Variable weather patterns and persistent drought are two contributors to the strain on U.S. beef production. Limited water, forages, and feedstuffs can lead to nutrient deficits that directly impact the health and well-being of livestock. Reduced caloric intake during early and mid gestation in beef cows causes intrauterine growth restriction (IUGR) of the fetus that may be attributed to disrupted placental growth and function (Long et al., 2009; Vonnahme and Lemley, 2011). These early life insults to the bovine fetus are linked to altered growth rates and metabolic profiles in the offspring (Long et al., 2009, 2010).
Skeletal muscle is the precursor of meat and profit for beef producers. Improvements in the efficiency of muscle deposition are necessary given the current economic and environmental challenges. Muscle begins to form in the bovine fetus by d 21 of gestation with primary myogenesis complete by d 90 (Robelin et al., 1991). Formation of secondary muscle fibers overlaps with primary myogenesis and continues throughout the later two-thirds of pregnancy such that at birth, the calf is born with a full complement of muscle fibers that do not experience postnatal hyperplasia (Picard et al., 1995). Thus, increasing the numbers of fibers and/or altering their hypertrophic efficiency in utero represents an opportunity to improve postnatal growth.
Nutrient restriction (NR) of ewes during early to mid gestation causes metabolic programming of the lamb in utero (Anthony et al., 2003). The resultant offspring are predisposed to increased adiposity in select depots and exhibit altered muscle profiles that include fewer but larger muscle fibers (Zhu et al., 2004). Lambs born to dams NR during early gestation (d 30 to 70) contained more slow-twitch muscle fibers and fewer fast-twitch fibers in some muscle groups, but not all, at birth (Fahey et al., 2005b). Pregnant beef cows maintained on poor quality pastures during mid to late gestation produced offspring that yielded lighter carcasses with less backfat (Underwood et al., 2010). The divergent effect of NR on muscle groups and adiposity is intriguing and remains unresolved.
The hypothesis that the detrimental impact of maternal NR on fetal muscle development is dependent on duration of the nutritional insult and directly levied on the muscle progenitor cell population was tested.
MATERIALS AND METHODS
All animal experimentation was completed in accordance with guidelines established and approved by the North Dakota State Animal Care and Use Committee.
Diets and Animals
Multiparous British crossbred cows were synchronized by injection of GnRH (100 g; Cystorelin; Merial, Duluth, GA) and a vaginal progesterone insert (Eazi-Breed CIDR; Pfizer Animal Health, New York, NY). Inserts were removed after 7 d and a single intramuscular injection of PGF2α (25 mg; Lutalyse; Pfizer Animal Health) was administered. Cows were artificially inseminated approximately 12 h after the onset of estrus.
Cows confirmed pregnant by transrectal ultrasonography at 30 d of gestation were randomly assigned to the following dietary treatments: cows maintained at 100% NE recommendations for maintenance and fetal growth (NRC, 2000) throughout the course of the study (CCC), cows nutrient restricted to 60% NE recommendations for maintenance and fetal growth (NRC, 2000) for 85 d followed by realimentation to 100% NE requirements for the remainder of the study period (RCC) and cows nutrient restricted to 60% NE requirements for 140 d followed by realimentation to 100% NE requirements for the remainder of the study (RRC). The diet was chopped grass hay [8.02% CP, 69.2% NDF, 41.5% ADF, and 57.9% TDN 9DM basis)] and was delivered by the Calan gate system (Northwood, NH). Cows were slaughtered at gestational d 85 (CCC, n = 6; RCC+RRC, n = 5), d 140 (CCC, n = 6; RCC, n = 5; RRC, n = 6) and d 254 (CCC, n = 6; RCC, n = 5; RRC, n = 6). The gravid uterus was removed and the fetus extracted. The left forelimb was excised from the fetus and immediately frozen at – 20 C. Fetus, organ and tissue weights will be reported in a separate manuscript (L.E. Camacho and K. A. Vonnahme, unpublished data).
The left forelimb infraspinatus (INF) was removed from fetal calves and a 1 by 1 cm portion was embedded in optimum cutting temperature media (Tissue Tek OCT; VWR International, Radnor, PA) before freezing in supercooled isopentane. The INF was selected for analysis due to its anatomical location and its increasing market value (flatiron steak). Ten micrometer cryosections were collected onto glass microscope slides (Superfrost Plus, VWR International). Sections were incubated with rabbit anti-dystrophin (1:500, PA1-37587; Thermo Scientific Inc., Waltham, MA) and anti-Pax7 [PAX7; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA] hybridoma supernatant overnight at 4°C. Cryosections were washed with PBS and incubated with the appropriate AlexaFluor488 and AlexaFluor564 labeled secondary antibodies (Invitrogen, Grand Island, NY) for 45 min at room temperature. Hoechst 33342 (1 μg/mL) was included with the secondary antibody for the visualization of nuclei. After extensive PBS washes, representative images were captured with a Nikon TI-U inverted microscope (Nikon, Lewisville, TX) equipped with an X-Cite 120XL epifluorescence illumination system (EXFO, Mississagua, Ontatio, Canada) and a DS-QI1Mc digital camera (Nikon). Fiber cross-sectional area (CSA) was measured as the region constrained by a dystrophin boundary. Myonuclear domain (MND) was calculated as myonuclei divided by fiber volume (CSA × 10 μm). Percentage Pax7 immunopositive was calculated as the number of Pax7-expressing cells divided by total nuclei.
Quantitative Reverse Transcription PCR
Approximately 1 g of muscle tissue was removed from the fetal INF and homogenized in Trizol (Invitrogen) for the isolation of total RNA. After centrifugation at 3,220 × g for 56 min at room temperature, the aqueous supernatant was passed through a silica spin column (Purelink Mini; Invitrogen) and the total RNA was collected with addition of 30 μL sterile, double-distilled water and centrifugation at 10,000 × g for 2 min at room temperature. Total RNA concentration and quality [absorbance (A) ratio at 260 and 280 nm] was quantified using a BioTek Eon microplate spectrophotometer (BioTek, Winooski, VT). All extractions yielded RNA with A260:A280 nm greater than 1.9. The A260:A230 nm was evaluated using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and all samples possessed ratios greater than 1.8. Total RNA was stored at –80°C.
Fifty nanograms of RNA was reverse transcribed (High Capacity cDNA Archive kit; Invitrogen) in a 20 μL reaction volume, according the manufacturers recommendations. One nanogram equivalent of total RNA was amplified with gene-specific primers (Table 1), DNA polymerase, and SYBRGreen chemistry (SYBR Select Master mix; Invitrogen) in a Realplex2 Mastercycler (Eppendorf, Hauppauge, NY) with the following conditions: a denature step of 95°C for 10 min and 40 cycles of 15 s at 95°C, 60°C for 15 s, and 68°C for 20 s. A final dissociation step included the parameters of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. Relative gene expression levels were calculated as 2–Ct gene of interest/2–Ct RPS9, in which Ct denotes threshold cycle. Amplicon fidelity was verified by DNA sequencing.
|Gene1||Forward primer (5′ to 3′)||Reverse primer (5′ to 3′)||Slope|
Data was sorted by day of gestation (d 85, 140, and 254) and analyzed using animal as the experimental unit. The PROC MIXED procedure (SAS Inst. Inc., Cary, NC) was used using dietary treatment as the fixed effect, and animal and fetus sex as the random effects. Pairwise comparisons between the least square means of the factor levels were computed using the PDIFF option of the LSMEANS statement. At a P ≤ 0.05 differences were considered significant and tendencies were considered at a P > 0.05 and P ≤ 0.10.
The effect of maternal NR for 85 (RCC) or 140 d (RRC) followed by dietary realimentation to recommended caloric intake (CCC) was examined in the fetal INF of the forelimb. Fetal INF cryosections were immunostained for dystrophin and muscle fiber size was measured. After an 85-d NR, the fetal INF contained larger (P < 0.05) muscle fibers than CCC (Fig. 1). Returning the dam to the recommended plane of nutrition after d 85 did not (P < 0.05) restore RCC fiber size equivalent to those found in the CCC fetus at d 140 of treatment. Continued nutrient deprivation of the dam through d 140 of gestation (RRC) caused a substantial reduction (P < 0.05) in INF fiber size by comparison to CCC and RCC. However, reestablishment of the recommended caloric intake to the pregnant RRC dams after d 140 resulted in fetal INF muscle fibers that were not different (P > 0.10) from CCC at d 254 of treatment (CCC = 345.2 ± 5.31 μm2 vs. RRC = 343.43 ± 5.15 μm2). These values were less (P < 0.05) than those found at d 254 for the RCC fetuses (RCC = 417.86 ± 5.37 μm2). No differences (P > 0.10) in the number of fibers per square millimeter were noted at d 254 (CCC = 530.54 ± 39.83 μm2, RCC = 512.39 ± 41.88 μm2, RRC = 541.52 ± 42.10 μm2). Representative photomicrographs of the fibers at the indicated time points are shown in Fig. 2.
The increase in INF fiber CSA after 85 d of NR was intriguing and suggestive of precocious differentiation. Limb muscles are formed through the concerted actions of Paired box 3 (Pax3) and Paired box 7 (Pax7; Relaix et al., 2005) with Pax7 required for fetal myoblast formation (Hutcheson et al., 2009). The numbers of Pax7 immunopositive myogenic precursors were enumerated in the fetal INF. Immunocytochemical localization of dystrophin was performed to ensure correct positioning of the myoblasts within the muscle bed. Representative photomicrographs CCC and NR (d 85) are shown in Fig. 3A. Results indicate that maternal NR for 85 d results in a reduction (P < 0.05) in the percentage of Pax7 immunopositive nuclei within the INF (Fig. 3B). Control INF muscle contain approximately 66% of the total muscle nuclei as Pax7 myogenic precursors, substantially more than the 46% found in the NR INF. The percentage of Pax7 positive cells remained constant in RCC and RRC at d 140, which did not differ from CCC. In all groups, the numbers of Pax7 cells declines to approximately 12.7% of total muscle nuclei at d 254. The rapid loss in myogenic precursors with caloric restriction at d 85 is unlikely a product of cell death or transdifferentiation of the myogenic precursors as the MND size remained unchanged independent of treatment at d 85 (Table 2). A significant increase (P < 0.05) in MND was apparent in the RRC fetuses at d 140. A larger cytoplasmic region governed by fewer myonuclei suggests that between d 85 and 140 of nutrient deprivation, there was a suppression of myoblast fusion or a loss of myoblasts capable of fusion. However, the effect was transient as no differences (P > 0.10) in myonuclei number and MND were evident at d 254 between the groups.
|Diet1||MN2||MND,3 × 103 μm3||MN||MND, × 103 μm3||MN||MND, × 103 μm3|
|CCC||0.841 ± 0.09||30.0 ± 5.0||0.617 ± 0.06||32.2 ± 5.5a||0.423 ± 0.06||11.5 ± 5.0|
|RCC4||0.973 ± 0.10||22.7 ± 6.6||0.594 ± 0.07||33.5 ± 5.0a||0.561 ± 0.06||13.5 ± 5.0|
|RRC||0.647 ± 0.05||42.3 ± 5.5b||0.452 ± 0.06||12.0 ± 5.0|
Larger MND and fewer Pax7 immunopositive cells may be attributed to fewer muscle progenitors and greater numbers of nonmyogenic cells. Relative numbers of connective tissue fibroblasts were examined by quantitative PCR for expression of the collagen and extracellular matrix modifying enzymes, lysyl oxidase (LOX), bone morphogenetic protein 1 (BMP1), and cystatin-C (CYS; Trackman, 2005). Results indicate that 85 d of NR causes (P < 0.05) an upregulation of LOX and BMP1 (Fig. 4). Transcript abundance for the enzymes in RCC and RRC fetal INF did not differ from CCC at either d 140 or 254.
The IGF family is implicated in pre- and postnatal skeletal muscle growth and development in mammals (Holt et al., 2012). Due to the increased size of the primary fibers and the compensatory growth of secondary fibers in the NR fetuses, the mRNA abundance of the IGF and their cognate receptors were measured. Maternal NR for 85 d caused a decrease (P < 0.05) in IGF1 transcript abundance with a corresponding increase (P < 0.05) in IGF2 mRNA content (Fig. 5). Relative amounts of IGF2 were decreased (P < 0.05) with continued NR through d 140 (RRC) by comparison to CCC. Muscle content of IGF1 and IGF2 tended to be reduced in RCC at d 140 by comparison with CCC (P = 0.06 and P = 0.09, respectively). No differences amongst the groups were found for either ligand at d 254. Message abundance for IGF1 receptor (IGF1R) and IGF2 receptor (IGF2R) were not impacted (P > 0.10) by NR or realimentation.
Skeletal muscle originates from the dermomyotome compartment of the somite with regions within the anatomical structure giving rise to distinct subsets of myogenic cells and muscles (Messina and Cossu, 2009). The paralogs, Pax3 and Pax7, are required for all embryonic, fetal and adult muscle formation; genetic ablation of Pax3 and Pax7 results in a complete absence of muscles in the trunk and limbs, with the exception of the primary embryonic myotome (Relaix et al., 2005). Lineage tracing and gene ablation studies in mice reveal that Pax3 expression is required for the initial stages of embryonic myogenesis whereas both Pax3 and Pax7 are required for fetal and adult muscle formation (Relaix et al., 2004, 2006). During later stages of fetal muscle development, the Pax3 and Pax7 cell populations adopt the characteristic satellite position atop the muscle fibers to form the adult muscle progenitor pool at birth (Kassar-Duchossoy et al., 2005). Similar to mice, bovine fetuses contain a robust pool of Pax7 immunopositive muscle precursor cells at the early stages of fetal muscle formation. The cells are loosely associated with the muscle fibers at d 85 of gestation and continue to be found adjacent to the fiber through the later stages of fetal myogenesis. Enumeration of the cells indicates a steady decline in numbers from approximately 65% of the total muscle bed nuclei to slightly more than 15% by late gestation. To our knowledge, this is the first in-depth examination of muscle progenitor numbers in the bovine fetus. The myogenic precursors are fewer in number by nearly one-half by comparison to rodent numbers at birth (16 vs. 30% of total nuclei; Allen and Rankin, 1990). This may be attributed to the anatomical location of the muscle. Mouse forelimb muscles contain threefold more Pax3 than Pax7 muscle progenitors (Relaix et al., 2004). By extrapolation, the majority of the muscle progenitors in the fetal bovine forelimb may be present as Pax3 expressing cells with Pax7 denoting a smaller pool. Our ability to detect and enumerate Pax3-expressing cells is hampered by the lack of a high-affinity antibody to the protein.
Given the critical need for Pax3/7 cells for muscle development and growth, it is imperative that these cells remain available to the fetus throughout gestation and adult life. Maternal nutrient deprivation disrupted the initial stages of fetal myogenesis as evidenced by a reduction of approximately 30% in the numbers of Pax7-expressing cells. The pool size was not further reduced by continued maternal NR through d 140 of gestation. Indeed, all fetuses contained equal numbers of muscle progenitors independent of maternal diet at d 140 and 254 suggesting that the muscle progenitor population incurred no irrevocable harm. The basis for fewer Pax7 cells after 85 d of NR may be a product of precocious differentiation. Tissue morphology of the NR fetuses at d 85 demonstrates a population of large myofibers, presumably primary fibers, with several associated Pax7 cells. An effective stimulus for myoblast differentiation and fiber formation in vitro is mitogen deprivation. Maternal NR may act in a similar manner to cause premature differentiation of the progenitor pool and their fusion into primary fibers. Myoblasts isolated from IUGR fetuses display perturbed proliferation and differentiation kinetics in vitro suggesting that nutrient deprivation may affect myogenesis (Yates et al., 2012). However, we cannot rule out the possibility that the reduced numbers of Pax7 cells is a consequence of apoptosis or transdifferentiation to a nonmyogenic lineage. Fetuses and newborn lambs from nutrient restricted ewes have altered muscle formation with an increase in connective tissue and intramuscular (IM) adiposity (Zhu et al., 2004, 2006). The greater amount of postnatal IM fat may be a product of lineage conversion from the Pax7 myogenic progenitor to an adipocyte. Evidence for spontaneous and permissive transdifferentiation of Pax7 cells exists in mouse isolated myofiber cultures (Asakura et al., 2001; Shefer et al., 2004). Alternatively, the greater size of the IM fat depot and fascicular connective tissue depots may be a result of greater fibrogenic cell numbers and/or activities. Our results demonstrate an increase in LOX and BMP1, two genes that are key participants in procollagen processing and intermolecular collagen crosslinking, in d 85 fetal forelimb muscle (Lucero and Kagan, 2006; Maruhashi et al., 2010). Increased expression of the genes is suggestive of an increase in the numbers of connective tissue fibroblasts. Indeed, a greater RRC MND at d 140 provides evidence for fewer myoblasts capable of fusing with the adjacent fiber. Because the RCC MND did not differ from CCC at d 140, it is unlikely that the muscle precursor pool was lost or irreplaceably damaged during the shorter duration of NR. Long-term damage to the myoblast pool, therefore, requires sustained NR beyond the first trimester. The issue of altered fibrogenic and myogenic populations requires a more thorough investigation in the temporal and spatial accumulation of said cell types in the normal gestating fetus before overlaying a nutritional insult.
The effects of early gestational nutrient deprivation on fetal sheep muscle formation include a reduction in secondary fiber number that is reflected in a reduced secondary:primary fiber ratio (Zhu et al., 2004; Quigley et al., 2005). After 85 d of NR, prominent primary fibers were evident with much smaller secondary fibers. Although not calculated, a skewed fiber ratio likely is present in these fetuses. Realimentation allows for dramatic catch-up growth of the secondary fibers such that no differences in fiber density exist at d 254 of gestation. A striking increase in d 254 fiber CSA is apparent in RCC fetal INF that is not evident in either CCC or RRC. The divergent response between RCC and RRC argues that a critical window relating to hypertrophy and fiber size is present within the first trimester of pregnancy. In a similar ovine model of maternal NR, muscle-specific responses were observed (Fahey et al., 2005b). Restriction during the first trimester followed by realimentation caused an increase in diameter of fast muscle fibers in the LM and vastus lateralis but not the semitendinosus; slow fiber CSA was unaffected by NR in the examined muscles. The compensatory growth of the fetal fibers argues that restriction during the first trimester imparts a heightened sensitivity to nutrient load to the fetus, which is retained throughout gestation. Prolonged NR through d 140 of gestation eliminates the beneficial effects on fetal fiber hypertrophy. Closer examination of the increase rate of fiber hypertrophy in the RCC and RRC groups after realimentation is remarkable and points to elevated protein synthesis rates within the myofiber. To meet the metabolic demands of the growing fiber, nutrient transporters and/or anabolic signaling systems may display a heightened sensitivity thereby allowing increased substrate supply for protein synthesis. This issue remains unresolved.
The IGF growth factor signaling system is key to proper metabolism and muscle growth. In general, systemic IGF2 controls prenatal growth and postnatal growth is controlled by IGF1 (Bass et al., 1999). Both transcripts are expressed in a temporal manner in ruminant skeletal muscle with peak expression occurring near the beginning of the second trimester (Listrat et al., 1999; Fahey et al., 2005a). Nutrient restriction of ewes through d 80 of gestation followed by realimentation does not impair IGF1 expression in the fetal quadriceps by comparison to control fetuses from well-nourished ewes (Brameld et al., 2000). This dietary regiment does cause alterations in IGF2 mRNA content with lower amounts of the growth factor found in realimented fetal muscles at near term (d 140). However, the differences in expression may be a reflection of overfeeding and not a blunted transcriptional response as IGF2 mRNA did demonstrate the expected temporal decline in abundance whereas the well-nourished controls did not. In runt piglets, LM IGF1 mRNA content does not differ from normal littermates at d 65 of gestation (Tilley et al., 2007). However, the transcript abundance increased through late gestation by comparison with the decline observed in normal piglets. These results imply that the autocrine IGF system is attempting to overcome growth retardation by increasing hypertrophic signals. Bovine fetuses experiencing NR through the first trimester demonstrate a reduction in IGF1 and an increase in IGF2 mRNA content. Decreased IGF1 content is in agreement with reports of reduced IGF1 mRNA abundance found in the quadriceps of ovine fetuses from ewes suffering placental insufficiency (Kind et al., 1995). The detriment of decreased IGF1 during embryonic myogenesis remains unresolved. Insulin-like growth factor 2 is regarded as a potent fetal mitogen as the mice null for the mutation are 60% smaller than wild-type and excessive production of the growth factor causes overgrowth (Gicquel and Le Bouc, 2006). Increased fiber size of the RCC group at d 254 may be explained by the modest increase in IGF2 expression experienced during the first trimester. Alternatively, improved ligand-initiated signaling may underlie the increased fiber size in the d 254 RCC fetus. The IGF2 receptor is a nonsignaling molecule whose expression at the cell surface regulates the bioavailability of the IGF2 ligand (Brown et al., 2009). The majority of IGF2 effects likely originate from invoking the IGF1R, a mechanism that is prevalent in skeletal muscle (Alzhanov et al., 2010). Measurement of muscle IGF1R and IGF2R mRNA at the various gestational time points demonstrated no differences between control and NR fetuses. These results differ from sheep models of IUGR, which demonstrate that fetal muscle IGF1R and insulin receptor protein are greater at mid gestation in ewes suffering placental insufficiency (Muhlhausler et al., 2009). No change in receptor with an increase in ligand argues for a potentiation of intracellular signals that modulate the hypertrophic response.
In summary, NR of pregnant beef cows during the first trimester does impact the development of fetal muscle. Realimentation of the dam allows for compensatory growth of the fetal muscle possibly due to autocrine IGF2 activity. The duration of NR dictates the long-term consequences on the fetal musculature, though, as NR through the second trimester likely increases the numbers of connective tissue cells, which may impact substantially meat quality of the offspring.