Development and in Vitro Evaluation of Ferti-Max for Extended Liquid Storage of Broiler Breeder Rooster Semen

This study describes the formulation and in vitro evaluation of FERTI-MAX, a proprietary extender developed through iterative, osmolality-guided optimization to approximate the osmotic and pH environment of chicken seminal plasma. Successive prototypes were narrowed from an initial hypertonic range of approximately 520-750 mOsm/kg to a final formulation at pH ~7.2 and osmolality 342-362 mOsm/kg, incorporating D-fructose, TES buffer, PVP 30, L-glutamic acid, D-serine, L-ascorbic acid, and antibacterial and antifungal agents. Pooled broiler breeder rooster semen diluted with FERTI-MAX at 1:1, 1:2, and 1:4 was stored at 4°C and assessed daily for 16 days. Motility remained at or above 85% across all three dilutions through Day 7 and at or above 80% through Day 8, then declined in a dilution-dependent manner, with the 1:1 dilution retaining ≥40% motility through Day 14. FERTI-MAX therefore supports a high-motility storage window of approximately one week at 4°C. Fertility and hatchability were not assessed. 1. INTRODUCTION Artificial insemination (AI) is a cornerstone reproductive technology in commercial broiler breeder operations, improving genetic dissemination and overcoming mating incompatibilities between sire and dam lines of divergent body size [1]. Because fresh avian semen loses fertilizing capacity rapidly outside the reproductive tract, extenders are used to stabilize the osmolality, pH, and energy supply that spermatozoa require during storage [7]. Osmolality is among the most critical of these determinants, chicken seminal plasma lies close to 325 mOsm/kg, and extenders formulated near this value preserve motility and membrane integrity during cold storage considerably better than hypertonic or hypotonic media [2]. Oxidative stress and bacterial contamination further shorten the usable life of extended semen [3][4]. Established extenders such as Lake's diluent, EK, IGGKPh, and BPSE, validated largely in layer or indigenous lines, typically support motility for 24-72 hours of cold storage [5][6][8]. This study reports the osmolality-guided development of FERTI-MAX and its in vitro evaluation for preserving broiler breeder rooster sperm motility during extended cold storage. 2.

Materials And Methods

2.1 FORMULATION FERTI-MAX was developed between March and August 2025 at RR Animal Health Care Ltd. (Hyderabad, India). Successive candidate formulations were prepared in autoclaved deionized water, adjusted to pH 6.9-7.2 with orthophosphoric acid, and their osmolality measured by freezing-point osmometry (Knauer K-7400S; VCult Life Sciences, Bangalore, India). The composition of the final formulation is summarized in Table 1; concentrations of the core components are not disclosed for proprietary reasons. Table 1. Functional composition of the final FERTI-MAX formulation. Component Category / function Concentration D-Fructose Energy substrate for sperm metabolism Proprietary TES buffer (with physiological salts) pH buffering Proprietary PVP 30 (polyvinylpyrrolidone) Osmoprotective, non-permeating polymer Proprietary L-Glutamic acid; D-Serine Amino acids; osmotic/metabolic support Proprietary L-Ascorbic acid (vitamin C) Antioxidant Proprietary Gentamicin; Chlortetracycline Antibacterial 200 mg/L; 40 mg/L Fluconazole; Amphotericin B Antifungal 25 mg/L; 0.5 mg/L 2.2 EXPERIMENTAL

Design

Semen was collected from broiler breeder roosters by abdominal massage, pooled across several roosters, and processed within minutes. Early prototype formulations were screened at 34°C (15-120 min) and 4°C (6-84 h) across dilutions of 1:1 to 1:8. For final validation, semen was diluted with the final FERTI-MAX formulation at 1:1, 1:2, and 1:4 (semen:extender, v/v), stored at 4°C, and assessed daily from Day 0 to Day 16. Refrigerated samples were re-equilibrated for 2 min at 37°C before each assessment. 2.3 MOTILITY ASSESSMENT A 10 µL drop of semen was placed on a pre-warmed glass slide and the percentage of progressively motile spermatozoa was estimated subjectively by a trained analyst under brightfield microscopy (trinocular compound microscope, 40×; Supplementary Figure S1). Data from pooled, unreplicated samples are presented descriptively, without inferential statistical testing. 3.

Results

3.1 OSMOLALITY Across successive iterations, osmolality was reduced from approximately 520-750 mOsm/kg toward the physiological value of chicken seminal plasma (~325 mOsm/kg), converging to 342-362 mOsm/kg at pH ~7.2 (Figure 1). The final value was independently confirmed by freezing-point osmometry at 362 mOsm/kg. 3.2 PROTOTYPE SCREENING With an early prototype at 34°C, the 1:1 dilution sustained the highest motility over 120 min (85-100%), outperforming neat semen (85% to 60% within 60 min), whereas the 1:4 and 1:8 dilutions remained low (30-50%). At 4°C, the same prototype maintained the 1:2 dilution at 90% for 84 h, while the 1:4 and 1:8 dilutions declined to 40% and 15%, respectively (Supplementary Tables S1 and S2). Figure 1. Osmolality of successive FERTI-MAX prototypes converging toward the physiological target range (shaded band). 3.3 FINAL VALIDATION With the final formulation, motility remained at or above 85% across all three dilutions through Day 7 at 4°C and at or above 80% through Day 8 (Figure 2; Supplementary Table S3). Motility then declined in a dilution-dependent manner: the 1:2 and 1:4 dilutions fell to 40% and 20% on Day 9 and into single digits by Day 12, whereas the 1:1 dilution declined gradually, holding at 50% on Day 11 and 40% through Days 12-14 before reaching 10% on Day 16. 4.

Discussion

The final osmolality of 342-362 mOsm/kg sits modestly above the ~325 mOsm/kg of chicken seminal plasma but within the range described as functional for established poultry extenders [2][5]. Its composition targets the main causes of motility loss: D-fructose supplies a glycolysable substrate for ATP-dependent motility [9], PVP 30 supports membrane integrity [10], as polymers such as poloxamer 188 protect rooster sperm during processing [11], ascorbic acid counters oxidative stress [4], and the antimicrobial system limits contamination [3]. Figure 2. Motility of FERTI-MAX-extended semen during 16-day storage at 4°C; shading marks the ≥85% window common to all dilutions. This one-week window at 4°C compares favourably with the 24-72 hours typical of widely used poultry extenders [5][7], although the comparison is indirect. The divergence after Day 8 indicates a practical trade-off: higher dilutions yield more insemination doses per ejaculate but narrow the storage window. Under the company's field protocol (1:2 dilution, 60-100 µL per hen), FERTI-MAX-extended semen should therefore be used within roughly one week of dilution. 5.

Conclusion

FERTI-MAX (pH ~7.2; 342-362 mOsm/kg) maintained broiler breeder rooster sperm motility at or above 85% for seven days at 4°C across 1:1 to 1:4 dilutions, with the 1:1 preparation retaining usable motility longest. These descriptive, single-sample findings are preliminary; replicated AI trials with fertility and hatchability endpoints, computer-assisted motility assessment, and a direct comparison against an established commercial extender are recommended. REFERENCES 1. Getachew, T. (2016) 'A review article of artificial insemination in poultry', World's Veterinary Journal, 6(1), pp. 25-33. 2. Woelders, H., de Wit, A.A.C., Engel, B., Hulsegge, B., Grasseau, I., Blesbois, E., Bernal, B. and Santiago-Moreno, J. (2022) 'Freezing chicken semen: influence of base medium osmolality, cryoprotectants, cryoprotectant concentration, and cooling rate on post-thaw sperm survival', Cryobiology, 108, pp. 67-77. https://doi.org/10.1016/j.cryobiol.2022.06.003 3. Tvrdá, E., Petrovičová, M., Ďuračka, M., Benko, F., Slanina, T., Galovičová, L. and Kačániová, M. (2023) 'Short-term storage of rooster ejaculates: sperm quality and bacterial profile differences in selected commercial extenders', Antibiotics, 12(8), 1284. https://doi.org/10.3390/antibiotics12081284 4. Díaz Ruiz, E., Navas González, F.J., León Jurado, J.M., Arando Arbulu, A., Delgado Bermejo, J.V. and González Ariza, A. (2024) 'Effects of supplementation of different antioxidants to cryopreservation extender on the post-thaw quality of rooster semen - a meta-analysis', Animals, 14(20), 2936. https://doi.org/10.3390/ani14202936 5. Buathalad, K., Koedkanmark, T., Boonkum, W. and Chankitisakul, V. (2025) 'Optimizing rooster semen preservation: effect of oxygen exposure, sample rotation, and HEPES buffer supplementation', Animals, 15(16), 2391. https://doi.org/10.3390/ani15162391 6. Getachew, T. (2023) Semen quality, fertility and hatchability of in vitro liquid and cryopreserved semen using commercial and homemade extenders from indigenous Horro chicken breed. MSc thesis. Addis Ababa University. 7. ICAR-Central Avian Research Institute (no date) ICAR-CARI poultry semen diluent for 24-48 hours storage of sperm. Available at: https://icar.org.in. 8. Balogun, A.S., Akinosun, A.A., Lawal, I.A., Hassan, U.O. and Hammed, B.B. (2024) 'Optimizing the liquid storage preservation ability of natural formulated poultry semen extender for turkey breeding', Proceedings of the 49th Annual Conference of the Nigerian Society for Animal Production. 9. Slanina, T. et al. (2013) 'Fructose supplementation of turkey semen extenders: effects on sperm motility', Journal of Microbiology, Biotechnology and Food Sciences, 2(Special Issue 1), pp. 1373-1385. 10. Rakha, B.A., Ansari, M.S., Akhter, S., Zafar, Z., Hussain, I., Santiago-Moreno, J. and Blesbois, E. (2017) 'Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen with polyvinylpyrrolidone', Cryobiology, 78, pp. 27-33. https://doi.org/10.1016/j.cryobiol.2017.07.006 11. Mehdipour, M., Daghigh Kia, H. and Martín