BackgroundWhen prepubertal lambs are superovulated, the ovarian response to gonadotropin stimulation has great individual difference and the collected oocytes have lower developmental ability than that of adult ewes. Over the years, growth hormone (GH) has been used in assisted reproduction because it can improve the reproductive performance in humans and animals. However, the effect of GH on ovaries and oocytes of prepubertal lambs remains unclear.MethodsBefore and during follicle-stimulating hormone (FSH) superovulation of prepubertal lambs (4‒6-week-old), the lambs were treated with high (50 mg) or low dose (25 mg) of ovine GH in a long (5 days) or short (2 days) period. The recovered oocytes were used for in vitro maturation and fertilization, and several parameters of oocyte quality and development capacity were evaluated. The possible underlying mechanisms of GH action were explored by analysis of granulosa cell (GC) transcriptome, ovarian proteome and follicular fluid metabolome.ResultsTreatment of lambs with 50 mg GH over 5 days (long treatment) potentially promoted the response of lambs to superovulation and improved the development capacity of retrieved oocytes, consequently increasing the high quality embryo yield from lambs. A number of differently expressed genes or proteins were found in ovaries between GH-treated and untreated lambs. Cellular experiments revealed that GH reduced the oxidative stress of GCs and promoted the GC proliferation probably through activation of the PI3K/Akt signaling pathway. Finally, analysis of follicular fluid metabolome indicated that GH treatment altered the abundance of many metabolites in follicular fluid, such as antioxidants and fatty acids.ConclusionsGH treatment has a beneficial role on function of lamb ovaries, which supports the development of follicles and oocytes and improves the efficiency of embryo production from prepubertal lambs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13048-023-01209-y.