Rural sheep farmers experience considerable economic difficulties stemming from multiple challenges, including harsh environmental conditions, limited grazing areas, nutritional deficiencies, and prolonged postpartum anestrus periods (Nel et al., 2023; Ngcobo et al., 2026). In an economically sustainable sheep production system, two lambings per year can be achieved through effective reproductive control. However, to enhance reproductive parameters, it is essential to provide the ewes with an adequate and high-quality nutritional regimen (Aouina et al., 2025). Livestock intensively farmed under modern production systems typically have limited or no access to fresh pasture. Moreover, preserved forage contains low concentrations of polyunsaturated fatty acids; therefore, animal diets are frequently supplemented with fats derived from oilseeds rich in linoleic acid (Wathes et al., 2007).

Nutritional strategies, including dietary lipid supplementation, have been widely investigated as practical approaches to improve reproductive performance in ruminants (Yadav et al., 2019). Lipid supplementation may influence reproductive function through several mechanisms involving the hypothalamus, anterior pituitary, ovary, and uterus (Rahbar et al., 2014). Polyunsaturated fatty acids (PUFAs), especially n-6 and n-3 fatty acids, are essential nutrients because animals cannot synthesize them de novo and must obtain them from the diet (Wathes et al., 2007). Among these, linoleic acid is an essential n-6 PUFA that may be desaturated and elongated to arachidonic acid after escaping ruminal biohydrogenation and being absorbed from the digestive tract. Arachidonic acid is the main precursor for prostaglandin F2α synthesis and may therefore influence uterine prostaglandin secretion and circulating concentrations of 13,14-dihydro-15-keto prostaglandin F2α (PGFM), the major metabolite of prostaglandin F2α (PGF2α) (Funston, 2004; Weems et al., 2006).

The possible reproductive effects of lipid supplementation are not limited to prostaglandin metabolism. Dietary fat may also affect ovarian steroidogenesis by increasing the availability of cholesterol, which is the primary substrate for steroid hormone synthesis, including estradiol (Grummer and Carroll, 1991; Bao et al., 1995). Therefore, oilseed supplementation rich in unsaturated fatty acids may potentially alter endocrine responses related to follicular activity, estradiol secretion, luteal function, and uterine prostaglandin production. Previous studies have reported that dietary sources rich in n-6 fatty acids may influence prostaglandin secretion and PGFM concentrations in ruminants (Petit et al., 2004; Grant et al., 2005; Silvestre et al., 2011). However, the magnitude of these effects may depend on species, physiological status, lipid source, dose, duration of supplementation, and the use of hormonal synchronization protocols.

Safflower (Carthamus tinctorius L.) is an ancient oilseed plant belonging to the Compositae family and has become increasingly important as a drought-resistant crop, particularly under conditions associated with climate change (Gümüş and Küçükersan, 2016). Safflower seeds contain approximately 20 %–40 % oil, and oleic, linoleic, stearic, and palmitic acids constitute 96 %–99 % of safflower oil (Coşge et al., 2007; Knowles, 1982; Sabzalian et al., 2008). Depending on the variety, the proportions of oleic and linoleic acids may vary between 10 %–32 % and 58 %–81 %, respectively (Coşge et al., 2007; Sabzalian et al., 2008). Because of its high linoleic acid content, safflower seed may represent a practical dietary source of n-6 PUFA for ruminants. In this context, safflower supplementation may have the potential to influence prostaglandin metabolism through the linoleic acid–arachidonic acid–PGF2α pathway and steroid hormone synthesis through lipid-related changes in ovarian function.

In addition to its fatty acid profile, safflower contains several polyphenolic compounds, including lignans, flavones, and serotonin derivatives. Lignans and flavones are known to possess phytoestrogenic activity and may interact with estrogen receptors (Hong et al., 2002; Cho et al., 2011). Therefore, these compounds may influence estrogen-dependent reproductive processes, including follicular activity, estrus behavior, reproductive tract function, and circulating estradiol concentrations. Safflower-derived compounds have previously been associated with estrogen-related biological effects, particularly in models of estrogen deficiency and bone metabolism (Cho et al., 2011; Yuk et al., 2002). Moreover, safflower has traditionally been used for menstrual disorders and female infertility in some traditional medicine practices (Yang et al., 1993; Dajue and Mündel, 1996). Although most of these effects have been reported in non-ruminant or biomedical models, they provide a biological basis for investigating whether safflower supplementation can influence estradiol concentrations and reproductive outcomes in synchronized ewes.

Despite the potential effects of safflower seed on prostaglandin metabolism, steroidogenesis, and estrogen-related reproductive processes, there is limited information on its use as a nutritional supplement for improving reproductive performance in ewes. In particular, the effects of short-term whole safflower seed supplementation during progestagen-based estrus synchronization in anestrous ewes remain unclear. Because the non-breeding season is characterized by reduced reproductive activity, it is important to determine whether a short-term nutritional intervention during synchronization can support endocrine responses and reproductive outcomes.

Based on these mechanisms, we hypothesized that short-term dietary supplementation with safflower seed during progestagen-based synchronization may modulate circulating E2 and PGFM concentrations and thereby potentially influence reproductive outcomes. Therefore, the objective of the present study was to determine whether supplementation with whole safflower seed during the 7 d sponge period affects estrus response, pregnancy and lambing outcomes, and circulating E2 and PGFM concentrations in anestrous Hungarian Merino ewes synchronized with a short-term progestagen protocol.

Estrus rate, pregnancy rate, lambing rate, number of lambs, and litter size were determined in the control and safflower groups at the first, second, and overall service. The reproductive parameters are presented in Fig. 1 and Table 3.

There were no statistically significant differences in any of the reproductive parameters at the first, second, and overall services between the groups (p > 0.05).

Serum E2 and PGFM concentrations measured on day 0, day 7, and the day of mating are presented in Table 4 and Fig. 2. For E2, there was a significant effect of time (p < 0.0001), but no significant effect of treatment group (p=0.489) or treatment group × time interaction (p=0.194). For PGFM, there was a significant effect of time (p < 0.0001), but no significant effect of treatment group (p=0.571) or treatment group × time interaction (p=0.093). Serum E2 concentrations increased from day 0 to the day of mating in both groups, whereas serum PGFM concentrations decreased markedly on the day of mating. Overall, no significant effect of safflower supplementation on serum E2 or PGFM concentrations was detected during the synchronization period.

Safflower (Carthamus tinctorius L.) is a drought-tolerant forage and oilseed whose seeds characteristically contain high proportions of polyunsaturated fatty acids (PUFAs) – most notably linoleic acid (n-6) – together with polyphenolic constituents (Yaginuma et al., 2002; Landau et al., 2005; Peiretti, 2009). As a feed ingredient, safflower and its products have been evaluated under field conditions for their effects on growth, fetal development, neonatal survival, and milk composition (Bolte et al., 2002; Bouattour et al., 2006; Peiretti, 2009; Afshar et al., 2021; Bottger, 2001). Although palatability can be lower than that of other oilseeds, this drawback is commonly alleviated when safflower is incorporated into mixed rations (Smith, 1996; Sudhamayee et al., 2004). This study investigated whether short-term supplementation with whole safflower seeds, a linoleic-acid-rich oilseed, modulates reproductive performance and circulating hormonal dynamics in anestrous ewes synchronized with a progestagen protocol. Across first, second, and overall services, estrus, pregnancy, and lambing rates were comparable between the safflower-supplemented and control groups, and no pregnancy loss was observed between pregnancy diagnosis and lambing (Table 3). Regarding endocrine responses, serum E2 showed a significant time effect over the synchronization period but no group effect, while the prostaglandin F metabolite (PGFM) declined markedly toward mating in both groups, again, with no statistically significant between-group differences detected (Table 4). A slight, non-significant increase in PGFM after 7 d of safflower feeding was noted but was not associated with measurable improvements in fertility outcomes.

The lack of improvement in reproductive indices in our anestrous ewes appears to differ from some previous reports in cattle and goats in which lipid supplementation, particularly n-6-rich sources, has been linked to enhanced luteal function, shifts in eicosanoid profiles, and higher conception rates (Grummer and Carroll, 1991; El Banna et al., 2005; Grant et al., 2005; Ravikiran et al., 2022). For instance, diets high in n-6 fatty acids may favor series-2 prostaglandin production (Petit et al., 2004), and safflower oil supplementation during the transition period increased serum PGFM in dairy cows (Silvestre et al., 2011). In ewes, pre-mating n-6 supplementation has likewise been associated with higher plasma progesterone and PGFM (Soydan et al., 2020). Species-level differences, physiological state (postpartum versus anestrus), supplementation duration, and the physical form and dose of the supplemental lipids may have contributed to these divergent outcomes.

Dietary linoleic acid that escapes ruminal biohydrogenation can be elongated and desaturated to arachidonic acid – the substrate for PGF2α synthesis – thereby potentially influencing luteolysis, ovulation, and uterine function (Funston, 2004; Weems et al., 2006). Fat supplementation can also elevate circulating cholesterol, a precursor for steroidogenesis, and modulate lipoprotein delivery to ovarian cells (Grummer and Carroll, 1991; Bao et al., 1995). In the present study, however, the supplementation protocol (75 g ewe⁻¹ d⁻¹ of whole safflower seed for 7 d) may not have provided sufficient exposure to linoleic acid to induce measurable changes in systemic lipid metabolism, steroidogenic activity, or uterine eicosanoid production within the hormonal milieu of the progestagen-based synchronization protocol.

The endocrine responses observed in the present study are more likely to reflect protocol-driven physiological changes rather than an effect of safflower supplementation. Serum E2 concentrations increased toward mating in both groups, most likely reflecting follicular development stimulated after sponge removal and eCG administration. However, this increase was not influenced by safflower supplementation as no significant treatment effect or treatment × time interaction was detected. Conversely, serum PGFM concentrations declined markedly on the day of mating in both groups, suggesting that PGFM dynamics were primarily time-dependent and were not significantly modified by safflower seed supplementation. The absence of detectable endocrine and reproductive responses may also be related to the relatively short supplementation period. In addition, the use of whole safflower seed may have limited the intestinal availability of linoleic acid because a substantial proportion of dietary unsaturated fatty acids undergoes ruminal biohydrogenation before absorption. Consequently, the amount of biologically available n-6 polyunsaturated fatty acids reaching the small intestine may have been insufficient to induce measurable endocrine or reproductive responses during the synchronization period.

Several design features likely limited the detectable effect size: (1) the supplementation (7 d during sponge insertion) was short compared with studies reporting endocrine or fertility benefits from lipid supplementation; (2) the use of whole seeds rather than rumen-protected forms likely increased ruminal PUFA hydrogenation and reduced post-ruminal delivery; (3) the safflower used contained linoleic acid at the lower end of reference ranges, potentially attenuating the expected n-6 effects; (4) the strong exogenous control of a progestagen–eCG synchronization regimen may have overshadowed nutritionally mediated changes; and (5) although the groups were balanced (n=22 per group), the present sample size may have limited the ability to detect moderate treatment effects. Because reproductive responses are influenced by multiple nutritional and endocrine factors, a short-term increase in dietary linoleic acid may not be sufficient to overcome the physiological constraints associated with seasonal anestrus, particularly when combined with a hormonally controlled synchronization protocol.

Within the constraints tested here, supplementation with 75 g ewe⁻¹ d⁻¹ of whole safflower seed for 7 d during progestagen synchronization in anestrous ewes was not associated with measurable increases in estrus expression, conception, or lambing. To better delineate safflower's potential in small-ruminant reproduction, future trials should extend supplementation to 3–6 weeks prior to and through synchronization with dose–response evaluation, compare whole seeds with rumen-protected lipid sources and high-linoleic with high-oleic cultivars, incorporate mechanistic endpoints (lipid profiles, mid-luteal progesterone, serial PGFM, LH pulses, luteal blood flow), stratify by physiological state, and increase sample size based on predefined minimal clinically important differences.

Short-term supplementation with 75 g ewe⁻¹ d⁻¹ of whole safflower seed during a 7 d progestagen-based synchronization protocol did not result in statistically detectable improvements in estrus response, pregnancy rate, lambing outcome, litter size, or circulating E2 and PGFM concentrations in anestrous Hungarian Merino ewes under the present experimental conditions. These findings suggest that the supplementation protocol evaluated in this study did not provide measurable reproductive or endocrine benefits. However, further studies using longer supplementation periods, rumen-protected lipid sources, dose–response designs, and larger sample sizes are needed to better define the potential role of safflower supplementation in improving reproductive performance in ewes.