Changes in placental development have been associated with foetal abnormalities after in vitro embryo manipulations. This study was designed to investigate bovine conceptus development and substrate levels in plasma and fluids in in vivo-and in vitro-produced (IVP) concepti and neonates. In vivo-produced and IVP embryos were derived by established embryo production procedures. Pregnant animals from both groups were slaughtered on days 90 or 180 of gestation, or allowed to go to term. Conceptus and neonatal physical traits were recorded; foetal, maternal and neonatal blood, and foetal fluids were collected for the determination of blood and fluid chemistry, and glucose, fructose and lactate concentrations. Placental transcripts for specific glucose transporters were determined by quantitative RT-PCR. No significant differences in uterine and conceptus traits were observed between groups on day 90. On day 180, larger uterine, placental and foetal weights, and an increase in placental gross surface area (SA) in IVP pregnancies were associated with increased glucose and fructose accumulation in foetal plasma and associated fluids, with no differences in the expression of components of the glucose transporter system. Therefore, the enlarged placental SA in IVP pregnancies suggests an increase in substrate uptake and transport capacity. Newborn IVP calves displayed higher birth weights and plasma fructose concentrations soon after birth, findings which appeared to be associated with clinical and metabolic distress. Our results indicated larger concepti and increased placental fructogenic capacity in mid-to late IVP pregnancies, features which appeared to be associated with an enhanced substrate supply, potentially glucose, to the conceptus.

Although a majority of clones are born normal and apparently healthy, mortality rates of nearly 30% are described in many reports. Such losses are a major limitation of cloning technology and represent substantial economic investment as well as justifiable animal health and welfare concerns. Prospective, controlled studies are needed to understand fully the causes of neonatal mortality in clones and to develop preventive and therapeutic strategies to minimize losses. We report here the findings of studies on the hematologic and biochemical profiles of cloned and control calves in the immediate 48-h postpartum period. Cloned calves were similar to control calves for a majority of parameters studied including blood gases, concentrations of plasma proteins, minerals and electrolytes, and white blood cell, neutrophil, lymphocyte, and platelet counts. The most notable differences between clones and controls in this study were reduced red- and white-blood cell counts in clones at birth and 1 h of age. As a group, plasma electrolyte concentrations were more variable in clones, and the variability tended to be shifted either higher (sodium, chloride) or lower (potassium, bicarbonate) than in controls. Previously, we noted differences in carbohydrate parameters, the length of time required for clones to make the neonatal adaptation to life ex utero, and morphology of the cloned placenta. Taken together, our findings suggest that cloned calves experience greater difficulty adjusting to life ex utero and that further research is warranted to determine the nature of the relationship between the physiological differences noted here in clones at birth and concomitant abnormal placental morphology.

The period immediately after birth is a vital time for all newborn calves as the cardiovascular, respiratory, and other organ systems adapt to life ex utero. Reported neonatal mortality rates suggest this period to be especially critical in cloned calves; yet prospective, controlled studies on the physiological status of these calves are lacking. The objectives of this study were to compare neonatal (birth to 48 h of age) physical and clinical characteristics and placental morphology of cloned and embryo transfer control calves delivered by cesarean section after induced labor. All calves were raised under specialized neonatal-care protocols at a large-animal veterinary research and teaching hospital. Cloned calves were similar to controls for many parameters studied. Notable exceptions included developmental delays of important physical adjustment parameters and enlargement of the umbilical region. Placentas associated with cloned calves contained fewer total placentomes, a twofold increase in surface area and mass per placentome, and a shift in placentome morphology toward larger, flatter placentomes. The most striking clinical variations detected in clones were hypoglycemia and hyperfructosemia, both measures of carbohydrate metabolism. Because the placenta is known to be the source of plasma fructose in newborn calves, increased fructose production by the cloned placenta may be an important factor in the etiology of umbilical and cardiac anomalies in clones observed in this and other studies.

The timing of the transition from maternal to zygotic control of development (MZT) and the initiation of transcription was studied in domestic cat embryos to determine if there is a temporal association between these phenomena and the developmental block observed in cat embryos fertilized in vitro. Embryos were derived from in vitro‐matured, in vitro‐fertilized (IVM/IVF) oocytes. In Experiment 1, embryos (n = 52) were cultured continuously in the presence of 10 μg/ml α‐amanitin (a transcriptional inhibitor) from 12‐hr postinsemination (hpi), and cleavage stage was evaluated every 24 hr. The proportion of embryos cleaving to at least the 5–8‐cell stage in the presence of α‐amanitin (32/52) was similar (P > 0.05) to that of controls cultured without α‐amanitin (25/50). In contrast, only 7.7% of α‐amanitin‐treated embryos cleaved to the 9–16‐cell stage, compared with 38.0% of the controls (P < 0.05), indicating that products of embryonic transcription were required for cleavage beyond the 5–8‐cell stage. In Experiment 2, embryos were cultured in the presence of 20 μM 3H‐uridine for 12 hr beginning at 24, 36, 48, or 60 hpi and subjected to autoradiography. Embryos of 5–8‐cell and 9–16‐cell stages (14 of 27 and 8 of 12, respectively) clearly demonstrated nuclear labeling, a finding also confirmed by computer‐aided densitometry. It is concluded that embryonic transcription and the MZT occur by the 5–8‐cell stage of IVM/IVF domestic cat embryo development and the MZT is not directly related to the partial morula‐to‐blastocyst developmental block observed in cultured IVF cat embryos. Mol. Reprod. Dev. 48:208–215, 1997. © 1997 Wiley‐Liss, Inc.

Potential applications of somatic cell nuclear transfer to agriculture and medicine are currently constrained by low efficiency and high rates of embryonic, fetal, and neonatal loss. Nuclear transfer efficiency in cattle was compared between three donor-cell treatments from a single animal, between four donor-cell treatments in sequential stages of differentiation from a single cell lineage and genotype, and between the same cell type in two donors. Cumulus and granulosa donor cells resulted in a greater proportion of viable day-7 embryos than ear-skin cells; pregnancy rate and losses were not different among treatments. The least differentiated cell type in the follicular cell lineage, preantral follicle cells, resulted in fewer cloned blastocysts (11%) than cumulus (30%), granulosa (23%), and luteal (25%) donor cells. Cloned blastocysts that did develop from preantral follicle cells (75%) were more likely to progress through implantation into later stages of pregnancy than cloned blastocysts from cumulus (10%), granulosa (9%), and luteal (11%) donor cells (p < 0.05). Day-7 embryo development from granulosa cells was similar between two donors (19 vs. 24%) and proved to be a poor indicator of further development as day-30 pregnancy rates varied threefold between donors (48 vs. 15%, p < 0.05). Results reported here emphasize the crucial role of the nuclear donor cell in the outcome of the nuclear-transfer process.

Embryonic mortality and abnormal placental morphology have been reported by most researchers studying nuclear transfer (NT), and it now is accepted that placental anomalies and poor development of cloned embryos are related. As early as day 50 of gestation, cloned bovine concepti exhibit poor structural organization of the developing placentomes. These experiments were designed to identify alterations in maternal-fetal interactions during establishment of the placentas of NT-derived embryos at day 30 of gestation. Bovine NT embryos were produced using cultured fibroblast cells from a single Hereford donor cow, and control embryos were derived from in vitro fertilization (IVF). Following in vivo culture in ligated sheep oviducts, day-8 blastocysts were transferred to synchronized recipient heifers. Tissues recovered from viable day-30 pregnancies were analyzed by real-time RT-PCR, immunohistochemistry, and quantitative histological techniques. Immunoperoxidase staining of caruncular tissue from NT- and IVF-derived pregnancies revealed no significant differences in expression of the extracellular matrix proteins, collagen type IV and laminin, or the receptor subunits, integrins alpha1 and alpha3, suggesting that altered expression of these proteins at day 30 of gestation is not a primary cause of abnormal placentome structure in cloned concepti. Percentage of binucleate cells (BNC) within the trophoblast also was similar in NT- and IVF-derived pregnancies; however, expression of the BNC-specific placental lactogen (PL) transcript was elevated in NT-derived concepti (p < 0.05). These results indicate that regulation of PL transcription was altered in cloned day-30 placental tissues, suggesting the presence of irregular fetal-maternal signaling patterns that might undermine continued development of NT-derived concepti.

Health problems and mortality rates of cloned calves are major limitations of cloning technology and represent substantial economic losses as well as justifiable animal health and welfare concerns. The objectives of this study were to compare neonatal viability and physiological status of cloned and control calves. Cloned (Holstein, n = 5; Hereford, n = 3) and control (embryo transfer: Holstein, n = 3; Hereford, n = 3) calves were carried in the same group of Hereford × Angus crossbred recipient dams and were delivered by Cesarean section at term (Days 273–280) following induced labor. Additional calves (Holstein, n = 3; Hereford, n = 2) resulting from AI and delivered vaginally by their natural dams (Days 269–279) following spontaneous initiation of parturition were included as normal controls to evaluate the effects of the induction procedure. Physical evaluations and measurements of blood biochemistry (19 parameters), gases and electrolytes (9 parameters), and complete blood counts (18 parameters) were performed within 10 min of birth and at 1, 6, 12, 18, 24, 36, and 48 h after birth. Cloned calves were observed with increased occurrence of flexural limb deformities (4/8 clones, 0/9 controls; P < 0.05) and developmental delays of physical adjustment parameters such as time to suckle and stand (5/8 clones requiring >3 h; P < 0.05). Cloned calves were more variable than, but not different from, controls for most blood parameters measured. Compared with controls, at birth clones exhibited reduced red blood cell counts (6.8 and 8.6 × 109 cells/mL, clones and controls, respectively; P < 0.01), plasma bicarbonate (23.1 vs. 26.2 mmol/L; P < 0.05), and plasma glucose (39.4 vs. 73.6 mg/dL; P < 0.05). Blood urea nitrogen concentrations in clones tended to be elevated beginning 24 h after birth and were significantly greater than those in controls by 48 h (13.4 vs. 7.4 mg/dL; P < 0.01). Echocardiographic measurements at 24 h of age varied between groups (Table 1) and were indicative of circulatory abnormalities likely originating in utero for clones. The results of this study identified the physiological differences between clones and controls at birth and may be useful in the development of clinical-care protocols to maximize the health and survival of cloned calves. Table 1. Echocardiographic characteristics of cloned and control calves

Somatic cell nuclear transfer is associated with high incidence of fetal loss, late-term pregnancy complications, perinatal mortality, and abnormal placental development. Several groups have described abnormalities of early and mid-gestation cloned placentas (Hill et al. 2000 Biol. Reprod. 63, 1787–1794; Lee et al. 2004 Biol. Reprod. 70, 1–11). The objective of our study was to characterize differences in the placentas of clones and control calves at term delivery. Clones were produced from ovarian cell lines from two donors (Holstein, n = 5; Hereford, n = 2). Breed-matched controls included AI (Holstein, n = 3) and embryo transfer (Holstein, n = 3; Hereford n = 3) calves. All calves were delivered alive with no visible birth defects between Days 273 and 280 of gestation, and placentas were recovered for measurement and morphological analysis. When possible, pregnancies were delivered via caesarian section, and the entire uterus was recovered for classification of anatomical shape of placentomes. Each placentome was measured, weighed, and classified by type as (A) engulfing mushroom-like; (B) sub-engulfing mushroom-like; (C) flattened, non-engulfing; and (D) convex (adapted from Penninga and Longo 1998 Placenta 19, 187–193, for sheep). Mean number of placentomes per placenta was significantly greater in controls than clones, while total mass of placentomes in the pregnant horn was significantly greater in clones than in controls (Table 1). Total surface area of placentomes in the pregnant horn tended to be larger and more variable in clones (range: 2710–7450 cm2) than in controls (range: 3120–5030 cm2; P < 0.10). A two-fold increase was observed in cloned placentas, as compared with control placentas, in mean surface area per placentome and mass per placentome. Anatomically, cloned placentas differed from controls in the percentage of placentomes classified Type A (controls > clones) and Type C (clones > controls). Other abnormalities noted in cloned placentas included moderate to severe edema, teratomas, enlarged vessels, and large areas devoid of placentation. All clones and 2/9 controls displayed enlarged umbilical vessels. Significant placental abnormalities were observed in all cloned pregnancies. Table 1. Placental characteristics of term cloned and control pregnancies