Assisted Reproductive Technologies in Sheep

= technologies that enhance or improve fertility e.g. ~ AI (insemination of semen into repro tract) ~ semen cryopreservation (freezing of sperm) ~ embryo transfer (transfer of embroyo from doner to recipient ewe) ~ cloning ~ transgenesis (DNA of one organism into another) ~ IVF ~ in vitro embryo production (fertilised oocyte cultured in lab until point of embryo) (quick turnaround for donor) ~ store semen (chilled, frozen) ~ oestrus synchronisation (all animals brought into oestrus at same time)

• length = 17 days • ovilation occurs between day 0 and 2 • day 2 = corpus luteum begins to be formed • day 14 = luteolysis (structual demise of CL) (diagram in folder) • oestrodidol does most functions for oestrogen

• oestrus last 24-42 hours ~ signs - restlessness, stamping feet, sniffing male- - 6-8% ewes in oestrus each day of breeding season • at least 17 days to inseminate a flock naturally cycling ewes • ewes can be mised and labor intensive

= use of synthetic hormones to syncronise oestrus in a flock

• well established method • determine expected time of ovulation (less labour) • multiple insemination at one time point • ewes lamb in unison = facilitating management during preg/parturition, all lambs in short time span,manage nutrition at same stage • stimulate ovulation = increase fertility (more ewes preg) and fecundity (more offspring per female) • out of season breeding (mar-sept) • no need to observe and draft off females in oestrus • no teaser male required to detect oestrus

• can be costly • reduced fertility (if not managed properly) ~ e.g. 300 ewes brought into oestrus but not used for artificial insemination program ~ put out for natural ating with few rams = unable to mate with all = lower fertility rates

(diagram in folder) • adv - cheap (£4-5 per ewe) • quickest (days to ovulation) and most reliable method of synchronisation • dis - enviro contamination: residue progesterone/antibiotics (accumulate in tissues) not ethical/green/clean (waste disposal important) • preg rates lower than untreated ewes

= the deposition of semen into the female reproductive tract by artificial means - ease of transport (frozen semen), cheaper to transport than whole animal ~ allow transport of sperm between countries that dont allow import/export of live animals - increase breeding efficiency = remove sub-fertile rams (collect and assess semen) and inseminate all females - lower ram maintenance - prevention/control disease (no male/female contact, less transport rams) - use of incapacitated males e.g. old, injured ~ identify why cant mate (hereditary/genetic trait) - synchronised breeding - out of season breeding = use in semen semen (impaired func/integrity if take sperm out of season, lower fertility rate) - increase rate of genetic gain (ram naturally 50 ewes/years vs AI 1000 ewes/2-3 weeks)

• Inbreeding ~ More prevalent in a small flock or herd. ~ Care must be made to ensure selection intensity is not too high. ~ Can have opposite effect (more sires available). • Reduced fertility ~ Pregnancy rates can be less than with natural mating. ~ Can be caused by incorrect handling of semen or unsuccessful oestrous synchronisation. ~ ability of person undertaking AI affect • Cost ~ Technician, drugs, hormones, semen. ~ Cost per insemination relatively low, cost of labour, drugs and hormones is greatest.

- laproscopic ~ 20 million sperm ~ 70% fertility with FT sperm - Cervical ~ up to 180m sperm ~ 70% fertility with fresh sperm ~ less than 20% with frozen - Vaginal ~ 300m fresh sperm ~ 70% fertility (seen 80-90% commonly) ~ FT semen not effective

• Timing of semen deposition relative to ovulation is critical • Timing of AI dependent on site of semen deposition • if too early = sperm sat in repro tract, frozen sperm die quickly ~ Cervical AI: optimal fertility 55h after pessary (sponge/CIDR) removal (further to travel to fertilise oocyte) ~ Intra-uterine AI: Optimal fertility 60-66h after sponge removal • Should AI prior to but close to ovulation ~ Fresh sperm viable for 3-5d in repro tract ~ Cryopreserved semen have reduced fertility (max. 12 h) ~ Oocyte viable for 24h post-ovulation ~ need to ensure sperm already moving in repro tract when ovulating ~ Advantages to using ovulation-inducing agents (GnRH/PMSG) = ovulation occur at time expected to

• Using fresh semen: ~ Fertility rates average 70% ~ BUT fresh-semen is only viable for 24 hours (some advances with cooled semen) • Using cryopreserved (or frozen-thawed) semen so stored for years: ~ Fertility rates approx. 20% ~ Lambing rates range from 6.7 – 57 % (not commercially viable) • better results cryopreserved rather than chilled

• Sperm storage reduces sperm integrity and function • Sperm storage reduces motility in utero (4h post-AI) however stored sperm still give viable fertility rates • Sperm storage reduces movement of sperm through the cervix • Cervical AI with stored sperm is unviable (low fertility rates) = Transit of stored sperm through the cervix is impaired

• Changes to cervical mucus composition with oestrous synchronisation ~ sperm interact and need to get through mucus • Changes to sperm membrane structure and biochemistry during storage

• Will by-pass the cervix • Costly at approx. £20/ewe (AB Europe 1-10 ewes) cost reduced if more ewes • Time consuming • Requires technical ability • Not considered welfare friendly = anaesthesia, put into cradal upside down, rumen push on lungs, vomiting, injected directly into uterine horn • need to be upside down so rumen goes down and visualise uterus easily • Can only be used in recognised sheep breeding schemes and performed by a veterinarian/technician

- Intrauterine ~ 73% (comparable to laproscopic ai) - Deep cervical (2.5-4cm deep) ~ 61% - Mid-cervical (1.5-2.5cm deep) ~ 47% - Superficial cervical (0.5-1.5cm deep) ~ 39% - cervical os ~ 19%

• cervical trauma • anatomy of cervix ~ narrow lumen ~ cervical rings interlock and missaligned (espe 2nd/3rd rings - lumen 2-3cm) ~ difficult to pass inseminating pippete through cervix • grade 1 = cervical rings aligned and typically able to penetrate cervix at around 4cm • grade 2 = some missalignment but still penetrate well in depths of cervix • grade 3 = mass interlocking rings prevent insemination pippete reaching cervix , less than 30cm, hard to locate cervical opening

• Modify the inseminating pipette ~ Flexible pipettes but inconsistent effects on lambing and fertility rates ~ Wulster-Radcliffe, M.C., and Lewis, G.S. 2002 ~ flexible more trauma = move around more and need more pressure to get in • Relax the cervix prior to insemination ~ Natural relaxation at oestrous. Prostaglandins, oxytocin and FSH (with varying results = some allowed penetration but pregnancy rate impaired) Falchi, L. et al., 2012. ~ prostaglandin e2 to make cervix relax • Focus on the sperm ~ Alterations occur on the sperm membrane during storage. Can we mitigate these? Take an integrated approach ~ Sperm transit in the reproductive tract. ~ Which proteins/signalling pathways are important?