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Basics ⬇

Outline

BASICS

Definition!!navigator!!

  • Intrauterine placement of extended fresh, cooled, frozen semen; aseptic technique
  • Standard AI—min. 300–1000 × 106 PMS, fresh/cooled transported semen; 200–250 × 106 PMS, frozen semen; deposited into uterine body
  • DHI for standard or lower doses—1–25 × 106 PMS; low volume deposited into tip of uterine horn (ipsilateral to DF)
  • HI for LDI—1–3 × 106 PMS to tip of uterine horn (ipsilateral to DF)

Pathophysiology!!navigator!!

Advantages

  • Prevent stallion overuse
  • Efficient use of ejaculate—breed more mares per season (AI, 120+; live cover, 40–80)
  • Wider stud use—older with problems: musculoskeletal, behavioral
  • Ship semen across all borders—eliminate mare and foal transport, ↓stallion injuries; ↓trauma to mares (recent genital surgery, genital abnormalities, rejecting stallion's advance)
  • Extender antibiotics prevent many infections/transmission of VD
  • Assess semen before AI
  • LDI—stallion with limited availability/showing, large book/short supply, subfertile/poor quality, or costly (sex-sorted)
    • Divide frozen AI dose to produce more than one foal
    • Stallion is dead or epididymal spermatozoa collected at the time of castration or stallion's death

Systems Affected!!navigator!!

Reproductive

Signalment!!navigator!!

  • Thoroughbreds—only live cover
  • Other breed registries allow AI

Signs!!navigator!!

Historical Findings

Records of prior cycles/pregnancies—predict days in heat, time of ovulation, selection of stallion, and semen to be used.

Teasing and Physical Examination Findings

  • Ovulation timing is critical
    • Predict by history, teasing record, previous TRP/US
    • During estrus—tease daily; min. every other day
    • On second day of estrus, daily or every other day TRP/US
    • Perform US, as needed, to determine optimal time to breed
  • TRP—DF (35 + mm), ↓uterine and cervical tone
  • Follicle size/growth rate, edema of endometrial folds (peaks preovulation 72–96 h, decreased/absent 24–48 h in young, normal mares)
  • DF often pear-shaped 12–24 h preovulation
    • Low uterine edema plus DF (≥40 mm)—indicates ovulation is close
  • Ovulation depression, corpus haemorrhagicum, corpus luteum—evidence of ovulation

Diagnosis ⬆ ⬇

Outline

DIAGNOSIS

Procedural Issues!!navigator!!

Timing and Frequency of Breeding

  • Semen longevity varies by stallion, preservation method
  • Equine ova—short viability (8–18 h post ovulation)

Teasing and Examinations

  • AI as close to ovulation as possible
  • OIA—GnRH analog, hCG, or combination when DF is >35 mm induces ovulation
  • US 4–6 h post AI for DUC (especially if using frozen semen) and ovulation
  • Evaluate normal, fertile mares 24–48 h after AI for ovulation
  • Oxytocin IM/IV 4–6 h post AI. Can be given by trained personnel to ensure uterine clearance

Fresh (Raw or Extended) Semen

  • Routine breeding—every other day if not using OIA, stallion has a small book, or mare is normal
    • Begin day 2 or 3 of estrus until mare teases out or when a DF follicle is detected by TRP/US
    • Can use OIA
  • Inseminate within 48 h preovulation (acceptable pregnancy rates)

Cooled Transported Semen

  • More mare examinations; fertility of some cooled semen decreases markedly >24 h
  • OIA when DF is ≥35 mm to induce ovulation; order semen
  • Semen arrives 24 h after ovulatory drug administration and 12–18 h before expected ovulation
  • AI ≤ 12–24 h preovulation, min. dose of 500 × 106 PMS for acceptable conception rates
  • Semen with poor post-cooling fertility should be sent counter to counter (i.e. airline transport)
    • Use only one AI dose if good quality semen; mare is predisposed to DUC/PMIE
    • No advantage to keeping second AI dose to rebreed next day in normal mares; uterus is best incubator for sperm

Frozen Thawed Semen

  • Precise timing of AI; post-thaw longevity is reduced to ≤12–24 h
  • Mare management—serial, daily teasing, TRP/US
  • 2 different protocols for AI depending if multiple doses or a single dose of semen; fertile mare or predisposed to DUC
  • Timed AI:
    • OIA when DF ≥ 35 mm
    • AI at 24 h; repeat at 40 h after OIA; ensure viable sperm available during ovulatory period
    • Treat mare if intrauterine fluid 4–6 h after AI
  • Alternatively:
    • OIA when DF ≥ 35 mm
    • TRP/US—TID–QID, ensure AI very close to preovulation, or, most important, ≤6–8 h post ovulation
    • Treat mare if intrauterine fluid 4–6 h after AI
  • Pregnancy rate for timed AI (2 doses) is equal to a one-time AI 6–8 h post ovulation, less intensive labor, fewer mare examinations; trade-off is increased endometrial irritation
  • Combined methods for AI—timed but using one dose
    • OIA when DF ≥ 35 mm at 8–10 PM (preferably combination of OIA and when follicle <40 mm to have more control over induction)
    • TRP/US only once or twice the next day and again at 8 AM, 34–36 h post induction to assure the presence of a DF
    • TRP/US 6 h later, 40–42 h post induction; >90% of mares ovulate within this window
    • If mare has ovulated, proceed to AI; if not, keep performing TRP/US every 6 h until ovulation
    • This method assures AI within 2–6 h of ovulation, limiting the waste of an AI dose if ovulation does not occur
    • Treat mare if DUC 4–6 h after AI

Low-Dose Insemination

  • Allows use of a reduced dose of semen (fresh, cooled, frozen)
    • Acceptable pregnancy with doses as low as 25 × 106 PMS in volumes of 20–1000 µL (1 mL)
    • Deposit semen at UTJ, ipsilateral to DF
  • Can be either HI or DHI
    • Similar pregnancy rates when using >5 × 106 PMS
    • HI may provide advantage when AI dose is 1–3 × 106 PMS
    • DHI is inexpensive; requires less personnel and skills
  • Mare management varies according to method of semen preservation and severity of her endometrial inflammatory reaction

General Comments

  • If ovulation has not occurred within recommended times for fresh (48 h), cooled (24 h), or frozen (6–12 h) semen, rebreed
    • Consider it may be an anovulatory follicle if mare is unresponsive to the OIA
  • Older ova or semen, poor timing, percent EED increases

Laboratory Tests!!navigator!!

Progesterone level of >1 ng/mL confirms ovulation.

Imaging!!navigator!!

US

Other Diagnostic Procedures!!navigator!!

Semen Analysis

  • Min. parameters—volume, motility, concentration
  • Morphology—optional, of particular use if stallion has fertility problems
  • Small sample of cooled/frozen semen should be saved and warmed (37°C); evaluate immediately post AI
    • For DHI with frozen semen—after thawing straws, remove the sealed end, place one drop of semen on a slide for evaluation pre-AI
    • Pipet and stylet—completely empty straws unless purposely saving 10% of the last straw
  • Slide, coverslip, pipet—prewarm to avoid cold shock
  • Min. number of sperm—300–1000 × 106 PMS (fresh/chilled); 200–250 × 106 PMS (frozen)

Stallion's Disease Status

Should be negative for equine infectious anemia, equine viral arteritis, contagious equine metritis, VD.

Mare Selection

  • Her fertility matters even more if using frozen semen or its quality is poor
  • Reproductive history ± normal estrous cycles, uterine cultures/cytology, DUC
  • Fertility is best—normal, pluriparous/maidens; less, older pluriparous; older maiden/barren mare

Prebreeding Uterine Culture and Cytology of Mare

  • All, except young maiden mares—at least one negative uterine culture and cytology prebreeding
    • Avoid disease transmission to the stallion
    • Early identification of possible problems
  • Pregnancy rates lower, EED higher if treated for an infection in same cycle as AI

Treatment ⬆ ⬇

TREATMENT

Prebreeding

  • Presence of ≥2 cm uterine fluid prebreeding, LRS uterine lavage up to 1 h before AI
  • Use 10 IU oxytocin IV post lavage only if performed >4 h prebreeding
  • Should not affect fertility

AI Technique

  • Sterile, disposable equipment. Mare restrained, rectum free of manure, perineal area thoroughly cleansed (mild detergent/antiseptic solution/soap); min. three rinses to remove any residue
  • Sterile sleeve; non-spermicidal lubricant applied to dorsum of gloved hand
    • 50–56 cm AI pipet carried in the gloved hand for fresh and cooled semen, less frequent for frozen semen
    • Index finger first passes through cervical lumen, serves as a guide by which the pipet can readily be advanced to a position no more than 2.5 cm into the uterine body
    • Syringe, non-spermicidal plastic plunger, e.g. Air-Tite, containing the extended semen is attached to the pipet, semen is slowly deposited into uterus; remaining semen in the pipet is rapidly delivered by using a small bolus of air (1–3 mL) in the syringe

Fresh Extended Semen

  • AI immediately after collection
  • Semen mixed with appropriate extender; immediate AI; semen-to-extender ratio (1:1 or 1:2), if small ejaculate volume and high concentration or mare predisposed to PMIE

Cooled Transported Semen

  • Before first shipment, perform longevity test to determine best semen extender
  • Collect, dilute in extender, cool to 4–6°C for 24–48 h. With transport, can be modest decrease of fertilizing capacity (stallion dependent)
  • Semen-to-extender ratio of 1:3 or 1:4 is acceptable; may be as high as 1:15 (dependent on original concentration); semen longevity optimized by extending to 25–50 × 106 spermatozoa/mL
  • Ship minimum 1 × 109 PMS; approx. 50% loss in shipment; so 500 × 106 PMS remain for AI at 24 h

Frozen Thawed Semen

  • Frozen semen packed in 0.5, 2.5, or 5 mL straws; stored in liquid nitrogen
  • A 5 mL straw contains 600–1000 × 106 sperm cells
  • Dependent on post-freeze sperm motility and AI method, 1–4 + straws may be needed
  • A 0.5 mL straw contains 200–800 × 106 sperm cells
    • Thawing protocols vary; reported best paired with a particular freezing method. Seek specific information regarding thawing. In absence of a recommended protocol, 37°C for 30–60 s may be an acceptable alternative
  • Post thaw, semen should be in the mare within 5 min
  • Methods of frozen semen AI:
    • Into uterine body, regular AI pipet
    • DHI—flexible pipet and stylet (0.5 mL straws, LDI flexible pipet, and inner catheter (2.5–5 mL straws))
    • HI (LDI, especially when <1–3 × 106 PMS)
  • Advantages of DHI and HI over uterine body—reduction in sperm transport time; increased number of sperm to colonize the oviduct ipsilateral to the DF (77% vs. 54%)
  • Post-AI uterine treatment, strongly recommended (high concentration of spermatozoa in thawed straw + absence of seminal plasma may induce an acute PMIE)
  • No scientific evidence that reducing spermatozoa number decreases the PMIE

Procedures

  • Sedation of the mare is recommended for HI but is rarely needed for DHI. Procedure should be performed quickly (≤10 min) to avoid inducing uterine trauma
  • DHI—flexible 65–75 cm AI pipet through the cervix, to tip of the uterine horn ipsilateral to DF:
    • Attach 3 mL syringe to the external pipet end (avoid introducing air during the procedure)
    • Hold sterile pipet in a 45° curve before the procedure, the bend helps direct its tip into the desired horn
    • Once through the cervix, remove the AI hand from the vagina, place it in the rectum to transrectally guide the pipet tip rostral and slightly ventral into the uterine horn, toward its tip
    • Manual transrectal elevation of the uterine horn tip may help pipet passage
    • Insert the 0.5 mL straw into the pipet; a flexible steel stylet pushes the straw through the pipet to the nipple at the tip; deposit the semen close to or onto the UTJ. If more than one straw is used, remove the stylet, its bulbous end grabs the straw, the next straw is then introduced
  • A similar pipet, without stylet, with an inner tube, is used for AI of frozen semen from 2.5–5 mL straws. This pipet also used for AI of cooled shipped semen after centrifugation (poor quality, very dilute)
  • HI—introduction of an endoscope into the mare's uterus:
    • Approach and visualize the UTJ/oviductal papilla ipsilateral to DF
    • Pass small catheter loaded with semen through the channel, deposit semen slowly on the UTJ
    • Rapidly remove endoscope
    • No significant irritation if performed in <5 min
    • Volumes >1 mL tend to run down the air-distended uterine horn
  • HI compared with DHI—↑expensive, ↑skill, ↑labor, ↑personnel. The method of choice if AI of 1–3 × 106 PMS

Post Breeding

  • US 4–6 h after AI for DUC
    • If fluid, uterine lavage (sterile saline/LRS), followed by oxytocin 4–6 h after AI
    • Repeat oxytocin at 3–4 h intervals, if fluid persists; again at 12–24 h until inflammation resolves

Medications ⬆ ⬇

Outline

MEDICATIONS

Drug(s) of Choice!!navigator!!

  • OIA most effective if follicle is ≥35 mm; within 36–42 h with:
    • hCG (1500–3000 IU IV/IM; range 36–72 h)
    • GnRH analog—Sucromate (deslorelin acetate 1.8 mg IM)
    • Compounded GnRH analogs (deslorelin 1.5 mg IM, histrelin 1 mg IM)
    • Compounded GnRH analog + hCG combo (deslorelin 1.5–2 mg + hCG 1500–2500 IU IM)
  • Ecbolic drugs—to treat PMIE and DUC
  • Prostaglandins—misoprostol for cervical relaxation

Contraindications!!navigator!!

See chapter Endometritis.

Precautions!!navigator!!

See chapter Endometritis.

Follow-up ⬆ ⬇

Outline

FOLLOW-UP

Patient Monitoring!!navigator!!

  • Begin teasing by 11 days post ovulation
    • Suspect endometritis if a shortened cycle; owing to endogenous prostaglandin release
  • US for pregnancy 14–15 days post ovulation; rule out potential twins vs. lymphatic cyst
  • Follow-up TRP/US—24–30 days (confirm embryo and heartbeat)
  • Serial TRP examinations—45, 60, 90, 120 days gestation

Possible Complications!!navigator!!

  • Artificial vagina preparation, handling, maintenance
  • Semen evaluation at collection—ship adequate AI dose and/or send correct number of semen straws
  • Shipping methods—Equitainer, reusable box cooling containers, vapor tank
    • Cooled shipments—entire breeding program is at mercy of airlines/couriers
  • Operator skills
    • To manipulate and place semen through the cervix, into the uterine lumen, or to the tip of the horn (proper and timely)
    • Handling storage, straw transfer from main tank, water bath, vapor shipper, thawing and evaluation of frozen semen
  • Misidentification of stallions/mares

Miscellaneous ⬆ ⬇

Outline

MISCELLANEOUS

Pregnancy/Fertility/Breeding!!navigator!!

Cooled Semen

Per cycle pregnancy rates almost equal to on-farm fresh semen AI (60–75%) if quality is good after cooling for 24 h at 5–6°C.

Frozen Semen

  • Pregnancy rates per cycle using frozen semen are 5–10% lower than cooled shipped semen for most stallions
  • Spermatozoa are stressed; loss of ≅50% at freezing and thawing
  • First-cycle pregnancy rates 30–45% (range 0–70%); wide range between stallions
    • Requires greatest management. Good quality of semen has positive impact on pregnancy rate
  • Candidate selection for frozen semen breeding
    • Most fertile—young pluriparous mares, young maidens
    • Least fertile—old pluriparous mares, old maidens, barren mares
  • Older eggs or semen due to poor timing—decreased conception rate, increased EED

Synonyms!!navigator!!

Artificial breeding

Abbreviations!!navigator!!

  • AI = artificial insemination
  • DF = dominant follicle
  • DHI = deep horn insemination
  • DUC = delayed uterine clearance
  • EED = early embryonic death
  • GnRH = gonadotropin-releasing hormone
  • hCG = human chorionic gonadotropin
  • HI = hysteroscopic insemination
  • LDI = low-dose AI
  • LRS = lactated Ringer's solution
  • OIA = ovulation induction agent
  • PMIE = post-mating-induced endometritis
  • PMS = progressively motile sperm
  • TRP = transrectal palpation
  • US = ultrasound
  • UTJ = uterotubal junction
  • VD = venereal disease

Author(s) ⬆

Author: Maria E. Cadario

Consulting Editor: Carla L. Carleton