Increasing palmitic acid and reducing stearic acid content of supplemental fatty acid blends improves production performance of mid-lactation dairy cows

We determined the effects of altering the ratio of palmitic (C16:0) and stearic (C18:0) acids in supplemental fatty acid (FA) blends on production responses of mid-lactation dairy cows. Twenty-four multiparous Holstein cows (mean ± standard deviation; 47.1 ± 5.8 kg of milk yield, 109 ± 23 DIM) were randomly assigned to treatment sequences in a replicated 4 × 4 Latin square design with 21-d periods. Treatments were a control diet not supplemented with FA (CON), and 3 diets incorporating 1.5% of dry matter (DM) FA supplement blends containing 30% C16:0 + 50% C18:0, 50% C16:0 + 30% C18:0, and 80% C16:0 + 10% C18:0. Additionally, the FA blends were balanced to contain 10% of oleic acid ( cis-9 C18:1). The FA blends replaced soyhulls in the CON diet. Diets were formulated to contain (% of DM) 31.0% neutral detergent fiber, 27.0% starch, and 16.9% crude protein. The statistical model included the random effect of cow within square and the fixed effects of period, treatment, and their interaction. Preplanned contrasts included CON versus overall effect of FA supplementation and the linear and quadratic effects of increasing C16:0 in FA blends. Overall FA treatment had no effect on dry matter intake (DMI), but increasing C16:0 linearly increased DMI. Compared with CON, overall FA treatment increased yields of milk, 3.5% of fat-corrected milk, energy-corrected milk, and milk fat but did not affect milk protein yield. Increasing C16:0 linearly increased milk fat yield and tended to linearly increase the yields of 3.5% of fat-corrected milk and energy-corrected milk. Fatty acid supplementation decreased the yield of de novo milk FA but increased yields of mixed and pre-formed milk FA compared with CON. Increasing C16:0 in FA treatments did not affect the yield of de novo milk FA, linearly increased the yield of mixed, and decreased the yield of preformed milk FA. In summary, feeding FA supplements containing C16:0 and C18:0 increased milk production responses with no effect on DMI compared with a control diet. Mid-lactation cows producing ~40 to 50 kg/d milk yield responded best to increasing supplemental C16:0 in FA supplements, demonstrating that FA supplements higher in C16:0 and limited in C18:0 improves production responses.


INTRODUCTION
Due to increasing nutrient and energy demands of dairy cows as milk production increases, dietary approaches to meet these demands must be investigated.A common approach to achieve this is through fatty acid (FA) supplementation, and the use of FA supplements has become common practice in dairy cow nutrition to help support milk production and milk fat yield (Rabiee et al., 2012;dos Santos Neto et al., 2021a,b).Palmitic (C16:0), stearic (C18:0), and oleic (cis-9 C18:1) acids are the most common FA in commercially available fat supplements.Previous research has highlighted that individual FA and combinations of FA have different effects on nutrient digestibility and production responses.Evaluating the effects of highly enriched FA supplements of either C16:0 or C18:0, milk fat yield was increased (Lock et al., 2013;Piantoni et al., 2013Piantoni et al., , 2015)), and combinations of these FA have been found to affect production responses differently (de Souza et al., 2018;Western et al., 2020a).Additionally, the physiological state of the cow is a factor that can interact with nutrition, thus affecting production responses (Harvatine and Allen, 2005;Boerman et al., 2015b), and cows at varying production levels respond differently to combinations of FA (de Souza et al., 2019;Western et al., 2020a;Burch et al., 2021).Understanding which FA combinations can optimize production responses of cows at different production levels is important to consider for nutritionists and producers.
We have examined the effects of altering ratios of C16:0 + cis-9 C18:1 for cows at different production levels and observed that high-producing cows (≥55 kg/d) increase production with higher levels of cis-9 C18:1, whereas lower-producing cows (<50 kg/d) respond better to more C16:0 (de Souza et al., 2019;Western et al., 2020a).Burch et al. (2021) compared FA blends of 60% C16:0 + 30% C18:0 versus 60% C16:0 + 30% cis-9 C18:1 within different production levels and observed that low cows (average 42.5 kg/d milk yield) responded best to the C16:0 + C18:0 blend.Considering previous research evaluating effects of C16:0 and C18:0 on production level, Piantoni et al. (2015) found no significant effect of feeding a C18: 0 -enriched supplement to low-producing cows.Additionally, Rico et al. (2014) observed that a C16: 0 -enriched supplement improved milk production compared with an C18: 0 -enriched supplement, regardless of production level.Most commercially available saturated FA supplements are enriched in C16:0 (≥80%) or a mix of C16:0 + C18:0 (typically 55% and 30%, respectively).The effects of these commercial products on production responses have been examined (Western et al., 2020b;dos Santos Neto et al., 2021a) with supplements containing more C16:0 improving milk production compared with C16:0 + C18:0, but production level was not considered.It is also important to note that none of these experiments varied the ratios of C16:0 + C18:0, as all ratios were of fixed levels with only single comparisons (de Souza et al., 2018;Western et al., 2020b;Burch et al., 2021) and to maximize milk production responses, it is necessary to evaluate different ratios of C16:0 + C18:0.To establish an optimum FA ratio, the objective of our study was to determine how altering the ratio of C16:0 and C18:0 in FA blends affects milk production responses of dairy cows producing ~40 to 50 kg/d milk yield; a range that is similar to the mid-lactation cows used in our aforementioned studies (de Souza et al., 2019;Western et al., 2020b;Burch et al., 2021).We hypothesized that as the ratio of C16:0 increased in the FA blends, DMI and milk production responses would increase.To our knowledge, multiple combinations of FA supplement blends containing different ratios of C16:0 + C18:0 that are commercially pertinent for the dairy industry have not been evaluated for midlactation dairy cows in a single study.

Design and Treatments
All experimental procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University (East Lansing, Michigan).Twenty-four mid-lactation, multiparous Holstein cows from the Michigan State University Dairy Cattle Teaching and Research Center were randomly assigned to a treatment sequence in a replicated 4 × 4 Latin square design balanced for carryover effects in four 21-d periods.All animals received a common diet with no FA supplementation during a 7-d preliminary period to obtain baseline values that were used to assign cows to treatment sequences.The starting average for all animals, with mean ± standard deviation, were 51.9 ± 5.8 kg of milk yield, 100 ± 23 DIM, and 681 ± 66 kg of BW.
The treatments consisted of (1) control diet (CON; diet with no supplemental FA), (2) FA blend containing 30% C16:0 and 50% C18:0 (L-PA), (3) FA blend containing 50% C16:0 and 30% C18:0 (M-PA), and (4) FA blend containing 80% C16:0 and 10% C18:0 (H-PA).All FA blends were balanced to contain 10% cis-9 C18:1.The FA blends were fed at 1.5% FA (DM basis) of the diet and replaced soyhulls from the control diet.To get our desired ratios of C16:0 and C18:0, 3 commercially available products were used (Table 1).The proportion of each of these supplements and the FA profile of the final FA treatment blends are shown in Table 2.All experimental diets were formulated to meet the nutrient requirements of the average cow (Table 3; NRC, 2001;NASEM, 2021).The DM concentration of forages was determined twice weekly, and diets were adjusted when necessary.A base diet, containing corn silage, alfalfa silage, corn grain, high-moisture corn, soybean meal, mineral mixes, whole cottonseed, and soybean meal, was mixed in a wagon daily.Then, soyhulls, FA blends, and the base diet were mixed in a tumble-mixer for each treatment diet.Cows were fed 115% of expected intake at 1000 h daily.Feed access was blocked from 0800 to 1000 h for orts collection and offering of new feed.Cows were housed in individual tiestalls throughout the experiment, with water available ab libitum in each stall, which were bedded with sawdust and cleaned twice daily.

Data and Sample Collection
Samples and production data were collected during the last 5 d of each treatment period (d 17-21).Samples of all diet ingredients (0.5 kg) and orts (12.5% of refusals) were collected daily and composited by cow per period for analysis.Milk yield was recorded, and 2 milk samples were collected at each milking.The first aliquot was collected in a sealed tube with preservative and stored at 4°C for milk component analysis.The second aliquot was stored without preservative at −20°C until analyzed for FA composition.Blood (~15 mL) samples were collected every 15 h resulting in 8 samples per cow per period and stored on ice until centrifugation at 2,000 × g for 15 min at 4°C.Plasma was transferred into microcentrifuge tubes and stored at −20°C until composited by cow per period.Body weight was measured 3 times per week following the afternoon milking with changes in BW determined according to Boerman et al. (2015b).On the last day of each period, BCS was determined by 3 trained investigators on a 5-point scale in 0.25 increments (Wildman et al., 1982).

Sample Analysis
Milk samples were analyzed for fat, true protein, and lactose concentrations by mid-infrared spectroscopy (AOAC, 1990;method 972.160) by the Michigan DHIA (Central Star DHI, Grand Ledge, MI).Yields of milk components, 3.5% of FCM, and ECM were calculated using milk yield and component concentrations for each milking, summed for a daily total, and averaged for each period.Milk samples used for analysis of FA composition were composited based on milk fat yield (d 17-21 per period).Milk lipids were extracted, FAME-prepared, and analyzed by GC as described previously (Lock et al., 2013).Yields of individual FA (g/d) in milk fat were calculated using milk fat yield and FA concentration to determine yield on a mass basis using the molecular weight of each FA while correcting for glycerol content and other milk lipid classes (Piantoni et al., 2013).
Dietary ingredients and orts were dried at 55°C in a forced-air oven for 72 h for DM determination.Dried samples were ground in a Wiley mill (1-mm screen; Arthur H. Thomas, Philadelphia, PA).Samples of feed ingredients and orts were analyzed for NDF, starch, and CP according to Boerman et al. (2017).Fatty acid contents of feed ingredients were analyzed using a 2-step method adapted from Jenkins (2010).Driedground samples were weighed to yield ~10 to 50 mg of FA, and cis-10 C17:1 (1:1 mg/mL toluene) was added as the internal standard.Samples were dissolved in sodium methoxide at twice the volume of the internal standard in toluene solution (2:1 mL/mL).Samples were then incubated for 10 min at 50°C, cooled for 5 min, and a 5% methanolic HCl solution (1 mL of acetyl chloride:10 mL of cold methanol) was added to double Average (n = 4) based on samples taken during the last 5 d of the experimental period.
sample volume.Samples were further incubated at 80°C for 10 min and cooled for another 10 min.Once at room temperature, a 6% aqueous potassium carbonate solution and n-hexane were added to samples and vortexed.The solvent layer containing n-hexanes and FAME was added to a tube containing 2 g of anhydrous sodium sulfate.The FAME were filtered through silica gel and charcoal, solvents were removed under nitrogen flux at 37°C, the FAME were weighed, and a 1% solution with n-hexane was prepared on a weight basis, which was used for GLC analysis.Feed FAME were determined on a GC-2010 Plus gas chromatograph (Shimadzu) equipped with a split injector (1:100 split ratio) and a flame ionization detector using a CP8827 WCOT fused-silica column (30 m × 0.32 mm i.d.× 0.25 μm film thickness; Varian Inc.).Hydrogen was used as the carrier gas at a flow rate of 1 mL/min and for the flame ionization detector at 40 mL/min.The other flame ionization detector gases were purified air at 400 mL/min and nitrogen-makeup gas at 30 mL/min.Injector and detector temperatures were kept at 270°C.The oven program was as follows: initial temperature of 140°C and held for 1 min, programmed at 5°C/min to 225°C, then programmed at 50°C/min to 250°C held for 5.5 min.Injection volume was 1 μL.Individual FAME were identified by comparison of retention times with known FAME standards (GLC reference standard 63-A and GLC reference standard 455 from Nu-Chek Prep Inc.).
Plasma insulin was determined by ELISA (Bovine Insulin ELISA; Mercodia AB, Uppsala, Sweden) and nonesterified FA (NEFA) and BHB were analyzed using an Olympus AU640e chemistry analyzer (Olympus America, Center Valley, PA), all at the Diagnostic Center for Population and Animal Health at Michigan State University (East Lansing, MI).

Statistical Analysis
All data were analyzed using the GLIMMIX model procedure of SAS (version 9.4, SAS Institute, Cary, NC).Data were analyzed using the following model: where Y ijkl = the dependent variable, μ = the overall mean, C(S) i(j) = random effect of cow nested within square (i = 1 to 4; j = 1 to 6), P k = fixed effect of period (k = 1 to 4), T l = fixed effect of treatment (l = 1 to 4), P k × T l = the interaction of period and treatment, and e ijkl = residual error.P k × T l was not significant for all variables and was removed from the model.Main effects were declared significant at P ≤ 0.05 and tendencies P ≤ 0.10.Three pre-planned contrasts evaluated (1) the overall effect of FA supplements (CON vs. the average of the FA treatments [FAT]; [1/3 (L-PA + M-PA + H-PA)]), (2) the linear effect of increasing C16:0 in FA blends, and (3) the quadratic effect of increasing C16:0 in FA blends.

DMI and Production Responses
Overall, FAT did not affect DMI (P = 0.44; Table 4); however, FAT increased yields of milk, 3.5% FCM, ECM, milk fat, milk lactose (all P < 0.05), milk fat content (P < 0.05), and feed efficiency (ECM/DMI; P < 0.05).The FAT did not affect milk protein yield (P = 0.36) but decreased milk protein content (P < 0.05), due to the increase in milk yield without a change in milk protein yield.The FAT tended to decrease BCS change (P = 0.10) but did not affect milk lactose content, BW, BW change, or BCS (all P > 0.16).
Increasing C16:0 in FA treatments linearly increased DMI, milk fat yield, milk fat content, BCS, and BCS change (all P < 0.05) and tended to linearly increase yields of 3.5% FCM and ECM (both P = 0.10).We observed a quadratic effect of increasing C16:0 in FA treatments for ECM/DMI (P < 0.05).Increasing C16:0 in FA treatments did not affect yields of milk, milk protein, or milk lactose, milk protein content, BW, or BW change (all P > 0.17).

Milk FA Contents and Yield
Milk FA are derived from 2 sources: <16 carbon FA (de novo) from de novo synthesis in the mammary gland and >16 carbon FA (preformed) originating  (Tyrrell and Reid, 1965).
from extraction from plasma.Mixed-source 16-carbon FA (C16:0 and cis-9 C16:1) originate from de novo synthesis in the mammary gland and extraction from plasma.
Overall FAT decreased the yield of de novo milk FA and increased the yields of both mixed and preformed milk FA (all P < 0.01; Table 5).Additionally, FAT increased the yields of C4:0 and cis-9 C18:1 and decreased the yields of 8 to 14 carbon milk FA (all P < 0.05).We observed no linear effect of increasing C16:0 in FA treatments on the yield of de novo milk FA (P = 0.14), but increasing C16:0 in FA treatments linearly increased the yield of mixed milk FA (P < 0.01) due to increasing both C16:0 and cis-9 C16:1 yields (both P < 0.01).In contrast, increasing C16:0 in FA treatments linearly decreased the yield of preformed milk FA (P < 0.01) due to a decrease in the yields of some 18-carbon FA, predominantly C18:0 (P < 0.01).
On a content basis, overall FAT decreased de novo milk FA and increased both mixed and preformed milk FA (all P < 0.01; Supplemental Table S1; https: / / doi .org/ 10 .5281/zenodo .10058735).Increasing C16:0 in FA treatments linearly decreased the content of de novo (P = 0.01) and preformed (P < 0.01) milk FA but increased the content of mixed milk FA (linear; P < 0.01).

DISCUSSION
Continuing interest in fat supplementation strategies has given more focus to the FA profile of supplements and how different FA and combinations of FA affect digestibility, metabolism, and production responses in dairy cows.As highlighted in our recent meta-analysis, C16:0 supplementation versus diets without FA supplementation increases energy partitioning toward milk with increases in the yields of milk and milk fat (dos Santos Neto et al., 2021a).Additionally, when directly comparing commercial products containing 84% C16:0 versus 33% C16:0 + 53% C18:0, the FA supplement with more C16:0 increased milk energy output as well as 3.5% FCM and ECM yields (Western et al., 2020a).When blending FA supplements to achieve dif-  2021) included 30% C18:0, thus it is unclear which level of C18:0 in a FA blend would be appropriate for increasing production responses.Considering these blend studies and previous results with cows producing <50 kg/d milk yield, it remained to be determined which FA ratio of C16:0 + C18:0 would maximize milk responses of midlactation cows.Thus, the goal of our current study was to alter the ratio of C16:0 and C18:0 in supplemental FA blends and evaluate their effects on milk production responses to identify the best ratio of C16:0 + C18:0 for cows producing ~40 to 50 kg/d.We observed variation in DMI responses with FA supplementation, potentially due to the profile of the FA supplement and the amount supplied (Rabiee et al., 2012), with our results supporting this as we observed that increasing C16:0, and lowering C18:0, increased DMI by 0.60 kg/d in FA blends.However, we observed no overall effect on DMI for CON versus FAT, which agrees with a recent meta-analysis where DMI was not affected by SFA supplements containing C16:0 or C16:0 + C18:0 compared with non-FA control diets (dos Santos Neto et al., 2021a).It is important to note that cis-9 C18:1 was kept constant at 10% in our FA blends, thus any hypophagic effect due to unsaturated FA (Harvatine and Allen, 2006) would likely be similar across FA treatments.In contrast to our results, Shepardson and Harvatine (2021) observed an increase in DMI with a 45% C16:0 + 49% C18:0 and 93% C18:0 compared with a treatment containing 91% C16:0.Boerman et al. (2017) increased dietary inclusion of a C18: 0 -enriched FA supplement (93% C18:0) up to 2.3% DM and observed an increase in DMI, along with a marked decrease in FA digestibility and no benefits on production responses.We observed many factors that affect DMI, such as energy demands for milk production, physiological state, and rumen fill, along with interactions with other dietary ingredients (NASEM, 2021).We observed little evidence in the literature, however, that C16: 0 -enriched supplement (80-90% C16:0) decreases DMI (dos Santos Neto et al., 2021a), and in fact, in our study, increasing C16:0 in FA treatments increased DMI.
Supplementation of C16:0 and combinations of C16:0 + C18:0 have been observed to increase milk yield compared with a non-FA control diet (dos Santos Neto et al., 2021a).Our observations support this, as overall FAT increased milk yield compared with CON.A potential mechanism could be due to high-fat diets often decreasing de novo FA synthesis, which may spare mammary glucose that can be used for lactose synthesis (Cant et al., 1993;Palmquist, 2006).Our current study supports this suggestion, as overall FAT increased yields of both milk and milk lactose and decreased de novo milk FA yield compared with CON.Increasing C16:0 content in our FA blends linearly increased yields of 3.5% FCM, ECM, milk fat, and milk fat content, similar to a recent meta-analysis and study performed in our laboratory that also observed greater milk production increases with more C16:0 in FA supplements (Western et al., 2020b;dos Santos Neto et al., 2021a).The increase in milk energy output observed with FA supplements higher in C16:0, compared with a mix of C16:0 + C18:0, is indicative of the increase in dietary C16:0 content, providing more nutrients to the mammary gland, specifically for FA synthesis (dos Santos Neto et al., 2021a).In contrast to our results and those of Western et al. (2020b), Shepardson and Harvatine (2021) reported no difference in yields of milk fat and ECM yields with supplements containing either 91.0%C16:0 and 45% C16:0 + 49% C18:0, which may be attributed to the 1.2-kg difference in DMI between treatments.
It was previously hypothesized that feeding a combination of C16:0 + C18:0 would be the most favorable for improving milk production responses (Loften et al., 2014), but results from our study do not support this, as increased C16:0 supplementation increased milk production performance compared with more C18:0.This is also supported by de Souza et al. (2019) and Western et al. (2020a,b), as they reported that cows with similar production responded best to a ratio of 80% C16:0 + 10% cis-9 C18:1.Our FA blends contained 10% cis-9 C18:1, suggesting that cows producing ~40 to 50 kg/d milk yield would increase milk production with a combination that contained 80% C16:0 and 9-10% cis-9 C18:1.Results from our current experiment, and evidence from the meta-analysis by dos Santos Neto et al. (2021a) reviewing SFA supplements, conclude that we observed no recent research to support the use of FA supplements containing notable amounts of C18:0.
The increase in milk fat yield observed with FAT was due to an increase in both mixed and preformed milk FA yields compared with CON, similar to results reported by dos Santos Neto et al. (2021a).This response would be expected as overall FAT increased the supply of long-chain FA available for absorption and utilization.Overall, FAT decreased de novo milk FA yield, which could be explained by a substitution effect where preformed FA substitute de novo FA in diets supplemented with FA (Glasser et al., 2008).Additionally, FAT increased the yields of C4:0 and cis-9 C18:1 milk FA, an indication of shifts in FA sources to ensure the regulation of milk fat fluidity (Barbano and Sherbon, 1980;Jensen, 2002).The melting point of milk fat influences fluidity and is increased with carbon chain length and lowered with increased degree of unsaturation (Dils, 1986).The esterification of specific combinations of FA on the triglyceride backbone keeps the melting point close to 39°C, which provides the mammary gland with plasticity to secrete triglycerides into the milk fat globules to be incorporated into milk and be fluid at body temperature of the cow (Timmen and Patton, 1988).Increasing C16:0 in FA blends had no effect on de novo milk FA yield, linearly increased mixed milk FA yield and linearly decreased preformed milk FA yield.The differences observed between the mixed and preformed milk FA yields are expected, due to increasing C16:0 in FA blends increasing the supply of 16-carbon FA for utilization by the mammary gland.The lack of effect on de novo milk FA yield with increased C16:0 could be explained by positional distribution on the milk triglyceride, as C16:0 has high affinities for the sn-1 (34.0 mol/100 mol) and sn-2 (32.3 mol/100 mol) positions whereas C4:0 and C6:0 are positioned on sn-3 (34.5 and 12.9 mol/100 mol, respectively; Jensen et al., 1991).Additionally, there was a linear decrease in C14:0 yield as C16:0 was increased in the FA blends, possibly due to competition for the sn-2 position, as C14:0 also has a high affinity for this position (17.1 mol/100 mol; Jensen et al., 1991).The likely shift in distribution of individual FA, such as C4:0, C14:0, and cis-9 C18:1, on milk triglycerides observed in our study indicate mechanisms to ensure milk fat fluidity, as well as efficient utilization of supplied FA versus synthesized FA.
Our current study evaluated mid-lactation cows that are in positive energy balance, which could explain the lack of effect of treatments on BW gain, similar to results reported in studies comparing SFA supplements (Western et al., 2020b;dos Santos Neto et al., 2021a).There was a difference for BCS and BCS change, as increasing C16:0 in FA treatments increased BCS, but the difference was less than a 0.25-point change, suggesting that longer treatment periods are needed to focus on changes in energy partitioning.The difference observed in BCS and BCS change could potentially be due to more C16:0 in the FA blends increasing FA digestibility as it is well established that C18:0 decreases FA digestibility compared with C16:0 and other FA (Boerman et al., 2015a;dos Santos Neto et al., 2021a).Western et al. (2020b) determined energy intake using bomb calorimetry and observed that a 84% C16:0 supplement increased gross energy digestibility and digestible energy intake compared with a supplement containing a mix of C16:0 + C18:0 (33% and 53%, respectively).Digestibility was not determined for our current study, as samples were spoiled due to a freezer malfunction, but we would expect the treatments containing more C16:0 to increase FA digestibility, based on studies utilizing FA supplements containing C16:0 and C16:0 + C18:0 (Western et al., 2020b;dos Santos Neto et al., 2021a;Shepardson and Harvatine, 2021).We would also presume that increasing C16:0 would improve NDF digestibility, as dietary C16:0 has been suggested to increase retention time in the rumen due to an increase in the gut peptide cholecystokinin (Piantoni et al., 2013).Additionally, supplementation of C16:0 to nonlactating Holstein cows and young Holstein dairy bulls increased rumen bacteria populations (Vargas-Bello-Pérez et al., 2016;Zhang et al., 2020) that contain cellulolytic and hemicellulolytic activities (Wang and McAllister, 2002;Krause et al., 2003;Russell et al., 2009).dos Santos Neto et al. (2021a) hypothesizes the potential for C16:0 to spare ATP molecules when incorporated in rumen bacterial membranes (Hackmann and Firkins, 2015), possibly sparing energy that favors bacteria growth by incorporation of C16:0 into the cell membranes and positively affecting fibrolytic bacteria (Hauser et al., 1979;Mackie et al., 1991).Further research should be conducted to evaluate the mechanisms in which C16:0 supplementation effects NDF digestibility and rumen bacteria.
There were some limitations to our study, as we blended commercially available products to achieve our desired ratios instead of a single, prilled product.The SFA supplements are mostly prills, but our blends were a mix of prills and a Ca-salt of palm FA.It is worth considering dissociation of Ca-salts and subsequent biohydrogenation of unsaturated FA, but there were similar inclusion levels of Ca-salts across FA treatments (Table 2) that resulted in similar cis-9 C18:1 contents for all treatment diets, thus biohydrogenation of unsaturated FA to C18:0 should be comparable.Due to the similar ratio of prilled supplement to Ca-salt inclusion in our FA blends, we do not believe that differences in production responses are due to the form of the FA blends, but ultimately due to FA profile of each blend.A recent study evaluating form versus profile of FA supplements suggested that the form of the supplement was more important, as a free FA product (80% C16:0 + 10% cis-9 C18:1) increased production responses compared with a Ca-salt containing similar FA profile (Shpirer et al., 2023).However, results should be interpreted with caution due to considerable diet ingredient alterations between treatments that could be a potential factor in treatment differences as well as discrepancies in the methodologies used for FA analysis of feed and fecal samples.

CONCLUSIONS
Our current study is unique, as we were able to evaluate differences across a range of ratios of C16:0 and C18:0 in FA blends in a single study, which previously has not been reported.Overall, FA supplementation increased milk production responses compared with a non-FA supplemented control.Increasing C16:0 and decreasing C18:0 in FA blends increased DMI, yields of 3.5% FCM, ECM, and milk fat and milk fat content, indicating that mid-lactation cows averaging ~40 to 50 kg/d of milk yield responded best to a FA ratio of 80% C16:0 + 10% C18:0.These results provide further support to recommend increasing C16:0 content and limiting C18:0 content in FA supplements.This study furthered our knowledge of the importance of FA profile of fat supplements and the potential for grouping cows according to production level.Results from this experiment can be applied on-farm and will allow for the targeted nutrition of these production levels to allow for maximum production performance, thus potentially increasing farm profitability.Overall, our study, along with other recent research, supports the use of C16: 0 -enriched supplements and suggests there is little to no benefit of high levels of C18:0 in FA supplements.
Bales et al.: INCREASING PALMITIC ACID IMPROVES PRODUCTION

Table 1 .
Bales et al.:INCREASING PALMITIC ACID IMPROVES PRODUCTION Composition of fatty acid (FA) supplements used to make the FA blends 1 2Average (n = 4) composition of FA supplements based on samples taken during the collection period.

Table 2 .
Proportion of fatty acid (FA) supplements and FA profile of each FA blend