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Transfer of fat-soluble vitamins and PCBs from mother to pups in grey seals (Halichoerus grypus)
C’omp. Biorhem. fllwiol. Vol. 109C. No. 2, pp. I I I-I 17, 1994 Copyright (? 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742-8413194 $7.00 + 0.00
Pergamon 0742~8413(94)00055-7
Transfer of fat-soluble vitamins and PCBs from mother to pups in grey seals (Halichoerus grypus) Florian
J. Schweigert*“f and Wayne T. Stobo$
*Department of Physiology, Physiological Chemistry and Nutrition Physiology, Veterinary Faculty, Ludwig-Maximilians-Universitgt Miinchen, Veterinlrstr. 13, D-80539 Miinchen 22, Germany; and $Department of Fisheries and Oceans, Marine Fish Division, Bedford Institute of Oceanography, Dartmouth, NS, Canada B2Y 4A2 Lactation in seals is characterized by a rapid and enormous lipid transfer from mother to pups within a milk rich in lipids. Since grey seals do not feed during lactation, all milk constituents are solely derived from body stores. Monitoring levels of fat-soluble vitamins as well as PCBs in blubber and milk may give an insight into the mechanisms involved in their mobilization from blubber, transfer into milk and deposition in the blubber of pups. During lactation, total lipids in milk increased from 261 to 601 g/l. While the level of PCBs in milk per g lipid remained constant throughout lactation, vitamin E, as well as vitamin A and cholesterol, showed a marked decrease during lactation when expressed as quantity per unit lipid. An incomplete transfer of all components from maternal blubber to milk was observed, except for vitamin E. The milk of pregnant females had vitamin E levels per unit lipid which were three times higher than that in blubber, indicating a mobilization of vitamin E from the liver. During the later stages of lactation, there were no differences between the levels of vitamins A and E per unit lipid in the milk and the blubber of suckling pups. The close correlation of PCBs with total milk lipids and the drastic decrease in all other monitored fat-soluble components in seal milk with the progress of lactation point to different mechanisms of mobilization and transport for triglycerides and PCBs compared to fat-soluble vitamins and cholesterol. Key words: Lipid metabolism; Fat-soluble Mother-pup transfer; Seal; Marine mammal. Comp. Biochem.
Physiol.
vitamins;
Fasting;
Lactation;
Milk;
109C, 11 l-l 17, 1994.
Introduction As part of their annual grey seals (Halichoerus
reproductive cycle gr$pus) aggregate
on isolated islands and shorelines, where pupping and subsequent mating occur. On Sable Island, an island 160 km east of Nova Scotia (Canada) in the Northwest Atlantic Ocean, this activity persists from midDecember to early February, although individual seals remain on the island for shorter durations (Boness and James, 1979). The lactation strategy of grey seals, and phocids generally, is characterized by adult fasting and a relatively short intense lactation
Correspondence to: F. J. Schweigert, Department of Physiology, Veterinary Faculty, University of Leipzig, Semmelweisstr. 2. D-04103 Leipzig, Germany. Tel. (0341) 8827-277/276; Fax: (0341) 8827-277/44X tPresent address: Department of Physiology, Veterinary Faculty, University of Leipzig, Semmelwisstr. 2, D-04103 Leipzig, Germany. Received 12 October 1993; revised 19 July 1994; accepted 2 August 1994. III
112
F. J. Schweigert
period of about 18 days (Bonner, 1984) after which the pups are abruptly weaned. This strategy requires that a large store of nutrients be accumulated in maternal tissue such as the blubber (subcutaneous adipose tissue) prior to parturition. Pups are nursed with a milk consisting of over 50% of lipids (Amoroso and Mathews, 1951) and consequently high in energy. During the 2-3week fasting period, lipid oxidation satisfies the mother’s energy requirements both for maintenance and milk production. Lactating females lose about 65 kg of their original body mass during lactation, primarily as lipids from the blubber. This corresponds to a total energy expenditure of 126 MJ per day, 57% of which are incorporated into the milk (about 22.3 MJjkg milk) (Fedak and Anderson, 1982). Milk, however, not only supplies the newborn with energy but also with essential immunoglobulins and fat-soluble components such as vitamins, cholesterol and phospholipids. This transfer is of importance to the nursing pup, since the placenta limits the intrauterine transfer of these components during fetal development (Butler, 1974; Bates, 1983). While differences in the concentration of fat-soluble vitamins and cholesterol in colostrum and mature milk are well-established for terrestrial animals and man (Bitman et ul., 1986; Schweigert, 1990; Boersma et al., 1991) nothing is known for seals or marine mammals in general. In this paper we examine both the changing composition of milk and changes in the blubber composition of mother and pups from just prior to parturition until weaning.
Materials and Methods Milk and blubber samples were obtained from five pregnant and 19 lactating Atlantic grey seal (Halichoerus grypus) mother-pup pairs at different stages of lactation for this and other studies with agreement of the Canadian Government in January 1987, from the pupping colony on Sable Island (Canada) in the Northwest Atlantic Ocean. The animals were shot with a high-powered rifle. Instantaneous death was confirmed by the examination of the eye reflexes. Morphometric measurements were obtained according to the American Society of Mammalogists (1967). The age of the adults
and W. T. Stobo
was determined by the counting of dental annuli in thin cross-sections of the lower left canines (Mansfield, 1991). The average ages of pregnant animals, and those of stages I, II and III of lactation, were 14.0 * 3.4, 8.9f 1.6, 13.0f 1.3 and 8.8 f 1.6 years, respectively. The estimated stage of lactation was based on the developmental stage of the pup. Like several other seal species, grey seal pups pass through several identifiable stages during the lactation period. involving changes in pelage and body fatness (Kovacs and Lavigne, 1986). Each stage represents approximately 5 days of development (Radford et al., 1978) adding up to 15-18 days for the whole period of lactation. Pregnant animals were estimated to be 2-3 days before parturition, because they come ashore only a few days prior to parturition. To obtain uncontaminated and sufficient 90 IU oxytocin (Hydromilk samples, Chemie Munchen, Germany; commercially available oxytocin was concentrated from 10 IU/ml to 30 III/ml by lyophilization) were injected using a blow pipe system (Teleinject, Germany) 10 min before the animals were shot. The milk was obtained by opening the seal in the median abdominal region, then cutting between the skin and subcutaneous adipose tissue (blubber) towards the ductus of the mammary gland. Using this procedure, uncontaminated milk (i.e. free from blood) could be collected. Oxytocin could not be administered to pregnant animals prior to death because females which are disturbed prior to parturition immediately return to the ocean. Nevertheless, in the case of the five animals sampled, we were still able to collect sufficient uncontaminated milk by the above dissection procedure. Based on a study of Oftedal et al. (1988) the administration of oxytocin has no effect on the lipid composition of milk. Therefore, an effect on fat-soluble vitamins and cholesterol is not probable and milk samples of animals shortly before parturition (no oxytocin) can be compared with those obtained during lactation (oxytocin). The colour of fresh grey seal milk ranged from reddish or beige (pregnant animals) to white or greyish-blue (end of lactation). Viscosity of the milk increased, being like thick cream at the end of lactation. The milk samples had a strong
113
Vitamins and lactation in seals
fishy odour and taste. Blubber sections were obtained from the mid-dorsal region of mothers and pups. All samples were stored at -20°C and shipped to Germany on dry ice for chemical analysis within 6 months. Lipids were extracted from milk and blubber according to Radin (198 1). To ensure complete extraction of lipid from the blubber or milk, an average of 3 g samples was extracted three times in a mixture of n -hexane-isopropanol (3 : 2, v/v, 10 ml). The supernatants of the three extractions were combined, 0.1 M NaCl (10 ml) were added and phase separation was obtained within 30 min. At this point, lipid content of tissue and milk samples was determined gravimetrically in the dried organic extract. For the determination of cholesterol and fat-soluble vitamins, an aliquot of the upper organic phase was dried and saponified under nitrogen with a volume of ethanolic KOH (5 g KOH, 5 ml H20, 50 ml ethanol) three times that of the aliquot for 30 min at 70-75°C (Frolik and Olson, 1984). The organic extract (three extractions in n-hexane, total 4 ml) of the saponified sample was then dried and reconstituted in methanol-ethanol (80: 20). Reconstituted samples were directly injected on to isocratic rp-HPLC and analyzed for fatsoluble vitamins in milk and blubber, and cholesterol in milk only, as described elsewhere (Stump et al., 1984; Schweigert, 1990). PCBs were determined by glass capillary gas-chromatography as described (Stijve and Cardinale, 1974; Beck and Mathar, 1985) and calculated as Clophen A60.
Table I. Lipids, fat-soluble
Component Lipids mg/gt PCB pglg Cholesterol pg/g Vitamin A Ilg/g Vitamin E pg/g
Results While the stage of lactation of mothers and pups had no effect on the body length of the animals, all morphometric measurements related to body weight changed with the progression of lactation. In lactating animals, girth and blubber error) thickness (mean &-standard decreased (151 f 3cm to 124f 2cm and 5.5 k 0.4 cm to 2.6 f 0.2 cm, respectively). In pups, both parameters and body weight increased with the duration of sucking (girth: 52 + 1 to 91 + 3 cm, blubber thickness: 0.5 f 0.04 to 4.1 f 0.5 cm and weight: 18 + 1 to 42 + 4 kg). These data are described in more detail elsewhere (Schweigert, 1993). Data on lipid content and concentration of fat-soluble vitamins and PCBs in milk samples of different stages of lactation are summarized in Table 1. The concentration of total lipids was closely correlated with stage of lactation (Y = 0.95, P < 0.001) and, together with the PCBs in the milk, increased more than 2-fold, while vitamins A and E and cholesterol decreased. This decrease was even more impressive, when these components were expressed as quantity per unit lipid (Fig. 1). In colostrum, levels of vitamins A and E as well as cholesterol per unit lipid were 3.6, 11.2 and 4.4 times higher, respectively, than in milk obtained at the end of the lactation period. Vitamin A, vitamin E and cholesterol were negatively correlated with total lipids (Y = -0.61, - 0.85, - 0.67, respectively, all P < 0.004) and the stage of lactation (r = -0.48, -0.78, -0.78, respectively, all
vitamins, and PCBs per unit weight of grey seal milk at different of lactation (mean k standard error) Pregnant (3/3/3/4/4)* 163-303-318: 0.674.76-1.13* 657-667-692; 3.3 f 1.0 40.3 f 2.3
stages
Stage I (4/-161717)
Stage II (6/-161616)
Stage III (6/6/616/6)
384 + 52 ND 417 & 59 3.1 * 0.4 31.2k4.5
492 k 35 ND 225 f. 25 2.0 * 0.4 9.7 k 1.6
601 2.26 290 2.9 9.1
& + k * *
22 0.48 37 1.0 1.2
*Values in brackets represent the respective number of animals sampled. Differences in the number of samples investigated are due to the limited amount of samples available. tDue to the high viscosity of the sample concentrations had to be determined based on weight. $Individual results. ND = not determined.
F. J. Schweigert and W. T. Stobo
114
Table 2. Fat-soluble
Component PCBs Vitamin Vitamin
A E
vitamins and PCBs per unit weight of adipose tissue (@g/g) of grey seal mother and pups at different stages of lactation (mean + standard error)
Pregnant Mother Fetus (5/5/5)* (-1515) 9.3 + 5.0 45.6k13.5 37.8 f 8.0
ND 4.8kl.O 2.3 + 0.2
Stage I Mother Pup (-1717) (-1717) ND 43.lk3.9 48.3 k 7.0
Stage II Mother Pup (-/6/6) (-/6/6)
ND ND 3.9kO.3 41.4k3.3 9.4 + 1.7 47.3 t_ 7.3
*Values in brackets represent the respective number of animals sampled. investigated are due to the limited amount of samples available. ND = not determined.
P < 0.001). There was a significant positive correlation between the declines in vitamin A and vitamin E (Y = 0.84, P < O.OOl), vitamin A and cholesterol (r = 0.47, P < 0.05) and vitamin E and cholesterol (r = 0.92, P -c 0.001). Neither vitamin E nor vitamin A levels in blubber of lactating females changed with the progression of lactation (Y = 0.31;
Fig. 1. The effect of the stage of lactation on levels of vitamins A and E in maternal blubber ( ?? ). milk (Q)” and in the blubber of the suckling pup (H) in pg/g fat (mean & standard error)h. “Individual values for the three milk samples of pregnant animals were 6. I, 7.2 and 38.8 pg vitamin A/g lipid and 111.7, 130.3 and 276.3 pg vitamin E/g lipid. hNumber of samples: pregnant: 5-3-5; stage I: 7-4-7: stage II: 6-6-6; stage III: 6-6-6.
ND 2.5kO.2 14.9 k 1.2 Differences
Stage III Mother Pup (11b/6) (91616) 7.5 * 1.4 50.3+15.1 59.4f 5.8
3.2 + 0.9 2.3kO.4 14.9 & 0.9
in the number of samples
P < 0.190; Y = 0.01, P < 0.954). PCB levels in blubber were lower at the end of lactation. In suckling pups (Fig. l), levels of vitamin A in blubber decreased (r = - 0.70, P < 0.002) and those of vitamin E increased during the period of suckling (r = 0.66, P < 0.005).
Discussion Changes in morphometric date related to body weight (girth and blubber thickness) reflect energy mobilization during lactation as well as energy transfer to the pup with a milk rich in lipids. This energy transfer into milk is estimated to be 22.3 MJjkg milk and females produce 2-3 kg milk a day, which accounts for about 57% of the total daily energy expenditure (Fedak and Anderson, 1982). The comparable weight increase in suckling pups of 11-12 kg between each stage (I, II and III), despite a marked increase in energy content of milk, is due to the fact that, with increasing energy density of the milk, milk uptake of the pups decreases (Fedak and Anderson, 1982). Extraordinarily high lipid levels in grey seal milk are known for this species as well as for other seal species (Amoroso and Mathews, 1951). At the end of lactation (stage III) the milk of one animal contained 701 g lipids per kg milk, the highest level of milk fat ever reported for terrestrial or marine mammals. The observed increase in lipids during lactation corresponds to results obtained for Weddell seals (Kooyman and Drabek, 1968), elephant seals (Riedman and Ortiz, 1979) and harp seals (Lavigne et al., 1982). Contrary to this, no changes in lipid levels were observed during the very brief lactation period (4 days) in the hooded seal (Oftedal et al., 1988); and in the South American sea lion, lipid levels
Vitamins
and lactation
were found to decrease (Trillmich and Lechner, 1986). Numerous papers have investigated lipid content and lipid composition of marine mammal milk, but very little is known for the secretion of vitamins A and E or cholesterol with the milk. The levels of vitamin A found in our study correspond to levels et al. (1955) in three found by Gregory whales, and by Rodahl and Davies (1949) in a single harp seal. We found that the levels of these fat-soluble vitamins, especially vitamin E and cholesterol, were much higher than those found in the milk of terrestrial mammals, but when expressed as quantity per unit lipid, the levels were in the same order of magnitude or even lower (vitamin A) as in these species. The marked decreases in fat-soluble vitamins A and E and cholesterol during the lactation period correspond to observations in terrestrial mammals and in man (Bitman et al., 1986; Schweigert, 1990; Boersma et al., 1991). To our knowledge the milk of seals has not been investigated during the colostrum phase, that period shortly before or at parturition. This period is of special interest, because the colostrum composition is specific to the needs of the new-born with regard to lipids, fat-soluble vitamins, proteins and/or peptides as well as hormones (West, 1989) and is known to differ markedly from that reported for mature milk in terrestrial mammals and man (Butler, 1974; Lammi-Keefe and Jensen, 1984; Bitman et al., 1986; Schweigert, 1990; Boersma et al., 1991). Interestingly, the level of total lipids, or triglycerides, in colostrum is similar to, or even lower than, that in mature milk (Herting and Drury, 1969; Lammi-Keefe and Jensen, 1984; Wood and Bitman, 1986; Boersma et al., 1991). Our study indicates that grey seals are similar to other mammals in that respect. The increase in total lipids and PCBs in milk coincides with a positively correlated decrease in fat-soluble vitamins and cholesterol. This difference indicates that two different mechanisms are involved in the transfer of lipids from blood into the mammary gland (Harzer and Haug, 1985; Schweigert, 1990). Due to the limited number of samples, however, further studies for generalization would be necessary. Previous studies in dairy cows (Schweigert, 1990)
in seals
115
showed a drastic decrease in plasma low density lipoproteins (LDL) associated with the formation of colostrum. It was hypothesized that the decrease was associated with an increase in LDL-receptors which facilitated the transfer of vitamin E, cholesterol and phospholipids. We suggest that this is one of the mechanisms functioning in grey seals, the other one being the transfer of triglycerides via very low density lipoproteins (VLDL) and the lipoprotein-lipase pathway into milk (Dils, 1986). SpindlerVomachka and Vodicnik (1984) found that PCBs are predominately associated with the VLDL fraction in rats. In seals, a similar situation may exist, this explaining the increase in PCBs coincident with triglycerides. Female grey seals on Sable Island fast throughout lactation. Therefore, all milk components are exclusively derived from body reserves. The similarity in fatty acid composition of blubber and milk in seals (Ackman and Burgher, 1963) indicates that the de nozm synthesis of lipids contributes only marginally to total lipids in milk. Therefore, the majority of milk lipids originate from the blubber and concentrations of vitamins A and E and PCBs per unit lipid in milk and blubber can be directly compared (Fig. 1). Throughout the lactation period, the concentration of vitamin E in adult females increased in blubber and decreased in milk. The concentration of vitamin E in the early stages of lactation was very high compared to that in blubber, suggesting mobilization from other organs. One source could be the liver, in which the concentration of vitamin E decreased by 60% during lactation (Schweigert, Luppertz and Stobo, in preparation). In the later stages of lactation, an incomplete mobilization was evident. This has also been observed in the guinea-pig (Machlin et al., 1979) and man (Schaefer et al., 1983). The vitamin A concentration also increased in blubber while decreasing in the milk of adult females. But, contrary to the status of vitamin E, at all stages of lactation the concentrations of vitamin A in milk were much lower than that in blubber. Thus an incomplete mobilization of vitamin A from blubber is also indicated. In pups, the level of vitamin A decreased throughout the lactation period in a
116
F. J. Schweigert and W. T. Stobo
manner similar to that observed in the milk. This coincident decrease indicates that pups are highly efficient in conserving the vitamin A from milk and storing it in blubber. The situation for vitamin E in the blubber of pups differs from that of vitamin A. Early in lactation, the amount of vitamin E being stored in the blubber is a small proportion of that observed in the milk but it increases throughout the lactation period. In the later stages of lactation, the amount of vitamin E being deposited in the blubber exceeds that being transferred via the milk. In conclusion, this study indicates that the fat-soluble vitamins (vitamins A and E), cholesterol and PCB composition of seal milk changes dramatically as lactation progresses. It also indicates that grey seals are similar to terrestrial mammals and man in the differences observed between colostrum and mature milk, thus indicating that the physiological processes are the same although the absolute quantities differ quite dramatically. This indicates that, in seals. two different mechanisms for the transfer of lipophilic components may also exist-one for the transfer of the triglycerides and possibly PCBs, and a separate one for other fat-soluble components such as vitamins and cholesterol. The higher concentrations in colostrum emphasize their importance in the nutrition of the newborn pup. Acknowledgements-We thank Brian Beck for his assistance in the field work and the age determination of the adult animals. Total lipids in milk were kindly determined by Dr Precht (Bundesforschungsanstalt fiir Milchforschung). Financial support for this study was provided by the Deutsche Forschungsgemeinschaft (Sch 373/l-l), the Miinchener Universitlts Gesellschaft, the Hanns Seidel Stiftung e. V.. Germany and the Department of Fisheries and Oceans, Canada.
References Ackman R. G. and Burgher R. D. (1963) Component fatty acids of the milk of the grey (Atlantic) seal. Can. J. Biochem. Physiol. 41, 2501-2505. American Society of Mammalogists (1967) Standard measurements of seals. J. Mammal. 48, 459-462. Amoroso E. C. and Mathews J. H. (1951) The growth of the grey seal (Halichoerus grypus (Fabricius)) from birth to weanine. J. Anat. 85. 427. Bates C. J. (1983) Vi&min A in pregnancy and lactation. Proc. Nutr. Sot. 42, 65-79. Beck H. and Mathar W. (1985) Analysenverfahren PCBzur Bestimmung von ausgewlhlten
Einzelkomponenten sundhbl.
28,
in Lebensmitteln.
Bundesge-
l-12.
Bitman J., Freed L. M., Neville M. C.. Wood D. L., Hamosh P. and Hamosh M. (1986) Lipid composition of prepartum human mammary secretion and postpartum milk. J. Pediatr. Gastroenterol. Nutr: 5, 608-615. Boersma E. R., Offringa P. J., Muskiet F. A. J., Chase W. M. and Simmons I. J. (1991) Vitamin E, lipid fractions, and fatty acid composition of colostrum. transitional milk, and mature milk: an international comparative study. Am. J. clin. Nutr. 53, 1197-1204. Boness D. J. and James H. (1979) Reproductive behaviour of the grey seal (Halichoerus grypus) on Sable Island, Nova Scotia. J. Zool., Lond. 188, 477-500. Bonner W. N. (1984) Lactation strategies in pinnipeds: problems for a marine mammalian group. Svmp.
Zool.
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51, 253-272.
Butler J E (1974) Immunoglobulins of the mammary secretions. In Lactation: A Comprehensive Treatise Vol. III (Edited by Larson B. and Smith V. R.), pp. 217-253. Academic Press, Ney York. Dils R. R. (1986) Comparative aspects of milk fat synthesis. J. Dairy Sci. 69, 904-910. Fedak M. A. and Anderson S. S. (1982) The energetits of lactation: accurate measurements from a large wild mammal, the grey seal (Halichoerus gwppus). J. Zool.
198, 473-479.
Frolik C. A. and Olson J. A. (1984) Extraction, separation and chemical analysis of retinoids. In The Retinoids (Edited by Sporn M. B., Roberts A. B. and Goodman D. S.), pp. 181-233. Academic Press, Orlando, FL. Gregory M. E., Kon S. K., Rowland S. J. and Thompson S. Y. (1955) The composition of the milk of the blue whale. J. Dairy Res. 22, 108-l 12. Harzer G. and Haug M. (1985) Correlation of human milk vitamin E with different lipids. In Composition and
Ph?,siological
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cf‘ Human
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(Edited by Schaub J.), pp. 247-254. Elsevier Science, Amsterdam. Herting D. C. and Drury E.-J. E. (1969) Vitamin E content of milk. milk products, and simulated milks: relevance to infant nutrition. Am. J. Clin. Nutr. 22,
147-155.
Kooyman G. L. and Drabek C. M. (1968) Observations on milk. blood, and urine constituents of the Weddell seal. Physiol. Zool. 41, 187-194. Kovacs K. M. and Lavigne D. M. (1986) Growth of grey seal (Halichoerus grypus) neonates: differential maternal investment in the sexes. Can. J. Zool. 64, 1937-1943. Lammi-Keefe C. J. and Jensen R. G. (1984) Lipids in human milk: a review 2. Composition and fat-soluble vitamins. J. Pediat. Gastroenterol. Nutr. 3, 172-198.
Lavigne D. M.. Stewart R. E. A. and Fletcher F. (1982) Changes in composition and energy content of harp seal milk during lactation. Physiol. Zool. 55, I-9. Machlin L. J., Keating J., Nelson J.. Brin M., Filipski R. and Miller 0. N. (1979) Availability of adipose tissue tocopherol in the guinea-pig. J. Nutr. 109, 105-109.
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Mansfield A. W. (1991) Accuracy of age determination in the grey seal Halichoerus gryppus of Eastern Canada. Mar. Mammal Sci. 7, 44-49. Oftedal 0. T., Boness D. J. and Bowen W. D. (1988) The composition of hooded seal (Cystophora for postnatal fattencrisfafa) milk: an adaptation ing. Can. J. Zool. 66, 318-322. Radford P. J.. Summers C. F. and Young K. M. (1978) A statistical procedure for estimating grey seal pup production from a single census. Mammal Rev. 8, 35-42. Radin N. S. (1981) Extraction of tissue lipids with a solvent of low toxicity. Merh. Enzym. 72, 5-7. Riedman M. and Ortiz C. L. (1979) Changes in milk composition during lactation in the northern elephant seal. Physiol. Zool. 52, 240-249. Rodahl K. and Davies A. W. (1949) Vitamin A in seals. Biochem. J. 45, 408-412. Schaefer E. J., Woo R., Kibata M.. Bjornsen L. and Schreibman P. H. (1983) Mobilization of triglyceride but not cholesterol or tocopherol from human adipocytes during weight reduction. Am. J. Clin. Nutr. 37, 749-754. Schweigert F. J. (1990) Effect of gestation and lactation on lipoprotein pattern and composition in dairy cows. J. Anim. Physiol. Anim. Nutr. 6, 75-83.
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Schweigert F. J. (1993) Effects of energy mobilization during fasting and lactation on plasma metabolites in the grey seal (Halichoerus grypus). Camp. Biothem. Physiol. 105A, 347-352. Spindler-Vomachka M. and Vodicnik M. J. (1984) Distribution of 2,4,5,2’,4’,5’-hexachlorobiphenyl among lipoproteins during pregnancy and lactation in the rat. J. Pharmac. exp. Ther. 74, 263-268. Stijve T. and Cardinale E. (1974) Rapid determination of chlorinated pesticides, polychlorinated biphenyls and a number of phosphated insecticides in fatty foods. Mitt. Lebens. Hyg. 65, 131-l 50. Stump D. D.. Roth F. E. Jr and Gilbert H. S. (1984) Simultaneous determination by high-performance liquid chromatography of tocopherol isomers, a-tocopheryl quinone, and cholesterol in red blood cells. J. Chromat. 306, 371-376. Trillmich F. and Lechner E. (1986) Milk of the Galapagos fur seal and sea lion, with a comparison of the milk of eared seals (Otariidae). J. Zool. 209, 271-277. West D. W. (1989) The origin, transport and function of hormones and growth factors in milk. Exp. Clin. Endocr. (Life Sci. Adv.) 8, 145-156. Wood D. L. and Bitman J. (1986) Cholesteryl esters of cows’ milk. J. Dairy Sci. 69, 2203-2208.
Pergamon 0742~8413(94)00055-7
Transfer of fat-soluble vitamins and PCBs from mother to pups in grey seals (Halichoerus grypus) Florian
J. Schweigert*“f and Wayne T. Stobo$
*Department of Physiology, Physiological Chemistry and Nutrition Physiology, Veterinary Faculty, Ludwig-Maximilians-Universitgt Miinchen, Veterinlrstr. 13, D-80539 Miinchen 22, Germany; and $Department of Fisheries and Oceans, Marine Fish Division, Bedford Institute of Oceanography, Dartmouth, NS, Canada B2Y 4A2 Lactation in seals is characterized by a rapid and enormous lipid transfer from mother to pups within a milk rich in lipids. Since grey seals do not feed during lactation, all milk constituents are solely derived from body stores. Monitoring levels of fat-soluble vitamins as well as PCBs in blubber and milk may give an insight into the mechanisms involved in their mobilization from blubber, transfer into milk and deposition in the blubber of pups. During lactation, total lipids in milk increased from 261 to 601 g/l. While the level of PCBs in milk per g lipid remained constant throughout lactation, vitamin E, as well as vitamin A and cholesterol, showed a marked decrease during lactation when expressed as quantity per unit lipid. An incomplete transfer of all components from maternal blubber to milk was observed, except for vitamin E. The milk of pregnant females had vitamin E levels per unit lipid which were three times higher than that in blubber, indicating a mobilization of vitamin E from the liver. During the later stages of lactation, there were no differences between the levels of vitamins A and E per unit lipid in the milk and the blubber of suckling pups. The close correlation of PCBs with total milk lipids and the drastic decrease in all other monitored fat-soluble components in seal milk with the progress of lactation point to different mechanisms of mobilization and transport for triglycerides and PCBs compared to fat-soluble vitamins and cholesterol. Key words: Lipid metabolism; Fat-soluble Mother-pup transfer; Seal; Marine mammal. Comp. Biochem.
Physiol.
vitamins;
Fasting;
Lactation;
Milk;
109C, 11 l-l 17, 1994.
Introduction As part of their annual grey seals (Halichoerus
reproductive cycle gr$pus) aggregate
on isolated islands and shorelines, where pupping and subsequent mating occur. On Sable Island, an island 160 km east of Nova Scotia (Canada) in the Northwest Atlantic Ocean, this activity persists from midDecember to early February, although individual seals remain on the island for shorter durations (Boness and James, 1979). The lactation strategy of grey seals, and phocids generally, is characterized by adult fasting and a relatively short intense lactation
Correspondence to: F. J. Schweigert, Department of Physiology, Veterinary Faculty, University of Leipzig, Semmelweisstr. 2. D-04103 Leipzig, Germany. Tel. (0341) 8827-277/276; Fax: (0341) 8827-277/44X tPresent address: Department of Physiology, Veterinary Faculty, University of Leipzig, Semmelwisstr. 2, D-04103 Leipzig, Germany. Received 12 October 1993; revised 19 July 1994; accepted 2 August 1994. III
112
F. J. Schweigert
period of about 18 days (Bonner, 1984) after which the pups are abruptly weaned. This strategy requires that a large store of nutrients be accumulated in maternal tissue such as the blubber (subcutaneous adipose tissue) prior to parturition. Pups are nursed with a milk consisting of over 50% of lipids (Amoroso and Mathews, 1951) and consequently high in energy. During the 2-3week fasting period, lipid oxidation satisfies the mother’s energy requirements both for maintenance and milk production. Lactating females lose about 65 kg of their original body mass during lactation, primarily as lipids from the blubber. This corresponds to a total energy expenditure of 126 MJ per day, 57% of which are incorporated into the milk (about 22.3 MJjkg milk) (Fedak and Anderson, 1982). Milk, however, not only supplies the newborn with energy but also with essential immunoglobulins and fat-soluble components such as vitamins, cholesterol and phospholipids. This transfer is of importance to the nursing pup, since the placenta limits the intrauterine transfer of these components during fetal development (Butler, 1974; Bates, 1983). While differences in the concentration of fat-soluble vitamins and cholesterol in colostrum and mature milk are well-established for terrestrial animals and man (Bitman et ul., 1986; Schweigert, 1990; Boersma et al., 1991) nothing is known for seals or marine mammals in general. In this paper we examine both the changing composition of milk and changes in the blubber composition of mother and pups from just prior to parturition until weaning.
Materials and Methods Milk and blubber samples were obtained from five pregnant and 19 lactating Atlantic grey seal (Halichoerus grypus) mother-pup pairs at different stages of lactation for this and other studies with agreement of the Canadian Government in January 1987, from the pupping colony on Sable Island (Canada) in the Northwest Atlantic Ocean. The animals were shot with a high-powered rifle. Instantaneous death was confirmed by the examination of the eye reflexes. Morphometric measurements were obtained according to the American Society of Mammalogists (1967). The age of the adults
and W. T. Stobo
was determined by the counting of dental annuli in thin cross-sections of the lower left canines (Mansfield, 1991). The average ages of pregnant animals, and those of stages I, II and III of lactation, were 14.0 * 3.4, 8.9f 1.6, 13.0f 1.3 and 8.8 f 1.6 years, respectively. The estimated stage of lactation was based on the developmental stage of the pup. Like several other seal species, grey seal pups pass through several identifiable stages during the lactation period. involving changes in pelage and body fatness (Kovacs and Lavigne, 1986). Each stage represents approximately 5 days of development (Radford et al., 1978) adding up to 15-18 days for the whole period of lactation. Pregnant animals were estimated to be 2-3 days before parturition, because they come ashore only a few days prior to parturition. To obtain uncontaminated and sufficient 90 IU oxytocin (Hydromilk samples, Chemie Munchen, Germany; commercially available oxytocin was concentrated from 10 IU/ml to 30 III/ml by lyophilization) were injected using a blow pipe system (Teleinject, Germany) 10 min before the animals were shot. The milk was obtained by opening the seal in the median abdominal region, then cutting between the skin and subcutaneous adipose tissue (blubber) towards the ductus of the mammary gland. Using this procedure, uncontaminated milk (i.e. free from blood) could be collected. Oxytocin could not be administered to pregnant animals prior to death because females which are disturbed prior to parturition immediately return to the ocean. Nevertheless, in the case of the five animals sampled, we were still able to collect sufficient uncontaminated milk by the above dissection procedure. Based on a study of Oftedal et al. (1988) the administration of oxytocin has no effect on the lipid composition of milk. Therefore, an effect on fat-soluble vitamins and cholesterol is not probable and milk samples of animals shortly before parturition (no oxytocin) can be compared with those obtained during lactation (oxytocin). The colour of fresh grey seal milk ranged from reddish or beige (pregnant animals) to white or greyish-blue (end of lactation). Viscosity of the milk increased, being like thick cream at the end of lactation. The milk samples had a strong
113
Vitamins and lactation in seals
fishy odour and taste. Blubber sections were obtained from the mid-dorsal region of mothers and pups. All samples were stored at -20°C and shipped to Germany on dry ice for chemical analysis within 6 months. Lipids were extracted from milk and blubber according to Radin (198 1). To ensure complete extraction of lipid from the blubber or milk, an average of 3 g samples was extracted three times in a mixture of n -hexane-isopropanol (3 : 2, v/v, 10 ml). The supernatants of the three extractions were combined, 0.1 M NaCl (10 ml) were added and phase separation was obtained within 30 min. At this point, lipid content of tissue and milk samples was determined gravimetrically in the dried organic extract. For the determination of cholesterol and fat-soluble vitamins, an aliquot of the upper organic phase was dried and saponified under nitrogen with a volume of ethanolic KOH (5 g KOH, 5 ml H20, 50 ml ethanol) three times that of the aliquot for 30 min at 70-75°C (Frolik and Olson, 1984). The organic extract (three extractions in n-hexane, total 4 ml) of the saponified sample was then dried and reconstituted in methanol-ethanol (80: 20). Reconstituted samples were directly injected on to isocratic rp-HPLC and analyzed for fatsoluble vitamins in milk and blubber, and cholesterol in milk only, as described elsewhere (Stump et al., 1984; Schweigert, 1990). PCBs were determined by glass capillary gas-chromatography as described (Stijve and Cardinale, 1974; Beck and Mathar, 1985) and calculated as Clophen A60.
Table I. Lipids, fat-soluble
Component Lipids mg/gt PCB pglg Cholesterol pg/g Vitamin A Ilg/g Vitamin E pg/g
Results While the stage of lactation of mothers and pups had no effect on the body length of the animals, all morphometric measurements related to body weight changed with the progression of lactation. In lactating animals, girth and blubber error) thickness (mean &-standard decreased (151 f 3cm to 124f 2cm and 5.5 k 0.4 cm to 2.6 f 0.2 cm, respectively). In pups, both parameters and body weight increased with the duration of sucking (girth: 52 + 1 to 91 + 3 cm, blubber thickness: 0.5 f 0.04 to 4.1 f 0.5 cm and weight: 18 + 1 to 42 + 4 kg). These data are described in more detail elsewhere (Schweigert, 1993). Data on lipid content and concentration of fat-soluble vitamins and PCBs in milk samples of different stages of lactation are summarized in Table 1. The concentration of total lipids was closely correlated with stage of lactation (Y = 0.95, P < 0.001) and, together with the PCBs in the milk, increased more than 2-fold, while vitamins A and E and cholesterol decreased. This decrease was even more impressive, when these components were expressed as quantity per unit lipid (Fig. 1). In colostrum, levels of vitamins A and E as well as cholesterol per unit lipid were 3.6, 11.2 and 4.4 times higher, respectively, than in milk obtained at the end of the lactation period. Vitamin A, vitamin E and cholesterol were negatively correlated with total lipids (Y = -0.61, - 0.85, - 0.67, respectively, all P < 0.004) and the stage of lactation (r = -0.48, -0.78, -0.78, respectively, all
vitamins, and PCBs per unit weight of grey seal milk at different of lactation (mean k standard error) Pregnant (3/3/3/4/4)* 163-303-318: 0.674.76-1.13* 657-667-692; 3.3 f 1.0 40.3 f 2.3
stages
Stage I (4/-161717)
Stage II (6/-161616)
Stage III (6/6/616/6)
384 + 52 ND 417 & 59 3.1 * 0.4 31.2k4.5
492 k 35 ND 225 f. 25 2.0 * 0.4 9.7 k 1.6
601 2.26 290 2.9 9.1
& + k * *
22 0.48 37 1.0 1.2
*Values in brackets represent the respective number of animals sampled. Differences in the number of samples investigated are due to the limited amount of samples available. tDue to the high viscosity of the sample concentrations had to be determined based on weight. $Individual results. ND = not determined.
F. J. Schweigert and W. T. Stobo
114
Table 2. Fat-soluble
Component PCBs Vitamin Vitamin
A E
vitamins and PCBs per unit weight of adipose tissue (@g/g) of grey seal mother and pups at different stages of lactation (mean + standard error)
Pregnant Mother Fetus (5/5/5)* (-1515) 9.3 + 5.0 45.6k13.5 37.8 f 8.0
ND 4.8kl.O 2.3 + 0.2
Stage I Mother Pup (-1717) (-1717) ND 43.lk3.9 48.3 k 7.0
Stage II Mother Pup (-/6/6) (-/6/6)
ND ND 3.9kO.3 41.4k3.3 9.4 + 1.7 47.3 t_ 7.3
*Values in brackets represent the respective number of animals sampled. investigated are due to the limited amount of samples available. ND = not determined.
P < 0.001). There was a significant positive correlation between the declines in vitamin A and vitamin E (Y = 0.84, P < O.OOl), vitamin A and cholesterol (r = 0.47, P < 0.05) and vitamin E and cholesterol (r = 0.92, P -c 0.001). Neither vitamin E nor vitamin A levels in blubber of lactating females changed with the progression of lactation (Y = 0.31;
Fig. 1. The effect of the stage of lactation on levels of vitamins A and E in maternal blubber ( ?? ). milk (Q)” and in the blubber of the suckling pup (H) in pg/g fat (mean & standard error)h. “Individual values for the three milk samples of pregnant animals were 6. I, 7.2 and 38.8 pg vitamin A/g lipid and 111.7, 130.3 and 276.3 pg vitamin E/g lipid. hNumber of samples: pregnant: 5-3-5; stage I: 7-4-7: stage II: 6-6-6; stage III: 6-6-6.
ND 2.5kO.2 14.9 k 1.2 Differences
Stage III Mother Pup (11b/6) (91616) 7.5 * 1.4 50.3+15.1 59.4f 5.8
3.2 + 0.9 2.3kO.4 14.9 & 0.9
in the number of samples
P < 0.190; Y = 0.01, P < 0.954). PCB levels in blubber were lower at the end of lactation. In suckling pups (Fig. l), levels of vitamin A in blubber decreased (r = - 0.70, P < 0.002) and those of vitamin E increased during the period of suckling (r = 0.66, P < 0.005).
Discussion Changes in morphometric date related to body weight (girth and blubber thickness) reflect energy mobilization during lactation as well as energy transfer to the pup with a milk rich in lipids. This energy transfer into milk is estimated to be 22.3 MJjkg milk and females produce 2-3 kg milk a day, which accounts for about 57% of the total daily energy expenditure (Fedak and Anderson, 1982). The comparable weight increase in suckling pups of 11-12 kg between each stage (I, II and III), despite a marked increase in energy content of milk, is due to the fact that, with increasing energy density of the milk, milk uptake of the pups decreases (Fedak and Anderson, 1982). Extraordinarily high lipid levels in grey seal milk are known for this species as well as for other seal species (Amoroso and Mathews, 1951). At the end of lactation (stage III) the milk of one animal contained 701 g lipids per kg milk, the highest level of milk fat ever reported for terrestrial or marine mammals. The observed increase in lipids during lactation corresponds to results obtained for Weddell seals (Kooyman and Drabek, 1968), elephant seals (Riedman and Ortiz, 1979) and harp seals (Lavigne et al., 1982). Contrary to this, no changes in lipid levels were observed during the very brief lactation period (4 days) in the hooded seal (Oftedal et al., 1988); and in the South American sea lion, lipid levels
Vitamins
and lactation
were found to decrease (Trillmich and Lechner, 1986). Numerous papers have investigated lipid content and lipid composition of marine mammal milk, but very little is known for the secretion of vitamins A and E or cholesterol with the milk. The levels of vitamin A found in our study correspond to levels et al. (1955) in three found by Gregory whales, and by Rodahl and Davies (1949) in a single harp seal. We found that the levels of these fat-soluble vitamins, especially vitamin E and cholesterol, were much higher than those found in the milk of terrestrial mammals, but when expressed as quantity per unit lipid, the levels were in the same order of magnitude or even lower (vitamin A) as in these species. The marked decreases in fat-soluble vitamins A and E and cholesterol during the lactation period correspond to observations in terrestrial mammals and in man (Bitman et al., 1986; Schweigert, 1990; Boersma et al., 1991). To our knowledge the milk of seals has not been investigated during the colostrum phase, that period shortly before or at parturition. This period is of special interest, because the colostrum composition is specific to the needs of the new-born with regard to lipids, fat-soluble vitamins, proteins and/or peptides as well as hormones (West, 1989) and is known to differ markedly from that reported for mature milk in terrestrial mammals and man (Butler, 1974; Lammi-Keefe and Jensen, 1984; Bitman et al., 1986; Schweigert, 1990; Boersma et al., 1991). Interestingly, the level of total lipids, or triglycerides, in colostrum is similar to, or even lower than, that in mature milk (Herting and Drury, 1969; Lammi-Keefe and Jensen, 1984; Wood and Bitman, 1986; Boersma et al., 1991). Our study indicates that grey seals are similar to other mammals in that respect. The increase in total lipids and PCBs in milk coincides with a positively correlated decrease in fat-soluble vitamins and cholesterol. This difference indicates that two different mechanisms are involved in the transfer of lipids from blood into the mammary gland (Harzer and Haug, 1985; Schweigert, 1990). Due to the limited number of samples, however, further studies for generalization would be necessary. Previous studies in dairy cows (Schweigert, 1990)
in seals
115
showed a drastic decrease in plasma low density lipoproteins (LDL) associated with the formation of colostrum. It was hypothesized that the decrease was associated with an increase in LDL-receptors which facilitated the transfer of vitamin E, cholesterol and phospholipids. We suggest that this is one of the mechanisms functioning in grey seals, the other one being the transfer of triglycerides via very low density lipoproteins (VLDL) and the lipoprotein-lipase pathway into milk (Dils, 1986). SpindlerVomachka and Vodicnik (1984) found that PCBs are predominately associated with the VLDL fraction in rats. In seals, a similar situation may exist, this explaining the increase in PCBs coincident with triglycerides. Female grey seals on Sable Island fast throughout lactation. Therefore, all milk components are exclusively derived from body reserves. The similarity in fatty acid composition of blubber and milk in seals (Ackman and Burgher, 1963) indicates that the de nozm synthesis of lipids contributes only marginally to total lipids in milk. Therefore, the majority of milk lipids originate from the blubber and concentrations of vitamins A and E and PCBs per unit lipid in milk and blubber can be directly compared (Fig. 1). Throughout the lactation period, the concentration of vitamin E in adult females increased in blubber and decreased in milk. The concentration of vitamin E in the early stages of lactation was very high compared to that in blubber, suggesting mobilization from other organs. One source could be the liver, in which the concentration of vitamin E decreased by 60% during lactation (Schweigert, Luppertz and Stobo, in preparation). In the later stages of lactation, an incomplete mobilization was evident. This has also been observed in the guinea-pig (Machlin et al., 1979) and man (Schaefer et al., 1983). The vitamin A concentration also increased in blubber while decreasing in the milk of adult females. But, contrary to the status of vitamin E, at all stages of lactation the concentrations of vitamin A in milk were much lower than that in blubber. Thus an incomplete mobilization of vitamin A from blubber is also indicated. In pups, the level of vitamin A decreased throughout the lactation period in a
116
F. J. Schweigert and W. T. Stobo
manner similar to that observed in the milk. This coincident decrease indicates that pups are highly efficient in conserving the vitamin A from milk and storing it in blubber. The situation for vitamin E in the blubber of pups differs from that of vitamin A. Early in lactation, the amount of vitamin E being stored in the blubber is a small proportion of that observed in the milk but it increases throughout the lactation period. In the later stages of lactation, the amount of vitamin E being deposited in the blubber exceeds that being transferred via the milk. In conclusion, this study indicates that the fat-soluble vitamins (vitamins A and E), cholesterol and PCB composition of seal milk changes dramatically as lactation progresses. It also indicates that grey seals are similar to terrestrial mammals and man in the differences observed between colostrum and mature milk, thus indicating that the physiological processes are the same although the absolute quantities differ quite dramatically. This indicates that, in seals. two different mechanisms for the transfer of lipophilic components may also exist-one for the transfer of the triglycerides and possibly PCBs, and a separate one for other fat-soluble components such as vitamins and cholesterol. The higher concentrations in colostrum emphasize their importance in the nutrition of the newborn pup. Acknowledgements-We thank Brian Beck for his assistance in the field work and the age determination of the adult animals. Total lipids in milk were kindly determined by Dr Precht (Bundesforschungsanstalt fiir Milchforschung). Financial support for this study was provided by the Deutsche Forschungsgemeinschaft (Sch 373/l-l), the Miinchener Universitlts Gesellschaft, the Hanns Seidel Stiftung e. V.. Germany and the Department of Fisheries and Oceans, Canada.
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