Insulin secretion, body composition and pig performanceare altered by feeding pattern

Ronald E. NewmanA, Jeffery A. DowningA, Peter C. ThomsonA, Cherie L. CollinsB,David J. HenmanB and Stuart J. WilkinsonA,C

AFaculty of Veterinary Science, University of Sydney, Camden, NSW 2570, Australia.BRivalea Australia Pty Ltd, Corowa, NSW 2646, Australia.CCorresponding author. Email: stuart.wilkinson@sydney.edu.au

Abstract. Three studies investigated the effect of feeding strategy on production performance and endocrine status ofgrowing pigs. For Experiment 1, 20 entire male pigs (70.0 � 4.6 kg) were allocated randomly to individual pens in one offour climate-controlled rooms. Pigs were fed for 23 days either ad libitum or entrained to feed bi-phasically for two 90-minperiods. For Experiment 2, 20 entire male pigs (41.2� 3.5 kg) were housed as per Experiment 1. Pigs were fed for 49 dayseither ad libitum or fed bi-phasically for two 60-min periods. For Experiment 3, 100 female pigs (66.1 � 3.5 kg) wererandomly allocated to individual pens within a commercial piggery and fed for 42 days either ad libitum or bi-phasically fortwo 60-min periods. Ear vein catheters were inserted into 10 pigs from each group and hourly blood samples were collectedfor 24 h in Experiments 1 and 2 and for 11 h in Experiment 3. Plasma insulin, non-esterified fatty acid and glucoseconcentrations were determined in Experiments 1 and 2, and glucose and insulin concentrations in Experiment 3. Feedintake and performance were recorded in all experiments and carcass composition was assessed by computed tomographyfor Experiment 2. There were no differences in final liveweight between the two treatment groups for all experiments. Pigsfed for two 90-min periods (Experiment 1) showed no difference in feed intake when compared with feeding ad libitum.Pigs in Experiment 2 fed for two 60-min intervals consumed 2.49 kg/pig.day compared with those fed ad libitum thatconsumed 2.68 kg/day (P = 0.057). In Experiment 3, pigs fed twice daily consumed 2.82 kg/pig.day compared with2.91 kg/pig.day in ad libitum-fed pigs (P = 0.051). Bi-phasic fed pigs in Experiment 2 had improved (P < 0.05) feedconversion efficiency compared with pigs fed ad libitum. For all experiments, there was no difference in plasma glucoseconcentrations between the two treatments. In all three experiments, the circulating insulin concentrations for pigs fedad libitum remained at a constant level throughout the sampling period. However, plasma insulin concentrations for thebi-phasic fed pigs significantly increased ~1 h after both feeding periods during all three experiments. Insulin secretion ofpigs fed for two 90-min periods differed from that of pigs fed for two 60-min periods. Plasma insulin concentration increasedfive-fold following feeding for 60 min, compared with that in pigs fed for 90 min, which increased two-fold. Bi-phasic-fedpigs fromExperiment 2 had reduced (P < 0.05) total carcass fat and significantly increasedmuscle when comparedwith pigsfed ad libitum. The data showed that feeding pigs at two succinct periods aligned insulin secretion to the time of feeding.Pigs fed for 60 min, unlike those fed for 90-min intervals, had reduced feed intake in comparison to those fed ad libitum.This may suggest that the duration of the feeding bout is important for this response and this may in turn influence bothenergy balance and the way energy is partitioned.

Additional keywords: carcass composition, feed efficiency, feed intake, pigs.

Received 27 March 2013, accepted 8 July 2013, published online 28 August 2013

Introduction

Ad libitum feeding is the most common feeding pattern used incommercial pig production and a major management strategyused to optimise both pig performance and efficiency of feedutilisation. A principal objective for most pig producers is toincrease lean gain while limiting fat tissue deposition (Quiniouet al. 1999). Feeding pattern has been shown to have effects notonly on feed efficiency (Cohn et al. 1962; Scrimgeour et al. 2008)but also on carcass composition (Leveille 1970; Partridge et al.1985; Persson et al. 2008).

For optimal protein deposition in growing animals, a balancedsupply of amino acids and energy is required (van den Borneet al. 2007).However, it is themetabolic hormones such as insulinthat regulate metabolism, which has an important role in themetabolism of glucose, fats and proteins (Stockhorst et al. 2004).Metabolic hormone secretion patterns and concentrations,particularly growth hormone and insulin, are altered withdiffering feeding strategies, and this appears to have importantconsequences for performance (Steffens 1967; Scrimgeouret al. 2008). A recent study in pigs fed ad libitum identified

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that insulin secretion was not aligned to either feeding behaviouror to plasma glucose concentrations (Scrimgeour et al. 2008).Insulin concentrations remained relatively constant throughoutthe 24-h sampling period. However, when pigs were entrainedto feed either twice daily or once daily, the pattern of insulinsecretion responded to the period of feeding, with significantincreases occurring ~1 h after the onset of feed ingestion(Scrimgeour et al. 2008). In the same study, feed efficiency(FCR) was also improved in pigs fed twice daily, whencompared with those fed ad libitum. However, the fact thatthe treatments were applied sequentially using the sameanimals may have confounded the results because of the timefactor.

As insulin is an important metabolic hormone involvedin promoting a range of growth processes including feedingbehaviour (Benoit et al. 2004), protein deposition (Fuller et al.1977) and metabolism (Koopmans et al. 2005), the resultsobtained by Scrimgeour and colleagues (2008) suggest thattwice daily feeding may promote more efficient utilisation ofenergy substrate for metabolism and growth in pigs. Therefore,the objective of the present study was to investigate theinfluence of feeding regimen on energy utilisation in thegrowing pig. To meet this objective, a series of experimentswas conducted, first, to determine the optimal time of eachfeeding bout in respect to improved performance. Second, toinvestigate the effect of feeding pattern in a larger cohort ofanimals housed in a research facility within a commercialpiggery. Together, these studies tested the hypothesis thatfeeding pigs at two succinct intervals aligns insulin secretionmore closely to the time of feeding, resulting in improvedproductivity.

Materials and methods

Animals and housingAll procedures involving animals for Experiments 1 and 2 wereapproved by the Animal Ethics Committee of the ElizabethMacarthur Agricultural Institute, Menangle, New South Wales,Australia. The animal procedures for Experiment 3 wereapproved by the Animal Care and Ethics Committee ofRivalea Australia, Corowa, New South Wales, Australia. BothAnimal Ethics Committees complied with the Australian codefor use of animals for scientific purposes (National Health andMedical Research Council 2004).

Experiments 1 and 2For Experiment 1, 20 entire male pigs (Large white ·

Landrace) with a liveweight of 70.0 � 4.6 kg (mean � s.e.m.)were sourced from the University of Sydney piggery. ForExperiment 2, 20 entire male pigs (Large white · Landrace)with a liveweight of 41.2 � 3.5 kg were sourced from the samepiggery. On arrival at the experimental facility, pigs for bothexperiments were weighed and allocated to individual penslocated in one of four separate climate-controlled rooms, withtwo rooms sharing one of two common air spaces. Each air spacewas provided with 100% fresh air, with ammonia concentrationsbeing kept below 2 · 10–3 g/L. All four rooms were maintainedat 23 � 1�C and pigs were exposed to a 12 : 12 light : darkphotoperiod, with lights being turned on at 0600 hours.

Experiment 3To test the hypothesis in female grower/finisher pigs, 100

female pigs (Large white · Landrace, PrimeGro Genetics,Corowa, NSW, Australia) with a starting weight of 58.8 �0.5 kg (mean � s.e.m.) were randomly allocated to singlepens. Pens were partially slatted in an uninsulated building,with side shutters controlled by temperature. Pigs wereacclimatised to the facility and their allocated feeding pattern,ad libitum (n = 50) or bi-phasically (n = 50), for a period of7 days before the start of the test period and fed a commercialgrower diet during this time (Table 2).

Experimental proceduresTreatments and measuresExperiments 1 and 2. Pigs in Experiment 1 were fed a

commercial grower/finisher diet (Table 1), while pigs used inExperiment 2 were fed a commercial grower diet (Table 1).Each pig was fed either ad libitum with access to feed 24 hper day or a similar amount of feed was offered with unrestrictedaccess for two discrete periods, one in the morning and one in

Table1. Dietary ingredients (g/kg)andnutrient specifications, onas-fedbasis (Experiment 1 and Experiment 2)CP, crude protein; DE, digestible energy

Parameter Experiment 1 Experiment 2

Ingredients (g/kg)Wheat 645Wheat (12.5% CP) 668Barley 125Canola meal full fat 50Meat meal(55% CP) 84Soybean meal (48% CP) 83Blood meal (90% CP) 10Dicalcium phosphate 17Limestone 3.0 11Millrun 75Millrun (15% CP) 163Salt 3.5 2.5Pig breeder premixA 2.0 2.5Choline chloride 0.4 0.4Lysine-HCl 2.0

Nutrient specification (calculated; g/kg) (SCA 1987)DE (MJ/kg) 188 134Protein 210 169Fat 41 25Fibre 40 40Available P 4.0 5.0Available lysine 10 10

AIn Experiment 1, pig breeder premix provided the following quantities ofvitamin and minerals per kg of complete diet: Vitamin A, 5600 IU; VitaminD3, 1200 IU; Vitamin E, 36 IU; Vitamin K, 1.6 mg; Vitamin B12, 0.01 mg;riboflavin, 4.8 mg; pantothenic acid, 8.8 mg; biotin, 0.08 mg; niacin, 12 mg;folic acid, 0.8mg;pyridoxine, 2.4mg; iron,40mg; zinc,128mg;manganese,32 mg; copper, 128 mg; cobalt, 0.8 mg; iodine, 1.2 mg and selenium, 0.16mg. In Experiment 2, pig breeder premix provided the following quantitiesper kg of complete air-dry diet: Vitamin A, 2500 IU, Vitamin D3, 1000 IU;Vitamin E, 30mg; Ca-D-pantothenate 5 mg; riboflavin, 2 mg; Vitamin B12,0.005mg; selenium, 0.2mg; copper, 10mg; iron, 60mg;manganese, 25mg;zinc, 50 mg, iodine, 0.2 mg and Endox, 20 mg.

B Animal Production Science R. E. Newman et al.

the afternoon. The duration of both morning and afternoonfeeding periods varied between experiments. Experimentalconditions were similar to those described by Scrimgeouret al. (2008), but to determine whether the productionresponses could be improved, the period of feeding wasincreased by 30 min for Experiment 1, to 90 min twice daily(0900 hours to 1030 hours and 1600 hours to 1730 hours). Pigswere fed for two 60-min periods in Experiment 2. For bothexperiments, each treatment was equally represented in each ofthe two air spaces. Feed was offered to maintain ~2 kg in eachtrough and residues were recorded daily or after each feedingperiod. Water was provided using nipple drinkers. Pigs weremaintained on these two feeding patterns for 23 days forExperiment 1 and 49 days for Experiment 2. Estimates ofdaily feed intake for the ad libitum-fed pigs were obtained bysubtracting the daily residue weight from the weight of the feedoffered the previous day. Similarly, feed residues collected at1030 hours (Experiment 1) or 1000 hours (Experiment 2) and1730 hours (Experiment 1) or 1700 h (Experiment 2) weresubtracted from feed offered at 0900 hours and 1600 hours,respectively, during twice daily feeding. Bodyweights of pigswere recorded weekly. On Day 21 for Experiment 1, a catheterwas placed into the external jugular vein of each pig via an earvein (Anderson andElsley1969) andonDay45 forExperiment 2.On Day 22 (Experiment 1) and Day 46 (Experiment 2), bloodsamples (3 mL) were collected in tubes containing K3 EDTA,from each pig at hourly intervals for 24 h. Blood samplingcommenced at 1100 hours following food withdrawal from thebi-phasic-fed pigs. The blood was centrifuged at 1048g for20 min at 4�C and plasma was stored immediately aftersampling at �20�C until assayed. All pigs from Experiment 2were transported to a commercial abattoir onDay 50, slaughteredand P2 was determined by a commercial operator. The dressweight for each pig was recorded and the carcasses splitlongitudinally through the spine and kept refrigerated at 4�Cuntil analysed by computed tomography (CT) scanning.

Experiment 3. Pigswere randomly allocated to anad libitumor bi-phasic feeding pattern. The bi-phasic feeding patternconsisted of two 60-min feeding periods per day, one in themorning and the other in the afternoon, 0800 hours to 0900 hoursand 1400 hours to 1500 hours, respectively. To prevent accessto feed between feeding sessions, boards were secured in front ofthe feeders to prevent pig access. Pigs were offered a commercialgrower diet to Day 14 of the test period, and a commercialfinisher diet thereafter to slaughter (Table 2). Feed intakes andbodyweights were measured at 2-weekly intervals for 42 days(Days 0–14, 14–28 and 28–42). On Day 43, a subsample of pigsfrom both treatment groups fed either ad libitum (n = 8) orbi-phasically (n = 8) were randomly selected and the externaljugular vein of each pig was catheterised via an ear vein(Anderson and Elsley 1969). On Day 44, blood samples(3 mL) were collected from each pig at hourly intervals for 11h, commencing at 0700 hours in tubes containing K3 EDTA.

Computed tomography (CT) scanningComputed tomography (CT) scanning was utilised in

Experiment 2 to assess changes in body composition.Carcasses were analysed for proportions of lean tissue, adiposetissue, bone and free water by using the methods of (Giles et al.

2009) and a Picker PQ 2000 spiral CT scanner (Model PQ 2000,PhilipsMedical Systems, Picker International,HighlandHeights,Ohio, USA).

Determination of hormone and metaboliteconcentrationsPlasma insulin concentrationsweredetermined induplicate by

using a commercial double-antibody radio-immunoassay, with aprimary antibody raised against porcine insulin and 125I-labelledinsulin as the tracer (kit #PI-12K; Linco Research, Saint Charles,MO, USA). Concentrations of circulating non-esterified fattyacids (NEFA)were determined by the acyl-CoA synthetase/acyl-CoA oxidase method (NEFA C-test; Wako Chemicals USA,

Table2. Dietary ingredients (g/kg)andnutrient specifications, onas-fedbasis (Experiment 3)DE, digestible energy

Parameter Grower diet Finisher diet

Ingredient (%)Wheat 710 568Canola meal (36%) 124 41Barley 100Mill mix 50 200Meat meal 45 17Blood meal 22Tallow 20 20Water 20 10Tallow enzyme 17Limestone 11 17Lysine-HCl 3.5 4Salt 2.0 2Copper proteinate 1.0 1Rumensin 100A 1 1Betaine 1Threonine 0.8 1.3Vitamin mineral premix 0.7B 0.7C

DL-methionine 0.2Porzyme 9310D 0.2Natuphos 5000E 0.1 0.1

Nutrient specification (calculated; g/kg) (SCA 1987)DE (MJ/kg) 138 138Protein 186 136Fat 36 53Fibre 40 43Available P 4.4 3.4Available lysine 9.7 7

ACoccidiostat (Elanco Animal Health, West Ryde, New South Wales,Australia).

BProvided the following quantities of vitamin and minerals per kg of air-drydiet: Vitamin A, 2500 IU, Vitamin D3, 1000 IU; Vitamin E, 30 mg; Ca-D-pantothenate, 5 mg; riboflavin, 2 mg; Vitamin B12, 0.005mg; selenium, 0.2mg; copper, 10mg; iron, 60mg; manganese, 25mg; zinc, 50mg, iodine, 0.2mg and Endox, 20 mg.

CProvided the following quantities of vitamin and minerals per kg of air-drydiet: Vitamin A, 1500 IU; Vitamin D3, 500 IU; Vitamin E, 10 mg; Ca-D-pantothenate, 2.5 mg; riboflavin, 2 mg; selenium, 0.15 mg; copper, 10 mg;iron, 60 mg; manganese, 10 mg; zinc, 50 mg; iodine, 0.2 mg and Endox, 20mg.

DXylanase (Danisco Animal Nutrition, UK).%Phytase (BASF Australia, Southbank, Victoria, Australia).

Feeding patterns and pig performance Animal Production Science C

Richmond, Virginia, USA). Plasma glucose measurements weredetermined by the glucose oxidase method (Huggett and Nixon1957) for Experiments 1 and 2. For Experiment 3, plasma glucoseconcentrations were measured using a Konelab 20XTibiochemical analyser (Thermo Electron, Clinical Diagnostics,Clinical Chemistry & Automation Systems, Ratastie, Vantaa,Finland) with a reagent kit from Thermo Trace (Melbourne,Victoria, Australia), that used the hexokinase/glucose-6-phosphate dehydrogenase method.

Statistical analysesLinear mixed models were used for all three experiments toanalyse all the traits, with logarithmic transformations usedwhere necessary (FCR). Models included fixed effects fortreatment (ad libitum v. bi-phasic feeding regimens), day offeeding, and their interaction (to test for different-shaped timeprofiles for the two feeding patterns). In addition, random termswere included for Room and Pen (nested within the Room). Allanalyseswere undertaken using theREMLprocedure ofGENSTAT

Release 11 (VSN International, Hemel Hemstead, UK). Theexperimental unit for all analyses was the individual animal.Student’s t-tests were performed to identify differences in totalfeed consumption between treatments for Experiment 3.

Results

Growth performance

Experiment 1

There was no significant difference in bodyweight betweenthe two treatment groups over the experimental period (Table 3).Pigs fed bi-phasically consumed significantly (P < 0.05) less feedfor the first 4 days of the experiment than did their ad libitum-fedcounterparts. A result attributed to pigs acclimatising to thisfeeding regimen. Pigs from the bi-phasic-fed group consumeda similar amount of feed over the experimental period as did thosefed ad libitum (3.1 � 0.17 and 3.3 � 0.17 kg/day, respectively)resulting in a non-significant difference in the efficiency of feedutilisation (Table 3).

Experiment 2

Bodyweights increased over the experimental period butthere were no significant differences in bodyweight betweenthe two treatments. The final liveweight and the dress carcassweight for the bi-phasic-fed and ad libitum-fed pigs weresimilar, with no significant difference in average daily gain

between the two treatment groups (Table 4). There was adecrease (P = 0.057) in the amount of feed consumed whenpigs were fed bi-phasically compared with pigs fed ad libitum,with the difference being ~200 g/day per pig over the 49-daytreatment period (Table 4). The efficiency of feed utilisation wasimproved with the bi-phasic feeding regime (P < 0.05). Althoughnot significant (P = 0.18), the depth of back fat at the P2 positionwas lower in the bi-phasic-fed pigs than in pigs fed ad libitum(Table 4). There was a reduction (P < 0.05) in the percentage oftotal carcass fat and an increase (P < 0.05) in the total percentageof muscle, as determined by CT analysis for the bi-phasic-fedpigs, when compared with those fed ad libitum (Table 4). Therewere no significant differences in the total percentage of bone,skin or free water (Table 4).

Experiment 3

As forExperiments 1 and2, therewas no significant differencein bodyweight for the two treatment groups (Table 5). However,similar to Experiment 2, feed intakes over the experimentalperiod for pigs fed bi-phasically were reduced (P = 0.051)

Table 3. Performance characteristics for pigs fed ad libitum or twicedaily (bi-phasic) for 23 days (Experiment 1)

Bi-phasic group entrained to two 90-min feeding periods (from 0900 hours to1030 hours and from 1600 hours to 1730 hours). ADG, average daily gain;

FCR, feed conversion ratio

Parameter Ad libitum Bi-phasic P-value

n 10 10Final liveweight (kg) 112.0 ± 1.8 110 5 ± 1.8 0.61Daily feed intake (kg/day) 3.3 ± 0.2 3.1 ± 0.2 0.51FCR (kg/kg) 2.52 ± 0.1 2.37 ± 0.1 0.34ADG (kg) 1.51 ± 0.65 1.43 ± 0.65 0.47

Table 4. Performance and carcass composition for pigs fed ad libitumor twice daily (bi-phasic) for 49 days (Experiment 2)

Bi-phasic group entrained to two 60-min feeding periods (from 0900 hoursto 1000 hours and from 1600 hours to 1700 hours). Backfat thickness wasmeasured at the P2 position (left side of the 10th rib and 6 cm away from the

spine). ADG, average daily gain; FCR, feed conversion ratio

Parameter Ad libitum Bi-phasic P-value

n 10 10Final liveweight (kg) 92.7 ± 1.1 92.5 ± 1.1 0.990Daily feed intake (kg/day) 2.68 ± 0.07 2.49 ± 0.07 0.057FCR (kg/kg) 2.63 ± 0.03 2.39 ± 0.03 0.032ADG (kg) 1.04 ± 0.3 1.05 ± 0.4 0.880Dress weight (kg) 66.0 ± 1.6 65.9 ± 1.9 0.960Backfat thickness (mm) 14.8 ± 0.82 12.2 ± 0.87 0.180Bone (%) 11.1 ± 0.40 11.2 ± 0.35 0.890Fat (%) 14.7 ± 0.84 12.3 ± 0.75 0.027Muscle (%) 63.8 ± 0.78 66.4 ± 0.70 0.015Skin (%) 2.78 0. ± 14 2.96 ± 0.12 0.360Free water (%) 7.54 0. ± 38 7.22 ± 0.34 0.530

Table 5. Performance characteristics for pigs fed ad libitum or twicedaily (bi-phasic) for 42 days (Experiment 3)

Bi-phasic group entrained to two 60-min feeding periods (from 0900 hours to1000 hours and from 1600 hours to 1700 hours). Backfat thickness wasmeasured at the P2 position (left side of the 10th rib and 6 cm away from the

spine). ADG, average daily gain; FCR, feed conversion ratio

Parameter Ad libitum Bi-phasic P-value

n 50 50Final liveweight (kg) 107.6 ± 1.1 107.0 ± 0.8 0.630Daily feed intake (kg/day) 2.91 ± 0.02 2.82 ± 0.01 <0.001Total feed consumption (kg) 122 ± 0.92 118.68 ± 0.85 0.051FCR (kg/kg) 3.05 ± 0.03 2.86 ± 0.05 0.460ADG (kg) 0.97 ± 0.01 1.00 ± 0.2 0.300Backfat thickness (mm) 9.52 ± 0.12 9.40 ± 0.13 0.650

D Animal Production Science R. E. Newman et al.

when compared with those of pigs fed ad libitum (Table 5).There was no significant improvement in feed utilisation nor asignificant reduction in back fat depth when compared withfeeding ad libitum (Table 5).

Hormones and metabolites

Experiment 1

The post-prandial insulin concentrations in pigs fed twicedaily significantly (P< 0.05) increased 1 h after each feeding bout(Fig. 1). In contrast, plasma insulin was relatively constant overthe 24-h period for animals fed ad libitum. However, circulatinginsulin concentrationswere significantly increased 1 h after lightswere turned on and although declining, thereafter remainedsignificantly elevated until sampled at 1000 hours.

Plasma glucose concentrations for animals fed ad libitumremained constant throughout the sampling period (Fig. 4).In contrast, plasma glucose concentrations were significantly(P < 0.05) reduced for animals fed bi-phasically at the onset ofthe sampling period (1100 hours) and before and during thetwo feeding periods from 0900 hours to 1030 hours and from1600 hours to 1730 hours.

There were no significant differences between the treatmentgroups for plasma NEFA concentrations (Fig. 7). Pigs from bothgroups showed increased concentrations at the commencementof the sampling period (1100 hours) and this was significant(P < 0.05) only for pigs fed ad libitum.

Experiment 2

Plasma insulin concentrations for the ad libitum-fed pigsremained relatively constant over the 24-h sampling period,with a mean value of 15.9 � 1.08 mU/mL (Fig. 2). In contrast,insulin concentrations for the bi-phasic-fed pigs were higher(P < 0.05) ~1 h after each of the two feeding periods,increasing from 7.64 � 2.52mU/mL at 1600 hours to 31.27 �5.38 mU/mL at 1700 hours and from 5.81� 0.95 mU/mL at 0900hours to 39.34 � 5.21 mU/mL at 1000 hours. Insulinconcentrations for the bi-phasic-fed pigs were lower than thosefor animals fed ad libitum from 0400 hours to the onset of thefeeding period (from 0900 hours to 1000 hours), with a meanvalue for this period of 7.21 � 0.71 mU/mL for the bi-phasic-and 16.74 � 2.44 mU/mL for the ad libitum-fed pigs. However,

this difference failed to reach significance at any single samplingtime.

There was no significant difference in plasma glucoseconcentrations for pigs fed bi-phasically or ad libitum over the24-h sampling period (Fig. 5). However, plasma glucoseconcentrations tended to rise during the period of darkness forthe bi-phasic-fed pigs.

Therewere no significant differences inNEFA concentrationsfor pigs fed bi-phasically or ad libitum over the 24-h samplingperiod (Fig. 8). However, circulating NEFA concentrationsappeared more variable for pigs fed ad libitum than those forthe bi-phasic-fed pigs between 0200 hours and 0400 hours.

Experiment 3

Over the 11-h sampling period, the plasma insulin profiles forboth the bi-phasic- and ad libitum-fed pigs showed a similarresponse in circulating insulin concentrations as did those for pigsfed either bi-phasically or ad libitum from Experiments 1 and 2(Fig. 3). The plasma insulin profile for animals fed ad libitumremained relatively constant throughout the sampling period,whereas the post-prandial insulin concentrations for pigs fedbi-phasically were significantly (P < 0.05) elevated 1 hfollowing each feeding bout. However, unlike in the previousexperiments, plasma insulin concentrations following themorning feed were significantly (P < 0.05) elevated in the bi-

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Fig. 1. Circulating concentrations of plasma insulin for pigs inExperiment 1fed at two 90-min feeding periods (&) or ad libitum (~). Standard error of themean (s.e.m.) is shown as the error bar.

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Feeding patterns and pig performance Animal Production Science E

phasic pigs, compared with the insulin response following theafternoon feed.

Although plasma glucose concentrations were reduced forthe bi-phasic-fed group, the glucose profiles for both treatmentswere similar, with little variation over the 11-h sampling period(Fig. 6).

Discussion

A significant observation from the present study has been thereduction in feed intake but similar weight gain, resulting in animproved FCR for pigs fed for two 60-min periods, one in themorning and one in the afternoon, compared with pigs fedad libitum. This finding was confirmed in the third studywhere a larger cohort of animals was used and maintainedunder less controlled environmental conditions. A secondimportant finding has been the difference in energypartitioning for pigs on the two feeding regimens observed inExperiment 2. Feeding pigs bi-phasically significantly decreasedthe proportion of fat deposited in the carcass and significantlyincreased the percentageofmuscle.A third important observationwas to show that by increasing the feeding period for the bi-phasic-fed pigs from two 60-min bouts to two 90-min periodsnegated any improvement in production as observed for

Experiments 1 and 3 with two 60-min feeding bouts. Thisfinding suggests that the duration of the feeding bout may becritical for this bi-phasic feeding pattern to have a positive effecton production outcomes.

The changes in animal performance were associated withdistinct differences in the plasma insulin secretory profile. In

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Fig. 4. Circulating concentrations of plasma glucose for pigs in Experiment1 fed at two 90-min feeding periods (&) or ad libitum (~). Standard error ofthe mean (s.e.m.) is shown as the error bar.

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Fig. 5. Circulating concentrations of plasma glucose for pigs in Experiment2 fed at two 60-min feeding periods (&) or ad libitum (~). Standard error ofthe mean (s.e.m.) is shown as the error bar.

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Fig. 6. Circulating concentrations of plasma glucose for pigs in Experiment3 fed at two 60-min feeding periods (&) or ad libitum (~). Standard error ofthe mean (s.e.m.) is shown as the error bar.

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Fig. 7. Circulating concentrations of plasma non-esterified fatty acids(NEFA) for pigs in Experiment 1 fed at two 90-min feeding periods (&)or ad libitum (~). Standard error of themean (s.e.m.) is shownas the error bar.

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Fig. 8. Circulating concentrations of plasma non-esterified fatty acids(NEFA) for pigs in Experiment 2 fed at two 60-min feeding periods (&)or ad libitum (~). Standard error of themean (s.e.m.) is shownas the error bar.

F Animal Production Science R. E. Newman et al.

all three experiments, insulin secretion for the bi-phasic fed pigsresulted in a substantial spike in plasma insulin following theonset of each feeding period. However, feeding pigs ad libitumresulted in little variation in circulating plasma insulinconcentrations over the 24-h or 11-h periods. This differencein the insulin secretory profile between the two treatment groupsmaymodify insulin action and provide a link between the feedingpattern and the way the energy is partitioned. Importantly, oneof the major differences observed in these studies has been thepattern of insulin secretion. The post-prandial rise in plasmainsulin was substantially increased for animals fed for two 60-min periodswhen comparedwith feeding for two 90-min periods.The magnitude of this increase from pre-prandial insulinconcentrations for the morning feed bout was five-fold forExperiment 2 and a 20-fold increase for the morning feed boutwas observed for Experiment 3. Interestingly, when both feedingbouts were increased from 60-min to 90-min duration(Experiment 1), the increase in plasma insulin was only two-fold when compared with the pre-prandial concentrations.

Insulin plays a significant role in the central regulation ofboth short- and long-term homeostasis by providing a negativefeedback loop for the post-prandial inhibition of food intake,with subsequent effects on feeding behaviour and the controlof bodyweight (Gerozissis 2003, 2008). Insulin and insulinreceptors are widely distributed in the brain (Gerozissis 2003),including the arcuate nucleus of the hypothalamus, a regionthat regulates energy balance via neurons that control feedingbehaviour (Baskin et al. 1985). In mice, insulin concentrationsare greater in the hypothalamus immediately before feeding, withthe concentrations further increasing if the animals remain unfed(Orosco et al. 1995). Brain insulin is thought to be exclusivelyof pancreatic origin (Banks 2004), although it has also beenproposed that the brain itself synthesises some amount of theinsulin (Gerozissis 2003; Schechter et al. 2005), with thisproduction being affected by both genetic and environmentalfactors (Gerozissis 2008). The differences in feed intake betweenthe two treatment groups observed in the current studymay be theresult of an alteration in the central regulation of energy balancevia peripheral insulin. The peaks of insulin following bi-phasicfeeding could change brain insulin concentrations as insulincrosses the blood brain barrier in proportion to its plasmaconcentration (Margolis and Altszuler 1967; Woods and Porte1977). This notion is corroborated by our findings fromExperiments 2 and 3, where a substantial rise in plasma insulinwas observed following each 60-min feeding bout. Therefore,it appears that a specific threshold in peripheral insulinconcentrations may be required to influence feeding behaviour,presumably via the central feedback mechanisms that controlsatiety.

Satiety-regulating peptides, such as islet amyloid polypeptideor amylin (Arnelo et al. 1996; Olsson et al. 2007), are co-releasedwith insulin in response to feeding (Cooper 1994; Arnelo et al.1998) and these may also be involved. It is conceivable that thereduced feed intake observed for the bi-phasic-fed pigs may bethe result of a tighter regulation of energy balance via the releaseof insulin and or satiety-regulating peptides that follow ameal. Our results showed that insulin secretion for pigs fed bi-phasically followed the classical post-prandial response, withincreased plasma concentrations after the onset of feeding,

whereas insulin concentrations for pigs fed ad libitum tendedto oscillate over the sampling period, without any pronouncedchange in the secretory profile.

Emerging evidence has suggested that peripheral hormonesact on midbrain dopaminergic systems to play regulatory rolesin controlling feed intake (Narayanan et al. 2010). Insulin andleptin receptors have been located in the mesolimbic systemof the ventral tegmental area of the brain and mesolimbicsignalling has been proposed to be an important factor inmediating reward-related behaviours such as feed intake(Davis et al. 2010). Research has also shown that insulinexerts influence on dopamine circuits and mediates themotivational and reward aspects of feed consumption(Narayanan et al. 2010). This mechanism has beendemonstrated in rats whereby administration of insulin into theventral tegmental area (VTA) led to a decrease in sucrose intakeand it has been proposed that insulin signalling within the VTAleads to a cessation in feed intake of palatable foods (Figlewiczet al. 2006). Observations from the present study indicated thatpigs when fed twice daily for 60-min periods ate continuously forthe first 30 min and thereafter remained inactive. It is possiblethat pigs fed for 60 min achieved post-prandial plasma insulinconcentrations required to provide ‘food reward’ which led to acessation of feeding as well as a period of subsequent inactivity.This is in contrast to pigs that were fed ad libitum who had nodiscernable pattern of insulin secretion and were more activeduring light hours. These observations are in agreement withthose of Scrimgeour et al. (2008) who reported that pigs fedtwice daily spent more time resting than those fed ad libitum.Increased periods of rest between feeding bouts would lead to areduction in the amount of energy expended during activity andthis may have also contributed to the improved performance.

Another mechanism thought to contribute to the regulation offeed intake is that of gastric distension or gut fill (Gregory et al.1989). The volume of a meal consumed during feeding activatesreceptive elements in the stomach and small intestine, resulting inalterations in digesta transit time and digestibility of feed (Moranet al. 2001). In the present study, pigs that were fed twice dailyconsumed two larger meals that were in contrast to ad libitum-fedpigs who consumed smaller quantities during each feeding bout.This observation is consistent with the findings of Scrimgeouret al. (2008). However, although distension of the stomach hasbeen shown to depress feed intake, it does not completely abolishfeeding (Forbes 2007). In a similar design to the current study,Gregory et al. (1989) showed that pigs fed twice daily consumedsimilar quantities of feed at each feeding session, and althoughgastric volume may influence feed intake, it was not the majordeterminant in terminating feeding. The authors furtherpostulated that the change in gastric volume within a mealmay be more important than gastric volume per se in theregulation of feed intake. This proposed mechanism may alsohave contributed to the reduced feed intake in pigs that were fedfor the two 60-min periods where the change in gastric volumefollowing the two feeding periods may have led to a cessationof feeding.

The finding that feed intakes were substantially reduced forthe bi-phasic-fed pigs while achieving similar bodyweights asfor theirad libitum counterparts is of great interest, particularly forthe pig industry because feed is themajor cost of pork production.

Feeding patterns and pig performance Animal Production Science G

In the current study, the bi-phasic-fed pigs had access to a similarquantity of feed as did those fed ad libitum, but the feed wasspread over two 60-min periods. These pigs consumed ~92.5%of the corresponding ad libitum intake over the experimentalperiod (0–49 days) in Experiment 2. Leymaster and Mersmann(1991) reported that when pigs were restrictively fed to a similarlevel as observed in the present study, daily gain was reducedbut both FCR and body composition (increased lean tissuecontent and decreased fat deposition) were improved.Although our data showed similar findings in respect to FCRand carcass composition, there was no difference in the growthrates of pigs. This dissimilarity between the two studies mayrelate to the feeding regimens, with this being a major variationin experimental design. Leymaster and Mersmann (1991) fedpigs the restricted ration throughout the day, whereas in thecurrent study, pigs were fed at two discrete 60-min periods.

The bi-phasic feeding pattern used in Experiment 2 resultedin pigs having a greater proportion of muscle (2.8%) and a lowerproportion of fat (2.6%) than for pigs fed ad libitum. Thisimproved nutrient utilisation may also be a response to thedifference in insulin status for these two treatment groups.Studies in growing animals have shown that the release ofinsulin and amino acids after a meal plays a pivotal role in thestimulation of skeletal muscle protein synthesis (Garlick et al.1983; Davis et al. 1996; O’Connor et al. 2003; Orellana et al.2006). Studies in neonatal pigs have shown that elevatedconcentrations of circulating insulin, similar to those found infed animals, are required for the phosphorylation of skeletalmuscle insulin receptors and the stimulation of proteinsynthesis (Suryawan et al. 2004). In addition, studies in olderanimals have also shown that amino acids act independently orin conjunction with infused insulin to stimulate protein synthesisand act as primary regulators of protein synthesis in skeletalmuscle (Bennet et al. 1990; Giordano et al. 1996; Volpi et al.1998). A specific threshold in insulin concentration appears tobe necessary to initiate protein synthesis. Studies in humans byCahill (1971) have shown that concentrations of insulin greaterthan 10mU/mL are required for both lipogenesis and proteinsynthesis. Therefore, a bi-phasic feeding regimen, where thecirculating insulin concentrations exceed this value, may beinstrumental in the stimulus of protein synthesis.

Although insulin per se has been shown to stimulate whole-body protein synthesis in severalmammalian species (Davis et al.2002; O’Connor et al. 2003), insulin sensitivity may also have asignificant role. Recent studies in neonatal piglets (Bergeron et al.2007) and steers (Gingras et al. 2007) have shown that proteinanabolism is upregulated via enhanced insulin sensitivity inanimals fed dietary long-chain n-3 polyunsaturated fatty acids.In both studies, the increase in whole-body protein accretion wassuggested to be the result of a decrease in protein breakdown,withno alteration in its synthesis. In addition, studies in rodents haveshown an amino acid-enhanced sensitivity of skeletal muscleprotein synthesis to insulin, allowing for maximal rates of proteinsynthesis to be achieved at lower insulin concentrations whenamino acids are infused concurrently (Garlick and Grant 1988).Taken together, these above studies suggest a dynamic role forinsulin, insulin sensitivity and amino acids and collectively theymay act synergistically to stimulate protein synthesis.

However, other growth-promoting metabolic processes mayalso be involved in the improved performance for the bi-phasic-fed pigs observed in the current studies. The exogenousadministration of porcine somatotropin (pST) enhancesproduction responses that result in a greater weight gain,improved FCR and a preferential deposition of protein at theexpense of lipid accretion (Campbell et al. 1988, 1989; Klindtet al. 1995). The effectiveness of pST appears to be influencedby the method of its delivery. Daily injections of pST that resultin large daily peaks in circulating concentrations improvecarcass leanness and FCR, a result of both a decrease in feedconsumption and an increase in the rate of gain (Yen et al. 1990;Klindt et al. 1992). Whereas the delivery of pST via a sustained-release implant also improves both carcass leanness and FCR,but the improvement in FCR is a consequence of a decrease infeed consumption without a change in the rate of gain (Yen et al.2005). Interestingly, the production responses obtained from theuse of pST implants are similar to those observed for the bi-phasic-fed pigs in the current study. This improved productivitymay also be a response to an alteration in the plasma GH profilefor these pigs. Although not measured for these studies, we havepreviously shown a doubling in the number of GH peaks fromtwo to four over a 12-h period for pigs fed bi-phasically,compared with those fed ad libitum (Scrimgeour et al. 2008).Therefore, it is conceivable that the improved productionresponses we have observed for the bi-phasic-fed group maybe the result of heightened metabolic growth processes, in whichboth insulin and GH play a decisive role.

A feeding regimen where pigs are fed over a small number ofsuccinct intervals may indeed be a strategy for improving pigproduction. A recent study has also shown that feeding frequencyinfluences pig performance (Persson et al. 2008). In the presentstudy, pigs fed three times daily had a larger weight gain and agreater final liveweight and carcass weight than did pigs fed ninetimes per day. In addition, feeding frequency also altered carcasscomposition as the pigs fed three times daily had increased leanmeat content compared with those pigs fed more frequently(Persson et al. 2008).

It is important to note that in the experiments reported in thepresent study, pigs were individually housed and the effects of afeeding regime remain to be investigated in group-housed pigs.Competition for feed in group-housed pigs causes pigs to eatfaster and aggressive behaviours are caused by competition(Hessel et al. 2006). Further research is required to investigatethe appropriate feeder space, feeding intervals and duration ingroup-housed pigs.

In summary, feeding pigs at distinct morning and afternoonperiods stimulated insulin secretion, which resulted in a distinctspike following each feeding period, whereas plasma insulinconcentrations remained unresponsive in pigs fed ad libitum.Importantly, the duration of the feeding periodmay also influenceinsulin secretion and this may in turn influence productionoutcomes. This altered insulin status was associated withenhanced productivity, resulting in improvements in FCR andcarcass composition. The results from the present experimentssuggested that a bi-phasic feeding pattern may be beneficial tocommercial piggeries. However, the optimum feeding duration,effect of age as well as the practicalities of the implementation

H Animal Production Science R. E. Newman et al.

of such a feeding regimen in a group-housed environment willneed to be explored and warrant further investigation.

Acknowledgements

The authors are grateful to the Australian Pork Cooperative Research Centreforfinancial support throughProject 2F 101 ‘Effects of fatty acids and feedingstrategies on the performance and carcass composition of growing pigs’ andProject 2G 108 ‘Bi-phasic feeding to improve pig performance and bodycomposition’.

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