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

The discovery began with a lucky accident of mutagenesis. The researchers, led by Yushan Hou, Xuejiao Kong and Junjie Le of Northeast Agricultural University in Harbin, treated seeds of MK1, a naturally cold-tolerant japonica rice line, with a chemical mutagen that scatters random mutations across the genome. Among the resulting plants, they spotted one seedling that had lost MK1’s prized cold tolerance, wilting under chilling conditions that its parent shrugged off. They named this mutant cs1, for cold-sensitive. Genetic crosses revealed that the vulnerable phenotype was controlled by a single recessive nuclear gene, meaning one broken gene was enough to strip away the tolerance.

To find that gene, the team turned to MutMap, an elegant sequencing strategy that compares the genomes of mutant and parent to locate the causal mutation quickly. The mapping converged on a region of chromosome 9 containing OsERF5, which belongs to the large AP2/ERF family of transcription factors. These proteins bind specific DNA sequences and switch suites of target genes on or off, and in rice they have repeatedly surfaced as players in stress responses to drought, salt, heat and cold. Within OsERF5, the mutant carried a single nonsynonymous SNP in the AP2 domain, the part of the protein that grips DNA. That one-letter change in the genetic code swapped an alanine for a serine, altering the protein’s architecture precisely where it needed to function.

Expression patterns added weight to the suspicion that OsERF5 matters under stress. The gene is broadly active across the plant, but its activity surges in response to cold, salinity, osmotic stress and gibberellin, a plant hormone better known for driving stem elongation and germination. That gibberellin responsiveness turned out to be a crucial clue, hinting that OsERF5 sits at a junction between hormone signalling and the plant’s chemical armour against cold. The researchers then ran the decisive functional tests: knocking OsERF5 out made seedlings more sensitive to chilling, while overexpressing the gene or restoring a functional copy in the mutant enhanced tolerance. The results confirmed that OsERF5 is a genuine positive regulator of cold tolerance at the seedling stage, not a bystander.

How does a single transcription factor protect a seedling from cold? The answer lies in the chemistry of stress. When plants are chilled, their photosynthetic machinery and metabolism leak electrons, generating reactive oxygen species such as hydrogen peroxide. In modest amounts these molecules act as signals, but when they accumulate they oxidise membranes and proteins, causing cellular damage. Malondialdehyde, or MDA, is a classic fingerprint of that membrane damage. The team’s physiological analyses showed that plants with functional OsERF5 accumulated less hydrogen peroxide and less MDA under cold stress, indicating a strengthened antioxidant system that mops up reactive oxygen before it wreaks havoc. In the knockout lines, those protective responses were weakened, and the damage markers climbed.

The gibberellin connection gives this defence system an intriguing twist. Gibberellin is best known as a growth hormone, and breeders have long manipulated it, most famously in the semi-dwarf wheat and rice varieties of the Green Revolution. The new study links gibberellin signalling to reactive oxygen species homeostasis during cold stress, with OsERF5 associated with a GA-mediated antioxidant defence. In practical terms, the hormone does not merely tell the plant to grow; under chilling conditions it appears to help orchestrate the deployment of antioxidant capacity, and OsERF5 is a key intermediary in that command chain. This reframes gibberellin as part of the stress-response toolkit, not just a growth accelerator, and suggests that fine-tuning this pathway could yield seedlings that stay both vigorous and protected when temperatures plunge.

Beyond the lab bench, the study has an evolutionary story to tell. By scanning thousands of rice varieties, the researchers identified twelve haplotypes, distinct versions of the OsERF5 gene sequence carried by different accessions. One of them, Hap_I, stood out as the cold-tolerant version, and it is overwhelmingly predominant in temperate japonica rice, the subspecies grown in cooler regions of Japan, Korea, northern China and beyond. Population genetics analysis revealed that OsERF5 has undergone strong selection during rice domestication, meaning that ancient farmers and natural selection together favoured the cold-tolerant version as rice spread from its tropical origins into harsher climates. The gene thus carries the imprint of a millennia-long breeding programme conducted without anyone knowing the word genetics.

That history makes Hap_I immediately useful for modern breeders. Because the cold-tolerant haplotype already exists in cultivated germplasm, breeders can track it with DNA markers and introgress it into elite varieties that lack it, a strategy known as marker-assisted selection. This is far faster than waiting for plants to grow out under cold conditions, and it avoids dragging along unwanted traits from donor parents. For rice production in high-latitude regions such as northeastern China, where the research team is based, and in high-altitude zones where chilling nights are routine, a validated cold-tolerance haplotype is exactly the kind of resource that turns molecular knowledge into sturdier fields. The authors describe Hap_I as a valuable genetic resource for breeding cold-adapted rice, and the evidence supports that framing.

The study also slots into a rapidly growing catalogue of rice cold-tolerance genes. Earlier work identified COLD1, whose natural variation confers chilling tolerance, and more recent discoveries include COLD11, CTF1, CTB5 and CTB4a, each illuminating a different node of the cold-response network, from ion channels to flowering-stage resilience. OsERF5 adds a transcriptional regulator tied to hormone signalling and antioxidant defence, a layer that had been less well characterised at the seedling stage. Together these genes sketch a picture of cold tolerance as a distributed property, built from sensory machinery, hormonal cross-talk and enzymatic detoxification, any single component of which can be improved by breeding.

For a world where extreme weather increasingly disrupts crop production, the stakes are concrete. Cold damage at the seedling stage forces farmers to delay planting or resow, shrinking the growing season and yields in marginal regions. A gene like OsERF5, validated by knockout, overexpression and complementation, and backed by a clear haplotype signature and a plausible molecular mechanism, gives breeders a precise target rather than a statistical shadow. The work, funded by the National Natural Science Foundation of China and partner programmes, demonstrates how a chemical mutagenesis experiment in a single rice line can end up tracing the evolutionary history of a species and pointing the way to the next generation of cold-proof crops. As chilling temperatures continue to test the limits of the world’s rice paddies, the humble transcription factor on chromosome 9 may prove to be one of the quiet heroes of food security.

Subject of Research: A rice AP2/ERF transcription factor gene, OsERF5, that regulates seedling cold tolerance through gibberellin-mediated antioxidant defence

Article Title: OsERF5 enhances cold tolerance and is associated with GA‑mediated antioxidant defence in rice

Article References: Hou, Y., Kong, X., Le, J., Yang, R., Qi, N., Wang, J., Liu, H., Zheng, H., Xin, W., Zou, D., Lei, L., & Yang, L. (2026). OsERF5 enhances cold tolerance and is associated with GA‑mediated antioxidant defence in rice. Theoretical and Applied Genetics, 139(10), Article 273. https://doi.org/10.1007/s00122-026-05385-5

Image Credits: AI Generated

DOI: 10.1007/s00122-026-05385-5

Keywords: rice, cold tolerance, OsERF5, transcription factor, gibberellin, reactive oxygen species, antioxidant defence, haplotype, japonica rice, plant breeding, abiotic stress, MutMap