[Infusion sets in neonatology: What practices in France?] / Montages de perfusion en néonatologie : quelles pratiques en France ?

OBJECTIVES: Therapeutic management of ill newborns can require complex infusion practices using medical devices (MD). Currently, there does not exist any recommendations concerning these infusion practices. The objective of this work was to study and characterise French infusion methods neonatal and neonatal intensive care units. MATERIALS AND METHODS: The study was performed in 2019, during 6 months. French hospitals possessing high (type 3) or medium (type 2B) grade maternity ward were contacted and asked to complete a 5 part online survey, to gather general information about the hospital/ward, infusion methods (overall and detailed), and detailed information about the medications and MD used. RESULTS: The participation level was of 19.6 % Type 3 maternities use overall two-times more MD than those of type 2B. The vascular access device most commonly used was a single lumen catheter (80.6 % of infusion methods). 100 % of the hospitals having answered used multi-access devices (three-way tap, multiport infusion manifold, Y-extension lines) and 93.5 % used a pump-infusor. Lipidic filters for parenteral nutrition were used in 78.6 % of the hospitals. Two general standard of infusion methods were isolated: a simple version with two access points (type 2B hospitals), and a complex one with five access points (from hospitals with type 3 maternities). CONCLUSIONS: Neonatal infusion practices in France are very heterogeneous, thus exposing the patients to a degree of variability during their therapeutic management. This work is a first step forwards to help analyse and anticipate the risks of content/container interactions caused by infusion practices.

[Antimicrobial prophylaxis for caesarean delivery: changes in practice and administration before incision, feasibility study to paediatricians in Auvergne]. / Antibioprophylaxie pour césarienne: modification des pratiques et administration avant incision, enquête de faisabilité auprès des pédiatres du réseau de santé périnatale d'Auvergne (RSPA).

OBJECTIVE: To describe the knowledge of paediatricians regarding the practice of antimicrobial prophylaxis for caesarean section in reference to the Consensus Conference of the French Society of Anesthesia and Intensive Care (SFAR) and assess the feasibility of a change in attitude (injection of the antibiotic prior to incision) among paediatricians Perinatal Health Network of Auvergne (RSPA) working in maternity. STUDY DESIGN: Cross sectional study by survey. METHODS: First questionnaire was sent to 46 RSPA paediatricians working in maternity. Almost one-third of paediatricians who returned the questionnaire said they were not concerned. A second questionnaire was developed with two paediatricians of the CHU and sent to the same 46 paediatricians. The statistical part involved percentages. RESULTS: Response rates were respectively 61% and 67%. For the first questionnaire, only 25% of the paediatricians knew the antibiotic and the time for injection. For the second questionnaire, 87% were in favour of an administration before incision and 42% thought it will not affect the care of the newborn. For 35% of respondents, it could lead to a change in the duration of antibiotic therapy in cases of perinatal infection and for 13% only a delay in the implementation of antibiotic therapy in children. CONCLUSION: The RSPA paediatricians did not know the practices of antibiotic prophylaxis for caesarean section. However, they did not appear opposed to an administration before cord clamping as it would not delay the implementation of any antibiotics in the newborn.

[A lethal variant of Netherton syndrome in a large inbred family]. / Forme létale de syndrome de Netherton au sein d'une famille multiplex consanguine.

Netherton syndrome is a rare autosomal recessive disorder characterized by the triad of ichthyosiform erythrodermia, typical hair dysplasia, and severe atopic features. The broad range of variable expression of this disease is well described and 20% of complications occur during the neonatal period such as hypernatremic dehydration, electrolyte imbalances, recurrent or severe infections, and failure to thrive. Mutation of the SPINK5 gene has been identified as disease-causing in Netherton syndrome, but the pathophysiology still remains unclear. Almost all SPINK5 mutations result in the absence of the serine-protease inhibitor LEKTI protein in both keratinocytes and lymphocytes. In this study, we report on a severe form of Netherton syndrome observed in three patients within a large inbred Rom family. All of them died in the first months of life despite early treatment. They were found to be homozygous for the c.1431-12G>A SPINK5 gene mutation, which has not been associated with a lethal form of the disease thus far. This family illustrates the extreme phenotype of Netherton disease of neonatal onset. Molecular diagnosis allowed further genetic counseling and prenatal testing during other pregnancies.

[Congenital lobar emphysema: a rare etiology of hyperechoic lung]. / Une étiologie rare de poumon hyperéchogène: l'emphysème lobaire géant congénital.

Lobar congenital emphysema is a rare pulmonary malformation corresponding to progressive overinflation of a pulmonary lobe secondary to partial bronchial obstruction. Prenatal diagnosis is mainly based on lung hyperechoic area. Sonographic features are not specific highlighting the interest of fetal MRI or postnatal tomodensitometry. This case report describes prenatal detection including pitfalls and postnatal management.

Holocene elephant seal distribution implies warmer-than-present climate in the Ross Sea.

We show that southern elephant seal (Mirounga leonina) colonies existed proximate to the Ross Ice Shelf during the Holocene, well south of their core sub-Antarctic breeding and molting grounds. We propose that this was due to warming (including a previously unrecognized period from approximately 1,100 to 2,300 (14)C yr B.P.) that decreased coastal sea ice and allowed penetration of warmer-than-present climate conditions into the Ross Embayment. If, as proposed in the literature, the ice shelf survived this period, it would have been exposed to environments substantially warmer than present.

[Neonatal morbidity after conservative management followed by systematic delivery at thirty-four weeks of gestation in preterm rupture of membranes]. / Morbidité néonatale après attitude d'expectative suivie d'une naissance systématique à 34 semaines d'aménorrhée en situation de rupture prématurée des membranes.

OBJECTIVE: To evaluate the neonatal morbidity and its risks factors in case of uncomplicated preterm rupture of membranes managed conservatively with subsequent planned delivery at 34 weeks of gestation. PATIENTS AND METHODS: We studied retrospectively 42 consecutive neonates systematically delivered at 34 weeks of gestation after more than 48 hours of conservative management for uncomplicated preterm rupture of membranes. Conservative management was conducted in a single tertiary care center and consisted in corticotherapy and in antibiotherapy (amoxycilline during 7 days). We evaluated the neonatal mortality rate, the incidence of infection, respiratory distress, neurological disorders, and we looked for their prenatal risks factors. RESULTS: Forty-two neonates were included. The median gestational age at rupture was 31.1 weeks of gestation (from 25 to 33.9 weeks). The median duration of expectant management was 20 days (from 2.4 to 65 days). We observed 7 cases of neonatal infection but no septic failure, 18 cases of respiratory distresses among which 9 required a tracheal intubation for a mean duration of 3.7 days, no perinatal encephalopathy (5 cases of subependymal haemorrhage) and no neonatal death. We isolated one single risk factor that was the lowest gestational age at rupture in case of subsequent respiratory distress (29.6 vs 31.9 weeks; P=0.02). DISCUSSION AND CONCLUSION: Neonatal morbidity in this population consisted mainly in respiratory distresses with an increased incidence when gestational age at rupture decreased.

Organochlorine pesticides and polychlorinated biphenyls in California sea lions.

Concentrations of polychlorinated biphenyls (PCBs), DDTs, chlordanes, HCHs, hexachlorobenzene (HCB), dieldrin, heptachlor epoxide, tris(4-chlorophenyl)methane (TCPMe), and tris(4-chlorophenyl)methanol (TCPMOH) were measured in the blubber of California sea lions (Zalophus californianus) collected in 2000. DDTs were the most predominant contaminants, followed by PCBs, chlordanes, TCPMe, HCHs, TCPMOH, dieldrin, and heptachlor epoxide. Concentrations of PCBs and DDTs varied from a few microg/g to several hundreds of microg/g on a lipid weight basis. Concentrations of DDTs have declined by an order of magnitude over the last three decades in California sea lions; nevertheless, the measured concentrations of PCBs and DDTs in California sea lions are still some of the highest values reported for marine mammals in recent years. Concentrations of organochlorines were highly correlated with one another. Concentrations of PCBs and DDTs in the blubber of gray whale, humpback whale, northern elephant seal, and harbor seal, and in the adipose fat of sea otter, were lower than the levels found in California sea lions, and were in the range of a few to several microg/g on a lipid weight basis.

[Ventricular septal defect of the neonate]. / Communication interventriculaire du nourrisson.

Ventricular septal defect is the commonest congenital cardiac lesion and represents 30-40% of all congenital heart disease with a prevalence of 1.8 to 6.5 per 1000 births. The aim of this study was to evaluate the outcome of neonates with ventricular septal defects and to deduce from the echocardiographic appearances in the first year of life a relationship between the initial anatomy and the outcome. Between January 2001 and July 2003, 89 children from the Auvergne region were followed up prospectively for an average period of 7.6 months. The study showed that the majority of muscular ventricular septal defects with a diameter of 3 mm and less progress to a reduction in size and spontaneous closure more commonly and at an earlier stage than perimembranous ventricular septal defects.

Naloxone for shock.

BACKGROUND: There is pre-clinical evidence, involving several animal species, suggesting that opioid peptides play a role in the physiopathology of shock (endotoxic, hypovolemic, cardiogenic, spinal, anaphylactic). Many case reports have suggested that naloxone (an opiate antagonist) might be an effective treatment for shock in humans, but others have not supported such a point of view. This controversy led us to undertake a meta-analysis of the available evidence on the efficacy of naloxone as a treatment measure of shock in humans. OBJECTIVES: To evaluate the effectiveness and safety of naloxone in human shock and to estimate the methodological quality of the clinical trials. SEARCH STRATEGY: Computerized bibliographic search up to December 2002, review of references of all papers found on the subject and contact with primary investigators of eligible studies. SELECTION CRITERIA: Randomized controlled trials evaluating naloxone in human shock, regardless of the patient's age (adult, child or neonate). DATA COLLECTION AND ANALYSIS: Three independent reviewers extracted data on study design, intervention, outcome and methodological quality. MAIN RESULTS: Three independent readers reviewed 80 human publications and selected six clinical trials. Overall agreement on study selection was perfect (concordance: 100%). This meta-analysis includes six studies involving 126 patients with septic, cardiogenic, hemorrhagic or spinal shock. Naloxone therapy was associated with statistically significant hemodynamic improvement (odds ratio 0.24; 95% confidence interval [95%CI] 0.09-0.68). The mean arterial pressure was significantly higher in the naloxone groups than in the placebo groups (weighted mean difference: +9.33 mmHg; 95%CI 7.07-11.59). No heterogeneity was found for this outcome. The death rate was lower in the naloxone group (odds ratio 0.59; 95%CI 0.21-1.67) but this was consistent with the play of chance. A significant heterogeneity for the latter outcome was detected (p<0.05). REVIEWER'S CONCLUSIONS: Naloxone improves blood pressure, especially mean arterial blood pressure. However, the clinical usefulness of naloxone to treat shock remains to be determined, and additional randomized controlled trials are needed to assess its usefulness.

Three-dimensional movements and swimming activity of a northern elephant seal.

We attached a video system and data recorder to a northern elephant seal to track its three-dimensional movements and observe propulsive strokes of the hind flippers. During 6 h of recording, the seal made 20 dives and spent 90% of the time submerged. Average dive duration, maximum depth and swimming speed were 14.9 min+/-6.1 S.D., 289 m+/-117 S.D. and 1.1 m s(-1)+/-0.12 S.D., respectively. The distance swum during a dive averaged 925 m+/-339 S.D., and the average descent and ascent angles were 41 degrees +/-18 S.D. and 50 degrees +/-21 S.D., respectively. Dive paths were remarkably straight suggesting that the seal was navigating while submerged. We identified three modes of swimming based on the interval between propulsive strokes: continuous stroking; stroke-and-glide swimming; and prolonged gliding. The seal used continuous stroking from the surface to a mean depth of 20 m followed by stroke-and-glide swimming. Prolonged gliding started at a mean depth of 60 m and continued to the bottom of dives. For dives to depths of 300 m or more, 75% of the descent time was spent in prolonged gliding and 10% in stroke-and-glide swimming, amounting to 5.9-9.6 min of passive descent per dive. Average swimming speed varied little with swimming mode and was not a good indicator of propulsive effort. It appears that the seal can use prolonged gliding to reduce the cost of transport and increase dive duration. Energetically efficient locomotion may help explain the long and deep dives that routinely exceed the theoretical aerobic dive limit in this species.

Respiration and heart rate at the surface between dives in northern elephant seals.

All underwater activities of diving mammals are constrained by the need for surface gas exchange. Our aim was to measure respiratory rate (fb) and heart rate (fh) at the surface between dives in free-ranging northern elephant seals Mirounga angustirostris. We recorded fb and fh acoustically in six translocated juveniles, 1.8-2. 4 years old, and three migrating adult males from the rookery at Año Nuevo, California, USA. To each seal, we attached a diving instrument to record the diving pattern, a satellite tag to track movements and location, a digital audio tape recorder or acoustic datalogger with an external hydrophone to record the sounds of respiration and fh at the surface, and a VHF transmitter to facilitate recovery. During surface intervals averaging 2.2+/-0.4 min, adult males breathed a mean of 32.7+/-5.4 times at a rate of 15. 3+/-1.8 breaths min(-)(1) (means +/- s.d., N=57). Mean fh at the surface was 84+/-3 beats min(-)(1). The fb of juveniles was 26 % faster than that of adult males, averaging 19.2+/-2.2 breaths min(-)(1) for a mean total of 41.2+/-5.0 breaths during surface intervals lasting 2.6+/-0.31 min. Mean fh at the surface was 106+/-3 beats min(-)(1). fb and fh did not change significantly over the course of surface intervals. Surface fb and fh were not clearly associated with levels of exertion, such as rapid horizontal transit or apparent foraging, or with measures of immediately previous or subsequent diving performance, such as diving duration, diving depth or swimming speed. Together, surface respiration rate and the duration of the preceding dive were significant predictors of surface interval duration. This implies that elephant seals minimize surface time spent loading oxygen depending on rates of oxygen uptake and previous depletion of stores.

Breathing frequencies of northern elephant seals at sea and on land revealed by heart rate spectral analysis.

Elephant seals breathe episodically at sea and on land and surprisingly long apnoeas occur in both situations. An important difference is that recovery from apnoeic periods is much quicker at sea, which might be due, in part, to differences in the ventilatory response. Respiratory frequencies of juvenile northern elephant seals diving at sea and resting on land were estimated from time-frequency maps of the Wigner distribution of heart rate variability. Simultaneous direct measurement of respiration and estimation of respiratory frequency (fR) in the laboratory demonstrated that the error of estimation was small (mean +/- S.D.= 1.05+/-1.23%) and was independent of the magnitude of fR. Eupnoeic fR at sea was 2.4 times higher than on land (22.0+/-2.0 vs. 9.2+/-1.3 breaths min(-1), respectively), facilitating quick recovery from the preceding dive and allowing a 34% increase in time spent apnoeic at sea versus on land. The overall fR (no. of breaths in a eupnoea divided by the total time of the apnoea+eupnoea cycle) of 2.3+/-0.6 breaths min(-1) at sea was no different from the rate on land and was inversely related to the preceding dive duration, suggesting that metabolism on longer dives may be reduced.

Sink or swim: strategies for cost-efficient diving by marine mammals.

Locomotor activity by diving marine mammals is accomplished while breath-holding and often exceeds predicted aerobic capacities. Video sequences of freely diving seals and whales wearing submersible cameras reveal a behavioral strategy that improves energetic efficiency in these animals. Prolonged gliding (greater than 78% descent duration) occurred during dives exceeding 80 meters in depth. Gliding was attributed to buoyancy changes with lung compression at depth. By modifying locomotor patterns to take advantage of these physical changes, Weddell seals realized a 9.2 to 59.6% reduction in diving energetic costs. This energy-conserving strategy allows marine mammals to increase aerobic dive duration and achieve remarkable depths despite limited oxygen availability when submerged.

Therapy of shock with naloxone: a meta-analysis.

OBJECTIVES: To evaluate the effectiveness of naloxone in human shock; and to estimate the methodologic quality of the clinical trials. DATA SOURCES: Computerized bibliographic search on MEDLINE covering the period from January 1979 to July 1996, review of references of all papers found on the subject, and contact with primary investigators of eligible studies. STUDY SELECTION: To be included in this study, a paper should be a randomized, clinical trial published in a peer-reviewed journal evaluating naloxone in human shock, regardless of the patient's age (adult, child, neonate). Three independent readers reviewed 61 human publications and selected five clinical trials. Overall agreement on study selection was perfect (concordance: 100%). We excluded a posteriori two studies whose authors were unable to provide us with the raw data to complete contingency tables. This meta-analysis deals with three studies including 61 patients with septic shock. DATA EXTRACTION: Three independent reviewers extracted data on study design, intervention, outcome, and methodologic quality. The intraclass correlation coefficient was 0.7. The quality score of each study was 48, 60, and 61, on a scale of 104. DATA SYNTHESIS: Naloxone therapy was associated with statistically significant hemodynamic improvement (typical odds ratio: 0.241; 95% confidence interval: 0.08 to 0.68). The overall effect size was 0.89. However, a publication bias was possible. The case fatality rate was not decreased by naloxone (typical odds ratio: 0.60; 95% confidence interval: 0.21 to 1.67); a chi-square analysis detected significant heterogeneity for the latter outcome (p < .05). CONCLUSIONS: Naloxone improves blood pressure. However, the clinical usefulness of naloxone to treat shock remains to be determined and additional randomized clinical trials are needed to assess its usefulness.

Protein catabolism and renal function in lactating northern elephant seals.

Northern elephant seals, Mirounga angustirostris, fast completely from food and water during lactation. Previous investigations of maternal investment suggested physiological constraints on the level of energy expenditure during lactation. In this study, two components of phocid fasting physiology, protein sparing and reduced glomerular filtration rate, were examined for effects of changing body composition and lactation duration. Protein catabolism was estimated from 14C-urea turnover in five mid- and five late-lactation females. Body composition was determined by using an ultrasound scanner to measure blubber depth coupled with morphometrics. Glomerular filtration rate was measured in five females at mid- and late-lactation using plasma clearance of 3H-inulin. Protein catabolism increased significantly between measurements. The contribution of protein to metabolism varied with body composition and lactation duration. Mass-proportional glomerular filtration rate increased significantly between measurements. These data suggest that conflicting metabolic demands of lactation and fasting might constrain the duration and magnitude of maternal investment in northern elephant seals.

Genetic variability in Guadalupe fur seals.

The Guadalupe fur seal (Arctocephalus townsendi) population underwent one or two severe bottlenecks due to commercial sealing in the late 19th century. Since then the protected population has been growing steadily around their only rookery, Isla de Guadalupe, Mexico. We probed both nuclear and mitochondrial genomes using multilocus nuclear DNA profiling and mitochondrial DNA sequencing to estimate the level of genetic variability of the present population. Unlike other pinniped populations that have experienced similar historical bottlenecks, such as Hawaiian monk seals and northern elephant seals, high levels of genetic variability were found.

Effects of buoyancy on the diving behavior of northern elephant seals.

Marine mammals experience radical seasonal changes in body composition, which would be expected to affect their buoyancy in the water. The aim of this study was to examine the relationship between such changes in buoyancy and diving behavior in northern elephant seals Mirounga angustirostris. This was achieved by modifying the buoyancy of 13 juvenile elephant seals translocated from Año Nuevo State Reserve, CA, USA, and released at various sites in Monterey Bay, CA, USA. The buoyancy of each seal was calculated and was increased or decreased using syntactic foam or lead weights, and their diving behavior was recorded as they returned to Año Nuevo. The seals were divided into three groups: increased buoyancy (B+), reduced buoyancy (B-) and control seals (Bc). Mean descent rates were 0.77+/-0.3 ms-1 for the B+ seals, 0.82+/-0.2 ms-1 for the control seals and 0.87+/-0.3 ms-1 for the B- seals, and were significantly different. Mean ascent rates for the three treatments were 0.82+/-0.3 ms-1 for the B+ seals, 0.86+/-0.3 ms-1 for the control seals and 0.82+/-0.3 ms-1 for the B- seals. All the B+ seals ascended faster than they descended, while four of the five B- seals descended faster than they ascended. There was a significant negative correlation between buoyancy and descent rate, with less buoyant seals descending faster than more buoyant seals. There was, however, no correlation between ascent rate and buoyancy. This suggests that seals may use negative buoyancy to drift passively during descent, but that all seals may swim continuously during ascent. There was a significant correlation between buoyancy and the drift descent rate of C-type drift dives, including upwards drift in the most buoyant seal. Buoyancy was not correlated with diving depth, trip duration, dive duration or surface-interval duration. This study demonstrates that buoyancy plays a significant role in shaping diving behavior in northern elephant seals and that elephant seals may adjust their behavior to suit their buoyancy, rather than adjusting their buoyancy to suit a dive. This study also validated the truncated cones method of calculating body composition in this species by comparing it with body composition determined using tritium dilution.

Markers of cellular dysoxia during orthotopic liver transplantation in pigs.

OBJECTIVE: To characterize global, regional, and end-organ markers of cellular dysoxia during orthotopic liver transplantation and early reperfusion in pigs. DESIGN: Descriptive study. SETTING: University hospital research laboratory. ANIMALS AND INTERVENTIONS: 7 fasted, anesthetized, and mechanically ventilated Yorkshire pigs underwent orthotopic liver transplantation. Oxygen consumption (VO2) and oxygen delivery (DO2) were both calculated using standard formulae. Gastric interstitial pH and the gastroarterial partial pressure of carbon dioxide (PCO2) gradient were measured with a gastric tonometer. The following were determined from arterial blood samples: serum lactate to pyruvate ratio, serum 3-hydroxybutyrate to acetoacetate ratio, plasma free fatty acids, and plasma free and total carnitine levels. MEASUREMENTS AND RESULTS: Data were collected 1 h after induction of anesthesia (I), at the end of the anhepatic phase (A), and 1 h after reperfusion (R), Median (range) VO2 values obtained at the specified time points were: I 318 (206-1860), A 210 (152-408), R 330 (214-424) ml/kg per min, respectively (NS); DO2 values were: I 1828 (1382-3259), A 1219 (452-2492), R 1741 (1345-12,071) ml/kg per min, respectively (NS). The lactate to pyruvate ratio, reflecting the redox potential of the cytosol, progressively increased: I 22 (9-46), A 29 (16-68), R 43 (23-55), (p < 0.05). Gastric interstitial pH, as well as the gastroarterial PCO2 gradient values at the specified time points did not reach statistical significance. Levels of ketone bodies (3-hydroxybutyrate+acetoacetate) remained lower than 0.120 mmol/l. The ketone body ratio did not significantly vary over time (NS). Plasma esterified and free carnitine concentrations and free fatty acid values remained within normal limits (NS). Among these markers, the ketone body ratio presented the largest area under the receiver operating characteristic curve as a marker of postoperative mortality, with an inflexion point at 0.9. CONCLUSION: In this study, orthotopic liver transplantation was associated with significant variations over time in the redox potential of the cytosol. Postoperative mortality was, however, related to the redox state of the liver mitochondria. Our data suggest the occurrence of abnormal tissue oxygenation during liver transplantation.

Heart rate and oxygen consumption of northern elephant seals during diving in the laboratory.

Many techniques have been employed to measure metabolic and cardiovascular changes in diving marine mammals. Each of these methods has its advantages, but the methods also have drawbacks when applied to phocid seals. The aim of this study was to investigate heart rate and metabolic responses to diving in juvenile northern elephant seals that are not associated with forced changes in exercise state, and, secondarily, to investigate whether heart rate could be used as an indicator of metabolic rate in this species. Six seals were allowed to dive freely in a metabolic chamber while simultaneous measurements of heart rate and oxygen consumption were made. Within each dive cycle (dive and surface interval), the seals spent an average of 74% of the time submerged. Mean dive duration was 6.43+/-0.6 (SD) min. Mean oxygen consumption during diving was 3.32+/-0.4 mL O2 min-1 kg-1, a decrease of approximately 26% from baseline values. An inverse relationship was observed between oxygen consumption and the percentage of time spent submerged in each dive cycle. The total amount of oxygen consumed during the surface interval increased with increasing dive duration, while the duration of the surface interval itself did not change, indicating that seals alter the rate of O2 uptake rather than the time spent at the surface. Mean heart rate during diving was 34.5+/-6.2 beats min-1, 36% lower than resting values. Mean diving heart rate was independent of dive duration, percent time submerged, and oxygen consumption. Mean surface interval heart rate was 66.6+/-11.1 beats min-1 and was not correlated with oxygen consumption. Average heart rate over the entire dive cycle increased with increasing oxygen consumption in all of the seals, but there was only a significant relationship in two seals, which casts some doubt on the usefulness of heart rate as an indicator of metabolic rate in this species. While providing important information on the changes in heart rate and oxygen consumption during diving in northern elephant seals, a complete understanding of the diving metabolic rate of these animals will require a combination of approaches that can be used in concert with data on freely living animals.