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Long-term effects of foetal undernutrition on intermediary metabolism ingrowing lambsAli Kiania; Mette O. Nielsenb; Anne-Helene Tausonb; Malin P. Tygesenb; Sanne M. Hustedb; AndreChwalibogb

a Animal Sciences Group, Faculty of Agricultural Sciences, Lorestan University, Khoramabad, Iran b

Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University ofCopenhagen, Denmark

First published on: 10 January 2011

To cite this Article Kiani, Ali , Nielsen, Mette O. , Tauson, Anne-Helene , Tygesen, Malin P. , Husted, Sanne M. andChwalibog, Andre(2011) 'Long-term effects of foetal undernutrition on intermediary metabolism in growing lambs',Archives of Animal Nutrition, 65: 1, 46 — 54, First published on: 10 January 2011 (iFirst)To link to this Article: DOI: 10.1080/1745039X.2010.533551URL: http://dx.doi.org/10.1080/1745039X.2010.533551

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Long-term effects of foetal undernutrition on intermediary metabolism in

growing lambs

Ali Kiania*, Mette O. Nielsenb, Anne-Helene Tausonb, Malin P. Tygesenb,Sanne M. Hustedb and Andre Chwalibogb

aAnimal Sciences Group, Faculty of Agricultural Sciences, Lorestan University, Khoramabad,Iran; bDepartment of Basic Animal and Veterinary Sciences, Faculty of Life Sciences,University of Copenhagen, Denmark

(Received 25 May 2010; accepted 6 September 2010)

The objective of this study was to investigate the effects of foetal undernutritionon the metabolism in growing lambs. Seven-month-old lambs whose mothers hadbeen fed either restrictively (RN; n ¼ 14) or adequately (AN; n ¼ 6) in lategestation were fasted for three days. One hour before fasting and after 48 h and72 h fasting, changes in plasma concentrations of metabolites, i.e. glucose, non-esterified fatty acids (NEFA), 3-b-hydroxybutyrate (BOHB) and urea as well ashormones, i.e. insulin, the insulin-like growth factor (IGF-I) and leptin, weredetermined. Blood glucose, NEFA, urea, insulin, IGF-I and leptin were notdifferent between the two groups of lambs. Unexpectedly, at the end of the 3 dfasting, in spite of lower NEFA concentration (1.6 + 0.03 vs. 1.9 + 0.05 mM inGroups RN and AN, respectively), the BOHB concentration in RN lambs(0.94 + 0.02 mM) was significantly higher than that in AN lambs(0.78 + 0.04 mM). This higher rate of BOHB production might be interpretedas perturbations in ketone body metabolism potentially induced by under-nutrition during foetal life. However, more investigations are necessary to clarifythis interrelationship.

Keywords: metabolic programming; metabolites; fasting; sheep

1. Introduction

Undernutrition during foetal life causes alterations in endocrine function in thefoetus (Fowden et al. 2005, 2006). Prenatal adaptations in the endocrine systempresumably aim to ensure immediate survival in prenatal life under adverseintrauterine conditions. Prenatal adaptations induced by intrauterine undernutritionmay also improve prenatal metabolic efficiency for foetal development in sheep(Kiani et al. 2008). Undernutrition decreased energy expenditure of foetaldevelopment during late gestation (Kiani et al. 2008). It is believed that theseprenatal adaptations might produce individuals who would have better performanceunder poor nutritional conditions (Hales and Barker 2001; Gluckman et al. 2005)and higher metabolic efficiency (de Moura and Passos 2005) in postnatal life.

Fasting is a nutritional challenge during which a number of metabolicadjustments occur in order to spare glucose for vital organs, and the plasma

*Corresponding author. Email: arkashkia@gmail.com

Archives of Animal Nutrition

Vol. 65, No. 1, February 2011, 46–54

ISSN 1745-039X print/ISSN 1477-2817 online

� 2011 Taylor & Francis

DOI: 10.1080/1745039X.2010.533551

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concentration of many hormones and metabolites changes remarkably. Ourhypothesis was that lambs exposed to undernutrition during their foetal life wouldshow a different pattern of plasma hormones and metabolites in response to fasting.Therefore, the objective of this study was to investigate the effects of foetalundernutrition on circulating hormones and metabolites in growing lambs subjectedto three days of fasting.

2. Materials and methods

2.1. Animals and experimental protocols

All experimental procedures complied with the guidelines of and were approved bythe National Committee on Animal Experimentation, Denmark. Twenty Shropshirelambs of both sexes, born from 10 twin-bearing ewes in their second or third parity,were used in the experiment at seven months of age. The mothers were randomlyallocated to two feeding treatments for the last six weeks pre partum. They were fedeither adequately (AN; 100% energy and protein requirements) or restrictively (RN;about 60% of energy and protein requirements) according to the National ResearchCouncil (NRC 1985) (Table 1). The RN ewes were fed only hay silage (58% DM,10 MJ ME/kg DM, 8.2% crude protein (CP), 41.9% NDF, 9.2% ash and 1.6% fat).The AN ewes’ diet consisted of hay silage supplemented with barley (88.7% DM,10.4% CP, 2.2% ash and 2.3% fat) and a protein supplement (89.5% DM, 45.4%CP, 5.1% ash and 5.7% fat). After parturition, all ewes were fed ad libitum with haysilage plus 1000 g barley and 200 g protein supplement. The lambs’ birth weightswere recorded. All twin lambs were reared by their own dam until weaning at eightweeks of age. The lambs’ body weights were recorded biweekly until seven months ofage. From three weeks of age, all lambs were offered commercial concentrate(Faremix, DLG, Denmark; 88.8% DM, 14.0% CP, 6.78% ash and 3.2% fat) adlibitum. After weaning, the lambs were kept outdoors, grazing a good quality pasture

Table 1. Number of lambs, lambs’ birth weight, metabolisable energy (ME), crude protein(CP) intake and milk yield of ewes fed adequately or restrictively during the last six weeks ofgestation.*

Adequate feeding (AN) Restricted feeding (RN)

Number of lambs (females þ males) 6 (3 þ 3) 14 (7 þ 7)ME intake [MJ/d]Week 6–3 pre-partum 15.3a + 1.0 9.2b + 0.8Week 3–0 pre-partum 19.9a + 2.3 11.9b + 1.3

CP intake [g]Week 6–3 pre-partum 235a + 15 149b + 10Week 3–0 pre-partum 306a + 25 160b + 15

Body weight of ewes [kg]Day 50 pre-partum 90a + 8 83a + 5Day 7 pre-partum 91a + 7 78b + 4

Birth weight of lambs [kg]Mean + SD 4.3 + 0.28 3.9 + 0.18Range 4.0–5.0 2.4–5.2

Milk yield at 32 days post-partum [kg/d] 3.78a + 0.314 2.95b + 0.197

Notes: *Values are means + standard error. Mean values within a row with different superscript lettersare significantly different (p 5 0.05).

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with a daily supplement of 200 g of the commercial concentrate until seven monthsof age. During the three weeks prior to initiation of the experiment, the lambs werehoused indoors and fed ad libitum (fed state) a high quality silage (58% DM, 10 MJME/kg DM, 8.2% CP, 41.9% NDF, 9.2% ash and 1.6% fat). Fasting experimentswere performed at seven months of age. Lambs from both RN (n ¼ 14 [7 femalesand 7 males]) and AN (n ¼ 6 [3 females and 3 males]) groups were fasted for 72 hwith free access to water. The lambs were individually placed inside metabolic cages(temperature 10–158C, humidity 65–75%, 12 h light–dark cycle, cage 59 cm wide,160 cm long and 80 cm high) throughout the fasting period.

2.2. Blood sampling and assays

Blood samples were collected 1 h before the withdrawal of feed (0 h) and also after 48and 72 h of fasting. Blood samples were taken by puncture of the jugular vein, andblood was collected in 10 ml heparin-flourine vacuum tubes (Becton DickinsonVacuntainer System Europe, France) and 10 ml EDTA vacuum tubes (BectonDickinson Vacuntainer System, UK). Blood samples were immediately cooled on iceand then centrifuged at 1006 g for 15 min at 48C within 30 min after collection.Plasma samples were transferred to polystyrene tubes (Hounissen, Rossikov,Denmark) and frozen at 7208C pending analysis. In order to measure daily milkyield, lambs were separated from their dams twice daily (for about 3 h each time), thenewes were injected 1 ml Oxytocin (100 IU/ml, Novarties, Denmark) through thejugular vein. Directly after injection, ewes were hand milked. For each ewe, the weightof the collected milk was divided by separation time [h] and then multiplied by 24.

Plasma concentrations of glucose were analysed by a commercially availablespectrophotometric kit (17–25 InfinityTM, Sigma Diagnostic1, USA). Plasma insulinconcentration was determined by a sandwich-type time-resolved fluoroimmunoassay(DELFIA) as described by Ingvartsen et al. (1999). Plasma concentrations of non-esterified fatty acids (NEFA) were analysed by a spectrophotometric kit (WakoChemicals GmbH, Germany). Plasma concentration of 3-b-hydroxybutyrate(BOHB) was determined spectrophotometrically as an increase in absorbance at340 nm due to the production of NADH, at slightly alkaline pH in the presence of b-hydroxybutyrate dehydrogenase (Harano et al. 1985). Plasma urea-N analyses weredetermined enzymatically as described by Roch-Ramel (1967). Plasma samples forleptin were freeze-dried and analysed at the University of Western Australia, Perth.Leptin analyses were performed in duplicate by a double-antibody radioimmunoas-say using ovine leptin raised against bovine leptin as described by Blache et al.(2000). The limit of detection was 0.07 ng/ml. Plasma concentration of the insulin-like growth factor (IGF-I) was determined by double-antibody RIA with humanrecombinant IGF-I and antihuman IGF-I antiserum according to Breier et al.(1991).

2.3. Statistical analysis

Repeated measurements of plasma hormones and metabolites were analysed usingthe following linear mixed model:

Yijk ¼ m þ ai þ bj þ gk þ abð Þijþ bgð Þjkþ agð Þikþ abgð Þijkþ eijk;

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where m was the population mean, ai was the fixed effect of late gestation maternalnutrition (RN or AN), bj was the fixed effect of gender, gk was the fixed effect ofblood sampling time (0 h, 48 h, 72 h), and (ab)ij, (bg)jk, (ag)ik and (abg)ijk were theinteractions between fixed effects. eijk was the residual error (Littell et al. 2000). Ifany of the systematic interaction effects did not reach significance (p 4 0.05), theywas eliminated from the model.

Based on a likelihood ratio test, the covariance structure of the repeatedmeasurements was modelled as compound symmetry (CS), auto-regressive order 1(AR1) or unstructured (UN). Relative standard division (RSD) are presented unlessotherwise mentioned. Comparisons with p 5 0.01 are declared highly significant,p 5 0.05 significant and 0.05 5 p 5 0.10 are considered as trends.

3. Results

3.1. Birth weight and fasted body weight at seven months of age

Birth weights were not significantly different between female (4.2 + 0.2 kg, n ¼ 10)and male lambs (4.1 + 0.2 kg, n ¼ 10). Also, the birth weights of RN lambs werenot different from AN lambs (Table 1). In spite of the fact that both groups were fedad libitum with the same lactation diet from the day of parturition, lactating ewes inthe RN group had a significantly lower milk yield than the AN ewes (Table 1).Although at seven months of age the fasted body weight in both feeding groups oflambs was similar, males had significant higher fasted body weights (34 + 1.6 kg,n ¼ 10) than females (28 + 1.6 kg, n ¼ 10). The body weights of lambs up to sevenmonths of age are shown in Figure 1. The RN lambs were significantly lighter thanthe AN lambs throughout the first six weeks of post-natal life. After weaning theweight differences were no longer significant.

Figure 1. Birth weight and body weight of lambs whose mothers were fed either restrictively(n ¼ 14, �) or adequately (n ¼ 6, .) during their last six weeks of gestation. *p 5 0.05, **p 50.01.

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Table 2. Plasma metabolite and hormone concentrations measured in the fed state (0 h) andafter 48 and 72 h of fasting (48 and 72 h) in seven-month-old growing lambs whose motherswere fed either adequately or restrictively during the last six weeks of gestation.*

Maternal nutrition

p-valuesAdequate feeding

(AN, n ¼ 6)Restricted feeding(RN, n ¼ 14)

Fasting time 0 h 48 h 72 h SE 0 h 48 h 72 h SE MN{ Time Sex

Glucose [mM] 3.7a 2.7c 3.0b 0.08 3.5a 2.7c 3.1b 0.05 NSx 50.001 NSInsulin [ng/ml] 0.40a 0.05c 0.23b 0.04 0.37a 0.04c 0.20b 0.03 NS 50.001 NSNEFA# [mM] 0.1d 1.3c 1.9a 0.05 0.1d 1.2c 1.6b 0.03 NS 50.001 NSBOHB{ [mM] 0.79bc 0.69c 0.78bc 0.04 0.90ab 0.77c 0.94a 0.02 50.01 50.001 NSUrea [mM] 6.7c 11.9a 9.9b 0.45 7.0c 11.6a 9.3b 0.29 NS 50.001 NSLeptin [ng/ml] 1.7a 0.8b 0.8b 0.08 1.9a 0.9b 0.8b 0.05 NS 50.001 NSIGF-I [ng/ml] 47a 18b 12b 3.0 49a 20b 13b 1.8 NS 50.001 50.01

Notes: *Values are means + standard error (SE); {MN, Maternal nutrition; xNS, Non-significant (p 40.05); #NEFA, Non-esterified fatty acids; {BOHB, 3-b-hydroxybutyrate. Mean values within a row withdifferent superscript letters are significantly different (p 5 0.05).

3.2. Plasma concentrations of metabolites and hormones during fasting

In both groups of seven-month-old lambs, plasma glucose concentrations droppedsharply from the fed to fasted state (Table 2). The lowest level was reached at 48 h(2.7 + 0.07 mM); thereafter glucose concentrations gradually increased and almostreached the fed state values at 72 h fasting (3.1 + 0.07 mM). The kind of foetalnutrition did not affect the plasma concentration of glucose in growing lambs.Circulating insulin levels were highest at the fed state (on average 0.38 ng/ml) anddecreased rapidly to reach the lowest level after 48 h of fasting (about 0.04 ng/ml).After 72 h fasting, the insulin level increased towards the non-fasted level(0.22 + 0.03 ng/ml). The plasma concentrations of insulin in growing lambs werenot affected by different foetal nutrition (Table 2).

In both groups of lambs, the plasma concentration of NEFA increased sharplythroughout the fasting period. After 72 h fasting, the NEFA concentration in ANlambs was significantly higher compared to RN lambs (Table 2).

Interestingly, the mean plasma BOHB concentration in fed and fasted lambs wassignificantly higher in RN lambs (0.87 + 0.02 mM) than that in AN lambs(0.75 + 0.04 mM). In both groups, the plasma BOHB concentration dropped after48 h fasting in comparison to the intial values. However, after 72 h fasting, theBOHB concentration in plasma increased again (Table 2). Plasma urea concentra-tions did not differ between the AN and the RN lambs, but increased in response tofeed deprivation, reaching a peak level after 48 h fasting in both groups (Table 2).

Plasma concentrations of IGF-I (on average 25.5 + 3.0 vs. 27.5 + 1.8 ng/ml forAN and RN groups, respectively) and leptin (on average 1.1 + 0.08 vs.1.2 + 0.05 ng/ml for AN and RN groups, respectively) did not differ betweenfeeding groups. In both groups, concentrations of IGF-I and leptin decreasedsignificantly after 48 h fasting. The plasma leptin concentration did not decreasefurther; whereas the IGF-I concentration continued to decrease numericallythroughout the 72 h fasting period, but the decrease was not significant (Table 2).The concentration of IGF-I in plasma was significantly higher in males compared tofemales (32.2 + 2.3 vs. 20.8 + 2.4 ng/ml).

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4. Discussion

Restricted nutrient supply to the foetus is one of several stimuli that have beenproposed to cause small size at birth (Harding 2001; Stephenson and Symonds 2002;Luther et al. 2005; Wallace et al. 2005). In the present study, nutrient restriction didnot result in a smaller birth weight. It should be noted that late gestation feedrestriction in twin-bearing ewes does not necessarily affect the birth weight of twinsequally because large variations within-litter birth weight is usually observed intwins. In the present study, the lambs’ birth weights ranged between 2.4 and 5.2 kg inthe RN lambs, whereas for the AN lambs the range was only 4.0–5.0 kg. The largevariation between twins combined with a relatively modest dietary restriction (40%in energy and protein intake) may probably explain why the birth weights were notsignificantly different.

During the suckling period (first eight weeks of life), lambs born by restricted fedewes remained smaller compared to lambs from adequately fed ewes. This cannotsolely be ascribed to the nutrition level during late foetal life, because growth rateduring the suckling period is strongly associated with milk intake (Morgan 2005).This study is consistent with our previous study (Tygesen 2005), which showed thatfeed restriction in late gestation reduces milk production of the ewe in the subsequentlactation. On the other hand, the fact that nutrient restriction in late gestation affectssubsequent lactation means that the RN lambs were nutritionally restricted not onlyduring late foetal life but to some extent also during early postnatal life.

As expected, in both groups the fasting of lambs resulted in a reduction of plasmaglucose, insulin, IGF-I and leptin, and in an elevation of plasma levels of NEFA andurea within the first 48 h of fasting. This is similar to previous reports on ruminants(Heitmann et al. 1986; McCann and Hansel 1986; Ward et al. 1992; McGuire et al.1995; Amstalden et al. 2000; Chelikani et al. 2004). In the study of Heitmann et al.(1986) the splanchnic output of glucose was reduced by 40% and the hepatic uptake offree fatty acids increased three-fold. Consequently, this led to an increased hepaticrelease of ketone bodies in fasted ewes. In our study, when fasting was prolonged from48–72 h, NEFA concentrations increased even further, whereas glucose, insulin andurea concentrationswere normalised and stabilised at an intermediate level between the48 h fasting and pre-fasting values. This may be associated with a development ofinsulin resistance during a prolonged fasting period. Thereby, glucose can be spared formetabolism in non-insulin dependent organs. Consequently, metabolism in insulin-dependent tissues might shift away from glucose and more towards oxidation of fattyacids released from adipose tissue depots.

Plasma levels of the circulating hormones and metabolites measured in growinglambs were not affected by nutrient restriction in late gestation. However, foetalundernutrition caused higher BOHB concentrations in lambs. After three days offasting, lambs from restricted fed ewes showed higher BOHB and lower NEFAconcentrations compared to adequately nourished lambs. Plasma concentrations ofketone bodies in the restrictively nourished lambs were similar to those observed inhuman type-2 diabetes patients (Avogaro et al. 1996). These findings also agree withour previous study (Tygesen 2005) and other studies (Gardner et al. 2005), but theopposite has been found in rats, where offspring born to protein-restricted dams hada lower BOHB concentration during fasted state (Ozanne et al. 1998).

In fed ruminants, plasma BOHB originated mainly from butyrate formed duringmicrobial fermentation of carbohydrates in the forestomachs, which is subsequentlyconverted to BOHB in the ruminal epithelium and liver upon absorption (Katz and

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Bergman 1969; Bergman and Wolff 1971; Emmanuel 1980). Additionally, the liver isa net producer of ketone bodies in ketogenic processes, i.e. the incomplete oxidationof NEFA (Drackley et al. 1991a). An increased plasma concentration of NEFA isassociated with a proportional increased hepatic NEFA uptake (Alison 1986), aswell as with an increased hepatic ketone body formation, which also includes BOHB(Drackley et al. 1991b; Andersen et al. 2002). Hence, in the 3 d fasted sheep, plasmaBOHB is expected to be primarily related to plasma NEFA concentration andrates of hepatic ketogenesis, whereas the contribution from absorption of butyratefrom the ruminal epithelium is insignificant. In the present study, at 72 h of fasting,plasma concentrations of NEFA in RN lambs were lower than in AN lambs.Therefore, it was expected that RN lambs showed lower concentrations of BOHB.However, unexpectedly, RN lambs showed higher BOHB in spite of having lowerNEFA in their blood. These findings suggest that the higher BOHB level in the RNlambs could not be ascribed to a more extensive mobilisation of fat in these lambs,but rather to a higher rate of hepatic ketogenesis.

Insulin is one of the most important hormones determining the net rate of NEFArelease from adipose tissues, due to its strong anti-lipolytic action (Brown et al. 1998)and general anabolic effects on fat deposition. Furthermore, hyperinsulinemic-euglycemic studies in ruminants suggested that insulin can block the mitochondrialoxidation of NEFA and hence ketogenesis, probably due to reduced translocation ofNEFA into the mitochondria (Andersen et al. 2002). However, in the present study,insulin can hardly explain differences in BOHB plasma levels because both AN andRN lambs had similar plasma concentrations of insulin during feeding and fasting.

The present study therefore indicates that undernutrition in late gestation may becapable of inducing permanent changes in the hepatic ketogenic rate, and perhapsalso in peripheral ketone body metabolism. The underlying mechanism is yetunknown. Whether an increased hepatic ketogenic rate can be considered as anadaptation for survival in a nutritionally-challenged situation (e.g. three days of feeddeprivation) remains unclear.

In conclusion, the present study showed that growing lambs whose mothers werefeed-restricted during late gestation had higher plasma BOHB concentrations in spiteof lower plasma NEFA after three days of fasting. This higher hepatic ketogenicrate possibly points towards perturbations in hepatic ketone body metabolism, whichmay be induced by undernutrition during foetal life; however, this requires furtherinvestigations.

Acknowledgements

This study was financed by the Iranian Ministry of Research, Science and Technology, theResearch Council for Development Research, Denmark, and by the Faculty of Life Sciences,University of Copenhagen, Denmark. The authors wish to thank Ruth Jensen, Vibeke G.Christensen, Abdalla Ali and Kaj Thorhauge for their technical assistance. We thank theUniversity of Western Australia, Perth, for performing leptin and IGF-1 blood analyses.

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