ARTS

• Animals are the largest users of ARTs in Australia
• One semen sample will allow for insemination for 100s of ewes
• Can choose genetic qualities that are more favourable (mounting quality)
• Detecting/synchronising oestrus in females

  1. Timing is vital in AI programs

  2. Insemination must coordinate with ovulation

  3. Natural oestrus- harnessed wethers

  4. Synchronised oestrus- fixed time for mob

  5. Improves efficiency and control of synchronisation
• Methods of Semen Collection

  • Artificial vagina
  • Electroejaculation
  • Digital manipulation ("gloved-hand" method)
  • Penile vibrostimulation
  • Female condom
• Artificial Vaginas (AVs)


  • Stimulus to imitate natural service
  • Temperature: 42-45 C water
  • Pressure: inflated with air
  • Rubber liner must be clean and dry
  • Sperm will denature and die if they get too cold and wet
  • Ram is allowed to mount a teaser ewe
• Electro-ejaculation


  • Reliable method of semen collection
  • Must be performed or under the supervision of a veterinarian
  • Sedation and restraint of male required
  • Localised electrical stimulation of the nerves controlling ejaculation
  • Probe is inserted into rectum and series of electrical pulses released
  • increasing in voltage from 5-16 volts
  • Duration of 1-2 sec each
  • Rest period of 0.5-2 secs between pulses
  • Continues until ejaculation
• Extra fluid from electroejaculation

  Can alter component of the ejaculate and can impact the quality of the sperm
• Larger volume of ejaculate

  Concentration of sperm/mL will decrease
• Processing semen for inspection

  • Fresh
  • Chilled
  • Cryopreserved
  • Pellets
  • Straws
• Semen insemination dose


  • Dependant on type of semen used (species and type of oestrus: natural or synchronised)
  • Sperm quality decreases overtime, so insemination must occur close to ovulation to get the best possible chance of it reaching the egg
  • Or add more sperm to female to increase the chance in a similar way
  • Cryopreserved sperm require more sperm per dose due to having half the lifespan of fresh sperm
  • Dependant on where semen is deposited
• When to inseminate?


  • Majority of females will be in oestrus 36-48h and ovulate about 60h post pessary removal
  • Compromised sperm should be inseminated closer to ovulation
  • Seminal plasma plays a fundamental role in cervical transit/interaction of sperm with cervix in sheep, whether through immune function, cervical mucus interaction, specific protein-protein interaction
• Benefits of AI


  • Improves dissemination of valuable male genetics
  • Semen from males unable to mount
  • Enables domestic and international transport
  • Enhances breeding management
  • Facilitates progeny testing
  • Permits crossbreeding to change production trait
  • Safer and cheaper
  • Long-term storage of frozen-thawed semen
  • Reduces risk of disease transmission
• Embryo Transfer


  1. Synchronisation and superovulation

  2. Donor ewes: Treated to control oestrus and ovulation with progestogens, PMSG and GnRH, Treated to cause the release of a large (10-20) numbers of mature ova from ovaries with FSH

  3. Recipient ewes are treated to control oestrus and ovulation with progestogens and PMSG
• Embryo recovery


  • Recover at early embryo stages: Zygote to 8-cell (oviducts), Morula to blastocyst (uterine horns)
  • Surgical methods: Laparoscopy, Flush oviducts and uterine horns
  • Non-surgical methods: Foley catheter (uterine horns only)
• Insemination of embryos


  • Fresh embryos: Donor and recipient cycles must be within a day of each other, Different ovulation timing relative to P4 sponge removal
  • Frozen embryos: Recipients' cycles must be appropriate for stage of embryo, Often reduces chances of survival
• Benefits of ET


  • Allows greater dissemination of valuable female genetics
  • Particularly for monotocous species (cattle, sheep)
  • Females not able to be mounted (injured, old)
  • Overcomes problems associated with transporting valuable genetics
  • Breeding management tool
  • Exotic and endangered species
  • Circumvents seasonal limitations to breeding
  • Essential part of other advanced reproductive technologies
• Oocyte In Vitro Maturation (IVM)


  1. Recover immature oocytes (cumulus-enclosed)

  2. Stimulated donor ovaries (JIVET/MIVET)

  3. Abattoir-sourced ovaries (research)

  4. Incubate in maturation medium: Physiological pH and osmolarity, Gonadotrophins, Energy substrates, Growth factors, Anti-oxidants, Antibiotics

  5. Duration of incubation varies between species, Similar to interval between LH-surge and ovulation
• Factors Affecting Oocyte Quality


  • Maternal age
  • Follicle diameter
  • Oocyte diameter
  • Season
  • Glutathione content
  • Oocyte-secreted factors: GDF-9, BMP-15
  • Mitochondrial activity: ATP
• In Vitro Fertilisation (IVF)

  1. Sperm preparation: Wash to remove any toxic substances, Incubate in capacitating medium: Ca 2+, cAMP analogues, Albumin (BSA, HSA), Bicarbonate

  2. Oocyte preparation: Partially remove cumulus cells from mature (metaphase II stage) oocytes

  3. Gamete co-incubation: Fertilisation medium must maintain gamete function, Sperm: egg ratio typically 1000 x greater than that in vivo
• Intra-Cytoplasmic Sperm Injection (ICSI)

  • Overcomes male infertility problems: Oligospermia (low sperm count), Azoospermia (no sperm)
  • Used in nearly half of all human IVF treatments
  • Testicular sperm extraction (TESE)
  • Single sperm injected, Is the selected sperm healthy?
• JIVET


  • Harvesting eggs from pre-pubertal lambs (6-8 weeks) or calves via oocyte pick up
  • Culturing them to maturity and fertilizing with semen from elite sires
  • Advantages: Allows early progeny testing, Significantly reduces generation interval, Accelerates rate of genetic gain achievable
  • Disadvantages: Huge variation in response to ovarian stimulation, Technical expertise required, Cost
• MOET (Multiple Ovulation Embryo Transfer)

  • Involves a 5 week superovulation program, resulting in several eggs being released from the donor cow
  • The cow is inseminated and embryos are then flushed from the cow's uterus the following week
  • An average flush would produce 5 embryos
  • Advantages: Increase the reproductive rate of individuals or groups of animals, Non-surgical, Embryos can be transferred fresh, or frozen in our lab for use later, Entire procedure can be carried out on farm, Consider 'banking' embryos in case of future TB breakdown
• Benefits of IVP


  • Efficiency rivals collection of in vivo-derived embryos
  • Obtain offspring from sub-fertile or infertile females
  • Juvenile, injured or old animals
  • Use semen from valuable males more efficiently
  • One AI dose can be used to fertilise 100s of oocytes by IVF
  • In vitro fertility test
  • Gamete and embryo biology research
  • Important component of other reproductive technologies
• Sexed semen


  • Differentiates based on X and Y DNA chromosome content
  • Females have 2 X's, males have X and Y
• Sexed semen prevents issues with dairy cows having extra calves
• Choosing to make only male lambs to sell and make a larger profit in the sheep industry
• Use of sexed semen in industry

  • Cattle: First calves produced through sexed semen in 1999, In the 12 months preceding March 2020, 51.3% of all semen sales was sexed
  • Sheep: USyd responsible for developing the protocol for use in sheep, Sexed fresh semen recently released to industry
• Frozen sexed semen is still in development, compromised when frozen and inseminated in low doses
• Not sure whether it degrades quality or affects how sperm reaches egg in the AI process
• Benefits of using sexed semen in your AI program

  • Female selection: Replacement of dairy heifers, Avoid unwanted male calves
  • Male selection: Increase stud production of sires with desirable genes, Manage breeding of endangered species in captivity, Being able to transfer males to zoos and increase gene pools
  • Control sex-linked diseases in humans
• Improving the cryo-survival of spermatozoa

  • Use of rhino semen collected via EJ is very inefficient due to viscosity (sperm gets trapped in viscous fractions of the seminal plasma)
  • Approx. 2mLs of 70mLs is suitable for AI
  • Prevents: Use of semen for ARTs, Use of advanced semen assessment- CASA & flow cytometry
  • Hypothesised to be related to the increased presence of proteins in seminal plasma following EJ, targeting those proteins is being attempted
  • Alpacas also have viscous semen, student used papayas to break down those viscous proteins
• The Goal: Adapt to changing times, improve fertility and secure the future of artificial breeding to maintain the genetic progress of our animal industries