Sheep were fed fat sources to enhance the energy density of their diet. Many aspects of sheep production, especially reproductive performance, are affected by dietary fat supplementation (Hervás et al., 2021; Mirzaei-Alamouti et al., 2018). Total dry matter intake in this study met the recommendation of the NRC (NRC, 2007) which 970 g/head/day for pregnant ewes weighing ±20 kg. The fact that dry matter consumption did not differ from that of the control group suggests that the addition of palm oil and lemuru oil as sources of EPA and DHA to the concentrate did not affect palatability, although PFO and FO using lemuru oil are waste products from marine biomass and tend to have a fishy odor. Interestingly, these results also showed that high energy density using oil supplementation in flushing concentrate did not reduce ration consumption; the addition of oil only affected crude fat consumption. These data are similar to those of previous studies in sheep where oil-fat treatment was used (Behan et al., 2019; Candyrine et al., 2017; Núñez de González et al., 2020). However, this contradicts the findings of Dauber (Dauber et al., 2022), who stated that the addition of oils such as rice, canola, linseed, and safflower oil caused significant differences in DM intake; however, the explanation of how and why this occurred remains unexplained. Although previous studies have also mentioned that adding oil in the range of 3–3.5% does not affect palatability, those statement is consistent with the results of this study, higher fat consumption in late pregnancy due to the crude fat concentration during this period. Flushing concentrate had a crude fat content of 7%, whereas the control concentrate was 1.22%. Crude fat consumption in early to mid-pregnancy was not affected by flushing concentrate, as the animals received the same initial crude fat concentration.
Flushing with palm oil or lemuru oil had significantly lower values of triglycerides than the control. This may be due to the fact that the addition of PUFAs contains in palm and lemuru oils can directly inhibit the development of blood triglycerides in general (Moallem, 2018). Lower triglycerides level in FO thought to be because EPA and DHA are poor substrates for the liver enzymes involved in triglyceride synthesis, thus preventing the production of very low-density lipoprotein (VLDL) and triglycerides. It may also prevent the esterification of other fatty acids in the body that would otherwise be used to produce triglycerides (Teama and El-Tarabany, 2016). Another explanation for the significantly higher triglyceride levels in PO than other treatment could be the 10% higher BETN content of the diet (Table 2) compared to the other treatments, where it is known that triglycerides are formed from carbohydrates and fats (Sitaresmi et al., 2020).
Progesterone is a steroid hormone secreted from the corpus luteum (CL) and is responsible for establishing and maintaining gestation (Widayati et al., 2019). The data in this study is comparable to data on sheep with Garut ewes supplemented with sunflower seed oil (Khotijah et al., 2015) or with palm oil supplementation in goats (Tudisco et al., 2019). The opposite results were reported in Etawah goats supplemented with flaxseed and lemuru fish oil which stated the progesterone hormone was significantly higher than in control (Astuti et al., 2020). This can be interpreted that the addition of 3–6% lemuru oil in concentrate is not in line with the hypothesis that the addition of omega-3 EPA and DHA can increase progesterone hormone levels during pregnancy (Astuti et al., 2020; Moallem, 2018) or reduce progesterone levels (Hess et al., 2008). The different results can be attributed to several factors, including the animal, experimental design, fat source and type of fatty acids used, and concentration (Moallem, 2018). These results indicate that lemuru oil as a source of EPA and DHA (3–6%) and palm oil as a PUFA source in ewe rations did not interfere with the plasma progesterone profile during gestation. Another interesting and importance finding in this study was that the PFO and FO treatments maintained progesterone levels in the last period of gestation, which should have dropped sharply to zero just before delivery because of increasing luteolysis, but up to 6 days before delivery, progesterone concentrations in PFO and FO remained high. This may be because sheep fed Lemuru oil rich in DHA and EPA reduced luteal responsiveness through PGF2α activity and saved 54% of CL from functional regression, then maintained progesterone concentration from 0 to 48 h before partus or acted as an anti-luteolytic agent (Plewes et al., 2018).
The result data in this study are similar in goats (Mahla et al., 2017) and cattle (Bernal-Santos et al., 2010; Kraisoon et al., 2018) supplemented with fish oil. Fish oil supplementation with DHA and EPA decreases the luteolytic activity of prostaglandins in early pregnancy. It increases the production of PG-series 3 through competitive inhibition of A6 desaturase, which inhibits the synthesis of arachidonic acid (AA) and interferes with the activity of the cyclo-oxygenase-2 (COX-2) enzyme (Mahla et al., 2017). Higher concentrations of both EPA and DHA in Lemuru oils inhibit phospholipase A2 (PLA2) exertion within the uterus, which in turn decreases arachidonic acid (AA) metabolism (Bernal-Santos et al., 2010). The formation of series-2 PG, which has higher bioactivity, can be switched to series-3 PG, which has lower bioactivity, when EPA or DHA replaces AA in the membrane phospholipids. Mattos et al., 2003; also, DHA and EPA tend to decrease PGFM and PGEM in ewes during critical luteolityc windows and prevent early embryonic death (Ambrose et al., 2006; Mahla et al., 2017). Another study explained that fish oil supplementation significantly decreased the expression of StAr and P450scc after prostaglandin injection, preventing CL regression (Plewes et al., 2018).
Further interesting data in this study showed that flushing with palm oil (PO) resulted in the highest embryo loss rate. This is because oil flushing increases ovulation and the potential for multiple births (Greco et al., 2018). Still, palm oil flushing, in contrast to lemuru fish oil supplementation, could not reduce the potential activity of prostaglandins (plant oils did not show significant inhibition of PG in the endometrium) (Khotijah et al., 2022; Verma et al., 2018), and possibly the size of the CL formed is small compared to fish oil supplementation (Mahla et al., 2017), resulting inability to maintain pregnancy in PO treatment. However, in the control group, the resulting pregnancy is mostly single embrio, so it is better to maintain the pregnancy and cause the embryo loss rate to be lower than PO. FO had a larger corpus luteum than PO and CNT, and progesterone levels tended to be higher (Verma et al., 2018) and resulting be the lowest embrio loss during the study.
The resulting ratio of male to female lambs was typically 50:50 in the control group. Interestingly, flushing treatment (PO, PFO, and FO) changed the male-to-female ratio, giving birth to more male lambs, this higher proportion of male lamb may related to amount of energy in the feed. Similar results have been reported in livestock fed with high PUFA (Conway et al., 2018; Khotijah et al., 2015). The mechanism by which fatty acids affect offspring sex is complicated and poorly understood. Studies on deer in oil supplementation showed a male birth ratio of 75% compared with 46% in controls (Wauters and Lens, 1995) and similar data in mice (Dama et al., 2011; Rosenfeld et al., 2003). High-energy feeding like flushing treatments results in the birth of higher male lambs (Gharagozlou et al., 2016). In addition, the high number of male offspring may also be due to an increase in estrogen concentration in the plasma of ewes, which in turn has been linked to modulation of the ewes' immune system and signaling in the oviduct (Gharagozlou et al., 2016). This high-fat feed is associated with a high estrogen level (Mirzaei-Alamouti et al., 2018). High concentrations of estradiol in the culture medium increase the number of male embryos (Chang et al., 2022; Zhang et al., 2008). Cows with high estradiol concentrations have also been found to have a higher ratio of male calves (Emadi et al., 2014). The sex ratio may also be influenced by the likelihood of X and Y sperm reaching the oviduct (Gharagozlou et al., 2016). The consumption of EPA and DHA has been associated with the inhibition of PGF2α synthesis (Kraisoon et al., 2018) and results in certain immune responses, including antibody production, cell differentiation, proliferation, migration, and antigen presentation (Gutiérrez et al., 2019). Changes in the contraction rate of the fallopian tubes due to the inhibition of PGF2 production can lead to changes in the sex ratio of the offspring (Emadi et al., 2014).
The incidence of twin or triplet births was relatively high, both in treatment with lemuru oil or palm oil supplementation, as follicle diameters and quantity were increased (Nugroho et al., 2021), and the addition of lemuru fish oil (FO) resulted in the highest number of triplet or quadruplet births (P < 0.05). This study is similar to that of fish oil supplementation in goats by a previous study (Mahla et al., 2017; Verma et al., 2018), which showed that FO administration tended to produce more large follicles (de Graaf) and total ovulations than palm oil administration. This also supported the data in cows (Moussavi et al., 2007), but a detailed explanation of this phenomenon is yet to be determined. It is possible that the addition of omega-3 fatty acids in FO increases insulin sensitivity (Chanda et al., 2018; Huang et al., 2010), which in turn increases IGF-1 and follicular activity. The flush ratio of ewes had no significant effect (P > 0.05) on lamb birth weight. The lamb birth weights in this study ranged from 2.16 to 2.70 kg. The birth weight of lambs was almost identical to that of lambs, which administered flush rations with sunflower oil (Khotijah et al., 2015). The same birth weight between treatments suggests that lambs born on flush rations containing EPA and DHA in the PFO and FO treatments, although not singles, were born at the same weight as those in the control treatment, tending to be singles and twins.
In conclusion, this study found that using sardine fish processing waste by flushing with 6% Lemuru fish oil positively affected the reproductive performance of Garut sheep. EPA and DHA contained in Lemuru fish oil were shown to increase litter size and maintain pregnancy in sheep, as proven by the highest increase in multiple births. This is due to the fact that the addition of DHA and EPA improves insulin sensitivity, thereby increasing IGF-1 activity and follicular activity, which in turn increases the number of ovulations. In addition, fish oil has been shown to inhibit the hormone prostaglandin factor in early pregnancy, thereby preventing embryo loss. Other conclusions suggest that the use of lemuru oil from sardine waste can not only reduce potential environmental pollution but can also be used as a supplement to improve the reproductive performance of small ruminants.