ARTIFICIAL INSEMINATION ESTRUS SYNCHRONIZATION EMBRYO TRANSFER

Reproduction – A.I. Artificial Insemination, aka A.I. The deposition of spermatozoa in the female genitalia by artificial rather than by natural means. A.I. reported to be used as early as 3222 BC in horses. A.I. in dogs initially conducted in 1780. A.I. first used commercially in the United States in 1938, primarily in dairy cattle.

Reproduction – A.I. Artificial Insemination, aka A.I. (continued) Freezing bovine semen in liquid nitrogen success in the early 1950’s. All turkeys in the United States are bred by A.I., which started in the 1970’s, due to large breasted ‘toms’. Over 50% of U.S. dairy calves born in the US are from AI. 5 - 10% of U.S. beef cattle, sheep, domestic deer/elk and horses are bred by A.I. today.

Reproduction – A.I. The Principal Advantages of A.I. Are: More rapid genetic improvement through the use of superior sires. Reduction or elimination of cost and risk of maintaining a sire for a small herd or flock; and Reduced risk of spreading certain reproductive diseases.

Reproduction – A.I. Other Advantages of A.I.: Extend the usefulness of outstanding sires in order to maximize genetic improvement. For example, in an A.I. program, bulls can produce 200-700 units (straws) of semen/ejaculate; at 2 to 3 ejaculates (collections) per week. Control Semen Quality

Reproduction – A.I. Other Advantages of A.I. (continued): Thousands of offspring can be produced by a single sire compared to a few hundred from natural service. Offspring can be produced after the sire is dead or disabled. Generally it takes approximately 1.5 units (straws) of semen to produce each A.I.’ed calf born in the United States.

Reproduction – A.I. Semen Collection: Artificial Vagina (aka A.V.) – the training of the male is most crucial for success in utilizing this semen collection method. Electro-Ejaculator – the use of very low voltage delivered to the male via the rectum to bring about ejaculation for semen collection.

Reproduction – A.I. Semen Collection (continued): Vaginal Reclamation – the retrieval of semen from the vagina of the female used for the collection of semen. The male is allowed to breed the female naturally with this process. Straws, stainless steel spoons, pipettes, etc. can be utilized. Manual Massage – this method requires that the handler place his/her arm and hand inside the sire’s colo-rectal area and the massaging of the male’s ampullae, and in turn bring about the release (ejaculation) of the semen.

Reproduction – A.I. The Bovine Artificial Vagina:

Reproduction – A.I. Artificial Vaginas for Domestic Farm Animals:

Reproduction – A.I. Semen Collection Methods:

Reproduction – A.I. Collecting Semen via A.V. from Stallion:

Reproduction – A.I. Semen Analysis in the Field via Microscope.

Reproduction – A.I. Counting Chamber and Grid for Determining Sperm Concentration (center grid):

Reproduction – A.I. Semen Analysis:

Reproduction – A.I. Semen Evaluation & Assessment:

Reproduction – A.I. Semen Evaluation Criteria: Color Volume Motility Sperm Concentration (Density) Abnormalities pH

Reproduction – A.I. Cryogenic Semen Storage: In cattle, each unit should contain around 20 million motile normal spermatozoa. (post-thaw) Semen can be used ‘fresh’ (non-frozen), and if properly handled can remain viable from 1 to 2 days from collection. If semen is to be used ‘fresh’ it must be maintained at 400F. Semen (extended & diluted) is frozen in liquid nitrogen Liquid Nitrogen boils at -321°F and freezes solid at -346°F.

Reproduction – A.I. Cryogenic Semen Storage (continued): Stored in glass vials (ampules) or plastic straws. (1/4 or 1/2 ml) Cryogenically stored semen can last for many years. Poultry semen does not stand freezing and thawing well so is generally used within two (2) hours after collection. Extended, cooled boar semen provides good conception rates up to 72 hours.

Reproduction – A.I. Cryogenic Preservation of Semen (freezing in liquid Nitrogen)

Reproduction – A.I. Cryogenic Preservation of Semen (freezing in liquid Nitrogen)

Semen Quality

Reproduction – A.I. Semen Extenders – Diluents: Milk or Egg Yolk Electrolytes Chemical Buffers Antibiotics Glycerol

A.I. REQUIRES: Heat Detection Trained Technician Semen Quality Assurance Animal restraint facilities

Reproduction – A.I. Estrus Detection is Important in an A.I. Program: Standing Heat – female stands still when mounted by male or another female. Estrus – check twice daily, both AM and PM Observation – watch open females for at least 30 minutes to detect standing heat.

Reproduction – A.I. Signs of Estrus: Restlessness (In-heat females seek out males) Cows attempting to mount other cows. Clear mucus discharge from vagina. Sheep and goats are best checked utilizing a sterilized male, equipped with a breast marking harness. Gilts and sows in heat stand rigid with ears erect when hands are placed firmly on their backs. The vulva is usually red and swollen. Presence of boar and the resulting sounds and odors can help detect heat in swine.

Reproduction – A.I. Signs of Estrus (continued): Mares: Tail elevation Contractions of vulva (aka ‘Winking’) Spreading of legs Frequent urination

Reproduction – A.I. Signs of Estrus – Additional Considerations: Proper time of insemination is CRITICAL! Insemination time should be close to ovulation time erring a little early. A cow/heifer detected in estrus in AM, should be inseminated in PM. A cow/heifer detected in estrus in PM, should be inseminated the next morning. Bull sperm viable about 24 hrs in the female repro tract. In cattle, egg viable for 8 hrs post- ovulation.

The Equation of Reproduction A x B x C x D = % pregnancy Where: A=Heat Detection B= Inseminator Efficiency C=Fertility Level of the Cows D= Semen Fertility If any factor is less than 50% the results will be poor!

Good Work Heat Detection 50% Inseminator 95% Cow Fertility 80% Semen Fertility100% Preg Rate = 38%

Trouble Heat Detection 40% Inseminator 75% Cow Fertility 70% Semen Fertility 60% Preg Rate = 12.6%

Reproduction – A.I. A.I. in Cattle:

A.I.

Reproduction – A.I. ARTIFICIAL INSEMINATION OF DOMESTIC ANIMALS WITH FROZEN SEMEN Cattle Sheep Goats Swine Horses Turkeys Volume of inseminate (ml) 0.25-0.5 0.05-0.2 50 20-50 0.025-0.05 Number of motile sperm per inseminate (106) 20 120-150 100-150 5000 1500 100 Time of insemination 8-16 hr after onset of estrus 10-20 hr after onset of estrus 12-36 hr after onset of estrus 15-30 hr after onset of estrus Every 2 days during estrus Weekly Site of semen deposition Uterine Body Cervix Cervix or uterus Uterus Vagina

Heat Detection Aids Tail Chalk Pedometry Various devices to glue to tailhead (Decal, Ink filled, or electronic) Teasers Real-time monitors on front leg or neck. (Quite accurate)

Teaser Males or Androgenized females Males are vasectomized or otherwise altered to be sexually active but unable to ejaculate fertile sperm. Females are high on testosterone. Teasers are used to identify females in estrus (heat) which can be ready for A.I. Teasers have all of the risks and management issues of keeping intact males.

Reproduction – A.I. Estrous Synchronization: Is controlling or manipulating the ‘estrous’ cycle so that females express ‘estrus’ at approximately the same time. Is a useful part of an A.I. program, because heat checking and breeding animals under extensive management conditions is time-consuming and expensive. Is also an important and essential tool in ‘Embryo Transfer’ (aka E.T.) programs.

Reproduction – A.I. Estrous Synchronization (cattle): Endocrine and/or hormonal control is a must! Tools: 1. Prostaglandin or Prostaglandin F2α (PGF 2α) – a naturally occurring fatty acid that has a marked effect on reproductive systems. Prostaglandin destroys the CL, or source of progesterone(P4). Without P4 follicles are allowed to grow and ovulate. 2. MGA and Prostaglandin – MGA (melengestrol acetate) a feed additive that suppresses ‘estrus’ in heifers and is widely used in the feedlot industry.

Reproduction – A.I. Estrous Synchronization: CIDR Tools: 3. Ov-Synch – A combination of GnRH and prostaglandin. 4. CIDR® - (Controlled Internal Drug Release) Progesterone vaginal insert for estrous synchronization. CIDR

Synch Programs (Cattle) One Injection System (prostaglandin) Two Injection System (prostaglandin) MGA and Prostaglandin Pre-Synch GnRH- PG Ov-Synch / Co-synch MGA-GnRH-PG 7-11 / 6G6 CIDR Synch

CIDR Synch

Embryo Production and Transfer (Cattle) Purpose is to increase number of offspring from a “superior” female, results are very inconsistent -Ave. 5-6 transferrable embryos per donor -Ave. CR 60-70% fresh embryos -Ave. CR 50-60 % frozen embryos -Problem breeders may = problem donors

ET Standing Heat Protocol (Cow) Donors-FSH start 8-12 days following heat FSH injections am and pm for four days Day 4 donors are given PGF2a am and pm Recipients-PGF2a pm day 3 Recips and donors-heat pm day 5/ am day 6 Recipient heats should be recorded Donors-breed 3 X in 24 hours Donors-GnRH injection at first breeding Flush 7 days following insemination

ET CIDR Protocol (Cow) Donors can start at any time Donors receive an injection of estradiol when CIDR is implanted FSH injections start on day 4 after CIDR implantation Pull CIDRs from recips and donors at the last FSH injection Follow same instructions as above for prostaglandin and breeding

Transferring embryos (Cattle) Recips-healthy and proper BCS Recips-in heat within 24 hrs of donors Recips-palpate to ensure reproductive tract is in good condition & a 7-day CL on an ovary Embryo is placed midway up uterine horn on the side of the CL Frozen embryos can be stored in liquid Nitrogen indefinitely? Two methods of freezing/thawing (Follow Directions Exactly)

Equation of Success of Embryo Transfer Similar to Equation of Reproduction, but add several more steps to the equation including: injection accuracy, recip/donor timing, donor response to injections, ability of semen to fertilize multiple eggs, ET technician ability to recover, grade, and implant or freeze embryos

Follicular Aspiration/In-Vitro Fertilization Use FSH to cause multiple follicles to grow. Successful use of sexed semen (pre-freeze or post-thaw) Cattle average 18 aspirated oocytes resulting in 5 transferrable embryos (day 7) Donors can be pregnant (40 to 100 days) 45-50% Preg rates on fresh embryos

Embryo Options Fresh-ship Freeze Buy a pregnant Recip (60 days) Buy a live calf (30 days old)

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