Heat stress impact on placental growth and function

In sheep, the maximum placenta growth rate occurs between days 40 and 80 of gestational age (dGA), during which the placenta expands its surface area to form functional placentomes for maternal-fetal exchange.^{Reference Ehrhardt and Bell43} Reductions in placental growth and development were observed in mid-gestating ewes (75 dGA) following 25 d of maternal HS, even in the absence of reductions to fetal weight at this stage.^{Reference Vatnick, Ignotz, Mcbride and Bell20} The mechanisms by which HS restricts placental growth in livestock remain unclear. However, it is likely associated with a combination of physiological, vascular, hormonal, and cellular mechanisms that disrupt placental function and development. In HS animals, thermoregulatory adaptations redirect blood flow toward peripheral tissue, such as the skin, to enhance radiant heat loss.^{Reference Collin, Lebreton and Fillaut44} This adaptation is likely to reduce blood perfusion and cause cellular stress in visceral organs, including the placenta. It is well established that HS-induced placental growth restriction is associated with decreases in uterine and umbilical blood flows in late pregnancy.^{Reference Bell, Wilkening and Meschia21}

In mid-gestating ewes, placental growth restriction due to HS initially results in a compensatory increase in VEGF mRNA expression in the cotyledon after 15–20 d of HS.^{Reference Regnault, Orbus and de Vrijer45} However, prolonged exposure to HS from mid to late gestation results in reduced mRNA expression of VEGF and its receptor in the cotyledon,^{Reference Regnault, de Vrijer and Galan24} as well as lower cotyledonary or umbilical expression of endothelial nitric oxide synthase.^{Reference Galan, Regnault and Le Cras46–Reference Ziebell, Galan and Anthony48} In addition to vascular impairment, HS is linked to lower maternal concentrations of progesterone and ovine placental lactogen, indicating adverse effects on the development and function of trophoblast cells responsible for steroid production and metabolism.^{Reference Regnault, Orbus, Battaglia, Wilkening and Anthony49,Reference Bell, Mcbride, Slepetis, Early and Currie50} Additionally, HS has been associated with cellular-level changes, such as reduced placental cell numbers, lower DNA content, and decreased protein synthesis capacity in pregnant ewes.^{Reference Vatnick, Ignotz, Mcbride and Bell20,Reference Early, Mcbride, Vatnick and Bell51} These findings highlight that maternal HS disrupts placental growth and function through a multifactorial mechanism. However, it remains unclear whether these maladaptive changes provoke placental growth restriction or are simply a consequence of reduced placental mass in HS pregnant ewes.

The transport of oxygen, glucose, and amino acids across the placenta is mediated by passive diffusion, facilitated diffusion, and active transport, respectively.^{Reference Battaglia and Meschia52} Placental transport capacity for oxygen and glucose is reduced in HS ewes during late gestation, as a result of decreased placental permeability.^{Reference Bell, Wilkening and Meschia21,Reference Thureen, Trembler, Meschia, Makowski and Wilkening22,Reference Regnault, de Vrijer and Galan53} Additionally, HS ewes with placental insufficiency exhibit approximately 40% lower transplacental flux of amino acids from the maternal circulation to the fetus when adjusted for placental mass.^{Reference Anderson, Fennessey, Meschia, Wilkening and Battaglia23,Reference Ross, Fennessey, Wilkening, Battaglia and Meschia40} Although reduced blood flow can limit placental uptake and transfer of flow-limited substrates such as oxygen, it is unlikely to be the primary factor limiting glucose and amino acid transport in HS ewes.^{Reference Bell, Hay and Ehrhardt54} Rather, impaired placental transport capacity for glucose and amino acids is likely due to a reduced surface area and lower abundance of nutrient transporters per unit of placental weight, as both nutrients are transported via specific transporter proteins on placental membranes.^{Reference Ehrhardt and Bell55,Reference Regnault, de Vrijer and Battaglia56} In HS ewes, expression of the glucose transporter 8 (GLUT 8) is decreased during late gestation.^{Reference Limesand, Regnault and Hay25} Recent transcriptomic analyses in HS pregnant pigs have also revealed widespread downregulation of major placental nutrient transporter mRNA expression by mid-gestation.^{Reference Zhao, Liu and Marth57}

Fetal metabolic and endocrine response to heat stress-induced placental insufficiency

The developing fetus utilizes nutrients taken up from the umbilical circulation to meet two primary needs: supporting essential metabolic processes via oxidative metabolism and promoting tissue growth and protein accretion. In HS-induced FGR fetal sheep, there were lower weight-specific net fetal uptake rates for glucose,^{Reference Limesand, Regnault and Hay25,Reference Pendleton, Antolic and Kelly38,Reference Camacho, Davis and Kelly39} amino acids^{Reference Regnault, de Vrijer and Galan53,Reference Brown, Palmer and Teynor58} and oxygen despite an increased transplacental oxygen and glucose gradients.^{Reference Thorn, Regnault and Brown28,Reference Camacho, Davis and Kelly39,Reference Brown, Palmer and Teynor58} To explore the metabolic efficiency of fetal substrate utilization, the substrate/oxygen quotient was calculated (defined as the theoretical fraction of total fetal O₂ consumption required to completely oxidize the net umbilical uptake of a particular substrate). Combined substrate/oxygen quotients for glucose, lactate, and amino acids were reduced to levels that barely exceed basic energy requirements for oxidative metabolism in whole sheep fetuses^{Reference Regnault, de Vrijer and Galan53} or in the hindlimb,^{Reference Rozance, Zastoupil and Wesolowski35} which leaves little room for tissue accretion. This limited surplus of nutrients for energy reserves results in a 22% reduction in hindlimb linear growth rate in FGR fetuses compared to control fetuses.^{Reference Rozance, Zastoupil and Wesolowski35}

Fetal adaptation to systemic hypoxemia involves activation of the sympathetic nervous systems.^{Reference Jones, Roebuck, Walker and Johnston59} Sympathetic overactivity is maintained by adrenergic-mediated hormones, such as catecholamines. A negative association between blood oxygen content and plasma noradrenaline concentrations was observed in FGR fetal sheep.^{Reference Macko, Yates and Chen30,Reference Leos, Anderson, Chen, Pugmire, Anderson and Limesand60,Reference Limesand, Rozance, Zerbe, Hutton and Hay61} Higher concentrations of plasma noradrenaline inhibit insulin secretion via the α2-adrenergic receptors on β-cells.^{Reference Yates, Macko and Chen62} Moreover, chronic exposure to elevated catecholamines persistently inhibits β-cell function and reduces both basal and glucose-stimulated insulin secretion (GSIS) in FGR fetuses.^{Reference Camacho, Davis and Kelly39,Reference Leos, Anderson, Chen, Pugmire, Anderson and Limesand60,Reference Limesand, Rozance, Macko, Anderson, Kelly and Hay63} In addition to catecholamine-mediated inhibition of insulin secretion, low glucose and oxygen concentrations also decrease β-cell proliferation and insulin secretion.^{Reference Limesand, Rozance, Zerbe, Hutton and Hay61,Reference Limesand, Jensen, Hutton and Hay64} Together, these actions lower fetal insulin concentrations and limit fetal glucose uptake by peripheral tissues, which will conserve glucose for critical organs (e.g., brain and heart) under hypoglycemic conditions.^{Reference Hay65} We have demonstrated that surgical ablation of the fetal adrenal medulla prevents acute hypoxia-induced norepinephrine secretion in FGR fetal sheep and partially restores β-cell function and insulin secretion responsiveness.^{Reference Davis, Camacho and Pendleton37,Reference Macko, Yates and Chen66} These findings highlight the role of adrenergic signaling in regulating fetal metabolic adaptations to hypoxemia and suggest that targeting these pathways may offer therapeutic options to mitigate the endocrine and metabolic impairments observed in FGR fetuses.

The impact of heat stress-induced placental insufficiency on fetal muscle fiber myogenesis and proliferation

In livestock species, myogenesis occurs during embryonic and fetal development via satellite cell proliferation, differentiation, and fusion of myoblasts into multinucleated myofibers.^{Reference Picard, Lefaucheur, Berri and Duclos67,Reference Oksbjerg, Gondret and Vestergaard68} The absolute fiber number is established around the time of birth. Postnatal muscle regeneration and growth involve activation of quiescent satellite cells that reside between the sarcolemma and the basement membrane of myofibers. These quiescent satellite cells are activated only during hypertrophic growth and repair in postnatal skeletal muscle by fusing into existing muscle fibers.^{Reference Rhoads, Rathbone, Flann, Du and McCormick69} Although different animal species have distinct developmental timelines and rates of fetal myogenesis, the formation of skeletal muscle in livestock is characterized in a biphasic manner.^{Reference Picard, Lefaucheur, Berri and Duclos67,Reference Wigmore and Stickland70} The formation of primary muscle fibers starts from early to mid-gestation, followed by the formation of secondary and tertiary muscle fibers. For example, in sheep, the formation of primary muscle fibers starts at approximately day 30 (20 %) of gestation, whereas most secondary muscle fibers are formed at approximately day 85 (60 %) of gestation.^{Reference Wilson, McEwan, Sheard and Harris71,Reference Fahey, Brameld, Parr and Buttery72}

In the hindlimb of near-term fetal sheep, skeletal muscle constitutes approximately 40% of the total mass and exhibits both slow- and fast-twitch fibers, similar to other major muscle groups in the body.^{Reference Hicks, Beer and Herrera73,Reference Yates, Cadaret and Beede74} This similarity in muscle structure and composition makes the hindlimb an effective and representative sample for studies on muscle physiology. Hindlimb muscle from near-term fetal sheep exposed to maternal HS has fewer total myofibers and BrdU-positive myonuclei, indicating reduced myogenesis and fewer proliferating satellite cells.^{Reference Chang, Rozance and Wesolowski75} Similarly, in fetal piglets, a lower density of primary muscle fibers was observed as early as mid-gestation due to maternal HS.^{Reference Zhao, Liu and Bell76} Impaired myogenesis in FGR muscle is likely driven by decreased proliferative capacity of muscle satellite cells as the formation of new myofibers requires the fusion of activated satellite cells into myotubes.^{Reference Rhoads, Rathbone, Flann, Du and McCormick69} Studies have shown that hindlimb muscle from FGR fetuses proliferated at slower rates and expressed lower levels of cell cycle-related genes,^{Reference Soto, Blake and Wesolowski34,Reference Yates, Clarke and Macko77,Reference Zhao, Kelly and Luna-Ramirez78} resulting in fewer satellite cells and reduced DNA synthesis rates in FGR muscle.^{Reference Greenwood, Slepetis, Hermanson and Bell79,Reference Greenwood, Hunt, Hermanson and Bell80}

To further investigate cellular adaptation in fetal skeletal muscle, satellite cells (myoblasts) can be isolated from the hindlimb.^{Reference Allen, Temm-Grove, Sheehan and Rice81} When myoblasts were cultured for 3 d in growth media supplemented with serum collected from FGR fetuses (hormone-limited media), FGR myoblasts showed slower proliferation rates than control myoblasts.^{Reference Yates, Clarke and Macko77} Furthermore, when FGR myoblasts were cultured with serum from control fetuses, their proliferation rates increased but remained slower than controls with control serum.^{Reference Yates, Clarke and Macko77} In another study, when isolated myoblasts were cultured in nutrient-enriched media and stimulated with insulin for 5 d, FGR myoblasts had normal or higher proliferative rates.^{Reference Soto, Blake and Wesolowski34} These findings demonstrate that FGR myoblasts have intrinsic deficiencies in proliferation but remain responsive to sustained extrinsic anabolic stimulation. These observations also indicate that the reduced proliferation capacity in FGR myoblasts is, at least partially, reversible, highlighting the potential for nutrient or hormone supplementation to support muscle growth, as discussed in later sections.

The impact of heat stress-induced placental insufficiency on fetal muscle hypertrophy and protein synthesis

After myogenesis concludes, muscle fibers continue to grow through increases in diameter and length, a process known as hypertrophy. Muscle hypertrophy is driven by continued satellite cell activation to proliferate and fuse with established multinucleated myotubes during late fetal and postnatal life.^{Reference Rhoads, Rathbone, Flann, Du and McCormick69} Increases in DNA and myonuclei content contribute to increased muscle protein accretion. Skeletal muscle net protein accretion is associated with the balance between protein synthesis and degradation. When protein synthesis occurs at a faster rate than protein degradation, it leads to net protein accretion and myofiber hypertrophy.

Protein synthesis is stimulated by the PI3K/AKT/mTORC1 signaling pathway in response to anabolic nutrients and/or growth factors.^{Reference O’Connor, Bush, Suryawan, Nguyen and Davis82–Reference Brown, Rozance, Barry, Friedman and Hay84} Under conditions of adequate nutrients, growth factors such as insulin and IGF-1 bind to their respective tyrosine kinase receptors on the surface of a myocyte. The binding activity phosphorylates the receptor and PI3K/Akt kinases, which activates mTOR via inhibition of the tuberous sclerosis complex 2 (TSC2).^{Reference Inoki, Zhu and Guan85} The activation of mTORC1 then promotes the dissociation of 4E-binding protein 1 (4EBP1) from the eukaryotic translation initiation factor 4E (eIF4E) complex, enabling the formation of the active eIF4F translation complex and thus increasing protein synthesis.^{Reference Ma and Blenis86} Conversely, activated mTORC1 inhibits protein degradation by blocking the autophagy-lysosome pathway.^{Reference Zhao, Brault and Schild87} Under nutrient-restricted conditions, hypoxia, low energy availability (elevated AMP:ATP ratio), and reduced growth factor concentrations activate AMP-activated protein kinase (AMPK) signaling, often through HIF-1-dependent pathways. This activation suppresses mTORC1 activity, thereby inhibiting protein synthesis.^{Reference Brugarolas, Lei and Hurley88,Reference Liu, Cash, Jones, Keith, Thompson and Simon89} However, it is important to note that the specific role of AMPK in skeletal muscle of FGR fetuses remains uncertain. For instance, no significant differences in phosphorylated or total AMPK protein abundance were observed in FGR fetal muscle.^{Reference Thorn, Regnault and Brown28,Reference Stremming, Jansson, Powell, Rozance and Brown90} While this does not eliminate the possibility of AMPK involvement, further research is needed to determine whether these pathways are consistently activated in the muscle tissue of FGR fetuses exposed to HS-induced placental insufficiency.

Hindlimb muscles from FGR fetal sheep near term had uniformly smaller myofibers compared to normally grown fetuses, regardless of fiber type.^{Reference Yates, Cadaret and Beede74} Additionally, protein synthesis and accretion rates were 36 and 55% lower, respectively.^{Reference Rozance, Zastoupil and Wesolowski35} This reduction in muscle net protein accretion explains the slower growth rates observed in FGR fetuses.^{Reference Rozance, Zastoupil and Wesolowski35,Reference Galan, Hussey and Barbera91} To elucidate protein metabolism in developing fetuses, isotopically labeled essential amino acid tracer techniques were used.^{Reference Brown, Rozance, Thorn, Friedman and Hay29,Reference Carver, Quick, Teng, Pike, Fennessey and Hay92–Reference Limesand, Rozance, Brown and Hay94} These techniques allow for the simultaneous quantification of net protein synthesis and breakdown in the whole body or in the hindlimb specifically. Using tracer methodologies, studies have demonstrated reduced whole-body protein accretion in fetal sheep exposed to hypoxemia,^{Reference Milley93,Reference Milley95} hypoglycemia,^{Reference Carver, Quick, Teng, Pike, Fennessey and Hay92} and elevated circulating cortisol or norepinephrine concentrations,^{Reference Milley96,Reference Milley97} all of which are associated with the internal FGR fetal milieu.^{Reference Limesand, Camacho, Kelly and Antolic98} These studies demonstrate that the causes of slower muscle growth in FGR fetuses are multifaceted. While decreased protein synthesis is evident in FGR fetal sheep, it remains less certain whether muscle protein degradation is upregulated. We have recently shown increased expression of transcripts associated with the autophagic/lysosomal proteolysis pathway (e.g., LC3B, BNIP3L, and GABARAPL1) in FGR fetal sheep muscle.^{Reference Zhao, Kelly and Luna-Ramirez78} However, others have shown that total fetal or hindlimb muscle protein breakdown rates were unaffected in HS-induced ^{Reference Rozance, Zastoupil and Wesolowski35} or isovolemic hemodilution-induced FGR fetuses.^{Reference Rozance, Wesolowski, Jonker and Brown99} Despite these findings, it is important to consider that protein synthesis and degradation rates are not mutually exclusive processes. Reduced nutrient availability due to placental insufficiency can limit protein synthesis potential as discussed above, while cellular stress may activate autophagy and protein breakdown signaling pathways in skeletal muscle.^{Reference Zhao, Kelly and Luna-Ramirez78}

The impacts of heat stress-induced placental insufficiency on fetal skeletal muscle energy metabolism

Interestingly, under basal conditions, late gestation FGR fetal sheep (approximately 130 dGA) exhibited near-normal rates of weight-specific hindlimb glucose uptake^{Reference Rozance, Zastoupil and Wesolowski35} and whole-body glucose utilization,^{Reference Limesand, Rozance, Smith and Hay26,Reference Camacho, Davis and Kelly39} despite a reduction of approximately 30% in rates of net umbilical (fetal) glucose uptake.^{Reference Limesand, Regnault and Hay25,Reference Limesand, Rozance, Smith and Hay26,Reference Rozance, Zastoupil and Wesolowski35,Reference Davis, Camacho and Pendleton37,Reference Camacho, Davis and Kelly39} Given that basal and glucose-stimulated insulin concentrations were lower in FGR fetuses, near-normal rates of hindlimb glucose uptake and whole-body glucose utilization indicated increased peripheral insulin sensitivity.^{Reference Limesand, Rozance, Smith and Hay26,Reference Thorn, Regnault and Brown28,Reference Thorn, Brown, Rozance, Hay and Friedman100} The discrepancy between rates of umbilical glucose uptake and fetal glucose utilization indicates that there was endogenous hepatic glucose production in FGR fetuses, which was not affected by hyperinsulinemia and demonstrated central insulin resistance.^{Reference Thorn, Brown, Rozance, Hay and Friedman100} These findings reveal systemic adjustments in glucose and insulin homeostasis and signify potential reprogramming of skeletal muscle metabolism to facilitate glucose clearance and production rates in FGR fetuses.

Despite normal fetal glucose utilization rates, the fraction of glucose oxidized to carbon dioxide was less in FGR fetuses.^{Reference Limesand, Rozance, Smith and Hay26,Reference Brown, Rozance and Bruce31,Reference Camacho, Davis and Kelly39} Impaired oxidative metabolism in FGR fetuses is believed to be associated with mitochondrial dysfunction, especially in skeletal muscle.^{Reference Pendleton, Wesolowski, Regnault, Lynch and Limesand101} The metabolic intermediate acetyl-CoA produced from glucose (pyruvate), amino acids, and fatty acids converges in the tricarboxylic acid (TCA) cycle. The series of enzymatic reactions in the TCA cycle releases carbon dioxide and transfers electrons to energy carriers, NADH and FADH2.^{Reference Fernie, Carrari and Sweetlove102} These reducing equivalents are subsequently oxidized at the mitochondrial electron transport chain (ETC) to produce ATP. Thus, cellular respiration via the ETC is a vital process of mitochondrial function.^{Reference Brand and Nicholls103} Using an ex vivo fiber optic fluorescence system, we have demonstrated a lower glucose-derived mitochondrial oxygen consumption rate (OCR) in the hindlimb muscle of FGR fetal sheep.^{Reference Pendleton, Antolic and Kelly38,Reference Zhao, Kelly and Luna-Ramirez78} Although another study reported preserved mitochondrial OCR in permeabilized muscle fibers from FGR fetuses, it also found reductions in type I oxidative fiber expression, citrate synthase (CS) activity, and mitochondrial complex I subunit expression, indicating mitochondrial dysfunction.^{Reference Stremming, Chang and Knaub104} Consistent with these observations, ATP content was reduced in FGR fetal muscle.^{Reference Stremming, Jansson, Powell, Rozance and Brown90,Reference Stremming, Chang and Knaub104} In response to diminished ATP availability, ATP expenditure was downregulated, as evidenced by reduced activity of ATP-dependent Na+/K+ ATPase in FGR fetal muscle.^{Reference Stremming, Jansson, Powell, Rozance and Brown90} This coordinated reduction in ATP availability and expenditure represents an adaptive response that conserves energy under conditions of limited substrate availability but restricts energy-intensive processes essential for normal muscle growth.

Experimental evidence has shown that reduced glucose oxidation in FGR fetuses is associated with a combination of mitochondrial ETC dysfunction and inhibition of pyruvate flux into the mitochondrial matrix (Fig. 2). Biceps femoris muscle from FGR fetal sheep had lower protein abundance of subunit (NDUFB8) and reduced activity of mitochondrial complex I, the largest component of the respiratory chain.^{Reference Pendleton, Antolic and Kelly38,Reference Stremming, Chang and Knaub104} We also showed that FGR fetal muscle had a robust increase in mRNA and protein expression of NADH dehydrogenase 1 α subcomplex 4-like 2 (NDUFA4L2),^{Reference Pendleton, Antolic and Kelly38,Reference Zhao, Kelly and Luna-Ramirez78} a negative regulator of mitochondrial complex I activity. During hypoxia, NDUFA4L2 is upregulated by hypoxia-inducible factor 1a (HIF1a) and integrates into mitochondrial complex I and reduces the activity of the ETC^{Reference Tello, Balsa and Acosta-Iborra105}. The induction of NDUFA4L2 also partially explains reduced mitochondrial OCRs in cancer cells under hypoxic conditions.^{Reference Tello, Balsa and Acosta-Iborra105} In murine skeletal muscle, ectopic overexpression of NDUFA4L2 reduced mitochondrial respiration and caused a ∼ 20% reduction in muscle mass.^{Reference Liu, Chaillou and Santos Alves106} Thus, we hypothesize that hypoxic induction of mitochondrial NDUFA4L2 decreases complex I activity, which contributes to lower OCRs and further defines reduced muscle growth in FGR fetal muscle (Fig. 2).

Figure 2. Summary of fetal hindlimb skeletal muscle adaptations in heat stress-induced placental insufficiency and fetal growth restriction (FGR) in sheep. The upregulated and downregulated (FGR compared to control) metabolic pathways and substrate concentrations are indicated in purple and blue text, respectively, whereas the descriptive labels and unchanged events are shown in black text. FGR fetuses had lower circulating nutrient and anabolic hormone concentrations while having higher norepinephrine and lactate concentrations. Hindlimb muscle from FGR fetuses had lower satellite cell myogenesis and hypertrophy, contributing to lower muscle mass and protein accretion. There was reduced pyruvate-driven oxidation in the mitochondria of FGR fetuses, leading to the accumulation of intramuscular pyruvate. The impaired pyruvate oxidation capacity was associated with a combination of mitochondrial electron transport chain dysfunction (e.g., upregulation of NDUFA4L2) and inhibited pyruvate flux into the mitochondrial matrix and its conversion into the TCA cycle (e.g., downregulation of MPC2, PC, and CS). Additionally, there was reduced expression of TCA-related enzymes. These mitochondrial deficits were associated with lower mitochondrial oxygen consumption rates and reduced ATP production. HIF-1, hypoxia-inducible factor 1; NDUFA4L2, NADH dehydrogenase (ubiquinone) 1 α subcomplex, 4-like 2; GPT, glutamic-pyruvic transaminase (alanine aminotransferase); LDHB, lactate dehydrogenase B; MPC2, mitochondrial pyruvate carrier 2; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; CS, citrate synthase; ACO1, aconitase 1; IDH, isocitrate dehydrogenase; OGDH, oxoglutarate dehydrogenase; SUCLA2, succinate-CoA ligase ADP-forming subunit b; SDHC, succinate dehydrogenase subunit C; FH, fumarate hydratase; MDH, malate dehydrogenase; BCAT1 and 2, branched-chain amino acid transaminase 1 and 2.

In addition to mitochondrial ETC dysfunction, studies have shown downregulation of mitochondrial enzymes (e.g., pyruvate carboxylase (PC), CS, and pyruvate carrier 2 (MPC2)) that govern pyruvate’s flux into the mitochondrial matrix and conversion into the TCA cycle in FGR fetal muscle (Fig. 2).^{Reference Pendleton, Antolic and Kelly38,Reference Zhao, Kelly and Luna-Ramirez78,Reference Stremming, Chang and Knaub104,Reference Pendleton, Humphreys and Davis107} Despite lower mRNA expression of pyruvate dehydrogenase(PDH),^{Reference Chang, Wesolowski and Gilje33} which converts pyruvate to acetyl-CoA, there was increased PDH enzyme activity in FGR muscle.^{Reference Pendleton, Humphreys and Davis107} The greater PDH activity indicates a compensatory mechanism in response to deficiencies in pyruvate transport into the mitochondria to enter the TCA cycle. However, even with enhanced PDH activity, pyruvate-specific mitochondrial oxidation remained impaired, as evidenced by reduced mitochondrial OCR in permeabilized soleus muscle fibers from FGR fetuses.^{Reference Zhao, Kelly and Luna-Ramirez78} These findings suggest that mitochondrial entry of pyruvate may still be restricted, possibly due to downregulation of MPC2 or PC, leading to intramuscular pyruvate accumulation. In turn, excess pyruvate appears to be diverted to alternative pathways, such as transamination to alanine (Fig. 2).^{Reference Chang, Wesolowski and Gilje33}

Placental insufficiency adversely affects hindlimb muscle amino acid metabolism. Weight-specific net uptake rates for nearly all essential (e.g., lysine, threonine, and phenylalanine) and non-essential (e.g., alanine, arginine, glycine, and glutamate) amino acids in the hindlimb of FGR fetal sheep were reduced.^{Reference Chang, Wesolowski and Gilje33,Reference Rozance, Zastoupil and Wesolowski35} The lower amino acid uptake rate in FGR hindlimb correlated with reduced hindlimb size and muscle mass, indicating that diminished amino acid metabolism contributes to impaired muscle growth. Alongside reduced amino acid uptake, the expression of branched-chain amino acid transaminase 1 (BCAT1) and BCAT2, key enzymes responsible for branched-chain amino acid (BCAA) transamination, was significantly reduced in FGR muscle.^{Reference Chang, Wesolowski and Gilje33} This may have contributed to higher intramuscular concentrations of BCAA (e.g., lysine and isoleucine) and reduced utilization of nitrogen from BCAA catabolism (Fig. 2). It is proposed that FGR muscle shifted amino acid metabolism away from protein accretion and toward the production and release of the ammoniagenic amino acids, including alanine, glycine, and glutamine, into the circulation.^{Reference Chang, Wesolowski and Gilje33} These metabolic adaptations are likely mechanisms to conserve energy and maintain nitrogen balance under nutrient-stressed conditions, though at the cost of muscle protein synthesis and growth.

Strategies to improve muscle growth in growth-restricted animals

Intervention strategies are limited to attenuate the HS impact on gestating animals and fetal development, especially to improve placental function and fetal skeletal muscle growth. While cooling technologies, such as fans, misters, conductive cooling, and shade structures, have shown promising results in improving reproductive performance in livestock species,^{Reference Perano, Usack, Angenent and Gebremedhin108–Reference Johnson, Jansen and Galvin110} their implementation is often costly, energy-intensive, and limited to certain environments or facilities. Recent studies identifying genetic markers and pathways associated with improved heat tolerance and adaptive traits in HS pregnant ewes offer a complementary approach.^{Reference Luna-Nevárez, Pendleton, Luna-Ramirez, Limesand, Reyna-Granados and Luna-Nevárez111,Reference Luna-Ramirez, Limesand and Goyal112} Research over the past two decades has only begun to explore targeted nutritional or hormonal strategies to improve birth outcomes for growth-restricted fetuses. A critical initial step involves understanding how externally supplied nutrients are transferred to the fetus and how the fetus metabolically responds to these nutritional inputs. To address these challenges, research has focused on the effect of direct fetal nutrient delivery on fetal metabolism, hormone profiles, and growth. Here, we will summarize the current understanding of targeted fetal nutritional supplementation on fetal and muscle growth, primarily informed by studies on chronically catheterized normal or FGR fetal sheep. Although direct fetal infusions are not practical in farm practice or in human pregnancies, insights from these experiments could inform optimized maternal nutritional strategies to mitigate the adverse effects of HS on fetal and postnatal development.

Fetal oxygenation

Fetal hypoxemia alone causes FGR in farm animals.^{Reference Brain, Allison and Niu131–Reference Botting, Skeffington and Niu134} Thus, restoration of fetal oxygenation remains an attractive therapeutic strategy to improve the growth of FGR fetuses. To raise fetal oxygenation, maternal oxygen supplementation is commonly applied because oxygen can diffuse across the concentration gradient through the placenta. We have demonstrated that maternal tracheal insufflation of humidified oxygen during late gestation increased fetal oxygenation in FGR fetal sheep.^{Reference Camacho, Davis and Kelly39,Reference Macko, Yates and Chen66} In addition, fetal oxygenation restored GSIS in both fetal^{Reference Macko, Yates and Chen66} and neonatal^{Reference Cadaret, Posont and Swanson135} FGR lambs. At neonatal stages, FGR lambs from heat-stressed ewes that received intermittent oxygen therapy for 2 weeks during late gestation had larger birth weights, neonatal growth rates, and hindlimb mass at 1 month of age compared to untreated FGR lambs.^{Reference Cadaret, Posont and Swanson135} However, it was noted that acute fetal oxygenation alone neither lowered fetal plasma catecholamine concentrations nor attenuated fetal hypoglycemia in FGR fetuses.^{Reference Macko, Yates and Chen66} Thus, we postulate that other anabolic stimuli, coupled with fetal oxygenation, should be considered to fully restore the pathologies of FGR. We have recently demonstrated that maternal tracheal insufflation of humidified oxygen combined with fetal glucose infusion for 5 d at late gestation normalized fetal oxygen, glucose, and insulin concentrations in FGR fetal sheep.^{Reference Camacho, Davis and Kelly39} In skeletal muscle, the mRNA expression of NDUFA4L2 was increased 3-fold in FGR fetuses, and this upregulation was attenuated by chronic fetal treatment with oxygen and glucose (Fig. 3). Given the negative association between NDUFA4L2 and mitochondrial complex I activity in hypoxia,^{Reference Tello, Balsa and Acosta-Iborra105} the normalization of NDUFA4L2 indicates improvement in fetal oxygenation and mitochondrial respiratory function in FGR muscle. Further studies are needed to investigate whether oxygen and glucose administration is sufficient to improve the oxidative metabolism of glucose and spare amino acids for tissue accretion in skeletal muscle.

Figure 3. Fetal oxygen and glucose treatment for 5 d normalizes NDUFA4L2 expression in heat stress-induced placental insufficiency and fetal growth restricted (FGR) muscle. Expression levels of NADH dehydrogenase 1 α subcomplex 4-like 2 (NDUFA4L2) mRNA were determined in the biceps femoris muscle of FGR-air and saline (FGR-AS; n = 7), FGR-oxygen and glucose (FGR-OG; n = 7), and control (CON; n = 8) fetuses. Quantitative polymerase chain reaction (PCR) results are presented as the log₂ fold change. Each data point represents the value from an individual fetus within its respective experimental group. Box plots show the interquartile range and median (horizontal line), with whiskers indicating minimum and maximum values. Groups were analyzed with an ANOVA. **denotes P < 0.01 differences between groups. The figure is based on unpublished experimental data.

Other prenatal nutritional strategies

Increasing evidence suggests that oxidative stress and inflammation contribute to the pathogenesis of FGR, supporting the potential of antioxidant and anti-inflammatory therapies as intervention strategies.^{Reference Botting, Skeffington and Niu134,Reference Thompson and Al-Hasan141,Reference Brain, Allison and Niu142} Maternal or fetal antioxidant treatments, such as resveratrol,^{Reference Darby, Saini and Soo143} melatonin,^{Reference Thakor, Herrera, Serón-Ferré and Giussani144–Reference McCarty, Owen and Hart146} and vitamin C^{Reference Thakor, Herrera, Serón-Ferré and Giussani144} improved uteroplacental blood flow in pregnant sheep or cattle with normal pregnancies. In compromised pregnancies, maternal antioxidant supplementation improved fetal growth or birth weight in livestock species.^{Reference Brain, Allison and Niu142,Reference Parraguez, Atlagich and Araneda147–Reference Vazquez-Gomez, Garcia-Contreras and Torres-Rovira150} In addition to oxidative stress, studies also demonstrated enhanced fetal systemic or muscle-specific inflammatory cytokine signaling in compromised pregnancies (e.g., maternal HS or inflammation).^{Reference Cadaret, Merrick and Barnes151–Reference Zhao, Green and Marth153} Daily fetal infusion of anti-inflammatory ω-3 polyunsaturated fatty acids for 5 d during late gestation alleviated fetal hypoglycemia and hypoxemia and recovered hindlimb muscle growth in HS-induced FGR fetal sheep.^{Reference Beer, Lacey and Gibbs154,Reference Beer, Lacey and Gibbs155}

Challenges to restore growth performance in FGR offspring

It remains a major challenge to fully restore the growth of FGR offspring. Most of the abovementioned studies attempted nutrient, oxygen, and hormone replacement strategies at around 90% of gestation, or near term. It may be that toward the end of gestation, adaptations to placental insufficiency become fixed and difficult to reverse, requiring interventions earlier in gestation. After birth, FGR neonatal animals may continue to demonstrate limitations in the capacity to exhibit normal trajectories of postnatal growth and additionally have more difficulties ingesting sufficient colostrum or milk due to their lower vigor. Thus, they have lower energy reserves that affect survival and growth.^{Reference Hole, Ayuso and Aerts156} Low-birth-weight animals are commonly provided with compensatory nutrition to promote catch-up growth.^{Reference Sarr, Gondret, Jamin, Huërou-Luron and Louveau157–Reference Hu, Han and Chen160} However, this intervention can increase the risks of metabolic syndrome in the offspring, which can manifest as excess fat deposition,^{Reference Sarr, Louveau, Le Huërou-Luron and Gondret161,Reference Liu, Yang and He162} abnormal immune function,^{Reference Han, Hu and Xuan163} and muscle mitochondrial dysfunction.^{Reference Liu, Chen and Yao164} Therefore, it is essential to investigate the mechanisms behind how both FGR fetuses and neonates respond to nutrient stimuli and the long-term consequences. For instance, FGR offspring often exhibit impaired gastrointestinal development, which can limit nutrient absorption and digestibility, as observed in pigs^{Reference D’Inca, Gras-Le Guen, Che, Sangild and Le Huërou-Luron165–Reference Tang and Xiong168} and lambs.^{Reference Avila, Harding, Rees and Robinson169–Reference Gao, Hou and Liu171} Additionally, the intrauterine environment may also program skeletal muscle anabolic resistance, as indicated by blunted insulin- and amino acid-induced muscle protein synthesis in preterm neonatal piglets.^{Reference Naberhuis, Suryawan and Nguyen172,Reference Rudar, Naberhuis and Suryawan173} Thus, optimizing nutrient absorption efficiency while avoiding the metabolic dysfunctions commonly associated with excessive catch-up growth is crucial and warrants further investigation when considering nutritional strategies for FGR neonates.