Current Achievements in Artificial Current Achievements in Artificial

Breeding of Horses in Subtropical TaiwanBreeding of Horses in Subtropical Taiwan

Jacky P. ChanJacky P. Chan

Veterinary Medicine Teaching Hospital, Veterinary Medicine Teaching Hospital, Department of Veterinary Medicine,Department of Veterinary Medicine,

College of Veterinary MedicineCollege of Veterinary MedicineNational Chung-Hsing UniversityNational Chung-Hsing University

Danehill Danehill dove orchiddove orchid

Danehill Danehill Great vintageGreat vintage

Danehill Danehill champagechampage

Stallion Stallion DanehillDanehill

Influence of the Influence of the length of day light on length of day light on the ovary function in the ovary function in

maresmares

Photoreceptors in the eyes

Pineal

Hypothalamus

Anterior pituitary

Ovaries

increasing day light

decreasing day lightneurotransmission

portal system

blood circulation

MelatoninMelatonin

GnRHGnRH

GonadotropineGonadotropine

Eva's cycle

0

1

2

3

4

5

6

-16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 1 3 5 7 9 11 13 15 17 19 21 23 25 0 2 4 6 8 10 12 14 16 18 20 22 0Days of cycle

Siz

e(cm

)

Dominant Follicle Subordinate Follicle

CL1 CL2

OvulationOvulation

OvulationOvulation

2nd Ovulation

2nd Ovulation

Mares in subtropical Taiwan showed estrus cycles accompanied by

ovulation throughout the year, but some of them complete ovulation

mainly during March through October.

Northern semi-sphereNorthern semi-sphere

Subtropical TaiwanSubtropical Taiwan

These mares are not only possible defined seasonal breeders, but

also year-round breeders that could be bred throughout the year in

Taiwan.

Large amounts of equine LH are released during an 8- to 9-day Large amounts of equine LH are released during an 8- to 9-day

period with ovulation occurring on the 3rd to 5th day.period with ovulation occurring on the 3rd to 5th day.

Date of AI and Pregnancy RateDate of AI and Pregnancy Rate

Date of AI with fresh semen

Days prior to Ovulation Ovulation

Day after the Ovulation

No. of AI

Pregnancy Rate(%)

4

12

33

3

15

60

2

51

65

1

9

89

0

94

52

1

70

6

(Woods et al., 1990,)

0D -1D -2D -3D -4 0-6 hrs

Fertility Low High Low

Ovulation

D -5

Fertility curve

12 hrs pre- and 6 12 hrs pre- and 6 hrs postovulationhrs postovulation

Effect of post-ovulatory AI on pregnancy Effect of post-ovulatory AI on pregnancy rates and embryonic mortalityrates and embryonic mortality

0

20

40

60

80

0-6 6-12 12-18 18-24 24-30 30-36 36-42 42-48

AI after the ovulation (hours)

Preg

nanc

y ra

te (%

)

No. of embryonic losses

3 3 3 4 1

(11/14)

(13/20)

(7/14)

(7/21)

(3/12)

(0/10) (0/12) (0/9)

No. Pregnancies/AI

(Woods et al., 1990)

Ovulation distribution after hCG Ovulation distribution after hCG administrationadministration

• 0-12 h: 6%0-12 h: 6%

• 13-24 h: 10%13-24 h: 10%

• 25-36 h: 33%25-36 h: 33%

• 37-48 h: 42%37-48 h: 42%

• > 48 h: 9%> 48 h: 9%

0-12 h

13-24 h

25-36 h

37-48 h

> 48 h

33%

9% 6% 10%

(Barbacini S. et al. 2000)

Totally 1040 cycles

Ovulation occurs within 48 hrs in Ovulation occurs within 48 hrs in 91% 91% （（ 947/1040 cycles947/1040 cycles ）） of cases and in the of cases and in the period 25-48 hrs in 75%.period 25-48 hrs in 75%.

42%

Influence of season on ovulation Influence of season on ovulation distribution after hCG administrationdistribution after hCG administration

• Feb. Mar. Apr.Feb. Mar. Apr.

0-24 h: 9%0-24 h: 9%

25-48 h: 83%25-48 h: 83%

> 48 h: 8%> 48 h: 8%

• May Jun. Jul.May Jun. Jul.

0-24 h: 21%0-24 h: 21%

25-48 h: 70%25-48 h: 70%

> 48 h: 9%> 48 h: 9%

(Barbacini S. et al. 2000)

0

20

40

60

80

100

Early season Late season

0-24 h

25-48 h

> 48 h

Totally 1040 cycles

Iris's cycle

0

1

2

3

4

5

6

-15 -13 -11 -9 -7 -5 -3 -1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 0 2 4 6 8 10 12 14 16 18 20 22 24 1 3 5 7 9 11 13 15 17 19 21 23 25 27

Days of cycle

Size

(cm

)Dominant Follicle Subordinate Follicle1

Subordinate Follicle2 CL1

HCG

Ovulation

OvulationOvulation

Natural mating or artificial Natural mating or artificial insemination??insemination??

Pregnancy rate in maresPregnancy rate in mares• The optimal time for insemination with The optimal time for insemination with

frozen-thawed semen include a shorter frozen-thawed semen include a shorter interval than if fresh semen is used.interval than if fresh semen is used.

– chilled semen: 55-70% per cyclechilled semen: 55-70% per cycle

– frozen semen: 35-50% per cyclefrozen semen: 35-50% per cycle

• overall pregnancy rates at the end of the overall pregnancy rates at the end of the season: 50-90% (75%)season: 50-90% (75%)– live foal rate: 65%live foal rate: 65%

(Fonateban F. Pycock, 1999)(Fonateban F. Pycock, 1999)

AI programAI program

• The mares should be inseminated twice or The mares should be inseminated twice or more at a minimal number of more at a minimal number of 150 x 10150 x 1066 total total spermatozoa per cycle.spermatozoa per cycle.

• Insemination into the Insemination into the uterine bodyuterine body provided provided higher pregnancy rates than insemination higher pregnancy rates than insemination deep into the uterine horn. deep into the uterine horn.

(Vidament M. et al. 2000)

(Sieme H. et al. 2004, Squires EL. et al. 2004 )

bacterial flora in the genital tractbacterial flora in the genital tract

• Escherichia coli Escherichia coli (40%, 12/30)(40%, 12/30)

• Streptococcus zooepidemicus Streptococcus zooepidemicus (13.3, 4/30)(13.3, 4/30)

• Klebsiella pneumoniae Klebsiella pneumoniae (3.3%, 1/30) (3.3%, 1/30)

• Pseudomonus aeruginosa Pseudomonus aeruginosa (3.3%, 1/30)(3.3%, 1/30)

• A pre-breeding culture was suggested for a A pre-breeding culture was suggested for a routine AI . routine AI .

problem in the clinical caseproblem in the clinical case

What is the optimal time for AI after a single What is the optimal time for AI after a single examination?examination?

• detection of estrous behaviordetection of estrous behavior• an ovulatory follicle an ovulatory follicle 35 mm 35 mm• an visible edema pattern of endometrial foldan visible edema pattern of endometrial fold

Prediction of ovulationPrediction of ovulation• changes in size and shape of the preovulatory changes in size and shape of the preovulatory

follicles follicles – Diameter (31-49 mm)Diameter (31-49 mm)– Incidence of non-spherical shapeIncidence of non-spherical shape

D -4D -4 D -3D -3

D -2D -2

D -1D -1

Changes in size and shape of the Changes in size and shape of the preovulatory folliclespreovulatory follicles

• Diameter (31-49 mm)Diameter (31-49 mm)

26 x 28 mm 38 x 45 mm26 x 28 mm 38 x 45 mm

50 x 58 mm

> 40mm, 74%

ovulation

atresiaatresia

> 35mm, 21%

(Gastal et al. 1998)

Changes in size and shape of the preovulatory follicles

• Incidence of non-spherical shapeIncidence of non-spherical shape

(Townson et al. 1989)

The shape change begins on The shape change begins on 2-3 days prior to ovulation!!2-3 days prior to ovulation!!

should be checked every 6 should be checked every 6 hours. hours.

decrease in fluid pressure within the antrum

84-89%

Prediction of ovulationPrediction of ovulation

• digital analysis of echotextural changes in the digital analysis of echotextural changes in the wall of preovulatory folliclewall of preovulatory follicle

– echogenicity of the apparent echogenicity of the apparent granulosa granulosa layer (GL)layer (GL) and prominence of the and prominence of the anechoic anechoic layer (AL)layer (AL) beneath the granulosa beneath the granulosa

-to quantify the echotextural changes in the preovulatory -to quantify the echotextural changes in the preovulatory wall of mares using computer-assisted image analysiswall of mares using computer-assisted image analysis

GL

AL

Echotexture indicator would have been more efficient for Echotexture indicator would have been more efficient for initiation of breeding than the diameter indicators.initiation of breeding than the diameter indicators.

(Gastal et al. 1998)

1.1. Pixel values along a line traversing the follicle wall from the Pixel values along a line traversing the follicle wall from the peripheral antrum, GL, AL, to the stroma were measured. peripheral antrum, GL, AL, to the stroma were measured.

2.2. Echotexture values for the echogenicity of the GLEchotexture values for the echogenicity of the GL （（ anechoic anechoic and dark gray to echoic and whiteand dark gray to echoic and white ）） and ALand AL （（ gray and thin gray and thin to black and thickto black and thick ）） were subjectively scored from 1 to 3.were subjectively scored from 1 to 3.

Evaluating the changes of follicular wallEvaluating the changes of follicular wall

The pixel values along the curveThe pixel values along the curve （（ PP00, P, P11, P, P22 ）） were were

used to obtain the slope of a regression line of the fall used to obtain the slope of a regression line of the fall segment.segment.

4 days before ovulation

0

50

100

150

200

1 11 21 31 41

Distance (Pixel)

Inte

nsity

Score GL = 2 AL = 1.5Slope = 17.1

0

50

100

150

200

1 11 21 31 41

Distance (Pixel)

Inte

nsity

Score GL = 2 AL = 2Slope = 18.6

2 days before ovulation

Score GL = 3 AL = 2.5Slope = 21.5

18 hours before ovulation

0

50

100

150

200

1 11 21 31 41

Distance (Pixel)

Intens

ity

Evaluating the changes of follicular wallEvaluating the changes of follicular wall

• As ovulation approached, the granulosa As ovulation approached, the granulosa echogenicity and the prominence of AL echogenicity and the prominence of AL revealed significant changes on D-1.revealed significant changes on D-1.– 81.3% slope 81.3% slope 19.0 on D-1 19.0 on D-1– 100% GL score 100% GL score 2 on D-1, 96.9% AL score 2 on D-1, 96.9% AL score

2 on D-1 2 on D-1

Both methods were effective for predicting Both methods were effective for predicting the occurrence of ovulation.the occurrence of ovulation.

Evaluating the changes of follicular wallEvaluating the changes of follicular wall

• The slope criterion could be used as an The slope criterion could be used as an indicator for close prediction of impending indicator for close prediction of impending ovulation within one day.ovulation within one day.

• The criterion of the 2 echotexture changes The criterion of the 2 echotexture changes could enhance the reliability of prediction.could enhance the reliability of prediction.– The increased granulosa echogenicity was The increased granulosa echogenicity was

indicative of follicle growth. indicative of follicle growth. – The development of an AL could distinguish the The development of an AL could distinguish the

future dominant follicle from the other follicles.future dominant follicle from the other follicles.

Evaluating the changes of follicular wallEvaluating the changes of follicular wall

• An anovulatory large follicleAn anovulatory large follicle （（ 30 30 mmmm ）） might be recognized through this might be recognized through this method in advance.method in advance.

Regime I for timing of inseminationRegime I for timing of insemination

3,000 IU hCG, IV3,000 IU hCG, IV75-85% of mares will ovulate 75-85% of mares will ovulate 24-48 hours24-48 hours

after injectionafter injectionAI during this period!AI during this period!

Regime II for timing of inseminationRegime II for timing of insemination

1. to examine the mare 1. to examine the mare every 6 hoursevery 6 hours as as ovulation approachesovulation approaches

2. A single insemination 2. A single insemination dose can be used until dose can be used until ovulation is detected.ovulation is detected.

Regime III for timing of inseminationRegime III for timing of insemination

3,000 IU hCG, IV3,000 IU hCG, IV 75-85% of mares will ovulate 24-48 hours after 75-85% of mares will ovulate 24-48 hours after

injectioninjection• breeding when the breeding when the slope value slope value 19.0 19.0 and and a score a score

of both GL and AL of both GL and AL 2 2• AI into the AI into the uterine bodyuterine body with only one dose of with only one dose of

frozen/thawed semen containing frozen/thawed semen containing 500 x 10500 x 1066 spermatozoaspermatozoa

• monitoring the mares at 12-hour intervals until monitoring the mares at 12-hour intervals until ovulationovulation

Jacky’s Family

Best regards!!

Thank you for your

attentions!!