Biol. Neonate 3 6 : 63-69 (1979)
Studies on the Effect of Acute Asphyxia on the Fetal Pig
in utero G.C.B. Randall' Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (E), Ottawa, Ont.
Key Words. Asphyxia • Fetal pig ■Stillbirth Abstract. 8 term fetal pigs (110—112 days gestation) and one 97-day fetus were asphyxiated in
útero by occlusion of the umbilical cord. Mean times to last gasp and last heart beat were 5.1 and 22.4 for term and 5.4 and 30.4 min for the 97-day fetus. Cord occlusion was followed by profound bradycardia and an increase in blood pressure which was maintained until gasping ceased. Profound acidemia, hypercapnia and hyperlactacidemia developed in all animals and values following asphyxiation were comparable with those seen in stillborn piglets. Liver and cardiac glycogen levels were lower in asphyxiated fetuses than in littermates but muscle glycogen levels were similar in both groups.
Previous studies on blood gases, pH and lactic acid levels indicated that asphyxia was a major cause of stillbirth in the pig (19,20) and that premature rupture of the umbilical cord might be a predisposing cause (11,20). Although the sequence of changes following asphyxia is similar (18), variations in the dura tion of survival are known to occur between species (12). Miller and Miller (17) showed that newborn piglets had a short time to last gasp 1 The author wishes to acknowledge the skilled technical help of Mrs. J. Hierlihy and Messrs. Y. Barbeau, G. Raby, IV. G. Addison and R.L. Marenger.
when placed in a mixture of 95% N2 + 5% C 0 2, but few other studies are available for this species. In this paper changes occurring in the unanesthetized fetal pig during anoxia, resulting from occlusion of the umbilical cord at term, are described and compared with observations made on naturally asphyxiated piglets.
Materials and Methods 10 Yorkshire gilts (127-200 kg body weight) were used. Catheters and electrodes were implanted into 13 fetuses under halothane: oxygen anesthesia (Fluothane, Ayerst), following induction with thio pental sodium (Pentothal, Abbott). Fetal cannulation was performed at 108-111 days gestation (mean
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Introduction
64
Fetal Asphyxiation The sow was moved to a crush and allowed time to settle; most animals lay quietly during the study. The fetal catheters and electrodes were connected to a polygraph and records taken for 1-2 h to establish base levels. Towards the end of this period fetal blood samples were taken for blood gas, glucose, fructose and lactic acid estimation. The occluder was then inflated and fetal recording continued until the cessa tion of regular heart beats from the EKG trace. A second blood sample was collected either just preced ing or following cessation of fetal heart beat. The sow was then killed with barbiturate. Tissue samples were removed immediately from the asphyxiated piglet, and one or two littermates, and frozen in liquid N2 in the following order: cardiac ventricles, liver and thigh muscle. Samples were stored at -7 9 °C until analyzed. Similar samples were taken from three naturally oc curring ‘stillborn’ piglets prior to cessation of heart beat. In this paper a ‘stillborn’ piglet is one in which there is a slow heart beat at the time of delivery which ceases within a few minutes of birth, but no respira tory efforts are made (20).
Absolute pressures were monitored with trans ducers (Statham, model P23AC or P23DC) positioned at the level of the dorsal line of the sow’s mammary glands. The carotid cannula was infused with hepa rinized saline (0.2 ml/h) during recording to reduce clotting. True fetal blood pressure was obtained by subtracting cither amniotic fluid or tracheal pressure from absolute blood pressure. Heart rate was taken from the carotid pulse or the QRS complex of the EKG trace. Blood pH, p 0 2 and pC02 were determined at 38.5 °C using a BMS3 blood microsystem with a PHM71 acid base analyzer (Radiometer, Copenhagen). Samples were stored in iced water until analyzed. Glucose, fructose and lactic acid levels were estimated in whole blood following deproteinization of 100 ix\ in ice-cold uranyl acetate or perchloric acid. Glucose was estimated using a commercial glucose oxidaseperoxidase method (Boehringer, Mannheim), fructose using Seliwanof reagents (5) and lactic acid by a microdetermination method (13). Tissue glycogen was estimated using anthrone reagent as described pre viously (22).
Results
9 of 13 fetuses survived surgery, and in the other 4 death appeared to have resulted from pressure of the occluder on the umbilical cord following entanglement of its actuating tubing in the felal limbs. Cardiovascular and Respiratory Changes A regular pattern was seen in the term fetuses although variations in magnitude or duration of changes occurred between individ uals. As an example, the changes in one fetus are shown in figure 1. A profound bradycardia occurred within 15 sec of cord occlusion (Fig. 1) reaching its lowest rate 30-45 sec after occlusion, at which time fetal heart rate was 32.2% (range 26.5-39.5%) of preasphyxia rates. It then increased towards resting values (39.8—93.6%), peaking 2—3 min after occlu sion, and then fell rapidly during the later
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109.6) in 12 fetuses from 9 sows and at 96 days gestation in 1 fetus. With two exceptions (48 and 96 h), fetal asphyxiation studies were made 24 h after surgery. A polyvinyl cannula (0.58 mm i.d. X 0.96 mm o.d., Bolab Inc., Derry, New Hampshire) was placed in the right-hand fetal carotid artery using techniques described elsewhere (21). A similar catheter (length 2 -3 cm) was inserted into the fetal trachea through a hole made with a hypodermic needle immediately behind the larynx. A polyvinyl amniotic fluid catheter (1.0 mm i.d. X 2.0 mm o.d.) was sutured to the piglet’s neck close to the carotid catheter and, in most animals, two subdcrmal platinum EKG electrodes (Grass Instruments, Quincy, Mass.) were attached to the anterior chest wall. Finally, a 14-mm blood vessel occluder (Model V 0-4, Rhodes Medical Instruments, Woodland Hills, Calif.) was tied loosely around the umbilical cord and the uterus closed with 20 cm of each catheter within the uterus. Capped hypodermic needle hubs were bonded into the external ends of the catheters and these, together with the electrode leads and the occluder actuating tubing, were stored aseptically in a plastic bag within a pouch sutured to the sow’s flank. Cannulac were filled with sterile heparinized saline (100 lU/ml) between samples.
Randall
Asphyxia of Fetal Pig
65
Fig. 1. Changes in heart rate and mean, systolic and diastolic blood pressures following occlusion of the umbilical cord in one fetus.
stages of fetal gasping. During the terminal stages the heart rate was often irregular and extrasystolic beats were also present. Mean fetal blood pressure rose during the first 15—30 sec with increases in both systolic and diastolic pressures. In most animals there was a transi tory ‘fall’ in mean blood pressure —as has been seen in sheep (9) — 30—60 sec after occlusion. This was coincident with the trough in heart rate and a low diastolic pressure between beats although systolic pressure continued to rise.
Gestational age, days
TLG, min
TLHB, min
110-112 97
5.1 (4.1-6.1) 5.4
22.4 (14.2-34.3) 30.4
Thereafter, mean blood pressure rose to a peak 2 -3 min after occlusion before falling rapidly as fetal breathing ended. Mean times to last heart beat are shown in table I. In the 97-dayold fetus changes in heart rate were similar to term fetuses, but changes in blood pressure were less marked. The increase in mean blood pressure was smaller and of shorter duration than that seen in term piglets. Within 15-30 sec of cord occlusion the fetuses gasped rapidly (8—12/min) for about 1 min. The early gasps were followed by slower, deeper gasps (45—55 mm Hg) at 2/min which often became more frequent (4/min) and shallower terminally. No distinct period of primary apnea was observed although a short delay preceding the deeper gasps was noted in a few animals. Blood pH, p 0 2, pC 02, glucose, lactic acid and hematocrit values for asphyxiated piglets and stillborn piglets are shown in table II. Values for the 97-day fetus were similar to those of term fetuses and were included with them. Changes were similar for all parameters except glucose, where marked individual dif ferences were seen and values increased by 25—356% over preasphyxia levels. Tissue glycogen levels from asphyxiated piglets, un asphyxiated littermates and stillborn piglets are presented in table III. In the three piglets where fructose levels were estimated in the fetus
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Table I. Mean (and range) of time to last gasp (TLG) and time to last heart beat (TLHB) in nine term pig fetuses and one 97-day fetus.
Randall
66
Table II. Mean (t SE) blood pH p 0 2, pC02, hematocrit glucose and lactic acid levels in term pig fetuses before and after asphyxiation, and in stillborn fetuses at delivery
Preasphyxiation
Postasphyxiation
Stillborn piglets'
PH
7.43 ± 0.013 (6)
6.66 ± 0.020 (6)
6.69 ± 0.039 (22)
p 0 2, mm Hg
22.9 ± 0.60 (6)
1.6 ± 0.22 (6)
-
pC02, mm Hg
45.1 ± 1.41 (6)
170.8 ± 5.23 (6)
Glucose, mg/100 ml
50.7 ± 4.57 (6)
146.6 ± 35.61 (6)
Lactic acid, mg/100 ml
39.9 ± 6.64 (6)
185.5 ± 19.73 (6)
Hematocrit, %
32.1 ± 1.18 (5)
33.5 ± 1.22 (S)
156.4 t 4.4 (21) 159.3 ± 4.4 (6) —
before and after asphyxiation, levels fell (in all three) from a mean of 121.6 to 105.9 mg/ 100 ml.
Discussion
Most studies on neonatal asphyxia have been made on ‘newborns’ exposed to N2 or N2 + C 02 mixtures at temperatures similar to those in utero (4, 16, 24), asphyxia upon delivery (3, 8), asphyxiation of the exposed fetus by cord occlusion (9) or reduction in maternal 0 2 (2); variations between methods have been dis cussed previously (7). A preliminary study in dicated that asphyxia at delivery of the neonatal pig would be difficult to control and that the sow is not a good candidate for prolonged local anesthesia. The present tech nique enabled a number of parameters to be
measured in conscious fetuses at uterine temperatures and, since asphyxia can be as sociated with premature cord rupture in the pig (11, 20), it also provided a close model for naturally occurring stillbirth. Most experiments were carried out 24 h after surgery. At that time fetal preparations were probably not stable (6, 15) and fetal and maternal pC02 levels were lower and fetal lactate levels slightly higher than those seen in chronic preparations (21). However, longer intervals increased the chance of the umbilical-cuff actuating tube becoming entangled in the limbs and, while the stress of surgery may have influenced survival time, the times for the two fetuses asphyxiated 48 and 96 h postsurgery were similar to those at 24 h. The pattern of events following occlusion of the umbilical circulation was similar to that seen in other neonates (18) although minor
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Values in parentheses are number of animals sampled. 1 Data from Randall (19) and Randall and Penny (23).
67
Asphyxia of Fetal Pig
Table III. Mean (± SE) tissue glycogen levels (g/100 g wet weight) in asphyxiated piglets, their littermates and stillborn piglets at delivery
Asphyxiated piglet(s)
Littcrmate(s)
term
97-day
term
97-day
Heart
0.24 ± 0.05 (7)
0.16 (1)
1.29 i 0.19 (9)
1.88 (1)
0.29 ± 0.06 (3)
Liver
8.6 ± 1.03 (7)
1.21 (1)
12.9 ± 0.68 (9)
7.87 (1)
8.0 ± 0.81 (3)
Muscle
8.0 ± 0.42 (7)
4.0 (1)
8.3 ± 0.59 (9)
5.0 (1)
7.3 ± 0.29 (3)
acid levels were higher, perhaps as a result of different timing of the samples. Hyperglycemia was present in all asphyxiated piglets although marked differences (unrelated to survival time) were seen between individuals. Variations in the degree of hyperglycemia present in newborn calves exposed to transient hypoxia have been reported (10). In mature sheep fetuses glucose levels fell after the cord was clamped, but rose as the period of asphyxia became prolonged (9); similar findings have been reported in rhesus monkeys and calves (10, 24). The present study would have missed such a fall, if present, since all samples were taken terminally. It seems probable that the high glucose levels resulted from the mobilization of liver glyco gen, as in the sheep and calf (9, 10), and liver glycogen levels were lower than those of un asphyxiated controls. Whether the differences in glucose levels could be attributed to variation in initial levels of liver glycogen or to changes in hepatic circulation due to catecholamines (14) remains speculative. However, a marked hyper glycemic response was seen in the 97-day fetus where liver glycogen levels would be compara tively low (22). In 3 fetuses fructose levels fell during asphyxia. In the sheep, levels have been
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differences were seen. Primary apnea following the initial burst of gasping was not distinct and the slower gasping often followed directly from the initial rapid gasps. This was probably related to the short time to last gasp in this species. Miller and Miller (17) reported an interval of 4 min for time to last gasp for newborn piglets asphyxiated in 95% N2 + 5% C 0 2 at 39 °C compared with 5.1 min in the present study. The difference might be due to variations in age but the time to last gasp in the 97-day fetus was similar to that of the older group. The short times to last gasp and heart beat indicate that the pig, as with other species in which the fetus is relatively mature at birth, is very susceptible to asphyxia. However, within this limitation considerable variation was seen between individuals, suggesting that some fetuses within a litter may be more susceptible to asphyxia than others. Stretching of the umbilical cord during delivery would tend to decrease umbilical circulation prior to the cord breaking, and the survival time following rupture of the cord during delivery might be shorter than that reported here. Mean blood pH values were similar those of stillborn piglets (table II) but pC 02 and lactic
Stillborn piglets
Randall
68
References 1 Aherne, F.X.; Hays, V.W.; Ewan, R.C., and Speer, V.C.: Glucose and fructose in the fetal and newborn pig. J. Anim. Sci. 29: 906-911 (1969). 2 Britton, H.G.; Nixon, D.A., and Wright, G.H.: The effects of acute hypoxia on the sheep foetus and some observations on recovery from hypoxia. Biol. Neonate 11: 277-301 (1967). 3 Campbell, A.G.M.; Cross, K.W.; Dawes, G.S., and Hyman, A.I.: A comparison of air and 0 2, in a hyperbaric chamber or by positive pressure ventila tion, in the resuscitation of newborn rabbits. J. Pediat. 68: 153-163 (1966). 4 Cassin, S.; Swann, H.G., and Cassin, B.: Respira tory and cardiovascular alterations during the process of anoxic death in the newborn. J. appl. Physiol. 15: 249-252(1960). 5 Chinard, F.P.; Danesino, V.; Hartmann, W.L.; Huggett, A.S.-G.; Paul, W., and Reynolds, S.R.M.: The transmission of hexoses across the placenta in the human and the rhesus monkey (Macaca mulatto). J. Physiol., Lond. 132: 289-303 (1956). 6 Comline, R.S. and Silver, M.: Daily changes in foetal and maternal blood of conscious pregnant ewes, with catheters in umbilical and uterine vessels. J. Physiol., Lond. 209: 567-586 (1970). 7 Dawes, G.S.: Foetal and neonatal physiology (Year Book, Chicago 1968). 8 Dawes, G.S.; Jacobson, H.N.; Mott, J.C.; Shelley, H.J., and Stafford, A.: The treatment of asphyxiated mature foetal lambs and rhesus monkeys with intravenous glucose and sodium carbonate. J. Physiol., Lond. 169: 167-184 (1963). 9 Dawes, G.S.; Mott, J.C., and Shelley, H.J.: The importance of cardiac glycogen for the main tenance of life in foetal lambs and newborn animals during anoxia. J. Physiol., Lond. 146: 516-538 (1959). 10 Edwards, A.W. and Silver, M.: The effect of asphyxia on the plasma glucose concentration in newborn calves. Biol. Neonate 14: 1-7 (1969). 11 English, P.: Preliminary report to PI DA (School of Agriculture, University of Aberdeen, Aberdeen 1968). 12 Fazekas, J.F.; Alexander, F.A.D., and Himwich, H.E.: Tolerance of the newborn to anoxia. Am. J. Physiol. 134: 281-288 (1941).
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reported as either falling (9) or unchanged (2), although in both cases fructose was thought to contribute little towards fetal energy supplies during asphyxia. The pig fetus generally metab olizes little fructose (1) but it might act as a substrate during asphyxia. Cardiac and liver glycogen levels of as phyxiated piglets were significantly lower than those of unasphyxiated littermates and term fetuses (22). In occasional fetuses cardiac glyco gen was depleted after asphyxiation, although a small quantity was still present in most animals at the time of death. In contrast, muscle glyco gen levels were apparently unaffected by asphyxia and in some instances levels in as phyxiated piglets were higher than those in control littermates. These findings are in agree ment with those of other studies on term fetuses of different species (2, 9). It has been suggested that the time to last gasp is related to the initial cardiac glycogen levels (9). The pig and sheep fetus both have somewhat similar cardiac glycogen levels during late gestation (2, 9, 22), and times to last gasp are also similar (9, this study). It is of interest that both survival time and cardiac glycogen decrease with advancing gestational age in the sheep (9), whereas the time to last gasp for the 97-day pig fetus was similar to that of term fetuses. There is no fall in cardiac glycogen in the second half of gestation in the pig (22). The present study indicates that the pig fetus, like other species in which the fetus is mature at birth, is relatively susceptible to asphyxiation, and it has been suggested that premature rupture of the umbilical cord may be a predisposing factor to asphyxia (20). The strength of the umbilical cord appears to vary between litters and investigations into the cause of this variation might provide a more practical solution to reducing losses at birth than at tempts to resuscitate newborn piglets.
13 Harrower, J.R. and Brown, C.H.: Blood lactic acid: a micromethod adapted to field collection of microliter samples. J. appl. Physiol. 33: 709-711 (1972). 14 Levine, R.A.: Effect of glycogenolytic agents on phosphorylase activity of perfused rat liver. Am. J. Physiol. 208: 317-323 (1965). 15 Meschia, G.; Cotter, J.R.; Breathnach, C.S., and Barron, D.H.: The hemoglobin, oxygen, carbon dioxide and hydrogen ion concentrations in the umbilical bloods of sheep and goats as sampled via indwelling plastic catheters. Q. J1 exp. Physiol. 50: 185-195 (1965). 16 Miller, J.A.: in Windle, Neurological and psycho logical deficits of asphyxia neonatorum with con sideration of use of primates for experimental investigations (Springfield 1958). 17 Miller, J.A. and Miller, F.S.: Studies on prevention of brain damage in asphyxia. Devi Med. Child Neur. 7: 607-619 (1965). 18 Mott, J.C.: The ability of young mammals to withstand total oxygen lack. Br. med. Bull. 17: 144-147 (1961). 19 Randall, G.C.B.: The relationship of arterial blood pH and pC02 to the viability of the newborn piglet. Can. J. comp. Med. 35: 141-146 (1971).
69
20 Randall, G.C.B.: Observations on parturition in the sow. 11. Factors influencing stillbirth and perinatal mortality. Vet. Rcc. 90: 183-186 (1972). 21 Randall, G.C.B.: Daily changes in the blood of conscious pigs with catheters in foetal and uterine vessels during late gestation. J. Physiol., Lond. 270: 719-731 (1977). 22 Randall, G.C.B. and L’Ecuyer, C.: Tissue glycogen and blood glucose and fructose levels in the pig fetus during the second half of gestation. Biol. Neonate 28: 74-82 (1976). 23 Randall, G.C.B. and Penny, R.H.C.: Stillbirths in pigs: observations on blood lactic acid levels. Vet. Rec. 83: 57 (1968). 24 Rivera, A.; Brann, A.W.; Martinez-de Jesus, J., and Myers, R.E.: Glycogen content of vital organs of newborn monkeys recovering from asphyxia. Biol. Neonate 20: 22-39 (1972).
G.C.B. Randall, Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (E), Box 11300, Station H, Ottawa, Ont., K2H 8P9 (Canada)
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Asphyxia of Fetal Pig
Studies on the Effect of Acute Asphyxia on the Fetal Pig
in utero G.C.B. Randall' Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (E), Ottawa, Ont.
Key Words. Asphyxia • Fetal pig ■Stillbirth Abstract. 8 term fetal pigs (110—112 days gestation) and one 97-day fetus were asphyxiated in
útero by occlusion of the umbilical cord. Mean times to last gasp and last heart beat were 5.1 and 22.4 for term and 5.4 and 30.4 min for the 97-day fetus. Cord occlusion was followed by profound bradycardia and an increase in blood pressure which was maintained until gasping ceased. Profound acidemia, hypercapnia and hyperlactacidemia developed in all animals and values following asphyxiation were comparable with those seen in stillborn piglets. Liver and cardiac glycogen levels were lower in asphyxiated fetuses than in littermates but muscle glycogen levels were similar in both groups.
Previous studies on blood gases, pH and lactic acid levels indicated that asphyxia was a major cause of stillbirth in the pig (19,20) and that premature rupture of the umbilical cord might be a predisposing cause (11,20). Although the sequence of changes following asphyxia is similar (18), variations in the dura tion of survival are known to occur between species (12). Miller and Miller (17) showed that newborn piglets had a short time to last gasp 1 The author wishes to acknowledge the skilled technical help of Mrs. J. Hierlihy and Messrs. Y. Barbeau, G. Raby, IV. G. Addison and R.L. Marenger.
when placed in a mixture of 95% N2 + 5% C 0 2, but few other studies are available for this species. In this paper changes occurring in the unanesthetized fetal pig during anoxia, resulting from occlusion of the umbilical cord at term, are described and compared with observations made on naturally asphyxiated piglets.
Materials and Methods 10 Yorkshire gilts (127-200 kg body weight) were used. Catheters and electrodes were implanted into 13 fetuses under halothane: oxygen anesthesia (Fluothane, Ayerst), following induction with thio pental sodium (Pentothal, Abbott). Fetal cannulation was performed at 108-111 days gestation (mean
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Introduction
64
Fetal Asphyxiation The sow was moved to a crush and allowed time to settle; most animals lay quietly during the study. The fetal catheters and electrodes were connected to a polygraph and records taken for 1-2 h to establish base levels. Towards the end of this period fetal blood samples were taken for blood gas, glucose, fructose and lactic acid estimation. The occluder was then inflated and fetal recording continued until the cessa tion of regular heart beats from the EKG trace. A second blood sample was collected either just preced ing or following cessation of fetal heart beat. The sow was then killed with barbiturate. Tissue samples were removed immediately from the asphyxiated piglet, and one or two littermates, and frozen in liquid N2 in the following order: cardiac ventricles, liver and thigh muscle. Samples were stored at -7 9 °C until analyzed. Similar samples were taken from three naturally oc curring ‘stillborn’ piglets prior to cessation of heart beat. In this paper a ‘stillborn’ piglet is one in which there is a slow heart beat at the time of delivery which ceases within a few minutes of birth, but no respira tory efforts are made (20).
Absolute pressures were monitored with trans ducers (Statham, model P23AC or P23DC) positioned at the level of the dorsal line of the sow’s mammary glands. The carotid cannula was infused with hepa rinized saline (0.2 ml/h) during recording to reduce clotting. True fetal blood pressure was obtained by subtracting cither amniotic fluid or tracheal pressure from absolute blood pressure. Heart rate was taken from the carotid pulse or the QRS complex of the EKG trace. Blood pH, p 0 2 and pC02 were determined at 38.5 °C using a BMS3 blood microsystem with a PHM71 acid base analyzer (Radiometer, Copenhagen). Samples were stored in iced water until analyzed. Glucose, fructose and lactic acid levels were estimated in whole blood following deproteinization of 100 ix\ in ice-cold uranyl acetate or perchloric acid. Glucose was estimated using a commercial glucose oxidaseperoxidase method (Boehringer, Mannheim), fructose using Seliwanof reagents (5) and lactic acid by a microdetermination method (13). Tissue glycogen was estimated using anthrone reagent as described pre viously (22).
Results
9 of 13 fetuses survived surgery, and in the other 4 death appeared to have resulted from pressure of the occluder on the umbilical cord following entanglement of its actuating tubing in the felal limbs. Cardiovascular and Respiratory Changes A regular pattern was seen in the term fetuses although variations in magnitude or duration of changes occurred between individ uals. As an example, the changes in one fetus are shown in figure 1. A profound bradycardia occurred within 15 sec of cord occlusion (Fig. 1) reaching its lowest rate 30-45 sec after occlusion, at which time fetal heart rate was 32.2% (range 26.5-39.5%) of preasphyxia rates. It then increased towards resting values (39.8—93.6%), peaking 2—3 min after occlu sion, and then fell rapidly during the later
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109.6) in 12 fetuses from 9 sows and at 96 days gestation in 1 fetus. With two exceptions (48 and 96 h), fetal asphyxiation studies were made 24 h after surgery. A polyvinyl cannula (0.58 mm i.d. X 0.96 mm o.d., Bolab Inc., Derry, New Hampshire) was placed in the right-hand fetal carotid artery using techniques described elsewhere (21). A similar catheter (length 2 -3 cm) was inserted into the fetal trachea through a hole made with a hypodermic needle immediately behind the larynx. A polyvinyl amniotic fluid catheter (1.0 mm i.d. X 2.0 mm o.d.) was sutured to the piglet’s neck close to the carotid catheter and, in most animals, two subdcrmal platinum EKG electrodes (Grass Instruments, Quincy, Mass.) were attached to the anterior chest wall. Finally, a 14-mm blood vessel occluder (Model V 0-4, Rhodes Medical Instruments, Woodland Hills, Calif.) was tied loosely around the umbilical cord and the uterus closed with 20 cm of each catheter within the uterus. Capped hypodermic needle hubs were bonded into the external ends of the catheters and these, together with the electrode leads and the occluder actuating tubing, were stored aseptically in a plastic bag within a pouch sutured to the sow’s flank. Cannulac were filled with sterile heparinized saline (100 lU/ml) between samples.
Randall
Asphyxia of Fetal Pig
65
Fig. 1. Changes in heart rate and mean, systolic and diastolic blood pressures following occlusion of the umbilical cord in one fetus.
stages of fetal gasping. During the terminal stages the heart rate was often irregular and extrasystolic beats were also present. Mean fetal blood pressure rose during the first 15—30 sec with increases in both systolic and diastolic pressures. In most animals there was a transi tory ‘fall’ in mean blood pressure —as has been seen in sheep (9) — 30—60 sec after occlusion. This was coincident with the trough in heart rate and a low diastolic pressure between beats although systolic pressure continued to rise.
Gestational age, days
TLG, min
TLHB, min
110-112 97
5.1 (4.1-6.1) 5.4
22.4 (14.2-34.3) 30.4
Thereafter, mean blood pressure rose to a peak 2 -3 min after occlusion before falling rapidly as fetal breathing ended. Mean times to last heart beat are shown in table I. In the 97-dayold fetus changes in heart rate were similar to term fetuses, but changes in blood pressure were less marked. The increase in mean blood pressure was smaller and of shorter duration than that seen in term piglets. Within 15-30 sec of cord occlusion the fetuses gasped rapidly (8—12/min) for about 1 min. The early gasps were followed by slower, deeper gasps (45—55 mm Hg) at 2/min which often became more frequent (4/min) and shallower terminally. No distinct period of primary apnea was observed although a short delay preceding the deeper gasps was noted in a few animals. Blood pH, p 0 2, pC 02, glucose, lactic acid and hematocrit values for asphyxiated piglets and stillborn piglets are shown in table II. Values for the 97-day fetus were similar to those of term fetuses and were included with them. Changes were similar for all parameters except glucose, where marked individual dif ferences were seen and values increased by 25—356% over preasphyxia levels. Tissue glycogen levels from asphyxiated piglets, un asphyxiated littermates and stillborn piglets are presented in table III. In the three piglets where fructose levels were estimated in the fetus
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Table I. Mean (and range) of time to last gasp (TLG) and time to last heart beat (TLHB) in nine term pig fetuses and one 97-day fetus.
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Table II. Mean (t SE) blood pH p 0 2, pC02, hematocrit glucose and lactic acid levels in term pig fetuses before and after asphyxiation, and in stillborn fetuses at delivery
Preasphyxiation
Postasphyxiation
Stillborn piglets'
PH
7.43 ± 0.013 (6)
6.66 ± 0.020 (6)
6.69 ± 0.039 (22)
p 0 2, mm Hg
22.9 ± 0.60 (6)
1.6 ± 0.22 (6)
-
pC02, mm Hg
45.1 ± 1.41 (6)
170.8 ± 5.23 (6)
Glucose, mg/100 ml
50.7 ± 4.57 (6)
146.6 ± 35.61 (6)
Lactic acid, mg/100 ml
39.9 ± 6.64 (6)
185.5 ± 19.73 (6)
Hematocrit, %
32.1 ± 1.18 (5)
33.5 ± 1.22 (S)
156.4 t 4.4 (21) 159.3 ± 4.4 (6) —
before and after asphyxiation, levels fell (in all three) from a mean of 121.6 to 105.9 mg/ 100 ml.
Discussion
Most studies on neonatal asphyxia have been made on ‘newborns’ exposed to N2 or N2 + C 02 mixtures at temperatures similar to those in utero (4, 16, 24), asphyxia upon delivery (3, 8), asphyxiation of the exposed fetus by cord occlusion (9) or reduction in maternal 0 2 (2); variations between methods have been dis cussed previously (7). A preliminary study in dicated that asphyxia at delivery of the neonatal pig would be difficult to control and that the sow is not a good candidate for prolonged local anesthesia. The present tech nique enabled a number of parameters to be
measured in conscious fetuses at uterine temperatures and, since asphyxia can be as sociated with premature cord rupture in the pig (11, 20), it also provided a close model for naturally occurring stillbirth. Most experiments were carried out 24 h after surgery. At that time fetal preparations were probably not stable (6, 15) and fetal and maternal pC02 levels were lower and fetal lactate levels slightly higher than those seen in chronic preparations (21). However, longer intervals increased the chance of the umbilical-cuff actuating tube becoming entangled in the limbs and, while the stress of surgery may have influenced survival time, the times for the two fetuses asphyxiated 48 and 96 h postsurgery were similar to those at 24 h. The pattern of events following occlusion of the umbilical circulation was similar to that seen in other neonates (18) although minor
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Values in parentheses are number of animals sampled. 1 Data from Randall (19) and Randall and Penny (23).
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Asphyxia of Fetal Pig
Table III. Mean (± SE) tissue glycogen levels (g/100 g wet weight) in asphyxiated piglets, their littermates and stillborn piglets at delivery
Asphyxiated piglet(s)
Littcrmate(s)
term
97-day
term
97-day
Heart
0.24 ± 0.05 (7)
0.16 (1)
1.29 i 0.19 (9)
1.88 (1)
0.29 ± 0.06 (3)
Liver
8.6 ± 1.03 (7)
1.21 (1)
12.9 ± 0.68 (9)
7.87 (1)
8.0 ± 0.81 (3)
Muscle
8.0 ± 0.42 (7)
4.0 (1)
8.3 ± 0.59 (9)
5.0 (1)
7.3 ± 0.29 (3)
acid levels were higher, perhaps as a result of different timing of the samples. Hyperglycemia was present in all asphyxiated piglets although marked differences (unrelated to survival time) were seen between individuals. Variations in the degree of hyperglycemia present in newborn calves exposed to transient hypoxia have been reported (10). In mature sheep fetuses glucose levels fell after the cord was clamped, but rose as the period of asphyxia became prolonged (9); similar findings have been reported in rhesus monkeys and calves (10, 24). The present study would have missed such a fall, if present, since all samples were taken terminally. It seems probable that the high glucose levels resulted from the mobilization of liver glyco gen, as in the sheep and calf (9, 10), and liver glycogen levels were lower than those of un asphyxiated controls. Whether the differences in glucose levels could be attributed to variation in initial levels of liver glycogen or to changes in hepatic circulation due to catecholamines (14) remains speculative. However, a marked hyper glycemic response was seen in the 97-day fetus where liver glycogen levels would be compara tively low (22). In 3 fetuses fructose levels fell during asphyxia. In the sheep, levels have been
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differences were seen. Primary apnea following the initial burst of gasping was not distinct and the slower gasping often followed directly from the initial rapid gasps. This was probably related to the short time to last gasp in this species. Miller and Miller (17) reported an interval of 4 min for time to last gasp for newborn piglets asphyxiated in 95% N2 + 5% C 0 2 at 39 °C compared with 5.1 min in the present study. The difference might be due to variations in age but the time to last gasp in the 97-day fetus was similar to that of the older group. The short times to last gasp and heart beat indicate that the pig, as with other species in which the fetus is relatively mature at birth, is very susceptible to asphyxia. However, within this limitation considerable variation was seen between individuals, suggesting that some fetuses within a litter may be more susceptible to asphyxia than others. Stretching of the umbilical cord during delivery would tend to decrease umbilical circulation prior to the cord breaking, and the survival time following rupture of the cord during delivery might be shorter than that reported here. Mean blood pH values were similar those of stillborn piglets (table II) but pC 02 and lactic
Stillborn piglets
Randall
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References 1 Aherne, F.X.; Hays, V.W.; Ewan, R.C., and Speer, V.C.: Glucose and fructose in the fetal and newborn pig. J. Anim. Sci. 29: 906-911 (1969). 2 Britton, H.G.; Nixon, D.A., and Wright, G.H.: The effects of acute hypoxia on the sheep foetus and some observations on recovery from hypoxia. Biol. Neonate 11: 277-301 (1967). 3 Campbell, A.G.M.; Cross, K.W.; Dawes, G.S., and Hyman, A.I.: A comparison of air and 0 2, in a hyperbaric chamber or by positive pressure ventila tion, in the resuscitation of newborn rabbits. J. Pediat. 68: 153-163 (1966). 4 Cassin, S.; Swann, H.G., and Cassin, B.: Respira tory and cardiovascular alterations during the process of anoxic death in the newborn. J. appl. Physiol. 15: 249-252(1960). 5 Chinard, F.P.; Danesino, V.; Hartmann, W.L.; Huggett, A.S.-G.; Paul, W., and Reynolds, S.R.M.: The transmission of hexoses across the placenta in the human and the rhesus monkey (Macaca mulatto). J. Physiol., Lond. 132: 289-303 (1956). 6 Comline, R.S. and Silver, M.: Daily changes in foetal and maternal blood of conscious pregnant ewes, with catheters in umbilical and uterine vessels. J. Physiol., Lond. 209: 567-586 (1970). 7 Dawes, G.S.: Foetal and neonatal physiology (Year Book, Chicago 1968). 8 Dawes, G.S.; Jacobson, H.N.; Mott, J.C.; Shelley, H.J., and Stafford, A.: The treatment of asphyxiated mature foetal lambs and rhesus monkeys with intravenous glucose and sodium carbonate. J. Physiol., Lond. 169: 167-184 (1963). 9 Dawes, G.S.; Mott, J.C., and Shelley, H.J.: The importance of cardiac glycogen for the main tenance of life in foetal lambs and newborn animals during anoxia. J. Physiol., Lond. 146: 516-538 (1959). 10 Edwards, A.W. and Silver, M.: The effect of asphyxia on the plasma glucose concentration in newborn calves. Biol. Neonate 14: 1-7 (1969). 11 English, P.: Preliminary report to PI DA (School of Agriculture, University of Aberdeen, Aberdeen 1968). 12 Fazekas, J.F.; Alexander, F.A.D., and Himwich, H.E.: Tolerance of the newborn to anoxia. Am. J. Physiol. 134: 281-288 (1941).
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reported as either falling (9) or unchanged (2), although in both cases fructose was thought to contribute little towards fetal energy supplies during asphyxia. The pig fetus generally metab olizes little fructose (1) but it might act as a substrate during asphyxia. Cardiac and liver glycogen levels of as phyxiated piglets were significantly lower than those of unasphyxiated littermates and term fetuses (22). In occasional fetuses cardiac glyco gen was depleted after asphyxiation, although a small quantity was still present in most animals at the time of death. In contrast, muscle glyco gen levels were apparently unaffected by asphyxia and in some instances levels in as phyxiated piglets were higher than those in control littermates. These findings are in agree ment with those of other studies on term fetuses of different species (2, 9). It has been suggested that the time to last gasp is related to the initial cardiac glycogen levels (9). The pig and sheep fetus both have somewhat similar cardiac glycogen levels during late gestation (2, 9, 22), and times to last gasp are also similar (9, this study). It is of interest that both survival time and cardiac glycogen decrease with advancing gestational age in the sheep (9), whereas the time to last gasp for the 97-day pig fetus was similar to that of term fetuses. There is no fall in cardiac glycogen in the second half of gestation in the pig (22). The present study indicates that the pig fetus, like other species in which the fetus is mature at birth, is relatively susceptible to asphyxiation, and it has been suggested that premature rupture of the umbilical cord may be a predisposing factor to asphyxia (20). The strength of the umbilical cord appears to vary between litters and investigations into the cause of this variation might provide a more practical solution to reducing losses at birth than at tempts to resuscitate newborn piglets.
13 Harrower, J.R. and Brown, C.H.: Blood lactic acid: a micromethod adapted to field collection of microliter samples. J. appl. Physiol. 33: 709-711 (1972). 14 Levine, R.A.: Effect of glycogenolytic agents on phosphorylase activity of perfused rat liver. Am. J. Physiol. 208: 317-323 (1965). 15 Meschia, G.; Cotter, J.R.; Breathnach, C.S., and Barron, D.H.: The hemoglobin, oxygen, carbon dioxide and hydrogen ion concentrations in the umbilical bloods of sheep and goats as sampled via indwelling plastic catheters. Q. J1 exp. Physiol. 50: 185-195 (1965). 16 Miller, J.A.: in Windle, Neurological and psycho logical deficits of asphyxia neonatorum with con sideration of use of primates for experimental investigations (Springfield 1958). 17 Miller, J.A. and Miller, F.S.: Studies on prevention of brain damage in asphyxia. Devi Med. Child Neur. 7: 607-619 (1965). 18 Mott, J.C.: The ability of young mammals to withstand total oxygen lack. Br. med. Bull. 17: 144-147 (1961). 19 Randall, G.C.B.: The relationship of arterial blood pH and pC02 to the viability of the newborn piglet. Can. J. comp. Med. 35: 141-146 (1971).
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20 Randall, G.C.B.: Observations on parturition in the sow. 11. Factors influencing stillbirth and perinatal mortality. Vet. Rcc. 90: 183-186 (1972). 21 Randall, G.C.B.: Daily changes in the blood of conscious pigs with catheters in foetal and uterine vessels during late gestation. J. Physiol., Lond. 270: 719-731 (1977). 22 Randall, G.C.B. and L’Ecuyer, C.: Tissue glycogen and blood glucose and fructose levels in the pig fetus during the second half of gestation. Biol. Neonate 28: 74-82 (1976). 23 Randall, G.C.B. and Penny, R.H.C.: Stillbirths in pigs: observations on blood lactic acid levels. Vet. Rec. 83: 57 (1968). 24 Rivera, A.; Brann, A.W.; Martinez-de Jesus, J., and Myers, R.E.: Glycogen content of vital organs of newborn monkeys recovering from asphyxia. Biol. Neonate 20: 22-39 (1972).
G.C.B. Randall, Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (E), Box 11300, Station H, Ottawa, Ont., K2H 8P9 (Canada)
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Asphyxia of Fetal Pig
