J Sci Food Agric 1997, 75, 212È216

The Relationship Between the a-TocopherolContent of the Yolk and its Accumulation in theTissues of the Newly Hatched ChickPeter F Surai, Tibor Gaal, Raymond C Noble and Brian K Speake*

Department of Biochemical Sciences, Scottish Agricultural College, Auchincruive, Ayr, KA6 5HW, UK

(Received 13 August 1996 ; revised version received 8 January 1997 ; accepted 14 March 1997)

Abstract : The e†ect of a range of concentrations of a-tocopherol in the initialyolk on the subsequent levels of this vitamin in the tissues of the newly hatchedchick was investigated. Four batches of fertile eggs (Ross 1 broiler-breeder type)with mean yolk a-tocopherol concentrations (kg g~1 yolk) of 60É8, 155É3, 272É2and 376É6 were incubated and the levels of the vitamin in the liver, brain, heartand kidney of the resultant chicks were measured. The liver of the day-old chickcontained far higher levels of the vitamin than were found in the other tissuesstudied. The concentration of a-tocopherol in the liver was directly proportionalto the initial level of this vitamin in the yolk. The concentrations of a-tocopherolin the brain were only about 1% of the corresponding liver values but were alsodirectly proportional to the levels initially present in the yolk. The concentra-tions of a-tocopherol exhibited by the heart and kidney were a†ected by theinitial yolk values but the response was not linear over the range of yolk a-tocopherol concentrations studied. The results indicate that the initial concentra-tion of a-tocopherol in the yolk has a profound e†ect on the subsequent levels ofthis vitamin in the tissues of the chick. In a separate experiment, the changes inthe concentration of a-tocopherol in the liver were studied over the Ðrst 9 daysafter hatching. The vitamin was found to be rapidly depleted from the liver afterhatching.

J Sci Food Agric 75, 212È216 (1997)No. of Figures : 2. No. of Tables : 1. No. of References : 30

Key words : a-tocopherol, yolk, chick

INTRODUCTION

The tissue lipids of the newly-hatched chick are mark-edly enriched in highly polyunsaturated fatty acids(Anderson et al 1989, 1990 ; Lin et al 1991 ; Cherian andSim 1992 ; Speake et al 1993 ; Maldjian et al 1995 ;Farkas et al 1996). Although these fatty acids haveimportant functions in the developing animal(Neuringer et al 1988 ; Innis 1992 ; Crawford 1993), theirpresence imparts a high degree of susceptibility to free-radical induced tissue damage as a result of their pro-

* To whom correspondence should be addressed.Contract grant sponsor : Scottish Office Agriculture, Environ-ment and Fisheries Dept.Contract grant sponsor : British Council.

pensity to lipid peroxidation (Sevanian and Hochstein1985 ; De Man 1992 ; Niki et al 1993). The hatching andearly neonatal periods may represent a particularly haz-ardous time as the emergent chick becomes exposed toatmospheric oxygen and also exhibits a dramaticincrease in its rate of oxidative metabolism (Freemanand Vince 1974). Defence against lipid peroxidation isprovided by a range of antioxidant components presentin the tissues of the hatchling (Gaal et al 1995). Principalamongst these is a-tocopherol (vitamin E), the majorlipid-soluble, chain-breaking antioxidant of animaltissues (Packer 1992 ; Buettner 1993 ; Sies 1993 ; MacP-herson 1994). This vitamin is pre-packaged in the yolkduring oocyte maturation (Griffin et al 1984) and, fol-lowing fertilisation and the initiation of embryogenesis,is distributed to the developing tissues of the embryo

2121997 SCI. J Sci Food Agric 0022-5142/97/$17.50. Printed in Great Britain(

a-T ocopherol in the chick 213

via the yolk sac membrane and the circulation (Noble etal 1993 ; Gaal et al 1995 ; Speake et al 1996).

Encephalomalacia is a recognised clinical conse-quence of a-tocopherol deÐciency during chick develop-ment (Dror et al 1976). The further possibility that theaetiology of a proportion of the mortalities during theembryonic, peri-hatching and neonatal periods of chickdevelopment may lie in lipid peroxidative eventsremains to be evaluated. However, such considerationsraise the potential for increasing chick viability by sup-plementing the laying henÏs diet with a-tocopherol inorder to elevate the level of this antioxidant in the yolk.A key question which then arises is the degree to whichan increase in the concentration of a-tocopherol in theyolk is able to inÑuence the subsequent level of thisvitamin in the tissues of the hatchling. For instance, it isconceivable that the tissues may be limited in theirability to take up and store the vitamin and also anyexcess a-tocopherol may simply be eliminated via excre-tion in the bile (Kayden and Traber 1993). The purposeof the present study was to evaluate the e†ects of varia-tions in the level of a-tocopherol in the initial yolk onthe accumulation of the vitamin by the tissues of thechick.

EXPERIMENTAL

Experimental material

Batches of fertile eggs (Ross 1 broiler-breeder type) wereobtained from commercial hatcheries. The eggs wereincubated at 37É5¡C and 60% relative humidity in aforced draught incubator with automatic egg turning.On hatching (after 21 days of incubation), the chickswere maintained in the incubator for 1 or 2 days withfree access to drinking water but with no food provi-sion. Livers, brains, hearts and kidneys were collectedfor analysis.

In one experiment, designed to measure post-hatchchanges in the a-tocopherol content of the liver, chickswere maintained for up to 9 days with free access to astarter diet for broilers which contained 32 mg a-tocopherol kg~1 feed.

Determination of a-tocopherol

a-Tocopherol was determined by the method ofMcMurray et al (1980) as modiÐed by Gaal et al (1995).In brief, the samples were saponiÐed with ethanolicKOH in the presence of pyrogallol and then a-tocopherol was extracted from the mixture with pet-roleum spirit. The extract was dried under nitrogen,redissolved in methanol and injected onto a Spherisorbtype S30DS2 3 km reverse-phase high-performanceC18liquid chromatography column, 15 cm ] 4É6 mm

(Phase Separations, Clwyd, UK). Chromatography wasperformed using a mobile phase of methanol/water(97 : 3, v/v) at a Ñow rate of 1É1 ml min~1. Fluorescencedetection of a-tocopherol utilised excitation and emis-sion wavelengths of 295 and 330 nm, respectively.Calibration was performed using standard solutions ofa-tocopherol (Fluka, Gillingham, Dorset, UK) inmethanol.

Presentation of results

All results are the means ^ SEM of measurements onÐve yolks or on tissues from Ðve chicks.

RESULTS

The concentrations of a-tocopherol in the yolks of eightseparate batches of fertile eggs obtained from com-mercial hatcheries were determined. All these batches ofeggs were of the same (Ross 1) broiler-breeder strain.For Ðve of these batches, the concentrations lay withinthe range between 110 and 165 kg g~1 yolk ; namely,110É9 ^ 8É3, 127É5 ^ 10É1, 155É3 ^ 12É1, 164É8 ^ 12É6and 153É8 ^ 21É9. However, for the remaining threebatches, the yolk concentrations of the vitamin fellmarkedly outwith this range ; namely, 60É8 ^ 5É4,272É2 ^ 24É6 and 376É6 ^ 38É8. This variation providedan opportunity to investigate the relationship betweenthe concentration of a-tocopherol in the initial yolk andthe subsequent levels of this vitamin in the tissues of thenewly-hatched chick. Eggs from these three batches andfrom one of the batches in the “normalÏ range (153É3 kga-tocopherol g~1 yolk) were incubated and the concen-trations of a-tocopherol in the tissues of the day-oldchick were determined.

The levels of a-tocopherol in the liver of the day-oldchick (Fig 1) were between one and two orders of mag-nitude greater than the concentrations measured in theother tissues studied (Fig 2). A linear relationship wasobserved between the concentration of this antioxidantin the initial yolk and in the liver of the hatchling andthese parameters were very highly correlated(r \ 0É999). The levels of a-tocopherol in the brain wereonly about 1% of the values for the liver in all cases(Fig 2). However, there was again a clear linear relation-ship (r \ 0É990) between the initial yolk concentrationsof the vitamin and the subsequent levels in the brain.The di†erence in the initial yolk a-tocopherol concen-trations between the lowest (60É8 kg g~1) and thehighest (376É6 kg g~1) values used in this study resultedin a 3É2-fold increase in the concentration of the vitaminin the brain compared with an increase of 4É8-fold in theliver.

The concentrations of a-tocopherol in the heart andkidney of the day old chick (Fig 2) were also dependent

214 P F Surai et al

Fig 1. Relationship between the a-tocopherol concentrations in the initial yolk and in the liver. Error bars represent SEM ofmeasurements on Ðve yolks or chicks.

on the initial yolk level of the vitamin. However, alinear relationship was not observed for these tissues asthe response was blunted at the higher initial yolkvalues.

In a separate experiment, using the batch of eggs withan initial yolk concentration of a-tocopherol of164É8 ^ 12É6 kg g~1, the changes in the tissue levels ofthe vitamin were determined during the Ðrst nine daysafter hatching. The level of a-tocopherol in the liverdecreased dramatically after hatching to such an extent

that the concentration at day 9 was only 4É7% of that atday 1 (Table 1).

DISCUSSION

The variation in the a-tocopherol content of the yolksfrom the di†erent batches of eggs presumably reÑectsdi†erences in the level of this vitamin in the feed provid-ed to the laying Ñocks. The variation is not due to

Fig 2. Relationship between the a-tocopherol concentrations in the initial yolk and in the brain heart and kidney(=), (>) (…).Error bars represent SEM of measurements on Ðve yolks or chicks.

a-T ocopherol in the chick 215

TABLE 1Changes in the concentration of a-tocopherol in the liver after

hatchinga

Days post-hatch a-T ocopherol (kg g~1 liver)

1 566É2 ^ 41É12 310É4 ^ 10É95 143É2 ^ 11É79 26É7 ^ 1É4

a Initial a-tocopherol concentration in the yolk was164É8 ^ 12É6 kg g~1. After hatching, the chicks were allowedfree access to a starter diet for broilers which contained 32 mga-tocopherol kg~1 feed. Results are means^ SEM of mea-surements on Ðve chicks at each stage.

strain di†erences since all batches of eggs were obtainedfrom Ñocks of the same (Ross 1) broiler-breeder type.The present results indicate that a 6É2-fold increase inthe concentration of a-tocopherol in the yolk results inincreases of 4É8- and 3É2-fold in the liver and brain,respectively. SigniÐcant but non-linear increases in a-tocopherol content were also observed for the heart andkidney. Thus it is clear that increases in the yolkcontent of the vitamin have profound e†ects on the sub-sequent levels in the tissues of the chick. Bartov et al(1965) previously reported that the level of a-tocopherolin the liver of the newly hatched chick was directly pro-portional to the content of this vitamin in the breederdiet. The consequences of such a fortiÐcation of thechickÏs antioxidant capacity on the susceptibility of thetissues to lipid peroxidation and on chick viabilityremain to be established. The relatively low levels of a-tocopherol in the brain of the chick in comparison withthe other tissues has been previously noted (Gaal et al1995 ; Speake et al 1996). This apparent deÐciency in thebrainÏs antioxidant defence capacity is further com-pounded by the relatively low levels of vitamin A, Se,glutathione peroxidase (Gaal et al 1995) and carot-enoids (Speake et al 1996) which are characteristic ofthis tissue. In spite of the fact that the brain of the chickcontains high levels of ascorbic acid (Wilson 1990 ;Speake et al 1996), there does appear to be a generaldeÐcit in this tissueÏs endowment with antioxidant com-ponents. This deÐcit may be particularly deleterious inthe light of the very high levels of polyunsaturated fattyacids in the neuronal phospholipids (Anderson et al1989 ; Cherian and Sim 1992 ; Maldjian et al 1996) andalso since the metabolism of brain tissue generates espe-cially high levels of free radicals (Reiter 1995). Thus thepresent observation that increases in the a-tocopherolcontent of the yolk are subsequently reÑected in theconcentration of this antioxidant in the brain may be ofparticular signiÐcance when considering the means bywhich the resistance of the nervous system to lipid per-oxidation may be enhanced. Somewhat analogousresults have been obtained by feeding weanling rats

with elevated levels of a-tocopherol (Vatassery et al1988) ; an increase in the level of a-tocopherol in thebrain was observed although a greater response to thisdietary supplementation was displayed by the liver.

The liver is by far the major site of a-tocopheroldeposition during chick embryo development, presum-bly due to the uptake of lipoprotein remnants (Gaal etal 1995). This hepatic sequestration of a large propor-tion of the yolk-derived a-tocopherol inevitably limitsthe uptake of this antioxidant by the other embryonictissues. However, it is possible that, after hatching, theliver may secrete a proportion of its accumulated a-tocopherol into the circulation in the form of very lowdensity lipoprotein (Kayden and Traber 1993), thusacting as a source of supply for the non-hepatic tissuesof the chick. The dramatic decrease in the level of a-tocopherol in the liver between days 1 and 9 afterhatching (Table 1), in spite of the provision of substan-tial dietary levels of this vitamin (32 mg kg~1 feed)during this time, clearly supports this possibility. Thus,it is feasible that the elevated accumulation of a-tocopherol in the liver as a consequence of increasedlevels of the vitamin in the yolk may ultimately enhancethe antioxidant capacity of the other tissues of thechick.

In conclusion, it is suggested that enhancing the a-tocopherol content of the yolk by dietary supplementa-tion of the laying hen may represent a simple means ofimproving the resistance of the chick to lipid peroxi-dation.

ACKNOWLEDGEMENTS

The authors are grateful to the Scottish Office Agricul-ture, Environment and Fisheries Department and to theBritish Council for funding this work.

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