DOI: 10.1111/jpn.12108

ORIGINAL ARTICLE

Low protein provision during the first year of life, but not during foetal life, affects metabolic traits, organ mass development and growth in male mink (Neovison vison) K. Vesterdorf1, D. Blache2, A. Harrison1, C. F. Matthiesen1 and A.-H. Tauson1 1 Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark, and 2 UWA Institute of Agriculture (Animal Production) M085, The University of Western Australia, Crawley, WA, Australia

Summary Low protein provision in utero and post-partum may induce metabolic disorders in adulthood. Studies in mink have mainly focused on short-term consequences of low protein provision in utero whereas the long-term responses to low protein (LP) provision in metabolically programmed mink are unknown. We investigated whether low protein provision in utero affects the long-term response to adequate (AP) or LP provision after weaning in male mink. Eighty-six male mink were exposed to low (19% of ME from CP; crude protein) or adequate (31% of ME from CP) protein provision in utero, and to LP (~20% of ME from CP) or AP (30–42% of ME from CP) provision post-weaning. Being metabolically programmed by low protein provision in utero did not affect the response to post-weaning diets. Dietary protein content in the LP feed after weaning was below requirements; evidenced by lower nitrogen retention (p < 0.001) preventing LP mink from attaining their growth potential (p < 0.02). LP mink had a lower liver, pancreas and kidney weight (p < 0.05) as well as lower plasma IGF-1 concentrations at 8 and 25 (p < 0.05) weeks, and a higher incidence of hepatic lipidosis at 25 weeks (p < 0.05). Furthermore, LP mink had a higher body fat (p < 0.05) and lower body CP content (p < 0.05) at 50 weeks of age. It is concluded that some effects of low protein provision in utero can be alleviated by an adequate nutrient supply post-partum. However, long-term exposure to low protein provision in mink reduces their growth potential and induces transient hepatic lipidosis and modified body composition. Keywords foetal programming, post-weaning malnutrition, nitrogen metabolism, hepatic lipidosis, plasma hormones, body composition Correspondence Prof. A.-H. Tauson, Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 3, 1870 Frederiksberg C, Denmark. Tel: +45 3533 3039; Fax: +45 3533 3020; E-mail: [email protected] Received: 1 March 2013; accepted: 27 June 2013

Dietary low protein provision during foetal life has been shown to cause metabolic and endocrine adaptations with long-term health implications (Hales and Barker, 2001). Moreover, dietary low protein provision is associated with limited growth in the neonate (Owens et al., 1989), which in turn may lead to the development of type 2 diabetes, hypertension and cardiovascular disease in adult life (Barker et al., 1989, 1990; Hales et al., 1991; Hales and Barker, 1992). Adaptive metabolic changes in response to nutritional challenges in utero using low protein (LP) diets have previously been demonstrated in various mammalian experimental models. Low protein provision in utero in the rat provoked long-term changes in both glucose and lipid metabolism (Desai et al., 1995) giving rise to restricted growth and adaptive changes in organ size

(Desai et al., 1996). In mink, a strict carnivore and seasonal breeder, low protein provision in utero resulted in decreased birth weights (Matthiesen et al., 2010a; Vesterdorf et al., 2012) and affected the expression of key hepatic enzymes (Matthiesen et al., 2010a), suggesting that the mink may be a useful alternative animal model in which to study LP challenges. The ‘thrifty phenotype’ hypothesis suggests that exposure to a poor nutrient environment postnatally similar to that experienced in utero may give a short-term advantage for the neonate, but may be detrimental for adult animal health (Hales and Barker, 2001). As such, the nutrient environment after birth seems to have a direct impact on the extent to which effects of intrauterine metabolic programming are lost, diminished or amplified in later life (Newnham et al., 2002). Studies in mink indicate that although some effects of metabolic programming

Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH

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Introduction

Low protein provision affects male mink kit metabolism and development

K. Vesterdorf et al.

in utero seen early in life, such as reduced growth, may be alleviated post-natally with a return to an adequate nutrient supply (Matthiesen et al., 2010a; Vesterdorf et al., 2012), other metabolic adaptations may persist. Just such a persistent effect is reduced protein oxidation (OXP), another being modified hepatic enzyme expression in weanling male mink when returned to an adequate protein (AP) supply post-natally (Matthiesen et al., 2012). In rats, a similar treatment had permanent effects on renal enzyme expression (Brennan et al., 2006), as well as altered pancreatic growth (Desai et al., 1996), pancreatic morphology (Snoeck et al., 1990), and insulin production causing impaired glucose tolerance (Chamson-Reig et al., 2006). When exposed to an excess diet after birth, rats exposed to a low protein diet during foetal life became obese (Jones et al., 1984). Likewise, the timing of a dietary intervention seems to have a direct impact on the extent to which metabolic programming affects an animal’s health (Desai et al., 1996, 2007). Exposure to a LP diet for neonatal rat pups affected growth (Desai et al., 1996; Zambrano et al., 2006) and had an adverse effect on glucose metabolism, which persisted into adulthood (Zambrano et al., 2006). Exposing mink kits to a LP diet after weaning likewise decreased growth performance (Matthiesen et al., 2012), while long-term low protein provision from weaning until pelting increased hepatic lipid infiltration and mortality rate (Damgaard et al., 1998a). While studies in mink have mainly focused on the short-term consequences of exposure to low protein provision during foetal life (Matthiesen et al., 2010a; Vesterdorf et al., 2012), the long-term response to a LP diet in metabolically programmed mink remains unexplored. The present study therefore investigated the long-term physiological response to low protein provision after weaning on mink metabolism and development. The hypothesis tested was that mink that have been protein restricted during foetal development have a better ability to adapt to low protein provision after weaning compared with mink kits that have been exposed to an adequate maternal diet during foetal life.

Eighty-six male mink kits born to dams fed an adequate protein diet (FA; crude protein/fat/carbohydrate, CP/fat/CHO, ratio of 31:55:14% of metabolizable energy, ME, n = 47) or a low protein diet (FL; 19:49:32% of ME, n = 39) the last 21.2  3.3 days before parturition were used (Vesterdorf et al., 2012). Dams and offspring were fed a conventional mink diet from parturition until weaning at 7 weeks of age. Immediately after weaning, the male kits from each foetal dietary treatment group (FA and FL) were randomly placed on either an adequate protein (AP, n = 44) or a low protein diet (LP, n = 42) to give four treatment groups: foetal adequate protein/ post-weaning adequate protein (n = 24), foetal adequate protein/post-weaning low protein provision (n = 23), foetal low protein provision/post-weaning adequate protein (n = 20) and foetal low protein provision/post-weaning low protein provision (n = 19). The mink were kept on their designated diet until euthanasia. The study comprised four experimental periods of balance and respiration experiments from 8 to 50 weeks of age. The experimental periods and mean mink age in weeks were as follows: Period I. End June, 8 weeks of age; Period II. Mid-September, 19 weeks of age; Period III. Mid-October, 25 weeks of age; and Period IV. Mid-April, 50 weeks of age. Twelve male mink were used in all four consecutive experimental periods (Core mink; n = 3 per dietary treatment group) and were euthanized at the end of Period IV. One mink died two weeks before Period IV due to unknown causes. In each of the first three experimental periods, an additional 12 males were randomly selected for participation in that balance experiment period and euthanized at the end of that experiment (n = 3 per dietary treatment group; total n = 36). Thus, a total of 48 male mink were used for the balance and respiration experiments. Euthanized mink were used for organ- and body composition studies.

Materials and methods

Animals and housing

The experiment was performed in accordance with the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and the Animal Experimentation Act under Danish national legisla-

The 86 mink were kept under standard mink farming conditions (Rørrendeg ard Experimental Mink Farm, T astrup, Denmark). Mink that were taking part in the balance and respiration experiments were transferred from the mink farm to the animal laboratory facility,

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tion. Experimental procedures were approved by the Danish Animal Experiments Inspectorate, Licence Number 2005/561-994. Experimental design

Low protein provision affects male mink kit metabolism and development

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where they were kept in metabolism cages under natural daylight conditions (55°N 12°) at an ambient temperature of 15–20 °C. Once during each balance period, each animal was transferred to a respiration unit in an adjacent laboratory unit for an indirect calorimetry experiment. The mink were weighed weekly from 8 weeks of age until euthanasia, or until they were 27 weeks of age. Core mink were weighed also at 50 weeks of age. Post-weaning dietary treatment

The animals were fed ad libitum once a day and had free access to drinking water. The base of the two different dietary treatments after weaning was identical. The adequate protein diet (AP) consisted only of conventional mink farm feed (Sjaellands Pelsdyrfoder A.m.b.a., St arup, Denmark), while the low protein diet (LP) was prepared on site by mixing AP feed with additional feedstuffs using a food mixer (Bjørn Varimixer, Denmark) to reach the desired lower level of CP and higher level of CHO (Table 1). Hence, the LP diet was achieved mainly by adding dietary CHO and some fat so that the CP content ranged from 30 to 42% of ME in the AP diet and from 20 to 24% of ME in the LP diet. The nutrient composition of the LP and AP diets in each of the four experimental periods is given in Table 2. Each LP feed batch was prepared on the day AP feed was delivered from the manufacturer; the LP and AP feed batches were frozen immediately in plastic bags apportioned by day ration weight and kept at 20 °C. The feed was removed from the freezer the day before required and thawed overnight at room temperature. Balance and respiration experiments

The animals were acclimatized to the metabolic cage and the laboratory facilities for a three-day period prior to the balance experiments, which were Table 1 Mean dietary composition of the experimental low protein (LP) diet during the first year of life for male mink after weaning. The LP diet is based on an adequate protein (AP) diet with additional feedstuffs LP diet LP composition, g/kg AP diet Corn oil Pre-cooked corn starch Potato mash powder Sugar beet pulp Water

730 50 50 50 10 110

Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH

performed over a four-day period. During the acclimatization and balance periods, the animals were fed ad libitum once daily and had free access to drinking water. Feed supply was weighed and recorded, and feed refusals in the bowl and spillage in the cage were collected and weighed. The feed spillage was subtracted from the feed supply to calculate the feed intake. Faeces and urine were quantitatively collected, weighed, and stored at 20 °C. Urine was quantitatively collected in 10 ml of 5% sulphuric acid solution. After removal of faeces, residues were collected by rinsing the collection screens and funnels in a 1% citric acid solution to minimize nitrogen losses. Feeding, change of water and collection was performed daily between 08:00 and 10:00 h. Respiration experiments by indirect calorimetry in an open-air circulation unit (Chwalibog et al., 2004) were performed on all animals once per balance period, over a period of 22 h. The average temperature and relative humidity of the respiration chambers were 17 °C and 67% respectively. In the first balance period at 8 weeks of age, two male mink kits were measured together per cage due to the relatively small body size. Heat production for each animal was therefore calculated according to the ratio of the metabolic body size of that animal in relation to the sum of the metabolic body size of the two mink kits per cage. At 19, 25 and 50 weeks of age, there was only one male mink per respiration chamber. The mink were weighed at the beginning and the end of each balance period, as well as on the day of the 22-h respiration experiment. Plasma collection

Blood was sampled by venipunture of the vena cephalica antebrachii (Blixenkrone-Møller et al., 1987) of non-anaesthetized Core mink at the end of each balance experiment. All blood samples were collected in heparinized tubes, centrifuged at 3300 g for 15 min and the blood plasma was stored at 20 °C until used. The blood plasma concentrations of insulin-like growth factor 1 (IGF-1), insulin and leptin were analysed by radioimmunoassay (RIA) at The University of Western Australia, Australia. All plasma hormones were assayed in duplicate in a single assay for each hormone using double-antibody RIA methods previously described and validated for mink plasma samples (Matthiesen et al., 2010a). The detection limits were 0.39 lU/ml for insulin, 0.05 ng/ml for leptin and 0.04 ng/ml for IGF-1. IGF-1 was diluted by 40 to be measured in the standard curve. The coefficients of variation were 4.5% at 9.45 lU/ml, 5.1% at 4.56 lU/ ml and 7.6% at 1.89 lU/ml for insulin, 5.9% at 3

Low protein provision affects male mink kit metabolism and development

K. Vesterdorf et al.

Table 2 Chemical composition of experimental diets: adequate protein (AP) and low protein (LP) diets at four experimental periods during the first year of life for male mink after weaning. Nutrient distributions as a percentage of metabolizable energy (% of ME) in the diets are given for crude protein, fat, and carbohydrate (CP/fat/CHO) Period I 8 weeks of age

Dry matter (DM), g/kg Ash, g/kg DM CP, g/kg DM Fat, g/kg DM CHO, g/kg DM Gross energy, MJ/kg DM ME*, MJ/kg DM CP/fat/CHO (% of ME)

Period II 19 weeks of age

Period III 25 weeks of age

Period IV 50 weeks of age

AP

LP

AP

LP

AP

LP

AP

LP

330 103 465 210 222 23.4 17.3 42:45:13

374 71 290 257 381 23.7 19.4 24:50:26

421 61 397 269 273 25.1 19.6 31:53:16

463 43 273 318 367 25.4 21.4 20:57:23

442 70 366 254 311 23.9 19.0 30:51:19

472 54 259 279 408 24.3 20.4 22:52:26

333 115 428 224 232 23.3 16.0 40:52:8

424 80 243 283 394 24.0 20.2 22:54:24

*ME was determined as: ME = GEFEUE.

2.24 ng/ml, 4.9% at 1.21 ng/ml and 7.2% at 0.62 ng/ ml for leptin, and 7.6% at 1 ng/ml and 6.3% at 0.16 ng/ml for IGF-1. Organ and carcass collection

At the end of each experimental period, three animals per treatment group were euthanized. Anaesthesia was achieved by intramuscular injection of 1.5 ml/kg BW of Ketaminol and 1.0 ml/kg BW of Narcoxyl (50 mg/ ml and 20 mg/ml, respectively, Intervet International B.V. Boxmeer, The Netherlands). Upon loss of interdigital-, palpebral- and corneal reflexes, the animal was euthanized by cardiac excision. The pancreas, liver and kidneys used for organ development analysis were retrieved and weighed. Only livers from Period III and IV (N = 23) were used to analyse liver chemical composition. The mink carcasses, including skins and subcutaneous fat but without the retrieved organs, were frozen and kept at 20 °C until used for body composition analysis. Carcasses from Period II, III and IV were used: In Period III, two carcasses per treatment group were used, and in Period IV 11 carcasses were used (only two carcasses from mink on a foetal low protein provision and an adequate protein provision after weaning). The frozen carcasses were homogenised using a Bizerba meat mincer (Bizerba FW-N 82 System Unger, Bizerba GmbH KG, Ballingen, Germany) using the 8-mm setting, while liver was homogenised using the 4-mm setting, for body and liver chemical composition analysis.

and the homogenised liver and carcasses, while DM, and then ash content, was analysed in wet samples of the feed residues. It was assumed that feed residues held the same amount of organic matter as that of the various diets. Samples of diets, faecal matter, and the liver and carcass samples were freeze-dried and analysed for DM, ash, fat, and gross energy. Urine and citric acid rinse were analysed for content of N. Dry matter was found by evaporation at 105 °C overnight (10 h), and ash was then determined by incineration overnight at 525 °C (10 h). Fat content was determined by hydrolysis in 3 M hydrochloric acid and subsequent fat extraction with petroleum ether using the Soxtec system 1047 hydrolysing unit Tecator (Foss, H€ ogan€ as, Sweden) and a 1043 extraction unit (Foss) respectively. Samples were analysed for N content by the micro-Kjeldahl analysis technique using a 2020 Digestor at 420 °C and a 2200 Kjeltec auto distillation unit (Foss). The content of crude protein was calculated as N x 6.25. Carbohydrate content was calculated by difference: CHO = DMAshCPFat. Gross energy content was determined in an adiabatic bomb calorimeter using the IKA Calorimeter System (IKA Gmbh KG, Staufen, Germany). Calculations

Dry matter (DM) and nitrogen (N) content were analysed in wet samples of the experimental diets, faeces,

Calculations of metabolizable energy (ME), retained energy (RE), retained N (RN) and the nonprotein respiratory quotient (RQnp) were performed as described previously (Matthiesen et al., 2010a). Urinary energy was calculated as UE ¼ 27:115kJg1  UNg  0:847kJ (A.-H. Tauson, unpublished). Utilization of N for retention (RNDN) was calculated as the fraction RN of digested N (DN) and presented as a

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Chemical analyses

K. Vesterdorf et al.

percentage. The heat production (HE) calculation was based on Brouwer (1956), while calculations of the oxidation of protein, fat and carbohydrate (OXP, OXF and OXCHO respectively) as a percentage of HE were based on Chwalibog et al. (1992). Substrate oxidation formulas were validated for RQnp values between 0.707 and 1.0 and above 1.0 by Chwalibog et al. (1992) and below 0.707 by Chwalibog et al. (2004). Where RQnp was below 0.707 the OXCHO was set to zero. Fat oxidation was consequently calculated as the net value of CHO oxidation plus the value of fat oxidation (Chwalibog et al., 2004). Average weekly weight gain was calculated as AWG = Wn+1Wn, where Wn and Wn+1 are body weights in g at the beginning and end of that week. Statistical analysis

Tests for statistical significance between means were performed using the Mixed Procedure in SAS as repeated measures (SAS/STATâ software, version 9.2, SAS Institute, Cary, NC, USA) on data, which were normally distributed and of equal variance. Thus, the statistical model used was

Yijkl ¼ l þ ai þ bj þ @k þ ðabÞij þ ða@Þik þ ðb@Þjk þ ðab@Þijk þ ijkl

Low protein provision affects male mink kit metabolism and development

of the low protein provision experienced during foetal life on male mink body weights, nutrient intake, energy- and nitrogen metabolism, organ development or body and liver composition at any of the time points studied after weaning. In the following, the data have therefore been pooled according to the dietary treatment after weaning. Results related to nutrient intake and metabolic parameters are given in relation to metabolic body size for comparative purposes. Results concerning organ development are given as organ weight to body weight in per cent. Body and liver chemical composition are given as chemical content to dry matter in per cent. Nutrient intake

The intake of CP, fat and CHO reflected the different nutrient composition of the diets (all p < 0.01). Crude protein intake of LP mink decreased from 17.6  1.0 g/kg0.75/day at 8 weeks of age to 6.4  0.6 g/kg0.75/day at 50 weeks of age (p < 0.001) and was lower than that of AP mink (p < 0.001). In AP mink, CP intake decreased from 29.3  1.0 g/ kg0.75/day at 8 weeks of age to 15.8  0.9 g/kg0.75/ day at 25 weeks of age (p < 0.001), but was 44% higher (p < 0.001) at 50 than at 25 weeks of age. Nitrogen metabolism

where l represents the general mean, ai represents the fixed effect of experimental period, ßj is the fixed effect of the dietary intervention during foetal life (FA or FL), ok is the fixed effect of the dietary intervention post-weaning (AP or LP), (aß)ij, (ao)ik, (ßo)jk, and (aßo)ijk are the interactions between fixed effects, and eijkl represents the residual error ~ N (0, r2i ). Animal was the subject in the repeated measures analysis. Model reduction was performed for non-significant effects. Covariance parameters were estimated using the REML method. Pairwise comparison of the LSmeans was carried out using the PDIFF option. Data are presented as LSmeans  SE. Differences between means were considered significant when p < 0.05 and a tendency when p < 0.1.

Decreasing CP intake resulted in a 59% decrease in DN (p < 0.001, Table 4) from 8 through 25 weeks of age in the LP mink. LP provision affected the N metabolism such that RN was lower at 8 weeks of age (p < 0.01), while in older mink RN was similar irrespective of dietary treatment as relatively lower amounts of N were excreted with the urine in the LP mink (p < 0.001). Nitrogen utilization for retention (RNDN) was affected by age but not by protein provision (p < 0.001). At 8 weeks of age, RNDN was 49.4  4.1% and 44.2  3.3% for LP and AP mink, respectively, indicating a high demand for protein for growth. From 25 through 50 weeks of age, RN was close to zero, irrespective of dietary treatment, indicating that growth was completed in the animals.

Results

Energy metabolism

Low protein provision during foetal life was found to cause a significant reduction in body weights at birth (Vesterdorf et al., 2012). The most important metabolism-, organ development-, and body composition traits as an effect of foetal dietary treatment are given in Table 3. There was no statistically significant effect

Metabolizable energy intake (Table 5) in AP mink was lower at 25 than at 8 weeks of age (p < 0.001), but higher at 50 weeks of age (p < 0.001). In contrast, ME intake in LP mink decreased from 8 through 50 weeks of age (p < 0.001). ME intake was similar in both treatment groups from weaning until 19 weeks of

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Table 3 Main results of energy and nitrogen (N) metabolism, substrate oxidation, organ development, and chemical composition of the body of male mink during the first year of life as a function of foetal (F) dietary treatment. Intake is given as dry matter (DM). Energy metabolism is given as metabolizable energy (ME), heat production (HE), retained energy (RE) and oxidation of protein (OXP), fat (OXF) and carbohydrate (OXCHO) as a percentage of HE. Nitrogen metabolism is given as retained N (RN) and utilization of digested N (RNDN). The mink had been exposed to either an adequate (FA) or a low protein (FL) diet during the last 21.2  3.3 days of foetal life and were fed either an adequate or a low protein diet from weaning. Values are mean  SE per animal Age, weeks 8 (n = 24) Body weight, kg FA 0.81  0.04a* FL 0.98  0.03a Intake DM, g/kg0.75/day FA 66  3a FL 57  2a Energy metabolism ME, kJ/kg0.75/day FA 1248  55a FL 1080  36a HE, kJ/kg0.75/day FA 916  8a FL 894  10a RE, kJ/kg0.75/day FA 332  58a FL 185  39ab Substrate oxidation OXP, % of HE FA 22  3a FL 18  3a OXF, % of HE FA 54  3a FL 57  3a OXCHO, % of HE FA 24  5a FL 25  4a Nitrogen metabolism RN, g/kg0.75/day FA 1.46  0.22a FL 1.28  0.15a RNDN, % FA 46  4a FL 48  3a Organ development Liver, % of BW FA 3.37  0.19a FL 3.27  0.20a Kidney, % of BW FA 0.35  0.01a FL 0.36  0.02a Pancreas, % of BW FA 0.30  0.02a FL 0.31  0.03a Body chemical composition Crude protein, % of DM FA N/A‡ FL N/A

6

19 (n = 24) 2.59  0.07bd 2.59  0.06b

25 (n = 24)

50 (n = 11)

p-value, effect of† F-diet

2.94  0.14c 2.90  0.13c

2.74  0.09d 2.93  0.14c

NS

47  2b 48  2b

38  3c 34  3c

38  6bc 37  7c

NS

949  39b 968  38a

725  58c 669  54b

660  96c 656  109b

NS

696  12b 643  30b

635  24c 633  28b

678  36bc 666  36b

NS

253  44a 325  57a

90  46b 36  51b

18  121b 10  116c

NS

26  3a 29  2b

26  3a 26  2ab

31  8a 28  8ab

NS

33  4b 24  4ac

53  5ac 53  3b

61  7ac 62  5ac

NS

42  3b 47  4b

21  3ac 21  3ac

8  3d 10  6ac

NS

0.42  0.03b 0.37  0.05b

0.02  0.06c 0.03  0.06c

0.00  0.07c 0.05  0.06c

NS

45  2a,A 19  1b,B

2  6b 9  7c

2  8b 8  6c

NS

1.81  0.07b 1.80  0.07b

2.52  0.28c 2.31  0.38bc

2.13  0.09c 3.04  0.15c

NS

0.24  0.01b 0.24  0.01b

0.18  0.01c 0.20  0.01c

0.20  0.01c 0.19  0.02c

NS

0.16  0.02b 0.17  0.01b

0.11  0.01c 0.14  0.01b

0.16  0.02bc 0.16  0.02b

NS

37  1a 38  2a

38  2ab 39  2a

48  4b 45  3a

NS

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Table 3 (Continued) Age, weeks 8 (n = 24) Fat, % of DM FA FL

19 (n = 24)

25 (n = 24)

58  2a 57  2a

N/A N/A

p-value, effect of† F-diet

50 (n = 11)

57  2ab 56  3a

45  4b 49  4a

NS

*Different lowercase superscripts (a, b and c) denote statistical differences within the same treatment group between age periods; p < 0.01. †The effect of foetal (F) diet is given in the table for each measured variable. NS denotes no statistically significant effect. ‡No values are available for body chemical composition of 8-week-old mink due to technical difficulties.

Table 4 Nitrogen (N) metabolism in male mink during the first year of life as a function of post-weaning (PW) dietary treatment. Nitrogen metabolism is given as digested N (DN), N contained in urine (UN), retained N (RN) and utilization of digested N (RNDN). The mink had been exposed to either an adequate or a low protein diet during the last 21.2  3.3 days before parturition and were fed either an adequate (AP) or a low (LP) protein diet from weaning. Values are mean  SE per animal Age, weeks 8 (n = 24)

p-value, effect of 19 (n = 24)

25 (n = 24)

50 (n = 11)

PW-diet

Age

PW-diet 9 Age

0.75

DN, g/kg /day AP 3.75  LP 2.18  UN, g/kg0.75/day AP 2.07  LP 1.09  RN, g/kg0.75/day AP 1.66  LP 1.09  RNDN, % AP 44.2  LP 49.4 

0.20a*,A† 0.14a,B

2.46  0.08b,A 1.48  0.06b,B

1.95  0.11c,A 0.95  0.08c,B

2.77  0.14d,A 0.77  0.06c,B

***

***

***

0.16a,A 0.08a,B

1.91  0.06a,A 1.10  0.06a,B

1.61  0.09a,A 0.76  0.06a,B

2.65  0.11b,A 0.76  0.08a,B

***

**

***

0.15a,A 0.13a,B

0.47  0.04b 0.32  0.03b

0.03  0.05c 0.05  0.07c

0.01  0.04c 0.06  0.08c

***

***

**

3.3a 4.1a

19.2  1.5b 22.1  1.8b

1.4  2.4c 11.6  9.0c

0.2  1.6c 8.9  9.3c

NS

***

NS

*Different lowercase superscripts (a, b and c) denote statistical differences within the same treatment group between age periods; p < 0.01. †Different uppercase superscripts (A and B) denote statistical differences between treatment groups within the same age period; p < 0.01. The effect of post-weaning diet (PW-diet), age, and the interaction between diet and age are given in the table for each measured variable, where *p < 0.05, **p < 0.01 and ***p < 0.001. NS denotes no statistical significance. There was no effect of foetal life dietary treatment on any of the measured parameters.

age; however, LP mink had a lower ME intake cf AP mink at 25 and 50 weeks of age (both p < 0.001). Heat production was highest at 8 weeks of age (both treatment groups: p < 0.001) and remained constant from 19 through 50 weeks of age, irrespective of dietary treatment. At 8 weeks of age, the positive energy balance of the AP mink was largely supported from energy retention in protein; however, by 19 weeks of age, RE depended on a higher percentage of fat than of protein energy retention; 77% from RFE (p < 0.01) and 23% from RPE (p < 0.001). In LP mink, however, the fat to protein energy retention was 43% to 57% of total RE at 8 weeks of age. The ratio changed at 19 weeks of age to 82% RFE and 18% RPE of RE (both p < 0.001). Heat production was similar in LP mink at 25 and at

The substrate oxidation patterns reflected the nutrient compositions of the diets in each experimental period (Table 5). As such, OXP was consistently lower (p < 0.001) in LP cf AP mink. At 8 weeks of age, LP provision resulted in a higher rate of carbohydrate oxidation (OXCHO; p < 0.01) and a higher RQnp (p < 0.001) compared with controls. In 25 weeks old mink, OXF was higher compared to that at 19 weeks

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7

50 weeks of age; however, the low protein provision caused a lower ME intake, which resulted in a negative RE in this period (p < 0.05 and p < 0.001, respectively). At 50 weeks of age, the LP mink mobilized fat evidenced by the negative RFE. Substrate oxidation

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K. Vesterdorf et al.

Table 5 Energy metabolism in male mink during the first year of life as a function of post-weaning (PW) dietary treatment. Intake is given as dry matter (DM) and gross energy (GE). Energy metabolism is given as metabolizable energy (ME), heat production (HE), retained energy (RE), energy retained in protein (RPE) and in fat (RFE), utilization of ME for retention (RE/ME, %), non-protein respiratory quotient (RQnp), and oxidation of protein (OXP), fat (OXF) and carbohydrate (OXCHO) as a percentage of HE. The mink had been exposed to either an adequate or a low protein diet during the last 21.2  3.3 days of foetal life and were fed either an adequate (AP) or a low (LP) protein diet from weaning. Values are mean  SE per animal Age, weeks 8 (n = 24) Body weight, kg AP 0.81  0.03a* LP 0.91  0.04a Intake DM, g/kg0.75/day AP 63  2a LP 60  3a GE, kJ/kg0.75/day AP 1493  50a LP 1433  81a Energy metabolism ME, kJ/kg0.75/day AP 1150  43a LP 1178  72a 0.75 HE, kJ/kg /day AP 912  7a LP 898  12a RE, kJ/kg0.75/day AP 237  41a,c LP 280  72a RPE, kJ/kg0.75/day AP 247  23a,A LP 162  19a,B RFE, kJ/kg0.75/day AP 0  40a LP 122  56a RE/ME, % AP 20  3a LP 22  5a RQnp AP 0.77  0.00a,A LP 0.82  0.00a,c,B Substrate oxidation OXP, % of HE AP 26  2a,A LP 14  1a,B OXF, % of HE AP 58  2a LP 53  3a OXCHO, % of HE AP 16  2a,A LP 33  2a,B

p-value, effect of‡ 19 (n = 24)

25 (n = 24)

2.57  0.08b 2.61  0.05b

3.18  0.11c,A† 2.66  0.11b,B

50 (n = 11) 2.65  0.13b 2.97  0.07b

PW-diet

Age

PW-diet x Age

*

**

*

50  2b 44  2b

43  2c,A 29  2c,B

53  2b,A 26  3c,B

***

***

***

1260  44b 1127  50b

1048  58c,A 731  54c,B

1251  54b,A 604  61c,B

***

***

***

990  35b 927  40b

807  46c,A 588  46c,B

872  39d,A 480  48c,B

***

***

***

678  22b 661  26b

677  25b 591  20b

646  25b 695  39b

NS

***

NS

312  46a 266  56a

129  50b,A 3  39b,B

226  14b,c,A 215  78c,B

***

***

***

71  6b 48  4b

5  7c 7  10c

2  7c 9  12c

***

***

**

241  44b 217  54a

124  49b,A 4  34b,B

224  10b,A 205  81c,B

***

**

***

30  5ac 27  5a

13  6b,A 7  8b,B

26  1c,A 57  27b,B

**

*

NS

0.88  0.00a 0.89  0.00a

0.79  0.01a 0.78  0.01b,c

0.71  0.02b 0.75  0.01b

***

*

NS

34  2b,A 21  1b,B

33  2b,A 19  1b,B

48  1c,A 14  2a,B

***

***

***

27  3b 30  5b

47  4c,A 59  4a,B

49  2c,A 72  3c,B

*

***

*

39  2b,A 49  4b,B

20  3a 21  3c

3  2c 14  4c

***

***

NS

*Different lowercase superscripts (a, b and c) denote statistical differences within the same treatment group between age periods; p < 0.05. †Different uppercase superscripts (A and B) denote statistical differences between treatment groups within the same age period; p < 0.05. ‡The effect of post-weaning diet (PW-diet), age and the interaction between diet and age are given in the table for each measured variable, where *p < 0.05, **p < 0.01 and ***p < 0.001. NS denotes no statistical significance. There was no effect of foetal life dietary treatment on any of the measured parameters.

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of age in both treatment groups (both p < 0.001), and was higher in LP mink cf AP mink (p < 0.05). However, OXCHO was lower in both treatment groups (both p < 0.001) compared to 19 weeks of age, which explains the lower RQnp for the 25-week-old mink. In spring, at 50 weeks of age, low protein provision resulted in an even lower rate of OXP (p < 0.05), while OXF was higher (p < 0.02) than in 25-week-old LP mink. Body weights

The body weight of mink participating in the balance experiment (n = 24) at 8 weeks of age was not affected by foetal or post-weaning dietary treatments (Tables 3 and 5). The number of animals weighed decreased with the season due to organ collection, and dietary treatment animals not participating in the balance experiments were euthanized for their fur in the turn of the months November – December. Body weights (Fig. 1) increased from 11 to 21 weeks of age in LP animals (p < 0.001) and from 11 to 26 weeks of age in AP mink (p < 0.02). Between 21 and 27 weeks of age, the low protein provision resulted in a decrease in weight gain (p < 0.05) causing LP mink to reach a maximum body weight, which was lower cf AP mink (p < 0.02). In contrast, the body weight of AP mink

Low protein provision affects male mink kit metabolism and development

reached a maximum at 26 weeks of age. The animals were not weighed between 27 and 50 weeks of age. However, the body weight of AP mink was 16% lower by 50 weeks of age (p < 0.05), whereas LP body weight tended to be higher (p < 0.1) than at 25 weeks of age. Furthermore, LP mink weighed 12% more than AP mink (Table 5; p = 0.05) at 50 weeks of age. Plasma hormone concentrations

Low protein provision caused lower plasma concentrations of insulin-like growth factor 1 (IGF-1; Fig. 2A) at 8 (18%, p < 0.01) and at 25 (34%,

(a)

(b)

(c)

Fig. 1 Body weights of male mink in the post-weaning growth period. Body weights from 11 to 27 weeks of age in male mink exposed to adequate (AP) or low protein (LP) diet after weaning. The number of animals in each group decreased with time such that in the AP group, n = 38 from week 11–20, n = 32 from week 21–26 and n = 26 in week 27. In the LP group, n = 36 from week 11–12, n = 35 in week 13, n = 34 from week 14–20, n = 27 from week 21–23, n = 26 in week 24, n = 25 in week 25–26 and n = 19 in week 27. The bar denotes differences between dietary treatment groups; p < 0.02.

Fig. 2 Blood plasma concentrations of (A) insulin-like growth factor 1 (IGF-1), (B) insulin and (C) leptin in growing male mink. The mink were exposed to adequate (AP) or low protein (LP) diet after weaning, and blood was sampled at 8, 19 and 25 weeks of age. For each dietary group, n = 6 (N = 12) mink at 8, 19 and 25 weeks of age. Different letters denote statistically significant differences within age groups; p < 0.05. Bars denote statistically significant differences between treatment groups; *p < 0.05 and **p < 0.01.

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Low protein provision affects male mink kit metabolism and development

p < 0.05) weeks of age cf AP mink. Plasma IGF-1 concentrations were lower at 25 than at 8 weeks of age in both treatment groups (AP: 45% and LP: 56%; p < 0.001). Dietary protein provision and age did not affect the insulin plasma concentration (Fig. 2B). The leptin plasma concentration was 60% lower in LP mink cf AP mink (p < 0.01) at 25 weeks of age (Fig. 2C). Organ development

In both treatment groups, the ratios of organ weight to body weight (Fig. 3) were highest in the youngest mink (8 weeks of age; p < 0.05), whereas the ratios were lower and remained constant from full body (a)

(b)

(c)

K. Vesterdorf et al.

length, which had been reached around 19 weeks of age and through to 50 weeks of age. Low protein provision affected organ growth negatively, such that at 8 weeks of age all measured organ to body weight ratios were lower (all p < 0.02), and at 19 weeks of age liver- and pancreas ratios were lower (p < 0.01 and p < 0.05 respectively) than in AP mink. The ratio of liver to body weight ratio tended to be higher in LP mink at 25 than at 19 weeks of age (p < 0.1); however, at 50 weeks of age liver to body weight was not affected by the dietary treatment. Body- and liver chemical composition

The body chemical composition of the mink was not affected by low protein provision until at 50 weeks of age (Fig. 4) where LP mink bodies contained less CP (p < 0.05; Fig. 4A), more fat (p < 0.05; Fig. 4B) and less ash (p < 0.01; Fig. 4C) than AP mink. The body content of fat, ash and CP in the LP mink did not change with age; however, at 50 weeks of age the fat content of AP mink was lower (p < 0.05), ash was higher (p < 0.05) and CP tended to be higher (p < 0.1) than in younger AP animals. Low protein provision affected liver fat content at 25 weeks of age (p < 0.05; Fig. 5) such that LP livers contained 54  11% of fat (ranging from 24% to 83% of DM; Fig. 5B) while AP livers contained 20  2% fat (ranging from 14% to 27% of DM), thus indicating some degree of fatty infiltration in 67% of the LP livers. While fat content increased CP content decreased in the LP livers at this age (A; LP: 41  11% of DM cf AP: 75  2% of DM; p < 0.05; Fig. 5A). Liver chemical composition was not affected by low protein provision at 50 weeks of age, but the content of fat (19  2% of DM; p < 0.05) was lower while CP content was higher (76  2% of DM; p < 0.05) than in livers of 25-week-old animals. Discussion

Fig. 3 Organ weights of growing male mink. Organ weight in relation to body weight (%) of male mink exposed to adequate (AP) or low protein (LP) diet after weaning. (A) liver, (B) pancreas and (C) kidney. Different letters denote statistically significant differences within age groups; p < 0.05. Bars denote statistically significant differences between treatment groups; *p < 0.05. The liver weight relative to body weight tended to be higher in LP than in AP mink at 25 weeks of age (p < 0.1).

The present study investigated the long-term response to low protein provision after weaning on mink metabolism and development in mink kits that had been exposed to low- or adequate protein provision in foetal life. Moreover, this study investigated whether mink that have been protein restricted during foetal development are better able to adapt to low protein provision, with a specific focus on the ability to conserve protein measured by protein oxidation and efficiency of N utilization. According to the ‘thrifty phenotype’ hypothesis (Hales and Barker, 1992), poor nutritional supply in utero increases the capacity for

10

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Low protein provision affects male mink kit metabolism and development

(a)

(a)

(b)

(b)

(c)

(c)

Fig. 4 Body chemical composition of male mink. The chemical composition is presented as percentage of (A) crude protein (CP), (B) fat and (C) ash content in dry matter (DM) in bodies of male mink aged 19, 25 or 50 weeks. The mink had been exposed to adequate (AP) or low protein (LP) diet after weaning. Significant differences within chemical fraction between age groups are denoted by different lower case letters (a and b), and significant differences between dietary treatments in each age group are denoted by *p < 0.05.

Fig. 5 Liver chemical composition of male mink. The chemical composition is presented as percentage of (A) crude protein (CP), (B) fat and (C) ash content in dry matter (DM) in livers of male mink aged 25 or 50 weeks. The mink had been exposed to adequate (AP) or low protein (LP) diet after weaning. Significant differences within chemical fraction between age groups are denoted by different letters and significant differences between dietary treatments in each age group are denoted by *p < 0.05.

nutrient utilization in the face of a poor nutritional environment post-natally. However, in our study, although low protein provision during foetal life resulted in a significant decrease in body weights at birth and until three weeks of age (Vesterdorf et al., 2012), no effect of the low protein provision during foetal life on post-weaning responses to an adequate or a low protein provision was observed with regard to metabolism and development. Hence, the mink that had been protein restricted during foetal life were not observed to be better able to conserve protein after

weaning than mink that had been exposed to an adequate diet in utero. Furthermore, mink fed a low protein provision after weaning adapted their OXP in accordance with the CP supply, but were unable to completely compensate for the lower availability of N by increasing their efficiency of utilization of N for retention. Most of our documented changes corroborate findings in previous studies on effects of low protein provision in mink, and it is therefore assumed that the observed metabolic changes were caused primarily by the low protein provision rather than by differences in

Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH

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Low protein provision affects male mink kit metabolism and development

the fat and CHO supply. Moreover, CHO and fat were generally within the recommended limits for the growth and furring periods (Lass en et al., 2012).

K. Vesterdorf et al.

kits were exposed to an adequate nutritional environment until weaning. Effects of post-weaning diet on measured variables

Effects of pre-natal diet on measured variables

Providing pregnant mink dams with 19% of ME from CP resulted in lower body weights at birth in the male mink offspring used in the present study (Vesterdorf et al., 2012). When lactating dams were fed an adequate protein supply, the male offspring experienced a period of catch-up growth such that body weights were similar to controls by 3 weeks of age (Vesterdorf et al., 2012). A previous study, which provided mink foetuses with only 14% of ME from CP, observed lower body weights at birth and slightly slower catchup growth (Matthiesen et al., 2010a). In those mink, the severe low protein provision during foetal life resulted in lower OXP in the male mink exposed to an AP diet early after weaning (Matthiesen et al., 2012). However, the present study shows that a more moderate low protein provision during foetal life (Vesterdorf et al., 2012) does not affect metabolic traits after weaning in male mink. It is known that the time of nutritional rehabilitation is of utmost importance to correct the effects of foetal nutritional restriction (Desai et al., 1996). Our findings suggest that the post-natal rather than the pre-natal period is the most critical for overall growth in mink kits that are born altricial. Indeed, low protein provision during foetal life did not affect body and organ growth in the 8- or 50-week-old mink used in the present study, which agrees with published data for young mink (Matthiesen et al., 2012) and rats (Desai et al., 1996) and yearling female mink (Matthiesen et al., 2010b) respectively. Plasma IGF-1, leptin and insulin levels were not affected by low protein provision during foetal life in the present study, a finding that concurs with previous results in weanling male mink (Matthiesen et al., 2012) and young rats (Pinheiro et al., 2008; Gosby et al., 2009). Changes in key regulatory hormones induced by low protein provision during foetal life are age-specific in rats (Ozanne and Hales, 2002). As such, low protein provision during foetal life was found to affect plasma hormone levels in adolescent male rats exposed to a low protein diet during lactation (Zambrano et al., 2006), whereas adequate nutrient supply after birth alleviated these changes in the hormone profile (Dahri et al., 1995). We suggest that any effects of the pre-natal diet on body and organ growth and on plasma hormone levels were alleviated early in post-natal life as the 12

Nitrogen metabolism

Low protein provision caused a lower retention of N compared to the AP mink early after weaning, as was previously observed in similar aged mink (Larsson et al., 2012; Matthiesen et al., 2012). Hence, the animals were not able to retain N to their full capacity, which indicates that the CP supply of 24% of ME from CP was below the protein requirement for this period of rapid growth. Whereas exposure of similar aged male mink kits to 30% of ME from CP resulted in a lower level of retention of N, but a better efficiency in terms of utilizing the digested N for retention (Larsson et al., 2012), the present study shows that the LP mink were not able to compensate for the lower N provision by increasing the efficiency of utilization of N for retention when exposed to a dietary CP supply of 24%. Our finding therefore supports the conclusion of Larsson et al. (2012) that the recommended 40% of ME from CP during the period of rapid growth remains valid (Lass en et al., 2012). Energy metabolism, substrate oxidation and whole body composition

Energy balance was not affected by the low protein provision during the period of rapid growth at 8 weeks of age. However, as expected, low protein provision resulted in more energy being retained in fat than was observed in the AP mink. Mink skeletal development is complete by ~18 weeks of age (Charlet-Lery et al., 1980) and the requirement for CP for muscle mass growth decreases, which in the present study was reflected in higher RFE and lower RPE, irrespective of dietary treatment. Body weight gain from 19 weeks onwards was thus accomplished through a higher accretion of fat, which concurs with previous findings in mink (Hellwing et al., 2005). Because low protein provision caused a 27% lower ME intake at 25 weeks of age, but did not affect HE, the energy balance approached zero, which explains the plateau in body weights. The cat, another strict carnivore, can adapt its protein oxidation to the dietary protein intake, provided that the protein requirement is met (Green et al., 2008). Because the nitrogen balance of the LP mink was similar to that of AP mink from 19 through 50 weeks of age, it can be inferred that the protein Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH

K. Vesterdorf et al.

requirement was met by the LP diet in the older mink kits, and further, that the LP mink met the low level of protein intake by adapting the rate of protein oxidation accordingly, as has been shown previously in cats (Green et al., 2008) and mink (Fink et al., 2004; Matthiesen et al., 2010a). Our study shows that prolonged exposure to low protein provision throughout the first year of life for male mink caused changes to their metabolism, as evidenced by a reduced lean mass at 50 weeks of age. Similar findings have been reported for proteinrestricted adult rats (Desai et al., 1996; Gosby et al., 2009) and pigs (Close et al., 1983). Thus, it is possible to alter the body composition of adult mink by longterm exposure to a low protein provision from the time of weaning. Body weights

The AP diet met the practical CP recommendations outlined for growing mink (Lass en et al., 2012), and the AP mink exhibited a normal growth as a consequence, thus confirming previous studies in mink (Skrede, 1978; Glem-Hansen, 1980; Ty€ opp€ onen et al., 1986; Damgaard et al., 1998b; Hellwing et al., 2005). The effect of low protein provision on kit growth performance was not observed until 21 weeks of age and supports similar findings in protein-restricted mink (Glem-Hansen, 1980; Damgaard et al., 1998b). An adequate level and quality of nutrition is of the utmost importance if an animal is to express its potential for growth; however, because the LP mink were fed a low protein supply, the subsequent lower N balance was reflected in a higher degree of fat retention in these animals cf AP mink. Low protein provision thus resulted in lower maximal body weights, which were reached earlier in the growth period than in AP mink. This finding concurs with previous studies (Ty€ opp€ onen et al., 1986; Damgaard et al., 1998b) and indicates that the low protein provision prevented the LP mink from reaching their full growth potential.

Low protein provision affects male mink kit metabolism and development

et al., 2009). The lower plasma IGF-1 levels in LP males at 25 weeks of age were thus explained by the lower feed intake and restricted growth at this age, supporting the findings of Gosby et al. (2009). Low protein provision had no effect on circulating levels of insulin, which is in agreement with similar findings in weanling male mink (Matthiesen et al., 2012). Contrary to the findings of Matthiesen et al. (2012), however, there was no effect of low protein provision on plasma leptin levels in the 8-week-old kits of the present study, whereas reduced plasma levels were observed at 25 weeks of age. Plasma leptin levels reflect energy intake and body weight changes in mink (Tauson and Forsberg, 2002), and are related to body fat mass (Blache et al., 2000), which explains the lower plasma leptin levels at this age. The plasma leptin levels observed in the present study were higher than those observed in mink females (Tauson et al., 2004; Matthiesen et al., 2010a), agreeing with studies in rats where a similar gender difference was observed (Zambrano et al., 2006). Organ development and liver composition

Plasma IGF-1 concentrations were affected by the low protein provision during the period of rapid growth in 8-week-old kits, which concurs with a previous study in rats (Gosby et al., 2009). IGF-1 regulates foetal and post-natal growth (Baker et al., 1993), and because the nutritional plane of the individual influences IGF1 levels (Underwood et al., 1994), growth restriction in rats exposed to low protein provision after weaning was explained by low concentrations of IGF-1 (Gosby

Pancreas and kidney mass was negatively affected by the low protein provision during growth, agreeing with findings in male mink kits (kidney: Larsson et al., 2012) and rats (pancreas and kidney: Desai et al., 1996). Selective organ growth occurred such that kidney regained mass before liver and pancreas, as has been found previously in the rat (Desai et al., 1996). A low protein provision resulted in reduced liver mass at 8 weeks of age, which concurs with previous findings in similar aged mink (Larsson et al., 2012; Matthiesen et al., 2012), in pregnant (Matthiesen et al., 2010a) and lactating mink dams (Fink et al., 2007), and in rats (Desai et al., 1996) and cats (Park et al., 1999). Taken together with the nitrogen and energy balance results, our findings indicate that reduced liver growth may be related to the low protein provision, as less liver mass was needed for metabolizing the protein in the LP diet during the growth period. Hence, differences in the ability to regulate OXP and to adapt to different levels of CP supply were likely related to differences in liver mass. The fat content in livers from 25–week-old LP mink was higher cf livers from controls. Our finding that hepatic lipidosis was related to increased liver mass confirms a previous observation in similarly aged LP mink (Damgaard et al., 1998a) and is supported by findings in cats where prolonged nutrient deficiency resulted in hepatic lipidosis (Biourge et al., 1994).

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Plasma hormone concentrations

Low protein provision affects male mink kit metabolism and development

Nutrient deficiency in cats results in increased lipolysis in the peripheral tissues (Armstrong and Blanchard, 2009). The mobilized free fatty acids are taken up in the hepatocytes (Hall et al., 1997) where they may be converted into triglycerides. In the cat, however, protein malnutrition has been found to cause limited synthesis of and modifications to the hepatic lipoproteins (Alpers and Sab esin, 1987; Pazak et al., 1998, respectively) responsible for removing triglycerides from the liver (Dimski, 1997), thus causing hepatic accumulation of lipids (Blanchard et al., 2004). Hence, the occurrence of hepatic lipidosis in the LP mink may have been caused by the long-term effect of protein deficiency during the growth period and prolonged low feed intake, resulting in accumulation of lipids in the liver. Further studies are now needed to investigate the effect of low protein provision on liver metabolism.

K. Vesterdorf et al.

tein requirement during the period of rapid growth. The protein supply of the LP diet was therefore too low to allow maximum protein retention during growth and prevented the mink from reaching their full growth potential. Our results indicate that the ability to regulate the oxidation of protein, thus adapting to different levels of protein supply, was likely caused by differences in liver mass. We suggest that the occurrence of hepatic lipidosis at the end of the growth period was caused by a long-term effect of low protein provision and prolonged low feed intake during growth. Further studies are now needed to investigate the effect of low protein provision on liver metabolism, and to elucidate whether any metabolic modifications may be expressed in the F2-generation from male mink that have been protein restricted during foetal life. Acknowledgements

Conclusions Exposure to a low protein diet (19% of ME from CP) in utero did not affect the physiological responses to an adequate or low protein provision after weaning in male mink, and hence did not increase the ability to conserve protein. Our findings suggest that any modifications caused by foetal low protein provision observed in perinatal life were alleviated by the time of weaning when lactating dams were exposed to an adequate level of nutrition post-partum. Our results indicate that ~20% of ME from CP was below the proReferences Alpers, D. H.; Sab esin, S. M., 1987: Fatty liver: Biochemical and clinical aspects. In: L. Schiff, E. R. Schiff (eds), Disease of the Liver, 6th edn. Lippincott, Philadelphia, Pensylvania, USA. Armstrong, P. J.; Blanchard, D., 2009: Hepatic lipidosis in cats. Veterinary Clinical North American Small Animal Practice 39, 599–616. Baker, J.; Liu, J. P.; Robertson, E. J.; Efstratiadis, A., 1993: Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75, 73–82. Barker, D. J.; Winter, P. D.; Osmond, C.; Margetts, B.; Simmonds, S. J., 1989: Weight in infancy and death from ischaemic heart disease. Lancet 334, 577–580. Barker, D. J.; Bull, A. R.; Osmond, C.; Simmonds, S. J., 1990: Fetal and placental size and risk of hypertension in

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The authors wish to thank Maria Larsson for assistance with balance experiments, Ebba de Neergaard Harrison, Inge Mejdahl and Merethe Stubgaard for their technical assistance with the chemical analyses; Boye Pedersen for animal handling; and Abdalla Ali for the Respiration Unit operation at the University of Copenhagen. Furthermore, the authors wish to thank Margaret Blackberry for her RIA work at the University of Western Australia. The project was funded by the Danish Agency for Science Technology and Innovation and Danish Fur Breeders Research Centre.

adult life. British Medical Journal 301, 259–262. Biourge, V. C.; Groff, J. M.; Munn, R. J.; Kirk, C. A.; Nyland, T. G.; Madeiros, V. A.; Morris, J. G.; Rogers, Q. R., 1994: Experimental induction of hepatic lipidosis in cats. American Journal of Veterinary Research 55, 1291–1302. Blache, D.; Tellam, R. L.; Chagas, L. M.; Blackberry, M. A.; Vercoe, P. E.; Martin, G. B., 2000: Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep. Journal of Endocrinology 165, 625–637. Blanchard, G.; Paragaon, B. M.; S erougne, C.; F er ezou, J.; Milliat, F.; Lutton, C., 2004: Plasma lipids, lipoprotein composition and profile during induction and treatment of hepatic lipidosis in cats and the metabolic effect of one daily meal in healthy cats. Journal of Animal Physiology and Animal Nutrition 88, 73–87.

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Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH

Low protein provision during the first year of life, but not during foetal life, affects metabolic traits, organ mass development and growth in male mink (Neovison vison).

Low protein provision in utero and post-partum may induce metabolic disorders in adulthood. Studies in mink have mainly focused on short-term conseque...
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