Plasma and erythrocyte fatty acids in captive Asian

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Plasma and erythrocyte fatty acids in captive Asian (Elephas maximus) and African (Loxodonta africana) elephants M. Clauss, Y. Wang, K. Ghebremeskel, C. E. Lendl, W. J. Streich The fatty acid components of the plasma triglycerides and the phospholipid fractions of the red blood cells of a captive group of two African (Loxodonta africana) and four Asian (Elephas maximus) elephants were investigated. All the animals received the same diet of hay, fruits and vegetables, and concentrates. A comparison with data from free-ranging African elephants or Asian work-camp elephants showed that the captive elephants had lower proportions of polyunsaturated fatty acids (PUFAs), and for several lipid fractions a higher n-6:n-3 ratio, than their counterparts in the wild or under the more natural, in terms of diet, work-camp conditions. The difference in PUFA content was smaller in the African than in the Asian elephants. The captive Asian elephants tended to have lower levels of n-3 and total unsaturated fatty acids in their red blood cells than the captive African elephants.

ARACHIDONIC (20:4n-6) and docosahexaenoic (22:6n-3) acids, the major metabolites of the n-6 and n-3 fatty acid families, respectively, are vital components of cellular and subcellular membranes. Both acids play a prominent role in the control and regulation of crucial cellular functions. Dihomogammalinolenic (20:3n-6), arachidonic and eicosapentaenoic acid (20:5n-3) are precursors of the hormone-like eicosanoids. These are a complex group of biologically active compounds which are involved in the control and regulation of blood flow, cell-mediated immunity and inflammation, insulin release, and reproduction. Linoleic acid and α-linolenic acid, which are the parent compounds of the n-6 and n-3 fatty acid families, respectively, cannot be synthesised by animals and have to be obtained from the diet. However, animals can synthesise the various metabolites of these two fatty acids through a series of elongation and desaturation steps. Over the past five decades, there has been a large increase in the human consumption of n-6 fatty acids, and a concomitant decrease in the consumption of n-3 fatty acids, which has led to an imbalance of n-6:n-3 in cell membranes. Current evidence indicates that this imbalance may be associated with various chronic disorders, including diabetes, cardiovascular and cerebrovascular diseases, some types of cancers and allergic hypersensitivity (Simopoulos 1991, Okuyama and others 1997, Leonard 1999, Connor 2000, O’Keefe and Harris 2000). An imbalance of n-6:n-3 is also observed in animals reared on hay, grains and concentratebased feeds (Koizumi and others 1991, French and others 2000, Cordain and others 2002). Marchello and others (1996) reported an n-6:n-3 ratio of 4:1 in grass-fed bison and 21:1 in grain-fed bison. Exotic animals in zoological gardens, especially herbivores, are fed different foods from those they would consume in their natural habitat. In contrast with the plants eaten by wild herbivores, which are rich in n-3 fatty acids (Davidson 1998), the foods fed in zoological gardens are higher in saturates, monounsaturates and n-6 polyunsaturates, and are low in n3 polyunsaturates (Grant and others 2002). Signs of n-6 and n-3 imbalance have been observed in fish (Van Vliet and Katan 1990), reptiles (Noble and others 1993), birds (Simopoulos and Salem 1989, Noble and others 1996) and mammals (Crawford 1968, Crawford and others 1969, 1970, 1991, Miller and others 1986, Florant and others 1990, Koizumi and others 1991). No comprehensive data on the fatty acid status of captive and wild elephants are available, and it is therefore not known whether the diets fed to elephants in captivity have resulted in their having an abnormal ratio of fatty acids in their cell 54

membranes. It is possible that some of the vascular and skin disorders observed in elephants living outside their natural habitats could be due to changes in their membrane fatty acid composition. The aims of this study were to investigate the levels of n-6 and n-3 fatty acids in the plasma and red cell lipids of captive African and Asian elephants, and to compare the results with the data available in the literature (Moore and Sikes 1967, McCullagh 1973, Cmelik and Ley 1977, Sreekumar and Nirmalan 1990). MATERIALS AND METHODS Animals Two African and four Asian captive adult female elephants which were fed on a diet of grass hay, produce (mostly carrots and apples, with a variety of other fruits and vegetables) and concentrates (a commercial horse concentrate) were studied. The animals received the hay ad libitum throughout the day and were given one morning and one evening meal of produce and concentrates. The estimated food intake per animal per day was 60 kg grass hay, 10 kg concentrates and 10 kg produce. The African elephants were 17 and 19 years old and were both estimated to weigh 2000 kg; the Asian elephants ranged between 25 and 35 years old and had estimated bodyweights between 3053 and 3735 kg. Samples and analyses Blood samples were taken from an ear vein as part of a regular veterinary health check. Plasma and red blood cells (RBCs) were separated by centrifugation at 1000 g for 15 minutes at 4°C and stored at –80°C until analysis. The plasma and RBC total lipids were extracted by the method of Folch and others (1957). The samples were homogenised in chloroform and methanol (2:1 v/v) containing 0·01 per cent butylated hydroxytoluene (BHT) as an antioxidant under nitrogen. The phosphoglyceride classes were separated by thin-layer chromatography on silica gel plates by the use of chloroform/ methanol/acetic acid/formic acid/water (70:30:12:4:2) containing 0·01 per cent BHT. The developing solvents – petroleum spirit/ether/formic acid/methanol (85:15:2·5:1) containing 0·01 per cent BHT – were used for the separation of the triglycerides. Bands were detected by spraying with a methanolic solution of 2,7-dichlorofluorescein (0·01 per cent w/v) and identified by the use of standards as described by Ghebremeskel and others (1995). Fatty acid methyl esters were prepared by heating the lipid fractions with 4 ml 15 per cent acetyl chloride in methanol in The Veterinary Record, July 12, 2003

Veterinary Record (2003) 153, 54-58 M. Clauss, MSc, DrMedVet, Institute of Animal Physiology, Physiological Chemistry and Animal Nutrition, Veterinary Faculty, LudwigMaximilians University of Munich, Veterinärstrasse 13, D-80539 Munich, Germany Y. Wang, BSc, MPhil, K. Ghebremeskel, BSc, MSc, PhD, MIBiol, Institute of Brain Chemistry and Human Nutrition, University of North London, London N7 8DB

C. E. Lendl, DrMedVet, CertVA, MRCVS, Erben, Fitz and Partners Veterinary Clinic, Gessertshausen, Germany W. J. Streich, DrRerNat, Institute of Zoo Biology and Wildlife Research (IZW), Berlin, Germany

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TABLE 1: Percentage composition of serum/plasma triglycerides or total lipid fatty acid (FA) of elephants from different studies

Source Species

McCullagh (1973)

Cmelik and Ley (1977)

Sreekumar and Nirmalan This study (1990) This study

Loxodonta Loxodonta Loxodonta Loxodonta africana africana africana africana Free-ranging Free-ranging Free-ranging Captive Serum Serum Plasma Plasma

Habitat Sample Fraction Number 12:0 14:0 14:2 15:0 16:0 16:1 16:2 17:0 18:0 18:1 18:2n-6 18:3n-3 20:1 20:2n-6 20:3n-6 20:4n-6 22:6n-3 24:1 Saturated FAs Monounsaturated FAs PUFAs Saturated/ unsaturated FAs n-6:n-3 DBI

Moore and Sikes (1967)

TG

TG

TG

2 – 3·2 – – 43·1 2·2 – 0·7 8·6 38·8 0·6 2·8 – – – – – – 55·6

6 – 2·6 – – 40·5 2·6 – 2·4 10·2 31·9 5·2 0·2 – – 0·9 0·9 – – 55·7

5 – 4·4 1·2 – 30·5 4·0 2·3 1·3 6·4 42·3 3·1 3·8 – 0·5 – 0·2 – – 42·6

2 0·0 2·9* – – 42·8* 1·8 – – 12·6 33·0 1·8 1·5 0·0 0·0 0·0 0·0 0·0 – 58·2

Elephas maximus Work camp Plasma Total lipids 9 – 1·4 – 0·3 21·1 – – 1·2 11·2 16·3 25·6 5·5 0·3 0·5 – 8·8 – 0·9 33·9

41·0 3·4

34·5 7·2

46·2 11·1

34·8 3·3

16·8 40·0

TG

1·25 0·21 0·506

1·34 30·5 0·518

0·75 0·85 0·725

1·54 1·22 0·428

0·62 6·44 1·199

Elephas maximus Captive Plasma

24·8 4·3

TG

4 0·4 4·4* – – 52·5* 1·6 – – 10·0 23·2 3·0 1·3 0·0 0·0 0·0 0·0 0·0 – 67·3

2·48 2·43 0·346

* Statistically significant difference between the results for the African and Asian elephants in this study PUFA Polyunsaturated fatty acid, DBI Double bond index, TG Triglyceride

a sealed vial at 70°C for three hours under nitrogen. The esters were separated by gas-liquid chromatography (HRGC MEGA 2 Series; Fisons Instruments) equipped with a flame ionisation detector and fitted with a capillary column (25 m x 0·32 mm internal diameter, 0·25 µm film, BP20). Hydrogen was used as the carrier gas, and the injector, oven and detector temperatures were 250, 200 and 280°C, respectively. The fatty acid methyl esters were identified by comparing their retention times with those of authentic standards, and by the calculation of equivalent chain-length values. Peak areas were quantified by a computer chromatography data system (EZChrom Chromatography Data System; Scientific Software). The data are presented as means (sd). As comparative measurements, the ratios of saturated:unsaturated fatty acids and n-6:n-3 PUFAs, and the double bond index, were calculated. For each variable, the African and Asian elephants were compared by a t test. In addition, the retrospective power of the t test for variables with large differences between the two species was calculated. The calculations were made by using SPSS 9.0 (SPSS) and Power and Precision 2.1 (Biostat) software. RESULTS The African elephants had a total lipid content in plasma of 1·30 to 1·90 g/litre and in RBCs of 2·10 to 3·70 g/litre; in the Asian elephants the respective averages were 1·22 (0·28) g/litre and 2·60 (0·90) g/litre. The fatty acid composition of the plasma triglycerides is shown in Table 1 with comparative literature data, and the fatty acid composition of the choline and ethanolamine phospholipid fraction of the RBCs is shown in Table 2. The captive elephants had lower proportions of PUFAs, and, for several lipid fractions, a higher n-6:n-3 ratio. The Veterinary Record, July 12, 2003

The difference in PUFA content was smaller in the African than in the Asian elephants. The captive Asian elephants tended to have lower levels of n-3 fatty acids and total unsaturated fatty acids, and for the ethanolamine phospholipid fraction of the RBCs the difference between the saturated:unsaturated fatty acids ratios was significant. Other significant differences between the two elephant species are shown in the tables. DISCUSSION Owing to the small sample size, this study must be considered as a pilot investigation. With the exception of 20:5n-3, the power of discrimination for the variables with considerable mean differences (at least 30 per cent) but non-significant t tests ranged between 10 and 40 per cent. The non-significant results may therefore have been due to the small sample size. Together with a similar study of even smaller numbers of samples of spermatozoa (Swain and Miller 2000) and a reference to liver tissue in a footnote in Crawford and others (1991), these data constitute the only information available on the fatty acid composition of the plasma and RBCs of captive elephants. The data can be compared with other investigations of freeranging African elephants. For the Asian elephants, the plasma triglyceride composition was compared with the total plasma lipid composition studied by Sreekumar and Nirmalan (1990); the triglycerides constitute the majority of the total plasma lipids, and this comparison therefore seems valid. The accuracy of the analyses of fatty acids in this and the earlier studies may be expected to differ; however, fatty acid data are generally expressed as percentages of the total fatty acids measured, and are thus less susceptible to changes in the sensitivity of the analytical methods than are absolute concentration data. The Asian elephants had much lower levels of unsaturated fatty acids. The difference was most pronounced in the n-6 acids, resulting in a lower n-6:n-3 plasma ratio in the captive group, in contrast with the pattern usually observed in zoo animals. The African elephants were generally lower in unsaturated fatty acids than samples from free-ranging elephants, but the difference was not as great. Apart from the serum levels observed by Moore and Sikes (1967), the captive African elephants had higher n-6:n-3 ratios, even for comparable phospholipid fractions, in their RBCs than the ratios in the samples of serum/plasma from other studies. In general, therefore, the usually observed differences in fatty acid composition between free-ranging and captive wild animals were also observed in these elephants. Similarly, Crawford and others (1991) found that the percentage of n-3 PUFAs in liver cell membranes was 4·3 per cent in free-ranging African elephants but only 0·3 per cent in captive animals. The lower content of unsaturated fatty acids in the freeranging African elephants than in the Asian work-camp elephants is probably due to the more arid conditions of the African habitat. McCullagh (1973) questioned whether African elephants were deficient in essential fatty acids. Anatomical studies of African elephants living in different habitats in East Africa have shown that stress and deterioration of the vegetation have had a marked effect on the prevalence of arterial disease (Sikes 1968a, b, 1969). Comprehensive studies by McCullagh and Lewis (1967) and McCullagh (1969a, b, 1975) confirmed these findings: elephants forced to live in national parks consisting mainly of open grassland had a high prevalence of arterial disease, whereas animals living in forest areas were virtually free from it. Analyses of their diets have shown that the vegetation in the forests provides considerably larger quantities of PUFAs than the grassland vegetation (McCullagh 1972, 1973). Atherosclerosis was also recorded by Dilman and Carr (1970) in free-ranging African elephants, but there have been no comparable studies on atherosclerosis in Asian elephants. 55

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TABLE 2: Percentage composition of plasma or red blood cell (RBC) phospholipid (PG) or choline (CPG) and ethanolamine phospholipid (EPG) and of the spermatozoal membranes of elephants

Source Species

Moore and Sikes (1967)


Loxodonta Loxodonta Loxodonta africana africana africana Free-ranging Free-ranging Free-ranging Serum Plasma Plasma

Habitat Sample Fraction Number 12:0 14:0 14:1 14:2 15:0 16:0 16:1 16:2 17:0 18:0 18:1 18:2n-6 18:3n-6 18:3n-3 20:0 20:1 20:2n-6 20:3n-6 20:4n-6 20:5n-3 22:2n-9 22:3n-6 22:4n-6 22:4n-3 22:5n-6 22:5n-3 22:6n-3 24:0 Saturated FAs Monounsaturated FAs PUFAs Saturated/ unsaturated FAs n-6:n-3 DBI


This study

This study

This study This study

Loxodonta africana Captive

Loxodonta africana Captive

Elephas maximus Captive

Elephas maximus Captive

PG

CPG

EPG

RBC CPG

RBC EPG

RBC CPG

RBC EPG

6 – 0·9 – – – 15·4 0·9 – 1·1 32·6 12·2 12·3 – 0·7 1·4 1·4 1·2 6·2 6·3 – – – – 8·0 – 4·0 0·8 – 51·4

5 – 2·5 – 0·1 – 14·1 – 2·7 – 49·4 10·1 8·1 0·8 2·8 – – 0·7 2·7 1·9 – 3·3 – – – – – 0·6 – 66·1

3 – 3·8 0·5 – 0·1 18·7 0·4 1·8 – 23·8 15·1 5·6 5·2 – 0·5 – 3·5 0·5 6·1 – 6·0 – – – – 2·3 1·3 – 47·0

2 – 0·0 – – – 14·9* 1·0 – – 31·4 13·8 13·2 0·0 1·2 – 0·0 0·6 6·4* 8·3* 1·1* – – 0·7 – 0·0 2·4* 0·0 0·0 46·2

2 – 0·0 – – – 10·9 2·0* – – 9·7* 44·6* 9·8* 0·0 1·5 – 0·7 0·0 2·3 7·7* 1·8 – – 0·0 – 0·0 1·0 0·0 0·0 20·6*

4 – 0·5 – – – 19·9* 0·7 – – 31·9 12·8 16·7 0·0 1·4 – 0·1 0·8 3·8* 5·4* 0·3* – – 0·4 – 0·0 1·1* 0·0 0·4 52·7

4 – 0·0 – – – 12·8 1·4* – – 12·1* 39·7* 15·5* 0·0 2·2 – 0·6 0·1 2·0 5·1* 1·0 – – 0·0 – 0·0 1·0 0·0 0·0 24·8*

14·5 39·5

10·1 23·8

16·1 33·0

14·8 33·9

47·2* 24·1

13·6 29·9

41·8* 26·9

0·95 1·93 1·442

2·25 4·18 0·702

0·96 5·81 1·130

0·96 6·21 1·179

0·29* 4·60 1·229

1·25 9·68 0·941

0·36* 5·40 1·156

Swain and Miller (2000)

Swain and Miller (2000)

Loxodonta Elephas africana maximum Captive Captive Spermatozoa Spermatozoa – – 3 2 5·0 3·1 3·9 12·0 0·5 0·4 – – – – 12·5 16·1 0·1 0·6 – – – – 1·8 3·9 3·1 6·9 0·9 1·7 0·0 0·0 – – – – – – – – – – 1·3 1·9 – – – – 0·2 0·7 1·7 5·0 – – 1·8 0·0 – – 68·1 42·9 – – 23·2 35·0 3·6 73·9 0·30 0·09 4·353

7·9 52·2 0·49 0·22 2·986

* Statistically significant difference between the results for the African and Asian elephants in this study PUFA Polyunsaturated fatty acid, DBI Double bond index

Fatty acids are selectively allocated to different body tissues (Crawford and others 1976). From the data on captive elephants it can be assumed, for example, that long-chain PUFAs are selectively allocated to spermatozoa (Table 2), and a similar priority for brain and neural tissue can be postulated. The composition of the elephant’s brain resembles that of other animals in its high content of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids (Crawford and others 1976), and their large brain – Crile and Quiring (1940) reported 5·7 kg of brain tissue in a 6700 kg African elephant – would require large amounts of long-chain PUFAs for brain tissue alone. Accordingly, these fatty acids would need to be spared in other tissues, and this sparing could explain the absence of a high proportion of long-chain PUFAs in the plasma and RBC fractions measured (Tables 1 and 2) and in elephant milk. The milk of elephants contains large proportions of capric acid (10:0) and only small amounts of longerchain fatty acids (McCullagh and Widdowson 1970, Peters and others 1972). Whether this should be interpreted as a response to the special requirements of elephant neonates (Sheldrick 1990) or a response to a limited supply of PUFAs from the natural diet is uncertain. For other mammals, PUFAs are essential for the development of newborn animals (Simopoulos 1991), and especially for the brain. The concentration of capric acid in elephant milk increases, and that of longer chain fatty acids decreases, throughout lactation 56

(McCullagh and Widdowson 1970). Few other mammals have to give milk to their offspring for as long as the elephant does, and the supply of PUFAs may be specially controlled so that their concentration in the milk can gradually be reduced as the offspring grows. Swain and Miller (2000) found a difference in the fatty acid composition of the membrane of the spermatozoa of elephants of different species, African elephants having higher levels of PUFAs in general and n-3 fatty acids in particular. The animals used in their study were kept at different facilities, and their feeding regimens were not documented. In this study, a similar difference between the species can be detected in the content of unsaturated fatty acids and the n-6:n-3 ratio in all the lipid fractions investigated (Tables 1, 2), although differences in the feeding patterns of the animals – which all received the same diet – seemed unlikely. In a comparative study of the digestive physiology of captive Asian and African elephants, Hackenberger (1987) observed that African elephants had a significantly faster passage rate. The presence of bacteria in the small intestine (Eloff and Van Hoven 1980, Van Hoven and others 1981) coupled with a longer retention time of digesta in this compartment might therefore result in slightly more fatty acid hydrolysis, and hence desaturation, before absorption, and reduce the relative availability of unsaturated fatty acids in Asian elephants on comparable diets. The Veterinary Record, July 12, 2003

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A deficiency of PUFAs may be of relevance in captive elephants, because they have repeatedly been observed to suffer from atherosclerosis (Lindsay and others 1956, Ratcliffe and Cronin 1958, Vastesaeger and Delcourt 1961, Finlayson 1965, Mikota and others 1994). In addition, a deficiency of PUFAs could have an effect on their reproductive performance. Up to now, it has not been possible to cryopreserve sperm from Asian elephants successfully (O’Brian and others 1998). Essential fatty acid deficiency has been shown to impair the development of spermatozoa (Alfin-Slater and Bernick 1958, Whorton and Coniglio 1977, Marzouki and Coniglio 1982, MacDonald and others 1984). Mammalian and avian sperm contain large proportions of PUFAs (Scott 1973, Parks and Lynch 1992), and the PUFA composition of sperm is, to a certain extent, influenced by the PUFA composition of the diet (Paulenz and others 1995, Blesbois and others 1997, Kelso and others 1997a). Sperm of low fertility is often characterised by a high n-6:n-3 ratio, but sperm of higher quality generally has higher PUFA levels (Cerolini and others 1997) and a lower n6:n-3 ratio, that is, it contains more n-3 PUFAs (Nissen and others 1981, Nissen and Kreysel 1983, Sebastian and others 1987, Blesbois and others 1997, Connor and others 1997, Kelso and others 1997b, c, Zalata and others 1998, Cerolini and others 2000). It has been shown that feeding a n-3 PUFArich diet can improve sperm quality and fertility in fowl (Blesbois and others 1997) and men (Kiriakova and others 1998).These relationships suggest that the fatty acid composition of elephants should be investigated more thoroughly.

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sition of nine plants consumed by two African herbivores. Lipids 33, 109-113 DILMAN, J. S. & CARR, W. R. (1970) Observations on arteriosclerosis, serum cholesterol and serum electrolytes in the wild African elephant. Journal of Comparative Pathology 80, 81-87 ELOFF, A. K. & VAN HOVEN, W. (1980) Intestinal protozoa of the African elephant. South African Journal of Zoology 15, 83-90 FINLAYSON, R. (1965) Spontaneous arterial disease in exotic animals. Journal of Zoology (London) 147, 239-343 FLORANT, G. L., NUTTLE, L. C., MAULLINEX, D. E. & RINTOUL, D. A. (1990) Plasma and white adipose tissue lipid compositions in marmots. American Journal of Physiology 258, R1123-R1131 FOLCH, J., LEES, M. & SLOAN-STANLEY, G. H. (1957) A simple method for isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497-509 FRENCH, P., STANTON, C., LAWLESS, F., O’RIORDAN, E. G., MONHAN, F. J., CAFFREY, P. J. & MOLONEY, A. P. (2000) Fatty acid composition, including conjugated linoleic acid, of intramuscular fat from steers offered grazed grass, grass silage, or concentrate-based diets. Journal of Animal Science 78, 2849-2855 GHEBREMESKEL, K., LEIGHFIELD, M., LEAF, A., COSTELOE, K. & CRAWFORD, M. A. (1995) Fatty acid composition of plasma and red cell phospholipids of preterm babies fed on breast milk and formulae. European Journal of Pediatrics 154, 46-52 GRANT, J. B., BROWN, D. L. & DIERENFELD, E. S. (2002) Essential fatty acid profiles differ across diets and browse of black rhinoceros. Journal of Wildlife Diseases 38, 132-142 HACKENBERGER, M. K. (1987) Diet digestibilities and ingesta transit times of captive Asian and African elephants. MS Thesis. Guelph, Canada, University of Guelph KELSO, K. A., CEROLINI, S., NOBLE, R. C., SPARKS, N. H. C. & SPEAKE, B. K. (1997a) The effects of dietary supplementation with docosahexaenoic acid on the phospholipid fatty acid composition of avian spermatozoa. Comparative Biochemistry and Physiology 118B, 65-69 KELSO, K. A., CEROLINI, S., SPEAKE, B. K., CAVALCHINI, L. G. & NOBLE, R. C. (1997b) Effects of dietary supplementation with alpha-linolenic acid on the phospholipid fatty acid composition and quality of spermatozoa in cockerels from 24 to 72 weeks of age. Journal of Reproduction and Fertility 110, 53-59 KELSO, K. A., REDPATH, A., NOBLE, R. C. & SPEAKE, B. K. (1997c) Lipid and antioxidant changes in spermatozoa and seminal plasma throughout the reproductive period of bulls. Journal of Reproduction and Fertility 109, 1-6 KIRIAKOVA, N., KIRIAKOV, A., SCHNEIDER, E. & BONEV, A. (1998) Therapeutic effect of essential phospholipids on functional sexual disorders in males. Journal of the European Academy of Dermatology and Venereology 11, 191-193 KOIZUMI, I., SUZUKI, Y. & KANEKO, J. J. (1991) Studies of the fatty acid composition of intramuscular lipids of cattle, pigs and birds. Journal of Nutritional Science and Vitaminology 37, 545-554 LEONARD, E. C. (1999) Dietary fatty acids: effect of n:6/n-3 balance in human nutrition and health. Lipid Technology 11, 110-114 LINDSAY, S., SKAHEN, R. & CHAIKOFF, I. L. (1956) Arteriosclerosis in an elephant. Archives of Pathology 61, 207-218 MCCULLAGH, K. G. (1969a) The growth and nutrition of the African elephant. I. Seasonal variations in the rate of growth and the urinary excretion of hydroxyproline. East African Wildlife Journal 7, 85-90 MCCULLAGH, K. G. (1969b) The growth and nutrition of the African elephant. II. The chemical nature of the diet. East African Wildlife Journal 7, 91-97 MCCULLAGH, K. G. (1972) Arteriosclerosis in the African elephant. I. Intimal atherosclerosis and its possible causes. Atherosclerosis 16, 307-335 MCCULLAGH, K. G. (1973) Are African elephants deficient in essential fatty acids? Nature 242, 267-268 MCCULLAGH, K. G. (1975) Arteriosclerosis in the African elephant. II. Medial sclerosis. Atherosclerosis 21, 37-59 MCCULLAGH, K. G. & LEWIS, M. G. (1967) Spontaneous arteriosclerosis in the wild African elephant. Its relation to the disease in man. Lancet ii, 492-495 MCCULLAGH, K. G. & WIDDOWSON, E. M. (1970) The milk of the African elephant. British Journal of Nutrition 24, 109-117 MACDONALD, M. L., ROGERS, Q. R., MORRIS, J. G. & CUPPS, P. T. (1984) Effects of linoleate and arachidonate deficiencies on reproduction and spermatogenesis in the cat. Journal of Nutrition 114, 719-726 MARCHELLO, M. J., HADLEY, M., SLANG, E. R., MILNE, D. B. & DRISKELL, J. A. (1996) Nutrient composition of fed bison. Bison World 31, 27-32 MARZOUKI, Z. M. H. & CONIGLIO, J. G. (1982) Effect of essential fatty acid deficiency on lipids of rat sertoli and germinal cells. Biology of Reproduction 27, 312-315 MIKOTA, S. K., SARGENT, E. L. & RANGLACK, G. S. (1994) The cardiovascular system. In Medical Management of the Elephant. West Bloomfield, Indira Publishing House. pp 107-118 MILLER, G. J., FIELD, R. A., RILEY, M. L. & WILLIAMS, J. C. (1986) Lipids in

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wild ruminant animals and steers. Journal of Food Quality 9, 331-343 MOORE, J. H. & SIKES, S. K. (1967) The serum and adrenal lipids of the African elephant. Comparative Biochemistry and Physiology 20, 779-792 NISSEN, H. P. & KREYSEL, H. W. (1983) Polyunsaturated fatty acids in relation to sperm motility. Andrologia 15, 264-269 NISSEN, H. P., KREYSEL, H. W. & SCHIRREN, C. (1981) Composition of the lipid-bound fatty acids of human semen in relation to its fertility values. Andrologia 13, 444-451 NOBLE, R. C., MCCARTNEY, R. & FERGUSON, M. W. (1993) Lipid and fatty acid compositional differences between eggs of wild and captive-breeding alligators: an association with reduced hatchability? Journal of Zoology (London) 230, 639-649 NOBLE, R. C., SPEAKE, B. K., MCCARTNEY, R., FOGGIN, C. M. & DEEMING, D. C. (1996) Yolk lipids and their fatty acids in the wild and captive ostrich. Comparative Biochemistry and Physiology 113B, 753-756 O’BRIAN, E., HOFFMAN, S., SCHAWANG, T., SUEDMEYER, W. K., EASTON, E., SCHMIDT, D. & LOSKUTOFF, N. (1998) An update to improve the shortand long-term storage of elephant semen. Proceedings of the Third International Elephant Research Symposium. Springfield, USA, June 6 to 7, 1998. p 7 O’KEEFE, J. H. Jr & HARRIS, W. S. (2000) From Inuit to implementation: omega-fatty acids come of age. Mayo Clinic Proceedings 75, 607-614 OKUYAMA, H., KOBAYASHI, T. & WATANABE, S. (1997) Dietary fatty acids – the n-6/n-3 balance and chronic elderly diseases. Excess linolenic acid and relative n3 deficiency syndrome seen in Japan. Progress in Lipid Research 35, 409-457 PARKS, J. E. & LYNCH, D. V. (1992) Lipid composition and thermotropic phase behavior of boar, bull, stallion and rooster sperm membranes. Cryobiology 29, 255-266 PAULENZ, H., TAUGBOL, O., HOFMO, P. O. & SAAREM, K. (1995) A preliminary study on the effect of dietary supplementation with cod liver oil on the polyunsaturated fatty acid composition of boar semen. Veterinary Research Communications 19, 273-284 PETERS, J. M., MAIER, R., HAWTHORNE, B. E. & STORVIK, C. A. (1972) Composition and nutrient content of elephant milk. Journal of Mammalogy 53, 717-724 RATCLIFFE, H. L. & CRONIN, M. T. (1958) Changing frequency of arteriosclerosis in mammals and birds at the Philadelphia zoological gardens.

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Circulation 18, 41-52 SCOTT, J. W. (1973) Lipid metabolism of spermatozoa. Journal of Reproduction and Fertility Supplement 18, 65-76 SEBASTIAN, S. M., SELVARAJ, S., ARULDHAS, M. M. & GOVINDARAJULU, P. (1987) Pattern of neutral and phospholipids in the semen of normospermic, oligospermic and azoospermic men. Journal of Reproduction and Fertility 79, 373-378 SHELDRICK, D. (1990) Raising baby orphaned elephants: part II. Swara 13, 23-31 SIKES, S. K. (1968a) Observations on the ecology of arterial disease in the African elephant in Kenya and Uganda. Proceedings of the Symposium of the Zoological Society of London 21, 251-273 SIKES, S. K. (1968b) Habitat stress and arterial disease in elephants. Oryx 9, 286-292 SIKES, S. K. (1969) Habitat and cardiovascular diseases. Observations made on elephants and other free-living animals in East Africa. Transactions of the Zoological Society of London 32B, 1-104 SIMOPOULOS, A. P. (1991) Omega-3 fatty acids in health and disease and in growth and development. American Journal of Clinical Nutrition 54, 438-463 SIMOPOULOS, A. P. & SALEM, N., Jr (1989) n-3 fatty acids in eggs from rangefed Greek chickens. New England Journal of Medicine 321, 1412 SREEKUMAR, K. P. & NIRMALAN, G. (1990) The fatty acid composition of plasma lipids in the Indian elephant (Elephas maximus). Veterinary Research Communications 14, 427-431 SWAIN, J. E. & MILLER, R. R. (2000) A post-cryogenic comparison of membrane fatty acids of elephant spermatozoa. Zoo Biology 19, 461-473 VAN HOVEN, W., PRINS, R. A. & LANKHORST, A. (1981) Fermentative digestion in the African elephant. South African Journal of Wildlife Research 11, 78-86 VAN VLIET, T. & KATAN, M. B. (1990) Lower ratio of n-3 to n-6 fatty acids in cultured than in wild fish. American Journal of Clinical Nutrition 51, 1-2 VASTESAEGER, M. M. & DELCOURT, R. (1961) Spontaneous atherosclerosis and diet in captive animals. Nutritio et Dieta 3, 174-188 WHORTON, A. R. & CONIGLIO, J. G. (1977) Fatty acid synthesis in testes of fat-deficient and fat-supplemented rats. Journal of Nutrition 107, 79-86 ZALATA, A. A., CHRISTOPH, A. B., DEPUYDT, C. E., SCHOONJANS, F. & COMHAIRE, F. H. (1998) The fatty acid composition of phospholipids of spermatozoa from infertile patients. Molecular Human Reproduction 4, 111-118

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