The study was done at the veterinary teaching hospital of the University of Montreal
Neonatal Morbidity and Mortality of 31 Calves Derived from Somatic Cloning
Version of Record online: 19 JUN 2013
Copyright © 2013 by the American College of Veterinary Internal Medicine
Journal of Veterinary Internal Medicine
Volume 27, Issue 5, pages 1218–1227, September/October 2013
How to Cite
Brisville, A.-C., Fecteau, G., Boysen, S., Desrochers, A., Dorval, P., Buczinski, S., Lefebvre, R., Hélie, P., Blondin, P. and Smith, L.C. (2013), Neonatal Morbidity and Mortality of 31 Calves Derived from Somatic Cloning. Journal of Veterinary Internal Medicine, 27: 1218–1227. doi: 10.1111/jvim.12129
- Issue online: 13 SEP 2013
- Version of Record online: 19 JUN 2013
- Manuscript Accepted: 14 MAY 2013
- Manuscript Revised: 22 FEB 2013
- Manuscript Received: 17 JUL 2012
- Strategic Grant of the NSERC (Natural Sciences and Engineering Research Council)
- Physical congenital defect;
- Umbilical infection
The neonatal period is associated with high morbidity and mortality in cloned calves.
To describe morbidity and mortality in cloned calves from birth to 2 years of age.
Thirty-one somatic cell-derived Holstein calves delivered at a veterinary teaching hospital.
Medical files were retrospectively analyzed.
Four calves were stillborn. Five calves born alive had physical congenital defects. Twenty-three calves had an enlarged umbilical cord. Laboratory abnormalities included acidemia, respiratory acidosis, hyperlactatemia, anemia, stress leukogram, decreased total protein, albumin and globulins, and increased creatinine. Twenty-five calves survived the 1st hour of life. Among them, 11 stood without assistance within 6 hours of birth, 10 calves took longer than 6 hours to stand, and 4 never stood. Twenty-two calves suffered from anorexia. Twelve calves had complications arising from umbilical cord infections. Three calves developed idiopathic hyperthermia (>40°C). Eight calves suffered from gastrointestinal problems, including ruminal distension, abomasal ulcers, neonatal enteritis, intussusception, and abomasal displacement. Mortality between birth and 3 weeks of age was 32% (10/31). Causes of death and reasons for euthanasia included stillbirths, respiratory failure, and limb deformities. Mortality between 3 weeks and 2 years of age was 19% (4/21), with deaths in this group attributed to generalized peritonitis and complications arising from umbilical infections. Overall, mortality rate within 2 years of age was 14/31 (45%).
Conclusion and Clinical Importance
Respiratory problems, limb deformities, and umbilical infections were the most common causes of morbidity and mortality in these cloned calves.
complete blood cells count
Centre Hospitalier Universitaire Vétérinaire
arterial partial pressure of carbon dioxide
venous partial pressure of carbon dioxide
arterial partial pressure of oxygen
positive pressure ventilation
venous partial pressure of oxygen
- Sat O2
hemoglobin saturation of oxygen
Over the past 25 years, several animal species have been successfully cloned from somatic cell lines including cattle.[1, 2] Unfortunately, neonatal mortality rates of cloned calves remain high.[1-9] The main complications reported at birth include overweight or large offspring syndrome, limb flexural deformity, respiratory problems, and metabolic abnormalities.[12, 13] Reports concerning cloned calf neonatal health are based on experimental studies comparing cloned calves with calves born from artificial insemination or embryo transfer,[7, 12-14] or observational studies reporting clinical and pathologic features of cloned calves.[7, 9, 11] These reports have not addressed interventional therapies or intensive management of numerous cloned calves with details.
The objectives of this study were to report the causes of morbidity and mortality in cloned calves and to describe their management in a veterinary hospital facility. Our hypotheses were that cloned calves would develop life-threatening neonatal complications, including respiratory difficulties, and would need intensive management in a dedicated calf neonatal intensive care unit. Respiratory difficulties have already been reported and will be summarized in this article.
Material and Methods
Between 2004 and 2007, the Centre Hospitalier Universitaire Vétérinaire (CHUV) of the University of Montreal in Saint-Hyacinthe was involved with the birth and the first few days of postpartum care of newborn calves derived from somatic cloning.
Evaluation of Health at Birth
Medical records were retrospectively reviewed. Abnormalities concerning calving, the mother, and the fetal membranes were noted when available. The calves' clinical variables at birth were recorded (congenital physical abnormalities, weight, heart rate, respiratory rate and effort, rectal temperature, and awareness). Complications that arose (umbilical bleeding, apnea, dyspnea) and supportive techniques used at the time of birth also were recorded. Blood was collected from the jugular vein of live calves within 5 minutes of birth. Serum biochemistry, complete blood cells count (CBC), and venous blood gas analyses were performed.
Evaluation of Health after Birth
The observation period extended from birth until 2 years of age. In this study, the hospitalization period referred to the period from birth to discharge from the hospital (or death if the calf was not discharged). The follow-up period referred to the period between discharge of the calf from the hospital and 2 years of age.
Medical records from the hospitalization period were retrospectively reviewed with particular emphasis on physical examination findings (heart rate, respiratory rate, rectal temperature, size, and appearance of the umbilicus) and behavior (time needed for the calf to stand without assistance, awareness, suckle reflex, and appetite). The results of laboratory tests carried out (complete blood count, serum biochemistry panel, bacteriological culture of umbilical remnants, thoracic radiographs, and blood gas analyses) were used to identify the major abnormalities and assess their progression.
During the follow-up period, phone contact was maintained with the owner to determine if calves remained healthy and gained weight appropriately (based on visual examination of appetite and general behavior). If calves were readmitted to the hospital, their records were reviewed to determine the cause of admission, the suspected diagnosis, and the outcome. Physical examination findings, diagnostic tests, and treatments were recorded.
A respiratory problem was considered when at least one of the following clinical signs was present: dyspnea (increased respiratory effort, abdominal effort), apnea (absence of spontaneous breathing), bradypnea or tachypnea, hypoxemia or hypercapnia. Anorexia was defined as a calf refusing to nurse and needing to be fed via an esophageal tube. An enlarged umbilicus was subjectively diagnosed when the attending clinician made note of an enlarged umbilicus in the medical record at the time of birth. An umbilical infection was suspected based on the presence of a swollen, painful, malodorous and wet navel, sonographic evidence of an enlarged internal umbilical structure with heterogeneous material in the lumen, or some combination of these findings. An umbilical infection was diagnosed when bacterial cultures (taken at the time of surgical excision of the umbilical remnants) were positive. Other abnormalities were diagnosed when recorded variables fell outside normal reference intervals for healthy newborn calves (hyper- or hypothermia, for example).
Contaminated colostrum may be associated with decreased immunoglobulin absorption and subsequently contribute to failure of passive transfer.[16, 17] To determine the number of bacterial colony-forming units (CFU) per ml of colostrum, thawed samples of frozen colostrum were submitted for bacterial culture. Results of bacterial culture were recorded.
All available necropsy reports were reviewed.
Neonatal calf management was described based on the review of individual medical records, archived photographs, and videos. The management goal was to establish protocols for intensive care facility set-up, serial evaluations, diagnostic testing, interventions, and therapies.
Variables of interest were reported as mean and SD or median, 5–95% percentiles, and range (when sample size was smaller than 10 or data were not normally distributed). The mode also was used once.
Animals (n = 31)
Thirty-one cloned calves were delivered in 5 groups or cohorts between August 2004 and June 2007 (Table 1). All surrogate mothers were Holstein heifers. The lineage of 4 calves from the 1st cohort was not available, whereas the remaining 27 calves from various cohorts were derived from 12 different genetic lineages (1–5 calves per line, Table 1).
|Genetic Line Cell||Not Available||A||B||C||D||E||F||G||H||I||J||Total|
Three heifers of the 1st cohort were admitted to the hospital on an emergency basis in advance of their anticipated due dates: 2 had severe hydrallantois (at day 262 and 267 of gestation) and the third showed signs of premature parturition (at day 269 of gestation). They had emergency cesarean sections to deliver 3 stillborn calves.
A cesarean section was carried out for each of the other 28 surrogate heifers, before natural delivery, at 274 ± 2.5 d gestation. Each heifer received 20 mg of dexamethasone1 and 25 mg of prostaglandin F2α2 24 hours before surgery. A left flank cesarean section was performed before the heifers entered labor. Cesarean sections were performed using a 2% lidocaine3 regional block (proximal paravertebral block of nerves T13, L1, and L2) with the animals standing. Before surgery, the heifers received penicillin procaine G4 (21,000 UI/kg, IM) and flunixin meglumine5 (1.1 mg/kg, IV). Twenty-eight cloned calves were delivered: 1 stillborn calf with anasarca and 27 live calves.
Clinical and Physical Characteristics of Cloned Calves Alive at Birth (n = 27)
The average birth weight was 56 ± 9.1 kg. Five calves were female (median, 65 kg; range, 35–82 kg; percentiles 5–95%, 39–78.6 kg) and 22 were male (median, 54 kg; range, 45–72 kg; percentiles 5–95%, 49–64.8 kg; Fig 1).
Seven calves were severely bradypneic (respiratory rate <10 breaths per minute) or apneic at birth. Orotracheal tubes were passed and positive pressure ventilation (PPV), using an Ambu Bag,6 was initiated in these 7 calves. One of these calves experienced cardiopulmonary arrest and underwent successful closed chest cardiopulmonary resuscitation, which included a single IV dose of epinephrine6 (0.15 mg/kg, once, IV). Unfortunately, this calf therefore was euthanized at 1 day of age. Two calves that remained bradycardic, despite intubation and PPV, received a single dose of IV epinephrinef (0.15 mg/kg, once, IV). Two of the 7 calves received a single dose of caffeine8 (100 mg/calf, once, PO) 2 hours after birth. Four of the 7 calves were successfully weaned from PPV and extubated within 15–60 minutes of starting PPV (including the two that received IV epinephrine for persistent bradycardia). Three of the 7 calves remained apneic and became hypercapneic when trying to wean them from PPV, and were switched to mechanical ventilation with 100% oxygen.
Five calves had flexural limb deformities of variable severity. Two were severely affected and subsequently euthanized within the 1st hour after birth. A 3rd calf was euthanized at 24 hours of age attributable to both moderate flexural limb deformities and respiratory failure that did not improve after mechanical ventilation. Two calves suffered moderate carpal flexure deformities that resolved with physical treatment.
The attending clinician reported that the umbilical cords of 23 cloned calves were enlarged (information on the umbilical cord was not available for 4 calves in the first cohort). The size of the umbilical cords was estimated to be up to 5 cm in diameter (based on photographs), but a systematic umbilical cord measurement was not performed or reported in the records. For 10 calves (from the 2nd and 3rd cohorts), the surgeon stretched the cord manually during the cesarean section, tearing it within approximately 3 cm of the abdominal wall. For 13 calves (from the 4th and 5th cohorts), the surgeon ligated the umbilical cord with 2 plastic sterile self-tightening clamps, then an emasculator was positioned on the umbilical cord before it was cut with a scalpel 7–10 cm from the abdominal wall. Five of these calves had recurrent umbilical bleeding within 6 h of birth. Additional plastic clamps were placed proximal to the previously applied clamps. Information regarding umbilical cord management was not available for 4 calves (1st cohort).
Venous blood gas analyses, CBC, and biochemical results are presented in Tables 2-4. Cloned calves were acidotic, suffering from both respiratory and lactic acidosis. They also suffered from normocytic hypochromic anemia, stress leukogram (leukocytosis, neutrophilia, and lymphopenia), hypoproteinemia (with both hypoalbuminemia and hypoglobulinemia), and had increased serum creatinine concentration.
|Parameters||Cloned Calves (n = 27)||Reference Range|
|pH||7.18 ± 0.11||7.35–7.45|
|PvO2 (mmHg)||23.5 ± 6.8||nd|
|PvCO2 (mmHg)||70.6 ± 10.1||35–45|
|Sat O2 (%)||35.2 ± 16.5||nd|
|Lactate (mmol/L)||8.67 ± 2.24||<2|
|HCO3− (mmol/L)||29.7 ± 1.8||22–28|
|Parameter||Cloned Calves||Reference Range|
|Erythrocytes (×106/μL)||5.9 ± 1.1||5–10|
|Hemoglobin (g/dL)||7.42 ± 1.93||8–15|
|Hematocrit (%)||26.3 ± 6.0||24–46|
|Mean cell volume (fL)||44 ± 5.6||40–60|
|Mean cell hemoglobin concentration (g/L)||281 ± 16.5||300–360|
|Leukocytes (×103/μL)||12.2 ± 4.4||4–12|
|Neutrophils (×103/μL)||9.4 ± 3.8||0.6–4|
|Lymphocytes (×103/μL)||1.3 ± 0.5||2.5–7.5|
|Monocytes (×103/μL)||0.3 ± 0.4||0–0.8|
|Eosinophils (×103/μL)||0 ± 0.01||0–0.9|
|Platelets (×103/μL)||548 ± 174||100–800|
|Fibrinogen (mg/dL)||230 ± 130||0–500|
|Total solids (g/dL)||5.08 ±0.42||4.2–5.4|
|Parameters||Cloned Calves (n = 27)||Reference Range|
|Glucose (mg/dL)||50.4 ± 25.2||47–88|
|Blood urea nitrogen (mg/dL)||10.8 ± 1.8||4.5–18.2|
|Creatinine (mg/dL)||2.04 ± 0.43||0.6–1.4|
|Bilirubin (mg/dL)||0.54 ± 0.15||0–0.8|
|AST (UI/L)||16 ± 5||30–104|
|GGT (UI/L)||8 ± 2||9–39|
|CK (U/L)||120 ± 182||0–310|
|Protein (g/dL)||4.4 ± 0.4||5.9–8|
|Albumin (g/dL)||2.2 ± 0.2||2.7–4|
|Globulins (g/dL)||1.8 ± 0.2||2.6–4.5|
|Sodium (mEq/L)||143 ± 3||134–147|
|Chloride (mEq/L)||99.2 ± 3.6||96–109|
|Potassium (mEq/L)||4.7 ± 0.5||3.8–5.3|
|Total calcium (mg/dL)||12.8 ± 0.8||8.8–10.8|
|Phosphorus (mg/dL)||6.8 ± 0.6||3.4–8.7|
|Total CO2 (mEq/L)||23.8 ± 4.3||22–33|
|Anion gap (mEq/L)||25 ± 4||7–18|
Observations after Birth during the Hospitalization Period (n = 25)
Twenty-five of the cloned calves were alive an hour after birth.
Seventeen calves (median weight, 53 kg; percentiles 5–95%, 45–61 kg; range, 45–61 kg) stood without assistance within 24 hours of birth: 11 within 6 hours (median weight, 51 kg; percentiles 5–95%, 45–60 kg; range, 45–60 kg) and 6 between 6 and 24 hours of age (median weight, 58 kg; percentiles 5–95%, 54–61 kg; range, 53–61 kg). Four calves (median weight, 61 kg; percentiles 5–95%, 51–79 kg; range, 50–82 kg) stood without assistance between 3 and 5 days of age: they suffered from muscle weakness and received respiratory support (oxygen treatment, mechanical ventilation, or both). Four calves (median weight, 49.5 kg; percentiles 5–95%, 37–63 kg; range 35–65 kg) never stood: they suffered from severe respiratory disease that resulted in death or euthanasia. One of them also had mild flexural limb deformities.
Twelve calves had a strong suckle reflex and drank colostrum with vigor, whereas 12 others had to be fed via an esophageal tube. Pooled frozen colostrum from several animals was provided by the owner. Samples of thawed colostrum from the last 3 cohorts were submitted for bacterial culture. Bacterial growth was found in 14 of the 15 samples submitted: 1 to 4 different bacteria were isolated from each sample analyzed. The isolated bacteria were mammary pathogens including Staphylococcus aureus (5/15), normal inhabitants of bovine skin or mucosa including Staphylococcus spp. (9/15), Corynebacterium spp. (2/15) and Streptococcus spp. (1/15), fecal contaminants including Enterobacter spp. (3/15), Klebsiella spp. (1/15) and Escherichia coli (1/15), and environmental contaminants including Pseudomonas spp. (6/15), Bacillus spp. (3/15), Micrococcus spp. (1/15), and gram-negative rods (1/15). Overall, 10/15 colostrum samples had total counts >5,000 CFU/ml of colostrum.
The smallest calf (35 kg) did not receive colostrum within the first 24 hours of birth because esophageal intubation was not possible. It was transfused with 1 L of fresh plasma and treated with isotonic IV fluids and dextrose. It died at 1 day of age.
After initial administration of colostrum, 24 calves were bottle-fed 2–2.5 L of fresh milk 3 times per day (8:00 am, 4:00 pm, and 12:00 am). It corresponded to 10–13% of body weight of milk per day. Only 3 calves nursed normally during the hospitalization period. Twenty-one calves refused to nurse and were subsequently fed via an esophageal tube. They required intubation for a median of 7 feedings (min-max, 1–12; mode, 9 feedings). The calves were always encouraged to nurse before esophageal tube feeding.
Three calves showed no clinical or physical abnormalities during the hospitalization period, required no further medical care, and subsequently were discharged at 1 and 2 days of age. The other 22 calves suffered at least 1 pathological abnormality during the hospitalization period.
Twenty-two calves suffered from respiratory problems during the hospitalization period: 7 at birth, 9 within 12 h of age, and 6 later than 24 hours of age. These findings have been previously published. Respiratory problems included dyspnea (22/22), tachypnea (14/22), and bradypnea or apnea (7/22). Nineteen calves initially were treated with intranasal oxygen supplementation. Ten of these had arterial blood gas analysis before oxygen supplementation, which documented hypoxemia in 5 calves (PaO2 < 55 mmHg; reference range, 55.3–88.5 mmHg). Arterial blood gas analysis was performed in 17/19 calves receiving oxygen supplementation within 6 hours of placing the intranasal lines. Fifteen calves had PaO2 > 80 mmHg and 2 calves remained hypoxemic (PaO2, 33.2 and 41.5 mmHg) despite oxygen supplementation. These 2 calves subsequently were started on mechanical ventilation. Overall, 9 calves were mechanically ventilated during the hospitalization period: 3 at birth because of apnea, 2 because they remained hypoxemic despite intranasal oxygen treatment (one of which was also severely hypercapnic with PaCO2 of 103.9 mmHg), and 4 because they suffered from severe hypercapnia (PaCO2 > 80 mmHg). Five of the 9 mechanically ventilated calves were successfully weaned from the ventilator and 4 died or were euthanized because of respiratory failure. Their last blood gas analysis and respiratory parameters are presented in the Table 5. Calf 2 was euthanized because of poor prognosis associated with its respiratory problem and the flexural limb deformities.
|Calf 1 (4-day-old)||Calf 2 (1-day-old)||Calf 3 (1-day-old)||Calf 4 (1-day-old)|
|Respiratory rate (breath per min)||120||28||52||n/d|
|Respiratory support|| |
The umbilical vessels of 7 calves failed to retract into the abdomen within 24 hours of birth. They were surgically shortened to 5 cm from the abdominal wall within the 1st week of birth. The umbilical stump was dipped with a 5% iodine solution every 6 hours for 36 hours to help with infection control and promote drying of the umbilicus.
Three calves of the last cohort developed omphalitis during the hospitalization period with extension to ≥1 internal umbilical structures as determined by physical findings (ie, swollen, painful, malodorous, and wet navel), ultrasonography findings (ie, internal umbilical structure >1 cm diameter with heterogenous hyperechoic material in the lumen), and CBC results (increased neutrophil count and fibrinogen concentration). The 3 calves were discharged from the hospital at the age of 1 week with antimicrobial treatment for 10 days until repeated ultrasound examination. One of them died suddenly at the owner's facility the day before ultrasound reevaluation. Necropsy determined that an hepatic abscess had ruptured inside an hepatic venous sinus, causing septic shock and death. The remaining 2 calves' umbilical cords were surgically managed during the follow-up period.
Assessment of Passive Transfer of Immunity
Total solids and globulins were measured at birth and at 48 hours of age to assess passive transfer of immunity. Total solids increased from 50.6 ± 4.3 g/L at birth to 61.6 ± 5.1 g/L at 48 hours of age, and the mean plasma globulin concentration increased from 18 ± 2.7 g/L at birth to 31.4 ± 8 g/L at 48 hours of age. The calf that did not receive colostrum and was transfused with 1 L of fresh plasma (from a male donor) was excluded from the calculation of the mean. Its total solids increased from 43.1 to 55 g/L and globulins increased from 17.7 to 21.6 g/L, respectively.
Other Pathologic Findings
One calf was diagnosed with bronchopneumonia based on physical findings (eg, fever, dyspnea, increased respiratory rate, abnormal pulmonary auscultation), radiographs (alveolar pattern), CBC results (increased fibrinogen concentration of 900 mg/dL), and positive bacterial culture from a tracheal swab (E. coli and Klebsiella spp.) Another calf was suspected to suffer from sepsis based on abnormal behavior (eg, lethargy, weakness, anorexia), physical findings (eg, fever, diarrhea, tachycardia, tachypnea), and CBC results (increased fibrinogen concentration of 600 mg/dL, neutrophilia with a left shift and toxic changes, lymphopenia). However, the blood culture was negative. Two calves suffered from diarrhea between 2 and 4 days of age. Fecal sample analyses for bacteria and parasites were negative. The calves received isotonic fluids9 IV and the diarrhea resolved within 2 days. One calf had a swollen carpus. Based on physical examination (ie, swelling without articular distension) and CBC results (increased fibrinogen concentration of 800 mg/L), the calf was suspected to suffer from peri-arthritis. Three calves of the last cohort developed lameness and swelling around the growth plates of the metatarsus. Based on radiographs, they were diagnosed with panosteitis possibly associated with their rapid growth. They were treated with nonsteroidal anti-inflammatory drugs and the amount of milk per day was decreased to 10% of their body weight. Lameness resolved within 2 weeks, but radiography was not repeated.
Calf Rectal Temperatures
During hospitalization, 10 calves developed hyperthermia (>40°C). Four had concomitant infections (ie, bronchopneumonia, peri-arthritis, diarrhea, septicemia) and were considered febrile. Three calves had enlarged umbilical stumps and normal CBC at the time hyperthermia was noted. The increased body temperature may be interpreted as fever (based on the later development of omphalitis) or as hyperthermia because they had no other systemic signs of inflammation. In addition, their body temperature decreased when the calves were exposed to ice, fan, and cold wet towels. The remaining 3 hyperthermic calves had no identifiable source of infection, and a normal CBC was noted at the time of hyperthermia. They were diagnosed with idiopathic hyperthermia and treated with ice, cold wet towels, and convection (use of a fan).
Part of our facility was dedicated to cloned calves. Access to the room where cloned calves were housed was controlled to minimize the number of people in contact with them. Individual stalls were bedded with straw and equipped to provide intranasal oxygen. A team of specialized veterinarians, nurses, students, and barn crew was formed. The student team was trained to take care of newborn calves. The board-certified specialists were large animal internists, surgeons, critical care specialists, and theriogenologists. An internist and a critical care specialist were always available during hospitalization.
The caesarian sections were scheduled before heifer's due dates to allow all of the team to be ready. One to 2 calves were delivered per day. The surgery was performed by a board-certified surgeon. As soon as the calf was delivered, it was examined by an internist and a critical care specialist. Resuscitation was attempted if needed. It was then transferred to its stall. Nurses and students assisted the veterinarians with neonatal care: umbilical stump dipping, regular recording of temperature, heart and respiratory rates (every 1–2 hours during the first 6 hours, then at least every 4 hours), assistance to stand and nursing. Thoracic radiographs were routinely taken the 1st and 3rd day.
The calves were observed 24 hours a day by a nurse or a student. When a calf needed mechanical ventilation based on the critical care specialist's evaluation, it was provided via nasotracheal tube to allow the calf to nurse during the period of mechanical ventilation. A person was dedicated exclusively to monitor those calves. When a calf needed a catheter to receive either fluids or drugs, a blood culture was taken beforehand. All of the blood cultures were negative. An arterial catheter was installed in all calves suffering from respiratory difficulties. Blood analyses (CBC and biochemistry) were performed at birth and at least every 2–3 days depending on the health of each calf. Blood gas analyses were performed at least every 12 hours for calves suffering from respiratory difficulties, and as often as every half-hour when they were mechanically ventilated.
Observations during the Follow-up Period (n = 21)
In total, 12 calves developed omphalitis (ie, swollen, painful, wet navel): 3 beginning during the hospitalization period and 9 beginning during the follow-up period at the owner's facility. These calves were examined for umbilical infection (physical examination, CBC, serum biochemistry profile, and abdominal ultrasound examination) at the CHUV every 2–3 weeks after discharge from the hospital. In all 12 calves, umbilical infection extended to ≥1 internal umbilical structures as determined by ultrasonography: 6 calves had an infected urachus (and 1 among them had pyelonephritis), 7 calves had omphaloarteritis, 3 calves had omphalophlebitis, and 5 calves had >1 infected structure. The incidence of umbilical complications based on the method used to sever the umbilical cord at birth is presented in Table 6. Umbilical care is described in Figure 2. Eight calves received antibiotics before the development of umbilical infection (as prophylaxis when the calves were intubated and mechanically ventilated or to treat an infection (of the umbilicus or elsewhere). The antibiotics used were as follows: a combination of ampicillin10 (20 mg/kg IV q8 h) and trimethoprim/sulfadoxine11 (5 ml/45 kg IV q12 h), ceftiofur sodium12 (5–10 mg/kg IV q8 h to q12 h) or enrofloxacin13 (10 mg/kg IV q24 h). All calves eventually received antibiotics when the diagnosis of umbilical infection was made. Eleven of the 12 calves underwent surgical resection of the infected umbilical structures (Fig 2). Six calves also had a partial cystectomy secondary to an infected urachus. During the same surgery, one of them had a unilateral left nephrectomy performed because of pyelonephritis (Fig 2). The calf was still alive at 2 years of age. The surgeries were performed between 3 weeks and 3 months of age depending on the response of the umbilical infection to medical treatment. The calf that did not undergo surgery was scheduled for surgical removal of the umbilicus, but died acutely before the surgery could be performed (Fig 2). Bacterial culture of the infected structures at the resection site was available for 9 calves. The most frequent bacterial isolates included E. coli (n = 6), anaerobic bacteria (n = 5), and Trueperella progenies (formerly Arcanobacterium pyogenes; n = 4). Secondary complications were observed in 7 calves. Two calves that had surgery developed postoperative incisional hernias that were surgically corrected without further complication (Fig 2). Five calves developed wound infections (subcutaneous abscesses, abdominal abscesses, or both) with or without an incisional hernia. They underwent a 2nd surgery to drain or remove the abscesses. One of them was euthanized after 4 weeks of treatment (drainage and antibiotics) because the abscesses were still very extensive. Another one of these calves died suddenly at the age of 2 months. Necropsy identified abscessed umbilical arterial remnants, numerous adhesions between these abscesses and the bladder and small intestines, abdominal wall abscesses at the site of the umbilical surgery, and an hemorrhagic diathesis of unknown origin. The cause of death of this calf remained unclear. Disseminated intravascular coagulation was a possible explanation.
|Method Used to Rupture the Umbilical Cord at Birth|
Gastrointestinal complications were observed in several cloned calves. Three calves suffered recurrent ruminal bloat at approximately 2 months of age. Bloat was relieved with a 2-cm-diameter rumen fistula made at the left paralumbar fossa. Two calves developed abomasal ulcers at 3 weeks and 1½ months of age, respectively. Both died after ulcer perforation and subsequent septic peritonitis. Other gastrointestinal disorders included enteritis (n = 3) and intussusception of the spiral colon associated with abomasal displacement (n = 1). These calves were treated successfully for these complications. The intussusception was treated surgically by removing the loop where the centripetal spiral colon becomes the centrifugal spiral colon, and anastomosing the centrifugal colon with the centripetal colon. The abomasal displacement corrected itself after surgical correction of the intussusception.
Fourteen of the cloned calves died or were euthanized. Four calves were stillborn. Necropsy reports were not available for these calves. Two calves were euthanized shortly after birth because of arthrogryposis. Necropsy reports identified flexural limb deformities (especially of the forelimbs), moderate to extensive pulmonary atelectasis, and the presence of squamous cells in the alveoli consistent with aspiration of amniotic fluid. The lungs were considered normal for 1-hour-old calves. Four calves died or were euthanized because of respiratory problems during the hospitalization period (between 1 and 4 days of age). From these 4 calves, 3 necropsy reports were available. The main findings included moderate diffuse fibrinous-histiocytic pneumonia associated with moderate atelectasis, interstitial edema, and hepatic dysplasia; marked pulmonary atelectasis associated with damage to the alveoli and the presence of hyaline membranes within the alveoli (compatible with neonatal respiratory distress syndrome), moderate forelimb arthrogryposis, and patent foramen ovale (1 cm diameter); and diffuse interstitial pneumonia associated with marked diffuse atelectasis and the presence of hyaline membranes within the alveoli (compatible with neonatal respiratory distress syndrome), and mild lymphoid depletion of the thymus. Four calves died during the follow-up period. Two of them suffered from acute peritonitis either because of perforation of abomasal ulcers (n = 1) or the rupture of abscessed residual umbilical structures (n = 1, omphalophlebitis). The 3rd calf died suddenly and necropsy identified abscessed umbilical arterial remnants, abscesses in the abdominal wall at the site of the umbilical surgery, and a hemorrhagic diathesis of unknown origin. The cause of death of this 3rd calf remained unclear. The 4th calf was euthanized because the abdominal and subcutaneous abscesses secondary to the umbilical surgery were extensive.
The neonatal period for cloned calves is critical with morbidity rates reaching as high as 67–100%.[7, 11-13, 18] In this study, the morbidity rate among cloned calves born alive was 89%, which is consistent with previous studies. This retrospective study completes a series of studies on the health of cloned calves at birth,[6, 7, 9, 11-14, 19, 20] and documents common health complications of cloned calves during adaptation to extrauterine life.
Weakness, lack of appetite, respiratory problems, flexural limb deformities, and increased umbilical vessel size were the main pathologic findings in our study, which is similar to what has been reported previously in cloned calves.
The most common abnormality observed was enlarged umbilical vessels, which has been reported in 25–100% of cloned calves.[7, 11, 12] Some authors have linked increased umbilical vessel size with placental abnormalities, which frequently are observed during the gestation of cloned calves, including placentomes that are larger than normal, of irregular shape, or fewer in number. Twelve calves (12/25, 48%) had an infection of ≥1 of the umbilical structures, and 3 died of umbilicus-related complications. The incidence of umbilical infections is higher in our cloned calves than what has been reported in dairy calves (14.2%). The size of the umbilical vessels may have been a risk factor for umbilical infection in these cloned calves. There are several hypotheses that may explain this high rate of umbilical infection. The size of the umbilical vessels prevented their normal retraction at birth. As a result, several of the umbilical vessels needed to be clamped. The use of tightening clamps may have contributed to the risk of contamination by preventing drainage of accumulated blood from the umbilical tissue, favoring bacterial growth. We observed more umbilical complications in calves whose umbilical cords were clamped compared with calves whose umbilical cords were stretched and torn. Based on our observations, we recommend umbilical cord clamping be avoided until additional studies can be conducted to support or refute our findings. In cases when the umbilical cord is too large to be stretched and torn, it may be preferable to remove the clamps used to control hemorrhage within a few hours of birth, once bleeding has ceased. Surgical excision of the umbilicus with abdominal wall closure, once the calf is stabilized after delivery, may be another alternative. A poorly developed immune system also may have contributed to the high incidence of umbilical infections observed. The incidence of infection remained high despite efforts to keep the calves in a clean isolated environment where traffic was limited, despite monitoring and treating the umbilicus every 6 hours, and despite the calves receiving antibiotics. In addition, the incidence of surgical site infection and secondary incisional hernias (4/11) was higher in these cloned calves than in other patients treated at our hospital (personal communication) or compared with previously published data concerning umbilical surgery on calves (1/90). A few studies have reported cases of thymic aplasia and immune deficiency in cloned calves,[4, 23, 24] but a recent study showed no difference in immune function in 1-year-old cloned calves compared with a control group of calves of the same age. Extrapolation of these results to our study is difficult because the calves of our study were newborn calves.
Respiratory abnormalities were another common cause of morbidity in our study, with hypoxemia being the most frequently documented finding. Hypoxemia was suspected to be secondary to delayed resorption of pulmonary fluid, right-to-left shunting because of persistent fetal circulation or neonatal distress syndrome caused by surfactant deficiency. The reader is referred to Brisville et al for a more detailed discussion concerning respiratory problems in cloned calves. The timing of cesarean sections could have influenced the incidence of neonatal distress syndrome. In our study, cesarean sections were performed 24 h after induction, before the start of the calving process, which allowed a multidisciplinary team (surgeons, internists, intensive care specialists, nurses, laboratory technicians) to be ready to intensively manage cloned calves at birth. Performing a cesarean section after the onset of labor may improve respiratory function of newborn cloned calves.
The colostrum used in this study often was contaminated above 5,000 CFU/mL, which may have challenged the cloned calves' immune system. A recent study concluded that calves fed heat-treated colostrum had lower risk of illness than calves fed fresh colostrum. The heat treatment decreased the total plate count and total coliform count of the colostrum and resulted in better absorption of colostral IgG by the calves. Therefore, contamination of colostrum may be a risk factor for morbidity in calves. However, 94% of colostrum given to neonatal calves in Quebec's commercial dairy farms is contaminated with a confidence interval of 15,135–34,674 CFU/mL. Although use of contaminated colostrum is not ideal, cloned calves were not subjected to different colostrum management strategies than dairy calves in Quebec, and therefore the use of contaminated colostrum alone does not fully explain the increased incidence of infection seen in our cloned calves. In addition, of the 8 calves for which both colostrum and umbilical vessel remnant cultures were available, only 1 had the same bacteria isolated: Pseudomonas aeruginosa. However, this bacterium also is commonly present in the environment. Thus, contaminated colostrum did not seem to be the source of the umbilical infection, but may have challenged the immune system during the neonatal period. A challenged immune system may have facilitated conversion of umbilical contamination into umbilical infection.
Cloned calves in this study often had hematologic values that fell outside of reference intervals established by our laboratory. However, these reference intervals were established based on adult dairy cattle. Respiratory acidosis, hypoproteinemia, and increased serum creatinine concentration have already been reported in healthy newborn calves. However, blood lactate concentration was higher in cloned calves compared with healthy newborn calves. The marked lactic acidosis in cloned calves that have significantly lower erythrocyte count, hemoglobin concentration, hematocrit and mean cell hemoglobin concentration (compared with newborn calves derived from natural fertilization) may have resulted in decreased oxygen delivery to tissues. The resulting hypoxia may explain the weakness observed in some cloned calves that demonstrated delayed nursing, standing up or both.
A previous study compared the hematologic values of 8 cloned calves with a control group derived from in vivo embryo transfer raised under the same conditions. The investigators found that the erythrocyte count and hematocrit were significantly lower than those of the controls. Another study reported similar observations. Anemia during the neonatal period has been reported in normal calves as well. It is hypothesized that vascular volume expansion after the first nursing, decreased production of erythrocytes by bone marrow in response to increased oxygen in the blood, destruction of fetal erythrocytes, and iron or erythropoietin deficiency all could contribute to the anemia noted in normal calves. However, most of these mechanisms cannot explain the anemia observed immediately after birth, before the ingestion of colostrum. We believe that blood loss during birth may have contributed to the anemia noted in our study. Because of the enlarged umbilical vessels noted in our calves, we speculate that a substantial quantity of blood was sequestered in these vessels, and was subsequently lost during surgery or sequestrated inside the umbilical cord after placing plastic sterile self-tightening clamps.
Some cloned calves in this study had episodes of idiopathic hyperthermia, not related to any identified infection. This finding is similar to another study, which found cloned calves to have higher temperatures than control calves raised concurrently under the same conditions. The origin of hyperthermia in cloned calves has not been determined. Hypotheses include higher metabolic rates, thyroid abnormalities,[33, 34] higher brown adipose tissue, and higher catecholamine concentrations that can stimulate heat production by brown adipose tissue. The latter is less likely based on results of a recent study. Although we could not further determine the cause of hyperthermia in our calves, we did find that hyperthermia could be managed and that it eventually resolved on its own. In our study, the application of ice, cold wet towels, and fans helped to control episodes of hyperthermia. Environmental conditions were not believed to be a contributing factor to this idiopathic hyperthermia because the climate was temperate (18–20°C) and well ventilated.
Morbidity was high in cloned calves during the neonatal period in our study. Enlarged umbilical vessels associated with a high incidence of umbilical infections and postoperative complications are common. Respiratory complications also are common and require intensive management including oxygen treatment and mechanical ventilation. Finally, anorexia may be observed and should be managed by esophageal feedings. Based on our observations, we recommend close monitoring of cloned calves during the neonatal period to allow early identification of complications and timely intervention.
Semex Alliance for their financial support of this study. Carl Bernard, for daily care of the cloned calves. Guy Beauchamp, for assistance with the statistical analysis.
The study was supported by a Strategic Grant of the NSERC (Natural Sciences and Engineering Research Council) of Canada.
Conflicts of Interest: Authors disclose no conflict of interest.
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- 24Pathology of diseases in calves cloned by nuclear transfer. Cloning Stem Cells 2002:281..