Effects of adding rumen-protected palm oil in diet on milk fatty acid profile and lipid health indices in Kivircik ewes

This study aimed to determine the effect of the addition of rumen-protected palm oil making up 3% of the ration on lipid health indices and milk fatty acid composition of Kivircik ewes’. Kivircik ewes at two years of age, the same parity, lactation stage, and the same bodyweight (52.57 ± 5.80 kg) were chosen for this purpose. Two groups were formed, in which the control group was fed a basal diet without feed supplementation, whereas the treatment group received rumen-protected palm oil which corresponded to 3% of the ration. In order to protect palm oil, it was coated with calcium salts. Treatment increased the palmitic acid (C16:0) content of milk compared to the control group (P < 0.05) and tended to increase saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) (P = 0.14). An increase in SFA and MUFA was attributed to an increase in palmitic acid and oleic acid (C18:1), respectively (P < 0.05). Results indicated that the omega-6/omega-3 ratio (n-6/n-3) ranged between 0.61 and 2.63. The inclusion of palm oil in the diet tended to increase desirable fatty acids (DFAs) regardless of the week of milk sampled (P = 0.42). Treatment did not improve the atherogenicity index (AI), thrombogenicity index (TI), health-promoting index (HPI), and hypocholesterolemic/hypercholesterolemic (h/H) ratio. Results showed that adding rumen-protected palm oil is a plausible method to meet the energy intake of ewes required during lactation without negatively affecting lipid health indices.


Introduction
Even though dairy ewes' milk fat is critical for economies in the Middle East and Mediterranean countries where it is primarily used for fermented dairy products, practices regarding the appropriate dietary treatments to increase potentially beneficial fatty acids in ewes' milk fat remain unclear (Boyazoglu and Morand-Fehr 2001). Regional animal breeding programs around the world are implemented to increase ewes' milk and meat yields, and new technological developments are being integrated into the field. In addition to cow milk, ewe, and goat milk contributes to total milk production in Turkey. Ewes are typically reared by small family farmers and villagers and are typically kept on natural pastures. In recent years, the sustainability of ewe husbandry has declined due to problems in animal feeding, feed costs, subsidies, and market prices.
The Kivircik breed, which is widely grown in Bulgaria, Greece, Turkiye (mostly in the Marmara region), and some Aegean and Mediterranean provinces, is distinguished by its thin tail and tasty meat. It is a breed that is well-adapted to adverse climatic conditions (Ekiz et al. 2022). The high number of ewes and abundant pasture resources in Turkiye explain the ewes' product potential. According to 2022 data, the ewes' population was 46.122.627 heads, making Turkiye 8 th in the world (Turkish Statistical Institute (TUIK) 2022). Ewes' milk, which is the most valuable and sold in large quantities, is processed into dairy products such as yogurt and high-quality cheeses. Turkey produces 22.960.379 tons of milk, of which 1.521.455 tons (6.6%) are ewes' milk (Turkish Statistical Institute (TUIK) 2022).
Factors affecting both the amount and composition of ewes' milk and genetic variation are the basic selection 159 Page 2 of 9 criteria in regions where dairy ewes' milk production is important. Environmental factors such as feeding should be considered when determining a ewe's milk yield capacity (Park et al. 2007). During lactation, dairy ewes' have significant energy needs resulting in proper feeding to meet these demands. As feed intake capacity is a limiting factor, during lactation ewes need additional fat sources to meet their high energy requirements and to maintain minimum dietary fiber intake for rumen fermentation (Bianchi et al. 2018a). Studies showed that vegetable oils, particularly seed, and essential oils, are used to meet dairy animals' energy requirements. This method may be ineffective in some cases, as high dietary fat levels reduce the digestion of dry matter in the rumen, resulting in reduced energy availability (Ferrer et al. 2005). One way to decrease the adverse effect of fat on dry matter digestion is to use it in a protected form. Ruminant diets used in milk production systems frequently contain palm oil as an energy source (Moallem 2018).
The bioactive components of ewes' milk can make this dairy product a functional food. Since ewes milk has a high concentration of short and medium-chain fatty acids, it has a beneficial characteristic for lipid malabsorption syndrome. On the other hand, SFA, including lauric acid (C12:0), myristic acid (C14:0), and palmitic acid (C16:0), form the majority of the fatty acid content of ewe milk. These fatty acids are hypercholesterolemic, which raises the risk of cardiac disorders and compromises the use of dairy products produced from ewe milk (Zhang et al. 2006). Recent studies have focused on the fatty acid content of milk and its various effects on human health. For different situations, such as fat supplementation and feed sources, animal studies integrating ruminant nutrition and milk fatty acids composition are required. According to the studies, adding palm oil to ruminant diets increased milk yield and milk fat (Mosley et al. 2007;Purushothaman et al. 2008;Bianchi et al. 2014;Kirovski et al. 2015). In a study designed to determine the effect of feeding rumen-protected palm oil fatty acids on the performance of lactating crossbred cows, Purushothaman et al. (2008) reported an increase of unsaturated fatty acids like oleic acid, linoleic and linolenic acid in milk which in turn resulted in an increase in unsaturated fatty acids that may be beneficial to consumers for cardiovascular health. Palm oil is a preferred fat supplement by producers and feed manufacturers due to its lower cost when compared to other fat sources used in ruminant nutrition. Supplementing ration with rumen-protected fat significantly increases milk production and composition (Kirovski et al. 2015). As a result, more research is needed to identify additional sources of variation in response to rumen-protected fat supplementation. Furthermore, studies should be conducted to determine the effect of fat supplementation on promoting the health properties of sheep milk. Thus, the purpose of this study was to determine the impact of the addition of rumen-protected palm oil making up 3% of the ration on lipid health indices and milk fatty acid composition of Kivircik ewes' milk.

Animals and dietary treatments
In the study, 14 Kivircik ewes at two years of age and with approximately the same bodyweight (52.57 ± 5.80 kg) were used. Two groups, each consisting of 7 ewes were formed and housed in individual boxes and fed individually. The control group was fed a basal diet without feed supplementation, whereas the treatment group received rumen-protected palm oil which was bought from Royal Feed Add. Co. (Kayseri, Turkiye; Table 1) which corresponded to 3% of the ration which was sprinkled on the ration. The level of adding rumen-protected palm oil was chosen as 3% as high levels of dietary fat levels reduce the digestion of dry matter in the rumen, resulting in reduced energy availability (Ferrer et al. 2005). The ration was fed twice a day and consisted of 40% of roughage and 60% concentrate. Wheat straw was fed as a roughage source whereas manufactured feed having 16% crude protein, 2.2% crude oil, and 10% crude fiber was the concentrate source ( Table 2). The manufactured concentrate feed label indicated that it included wheat bran, corn, sunflower meal, barley, wheat, molasses, dried distillers grain solubles (DDGS), soybean, limestone, vitaminmineral premixes. The experiment lasted for 4 weeks during which ewes were weighed every week. Ewes were milked twice a day once in the morning and once in the evening by hand at the same hours throughout the study and the remaining milk was suckled by lambs. At evening milking of the second and fourth week, 100 ml of milk was obtained for further analysis.

Fatty acid analysis
Milk samples were obtained and stored at − 20 °C until further fatty acid analysis. During the analysis, it was first left to thawing at 4 ± 1 °C and after homogenization, it was prepared for the extraction and esterification of lipids. Milk fat extraction was conducted by using a 2:1 mixture of chloroform and methanol. The milk fat was collected and overnight dried at 50 °C in a vacuum rotary evaporator. The percentage of total milk fat in samples was expressed as fat (w/w) per 100 g of milk.
Using ISO 12966 methods, the methyl esterification of fatty acids (FAMEs) operation was carried out (ISO 12966-2, 2011, Geneva, Switzerland). A Perkin Elmer Gas Chromatography (GC) system (Auto system GLX, Shelton, USA) equipped with a flame ionization detector (FID) and a fused silica capillary column (SPTM-2380, 100 m × 0.20 mm × 0.25 m ID) was used to determine the composition of FAME and quantify each FAME. The following GC conditions were carried out: Helium was the carrier gas at a flow rate of 0.5 mL/min. Temperatures for the injector and detector were set at 260 °C and 280 °C, respectively. The oven's temperature was programmed to rise from 120 to 220 °C over the course of 10 min, increasing by 5 °C/min. The retention durations of FAME standards were used to identify each FAMEs, and the calibration curves created for each FAME were used to quantify those FAMEs (Domínguez et al. 2022). Areas and retention times were calculated automatically. By comparing the relative retention times of their peaks from the standards, FAME samples were identified (Sigma-Aldrich, St Louis, MO, USA). To determine the fatty acids in milk samples, the relative percentage of each fatty acid as a percentage of the sum of all fatty acids found in the sample was used. Using the fatty acids data, other known fatty acid combinations and ratios, such as total UFA, total SFA, total MUFA, and total PUFA, were calculated.

Health lipid indices
The indices used for healthy milk fat shown below were calculated by using the milk fatty acid profile. In various studies, many nutritional lipid indices have been employed to assess the health benefits of milk and other dairy products. PUFA/SFA ratio is an index typically used to evaluate how diet affects cardiovascular health (CVH). The linoleic acid (LA, C18:2 n-6)/α-linolenic acid (ALA, C18:3 n-3) ratio was also calculated (n-6/n-3). To prevent lifestyle diseases like cancer and coronary heart disease, the diet should generally have a PUFA/SFA ratio over 0.45 and an n-6/n-3 ratio below 4.0 (Simopoulos 2002). Desirable fatty acids (DFAs) were calculated as the sum of UFA and C18:0 (stearic acid) using the following formula (Medeiros et al. 2014): The atherogenicity index (AI) and thrombogenicity index (TI) indices were calculated using the Ulbricht and Southgate formula (1991). The health-promoting index (HPI), which focuses on the impact of FA composition on cardiovascular disease, was developed by Chen et al. in 2004 to evaluate the nutritional value of milk fat.
The hypocholesterolemic/hypercholesterolemic fatty acid ratio (h/H) ratio was found according to the formula by Chen and Liu (2020).

Statistical analysis
Individually housed ewes were used as the experimental unit. Statistical analysis was performed using the STATIS-TICA package (version 26.0). The Mann-Whitney nonparametric multivariate test was run. For unexpected variables, descriptive statistics were used. Mean and min. and max. values were calculated from the scattered data, and central tendency variables were evaluated with a normality test. Multiple comparisons between the weeks, control, and treatment groups were done using the Bonferroni test. The Kruskal-Wallis test was performed to check the difference between 2 and 4 weeks of lactation. The data were presented as mean and pooled standard error of mean is provided. The significance level was set at P < 0.05 and significance levels P ≤ 0.09 were reported as a tendency.

Results
The effect of treatment on the fatty acid composition of Kivircik ewes milk is provided in Table 3. When rumenprotected palm oil was added to the ration, it increased the palmitic acid (C16:0) content of milk (P < 0.05). In both treatments, compared to the 2 nd week, linoleic (C18:2n2), linolenic acid (C18:3), and eicosatrienoic acid (C20:3 n6) content increased in the 4 th week (P < 0.05).
Saturated fatty acid, unsaturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid values are given in Table 3. Even though there was no significant difference in SFA and MUFA, the treatment group tended to have higher SFA and MUFA content than the control group and regardless of treatment, SFA and MUFA content tended to decrease in the 4 th week (P = 0.14). Even though treatment did not affect PUFA content, PUFA content increased in the 4 th week (P < 0.05).
The effect of treatment on health lipid indices (HLI) of Kivircik ewes' milk is provided in Table 4. Despite the fact that treatment had no effect on PUFA/SFA content in the current study, PUFA/SFA content increased in the fourth week (P < 0.05) and ranged from 0.10 to 0.33. Treatment had no effect on n-6/n-3 ratio which ranged between 0.61 and 2.63, thus indicating that it was healthy from health standpoint. In this study, including palm oil in the diet tended to increase DFA levels regardless of the week of milk sampled (P = 0.42). Regardless of treatment, ewe milk from all treatment groups had a similar AI, even though incorporating palm oil into the ration at 3% tended to increase AI within the same week (P = 0.18). Thrombogenicity index (TI) values are given in Table 4. Regardless of treatments, ewes' milk from all treatment groups had similar TI even though adding 3% palm oil into the ration tended to decrease TI in the second week, whereas tended to increase in the 4 th week (P = 0.16). Regardless of treatments, ewes' milk from all treatment groups had similar HPI even though adding 3% palm oil into the ration tended to decrease HPI within the same week (P = 0.11), and it ranged between 0.40 and 0.52 (Table 4). Hypocholesterolemic/hypercholesterolemic (h/H) values are given in Table 4. Regardless of treatment, milk from all treatment groups had similar HPI, even though incorporating 3% palm oil into the ration tended to lower HPI within the same week.

Discussion
Findings in the current study showed that adding rumenprotected palm oil to the ration increased the palmitic acid (C16:0) content of milk (P < 0.05). In a study determined to examine the impact of various palm oil fatty acid amounts on feed consumption and milk yield in Holstein cows, Mosley et al. (2007) found that milk palmitic acid concentration increased linearly as the intake of palm oil increased. Steele and Moore (1968) found that increased dietary palmitic acid intake significantly increased milk palmitic acid content (38.7% vs 60.7%). In the current study, adding rumen-protected palm oil into the ration at 3% increased palmitic acid concentration in milk from 28 to 36% (control vs treatment) corresponding to a 29% increase in palmitic acid concentration in milk. Other studies also found that adding palmitic acid to rations increased the concentration of palmitic acid in milk (Bodas et al. 2010;Castro et al. 2009;Hervás et al., 2021). In this study, we used rumen-protect palm oil to make it available in the small intestine. For a similar purpose to make palmitic acid available in the small intestine by using duodenal infusions of 500 g of palmitic acid (98% purity), Enjalbert et al. (2000) reported that palmitic acid concentrations in milk increased by 30% compared to the control group. Regardless of treatment, compared to the 2 nd week, linoleic, linolenic acid, and eicosatrienoic acid content increased in the 4 th week (P < 0.05). Similar results Means with a different superscript in the same row within the same treatment in each week differ (P < 0.05)

Item
Week 2 Week 4 SEM C + palm oil (3%) C + palm oil (3%) Butyric (  were also reported by Bianchi et al. (2014) who found that the inclusion of palm oil in the diet of dairy ewes increased linoleic (C18:2n2), linolenic acid (C18:3) content of milk as days on feed progressed (60 vs 120 days). Oleic and linoleic acids were the other major unsaturated fatty acids in rumen-protected palm oil used in the current study (Table 1), accounting for 11.78 and 2.28% of the total fatty acid composition, respectively. As the rumen-protected palm oil used in this study escapes the rumen and becomes available in the small intestine, readily available palmitic, oleic, and linoleic acid in the duodenum could be the reason for treatment group has higher concentrations of these fatty acids. Treatments SFA and MUFA contents were comparable, and regardless of treatment, SFA and MUFA content tended to decrease in the fourth week (P = 0.14; Table 3). According to Mosley et al. (2007), when the intake of palm oil increased, this increased saturated fatty acid concentration and decreased total MUFA in Holstein cow milk. Their results partially agree with our findings, as in this study adding rumen-protected palm oil to the ratio increased both SFA and MUFA. The addition of rumen-protected palm oil to the ration would be expected to increase SFA as it significantly increased the concentration of palmitic acid in milk (Table 3). The increase in MUFA could be attributed to oleic acid (C18:1), but the exact cause of this increase is unknown. Bianchi et al. (2018b) reported similar findings, revealing that adding palm oil to dairy ewes' diets increased SFA and MUFA levels in their milk. In a study, it has been observed that the total content of SFA, which may also have adverse effects on human health increased after 6% palm oil was added. Some SFAs have been linked to coronary heart disease, insulin resistance, and increases in low-density lipoprotein (Bianchi et al. 2018a). Although the total content of SFAs increased, Zong et al. (2016) discovered that the content of lauric (12:0) and myristic (14:0) acids decreased, which may benefit the consumers as these fatty acids are linked to a reduction in coronary heart disease. Despite the fact that treatment had no effect on PUFA content, PUFA content increased in the fourth week (P < 0.05). Mosley et al. (2007) discovered that palm oil consumption increased PUFA in Holstein cow milk in a similar manner. In another study conducted on dairy ewes, Bianchi et al. (2018b) found that adding palm oil to the diet increased PUFA levels as the days on feed progressed (60 days vs. 120 days).
The nutritional value of dairy products from the perspective of health standpoint is determined by the fatty acid composition profile. There are different indices used for assessing diet nutrition value and consumer health. The most important and commonly used HLI that are used in the current study are PUFA/SFA, n-6/n-3, desirable fatty acids (DFAs), atherogenicity index (AI), thrombogenicity index (TI), health-promoting index (HPI), and h/H (hypocholesterolemic/ hypercholesterolemic acids) ( Table 4). One of the important indices used for evaluating the nutritional quality of foods and the effects of diet on cardiovascular health is PUFA/SFA. It is stated that PUFA and SFA have the opposite effect on cholesterol metabolism, where PUFA in the diet decreases low-density lipoprotein (LDL) cholesterol thus serum cholesterol level, and SFA increases serum cholesterol. As a result, a higher PUFA/SFA ratio is preferred (Chen and Liu 2020). Even though treatment did not affect PUFA/SFA content in the current study, PUFA/ SFA content increased in 4 th week (P < 0.05). According to Simopoulos (2002), a PUFA/SFA ratio greater than 0.45 in the diet is recommended for fighting coronary heart disease and cancer. The PUFA/SFA ratio in the current study ranged between 0.10 and 0.33, which was lower than the recommended ratio. This is due to the greater contributions of C14:0, C16:0, and C18:0 acids to total SFA. Mierlita (2018) discovered that the PUFA/SFA ratio in Turcana Means with a different superscript in the same row within the same week in each treatment differ (P < 0.05) Means with a different superscript in the same row within the same treatment in each week differ (P < 0.05) Treatment Item Week 2  dairy ewes grazed part-time or supplemented with hemp seed ranged between 0.106 and 0.175. The n-6/n-3 ratio is another commonly used ratio to evaluate the nutritional quality and health benefits of animal fat (Pilarczyk et al. 2005), and a level of less than 4.0 is recommended for human consumption to combat lifestyle diseases such as coronary heart disease and cancers (Simopoulos 2002). The n-6/n-3 ratio of 0.61-2.63 indicates that the milk fat produced in this study was beneficial to health. Mierlita (2018) discovered that milk from Turcana dairy ewes fed indoors or part-time grazed and supplemented or not with hemp seed had an n-6/n-3 ratio ranging from 0.86 to 1.37. Sharma et al. (2018) discovered that indigenous Indian cow milk had a lower n-6/n-3 ratio than exotic and crossbreed counterparts, demonstrating that indigenous cow milk was superior to other milk. Desirable fatty acids (DFAs) are the sum of UFAs and stearic acid and are considered beneficial as they lower plasma cholesterol and triacylglycerols (Mensink et al. 2003). A higher DFA value indicates a healthier product. Atti et al. (2006) and Kasapidou et al. (2021) found comparable results to ours, reporting that ewes' milk from semi-intensive production or pasturebased systems had higher DFA values. The relationship between total UFAs, which are anti-atherogenic as they prevent the accumulation of plaque and lower levels of phospholipids, cholesterol, and esterified fatty acids, and the sum of C12:0, C14:0, and C16:0, which are thought pro-atherogenic as they promote the adhesion of lipids to cells of the circulatory and immune systems, could be seen from the AI formula. Thus, low levels of AI are regarded as a healthy situation from a health standpoint (Yurchenko et al. 2018). The inclusion of palm oil in the current study increased AI because it significantly increased the 16:0 content (P < 0.05) and ranged from 1.90 to 2.51, which was within the range values (1.42-5.13) reported by Chen and Liu for dairy products (2020). Research showed that the main factor influencing AI is dietary treatment (Chen and Liu 2020) however, the stage of lactation, grazing season, and some other factors may affect AI in dairy products (Lauciene et al. 2019). The thrombogenic index is calculated by dividing pro-thrombogenic FAs (C12:0, C14:0, and C16:0) by anti-thrombogenic FAs (C12:0, C14:0, and C16:0) (MUFAs and the n-3 and n-6 families). Consuming foods with a lower TI is recommended as research showed that it is beneficial to cardiovascular health (Ulbricht and Southgate 1991). Chen and Liu (2020) discovered that the TI of dairy products ranged from 0.39 to 5.04. In comparison to the values discovered by Chen and Liu (2020), our results were within the range and could be considered healthy. Health-promoting index (HPI) is the inverse of AI, and dairy products with a higher HPI are thought to be healthier. Various studies on dairy products such as milk and cheese revealed that in these products HPI ranged from 0.16 to 0.68. (Chen et al. 2004;Bobe et al. 2007;Bonanno et al. 2016;Giorgio et al. 2019). In the current study, HPI ranged between 0.40-0.52, which was consistent with other researchers' findings. Kasapidou et al. (2021) discovered that milk from semi-intensive farms had higher HPI than milk from intensive farms (0.359 vs 0.465). Hypocholesterolemic/hypercholesterolemic (h/H) values are given in Table 4. According to Santos-Silva et al. (2002), higher values of the h/H ratio are preferred since they are linked to the impact of fatty acid content on cholesterol and the risk of cardiovascular disease. Even though the inclusion of palm oil into the ration tended to decrease the h/H ratio (P = 0.42) in the current study, the values were still in the range reported in the literature as they ranged between 0.32-1.19 in dairy products (Ivanova et al. 2015;Sinanoglou et al. 2015;Mierlita 2018;Salles et al. 2019;Ahmad et al. 2020).

Conclusion
Adding rumen-protected palm oil which accounts for 3% of the ration increased the milk fat percentage in Kivircik ewes. The inclusion of rumen-protected palm oil into the ration both increased numerically SFA and MUFA due to increases in palmitic acid (16:0) and oleic acid (C18:1), respectively. Our results showed that adding rumen-protected palm oil improved the health profile of ewes' milk. Considering the n-6/n-3, DFA values, and AI, TI, HPI, and h/H indices which were in the range reported in the literature. Results showed that to increase the energy intake of ewes, rumen-protected palm oil can be added to their diet without negatively affecting nutritional health indices.
Author contribution GS contributed to all tasks, carried out the chemical analysis, authored the initial draft of the manuscript, and completed the final version. UA conducted the feeding trial, and the manuscript drafting. MY was involved in the study's conception, planning, and coordination. HK contributed to the statistical analysis and initial and final draft of the manuscript. The final manuscript was read and approved by all authors.
Funding This project was financially supported by the Scientific Research Commission of Suleyman Demirel University (Project #TBY-2018-5559).

Data availability
The datasets generated and analyzed during this study are available upon request from the corresponding author.

Declarations
Ethical approval This study was carried out on a commercial ewes' farm outside the university and all procedures were components of routine management, thus animal ethics application was not required and waived.