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Veterinary Gynaecology: Comprehensive Notes

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1. MOET (Multiple Ovulation and Embryo Transfer)

Definition

Objectives

• Maximize offspring from genetically elite donors
• Reduce generation interval
• Increase selection intensity on the female side
• Accelerate genetic improvement in livestock (cattle, sheep, goat, buffalo)
• Note: Horses cannot be superovulated; MOET is inapplicable in equines

Step-by-Step MOET Procedure

• Genetically superior female, good body condition score (BCS 3-3.5)
• Disease-free (BVD, IBR, leptospirosis, brucellosis negative)
• Normal reproductive history, no repeat breeding

• Similar size to donor
• Normal cyclicity; synchronized with donor (±12 hours)
• BCS 2.5-3.5

• Both donor and recipients are synchronized using prostaglandin F2α (PGF2α) or progesterone + estradiol protocols (CIDR-based)
• Synchronization ensures recipients are at the correct luteal stage at time of transfer

• FSH (Follicle Stimulating Hormone) administered in twice-daily decreasing doses over 4 days (days 9-12 of cycle, or after CIDR removal)
• Common FSH protocol: 50, 50, 40, 40, 30, 30, 20, 20 mg (total ~320 mg) IM twice daily
• PGF2α given on Day 3 of FSH treatment to lyse CL and induce estrus
• Target: 8-15 ovulations per donor (compared to 1 normally)
• Bos indicus cattle: require lower FSH doses than Bos taurus

• Donor inseminated twice: at estrus and 12 hours later
• Semen from genetically superior bull (fresh, chilled, or frozen-thawed)

• Performed Day 6-8 post-estrus (Day 7 most common) - at morula/early blastocyst stage
• Non-surgical method: Foley catheter (14-16 Fr) inserted through cervix into each uterine horn
• Uterine horns flushed with PBS (phosphate buffered saline) or Dulbecco's PBS, approximately 500 mL total
• Flush recovered in filter (75 µm mesh); searched under stereo microscope (10-40x)

• Fresh (same day) or cryopreserved embryos
• Non-surgically transferred into ipsilateral uterine horn of recipient
• Recipient must be at Day 7 of diestrus (matching donor's cycle day)
• Average pregnancy rate: 50-70% with Grade 1 embryos

Factors Affecting MOET Success

• Donor age and health status
• FSH type and protocol
• Insemination timing
• Technician skill
• Recipient uterine environment
• Season (better response in non-summer months)

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2. IVF (In Vitro Fertilization) in Veterinary Practice

Definition

IVF vs MOET

IVF Procedure

• OPU (Ovum Pick-Up): Transvaginal ultrasound-guided follicular aspiration
• No FSH stimulation needed (though priming with FSH improves oocyte numbers)
• Can be performed in pregnant cows; repeat every 7-14 days
• Alternatively, oocytes from slaughterhouse ovaries (abattoir-derived)

• Oocytes cultured in maturation medium (TCM-199 + hormones + serum) for 22-24 hours at 38.5°C, 5% CO2
• Cumulus-oocyte complexes (COCs) mature from GV to MII stage

• Capacitated spermatozoa (heparin or ionophore capacitation) added to matured oocytes
• Co-incubated for 18-22 hours
• Sperm concentration: 1-2 × 10⁶ spermatozoa/mL

• Fertilized oocytes (presumptive zygotes) stripped of cumulus cells
• Cultured in synthetic oviduct fluid (SOF) or CR1aa medium for 6-7 days
• Blastocyst rate: 30-50% of cleaved embryos

• Fresh blastocysts transferred on Day 7
• Or vitrification/slow freezing for storage

Advantages of IVF over MOET

• No need for superovulation (avoids hormonal stress)
• Can be used in pregnant cows
• More frequent oocyte collection
• Useful in prepubertal, old, or non-cycling animals
• Better for Bos indicus breeds

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3. Pregnancy Diagnosis in Veterinary Animals

A. Methods of Pregnancy Diagnosis

1. Non-Return to Estrus (Indirect Method)

• Simplest presumptive method
• Absence of estrus 18-24 days after mating suggests pregnancy
• Accuracy: ~75% (low - some non-pregnant animals show silent heat)

2. Rectal Palpation (Cattle, Equine)

• Membrane slip: Earliest reliable sign (~30 days) - allantochorion slips between fingers
• Positive fremitus: Palpable vibration in ipsilateral uterine artery - reliable from 4th month onward

3. Ultrasonography (B-mode real-time)

• Most accurate and earliest non-destructive method
• Cattle: Pregnancy detectable at 25-28 days (heartbeat visible at 28 days)
• Canine: Gestational sac at 18-25 days, heartbeat at 25 days, fetal number estimate best at 28-35 days
• Equine: Conceptus vesicle at 10-12 days (transrectal probe)
• Small ruminants: Transabdominal probe, 40-50 days minimum
• Transducer: 5-7.5 MHz for small animals; 3.5-5 MHz for large animals

4. Progesterone Assay

• Blood/milk progesterone measured at Day 21-24 post-mating
• P4 >1 ng/mL (milk) or >2 ng/mL (serum) = suggestive of pregnancy (or diestrus)
• High sensitivity (~95%) but moderate specificity (can be high in non-pregnant diestrus)
• Most useful as a non-pregnancy test: P4 < 1 ng/mL = definitely not pregnant

5. Estrone Sulphate (Fetoplacental Hormone)

• Produced by viable fetoplacental unit
• Cattle: Detectable from Day 100 onward in urine/blood/milk
• More specific than progesterone; confirms fetal viability

6. Pregnancy-Specific Protein B (PSPB) / Pregnancy-Associated Glycoproteins (PAGs)

• Produced by binucleate trophoblast cells (ruminants)
• Cattle PAG ELISA: accurate from Day 26-30
• Accurate for pregnancy detection and fetal viability

7. Vaginal Biopsy (Small Ruminants)

• Caruncular biopsy - proliferative changes confirm pregnancy
• Not routinely used; semi-invasive

8. Ballottement / External Palpation

• Late pregnancy (5th month onward in cattle)
• Fetal bump felt on right paralumbar fossa
• Squeezing abdomen: fetal rebound (ballottement)

Pregnancy Diagnosis in Canine (Bitch) - Special Notes

• Relaxin is produced by the canine placenta - only reliable blood pregnancy marker in the dog (species-specific)
• Fetal number: Radiography at Day 45+ is most accurate for counting puppies

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4. Estrous (Estrus) Cycle in Canine

Overview

• Dogs are monoestrous (one to two cycles per year)
• Average inter-estrous interval: 6-7 months (range 4-12 months)
• First estrus: 6-24 months (small breeds earlier, giant breeds later)
• Basenji and Tibetan Mastiff: only one cycle per year (fall/autumn)

Four Stages of the Canine Estrous Cycle

1. Proestrus

• Duration: Average 9 days (range 3-27 days)
• Hormones: Rising estrogen, low progesterone, FSH/LH beginning to rise
• Signs:
  • Vulval swelling (turgid, edematous)
  • Serosanguineous (blood-tinged) vaginal discharge
  • Male dogs attracted to female but female refuses mating (no lordosis)
  • Frequent licking of vulva
• Vaginal cytology: Increasing cornification; RBCs +++, parabasal and intermediate cells, then superficial cells
• LH: Begins to rise toward end of proestrus

2. Estrus (Standing Heat)

• Duration: Average 9 days (range 4-24 days)
• Hormones: LH surge (marks start of estrus), estrogen drops, progesterone rises
• Ovulation: Occurs 24-72 hours after LH surge (primary oocytes released - require 48-72h maturation before fertilization)
• Fertile period: Days 3-7 of estrus (ovulation + oocyte maturation window)
• Signs:
  • Female stands for mating (lordosis, tail deflection "flagging")
  • Discharge becomes straw-yellow/clear (less blood)
  • Vulva softens slightly
• Vaginal cytology: >80-90% cornified (anuclear superficial) squamous cells - KEY finding
• Progesterone level at ovulation: ~5-10 ng/mL (serum) - used clinically to time breeding

3. Diestrus (Metestrus)

• Duration: ~60-90 days (whether pregnant or not)
• Hormones: Progesterone peaks at 3-4 weeks post-ovulation (15-90 ng/mL), then gradually declines
• Signs:
  • Female refuses mating
  • Vulva returns to normal
  • If pregnant: gestation (63 days from ovulation)
  • If not pregnant: pseudo-pregnancy (false pregnancy) possible due to luteolytic failure
• Vaginal cytology: Abrupt shift - return of parabasal cells, metestrum cells (neutrophils), discharge stops

4. Anestrus

• Duration: Average 4 months (range 2-10 months)
• Hormones: Low estrogen, low progesterone (basal levels)
• Signs: No vulval swelling, no discharge, no male attraction
• Purpose: Uterine involution and preparation for next cycle
• Vaginal cytology: Parabasal cells predominate, inactive smear

Summary Table: Canine Estrous Cycle

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5. Estrus Synchronization Methods

A. In Cattle

1. Prostaglandin F2α (PGF2α) Protocol

• Luteolytic agent - causes CL regression in mid to late diestrus (Days 6-17)
• Single injection: Administer to cyclic cows; ~65% respond (those with CL)
• Double injection: PGF2α given; repeat after 11-14 days - nearly 100% response
• Dose: 25 mg dinoprost (Lutalyse) or 500 µg cloprostenol IM
• Estrus expected within 48-96 hours

2. Progesterone-Based Protocols

• CIDR (Controlled Internal Drug Release): Intravaginal progesterone device (1.38 g P4); inserted for 7 days
• CIDR + estradiol at insertion + PGF2α at removal
• Estrus: 48-72 hours post-CIDR removal
• Useful in acyclic/anovulatory cows

3. Ovsynch Protocol (GnRH-PGF2α-GnRH)

• Day 0: GnRH 100 µg (ovulation or luteinization of dominant follicle)
• Day 7: PGF2α (lyse CL)
• Day 9: GnRH (induce LH surge and ovulation)
• Day 9.5 (16 h after 2nd GnRH): Timed AI (TAI)
• Pregnancy rate: 25-35% (allows fixed-time AI without estrus detection)

4. Select Synch / Co-Synch

• Modifications of Ovsynch
• Co-Synch: AI done at time of 2nd GnRH (simplifies handling)

B. In Small Ruminants (Sheep/Goats)

1. Progesterone Sponges/CIDRs

• Intravaginal sponge impregnated with MAP (medroxyprogesterone acetate, 60 mg) or FGA (fluorogestone acetate, 45 mg)
• Inserted for 12-14 days, then removed
• PMSG (300-500 IU) IM given at sponge removal to improve ovulation rate
• Estrus: 24-48 hours post-sponge removal

2. Ram/Buck Effect (Biostimulation)

• Introduction of male after prolonged separation during anestrus
• Female estrus induction within 4-7 days
• Particularly effective in seasonally breeding species

3. Melatonin Implants

• Used in seasonal breeders to mimic short-day photoperiod
• Advances the breeding season in sheep and goats

C. In Canine (Bitch) - Estrus Synchronization/Induction

1. GnRH Agonists

• Deslorelin implant (Suprelorin 4.7 mg or 9.4 mg SC) - GnRH agonist
  • Initial stimulatory phase (3-7 days): can induce estrus
  • Prolonged use: causes downregulation - used for contraception
• Buserelin, leuprolide, fertirelin: Also used for estrus induction (pulsatile administration)

2. Dopamine Agonists (to end anestrus)

• Cabergoline: 5 µg/kg/day PO for 7-14 days (reduces prolactin, allows GnRH secretion)
• Bromocriptine: 10-20 µg/kg/day - older agent, more side effects (nausea)
• Useful for abbreviating anestrus and inducing early estrus
• Success rate: ~70-80%

3. Exogenous Gonadotropins

• PMSG (eCG) + hCG protocol:
  • PMSG 20-44 IU/kg IM daily for 9-10 days (FSH-like activity - follicular development)
  • hCG 25-100 IU/kg IM on Day 10 or Day 1 of estrus (LH-like activity - induce ovulation)
  • Estrus induction: 80-100%; pregnancy rate: ~50-60%
• FSH alone: Less commonly used due to short half-life

4. Synthetic Estrogens

• Diethylstilbestrol (DES): Historical; used in some countries
• Can induce proestrus but risk of pyometra and aplastic anemia (now largely abandoned)

D. In Equine

1. PGF2α

• Effective only in mid-late diestrus (Days 5-14)
• Estrus in 2-5 days after CL regression
• Common: 5 mg dinoprost or 250 µg cloprostenol IM

2. Progesterone + Estradiol (P+E)

• 10-14 days of progesterone (suppress follicular activity) + estradiol
• Synchronous estrus after withdrawal

3. hCG or Deslorelin (Ovulation Induction)

• hCG 2500 IU IV or deslorelin SC induces ovulation within 36-48 hours
• Used when dominant follicle ≥35 mm is present

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6. ICD (Induced Corpus Luteum / Induced CL)

Definition

Methods of Inducing a Corpus Luteum

In Cattle

• GnRH (100 µg IM): Triggers LH surge → ovulation of dominant follicle → CL formation within 5-7 days
• hCG (2500-3000 IU IM/IV): Direct LH-like action → ovulation and CL formation
• Used in the Ovsynch protocol (1st GnRH injection) to synchronize follicular wave and create a new CL

In Canine

• hCG (500-1000 IU IM): Induces ovulation of mature follicles → CL formation
• Used therapeutically to:
  • Treat cystic ovaries (follicular cysts that failed to ovulate)
  • Create an ICD for progesterone supplementation
  • As part of estrus induction protocols

In Equine

• hCG 2500 IU IV or IM when dominant follicle ≥35 mm
• Ovulation within 24-48 hours; CL detectable by Day 5-7

Characteristics of Induced CL vs Natural CL

Clinical Significance of ICD

• In Ovsynch: The first GnRH creates an ICD that serves as the target for subsequent PGF2α lysis, allowing timed AI
• Cystic Ovarian Disease (Follicular Cysts): GnRH or hCG given to induce ovulation and CL formation (luteinization) - converting follicular cysts to luteal cysts or normal CL
• Progesterone support: ICD produces progesterone essential for uterine preparation and early embryo development
• Embryo transfer programs: ICD used to synchronize recipients at the correct stage

ICD in Context of Cystic Ovarian Disease (COD) in Cattle

• Follicular cyst: Anovulatory follicle >25 mm persisting >10 days without CL
• Treatment: GnRH 100 µg IM → induces LH surge → luteinization/ovulation → ICD formation
• After ICD formation (~10-14 days), PGF2α given to lyse CL → synchronized estrus
• This is the GnRH-PGF2α protocol for treatment of COD with subsequent breeding

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Quick Reference: Key Hormones in Veterinary Reproduction

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