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Biology of Reproduction 66, 1596-1603 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Clinical, Hormonal, and Hematologic Characteristics of Bovine Calves Derived from Nuclei from Somatic Cells

P. Chavatte-Palmer1,,a, Y. Heymana, C. Richardb, P. Mongetc, D. LeBourhisd, G. Kanne, Y. Chilliardf, X. Vignona, and J.P. Renarda

a Biologie du Développement et Biotechnologies, Unité Mixte de Recherche Institut National de la Recherche Agronomique/Ecole Nationale Vétérinaire d'Alfort, Domaine de Vilvert, 78352 Jouy en Josas cedex, France b Institut National de la Recherche Agronomique, Unité Commune d'Elevage des Animaux de Bressonvilliers, 91630 Leudeville, France c Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université F. Rabelais de Tours, Physiologie de la Reproduction et des Comportements, UMR 6073, 37380 Nouzilly, France d Union Nationale des Coopératives Agricoles d'Elevage et d'Insémination Artificielle, Technical Services, 94 703 Maisons-Alfort, France e Institut National de la Recherche Agronomique, Unité de Physiologie Animale, 78352 Jouy en Josas cedex, France f Unité de Recherche sur les Herbivores, Equipe Tissu Adipeux et Lipides du Lait, Institut National de la Recherche Agronomique-Theix, 63122-St-Genes Champanelle, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although healthy animals are born after nuclear transfer with somatic cells nuclei, the success of this procedure is generally poor (2%–10%) with high perinatal losses. Apparently normal surviving animals may have undiagnosed pathologies that could develop later in life. The gross pathology of 16 abnormal bovine fetuses produced by nuclear transfer (NT) and the clinical, endocrinologic (insulin-like growth factors I and II [IGF-I and IGF-II], IGF binding proteins, post-ACTH stimulation cortisol, leptin, glucose, and insulin levels), and biochemical characteristics of a group of 21 apparently normal cloned calves were compared with those of in vitro-produced (IVP) controls and controls resulting from artificial insemination. Oocytes used for NT or IVP were matured in vitro. NT to enucleated oocytes was performed using cultured adult or fetal skin cells. After culture, Day 7, grade 1–2 embryos were transferred (one per recipient). All placentas and fetuses from clones undergoing an abnormal pregnancy showed some degree of edema due to hydrops. Mean placentome number was lower and mean placentome weight was higher in clones than in controls (69.9 ± 9.2 placentomes with a mean weight of 144.3 ± 21.4 g in clones vs. 99 and 137 placentomes with a mean individual weight of 34.8 and 32.4 g in two IVP controls). Erythrocyte mean cell volume was higher at birth (P < 0.01), and body temperature and plasma leptin concentrations were higher and T4 levels were lower during the first 50 days and the first week (P < 0.05), respectively, in clones. Plasma IGF-II concentrations were higher at birth and lower at Day 15 in clones (P < 0.05). Therefore, apparently healthy cloned calves cannot be considered as physiologically normal animals until at least 50 days of age.

developmental biology, leptin, parturition, placenta, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has now been established that normal fertile offspring can be produced from nuclear transfer (NT) of somatic cells [1–4]. A striking feature of NT and to a lesser extent in vitro-produced (IVP) embryos in domestic species is the high incidence of gestational losses in early pregnancy and in the late fetal and perinatal periods. In cows and sheep, the majority of losses are observed in the first third of pregnancy, and recent studies suggest that they may be associated with placental vascularization deficiencies [5–7].

The large offspring syndrome (LOS) has been described in late gestation and causes important perinatal deaths in IVP embryos with specific culture conditions (coculture and use of serum-supplemented medium) and in clones [8, 9]. In this syndrome, late fetal losses are associated with excess fetal size, abnormal placental development (i.e., hydroallantois, enlarged edematous placentomes in reduced numbers), and asynchronous growth of organs [10–15]. There is a higher incidence of LOS in clones produced from somatic cells compared with clones produced from embryonic cells and in all clones compared with embryos. This is true even when embryos are cultured under the same conditions up to the blastocyst stage, thus reflecting long-lasting effects of nuclear reprogramming [16]. A recent study indicates that, in sheep, the insulin-like growth factor receptor 2 (IGF-R2) is suppressed in affected fetuses produced by in vitro fertilization, thus confirming prior hypotheses suggesting that a perturbation in the imprinting process is affected [17].

In the case of clones, prolonged gestations are common, and live offspring occasionally exhibit a respiratory distress syndrome and several types of abnormalities that may hinder their survival [2, 11, 13, 15, 18, 19]. There is some homogeneity in the cause of neonatal death in cloned animals: left heart insufficiency and respiratory distress have often been documented. One study gives very thorough details on the clinical course of 13 live calves born from fetal somatic cloning [15]. The 13 calves showed clinical symptoms ranging from normal (4 calves) to severe respiratory distress syndrome and left-sided cardiopathy with ventricular dilation and pulmonary hypertension. The calves that were abnormal at birth all had an edematous placenta. In our laboratory, several calves have shown a similar pathologic pattern at birth with hyperventilation, left ventricular dilation and tachycardia, and lack of a suckling reflex, together with an abnormal placenta (unpublished data). Enlarged umbilical vessels necessitating surgical removal of the stump to prevent infection are also a common finding [20, 21]. The death of neonatal cloned calves due to amniotic fluid inhalation described by others may be part of the same respiratory distress syndrome [3]. It is assumed that these symptoms are a consequence of the LOS during pregnancy.

A wide range of other illnesses, however, have been reported in clones, including infections, such as ruminitis and abomasomitis, which were the cause of death in a 2-day-old calf in New Zealand [22], or coccidiosis and infection after trauma [21, 23]. We have reported the case of one somatic clone diagnosed with thymic aplasia directly related to the cloning process, as its identical embryonic clones (one of which gave the donor cell for the first clone) were normal [19]. Moreover, one calf died at 1 day of age because of acute renal insufficiency in our laboratory, and we have frequently observed paradoxical hyperthermia. These pathologies may not be directly related to disturbances in imprinting processes.

The aim of this study was to assess if apparently normal cloned calves share similar clinical and endocrinologic characteristics with normal controls produced by artificial insemination (AI). Clinical parameters such as body weight and body temperature were recorded together with hematologic and biochemical factors. Factors involved in growth, such as concentrations of IGF-I and IGF-II, IGF binding proteins (IGFBP), growth hormone (GH), insulin, and glucose, were also measured. Leptin was measured because of its important role in the regulation of body weight and insulin and glucose regulation [24]. Thyroxine (T4) was measured because of its role in growth and the regulation of body temperature, whereas the cortisol response to ACTH stimulation was measured to assess calf adrenal maturity. The second aim of this study was to describe the gross pathologic findings in cloned fetuses from pathologic pregnancies in the last two thirds of the gestation to try to assess if all pathologic cases share similar lesions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Embryos

All blastocysts were produced in vitro by somatic NT (clones) or by in vitro fertilization (IVF) (controls). All embryos were produced and cultured in the same culture conditions as described previously [16].

Oocyte preparation Bovine ovaries were collected from two different abattoirs, washed several times with fresh saline, and then transported in sterile PBS at 33°C to the laboratory within 3 h of collection. Cumulus oocyte complexes (COCs) were aspirated from follicles 2–7 mm in diameter, washed, and selected morphologically for in vitro maturation according to their density of cumulus cell layers. For in vitro maturation, groups of 30–40 COCs were incubated in tissue culture medium 199 (Sigma, St. Louis, MO) supplemented with 10% (v/v) fetal calf serum (FCS) (Life Technologies, Paisleys, Scotland, U.K.), 10 µg/ml FSH, and 1 µg/ml LH (Stimufol; Merial, Lyon, France) for 22 h at 39°C in a humidified atmosphere of 5% CO2 in air. After maturation, oocytes were used either for preparation of recipient cytoplast for cloning or for IVF to provide the control group of embryos. The metaphase II and polar body chromatin were removed from oocytes for NT.

Somatic NT For somatic cloning, donor cells from fetal or adult skin biopsies were cultured over several passages to obtain either a growing or a quiescent population of cells on the day of NT. Two fetal and one adult cell line were used. The cells were mechanically scrapped, pelleted at 1200 x g for 5 min, and resuspended in fresh medium before NT. Each isolated cell was inserted under the zona pellucida of the recipient cytoplast and fused by electrostimulation [23].

All of the reconstituted embryos were cultured in microdrops of 50 µl B2 medium (CCD, Paris, France) with 2.5% FCS and seeded with vero cells. The droplets were overlaid with mineral oil (M8410; Sigma, Rhône-Mérieux, Lyon, France) and incubated for 7 days at 39°C under 5% CO2. Cleavage was assessed at Day 2 after fusion, and blastocyst formation was evaluated at Day 7. Expanding or early hatching blastocysts by Day 7 were transferred into recipient heifers. Evaluation of the cell number by counting nuclei in a sample of these blastocysts indicated a mean number of 102 ± 17 nuclei, which was similar to that of control IVF embryos.

Control IVF embryos Control groups of IVF embryos were obtained from the same batches of in vitro-matured oocytes. Twenty-four hours after the onset of maturation, oocytes were coincubated with heparinized, capacitated, freeze-thawed spermatozoa in Tyrode albumin lactate pyruvate medium (Sigma) for 18 h according to a standard technique [16]. A single batch of frozen sperm from the same Holstein bull was used throughout the experiment. After IVF, presumptive zygotes were freed of their cumulus cells by vortexing and pipetting and were cultured until Day 7 under the same conditions as those for NT embryos. By Day 7, grade 1 and 2 blastocysts were transferred to recipients.

Recipients

Recipient animals were normally cycling Charolais or cross-bred heifers raised in the same conditions and transported to the experimental farm by the age of 12–14 mo after several serologic tests to establish the absence of any infectious disease (Genicam, Aron, France). They were synchronized for embryo transfer by the age of 15–18 mo. After estrus was detected in the heifers, heifers that were synchronous ±24 h of the embryo age and carrying a palpable corpus luteum were selected for embryo transfer. Day 7, grade 1 and 2 blastocysts (IVP or NT, single transfer) were transferred nonsurgically into the uterine horn ipsilateral to the corpus luteum using the miniaturized embryo transfer syringe and sheath (IMV, L'Aigle, France) under light epidural anesthesia.

AI pregnancies were obtained by AI of Holstein dairy cows with Holstein semen in the same farm.

Monitoring of Pregnancy

All recipients were examined for the presence of plasma progesterone at 21 days postovulation. The presence of a viable fetus was detected by 35 ± 2 days using transrectal ultrasonography (Pie Medical ultrasound [Hospimedi, Pouilly, France] with 5.0-MHz probe). The transfer of IVF embryos and fetal or adult somatic clones resulted in the same range of initiated pregnancy rates (55.6%–62.7%), but the pregnancy rate was significantly lower in somatic adult clones (33.8%) and somatic fetal clones (27.5%) compared with controls (IVF, 52.9%) (P < 0.01 as detailed elsewhere [16]).

The viability of the fetus as well as the ultrasonographic aspect of the placenta were monitored as previously described [16]. This monitoring was performed to detect any pathologic development of pregnancy such as severe hydroallantois. If such complications occurred, the pregnant recipient was slaughtered, the uterus was retrieved, and samples from the fetus and placenta were recovered. Examination of the fetuses and placentas included gross examination and weighing of organs.

Calving and Monitoring of Calves

Calving Pregnant recipients were kept indoors until calving. Cloned calves were delivered by cesarean delivery when natural calving had not occurred by Day 282 of pregnancy, in which case an i.m. injection of 30 mg dexamethasone (Dexadreson; Intervet, Angers, France) was administered 36 h before surgery. Newborn calves were given pooled colostrum produced on the farm within 2 h after calving. Birth weight, sex, and gestation length were recorded for each group. Calves were isolated and followed up to the age of 2 mo. Blood samples were taken by jugular venipuncture at daily intervals for the first week and weekly intervals for the first month and every 2 wk thereafter unless the calves were ill, in which case more samples were obtained. An aliquot was used for immediate measurement of glycemia before each suckling for the first week. Control animals were born naturally except for two for which an elective cesarean delivery was performed.

Animals

Because of the time span over which these experiments were performed (almost 3 yr), not all animals were subjected to all sampling protocols. The number of animals used for each assay is given with the results.

Fetuses and stillborn calves Twelve cloned (n = 2 fetal from one cell line; n = 10 adult somatic clones from one cell line) Holstein fetuses were recovered (9 after slaughter and 3 after spontaneous abortion) between 154 and 245 days of gestation (mean, 204.1 ± 28.8 days of gestation). Five more cloned Holstein calves (n = 1 fetal and n = 4 adult somatic clones from the same cell line) were recovered at term (274.4 ± 2.6 days of gestation). Three IVF calves were recovered at 174, 217, and 242 days of gestation. These calves shared the same genetic father as the clones. Postmortem examination of the fetuses and placentas included gross examination and weighing of organs.

Live calves Altogether, 7 fetal (6 from one cell line and 1 from another cell line) and 14 adult (all from one cell line) somatic clones and 196 contemporary Holstein controls (n = 20 IVP mainly used for bodyweight; n = 176 calves conceived by AI) from Institut National de la Recherche Agronomique (INRA, Nouzilly, France) were used. Cloned animals were born at term (n = 18) and 1 wk before term (n = 2) by elective cesarean delivery as described previously. One was delivered vaginally without help. All calves survived over the age of 2 mo. Control calves were born naturally except for 2 animals born at term by elective cesarean delivery.

Measurements and Assays

Clinical parameters Rectal temperature was measured at least twice a day for the first week of life and at less regular intervals thereafter. Complete blood counts and biochemical analyses were performed in a commercial medical laboratory using conventional methods. Briefly, total cell counts were performed automatically on a compact automated hematology analyzer designed for the general hematology laboratory (Cell Dyn 3000; Abbot Laboratories, Rungis, France), and differential leukocyte analyses were performed manually on eosin-nigrosin stained slides by counting 100 cells. Fibrinogen concentrations were measured using a chronometric method with Fibrinomat reagent (Hémolab Laboratory, Lyon, France) on a coagulation apparatus (Option 4; Biomérieux SA, Marcy l'Etoile, France). Biochemical analyses were performed using a Koné 30i analyzer (Koné Instruments, Espoo, Finland) with Koné reagents. For aspartate aminotransferase (AST) and alanine aminotransferase (ALT), a kinetic method was used at 37°C using glutamic-oxaloacetic transaminase IFCC Koné and trans-glutamine phosphate (TGP) IFCC Koné reagents, respectively. An enzymatic method using urease glutamate dehydrogenase and Koné urea was used for measuring urea concentrations and creatinine was assayed using a colorimetric method (Jaffé method).

Thyroxine The T4 concentration was measured with the Enzymun-Test FT4 from Roche Diagnostics (Meylan, France). All samples were assayed in the same run. The intraassay variation was 1.97% at 14.25 pmol/L (n = 6).

Plasma IGF-I and IGF-II Plasma IGF-I and IGF-II concentrations were measured by RIA using recombinant DNA-derived human IGF-I (hIGF-I) and hIGF-II obtained from Ciba-Geigy (Basel, Switzerland). Human IGF-I and hIGF-II were iodinated using the iodogen method, followed by purification on Sephadex G50 fine (Pharmacia, Saint-Quentin-en-Yvelines, France), as described previously [25]. Rabbit antiserum prepared against hIGF-I was donated by Dr. Jean Closset (Université de Liège, Belgium). Monoclonal antiserum prepared against hIGF-II was purchased from Amano Corp. (Nagoya, Japan). IGFs were extracted according to a procedure proposed by Le Bouc (Hôpital Trousseau, Paris, France) for human plasma [26]. Plasma samples (25 µl) were diluted in HCl 0.01 N and ultrafiltered on Centricon 30 membranes (Millipore Corp., Saint-Quentin en Yvelines, France). The eluates (4 ml) containing IGFs (C-30 filtrate) were subsequently frozen in liquid nitrogen, lyophilized, and stored desiccated. The samples were reconstituted before assaying in RIA buffer (0.03 M NaH2PO4, 500 µl/L Tween-20, 200 mg/L protamine sulfate, 200 mg/L NaNO3, and 3.72 g/L EDTA, pH 7.4, in a final volume of 0.5 ml). The RIA conditions were similar to those previously described for human [26], mouse [27], and chicken [28] IGF RIAs, and this method was validated in those species. The intraassay coefficient of variation was 14.1% and 12.4% for IGF-I and IGF-II, respectively (n = 5). For each peptide, all samples were first processed together to eliminate possible interassay variations and assayed undiluted (50 µl for IGF-I, 25 µl for IGF-II) in triplicate. In most cases (188 for IGF-I and 151 for IGF-II out of 203 samples), the dilution fell within the linear dilution range (30–1000 pg per tube for IGF-I and 30–2000 pg per tube for IGF-II). For the other samples, one supplementary assay was performed with higher assay volumes for samples with low concentrations.

IGF binding protein To detect and quantify circulating IGFBP, nonextracted plasma samples were subjected to SDS-PAGE and Western ligand blotting using 125I-hIGF-II as described previously [28]. To check interexperimental variability, the same two control samples were run in each gel. Quantification was performed using the phosphoimager Storm 840 (Molecular Dynamics, Bondoufle, France).

Leptin Leptin concentration was measured using a specific RIA previously validated in the ewe [29]. All samples were assayed together, and the intraassay coefficient of variation was 13%.

Growth hormone GH was measured in all samples in a single specific RIA as described previously [30]. The intraassay coefficient of variation was 8.4%.

Glucose and insulin An insulin and glucose response test to feeding 2 L of milk replacer was performed at 2 and 7 days of age at 1000 h. Plasma samples were obtained before and every 15 min for 2 h, and then at 3 and 4 h postsuckling for glucose and insulin assays. Insulin was measured by RIA using a Kit Insulin-CT (Cis Bio International, Oris, France) as described previously [31]. Intraassay and interassay coefficients of variation were 9% and 13%, respectively. Blood glucose concentrations were measured on whole blood immediately after sampling using an automated PrecisionPlus MediSense reader (MediSense France, Schlitingheim, France).

Cortisol An ACTH stimulation test with 0.125 mg ACTH (Synacthène; Novartis Pharma SA, Rueil-Malmaison, France) was performed at 1, 7, and 30 days of age at 1000 h. Plasma samples were obtained by jugular venipuncture just before and 1 and 2 h after ACTH stimulation. Cortisol was measured by RIA with a rabbit antibody validated for bovine plasma. The antibody was provided by N. Poulain, Institut National de la Recherche Agronomique. Intraassay and interassay coefficients of variation were 9.3% and 7.6%, respectively, for 3.9 ng/ml and 5.2% and 9.8%, respectively, for 16.6 ng/ml.

Statistical Analysis

All results are presented as the mean ± SEM. Data were analyzed with ANOVA, General Linear Analysis with repeated measurement analysis, as appropriate, using Systat 7.0 Software (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fetuses

Eleven of 12 cloned Holstein fetuses were undergoing a pathologic gestational process. The apparently normal one was obtained purposely to examine a "normal" fetal clone. The 5 term clones were stillborn (274.4 ± 2.6 days of gestation). One IVP calf had an abnormal hydroallantoic gestation (LOS) and was recovered at 242 days. The other two had an apparently normal gestation and were obtained on purpose to serve as control material.

In most cloned fetuses and in the IVP calf from abnormal pregnancies, some degree of abdominal ascites and edema was found. When fetal membranes were recovered (n = 7 clones; n = 1 IVP), large, edematous cotyledons were often found. The mean number of placentomes was lower and the mean and median weight of the placentomes was higher than those reported in the literature for normal pregnancies [32] and those of the controls (n = 69.9 ± 9.2 placentomes with a mean weight of 144.3 ± 21.4 g in abnormal clones; n = 111 placentomes with a mean weight of 72.9 ± 4.8 g in the normal clone; versus 99 and 137 placentomes with a mean individual weight of 34.8 and 32.4 g in the two normal IVP controls). The placenta was not recovered from the abnormal IVP pregnancy. Individual organ weights in relation to body weight did not reveal any obvious difference between controls and clones, but there was a high variability for allometric measurements between clones. In two cases (one fetus and one stillborn calf), an abnormal kidney was noted (one disproportionately large kidney in the fetus, both kidneys autolyzed in the stillborn calf), and the kidney from the "normal" fetal clone seemed very small. Gross morphologic abnormalities in other organs were not observed apart from one large fatty liver in one fetus and a seemingly large amount of fat surrounding abdominal organs in several fetuses.

Calves

No significant differences were found between clones from fetal or adult cells. The data from these two groups were subsequently pooled in the results.

Body weight Body weight at birth was significantly higher in clones compared with IVP and AI calves of Holstein breed born on the farm in the same time period (55.1 ± 2.7 kg, n = 16; 45.7 ± 1.5 kg, n = 20; and 43.7 ± 0.5 kg, n = 176; respectively) (P < 0.001 for clone vs. AI and clone vs. IVP). However, there was no statistical difference between IVP and AI animals. Figure 1 illustrates the shift to the right of body weight in the cloned calves. At birth, 12.5% of the clones were heavier than the heavier of the controls.



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  		FIG. 1. Repartition of the birth weight of the calves born after AI, IVP, or somatic cloning. Body weight is shifted to the right in clones

Body temperature Mean body temperature in clones and controls from 0 to 7 days is shown in Figure 2. This experiment was run on 10 clones and 10 contemporary controls (n = 8 AI; n = 2 IVP). The mean rectal temperature of clones was significantly higher than that of controls (P < 0.001) in the first week and until 50 days of age. Characteristic peaks of rectal temperature up to 41°C were seen commonly in clones without any clinical signs and unrelated to outside temperature. They did not respond to any nonsteroidal anti-inflammatory treatment, and temperature was lowered using wet towels and ventilation. Despite aggressive cooling, elevated temperature spikes could last for 24–36 h.



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  		FIG. 2. Mean ± SEM body temperature in 10 AI control and 10 contemporary cloned calves from birth to 7 days

Hematologic parameters Hematologic parameters were measured sequentially in 21 clones and 8 AI controls. Red blood cell count, hematocrit, hemoglobin, white blood cell count and differential counts, and clinical biochemistry (urea, creatinine, AST, and ALT) were within the normal range in all clones but for one animal with lymphoid aplasia that had reduced lymphocyte and red blood cell counts [19]. Mean cell volume (MCV), however, was significantly higher in clones compared with controls (50.07 ± 1.29 fl in clones vs. 43.59 ± 0.60 fl in controls, P < 0.01). It is known that MCV decreases with gestational age in most animals, but gestational age was not significantly different between clones and controls. In contrast, the neutrophil:lymphocyte ratio at birth, which reflects cortisol increase at birth and therefore adrenocortical maturation, was significantly higher in clones compared with controls (6.28 ± 0.9 vs. 3.14 ± 1.1; P = 0.05).

Thyroxine Plasma T4 was measured in 7 clones and 4 AI controls for 2 mo to test whether an abnormal regulation of thyroid hormones could be the cause of fluctuations in body temperature. Plasma T4 was significantly lower in clones than in controls during the first 15 days (P < 0.05) (Fig. 3).



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  		FIG. 3. Mean ± SEM T4 concentrations in 4 AI control and 7 contemporary clones from birth to 7 wk. Concentrations of T4 were significantly lower in clones in the first 14 days after birth (P < 0.05)

IGF-I, IGF-II, and IGFBP IGF-II and IGF-I are known to play a role in fetal and postnatal growth, respectively, and their concentrations in seven clones and five AI controls are shown in Figures 4 and 5. Overall, there was no significant difference between clones and controls in IGF-I concentrations. Concentrations of IGF-II were statistically higher in clones at birth (P < 0.05) and were statistically lower compared with those in controls at Day 15 (P < 0.05). There were no noticeable difference in IGFBP profiles between apparently normal clones and controls.



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  		FIG. 4. Mean ± SEM plasma IGF-I concentrations in 7 clones and 5 AI controls until 80 days after birth. There was no significant difference between groups

Leptin Plasma leptin concentrations were statistically higher in six clones than in five AI controls in the first week after birth (P < 0.01) (Fig. 6).



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  		FIG. 6. Mean ± SEM leptin concentrations in 6 clones and 5 AI controls over 4 wk. Plasma leptin concentrations were statistically higher in clones compared with controls (P < 0.01) during the first week

Growth hormone Plasma GH was also measured because of the important role of GH in growth, but there was no statistical difference between plasma GH concentrations between clones and controls (n = 5 and 6, respectively, same animals as for the leptin assay).

Insulin and glucose responses to feeding Insulin and glucose responses to feeding were measured as indicators of metabolic disturbances, which could also explain the increased size of the cloned calves. There was no statistical difference, however, at 1 and 8 days of age between insulin concentrations in clones (n = 6) and AI controls (n = 6) before and after the meal and between glucose concentrations before and after the meal in three clones and four AI controls for glucose concentrations.

Cortisol The increase in plasma cortisol in response to ACTH stimulation reflects adrenal function and adrenal maturation. In premature unstressed animals, the adrenal response to ACTH is decreased. Twelve clones (11 born by cesarean delivery, 1 delivered naturally without assistance), 6 AI controls born naturally, and 2 controls delivered by cesarean delivery in the same conditions as clones were used to determine if adrenocortical maturation was deficient in clones. Absolute values for basal (T0) and poststimulation cortisol concentrations were significantly lower in clones and controls born by cesarean delivery at 1 day of age compared with naturally born controls (P < 0.05) as shown in Figure 7. Cortisol response (percent increase after stimulation, data not shown) was not statistically different in clones and controls. These data confirm that the adrenocortical response to ACTH was normal, although basal levels were lower in clones and controls born by cesarean delivery. The lower basal cortisol values were probably due to the cesarean procedure rather than to cloning itself.



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  		FIG. 7. Cortisol response to ACTH (125 µg Synacthène) stimulation in clone (n = 12), AI controls born by cesarean delivery (n = 2), and AI controls born naturally (n = 6) at 1, 7, and 30 days of age. T0 indicates the time just before injection; T1 and T2, 1 and 2 h after injection, respectively. Absolute values for basal (T0) and poststimulation cortisol concentrations were significantly lower in clones and controls born by cesarean delivery at 1 day of age compared with those in naturally born controls (P < 0.05), but there was no significant difference between groups in the cortisol increase after treatment at any time point


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Disturbances in the imprinting process have been shown to be at the origin of the severe abnormalities that can be found in animals with LOS [17]. In the present study, aberrant expression or methylation patterns were not evaluated, but the pathologic findings in abnormal animals fit well with an incomplete reprogramming of the nuclear DNA leading to abnormal methylation patterns of some clustered genes such as IGF-II. It is not certain, however, that other pathologic processes could not contribute to the syndrome or be present in apparently normal cloned animals. The data collected over more than 2 yr in a large number of controls and cloned animals indicate that there are several differences between apparently clinically normal clones and contemporary controls.

The postmortem findings in pathologic clones are in agreement with those described by others in cloned sheep and cattle with the presence of hydroallantois, hydramnios, and edematous placentas [3, 9, 12, 14, 20]. Differences in body weight at birth have been previously reported for clones and IVP calves [9, 12–14], and the use of coculture or serum in culture medium has been pointed out as a possible cause for the LOS in sheep [33] and cattle [34–37]. Other studies, however, failed to produce offspring with heavy birth weights using bovine serum in culture [38–41]. In the present study, cloned calves were significantly heavier than controls. The increase in birth weight was specific to the NT procedure and not to the current use of coculture and FCS for the culture of embryos because all embryos were cultured in the same conditions. Nevertheless, the prolonged use of serum for the culture of donor cells may be important in the initiation of LOS in cloned fetuses. Significant differences in allometric measurements were not found in fetuses, but in term cloned calves, organ weights seemed higher than those reported previously in normal and oversized IVP calves [36] (e.g., liver weight, 1.80 ± 0.29 kg for clones in our study compared with 1.46 ± 0.59 kg for IVP calves [36]). These data are in agreement with those of a recent study of cloned sheep fetuses that showed that cloned sheep fetuses already have an enlarged liver at Day 35 of gestation [7]. Alterations of the processes of imprinting during early development have been pointed out as probably a major cause for LOS, with the genes for IGF-II and IGFBP-2 being candidate genes [41–43]. Fetal overgrowth, kidney dysplasia, and visceromegaly have been described in human infants with the Beckwith-Wiedemann or Simpson-Golabi-Behmel syndromes with clinically variable symptoms [44–46]. These syndromes share many common traits with our findings in calves. They are due to excess IGF-II expression due to perturbations of imprinting during fetal development. Other anomalies described in sheep, such as dermal hemorrhaging and accumulation of liquid in the fourth ventricle of the brain, were not observed, possibly because fetuses with these malformation could not develop much further in gestation [7]. Finally, the number and mean weight of placentomes in term fetuses were described in older reports to be 90.4 and 47 g, respectively [32], which are comparable to those found in the two controls. The lower number of placentomes and their increased weight in clones suggest that there was compensatory placentome growth as described in an experiment in which carunculectomies were performed [47]. There were no statistically significant differences between groups in IGF-I, IGFBP, and GH concentrations, suggesting that these genes are normally expressed in apparently clinically normal cloned calves after birth. Concentrations of IGF-II were higher at birth in clones, and this might indicate that IGF-II concentrations are elevated during fetal life but that they decrease rapidly to normal levels after separation from the placenta. This rapid decrease of IGF-II concentrations has also been described in fetal sheep [48]. In normal human infants, however, IGF-I concentrations decrease very rapidly on Day 1 after birth, whereas IGF-II concentrations do not significantly change during the first week after birth [49].

Increased body temperature at birth has been reported in IVP calves [40]. In the present study, body temperature was not increased at birth but increased in spikes of 12–48 h within the 3 wk after birth. This paradoxical hyperthermia did not respond to traditional pharmacologic treatment (nonsteroidal anti-inflammatory drugs) and, to our knowledge, it has not been associated with any genetic disturbance. Thyroid hormones are known to play a major role in temperature regulation [50]. Plasma concentrations of T4 were lower in clones, which can be consistent with increased temperatures [51]. Catecholamines and other thyroid hormones such as triiodothyronine have not been measured and may be increased [50–52]. Despite their high body temperature, the calves remained alert and had a normal behavior with no modification of hematologic or biochemical parameters, and they did not require antibiotic treatment, which rules out the possibility of infectious causes.

Sangild and collaborators [40] have reported that in the immediate postnatal period, IVP calves show reduced blood glucose levels. Alterations in glucose metabolism were not observed at basal concentrations or during postprandial conditions, and no differences were found in insulin responses after a meal in clones. In a few other cases, however, (not those in which the insulin tests were performed) hypoglycemia and hypothermia were present in the neonatal period (first 24 h), necessitating heating and infusion of glucose solutions despite correct colostrum intake at birth. The presence of hypothermia suggests that this phenomenon is not related to the hyperthermia previously described. Concentrations of GH were not different between clones and controls either, suggesting that these metabolic pathways are not affected after birth and that the reported elevated leptin concentrations did not affect glucose metabolism.

Decreased basal cortisol levels were probably due to the effect of the elective cesarean delivery in clones (see Materials and Methods) as the two control calves born by elective cesarean delivery had low basal cortisol concentrations on Day 1, similar to the clones. Because a lack of adrenal response to endogenous ACTH is unlikely to be the cause of the prolonged gestations described in clone and LOS pregnancies, a lack of endogenous ACTH release by the fetus or a placental dysfunction (lack of response to fetal cortisol) may be responsible. In agreement with these data, Sangild et al. [40] did not find any differences in adrenal cortex function in late-gestation IVP fetuses that were catheterized in vivo, but these animals did not have any symptoms of LOS. Placental edema due to LOS and secondary to alterations in parental imprinting of genes may impair fetomaternal communication and be the origin of the prolonged gestations. Finally, the normal cortisol responses to ACTH imply that the calves were not premature.

The elevated leptin concentrations described in this study may be due to increased adipose tissue and increased birth weight [53] and placental weight [54]. In fetal and adult sheep, plasma leptin concentrations are known to increase with body fatness and body score [29, 53]. Indeed, subjective observations made at postmortem in our laboratory suggest that the cloned fetuses and newborn calves seemed to have more fat surrounding the intra-abdominal organs compared with controls. Leptin concentrations are generally higher in female neonates than in male neonates [55, 56], but in the present case, all controls and most clones were females, thus excluding gender as a cause for increased plasma leptin levels. In humans, a rapid postnatal decrease in plasma leptin levels occurs as a result of the sudden loss of the placental contribution to high fetal leptin concentrations. This is in contrast with our data in the bovine species, as leptin concentrations seemed stable after birth in controls. Apart from influencing fetal growth, leptin has also been proposed as a regulator for both hematopoiesis and angiogenesis during pregnancy [55]. Altered leptin concentrations in early pregnancy may play a role in the angiogenesis and hematopoiesis abnormalities described by others in failed cloned pregnancies in the first trimester [6, 7, 57]. The increased MCV seen in clones seems to indicate that hematologic maturation is not complete in these calves and could be related to disturbances in leptin concentrations. Elevated leptin concentrations in calves could also be the cause for increased thermogenesis [58]. Finally, leptin is not known to be an imprinted gene, and this may represent another mechanism by which development is affected after NT.

In conclusion, in contrast with the placentomegaly, somatic overgrowth, and omphalocele present in sick clones that are suggestive of perturbation in the imprinting process for IGF-R2, as suggested by other investigators [17], apparently normal clones show some physiologic particularities (alterations in temperature regulation, increased abdominal fat and leptin concentrations, higher MCV) in the first few weeks after birth that are not typical of overgrowth syndromes due to IGF-II overexpression. Moreover, the increased length of gestation noted for clones is not due to fetal adrenal dysfunction.



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  		FIG. 5. Mean ± SEM plasma IGF-II concentrations in 18 clones and 5 AI controls according to postnatal age. Concentrations of IGF-II were significantly higher in clones at birth (P < 0.05) and lower at Day 15 (P < 0.05) than those in controls


   ACKNOWLEDGMENTS
 
The authors thank Patrice Laigre and the staff from Bressonvilliers for their help with the animals and Andrew Ponter for his help with the T4 assays.


   FOOTNOTES
 
First decision: 17 October 2001.

1 Correspondence: Pascale Chavatte-Palmer, Biologie du Développement et Biotechnologies, UMR INRA/ENVA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France. FAX: 00 33 0 1 34 65 26 77; chavatte{at}jouy.inra.fr Back

Accepted: December 19, 2001.

Received: September 19, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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