; 4 FCR = feed conversion ratio; CV (%) = coefficient of variation; L = linear effect; Q = quadratic effect.
Table 5 shows the pigs' plasma levels of αtocopherol and vitamin B12 at the end of the experimental period.There was a linear increase (P<0.05) in the plasma concentrations of α-tocopherol and vitamin B 12 because of increasing the vitamin supplementation in the diet of pigs (40-90kg).Y = -0.002065x+ 2.733637 0.0016 0.037 82.93 1 FAW = final average weight; 2 ADFI = average daily feed intake; 3 ADWG = average daily weight gain; 4 FCR = feed conversion ratio.

DISCUSSION
In the present study, was observed that increasing dietary vitamin supplementation for pigs provided improvement in performance parameters, especially FCR.Similarly, Chae et al. (2000) observed a linear effect on ADWG and FCR of growing pigs with the inclusion of 150% vitamins in relation to the NRC (Nutrient…, 1998) recommendations.Choi et al. (2001) also observed a quadratic effect on ADWG and FCR due to supplementation up to 200% of the NRC requirements (Nutrient…, 1998) for pigs four weeks before slaughter.Cho et al. (2017) conducted three separate experiments with different ages, types, and vitamin levels.In experiment 1, a linear effect was found in ADWG and ADFI due to increased supplementation of fat-soluble and B-complex vitamins up to 28 days post-weaning of piglets.However, in experiment 2 the increase in vitamin supplementation did not influence the performance of pigs until 67 days of age.However, in experiment 3 a quadratic effect was observed on ADWG and feed efficiency of pigs fed diets containing up to 470% of the NRC requirements (Nutrient…, 1988) for B-complex vitamins.Similarly, Mahan et al. (2007) observed improvement of ADWG, ADFI and feed efficiency of pigs in the phase from 23 to 55kg, due to an increase of B-complex vitamins in the diet.According to these authors, there was also an increase in the FAW, ADWG, and ADFI of the pigs in the subsequent phase, from 55 to 85kg.However, for the final phase, with pigs from 85 to 120kg, no performance improvement was observed.
Currently, the NRC (Nutrient…, 2012) vitamin recommendations may not reflect the requirements of modern commercial hybrids for the expression of their maximum genetic potential (Isabel et al., 2012;Dalto & Silva, 2020).Based on this, Yang et al. (2020) observed that supplying vitamins above the levels recommended by the NRC resulted in an increase in ADWG and feed efficiency of piglets during a period of 28 days post weaning.However, Shaw et al., (2002), Gaudré andVautier (2009) andSilva et al., (2015) did not observe improved performance due to de increase of vitamins in the diet of pigs in the growth phase.For the finishing phase, Mavromichalis et al. (1999), Tian et al. (2001) and Groesbeck et al. (2007) did not observe positive effect on pig performance due to the increase of vitamin supplementation.
In the present study, an increase in vitamin plasma levels and performance of pigs was observed due to increase of vitamin in the diets.Among the fat-soluble vitamins, vitamin A acts beyond the interconversion processes of rhodopsin by light uptake, improved phospholipid membranes and stimulates osteoclasts and can accelerate bone development (Isabel et al., 2012).However, Ching et al. (2002) found that excess of vitamin A in the diet reduced by 30% of blood tocopherol.Regarding the antioxidant effect of vitamin E, Silva-Guillen et al. (2020) observed a reduction in malonaldehyde and a tendency to reduce the interleukins (1β, 1ra, 2 and 4) in piglets fed a diet containing peroxidized oil and supplemented with 33 IU/kg DL-α-tocopherol.Thus, the increase in plasma levels of α-tocopherol observed in our study may have contributed to the improved immune status of pigs.Furthermore, it can be inferred that the concentration of vitamin A provided in the diet did not impair the vitamin E absorption, indicating that there was a balance between the supplementation levels of these vitamins.
Vitamin D 3 is another that can positively impact performance, calcium and phosphorus homeostasis, bone mineralization, and immune system, especially, for pigs raised in confinement without access to sunlight (Kolp et al., 2017).According to Witschi et al. (2011) andPanisson et al. (2021) the supplementation of 50µg/kg of vitamin D 3 or 25(OH)D 3 in the diet of pregnant sows provided improvement in bone characteristics and increase in performance of piglets.Regarding vitamin K, Monegue (2013) observed an increase in prothrombin activity with the supplementation of 0.5 ppm of vitamin K 3 in pig diets formulated with corn containing mycotoxins.
As described earlier, B-complex vitamins are enzyme cofactors of numerous metabolic reactions.Vitamin B 1 (thiamine) acts on the decarboxylation of α-ketoacids and glucose metabolism accelerating the Krebs cycle (Isabel et al., 2012).And vitamin B 2 (riboflavin) optimizes the of electrons and protons in the respiratory chain in the enzymatic form of flavin adenine dinucleotide and removes nitrogen from amino acids making the carbon chain available to be used as an energy source (Combs Jr, 2008).Based on this, Lutz and Stahly (1998) observed an increase in ADWG, feed efficiency and lean meat content in the carcass of pigs fed diets containing 3.7 to 7.4 mg/kg riboflavin.
Although it can be synthesized from tryptophan, vitamin B 3 (nicotinic acid) also acts in electron transport by converting nicotinamide adenine dinucleotide (Isabel et al., 2012).Its efficacy, however, depends on the combination between vitamin B 2 and vitamin B 6 (pyridoxine).Real et al. (2002) found an increase in ADWG and feed efficiency of growing and finishing pigs fed diets supplemented with niacin at 13 to 55 mg/kg.According to Saddoris et al. (2005), using the ideal protein concept in formulating diets increases the requirements of B vitamins, especially in diets formulated with wheat and sorghum.According to these authors, supplementation of 13.2 ppm of pantothenic acid (vitamin B 5 ) in pig diets increased the loin area and reduced the last rib fat, producing higher lean meat content in the carcass.This is because pantothenic acid is part of coenzyme A with a direct influence on basal metabolism, with increase in protein acetylation, on Krebs cycle Folic acid, pyridoxine (B 6 ) and cyanocobalamin (B 12 ) are also mainly linked to protein and amino acid metabolism.Vitamin B 6 influences transaminations, glycogenolysis and synthesis of neurotransmitters (serotonin and noradrenaline) (Isabel et al., 2012).For its activation, vitamin B 12 depends on microbial synthesis and presence of cobalt.In its active form, vitamin B 12 acts in the recycling of folic acid, in the synthesis of methionine from homocysteine with isomerization of the methyl-malonyl coA enzyme, which, in turn, will act in the degradation of amino acids and fatty acids with odd number of carbon (Combs Jr, 2008;Isabel et al, 2012).Folic acid, on the other hand, participates in methylation reactions and receives carboxyl groups from serine, glycine, and histidine for the synthesis of other amino acids.The increase in vitamin B 12 concentration in swine plasma due to increasing vitamin supplementation indicates optimization in amino acid metabolism, especially sulfur amino acids.
As to vitamin B 6 supplementation, Woodworth et al. (2000) reported an increase in ADWG of piglets fed diets with 3.3 mg/kg.In contrast, Yu et al. (2010) observed increased ADWG and ADFI of piglets fed diets supplemented with 2.5 mg/kg folic acid.Wang et al. (2021) also found a linear increase in ADWG and ADFI with folic acid supplementation of piglets in the postweaning phase.
Although it is not considered to be a vitamin, choline also has a synergistic effect with B vitamins with acetyl co A formation and donation of methyl groups for synthesis of homocysteine, for example.Li et al. (2018) reported that choline chloride supplementation for pigs from weaning to slaughter reduced serum concentrations of free fatty acids, triglycerides, and lipoprotein lipase expression, indicating optimization in lipid metabolism.
In the production process countless variations coexist regarding the commercial hybrid adopted, sex, management, age, rearing density, environmental factors (temperature, humidity, and health challenges), stressors, feed quality, and combination of ingredients in the formulation, storage and physical form of the diet, processing of ingredients and, mainly, source and vitamin supplementation levels (Combs Jr 2008;Isabel et al., 2012).The variation among the aforementioned factors and the differences in the experiments performed to determine vitamin supplementation requirements explain the divergence among the studies.
Table 7 shows the recommendation for vitamin supplementation for modern hybrid swine in the growing phase (I and II) and finishing I.In general, the average vitamin levels suggested in the latest edition of the Brazilian Tables for Poultry and Swine (Rostagno et al., 2017) are, on average, 3.86% higher than the levels in the present study.When analyzing the vitamin classes separately, it turns out that Rostagno et al. (2017) recommend an increase of 1.98 and 4.70% for fat-soluble and water-soluble vitamins, respectively, in relation to the levels observed in the present study.When separately comparing the data from the present study with the levels of Rostagno et al. (2017), the fatsoluble vitamins with higher increase were D 3 and E, 2.03 and 4.23%, respectively.As for the water-soluble vitamins, the ones that increased the most were folic acid and vitamin B 2 , 8.65 and 21.61%, respectively.Thus, recent research and recommendations from independent nutritionists indicate that vitamin requirements based on higher levels may be a trend aimed not only at improving performance, but also to increase gut health, immune status, and meat quality of modern pigs that directly impact the productive efficiency of animals.

Table 3 .
Performance of pigs fed diets with increasing levels of vitamin supplementation

Table 4 .
Performance of pigs fed diets with increasing levels of vitamin supplementation