The Fat from Frozen Mammals Reveals Sources of Essential Fatty Acids Suitable for Palaeolithic and Neolithic Humans

Abstract

The elucidation of the sources of n-3 fatty acids available for the humans in the Upper Palaeolithic and Neolithic is highly relevant in order to ascertain the availability of such nutrients in that time frame as well as to draw useful conclusions about healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen mammals found in the permafrost of Siberia (Russia). A total of 6 specimens were included in this study: 2 mammoths, i.e. baby female calf called "Lyuba" and a juvenile female called "Yuka", both specimens approximately from the same time, i.e. Karginian Interstadial (41,000 and 34,000 years BP); two adult horses from the middle Holocene (4,600 and 4,400 years BP); and two bison very close to the Early Holocene (8,200 and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass spectrometry (GLC-MS) and GLC-flame ionization detector (GLC-FID). As demonstrated in this work, the fat of single-stomached mammals often consumed by Palaeolithic/Neolithic hunters contained suitable amounts of n-3 and n-6 fatty acids, possibly in quantities sufficient to meet the today's recommended daily intake for good health. Moreover, the results also suggest that mammoths and horses at that time were hibernators.

Full text

The Fat from Frozen Mammals Reveals Sources of
Essential Fatty Acids Suitable for Palaeolithic and
Neolithic Humans
Jose
´L. Guil-Guerrero
1
*, Alexei Tikhonov
2
, Ignacio Rodrı
´guez-Garcı
´a
3
, Albert Protopopov
4
,
Semyon Grigoriev
5
, Rebeca P. Ramos-Bueno
1
1Food Technology Division, CeiA3, University of Almerı
´a, Almerı
´a, Spain, 2Zoological Institute, Russian Academy of Sciences, Saint-Petersburg, Russian Federation,
3Organic Chemistry Division, CeiA3, University of Almerı
´a, Almerı
´a, Spain, 4Department of Mammoth Faunal Studies, Sakha (Yakutia) Republic Academy of Sciences,
Yakutsk, Russian Federation, 5Yakutsk Scientific Research Institute of Applied Ecology of the North, North-Eastern Federal University, Yakutsk, Russian Federation
Abstract
The elucidation of the sources of n-3 fatty acids available for the humans in the Upper Palaeolithic and Neolithic is highly
relevant in order to ascertain the availability of such nutrients in that time frame as well as to draw useful conclusions about
healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen mammals found in the
permafrost of Siberia (Russia). A total of 6 specimens were included in this study: 2 mammoths, i.e. baby female calf called
‘‘Lyuba’’ and a juvenile female called ‘‘Yuka’’, both specimens approximately from the same time, i.e. Karginian Interstadial
(41,000 and 34,000 years BP); two adult horses from the middle Holocene (4,600 and 4,400 years BP); and two bison very
close to the Early Holocene (8,200 and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass
spectrometry (GLC-MS) and GLC-flame ionization detector (GLC-FID). As demonstrated in this work, the fat of single-
stomached mammals often consumed by Palaeolithic/Neolithic hunters contained suitable amounts of n-3 and n-6 fatty
acids, possibly in quantities sufficient to meet the today’s recommended daily intake for good health. Moreover, the results
also suggest that mammoths and horses at that time were hibernators.
Citation: Guil-Guerrero JL, Tikhonov A, Rodrı
´guez-Garcı
´a I, Protopopov A, Grigoriev S, et al. (2014) The Fat from Frozen Mammals Reveals Sources of Essential
Fatty Acids Suitable for Palaeolithic and Neolithic Humans. PLoS ONE 9(1): e84480. doi:10.1371/journal.pone.0084480
Editor: Wolf-Hagen Schunck, Max Delbrueck Center for Molecular Medicine, Germany
Received July 21, 2013; Accepted November 14, 2013; Published January 8, 2014
Copyright: ß2014 Guil-Guerrero et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors acknowledge the funding received to perform this work by the ‘‘Plan Propio de Investigacio
´n’’ of the University of Almerı
´a, made to the
Research Group ‘‘Chemistry of Biomolecules and Food Processing (FQM-010)’’. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jlguil@ual.es
Introduction
The intake of n-3 (omega-3) fatty acids (FAs) during the
Palaeolithic has been recently studied in terms of the n-6:n-3 ratio
in the human diet, with an increase in this ratio being observed in
the present day. This fact is of particular relevance, because an
understanding of ancestral human experience over evolution may
provide key knowledge on human dietary needs to maintain and
improve health and avoid chronic illnesses [1], [2]. Moreover, the
sources of n-3 available to Palaeolithic humans have been subject
to controversy [3]; thus the elucidation of the sources of n-3 FAs
that may have fed Palaeolithic humans in critical evolutionary
stages can help clarify human evolution.
With its low plant-animal subsistence ratio for northern hunter-
gatherers, the Palaeolithic diet was probably dominated by animal
foods, especially during the cold season when plant resources
decline [4]. Among Palaeolithic animals, woolly mammoths
(Mammuthus primigenius Blumenbach, 1799), were good options for
human consumption. Mammoths were monogastric herbivores,
having a digestive physiology and diet similar to that of the woolly
rhinoceros and the horse [5]. Although the consumption of
mammoths by Palaeolithic humans has been controversial in
comparison with mass hunting of species such as horses and bison,
several authors have found evidence of this dietary behaviour, as
has been deduced from Valea-Morilor excavations [6]. Probably,
the hunting of mammoths was possible by tracking such
pachyderms; Palaeolithic hunters could have caused stampedes
as a hunting strategy, and attacked animals that could not
maintain keep up with the herd. Mainly calves and some older
individuals could have been killed in this way, thereby providing
significant amounts of fat to ancestral hunters [7]. Moreover,
through combined climate and population models, it has been
shown that mammoth hunting pressure was clearly involved in the
extinction of this pachyderm [8], and evidence of hunting with
stone weapons during the Palaeolithic has been found [9], [10],
[11]. The prey or scavenged products of mammoths would have
been dragged or carried to the caves where Palaeolithic humans
lived, as shown in the cave of Spy, which yields almost 10,000
remains from Ice Age mammals, the most frequent species being
horse, cave hyena, and woolly mammoth [7].
Some of the present authors have previously described the
presence of thick layers of subcutaneous fat and even humps on
the neck in mummified carcasses of the mammoths found in the
permafrost of Siberia (Russia) [12], [13]. The use of the mammoth
fat, which is a large organ rich in energy, could have provided
substantial benefits to ancestral hunters. That is, one medium-
PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e84480

sized mammoth could have nourished a group of 50 humans
(either Cro-Magnon or Neanderthal) for at least 3 months [14],
while at the low temperatures in which they lived, would facilitate
the conservation of the carcasses. Furthermore, by eating the fat of
mammoths, Palaeolithic humans could have obtained clean, low-
protein energy for several days.
This paper reports on the FA profiles of the fat of some animals
from the Ice Age to the Neolithic, discussing the possibility of fat
use as an n-3 source for such hunters. Also, the possibility that
some of these mammals were hibernating is discussed, as suggested
by some of the FAs found.
Materials and Methods
Samples
Permission was received to examine the relevant specimens
from museum collections. Samples from the frozen mummies were
donated by the Salekhard Museum, the Museum of the Mammoth
of North-Eastern Federal University, and the Academy of Sciences
of the Yakutia Republic in Yakutsk (Russia). A total of 6 specimens
were included in this study (Table 1): 2 mammoths, i.e. a female
calf called ‘‘Lyuba’’ from Yamal Peninsula (north-western Siberia)
and a young female called ‘‘Yuka’’ from the banks of Laptev strait
(north-eastern Siberia), both specimens approximately from the
same time period (Karginian Interstadial, 41,000 and 34,000 years
BP, respectively). Two adult horses from Yakutia, one (horse
Yukagir) from the same place as Yuka and the other one (horse
Batagay) from the locality near settlement Batagay in the middle
stream of the Yana River. Both carcasses were surprisingly of the
same approximate age, from the middle Holocene (4,600 and
4,400 years BP). Two bison, i.e. a baby bison from Batagay (bison
‘‘Batagay’’) and a complete body of an adult male from the same
region as Yuka and the Yukagir horse (bison Yukagir), were both
again very close in time, from the Early Holocene (8,200 and
9,300 years BP).
The dried and conserved baby mammoth Lyuba is currently on
display at the Salekhard Museum, while the other individuals are
kept in the freezers of Museum of the Mammoth of North-Eastern
Federal University and Academy of Sciences of Yakutia Republic
in Yakutsk. The samples were kept in the freezers of Zoological
Institute Russian Academy of Sciences in Saint Petersburg. Sterile
conditions were maintained during the dissection procedures and
using a special drill on the frozen carcasses. During the dissection,
fat from Lyuba was taken from four different positions, including
the hump on the neck. The samples from the other specimens
were taken from the layers under the skin in the best preserved
areas (Table 1).
Oil extraction and transesterification
Simultaneous oil extraction and transesterification was done
according to previous works [15]. From each sample, 50 mg were
weighed in test tubes and n-hexane (1 mL) was added to each one.
FA methyl esters (FAMEs) were obtained after adding 1 mL of the
methylation mixture, which was composed by methanol:acetyl
chloride (20:1 v/v), and then heated at 100uC for 10 min. After
cooling at room temperature, 1 mL of distilled water was added in
each tube, after which the tubes were centrifuged at 3500 rpm for
5 min. The Upper hexane layer was removed for gas-liquid
chromatography (GLC) analyses.
GLC Analyses
Firstly, FAMEs were analysed by using a Focus GLC (Thermo
Electron, Cambridge, UK) equipped with Flame Injection
Detector (FID) and a Omegawax 250 capillary column
(30 m60.25 mm i.d. 60.25 mm film thickness; Supelco, Belle-
fonte, PA, USA). The temperature programme was: 1 min at
90uC, heating until 200uC at a rate of 10uC/min, constant
temperature at 200uC (3 min), heating until 260uC at a rate of
6uC/min and constant temperature at 260uC (5 min). The injector
temperature was 250uC with a split ratio 50:1. The injection
volume was 4 mL and the detector temperature was 260uC.
Nitrogen was used as the carrier gas (1 mL/min) and peaks were
identified by retention times determined for known FAME
standards (PUFAs No. 1 from Sigma, St. Louis, USA), while FA
contents were estimated by using methyl pentadecanoate (17:0) as
internal standard.
All samples were subjected to a second round of analyses by
GLC-mass spectrometry (GLC-MS) at the Scientific Instrumen-
tation Centre of the University of Granada (Spain). Samples were
injected (2 ml) into an Agilent 7890A gas chromatographer with an
apolar column in split mode, coupled with a Quattro micro GLC
mass spectrophotometer (Waters, UK), with a positive electron
impact source (70 eV) and full scan spectra acquisition. All FAs
were detected and quantified by comparison of retention times
and mass spectra with external standards, which were run at three
different concentrations. The full dataset of the GLC-MS analysis
is available upon request.
Experiments for all samples were conducted at least in triplicate.
Results are expressed as mean value 6S.D in Tables 2 and 3.
Table 1. Sample characteristics of frozen mammals.
Sample code Animal Organ Years BP
MY Juvenile mammoth ‘‘Yuka’’ Fat from left hind leg 34,300
ML1 Baby mammoth ‘‘Lyuba’’ Fat from intestines 41,000
ML2 Baby mammoth ‘‘Lyuba’’ Fat from hump 41,000
ML3 Baby mammoth ‘‘Lyuba’’ Fat from abdominal wall 41,000
ML4 Baby mammoth ‘‘Lyuba’’ Fat under the skin of belly 41,000
HY Horse ‘‘Yukagir’’ Fat from hind leg 4,600
HB Horse ‘‘Batagay’’ Fat from hind leg 4,400
BY Bison ‘‘Yukagir’’ Fat under the skin on the belly 9,300
BB Baby bison ‘‘Batagay’’ Fat under the skin on the belly 8,200
doi:10.1371/journal.pone.0084480.t001
Fatty Acids from Frozen Mammals
PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e84480

Results and Discussion
The animals sampled in this study are representative of those
eaten by Upper Palaeolithic and Neolithic hunters [4], [7], [11],
[14]. Two species, mammoths and horses, were monogastric,
while bison was a ruminant; thus, differences were expected in the
FA profiles of their fat, because monogastric animals can to a
larger extent assimilate most FAs from the food they eat [16]. A
detailed analysis of the content of intestinal samples from a one-
month-old female Siberian mammoth calf Lyuba, revealed the
presence of large amounts of herbaceous plants, e.g. species
belonging to Cyperaceae, Asteraceae, Salicaceae, Chenopodia-
ceae, Poaceae [17]. Some of these plant species are known to be
good sources of polyunsaturated FAs (PUFAs) belonging to the n-3
series [18]. Therefore, the subcutaneous fat of mammoths should
include suitable concentrations of a-linolenic acid (ALA, 18:3n-3),
which is the major FA in leaf tissues, and other n-3 FAs.
We hypothesised that the fat of monogastric animals constituted
a raw source of n-3 and n-6 FAs for Upper Palaeolithic and
Neolithic people. For this, we analysed the fat from various body
parts of the mammoth Lyuba, and the juvenile female Yuka, two
horses, and two bison (Table 1). Lyuba samples were collected
from different areas in her body due to the discovery of the fat
concentration (hump) on the neck of mammoth calves (Figure 1)
[12], [13]. There is clear evidence that mammoths, even at a
young age, stored large amounts of fat to survive during the long
Arctic winter. Therefore, another purpose of this research was
connected with the possibility of finding differences between the
hump fat and the fat from other areas of the body. On the other
hand, the fat from the most recent carcasses of extinct bison and
horses (8,200–9,300 years BP and 4,400–4,600 years BP,
respectively) in Siberia can provide information concerning
similarities with modern Canadian forest bison and Yakutian
horses.
A chromatogram of the FAs taken from under the belly skin of
Lyuba (ML4) is plotted in Figure 2, while the mass spectra of a
selection of PUFA showing their characteristic fragmentation
patterns are displayed in Figure 3. The FA profiles shown here
(Tables 2 and 3) are similar to others commonly found in the
subcutaneous fat of present-days grass-feeding animals, such as
elephant [19], horse [20], [21], and bison [22], although some
differences in the FA percentages were found, as discussed below.
On the other hand, hydroxylated FAs, which are associated with
fat immersion in water, and that have previously been described in
other frozen fats [23], were not detected here.
The subcutaneous fat of the baby mammoth Lyuba (ML1-ML4)
contains noticeable amounts of saturated even-chain FAs: myristic
acid (MA, 14:0), palmitic acid (PA, 16:0), and stearic acid (SA,
18:0), and minor amounts of C11-17 saturated odd-FAs. In
addition, quantifiable amounts of unsaturated even-FAs were
present in all samples: palmitoleic acid (POA, 16:1n-7), oleic acid
(OA, 18:1n-9), linoleic acid (LA, 18:2n-6), gondoic acid (GOA,
20:1n-9), ALA, eicosadienoic acid (EDA, 20:2n-6), and eicosa-
trienoic acid (ETE, 20:3n-3). Other FAs, such as erucic acid (ERA,
22:1n-9), dihomo c-linolenic acid (DHGLA, 20:3n-6), arachidonic
acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), and
nervonic acid (NVA, 24:1n-9) were detected in some fatty tissues.
The juvenile mammoth Yuka (MY) contains only C17 as
saturated odd-FA, high amounts of MA, PA and SA, and
registered higher percentages of OA, LA, and POA than did the
baby mammoth Lyuba, as well as minor amounts of ALA and AA.
As in the mammoths, the two bison had different FA profiles: the
one called bison Yukagir (BY) had a relatively high percentage of
OA, while the one called bison Batagay (BB) lacked significant
amounts of this FA, and registered higher amounts of saturated
FAs than did bison Yukagir, both animals showing low amounts of
PUFAs. Finally, both horses had high percentages of PA and OA,
the one called horse Yukagir (HY) exhibiting several PUFAs: LA,
ALA, ETE, and EPA.
The samples analysed differed in terms of FA preservation,
although, given the high amounts of unsaturated FAs detected, the
samples were generally better preserved than expected. However,
following the above-mentioned analytical methodology, some
other PUFAs were not detected, i.e., docosapentaenoic acid (DPA,
22:5n-3), docosatetraenoic acid (DTA, 22:4n-6) and docosahex-
aenoic acid (DHA, 22:6n-3). This is a normal situation, considering
the high degree of unsaturation of these latter PUFAs, which
induces rapid degradation. In any case, we do not ruled out their
appearance in other samples from frozen mammoths or horses
having better preservation status than the present ones.
It was noticeable that most samples contained total FA amounts
consistent with those that contemporary animals have in the same
organs. However, two animals, the mammoth Yuka and the horse
Batagay, yielded very low percentages. This could be due to the
degradation of the samples and also to contamination of the
original tissues by foreign substances such as hair and fur.
Table 2. Total fatty acids and saturated fatty acids composition of the fat from frozen mammals
a
.
FAs % of total FAs
Samples
Total FAs
g/100 g tissue 10:0 11:0 12:0 13:0 MA 14:0 15:0 PA 16:0 17:0 SA 18:0 20:0 22:0
MY 0.660.1 ----7.160.4 - 39.161.5 0.260.0 14.361.1 - -
ML1 21.560.7 0.560.1 0.160.0 2.260.2 0.260.0 8.860.3 1.560.1 75.362.1 0.960.2 4.060.3 - 0.02
ML2 23.660.9 0.560.0 0.160.0 2.160.1 0.160.0 7.460.6 1.360.2 75.461.5 0.860.1 2.960.3 - -
ML3 28.561.2 0.560.1 0.160.1 2.060.3 0.260.0 8.660.6 1.560.2 74.861.6 0.760.2 2.660.2 - -
ML4 18.560.8 0.460.1 0.160.0 2.560.2 0.260.1 9.960.5 2.060.1 70.662.2 1.060.1 4.460.3 0.260.1 0.02
HY 23.861.0 - - 0.160.0 - 3.260.4 0.260.0 36.962.0 0.160.0 3.860.2 - -
HB 1.060.1 ----4.660.3 0.260.1 48.763.0 - 7.160.5 - 0.02
BY 16.361.2 ----8.060.2 - 48.062.8 0.160.0 28.061.7 - -
BB 15.760.9 - - 0.460.1 - 5.760.3 0.660.0 48.763.2 0.260.0 37.462.0 1.160.1 0.06
a
Mean 6SD of three independent determinations performed by GLC-MS.
doi:10.1371/journal.pone.0084480.t002
Fatty Acids from Frozen Mammals
PLOS ONE | www.plosone.org 3 January 2014 | Volume 9 | Issue 1 | e84480

In mammoth tissues, besides OA and LA, ALA and ETE were
found in almost all samples. The occurrence of these FAs in the
plants that all these animals presumably ate is quite clear; these
FAs also reach relatively high percentages in some lichen species
characteristic of the Siberian tundra, such as Cladina arbuscula and
C. stellaris, with concentrations above 1%, these species being
consumed today by reindeer [24]. Other lichens that might have
been consumed by mammoths, such as Leptogium saturninum, also
contain concentrations of these PUFAs at the same level [25].
Lyuba shows an FA profile (samples ML1-ML4) that was
probably influenced by milk intake, as it contained all the
saturated C10-18 FAs, similar to those present in the milk of the
current Indian elephant [26]. Furthermore, it has been cited that
the milk of such pachyderms contains detectable amounts of ALA
and ETE, which probably came directly from the animal’s diet
[26]. Therefore, assuming that mammoths had a metabolism
similar to those of their current relatives, we can deduce that the
FAs found in the subcutaneous fat of Lyuba came from mammoth
milk, which would contain the FAs indicated above due to the
mother’s intake of lichens, mosses, and several plants.
It is well established that single-stomached animals, such as
elephants, rhinoceroses and horses, are susceptible to changes in
the FA composition in their adipose tissue as a result of the intake
of fats having different FA profiles [15]. In fact, the carbon-carbon
double bonds present in PUFAs are not hydrogenated during
digestion and, hence, PUFAs can be incorporated into depot fat
without modification [27], as recently established in grass-fed
Yakutian and Galician horses [20], [21]. Thus, as in horses or
elephants, the PUFA ingested by mammoths could have been
incorporated into their body tissues, as in modern elephants, in
which the PUFA content in the subcutaneous fat can reach nearly
25% of total FAs [19].
Although in this study all the FAs usually present in the depot
fat of mammals have been detected, their relative proportions
clearly differ from the original. It is widely accepted that frozen fats
undergo a transformation in which the unsaturated FAs tend to
disappear in favour of two units of shorter saturated FAs [28].
Thus, the FA profiles of the samples shown in Tables would be due
to a combination of certain factors: the foods consumed by the
animals, the physiology of their digestive system, and the extent of
the FA transformation. By considering the FA profiles of the
current relatives to the frozen animals studied here, the original
FA profiles have been deduced (Table 4). The principle underlying
Table 3. Unsaturated fatty acids composition of the fat from frozen mammals
a
.
FAs % of total FAs
OA 18:1
Samples 14:1
POA
16:1 17:1
n-9E n-7 n-9Z
Total
18:1
LA
18:2
n
-6
GOA
20:1
n
-9
ALA
18:3
n
-3
EDA
20:2
n
-6
ERA
22:1
n
-9
DHGLA
20:3
n
-6
ETE
20:3
n
-3
AA
20:4
n
-6
EPA
20:5
n
-3
NVA
24:1
n
-9
MY - 7.160.5 - - - 28.561.8 28.561.8 3.660.3 - 0.01 - - - - 0.01 - -
ML1 - 0.460.1 0.260.0 2.160.3 0.460.1 3.060.2 5.460.6 0.260.0 0.160.0 0.08 0.04 0.03 0.02 0.07 0.02 0.09 0.160.0
ML2 - 0.960.2 0.360.1 1.460.2 0.760.2 5.860.6 7.960.6 0.160.0 0.160.0 0.09 0.03 - - 0.08 - - -
ML3 - 0.660.1 0.360.0 1.960.3 0.860.1 4.960.3 7.660.4 0.260.0 0.160.0 0.09 0.03 - - 0.07 - - -
ML4 - 0.660.2 0.360.1 4.360.3 - 3.460.4 7.660.5 0.360.1 0.160.1 0.08 0.04 0.03 - 0.04 0.02 - -
HY 0.260.1 7.160.5 0.460.0 - 1.560.2 40.160.5 41.662.4 1.860.2 0.360.0 4.260.2 - - - 0.01 - 0.01 -
HB - 2.160.2 - - 0.760.1 36.762.0 37.461.1------- - - -
BY-----16.061.3 16.060.9 - 0.160.1 0.960.1 - - - 0.260.1 - 0.02 -
BB - - - 0.760.2 - 4.660.5 5.360.4 - - - - - - 0.06 - - -
a
Mean 6SD of three independent determinations performed by GLC-MS.
Figure 1. Mammoth calf Lyuba had a hump on the neck
consisting of special fat cavities.
doi:10.1371/journal.pone.0084480.g001
Fatty Acids from Frozen Mammals
PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e84480

this FA-profile reconstruction is that the amounts of PA and SA in
the samples must be increased with the amounts coming from the
transformed C18 PUFA (to PA) and C20 PUFA (to SA), as
discussed above.
In the case of the juvenile mammoth Yuka, the high amounts of
OA found indicate that such a percentage is closely related to the
original, whereas the PA percentage is clearly higher due to the
transformation of pre-existent PUFAS —that is, LA and ALA
became PA. Meanwhile, for SA this increase was due to the
transformation of variable amounts of GOA, EDA, DHGLA,
ETE, AA, and EPA. Other FAs encountered in high amounts in
this mammoth were MA and POA, but in values similar to those
usually found in hibernating animals such as badger, bear, and
beaver [29]. In addition, as occurs in hibernating mammals, this
mammoth shows minor amounts of several PUFAs, although the
original PUFA percentages should be higher, as a large amount of
these had to be transformed, as indicated above. Therefore, high
percentages of LA and ALA should have been present in the
original fat, which would have had strong effects on mammoth’s
hibernation [30]. This behaviour could have given an evolutionary
advantage to mammoths, since the animals that hibernate have
better chances of surviving the winter, when temperatures are low
and food supplies are virtually nonexistent [30].
Such deductions concerning the possibility of mammoth
hibernation are strongly supported by the behaviour of modern
Yakutian horses; that is, during the winter and strong frosts,
although they move somewhat, they stay mainly in sleeping
position with minimal feeding. Also, this horse has an unusual
thick layer of fat under the skin and in the abdominal cavity.
Presumably, the woolly mammoth in the Arctic zone of Siberia
showed a similar behaviour. In such areas, during the long and
dark winter, the animals could semi-hibernate with minimal
activity due to the presence of that special kind of fat in their
tissues.
For the baby mammoth Lyuba, the original FA profile would be
highly speculative to deduce, given both the animal’s infant status
and the high content of PA, which would come from either OA,
LA, and/or ALA. On the other hand, the tissues of this animal
contain ETE, a PUFA usually found in fish oil and in hibernating
mammals [31]. The difference between the two specimens of
mammoth may be related not only to their individual ages but also
to the season of their death. Lyuba died at the beginning of
summer (1–2 months old), but Yuka, as the wool covering with
thick underwool suggests, died during the cold period (late autumn
to winter).
To reconstruct the original FA profile of bison and horses, we
undertook a process similar to that explained above (Table 4). In
Figure 2. Gas-liquid chromatogram of fatty acid methyl esters from under skin fat from the belly of mammoth Lyuba. As noted in the
chromatogram, palmitic acid (16:0) is the main FA component, but the peaks due to the monounsaturated 18:1n-9Eand 18:1n-9ZFAs can be clearly
seen too.
doi:10.1371/journal.pone.0084480.g002
Fatty Acids from Frozen Mammals
PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e84480

Figure 3. Selection of mass spectra of polyunsaturated fatty acids methyl esters. The typical vfragmentation peaks at m/z 108 due to n-3
terminal groups, as well as the afragments (at m/z 236 for 18:3n-3, m/z 264 for 20:3n-3, and m/z 180 for 20:4n-6) are clearly seen. Note that the
methyl group is a consequence of the derivatization, and was not present in the original sample.
doi:10.1371/journal.pone.0084480.g003
Fatty Acids from Frozen Mammals
PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e84480

the case of horses, LA and ALA percentages were partially
subtracted from PA ones, and distributed according to the
proportions detected in the subcutaneous fat of other living horses
related to those considered here [20], [21]; with the assumption
that the current OA percentage is probably very close to the
original. In the case of the bison, the ratios among FAs were taken
from literature [22].
In addition to this reconstruction, a remarkable finding was
made in the fat of horse Yukagir (HY), which is the high amount of
ALA detected (4.2%). Such a quantity had never been detected in
any sample from frozen prehistoric animals or humans; only in
some cases have minor amounts of LA been discovered, as in the
Tyrolean Iceman [23]. This unexpected finding suggests a high
intake of ALA by these animals, leading to percentages of around
20% in their subcutaneous fat (Table 3), which could have
contributed to fulfil the daily needs of n-3 FAs for hunters at the
Neolithic, i.e. the time in which these animals lived. On the other
hand, the reconstructed FA profiles of both horses are closely
similar to that shown by current Yakutian horses [20], suggesting
that this ancestor also semi-hibernated.
The FA profiles described here have clear implications for Stone
Age humans. Although most long-chain PUFAs have been
transformed, the remaining amounts indicate the ability of fatty
tissues to store them. Given the high proportion of PUFAs
deduced for these frozen animals, despite the difficulty of
determining the exact quantity of fat in the unaltered carcasses,
and under the assumption that mammoths would have required a
large amount of subcutaneous fat in their bodies to survive in
extremely cold environments, the mammoths could have satisfied
the daily n-3 needs of Palaeolithic hunters. The same deduction
could be proposed for horse, but in this case for humans at the
middle Holocene, i.e. the age to which the analysed samples have
been attributed. An added advantage of the use of this organ as a
PUFA source might have been that the fat obtained from carcasses
could have been preserved for long periods of time until
consumption.
The results of this study indicate that the monogastric animals
analysed, i.e. the woolly mammoth and the horse, might have had
a hibernating or semi-hibernating behaviour, while their subcu-
taneous fat could have been consumed by Stone Age hunters to
fulfil the daily needs in essential FAs.
Author Contributions
Conceived and designed the experiments: JLGG. Performed the
experiments: JLGG IRG AT AP SG RPRB. Analyzed the data: JLGG.
Contributed reagents/materials/analysis tools: JLGG IRG AT AP SG
RPRB. Wrote the paper: JLGG AT IRG.
References
1. Eaton SB, Konner M (1985) Paleolithic nutrition.A consideration of its nature
and current implications. New Eng J Med 312: 283–289.
2. Crawford MA, Bloom M, Broadhurst CL, Schmidt WF, Cunnane SC, et al.
(1999) Evidence for the unique function of docosahexaenoic acid during the
evolution of the modern hominid brain. Lipids 34: 9–47.
3. Cordain L, Watkins BA, Mann NJ (2001) Fatty Acid Composition and Energy
Density of Foods Available to African Hominids. Evolutionary Implications for
Human Brain Development. World Rev Nutr Diet 90: 44–161.
4. Svoboda J, Pe´an S, Wojtal P (2005) Mammoth bone deposits and subsistence
practices during Mid-Upper Palaeolithic in Central Europe: three cases from
Moravia and Poland. Quater Int 126/128: 209–221.
5. Bocherens H (2003) Isotopic biogeochemistry and the paleoecology of the
mammoth steppe fauna. Deinsea 9: 57–76.
6. Obada T, van der Plicht J, Markova A, Prepelitsa A (2012) Preliminary results of
studies of the Valea Morilor Upper Palaeolithic site (Chisinau, Republic of
Moldova): A new camp of mammoth hunters. Quater Int 276–277: 227–241.
7. Germonpre´ M, Udrescu M, Fiers E (2012) Possible evidence of mammoth
hunting at the Neanderthal site of Spy (Belgium). Quater Int doi: doi:10.1016/
j.quaint.2012.10.035.
8. Nogue´s-Bravo D, Rodriguez J, Hortal J, Batra P, Araujo MB (2008) Climate
change, humans, and the extinction of the woolly mammoth. PloS Biol 6: 685–
692.
9. Mussi M, Villa P (2008) Single carcass of Mammuthus primigenius with lithic
artefacts in the Upper Pleistocene of northern Italy. J Archaeol Sci 35: 2606–
2613.
10. Zenin V, Leshchinskiy S, Zolotarev K, Grootes P, Nadeau MJ (2006) Lugovskoe:
geoarcheology and culture of a Paleolithic site. Archaeol Ethnol Anthropol
Eurasia 25: 41–53.
11. Basilyan A, Anisimov M, Nikolskiy P, Pitulko V (2011) Woolly mammoth mass
accumulation next to the Palaeolithic Yana RHS site, Arctic Siberia: its geology,
age and relation to past human activity. J Archaeol Sci 38: 2461–2474.
12. Boeskorov G, Tikhonov A, Lazarev P (2007) A new find of a mammoth calf.
Dokl Biol Sci 417: 480–483.
13. Fisher DC, Tikhonov AN, Kosintsev PA, Rountrey AN, Buigues B, et al. (2011)
Anatomy, death, and preservation of a woolly mammoth (Mammuthus primigenius)
calf, Yamal Peninsula, northwest Siberia. Quater Int 255: 94–105
14. Brand-Miller J, Mann N, Cordain L (2009) Paleolithic nutrition: what did our
ancestors eat? In: Selinger A, Green A, editors. ISS 2009 Genes to Galaxies.
Sydney: University Publishing Service. pp. 28–42.
15. Rincon Cervera MA, Venegas Venegas E, Ramos Bueno RP, Guil-Guerrero JL
(2012) Synthesis and purification of structured triacylglycerols from evening
primrose and viper’s bugloss seed oils. Food Res Int 48: 769–776.
16. Doreau M, Ferlay (1994) A Digestion and utilisation of fatty acids by ruminants.
Anim Feed Sci Tech 45: 379–396.
17. Kosintsev PA, Lapteva EG, Trofimova SS, Zanina OG, Tikhonov AN, et al.
(2012) Environmental reconstruction inferred from the intestinal contents of the
Yamal baby mammoth Lyuba (Mammuthus primigenius Blumenbach, 1799).
Quarter Int 255: 231–238.
18. Guil JL, Torija ME, Gime´nez JJ, Rodrı
´guez I (1996) Identification of fatty acids
in edible wild plants by gas chromatography. J Chromat A 719: 229–235.
19. Meyer HHD, Rowell A, Streich WJ, Stoffel B, Hofmann RR (1998)
Accumulation of polyunsaturated fatty acids by concentrate selecting ruminants.
Comp Biochem Physiol A Mol Integr Physiol 120: 263–268.
20. Mordovskaya VI, Krivoshapkin VG, Pogozheva AV (2006) The role of omega-3
polyunsaturated fatty acids found in young horse meat in the prevention of
atherosclerosis among the indigenous population of the Repub lic Sakha
(Yakutia). ICCH13 Proceedings. pp. 12–16.
21. Guil-Guerrero JL, Rinco´ n-Cervera MA, Venegas-Venegas CE, Ramos-Bueno
RP, Sua´rez-Medina MD (2013) Highly Bioavailable a-linolenic Acid from the
Subcutaneous Fat of the Palaeolithic Relict ‘‘Galician horse’’. Int Food Res J 20:
3249–3258.
22. Turner TD (2005) Evaluation of the effect of dietary forage and concentrate
levels on the fatty acid profile of bison tissue. Doctoral dissertation: University of
Table 4. Approximate original fatty acid profiles calculated
for frozen mammals.
FAs % of total FAs
a,b
16:0
PA
18:0
EA
18:1
n
-9
OA
18:2
n
-6
LA
18:3
n
-3
ALA
C20
PUFA
MY
c
18
39.1
8
14.3
28.5 7
3.6
18
0.0
,6
HY
d
18
36.9
2
3.8
41.6 7
1.8
18
4.2
,2
HB
d
19
48.7
4
7.1
37.4 8
0.0
22
0.0
,3
BY
e
32
48.0
27
28.0
29
16.0
2
0.0
1
0.0
,1
BB
e
27
48.7
36
37.4
24
5.3
2
0.0
1
0.0
,1
a
Only the major FAs have been considered.
b
A superscript number indicates the FA percentages found in frozen samples,
while all derived percentages appear in italics. In reconstructed figures, C18
PUFA percentages are partially subtracted from PA figures; C20 PUFAs from EA.
For both bison, reconstructed OA percentages are partially subtracted from PA.
c
PUFA ratios and PA percentage in agreement with those of grass-fed elephants
[19].
d
PUFA ratios and PA percentage as grass-fed Siberian [20] and Galician horses
[21].
e
PUFA ratios and PA percentage as grass-fed Bison subcutaneous fat [22].
doi:10.1371/journal.pone.0084480.t004
Fatty Acids from Frozen Mammals
PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e84480

Saskatchewan. 187 p. Available: http://ecommons.usask.ca/bitstream/handle/
10388/etd-01042006-114843/Bison-FA-study.pdf. Accessed 2013 Sep 25.
23. Makristathis A, Schwarzmeier J, Mader RM, Varmuza K, Simonitsch I, et al.
(2002) Fatty acid composition and preservation of the Tyrolean Iceman and
other mummies. J Lipid Res 43: 2056–2061.
24. Sampels S (2005) Fatty Acids and Antioxidants in Reindeer and Red Deer.
Emphasis on Animal Nutrition and Consequent Meat Quality. Doctoral thesis.
Upsala: Swedish University of Agricultural Sciences.
25. Rezanka T, Dembitsky VM (1999) Fatty Acids of Lichen Species from Tian
Shan Mountains. Folia Microbiol 44: 643–646.
26. Glass RL, Jenness R (1971) Comparative biochemical studies of Milk-VI.
Constituent fatty acids of milk fats of additional species. Compar Biochem
Physiol B: Compar Biochem 38: 353–359.
27. Wood JD, Enser M, Fisher AV, Nute GR, Sheard PR, et al. (2008) Fat
deposition, fatty acid composition and meat quality: A review. Meat Sci 78: 343–
358.
28. Morgan ED, Titusz L, Small RJI, Edwards C (1983) The Composition of Fatty
Materials from a Thule Eskimo Site on Herschel Island. Arctic 36: 356–360.
29. Zalewski K, Martysiak-Z
˙urowska D, Iwaniuk M, Nitkiewicz B, Stołyhwo (2007)
A Characterization of Fatty Acid Composition in Eurasian Badger (Meles meles).
Polish J Environ Stud 16: 645–650.
30. Florant GL (1998) Lipid Metabolism in Hibernators: The Importance of
Essential Fatty Acids. Am Zoologist 38: 331–340.
31. Ka¨ kela¨ R, Hyva¨ rinen H (1996) Fatty acids in extremity tissues of Finnish beavers
(Castor canadensis and Castor fiber) and muskrats (Ondatra zibethicus). Comp Biochem
Physiol B l13: 113–124.
Fatty Acids from Frozen Mammals
PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e84480