Supplementation of Prill Fat Derived from Palm Oil on Nutrient Digestibility and Dairy Cow Performance

Study Program Nutrition and Feed Technology, Department of Nutrition and Feed Technology, Faculty of Animal Science, IPB University (Bogor Agricultural University), Jl. Agatis, Kampus IPB Dramaga, Bogor, 16680, West Java, Indonesia Department of Nutrition and Feed Technology, Faculty of Animal Science, IPB University, (Bogor Agricultural University), Jl. Agatis, Kampus IPB Dramaga, Bogor, 16680, West Java, Indonesia


Introduction
Dairy cow production is likely to be adversely affected by feed nutrients and adequate nutrient requirements. The insufficiency of feed availability and quality for its production cause low productivity of dairy cows (Hasanah et al., 2017). Dairy cows in Indonesia mostly fed with Napier grass and concentrate which produced by cooperative (Zahera and Permana, 2015). Napier grass is a productive forage (250 ton/ha/year) (Despal et al., 2019) but it has a low quality (55% Total Digestible Nutrient(TDN), 8-12% Crude Protein (CP)) (Riestanti et al., 2020) and is influenced by seasons (Sajimin and Purwantari, 2019). To fulfil the requirement of dairy cattle average production (13.5 L/head/day) that require 60.9% TDN and 12.20% CP (NRC, 2001), dairy farmers often added concentrate. Concentrate that used by a traditional dairy farmer contained <60% TDN. Lack of nutrient fulfilment has become bigger during the dry season when forage quality rapidly decreased (Retnani et al., 2014) as an increased of livestock requirement which driven by the higher maintenance requirement of dairy cattle (Sutarno and Setyawan, 2016). Supplementation with the other energy sources should be considered to fulfil the lack of energy in dairy ration.
Fat can be used as an alternative for low energy ration supplementation (Riestanti et al., 2020). Fat supplementation in dairy ration can be used as a high energy density source for ruminants with a low heat increment (Santos et al., 2017). Fat can be used as a post-rumen energy source which could overcome the low quality of forages and increase milk production (Naik, 2013). However, in fat supplementation, level and techniques should be considered to avoid rumen metabolic disturbance. The addition of fat in dairy rations often causes termination in the rumen fermentation due to the nature of fat that surrounds protozoa (Firkins et al., 2007), so that protozoa immobilization will be agitated. The problems occur mainly due to the high utilization level of fat in form of oil (more than 6-7% total fat in ration) (NRC, 2001).
The technique of fat supplementation was aimed at protecting fat (rumen bypass). Ca-soap, hydrogenated partially process, formaldehyde, are among the popular bypass fat protection technique. Currently, prill fat has become popular that used for temperate dairy cattle especially, during the summer season to increase energy ration and decrease heat increment.
Prill fat is a hydrogenated vegetable oil which contain more than 85% palmitic fatty acid with high melting point. Due to this reason, prill fat does not melt at rumen temperature and bypass rumen degradation so that these fatty acids are absorbed directly in the small intestine and digested in small intestine by lipase enzyme (Kundu et al., 2014).
Prill fat supplementation was reported to increase energy consumption and known to have a significant effect on increasing milk production. It has been found that hydrogenated palm oil provides a better energy supply for lactating dairy cows than calcium soap of palm oil fatty acid (Karcagi et al., 2010). Prill fat is more effective to work as a source of energy to maintain the energy balance in lactating ruminants during the summer conditions (Somagond et al., 2020). Accordingly, many studies have been conducted with the aim of fat as an energy source using prill fat high in palmitic fatty acid was reported by Kundu et al. (2014), de Souza et al. (2017 and Mathews et al. (2016), which stated that there was an increase in milk production and milk fat in dairy cow, but had no effect on body weight gain and dry matter intake of dairy cows.
Utilizations of prill fat in Indonesia are still limited. Our previous research showedthatutilization of 2% prill fat contained 95% palmitic acid improved the total VFA concentration without interfering the rumen fermentation and digestibility (Riestanti et al.,2020). Study on utilization of 2% prill fat in dairy ration with different palmitic acid content showed that prill fat with 86% palmitic acid has improved milk production and quality. So far, there is no information on the optimum level of 86% palmitic acid prill fat supplementation in the dairy ration.
Therefore, this study was aimed to determine the optimum level of prill fat (86% palmitic acid) supplementation in dairy ration on fermentation characteristics, feed digestibility, milk production, milk components and milk fatty acid profiles.

Ration Preparation
The study was conducted from February 2020 to August 2020 at the Laboratory of Dairy Nutrition, IPB University and KUNAK, Cibungbulang, Dairy Farm. Dairy cows ration was constructed using elephant grass, concentrate and soybean curd with 58.28%: 33.62%: 8.10% ratio of DM. The ratio of feed was made based on the provision which made at KUNAK dairy farm, Cibungbulang, Bogor. Table 1 showed the composition and nutrient content of feed ingredients for dairy cow.
The dietary nutrient requirement and the maintenance needs of the dairy cows were calculated in accordance with NRC (2001), where the needs of dairy cow with an average body weight of 417 kg and the 12 L of milk production need of 12.20% crude protein and 61.30% TDN. Based on the calculations, crude protein and TDN were not fulfilled if we provided with the local feed only. Supplementing with prill fat is expected to improve the energy. Prill fat is needed to fulfill the energy of the dairy cow which has a low energy in their ration. The ration composition and prill fat composition of palm oil byproduct was presented in Table 2 and 3. respectively.

In Vitro Trial
In vitro analysis in this study was carried out following Tilley and Terry (1963) method. The rumen fluid was drawn from two fistulated dairy cows for in vitro analysis, following standard laboratory procedures. Sample of the ration (0.5 g) and McDougall solution (40 mL) for each treatment was poured into fermenter tubes. Then, the rumen fluid from different cows (10 mL) added to the tube followed by the addition of CO2 (anaerobic condition). Dry Matter Digestibility (DMD) and Organic Matter Digestibility (OMD) measurements were conducted by incubating the fermenter tubes filled with the sample of ration at 39°C for 48 h in the shaker water bath. pH, ammonia (NH3)N concentration and total VFA were carried out in 4 h after the incubation.
Research variables observed consisted of fermentation characteristics (pH, microbe's activity, ammonia (NH3)N concentration and total VFA production), nutrient digestibility (dry matter and organic matter), NDF and ADF digestibility.

Collection and Measurement Samples of In Vitro Analysis
Characteristics of rumen fermentation observed in this study were the concentration of ammonia which analyzed through Conway micro diffusion method (Conway, 1962), pH (analyzed by pH meter) and steam distillation method used to analyze the total VFA concentration. Measurement of the total population of protozoa and bacterial was carried out using the Ogimoto and Imai (1981) method. Nutrient digestibility (dry matter digestibility and organic matter digestibility were measured after 48 h of incubation. The 2 drops of HgCl2 were added to the substrate after 48 h incubation period (to stop microbe's activity), then it was centrifuged at 3000 rpm in 15 min ato separate the supernatant and solid component. Supernatants were eliminating and 50 mL of pepsin solution were added to the solid component in each tube. Tubes were incubated in the shaker water bath at 39 o C for 48 h. Then, supernatants were eliminated and the solids were washed with hot water and filtered using vacuum pump and Whatman filter paper. Substrate placed in the porcelain cup and dried at the oven (105°C in 24 h) for dry matter measurements. Then, substrates were incinerated in the furnace at 6 h (600°C) for the organic matter measurements.

Neutral Detergent Fiber Digestibility (NDFD) and Acid Detergent Fiber Digestibility (ADFD)
The residue from in vitro was analyzed according to the method described by Van Soest et al. (1991) using ANKOM 200 fiber analyzer. The in vitro residue was put into F57 fiber bag and then heated to a temperature of 100°C in neutral detergent solution for 75 min and acid detergent solution for 60 min to get the residual weight after the extraction process. NDF and ADF values are calculated from the residual weight after extraction divided by the initial sample weight.

In Vivo Trial
The study was conducted for 49 days, located in KUNAK, Cibungbulang dairy farm. A total of 20 multiparous Holstein cows in early and mid-lactation were managed under the intensive condition and adjusted to the breeder preference, researchers only added feed supplements in the morning. The feed consumption calculated every day. The forage and concentrate used are weighed using a hanging scale with a of 100 kg capacity. Milking is done 2 times a day, at 06.30-08.00 WIB in the morning and at 15.30-17.00 WIB in the afternoon. The milk production is measured using a 3000 mL measuring cup. Dairy cow milk production is collected in the morning and afternoon during milking. Analysis of the quality of milk is carried out using Lactoscan.

Analysis of the Fatty Acid Milk Profile
The milk fatty acid profile was analyzed on the last day of observation using Near-Infared Spectroscopy (NIRS), Animal Logistic Indonesia Netherland (ALIN) Laboratory. Milk samples were put into a petri dish, then placed on a device for further spectrum analysis using NIRS.

In Vivo Trial
The in vivo trials were designed by T-Test using the SASUniversity edition. This study was carried out with 2 treatments and 5 replications with each treatment consisting of 2 lactating dairy cows, as follows: T0 = Control ration in the form of forage + concentrate + soybean curd T1 = T0 +2% palm oil prill fat (optimum results in in vitro tests) The data were analyzed using ANOVA and the significant different among treatments were further tested by Duncan multiple range test using the SAS University edition. Differences among treatments were considered significant at p<0.01 and p<0.05.  (Wardeh, 1981)

Characteristics of Ruminal Fermentation and Rumen Microbial Activity (In Vitro Trial)
Prill fat supplementation had no effect on pH, ammonia concentration, and total rumen bacteria. However, there was a significant effect of prill fat supplementation on total Volatile Fatty Acid (VFA) concentration (p<0.01) along with the high level of prill fat addition in the ration. The addition of prill fat at different levels had a significant effect (p<0.05) on the digestibility of Dry Matter (DM), organic matter (OM), Neutral Detergent Fiber Digestibility (NDFD), Acid Detergent Fiber Digestibility (ADFD), Hemicellulose Digestibility (HSD), and cellulolytic bacteria population. The fermentation characteristics of the ration supplemented with prill fat showed in Table 4 and the effects of prill fat supplementation on nutrient digestibility values showed in the Table 5.
Based on the results of the polynomial test, the optimum level of prill fat supplementation of palm oil by-product in dairy cattle rations was 2% with a linear equation. This value indicates that the optimum level of prill fat addition did not impair the ruminal fermentability. Regression equation were used to examine the effect of prill fat addition on the ruminal fermentability. The regression equation of prill fat addition on the fermentability characteristics were shown in Fig. 1 to 3 respectively. The resulting prediction were shown below.
The protozoa prediction equation: Y = 6.5969-0.0337(x), (Y was the protozoa value and x was the prill fat level).
The total bacteria prediction equation: Y = 9.983-0.0422(x), (Y was the total bacteria value and x was the prill fat level) The cellulolytic bacteria prediction equation: Y = 9.783+0.004(x), (Y was the cellulolytic bacteria value and x was the prill fat level) The amylolytic bacteria prediction equation: Y = 9.7403-0.0936 (x), (Y was the amylolytic bacteria value and x was the prill fat level) The proteolytic bacteria prediction equation: Y = 9.0955-0.0604(x), (Y was the proteolytic bacteria value and x was the prill fat level) The Volatile Fatty Acid (VFA) prediction equation: Y = 105.62-3.0246(x), (Y was the VFA value and x was the prill fat level) The NH3(ammonia) prediction equation:

In Vivo Trial Dry Matter Intake
Feed intake is a description of the amount of feed that can be consumed by cattle for maintenance and production (NRC, 2001). The results showed that dairy cows consumed 16 kg of DM head/day. There was no significant effect of prill fat addition in dairy ration. However, milk yield of dairy cows was affected by the addition of prill fat in the ration (p<0.05). Table 6 showed the dry matter intake of dairy cows in KUNAK, Cibungbulang.

Dairy Cow's Milk Yield
The supplementation of 2% prill fat in the ration between delta (Δ) showed significant effect (p<0.05) on the enhancement of milk production. This study showed that a decrease of milk yield in control (T0) was from 12.10 to 10.75 L during the trial period. However, there was an enhancement of milk yield in T2 treatment, from 8.90 to 10.45 L. The milk yield of dairy cows in KUNAK, Cibungbulang shown in Table 7.

Dairy Cow's Milk Components
The addition of prill fat in the ration during maintenance between delta (Δ) did affect (p<0.05) the milk fat component. Supplementation of 2% prill fat had no effect on the other milk component (solid non fat, lactose, protein and total solid). The milk components in KUNAK before prill fat supplementation did not fulfill the National StandardsOrganization (BSN, 2011), especially in the fat component and total solid. Table 8 showed the milk component of dairy cows in KUNAK, Cibungbulang during the supplementation.

Dairy Cow's Milk Fatty Acid Profile
The addition of prill fat in the rations affected (p<0.05) three types of milk fatty acids from 19 fatty acids analyzed using NIRS. The result shows that caproic acid, palmitoleate and conjugated linoleic acid (CLA) are fatty acids that have significant results on the effect of prill fat addition in dairy cow rations (p<0.05). Table 9. showed the result of the milk fatty acids profile supplemented with prill fat in dairy cow ration.            Treatments  (Ulbricht and Southgate, 1991); HH: [(cis,C18:1 + ∑PUFA)/C12:0 + C14:0 + C16:0)] (Santos-Silva et al., 2002) a, b Different superscripts on the same row show significant differences (p<0.05)

Characteristics of Ruminal Fermentation and Rumen Microbial Activity (In Vitro Trial)
The in vitro fermentation process can be optimal if the rumen fluid is in a pH condition that is suitable with the rumen microbial environment. The pH value in all treatments was in the range of 6.8 which indicates that this value was in the normal conditions to support fermentation activity in the rumen (McDonald et al., 2011). The protozoa population from the study decreased along with the increase in the percentage of prill fat addition. This condition can be predicted that protozoa had no lipolytic activity as bacteria in entangled conditions with fat particles, so that the metabolic activity of protozoa tend to decrease when higher prill fat levels are added in dairy ration. The result was consistent with Behan et al. (2019) which reported that the decrease of total ruminal protozoa population in ruminant is caused by the addition of prill fat in the ration.
The rumen bacteria population decreased along the increasing level of prill fat added to the ration that is in the normal population range according to McDonald et al. (2011), was 9-10 log cfu/mL. These results were consistent with Pantoja et al. (1994) that fat in the rumen was associated with ration particles with a physical surface covering and inhibiting rumen microbial metabolism.
Similar with the total rumen bacteria count, the effect of prill fat addition in the dairy ration had a significant effect (p<0.05) on the cellulolytic bacteria population. This can be caused by the surface covering of prill fat, so that cellulolytic bacteria unable to digest fiber when the ration has a high-fat content. Cellulolytic bacteria are bacteria that break down cellulose into glucose which is used for the synthesis of macromolecules and microbial cells (Samsu et al., 2010). Cellulolytic bacteria had a slow growth than amylolytic bacteria (Samsu et al., 2010) which caused a decrease in the ability of bacteria to maintain their biomass in digesting rations with high-fat content.
The use of prill fat in different levels did not significantly affect the amylolytic and proteolytic bacteria population. It may be due to the ration that had an equal protein content in each treatment which did not affect the biomass of proteolytic bacteria. Amylolytic bacteria had a faster growth phase to support rumen fermentation than cellulolytic bacteria which can be reflected in a balanced population after the addition level of prill fat.
Proteolytic bacteria are bacteria that break down proteins, amino acids and other peptides into ammonia (Orskov, 1982) and produce intermediate compounds and other end products that vary widely. According to Czerkawski (2013), the type of feed that consumed by livestock will affect the bacterial population and the proportion of each microbial species.
The average ammonia concentration of the rumen fluid with prill fat supplementation in this study ranged from 8.56-10.14 mM. McDonald et al. (1995) stated that ammonia (NH3)Nconcentration in the rumen was around 5-17.65 mM. In this study, prill fat supplementation had no effect on ammonia (NH3)Nconcentrations. This was due to the absence of protein content in prill fat. This study was in accordance with Riestanti et al., (2020) that prill fat supplementation did not affect ammonia (NH3)Nconcentrations. Study of Montgomery et al. (2008) reported that lipid supplementation did not affect N metabolism in the rumen. Furthermore, the activity of proteolytic bacteria is not affected by the addition of prill fat in term of the non-existence of protein content in prill fat.
VFA roles as an energy source for livestock and a carbon source for microbial protein synthesis (Rodríguez et al., 2007). The average value of total VFA concentration with prill fat supplementation ranged from 107.78-127.14 mM. This value was still in the range of normal VFA concentrations that found in ruminants. According to McDonald et al. (1995), VFA concentrations that support microbial growth ranged from 80-160 mM. Addition of 6% prill fat (P4) treatment resulted in the highest level of total VFA concentration. This condition can be predicted that the addition of fat will increase the total VFA due to the enhancement of feed degradation processes by rumen microorganisms. Jenkins et al. (2008) stated that lipids which enter the rumen will undergo lipolysis which causes fat to be degraded into fatty acids and glycerol, then glycerol will be converted into VFA.
Recent study conducted by Riestanti et al., (2020) reported that the concentration of total Volatile Fatty Acids (VFA) was influenced by the level of prill fat addition at different levels of palmitic acid. Prill fat that contained 96% Palmitic Acid (PA) produced the highest VFA value compared to the control and the other treatments (76 and 86% PA content), but the total VFAconcentration of all treatments was still in the normal range of VFA concentrations in the rumen, so the prill fat addition at the level of 2% was not interfering the rumen fermentation activity.
Research by Naik et al. (2010) showed that the addition of bypass fat in the form of Calcium salts of Long Chain Fatty Acids (Ca-LCFA) 300 g/day with a ratio roughage: Concentrate 65:35 using in vivo did not affect the ammonia concentration in the rumen. Chalupa et al. (1986) also reported that the addition of 5-15% bypass fat had no effect on the rumen fermentation characteristics of dairy cows. Naik et al. (2009) reported that the addition of protected fat in the form of Ca-LCFA did not affect Dry Matter Digestibility (DMD), ammonia concentration and pH. The addition of protected fat in the ration showed an effect on the total VFA concentration due to an increase in energy by fat.

Nutrient Digestibility the Dairy Ration Supplemented of Prill Fat
The dairy ration digestibility produced in this study was in the normal digestibility according to Lestari and Abdullah (2015), it was above 60%, although there was a depression in the dry matter digestibility and organic matter digestibility along with the increasing level of prill fat addition. This condition caused by an interfere of palm oil prill fat with the microbial activity of the rumen due to the nature of the fat that can surrounded the feed particles. This result can be seen in the cellulolytic bacterial population (Table 4) which decreased significantly along with the enhancement of prill fat addition in the ration.
The highest NDF digestibility was in control treatment with 59.03% and the digestibility decreased along with the increase in the level of prill fat addition in the ration. This can be caused by a decrease of rumen bacteria activity, especially the cellulolytic bacterial population, which has decreased significantly to the addition of prill fat levels. It can also be predicted due to the physically closure of the feed particles by fat, so that the bacteria cannot maintain the ecosystem conditions in the rumen. Adesogan et al. (2019) stated that lignocellulose bonds also become a limitation in the use of feed ingredients in dairy rations because they could reduce the digestibility level in the nutritional value of the feed.
In addition, there were drawbacks of the in vitro method of Tilley and Terry (1963), which was unable to measure the post-rumen digestive system. Protected fat will pass through the rumen without changing the postrumen digestive system and resulting the absorption of fatty acids in the jejunum area of the small intestine (Lock et al., 2006). Measurement of post-rumen digestion of fat can be carried out using the in vivo method or the addition of lipase enzymes separately in the in vitro method. Therefore, for further research, the addition of lipase enzyme should be done to measure post-rumen fat digestion.

Dairy Cow Performance in KUNAK, Dairy Farm, Cibungbulang (In Vivo Trial) Dry Matter Intake
Dry matter intake is the amount of feed that can be consumed by cattle for cattle production. Dry matter intake in this study was lower than the study by Despal et al. (2013) which was 23 kg DM. In this study, the addition of prill fat to the ration had no effect on the amount of Dry Matter (DM) intake. This can be predicted that prill fat isa hydrogenated fatty acid. Prill fat contains fewer double bonds (6%), so their addition did not affect feed consumption. Prill fat contains high levels of saturated fatty acids (palmitic) so that it does not affect gastrointestinal motility. The degree of saturation of a fatty acid that reaches the small intestine in dairy cattle, will cause disturbances to gastrointestinal motility and will cause a decrease in dry matter intake (Drackley et al., 1992). Relling and Reynolds (2007) reported that the decrease in dry matter consumption is caused by the fat supplemented into the ration, which will cause an association with an increase in the concentration of peptide-1 in plasma, i.e., glucagon (a peptide hormone in the small intestine with a hypophagic effect.

Dairy Cow's Milk Yield
The enhancement of milk production after prill fat supplementation can be caused by an increase in de novo lipogenesis and pre-formed uptake of fatty acids (Mathews et al., 2016).
The increase in milk production can also be caused by the substitution of starch by protected fat so that there was a positive energy balance in dairy cattle, thereby changing the metabolism in the mammary glands (Wina and Susana., 2013). The energy that used by the dairy cow is obtained from their rations and the mobilization of fat in their bodies which can be maintained for production.
The depression of milk yield in Control (T0) was a normal condition for dairy cows, where the normal persistence in dairy cows is 89-92% which indicates that milk production has decreased by 8% every 30 days (Lowry, 1990). The persistence of milk production in the control treatment was 89.8%. This result showed that there was a decrease of 11% every 30 days. The enhancement of milk yield in Treatment (T1) with a persistence value of 110.6% after the peak was not normal, this can be caused by a nutrient improvements or recovery from disease.

Dairy Cow's Milk Components
Milk fat is one of the most important components in milk and the addition of prill fat in the ration can enhance the total of milk fat. This can be caused by the precursors for the synthesis of milk fat is fatty acids which derived from the rumen fermentation, mobilization of body fat and feed (Despal et al., 2019). According to Maheswari (2004), milk fat content was influenced by feed because most of the milk components were synthesized in udders from simple substrates.
The enhance of the fatty acids availability in feed will increase the rate of milk fatty acid synthesis which can be done by provide the sufficient of energy. It can be reflected in the enhancement of milk fat levels inlactating dairy cows which are given the prill fat in the ration.
Bypass fat supplementation reported to increase energy consumption and known to have a significanteffect on increasing milk production. Accordingly, many studies have been conducted with the aim of fat as an energy source using prill fat was reported by Kundu et al. (2014), Piantoni et al. (2015), Chamberlain andDePeters (2017), de Souza et al. (2017) and Mathews et al. (2016), which stated that there was an increase in milk production and milk fat in dairy cow, but had no effect on body weight gain and dry matter intake of dairy cows.

Dairy Cow's Milk Fatty Acid Profile
The enhancementof fatty acid content in milk is influenced by the presence of sufficient energy supply for fatty acid synthesis in the udder glands, in addition to milk production. The addition of Saturated Fatty Acids (SFA) to the ration was reported to increase the circulation of Non-Esterified Fatty Acid (NEFA) on dairy cattle (Piantoni et al., 2013). The ability of saturated fatty acids to increase NEFA synthesis is due to the high supplementation of palmitic acid (C:16) in the ration (Mathews et al., 2016). Long chain fatty acids (C >12) are converted to CoA and the triacylglycerol (TAG) has re-esterification during the absorption process. TAG then broken down into chylomicrons with the support of lipoprotein lipase in the tissues which then contributes to the circulation of NEFA in ruminants and it depends on lipase activity in certain tissues (Bickerton et al., 2007).
Atherogenicity Index (AI) was in the part of milk fatty acid profile which became an important value in functional food. Based on this study, the AI of milk for dairy cows in KUNAK Cibungbulang was in the low AI value according to Sharma et al. (2018) with AI values for normal dairy cows ranging from 1.6 to 3.79. AI in the control were 2.41 and 2.82 in the 2% prill fat treatment. Nantapo et al. (2014) examined the fatty acid composition during lactation at different periods and found that the lowest AI was resulted a value range from 4.08 to 5.13. The ratio of Hypocholesterolemic/Hypercholesterolemic (HH) is more accurate in reflecting the effect of fatty acid composition on Cardiovascular Disease (CVD) than the PUFA or SFA ratio. The value of HH ratio in dairy cow's milk according to Salles et al. (2019) ranging from 0.406 to 0.573. In this study, the value of HH ratio in dairy cow's milk in KUNAK Cibungbulang was 0.46 in control and treatment. These results indicate that 2% prill fat supplementation into the ration did not have a negative effect on the AI index and HH ratio of dairy cow.

Conclusion
Prill fat supplementation of 86% palmitic fatty acid at 2% level in dairy cow's ration did not interfere on rumen ferment ability and in vitro digestibility. Supplementation of 2% prill fat on in vivo enhanced the milk yield, milk persistence and improve milk fat profile as indicated by the alteration of PUFA and CLA.