Assisted Repor: Artificial Insemination (A.I.) and Embryo Transfer (E.T.) Flashcards
Artificial insemination
Bovine, Equine, Canine, Ovine, and other species
Sperm is collected from the maile and placed into the female reproductive tract to join with a naturally ovulated egg
Sex-Selected semen
Bovine and Equine
Sperm is sorted by flow cytometry into predominantly X-bearing sperm and predominantly Y-bearing sperm aliquots
the sorted sperm is then used for AI
Embryo Transfer
Bovine and equine most commonly
AI or natural service is used to inseminate a female
The resulting embryo(s) are collected prior to the time of “hatching” from the zona pellucida and transferred to the different female (recipient) ofr the rest of gestation
In vitro Fertilization
Becommong Common
A single sperm cell is inserted into an egg in a laboratory environment
Embryo is cultured on artificial bedia of several days and if the embryo proceeds to the blastocyst stage - it is transferred inot a recepient (donor or another female) for the rest of gestation
Gamete Intra-Fallopian Transfer (GIFT)
Mostly experimental
An egg is aspirted from a donor female’s ovary prior to ovulation and then planced in the uterine tube of a recipient
THe recipent is bred naturally or via AI
Embryo-splitting
Bovine and Equine
Identical twins (or more) can be produced by splitting a day -6 embryo (blastocyst before hatching from ZP) – and placing the fragments into empty ZP shells and then transferring them into recipients
Cloning (Nuclear transfer)
Bovine, ovine, canine, and equine
Multiple genetically identical animals can be produced by taking cells from an embryonic blastocyst and implanting them into enucleated oocytes which are transferred to a recipient
Cloning (Somatic Cell Nuclear Transfer)
Ovine, canine, bovine, and equine
Multiple genetically identical animals can be produced by taking an adult cell and “re-programming” it to allow differentiation into an entire organism when implanted into enucleated eggs
Artificial Insemination
Advantages
Genetic Improvement - Widespread use of males likely to produce valuable offspring (based on the sire’s performance) or proven to have valuable offspring (based on many documented offspring)
Can use males form anywhere in the world
Don’t have to have as many males
Remove/reduce risk of venereal disease
Artificial Insemination
Disadvantages
Skill of estrus detection
Trained insemination - skill that must be hired or learned
Time and skill to commit to herd/animal reproduction
Increased cost per pregnancy
Collecting Semen for AI
Semen can be purchased form commercial suppliers and directly from males owners
Semen can be collected on-site and used to mate many more females and at decreased risk of injury than via natural service
Bull, Dog, and stallion semen is frequently frozen - other species primarily use fresh or chilled semen
THe semen of some males does not freeze well - this is not necessarily related to fertility in natural service
Collecting semen for AI
Artificial Vagina
Males that are trained to mount a dummy or teaser animal
Bulls, Stallions
Stallion (temperature / pressure ejaculator) - AV has a jacket that is filled with warm water
Bulls (Pressure ejaculator) - AV does not need a warm water jacket
Collecting semen for AI
Electroejaculation
Males that are not trained to mount a dummy or teaser animal
Bulls, bucks, Wildlife
Specialized probe placed into the rectum applies a series of low-voltage pulses to the pelvic nerves involved in erection and ejaculation
Collecting Semen for AI
manual Stimulation
Pressure ejaculators - massage the penis
Dogs, boars, whales
Collection tube rather then true AV
Collecting Semen for AI
DO NOT USE
latex, plasitc, and lubricants can be detrimental to sperm viablility
Limit their use and contact time
Cryopreservation of sperm
Adequate fertility post-thaw requires:
Appropriate diluents / extenders
Non-penetrating cryoprotectant
Penetrating cryoprotectant to protect from ice cyrstal formation
Buffer to prevent pH change
Sugar to provide an energy source
Salt to provide proper osmolality
Antibiotic to kill pathogens
Cryopreservation of sperm
Adequate fertility post-thaw requires:
Appropriate Sperm Dilution
Bulls: 20 million sperm per dose - assume 50% live sperm post-thaw
Can use a lot less - but must thaw and place semen very well
Cryopreservation of sperm
Adequate fertility post-thaw requires:
Appropriate Cooling and Thawing Speed
Rapid Cooling - but in steps
Add some extender ingredients and cool from 102F to 40F - refrigeratation temp
Add cyroprotectants and package into 0.5 mL straws - hold at 40F for a few hours
Freeze in liquid nitrogen vapor at -238F
Store in liquid nitrogen until used -320F
Cryopreservation of sperm
Adequate fertility post-thaw requires:
Rapid Warming
Semen thawed at 95F for 12 seconds to one minute had best post-thaw viability
Semen thawed at 68F for 12 seconds to one minute has acceptable post-thaw viability
Semen thawed at 41F water bath or in a shirt pocket has lowest post-thaw viability
Artificial Insemination
Detection of Estrus
Used to estimate timing of ovulation
Difficult - great species-to-species viariability and animal-to-animal variability within a species
Artificial Insemination
Timing of Insemination
Canine
Fresh Semen
Ovulation occurs 1 to 2 days after LH surge
Wait longer to breed after ovulation
Fresh semen: lives 5-6 days in uterus
Breed 4-6 days after LH surge
Artificial Insemination
Timing of Insemination
Canine
Chilled Semen
LIves about 1-3 days after ejaculaiton
Will lose at least 1 day in shipping
Breed 4-5 days after LH surge
If breeding 2 times, breed at days 3 and 5; 4 and 6 post LH peak
Must plan ahead - order semen when she ovulates
Artificial Insemination
Timing of Insemination
Canine
Frozen semen
Lives about 12 hours in uterus
Breed 5-6 days after LH surge
no Problem with shipping at the last minute - have the semen on hand far in advance of breeding
Intrauterine insemination - surgical or transcervical catheterization
Artificial Insemination
Timing of Insemination
Bovine
Almost always utilize frozen semen
Successful breeding with frozen semen since 1940s
Fertility using forzen semen in equal to natural service
Semen is placed in the uterus - pipette is passed through the cervix
Breed approximately 12 h after first detection in esturs
Artificial Insemination
Timing of Insemination
Bovine
Frozen straws
Handle with care
Canister in the liquid tank contains several “canes” filled with several “straws” of semen
The straw you are removing is not the problem - be careful not to let the staws around it to warm up
Once thawed - do not let the straw cool
Artificial Insemination
Timing of Insemination
Equine
Fresh semen us used immediatley does not have to be extended
Fresh semen used 10 minutes or longer after collection should be extended
Cold shold causes plasma membrane damage and acrosome changes - reduced fertility
Extended semen used within 6-12 horus can be stored at room temp
Removing seminal plasma delays capacitation - extends life of sperm for transport
To maintain viability for longer than 12 hours - cool to 40-45F over a 2-3 hour period
Ship in container designed to maintian steady temperature
Artificial Insemination
Timing of Insemination
Equine
Chilled semen
Do not expect to remain viable longer than 60-72 hours
Inseminate mares within 24 hours prior to ovulation
1.8mg deslorelin acetate cna be used in mares with a >35mm follicle to induce ovulation
~85% will ovulate w/n 24-48 hours
Sucromate equine is in FDA appreved frug contianing deslorelin and therefore it is not legal to use a compounded deslorelin
Artificial Insemination
Timing of Insemination
Ovine
Ram effect is utilized to aid in synchronization
AI si not commonly done in ewe becuase it is dufficult to detect estrus
AI is not commonly done in the ewe becuase it is difficult to pass a catheter through the cervix
Artificial Insemination
TIming of Insemination
Ovine
Cervical AI - speculum used to visualize the cervical os and fresh or chilled semen is deposited in the cervix
Artificial Insemination
TIming of Insemination
Ovine
Transcervical AI - special forceps are used to pull the cervix into the vagina where another specialized scope is used to guide a catheter through the cervix
Artificial Insemination
TIming of Insemination
Ovine
Laparoscopic AI
surgical endoscopic approach to deposit semen directly into uterus
