Current Achievements in Artificial Breeding of Horses in Subtropical Taiwan Jacky P. Chan Veterinary Medicine Teaching Hospital, Department of Veterinary Medicine, College of Veterinary Medicine National Chung-Hsing University

Danehill dove orchid Danehill Great vintage Danehill champage Stallion Danehill

Influence of the length of day light on the ovary function in mares Photoreceptors in the eyes Pineal Hypothalamus Anterior pituitary Ovaries increasing day light decreasing day light neurotransmission portal system blood circulation Melatonin GnRH Gonadotropine

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

Date 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(%) (Woods et al., 1990,)

0D -1D -2D -3D hrs Fertility LowHighLow Ovulation D -5 Fertility curve 12 hrs pre- and 6 hrs postovulation

Effect of post-ovulatory AI on pregnancy rates and embryonic mortality No. of embryonic losses (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 administration 0-12 h: 6%0-12 h: 6% h: 10%13-24 h: 10% h: 33%25-36 h: 33% h: 42%37-48 h: 42% > 48 h: 9%> 48 h: 9% 33% 9%6% 10% (Barbacini S. et al. 2000) Totally 1040 cycles Ovulation occurs within 48 hrs in 91% （ 947/1040 cycles ） of cases and in the period hrs in 75%. 42%

Influence of season on ovulation distribution after hCG administration Feb. Mar. Apr.Feb. Mar. Apr h: 9% 0-24 h: 9% h: 83% h: 83% > 48 h: 8% > 48 h: 8% May Jun. Jul.May Jun. Jul h: 21% 0-24 h: 21% h: 70% h: 70% > 48 h: 9% > 48 h: 9% (Barbacini S. et al. 2000) Totally 1040 cycles

Natural mating or artificial insemination??

Pregnancy rate in mares The optimal time for insemination with frozen-thawed semen include a shorter interval than if fresh semen is used.The optimal time for insemination with frozen-thawed semen include a shorter interval than if fresh semen is used. –chilled semen: 55-70% per cycle –frozen semen: 35-50% per cycle overall pregnancy rates at the end of the season: 50-90% (75%)overall pregnancy rates at the end of the season: 50-90% (75%) –live foal rate: 65% (Fonateban F. Pycock, 1999)

AI program The mares should be inseminated twice or more at a minimal number of 150 x 10 6 total spermatozoa per cycle.The mares should be inseminated twice or more at a minimal number of 150 x 10 6 total spermatozoa per cycle. Insemination into the uterine body provided higher pregnancy rates than insemination deep into the uterine horn.Insemination into the uterine body provided higher pregnancy rates than insemination deep into the uterine horn. (Vidament M. et al. 2000) (Sieme H. et al. 2004, Squires EL. et al )

bacterial flora in the genital tract Escherichia coli (40%, 12/30)Escherichia coli (40%, 12/30) Streptococcus zooepidemicus (13.3, 4/30)Streptococcus zooepidemicus (13.3, 4/30) Klebsiella pneumoniae (3.3%, 1/30)Klebsiella pneumoniae (3.3%, 1/30) Pseudomonus aeruginosa (3.3%, 1/30)Pseudomonus aeruginosa (3.3%, 1/30) A pre-breeding culture was suggested for a routine AI.A pre-breeding culture was suggested for a routine AI.

problem in the clinical case What is the optimal time for AI after a single examination? detection of estrous behaviordetection of estrous behavior an ovulatory follicle  35 mman ovulatory follicle  35 mm an visible edema pattern of endometrial foldan visible edema pattern of endometrial fold

Prediction of ovulation changes in size and shape of the preovulatory follicleschanges in size and shape of the preovulatory follicles –Diameter (31-49 mm) –Incidence of non-spherical shape D -4 D -3 D -2 D -1

Changes in size and shape of the preovulatory follicles Diameter (31-49 mm)Diameter (31-49 mm) 26 x 28 mm 38 x 45 mm 50 x 58 mm > 40mm, 74% ovulation atresia > 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 2-3 days prior to ovulation!! should be checked every 6 hours. decrease in fluid pressure within the antrum 84-89%

Prediction of ovulation digital analysis of echotextural changes in the wall of preovulatory follicledigital analysis of echotextural changes in the wall of preovulatory follicle –echogenicity of the apparent granulosa layer (GL) and prominence of the anechoic layer (AL) beneath the granulosa

-to quantify the echotextural changes in the preovulatory wall of mares using computer-assisted image analysis GL AL Echotexture indicator would have been more efficient for initiation of breeding than the diameter indicators. Echotexture indicator would have been more efficient for initiation of breeding than the diameter indicators. (Gastal et al. 1998)

1.Pixel values along a line traversing the follicle wall from the peripheral antrum, GL, AL, to the stroma were measured. 2.Echotexture values for the echogenicity of the GL （ anechoic and dark gray to echoic and white ） and AL （ gray and thin to black and thick ） were subjectively scored from 1 to 3.

Evaluating the changes of follicular wall The pixel values along the curve （ P 0, P 1, P 2 ） were used to obtain the slope of a regression line of the fall segment.

4 days before ovulation Score GL = 2 AL = 1.5 Slope = 17.1

Score GL = 2 AL = 2 Slope = days before ovulation

Score GL = 3 AL = 2.5 Slope = hours before ovulation

Evaluating the changes of follicular wall As ovulation approached, the granulosa echogenicity and the prominence of AL revealed significant changes on D-1.As ovulation approached, the granulosa echogenicity and the prominence of AL revealed significant changes on D-1. –81.3% slope  19.0 on D-1 –100% GL score  2 on D-1, 96.9% AL score  2 on D-1 Both methods were effective for predicting the occurrence of ovulation. Both methods were effective for predicting the occurrence of ovulation.

Evaluating the changes of follicular wall The slope criterion could be used as an indicator for close prediction of impending ovulation within one day.The slope criterion could be used as an indicator for close prediction of impending ovulation within one day. The criterion of the 2 echotexture changes could enhance the reliability of prediction.The criterion of the 2 echotexture changes could enhance the reliability of prediction. –The increased granulosa echogenicity was indicative of follicle growth. –The development of an AL could distinguish the future dominant follicle from the other follicles.

Evaluating the changes of follicular wall An anovulatory large follicle （  30 mm ） might be recognized through this method in advance.An anovulatory large follicle （  30 mm ） might be recognized through this method in advance.

Regime I for timing of insemination  3,000 IU hCG, IV  75-85% of mares will ovulate hours after injection  AI during this period!

Regime II for timing of insemination 1. to examine the mare every 6 hours as ovulation approaches 2. A single insemination dose can be used until ovulation is detected.

Regime III for timing of insemination  3,000 IU hCG, IV  75-85% of mares will ovulate hours after injection breeding when the slope value  19.0 and a score of both GL and AL  2breeding when the slope value  19.0 and a score of both GL and AL  2 AI into the uterine body with only one dose of frozen/thawed semen containing 500 x 10 6 spermatozoaAI into the uterine body with only one dose of frozen/thawed semen containing 500 x 10 6 spermatozoa monitoring the mares at 12-hour intervals until ovulationmonitoring the mares at 12-hour intervals until ovulation

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