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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
* Tasmanian Institute of Agricultural Research (TIAR), Mt. Pleasant, Tasmania, 7250, Australia;
and
Department of Primary Industries and Water (DPIW), Mt. Pleasant, Tasmania, 7250, Australia; and
School of Animal Biology, University of Western Australia, Crawley, Western Australia, 6009, Australia
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: behavior • dexamethasone • maturity • neonate • sheep • survival
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In winter lambing systems, the ability of the newborn to implement nonshivering thermogenesis and rapidly bond with its mother is vital to survival immediately postpartum (Dwyer et al., 2001). Enhanced neonatal lamb survival may be related to improved metabolic and endocrine maturity at birth, as indicated by a greater reliance on glucose and NEFA than on AA for energy metabolism (Greenwood et al., 2002; Thompson et al., 2006), and improved behavioral competency and thermoregulation ability (Dwyer and Morgan, 2006).
Cortisol and the thyroid hormones play an important role in the maturation of the fetus (Liggins, 1994), with significant individual variation in the timing and magnitude of the prepartum cortisol surge (Magyar et al., 1980; Schwartz and Rose, 1998). Maternal glucocorticoid treatment is used to stimulate fetal cortisol secretion, with cortisol or glucocorticoid administration in late gestation increasing glycogen and glucose availability (Fowden et al., 1993; Liggins, 1994; Franko et al., 2007) and capacity for nonshivering thermogenesis (Bispham et al., 1999). Prenatal infusion of glucocorticoids also induces a transient increase in leptin (Forhead et al., 2002), a hormone with numerous actions on fetal growth and development (for review see Henson and Castracane, 2006). Time of administration and dose rate must be managed to avoid the risk of premature parturition (Thorburn, 1991), birth weight reductions (Fowden et al., 1996; Jobe et al., 1998), and adverse programming effects on growth (Sloboda et al., 2002). Alterations in fetal growth may also change ghrelin concentrations at birth, with circulating ghrelin related to gestational age in human fetuses (Farquhar et al., 2003; Chiesa et al., 2008).
The response of ghrelin and leptin to a single injection of dexamethasone has not been investigated in lambs, and little is known about whether prenatal glucocorticoid administration will have a positive effect on lamb survival after a normal delivery. The experiment described here investigates the effect of rate and timing of glucocorticoid administration to late-gestation Merino ewes on neonatal lamb metabolic and endocrine maturity, behavior, survival, and growth.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mating Management
Multiparous fine wool Merino ewes (n = 352) were weighed (49 ± 6.2 kg; mean ± SD) and BCS recorded (2.8 ± 0.57, 1 = emaciated, 5 = grossly fat at 0.5 intervals, Russel et al., 1969) 3 d before a 6-wk mating period with 7 poll Dorset rams during late February and March 2007. Ewes were grazed at Cressy Research Station (41°44'13''S, 147°04'43''E) on perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) dominant pastures during the mating period. Rams had marking harnesses attached, and mating date was estimated based on ewe rump crayon markings observed twice a week.
Prelambing Management and Treatments
Pregnancy scanning by an experienced commercial operator occurred 91 d after the commencement of mating, with ewes shorn around the back legs and udder on the following day. Due to limited pasture supply during pregnancy, the ewes were supplemented with oats (12.7 MJ of ME/kg of DM, 11.1% CP, 18.1% ADF) or barley grain (13.1 MJ of ME/kg of DM, 10.4% CP, 6.2% ADF) every 2 d at about 600 to 1,000 g per ewe, with an average quality grass hay (7.6 MJ of ME/kg of DM, 11% CP, 62.4% NDF) provided ad libitum.
Ewes (n = 150) were divided into 3 lambing groups according to the date the ewes were crayon marked (14, 17, or 21 d after ram introduction). Each group of 50 ewes had the same number of ewes bearing single or twin lambs. In each of the 3 lambing groups, 5 single-bearing and 5 twin-bearing ewes were randomly allocated to 1 of 5 treatment groups, ensuring groups were balanced for BW and BCS during mating and pregnancy. Treatments (n = 30 ewes in total with 15 single-bearing and 15 twin-bearing) included a saline control [1 mL, intramuscular in neck] or dexamethasone (DEX, 2 mg/mL as the sodium phosphate, Dexadreson, Intervet Australia Pty. Ltd., Bendigo East, Victoria, Australia) injected at 1 of 2 dose rates (1.5 or 3.0 mg intramuscular in neck) at d 130 or 141 of gestation [estimated as halfway during the relevant crayon marking period (i.e., d 12, 16, and 19 after ram introduction)]. One-half of the control ewes were saline injected at d 130 and the remainder at d 141.
Two weeks before the commencement of lambing, ewes (51 ± 5.8 kg of BW, 2.6 ± 0.41 BCS) were vaccinated (2 mL of Coopers Guardian 6 in 1 for sheep and lambs, Schering-Plough Pty. Ltd., Baulkham Hills, New South Wales, Australia), injected (3.5 mL of Cydectin Long Acting injection for Sheep, 20 g/L of moxidectin, Fort Dodge Australia Pty. Ltd., Baulkham Hills, New South Wales, Australia), and grazed as a single flock in a 4-ha paddock. The ewes were handled quietly and habituated to the presence of people.
Pasture height was determined using a 315-mm square rising plate meter (I.K.J. & M.A. Englund, Werribee, Victoria, Australia) and at least 250 placements across the paddock. Pasture samples (at least 12 samples per ha) were cut to ground level using hand shears, dried at 60°C for determination of DM content, and then ground (1-mm screen) for nutritional analysis using NIR spectroscopy (Forage Quality Package, FeedTest, Hamilton, Victoria, Australia). A subsample was hand-separated before drying for determination of botanical composition (DM basis). The pasture averaged 4 cm in height, was predominantly perennial ryegrass (50% of DM) with some (2% of DM) clover and was of reasonable quality (27% DM, 10.1 MJ of ME/kg of DM, 22.5% CP, 53.6% NDF).
Lambing Management
At the commencement of the lambing period, ewes were drafted into the 3 lambing groups, and each group was placed in 1 of 3 pasture subdivisions (each about 1.85 ha) within a 5.75-ha lambing paddock. Pregrazing pasture (10.8 MJ of ME/kg, 22.3% CP, 51.9% NDF) averaged 5.5 cm in height and consisted predominantly of perennial ryegrass (49% of DM) and orchardgrass (Dactylis glomerata, 28% of DM) with very little clover (1% of DM). Lambing was completed within 20 d and ewes grazed within the lambing paddock for a total of 1 mo.
Weather Conditions During Lambing
Weather records were collected every 30 min during the lambing period at an official meteorology recording site located on the research station. During the lambing period the mean air temperature was 8°C (range –1.7 to 14.8°C), mean wind speed was 19.3 km/h (range 0 to 54 km/h), and mean 24-h rainfall was 3.6 mm (range 0 to 18.2 mm), resulting in a mean chill index of 1,120 kJ/m2 per h (range 1,015 to 1,320 kJ/m2 per h; Kleemann and Walker, 2005).
Behavioral Observations
A movable 5-m high tower was used to facilitate 24-h behavioral observations during lambing with a 400-W Metal Halide floodlight mounted for use during low ambient light conditions. Ewes were allowed to lamb unaided wherever possible; however, assistance was rendered (n = 34) in cases of dystocia, generally 2 to 3 h after appearance of membranes, and the ewe identity recorded. Behavioral observations at lambing were recorded based on Dwyer and Morgan (2006), including date and time(s) of first signs of prelambing behavior (bleating, spatial separation, circling, agitation), of appearance of fluids or membranes, when the lamb was fully expelled, when the ewe first groomed the lamb (licking the lamb, cleaning away afterbirth), the first standing attempt of the lamb (elevation of BW on at least one foot), the first standing of the lamb (standing on all 4 legs for more than 5 s), the first seeking of the udder of the lamb (nuzzling or butting ewe flanks or hindquarters near the udder), the first suckling of the lamb (lamb in parallel inverse position, correct head presentation for teat access, may coincide with tail wagging), and the first bleating of the lamb (audible vocalization).
Lamb Measurements
The lamb(s) were identified with the mother and caught by gloved personnel before suckling, their sex recorded, and birth weight, crown-rump length, girth at the heart, and rectal temperature measurements taken. Then, on average 29 ± 15.5 min after birth, a 5-mL blood sample was collected by jugular venipuncture (19 gauge, 1'' needle) into blood tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) containing K3EDTA (0.117 mL of 15% EDTA) for subsequent glucose, NEFA, β-hydroxy-butyrate (BHB), urea and ghrelin analysis, or sodium heparin (143 USP units) for subsequent leptin analysis. Tubes were inverted 8 times and kept on ice before centrifugation (3,000 x g, 10 min, and 4°C) and plasma aspiration. For ghrelin analysis, 0.6 mL of plasma was stored in tubes containing 175 µL of a fresh phenylmethylsulfonyl fluoride (PMSF) solution (10 µL of 200 mM stock solution added to 990 µL of anhydrous methanol) and 30 µL of 1 N HCl. All plasma samples were stored at –20°C and then at –80°C.
During the lamb recording and blood sampling procedure, the maximum distance the ewe moved from the lamb was estimated visually. After blood sampling an eartag was inserted to individually identify the lamb(s), and then personnel retired from the tagging site and the time taken for the ewe to return to the lamb was recorded. As much as possible to minimize disruption to the ewe and lamb bonding process, lamb recording did not take place until after grooming and the lamb had commenced to seek the udder.
Autopsies
The time of lamb death was recorded, lambs collected, and autopsies (Holst, 2004) performed by qualified veterinary surgeons. Observations included whether the lamb had breathed, walked, fed, or been cleaned, the presence of external abnormalities, edema (head, neck, or legs) or hemorrhages of the brain, petechiation, liver damage, metabolism of fat stores, and the weight of the thyroid gland. These observations were used to divide cause of death into dystocia or mismothering- and starvation-related cases. Given prevailing cold environmental conditions and animal welfare considerations, any abandoned lambs that were considered nonviable were killed (n = 16) and autopsied.
Postlambing and Weaning
Ewes were moved from the lambing paddock 32 d after the onset of lambing onto perennial ryegrass-based pastures, with average quality grass hay (7.6 MJ of ME/kg of DM, 11% CP, and 62.4% NDF) provided ad libitum. Lambs were vaccinated (2 mL, Coopers Guardian 6 in 1 Vaccine for Sheep + Selenium for lambs, Schering-Plough Pty. Ltd.), castrated, and tail rings applied 43 d after the start of lambing. Lamb BW, crown to rump length, and girth at the heart were measured 75 d after the commencement of lambing, which was 3 d before weaning. At weaning, ewes and lambs were collected at 0830 h and jugular blood samples collected from the lambs in random order while with the ewes. Jugular blood (10 mL) was collected into evacuated blood tubes (Vacutainer), centrifuged at 1,120 x g for 10 min at 4°C, and the plasma aspirated. The storage tubes for ghrelin analysis contained (per mL of plasma) 10 µL of PMSF solution (10 mg/mL) and 50 µL of 1 N HCl. Then, lambs were separated from the ewes and grazed together for 69 d on perennial ryegrass dominant pastures, also including annual grasses and limited subterranean (Trifolium subterraneaum) and white (Trifolium repens) clover, and then for 4 d on a dual-purpose oat crop before weighing.
Plasma Analysis
Plasma BHB was determined with a kinetic enzyme method (Ranbut D-3-Hydroxybutyrate kit - RB 1007, Randox Laboratories Ltd., Crumlin, UK, ±0.055 at 1.23 mmol/L, 95% confidence interval). Glucose analyses were performed using an enzymatic UV test (hexokinase method, Olympus kit, OSR6121, Europa GmBH, Hamburg, Germany, ±0.14 at 15.6 mmol/L). Samples with hemolysis concentrations greater than 50 mg/dL were not included in the analysis (n = 19). Nonesterified fatty acids were analyzed using an enzymatic colorimetric method (ACS ACOD method, NEFA C-Test kit 279–75401, Wako Chemical. Co., Osaka, Japan, ±0.04 at 0.91 mmol/L) and urea with a kinetic UV test (Olympus test kit - OSR6134, ±0.35 at 7.75 mmol/L). All tests were performed on an Olympus AU 400 autoanalyzer (Olympus, Hamburg, Germany).
Plasma ghrelin was measured in duplicate by a modified double-antibody RIA method based on the Linco total Ghrelin RIA kit (GHRT-89HK, Linco, St. Charles, MO). Highly purified human ghrelin (cat No. 031–30, Phoenix Pharmaceuticals Inc., Burlingame, CA) was iodinated using the chloramine T method (Greenwood et al., 1963), then the labeled hormone was purified using a Sepak C-18 column (Alltech Australia, Baulkham Hills, New South Wales, Australia) activated with methanol (5 mL) followed by double deionized water (5 mL) and primed with 10% acetonitrile (5 mL) followed by 60% acetonitrile containing 0.2% triflouro acetic acid (5 mL). The iodinated hormone was eluted with the 60% acetonitrile containing 0.2% triflouro acetic acid. On d 1 of the assay, a stock solution of highly purified human ghrelin (cat No. 031–30 Phoenix Pharmaceuticals Inc.) was serially diluted in assay buffer to the following concentrations of standards: 5,000, 2,500, 1,250, 625, 312, 156, 78, 39, and 19.5 pg/mL. Duplicate 100-µL plasma samples or standards were diluted to 200 µL with assay buffer (0.01 M phosphate, 0.01 M EDTA, 0.08% sodium azide, and 0.1% gelatin in 1 L of double deionized water; pH 8.5). After addition of 100 µL of rabbit anti-ghrelin (Human Ghrelin T-4745, Peninsula Laboratories, San Carlos, CA, diluted as recommended 1 in 50 in assay buffer), the tubes were vortexed and incubated at 4°C overnight. Cross-reactions were 100% with human ghrelin, 100% with (des-octanoyl)-ghrelin, and 0% with human growth hormone releasing factor, orexin A and B, secretin, VIP, and galanin. On d 2, 100 µL of labeled hormone (~10,000 cpm in assay buffer containing 0.025% Triton-X100 and normal rabbit serum diluted 1 in 500) was added, tubes were vortexed, and incubation was continued for 24 h at 4°C. On d 3, 100 µL of donkey anti-rabbit serum (Ooopsie/91, RIA Laboratory, University of Western Australia, Crawley, Australia, 1:10 in assay buffer) was added and the tubes were vortexed. After incubation overnight at 4°C, 1.0 mL of 5% polyethylene glycol (PEG 8000, BDH, Poole, UK) in assay buffer was added to the tubes (except total count tubes) before centrifugation at 1,500 x g for 25 min at 4°C (Beckman, J-6M/E, Meriden, CT). The supernatant was decanted, and the pellets were left to dry overnight before the activity of the precipitate was determined on a gamma counter (Packard Cobra-II, Auto Gamma, Meriden, CT). The assay was validated for sheep plasma by checking for parallelism using a serial dilution of pooled samples of sheep plasma. All samples were processed in a single assay, and the limit of detection was 25 pg/mL. The assay included 6 replicates of 3 control samples containing 689, 1,324, and 2,299 pg/mL, which were used to estimate the intraassay CV of 4.1, 1.8, and 3.8%, respectively.
Plasma leptin was measured in duplicate by a double-antibody radioimmunoassay (Blache et al., 2000). All samples were processed in a single assay, and the limit of detection was 0.05 ng/mL. The assay included 6 replicates of 3 control samples containing 0.40, 0.91, and 1.79 ng/mL, which were used to estimate the intraassay CV of 7.9, 4.0, and 4.9%, respectively.
Statistical Analysis
One control ewe of the third lambing group had a prolapsed vagina 5 d before lambing, was treated by a veterinarian, and subsequently removed from the trial before DEX treatment. One ewe scanned as single-bearing failed to lamb (3-mg treatment, d 141, not included in the analysis), 2 ewes scanned as single-bearing had twins (1.5 mg, d 141; 3 mg, d 130, included in the analysis), and 2 ewes scanned as twin-bearing had single lambs (1.5 mg, d 130; 0 mg, d 141, included in the analysis). Where uncertainty existed about a behavioral or autopsy observation, these were not included in the final data set, nor were plasma samples where uncertainty existed as to whether the lamb had suckled or not before collection. Lamb survival data were categorized into lamb mortality at birth, and those dying by 12, 24, 48, and 72 h after birth.
All statistical analyses were carried out using the SAS/STAT software (Cary, NC). Normality of data was tested using PROC UNIVARIATE, and data transformed (natural log) where required before statistical analysis. PROC UNIVARIATE was also used to calculate median and quartile values for the behavioral observations. Continuous data on birth characteristics and behaviors, lamb blood measures, and lamb growth were analyzed using PROC GLM using a model that included lambing group, litter size (singleton or twin bearing), treatment, and level and time of DEX injection nested within treatment and their interaction terms. Ewe BW at mating was included as a covariate factor to account for maternal effects (Edwards and McMillen, 2002).
Where litter size was a significant factor, treatment effects were examined separately within singleton or twin lamb data sets. Because the prepartum surge in cortisol is related to fetal sex (Edwards and McMillen, 2002), treatment effects were also examined separately within female and male lamb data sets. Rectal temperature was divided into 2°C groups (n = 4) from 
35 to >39°C, and heart girth groups (n = 3) were divided at approximately ± 1 SD from the mean. Blood plasma data were grouped into low and high groups >1 SD from the mean, and a third group within ± 1 SD from the mean. Least squares means were estimated for litter size, treatment combination, and assistance during lambing. Differences among means were analyzed using a protected F-test by means of t-tests, where significance was declared at P < 0.05 and trends discussed at P < 0.10.
Categorical data from lambing, lamb survival, and autopsy findings were analyzed using PROC GENMOD, assuming a binomial distribution and logit link function, and the model described previously. Where the 
2 test was significant (P < 0.05), PROC FREQ was used to determine frequency distributions. Relationships among lamb survival and weather, birth, blood, and behavior characteristics were tested individually using PROC GENMOD and declared significant where P 2 0.05. A Fisher’s exact test was used for low-frequency data analysis. For survival and autopsy analysis, lamb birth weight group was included in the model where birth weight was divided into 5 x 1 kg groups, starting at 3 kg and ranging up to >6 to 7 kg. Due to missing behavioral data, valid analyses of lamb survival for all periods and some behavioral attributes were not possible.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The DEX treatment had no effect on lamb survival to 72 h after birth or on the number of lambs requiring assistance at birth (Table 1). There was no relationship between birth weight or litter size and lambs euthanized; however, a greater proportion (P 2 < 0.01) of the lambs dying during lambing were euthanized when born to ewes handled at d 141 (31%, n = 42 total) compared with ewes handled at d 130 (7%, n = 43 total). There tended (P = 0.09) to be a lesser proportion of lambs born to treated ewes dying due to dystocia (20%, 6 of 30 for 1.5 mg; 31%, 11 of 35 for 3 mg) than for lambs born to untreated ewes (43%, 6 of 14). The DEX treatment had no effect (P = 0.11) on the number of lambs dying due to mismothering or starvation-related causes.
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Two weeks before the commencement of lambing, ewes subsequently treated with DEX tended (P = 0.07) to have a lesser BCS than the control ewes, although BW was not different (Table 2). Maternal DEX treatment did not alter the estimated gestation length compared with untreated ewes and did not affect presuckling plasma glucose, NEFA, urea, or leptin concentrations. There was an interaction (P < 0.01) between dose rate and time of maternal DEX treatment such that the 3-mg treatment applied at d 130 tended to increase presuckling BHB concentrations relative to the control (P = 0.12) and the 1.5-mg treatment (P = 0.06), whereas the 3-mg treatment applied at d 141 decreased presuckling BHB concentrations relative to the control and the 1.5-mg treatment (P 0.03).
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Sex Effects. Whereas male lamb birth weights were reduced for the 3-mg treatment relative to the 1.5-mg treatment (4.5 ± 0.10 kg and 4.8 ± 0.11 kg, respectively; P < 0.05), the difference in female birth weights was not significant (4.3 ± 0.12 kg and 4.5 ± 0.11 kg, respectively; P = 0.18). Female lambs born to ewes treated with 3 mg of DEX had lower rectal temperatures (36.9 ± 0.37°C, P < 0.05) than the control and 1.5-mg treated female lambs (38.2 ± 0.60°C and 37.9 ± 0.31°C, respectively), but this effect was not significant in male lambs (P = 0.47). Sex of the lamb did not significantly alter the treatment effect on plasma BHB. The rise in ghrelin concentrations for treatment at d 141 was seen in male lambs (470 ± 34.9 vs. 287 ± 63.9 pg/mL, respectively; P < 0.05) but was not significant in female lambs (421 ± 39.0 vs. 350 ± 98.3 pg/mL, respectively; P = 0.81). Sex of the lamb per se did not affect (P > 0.10) period of gestation, BW, length, or girth at birth, or BW gain to marking and weaning.
Behavioral Observations at Lambing
Ewes treated with DEX at d 130 produced lambs that took longer to bleat than lambs born to control ewes (mean 20 ± 3.0 vs. 10 ± 4.7 min, respectively; P < 0.05). Other behavioral interactions between ewes and neonatal lambs were not affected by DEX treatment or litter size. From expulsion of the lamb, ewes took 1 min (0.5 to 2.0 min, median with inter-quartile range, n = 126, 2 ± 0.3 min, mean ± SE) to commence grooming, with 7 min (4 to 14 min, n = 112, 11 ± 1.0 min) elapsing from expulsion until lambs attempted to stand, 14 min (4 to 24 min, n = 102, 17 ± 1.8 min) to commence bleating, 14 min (9 to 23 min, n = 105, 18 ± 1.4 min) to stand, and 21 min (14 to 36 min, n = 77, 28 ± 2.3 min) to commence seeking the udder. Lambs first suckled 58 ± 3.7 min after expulsion (n = 63), but due to the potentially disruptive influence of lamb data recording and blood sampling, this figure should be viewed with caution. The maximum distance ewes moved away during lamb sampling was 15 m (median, range 0 to 150 m), and the time for ewes to return to the lamb after sampling (median 1 min, n = 145) was unaffected (P > 0.26) by litter size or treatment.
BW and Growth Rate
There were interactions (P < 0.05) between level and time of treatment on lamb growth performance to weaning (Table 5), where singleton lambs born to ewes treated with DEX at 1.5 mg on d 141 exhibited lighter BW at weaning (Figure 1), and growth rates from birth to weaning (272 ± 9.6 g/d; n = 11), than the comparable controls (328 ± 12.5 g/d; n = 7) or lambs born to ewes treated at 3 mg (307 ± 9.6 g/d; n = 12). Length at weaning was also less (P < 0.001) for singleton lambs within the 1.5-mg treatment group (83.5 ± 0.85cm) compared with the control group (90.2 ± 1.09 cm) or the 3-mg treatment group (87.9 ± 0.82 cm). In contrast to the effects on singleton lambs, twin lamb growth to weaning was unaffected by treatment (P > 0.10).
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There was an interaction (P < 0.01) between rate and time of DEX treatment on postweaning growth rate such that lambs born to ewes treated with DEX at 1.5 mg on d 141 exhibited greater (P < 0.05) average growth rates (178 ± 7.0 g/d) compared with the lambs born to ewes treated with 3 mg (154 ± 6.8 g/d), but did not differ from controls (160 ± 10.4 g/d). This offset treatment differences before weaning, resulting in no DEX treatment effects on BW 73 d after weaning.
Sex Effects on Growth to Weaning. Female lambs born to ewes treated with 3 mg of DEX on d 141 of gestation had greater (P < 0.01) growth rates to weaning (316 ± 10.2 g/d) than untreated female lambs (241 ± 15.7 g/d) or 1.5-mg treated female lambs (269 ± 7.4 g/d). This was reflected in BW at weaning (Figure 2). In contrast, female lambs born to ewes treated with 3 mg on d 130 presented with lighter BW at weaning (20.8 ± 0.59 kg) than the 1.5-mg treated female lambs (22.5 ± 0.52 kg; P < 0.05). There was no effect of treatment on male lamb BW at weaning (P > 0.34).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Survival and Thermoregulation
Maternal DEX treatment had no overall effect on lamb survival under field conditions to 72 h after birth. This is consistent with the results of Moss et al. (2001), who reported no change in postnatal lamb survival with a single dose of betamethasone (0.5 mg/kg) given at d 104 of gestation. In contrast, an earlier study (Restall et al., 1976) investigating the induction of parturition in sheep supplied DEX (8 and 16 mg) around d 141 of gestation and found that the group treated with 8 mg had a mortality rate of 10.5% (n = 2 of 19) compared with 25% for control lambs (n = 5 of 20). Birth weights were 4.2 to 4.6 kg and unaffected by treatment. Although only small numbers of lambs were involved in that study, which focused on induction of parturition, the reported survival benefit of the 8-mg DEX treatment was not observed for the dose rates examined here.
However, there tended to be a reduction in the incidence of dystocia in treated ewes. This may be related to the dose-dependent reductions in lamb heart girth, as well as birth weight in twin and male lambs. Glucocorticoids act in rate- and tissue-dependent ways to promote cellular differentiation and organ maturation in the fetus (Liggins, 1994; Fowden et al., 1998; Franko et al., 2007), slowing the rate of fetal growth when supplied in pharmacological doses (Fowden et al., 1996; Jobe et al., 1998) and often expressed as reduced birth weights (Clarke et al., 1998; Moss et al., 2001). Despite potential reductions in dystocia incidence, in cold environments increased surface area to BW ratios resulting from heart girth reductions would be likely to increase relative heat losses (Alexander and McCance, 1958), reducing the likelihood of survival. High mortality rates of twin lambs have been assigned to hypothermia, poor cold tolerance, and low birth weights (Alexander and McCance, 1958; Alexander et al., 1980; Donnelly, 1984; Obst et al., 1991). Small DEX dose rates were selected to minimize the risk of birth weight reductions while potentially enhancing metabolic maturity. Our results indicate that the 1.5-mg dose rate when maternally administered at 130 and 141 d of gestation was not sufficient to alter the gross morphology of newborn lambs.
In addition to birth weight reductions, we observed that twin and ewe lambs born to 3 mg treated ewes had about 1°C lesser presuckling rectal temperatures than control animals. There is a positive relationship between birth weight and rectal temperature within 1 h of birth, particularly in lambs of lighter birth weights (Alexander and McCance, 1958; Dwyer and Morgan, 2006). Consistent with the results presented here, maternal DEX treatment (16 mg, d 138) has resulted in a decreased rectal temperature (about 1°C) in premature lambs 0.5 to 1 h after caesarean delivery, and increased the time taken for lambs to restore rectal temperatures after birth in a study presented by Clarke et al. (1998). However, by 6 h after birth these lambs had greater temperatures than control lambs. This was thought to be associated with greater free fatty acid concentrations, which were indicative of increased rates of lipolysis, and potentially enhanced nonshivering thermogenesis (Clarke et al., 1998). Due to concerns about mismothering of ewes and lambs under field conditions in the present study, rectal temperatures were not recorded 6 h after birth. Because survival rates were not reduced, it is reasonable to assume that the treatment-induced reduction in body temperature was not maintained for an extended period. We also did not find any change in presuckling plasma NEFA concentrations with DEX treatment.
Compared with twin lambs, the lack of a reduction in presuckling rectal temperature in singleton lambs with DEX treatment may relate to greater physiological maturity of singleton lambs at the same gestational age as twins. Whereas there is significant individual variation in the timing and magnitude of the prepartum surge in cortisol (Magyar et al., 1980), it occurs earlier for singleton (d 128 ± 1.7 of gestation) than for twin-born lambs (d 135 ± 1.2; Edwards and McMillen, 2002). Basal adrenocortical function is relatively reduced or delayed in twin compared with singleton pregnancies, possibly as a mechanism to maximize neonate viability (Gardner et al., 2004). This earlier cortisol rise may have altered the responses to the 3-mg DEX treatment between singleton and twin lambs. In our study, singleton lambs had greater presuckling plasma glucose and lesser NEFA concentrations, and recorded greater rectal temperatures, than twin lambs. Consistent with these results, increased metabolic maturity at birth has been associated with a greater reliance on glucose than AA for energy metabolism (Greenwood et al., 2002; Thompson et al., 2006).
Survival and Behavior
The doubling of the time taken to vocalize by lambs born to ewes treated with DEX at d 130 of gestation did not alter survival rates for those lambs. There was no change in the time taken to stand or suckle in treated lambs, even though decreased rectal temperatures 1 h after birth have been associated with slow behavioral progression to standing and suckling (Dwyer and Morgan, 2006). Lambs that are faster to stand and suckle have been reported to have improved survival rates (Cloete and Scholtz, 1998; Dwyer et al., 2001). Under winter lambing systems the newborn is exposed to excessive cold stress, making an ability to rapidly bond with and suckle its mother vital for neonate survival immediately postpartum (Alexander, 1962; Dwyer and Morgan, 2006). Although resulting in a reduced rectal temperature, DEX treatment did not alter behaviors promoting neonatal survival.
Physiological and Metabolic Effects
We did not observe any change in estimated gestation length, although maternal glucocorticoid treatment can induce parturition by mimicking the prepartum surge in fetal cortisol that initiates the cascade of enzymatic and hormonal changes leading to the induction of labor (Silver, 1990; Thorburn, 1991). The dose rates of DEX treatment in this study were less than the amount (8 mg) previously reported to induce parturition in ewes when administered at d 141 of gestation (Restall et al., 1976).
The DEX treatments examined in the current study resulted in no change in the presuckling plasma concentrations of NEFA, glucose, or urea, implying that treatment did not alter the initial availability or usage of energy reserves. Whereas free fatty acids remain at reduced concentrations during imposed changes in fetal plasma cortisol concentrations (Mostyn et al., 2003), glucocorticoid administration in late gestation can cause increases in the accumulation of fetal liver glycogen, enhanced fetal hepatic and renal gluconeogenic enzyme activities, and can increase plasma glucose concentrations (Liggins, 1994; Fletcher et al., 2000; Franko et al., 2007). The lack of change in plasma glucose in this study may be a result of the smaller dose rates used and the timing of injections. Consistent with a lack of effect of treatment on energy metabolism, no change in presuckling plasma leptin concentrations were observed after a full-term delivery. This is also consistent with Bispham et al. (2002), who reported that DEX administration at 8 mg on d 138 of gestation had no effect on leptin mRNA abundance in lambs delivered at d 140 by caesarean section.
Single lambs had lesser circulating ghrelin concentrations compared with twins. In human neonates, ghrelin concentrations are elevated in cord blood from small-for-gestational-age subjects (Farquhar et al., 2003; Kitamura et al., 2003; Chiesa et al., 2008). In our study, increased litter size increased plasma ghrelin, which indicates a similar relationship between ghrelin and size at birth exists in sheep because twins had a smaller BW than single lambs. As ghrelin acts to stimulate feed intake (Sugino et al., 2004), this may represent a protective response to promote nutrient ingestion and survival in smaller neonates.
The timing of administration of glucocorticoid seems to be crucial in activating ghrelin secretion. Increases in plasma ghrelin concentrations were observed in singleton and male neonates with treatment on d 141 of gestation; however, treatment at d 130 did not induce as great a response. This observation is in agreement with reports that in normally developed human infants at birth, ghrelin concentrations are positively related to gestational age (Farquhar et al., 2003; Chiesa et al., 2008). Plasma ghrelin and leptin concentrations were also elevated by multiple DEX treatment in normoxic newborn rodents, but only ghrelin responses were seen in hypoxic rodents (Bruder et al., 2005). Overall, the ghrelin response to prenatal DEX suggested that 1) regulation of ghrelin secretion is very sensitive to glucocorticoids because it was the only indicator of endocrine and physiological maturity that was affected by treatment, and 2) that DEX treatment can be used to elevate circulating ghrelin in the neonate, depending on timing of administration. More research is indicated to investigate the role of glucocorticoid treatment in promoting neonate maturation at birth and, tentatively, improve its subsequent chance of survival.
Growth up to and after Weaning
Treatment with DEX did not affect measured blood metabolite and hormone concentrations of lambs at weaning; however, there were rate and time-of-treatment-dependent increases and decreases in BW gain to weaning and BW and size of lambs at weaning. Glucocorticoid treatment can reprogram the hypothalamic-pituitary-adrenal axis and cardiovascular, glucose, and stress responses in offspring and may affect growth in the postnatal period (Moss et al., 2001; Sloboda et al., 2002; Moritz et al., 2005). Restall et al. (1976) reported DEX (8 and 16 mg) supplied around d 141 of gestation did not affect lamb growth rates to 1 mo postparturition, and although Moss et al. (2001) observed reduced lamb BW at 3 mo of age after 4 maternal injections of betamethasone between d 104 and 125 of gestation, these BW differences did not persist at 6 mo of age. In contrast, Roussel and Hemsworth (2002) reported that mild prenatal stress increased BW at birth and tended to produce greater BW at 1 and 8 mo of age. The effects of glucocorticoid administration appear dependent on time of administration and dosage (Sloboda et al., 2002; Moritz et al., 2005) and potentially sex of the offspring (Moritz et al., 2005; Kapoor et al., 2006).
In conclusion, it was thought that administration of small doses of glucocorticoids to ewes in late gestation may promote metabolic and endocrine maturity in the neonate, resulting in improved thermoregulation and behavioral responses at birth, and so promote survival over the critical first few days of life. Because maternal DEX treatment did not increase presuckling rectal temperatures or speed to suckling, it can be concluded that the treatment did not improve the thermoregulatory capacity of the lamb. Indeed the 3-mg treatment of twin-bearing ewes may potentially have adverse effects on lamb survivability through reductions in birth weight, heart girth, and presuckling rectal temperature. Although the environmental challenge was large, there was no benefit to, or impairment of, lamb survival by maternal DEX treatment under the conditions of this study. The apparent sensitivity of ghrelin to maternal glucocorticoid treatment at d 141 of gestation and its potential to influence neonate maturity at birth and improve lamb survival warrants further investigation.
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3 Current address: Animal Science, DairyNZ, Hamilton, 3240, New Zealand.
2 Corresponding author: Dale.Miller{at}dpiw.tas.gov.au
Received for publication January 23, 2009. Accepted for publication June 10, 2009.
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