Sheep play an important role in the livelihood and nutritional security of small and marginal farmers globally. The reproductive efficiency of sheep is compromised in subtropical climates because of nutritional and environmental factors. Improving the reproductive performance of sheep is important to improve and utilize limited animal resources and meet humans' need for quality animal protein. It has been previously reported that flushing ewes before breeding can improve folliculogenesis, ovulation, and lambing rates in sheep (1, 2). Nutritional interventions are preferred over hormonal interventions to augment the reproductive efficiency in livestock. It is generally accepted that nutrition not only increases the strength of animals but also determines their reproductive efficiency depending on the fatty acid (FA) composition of the diet (3, 4). Liel et al. (5) reported that FA calcium soap supplementation increases the number and size of ovarian follicles and serum progesterone concentrations in cyclic sheep. The addition of fatty foods to the diet has long been used to reduce postpartum depression, improve energy balance to increase milk production, and significantly improve fertility in lactating cows (6–8). Therefore, attention has been focused on determining the reproductive effects of various FAs, particularly long-chain polyunsaturated fatty acids (PUFAs), such as omega-3 (ω-3) and omega-6 (ω-6) FAs (8, 9).

Previous studies have shown that dietary supplementation of ω-3 PUFAs may have beneficial effects on the developmental processes of humans and animals (10). Mattos et al. (3) showed that ω-3 PUFAs can reduce uterine prostaglandin F2α (PGF2α) release and reduce the sensitivity of the corpora lutea (CL) to PGF2α, potentially aiding in preventing luteolysis and fostering the establishment of cow pregnancy. Omega-3 PUFA supplementation is associated with an increased conception rate, follicular turnover and growth, ovulation, CL size, and steroidogenesis (11, 12). Moreover, increasing the long-chain ω-3 PUFA intake in the later stages of pregnancy prolongs the pregnancy and prevents behavioral abnormalities, such as the time taken for newborns to stand and breastfeed (13, 14). Other studies have also highlighted the role of nutrition in improving the performance of farm animals such as buffalos (15–17), horses (18), and pigs (19, 20). Omega-3 PUFAs used for animals to enhance their fertility can be obtained from varied sources. Flaxseed or linseed is one of the commonly available ingredients that are rich in ω-3 FAs and short-chain PUFAs (21). Although flaxseed supplementation in cows, buffalos, and pigs has been extensively researched, information on its effects on nulliparous sheep is limited. In addition, studies on the effect of flaxseed supplementation on sheep reproduction in subtropical climates are lacking (2). Therefore, this study aimed to examine the effects of flaxseed supplementation on the fertility, endocrine profile, hematological profile, and serum FA profile of sheep in a subtropical climate.

Previous studies that investigated the effects of ω-3 PUFA on the reproductive performance of pigs, cows, buffalos, sheep, and goats reported contradictory results. These inconsistencies in the results are primarily caused by differences in the animal species, the source and dosage of ω-3 FA, and the duration of feeding. Despite the presence of abundant literature, there is little evidence that linseed supplementation affects sheep reproduction in tropical and subtropical climates. Thus, this study aimed to evaluate the effects of dietary supplementation of ω-3 PUFA derived from linseed on the reproductive parameters, critical hormonal profiles, and biochemistry in sheep during the breeding season in subtropical climates.

In the present study, the estrus induction rate was significantly higher in all treatment groups than in the control group. Similarly, the estrus induction interval was significantly shorter in group III. The control ewes recorded significantly lower conception rates, and the lambing rates were significantly higher in linseed-supplemented sheep. Mahla et al. (2) demonstrated that supplementation of fish oil with n-3 PUFAs for ewes improved their follicle numbers, ovulation rates, and twinning percentages. El-Shahata and Abo-Elmaaty (25) reported increased ovarian preovulatory follicles and ovulation rates in ewes after supplementation with calcium salts of long-chain FAs. According to Mahla et al. (26), supplementing ω-3 PUFA-rich fish oil in goats significantly increased their follicle count, ovulation rate, and kidding rate as well as the follicle size. In a study conducted by Garcia-Bojalil et al. (27), supplementation of calcium salts of long-chain FAs increased the number of CLs, decreased the time for the initial increase in progesterone, and restored the pattern of accumulated plasma progesterone concentrations. Moreover, the use of FA calcium soaps during flushing has been confirmed to be effective in improving fertility and lambing rate (28). Similarly, n-3 PUFAs improved the conception rate, follicular turnover, and growth in cattle (11, 12, 29). Some studies also reported that supplementation with n-3 PUFA increased the number of small (30, 31), medium (29, 32), and large (33) follicles in cattle, indicating that n-3 PUFA affects the follicular dynamics. Cattle supplemented with flaxseed oil and fish oil were found to have increased number of small follicles (34, 35). According to Ambrose et al. (11), cows fed with flaxseed oil showed a greater conception rate in a fixed timed artificial insemination (AI) than cows fed with sunflower oil. Similarly, Dirandeh et al. (36) observed a 66.7% increase in the conception rate in cows supplemented with flaxseed compared to that in control cows. Nazir et al. (16) observed increased conception rates in buffaloes supplemented with flaxseed on day 63 post-AI than in the control buffaloes. Akbarinejad et al. (37) reported no change in their findings, which is in contrast to the findings of the present study. This difference in results could be attributed to multiple factors, including the duration of PUFA supplementation, the source of PUFA, climate variation, and breed differences.

PUFAs are precursors of PGs, which play multiple roles in animal reproduction (9). Mammals must obtain PUFAs from their diet because they lack the enzymes essential to synthesize PUFAs internally (38). Arachidonic acid (AA), an n-6 PUFA, serves as the precursor for pro-inflammatory 2-series PGs. Conversely, animals can also experience anti-inflammatory effects by consuming food rich in n-3 PUFAs such as eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3). According to Abayasekara and Wathes (39), PG metabolism can be affected by the ratio of n-6 to n-3 PUFAs in the diet. In vivo and in vitro experiments have established that n-3 PUFAs reduce endometrial PG synthesis, thereby contributing to embryo survival (40–42). PUFAs are involved in a number of processes that play crucial roles in the normal fluidity of cell membranes and intracellular signaling (9), as well as in ovulation, fertilization, and parturition (39).

The present study demonstrated that ω-3 PUFA-rich linseed supplementation did not affect gestation length in sheep. This finding is consistent with studies in sows supplemented with ω-3 PUFA-rich flaxseed around the breeding time, which did not alter the pregnancy length (20, 43). Mattos et al. (44) revealed that supplementing cows with fish oil during the peri-partum period had no effect on the gestation length. By contrast, the gestation length was extended by 2 days (147.5 vs. 145.5 days) in ewes supplemented with fish oil in the late gestation period compared to those supplemented with Megalac (14). These findings may be explained by the fact that, in the present study, diets rich in ω-3 PUFA were supplemented during the early gestation period, whereas when ω-3 PUFA was supplemented during the late gestation period, prolonged gestation has been observed. A diet supplemented with PUFAs can affect the gestation length by altering the types and quantities of PGs synthesized, which are essential for parturition (45).

The present study demonstrated that the serum estrogen and progesterone concentrations were significantly affected by linseed supplementation. These results are in accordance with previous studies conducted on PUFA-fed sheep (25, 46) and goats (47). Sklan et al. (48) showed that cows fed with calcium salts of FAs produced higher progesterone during the estrous cycle than control cows. According to Petit and Twagiramungu (12) and Dirandeh et al. (36), cows that received flaxseed supplements had higher P₄ levels. An increased number of CLs and improved ovarian steroidogenesis may account for this increase in progesterone levels. According to Hawkins et al. (49), increased P₄ in cows fed with calcium salts of long-chain FAs may be related to reduced P₄ clearance from the plasma rather than increased luteal cell uptake of cholesterol. Nevertheless, Robinson et al. (50) observed lower plasma progesterone in cows treated with PUFAs in the early luteal phase. Higher proliferator-activated receptors have been linked to higher plasma concentrations of eicosapentaenoic acid (51), which could lead to a decrease in P₄ clearance (52). Conception rates may also increase with PUFA feeding because of the increased production P₄ due to increased CL and decreased embryo mortality (8, 12, 53, 54). FA supplementation in cyclic ewes increased the levels of progesterone in the blood and the size and number of ovarian follicles (5). According to Rawlings et al. (55) and Bartlewski et al. (56, 57), feeding an FA-concentrated diet increased the mean serum estrogen (pg/ml) content in sheep by three to four times. The unsaturated FA content of linseed has been shown to improve the physical properties of follicles and oocytes (58, 59).

In the present study, the hemoglobin concentrations in the treatment groups increased significantly on day 60 compared to that in the control group; however, no effect was recorded on day 30. Unfortunately, studies on sheep are not available in the literature that could be used for comparison. However, the pattern of change in the hemoglobin level was similar to that of the observations of other studies that used flaxseed oil in rats (60, 61). The addition of linseed oil to broiler chicks produced a non-significant shift in the concentrations of hemoglobin (62). In terms of PCV, the results of the present study showed that the percentage of PCV increased on days 30 and 60 of linseed supplementation. Improvement of PCV due to linseed supplementation could be due to its high content of PUFA, which protects the cell membrane of red blood corpuscles. In another study on rats, flaxseed oil improved the TEC and PCV owing to its high content of n-3 PUFA (63). Similarly, in the case of the TLC and TEC, a significant effect of linseed supplementation was observed on day 60. Similar profiles of the TEC were recorded in other studies on linseed-fed lambs (64–66). Furthermore, a study on Iraqi Awassi lambs fed with flaxseed oil showed improved hemoglobin concentration, red blood cell count, and blood glucose levels as well as decreased TLC after oral dosing (67). Supplementing with linseed may result in reticulocytosis, induce polycythemia, and boost erythropoietic activity in the liver and spleen. Shunthwal et al. (68) showed that feeding broiler chicks linseed oil enhanced their blood parameters and, consequently, the n6/n3 ratio of FAs in the body system.

The findings of the present study indicated a statistically significant increase in blood glucose levels in the treatment groups on days 45 and 60 compared to the control group. These findings are consistent with the results of Kia and Safdar (46). However, fish oil did not significantly affect the blood glucose levels in sheep, according to Mahla et al. (2). FAs can have a direct impact on insulin secretion triggered by glucose. Saturated FAs are insulinogenic, implying that they have a stronger effect on insulin secretion than unsaturated FAs (69, 70). Furthermore, lipid supplements with various FA compositions can alter the release of gastrointestinal peptides such as glucagon-like peptide 1 and glucose-dependent insulinotropic peptide 1. Thus, as observed in Holstein cows, this alteration may improve glucose-mediated insulin secretion (71). Cows fed diets enriched with flaxseed (linseed) and fish oil had higher plasma glucose levels (29, 72). Conversely, ewes fed diets enriched with flaxseed oil did not show a significant change in glucose levels (73).

In the present study, linseed supplementation significantly reduced serum MUFA concentration but significantly increased serum PUFA concentration from day 15 of supplementation. There is a lack of literature on the effect of feeding linseed to sheep on their serum MUFA and PUFA profiles. Mahla et al. (2) found that feeding PUFA to ewes significantly affected their plasma total cholesterol as well as high- and low-density lipoprotein-cholesterol concentrations. Similarly, Abuelfatah et al. (74) found that feeding whole linseed to goats increased the concentration of PUFA in the rumen. According to Singh et al. (75, 76), feeding boars linseed oil can boost antioxidant indicators and significantly reduce lipid peroxidation in their seminal plasma and serum. Furthermore, because linseed oil contains a higher concentration of these FAs than other oils, feeding sheep linseed oil may alter their serum PUFA and MFA profiles. This finding is consistent with the finding of Petit et al. (29). Although the present study provides the first comprehensive results on the reproductive performance of sheep fed linseed in subtropical climates, it has limitations related to a small number of animals and a short duration of study. It would be interesting to investigate the long-term effects of flaxseed supplementation. Furthermore, future studies focusing on follicular dynamics, follicular atresia, and embryonic development would be interesting.

Supplementing linseed to nulliparous ewes for 2 months during the breeding season in subtropical climates improved their reproductive efficiency by modulating the hormonal and biochemical milieu. Ewes that were fed linseed (15%) had a significantly higher rate of estrus induction, a shorter estrus induction interval, and an increased rate of conception and twinning. Simultaneously, linseed supplementation resulted in increased serum P₄ concentration, decreased serum MUFA levels, and increased serum PUFA levels. The study suggests that strategic dietary adjustments, such as the inclusion of 15% linseed during the breeding season, can significantly enhance reproductive outcomes in sheep farmed in subtropical climates.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was approved by Institute Animal Ethics Committee, NDVSU-College of Veterinary Science and Animal Husbandry. The study was conducted in accordance with the local legislation and institutional requirements.

PA: Writing – original draft, Conceptualization, Data curation, Investigation, Validation. JR: Conceptualization, Writing – original draft, Project administration, Resources, Supervision. AS: Conceptualization, Writing – original draft, Data curation, Investigation. BO: Investigation, Writing – original draft, Methodology, Supervision, Validation. AJ: Investigation, Methodology, Writing – original draft, Formal analysis. AB: Investigation, Writing – original draft. NB: Investigation, Writing – original draft, Conceptualization, Methodology. MA: Conceptualization, Writing – original draft. AR: Writing – original draft, Methodology. AS: Writing – original draft, Data curation, Investigation. SP: Writing – original draft, Formal analysis, Writing – review & editing. AM: Writing – review & editing. RK: Writing – review & editing. JC: Writing – review & editing. MS: Writing – review & editing.