Thyroxine and Nano-Melatonin Team Up to Boost Frozen Bull Sperm Quality and Fertility

Freezing bull semen is one of the cornerstones of modern cattle breeding, allowing prized genetics to be shipped across continents and used for decades after a bull’s death. Yet the process comes at a cost. Ice formation, osmotic shock and a surge of reactive oxygen species during cryopreservation batter sperm cells, damaging their membranes, their DNA and the mitochondria that power their journey to the egg. A new study published in Veterinary Medicine and Science suggests that a surprisingly simple two-hormone cocktail, thyroxine combined with a nanoscale form of melatonin, can dramatically blunt that damage and even raise pregnancy rates on the farm.

The research, conducted on Simmental bulls at an Egyptian livestock training centre, is the first to test this particular pairing in cryopreserved bull semen. Thyroxine, the thyroid hormone better known for regulating metabolism, has previously been shown to kick sperm into a state of hyperactive flagellar motion and to improve the recovery of morphologically normal cells. Melatonin, the pineal gland hormone that governs sleep cycles, is a potent antioxidant that scavenges free radicals and bolsters the cell’s own enzymatic defences. By shrinking melatonin down to nanoparticles roughly 22 nanometres across, the team aimed to squeeze far more protective power out of far smaller doses.

The experimental design was thorough. Over eight weeks, the researchers collected 96 ejaculates from six healthy, fertile Simmental bulls aged three to four years, twice weekly using an artificial vagina warmed to 40 to 42 degrees Celsius. Only samples meeting strict quality thresholds, at least 75 percent progressive motility, a volume of five millilitres or more, fewer than 10 percent abnormal sperm and a concentration of at least one billion sperm per millilitre, were accepted for freezing. Ejaculates from all six bulls were pooled on each collection day to smooth out individual variation, and each pool was treated as a single experimental replicate.

The melatonin nanoparticles themselves were synthesised from pharmaceutical-grade melatonin dissolved in double-distilled water, stirred, ultrasonicated, dried at 45 degrees Celsius and ground for ten hours into a fine homogeneous powder. X-ray diffraction confirmed the crystal structure, field-emission scanning electron microscopy revealed spherical particles with a non-smooth surface, and energy-dispersive X-ray analysis verified purity. The average grain diameter fell from 72 nanometres for bulk melatonin to 22 nanometres for the nano-formulation, and a zeta potential of minus 39.5 millivolts indicated good electrostatic stability in solution, a key factor in keeping nanoparticles from clumping together.

Semen was diluted into a standard Tris-egg yolk-glycerol extender and split into ten treatment groups: an untreated control, three thyroxine concentrations (0.5, 0.75 and 1.0 micrograms per decilitre), three nano-melatonin concentrations (30, 45 and 60 micrograms per decilitre) and three combination groups pairing the corresponding doses. The samples were cooled to 4 degrees Celsius over four hours, loaded into 0.25-millilitre straws and frozen in a programmable biofreezer, plunging at minus 30 degrees per minute down to minus 15 degrees and then minus 50 degrees per minute to minus 140 degrees, before storage in liquid nitrogen. Thawing took just 30 seconds in a 37-degree water bath.

When the thawed sperm were examined with a computer-assisted sperm analysis system tracking roughly 1,500 cells per sample at 60 frames per second, one combination stood out clearly. The pairing of 0.5 micrograms per decilitre thyroxine with 30 micrograms per decilitre nano-melatonin, dubbed T0.5 plus NM30, delivered the highest total motility at 58.31 percent and progressive motility at 52.38 percent, against 41.23 and 36.54 percent respectively in untreated controls. Sperm in this group also swam faster and straighter, posting top values for curvilinear velocity, straight-line velocity, straightness, linearity and beat cross frequency.

Functional tests told the same story. The winning combination produced the best viability at 60.69 percent, acrosome integrity at 75.69 percent, plasma membrane integrity measured by the hypo-osmotic swelling test at 57.23 percent, and mitochondrial membrane potential assessed with JC-10 flow cytometry at 58.77 percent. DNA integrity, evaluated with acridine orange staining, reached 92 percent, the highest of any group. Notably, higher thyroxine doses of 0.75 and 1.0 micrograms per decilitre performed worse than the lowest dose, echoing earlier reports that excessive thyroxine can inhibit cytochrome c oxidase and impair mitochondrial function.

The biochemical data revealed why the combination works. In the T0.5 plus NM30 group, glutathione peroxidase activity climbed to 21.31 nanomoles per minute per millilitre, superoxide dismutase reached 42.08 units per millilitre and total antioxidant capacity hit 2.25 millimolar, all significantly above controls, while malondialdehyde, the classic marker of lipid peroxidation, dropped to 0.61 micromoles per millilitre from 1.29 in controls. Annexin V and propidium iodide flow cytometry showed the combination also shifted cells away from death: viable non-apoptotic sperm rose to 63 percent, while late apoptotic and necrotic fractions fell to 18.46 and 8.39 percent respectively. The authors attribute this to complementary mechanisms, with thyroxine binding receptors outside the nucleus to trigger cyclic AMP and calcium release that fuels motility, and nano-melatonin neutralising reactive species, blocking the mitochondrial permeability transition pore and suppressing caspase-3 activation through pathways including SIRT-1/FOXO-1 and Nrf-2/HO-1.

The most striking result, however, came from the field. Because the lowest-dose combination had performed best in the laboratory, the team reserved it for a live fertility trial. One hundred and forty healthy cyclic cows were synchronised with a GPG hormone protocol and inseminated in August 2025 with frozen-thawed semen from four of the treatment groups, 35 cows each, with duplicate inseminations morning and evening. Pregnancy was confirmed by ultrasound on day 45. Cows receiving the combination semen achieved a conception rate of 82.86 percent, compared with 77.14 percent for nano-melatonin alone, 57.14 percent for thyroxine alone and just 48.57 percent for the control extender, a difference the authors describe as statistically significant.

The implications for the global artificial insemination industry could be considerable, since even modest gains in conception rates translate into substantial economic returns for dairy and beef producers. The nano-formulation is also appealing because it achieves strong effects at doses far below conventional melatonin supplements, which earlier studies have applied at up to 0.7 milligrams per millilitre. The authors caution that their study has limitations, including the use of pooled semen, the absence of molecular validation of the signalling pathways involved and relatively limited nanoparticle characterisation, and they call for future work to unpick the mechanisms underlying the synergy. Even so, the message is clear: a dash of thyroid hormone and a pinch of sleep-hormone nanoparticles may be exactly what frozen bull sperm needs to survive the deep freeze and deliver in the field.

Subject of Research: Effects of thyroxine and nano-melatonin supplementation on cryopreserved Simmental bull semen quality and fertility

Article Title: Synergistic Effects of Thyroxine and Nano‐Melatonin on Post‐Thaw Sperm Quality, Antioxidant Defence and Fertility of Cryopreserved Simmental Bull Semen

Article References: Abonagy, M., Arisha, A., Salama, M., Ashour, M., Shukry, M., Shedeed, S., Shehabeldin, A., El‐Shafai, N., Ibrahim, Z., Mahmoud, S., Saad, M., Alameen, A., Elkhidr, R. Y., Alif, O. H., Khalphallah, A., Elmeligy, E., Khesruf, K. A., Abdelhafez, E. A., & Algabary, A. (2026). Synergistic Effects of Thyroxine and Nano‐Melatonin on Post‐Thaw Sperm Quality, Antioxidant Defence and Fertility of Cryopreserved Simmental Bull Semen. Veterinary Medicine and Science, 12(6), Article e71252. https://doi.org/10.1002/vms3.71252

Image Credits: AI Generated

DOI: 10.1002/vms3.71252

Keywords: cryopreservation, bull semen, thyroxine, nano-melatonin, antioxidant defence, oxidative stress, sperm motility, mitochondrial membrane potential, apoptosis, artificial insemination, Simmental cattle, fertility