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REPRODUCTION
RESEARCH
Dietary unsaturated fatty acids influence preovulatory
follicle characteristics in dairy cows
M Zachut
1,2
, A Arieli
2
, H Lehrer
1
, N Argov
2
and U Moallem
1
1
Department of Dairy Cattle, Institute of Animal Sciences, Volcani Center, PO Box 6, Bet-Dagan 50250, Israel and
2
Department of Anima l Science, Faculty of Agriculture, Hebrew University, Rehovot 76-100, Israel
Correspondence should be addressed to U Moallem; Email: uzim@volcani.agri.gov.il
Abstract
Dietary unsaturated fatty acids (UFAs) have been implicated in several reproductive processes in dairy cows through a variety of mechanisms.
This study examined the effects of periparturient supplementation of rumen bypass fats low or high in proportion of UFAs (oleic and linoleic)
on preovulatory follicle characteristics. Forty-two 256-day pregnant dairy cows were divided into three groups and were fed a control diet
(nZ14) or supplemented with fats either low (LUFA; nZ14) or high (HUFA; nZ14) in UFAs. At 14–15 days following behavior estrus, the
cows received a prostaglandin F
2a
injection and 48 h later O7 mm follicles were aspirated. Progesterone (P
4
), androstenedione (A
4
), and
estradiol (E
2
) were determined in the follicular fluid. Out of 75 follicles, 37 follicles that were aspirated between 55 and 70 days post partum
were regarded as E
2
-active follicles (E
2
/P
4
ratio O1) and subjected for further analysis. The diameter of preovulatory follicles was greater in
cows fed HUFA than in those fed control or LUFA. The concentrations and content of A
4
and E
2
in follicles and E
2
/P
4
ratio were higher in the
HUFA group than in the other two groups. The P450 aromatase mRNA expression in granulosa cells that were collected from the aspirated
preovulatory follicles was also higher in the HUFA cows than in the other groups. A significant correlation was observed between E
2
concentrations in preovulatory follicles and E
2
concentrations in plasma at aspiration. In conclusion, dietary UFA increased the size of and
elevated steroid hormones in preovulatory follicles, which may be beneficial to consequent ovarian function.
Reproduction (2008) 135 683–692
Introduction
Dietary fat has been shown as beneficial to the
reproductive system in dairy cows (Staples et al.1998).
Recently, it has been accepted that the composition offatty
acids (FAs) in the supplemented fat has a crucial role in
determining the effect on reproduction (Mattos et al.2000,
Lucy 2001). There is some evidence that dietary unsatu-
rated FAs (UFAs) positively affect ovarian function in dairy
cows, although the precise mechanism is unknown
(Staples et al. 1998, Robinson et al. 2002). Leroy et al.
(2005) demonstrated that the saturated FAs palmitic
(C16:0) and stearic (C18:0) had reduced the cleavage
rate and the development rate of blastocysts in vitro.
UFAs are essential components of all cell membranes
and the proportion of different UFAs in tissues of the
reproductive tract reflect dietary consumption (Wathes
et al.2007). UFAs can influence reproductive processes
through a variety of mechanisms; they provide the
precursors for prostaglandins (PGs) synthesis and can
modulate the expression patterns of many key enzymes
involved in both PG and steroid metabolism (Wathes et al.
2007). It has been shown by Elmes et al. (2004) in pregnant
ewes that increased consumption of linoleic acid (C18:2)
elevated the proportion of arachidonic acid (C20:4)
in maternal plasma and fetal tissues and enhanced the
placental PGs production. Supplementation of rumen
bypass polyunsaturated FA to ewes increased the number
of follicles and oocytes in the ovaries and enhanced the
number of high-qualityoocytes(Zeron et al. 2002). Dietary
linolenic acid (C18:3) has been shown to increase follicle
diameter and elevate plasma estradiol (E
2
) concentrations
during the follicular phase (Robinson et al.2002). These
results suggest that dietary UFA may have a pivotal role in
modulating follicular development, PGs secretion, and
steroidogenesis.
Our hypothesis was that specific dietary FAs might
have a role in regulation of steroidogenesis in pre-
ovulatory follicles. The objective of the present study was
to compare the effects of periparturient supplementation
of two rumen bypass dietary fats containing either a high
or low proportion of UFAs (oleic and linoleic) on the
characteristics of preovulatory follicles in dairy cows.
Results
Milk production and dry matter intake
The average milk production during the first 70 days post
partum was 5% higher in the high proportion of UFAs
(HUFA) and low proportion of UFAs (LUFA) groups as
q 2008 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0556
ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org
compared with control; 41.2, 43.3, and 43.1 kg/day
for control, LUFA, and HUFA respectively (pooled
S.E.M.Z0.6; P!0.02), with no significant differences
in 3.5% fat-corrected milk production. Daily average
dry matter intake until 70 days post partum was
similar between groups; 23.6, 23.2, and 23.6 kg for
control, LUFA, and HUFA groups respectively (pooled
S.E.M.Z0.2). No differences between groups were
observed in the calculated net energy balance of the
cows until 70 days in lactation. The interval between
calving and time of positive energy balance was
longer in the HUFA group than in the control group
(P!0.04) but not from the LUFA group (26.9, 41.6,
and 44.2 days for control, LUFA, and HUFA groups
respectively; pooled
S.E.M.Z5.7).
Preovulatory follicles characteristics and hormones
The average plasma progesterone (P
4
) concentration was
5.12 ng/ml at the day of PGF
2a
injection and 0.11 ng/ml
at the day of aspiration with no differences between
groups. Three cows that had a different pattern of plasma
P
4
concentrations from the expected (according to
the experimental schedule) were excluded from the
analysis. Aspirations that were conducted between 55
and 70 days post partum were subjected to further
analysis.
Follicles were regarded as E
2
active whenever the
E
2
/P
4
ratio in FF was O1 and regarded as E
2
inactive
whenever the E
2
/P
4
ratio was %1(Ireland & Roche
1982). Thirty-seven out of seventy-five follicles larger
than 7 mm (w50%) that were aspirated were defined as
E
2
active and these follicles were used for further
analysis. The average number of E
2
-active follicles that
were aspirated was 1.3 per cow in the control and LUFA
groups, and 1.0 in the HUFA group with no significant
differences between groups. The average diameter of
the follicles aspirated from the HUFA group was
larger than in the control and LUFA groups (P!0.03;
Table 1). The volume of the follicles in the HUFA group
tended to be larger than in the control and LUFA groups
(P!0.06; Table 1).
The concentrations of P
4
, androstenedione (A
4
), and E
2
in FF of E
2
-active follicles are presented in Fig. 1.TheP
4
concentrations and content in FF did not differ between
groups (Table 1; Fig. 1). The A
4
concentrations in the HUFA
group were 69% higher than in the control group
(P!0.04), and 4.8-fold higher than in the LUFA group
(P!0.001). The A
4
content (Table 1) was 3-fold higher in
the HUFA group than in the control (P!0.001), and 11.5-
fold higher than in the LUFA group (P!0.001). The A
4
concentrations were lower in LUFA as compared with the
control (P!0.04), but the A
4
content of the FF did not differ
between these two groups.
The FF concentrations of E
2
were 40% higher in the
HUFA group than in the control group (P!0.01), and
threefold higher than in the LUFA group (P!0.001;
Fig. 1). The E
2
content in HUFA was 2.4-fold higher than
in the control (P!0.01) and 4.1-fold higher than in the
LUFA group (P!0.003; Table 1). The E
2
concentrations
in the LUFA group were 53% lower than in the control
(P!0.001), with no differences in E
2
content among
these groups. The E
2
/P
4
ratio was 47% higher in the
HUFA group than in the control (P!0.03) and 2.5-fold
higher than in the LUFA group (P!0.001). The E
2
/P
4
ratio was 41% lower in LUFA than in control (P!0.05).
Concentrations and content of non-esterified fattyacids
(NEFA) and insulin in FF are shown in Table 1. NEFA and
insulin concentrations in FF did not differ between
groups, but NEFA and insulin contents in FF tended to
be higher in HUFA as compared with control (P!0.09).
Plasma E
2
concentrations at the day of follicle aspiration
The concentration of E
2
in plasma at the day of follicle
aspiration was examined in a subgroup of 14 randomly
selected cows (four to five cows from each treatment).
The concentration of E
2
in plasma at the day of follicle
aspiration was numerically but not significantly higher in
HUFA cows than in the control and LUFA cows (3.5, 4.9,
and 5.1 pg/ml for control, LUFA, and HUFA groups
respectively; pooled
S.E.M.Z0.1).
A positive correlation between E
2
concentrations in
plasma at the day of follicle aspiration and E
2
concentrations in FF was observed (rZ0.55, P!0.01).
Furthermore, the correlation between E
2
concentrations
in plasma and the E
2
content (ng) in FF was also
significant (rZ0.48, P!0.03). No significant correlation
was found between the diameter of the follicle and E
2
in
plasma (rZ0.32, P!0.19).
Table 1 Characteristics of preovulatory follicles from cows supple-
mented with fat high or low in unsaturated fatty acids (UFAs) proportion.
Treatments
c
Control LUFA HUFA S.E.M.
n, cows
d
12 9 9
n, follicles 16 12 9
Follicles per cow 1.3 1.3 1.0 0.2
Diameter (mm) 11.8
b
10.5
b
15.4
a
1.0
Volume (ml) 1.1 1.0 2.2 0.4
Progesterone (ng) 136.9 136.8 211.6 75.7
Androstenedione (ng) 136.9
b
36.0
b
416.7
a
70.6
Estradiol (ng) 1490.5
b
867.6
b
3523.6
a
539.0
NEFA (mEq/l) 219.8 211.8 178.9 39.0
NEFA (mEq) 198.3 228.5 416.5 87.6
Insulin (pg/ml) 101.7 86.3 115.9 22.9
Insulin (pg) 96.7 95.7 225.3 53.3
a,b
Within rows, means with different superscript letters are statistically
different (P!0.05). NEFA, non-esterified fatty acids.
c
Treatments were: cows fed from 256 days of pregnancy a dry cow
ration and post partum fed a lactating cow diet (CTL) or supplemented
with a fat either containing a low (LUFA) or high (HUFA) proportion of
unsaturated fatty acids.
d
Cows with at least one E
2
-active follicle.
684 M Zachut and others
Reproduction (2008) 135 683–692 www.reproduction-online.org
P450 aromatase mRNA expression in granulosa ce lls
The mRNA expression of P450 aromatase in granulosa
cells was determined and the relative expression is
presented in Fig. 2. The P450 aromatase mRNA expression
in granulosa cells was similar between control and LUFA
and was increased by one order of magnitude in HUFA
cows in comparison with control (P!0.016).
FA profile in plasma and follicular fluid (FF)
As shown in Table 2, the percentage of linoleic acid
(C18:2) in plasma was higher in the HUFA group than in
the LUFA group (P! 0.05) and similar to the control
group. The total percentage of PUFA (C18:2 and longer
FAs) was higher in HUFA than in LUFA (Table 2;
P!0.05), and similar to the control group (P!0.12).
The total percentage of saturated FAs tended to be higher
in LUFA compared with HUFA (Table 2; P!0.08).
The FA profile in FF was analyzed in 30 out of 37
E
2
-active follicles, since not all follicles had sufficient
volume of FF for analysis. As shown in Table 3, the FA profile
in FF did not differ between treatments, except for the
percentage of arachidonic acid (C20:4), which was lower in
the LUFA group than in the control group (P!0.05), but was
not different from that in the HUFA group.
Relationship between FF hormones, insulin, a nd NEFA
Across treatments, correlation coefficients between FF
hormones, insulin, and NEFA are presented in Table 4.The
content of P
4
,A
4
,andE
2
in FF was positively correlated
with insulin and NEFA contents in FF. Furthermore, E
2
content in FF was positively correlated with P
4
and A
4
contents in FF. E
2
concentrations in FF was positively
correlated with NEFA, A
4
content, and P
4
concentrations
Figure 1 Concentrations of (A) progesterone, (B) androstenedione,
(C) estradiol and (D) estradiol/progesterone ratio in FF of cows fed from
256 days of pregnancy a dry cow ration and post partum fed a lactating
cow diet (CTL) or supplemented with a fat either containing a low
(LUFA) or high (HUFA) proportion of unsaturated fatty acids.
a,b,c
Means
with different superscript letters are statistically different (P!0.05).
Figure 2 P450 aromatase mRNA relative expression in granulosa cells
obtained from preovulatory follicles of cows fed from 256 days of
pregnancy a dry cow ration and post partum fed a lactating cow diet
(CTL) or supplemented with a fat either containing a low (LUFA) or high
(HUFA) proportion of unsaturated fatty acids.
a,b
Means with different
superscript letters are statistically different (P!0.05).
Table 2 Fatty acid profile in plasma at the day of follicle aspiration.
Treatments
c
Control LUFA HUFA S.E.M.
n 12 9 9
C14:0 1.66 1.53 1.49 0.09
C14:1 0.72 0.71 0.68 0.08
C16:0 13.31 13.91 13.14 0.41
C16:1 1.23 1.46 1.27 0.19
C18:0 12.56 13.35 12.29 0.47
C18:1 8.65 8.99 8.57 0.39
C18:2 55.36
a,b
53.68
b
56.24
a
0.87
C18:3 1.80 1.73 1.71 0.10
C20:3 2.35 2.54 2.55 0.16
C20:4 2.18 2.10 2.05 0.22
Saturated fatty acids 27.53
a,b
28.79
a
26.92
b
0.69
Polyunsaturated fatty acids 61.69
a,b
60.05
b
62.55
a
0.88
a,b
Within rows, means with different superscript letters are statistically
different (P!0.05).
c
Treatments were: cows fed from 256 days of pregnancy a dry cow
ration and post partum fed a lactating cow diet (CTL) or supplemented
with a fat either containing a low (LUFA) or high (HUFA) proportion of
unsaturated fatty acids.
Dietary fatty acids and follicular steroids
685
www.reproduction-online.org Reproduction (2008) 135 683–692
in FF. The NEFA content in FF was positively correlated
with insulin content and P
4
concentrations in FF. Other
correlations were not statistically significant.
Relationship between follicul ar fluid FA concentrations
and hormones
Correlations were examined between follicular fluid P
4
,
A
4
, and E
2
concentrations and specific FA concen-
trations (Table 5). A positive correlation was found
between P
4
and E
2
concentrations in FF and concen-
trations of C16:0, C18:1, C18:2, C18:3, and total FA in
FF (P!0.05). A
4
concentrations in FF had a positive
correlation with the concentrations of C16:0, C18:2,
and total FA in FF (P!0.05). The concentration of
C20:4 in FF was positively correlated with the
concentrations of P
4
(P!0.03).
NEFA and insulin in E
2
-active or E
2
-inactive follicles
A total of 75 follicles were aspirated; 37 follicles were
regarded as E
2
active and 38 follicles were regarded as E
2
inactive. Across treatments, analysis showed that insulin
concentration was 4.2-fold (P!0.06) and insulin content
was 2.9-fold (P!0.0001) higher in FF from E
2
-active
follicles as compared with E
2
-inactive follicles (Table 6).
Furthermore, NEFA concentrations and content were
3.3- and 4.9-fold higher in E
2
-inactive follicles than in
E
2
-active follicles respectively (Table 6; P!0.0001).
Discussion
To our knowledge, this is the first study in which dietary
UFA had an effect on preovulatory follicle characteristics
in dairy cows. In the present study, the concentrations
and content of A
4
and E
2
in preovulatory follicles of cows
supplemented with fat that contained high proportion of
UFA were enhanced compared with control or cows that
were supplemented with fat that contained low proportion
of UFA. The mRNA expression of P450 aromatase was also
increased in granulosa cells obtained from HUFA cows
than in the control or LUFA cows.
In the current study, no significant correlation was
observed between preovulatory follicles size and E
2
concentrations in plasma (rZ0.32; P!0.19), which is in
agreement with a report by Wiltbank et al. (2006).
However, a significant correlation was detected between
E
2
concentrations in plasma and E
2
concentrations or E
2
content in follicles. The concentration of E
2
in plasma
has been positively correlated with estrus duration and
behavior in cattle (Mondal et al. 2006, Wiltbank et al.
2006). Pregnancy rates were related to the diameter
of the preovulatory follicles and E
2
concentration in
plasma in dairy cows (Lopes et al. 2007). It was also
demonstrated by Botero-Ruiz et al. (1984) that follicles
aspirated from women who conceived after in vitro
fertilization had significantly higher E
2
concentrations
than similar follicles from women who failed to
conceive. Furthermore, a relationship between peak
serum E
2
concentrations and pregnancy rate after
embryo transfer on day 5 was also observed in human
(Chen et al. 2003). In an in vitro study, Tesarik &
Mendoza (1995) demonstrated a direct effect of E
2
on
fertilization and cleavage rate of mature oocytes. They
also suggested a model in which E
2
impacts the cell
surface by increasing the intracellular free [Ca
2C
], which
serves as a second messenger and contributes to
capacitation and early post-fertilization development
(Tesarik & Mendoza 1995). Collectively, these findings
indicate positive effects of E
2
concentrations in FF on
Table 3 Fatty acid profile in follicular fluid of estradiol (E
2
)-active
follicles.
Treatments
c
Control LUFA HUFA S.E.M.
n 11 10 9
C14:0 2.40 2.80 2.86 0.28
C14:1 1.12 2.50 1.10 0.15
C16:0 15.52 14.03 16.24 0.86
C16:1 1.74 1.65 1.70 0.15
C18:0 11.43 11.46 11.46 0.42
C18:1 9.43 9.78 9.15 0.39
C18:2 51.74 52.47 51.99 0.95
C18:3 1.67 1.57 1.66 0.09
C20:3 1.88 1.96 1.97 0.10
C20:4 2.25
a
1.79
b
1.87
a,b
0.17
Saturated fatty acids 26.95 25.49 27.70 1.03
Polyunsaturated fatty acids 57.55 57.78 57.48 0.97
a,b
Within rows, means with different superscript letters are statistically
different (P!0.05).
c
Treatments were: cows fed from 256 days of pregnancy a dry cow
ration and post partum fed a lactating cow diet (CTL) or supplemented
with a fat either containing a low (LUFA) or high (HUFA) proportion of
unsaturated fatty acids.
Table 4 Across treatments (nZ37) correlation coefficients between follicular fluid hormones, NEFA and insulin in E
2
-active follicles.
Item P
4
(ng) A
4
(ng) E
2
(ng) NEFA (mEq) Insulin (pg) E
2
(ng/ml)
P
4
(ng) – NS 0.76
†
0.88
†
0.40* NS
A
4
(ng) NS – 0.77
†
0.40* 0.34* 0.53
†
E
2
(ng) 0.76
†
0.77
†
– 0.79
†
0.60
†
–
NEFA (mEq) 0.88
†
0.40* 0.79
†
– 0.53
†
NS
NEFA (mEq/l) NS NS NS – NS 0.31*
P
4
(ng/ml) – NS 0.45
†
0.49
†
NS 0.41*
P
4
, progesterone; A
4
, androstenedione; E
2
, estradiol; NEFA, non-esterified fatty acid. *P!0.05;
†
P!0.0001.
686 M Zachut and others
Reproduction (2008) 135 683–692 www.reproduction-online.org
estrus characteristics in cattle and fertilization, cleavage
rate and pregnancy rate in human. These findings make
the enhancement of E
2
concentrations in FF of special
interest, since it may increase the potential of successful
pregnancy.
There is littleinformation on the effects of dietary UFA on
ovarian steroidogenesis. It has been found in two studies
that feeding UFA increased E
2
levels in plasma; Robinson
et al. (2002) supplemented dairy cows with fats containing
either high concentrations of C18:3 or high concentrations
of C18:2 and observed higher E
2
concentrations in
plasma in the C18:3 treatment than in the control, with
intermediate values in the C18:2 group. Lammoglia et al.
(1997) also found increased E
2
concentrations in plasma
throughout the first, but not the second estrous cycle after
fat supplementation using rice bran (rich in C18:2). There
is some evidence that dietary fat could increase E
2
content
or concentrations in FF. Cows that were supplemented
with fat that contained calcium salts of FA (6.5% C18:2)
had higher E
2
content in E
2
-active follicles as compared
with the control group (Moallem et al. 1999). In our
previous report that focused on the follicular development
in early lactation, a tendency for higher concentrations of
E
2
in FF from E
2
-active follicles that were aspirated on day
12 post partum wasobservedincowsthatwere
supplemented with HUFA as compared with LUFA or
control cows (P!0.1; Moallem et al. 2007). In the current
study, cows that were supplemented with HUFA that
contained 33.6% of C18:1 and 30.5% of C18:2 had higher
concentrations and content of E
2
in follicles than control or
LUFA supplemented cows.
We also observed higher E
2
/P
4
ratio in follicles from the
HUFA group than in the control and LUFA groups (1.5- and
2.5-fold higher respectively). It is well established that the
E
2
/P
4
ratio is one of the most precise indicators of follicles
health (McNatty et al.1979, Ireland & Roche 1982).
Andersen (1993) demonstrated a significant correlation
between the pregnancy potential of human oocytes after
in vitro fertilization and the E
2
/androgen ratio in FF.
The enhanced E
2
concentrations and content and the
higher E
2
/P
4
ratio in preovulatory follicles of the HUFA
cows in the current study may contribute to an increased
pregnancy potential as explained previously.
Although we did not observe differences in the
percentage of C18:2 in FF between groups, across
treatment data showed a positive correlation between
the C18:2 and E
2
concentrations in FF (rZ 0.43;
P!0.02). The mechanism behind the enhanced E
2
synthesis when feeding UFA is still unclear, however,
several explanations have been suggested. Steroid
synthesis requires increased expression of StAR protein,
which mediates transfer of cholesterol from the cytosol
to the inner mitochondrial membrane (Stocco & Clark
1996). Linoleic acid can be converted to arachidonic
acid that increases the expression of StAR (Wang et al.
2000). Wathes et al. (2007) suggested that arachidonic
acid and its metabolites may indirectly affect the
steroidogenic machinery via PGs. It was demonstrated
by Wang et al. (2003) that inhibition of specific PGs
endoperoxide synthase-2 (PTGS2) was associated with
increased StAR expression and steroid output, which
could be caused by a decrease in PGF
2a
that inhibits
StAR protein expression. However, in the current study,
we did not observe differences in P
4
concentrations and
content between groups, which may not support this
theory of elevated StAR activity. Elmes et al. (2004) and
Cheng et al. (2005) reported that feeding ewes a diet with
high C18:2 increased the in vitro and in vivo synthesis of
PGE
2
in placental membranes. This finding indicated
that C18:2 plays a role in regulation of PGs synthesis and
thus could affect steroidogenesis. It was reported by Sarel
& Widmaier (1995) that C18:2 stimulated the adrenal
corticosterone synthesis, which demonstrated the effects
of UFA on steroidogenesis in a variety of tissues. It was
also suggested by Wathes et al. (2007) that UFA may alter
the function of transcription factors and thus affect
cellular enzymes that regulate PGs and steroids
synthesis. Indeed, the expression of P450 aromatase
mRNA was higher in granulosa cells of UFA cows
compared with the control, which supports this theory.
Although there is some evidence for the implication of
UFA in steroidogenesis regulation, the mechanism
behind this regulation is not clear and further research
is required to elucidate this issue.
Preovulatory follicles from the HUFA group were
larger and tended to have larger volume compared with
Table 5 Across treatments (nZ30) correlation coefficients between follicular fluid fatty acids concentrations and hormones.
Item C16:0 C18:1 C18:2 C18:3 C20:3 C20:4 Total FA
P
4
(ng/ml) 0.49
†
0.35* 0.45* 0.34* NS 0.40* 0.46*
A
4
(ng/ml) 0.47
†
NS 0.32* NS NS NS 0.35*
E
2
(ng/ml) 0.58
†
0.39* 0.43* 0.36* 0.49
†
NS 0.48
†
P
4
, progesterone; A
4
, androstenedione; E
2
, estradiol; FA, fatty acid. *P !0.05,
†
P ! 0.008.
Table 6 Across treatments insulin and non-esterified fatty acids (NEFAs)
in estradiol (E
2
)-active and E
2
-inactive follicles.
E
2
active E
2
inactive S.E.M.
n, follicles 37 38
Insulin (pg/ml) 91.97
a
21.64
b
11.92
Insulin content (pg) 117.55
a
41.12
b
28.43
NEFA (mEq/l) 206.81
b
682.72
a
36.94
NEFA content (mEq) 260.12
b
1280.18
a
194.7
a,b
Within rows, means with different superscript letters are statistically
different (P!0.05). NEFA, non-esterified fatty acids.
Dietary fatty acids and follicular steroids
687
www.reproduction-online.org Reproduction (2008) 135 683–692
the control and LUFA follicles. There are some other
reports in which increased diameter of preovulatory
follicles were observed as a result of fat supplementation
in dairy cows (Lucy et al. 1991, Moallem et al. 1999). It
was shown by Bilby et al. (2006) that cows fed a diet
enriched with C18:2 had larger follicles, which is in
agreement with the current study.
Lower concentrations of A
4
and E
2
in preovulatory
follicles of the LUFA cows than in the control was observed
in the current study, with no differences in A
4
and E
2
contents. The E
2
/P
4
ratio was also lower in LUFA than in
control. Vanholder et al.(2005)showed that saturated FA
had a toxic effect on bovine granulosa cell growth and
function in vitro, and similar effects were observed in
human granulosa cells (Mu et al. 2001). It was also
reported by Mu et al. (2001) that supplementation of
arachidonic acid antagonized the saturated FA-induced
apoptosis in granulosa cells. Leroy et al. (2005) demon-
strated that in vitro addition of saturated FA (C16:0 and
C18:0) to oocytes during maturation had negative effects
on maturation, fertilization, cleavage rate, and blastocysts
yield. These findings indicate that saturated FA may have
an adverse effect on granulosa cells, which might partly
explain the lower concentrations of A
4
and E
2
in the
preovulatory follicles of the LUFA cows. In the current
study, we indeed found that the percentage of saturated
FA in plasma was higher in the LUFA group than in the
HUFA group, but not than that of the control. When
examining the FA profile in FF, we also observed lower
percentage of arachidonic acid (C20:4) in FF of LUFA than
in control. As mentioned above, arachidonic acid has a
positive effect on steroidogenesis and it might be that the
LUFA follicles had insufficient arachidonic acid and
consequently lower steroidogenic hormones compared
with the control.
Cell culture studies demonstrated a dependence of
bovine granulosa cells on the presence of physiological
concentrations of insulin (Gutierrez et al. 1997).
Armstrong et al. (2002) observed a correlation between
circulating insulin concentrations and E
2
production in
cultured granulosa cells. In the current study, we
observed a tendency for higher content of insulin in
HUFA follicles that had higher concentrations of E
2
compared with the control follicles. Insulin content was
also positively correlated with P
4
,A
4
, and E
2
contents in
FF. Our results confirm these previous findings in which
insulin plays a significant role in follicle function.
Data from the present study also showed that the E
2
-
inactive follicles had 3.3-fold higher concentration and
4.9-fold higher content of NEFA than E
2
-active follicles.
The content of the FF is assumed to be derived from the
vasculature in the surrounding thecal layers (Clarke et al.
2006). However, the NEFA concentration in plasma of
cows at that stage of lactation (55–70 days post-calving)
is generally lower than that observed in the E
2
-inactive
follicles in the current study (Leroy et al. 2005).
Moreover, Leroy et al. (2005) did not observe a constant
association between NEFA concentrations in plasma and
FF. In advanced stages of atresia, the granulosa cells have
degenerated, and the FF is filled with cellular debris
(Van Wezel et al. 1999). Therefore, we suggest that the
increase in NEFA observed in E
2
-inactive follicles may be
due to the disintegration of granulosa cells, which
causes a leakage of NEFA and other cellular content
into the FF. However, when examining the E
2
-active
follicles in the current study, we observed a positive
correlation between NEFA and P
4
,A
4
, and E
2
contents,
which indicates that NEFA is not necessarily a negative
indicator for steroidogenesis potential of the follicle.
In conclusion, dietary UFA increased the diameter and
tended to increase the volume of preovulatory follicle
that were aspirated between 55 and 70 d post partum.
No differences were observed between groups in P
4
concentrations and content in follicles, however, A
4
and
E
2
concentrations and content were higher in cows that
were supplemented with HUFA than control or LUFA.
Higher P450 aromatase mRNA expression was also
demonstrated in granulosa cells that were collected from
the aspirated follicles. The findings of the current study
indicate beneficial effects of dietary UFA on the
preovulatory follicles size and steroidogenesis, although
the mechanism is not fully clear.
Materials and Methods
Cows and treatments
The experimental protocol of the study was approved by the
Volcani Center Animal Care Committee and was conducted at
the Volcani Center experimental farm in Bet Dagan, Israel. The
study was conducted from September to April to avoid heat
stress effects. Forty-two multiparous Israeli-Holstein dry dairy
cows, 249-day pregnant (average live body weightZ648G8 kg),
were group-housed in covered loose pens with adjacent outside
yards, which were equipped with a real-time electronic
individual feeding system. The cows were stratified into three
groups on the basis of previous lactation milk and fat, parity, body
weight, and body condition score. All treatments commenced
prepartum at 256 days of pregnancy as follows: (1) Control were
fed a dry cow diet and post partum were fed a lactating cow diet
according to National Research Council (NRC 2001) recommen-
dations (nZ14); (2) LUFA were supplemented with 230 g/day per
cow of a rumen-protected fat that contained a low proportion of
UFAs (Energy Booster 100, Milk Specialties, Dundee, IL, USA),
until 100 days post par tum (nZ 14) and (3) HUFA were
supplemented with 215 g/day per cow of a rumen-protected fat
that containeda high proportionof UFAs (Megalac-R, Churchand
Dwight, Princeton, NJ, USA) until 100 days post partum (nZ14).
The composition and content of the pre- and postpartum diets are
presented in Tables 7 and 8 respectively. The cows were fed ad
libitum pre-andpost partum. TheFA profile of Energy Booster100
was 28.2% palmitic acid (C16:0), 51.2% stearic acid (C18:0),
8.4% oleic acid (C18:1), 1.5% linoleic acid (C18:2), 0.1%
linolenic acid (C18:3), and 10.6% other FAs (measured as a
percentage of total FA). The FA profile of Megalac-R was 17.4%
688 M Zachut and others
Reproduction (2008) 135 683–692 www.reproduction-online.org
palmitic acid (C16:0), 2.1% stearic acid (C18:0), 33.6% oleic
acid (C18:1), 30.5% linoleic acid (C18:2), 2.4% linolenic acid
(C18:3), and 14% other FAs. The diets were formulated to be
isoenergetic. The energy contents of the supplements that were
used in thisstudy werecalculated accordingto the manufacturer’s
specifications, which were 6.0 and 6.6 NE
L
Mcal/kg DM for
Energy Booster 100 (LUFA) and Megalac-R (HUFA) respectively.
Cows were milked thricedaily and milk production was recorded
electronically (SAE, Kibbutz Afikim, Israel). The cows were
weighed automatically thrice daily after each milking with a
walking electronic scale. The individual daily energy balance
was calculated as described previously (Moallem et al.2007).
Follicular fluid aspiration
At 38–40 days post partum, ovaries of all cows were examined
once for the presence of corpus luteum (CL) by ultrasonography
(Scanner 200; Pie Medical, Maastricht, The Netherlands). Cows
with ovaries that had a CL were injected with 2.5 ml PGF
2a
analog (Estrumate; Coopers Animal Health Ltd, Berkhamsted,
UK) to facilitate estrus. Cows without a CL were injected with
5 ml gonadotropin-releasing hormone analog (0.02 mg buser-
elin; Receptal, Intervet International B.V. Boxmeer, Holland) to
induce ovulation of large follicles, followed 7 days later by
injection of 2.5 ml PGF
2a
. Cows that were observed for signs of
estrus visually or by pedometers (Computerized Dairy Manage-
ment Systems, SAE AFIKIM, Afikim, Israel), received
14–15 days later a second injection of 2.5 ml PGF
2a
to cause
luteolysis and to enable preovulatory follicular development.
Forty-eight hours after the second PGF
2a
injection, aspiration of
FF was conducted using the ovum pick up procedure (Moallem
et al. 1999). The FF aspirations were performed as follows:
cows were sedated with an i.m. injection of 1 ml of 2%
Rompun (XYL-M2 Veterinary, xylazine base 20 mg/ml; VMD,
Arendonk, Belgium) and were given a local anesthesia of 5 ml
of 2% lidocaine HCl (2% esracain, 200 mg/10 ml; Rafa
Laboratories, Jerusalem, Israel) injected epidurally between
the last sacral and first caudal vertebrae. Ovaries were
examined and the diameters of the large follicles were
measured; follicles R7 mm were aspirated. Each follicle was
aspirated into a single tube, centrifuged, and the FF was stored
at K18 8C until analysis. After the separation of the FF, 1 ml
RNAlater (Sigma–Aldrich Inc.) was added to the residue of each
tube and then they were frozen at K18 8C until determination
of mRNA of P450 aromatase in the granulosa cells.
Blood samples for determination of P
4
were taken at the
day of the second PGF
2a
injection (48 h prior to follicular
Table 8 Basal lactating cow diet ingredients and chemical composition.
Treatments
a
Ingredients Control LUFA HUFA
Percentage of dry matter
Corn grain ground 18.0 15.3 15.4
Barley grain rolled 18.2 18.5 18.5
Rapeseed meal 1.0 1.0 1.0
Corn gluten meal 3.1 3.2 3.2
Soybean meal 5.0 5.1 5.2
Sunflower meal 1.7 1.8 1.8
Corn gluten feed 7.5 7.6 7.6
Cottonseed 4.6 4.7 4.7
Wheat silage 12.1 12.4 12.4
Corn silage 11.7 11.9 11.9
Dried distillers grain 3.8 3.9 3.9
Molasses 0.6 0.6 0.6
Vetch hay 2.1 2.2 2.2
Oats hay 8.6 8.8 8.8
Soybean oil 0.1 0.1 0.1
Salt 1.4 1.4 1.4
Calcium bicarbonate 0.5 0.5 0.5
Vitamins and minerals 0.1 0.1 0.1
Energy Booster 100 1.0
Megalac-R 0.9
Chemical composition
NE
L
(Mcal/kg) 1.78 1.78 1.78
Crude protein 17.2 17.0 17.0
RUP 6.0 6.0 6.0
ADF 19.4 19.4 19.4
NDF 31.7 31.7 31.7
Ether extract 3.55 4.45 4.55
Ca 0.9 0.9 0.9
P 0.4 0.4 0.4
NE
L
, net energy for lactation; RUP, rumen undegradable protein;
ADF, acid detergent fiber; NDF, neutral detergent fiber.
a
Treatments were: cows fed from 256 days of pregnancy a dry cow
ration and post partum fed a lactating cow diet (CTL) or supplemented
with a fat either containing a low (LUFA) or high (HUFA) proportion of
unsaturated fatty acids.
Table 7 Ingredients and chemical composition of diets.
Treatments
a
Ingredients Control LUFA HUFA
Percentage of dry matter
Corn grain ground 12.9 4.9 4.8
Barley grain 6.9 5.9 5.8
Rapeseed meal 0.4 0.3 0.3
Gluten meal 1.2 1.0 1.0
Soybean meal 1.9 1.6 1.6
Sunflower meal 0.7 0.6 0.6
Gluten feed 2.9 2.4 2.4
Cottonseed 1.7 1.5 1.5
Wheat silage 4.6 3.9 3.9
Corn silage 4.5 3.8 3.7
Dried distillers grain 1.4 1.2 1.2
Legume hay 0.8 0.7 0.7
Oats hay 59.0 69.6 70.0
Salt 0.5 0.4 0.4
Calcium bicarbonate 0.2 0.2 0.2
Vitamins and minerals 0.04 0.03 0.03
Supplements
Energy Booster 100 1.9
Megalac-R 1.7
Chemical composition
NE
L
, Mcal/kg 1.48 1.49 1.49
Crude protein 12.0 12.0 12.0
Ether extract 1.5 3.0 2.9
Crude NDF 45.0 48.0 49.0
Ca 0.6 0.6 0.8
P 0.3 0.3 0.3
NE
L
, net energy for lactation; NDF, neutral detergent fiber.
a
Treatments were: cows fed from 256 days of pregnancy a dry cow
ration and post partum fed a lactating cow diet (CTL) or supplemented
with a fat either containing a low (LUFA) or high (HUFA) proportion of
unsaturated fatty acids.
Dietary fatty acids and follicular steroids
689
www.reproduction-online.org Reproduction (2008) 135 683–692
aspiration) and at the day of follicle aspiration from the
jugular vein into vacuum tubes with lithium heparin
(Becton Dickinson Systems, Cowley, England). Plasma was
separated immediately from blood samples and stored at
K18 8C until analysis.
Chemical analysis
Total mixed rations were sampled weekly and dry matter,
crude protein, neutral detergent fiber (NDF), acid detergent
fiber (ADF), calcium, and phosphate were determined.
Feed samples were dried at 65 8C for 24 h and then ground
to pass through 1.0 mm screen (Retsch S-M-100). The
ground samples were dried at 100 8C for 24 h and analyzed
for N (AOAC, 1990; method 984.13; Kjeltec Auto 1030
Analyzer, Tecator, Hoganas, Sweden), Ca (AOAC, 1990;
method 935.13), P (AOAC, 1990; method 964.06), and
NDF and ADF contents were determined with Ankom
equipment (Ankom Technology, Fairport, NY, USA; NDF,
using a-amylase and sodium sulfite). Net energy for
lactation (NE
L
) values was calculated using the NRC
(2001) values except for the fat supplements. The rumen
undegradable protein (RUP) values of most of the feedstuffs
were from Arieli et al. (1989). The NRC (2001) RUP values
were used for feedstuffs that were not examined in Arieli
et a l. (1989).
Concentrations of P
4
and E
2
in FF, and plasma P
4
were
determined by RIA (Diagnostic Products, Los Angeles, CA,
USA) as well as FF A
4
concentrations (Diagnostic Systems
Laboratories, Webster, TX, USA). Concentrations of non-
esterified FA (NEFA) in FF were determined by a NEFA kit
(Wako NEFA C test kit; Wako Chemicals GmbH, Neuss,
Germany). Concentrations of insulin in FF were determined by
RIA (Diagnostic Products).
To determine the concentrations of plasma E
2
, 1 ml plasma
samples were extracted with diethyl ether (HPLC, Bio Lab Ltd,
Jerusalem, Israel) and then E
2
was determined by RIA kit (Third
Generation Estradiol kit, DSL-39100; Diagnostic Systems
Laboratories).
FAs in plasma and FF were extracted (Moallem et al. 1999)
and analysis of FA was performed with 5890 series 2 gas
chromatograph (Hewlett–Packard) equipped with a capillary
column (30 m!0.53 mm, 0.5 mm; Agilent Technologies, Santa
Clara, CA, USA) and an FID detector. The column was
maintained at 160 8C isothermal. Nitrogen was used as carrier
gas with a linear velocity of 22 cm/s; injection volume was 2 ml.
The injection port was maintained at 230 8C and the detector at
235 8C. Detector air flow was 400 ml/min and the hydrogen
flow was 33 ml/min. A known amount of C17:0 was added to
the samples prior to extraction in order to determine the FA
concentrations in the FF.
P450 aromatase mRNA expression in granulosa cells
Total RNA isolation
Total RNA was isolated from granulosa cells obtained from the
aspirated preovulatory follicles using TRI reagent (10 ml/g
tissue) according to the manufacturer’s protocol (MRC
Molecular Research Center, Cincinnati, OH, USA). RNA quality
and quantity were assessed by spectrophotometric measure-
ments at 260 and 280 nm. Only high-purity (260–280 ratio
between 1.75 and 2) RNA was used.
mRNA analysis
First-strand cDNAs were synthesized from 5 mg total RNA from
each follicle using oligo(dT)
18
as the primer in the presence of
MLV reverse transcriptase (Fermentas Inc., Hanover, MD, USA)
for 1 h at 42 8C. The cDNA was purified from the PCR mix
using High Pure PCR Product Purification kit (Roche
Diagnostics GmbH).
Real-time PCR
Expression of P450 aromatase mRNA in preovulatory follicles
was determined by real-time PCR carried out following the
manufacturer’s specifications using a GeneAmp 5700
sequence detection system (Applied Biosystems, Foster City,
CA, USA). mRNA samples were reverse transcribed as
described in the mRNA analysis section and 5 ml cDNA was
used in 50 ml final volume for the PCR. cDNA was amplified
by qPCR Mastermix for SYBR Green I, using primers designed
by the Primer Express version 2.0.0 software (Applied
Biosystems) as follows: forward 3
0
-GCCAAGAGCAACAAGCA-
TATCAG-5
0
and reverse 3
0
-CTTGGAAAATTCACTCAT-
CTGTTTGA-5
0
.
Relative mRNA expression of P450 aromatase was
determined by the DDC
T
quantification method as described
previously by Livak & Schmittgen (2001), using the relative
expression of GAPDH mRNA as a reference. C
T
stands for
the threshold cycle, that is, the PCR cycle in which an increase
in reporter fluorescence above a baseline signal can first
be detected.
Statistical analysis
The continuous variables (milk and dry matter intake) were
analyzed as repeated measurements using Proc Mixed of SAS
software (version 8.1, SAS User’s Guide 2000). The final model
used was
Y
ijklm
Z m C T
i
C L
j
C CðT !LÞ
ijk
C DIM
ijkl
C DIM
ijkl
!DIM
ijkl
C DIM
ijkl
!DIM
ijkl
!DIM
ijkl
C E
ijklm
where mZoverall mean, T
i
Ztreatment effect,
i
Z1–3, L
j
Z
parity,
j
Z2orO2, C(T!L)
ijk
Zcow
k
nested in treatment
i
and
cow nested in parity
j
, DIM
ijkl
Zday in milk as continuous
variable, and e
ijklm
Zrandom residual. Whenever the quadratic
or cubic effects were not significant, they were excluded
from the model and the model was rerun. Milk production
was analyzed using the previous lactation data as co-variable.
Dry matter intake was analyzed using the pre-treatment
body weight as co-variable. The follicle diameter, volume,
hormones, NEFA, and FA profile and concentrations were
690 M Zachut and others
Reproduction (2008) 135 683–692 www.reproduction-online.org
analyzed using the general linear models procedure of SAS
(2000). Across treatments correlations analysis were per-
formed using the Proc REG procedure of SAS software
(2000). The analysis of mRNA expression was carried out
using the JMP (version 5.0.1, SAS Institute, Cary, NC, USA).
Gene expression levels were compared using one-way
ANOVA.
Least squares means and adjusted
S.E.M. are presented in the
tables and P!0.05 was accepted as significant unless
otherwise stated.
Acknowledgements
This research was supported by Research Grant No. US-3422-03
R from BARD, The United States – Israel Binational Agricultural
Research and Development Fund. The authors gratefully
acknowledge the kind donation of Megalac-R by Church &
Dwight (Princeton, NJ, USA) and Energy Booster 100 by Milk
Specialties (Dundee, IL, USA). They also thank the experimental
dairy farm’s team at the Volcani Center, Bet Dagan, Israel for their
assistance with animal care. The authors declare that there is no
conflict of interest that would prejudice the impartiality of this
scientific work.
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Received 13 December 2007
First decision 29 January 2008
Accepted 1 February 2008
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Reproduction (2008) 135 683–692 www.reproduction-online.org
