Preliminary result of this study was presented as a poster at the ACVIM Forum 2006, Seattle. This work was performed at the University Veterinary Teaching Hospital at The Faculty of Veterinary Medicine in Saint-Hyacinthe—Université de Montréal.
Corresponding author: Anne-Claire Brisville, Dre Med Vet, IPSAV, Hopital des animaux de la Ferme, Centre Hospitalier Universitaire Vétérinaire, Faculté de Médecine Vétérinaire—Saint-Hyacinthe, Université de Montréal, 3200 rue Sicotte, CP 5000, Saint-Hyacinthe, QC, Canada J2S 7C6; e-mail: email@example.com.
Background: Numerous clinical abnormalities occur in cloned calves during the neonatal period.
Methods: Retrospective clinical study of the cloned calves born at the Veterinary Teaching Hospital, Saint-Hyacinthe, QC.
Results: Records of 31 cloned calves were reviewed. Twenty-five records were included. Four stillborn calves and 2 calves euthanized at birth were excluded. Twenty-two calves suffered from respiratory diseases. Nineteen calves received intranasal oxygen treatment (INO). They were tachypneic (78 breaths per minute) and 5 of them were hypoxemic (PaO2 < 55 mmHg). Two of 19 calves remained hypoxemic despite INO. Thirteen calves were weaned from INO after a median of 70 hours and were discharged at a median of 5 days of age. Nine calves required ventilatory support: 3 from birth and 6 after INO. Five were successfully weaned from the ventilator after a median of 32 hours and were discharged at a median of 8 days of age. Three calves died and 1 was euthanized because of respiratory disease. Necropsy revealed atelectasis, pulmonary congestion, and alveolar damages.
Conclusion and Clinical Importance: Respiratory disease occurs frequently in cloned calves. The most frequent abnormality is hypoxemia because of V/Q mismatch. It is possible to successfully support these calves by INO and mechanical ventilation.
partial pressure of carbon dioxide in arterial blood (in mmHg)
partial pressure of oxygen in arterial blood (in mmHg)
positive pressure ventilation
respirations per minute
Nuclear transfer is a reproductive technology used to produce genetically identical individuals or clones. This technique, which has enabled the birth of healthy individuals in several animal species including cattle,1,2 has many applications.3 However, the effectiveness of this technique is limited. Overall efficiency of somatic cell nuclear transfer was estimated to 4% (number of newborn animals in good health per reconstructed embryos), regardless of species.4 Another reported problem for cattle is high neonatal death rates (from 12.5 to 42%).5–7 Among the main problems at birth are high birth weight or large offspring syndrome,8 myo-arthro-skeletal malformations,9 respiratory difficulties,6 and metabolic abnormalities.10,11
Between 2004 and 2007, the University Veterinary Teaching Hospital, at Université de Montréal in Saint-Hyacinthe, was involved with the birth and the first few days of postpartum care in newborn calves derived from somatic cloning. Respiratory abnormalities were one of the main problems observed. The first objective of this study was to describe the abnormalities in respiratory function of cloned calves at birth and throughout the neonatal period. The second objective was to describe the indications, methodology, calves' response and complications of 2 interventions uncommonly used in cattle: intranasal oxygen treatment (INO) and mechanical ventilation.
Materials and Methods
The medical records of all cloned Holstein calves born at the University Veterinary Teaching Hospital between 2004 and 2007 were studied retrospectively. Observations were divided into a “Birth Period” (from birth to 1 hour of age) and a “Neonatal Period” (from 1 hour of age to discharge from the hospital). The duration of gestation, induction technique (if used), and type of parturition were recorded (vaginal versus cesarean section). Body weight, heart rate, behavior (alert or depressed), appetite, respiratory rate (RR), and effort were extracted from the physical examination at birth and at specific times during the neonatal period. Therapies used to support pulmonary function (INO, manual or mechanical positive pressure ventilation [PPV]), the time they were implemented, the duration of their administration, and the calves' response to treatment (improvement or deterioration of behavior, RR, complications, and survival) were all recorded. Additional treatments were also recorded.
Hematology results, thoracic radiographs, and results of blood gas analyses were reviewed. The hematologic parameters recorded included red cell count, hemoglobin, and hematocrit levels at birth (<10 minutes after umbilical cord section). Radiographically visible pulmonary lesions were characterized by board-certified radiologists, according to pattern (interstitial or alveolar), severity (mild, moderate, or severe), and distribution (localized or diffuse). When available, serial thoracic radiographs were characterized as improved, unchanged, or worse relative to previous radiographs. Blood gas parameters selected to characterize calves' respiratory diseases or calves' response to treatment included pH, partial pressure of oxygen and carbon dioxide (PaCO2 and PaO2), and blood levels of bicarbonate and lactate in either venous or arterial blood. The alveolar-arterial oxygen gradient (Aa) was calculated from the PaO2 and before oxygen supplementation using the formula Aa = [0.21 × (750–47)−PaCO2/0.8]−PaO2.12 Barometric pressure in Saint-Hyacinthe is 750 mmHg). The PaO2/FIO2 ratio was not used because the percentage of inspired oxygen was unknown when the animals were receiving intranasal oxygen. Calves were considered survivors if they were discharged from the hospital. Necropsy reports of the nonsurviving calves were reviewed when available.
Normal blood gas values for newborn calves13 were used as a the reference range. A calf was considered tachypneic if the RR was higher than 60 respirations per minute (rpm)14 and bradypneic if RR was below 30 rpm.14 Signs of dyspnea included increased respiratory effort and abdominal breathing.15
Arterial blood was collected from the medial branch of the caudal auricular artery via a 24 G, 0.75-in. catheter. The samples were analyzed within 2 minutes with a blood gas analyzer.a
INO and mechanical PPV (mechanical PPV) techniques were described.
Continuous data were summarized by mean and standard deviation, or median and range when not normally distributed. Noncontinuous data were described using frequencies. T-test for inequal variance was used to compare the analyses of blood gases and CBC at birth between groups of cloned calves.
Thirty-one medical records were studied. Six of them were excluded: 4 dealt with stillborn animals and the other 2 with animals euthanized <6 hours after birth because of severe physical deformities such as arthrogryposis (severe contracture of all limbs). Twenty-five cases were finally included. The mean gestation period was 274 days (standard deviation: 2.6 days, between 269 and 279 days). Parturition was induced by intramuscular injections of dexamethasone (20 mg)b and cloprostenol (25 mg).c All calves were born by left flank cesarean section 24 hours after induction. The mean birth weight was 56 kg (standard deviation = 9.1 kg). There were 5 females (median = 65 kg; from 35 to 82 kg) and 20 males (median = 54 kg; 45 to 72 kg).
Abnormalities of Respiratory Function during Birth Period
Eighteen of the 25 calves were breathing spontaneously at birth (Fig 1) with an effective RR, whereas seven had severe bradypnea (<10 bpm) or apnea: they were intubated at birth with an orotracheal tube and manually ventilated. Four of them were extubated after 15–60 minutes of manual PPV when they demonstrated an effective respiration. In 3/7 calves, respirations did not improve and these calves were switched from manual PPV to mechanical PPV with an initial FIO2 set at 100%.
An analysis of venous blood taken from the jugular vein was performed on all calves within 5 minutes of birth and before any treatment was initiated (Table 1). Calves with a low RR at birth had statistically lower pH (P= .002), bicarbonates (P= .01), hemoglobin (P= .001), and hematocrit (P= .01) and statistically higher partial pressure of carbon dioxide in venous blood (in mmHg) (P= .01). The different mean lactate concentration was not statistically significant (P= .057).
Table 1. Analysis of venous jugular blood samples from neonatal cloned calves.a
Calves with an Effective Respiratory Rate at Birth (n = 18)
Calves with a Noneffective Respiratory Rate at Birth (n = 7)
PvCO2, partial pressure of carbon dioxide in venous blood.
Values are reported as mean ± standard deviation.
Samples were obtained prior to any treatment and within 5 minutes of birth.
pH (P= .002)
7.22 ± 0.08
7.06 ± 0.08
PvCO2 (mmHg) (P= .01)
68.9 ± 9.0
79.8 ± 7.8
Lactate (mmol/L) (P= .057)
9.1 ± 2.8
13.1 ± 4.4
HCO3− (mmol/L) (P= .01)
28.1 ± 3.5
22.1 ± 4.0
Red cell count (× 106/μL)
6.08 ± 1.02
5.31 ± 1.18
Hemoglobin (g/dL) (P= .001)
8.0 ± 1.8
5.6 ± 1.2
Hematocrit (%) (P= .01)
27.8 ± 5.9
21.6 ± 4.2
Abnormalities of Respiratory Function during Neonatal Period
Among the 18 calves that had an effective RR at birth, 9 developed respiratory diseases within the first 12 hours of age (mostly within 2 hours of age except for 2 calves at 4 and 9 hours of age) whereas 6 developed respiratory diseases after 24 hours of age. The remaining 3 calves remained healthy and were discharged from the hospital within 2 days of birth. Of the 7 calves that had an ineffective RR at birth, 3 required prolonged mechanical ventilation during the neonatal period, 3 had persistent respiratory diseases after initial cessation of manual PPV, and 1 was able to breath normally after initial manual PPV, but developed respiratory abnormalities after 24 hours of birth. Respiratory abnormalities observed included dyspnea (22/22), tachypnea (14/22), bradypnea (3/22), hypoxemia (5/22), and hypercapnia (19/22 calves, of which 8 had a PaCO2 >80 mmHg).
Thoracic radiographs were performed in 21/22 calves within 6 hours after birth (Table 2). Thoracic radiographs could not be performed in 1 calf because it was receiving mechanical PPV and was considered too unstable to be transported to the imaging room. This calf eventually died. Thoracic radiographs were repeated at 1 day of age in 17 calves, all of which had previous radiographic abnormalities. Comparing radiographs at birth and 1 day of age in the calves that died, all calves had severe progressive radiographic lesions.
Table 2. Comparison of thoracic radiographic lesions and outcome in 21 cloned calves diagnosed with respiratory problems.
Classification of Thoracic Radiograph Lesions
Calves that Lived (n = 18)
Calves that Died (n = 3)
Normal thoracic radiographs
RR was recorded for 18/19 calves immediately before oxygen treatment. Their mean RR was 74 ± 14 rpm, with 14/18 being tachypneic. An arterial blood gas analysis was performed in 10 calves before receiving intranasal oxygen (Figs 2 and 3). All of them were tachypneic. Five had a PaO2 lower than 55 mmHg (reference range = 55.3–88.5 mmHg) and 6 (including 5 hypoxemic calves) had a moderate degree of hypercapnia (Table 3). The Aa gradient was >30 mmHg for the 5 hypoxemic calves.
Table 3. Arterial blood gas analysis of 10 neonatal cloned calves before the initiation of intranasal oxygen therapy.
Number of Cloned Calves out of Reference Range
Aa, alveolar-arterial oxygen gradient; PaO2, partial pressure of oxygen in arterial blood; PaCO2, partial pressure of carbon dioxide in arterial blood.
Values are reported as median (minimum-maximum).
Reference from Bleul13 are reported as mean ± 2 standard deviations.
23.5 (calculated mean)
5/10 > 30
Therapies to Support Pulmonary Function
All 22 calves that developed respiratory diseases received supplemental oxygen treatment, either intranasally or via a respirator (Fig 1).
All calves suspected to have respiratory diseases received INO as a first-line treatment.
INO was administered via 1 or 2 intranasal flexible plastic tubes attached to a halter and inserted into the nostrils at a length of about 8–10 cm, depending on the size of the calf. The end of the plastic tubes had additional holes added to prevent occlusion of the tubes and to decrease the risk of “jet lesions” at high oxygen flow rates. The oxygen flow per tube ranged from 1 to 15 L/min (or 30 L O2/min per animal). The administered oxygen was humidified at room temperature and the positions of the tubes were verified every hour.
An arterial blood gas analysis was obtained from 17/19 calves receiving INO within 6 hours of initiating treatment. Fifteen calves had a PaO2 above 80 mmHg while receiving oxygen flow rates between 67 and 421 mL O2/kg/min. Their mean PaCO2 was 59.8 ± 10.1 mmHg and 10/15 were hypercapnic. Their mean RR was 67 ± 21 rpm and 8/15 were tachypneic. Two calves remained hypoxemic (PaO2= 33.2 and 41.5 mmHg) while receiving 238 and 200 mL O2/kg/min, respectively. Their respective PaCO2 was 55.7 and 103.9 mmHg. Their respective pH was 7.23 and 7.04. The former's RR was 72 rpm and the latter's was not available. These 2 calves were subsequently treated with mechanical PPV. Of the 19 calves treated with INO as a first-line treatment, 13 calves were successfully weaned off oxygen after a median of 70 hours (from 54 to 283 hours). They were subsequently discharged from the hospital. The remaining 6 calves were treated with mechanical PPV.
The complications observed during the administration of intranasal oxygen were minor. The removal of the tubes was observed with all calves either because the shape of the tube did not conform to their nasal morphology or because the tube was too short. Moreover, even when a tube was properly adjusted, the calves were able to dislodge the tubes via the process of licking. Another complication observed less frequently (n = 2) was the obstruction of the tubes by dried secretions. These complications were prevented by regularly checking the position of the tubes (every hour), adding additional holes to the tubes, adapting the shape of the tubes to the morphology of the calves, fixing the tubes in place with halters, and cleaning them and checking the permeability of the tubes, daily if necessary, by withdrawing them from the nose.
Low RR at birth, persistent hypoxemia despite INO, and hypoventilation, based on severe hypercapnia (PaCO2 > 80 mmHg), associated with acidemia (pH<7.2) were indications for mechanical PPV.
During mechanical PPV, calves were intubated with a nasotracheal tube (6–7.5 mm inner diameter) and placed on a respirator.d The calves were not sedated during mechanical ventilation unless necessary. Nasotracheal intubation allowed the calves to move freely and to be fed PO. Blood gas analysis was performed at intervals varying from 15 minutes to 2 hours and allowed the respirator to be adjusted to the needs of each calf. The objectives of mechanical ventilation were similar to those previously published in cloned calves.16 A Synchronized Intermittent Mandatory Ventilation (SIMV) mode was used in all calves. Delivered mechanical breaths per minute ranged between 24 and 42, dependent on PaCO2 and pH values. The FiO2 setting ranged between 30 and 100% and was adjusted to the minimal value that maintained PaO2 > 100 mmHg. The initial tidal volume was set at 5 mL/kg and was increased in 2 mL/kg increments up to 10 mL/kg, based on PaCO2 and pH values. Positive end-expiratory pressure was used to prevent alveolar collapse, improve alveolar recruitment, and decrease alveolar sheer stresses, and ranged between 3 and 7 cmH2O. Positive inspiratory pressures ranged between 20 and 30 cmH2O. The respirator settings were adjusted to maintain pH > 7.2, PaCO2 < 60 mmHg, and PaO2 above 100 mmHg.
Nine calves were mechanically ventilated during their neonatal period at the hospital (Fig 1): 3 at birth because of noneffective RRs, 2 because they remained hypoxemic despite INO, and 4 because they suffered from severe hypercapnia associated with acidemia later than 6 hours after initiating INO. Arterial blood gas analyses were obtained for 6 calves before starting mechanical PPV (Table 4). All 6 of these calves were receiving INO at the time blood gases were drawn.
Table 4. Arterial blood gas analysis of 6 neonatal cloned calves before mechanical positive pressure ventilation (PPV).
Number of Cloned Calves out of Reference Range
PaO2, partial pressure of oxygen in arterial blood; PaCO2, partial pressure of carbon dioxide in arterial blood.
Values are reported as median (minimum-maximum).
Reference from Bleul13 are reported as mean ± 2 standard deviations.
2/6 higher and 2/6 lower
Of the 9 calves mechanically ventilated, 5 calves were successfully weaned from the ventilator after a median duration of 32 hours (18–48 hours). One calf died after an episode of severe hypoventilation (PaCO2= 85.2 mmHg) 10 hours after the initial weaning from the respirator. The owner declined the recommendation to reinstitute mechanical PPV. Three calves were never weaned from the respirator. Two died of respiratory failure and the third was euthanized because of moderate arthrogryposis and lack of improvement in respiratory function despite mechanical PPV. They remained on a mechanical PPV with 100% oxygen for 13, 38, and 27 hours, respectively.
Few complications were observed with mechanical PPV. One calf struggled against the ventilator and had to be weaned early. Two calves extubated themselves during periods of agitation. They were reintubated immediately via the nasotracheal route and temporarily sedated with diazepam (0.1 mg/kg IV single dose).e Finally, partial obstruction of the nasotracheal tube by dry bronchial secretions was observed in 2 calves, which were mechanically ventilated for more than 24 hours. The tracheal tubes were cleaned via aspiration or replaced.
Four calves died, 3 spontaneously and 1 was euthanized (Fig 1). Necropsy reports were available for 3 of the calves. One or more of the following lung lesions were present in each of these calves: pulmonary congestion, atelectasis, or diffuse alveolar damages with presence of material in the alveolar space (fibrin [n = 2], keratinized cells [n = 1], hyaline membranes [n = 1], and meconium [n = 1]). In addition emphysema (n = 1), thoracic and pericardial effusion (n = 1), hepatic congestion (n = 2), and a patent foramen ovale (n = 1) were also observed. A presumptive diagnosis of hyaline membrane disease or pulmonary immaturity was described for each of the 3 necropsies. The calf whose necropsy report was not available died of suspected respiratory failure (last arterial blood gas levels on mechanical PPV: pH = 7.12, PaO2= 36.2 mmHg, PaCO2= 92.1 mmHg, bicarbonates = 29.3 mmol/L, lactate = 2.5 mmol/L). Furthermore, the 4 stillborn calves had a postmortem examination. The findings included signs of chronic lung inflammation (macrophages were observed within the pulmonary lesions).
The other 21 calves (Fig 1) left the hospital in good health. The mean duration of hospitalization for the cloned calves was 6.3 days (standard deviation: 4.1, between 1 and 21 days). The 3 calves that did not suffer from respiratory diseases were discharged after a median hospital stay of 1 day (1–2 days). The calves that received only intranasal oxygen left the hospital after a median hospital stay 5 days (3–14 days) and 10/13 remained hospitalized for less than a week. Finally, the calves mechanically ventilated were discharged from hospital after 6, 5, 8, 14, and 16 days.
Respiratory diseases in the neonatal period have been reported with an incidence varying from 0 to 90% in neonatal cloned cattle.6,7,9–11 Our observations are consistent with the literature that respiratory diseases are a limiting factor in the survival of cloned calves during the neonatal period. Excluding calves euthanized because of malformations, respiratory diseases were the only cause of death among calves during their hospitalization.
Respiratory diseases affecting cloned calves were characterized by varying degree of hypoxemia, hypercapnia, and tachypnea. There are a number of possible underlying conditions that have been reported in the bovine, equine, and human literature that might explain the respiratory diseases observed in this study.
One pattern of respiratory disease observed in cloned calves was a combination of hypoxemia, mild hypercapnia, and elevated Aa. The condition developed within 1–24 hours after birth, and a favorable response to INO was observed. These blood gas abnormalities were consistent with a moderate degree of hypoventilation and ventilation-perfusion mismatch (V/Q mismatch). The pattern could be comparable to transient tachypnea of the newborn in human neonatalogy17 or idiopathic transient tachypnea in equine neonatalogy.18 In human newborns, moderate hypoxemia and tachypnea developed within 6 hours of birth, secondary-to-delayed resorption of fetal pulmonary fluid.17 Disease is self-limiting and resolves in 3–4 days.17 In equine neonatalogy, tachypnea developed in hyperthermic foals in hot and humid environments, as a result of presumptive imbalance of thermoregulation.18 Cloned calves from this study showed no increase in rectal temperature at the onset of respiratory abnormalities and the ambient temperature was controlled. Delayed resorption of pulmonary fluid is a plausible hypothesis. It can be further noted that cesarean births have been identified as a risk factor for the onset of transient tachypnea of the newborn in human neonatalogy.17
A 2nd pattern of respiratory disease observed in cloned calves was a severe hypoxemia that did not improve with INO. This pattern could be consistent with a right to left shunt resulting from a persistent fetal circulation. Failure of closure of the foramen ovale and ductus arteriosus, resulting in a persistent pulmonary hypertension, is described in human,19 equine,18 and bovine neonates.7,20 In calves, clinical signs include hypoxemia, mild hypercapnia, tachypnea, and increased pulmonary artery pressure.7,20,21 Signs of pulmonary hypertension have previously been reported in one cloned calf that died of respiratory diseases in the neonatal period.7 The pulmonary artery pressure was not measured in the cloned calves of this study because of the invasiveness of the procedure.
A 3rd pattern of respiratory disease observed in cloned calves was a combination of hypoxemia and severe hypercapnia that developed within 6 hours of birth. These blood gas abnormalities were consistent with hypoventilation and V/Q mismatch. The importance of V/Q mismatch was difficult to assess as no arterial blood gas analysis was available before oxygen supplementation and alveolar-arterial gradients were not estimated. However, this pattern could be comparable to neonatal respiratory distress syndrome described in human22,23 and bovine neonates.24,25 Respiratory distress syndrome result from surfactant deficiency22 and occurred in premature newborns or secondary to inactivation of surfactant by meconium or lung inflammation.26 Without functional and sufficient surfactant, the pulmonary alveoli collapse, attributable to surface tension, and therefore breathing effort increases.22 The newborn progresses to a state of exhaustion and breathing become inefficient. In human neonatology, neonatal respiratory distress syndrome is self-limiting and mechanical ventilation can support respiratory function until the newborn produces surfactant in sufficient quantity and quality.22
The administration of INO is a common supportive treatment used in large animals.16,27–29 In this retrospective study, the decision to initiate the INO was based on an analysis of arterial blood gases when available. When arterial blood gas analysis was not available, the following clinical parameters were used: alertness, quality of breathing, RR, interest in their surroundings, and ability to stand or suckle. Although these clinical parameters are subjective, the clinical experience of this study suggests they are important clues to the onset of respiratory diseases in newborn cloned calves. Other teams who have worked with cloned calves have chosen to systematically administer intranasal oxygen at birth for at least 30 minutes.7,10 The incidences of respiratory diseases in these studies were 37.57,10 and 50%,7 which are lower than in our study (88%, 22/25). Thus the routine use of intranasal oxygen appears to be a valuable option in the management of cloned calves during the neonatal period.
The use of mechanical ventilation has been reported in equine neonatal intensive care units.29–31 There are only two publications reporting the use of mechanical ventilation in cattle, both in newborn cloned calves.7,16 Newborn calves tolerated mechanical ventilation without sedation and nasotracheal intubation allows calves to continue suckling while on a respirator. Animals can therefore be maintained on a respirator for extended periods of time without the risks associated heavy sedation or anesthesia.
A final interesting observation concerning this study is the advantages and limits of arterial blood gas analyses. An increase in RR and effort prompted clinicians to supplement these animals with intranasal oxygen upon clinical suspicion of hypoxemia. The treatment led to clinical improvement, including the return of a suckling reflex. For some of these animals, an arterial blood gas analysis was available just before the start of oxygen treatment. Half of these calves had normal PaO2 values compared with published reference ranges.13 Two hypotheses may explain the difference between clinical suspicion and laboratory analysis. First, the published values,13 which mainly refer to Simmental and Brown Swiss calves born after natural parturition, might be inadequate as a reference range for Holstein cloned calves born by cesarean section. Breed, prematurity, and birth method could be factors influencing PaO2 in the neonatal period. Blood gas values obtained from healthy newborn Holstein calves without respiratory difficulties, born by cesarean section, may be more comparable references for the cloned calves in our study. Second, cloned calves may have suffered from some degree of hypoxia despite a PaO2 considered normal according to Bleul et al.13 Severe anemia can cause hypoxia independent of PaO2 and oxygen saturation values. Also, a structural abnormality of hemoglobin, modifying its dissociation curve, would decrease oxygen delivery to the tissues. Anemia was observed in the cloned calves of this study, but it is difficult to determine if it was severe enough to cause hypoxia. In addition, the calves were weaned from oxygen after approximately 3 days and without transfusion treatment. This duration seems insufficient to coincide with the correction of anemia.
The incidence of respiratory problems in cloned calves is high. Different patterns of respiratory problems were observed. It is plausible that more than one disease affects cloned calves, and the exact causes of respiratory problems still needs to be determined. In addition, INO and mechanical ventilation could be used in neonatal cloned calves without severe complications. Attentive monitoring is still required and may include arterial blood gas analysis and radiography.
aStat profile M, Nova Biomedical, Mississauga, ON
bDexamethasone 5, Vétoquinol, Lavaltrie, QC
cLutalyse, Pfizer, London, ON
dEsprit Ventilator, Respironics Inc, Vista, CA
eValium, Roche, Nutley, NJ
The study was supported by CRSNG (Natural Sciences and Engineering Research Council of Canada).
The authors thank The Semex Alliance for their support in this study, Carl Bernard for his technical help during the hospitalization of the cloned calves, and Guy Beauchamp, who performed the statistical analysis of this study.