Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 14 Workshop Abstracts hippelaphus) and identification of genes for earlier calving traits. Recognition is also given to environmental and genetic effects on gestation length of red deer. Perinatal calf mortality, which averages 8-12% of calves born, is arguably the greatest source of reproductive wastage. It is mainly attributable to calf abandonment and dystocia due to inappropriate calving environments that lead to adverse social interactions between parturient hinds. Risk factors include lack of low vegetative cover for parturition and calf concealment, high stocking intensities and high calving synchrony, leading to inter-hind conflicts and disturbance around parturition. A recent trend towards use of extensive high-country rangelands for breeding units has been associated with improved calf survival due to the provision of better calving environments. Pubertal failure in 16-month-old hinds has been seen as a major source of reproductive wastage over the last decade. While various casual factors have been proposed, only the genetic introgression of the larger Wapiti subspecies (e.g. nelsoni, manitobensis, roosevelti) has been definitively shown to influence female puberty. Recent studies have noted that crossbred females (>30% wapiti parentage) often fail to attain sufficient liveweight by 16 months of age to enter puberty in their second year. This mainly reflects inappropriate nutritional and health management for the larger crossbred genotypes but may also be indicative of later maturation characteristics of the Wapiti subspecies. WS08-2 Development of large scale commercial AI for genetic improvement in farmed red deer in New Zealand McMillan, W William McMillan Consultancy Limited, New Zealand Highly invasive and labour intensive methods of artificially inseminating red deer have been replaced by trans-cervical methods using experienced teams of experienced bovine AB technicians on a large scale. Semen can be harvested from stags of all ages, including yearling stags from 4-6 weeks before the start of the female breeding season. Fresh semen can be routinely used for 3 days after collection. A deer specific intra-vaginal progesterone releasing device has been developed to facilitate fixed time AI. Mean pregnancy rates of 60- 75% are achieved, although considerable herd to herd variation exists. Pregnancy rates do not appear to be affected by the interval from the end of synchrony treatment to insemination. Yearling hinds (15-16 months of age) can be induced to breed at least a month earlier than usual with acceptable pregnancy rates. These technologies are being combined to increase the rate of genetic gain in weight-for-age in red deer in New Zealand as well as managing disease by eliminating the need to bring in outside animals. WS08-3 In vitro production of cervid embryos: results, limits and future perspectives regarding endangered species conservation Locatelli, Y MNHN, France Despite conservation actions, more than five thousand vertebrates species are threatened with extinction according to 2007 IUCN evaluation. Conservation of animal species relies when possible on conservation programs performed in or ex situ. For mammals, ex situ programs relies on captive breeding in zoos or similar institutions and are aimed in maintaining genetic variability of species. Within cervids, about 40 subspecies are facing extinction (IUCN red list). Reproductive technologies developed for cattle (sperm freezing, artificial insemination, superovulation, in vitro production of embryos, embryo transfer…) were proposed for deer as efficient tools to increase number of breed per hind and for genetic preservation/exchange between spots of conservation. If some biotechnologies were quite easily transposed to deer species, it now clearly appears that understanding of reproductive physiology of each species is a key factor for successful transposition. Achievability of in vitro embryo production methods was investigated in sika deer and red deer as models for management of endangered deer species. After in vitro maturation of cumulus-oocytes complexes, our recent findings (Locatelli et al. 2005, 2006) indicates a 60-80 % fertilisation rate for both species when synthetic oviduct fluid (SOF) medium supplemented with 20% oestrous sheep serum was used for in vitro fertilisation. Developmental competence was assessed for both red and sika deer embryos in two different culture systems. Embryos were cultured in SOF medium in the presence or absence of ovine oviduct epithelial cells (oOEC). In contrast with classical SOF culture, high proportion of embryos reached the blastocyst stage in presence of oOEC. Theses results may indicate specific needs of deer embryo as compared with sheep, goat or cow embryos. Viability of frozen and vitrified red deer embryos produced in vitro on oOEC was confirmed by successful embryo transfer to synchronised red deer recipients. Viability of sika deer blastocysts was assessed by transferring 14 frozen/thawed embryos to 7 red deer recipients. One of the seven red deer recipients was diagnosed pregnant by ultrasonography on day 56 after sika deer embryo transfer. A healthy male sika deer fawn was born unassisted after 224 days of gestation (Locatelli et al. 2008). Presents results illustrate achievability to produce viable embryos in vitro in two deer species. Furthermore confirmation of interspecific embryo pregnancy in deer may offer new possibilities for conservation of rare deer species in future. WS08-4 The ART of red deer (Cervus elaphus) cloning Berg, D 1 *; Berg, M 1 ; Mackintosh, C 2 ; Li, C 3 ; Asher, G 4 1Reproductive Technologies 2Animal Health, 3Growth and Development, 4AgSystems, AgResearch, New Zealand In New Zealand a unique opportunity existed to use antlerogenic periosteum (AP) cells from red deer as nuclear donors for cloning by somatic cell nuclear transfer (SCNT). AP cells, found only in the deer, are stem cells that give rise to antlers in stags. They are proposed to be retained foci of embryonic tissue and their multipotent nature (Anat Embryol 204:375-388, 2001) makes them attractive for SCNT. SCNT in all species cloned so far is associated with high incidences of pregnancy failure throughout gestation (>90%), poor maternal preparation for parturition and high incidence of neonatal mortality (~67% of live offspring surviving to weaning). In cattle particularly, standard farm management practices have been adapted to deal with these issues. However, these farm practices may not be appropriate when applied to a pastoral semi-domesticated species such as deer. In preparation for red deer SCNT cloning, we adapted methods developed for cattle cloning such as non-surgical embryo transfer, intensive fortnightly pregnancy monitoring by trans-rectal and transabdominal ultrasound, from Day 35 to Day 290 of gestation, and rectal palpation to assess cervical dilation close to parturition and applied them to deer pregnancies generated by in vitro fertilisation (IVF). These were applied to two sets of SCNT (Biol Reprod 77: 384- 394, 2007): a preliminary trial using 14 recipients accustomed to intensive handling, followed by a pastoral field trial involving the non-surgical transfer of 70 single SCNT blastocysts. Gestation lengths and birth weights were compared with contemporaneous naturally mated half-siblings or AI offspring. The health of newborn calves in all treatment groups were assessed by hematological and blood chemistry analysis. Overall, the pregnancy rate at Day 35 was 39% (33/84). However, only 11/84 surrogate dams were pregnant at Day 150, but there were no further pregnancy losses beyond this. Three of these failed pregnancies required the manual extraction of mummified fetuses. All SCNT surrogates exhibited normal signs of parturition and calved without induction. The mean gestation length and birth weight of SCNT calves were not different to the controls. All SCNT calves were reared by their surrogate dams. No neonatal mortality occurred for calves born in the preliminary trial (0/3); however 3/8 calves were lost within the first week in the field trial. The remaining eight clones are now 2 and 4 years of age.
16 t h International Congress on Animal Reproduction Workshop Abstracts 15 WS08-5 The strict timing of reproductive capacity in roe buck – an ideal model system for seasonally reproducing cervidae Schoen, J 1 *; Blottner, S 2 1Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany; 2Leibniz-Institute for Zoo- and Wildlife Research, Berlin, Germany The roe deer is a typical seasonal breeder with a short sexually active period lasting from mid July to mid August. Activity of tissues which are relevant for reproduction is very strictly regulated compared to other cervids. Exogenous signals from the environment cause manifest cyclic changes in the endocrine system of the animal, which result in distinct alterations of morphological and functional parameters within all reproductive organs. In the male, these alterations concern sperm production within the testis as well as structure and function of the epididymis and accessory sex glands. Of course they also greatly concern behavioural aspects, which we will however leave aside here. Only at the time of rut, functionality of the reproductive tract is optimized so that sperm output of high quality and quantity can be ascertained. Even in phases before and after the mating period, spermatozoa are produced to a certain degree, but they are morphologically and functionally of low quality, due to suboptimal conditions in the reproductive tract. The gonadotropins show cyclical releasing patterns throughout the year with an LH peak at the beginning of spermatogenesis activation in March. This causes modulations in the differentiation status of Leydig cells followed by an increase of testosterone secretion. The endocrine variations trigger the seasonal up and down of germ cell proliferation and differentiation in the testis, which is mirrored by extreme changes in tubulus diameter and testis mass. In the epididymis remarkable alterations in the tissue composition of the duct as well as in its expression patterns are necessary to assure controlled maturation of spermatozoa during the rut. Lastly, the cyclic changes in volume and exocrine activity of the accessory glands cause a seasonally modified composite of the seminal fluid. All of this leads to enormous variations in quality and quantity of sperm cells in the ejaculate. The variability of the reproductive capacity of the roe buck is based on the combined actions and the subtle interplay of the endocrine system, germinative and somatic testicular cells, the epithelium of the epididymis and the accessory glands. This fragile interaction is necessary to facilitate the optimal timing of male reproductive fitness towards rutting season. The non domesticated roe deer is an absolutely fascinating model that enables us to study the different levels of seasonally determined regulation of reproduction with hardly any artificial influence. Workshop 09 - Cryopreservation of gametes and embryo Moderator: Sandor Cseh (Hungary) WS09-1 Basic Cryobiology of Gametes and Embryos Leibo, SP Department of Biological Sciences, University of New Orleans; Audubon Center for Research of Endangered Species, New Orleans, LA U.S.A. The ability to cryopreserve spermatozoa, oocytes and cleavage-stage embryos is essential for the efficient husbandry of all mammalian species. Live offspring of more than fifty species have been produced by artificial insemination of females or by in vitro fertilization of oocytes with cryopreserved spermatozoa. Live young of at least twenty-five species have resulted from transfer of cryopreserved embryos or oocytes. For some species, offspring have been produced from gametes or embryos that have been preserved in the frozen state for at least twenty-five years, leading to speculation that cryopreserved germplasm can probably be stored at least for millennia. Despite the diversity of species that have been successfully preserved, the basic principles from which the procedures to cryopreserve them have evolved are very similar. Gametes or embryos are suspended in a solution of a cryoprotective additive (CPA) that causes the cells to undergo partial dehydration by loss of cell water. Then, the cells are either cooled to low subzero temperatures at high rates to “capture” the cells in the dehydrated state, or the cells are first cooled rather slowly to intermediate subzero temperatures causing dehydration in response to the increasingly concentrated solution as extracellular water is removed in the form of ice. In both cases, the purpose is to prevent cell water from crystallizing within the cell. After storage, cryopreserved cells are warmed to physiological temperatures and the CPA is removed. In general, if cells have been cryopreserved by being rapidly cooled, they are warmed rapidly; if preserved by slow cooling, the cells are warmed relatively slowly. Literally, thousands of experimental reports have been published in which details of methods to cryopreserve gametes and embryos are described. Various low molecular weight compounds have been used successfully as cryoprotectants. Cooling and warming rates ranging from less than 0.5ºC/minute to more than 10,000ºC/minute have been used to cryopreserve germplasm. A wide variety of devices and containers have been used to cryopreserve cells. In general, it appears that it is not the actual cooling or warming procedure or the storage temperature that damages germplasm. Rather, most evidence indicates that the key to successful cryopreservation is the composition of the suspending medium. Consequently, efforts continue to determine the effects of such media on gametes and embryos and to seek better media for cell preservation. WS09-2 Update on Vitrification as a novel Cryopreservation Technique for Oocytes and Embryos Liebermann, J Fertility Centers of Illinois, Chicago, IL, USA The impact of cryopreservation on the growth and improved efficiency of assisted reproduction in humans is becoming increasingly appreciated, with close to a fifth of all births following in vitro fertilization and embryo transfer Worldwide arising from cryopreservation of supernumerary embryos. As culture techniques and embryo quality improve, the ratio of fresh to cryopreserved embryo babies will approach a more equal ratio, indicative of an increased reliance on the use of embryos following cryostorage. One of the most commonly discussed issues in cryobiology is whether slow-cooling or rapid-cooling protocols satisfy the fundamental cryobiological principles of reducing damage by ice crystal formation during freezing and thawing. One way to achieve this target is by attempting to establish a glassy or vitreous state, wherein molecular translational motions are arrested without structural reorganization of the liquid. Such recent studies have opened the door to a potentially highly successful and consistent alternative to overcoming the problems of slow-freezing and thawing cells, named vitrification. Although initially reported in 1985 as a successful cryopreservation approach for mouse embryos, vitrification has taken a backseat to the much more widely adopted conventional freezing technology applied to both gametes and embryos in animal and human assisted reproduction. Recent years have seen a resurgence of interest in this ultra-rapid cryopreservation technology, as the limitations of slowrate freezing have become more evident in the clinical arena. Vitrification is a simple technology, potentially faster, and inexpensive. Furthermore, vitrification has proven to be consistent and reliable when carried out properly, and it allows also more individual patient flexibility. Vitrification has so far been used for gametes and embryos (developmental days 1 to 5). However, its use has been more advantagous with chill-sensitive cells such as oocytes and blastocysts. Successful vitrification of human spermatozoa without cryoprotectants was recently described. There is very few data available concerning the vitrification of human ovarian tissue. Although some problems remain to be fully addressed with vitrification as a routine cryopreservation technique, we believe that it shows much promise as a viable alternative to conventional freezing technology. The issues with vitrification are well defined and limited in number, and to our way of thinking easily surmountable.
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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