Evaluation of Digestible lysine levels in diets with high energy density for finishing pigs

Objective. To evaluate the effects of different levels of digestible lysine in diets with high energy density on productive performance and carcass characteristics of finishing pigs. Materials and Methods. Seventy crossbred barrows (initial body weight of 83.36 kg) were used and allotted in a randomized block design with five treatments, seven replications and two pigs per experimental unit. Pigs were fed ad libitum with diets containing 3.5 kcal/kg of ME and five levels of digestible lysine (0.46, 0.52, 0.58, 0.64 and 0.70%) during four weeks. Final live weight (FLW), daily feed intake (DFI), daily weight gain (DWG), feed conversion (FC), daily lysine intake (DLI), and the amount of lysine per body weight gain (DLI/DWG), were evaluated. At the end of the experiment, blood samples were taken from each pig to determine urea nitrogen concentration (UN) in serum and slaughtered to evaluate quantitative and qualitative carcass characteristics. Results. The FLW increased linearly (p<0.05).There were no differences among treatments for DFI, DWG, FC, carcass characteristics and UN. The DLI and DLI/DWG varied significantly (p<0.001) and increased linearly (p<0.001) with each lysine level. Pigs that consumed the limiting diet in lysine (0.46%) showed less DLI and DLI/DWG (p<0.001) than pigs fed the other diets. Conclusions. The amount of DLI/DWG increased with the evaluated levels of digestible lysine in diets with high energy density, without effects on productive performance and carcass characteristics of finishing pigs.


INTRODUCTION
Modern pork production is based on using breeds whose optimal performance is determined, principally, by the quality of the diet.Genetic improvement has led to a selection of pigs with greater protein deposition and muscle development (1), which deserves to evaluate amino acids requirements for adequate diet formulation.Also, the amount of an amino acid and energy density in these diets determine the deposition of protein and fat in the pork carcass (2).Lysine is the first limiting amino acid in pig diets, and is used as a reference in the formulation, considering the recommended levels of standardized digestible lysine to estimate the requirements of other essential amino acids and guarantee optimum growth and yield in lean meat (3)(4)(5).For finishing pigs between 80 and 120 kg of weight, 0.52% dietary digestible lysine is recommended (6).
Additionally, feed intake is determined by dietary energy density (6), and it is therefore appropriate to adjust the proportion of amino acids if the energy density or feed intake changes (7).Previous studies in temperate climates (8)(9)(10)(11) have evaluated the growth and carcass characteristics of pigs in response to different levels of lysine in high energy density diets without evidence of negative effects on these characteristics.On the other hand, it has been demonstrated that high energy density diets limiting in lysine do not have deleterious effects Colina -Digestible lysine in pigs on growth but increase the fat content in the carcass of finishing pigs (12), while pigs fed during this phase with diets low in lysine and energy have increased deposition of back and intramuscular fat in the carcass (13).
In Venezuela, studies have been conducted to evaluate the carcass characteristics of pigs (14,15) without considering the effect of digestible lysine on that.In this sense, it is necessary to investigate this highly relevant point in the tropic, since climatic conditions affect the requirements of amino acids and the productive performance of pigs (4) while genetic factors and variations between animals also influence the results obtained (2).
The objective of this study was to evaluate the effects of different levels of digestible lysine in diets with a high energy density on the productive performance and carcass characteristics of finishing pigs.

MATERIALS AND METHODS
Bioethical aspects.The Bioethics Committee of the Faculty of Veterinary Sciences at the Universidad Central de Venezuela (FCV-UCV) endorsed this study, which was within established protocol when using experimental animals.

Study location.
The study was performed in the Laboratory of the Swine Section of the Institute of Animal Production in the Agronomy Faculty at the Universidad Central de Venezuela (UCV) in Maracay, Aragua State.The climatic conditions of the area: (67°36'36'' longitude, 10°16'20'' latitude, and 443 meters above sea level), average temperature of 25.1°C and average annual rainfall of 1.063 mm.Two experimental barns and 35 pens were used.In one barn 20 pens were used (4 m 2 each), with a concrete floor and separated by walls; and in the second barn 15 pens were used (4.2 m 2 each), with half solid flooring and half drainage, with concrete divisions.Both barns had aluminum sheeting roofs and natural light.Each pen had a fixed self feeder and a stainless steel nipple waterer to provide feed and water ad libitum, respectively.Animals.Seventy crossbred barrows (Yorkshire × Landrace × Duroc) with an average initial body weight of 83.36 kg, from a commercial farm were used.Pigs were randomly allotted, two per pen, to one of five experimental diets (treatments) for four weeks. 1 shows the ingredients and chemical composition of the experimental diets, formulated to obtain five different levels con alta densidad energética sin evidencia de efectos negativos sobre estas características.Por otra parte, se ha demostrado que el consumo de dietas con alta densidad energética y limitantes en lisina no tiene efectos deletéreos sobre el crecimiento pero aumenta el contenido de grasa en la canal de cerdos en etapa de finalización (12), mientras que los cerdos alimentados en esta etapa con dietas de bajo contenido de lisina y energía han resultado en incrementos en la deposición de la grasa dorsal e intramuscular en las canales obtenidas (13).
El objetivo de este estudio fue evaluar los efectos de diferentes niveles de lisina digestible en dietas con alta densidad energética sobre el desempeño productivo y características de la canal de cerdos en finalización.
Variables de desempeño productivo.Los cerdos fueron pesados semanalmente utilizando una báscula electrónica (Tru-Test ® Speedrite, Serie EC 2000) con capacidad para 1500 kg.El consumo diario de alimento (CDA) se determinó a través del pesaje y registro del alimento colocado en los comederos menos el alimento no consumido semanalmente para cada unidad experimental.El peso vivo inicial (PVI) y final (PVF) promedio de los cerdos en cada unidad experimental permitieron calcular la GDP y se estimó la conversión de alimento (CA=CDA/ GDP).Adicionalmente, se estimó el consumo diario de lisina (CDLis) considerando el contenido de lisina obtenido mediante análisis de cada dieta formulada y el consumo de lisina por cada kilogramo de peso vivo ganado (CDLis/GDP) de acuerdo con lo señalado en otros estudios (7,10).Determinación de nitrógeno ureico.Para determinar la concentración de nitrógeno ureico (NU) en suero sanguíneo, se extrajeron muestras de sangre a todos los cerdos, entre las 08:00 y 10:00 h el último día del experimento, para ello, se consideraron las normas de bioética para la utilización de animales con fines experimentales en Venezuela (17). of digestible lysine (0.46; 0.52; 0.58; 0.64 y 0.70%).The increase in each level of digestible lysine was obtained by adjusting the amount of corn, soybean meal and synthetic lysine in each one of the diets, which contained 5% raw palm oil, obtaining 3.50 Mcal/kg of metabolizable energy (ME).The content of the rest of the nutrients was adjusted according to the nutritional needs (6).The chemical composition of the ingredients used (corn and soybean meal) and the experimental diets was determined by analyzing the dry matter (DM:105°C/24 h) and crude protein (N Kjeldahl × 6.25) according to the procedures described (16).The content of lysine in experimental diets was determined using near infrared spectroscopy (NIRS).Productive performance variables.The pigs were weighed weekly using an electronic scale (Tru-Test ® Speedrite, Series EC 2000) with a 1500 kg capacity.The daily feed intake (DFI) was determined through weighing and recording the feed placed in the feeders minus the feed not consumed weekly by each experimental unit.The average initial live weight (ILW) and the final live weight (FLW) of pigs in each experimental unit, were used to calculate the daily weight gain (DWG) and the feed conversion (FC= DFI/DWG) was estimated.Additionally, the daily intake of lysine (DLI) was estimated considering the content of lysine obtained through analysis of each diet formulated and the intake of lysine for each kilogram of live weight gained (DLI/DWG) according to what suggested in other studies (7,10).

Colina -Digestible lysine in pigs
Determining urea nitrogen.To determine the concentration of serum urea nitrogen (UN), blood samples were taken from each pig between 08:00 y 10:00 h the last day of the experiment considering the bioethical standards for using experimental animals in Venezuela (17).
Blood samples were extracted in duplicate by puncture of external jugular vein using 18 G x 1 ½'' needles and 5 mL vacuum tubes (Gel Clot Activator, Vacuum Diagnostics, Zhejiang, China).The samples were transported to the Laboratory of Clinical Pathology of the FCV-UCV, where the serum was separated by centrifuge at 1.200 g for 20 min at 4°C within 8 h after collection and were conserved in 2 mL aliquots at -20°C until analysis.The analysis was done in duplicate using a quantitative kinetic determination kit (Chemroy ® , procedure CR245, Texas, U.S.A.) and the concentration of UN in serum was expressed in mg/dL.

Carcass evaluation.
When the pigs reached slaughter weight (an average of 102.81 kg), were subjected to a 24 h period of fasting and were moved to the commercial slaughter facility; after 6 h of rest, the pigs were electrically stunned and slaughtered by exsanguination, according to the procedures established in the code for hygiene practices in Venezuela (18).The hot carcass weight was recorded (HCW, with the head and feet), carcass yield (CY) was calculated, and the carcass were later refrigerated (4-6°C) for 16-24 h.After this period the head was removed and the carcass was cut longitudinally into two equal halves, allowing the quantitative characteristics to be evaluated.The backfat thickness (BFT) was measured with a metric ruler in three places: opposite to the first rib, last rib, and last lumbar vertebra, respectively, obtaining an average of the three measurements.A transverse cut between the 10 th and 11 th intercostal space was performed to measure the Longissimus dorsi muscle area (LDMA) using a grid; the depth of the back fat (BFD) that covers the LDMA was measured with a metric ruler in three positions of the muscle area (1/4, 1/2, and 3/4).The values obtained of the LDMA and the BFD were used to calculate the lean carcass yield (LCY) using prediction equations (6,19).
The qualitative characteristics evaluated in the carcass were pH, temperature and water holding capacity (WHC).
No se evidenció el incremento de la GDP por cada unidad del contenido de lisina digestible en la dieta como era de esperarse.El CDLis y la CDLis/ GDP variaron significativamente (p<0.001)entre tratamientos e incrementaron linealmente (p<0.001)con el contenido de lisina digestible de la dieta (Tabla 2).En tal sentido, los cerdos que consumieron la dieta limitante en lisina (0.46%) mostraron menor CDLis (p<0.001),conjuntamente con la menor cantidad (p<0.001)requerida de este aminoácido por kilogramo WHC was determined using the methodology applied by Jerez-Timaure et al (20), using 1.6 g of the sample of a fresh steak obtained from the Longissimus dorsi muscle at the 10 th intercostal space.The samples were subjected to refrigerated centrifugation to obtain the percentage of water lost due to differences in weight.
Additionally, it was estimated through a frequency distribution analysis, the proportion (%) of pale, soft and exudative meat (PSE); dry, firm and dark (DFD); and normal for each treatment, according to the classification suggested by Castrillón et al (21).

Experimental design and statistical analysis.
The pigs were distributed in a randomized block design (two blocks represented by each barn) with five treatments (levels of digestible lysine), seven replications, and two pigs per experimental unit.The results were expressed as an average of weekly measurements of the DFI, DWG, FC, DLI and DLI/DWG variables of the same experimental unit.The values considered in the analysis of the ILW, FLW, UN variables and the carcass characteristics corresponded to a single measurement.The statistical analysis for all the variables was performed using the Proc Mixed of SAS software (22).
A regression analysis was used to observe the linear and quadratic effect on the levels of digestible lysine on each variable.Probability values of p≤0.05 were considered significant.

RESULTS
Under the evaluated conditions, significant differences in the productive performance were not detected in FLW, DFI, DWG and FC of the pigs due to the effect of digestible lysine levels, as is shown in table 2. The quadratic effect of the level of digestible lysine was not significant for any of the variables, therefore only the linear effect is presented.The FLW increased linearly (p<0.05) while the FC tended to decrease (p=0.08) with the increase in the level of lysine in the diet.There was no evidence of the increase of DWG per unit of digestible lysine in the diet, as was expected.
The DLI and the DLI/DWG varied significantly (p<0.001)among treatments and increased linearly (p<0.001) with the content of digestible lysine in the diet (Table 2).In that sense, the pigs that consumed the diet limiting in lysine (0.46%) had less DLI (p<0.001), in conjunction with the lower quantity (p<0.001)required of this amino acid per kilogram of weight gained, while the pigs that consumed the diet with 0.70% of digestible lysine showed the greatest DLI (p<0.001).However, the DLI/DWG of the pigs fed with the diet that contained 0.52% of digestible lysine was not significantly different from that observed in the pigs whose diets contained a greater amount of lysine.
The concentration of UN (Table 2) did not significantly change among treatments but decreased linearly (p<0.05) with the increase of digestible lysine in the diet.
There were not significant differences among treatments for the quantitative carcass characteristics (Table 3), however the BFD tended to be lower (p=0.07) in the carcass of pigs whose diets contained the greatest level of lysine (0.70%).The HCW tended to increase (linear effect, p<0.06), with the level of lysine in the diet.The qualitative carcass characteristics of the evaluated pigs did not show differences among treatments (Table 4).
The result of the frequency analysis to estimate the proportion of PSE, DFD and normal meats (Table 5) showed a greater proportion of meats with normal pH in the pigs fed with the experimental diets; however, within each treatment group and for the total of the samples, the percentage of meats with pH values below normal (PSE) oscillated between 15 and 28%.
El resultado del análisis de frecuencia para estimar la proporción de carnes PSE, DFD y normal (Tabla 5), evidenció en los cerdos alimentados con las dietas experimentales, una mayor proporción de carnes con pH normal; sin embargo dentro de cada grupo de tratamiento y para el total de las muestras, el porcentaje de carnes con valores de pH por debajo de lo normal (PSE) osciló entre 15 y 28%.

DISCUSSION
The results indicate that it is probable that the levels of digestible lysine evaluated were not adequate to detect the differences in the DWG, DFI, FC and FLW variables due to the treatment.Studies conducted on finishing pigs fed with deficient diets or ajusted to the lysine requirement and containing 3.4 Mcal/ kg of EM have also shown that there is no effect of dietary lysine level on DFI and DWG (12).Even in growing pigs, fed with lower energy density diets, the effect of lysine on the productive performance has not been observed (3,23).According to those reported by several authors (4,12,13,23,24), the DFI is a variable that generally is not affected by the content of lysine in the diet, response that is observed when the diets are isocaloric and the daily intake of lysine increases linearly with the content of lysine in the diet, such as in the case of the present study.Although this response was evident without significant variations in the DFI among treatments, it is indicative that the pigs do not regulate this intake in function to the amino acids present in the diet (5) but the increase of lysine intake is proportional to the content of this amino acid in it (3,23,24).This argument explains the differences in DLI with each increase in the level of digestible lysine.
Previous studies have observed significant variations in DWG and FC by increasing the levels of lysine in diets with 3.6 Mcal/kg (9) and 3.48 Mcal/kg of EM (10), however, the response could be influenced by the ingredients used.Other studies (9,10) used choice white grease to maintain high the energy density of the diets and increasing the content of lysine,
while in the present study raw palm oil was used.This energetic ingredient, an alternative to traditional lipid sources in the tropics, can be added in a diet for finishing pigs without effects on productive performance (25).
It was observed that DLI/DWG in the diet of 0.52%, corresponding to the recommended value (6), did not significantly differ from that obtained with diets that contained a greater amount of lysine.The pigs in this study consumed between 16.34 and 22.44 g of lysine/kg of DWG, values within the range observed in the literature for pigs of similar weight (8)(9)(10).It has also been shown that the quantity of DLI/DWG increases linearly and the DWG does not significantly change by increasing the content of lysine in the diet (4), response that also coincides with the results.
T h e U N c o n c e n t ra t i o n d i d n o t c h a n g e significantly among treatments, which is explainable since the diets had similar values of crude protein.On the other hand, the regression analysis showed a linear reduction of UN with the increase of the digestible lysine levels in the diet.In that sense, it is probable that the digestible lysine levels used were very close to each other, impeding noticeable differences among treatments.It has been highlighted that the quantity of lysine consumed is used for maintenance and protein deposition (6), after which it is catabolized and the resulting nitrogen is excreted as urea.
The excess of amino acids in the diet has been related to high UN concentrations (26) and therefore the concentration of urea in blood is an indicator of protein metabolism.In this study the concentration of UN decreased linearly, which indicates that there was no excess of amino acids.Respecting the results obtained in the UN, other studies (24,26) have not observed differences among treatments by increasing the levels of digestible lysine in the diet.
Re f e r r i n g t o t h e q u a n t i t a t i v e c a r c a s s characteristics, although there was a linear trend of HCW to increase with the lysine content, overall no significant differences were observed among treatments.This result indicates that the increase of HCW was associated to the simultaneous increase of the FLW.According to the results of the current study, increasing the levels of digestible lysine in the diet does not affect the quantitative carcass characteristics of finishing pigs (8,24) nor have significant effects been detected when changing the nutritional plan by adjusting those levels in the diets on carcass yield or lean carcass yield (5).
Referente a las características cuantitativas de la canal, aunque se observó una tendencia lineal del PCC a aumentar con el contenido de lisina,de manera general,no se observaron diferencias significativas entre tratamientos para estas características.Este resultado indica que el aumento en el PCC estuvo asociado al aumento simultáneo del PVF.De acuerdo con los resultados del presente estudio, se ha evidenciado que el incremento de los niveles de lisina digestible en la dieta no afecta las características cuantitativas de la canal de cerdos en etapa de finalización (8,24), así como tampoco Regarding the qualitative carcass characteristics of finishing pigs fed with different levels of lysine, it has been shown that characteristics such as pH (2,3,12) and WHC (2) are not affected.It has been noted (3,24) that carcass characteristics vary little in response to variations in the content of lysine in the diet, which may be attributed in this study to the fact that the feed intake was similar among different treatments and when experimental diets were formulated, a similar energy density was maintained, and the digestible amino acid requirements were considered.Moreover, these aspects also explain the invariable response in the productive performance of the pigs evaluated.
With respect to the frequency distribution analysis to estimate the proportion of PSE, DFD and normal meat, there was greater proportion of meat with normal pH in all the carcass evaluated.Lysine levels in the diet did not affect the pH or temperature of the carcass, so that the occurrence of PSE meat can be attributed to chronic stress just before slaughter (27) or the handling of pigs, especially while stunning (20).
The PSE meat is a defect associated with a rapid rate of postmortem glycolysis, which is characterized by a high acidification rate in the meat; while DFD meats are due to prolonged stress that consumes the reserves of muscular glycogen before slaughter, resulting in a low production of lactic acid (1).Studies in Mexico have found a proportion of PSE pork meat between 3.5 and 8.0% (1), while in Venezuela values between 8 and 10% have been observed, and a greater percentage of meats with pH 24 values that have oscillated between 5.5 and 6.0 (20).However, the proportion of PSE meats was lower when compared with that shown in this study, while the pH 24 values are slightly lower than those observed in the country (15) (20).
The results allow to conclude that the levels of digestible lysine between 0.46 and 0.70% in diets with 3.5 Mcal/kg of ME do not affect the productive performance and carcass characteristics of finishing pigs.However, the amount of lysine consumed per unit of weight gained increased with the level of digestible lysine in the diet.

Colina -Digestible lysine in pigs
The linear increase of final live weight per unit of lysine was noted without the concomitant increase in weight gain, and without differences among treatments, which may be associated with the fact that lysine levels evaluated were not wide enough each other to detect it.In this regard, conducting further studies to evaluate digestible lysine levels of greater amplitude at various levels of metabolizable energy is recommended.con 3.5 Mcal/kg de EM no afectan el desempeño productivo y las características de la canal de los cerdos en etapa de finalización.No obstante, la cantidad de lisina consumida por unidad de peso ganado aumenta con el nivel de lisina digestible en la dieta.
El incremento lineal del peso vivo final por cada unidad del contenido de lisina se evidenció sin el concomitante aumento en la ganancia de peso, y sin diferencias entre tratamientos lo que podría asociarse a que los niveles de lisina evaluados no fueron lo suficientemente amplios entre sí para detectarlas.En tal sentido, se recomienda la realización de nuevos estudios dirigidos a evaluar niveles de lisina digestible de mayor amplitud a varios niveles de energía metabolizable.

Table 2 .
Effect of digestible lysine levels in high energy density diets on the productive performance and blood urea of finishing pigs.
SE:Standard error; ILW: initial live weight; FLW: final live weight; DFI: daily feed intake; DWG: daily weight gain; FC: feed conversion (DFI/DWG).The DFI, DWG and FC values correspond to the average over four weeks.DLI: daily lysine intake; DLI/DWG: Lysine intake per kg of live weight gained; UN: concentration of urea nitrogen in serum.abcdeDifferent letters in the same row indicate significant differences according to the value of probability corresponding to the effect of the level of digestible lysine (treatment).fProbability value corresponding to the effect of the level of digestible lysine (treatment) and the linear effect of digestible lysine as the result of the regression analysis, respectively.

Table 3 .
Effect of digestible lysine levels in high energy density diets on the quantitative carcass characteristics of finishing pigs.
a Probability value corresponding to the effect of the level of digestible lysine (treatment) and the linear effect of digestible lysine as the result of the regression analysis, respectively.

Table 4 .
Effect of digestible lysine levels in high energy diets on the qualitative carcass characteristics of finishing pigs.Standard error; pH 45 : pH measured at 45 min postmortem; pH 24 : pH measured at 24 h postmortem; T 45 : temperature measured at 45 min postmortem; T 24 : temperature measured at 24 h postmortem; WHC: water holding capacity, expresses the water lost by effect of centrifugation.a Probability value corresponds to the effect of the level of digestible lysine (treatment) and the lineal effect of digestible lysine as the result of regression analysis, respectively.

Table 5 .
Proportion (%) of pale, soft and exudative meat (PSE), dry, firm and dark meats (DFD) and normal ones for each treatment.