metabolism in sheep Ontogeny of fetal hepatic and

Ontogeny of fetal hepatic and placental growth and metabolism in sheep I. Vatnick and A. W. Bell

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on the following topics: Biochemistry .. Metabolism Biochemistry .. Oxygen Consumption Physiology .. Pregnancy Medicine .. Liver

Ontogeny of fetal hepatic and placental and metabolism in sheep I. VATNICK Department

AND A. W. BELL of Animal Science, Cornell

University,

growth

Ithaca, New

York 14853

were scanneddaily with a real time ultrasonic device (Johnson & Johnson Technicare 210 DX, New Brunswick, NJ) from day 45 postcoitus (PC) to verify litter size and gestationalage.From 50 days PC the eweswere individually fed once daily a total mixed ration containing 2.5 Meal metabolizable energy (ME) and 150 g crude protein (CP) per kilogram dry matter to fulfill estimated nutrient requirements depending on body weight, stage of pregnancy, and litter size according to National ResearchCouncil recommendations(20). Six twin-pregnant eweswere housedin individual floor pens from 50 days PC until slaughteredat 75 days PC. Another group of six twin-pregnant eweswas maintained under similar conditions from 50 days until slaughteredat 136 days PC. This group underwent surgery on day 100 PC when one placentome was excisedfor the in vitro vo2 studies,asdescribedpreviously (23). This procedure had no apparent detrimental effects on subsequent fetal and placental growth or grossmorphology. The six single-pregnant eweswere similarly housedfrom 50 days PC until slaughter at 136 days PC. Dissection and tissue sampling procedures. At slaughterewes were stunnedwith a captive-bolt pistol and exsanguinatedusing procedures approved by the Cornell University Institutional Animal Care and Use Committee. The pregnant uterus was rapidly removed from the abdominal cavity, weighed, and dissectedto separateeachfetus and placenta. Placental weight was THE PLACENTA and fetal liver are vital organs for nutriconsideredto be the aggregateweight of all placentomesfor each ent supply and metabolism in the growing fetus. The fetus, trimmed of endometrium and fetal membranes.Fetuses anatomic arrangement of these organs ensures intimate relations between their functions, similar to those be- were towel dried and weighed,and fetal livers were removedand tween liver and gut in postnatal life. However, the ges- weighed. Tissue samplesfrom the placenta and liver of one fetus per tational patterns of growth of the fetal liver and placenta ewe were used for in vitro metabolic measurements.A single vary considerably. In sheep, placental weight remains placentomeand liver werewashedin salineto removeblood and static or declines through the latter half of gestation placed in complete media (M 199, Sigma Chemical, St. Louis, (3,5,22), whereas the fetal liver continues to grow albeit MO) saturated and continuously bubbled with 95% 02-5% CO2 at a slower rate than the rest of the fetus (6, 12). In vivo at 4°C. During dissectionone placentomewaspinned to a dissection dish placedon ice, coveredwith M 199and continuously measurement of uteroplacental oxygen consumption bubbled with 95% 02-5% CO,. Predominantly maternal tissue (Vo2) during mid and late pregnancy suggests that placental metabolic rate does not change appreciably, de- was excised from the very outer portion of the placentome, an spite a major increase in functional capacity of the pla- area poor in trophoblastic interdigitation asjudged by histological examination of many other placentomes. Predominantly centa over this period (7, 8). Also, we have previously fetal tissue was excised from the center of the fetal portion of postulated that the gestational decline in relative weight the placentome near the chorionic surface. Tissue sections of the liver may account for much of the decline in (25-50 mg) were blotted, weighed, and kept in individual 5-ml weight-specific metabolic rate of the whole fetus during test tubes filled with M 199 saturated with 95% 02-5% CO2at the latter half of pregnancy, implying little change in 4°C until placed in the oxygen electrodechamber. The remainweight-specific metabolic rate in the fetal liver (6). How- ing placentomeswere homogenizedin a Waring blender and the ever, gestational changes in the metabolic rates of these homogenatewas stored at -2OOC. Sections of fetal liver (25-50 mg) were dissectedfrom the organs, interrelations between them, and their conseright lobe under the sameconditions. Becauseof the small size quences for energy expenditure in the whole conceptus of livers from 50-day fetuses,dry matter determinations were have not been systematically studied. done on livers obtained from six other fetusesat the samegesTherefore, in the present study we have measured tational age,and the averageof these valueswas applied to the placental and fetal VO, in vitro in ovine tissues obtained measurements. at different stages of gestation and have related these VO,AnalyticaL procedures. Oxygen uptake of fetal and maternal metabolic rates to rates of growth of these organs be- placenta and fetal liver sections was measuredwith a Clark tween mid and late pregnancy. polarographic electrode in a biological oxygen monitoring system (YSI 5300, Yellow Springs Instruments, Yellow Springs, METHODS OH). Sections of tissue (25-50 mg) were placed in a chamber Animals and feeding. Twelve twin-pregnant and six single- containing 4 ml of M 199 saturated with air and equilibrated to pregnant Dorset and Finn-Dorset ewesof known mating date 37°C and pH 7.4. Oxygen consumption wasmonitored for 8-12 0363-6119/92

$2.00

Copyright

0 1992 the American

Physiological

Society

R619


Vatnick, I., and A. W. Bell. Ontogeny of fetal hepatic and placental growth and metabolismin sheep.Am. J. Physiol. 263 (Regulatory Integrative Cornp. Physiol. 32): R619-R623, 1992. -Ontogeny of fetal hepatic and placental growth and in vitro oxygen consumption (i70,) wasinvestigated in fetal lambsat 75, 100, and 136days postconception. Fetal hepatic relative weight and placental absoluteand relative weights declined during this period. Oxygen consumption per gram dry weight of fetal liver and maternal placenta declined betweenmid and late gestation while fetal placental i702 was unchanged.Estimated iTo, of the whole placenta did not changewhile the estimatedtotal hepatic VO, increasedmore than threefold between 75 and 136 days. Total hepatic ire, was highly correlated with total placental VO, at 136days (r = 0.84). The resultssuggestthat the placenta reachesits maximum growth and metabolic capacity before 100 days and possibly at or before midgestation. Changesin hepatic weight-specific total i702, in addition to the declining relative size of the fetal liver, must contribute to the progressivedecline in metabolic rate of the whole fetus during the secondhalf of pregnancy. Correlations between placental and fetal liver weights and metabolic rates suggestthe possibility of placental regulation of fetal hepatic growth and metabolism. oxygen consumption; fetal liver; placenta

R620

FETAL

HEPATIC

AND

PLACENTAL

RESULTS

Except where noted, the following results refer only to the 12 twin-pregnant ewes studied at 75,100, or 136 days PC ilacental and fetal hepatic growth. Fetal weight increased more than 15-fold from 232 t 7 (SE) g at 75 days PC to 3,583 t 228 g at 136 days PC. Placental wet weight declined 300 g from mid to late gestation, while dry weight did not change during this time. Total placental DNA content also remained constant between 75 and 136 PC days (Table 1). Placental weight was 2.75 times that of fetal Table 1. Fetal whole body, hepatic and placental weights 75 Days

IN SHEEP

weight at 75 days PC but was only 9% of fetal weight at 136 days PC (Table 1). Fetal hepatic weight increased approximately sixfold between 75 and 136 days PC. Fetal liver was 6.4% of total fetal weight at 75 days PC but only 2.4% at 136 days PC (Table 1). Placental oxygen consumption. Dry weight-specific iTo,, and ouabain-sensitive and ouabain-insensitive VO, of fetal placenta remained unchanged between mid and late pregnancy (Table 2). Maternal total placental VO, declined from 53 ~1 min-l l g dry tissue- l at 75 days PC to 44 at 100 days PC and further declined to 31 ~1. mine1 lg-l at 136 days PC (P < 0.05, Table 2). Similarly, maternal ouabaininsensitive placental \io, declined from 41 ~1. min-l l g dry tissue-l at 75 days PC to 34 at 100 days PC and further to 26 ~1l rein-l l g- 1 at 136 days PC (P < 0.05). Ouabain-sensitive VO, remained unchanged during this time (Table 2). Hepatic oxygen consumption. Hepatic weight-specific total vo2 tended to be higher (P < 0.1) at 50 days than at 75 days and declined sharply (P < 0.05) by 136 days PC (Fig. 1). Ouabain-sensitive Vo2 remained unchanged between 50 and 75 days and tended to be lower (P < 0.1) at 136 days than at 75 days. Ouabain-insensitive Tjo2 also remained unchanged between 50 and 75 days but declined at 136 days PC (P < 0.05, Fig. 1). Correlations. Weight-specific fetal hepatic Vo2 was not significantly correlated with placental weight at 75 or 136 days. There was also no relation between fetal liver and placental weights or between total fetal hepatic iTog and placental weight at 75 days. Fetal hepatic weight was highly correlated with placental weight (r = 0.87; Fig. 2A) and total fetal hepatic I-70, with total placental iTo, (r = 0.84; Fig. 2B) at 136 days. These correlations include individual data from a group of single-pregnant ewes not included in the ontogenic studies. l

136 Days

DISCUSSION Fetal Wet wt, g Hepatic Wet wt, g Dry wt, g % Fetal wt (wet) Placental Wet wt, g Dry wt”, g % Fetal wt (wet), g Total DNA content*, Values P > 0.1.

are means

g

232.2t6.9

3,583.0+228.2

14.8t0.5 2.9tO.l 6.4t0.2

88.0k7.3 17.2t1.4 2.4k0.1

635.0t39.1 57.4t3.1 274.6t16.9 1.221kO.08

332.5t41.2 55.2k7.3 8.9t0.8 1.30t0.30

k SE; n = 12 fetuses.

* Not

significantly

different,

Despite many studies involving measurement of placental size in late pregnancy there is a lack of precise information on the gestational pattern of macroscopic and cellular growth of this organ in sheep and other species. In particular, the timing and metabolic basis for the abrupt cessation of hyperplastic ovine placenta growth are poorly defined. It is widely accepted that the formation of placentomes from cotyledonary and caruncular tissue is virtually complete by 40 days PC (2, 5) and that the placenta reaches its maximum weight sometime

Table 2. In vitro oxygen consumption of fetal and maternal placenta Gestational 75

P

Age, days 100

136

PSE G

F

M

F

36.8 25.2 11.2

52.8 41.2 12.7

39.2 28.8 11.4

M

Vo2, phnin-l-g Total Ouabain Ouabain

dry 37.2 29.3 7.6

S

G x S*

M

tissuewl

31.4 3.2 co.05 26.1 2.9 O.l tissue;

co.01 co.005 >O.l and M, maternal


min then ouabain, a specific inhibitor of Na+-K+-adenosinetriphosphatase (ATPase; 15), was added to the chamber to a final concentration of 10m4 M, and iTg2 was monitored for an additional 8-12 min. This dose was shown to produce a maximal response in preliminary experiments (B. W. McBride and I. Vatnick, unpublished observations). The difference between VO, rates before and after addition of ouabain is defined as ouabain-sensitive respiration and is considered to represent the energy cost of Na+-K+-ATPase activity (16). Tissue sections respired linearly over a period of 2 h, and there was parallelism with doubling of section weights. Placental and hepatic dry weights were determined by placing subsamples of wet tissues in a vacuum drying oven at 60°C for 48 h. Tissue DNA concentration was measured by a modification of the method of Munro and Fleck (19). Statistics. Least squares regression analysis was used to determine relations between independent variables. Analysis of variance (ANOVA) with Scheffe’s modification was used for multiple comparisons of mean fetal and organ weights and hepatic respiration among all groups. A split plot ANOVA was used to analyze the in vitro placental respiration data.

Vo,

FETAL

HEPATIC

AND

PLACENTAL

a l-

76.2%

b 78.9%

66.4%

,

7=-l

136 Gestational

age

(d)

Fig. 1. Effect of gestational age on fetal hepatic oxygen consumption. Histograms represent means for ouabain-insensitive (open) and ouabain-sensitive (closed) oxygen consumption. Vertical bars are SE; n = 6 fetuses. Values with superscripts a, b are different at P < 0.05.

2001A

100

I

200

I

300 Placental

t

400 weight

I

500

1

600

(g)

1250 E

.-0 z e =

250 1000 Platen

tal

15 00 oxygen

2000 uptake

2500 (pl/min)

Fig. 2. Relations between fetal liver weight and placental weight (A, y = 19.1 + 0.270x, r = 0.87, P < 0.01) and fetal hepatic oxygen uptake and placental oxygen uptake (B, y = 276 + 0.291x, r = 0.84, P < 0.01) in single-pregnant (0) and twin-pregnant (A) ewes at 136 days.

before 90 days, then declines in weight thereafter (3, 5). Recently communicated data from our laboratory (14) show that the sheep placenta reaches its maximum weight as early as 75 days PC and that by then nuclear proliferation and, by inference, hyperplastic growth has ceased because there was no increase in total DNA content between 75 and 100 days. Those findings are entirely consistent with the present results, which show unchanged placental DNA content between 75 and 136 days. The decline in relative weight of the liver during fetal life has been thoroughly characterized (6, 12). In this study relative weights are expressed on a wet weight basis, since fetal dry weight was not measured. Like all fetal organs and tissues, the liver steadily dehydrates during the latter half of gestation, but the pattern of change in hydration differs from that of the whole body (6). Therefore, relative weight of fetal liver expressed on a dry mat-

IN

SHEEP

R621

ter basis would be somewhat different from present values for relative wet weight. The vascular anatomy of the placenta precludes direct in vivo measurement of placental V02 in conscious animals. Although in vivo measurements of uteroplacental Vo2 can be made (US), these measurements include metabolism of nonplacental uterine tissues. More specific measurements have been made on anesthetized ewes but values for placental Vo2 were much lower than those for uteroplacental \io, obtained in conscious animals (10, 18). In vitro measurements of highly active organs such as the placenta or liver inevitably underestimate metabolic rates in vivo (16) possibly because of inadequate oxygen delivery. The present data may at best provide an approximation of the relative developmental changes that occur in vivo. Calculations based on in vivo measurements of uteroplacental (8) and hepatic (9) VO, in late gestation indicate in vitro estimates of VO, for these organs at - 15 and 20% of in vivo measurements for the whole placenta and liver, respectively (see APPENDIX). The value for total placental Vo2 obtained in this study is similar to that obtained in a study of placental Vo2 in situ in anesthetized ewes (10). Weight-specific VO, of fetal placenta remained unchanged during the second half of gestation, whereas VO, of maternal tissues declined. These results contrast with those briefly reported by Reynolds and Redmer (21) who, using similar in vitro methodology, found an increase in weight-specific VO, of placental tissues over the same period. However, these authors expressed their results on a wet weight basis, disregarding the marked increases in placental dry matter content between mid and late pregnancy. Placental dry matter content is -9% at 75 days PC and 17% at 136 days PC, which could account for the apparent gestational increase in total wet weight-specific Vo2 reported by these authors. Weight-specific \io, of human placenta in vitro declined 50% from early to late gestation (24). Ouabain-sensitive placental respiration remained constant through gestation and accounted for 20-30% of total vo2 in fetal placenta and M-24% in maternal placenta. Ouabain-sensitive respiration is considered to be an estimate of the metabolic costs of the Na+-K+ATPase pump and therefore a measure of the energy costs for ion pumping (16). The decline in maternal weight-specific total VO, was apparently accounted for entirely by ouabain-insensitive \io,, i.e., by energy costs other than ion pumping. This interpretation assumes that ouabain at 10s4M does not cause nonspecific depression of other enzymes (15, 16). Estimated Oo, of the whole placenta (ml/min) remained constant between 75 and 136 days PC despite a tremendous increase in fetal demands for oxygen and nutrients. The high rate of placental metabolism in late pregnancy is presumably explained by the energy costs of meeting these demands. At midgestation the fetal nutrient requirements are much smaller and probably cannot account for the high metabolic rate of the placenta. The surprisingly high rate of placental VO, at this time may be more a consequence of the rapid growth of the placenta than of its functional energy costs. Although present in vitro values are quantitatively much lower than in vivo


0:

0

iTo,

R622

FETAL

HEPATIC

AND

PLACENTAL

APPENDIX).

Despite the tendency of hepatic ouabain-sensitive Vo2 to decline from early to late pregnancy, the fractional energy cost of ion pumping increased in late pregnancy. The reciprocal decline in contribution of ouabain-insensitive Vo2 to total weight-specific Vo2 suggests that processes other than ion pumping account for most of the decline in hepatic metabolic activity (Table 1). The liver’s role in erythropoiesis diminishes substantially between mid and late pregnancy (12), but whether this is involved in the decrease in \jo2 during this time is not clear. Certainly, the fractional rate of protein synthesis in the whole fetus decreases markedly between mid and late gestation (17); it is likely that a similar decline occurs in the fetal liver. The high correlation between placental and fetal hepatic weights in late gestation appears to account for a similar degree of correlation between placental and hepatic metabolic rates. This suggests that fetal hepatic growth and metabolism may be closely related to the ability of the placenta to supply nutrients, as indicated by its size in late pregnancy, and may explain the special sensitivity of hepatic growth to placental insufficiency in growth-retarded fetuses (1). The anatomic basis for such a relation is strong because much of the umbilical venous blood leaving the placenta perfuses the liver before reaching the fetal systemic circulation (13). This provides a direct means of communication and coordination of metabolic activity of these two vital organs. Specific examples of interrelated metabolic processes include cycling of amino acids and their products or precursors between the placenta and fetal liver (e.g., glutamate-glutamine, serineglycine), and hepatic conversion to urea of ammonia originating from placental amino acid catabolism (4, 11). In conclusion, major findings of this study are first, that there is surprisingly little change in placental Vo2 on a dry weight-specific basis between mid and late gestation despite major increases in functional activity and associated metabolic demands over this period. The source(s) of

IN

SHEEP

the relatively high metabolic rate of the midgestation placenta warrants further study. Second, we found that in contrast to that of the placenta, weight-specific iTop of the fetal liver declines markedly from mid to late gestation. Our suggestion that the size, metabolic rate, and, presumably, functional properties of the fetal liver near term may be directly influenced by size and functional capacity of the placenta requires direct investigation. This should be possible with the availability of techniques for simultaneously measuring fetal liver metabolism (9) and functional characteristics of the placenta (8) in vivo in normal and placentally insufficient fetuses. APPENDIX

Calculations

and Assumptions:

Liver

I) Fetal hepatic vo2 in vivo in late gestation is 4 ml. min-lo 100 g-l, accounting for 17% of total VO2 (9). 2) Fetal hepatic vo2 in vitro in late gestation is 0.75 ml=min-l 100g-l (estimated from Tables 1 and 3), i.e., 19% of in vivo value. 3) Fetal hepatic VO, in vitro in midgestation is 1.34 ml mine1 100g-l X 14.8 g = 0.2 ml/min (estimatedfrom Tables 1 and 3). 4) Fetal hepatic VO, in vitro in midgestation (75 days PC) is assumedto be also 19% of in vivo value. Therefore estimatedin vivo fetal hepatic VO, = 0.2/0.19 = 1.1 ml/min. 5) Total fetal \io, in vivo at 75 days is - 10.5ml min-l kg-l (6). Therefore estimated total vo2 = 10.5 X 0.232 = 2.44 ml/ min. 6) Estimated contribution of hepatic to total iTo in midgestation = M/2.44 = 45%. l

l

l

l

Calculations

and Assumptions:

l

Placenta

I) Estimates of uteroplacental VO, in vivo at late gestation range between 12 and 22 ml/min (assumemean of 16 ml/min; Refs. 8 and 18). Placental To2 is assumedto account for -80% of uteroplacental oxygen consumption (18); therefore, placental VOg = 0.8 X 16 = 12.8 ml/min. 2) Placental i70, in vitro was measuredin placental slices that included both fetal and maternal tissue. Assuming equal weights of these tissues, mean placental vo2 in vitro is 34 ~1 min-l l g dry tissue-l (Table 2). From this and placental dry weight (Table I), calculated vo2 of the whole placenta in vitro is 1.89ml/min = 1.89/12.8= 15% of the estimatedin vivo value. l

The technical assistance of Ramona Slepetis is gratefully acknowledged. This study was supported by the New York Agricultural Experiment Station. Present address of I. Vatnick: Dept. of Biological Sciences, SUNY, Binghamton, NY 13901. Address for reprint requests: A. W. Bell, Morrison Hall, Dept. of Animal Science, Cornell University, Ithaca, NY 14853-4801. Received

26 July

1991; accepted

in final

form

17 March

1992.

REFERENCES

Alexander,

G. Birth weight of lambs: influences and consequences. In: Size at Birth, edited by K. Elliot and J. Knight. Amsterdam: Elsevier, 1974, p. 215-245. Alexander, G. Factors regulating the growth of the placenta: with comments on the relationship between placental weight and fetal weight. In: Abnormal Fetal Growth: Biological Bases and Consequences, edited by F. Naftolin. Berlin: Dahlem Konferenzen, 1978, p. 149-164. Barcroft, J. Researches on PrenataZ Life. Springfield, IL: Thomas, 1947.


estimates of placental \io,, it is notable that the latter also do not indicate a major change between mid and late gestation (7, 8). In fetal sheep, weight-specific metabolic rate declines between mid and late gestation. The rate of this decline parallels that of the decline in the relative growth of the metabolically active organs, the liver in particular (6). This led to our suggestion that changes in relative organ size may have more influence on whole body metabolic rate than do changes in weight-specific cellular metabolism. Such a notion is also consistent with the proposal that the gestational decline in relative growth of the liver is due to a decrease in functional demand (12). However, present data suggest that changes in weight-specific hepatic VOW, in addition to declining relative size of the fetal liver, must contribute to the progressive decline in metabolic rate of the whole fetus during the second half of pregnancy. Measurements in vivo indicate that liver iTo accounts for - 17% of fetal total VO, in late gestation (9), a value less than half of our estimate for the midgestation fetus, assuming that the ratio of in vitro to in vivo values for hepatic VO, remain unchanged through gestation (see

Vo,

FETAL

HEPATIC

AND PLACENTAL

4. Battaglia, F. C., and G. Meschia. Fetal nutrition. Annu. Reu. Nutr. 8: 43-61, 1988. A. W. Factors controlling placental and foetal growth and 5. Bell, their effects on future production. In: Reproduction in Sheep,

edited by D. R. Lindsay and D. T. Pierce. Cambridge UK: Cambridge Univ. Press, 1984, p. 144-152. 6. Bell, A. W., F. C. Battaglia, and G. Meschia. Relation between metabolic rate and body size in the ovine fetus. J. Nutr. 117:

R. A., R. Slepetis, L. Klei, and A. W. Bell. Protein and DNA synthesis during and after proliferative growth of the ovine placenta in mid pregnancy (Abstract). J. Anim. Sci. 69,

14. Ehrhardt,

suppz. 1: 314, 1991. 15. Glyn, I. M. The action of cardiac Pharmacol. Reu. 16: 381-407, 1964. 16. Kelly, J. M., and B. W. McBride.

glycosides on ion movements.

The sodium pump and other mechanisms of thermogenesis in selected tissues. Proc. Nutr. Sot.

49: 185-202, 17. Kennaugh, C. Battaglia.

1990. J. M.,

A. W. Bell,

C. Teng,

G. Meschia,

and

F.

Ontogenetic changes in the rates of protein synthesis and leucine oxidation during fetal life. Pediatr. Res. 22:

688-692, 18. Meschia,

Utilization Proc.

1987. G., F. C. Battaglia,

W. W. Hay,

and

J. W. Sparks.

of substrates by the ovine placenta in vivo. Federation

39: 159-164,

1980.

19. Munro, H. N., and A. Fleck. Analysis of tissues and body fluids for nitrogenous constituents. In: Mzmmuliun Protein Metabolism, edited by H. N. Munro. New York: Academic, 1969, p. 423-525. 20. National

Research

Council.

Nutrient

Requirements

of Sheep

(6th ed.). Washington,

DC: Natl. Acad. Press, 1985. 21. Reynolds, L. P., and D. A. Redmer. Oxygen consumption of ovine placental tissues at several stages of gestation (Abstract). J. Anim. Sci. 65, Suppl. 1: 413, 1987. 22. Stegeman, J. H. J. Placental development in the sheep. Bijdr. Dierkd. 44: 4-72, 1974. 23. Vatnick, I., P. A. Schoknecht, R. Darrigrand, and A. W. Bell. Growth and metabolism of the placenta after unilateral fetectomy in twin-pregnant ewes. J. Deu. Physiol. 15: 351-356, 1991. 24. Villee, C. A. The metabolism of the human placenta in vitro. J. BioZ. Chem. 205: 113-123, 1953.


1181-1186, 1987. 7. Bell, A. W., J. M. Kennaugh, F. C. Battaglia, E. L. Makowski, and G. Meschia. Metabolic and circulatory studies of fetal lamb at midgestation. Am. J. Physiol. 250 (Endocrinol. Metab. 13): E538-E544, 1986. 8. Bell, A. W., R. B. Wilkening, and G. Meschia. Some aspects of placental function in chronically heat-stressed ewes. J. Deu. Physiol. 9: 17-29, 1987. 9. Bristow, J., A. M. Rudolph, A. M. Itskovitz, and R. Barnes. Hepatic oxygen and glucose metabolism in the fetal lamb. J. CZin. Inuest. 71: 1047-1061, 1983. 10. Campbell, A. G. M., G. S. Dawes, A. P. Fishman, A. I. Hyman, and G. B. James. The oxygen consumption of the placenta and foetal membranes in the sheep. J. Physiol. Lond. 182: 439-464, 1966. 11. Carter, B. S., R. R. Moores, Jr., and F. C. Battaglia. Placental transport and placental metabolism of amino acids. J. Nutr. Biochem. 2: 4-13, 1991. A. T. Growth and function of fetal liver. J. Embryol. Exp. 12. Dick, Morphol. 4: 97-109, 1956. 13. Edelstone, D. I., A. M. Rudolph, and M. A. Heymann. Liver and ductus venosus blood flows in fetal lambs in utero. Circ. Res. 42: 426-433, 1978.

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\joZ IN SHEEP