The form more than the fatty acid profile of fat supplements influences digestibility but not necessarily the production performance of dairy cows

The form of a lipid supplement, its degree of saturation, and its fatty acid (FA) profile greatly influence di-gestibility and cow productive response. The objective in this study was to examine the effect of fat supplements that differ in their form or FA profile on nutrient digestibility and cow performance. Forty-two mid-lactation cows (128 ± 53 d) were assigned to 3 treatment groups according to milk yield, days in milk, and body weight. For 13 wk, the cows were fed rations that contained (on a dry matter basis) (1) 2.4% of calcium salts of fatty acids (CSFA) consisting of 45% palmitic acid (PA) and 35% oleic acid (OA; CS45:35); (2) 2.4% of CSFA consisting of 80% PA and 10% OA (CS80:10); or (3) 2.0% of free FA consisting of 80% PA and 10% OA (FF80:10). Rumen samples were taken to measure the ammonia and volatile FA concentrations, and fecal samples were taken to measure the digestibility. Pre-planned comparisons were CS45:35 versus CS80:10 to assess 2 CSFA supplements with different FA profiles, and CS80:10 versus FF80:10 to assess similar FA profiles in different forms. Compared with CS45:35, CS80:10 decreased the milk yields, increased the fat percentage, and tended to increase the energy-corrected milk (ECM) yields. The fat percentage of milk was highest in the FF80:10 cows (4.02%), intermediate in the CS80:10 cows (3.89%), and lowest in the CS45:35 cows (3.75%). Compared with CS80:10, FF80:10 increased milk yields (50.1 vs. 49.4 kg/d, respectively), tended to increase fat percentage, and increased 4% fat-corrected milk (4% FCM; 49.1 vs. 47.7 kg/d, respectively) and ECM yields (49.5 vs. 48.2 kg/d, respectively). Treatment had no effect on dry matter intake (DMI), and compared with CS80:10 cows, the calculated energy balance was lower in the FF80:10 cows. The 4% FCM/ DMI and ECM/DMI ratios were higher in the FF80:10 group compared with the CS80:10 group. Compared with the CS80:10 cows, the FF80:10 cows had a lower rumen pH, higher propionate, lower acetate/propionate ratio, and higher total VFA. Compared with CS45:35 cows, the apparent total-tract digestibilities of neutral detergent fiber and acid detergent fiber were higher in CS80:10 cows; whereas, the apparent total-tract digest-ibilities of dry matter, organic matter, protein, neutral detergent fiber, and acid detergent fiber were higher in the CS80:10 cows compared with the FF80:10 cows. Compared with the CS80:10 group, the apparent di-gestibility of total FA was 13.0 percentage points lower in the FF80:10 cows (79.1 vs. 66.1%, respectively), and similarly, the digestibilities of 16-carbon and 18-carbon FA were lower in the FF80:10 cows than in the CS80:10 cows. In conclusion, the form, more than the FA profile of fat supplements, influenced digestibility. Further, the CSFA supplements were more digestible than the free fatty acids, regardless of the FA profile. However, energy partitioning toward production appeared to be higher in the FF80:10 cows, although the digestibility of nutrients was lower than in the CSFA product with a similar FA profile.


INTRODUCTION
Several attempts have been made to decrease the impact of fatty acids (FA) on rumen digestion.The predominant type of lipid in the basal diet of the dairy cow is plant-sourced PUFA, which are mostly triglycerides and glycolipids.The form of the lipid supplement [triglyceride or free FA (FFA), calcium salt, or prilled fat], its degree of saturation, and its FA profile greatly influence the cows' productive response (Lock et al., 2013).The 2 most popular rumen-inert fat supplements are calcium salts of fatty acids (CSFA) and prilled saturated FFA.Previous studies have shown an increase in both milk yield and milk fat in cows fed CSFA, compared with control cows without added fat (Moallem et al., 2000), and a lack of negative effects on the digestibility of dietary components, compared with cows fed prilled hydrogenated fat (Harvatine and Allen, 2006a).However, CSFA were found to consistently decrease DMI (Allen, 2000).Furthermore, CSFA rich in UFA have been suggested to be much more prone to dissociate in the rumen than are CSFA rich in SFA, especially under decreasing pH conditions (Sukhija and Palmquist, 1990;Ferlay et al., 1992).In several studies, long-chain SFA were fed to dairy cows, and increases in milk yield and milk fat yield were observed, along with better feed efficiency, compared with cows fed CSFA containing UFA (Harvatine and Allen, 2006a) or control cows with no supplemental fat (Lock et al., 2013;Piantoni et al., 2013).Harvatine and Allen (2005) observed lower DMI with feeding CSFA, compared with SFA (prilled, hydrogenated FFA), but similar milk yields.
In recent years, research has emerged on how the ratio of palmitic to oleic FA in protected fat supplements affects digestibility and cow performance.In a study by de Souza et al. (2019), cows were fed diets with different palmitic acid (PA) to oleic acid (OA) ratios; the authors found that increasing the OA proportion in the diet increased the FCM, the ECM, and the milk energy output in very high-producing cows (60.0 ± 1.9 kg/d).In addition, in high-producing cows, increasing the OA proportion in the diet increased the milk fat yield owing to an increase in de novo and preformed milk FA.In another study, a PA-enriched supplement increased NDF, FA, gross energy digestibility, ECM, and milk fat yield, compared with a supplement containing both PA and stearic acid (SA; Western et al., 2020).Further, dos Santos Neto et al. ( 2021) performed a series of meta-analyses to evaluate the effects of diets supplemented with SFA compared with control diets without fat supplementation on nutrient digestibility and production.They reported a higher milk fat percentage in diets enriched with PA (≥80%), compared with diets enriched with PA+SA supplements included at ≤3%.
Most previous studies on the effects of the FA profile on digestibility and cow performance involved a blend of fat supplements in different forms.Moreover, in NAS-EM (2021) the total-tract digestibility (Table 4-1) was categorized by FA sources, divided according to form and FA profile.However, the impact of the combination between form and FA profile on total-tract digestibility was not discussed, most probably because of a lack of research addressing precisely this issue.Therefore, a comparative study examining the effects of a similar FA profile of the fat supplement but in different forms or a similar form but with a different FA profile is required to distinguish between the effect of form and that of the FA profile of the supplemental fats.We examined the effects of fat supplements differing in form (CSFA or FFA) and in the FA profile (45% PA and 35% OA, or 80% PA and 10% OA) on production, feed efficiency, and digestibility of nutrients.

MATERIALS AND METHODS
The experimental protocol for the study was approved by the Volcani Center Animal Care Committee (approval number IL 911/21), and the study was performed in accordance with the relevant guidelines and regulations.The experiment was conducted at the Volcani Center experimental farm in Rishon Lezion, Israel.Forty-two multiparous high-yielding Israeli-Holstein dairy cows were housed in a covered loose pen with adjacent outside yards that were equipped with a real-time electronic individual feeding system.Each feeding station was equipped with an individual identification system (ID tag, S.A.E.Kibbutz Afikim, Israel) that allowed each cow to enter a specific feeding station and automatically recorded each meal.After 10 d of adaptation to the feeding stations, cows were divided into 3 treatment groups, each with 14 cows.Cows were stratified randomly within stratum and strata of milk production, DIM, parity, and BW.The cows were fed a milking-cow ration supplemented with the following treatments: (1) CS45:35, which contained 2.4% (DM basis) of CSFA consisting of 45% palmitic and 35% oleic acids; (2) CS80:10, which contained 2.4% (DM basis) of CSFA consisting of 80% palmitic and 10% oleic acids; and (3) FF80:10, which contained 2.0% (DM basis) of FFA consisting of 80% palmitic and 10% oleic acids.The CS45:35 was produced by Poliva Ltd. (Ramla, Israel) from palm fatty acid distillate, and the CS80:10 and FF80:10 supplemental fats were produced by Nutrion Internacional S.L.U from the same raw material from fractionated/distillate oil.
The average parameters (mean ± SD) at the start of the study of the CS45:35, CS80:10, and FF80:10 groups, respectively, were as follows: milk with 55.0 ± 4.9, 55.0 ± 6.6, and 55.0 ± 5.9 kg/d; parity number with 3.0 ± 1.1, 3.3 ± 1.4, and 2.9 ± 1.0; DIM with 123 ± 54,125 ± 58,and 133 ± 49 d;and BW with 654 ± 51,650 ± 59,and 665 ± 56 kg.Cows were fed once a day at 1000 h at 105% of the expected intake, which was adjusted daily according to the previous day's intake.The composition and content of the diets are presented in Table 1, and the FA composition of the fat supplements and the TMR are presented in Table 2.The study continued for 13 wk as a continuous experiment.Cows were milked 3 times daily and yields were recorded electronically.Cows were weighed automatically after each milking with a walk-on electronic scale.Health events were recorded throughout the entire study period.In addition, milk samples were collected from 3 consecutive milkings about every 10 d (in total, 9 milk tests) and analyzed for milk fat, protein, lactose, and urea by infrared (standard IDF 141C:2000) at the laboratories of the Israeli Cattle Breeders' Association.Somatic cell counts were also determined in the same laboratory.
The cows were equipped with collar-mounted tags (HR-Tags; SCR Engineers Ltd.) that monitored and transmitted the rumination time.Rumination data were recorded by a special microphone that detected chewing actions by analyzing the vocal signals.Data were stored in 2-h blocks and recorded 3 times daily at the milking parlor.In addition, the cows were equipped with another sensor (Plus, S.A.E.Afikim) that moni-tored the activity (steps per hour) and the lying time (minutes).
The energy content in milk was calculated according to the National Research Council (NRC, 2001)  Blood samples for metabolite analysis were taken at d 0, 34, 64, and 87 of the study period after the morning milking.Blood samples were collected from the jugular vein into vacuum tubes containing lithium heparin (Becton Dickinson Systems) and were immediately put on ice.Plasma was then collected after centrifugation  at 1,500 × g for 20 min at 4°C and was stored at −32°C pending analysis.

Rumen Sampling and Analysis
In wk 8 of the study, 12 cows from each group were randomly selected for collection of rumen fluid samples for analysis of ammonia and VFA concentrations.The samples were collected by trained personnel using a stomach vacuum at 2 and 5 h postfeeding.Precautions were taken to avoid contamination of rumen samples with saliva.First, a vacuum pump was turned on only after ensuring that the tube was positioned inside the rumen; in addition, the first 250 mL of fluid collected was dumped.Filtered rumen fluid (5 mL) was immediately mixed with 20% trichloroacetic acid (1:1 vol/vol) and frozen at −20°C.These rumen samples were later used to determine the ammonia concentration by the phenol procedure (Chaney and Marbach, 1962).Next, 700 μL of 10% HgCl 2 solution was added to another 10 mL of filtered ruminal fluid, which was then centrifuged at 1,008 × g at 15°C for 10 min, before being stored at −20°C.The VFA in the centrifuged ruminal fluid samples were determined using a 5890 series 2 gas chromatograph (Agilent Technologies) equipped with a capillary column (30 m × 0.53 mm, 0.5 mm i.d.; Agilent Technologies) and a flame ionization detector.The injection port, column, and detector were maintained at 175, 130, and 165°C, respectively.

Digestibility Measurements
In wk 9 of the study, 12 cows were randomly selected from each group for the apparent total-tract digestibility measurements.Fecal samples were collected from the rectum at 3-h intervals over a 24-h period spanning 2 consecutive days, for a total of 8 fecal samples per cow.The fecal samples were then air-dried to a constant weight, at 60°C for 7 d.The dried fecal samples were coarsely ground and combined on an equal DM basis into one representative fecal sample per cow.For in situ digestibility measurements, 5 g was taken from each representative sample, ground, and placed in a 6 × 12 cm Dacron bag with 42-to 44-μm pores.Three replicate Dacron bag samples were prepared for each cow and incubated in the rumen for 192 h.Simultaneously, during the 2 d of fecal sampling, 3 samples of the 3 experimental diets were taken.The samples were oven-dried at a temperature below the melting point of the added fat supplements (48°C for 6 d).The dried samples were then ground, and equal amounts from each of the 3 sampling days were combined to form one representative dietary sample.The dried samples were then analyzed for DM, CP, ash, total FA, and several specific FA.In addition, 5-g aliquots were taken from the diet samples (5 replicates for each diet), put into Dacron bags, and placed in the rumen for 192 h to determine indigestible NDF.After the fecal and dietary samples were removed from the rumen, they were airdried at 48°C for 6 d.The indigestible NDF was measured and used as a marker for the apparent total-tract digestibility analysis.The digestibilities of DM, OM, protein, fat, NDF, and ADF were determined.

Fatty Acids in TMR and Feces Samples
The analyses of the total FA and FA profile of the TMR and fecal samples were performed at Cargill-Nutral laboratory.For the analysis of total FA in the TMR and fecal samples, all samples were saponified, extracted, and acidified, and FA methyl esters were analyzed by GC.
In short, the method consisted of saponification of the glycerides and phospholipids, followed by extraction by the Soxhlet method (Foss extraction equipment), using petroleum ether and gravimetry (based on ISOO 6492 and regulation of the European Commission EC, No. 152/2009).For the Soxhlet method, TMR or fecal samples were weighed in a cellulose cartridge and inserted into the extraction unit.Following saponification, petroleum ether contained in the extraction vessel extracted fat from the sample in a 2-stage process followed by a third stage of solvent recovery.The glass extraction was dried and weighed for calculation of the percentage of fat in the sample.The released FA were esterified in the presence of boron trifluoride methanol.
Then, the hydrolysis procedure was used, in which the samples were acidified with 3 N hydrochloric acid to remove covalent bonds between fats or lipids and lipoproteins, polysaccharides, or metallic salts.This procedure was used because the samples (feeds and feces) included CSFA.
For methylation of FA, following homogenizing and melting, 200 to 250 mg of samples were methylated with 4 mL of methalonic sodium hydroxide for 10 min.Next, 5 mL of boron trifluoride methanol was added for Shpirer et al.: FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS 2 min, followed by 5 mL of hexane for 1 min, while gently boiling under reflux.Then, a solution of saturated sodium chloride was added and gently rotated until the hexane phase was at the neck of the volumetric flask.The solution was left to cool, after which a portion of the upper organic phase containing methyl esters was transferred to a vial containing anhydrous sodium sulfate and stored until GC analysis.
The quantification of the FA profile was carried out using a Perkin Elmer Model 8400 Gas Chromatograph, equipped with a semi-capillary column (ref.

Analysis of Metabolites
The plasma BHB concentration was determined with a RANBUT D-3-Hydroxybutyrate kit (Randox), in which a reaction between 3-hydroxybutyrate and dehydrogenase generates a UV emission correlated with the sample BHB concentration.The samples were examined at 340 nm with an optical density reader (spectro V-11D, MRC), and the results were calibrated against a known BHB concentration.The intra-and interassay coefficients of variation for the BHB assay were 1.3 and 1.8%, respectively.
The plasma concentration of nonesterified fatty acids (NEFA) was determined with a NEFA C Test Kit (Wako Chemicals GmbH).The intra-and interassay coefficients of variation for the NEFA assay were 5.9 and 6.2%, respectively.The plasma concentrations of glucose, triglyceride, cholesterol, alkaline phosphatase (ALP) catalytic activity, and aspartate aminotransferase (AST) catalytic activity were determined using a Cobus C111 autoanalyzer (Roche Holding GmbH) with specific reagents.All results were calibrated against a known metabolite concentration or activity standard of enzymes.Glucose was determined using the reagent set GLUC2, which used 2 enzymatic reactions with hexokinase and glucose-6-phosphate dehydrogenase to generate a UV emission that was correlated with the sample glucose concentration.The triglyceride concentration was determined using 4 enzymatic reactions with lipase, glycerol kinase, glycerol phosphate oxidase, and peroxidase to generate a UV emission that was correlated with the sample triglyceride concentration.The plasma cholesterol concentration was determined using enzymatic reactions with cholesterol esterase, cholesterol oxidase, and peroxidase to generate a UV emission that was correlated with the sample cholesterol concentration.The plasma ALP catalytic activity was examined using ALP to convert p-nitrophenyl phosphate and water to phosphate and p-nitrophenol.The p-nitrophenol released was directly proportional to the catalytic ALP activity; it generated a UV emission that was correlated with the sample ALP catalytic activity.The plasma AST catalytic activity was examined using AST to convert l-aspartate and 2-oxoglutarate to oxaloacetate and l-glutamate.The oxaloacetate was then reacted with NADH in the presence of malate dehydrogenase to form NAD+. The rate of the NADH oxidation was directly proportional to the catalytic AST activity, and the UV emission generated was correlated with the sample AST catalytic activity.

Statistical Analysis
Continuous variables (milk, milk solids, DMI, and efficiency variables) were analyzed as repeated measurements with the MIXED Procedure of SAS (version 9.2; SAS Institute Inc.), according to the following model: where μ = the overall mean, T i = the fixed effect of treatment (i = 1-3), L j = the fixed effect of parity (j ≥ 2), C(T) ik = the random effect of cow k nested within treatment i, D l = the fixed effect of DIM, R m = the fixed effect of time, R m × T i = the fixed effect of the interaction between time and treatment, and E ijklm = the residual error.When relevant, variables were analyzed with the specific data of the pretreatment period as covariates.
The interactions between parity and treatment and between DIM and treatment were initially included in the model, but were not significant (P > 0.20) and therefore ultimately excluded from the model.The autoregressive order 1 was used as a covariance structure in the model because it resulted in the lowest Bayesian information criterion for most of the variables that were tested.
Rumen measurements (ammonia and VFA concentrations) were analyzed as repeated measurements with the MIXED Procedure of SAS (version 9.2).Nutrient digestibility was analyzed with the General Linear Models procedure of SAS (version 9.2).Least squares means and the adjusted standard error of the mean (SEM) are presented in the tables.Contrasts were declared significant at P ≤ 0.05 and tendencies were reported at 0.05 < P ≤ 0.10, unless otherwise stated.

Fat Components Intake and Digestibility
The total FA intake was not significantly different between groups (Table 7); however, compared with CS45:35 cows, the CS80:10 group showed increased intake of 16-carbon FA (18.8%;P = 0.03) and tended to have decreased 18-carbon FA intake (P = 0.08).Com-pared with CS80:10 cows, the FF80:10 group tended to have increased 18-carbon FA intake (P = 0.08).No differences were observed between the CS45:35 and CS80:10 groups in the digestibility of all fat components of the diet.However, compared with FF80:10 cows, the CS80:10 group had increased apparent digestibilities of total FA by 19.7% (P = 0.02), 16-carbon FA by 20.1% (P = 0.004), and 18-carbon FA by 19.3% (P < 0.001).The digested amount of 16-carbon FA was higher in the CS80:10 group than in the CS45:35 group (P = 0.04).Compared with the FF80:10 cows, the CS80:10 cows showed a tendency for increases in the digested amount of total FA (P = 0.07) and an increase in the 16-carbon FA (P = 0.008).

Metabolite Concentrations in Blood
Treatments had no effect the plasma concentration of glucose, NEFA, or triglycerides (Table 8).The concentration of ALP (P = 0.04) was higher and that of AST (P = 0.09) tended to be higher in the CS80:10 cows compared with the CS45:35 cows.Compared with CS80:10 cows, the FF80:10 group tended to have a higher plasma BHB concentration (P = 0.10) and the cholesterol concentration was lower (P = 0.04).

DISCUSSION
In this study, we examined the form and FA profile of fat supplements in relation to digestibility and performance in high-yielding dairy cows.We compared 2 CSFA supplements with different FA profiles (45% PA and 35% OA; 80% PA and 10% OA) and an FFA supplement with 80% PA and 10% OA.We found that the form of the fat supplement, more than its FA profile, influenced the digestibility of FA and other diet components.However, energy partitioning toward production appeared to be higher in cows in the FF80:10 group, although the digestibility of nutrients was lower than in the group receiving a supplement with the same FA profile but in the CSFA form (CS80:10).

Milk and Milk Solids Production
The milk yield was 1.4% higher in cows fed the 80:10 ratio in the form of FFA (FF80:10), compared with cows fed the same FA ratio but in the form of CSFA (CS80:10).However, the milk yield was higher in cows fed a supplement in the same form (CSFA), but differing in the FA profile; cows fed with a lower PA but a higher OA (CS45:35) produced more milk.This result suggests that both the form and the FA profile of a supplement may affect milk production.In general, the effect of lipid supplements with a different form or FA profile on milk yield varied.Western et al. (2020) reported that the milk yield of cows fed a supplement enriched with PA did not differ from that of cows fed a mix of PA and SA.Similarly, Shepardson and Harvatine (2021) did not observe any differences in milk yield between cows fed lipid supplements differing in their PA and SA ratio.Feeding increasing amounts of SA in the diet did not influence milk yield in a study by Boerman et al. (2017), andde Souza et al. (2019) reported that increasing the amounts of OA at the expense of PA tended to increase the milk yield, which is similar to our findings (CS45:35 vs. CS80:10).Warntjes et al. (2008) found that feeding high amounts of PA tended to increase the milk yield, and Rico et al. (2017) reported a tendency for lower milk yield with an increasing dose of PA in the diet.In a meta-analysis, Rabiee et al. (2012) reported that CSFA or prilled fat markedly, but not significantly increased milk production as compared with diets with lower fat content.As previously mentioned, we found that the milk production for cows fed the same FA profile (80:10) in different forms was higher for cows fed free FA compared with CSFA.It can be concluded that the effect of fat supplementation in either form or FA profile is inconsistent.
In the current study, either the form or the FA profile of the dietary lipid supplement significantly affected the milk fat percentage.Increasing the PA proportion in the supplement from 45 to 80% in the CSFA form increased the milk fat content (3.89 vs. 3.75%, respectively).In addition, the high PA in the FFA form increased the milk fat content more than when the CSFA form was used (4.02 vs. 3.89%, respectively).In de Souza et al. (2019), altering the PA/OA ratio toward a high PA and low OA increased the milk fat content in the milk.However, it should be noted that the effect of the PA to OA ratio on milk fat content could be attributable to the source of supplemental fats, which was a blend of 2 forms of fat supplements: prilled FFA and CSFA.In Western et al. (2020), increasing the PA content (prilled FFA and CSFA) at the expense of SA increased the fat percentage.Feeding cows a saturated FFA rich in PA at a high rate (16.6% of DM) decreased the milk fat percentage (Warntjes et al., 2008).In a meta-analysis, dos Santos Neto et al. ( 2021) reported a higher fat percentage in diets enriched with PA (≥80%), compared with diets enriched with PA+SA supplements included at ≤3%.Rico et al. (2014) observed higher milk fat content when cows were fed a high-PA supplement in the form of FFA, compared with CSFA of palm oil, which is in agreement with our results.Collectively, in most studies, enriching the lipid supplements with PA increased the milk fat percentage.In our study, we demonstrated the same trend of increased milk fat upon increasing the PA ratio in the supplement, but we also showed that the form (FFA vs. CSFA) had an effect on the milk fat percentage.In addition, we observed higher fat yields in cows fed FF80:10 than in cows fed CS80:10.Other researchers reported a variety of impacts on the milk fat yield in response to fat supplements enriched with PA (Warntjes et al., 2008;de Souza et al., 2019;Rico et al., 2014;Western et al., 2020).
In our study, dietary treatment did not affect the protein percentage and yields.Western et al. (2020) found that increasing the PA in the fat supplement did not influence the protein percentage and yield, but Warntjes et al. (2008) found that feeding a supplement rich in PA tended to increase the protein percentage and increased the protein yield.In the study by de Souza et al. (2019), increasing the PA at the expense of OA did not affect the protein percentage, but it tended to decrease the protein yield.In Rico et al. (2014), cows fed palm oil CSFA had a tendency for a lower protein percentage, but not lower yields, among low-producing cows compared with cows fed a high-PA supplement in the form of FFA.The form of the lipid supplements also affected the protein percentage in other studies.The inclusion of a CSFA supplement decreased milk protein concentration relative to the inclusion of a fat supplement rich in PA (79%) in the form of triglycerides (Oyebade et al., 2020), which is in agreement with the reports of Grummer (1988), Weiss and Wyatt (2004), and de Souza and Lock (2018) regarding cows fed CSFA compared with a PA-enriched triglyceride supplement.Furthermore, Rabiee et al. (2012) reported that relative to other fat supplements, CSFA elicited the largest negative effect on the milk protein percentage, regardless of the FA profile of the supplement; however, in our study, compared with the CS80:10 cows, the FF80:10 cows had increased casein, but not crude milk protein percentage.
The treatment did not affect the lactose percentage or lactose yields.Increasing the PA at the expense of OA in de Souza et al. (2019) decreased the lactose per-centage and yields, which is in accordance with the findings of Rico et al. (2014).However, other studies did not observe any differences in the lactose percentage or yields with a higher PA proportion in the lipid supplement (Rico et al., 2017;Western et al., 2020), in any form.
Compared with CS80:10, the FF80:10 supplement increased the 4% FCM by 2.9%.In a study using a blend of products, increasing the PA proportion in the lipid supplement did not affect the 3.5% FCM yields (de Souza et al., 2019); however, Western et al. ( 2020) observed higher FCM yields when feeding with a supplement enriched with PA at the expense of SA, and Rico et al. (2017) reported that increasing the PA dose in the diet increased the FCM yields.However, in our study, the increased FCM yields with an increase in the PA proportion of the supplement was observed only in the FFA form, but not in the CSFA form.
Compared with CS80:10, the FF80:10 supplement increased the ECM yields.Interestingly, the yields of FCM and ECM were higher in the supplement that was enriched with PA in the FFA form, but not in the CSFA form.This outcome may suggest that the energy partitioning toward production is higher in the FFA form than in the CSFA form, possibly because of postabsorptive differences between products.More research is required to establish this hypothesis.

Feed Intake, Efficiency, and BW Changes
We did not observe any differences in DMI between treatments, which is in agreement with other reports with a higher PA proportion in the lipid supplement (Warntjes et al., 2008;de Souza et al., 2019;Western et al., 2020).Feed efficiency for 4% FCM and ECM yields of cows fed a supplement enriched with PA in the form of FFA was higher than those of cows fed a supplement with the same FA profile but in the form of CSFA.However, the efficiency for FCM and ECM production was not affected by the FA profile in the form of CSFA.Compared with CS80:10, the EB of cows fed the FF80:10 diet was lower than in both other treatments, with a numerically but not significantly lower BW gain.This outcome may indicate that the partitioning of FA and energy in supplemental fats in the FFA form differs from those of the CSFA form in any FA profile: more for milk production and less for body mass gain.
We observed significant differences in rumination time between treatments, with the cows fed both supplements enriched with PA having higher rumination times.Oyebade et al. (2020) found that the rumination time of cows fed palm oil CSFA was lower than that of cows fed PA-enriched supplement in the form of triglycerides.Although rumination time is associated with digestibility, the total-tract digestibility of DM and OM was lower in cows fed the PA-enriched supplement in the study by Oyebade et al. (2020).

Rumen Measurements and Digestibility
Cows fed the fat supplement in the FFA form had a lower rumen pH than cows fed a supplement with the same FA profile but in the CSFA form.However, the rumen pH was within the normal range for both groups.The pKa of the FA complex of the supplement is an important factor influencing the dissociation in the rumen (Sukhija and Palmquist, 1990).In this regard, CSFA rich in UFA have been suggested to be much more prone to dissociation in the rumen than CSFA rich in SFA (Sukhija and Palmquist, 1990;Ferlay et al., 1992); however, this was not demonstrated in our study, which showed that the rumen pH values of both groups fed CSFA were similar, although the ratio between SFA and UFA was altered.It should be noted that the rumen acidity is determined mainly by the fermentation rate of carbohydrates, rather than by lipids.However, the metabolic interrelationship between fat and carbohydrates can affect rumen acidity, especially when energy from fat sources replaces carbohydrates.
Acetate concentrations were higher in the CS45:35 cows than in the CS80:10 cows, and compared with the CS80:10 group, the FF80:10 cows had higher propionate levels, which led to a lower C2/C3 ratio in the FF80:10 group.In addition, compared with the CS45:35 and FF80:10 cows, the CS80:10 cows had a lower total VFA concentration.Several studies determined how the inclusion of a variety of lipid supplements in the diet influenced rumen VFA, with diverse results.Grummer (1988) reported a higher butyrate concentration for cows fed CSFA, compared with prilled fat, without differences in other VFA.Harvatine and Allen (2006b) observed differences in the VFA concentration between cows supplemented with different fat supplements and amounts.They found that the total ruminal VFA concentration tended to decrease linearly with increasing SFA (Harvatine and Allen, 2006b), which is in accordance with our findings that the total VFA in the CS80:10 group was 6.8% lower than in the CS45:35 group.From our results, it can be concluded that the form and FA profile of lipid supplements can affect fermentation in the rumen, but the direction is not consistent.Moreover, the fat level in the diets and the crude fat intake in our study were similar between groups, and with such protected fat supplements with negligible solubility in the rumen, the effects on carbohydrate fermentation are likely to be minimal.

Apparent Total-Tract Digestibility of Nutrients
Compared with FF80:10 cows, the CS80:10 cows had increased apparent total-tract digestibility of DM, OM, NDF, and ADF.In addition, the NDF and ADF digestibility was higher in the CS80:10 cows than in the CS45:35 cows.In the study by Piantoni et al. (2013), the DM and OM digestibility was higher in cows supplemented with 2% PA (99% PA) than in control cows, which was not observed in our study in the CS80:10 group as compared with the CS45:35 group.In Western et al. (2020), DM digestibility was higher in cows fed higher PA than in cows fed a diet with high SA.In de Souza et al. ( 2019), increasing the PA at the expense of OA did not enhance the DM digestibility, which is in accordance with our findings.In several studies, the NDF digestibility was enhanced by increasing the PA in the supplement (Warntjes et al., 2008;Piantoni et al., 2013;Rico et al., 2017;Western et al., 2020;), but other studies did not have the same outcome (Boerman et al., 2017;de Souza et al., 2019).Importantly, we observed an increase in NDF or ADF digestibility with feeding the CS80:10, but not with the FF80:10; this result means that the form has a much more dominant influence on these nutrients' digestibility than the FA profile of the supplement.Moreover, the digestibility of all diet components was lower for the FF80:10 diet than for the CS80:10 diet.In a meta-analysis by Dos Santos Neto et al. ( 2021), an increase of 4.50 percentage units in NDF digestibility was found in diets that included ≤3% FA supplements with ≥80% PA in the FFA form; however, they were compared with non-fat-supplemented control diets, which was not the case in our study.Moreover, no information was provided about the remaining 20% of FA of the supplement, which might also have influenced the digestibility.
As compared with CS: 80: 10, the digestibility of the total FA, 16-carbon FA, and 18-carbon FA was not affected by increasing the PA content in the CSFA form (CS80:10).However, the digestibility of total FA was ~18% higher in cows fed a supplement enriched with PA in the CSFA form (CS80:10) than the FFA form (FF80:10), with a similar trend of lower digestibilities for 16-carbon and 18-carbon FA in the FFA form.The 18-carbon FA digestibility was 3.3 to 7.6 percentage points lower than that for the 16-carbon FA in all groups, which was also found by Boerman et al. (2017), but not by Piantoni et al. (2013).In de Souza et al. (2019), increasing the OA proportion in the supplemental fat at the expense of PA increased the digestibilities of total, 16-carbon, and 18-carbon FA; however, this outcome was not observed in our study when both CSFA supplements were compared.Moreover, it should be noted that in the study of de Souza et al. (2019), the treatment groups were fed combinations of 2 supplements in different forms, prilled fat and CSFA, which may have influenced the digestibility, regardless of the FA profile of the combination.In Harvatine and Allen (2006b), the total 16-carbon and 18-carbon FA digestibilities increased linearly as the proportion of the unsaturation of supplemental fat increased.In our study, a higher OA proportion in the supplements did not affect the supplemental fat digestibility, although in several other studies UFA had a higher digestibility than did SFA (Weiss and Wyatt, 2004;Harvatine and Allen, 2006b;Weiss et al., 2011;Boerman et al., 2015).The interrelationships between the diet components, such as the forage and crude fat concentrations, may affect the FA digestibility.Boerman et al. (2015) suggested that total FA digestibility is affected not only by the type of the supplemental fat, but also by the interaction between DMI and the dietary fat concentration.In our study, the DMI was identical between groups, which enabled us to isolate the differences in digestibility between form and FA profile of the supplemental fats.In our study, in a comparison of both supplements enriched with PA, the crude fat as well as the 16-carbon and 18-carbon FA digestibility was ~19% higher in the CSFA than in the FFA PA-enriched supplement, with similar digestibilities of these parameters between the CSFA supplements, regardless of the FA profile.This interesting finding indicates that the form of the fat supplement influences the digestibility more than the FA profile does, and that fat supplement in the FFA form has a lower digestibility.
Although the digestibility of all diet components was markedly lower, and the digested total FA or 16-carbon FA in the PA-enriched supplement in the FFA form (FF80:10) was lower than in the CSFA form (CS80:10), the production performance was higher in the FF80:10 cows.In addition, the feed efficiencies for FCM and ECM production were significantly higher in the FF80:10 cows.However, the EB was lower in the FF80:10 cows than in the CS80:10 cows, with no significant differences in BW gain between groups, which is in agreement with the findings of de Souza et al. (2019).This discrepancy between digestibility outcomes and production performance can be explained by differences in energy partitioning or utilization, whereby more energy was directed to production in the FF80:10 group.However, this assumption was not supported by the BW gain results between the groups in our study, and it needs further investigation.

Blood Metabolites
The concentrations of BHB tended to be higher in cows fed with PA-enriched supplement in the FFA form than in the CSFA form.In de Souza et al. ( 2019), decreasing the PA in the supplemental fat decreased the BHB concentrations, which was not found in our study when we compared CS45:35 with CS80:10.In Piantoni et al. (2013), a diet enriched in PA increased the NEFA concentration, which was not observed in our study for either of the PA-enriched supplemental fats.The blood cholesterol concentration was higher in the CS80:10 cows, compared with the FF80:10 cows, which might be explained by the lower linoleic acid in the diet of the CS80:10 cows, as was found in humans for diets rich in PA, but low in linoleic acid.

CONCLUSIONS
We compared 2 CSFA supplements that had different FA profiles, either 45% PA and 35% OA or 80% PA and 10% OA, in 2 different forms, CSFA and FFA.This study design enabled us to differentiate between the effect of the form and the FA profile of the supplemental fats.We observed higher milk yields, higher milk fat percentages and yields, and a higher feed efficiency for 4% FCM and ECM, but a lower EB in cows fed the FF80:10 diet compared with cows fed a supplement with the same FA profile, but in the CSFA form.This improved performance occurred, although the digestibility of most diet components, including fat, was lower in the FF80:10 cows.In addition, DM, OM, protein, and fat digestibilities were similar between groups fed supplements different in FA profile, but similar in form (CSFA).These findings mean that the form, more than the FA profile of the fat supplements influenced the digestibility of FA and other diet components.However, it appears that energy partitioning toward production was higher in the FF80:10 diet, although the digestibility of nutrients was lower than the CSFA product.Further research along these lines is required to determine the optimal FA form and profile needed to achieve high production and to better define the energy partition in such diets.
equations.The energy balance (EB) was calculated daily according to the following equations (NRC, 2001):NE c = NE L per kilogram of DM × DMI (calculated using the values from NRC, 1989), NE m = BW 0.75 × 0.08, NE p = milk (kg) × {[0.0929 × (fat %)] + [0.0547 × (protein %)] + [0.0395 × (lactose %)]}, EB = NE c − (NE m + NE p ), where NE c = the net energy consumed; NE m = the net energy required for maintenance; and NE p = the net energy output in milk.The conversion rate of the feed DMI to milk was calculated from the daily individual data.
Shpirer et al.: FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS

Table 1 .
Shpirer et al.: FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS Ingredients and the chemical composition of the experimental diets

Table 2 .
The main fatty acids (as % of total fatty acid) in the fat supplements and diets

Table 3 .
Shpirer et al.: FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS Milk and milk solid yields as affected by fat supplements 2Preplanned contrasts included CS45:35 versus CS80:10 to compare 2 CSFA supplements with different fatty acid profiles, and CS80:10 versus FF80:10 to compare similar fatty acid profiles in different forms.

Table 4 .
Shpirer et al.: FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS Least squares means of DMI, energy balance (EB), and efficiency calculations 2Preplanned contrasts included CS45:35 versus CS80:10 to compare 2 CSFA supplements with different fatty acid profiles, and CS80:10 versus FF80:10 to compare similar fatty acid profiles in different forms.3Calculated energy balance.

Table 5 .
Least squares means of rumen pH and the concentrations of ammonia and VFA

Table 6 .
Shpirer et al.:FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS Least squares means of the apparent total-tract digestibility and digestible intake of diet components as affected by fat supplements 2Preplanned contrasts included CS45:35 versus CS80:10 to compare 2 CSFA supplements with different fatty acid profiles, and CS80:10 versus FF80:10 to compare similar fatty acid profiles in different forms.

Table 8 .
Shpirer et al.: FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS The least squares means of metabolites and enzyme concentrations 3Nonesterified fatty acids.4Triglycerides.5Alkalinephosphatase.6Aspartatetransaminase.
Shpirer et al.: FORM AND FATTY ACID PROFILE OF FAT SUPPLEMENTS