UDAA2214 Group 1: Human intervention in farm animal reproduction (From lab to farm) by Jean K

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Equine IVF

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Lab-based Equine Assisted Reproductive Technologies (ARTs)

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In Vitro Fertilization (IVF) is the process of creating embryos from oocytes (unfertilised egg cells) by fertilising them with sperms on a petri dish (Hinrichs, 2010).

However, standard in vivo fertilization is not yet repeatable in the horse, the fertilisation process can also be performed by using Intra Cytoplasmic Sperm Injection (ICSI) which is the injection of one spermatozoa into oocyte cytoplasm.

Step 1: Oocyte collection

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There are 2 approaches to collect immature oocytes (cumulus-oocyte complexes) which are from live donor animals or directly from ovaries obtained from an abattoir (Carter et al., 2002).

(A) From live donor animals - The ovum pick-up technique (OPU)

The donor female is restrained in a suitable holding chute and sedated with detomidine. The faeces of mare are removed. The extra genital area and perineum are cleaned. The mare is then administered epidural anaesthesia.
The vaginal canal is inserted with the ultrasound probe for visualization of ovaries on the ultrasound. The ovary is grasped via the rectum and held against the vaginal wall.
Follicles are identified as black (hypo echoic) circular shapes on the monitor screen.
A 12 gauge double lumen needle with 60 cm long is inserted through the needle guide in the plastic handle to aspirate and inject fluid at the same time.
The needle is connected to a vacuum pump, passing into a 500 ml glass bottle to collect follicular fluid containing the oocytes.
The fluid of 1 cm or bigger follicle is aspirated and flushed repeatedly with commercial embryo flushing medium supplemented with heparin while scraping the follicular wall for the enhancement of oocyte recovery.
The follicular fluid is then passed through the standard bovine embryo filter.

(B) From an abattoir

The ovaries are removed from the mare reproductive tract after the extraction of internal organs from the carcass. The ovaries are placed into saline containers.
The ovaries are then transferred to the second container with saline concentration and placed in the thermos.
The ovaries are transported to the lab in physiological saline that consists of antibiotics to prevent bacterial contamination at 22-24°C.
The best results are obtained when oocytes are collected within 4-6 hrs after slaughter.
Oocytes can be collected by aspiration using a syringe and needle or by slashing the ovary surface with a scalpel blade and collecting it into a beaker by cell strainer method.

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Step 2: In Vitro Maturation (IVM) of oocytes

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The oocytes are cultured in a maturation medium (TCM-199) to finish the first meiotic division and stop at the metaphase II so that oocytes are ready to be fertilised.
The maturation medium is incubated for about 30 hours at 38.5°C in a humidified atmosphere of 5% CO₂ in air.
The oocytes are assessed under the microscope for the evaluation of maturity. The extrusion of the first polar body can be determined and the oocytes are ready for ICSI.

References list:

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Carter, C. and Greening, L., 2012, July. Auditory stimulation of the stabled equine; the effect of different music genres on behaviour. Applied Animal Behaviour Science. [online] Available at: [Accessed 1 September 2020].

Carter, J.A., Bellow, S., Meintjes, M., Perez, O., Ferguson, E. and Godke, R.A., 2002. Transvaginal ultrasound-guided oocyte aspiration for production of embryos in vitro. Archives Animal Breeding. [online] Available at: [Accessed 31 August 2020].

Choi, Y.-H., Ross, P., Velez, I.C., Macias-Garcia, B., Riera, F.L. and Hinrichs, K., 2015. Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations. Reproduction, 150(1), pp.31-41.

Hanevik, H. I., Hessen, D. O., Sunde, A., and Breivik, J., 2016. Can IVF influence human evolution? Human Reproduction, [e journal], 31(7), pp. 1397–1402. 10.1093/humrep/dew089

Hinrichs, K., 2010. In Vitro Production of Equine Embryos: State of the Art. Reproduction in Domestic Animals. [online] Available at: [Acessed 31 August 2020].

Hinrichs, K., 2018. Assisted reproductive techniques in mares. Reproduction in Domestic Animals, [e journal], 53, pp. 4–13 10.1111/rda.13259

Kraemer, D.C., 2013. A history of equine embryo transfer and related technologies. Journal of Equine Veterinary Science, 33(5), pp.305-308.

McCue, P.M., n.d. Embryo Transfer. [online] Available at: [Accessed 1 September 2020].

Menon, S., 2018. Changing lives one ride at a time. The Star, [online] 5 August. Available at: [Accessed 1 September 2020].

Price, T. M., Murphy, S. K., and Younglai, E. V., 2006. Perspectives: The Possible Influence of Assisted Reproductive Technologies on Transgenerational Reproductive Effects of Environmental Endocrine Disruptors. Toxicological Sciences, [e journal], 96(2), pp. 218–226. 10.1093/toxsci/kfl196

Sertich, P.L., 2014. Reproductive cycle in horses. [online] Available at: [Accessed 2 September 2020].

Smits, K., Hoogewijs, M., Woelders, H., Daels, P. and Van Soom, A., 2012. Breeding or assisted reproduction? Relevance of the horse model applied to the conservation of endangered equids. Reproduction in Domestic Animals, 47, pp.239-248.

Stout, T.A.E., 2010. Equine embryo transfer: review of developing potential. Equine Veterinary Journal, 38(5), pp.467-478.

Troxel, T.R., n.d. Embryo Transfer in Cattle. [online] Available at: [Accessed 1 September 2020].

University of Wisconsin-Madison, n.d. Assisted Reproductive Technology. [online] Available at:< http://www.ansci.wisc.edu/jjp1/equine/technology/intro.html> [Accessed 7 September 2020]

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Opinion on the effects of ARTs

Animal perspective

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Environment perspective

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Economical perspective

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Equine Embryo Transfer (ET)

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In 1972, First report on equine and donkey offspring produced through ET was published by Allen and Rowson. 81 years after first successful mammalian case, rabbit's ET. 39 years after sheep and goat and 21 years after cattle and pigs.

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Increase in production
Enables the production of foals from infertile animals

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Embryo Culture

wiiliamtong299 — 2020-09-01 12:43:39 UTC

The development of equine embryos in vitro starts with the formation of a morula (8 to 16 cell stage) which usually occurs after 3 to 4 days of fertilization and can be supported by a variety of media.
However, the development of equine blastocyst requires the embryos to be cultured in DMEM⁄F-12 medium with 10% fetal bovine serum, at a ratio of 1 μl medium per embryo.
The atmospheric air mixture should consist of 5% O₂, 5% CO₂ and 90% N₂ at 38.2℃.
DMEM⁄F-12 contains 17 mM glucose which acts as a predominant energy substrate in the blastocyst.
The embryos will start to develop to the blastocyst stage at the 7 days through 11 days of culture.
Hence, the embryos should be transferred to recipient mares once the blastocyst starts to develop even at an early stage.

(Hinrichs, 2010)

What is Embryo Transfer?

wiiliamtong299 — 2020-09-01 12:47:35 UTC

Embryo transfer is a process where one or more embryos are being removed (flushed) from the uterus located in the reproductive trait of a donor female (e.g., mare) and transferring the embryos into one or more recipient females (e.g., mare) (McCue, n.d.). However, laboratory techniques such as in vitro fertilization can also produce embryos that involve the fertilization of oocytes with sperm cells in a dish (Troxel, n.d.). Later, the embryos produced in vitro will be transferred into the uterus of a female to enable pregnancy to begin.

What is Intracytoplasmic Sperm Injection (ICSI)?

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To fertilise the egg in IVF by using ICSI, a micropipette is used to inject a single sperm into the centre of the egg (Hinrichs, 2018). The diagram below illustrates the injection of sperm into an egg.

ICSI is performed during embryo genetic testing as it can prevent sperm that failed to fertilise the egg from attaching to the exterior of the egg, causing inaccurate test results.

Why is ICSI more preferred than IVF?

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ICSI is able to assist the following infertility problems

Low sperm count
Poor sperm movement
Low quality sperm
Failure of insemination
Abnormal sperm

(Hinrichs, 2018)
The diagram below illustrates the types of abnormal sperm

Fun fact: How fast does a horse run?

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Quickest recorded sprinting speed: 88 km/h (55 mph)

Gallop: around 44 km/h (27mph)

Advantages of ICSI

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The cumulus cells that surrounds the egg after being removed from the ovary makes it difficult to be located under the microscope and makes it difficult to determine the maturity of the egg and as immature eggs cannot be fertilised, it will cause the failure of fertilisation.

During ICSI, the cumulus cells are stripped away, allowing the scientists to confirm the maturity of the egg. The Figure below illustrates the cells surrounding the egg.

♩♪♫ Horses like classical and country music ♩♪♫

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A study on the effect of different music genres on stabled horses was done by Carter and Greening (2012).

Jazz and rock are not the best genres to play for equine.

Minor key effect stimulate adverse behavioural responses.
Display alert behaviours such as stamping, snorting, head tossing and whinnying.

They prefer music with short melodies and strong rhythmic patterns such as those found in classical and country music.

Rest and relaxed behaviours.

The horses respond to the vibrations, not the words. They won’t get bored like we do. Thus, it is ideal to have music playing on repeat.

Music has also been used during post-surgery recovery of equine.
Benefits: calming effect, a sense of safety, masking jarring sounds

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Then, Equine's ET techniques were improved for commercialization 10 years after the first succession.

First successful hybrid.

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First ET case was performed surgically between horses and donkeys, producing one mule and one hinny in 1974. For horses, 18 embryos from pony mares were collected and 15 blastocysts were transferred into uteri of 15 mares using non surgical ways. 4 off spring were successfully produced from 6 pregnancies. (Kramer, 2013).

Choices of horses for ET

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Superior sports horse mares are usually the primary choice for ET to produce offspring to ensure their competitive careers. More then one offspring can be produced in a single breeding session to ensure the economic values. Older mares that are not able to pregnant and those that have degeneration in endometrium while possessing values can look for healthier recipient mare.

Non surgical ways of ET

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Three plastic tubes that is one inside another was guided into
the 🤬's anterior fornix. A needle in the middle tube was penetrated through vaginal wall into the lumen of uterine. Apparatus was manipulated and guided into position through rectal wall. CO₂was channeled into uterus to determine location of the tip of the needle. Rubber bulb was installed at opposite end of the hypodermic needle for transferring of eggs. Tubes with eggs were passed into uterine lumen which is the location that the eggs was deposited together with 0.2 ml of medium.

Country song

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Classical song

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Jazz song

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Rock song

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Connemara Pony

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Trakehner Stallion

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Factors Affecting Transfer Success

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Embryo quality
Transfer technique
Donor-recipient synchrony
Recipient suitability (age, normal cyclicity, no signs of reproductive tract pathology, relative size between mare and stallion, general health and body condition).

(Stout, 2010)

Irish Sport Horse

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The Akhal-Teke

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Embryo Transfer

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The horse embryos in a ‘holding medium’ can be transferred to the uterus of the recipient mare.
Transcervical transfer of equine embryos can be performed using a transfer pipette after loading the embryo into either a 0.25- or 0.50- mL straw.
The straw is loaded with three columns of fluid separated by air spaces. i. First fluid column serves to lubricate the pipette exit. ii. Second fluid column is the embryo. iii. Third fluid column ensure the embryo is flushed out from the pipette.
The pipette is enclosed with a sterile plastic sheath to prevent contamination from the recipient’s 🤬 or 🤬 when entering the uterus.
Upon reaching the cervix, the sheath is pulled slowly and firmly backwards while maintaining the pipette is in the correct place.
In addition, the pipette should manoeuvre through the constricted cervical canal without causing any excessive dilation or trauma.
Hence, the protruding portion of the cervix can be straightened by holding it firmly between two fingers and pulling it backwards, or using a modified rat-toothed Velsellum forceps to grasp and straighten it.

(Stout, 2010)

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Genetic improvement

Allow genetically valuable animals to procreate more extensively

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Enables breeding between animals in different geographic locations

Ovaries collected can be shipped to specialized labs in other countries to perform ICSI.

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Preservation of endangered species, such as the Przewalski's horse

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One commonality for ARTs is expensive, thus constricting the widespread commercial adoption of ARTs.

Equine therapy/ Hippotherapy

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A treatment that involves activities with horses to promote occupational, physical and emotional growth in persons.

It has been proven to be medically beneficial for conditions such as cerebral palsy, traumatic brain injury, sensory deficit in children, autism and hyperactivity (Menon, 2018).

Hippotherapy in Malaysia: Happy Farms Hippotherapy Malaysia, Green Apple Hippotherapy
Riding for Disabled Association: RDA Penang Turf Club, Selangor Turf Club and Equestrian Centre

Feeling down? Saddle up!

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wiiliamtong299 — 2020-09-02 01:13:26 UTC

Ensures the sustainability of the desirable phenotype to produce high quality and safe meat.

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Mexico, Switzerland, Kazakhstan, Belgium, Japan, Germany, Indonesia, Poland and China are among the common horse consuming countries.

But what happens after IVF and ICSI?

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wiiliamtong299 — 2020-09-02 03:25:48 UTC

10% fetal bovine serum

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DMEM/F-12 medium

ICSI Procedure

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The development of IVF and ICSI has eliminated the natural breeding and natural selection during fertilisation as they are able to assist the males with dramatic sperm abnormalities to reproduce (Price, Murphy and Younglai, 2006.)
This may cause a shrinkage in the gene pool as only the few breeds that have the superior genes will be chosen to be bred.

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→ Sonogram of a dominant follicle of mare

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(Sertich, 2014)

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huiqian — 2020-09-02 06:55:09 UTC

Increases the reproductive fitness by removing naturally occurring selective barriers (Hanevik, et al., 2016).
The use of ARTs has influenced the evolution of organisms (Hanevik, et al., 2016)
Due to the selection pressure during IVF, a study has shown that there is a significant effect of evolution as it directly influences reproductive success (Hanevik, et al., 2016)

Equine Therapy Helps Veterans Cope With Post-traumatic Stress Disorder

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“Words don’t need to be said to express who you are.”

Horses are helping teens' mental health

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Horses heal invisible wounds

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"It reminds you to settle down, breathe, get grounded and everything will be okay."

Evolution of horse

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There are more than 350 different breeds of horses !

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Fun Fact: Do you know that The Queen of England loves her horses?

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The Windsor Grey and the Cleveland Bay stallions are among the two breeds chosen to be bred by the Royal Family
The Windsor Grey has been bred since the 19th century and is preferred by the Royal Family because of its beautiful coat colour
The Windsor Grey were used in many occasions such as royal weddings, the queen's birthday, the queen's coronation and so on.
The late George Duell bred the Cleveland Bay stallion Mulgrave Supreme in North Yorkshire in 1961 and was later purchased by The Queen.

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Members of the Royal Family and the Monarchs have been using the Windsor Grey Horses to pull their carriages for ceremonial purposes ever since 1837.

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Generally the Royal Family LOVES their horses (especially the queen)

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Group members

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🐎Johnson Wong Xiu Yang
🐎 Lee Hui Qian
🐎 Ngu Hui En
🐎 Wiiliam Tong Meng Yang

History of Assisted Reproductive Technology(ART) in mare.

atfensterr1 — 2020-09-07 13:15:44 UTC

First Generation:- Discovered six decades ago, artificial insemination with frozen fresh sperm methods is still the horse management technique in general.
Second Generation:- Comes into people sight around 4 decades ago with techniques of embryo transfer(ET) and ultrasonography. This allow the imaging for reproductive organs and sexing for fetuses.
Third Generation:- Blooming of technologies and methods where techniques and methods include oocyte recovery, zygote intrafallopian transfer (ZIFT), embryo sexing, gamete intrafallopian transfer (GIFT) and intracytoplasmic sperm injection (ICSI) were discovered not longer than 20 years ago.

Scientific Classification for Genus Equus.

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Which techniques are not preferred?

atfensterr1 — 2020-09-07 13:33:25 UTC

The Second Generation method of ET is not used commonly since it is not able to collect more than one embryo per cycle per mare and the costs are quite high.The Third Generation are also not preferred due to the inefficiencies, high costs, and the incomplete registry recognition on breed of equine. (University of Wisconsin-Madison, n.d.).

atfensterr1 — 2020-09-07 13:51:38 UTC

A warm welcome to you all for visiting the blog!!! Lets start the experience joyously with some amazing NEIGH asmr ! Because..... Why not? Do loop it during the reading process ! This blog is all about Equine and Assisted Reproductive Technique (ARTs) involved.

Evolution of horses.

atfensterr1 — 2020-09-07 13:59:41 UTC

Approximately 50 million years ago, horses underwent evolution from Hyracotherium or Eohippus (the dawn horse) in the northern hemisphere. (Smits, et al, 2012).

Yup, it's this small doggo looking animal.

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Facts on equines.

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Equines have the abilities to interbreed freely with species from the genus Equus and produce offspring even though most of them are infertile. Mule and hinny are ideal examples of the breeding. Equines are also known for their abilities to show xenogeneic extraspecies pregnancies through ET, which means that they support pregnancy after ET and acts as recipient for exotic species of equine's embryos include Przewalski’s horse and Grant’s zebra. (Smits, et al, 2012).