The inefficient transfer of maternally fed alcohol to nursing rats

Alcohol, Vol, ~1,pp, 169,-174, 191~, e Ankho lntem#tlon#l lnc, Printed In the U,S,A,

0741,S~9/11~ $~,00 + ,00

The Inefficient Transfer of Maternally Fed Alcohol to Nursing Rats K E N N E T H R, SWIATEK, ~ OEOROE J, DOMBROWSKI JR, AND KUEN-LAN CHAO

Institute for the Study of Developmental Disabilities University of llllnols at Chlealto, Chlcalto, IL 60608 Received 22 April 198Y SWIATI]K, K, A,, O, J, DOM~ROW~IKI, JR. AND K,.L, CHAO, The Ind~ilc/¢nt~mn#er ~m.lePnell_y/ed ~lcohol ~ n.r~lne m~, ALCOHOL ~(~)16~174, l¢8~,~Tho growth of rat pupsn.rsed by ¢thBol.drinklnl mother~or pupse~POHd daily to othanol/v~l~orwM monitored for the Mr~t three week~ of life, P~ps n.reed by mothers t~d either ~ M~ ethemol ~ontainlng liquid diet or ~ 109~eth~no]/w.termixture h~l ~lgnlflo~ntly lower body waiitht~ ~er the ~nd postnatalweek th~ oontrol~,Thb deere~o In pup |rowth ooo.rred despite pup blood alcohol levels that did not oxooed|0 ml~, Only a smallfmotlonof the ~J~oholfed to mothers ever re#ohods.oklin| nnlmais,In oontr~t, pups exposeddaily to ethanol vapor regularly nohlevedblood alcoholgoneontr~tlon~In exee~sof |J0 mi~, but experiencedonly minimal growth retsrdatlon. These re~ult~sulle~t that m~temnl~l~oholf~eding~nnnotbe .ned to ~todythe effects of ethnnol upon postnatal develop. ment, Al~ohol Postnataldevelopment 9loud eth~mol Milk eth~mol

Llq.id dlot~

L~et~tion

THE deleterious effects of postnatal ethanol exposure upon the body nnd brain Srnwth of n.rslns rodents are cited frequently, In many of these reports Impaired Srowth In Indexed to alcohol dose ~ d used as evidence to support the validity of the particular doslns paradlsm employed [1,?], This re. tlonale Is dlscongertlns slnoe the extent of body Srowth Is. palrment associated with postnatal alcohol treatment varies Sreatly In the literature, Some lnvestls#tors do not find a slsnlflc~t d~erence between the Srowth rates of ethnnol treated and control pups [19, 20, 23, 26], Others report thnt body Srowth tn moderately impaired [l, 2, 28], and in a few canes the body welsht of ethanol treated pups was shown to iU behind that of controls by more th~n 30~ ['/,9], Since substantial qu~tltles of alcohol appear to re~h suckllns pups in all these sltu#tlons, ~ t o r s other than ethanol must be rosponsbile for these wide r&nsins Srowth effects, Several paredl|ms are currently .sod to treat immature animals with alcohol. N.rslns rodents can be dosed throush the milk supplied by a mother fed alcohol as part of her diet [4, 9, 20], throush an lntruastrtc tube [23,24], implanted cannula [$, 11] or by exposure of pups to an ethanol vapor/air mixture [2], Thele procedures can be classiflod Into two broad cat.;odes: (a) Indirect ethanol doslns via matern#l ~lministration, or (b) direct p.p dosins, l]ody woisht Srowth dolldte of 3..12~ are found for experiments where ~dcohol In ~mlnbtered directly to pups, ]n contrast, when alcohol in fed to the mother throushout the nurslns period a much Sreater ranp of pup body welsht deMdts (09~.60~) can be observed [?, 19],

R~t

Undarnutrltlon

Orowth

AltbouBh matern#l ethanol Mmlnlmtratlon lm appe~lns as a method for easily ~edinl idoohol to small s~klins ~lmals, such wide variation In postnatal Srowth effects suMIost that this technique may be problem#tic, In order to be experi. mentally valid, the pamdlBm requires that larse amounts of alcohol be effldentIy transl~rrod from the mother to off. sprlns through the milk, While ethanol may be present in milk [10,17], little resm~l Is liven to the t ~ t that any Inferrer. once with milk production will ~oncomitm|tly alter the amount of ethanol a suckllns animal Insure, In addition, the amount of ~cohol a nursllns recelvos Is also a ~nctlon of the rate of maternal ethanol catabolism and this in turn can be readily modulated by chanses in maternal nutritional stetus [18,21], An appredation of these f~tors has lncreasinSly led In. vestijatorl to use direct ethanol doslns schemes for postnatal studies, However, many workers still employ maternal eth#nol foodlns M a moans of doslns suckllns Mime.Is, Recent reports h#ve even suSSestedthat many of the prob. less assodated with this paredllm c&n be overcome by uslns nutritionally ferried diets [28], It Is undermtoodthat fudins alcohol to anho~ls In Lleher.DoCarll formulas [16] or ~1~" cue solutions has hecome an #ccepted technique for studies lnvolvlns adult and prqnant rats, However, the effective. nose of maternnl ethanol feedlns as n meBs of reliablyIntoxleatlns suckllns #nimnls In postnatal studies has never been adequately studied, The present study was undortekon to excise the relationship between the mode of maternal Alcohol Klmlnbtr#.

~Requem for repdntm mho,ld be nddreAoedto Kenneth R, Swlatek, Illinolm lmtitote for DevelopmentalD|Nbllltiea, 1640W, Roolevelt Ao~I, Chlcqfo, IL 60~,

169

170

S W I A T E K , DOMBROWSK1 AND CHAO TABLE 1 TREATMENT METHODS USEDTO DETERMINE THE EFFECTS OF ETHANOL UPON POSTNATALDEVELOPMENT" Dosing Method

Maternal Diet

Direct Pup Treatments

Maternal Liquid Maternal Dosage Consumption of Ethanol (ml/kg/day) (g/kg/day)

Total Caloric Intake (kcal/kg/day)

Liquid Diets

Sego Control (S-C) Sego + 3% ETOH (S-3) Sego + 6% ETOH (S-6) Sego Pair-Fed (S-PF)

None None None None

429 387 230"t 230t

0 11.60 13.83 0

47 I 440 ( 17)$ 254t (40)~: 254+

Water + Ethanol

Chow§ + Water Alone (W-C) Chow§ + 6%ETOH/Water(W-6) Chow§ + 10% ETOI-UWater (W-10)

None None None

195 217 204

0 13.01 20.40

491 535(18)~ 459 (32)$

Ethanol

Chow§ + Water Alone (EV)

Ethanol Vapor (Twice/Day)

195

0

Vapor

491

*Treatment protocols were implemented as described in the Method section. The numbers of litters used for each determination were: S-C, 12; S-3, 8; S-6, 15; S-PF, 5; W-C, 8; W-6, 3; W-10, 4; and EV, 8.tlndicates a significant difference from controls (p<0.05). SThe percentage of ethanol derived calories consumed. §Chow refers to the stock diet, Purina Chow #5012.

tion, maternal blood and milk ethanol content, nursling growth, and nursling blood alcohol. The effects produced on postnatal growth by maternal alcohol consumption are contrasted with those obtained when suckling animals are directly dosed by exposure to ethanol vapor [2]. METHOD Pregnant Sprague-Dawley rats were housed in separate cages and maintained on a stock diet of Purina Chow No. 5012 (ad lib) until the pups were born. Immediately after parturition litters were culled to 6 pups per mother (250-325 g). Control litters were allowed free access to Purina Chow and water. Litters were randomly assigned to the treatment groups on the first postnatal d a y , T r e a t m e n t s were continued until either the pups were killed or the animals were weaned on the 21st postnatal day. Animals were maintained on a 12/12 hr light/dark cycle. Food and liquid consumption were monitored daily. Unless stated otherwise blood samples for ethanol assays were obtained during the last half of the dark cycle. Rats are nocturnal animals and have been shown to eat heavily during this period [27]. Tail vein samples were collected from mothers and trunk blood samples were collected from the severed necks of pups. Statistical significance was evaluated with the Student's t-test. The Bonferroni correction [15] was applied in the case of multiple comparisons.

Ethanol~Liquid Diet Experiments Sego Vanilla Liquid Diet (PET Inc., St. Louis, MO) served as the base vehicle for the formulation of both control and test diets. Four maternally fed liquid diets were used: control (S-C), 3% (w/v) ethanol (S-3), 6% (w/v) ethanol (S-6) and control pair-fed (S-PF) to match the consumption of mothers given the 6% ethanol diet. Each diet contained 90.2% (w/v) of the Sego product. The control and 3% ethanol containing diets were adjusted to be isocaloric with the 6% ethanol diet (1.105 kcal/ml) by the addition of sucrose. Each

diet contained 3.4% (w/v) protein, 20.6% (w/v) carbohydrate and 1.5% (w/v) fat. When possible, feed bottles were washed and filled with fresh diet on a daily basis. For situations where the diet could not be changed daily, an excess of diet was supplied and 0.4%--0.5% sodium benzoate added to retard bacterial growth. In either case, fresh diet was always supplied within 72 hr. Except for the S-PF controls, mothers were allowed ad lib access to food.

Ethanol~Water Treatments Lactating dams fed the stock diet were given one of 3 liquids to drink ad lib while nursing: water only (W-C), 6% (w/v) ethanol/water (W-6), or 10% (w/v) ethanol/water (W-10).

Ethanol Vapor Experiments Suckling pups (3-20 days of age) nursed by mothers fed the Purina Chow were exposed twice daily to an ethanol vapor/air mixture (EV). The mixture (3.5-4.2% v/v ethanol) was prepared by passing air at 25 ° into a series of flasks containing 95% ethanol. The gas mixture was filtered through cotton and directed to a chamber where pups were temporarily placed during the treatment periods. After the first exposure to the ethanol vapor/air mixture (1.5 hr), the pups were removed from the treatment cage and placed with control litterrnates for the next 3 hours. At the end of this period, the pups were again placed into the treatment cage for 1 additional hour. Tail vein blood samples were obtained immediately after the second daily treatment from randomly selected pups. Ethanol content of the air in the treatment cage was routinely monitored with a solid state vapor sensor (Model TGS No. 812, Figaro Engineering Inc., Toyanaka City, Osaka 560, Japan) coupled to a strip chart recorder. The sensor was calibrated by measuring its response to ethanol vapor/air standards prepared by pipetting a known quantity of 100% ethanol into the bottom of a narrow-mouth flask, the volume of which was determined by the displacement of water. The flask containing the sensor was stop-

$WIATEK, DOMBROWSKI AND CHAO

172

TABLE 2 Till[ R~BCT OFTRRATMBNTPAKADIOMONTHEBTHANOL

CONTBNTOF MATSRNALBLOODAND MILKAND PUPBLOOD'

Treatment Oroupe Sampb Source Mother's Blood

Liquid Dlete AqueousBthenol BtiumolVapor $-3 $-6 W-6W-10 EV 2($,1 147,4 ±9.1 ±23.6

18,7 ±6.7

40.86 ±11.6

(7)

(5)

21.4 92,1 ±13.1±25.4 (2) (9)

18.8 ±6.5 (4)

15,5

6.9 3.4 ±2.5 ±2.1

8.5 ±3.2

19.I ±2.7

(3)

(6)

(5)

(3) Mother's Milk

Pup Blood

(15)

(13)

(I)

273.4 ~-34,5 (11)

'Alcohol doaln8 perKligmsare a8 listed in Table 1, The experimental conditions sad the collection sad analysis of samples are de~rlhed in the Materials sad Method section, Each entry represents the mesa :t: the ltandard error. Valuer enclosed by parentheses Indicate the number of litters sampled.

In accord with previous studies [7,9], the body weight of pups nursed by S-6 mothers was sign/flcantly less than of S-C controls after the llth day of suckling (Fig, 1), Growth deficits observed in litters treated with alcohol containing liquid diets are usually attributed entirely to the effects of ethanol, because It is pnerally presumed that the caloric intake of treated litters is equal to that of the liquid diet controls. However, this Is not necessarily true, As can be seen in Table 1, pupa ralxd by pair-fed mothers ($-PF) were found to be severely growth retarded when compared to the pups of the control diet fed mothers (S-C). The depressed growth curve of the pair-fed litters suggests that the reduced caloric intake of animals fed the 6% ethanol liquid diet is insufficient to sustain the level of lactation required for even near normal postnatal growth [7]. In contrast, at 3% ethanol concentration in the maternally administered liquid diet, the growth rate of pups cannot be distinguished from that of the ad lib control fed litters, even though their daily maternal ethanol consumption matches that of the mothers fed the 6% ethanol containing liquid diet (Table 1). Apparently rat mothers will consume a liquid diet containing 3% ethanol, but find the 6% ethanol/Sego combination unpalatable. The reduction in food intake and not the amount of ethanol ingested accounts for the poor growth of pups nursed by mothers fed the S-6 diet. Pup weight was significantly decreased below that of controis for litters nursed by mothers fed 10% aqueous ethanol (-31%) and in the groups exposed to ethanol/vapor (-20%) during the last week of suckling (Fig. 2). The W-6 pups showed a tendency toward lower body weights (-5%) than found for controls, but the difference was not significant. These growth deficits are less than those found for litters fed the liquid diet containing 6% ethanol, which were 39% below S-C pups. The W-10 mothers consumed 1.6 times more alcohol than the W-6 animals, and the larger growth deficits of the W-10 litters as compared to that of the Y¢-6 litters appear to be positively correlated with the amount of ethanol ingested (Table 1). However, alcohol consumption is not the

only factor which could have affected postnatal growth in these experiments, Although the W-6 mothers iugestad nearly the same amount of the stock diet as controls, the mothers that drank the 10~ ethanol solution consumed only 6 ~ as many chow.darivad calories as the W-C controls. Even though total caloric intake (alcohol + chow) by the W-10 dams w u adequate, the substantial reduction in protein as well as calorie-linkad nutrients (vitamins an minerals) could have adversely affected lactation in these mothers and secondarily the growth of their pups. The results obtained with the liquid diet paradigm parallel those found with aqueous ethanol in that attempts to feed larger quantities of ethanol caused a reduction in the consumption of non-alcohol derived calories. The S-6 rats reduced their food intake because the diet was unpalatable, whereas for the W-10 mothers the amount of alcohol (7.1 kcal/g) consumed reduced their need for other fuels. In either case, the maternal diet was altered to an extent where nutritional factors could seriously confound observations on postnatal development. The maternal diet was also affected when lower alcohol concentrations were used (S-3, W-6). However, the near normal postnatal growth curves obtained with these methods suggests that rat mothers do have some dietary flexibility in nutrient mix with rep.rd to ethanol derived calories. Additionally, it must be noted that mothers which are well nourished prior to parturition probably have a reserve capacity to temporarily buffer a small nutrient imbalance. In order to better understand the reasons why different ethanol dosing paradigms produce such varied effects on postnatal growth, maternal blood and milk alcohol measurements were performed and their values compared to the blood ethanol levels of pups suckled by these mothers (Table 2), Appreciable quantities of alcohol were not found in the blood of pups nursed by mothers consuming either an

ethanol containing liquid diet or one of the ethanol/water mixtures. Of the four treatment methods studied where ethanol was administered to suckling pups by maternal feeding, the 6% liquid diet (S-6) resulted in the lowest pup blood alcohol levels. The 3% ethanol liquid diet and the 6% ethanol/water mixtures gave slightly higher but similar pup blood ethanol levels, while the 10% ethanol/water formula resulted in the greatest pup blood alcohol concentrations at 19 rag%. Another report has also documented low pup blood alcohol levels with maternally fed alcohol [6]. It should be noted that, the high concentration of alcohol in the blood and milk of S-6 mothers (13.8 g ethanol/kg daily dose) as compared to the low blood and milk alcohol content of S-3, W-6 or W-10 mothers (11.6-20.4 g ethanol/kg daily dose) suggests that undernutrition and dehydration in the S-6 dams has impaired their ability to metabolize ethanol (Tables 1 and 2). The rate of ethanol catabolism has previously been shown to be dependent upon the nutritional state of an animal [18,21]. Rogers et al. [21] have shown protein malnourished adult rats to consume 13.2 g ethanol/kg body wt./day and attain a blood alcohol of 146 mg%, whereas the corresponding blood alcohol for a well fed group ingesting 17.3 g ethanol/kg/day was only 66 rag%. The low blood ethanol levels found in pups nursed by mothers fed either ethanol/water mixtures or the 3% ethanol liquid diet mimick the low maternal blood and milk alcohol concentrations obtained with these dosing paradigms. The results obtained with the 6% ethanol liquid diet experiment (S-6) are an exception in that the low pup blood alcohol levels found with this paradigm do not parallel the high

MATERNAL E T H A N O L DOSE AND RAT PUP GROWTH

I:~

40

50 (0) Control, S-C

o) IZ

(O) Control, W-C

(e) 3% Ethanol, S-3

A

30

(zx) 6% Ethanol, S-6 (x) Pair-Fed, $-PF

m

t

/

~ ~

40

[

./~[ u,I

2o >. o O m

(0) 6% Ethanol, W-6

/t

>"

20

0

II)

i0

lO

0

BIRTH

I

4

I

8

I

12

I

16

I

20

BIRTH

4

AGE IN DAYS

8

1'2

1~5

2'0

A G E IN DAYS

FIG. 1. Body growth of rat pups nursed by mothers fed liquid Sego diets containing different ethanol concentrations. Treatments are as indicated in Table 1. Protocols were implemented as described in the Method section. Since the growth of S-3 litters was indistinguishable from that of S-C litters, body weight data from these two groups were pooled and the resulting mean values used for comparisons with the S-6 and S-PF treatment groups. The numbers of litters averaged to each data point at 10, 12, 15 and 20 days were: 3, 3, 5 and 5 for the S-C+S-3 means; 6, 9, 11 and 4 for the S-PF treatment; and 2, 6, 13 and 4 for the S-6 condition, respectively. The vertical bars indicate one standard error about the mean. All other points represent the mean pup weight derived from at least 2 litters. Significant differences were found between the Controls S-3+S-C and the S-6 or S-PF groups at 12, 15, and 20 days of age (p<0.05).

FIG. 2. The body growth of rat pups exposed to alcohol through maternally ingested ethanol/water solutions and by the direct inhalation of an ethanol vapor/air mixture. Abbreviations are as in Table 1 and the experimental conditions are described in the Method section. The numbers of litters averaged for each data point at 12, 15 and 18 days were: 7, 20 and 14 for the W-C treatment; 5, 5 and 2 for the W-6 condition; 4, 14 and 11 for the EV curve; and 4, 7 and 3 for the W-t0 group, respectively. All other points represent the mean pup weight for at least 2 litters. The vertical bars represent one standard error about the mean. EV and W-10 treated litters were significantly different from the W-C controls at 15 and 18 days of age (p<0.01).

pered and set aside for 1-2 hr. A small open narrow tube placed in the stopper equalized the pressure inside the flask with the external barometric pressure. This procedure was cautiously used only for standards with a final partial pressure less than the vapor pressure of ethanol at the ambient temperature. Ethanol content of the gas mixture was also calculated from the measured weight loss of the aspirated liquid ethanol per unit time and the known air flow rate. Sensor measurements and calculations were verified by enzymatic assay of the aqueous ethanol solution prepared by bubbling a measured volume of the ethanol/air mixture into distilled water.

Ethanol Assays

Milk Collection Milk samples from animals fed ethanol were obtained during the last half of the dark cycle. Before milking, each mother was injected with 5-10 units of oxytocin (IP) and the litter either killed or temporarily removed to another cage. After 2-3 hr, the lactating female was lightly anesthesized with nembutal (IP) and milked by gently hand-stripping each teat [13]. The milk was siphoned into a 50 p.l pipette which was emptied into a larger capped tube. Samples (10-100/zl) were then withdrawn and added to a previously cooled tube containing 0.50 ml water. This procedure minimized the evaporation of ethanol from the small sized samples. A blood sample was simultaneously taken from the tail vein. Two qualitative measures were employed to assess the quantity of milk available to suckling pups: (a) the relative ease with which milk could be collected, and (b) the amount of milk curd found in the stomachs of pups.

Ethanol was assayed at 340 nm using alcohol dehydrogenase and NAD + (Sigma Chemical, St. Louis, MO) [3]. RESULTS

AND

DISCUSSION

Table 1 lists the combinations of maternally fed diets employed in this study together with the per diem caloric consumption and maternal ethanol dose. There was no significant difference in the total caloric intake between the liquid diet (S-C) and Purina Chow (W-C) fed controls. Among the liquid diet groups, the addition of 6% alcohol (S-6) caused a marked 46% reduction in caloric consumption as compared to that of controls (S-C). Pair-feeding experiments (S-PF) were initiated to control for any nutritional deficits which may have been caused by such a large decline in food intake. Within the Purina Chow fed groups, the consumption of ethanol did not significantly alter the total caloric intake of either the W-10 or W-6 mothers. The quantity of alcohol ingested by W-10 mothers was greater than the daily maternal ethanol dose for either the 6% or 3% ethanol liquid diet experiments. Figures 1 and 2 depict the growth curves for rat pups reared using the protocols described in Table 1. The liquid diet fed controls had significantly lower body weights at 15 days of age as compared to Purina Chow fed controls (0<0.005). This difference in growth suggests that the liquid Sego diet is not optimally formulated to support lactation. The nutritional adequacy of some of the commercially available liquid diets chosen to support lactating dams has been questioned in the literature [28].

MATERNAL ETHANOL DOSE AND RAT PUP OROWTH

173

ethanol concentrations observed in the mothers' blood and milk. One possible explanation for the low pup blood ethanol levels as well as their severely retarded growth could be insufficient maternal milk production. When S.6 pupa were

69t ethanol containing liquid diet or the 109$ aqueous ethanol can be attributed to ethanol, It Is difficult to ascertain pre. cisely the degree to which ethanol alone, the liquid diets, or the ethanol/water mixtures produced a deficit in nutrition

dissected, their stomachs were found to be empty, For all other treatment protocols listed in Table 1, substantial quantities of milk curd was found in the pup stomachs, Consistantiy ~ levels of blood alcohol (270 ml~) were attained daily in pups exposed to an ethanol vapor/air mixture (EV). Time course experiments indicate that the blood alcohol concentration can be maintained in excess of 90 m f ~ for up

and consequently pup growth. Even when pups are dosed

to 5 hr (results not shown). Despite this high daffy ethanol dose, body jrowth is retarded only 15-209t by the 3rd postnatal week. Even smaller growth deficits (8..11%) have also been reported by others who have used this treatment strategy [2] as well as by those who have administered alco. hol by intruutric intubation [23]. Whenever high maternal blood ethanol levels are maintained, lactation can also be suppressed by inhibition of the oxytocin modulated milk ejection reflex [5,10]. Furthermore, it is pot|ibis for ethanol mediated dehydration to cauls a decrease in milk production [12,22], The combination of these effects can diminish the quantity of milk available to pups and compromiN their growth. Postnatal undernutrition appears to be the major factor responsible for the poor growth of pups in the S-6 and W-10 studies. Moreover, although rat mothers will readily consume fluids containing moderate amounts of ethanol (S-3, W-6), they are able when well fed, to catabolize and eliminate most of the alcohol before it reaches the pups (Table 2), Adult rats have 2--3 times greater capacity to metabolize ethanol than humans [25], In addition, these observations agree with those of a previous study where it was estimated that the amount of ethanol an infant would receive from an acutely intoxicated mother is less than 295 of the alcohol consumed [14],

These experiments demonstrate that only part (<40% of the growth deficit observed in pup~ nursed by mothers fed a

directly through exposure to ethanol vapor or by intrtlllstric lntuhation some deficit in pup growth occurs [2,23], However, it is clear that pups can achieve high blood ethanol levels daffy without experiencing large deficits in growth (EV), Certainly any postnatal ethanol treatment paradigm which severely retards growth should be re.evaluated, Although nutritional issues are important, of equal concern is the amount of alcohol that suckling pups receive from an ethanol fed mother. The feeding of alcohol to a lactating dam can have a whole range of effects on pup growth, from severe retardation (S-6) to almost none at all (S-3, W-6). In either case, these data show that despite the presence or absence of growth effects only small amounts of alcohol are found in pup blood when maternal dosing paradigms are employed (Table 2), This finding casts serious doubt on the utility of using maternal ethanol feeding regimes in efforts to study 'alcohol effects' on postnatal development, It is probably impossible to completely separate postnatal ethanol exposure from real- and/or undernutrition, However, nutritional effects appear to be minimized in experiments where ethanol is directly administered to suckling animals. Since maternally ingested ethanol does not easily find its way to suckling animals, treatment protocols which depend on maternal dosing regimes can be expected to produce results which either have no direct relationship to the alcohol dose or are severely confounded by nutritional deficiencies, In order to fully appreciate the effects of an ethanol dosing regime and to assure that reasonable concentrations of

ethanol are at least achieved in nurslings, it is sululelted that their blood alcohol concentration be monitored and re.

ported,

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9. Druse, M. J. ancl J. H. Hofteig. The effect of chronic maternal alcohol consumption on the development of central nervous system myelin subfractions in rat offspring. Drug Alcohol Dep 2: 421-429, 1977. 10. Fuchs, A. R. and G. Wagner. The effect of ethyl alcohol on the release of oxytocin in rabbits. Acta Endocrinol 44: 593-605, 1963. 11. Grant, K. A. and H. H. Samson. Ethanol and tertiary butanol induced microcephaly in the neonatal rat: Comparison of brain growth parameters. Neurobehav Toxicol Teratol 4: 315--321, 1982. 12. Healy, M. Suppressing lactation with oral diruetics. Lancet 1: 1353--1354, 1961. 13. Keen, C. L., B. Lonnerdal, M. Ciegg and L. S. Hurley. Developmental changes in composition of rat milk. Trace elements, minerals, protein, carbohydrate and fat. J Nutr 111: 226--230, 1981. 14. Kesaniemi, Y. A. Ethanol and acetaldehyde in the milk and peripheral blood of lactating women after ethanol adminstration. Am J Obstet Gynecol 81: 84-86, 1974. 15. Kirk, R. E. Multiple Comparison Tests. In: Experimental Design: Procedures for the Behavioral Sciences. Belmont, CA Wadsworth Publishing Company, 1968, pp. 69-98.

174 16. Lieber, C. S. and L. M. DeCarli. The feeding of alcohol in liquid diets. Two decades of applications and 1982 update. Alcohol Clin Exp Res 6: 523-531, 1982. 17. Olow, J. Uber den ubergang des athylakohols yon der mutter zur frucht. Bioehem Z 134: 407-414, 1923. 18. Owens, A. H. and E. K. Marshall, Jr. The metabolism of ethyl alcohol in the rat. J Pharma~ol 115: 360-370, 1955. 19. Rawat, A. K. Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis. Biochem J 160: 653661, 1976. 20. Rawat, A. K. and S. Kumar. Effects of maternal ethanol consumption on the metabolism of dopamine in rat fetus and neonate. Res Commun Psyehol Psychiatr Behav 2:117-129, 1977. 21. Rogers, J., S. G. Wiener and F. E. Bloom. Long-term ethanol administrtion methods for rats. Advantages of inhalation over intubation of liquid diets. Behav Neural Bio! 27: 466-486, 1979. 22. Shorey, R. L., P. A. Terranella and W. Shive. Effects of ethanol on growth, consumption of food and body composition of weanling rats. ,I Nutr 107: 614-620, 1977. 23. Sonderegger, T. B., H. Calmes, S. Corbitt and E. G. Zimmermann. Lack of persistent effects of low-dose ethanol administered postnatally in rats. Neurohehav Toxi¢'ol Terutol 4: 463468, 1982.

SWIATEK, DOMBROWSKI AND CHAO

24. Sonderegger, T. B., D. Colbern, H. Calmes, S. Corbitt and E~ Zimmerman. Methodological note: Intragstric intubation of ethanol to rat pups. Neurobehav Toxic'ol Teratol 4: 477-481, 1982. 25. Wallgren, H. and H. Barry, 111. Some basic data on the chemistry and pharmacology of ethyl alcohol. In: Actions ojAl~ohol, Vol /. New York: Elsevier Publishing Company, 1970, pp, 46-49. 26. West, J. R., K. M. Hamre and D. R. Pierce. Delay in brain growth induced by alcohol in artificially reared rat pups. Alcohol 1: 213-222, 1984. 27. Wiener, S. G., W. J. Shoemaker, L. Y. Koda and F. E. Bloom. Interaction of ethanol and nutrition during gestation: Influence on maternal and offspring development in the rat..I Pharrnacol Exp Ther 216: 572-579, 1981. 28. Yeh, L. C. and F. L. Cerklewski. Formulation of a liquid diet tbr ethanol studies involving gestation and lactation in the rat..I NHtr 114: 634-637. 1984.