Ovarian Dysfunction in

Lactating Cows
James D. Ferguson, VMD, MS ACT, ACVN
University of Pennsylvania
School of Veterinary Medicine

What do we mean?
Cows that have failed to ovulate by 40 to 60 days post
calving
By the voluntary waiting period they have had no normal
estrous cycles
The later the VWP the fewer cows will be anovulatory,
therefore VWP of 70 DIM have been proposed for higher
producing cows

Cows that initiate ovulation and then enter a phase of

anovulation prior to the breeding period
Ovulate a follicle, but then either have a prolonged CL lifespan
(progesterone is elevated for more than 16 to 18 days) or a
prolonged interval between periods of elevated progesterone

Who are the players?

s coordination between structures in the brain and the ovary (and the uterus)

thalamus is the driver: releases pulses of GnRH, which drives the

ary Gland, which is an amplifier, and releases FSH and LH, which stimulates the

y produces waves of follicles; dominant ovulates and forms a Corpus Luteum

dominant follicle produces estrogen, inhibin, among other molecules
which inhibits the growth of other follicles, causing them to regress
CL produces progesterone

us produces prostaglandin F-2 which induces regression of the CL

based on a timed sequence of 16 days or so; unless an embryo
is present at day 14-17 days and produces a protein to stop the process

ation occurs every 21 d (18 to 24 days)

or three follicular waves occur during each estrous cycle

Some definitions
Follicular waves emergence of a group of follicles on the ovary > 4mm in diameter
FSH surge initiates follicular waves
Recruitment of a cohort of follicles, usually 4 to 6 in a wave or more
Occurs every 7 to 10 days depending if a cow has 2 or 3 waves per estrous cycle

Divergence a follicle within the cohort achieves 8.5 mm in size

Becomes LH sensitive
Produce estrogen and inhibin and causes regression of other follicles
Usually develops 2 to 3 days after emergence

Dominance a follicle > 10 mm in size and has the potential to ovulate

Ovulatory follicles are anywhere from 13 mm to 20 mm in size
If no LH surge (Progesterone > 2 ng/ml), follicle regresses in 2 to 3 days

Follicular waves during an estrous cycle cows have either 2 or 3 waves of follicles
which influences estrous cycle length
2 waves: estrous cycle 19 20 days (Pring et al. 2012)
3 waves: estrous cycle 21 22 days (Pring et al. 2012)
Cows tend to be consistent in the follicular waves they have

Hormonal changes in the estrous

cycle

Whats the incidence of ovulatory

problems?
Varies by herd but ranges any where from 10% to 30% of cows

Primiparous cows 49 71 DIMrange 28% to 54.1% four studies

Multiparous cows 49 - 71 DIM range 15% to 31.5% four studies
Greater in first lactation cows than older cows
Greater with greater body condition loss (>=1 unit) and cows < 2.5 in BCS
There has not been a strong association with milk production

Some of the variation in the literature is the days post calving used to
define anovulation (the varying VWP across herds)
For example Roth followed 47 cows to 100 days post calving
30 ovulated by 40 DIM; 17 had not
17 cows
4 ovulated by 50 days
4 ovulated by 60 days
8 were cystic (fluid structure>20 mm) but ovulated by 62 days (6 cows) and 1 cow by 99 days
1 cows failed to ovulate by 100 days

Lamming followed 505 cows with sequential

progesterone concentrations every 3 to 5 days
Days N ovulating
(%)
1-10
13
11-20 240
21-30 157
31-40
54
41-50
16
51 160 22

Cumulative percent (%) Percent

2.6
50.4
81.7
92.4
95.6
100.0

2.6
47.8
31.3
10.7
3.2
4.4

Whats the fertility in anovulatory

cows on TAI
Typical Literature
Ovulatory cows
Anovulatory cows

CR, %
32, 32, 34, 35, 35, 40

24, 9, 21, 21, 22, 27

Based on progesterone profiles being low prior to insemination

OR of pregnancy is about 2.04 for ovulatory cows versus anovulatory cows
Delays in ovulation are associated with reduced fertility

Anestrus delay in resumption of

ovarian cycles
Delay in first ovulation beyond 40 days postpartum
Effects 10% to 30% of cows

Not talking about silent heats cows cycling and not

seen in heat
Abnormal ovarian function

inactive ovaries lima beans

Growth of dominant follicles with no ovulation
Persistent large follicular structure Cystic Ovarian Disease
Persistent CL

Anestrous Conditions (AT Peters et

al. 2009)
Ovulation but prolonged CL lifespan
No ovulation large persistent structure (Cyst)
Follicular
Pool
No ovulation growth followed by atresia of dominant follicle
No deviation small follicles < 9 mm and smooth ovaries

Whats the incidence on

classification?
Follicular cyst versus inactive ovaries versus persistent CL
Ovarian Cysts (Follicular and Luteal)

Garverick reported 5.6% to 18.8% in the literature in a review paper in 1999

2.7% Argentina in 9,156 cows more recent report
Data from Norway 0.8%
Other reports 6% to 23% in literature
Personal observation 1.8% (five year period 35 herds, >5,000 cows)

Inactive ovaries - Failure to ovulate by the voluntary waiting period

10% is typical for most reports but can be up to 20% to 30%

Other ovarian dysfunctions

Prolonged CL: luteal phases longer than 15 - 25 days especially early postpartum 1.6% to
8%
Prolonged interluteal interval: longer than 12 days, long follicular period 13%

Whats the difference between a

cyst and failure to ovulate
Size of the anovulatory structure
Cysts have variable definitions on size
Classic definition > 25 mm, but now >17 to 20 mm are used

Anovulatory follicles <= 20 mm

Persistence on the ovary

cysts fail to ovulate and fail to regress
Cysts classic definition > 10 days; now some are using >6 days

Anovulatory follicles fail to ovulate but regress

persist less than 10 days regress normally

Absence of a CL
In both cases there is no CL present on the ovaries

Presence of other follicles

In Cystic cows follicle recruitment is depressed so there are few follicles > 5mm
In anovulatory cows follicular waves still occur

Why does a cyst develop?

It is unknown at this time
It is apparent for both conditions there is a disruption in the feedback
in the hypothalamic-pituitary-axis such that there is a failure in
estrogen to elicit an LH surge and induce ovulation
In normal cases the dominant follicle will regress, estrogen
production declines, FSH peaks and a new follicular wave emerges
and this repeats until the system works and ovulation ensues
In cystic cases there seems to be a disruption in the process of
apoptosis and growth so regression of the follicle does not occur,
estrogen continues to be produced, LH stimulates continued growth
of the follicle, FSH is depressed, no new follicular waves emerge and
the system freezes so to speak

Alterations in the system

Both a disruption in the production of LH, FSH, estrogen
and progesterone due to feedback mechanisms and the
influence of extra-reproductive hormones (insulin, IGF1)
Disruption in local receptors and regulators in granulosa
cells and theca cells altering receptor proteins and
enzymes associated with altered gene transcription
influence response to gonadotropins and production of
steroid hormones
Systemic factors and local factors and the balance
between them are at play

Ovulation - what should happen

Five days post calving FSH should increase and a follicular wave is
initiated
A cohort of follicles emerge > 4 mm in size (4 to 6 follicles)
FSH dependent

Emergence of a dominant follicle at 8.5 mm in size in 2 to 3 days

Dominant follicle becomes LH dependent with expression of LH receptors
Estrogen, Inhibin, Actin produced by dominant follicle depress FSH and cause atrophy of
other follicles in the cohort

Increasing estrogen produced by the

dominant follicle triggers an LH surge and
ovulation (FSH also peaks prior to ovulation)
Progesterone is produced by granulosa
and theca cells of the collapsed follicle
Waves of follicles continue during the estrous
cycle 2, or 3 waves of follicles

Postpartum what should happen

Uterine involution
Complete prior to 50 days

Ovarian function
First ovulation of first dominant follicle day 15 to 21
postpartum
Second ovulation by 32 to 42 days postpartum

So by 50 days postpartum the reproductive axis is fully

functional
Uterus is fully involuted and ovarian activity has been fully
established
Conception rates can exceed 40% at first breeding

But what happens

Ovulation of first dominant follicle by 15 to 21 days
happens in only 42% to 75% of cows will ovulate first
dominant follicle (very variable across herds)
Ovulation may not occur with emergence of first dominant
follicle
Regress with continuous follicular waves or develop a cyst which
freezes the system for a period

Metabolic and hormonal signals determine fate of first follicle

Related to Energy Balance, Age, Breed, Milk Production,

Season, Health

What can go wrong

Uterus fails to clear infection
Primarily a concern with Trueperella pyogenes

Ovarian function is dampened

Low production of estradiol and progesterone low fertility
Low LH amplitude and frequency of LH production

Normal ovarian function is disrupted

Failure of resumption of ovulation due to failure to trigger an LH surge

Cystic Ovarian Disease

Failure of a dominant follicle to ovulate with normal follicular waves
Cessation of cycling after it begins
Prolonged interestrus interval due to retained CL or delayed follicular recruitment after
ovulation

Anovulatory conditions with no CL

Anovulatory conditions (Wiltbank et al. Therio. 57:2002, Lopez et al. 2010, Peters 2009)
1. Cystic Ovarian Disease
Classically a follicular structure on the ovary >=25mm, which persists for at least 10 days in the absence of a
Corpus Luteum (>17 mm persists for 6 days; others use 20 mm and 10 days)
Follicular cysts often undergo luteinization and become a luteal cyst
A cystic CL is a normal ovarian structure typically formed after ovulation and is an immature CL usually 2 to 7 days
old

2. Follicles 16 to 24 mm in size with no ovulation (not considered cystic by some)

High circulating estrogen but no LH surge to cause ovulation of a dominant follicle
Still have follicular waves on a regular basis
Most common anovular condition (Lopez et al.2010)

3. Small follicles maximal size of only 9 to 15 mm and no ovulation

Common in cows early postpartum 25% may fail to ovulate first dominant follicle
Common with more negative energy balance
Deficiency of LH pulses frequency and amplitude inadequate follicular development of dominant follicle
Low estrogen production (or high liver clearance) dampens GnRH/LH pulses and leads to failure to ovulate

4. A cow with follicular growth only to emergence small follicles <9 mm

Wiltbank reports that they have observed this only in 3/1000 cases
My experience is this is not common small ovaries with no large follicular or luteal structures

What is the difference: cyst vs

anovulation?
Anovulatory condition absence of CL over 10 day period
Dominant follicles arise but dont ovulate persist 6 days or so
Follicle waves continue every 7 to 9 days

Cystic follicle absence of CL over a 10 day period

Large follicular structure that persists for 13 to 20 days
Wall thickness < 3mm (Luteal cyst wall thickness > 3mm)

Follicular waves are depressed and appear to arise only when the cyst
stops producing estrogen - every 15 to 21 days but a new cyst may form
Cysts do turnover and are replaced by other cysts

Both conditions have higher prevalence in first 40 days

postpartum

What is the defect leading to

anovulation?
Failure of LH surge to cause ovulation

Estrogen fails to induce an LH surge to cause ovulation

Hypothalamus is unresponsive to estrogen

Failure of adequate LH pulse frequency and amplitude to

cause maturation of a dominant follicle
Low estrogen synthesis so insufficient estrogen to elicit an LH
surge

Increased estrogen clearance by the liver inhibiting LH

surge

What is cause of anovulation - Cyst

Cystic structure
Low progesterone (0.1 to 1.0 ng/ml) can block LH surge but not suppress pulsatile
LH
About 60 to 75% of cystic cows have marginal progesterone concentrations

Follicular structure responds to LH and continues to grow and produce estrogen and
inhibin delaying follicular wave recruitment by depressing FSH
Continues to grow beyond 20 mm in size due to LH stimulation
Cysts will turnover and new cyst arise
Cysts cause a depression of follicular waves and a long period between recruitment

Hypothalamus cite of the major

defect
Hypothalamus is not responsive to estrogen feedback
Give estrogen and no GnRH is released to cause LH release from pituitary
Low concentrations of progesterone 0.1 to 1 ng/ml late in the luteal and
early follicular phase block LH release in response to rising estrogen
Hypothalamus seems insensitive to feedback
May need progesterone concentrations above 2 ng/ml to condition hypothalamus

If progesterone drops <0.1 ng/ml then spontaneous ovulation

Remove ovary and ovulation occurs within 6 weeks suggests feed back
from ovary is influencing hypothalamus (low, above basal progesterone?)
Give progesterone to increase blood level and hypothalamus regains
responsiveness

What modifies the response of the

system

Metabolic
Uterine infection
Stress
Genetic but very low heritability (Sweden!!)

Reproductive System

Metabolic System

Linkage of the reproductive syste

And the metabolic system
Critical linkages

Insulin
Insulin like growth factor I,I
IGF-I, II
Binding proteins (IGFBP, 1
Nutrients
fatty acids
amino acids
glucogenic precursors
Trace minerals
Vitamins
Modify the reproductive organs
Garnsworthy et al. Animal 2008

NE Balance

Milk Production

10
180
160

140
120

0
0

50

100

150

200

250

300

100
80

-5

60
40

-10

20
0

-15

0
Days in milk

NE Balance

50

100

150
Days in milk

200

250

300
Milk

MP Balance
800
600
400
200
0
-200

50

100

150

200

250

300

-400

Patterns post-calving in a group of cows

-600
Days in milk

MP Balance

Study by Taylor et al. 2003 in first lactation cows and ovulation postpartum
Normal first progesterone rise by 17 days postpartum
DOV first progesterone rise by 71 days postpartum
PCL1 extended first luteal phase ovulation day 19 with luteal length of 46 days
Schematic based on Taylor et al. 2003
10

00

20

40

60

80

100

Energy Balance, mcal/day

-5

-10

-15
DIM
Normal n=17

DOV n=9

PCL1 n=6

120

140

160

What influences ovulation?

Metabolic
Negative energy balance small follicles
Low serum insulin, low IGF-1
Low insulin associated with delayed ovulation and cystic ovarian disease

Fewer recruited follicles and sensitivity to gonadotropins is reduced

Reduced GnRH output and LH production

Uterine infection endotoxin release

Delays folliculogenesis dampens GnRH output
High uterine production of PGF-2 suppresses ovarian activity

Stress
Cortisol inhibits LH surge and prevents ovulation
Sequential ACTH injections will lead to ovarian cyst formation

Additional influences: Acute Dietary

Restriction
Dietary restriction
reduced estrogen synthesis, reduced responsiveness to FSH and IGF
signaling in granulosa cells
LH responsiveness in theca cells is reduced

Amplitude and frequency of LH pulses is reduced

The preovulatory surge of LH is reduced
Cows that ovulate: steroidal hormonal output reduced
Decline in hormonal production of estrogen and progesterone influencing
sequential follicular development and hypothalamus and pituitary function

Cows that dont ovulate: reduced steroidal hormonal output and a

reduction in transcription of mRNA reducing LH receptors on GC
Decline in production and responsiveness of system

Influences: Systemic and Local

factors
Systemic factors dampening of the hypothalamuspituitary-ovarian axis

Reduction in hormonal outputs and feedback regulation

Modified by insulin and IGF system of IGF-I, II and IGF binding
proteins (IGFBP1-6) which influence IGF availability

Local factors in the ovarian follicle

IGF-I, II and binding proteins
Inhibin, activin, follistatin
Receptor levels and gene transcription influences response to
hormonal inputs and output of steroidal hormones

Nutritional status modifiers

Item Nutritional InfluencePossible Signal
Release rate of GnRH
Release rate of LH

Energy Balance

Neuronal

Energy Balance

Insulin, IGF-1

Release rate of FSH Energy Balance

Insulin, IGF-1

Clearance rate of LH Dry matter intakeLiver blood flow

Clearance rate of FSH

Clearance rate of estrogen

Clearance rate of progesterone

Follicle sensitivity to FSHEnergy balance

Insulin

Follicle growth to FSH

Insulin

Energy balance

Growth of selected follicles Energy balance, dietary fat Insulin, NEFA

Growth of dominant follicle Energy balance, dietary fat Insulin, NEFA
Pring et al. 2012

How do you diagnose it?

Palpation low sensitivity and specificity on one time examination of structures
West Virginia 28 herds, 10 vets 40 cows with cyst; saline vs GnRH no difference in response
Follicular cysts 70% - 85%; Luteal cysts 41% - 52% (>=25 mm structure criteria)

Ultrasound improves sensitivity and specificity

Follicular cysts 72% to 92%; Luteal cysts 74% to 88%

One time exam for each is fraught with errors need two examinations 7 to 14 days
apart
Remember any diagnosis has to lead to a treatment to improve the likelihood of pregnancy
sooner than if no diagnosis had been made

Progesterone profiles
Daily up to every 3 days with milk recording systems or kits
ELISA or Biometallics Target test kits

Presynch OvSynch Protocol progesterone check

Problem with diagnosis on one

observation

What is a cyst? Definition has varied from >17 mm to >25mm

for 6 to 10 days
Large follicle on the ovary(ies) for 45% of the time within a cycle
10 to 20 mm in size; waxing or waning

Early CL 28% of the days of a cycle (Corpora hemorrhagicum)

Poorly formed and globular and mushy

73% of the time of a cycle the ovary may have a structure that
appears abnormal cystic
Spontaneous cure
Cysts are observed most frequently 14 d to 40 d postpartum and many
cows initiate ovulation with no treatment

For Example
Hatler et al. Follicular Cyst Criteria - > 17 mm for 6 days no CL
32 cows diagnosed
6 cows ovulated 7 days later

26 cows
13 of the 26 cows ovulated in an average of 19 days (range 6 to 41)

13 cows (17/36 = 33%) from initial observation

The spontaneous and transient nature of ovarian structures make diagnosis
and prospective studies difficult
Low frequency condition after 40 days
Errors of diagnosis on one examination (very high with a one time exam)
63% cows coming in estrus in Polish study diagnosed with a cyst

High spontaneous cure rate >60% reported in literature

Prior to 30 to 40 days post-calving up to 30% of cows may have a follicular cyst
Observe luteinization, rise in progesterone, and initiation of ovulation in >90% of cases

Prospective studies would require many observations to document longitudinal

changes

Using Presynch Ovsynch to check

ovulation
Combined
with progesterone tests
Typical injection
schedule for presynch-ovsynch

PGF ------14 days ----PGF ------14 (11) days------GnRH ------7 days--------PGF ---2 days---GnRH 1/2 day TAI

PGF1
PGF2
PGF3
f cows are cycling the following should be observed sampling blood or milk:

60% of cows P4>1 ng/ml

80% of cows P4>1 ng/ml
>80% of cows P4> 1ng/ml

The key sample alone is the PGF3 - >=80% of cows should have high progesterone by this injection
Sample a group of cows going through the protocol at the time of the PGF3 injection

So what do you do?

If a cow is cystic versus anovulatory does the type of treatment matter?
Probably not

Options
GnRH injection induce ovulation or luteinization estrus in 21+ days
Cause a rise in LH and ultimately a rise in progesterone to reset the hypothalamus

hCG human chorionic gonadotropin estrus in 21 days

LH like activity does the same as GnRH

Progesterone supplement CIDR/PRID for 7 - 12 days and removal estrus 4

days
Intravaginal device to increase progesterone to reset hypothalamus responsiveness to
estrogen

GnRH and prostaglandin either in combination at GnRH injection or followed by

PGF 7 to 14 days later
GnRH and implant a CIDR followed by CIDR removal and PGF 7 days later
Presynch Ovsynch protocol

Problems you may encounter

Treatment responses for cysts are reported > 80%
Fertility is often lower at first estrus

Progesterone therapy alone has been variable for initiation of cycles; fertility is
low at first ovulation following treatment
Marginal progesterone concentrations at time of treatment with GnRH

0.1 to 1.0 ng/ml blunt hypothalamus release of GnRH and subsequent LH release
Give PGF to regress any residue luteinized structure
Use a CIDR to increase progesterone above 1 to 2 ng/ml to reset hypothalamus
PGF combined with GnRH at treatment has had variable improvement in response

GnRH + 2 CIDRs in a timed TAI protocol in anovulatory cows has shown an

improvement in CR versus GnRH alone
2 CIDRs to increase blood progesterone above 1 ng/ml (closer to 2 ng/ml) to reset the
hypothalamus

Observed Data Responses

Presynch - OvSynch

0 d +14
+28
+35
Injection
PG1
PG2
GnRH PG3
N FSTCR %
Progesterone <>1 ng/ml
Anov.
LowLow
Low19 4/19
21.1
No GnRH resp LowHigh
Low 9 5/9 55.6
No GnRH resp High
Low
Low15 5/15
33.3
No GnRH resp High
High
Low20 6/20
30.0
Out of synch
63 20/63 31.7
Late Ov./
Early Ov./cycle
Delay Ov.
Early Ov.
In Synch
All

LowLow
High
13 4/13
30.8
High
Low
High
24 8/24
33.3
LowHigh
High
50 21/50 42.0
High
High
High
55 24/55 43.6
142
57/142 40.1
205

77/205 37.6

Florida Protocol (Bisinotto et al.

2015)
U/S
at GnRH(1) 38 - 44 DIM
U/S
U/S

No CL@GnRH PGF1 14 d --- PGF2 --- 11 d --- GnRH ---- 7 d --- PGF3 ---- 21/2 d
GnRH 1/2 d TAI
No CL@ GnRH PGF1 14 d --- PGF2 --- 11 d --- GnRH ---- 7 d --- PGF3 ---- 21/2 d
GnRH 1/2 d TAI
2 CIDR+
2CIDR out
CL at@GnRH
PGF1 14 d --- PGF2 --- 11 d --- GnRH ---- 7 d --- PGF3 ---- 21/2 d
GnRH 1/2 d TAI
No CL control (649)
No CL 2CIDR (633)
CR%, 32 d 31.3%
42.2
38.4
Preg, 60 d 28.9
37.2
33.9
Preg loss
8.5%
11.4
8.8

CL (640)

New CL at PGF342.6 (371)

46.8 (354)
48.0 (229)
No New CL@PGF3 18.6 (149)
35.0 (176)
38.3 (274)
anovular cows---------------------------------ovular cows
Cows with no CL at GrRH(1) was 27.0% across five herds

Conclusion
Anovulatory cows by the VWP may be 20% to 30% of cows
Diagnosis involves sequential observations or a protocol to create a
high prevalence of cows with a CL (Presynch-Ovsynch Protocol at
PGF3)
Use progesterone or US exam at this time to determine if CL/Progesterone
is present

Treatments utilize GnRH to elicit an LH surge and ovulation of a

dominant follicle or luteinization of a cyst
GnRH combined with prostaglandin or progesterone vaginal inserts
may enhance fertility
The most effective approach is to incorporate cows in a TAI program