The effect of lysine restriction during grower period on productive performance, serum metabolites and fatness of heavy barrows and gilts

Livestock Science 171 (2015) 36–43

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The effect of lysine restriction during grower period on productive performance, serum metabolites and fatness of heavy barrows and gilts J. Suárez-Belloch, J.A. Guada, M.A. Latorre n IUCA, Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, C/Miguel Servet, 177, 50013 Zaragoza, Spain

a r t i c l e in f o

abstract

Article history: Received 19 June 2014 Received in revised form 29 October 2014 Accepted 6 November 2014

A total of 200 Duroc cross (Landrace
Large White) pigs, 50% barrows and 50% gilts, of 26.3 7 0.55 kg body weight (BW) were used to study the effect of Lysine (Lys) restriction in the grower period on subsequent performance, serum metabolites and fatness. Four diets with Lys contents of 11.0, 9.1, 7.8 and 5.2 g/kg were offered to animals during the grower phase (45 d) in a two (sex)
four (diet) randomized block factorial design with five pens (replicates) per treatment and five pigs each. At the end of the restricted period, one pig per pen (five pigs per treatment) was slaughtered and the rest were fed a common finishing diet with 9.1 g/kg Lys until slaughter at 123 kg 7 2.35 kg BW. During the grower phase, Lys restriction reduced average daily gain (ADG) (Po 0.001), average daily feed intake (ADFI) (P¼ 0.008) and feed conversion ratio (FCR) (P ¼ 0.003). The responses were quadratic and similar for both sexes. At the end of this period, restricted pigs showed lower backfat thickness (P o 0.001) because they were lighter (47 vs. 64 7 0.7 kg BW) but had higher serum concentrations of triglycerides (linear; P ¼ 0.001) and cholesterol (quadratic; P¼ 0.039). Also, serum albumin decreased quadratically (P ¼ 0.004) with Lys restriction while urea increased with the highest level of restriction. During the finisher phase, ADG increased linearly (P o 0.001) in response to previous Lys restriction which was related with a trend to higher ADFI (P ¼0.0.97), resulting in a linear increase of FCR (P ¼ 0.002). However, the compensatory growth was incomplete and, during the overall period, there was a reduction of ADG (quadratic; P ¼ 0.041) and of FCR (linear; P ¼ 0.001) as dietary Lys content decreased, whereas fat depth at the gluteus medius (GM) muscle increased linearly (P ¼0.004). This entailed a delay to reach the slaughter BW of 1.87 0.43 d per g Lys restriction/kg of diet which was related to worse FCR by 0.034 70.0095 and to an increase of fat thickness at GM muscle of 0.517 0.168 mm. At the end of the trial, barrows grew faster, ate more feed and were fatter than gilts (P o 0.001). These results confirm that the Lys restriction during the grower period promoted an incomplete compensatory growth and might be a useful strategy to increase the fatness albeit with a cost in terms of FCR in production systems of heavy barrows and gilts intended for dry-cured products. & 2014 Elsevier B.V. All rights reserved.

Keywords: Fatness Growth performance Heavy pigs Lysine restriction Serum metabolites

n

Corresponding author. Tel.: þ34 876554168; fax: þ 34 976761590. E-mail address: [email protected] (M.A. Latorre).

http://dx.doi.org/10.1016/j.livsci.2014.11.006 1871-1413/& 2014 Elsevier B.V. All rights reserved.

J. Suárez-Belloch et al. / Livestock Science 171 (2015) 36–43

1. Introduction A certain level of body fatness is desirable in pig fattening systems intended for dry-cured meat production, such as ham, due to the beneficial effects on processing and eating quality (Ruíz et al., 2002). In fact, a backfat (BF) thickness threshold is required to claim for label, which is difficult to attain with current lean genotypes even at heavy market weights (Latorre et al., 2008). Improved genotypes are more efficiently converting feed into live weight gain and therefore they have replaced early maturing genotypes in the pork industry, even in the case of heavy pigs despite the economic losses caused by lack of fat covering (Latorre et al., 2009). As lysine (Lys) is the first limiting aminoacid (AA) for growth, a restricted supply decreases the rate of protein synthesis, increasing the proportion of energy retained as fat although with negative influence on performance traits (Adeola and Young, 1989) and serum profile (Mule et al., 2006). However, if the restriction is conducted during the juvenile phase, it may be compensated by an acceleration of growth thereafter without detrimental effects on performances along the overall growing period (Chiba, 1995; Fabian et al., 2002). This compensatory growth does not seem to be due to better utilization of nutrients (Lovatto et al., 2006) but to variations in tissues accretion (Bikker et al., 1996) with the result of higher carcass fatness (Degreef et al., 1992; Heyer and Lebret, 2007). However, the effect may depend on the length of restriction period (Kamalakar et al., 2009). Therefore, limiting the Lys content in the grower feed can be a useful strategy to manipulate body fatness at slaughter, easily implementable in practical conditions. In order to test this hypothesis and to define the appropriate level of restriction, an experiment was carried out aiming to quantify the effect of different dietary Lys levels during the grower phase on performances, carcass fatness and serum metabolites of crossbreed heavy pigs. 2. Material and methods All the experimental procedures used in this study were in compliance with the Spanish guidelines for the care and use of experimental animals in research (Boletin Oficial Estado, 2007). 2.1. Animal Husbandry and experimental design A total of 200 pigs, 50% barrows and 50% gilts, of 26.370.55 kg of body weight (BW) and 73 73 d of age for both sexes, were used. All animals were the progeny of Duroc sires and Landrace
Large White dams (PIC, Sant Cugat del Valles, Spain). Males had been castrated at 5 d of age. At the arrival to the farm, pigs were individually identified, weighed and housed in 40% slatted floor pens of 9 m2 (3 m
3 m) provided with individual feeders (five spaces) and an automatic drinking device, in a natural environment barn. Pigs were allotted by sex, in groups of five, to blocks of increasing BW and distributed within each block to the experimental treatments.

37

Four diets with decreasing levels of total Lys (11.0, 9.1, 7.8 and 5.2 g/kg fresh matter) were offered to barrows and gilts during the grower period (45 d). Therefore, there were eight experimental treatments (2 sexes
4 diets) and each treatment was replicated five times being the replicate a pen with five pigs allotted together. Diets were formulated to be isoenergetic and that with 11.0 g Lys/kg intended for meeting the Lys recommendations for 25–50 kg BW pigs (NRC, 2012), was considered as the control diet. The other diets were formulated by the progressive replacement of soybean meal by barley and synthetic Lys was not added in any case. An attempt was made to maintain a constant relationship with Lys of the remaining essential AAs, following the ideal protein concept (NRC, 2012). However, for diet containing 5.2 g Lys/kg, it was not possible with the same ingredients without adding synthetic AAs, so it was preferred to maintain an excess of non-limiting essential AAs which carried out a lower ideal protein content. After the grower period and until the slaughter, with 123.072.35 kg BW, all pigs received a common diet with 9.1 g Ly/kg, according to NRC (2012) recommendations for 50–135 kg BW pigs. The change of diet was carried out at a fixed age, rather than constant BW, to simulate commercial management conditions. The ingredient composition, estimated energy content (Fundación Española Desarrollo Nutrición Animal, 2010) and determined nutrient composition of diets are shown in Table 1. 2.2. Feed supply and analyses Feed, in mash form, and water were provided ad libitum through the trial. Weekly samples of each diet were pooled at the end of each experimental period and the composite samples preserved at room temperature to be analysed following the procedures of AOAC (2005). The dry matter was determined by oven drying method (930.15), organic matter and total ash by muffle furnace (942.05), crude protein by Kjeldahl method (984.13) and ether extract by Soxhlet analysis (920.39). The neutral detergent fiber was determined with an ANKOM 220 Fiber Analyser on dried samples (608 1C for 48 h), as described Mertens (2002), and were expressed as ash-free residues. The starch was analysed by polarimetry after hydrolysis with ethanol and HCl (Commission of the European Communities, 1999). The AAs composition was determined by HPLC-Fluorescence (PNT-M-109), with the exception of tryptophan and cystine that were analysed by HPLC-UV and gas chromatography, respectively. 2.3. Measurements, sampling and blood analyses Mortality and the possible presence of pathologies were daily checked. Individual BW and feed consumption per pen were recorded every two weeks and used to calculate average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) per replicate for each period (grower and finisher) and also for the overall trial. The last day of the grower period (day 45 of the trial), the BF depth at the last rib level was measured by the

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J. Suárez-Belloch et al. / Livestock Science 171 (2015) 36–43

Table 1 Ingredient composition and estimated and determined analyses of the experimental diets (g/kg as-fed basis, unless otherwise indicated). Grower diet (during 45 d), g Lys/kg(from 26 kg BW, during 45 days)

Ingredients Corn Wheat Barley Bakery meal Soybean meal 44%CP Rapeseed meal 00 Blended fat Calcium carbonate Dicalcium phosphate Sodium chloride Vitamin and mineral complexa Calculated nutrientsb Metabolizable energy (MJ/kg) Analyzed nutrients Dry matter Total ash Crude protein (CP) Ether extract Neutral detergent fiber Starch Total amino acids Lysine Methionine Methionine þcystine Threonine Tryptophan Standardized ileal digestible amino acidsc Lysine Methionine Methionine þcystine Threonine Tryptophan Ideal protein, g/kg CPc

11

9.1

7.8

5.2

240.0 220.0 81.5 60.0 311.8 30.0 30.0 10.0 8.7 5.0 3.0

240.0 220.0 167.4 60.0 225.9 30.0 30.0 10.0 8.7 5.0 3.0

240.0 220.0 250.4 60.0 142.9 30.0 30.0 10.0 8.7 5.0 3.0

240.0 220.0 333.4 60.0 59.9 30.0 30.0 10.0 8.7 5.0 3.0

13.63

13.63

13.63

13.63

Finisher diet (until 23 kg BW)

240.0 220.0 167.4 60.0 225.9 30.0 30.0 10.0 8.7 5.0 3.0 13.63

900.0 72.4 240.0 43.2 111.0 348.0

914.0 69.8 193.0 41.5 111.0 368.0

912.0 67.3 161.0 44.5 117.0 405.0

911.0 52.1 148.0 43.0 124.0 438.0

914.0 69.8 193.0 41.5 111.0 368.0

11.0 3.4 7.2 8.1 2.3

9.1 2.6 6.4 6.8 2.3

7.8 2.6 5.6 5.7 1.7

5.2 1.9 3.9 4.7 1.4

9.1 2.6 6.4 6.8 2.3

9.5 3.5 7.4 6.7 2.0

7.8 2.3 7.4 6.7 2.0

6.5 1.7 3.6 4.6 1.5

4.2 1.6 3.3 3.7 1.2

7.8 2.3 7.4 6.7 2.0

530.0

540.0

540.0

380.0

720.0

a Provided per kilogram of complete diet: 7000 IU Vitamin A; 1300 IU Vitamin D3; 10 IU Vitamin E; 0.4 mg Vitamin K3; 0.8 mg Vitamin B1; 3 mg Vitamin B2; 1 mg Vitamin B6; 15 mg Vitamin B12; 12 mg nicotinic acid; 8 mg calcium pantothenate; 10 mg choline chloride; 1 mg Biotine; 15 mg Cu (copper sulfate); 80 mg Fe (ferrous carbonate); 35 mg Mn (manganese sulphate); 80 mg Zn (zinc oxide); 0.1 mg Co (cobalt carbonate); 0.3 mg Se (sodium selenite) and 0.3 mg I (potassium iodate). b According to Fundación Española Desarrollo Nutrición Animal (2010). c According to NRC (2012).

same operator, on the right side of all animals and over the skin without clipping the fleece, by an ultrasound apparatus (series 12 S/N: 45000, Renco Lean-Meater, Minneapolis, USA). On the same day, from three pigs randomly selected per pen, a sample of 3.5 ml of blood was taken by jugular venipuncture into disposables tubes BD Vacutainer SSTTM II Advance Plus (Becton Dickenson, Belliver Industrial Estate, Plymouth, UK). After centrifugation (3000
g, 10 min), serum was stored at  20 1C until analyses. Blood analyses were carried out using a GernonStar equipment (RAL, Barcelona, España) in an external laboratory (Albeitar, Zaragoza, Spain). A colorimetric test based on green bromocresol (Human, Wiesbaden, Germany) was used to analyse albumin, whereas enzymatic kits (RAL, Barcelona, Spain) were utilized for the determination of urea (with urease and glutamate dehydrogenase), triglycerides (using glycerol-3-phosphate oxidase) and cholesterol (by peroxidase).

After blood sampling, a total of 40 pigs (one per pen randomly chosen) were moved 80 km to a commercial abattoir (Agroalimentaria de Teruel S.A., La Mata de los Olmos, Teruel, Spain), where they were kept in lairage for 15 h with full access to water but not to feed. Animals were stunned by exposure to CO2 (83% mean atmosphere concentration of gas; CO2 cycle of 90 s, with 60 s interval between discharges and sticking). After stunning, pigs were exsanguinated, scalded, dehaired, eviscerated and split down the midline according to standard commercial procedures. The BF thickness between 3rd and 4th ribs and also at level of gluteus medius (GM) muscle was measured, with a rule of millimetric precision, on the left side of each carcass. After, samples of subcutaneous fat at last rib level were taken from each pig carcass and immediately kept at  20 1C until analyses. One month later, the fat samples were thawed and stored in 4% buffered formaldehyde and embedded in paraffin. Sections (4 mm) were stained with

J. Suárez-Belloch et al. / Livestock Science 171 (2015) 36–43

haematoxylin and eosin. The number of adipocytes was counted with the 10
objective in 1.6 mm2 (10 areas of 0.16 mm2 each), using an Axiolab drb KT microscope (Jenna, Germany), for counting more than 2000 adipocytes and with a random distribution along of sections. When the average BW of each pen was approximately 123 kg, from all experimental animals (160), in vivo BF measurements were taken and blood samples were collected and analysed as described above. In the same day, pigs were moved to the abattoir and slaughtered following the same protocol used for younger animals. At 45 min post mortem, the BF depth at 3rd–4th ribs and at level of GM muscle was measured on the same carcass side as was previously detailed. 2.4. Statistical analyses Data were analysed as a randomized block design with treatments arranged factorially (2
4) using the GLM procedure of SAS (version 9.2). The model included block, sex (barrows and gilts) and diet (11.0, 9.1, 7.8 and 5.2 g Lys/kg) as main effects, as well as the interaction sex
diet. The linear and quadratic effects of dietary Lys content were analysed by orthogonal polynomials. The experimental unit was the pen for all the parameters. A P-value o0.05 was considered as a significant difference whereas a Pvalue between 0.05 and 0.10 as a trend. In addition, the Lys requirement during the grower period was estimated by adjusting pen means data of response criteria from barrows and gilts to broken-line models, using the NLmixed procedure of SAS (Robbins et al., 2006). Linear and quadratic models were evaluated including or not the effect of block. The linear broken-line equation was y ¼LþU
(R  X) and the quadratic brokenline equation was y¼L þU
(R X)
(R X), where L is the ordinate, U is the slope, R is the abscissa of the breakpoint and the value of R  X is zero at values of X 4R. The breakpoint between the slope and plateau phases of the model was changed iteratively until the Akaike information criterion was minimized. 3. Results No significant interaction between sex and diet was detected for any of the variables studied. Therefore, only main effects are shown in tables. 3.1. Growth performance During the grower period, when the Lys restriction was carried out, barrows grew faster than gilts (P¼0.047) but the ADFI and FCR were similar for both sexes (Table 2). The Lys restriction affected quadratically all growth performance traits, reducing ADG (Po0.001) and ADFI (P ¼0.008) but increasing FCR (P ¼0.003). Due to the decrease in ADG, at day 45 of the trial (end of the grower period), pigs fed low Lys levels were lighter than those fed high Lys levels (P o0.001) and when ADFI was expressed per kg of the average metabolic BW, the differences due to Lys restriction were removed.

39

The curvilinear response of ADG and FCR to dietary Lys content allows estimating Lys requirements as the minimum Lys concentration required to maximize ADG and FCR. For this aim, broken-line models were adjusted to data, irrespective of sex as the diet effect was independent of it. The best fitting was obtained with a linear plateau model (Fig. 1), with breakpoints at 9.7 70.22 (R2 ¼ 0.94) and 9.5 70.25 g Lys/kg (R2 ¼0.91) to maximize ADG and to minimize FCR, respectively, without differences due to sex. During the finisher period, when all experimental animals received a common diet, the differences in ADG between sexes were maintained (Po0.001) but barrows also had higher ADFI (P o0.001) without any difference in FCR. A compensatory increase of BW gain was observed (P o0.001) in response to Lys reduction in the grower diet. The effect was linear and associated to a higher ADFI (P ¼0.097), which was also observed when was express as metabolic BW (Po0.001), giving finally a linear decrease of FCR (P ¼0.002). For the overall trial (grower þfinisher), barrows had higher ADG and ADFI than gilts (P o0.001) being younger at slaughter (109 vs. 117 d; P o0.001). Also, the ADG showed a quadratic reduction (P¼0.041) as Lys content decreased in the grower phase whereas FCR increased linearly (P ¼0.001), especially for pigs fed diets with 7.8 and 5.2 g Lys/kg, being those animals older at slaughter (P o0.001) at similar BW.

3.2. Fatness and serum metabolites At the end of the grower period, no differences between barrows and gilts were detected in the albumin, urea, triglycerides and cholesterol concentrations (Table 3). Also, there was no effect of sex in the count of adipocytes from subcutaneous fat although barrows resulted fatter detecting the effect at last rib level (P ¼0.038). The dietary Lys restriction promoted a quadratic reduction of albumin (P ¼0.004), a quadratic increment of urea (P ¼0.009) and cholesterol (P ¼0.039) and a linear increase of triglycerides (P ¼0.001). The fitting of broken line models to these data showed that the estimated requirements of Lys were 10.8 70.53 g/kg to maximize serum albumin content and 9.6 70.71 g/kg to minimize cholesterol level (albumin (mg/dl) ¼3.97– 0.295
(10.8-g Lys/kg)2, R2 ¼ 0.79; and cholesterol (mg/dl) ¼90.71–5.14
(9.6-g Lys/kg), R2 ¼0.53). In addition, the Lys restriction decreased linearly carcass fatness at the end of the grower period although the effect was only significant at last rib measured by ultrasounds (P o0.001). At the end of the finisher period, blood from barrows had higher albumin (P ¼0.005) and urea (P ¼0.002) contents than that from gilts and carcasses from barrows were again fatter being observed this effect as much in vivo, at last rib level (P ¼0.001), as post mortem, at 3rd–4th ribs (P ¼0.035) and over the GM muscle (Po0.001). The effects of dietary Lys restriction on blood parameters disappeared at the end of the trial (P40.10) but the fat thickness at GM muscle level increased linearly (P ¼0.004) with Lys restriction.

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J. Suárez-Belloch et al. / Livestock Science 171 (2015) 36–43

Table 2 The effect of sex and Lys restriction in grower diet on productive performance variables of heavy pigs.a Sex

SEM (n¼ 20)

Barrows Gilts

Dietary Lys (g/kg) 11

9.1

7.8

SEM (n¼10) 5.2

P-valueb Sex

Lys Linear

Body weight (kg) Initial (at day 0) End of grower period (at day 45) End of finisher period (at slaughter) Grower period ADG (g/d) ADFI (kg/d) ADFI (g/kg mean BW0.75) FCR (g/g) Finisher period ADG (g/d) ADFI (kg/d) ADFI (g/kg mean BW0.75) FCR (g/g)

25.8 57.6 122.6

26.9 57.3 123.5

716 687 1.70 1.70 103.4 102.7 2.48 2.56

0.18 0.45 0.67

26.2 63.9 122.4

9.6 0.029 1.58 0.038

857 819 667 1.80 1.77 1.74 103.4 102.3 107.4 2.10 2.17 2.60

1006 893 11.9 2.97 2.62 0.034 101.9 89.4 1.21 2.97 2.93 0.040

26.7 63.0 123.3

26.1 55.8 123.4

26.3 46.9 123.1

0.25 0.73 1.26

462 13.7 1.48 41.1 99.1 2.23 3.21 0.054

Quadratic

0.001 0.828 0.616 0.686 o0.001 o0.001 0.484 0.721 0.664

0.047 o0.001 o0.001 0.924 o0.001 0.008 0.764 0.303 0.127 0.153 o0.001 0.003

891 934 956 1018 17.0 2.69 2.85 2.80 2.83 48.7 89.9 95.2 96.2 101.2 1.71 3.03 3.06 2.93 2.79 0.056

o 0.001 o0.001 o 0.001 0.097 o 0.001 o0.001 0.540 0.002

0.567 0.195 0.661 0.124

Overall period ADG (g/d) ADFI (kg/d) ADFI (g/kg mean BW0.75) FCR (g/g)

921 843 2.50 2.32 98.8 91.0 2.71 2.76

8.050 0.022 0.89 0.027

892 918 872 2.37 2.44 2.44 93.7 95.7 96.0 2.66 2.66 2.80

848 11.7 2.40 31.7 94.3 1.26 2.83 0.038

o 0.001 o 0.001 o 0.001 0.208

0.002 0.548 0.792 0.001

0.041 0.094 0.151 0.661

Grower-finishing period (d)

109

1.1

110

119

o 0.001 o0.001

0.073

a b

117

108

115

1.7

ADG: average daily gain; ADFI: average daily feed intake; FCR: feed conversion ratio. No significant interaction (sex
diet) was found.

Fig. 1. Response of average daily gain (ADG; ■ □) and feed conversion ratio (FCR; ▲ Δ) to total lysine content (Lys) of the feed during the growing period fitted to a linear broken line model (Robbins et al., 2006). Each point is the mean of 5 pen replicates, each with 5 barrows (■ ▲) or gilts (□ Δ). Breakpoints for ADG and FCR were 9.7 7 0.22 and 9.5 70.25 g Lys/kg (ADG ¼ 857–898.8
(9.7-g Lys/kg) R2 ¼ 0.94; FCR¼ 2.096 þ 2.615
(9.5-g Lys/kg) R2 ¼ 0.91).

4. Discussion 4.1. Growth performance and fatness The clear response of performance traits to dietary Lys levels confirms previous observations on the effectiveness of Lys to manipulate the pattern of growth (Chiba, 1995; Fabian et al., 2002; Kyriazakis et al., 1991). The similar

response to Lys of barrows and gilts suggests that differences in fat to lean accretion ratio were negligible to be detected during the early phase of growth, when energy intake is limiting the protein deposition (Whittemore, 1986). However, across Lys levels, barrows showed higher BF thickness, measured by ultrasounds, suggesting higher potential for fat deposition than females in agreement with previous reports (Latorre et al., 2008; RodríguezSánchez et al., 2011). To optimize ADG and FCR, the best fitting was obtained with breakpoints at 9.7 70.22 and 9.5 70.25 g Lys/kg, respectively, without differences due to sex. It means that the resulting Lys levels of restriction during this experimental period were, as average, 7, 20 and 47% of requirements for the three restriction treatments (7.8, 6.5 and 4.2 g Lys/kg, respectively). The average Lys concentration estimated as requirement, equivalent to 0.715 g Lys/MJ of metabolizable energy (ME) or 0.590 g digestible Lys/MJ ME, assuming a standardized ileal digestibility (SID) of 0.825 according to the AA digestibility of ingredients in the diet (NRC, 2012), was lower than the NRC allowance for a mix of barrows and gilts (0.813 g Lys/MJ ME). It was also lower than recent estimates (ranging 0.741 to 0.908 g SID Lys/MJ ME) based on performance traits (Shelton et al., 2011; Moore et al., 2013) or N balance data (Heger et al., 2009) for males or females of modern lean genotypes of comparable BW. However, it was close to values (0.621– 0.717 g Lys/MJ ME) estimated in previous studies with crossbred barrows of lower growth potential (Coma et al., 1995b; Zhang and Kim, 2013).

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Table 3 The effect of sex and Lys restriction in grower diet on serum metabolites and fatness traits of heavy pigs. Sex

SEM (n¼ 20)

Barrows Gilts

Dietary Lys (g/kg) 11

9.1

SEM (n ¼10) 7.8

5.2

P-valuea Sex

Lys Linear

At the end of the grower phase Albumin (mg/dl) Urea (mg/dl) Triglycerides (mg/dl) Cholesterol (mg/dl) Number of adipocytesb Fat depth at last rib (mm)c Fat depth at 3rd–4th ribs (mm)d Fat depth at gluteus medius muscle (mm)d At the end of the finisher phase Albumin (mg/dl) Urea (mg/dl) Triglycerides (mg/dl) Cholesterol (mg/dl) Fat depth at last rib (mm)c Fat depth at 3rd–4th ribs (mm)d Fat depth at gluteus medius muscle (mm)d

3.37 3.35 0.043 33.8 31.9 0.86 51.6 49.8 2.32 100.2 98 1.9 2542 2486 133.3 9.6 9.0 0.175 10.9 10.2 0.65 9.7 9.5 0.524

4.08 38.7 46.1 98.3 18.7 31.0 18.8

3.82 34.1 43.7 99.7 16.6 27.6 15.7

3.98 3.82 3.09 2.55 0.061 32.9 31.9 30.7 36.0 1.24 43.1 46.6 52.3 60.0 3.28 90.0 94.6 98.0 113.8 2.71 2677 2460 2418 2501 226.3 10.2 9.5 9.5 8.0 0.25 10.2 11.7 10.7 9.7 1.08 9.6 10.1 10.1 8.6 0.89

0.057 0.95 2.27 6.5 0.40 0.76 0.75

3.93 36.8 44.0 92.8 17.2 28.8 15.8

4.09 37.4 42.1 114.7 17.8 28.8 16.4

3.96 36.6 46.4 93.3 17.6 28.8 18.2

3.81 34.8 47.0 95.2 18.1 30.9 18.6

0.081 1.35 3.22 9.24 0.56 1.18 0.72

Quadratic

0.826 o 0.001 0.004 0.127 0.170 0.009 0.605 0.001 0.523 0.313 o 0.001 0.039 0.700 0.218 0.877 0.038 o 0.001 0.181 0.330 0.436 0.093 0.890 0.511 0.285

0.005 0.002 0.465 0.889 0.001 0.035 o0.001

0.177 0.244 0.353 0.731 0.332 0.076 0.004

0.073 0.381 0.696 0.293 0.912 0.722 0.946

a

No significant interaction (sex
diet) was found. Measured by microscope in 1.6 mm2 (10 areas of 0.16 mm2) with a 10
objective. Measured in vivo by ultrasounds. d Measured post-mortem by rule. b c

When Lys concentration was below this threshold, a decline in ADG was observed during the grower period associated to lower ADFI. The reduction in ADFI can be attributed to the lower BW at the end of this period. In fact, when it was expressed in relation to the average metabolic BW, it was similar in restricted and unrestricted treatments (102.9 vs. 103.4 72.23 g/kg BW0.75). The response of ADFI to protein restriction during early growth seems to be variable depending on the starter diet (Chiba, 1995), reporting some authors an increment (Fabian et al., 2002; Ferguson and Theeruth, 2002; Kyriazakis et al., 1991) while others did not find any effect (O'Connell et al., 2006; Kamalakar et al., 2009). However, independently of the response of feed intake, there is more agreement on the effect of protein restriction enhancing body fatness at the end of the depletion period, well when fat was measured as a body component (Ferguson and Theeruth, 2002; Kyriazakis et al., 1991; Whang et al., 2003) or as in vivo BF (Chiba, 1995; Fabian et al., 2002; Kamalakar et al., 2009). In the current experiment, this effect was likely masked by the differences in BW and the results suggest that the influence of live weight on BF measured by ultrasounds must be at least as large as that induced by nutrition. The BF depth is an early maturing component of total body fat and, from allometric relationships reported by Tess et al. (1986), around 30 percentage units of increase in carcass BF can be estimated for recorded differences in BW at the end of the grower period between extreme dietary treatments (46.9 and 63.9 kg BW). This can be compared with increments of 11 to 47 percentage units in ultrasounds BF promoted by protein restriction

levels of similar extent (40 to 51 percentage units) to those of this experiment (Chiba, 1995; Fabian et al., 2002; Kamalakar et al., 2009). During the finisher period, when a common diet was provided, the effects described before were reversed. Pigs previously restricted had higher ADG and were more efficient converting feed into gain, confirming the existence of compensatory growth as previously observed in response to restriction of energy (Critser et al., 1995; Heyer and Lebret, 2007) or protein (Chiba, 1995; Fabian et al., 2002; O’Connell et al., 2006) during early growth. Although in some of these experiments a complete compensation for the overall fattening period was recorded (Chiba, 1995; Fabian et al., 2002), we found only a full recovery with the lightest level of Lys restriction (9.1 g/kg), whereas pigs more restricted (20 and 47% of requirements) had lower ADG and worst FCR throughout the whole period even though the ADFI was not affected. This entailed a delay to reach the BW at slaughter of 1.8 70.43 d per each g/kg Lys restriction in the diet which was associated with an impairment of FCR by 0.03470.0095, but also with an increasing of fat thickness at GM muscle by 0.51 70.168 mm. A higher carcass fat coverage is a desirable trait for ham producers which, according to the above responses, have a cost in terms of FCR equivalent to 0.066 per mm of improvement whenever it would be required. The increase in ADFI during the realimentation period is a finding more frequent in experiments limiting energy intake during youth than in those limiting protein (Critser et al., 1995; Heyer and Lebret, 2007; Skiba et al., 2006).

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In pigs fed protein restricted diets, compensatory growth commonly occurs in absence of an increase in voluntary feed intake (Chiba, 1995; Fabian et al., 2002; O’Connell et al., 2006). It is due to a high protein accretion and efficiency of utilization (Whang et al., 2003; O’Connell et al., 2006) during the realimentation period at the expense of fatty tissue growth in an attempt to re-establish the composition of non-restricted pigs (Ferguson and Theeruth, 2002). However, the compensation may not be complete and differences in carcass fat deposition may persist, particularly at light BW (Kyriazakis et al., 1991; Whang et al., 2003). Moreover, the effect of realimentation is variable, depending on experimental animal age and severity of restriction (Skiba et al., 2006; Kamalakar et al., 2009), as well as on changes in feed intake, according to the type of AA imbalance (Henry, 1995). Thus, the higher fattening promoted by protein restriction from 28 to 65 kg BW persisted at 105 kg BW, although alleviated by an incomplete compensation (Degreef et al., 1992). In the present trial, the compensatory growth was not complete and differences in fat thickness at GM muscle were recorded. It agrees with the higher deposition rate of subcutaneous fat in ham observed by Heyer and Lebret (2007) during the realimentation phase of barrows and gilts after a period of feed restriction from 30 to 70 kg BW. This was associated to an increase in ADFI during realimentation and the subsequent response of fat and protein deposition rates to energy intake (Bikker et al., 1996). A trend to increase ADFI during the finisher period was also recorded in the present experiment which may contribute to explain the higher fat coverage of ham promoted by the protein restriction during growth. Similarly, the higher ADFI showed by barrows compared with gilts during the finisher phase and the overall experimental period was also associated to higher ADG and fatness, either measured in vivo or post mortem, without a negative effect on FCR as could be expected (Latorre et al., 2009; Rodríguez-Sánchez et al., 2011). 4.2. Serum metabolites The differences detected in all serum metabolites during the grower period, which were similar in both sexes, indicate a metabolic change promoted by dietary Lys restriction. The levels were re-established at the finisher period as was suggested by the lack of significant differences. Both, triglycerides and cholesterol increased in response to Lys restriction whereas albumin decreased, denoting the change in energy partitioning toward fat synthesis as previously reported by Kamalakar et al. (2009), Mule et al. (2006) and Whang et al. (2003). However, this modification of the metabolic status was not confirmed by a higher number of adipocytes or body fatness. This is in contrast to the positive association between cholesterol and ultrasound carcass BF recorded by Mule et al. (2006) and Kamalakar et al. (2009) in 50 kgpigs subjected to Lys restriction from 19 to 23 kg BW. The differences in live weight, recorded at the end of the grower period in our experiment, could explain these opposite results on carcass fat retention by confusing the effects of BW and Lys restriction.

Whereas the response of triglycerides to dietary Lys reduction was linear, the response of cholesterol and albumin was quadratic, allowing the identification of an inflexion point by broken-line models. Although the accurate of fitting was lower for both, albumin and cholesterol (R2 ¼0.79 and 0.53), than for ADG and FCR (R2 ¼0.94 and 0.91), the estimated requirements of Lys maximizing serum albumin and minimizing cholesterol did not differ from those estimated to optimize performance traits. These results confirm the long-standing recognition that has the albumin as the most sensitive fraction of serum total protein to protein malnutrition (Lowrey et al., 1962). The slight but significant increase in the albumin concentration in barrows compared to gilts might be explained by the greater ADG of barrows in agreement with the value attributed to albumin as index of efficient utilization of AAs (Mule et al., 2006), whereas the concomitant increase in serum urea may be related to the higher ADFI and therefore protein. Blood urea concentration has been used to define Lys requirements (Coma et al., 1995a; Roux et al., 2011) although not always successfully (Cameron et al., 2003), depending on the supply of non-limiting AAs relative to Lys. In the current experiment, the formulation of diets to get similar content of ideal protein prevented the response of blood urea to Lys concentration with the exception of the diet containing 5.2 g with lower proportion of ideal protein. Anyway, a significant increase in blood urea was noticed in the diet exceeding Lys requirements (11.0 g) confirming the usefulness of blood urea for estimating AA requirements, providing all diets ensure excess of nonlimiting AAs. Similar results were also reported by Fabian et al. (2002) and Whang et al. (2003).

5. Conclusions The results of the current trial confirm the ability of young pigs to exhibit compensatory growth in response to Lys or ideal protein restriction and the potential of this practice to increase fat coverage of ham. Although these effects are associated to incomplete compensatory growth, and have a cost in terms of FCR, they can be achieved at constant slaughter BW and with lower feed cost.

Conflict of interest statement There is no conflict of interest.

Acknowledgements This work was funded through the CDTI Project IDI20090836 of the Ministry of Science and Technology (Spanish Government), with participation of the Department of Industry and Innovation of the Government of Aragón and the European Social Fund. Jesús Suárez Belloch was granted by the University of Zaragoza.

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References Adeola, O., Young, L.G., 1989. Dietary protein-induced changes in porcine muscle respiration, protein synthesis and adipose tissue metabolism. J. Anim. Sci. 67, 664–673. AOAC, 2005. Official Methods of Analysis. In: Horvitz, W., Latimer, G.W. (Eds.), 18th ed . Bikker, P., Verstegen, M.W.A., Campbell, R.G., 1996. Performance and body composition of finishing gilts (45 to 85 kg) as affected by energy intake and nutrition in earlier life: II. Protein and lipid accretion in body components. J. Anim. Sci. 74, 817–826. Boletin Oficial Estado, 2007. (Law 32/2007 of 7th November for farm animal care, transport, experimentation and slaughtering). Ley 32/ 2007 de 7 de noviembre para el cuidado de los animales en su explotación, transporte, experimentación y sacrificio. Bol. of. Est. 268, 45914–45920. Cameron, N.D., McCullough, E., Troup, K., Penman, J.C., 2003. Serum urea concentration as a predictor of dietary lysine requirement in selected lines of pigs. J. Anim. Sci. 81, 91–100. Chiba, L.I., 1995. Effects of nutritional history on the subsequent and overall growth performance and carcass traits of pigs. Livest. Prod. Sci. 41, 151–161. Coma, J., Carrion, D., Zimmerman, D.R., 1995a. Use of plasma urea nitrogen as a rapid response criterion to determine the lysine requirement of pigs. J. Anim. Sci. 73, 472–481. Coma, J., Zimmerman, D.R., Carrion, D., 1995b. Interactive effects of feedintake and stage of growth on the lysine requirement of pigs. J. Anim. Sci. 73, 3369–3375. Commission of the European Communities, 1999. Determination of starch. Polarimetric method. J. Eur. Commun. 209, 25–27. Critser, D.J., Miller, P.S., Lewis, A.J., 1995. The effects of dietary-protein concentration on compensatory growth in barrows and gilts. J. Anim. Sci. 73, 3376–3383. Degreef, K.H., Kemp, B., Verstegen, M.W.A., 1992. Performance and bodycomposition of fattening pigs of 2 strains during protein-deficiency and subsequent realimentation. Livest. Prod. Sci. 30, 141–153. Fabian, J., Chiba, L.I., Kuhlers, D.L., Frobish, L.T., Nadarajah, K., Kerth, C.R., McElhenney, W.H., Lewis, A.J., 2002. Degree of amino acid restrictions during the grower phase and compensatory growth in pigs selected for lean growth efficiency. J. Anim. Sci. 80, 2610–2618. Ferguson, N.S., Theeruth, B.K., 2002. Protein and lipid deposition rates in gr 4 owing pigs following a period of excess fattening. S. Afr. J. Anim. Sci. 32, 97–105. Fundación Española Desarrollo Nutrición Animal, 2010. (FEDNA Standards for Feed Compound Formulation). Normas FEDNA para la formulación de piensos compuestos. In: De Blas, C., Mateos, G.G., García Rebollar, P. (Eds.), , FEDNA, Madrid, Spain. Heger, J., Křížová, L., Šustala, M., Nitrayová, S., Patráš, P., Hampel, D., 2009. Individual response of growing pigs to lysine intake. J. Anim. Physiol. Anim. Nutr. 93, 538–546. Henry, Y., 1995. Effect of a dietary amino-acid deficiency or imbalance during the initial period of growth in pigs on subsequent performance at slaughter. Ann. Zootech. 44, 3–28. Heyer, A., Lebret, B., 2007. Compensatory growth response in pigs: effects on growth performance, composition of weight gain at carcass and muscle levels, and meat quality. J. Anim. Sci. 85, 769–778. Kamalakar, R.B., Chiba, L.I., Divakala, K.C., Rodning, S.P., Welles, E.G., Bergen, W.G., Kerth, C.R., Kuhlers, D.L., Nadarajah, N.K., 2009. Effect of the degree and duration of early dietary amino acid restrictions on subsequent and overall pig performance and physical and sensory characteristics of pork. J. Anim. Sci. 87, 3596–3606. Kyriazakis, I., Stamataris, C., Emmans, G.C., Whittemore, C.T., 1991. The effects of food protein-content on the performance of pigs previously given foods with low or moderate protein contents. Anim. Prod. 52, 165–173. Latorre, M.A., Garcia-Belenguer, E., Ariño, L., 2008. The effects of gender and slaughter weight on growth performance and carcass traits of

43

pigs intended for dry-cured ham from Teruel (Spain). J. Anim. Sci. 86, 1933–1942. Latorre, M.A., Ripoll, G., García-Belenguer, E., Ariño, L., 2009. The increase of slaughter weight of gilts as strategy to optimize de production of Spanish high quality dry-cured hams. J. Anim. Sci. 87, 1464–1471. Lovatto, P.A., Sauvant, D., Noblet, J., Dubois, S., Milgen, J.V., 2006. Effects of feed restriction and subsequent refeeding on energy utilization in growing pigs. J. Anim. Sci. 84, 3329–3336. Lowrey, R.S., Barnes, R.H., Krook, L., Pond, W.G., Loosli, J.K., 1962. Influence of caloric level and protein quality on manifestations of protein deficiency in young pigs. J. Nutr. 78, 245–253. Mertens, D.R., 2002. Gravimetric determination of amylase treated neutral detergent fibre in feeds with refluxing beakers or crucibles: collaborative study. J. AOAC Int. 85, 1217–1240. Moore, K.L., Mullan, B.P., Campbell, R.G., Kim, J.C., 2013. The response of entire male and female pigs from 20 to 100-kg liveweight to dietary available lysine. Anim. Prod. 53, 67–74. Mule, H.R., Chiba, L.I., Fabian, J., Kuhlers, D.L., Jungst, S.B., Frobish, L.T., Nadarajah, K., Bergen, W.G., Welles, E.G., 2006. Effect of early dietary amino acid restrictions on serum metabolites in pigs selected for lean growth efficiency. Can. J. Anim. Sci. 86, 489–500. NRC, 2012. Nutrient Requirements of Swine, 11th rev. ed. National Academy Press, Washington, DC. O'Connell, M.K., Lynch, P.B., O’Doherty, J.V., 2006. The effect of dietary lysine restriction during the grower phase and subsequent dietary lysine concentration during the realimentation phase on the performance, carcass characteristics and nitrogen balance of growing -finishing pigs. Livest. Sci. 101, 169–179. Robbins, K.R., Saxton, A.M., Southern, L.L., 2006. Estimation of nutrient requirements using broken-line regression analysis. J. Anim. Sci. 84, 155–165. Rodríguez-Sánchez, J.A., Sanz, M.A., Blanco, M., Serrano, M.P., Joy, M., Latorre, M.A., 2011. The influence of dietary lysine restriction during the finishing period on growth performance and carcass, meat, and fat characteristics of barrows and gilts intended for dry-cured ham production. J. Anim. Sci. 89, 3651–3662. Roux, M.L., Donsbough, A.L., Waguespack, A.M., Powell, S., Bidner, T.D., Payne, R.L., Southern, L.L., 2011. Maximizing the use of supplemental amino acids in corn-soybean meal diets for 20- to 45-kilogram pigs. J. Anim. Sci. 89, 2415–2424. Ruíz, J., García, C., Muriel, E., Andrés, A.I., Ventanas, J., 2002. Influence on sensory characteristics on the acceptability of dry-cured ham. Meat Sci. 61, 247–254. SAS (version 9.2). Statistical Analysis Systems Institute. SAS Institute Inc., Cary, NC, USA. Shelton, N.W., Tokach, M.D., Dritz, S.S., Goodband, R.D., Nelssen, J.L., DeRouchey, J.M., 2011. Effects of increasing dietary standardized ileal digestible lysine for gilts grown in a commercial finishing environment. J. Anim. Sci. 89, 3587–3595. Skiba, G., Raj, S., Weremko, D., Fandrejewski, H., 2006. The compensatory response of pigs previously fed a diet with increased fibre content. 1. Growth rate and voluntary feed intake. J. Anim. Sci. 15, 393–402. Tess, M.W., Dickerson, G.E., Nienaber, J.A., Ferrell, C.L., 1986. Growth, development and body composition in three genetic stocks of swine. J. Anim. Sci. 62, 968–979. Whang, K.Y., Kim, S.W., Donovan, S.M., McKeith, F.K., Easter, R.A., 2003. Effects of protein deprivation on subsequent growth performance, gain of body components, and protein requirements in growing pigs. J. Anim. Sci. 81, 705–716. Whittemore, C.T., 1986. An approach to pig growth modeling. J. Anim. Sci. 63, 615–621. Zhang, Z.F., Kim, I.H., 2013. Determining of the effect of lysine:calorie ratio on growth performance and blood urea nitrogen of growing barrows and gilts in hot season and cool season in a commercial environment. Asian–Australas. J. Anim. Sci. 26, 401–407.