Ultrasonographic fetal well-being assessment, neonatal and postpartum findings of cloned pregnancies in cattle: A preliminary study on 10 fetuses and calves

Abstract

Cloned pregnancies in cattle are considered to be at risk due to a variety of fetal or adnexal abnormalities. Data is lacking concerning the possibility of transabdominal ultrasonography in the assessment of these high risk pregnancies. Transabdominal ultrasonography has rarely been reported in the assessment of bovine cloned pregnancies. Ten Holstein heifers carrying 8-month-old cloned fetuses were assessed by transabdominal ultrasonographic examination during the 3rd trimester of pregnancy. Fetal heart rates (FHR), movements, adnexal appearance, and placentome size were recorded. The outcome of the pregnancies was also noted and potential indicators of fetal demise recorded. Survival rate 1 week after birth was 30%. Mean FHR was 113 beats per minute (range: 92 to 128 bpm) during the fetal ultrasonography. No correlation between FHR and fetal activity was found. Fetal hyperactivity and imaging of hyperechoic particles in both allantoic and amniotic fluids were possible signs of fetal distress. Despite the size of the fetus and the deep bovine abdomen, fetal transabdominal ultrasonography can be performed in cattle. This preliminary study points to the necessity of further larger studies for defining normal and abnormal findings in bovine late pregnancy.

Introduction

Cloning is arguably one of the most important discoveries of modern biology. Somatic cell cloning has been described in sheep (1), goats (2), pigs (3), cattle (4), horses (5), cats (6), and recently, dogs (7). In ruminants, successful cloning has been achieved, using different techniques (8–15). Cloning and other in vitro manipulations of early stage embryos are associated with important losses at different stages of pregnancy (10,15–20). Cloned calves also have a higher rate of neonatal mortality than do normal calves or in vitro-produced calves (8,11,21). Various abnormalities of newborns and their adnexa (hydrallantois) have been described (8,11,17,18,21). The large offspring syndrome (LOS) appears to be the most common anomaly and its precise etiology remains uncertain (22,23). Underdevelopment of the placenta during the 1st trimester and compensatory overgrowth of the remaining placentomes is a possible explanation (11,19,21,24). The most challenging task in cloning cattle is to reduce fetal mortality, neonatal abnormalities, and recipient diseases by early assessment and recognition of fetal distress, allowing for strategic intervention (20,21,25).

Although limited data are available on the evaluation of the near term bovine cloned fetus, some reports have focused on ultrasonographic assessments that describe fetal heart rate (FHR), aortic diameter, and morphological aspects of fetal adnexa (17,18,26). Fetal movements, a reliable predictor of fetal well-being in other species, have not been studied in cattle (27–29). Although the fetal biophysical profile has not been described and validated in cattle until now, normal and abnormal pregnancy findings have been described in horses and used to establish an equine biophysical profile (27,28).

The objective in reporting the results of this preliminary study is to describe the ultrasonographic findings in a cohort of near term, bovine cloned pregnancies. Clinical and pathological findings, ultrasonographic observations of bovine cloned pregnancies, and the outcome of the pregnancies are described.

Materials and methods

All blastocysts were produced in vitro by somatic cell nuclear transfer, using donor skin fibroblasts derived from 3 mature Holstein bulls and host oocytes derived from slaughterhouse-derived Holstein ovaries. Recipients were healthy Holstein heifers that had been synchronized for embryo transfer, 8 d after estrus. All recipient heifers were housed in tie stall barns and fed hay ad libitum and concentrate (2 kg/d). The management of the animals did not differ from that of normal cloned pregnancies using clones from client-owned animals classically performed at the Faculté de Médicine Vétérinaire, Université de Montréal.

Pregnancy monitoring

The ultrasonographic protocole was previously accepted by the institutional animal care committee (Comité d’Éthique et d’Utilisation des Animaux). An ultrasonographic assessment of the near term fetus and uterus was performed by transabdominal ultrasonography, using a 3.5 MHz sector probe (Aloka 1700; Aloka, Tokyo, Japan). The examination lasted approximately 30 min. The fetal heart rate (FHR) was measured 3 to 5 times at 5-minute intervals. Initially, the fetal respiratory movements were observed, but, eventually, this parameter was abandoned, since it was impossible to obtain reliable information. The pregnant uterus was divided into 4 quadrants (left and right cranial, left and right caudal). The maximum length and width of the easiest placentome to measure from each quadrant was obtained, allowing the estimation of an arbitrary surface area for each placentome (width × length), considering a near rectangular form of the placentome. A mean placentome surface area was obtained by using the 4 measurements. The fetal activity during the examination period was graded from 0 to 3, using the scale developed previously in equine studies (27,28). Fetal activity was graded 0 if no movement was detected, 1 if only a few movements were detected (showing activity for less than one-third of the examination period), 2 if the fetus was fairly active (showing activity for more than one third but less than two-thirds of the examination period), 3 if the fetus was very active (showing activity for more than two-thirds of the examination period, almost consistently active). The difficulty in visualizing the entire fetus made it impossible to specify the type of movement or the complexity of the fetal activity. The maximal depth of allantoic or amniotic fluid and its echogenicity were also noted in order to be able to eventually detect oligohydramnios by decreased fluid accumulation (29). This maximal depth of amniotic or allantoic fluid was determined during the examination at the site where the maximum quantity of allantoic or amniotic fluid was observed. This measurement was independent of the position of the fetus. The amniotic fluid was easily identified by its relatively higher echogenicity compared with that of allantoic fluid, which is almost anechoic (27,28). An echogenicity scale was also used to assess fetal fluid based on published reports for the horse (27,28). The fetal fluids were graded 0 if they were anechoic, 1 if only a small amount of echogenic particles was detected, 2 if a moderate number of echogenic particles was observed, and 3 if echogenic particles were present continuously throughout the examination. Any suspected anomaly was also recorded during the ultrasonographic examination.

On the day of assessment, recipient whole blood samples were collected and submitted for a serum biochemical profile and a complete blood (cell) count (CBC). The body score (BS) of the heifers was also estimated, using a scale from 0 to 5 (30).

Calving procedures

The initial plan was to induce parturition by administering dexamethasone (Dexamethasone 5; Vétoquinol, Lavaltrie, Quebec), 25 mg, IM, 24 to 36 h prior to the day of cesarean section, in order to decrease the occurence of neonatal respiratory distress (31). The injections were spread over 3 d to permit 3 cesarean sections per day at 276, 277, and 278 d gestation. Cesarean sections and neonatal care were performed at the Centre Hospitalier Universitaire Vétérinaire (CHUV) de l’Université of Montréal.

Macroscopic abnormalities of the placentomes (reduced number, increased size, or edema) were recorded during the cesarean section. Cloned calves were examined promptly after birth to assess respiratory function. Neonatal care was based on the initial clinical assessment. Nasal and pharyngeal secretions were removed by suction, and cold water was applied to the neck and in the ears. The FHR and rhythm were then assessed. If persistent bradycardia (less than 80 beats/min) and/or bradypnea (less than 10 breaths/min) were observed, respiratory assistance was provided (first, with intranasal oxygen; then, after orotracheal intubation, by manual or mechanical ventilation). Umbilical care included disinfection with a 5% iodine tincture solution every 2 h until the umbilicus was dry. Frozen colostrum from healthy donor cows was offered to the calves within 2 h of birth. A serum biochemical panel was performed on days 1 and 2 to assess passive immunity transfer by checking total proteins, albumin, globulin, and gamma-glutamyl transferase activity values. The feeding schedule consisted of administering 2 L of whole milk, by bottle, every 8 h. Orogastric intubation was used if the calf failed to nurse on its own (1L, q46h). Clinical assessment of the calf was repeated q15min if cardiorespiratory parameters were abnormal or respiratory assistance was required, then q1h for 24 h, and then q12h when the animal was considered stable. Antimicrobial drugs were administered if sepsis was suspected, and dextrose-containing fluids were administered if blood glucose levels were < 4 mmol/L. Animals were discharged when they were standing and nursing independently, had normal vital signs, and further hospital care was no longer considered necessary. Dead calves and fetuses were submitted for postmortem examination. Abnormal organs were sampled and histologically examined. All medical complications occurring during the first 6 mo of life were recorded and the survival rate at 1 y was obtained.

Results

On the day of ultrasonographic examination, 9 heifers carrying freshly transferred (unfrozen) cloned embryos were confirmed to be pregnant at day 255; 1 heifer carrying a frozen-thawed cloned embryo was confirmed at day 220. Some complications developed prior to the expected calving date. Heifer # 1 had severe abdominal distension, weakness, and premature rupture of the fetal membranes on day 227 of pregnancy. The diagnosis was hydrallantois and the fetus was dead. The heifer was euthanized.

Heifers # 6 and 8 were referred to the veterinary hospital for poor general status and suspected hydrops on day 255 of pregnancy.

Heifers # 3, 4, and 7 were hospitalized on days 266, 265, and 264 of pregnancy, respectively for premature rupture of the fetal membranes and early parturition.

Heifers # 2, 5, 9, and 10 had parturition induced, according to the defined protocol, and cesarean sections performed at the veterinary hospital at 278, 278, 279, and 277 d gestation, respectively.

Health status of the heifers during the ultrasonographic fetal assessment

The recipients’ BS at examination is presented in Table 1. The median BS was 3.0 (range: 2.75 to 3.25). Increased levels of beta hydroxybutyrate (BHB) (n = 5), hyperfibrinogenemia (n = 6), and lymphopenia (n = 3) were the most commonly encountered abnormalities. Heifers with increased levels of BHB were administered propylene glycol, 250 mL/heifer, PO, q12h until the day of calving. Hypoglycemia and a high concentration of BHB (>3000 μmol/L) occurred in heifers # 7 and 8. Both heifers had premature delivery and hydrallantois, and their fetuses died. All heifers with an elevated anion gap delivered either a dead fetus or a calf that died shortly after birth.

Table 1.

Health status of the dam, evolution of the pregnancy, and outcome of cloned calves/fetuses

Heifer number	Body condition score at 8 months of gestationa	Biochemical panel, 8 months (abnormal values)	CBC of the dam	Evolution of the dam and the pregnancy	Chemistry panel if readmitted to the hospital	CBC of the dam if readmitted to the hospital	Outcome of the calf or fetus
1	3.0	—	—	Euthanized at 8 months of gestation	—	—	Euthanized on day 250 because of hydrallantois, anorexia, and recumbency.
2	2.75	➚BHB 1616 μmol/L	➚F 6 g/L ➘Lymph 2.2 × 109/L	Cesarean section performed on day 278	BHB 511 μmol/L	—	Dyspnic and hypercapnic, mechanically ventilated after birth. Cryptosporidiosis on day 10, anemia. Perforated abomasal ulcer and death at 1 month.
3	3.0	➚Glob 45.5 g/L ➚Anion gap 19.9 mmol/L	➚F 7 g/L	Premature rupture of fetal membranes on day 266	—	—	Dead fetus, hemorrhagic placenta, congenital hepatic fibrosis, myocardial degeneration.
4	3.25	➘Ca 2.19 mmol/L	➚F 6 g/L ➘Lymph 2.0 × 109/L	Admitted on day 265 for anorexia, weakness, cesarean section on day 268	—	—	Assisted ventilation under general anesthesia, euthanized when 1 day old. Slight portal hepatic fibrosis, left ventricular hypertrophy.
5	3.0	➚ BHB 169 1 μmol/L	➚F 6 g/L	Cesarean section on day 278, no abnormalities noted with the dam	—	—	Intranasal oxygen at birth, diarrhea when 10 days old, spiral colonic intussusception when 1 month old, free gas bloat at 2 months.
6	2.75	➚ BHB 1702 μmol/L ➘Ca 2.19 mmol/L ➘Glob 24.9 g/L ➘TotProt 58.1 g/L	➚F 6 g/L ➘Lymph 2.2 × 109/L	Cesarean section on day 267, placental edema, large placentomes	➚ BHB 2630 μmol/L ➚GGT 46 IU/L	—	Assisted ventilation under general anesthesia, died at 1 day of age. Pulmonary aspiration of amniotic fluid (subacute placental insufficiency).
7	3.25	➘Glu 2.2 mmol/L ➘Ca 2.14 mmol/L ➚ BHB 3267 μmol/L ➚ Anion gap 19.1 mmol/L	➚F 6 g/L	Premature rupture of the fetal membranes, abdominal distension of the heifer. Cesarrean section on day 264	➘TotProt 56.6 g/L, ➘Alb 23.6 g/L	➚F9 g/L, ➚Band neutrophils 0.2 × 109/L	Hydrallantois, placental edema, dead fetus, aspiration of amniotic fluid.
8	3.25	➘Glu 2.4 mmol/L ➘HCO3 20.7 mmol/L ➚ BHB 3627 μmol/L ➚ Anion gap 19.1 mmol/L	—	Readmitted on day 261 for abdominal distension, anorexia, fever and weakness. Cesarean section on day 268	➚ BHB 3060 μmol/L	➚F 8 g/L ➘Neu 4.6 × 109/L	Hydrallantois, placental edema. Bradycardia with absent spontaneous respirations, immediate death, subacute to chronic aspiration of amniotic fluid.
9	3.0	➘TotProt 58.7 g/L ➘Glob 23.5 g/L ➚ Cl 112 mmol/L	—	Uncomplicated pregnancy	➘PT54 g/L, ➘Glob 22.9 g/L	—	Healthy calf with no detectable abnormalities.
10	3.25	—	—	Suspected anasarca, premature rupture of fetal membranes. Cesarean section on day 277	—	F5 g/L, ➚Eosinophils 0.7 × 109/L	Anasarca fetalis.

^a Note: case 1 was only 220 days pregnant when examined.

Body condition score based on the (0–5) scale by Heinrich and Radostits (30); ➚ — increased; ➘— decreased; Alb — albumin; BHB — beta-hydroxybutyrate; CBC — complete blood cell count; Ca — calcium; Cl — chloride; F — fibrinogen; GGT — gamma-glutamyltransferase; Glob — Globulin; Glu — Glucose; HCO₃ — Bicarbonate; Lymph — Lymphocytes; Neu — neutrophils; TotProt — Total proteins.

Ultrasonography on the farm

The results of the transabdominal ultrasonographic examination are summarized in Table 2. The examination was performed easily without sedation. Imaging of the fetal thorax was possible on the 1st attempt in 7 cases. In 3 cases, a 2nd examination was necessary.

Table 2.

Results of ultrasonographic assessment of late pregnancy cloned fetuses/calves

Heifer	Days gestation when ultrasonography performed	Fetal heart rate, beats per minute (range)	Fetal activitya	Mean placentome size (cm2)	Maximal depth of uterine fluidsb (cm)	Body score of the heiferc	Gestation duration (days)	Birth weight (kg)	Echogenicity scored	Observation	Outcome
1	220	130 (120–140)	1	29	>20	3.0	—	ND	3	Decreased number of placentomes	Hydrallantois, euthanized at 8 months of pregnancy.
2	255	120 (112–124)	1	43	6.2	2.75	278	56	1	Small quantity of liquid	Fetus mechanically ventilated, died with abomasal perforated ulcer when 1 month old.
3	255	110 (92–124)	3	33	15.5	3.0	266	66	1	Very active fetus	Dead fetus.
4	255	110 (104–116)	2	22	19.1	3.25	268	51	1	—	Respiratory distress, mechanical ventilation, died 12 h after birth.
5	255	106 (104–108)	1	49	18.4	3.0	278	50	1	—	Intranasal oxygen, healthy 1 year later.
6	255	110 (100–120)	1	26	18.1	2.75	267	55	2	Chorioallantoic edema	Calf ventilated under general anesthesia, died 24 h after birth.
7	255	110 (104–120)	1	32	15.2	3.25	264	50	1	—	Hydrallantois, aspiration of amniotic liquid.
8	255	118 (116–120)	3	45	17.6	3.25	268	53	3	Large amount of debris in the amniotic fluid	Hydrallantois, placental edema, bradycardia with absent spontaneous respirations, died 30 minutes after birth.
9	255	120 (112–124)	1	25	14.1	3.0	279	44	1	—	Healthy at birth and 1 year later.
10	255	110 (96–112)	1	40	18.6	3.25	277	60	1	Anasarca, free liquid in the abdomen	Anasarca, dead fetus.

^a Fetal activity : 1 — fetus active less than 33% of the examination time (ET); 2 — fetus active between 33% and 66% of the ET; 3 — fetus active more than 66% of the ET.

^b Maximal depth of uterine fluid: deepest pocket of allantoic or amniotic fluid that can be assessed when the ultrasonographic examination was done.

^c Body condition score based on the (0–5) scale by Heinrich and Radostits (30).

^d Echogenicity score: 1 — small quantity of echogenic particles in the allantoic and amniotic fluid; 2 — moderate amount of echogenic particles in the allantoic and amniotic fluid; 3 — large amount of echogenic particles in the allantoic and amniotic fluid.

The mean FHR at 255 d of pregnancy was 113 beats/min (range: 106 to 130 beats/min). It was not possible to associate fetal activity with increased FHR. Fetal activity was always observed throughout the examination. There was no detectable sleeping phase during the entire examination time. Fetuses were considered active for less than one-third of the total examination time in 7 out of 10 cases. Two fetuses (carried by heifers # 3 and 8) were moving consistently during the examination and were allocated a score of 3 for fetal activity; both had a negative outcome (Table 2).

The maximal depth of uterine fluids was highly variable, ranging from 6.2 cm to more than 20 cm. The fetus with the smallest depth of uterine fluid (in heifer # 2) had serious cardiorespiratory problems at birth. Echogenicity of allantoic and amniotic fluid was considered normal (score of 1) in 7 out of 10 cases. Moderate hyperechoic particles (score of 2) were detected in the fetal fluid of heifer # 6, and high number of hyperechoic particles (score of 3) was seen in heifers # 1 and 8. Both cases with the echogenicity score of 3 were diagnosed with hydrallantois and their fetuses died.

Measurement of the thickness of the amniotic membrane showed important differences between normal pregnancies (0.8 to 1 cm) (heifers # 4 and 6) and those with hydrallantois (2 cm) (heifer # 8) (Table 3). The mean estimated placentome surface was 35 cm² (range from 22 to 45 cm²). In heifer # 10, ascites was observed in the fetal abdomen; fetal anasarca was diagnosed and confirmed at the time of caesarean section.

Table 3.

Comparison of ultrasonographic findings in heifers # 4, 6, and 8, on which more than 1 ultrasonographic examination was performed

Case	Days gestation when ultrasonography performed	Fetal heart rate beats per minute (range)	Fetal activitya	Mean placentome size (cm2)	Maximal depth of uterine fluidsb (cm)	Body score of the heiferc	Length of gestation (days)	Birth weight (kg)	Echogenicity scored	Observation	Outcome
4	255	110 (104–116)	2	22	19.1	3.25	268	51	1	—	Respiratory distress, mechanical
4	265	120 (108–130)	1	33	19.1	2.75	268	51	2	Amniotic membrane 1 cm	ventilation, died 12 h after birth aspiration of amniotic fluid.
6	255	110 (100–120)	1	26	18.1	2.75	267	55	2	Amniotic membrane edema	Calf ventilated under general anesthesia, died 24 h after birth.
6	260	114 (106–120)	1	41	>20	2.5	267	55	2	Turbidity of allantoic fluid.
6	264	114 (112–116)	1	37	>20	2.5	267	55	2	Amniotic membrane 0.8 cm
8	255	118 (116–120)	3	45	17.6	3.25	268	53	3	Large amount of debris in the amniotic fluid	Hydrallantois, placental edema, bradycardia with absent spontaneous
8	261	110 (108–120)	1	39	>20	3.0	268	53	2	Amniotic membrane 2.0 cm	respirations, died 30 min after birth.
8	264	128 (128–132)	2	40	>20	3.0	268	53	2	Amniotic membrane 2.0 cm

^a Fetal activity : 1 — fetus active less than 33% of the examination time (ET); 2 — fetus active between 33% and 66% of the ET; 3 — fetus active more than 66% of the ET.

^b Maximal depth of uterine fluid: deepest pocket of allantoic or amniotic fluid that can be assessed when the ultrasonographic examination was done.

^c Body condition score based on the (0–5) scale by Heinrich and Radostits (30).

^d Echogenicity score: 1 — small quantity of echogenic particles in the allantoic and amniotic fluid; 2 — moderate amount of echogenic particles in the allantoic and amniotic fluid; 3 — large amount of echogenic particles in the allantoic and amniotic fluid.

Evolution of the pregnancies

Heifer # 1 was diagnosed with hydrallantois, aborted on day 250, and was euthanized. Heifers # 4, 6, and 8 were referred to the CHUV on day 255 because of a decrease in appetite and abdominal distension. Their body condition had deteriorated since the 1st examination (Table 3) and they were profoundly depressed. Heifer # 4 was recumbent. In heifer # 6, the serum biochemical profile indicated an elevation of BHB concentration levels and an increase in gamma glutamyl-transferase (GGT) activity (46 IU/L; reference range: 0 to 39 IU/L) and a presumptive diagnosis of hepatic lipidosis was made. Heifer # 8 had a distended uterus with no palpable placentome or fetus upon transrectal examination. The initial treatment included continuous IV fluid therapy (dextrose 2.5% and NaCl 0.45%, 20 L per day) combined with insulin injections (200 IU, SC, q48h). All 3 heifers had repeated abdominal fetal ultrasonographic examinations (Table 3). The FHRs at subsequent examinations were not significantly different from those at the 1st examination. An interesting feature was observed in heifer # 8: its amniotic membrane separating the amniotic and allantoic fluid appeared thickened and measured 2.0 cm; it was thicker than in heifer # 4 (1.0 cm) and heifer # 6 (0.8 cm), which did not have hydrallantois. The amniotic and allantoic fluids had an echogenicity of 2 in all 3 cases. The fetal activity was observed during 33% to 66% of the examination time, leading to a score of 2 in all 3 cases. Parturition was induced to allow a cesarean section to be performed on day 267 for heifer # 6 and on day 268 for heifers # 4 and # 8.

Heifer # 6 delivered a 48 kg calf with enlarged umbilical structures. The calf experienced respiratory difficulties with persistent bradycardia (40–50 beats/min at birth to 25 beats/min 10 min after birth), so mechanical ventilation was initiated, after propofol induction (Rapinovet; Schering-Plough, Pointe-Claire, Quebec), 5 mg/kg BW, IV, to avoid the calf ’s resistance to mechanical ventilation. Auricular arterial blood gas (ABG) analysis revealed an increased PaCO₂ (70 mmHg: reference interval: 40 to 45 mmHg), hypoxemia (PaO₂ = 52 mmHg; reference interval: 80 to 100 mmHg), and hyperglycemia (15 mmol/L; reference interval: 5 to 9 mmol/L). Response 2 h after initiation of assisted ventilation (Esprit ventilator; Respironics, Pittsburgh, Pennsylvania, USA) was good, with a decrease in the PaCO₂(55 mmHg) and an increase in the PaO₂ (156 mmHg) values. Twelve hours after birth, the level of anesthetic was decreased to avoid the negative effects of long-term anesthesia (anorexia and ileus). A deterioration of respiratory function, based on results of repeated blood gas analysis, was observed. The deterioration in respiratory function was believed to be associated with the calf’s resistance to mechanical ventilation. The calf was euthanized 20 h after birth on account of the poor prognosis. Necropsy revealed a subacute pneumonic aspiration of amniotic fluid.

Heifer # 4 delivered its calf on day 268. The calf was tachypneic and tachycardic. Umbilical structures were grossly enlarged. Thoracic wall movements were superficial and the calf was considered to be in respiratory distress. A 10 mg/kg BW-bolus of caffeine was administered, PO, without success. Manual assisted ventilation was initiated following orotracheal intubation. The partial pressure of arterial CO₂ decreased temporarily, but it increased as soon as ventilation was terminated. The calf was then ventilated mechanically, after induction with propofol, 5 mg/kg BW, IV. Initially, the calf responded well to mechanical ventilation and showed a normal ABG analysis after 2 h of ventilation. Ten hours later the mechanical ventilation was stopped, whereupon the calf’s condition deteriorated and it was euthanized. Necropsy revealed a subacute pneumonic aspiration of amniotic fluid, congenital hepatic portal fibrosis, and left ventricular hypertrophy.

Heifer # 8’s calf was delivered by cesarean section on day 268. The uterus was distended and hydrallantois was suspected. Placentomes were large, edematous, and reduced in number. The calf was bradyarrhythmic (fewer than 20 beats/min) and severely dyspneic; it died shortly after birth. Necropsy revealed lesions compatible with a subacute pneumonic aspiration of amniotic fluid and hepatocyte vacuolization.

Heifers # 3 and 7 were referred to the CHUV immediately after the owner had observed premature rupture of the fetal membranes on days 264 and 266, respectively. Heifer # 3 was delivered of a large dead fetus (66 kg) via cesarean section. The placenta was hemorrhagic. Necropsy findings included multiple nodules in the liver and histologic lesions compatible with congenital hepatic fibrosis, myocardial degeneration, and moderate pneumonia, secondary to chronic aspiration of amniotic fluid due to fetal distress. The surgery performed on heifer # 7 revealed large edematous placentomes and an increased amount of allantoic fluid, compatible with hydrallantois. The fetus was dead. Necropsy findings included a subacute pneumonia, secondary to chronic aspiration of amniotic fluid.

Deliveries with no obvious disease of the dam

In 4 heifers # 2, 5, 9, and 10, parturition was induced in order to perform the cesarean sections on days 278 and 279. Heifer # 10, carrying a fetus with suspected anasarca, ruptured her fetal membranes prematurely on day 277, so a cesarean section was performed immediately. A large, dead, bulldog calf was delivered with at least 10 L of ascites and a swollen face, compatible with fetal anasarca.

Heifer # 5’s calf was born on day 278. Six hours after birth, analysis of ABG, sampled via an auricular catheter, revealed hypercapnia (PaCO₂ = 63 mmHg) and hypoxemia (PaO₂ = 54 mmHg). The respiratory rate was elevated (from 70 to 100 breaths/min). Thoracic radiographs showed a mixed interstitial and alveolar pattern in the lungs. Intranasal oxygen was given for 24 h (6 L/min). Two hours after initiating the oxygen therapy, the respiratory function markedly improved. The oxygen therapy was progressively reduced on day 2 without any deterioration in the calf’s respiratory condition. The rectal temperature increased (from 39°C to 40.3°C) on day 2 and remained elevated despite treatment with flunixin meglumine, (Banamin; Schering-Plough, Pointe-Claire, Quebec), 1 mg/kg BW, IV, q24h. The rectal temperature returned to normal on day 4. The calf was discharged on day 5.

Heifer # 2’s calf was delivered by cesarean section on day 278 and weighed 56 kg. Initial respirations were spontaneous, but they rapidly became superficial and labored. The venous blood gas, taken 30 min after birth, showed hypercapnic acidosis pH = 7.08 (reference interval: 7.35 to 7.45); PvCO₂ = 77 mmHg (reference interval: 35 to 47 mmHg); base excess (BE) = −9 mmol/L (reference interval: 0 to −6.1 mmol/L). One hour later, intranasal administration of oxygen was initiated (8 L/min). The calf’s general condition deteriorated 10 h later. The calf was tachypneic (80 breaths/min) with increased respiratory efforts and he was intubated nasotracheally without sedation. Mechanical ventilation was then initiated. The ABG and general condition of the calf improved over the next 10 h. Twenty hours later an ABG analysis revealed a major improvement (pH = 7.38; PaCO₂ = 55 mmHg; PaO₂ = 294 mmHg; BE = 8 mmol/L) in the respiratory function. Mechanical ventilation was continued for 2 d and the calf was weaned progressively from the ventilator on day 4. He was discharged from the hospital on day 6 with instructions to the owner to administer ceftiofur (Excenel; Pfizer, Kirkland, Ontario), 5 mg/kg BW, IM, q12h for 10 d.

Heifer # 9’s calf was born by cesarean section on day 279. The calf was considered healthy with no detectable abnormalities. He was kept under observation for 3 d and then discharged on day 4.

Evolution of the 3 surviving calves

The 3 calves that were discharged from CHUV did well for 1 wk. During their 2nd week of life, they developed cryptosporidial diarrhea and severe metabolic acidosis that was treated symptomatically. Six weeks later, heifer # 5’s calf developed abdominal distension and severe hypokalemia (1.9 mmol/L; reference range: 3.86 to 5.28 mmol/L) associated with a colocolic intussusception of the spiral colon that was surgically treated with laterolateral anastomosis and resection of the devitalised spiral colon. The calf was discharged after 15 d. The calf was eventually readmitted at 12 wk of age for chronic bloat. A long-term rumen fistula was placed and ceftiofur (Excenel; Pfizer), 1 mg/kg BW, IM, q12h was administered for 7 d. The calf then had an uneventful recovery.

Heifer # 2’s calf was weak, unthrifty, anemic, and slightly depressed. He was treated symptomatically with IV fluids for 3 d, but he remained depressed. His PCV dropped progressively (from 0.19 to 0.16 L/L; reference interval: 0.26 to 0.42 L/L). A nonregenerative anemia of unknown origin was diagnosed on the basis of a CBC. The calf progressively deteriorated and died 2 wk later. Multiple perforated abomasal ulcers and severe diffuse peritonitis were diagnosed on postmortem examination. Calves out of heifers # 5 and 9 were considered healthy at 1 y of age. Their semen has been evaluated and considered normal.

Discussion

Somatic cloning techniques are associated with multiple losses throughout pregnancy (13,17,19,21), most of which occur in the 1st trimester of gestation (17,32). In the 3rd trimester of gestation, losses have been reported as being 6.2% (17) and the mortality rate in the week 1 of life has been variously reported as 33% (17), 51% (33), or 87.5% (11). The overall mortality rate observed in this study (7 of 10 calves, 70%) was associated with various abnormalities of the dam, the fetus, and/or the placenta.

Abnormal placentation is recognized as an important limiting factor in cloning (4,19,22,32,33). Decreased numbers of placentomes have characterized the placental insufficiency in the 1st trimester of pregnancy and have been associated with compensated overgrowth of the remaining placentomes (22,32). Placental abnormalities have been associated with fetal fluid anomalies; the exact origin of these anomalies is unknown, but different authors have hypothesized that deficient angiogenesis and allantoic development during placentation may be involved (24,32). In late pregnancy, the most frequently reported abnormality is hydrallantois (11,14,17,22,34,35), as was observed in 30% of the cases in this study. The prognosis for the fetus should be guarded to poor, based on our findings and those in other reports (11,17,20,21).

Large offspring syndrome was also diagnosed in this study. Although the exact origin of the LOS remains unknown, large fetuses can have a detrimental effect on their dam, as shown by the relatively low BS of the recipient heifers in this study. An adequate nutrition program is necessary to maintain normal fetal growth. Maternal protein restriction in early to mid pregnancy in cattle may enhance fetal and placental growth (36). Large fetuses have more demanding metabolic requirements (37). These high metabolic requirements could have a negative effect on the health status of the dam because of metabolic deviation from the dam toward the fetus (8). Ketosis and hypoglycemia of the dam may have contributed to the poor outcome of the calves (38). Despite the increase in the frequency of feeding and amount of feed and the oral propylene glycol treatment instituted for the ketotic heifers, their general condition continued to deteriorate. Other reports have pointed out that dams carrying cloned fetuses may suffer from malnutrition and secondary pregnancy toxemia (8,11). The importance of closely monitoring the nutritional program of dams before the appearance of metabolic disorders is supported by this study.

Various congenital defects have been reported in cloned calves (11,12,17,39,40). Cardiac defects are the most frequent, with left heart failure, right ventricular dilation, and patent foramen ovale being commonly noted (8,11,21,39). Common hepatic defects in cloned calves have consisted of liver congestion, hepatic lipidosis (21,39), and congenital hepatic fibrosis (40). The precise etiology of these various anomalies remains unknown. Interaction between in vitro culture media and the early embryo, asynchronies between the uterus of the recipient and the implantation date, mitochondrial DNA interactions or mutation during cell divisions, and genomic imprinting are all potential causes (21,23). A recent study revealed aberrant gene expression in organs of cloned calves that died prematurely (39). The myocardial tissue gene expression was most frequently perturbed, which is concordant with the high rate of heart disorders in cloned calves (39).

Primary respiratory congenital defects are rare in cloned calves; however, respiratory distress is frequent (8,11,20,41). Depending on the study, different factors have been implicated in respiratory distress, including pulmonary hypertension (41), immaturity (11), and pulmonary infection (8). Chronic amniotic fluid aspiration was the most common postmortem diagnosis in this case series. It was suspected that amniotic fluid had been aspirated by the fetus for several days prior to birth, as macrophage infiltration was present in the lungs. We believe that this is associated with placental insufficiency and fetal distress. The most common clinical manifestation of respiratory distress was hypercapnic respiratory failure, which could be due to underexpanded lungs. It was diagnosed in 3 cases and successfully managed by mechanical ventilation in heifer # 2’s calf (42).

Other reported anomalies, such as persistent hyperthermia (21,26), anemia (12,21,43), hypoglycaemia (8,21,44), and hyperglycemia (45), were also identified in this study. Although the exact cause of these anomalies remains unknown, in calves with LOS, an increased circulating blood volume without a concurrent increase in the number of red blood cells could explain a relative anemia (43). Endocrine profiles have shown an increase of thyroid hormone in cloned calves (26). This primary hyperthyroidism could explain hyperthermia due to hypermetabolism. Increased insulinemia (8), decreased cortisolemia (46), or variations in insulin growth factors (26,46) could also interfere with carbohydrate metabolism. Due to the various differences between cloned and other calves, it has been suggested that cloned calves be considered physiologically distinct from other calves until 2 mo of age (43).

Ultrasonographic assessment of the conceptus in late gestation provides enlightening information. Although ultrasonogrpahic data on bovine fetal well-being are limited, the value of ultrasonography has been investigated in humans (29) and horses (27,28). However, direct extrapolation of this information to cattle is not possible. In general, the examination technique was easy to perform and well tolerated by the heifers. Parameters that were included were similar to those included in equine biophysical profiles; however, the placentome appears to be the easiest unit to measure, as results from previous studies evaluating clone pregnancies have suggested (17,26). The mean estimated placentome surface area was larger than previously reported (35 cm² in our study vs 27 cm²) (17). Ultrasonography allowed the early identification of fetal anasarca, which indicates the potential of this technique for early detection of fetal abnormalities. Although this preliminary study emphasizes the potential value of ultrasonographic assessment of fetal well-being, further studies still have to be performed in normal pregnancies to describe what is a healthy fetus, as well as to assess the repeatability of this noninvasive assessement.

Assessment of fetal movements is limited in cows, because the depth of the maternal abdomen and the large size of the fetus prevent complete ultrasonographic visualization of the type of movement (47). Therefore, the proportion of time that the fetus was moving over the total observation period was used. All pregnancies in which fetuses obtained a score of 3 (moving more than 66% of the time) were born dead or died within 30 min. No fetus was totally inactive (score of 0). Hyperactivity may represent an abnormal sign and should be included in a bovine fetal well-being profile.

Fetal heart rate variability (FHRV) and fetal movements (FM) are considered important criteria in the assessment of the human fetal well-being (29,48). Fetal heart rate variability was also used to assess fetal well-being in a sheep model of compromised pregnancy (49). Short periods of fetal tachycardia are normal after FM in the bovine or equine species (22,50,51). However, persistent tachycardia or persistent bradycardia is associated with a poor neonatal outcome in the equine and ovine species (50,52). Fetal bradycardia was also an indicator of imminent fetal death in aborting Angora does (53). Mean estimated FHR was not correlated with fetal activity levels in the present study. Continuous FHR recording could be more useful than obtaining 3 to 5 measurements over a 30-minute period, as in this study, since heart rate variability (acceleration and deceleration slopes) and its correlation with fetal movement could then be studied (54).

Reduced maximal depth of fetal fluid was associated with respiratory distress in heifer # 2. This finding may suggest that oligohydramnios is associated with fetal distress, as has been noted in humans and horses (28,29). Therefore, reduction in the quantity of fetal fluid near term in pregnancy should be considered a possible abnormal finding. Echogenic particles in bovine fetal fluids have classically been associated with severe fetal distress or fetal death (47). Fetal fluid scores of 2 or 3 were associated with a poor outcome (hydrallantois, neonatal mortality, or severe cardiorespiratory distress). Moderate amounts of particulate matter in the fetal fluid should be monitored over time; when they are present, complications should be anticipated.

The measurement of amniotic membrane thickness appeared to be an important criterion in the diagnosis of hydrallantois. This membrane is easily observed between the allantoic and amniotic fluids. The amniotic membrane was thickened in the case of hydrallantois. Edema of fetal membranes has been noted macroscopically during cesarean-sections performed on cases of hydrops (35,55).

This study confirms the current problems encountered in bovine somatic cloning. These various problems are associated with poor maternal condition, placental anomalies, and various neonatal problems. This is the first attempt to describe a bovine fetal biophysical profile in late pregnancy. The ultrasonographic findings provide information in regard to fetal well-being that appears to be promising for the detection of fetal congenital anomalies, as well as fetal distress. As in other species, the ultrasonographic assessment of late pregnancy in cattle could help the veterinarian to manage high risk pregnancies.