Comparison of rumen and abomasal infusions of an exogenous emulsifier on fatty acid digestibility of lactating dairy cows

We evaluated the effects of infusing an exogenous emulsifier (polysorbates-C18:1) either into the rumen or abomasum on fatty acid (FA) digestibility and production responses of lactating dairy cows. Nine ruminally cannulated multiparous Holstein cows (170 ± 13.6 d in milk) were assigned to a treatment sequence in replicated 3 × 3 Latin squares with 18-d periods consisting of 7 d of washout and 11 d of infusion. Treatments were abomasal infusions of water carrier only into the rumen and abomasum (control, CON), 30 g/d polysorbate-C18:1 (T80) infused into the rumen (RUM), or 30 g/d T80 infused into the abomasum (ABO). Emulsifiers were dissolved in water and delivered at 6-h intervals (total daily infusion was divided into 4 equal infusions per day). Cows were fed the same diet that contained [% diet dry matter (DM)] 32.2% neutral detergent fiber (NDF), 16.1% crude protein, 26.5% starch, and 3.41% FA (including 1.96% FA from a saturated FA supplement containing 28.0% C16:0 and 54.6% C18:0). Two orthogonal contrasts were evaluated: (1) the overall effect of T80 {CON vs. average of the T80 infusions [1/2 (ABO + RUM)]}, and (2) the effect of ABO versus RUM infusion. Compared with CON, infusing T80 increased the digestibilities of NDF (2.85 percentage units), total (4.35 percentage units), 16-carbon (3.25 percentage units), and 18-carbon FA (4.60 percentage units), and tended to increase DM digestibility and total and 18-carbon FA absorption. Compared with RUM, ABO decreased the intakes of total (28 g/d), 16-carbon (7 g/d), and 18-carbon FA (19 g/d); tended to increase the digestibility of total and 18-carbon FA; and had no effect on the absorption of total, 16-car-bon, or 18-carbon FA. Production responses did not change among our treatments. In conclusion, infusing 30 g/d polysorbates-C18:1 increased NDF and total, 16-carbon, and 18-carbon FA digestibility. Compared with RUM, ABO tended to increase the digestibilities of total and 18-carbon FA; however, this may be re-lated to the fact that ABO reduced the intakes of total, 16-carbon, and 18-carbon FA, not necessarily due to better emulsifying action per se. In summary, ABO and RUM both improved FA absorption.


INTRODUCTION
Fatty acid (FA) supplements are commonly used in the diets of dairy cows to increase energy content and support energy requirements for milk production (Rabiee et al., 2012;dos Santos Neto et al., 2021a,b).However, it is well known that the digestibility of FA decreases as the amount of FA reaching the small intestine increases (Boerman et al., 2015).Although total FA flow is an important factor affecting FA digestibility, FA profile also has a great influence.de Souza et al. (2018) reported that feeding a FA blend containing 45% C16:0 and 35% cis-9 C18:1 increased FA digestibility by 11.2 percentage units compared with a FA blend containing 40% C16:0 and 40% C18:0.Including a C18: 0 -enriched supplement at around 1.5% diet DM reduced FA digestibility by around 15 percentage units (Boerman et al., 2017;Prom and Lock, 2021).Furthermore, increasing a C16: 0 -enriched supplement from 0 to 2.25% of diet DM reduced FA digestibility in a soyhull basal diet but had no effect in a whole cottonseed basal diet (Rico et al., 2017a).Potential reasons for the reduction in FA digestibility in ruminants with increasing SFA flow to the small intestine include emulsifying limitations, reduced uptake and re-esterification in enterocytes, and a faster saturation of absorptive sites (Ockner et al., 1972;Glasser et al., 2008;dos Santos Neto et al., 2021b).Therefore, overcoming or improving low FA digestibility should increase nutrient absorption and production responses of dairy cows.
Polysorbates are a class of emulsifiers consisting of one polyethoxylated sorbitan esterified with FA.Their use is common in the pharmaceutical (Kaur and Mehta, 2017) and food (Kralova and Sjöblom, 2009) industries, as well as in calf milk replacers (Jenkins and Emmons, 1984;de Souza et al., 2020).Recently, we performed a series of studies to evaluate the effects of polysorbates on FA digestibility of lactating dairy cows.We observed a quadratic response when abomasally infusing polysorbate-C18:1 from 0 to 45 g/d, where 30 g/d was the optimum dose (de Souza et al., 2020).In addition, 30 g/d polysorbate-C18:1 had the best responses compared with polysorbates esterified with SFA (Prom et al., 2022).Polysorbate also improved FA digestibility to a similar extent as 60 g/d of a highly purified source of cis-9 C18:1 (dos Santos Neto et al., 2022).Despite these positive results, further studies are needed to determine the reliability and practical use of polysorbates as emulsifiers for cows.In our previous studies, we used abomasal infusions to postruminally deliver the exogenous emulsifier.However, to our knowledge, no study has compared infusing polysorbates into the rumen versus the abomasum.Understanding whether ruminal metabolism impacts the effect of polysorbates on FA digestibility is fundamental to paving the way to their inclusion in the diet of dairy cows.
Therefore, the objective of our study was to evaluate the effects of infusing polysorbate-C18:1 either into the rumen or abomasum.We hypothesized that using polysorbate-C18:1 would increase FA digestibility.We further hypothesized that the ruminal infusion would be as effective as the abomasal infusion.

Design and Treatments
All experimental procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University (East Lansing, MI).Nine ruminally cannulated multiparous Holstein cows averaging (mean ± SD) 170 ± 13.6 DIM, 43.6 ± 5.20 kg of milk, and 660 ± 32.6 kg of BW were blocked by milk production and balanced for parity and BCS in 3 squares (each with 3 cows) and then randomly assigned to treatment sequence in a replicated 3 × 3 Latin square design.Each 18-d treatment period consisted of a 7-d washout period and an 11-d infusion period, with sampling during the last 4 d of infusions.Infusions consisted of water carrier only into the rumen and abomasum (control, CON), 30 g/d polysorbate-C18:1 (Tween-80, Sigma-Aldrich; T80) infused into the rumen (RUM), or 30 g/d T80 infused into the abomasum (ABO).The acronyms ABO, RUM, and T80 only refer to the abomasum treatment, rumen treatment, and overall effect of T80, respectively, in the current study.The FA composition of the polysorbate is shown in Table 1.The dose of T80 was chosen based on results from our dose-response study using polysorbate-C18:1 (de Souza et al., 2020).Daily doses of T80 were dissolved in 200 mL of water in individual glass jars.The infusate solutions were divided into 4 infusions per day occurring every 6 h.Stainless steel abomasal infusion devices as described by Westreicher-Kristen and Susenbeth (2017), with the addition of a circular, flexible rubber flange, were inserted into the abomasum 5 d before the start of the study.Infusion lines attached to infusion devices (0.5-cm-diameter polyvinyl chloride tubing) passed through the rumen fistula and sulcus omasi into the abomasum (Tyburczy et al., 2008).Lines were checked daily throughout the study to ensure proper placement.Infusate solutions were delivered into infusion lines using 60-mL plastic syringes.
All animals received a common diet that was formulated to meet the requirements of the animals as determined by NRC (2001; Table 2).The diet included a commercially available C18: 0 -enriched FA supplement (Energy Booster 100, Milk Specialties Global; Table 2) at 1.9% diet DM to better evaluate the potential for T80 to improve FA digestibility.This diet was fed during a 7-d preliminary period and throughout the experiment.Dry matter concentration of forages was determined twice weekly, and diets were adjusted when necessary.Cows were housed in individual tie-stalls throughout the experiment and milked twice daily (0400 and 1500 h).Access to feed was blocked from 0800 to 1000 h for collection of orts and offering of new feed.Feed intake was recorded and cows were offered 115% of expected intake at 1000 h daily.Water was available ad libitum in each stall, and stalls were bedded with sawdust and cleaned twice daily.

Data and Sample Collection
Samples were collected during the last 4 d of each treatment period (d 15 to 18).Samples of all diet ingredients and orts from each cow were collected daily and composited by period for analysis.Milk yield was recorded and 2 milk samples were collected at each milking.One aliquot was collected in a sealed tube with preservative (Bronopol tablet; D and F Control Systems) and stored at 4°C for milk component analysis.The second aliquot was stored without preservative at −20°C for FA composition analysis.Fecal (~400 g) and blood (~15 mL) samples were collected every 9 h over the last 4 d of each period, totaling 8 samples per cow per period.The 9-h interval over 4 d simulates sampling every 3 h over a 24-h period to account for diurnal variation.Feces were stored in sealed plastic cups at −20°C.Blood was stored on ice until centrifugation at 2,000 × g for 15 min at 4°C (within 30 min of sample collection).Plasma was transferred into microcentrifuge tubes and stored at −20°C.
Body weight measurements were taken daily during the sampling period following the afternoon milking.
On the last day of the preliminary period and last day of each treatment period, 3 trained investigators determined BCS on a 5-point scale in 0.25-point increments (Wildman et al., 1982).

Sample Analysis
Diet ingredients, orts, and fecal samples were dried at 55°C in a forced-air oven for 72 h for DM determination.Dried fecal samples for each cow were then composited by period.Dried samples were ground with a Wiley mill (1 mm-screen; Arthur H. Thomas).Feed ingredients, orts, and feces were analyzed for ash, NDF, indigestible NDF, CP, and starch as described by Boerman et al. (2017).Fatty acid content of feed ingredients, orts, and feces was determined as described by Lock et al. (2013).Absolute DM was determined by drying samples in an oven at 105°C using the National Forage Testing Association reference method (Shreve et al., 2006).Indigestible NDF was used as an internal marker to estimate fecal output to determine apparent total-tract digestibility of nutrients (Cochran et al., 1986).The amount of FA in the infusate was considered for the intake, digestibility, and absorption of FA.
Milk samples were analyzed for fat, true protein, and lactose content by mid-infrared spectroscopy (AOAC, 1990;method 972.160;NorthStar Michigan Lab).Yields of milk components, 3.5% FCM, and ECM were calculated using milk yield and component contents from each milking, summed for a daily total, and averaged for each collection period.Milk samples used for analysis of FA composition were composited based on milk fat yield (d 15-18 of each period).Milk lipids were extracted and FAME prepared and quantified using gas-liquid chromatography according to Lock et al. (2013).Yields of individual FA (g/d) in milk fat were calculated by using milk fat yield and FA content to determine yield on a mass basis using the molecular weight of each FA and correcting for glycerol content and other milk lipid classes (Piantoni et al., 2013).
Plasma samples from each cow were composited by period before analysis.All plasma samples were analyzed in duplicate with a coefficient of variation of <5% between duplicates.Plasma samples were analyzed at the Michigan State University Veterinary Diagnostic Laboratory (Lansing, MI).Nonesterified FA were quantified using a Beckman Coulter AU series chemistry analyzer (Beckman Coulter).Insulin was determined with a bovine insulin ELISA using a solid-phase 2-site enzyme immunoassay (Mercodia).Glucose was quantified using a glucose oxidase method (PGO Enzyme Product No. P7119, Sigma Chemical Co.).

Statistical Analysis
All data were analyzed as a replicated 3 × 3 Latin square design using the mixed model procedure of SAS (version 9.4, SAS Institute Inc.) according to the following model: where Y ijkl = dependent variable, μ = overall mean, C(S) i(j) = random effect of cow within square (i = 1 to 3), S j = fixed effect of square (j = 1 to 3), P k = fixed effect of period (k = 1 to 3), T l = fixed effect of treatment (l = 1 to 3), and e ijkl = residual error.Three squares, each with 3 cows, were formed with lower (mean ± SD; milk yield 38.9 ± 0.96 kg/d), medium (milk yield 43.5 ± 2.37 kg/d), and higher (milk yield 50.9 ± 1.00 kg/d) production cows.The interactions period × treatment, period × square, and square × treatment were initially included in the model and removed when P > 0.20 (de Souza et al., 2020;Prom et al., 2021Prom et al., , 2022)).Normality of the residuals was checked with normal probability and box plots and homogeneity of variances with plots of residuals versus predicted values.Two orthogonal contrasts were evaluated: (1) the overall effect of T80 {CON versus the average of the T80 infusions [1/2(ABO + RUM)]}, and (2) the effect of ABO versus RUM infusion.Contrasts were declared significant at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10.All data are expressed as least squares means and standard errors of the means.

DISCUSSION
Improving FA digestibility is important for maximizing nutrient absorption and utilization.Our previous studies have consistently demonstrated that the abomasal infusion of polysorbate-C18:1 increases FA digestibility in lactating dairy cows (de Souza et al., 2018;Prom et al., 2021Prom et al., , 2022)).These studies all used abomasal infusions to evaluate emulsifier effects without possible alterations due to ruminal metabolism.However, understanding whether polysorbates are affected by the ruminal environment is essential to help determine their potential practical use.To our knowledge, no study has previously evaluated the effects of infusing polysorbates into the rumen and abomasum.
Our study provides evidence that FA absorption is improved when infusing polysorbate-C18:1 into the rumen and abomasum.
Compared with CON, infusing T80 did not affect DM or NDF intake, tended to increase DM digestibility, and increased NDF digestibility.Although polysorbate-C18:1 contains UFA (primarily cis-9 C18:1) associated with reductions in intake (Allen, 2000), its hypophagic effects are dose related, as de Souza et al. ( 2020) observed that infusing polysorbate-C18:1 decreased DMI at 45 g/d but not at 30 g/d.Similarly, Prom et al. (2022) anddos Santos Neto et al. (2022) did not observe any effect of infusing 30 g/d polysorbate-C18:1 on DMI.Our results also agree with other research evaluating the dietary inclusion of exogenous emulsifiers such as lysophospholipids to dairy cows (Rico et al., 2017b;Lee et al., 2019) or soy lecithin to beef calves (Shain et al., 1993).Interestingly, despite emulsifiers primarily affecting FA digestibility, we have observed a consistent positive effect of abomasal infusion of polysorbate-C18:1 on NDF digestibility, leading us to speculate this could be driven by an increase in rumen retention or hindgut digestion (dos Santos Neto et al., 2022;Prom et al., 2022).Nonetheless, NDF digestibility did not differ between ABO and RUM in the current study, suggesting that both contributed equally to the overall increased NDF digestibility observed for T80 compared with CON.This might indicate that the increased NDF digestibility is related to an increase in total-tract retention time, even with no changes in DMI.In fact, Piantoni et al. (2013) observed that including a C16:0 supplement in the diets of dairy cows did not affect DMI, and they associated the increase in NDF digestibility with a greater retention time regulated by an increase in cholecystokinin secretion.Additionally, in vitro experiments have reported that polysorbates positively affect microbial growth rates and the activities of cellulase and protease enzymes (Kamande et al., 2000;Goto et al., 2003;Lee and Ha, 2003).This topic deserves further attention.
As expected, T80 increased the digestibilities of total, 16-carbon, and 18-carbon FA compared with CON.Along with our previous findings, where we also observed that abomasal infusion of polysorbate-C18:1 at 30 g/d improved FA digestibility in dairy cows (de Souza et al., 2020;dos Santos Neto et al., 2022;Prom et al., 2022), the present results reinforce our postulation that increasing emulsification capacity can increase FA digestibility (de Souza et al., 2020;Prom et al., 2021Prom et al., , 2022)).Polysorbates are nonionic surfactants; therefore, their mechanism of action likely involves disrupting surface tension with the formation and stabilization of the emulsion, which allows the combination of immiscible substances (Carey and Small, 1970;Kralova and Sjöblom, 2009).Additionally, many surfactants self-aggregate, forming micelles whose polar heads face outward and nonpolar hydrophobic tails face inward (Carey and Small, 1970).Hence, nonpolar FA such as C16:0 and C18:0 can be confined inside the micelle and suspended in aqueous environments.Overall, micelles are important not just for SFA but they also play a crucial role in transporting all FA from feed particles in the lumen of the small intestine through the unstirred water layer to be absorbed by enterocytes (Noble, 1978;Doreau and Ferlay, 1994).We have demonstrated that 30 g/d of polysorbate-C18:1 consistently increases FA digestibility; however, the magnitude of change has varied across studies.In the current study, we observed an overall increase of 4.7 percentage units, whereas total FA digestibility was previously shown to increase by 10.2 (de Souza et al., 2020), 5.4 (Prom et al., 2022), and 3.4 percentage units (dos Santos Neto et al., 2022).These differences may be related to individual animal variation, differences in diet composition, DMI, total flow of FA intake, or stages of lactation.Further studies are needed to evaluate how these factors affect responses to polysorbate-C18:1 and other exogenous emulsifiers.
Although both RUM and ABO increased FA digestibility compared with CON by 3 and 5.7 percentage units, respectively, ABO tended to increase the digestibilities of total and 18-carbon FA compared with RUM.These results may suggest that ruminal infusion is less efficient than abomasal infusion.Polysorbate-C18:1 is an oleate ester of sorbitol; thus, ruminal esterases can hydrolyze the ester bonds releasing cis-9 C18:1 into the rumen environment (McAllister et al., 2000) and exposing it to biohydrogenation.As mentioned previously, cis-9 C18:1 itself can act as an emulsifier.However, we should also consider that, usually, FA digestibility decreases as FA intake increases (Boerman et al., 2015), and ABO had a DMI numerically lower than that of RUM, which resulted in lower intakes of total, 16-carbon, and 18-carbon FA.Therefore, it is possible that the observed differences for ABO compared with RUM were related to FA intake, not necessarily better emulsifying action due to bypassing the rumen.Ultimately, FA absorption was not different between ABO and RUM, indicating that they had similar effects by increasing total and 18-carbon FA absorption compared with CON.Our findings are novel and can have practical implications for the dairy industry.Nonetheless, future studies should examine potential strategies for utilizing polysorbate-C18:1 in diets of lactating dairy cows, including evaluating interactions between polysorbate-C18:1 and different dietary ingredients.
Production responses did not differ among our treatments.Previously, we observed that polysorbate-C18:1 increased FA absorption and the yields of milk fat and 3.5% FCM in studies in which cows averaged (mean ± SD) 109 ± 18 (de Souza et al., 2020), 96 ± 23 (dos Santos Neto et al., 2022), or 138 ± 52 DIM (Prom et al., 2022) at the beginning of the trial.In our current study, cows started the experiment at 170 ± 13.6 DIM, which may have influenced the lack of effects on production responses.It also explains the lower milk yield average in our current study compared with our previous studies (40.6 vs. 46.8 kg/d).Because lactation stage alters energy partitioning in dairy cows, it is possible that the additional absorbed FA could be partitioned to body reserves.However, our short-term Latin square study was primarily designed to examine nutrient digestibility and may not have had adequate power or period lengths to properly evaluate production and BW-related variables (Prom et al., 2021;dos Santos Neto et al., 2022).There is high variability in assessing BW and BW change, mainly due to gut fill (Prom et al., 2021).In addition, insulin increases nutrient uptake by muscle and adipose tissue (Bauman and Elliot, 1983), and we did not observe changes in plasma insulin or any other blood parameter.

CONCLUSIONS
Both RUM and ABO infusions of polysorbate-C18:1 increased the digestibilities of total, 16-carbon, and 18-carbon FA.Compared with RUM, ABO tended to increase the digestibilities of total and 18-carbon FA; however, this may be related to the fact that ABO reduced the intakes of total, 16-carbon, and 18-carbon FA, not necessarily due to better emulsifying action per se.We observed that ABO and RUM had similar FA absorption, indicating they had similar effects by increasing total and 18-carbon FA absorption compared with CON.Polysorbate-C18:1 increased NDF digestibility compared with CON, with no differences between ABO and RUM.Our results suggest that polysorbate-C18:1 has the potential to increase FA digestibility without the need for it to be rumen protected.Although there are possible practical implications for the dairy industry, further studies are warranted and should investigate the dietary inclusion of polysorbate-C18:1 and the best strategy for its use on farms, considering other management factors.

dos
Santos Neto et al.: OPPORTUNITIES FOR IMPROVING FATTY ACID DIGESTIBILITY

Table 1 .
Fatty acid (FA) profile and total FA content of the polysorbate supplement infused during treatment periods

Table 4 .
dos Santos Neto et al.: OPPORTUNITIES FOR IMPROVING FATTY ACID DIGESTIBILITY Dry matter intake, milk yield, milk composition, BW, and BCS of cows on treatment diets (n = 9)

Table 5 .
Milk fatty acid yields and contents by source of cows infused with treatments (n = 9)