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Reclamation of disturbances from oil sands mining requires effective soil management to ensure successful plant establishment and to promote recovery of native plant communities. In this study we investigated the effects of salvage depths (shallow vs. deep) and placement depths (shallow vs. deep) of forest topsoil on plant establishment, species richness, and soil properties in two substrate types (sand and peat-mineral). Shallow salvage led to greater tree stem densities and higher canopy cover for most plant groups, although there was no significant difference in species richness between shallow and deep salvages. Deep placement generally resulted in greater canopy cover, while its effect on plant density was very small for most plant groups. On peat-mineral substrate, fewer differences were detected between shallow and deep salvage, and multiple treatments resulted in greater cover. Findings suggest that a balance between maximizing the area over which propagules are redistributed and providing sufficient resources for successful plant establishment is necessary. Forest topsoil from shallow salvages and deep placements is recommended when targeting increased site productivity and species diversity. In contrast, deep salvage should be used when the primary objective is to obtain maximum reclamation material volume. Salvage depth effects may be influenced by substrate type, with peat-mineral substrate providing more favourable conditions for plant establishment. Further research is needed to assess the long-term impacts of different salvage and placement depths on plant community development and the potential effects of substrate properties on soil and plant response., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mackenzie, Naeth. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

During mining topsoil is salvaged and stockpiled until ready for reclamation, stockpiling can have detrimental effects on seed viability and soil quality. Research has assessed effects of salvage and placement depth of forest topsoil on plant community establishment, with little work on effects of storage, particularly in the boreal forest. Our research assessed boreal forest topsoil storage methods to determine effects on soil chemical and physical properties, native seed viability and germination and rhizome viability and emergence. Factors were topsoil stockpiling length, stockpile size, season of construction and soil texture. Four replicates of large and small stockpiles were constructed in the mineable oil sands, in northeastern Alberta. During construction seeds and rhizomes from a variety of native boreal plant species were buried within large (0.05, 1.0, 2.0, 4.0, 6.0 m) and small (0.05, 1.0, 3.0 m) stockpiles. Soil gas probes were installed at similar depths as seed and rhizomes were placed. Seeds and rhizomes were extracted eight months and sixteen months after construction; during that time soil samples were collected for various chemical analyses. Irrespective of stockpile size, the majority of species seeds and rhizomes buried below 1 m lost viability and did not germinate after eight months. Anaerobic soil conditions developed soon after construction and persisted at depths below 1.0 m in large stockpiles, and over time anaerobic conditions developed in smaller stockpiles. Only seeds of Geranium bicknellii and Dracocephalum parviflorum had a high survival rate in stockpiles; both species have hard seed coats and are physically dormant. Various soil nutrients increased in concentrations in their soluble forms after stockpiling. Direct placement of topsoil is a preferred soil handling technique; however, if topsoil has to be stockpiled increasing the surface area of stockpiles will help preserve some seed and rhizome viability., Competing Interests: The study received funding from the following commercial sources: Syncrude Canada Ltd., Suncor Energy Ltd., Shell Canada Limited Canadian Natural Resources Ltd., Total E&P Canada Ltd., Fort Hills Energy LP, General Petro-Canada Oil Sands Inc. The funding was in the form of a grant to Dr M Anne Naeth through the University of Alberta. The companies contributed towards an unrestricted and unencumbered grant provided for Dr Naeth to conduct the research for Dr Mackenzie’s PhD program. There were no expectations or requirements from the industrial funders other than to receive copies of the final thesis with results of the research. Following the completion of this research project, Dr Mackenzie joined Vertex Resource Group Ltd. as an employee. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development, or marketed products to declare.

In temperate climates, the cost of providing feed is greater in winter than in other seasons, causing ewes to be fed restricted rations during some periods of pregnancy. Epidemiological information indicates that undernutrition of the fetus may affect its health and performance in later life (i.e., fetal programming), and these effects may be passed between generations. The primary focus of the results presented in this paper is to examine the effects of feeding levels during pregnancy on a variety of traits from offspring at the fetal stage to 3.5 yr of age and also traits in the grand-offspring. Two studies are reported in which ewes were fed restricted diets during pregnancy, with a variety of fetal traits, offspring traits up to 3.5 yr of age, or grand-offspring traits up to 8 mo of age being measured. Study 2 also considered differences in dam size (heavy vs. light). In study 1, several fetal mammary gland measures indicated that milking ability may be enhanced in offspring from dams fed ad libitum during pregnancy. However, study 2 showed that mammary mass was greater in fetuses from dams fed at maintenance during pregnancy and that contemporaries of these fetuses produced greater protein and lactose yields in their first lactation. In the second lactation, the advantages in protein and lactose yields did not reoccur and ewes from ad libitum-fed dams produced greater fat yield. In study 2, grand-offspring whose granddams were fed at maintenance levels during pregnancy were lighter at birth in both the first and second parturitions than those whose granddams were fed ad libitum during pregnancy. First-parity grand-offspring whose granddams were fed maintenance levels during pregnancy achieved heavier BW by 40 to 50 d of age in the first lactation, which reflected the greater protein and lactose yields; however, no BW differences were present in second-parity lambs at the same age. A smaller proportion of first-parity ewe grand-offspring from heavy granddams that were fed ad libitum during pregnancy reached puberty at approximately 8 mo of age relative to the other granddam size and feeding groups. These results indicate that dam nutrition can affect the yield and composition of milk in their offspring and the BW and reproductive capability of their grand-offspring. Molecular and physiological mechanisms for these changes are being sought.

It was hypothesized that exposure of the fetus to adverse conditions in utero due to either maternal constraint or nutrition may result in developmental adaptations altering metabolism and postnatal growth of the offspring. Heavy (H) and light (L) Romney dams (G0) were allocated to ad libitum (A) or maintenance (M) nutritional regimens, from day 21-day 140 of pregnancy. Female twin-born offspring (G1) born to the dams in the four treatment groups will be referred to as HA-ewes, LA-ewes, HM-ewes and LM-ewes. At 16 months of age, offspring were catheterized and given intravenous insulin tolerance test (ITT), glucose tolerance test (GTT) and epinephrine tolerance test challenges to assess their glucose and fat metabolism in relation to their birth weight and postnatal growth. In HA-ewes, the regression coefficients of growth rates prior to puberty on insulin and glucose curves in response to GTT (InsAUCGTT) and ITT (GluAUCITT), respectively, were different from 0 (P < 0.05) and were different from the regression coefficients of HM-ewes. This may indicate that HA-ewes may have showed puberty-related insulin resistance at 16 months of age with increasing growth rates prior to puberty compared to HM- or LM-ewes. In HM-ewes, the regression coefficients of growth rates after puberty on InsAUCGTT and GluAUCITT were different from 0 (P < 0.05) and were different from those of HA-ewes. These results may indicate that offspring born to heavy dams fed maintenance during pregnancy and with greater postnatal growth rates after puberty could develop glucose intolerance and insulin resistance in later life.

Many environmental factors applied postnatally are known to affect milk production of the dam, but to date, the effects of different fetal environments on subsequent first lactational performance of the offspring have not been reported. Four hundred fifty heavy (H; 60.8 kg +/- 0.18) and 450 light (L; 42.5 kg +/- 0.17) dams were randomly allocated to ad libitum (A) or maintenance (M) nutritional regimens from d 21 until d 140 of pregnancy, under pastoral grazing conditions (HA, n = 151; HM, n = 153; LA, n = 155; LM, n = 153). At d 100 of pregnancy, a sub-group of twin-bearing dams was killed and fetal mammary glands collected. From 1 wk before lambing, all remaining dams were fed ad libitum until weaning. After weaning, female progeny were managed and fed under pastoral conditions as 1 group. At 2 yr of age, 72 twin-rearing ewe offspring were milked once a week for 7 wk. Fetuses from M-dams had heavier mammary glands (P = 0.03) compared with A-fetuses. Fetuses from H-dams had greater (P = 0.0008) mammary duct area compared with L-fetuses. At 2 yr of age, M-offspring had greater milk yields at d 7 (P = 0.02) and d 28 (P = 0.09) of lactation and tended to have greater accumulated milk yields (P = 0.11) compared with A-offspring. Ewes born to M-dams showed greater lactose percentage at d 14 (P = 0.002), d 21 (P = 0.06), and d 28 (P = 0.07) of lactation and greater (P = 0.049) accumulated lactose yields and CP (P = 0.06) yields compared with A-offspring. Ewes born to H-dams displayed greater milk yields at d 14 (P = 0.08) and d 21 (P = 0.02) and had greater accumulated milk yield (P = 0.08) and lactose yield (P = 0.04) compared with L-offspring. Lambs born to M-offspring were heavier at birth (P = 0.02) and grew faster until weaning (P = 0.02), matching the milk yield and composition data, compared with their ad libitum counterparts. Birth weight was not affected (P > 0.10) by grand dam size; however, lambs born to H-offspring grew faster from birth until d 49 of age (P = 0.03). In conclusion, dam nutrition during pregnancy affected the resulting milk production of the offspring and composition and growth of their lambs. In addition, dam size affected the milk production of the offspring, lactose yield, and growth of their lambs. These findings are important for furthering our understanding of how the environment to which the female fetus is exposed can affect her subsequent development and her ability to nourish the next generation.

Insulin plays an important role in regulating the partitioning of nutrients to the mammary gland, particularly in lactating ruminants fed concentrate-based diets. There is evidence that the nutritional status of the animals might also affect their response to insulin. This is largely untested in early lactating ruminants fed fresh forage. To investigate nutritional effects on insulin response, 12 lactating sheep, housed indoors, were allocated to one of two treatment groups (hyperinsulinaemic euglycaemic clamp (HEC) or control) in a randomised block design and fed perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture. Mammary amino acid (AA) net uptake from plasma and utilisation for milk protein synthesis was measured during the 4th day of the HEC using arterio-venous concentration differences, and 1-13C-leucine was used to estimate whole body and mammary gland leucine kinetics. There was no change in feed intake, milk protein output and mammary blood flow during the HEC (P > 0.1). The HEC decreased (P < 0.1) the arterial concentrations of all essential AA (EAA) except histidine. The mammary net uptake of some EAA (isoleucine, leucine, methionine and phenylalanine) was reduced by the HEC (P < 0.1). Leucine oxidation in the mammary gland was not altered during the HEC (P > 0.1) but mammary protein synthesis was reduced by the HEC (P < 0.05). These results show that sheep mammary gland can adapt to changing AA precursor supply to maintain milk protein production during early lactation, when fed fresh forage. How this occurs remains unclear, and this area deserves further study.

Bone formation and loss are related to the strain imposed on bone by muscle forces. Bone mineral content (BMC) and lean mass (LM) of fetal lambs was determined at day 140 of pregnancy in 8 groups of ewes, which were of either large or small body size, on either high (ad libitum) or maintenance pasture intake from day 21 of pregnancy, or carrying either singletons or twins. BMC and LM (using DXA scanning) of fetal hindquarters/spine were corrected to leg length. BMC and LM were less in twin than singleton groups (P < 0.001). Large ewes on high intake produced single fetuses with a (group mean) BMC/LM ratio that was higher (P < 0.002) than that in fetuses of large ewes with singletons on maintenance intake or twins on either high or maintenance intakes, the ratios of which were not different. In single fetuses from small ewes on high intake, the BMC/LM ratio was higher than those from small ewes with singletons on maintenance intake or twins on either high or maintenance intakes, the ratios of which were not different. The ratio was not different in singleton fetuses of ewes on high intake, whether they were large or small. Different fetal environments resulted in a given amount of muscle being associated with a higher or lower bone mass. Dietary intake during pregnancy was more important than maternal size in affecting the ratio. We conclude that intrauterine environmental factors may be important in determining bone mass postnatally, and possibly later in life.

Mice in mid lactation (n=6 per group) were fed a control diet (1.19% fat), or diets containing safflower oil (25% w/w) or olive oil (25% w/w) for 7d. Mammary and liver stearoyl CoA desaturase (SCD) mRNA levels and mammary SCD activities were higher in lactating mice fed the control diet than in those fed the oil-supplemented diets. Further, mammary SCD mRNA was directly related to mammary SCD activity. Milk fat composition was influenced by dietary fat composition. The olive oil diet, high in 18:1, led to high levels of this fatty acid in milk and the safflower oil diet, high in 18:2, resulted in a milk fat with high levels of 18:2. These results show that there is regulation of SCD at the transcriptional level, associated with changes in enzyme activity and in milk fat composition.

Keratinocyte growth factor (KGF) is a stroma-derived mitogen mediating epithelial-stromal interactions. We investigated the role of KGF during epithelial-stromal interactions accompanying ruminant mammogenesis. Target-specificity of KGF was demonstrated in that KGF-stimulated proliferation of bovine mammary epithelial, but not ovine mammary stromal cells. Consistent with a paracrine function, 4.6, 2.4, 1.5 and 0.9 kb mRNA transcripts were expressed by bovine stromal, but not epithelial cells. Within the ovine mammary gland, 2.4 and 1.5 kb KGF mRNAs were expressed in the fat pad while only the 2.4 kb transcript was transcribed in parenchyma. The abundance of KGF mRNA was greater in the extra-parenchymal fat pad than in the contralateral epithelium-free fat pad prior to puberty, and was less in parenchyma than in the intact or epithelium-free fat pads. Ovariectomy tended to increase KGF transcription while estrogen reduced expression. Of several tissues, mammary parenchyma expressed a 2.4 kb mRNA while adipose tissues expressed a 1.5 kb transcript. These results demonstrate local and systemic regulation of KGF transcription and support a paracrine role for KGF during ruminant mammogenesis.

The objective of this study was to develop a surgical procedure for the preparation of an epithelium-free mammary fat pad (cleared mammary fat pad; CFP) in ewes. At 7 to 10 d of age, ewe lambs (n = 43, mean BW 9.2 +/- .2 kg at 14 d) were sedated and one mammary gland was locally anesthetized. An incision circumscribing the base of the teat was made and blunt dissection was performed through the extraneous mammary fat pad tissue to enable the parenchyma and teat to be wholly removed. Failure to completely remove the epithelium enabled it to regenerate and grow into the mammary fat pad. Mean diameter of the parenchymal rudiment at 7 to 10 d of age was 8.9 +/- .5 mm (range of 5 to 16 mm). The excision site was closed with wound clips and recovered lambs returned to their dams. The contralateral mammary gland remained intact, allowing it to undergo normal development. Live weight gain was unaffected by this procedure. Ewes were subsequently slaughtered in groups at various stages of prepuberty, puberty, gestation, and lactation. Of 39 operated glands recovered, only one demonstrated epithelial outgrowth within the CFP. Parenchyma within the contralateral, intact gland underwent phases of rapid growth in prepuberty, puberty, and late gestation and was capable of milk synthesis after steroid induction or parturition. Change in weight of the CFP paralleled that of the intact mammary gland to 100 d of pregnancy. Sham CFP surgery was performed on four additional ewes wherein the parenchyma was completely excised and immediately replaced. Sham-operated epithelium populated the mammary fat pad and synthesized milk that could be expressed from the teat. A CFP in sheep will be a useful model for future investigations into the local growth regulatory mechanisms associated with ruminant mammogenesis.

The ontogeny of hepatic growth hormone (GH) receptors (GHR), as measured by responses of both plasma insulin-like growth factor-I (IGF-I) and hepatic GHR to an exogenous bGH stimulus, was examined using sheep of different ages (Days 1-7, 14-21, 28-35, and 56-63 of life, and yearlings). The IGF-I response to bGH was first examined in yearling sheep using two doses of bGH (0.1 and 0.2 mg/kg LW/d). Based on these results, lambs in four groups up to Day 63 of life were treated for 5 d with bGH (n = 10) at a dose of 0.15 mg/kg LW/d or with saline (n = 10). Jugular blood samples were taken once daily on Days - 1, 4, and 5 of treatment. bGH treatment in lambs up to Day 63 of life had little effect on plasma concentrations of GH, insulin, glucose or urea, but significantly (P < 0.05) increased circulating concentrations of IGF-I at all ages and of NEFA at Day 62/63 of life. In contrast, bGH treatment at either dose in yearlings significantly increased these parameters, except for plasma urea concentrations which were decreased in bGH-treated yearlings. However, the responses of plasma IGF-I concentration to bGH stimulus in lambs up to Day 63 of life were small compared to those in yearling sheep. Consistent with this, bGH treatment failed to affect hepatic GH binding in young lambs, but up-regulated it in yearling sheep. Furthermore, basal (unstimulated) GH binding did not differ between sheep of 7 vs. 63 vs. 365 d of age, despite the greater IGF-I responses to bGH in the latter group. It is suggested that hepatic GHR in lambs up to Day 63 of life are not fully functional compared to the situation in yearlings.

The effect of selective vagotomy of the abomasum, pylorus, duodenum and liver on insulin release during the cephalic phase of digestion was investigated in wethers and lactating ewes. Electrical stimulation of the cervical vagus nerves was carried out to test the completeness of the vagotomies performed. In experiment 1, using wethers, the abomasal, pyloric and duodenal branches (ADV; n = 7) or the hepatic, abomasal, pyloric and duodenal branches (HADV; n = 10) of the ventral and/or dorsal vagus nerves were cut; a third group of wethers underwent sham-operation (SO; n = 8). In experiment 2, vagotomy (ADV; n = 5) or sham-operations (SO; n = 5) were carried out in lactating ewes. Jugular blood was drawn before and after presentation of food for glucose and insulin determination (experiments 1 and 2) or before, during and after the electrical stimulation of the peripheral ends of the cut cervical vagus nerves in randomly selected lactating ewes (experiment 3: ADV = 3, SO = 3) and wethers (experiment 4: ADV = 4, HADV = 4, SO = 4), for determination of insulin only. Presentation of food caused an immediate and significant (P < 0.05) rise in plasma insulin levels in SO animals compared with ADV or HADV wethers (experiment 1) or ADV ewes (experiment 2) without any significant change in blood glucose concentrations. In comparison with the SO group the baseline-corrected areas under the insulin response curve were significantly (P < 0.05) smaller for the respective vagotomized groups for periods 1-2, 2-4 and 4-6 min (experiment 1) and 1-2 and 2-4 min (experiment 2) after presentation of food. Total area under the response curve for 10 min was significantly (P < 0.05) lower (experiment 1) and tended (P < 0.10) to be lower (experiment 2) for the vagotomized groups compared with that of the control groups. Direct electrical stimulation of the cervical vagus nerves raised plasma insulin concentrations to significantly (P < 0.05) higher levels in the SO ewes but not in the ADV ewes (experiment 3). It was also evident that in experiment 1, HADV did not have any additive effect over that achieved by ADV alone. These results indicate that the vagal innervation of the gut mediates insulin release during the cephalic phase of feeding in sheep. It is concluded that insulin secretion from the pancreatic -cells in response to either food-related reflex activation of the vagal nuclei in the hypothalamus or direct cervical vagus nerve stimulation is mediated through the vagal efferent fibres carried in the abomasal, pyloric and duodenal branches of the vagus nerves in sheep.

The effects of recombinant bovine growth hormone (bGH) treatment of pregnant ewes on maternal metabolism, placental development and fetal growth were examined in two studies. In a preliminary study (experiment one), single-bearing ewes were treated by twice-daily subcutaneous injection for 7 days with bGH (n = 8) at a dose of 0.15 mg/kg LW/day or with saline (n = 8) between days 101 and 107 of gestation inclusive. In experiment two, single- and twin-bearing ewes were treated for 14 days with bGH (0.15 mg/kg L W/day) (n = 10) or saline (n = 10) between days 70 and 83 or days 98 and 111 of gestation inclusive. Ewes were killed on the day following termination of bGH treatment and fetal and placental measurements recorded. Maternal plasma concentrations of GH, IGF-I and insulin were higher (P < 0.001) in bGH-treated ewes relative to saline-treated ewes in both experiments. Consistent across experiments was an increase (P < 0.05) in the weight of the myoendometrium in bGH-treated ewes. Treatment with bGH also increased the total weight of the gravid uterus (P < 0.05) in both experiments. Weights of the uterine fluids were increased by bGH in experiment one (P < 0.05), but an effect of the same magnitude could not be repeated in experiment two. In experiment one, there was a tendency towards increased mean fetal body weights after growth hormone treatment, although the effect was non-significant. In experiment two, treatment with bGH was associated with significantly (P < 0.05) higher fetal weights, but only at the later stage of gestation (day 112). This effect was additive with that of fetal rank. Exogenous bGH treatment had little discernible effect on measures of placental size. It is concluded that administration of exogenous bGH to pregnant ewes can stimulate fetal growth, but only after about day 100 of gestation. This response seems most likely to reflect changes in maternal nutrient partitioning or placental function, rather than placental size. These studies suggest a role for growth hormone of maternal or placental origin in the regulation of fetal growth.

The ability of ovine placental lactogen (oPL) to bind to the growth hormone receptor (GHR) raises the possibility that oPL may exert a growth hormone (GH)-like action on galactopoiesis. We have compared the effects of treating lactating ewes for 5 days with an equimolar dose (0.1 mg/kg/day, administered as two equal doses 12 hourly) of either bovine growth hormone (bGH) (n = 10), oPL (n = 10) or saline (n = 9) on hepatic and mammary GHR, insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3) gene expression and hepatic GHR number. Hepatic GHR and IGFBP-3 mRNA were unaltered by bGH or oPL treatment. Hepatic IGF-I mRNAs increased following bGH (P < 0.05) but not oPL treatment. GHR gene expression was greater in liver compared to mammary gland extracts. There was no effect of either bGH or oPL treatment on mammary GHR, IGF-I or IGFBP-3 mRNA or hepatic GHR number. These studies confirm the galactopoietic effects of bGH in lactating ruminants and suggest that the mechanism of this action is not via increased hepatic GHR number or gene expression. In addition, the increase in hepatic but not mammary IGF-I mRNA with bGH treatment suggests an endocrine action of IGF-I on milk synthesis. These studies also demonstrate that an equimolar dose of oPL is not galactopoietic or somatogenic in the lactating ewe.

The ability of the murine mammary fat pad to directly stimulate the growth of mammary epithelial cells and to modulate the effects of various mammogenic agents has been investigated in a newly described, hormone- and serum-free coculture system. COMMA-1D mouse mammary epithelial cells were cultured for 5 or 7 d with various supplements in the absence or presence of epithelium-free mammary fat pad explants from virgin female BALB/c mice. Cocultured fat pad stimulated increases in the DNA content of COMMA-1D cultures by two- to threefold or six- to eightfold after 5 or 7 d, respectively. The mitogenic effect was additive to that of 10% fetal calf serum and could not be attributed to the release of prostaglandin E2 or synthesis of prostaglandins by epithelial cells. In addition, bovine serum albumin attenuated (P < 0.05) the mitogenic effect of cocultured mammary fat pad. Added alone, insulinlike growth factor-I, epidermal growth factor, and insulin increased (P < 0.05) total DNA of COMMA-1D cultures by 2.5-, 3.7-, and 2.3-fold, respectively. Cocultured mammary fat pad markedly interacted (P < 0.01) with these mitogens to yield final DNA values that were 21.2-, 13.3-, and 22.1-fold greater than in basal medium only. Associated with this proliferation was the formation of numerous domes above the COMMA-1D monolayer. There was no proliferative response to growth hormone or prolactin in the absence or presence of cocultured fat pad (P > 0.05). Whereas hydrocortisone did not alter cell number, it attenuated (P < 0.05) the mitogenic effect of cocultured mammary fat pad. These results indicate that the murine mammary fat pad is not only a direct source of mitogenic activity, but also modulates the response of mammary epithelial cells to certain mammogens.

Although the insulin-like growth factors (IGF-I and -II) have been implicated in the stimulation of mammogenesis, little is known of their regulation in the mammary gland. In this study we removed epithelial tissue from one of the two mammary glands of 1-week-old ewe lambs and examined IGF-I and -II mRNA expression during postnatal development in both the intact mammary gland and in the gland cleared of epithelial tissue. Expression of IGF-I mRNA was highest at 6 and 10 weeks of age, coincident with the prepubertal phase of rapid mammary growth, then declined and remained low until expression increased during late pregnancy. IGF-I mRNA was more abundant in the mammary fat pad adjacent to parenchyma (MFP) than in the contralateral fat pad that had been surgically cleared of epithelium (CFP). The level of IGF-II mRNA in parenchyma was highest at 1-23 weeks of age due to an increase in the abundance of specific mRNAs. Expression was lower in the fat pads, with generally higher levels in the intact MFP than the CFP, and in these tissues IGF-II expression was shown to increase with age between 6 and 23 weeks. We also investigated the influence of the ovary and estrogen on the expression of IGFs. While IGF-I mRNA abundance was unaffected by ovariectomy, exogenous estrogen resulted in higher levels of expression in the MFP of ovariectomized ewes and tended to increase its level in the parenchyma of intact ewes. Ovariectomy increased IGF-II mRNA within mammary parenchyma whereas estrogen suppressed levels in both the parenchyma and MFP. These findings demonstrate that IGF-I and -II mRNAs are expressed locally within the developing ovine mammary gland and are regulated by stage of ontogeny, ovarian hormones, and epithelial stromal interaction.

The effects of recombinant ovine placental lactogen and bovine growth hormone on milk yield, milk composition, and concentrations of blood hormones and metabolites were compared in ewes during an established lactation. Beginning on d 17 of lactation, ewes were treated for 5 d with twice daily subcutaneous injections of ovine placental lactogen (n = 9), bovine growth hormone (n = 10) at a dose of 0.10 mg/d per kg of body weight, or saline (n = 10). Circulating concentrations of ovine placental lactogen were 24.6 +/- 1.6 ng/ml on d 5 for ewes treated with ovine placental lactogen, but concentrations of ovine placental lactogen were undetectable in ewes treated with either saline or bovine growth hormone. Treatment with bovine growth hormone increased circulating concentrations of growth hormone, insulin-like growth factor-I, and nonesterified fatty acids and decreased urea concentrations relative to those in ewes treated with ovine placental lactogen or saline. Compared with saline treatment, no parameters were affected by treatment with ovine placental lactogen. Treatment with bovine growth hormone or ovine placental lactogen treatment had no significant effects on plasma concentrations of insulin, glucose, or creatinine. Treatment with bovine growth hormone, but not ovine placental lactogen, increased yields of milk, fat, and lactose. Weight of the mammary gland was increased by bovine growth hormone, but not by ovine placental lactogen. Despite the fact that ovine placental lactogen is a potent somatogen, it does not appear to exhibit the same galactopoietic activity as bovine growth hormone in lactating ewes.

The growth-promoting and metabolic effects of recombinant ovine placental lactogen (oPL) were compared with those of recombinant bovine growth hormone (bGH) in young lambs. Lambs were treated by twice daily subcutaneous injection with oPL (n = 16) or bGH (n = 16) at a dose of 0.1 mg/kg live weight/day or with saline (n = 16) for 21 days commencing on day 3 of life. Jugular blood samples were taken on days 0, 10 and 20 of treatment. Half the lambs in each group were slaughtered at 24 days, and the other half at 9 months of age. Both bGH and oPL treatments induced small but significant (P < 0.05) increases in circulating concentrations of insulin-like growth factor-I (IGF-I) on day 10 of treatment, but not on day 20. Neither treatment altered plasma concentrations of glucose, non-esterified fatty acids, urea or creatinine compared to those in saline-treated lambs. Relative to those of bGH-treated (0.24 +/- 0.01 kg/day) or saline-treated (0.25 +/- 0.01 kg/day) lambs, live weight gains of oPL-treated lambs (0.28 +/- 0.01 kg/day) were significantly (P < 0.05) increased during treatment and differences in live weight were still apparent at 9 months of age. Similarly, treatment with oPL, but not bGH, significantly (P < 0.01) increased daily energy intake. It is concluded that placental lactogen and growth hormone do not have identical biological actions. While oPL is growth-promoting in young lambs, this effect may be mediated by stimulating voluntary feed intake rather than by elevating circulating concentrations of IGF-I.

The role of placental lactogen (PL) in the regulation of maternal metabolism and fetal growth is not understood. Both PL and growth hormone (GH) have been suggested as possible regulators of mammogenesis. Our aim was to compare the effects of recombinant ovine placental lactogen (oPL) and bovine growth hormone (bGH) on maternal mammary gland development and fetal growth. Pregnant ewes were treated from day 101 to 107 of gestation with twice daily subcutaneous injections of recombinant oPL (n = 7), bGH (n = 8) (0.15 mg/kg live weight/day) or saline (n = 8). On day 108 of gestation, fetal and maternal tissues were collected. The relative abundance of growth hormone receptor (GHR), insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) mRNA was assessed in mammary gland, maternal liver and heart, and in fetal and placental tissues. There was no detectable change in mammary tissue GHR, IGF-1 or IGFBP-3 gene expression with either bGH or oPL treatment. Maternal administration of bGH, but not oPL, during pregnancy caused an increase in maternal hepatic IGF-1 gene expression (P < 0.005). Treatment with oPL, but not bGH, resulted in a significant increase (P < 0.025) in the relative abundance of fetal hepatic IGFBP-3 mRNA. Maternal hepatic GHR gene expression was not affected by treatment. This study suggests that while bGH treatment of pregnant ewes induces characteristic somatogenic responses, oPL treatment does not have comparable effects. However, oPL may indirectly influence the fetal somatotropic axis by altering fetal hepatic IGFBP-3 production.

We have shown previously that chronic administration (8 weeks) of insulin-like growth factor-I (IGF-I) has little growth-promoting effect in well fed sheep. The aim of this study was to investigate the anabolic effects of IGF-I in energy-restricted conditions in which circulating concentrations of IGF-I in control animals were expected to be low. Young castrate male sheep were offered chaffed lucerne at a rate equivalent to 110% maintenance and were treated by sc injection three times per day for either 8 or 12 weeks with recombinant human IGF-I (150 micrograms/kg live wt x day) or saline in a 2 x 2 factorial design (eight animals per cell). IGF-I treatment significantly increased plasma IGF-I concentrations, but reduced plasma concentrations of IGF-II, GH, urea, and creatinine. Treatment with IGF-I also decreased (P < 0.1) GH secretion in response to a GRF load, but significantly (P < 0.05) increased the nonesterified fatty acid response to an epinephrine load. The reduction in circulating GH levels was accompanied by a suppression of [125I]oGH binding to hepatic microsomal membranes. This effect, if apparent in other tissues, may act as a feedback mechanism to limit the local synthesis of IGF-I and could explain why IGF-I treatment had little effect on the growth rate of the sheep, although it did increase nitrogen digestibility of the feed consumed and decreased the fat content of the hind leg. It also differentially promoted the growth of the spleen, thymus, and mandibular salivary gland and increased blood counts of eosinophils. It is concluded that IGF-I does not have marked effects on growth rate or body composition in sheep fed a near-maintenance diet. Possible reasons include the associated suppression of GH secretion and action, which limits the ability of treated animals to repartition absorbed nutrients.

Binding proteins for the insulin-like growth factors (IGFBPs) modulate the actions of IGF I and IGF II. IGFBP-2 is particularly high in plasma of pregnant and fetal animals and in milk. We investigated the peri-lactational control of IGFBP-2 expression and secretion. Fifteen singleton-bearing pregnant ewes at day 101 of gestation were injected sc twice daily for 8 days with bovine growth hormone (bGH) or ovine placental lactogen (oPL) both at 0.15 mg.kg-1.d-1 or saline. A further fifteen ewes at day 17 of lactation were injected sc twice daily for 5 days with bGH or oPL at 0.1 mg.kg-1.d-1 or saline. On the last day of injection blood samples were taken and the animals were sacrificed. Liver and mammary tissue samples were immediately frozen and subsequently extracted to provide total RNA for evaluation by Northern blot analysis using a rat IGFBP-2 cDNA probe. Plasma samples were analysed by Western ligand blotting for IGFBP-2. The comparison of the two saline-treated groups (pregnant vs lactating ewe) revealed no difference in the plasma concentrations of IGFBP-2. IGFBP-2 mRNA expression in the liver of the lactating ewes was markedly increased compared to that in the pregnant ewes. In contrast, in mammary tissue the expression was significantly lower in lactating than in pregnant sheep. In pregnant animals treatment with bGH, but not oPL, decreased the expression of IGFBP-2 in liver. There was a similar trend in the lactating ewe.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth allometry was examined over the range 6 to 112 days of age in male and female mice from lines selected for low (L) or high (H) plasma concentrations of insulin-like growth factor-I (IGF-I). Plasma IGF-I concentrations were greater in the H line than in L line mice from 28 days of age. H line mice also had greater liveweights and weights of the heart, kidneys, pancreas, lungs, liver, brain and testes from 21-28 days of age. Changes in weights of these organs reflected the general pattern of body growth, there being no consistent effects of selection line on allometric growth coefficients. Ovarian weights were not different between the lines. For muscle weights, (gastrocnemius, quadriceps femoris), the weights or lengths of bones (tibia, femur) and the nose-anus and anus-tail lengths, allometric growth coefficients were generally higher (P less than .05) in L line females than in L line males or H line mice of either sex. The allometric growth coefficient for spleen was significantly (P less than .05) greater in H line mice than in L line mice with the result that spleen weights were 30% higher in H line mice from 28 days of age. This is consistent with results from mice treated with, or transgenic for, IGF-I and suggests a specific effect of this hormone on growth of the spleen. Thymus weights were also greater in H line than in L line mice and developmental patterns of thymus weight closely paralleled those of circulating IGF-I.

Basal hormone/metabolite concentrations and responses to intravenous challenges of glucose, insulin and epinephrine were examined in Friesian cows from selection lines of low or high genetic merit treated with recombinantly-derived bovine somatotropin (bST) or control formulation in a 2 x 2 factorial design. Cows from the low genetic merit (low breeding index, LBI) line had previously been shown to be more responsive to the galactopoietic effects of bST (50 mg/day) than those from the high breeding index (HBI) line. Despite this, comparisons of metabolic differences were not confounded by differences in energy balance because bST treatment had also caused an increase in voluntary intake of cut pasture. Circulating levels of somatotropin, insulin-like growth factor-I (IGF-I) and insulin were greater in bST-treated than control cows but neither bST treatment nor selection line influenced basal concentrations of glucose, non-esterified fatty acids (NEFA), beta-hydroxybutyrate, urea or creatinine. Treatment with bST produced a small increase in sensitivity of cows to the lipolytic effects of epinephrine and this effect was similar in both selection lines. HBI cows had greater circulating insulin levels following the glucose challenge than LBI cows but bST treatment did not affect the insulin response to exogenous glucose. Whereas bST treatment retarded the glycogenolytic response to epinephrine and the clearance of blood glucose in response to insulin in LBI cows, it had no effect on epinephrine-stimulated glycogenolysis, and caused enhanced glucose clearance in response to insulin, in HBI cows. Results are consistent with bST altering the homeorhetic control of metabolism but do not adequately explain the greater responsiveness of LBI cows to the galactopoietic effects of bST.

A study was conducted to investigate developmental patterns of plasma concentrations of insulin-like growth factor-I (IGF-I), body growth and body composition in mice from lines selected for seven generations on the basis of low (L) or high (H) plasma IGF-I, and in a random-bred control (C) line. Litter size was standardized to eight individuals with equal sex ratios (as far as possible) within 48 h of birth. Pups were weaned at an average of 21 days and separated on the basis of sex. Blood samples were collected from one male and one female of each litter on days, 21, 42, 63 and 105 for analysis of plasma concentrations of IGF-I. The animals were then killed and analysed for water, fat and crude protein content. The plasma concentration of IGF-I was influenced by line (P less than 0.05) but not by sex. Significant (P less than 0.001) differences in liveweight between mice from L and H lines were first evident at 21 days of age. From 28 until 105 days of age the H line was significantly (P less than 0.001) heavier than both L and C lines, but differences between C and L lines were inconsistent and mostly non-significant. The growth velocity of the H line was significantly greater than that of C or L lines between 14 and 42 days of age, but differences in growth velocities of C compared with L lines were generally non-significant.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for the isolation of lobules of acini from bovine mammary gland and their storage in liquid nitrogen is described. After further dissociation of freshly prepared or frozen lobules, clumps of cells are obtained which attach to collagen gels and give rise to colonies which, on morphological criteria, appear predominantly epithelial. Storage for up to 6 months did not adversely affect viability. Increase in colony area involved cell division, was more rapid in air than in 95% oxygen and was enhanced by fetal calf serum.

A divergent selection experiment with mice, using plasma concentrations of insulin-like growth factor-1 (IGF-1) at 42 days of age as the selection criterion, was undertaken for 7 generations. Lines were not replicated. To obtain sufficient plasma for the IGF-1 assay, blood from four individuals was volumetrically bulked to obtain a litter mean IGF-1 concentration. This necessitated the use of between family selection. Although inbreeding accumulated in a linear fashion in each of the high, control and low lines, the rates were different for each line (3.6, 1.6 and 5.3% per generation for the high, control and low lines, respectively). As a consequence, the effects of selection and inbreeding are confounded in this experiment. Divergence between the high and low lines in plasma concentrations of IGF-1 continued steadily until generation 5. In generations 6 and 7, there was a reduced degree of divergence and this contributed towards the low realized heritability value of 0.15 +/- 0.12. Six-week liveweight showed a steady positive correlated response to selection for or against plasma concentrations of IGF-1 until generation 4 (high-low difference = 1.7 g = 12%). In generation 5, a substantial drop in 6-week liveweight in the low line relative to both the high and control lines occurred (high-low difference, 3.9; g, 25%). This difference was maintained until generation 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Reproductive performance, mammary gland weight and plasma concentrations of insulin-like growth factor-1 (IGF-1) were examined in 18-day-pregnant mice from lines divergently selected on the basis of plasma IGF-1 concentration. Females of the high IGF-1 (H) line were 14% heavier than those of the low IGF-1 (L) line at mating but did not differ in conception rate during a 15-day mating period. H-line females produced significantly larger litters by an average of 1.5 fetuses (19%), heavier fetuses (7%), greater total fetal weight (30%), heavier placental discs (15%), greater total placental weight (35%) and heavier mammary glands (18%). Plasma IGF-1 values were 12% greater in H-line than L-line females at Day 19 of gestation but the line difference was not significant. It is concluded that differences between the lines in litter size and mammary gland weight are most likely due to differences in maternal bodyweight (which are in turn a consequence of selection for plasma IGF-1 at puberty). Whether the difference in fetal weight is a function of fetal capacity to grow in utero or ability of the dam to provide nutrients for fetal growth is yet to be determined.

Bovine mammary epithelial cells cultured on floating gels of rat tail collagen showed two principal cell types, columnar and squamous, with ultrastructural features resembling secretory and myoepithelial cells respectively. Cultures of freshly prepared cells released alpha-lactalbumin into the culture medium and in some cases contained fat droplets, although these did not appear to be released. No ultrastructural evidence of casein synthesis was observed. A notable feature was the failure to secrete a continuous basement membrane. Intermediate filaments were present in abundance in squamous epithelial cells.

Renal clearance experiments were performed on unanesthetized winter flounder from which bladder urine was collected continuously and caudal vein blood was sampled periodically; renal tissue was also obtained terminally for comparison of test organic acid content in vivo and after incubation in vitro. Urine flow rates and inulin U/P (urine to plasma concentration) ratios were relatively constant for a given fish and averaged 1.0 ml/hr X kg fish and 2.6, respectively. In contrast, U/P ratios for all three test acids cycled from minima of near 100 to maxima of over 1000 roughly every 24 hr when plasma concentrations of unbound acid were below 1 micron; correction of plasma protein binding was required in the case of chlorphenol red, but not PAH or Diodrast. Both in vivo and in vitro the organic acid content of renal tissue was intermediate between plasma and urine concentrations. These results demonstrate that kidneys of intact flounder exhibit the remarkable concentrative capacity for exogenous organic acids previously observed with isolated tubules and suggest that the tubular urine concentration is established in two steps by cell transport first at the peritubular and second at the luminal membrane. The anterior kidney position and the magnitude of maximal PAH and Diodrast clearances, about 1000 ml/hr X kg flounder, are consistent with most of the cardiac output returning to the heart through the renal portal circulation; a regulatory shunt bypassing the peritubular capillaries is proposed to explain cycling of organic acid clearances to minimal values.

Three experiments were undertaken to examine the degree and causes of variation in plasma concentrations of insulin-like growth factor-1 (IGF-1) in mice. The relationship between IGF-1 concentrations and liveweight was also examined. In all three experiments, a number of non-genetic factors were found to contribute significantly to the variation in IGF-1 concentrations, the most important of these being sex and litter size. In one experiment, where pups from 16 litters were cross-fostered to avoid the confounding of maternal and direct genetic effects, a heritability of 0.40 +/- 0.27 was estimated for plasma IGF-1 concentration at 35 days of age. To examine further the existence of genetic variation in plasma concentrations of IGF-1 and the genetic covariation between plasma IGF-1 levels and other body traits, a selection experiment with mice has been initiated. Moderate to strong phenotypic correlations between IGF-1 concentrations and weight at an early age have been found in all three experiments.

Substantial responses in the 6-week and mature body-weights of mice occurred after 7 generations of selection for or against plasma levels of Insulin-like Growth Factor-1 (IGF-1). Plasma levels of IGF-1 were also significantly different after 7 generations of selection (high line = 85 +/- 2 ng/ml, low line = 58 +/- 2 ng/ml). The average 6-week weight in the line selected for high plasma IGF-1 was 22.5 +/- .2 g compared with 18.5 +/- .2 g in the low plasma IGF-1 line, after 7 generations of selection. The difference between lines was maintained at 20 weeks of age. These data provide further evidence for the roles of IGF-1 in the regulation of somatic growth and as a mediator of a genetic component of growth.

1. The proportions of carbon in individual milk constituents derived from feed and body tissues in dairy cows, were estimated by taking advantage of the natural variations which occur in the ratio, 13C:12C present in C3- and C4-plant species. 2. Four cows, which had previously grazed C3 plants (ryegrass (Lolium spp.) and white clover (Trifolium repens)), were accustomed to indoor feeding on a ration of C3-plant material (cut pastures and barley meal). The ration was then changed abruptly to one of C4-plant material (paspalum (Paspalum dilatatum) hay, maize silage and meal) for a period of 8 or 9 d in early and again in late lactation. 3. During early lactation it was estimated that 54% of the C in milk fat was derived from the body fat reserves of high genetic merit cows. Corresponding values for casein and lactose were 34 and 24% respectively, if it is assumed they were derived from body protein reserves. In contrast steam-volatile fatty acids in milk fat were almost entirely derived from dietary sources. 4. The proportional contribution of body-tissue C to individual milk constituents varied considerably between animals, possibly associated with genetic merit or the size of the body reserves available for mobilization. 5. In late lactation, when cows were close to energy and protein balance, contributions of body-tissue C to milk fat, casein and lactose ranged up to 19, 19 and 8% respectively. 6. Estimates of endogenous losses of C in faeces averaged 12 and 9% in early and late lactation respectively, and corresponding values for endogenous urinary C were 23 and 15%.

A study was undertaken to investigate the role of testosterone in regulating growth and circulating levels of insulin-like growth factor-I in male mice from lines divergently selected on the basis of plasma IGF-I. Controls of each lines were sham-operated at 10 days of age and treated with peanut oil from day 14 to day 70. A second group, which was castrated at 10 days and treated with testosterone enanthate (0.5 micrograms.(g body weight)-1.day-1) from day 14 to 70, did not differ from controls in body weight but had higher plasma IGF-I concentrations. Delaying testosterone therapy until day 42 in a third group retarded growth, with body weights being significantly lower than those of other two groups from days 35 to 56. However, plasma IGF-I levels in this group were not different from those of controls. Effects of line and treatment were additive. It is concluded that the greater pubertal growth of high-line compared to low-line males is not due to greater stimulation of circulating IGF-I by testosterone. Furthermore, testosterone does not appear to influence pubertal growth by acting on circulating levels of IGF-I.