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CREDIT

SEMINAR
O
n

ROLE OF POLYUNSATURATED
FATTY ACIDS oN BOVINE
FOlLICULOGENESIS

INTRODUCTION
Dietary fats influence the reproductive functions
Increasing energy balance
Direct action on reproductive processes
Thatcher et al., 2003

Polyunsaturated fatty acids constitute the major


portion of the fatty acid content of the follicular
fluid in ovarian follicles
Changing the fatty acid content of the diet was
found to reflect on the fatty acid distribution in
(Bilby et al., 2006)
reproductive tissues

Sources of major fatty acids


OIL (w/w, % FA) C 18:0

C 18:1

C 18:2

C 18:3

Safflower

12

77

<1

Cottonseed

25

21

50

Linseed (flax)

19

14

58

Corn

25

60

Tallow

15

41

Fishmeal

25

45

Olive

76

Soyabean

24

53

Sunflower

20

69

1
(Staples and Thatcher, 2005)

Pathway of Desaturation and Elongation of n-3


and n-6 fatty acids

(Mattos et al., 2004)

Metabolism of fatty acids to series


1, 2 or series 3 eicosanoids

Fortier et al., 2008

Effect of omega-3 on reproductive


hormones

Progesterone concentrations in the follicular


fluid were greater (p<0.001) for ewes offered
the n-3 diet than the n-6 diet
(Wonnacott et al., 2010
Cows fed diet high in the LA decreased the
level of progesterone during the luteal phase of
estrous cycle
(Robinson et al., 2002)

Plasma progesterone concentrations of cows


fed SFA were lower (p < 0.01) than those of
(Ambrose et al., 2006
cows fed Flaxseed and Sunflower
Progesterone concentrations were signicantly
higher in cows fed Flaxseed or Sunflower than
(Thangavaleu et al., 200
in those fed SFA.

Plasma Progesterone
concentration

SAT; Energy Booster; 3.75% DM,


whole flaxseed (FLX); 10% DM,
whole sunflower seed (SUN); 10.5% DM.

(Thangavaleu et al., 2007)

Fewer no. of small follicles in cows


supplemented with flaxseed (rich in C18:3n-3)
than in cows fed soybean (rich in C18:2n-6)
(Ponter et al., 2006)

The mean diameter of the ovulatory follicle


and CL was higher when dairy cows were fed
diets high in n-3
(Ambrose et
al., 2006)
Increase in the number of large follicles
and
their diameter with PUFA supplementation

(Robinson
Cows that were fed sh oil increased
theet al., 2002)
number of medium follicles (510 mm in
diameter)

(Moussavi et al., 2006

The ovulatory follicle was larger in cows


fed Flaxseed compared with those fed
Sunflower (16.9 VS 14.1 mm) (Ambrose et al., 2006)
Increase in diameter of largest follicle,
diameter of CL , increase in no. of medium
sized follicles, decrease in no. of small
follicles in n-3 supplementation as
compared to Megalac.

(Petit et al., 2002

Higher fertility was reported in cows


ovulating larger follicles even without an
increase in progesterone concentrations in
the subsequent luteal phase (Peters and Pursley.
2003)

Effects of dietary fats differing in n-6:n-3


ratio fed to high-yielding dairy cows on
oocyte quality
Item

Control

E-FLAX

E-SUN

Cows, n

Oocytes, n/cow

5.20

4.88

3.75

Grade 1 oocytes, n

3.74

3.45

2.50

Grade 1 + 2 oocytes, n

4.12

3.98

3.02

Oocytes, n/cow for IVF

3.05

3.29

2.16

49.73

47.07

Cleaved, % of oocytes 41.49

Encapsulated sunflower; 3.8 %DM,260.0 g of


C18:2n-6
Encapsulated flaxseed ; 3.8 % DM, 242.2 g

(Zachut et al., 2010

No. of class 2
follicles

Size of the CL
MEG; 2.8%DM, EE
6.5%
(FW)LIN; 6.1%, EE
5.2%
FIS ; 3.4%, EE 5.4%
OIL. (L); 500gm, EE
4.9%
(Petit et al., 2002)

Effects of different proportion of dietary n-6 and


n-3 PUFA on ovarian function in lactating dairy
cows
Parameter

Control

ALA (n-3)

LA (n-6)

n-6: n-3 ratio

0.59

0.36

1.66

Number of cow

No. of follicles (5-10 mm)

1.8

3.4

3.5

Maximum diameter of 1st dominant


follicle (mm)

13.3

15.6

16.9

ALA: LinPreme; xylose treated cracked linseed under steam;1.3kg/day


LA: SoyPreme; xylose treated cracked soybean under steam;2.8kg/day

(Robinson et al., 2002)

Treatment
Variables

Control

FLX

FO

P<

Total number of
oocytes aspirated

248

361

306

0.05

Oocytes
recovered/cow per
session

3.3

4.8

3.9

0.03

Cleavage rate (%)

35

52

48

0.01

No. of cleaved
oocytes/cow per
session

1.06

1.97

1.66

0.04

Control-Encapsulated SFA 1.9% and 2.2%, Encapsulated


FLX oil 2.4%, 2.9% and Encapsulated FO2.5%, 2.9%
(DM) prepartum and Postpartum, respectively

(Moallem et al.,
2013)

Effect of PUFA on fatty acid


concentrations in reproductive
tissues

Fatty acid profile in follicular fluid obtained


from pre-ovulatory follicles (% of total fatty
acids)
FA (%)

SFA

FLX

FO

P value

C18:2 n-6

45.38

45.48

40.99

NS

C18:3 n-3

2.20

5.69

1.90

0.001

C20:5n-3

0.29

0.32

0.76

0.03

C22:6n-3

0.03

0.03

0.28

0.001

n-3

2.78

6.10

3.53

0.001

n-6

48.12

47.25

42.41

NS

Fatty acid prole of granulosa cells (% of total fatty


acids)
n-3

2.64

4.71

2.99

0.001

n-6

41.22

33.80

31.13

0.03

Moallem et al., 2013

Effect of n-3 PUFA supplementation on


bovine endometrial fatty acid
concentrations

Fatty acid (% of total


FA)

Control

High PUFA (n- P value


3)

C18:2 Linoleic acid

7.39

4.53

<0.0001

C18:3 Linolenic acid

0.61

0.38

0.001

C20:4 Arachidonic acid

7.61

5.11

<0.001

C20:5 Eicosapentaenoic
acid

0.73

4.22

<0.0001

C22:6 Docosahexaenoic
acid

2.85

3.78

<0.01

Omega 6

15.01

9.64

<0.0001

Omega 3

4.20

8.39

<0.0001

CON; Palmit 80 Prills (80% palmitic acid) ; 310 g/Kg as fed.


Treatment; n-3 PUFA supplement; 667 g/Kg as fed.
(Coyne et al., 2008)
Both were partially rumen protected.
EE; CON; 27g/Kg DM. Treatmnt; 31g/Kg DM.

Effect of n-3 PUFA supplementation on


bovine endometrial gene expression

Not affected(Caldari et al., 2006)


Not affected

PGE2 considered a
luteotrophic mediator
and thus facilitates
the
Establishment of
pregnancy

Not affected

(McLaren et al.,

2006)

(Coyne et al., 2008)

Effects of PUFA supplementation


on invitro
PG production by ovine amnion
cells
Mean percentage of
each isoform of PGE
in samples

PUFAs

PGE1

PGE2

PGE3

Total PGE ng/ml per


45 h

CONT

36.0

30.7

33.0

31.8

DGLA, 20:3,
n-6

76

8.7

15.3

125.5

AA, 20:4, n-6

20.7

64.3

14.3

85.1

EPA, 20:5, n3

29.7

28.7

41.0

16.1
Kirkup et al., 2011

Oocytes quality and maturation

Fatty acids may affect oocyte maturation


directly, through altering the fatty acid
composition of oocytes lipids (Bender et al., 2010)
indirectly, by influencing the
concentrations of PG and other
metabolites in the follicular(Fouladi-Nashta
fluid
et al., 2009
surrounding the oocyte
Oocyte maturation both in vitro and in
vivo was improved following the inclusion
of short-chain (ALA) or long-chain (sh
(Zeron et al., 2002)
oil) n-3

Supplementation with n-6 may be


detrimental to oocyte maturation as they
inhibit the resumption of meiosis at the
germinal vesicle stage preventing the oocyte
from further maturation
(Marei et al., 2010)
The number of blastomeres of morula tended
to be higher (P < 0.09) in cows fed Flaxseed
than in those from cows fed SFA or Sunflower

(Thangavaleu . 2007)

Higher numbers of grade 1 oocytes were


collected from ewes supplemented with PUFA
(Zeron et al., 2002)

Diets enriched in unsaturated fatty acids


enhance early embryonic development in
lactating Holestein cows
Stage of
embryo

Morula
Blastocyst
Expanded
Blastocyst

Dietary groups
SAT

FLX

SUN

P
value

64.4
77.5
89.3

76.3
88.6
115.4

65.6
93.7
132.3

0.09
0.07
0.02

93.4

97.1

0.01

All embryo stages 77.1

Mean total number of Blastomere nuclei of embryos

SAT; Energy Booster 3.75% DM,


whole flaxseed (FLX) 10% DM,
whole sunflower seed (SUN); 10.5% DM.

(Thangavaleu .2007)

Impact of Dietary Fatty Acids on Oocyte


Quality and Development in Lactating Dairy
Cows
Variable

Low-fat

High-fat P
value

No. of aspirated oocytes

570

481

In vitro-fertilized oocytes

495

418

Oocyte recovery rate (%)

53.3

57.8

0.47

Mean small follicles (<4 mm)

7.1

6.1

0.021

Mean medium follicles (410


mm)

9.3

7.9

0.051

RIF (Megalac) ;
Low fat; 200g/day
High fat ; 800g/day
40-60 days after
calving

The fatty acid composition of RIF was


47% palmitic, 5% stearic, 38% oleic,
9% linoleic,
and 1% linolenic acids
(Fouladi-Nashta et al., 200

Effect of dietary fats on oocyte quality and


development
Variable
Low-fat
High-fat
P value
Oocytes quality
Grade 1

0.86 (11.6%) 0.86 (10.8%)

0.557

Grade 2

3.3
(35.6%)

2.36 (40.3%)

0.082

Grade 3

2.3
(32.2%)

2.1

Total cells

132.5

150.5

0.043

ICM

33.8

36.4

0.46

Cleavage rate

72.06

66.6

0.076

Blastocyst/IVF

19.4

27.4

0.004

38

0.017

(28.2%) 0.147

Development (%)

Blastocyst/cleav 29.1
ed

(Fouladi-Nashta et al., 2007

Impact of LA and ALA on bovine oocyte


maturation and embryo development
LA supplementation after
24 h

ALA supplementation after


24 h

(Waleed et al., 2009)

Effect of LA and ALA on


percentage of oocytes at MII stage
LA supplementation after
24 h

ALA supplementation
after 24 h

(Waleed et al., 2009)

Effect PUFA on PG
ConcentrationProduction
in spent
media
after maturation for 24 h
in the presence of LA
(mM)

Linolenic acid (50 M) resulted in a


signicant increase in the concentration of
PGE2 that of the control
PGE is an autocrine/ paracrine
Act via G-protein coupled receptors
(PTGER) on cumulus cells
receptors (PTGER2 and PTGER3) are
expressed in bovine COCs
cAMP, 2
messenger

Increased PGE2 production


could not overcome the
inhibitory effect of LA on
cumulus cell expansion and
oocyte maturation
(Waleed et al., 2009)

Critical mediator of oocyte maturation


and cumulus expansion
Knocking out genes encoding for PTGER
attenuates cummulus cell expansion and
oocyte nuclear maturation

Intracellular cAMP
concentration in COCs
LA supplementation(100 M)

50 M ALA, 3 M forskolin,
or both

Addition of forskolin increased cAMP


reaching a higher peak at 6 h.

Luteinizing hormone
stimulation
high cAMP
Dekel, 1988
selective activation of PKA type II by cAMP in
cumulus cells
selective inhibition of phosphodiesterase enzyme type 4
is needed for resumption of meiosis
(Luciano.
2004)
The concentration of cAMP in cumulus cells at 3 h is an important
regulator of
cumulus expansion and oocyte maturation

low cAMP in bovine COCs resulted in premature interruption of


cumulus oocyte gap junction communication and inhibition of
cumulus cell expansion, leading to defective oocyte maturation and
development
(Modina et al., 2001

MAPK1 AND MAPK3 ACTIVATION


LA (100M)
supplementation

ALA (50M)

Sustained high levels of MAPK activity at 24 h are


essential for maintaining the oocyte in MII arrest
because of their involvement in the regulation of
microtubule organization and meiotic spindle
assembly
(Tian et al., 2002)

its inactivation after that is a prerequisite for


pronuclear formation after fertilization
(Fan & Sun 2004)

AKT
PHOSPHORYLATION

Treatment of COCs

The effect of LA (100 mM)


on AKT phosphorylation.

The effect of ALA (50 M)


on AKT phosphorylation.

Akt is an important protein kinase which


stimulates transition of bovine oocytes from MI
to MII stage

(Tomek & Smiljakovic 2005

Akt is also important regulator of


translation and
protein synthesis in bovine oocytes
(Tomek et al., 2002)

EFFECT OF PUFA SUPPLEMENTATION ON OOCY

(Waleed et al.,
2009)

o The effect of high n-3 diets on ovulation


rate remain unclear
o Non-signicant alteration in ovulation
rate in heifers fed n-3 or n-6 diets may
have been masked by the superovulation induced by follicle stimulating
hormone (FSH) treatment
(Childs et al., 2008a)
o In addition, low numbers of animals in
some studies prevents a meaningful
(Burke et al., 1996)
interpretation of ovulation rate data

Embryo survival

Diets high in n-3 may reduce PGF2


synthesis which may prevent regression
of the CL, allowing continued secretion
of P4 that may improve embryo survival
Positive effect of n-3 on embryo survival
through reduced PGF2 secretion by
BEND cells in vitro
(Caldari-Torres et al., 2006
Embryo mortality was lower (P= 0.07)
following supplementation with the short
chain n-3 ALA from linseed in dairy cows

(Petit and Twagiramungu, 2006

The number of degenerate embryos


et al., 2008a)
was reduced following dietary(Childs
inclusion
of n-3
PUFA could improve Blastocyst quality
(Thangavelu
when compared with palmitic
and et al., 2007
stearic acids
increased conception rates in cows
given formaldehyde-treated flaxseed,
Petit et al., (2001)
compared with cows given a control
ration (low in ALA) supplemented with
Megalac.

Effect of dietary PUFA on super-ovulation


response, embryo recovery rate and
development in cattle
Control

n-3 PUFA

P-value

Estimated ovulations

17.50

15.06

NS

No. of non-fertilised

1.063

1.69

NS

No. of degenerate embryos 4.06

1.25

<0.05

No. of morula

0.94

1.81

NS

No. of early blastocysts

4.18

3.81

NS

No. of transferable
embryos (grades 1 and 2)

6.60

5.88

NS

CON; Palmit 80 151g/Kg DM ; n-3 PUFA


supplement,334g/ Kg DM

Childs et al., 2008

Lower Pregnancy Losses in Lactating Dairy


Cows Fed a Diet
Enriched in -Linolenic Acid
Observation (%)
FLAX
SUNF
Cows conrmed pregnant (n)
42
35
(32 d)
Early pregnancy loss (32 to 90
d)

4.8

11.4

Late pregnancy loss (90 d to


term)

5.1

17.2

Overall pregnancy loss (early


and late)

9.8

27.3

Sunflower
seed,of
rolled;
8.7
%
Proportion
cows
calving
Even though
cows received
90.2
72.7the
DM Flaxseed, rolled ; 9.0%
experimental diets for only 60 d (i.e.,
DM
from 28 d before TAI to 32 d after
EE, %DM; 7.15%
TAI), the ALA-enriched diet during
(SUNF),7.23%(FLAX)
Ambrose et al., 2006

early gestation improved


maintenance of pregnancy

Specic effects of n-3 and n-6 on


pregnancy rates in ruminants are variable
Pregnancy rates were higher and
pregnancy losses were lower when dairy
cows were fed diets high in n-3 compared
with n-6 or saturated Santos
fat et al., 2008, Wathes et
al., 2007)

Pregnancy rates were lower when beef


(Hess et al., 2008)
cows were fed diets high in the n-6 LA
Lower pregnancy rates may result from a
reduction in the number of functional CL
caused by increased concentrations of
luteolytic PGF2 from high n-6 diets
(Hess et al., 2008)

Gestation length and


parturition

In sheep, gestation length was extended by


two days when ewes were supplemented with
sh oil in the third trimester compared with
Megalac

(Capper et al., 2006)

Sheep fed a diet rich in n-6 PUFAs during late


gestation had higher circulating
concentrations of 2-series PGs in uterine and
placental tissues, and appeared to be more
susceptible to preterm parturition (Elmes et al.,
2004)
Betamethasone-induced preterm delivery
at
day 125 was delayed when ewes were infused
with a lipid high in n-3 from sh oil compared
with n- 6
(Ma et al.,
2000).

Gestation length was also longer (p =


0.08) when ewes received an algal
supplement high in DHA
(Pickard et al., 2008)
Dietary n-3 PUFAs in the timing of
parturition with a diet devoid of sh
during pregnancy being associated
with a
(Olsen et al. 2006)
higher risk of pre-term delivery in humans

Increased concentrations of EPA and DHA


in caruncular tissue are also associated
(Mattos et al., 2004)
with later calving

Raised dietary n-6 PUFA


and PG synthesis during
labour
in
ewes
TIME OF LABOUR ONSET
Early labour

LA supplemented
(n=8)

132d
138d
132d

Dexa- induced
labour

Control

40 hours

LA supplemented

32 hours

The basal concentration of both maternal PGFM and fetal PGFM


was nearly double in LA supplemented ewes on 139d before
Dexamethasone infusion

CONT; Megalac
-18g/day/sheep
TRET; SoyPreme190g/day/sheep

(Elmes et al., 2005)

Effects of the different feeding


treatments on gestation length and
measures of lamb viability

C
Gestation Length
(days)

145.2

Birth weight (kg)

5.2

3 week

6 week 9
week.

P
value

147.1

147.5

148.0

0.08

5.4

5.2

5.3

NS

21.6

22.6

0.05

Time to stand
31.0 27.9
(min)
C; Vegetable oil ; 32 g/ewe per day

,
S; 64 g algae /ewe per day, to provide 12 g

(Pickard et al., 2008

Mechanisms of inhibition of prostaglandin


secretion
1. Reduced synthesis of arachidonic
acid
Dietary PUFAs, particularly EPA and DHA are major

inhibitors of desaturation and elongation of FA


The 5 and 6 desaturase activity of rat hepatoma cells
was reduced when they were incubated with EPA
and DHA
(Larsen
et al., 1997

2. Competition of n-3 fatty acids for


desaturase activity

Presence of high concentrations of linolenic acid in the diet


would compete with linoleic acid for binding with 6
desaturase and reduce the conversion of linoleic acid to
arachidonic acid

3. Altered fatty acid profile in the plasma


membrane

Reduced availability of arachidonic acid for incorporation into


plasma membrane will result in greater incorporation of other fatty
acids, which may or may not be precursors of other eicosanoids
(Mattos,
2000)

4. Competition of PUFAs
for PGHS
Increased amounts of EPA could result in increased
synthesis of prostaglandins of the 3 series, and
reduced synthesis of PGF2
5. Effects on gene
expression
PUFAs involve activation of nuclear transcription
factors such as peroxisome proliferator activated
receptors (PPARs)

The PPARs interact with peroxisome proliferator


response elements (PPREs) in the regulatory
(Jumpregion
et al., 1996)
of target genes to activate or repress transcription

PUFAs bind to PUFA-binding proteins. The PUFA


PUFA binding protein complex then binds to
(Sessler and Ntambi, 1998
response elements to repress gene transcription

Proposed model of effect of diet rich in


n-3 fatty acids
on the synthesis of eicosanoids

(Mattos et al., 2000)

Model linking omega-3 polyunsaturated fatty acids


with
prostaglandins, hormones and reproduction success
in ruminants

CONCLUSION
Diets enriched in PUFA accelerates early embryonic
development in dairy cows, compared to a diet
enriched in saturated fatty acids.
Dietary fats influence the reproductive functions by
improving energy balance or direct action on
reproductive processes
PUFA has benecial effect on reproductive hormones,
oocytes
number,
quality,
maturation
and
development, decrease in early embryonic mortality,
enhanced embryo survival and neonatal behaviour.
PUFA affect the expression of genes involved in
many reproductive processes.
So the strategic supplementation of PUFA may be
used to increase the fertility in dairy animals

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