Effect of vitamin D3 supplementation on lysine utilization in growing rabbits

Animal Feed Science and Technology 254 (2019) 114221

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Effect of vitamin D3 supplementation on lysine utilization in growing rabbits

T

⁎

E.A. Elwakeela, , E. Abd El-khalekb, A.M. Abd El-Hadyb, M.G. Ahmeda, O.A. Hassanb a b

Department of Animal and Fish Production, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, 21545, Egypt Department of Poultry Production, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, 21545, Egypt

A R T IC LE I N F O

ABS TRA CT

Keywords: ADG FCR Nitrogen balance Blood metabolites Vitamin D3 Lysine

The effect of vitamin D3 (VD3) on lysine utilization in growing rabbits was studied in two experiments (Exp.). In Exp. 1, 162 growing rabbits (491 ± 38 g) were fed basal diets containing 16% crude protein ad libitum. The treatments were varying levels of VD3 (0, 500, and 1000 IU) and lysine (5.5, 6, and 6.5 g/kg dry matter; DM) in a 3 × 3 factorial design with six replicates in each treatment occurring for 5 weeks, and the experimental units were the cages (3 rabbits/ cage). Nine experimental VD3+lysine diets, (0 + 0), (0 + 0.5), (0 + 1), (500 + 0), (500 + 0.5), (500 + 1), (1000 + 0), (1000 + 0.5) and (1000 + 1), were formulated. A basal diet (0 + 0) was formulated to be deficient in lysine (5.5 g/kg DM) and VD3 (0.0 IU). Lysine significantly (P < 0.01) affected the final body weight (FBW), average daily gain (ADG) and feed conversion ratio (FCR) but did not affect feed intake. VD3 tended (P = 0.06) to improve the FCR with no significant effect on FBW and ADG. VD3 and lysine exhibited no effect on feed intake. The highest mortality rate (38.9%, 7/18) was observed for the 0 + 0 (control) treatment, and the lowest mortality rates of 5.6% and 0.0% were observed for the 1000 + 0 and 1000 + 0.5 treatments, respectively. Exp. 2, the nutrient digestibility and nitrogen balance trial, was conducted with 45 rabbits (n = 5/treatment) randomly chosen from Exp. 1. Rabbits were housed in metabolic cages for 8 d and offered 180 g/d of each respective diet described in Exp. 1. A significant VD3 × lysine interaction was observed for N intake (P < 0.05). VD3 tended to increase N intake (P = 0.08) and improved (P < 0.05) dry matter digestibility (DMD) and organic matter digestibility (OMD). Lysine increased (P < 0.05) N intake and retained N (P < 0.01), decreased (P < 0.01) urine N, and tended to improve crude protein digestibility (CPD) (P = 0.08) and OMD (P = 0.10). Lysine increased the plasma total protein (TP), albumin (Alb; P < 0.05) and calcium (Ca; P < 0.01) contents and decreased plasma cholesterol (Cho), while VD3 and lysine decreased the urea concentration. A significant (P < 0.01) VD3 × lysine interaction was observed for plasma phosphorus (P) and growth hormones (GHs). VD3 increased P (P < 0.01) and tended (P = 0.08) to increase GHs, whereas lysine decreased (P < 0.05) P at the 1.0 g level but caused a linear and quadratic (P < 0.05) increase in GHs. Overall, the supplementation of diets with VD3 did not improve lysine utilization in growing rabbits; however, lysine increased N intake, retained N, the FBW, and the ADG and improved the FCR.

⁎ Corresponding author at: Dept. of Animal and Fish Production, Fac. Of Agriculture, Alexandria University, El-Shatby, P.O. Box 21545, Alexandria, Egypt. E-mail address: [email protected] (E.A. Elwakeel).

https://doi.org/10.1016/j.anifeedsci.2019.114221 Received 30 September 2018; Received in revised form 9 June 2019; Accepted 14 July 2019 0377-8401/ © 2019 Elsevier B.V. All rights reserved.

Animal Feed Science and Technology 254 (2019) 114221

E.A. Elwakeel, et al.

1. Introduction Nitrogen excretion by farm animals is a major concern for environmental pollution (NRC, 2001). Improving the N utilization of animals by directing dietary nitrogen for protein deposition is the main goal for most nutritional scientists (NRC, 2001 and Titgemeyer, 2003). Lysine amino acid is the first limiting amino acid for the growth of rabbits and play a vital role in meat deposition in rabbits (Monteiro-Motta Scapinello et al., 2013). Improved lysine utilization in growing rabbits can improve animal performance by improving protein utilization and reducing excreted N. Rabbits that are raised and housed indoors may suffer a severe lack of vitamin D3 (VD3) and endure markedly diminished health and growth (Kanekar et al., 2010). VD3 is important for not only bone growth through its effect on calcium and phosphorus metabolism but also its effect on protein metabolism and muscle growth (Sugai and Matsuda, 1968). The mode of action of VD3 on protein metabolism is unclear; however, studies have shown that VD3 stimulates muscle cell proliferation and growth (Buitrago et al., 2001, 2003) through the activation of mitogen-activated protein kinase A (MPA) pathways in rats (Wu et al., 2000). Several studies in humans have shown that a VD3 deficiency causes weakness of muscles and repeated falls (Pfeifer et al., 2009; Janssen et al., 2010; Mason et al., 2016). Shimura et al. (1975) reported that rats fed a diet supplemented with 1.45% lysine and VD3 grew faster than rats fed diets supplemented only with lysine, and the effects of both supplemented diets surpassed those of the diet that was deficient in lysine, regardless of whether VD3 was added. The relationship between VD3 and lysine amino acid utilization in growing rabbits requires further investigation. We hypothesize that VD3 can improve lysine utilization in growing rabbits. The main objective of this research was to investigate the effects of VD3 and lysine amino acid supplementation on the performance, nutrient digestibility, nitrogen balance and blood metabolites of growing rabbits. 2. Materials and methods This study was conducted at the Department of Poultry Production, Faculty of Agriculture, Alexandria University, Egypt (31.20 °N, 30 °E). Procedures for this research were in accordance with the guidelines of animal welfare of the European Parliament (2010/63/EU) of the Council of 22 September 2010. Rabbits were housed in galvanized wire cage batteries with standard dimensions of 50 × 45 × 40 cm with an elevation of 110 cm from the floor in a well-ventilated rabbitary. The ranges of ambient temperature and relative humidity during the experimental period (5 weeks) were 12–15 °C and 64–65%, respectively. The experimental diets were offered to rabbits for 5 weeks. 2.1. Experiment 1 (Growth trial) A total of 162 newly weaned V-line rabbits (male and female) at 4 weeks of age and with an average weight of 491 ± 38 g were distributed randomly in a completely randomized 3 × 3 factorial arrangement design (18 animals per treatment) in 6 cages (n = 3 animals/cage). The cage was the experimental unit. Three concentrations of VD3 (0, 500, and 1000 IU/kg dry matter (DM) diet) and three concentrations of lysine (5.5, 6.0, and 6.5 g/kg) were used to formulate nine diets. The nine formulated diets contained VD3 (IU/kg DM) + lysine (g/kg DM) as follows: (0 + 0), (0 + 0.5), (0 + 1), (500 + 0), (500 + 0.5), (500 + 1), (1000 + 0), (1000 + 0.5) and (1000 + 1). L-Lysine HCl (99%) that was added to the diets was a white to pale yellow crystal produced by AJINOMOTO EUROLYSINE Co. S.A.S., Paris, France, import Multi Vita Animal Nutrition Company, 6th of October City, West Cairo, Egypt. A commercial source of 25-hydroxy cholecalciferol (96.59%) was used, and the crystal product contained 40,000,000 IU/g of VD3 and was produced in Ningbo, China. Rabbits were housed in galvanized wire cage batteries with standard dimensions of 50 × 45 × 40 cm with an elevation of 110 cm from the floor in a well-ventilated rabbitary. The basal diet (0 + 0) was designed to be limiting in VD3 and lysine but not in any other amino acid. To ensure that the diet was deficient only in lysine, other amino acids were added to the basal diet (Table 1). Diets were offered ad libitum at 09:00 am with manual feeders, and feed intake was recorded once weekly for each cage. Fresh drinking water was provided with automatic drinkers. Rabbits were weighed individually on a weekly basis. Mortality was recorded daily, and feed intake was adjusted to the number of rabbits remaining in the cages. The chemical analysis of the basal diet (Table 1) was analysed based on AOAC (1995) assays for DM (ID number 930.15), organic matter (OM; ID number 942.05), crude protein (CP; as 6.25 × N; ID number 954.01) crude fibre (ID number 920.85), ether extract (ID number 920.39), calcium (ID number 927.02), and phosphorus (ID number 995.11). The amino acid content of the basal diet was measured by an automatic amino acid analyser (AAA 400. Ingos Ltd., Czech Republic) 2.2. Experiment 2 2.2.1. Digestibility and nitrogen balance trial A total of 45 male rabbits that were fed the same diet used in Exp. 1 were distributed individually into metabolic cages. The metabolic cages had the same dimensions as those used in Exp. 1 with the ability to separate urine and faeces. Rabbits were allowed to adapt to individual feeding for 4 d, and then the orts, faeces and urine were collected and recorded daily for an additional 4 d. Concentrated sulfuric acid (20 mL) was added to the urine bucket to ensure that ammonia was not lost. Urine and faeces were kept at 4 °C until the end of the study. Samples were pooled, and 10% of faeces were taken for each rabbit and then kept at −20 °C for later analyses. Feed and faecal samples were dried at 55 °C for 36 h using a forced-air oven and then ground finely for passage through a 1mm screen. Then, the samples were chemically analysed according to AOAC (1995) assays for DM (ID number 930.15), OM (ID 2

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Table 1 Feed constituents, proximate analysis of the experimental diets. Ingredients

g/kg

Sunflower meal (36%) Yellow corn Barley Wheat bran Alfalfa hay Straw Lime stone Minerals Di-calcium phosphate Salt Molasses L-Lysine-HCl (99%)1,2 DL-Methionine (99%) L-Threonine (98.5%) L-Tryptophan (98.5%) Vitamin D32 Feed analysis of basal diet Crude protein (g/kg) DM (g/kg) OM (g/kg) Crude fibre (g/kg) Ether extract DE (Kcal/kg)* Lysine (g/kg) Total methionine (g/kg) Total threonine (g/kg) Total tryptophan (g/kg) Calcium (g/kg) Phosphorus (g/kg)

90 160 150 320 130 120 6 2 4.43 4 10 0 or 0.5 or 1.0 1.84 1.32 0.41 0, or 500 IU or 1000 IU 160 981 877 141 30.8 2460 5.5 4.0 6.4 2.0 10 5

1

L-Lysine HCl 99% (white to pale yellow crystals), AJINOMOTO EUROLYSINE Co. S.A.S., Paris, France, import Multi Vita Animal Nutrition Company, 6th of October City, West Cairo, Egypt. L-Lysine HCl was added to provide 0.0, 0.5, or 1.0 g lysine per kg diet. 1,2 Lysine HCl and vitamin D3 were supplemented as feed additives and not as basic components of the experimental diet. * estimated.

number 942.05), and CP (as 6.25 × N; ID number 954.01).

2.2.2. Blood metabolites On the final day of the study, blood samples (5 mL) were extracted from rabbits before feeding from the marginal ear vein. Blood samples were centrifuged immediately at 2000 × g for 20 min, and plasma samples were harvested and stored at −20 °C for later analysis. Plasma concentrations of total protein (TP), albumin (Alb), glucose (G), urea and cholesterol (Cho) were analysed using commercial enzymatic colorimetric kits (Diamond Diagnostics, Egypt). Plasma triglyceride concentrations were determined using a commercial kit (Bio Systems S.A. Costa Brava 30, Barcelona, Spain). Globulin concentrations were calculated by determining the difference between TP and Alb. Blood urea levels were measured according to the method of Patton and Crouch (1977). Plasma calcium and phosphorus levels were determined calorimetrically using commercial kits (Bio-Merieux, France, and Stanbio, USA). Plasma rabbit growth hormones (GHs) were measured by radioimmunoassay (RIA) using the method described by the assay manufacturer. The intra- and inter-assay coefficients of variation (CVs) were 6% and 10%, respectively.

2.3. Statistical analysis Data from Exp. 1 were analysed using the mixed procedure of SAS System 9.0 for Windows (SAS Inst. Inc., Cary, NC). Fixed effects in the model included VD3 (0.0, 500 and 100 IU/kg DM diet), lysine (0.0, 0.5 and 1.0 g/kg DM diet), VD3 × lysine interaction, and time (weeks). The cage (each including 3 rabbits) was included as a random effect. Linear and quadratic effects of VD3 and lysine inclusions and their interactions were evaluated using orthogonal polynomial contrasts. Treatment means were calculated using the LSMEANS option. The digestibility and nitrogen balance data from Exp. 2 were analysed using the mixed procedure of SAS (SAS INST., INC., Cary, NC, SAS, 2004) with treatment included as a fixed effect and rabbits as a random effect. The linear and quadratic effects of VD3 and lysine and their interactions were evaluated using orthogonal polynomial contrasts. Significance was considered at p ≤ 0.05, and tendencies were considered at 0.05 < P ≤0.01. 3

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Table 2 Effect of vitamin D3 and lysine supplementation in the diets of growing rabbits on final body weight (g), average daily gain (g/d), feed intake (g DM/d) and feed conversion ratio (g DMI/g gain). Diets

Initial BW

Final BW

ADG1

Feed intake2

FCR3

Control (0) VD3 500 1000 Lys 0.5 1.0 VD3+Lys 500 + 0.5 500 + 1.0 1000 + 0.5 1000 + 1.0 SEM4 Probability (p-values) VD3 effect Linear Quadratic Lys effect Linear Quadratic VD3 × Lys Means for main effects VD3 0 500 1000 Lys 0 0.5 1.0

495.5

1595.4

31.43

116

4.28

501.9 484.3

1725.4 1667

34.96 34.59

114.6 116.9

4.03 3.68

508.6 518.8

1667.7 1834.9

33.4 37.6

115.6 115.4

3.87 3.49

467.1 484.3 519.8 477.2 –

1705 1767 1738.2 1809 32.4

35.37 36.7 34.8 38.1 1.33

116.8 117.3 115.9 116.3 0.83

3.58 3.51 3.57 3.13 0.21

– – – – – – –

0.377 0.195 0.598 < 0.0001 0.106 < 0.0001 0.064

0.262 0.138 0.477 0.003 0.437 0.001 0.574

0.577 0.324 0.711 0.793 0.702 0.571 0.190

0.060 0.019 0.823 0.004 0.068 0.003 0.834

– – –

1702.6 1732.6 1738.1

34.14 35.66 35.82

115.7 116.2 116.3

3.88 3.71 3.46

– – –

1662.6b 1707.0b 1803.7a

33.66b 34.53b 37.43a

115.8 116.1 116.3

4.00a 3.67b 3.38b

4 To obtain the SEM for the control, values were multiplied by 1.1. 1 Interaction (VD3 × week) for ADG at P = 0.0019 (weeks 2 and 4). 2 Interaction for DMI (VD3 × week) at P = 0.003 (week 3). 3 Interaction for FCR (VD3 × week) at P = 0.023 (weeks 1 and 4). 3 Interaction for FCR (lysine × week) at P = 0.012 (week 1). a, b Means within columns with different superscripts differ significantly.

3. Results 3.1. Experiment 1 (Growth trial) 3.1.1. Effect of VD3 Data for the effect of VD3 on body weight (BW; g), average daily gain (ADG; g/d), feed intake (g/d), and feed conversion ratio (FCR; g DMI/g daily gain) of the growing rabbits are presented in Table 2. Supplementation of VD3 at 0.0, 500 and 1000 IU/kg DM diet to growing rabbits did not affect their ADG (P = 0.262); however, an interaction (P < 0.01) was detected between VD3 × time (week). An improvement occurred only at weeks 2 and 4, as rabbits supplemented with 500 and 1000 IU VD3 gained more (P < 0.05) weight than rabbits given 0.0 IU VD3. VD3 had no effect on daily feed intake (P = 0.577); however, a VD3 × time (week) interaction was detected (P < 0.05), as VD3 improved feed intake at week 3 for rabbits fed diets supplemented with 500 and 1000 IU VD3 compared to those fed diets with 0.0 IU VD3. VD3 had no effect (P = 0.377) on the final body weight (FBW) of the rabbits, but it tended (P = 0.06) to improve FCR to 3.7 (500 IU VD3) and 3.5 (1000 IU VD3) compared to 3.9 for the control (0.0 IU VD3) (Table 2). 3.1.2. Effect of lysine A lysine supplement of 1.0 g improved (P < 0.01) ADG compared to the effects of 0.0 and 0.5 g lysine supplements, with a quadratic (P < 0.01) effect. No difference in effect on ADG was detected between 0.0 and 0.5 g lysine supplementation. Compared to the 0.0 g lysine supplementation, the 0.5 and 1.0 g lysine supplementations had no effect on the daily feed intake (P = 0.793) of the rabbits. The improvement in FCR was significant (P < 0.01) with lysine supplementation, and this improvement was quadratic (P < 0.01). FCR decreased from 4.00 (0.0 g level) to 3.67 (0.5 g level) and 3.38 (1.0 g level). Lysine supplemented at 1.0 g improved (P < 0.01) FBW compared to the effects of 0.0 and 0.5 g lysine supplementation, with quadratic (P < 0.01) effects. There was no VD3 × lysine interaction for FBW, ADG, feed intake, and FCR (Table 2). The highest mortality rate (38.9%, 7/18) was recorded for the 0+0 (control) group, while the lowest mortality rates were 5.6% and 0.0% in the 1000+0 and 1000+0.5 groups, respectively (Fig. 1). 4

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Fig. 1. Effect of vitamin D3 and lysine supplementation on the mortality rates of growing rabbits.

3.2. Experiment 2 (Digestibility and nitrogen balance trial) 3.2.1. Effect of VD3 VD3 tended to increase N intake (P = 0.08), as 500 and 1000 IU VD3 caused a numerically higher N intake than 0.0 IU VD3. VD3 had no effect on urine N (P = 0.51), faecal N (P = 0.50), retained N (P = 0.47) and crude protein digestibility (CPD) (P = 0.28). However, VD3 supplementation improved (P < 0.05) dry matter digestibility (DMD) and organic matter digestibility (OMD), with a quadratic effect (P < 0.05) (Table 3).

3.2.2. Effect of lysine Lysine supplementation at 0.5 or 1.0 g increased (P < 0.05) N intake compared to the effect of the 0.0 g level. Lysine supplementation at 1.0 g reduced (P < 0.01) urine nitrogen excretion compared to the effects of 0.0 and 0.5 g lysine supplementations in a Table 3 Effect of vitamin D3 and lysine supplementation in the diets of growing rabbits on nutrient digestibility and nitrogen balance. Diets Control (0) VD 500 1000 Lys 0.5 1.0 VD3+Lys 500 + 0.5 500 + 1.0 1000 + 0.5 1000 + 1.0 SEM* Probability (p-values) VD3 effect Linear Quadratic Lys effect Linear Quadratic VD3 × Lys Means for main effects VD3 0 500 1000 Lys 0 0.5 1.0

N Intake

Urine N

Faecal N

Retained N

CPD

DMD

OMD

B

3.76

0.920

1.00

1.84

73.2

60.9

63.3

3.96A 4.04A

0.980 0.900

0.94 1.04

2.04 2.08

76.2 74.2

67.6 63.1

69.7 65.1

3.96A 4.08A

0.880 0.580

1.00 0.90

2.06 2.62

74.6 78.5

62.2 67.0

65.3 69.2

4.2A 3.96A 4.04A 4.10A 0.07

0.900 0.720 0.860 0.460 0.13

0.82 0.98 0.90 1.00 0.07

2.46 2.26 2.24 2.62 0.14

80.3 75.6 77.3 75.7 1.6

69.6 66.7 68.1 67.7 1.89

71.8 68.6 70.3 69.4 1.98

0.083 0.039 0.402 0.037 0.018 0.304 0.049

0.512 0.627 0.296 0.006 0.627 0.0017 0.940

0.503 0.824 0.253 0.352 0.154 0.848 0.467

0.468 0.223 0.919 0.0003 0.024 0.0004 0.085

0.281 0.800 0.118 0.082 0.031 0.576 0.117

0.039 0.104 0.045 0.123 0.111 0.191 0.290

0.047 0.146 0.041 0.104 0.063 0.290 0.289

3.93 4.04 4.06

0.783 0.867 0.740

0.97 0.91 0.98

2.17 2.25 2.31

75.4 77.4 75.8

63.4b 67.97a 66.3ab

65.9b 70a 68.3ab

3.92b 4.07a 4.05a

0.920a 0.88a 0.587b

0.99 0.91 0.96

1.99c 2.25b 2.50a

74.5 77.4 76.6

63.8 66.6 67.2

66.0 69.1 69.1

−C Means within columns with different superscripts differ significantly. a, b Means within columns with different superscripts differ significantly.

A

5

Animal Feed Science and Technology 254 (2019) 114221

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Table 4 Effect of vitamin D and lysine supplementation in the diets of growing rabbits on plasma total protein (TP), albumin (Alb), globulin (Glob), urea, glucose (G), cholesterol (Cho), triglycerides (TG), calcium (Ca), phosphorus (P) and growth hormone (GH) concentrations. Diets Control (0) VD3 500 1000 Lys 0.5 1.0 VD + Lys 500 + 0.5 500 + 1.0 1000 + 0.5 1000 + 1.0 SEM Probability (p-values) VD3 effect Linear Quadratic Lys effect Linear Quadratic VD3 × Lys Means for main effects VD3 0 500 1000 Lys 0 0.5 1.0

TP (g/dl)

Alb (g/dl)

Glob (g/dl)

Urea (mg/dl)

G (mg/dl)

Cho (mg/dl)

TG (mg/dl)

Ca (mg/dl)

P (mg/dl) c

GH (ng/ml)

5.2

3.35

1.86

29.5

104

135.1

151.3

6.93

5.43

22.7C

5.37 5.57

3.32 3.67

2.06 1.91

25.3 24

112 99.3

133.4 146

151 142

7.46 7.37

6.5a 6.04ab

23C 21.8C

5.66 5.7

3.8 3.8

1.86 1.90

22.8 21.3

108 105.5

133.3 119.3

152 151

7.42 7.28

6.03ab 5.33c

22C 25.7B

5.57 5.7 5.48 5.88 0.16

3.69 3.51 3.57 3.82 0.12

1.88 2.19 1.90 2.06 0.16

22.5 14.3 15.3 18.8 2.4

110.8 108.5 105.8 140·8 4.9

125.5 119.8 122.3 130.5 5.03

151 156 150 155 5.2

7.73 7.8 7.85 8.28 0.16

5.58bc 6.28a 6.48a 5.25c 0.19

25.2B 22.1C 25B 27.9A 0.9

0.616 0.352 0.766 0.025 0.164 0.017 0.53

0.162 0.711 0.063 0.019 0.019 0.099 0.144

0.442 0.526 0.270 0.445 0.662 0.236 0.937

0.033 0.013 0.461 0.007 0.004 0.005 0.214

0.216 0.527 0.104 0.744 0.451 0.915 0.907

0.283 0.373 0.188 0.003 0.011 0.013 0.183

0.642 0.532 0.485 0.378 0.470 0.232 0.791

0.002 < 0.0001 0.23 0.001 0.004 0.008 0.293

0.008 0.046 0.013 0.026 0.807 0.008 0.002

0.077 0.05 0.241 0.003 0.038 0.004 0.0006

5.52 5.55 5.64

3.65 3.50 3.69

1.87 2.04 1.96

24.5a 20.7b 19.3b

105.8 110.4 103.3

129.2 126.3 132.9

152 153 149

7.21b 7.66a 7.83a

5.60b 6.12a 5.92a

23.4 23.4 24.9

5.38b 5.57ab 5.76a

3.44b 3.69a 3.71a

1.94 1.88 2.05

26.3a 20.2b 18.1c

105.1 108.2 106.3

138.2a 127b 123.2b

148 151 154

7.25b 7.67a 7.78a

5.99a 6.03a 5.62b

22.5b 24.1a 25.2a

A−C a, b

Means within columns with different superscripts differ significantly. Means within columns with different superscripts differ significantly.

quadratic (P < 0.01) fashion. Retained N was increased significantly (P < 0.01) in a linear (P < 0.05) and then quadratic (P < 0.01) fashion with lysine supplementation. Lysine tended (P = 0.082) to improve CPD. Lysine supplementation had no effect (P > 0.05) on faecal N, DMD and OMD. No VD3 × lysine interaction was observed for urine N, faecal N, retained N, CPD, DMD and OMD (Table 3). 3.3. Blood metabolites The effects of VD3 and lysine on the contents of plasma TP, Alb, globulin (Glob), urea, G, Cho, triglycerides (TGs), calcium (Ca), phosphorus (P) and GH are presented in Table 4. Supplementation with VD3 had no effect on the plasma TP (P = 0.62), Alb (P = 0.16) and Glob (P = 0.442), G (P = 0.216), Cho (P = 0.283), and TG (P = 0.642) content and tended to increase only the GH content (P = 0.08). Supplementation with VD3 at 500 and 1000 IU increased (P < 0.01) P concentrations compared to the effect of 0.0 IU VD3. However, TP (P < 0.05), Alb (P < 0.05), Ca (P < 0.01) and GH (P < 0.01) were significantly increased upon lysine supplementation compared to the 0.0 g level. The effect of lysine supplementation was quadratic for TP (P < 0.02), linear for Alb (P < 0.02), linear (P < 0.01) and quadratic (P < 0.01) for Ca, quadratic (P < 0.01) for P and linear (P < 0.05) and quadratic (P < 0.01) for GHs. Lysine supplementation at the 1.0 g level decreased P (P < 0.05). Additionally, VD3 (P < 0.05) and lysine (P < 0.01) reduced plasma urea concentrations. The effect of VD3 on plasma urea was quadratic (P < 0.05), but the effect of lysine on plasma urea was linear and quadratic (P < 0.01). However, lysine reduced (P < 0.1) plasma Cho in a linear and quadratic fashion (P < 0.05), but VD3 did not affect plasma Cho concentrations. There was no VD3 × lysine interaction for plasma TP, Alb, Glob, urea, G, Cho, TG, Ca and GH concentrations. 4. Discussion 4.1. Experimental design The basal diet was formulated to be deficient in the amino acid lysine. To ensure that lysine was the only limiting amino acid in the diet, other amino acids were added to the diet to confirm that any change in body weight gain or retained N was due to lysine supplementation (Batista et al., 2016). The basal diet contained 5.5 g lysine/kg DM diet, and the highest lysine supplementation was 1.0 g to provide a final lysine content of 6.5 g/kg DM diet. Based on previous research, the optimal requirement of lysine to produce 6

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the highest nitrogen retention of growing rabbits is 7.0–7.5 g/kg DM diet (De Blas and Wiseman, 1998; Lebas, 2004). The reason for providing growing rabbits a lower lysine supplementation than their requirement in this study was to ensure that the response was within the linear response for body weight gain or nitrogen retention (Hussein et al., 2016). A lysine supplement that exceeded the normal lysine requirement would have caused the underestimation of lysine utilization (Hussein et al., 2016). However, in our study, a linear response for ADG was not observed, and instead, a quadratic response was recorded. Although a numerical increase in ADG was obtained at a lysine supplementation level of 0.5 g/kg DM, significant linear and quadratic responses for nitrogen retention with increasing doses of lysine supplementation were recorded. The nonlinear response in ADG (0 and 0.5 g/kg DM lysine) compared to the linear and quadratic effect of retained N in the current study could be due to 2 reasons: 1) Rabbits in the growth study (Exp. 1) were fed in groups and thus their feeding habits were different from the individually fed rabbits in the nitrogen balance trial (Exp. 2). Colina et al. (2003) reported that piglets fed in groups consumed less food than individually fed piglets. 2) In the nitrogen balance trial, only male rabbits were used, and it is well known that males usually need more amino acids for protein deposition, and their efficiency of nitrogen utilization is higher than that of females (Colina et al., 2003); this is in contrast to the growth study in the current study, in which both male and female rabbits were used. 4.2. Growth and nitrogen balance study The effect of the interaction between VD3 and lysine was not observed in the growth parameter and nitrogen balance trials, indicating that both VD3 and lysine were independent factors. However, a tendency for the VD3 × lysine interaction was observed only for FBW (P = 0.06) and retained N (P = 0.09), with no clear explanation for this interaction. In contrast to the current findings, Shimura et al. (1975) found that growth performance was improved in rats fed lysine plus VD3 compared with rats fed only lysine without VD3. The response to VD3 and lysine supplementation in rats could be due to the highly lysine-deficient diet provided or that rats responded differently than rabbits to lysine supplementation. To our knowledge, no research on the effect of VD3 on lysine utilization in growing rabbits is available to date. Although the overall effect of VD3 on ADG was not significant, a strong interaction was detected between VD3 and time (week, P < 0.01), which indicates that the effect of VD3 on growth is age dependent, as VD3 increased ADG only in weeks 2 and 4. Additionally, compared to the control diet (0 + 0), VD3 supplementation to diets lacking lysine (500 + 0, P = 0.08; and 1000+0, P = 0.12) numerically improved ADG. The tendency for improvement in FCR because of VD3 supplementation was a result of such a numerical improvement in ADG. Studies have shown that VD3 stimulates muscle cell proliferation and growth (Buitrago et al., 2001, 2003) through the activation of MPA pathways in rats (Wu et al., 2000). In the nitrogen balance trial, the tendency (P = 0.08) for VD3 to increase N intake at 500 and 1000 IU concentrations may be due to its ability to increase OMD. Many studies have shown that VD3 is anti-inflammatory (Lappe et al., 2007) and responsible for the integrity of intestinal epithelium (Kong et al., 2008). Therefore, VD3 could improve digestibility by maintaining a healthy gut. The observed mortality rate was higher in the 0 + 0 group (38.8%, 7/ 21) than in the other groups, indicating that the basal diet was deficient in both VD3 and lysine. Lysine supplementation increased retained N in linear and quadratic fashions, which indicates that the experimental diet was deficient in lysine. Therefore, supplemented lysine was used as retained N and thereby reduced the catabolism of other amino acids, as indicated by the reduction in urine nitrogen excretion, which was observed in the current study. Lysine supplementation did not affect faecal N among treatments but increased N intake, and although it was significant (P < 0.05), the increase in N intake was neither substantial (from 3.9 to 4.1 g/d) nor expected. The increase in N intake could be a result of the tendency (P = 0.12) of lysine to improve OMD and hence CPD (P < 0.05), which resulted in increased feed intake (P < 0.05; data are not shown) and hence N intake. Although no effect of lysine on feed intake was observed during the growth study (P = 0.331), the differences between the growth trial and nitrogen balance trial were previously discussed. However, VD3 did not improve retained N in the diet supplemented with lysine, indicating that VD3 did not improve lysine utilization. 4.3. Blood parameters Blood metabolites are considered indicators of changes in dietary supplementation, including modulation of enzymes or hormones responsible for N or lipid metabolism (Clarke and Abraham, 1992; McNeel and Mersmann, 2000). The increase in TP and Alb, as a response to lysine supplementation, indicates that lysine and other amino acids were used for protein deposition, and these results are in agreement with those of Yang et al. (2008) and Kamalakar et al. (2009), who reported that a lysine-deficient diet offered to piglets reduced plasma TP. In contrast, VD3 had no effect on TP and Alb. Additionally, the reduction in the urea concentration with VD3 and lysine supplementation reflects the use of amino acids for protein deposition, and fewer amino acids were catabolized (Hussein et al., 2016; Batista et al., 2016). VD3 has been reported for its importance in muscle strength and mass (Endo et al., 2003), and the mode of action by which VD3 affects protein metabolism is unclear (Agarwal et al., 2011). Many studies have reported a correlation between VD3 and GH (Ciresi and Giordano, 2017). In our study, VD3 tended (P = 0.08) to increase GH, while lysine significantly (P < 0.01) increased the GH concentration. Cree and Schalch (1985) reported that the GH content was lower in growing rats fed a lysine-deficient diet than in rats fed a control diet. In the present study, the interaction between VD3 and lysine supplementation on plasma GH (P < 0.01) concentration is not understood. Similar to our result, Hussein et al. (2016) found no effect of an abomasal infusion of lysine on blood glucose in growing steers. Similar results for blood glucose were obtained by Sai et al. (2014) who fed rumen-protected lysine and methionine to calves. In our study, lysine supplementation reduced the plasma Cho concentration. Lysine and methionine are known as precursors for carnitine production, which is a very important ammonium compound for fat oxidation and hence lowers the plasma Cho concentration 7

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(Steiber et al., 2004). Both VD3 and lysine increased (P < 0.01) the plasma calcium concentration linearly (P < 0.01), with no interaction detected between VD3 and lysine. VD3 is well known for increasing calcium absorption from the intestine (Christakos et al., 2011). Another mode of action of VD3 for reducing mortality and the numerical improvement of ADG when added to a diet without supplementation in the current study could be related to increased intestinal lysine availability due to increased calcium absorption. It has been shown that calcium increases lysine absorption through the intestinal epithelium in humans (Civitelli et al., 1992), while conversely, lysine can also increase calcium absorption (Bihuniak et al., 2014). In contrast to the effect of treatment on plasma calcium, VD3 increased plasma phosphorus concentrations in a quadratic response and with a strong interaction between VD3 and lysine (P < 0.01). This interaction is complex and difficult to explain. VD3 is well known for its effect on phosphorus absorption in the duodenum and jejunum (Marks et al., 2006). However, lysine supplementation led to a reduction in the P concentration only at a high level (1.0 g lysine). The reduced P concentrations in response to lysine supplementation are neither expected nor do they have a biological value for the purpose of the present study. More research is needed to understand the relationship between lysine and VD3 on plasma P concentration. 5. Conclusions Although VD3 reduced the mortality rate and blood urea content and tended to increase the GH content in rabbits, it did not improve lysine utilization in a lysine-supplemented diet. On the other hand, lysine supplementation improved ADG, retained N and GH levels regardless of whether VD3 was supplemented, suggesting that lysine supplementation can compensate for the deficiency in VD3 without adverse effects on rabbit performance. More research is needed to understand the interaction between VD3 and lysine in the effect of VD3 on lysine utilization. Acknowledgements The authors acknowledge the Department of Animal and Fish Production and the Department of Poultry Production, Faculty of Agriculture, Alexandria University for providing technical and financial support for the research and use of research facilities. References Agarwal, R., Hynson, J.E., Hecht, T.J., Light, R.P., Sinha, A.D., 2011. Short-term vitamin D receptor activation increases serum creatinine due to increased production with no effect on the glomerular filtration rate. Kidney Int. 80 (10), 1073–1079. AOAC, 1995. Official Methods of Analysis, 18th ed. Association of Analytical Chemists, Gaithersburg, MD, USA. Batista, E.D., Hussein, A.H., Detmann, E., Miesner, M.D., Titgemeyer, E.C., 2016. Efficiency of lysine utilization by growing steers. J. Anim. Sci. 94 (2), 648–655. https://doi.org/10.2527/jas.2015-9716. Bihuniak, J.D., Sullivan, R.R., Simpson, C.A., Caseria, D.M., Huedo-Medina, T.B., O’Brien, K.O., Kerstetter, J.E., Insogna, K.L., 2014. Supplementing a low-protein diet with dibasic amino acids increases urinary calcium excretion in young women. J. Nutr. 144 (3), 282–288. https://doi.org/10.3945/jn.113.185009. Buitrago, C., Vazquez, G., De Boland, A.R., Boland, R., 2001. The vitamin D receptor mediates rapid changes in muscle protein tyrosine phosphorylation induced by 1,25(OH)(2)D(3). Biochem. Biophys. Res. Commun. 289 (5), 1150–1156. Buitrago, C.G., Pardo, V.G., de Boland, A.R., Boland, R., 2003. Activation of RAF-1 through Ras and protein kinase Calpha mediates 1alpha,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells. J. Biol. Chem. 278 (4), 2199–2205. Christakos, S., Dhawan, P., Porta, A., Mady, L.J., Seth, T., 2011. Review, Vitamin D and intestinal calcium absorption. Mol. Cell. Endocrinol. 347 (1–2), 25–29. https:// doi.org/10.1016/j.mce.2011.05.038. Ciresi, A., Giordano, C., 2017. Vitamin D across growth hormone (GH) disorders: from GH deficiency to GH excess. Growth Horm. IGF Res. 33, 35–42. https://doi.org/ 10.1016/j.ghir.2017.02.002. Civitelli, R., Villareal, D.T., Agnusdei, D., Nardi, P., Avioli, L.V., Gennari, C., 1992. Dietary L-lysine and calcium metabolism in humans. Nutr. 8 (6), 400–405. Clarke, S., Abraham, S., 1992. Gene expression: nutrient control of pre-and posttranscriptional events. FASEB J. 6, 3146–3152. Colina, J., Miller, P.S., Lewis, A.J., Fischer, R., 2003. Influence of Crystalline or Protein-Bound Lysine on Lysine Utilization for Growth in Pigs. Digital Commons at University of Nebraska - Lincoln Nebraska. https://digitalcommons.unl.edu/coopext_swine/56/. Cree, T.C., Schalch, D.S., 1985. Protein utilization in growth: effect of lysine deficiency on serum growth hormone, somatomedins, insulin, total thyroxine (T4) and triiodothyronine, free T4 index, and total corticosterone. Endocrinology 117 (2), 667–673. https://doi.org/10.1210/endo-117-2-667. De Blas, C., Wiseman, J., 1998. The Nutrition of the Rabbit, 2nd ed. CABI Publishing, Cambridge 344p. Endo, I., Inoue, D., Mitsui, T., Umaki, Y., Akaike, M., Yoshizawa, T., Kato, S., Matsumoto, T., 2003. Deletion of vitamin D receptor gene in mice results in abnormal skeletal muscle development with deregulated expression of myoregulatory transcription factor. Endocrinology 144, 5138–5144. Hussein, A.H., Batista, E.D., Miesner, M.D., Titgemeyer, E.C., 2016. Effect of ruminal ammonia supply on lysine utilization by growing steers. J. Anim. Sci. 94, 656–664. Janssen, H.C., Samson, M.M., Verhaar, H.J., 2010. Muscle strength and mobility in vitamin D-insufficient female geriatric patients: a randomized controlled trial on vitamin D and calcium supplementation. Aging Clin. Exp. Res. 22, 78–84. 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. Kanekar, A., Sharma, M., Joshi, V.R., 2010. Vitamin d deficiency-a clinical spectrum: is there a symptomatic nonosteomalacic state? Int. J. Endocrinol. 521457. https://doi.org/10.1155/2010/521457. Kong, J., Zhang, Z., Musch, M., Ning, G., Sun, J., Hart, J., Bissonnette, M., Li, Y.C., 2008. Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier. Am. J. Physiol. Gastrointest. Liver Physiol. 294, G208–G216. https://doi.org/10.1152/ajpgi.00398.2007. Lappe, J.M., Travers-Gustafson, D., Davies, K.M., Recker, R.R., Heaney, R.P., 2007. Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial. Am. J. Clin. Nutr. 85, 1586–1591. Lebas, F., 2004. Reflections on Rabbit Nutrition With a Special Emphasis on Feed Ingredients Utilization. 8th World Rabbit Congress Puebla Mexico, WRSA Ed., pp. 686–736. Marks, J., Srai, S.K., Biber, J., Murer, H., Unwin, R.J., Debnam, E.S., 2006. Intestinal phosphate absorption and the effect of vitamin D: a comparison of rats with mice. Exp. Physiol. 91 (3), 531–537.

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E.A. Elwakeel, et al.

Mason, C., Tapsoba, J.D., Duggan, C., Imayama, I., Wang, C.Y., Korde, L., McTiernan, A., 2016. Effects of vitamin D3 supplementation on lean mass, muscle strength and bone mineral density during weight loss: a double-blind randomized controlled trial. J. Am. Geriatr. Soc. 4 (4), 769–778. McNeel, R., Mersmann, H., 2000. Nutritional deprivation reduces the transcripts for transcription factors and adipocyte-characteristic proteins in porcine adipocytes1. J. Nutr. Biochem. 11, 139–146. Monteiro-Motta Scapinello, C., Oliveira, A.F.G., Figueira, J.L., Catelan, F., Sato, J., Stanquevis, C.E., 2013. Levels of lysine and methionine+cystine for growing New Zealand white rabbits Ana Carolina R. Bras. Zootec. 42 (12), 862–868. National Research Council, 2001. Nutrient Requirements of Dairy Cattle. Natl. Acad. Press, Washington DC. Pfeifer, M., Begerow, B., Minne, H., Suppan, K., Fahrleitner-Pammer, A., Dobnig, H., 2009. Effects of a long-term vitamin D and calcium supplementation on falls and parameters of muscle function in community-dwelling older individuals. Osteoporos. Int. 20, 315–322. Sai, S., Thakur, S.S., Kewalrmani, N., 2014. Effect of supplementation of rumen protected methionine plus lysine on growth performance, nutrient utilization and blood metabolites in calves. Indian J. Anim. Nutr. 31 (1), 1–7. SAS Institute, 2004. SAS/STAT User’s Guide. Statistics, Version 9.0. SAS Institute, Cary, NC. Shimura, F., Moriuchi, S., Hosoya, N., 1975. The effect of lysine and vitamin d on the intestinal absorption of calcium. J-STAGE. 28 (3), 159–163. https://doi.org/10. 4327/jsnfs1949.28.159. Steiber, A., Kerner, J., Hoppel, C.L., 2004. Carnitine: a nutritional, biosynthetic, and functional perspective. Mol. Aspects Med. 25, 455–473. Sugai, M., Matsuda, I., 1968. The effect of vitamin D on transport of L-histidine by intestine. Chin. J. Biochem. Biophys. 170, 474–475. Titgemeyer, E.C., 2003. Amino acid utilization by growing and finishing ruminants. In: D’Mello, J.P.F. (Ed.), Amino Acids in Animal Nutrition, 2nd ed. CAB Int., Wallingford, UK, pp. 329–346. Wu, Z., Woodring, P.J., Bhakta, K.S., Tamura, K., Wen, F., Feramisco, J.R., Karin, M., Wang, J.Y., Puri, P.L., 2000. p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps. Mol. Cell. Biol. 20 (11), 3951–3964. Yang, Y., Jin, Z., Yoon, S., Choi, J., Shinde, P., Piao, X., 2008. Lysine restriction during grower phase on growth performance, blood metabolites, carcass traits and pork quality in grower finisher pigs. Acta. Agric. Scand. Section A-Anim. Sci. 58, 14–22.

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