Invited review: impact of specific nutrient interventions during mid-to-late gestation on physiological traits important for survival of multiple-born lambs

To improve production efficiency, the sheep meat industry has increased flock prolificacy. However, multiple-born lambs have lower birth weights, increased mortality and reduced growth rate compared with single-born lambs. Lamb mortality is a major issue for livestock farming globally and solutions are required to increase survival to realise the value of increased flock fecundity. Nutrition during gestation can influence maternal–foetal placental nutrient transfer and thus foetal growth and organ/tissue development, as well as improve postnatal productivity. This review covers the challenges and opportunities associated with increased prolificacy, highlights gaps in our knowledge and identifies some opportunities for how targeted intervention with specific nutrients during mid-to-late pregnancy may influence lamb survival and productivity with a specific focus on pasture-based systems. This time frame was selected as intervention strategies in short-time windows post-pregnancy scanning and before lambing to improve lamb survival in high-risk groups (e.g. triplets) are likely to be the most practical and economically feasible options for pasture-based extensive farming systems.     

Litter-size-dependent intrauterine growth restriction in sheep

Regulation of foetal development in sheep depends on interactions between the intrinsic capacity of the foetus for growth and the maternal environment. Lambs born in multi-foetus litters have relatively small placentae with fewer cotelydons, and lower birth weights. Litter-size-dependent intrauterine growth restriction (IUGR) is evident at mid gestation when metabolic needs of the conceptus are moderate, and overnutrition of ewes with multiple foetuses does not promote growth of their foetuses to the size of singletons. Those observations suggest that placental and conceptus growth in multi-foetus pregnancies is reprogrammed at mid gestation by an as yet undefined mechanism to attenuate foetal growth. This may protect the foetus from severe nutritional insult during late gestation, when its daily growth rate is at a maximum. In that way, lambs born in large litters with relatively lower birth weights may not experience the long-term physiological insults that can be observed in small lambs born to undernourished ewes.     

Foetal age determination and development in elephants

Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown-rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85-90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.     

Changes in concentrations of follistatin in maternal plasma and foetal fluids during pregnancy and parturition in sheep

Studies have shown that follistatin may be involved in the regulation of ovarian development, pregnancy and parturition. The aim of the present study was to measure follistatin levels in maternal plasma and foetal fluids during pregnancy and parturition in sheep. Using a previously described follistatin radioimmunoassay, we found that follistatin in foetal plasma and allantoic fluid was higher in the female than in the male at days 50-75 of gestation. Follistatin concentrations in maternal plasma declined from -9 to -3h before the completion of lamb delivery, and increased from 21 to 39 h after parturition. These results suggest that follistatin may play a role in femaleness development, and that follistatin may be involved in the mechanism of normal parturition and in regulation of the tissue repair after parturition.     

Ultrastructural changes in the placenta of the ewe following foetal infusion of cortisol.

The placental changes which followed continuous infusion of cortisol into the sheep foetus in the later stages of gestation were, like the hormonal changes, broadly similar to those of spontaneous parturition. There was, however, a premature separation of foetal and maternal tissues in certain areas of the placental cotyledons, and this separation appeared to protect the foetal epithelium from the degenerative changes which normally take place in the short space of time between the birth of the lamb and the delivery of the foetal membranes. The results suggest that an experimental model in which premature labour is induced by the administration of cortisol to the foetus is probably incomplete, and that additional factors almost certainly contribute to the cascade phenomenon of spontaneous parturition.     

Prenatal sonographic diagnosis of skeletal dysplasias. A report of 47 cases

The purpose of this study was to evaluate the foetal sonographic efficiency for prenatal diagnosis of osteochondrodysplasias. Forty-seven prenatal and postnatal cases diagnosed between January 1993 and December 1998 in the referral sonographic centres of Strasbourg were studied. All cases were reviewed retrospectively and the prenatal ultrasound findings and diagnosis were compared to the postnatal or post-mortem diagnosis. Each case was studied by ultrasonographers, geneticists, radiologists, and foetopathologists. Final diagnosis was based on clinical examination, skeletal survey and molecular testing as deemed necessary. Routine screening and dating was the indication for foetal sonography in 72% (32/47) of our cases. The most likely time of diagnosis was between 16 and 24 weeks of gestation (17 out of 47 cases, 36%), which corresponds to the time of foetal anomaly sonographic scan in France. The other cluster of cases (12 among 47, 26%) was disclosed before 16 weeks of gestation. These results illustrate the importance of a detailed evaluation of the limbs during sonographic examinations of first and second trimesters of pregnancy. While the identification of skeletal dysplasias was relatively easy in our study, the ability to make an accurate specific antenatal diagnosis was more difficult. An accurate diagnosis was proposed in 28 of the 47 cases (60%). In 19% of the cases (9/47), the prenatal diagnosis was not accurate; in 21% of the cases (10/47), the prenatal diagnosis was imprecise. In 45 of the 47 cases (96%) prenatal foetal scan correctly predicted the prognosis.     

Aspects of adipose-tissue metabolism in foetal lambs.

1. The mean volume of adipocytes, the rates of fatty acid and acylglycerol glycerol synthesis from various precursors (in vitro), the rates of oxidation of acetate and glucose (in vitro) and the activities of lipoprotein lipase and various lipogenic enzymes were determined for perirenal adipose tissue from foetal lambs during the last month of gestation. 2. The fall in the rate of growth of perirenal adipose tissue during the last month of gestation is associated with a diminished capacity for fatty acid synthesis and lipoprotein lipase activity, but no change in the rate of acylglycerol glycerol synthesis was observed. There was no fall in the activities of cytosolic acetyl-CoA synthetase or the NADP-linked dehydrogenases, suggesting that the decrease in the rate of fatty acid synthesis was due to an impairment at the level of acetyl-CoA carboxylase or fatty acid synthetase. 3. The rate of fatty acid synthesis from acetate was greater than that from glucose. The rate of fatty acid synthesis from glucose per adipocyte of foetal lambs was similar to that of young sheep. The characteristic metabolism of adipose tissue of the adult sheep is thus present in the foetus, despite the relatively large amounts of glucose in the foetal 'diet'.     

Quantification of glial fibrillary acidic protein in developing sheep brain cortex, cerebellum and stem

1. The glial fibrillary acidic protein (GFAP) content of foetal, young (lamb) and adult sheep brain white (stem and cerebellum) and grey (cortex) matter-enriched regions has been determined by means of an improved ELISA using one layer of anti-human GFAP monoclonal antibody. 2. The order of GFAP concentration in brain regions was as follows: brain stem greater than cerebellum greater than cortex. 3. Postnatal brain development accounts for an increase of GFAP in all the regions. The most important increase in GFAP was observed in the adult brain and was proportionally more significant in the grey matter-enriched cortex.     

Endocrine and metabolic regulation of muscle growth and body composition in cattle

Muscle metabolism (in interaction with other organs and tissues, including adipose tissue) plays an important role in the control of growth and body composition. Muscle ontogenesis has been described in different genotypes of cattle for myofibres, connective tissue and intramuscular depots. The ontogenesis or the action of putatively important factors controlling muscle development (IGF-II expression, IGF receptors, growth hormone (GH) receptor, myostatin, basic fibroblast growth factor, transforming growth factor-β1, insulin and thyroid hormones) has also been studied on bovine foetal muscle samples and satellite cells. The glucose/insulin axis has been specifically studied in both the bovine adipose tissue and heart. Clearly, cattle, like sheep, are mature species at birth based on their muscle characteristics compared to other mammalian or farm animal species. The different myoblast generations have been well characterised in cattle, including the second generation which is liable to be affected by foetal undernutrition at least in sheep. Interesting genotypes, for example, double-muscled genotype, have been characterised by an altered metabolic and endocrine status associated with a reduced fat mass, specific muscle traits and different foetal characteristics. Finally, the recent development of genomics in cattle has allowed the identification of novel genes controlling muscle development during foetal and postnatal life. Generally, a high muscle growth potential is associated with a reduced fat mass and a switch of muscle fibres towards the glycolytic type. The possibility and the practical consequences of manipulating muscle growth and, hence, body composition by nutritional and hormonal factors are discussed for bovines based on our current biological knowledge.     

Comparison of noninvasive methods for the evaluation of female reproductive condition in a large viviparous lizard,Tiliqua nigrolutea

We compared the diagnostic value of three noninvasive methods (radiography, ultrasound, and palpation) of evaluating reproductive status in females of a large viviparous skink, the blotched blue‐tongued lizard (Tiliqua nigrolutea). Radiography could be used to identify postovulatory females during the periovulatory period. However, in early to mid‐gestation, radiography did not allow differentiation of the relatively homogenous structures within the abdomen. In late gestation, once calcification of the embryonic skeleton had occurred, gestation could be diagnosed and embryos counted. Ultrasound allowed differentiation between preovulatory, postovulatory, and nonreproductive females during the periovulatory period. Ultrasound also allowed visualisation of follicles during the early and middle stages of gestation. However, the level of operator experience, and the misdiagnosis of artefacts limited the accuracy of diagnosis. In the later stages of gestation, ultrasound did allow the detection of identifiable features of the developing foetal unit (fluid‐filled foetal membranes, independent movement of the embryo, and visualisation of the heartbeat). Colour Doppler imaging could be used to visualise embryonic blood flow. Ultrasonography was not useful in detecting the number of embryos present. Palpation during the periovulatory period could be used to detect follicles and determine whether ovulation had occurred. However, during the remainder of the gestation period, palpation was unable to differentiate reproductive condition. This study provides a basis for the diagnosis of reproductive condition and problems in viviparous reptiles. Each of the diagnostic techniques is useful at different stages of gestation, and the use of a combined approach will give the most information throughout the reproductive cycle. Zoo Biol 21:253–268, 2002. © 2002 Wiley‐Liss, Inc.     

Analysis of hepatic copper, zinc, metallothionein and metallothionein-Ia mRNA in developing sheep

The concentrations of zinc, copper, metallothionein and metallothionein-Ia mRNA in sheep livers during development was determined. It was found that early sheep foetuses (30-40 days gestation) had very high concentrations of hepatic zinc (2305 +/- 814 micrograms/g dry mass), and that these levels declined steadily to 644 +/- 304 micrograms/g near to term. The copper concentrations in the foetal livers were not higher than those in the adult. The concentrations of metallothionein and metallothionein-Ia mRNA were also very high in the foetal livers and declined steadily during gestation from 261 +/- 94 molecules/pg RNA to 71 +/- 18 molecules/pg near to term. Metallothionein-Ia mRNA concentrations were closely correlated with hepatic zinc concentrations but not with copper. Metallothionein concentrations also decreased during gestation: e.g. 3044 micrograms/g (wet mass) in one foetus on day 34 of gestation to 862 micrograms/g on day 125. After birth, however, the concentrations of metallothionein declined to less than 100 micrograms/g and this decline occurred despite the presence of significant quantities of mRNA. The ratio of metallothionein/metallothionein-Ia mRNA decreased from 1.3 to 3.2 x 10(5) molecules metallothionein/molecule of metallothionein-Ia mRNA during gestation to between 0.28-0.64 x 10(5) molecules/molecule in the postnatal animals. We conclude that the major function of metallothioneins in the foetal liver is protection of the liver against the potentially toxic accumulation of zinc. In the postnatal sheep there appears to be a decreased synthesis or increased degradation of metallothionein.     

Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child

Due to the risks to the foetus with invasive prenatal diagnosis, non-invasive prenatal diagnosis (NIPD) is gaining tremendous interest but no reliable method that can be widely used has been developed to date. Manipulation of foetal cells and foetal cell-free genetic material in the maternal blood are two promising approaches being researched. The manipulation of foetal cells in the maternal circulation is more popular as it can provide complete genetic information of the foetus particularly the diagnosis of aneuploidies. However, the foetal cell numbers in the maternal circulation are small and their enrichment and ex vivo culture remain two major challenges for NIPD. Primitive foetal erythroblasts (pFEs) have been considered as a good potential candidate for early first trimester NIPD but their nature, properties and manipulation to provide adequate cell numbers remain a challenging task and several approaches need to be meticulously evaluated. In this review we describe the current status of NIPD and suggest some novel approaches in manipulating pFEs for future clinical application of NIPD. These novel approaches include (1) understanding the pFE enucleation process, (2) enriching pFE numbers by individual pick-up of pFEs from maternal blood using micromanipulation and microdroplet culture, (3) expansion of pFEs using mitogens and (4) decondensation of the pFE nucleus with histone deacetylase (HDAC) inhibitors followed by reprogramming using gene delivery protocols with/without small reprogramming molecules to improve reprogrammed pFE proliferation rates for successful NIPD.     

Fecal counts of lungworm larvae and reproductive effort in bighorn sheep, Ovis canadensis

Because parasite resistance and reproduction require metabolic resources, life-history models predict a tradeoff between current reproduction and parasite load. These tradeoffs have been widely studied in birds, but few studies have been conducted on mammals. We monitored lungworm ( Protostrongylus spp.) larvae counts in bighorn sheep ( Ovis canadensis ) over four years to examine how individual differences in fecal output of lungworm larvae (LPG) by yearlings and adults were affected by season, sex, body mass, age and reproductive effort. We also compared lamb mass at six months and LPG. Overall, we found that LPG varies seasonally, peaking in females prior to lambing and in males during the rut. Age had no effect on LPG for either sex. During autumn, we found no effect of age or mass on LPG for sheep one year and older. Lamb body size or sex did not affect LPG. Females that weaned a lamb had higher counts than females that did not produce a lamb or females whose lamb died during summer. For rams, social rank and testosterone levels were not related to LPG but LPG increased with time spent searching for estrous ewes during the rut. Our results suggest a tradeoff between parasite resistance and reproductive effort in bighorn sheep of both sexes.     

Decline with age in the rate of reduction of progesterone to 20 alpha-hydroxypregn-4-en-3-one in the blood of perinatal ruminants

The activity of the enzyme 20 alpha-hydroxysteroid dehydrogenase present in erythrocytes of foetal and new-born ruminants has been determined by incubating 0.1 ml blood with 0.16 mumol [4-14C]-progesterone for 15 min at 39 degrees C in a final volume of 2 ml buffered saline. It was found that the activity, measured as mumol 20 alpha-hydroxypregn-4-en-3-one produced from progesterone per millilitre of erythrocytes per hour, declined from levels at birth as high as 1.50 mumol for sheep, 0.50 mumol for goats and 0.43 mumol for cattle to levels of around 0.11, 0.08 and 0.04 mumol respectively by 30-60 days of age. This decline in activity was also apparent in blood taken from sheep foetuses in which longitudinal studies were possible and appeared to have begun prior to 35 days before term. The highest activity obtained was 2.59 mumol for foetal sheep blood taken at 115 days of gestation. It is suggested that the observed decline in 20 alpha-hydroxysteroid dehydrogenase activity is a function of the replacement of foetal erythrocytes with adult-type erythrocytes which begins around 120 days of gestational age and that the role of the enzyme is to maintain an appropriate progestational environment within the foetoplacental unit.     

Lung liquid production and composition in the "in utero" foetal lamb.

Experiments were carried out on foetal lambs in utero from 103 to 133 days' gestation, to measure the daily flow and solute composition of liquid from the foetal lung. The mean daily flow from foetuses with a tracheal fistula was 231 ml/day (9.6 ml/h), while the mean hourly flow in foetuses with a tracheal loop was 5.1 ml/h. The pattern of flow of lung liquid was intermittent and irregular, with short periods of rapid flow interspersed with longer periods of slow and no flow. The Na+, Ca2+, Mg2+, Cl- and urea concentrations showed no significant change with increasing gestational age, but the K+ concentration showed a significant rise. It is concluded that the lung of the foetal lamb behaves as an exocrine gland, secreting a substantial volume of liquid of a relatively constant composition.     

Maternal behaviour and lamb survival: from neuroendocrinology to practical application

Parental care promotes offspring survival and, for livestock species, this care is provided solely by the mother. Maternal behaviour in the sheep has been exceptionally well-studied compared with other species and many of the underpinning biological processes leading to the expression of maternal care are known. In this review the current state of play with regard to the biology of maternal care will be reviewed, and its application to provide practical solutions to reduce lamb mortality considered. For maternal care to be elicited at birth the ewe requires elevated circulating oestradiol in late gestation, which stimulates the expression of oxytocin receptors in both peripheral and central areas (particularly the hypothalamic and limbic areas of the brain). At birth stretching of the vaginocervical canal elicits a spinal reflex which triggers the release of oxytocin primarily from neurones within the paraventricular nucleus of the hypothalamus. Oxytocin release causes an increase in the neurotransmitters noradrenaline, acetylcholine, glutamate and γ-aminobutyric acid (GABA) in the olfactory bulb, and other brain regions important for maternal behaviour. Finally, sensory cues provided by the lamb, in particular the amniotic fluids surrounding it, lead to the expression of maternal behaviours (licking, low-pitched bleats, acceptance of the lamb at the udder and suckling). This allows the expression of the two facets of maternal behaviour in the ewe: nurturance of the young and maternal selectivity, whereby a specific olfactory memory for the ewes own lamb is formed and the expression of maternal care is restricted to this lamb. Variation in the expression of maternal care has been demonstrated in primiparous ewes compared with multiparous, in different sheep genotypes, with undernutrition, stress in pregnancy, following a difficult delivery, and may occur with variation in ewe temperament. An understanding of the importance of the timing of various events in late pregnancy and during parturition, as well as the factors that can disrupt these events, can help to design management activities to minimise risks to the successful onset of maternal behaviour. Management practices that work with the biology of the ewe will be the most successful in ensuring that maternal care is expressed, so improving the welfare of the ewe and lamb, and the profitability of the farm.     

ANALYSIS OF PROLIFERATION AND DIFFERENTIATION OF FOETAL GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN HAEMOPOIETIC TISSUE*

Analysis of in vitro colony formation in agar cultures of foetal haemopoietic tissues of eight mammalian species has shown that granulocyte-macrophage progenitor cells are present in foetal liver, yolk sac, marrow and spleen in numbers approaching the incidence in adult marrow. Such characteristics as buoyant density, growth rate and differentiation served to distinguish foetal from adult colony forming cells (CFCs). Cell cycle analysis performed by exposing haemopoietic cells to high doses of tritiated thymidine in vitro showed that foetal CFC proliferation in species of short gestation (rabbit, rat, mouse) approached or exceeded that observed in adult marrow. In contrast, in species of long gestation (human, monkey, calf, lamb, guinea-pig) a period of variable duration was observed when foetal liver CFCs entered a non-cycling G₀ or blocked G₁ phase. In these species foetal liver CFCs were found to be proliferating actively early in gestation and following the non-cycling phase again re-entered a proliferative state associated with onset of active granulopoiesis in foetal marrow and possible migration of CFC from liver to marrow. These results indicate the existence of granulocyte-macrophage progenitor populations displaying foetal characteristics and adapted to particular stages of haemopoietic development, a situation which closely parallels that reported for erythropoiesis.     

The Boer goat. II. Growth, nutrient requirements, carcass and meat quality

Growth rates of Boer goats were generally lower than sheep and, under favorable nutritional conditions, weight gains of more than 200 g per day were obtained, against values of up to 176 g per day under extensive subtropical conditions. Lactation and pregnancy had a marked effect on ME intake, and the latter had an improved feed conversion ratio (6.06 kg/kg) in comparison to that of virgin does (10.96 kg/kg). Below 6% crude protein in the diet, feed intake is reduced and has negative effects on birth weights, growth and milk production. Little information is available on mineral requirements of goats. The carcass of Boer goats is generally leaner, less compact and has different carcass proportions than sheep. The relatively high collagen contents with lower solubility of Boer goat meat, has meant that the eating quality has been regarded as inferior to that of lamb and mutton. Breeding holds the key to improving tenderness of goat meat; different slaughtering techniques can be used as well. Boer goats have high potential as meat animals when yielding three kid crops in 2 years and when fed to gain more than 200 g/day.     

Reducing the level of nutrition of twin-bearing ewes during mid to late pregnancy produces leaner prime lambs at slaughter

The Australian prime lamb industry is seeking to improve lean meat yield (LMY) as a means to increasing efficiency and profitability across the whole value chain. The LMY of prime lambs is affected by genetics and on-farm nutrition from birth to slaughter and is the total muscle weight relative to the total carcass weight. Under the production conditions of south eastern Australia, many ewe flocks experience a moderate reduction in nutrition in mid to late pregnancy due to a decrease in pasture availability and quality. Correcting nutritional deficits throughout gestation requires the feeding of supplements. This enables the pregnant ewe to meet condition score (CS) targets at lambing. However, limited resources on farm often mean it is difficult to effectively manage nutritional supplementation of the pregnant ewe flock. The impact of reduced ewe nutrition in mid to late pregnancy on the body composition of finishing lambs and subsequent carcass composition remains unknown. This study investigated the effect of moderately reducing ewe nutrition in mid to late gestation on the body composition of finishing lambs and carcass composition at slaughter on a commercial scale. Multiple born lambs to CS2.5 target ewes were lighter at birth and weaning, had lower feedlot entry and exit weights with lower pre-slaughter and carcass weights compared with CS3.0 and CS3.5 target ewes. These lambs also had significantly lower eye muscle and fat depth when measured by ultrasound prior to slaughter and carcass subcutaneous fat depth measured 110 mm from the spine along the 12th rib (GR 12th) and at the C-site (C-fat). Although carcasses were ~5% lighter, results showed that male progeny born to ewes with reduced nutrition from day 50 gestation to a target CS2.5 at lambing had a higher percentage of lean tissue mass as measured by dual energy X-ray absorptiometry and a lower percentage of fat during finishing and at slaughter, with the multiple born progeny from CS3.0 and CS3.5 target ewes being similar. These data suggest lambs produced from multiple bearing ewes that have had a moderate reduction in nutrition during pregnancy are less mature. This effect was also independent of lamb finishing system. The 5% reduction in carcass weight observed in this study would have commercially relevant consequences for prime lamb producers, despite a small gain in LMY.