Targeting Heightened Inflammatory Tone in IUGR Neonatal Lambs via Daily Oral Supplementation of ω-3 PUFA Improved Growth Rates, Muscle Mass, and Adiponectin Signaling

TL;DR

Supplementing ω-3 PUFA in IUGR lambs improved growth, muscle mass, and adiponectin signaling by reducing inflammation.

Contribution

Daily oral ω-3 PUFA supplementation resolves growth deficits in IUGR lambs by targeting neonatal inflammation.

Findings

ω-3 PUFA supplementation fully resolved bodyweight deficits in IUGR lambs by day 28.

ω-3 PUFA improved subcutaneous fat, visceral organ growth, and skeletal muscle mass in IUGR lambs.

ω-3 PUFA restored myoblast populations and AdipoR2 content, resolving asymmetric growth restriction.

Abstract

Background/Objectives: Circulating inflammatory cytokines and tissue sensitivity are both elevated following heat stress-induced intrauterine growth restriction (IUGR). Cytokines disrupt myoblast function and muscle growth, and thus we hypothesized that suppressing inflammatory tone in IUGR-born lambs by supplementing anti-inflammatory nutraceuticals would improve early postnatal growth. Methods: IUGR lambs produced by maternal heat stress were supplemented daily with 42 mg/kg oral omega-3 polyunsaturated fatty acid (ω-3 PUFA) Ca2+ salts or placebo from birth to 28 days of age. Results: By day 28, the 21% lighter bodyweights for IUGR lambs were fully resolved by ω-3 PUFA due to the complete recovery of average daily gain. Subcutaneous fat deposition and visceral organ growth were modestly diminished in IUGR-born lambs, but skeletal muscle mass was more markedly restricted. This coincided with 63% less muscle AdipoR2 but 27% greater circulating adiponectin. ω-3 PUFA reduced or eliminated deficits in subcutaneous fat, visceral organs, and five of the six individual muscles assessed, which corresponded with rescue of myoblast populations and AdipoR2 content. In turn, asymmetric growth restriction was resolved at one month of age. Conclusions: These findings show that targeting heightened inflammatory tone during the neonatal period in IUGR-born offspring can recover early growth in skeletal muscle and other soft tissues.

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1. Introduction

Maternal heat stress during peak placental development leads to intrauterine growth restriction (IUGR) of the fetus due to placental stunting [1]. Sustained fetal hypoxemia and hypoglycemia caused by placental insufficiency induce adaptive fetal programming aimed at nutrient sparing [2,3,4]. These programming mechanisms limit the growth of muscle and other soft tissues to below genetic potential [3,5,6], which benefits the undernourished fetus but is maladaptive after birth when O_2_ and nutrients are no longer limited [7,8]. Consequently, IUGR-born offspring are characterized by lighter birthweight, metabolic dysfunction, and poor body composition [7,8]. In livestock, these deficits compromise growth efficiency, carcass quality, and meat yield [9,10]. In humans, they increase the risk of obesity, hypertension, type 2 diabetes, and other metabolic disorders [11,12]. Recent studies show that fetal hypoxic stress stimulates inflammatory cytokine secretion and enhances inflammatory signaling pathways in muscle and other tissues [13,14,15,16]. Moreover, this heightened inflammatory tone persists in IUGR-born offspring [7,17,18,19]. We recently documented the role of inflammatory programming in skeletal muscle-centric metabolic dysregulation in IUGR fetuses and offspring [8,20,21]. Resolving heightened inflammatory tone in IUGR fetuses improved muscle growth and myoblast function near term [15,22], but the impact of postnatal inflammatory tone on growth restriction has not been explored. Therefore, we sought to determine whether mitigating inflammation by supplementation of ω-3 polyunsaturated fatty acids (ω-3 PUFA), which have well-documented anti-inflammatory properties [23,24,25,26], would improve early lean growth and body composition in IUGR-born lambs.

2. Materials and Methods

2.1. Animals and Experimental Design

These studies were approved by the Institutional Animal Care and Use Committee at the University of Nebraska–Lincoln, which is accredited by AAALAC International. IUGR lambs were produced via maternal hyperthermia as previously described [7,20,21]. Details of the experimental design for this study as well as metabolic and health outcomes were described previously [21]. In brief, Polypay ewes were timed-mated to a single sire and housed at 40 ± 1 °C, 35 ± 5% relative humidity from day 40 to 95 of gestation to produce placental insufficiency-induced IUGR lambs. Control lambs (n = 12) were produced from pair-fed ewes housed under thermoneutral conditions (19 ± 1 °C) throughout gestation. Lambs were weaned at birth, fed pooled colostrum, and hand-raised on ad libitum milk replacer (Land O’Lakes, Arden Hills, MN, USA) until 28 days of age. Beginning at birth, IUGR lambs were randomly assigned to receive daily oral boluses of molasses carrier containing no additive (IUGR; n = 11) or ω-3 PUFA Ca^2+^ salt (42 mg/kg, Strata, Virtus Nutrition, Corcoran, CA, USA) (IUGR + ω3; n = 12), as previously described [21]. This product is 16% eicosapentaenoic acid and docosahexaenoic acid (EPA + DHA). The 7 mg/kg daily EPA + DHA dose was from previous findings [27] and manufacturer recommendations. Lambs were euthanized via barbiturate overdose and necropsied at 28 days of age.

2.2. Biometrics

Lambs were weighed daily at 0800, just prior to the first feeding of the day. Crown circumference, hindlimb cannon bone length, abdominal circumference, and crown–rump length (i.e., body length) were measured at birth and weekly thereafter. At necropsy, organs, whole hindlimbs, and flexor digitorum superficialis, biceps femoris, semitendinosus, gastrocnemius, soleus, and longissimus dorsi muscles were weighed.

2.3. Estimated Muscle Mass and Body Composition

Bioelectrical impedance analysis was performed in live lambs at 23 days of age, as previously described [7]. Briefly, two sets of equally spaced electrodes were connected to 20G MONOJECT aluminum-hub needles placed subcutaneously over the longissimus dorsi muscle. Reactance, resistance, and phase angle were measured via a Quantum V (RJL Systems, Clinton Township, MI, USA). One electrode pair was placed 5 cm behind the scapula, and the other was placed 5 cm in front of the leading edge of the pelvis. Four consecutive 5-second measurements were recorded and averaged. Ultrasonic measurements of the loin eye area, loin depth, and backfat thickness were performed at 23 days of age, as previously described [7]. Images were captured between the 12th and 13th ribs using an IBEX PRO ultrasound (E.I. Medical Imaging, Loveland, CO, USA) with an L6.2 12-cm linear transducer. Vegetable oil was applied as a couplant, and the transducer was placed at an approximate 45° proximal angle. Measurements were determined utilizing the caliper tracing mode and averaged across 3 images/lamb. At necropsy, the longissimus dorsi muscle was frozen, and commercial proximate analyses were performed to determine moisture, protein, fat, ash, carbohydrate, and calorie content (Midwest Laboratories, Omaha, NE, USA; ISO/IEC 17025:2017), as previously described [7].

2.4. Circulating IGF-1 and Adiponectin

Blood samples were collected at birth and just before necropsy via jugular venipuncture into EDTA tubes as previously described [7,17]. Whole blood was centrifuged (14,000× g, 5 min, room temperature) to isolate plasma, which was stored at −80 °C. Commercial ELISA kits were used to quantify plasma concentrations of IGF-1 (ALPCO, Salem, NH, USA) and adiponectin (Biomatik, Kitchener, ON, Canada) in duplicate. Inter-assay and intra-assay coefficients of variation were less than 15% for both assays.

2.5. Skeletal Muscle Adiponectin Receptors and Fiber Types

At necropsy, semitendinosus muscle samples were collected and frozen in liquid nitrogen, and total protein was isolated as previously described [21]. Muscle samples were homogenized via sonication (3 × 5 s) in a low-salt TRIS-NaCl buffer + 2.5% protease + 2.5% phosphatase inhibitor and then centrifuged (14,000× g, 5 min, 4 °C). Total protein was quantified from the supernatant using a Pierce BCA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). 50-μg protein aliquots were mixed with Bio-Rad 4× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) and heated at 95 °C for 5 min. Samples were brought to room temperature, separated by SDS-PAGE, and transferred to Bio-Rad poly-vinylidene fluoride low-fluorescent membranes. Membranes were washed with tris-buffered saline with tween (TBS-T) and incubated with Bio-Rad EveryBlot Blocking Buffer at room temperature for 10 min. Membranes were then incubated at 4 °C overnight with rabbit anti-serum raised against T-cadherin (CDH-13; 1:1000, MilliporeSigma, Burlington, MA, USA) or rabbit anti-serum raised against adiponectin receptor 2 (AdipoR2; 1:1000, Abcam Ltd., Cambridge, UK). Finally, membranes were washed in TBS-T and incubated at room temperature for 1 h with goat anti-rabbit IR800 IgG secondary anti-serum (LI-COR Biosciences, Lincoln, NE, USA). Membranes were scanned using a LI-COR Odyssey Infrared System, and protein bands were analyzed with LI-COR Image StudioLite Software 5.2. Each protein of interest was normalized to total protein. Muscle fiber-type ratios were estimated from myosin heavy chain (MyHC) proportions determined via electrophoresis, as previously described [7]. In brief, semitendinosus samples were homogenized via sonication and centrifuged as above, and total protein concentrations were quantified from supernatant utilizing the Pierce BCA Assay Kit. A 40-μg aliquot of protein was combined with Bio-Rad 4× Laemmli Sample Buffer to make a 1× solution, which was incubated at room temperature for 10 min, heated to 70 °C for 10 min, and loaded into a Bio-Rad 4–15% Mini-PROTEAN TGX Protein Gel at 40 μg/well. MyHC isoforms were separated by SDS-PAGE. Electrophoresis was performed on a Bio-Rad Mini-PROTEAN Tetra Cell at 110 V for 3.5 h at room temperature. Gels were stained overnight with Gel-Code Blue (Thermo Fisher), destained in deionized water, and imaged on a LI-COR Odyssey infrared imaging system. MyHC-I, MyHC-IIa, and MyHC-IIx bands were quantified by LI-COR Image Studio Lite Software Ver 5.2.

2.6. Muscle Histology

Myoblast populations within semitendinosus muscles were evaluated via immunofluorescent staining, as previously described [7]. In brief, cross-sectional samples of the semitendinosus were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), embedded in OCT compound, and stored at −80 °C. Cryosections (10 µm) were mounted on charged microscope slides (Thermo Fisher) and dried for 30 min at 37 °C. Once dried, slides were rehydrated in PBS, boiled for 20 min in 10 mM citric acid, and allowed to cool to room temperature for antigen retrieval. Non-specific staining was blocked via incubation in 0.5% blocking reagent (Akoya Biosciences, Marlborough, MA, USA) for 1 h at room temperature in a humidified container. Slides were incubated overnight at 4 °C with primary antibodies diluted in PBS + 1% bovine serum albumin (MilliporeSigma). Negative controls were incubated in PBS + 1% bovine serum albumin only. Sections were incubated with mouse anti-serum raised against pax7 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) to identify myoblasts and were counterstained with rabbit antiserum raised against Ki-67 (1:400, Cell Signaling Technology, Inc., Danvers, MA, USA) to identify proliferating cells. Immunocomplexes were detected with affinity-purified immunoglobulin antiserum conjugated to AlexaFluor 555 or AlexaFluor 594 (1:1000; Cell Signaling). Immunofluorescent images were visualized on an Olympus IX73 and digitally captured with a DP80 microscope camera (Olympus Corp., Center Valley, PA, USA). Images were analyzed with ImageJ 1.54 Software (National Institutes of Health, Bethesda, MD, USA) to assess myoblasts. Populations within each muscle were quantified from a minimum of 1000 nuclei across 4 non-overlapping fields of view.

2.7. Statistical Analysis

Data collected at necropsy were analyzed by ANOVA using the mixed procedure of SAS 9.4 (SAS Institute, Cary, NC, USA) for the fixed effects of experimental group, sex, and birth number (i.e., singletons, twins, ortriplets). Fisher’s LSD test was used for mean separation. Weekly growth metrics and blood hormones were analyzed using the mixed procedure with repeated measures to analyze the effects of experimental group, age, and group × age interaction, as well as sex and birth number. Best-fit statistics were used to select appropriate covariance structures. Lamb was considered the experimental unit. Significant differences among means were declared at p ≤ 0.05 and tendencies at p ≤ 0.10. All data are presented as least-squares means ± standard error of the means.

3. Results

3.1. Inflammatory Indicators

Blood and tissue indicators of inflammation were previously reported for these lambs [21]. Briefly, plasma eicosapentaenoic acid (EPA) at birth was 22% less (p ≤ 0.05), plasma TNFα throughout the study was 31% greater (p ≤ 0.05), and muscle TNFα Receptor 1 (TNFR1) and IL-6 Receptor (IL6R) content at necropsy were 2.7-fold and 1.6-fold greater (p ≤ 0.05), respectively, for IUGR lambs than for controls. The increases in plasma TNFα and muscle IL6R were resolved in IUGR + ω3 lambs, but muscle TNFR1 remained elevated. IUGR-born lambs were also hyperlipidemic, as circulating triglyceride concentrations were 40% greater (p ≤ 0.05) than in controls at one month of age and were not resolved in IUGR + ω3 lambs.

3.2. Postnatal Growth

3.2.1. Milk Consumption and Bodyweight

No experimental group × age interaction was observed for daily milk intake, which was less (p ≤ 0.05) for IUGR lambs (3030 ± 63 mL/day) than controls (3894 ± 56 mL/day) and was intermediate for IUGR + ω3 lambs (3182 ± 66 mL/day) (Supplemental Figure S1). However, milk intake normalized to bodyweight did not differ among controls, IUGR, and IUGR + ω3 lambs (588 ± 29, 507 ± 27, and 492 ± 31 mL/kg/day). An experimental group × age interaction was observed (p ≤ 0.05) for bodyweight but not for other growth metrics. At birth, bodyweights were less (p ≤ 0.05) for IUGR and IUGR + ω3 lambs than for controls (Figure 1A, Supplemental Figure S2). On day 7, 14, and 21, bodyweights were less (p ≤ 0.05) for IUGR lambs than for controls and were intermediate for IUGR + ω3 lambs. At 28 days, bodyweights were less (p ≤ 0.05) for IUGR lambs but not IUGR + ω3 lambs compared to controls. Adjusted 30-day bodyweights were less (p ≤ 0.05) for IUGR lambs (10.1 ± 0.5 kg), but not IUGR + ω3 lambs (11.6 ± 0.5 kg), compared with controls (12.3 ± 0.2 kg). However, bodyweight changes from birth did not differ among groups (Figure 1B). Average daily gain between birth and day 7, between day 8 and 14, and between day 15 and 21 did not differ among groups (Table 1). Average daily gain between day 22 and 28 and from birth to day 28 tended to be less (p ≤ 0.10) for IUGR but not IUGR + ω3 lambs than for controls.

3.2.2. Biometrics

No experimental group × age interactions were observed for any biometric measures. Crown circumference, abdominal circumference, and hindlimb cannon bone length were less (p ≤ 0.05) for IUGR lambs than for controls and were intermediate for IUGR + ω3 lambs (Figure 2A–C). Body length was less (p ≤ 0.05) for IUGR lambs but not for IUGR + ω3 lambs, compared to controls (Figure 2D). Crown circumference-to-bodyweight, abdominal circumference-to-bodyweight, and hindlimb cannon bone length-to-bodyweight ratios were greater (p ≤ 0.05) for IUGR lambs but not for IUGR + ω3 lambs compared to controls (Figure 3A–C). Body length-to-bodyweight ratios were greater (p ≤ 0.05) for IUGR lambs than for controls and were intermediate for IUGR + ω3 lambs (Figure 3D).

3.2.3. Skeletal Muscle Mass

Whole hindlimb, flexor digitorum superficialis, longissimus dorsi, biceps femoris, and semitendinosus muscles were lighter (p ≤ 0.05) for IUGR lambs but not for IUGR + ω3 lambs compared to controls (Table 2). Soleus muscles were lighter (p ≤ 0.05) for IUGR and IUGR + ω3 lambs compared to controls. Gastrocnemius muscles were lighter (p ≤ 0.05) for IUGR lambs than for controls and were intermediate for IUGR + ω3 lambs.

3.2.4. Organ Weights

Kidney and liver weights were less (p ≤ 0.05) for IUGR lambs but not for IUGR + ω3 lambs compared to controls (Table 3). Lung and heart weights were less (p ≤ 0.05) for IUGR lambs than for controls and were intermediate for IUGR + ω3 lambs. Brain weights were not different between IUGR lambs and controls but were greater (p ≤ 0.05) for IUGR + ω3 lambs than for IUGR lambs. Heart-to-bodyweight ratios were not different among groups. However, brain-to-bodyweight ratios were greater (p ≤ 0.05) for IUGR lambs than for controls and were intermediate for IUGR + ω3 lambs.

3.3. Body Composition Estimates

3.3.1. Live-Animal Body Composition

BIA-estimated fat-free lean mass, fat-free soft tissue mass, total sum of the leg, sirloin, rack, shoulder, neck, riblets, shank, and lean trim mass (SUM), sum of the leg, sirloin, loin, rack, and shoulder mass (LSRLS), and sum of the leg, sirloin, and loin mass (LSL) did not differ among experimental groups (Table 4). Ultrasound-estimated back fat thickness (Figure 4A), loin eye area (Figure 4B), and loin depth (Figure 4C) between the 12th and 13th ribs were less (p ≤ 0.05) for IUGR lambs but not for IUGR + ω3 lambs compared to controls.

3.3.2. Skeletal Muscle Composition

Longissimus dorsi muscle protein content was less (p ≤ 0.05) for IUGR and IUGR + ω3 lambs than for controls (Figure 5A). Muscle fat content and fat-to-protein ratios tended to be greater (p ≤ 0.10) for IUGR and IUGR + ω3 lambs than for controls (Figure 5B,C). Muscle carbohydrate content was greater (p ≤ 0.05) for IUGR lambs but not for IUGR + ω3 lambs compared to controls (Figure 5D). Muscle moisture content (76.6 ± 0.3%), ash content (1.14 ± 0.07%), and caloric content (1.01 ± 0.02 Cal/g) did not differ among groups.

3.4. Muscle Fiber Type Ratios and Myoblast Profiles

Proportions of semitendinosus MyHC-I (60.9 ± 3.3%), MyHC-IIa (25.9 ± 2.8%), and MyHC-IIx (12.8 ± 2.3%) did not differ among groups. Pax7^+^ nuclei in the semitendinosus were less abundant (p ≤ 0.05) for IUGR lambs but not for IUGR + ω3 lambs compared to controls (Figure 6A). Abundance of Ki67^+^/pax7^+^ nuclei did not different among groups (Figure 6B).

3.5. Circulating IGF-1 and Adiponectin Signaling

No experimental group × age interactions were observed for circulating IGF-1 or adiponectin concentrations. Plasma IGF-1 did not differ among experimental groups (Figure 7A). Plasma adiponectin was greater (p < 0.05) for IUGR lambs than for controls and was intermediate for IUGR + ω3 lambs, regardless of day (Figure 7B). Both IGF-1 and adiponectin concentrations were greater (p < 0.05) at necropsy than at birth, regardless of experimental group. Skeletal muscle AdipoR2 content tended to be less (p < 0.10) for IUGR lambs than for controls and was intermediate for IUGR + ω3 lambs (Figure 7C). Skeletal muscle CDH-13 content did not differ among experimental groups (Figure 7D). Representative gel images for each protein of interest are provided below the respective graphs.

4. Discussion

In this study, we found that mitigating heightened inflammatory tone in IUGR-born offspring improved poor body composition by recovering skeletal muscle, adipose tissue, and organ mass. We previously documented systemic inflammation and enhanced inflammatory sensitivity in IUGR-born lambs [17,21]. Here, we show that targeting it from birth with daily oral supplementation of an anti-inflammatory nutraceutical rescued early soft tissue growth. The most profound effects of IUGR occurred in muscle hypertrophy and predictably coincided with reduced populations of hypertrophy-facilitating myoblasts. More modest restriction was observed for visceral organ growth and subcutaneous fat deposition, and only slight restriction was observed for skeletal growth. Inflammatory amelioration improved postnatal growth for each of these tissues, which essentially resolved bodyweight deficits and hallmark asymmetrical body composition by one month of age. Although this study does not explicitly rule out any potential indirect benefits of supplementing ω-3 PUFA, including mechanisms unrelated to inflammation, the strongest indication from the present findings is that targeting the intensified inflammatory tone in IUGR-born offspring is a promising strategy for recovering many of the inherent deficits in lean growth and body composition.

Better growth rates and improved lean mass observed when inflammatory tone was mitigated demonstrate the underlying mechanistic role of inflammatory adaptations in IUGR outcomes [15,17,21]. Despite being born 25% lighter, reestablishing normal rates of gain culminated in fully recovered body weights shortly before the typical weaning age for lambs. In contrast, unsupplemented IUGR lambs were still almost 18% lighter than normal at this age. Thus, without inflammatory abatement, the postnatal catch-up growth often observed following IUGR [8,28] recovered only about one-fourth of the original size disparity present at birth. Lighter bodyweights were the product of cumulative growth restriction in several soft tissues, most of which were partially or fully recovered when inflammatory tone was tempered after birth. Suppression of peripheral soft tissue growth facilitates brain- and bone-sparing nutrient repartitioning that is often necessary for the IUGR fetus to survive but is unnecessarily sustained postnatal [7,20,22]. Consequently, the brain and skeleton do not develop the same degree of growth restriction as most of the peripheral soft tissues [29]. In the present study, this was reflected in little or no reduction in absolute size of the brain, cranium, and lower leg bones and greater than normal proportional size of each relative to bodyweight. Likewise, the return of normal size-to-bodyweight ratios following ω-3 PUFA supplementation was due to the effects on bodyweight more so than on brain mass or bone length proper. These findings extend our previous observations implicating prenatal inflammation in fetal growth restriction [8,28] by confirming the role of persistent inflammatory programming in the permanent restriction of soft tissue growth.

Disproportionate reduction of muscle mass in IUGR-born lambs at one month of age illustrates the problematic persistence of muscle-centric programming resulting from intrauterine stress [30,31,32,33]. Despite the resolution of hypoxemia and hypo-nutrition at birth, IUGR loin eye areas remained 22% smaller, and the six muscles assessed in this study were on average almost 25% lighter. By comparison, IUGR-born lambs exhibited only 18% lighter bodyweights, 14% less subcutaneous fat, and 2–18% lighter organs. With the exception of the soleus, ω-3 PUFA supplementation partially or fully rescued skeletal muscle mass, which illustrates intrinsic inflammatory suppression of muscle growth capacity. Although postnatal β2 adrenergic stimulation improved muscle hypertrophy and lean mass in a previous study of IUGR lambs, it was conspicuously associated with evidence of a direct anti-inflammatory effect [7]. Moreover, the recovery of muscle mass by inflammatory mitigation in the present study was more profound than that previously observed with the β2 agonist. This implicates inflammatory programming as the likely major driver of skeletal muscle-centric growth restriction, despite IUGR muscle exhibiting both inflammatory and adrenergic dysregulation [7,8,15,16,17,21]. Improvements in growth following inflammatory mitigation were not uniform across all of the muscles assessed in this study. The benefits were of greatest magnitude for muscles previously shown to be made up primarily of fast-twitch or mixed fiber types [30,31,32,33]. In fact, the slow-twitch soleus was the only muscle in our study for which size was not at least partially recovered by ω-3 PUFA supplementation. These differential improvements were perhaps due to the more rapid assimilation of ω-3 PUFA into fast-twitch muscle fibers [32,34], which produced similarly disparate increases in protein synthesis in a previous study [35].

Improved muscle mass following the tempering of inflammatory tone in IUGR-born lambs corresponded with the restoration of myoblast populations. Myoblasts are the source of the myonuclei that accumulate within fibers to drive hypertrophic postnatal muscle growth [36]. Because myoblast function is rate-limiting for hypertrophy [37,38], the smaller populations observed in IUGR muscle at one month of age help explain the corresponding reductions in muscle mass. Likewise, the observed recovery of myoblast numbers presumably facilitated the improvements in muscle growth capacity exhibited by ω-3 PUFA-supplemented IUGR lambs. Our previous studies that identified intrinsic functional deficits in IUGR myoblasts had indicated a potential role for inflammatory programming [15,16,38], since inflammatory cytokines impair myoblast progression by locking the cells into eccentric proliferation patterns [16,39,40]. Although the present study offers no insight into the potential effect of ω-3 PUFA on muscle growth in uncompromised neonates, the present findings seem to confirm the role of inflammatory suppression by showing that recovery of myoblast numbers and muscle growth occurred in concert following inflammatory mitigation.

The 63% reduction in AdipoR2 observed in the semitendinosus muscle of IUGR-born lambs may help explain their diminished myoblast populations. Genetic animal models and cell culture studies have documented the role of adiponectin signaling in β-catenin-mediated proliferation and differentiation of myoblasts [41,42]. The increase in circulating concentrations of adiponectin was paradoxical and did not correspond to circulating IGF-1 concentrations as we had expected based on previous studies in humans [43,44]. However, we postulate that this increase in circulating adiponectin was a compensatory response to the large reduction in muscle adiponectin receptors and to the well-documented insulin resistance in IUGR offspring [7,45], as it is a powerful insulin sensitizer [46]. Moreover, circulating adiponectin concentrations in IUGR-born children were inconsistent across studies and may have been highly influenced by the severity of IUGR, sex of the child, and age at assessment [43,44,47,48,49,50,51]. Nevertheless, inflammatory mitigation in the present study resulted in partial rescue of adiponectin concentrations and muscle adiponectin receptor content in our IUGR-born lambs, demonstrating responsiveness of adiponectin signaling to inflammatory programming.

Diminished subcutaneous adiposity in IUGR-born lambs concomitant with hyperlipidemia and intramuscular lipid accumulation was indicative of programmed changes in lipid flux. In studies of IUGR fetuses, greater blood and intracellular lipids coincided with elevated circulating acylcarnitines, which are biomarkers for impaired fatty acid oxidative metabolism [52,53]. It is reasonable to speculate that dysfunctional fatty acid metabolism may have persisted after birth to produce the abhorrent pattern of lipid flux observed in our IUGR-born lambs. However, we cannot rule out the possibility that diminished subcutaneous fat and increased intramuscular lipids were due to greater lipid utilization by muscle as a way to offset the previously documented impairments in glucose metabolism [21]. When inflammatory tone was mitigated, subcutaneous fat deposits returned to normal in our IUGR-born lambs, but intramuscular fat content of the loin did not. Although somewhat unexpected, this disparity was not necessarily surprising. Elevated inflammatory cytokines are generally lipolytic [54,55,56], but studies in farm animals show that intramuscular fat depots and subcutaneous fat depots respond to regulatory factors with different sensitivities [53,57]. Subcutaneous fat is naturally accreted at much higher rates than intramuscular fat [58,59], and the effect of IUGR on intramuscular fat accretion in pigs differed markedly among specific muscles, with the loin being particularly unresponsive [60]. It is also possible that the programming mechanisms responsible for reduced myoblast populations increased intramuscular adipocyte populations by proxy, as both progenitors originate from the same stem cell community [53,57]. Although this study did not attempt to quantify intramuscular adipocytes, the timing of lineage determination coincides with the progression of fetal pathophysiologies and the appearance of myoblast dysfunction in the IUGR fetus [61].

5. Conclusions

From this study, we can conclude that the heightened inflammatory tone previously observed in the IUGR fetus persists in IUGR-born offspring and is a key component of programmed growth deficits in skeletal muscle and other soft tissues. Instituting a daily anti-inflammatory supplement regimen at birth was an effective restorative approach for soft tissue growth following heat stress-induced IUGR. The improvement in muscle mass was particularly noteworthy and coincided with recovery of myoblast populations, which are rate-limiting for muscle growth, and with evidence of improved adiponectin signaling. In addition, inflammatory mitigation generally improved visceral organ growth and subcutaneous fat deposition, although the deficits from IUGR were less severe for these tissues than for muscle. The role of inflammatory programming on postnatal growth established by this study aligns with the previously reported role in metabolic dysfunction, which was also muscle-centric. From a practical standpoint, the better growth outcomes show that strategic supplementation of anti-inflammatory nutraceuticals may be an effective and practical approach for improving early growth and efficiency outcomes in meat animals following stress-induced IUGR.