Fatty acid profile and cholesterol content of Ghezel sheep milk during lactation period

TLDR: The chemical composition, fatty acid profile, and cholesterol content of milk fat were analysed during the lactation period of thirty Iranian Ghezel sheep when they were fed dry hay for the first three months and grazed on fresh grass to the end of lactation.

Abstract: The chemical composition, fatty acid profile, and cholesterol content of milk fat were analysed during the lactation period of thirty Iranian Ghezel sheep. They were fed dry hay for the first three months and then grazed on fresh grass to the end of lactation, along with barley and wheat middling during the whole period. Fatty acid profile analysis showed palmitic acid to be the dominant fatty acid (45.24±1.88%). During lactation C6:0, C8:0, C10:0, C12:0, and C14:0 contents decreased, while C18:0, C18:1, C18:2, and CLA increased significantly, which can be associated with the change of nutrition from hay to fresh grazing. The cholesterol content of the sheep milk reached 14.88 mg/100 ml milk or 283.43 mg/100 g fat as an average for the whole period of milking. Regression analysis showed a significant increase in cholesterol from 5.42 to 32.87 mg/100 g milk during the lactation period.

Figures

Table 3. Sheep milk cholesterol content during lactation period

Table 2. Sheep milk fatty acids profi le during six months of lactation period

Table 1. Sheep milk chemical composition across the 6 months of lactation

Full text

0139–3006 © 2017 Akadémiai Kiadó, Budapest
Acta Alimentaria, Vol. 46 (4), pp. 457–463 (2017)
DOI: 10.1556/066.2017.46.4.8
FATTY ACID PROFILE AND CHOLESTEROL CONTENT OF GHEZEL
SHEEP MILK DURING LACTATION PERIOD
A. ALIZADEH
a
*, M.R. EHSANI
b
and L. ROFEHGARINEJAD
a
a
Department of Food Science and Technology, Tabriz branch, Islamic Azad University, Tehran Rd., 5157944533,
Tabriz. Iran
b
Department of Food Science and Technology, Science and Research Branch, Islamic Azad University,
Daneshgah Blvd, Simon Bulivar Blvd, 1477893855, Tehran. Iran
(received: 3 December 2016; accepted: 23 January 2017)
The chemical composition, fatty acid profi le, and cholesterol content of milk fat were analysed during the lactation
period of thirty Iranian Ghezel sheep. They were fed dry hay for the fi rst three months and then grazed on fresh grass
to the end of lactation, along with barley and wheat middling during the whole period. Fatty acid profi le analysis
showed palmitic acid to be the dominant fatty acid (45.24±1.88%). During lactation C6:0, C8:0, C10:0, C12:0, and
C14:0 contents decreased, while C18:0, C18:1, C18:2, and CLA increased signifi cantly, which can be associated
with the change of nutrition from hay to fresh grazing. The cholesterol content of the sheep milk reached 14.88
mg/100 ml
milk or 283.43 mg/100 g fat as an average for the whole period of milking. Regression analysis showed
a signifi cant increase in cholesterol from 5.42 to 32.87 mg/100 g milk during the lactation period.
Keywords: chemical composition, cholesterol, fatty acid, milk, sheep
Milk has an important role in human nutrition due to its complex nutrients that meet the
metabolic and growth needs. Although milking cows produce a signifi cant proportion of
worldwide milk production, small ruminants, such as goats and sheep, have a special place
regarding the specifi c composition of their milk and its impact on health (STRZALKOWSKA et
al., 2009). Considering that dairy goat and sheep farming are a vital sector in some
Mediterranean and Middle Eastern economies, they require development towards becoming
more industrial. Information on the composition of sheep milk and related products would be
essential for this to occur (PARK & HAENLEIN, 2006). Milk composition is affected by several
factors such as breed, stage of lactation, milking system, health, environment, and feeding
(BOCQUIER & CAJA, 1999; KUCHTIK et al., 2008). The effect of lactation period on milk
composition in different sheep breeds have been studied by several authors (KUCHTIK et al.,
2008), but the results do not support each other probably because of different factors that may
change sheep milk composition (NUDDA et al., 2002)
According to the FAO (2014) reports, Iran ranks eighth in the world for sheep milk
production, which is about 5% of the total milk production by country. The sheep in the
region are mostly, more than 95%, of the Ghezel breed, which has been noted as one of the
two distinctive milk type breeds in Iran (VALIZADEH, 2010). The population of this breed is
about 1.8 million in Iran, and classifi ed as fat-tail, heavy weight ones with mean lactation
yield of 100–220 kg (IZADIFARD & ZAMIRI, 1997; BANEH et al., 2013). Sheep milk in the region
is mostly processed as cheese. Lighvan is a village in a mountainous area in the province of
* To whom correspondence should be addressed.
Phone: +98 912 3309182; e-mails: a.alizadeh@iaut.ac.ir, ainaz_alizadeh@hotmail.com

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ALIZADEH et al.: FATTY ACID AND CHOLESTEROL CONTENT OF SHEEP MILK
Acta Alimentaria 46, 2017
Azerbaijan in Iran, and is famous for its specifi c semi-hard sheep-milk cheese. Although
Lighvan cheese's composition has been analysed in several papers, there is no study that
investigates this breed's milk composition. Meanwhile, studies on the cholesterol content of
sheep’s milk is limited to just a few studies and needs to be distinguished more with details
of different breeds.
Therefore, the aim of the present study is to evaluate the major chemical components,
fatty acid profi le, and cholesterol content of sheep milk throughout the lactation period in
order to report an average for the content and needed changes in the procedure during the
lactation period to help dairy technologists, cheese producers, nutritionists, and physicians.
1. Materials and methods
1.1. Animals feeding and sampling
The study was carried out on thirty Iranian Ghezel sheep, maintained in the most experienced
and biggest traditional farm of the region, Eidi. The manuscript contains experimental
animals, the study was conducted in accordance with the internationally accepted principles
for laboratory animal use and care, and our ethical committee on animal care approved the
protocol. Research has been done in the veterinary Faculty, Tabriz branch, Islamic Azad
University that is under supervision of the Science minister of Iran. The sheep were selected
with the help of a veterinarian in charge of milking the region’s animals. All of the selected
animals were in good condition and clinically healthy. They were numbered and fed the same
diet as all cattle, a prevalent way of feeding in the region. For the fi rst three months of
lactation (February–April), feeding was with dry hay in caves, but throughout the second
three months (May–July), hay was substituted with free fresh grazing. Barley and wheat
middling were also added to their diet during the period of lactation. This method of feeding
was similar to that of the other sheep keepers in the region, without any changes made in
order to attain the actual milk composition in the area. Milking was carried out by a combined
lamb-suckling and hand-milking procedure during the suckling period and by twice daily
hand-milking from weaning to the end of lactation. Lambs were weaned about 100 days after
lambing. Milk samples were gathered from each encoded sheep every month from two weeks
of parturition after the colostrum stage to the end of lactation lasting about six months. Milk
samples were kept in a freezer at –45 °C for analysis.
1.2. Chemical composition analysis
Milk samples were analysed for fat, protein, lactose, and total solids (TS) contents using
MilkoScan (Minor 78100, FOSS, Denmark), which was calibrated each month before the
tests. Soluble nitrogen content was detected by the standard Kjeldahl method according to the
AOAC 16.041 (1990).
1.3. Fatty acid profi le
Fatty acid composition of the frozen milk samples was determined with the direct
transesterifi cation method as proposed by LEPAGE and ROY (1986). A 100 ml milk sample was
mixed with 2 ml methanol:benzene (4:1) and then 200 ml acetyl chloride was added. The
methanolised sample was kept at 45 °C
for 1 h, 5 ml K
2
CO
3
was added to cooled vials, and
fi nally it was centrifuged at 2000 g. The upper phase was injected to GC.

459ALIZADEH et al.: FATTY ACID AND CHOLESTEROL CONTENT OF SHEEP MILK
Acta Alimentaria 46, 2017
1.3.1. Gas chromatography (GC). GC (Model 610, Buck Scientifi c, USA) was equipped
with a fl ame ionization detector and capillary column (TR-CN100, fused silica, 60 m ×
0.25 mm × 0.2 μm fi lm thickness, Teknokroma, Italy). The injection volume was 1 μl and the
split ratio was set at 1:100. Injection and detection temperatures were 240 °C and 260 °C,
respectively. The initial temperature of the oven was set at 190 °C for eight min, and then
increased with a rate of 1 °C min
–1
to reach 260 °C. Helium was used as the carrier gas with
a fl ow rate of 2 ml min
–1
. Fatty acid methyl esters were identifi ed by comparison of their
retention times with authentic standards analysed under the same conditions. Results were
expressed as % w/w of total fatty acid.
1.4. Cholesterol analysis
1.4.1. Sample preparation. Five ml KOH was added to 0.5 g milk and mixed for 15 s. The
lower phase was heated at 80
°C
for 15 min and vortexed every fi ve minutes for 10 s. Then, 1
ml of water and 5 ml hexane were added to the sample and shaken vigorously for 1 min. After
centrifugation at 2000 g, the upper phase was separated for injection to GC (F
LETOURIS et al.,
1998).
1.4.2. GC condition for cholesterol analysis. The column was capillary TRB sterol (30
m × 0.22 mm × 0.22 μm) (Teknokroma, Italy). The detector was FID adjusted at 300 °C.
Oven temperature was set at 285
°C,
while injection temperature was 300 °C. Carrier gas was
helium with a fl ow rate of 3 ml min
–1
. Injection volume was 1 μl.
1.4.3. Quantitative analysis. Equal concentrations of standard cholesterol and alpha-
cholestane (as internal standard) were prepared and injected to GC. The resulted peak areas
were divided to reach a factor (F).
An identifi ed quantity of internal standard (50 μg) was added to the sample and injected
to GC. The fi nal concentration of cholesterol was quantifi ed by multiplying 50 F and
cholesterol peak area, which accordingly divided by internal standard peak area.
1.5. Statistical analysis
One-way analysis of variance and subsequent comparison of means by the least signifi cant
difference (LSD) method at 5% probability level was carried out. Chemical composition was
assessed by regression model. Software including SAS and SPSS were used for statistical
calculations.
2. Results and discussion
2.1. Milk composition
Chemical composition of milk samples from thirty sheep (Ghezel breed) gathered during the
six months of the lactation period is shown in Table 1. Total average chemical composition
was obtained as: fat=5.41±1.48, protein=6.58±0.78, lactose=4.79±0.80, and total solids
17.52±2.03 (% w/w) (mean ± SD). Comparing these data with literature revealed an
unexpected result related to protein content, which was more than fat content, while most
studies have reported the opposite (PARK et al., 2007). In order to analyse this result, soluble
nitrogen and, accordingly, the casein content of the samples were detected. Casein content

460
ALIZADEH et al.: FATTY ACID AND CHOLESTEROL CONTENT OF SHEEP MILK
Acta Alimentaria 46, 2017
was 79±3% (mean ±SD) of the total protein, which was quite normal. This means that the
higher proportion of protein content in the sheep milk did not correlate with whey proteins
that might originate from diseases or other factors. Higher protein content could be related to
nutrition, in that a high level of nutrition and energy balance will decrease fat content and
increase protein content in sheep milk (BOCQUIER & CAJA, 1999). Furthermore, cereals in the
diet reduce acetate synthesis in the rumen, which is a precursor of fat (PULINA et al., 2006).
Breed could also be an important factor that identifi es sheep milk composition (BOCQUIER &
CAJA, 1999). In the fi rst month, the average fat content of the samples in this study was very
low. Samples were aimed to be gathered after second weeks of parturition, where there was
a sharp increase in milk yield as reported by IZADIFARD and ZAMIRI (1997) in the second week
from lambing for Ghezel sheep.
Table 1. Sheep milk chemical composition across the 6 months of lactation
Lactation
months
Fat (%) Protein (%) Lactose (%) TS (%)
Mean SD Mean SD Mean SD Mean SD
1
st
2.61 0.83 5.46 0.65 5.57 0.15 14.42 0.83
2
nd
4.91 1.43 5.78 0.54 5.31 0.24 16.64 1.23
3
rd
5.16 1.15 6.65 0.66 5.07 0.22 17.46 1.06
4
th
5.60 1.08 7.09 0.70 4.65 0.29 18.04 1.40
5
th
5.27 1.01 7.25 0.56 4.82 0.15 17.89 1.06
6
th
7.93 1.24 9.03 2.46 3.29 1.44 20.67 2.21
LSD (5%) 1.02 1.03 0.55 1.23
Data are mean±SD (n=30). LSD (5%): Least Signifi cant Difference at P<0.05
Milk composition changes during lactation were signifi cant (P<0.05), the results of
which are shown in Table 1. It can be concluded that fat, protein, and TS contents increased,
while lactose content decreased during the lactation period. In milk of sheep and other cattle,
at the peak of lactation with maximum lactose synthesis, protein and fat synthesis cannot
keep up with the lactose increase (NUDDA et al., 2002). It was obvious in our study that at the
fi rst half of lactation lactose synthesis was high, but it decreased towards the end, because of
the lower milk yield. Sharp changes in sheep milk composition at the sixth month were
related to the very low daily milk yield that decreased from 1600 g at fi rst month to about 200
g at sixth month. It would therefore be advisable to study a fi ve-month period of lactation in
future studies. Fat content did not change signifi cantly between the second and fi fth month,
while the maximum protein content was reported for the fourth and fi fth months (P<0.05)
excluding the sixth month. The lowest and highest lactose contents occurred at the fourth and
fi rst months, respectively, and fi nally, the lowest total solids content was recorded for the fi rst
month (P<0.05).
2.2. Fatty acid profi le
Fatty acid profi les of the sixty milk samples gathered during the six months of lactation are
shown in Table 2. Palmitic acid was the dominant fatty acid. Although, high palmitic acid is

461ALIZADEH et al.: FATTY ACID AND CHOLESTEROL CONTENT OF SHEEP MILK
Acta Alimentaria 46, 2017
a feature of the majority of mammals, in this study the content was more than twice the oleic
acid content (45.24 vs. 21.08%), while most studies have reported approximately equal
percentages of the two fatty acids (JANDAL, 1996; PARK et al., 2007; CARLONI et al., 2009).
Medium-chain fatty acids (MCFAs) of C10:0, C12:0, C14:0, and C16:0 contributed to 62%
of the total in this study, while the short-chain fatty acid (SCFA) content of C4:0, C6:0, and
C8:0 was 4.05%. Obviously, C4:0 and C10:0 percentages were less and palmitic acid contents
were higher than previously reported by PARK and co-workers (2007). Such results could
relate to nutrition and the cereals rich in palmitic acid that were added to the sheep’s diet.
Other signifi cant factors such as race, climate, and some other minor elements could also be
important.
Table 2. Sheep milk fatty acids profi le during six months of lactation period
Fatty
acid
(%)
Months of lactation
1st
Mean ±SD
2nd
Mean ± SD
3rd
Mean ±SD
4th
Mean ±SD
5th
Mean ± SD
6th
Mean ± SD
LSD
5%
C4:0 0.97 0.14 0.86 0.28 0.68 0.21 0.83 0.22 0.81 0.29 0.81 0.36 0.13
C6:0 2.73 0.66 2.37 1.00 1.89 0.56 1.12 0.36 1.24 0.24 0.95 0.46 0.18
C8:0 2.59 0.48 2.13 0.87 1.75 0.37 0.97 0.26 1.12 0.24 0.60 0.32 0.16
C10:0 4.85 1.17 4.17 1.46 3.78 1.12 1.69 0.52 2.02 0.47 0.99 0.55 0.23
C12:0 3.58 0.96 3.11 0.84 3.24 1.04 1.52 0.40 1.81 0.30 1.08 0.39 0.19
C14:0 13.30 1.19 14.42 1.26 14.87 2.13 8.83 1.84 11.42 0.72 9.14 3.08 0.25
C15:0 0.85 0.38 0.90 0.35 0.79 0.13 0.84 0.17 1.10 0.17 0.84 0.20 0.11
C16:0 45.64 2.99 46.36 4.14 45.74 2.50 41.85 3.67 47.26 1.56 44.60 2.98 0.21
C16:1 1.34 0.24 1.24 0.23 1.45 0.29 2.23 0.68 1.45 0.46 1.82 0.31 0.14
C18:0 6.90 1.14 7.29 1.05 7.19 1.11 9.24 2.32 9.10 0.77 9.06 2.51 0.24
C18:1 15.85 2.39 15.76 1.97 17.17 2.60 28.84 3.78 20.64 1.85 28.26 5.09 0.30
C18:2 1.33 0.33 1.20 0.21 1.27 0.23 1.75 0.29 1.62 0.32 1.60 0.30 0.11
CLA 0.09 0.05 0.20 0.12 0.18 0.08 0.29 0.17 0.42 0.15 0.28 0.10 0.10
Data are mean±SD (n=30). LSD (5%): Least Signifi cant Difference at P<0.05
Changes in fatty acid profi le during the lactation period were signifi cant except for C4:0,
C15:0, C16:0, and C16:1 (P<0.05). Regression analysis for the fatty acids indicated that
during lactation, C6:0, C8:0, C10:0, C12:0, and C14:0 contents decreased, while C18:0,
C18:1, C18:2, and CLA contents increased signifi cantly (P<0.05). This should be in
accordance with the change of nutrition from hay to fresh grazing.
Fatty acids of 4–14 carbon atoms are synthesized de novo in the mammary gland, while
C18 acids are take in from the circulating blood. C16:0 originates from both, and is less
altered (HUPPERTZ et al., 2009).
Polyunsaturated fatty acid (PUFA) content is very important, as these fatty acids have
an impact on health. In particular, CLA has such biological activity (STRZALKOWSKA et al.,
2009). Milk is an important source of CLA that could respond to more than 75% of human

Frequently asked questions

Q1. What are the contributions in "Fatty acid profile and cholesterol content of ghezel sheep milk during lactation period" ?

In this paper, the chemical composition, fatty acid profi le, and cholesterol content of milk fat were analyzed during the lactation period of thirty Iranian Ghezel sheep. 

Q2. What is the significance of the higher protein content in sheep milk?

Higher protein content could be related to nutrition, in that a high level of nutrition and energy balance will decrease fat content and increase protein content in sheep milk (BOCQUIER & CAJA, 1999). 

Q3. How was the feeding of the goats?

For the fi rst three months of lactation (February–April), feeding was with dry hay in caves, but throughout the second three months (May–July), hay was substituted with free fresh grazing. 

Q4. How was the methanolised milk sample prepared?

The methanolised sample was kept at 45 °C for 1 h, 5 ml K2CO3 was added to cooled vials, and fi nally it was centrifuged at 2000 g. 

Q5. What was the fatty acid content of the milk samples?

Medium-chain fatty acids (MCFAs) of C10:0, C12:0, C14:0, and C16:0 contributed to 62% of the total in this study, while the short-chain fatty acid (SCFA) content of C4:0, C6:0, and C8:0 was 4.05%. 

Q6. What was the purpose of the study?

The manuscript contains experimental animals, the study was conducted in accordance with the internationally accepted principles for laboratory animal use and care, and their ethical committee on animal care approved the protocol. 

Q7. What is the reason for the increase in cholesterol content of sheep milk?

It can be concluded that with the progress of lactation, the fat, protein, and total solids content of sheep milk increased, while lactose content decreased signifi cantly. 

Q8. What was the effect of the lower milk yield?

It was obvious in their study that at the fi rst half of lactation lactose synthesis was high, but it decreased towards the end, because of the lower milk yield. 

Q9. What was the average chemical composition of the samples?

Total average chemical composition was obtained as: fat=5.41±1.48, protein=6.58±0.78, lactose=4.79±0.80, and total solids 17.52±2.03 (% w/w) (mean ± SD). 

Q10. How was the temperature of the milk sample determined?

The initial temperature of the oven was set at 190 °C for eight min, and then increased with a rate of 1 °C min–1 to reach 260 °C. 

Q11. How was the cholesterol concentration quantifi ed?

The fi nal concentration of cholesterol was quantifi ed by multiplying 50 F and cholesterol peak area, which accordingly divided by internal standard peak area. 

Q12. What is the reason for the increase in fat content of sheep milk?

Increase in fat content during this period could be a reason for the cholesterol content increase as discussed by WOJTOWSKI and co-workers (2001) for an increase from 23.5 to 30 mg/100 ml milk.