ArticlePDF Available
Subclinical, chronic intramammary infection lowers steroid
concentrations and gene expression in bovine
preovulatory follicles
Y. Lavon
a
, G. Leitner
b
, E. Klipper
a
, U. Moallem
c
, R. Meidan
a
, D. Wolfenson
a,
*
a
Department of Animal Science, Faculty of Agriculture, Food and Environment, Hebrew University, Rehovot, Israel
b
Mastitis Laboratory, Veterinary Institute, Bet Dagan, Israel
c
Institute of Animal Science, Agricultural Research Organization, Bet Dagan, Israel
Received 22 July 2010; received in revised form 14 September 2010; accepted 16 September 2010
Abstract
Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit
delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory
follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic
cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were
caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2
; 42 h later,
follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively.
Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n 28) were 40% lower (P0.05) than those
in uninfected cows (n 24) and lower than in past clinical mastitic cows (n 9). Distribution analysis revealed a clear divergence
among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all
uninfected (P0.01) and most clinical cows (P0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows
had twofold higher (P0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P0.05) follicular andro-
stenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was
not affected. Reduced expression (P0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1
in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical
low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular
growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a
considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the
reproductive failure associated with this disease.
© 2011 Elsevier Inc. All rights reserved.
Keywords: Ovary; Follicle; Mastitis; Steroid; Theca cell; Granulosa cell
1. Introduction
Pathogenic stresses disrupt reproductive responses
and lower conception rates in cows [1]. Among these
stresses, mastitis is considered one of the most common
diseases affecting dairy cattle health and performance
* Corresponding author: Department of Animal Science, Faculty of
Agriculture, Food and Environment, Hebrew University, Rehovot
76100, Israel. Tel.: 972-8-948-9393; fax: 972-8-948-9868.
E-mail address: wolf@agri.huji.ac.il (D. Wolfenson).
Available online at www.sciencedirect.com
Domestic Animal Endocrinology 40 (2011) 98 –109
www.domesticanimalendo.com
0739-7240/11/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.domaniend.2010.09.004
and is therefore a frequent cause for culling [2], which
is attributed mainly to the development of chronic sub-
clinical mastitis. The incidence of subclinical mastitis
in developed countries varies from 20% to 50%. Epi-
demiological studies have associated clinical and sub-
clinical intramammary infection (IMI) before and after
artificial insemination with low fertility [3–5]. Acute
clinical cases of IMI last several days and are caused
mainly by Gram-negative bacteria. Subclinical IMI is
not characterized by visible clinical signs or typical
acute-phase responses. It lasts for several months and is
caused mainly by Gram-positive bacteria [6]. Indeed,
different inflammatory responses were documented
during induction of IMI by Gram-positive Staphylococ-
cus aureus or by Gram-negative lipopolysaccharide
(LPS) endotoxin [7].
Several studies have examined the immediate, acute
effects of pathogenic stress on the hypothalamic-pitu-
itary-ovarian axis [8]. A widely used acute model for
clinical mastitis employs the administration of Gram-
negative LPS endotoxin, mainly of E. coli origin [9].
LPS-induced or naturally occurring clinical IMI is char-
acterized by a transient surge of inflammatory media-
tors. Administration of LPS during the follicular phase
[8] or at onset of estrus [10] delayed the preovulatory
luteinizing hormone (LH) surge and ovulation in one-
third of cows. Others have shown that LPS or cytokines
depress steroid production in theca and granulosa cells
[11,12]. However, these experimental models do not sim-
ulate subclinical mastitis, which lasts several months and
is rather common in dairy farms. By its chronic nature,
subclinical IMI may have the potential to disrupt long-
term processes, such as follicular growth and develop-
ment, even more than acute clinical events.
We have recently shown [13] that 30% of subclini-
cal mastitic cows, which exhibited estrus at the normal
time after prostaglandin F
2
(PGF
2
), had delayed ovu-
lation This may be related to low or delayed preovula-
tory LH surge, resulting from lower than normal estra-
diol levels [14,15]. The latter was not associated with
any change in pulsatile LH or cortisol concentrations in
subclinical IMI or past clinical IMI [13]. In contrast,
disruption of estradiol secretion by acute exposure to
endotoxin is associated primarily with depressed pul-
satile LH secretion [8,16], which may be related to
activation of the adrenal axis [10,17]. The above infor-
mation suggests that the low estradiol level in one-third
of subclinical IMI cows is not caused by altered secre-
tion of LH pulses, but rather by the effect of one or
more as yet unidentified inflammatory mediators capa-
ble of disrupting steroidogenesis. To our knowledge,
there are no reports relating subclinical mastitis or past
clinical events to steroidogenesis. Here we examined
impairment of preovulatory follicle functions in cows
with subclinical IMI before and during the study and in
cows that had been exposed to a past clinical event of
IMI. Based on our previous studies [13], we expected a
divergence of the subclinical IMI cows into groups
exhibiting normal and low steroidogenic capacity. We
examined preovulatory follicle characteristics prior to
estrus manifestation and LH surge, including mRNA
levels of genes associated with steroidogenesis in the
granulosa and theca cells, follicular growth dynamics,
and follicular and circulating steroid concentrations.
2. Materials and methods
2.1. Experimental design
The experiment was approved by the local ethics
committee of Hebrew University. The study included 2
experiments. Experiment 1 (n 41) examined the
effect of subclinical and clinical mastitis on follicular
responses with an emphasis on granulosa cell re-
sponses. Experiment 2 (n 20) concentrated on re-
sponses of theca cells under subclinical IMI (clinical
mastitis was not included in experiment 2 because we
did not observe any effect on follicular responses in
experiment 1). In both experiments, follicular growth
and steroid concentrations were analyzed in cyclic Hol-
stein cows in their first to fifth lactations. In both
experiments, follicular cells and fluids were retrieved
from preovulatory follicles following the same syn-
chronization protocol and time interval (42 h) from
PGF
2
administration (prior to estrus, normally occurs
in high-milk-yielding cows 56 h or more after PGF
2
).
This made it possible to combine relevant data from the
2 experiments.
2.2. Animals and experimental groups
Cows were kept in an open shed with access to an
adjacent yard and fed a complete mixed ration contain-
ing 16.9% protein and 1.77 Mcal dry matter/kg. The
mean maximal and minimal air temperatures and rela-
tive humidities during the study were 26.1 °C and 15.6
°C and 85.8% and 43.6%, respectively. Cows were
monitored 2 or 3 times monthly for somatic cell counts
(SCC; Foss Electric, Hilleröd, Denmark). Two addi-
tional milk samples that were taken aseptically from
each of the 4 quarters 1 wk before and 1 d before
starting the experiment were processed as previously
described [18] and analyzed for SCC as well as for
99Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
bacteriological determination. Bacterial examinations
were carried out according to International Dairy Fed-
eration procedures and the bacteriological typing was
performed according to accepted standards [19]. SCC
was determined with a Coulter cell counter (Z1 Model,
Coulter Electronics Ltd, Luton, UK) as described [20].
Cows were divided into 3 (experiment 1) or 2 (ex-
periment 2) groups according to their SCC before the
study. The first group (experiments 1 and 2) consisted
of uninfected cows with SCC 150,000 cells/mL milk
and no detected bacteria. The second group (experi-
ments 1 and 2) consisted of typical, long-term subclin-
ical IMI cows with SCC 150,000 cells/mL milk in all
milk samples taken, with IMI in at least 1 quarter
associated with various bacteria, prior to and close to
the experiment. The third group (in experiment 1 only)
consisted of cows that had exhibited typical symptoms
of a short-term clinical mastitic event (lasting on the
order of days) 40 6 d prior to the study. These past
clinical events typically included a transient drop in
milk yield and a rise in milk conductivity (Afimilk,
Afikim, Israel) and a high SCC of at least 10
6
cells/mL milk with bacterial identification (in a milk
sample taken during the clinical event). Those cows
were routinely treated with antibiotics and anti-inflam-
matory medication for 3 d and had no infection during
the study. Experimental cows did not exhibit any other
diseases during the study period.
2.3. Synchronization protocol
The estrus cycles were synchronized by 2 injections
of PGF
2
analogue (500
g Cloprostenol, Estroplan,
Parnell Laboratories, Alexandria, Australia) given 13 d
apart. Forty-eight hours after the second PGF
2
injec-
tion, cows were injected with gonadotropin-releasing
hormone analogue (200
g Gonadorelin, Gonabreed,
Parnell Laboratories) to induce ovulation (day of gona-
dotropin-releasing hormone analogue day 0). On day
6 of the cycle, 2 doses of PGF
2
were given 10 h apart
to ensure complete regression of the corpus luteum in
all treated cows. Then, 42 h after PGF
2
(first dose), on
day 8 of the cycle, the preovulatory follicles were
aspirated (experiment 1) or cows were slaughtered and
their ovaries removed for further analysis (experiment
2). Ultrasonographic scanning (7.5-MHz linear probe
with an SSD-900 Aloka instrument, Tokyo, Japan) was
performed during synchronization and confirmed the
normal appearance of structures in the ovaries that were
typical for cyclic cows (first PGF
2
dose) and for cows
prior to the induced follicular phase (second PGF
2
dose). Additional scanning was performed to confirm
ovulation and later to follow the follicular growth dy-
namics of the first-wave dominant follicle until the day
of aspiration (experiment 1, day 8) or 2 d prior to
slaughter (experiment 2, day 6). Blood samples (exper-
iment 1) were collected on day 6 prior to PGF
2
injec-
tion and 24, 36, and 42 h later. Blood samples were
centrifuged and plasma was stored at 20 °C.
2.4. Follicular aspiration and collection of
granulosa cells
Cows were sedated with 14 mg xylazine hydrochlo-
ride (Sedaxylan; Eurovet Animal Health BV, Bladel,
Holland) to simplify handling of the animals and their
ovaries. Caudal epidural anesthesia was induced using
5 mL of 2% lidocaine to prevent abdominal and anal
strain. Follicular fluid and granulosa cells were aspi-
rated as previously described [21,22]. Briefly, an ultra-
sound scanner (Pie Medical, Maastricht, The Nether-
lands) was used with a 7.5-MHz vaginal transducer
equipped with a needle guide, which was attached to a
20-gauge needle and connected to a 5-mL sterile sy-
ringe. The pipeline and attached syringe were filled
with an exact volume of 2.7 mL sterile saline solution.
The contents of the preovulatory follicle were aspi-
rated, with repumping 2 or 3 times to detach the gran-
ulosa cells from the follicle wall, and the pipeline was
flushed with an additional 1 mL of saline (total volume
of saline used was taken into account when steroid
levels were calculated). For each cow, follicular fluid
and granulosa cells were collected from the preovula-
tory follicles. Because of technical difficulties during
the ultrasound-guided aspiration of the granulosa cells,
30% of the samples collected (evenly across all groups)
did not render a sufficient amount of total RNA for the
real-time polymerase chain reaction (PCR). Granulosa
cells were collected by centrifugation and follicular
fluid was stored at 20 °C. The cells were washed with
2 mL of sterile phosphate-buffered saline, snap frozen
in liquid nitrogen, and stored at 80 °C.
2.5. Collection of theca cells
Bovine ovaries were collected immediately after
slaughter (42 h after PGF
2
) at a local abattoir and
placed in ice-cold phosphate-buffered saline (Sigma
Chemical Co, St Louis, MO, USA). At the laboratory,
follicular fluid was aspirated from preovulatory folli-
cles using a syringe with a 22-gauge needle and stored
at 20 °C until analysis for estradiol, androstenedione,
and progesterone. Thecal cell layers were isolated from
the preovulatory follicles as described previously [23].
Briefly, follicles were opened by making a small inci-
100 Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
sion on the surface. Granulosa cells were removed by
gentle scraping and the thecal layers were peeled away
from each follicle, placed in a 2-mL tube, and stored at
80 °C until mRNA and protein determination.
2.6. Hormone determinations
Estradiol was measured in extracted plasma samples
using an ultrasensitive radioimmunoassay (RIA) kit
(DSL-4800, Diagnostic Systems Laboratory, Webster,
TX, USA). The standard curve was generated from
charcoal-stripped bovine plasma [24]. Cross-reactivity
of the assay was 2.4% for estrone, 0.64% for estriol,
and undetectable for testosterone and progesterone. The
minimum detectable amount was 0.5 pg/mL, and the
intra- and interassay coefficients of variation were 3%
and 5%, respectively. Plasma and follicular fluid pro-
gesterone concentrations were analyzed with a solid-
phase RIA kit (Diagnostic Products Corp, Los Angeles,
CA, USA) against a standard curve prepared from
ovariectomized cow plasma [25]. The minimum detect-
able amount was 0.2 ng/mL and the intra- and interas-
say coefficients of variation were 8.6 and 9.9%, respec-
tively. Follicular fluid samples were appropriately
diluted and estradiol concentrations analyzed using a
solid-phase RIA kit (Diagnostic Products Corp). The
assay sensitivity was 8 pg/mL and the intra-assay co-
efficient of variation was 3%. Androstenedione concen-
tration in the follicular fluid was analyzed by means of
a kit (androstenedione RIA DSL-4200, Diagnostic Sys-
tems Laboratory), following the manufacturer’s in-
structions. The minimum detectable amount was 0.02
ng/mL and the intra-assay coefficient of variation was
4.3%.
2.7. RNA isolation and real-time PCR
Total RNA was isolated from the cells with TRI
reagent (MRC, Cincinnati, OH, USA) according to the
manufacturer’s instructions. PCR was performed using
a PE Biosystems GeneAmp 5700 sequence-detection
system, with the SYBR Green I PCR kit, as previously
described [22]. Briefly, each real-time reaction (18
L)
contained the SYBR Green Master Mix that comprised
the ROX 6-carboxy-X-rhodamine passive reference, 1.5
mM deoxynucleotide triphosphates (Bioline GmbH,
Luckenwalde, Germany), including deoxyuridine 5=-
triphosphate, 5 mM MgCl
2
, uracil N-glycosylase, and
Amplitaq HotGoldStar DNA polymerase, 0.54
Lofa
1:10,000 dilution of SYBR Green stock solution, 10
nM of each primer, and cDNA. The
-actin (ACTB)
gene was used as a standard for mRNA in follicular
cells. Dissociation-curve analysis was run following
each real-time experiment to confirm the presence of
only one product and the absence of primer dimer
formation. The threshold cycle number (Ct) for each
tested gene X was used to quantify the relative abun-
dance of that gene: 2
–(Ct gene X-Ct actin)
1,000. Table 1
presents a list of the primers used, synthesized by
Sigma-Genosys (Rehovot, Israel).
Table 1
Primers used in real-time PCR
Gene Primer Sequence Product length (bp)
LHCGR Forward
Reverse
5=-GCTGATTTCCCTGGAGCTGAAG-3=
5=-CTGAATGGACTCTAGCCCGTAGG-3
147
CYP19A1 Forward
Reverse
5=-TGGTGATGATGAAGGTCGTCC-3=
5=-CGAGGCACTTGTCTGAATTTCT-3=
176
FSHR Forward
Reverse
5=-TGGCAAGTGCTTAATACCTGT-3=
5=-GCAAACGTGTTCTCCAACC-3=
149
CYP11A1 Forward
Reverse
5=-CTG CAA ATG GTC CCA CTT CT-3=
5=-CAC CTG GTT GGG TCA AAC TT-3=
209
CYP17A1 Forward
Reverse
5=-TGG ATC GTG GCC TAC CTC CT-3=
5=-AGG TCG CCA ATG CTG GAG TC-3=
215
HSD3B1 Forward
Reverse
5=-TCC ACA CCA GCA CCA TAG AA-3=
5=-AAG GTG CCA CCA TTT TTC AG-3=
118
StAR Forward
Reverse
5=-GTG GAT TTT GCC AAT CAC CT-3=
5=-TTA TTG AAA ACG TGC CAC CA-3=
203
ACTB Forward
Reverse
5=-CGGGSCCTGACGGACTACCTC-3=
5=-GCCATCTCCTGCTCGAAGTCC-3=
139
LHCGR, LH receptor; CYP19A1, cytochrome P450 aromatase; FSHR, FSH receptor; CYP11A1, cytochrome P450 side-chain cleavage; CYP17A1,
cytochrome P450 17-
-hydroxylase/17,20-lyase; HSD3B1,3
-hydroxysteroid dehydrogenase; StAR, steroidogenic acute regulatory protein;
ACTB,
-actin.
101Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
2.8. Western blot analysis
Western blot analysis was carried out as previously
described [26]. The thecal layers were homogenized in
lysis buffer (25 mM Tris–HCl, 100 mM NaCl, 0.5%
deoxycholate, 0.5% Nonidet P-40, 5 mM ethylenedia-
minetetraacetic acid, pH 7.5, and 10% protease inhib-
itor cocktail). Cell extracts were sonicated on ice for
10 s at low speed. Protein concentration was deter-
mined using Bio-Rad DC reagents. All steps were per-
formed on ice and samples were kept at 80 °C until
use. Proteins were electrically transferred to nitrocellu-
lose membranes. After2hofblocking in Tris-buffered
saline–Tween 20 5% lowfat milk, membranes were
incubated with cytochrome P450scc antibody diluted
1:5,000 (the antisera for cytochrome P450scc was a
generous gift from Dr. S. Silavin, Adenza Biochemical,
Sunnyvale, CA, USA). Membranes were washed 3
times and incubated with horseradish peroxidase– con-
jugated goat anti-rabbit IgG diluted 1:10,000 (Sigma)
for1hatroom temperature. Total p42/p44 mitogen-
activated protein kinase (MAPK; Sigma) diluted
1:50,000 was used as the loading control [27]. A chemi-
luminescent signal was generated with SuperSignal and
the membranes were exposed to x-ray film.
2.9. Data analyses
Initial analysis compared follicular fluid steroid con-
centrations in the 3 experimental groups (uninfected,
clinical, and subclinical). In a subsequent statistical
analysis, the concentration of estradiol in the follicular
fluid was used to classify the cows, within each exper-
imental group, to cows with normal or low concentra-
tions of estradiol in the follicular fluid. Based on several
studies in which preovulatory estradiol concentrations
were documented [28,29], cows with a follicular fluid
estradiol concentration 400 ng/mL were defined as nor-
mal-estradiol cows, and cows with estradiol concentration
400 ng/mL were defined as low-estradiol cows. Conse-
quently, 2 experimental groups were defined as normal-
estradiol groups: (1) the uninfected control group (all
cows were defined as normal estradiol) and (2) the
group of cows exhibiting a clinical mastitic event 40 d
prior to the study (designated past clinical; all cows
exhibited normal estradiol levels, except 1 cow with an
intermediate (low) level that was not included in the
analysis). The subclinical mastitic cows, on the other
hand, were defined as either (3) subclinical-normal es-
tradiol cows exhibiting a normal level of estradiol in the
follicular fluid or (4) subclinical-low estradiol cows
exhibiting low estradiol levels in the follicular fluid.
Thus, the subclinical group was the largest experimen-
tal group, so as to have a sufficient number of cows in
the normal- and low-estradiol subgroups to perform a
solid analysis. For SCC and bacteriological data, fol-
licular growth, and steroid levels in the follicular fluids,
the datasets of experiments 1 and 2 were combined
(data in these experiments did not differ). For the other
variables, data of experiment 1 (mRNA in granulosa
cells and plasma estradiol concentrations) or experi-
ment 2 (mRNA in theca cells and concentrations of
CYP11A1) are presented. For SCC analysis, data were
log-transformed to ensure normal distribution. For de-
terminations of estradiol concentration in the plasma
and follicular growth, cows served as experimental
units with repeated measurements within cows, and
data were analyzed using the general linear model pro-
cedure of SAS (Statistical Analysis Systems, Inc, http://
www.sas.com). The model included groups, cows
(within a group), day, and group day interaction.
Datasets that consisted of 1 piece of data per cow were
tested by one-way ANOVA and the Tukey–Kramer test
was used to determine the statistical difference between
groups. These datasets included gene expression data,
steroids in the follicular fluid, and protein levels. The
distribution of cows within experimental groups into
normal- or low-estradiol subgroups was tested by Fisher’s
exact test. Data are presented as means and SEM.
3. Results
3.1. Steroid hormones in the follicular fluid
Concentrations of steroids in the follicular fluids of
uninfected, past clinical, and subclinical mastitic
groups are presented in Table 2. Follicular estradiol and
androstenedione concentrations and the estradiol-to-
Table 2
Steroid concentrations and estradiol-to-progesterone (E/P) ratio in
the follicular fluid of preovulatory follicles obtained from
uninfected, subclinical, and past clinical mastitic cows by in vivo
aspiration (experiment 1) or following slaughter (experiment 2) on
day 8 of the cycle, 42 h after PGF
2
administration
Uninfected Subclinical Past clinical
a
n
b
24 28 9
Estradiol (ng/mL) 1,170 100
1
673 83
2
1,115 126
1
Androstenedione
(ng/ml)
196 32
1
121 21
2
137 43
1,2
Progesterone (ng/mL) 72 66568212
E/P ratio 19 2
1
12 2
2
15 2
1,2
Data are presented as means SEM. Means within a row with
different superscripts differ (P0.05).
a
Clinical events occurred 40 6 d before aspiration.
b
Each cow supplied 1 follicle.
102 Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
progesterone ratio were reduced in the subclinical
groups to 60% of values in uninfected cows (P0.05).
Similarly, follicular estradiol was higher in the past
clinical than in the subclinical group (P0.05; Table
2). Progesterone concentrations in the follicular fluids
and the volume of aspirated follicular fluid (2.05
0.25 mL) did not differ among groups. The variables
milk yield (38.1 1.2 kg/d), body condition score
(2.7 0.1, on a scale of 1 to 5), days in lactation (169
15 d), and parity number (3.1 0.2) did not differ
among uninfected, past clinical, and subclinical mas-
titic groups, and none of these variables was associated
with follicular estradiol concentration (normal or low).
Distribution of the cows within each group, accord-
ing to follicular estradiol levels, is presented in Table 3.
All 24 uninfected cows had normal follicular fluid es-
tradiol concentrations (400 ng/mL). In contrast, sub-
clinical cows showed a clear divergence: 32% (9/28)
exhibited low follicular estradiol concentrations (400
ng/mL), and the remaining two-thirds (19/28) exhibited
normal estradiol levels (400 ng/mL; Table 3;P
0.01, Fisher’s exact test compared with the uninfected
group, and P0.08 compared with the past clinical
mastitic group). The proportion of subclinical-low es-
tradiol cows was almost identical in experiments 1 and
2(Table 3). Cows that had had clinical mastitis 40 d
prior to the study (n 9) but were healthy at the time
of the experiment exhibited normal follicular estradiol
levels (with the exception of 1 cow with a low estradiol
level). Mean follicular estradiol concentration in the
subclinical-normal estradiol group did not differ from
that of the uninfected group. Androstenedione in the
follicular fluid of the subclinical-normal estradiol cows
was sevenfold higher than that in the subclinical-low
estradiol group (Table 4;P0.05), with the former
being similar to that in the uninfected and past clinical-
normal estradiol cows (Tables 2 and 4). The estradiol-
to-progesterone ratio was 1 in all groups; however,
the ratio for the subclinical-low estradiol group was
considerably lower than that obtained for the other 3
normal estradiol groups (Tables 2 and 4,P0.05).
Follicular progesterone levels did not differ among
groups (Tables 2 and 4).
3.2. Pathogens and SCC
The mastitis pathogens isolated from the mammary
glands of the infected cows are summarized in Table 5.
All clinical infections, occurring 40 6 d prior to the
study, were caused by E. coli. Most of the subclinical
infections were caused by Streptococcus dysgalactiae
and coagulase-negative staphylococci, and no associa-
tions were found between bacterial type or SCC and
normal or low follicular estradiol levels among subclin-
ical cows. The SCC levels were lower in the uninfected
cows than in the subclinical cows (P0.05). At the
time of the study, the past clinical cows still maintained
slightly higher SCC values than the uninfected cows
(P0.05; Table 5).
3.3. Steroid hormones in the circulation
All groups had similar plasma progesterone concen-
trations of around 2.9 0.4 ng/mL on day 6 of the
Table 3
Distribution of uninfected and mastitic cows according to estradiol concentration in the follicular fluid—normal or low
a
—in experiments 1
and 2
Uninfected Subclinical Past clinical
b
Normal Low Normal Low Normal Low
Experiment 1, granulosa (n) 16 0 11 5 (31%) 8 1
Experiment 2, theca (n) 8 0 8 4 (33%)
Total (experiments 1 and 2) (n)
c,d
24 0 19 9 (32%) 8 1
Estradiol (ng/mL) 1,170 100 925 67 138 51 1,115 126 —
a
Normal estradiol, 400 ng/mL; low estradiol, 400 ng/mL.
b
Clinical events occurred 40 6 d before aspiration.
c
P0.01 and
d
P0.08 for distribution of cows among groups according to follicular estradiol level in uninfected and clinical groups,
respectively, compared with the subclinical group by Fisher’s exact test.
Table 4
Follicular fluid concentrations of androstenedione and progesterone
in the preovulatory follicles and estradiol-to-progesterone (E/P)
ratio in subclinical mastitic cows (n 28) exhibiting either normal
or low estradiol in the follicular fluid
Subclinical
Normal Low (%)
Androstenedione (ng/mL) 167 24
1
23 6
2
Progesterone (ng/mL) 70 85110
E/P ratio 16 2
1
41
2
Data are presented as means SEM. Means within a row with
different superscripts differ (P0.05).
103Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
cycle (before PGF
2
injection), which dropped to basal
values of about 0.2 0.04 ng/mL on the day of fol-
licular aspiration (day 8; data not shown). In agreement
with the follicular concentrations, the plasma estradiol
curve was lowest in the subclinical-low estradiol group
but did not differ statistically from the other groups
(Fig. 1). However, just before aspiration, the mean
concentration of estradiol in the plasma from the sub-
clinical-low estradiol group was about half that in un-
infected, past clinical-normal, and subclinical-normal
estradiol cows (Fig. 1;P0.05).
3.4. Follicular growth dynamics
The growth patterns of the largest follicles during
the final stages of synchronization (day 2 to day 0), as
well as during the first follicular wave (day 2 to day 8),
were similar in all groups (Fig. 2). The diameters of the
preovulatory follicles just before the time of aspiration
(day 8, experiment 1) in the uninfected, past clinical-
normal, subclinical-normal, and subclinical-low estra-
diol groups were similar as well (15.3 0.5, 16.7
0.7, 16.0 0.6, and 15.6 0.4 mm, respectively; Fig.
2). The number of medium-size follicles (6 to 9 mm in
diameter) on days 2, 4, and 6 also did not differ among
groups (2.4 0.4, 2.5 0.2, and 1.6 0.2, respec-
tively). As expected, none of the cows manifested signs
of estrus before aspiration or slaughter 42 h after PGF
2
administration.
Table 5
Means SEM of nontransformed somatic cell count (SCC) and
number of cows with positive identification of pathogens isolated
in the 3 experimental groups classified according to intramammary
infection status (uninfected, subclinical, and past clinical) and
estradiol concentration in the follicular fluid (normal, low)
Uninfected
a
Subclinical
a
Past
clinical
b
Estradiol Normal Normal Low Normal
SCC (10
3
)/mL
milk
79 6* 646 141 772 261 277 84
S. dysgalactiae 5 2 0
E. coli 3 1 9
Coagulase-negative
staphylococci
860
Proteos 2 0 0
No bacterial
growth
100
*P0.05, uninfected group differed from the past clinical and
subclinical groups.
a
SCC values for uninfected and subclinical cows are the mean of
2 or 3 monthly milk tests and 2 additional samples taken within
1 wk before aspiration.
b
Mean values of 2 samples taken within 1 wk before aspiration.
During the clinical event, 40 6 d before aspiration, the mean
SCC value was 2,812 779 (10
3
). One organism was isolated
from each cow.
Fig. 1. Mean concentrations of estradiol in the plasma during the
follicular phase, from PGF
2
injection on day 6 until follicular aspi-
ration, 42 h later on day 8 (experiment 1), of cows exhibiting normal
or low estradiol in the follicular fluid: uninfected-normal (n 16, ),
subclinical-normal (n 11, ), subclinical-low estradiol (n 5, ),
and past clinical-normal (n 8, ) cows. Pooled SEM for the
respective groups were 0.56, 0.72, 1.08, and 0.7 pg/mL. *The con-
centration in the subclinical-low estradiol group just before time of
aspiration was lower (P0.05) than in uninfected, past-clinical-
normal, and subclinical-normal cows.
Fig. 2. Growth patterns of the large follicle, in experiments 1 and 2,
during final stages of estrus synchronization (day 2 to day 0) and
those of the first-wave dominant follicle (days 2 to 8 in experiment 1
and days 2 to 6 in experiment 2) of cows exhibiting normal or low
estradiol in the follicular fluid: uninfected-normal (n 24, ),
subclinical-normal (n 19, ), subclinical-low estradiol (n 9, ),
and past clinical-normal (n 8, ) cows. PGF
2
was given on day
6 and preovulatory follicles were aspirated in vivo (experiment 1) or
following slaughter (experiment 2) on day 8. Pooled SEM for the
respective groups: 1.16, 1.35, 1.05, and 0.92 mm.
104 Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
3.5. Levels of mRNA of gonadotropin receptors and
steroidogenic genes in the theca and granulosa cells
Levels of LH receptor mRNA (LHCGR) in the the-
cal cell layer were lowest in the subclinical-low estra-
diol group compared with the uninfected and subclini-
cal-normal cows (Fig. 3;P0.05). Similarly, the
expression of genes associated with steroidogensis in
the theca cells was lower in the subclinical-low estra-
diol group (n 4). This was evident for cytochrome
P450 side-chain cleavage (CYP11A1) and cytochrome
P450 17-
-hydroxylase/17,20-lyase (CYP17A1) rela-
tive to uninfected (n 8) and subclinical-normal cows
(n 8; P0.05; Fig. 3). In accordance with the
mRNA data, the level of CYP11A1 protein was re-
duced in the subclinical-low estradiol group (n 4)
relative to the uninfected cows (n 7; P0.04; Fig.
4). Values of subclinical-normal cows were intermediate
(n 8; Fig. 4). Unlike CYP11A1 and CYP17A1, steroi-
dogenic acute regulatory protein (StAR) and 3
-hydrox-
ysteroid dehydrogenase (HSD3B1) gene expression in the
theca cells did not differ among groups (Fig. 3).
Levels of cytochrome P450 aromatase mRNA
(CYP19A1) in the granulosa cells were about fivefold
higher (P0.05) in the uninfected (n 13) and past
clinical mastitic cows (n 4) and tended to be higher
(P0.1) in the subclinical-normal estradiol cows (n
7) than in the subclinical-low estradiol cows (n 4;
Fig. 5). As in the theca cells, LHCGR expression in the
granulosa cells was low in subclinical-low estradiol
cows (P0.05 or P0.08); however, follicle-stim-
ulating hormone (FSH) receptor (FSHR) did not differ
among groups (Fig. 5). Importantly, CYP19A1 and
FSHR were not detected in the theca layer, indicating
the absence of granulosa cell contamination (data not
shown).
4. Discussion
Data from these experiments indicate an association
between subclinical mastitis and a pronounced reduc-
tion of follicular estradiol and androstenedione in the
preovulatory follicles of one-third of the infected cows.
Low follicular steroid levels in these cows apparently
result from abnormally low expression of LHCGR and
steroidogenic genes in both the theca and the granulosa
cell layers.
Depression of estradiol in the follicles of the sub-
clinical-low estradiol group was directly caused by re-
duced CYP19A1 expression in the granulosa cells, and
reduced androstenedione level was caused by a lower
expression of both CYP11A1 and CYP17A1 in the theca
cells. A decrease in these 2 enzymes, considered rate-
limiting steps in androgen production in the theca cells,
reduced availability of the aromatization substrate, re-
inforcing the effect of the already low CYP19A1 levels.
Gene expression in the remaining two-thirds of the
subclinical cows (exhibiting normal follicular steroid
levels) was as high as in the uninfected cows. Interest-
Fig. 3. Expression of mRNA for gonadotropin receptor and steroidogenic genes in theca cells collected from preovulatory follicles (experiment
2): (A) LHCGR, (B) StAR, (C) CYP11A1, (D) CYP17A1, and (E) HSD3B1. RNA was extracted from cows exhibiting normal or low estradiol in
the follicular fluid: uninfected-normal (n 8), subclinical-normal (n 8), and subclinical-low estradiol (n 4) cows, followed by reverse
transcription and real-time PCR analysis. All animals were treated with PGF
2
42 h prior to collection of the ovaries. Data are presented as
means SEM. Different letters denote significant differences at P0.05.
105Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
ingly, expressions of StAR,HSD3B1, and FSHR were
not affected in the subclinical-low estradiol theca and
granulosa cells. The reason for the differential re-
sponses of the various steroidogenic genes to IMI is
still unclear.
Reduced LHCGR in both granulosa and theca cells
in the subclinical-low estradiol group appears to be a
major determinant in abnormal preovulatory follicle
function. Several lines of evidence demonstrate the
essential roles of LH signaling. First, LH upregulates
steroidogenic enzyme expression in both the granulosa
and the theca cell layers [30]. Second, during follicle
selection and deviation, as well as dominance acquisi-
tion, there is a transition from FSH to LH dependency
in granulosa cells, allowing continued steroid produc-
tion in the dominant follicle [31]. Therefore, reduced
LHCGR may indicate reduced LH support of the dom-
inant follicle [32]. Third, increased LHCGR expression
in the granulosa cells of dominant follicles has been
suggested to induce genes involved in granulosa and
thecal cell survival [30]. Interestingly, unlike the pos-
sible involvement of LHCGR reduction in attenuated
steroidogenesis, lower LHCGR did not affect the
growth of the preovulatory follicle in the subclinical-
low estradiol group.
Secretion of LH during the follicular phase most
probably does not play a direct role in LHCGR expres-
sion in subclinical-low estradiol follicles because LH
pulses are not affected by naturally occurring subclin-
ical or past clinical IMI [13]. Although we cannot rule
out the possible involvement of lower circulating FSH
in LHCGR levels, this is less likely because the induc-
tion of inflammation or uterine metritis does not appear
to alter pituitary secretion or circulating levels of FSH
[33,34]. Interestingly, in this study, the decreased ex-
pression of LHCGR was not accompanied by a reduc-
tion in FSHR in the granulosa cells. However, FSHR
could have already been down-regulated at the time of
follicular aspiration.
The endocrine status of follicles in the subclinical-
low estradiol group is of dominant preovulatory folli-
cles with reduced steroidogenic capacity. This is based
on the following: (a) all subclinical-low estradiol cows
exhibited a single large, first-wave, dominant preovu-
latory follicle, with growth dynamics that was nearly
identical to that of their counterparts in the uninfected
Fig. 4. Protein concentrations of CYP11A1 in theca cells (experiment
2) obtained from preovulatory follicles of uninfected-normal (n 7),
subclinical-normal (n 8), and subclinical-low estradiol cows (n
4). Protein extracts were processed for western blotting using specific
CYP11A1 antibodies as detailed under Materials and methods. The
membranes were also probed with anti-total mitogen-activated pro-
tein kinase (MAPK; p44/42) antibody. (A) Representative western
blot for CYP11A1 with a major band at 49.5 kDa and MAPK (42– 44
kDa) as a protein loading control. (B) Densitometric quantification of
CYP11A1 content. Bars represent the mean SEM. Different letters
denote significant differences at P0.05.
Fig. 5. mRNA expression of gonadotropin receptor and steroidogenic
genes in granulosa cells collected from preovulatory follicles (exper-
iment 1): (A) CYP19A1, (B) LHCGR, and (C) FSHR. RNA was
extracted from cows exhibiting normal or low estradiol in the follic-
ular fluid: uninfected-normal (n 13), subclinical-normal (n 7),
subclinical-low estradiol (n 4), and past clinical-normal (n 4)
cows, followed by reverse transcription and real-time PCR analysis.
All animals were treated with PGF
2
42 h prior to aspiration. Data are
presented as means SEM. Different letters denote significant dif-
ferences at P0.05. The subclinical-normal group tended to differ
from the subclinical-low estradiol group,
P0.08 or
#
P0.1.
106 Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
and mastitic-normal cows; this follicle was eventually
aspirated; (b) the lack of increase in progesterone in the
follicular fluid indicates that these follicles were not
atretic; and (c) although follicular estradiol was low,
the estradiol-to-progesterone ratio was 1, indicating
that the follicles in this group were estrogenically active
preovulatory follicles and not subordinate follicles. Im-
portantly, based on their growth profile and the low
progesterone concentrations in the follicular fluid, the
follicles were not cystic [35].
The involvement of factors other than IMI in the
impairment of follicular responses in the subclinical-
low estradiol group was disproven by the following: the
study was performed almost 6 mo after calving to
prevent possible effects of postpartum uterine disorders
or short cycles; all experimental groups manifested
normal cyclicity prior to the study and normal appear-
ance of ovarian structures was documented, from syn-
chronization through ovulation and growth of the first-
wave dominant follicle; no major veterinary problems
were recorded; and none of the herd variables listed
earlier differed among groups.
The rationale for examining the possible carryover
effect of past clinical IMI, occurring weeks or months
earlier, on reproductive responses is that early postpar-
tum cows are highly susceptible to bacteria that cause
clinical mastitis [36]. Therefore, a high proportion of
cows that have had clinical IMI early postpartum may
carry its potentially damaging effect on follicular func-
tion to time of breeding (or insemination) at later
stages. The current study shows that short-term clinical
events of mastitis occurring approximately 40 d prior to
the examination did not affect follicular function. An
epidemiological survey conducted recently supports
these findings (G. Leitner, E. Ezra, Y. Lavon, and D.
Wolfenson, unpublished data): the survey indicated that
clinical cases of IMI occurring within 2 wk prior to
insemination resulted in lower conception rate; how-
ever, earlier events, occurring 20 or 30 d before insem-
ination, did not affect conception. Collectively, these
studies indicate that the effects of short-term clinical
IMI on follicular responses fade with time and, specif-
ically, that ovarian follicles are unlikely to be affected
by a clinical event that occurred about 40 d earlier.
Unlike short-term clinical mastitic events, the long-
term nature of subclinical IMI can damage the long
process of follicular growth at various time points.
The proportion of one-third of IMI cows exhibiting
susceptibility to IMI vs two-thirds showing normal re-
productive responses has been observed, in 30 of a total
of 96 experimental cows, in 4 independent studies ex-
amining naturally occurring or induced subclinical or
clinical IMI [10,13,37, and the current study]. This
phenomenon therefore appears to hold for a broader
population. Many other studies have reported differen-
tial responses to endotoxin exposure. For instance, in
sheep, LPS disrupted LH pulse secretion in 50% of
ewes [16] and inhibited 36% to 54% of LH surges
[38,39]. In cattle, diverse responses to inflammation
and altered reproductive responses following injection
of Streptococcus uberis were reported [17]. It is unclear
what causes one-third of the subclinical IMI cows to be
susceptible while the other two-thirds remain unaf-
fected, showing a phenotype similar to that of unin-
fected cows. None of the following factors could be
related to the cows’ differential response: duration of
the disease before examination, severity of the infection
in terms of SCC levels, stage of lactation and number of
lactations, level of milk yield, body condition score,
and type of pathogen.
Because previous studies have ruled out the involve-
ment of circulating LH during the follicular phase, one
might assume that immune mediators affect ovarian
function. It is beyond the scope of the current study to
identify the specific immune mediator(s) that may be
associated with disruption of follicular steroidogenesis
in one-third of the subclincal cows. Several inflamma-
tory mediators have been shown to affect steroidogen-
esis, either directly or through interactions with the
insulin-like growth factor system or glucocorticoids
[40]. Among these, tumor necrosis factor
is a cyto-
kine that is frequently cited as being involved with
lower androgen production by theca cells and estradiol
production by granulosa cells [11,34]. Another proin-
flamatory cytokine, interleukin 1
, has been shown to
reduce FSH-induced estradiol production in large fol-
licles [41]. Others have shown attenuation of estradiol
production by interferon gamma and reduced CYP19A1
activity by interleukin 6 [11,40]. However, most, if not
all of these studies induced acute, short-term, experi-
mental pathogenic stress [9] and not subclinical,
chronic IMI. An example of the complexity of this
issue was demonstrated in a study in which no elevation
of tumor necrosis factor
could be detected in mastitis
induced by Gram-positive bacteria [7]. The question of
which inflammatory mediator(s) accounts for suppres-
sion of follicular steroidogenesis in subclinical IMI in
bovine therefore has yet to be resolved.
The reduced expression of several steroidogenic en-
zymes in both theca and granulosa cells not only re-
duced follicular steroid levels, but also resulted in low
circulating estradiol levels around estrus in the subclin-
107Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
ical-low estradiol cows. This was shown here and in
our previous study, in which a 50% lower circulating
estradiol concentration around estrus (compared with
uninfected controls) was associated with suppressed
LH surge and delayed ovulation [13]. Importantly, low
preovulatory estradiol concentration in the plasma did
not delay or abolish estrus manifestation in cows ex-
hibiting a longer than normal estrus-to-ovulation inter-
val [13,15,42].
In conclusion, the findings of the current study sug-
gest a possible mechanism by which a relatively large
group of subclinical mastitic cows manifest reduced
preovulatory estradiol output, which leads to low and
delayed LH surge and delayed ovulation, resulting in
low fertilization and conception rates.
Acknowledgments
The authors thank S. Jacoby, O. Krifucks, L. Weis-
blit, and G. Glick for their valuable help in performing
the study. This work was supported by the Israel Dairy
Board (Grant 820-216).
References
[1] Huszenicza GY, Jánosi SZ, Kulcsár M, Kóródi P, Reiczigel J,
Kátai L, Peters AR, De Rensis F. Effects of clinical mastitis on
ovarian function in post-partum dairy cows. Reprod Domest
Anim 2005;40:199 –204.
[2] Bascom SS, Young AJ. A summary of the reasons why farmers
cull cows. J Dairy Sci 1998;81:2299 –305.
[3] Santos JE, Cerri RL, Ballou MA, Higginbotham GE, Kirk JH.
Effect of timing of first clinical mastitis occurrence on lacta-
tional and reproductive performance of Holstein dairy cows.
Anim Reprod Sci 2004;80:31– 45.
[4] Schrick FN, Hockett ME, Saxton AM, Lewis MJ, Dowlen HH,
Oliver SP. Influence of subclinical mastitis during early lacta-
tion on reproductive parameters. J Dairy Sci 2001;84:1407–12.
[5] Pinedo PJ, Melendez P, Villagomez-Cortes JA, Risco CA. Ef-
fect of high somatic cell counts on reproductive performance of
Chilean dairy cattle. J Dairy Sci 2009;92:1575– 80.
[6] Pyorala S. Mastitis in post-partum dairy cows. Reprod Domest
Anim 2008;43(Suppl 2):252–9.
[7] Riollet C, Rainard P, Poutrel B. Differential induction of com-
plement fragment C5a and inflammatory cytokines during in-
tramammary infections with Escherichia coli and Staphylococ-
cus aureus. Clin Diagn Lab Immunol 2000;7:161–7.
[8] Suzuki C, Yoshioka K, Iwamura S, Hirose H. Endotoxin in-
duces delayed ovulation following endocrine aberration during
the proestrous phase in Holstein heifers. Domest Anim Endo-
crinol 2001;20:267–78.
[9] Bannerman DD, Paape MJ, Lee J-W, Zhao X, Hope JC, Rainard
P. Escherichia coli and Staphylococcus aureus elicit differential
innate immune responses following intramammary infection.
Clin Diagn Lab Immunol 2004;11:463–72.
[10] Lavon Y, Leitner G, Goshen T, Braw-Tal R, Jacoby S, Wolfen-
son D. Exposure to endotoxin during estrus alters the timing of
ovulation and hormonal concentrations in cows. Therio-
genology 2008;70:956 – 67.
[11] Spicer LJ, Alpizar E. Effects of cytokines on FSH-induced
estradiol production by bovine granulosa cells in vitro: Depen-
dence on size of follicle. Domest Anim Endocrinol 1994;11:
25–34.
[12] Herath S, Williams EJ, Lilly ST, Gilbert RO, Dobson H, Bryant
CE, Sheldon IM. Ovarian follicular cells have innate immune
capabilities that modulate their endocrine function. Reproduc-
tion 2007;134:683–93.
[13] Lavon Y, Leitner G, Voet H, Wolfenson D. Naturally occurring
mastitis effects on timing of ovulation, steroid and gonadotro-
phic hormone concentrations, and follicular and luteal growth in
cows. J Dairy Sci 2010;93:911–21.
[14] Roelofs JB, van Eerdenburg FJCM, Soede NM, Kemp B. Var-
ious behavioral signs of estrous and their relationship with time
of ovulation in dairy cattle. Theriogenology 2005;63:1366 –77.
[15] Bloch A, Folman Y, Kaim M, Roth Z, Braw-Tal R, Wolfenson
D. Endocrine alterations associated with extended time interval
between estrus and ovulation in high-yield dairy cows. J Dairy
Sci 2006;89:4694 –702.
[16] Battaglia DF, Krasa HB, Padmanabhan V, Viguie C, Karsch FJ.
Endocrine alterations that underlie endotoxin-induced disrup-
tion of the follicular phase in ewes. Biol Reprod 2000;62:45–53.
[17] Hockett ME, Almeida RA, Rohrbach NR, Oliver SP, Dowlen
HH, Schrick FN. Effects of induced clinical mastitis during
preovulation on endocrine and follicular function. J Dairy Sci
2005;88:2422–31.
[18] Leitner G, Krifucks O, Merin U, Lavi Y, Silanikove N. Inter-
actions between bacteria type, proteolysis of casein and
physico-chemical properties of bovine milk. Int Dairy J 2006;
16:648 –54.
[19] Oliver SP, Almeida RA, Gillespie BE, Headrick SJ, Dowlen
HH, Johnson DL, Lamar KC, Chester ST, Moseley WM. Ex-
tended ceftiofur therapy for treatment of experimentally-in-
duced Streptococcus uberis mastitis in lactating dairy cattle. J
Dairy Sci 2004;87:3322–9.
[20] Younis A, Krifucks O, Heller ED, Samra Z, Glickman A, Saran
A, Leitner G. Staphylococcus aureus exosecretions and bovine
mastitis. J Vet Med B 2003;50:1–7.
[21] Roth Z, Arav A, Bor A, Zeron Y, Braw-Tal R, Wolfenson D.
Improvement of quality of oocytes collected in the autumn by
enhanced removal of impaired follicles from previously heat-
stressed cows. Reproduction 2001;122:737– 44.
[22] Klipper E, Tatz E, Kisliouk T, Vlodavsky I, Moallem U,
Schams D, Lavon Y, Wolfenson D, Meidan R. Induction of
heparanase in bovine granulosa cells by luteinizing hormone:
Possible role during the ovulatory process. Endocrinology 2009;
150:413–21.
[23] Meidan R, Girsh E, Blum O, Aberdam E. In vitro differentiation
of bovine theca and granulosa cells into small and large luteal-
like cells: Morphological and functional characteristics. Biol
Reprod 1990;43:913–21.
[24] Turzillo AM, Fortune JE. Suppression of the secondary FSH
surge with bovine follicular fluid is associated with delayed
ovarian follicular development in heifers. J Reprod Fertil 1990;
89:643–53.
[25] Shaham-Albalancy A, Rosenberg M, Folman Y, Graber Y,
Meidan R, Wolfenson D. Two methods of inducing low plasma
progesterone concentrations have different effects on dominant
follicles in cows. J Dairy Sci 2000;83:2771– 8.
108 Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
[26] Levy N, Gordin M, Mamluk R, Yanagisawa M, Smith MF,
Hampton JH, Meidan R. Distinct cellular localization and reg-
ulation of endothelin-1 and endothelin-converting enzyme-1
expression in the bovine corpus luteum: Implications for lute-
olysis. Endocrinology 2001;142:5254 – 60.
[27] Klipper E, Levit A, Mastich Y, Berisha B, Schams D, Meidan
R. Induction of endothelin-2 expression by luteinizing hormone
and hypoxia: Possible role in bovine corpus luteum formation.
Endocrinology 2010;151:1914 –22.
[28] De Castro e Paula LA, Andrzejewski J, Julian D, Spicer LJ,
Hansen PJ. Oxygen and steroid concentrations in preovulatory
follicles of lactating dairy cows exposed to acute heat stress.
Theriogenology 2008;69:805–13.
[29] Zachut M, Arieli A, Lehrer H, Argov N, Moallem U. Dietary
unsaturated fatty acids influence preovulatory follicle charac-
teristics in dairy cows. Reproduction 2008;135:683–92.
[30] Mihm M, Baker PJ, Ireland JL, Smith GW, Coussens PM,
Evans AC, Ireland JJ. Molecular evidence that growth of dom-
inant follicles involves a reduction in follicle-stimulating hor-
mone dependence and an increase in luteinizing hormone de-
pendence in cattle. Biol Reprod 2006;74:1051–9.
[31] Ginther OJ, Beg MA, Bergfelt DR, Donadeu FX, Kot K. Fol-
licle selection in monovular species. Biol Reprod 2001;65:
638 – 47.
[32] Evans AC, Fortune JE. Selection of the dominant follicle in
cattle occurs in the absence of differences in the expression of
messenger ribonucleic acid for gonadotropin receptors. Endo-
crinology 1997;138:2963–71.
[33] McCann SM, Kimura M, Karanth S, Yu WH, Mastronardi CA,
Rettori V. The mechanism of action of cytokines to control the
release of hypothalamic and pituitary hormones in infection.
Ann N Y Acad Sci 2000;917:4 –18.
[34] Williams EJ, Sibley K, Miller AN, Lane EA, Fishwick J, Nash
DM, Herath S, England GCW, Dobson H, Sheldon MI. The
effect of Escherichia coli lipopolysaccharide and tumour necro-
sis factor alpha on ovarian function. Am J Reprod Immunol
2008;60:462–73.
[35] Braw-Tal R, Pen S, Roth Z. Ovarian cysts in high-yielding dairy
cows. Theriogenology 2009;72:690 – 8.
[36] Sordillo LM. Factors affecting mammary gland immunity and
mastitis susceptibility. Livest Prod Sci 2005;98:89 –99.
[37] Lavon Y, Leitner G, Meidan R, Moallem U, Klipper E, Wolfen-
son D. Immediate and carryover effects of Gram-negative or
Gram-positive toxin-induced mastitis on follicular functions in
cows. J Dairy Sci 2009;92:442.
[38] Adams TE, Sakurai H, Adams BM. Effect of stress-like con-
centrations of cortisol on estradiol-dependent expression of go-
nadotropin-releasing hormone receptor in orchidectomized
sheep. Biol Reprod 1999;60:164 8.
[39] Breen KM, Karsch FJ. Does cortisol inhibit pulsatile luteinizing
hormone secretion at the hypothalamic or pituitary level? En-
docrinology 2004;145:692– 8.
[40] Bornstein SR, Rutkowski H, Vrezas I. Cytokines and steroido-
gensis. Mol Cell Endocrinol 2004;215:135– 41.
[41] Baratta M, Basini G, Bussolati S, Tamanini C. Effects of inter-
leukin-1 beta fragment (163-171) on progesterone and estradi-
ol-17 beta release by bovine granulosa cells from different size
follicles. Regul Pept 1996;67:187–94.
[42] Saumande J, Humblot P. The variability in the interval between
estrus and ovulation in cattle and its determinants. Anim Reprod
Sci 2005;85:171– 82.
109Y. Lavon et al. / Domestic Animal Endocrinology 40 (2011) 98 –109
... With regard to the above, we recently found a similar proportion of subclinical mastitic cows that manifested impaired preovulatory follicular responses. A low follicular fluid estradiol level was associated with a low androstenedione level and with low expression of steroidogenic genes in granulosa cells in one-third of cows (Lavon et al., 2008b(Lavon et al., , 2009a. ...
... The diversity between individual mastitic cows in terms of exhibiting a normal or an extended E-O interval is unclear. Also unclear is the high variability in the pattern of the preovulatory LH surge, whether low and delayed, or normal and delayed, or when no surge was evident among the cows with an extended E-O (Lavon et al., 2008b, Lavon et al., 2009a. Our data support Burvenich et al. (2003), who stated that the severity of naturally occurring cases of clinical and subclinical mastitis and the variability in responses among individuals was largely related to cow factors. ...
Article
The effects of naturally occurring subclinical chronic or clinical short-term mastitis on time of ovulation, plasma steroid and gonadotropin concentrations, and follicular and luteal dynamics were examined in 73 lactating Holstein cows. Cows were sorted by milk somatic cell count and bacteriological examination into an uninfected group (n=22), a clinical mastitis group (n=9; events occurring 20+/-7 d before the study), and a subclinical chronic mastitis group (n=42). In addition, uninfected and mastitic cows were further sorted by their estrus to ovulation (E-O) interval. About 30% of mastitic cows (mainly subclinical) manifested an extended E-O interval of 56+/-9.2h compared with 28+/-0.8h in uninfected cows and 29+/-0.5h in the other 70% of mastitic cows. In mastitic cows with extended E-O interval, the concentration of plasma estradiol at onset of estrus was lower than that of uninfected cows or mastitic cows that exhibited normal E-O intervals (3.1+/-0.4, 5.8+/-0.5, and 5.5+/-0.5 pg/mL, respectively). The disruptive effect of mastitis on follicular estradiol probably does not involve alterations in gonadotropin secretion because any depressive effects of mastitis on pulsatile LH concentrations were not detected. Cortisol concentrations did not differ among groups. The preovulatory LH surge in mastitic cows with delayed ovulation varied among individuals, being lower, delayed, or with no surge noted compared with the normal LH surge exhibited by uninfected cows or mastitic cows with normal E-O interval (6.8+/-0.7 ng/mL). The diameter of the second-wave dominant follicle was larger and the number of medium follicles was smaller in uninfected and subclinical cows with normal intervals compared with subclinical cows with extended intervals (13.4+/-0.5 vs. 10.9+/-0.9mm, and 3.8+/-0.2 vs. 6.7+/-0.14 follicles, respectively). Mid-luteal progesterone concentrations were similar in uninfected and mastitic cows. These results indicate for the first time that around 30% of cows with subclinical chronic mastitis exhibit delayed ovulation that is associated with low plasma concentrations of estradiol and a low or delayed preovulatory LH surge.
... Lavon et al. (2010) identified similar long-term effects on follicular dynamics and steroids in cows with subclinical and resolved clinical mastitis. Interestingly, these authors also identified decreased androstendione and estradiol follicular fluid concentrations, as well as decreased expression of steroidogenic enzymes including P450c17 enzymes, in follicular cells from subclinical mastitic cows (Lavon et al., 2009). This would also imply that common pathways and mechanisms of action exist between uterine and mammary infections. ...
Article
Full-text available
The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.
Article
Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.
Article
Full-text available
The effects of separate infection with four major pathogens frequently associated with the occurrence of subclinical mastitis in cows (Staphylococcus aureus, S. chromogenes, Escherichia coli and Streptococcus dysgalactiae) on milk quality for cheese production were studied for quarters of the same animal. Infection increased somatic cell count (SCC), modified leucocyte distribution, decreased lactose concentration and increased proteolysis of casein. Regardless of bacteria type, the plasmin activity in milk from the infected glands increased ∼2 fold compared with uninfected quarters. These changes were associated with increased rennet clotting time and decreased curd firmness for milk from infected glands, indicating that milk quality for cheese production was negatively affected by infection. Although the general pattern of bacterial invasion was similar, each type of bacteria elicited the above-described responses in a specific manner. SCC, commonly used by the dairy industry as a measure of milk hygienic quality, provided the poorest prediction of milk quality for cheese production in comparison to indices of proteolysis of casein.
Article
Full-text available
Two experiments were conducted to investigate endocrine mechanisms by which the immune/inflammatory stimulus endotoxin disrupts the follicular phase of the estrous cycle of the ewe. In both studies, endotoxin was infused i.v. (300 ng/kg per hour) for 26 h beginning 12 h after withdrawal of progesterone to initiate the follicular phase. Experiment 1 sought to pinpoint which endocrine step or steps in the preovulatory sequence are compromised by endotoxin. In sham-infused controls, estradiol rose progressively from the time of progesterone withdrawal until the LH/FSH surges and estrous behavior, which began approximately 48 h after progesterone withdrawal. Endotoxin interrupted the preovulatory estradiol rise and delayed or blocked the LH/FSH surges and estrus. Experiment 2 tested the hypothesis that endotoxin suppresses the high-frequency LH pulses necessary to stimulate the preovulatory estradiol rise. All 6 controls exhibited high-frequency LH pulses typically associated with the preovulatory estradiol rise. As in the first experiment, endotoxin interrupted the estradiol rise and delayed or blocked the LH/FSH surges and estrus. LH pulse patterns, however, differed among the six endotoxin-treated ewes. Three showed markedly disrupted LH pulses compared to those of controls. The three remaining experimental ewes expressed LH pulses similar to those of controls; yet the estradiol rise and preovulatory LH surge were still disrupted. Our results demonstrate that endotoxin invariably interrupts the preovulatory estradiol rise and delays or blocks the subsequent LH and FSH surges in the ewe. Mechanistically, endotoxin can interfere with the preovulatory sequence of endocrine events via suppression of LH pulsatility, although other processes such as ovarian responsiveness to gonadotropin stimulation appear to be disrupted as well.
Article
Full-text available
The pattern and regulation of endothlin-2 (EDN2) expression and its putative roles in bovine ovaries were investigated. EDN2 mRNA was determined in corpus luteum (CL) and during folliculoluteal transition induced by GnRH in vivo. EDN2 was elevated only in the early CL and was not present in older CL. In the young CL, EDN2 mRNA was identified mainly in luteal cells but not endothelial cells that expressed the EDN1 gene. Similarly, in preovulatory follicles, EDN2 was expressed in the granulosa cells (GCs) and not in the vascular theca interna. LH and hypoxia are two major stimulants of CL formation. Therefore, GCs were cultured with bovine LH, under hypoxic conditions. GCs incubated with bovine LH resulted in increased EDN2 mRNA 42 h later. CoCl2, a hypoxia-mimicking agent, elevated EDN2 in GCs in a dose-dependent manner. Incubation of the human GC line (Simian virus 40 large T antigen) under low oxygen tension (1%) augmented EDN2 6 and 24 h later. In these two cell types, along with EDN2, hypoxia augmented VEGF. EDN2 induced in GCs changes that characterize the developing CL: cell proliferation as well as up-regulation of vascular endothelial growth factor and cyclooxygenase-2 (mRNA and protein levels). Human chorionic gonadotropin also up-regulated these two genes. Small interfering RNA targeting EDN-converting enzyme-1 effectively reduced its mRNA levels. This treatment, expected to lower the mature EDN2 peptide production, inhibited VEGF mRNA levels and GC numbers. Together these data suggest that elevated EDN2 in the early bovine CL, triggered by LH surge and hypoxia, may facilitate CL formation by promoting angiogenesis, cell proliferation, and differentiation.
Article
Full-text available
The objectives were to evaluate the effect of high linear somatic cell counts (LNSCC > or =4.5) during early lactation on reproductive performance and to estimate their association with the risk of abortion in a population of central-southern Chilean dairy cattle. The analysis included records from a population of 157 farms and considered 1,127,405 test-day records including 101,944 lactations that began between 1997 and 2006. After data edits, the analyses of calving to first service and calving to conception intervals consisted of 88,633 and 70,877 lactations, respectively. Once controlling for significant variables, time to first breeding was 21.8 d longer in cows with at least 1 high LNSCC before the first breeding compared with controls. Cows with at least 1 high LNSCC before the fertile breeding had an increment in time to conception of 48.7 d and required, on average, 0.49 more services to conceive. The odds of conception at first service in cows with a high LNSCC within 30 d before [after] breeding were 0.85 (0.81 to 0.89; 95% confidence interval ) [0.82 (0.78 to 0.87; 95% confidence interval)] times the odds of conception for cows without a high LNSCC during that period. The Cox proportional hazard model indicated that after correction by calving year, lactation number, and milk yield standardized to 305 d, the risk of pregnancy decreased by 44% if a high LNSCC occurred before breeding. Cows registering a high LNSCC during the first 90 d of gestation had an increased risk of abortion, being 1.22 (1.07 to 1.35; 95% confidence interval) times more likely to abort than nonaffected cows. It is concluded that subclinical mastitis, measured as LNSCC >/=4.5, had a significant effect on reproductive performance in Chilean dairy cattle.
Article
Full-text available
Pelvic inflammatory disease and metritis are important causes of infertility in humans and domestic animals. Uterine infection with Escherichia coli in cattle is associated with reduced ovarian follicle growth and decreased estradiol secretion. We hypothesized that this effect could be mediated by the bacterial lipopolysaccharide (LPS) or cytokines such as tumour necrosis factor alpha (TNFalpha). In vitro, bovine ovarian theca and granulosa cells were treated with LPS or TNFalpha and steroid secretion measured. In vivo, the effect of LPS or TNFalpha intrauterine infusion was determined by ovarian ultrasonography and measurement of hormones in cattle. Lipopolysaccharide reduced granulosa cell estradiol secretion, whilst TNFalpha decreased theca and granulosa cell androstenedione and estradiol production, respectively. In vivo, fewer animals ovulated following intrauterine infusion with LPS or TNFalpha. Lipopolysaccharide and TNFalpha suppress ovarian cell function, supporting the concept that pelvic inflammatory disease and metritis are detrimental for bovine ovarian health.
Article
Dairy cattle are more susceptible to mastitis during the periparturient period. It is well established that the incidence of mastitis with respect to lactation stage are directly related to changes in the composition, magnitude, and efficiency of the mammary gland defense system. There exist numerous genetic, physiological, and environmental factors that can compromise host defense mechanisms during the functional transitions of the mammary gland. For example, physiological stresses associated with rapid differentiation of secretory parenchyma, intense mammary gland growth, and the onset of milk synthesis and secretion are accompanied by a high energy demand and an increased oxygen requirement. This increased oxygen demand augments the production of oxygen-derived reactants, collectively termed reactive oxygen species (ROS). The excessive accumulation of ROS can lead to a condition referred to as oxidant stress that plays a central role in mediating uncontrolled inflammatory responses and causing tissue injury. While the last two decades have seen major progress in understanding the bovine mammary gland defense system and its function in preventing disease, diminished host defenses and increased susceptibility to mastitis continue to be a problem for dairy cattle during transition periods. This paper provides an overview of mammary immunobiology and describes those factors known to influence important mammary gland defenses during the periparturient period.
Article
Transition from the dry period to lactation is a high risk period for the modern dairy cow. The biggest challenge at that time is mastitis. Environmental bacteria are the most problematic pathogens around parturition. Coliforms are able to cause severe infections in multiparous cows, and heifers are likely to be infected with coagulase-negative staphylococci. During the periparturient period, hormonal and other factors make the dairy cows more or less immunocompromised. A successful mastitis control programme is focused on the management of dry and calving cows and heifers. Clean and comfortable environment, proper feeding and adequate supplementation of the diet with vitamins and trace elements are essential for maintaining good udder health. Strategies which would enhance closure of the teat canal in the beginning of the dry period and would protect teat end from bacteria until the keratin plug has formed decrease the risk for mastitis after calving. Dry cow therapy has been used with considerable success. Yet, a selective approach could be recommended rather than blanket therapy. Non-antibiotic approaches can be useful tools to prevent new infections during the dry period, in herds where the risk for environmental mastitis is high. Vaccination has been suggested as a means to support the immune defence of the dairy cow around parturition. In some countries, implementation of Escherichia coli core antigen vaccine has reduced the incidence of severe coliform mastitis after calving.
Article
This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF(2α) on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and 4; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, androstenedione, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about 40%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and 4, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and androstenedione were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P > 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and 4. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P > 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle 4 of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.
Article
The effects of naturally occurring subclinical chronic or clinical short-term mastitis on time of ovulation, plasma steroid and gonadotropin concentrations, and follicular and luteal dynamics were examined in 73 lactating Holstein cows. Cows were sorted by milk somatic cell count and bacteriological examination into an uninfected group (n=22), a clinical mastitis group (n=9; events occurring 20+/-7 d before the study), and a subclinical chronic mastitis group (n=42). In addition, uninfected and mastitic cows were further sorted by their estrus to ovulation (E-O) interval. About 30% of mastitic cows (mainly subclinical) manifested an extended E-O interval of 56+/-9.2h compared with 28+/-0.8h in uninfected cows and 29+/-0.5h in the other 70% of mastitic cows. In mastitic cows with extended E-O interval, the concentration of plasma estradiol at onset of estrus was lower than that of uninfected cows or mastitic cows that exhibited normal E-O intervals (3.1+/-0.4, 5.8+/-0.5, and 5.5+/-0.5 pg/mL, respectively). The disruptive effect of mastitis on follicular estradiol probably does not involve alterations in gonadotropin secretion because any depressive effects of mastitis on pulsatile LH concentrations were not detected. Cortisol concentrations did not differ among groups. The preovulatory LH surge in mastitic cows with delayed ovulation varied among individuals, being lower, delayed, or with no surge noted compared with the normal LH surge exhibited by uninfected cows or mastitic cows with normal E-O interval (6.8+/-0.7 ng/mL). The diameter of the second-wave dominant follicle was larger and the number of medium follicles was smaller in uninfected and subclinical cows with normal intervals compared with subclinical cows with extended intervals (13.4+/-0.5 vs. 10.9+/-0.9mm, and 3.8+/-0.2 vs. 6.7+/-0.14 follicles, respectively). Mid-luteal progesterone concentrations were similar in uninfected and mastitic cows. These results indicate for the first time that around 30% of cows with subclinical chronic mastitis exhibit delayed ovulation that is associated with low plasma concentrations of estradiol and a low or delayed preovulatory LH surge.
Article
We examined the hormonal and morphologic changes associated with ovarian cyst formation in high-yielding dairy cows. Follicle fluid was aspirated from 90 cysts and 15 preovulatory and 18 subordinate follicles and used for hormonal determination. Pieces of cystic wall were subjected to morphologic and immunohistochemical evaluation. Cysts were characterized by low concentrations of insulin, insulin-like growth factor-I (IGF-I), and glucose and high activity of IGF binding proteins (IGFBPs). Insulin and IGF-I levels were (mean+/-SEM) 205+/-22 pg/mL and 146+/-42 ng/mL in preovulatory follicles and 3+/-1 pg/mL and 61+/-6 ng/mL in cysts, respectively (P<0.001). Insulin-like growth factor-binding proteins activity was about 10 times higher in cysts than in preovulatory follicles. Cysts were classified into three types according to their estradiol-to-progesterone (E/P) ratio. Type 1 cysts (n=23) exhibited the highest E/P ratio (10.8+/-2.3), partial loss of granulosa cells, and severe morphologic changes in the theca interna. Expression of P(450) side-chain cleavage and P(450) 17 alpha-hydroxylase was noted in theca cells and expression of inhibin-alpha in granulosa cells. Type 2 cysts (n=35) had a low E/P ratio (0.07+/-0.02), and patches of luteal-like tissue in the cystic wall. Type 3 cysts (n=32) had an E/P ratio of 0.91+/-0.17, and no recognizable granulosa or theca cells. In summary, intrafollicular steroid levels as expressed by E/P ratio, together with IGF-I and insulin levels and morphologic changes in the follicular wall, may serve as accurate cyst-classification parameters. Because IGF-I and/or insulin play an essential role in the final stage of follicle development, it can be speculated that abnormal levels of these metabolic hormones might lead to follicle dysfunction, resulting in follicular regression or cyst formation.