Insulin-like growth factor (IGF) family genes are aberrantly expressed in bovine conceptuses produced in vitro or by nuclear transfer

Embryos produced through somatic cell nuclear transfer (NT) or in vitro production (IVP) are often associated with increased abortion and abnormalities thought to arise from disruptions in normal gene expression. The insulin-like growth factor (IGF) family has a major influence on embryonic, fetal and placental development; differences in IGF expression in NT- and IVP-derived embryos may account for embryonic losses during placental attachment. In the present study, expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-IIR mRNAs was quantitated in Day 7 and 25 bovine embryos produced in vivo, by NT, IVP, or parthenogenesis, to further understand divergent changes occurring during development. Expression of the IGF-I gene was not detected in Day 7 blastocysts for any treatment. However, there were no differences (P>0.10) among Day 7 treatments in the amounts of IGF-IR, IGF-II, and IGF-IIR mRNA. For Day 25 conceptuses, there was higher expression of IGF-I mRNA for NT and IVP embryonic tissues than for in vivo embryonic tissues (P<0.05). Furthermore, embryonic tissues from NT-derived embryos had higher expression of IGF-II mRNA than IVP embryonic tissues (P<0.05). Placental expression of IGF-IIR mRNA was greater for NT-derived than in vivo-derived embryos (P<0.05). There were no differences in IGF-IR mRNA across all treatments and tissues (P>0.10). In conclusion, these differences in growth factor gene expression during early placental attachment and rapid embryonic growth may directly or indirectly contribute to increased losses and abnormalities in IVP- and NT-derived embryos.     

Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses

The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30-90 of gestation. Fetal and placental characteristics were studied from Days 30-90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30-90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35-60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.     

Insulin-like growth factor-I and binding proteins 1, 2, and 3 in bovine nuclear transfer pregnancies

In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50-150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean +/- SEM, 30.3 +/- 2.3 ng/ml) compared with AI (19.1 +/- 5.5 ng/ml) or IVP (24.2 +/- 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.     

Implantation and placental development in somatic cell clone recipient cows

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.     

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.     

Evaluation of gestational deficiencies in cloned sheep fetuses and placentae.

Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface x Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.     

Perturbations in the biochemical composition of fetal fluids are apparent in surviving bovine somatic cell nuclear transfer pregnancies in the first half of gestation

Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO(4), glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO(4) levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO(4) acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.     

Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost during the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived including one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.     

Birth of African Wildcat cloned kittens born from domestic cats

In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo-matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro-matured oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two groups: recipients (n = 24) receiving < or =25 embryos and recipients (n = 26) receiving > or =30 embryos. Twelve recipients (46.2%) receiving > or =30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving < or =25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n = 17), versus day 5 (n = 4) or day 6 (n = 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.     

Efficient production of transgenic cloned calves using preimplantation screening

The genetic manipulation of donor cells before nuclear transfer (NT) enables prior selection for transgene integration. However, selection for genetically modified cells using antibiotic drugs often results in mixed populations, resulting in a mixture of transgenic and nontransgenic donor cells for NT. In this study, we attempted to develop efficient strategies for the generation of human bile salt-stimulated lipase (BSSL) transgenic cows. Preimplantation screening by either biopsy or green fluorescent protein (GFP) expression was used to detect NT-derived BSSL transgenic embryos to ensure that the calf born would be transgenic. We compared the development rates of NT-derived embryos from G418- and GFP-selected donor cells. There were no significant differences (P < 0.001) in cleavage rate (67.2% vs. 60.0%) and blastocyst formation rate (44.9% vs. 41.2%). We also compared the pregnancy rates of the G418/biopsy and GFP preimplantation screened NT-derived blastocysts. The Day 40 pregnancy rate of the G418/biopsy group (40%) was lower than that of the GFP group (57%), but the calf birth rate of the G418/biopsy group (40%) was higher than that of the GFP group (21%). Healthy BSSL transgenic calves were born after both screening processes. This is the first report of biopsy-screened cloned transgenic animals. The results suggest that both selection methods are useful for detecting transgenic NT embryos without negatively affecting their development into viable transgenic offspring.     

A paucity of structural integrity in cloned porcine blastocysts produced in vitro

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.     

Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones

Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG_67kDa), and two heterologous RIA with PAG_67kDa as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.     

Abnormal expression of trophoblast major histocompatibility complex class I antigens in cloned bovine pregnancies is associated with a pronounced endometrial lymphocytic response

Early embryonic losses are much higher in nuclear transfer (cloned) pregnancies, and this is a major impediment to improving the efficiency of cloned animal production. In cattle, many of these losses occur around the time of placental attachment from the fourth week of gestation. We studied the potential for altered immunologic status of cloned pregnancies to be a contributing factor to these embryonic losses. Expression of major histocompatibility complex class I (MHC-I) by trophoblast cells and distribution of endometrial T-lymphocyte numbers were investigated. Six 5-wk-old cloned pregnancies were generated, and 2 others at 7 and 9 wk were also included, all derived from the same fetal cell line. All 8 cloned placentas displayed trophoblast MHC-I expression. None of the 8 controls (4-7 wk old) showed any MHC-I expression. The percentage of trophoblast cells expressing MHC-I varied in the clones from 17.9% to 56.5%. Numbers of T lymphocytes (CD3(+) lymphocytes) were significantly higher in the endometrium of the majority of cloned pregnancies compared with controls. In the cloned pregnancies, large aggregates of T cells were frequently observed in the endometrium in addition to increased numbers of diffusely spread subepithelial lymphocytes. As trophoblast MHC-I expression is normally suppressed during early gestation, the observed MHC-I expression in the cloned pregnancies is likely to have induced a maternal lymphocytic response that would be detrimental to maintaining viability of the cloned pregnancy. These findings support a role for immunologic rejection in the syndrome of early embryonic loss in cloned bovine pregnancies.     

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal-fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.     

Fate of donor mitochondrial DNA in cloned bovine embryos produced by microinjection of cumulus cells

This study examined the fate of donor mitochondrial DNA during preimplantation development after nuclear transfer (NT) in cattle. Frozen-thawed cumulus cells were used as donor cells in the nuclear transfer. Mitochondrial DNA heteroplasmy in the nuclear transfer embryos was analyzed by allele-specific PCR (AS-PCR), direct DNA sequencing, and DNA chromatography. AS-PCR analysis for the detection of donor mitochondrial DNA was performed at the 1-, 2-, 4-, 8-, 16-cell, morula, and blastocyst stages of the embryos. The mitochondrial DNA from donor cells was detected at all developmental stages of the nuclear transfer embryos. However, mitochondrial DNA heteroplasmy was not observed in direct DNA sequencing of displacement-loop sequence from nuclear-transfer-derived blastocyst embryos. To confirm the mtDNA heteroplasmy in cloned embryos, the AS-PCR product from NT-derived blastocysts was analyzed by DNA sequencing and DNA chromatography. The nucleotides of NT-derived blastocysts were in accordance with the nucleotides from donor cells. These results indicate that the foreign cytoplasmic genome from donor cells was not destroyed by cytoplasmic events during preimplantation development that followed nuclear transfer.     

Evidence against humoral immune attack as the cause of sheep-goat interspecies and hybrid pregnancy failure in the doe

The failure of interspecies and hybrid pregnancies between the domestic sheep (Ovis aries) and goat (Capra hircus) is not completely understood. The sheep-goat hematopoietic chimera is a unique model for studying the role of the maternal immune response in failure of interspecies and hybrid pregnancies between these species. Hematopoietic chimeras were created by in utero transplantation of sheep fetal liver cells into goat fetuses. The resulting chimeric females were recipients of sheep demi-embryos genetically identical to their sheep cells and/or were bred to a ram to create a hybrid pregnancy. Pregnancy sera were analyzed for the presence of anti-species antibodies (Ab) using a lymphocyte microcytotoxicity assay. None of the concepti survived to term. Gross and histological evaluations of two interspecies sheep concepti revealed abnormal placentome formation. The humoral immune response of several hematopoietic chimeras to the challenging concepti differed from control animals. We observed delayed onset of Ab production, low and absent titers, and persistent Ab titers with delayed fetal death. Ultrasonography typically revealed normal fetal development associated with high volumes of placental fluids and retarded placentome development. We conclude that fetal death was associated with abnormal placental development that was not the result of maternal humoral immune attack.