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Effect of Calcium Soaps of Fatty Acids and Administration
of Somatotropin on Milk Production, Preovulatory Follicular
Development, and Plasma and Follicular Fluid Lipid Composition
in High Yielding Dairy Cows
U. MOALLEM,*
,
† Y. FOLMAN,* A. BOR,*
A. ARAV,* and D. SKLAN†
1
*Institute of Animal Science, Agriculture Research Organization,
The Volcani Center, Bet Dagan 50250, Israel
†Hebrew University Faculty of Agriculture, Rehovot 76100, Israel
ABSTRACT
The effect of fat and bovine somatotropin (bST) on
preovulatory follicular hormones and lipids was evalu-
ated by feeding cows for 150 d from parturition a con-
trol diet, a control diet plus 0.55 kg/d of calcium soaps
of fatty acids, or a control diet with 500 mg of bST
injected every 14 d. Fourteen days after a synchronized
or natural estrus, cows were injected with a PGF
2α
analogue; 48 h later, follicular fluid from all ovarian
follicles >8 mm was aspirated. Cows fed fat or injected
with bST produced more milk and milk solids than did
control cows, and cows on the bST treatment lost more
body condition after calving than did cows on the other
treatments. Both treatments changed the proportion
of estradiol-active follicles (>400 ng of estradiol/ml of
follicular fluid) and the correlation between follicular
fluid estradiol concentration and the total number
large follicles per cow. In follicles aspirated between 60
and 90 DIM the percentage of estradiol-active follicles
was 67, 40, and 0 for cows on the control, calcium soaps
of fatty acids, and bST treatments, respectively. After
90 DIM, no differences existed between treatments in
the percentage of estradiol-active follicles. Estradiol
concentration in follicular fluid was correlated with
DIM at follicle aspiration (r = 0.51). The proportion of
oleic acid in free fatty acids in plasma at 50 DIM was
lower in control cows and was lower in follicular fluid
of estradiol-active follicles. Both calcium soaps of fatty
acids and bST had a considerable effect on follicular
development and activity and the composition of fatty
acids in follicles.
(Key words: calcium soaps, bovine somatotropin, pre-
ovulatory follicles, fatty acids)
Received November 30, 1998.
Accepted June 14, 1999.
1
Please send correspondence to D. Sklan, Faculty of Agriculture,
PO Box 12, Rehovot, 76-100, Israel.
1999 J Dairy Sci 82:2358–2368 2358
Abbreviation key: BCS = body condition score,
CSFA = calcium soaps of fatty acids, E
2
= estradiol-
17β, E
2
-A = estradiol active, E
2
-I = estradiol inactive,
E
2
-LA = estradiol less active, P
4
= progesterone.
INTRODUCTION
The enhanced negative energy balance associated
with increased milk production depresses the fertility
of high yielding dairy cows (4). In our previous study,
cows treated with somatotropin (Sometribove, Mon-
santo Co., St Louis, MO) in early lactation produced
5 to 6 kg/d more milk than did control cows during the
first 150 DIM (22). This increase in production was
accompanied by a severe negative energy balance and
also by lower fertility in comparison with control cows.
Most of the trials with bST have been carried out from
60 DIM or later, and in some studies, but not in others,
lower reproductive performance of cows treated with
bST was reported (5, 7, 8, 9, 23, 32).
In our previous study (22), feeding calcium soaps
of fatty acids (CSFA) decreased, to some extent, the
negative effects of enhanced milk yield on reproductive
performance. The CSFA fed to cattle have been re-
ported to influence ovarian dynamics by increasing the
diameter of the preovulatory follicles and by changing
follicle turnover (1, 31). These effects of CSFA con-
sumption on reproductive function could be achieved
either through improving the energy balance of the
cows or by the direct influence of lipid components
on the reproductive system and especially developing
follicles in ovaries.
The objectives of this experiment were to character-
ize the preovulatory follicles and their steroidogenic
capacity as influenced by hormonal (bST) and nutri-
tional (CSFA) treatments, which affect milk yield and
energy balance. Specifically, the objectives were 1) to
investigate the effect of CSFA or bST on preovulatory
follicular development and 2) to study the effect of
CSFA or bST on lipid composition in the follicular fluid
FAT AND BOVINE SOMATOTROPIN ON FOLLICULAR HORMONES 2359
TABLE 1. Ingredients and nutrients of the control diet.
Composition % of DM
Corn grain 21.7
Barley grain 10.9
Soybean meal 4.4
Rapeseed meal 4.4
Sunflower meal 3.2
Gluten feed 1.3
Cottonseed 6.8
Wheat silage 8.0
Corn silage 19.8
Wheat bran 6.9
Citrus pulp 3.6
Pea hay 3.1
Oats hay 3.1
NPN (mixture)
1
0.5
Soybean oil 0.1
Salt/calcium 1.6
Fish meal 0.4
Vitamins and minerals
2
0.018
Nutrients
NE
L
3
(Mcal/kg) 1.72
CP 17.2
RUP
4
5.3
ADF 17.3
NDF 31.2
P 0.46
Ca 0.80
1
Contained 80% urea and 20% ammonium sulfate.
2
Contained 20,000,000 IU of vitamin A/kg, 2,000,000 IU of vitamin
D/kg, 15,000 mg of vitamin E/kg, 6000 ppm of Mn, 6000 ppm of Zn,
2000 ppm of Fe, 1500 ppm of Cu, 120 ppm of I, 50 ppm of Se, and
20 ppm of Co.
3
Calculated using NRC (24) values.
of preovulatory follicles and the possible relationship
with steroidogenesis.
MATERIALS AND METHODS
Fifty-four multiparous Israeli-Holstein cows of the
Bet Dagan (Israel) experimental farm were assigned
to three treatments. The cows were kept in covered,
loose pens with adjacent pen yards. The cows were
weighed, and body condition was determined 1 wk be-
fore expected parturition. Cows were blocked ac-
cording to parity, date of parturition, body weight,
body condition score (BCS), and daily FCM yield dur-
ing the first 150 d of the previous lactation into the
following three treatments: 1) control, fed the diet de-
scribed in Table 1; 2) CSFA, fed the control diet plus
0.55 kg of calcium soaps of fatty acid per cow per d
from parturition to 150 DIM (chemical composition of
CSFA: 47.4% palmitic acid, 5.2% stearic acid, 33.3%
oleic acid, 6.0% linoleic acid, and 8.1% calcium; Koffolk
Inc., Petach Tikva, Israel); 3) bST, fed the control diet
and injected subcutaneously with 500 mg of Zn-Some-
tribove every 14 d from 10 to 150 DIM. Groups were
filled within a 12-wk period, and the experiment was
Journal of Dairy Science Vol. 82, No. 11, 1999
carried out with cows calving between October and De-
cember.
All feeds were mixed and fed once daily from a mix-
ing wagon. Orts were removed and weighed daily.
Cows were milked three times daily, and milk yield
was recorded electronically. Milk composition was de-
termined from a composite of three consecutive milk-
ings every 2 wk until 150 DIM and then monthly until
the end of lactation. Fat, protein, and lactose determi-
nations of milk were carried out with infrared analysis.
From parturition, cows were weighed three times daily
after every milking with an automatic electronic scale.
The BCS on a 5-point scale was determined by one
experienced technician weekly from parturition to 200
DIM (6).
Blood samples were collected from the jugular vein
at 09:30 h every 2 d from 10 DIM into heparinized
vacutainers (Becton Dickinson Vacutainer Systems
Europe, Cowley, England) that were placed on ice,
centrifuged, and stored at –18°C until analysis.
Clusters of cows were given a single injection of 625
ng of the PGF
2α
analogue cloprostenol (Estrumate,
Coopers Animal Health Ltd., Berkhamsted, England)
commencing after 50 DIM to facilitate estrus detec-
tion. The interval from parturition to the first PGF
2α
injection was 68.7 ± 10, 70.1 ± 13, and 70.0 ± 8din
the control, CSFA, and bST groups, respectively. After
the PGF
2α
injection, cows were visually observed for
signs of estrus for 30 min twice daily. Cows were re-
garded as being in estrus if they manifested standing
estrus or mounted other cows that were not in estrus.
In addition, estrus was detected by measuring, with a
pedometer, the number of steps taken by cows between
milkings (Computerized Dairy Management Systems,
S.A.E. AFIKIM, Afikim, Israel). Whenever the number
of steps was 90% greater than the mean number of
steps during the previous 10 d, the cow was regarded
as being in estrus.
Thirteen cows that manifested estrus (7 control, 3
bST, and 3 CSFA), when a cluster of PGF
2α
-treated
cows were detected in estrus, were added to these clus-
ters without receiving the first PGF
2α
injection. Cows
that manifested estrus were allotted into a group
scheduled for aspiration of follicular fluid. Fourteen
days following estrus, cows were given a first or second
PGF
2α
injection to cause luteolysis and enable preovu-
latory follicular development. Forty-eight hours fol-
lowing the injection, aspiration of follicular fluid was
carried out. Cows that were not detected in estrus
following a PGF
2α
injection were given another PGF
2α
injection with the next cluster of cows. Within 5 mo,
eighteen clusters of cows were treated. As a result of
this procedure, the aspiration of follicular fluid was
carried out between 65 and 139 DIM, depending upon
MOALLEM ET AL.2360
the DIM when cows were seen in estrus and whether
cows responded to the first PGF
2α
injection.
Ovaries of cows were viewed by ultrasonography,
and follicular fluid was aspirated from follicles that
were ≥8 mm. The aspiration was by the ovum pickup
procedure (25), and needles were changed between
cows (Scanner 200; Pie Medical, The Netherlands).
Cows were given local anesthesia with 5 ml of 2%
lidocaine HCL injected epidurally between the last
sacral and first caudal vertebrae. Each follicle was
aspirated into a single calibrated tube where the vol-
ume of follicular fluid was determined. The fluid was
centrifuged and stored at –18°C until analysis. Sev-
enty-nine follicles were successfully aspirated from 46
cows out of the 54 that were included in the experi-
ment. In 5 cows, the anatomic structure of the repro-
ductive tract or the behavior of the cows did not enable
the aspiration of follicles, and 3 cows were not detected
in estrus.
Progesterone (P
4
) concentration in plasma and fol-
licular fluid was determined by radioimmunoassay as
previously described (30); estradiol-17β (E
2
) was also
determined (3). Lipids were extracted (10), and lipid
classes were separated by thin layer chromatography
and quantitated as previously described (30). Priority
was given to hormonal determinations, and in some
cases, insufficient material was aspirated for lipid
analyses. Concentration and composition of NEFA in
plasma were determined at 50 DIM and at 2 d before
follicle aspiration. Initiation of P
4
cyclicity was defined
as 5 d prior to the time when plasma P
4
concentrations
first reached 1 ng/ml after parturition.
Follicles were regarded as E
2
active (E
2
-A)orE
2
less active (E
2
-LA) whenever the E
2
(nanograms per
milliliter)/P
4
(nanograms per milliliter) ratio in follicu-
lar fluid was >1. Follicles were regarded as E
2
-inactive
(E
2
-I) whenever the E
2
/P
4
ratio was <1 (13). When the
frequency distribution of E
2
concentrations in 33 E
2
-
A dominant follicles was examined, it appeared that
the distribution was not normal, and that two separate
populations could be discerned. After logarithmic, x
2
and x
0.2
transformations of E
2
concentrations were car-
ried out, the boundary between the two populations
was within the range of 403 to 441 ng of E
2
/ml of
follicular fluid. We subsequently termed the two popu-
lations as E
2
-A and E
2
-LA. Thus, 25 cows with E
2
con-
centrations >400 ng/ml in follicular fluid had E
2
-A folli-
cles, and 8 cows with E
2
concentrations of <400 ng/ml
had E
2
-LA follicles. The classification of E
2
-A follicles
into two categories of more and less active enabled
further clarification of the effects of CSFA and bST
treatments on follicular development and E
2
syn-
thesis.
Journal of Dairy Science Vol. 82, No. 11, 1999
TABLE 2. Mean milk and milk solids production during the first 150
d of lactation.
Control
1
CSFA bST
(n = 18) (n = 18) (n = 18) SEM
Milk, kg/d 39.7
c
42.5
b
44.0
a
0.5
Fat, % 3.18 3.25 3.19 0.05
Fat, kg/d 1.27
b
1.39
a
1.39
a
0.02
Protein, % 2.98
a
2.92
b
2.92
b
0.03
Protein, kg/d 1.18
c
1.24
b
1.28
a
0.01
3.5% FCM, kg/d 37.8
b
40.9
a
41.9
a
0.5
a,b,c
Means within a row without a common superscript differ (P <
0.05).
1
Control, fed the control diet; CSFA, fed the control diet plus 0.55
kg/d of calcium soapsoffattyacids; and bST, control diet plus adminis-
tration of 500 mg of Zn-Sometribove (Monsanto Co., St. Louis, MO)/
14 d.
Statistical Analysis
Continuous variables were analyzed by ANOVA in
which the three treatments were the sources of varia-
tion using the general linear models procedure of SAS
(29) as previously described (22). Frequency data were
examined using the CATMOD procedure of SAS (29).
Least squares means and adjusted standard errors are
presented in the tables. Significance was at P < 0.05
unless otherwise stated.
RESULTS
Production of Milk and Milk Solids
The group mean DMI during the first 150 DIM was
24.0 ± 1.7, 23.3 ±2.3, and 24.7 ± 2.4 kg/d for the control,
CSFA, and bST groups, respectively. Milk, fat, and
protein yields during the first 150 DIM are shown in
Table 2. Cows fed CSFA and cows treated with bST
produced 2.8 and 4.3 kg/d, respectively, more milk
than did control cows. The milk protein percentage
was higher in control cows than in the other treat-
ments, but cows fed CSFA or treated with bST pro-
duced 5.1 and 8.5% more protein, respectively, than
did control cows. The FCM and fat yields of cows were
enhanced by CSFA and bST.
BW and BCS Changes
The BW changes from parturition until 180 DIM are
shown in Figure 1. The minimum BW was attained
later after calving for cows treated with bST than for
cows on the other treatments (P < 0.05). Cows on the
CSFA treatment lost slightly more BW than did cows
on the other treatments. Cows began gaining BW after
20, 30, and 48 DIM on control, CSFA, and bST treat-
ments, respectively. By 120 DIM, control and CSFA
cows had returned to their calving weights, whereas
FAT AND BOVINE SOMATOTROPIN ON FOLLICULAR HORMONES 2361
Figure 1. Changes in BW (top panel) and body condition score
(BCS) (bottom panel; 5-point scale where 1 = very thin to 5 = very
fat) during 180 DIM. Treatments were control (䉭), control plus 0.55
kg/d of calcium soaps of fatty acids (䊊), and control diet plus adminis-
tration of 500 mg of bST (Zn-Sometribove; Monsanto Co., St. Louis,
MO)/14 d (䊉).
bST cows returned to their parturition weights only
at 140 DIM. Changes in BCS are shown in Figure 1
and in Table 3. Cows on the bST treatment lost a
maximum of 1.10 U of BCS after calving compared
with 0.75 and 0.69 U for the CSFA and control cows,
respectively (P < 0.02). The minimum BCS of bST-
treated cows was attained 11 to 12 d later than for
cows of the other two groups, but this difference was
not statistically significant.
Frequency of Appearance of E
2
-A
and E
2
-I Follicles
The P
4
concentrations in plasma following the first
PGF
2α
injection or spontaneous estrus until 7 d after
aspiration are shown in Figure 2. During the 6 to 7 d
after aspiration, P
4
concentration in plasma did not
Journal of Dairy Science Vol. 82, No. 11, 1999
increase, indicating that corpora lutea had not devel-
oped following follicle aspiration.
Seventy-nine follicles larger than 8 mm in diameter
were aspirated from 46 cows. Twenty-four cows had,
at the time of aspiration, one E
2
-A follicle; 8 cows had
one E
2
-LA follicle; and 1 cow had one E
2
-A and one E
2
-
LA follicle. In 13 cows, all of the follicles >8 mm were
E
2
-I. The proportion of cows that had only E
2
-I follicles
was similar among treatments and ranged between 25
to 33% (Table 4). In the control group, all 4 cows with
E
2
-I follicles that were >8 mm had only one >8mm
follicle per cow, whereas in the CSFA and bST treat-
ments, 2 out of 4 cows and 3 out of 5 cows had two or
more E
2
-I follicles >8 mm per cow. Among cows that
had E
2
-A or E
2
-LA follicles, the proportion of cows that
had two or more follicles >8 mm was 82, 50, and 60%
for cows of the control, CSFA and bST groups, respec-
tively (P < 0.03 for control vs. bST and P < 0.12 for
control vs. CSFA). The data in Table 4 indicate that
the E
2
concentration in the follicular fluid of the E
2
-A
and E
2
-LA follicles was significantly correlated with
the total number of follicles >8 mm in the ovaries of
cows on the CSFA and bST treatments but not for
cows on the control treatment. This correlation was
negative in the CSFA treatment and positive in the
bST treatment. The following data are an illustration
of this opposite relationship between the E
2
concentra-
tion in follicular fluid and the number of follicles. All
6 cows in the control and bST groups with E
2
concen-
trations >1200 ng/ml in the follicular fluid had two or
three follicles >8 mm in their ovaries. All 5 cows in
the CSFA group with similar E
2
concentrations in the
follicular fluid had only one follicle >8 mm in their
ovaries.
Cows in the control and bST groups that had an E
2
-
A follicle had more follicles >8 mm than did cows of
the same treatments that had E
2
-I or E
2
-LA follicles
(Table 5). The opposite was true of the CSFA group.
In the control and bST groups, 80% or more of the
cows with E
2
-A follicles had two or more follicles >8
mm in contrast with only 33% of cows in the CSFA
group (P < 0.02).
Among the 21 cows from which follicles were aspi-
rated between 60 to 90 DIM, the proportion of cows
with E
2
-A follicles was 67 (6/9), 40 (2/5), and 0% (0/
7) (P < 0.02) for the control, CSFA, and bST groups,
respectively (Figure 3). The proportion of follicles aspi-
rated 60 to 90 DIM that were classified as E
2
-LA was
11, 40, and 57% for the same groups, respectively.
Thus, out of 8 cows that had E
2
-LA follicles, 7 had
aspirations before 90 DIM, and 6 of these were in the
CSFA and bST groups. The proportion of cows with
E
2
-A follicles was similar among treatments when as-
piration occurred later than 90 DIM (Figure 3.). After
MOALLEM ET AL.2362
TABLE 3. Mean changes in body condition score after parturition of cows with aspirated follicles.
Control
1
CSFA
(n = 15) (n = 16) bST (n = 15) SEM
BCS at parturition 3.15 3.14 3.19 0.13
Minimum BCS after parturition 2.46
a
2.39
a
2.09
b
0.10
DIM to minimum BCS 60.0 60.8 72.1 5.6
DIM to initiation of P
4
2
cyclicity 28.3 29.4 28.2 3.0
DIM at aspiration 89.0 98.5 93.2 4.1
BCS on day of aspiration 2.65
a
2.56
a
2.23
b
0.10
BCS at parturition –
BCS on day of aspiration –0.50
b
–0.59
b
–0.96
a
0.10
a,b
Means within a row without common superscripts differ (P < 0.05).
1
Control, fed the control diet; CSFA, fed the control diet plus 0.55 kg/d of calcium soaps of fatty acids;
and bST, control diet plus administration of 500 mg of Zn-Sometribove (Monsanto Co., St. Louis, MO)/14
d.
2
P
4
= Progesterone.
90 DIM, only 1 of 25 cows had an E
2
-LA follicle. The
follicles of control cows were aspirated earlier postpar-
tum (Table 6) than were follicles of other cows; follicles
from 60% of control cows versus 42% of cows on the
other two treatments were aspirated 60 to 90 DIM.
Despite the early aspiration of follicles from control
cows, the proportion of E
2
-A follicles during the whole
experiment was 67% (10/15), 56% (9/16), and 40% (6/
15) for the control, CSFA, and bST treatments, respec-
tively. Across treatments, 13 out of 21 (62%) cows
whose follicles were aspirated at <90 DIM did not have
E
2
-A follicles versus 8 of 25 (32%) cows aspirated after
90 DIM (P < 0.05).
Hormone Concentrations in Follicular Fluid
Across all treatments, the concentrations of andro-
stenedione and E
2
, but not that of P
4
, in follicular fluid
Figure 2. Plasma progesterone concentration before and after folli-
cle aspiration. Treatments were control; control plus 0.55 kg/d of
calcium soaps of fatty acids (CSFA); and bST, control diet plus admin-
istration of 500 mg of Zn-Sometribove (Monsanto Co., St. Louis, MO)/
14 d. Day 0 = day of follicle aspiration.
Journal of Dairy Science Vol. 82, No. 11, 1999
of E
2
-A follicles significantly increased between 60 and
139 DIM (Figure 4). The concentration of P
4
in follicu-
lar fluid, but not that of E
2
, was significantly higher
in CSFA cows than in the other two treatments (Table
6). The volume of follicular fluid and the amount of
P
4
and of E
2
in follicular fluid aspirated from cows
supplemented with CSFA was also significantly higher
than in control or bST-treated cows (Table 6).
NEFA Concentrations and Composition in Plasma
The total concentration of NEFA in plasma at 50
DIM and 2 d before aspiration was similar among all
treatments (Table 7). However, the composition of
NEFA in plasma was significantly different between
treatments both at 50 DIM and before aspiration. Con-
trol cows had a higher proportion of palmitic acid
(C
16:0
) and a lower concentration of stearic (C
18:0
) and
oleic (C
18:1
) acids than did cows of the other two groups.
The CSFA-fed cows had at 50 DIM a higher proportion
of linoleic acid (C
18:2
) than did the other two treat-
ments, whereas before follicle aspiration, bST cows
had the highest proportion of C
18:2
.
Across all treatments, the proportion of C
18:1
in
plasma at 50 DIM was 17.5 ± 1.1% in cows that had
only E
2
-I or E
2
-LA follicles compared with 14.1 ± 1.0%
in cows with E
2
-A follicles (P < 0.03). No significant
differences were found in proportions of other fatty
acids in plasma between cows that had only E
2
-I or
E
2
-LA follicles and cows that had E
2
-A follicles.
Concentrations and Fatty Acid Composition
of Lipid Fractions in Follicular Fluids
In follicular fluid of all aspirated follicles, the total
concentrations of NEFA and of fatty acids in phospho-
lipids and cholesterol esters were similar among treat-
ments (data not shown). In the phospholipid fraction,
control cows had a lower proportion of C
18:1
than did
FAT AND BOVINE SOMATOTROPIN ON FOLLICULAR HORMONES 2363
TABLE 4. Number of follicles >8 mm in ovaries of cows with different estradiol (E
2
) concentrations (nanograms
per milliliter) in follicular fluid of the dominant follicle.
E
2
active and less active
E
2
inactive 0–600 601–1200 >1201
Treatment n X
1
n X
1
n X
1
n X
1
r
2
Control
3
4 1.0 3 2.00 4 2.00 4 2.00 –0.12
CSFA 4 2.0 3 2.33 4 1.75 5 1.00 –0.80**
bST 5 1.6 4 1.25 4 2.00 2 2.50 0.62*
SEM 0.31 0.37 0.36 0.10
1
Number of follicles>8 mm per cow.
2
Correlation coefficient (r) between E
2
concentration in follicular fluid and number of follicles >8mmin
cows that had an E
2
-active or E
2
-less active follicle.
3
Control, fed the control diet; CSFA, fed the control diet plus 0.55 kg/d of calcium soaps of fatty acids;
and bST, control diet plus administration of 500 mg of Zn-Sometribove (Monsanto Co., St. Louis, MO)/14
d.
*P < 0.05.
**P < 0.005.
those on bST treatment and a higher proportion of
C
18:2
than did cows on CSFA treatment. The CSFA fed
cows had a higher proportion of C
18:0
than did cows
that were administered bST. There were no significant
differences between treatments in the composition of
NEFA and in the composition of fatty acids in the
cholesterol ester fraction. The content and composition
of fatty acids in follicular fluid of E
2
-A and E
2
-I or E
2
-
LA follicles is compared in Table 8. The phospholipid
fraction was at a higher concentration in the E
2
-A
follicles; in the two other lipid fractions, the concentra-
tions were similar in both classifications of follicles.
In the NEFA fraction, the proportion of palmitic acid
was higher and the proportion of oleic and linoleic
acids was considerably lower in E
2
-A follicles in com-
parison with E
2
-I and E
2
-LA follicles.
Correlations Among Follicular Fluid,
Fatty Acids, and Reproductive Hormones
Correlation coefficients between some fatty acids in
follicular fluid and plasma (50 DIM) and follicular fluid
TABLE 5. Mean number of follicles >8 mm in cows with different
follicular estradiol (E
2
) activities.
E
2
active E
2
inactive or less active
Treatment
1
n Xn X
Control 10 2.00
a,x
5 1.20
b,y
CSFA 9 1.33
b,y
7 2.14
a,x
bST 6 2.16
x
9 1.44
y
SEM 0.21 0.26
a,b
Means within a row without common superscripts differ (P <
0.05).
x,y
Within a column, number of follicles >8 mm for control and bST
groups differ from the CSFA group (P < 0.02).
1
Control, fed the control diet; CSFA, fed the control diet plus 0.55
kg/d of calcium soaps of fatty acids; and bST, control diet plus adminis-
tration of 500 mg of Zn-Sometribove (Monsanto Co., St. Louis, MO)/
14 d.
Journal of Dairy Science Vol. 82, No. 11, 1999
hormone concentrations are shown in Table 9. In follic-
ular fluid, the proportions of C
18:1
and C
18:2
were nega-
tively correlated with E
2
. In contrast, plasma (50 DIM)
C
18:2
correlated positively with E
2
in follicular fluid.
Oleic acid in follicular fluid and C
18:2
in plasma were
positively correlated with DIM. Also, at follicle aspira-
tion, E
2
and androstenedione concentrations in follicu-
Figure 3. Proportion of cows with E
2
-active (E
2
-A) follicles among
>8 mm dominant follicles at time of aspiration. Treatments were
control; control plus 0.55 kg/d of calcium soaps of fatty acids (CSFA);
and bST, control diet plus administration of 500 mg of Zn-Sometribove
(Monsanto Co., St. Louis, MO)/14 d.
MOALLEM ET AL.2364
TABLE 6. Mean concentrations and content of estradiol (E
2
), progesterone (P
4
), and androstenedione and
E
2
/P
4
ratio in follicular fluidofE
2
-active follicles.
Control
1
CSFA bST
(n=10) (n=9) (n=6) SEM
DIM at aspiration 90
b
107
a
106
a
6.0
Estradiol-17β, ng/ml 1218.9 1396.6 1029.3 191.6
Progesterone, ng/ml 33.0
b
55.4
a
30.0
b
6.3
Androstenedione, ng/ml 59.1 53.7 60.6 14.0
E
2
/P
4
43.9 33.0 42.6 10.3
Follicular fluid, ml 2.07
b
3.14
a
1.28
b
0.6
P
4
content of follicular fluid, ng 68.3
b
173.9
a
38.4
b
36.5
E
2
content of follicular fluid, ng 2525.1
b
4385.3
a
1317.4
b
621.6
a,b
Means within a row without a common superscript differ (P < 0.05).
1
Control, fed the control diet; CSFA, fed the control diet plus 0.55 kg/d of calcium soaps of fatty acids;
and bST, control diet plus administration of 500 mg of Zn-Sometribove (Monsanto Co., St. Louis, MO)/14
d.
lar fluid were positively correlated with DIM. Other
correlations were not statistically significant.
DISCUSSION
In this study, we have shown that both bST and
CSFA enhance milk yield while affecting follicular dy-
namics and follicular fatty acid concentrations. The
increase in milk yield, following CSFA feeding and bST
administration, found here was slightly lower than in
Figure 4. Mean concentrations of progesterone (P
4
), androstenedi-
one, and estradiol-17β (E
2
) and the E
2
/P
4
ratio in follicular fluid of
25 E
2
-active follicles of all treatments at different DIM of aspiration.
a,b,c
Means without common superscripts differ (P < 0.05). The left
axis is progesterone and androstenedione concentrations and the E
2
/
P
4
ratio, and the right axis is the estradiol concentration.
Journal of Dairy Science Vol. 82, No. 11, 1999
our previous report (22). Feed intake was similar in
both studies, whereas BCS at calving was somewhat
higher in the present study. Cows of the bST group
lost almost twice as much body condition between calv-
ing and follicle aspiration as did control cows (Table
3). As in our previous study, cows treated with bST
began to gain BW later and more slowly than did cows
of other treatments, and, therefore, they reached their
calving BW later in lactation. This greater BCS and
BW loss, together with the increase in milk yield and
similar DMI, may be considered an indication of a
more negative energy balance in cows of the bST group.
In contrast, cows fed CSFA lost a similar amount of
body condition as did control cows, despite increased
milk yield and somewhat lower feed intake.
Effects of CSFA and bST
on Follicular Development
The characteristics and numbers of follicles indi-
cated that CSFA and bST treatments considerably af-
fected follicular dynamics during the estrous cycle.
However, because sequential ultrasonographic de-
scription of the ovaries was not carried out in the pres-
ent study, the following discussion can only be of a
speculative nature.
The data shown in Tables 4 and 5 raise the possibil-
ity that feeding CSFA delayed or prevented follicle
turnover in some cows. Therefore, the dominant folli-
cles became older and larger (Table 6) and contained
greater amounts of E
2
in follicular fluid. This finding
may explain why only 36% of the E
2
-A cows fed CSFA
had two or more >8 mm follicles as compared with 80%
or more in the other two groups. Older, or perhaps
first wave, follicles produced more E
2
as they were
larger and, therefore, had a greater mass of theca and
granulosa cells, as suggested by Bigelow and Fortune
(2). The younger, second wave, follicles produced less
FAT AND BOVINE SOMATOTROPIN ON FOLLICULAR HORMONES 2365
TABLE 7. Mean composition (percentage) of NEFA in plasma at 50 DIM and at 2 d before follicle aspiration.
C
16:0
C
16:1
C
18:0
C
18:1
C
18:2
C
18:3
C
20:4
Total (µM)
n
50 DIM
Control
1
15 46.4
a
0.2 30.1
b
11.8
b
9.4
b
0.9
a
1.2
a
159.3
CSFA 16 34.6
c
0 34.2
a
17.6
a
12.9
a
0.6
b
0
b
151.7
bST 15 39.7
b
0 32.4
b
17.4
a
9.6
b
0.9
a
0
b
124.8
SEM 1.4 0.4 1.1 1.1 0.8 0.1 0.4 14.3
2 d before aspiration
Control 15 46.9
a
0.1 29.5
b
11.7
b
10.5
b
0.7 0.6 156.9
CSFA 16 36.6
b
0 34.7
a
17.7
a
10.4
b
0.6 0 143.0
BST 15 37.8
b
0 31.1
b
16.9
a
13.1
a
1.2 0 129.2
SEM 1.8 0 1.1 1.2 0.9 0.3 0.2 14.0
a,b,c
Within a column means without a common superscript differ (P < 0.05).
1
Control, fed the control diet; CSFA, fed the control diet plus 0.55 kg/d of calcium soaps of fatty acids;
and bST, control diet plus administration of 500 mg of Zn-Sometribove (Monsanto Co., St. Louis, MO)/14
d.
E
2
, hence the negative correlation between number of
follicles and follicular fluid concentration of E
2
in this
treatment (Table 4). It was previously reported that
feeding CSFA increased both the number and size of
medium-sized and large follicles (31). Lucy et al. (18,
19) reported that CSFA specifically increased preovu-
latory follicular size irrespective of its effect on energy
balance. The considerable increase in the volume of
follicular fluid of the CSFA-treated cows in the present
study (Table 6) was in agreement with those results.
The higher concentration of P
4
in the follicular fluid
of cows fed CSFA (Table 6) was similar to that found
in the study by Ryan et al. (28), who reported a greater
concentration of P
4
in the follicular fluid of beef cows
fed soybean oil. It was also previously reported that
feeding cows with CSFA increased cholesterol concen-
tration in plasma (22) and follicular fluid (33). The
increased P
4
concentration in follicular fluid was possi-
bly the result of the higher concentration of cholesterol
TABLE 8. Mean composition (percentage) of NEFA and fatty acids in phospholipids and cholesterol esters
in follicular fluids of estradiol (E
2
)-active (E
2
-A), E
2
-inactive (E
2
-I), and E
2
less active (E
2
-LA) follicles of all
treatments.
C
16:0
C
16:1
C
18:0
C
18:1
C
18:2
C
18:3
C
20:4
Total (ng/mL)
n
NEFA
E
2
-A 23 37.9
a
2.6 16.7 22.9
b
18.1
b
1.0 0 228.2
E
2
-I and E
2
-LA 33 31.0
b
1.2 14.7 26.0
a
26.1
a
3.4 0 242.3
SEM 2.5 1.8 1.0 1.3 2.0 2.6 0 26.1
Phospholipids
E
2
-A 23 19.5 0.6 26.4
a
18.5 31.5 0.8 2.5 556.3
a
E
2
-I and E
2
-LA 33 19.9 0.6 23.6
b
19.8 31.7 1.1 3.2 447.2
b
SEM 0.7 0.3 0.7 0.8 0.8 0.2 0.4 25.2
Cholesterol esters
E
2
-A 23 16.7 1.6 12.6 4.8 56.4 7.2 0.6 572.3
E
2
-I and E
2
-LA 33 14.7 1.5 13.7 5.2 55.4 8.8 0.8 579.5
SEM 1.2 0.3 1.0 1.0 1.8 1.3 0.3 43.2
a,b
Within a column means without a common superscript differ (P < 0.05).
Journal of Dairy Science Vol. 82, No. 11, 1999
precursor (11, 27, 34) and a prolonged life span of
dominant follicles (26) in a significant proportion of
CSFA-fed cows.
In the bST treatment, in contrast, E
2
concentration
was positively correlated with the number of >8mm
follicles (Table 4). This finding may suggest that fol-
lowing luteolysis, E
2
production was enhanced in
young follicles in cows with follicle turnover but was
impaired in older follicles in cows with less follicle
turnover. The older follicles in bST-treated cows possi-
bly were influenced by an earlier, more pronounced
negative energy balance. These follicles might have
had a reduced ability to produce E
2
during the preovu-
latory period compared with follicles of the same age
in control cows. It is also possible that the late appear-
ance of the first postpartum estrus and the first post-
partum AI previously reported (22) in bST treated
cows was a result of this reduction in E
2
synthesis.
MOALLEM ET AL.2366
TABLE 9. Correlation coefficients between NEFA (percentage) in follicular fluid (FF) and plasma (50 DIM)
and concentrations of hormones in FF of 25 estradiol (E
2
)-active follicles of all treatments.
Item 18:1 (FF) 18:2 (FF) 18:1 (Plasma) 18:2 (Plasma) DIM
Estradiol in FF –0.46** –0.36* –0.30 0.31* 0.51***
Androstenedione
in FF –0.26 –0.29 –0.18 0.27 0.44**
E
2
/P
4
in FF –0.39** –0.28 –0.29 0.19 0.30
DIM 0.46** –0.30 0.06 0.45**
*P < 0.05.
**P < 0.01.
***P < 0.001.
The results of the present and previous studies indi-
cated that high yielding dairy cows fed CSFA had, in
most cases, similar BW and BCS profiles and similar
or slightly improved conception rates compared with
control cows (22, 30, 31). The effects on follicular devel-
opment caused by CSFA reported in the present study
probably describe a similar situation to previous re-
ports (22, 30) in which CSFA did not significantly
change conception rates. However, administration of
bST early postpartum considerably affected BW and
BCS profiles and significantly reduced conception
rates (22).
DIM and E
2
Production
The large difference between the proportion of E
2
-
A follicles aspirated 60 to 90 d after calving of control
and bST cows (Figure 3) could be explained by the
greater negative energy balance of cows in the bST
group as indicated by the greater loss of body condition
(Table 3 and Figure 1). Negative energy balance has
been previously shown to adversely affect follicular
development (19, 20), whereas bST administered later
in lactation did not have such an effect. In cows treated
with bST after 60 DIM, the start of the second follicular
wave and the rise of E
2
levels following PG administra-
tion began earlier in the estrous cycle in comparison
with controls (16, 17). The levels of E
2
were higher in
bST-treated cows than in controls (18). The higher
levels of E
2
that were found in nonlactating versus
lactating cattle (19) might also have been the result
of a greater negative balance in lactating cows. Thus
in this study, the similarity in the proportion of E
2
-A
follicles after 90 DIM (Figure 3) and the gradual in-
crease in the follicular fluid concentration of E
2
be-
tween 60 and 139 d after calving (Figure 4) might
have been the result of improved energy balance in all
groups and, in particular, in bST-treated cows after
90 DIM. The data in Figure 1 show that BCS began
increasing between 60 to 75 DIM, whereas the increase
in BW began earlier. Because the development of ovar-
ian follicles takes longer than 40 d (21), the effects of
Journal of Dairy Science Vol. 82, No. 11, 1999
negative energy balance on follicular development
may become apparent only after the period of BW and
BCS decline.
Despite the considerable differences between the
control and bST groups in follicular development and
the effect that bST treatment had on fertility (22), the
interval from calving to first ovulation (as measured
by the first rise in P
4
values) both in this and our
previous study (22) was similar in all groups. These
results indicated that in high yielding cows the inter-
val to first ovulation was a poor indicator of follicu-
lar development.
Fatty Acid Composition and Follicular
Development
Changes in the composition of the lipid fractions in
plasma and follicular fluid were examined, and some
significant changes in NEFA composition in both
plasma and follicular fluid were observed. The reason
for the decrease of plasma palmitic acid and increase
of oleic acid in NEFA at 50 DIM and the increase
of linoleic acid before aspiration in cows of the bST
treatment is at present not clear. Differences between
the control and the CSFA groups could be explained
by the intake of increased amounts of saturated fatty
acids, which affect plasma levels. It is interesting that
the proportion of linoleic acid increased in cows on the
bST treatment but not in other groups between 50
DIM and 2 d before aspiration. The proportion of other
fatty acids did not differ between periods.
The smaller proportion of oleic and linoleic acids
were found in the NEFA fraction of follicular fluid in
E
2
-A follicles as compared with E
2
-I and E
2
-LA folli-
cles. A significant negative correlation coefficient be-
tween these acids and the E
2
concentration in follicular
fluid (Table 9) suggested that preovulatory growth was
accompanied by a decrease in these two acids and an
increase in the proportion of palmitic acid. These ef-
fects were similar to those of a previous report on the
composition of NEFA in porcine follicular fluid (34). It
was also previously shown (12) that the proportion of
FAT AND BOVINE SOMATOTROPIN ON FOLLICULAR HORMONES 2367
linoleic acid in the NEFA fraction of bovine follicular
fluid is inversely related to follicle diameter, and it
was suggested that linoleic acid may have a role in
preventing germinal vesicle breakdown in the bovine
oocyte (14, 15).
Polyunsaturated fatty acids are precursors of eico-
senoids, which have extensive effects on many aspects
of the reproductive cycle. The increase in the propor-
tion of saturated fatty acids at the expense of unsatu-
rated fatty acids in follicular fluid might be important
in this context. In the follicular fluid, changes in the
fatty acid composition of the phospholipids between
treatments were relatively small. However, for E
2
-A
and E
2
-I, changes in the linoleic acid levels of NEFA
were marked, as were changes in phospholipid concen-
trations. These changes might have influenced the
physiological function of these follicles.
CONCLUSIONS
Feeding CSFA or administration of bST to high
yielding dairy cows during the early postpartum pe-
riod considerably affected ovarian follicular dynamics
and estradiol production as well as fatty acid concen-
trations in follicles. Much of the early postpartum ef-
fect of bST on reproductive phenomena was through
increased milk yield, a greater loss in BCS, and a post-
partum delay in the ability of ovarian follicles to pro-
duce estradiol. These changes might have a consider-
able effect on the fertility of cows treated with bST
early postpartum.
ACKNOWLEDGMENTS
The authors thank Ofra Kedar for the lipid analysis,
Mara Heler for hormone analysis, and the staff of the
experimental farm at Bet Dagan (Israel) for their coop-
eration and technical assistance.
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