In the Merino ram, it is unclear whether cryopreserved sperm function and fertility is compromised when collected during the non-breeding season, when Merino ewes are seasonally anestrus. It was therefore investigated whether treatment with melatonin could improve sperm function or fertility when semen was collected during the period Merino ewes were seasonally anestrus. There were 16 Merino rams treated or not treated with melatonin implants during the non-breeding season of ewes (September). Ejaculates were collected before melatonin treatment (Week 0), during the period of melatonin release (Week 7) and subsequent breeding season (Week 23). In vitro sperm function was assessed before freezing, and at 0- and 3 -hs post-thaw. Fertility was determined through intrauterine insemination of ewes (n = 966) with frozen-thawed samples, during the breeding season. Compared to Week 0 values, spermatozoa from melatonin-treated rams had greater progressive motility at Week 7 (P = 0.019) and less DNA fragmentation (P = 0.003) at Weeks 7 and 23, whilst spermatozoa from non-treated rams were unchanged during these time-periods. There were no other treatment effects on sperm function or fertility (P > 0.05). In ejaculates collected during Week 23, there were no effects of treatment either before freezing or post-thawing. Sperm from ejaculates collected at Week 23, however, had lesser pre-freezing/post-thawing total motility and resulted in lower pregnancy rates (P < 0.05). It is concluded there are no effects of season on sperm quality or fertility of Merino rams and that melatonin treatment subtly improves quality of spermatozoa following cryopreservation.Copyright © 2020 Elsevier B.V. All rights reserved.