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Effects of maternal intake restriction during early pregnancy on fetal growth and bone metabolism in goats
Journal Pre-proof Effects of maternal intake restriction during early pregnancy on fetal growth and bone metabolism in goats Xilin Li, Hengzhi Li, Zhixiong He, Zhiliang Tan, Qiongxian Yan
PII:
S0921-4488(19)30236-6
DOI:
https://doi.org/10.1016/j.smallrumres.2019.106027
Reference:
RUMIN 106027
To appear in:
Small Ruminant Research
Received Date:
22 March 2019
Revised Date:
11 October 2019
Accepted Date:
22 November 2019
Please cite this article as: Li X, Li H, He Z, Tan Z, Yan Q, Effects of maternal intake restriction during early pregnancy on fetal growth and bone metabolism in goats, Small Ruminant Research (2019), doi: https://doi.org/10.1016/j.smallrumres.2019.106027
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Running title: maternal nutrition restriction on fetal bone metabolism Effects of maternal intake restriction during early pregnancy on fetal growth and bone metabolism in goats
a
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Xilin Li a,b, Hengzhi Li c , Zhixiong He a, Zhiliang Tan a,d,e , Qiongxian Yan a,d,e *
CAS Key Laboratory for Agro-Ecological Processes in Subtropical Region, National
Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and
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Poultry Production, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Hunan Provincial Engineering Research Center for
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Healthy Livestock and Poultry Production, Institute of Subtropical Agriculture, The
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Chinese Academy of Sciences, Hunan Provincial Key Laboratory of Nutritional Physiology and Metabolic Process Changsha, Hunan 410125, P.R. China University of Chinese Academy of Sciences, Beijing 100049, P.R. China
c
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing
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b
Hunan Co-Innovation Center for Utilization of Botanical Functional Ingredients,
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d
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210095, P.R. China
Changsha, Hunan 410128, P. R. China e
Hunan Co-Innovation Center of Animal Production Safety, CICAPS, Changsha, Hunan
410128, P.R. China
*Corresponding author E-mail address: [email protected](Qiongxian Yan).
Highlight
This study was designed to investigate the effect of maternal intake
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restriction on fetal growth and bone metabolism of goats. Maternal intake restriction during early gestation affected the thoracic
circumference, umbilical circumference, bone metabolic activities (INTP
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and CTX-1) and femur BGLAP mRNA expression of fetal goats.
This study provides a reference for the effects of nutrient limitation in
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early pregnancy on the bone development of fetal goats.
Abstract
The objective of this study was to investigate the effects of maternal
nutritional restriction on fetal growth and bone metabolism in early pregnancy. Twelve twin-bearing goats (Xiangdong black goats) in second pregnancy, with similar weight (31.2 ± 8.1 kg) and age (2.0 ± 0.3 year) were assigned to two dietary treatments in early
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gestation (26-65 days): control group (CON, 100% feed) and intake restriction group (IR, 60% of CON intake). The umbilical blood, right femur and liver of the fetuses were sampled on day 65 to determine the bone metabolism indicators and expression of genes
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involved in bone development. Maternal intake restriction did not affect the growth
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performance of pregnancies (P > 0.05), increased (P < 0.05) the body weight, body length,
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thoracic circumference and umbilical circumference of fetal goats, but decreased (P < 0.05) the concentrations of cross-linked N-terminal telopeptides of type I collagen and
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cross-linked C-terminal telopeptides of type I collagen, which involved in bone metabolic
gamma-carboxyglutamate
protein,
osteoprotegerin
and
mothers
against
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bone
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activity, in the fetal umbilical blood of the IR. In fetal femurs, the mRNA expression of
decapentaplegic homolog 1 both participating in osteoclast differentiation were increased (P < 0.05), whereas transcript of the low density lipoprotein receptor related protein 5 involved in osteoblast differentiation was decreased (P < 0.05), compared with the fetuses from the CON. These results indicate that nutrition restriction in early pregnancy
regulated the fetal growth and promoted formative activity of femur. The molecular mechanism of maternal intake restriction on bone metabolism needs further investigation.
Keywords Gestational nutrition; Fetal development; Femurs; Fetal goats
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1. Introduction
During pregnancy, the energy required for rapid fetal growth comes mainly from the placental absorption of glucose and amino acids in the maternal blood (Redmer et al.,
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2004; Priya et al., 2016). Therefore, the mother needs to provide sufficient protein and
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glucose to ensure normal fetal growth (Husted et al., 2008). Maternal malnutrition in pregnancy often leads to intrauterine growth retardation (IUGR), and short-term or long-
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term consequences, such as asymmetric growth of organs, hypoxia, acidemia, intrauterine death in the uterus and metabolic disorders, cardiovascular disease, diabetes, and obesity
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after birth (Wu et al., 2006; Decembrino et al., 2013).
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Bone is given a greater priority for nutrients than tissues such as muscle and fat during fetal life because bone plays a key role in protecting soft tissues, maintaining
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mineral balance, providing structural support, framework of the carcass, and supporting hematopoiesis (Florencio-Silva et al., 2015). The metabolic balance of bones is achieved through bone transformation that includes bone formation by osteoblasts and bone resorption by osteoclasts (Manolagas and Jilka et al., 1995). Generally, biochemical markers of bone metabolism are simple and effective methods to evaluate bone metabolic
activity, such as bone alkaline phosphatase (BALP) and bone gamma-carboxyglutamate protein (BGLAP, Lian et al., 1993), tartrate resistant acid phosphatase (TRAP, Minkin, 1982), cross-linked N-terminal telopeptides of type I collagen (INTP) and cross-linked C-terminal telopeptides of type I collagen (CTX-1, Banfi et al., 2010). Moreover, bone development is regulated by a variety of signaling pathways, such as the Wingless (Wnt)
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signaling pathway (Miller, 2002), the OPG/RANKL/RANK signaling pathway (Suda et al., 1999), and the mothers against decapentaplegic homologs (Smads) signaling pathway (Matsubara et al., 2008).
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Fetal bone development is susceptible to maternal nutrition disruption. In rats,
maternal protein restriction easily causes the reduction of femur weight and bone mineral
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content (Mehta et al., 2002) and the reduction of femoral head size with thinner and less
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dense trabeculae in off spring (Lanham et al., 2008). In sheep, the effects of maternal nutrients restriction on offspring’s bone are variable with either reduced offspring bone
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weight, length, or density (Tygesen et al., 2007; Kleemann et al., 2015). In fact, the phenomena of maternal malnutrition are very ubiquitous in ruminants fed on traditional
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grazing regions around the world, especially in dry or cold seasons because of the
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shortage of fodders. Because of this, fetal bone metabolism in conditions of malnutrition is getting more attention. Even so, only a few studies evaluating the eff ects of maternal nutrition restriction on off spring bone metabolism have been reported during late pregnancy (Tygesen et al., 2007; Fetoui et al., 2008). Fetal bone metabolism knowledges related to maternal malnutrition during early pregnancy are still scarce. Therefore, this
study was designed to investigate whether maternal nutrition restriction during early gestation affect fetal growth and bone metabolic activity via regulation of the Wnt, OPG/RANKL/RANK and Smads signaling pathways in goats.
2. Materials and methods
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This study was conducted according to the Animal Care and the Use Guidelines of the Animal Care Committee, Institute of Subtropical Agriculture, The Chinese Academy of
2.1.Animal management and dietary treatments
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Sciences, Changsha, China.
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Twenty goats (Xiangdong black, a local breed in Liuyang, Hunan Province, China)
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in second pregnancy, with similar weight (31.2 ± 8.1 kg) and age (2.0 ± 0.3 year) were selected as experimental animals. Synchronous estrus was conducted by intramuscular
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injection of progestogen followed by equine chorionic gonadotropin to ensure a consistent pregnancy date. The injections of equine chorionic gonadotropin and
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progestogen are both a mixture of 1000 units of powdered medicine diluted by 2 ml sterile
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physiological saline. All goats were inseminated directly into the uterus using frozenthawed semen from one buck. The day of artificial insemination was assumed as the first day of pregnancy. During early pregnancy (0-22 days), the goats were grazed on pasture freely. They were then individually fed in stainless steel bracket cages (1.2 × 0.91 m) to adapt the experimental diet for 3 days (23-25 days). Excluding goats that were not
pregnant or not twin bearing, twelve twin-bearing goats examined by ultrasonography were randomly allocated into two treatment groups: control group (CON, 100% feed, n=6), and intake restriction group (IR, 60% of CON intake, n=6). Ultrasonic examinations were arranged at day 25 and day 40 to insure the experimental goats were twin-bearing. The diagnostic criteria was the presence of two liquid dark areas of gestational sac and
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heartbeats, cotyledons and/or fetal parts recognized on B-ultrasonic scanner (Amer, 2010). Throughout pregnancy days 26-65, the control diet was supplied to meet 100% of metabolic energy and crude protein requirements for pregnant goats according to the
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Feeding Standard of Meat-Producing Sheep and Goats of Chinese agricultural industry
standards (HB, NY/T 816-2004). The parameters were set as followed: body weight,
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30kg; dry matter intake (DMI), 0.90 kg/d; digestible energy (DE), 12.34 MJ/d; metabolic
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energy (ME), 10.12 MJ/d; crude protein (CP), 89 g/d; calcium (Ca), 5.8 g/d; phosphorus (P), 3.9 g/d; NaCl, 4.5 g/d. The ingredients and nutrient composition of the experimental
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diet are shown in Table 1. Fresh Miscanthus sinensis was selected as forage, cut into small pieces with 3-5cm, and manually mixed with the concentrate before feeding. All goats
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were fed twice a day at 08:30h and 17:00h, and had free access to water. Goats in the
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CON were fed based on the Feeding standards above with the residues less than 5%, while goats in the IR were fed at 60% of CON intake. The feed amount for the CON was adjusted every week by extra 5% increments. The feed amount for the IR was modulated correspondingly. The feed intake of goats was recorded every day, and the orts of concentrate and forage were recorded separately after screening. The ratio of concentrate
to forage was 40:60.
2.2.Chemical Analysis
Feed samples including concentrate and Miscanthus sinensis were analyzed for dry matter (DM), crude protein (CP), calcium (Ca), phosphorus (P), neutral detergent fiber (NDF), acid detergent fiber (ADF) and crude fiber (CF) according to the procedures of
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Association of Official Analytical Chemists (Cunniff, 1995).
2.3.Blood and tissue samples collection
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At 65d, all the experimental goats were fasted overnight prior to weighing, bleeding
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and slaughtering. Post-slaughter the entire uterus was immediately removed. Fetuses from each treatment (CON, 5 male and 7 female; IR, 6 male and 6 female) were collected.
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After dissection, the umbilical cord attached to the uterine epithelial was cut and ligated, then the blood from the vein and arteries flowed into a 2 mL centrifuge tube by gravity.
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Blood samples were allowed to coagulate for 2 hours for the determination of bone
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metabolite indicators. Centrifugation was performed at 3000 g for 15 min at 4°C. The serum was aliquoted into 1.5 mL centrifuge tubes and stored in a -80°C freezer.
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Meanwhile, fetuses were weighed, and body length, thoracic and umbilical circumference were measured with tapeline. The femurs and livers in the right side of each fetus were collected, washed by normal saline and then immediately frozen in liquid nitrogen and stored in a -80°C freezer for gene expression analyses.
2.4.Metabolic marker activities determination
The contents of parathyroid hormone (PTH), bone alkaline phosphatase (BALP), bone gamma-carboxyglutamate protein (BGLAP), tartrate resistant acid phosphatase (TRAP), cross-linked N-terminal telopeptides of type I collagen (INTP) and cross-linked C-terminal telopeptides of type I collagen (CTX-1) in fetal umbilical cord serum were
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measured using colorimetric methods with a spectrophotometer (MB-530, Huisong technology development Co., Ltd, Shenzhen, China). The assay kits of PTH, BALP and BGLAP were purchased from Wuhan Huamei Bioengineering Co., Ltd. (Wuhan, Hubei,
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China). The assay kits of TRAP, INTP and CTX-1 were purchased from MyBioSource
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Inc. (San Diego, SC, USA). PTH (intra-assay precision: CV%<15%, inter-assay precision: CV%<15%, sensitivity: 25 pg/mL), BALP (intra-assay precision: CV%<8%, inter-assay
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Precision: CV%<10%, sensitivity: 1.95 mU/mL), BGLAP (intra-assay precision: CV%<8%, inter-assay precision: CV%<10%, sensitivity: 0.24 ng/mL), TRAP (intra-
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assay precision: CV%<15%, inter-assay precision: CV%<15%, sensitivity: 1.0 U/L), INTP (intra-assay precision: CV%<15%, inter-assay precision: CV%<15%, sensitivity:
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1.0 ng/mL) and CTX-1 (intra-assay precision: CV%≤8%, inter-assay precision: CV%≤
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12%, sensitivity: up to 0.05 ng/mL) were analyzed in duplicate.
2.5.Gene expression analyses
Total RNA was extracted from femurs and livers with the RNAiso Plus reagent (TaKaRa, JPN). The total RNA absorbance values of OD260 and OD280 were determined
with a nucleic acid analyzer (Nano Drop ND2000, USA). The RNA was used to reverse transcribe into cDNA (20μL) by Prime Script RT reagent Kit with gDNA Eraser (TaKaRa, JPN) to meet an OD260/OD280 ratio of 1.8 to 2.2, and evaluated by 1% agarose gel electrophoresis. The resulting cDNA template was stored at -80°C until analysis by realtime quantitative PCR. Primers shown in Table 2 were designed with premier 5.0 software
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according to the relevant gene sequences of goats in Genebank and synthesized by Shanghai Bioengineering Co., Ltd. (Songjiang, Shanghai, China). The specificity analyses of primers were performed preliminarily by using the Blast tool. Real-time
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quantitative PCR was proceeded by ABI Prism 7900 HT Fast Real-Time PCR System (ABI, CA) with the SYBR® Premix Ex TaqTM II (TaKaRa, JPN). The reaction system
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contained 5 μL SYBR® Premix Ex TaqTM (2x), 0.4 μL forward primer (10 μM), 0.4 μL
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reverse primer (10 μM), 0.2 μL ROX reference dye (50x), 1.0 μL cDNA and 3 μL sterilized ddH2O under the amplification procedure (95°C for 10 min to active the DNA
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polymerase, then cycled at 95°C for 5 s, and 60°C for 30 s for 40 cycles). The specificity of products was determined according to dissolution curve under the dissolution
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procedure (95°C for 15 s, 60°C for 15 s and 95°C for 15 s).
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The relative quantification of gene expression was calculated as a ratio of the target gene to the housekeeping gene by 2-ΔΔCt method (Livak and Schmittgen, 2001) under the premise of the same amplification efficiency of the target gene and the housekeeping gene, where –ΔΔCt = –[ (Ct target gene–Ct β-actin) IR group–(Ct target gene–Ct β-actin) CON group]. Ct was the number of experiment cycles when the amplification product reaches the fluorescence
threshold. In this study, we chose the β-actin as the housekeeping gene, which was manifested to be steadily expressed in samples. Other researchers also used the β-actin as the housekeeping gene in bone metabolic study for goats (Nemeth et al. 2017).
2.6.Statistical analysis
The data of this experiment was analyzed by one-way analysis of variance (ANOVA)
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using the MIXED procedure of SAS 9.2 (SAS Inst. Inc., Cary, NC, USA). The fixed effects of diet level and fetal sex were considered in the model. Because there were no
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interaction of diet level and fetal sex for all the indexes, the interaction effect was not
showed in the Tables. Maternal goat was considered as the random effect. Significance
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was declared at P < 0.05 and trends were considered at 0.05 ≤ P < 0.10.
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3. Results
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3.1.Growth performance and nutrient intake of maternal goats
As shown in Table 3, the final body weight and total body gain of the IR were not
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affected compared with the CON (P > 0.05). The dry matter intake of the concentrate in
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the IR was actually 61.68% of the CON in early gestation (P < 0.05), which was close to the 60% target. The dry matter intake of the forage in the IR was 70.39% of the CON (P < 0.05), and other nutrient components in the IR were between 61.68 to 70.39% of the CON (P < 0.05). The consumption of the CON and IR in relation to the feed offered was 95.83% and 94.94% respectively.
3.2.Growth of the fetuses
Compared with the CON, the body weight, body length, thoracic circumference and umbilical circumference of the IR fetuses were increased by intake restriction (P < 0.05) in early pregnancy (Table 4). These indicators were not changed by the fetal sex (P > 0.05).
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3.3.Bone metabolic marker activities of the fetal umbilical cord serum
The concentrations of INTP (P < 0.01) and CTX-I (P < 0.05) in the fetal umbilical
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cord serum of the IR were less than that of the CON. Other indexes of bone metabolism and conversion were not affected by intake restriction (P > 0.05) or fetal sex (P > 0.05,
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Table 5).
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3.4.Bone metabolic gene expression in the fetal liver and femur
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In the early pregnancy, the mRNA expression of alkaline phosphatase, liver/bone/kidney (ALPL), bone morphogenetic protein 2 (BMP2), and vitamin D (1, 25-
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dihydroxyvitamin D3) receptor (VDR) genes in the fetal livers of the IR were not affected by intake restriction (P > 0.05), but the mRNA expression of BGLAP was increased (P <
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0.10, Table 6). The mRNA abundance of ALPL, BGLAP, BMP2 and VDR were not influenced by fetal sex (P > 0.05). For the femurs of fetal goats, BGLAP, OPG and Smad1 mRNA in the IR were expressed at an increased amount (P < 0.05, Table 7 and 8), while low density lipoprotein receptor related protein 5 (LRP5) mRNA expression was less (P < 0.05) than that of the
CON (Table 8). Meanwhile, abundance of BGLAP, OPG, Smad1 and LRP5 mRNA were not changed by fetal sex (P > 0.05). No differences in the mRNA expression of ALPL, VDR, LRP6, β-catenin, Cnx43, CyclinD1, MMP16, RANKL, BMP2, Smad5 and Runx2 were observed neither by intake restriction (P > 0.05) nor fetal sex (P ＞ 0.05).
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4. Discussion
The aim of this experiment was to explore the effects of maternal malnutrition on fetal growth and skeletal metabolism in goats. Based on the results section, we indicated
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that DMI in the IR during early pregnancy was 66.40% of CON, which was close to our
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original expectation. According to our previous report (Yan et al., 2018; Li et al., 2018), the body weight of the fetuses in the IR group was about 2.3 times greater in CON and
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the body length was 1.4 times longer at 65 day of gestation. Simultaneously, thoracic and umbilical circumference of the fetuses were increased by 30% and 20% respectively in
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this study. These results were partly in agreement with a previous study (Osgerby et al., 2002), in which an increment in the fetus's thoracic circumference by nutritional
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constraint of ewes in early gestation was also observed. However, it was in contrast with
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the findings of Nishina et al. (2003) in Welsh Mountain ewes, where there were no overall differences in the fetal body weight and placental size between control (100% feed) and dietary restricted group (30% feed restriction). The difference could be ascribed to animal species and dietary ingredients. In addition, it has been reported that the absolute fetal growth is not evident before 90 days of gestation, but the relative growth rate is often very
distinct. For instance, the weight gain at gestational day 60 relative to day 45 is about 456.2%, and at gestational day 75 relative to day 60 is 429.2% (Zhao et al., 2012). The increase of fetal body weight could be ascribed to the nanny goats, under nutritional restriction, altering their energy metabolism through the changes in the expression of hormones and growth factors to adjust the distribution of nutrition (Coad et al., 2001).
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Skeletal development is in the process of dynamic balanceable metabolism of bone formation and bone resorption. Bone formation is mainly related to osteoblast metabolism, and bone resorption is mainly related to osteoclast metabolism. Enzymes or other
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decomposed products, derived from the bone cells and bone matrix, can be used as
markers to reflect the bone metabolic activity. For example, BALP and BGLAP proteins
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are associated with bone formation (Lian et al., 1993), whilst TRAP (Minkin, 1982), INTP
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and CTX-1 proteins (Banfi et al., 2010) are linked with bone resorption. Moreover, PTH is an important hormone of maintaining calcium and phosphorus metabolism and bone
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metabolism (Andress et al., 1989). In the current study, intake constraint of nanny goats greatly decreased the CTX-1 and INTP concentrations in the fetal umbilical cord blood.
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However, TRAP, BALP, BGLAP and PTH concentrations were unaffected. It is possible
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that the mixed blood from a vein and arteries of the umbilical cord was not appropriate to determine these metabolic markers in the fetuses, because it also contained blood from maternal circulation. These findings suggested that osteoclast metabolism of fetal goats was reduced and osteoblast metabolism was not affected by nutritional constraint, overall, resulting in the relative increase in osteogenic activity.
Fetal liver is the organ closely related to bone development and metabolism, and its various metabolic factors secreted regulate bone growth. The expression levels of ALPL, BGLAP and VDR are related with the regulation of calcium homeostasis and bone formation (Kollitz et al., 2016; Song et al., 2011). While BMP2 is a key factor in bone growth and development, and its expression in the liver also affects the growth and
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regeneration of the liver (Kan et al., 2009). In the current study, the mRNA expression of ALPL, BMP2 and VDR genes in the liver of fetuses were not affected by intake restriction
or fetal sex. Increase in fetal liver weight and decrease in DNA methylation was
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previously observed by Li et al. (2018), indicating that cell proliferation and epigenic modifications rather than fetal liver genes regulating bone formation are more sensitive
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to maternal nutrient restriction. Fortunately, BGLAP transcript tended to be increased by
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intake restriction. In the femur of fetuses, intake restriction increased the BGLAP expression, which revealed that the activity of osteoblast and bone formation were
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elevated. It also implies that regulatory mechanism of BGLAP transcript induced by nutrition restriction is different in visceral tissues and skeletal organs. Hence, it is
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reasonable to speculate that bone cells are more sensitive than other cells to nutritional
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restriction, considering BGLAP is mainly transcribed by osteoblasts (Lian et al., 1989), and some miRNAs (miR-125b and miR-30c2) might be involved in negatively regulating BGLAP expression in osteoblast (Laxman et al., 2015). The Wnt signaling pathway is an important regulator of osteoblast development and differentiation, and plays a major role in fetal bone formation (Miller, 2002). The classic
Wnt signaling pathway activates the downstream protein factor Dishevelled (Dsh) after binding to the Wnt receptor Frizzled and the collagenous receptor LRP5 / low density lipoprotein receptor-related protein 6 (LRP6) to form the Wnt-Frizzled-LRP5/Wnt-FzdLRP6 triplex, thereby inhibiting the phosphorylation of beta-catenin (β-catenin) by Glycogen synthase kinase 3β (GSK3β). Then β-catenin translocates into nucleus to
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activate the expression of downstream target genes (Mulholland et al., 2005), such as connexin 43 (Cnx43), CyclinD1 and matrix metalloproteinase 16 (MMP16) (Lyer et al.,
2013). In the present study, the mRNA expression of LRP6, β-catenin, Cnx43, CyclinD1
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and MMP16 were not affected, but the mRNA expression of LRP5 in the femur was
reduced by intake restriction. It suggested that only the LRP5 in the femur was sensitive
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to maternal nutritional restriction, and intake restriction or fetal sex did not alter the Wnt-
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Frizzled-LRP5 related pathway at the mRNA level. Whether proteins expression and post-translational modification in this pathway are changed by nutritional restriction
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needs to be further explored.
The OPG/RANKL/RANK signaling pathway is a key pathway through which
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osteoblasts regulate osteoclast differentiation and activation (Suda et al., 1999). The OPG
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and RANKL are mainly distributed in osteoblasts, meanwhile RANK is mainly distributed in
osteoclasts. Usually, RANKL binds to its receptor RANK, which
promotes the differentiation and maturation of osteoclasts. The OPG can also combine with RANKL to prevent the binding between RANKL and RANK and thus inhibit the formation and differentiation of osteoclasts (Khosla et al., 2001). The results of this
experiment showed that the expression of OPG was increased due to intake restriction, whereas the expression of RANKL was not affected, leading to the increment in the ratio of OPG to RANKL. Zhang et al. (2009) found dietary Ca restriction significantly upregulated the mRNA expression of OPG in proximal tibia of aged rats. The increase in this ratio indicated that the proliferation of osteoclasts was inhibited, thus promoting bone
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growth and development. BMP2-Smad-Runx2 axis plays an important role in the osteoblastogenesis (Matsubara et al., 2008). The BMP2 regulates osteoblast differentiation and bone
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formation by binding to type I and type II BMP2 receptors on the cell surface, and then
phosphorylates the Smads 1, 5 and 8 proteins in the cell nucleus. The phosphorylated
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protein forms a complex with Smad4, then transferred into the nucleus to combine with
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osteoblast to promote transcription factors such as Core Banding Factor 1 (Cbfa1), positively regulating the expression of Cbfa1, further enhancing the expression of
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osteoblast-specific genes and differentiation into osteoblast lines (Nishimura et al., 2002; Rosen, 2009). As one of the target genes of BMP2, Runx2 plays an important role in
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regulating osteoblast differentiation and bone development (Jeon et al., 2006; Byun et al.,
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2014). Our study found that the mRNA expression of genes BMP2, Smad5 and Runx2 were not affected by maternal nutrition restriction, while the expression of Smad1 mRNA in the femur was increased in the IR. This result showed that the upregulation of Smad1 transcript in the Smads signaling pathway may play an important role in manipulating the femur growth and development under maternal nutrition restriction. More investigation
should be focused on protein expression and post-translational modification in smad1 pathway response to maternal nutritional restriction.
5. Conclusions
In summary, the present results showed that maternal nutrition restriction in early
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pregnancy changed the fetal goats growth, altered the secretion of INTP and CTX-1 in umbilical cord blood, and upregulated the expression of BGLAP, OPG and Smad1 mRNA, down regulated the expression of LRP5 mRNA in femur, indicating that nutrition
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restriction during early gestion regulated the bone metabolic activity and promoted femur
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formation of fetal goats. It provides an important reference for nutrient regulation in early pregnancy for goats. The molecular mechanism of nutrient restriction on bone
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Acknowledgements
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development in goats needs to be clarified in the future.
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We thank Mr. Yuanwei Yang for providing technical assistance during livestock farming. This work was jointly supported by the National Natural Science Foundation of
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China (Grant No. 31402105, 31730092), National Key R&D Program of China (Grant No. 2018YFD0501903), and CAS Pioneer Hundred Talents Program.
Authors’ contributions
Qiongxian Yan and Zhiliang Tan designed the experiments. Hengzhi Li performed the experiments. Zhixiong He provided experimental technical help. Xilin Li and Hengzhi Li analyzed the data. Xilin Li wrote the main manuscript. All authors read and approved
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the final manuscript.
Conflict of interest
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The authors declare that they have no conflict of interests related to the submitted paper.
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ORCID X. L. Li https://orcid.org/0000-0002-1184-3551
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84, 2316-2337. Yan, Q., Xu, J., Wu, X., Su, D., Tan, Z., 2018. Stage-specific feed intake restriction differentially regulates placental traits and proteome of goats. Br. J. Nutr. 119, 11191132. Zhang, Y., Dong, X.L., Leung, P.C., Wong, M.S., 2009. Differential mRNA expression
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liaoning cashmere goats. China Herbivore Sci. 5, 008 (in Chinese).
Table 1 Ingredients and composition of the experimental diet (DM basis) Item Ingredients, % 60.00
Corn
26.80
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Miscanthus sinensis
Soybean meal
8.26
Fat powder
3.20 0.37
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Calcium bicarbonate
Premixa
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Sodium carbonate
0.39
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Calcium carbonate
0.18 0.80
Composition, %
11.51
Crude protein
11.46
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Metabolizable energy (MJ/kg)
a
Calcium
0.55
Phosphorus
0.20
Contained per kg of diet: 119 g MgSO4•H2O; 2.5 g FeSO4•7H2O; 0.8 g
CuSO4•5H2O; 3 g MnSO4•H2O; 5 g ZnSO4•H2O; 10 mg Na2SeO3; 40 mg KI; 30 mg CoCl2•6H2O; 95000 IU vitamin A; 17500 IU vitamin D; 18000 IU vitamin E.
Table 2 Primer sequences used for real-time quantitative PCR Genea
Primer sequences (5'-3')b
Product size
Gene Bank
110bp
XM_005677026.1
150bp
XM_013976665.1
F:GAACCGATGTGGAGTATGAGC ALPL R:GTGAGAGTGCTTGTGCTTCG
BGLAP R:CTCCTGGAAGCCGATGTG F:CCACAAGACCTACGACGACA VDR
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F:GCAGCGAGGTGGTGAAGA
130bp
F:ACGGCTCCGACGAACTCA LRP5
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R:GGAGGACGAGTTTCCAGAGA
XM_005680136.1
109bp
XM_013976027.1
149bp
XM_005680825.2
131bp
XM_005695574.1
153bp
XM_005684517.1
139bp
XM_005700049.1
135bp
XM_005689258.1
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R:TGAAGAGGGACAAGATGATGC F:AGGAGCGTCGTCAAGTAG
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LRP6
R:TGTAGGACCTGTGAGTGG F：TTACGGCAATCAAGAAAGCA
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β-catenin
R:CAGACAGCACCTTCAGCACT
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F:GGGCTTGCTGAGAACCTACAT
Cnx43
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R:GGAAACAGTCCACCTGATGC F:CTGCTTCTGTGGTGTGTGGA
Cyclin D1
R:CAAAGTATCACCGCCCACTT F:ACGGGCAGACCTTCGTATC
MMP16 R:CATCACCCTGTGGTTTCTCA
F:ATTTGGGCTCCTTCTAAC OPG
103bp
XM_005689133.2
125bp
XM_005687420.1
135bp
NM_001287564.1
R:CAGGGTCATGTCTATTCC F:CTTTGCCCATCTCACGATTA RANKL R:GTTTCCCATTGCTGAAGGTC F:TAACTCTAAGATTCCCAAGGC BMP2
F:ATCCCGAGTGGGTGTAGT Smad1 R:TCCTGGCGGTGGTATTCT
Smad5 R:TGCTCCCGCTCCACTCGTT
164bp
XM_013970504.1
137bp
XM_005682962.2
150bp
XM_005696518.1
111bp
NM_001009784.1
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F:ACCGCCGCCATCTCCACCTT
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R:TAACGACACCCACAACCC
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F:GCAGTTCCCAAGCATTTCAT Runx2
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R:GGTGGCAGGTAGGTGTGGTA F:CTTCCAGCCTTCCTTCCTG β-actin
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R:ACCGTGTTGGCGTAGAGGT ALPL = alkaline phosphatase, liver/bone/kidney; BGLAP = bone gamma
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carboxyglutamate protein; VDR = vitamin D (1, 25-dihydroxyvitamin D3) receptor;
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LRP5 = low density lipoprotein receptor related protein 5; LRP6 = low density lipoprotein receptor-related protein 6; β-catenin = beta-catenin; Cnx43 = connexin 43; MMP16 = matrix metalloproteinase 16; OPG = osteoprotegerin; RANKL = receptor activator for nuclear factor-κ B ligand; BMP2 = bone morphogenetic protein 2; Smad1 = mothers against decapentaplegic homolog 1; Smad5 = mothers against decapentaplegic homolog
5; Runx2 = runt-related transcription factor 2; β-actin = beta-actin. F = Forward primer; R = Reversed primer.
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b
Table 3 Effect of maternal intake restriction during early gestation on maternal goats growth performance and nutrient intake of the diets Itema
CONb
IRc
SEM
R/C(%)d
P value
30.55
27.20
1.77
-
0.38
31.99
27.69
1.28
1.44
0.49
0.81
402.58
248.33
521.29
Initial body weight
-
0.10
-
0.59
12.27
61.68
＜0.01
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Final body weight
36.14
70.39
0.02
94.94
-
-
-
Concentrate intake
Forage intake (g/d) Consumption
95.83
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rate (%)
924.99
614.21
40.81
66.40
＜0.01
CP (g/d)
163.46
103.50
4.723
63.32
＜0.01
GE (MJ/d)
15.68
10.31
0.64
65.72
＜0.01
NDF (g/d)
573.27
383.92
27.01
66.97
＜0.01
ADF (g/d)
185.81
130.53
12.77
70.25
0.02
CF (g/d)
223.89
155.99
14.28
69.67
＜0.01
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DMI (g/d)
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366.95
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(g/d)
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(day 65, kg) Total gain (kg)
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(day26, kg)
DMI = dry matter intake; CP =crude protein; GE = gross energy; NDF = neutral
detergent fiber; ADF = acid detergent fiber; CF = crude fiber. b
CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
d
R/C = IR/CON.
e
Consumption rate = the consumption of the two treatments in relation to the feed
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offered.
Table 4 Effect of maternal intake restriction during early gestation on the growth of fetal goats CONa
IRb
SEM
P diet
P sex
Body weight (g)c
33.28
76.13
5.02
<0.01
0.77
Body length (cm)d
9.85
13.86
0.53
<0.01
0.80
Thoracic circumference (cm)
6.44
8.56
0.27
<0.01
0.39
Umbilical circumference (cm)
1.28
1.50
0.05
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Item
CON = control group, n = 6.
b
IR = intake restriction group (60% of CON intake), n = 6.
0.57
Data were cited by Yan et al.(2018). The animals are the same as the Yan’s trials. Data were cited by Li et al.(2018).
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c
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a
<0.05
Table 5 Effect of maternal intake restriction during early gestation on bone metabolic markers in the umbilical cord serum of fetal goats IRc
SEM
P diet
P sex
PTH (ng/mL)
291.97
240.45
21.88
0.56
0.33
BALP (mU/mL)
4.49
4.00
0.76
0.49
0.41
BGLAP (ng/mL)
3.40
3.99
0.19
TRAP (U/L)
42.80
42.79
1.86
INTP (ng/mL)
0.11
0.04
0.01
CTX-1 (ng/mL)
0.66
0.50
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CONb
0.55 0.48
0.20
<0.01
0.15
-p 0.03
0.22
<0.05
0.58
PTH = parathyroid hormone; BALP = bone alkaline phosphatase; BGLAP = bone
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a
Itema
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gamma-carboxyglutamate protein; TRAP = tartrate resistant acid phosphatase; INTP = cross-linked N-terminal telopeptides of type I collagen; CTX-1 = cross-linked C-terminal
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telopeptides of type I collagen.
CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
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b
Table 6 Effect of intake restriction during early gestation on the liver mRNA expression of genes involved in bone metabolism of fetal goats IRc
SEM
P diet
P sex
ALPL
1.00
1.51
0.17
0.21
0.78
BGLAP
1.00
2.42
0.41
0.09
0.45
BMP2
1.00
1.31
0.25
VDR
1.00
1.18
0.19
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CONb
0.89
0.23
0.73
0.88
ALPL = alkaline phosphatase, liver/bone/kidney; BGLAP = bone gamma
-p
a
Itema
25-dihydroxyvitamin D3) receptor.
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carboxyglutamate protein; BMP2 = bone morphogenetic protein 2; VDR = vitamin D (1,
CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
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b
Table 7 Effect of maternal intake restriction during early gestation on the femur mRNA expression of genes involved in bone metabolism of fetal goats CONb
IRc
SEM
P diet
P sex
ALPL
1.00
0.97
0.10
0.85
0.63
BGLAP
1.00
1.69
0.11
<0.01
0.29
VDR
1.00
0.90
0.09
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a
Itema
0.44
0.37
ALPL = alkaline phosphatase, liver/bone/kidney; BGLAP = bone gamma
-p
carboxyglutamate protein; VDR = vitamin D (1, 25- dihydroxyvitamin D3) receptor. CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
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b
Table 8 Effect of maternal intake restriction during early gestation on the femur mRNA expression of genes involved in Wnt, OPG/RANKL/RANK, Smads signaling pathways of fetal goats Itema
CONb
IRc
SEM
P diet
P sex
1.00
0.64
0.05
LRP6
1.00
1.20
0.12
β-catenin
1.00
1.16
0.12
Cnx43
1.00
1.14
0.10
0.67
0.45
CyclinD1
1.00
1.10
0.06
0.48
0.67
MMP16
1.00
0.90
0.05
0.57
0.47
lP
re
LRP5
-p
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Wnt signaling pathway <0.01
0.28
0.39
0.36
0.53
0.93
OPG/RANKL/RANK signaling pathway 1.54
0.15
＜0.05
0.15
1.10
0.10
0.64
0.86
1.00
1.27
0.09
0.17
0.80
Smad1
1.00
1.83
0.16
＜0.05
0.24
Smad5
1.00
1.19
0.07
0.30
0.33
Runx2
1.00
0.88
0.07
0.48
0.93
1.00
na
OPG RANKL
1.00
ur
Smads signaling pathway
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BMP2
a
LRP5 = low density lipoprotein receptor related protein 5; LRP6 = low density
lipoprotein receptor-related protein 6; β-catenin = beta-catenin; Cnx43 = connexin 43; MMP16 = matrix metalloproteinase 16; OPG = osteoprotegerin; RANKL = receptor activator for nuclear factor-κ B ligand; BMP2 = bone morphogenetic protein 2; Smad1 = mothers against decapentaplegic homolog 1; Smad5 = mothers against decapentaplegic homolog 5; Runx2 = runt-related transcription factor 2. CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
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b
PII:
S0921-4488(19)30236-6
DOI:
https://doi.org/10.1016/j.smallrumres.2019.106027
Reference:
RUMIN 106027
To appear in:
Small Ruminant Research
Received Date:
22 March 2019
Revised Date:
11 October 2019
Accepted Date:
22 November 2019
Please cite this article as: Li X, Li H, He Z, Tan Z, Yan Q, Effects of maternal intake restriction during early pregnancy on fetal growth and bone metabolism in goats, Small Ruminant Research (2019), doi: https://doi.org/10.1016/j.smallrumres.2019.106027
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Running title: maternal nutrition restriction on fetal bone metabolism Effects of maternal intake restriction during early pregnancy on fetal growth and bone metabolism in goats
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Xilin Li a,b, Hengzhi Li c , Zhixiong He a, Zhiliang Tan a,d,e , Qiongxian Yan a,d,e *
CAS Key Laboratory for Agro-Ecological Processes in Subtropical Region, National
Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and
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Poultry Production, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Hunan Provincial Engineering Research Center for
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Healthy Livestock and Poultry Production, Institute of Subtropical Agriculture, The
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Chinese Academy of Sciences, Hunan Provincial Key Laboratory of Nutritional Physiology and Metabolic Process Changsha, Hunan 410125, P.R. China University of Chinese Academy of Sciences, Beijing 100049, P.R. China
c
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing
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b
Hunan Co-Innovation Center for Utilization of Botanical Functional Ingredients,
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d
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210095, P.R. China
Changsha, Hunan 410128, P. R. China e
Hunan Co-Innovation Center of Animal Production Safety, CICAPS, Changsha, Hunan
410128, P.R. China
*Corresponding author E-mail address: [email protected](Qiongxian Yan).
Highlight
This study was designed to investigate the effect of maternal intake
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restriction on fetal growth and bone metabolism of goats. Maternal intake restriction during early gestation affected the thoracic
circumference, umbilical circumference, bone metabolic activities (INTP
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and CTX-1) and femur BGLAP mRNA expression of fetal goats.
This study provides a reference for the effects of nutrient limitation in
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early pregnancy on the bone development of fetal goats.
Abstract
The objective of this study was to investigate the effects of maternal
nutritional restriction on fetal growth and bone metabolism in early pregnancy. Twelve twin-bearing goats (Xiangdong black goats) in second pregnancy, with similar weight (31.2 ± 8.1 kg) and age (2.0 ± 0.3 year) were assigned to two dietary treatments in early
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gestation (26-65 days): control group (CON, 100% feed) and intake restriction group (IR, 60% of CON intake). The umbilical blood, right femur and liver of the fetuses were sampled on day 65 to determine the bone metabolism indicators and expression of genes
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involved in bone development. Maternal intake restriction did not affect the growth
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performance of pregnancies (P > 0.05), increased (P < 0.05) the body weight, body length,
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thoracic circumference and umbilical circumference of fetal goats, but decreased (P < 0.05) the concentrations of cross-linked N-terminal telopeptides of type I collagen and
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cross-linked C-terminal telopeptides of type I collagen, which involved in bone metabolic
gamma-carboxyglutamate
protein,
osteoprotegerin
and
mothers
against
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bone
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activity, in the fetal umbilical blood of the IR. In fetal femurs, the mRNA expression of
decapentaplegic homolog 1 both participating in osteoclast differentiation were increased (P < 0.05), whereas transcript of the low density lipoprotein receptor related protein 5 involved in osteoblast differentiation was decreased (P < 0.05), compared with the fetuses from the CON. These results indicate that nutrition restriction in early pregnancy
regulated the fetal growth and promoted formative activity of femur. The molecular mechanism of maternal intake restriction on bone metabolism needs further investigation.
Keywords Gestational nutrition; Fetal development; Femurs; Fetal goats
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1. Introduction
During pregnancy, the energy required for rapid fetal growth comes mainly from the placental absorption of glucose and amino acids in the maternal blood (Redmer et al.,
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2004; Priya et al., 2016). Therefore, the mother needs to provide sufficient protein and
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glucose to ensure normal fetal growth (Husted et al., 2008). Maternal malnutrition in pregnancy often leads to intrauterine growth retardation (IUGR), and short-term or long-
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term consequences, such as asymmetric growth of organs, hypoxia, acidemia, intrauterine death in the uterus and metabolic disorders, cardiovascular disease, diabetes, and obesity
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after birth (Wu et al., 2006; Decembrino et al., 2013).
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Bone is given a greater priority for nutrients than tissues such as muscle and fat during fetal life because bone plays a key role in protecting soft tissues, maintaining
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mineral balance, providing structural support, framework of the carcass, and supporting hematopoiesis (Florencio-Silva et al., 2015). The metabolic balance of bones is achieved through bone transformation that includes bone formation by osteoblasts and bone resorption by osteoclasts (Manolagas and Jilka et al., 1995). Generally, biochemical markers of bone metabolism are simple and effective methods to evaluate bone metabolic
activity, such as bone alkaline phosphatase (BALP) and bone gamma-carboxyglutamate protein (BGLAP, Lian et al., 1993), tartrate resistant acid phosphatase (TRAP, Minkin, 1982), cross-linked N-terminal telopeptides of type I collagen (INTP) and cross-linked C-terminal telopeptides of type I collagen (CTX-1, Banfi et al., 2010). Moreover, bone development is regulated by a variety of signaling pathways, such as the Wingless (Wnt)
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signaling pathway (Miller, 2002), the OPG/RANKL/RANK signaling pathway (Suda et al., 1999), and the mothers against decapentaplegic homologs (Smads) signaling pathway (Matsubara et al., 2008).
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Fetal bone development is susceptible to maternal nutrition disruption. In rats,
maternal protein restriction easily causes the reduction of femur weight and bone mineral
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content (Mehta et al., 2002) and the reduction of femoral head size with thinner and less
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dense trabeculae in off spring (Lanham et al., 2008). In sheep, the effects of maternal nutrients restriction on offspring’s bone are variable with either reduced offspring bone
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weight, length, or density (Tygesen et al., 2007; Kleemann et al., 2015). In fact, the phenomena of maternal malnutrition are very ubiquitous in ruminants fed on traditional
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grazing regions around the world, especially in dry or cold seasons because of the
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shortage of fodders. Because of this, fetal bone metabolism in conditions of malnutrition is getting more attention. Even so, only a few studies evaluating the eff ects of maternal nutrition restriction on off spring bone metabolism have been reported during late pregnancy (Tygesen et al., 2007; Fetoui et al., 2008). Fetal bone metabolism knowledges related to maternal malnutrition during early pregnancy are still scarce. Therefore, this
study was designed to investigate whether maternal nutrition restriction during early gestation affect fetal growth and bone metabolic activity via regulation of the Wnt, OPG/RANKL/RANK and Smads signaling pathways in goats.
2. Materials and methods
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This study was conducted according to the Animal Care and the Use Guidelines of the Animal Care Committee, Institute of Subtropical Agriculture, The Chinese Academy of
2.1.Animal management and dietary treatments
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Sciences, Changsha, China.
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Twenty goats (Xiangdong black, a local breed in Liuyang, Hunan Province, China)
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in second pregnancy, with similar weight (31.2 ± 8.1 kg) and age (2.0 ± 0.3 year) were selected as experimental animals. Synchronous estrus was conducted by intramuscular
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injection of progestogen followed by equine chorionic gonadotropin to ensure a consistent pregnancy date. The injections of equine chorionic gonadotropin and
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progestogen are both a mixture of 1000 units of powdered medicine diluted by 2 ml sterile
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physiological saline. All goats were inseminated directly into the uterus using frozenthawed semen from one buck. The day of artificial insemination was assumed as the first day of pregnancy. During early pregnancy (0-22 days), the goats were grazed on pasture freely. They were then individually fed in stainless steel bracket cages (1.2 × 0.91 m) to adapt the experimental diet for 3 days (23-25 days). Excluding goats that were not
pregnant or not twin bearing, twelve twin-bearing goats examined by ultrasonography were randomly allocated into two treatment groups: control group (CON, 100% feed, n=6), and intake restriction group (IR, 60% of CON intake, n=6). Ultrasonic examinations were arranged at day 25 and day 40 to insure the experimental goats were twin-bearing. The diagnostic criteria was the presence of two liquid dark areas of gestational sac and
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heartbeats, cotyledons and/or fetal parts recognized on B-ultrasonic scanner (Amer, 2010). Throughout pregnancy days 26-65, the control diet was supplied to meet 100% of metabolic energy and crude protein requirements for pregnant goats according to the
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Feeding Standard of Meat-Producing Sheep and Goats of Chinese agricultural industry
standards (HB, NY/T 816-2004). The parameters were set as followed: body weight,
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30kg; dry matter intake (DMI), 0.90 kg/d; digestible energy (DE), 12.34 MJ/d; metabolic
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energy (ME), 10.12 MJ/d; crude protein (CP), 89 g/d; calcium (Ca), 5.8 g/d; phosphorus (P), 3.9 g/d; NaCl, 4.5 g/d. The ingredients and nutrient composition of the experimental
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diet are shown in Table 1. Fresh Miscanthus sinensis was selected as forage, cut into small pieces with 3-5cm, and manually mixed with the concentrate before feeding. All goats
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were fed twice a day at 08:30h and 17:00h, and had free access to water. Goats in the
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CON were fed based on the Feeding standards above with the residues less than 5%, while goats in the IR were fed at 60% of CON intake. The feed amount for the CON was adjusted every week by extra 5% increments. The feed amount for the IR was modulated correspondingly. The feed intake of goats was recorded every day, and the orts of concentrate and forage were recorded separately after screening. The ratio of concentrate
to forage was 40:60.
2.2.Chemical Analysis
Feed samples including concentrate and Miscanthus sinensis were analyzed for dry matter (DM), crude protein (CP), calcium (Ca), phosphorus (P), neutral detergent fiber (NDF), acid detergent fiber (ADF) and crude fiber (CF) according to the procedures of
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Association of Official Analytical Chemists (Cunniff, 1995).
2.3.Blood and tissue samples collection
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At 65d, all the experimental goats were fasted overnight prior to weighing, bleeding
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and slaughtering. Post-slaughter the entire uterus was immediately removed. Fetuses from each treatment (CON, 5 male and 7 female; IR, 6 male and 6 female) were collected.
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After dissection, the umbilical cord attached to the uterine epithelial was cut and ligated, then the blood from the vein and arteries flowed into a 2 mL centrifuge tube by gravity.
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Blood samples were allowed to coagulate for 2 hours for the determination of bone
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metabolite indicators. Centrifugation was performed at 3000 g for 15 min at 4°C. The serum was aliquoted into 1.5 mL centrifuge tubes and stored in a -80°C freezer.
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Meanwhile, fetuses were weighed, and body length, thoracic and umbilical circumference were measured with tapeline. The femurs and livers in the right side of each fetus were collected, washed by normal saline and then immediately frozen in liquid nitrogen and stored in a -80°C freezer for gene expression analyses.
2.4.Metabolic marker activities determination
The contents of parathyroid hormone (PTH), bone alkaline phosphatase (BALP), bone gamma-carboxyglutamate protein (BGLAP), tartrate resistant acid phosphatase (TRAP), cross-linked N-terminal telopeptides of type I collagen (INTP) and cross-linked C-terminal telopeptides of type I collagen (CTX-1) in fetal umbilical cord serum were
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measured using colorimetric methods with a spectrophotometer (MB-530, Huisong technology development Co., Ltd, Shenzhen, China). The assay kits of PTH, BALP and BGLAP were purchased from Wuhan Huamei Bioengineering Co., Ltd. (Wuhan, Hubei,
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China). The assay kits of TRAP, INTP and CTX-1 were purchased from MyBioSource
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Inc. (San Diego, SC, USA). PTH (intra-assay precision: CV%<15%, inter-assay precision: CV%<15%, sensitivity: 25 pg/mL), BALP (intra-assay precision: CV%<8%, inter-assay
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Precision: CV%<10%, sensitivity: 1.95 mU/mL), BGLAP (intra-assay precision: CV%<8%, inter-assay precision: CV%<10%, sensitivity: 0.24 ng/mL), TRAP (intra-
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assay precision: CV%<15%, inter-assay precision: CV%<15%, sensitivity: 1.0 U/L), INTP (intra-assay precision: CV%<15%, inter-assay precision: CV%<15%, sensitivity:
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1.0 ng/mL) and CTX-1 (intra-assay precision: CV%≤8%, inter-assay precision: CV%≤
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12%, sensitivity: up to 0.05 ng/mL) were analyzed in duplicate.
2.5.Gene expression analyses
Total RNA was extracted from femurs and livers with the RNAiso Plus reagent (TaKaRa, JPN). The total RNA absorbance values of OD260 and OD280 were determined
with a nucleic acid analyzer (Nano Drop ND2000, USA). The RNA was used to reverse transcribe into cDNA (20μL) by Prime Script RT reagent Kit with gDNA Eraser (TaKaRa, JPN) to meet an OD260/OD280 ratio of 1.8 to 2.2, and evaluated by 1% agarose gel electrophoresis. The resulting cDNA template was stored at -80°C until analysis by realtime quantitative PCR. Primers shown in Table 2 were designed with premier 5.0 software
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according to the relevant gene sequences of goats in Genebank and synthesized by Shanghai Bioengineering Co., Ltd. (Songjiang, Shanghai, China). The specificity analyses of primers were performed preliminarily by using the Blast tool. Real-time
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quantitative PCR was proceeded by ABI Prism 7900 HT Fast Real-Time PCR System (ABI, CA) with the SYBR® Premix Ex TaqTM II (TaKaRa, JPN). The reaction system
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contained 5 μL SYBR® Premix Ex TaqTM (2x), 0.4 μL forward primer (10 μM), 0.4 μL
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reverse primer (10 μM), 0.2 μL ROX reference dye (50x), 1.0 μL cDNA and 3 μL sterilized ddH2O under the amplification procedure (95°C for 10 min to active the DNA
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polymerase, then cycled at 95°C for 5 s, and 60°C for 30 s for 40 cycles). The specificity of products was determined according to dissolution curve under the dissolution
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procedure (95°C for 15 s, 60°C for 15 s and 95°C for 15 s).
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The relative quantification of gene expression was calculated as a ratio of the target gene to the housekeeping gene by 2-ΔΔCt method (Livak and Schmittgen, 2001) under the premise of the same amplification efficiency of the target gene and the housekeeping gene, where –ΔΔCt = –[ (Ct target gene–Ct β-actin) IR group–(Ct target gene–Ct β-actin) CON group]. Ct was the number of experiment cycles when the amplification product reaches the fluorescence
threshold. In this study, we chose the β-actin as the housekeeping gene, which was manifested to be steadily expressed in samples. Other researchers also used the β-actin as the housekeeping gene in bone metabolic study for goats (Nemeth et al. 2017).
2.6.Statistical analysis
The data of this experiment was analyzed by one-way analysis of variance (ANOVA)
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using the MIXED procedure of SAS 9.2 (SAS Inst. Inc., Cary, NC, USA). The fixed effects of diet level and fetal sex were considered in the model. Because there were no
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interaction of diet level and fetal sex for all the indexes, the interaction effect was not
showed in the Tables. Maternal goat was considered as the random effect. Significance
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was declared at P < 0.05 and trends were considered at 0.05 ≤ P < 0.10.
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3. Results
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3.1.Growth performance and nutrient intake of maternal goats
As shown in Table 3, the final body weight and total body gain of the IR were not
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affected compared with the CON (P > 0.05). The dry matter intake of the concentrate in
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the IR was actually 61.68% of the CON in early gestation (P < 0.05), which was close to the 60% target. The dry matter intake of the forage in the IR was 70.39% of the CON (P < 0.05), and other nutrient components in the IR were between 61.68 to 70.39% of the CON (P < 0.05). The consumption of the CON and IR in relation to the feed offered was 95.83% and 94.94% respectively.
3.2.Growth of the fetuses
Compared with the CON, the body weight, body length, thoracic circumference and umbilical circumference of the IR fetuses were increased by intake restriction (P < 0.05) in early pregnancy (Table 4). These indicators were not changed by the fetal sex (P > 0.05).
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3.3.Bone metabolic marker activities of the fetal umbilical cord serum
The concentrations of INTP (P < 0.01) and CTX-I (P < 0.05) in the fetal umbilical
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cord serum of the IR were less than that of the CON. Other indexes of bone metabolism and conversion were not affected by intake restriction (P > 0.05) or fetal sex (P > 0.05,
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Table 5).
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3.4.Bone metabolic gene expression in the fetal liver and femur
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In the early pregnancy, the mRNA expression of alkaline phosphatase, liver/bone/kidney (ALPL), bone morphogenetic protein 2 (BMP2), and vitamin D (1, 25-
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dihydroxyvitamin D3) receptor (VDR) genes in the fetal livers of the IR were not affected by intake restriction (P > 0.05), but the mRNA expression of BGLAP was increased (P <
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0.10, Table 6). The mRNA abundance of ALPL, BGLAP, BMP2 and VDR were not influenced by fetal sex (P > 0.05). For the femurs of fetal goats, BGLAP, OPG and Smad1 mRNA in the IR were expressed at an increased amount (P < 0.05, Table 7 and 8), while low density lipoprotein receptor related protein 5 (LRP5) mRNA expression was less (P < 0.05) than that of the
CON (Table 8). Meanwhile, abundance of BGLAP, OPG, Smad1 and LRP5 mRNA were not changed by fetal sex (P > 0.05). No differences in the mRNA expression of ALPL, VDR, LRP6, β-catenin, Cnx43, CyclinD1, MMP16, RANKL, BMP2, Smad5 and Runx2 were observed neither by intake restriction (P > 0.05) nor fetal sex (P ＞ 0.05).
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4. Discussion
The aim of this experiment was to explore the effects of maternal malnutrition on fetal growth and skeletal metabolism in goats. Based on the results section, we indicated
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that DMI in the IR during early pregnancy was 66.40% of CON, which was close to our
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original expectation. According to our previous report (Yan et al., 2018; Li et al., 2018), the body weight of the fetuses in the IR group was about 2.3 times greater in CON and
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the body length was 1.4 times longer at 65 day of gestation. Simultaneously, thoracic and umbilical circumference of the fetuses were increased by 30% and 20% respectively in
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this study. These results were partly in agreement with a previous study (Osgerby et al., 2002), in which an increment in the fetus's thoracic circumference by nutritional
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constraint of ewes in early gestation was also observed. However, it was in contrast with
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the findings of Nishina et al. (2003) in Welsh Mountain ewes, where there were no overall differences in the fetal body weight and placental size between control (100% feed) and dietary restricted group (30% feed restriction). The difference could be ascribed to animal species and dietary ingredients. In addition, it has been reported that the absolute fetal growth is not evident before 90 days of gestation, but the relative growth rate is often very
distinct. For instance, the weight gain at gestational day 60 relative to day 45 is about 456.2%, and at gestational day 75 relative to day 60 is 429.2% (Zhao et al., 2012). The increase of fetal body weight could be ascribed to the nanny goats, under nutritional restriction, altering their energy metabolism through the changes in the expression of hormones and growth factors to adjust the distribution of nutrition (Coad et al., 2001).
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Skeletal development is in the process of dynamic balanceable metabolism of bone formation and bone resorption. Bone formation is mainly related to osteoblast metabolism, and bone resorption is mainly related to osteoclast metabolism. Enzymes or other
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decomposed products, derived from the bone cells and bone matrix, can be used as
markers to reflect the bone metabolic activity. For example, BALP and BGLAP proteins
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are associated with bone formation (Lian et al., 1993), whilst TRAP (Minkin, 1982), INTP
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and CTX-1 proteins (Banfi et al., 2010) are linked with bone resorption. Moreover, PTH is an important hormone of maintaining calcium and phosphorus metabolism and bone
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metabolism (Andress et al., 1989). In the current study, intake constraint of nanny goats greatly decreased the CTX-1 and INTP concentrations in the fetal umbilical cord blood.
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However, TRAP, BALP, BGLAP and PTH concentrations were unaffected. It is possible
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that the mixed blood from a vein and arteries of the umbilical cord was not appropriate to determine these metabolic markers in the fetuses, because it also contained blood from maternal circulation. These findings suggested that osteoclast metabolism of fetal goats was reduced and osteoblast metabolism was not affected by nutritional constraint, overall, resulting in the relative increase in osteogenic activity.
Fetal liver is the organ closely related to bone development and metabolism, and its various metabolic factors secreted regulate bone growth. The expression levels of ALPL, BGLAP and VDR are related with the regulation of calcium homeostasis and bone formation (Kollitz et al., 2016; Song et al., 2011). While BMP2 is a key factor in bone growth and development, and its expression in the liver also affects the growth and
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regeneration of the liver (Kan et al., 2009). In the current study, the mRNA expression of ALPL, BMP2 and VDR genes in the liver of fetuses were not affected by intake restriction
or fetal sex. Increase in fetal liver weight and decrease in DNA methylation was
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previously observed by Li et al. (2018), indicating that cell proliferation and epigenic modifications rather than fetal liver genes regulating bone formation are more sensitive
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to maternal nutrient restriction. Fortunately, BGLAP transcript tended to be increased by
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intake restriction. In the femur of fetuses, intake restriction increased the BGLAP expression, which revealed that the activity of osteoblast and bone formation were
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elevated. It also implies that regulatory mechanism of BGLAP transcript induced by nutrition restriction is different in visceral tissues and skeletal organs. Hence, it is
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reasonable to speculate that bone cells are more sensitive than other cells to nutritional
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restriction, considering BGLAP is mainly transcribed by osteoblasts (Lian et al., 1989), and some miRNAs (miR-125b and miR-30c2) might be involved in negatively regulating BGLAP expression in osteoblast (Laxman et al., 2015). The Wnt signaling pathway is an important regulator of osteoblast development and differentiation, and plays a major role in fetal bone formation (Miller, 2002). The classic
Wnt signaling pathway activates the downstream protein factor Dishevelled (Dsh) after binding to the Wnt receptor Frizzled and the collagenous receptor LRP5 / low density lipoprotein receptor-related protein 6 (LRP6) to form the Wnt-Frizzled-LRP5/Wnt-FzdLRP6 triplex, thereby inhibiting the phosphorylation of beta-catenin (β-catenin) by Glycogen synthase kinase 3β (GSK3β). Then β-catenin translocates into nucleus to
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activate the expression of downstream target genes (Mulholland et al., 2005), such as connexin 43 (Cnx43), CyclinD1 and matrix metalloproteinase 16 (MMP16) (Lyer et al.,
2013). In the present study, the mRNA expression of LRP6, β-catenin, Cnx43, CyclinD1
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and MMP16 were not affected, but the mRNA expression of LRP5 in the femur was
reduced by intake restriction. It suggested that only the LRP5 in the femur was sensitive
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to maternal nutritional restriction, and intake restriction or fetal sex did not alter the Wnt-
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Frizzled-LRP5 related pathway at the mRNA level. Whether proteins expression and post-translational modification in this pathway are changed by nutritional restriction
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needs to be further explored.
The OPG/RANKL/RANK signaling pathway is a key pathway through which
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osteoblasts regulate osteoclast differentiation and activation (Suda et al., 1999). The OPG
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and RANKL are mainly distributed in osteoblasts, meanwhile RANK is mainly distributed in
osteoclasts. Usually, RANKL binds to its receptor RANK, which
promotes the differentiation and maturation of osteoclasts. The OPG can also combine with RANKL to prevent the binding between RANKL and RANK and thus inhibit the formation and differentiation of osteoclasts (Khosla et al., 2001). The results of this
experiment showed that the expression of OPG was increased due to intake restriction, whereas the expression of RANKL was not affected, leading to the increment in the ratio of OPG to RANKL. Zhang et al. (2009) found dietary Ca restriction significantly upregulated the mRNA expression of OPG in proximal tibia of aged rats. The increase in this ratio indicated that the proliferation of osteoclasts was inhibited, thus promoting bone
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growth and development. BMP2-Smad-Runx2 axis plays an important role in the osteoblastogenesis (Matsubara et al., 2008). The BMP2 regulates osteoblast differentiation and bone
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formation by binding to type I and type II BMP2 receptors on the cell surface, and then
phosphorylates the Smads 1, 5 and 8 proteins in the cell nucleus. The phosphorylated
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protein forms a complex with Smad4, then transferred into the nucleus to combine with
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osteoblast to promote transcription factors such as Core Banding Factor 1 (Cbfa1), positively regulating the expression of Cbfa1, further enhancing the expression of
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osteoblast-specific genes and differentiation into osteoblast lines (Nishimura et al., 2002; Rosen, 2009). As one of the target genes of BMP2, Runx2 plays an important role in
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regulating osteoblast differentiation and bone development (Jeon et al., 2006; Byun et al.,
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2014). Our study found that the mRNA expression of genes BMP2, Smad5 and Runx2 were not affected by maternal nutrition restriction, while the expression of Smad1 mRNA in the femur was increased in the IR. This result showed that the upregulation of Smad1 transcript in the Smads signaling pathway may play an important role in manipulating the femur growth and development under maternal nutrition restriction. More investigation
should be focused on protein expression and post-translational modification in smad1 pathway response to maternal nutritional restriction.
5. Conclusions
In summary, the present results showed that maternal nutrition restriction in early
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pregnancy changed the fetal goats growth, altered the secretion of INTP and CTX-1 in umbilical cord blood, and upregulated the expression of BGLAP, OPG and Smad1 mRNA, down regulated the expression of LRP5 mRNA in femur, indicating that nutrition
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restriction during early gestion regulated the bone metabolic activity and promoted femur
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formation of fetal goats. It provides an important reference for nutrient regulation in early pregnancy for goats. The molecular mechanism of nutrient restriction on bone
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Acknowledgements
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development in goats needs to be clarified in the future.
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We thank Mr. Yuanwei Yang for providing technical assistance during livestock farming. This work was jointly supported by the National Natural Science Foundation of
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China (Grant No. 31402105, 31730092), National Key R&D Program of China (Grant No. 2018YFD0501903), and CAS Pioneer Hundred Talents Program.
Authors’ contributions
Qiongxian Yan and Zhiliang Tan designed the experiments. Hengzhi Li performed the experiments. Zhixiong He provided experimental technical help. Xilin Li and Hengzhi Li analyzed the data. Xilin Li wrote the main manuscript. All authors read and approved
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the final manuscript.
Conflict of interest
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The authors declare that they have no conflict of interests related to the submitted paper.
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ORCID X. L. Li https://orcid.org/0000-0002-1184-3551
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Tygesen, M.P., Harrison, A.P., Therkildsen, M., 2007. The effect of maternal nutrient restriction during late gestation on muscle, bone and meat parameters in five month old lambs. Livest. Sci. 110, 230-241.
Wu, G., Bazer, F.W., Wallace, J.M., Spencer, T.E., 2006. Board-invited review: intrauterine growth retardation: implications for the animal sciences. J. Anim. Sci.
84, 2316-2337. Yan, Q., Xu, J., Wu, X., Su, D., Tan, Z., 2018. Stage-specific feed intake restriction differentially regulates placental traits and proteome of goats. Br. J. Nutr. 119, 11191132. Zhang, Y., Dong, X.L., Leung, P.C., Wong, M.S., 2009. Differential mRNA expression
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profiles in proximal tibia of aged rats in response to ovariectomy and low-Ca diet. Bone 44, 46-52.
Zhao, Y.J., Chang, Q., Jiang, H.Z., 2012. A preliminary study on the fetal development of
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liaoning cashmere goats. China Herbivore Sci. 5, 008 (in Chinese).
Table 1 Ingredients and composition of the experimental diet (DM basis) Item Ingredients, % 60.00
Corn
26.80
ro of
Miscanthus sinensis
Soybean meal
8.26
Fat powder
3.20 0.37
-p
Calcium bicarbonate
Premixa
lP
Sodium carbonate
0.39
re
Calcium carbonate
0.18 0.80
Composition, %
11.51
Crude protein
11.46
Jo
ur
na
Metabolizable energy (MJ/kg)
a
Calcium
0.55
Phosphorus
0.20
Contained per kg of diet: 119 g MgSO4•H2O; 2.5 g FeSO4•7H2O; 0.8 g
CuSO4•5H2O; 3 g MnSO4•H2O; 5 g ZnSO4•H2O; 10 mg Na2SeO3; 40 mg KI; 30 mg CoCl2•6H2O; 95000 IU vitamin A; 17500 IU vitamin D; 18000 IU vitamin E.
Table 2 Primer sequences used for real-time quantitative PCR Genea
Primer sequences (5'-3')b
Product size
Gene Bank
110bp
XM_005677026.1
150bp
XM_013976665.1
F:GAACCGATGTGGAGTATGAGC ALPL R:GTGAGAGTGCTTGTGCTTCG
BGLAP R:CTCCTGGAAGCCGATGTG F:CCACAAGACCTACGACGACA VDR
ro of
F:GCAGCGAGGTGGTGAAGA
130bp
F:ACGGCTCCGACGAACTCA LRP5
-p
R:GGAGGACGAGTTTCCAGAGA
XM_005680136.1
109bp
XM_013976027.1
149bp
XM_005680825.2
131bp
XM_005695574.1
153bp
XM_005684517.1
139bp
XM_005700049.1
135bp
XM_005689258.1
re
R:TGAAGAGGGACAAGATGATGC F:AGGAGCGTCGTCAAGTAG
lP
LRP6
R:TGTAGGACCTGTGAGTGG F：TTACGGCAATCAAGAAAGCA
na
β-catenin
R:CAGACAGCACCTTCAGCACT
ur
F:GGGCTTGCTGAGAACCTACAT
Cnx43
Jo
R:GGAAACAGTCCACCTGATGC F:CTGCTTCTGTGGTGTGTGGA
Cyclin D1
R:CAAAGTATCACCGCCCACTT F:ACGGGCAGACCTTCGTATC
MMP16 R:CATCACCCTGTGGTTTCTCA
F:ATTTGGGCTCCTTCTAAC OPG
103bp
XM_005689133.2
125bp
XM_005687420.1
135bp
NM_001287564.1
R:CAGGGTCATGTCTATTCC F:CTTTGCCCATCTCACGATTA RANKL R:GTTTCCCATTGCTGAAGGTC F:TAACTCTAAGATTCCCAAGGC BMP2
F:ATCCCGAGTGGGTGTAGT Smad1 R:TCCTGGCGGTGGTATTCT
Smad5 R:TGCTCCCGCTCCACTCGTT
164bp
XM_013970504.1
137bp
XM_005682962.2
150bp
XM_005696518.1
111bp
NM_001009784.1
-p
F:ACCGCCGCCATCTCCACCTT
ro of
R:TAACGACACCCACAACCC
re
F:GCAGTTCCCAAGCATTTCAT Runx2
lP
R:GGTGGCAGGTAGGTGTGGTA F:CTTCCAGCCTTCCTTCCTG β-actin
a
na
R:ACCGTGTTGGCGTAGAGGT ALPL = alkaline phosphatase, liver/bone/kidney; BGLAP = bone gamma
ur
carboxyglutamate protein; VDR = vitamin D (1, 25-dihydroxyvitamin D3) receptor;
Jo
LRP5 = low density lipoprotein receptor related protein 5; LRP6 = low density lipoprotein receptor-related protein 6; β-catenin = beta-catenin; Cnx43 = connexin 43; MMP16 = matrix metalloproteinase 16; OPG = osteoprotegerin; RANKL = receptor activator for nuclear factor-κ B ligand; BMP2 = bone morphogenetic protein 2; Smad1 = mothers against decapentaplegic homolog 1; Smad5 = mothers against decapentaplegic homolog
5; Runx2 = runt-related transcription factor 2; β-actin = beta-actin. F = Forward primer; R = Reversed primer.
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ro of
b
Table 3 Effect of maternal intake restriction during early gestation on maternal goats growth performance and nutrient intake of the diets Itema
CONb
IRc
SEM
R/C(%)d
P value
30.55
27.20
1.77
-
0.38
31.99
27.69
1.28
1.44
0.49
0.81
402.58
248.33
521.29
Initial body weight
-
0.10
-
0.59
12.27
61.68
＜0.01
re
Final body weight
36.14
70.39
0.02
94.94
-
-
-
Concentrate intake
Forage intake (g/d) Consumption
95.83
e
na
rate (%)
924.99
614.21
40.81
66.40
＜0.01
CP (g/d)
163.46
103.50
4.723
63.32
＜0.01
GE (MJ/d)
15.68
10.31
0.64
65.72
＜0.01
NDF (g/d)
573.27
383.92
27.01
66.97
＜0.01
ADF (g/d)
185.81
130.53
12.77
70.25
0.02
CF (g/d)
223.89
155.99
14.28
69.67
＜0.01
ur
DMI (g/d)
Jo a
366.95
lP
(g/d)
-p
(day 65, kg) Total gain (kg)
ro of
(day26, kg)
DMI = dry matter intake; CP =crude protein; GE = gross energy; NDF = neutral
detergent fiber; ADF = acid detergent fiber; CF = crude fiber. b
CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
d
R/C = IR/CON.
e
Consumption rate = the consumption of the two treatments in relation to the feed
Jo
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offered.
Table 4 Effect of maternal intake restriction during early gestation on the growth of fetal goats CONa
IRb
SEM
P diet
P sex
Body weight (g)c
33.28
76.13
5.02
<0.01
0.77
Body length (cm)d
9.85
13.86
0.53
<0.01
0.80
Thoracic circumference (cm)
6.44
8.56
0.27
<0.01
0.39
Umbilical circumference (cm)
1.28
1.50
0.05
ro of
Item
CON = control group, n = 6.
b
IR = intake restriction group (60% of CON intake), n = 6.
0.57
Data were cited by Yan et al.(2018). The animals are the same as the Yan’s trials. Data were cited by Li et al.(2018).
Jo
ur
na
lP
d
re
c
-p
a
<0.05
Table 5 Effect of maternal intake restriction during early gestation on bone metabolic markers in the umbilical cord serum of fetal goats IRc
SEM
P diet
P sex
PTH (ng/mL)
291.97
240.45
21.88
0.56
0.33
BALP (mU/mL)
4.49
4.00
0.76
0.49
0.41
BGLAP (ng/mL)
3.40
3.99
0.19
TRAP (U/L)
42.80
42.79
1.86
INTP (ng/mL)
0.11
0.04
0.01
CTX-1 (ng/mL)
0.66
0.50
ro of
CONb
0.55 0.48
0.20
<0.01
0.15
-p 0.03
0.22
<0.05
0.58
PTH = parathyroid hormone; BALP = bone alkaline phosphatase; BGLAP = bone
re
a
Itema
lP
gamma-carboxyglutamate protein; TRAP = tartrate resistant acid phosphatase; INTP = cross-linked N-terminal telopeptides of type I collagen; CTX-1 = cross-linked C-terminal
na
telopeptides of type I collagen.
CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
Jo
ur
b
Table 6 Effect of intake restriction during early gestation on the liver mRNA expression of genes involved in bone metabolism of fetal goats IRc
SEM
P diet
P sex
ALPL
1.00
1.51
0.17
0.21
0.78
BGLAP
1.00
2.42
0.41
0.09
0.45
BMP2
1.00
1.31
0.25
VDR
1.00
1.18
0.19
ro of
CONb
0.89
0.23
0.73
0.88
ALPL = alkaline phosphatase, liver/bone/kidney; BGLAP = bone gamma
-p
a
Itema
25-dihydroxyvitamin D3) receptor.
re
carboxyglutamate protein; BMP2 = bone morphogenetic protein 2; VDR = vitamin D (1,
CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
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b
Table 7 Effect of maternal intake restriction during early gestation on the femur mRNA expression of genes involved in bone metabolism of fetal goats CONb
IRc
SEM
P diet
P sex
ALPL
1.00
0.97
0.10
0.85
0.63
BGLAP
1.00
1.69
0.11
<0.01
0.29
VDR
1.00
0.90
0.09
ro of
a
Itema
0.44
0.37
ALPL = alkaline phosphatase, liver/bone/kidney; BGLAP = bone gamma
-p
carboxyglutamate protein; VDR = vitamin D (1, 25- dihydroxyvitamin D3) receptor. CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
Jo
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lP
re
b
Table 8 Effect of maternal intake restriction during early gestation on the femur mRNA expression of genes involved in Wnt, OPG/RANKL/RANK, Smads signaling pathways of fetal goats Itema
CONb
IRc
SEM
P diet
P sex
1.00
0.64
0.05
LRP6
1.00
1.20
0.12
β-catenin
1.00
1.16
0.12
Cnx43
1.00
1.14
0.10
0.67
0.45
CyclinD1
1.00
1.10
0.06
0.48
0.67
MMP16
1.00
0.90
0.05
0.57
0.47
lP
re
LRP5
-p
ro of
Wnt signaling pathway <0.01
0.28
0.39
0.36
0.53
0.93
OPG/RANKL/RANK signaling pathway 1.54
0.15
＜0.05
0.15
1.10
0.10
0.64
0.86
1.00
1.27
0.09
0.17
0.80
Smad1
1.00
1.83
0.16
＜0.05
0.24
Smad5
1.00
1.19
0.07
0.30
0.33
Runx2
1.00
0.88
0.07
0.48
0.93
1.00
na
OPG RANKL
1.00
ur
Smads signaling pathway
Jo
BMP2
a
LRP5 = low density lipoprotein receptor related protein 5; LRP6 = low density
lipoprotein receptor-related protein 6; β-catenin = beta-catenin; Cnx43 = connexin 43; MMP16 = matrix metalloproteinase 16; OPG = osteoprotegerin; RANKL = receptor activator for nuclear factor-κ B ligand; BMP2 = bone morphogenetic protein 2; Smad1 = mothers against decapentaplegic homolog 1; Smad5 = mothers against decapentaplegic homolog 5; Runx2 = runt-related transcription factor 2. CON = control group, n = 6.
c
IR = intake restriction group (60% of CON intake), n = 6.
Jo
ur
na
lP
re
-p
ro of
b