Intravenous clomiphene citrate formulation for enhancing equine follicular development and oocyte recovery

Background

Assisted reproductive technologies in equine medicine have become essential tools for improving genetic dissemination and managing fertility in sport and breeding horses. Intracytoplasmic sperm injection and in vitro embryo production rely on effective follicular development and reliable oocyte retrieval to maximize yield. However, mares present unique physiological challenges, including seasonal anestrus, variable endocrine profiles, and sensitivity to exogenous hormones. As demand grows for consistent outcomes in high-value genetic programs, there is a clear need to optimize protocols that promote uniform follicular growth and enhance oocyte availability without compromising mare health or welfare.

Traditional stimulation regimens typically involve multiple injections of gonadotropins such as eCG or FSH in combination with prostaglandins, often administered via intramuscular or subcutaneous routes. These methods suffer from unpredictable absorption kinetics, fluctuating plasma hormone concentrations, and individual mare variability, resulting in inconsistent follicle recruitment and suboptimal oocyte recovery. Oral treatments face poor bioavailability and variable metabolic clearance, while repeated invasive procedures increase stress and labor costs. Consequently, current protocols frequently yield low embryo production rates and necessitate extensive monitoring and intervention, underscoring the need for more reliable and efficient approaches.

Technology

This approach employs a stable intravenous formulation of clomiphene citrate complexed with hydroxypropyl-β-cyclodextrin, yielding a clear solution at pH 4.0–4.4 and osmolality of 270–350 mOsm that retains 90–105% assay integrity over six months. Mares undergo follicle ablation on day 0, receive 250 µg prostaglandin F2α on day 6, and are treated with 0.5% clomiphene citrate (2 g IV) daily from days 10–14, with oocyte aspiration performed on day 17. Daily ultrasonography and blood sampling track follicular growth, revealing an average of 11.3±1.2 follicles and a dominant follicle diameter of 40±5.1 mm. Hormonal profiles show significant correlations between AMH, LH, FSH, progesterone and estradiol, and pilot data demonstrate a 35% boost in embryo development, with a 56.6% maturation rate and 28.3% blastocyst formation in treated mares.

What sets this method apart is its ability to achieve therapeutic plasma concentrations that oral dosing cannot, thanks to cyclodextrin-enhanced solubility and a validated pharmacokinetic profile. The protocol’s synchronized timing of follicle ablation, prostaglandin administration and IV dosing optimizes follicular wave dynamics, while stability under varied storage conditions ensures reproducibility. Multivariate hormone correlation analyses and a robust biplot capturing over 85% of variability underline its scientific rigor, distinguishing it from existing equine in vitro embryo production techniques.

Benefits

• Improved follicular development with increased follicle count and size
• Enhanced oocyte recovery rates during transvaginal aspiration
• Higher embryo maturation and blastocyst formation rates (up to 35% improvement)
• Stable intravenous CC formulation maintaining 90–105% potency over 6 months
• Optimized IV dosing achieving reliable therapeutic plasma concentrations
• Synchronized ovum pick‐up timing via a controlled ablation and hormone protocol
• Favorable modulation of key reproductive hormones (AMH, FSH, LH, P4, E2)
• Potential to significantly boost in-vitro embryo production efficiency in mares

Applications

• Equine in vitro embryo production
• Commercial equine IVF clinics
• Mares superovulation treatment protocols
• Oocyte harvest veterinary services
• Embryo transfer horse breeding

Additional info

An intravenous formulation of clomiphene citrate complexed with hydroxypropyl-β-cyclodextrin remains stable (90–105% assay, pH 4.0–4.4, 270–350 mOsm) for six months. After follicle ablation and prostaglandin F2α synchronization, mares receive daily 2 g IV doses (0.5% CC) for five days, boosting follicle count (11.3 ± 1.2), modulating AMH, FSH, LH, P4, and E2 levels, and achieving 56.6% oocyte maturation and 28.3% blastocyst rates.