Asymmetric expression of proteins in the granules of the placentomal Binucleate cells in Giraffa camelopardalis†.

Mature granulated trophoblast binucleate cells (BNC) have been found in all ruminant placentas examined histologically so far. BNC are normally fairly evenly distributed throughout the fetal villus and all their granules contain a similar variety of hormones and pregnancy associated glycoproteins (PAGs). Only the Giraffe is reported to show a different BNC protein expression, this paper is designed to investigate that. Gold labelled Lectin histochemistry and protein immunocytochemistry were used on deplasticised 1 µm sections of a wide variety of ruminant placentomes with a wide range of antibodies and lectins. In the Giraffe placentomes, even though the lectin histochemistry shows an even distribution of BNC throughout the trophoblast of the placental villi, the protein expression in the BNC granules is limited to the BNC either in the apex or the base of the villi. Placental lactogens and Prolactin (PRL) are present only in basally situated BNC: PAGs only in the apical BNC. PRL is only found in the Giraffe BNC which react with many fewer of the wide range of antibodies used here to investigate the uniformity of protein expression in ruminant BNC. The possible relevance of these differences to ruminant function and evolution is considered to provide a further example of the versatility of the BNC system.

Placentation in Equids.

This chapter focuses on the early stages of placental development in horses and their relatives in the genus Equus and highlights unique features of equid reproductive biology. The equine placenta is classified as a noninvasive, epitheliochorial type. However, equids have evolved a minor component of invasive trophoblast, the chorionic girdle and endometrial cups, which links the equine placenta with the highly invasive hemochorial placentae of rodents and, particularly, with the primate placenta. Two types of fetus-to-mother signaling in equine pregnancy are mediated by the invasive equine trophoblast cells. First, endocrinological signaling mediated by equine chorionic gonadotrophin (eCG) drives maternal progesterone production to support the equine conceptus between days 40 and 100 of gestation. Only in primates and equids does the placenta produce a gonadotrophin, but the evolutionary paths taken by these two groups of mammals to produce this placental signal were very different. Second, florid expression of paternal major histocompatibility complex (MHC) class I molecules by invading chorionic girdle cells stimulates strong maternal anti-fetal antibody responses that may play a role in the development of immunological tolerance that protects the conceptus from destruction by the maternal immune system. In humans, invasive extravillous trophoblasts also express MHC class I molecules, but the loci involved, and their likely function, are different from those of the horse. Comparison of the cellular and molecular events in these disparate species provides outstanding examples of convergent evolution and co-option in mammalian pregnancy and highlights how studies of the equine placenta have produced new insights into reproductive strategies.

Placentation in the African Elephant (Loxodonta africana).

The female elephant shows a 3-week "follicular phase" to commence her 16-week estrous cycle at the end of which a second surge in pituitary luteinizing hormone (LH) release matures and ovulates an ovarian follicle in association with estrous behavior and mating, whereas the first LH surge at the start of the follicular phase causes luteinization of 3-5 partially developed follicles. The prolonged pregnancy of 22 months is supported by a zonary endotheliochorial placenta which secretes placental lactogen (ePL) from around 40 days of gestation in association with replacement of the lumenal epithelium of the endometrium by trophoblast and the development of large corpora lutea (CLs) in the maternal ovaries from the previously formed luteinized follicles in response to the first LH peak early in the follicular phase. The zonary placenta develops above, rather than within, the endometrium. The elephant placenta secretes neither estrogens nor progestagens throughout gestation, as pregnancy maintenance relies on 5α-dihyroprogesterone and other 5α reduced progestagens secreted by secondary CLs stimulated by ePL and the stromal tissue of the fetal gonads, which become extremely enlarged during the second half of the 22-month pregnancy. In female fetuses, this ovarian enlargement includes the development and subsequent regression of multiple primary and secondary follicles with a consequent substantial decline in primary follicle numbers at birth. During the next 8-9 years of pre-pubertal life, however, oocyte and primary follicle numbers recover to levels near those found in late gestation, which may be evidence of postnatal oogenesis occurring in the elephant.

Placentation in the plains zebra (Equus quagga).

The placenta and fetal gonads of 12 pregnant plains zebra (Equus quagga), estimated to be between 81 and 239 days of gestation, were examined. The diffuse, microcotyledonary zebra placenta appeared, developmentally, to be 3-4 weeks behind its counterpart in horse pregnancy and this, together with the presence of small and long-lived endometrial cups, low levels of zebra chorionic gonadotrophin in maternal serum and few accessory corpora lutea in the maternal ovaries during the first half of gestation, made zebra pregnancy more similar to donkey than horse pregnancy. Zebra fetal gonads enlarged after 80 days of gestation and their interstitial cells stained positively for 3ß hydroxysteroid dehydrogenase and 17,20 lyase steroid enzymes while the trophoblast stained for aromatase. This confirmed that zebra fetal gonads, like those of the horse and donkey, can synthesise C19 androgens, which can then be aromatised by the placenta to C18 oestrogens. It is remarkable that such unusual feto-placental mechanisms of production of gonadotrophic and steroid hormones has persisted unchanged within the genus Equus despite the many physical adaptations and the considerable loss of chromosomes that have occurred during the evolution of its member species.

The binucleate cell of okapi and giraffe placenta shows distinctive glycosylation compared with other ruminants: a lectin histochemical study.

The placenta of ruminants contains characteristic binucleate cells (BNC) with a highly conserved glycan structure which evolved early in Ruminant phylogenesis. Giraffe and Okapi placentae also contain these cells and it is not known whether they have a similar glycan array. We have used lectin histochemistry to examine the glycosylation of these cells in these species and compare them with bovine BNC which have a typical ruminant glycan composition. Two placentae, mid and near term, from Giraffe (Giraffa camelopardalis) and two term placenta of Okapi (Okapia johnstoni) were embedded in resin and stained with a panel of 23 lectins and compared with near-term bovine (Bos taurus) placenta. Significant differences were found in the glycans of Giraffe and Okapi BNC compared with those from the bovine, with little or no expression of terminal αN-acetylgalactosamine bound by Dolichos biflorus and Vicia villosa agglutinins which instead bound to placental blood vessels. Higher levels of N-acetylglucosamine bound by Lycopersicon esculentum and Phytolacca americana agglutinins were also apparent. Some differences between Okapi and Giraffe were evident. Most N-linked glycans were similarly expressed in all three species as were fucosyl residues. Interplacentomal areas in Giraffe and Bovine showed differences from the placentomal cells though no intercotyledonary BNC were apparent in Okapi. In conclusion, Giraffidae BNC developed different glycan biosynthetic pathways following their split from the Bovidae with further differences evolving as Okapi and Giraffe diverged from each other, affecting both inter and placentomal BNC which may have different functions during development.

Ovarian and placental morphology and endocrine functions in the pregnant giraffe (Giraffa camelopardalis).

Gross, histological and immunocytochemical examinations carried out on maternal and fetal reproductive tissues from two pregnant giraffes at an estimated 8 and 13.5 months of gestation (term=15 months) revealed a typically ruminant macrocotyledonary placenta with binucleate trophoblast cells scattered sparsely in the placentome where they stained intensely with a prolactin antiserum. Binucleate cells were present in greater numbers in the intercotyledonary allantochorion where they did not stain for prolactin whereas the uninucleate trophoblast still did. A single large corpus luteum of pregnancy and several small luteinised follicles were present in the maternal ovaries while the fetal ovaries at 13.5 months gestation showed an assortment of enlarging antral follicles and partially and completely lutenised follicles, the granulosa and luteal cells of which stained positively for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17,20 lyase, prolactin, progesterone receptor and androgen receptor, but negatively for aromatase. The uninucleate trophoblast of the placentome and intercotyledonary allantochorion, the epithelium of the maternal endometrial glands, the seminiferous epithelium in the fetal testis at 8 months of gestation and the zonae fasciculata and reticularis of the fetal adrenal at 13.5 months also stained positively for 3ß-HSD and negatively for aromatase. Endocrinologically, it appears that the giraffe placenta is more similar to that of the sheep than the cow with a placental lactogen as the likely driver of the considerable degree of luteinisation seen in both the maternal and the fetal ovaries.

Persistence of an immunoreactive MUC1 protein at the feto-maternal interface throughout pregnancy in the mare.

A polyclonal human mucin-1 (MUC1) antibody was used to stain immunohistochemically for the presence of MUC1 on the endometrium and fetal membranes in mares between 20 and 309 days of gestation. Western blot analysis demonstrated the presence of a protein equivalent in size to a human MUC1 isoform, MUC1/Y, in equine endometrium, allantochorion and amnion. At all stages of gestation examined immunoreactivity to the MUC1 antibody was detected on the apical surface of the lumenal epithelium of the endometrium and the epithelium lining the mouths and apical regions of the endometrial glands. Furthermore, it persisted unchanged on the surface of the lumenal epithelium lying beneath the highly-invasive chorionic girdle component of the trophoblast before, during and after development of the endometrial cups. The MUC1 immunoreactive protein was also present on the trophoblast and other components of the fetal membranes during the post-fixation, pre-attachment period of gestation (20-40 days) and it persisted on the apical surface of the non-invasive trophoblast of the allantochorion before, during and after attachment, microvillous interdigitation and development of the microcotyledonary epitheliochorial placenta. Hence, the delayed placentation response in mares appears to occur independently of the persistence of an immunoreactive MUC1 protein at the feto-maternal interface.

The progression of small-follicle reserves in the ovaries of wild African elephants (Loxodonta africana) from puberty to reproductive senescence.

This study aimed to determine whether the follicle reserve in the ovary of the African elephant declines progressively after puberty and whether its depletion constrains the fertility of older females. Elephant ovaries were fixed in 4% neutral buffered formalin and small-follicle counts made using stereological protocols. Excepting a slight rise in small-follicle numbers between 16 and 25 years of age, there was a trend for follicle numbers to fall from puberty to 70 years. Reproductive status did not impact significantly on small-follicle numbers (P=0.31). The number of early primary follicles, initially higher in number than true primary follicles, fell from post-puberty to nil at 45 years of age. Six of the seven oldest animals in the study showed signs of recent ovarian activity in the form of antral follicles, corpora lutea or large corpora nigra. The four oldest elephants (mean age 69 years) had a median small-follicle count of 11,113. In summary, it appears that the elephant ovary is capable of supplying oocytes for ovulation right up to the time of death at the age of maximum life expectancy, although the follicle reserve becomes depleted in some older elephants.

Comparative reproduction of the female horse, elephant and rhinoceros: implications for advancing Assisted Reproductive Technologies (ART).

Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant and rhinoceroses share some reproductive features with equids, we postulate that procedures such as OPU, ICSI, in vitro fertilisation (IVF) and embryo transfer (ET), which are well established in the horse, may represent a basis to develop protocols for endangered pachyderms. In this review, we summarize current knowledge on reproductive physiology relevant to ART. We discuss the current state of ART in all three families and the requirements for the successful implementation of OPU, ICSI, IVF and ET in these species.

Skeletal development in the African elephant and ossification timing in placental mammals.

We provide here unique data on elephant skeletal ontogeny. We focus on the sequence of cranial and post-cranial ossification events during growth in the African elephant (Loxodonta africana). Previous analyses on ossification sequences in mammals have focused on monotremes, marsupials, boreoeutherian and xenarthran placentals. Here, we add data on ossification sequences in an afrotherian. We use two different methods to quantify sequence heterochrony: the sequence method and event-paring/Parsimov. Compared with other placentals, elephants show late ossifications of the basicranium, manual and pedal phalanges, and early ossifications of the ischium and metacarpals. Moreover, ossification in elephants starts very early and progresses rapidly. Specifically, the elephant exhibits the same percentage of bones showing an ossification centre at the end of the first third of its gestation period as the mouse and hamster have close to birth. Elephants show a number of features of their ossification patterns that differ from those of other placental mammals. The pattern of the initiation of the ossification evident in the African elephant underscores a possible correlation between the timing of ossification onset and gestation time throughout mammals.

Luteal maintenance of pregnancy in the African elephant (Loxodonta africana).

The ovaries of eight African elephant foetuses and their mothers between 2 and 22 months of gestation, and those of two cycling and two lactating elephants, were examined grossly, histologically and immunocytochemically, with emphasis on the development and regression of accessory corpora lutea (CL) of pregnancy and the steroidogenic capacities of the accessory CL and the foetal ovaries. The results supported recent findings that the accessory CL form as a result of luteinisation, with and without ovulation, of medium-sized follicles during the 3-week inter-luteal period of the oestrous cycle. They enlarge significantly and become steroidogenically active around 5 weeks of gestation, probably in response to the placental lactogen which is secreted by the implanting trophoblast of the conceptus. The large luteal cells stained strongly for 3ß hydroxysteroid dehydrogenase (3ßHSD) activity throughout the 22-month gestation period although they showed vacuolation and other degenerative changes in the final months of gestation coincident with hypertrophy and hyperplasia of 3ßHSD-positive interstitial cells in the foetal gonads. It is proposed that the progestagens secreted by the enlarged gonads of the elephant foetus may function both to assist the maternal ovaries in supporting the pregnancy state and to induce torpor and intrauterine immobility of the rapidly growing foetus.

Development of the germinal ridge and ovary in the African elephant (Loxodonta africana).

The follicular reserve and its ontogeny in the elephant are of interest because elephants have the longest reproductive life of all land-based mammals. They also have the longest recorded pregnancy, which allows a protracted view of the series of significant events involved in the development of the embryonic and fetal gonads. The large elephant population of Zimbabwe provided the opportunity to collect conceptuses from elephants culled for management reasons and hunted professionally. Five embryos aged 76-96 days and the ovaries of four fetuses aged 4.8-11.2 months were fixed in 4% buffered formalin and studied by conventional histological sectioning and a stereological protocol to calculate the follicle reserve of each fetus. These observations enabled the conclusion that the migration of primordial germ cells into the indifferent gonad terminates at around 76 days of gestation while entry of oogonia into meiosis along with first follicle formation starts at around 5 months. Peak numbers of follicles are present by mid-gestation towards the end of the 6-month mitotic-meiotic transition period. It appears that the cortex of the elephant fetal ovary at mid-gestation (11 months) has already reached a developmental stage exhibited by the ovaries of many other mammals at full term.

Intravenous immunoglobulin therapy in the treatment of BK viremia and nephropathy in pediatric renal transplant recipients.

Polyoma BKVN is a significant cause of allograft dysfunction and loss in renal transplant recipients. Reduction in immunosuppression is accepted as first-line therapy to decrease viral load and prevent allograft injury and dysfunction. We report our experience with persistent BKV after reduction in immunosuppression followed by successful clearance of BKV in three pediatric renal transplant recipients and histological resolution of BKVN in a fourth patient following therapy with IVIG. Once BKV was detected, immunosuppression was reduced and BKV was monitored until clearance was achieved. All four patients were given IVIG in a dose of 2 g/kg. Allograft function remained stable in all patients. Early routine screening for BKV allows early intervention to prevent the development of BKVN and permanent allograft damage. While immunosuppression reduction is a logical first-line therapy, second-line therapy is not well established. IVIG seems to be an effective treatment for persistent BKV after reduction in immunosuppression and for BKVN and can therefore be considered as a therapeutic option in these patients.

Observations on the glycosylation of the term placenta of the Indo-Pacific Bottlenose Dolphin (Tursiops aduncus): A lectin histochemical study.

INTRODUCTION: Little is known about the glycosylation of placental villi and areolae of cetaceans. Term tissue from the delivered placenta of an Indo-Pacific Bottlenose Dolphin (Tursiops aduncus) was examined using lectin histochemistry to compare trophoblast glycosylation in these two locations. METHODS: Placental blocks fixed in 10% formalin were resin-embedded before semithin sections were stained with 24 biotinylated lectins and an avidin-biotin revealing system. RESULTS: Areolar trophoblast was composed of large, bulbous cells packed with numerous granules compared to the smaller, cuboidal cells clothing the chorionic villi, which had a sparser, mainly subapical granule population. Both were richly glycosylated; generally areolar cells were more heavily stained apart from poor binding to some N-acetylgalactosamine and N-acetylglucosamine termini. Most striking was the distribution of α1,2-linked fucosyl residues, weakly expressed in villous trophoblast but intensely stained in some areolar cells, also terminal sialic acids. Some lectins bound in a variable fashion. Staining of terminal α-d-mannose, which locates mainly to lysosomes, was heavy in areolar cells compared to scattered irregular foci in villous cells. DISCUSSION AND CONCLUSION: The many intracellular inclusions reflect ongoing lysosomal breakdown of histotroph in areolar cells which often show heterogeneous glycosylation staining unlike the uniformly stained villous cells, possibly reflecting partial breakdown of ingested sialoglycoprotein, cell turnover or regional variation in uptake of histotroph. Our results indicate that Dolphin areolae are functionally distinct from villous trophoblast, performing absorptive and phagocytic functions similar to other Artiodactyla.

The effect of embryo reduction and transfer on luteostasis in the mare.

This study investigated the effects of embryo reduction and transfer of Day 11 embryos, with or without subsequent reduction, on luteostasis in the mare. In Experiment 1, reduction of embryos at Days 10 (n = 15), 11 (n = 47), 12 (n = 36), 13 (n = 27), 14 (n = 5) and 16 (n = 2) of pregnancy resulted in luteostasis in 13%, 47%, 78%, 89%, 80% and 100% mares. Mares undergoing > 1 embryo reduction showed consistency in when luteostasis occurred. In Experiment 2, transfer of Day 11 embryos to recipient mares 10 (n = 9), 11 (n = 8), 12 (n = 9) and 13 (n = 8) days post ovulation resulted in luteostasis in 78%, 87.5%, 78% and 37.5% of mares. Only 22%, 37%, 0% and 12%, respectively, of these mares remained pregnant. In the Day 10, 11 and 12 recipients luteostasis occurred on at least one occasion when an embryo was detected at 24 h but not at 48 h post transfer. In the Day 12 recipients luteostasis occurred on three occasions (3/9;33%) when the transferred embryo was not detected at 24 h. In Experiment 3 reduction of a Day 11 embryo 24 h after transfer to a Day 10 (n = 4), 11 (n = 6), 12 (n = 6) or 13 (n = 6) recipient resulted in luteostasis in 100%, 83%, 100%, and 83% of mares. All five Day 11 recipients that had an embryo reduced 12 h post transfer became luteostatic. These results suggest there is plasticity overall, but individual rigidity, in the timing of maternal recognition of pregnancy. Furthermore, an intact embryo need only be present in the uterus for 12 h to cause luteostasis.

Functions of ectopically transplanted invasive horse trophoblast.

The invasive and fully antigenic trophoblast of the chorionic girdle portion of the equine fetal membranes has the capacity to survive and differentiate after transplantation to ectopic sites. The objectives of this study were to determine i) the survival time of ectopically transplanted allogeneic trophoblast cells in non-pregnant recipient mares, ii) whether equine chorionic gonadotropin (eCG) can be delivered systemically by transplanted chorionic girdle cells, and iii) whether eCG delivered by the transplanted cells is biologically active and can suppress behavioral signs associated with estrus. Ectopically transplanted chorionic girdle survived for up to 105 days with a mean lifespan of 75 days (95% confidence interval 55-94) and secreted sufficient eCG for the hormone to be measurable in the recipients' circulation. Immunohistochemical labeling of serial biopsies of the transplant sites and measurement of eCG profiles demonstrated that graft survival was similar to the lifespan of equine endometrial cups in normal horse pregnancy. The eCG secreted by the transplanted cells induced corpora lutea formation and sustained systemic progesterone levels in the recipient mares, effects that are also observed during pregnancy. This in turn caused suppression of estrus behavior in the recipients for up to 3 months. Thus, ectopically transplanted equine trophoblast provides an unusual example of sustained viability and function of an immunogenic transplant in a recipient with an intact immune system. This model highlights the importance of innate immunoregulatory capabilities of invasive trophoblast cells and describes a new method to deliver sustained circulating concentrations of eCG in non-pregnant mares.

Intrauterine administration of plant oils inhibits luteolysis in the mare.

REASONS FOR PERFORMING THE STUDY: The maternal recognition of pregnancy (MRP) signal in the mare has not been determined, although oestrogens have been proposed as a potential candidate. OBJECTIVES: To determine effects of intrauterine administration of oestrogen and various oils on cyclic luteolysis in the mare. HYPOTHESIS: Intrauterine oestradiol or fatty acids may suppress luteolysis in the cycling mare when administered during late dioestrus. METHODS: A single 1 ml dose of slow-release oestradiol (10 mg/ml) in fractionated coconut oil was infused into the uterine lumen of cycling mares on Days 6, 8, 10, 12 or 14 post ovulation (n=12 in each group). Four further groups, each of 12 mares, received an intrauterine infusion of either 1 ml of fractionated coconut oil, peanut oil, mineral oil or a slow-release preparation of oestradiol (10 mg/ml) in mineral oil on Day 10 post ovulation. Serial blood samples were assayed for progesterone concentrations to monitor luteal function. RESULTS: Intrauterine administration of oestradiol in fractionated coconut oil showed peak efficacy at Day 10 when luteolysis was delayed in 11/12 (92%) mares. The ability of the treatment to delay luteolysis was not significantly different when administered on Days 8 (9/12; 75%), 12 (10/12; 83%) or 14 (6/12; 50%) of dioestrus, but declined significantly when given on Day 6 (3/12; 25%). Oestradiol was not needed to initiate luteostasis since fractionated coconut oil alone or peanut oil administered at Day 10 induced the same high rate of luteal persistence (11/12; 92% for both oils). In contrast, mineral oil did not prolong luteal lifespan, either when administered alone (2/12; 17%) or combined with oestradiol (3/12; 25%). CONCLUSION: These results do not unequivocally rule out a possible involvement of embryonic oestrogens in MRP in the mare but suggest it is unlikely. The results demonstrate that plant oils can postpone luteolysis, suggesting they may modulate synthesis or release of prostaglandins from the mare's endometrium. POTENTIAL RELEVANCE: Administration of fractionated coconut or peanut oil on Day 10 post ovulation provides an effective and practical method of prolonging luteal function ('pseudopregnancy') thereby suppressing unwanted oestrous behaviour. Further studies to elucidate the mechanism by which this is achieved may increase understanding of both luteostasis and MRP signal in the mare.

Successful transfer of day 10 horse embryos: influence of donor-recipient asynchrony on embryo development.

A total of 78 day 10 horse embryos were transferred non-surgically to recipient mares that had ovulated 9, 7, 6, 5, 4, 3, 2 or 1 day after (negative asynchrony), on the same day (synchronous), or 2 or 4 days before (positive asynchrony) the donor (n=6 or 8 mares per group). Pregnancy rates between 100% (6/6) and 63% (5/8) were seen in recipient mares that were between +2 and -6 days asynchronous. Embryo survival to the heartbeat stage declined in recipients that were -7 days asynchronous and no embryos survived in recipients that were -9 days asynchronous. Irrespective of uterine asynchrony, cessation of embryo mobility and fixation at the base of a uterine horn occurred when the conceptus was approximately 17 days old. Conceptus growth and development was slowed when embryos were placed in negatively asynchronous uteri. At the greatest degree of negative asynchrony at which embryos survived to the heartbeat stage, i.e. -7 and -6 days, development of the embryo proper and allantois was retarded. Luteostasis was achieved in recipient mares when day 10 embryos were transferred to recipient mares at any stage of asynchrony between -9 and +2 days with respect to the donor. These results indicate that in the horse, there is a wide window for establishment of pregnancy following embryo transfer to asynchronous recipients. Although progesterone priming of the uterus to a stage equivalent to that of the transferred embryo does not appear to be a prerequisite for embryo survival, it does nonetheless influence embryonic development.

Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon.

REASONS FOR PERFORMING STUDY: Injury to the superficial digital flexor tendon (SDFT) is common in racing and sport horses and poor tendon regeneration leads to high reinjury rates. Autologous mesenchymal stromal cells (MSCs) are being used clinically to improve tendon regeneration but they have some practical limitations. Embryonic stem cells (ESCs) may overcome these limitations but their fate following injection into the damaged SDFT is unknown. OBJECTIVE: To inject MSCs and ESCs into distinct areas of damage in the SDFT and monitor their survival over a 3 month period. METHODS: MSCs and ESCs expressing different reporter genes were injected into separate sites of mechanically induced damage in SDFTs. Cell survival and distribution were examined post mortem after 10, 30, 60 and 90 days and host immune responses determined. RESULTS: Neither MSCs nor ESCs produced signs of cell-mediated immune response or tumour formation. ESC survival was high and numbers were maintained at a constant level over 90 days. ESCs were present at all sites of damage. In contrast, MSCs showed <5% survival at 10 days and numbers declined over the course of the experiment. MSCs were detected only at the site into which they were injected. CONCLUSIONS: ESCs survived in greater numbers than MSCs in the damaged tendon and did not induce an immune response, or form tumours at the injection sites in the 90 day time period studied. ESCs also demonstrated an ability to migrate to other areas of damage within the same tendon, whereas MSCs did not. POTENTIAL RELEVANCE: ESCs can be used allogeneically, therefore providing a possible 'off the shelf' source of cells for therapeutic use which overcomes the practical limitations of autologous MSCs. Furthermore, MSCs and ESCs have different survival rates and migration patterns in the damaged tendon, suggesting that they may produce different functional effects. This may have clinical relevance to treating tendon injuries in the horse.

Managed wildlife breeding-an undervalued conservation tool?

Knowledge of and the technologies and resources applied to the ex situ care for wildlife have improved greatly in recent years. This has resulted in numerous successes bringing back populations from the brink of extinction by the reintroduction or restoration of animals from conservation breeding programmes. Controlled breeding of wildlife by humans is discussed controversially in society and in scientific circles and it faces a number of significant challenges. When natural breeding fails, Assisted Reproduction Technologies (ART) have been postulated to increase reproductive output and maintain genetic diversity. Furthermore, technical advances have improved the potential for successful collection and cryopreservation of gametes and embryos in many wildlife species. With the aim of creating a better understanding of why ex situ and in situ conservation of threatened species must complement each other, and under which circumstances ART provide additional tools in the rescue of a threatened population, we elucidate the current situation here by using as examples three different megavertebrate families: elephantidae, rhinoceridae and giraffidae. These mammal families consist of charismatic species, and most of their members are currently facing dramatic declines in population numbers. On the basis of these and other examples, we highlight the importance of captive zoo and other managed wildlife populations for species survival in a human dominated world. Without the possibility to study reproductive physiology in trained or habituated captive individuals, major advances made in wildlife ART during the past 20 years would not have been possible. This paper reviews the benefits and future challenges of large mammal conservation breeding and examines the role of assisted reproduction in such efforts.