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Animal (2015), 9:6, pp 10081015 The Animal Consortium 2015

doi:10.1017/S1751731115000099
animal

Inuence of glucogenic dietary supplementation and reproductive


state of dairy cows on the composition of lipids in milk
R. Mesilati-Stahy1, H. Malka2 and N. Argov-Argaman1
1
The Department of Animal Science, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem Israel, PO Box 12,
Rehovot 76100, Israel; 2Extension Service, Ministry of Agriculture, Bet Dagan, Israel

(Received 18 April 2014; Accepted 3 December 2014; First published online 18 February 2015)

We studied the effects of the frequently used glucogenic dietary supplementation in dairy herds and the hormonal changes
occurring during the normal estrous cycle on the composition and concentration of milk lipid components. Holstein dairy cows
were synchronized with two injections of prostaglandin F2 (estrus = day 0). Animals were held as controls or drenched for
11 days (day 3 to day 8 of the cycle) with 850 ml/day liquid propylene glycol (treatment, n = 13 per group). Blood and milk
samples were collected on day 1 and 8 of the cycle. In both groups, plasma progesterone concentration increased 10-fold
between 1 and 8 days post-estrus. Milk fatty acid composition was associated primarily with estrous-cycle day: polyunsaturated
fatty acids increased by 16%, n-6 by 15% and n-3 by 1% from day 1 to 8 post-estrus. Polar lipid composition was also altered by
cycle day: phosphatidylethanolamine concentration was 2-fold and 1.5-fold higher on day 1 v. day 8 post-estrus in the control and
treatment groups, respectively. Phosphatidylserine concentration in milk was also affected by cycle day by treatment interaction
( P = 0.04). A progesterone level by treatment interaction inuenced the triglyceride-to-phospholipid ratio in the milk ( P = 0.02).
The results suggest that progesterone plays a role in modulating milk lipid composition and structure. Therefore, strategies
designed to alter milk lipid composition should consider the cows reproductive status.

Keywords: dairy cow, fatty acid, polar lipid, estrous cycle, propylene glycol

Implications cancer pathogenesis and even gut bacteria population


(Dillehay et al., 1994; Burgess et al., 2005). The concentra-
The dairy industry utilizes glucogenic dietary supplements to
tions of polar lipids in milk also have industrial implications
improve energy balance and, potentially, reproductive perfor-
due to their emulsifying properties, which are required for
mance. This study illustrates the interactions between the fre-
some food systems (Thompson and Singh, 2006). Hence,
quently used glucogenic supplement, liquid propylene glycol
understanding how milk polar lipid composition and yield are
and the animals reproductive status. The combined effect of
determined can provide novel means for improving dairy
the metabolic response to propylene glycol drenching and
product quality for human consumption.
estrous-cycle stage changed the milks fatty acid composition,
Milk polar lipid composition and concentration are closely
as well as phospholipid composition and concentration, and
associated with lactation stage (Bitman and Wood, 1990),
may induce structural modications of the milk fat globule.
genetic predisposition (Argov-Argaman et al., 2013), plasma
insulin concentration (Argov-Argaman et al., 2012) and diet
(Lopez et al., 2008). In general, milk polar lipid composition and
Introduction concentration are altered when systemic or local within the
Milk fat is secreted in a unique structure termed milk fat mammary gland changes in lipid metabolism are induced.
globule (MFG), which consists of a triglyceride core covered Two factors with a known effect on whole-body lipid metabo-
with three layers of polar lipids and proteins termed MFG lism are insulin and progesterone (Bauman and Currie, 1980;
membrane (Mather and Keenan, 1998). Polar lipids are Flint et al., 1984; Li et al., 2007). Although the concentrations of
considered health-promoting bioactive constituents in the both hormones are frequently altered by glucogenic dietary
human diet due to their effect on plasma lipid prole, colon supplementation in dairy cows and during the animals estrous
cycle, respectively, information about their individual and

E-mail: argov.nurit@mail.huji.ac.il combined effects on lipid composition in milk is still scarce.

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Estrous cycle associated with milk lipidome

Propylene glycol is a food industry by-product and a well- Material and methods
known glucogenic dietary supplement for dairy cows.
Cows and treatments
Propylene glycol is often used to improve dairy cows energy
The experimental protocol for the study was approved by the
balance at early lactation stages, primarily due to its ability
Volcani Center Animal Care Committee and was conducted at
to provide concentrated energy, which is rapidly absorbed
the Israel dairy farm, Israel. Thirty-seven Israeli Holstein cows,
and metabolized. In the rumen, propylene glycol adminis-
60 DIM in their rst, second or third lactation, were assigned to
tration elevates propionate and decreases acetate con-
the study. Only healthy cows were included as determined by
centrations (Shingeld et al., 2002). Post-absorption, the
the local veterinarian 2 weeks before the start of the study.
elevated propionate increases plasma glucose concentration
Body condition score was determined 1 week before expected
(Miyoshi et al., 2001; Rizos et al., 2008), which may lead to a
parturition and 40 days postpartum. The cows were free to
transient increase in plasma insulin concentration (Rizos
move around in covered pens with an adjacent yard and pro-
et al., 2008) and reduced plasma non-esteried fatty acid
vided total mixed ration, formulated to meet or exceed NRC
(NEFA) concentrations when applied at early lactation stages
requirements (2001), ad libitum (Table 1). The treatment group
(Burhans et al., 1997; Stokes and Goff, 2001). Fatty acids
was drenched with 850 ml/day liquid propylene glycol after the
absorbed from the plasma, including NEFA, are considered
morning milking, but before they were allowed to return to
to be the source of long-chain fatty acids (>16C) in milk
their stalls. Cows were milked three times a day at 8-h intervals
(Bauman and Griinari, 2003; Corl et al., 2006; Couvreur
and milk production was recorded electronically.
et al., 2007). Therefore, propylene glycol may alter milk lipid
Cows were given two injections (625 ng each) of the
composition by modulating NEFA availability to mammary
prostaglandin F2 (PGF2) analog cloprostenol (Estrumate,
gland cells and by altering the availability of lipogenic pre-
Coopers Animal Health Ltd, Berkhamsted, UK) 14 days apart,
cursors, for example, acetate, from the rumen, for lipogen-
beginning after 60 DIM to facilitate estrus detection. After
esis by mammary gland cells.
the second PGF2 injection, cows were visually observed for
Progesterone has been shown to modulate the partition-
signs of standing estrus. Alternatively, time of estrus was
ing of lipid precursors between mammary gland and adipose
determined by pedometric system (S.C.R. Engineers, Ltd,
tissue, primarily by inducing opposing changes in lipoprotein
Nethanya, Israel). Day of estrus was designated day 0.
lipase (LPL) activity and expression in these two tissues
Cows were blocked by body condition score at 40-day
(Bauman and Currie, 1980; Flint et al.,1984). LPL hydrolyzes
postpartum (Wildman et al., 1982) and for cows at second and
lipoprotein triglycerides, releasing fatty acids, which are
third lactation, according to their previous lactations milk
utilized by the mammary gland as preformed fatty acids.
yield. Cows were then assigned to control (n = 18) or treat-
Hence, changes in progesterone concentration might mod-
ment (n = 19) groups. Animals were held as controls or
ulate the availability of preformed fatty acids from the cir-
drenched for 11 days with 850 ml/day liquid propylene glycol.
culation to the mammary gland by changing LPL expression
To ensure that corpus luteum formation occurs in a high-insulin
and activity, and thus affect milk fatty acid composition.
state, treatment was begun 3 days before expected estrus and
In the present study, we hypothesized that uctuations in
lasted until 8 days post-estrus. Cows were excluded from the
progesterone concentration during the estrous cycle might
study if standing estrus was not detected. The nal number of
modulate lipid metabolism in the mammary gland and hence
animals reaching the conclusion of the experiment was 13 for
affect the composition of milk lipids. In addition, we studied
the control and 13 for the treatment group.
the effect of propylene glycol supplementation on milk lipid
composition, as well as its interaction with progesterone
concentration. To reduce the confounding effect of propylene Samples
glycol on plasma NEFA concentrations and subsequently on Blood samples were collected three times a day from 1 day
milk fatty acid composition, cows allocated to this study after the second PGF2 injection (day 3) till the end of the
were at 60 days in milk (DIM). At this stage of lactation, cows experiment. A 5-ml aliquot of blood was taken from the tail
are transitioning from negative to positive postpartum
energy balance (i.e. adipose lipolysis rate is decreasing) and
therefore, the effect of propylene glycol administration on Table 1 Chemical composition of cows diet
NEFA plasma concentration (Burhans et al., 1997; Stokes and
Nutrient analysis %a
Goff, 2001) is expected to be modest. In addition, at this
stage of lactation, the mammary gland utilizes more lipo- Non-degradable protein 5
genic precursors, such as acetate, and less NEFA for lipo- Total protein 15.7
genesis (Gross et al., 2011). To examine the effect of estrous- NDF 31.4
cycle day on milk lipid composition, cows were synchronized ADF 11.4
and plasma levels of insulin and progesterone were deter- Ether extracts 13.2
mined on day 1 and 8 post-estrus in both treated (with Ash 4.9
propylene glycol) and untreated cows. During this period, a Net energyb 1.76
spontaneous increase in plasma progesterone concentration a
Chemicals are expressed as % of dry matter.
is expected upon corpus luteum formation. b
MCal/kg dry matter.

1009
Mesilati-Stahy, Malka and Argov-Argaman

vein into vacuum tubes (Boston Dickinson Systems, Cowley, at 40C/min, holding at 250C for 5 min. Run time was
UK). The rst sampling was performed 17 h after food 37.9 min. Helium was used as the carrier gas at 2.21 ml/min.
distribution and 2 h before the morning milking. The second Peaks were identied by comparison with retention times of
blood sample was collected 5 h after food distribution. The two external standards: for polyunsaturated fatty acids
third blood sample was collected after the evening milking, (PUFA), PUFA-2 (Sigma Aldrich Israel Ltd, Rehovot, Israel),
8 h after food distribution. Plasma was separated and a FAME C8:0 to C24:0 mix (Supelco, Bellefonte, PA,
immediately from the blood samples and stored at 18C USA). The area of each fatty acid peak was recorded using
until analysis. ChemStation software (Agilent Technologies) and fatty acid
Milk samples were collected from the morning milking on weights were calculated using the internal standards area
days 1 and 8 post-estrus. Milk solids were monitored and under the curve and presented as g/kg fat.
recorded by the S.C.R. dairy herd management system (S.C.R.
Engineers, Ltd). Milk fat, protein and lactose contents were Analysis of polar and neutral lipids
determined by mean Iranalysis (standard IDF 141C, 2000) at the Polar and neutral lipids were identied and quantied
Israeli Cattle Breeders Association laboratory (Caesarea, Israel). by HPLC (HP 1200, Agilent Technologies) combined with
an evaporative light-scattering detector (ELSD; Agilent
Plasma hormone and metabolite measurements Technologies) as previously described (Argov-Argaman et al.,
Plasma insulin concentration was determined in the morning 2012). The separation process was managed by ChemStation
and evening samples by radioimmunoassay (Coat-A-Count software, which permitted data acquisition from the ELSD.
Insulin diagnostic kit, Siemens Medical Solutions Diagnostics, Lipid identication was performed using external standards:
Los Angeles, CA, USA) with intra- and interassay CV of 7.2% triacylglycerol, diacylglycerol, monoacylglycerol, free fatty
and 5.1%, respectively. Plasma progesterone concentration acids, cholesterol, phosphatidylethanolamine (PE), phos-
was determined by radioimmunoassay (TKPG1, Coat-A-Count phatidylinositol (PI), phosphatidylserine (PS), phosphati-
diagnostic kit, Siemens Medical Solutions Diagnostics) with a dylcholine (PC) and sphingomyelin (SM). Standard curves
sensitivity of 0.2 ng/ml, and intra- and interassay CV of 10% were used to calculate the weight percentage of each of the
and 10.2%, respectively. Plasma NEFA concentration was identied phosopholipids and cholesterol from the overall
determined in blood samples collected around noon by Wako weight of the identied membrane components (i.e. choles-
NEFA C test kit (Wako Chemicals GmbH, Neuss, Germany). terol, PE, PI, PS, PC, SM), as indicated below.

Extraction of total lipids and fatty acid methyl ester (FAME) HPLC/ELSD calibration
preparation The identication of phospholipids and SM was carried out by
Total lipids were extracted from the milk using the cold- comparison with the retention time of pure standards. To eval-
extraction procedure (Folch et al., 1957) with minor adapta- uate phospholipids and SM, ve calibration curves were deter-
tions as previously described (Argov-Argaman et al., 2012). mined from the area values obtain by injecting different amounts
Each sample was divided into two equal portions: one was of standard: cholesterol (2 to 15 g), free fatty acids (5 to 80 g),
used for lipid analysis by normal-phase liquid chromatography PE and PS (2 to 25 g), PI (5 to 80 g), PC (0.5 to 10 g) and SM
and the other was used for fatty acid analysis by gas chroma- (5 to 80 g). Calibration curves were calculated by applying the
tography. For liquid chromatography of polar lipids, total lipids equations of the power model to the area and concentration
were extracted from 0.5 ml of milk with methanol, chloroform values; cholesterol: y = 0.1016 0.49 (r 2 = 0.97), free fatty
and water (1 : 2 : 0.6, v/v), ltered and dissolved in 100 L acids: y = 1.73 0.41 (r 2 = 0.99), PE: y = 0.035 0.63 (r 2 =
chloroformethanol (3%). For gas chromatography analysis, 0.99), PI: y = 0.114 0.607 (r 2 = 0.99), PC: y =0.051 0.603
total lipids were extracted from 0.5 ml of milk with methanol, (r 2 = 0.99), SM: y = 0.11 0.62 (r 2 = 0.99), PS: y =
chloroform and water (1 : 2 : 0.6, v/v) and used to generate 0.0824 0.7645 (r 2 = 0.99). The sum of the glycerophospholipid,
FAME by incubation of the lipid extract in 2.5 ml of 5% (v/v) SM and cholesterol concentrations was regarded as the total
H2SO4methanol in a 65C water bath for 1 h. After incuba- membrane weight (100%). Each of the polar lipids weights in
tion, FAME were restored by the addition of 1.9 ml petroleum 1 ml of milk was calculated according to the standard curves,
ether and 3.5 ml double-distilled water. The upper phase was and weight percentage of total membrane lipids (PE + PI +
collected and evaporated under vacuum, and 100 L petro- PC + SM + PS + cholesterol) was determined.
leum ether was added before injection for analysis.
Statistical analysis
Gas chromatography analysis The statistical analyses were performed using JMP 7 (SAS
The analysis was performed in a gas chromatograph (Agilent Institute Inc., Cary, NC, USA). Differences between treatment
Technologies, Santa Clara, CA, USA) equipped with a fused- and control groups, and within and between days were
silica capillary column (60 m 0.25 mm ID, DB-23, Agilent compared by repeated measures analysis (for day 1 and 8
Technologies) as previously described (Argov-Argaman et al., post-estrus) using repeated measures ANOVA. The main effects
2012). Briey, oven temperature was programmed from were treatment, day in estrous cycle and progesterone plasma
130C to 170C at a rate of 27C/min, from 170C to 215C concentration, and treatment by days post-estrus and treat-
at 2C/min, holding at 215C for 8 min, from 215C to 250C ment by progesterone interactions, with individual cow nested

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Estrous cycle associated with milk lipidome

in the treatment. Parity number and the interactions between differ between groups and was not affected by cycle day or
parity number and treatment, parity number and progesterone treatment (P = 0.13 and 0.3, respectively).
concentration, and parity number and insulin were excluded
from the model after nding no signicant effect on milk yield Milk parameters
or composition. Data are presented as least square means and Milk yield was affected by the cycle day treatment inter-
standard deviation (s.d.). Signicance level was set at 0.05. action (P = 0.04), but no signicant differences were found
between groups within the same sampling day (Table 3).
Milk protein yield was affected by the interactions between
Results cycle day and treatment and progesterone and treatment
Plasma hormone and metabolite concentrations (P = 0.01 and 0.04, respectively), and was higher in the
Progesterone concentrations were affected by day of the estrous treatment group on day 8 post-estrus than in controls on day
cycle. Similar progesterone concentrations were found in both 8 post-estrus and the treatment group on day 1 post-estrus.
groups on day 1 post-estrus, and in both groups, progesterone
concentrations increased 10-fold from day 1 to 8 post-estrus. Milk polar lipid composition and diurnal yield
Drenching with propylene glycol did not change plasma Milk PE concentration was affected by both cycle day and pro-
insulin concentrations on either sampling day, or in morning v. gesterone concentration (P = 0.006 and 0.001, respectively),
evening samples (Table 2). NEFA concentrations measured in with a lower concentration on day 8 post-estrus in the
plasma collected at noon on days 1 and 8 post-estrus did not treatment group compared with the control group on day 1

Table 2 Plasma progesterone, insulin and NEFA concentrations in cows drenched with liquid propylene glycol (Trt) compared with non-drenched
controls (Ctrl) on days 1 and 8 post-estrus
Day 1 Day 8

Ctrl Trt Ctrl Trt P-value

Mean s.d. Mean s.d. Mean s.d. Mean s.d. Trt Cya Trt Cy P4 P4 Trt

P4 (ng/ml)b 0.1B 2.33 0.3B 2.33 3.4A 2.05 4.3A 2.05 0.74 <0.0001 0.27
Ins am (ng/ml)c 2.2 3.64 3.7 5.8 3.1 5.5 3.2 6.8 0.24 0.86 0.49 0.48 0.42
Ins pm (ng/ml)d 3.8 11.1 6.9 10.06 4 10.06 5.6 11.4 0.17 0.75 0.68 0.58 0.77
NEFA (Eq/ml) 348 483.3 445 379.7 572 387.5 460 455 0.3 0.13 0.18 0.33 0.36
Cy = cycle day; P4 = progesterone; Ins = insulin; NEFA = non-esteried fatty acids.
Values are least square means and s.d., n = 26.
a
Days post-estrous.
b
Concentration in plasma samples.
c
Concentration in morning blood samples.
d
Concentration in evening blood samples.
Different letters indicate signicant differences between groups and between days, P 0.05.

Table 3 Milk yield, and concentration of fat and protein in milk collected from cows drenched with liquid propylene glycol (Trt) compared with non-
drenched controls (Ctrl) on days 1 and 8 post-estrus
Day 1 Day 8

Ctrl Trt Ctrl Trt P-value

Mean s.d. Mean s.d. Mean s.d. Mean s.d. Trt Cya Trt Cy P4 P4 Trt

Milk yieldb 44.2 19.2 43.1 17.8 42.6 17.8 51 19.2 0.76 0.16 0.04 0.57 0.15
Fat (%) 3.6 2 3.4 2 3.8 2 2.9 2 0.4 0.77 0.1 0.95 0.1
Protein (%) 2.9 0.7 3.1 0.7 2.9 0.7 3.2 0.7 0.38 0.58 0.71 0.67 0.77
Fat (kg/day) 1.3 1.1 1.4 1 1.6 1 1.6 1.2 0.79 0.24 0.76 0.67 0.8
Protein (kg/day) 1.32AB 0.5 1.3B 0.5 1.26B 0.5 1.6A 0.6 0.87 0.07 0.01 0.6 0.04
Cy = cycle day; Ins = insulin; P4 = progesterone.
Values are least square means and s.d., n = 26.
a
Days post-estrus.
b
Mean daily milk yield, liter per day.
Different letters indicate signicant differences between groups and between days, P 0.05.

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Mesilati-Stahy, Malka and Argov-Argaman

Table 4 Polar lipid composition (weight %) of milk collected on days 1 and 8 post-estrus from cows drenched with propylene glycol (Trt) compared
with non-drenched controls (Ctrl)
Day 1 Day 8

Ctrl Trt Ctrl Trt P-value

Mean s.d. Mean s.d. Mean s.d. Mean s.d. Trt Cya Trt Cy P4 P4 Trt

Weight%
PC 8.8 11.6 10.3 11.6 10.7 9.6 9.1 13 0.57 0.84 0.46 0.39 0.83
PE 7.4A 6 5.4AB 5.2 4AB 4.9 2.4B 6 0.18 0.0065 0.86 0.0014 0.16
PI 14.7 37.7 21.8 37.7 20.4 35.6 21.3 39.7 0.37 0.48 0.4 0.1 0.67
PS 17.9AB 22.3 10.9B 22.4 18.1AB 18.3 27.9A 25.8 0.1 0.03 0.04 0.16 0.21
SM 23.4 22.6 28.9 22.6 24.3 19.2 18.9 25.4 0.24 0.2 0.12 0.71 0.14
Chol 30.2 23.3 24.08 24.6 23.9 23.34 23.8 26.7 0.22 0.34 0.37 0.22 0.46
Sum PLb 4.4 10.3 7.2 10.3 9 8.4 2.4 15.5 0.24 0.94 0.04 0.82 0.05
Tg : PLc 135.7 235.2 48.2 233.9 51.6 191.8 115.02 125 0.09 0.84 0.1 0.46 0.16
Cy = cycle day; Ins = insulin; P4 = progesterone; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine;
SM = sphingomyelin; Chol = cholesterol.
Values are least square means and s.d., n = 26.
a
Days post-estrus.
b
Daily yield of sum of polar lipids (g/day).
c
Weight ratio between triglycerides and phospholipids in the sample.
Different letters indicate signicant differences between groups and between days, P 0.05.

post-estrus (Table 4). The combined effect of cycle day and (C18:0) was affected by the interaction between cycle day
treatment affected milk PS concentration (P = 0.04) with a and treatment (P = 0.05) and was 25% lower in the treat-
2.5-fold higher concentration in the treatment group on ment v. control group on day 8 post-estrus. The concentra-
day 8 v. day 1 post-estrus. tion of oleic acid (C18:1n9) was also affected by cycle day
Polar lipid yield was strongly inuenced by the combined (P = 0.04) and was highest in the control group on day 8
effect of progesterone level and treatment. The daily yield of the post-estrus, and 20% lower in the control group on day 1
sum of polar lipids was affected by the combined effect of cycle post-estrus. The concentration of milk PUFA was mostly
day and treatment (P = 0.04) and the interaction between affected by either cycle day or progesterone level, or both.
progesterone concentration and treatment (P = 0.05). For example, the concentration of linoleic acid (C18:2n6) was
The weight ratio between triglycerides and phospholipids affected by cycle day (P = 0.01). Cycle day and progesterone
was determined (Table 4). The combined effect of treatment concentration affected the concentration of arachidonic acid
and progesterone inuenced the triglyceride-to-phospholipid (C20:4n6). The same pattern was found for docosahexaenoic
ratio in the samples (P = 0.02). No signicant differences acid concentration (C22:6n3), which was affected by cycle
were found between groups within the same day. day (P = 0.03). When fatty acids were grouped according to
their biochemical features, such as length and number of
Milk fatty acid composition unsaturated bonds, their concentration was mostly affected
Treating the cows with propylene glycol inuenced the con- by cycle day and progesterone concentration, and to a much
centration of odd-chain fatty acids, including tridecanoic, lesser extent by treatment. The concentration of total PUFA
pentadecanoic, heptadecanoic and heptadecanoyl fatty acids was affected by cycle day and progesterone concentration
(C13:0, C15:0, C17:0 and C17:1, respectively; Table 5). (P = 0.01 and 0.05, respectively) and was highest on day 8
Pentadecanoic acid concentration was highest in the treat- post-estrus in the treatment group, and 20% lower in the
ment group on day 8 post-estrus compared with the treat- control group on day 1 post-estrus. The concentration of
ment group on day 1 and control group on day 8 post-estrus. total n-6 fatty acids was also inuenced by cycle day and
The summed concentration of odd-chain fatty acids was progesterone concentration (P = 0.01), with a higher con-
affected by treatment (P = 0.03) and was highest in the centration in the treatment group on day 8 post-estrus than
treatment group on day 8 post-estrus relative to the control in the control group on day 1 post-estrus. The same was
group on days 1 and 8 post-estrus. found for the sum of all n-3 PUFA in the milk, which was
Cycle day and progesterone alone, or their interaction with affected by cycle day (P = 0.04) and was 75% higher in the
treatment, affected most of the milk fatty acids. For example, treatment group on day 8 post-estrus than in the control
cycle day inuenced palmitoleic acid (C16:1n7) concentra- group on day 1 post-estrus. The summed concentration of all
tions (P = 0.02) with a 45% higher concentration in the saturated fatty acids in the milk was affected by the inter-
treatment group on day 8 post-estrus than in the control action between cycle day and treatment (P = 0.05), with no
group on day 1 post-estrus. The concentration of stearic acid signicant differences between treatment groups.

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Estrous cycle associated with milk lipidome

Table 5 Concentration (g/kg fat) of fatty acids in milk collected on days 1 and 8 post-estrus from cows drenched with propylene glycol (Trt) compared
with non-drenched controls (Ctrl)
Day 1 Day 8

Ctrl Trt Ctrl Trt P-value

Fatty acids Mean s.d. Mean s.d. Mean s.d. Mean s.d. Trt Cya Trt Cy P4 P4 Trt

C10:0 0.27 0.032 0.18 0.032 0.14 0.03 0.1 0.04 0.25 0.09 0.65 0.06 0.66
C12:0 0.4 0.032 0.39 0.032 0.33 0.02 0.32 0.03 0.89 0.2 0.97 0.14 0.83
C13:0 0.01 0.001 0.02 0.001 0.01 0.001 0.02 0.001 0.09 0.94 0.72 0.95 0.95
C14:0 1.41 0.045 1.28 0.039 1.18 0.04 1.3 0.04 0.2 0.19 0.13 0.16 0.24
C14:1 0.08 0.006 0.09 0.006 0.08 0.006 0.09 0.006 0.46 0.9 0.83 0.56 0.98
C15:0 0.11AB 0.006 0.13BC 0.005 0.11C 0.005 0.15A 0.006 0.07 0.57 0.11 0.31 0.32
C15:1 0.02 0.001 0.02 0.001 0.02 0.001 0.02 0.002 0.42 0.56 0.75 0.71 0.92
C16:0 3.81 0.065 3.71 0.058 3.63 0.06 3.84 0.065 0.47 0.77 0.17 0.98 0.19
C16:1n7 0.15B 0.006 0.2AB 0.006 0.2AB 0.006 0.22A 0.006 0.06 0.02 0.68 0.06 0.7
C17:0 0.04 0.019 0.05 0.019 0.04 0.02 0.05 0.019 0.07 0.34 0.51 0.14 0.23
C17:1 0.01 0.001 0.02 0.001 0.02 0.001 0.02 0.001 0.09 0.18 0.55 0.1 0.36
C18:0 0.85AB 0.039 0.9AB 0.039 1.02A 0.04 0.76B 0.039 0.89 0.61 0.05 0.2 0.11
C18:1n7 0.11 0.006 0.1 0.006 0.07 0.006 0.07 0.006 0.8 0.22 0.92 0.44 0.99
C18:1n9 2.05B 0.058 2.2AB 0.052 2.46A 0.05 2.2AB 0.058 0.24 0.04 0.06 0.12 0.07
C18:1t 0.2 0.026 0.25 0.019 0.19 0.02 0.2 0.026 0.39 0.44 0.62 0.51 0.48
C18:2n6 0.34 0.01 0.35 0.009 0.36 0.01 0.39 0.01 0.74 0.01 0.3 0.01 0.3
C18:3n3 0.03 0.001 0.03 0.001 0.03 0.001 0.03 0.001 0.61 0.85 0.96 0.47 0.78
C20:4n6 0.017 0.001 0.02 0.001 0.023 0.001 0.024 0.001 0.29 0.05 0.74 0.02 0.87
C20:5n3 0.01 0.002 0.01 0.002 0.01 0.002 0.02 0.002 0.48 0.24 0.78 0.29 0.64
C22:1 0.01 0.003 0.01 0.003 0.01 0.003 0.02 0.002 0.57 0.55 0.83 0.64 0.82
C22:4n6 0.01 0.005 0.02 0.004 0.02 0.004 0.03 0.005 0.34 0.5 0.66 0.65 0.93
C22:6n3 0.005 0.003 0.011 0.003 0.018 0.003 0.022 0.003 0.31 0.03 0.89 0.73 0.85
C24:0 0.01 0.003 0.02 0.003 0.02 0.003 0.02 0.003 0.05 0.27 0.04 0.3 0.18
SFA 6.75 0.065 6.39 0.065 6.31 0.06 6.42 0.065 0.05 0.09 0.05 0.24 0.06
SAT < 16C 2.09 0.065 1.87 0.065 1.64 0.06 1.7 0.065 0.28 0.07 0.4 0.05 0.45
Odd chain 1.65 0.74 1.99 0.61 1.55 0.7 2.26 0.74 0.03 0.5 0.13 0.24 0.46
MUFA 2.53 0.065 2.79 0.065 2.89 0.06 2.68 0.065 0.12 0.26 0.04 0.45 0.05
PUFA 0.42B 0.013 0.46AB 0.013 0.57AB 0.01 0.51A 0.013 0.13 0.01 0.38 0.05 0.46
n-6 0.39B 0.013 0.43AB 0.013 0.46AB 0.01 0.48A 0.013 0.13 0.01 0.53 0.01 0.72
n-3 0.04B 0.004 0.05AB 0.004 0.05AB 0.004 0.07A 0.004 0.32 0.04 0.93 0.59 0.74
Cy = cycle day; Ins = insulin; P4 = progesterone; SFA = sum of saturated fatty acids in g/100 g fat; odd chain = sum of odd-chain SFA in g/100 g fat; MUFA = sum
of monounsaturated fatty acids in g/100 g fat; PUFA = sum of polyunsaturated fatty acids in g/100 g fat; O6 = sum of n-6 PUFA in g/100 g fat; O3 = sum of n-3 PUFA in
g/100 g fat.
Values are least square means and s.d., n = 26.
a
Days post-estrus.
Different letters indicate signicant differences between groups and between days, P 0.05.

Discussion glucogenic additives (van Knegsel et al., 2007), and speci-


cally propylene glycol, on milk production and reproductive
The results of the present study illustrate a strong effect of performance. In the present study, to avoid the confounding
the uctuations in progesterone concentration during the effect of lowering NEFA concentrations by propylene glycol
estrous cycle on milk lipid constituents. Moreover, the com- drenching, which would in itself alter milk lipid composition,
bination of drenching with propylene glycol and plasma the cows were at 60 DIM. Therefore, the massive mobiliza-
progesterone concentration had a strong effect on polar lipid tion of body reserves, in the form of NEFA, which at earlier
composition and yield. On the other hand, treatment alone lactation stages serves as the main source for milk lipid
(i.e. drenching dairy cows with propylene glycol) affected synthesis (Mcguire et al., 2004), was avoided. The fact that in
only a few minor constituents of milk fat. the current study treatment had no impact on plasma NEFA
Propylene glycol is a glucogenic dietary supplement, concentration validates our selected study design.
which has been used in the dairy industry for several dec- Drenching with propylene glycol did not increase insulin
ades. Recent interest in this product stems from its increased concentrations in either morning or evening plasma samples.
supply in the feed industry (Nielsen and Ingvartsen, 2004). Individual metabolic variations in terms of metabolic
Nevertheless, claims have been made regarding the effects of response to propylene glycol administration, as previously

1013
Mesilati-Stahy, Malka and Argov-Argaman

shown (Miyoshi et al., 2001), could explain the high standard compositions. For example, milk PUFA concentration was
deviations reported in each group and at each time point. higher on day 8 post-estrus than on day 1, under the regulation
This great variability resulted in a non-signicant increase in of cycle day and progesterone. The PUFA found in milk are
insulin in the evening samples. The fact that no differences dietary in origin. The mammary gland absorbs and incorporates
were found in the morning samples might be attributed to these fatty acids from the circulation into the milk fat by using
the proximity of blood sampling time to the propylene glycol LPL and fatty acid-binding proteins (Bauman and Davis, 1974;
drenching, because plasma insulin concentration is expected Lehner and Kuksis, 1996; Bernlohr et al., 1999). In rat mam-
to peak 2 h after propylene glycol administration (Studer mary gland, progesterone has been shown to increase LPL
et al., 1993; Grummer et al., 1994). activity (Hadsell et al., 2008). The major hormonal change
In a previous study, following alteration of lipid metabolism associated with cycle day is the transition from an estrogen-
in the mammary gland by hyperinsulinogenic clamp, PS was dominant environment at the follicular stage of the cycle
the main MFG membrane constituent to show changes in (days 02) to a progesterone-dominant environment in the
concentration (Argov-Argaman et al., 2012). Similarly, in the early luteal phase (days 78; Bauman and Davis, 1974). Hence,
present study, the combined effect of treatment and cycle day milk collected on day 1 post-estrus was most probably syn-
affected PS. Moreover, cycle day and progesterone concentra- thesized in a high estrogen/low progesterone environment,
tion also inuenced PE concentration in the milk. Altered milk whereas milk collected on day 8 post-estrus was synthesized in
phospholipid composition is associated with structural differ- a high progesterone/low estrogen one. It is therefore possible
ences for the MFG (Mesilati-Stahy and Argov-Argaman, 2014). that the changes in progesterone concentration during the
Accordingly, the altered composition of milk polar lipids found estrous cycle underlie the changes in milk PUFA concentration
here was associated with a triglyceride-to-phospholipid ratio in reported here.
the milk that indicated altered MFG diameter (Mesilati-Stahy Summing up, we examined the effect of propylene glycol
and Argov-Argaman, 2014). Such structural modications of drenching and progesterone concentration (i.e. day of
the MFG might have nutritional implications for human health estrous cycle) on milk fatty acid and lipid species composition
(reviewed by Michalski, 2007), as well as industrial implications and yield in dairy cows. Results indicate that propylene glycol
due to their effect on dairy products textural and physical administration 60 days postpartum affects the concentration
properties (Michalski et al., 2003; Lopez et al., 2011). A change of odd-chain fatty acids in milk. The PUFA and saturated fatty
in MFG mean diameter is expected to alter polar lipid yield, as acid concentrations were affected by cycle day, which
was found in the present study under the combined effect of suggests that the changes in the hormonal milieu during the
treatment and progesterone concentration. Taken together, the estrous cycle play a role in regulating milk lipid composition.
results show that the effect of drenching with propylene glycol Finally, changes in the composition and yield of milk polar
on polar lipid yield is dependent on the cows estrous-cycle lipids suggest that cycle day inuences the MFG structure.
stage and plasma progesterone levels. Therefore, protocols that This warrants further study due to its possible implications
are used to improve or modify milk lipid composition should for human health and the dairy industry.
consider the animals reproductive status, as this can change
the effect of the dietary supplement on milk lipid composition.
The major effect of drenching dairy cows with propylene Acknowledgments
glycol found in the present study was an elevated concentra- This publication was made possible in part by support from the
tion of odd-chain fatty acids. Administration of propylene Israeli Dairy Board under Cooperative Agreement No. 039820,
glycol has been shown to rapidly increase propionate and and the Israeli Agricultural Ministry Chief Scientist under
decrease acetate concentrations in the rumen (Christensen Cooperative Agreement No. 0396768. Sponsors had no role in
et al., 1997). The rapid elevation in propionate upon propylene study design, collection, analysis or interpretation of the data,
glycol administration (Emery et al., 1967; Shingeld et al., or in the writing or publication of the data.
2002) might induce de novo synthesis of odd-chain fatty acids
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