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Conspecific and xenospecific placental lactogen levels measured in EB478 and D5102, recipients of blastomere-aggregation embryos.
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Chimeric sheep-goat pregnancies were established in 24 ewes and 29 does by transferring 251 embryos, prepared by the blastomere-aggregation technique, to 52 ewes and 61 does. Fifteen does experienced early pregnancy failure; however, term offspring were delivered by 24 ewes (17 lambs, 3 kids, 6 chimeras) and 14 does (6 lambs, 9 kids, 6 chimeras). (...
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Context 1
... serum samples collected on Day 125 of preg- nancy were available from 2 of the 7 does that had elevated levels of both oPL and cPL. The sample from D4016 con- tained 165 ng cPL/ml and 117 ng oPL/ml; the sample from D4049 contained 105 ng cPL/ml and 29 ng oPL/ml. Weekly serum samples were available from the other 7 recipients (Table 2; Fig. 1). In the ewes, oPL increased gradually to peak levels on Day 133 of pregnancy while cPL peaked at much higher levels earlier in pregnancy. Similarly, in the does, peak oPL levels were observed up to 42 days before peak levels of cPL. Total xenospecific PL production was greater than total conspecific PL production in 5 of the 7 ...
Context 2
... cell chimerism was indicated by the presence of both conspecific and xenospecific PL in the serum of 2 ewes and 7 does ( Fig. 1; Table 2). In most recipients tested weekly between Days 42 and 140 of pregnancy, the levels of both hormones were consistently elevated throughout pregnancy. The temporal patterns of the conspecific hor- mone levels resembled those of dams experiencing normal ovine and caprine pregnancy [20,33], but levels were lower and more variable from ...
Citations
... TSCs retain their indefinite self-renewal and differentiation repertoire into all trophoblast subtypes of the placenta. This is revealed from the production of animal chimeras by transplanting into ICM cells or grafting TSCs into pre-implantation embryos in mice, sheep, and goats ( Ruffing et al. 1993;Latos and Hemberger 2016). Like other pluripotent stem cells, the TSCs serve as good model systems for studying trophoblast growth and differentiation ( Rielland et al. 2008), CRISPR/Cas9-mediated genome editing, biochemical analysis, investigating transcriptional networks, characterizing epigenetic signatures, and examining the protein-protein interactions ( Latos and Hemberger 2016). ...
Stem cells have sparked a revolution in biomedical and veterinary medicine. The past two decades have witnessed astounding innovations in pursuit of stem cell applications in livestock production and health. Stem cells are reported from various domestic animals. The stem cells in livestock species are important candidates for genomic testing, selection, genome engineering, and developing model animals for investigating human diseases. Mesenchymal stem cells, due to the ease of attainment, pluripotency, and better proliferation activity have emerged as clinically important cells for treating injuries in pet and companion animals. Improved cell culture techniques, culture media, and supplements, insights into gene-environmental interactions may solve current bottlenecks associated with segregation, description, and applications of stem cells in livestock.
... Dev. 57: 500-506, 2011) he blastomere-aggregation technique has been mainly used in the studies for cell allocation [1] and for the production of chimeric animals [2,3]. It has been also used to improve the developmental ability of embryos. ...
Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.
... The purpose of this study was to examine the efficacy of in utero HSC transplantation in a model using goats transplanted with sheep HSC. While sheep and goats have been successful recipients of allogeneic in utero HSC transfers and sheep of xenogeneic in utero HSC transfers, the only sheep-goat hematopoietic chimeras created are the result of embryo aggregation (29) and blastocyst injection techniques (30). Chimerism in these animals, however, can be found in various tissues of the body. ...
... However, unlike the donors and hosts of many other xenotransplantation models, the sheep and the goat are genetically similar. Sheep-goat chimeras produced by combining early embryonic cells (blastomeres) often express stable blood chimerism as shown in this study and previously reported (29,30,37). These data suggest that compatibility of the species' BM microenvironment exists to support hematopoiesis of either species. ...
Both allogeneic and xenogeneic hematopoietic chimera models have been developed, including fetal sheep models that demonstrated high levels of stable, multilineage engraftment created by in utero hematopoietic stem cell transplantation. The aim of this study was to test the efficacy of in utero transplantation to create xenogeneic sheep-goat hematopoietic chimeras. Fetal liver cells and T-cell-depleted adult bone marrow were tested as sources of hematopoietic stem cells. Donor cells were injected intraperitoneally into 130 recipient fetuses between 49 and 62 days of gestation. Groups 1 and 2 received crude fetal liver cell preparations. Group 3 received fetal liver cells that were incubated overnight in a phytohemagglutinin-stimulated lymphocyte-conditioned medium (PHA-LCM). In Group 4, hematopoietic stem cells were concentrated by using additional density separations. Group 5 fetal recipients received low-density, T-cell-depleted adult bone marrow cells. In Group 1, fetuses were accessed via hysterotomy. Hematopoietic stem cells were injected into Groups 2, 3, 4, and 5 without cutting through the uterine wall. Fetal survival in the five groups ranged from 56 to 100%. The percentage of chimeras from injected fetuses ranged from 43 to 92% by FACS and PCR analyses; however, levels of chimerism were low (<1%). The highest rates of chimerism were found among recipients of low-density fetal liver cells. Despite the pre-immunocompetent status of the fetal recipients and the genetic similarities between sheep and goats, high levels of engraftment were not observed. The consistently low levels of chimerism observed in this study, as well as the poor results recently reported by others using these procedures, indicate that significant barriers exist to transplanting hematopoietic stem cells in utero.
... In order to prevent the detection of anomalous antibodies associated with potential placental defects, a model must be selected in which the parameters of the pregnancy approach the normal limits observed in both species. The chimeric sheep-goat conceptus has been shown to develop normally to term in both species [7], even when the trophoblast is chimeric [8]. Moreover, the microscopic appearance of the fetal and recipient components of the placenta is normal [8] , and species-specific attributes of the conceptus permit the detection of chimerism in the tissues hypothesized to be responsible for recipient immunization, namely the trophoblast and the fetal blood cells. ...
... The chimeric sheep-goat conceptus has been shown to develop normally to term in both species [7], even when the trophoblast is chimeric [8]. Moreover, the microscopic appearance of the fetal and recipient components of the placenta is normal [8] , and species-specific attributes of the conceptus permit the detection of chimerism in the tissues hypothesized to be responsible for recipient immunization, namely the trophoblast and the fetal blood cells. Anti-parental antibodies have been observed in response to both sheep-goat interspecific and hybrid pregnancies, and in sheep-goat chimeric pregnancies resulting from injection of caprine inner cell masses into ovine blastocysts [6]. ...
... In addition, PBLs were isolated from the blood of the offspring between 30 and 60 days of age and tested for chimerism using species-specific sera in two CDC assays. Electrophoretic assays of species-specific isozymes of glucose phosphate isomerase and 6-phosphogluconate dehy- drogenase [8, 10] in RBC lysates and homogenates of placental tissues were used to detect chimerism of the RBCs, allantochorion, amnion, and umbilical cord. Chimerism in the binucleate cell population of the trophoblast was detected by applying RIAs for ovine and caprine placental lactogen (PL) to serum samples collected from the pregnant recipients [8,11,12]. ...
Antibody production was evaluated in 62 recipients of blastomere-aggregation sheep-goat embryos, including 23 multiparous ewes, 21 multiparous does, 16 primiparous does, and 2 virgin does. The reactivity of sera collected weekly after the embryo transfer surgery was compared to that of sera collected prior to the embryo transfer by means of 1) complement-dependent cytotoxicity tests against peripheral blood lymphocytes (PBLs) from the parents of the embryo(s) and from random-bred sheep and goats, 2) hemagglutination and hemolytic assays with red blood cells (RBCs) from the two sires of the embryo(s), and 3) assays with PBLs and RBCs following absorptions with RBCs and PBLs from the parents and offspring. Although cross-reactivity to ovine and caprine PBL antigens was present in the control sera of some recipients, xenogeneic immunization during pregnancy was detected in 20 of 30 recipients that experienced term pregnancy. The xenogeneic response involved the production of antibody that reacted with both PBLs and RBCs. Allogeneic responses to RBCs were not observed, but allogeneic responses to PBLs occurred frequently, beginning after the onset of the xenogeneic response in most recipients (98 +/- 28 vs. 57 +/- 15 days in ewes; 93 +/- 23 vs. 46 +/- 7 days in does; mean day of onset +/- SD). The onsets of the responses were examined in conjunction with data collected on fetal and placental chimerism to evaluate possible routes of immunization. The onsets of the allogeneic responses and the limited serum reactivity to third-party PBLs suggested that fetal lymphocytes leaking across the placenta immunized the recipients to parentally inherited polymorphic antigens. The xenogeneic responses were associated with placental chimerism and appeared to involve the recognition of a species-specific monomorphic antigen shared by PBLs and RBCs. Neither of the responses appeared to affect continuation of pregnancy to term.
... In order to prevent the detection of anomalous antibodies associated with potential placental defects, a model must be selected in which the parameters of the pregnancy approach the normal limits observed in both species. The chimeric sheep-goat conceptus has been shown to develop normally to term in both species [7], even when the trophoblast is chimeric [8]. Moreover, the microscopic appearance of the fetal and recipient components of the placenta is normal [8], and species-specific attributes of the conceptus permit the detection of chimerism in the tissues hypothesized to be responsible for recipient immunization, namely the trophoblast and the fetal blood cells. ...
... The chimeric sheep-goat conceptus has been shown to develop normally to term in both species [7], even when the trophoblast is chimeric [8]. Moreover, the microscopic appearance of the fetal and recipient components of the placenta is normal [8], and species-specific attributes of the conceptus permit the detection of chimerism in the tissues hypothesized to be responsible for recipient immunization, namely the trophoblast and the fetal blood cells. ...
... In addition, PBLs were isolated from the blood of the offspring between 30 and 60 days of age and tested for chimerism using species-specific sera in two CDC assays. Electrophoretic assays of species-specific isozymes of glucose phosphate isomerase and 6-phosphogluconate dehydrogenase [8,10] in RBC lysates and homogenates of placental tissues were used to detect chimerism of the RBCs, allantochorion, amnion, and umbilical cord. Chimerism in the binucleate cell population of the trophoblast was detected by applying RIAs for ovine and caprine placental lactogen (PL) to serum samples collected from the pregnant recipients [8,11,12]. ...
Antibody production was evaluated in 62 recipients of blastomere-aggregation sheep-goat embryos, including 23 multiparous ewes, 21 multiparous does, 16 primiparous does, and 2 virgin does. The reactivity of sera collected weekly after the embryo transfer surgery was compared to that of sera collected prior to the embryo transfer by means of 1) complement-dependent cytotoxicity tests against peripheral blood lymphocytes (PBLs) from the parents of the embryo(s) and from random-bred sheep and goats, 2) hemagglutination and hemolytic assays with red blood cells (RBCs) from the two sires of the embryo(s), and 3) assays with PBLs and RBCs following absorptions with RBCs and PBLs from the parents and offspring. Although cross-reactivity to ovine and caprine PBL antigens was present in the control sera of some recipients, xenogeneic immunization during pregnancy was detected in 20 of 30 recipients that experienced term pregnancy. The xenogeneic response involved the production of antibody that reacted with both PBLs and RBCs. Allogeneic responses to RBCs were not observed, but allogeneic responses to PBLs occurred frequently, beginning after the onset of the xenogeneic response in most recipients (98 +/- 28 vs. 57 +/- 15 days in ewes; 93 +/- 23 vs. 46 +/- 7 days in does; mean day of onset +/- SD). The onsets of the responses were examined in conjunction with data collected on fetal and placental chimerism to evaluate possible routes of immunization. The onsets of the allogeneic responses and the limited serum reactivity to third-party PBLs suggested that fetal lymphocytes leaking across the placenta immunized the recipients to parentally inherited polymorphic antigens. The xenogeneic responses were associated with placental chimerism and appeared to involve the recognition of a species-specific monomorphic antigen shared by PBLs and RBCs. Neither of the responses appeared to affect continuation of pregnancy to term.
This chapter provides the basic information of caprine reproduction needed by the veterinary practitioner. It includes the guidelines for handling infertility, abortions, and obstetric problems. The female goat is called a doe in polite circles or when discussing valuable or cherished animals. In temperate regions, goats are seasonally polyestrous. The duration of the period when regular estrous cycles occur varies with the region, breed, and herd. The chapter presents a summary of pregnancy diagnosis techniques for goats. Akabane virus has caused abortions and perinatal mortality of calves, lambs, and kids. A freemartin is a female rendered sterile by being co‐twin to a male fetus. The male goat is called a buck or billy. The buck is frequently described as half of the breeding herd. Posthitis, the inflammatory condition of the prepuce also termed “pizzle rot,” often results from overgrowth of urea‐splitting bacteria in the prepuce of males consuming excessive dietary protein.
Sheep and goat pregnancies were established by embryo transfer in three multiparous, interspecies chimeras (two sheep-goat chimeras and one sheep/goat hybrid-sheep chimera). At day 39 of gestation, placentomes were collected for immunohistochemical evaluation using anti-sheep and anti-goat antibodies. The degree of trophoblastic attachment to the maternal endometrium was determined for concepti of the two species, with special attention to areas in which the conceptus trophoblast was the same versus different species as the maternal caruncular epithelium. Goat concepti were dead and sheep concepti alive at sample collection. The goat concepti had fewer (P < 0.01) and smaller (P < 0.01) placentomes than the sheep concepti and the goat trophoblast was less adhered to the maternal epithelium than was the sheep trophoblast. Of particular interest were areas at the conceptus/caruncular interface in the sheep/goat hybrid-sheep chimera. The goat conceptus trophoblast had adhered to the hybrid but not to the sheep caruncular epithelium. The sheep conceptus trophoblast had adhered to areas of the sheep but not to the hybrid maternal epithelium. These results suggest that faulty attachment between trophoblast and maternal epithelium is a major factor in sheep-goat interspecies pregnancy failure. Since sheep-goat chimeras are tolerant to monomorphic sheep and goat species-specific antigens, the results are likely due to inappropriate signalling at the conceptus/matemal interface and not due to classical immune rejection. Selection of embryo/recipient combinations for successful interspecies pregnancies in ruminants, and development of strategies to overcome barriers to interspecies pregnancies that currently exist, might be aimed more productively at concerns for adequate placental formation than at concerns over maternal immunological rejection of the conceptus.
Semen from 11 male sheep-goat chimeras and one male sheep-goat hybrid-sheep chimera was evaluated on criteria commonly used as in vitro measures of semen quality. In addition, the species origin of germ cells was determined. Five males that were sex chimeras produced spermatozoa only from their XY cells. Three XY/XY sheep-goat males were shown by electrophoretic separation of sperm isozymes to produce both sheep and goat spermatozoa; one male produced live young from both ewes and does inseminated with his semen. The sheep-hybrid male produced only sheep spermatozoa. Significant variation in semen quality was observed among individual males, but semen characteristics were not strictly associated with the male's sex chromosome complement, the species origin of his gametes or the degree of somatic chimerism. These results demonstrate that male interspecies sheep-goat chimeras are fertile and that XY/XY chimeras can produce fertile gametes of both species.
The aim of the present review is to provide information to researchers and practitioners concerning the reasons for the altered viability and the medium- and long-term consequences of cryopreservation of manipulated mammalian embryos. Embryo manipulation is defined herein as the act or process of manipulating mammalian embryos, including superovulation, AI, IVM, IVF, in vitro culture, intracytoplasmic sperm injection, embryo biopsy or splitting, somatic cell nuclear transfer cloning, the production of sexed embryos (by sperm sexing), embryo cryopreservation, embryo transfer or the creation of genetically modified (transgenic) embryos. With advances in manipulation technologies, the application of embryo manipulation will become more frequent; the proper prevention and management of the resulting alterations will be crucial in establishing an economically viable animal breeding technology.
Different percentages of cells with a female sheep or male goat karyotype were found in kidney (12.0% vs. 88.0%) and lung (42.6% vs. 57.4%) cell cultures from a 10-year-old chimera. Skin biopsies from patches with goat hair or sheep wool showed different, age-related goat-to-sheep fibroblast ratios. Karyotypic analysis of lymphocytes obtained from peripheral blood of the chimera at 6 and 10 yr of age showed no chimerism. Two weeks after birth, however, lymphocytes with both sheep (54,XX) and goat (60,XY) karyotypes were apparent in the blood of this chimera. Twenty percent of the blood cells examined at 2 wk had a caprine karyotype; this proportion declined with time, until it was totally eliminated at age 6.
