Aug 4, 2009 - differentiation in the pig (Knight, et al. 1977). ...... Sterle JA, Boyd CK, Peacock JT, Koenigsfeld AT, Lamberson WB, Gerrard DE & Lucy MC 1998.
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Accepted Preprint first posted on 4 August 2009 as Manuscript JOE-09-0131
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Responses to maternal growth hormone or ractopamine during early-mid pregnancy are similar in
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primiparous and multiparous pregnant pigs.
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Authors: Kathryn L Gatford*†‡, Miles J de Blasio*†‡, Claire T Roberts*†‡, Mark B Nottle†‡, Karen L
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Kind*†#, William H E J van Wettere#, Robert J Smits¶, Julie A Owens*†‡
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* Research Centre for Early Origins of Health and Disease, Robinson Institute, University of
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Adelaide SA 5005, Australia
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†
Research Centre for Reproductive Health, Robinson Institute, University of Adelaide SA 5005,
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Australia
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‡
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University of Adelaide SA 5005, Australia
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#
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of Adelaide, Roseworthy SA 5371, Australia
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¶
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Australia
Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health,
Discipline of Agricultural and Animal Science, School of Agriculture, Food and Wine, University
Research and Development Unit, QAF Meat Industries Ltd., Redlands Rd, Corowa NSW 2646,
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Key words: growth hormone, ractopamine, fetal growth, muscle, placenta, pig
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Running head: Fetal responses to maternal GH, ractopamine and parity
1 Copyright © 2009 by the Society for Endocrinology.
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Abstract
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Fetal growth is restricted in primiparous pigs (gilts) compared to dams who have had previous
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pregnancies (sows), as in other species. In gilts, daily maternal pGH injections from d 25 to 50 of
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pregnancy (term ~115 d) increase fetal growth and progeny muscularity, and responses in sows are
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unknown. Whether feeding the β2-adrenergic agonist ractopamine during this period increases
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progeny growth rates in either parity, and fetal responses in gilts, have not been investigated. We
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hypothesised that fetal and placental growth and fetal muscle development would be increased more
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by maternal pGH and/or ractopamine during early-mid pregnancy in gilts than sows, since fetal
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growth is restricted in gilts causing lower birth weights. Large White x Landrace gilts and sows
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were injected daily with water (controls) or pGH (~15 µg.kg-1.d-1), or were fed 20 ppm
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ractopamine, between d 25 and 50 of pregnancy. Maternal pGH increased litter average fetal weight
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(11%, P=0.007) and length (3%, P=0.022), but not placental weight, at d 50 of pregnancy,
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irrespective of parity, and had greatest effects in the heaviest fetuses of each litter. Maternal
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ractopamine increased average fetal weight (9%, P=0.018), but not length. Muscle fibre diameter
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was increased by pGH in heavy littermates, and by ractopamine in median littermates. Similar fetal
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growth responses to pGH and ractopamine in gilts and sows suggest that these hormones increase
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fetal nutrient availability similarly in both parities. We therefore predict that sustained pGH
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treatment will increase progeny birth weight, postnatal growth and survival, in both sows and gilts.
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Introduction
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Primiparity and adolescent pregnancy each restrict fetal growth in humans and other species,
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including pigs (Ritter, et al. 1984) (Bryan & Hindmarsh 2006; Rasmussen & Fischbeck 1987).
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These may both contribute to the reduced birth weight and poorer subsequent performance of
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progeny of primiparous pigs (first pregnancy, gilts) compared to multiparous pigs (sows), since
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fetal growth and birth weight predict neonatal survival and postnatal growth and metabolism in pigs
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as in other species (Campbell & Dunkin 1982; Dwyer, et al. 1993; Fahmy & Bernard 1971;
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Wigmore & Stickland 1983; Winters, et al. 1947). Multiple factors can restrict fetal growth and
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birth weight in pigs; competing maternal demands in growing adolescent animals, large and
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variable litter size, and restricted maternal nutrition used in commercial pig production systems
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during pregnancy. Interventions to increase fetal growth may therefore be more effective in the gilt
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than the multiparous sow, where fetal growth and development are less constrained.
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Manipulation of nutrition in the pig shows that early-mid pregnancy is a critical period for fetal
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growth and development, when increasing fetal growth leads to improved postnatal growth and
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muscle gain (Dwyer, et al. 1994). This corresponds to the period of most rapid placental growth and
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differentiation in the pig (Knight, et al. 1977). We and others have shown that daily maternal
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injections of gilts with porcine growth hormone (pGH) during early-mid pregnancy increases fetal
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growth (Gatford, et al. 2000; Kelley, et al. 1995), numbers of muscle fibres in fetuses or progeny at
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birth (Rehfeldt, et al. 1993; Rehfeldt, et al. 2001b) and increases growth rates and muscle size of
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their postnatal progeny (Gatford, et al. 2003; Kelley et al. 1995). Some studies have reported the
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greatest responses to pGH are greatest in the smallest piglets within each litter (Rehfeldt & Kuhn
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2006; Rehfeldt, et al. 2001a; Sterle, et al. 1995), suggesting that the effects of pGH are greatest in
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the most restricted fetuses. Limited evidence from pigs and sheep also suggests that GH may act at
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least in part by increasing placental growth and/or function (Harding, et al. 1997; Rehfeldt et al. 3
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2001a; Sterle et al. 1995; Wallace, et al. 2004). Whether pGH promotes fetal growth in sows with
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lower competing nutrient demands for maternal growth is not known.
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Feeding pregnant pigs with β2-adrenergic agonists has been suggested as an alternate endocrine
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strategy to increase fetal and progeny growth and development. Progeny growth rates and carcass
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weight, but not birth weight, muscle size nor muscle fibre numbers, were increased in sows fed with
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the β2-adrenergic agonist ractopamine from d 25 to 50 of pregnancy (Hoshi, et al. 2005a; Hoshi, et
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al. 2005b). Similarly, feeding sows the β2-adrenergic agonist salbutamol in the first third of
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pregnancy increased muscle size and altered muscle fibre types of progeny (Kim, et al. 1994).
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Whether these changes in progeny muscle development are preceded by increased fetal and
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placental growth is not known, and parity differences in responses to β-adrenergic agonists have
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also not been explored.
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We therefore tested the hypotheses that daily pGH injections or feeding ractopamine during early-
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mid pregnancy (from d 25 to 50) would promote fetal and placental growth and fetal muscle
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development to a greater extent in gilts than in sows, and that these effects would be greatest in the
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smallest fetuses in each litter.
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Materials and Methods
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Animals
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The study was designed in accordance with the Australian Code of Practice for the Care and Use of
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Animals for Scientific Purposes (National Health and Medical Research Council of Australia 1997)
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and approved by the University of Adelaide Animal Ethics Committee. The in vivo studies were
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conducted at the University of Adelaide Roseworthy Piggery. Twenty-four Large White x Landrace
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gilts were mated at 23 weeks of age, and twenty-four mature Large White x Landrace, 3rd to 5th 4
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parity sows were mated at the first post-weaning estrus. Pigs were mated twice by artificial
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insemination using semen from Landrace or Large White boars, either in the morning and afternoon
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of the same day, or on the afternoon and morning of consecutive days. The day of second
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insemination was taken as d 0 of pregnancy. All animals were individually housed in stalls
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throughout the study.
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Nutrition and treatments
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Gilts and sows were fed ~1 kg of a dry sow diet (13.0 MJ DE/kg, 15.2% total protein, 0.65% total
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lysine) on the day of mating (d 0) and d 1. From d 2 until the end of the study, gilts were fed 2.2
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kg/d and sows were fed 2.5 kg/d of the same diet. Pregnancy was confirmed by ultrasound scanning
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at d 23 of pregnancy, and dams were randomly allocated within parity groups to control, pGH-
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injected or ractopamine-fed treatment groups (n=8 per treatment and parity). Control dams were
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injected i.m. daily with 1 mL sterile water from day 25 to day 50 of pregnancy. pGH-injected gilts
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and sows were injected i.m. daily with 1 mL sterile water containing 2.0 or 3.5 mg pGH
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respectively (recombinant porcine GH, Reporcin, OzBiopharm Pty Ltd, Knoxfield, Vic, Australia),
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which was calculated to provide a dose of ~15 µg pGH.kg-1.d-1 in both parity groups, based on
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previous live weight data from the herd. Ractopamine-fed dams were fed ractopamine at 20 ppm in
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the diet (added to the daily ration as Paylean, 2.2 g/day in gilts and 2.5 g/day in sows, Elanco
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Animal Health, USA). One gilt (pGH-injected) returned to oestrus in the first week of the treatment
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period and was removed from the study. Pregnant dams were weighed, and backfat depth measured
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by ultrasound at the P2 site (110 mm from the midline over the 13th rib, using B-mode live
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ultrasound), at the start of treatments and on the day of post-mortem (Gatford et al. 2000).
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Postmortem
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Maternal blood was collected by jugular venepuncture into EDTA tubes on the morning of post-
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mortem (day 50 of gestation), and placed on ice. Dams were then humanely killed, and a 5
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hysterectomy performed. Number of fetuses and visible resorption sites were counted, and
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individual fetuses and their intact placentae were dissected from the uterus. Placentae were cut
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lengthways, laid out flat, and digitally photographed on a board marked with a 1 cm grid for later
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measurement of placental area, made by tracing the perimeter of the placenta in VideoPro software
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(Leading Edge software, Adelaide). Fetal blood was collected into EDTA tubes from the umbilical
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cord immediately after removal of each fetus, and placed on ice. Blood was centrifuged and plasma
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collected and stored frozen at -20C for later analyses. Weights, crown-rump length, abdominal
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circumference, head width and sex of each fetus were measured and recorded. The fetal liver was
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dissected and weighed, and the left fetal hindlimb was removed and a mid-femur cross-section was
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taken through the upper hindlimb. Fetal muscle sections were fixed in 4% paraformaldehyde in PBS
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for 24 h, then washed in PBS, and embedded in paraffin wax. The maternal ovaries were removed
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and number of corpora lutea counted as a measure of ovulation rate. The proportion of fetuses plus
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visible implantations was calculated as the number of fetuses plus resorption sites divided by the
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number of corpora lutea. Fetal survival was calculated as the number of fetuses divided by the
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number of corpora lutea. Runt piglets were defined as fetuses with weight more than 2SD below the
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mean weight calculated using the mean and SD of weight for their litter.
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Hormone and metabolite analyses
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Growth hormone concentrations in maternal plasma were measured by radioimmunoassay. Growth
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hormone purified from porcine pituitaries (Lot# AFP10888C) was obtained from the National
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Hormone and Peptide Program (NHPP), NIDDK and Dr Parlow, and used as standard and for
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iodination as tracer. Rabbit anti-porcine GH (Lot# AFP422801), also obtained from the NHPP, was
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used as the primary antibody. All maternal samples were analysed for pGH in a single RIA with an
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intra-assay CV of 9.9%. IGFs were extracted from plasma by size exclusion high performance
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liquid chromatography at pH 2·5, using a modification of the original procedure (Scott & Baxter
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1986), as described previously (Owens, et al. 1990). IGF-I concentrations were determined by RIA 6
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of neutralised chromatography fractions devoid of IGF binding proteins, using the Conlon rabbit
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polyclonal antibody to human IGF-I (Francis, et al. 1989). All maternal plasma samples were
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extracted in a single HPLC run with a recovery of 125I-IGF-I of 87.5%, and assayed in a single RIA,
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with an intra-assay CV of 13.7% for a HPLC eluate fraction 3 pool containing 18 ng/ml IGF-I.
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Covariance for extraction and assay of a maternal porcine plasma QC containing 174 ng/ml IGF-I
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and included at the start, middle, and end of the HPLC run of maternal samples was 7.2% (n = 1
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HPLC runs, 3 measurements). Insulin in maternal plasma was measured in a single assay using a
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commercially-available RIA kit (Human insulin-specific RIA kit, HI-14K, Linco Research Inc, St
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Charles, USA) with an intra-assay CV of 6.4% and 100% cross-reactivity with porcine insulin.
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Glucose and urea in maternal and fetal plasma were measured by colorimetric enzymatic analysis
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on a Hitachi 912 automated metabolic analyser using Roche/Hitachi Glucose/HK and UREA/BUN
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kits respectively (Roche Diagnostics GmbH, Mannheim, Germany).
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Fetal muscle histology
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Fetal muscle fibre density and size were measured on the lightest (non-runt), median weight, and
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heaviest fetus of each litter. Cross-sections (7 µm) of the fetal muscle were cut from embedded
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blocks and mounted on electrostatically charged slides (Superfrost Plus, Menzel GmbH & Co KG,
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Germany). Sections were stained with haemotoxylin-eosin, and images digitally-captured using a
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40x lens and the NanoZoomer slide capture system (Hitachi, Japan). M. semitendinosus muscle
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cross-sectional area, muscle fibre density and muscle fibre diameters were measured on all sections
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where the M. semitendinosus was complete and fibres approximately perpendicular to the section.
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Cross-sectional area was measured by tracing the M. semitendinosus perimeter in the NanoZoomer
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viewing software (NDP View, Hitachi, Japan). For each section, twelve fields were captured at 20x
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resolution for later counting of muscle fibres using random-systematic sampling, starting in the
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upper left-hand quadrant and capturing fields with constant horizontal and vertical spacing. All
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fibres (primary and secondary combined) were counted in an area of 0.1055 mm2 per field using 7
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VideoPro image analysis software (Leading Edge, Adelaide). Fibre diameter of primary fibres was
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measured at x40 resolution using NDP View software for twenty fibres per field and 12 fields per
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M. semitendinosus (total 240 fibres per fetus), with diameter measured on primary fibres falling
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closest to points on a sampling grid.
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Statistical analyses
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One gilt returned to oestrus within 1 week of commencing treatment and was excluded from the
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study. The effects of maternal parity, treatment and their interaction on maternal and litter outcomes
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were analysed using a two-way analysis of variance model, with litter size (total number of fetuses)
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included as a covariate. Specific contrasts were performed to test the a priori hypotheses that
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maternal pGH treatment or ractopamine treatment would increase fetal growth and survival.
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Hormone and metabolite data were excluded for two pGH-treated sows that were not injected on
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the morning of post-mortem. Mixed model procedures were used to further evaluate weights of
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individual fetuses and placentae and fetal:placental weight ratios, to allow analysis of effects of
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fetal sex and also to compare responses to maternal factors in littermates of varying weight within
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each litter. All fetuses were ranked and assigned to a quartile of weight within their litter. Sire breed
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did not affect outcomes and was excluded as a factor in the models. These mixed models included
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maternal parity, maternal treatment, fetal sex, fetal weight quartile, and their interactions, and litter
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size was included as a covariate. Measures on each fetus within a litter were treated as repeated
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measures on the dam, and multiple comparisons between treatment groups for repeated measures
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models used the Bonferroni correction method. Where effects of parity or treatment varied with
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fetal weight quartile, effects of sex, maternal treatment and maternal parity were evaluated
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separately for fetuses of each weight quartile. Fetal M. semitendinosus characteristics were similarly
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evaluated, using a repeated measures model including maternal parity, maternal treatment, fetal size
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group (lightest, median or heaviest of litter), and their interactions. Fetal sex and litter size did not
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affect M. semitendinosus characteristics and were excluded from these models. Data were log8
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transformed before analysis where necessary to achieve equal variances and normality. All tests
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were carried out using SPSS v17.0 for Windows (SPSS Inc. USA) and are presented as mean ±
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SEM for reproductive data and estimated mean ± SEM where litter size was included in the model,
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unless otherwise indicated.
200 Results
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Maternal weight and backfat
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Prior to the start of treatments, maternal weight and maternal P2 backfat depth were greater in sows
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than in gilts (P
