principles of development for bovine oocytes and follicles

BrittBrittAAPBAAPB20052005

Folliculogenesis – Factors Which Folliculogenesis – Factors Which Affect Oocyte Competence.Affect Oocyte Competence.

Jack H Britt

University of Tennessee

[email protected]

Getting the Most Out of Embryo TransferGetting the Most Out of Embryo Transfer

AABP Preconference Seminar 14 -- September 21, 2005 -- Salt Lake CityAABP Preconference Seminar 14 -- September 21, 2005 -- Salt Lake City


Confused about follicular development?Confused about follicular development?


Take Home MessagesTake Home Messages

Jack H Britt, University of TennesseeJack H Britt, University of Tennessee

Oocytes and follicles are formed during fetal development – but many Oocytes and follicles are formed during fetal development – but many degenerate before puberty – degenerate before puberty – most oocytes are lost earlymost oocytes are lost early..

Among cattle, follicle populations differ among animals but are Among cattle, follicle populations differ among animals but are repeatable within animals over lifetime – repeatable within animals over lifetime – more follicles = more oocytesmore follicles = more oocytes..

Number of growing follicles is directly related to the size of the resting Number of growing follicles is directly related to the size of the resting pool of follicles -- pool of follicles -- larger pool = more oocyteslarger pool = more oocytes..

Number of FSH-responsive follicles within an individual is highly Number of FSH-responsive follicles within an individual is highly repeatable – repeatable – responsive heifers = responsive cowsresponsive heifers = responsive cows..

Folliculogenesis takes months and quality of follicles and oocytes is Folliculogenesis takes months and quality of follicles and oocytes is affected by environmental conditions that exist during this period – affected by environmental conditions that exist during this period – follicle environment affects oocyte qualityfollicle environment affects oocyte quality..


Atretic

Preantral Follicles

Ovarian Microscopic and Ovarian Microscopic and Macroscopic StructuresMacroscopic Structures


Follicle Development and ActivationFollicle Development and Activation

Take Home MessageTake Home MessageOocytes and follicles are formed during fetal development – but many Oocytes and follicles are formed during fetal development – but many

degenerate before puberty – degenerate before puberty – most oocytes are lost earlymost oocytes are lost early..


Follicle and Oocyte Populations Follicle and Oocyte Populations in the Bovine Ovary in the Bovine Ovary

Cushman, DeSouza, Hedgpeth & Britt. 1999. Biol. Reprod. 60: 349-354 Cushman, DeSouza, Hedgpeth & Britt. 1999. Biol. Reprod. 60: 349-354

Follicle PopulationsFollicle Populations

0° - Primordial0° - Primordial

1° - Primary1° - Primary

2° - Secondary2° - Secondary

(about 140,000 at birth)(about 140,000 at birth)

0°

2°

1°

0°

1°

2°

1°

Primordial to PrimaryPrimordial to Primary

• Granulosa cells become Granulosa cells become cuboidalcuboidal

• Oocyte growth occursOocyte growth occurs

Primary to SecondaryPrimary to Secondary

• Granulosa cell proliferationGranulosa cell proliferation

• Multiple LayersMultiple Layers

• Zona PellucidaZona Pellucida

• Theca cell differentiationTheca cell differentiation


0

2

4

6

8

10

12

0

2

4

6

8

10

12

A. Primordial Follicles

a

bb

b b

a aaa

a

B. Primary Follicles

a

b

b bb

a a a

a

a

in vitro in ovo

Day 10 Day 7 Day 4 Day 2 Day 0

Nu

mb

er o

f fo

llicl

es p

er s

ecti

on

(±

SE

M)

Bovine Follicles Activate SpontaneouslyIn Vitro, but not In Ovo

Cushman et al. 2002. Hum Reprod 17:48; Gigli et al. 2005 Mol. Reprod. Develop.71:480Cushman et al. 2002. Hum Reprod 17:48; Gigli et al. 2005 Mol. Reprod. Develop.71:480

Procedures:Pieces of bovine ovarieswere cultured in vitro ortransplanted to fertilized chicken eggchorio-allantois membrane (in ovo).

Therefore:Activation is inhibited in tissue supplied with blood-- suggesting a blood borneinhibitor of gonadal origin


Duration of FolliculogenesisDuration of Folliculogenesis in the Bovine in the Bovine

Primordial

PrimordialPrimordial

40 days40 days10 days10 days10 days10 days??

80 to 100 days

FactorsFactors

Steroids Steroids &&

Growth Growth FactorsFactors

Growth Growth FSH & LHFSH & LH


FSHFSHGrowth Growth

FactorsFactors

FSHFSH

GraafianGraafianAntralAntralPreantralPreantralPrimaryPrimary

Britt, J. H. 1992. The Bovine Practitioner Proceedings 24: 39-43 Britt, J. H. 1992. The Bovine Practitioner Proceedings 24: 39-43


Follicle Numbers are RepeatableFollicle Numbers are Repeatable

Take Home MessageTake Home MessageAmong cattle, follicle populations differ among animals but are Among cattle, follicle populations differ among animals but are

repeatable within animals over lifetime – repeatable within animals over lifetime – more follicles = more oocytesmore follicles = more oocytes


Number of Follicles is Repeatable Number of Follicles is Repeatable Across Waves and CyclesAcross Waves and Cycles

Used with permission: Used with permission: Burns et al. 2005. BOR 73: 54-62Burns et al. 2005. BOR 73: 54-62

• Ovaries ultrasounded 2X DailyOvaries ultrasounded 2X Daily

• Cows (heifers and lactating or Cows (heifers and lactating or nonlactating cows, n=30) were nonlactating cows, n=30) were ranked for low (ranked for low (≤15), ≤15), intermediate (16-20), high (21-intermediate (16-20), high (21-25) or very high (>25) follicles 25) or very high (>25) follicles per wave.per wave.

• Note: Note: Repeatability of number of Repeatability of number of follicles (bars) per wavefollicles (bars) per waver=.95; 98 waves; 30 animalsr=.95; 98 waves; 30 animals


ComparisonComparison Correlation coefficientCorrelation coefficient

Season: Spring, Summer, FallSeason: Spring, Summer, Fall .86 to .95.86 to .95

Waves per cycles: two or threeWaves per cycles: two or three .93 to .95.93 to .95

Lactation: lactating or dryLactation: lactating or dry .93 to .94.93 to .94

Ultrasound technicianUltrasound technician .86 to .95.86 to .95

Age: Heifers or Cows Age: Heifers or Cows (see footnote)(see footnote) .92 to .96.92 to .96

OverallOverall .95.95

Repeatability of Number of Repeatability of Number of Follicles Follicles ≥ 3 mm in Holsteins≥ 3 mm in Holsteins

Burns et al. 2005. Biol. Reprod. 73: 54-62Burns et al. 2005. Biol. Reprod. 73: 54-62

No. follicles in same animals: As heifers, 21.2±1 vs As cows, 16.4±1


Follicle Pool Size Dictates Follicle Pool Size Dictates Populations Developing Populations Developing

Take Home MessageTake Home MessageNumber of growing follicles is directly related to the size of the resting Number of growing follicles is directly related to the size of the resting

pool of follicles -- pool of follicles -- larger pool = more oocyteslarger pool = more oocytes..


How about the relationship between theHow about the relationship between thenumber of follicles and ovulation rate?number of follicles and ovulation rate?

Ovulatory response in the Ovulatory response in the remainingremaining ovary was ovary was compared with the number of follicles in the compared with the number of follicles in the

contralateralcontralateral ovary removed before superovulation. ovary removed before superovulation.

Twenty-eight non-lactating Holstein and Jersey Cows

Unilateral ovariectomy - Day 8 of cycle Ipsilateral to CL

Exogenous gonadotropin - Day 9 of cycle FSH-P (28 mg) for 4 days Lutalyse (25 mg) twice on third day Remaining ovary collected on Day 8-12 of subsequent cycle to

quantify CL



CL Primordial Primary Secondary Tertiary

<5 77.4a 64.8 4.1 1.6a

5-14 178.2b 137.3 9.1 3.8b

>14 173.7b 95.7 9.1 3.8b

SEM 22.4 22.1 1.7 0.6

a,bP < 0.05 within a column

MicroscopicMicroscopic Follicles in the Bovine Ovary Follicles in the Bovine Ovary in Cows with Differing Responses to FSHin Cows with Differing Responses to FSH



CL

Total

Small

(<3 mm)

Medium

(3-7 mm)

Large

(>7mm)

<5 6.1a 2.4a 2.5a 1.3

5-14 22.4b 11.6b 9.3b 1.6

>14 21.7b 8.3b 11.1b 2.3

SEM 3.1 1.9 2.4 0.7

a,bP 0.06 within a column

MacroscopicMacroscopic Follicles in the Bovine Ovary Follicles in the Bovine Ovary in Cows with Differing Responses to FSHin Cows with Differing Responses to FSH



Ultrasound Test to Predict Ultrasound Test to Predict Superstimulatory Response in CattleSuperstimulatory Response in Cattle

Singh et al. Theriogenology 2004. 62:227-243.Singh et al. Theriogenology 2004. 62:227-243.

121 cows121 cows

Synchronized 1Synchronized 1stst wave (E and P to synchronize) wave (E and P to synchronize)

Ultrasound to estimate number of follicles Ultrasound to estimate number of follicles ≥≥2 mm2 mm

Synchronized 2Synchronized 2ndnd wave in high (n=16) and low (n=20) end cows wave in high (n=16) and low (n=20) end cows

FSH 2X daily for 3 daysFSH 2X daily for 3 days

Number of follicles after FSHNumber of follicles after FSH

5-7 mm: High end = 16.85-7 mm: High end = 16.8±2.2 vs Low end = 8.1±0.9±2.2 vs Low end = 8.1±0.9

≥ ≥ 8 mm: High end = 22.7±4.1 vs Low end = 9.7±1.68 mm: High end = 22.7±4.1 vs Low end = 9.7±1.6

Best predictor of superstimulatory response was Best predictor of superstimulatory response was number of follicles ≥2 mm at wave emergence.number of follicles ≥2 mm at wave emergence.


Follicle Numbers are Repeatable Follicle Numbers are Repeatable Within IndividualsWithin Individuals

Take Home MessageTake Home MessageNumber of FSH-responsive follicles within an individual is highly Number of FSH-responsive follicles within an individual is highly

repeatable – repeatable – responsive heifers = responsive cowsresponsive heifers = responsive cows..


0 4 8 12 16

1

3

5

7

9

Tw

in P

air

Average Number of Oocytes Per Session

Twin 2Twin 1

Characteristics of responses among Characteristics of responses among monozygotic twins to OPUmonozygotic twins to OPU

Machado et al. Theriogenology 2005. pre-release Machado et al. Theriogenology 2005. pre-release

9 pair of twins, OPU at 3-4 day intervals, 195 OPU sessions (10 or 11 per donor)

Only this pair differed


Response

No.

follicles

No.

COC

No.

Embryos

Recovey

Rate %

Low (21%) 8.8a 6.3a 1.8a 72a

Medium (26%) 13.2b 10.4b 2.5b 79

High (19%) 22.4c 18.5c 5.6c 83b

Mixed (34%) 15.1b 12.0d 3.5d 79 a,b,cP 0.05 within a column

Responses among Low, Medium and Responses among Low, Medium and High Responders to OPUHigh Responders to OPU

De Roover et al Theriogenology 2005. 63:1902-1913. De Roover et al Theriogenology 2005. 63:1902-1913.

112 animals, 665 OPU sessions, 5.9 sessions per animal


Variable Control Twinner SEM P <

No. of animals 24 24

Total ovarian wt., g 19.2 26.6 1.3 0.01

No. of small follicles (5 mm) 43.5 60.8 5.1 0.05

No. of medium follicles 3.3 6.4 1.2 0.05

No. of large follicles (10 mm)

1.6 2.5 0.1 0.01

No. of CL 1.1 1.5 0.1 0.01

CL wt., g 2.9 2.6 0.2 NS

Total luteal wt., g 3.0 3.6 0.2 0.05

Echternkamp et al. 2004. JAS 78:1564Echternkamp et al. 2004. JAS 78:1564

Ovarian Characteristics of Cattle Ovarian Characteristics of Cattle Selected or Unselected for TwinsSelected or Unselected for Twins


Stage

Line n Primordial Primary Secondary

Control 7 104.7 58.7 6.3a

Twinner 7 153.7 84.3 12.9b

S.E.M. 27.4 17.3 2.0

a,bP < 0.05 within a column

Preantral Follicles in Cattle Preantral Follicles in Cattle Selected and Unselected for TwinsSelected and Unselected for Twins

Cushman, Hedgpeth, Echternkamp and Britt.2000. J. Anim. Sci. 78: 1564-1567.Cushman, Hedgpeth, Echternkamp and Britt.2000. J. Anim. Sci. 78: 1564-1567.


Environmental Effects on Environmental Effects on Follicle and Oocyte QualityFollicle and Oocyte Quality

Take Home MessageTake Home MessageFolliculogenesis takes months and quality of follicles and oocytes is Folliculogenesis takes months and quality of follicles and oocytes is affected by environmental conditions that exist during this period – affected by environmental conditions that exist during this period –

follicle environment affects oocyte qualityfollicle environment affects oocyte quality..


Hormonal and Other Factors Affecting Hormonal and Other Factors Affecting Number of Follicles and OocytesNumber of Follicles and Oocytes

Trait measured in 1990’s

Adapted from Lucy et al., 1998 and Bonczek et al. ,1988 Adapted from Lucy et al., 1998 and Bonczek et al. ,1988 *1960’s = semen from 1960’s bulls; 1990’s = semen from 1990’s bulls*1960’s = semen from 1960’s bulls; 1990’s = semen from 1990’s bulls

• Milk yield, 305 d, lbs.

• Insulin, µIU/ml

• Somatotropin, ng/ml

• IGF-I before day 14

• IGF-I after day 21

• Progesterone, ng/ml

• Luteal phase, d

16,282

22.5

3.7

lower

higher

4.4

20.0

1960’s Genetics*

21,731

18.8

4.5

higher

lower

3.2

16.6

1990’s Genetics*


Can one grow more follicles by short-Can one grow more follicles by short-or long-term treatment with hormones?or long-term treatment with hormones?

Primordial

FactorsFactors

Steroids Steroids &&


Growth Growth FSH & LHFSH & LH


FSHFSHGrowth Growth

FactorsFactors

FSHFSH


Long-Term Treatment with Estradiol Long-Term Treatment with Estradiol and bST to Grow More Folliclesand bST to Grow More Follicles

UnilateralOvex.

Con

E2

bST

E2bST 0 50 67

Days from Onset of Treatments

Cushman et al. 2001. BOR 65:851; Cushman et al. 2001. Therio 55:1533Cushman et al. 2001. BOR 65:851; Cushman et al. 2001. Therio 55:1533

Super-ovulate

Treatment with bST and EstradiolTreatment with bST and Estradiol


Serum EstradiolSerum Estradiol

Cushman et al. 2001. Theriogenology 55:1533Cushman et al. 2001. Theriogenology 55:1533

Two CompudoseTwo Compudose™ implants ™ implants inserted on Day 0.inserted on Day 0.


Serum Growth Hormone and IGF-ISerum Growth Hormone and IGF-I


PosilacPosilac™ (500 mg) ™ (500 mg) injected every 14 days.injected every 14 days.


n 0 1 2 3

Control 1620 62.9 32.8 3.0 1.2

E2 1611 48.4 48.0 2.6 1.0

bST 1575 56.7 38.0 3.6 1.7

E2+bST 1887 58.2 37.7 2.6 1.5

Percentages of Follicles within each Percentages of Follicles within each Stage for Cattle Treated for 70 dStage for Cattle Treated for 70 d

Cushman et al. 2001. BOR 65:851; Cushman et al. 2001. Therio 55:1533Cushman et al. 2001. BOR 65:851; Cushman et al. 2001. Therio 55:1533


Number of CL in Cattle Treated with E2, Number of CL in Cattle Treated with E2, bST or the CombinationbST or the Combination

02468

101214161820

Control E2 bST E2+bST

Nu

mb

er o

f C

L

Trial 1

Trial 2

Cushman et al. 2001. Therio 55:1533Cushman et al. 2001. Therio 55:1533


Transferable EmbryosTransferable Embryos


Treatment*Total Ova

TransferableOva

Develop.Rate, %

bST 7.9±1.3a 6.1±1.3a 94±9x

Saline 4.3±1.5b 2.5±1.5b 74±10y

E2 6.8±1.5 4.3±1.5 84±10

Blank 5.4±1.3 4.3±1.3 83±8

a,b P<.01; xy P<.09; Main effects, no interactions.


Transition from FollicleTransition from Follicle to Corpus Luteum to Corpus Luteum

• Provides environment for oocyteProvides environment for oocyte• Secretes estrogen Secretes estrogen • Estrogen causes estrusEstrogen causes estrus• Induces LH surge and ovulationInduces LH surge and ovulation

• Secretes progesteroneSecretes progesterone• Regulates follicle wavesRegulates follicle waves• Regulates embryonic growthRegulates embryonic growth• Required for pregnancyRequired for pregnancy

OvulationOvulation

The The FOLLICLEFOLLICLE is converted to the is converted to the CORPUS LUTEUMCORPUS LUTEUM at ovulation (26-30 hours after onset of estrus).at ovulation (26-30 hours after onset of estrus).


Postpartum Progesterone and Fertility

PostpartumAnestrus

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 12 24 36 48 60 72 84Days Postpartum

1 2 3

Increasing number and height of profiles is betterbefore AI.


-56 -28 0 28 56 84 112 140Days Postpartum

123456Ovulation No.

Follicle Development During the Postpartum Period


Ovulation No.

-8-6-4-2024

-56 -28 0 28 56 84 112 140Days Postpartum

Energy Balance

123456

Follicle Development and Energy Balance


Progesterone in Cows With Different Body Condition Changes Postpartum

Postpartum Cycle

2

3

4

5

6A

vg.

Lu

teal

Pro

ges

tero

ne,

ng

/mL

1 2 3 4 5

Maintained Condition 5-wk Postpartum

Lost Condition 5-wk Postpartum


0

20

40

60

80

100

Pe

rce

nta

ge

of

Fo

llic

les Calf (6 mo)

Open CowPreg Cow30 d PP Cow

Development of Cultured Preantral Development of Cultured Preantral Bovine FolliclesBovine Follicles

*

*

Figueiredo et al. Theriogenology 42: 1303-1310, 1994.Figueiredo et al. Theriogenology 42: 1303-1310, 1994.

*P < .05

Normal, d 1 Normal, d 5 Growing, d 5

**


Seasonal Differences in Fertility

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec20

25

30

35

40

45

50

55

60

Conception Rate, %

3,200 Primiparous Holstein cows Southern USA3,200 Primiparous Holstein cows Southern USA


Seasonal Differences in P4 Secretion from Dominant Follicle Thecal Cells

050

100150200250300350400

1 2 5 7 9

Day of Culture

P4

ng

/100

,000

cel

ls Winter

Summer

Wolfenson et al. Dom. Anim. Endocrinol. 22: 81-90, 2002.Wolfenson et al. Dom. Anim. Endocrinol. 22: 81-90, 2002.

Cells from Day 6 first wave dominant follicles.Cells from Day 6 first wave dominant follicles.


Seasonal Differences in P4 in Blood and Tissue in Holsteins in Israel

0

1

2

3

4

5

6

7

1 4 7 9 11 13 16 18 20

Day of Cycle

P4

ng

/mL

Winter

Summer

050

100150200250300350400

1 2 5 7 9

Day of Culture

P4

ng

/100

,000

cel

ls Winter

Summer

Effects of differences in P4Effects of differences in P4

Oocyte environmentOocyte environment

Follicle dominanceFollicle dominance

“ “Aged” oocyte and follicleAged” oocyte and follicle

Embryo survivalEmbryo survival

Wolfenson et al. Dom. Anim. Endocrinol. 22: 81-90, 2002.Wolfenson et al. Dom. Anim. Endocrinol. 22: 81-90, 2002.


FSH or bST during One Cycle Improved Oocyte Quality during Next Cycle

0%

20%

40%

60%

80%

100%

Control FSH Control bST

Treatment Groups

Gra

de

1 O

ocy

tes

(%)

Roth et al. J. Dairy Sci. 85: 1398-1405, 2002.Roth et al. J. Dairy Sci. 85: 1398-1405, 2002.

Holstein cows – Holstein cows – heat stressedheat stressed in Israel in summer and treated in Oct or Nov in Israel in summer and treated in Oct or NovAll visible follicles aspirated on Day 4 of Cycle 1 and Cycle 2All visible follicles aspirated on Day 4 of Cycle 1 and Cycle 2

1 2 21 21 21

Control vs FSHControl vs FSH2 x 200 mg of FSH2 x 200 mg of FSHDay 5 & 12 of Cycle 1Day 5 & 12 of Cycle 1

Control vs bSTControl vs bST500 mg Somatech (Posilac)500 mg Somatech (Posilac)Day 4 & 18 of Cycle 1Day 4 & 18 of Cycle 1


Not All Studies Show An Effect of bST on Number of Oocytes or Embryos

0

2

4

6

8

10

12

Cycle 1 Cycle 2 Cycle 3 Cycle 4

To

tal E

mb

ryo

s an

d O

ocy

tes

rbST

Control

0

1

2

3

4

5

6

Cycle 1 Cycle 2 Cycle 3 Cycle 4

Tra

nsfe

rab

le E

mb

ryo

s rbST

Control

Hasler et al. 2003. Theriogenology 59: 1919-1928.Hasler et al. 2003. Theriogenology 59: 1919-1928.

•57 primiparous Angus cows57 primiparous Angus cows•Four consecutive superovulation cyclesFour consecutive superovulation cycles•500 mg rbST every 14 days500 mg rbST every 14 days


Thank You !

The University of Tennessee’s Cloned Jersey HeifersThe University of Tennessee’s Cloned Jersey Heifers

principles of development for bovine oocytes and follicles

Education

responsive follicles

primary follicles

primordial follicles

quality of follicles

oocytesmore follicles

repeatablenumber of

brittbritt aapbaapb

resting pool of follicles