A tremendous amount is required of the oocyte cytoplasm to reprogram me a differentiated donor nucleus after somatic cell nuclear transfer so that it re-acquires a state of totipotency and can form a cloned individual. These reprogramming events must occur in a relatively short period after embryo reconstruction, quite unlike the situation during gametogenesis. It is remarkable that nuclear transfer can produce physiologically normal animals, but the process is highly prone to epigenetic errors. Aberrant patterns of gene expression are believed to contribute to the cumulative losses and abnormal phenotypes observed throughout development, from embryonic to adult stages. As a consequence, cloning efficiencies with current nuclear transfer technology are low and limit practical applications in farming. These applications depend upon the reliability with which nuclear transfer can reproduce specific genotypes and phenotypes in resulting offspring. Present animal welfare issues are a significant barrier for acceptance of nuclear transfer in agriculture. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. The safety of food products derived from clones also needs to be thoroughly evaluated. The current indication that the sexually derived offspring of nuclear transfer clones are normal provides confidence for the first applications of the technology in agriculture, namely the generation of cloned, genetically elite animals for subsequent breeding. Future improvements in cloning will come from both technical advances and a greater fundamental understanding of the molecular mechanisms of reprogramming with an aim to control this process better. Increased efficiencies should improve the acceptability and utility of cloning technology.
Evidence is presented that bovine somatotrophin (bST) treatment of lactating dairy cows enhances both expression of oviductal insulin-like growth factor II (IGF-II) mRNA and endometrial insulin-like growth factor binding protein 3 (IGFBP-3) mRNA between day 3 and day 7 of the oestrous cycle. mRNA encoding growth hormone (GH) receptor in endometrial tissues increased between day 3 and day 7 of the oestrous cycle. The changes induced by bST treatment may contribute to stimulation of embryo development and increase pregnancy rates in lactating dairy cows. Additive effects of bST and rb interferon tau (rbIFN-tau) to inhibit phorbol ester induction of prostaglandin F2alpha secretion in immortalized bovine endometrial cells indicates that there is interplay between their signal transduction pathways. Non-lactating dairy cows were killed at day 17 after oestrus to evaluate the effects of pregnancy status (cyclic versus pregnant) and bST (bST versus control) treatment on endometrial gene expression. Distinctly different mRNA and protein responses were detected between cyclic and pregnant cows that were related to luteolytic-antiluteolytic drive (that is expression of progesterone receptor, oxytocin receptor, oestradiol receptor alpha and prostaglandin GH synthase 2 (PGHS-2)). The bST-induced changes in PGHS-2 protein (+), oxytocin receptor mRNA (+) and oestrogen receptor alpha protein (+) may potentially affect the mechanisms associated with maintenance of pregnancy. Two experiments were conducted to evaluate whether ovarian follicular suppression induced by biodegradable deslorelin implants would reduce either early or late embryo losses. A 450 microg deslorelin implant used to induce ovulation in a timed insemination programme decreased subsequent follicular development and tended to reduce early embryo losses, whereas a 2.1 mg deslorelin implant failed to reduce late embryonic losses when inserted on day 27 of pregnancy.
It has been demonstrated that variations in litter size or ovulation rate in different breeds of sheep can be associated with the segregation of several major genes. This set of natural mutants constitutes a valuable resource to determine key points in the biochemical pathways controlling the development of ovarian follicles. The French genetic programmes were devised to identify two of these genes: the Booroola (FecB) and Lacaune genes. The FecB prolific mutation corresponds to a non-conservative mutation (Q249R) in the intracellular kinase-signalling domain of the bone morphogenetic protein receptor type IB (BMPR-IB) gene. The Lacaune gene is situated on ovine chromosome 11. Positional cloning is currently in progress to identify the relevant gene and mutation. A similar approach, limited to linkage testing of candidate genes, is proposed to classify the different prolificacy genes in sheep.
Melatonin, which is synthesized at night by the pineal gland, is present in the cerebrospinal fluid (CSF), but its entry site and its role in this compartment are not known. Using several approaches, we tested the hypothesis that melatonin enters the CSF through the pineal recess, an evagination of the third ventricle. CSF melatonin concentrations are higher near the pineal gland than in the anterior part of the third ventricle, and decrease markedly (80%) after sealing off the pineal recess. Moreover, ultrastructure and permeability analyses of the pineal-CSF interface showed that melatonin could reach the CSF either via delivery in situ by protruding pinealocytes that make direct contact with the CSF or via extracellular secretion and interstitial fluid draining into the ventricular lumen. These data indicate that melatonin in the CSF probably originates from a few pinealocytes of the basal part of the pineal gland neighbouring the pineal recess. Melatonin carried to the brain by the blood appears to be able to mediate the effects of photoperiod on reproduction, but it is unclear whether melatonin in CSF may fine-tune this response both in terms of timing and amplitude. It is critical to determine which pathway, blood or CSF, allows melatonin to reach its central targets more efficiently.
The reproductive efficiency of camels under their natural pastoral conditions is low. The reasons for this low reproductive efficiency include the short breeding season, the late age of reaching puberty and the long gestation period of 13 months. The introduction of controlled breeding programmes is important but several problems have to be considered. For example, oestrous behaviour is very vague and difficult to interpret, as it does not often relate to follicular development in the ovaries. In addition, all camelids are induced ovulators that normally ovulate only in response to mating, so alternative methods of inducing ovulation, such as injecting gonadotrophic hormones, have been investigated. The use of embryo transfer is becoming increasingly important but involves the necessity to superovulate the donors and synchronize the recipients so that they ovulate preferably 24 h after the donor. Superovulation can be achieved using exogenous gonadotrophins, although there is a high incidence of follicle luteinization before mating, of overstimulated ovaries and non-responsive females. The development of AI in camels is complicated by the difficulty of collecting semen and the gelatinous nature of the semen produced. However, diluting semen in Green Buffer and inseminating a minimum of 300 x 10(6) live spermatozoa has given encouraging results. The ability to control the follicular cycle of camels is leading to an improvement in reproductive efficiency.
The aim of this review is to pinpoint the areas that require further research for greatest impact to improve the efficiency of dairy and beef production. Increased knowledge about the principal causes of reduced fertility is essential. Increases in milk yield have been at the expense of reduced fertility in dairy cows and although diet has a major impact, the precise interaction between nutrition and reproduction still needs to be characterized in both beef and dairy cows. Furthermore, during periods of inadequate nutrition or stress, the intensity of oestrus is reduced by inadequate exposure to oestradiol. However, it is still unclear which pheromones are involved at oestrus, how synthesis is controlled and how pheromones are detected in herd-mates. GnRH may be involved in behaviour but the brain centres that translate hormonal messages are unknown. Attempts to overcome poor oestrous detection include measurements of milk progesterone, telemetric pressure detectors and devices that record the extra activity at oestrus. Substitution of heat detection by 'hormone treatment remedies' has met strong consumer resistance in Europe. The creation of larger cattle units and increased movements world-wide render herds more susceptible to infectious agents, such as Neospora, Leptospira, Trypanosoma and Bovine Viral Diarrhoea virus (BVDv), but it is unclear how other clinical conditions, such as lameness or endometritis, also interfere with ovarian function. The future of dominant follicles selected within 14 days after parturition is crucial--normal ovulation, prolonged persistence or atresia. Calving carries the greatest risk in the reproductive life of a cow and yet little work has focused on reducing the frequency of this event. For dairy cows, a greater understanding about induced or extended lactation is required. For beef animals, precise induction of twinning and nutritional adjustments could produce two offspring per pregnancy. At the start of pregnancy, the trophoblast produces interferon to prevent luteolysis, but the immunological implications are unknown and it is not clear how the rest of pregnancy is maintained. Profiles of pregnancy specific protein B (PSPB) have increased understanding of embryonic death. However, 25% of cows in abattoirs are pregnant, even though 30% of involuntary cullings are 'for failing to conceive'. Clearly, this is an area of wastage that requires urgent resolution. It is unknown why undernutrition at the time of insemination or in early pregnancy leads to delayed births, low fetal weights and later adverse health. At the end of pregnancy, the fetus controls the onset of parturition, but very little is known about the biochemical control of cervical dilation and placental separation. On the male side, bulls are selected for optimal freezability of semen; however, there is as yet no reliable predictor for semen fertility. Methods for accurately pre-determining the sex of both semen and embryos will revolutionize the dairy and beef industries.
The physical interface between the female germ line and enveloping somatic cells is dynamically modified throughout the course of folliculogenesis. How selective pathways for communication between the oocyte and granulosa cell are established and regulated remains to be determined, but insights into the structural basis for this communication are emerging. This review summarizes the available evidence that supports the notion that the integration of oogenesis with folliculogenesis is achieved by regulated cell interactions between oocytes and granulosa cells.
The development of nuclear transfer from tissue culture cells in livestock made it possible in principle to produce animals with subtle, directed genetic changes by in vitro modification of nuclear donor cells. In the short period since nuclear transfer was first performed, gene targeting in livestock has become a reality. Although gene targeting has immediate potential in biotechnology, it is unclear whether there are practical agricultural applications, at present. The first livestock targeting experiments have been directed at engineering animals either to render their organs immunologically compatible for human transplantation, or for improving the commercial production of recombinant proteins in the transgenic mammary gland. All successful examples of targeting have involved target loci that are expressed in the nuclear donor cell line. Two important barriers to the further development of this technology are adapting protocols for non-expressed genes and modifying procedures to enhance the lifespan of targeted cells in vitro. This review provides data that illustrate the difficulty in targeting non-expressed genes and discusses some of the practical issues associated with providing targeted nuclear donor cells that are competent for nuclear transfer.
The Booroola phenotype is associated with a point mutation in the kinase domain of the bone morphogenetic protein receptor 1 B (BMPR1 B), and is characterized by 'precocious' differentiation of ovarian follicles, leading to the production of large numbers of ovulatory follicles that are smaller in diameter than wild-type follicles. These smaller follicles attain differentiation markers, such as expression of mRNA for P450 aromatase and inhibin-betaA subunit, granulosa cell LH receptors and aromatase activity, earlier than follicles from wild-type ewes. However, the preovulatory follicles from mutant ewes collectively secrete similar quantities of oestradiol, androstenedione and inhibin A in exactly the same pattern as wild-type ewes, which result in similar concentrations of FSH. The available evidence strongly indicates that the Booroola mutation exerts its action at the ovary rather than by altering gonadotrophin secretion. The bone morphogenetic protein (BMP) receptors and putative ligands are ubiquitously expressed within the ovary and BMPs seem to be involved in the paracrine regulation of FSH action. Thus, if the mutation is causing a reduction in BMPR1 B signalling, it may act on an inhibitor of follicle differentiation. Further research in this area will concentrate on the elucidation of the natural ligands for BMPR1 B at different stages of follicle development and examine the effect of BMPR1 B mutation on the downstream signalling cascade.
Absent or irregular ovarian cycles in lactating dairy cows are caused by failure to ovulate the dominant follicle at the appropriate time. The follicle then either regresses or develops into a cyst. This process can be triggered by a variety of metabolic and disease factors that act at the hypothalamus and pituitary gland to inhibit pulsatile LH secretion and the LH surge, and at the ovary to reduce follicular growth and oestradiol production. Cows of poor energy status have low circulating concentrations of insulin-like growth factor I (IGF-I). Predisposing factors include calving difficulties, inappropriate diet, reduced intake of dry matter and a high rate of body condition score loss. Various stressors predispose the follicle to cyst development by inhibiting the LH surge and ovulation; these include common infections, such as mastitis. Even when ovulation does occur, poor follicular development may result in production of an inadequate corpus luteum. The timing of the increase in progesterone in the early luteal phase (days 4-5) appears to be a key determinant of fertility, probably because it alters the secretory activity of the reproductive tract, thus influencing embryonic growth and interferon-tau production. A period of negative energy balance after calving can reduce fertility even though metabolic parameters have apparently improved at the time of service.
The reproductive physiology of postpartum cows is different from that of heifers because of the combined effects of the past pregnancy and lactation. Neither lactation nor pregnancy has a major effect on postpartum fertility when calving is free from disease and lactation is moderate. Postpartum beef cows in good body condition have conception rates nearly equivalent to those of virgin heifers once their uteri are involuted and they initiate ovarian cycles. However, cows will experience infertility when nutrient requirements for maintenance and lactation exceed nutrient intake (postpartum beef cows) or when nutrients are specifically partitioned toward lactation (postpartum dairy cows). The subsequent loss of body fat that occurs in either case has effects on a variety of reproductive processes and reproduction, becomes less efficient. The mechanisms that lead to abnormal reproduction in nutritionally compromised postpartum cattle have been investigated intensively. Much of the effort has focused on the nature of the signal (endocrine or otherwise) that controls pituitary secretion of LH and FSH, the response of the ovary to LH and FSH, and other ovarian effects that are independent of gonadotrophins. Reproductive studies in ruminants have tended toward studies of follicular development and this focus relates back to solving the problem of anoestrus. Less work has been done on the effects of nutrition on the early embryo, the health of which may be predetermined by factors affecting the oocyte within the preovulatory follicle. Few studies have examined the effect of nutrition on uterine function in postpartum cattle. Solutions to postpartum reproduction will probably arise from a variety of approaches that include traditional physiology as well as more modern genomic and proteomic technologies.
Stress influences the activity of the reproductive system at several sites. One of the most significant effects is at level of the GnRH secretory system to reduce GnRH pulsatility and thus LH pulsatility. This in turn reduces the oestradiol signal that stimulates the GnRH-LH surge in the follicular phase. Three sequential phases have been identified in the induction of the GnRH-LH surge by oestradiol: (i) activation, (ii) transmission and (iii) surge secretion. There is evidence that administration of endotoxin prevents activation but not transmission, hypoglycaemia blocks both activation and transmission, whereas truck transport is effective during the late, but not early, transmission phase. Opioids mediate the suppressive effects of hypoglycaemia on both LH pulsatility and the delayed onset of the LH surge in ewes. The exact neurocircuitry used in sheep is yet to be identified but many of the connections that are proposed as important in rats are present in sheep. Corticotrophin-releasing hormone (CRH) neurones in the paraventricular nucleus that project axons to the median eminence probably do not directly inhibit GnRH, but either afferent or parallel central pathways are involved. New members of the CRH peptide and receptor families have been identified, but roles in the control of reproduction have yet to be determined.
Buffalo are of high economic importance for farmers in several developing countries but reproductive performance is poor. A large proportion of heifers attain puberty at 3-5 years of age. A good quality diet supplemented with extra nutrients reduces the age of puberty, whereas the effects of administration of exogenous GnRH or equine chorionic gonadotrophin (eCG) are equivocal. The incidence of anoestrus in buffalo ranges from 20 to 80% depending on season. Most buffalo cease ovarian cyclicity during hot summers probably due to the combined effects of nutrition, environment and management. Keeping buffalo cool by wallowing, water sprinklers or shade improves fertility. Supplementary feeding with Urea Molasses Multi-nutrient Blocks (UMMB) for 60 days before calving enhances the early onset of postpartum oestrus. Regular UMMB supplementation also improves pregnancy rates in anoestrous non-pregnant buffalo. Prepartum vaginal prolapse is hereditary and eradication can be achieved by genetic selective breeding programmes. Treatment with calcium, phosphorus and progesterone gives only transient relief to clinical cases. Uterine torsion is the most common cause of dystocia (70%). Deployment of Sharma's detorsion method and anti-stress measures increase survival rates in cases presented within 36 h. In conclusion, greater understanding about the effects of better year-round nutrition, improved management and markers for logical breeding programmes are essential to curtail the incidence of the reproductive disorders that reduce buffalo fertility.
The physiological mechanisms controlling ovulation rate in mammals involve a complex exchange of endocrine signals between the pituitary gland and the ovary, and a localized exchange of intraovarian hormones between the oocyte and its adjacent somatic cells. The discoveries in sheep of mutations in bone morphogenetic protein 15 (BMP15) and bone morphogenetic protein receptor type IB (BMPR-IB) together with recent findings on the physiological effects of growth differentiation factor 9 (GDF9) and BMP15 on follicular development and ovulation rate highlight some important differences in the way in which the oocyte may function in mammals with different ovulation rate phenotypes. In sheep, BMP15 and GDF9 have each been shown to be essential for the early and later stages of follicular development. In addition, ovulation rate is sensitive to changes in the dose of either of these two oocyte-derived growth factors. These findings are in contrast to those reported for mice in which GDF9, but not BMP15, is essential for follicular development. The evidence to date is consistent with the hypothesis that the oocyte plays a central role in regulating key events in the process of follicular development and hence, is important in determining ovulation rate. Moreover, it appears that the mechanisms that the oocyte uses to control these processes differ between species with low and high ovulation rate phenotypes.
In nuclear transfer reconstructed embryos, the co-ordination of donor nuclear and recipient cytoplasmic cell cycle phases is essential to maintain ploidy and prevent DNA damage. However, the stage of the cell cycle at the time of reconstruction and the method of reconstruction may also have a significant impact on the subsequent development of the embryo and fetus through a number of other mechanisms. This paper reviews some of the information currently available and proposes that consideration of the cell cycle may lead to improvement of methods for embryo reconstruction.
Animals adjust the time of year that they reproduce through their ability to perceive and respond to critical aspects of their environment, such as photoperiod, nutrition or the socio-sexual milieu, and their genotype determines the degree of response to each stimulus. Ultimately, information from environmental cues filters through to the GnRH neurones in the brain which are the primary regulator of fertility. Each of these cues has been studied in isolation and the mechanisms by which they affect GnRH secretion are now better, if not fully, understood. In the field, the brain centres that control GnRH must integrate information from all cues at any given time before 'formulating a reproductive decision'. In this review, the effect of this integration is illustrated by showing how the acute GnRH response to a nutritional signal can be modulated by genotype, photoperiod and social cues, to the point of being completely blocked under some circumstances. Candidate pathways that may mediate these modulatory effects at both the whole body and brain have been proposed, although none of these pathways are confirmed and some have not yet been studied. As a guide for further research, we propose a working model that integrates the inputs and explains the interactions between them.
Exposure of the sheep fetus to testosterone from day 30 to day 90 of a 147 day gestation causes the neurones that control GnRH secretion, the GnRH neuronal network, to become organized in a sex-specific manner. After androgen exposure in utero, GnRH neurones are activated in a sexually differentiated pattern by gonadal steroid hormones. Specifically, follicular phase concentrations of oestrogen trigger a GnRH 'surge' in ewes, but not in rams or females treated with androgen during fetal life. Furthermore, progesterone is a less potent inhibitor of GnRH release in rams or females treated with androgen during fetal life. The reasons for the sexual differentiation of these steroid feedback mechanisms probably reside in a dimorphism in steroid-sensitive neural inputs to GnRH neurones. The density of neurones containing oestrogen receptor alpha is sexually differentiated in areas of the ovine brain that are known to be involved in the steroidal regulation of GnRH. Furthermore, neurones in these regions are activated in a gender-specific pattern. A determination of the neural phenotype of these steroid-sensitive cells will form a basis for understanding the mechanisms by which the GnRH neuronal network is organized and activated in a sexually differentiated manner.
Intra-uterine growth retardation (IUGR), caused by maternal undernutrition or placental insufficiency, is usually associated with disproportionately large reductions in the growth of some fetal organs and tissues (thymus, liver, spleen, thyroid) and impaired cellular development of other tissues (small intestine, secondary wool follicles, skeletal muscle). Growth of other tissues, most notably brain, is relatively unimpaired. In our restudy of postnatal consequences of IUGR in the offspring of prolific ewes, growth-retarded newborn lambs tended to be hypoglycaemic and showed sluggish postnatal engagement of the growth hormone (GH)-insulin-like growth factor (IGF) system. When artificially reared in an optimum environment, low birth weight lambs grew at rates similar to those of normal lambs. However, low birth weight lambs were fatter at any given weight, apparently related to their high energy intakes, especially soon after birth, had low maintenance energy requirements, and limited capacity for bone and muscle growth. These growth characteristics were accompanied by higher plasma concentrations of GH and leptin, and lower concentrations of insulin-like growth factor I (IGF-I) during the first 2 weeks of postnatal life, and higher concentrations of insulin during subsequent growth up to 20 kg body weight. Emerging evidence indicates that in sheep, as in rodents, fetal programming of postnatal cardiovascular and metabolic dysfunctions is associated with IUGR and may be mediated partly by overexposure of the fetus to cortisol. Similar postnatal responses can be elicited by maternal undernutrition or cortisol treatment in early to mid-pregnancy without changing the growth of the fetus or placenta.
Intrauterine growth restriction (IUGR) is a significant health issue that not only affects infant mortality and morbidity, but may also predispose individuals to coronary heart disease, diabetes, hypertension and stroke as adults. The majority of IUGR pregnancies in humans are characterized by asymmetric fetal growth, resulting from inadequate nutrient transfer to the fetus. Furthermore, most of these pregnancies involve functional placental insufficiency, and may also show altered umbilical velocimetry. As the severity of IUGR increases, the fetus becomes increasingly hypoxic, hypoglycaemic and acidotic. In addition, placental transfer or utilization of some amino acids is known to be altered in IUGR pregnancies. Although a great deal has been learned from clinical studies of human IUGR, appropriate animal models are required to define completely the mechanisms involved in the development of IUGR. The pregnant sheep is a long-standing model for placental-fetal interactions, and fetal growth restriction can be induced in pregnant sheep by maternal nutrient restriction, maternal nutrient excess, administration of glucocorticoid, utero-placental embolization, carunclectomy and maternal hyperthermia. Although all of these sheep models are capable of inducing fetal growth restriction, the degree of restriction is variable. This review compares these sheep models of IUGR with the characteristics of human IUGR.
