Blastocyst production by conventional in vitro fertilization (cIVF) in horses: Effects of sperm storage method, incubation timing of cool-stored semen before gamete co-incubation, and comparisons between cIVF and intracytoplasmic sperm injection (ICSI).

This study tested whether a clinic-ready conventional IVF protocol for horses works equally well with fresh, cooled, or frozen semen and whether a shorter sperm preincubation is acceptable, and it compared the protocol’s performance to ICSI. Cleavage rates were similar across conditions, but cIVF yielded fewer blastocysts per cleaved embryo than ICSI with frozen semen, while embryos from both methods established early pregnancies after transfer.

What the researchers wanted to know

• Whether conventional IVF (cIVF) in horses can reliably produce blastocysts using practical sperm storage methods used in clinics (fresh, cool-stored, or frozen/thawed).
• Whether shortening the duration that cooled semen is incubated in capacitating medium (FERT-PHE) before mixing with oocytes affects outcomes.
• How cIVF compares with intracytoplasmic sperm injection (ICSI), the current clinical standard for equine in vitro embryo production.

Why this matters

• ICSI is the prevalent method in equine practice because traditional IVF has historically been unreliable in horses; a recently reported cIVF protocol using FERT-PHE shows promise.
• Demonstrating that cIVF performs well with common semen handling and shorter lab times could make it more accessible and efficient.
• Direct comparison with ICSI clarifies where cIVF currently stands in terms of efficiency and embryo developmental competence.

How the study was designed

• Oocytes: In vitro–matured equine oocytes obtained by transvaginal oocyte aspiration (TVA).
• Sperm capacitation: Tyrode’s Albumin Lactate Pyruvate medium supplemented with penicillamine, hypotaurine, and epinephrine (FERT-PHE).
• Primary outcomes:
  • Cleavage rate at day 5 (embryos with ≥8 blastomeres).
  • Blastocyst rate per cleaved embryo.
  • Blastocysts per mare TVA/cIVF cycle.

Experiment 1: Effect of sperm storage method on cIVF

• Compared fresh, cool-stored, and frozen/thawed semen from the same fertile stallion (OP).
• Cleavage rates:
  • Fresh: 68% (38/56)
  • Cool-stored: 61% (34/56)
  • Frozen/thawed: 54% (29/54)
• Blastocyst rate among cleaved embryos:
  • Fresh: 18% (7/38)
  • Cool-stored: 21% (7/34)
  • Frozen/thawed: 21% (6/29)
• Blastocysts per mare TVA/cIVF cycle: approximately 1 for all three groups.
• Statistical outcome: No significant differences among storage methods (P > 0.05).

Experiment 2: Shortening sperm preincubation in FERT-PHE for cooled semen

• Compared 22 hours versus 10 hours of sperm exposure to FERT-PHE before gamete co-incubation.
• Cleavage rate:
  • 22 h: 45% (29/64)
  • 10 h: 56% (47/84)
• Blastocyst rate among cleaved embryos:
  • 22 h: 28% (8/29)
  • 10 h: 19% (9/47)
• Blastocysts per mare TVA/cIVF cycle:
  • 22 h: 1.1
  • 10 h: 1.3
• Statistical outcome: No significant differences between 22 h and 10 h (P > 0.05).

Experiment 3: cIVF versus ICSI using frozen/thawed semen from two fertile stallions

• Stallions: OP and HW; methods compared within stallions and combined.
• Cleavage rates:
  • cIVF: OP 41% (16/39) vs HW 52% (34/65); no difference (P > 0.05).
  • ICSI: OP 34% (22/64) vs HW 53% (19/36); no difference (P > 0.05).
  • Combined: cIVF 48% (50/104) vs ICSI 41% (41/100); no difference (P > 0.05).
• Blastocyst rate among cleaved embryos:
  • cIVF: OP 13% (2/16) vs HW 26% (9/34); no difference (P > 0.05).
  • ICSI: OP 45% (10/22) vs HW 42% (8/19); no difference (P > 0.05).
  • Combined: cIVF 22% (11/50) vs ICSI 44% (18/41); significantly lower for cIVF (P < 0.05).
• Embryo transfer feasibility and viability:
  • Six vitrified-warmed blastocysts (3 cIVF, 3 ICSI) from stallion HW were transferred.
  • All recipients confirmed pregnant at days 14, 28, and 45 of gestation.

What the results mean

• cIVF performance is robust across common semen storage methods when using a fertile stallion: fresh, cooled, and frozen semen all yielded comparable cleavage and blastocyst outcomes.
• Shortening sperm capacitation in FERT-PHE from 22 h to 10 h with cooled semen did not compromise cleavage or blastocyst production, suggesting laboratories can reduce preparation time without loss of efficiency.
• Although fertilization/early cleavage efficiency was similar between cIVF and ICSI with frozen semen, fewer cIVF embryos progressed from cleavage to blastocyst overall, indicating a post-fertilization developmental gap relative to ICSI.
• Embryos from both cIVF and ICSI were capable of establishing early pregnancies after vitrification-warming and transfer, supporting clinical viability of embryos from either method.

Strengths and limitations

• Strengths:
  • Direct, clinically relevant comparisons of sperm storage methods and incubation durations.
  • Head-to-head evaluation of cIVF versus ICSI using the same frozen semen sources.
  • Demonstration of embryo transfer and early pregnancy establishment for both methods.
• Limitations:
  • Limited number of stallions (one in Experiments 1–2; two in Experiment 3), all fertile; results may not generalize to subfertile sires.
  • Sample sizes moderate; some differences might be underpowered.
  • Development assessed to blastocyst and early pregnancy only; no foaling outcomes reported.
  • Definitions of cleavage at day 5 and culture conditions may influence comparability with other labs.

Clinical and practical implications

• Clinics can consider cIVF with fresh, cooled, or frozen semen without expecting major differences in early outcomes when using a fertile stallion.
• Reducing FERT-PHE preincubation from 22 h to 10 h with cooled semen appears feasible, potentially simplifying scheduling and reducing lab time.
• When using frozen semen, ICSI remains more efficient than cIVF in converting cleaved embryos to blastocysts overall; centers prioritizing maximum blastocyst yield may prefer ICSI.
• Both cIVF- and ICSI-derived embryos can be vitrified and establish early pregnancies post-transfer, supporting their clinical utility.

Open questions and future directions

• Identify why cIVF embryos have lower blastocyst conversion post-cleavage with frozen semen (e.g., capacitation dynamics, sperm selection, oxidative stress, zona interactions, or embryo culture environment).
• Evaluate performance across a broader range of stallions, including subfertile sires and different semen freezing/cooling protocols.
• Optimize cIVF parameters further (capacitation timing windows shorter than 10 h, PHE concentrations, media formulations, oocyte handling) to close the gap with ICSI.
• Assess in vivo outcomes to term, foal health, and long-term performance for cIVF- versus ICSI-derived embryos.
• Compare costs, labor, and sperm use efficiency between cIVF and ICSI in routine clinical practice.

Key numbers at a glance

• cIVF cleavage with fresh/cool/frozen semen: 68% / 61% / 54% (no significant differences).
• cIVF blastocyst rate among cleaved embryos: 18% / 21% / 21% (no significant differences).
• Cooled semen FERT-PHE preincubation 22 h vs 10 h:
  • Cleavage: 45% vs 56% (no significant difference).
  • Blastocyst per cleaved: 28% vs 19% (no significant difference).
• Frozen semen, combined across two stallions:
  • Cleavage: cIVF 48% (50/104) vs ICSI 41% (41/100) (no significant difference).
  • Blastocyst per cleaved: cIVF 22% (11/50) vs ICSI 44% (18/41) (significantly lower for cIVF).
• Embryo transfer: 3 cIVF + 3 ICSI vitrified-warmed blastocysts → 6/6 pregnancies at days 14, 28, and 45.