Dietary lysine : calorie ratio on performance , blood parameters and apparent nutrient digestibility in pigs from 50 to 70

1Department of Animal Sciences. Universidade Federal da Grande Dourados. Dourados. MS. Brazil. 2Instituto de Zooteecnia IZ.. Nova Odessa. Sao Paulo. Brazil. 3Department of Animal Nutrition and Production. School of Veterinary Medicine and Animal Sciences. University of São Paulo (USP). Pirassununga. SP. Brazil. 4Department of Animal Production. FMVZ – University of Sao Paulo Estate “Júlio de Mesquita Filho”. Botucatu. Brasil.


INTRODUCTION
According to new concepts about human health and increase on protein demand there was significant improvement in carcass quality of pigs to supply mar-ket which seeks a pork lean meat.Thus, it should be considered some conditions that impose changes in nutritional requirements of pigs, such as genetic potential for meat production, gender and age for maximal performance.
Amino acid levels in most of diets are recommended from the data reviewed (National Research Council, 1998) and (Rostagno et al. 2011, p.252), whose most of results are based on diets with high levels of crude protein (CP).Thus the lysine requirement may be overestimated.However, lysine is a limiting amino acid in pig diets based on corn and soybean meal and its use to afford the maximum growth should be considered under all conditions that affect the genetic potential for meat deposition (Zangeronimo et al. 2009, p. 1507and Fix et al. 2010, p.108).
The body protein accretion reflects the efficiency of dietary nutrients utilization on protein anabolism process reflecting on pig growth as well as matches the diet energetic contribution.The energy intake should meet the real demands for maintenance and body mass deposition and the respective requirements change according to protein level (amino acids) of the diet (Urynek & Buraczewska, 2003, p.1227and Trindade Neto et al. 2008, p.103 ).
Adipocytes and skeletal muscle cells involved on immune system as flag antigens, through Toll-like receptors under an integrated manner, regulate the growth of mammals (Gabler & Spurlock, 2008, p.64).In midst of this complex growth system and immunological integrity the health and stress challenges may change the immune response (reflecting on WBC difference), therefore affecting the pig nutritional requirements (Williams et al. 1997a, p.2472and Salak-Johnson & McGlone, 2007, p.81).Thus, the objective of this study was to evaluate the possible effects of digestible lysine level and its ratio with metabolizable energy on pig performance, apparent nutrient digestibility, carcass characteristics and white blood cell counting.

MATERIAL AND METHODS
The experimental protocol was reviewed and approved by the Animal Care and Use Committee (CEUA), Faculty of Veterinary Medicine and Animal Science, UNESP, Botucatu.Performance and digestibility trials were carried out in Animal Science Institute of SAA, Sao Paulo, Brazil.

AnimAls, design And diets
A total of 144 crossbred (P76 x Näima P76 -Pen Ar Lan) were used in two trials as follow: 72 gilts (46.05±0.38 kg of BW) and 72 barrows (49.75±0.41kg of BW), both with an average of 91 d.Pigs were blocked based on BW and then assigned randomly to fed five dietary Lys levels as follow: 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 g/kg.Each dietary treatment was assigned to six replicate pens and pigs had unlimited access to feed and water.The experimental unit was two pigs by pen.
Pigs were housed in solid concrete floors (2x1m) separated by steel grates equipped and the experimental facility had windows on the sides to control ventilation.Daily environmental temperatures (maximum and minimum) were recorded 8am and 3pm.
Experimental diets were based on corn, soybean meal, soybean oil, minerals, vitamins and industrial amino acids (Table I).Total Lys levels analysed in corn and soybean meal were used to estimate digestible amino acid considering 88% as average rate of standardized digestibility whose index was based on weighted mean of lysine digestibility coefficients proposed by Rostagno et al. (2005), according to dietary inclusion levels of corn and soybean meal.
During first 10 days of initial experimental period the diets were supplied with Tiamulin (225g/ton) and Mebendazole (450g/ton).

PerformAnce And ultrAsound evAluAtion
Pigs were weighed at 16 th and 32 nd days of evaluation to calculate body weight (BW), average daily gain (ADG), relative daily gain (RDG), feed intake (FI), feed conversion ratio (FCR), crude protein intake (CPI) and crude protein efficiency (CPE) according to Gandra et al. (2012Gandra et al. ( , p. 2039)).All pigs were subjected to backfat thickness (BT) and loin area (LA) measurements by ultrasound on the last day of the experiment, around 70 kg of BW and 123 d age.Backfat thickness was measured 4 to 5 cm from midline on the right side at the 10 th rib using a real-time ultrasonic Vetko Plus Brand.

Blood PArAmeters
On day 32 nd day were sampled 5 mL blood from 4 gilts and 4 barrows by treatment via jugular venipuncture through heparinized syringes and placed on ice immediately after collection.Into the 20 min blood samples were centrifugated at 2,500 × g for 15 minutes.Plasma aliquots were refrigerated and sent to laboratory for white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), eosinophils (EOS), monocytes (MON) count.Lymphocytes and neutrophils ratio (NEU/LYM) was calculated to clarify if WBC increase was due to acute immune reaction (higher NEU) or chronic (higher LYM).
digestiBility AssAy A balance trial was conducted to evaluate the N retention, apparent digestibility and metabolizable energy of the diets used on performance evaluation.Environmental temperatures in the digestibility room were controlled by windows ventilation or "Split System" air conditioners.
A total of 24 crossbred pigs similar in ancestry with an initial BW of 49.075 ± 4.34 kg were randomly assigned as follow: 12 gilts and 12 barrows, individually housed in adjustable metabolism crates (1.4 x 0.6 m) to fed 4 diets used in performance evaluation, according to 4 x 2 factorial with the factors being Lys levels and sex, respectively.Diets were formulated to four Lys levels (7.00, 9.00, 10.00 and 12.00 g/kg) defined as two higher and intermediate levels used in pig performance trials.The experimental period lasted 10 d and first 5 d were for diet and cage adaptation, when also were fed with antibiotic and anthelmintic, as supplied in performance trials, and subsequent 5 d were for collection of feces and urine to determine N, DE and ME.
For collection phase pigs were fed their respective diets based on BW 0.75 d -1 .Pigs were fed twice daily at 8a.m. and 3p.m. and had free access to water.Feces and urine collections were performed according to Barbosa et al. (1999).Gilts urine collection occurred without probe use.

stAtisticAl AnAlyses
Performance, carcass ultrasound measurements and WBC variables were submitted to regression analysis through orthogonal polynomial considering the digestible lysine levels, using PROC MIXED of SAS (Version 9.1.3, SAS Institute, Cary, NC 2004), according to the model: Y ij = µ + A i + B j + e ij ,, where: Y ij : constant associated to all observations; µ: overall average of the variable; A i : effect of lysine level i, being i = 1, 2, ... and 6; B j : effect of block j, being j = 1, 2, ... and 6; e ij : random error associated to each observation.
Variables from N balance, DE and ME were submitted to regression analysis through orthogonal polynomial considering the digestible lysine levels, using PROC MIXED of SAS (Version 9.1.3,SAS Institute, Cary, NC 2004), according to the model: Y ij = µ + A i + Sj + Ai(Sj) + e ij , where: Y ij : constant associated to all observations; µ: overall average of the variable; Ai : effect of lysine level i, being i = 1, 2, 3 and 4; Sj: effect of sex j, being j = 1 and 2; Ai(Sj) : effect of interaction; e ij : random error associated to each observation.Considered effects for treatment was P < 0.05 and Lys level estimated as optimal was obtained from quadratic equation derived.

Blood PArAmetrs (gilts)
Results of WBC by regression analysis (Table IV) did not show effect (P>0.05) of Lys in WBC, NEU, LYM, EOS, MON and NEU/LYM.The performance of barrows (Table V) presented (P<0.05)linear increase in CPE (Y=1.830+0.079X,R 2 =0.46) from d 0 to 16 and quadratic response (Y=97.730+63.327X-3.472X 2 , R 2 =0.67) from d 0 to 32.The most important results in performance evaluation shown that lower studied level of Lys (7.00g/kg) was sufficient to meet FCR and ADG.

PerformAnce And ultrAsound evAluAtion (BArrows)
On BT and LA evaluations there was no effect of dietary Lys: ME ratio in barrows during experiment when pigs reached 75 kg BW (Table VI).However the BT increased linearly in gilts as response to dietary Lys: ME increase as well as there was quadratic effect on LA and estimated level was 9.86 g/kg Lys by MJ ME.

DISCUSSION
The linear improvement (P<0.05) on FCR and CPE observed from d 0 to 16 for gilts and CPI reduction from d 0 to 16 and d 0 to 32 are in agreement with other similar studies (Main et al. 2008, p.2190and Fix et al. 2010, p.108).The lysine level increase, relative to metabolizable energy, by adding synthetic amino acid DL-methionine and keeping the proportions of other amino acids to lysine for pigs have been reported by Main et al. (2008Main et al. ( , p.2190)).The present estimate of 10.14 g/kg Lys and 0.71 g/MJ Lys:ME is similar to the value 10.10 g/kg Lys or 0.67 g/MJ Lys:ME suggested by Main et al. (008) for pigs to meet maximum growth and feed efficiency.However, recommended levels by Rostagno et al. (2011, p.252) are lower (9.45 g/kg Lys or 0.77 g/MJ Lys:MJ) indicating that Lys level changes according to dietary energy.Furthermore, Fix et al. (2010, p.108) demonstrated that a modern feeding program for pigs using supplementation with synthetic amino acids results in better performance and carcass independently of genotype.
Observed ratio between BT and LA remained constant, around 0.21 to 9.86 g/kg Lys.De la Lata et al. ( 2002) also observed linear increase on BT, according to Lys increase but not found effect on LA for barrows.On the other hand, Main et al. (2008Main et al. ( , p.2190) ) verified linear decrease on LA by increasing of total Lys that changed from 7.1 to 12.2 g/kg.As discussed by Schinckel et al. (2002Schinckel et al. ( , p.1419)), backfat thickness and longissimus muscle obtained by ultrasound method are appropriated measurements, despite not represent similar values those collected directly in carcass after slaughter.Changes on fat and lean meat in carcass are due to nutritional adjustment of diet and genotype (Rosenvold & Andersen, 2003, p.2129) and probably the changes observed in present study are due to genotype and their intrinsic characteristics.
Innate immunity is always present under different degrees and may be strengthened or weakened by several factors such as: injuries, dehydration, nutritional status, genotype and stress (Carroll & Forsberg, 2007, p.105).
The authors reported that the immune system under good working protect body against most of pathogenic organisms and respective diseases propitiating physical barriers such as: skin, gastrointestinal mucosa, stomach acid and non-specific cells (WBC) that detect and immediately eliminate these agents.
Lys is directly involved in some defence functions of animal physical integrity as the regulation of nitric oxide synthesis, in antiviral activity (fight against herpes), protein methylation (e.g.trimethyl-lysine in calmodulin), among others, and, indirectly, in the structure of hydroxy-lysine molecule and collagen function (Wu, 2009, p.1).
Therefore, without evidence for any type of disease and no statistical effect in WBC of gilts due to experimental treatments, it was observed that genetic Table IV.White blood cell count of gilts (absolut values by microliter±standard deviation) at the end of the trial (123 days of age) according to Lys nutritional levels (g/kg) (Contagem de leucócitos de leitoas (valores absolutos micolitros±desvio padrão) no final do experimento (123 dias de idade) de acordo com os níveis de lisina).potential for growth was characterized, even with some white blood cell changes, whose values were above or below the reference postulated by Jain & Schalm's, (1986, p.240).

Variables
For barrows, the Lys effects were only observed on crude protein efficiency showing linear increase and on daily crude protein intake characterized by quadratic effect and estimation of 10.9g/kg Lys as optimal level.However, according to the other results for variables of higher interest, that showed similarity among treatments, it was recommending 7.00 g Lys/ kg for barrows from 46 to 72 kg of BW.Unlike happened with gilts, the estimated Lys level for barrows is below those suggested by Rostagno et al. (2011, p.252) who suggested 8.83 g/kg of Lys or 0.65 g Lys/MJ ME for pigs from 50-70 kg BW.Fix et al. (2010, p.108) compared two pig genotypes and two feeding programs (from 80's to current decade) and did not observe differences in backfat thickness using same feed formulation.However, there was an increase in rib eye area regardless the genetics.In contrast, Main et al. (2008Main et al. ( , p.2190) observed a linear reduction in backfat thickness and quadratic effect on loin eye area, when increased from 7.9 to 14.0 g/kg of dietary Lys and obtained 7.95 g/kg of Lys or 0.53 g/MJ of ME, as optimal for barrows.Monocytes are immature macrophages that participate in phagocytosis (Guyton & Hall, 2006, p.1264) and in innate immunity through nonspecific defence mechanisms that serve as the first defence line against infectious organisms and occur quickly, preceding the emergence of antigens in the body (Salak-Johnson & McGlone, 2007, p.81).The monocyte numbers of barrows were lower under the level 9.83 g/kg of Lys, showing that these pigs, probably, behaved better against any aggressive agent present in the experimental facilities.Although some values of WBC have been shown above or below those postulated by Jain & Schalm's (1986, p.240), the pigs did not show any disease clinical signs during the trial period.
Regarding to protein metabolism, differences between genders have been confirmed during digestibility trial, when gilts presented higher efficiency in nitrogen retention.The present study occurred under similar conditions to Trindade Neto et al. (2005Neto et al. ( , p.1980) ) who evaluated ME and Lys levels for a specific genotype and also observed that gilts are more efficient in nutrient use to meet performance needs, compared with castrated male pigs.Thus, generally the gilts have higher nutrient requirement than the barrows (Ekstrom, 1991, p.415) and from 30 kg of BW is possible verify effects of sex on pig performance until the end of finish phase.
As observed the maximum DE and ME were obtained at the levels of 10.12 and 10.23 g/kg of Lys, respectively and the mean value 10.17 g/kg of Lys, while N absorbed and retained increased linearly in response to Lys levels.Thus, it was confirmed that protein deposition in growing pigs is limited by energy intake (Bikker et al., 1994(Bikker et al., , p.1744) ) and there is an equilibrium or adjustment among ingestion, absorption and retention (Resende et al., 2006(Resende et al., , p.1101)).

CONCLUSIONS
Suggested Lys levels for gilts and barrows from 50 to 70 kg live weight are: 10.14 and 7.00 g/kg Lys or 0.71 and 0.49 g/MJ Lys:ME, respectively, according to experimental conditions this study.

Table VIII.
Apparent digestibility of barrows and gilts, with average body weight of 53.5 kg, according to Lys nutritional levels (g/kg) (Digestibilidade aparente de leitões e leitoas com média de peso vivo de 53,5 kg de acordo com os níveis de lisina (g/kg)).

Table I .
Calculated composition of the experimental diets for pigs from 50 to 70 kg of live weight (Composição das dietas experimentais para suinos de 50 a 70 kg de peso vivo).

Table II .
Gilts performance according to Lys nutritional levels (g/kg) (Desempenho de leitoas de acordo com os níveis

Table VII .
White blood cell count of barrows (absolut values by microliter±standard deviation) at the end of the trial (123 days of age) according to Lys nutritional levels (g/kg) (Contagem de leucócitos de leitõs (valores absolutos micolitros±desvio padrão) no final do experimento (123 dias de idade) de acordo com os níveis de lisina).