Effects of Dietary Potassium Carbonate and Fat Concentration in High Distiller Grain Diets Fed to Dairy Cows

EFFECTS OF DIETARY POTASSIUM CARBONATE AND FAT CONCENTRATION IN HIGH DISTILLER GRAIN DIETS FED TO DAIRY COWS

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Kathryn Cara Lamar, B.S.

Graduate Program in Animal Sciences

The Ohio State University

2013

Master’s Examination Committee

Dr. William P. Weiss, Advisor

Dr. Steven C. Loerch

Dr. Kristy M. Daniels

Kathryn Cara Lamar

2013 `

ABSTRACT

Distiller grains with solubles (DGS) can induce milk fat depression when included in dairy cow diets at greater than 20% DM. In vitro experiments have found that potassium (K) supplementation with potassium carbonate (K2CO3) decreased concentrations of biohydrogenation intermediates associated with milk fat depression

(MFD), such as trans-10, cis-12 conjugated linoleic acid (CLA). These intermediates are often produced when diets are fed to cows with high concentrations of polyunsaturated fatty acids, like those in DGS. We hypothesized that there would be an interaction between level of K and level of fat. We hypothesized that adding K2CO3 to a high fat diet based on DGS would alleviate MFD. We also hypothesized that the addition of K2CO3 to a low fat diet based on DGS would have no effect on milk fat percent because these diets would not cause MFD. Sixteen Holstein cows averaging 157 days in milk were placed into 4 blocks; each block comprised a 4x4 Latin square with 21 d periods and a

2x2 factorial arrangement of treatments. The basal diet (no added K or fat) contained

27% DGS, 47% corn silage, 22% starch, 32% NDF, 4.2% long chain fatty acid, and 1.2% K

(DM basis). Treatments were 0 or 2.3% added fat from corn oil (in high fat diets, DGS + corn gluten meal + corn oil = 27%) with 0 or 1% added K. Diets with added K had

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supplemental K in the form of K2CO3 from DCAD Plus® (Church & Dwight Co., Inc.,

Princeton, NJ). DCAD is a measure of the balance between cations and anions in the diet. DCAD over 20 meq/100 g DM improves performance for lactating cows, while low or negative DCAD benefit dry cows. Diets with added K had a DCAD of approximately 30, while diets without added K had a DCAD of 2. This low DCAD may have limited performance for cows fed diets without added K. Dry matter intake (DMI) decreased with added fat (21.0 vs. 22.5 kg/d; P<0.01) and tended to decrease with added K (21.4 vs. 20.1 kg/d; P<0.06). Milk yield decreased with added fat (30.5 vs. 32.3 kg/d; P<0.01), which may have been due to decreased DMI. No fat x K interaction was observed perhaps because MFD occurred with all diets. Milk fat percent increased with added K

(2.82% vs. 2.56%; P<0.01) and decreased with added fat (2.51% vs. 2.89%; P<0.01). Milk fat yield was affected similarly and tended to increase with added K (0.87 vs. 0.82 kg/d;

P<0.10) and decreased with added fat (0.76 vs. 0.93 kg/d; P<0.01). Trans-10, cis-12 CLA in milk decreased with added K (P<0.02) indicating that the additional K2CO3 was decreasing incomplete biohydrogenation. Trans-10, cis-12 CLA increased with added fat

(P<0.01) because excess unsaturated fatty acids in the diet results in increased incomplete biohydrogenation. Supplemental K2CO3 led to an increase in milk fat for both high fat and low fat diets indicating that it could be used to alleviate MFD, though values did not return to levels of a typical Holstein cow.

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ACKNOWLEDGEMENTS

Big thanks to Dr. Weiss for giving me a shot. Thanks for all the time you spent reading over my proposal, work plan, and thesis, watching me practice presentations, and teaching me how to be an all around better scientist and researcher. Thanks to Dr.

Loerch for being there when I needed help stringing words together for applications and cover letters and for all of his help and support. Thanks to Dr. Kristy Daniels for all of her advising while I was here and also for her help and support, as well.

Donna Wyatt, thank you so much for teaching me how to be in a lab. All of my techniques and knowledge are because of you and I can’t thank you enough for that. I will miss our talks greatly.

To Kevin and his farm crew, thank you for all the work you guys did for my experiment.

To my family and friends, I can’t even begin to express how invaluable your love and support were during this time. There’s no way I could have made it through all of this without you.

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VITA

September 28, 1990………………………………………Born – Columbus, Ohio

May 2008………………………………………………………Gahanna Lincoln High School

2011………………………………………………………………B.S. Agriculture, The Ohio State

University

2011 to present………………………………………………Graduate Research, Animal Sciences,

The Ohio State University

FIELDS OF STUDY

Major Field: Animal Sciences

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TABLE OF CONTENTS

Abstract ...... ii Vita ...... v Table of Contents ...... vi List of Tables ...... vii List of Figures ...... viii Chapter 1: Literature Review ...... 1 Introduction...... 1 Sodium ...... 2 Sodium Absorption ...... 3 Sodium Deficiency ...... 4 Sodium Toxicity ...... 5 Potassium ...... 5 Potassium Absorption ...... 7 Potassium Deficiency ...... 8 Potassium Toxicity ...... 8 Chloride ...... 9 Chloride Absorption ...... 9 Chloride Deficiency ...... 10 Chloride Toxicity ...... 11 Sulfur ...... 11 Sulfur Absorption ...... 12 Sulfur Toxicity ...... 13 Dietary Cation Anion Difference ...... 13 Hypocalcemia ...... 14 Summary ...... 17 Chapter 2: Introduction ...... 19 Chapter 3: Materials and Methods ...... 22 Chapter 4: Results and Discussion ...... 28 References ...... 63

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LIST OF TABLES

Table 1: Ingredient composition of diets, DM basis ...... 39 Table 2: Nutrient composition of diets ...... 40 Table 3: Nutrient composition of corn silage ...... 41 Table 4: Nutrient composition of distiller grains ...... 42 Table 5: Effects of treatment on DMI, milk production, and milk composition, all 3 wk of treatment ...... 43 Table 6: Effects of treatment on DMI, milk production, and milk composition, wk 3 ..... 44 Table 7: Effects of treatment on milk fatty acid concentrations, all 3 wk ...... 45 Table 8: Effects of treatment on milk fatty acid concentrations, 30 h into treatment .... 47 Table 9: Effects of treatment on milk fatty acid concentrations, d 21 of treatment ...... 49 Table 10: Effect of treatment on proportion of C 18:0 and C 18:2 relative to total concentration of C 18 fatty acids ...... 51 Table 11: Effect of treatment on estimated urine excretion (L/d) ...... 52 Table 12: Effect of treatment on mineral intake and excretion (g/day) ...... 53 Table 13: Effect of treatment on urine mineral excretion/mineral intake...... 55 Table 14: Effect of treatment on mineral milk concentration, g/kg ...... 56

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LIST OF FIGURES

Figure 1: Effect of level of dietary fat and K on DMI by week within period...... 57 Figure 2: Effect of level of dietary fat and K on DMI over the entire period...... 58 Figure 3: Effect of level of dietary fat and K on milk production over the entire period . 59 Figure 4: Effect of level of dietary fat and K on milk fat yield over the entire period ...... 60 Figure 5: Effect of level of dietary fat and K on milk fat percent over the entire period . 61 Figure 6: Correlation of milk fat percent to trans-10 cis-12 concentration ...... 62

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CHAPTER 1: LITERATURE REVIEW

Introduction

Dietary cation anion difference (DCAD) is the measure of the difference between cations, sodium (Na) and potassium (K), and anions, chloride (Cl) and sulfur (S), in dairy cows diets. These ions have the greatest effect on acid-base balance in the body. Adding cations increases DCAD value and adding anions decreases the DCAD value. DCAD levels in diets can affect many things including feed intake, acid-base levels in the body, blood and urine pH. The DCAD is generally expressed as milliequivalents (mEq) of [(Na+ + K+) –

(Cl– + S2–)]/100 g of dietary DM. It can also be expressed as mEq/kg. The DCAD equation

DCAD = (Na+ + K+) - (Cl- + S2-) is the most commonly used form of the equation in dairy cattle nutrition (Ender et al., 1962; Block, 1984). Lean et al., (2006) concluded that this equation was the best for predicting milk fever incidence in dairy cows. Horst et al.

(1997) recommended that other anions and cations be included in the equation. He proposed the equation DCAD = (0.38 Ca2+ + 0.3 Mg2+ + Na+ + K+) - (Cl- + S2-). Goff (2000) proposed a variation of this equation. His equation took into account the capacity of different salts to acidify urine and recommended DCAD = (0.15 Ca2+ + 0.15 Mg2+ + Na+ +

K+) - (Cl- + 0.25 S2- + 0.5 P3-). Tucker et al. (1991) suggested that the DCAD equation should be DCAD= (0.38 Ca2+ + 0.3 Mg2+ + Na+ + K+) - (Cl- + 0.6 S2- + 0.5 P3-) based on the

` research of Spears et al. (1985). The DCAD equation used in this thesis will be DCAD =

(Na+ + K+) - (Cl- + S2-).

Sodium

Sodium is the primary extracellular cation (Aitken, 1976). Heart function and nerve impulse conduction and transmission are dependent on the proper balance of Na and K. Sodium is also involved in the sodium-potassium pump, which enables transport of glucose, amino acids, and phosphate into cells, and hydrogen, calcium, bicarbonate,

K, and Cl ions out of cells (Lechene, 1988). Sodium is a major component of salts in saliva, which buffers acid from ruminal fermentation (Blair-West et al., 1970).

Milk production can increase with addition of sodium bicarbonate (NaHCO3).

Sodium bicarbonate produced small increases in DMI and milk yield (MY) (Canale and

Stokes, 1988). Sodium bicarbonate increased ruminal pH and MY response was maximal with 0.70% Na and 1.58% K (Stokes and Bull, 1986). The addition of Na2CO3 at 0.78% increased milk fat percent, age and yield, and 4% fat corrected MY (Belibasakis and

Triantos, 1991). Though, in one commercial herd where diets were based on alfalfa hay,

0.8% NaHCO3 reduced MY in second lactation and older animals (Canale and Stokes,

1988). Dairy cattle absorb dietary Na very efficiently, but only very small amounts are stored in a form that is readily available for metabolism. Concentration in milk is between 25 and 30 mEq/L. Sodium concentration increases during mastitis when serum leaks into milk, but is not significantly affected by dietary Na content (Kemp, 1964)

Typical concentrations of Na in blood plasma are 150 mEq/L and 160 to 180 mEq/L in saliva.

Empirical modeling of data from 15 experiments with lactating cows conducted in either cool or warm seasons showed that DMI and MY were improved by dietary concentrations of Na well above those needed to meet requirements (Sanchez et al.,

1994a,b). Dry matter intake (DMI) and MY responses over a range of dietary Na concentrations from 0.11 to 1.20% DM were curvilinear, with maximum performance at

0.70 to 0.80% DM. In hot weather, MY and DMI increased when Na was supplemented from 0.18% Na to 0.55% total dietary Na with either NaCl or NaHCO3 (Schneider et al.,

1986).

Sodium Absorption

Agricultural Research Council (1980) estimated that 91% of Na consumed by cattle was absorbed. Apparent absorption of Na by dairy cows fed fresh forages ranges from 77 to 95%, with an average of 85% (Kemp, 1964). Sodium chloride (NaCl) is most often used and the Na in NaCl is essentially 100% available. Sodium absorption occurs throughout the digestive tract by an active transport process in the reticulorumen, abomasum, omasum, and duodenum. Passive absorption occurs through the intestinal wall. Substantial active absorption against a sizable concentration gradient also occurs in the lower small intestine and large intestine (Renkema et al., 1962). Sodium concentrations in blood and tissues are maintained principally via reabsorption and excretion by the kidneys. There is close synchrony between the excretion of Na, K, and

Cl. Sodium is the central effecter of ion excretion and changes in renal resorption determine Na excretion. Endocrine control via tissue receptors and the renin- angiotensin system, aldosterone, and atrial natriuretic factor monitor and modulate Na concentrations in various tissues, which consequently control fluid volume, blood pressure, K concentrations, and renal processing of other ions. Kidneys are efficient in reabsorbing Na when dietary Na is deficient.

Sodium Deficiency

When Na is deficient, it is decreased in saliva and is reabsorbed in the kidneys.

Mallonee et al. (1982a) found when feeding a diet without supplemental NaCl (0.16%

Na) feed intake and MY began to decline within 1 to 2 wk. Pica and drinking of urine of other cows were also observed (Mallonee et al., 1982a). Although dietary Cl concentration was not measured in the study, potassium chloride (KCl) was supplemented (1.0% DM KCl), so Cl deficiency was not the cause of the condition.

Babcock (1905) fed a diet very low in Na to dairy cows and described intense craving for salt and general pica. Other deficiency signs include loss of appetite, rapid loss of body weight, haggard appearance, lusterless eyes, and rough hair coat (Underwood, 1981).

More extreme signs of deficiency include loss of coordination, shivering, weakness, dehydration, and cardiac arrhythmia leading to death. Feeding lactating dairy cows a diet with no supplemental NaCl (0.16% Na DM) resulted in marked depressions in DMI and MY after just 1 to 2 wk of feeding (Mallonee et al., 1982a).

Sodium Toxicity

The National Research Council (2001) states that the maximum tolerable dietary concentration of NaCl is 4% for lactating cattle, which equals about 1.6% Na. (National

Research Council, 2005). High intakes of NaCl can lead to an increase of incidence and severity of udder edema (Randall et al., 1974). Toxicity signs included severe anorexia, reduced water intake, dehydration, weight loss, and ultimately physical collapse.

Feeding diets with 0.88% Na from NaCl or NaHCO3 to mid-lactation Holstein cows did not cause toxicity or reduce feed intake and MY compared with 0.55 % Na (Schneider et al., 1986). With an adequate supply of clean drinking water, cattle can tolerate large quantities of dietary NaCl. Jaster (1978) provided drinking water with 0 or 2.5 g/L NaCl for a 28-d period to lactating cows and MY declined and water consumption increased.

Cattle drinking water that contained 0.7 to 1.5 mEq/L NaCl suffered from toxicosis

(Weeth et al., 1960; Weeth and Haverland, 1961).

Potassium

Potassium is the main intracellular cation and is the third most abundant mineral element in the body. It must be supplied daily in the diet because there is little storage in the body and the animal’s requirement for K is highest of all the mineral element cations. Milk contains about 38.5 mEq/L K. Saliva typically contains 10 mEq/L, whereas concentrations in ruminal fluid range from 40 to 100 mEq/L. Blood plasma contains 5 to

10 mEq/L K per liter. The majority of K in blood is located within red blood cells (Aitken,

1976; Hemken, 1983). Early research indicated that 0.70 to 0.75% dietary K was

` sufficient to meet requirements of early and mid- to late-lactation cows (Dennis et al.,

1976; Dennis and Hemken, 1978; Erdman et al., 1980), though the requirement is now set at 1.0% DM (National Resource Council, 2001).

Potassium is involved in osmotic pressure and membrane potential, acid-base regulation, water balance, nerve impulse transmission, muscle contraction, oxygen and carbon dioxide transport; phosphorylation of creatine, pyruvate kinase activity, as an activator or co-factor in many enzymatic reactions, cellular uptake of amino acids and synthesis of protein, carbohydrate metabolism, and in maintenance of normal cardiac and renal tissue (National Research Council, 2001; Stewart, 1981; Hemken, 1983).

Forages are generally higher in K than grains (National Research Council, 2001).

In an empirical modeling of data with 1,444 cow-period observations, DMI and

MY responses over a range of dietary K concentrations from 0.66 to 1.96 %, results were curvilinear, with maximum performance when diets contained 1.50% K in the cool season. In the warm season, DMI and MY increased as K% DM increased (Sanchez et al.,

1994a,b). Mallonee (1984) found no benefit of increasing dietary K from 1.07 to 1.58% on feed intake or lactational performance of mid-lactation Holstein cows. Feed intake and MY were reduced with the 4.6% K, and water intake, urinary excretion, and total K excretion were increased with increasing concentrations of K in a study by Fisher et al.

(1994). Many studies have found that feeding higher concentrations of dietary K than needed to meet National Research Council (2001) recommendations of lactating cows in thermoneutral environments increased feed intake and MY compared with cows fed

` lower dietary concentrations (Beede et al., 1983; Schneider et al., 1984; Mallonee et al.,

1985; Schneider et al., 1986; West et al., 1987; Sanchez, 1994a). A dietary K concentration of 1.5% DM during heat stress maximized lactational performance (Beede and Shearer, 1991). Scheider et al. (1986) showed greater production from heat- stressed cows if dietary K levels were above NRC recommended levels.

Potassium Absorption

Potassium in feeds exists as simple ions, which are readily available for absorption (Emanuele and Staples, 1990, 1991; Ledoux and Martz, 1990). Hemken

(1983) indicated that K is almost completely absorbed with a true absorption of 95% or greater for most feedstuffs. Average apparent absorption of K in eight forages fed to cattle and sheep was 85% (Miller, 1995). An absorption coefficient value of 90% for K is used for all types of feedstuffs and mineral sources of K (National Research Council,

2001). Potassium is mainly absorbed from the duodenum by simple diffusion, though some absorption also occurs in the jejunum, ileum, and large intestine. The main excretory route of excess absorbed K is via the kidneys. This route is lower concentrations of K in plasma and milk, higher blood hematocrit reading, and overall primarily under regulation by aldosterone, which increases Na resorption in the kidney with the concomitant excretion of K. Blood acid-base status also affects urinary excretion of K (McGuirk and Butler, 1980). With the onset of an alkalotic condition, intracellular protons are exchanged with K in blood plasma as part of the regulatory mechanisms to maintain acid-base equilibrium and blood pH, reducing K in blood. A

` large gradient exists between intracellular renal tubule concentrations of K and that of urine. This gradient affects the passage of K from the tubular cells into urine.

Potassium Deficiency

Signs of K deficiency include a decrease in feed and water intake, reduced body weight and MY, pica, loss of hair glossiness, decreased pliability of the hide, muscle weakness. Rate and severity of deficiency appeared to be related to milk production, where higher producing cows were affected more quickly and severely than lower producing cows. Signs of severe K deficiency were manifested in lactating dairy cattle fed diets with less than 0.15 % K (Pradhan and Hemken, 1968; Mallonee et al., 1982b).

In a trial with mid-lactation cows, 0.42% dietary K reduced DMI and MY; however, no differences in DMI or MY were noted for cows consuming diets with 0.69 or 0.97 % K

DM (Dennis and Hemken, 1978). Severe K deficiency can occur in diets with 0.06 to

0.15% K (Pradhan and Hemken, 1968; Mallonee et al., 1982b).

Potassium Toxicity

Absorbed K in excess of requirements is excreted mainly in urine. Feeding dietary

K above requirement can reduce magnesium absorption and may cause udder edema.

The maximum tolerable concentration in the diet for dairy cattle is 3.0% of DMI

(National Research Council, 2001). When 4.6% dietary K via supplemental potassium carbonate (K2CO3) was fed to cows during early lactation, DMI and MY were reduced, and water intake, urinary excretion, and total K excretion were increased (Fisher et al.,

1994).

Chloride

Depending on MY, the dietary requirement for Cl is between 0.25%-0.30% of dietary DM. Typical concentrations of Cl in blood plasma are between 90 and 110 mEq/L and 10 to 30 mEq/L in ruminal fluid. During lactation, Cl concentration is highest in colostrum, declines to normal levels, then rises again at the end of lactation and is present in milk at 32.5g/L on average (Agricultural Research Council, 1965). Chloride is the most common anion in extracellular body fluids of mammals (Fettman et al, 1983), making up more than 60 % of the total anion equivalents in extracellular fluid. As such,

Cl is also the major anion in gastric secretions in the form of hydrochloric acid (HCl), which is also known as gastric acid and is needed for digestion. Gastric acid is necessary for the activation of pepsin, which is required for protein digestion. Chloride is also essential for the activation of pancreatic amylase and is found in large concentration in bile and other intestinal juices (Phillipson, 1977). About 80 % of the Cl in the digestive tract arises from digestive secretions in saliva, gastric fluid, bile, and pancreatic juice.

Chloride Absorption

Chloride is absorbed throughout the digestive tract. The absorption coefficient for Cl from both feedstuffs and mineral sources is approximately 90% for dairy cattle

(Henry, 1995c). The Cl from HCl is absorbed in the small intestine by passive diffusion along an electric gradient by exchange with bicarbonate (Tucker et al., 1987). Chloride is transported across the ruminal wall to blood against a wide concentration gradient

(Sperber and Hyden, 1952). Chloride is co-transported actively with Na across the rumen

` wall (Martens and Blume, 1987). Excess Cl is excreted mainly in urine and feces as NaCl or KCl (Sanchez et al., 1994b). Regulation of the concentration of Cl in extracellular fluid and its homeostasis is coupled with Na. Chloride excretion is also influenced by the bicarbonate ion. If blood bicarbonate rises, a similar amount of Cl is excreted by the kidneys to maintain systemic acid-base balance.

Chloride Deficiency

Babcock (1905) offered free choice KCl instead of NaCl to a cow fed a diet with no supplemental NaCl and the cow ate a considerable amount of it, suggesting that the deficiency was for Cl, not NaCl. Though, it could also suggest that cattle preferred the taste of KCl to NaCl. Experiments that fed diets with 0.18% Cl (i.e, less than the current

NRC requirement) found that cows conserved Cl by dramatically reducing excretion of Cl in urine, feces, and milk (Coppock et al., 1979; Fettman et al., 1984). Coppock et al.,

(1979) also found that cows fed a low Cl diet consumed more salt block than cows fed

0.40% Cl. Fettman et al. (1984) found that cows fed 0.10 % Cl rapidly exhibited clinical signs of deficiency and poor performance compared with those fed medium and high concentrations of dietary Cl. Clinical signs of Cl deficiency are reduced appetite, weight loss, lethargy, emaciation, decreased lactation, constipation, cardiovascular depression, excessive thirst, and excessive urination. In advanced stages, cows can suffer from severe eye defects, reduced respiration rates, and blood and mucus in feces.

Metabolically, Cl deficiency resulted in severe metabolic alkalosis and low blood Cl which can result in low blood Na and K.

Chloride Toxicity

Maximum tolerable dietary concentration of Cl is 4.5% for non-lactating cows and 3.0% for lactating cows (National Research Council, 2005). Negative effects of excess dietary Cl include decreased DMI and MY, with effects being more pronounced in summer weather than in winter (Sanchez et al., 1994a). Empirical models with 1,444 cow-period means showed that increasing dietary Cl from 0.15 to 1.62% decreased DMI and MY of mid-lactation cows (Sanchez et al., 1994a). Negative effects of increased dietary Cl were more dramatic in summer weather than in winter (Sanchez et al.,

1994a). This is consistent with the results of Escobosa et al. (1984) showing profound exacerbating effects of high dietary Cl on acid-base balance and milk production during heat stress.

Sulfur

The S requirement for dairy cows is 0.20% of dietary DM. The dietary requirement of S provides enough substrate to ensure maximal microbial protein synthesis (Bouchard and Conrad, 1973 a, b). Sulfur is found in the amino acids methionine, cysteine, homocysteine, and taurine (National Research Council, 2001). It is also present in the B-vitamins, thiamin and biotin. Non-protein nitrogen, such as urea, added to diets cannot be incorporated into microbial protein unless adequate S is present for cysteine and methionine formation. The strong reducing environment within the rumen can reduce dietary sulfate, sulfite, and thiosulfate to sulfide (Lewis, 1954).

High concentrations of dietary S can decrease feed intake and overall performance of

` ruminants. While in the digestive tract, hydrogen sulfide gas, hydrogen sulfide in solution, sulfur dioxide gas, and pentathionic acid are produced (Lis, 1983). Sulfide absorbed from the rumen can be detoxified by oxygenated hemoglobin in the blood

(Evans, 1967) and the liver through the sulfide oxidase system (Anderson, 1956).

Not all bacteria in the rumen utilize all forms of S (Emery et al., 1957a).

Elemental S is not well utilized by many ruminal bacteria (Ishimoto et al., 1954). Emery

(1957a) and Emery et al. (1957b) reported that ruminal microbes produce twice as much cysteine as methionine from inorganic sulfate. Bryant (1973) found that the predominant ruminal cellulolytic bacteria, Fibrobacter succinogenes, could utilize sulfide or cysteine but not sulfate. Many strains of Ruminococcus grew in media containing only sulfide or sulfate-sulfur (Bryant, 1973).

Sulfur Absorption

Sulfur incorporated into microbial protein is absorbed from the small intestine as cysteine and methionine. Some dietary S is absorbed as the sulfate or sulfide anion.

Sulfate-sulfur is absorbed more efficiently in the small intestine than other sources of S

(Bird and Moir, 1971). Elemental S is much less available, probably because it is not very soluble (Fron et al., 1990). Lignin sulfonate is also a poorly utilized source of S (Bouchard and Conrad, 1973a). The sulfur-containing amino acids provide a major dietary source of

S for the ruminal microbes. Protection of proteins and amino acids from ruminal degradation could result in less S being available for microbial protein synthesis in the rumen, but will help the cow obtain amino acids required for her tissues. Methionine,

` methionine analogs, and sulfate salts are utilized equally well in meeting the dietary S requirements of the cow and ruminal microbes (Bouchard and Conrad, 1973a, b; Bull and Vandersall, 1973; Thomas et al., 1951).

Sulfur Toxicity

Studies by Beke and Hironak (1991) and Mcallister et al. (1997) found that polioencephalomalacia, a neurological disease associated with thiamine deficiency, occurs in beef cattle fed greater than 0.5% S. For dairy cattle, the NRC set maximum dietary S levels at 0.3% for high starch diets and 0.5% for high forage diets (National

Research Council, 2005). Symptoms of high concentration of dietary S are severe watery diarrhea, respiratory distress, muscle twitching, severe dehydration, congested lungs, acute enteritis, abdominal pain, and strong odor of hydrogen sulfide on the breath (Lis,

1983). Neurotoxic effects of sulfide are caused by eructation of hydrogen sulfide along with other gasses from the rumen. These gasses are then absorbed through the lungs

(Bird, 1972). Sulfates are less toxic, though they can cause an osmotic diarrhea and excess sulfate added to rations can reduce feed intake and performance (Kandylis,

1984).

Dietary Cation Anion Difference

Tucker et al. (1988) found that cows fed DCAD 20 mEq/100 g of DM yielded 9% more milk than those fed a DCAD of -10 mEq/100 g of DM. West et al. (1991) found in both hot and cool environments, increasing DCAD from -12 to 31 mEq/100 g of DM increased DM, MY, 4% FCM, and milk protein. Delaquis and Block (1995) reported that

` increasing DCAD from 6 to 26 mEq/100 g of DM increased milk production in early and mid-lactation, but not in late lactation. Wildman et al. (2007) fed diets providing DCAD of 25 or 50 mEq and higher DCAD improved DMI, MY, and concentrations of milk fat and protein. Erdman (2011) and Hu and Murphy (2004) found that maximal MY and DMI occurred at DCAD of 34-40 mEq/100 g of DM. In a study by Harrison, et al. (2012), increased milk production was achieved at a DCAD of 53 mEq/100 g of DM versus a

DCAD of 32 mEq/100 g of DM.

Hypocalcemia

The transition period in dairy cow, as defined by Grummer (1995), is the three wk before and after parturition. The transition period is considered the most difficult time for a dairy cow, determining the cow’s health, production, and reproduction in the subsequent lactation (Keady et al., 2001). During the transition period, the clearance of

Ca to the placenta ceases, but the lactational Ca demand increases rapidly (Roche et al.,

2003b). Though milk fever affects only a small percentage of cows, nearly all cows experience some decrease in blood calcium during the first days after calving, while their intestines and bones adapt to the calcium demands of lactation (Ender et al. 1971;

Ramberg, 1974).

Nearly all high producing dairy cows will undergo some degree of hypocalcemia within the first 2 d of parturition and subsequent onset of lactation (Ramberg et al.,

1984). Daily body turnover of Ca changes from approximately 10 g in non-lactating cows to greater than 30 g in lactating cows (Horst et al., 1997). In cows with hypocalcemia,

` the calcium homeostatic mechanisms, which normally maintain blood calcium concentration between 9 and 10 mg/dL, fail and the lactational drain of calcium causes blood calcium concentration to fall below 5 mg/dL. Normal physiological levels of plasma calcium range from 8.5 to 11.5 mg/dL and a normal decrease in plasma calcium of 2 mg/dL is expected at calving (Niedermeier et al., 1949). Hypocalcemia may be classified as subclinical, with levels from 7.5 to 8.5 mg/dL, or clinical, with levels from 5.0 to 6.0 mg/dL (Jorgensen, 1974). During the dry period calcium requirements are minimal and the mechanisms in place to replace calcium lost from the plasma pool are relatively inactive (Ramberg et al., 1984).

Hypocalcemia can impair muscle and nerve function enough so that cows are unable to rise. Cows that have had milk fever are more susceptible to other disorders such as lack of appetite, mastitis, displaced abomasum, retained placenta, and ketosis

(Curtis et al., 1985). Hypocalcemia at calving is a predisposing factor for dystocia, prolapsed uterus, retained placenta, and early metritis (Erb and Grohn, 1988; Grohn et al., 1989). Animals suffering from clinical hypocalcemia exhibit symptoms such as decreased appetite, tetany, inhibition of urination and defecation, lateral recumbency, and eventual coma and death if untreated (Horst et al., 1997). Milk production may suffer long after the transition period has passed (Block, 1984).

The adaptation to the onset of lactation during the critical first days of lactation is accomplished by release of parathyroid hormone (PTH), which reduces urinary calcium losses, stimulates bone calcium resorption, and increases 1,25-dihydroxyvitamin

D synthesis to enhance active intestinal transport of calcium. All three must be operational if hypocalcemia is to be minimized. Milk fever risk factors reduce the efficiency of one or more of these homeostatic mechanisms. By carefully regulating the amount of 1,25-dihydroxyvitamin D produced, the amount of dietary calcium absorbed can be adjusted to maintain a constant concentration of extracellular calcium (DeLuca,

1979; Bronner, 1987).

Metabolic alkalosis impairs the physiologic activity of PTH so that bone resorption and production of 1,25-dihydroxyvitamin D are impaired reducing the ability to successfully adjust to the calcium demands of lactation (Block, 1984; Block, 1994;

Gaynor et al., 1989; Goff et al., 1991; Phillippo et al., 1994). Evidence suggests that metabolic alkalosis induces conformational changes in the PTH receptor, which prevents tight binding of PTH to its receptor.

Prepartum DCAD dramatically affects Ca metabolism at parturition (Oetzel et al.,

1988; Goff et al., 1991). Diets with a low DCAD alter Ca homeostasis and increase plasma Ca concentration at parturition, which helps prevent hypocalcemia. Goff et al.

(1991) observed that Jersey cows fed anionic diets had an increased 1, 25-(OH) 2 D response per unit of decline in Ca in serum. Anionic diets increase the efficiency of Ca absorption from the gastrointestinal tract (Lomba et al., 1978). Studies demonstrated that addition of anions to the prepartal diet could prevent milk fever (Ender et al., 1971;

Block, 1984), though Hu and Murphy (2004) found no evidence of a direct relationship between blood Ca concentration and DCAD postpartum. Higher blood Ca concentrations

` were reported for cows fed anionic diets (Block, 1984; Joyce et al., 1997) as acidogenic diets increase serum Ca by increasing Ca mobilization from bone.

Summary

Dietary DCAD can have a large effect on dairy cows. For lactating cows, high levels of DCAD, from 30-40 mEq/100 g DM, can lead to an increase in DMI, milk production, and yield of milk components. For dry cows, a low or negative DCAD can decrease the risk of hypocalcemia, or milk fever, which decreases the risk of other significant illnesses as well. DCAD affects overall body acid-base balance, which affects the overall health of a dairy cow and balance of other minerals in the body, as well.

Understanding of the effects of the balance between the cations and anions in dairy cows can be beneficial for dairy farmers.

Potassium is one of the cations used to calculate DCAD. In dairy cows, it has recently been reported that addition of K in the form of K2CO3 can increase milk fat production by decreasing the incomplete ruminal biohydrogenation caused by unsaturated fat in the diet. We hypothesized that supplementing K2CO3 to a diet with a high concentration of fat from DGS would alleviate MFD, but would have no effect when supplemented to a diet with lower concentrations of fat because these lower fat diets would likely not induce MFD. Further, we hypothesized that the positive effect of K2CO3 would be caused by reducing the production of trans-10, cis-12 CLA caused by incomplete biohydrogenation of the dietary PUFA. The objective of this study was to

determine whether supplemental K2CO3 could alleviate the decrease in milk fat percent caused by an increase of unsaturated fat in a diet with DGS.

CHAPTER 2: INTRODUCTION

Dried distiller grains with solubles (DGS) are a byproduct of the ethanol industry and are traditionally fed as a protein supplement. Distiller grains with solubles are also an excellent source of energy due to their high concentration of digestible NDF and fat, specifically C 18:2 (Schingoethe et al., 2009). Griinari et al. (1998) demonstrated severe

MFD when unsaturated fat from corn oil, which is similar to the fat in DGS, was added to diets that contained 14.8 vs. 32.1% NDF. Leonardi et al. (2005) found slight, linear decreases in milk fat content as DGS increased from 0 to 15% of diet DM. In a study by

Kalscheur (2005), milk fat content was lower only in DGS diets that contained less than

50% forage and 22% forage NDF. Experiments by Abdelqader et al., (2008) and Leonardi et al. (2005) indicated that the effects of adding corn oil to diets is similar to those when fat from DGS is added, when levels of dietary fat are similar. Diets containing high concentrations of polyunsaturated fatty acids (PUFA), such as that found in DGS, can depress milk fat content. The predominant PUFA in dairy cow diets are linolenic, C 18:3, and linoleic, C 18:2, acids in plant lipids. Milk fat depression (MFD) occurs when milk fat yield is reduced, but milk volume and other components are not affected (Peterson, et al. 2003). Milk fat depression caused by abomasal infusion of trans-10, cis-12 CLA can decrease milk fat percent within 10 hours (Harvatine and Bauman, 2007b). Diet-induced

MFD develops over 7 to 18 days with the lowest point of milk fat depression occurring 18 days into treatment (Shingfield et al., 2006b).

Diets that induce MFD are known to alter ruminal lipid metabolism, leading to an increased production of specific trans and conjugated fatty acid isomers that are absorbed in the lower gastrointestinal tract and inhibit milk fat synthesis in lactating dairy cows (Shingfield and Griinari, 2007; Shingfield et al., 2010; Maxin et al., 2011).

Under some conditions of diet-induced MFD, rumen production, and milk fat content of trans-10, cis-12 conjugated linoleic acid (CLA) increases (Harvatine et al., 2009;

Shingfield et al., 2010; Maxin et al., 2011).

The addition of potassium carbonate (K2CO3) may alleviate the negative effects of the unsaturated fats in corn oil. Jenkins et al. (2011) found that the addition of K2CO3 to cultures of mixed ruminal microorganisms decreased production of trans-C18:1 and trans-10, cis-12 CLA (Jenkins et al., 2011). An in vitro study by Morris et al. (2012) showed that incomplete biohydrogenation induced by unsaturated fatty acid was alleviated by addition of 3% potassium (K) in the form of K2CO3, but not by addition of

3% K in the form of KCl, showing that K2CO3 was better at reducing incomplete biohydrogenation than KCl. Harrison et al. (2012) evaluated the dietary K requirements using K2CO3. Diets included K at levels of approximately 1.3% and 2.1% DM. The study found that diets with 2.1% K had increased dry matter intake (DMI), milk fat percentage,

milk yield (MY), and efficiency of milk production per unit of DMI when compared to cows fed the diet with 1.3% K. Though, MFD did not occur in this experiment.

We hypothesized that supplementing K2CO3 to a diet with a high concentration of fat from DGS would alleviate MFD, but would have no effect when supplemented to a diet with lower concentrations of fat because the low fat diets would not induce MFD.

Further, we hypothesized that the positive effect of K2CO3 would be caused by reducing the production of trans-10, cis-12 CLA caused by incomplete biohydrogenation of the dietary PUFA.

CHAPTER 3: MATERIALS AND METHODS

Animals and Diets

All procedures involving animals were approved by the Institutional Animal Care and Use Committee of The Ohio State University. Twelve multiparous (157+8 days in milk (DIM), 37+2 kg/d MY) and four primiparous (156+4 DIM, 29+1 kg/d MY) Holstein cows were used. Multiparous cows were blocked by MY. Each block comprised a 4x4

Latin square with 21 d periods and a 2x2 factorial arrangement of treatments. Cows were fed one of four diets (Table 1). Treatments were 0 or 1.9% added K2CO3 (DCAD

Plus®; Church & Dwight Co., Inc., Princeton, NJ) with 4.2 or 5.8% long chain fatty acids

(LCFA) (Table 2). All diets contained 47% corn silage (Table 3) and 27 % DGS (Table 4) or its equivalent from a mix of DGS, corn gluten meal, and corn oil. The mixture of DGS, corn gluten meal, and corn oil was used rather than a source of DGS with a higher fatty acid concentration because it minimized differences in nutrient composition between treatments that may have occurred if we had simply purchased a higher fat DGS to use for our high fat diets. Treatments were designated as low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added K

(HF+K). The inclusion rate for DGS in this experiment was chosen because previous research found decreased milk fat percentage and yield when cows were fed diets with

20% DGS (Hippen et al., 2004). For our experiment, K2CO3 included at 1.9% increased

dietary K by 1%. When K2CO3 was added to the diet, soybean hulls were decreased because this had the most minimal effect on differences of NEL between treatments.

Diets were formulated to meet nutrient requirements for 650 kg lactating dairy cows producing 40 kg milk/d (National Research Council, 2001). Cows were housed in tie stalls and fed once daily (0400h). Diets were offered ad libitum (feed refusals averaged

9.2%, as fed basis) as mixed rations. Amount of feed offered and refused were measured daily and were used to determine DMI. Diets were adjusted weekly to account for changes in corn silage DM concentration. Cows were milked twice daily

(0200 and 1300 h) and milk weights were recorded electronically at each milking. Cows were body condition scored (1= emaciated; 5=obese) by two independent people

(averaged) at the beginning of the experiment and on d 21 of each period. Cows were weighed at the beginning and end of the experiment and on d 21 of each period at 0800 h. Body weight change was calculated as the difference between the end of the period weight and the end of the previous period weight.

Sampling Collection and Analysis

Samples of fresh feeds were collected weekly and composited by period. Orts

(10% of wet weight) were taken once a wk, stored in the freezer for up to 2 wk, and composited within cow and period. Samples of DGS were collected from the feed facility once per period. Weekly samples of silage and refusals were analyzed for DM (100°C for

48 h). A composite of corn silage and each refusal sample were dried at 55°C and ground in a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA) through a 1-mm screen before

` nutrient analyses. Grains were not ground or oven-dried. Samples were analyzed for

DM (100°C oven for 24 h), ash (AOAC, 2000), starch (Weiss and Wyatt, 2000), NDF

(Ankom200 Fiber Analyzer, Ankom Technology, Fairport, NY) with sodium sulfite and amylase (Ankom #FAA), CP (Kjeldahl N x 6.25, (AOAC 984.13.4.2.09, 2000), and LCFA

(Weiss and Wyatt, 2003).

Initial analysis of concentrate samples with added K resulted in starch levels higher than expected based on formulation and the concentrations in the other concentrate mixes. The pH of the assay solutions containing samples of the K supplemented mixes after the 60 min water bath incubation at 90°C were 3 pH units higher than samples without added K (Table 1). This higher pH was a result of the additional K and was outside the optimal pH range for the alpha amylase. Several pH comparisons found that an addition of 50 uL 2M HCl lowered the pH so that proper starch analysis could occur.

Silage and refusal samples were dry-ashed and concentrate samples were perchloric acid digested. After digestion, mineral analyses were conducted using an inductively coupled plasma spectrograph (Service Testing and Research [STAR]

Laboratory, Ohio Agricultural Research and Development Center, Wooster, OH).

Chloride concentrations were analyzed by extraction with 0.5% nitric acid followed by potentiometric titration with silver nitrate using a Brinkman Metrohm 848 Titrino Plus.

(Brinkmann Instruments Inc., Westbury, NY) by Cumberland Valley Analytical Services

(CVAS; Hagerstown, MD). Samples of the four total mixed rations (TMR) were

` constructed from dried samples of forages and concentrates and were analyzed for 30-h in vitro NDF digestibility (Goering and Van Soest, 1970) by CVAS. Corn silage particle size distribution was measured on wet period composite samples using a Penn State Particle

Separator (PSPS) (Lammers et al., 1996). Particle size distributions of concentrate mix samples were measured by dry sieving using a vertical oscillating sieve shaker

(Analysette 3; Fritsch, Oberstein, Germany) equipped with a stack of sieves (W. S. Tyler,

Inc., Mentor, OH) arranged in descending mesh size. Sieve mesh sizes were 1.18, 0.6,

0.3, and 0.15 mm. The DCAD value of feeds was determined using the equation DCAD =

[(%Na × 43.5 + %K × 25.6) − (%Cl × 28.2 + %S × 62.5)] (Ender et al., 1962). Drinking water was collected from the farm once per period and was analyzed for mineral content using an inductively coupled plasma spectrograph (STAR Laboratory).

Milk samples (a.m. and p.m. milkings) were collected on d 3, 7, 10, 14, 17, and 21 of each period for determination of milk, fat, protein, lactose, (B2000 Infrared Analyzer

(Bentley Instruments, Chaska, MN) and MUN concentrations (Skalar SAN Plus segmented flow analyzer; Skalar Inc., Norcross, GA) by DHI Cooperative, Inc. (Columbus,

OH). Milk yields from d 3, 7, 10, 14, 17 and 21 were used to calculate yields of milk components. The energy concentration of milk was calculated from milk fat, protein, and lactose (NRC, 2001).

Milk samples (p.m.) were collected 30 h after the start of each period, and d 7,

14, and 21. Samples were stored at 4°C up to 24 hours until milk fat removal. Samples

` were centrifuged at 20,000xg for 20 min at 4°C. The fat layer was skimmed off and stored at -20°C until milk fat analysis by gas chromatography.

Milk samples (a.m.) were collected on d 21 for mineral analysis. The samples were stored at 4°C up to 1 wk until analysis. Samples were warmed by heating at 37°C for 15 minutes and homogenized by repeated (10x) pouring into a beaker. A 2-mL aliquot was digested with nitric acid and analyzed for mineral content using an inductively coupled plasma spectrograph (STAR Laboratory).

Urine samples (~100 mL) were collected by vulva stimulation on days 16 and 17 from each cow. Individual samples were analyzed and then averaged by cow by period.

Samples were stored at -20°C until analysis. Thawed samples were warmed by heating at 37°C for 15 min and homogenized by repeated (10x) pouring into a beaker. A 5-mL aliquot was nitric acid digested and analyzed for minerals using an inductively coupled plasma spectrograph (STAR Laboratory). Samples were also analyzed for creatinine

(Cayman Chemical Item Number 500701, Ann Arbor, MI) and Kjeldahl N (AOAC

984.13.4.2.09, 2000). Mineral excretion was determined using mineral analysis and calculated urine excretion. Urine excretion was estimated using creatinine as urine excretion (L/d) = (29 mg daily creatinine excretion/kg BW)/ (analyzed creatinine in sample) (Valaderes et al., 1999). Urine excretion was also estimated as urine excretion

(L/d) = (0.0259*kg BW * MUN (mg/dL))/ (analyzed N in sample) (Kauffman and St-Pierre,

2001).

Statistical Analysis

Data were analyzed with mixed models using the MIXED procedure of SAS

(version 9.3, SAS Institute, Inc., Cary, NC). Denominator degrees of freedom for all tests were adjusted using the Kenward-Rogers option.

One model included the effects of cow within square (random, df= 12), square

(random, df= 3), period (random, df= 3), dietary fat (fixed, df = 1), dietary K (fixed, df =

1), K x fat interaction (fixed, df= 1), and the residual error (random, df = 42). This model was used to analyze urine, milk minerals, and results that included data from week 3 only that had been averaged within cow within period.

Daily milk yield and DMI were averaged by week within cow and period and weekly means were analyzed using a model that included the effect of cow within square (random, df= 12), square (random, df= 3), period (random, df= 3), dietary fat

(fixed, df = 1), dietary K (fixed, df = 1), K x fat interaction (fixed, df= 1), effect of wk

(fixed, df=2), K x wk (fixed, df=2), fat x wk (fixed, df=2), and fat x K x wk (fixed, df=2), and the residual error. Week was a repeated effect. The model for milk composition was the same except day (df =5 for composition and 3 for milk fatty acids) replaced the wk terms in the model. The SLICE option of LSMEANS was used if a K by time or fat by time interaction occurred. If a time by treatment effect occurred for a specific dependent variable, then data from wk 3 of each period were averaged within cow and were analyzed using the above model.

CHAPTER 4: RESULTS AND DISCUSSION

Diet

Diet composition and analyses are shown in Tables 1 and 2. The DCAD was 2 mEq/100 g DM for diets without additional K and about 30 mEq/100 g DM for added K diets. Many studies have found that a DCAD of 20 mEq/100 g DM or higher result in higher milk production and DMI for lactating dairy cows (Tucker et al., 1988; Delaquis and Block, 1995; Wildman et al., 2007). In an analysis of 16 studies, Hu et al. (2007a) found that DMI, milk yield, and milk component production were maximized at 47 meq/kg. Dry cows benefit from a low or negative DCAD, which helps prevent hypocalcemia (NRC, 2001). The diets with added K had DCAD values that have been found to be adequate for lactating cows, but it is possible that the low DCAD values could have limited performance for the cows fed the diets without added K.

The total amount of dietary fat increased with the high fat diets, but the fatty acid profile of diets was similar between low and high fat diets. It is important to note that about 46% of the fatty acid present in the study was from C 18:2. The sulfur in the diets was high at 0.44% DM (National Research Council, 2001), which was likely due to the high inclusion of DGS (Table 4).

Corn silage 30 h NDF digestibility was determined because low corn silage digestibility can decrease DMI. Based on other studies, corn silage digestibility should have been adequate and should not have led to a decrease in DMI (Oba and Allen,

1999b). In vitro TMR digestibility and concentrate mix particle size were similar between diets, indicating that diets should have been digested similarly. A study by Rémond et al.

(2004) found that grain should be finely ground (particle size <1 mm) to prevent a decrease in total tract starch digestibility. In this study, grain particle size averaged less than 0.70 mm for all concentrate mixes, so starch degradability should have been adequate. Saliva is necessary for buffering of the rumen and saliva production is determined by chewing time. Chewing time was not assessed in this study, though based on studies by Lammers et al. (1996) and Kononoff et al. (2003), corn silage particle size should have been adequate for proper chewing and saliva production.

Cattle had free access to water which contained 99 mg/L Cl, 0.24 mg/L P, 1.7 mg/L K, 67 mg/L Ca, 30 mg/L Mg, and 20 mg/L S. These concentrations were typical and should have had little to no effect on DCAD or treatment (Solomon et al., 1995; National

Research Council, 2001; Beede, 2005; Castillo et al., 2013). The DGS contained 4.1% starch, 28% NDF, 30.8% protein, 7.8% LCFA, 0.98% S, and 1.27% K (Table 4), which is similar to typical DGS, though they were slightly lower in fat, as they were a reduced fat

DGS (Anderson et al., 2006).

Dry Matter Intake, Milk Production, and Milk Composition

Fat by week and K by week interactions were observed for DMI (Table 5; Figure

1). For cows fed high fat diets, DMI was low and constant over the 3 wk, but increased by the end of the period for cows fed low fat diets. Intake by cows fed diets with supplement K were affected by treatment (P<0.01), whereas cows fed diets without supplemental K was constant and higher over the 3 wk period (P<0.17). Figure 2 shows this K by day and fat by day interaction in more detail. Low fat diets were affected by wk

(P<0.01), but high fat diets were not (P<0.92). Fat by wk interactions were also observed for MY (Table 5; Figure 3). Milk yield for cows fed high fat diets were affected by treatment over time (P<0.01), but MY for cows fed low fat diets were unchanged

(P<0.24).

Fat by day interactions were observed for both milk fat percent and milk fat yield

(Table 5). As shown in Figures 4 and 5, these measurements followed similar patterns, with a lowest milk fat percent around day 18. Milk fat percent and fat yield were both unaffected by K by time (P<0.95 and P<0.67, respectively). Milk fat yield with low fat diets were unaffected by time (P<0.33), but the high fat treatment affected milk fat yield by the end of the period (P<0.01). The changes over time for production and milk composition data were indicative of the cows adjusting to treatments. Because of this assumed adjustment, we decided to look at solely week 3 to compare treatment effects.

Table 6 shows the production and milk composition results for the final wk of the periods. DMI decreased with added fat (P<0.01). This decrease in DMI with added fat was expected. Many studies have found that dietary unsaturated fats decrease DMI

(Pantoja et al., 1994; Pantoja et al, 1996; Firkins and Eastridge, 1994). Extensive biohydrogenation of FA occurs in the reticulorumen (Viviani, 1970) and should reduce the hypophagic effects of some sources of unsaturated fat, but biohydrogenation is reduced when the amount of unsaturated fat in the diet increases (Christensen et al.,

1998), so DMI is still reduced.

Diets with added K also tended to decrease DMI (P<0.06). This is contrary to a study by Harrison et al. (2012) that found the addition of K2CO3 at 2.1% DM versus 1.3% increased DMI. There were many differences between this study and Harrison et al.

(2012), but the biggest difference was the length of time that diets were fed. Harrison et al. (2012) fed his cattle the same diet, either 2.1% K or 1.3% K, for 12 wk, but 2.1% K diets did not increase DMI until 3 or 4 wk into the experiment. As the cows in this experiment were only on treatments for 3 wk, it is possible we would have seen this increase in DMI with added K diets we fed out treatments for a long period of time.

Milk NEL (Mcal/d) is a measure of the energy in daily milk excretion. It is calculated using yields of milk fat, milk protein, and milk lactose (National Research

Council, 2001). Milk NEL had a K by fat interaction (P<0.03) (Table 5) where adding fat did not statistically affect diets with added K, but did statistically affect diets with added

K. This difference in milk NEL between diets without added K was likely caused because milk fat yield increased with added K, leading to a decrease in NEL for diets with added K.

Yields of milk components for diets with added K were similar, resulting in similar NEL.

The HF-K diet did not have similar yields to the LF-K diet because it did not have the added K to increase in milk fat yield to values near the low fat diet.

As shown in table 6, MY decreased for high fat diets (P<0.01), which may have been a result of decreased DMI. Milk protein percent and yield decreased with added K

(P<0.01). This was not expected and we are not certain why this occurred. Milk protein percent increased with added fat, though milk protein yield was unaffected (P<0.12).

Because MY decreased with added fat and milk protein yield was unaffected, this increase in milk protein percent was likely due to a change in concentration of milk protein yield relative to MY.

Milk urea nitrogen (MUN) decreased with added K (P<0.01). As shown in Table

11, urine excretion increase with added K (P<0.01). This increase in urine is likely associated with an increase in water intake. MUN is directly related to the concentration of blood urea nitrogen (BUN), which is affected by the amount of water in the body.

Increasing water intake increases body water, thus diluting BUN and, ultimately, MUN.

Water intake was not measured in this study, but urine volume was estimated. Feeding excess K is associated with increased water intake and urine excretion (Fisher et al.,

1994). Table 11 shows that estimated urine excretion increased with added K (P<0.01), which may be indicative of an increase in water intake, which would increase body water and decrease BUN and MUN concentration.

Net energy of lactation (Mcal/kg milk) increased with added K (P<0.04) and decreased with added dietary fat (P<0.01). This is indicative of the decrease in milk fat

` with added fat, which decreases the energy in milk, and the increase in milk fat with added K, which increases the energy in milk. Body condition scores, body weights, and body weight changes were not affected by treatment (Table 4).

Milk fat percent increased with added K (P<0.01) and decreased with added fat

(P<0.01). Milk fat yield was affected similarly and tended to increase with added K

(P<0.10) and decreased with added fat (P<0.01). Adding K to the high fat diet resulted in increased milk fat as hypothesized, but we did not expect that this would also occur for the low fat diets. We expected added K to increase milk fat percent by decreasing incomplete biohydrogenation, which results in decreased milk production. We did not expect incomplete biohydrogenation to occur for the low fat diets, which is why we did not expect an increase in milk fat percent for the low fat diets. All diets caused milk fat depression. This may have occurred because of the high level of S in the diet. The high amount of S from the DGS could have decreased rumen pH (Felix and Loerch, 2011). A low rumen pH with the high amount of unsaturated fatty acid in both the low and high fat diets could have caused the milk fat depression with all diets. Ivancic and Weiss

(2004) found a decrease in milk fat percent when 0.4% S was fed vs. 0.2% S.

Milk Fatty Acids

Many of the milk fatty acids were affected by fat by time or K by time interactions (Table 7). We assumed that this may have been due to the cows adjusting to new diets at 30 h into each period, but when only data from d 7, 14, and 21 were analyzed, time interactions were still observed, which indicates that cows may still have

` been adjusting to diets on days 7 and 14. Because of these suspected adjustments, we looked at data from d 21 (Table 9), which follows along with the previous analysis of solely wk 3 of production and milk composition data.

Table 8 shows the milk fatty acid analysis of samples collected at 30 h into treatment. This 30 h collection represents the initial adjustments to the new diets and is especially essential for estimating changes in ruminal environment to the new diets.

Branched chain fatty acids have been found to have the potential to predict molar proportions of volatile fatty acids (VFA) in the rumen (Vlaeminck et al., 2006). Often when MFD occurs, the molar proportion of ruminal acetate decreases and proportion of ruminal propionate increases. Concentrations of iso C14:0 and iso C15:0 in milk fat are positively related with rumen proportions of acetate and negatively related with molar proportions of propionate (Vlaeminck et al., 2006). Iso C 14:0 increased with added K

(P<0.01) and decreased with added fat (P<0.01). Iso C 15:0 behaved similarly. It also increased with added K (P<0.03) and decreased with added fat (P<0.03). These results may indicate that ruminal concentrations of acetate were increasing and proportion of propionate was decreasing with added K. This may also indicate that proportion of acetate decreased with added fat and concentration of propionate increased with added fat.

Milk fat depression results in a decrease in proportion of short chain fatty acids

(SCFA) and an increase in the proportion of LCFA (Peterson et al., 2003). Short chain fatty acids, those less than 16 carbons, were increased with added K (P<0.01) and

` decreased with added fat (P<0.01) (Table 9). The C 16 fatty acids were increased with added K (P<0.02) and decreased with added fat (P<0.01). Long chain fatty acids, those greater than 16 carbons, decreased with added K (P<0.01) and increased with added fat

(P<0.01). The increase in LCFA with added fat may have been caused by the overall increase of LCFA in the diets. Trans isomers, which are included in the LCFA, lead to a decrease in SCFA, as well.

Many of the trans isomers, including trans-6+8 18:1, trans-9 18:1, trans-10 18:1 and trans-12 18:1 increased with added fat (P<0.01) and decreased with added K

(P<0.01) (Table 9). Trans-10, cis-12 CLA is a biohydrogenation intermediate that increases in concentration when incomplete biohydrogenation occurs. Table 9 shows that trans-10, cis-12 CLA in milk decreased with added K (P<0.02) indicating that the additional K is increasing milk fat production by decreasing incomplete biohydrogenation. Trans-10, cis-12 increased with added fat (P<0.01) because excess unsaturated fatty acids in the diets results in increased incomplete biohydrogenation. As

Figure 6 shows, there was a linear relationship between milk fat percent and trans-10, cis-12.

Biohydrogenation is the process of changing dietary unsaturated fatty acids, such as C 18:2, into saturated fatty acids, such as C18:0. Table 10 shows the effect of treatment on proportion of C 18:0 and C 18:2 relative to total concentration of C 18 fatty acids. Both C 18:0 and C 18:2 concentration were unaffected by added fat, though

C 18:0 increased with added K (P<0.01) and C 18:2 decreased with added K. This may indicate that adding K increased biohydrogenation of C 18:2 to C 18:0.

Mineral Excretion

Mineral intake and excretion was calculated for wk 3 because that is the week urine samples and milk samples for mineral analysis were taken. As shown in Table 11, urine excretion (L/d) estimated using both creatinine and MUN equations did not differ statistically (P<0.29). Estimated urine excretion increased with added K (P<0.01) and with added fat (P<0.01). The increase with added K was expected as excess K is associated with increased water intake and urine excretion (Fisher et al., 1994). For the creatinine method, fat had no significant effect on estimated urine excretion (P<0.16), but urine excretion increased with added fat for the MUN method (P<0.05).

Urine excretion of K increased with added K (P<0.01) as was expected, as an increase in K intake would lead to an increase in K in urine. Magnesium excretion in urine decreased with added K for both treatments (P<0.01) and added fat (creatinine,

P<0.01; MUN, P<0.02). Urinary mineral excretion was measured to evaluate the effects of K on Mg absorption. Assuming typical diets and DMI, diets with approximately 0.2%

Mg will usually meet Mg requirements for lactating cows (NRC, 2001); though adding K decreases Mg absorption (Newton et al., 1972). There are two equations suggesting additional Mg is required when excess K is fed. Weiss (2004) suggested an additional

0.08% Mg for each additional 1% dietary K, which calculates to a maximum of 0.30% Mg for our added K treatments. Schonewille et al., (2008) suggested an additional 0.02% for

` each additional 1% dietary K, which calculates to 0.23% Mg. We planned to meet requirements by preparing diets that supplied 0.30% Mg, though analysis shows that high fat without added K only contained 0.25% Mg. Though, for Weiss (2004) and

Schonewille et al., (2008), a maximum of 0.22% Mg would suffice for the without added

K treatments. Also, Mg being present in the urine is a clear indication that enough Mg was fed.

The decrease of Mg in urine with added fat may have been due to the decrease in intake or increased number of free fatty acids in the rumen. Increased dietary fat leads to a decrease in Mg absorption (Ramirez and Zinn, 2000). Magnesium binds to free fatty acids. The fatty acids are dissociated in the abomasum, which frees up the Mg, but

Mg is absorbed in the rumen only. Urinary S excretion decreased with added K (P<0.01) for both treatments and with added fat for the creatinine method. The decrease with added fat may have been due to the decrease in S intake, though the difference between S intakes with added K was not significant and does not explain this decrease in urine S excretion. Mineral values in urine are reasonable based on papers by Tucker and Hogue (1990) and Nennich et al., (2006). Table 13 shows the effect of treatment on the percentage of urine mineral excretion/mineral intake. Magnesium urine excretion/intake also decreased with added K, which was likely a result of K blocking Mg absorption.

Milk mineral excretion were affected by treatment, but concentrations of minerals in milk (Table 14) were normal (National Research Council, 2001). Though

` dietary fat did decrease concentrations of P, Ca, Mg, S, and Na in milk, which may have been caused by the decrease in mineral intake of all of these minerals with added fat diets (P<0.01).

Conclusion

Diets based on DGS that contained high concentrations of PUFA led to MFD. The addition of K2CO3 to the high fat diets increased milk fat percent and milk fat yield.

Though all diets led to milk fat depression, which may have been a result of the high amount of S in the diets due to the high S in the DGS. Milk fat analysis suggested that additional K2CO3 decreased incomplete biohydrogenation as trans-10, cis-12 CLA decreased with added K treatments. An increase in proportion of C 18:0 along with a decrease in C 18:2 may indicate that adding K led to an increase in biohydrogenation of

C 18:2 to C 18:0.

Table 1: Ingredient composition of diets, DM basis

Treatment1 Ingredient LF-K LF+K HF-K HF+K Corn Silage, % 47.0 47.0 47.0 47.0 Concentrate Mix, % 53.0 53.0 53.0 53.0 Dried distiller grains with solubles2, % 27.0 27.0 22.0 22.0 Corn Oil, % - - 2.3 2.3 Corn gluten meal, % - - 2.7 2.7 DCAD Plus3, % - 1.9 - 1.9 Corn (ground), % 8.5 8.5 8.5 8.5 Soybean meal 48% CP, % 6.5 6.5 6.5 6.5 39 Soybean hulls, % 6.9 5.0 6.9 5.0 Calcium carbonate, % 2.78 2.78 2.78 2.78 Magnesium oxide, % 0.13 0.13 0.13 0.13 Trace mineral salt, % 0.64 0.64 0.64 0.64 Mineral and vitamin premix4, % 0.55 0.55 0.55 0.55

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Dakota Gold® Dried Distiller Grains with Solubles (Poet Nutrition, Sioux Falls, SD). 3DCAD Plus®; Church & Dwight Co., Inc., Princeton, NJ 4Contained 71.4 % biotin (220mg/kg) premix (DSM Nutritional Products, Inc., Parsippany, NJ), 11.3% Selenium premix (200mg/kg), 0.4% copper sulfate, 9.4% Vitamin E premix (44 IU/g), 5.6% Vitamin D premix (3,000 IU/g), and 1.9% Vitamin A premix (30,000 IU/g).

Table 2: Nutrient composition of diets

Treatment1 Nutrient LF-K LF+K HF-K HF+K Long chain FA (LCFA), %DM 4.2 4.3 5.8 5.9 C 16:0, g/100 g of LCFA 7.4 7.4 6.9 7.0

C 18:0, g/100 g of LCFA 2.7 2.6 2.5 2.4

C 18:1, g/100 g of LCFA 22.4 22.1 23.2 23.3

C 18:2, g/100 g of LCFA 46.0 45.1 47.3 47.2 Other and unidentified, g/100 g of LCFA 21.4 22.7 20.1 20.1 Starch, %DM 23.4 24.9 23.3 25.3 NDF, %DM 32.1 30.2 31.0 30.0 Forage NDF, %DM 17.9 17.9 17.9 17.9 CP, %DM 17.2 17.3 17.4 17.5 Ash, %DM 5.6 7.9 5.4 7.5 K, %DM 1.24 2.29 1.19 2.20 Na, %DM 0.21 0.24 0.20 0.22 Cl, %DM 0.48 0.47 0.50 0.47 S, %DM 0.40 0.40 0.36 0.37 Mg, %DM 0.32 0.32 0.25 0.29 Ca, %DM 0.92 0.89 0.69 0.86 P, %DM 0.46 0.45 0.44 0.46 DCAD, (mEq/100 g DM)2 2 31 2 29 30 hour NDF Digestibility, %NDF 67.9 69.8 67.3 68.6 Concentrate mix mean particle size, mm 0.60 0.59 0.62 0.65 Concentrate mix pH 4.56 7.42 4.60 7.54 3 NEL, Mcal/kg 1.63 1.59 1.70 1.68 MP allowable milk, kg/d 33.5 32.4 34.0 34.2

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2DCAD, mEq/100 g DM = [(%Na × 43.5 + %K × 25.6) − (%Cl × 28.2 + %S × 62.5)]. 3NRC (2001)

Table 3: Nutrient composition of corn silage (n=4)

Item Mean SD DM % 41.2 1.0 Starch, %DM 33.5 2.2 NDF, %DM 37.8 2.7 CP, %DM 7.7 0.5

Ash, %DM 3.57 0.52 30 hour NDF Digestibility, %NDF 54.7 0.29 Particle size, as fed1 Top Screen, % as fed 6.3 2.7 Middle Screen, % as fed 69.7 1.8 Pan, % as fed 24.0 2.2

1Penn State Particle Separator (Lammers et al., 1996)

Table 4: Nutrient composition of distiller grains (n=4)

Item Mean SD Starch, %DM 4.1 0.62 NDF, %DM 28.0 0.51 CP, %DM 30.8 0.64 LCFA, %DM 7.8 0.39 C 16:0, g/100 g of LCFA 13.1 0.40 C 18:0, g/100 g of LCFA 2.4 0.07 C 18:1, g/100 g of LCFA 23.9 0.17 C 18:2, g/100 g of LCFA 50.5 1.34 Other and unidentified, g/100 g of LCFA 10.1 1.70 Ash, %DM 5.38 0.10 K, %DM 1.27 0.04 Na, %DM 0.23 0.02 Cl, %DM 0.21 0.02 S, %DM 0.97 0.02 Mg, %DM 0.41 0.01 Ca, %DM 0.03 0.002 P, %DM 0.89 0.02 Grain pH 3.59 0.04

Table 5: Effects of treatment on DMI, milk production, and milk composition, all 3 wk of treatment (n=16 cow period per treatment)

1 Treatment P< 2 Item LF-K LF+K HF-K HF+K SEM K Fat KxFat KxTime FatxTime KxFatxTime DMI, kg/d 23.0 21.3 21.6 20.5 1.04 0.01 0.01 0.34 0.02 0.01 0.59 Milk yield, kg/d 32.8 31.1 31.5 31.1 3.40 0.09 0.27 0.25 0.76 0.01 0.97 3 Milk NEL , Mcal/kg 0.64 0.66 0.62 0.65 0.018 0.01 0.01 0.28 0.87 0.01 0.92

Milk NEL, Mcal/d 21.4 20.5 19.5 20.3 2.17 0.82 0.01 0.03 0.33 0.01 0.93 Milk fat, % 2.86 3.08 2.63 2.93 0.19 0.01 0.01 0.47 0.96 0.01 0.96 Milk fat, kg/d 0.97 0.96 0.84 0.92 0.12 0.07 0.01 0.05 0.68 0.01 0.64 Milk protein, % 3.39 3.33 3.48 3.37 0.092 0.03 0.06 0.43 0.08 0.03 0.68 Milk protein, kg/d 1.12 1.03 1.08 1.05 0.096 0.01 0.57 0.22 0.75 0.32 0.56 Lactose, % 4.83 4.78 4.82 4.85 0.079 0.85 0.17 0.06 0.73 0.76 0.52

43 Lactose, kg/d 1.60 1.49 1.52 1.52 0.16 0.08 0.32 0.08 0.75 0.13 0.74 MUN, mg/dL 15.7 13.7 16.4 14.6 0.45 0.01 0.01 0.53 0.24 0.16 0.28

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2DMI and milk yield samples were collected weekly. Milk composition data was collected on d 3, 7, 10, 14, 17, and 21. 3Calculated from milk fat, protein, and lactose yield (NRC, 2001)

Table 6: Effects of treatment on DMI, milk production, and milk composition, wk 3 of treatment (n=16 cow period per treatment)

Treatment1 P< Item LF-K LF+K HF-K HF+K SEM K Fat KxFat DMI, kg/d 22.9 22.1 21.3 20.7 0.98 0.06 0.01 0.88 Milk yield, kg/d 33.0 31.6 30.6 30.4 3.6 0.24 0.01 0.34 2 Milk NEL , Mcal/kg 0.63 0.65 0.59 0.62 0.020 0.04 0.01 0.43

Milk NEL, Mcal/d 21.0 20.2 17.8 18.9 2.08 0.75 0.01 0.11 Milk fat, % 2.74 2.99 2.39 2.64 0.13 0.01 0.01 1.00 Milk fat, kg/d 0.92 0.94 0.72 0.80 0.98 0.10 0.01 0.27 Milk protein, % 3.41 3.33 3.56 3.41 0.085 0.01 0.01 0.33

44 Milk protein, kg/d 1.12 1.03 1.07 1.03 0.10 0.01 0.12 0.25

Lactose, % 4.83 4.77 4.84 4.86 0.086 0.69 0.17 0.28 Lactose, kg/d 1.60 1.49 1.47 1.48 0.16 0.10 0.02 0.05 MUN, mg/dL 16.0 13.9 16.0 14.6 0.63 0.01 0.24 0.22 BCS 3.0 3.2 3.2 3.1 0.20 0.43 0.47 0.08 BW, kg 671 673 676 671 16.45 0.75 0.62 0.32 BW Change, kg/d 0.68 0.45 0.22 0.38 0.21 0.88 0.22 0.37

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Calculated from milk fat, protein, and lactose yield (NRC, 2001)

Table 7: Effects of treatment on milk fatty acid concentrations, all 3 wk (n=16 cow period per treatment)

Treatment1 P< Fatty Acid, g/100g fatty acids2 LF-K LF+K HF-K HF+K SEM K Fat KxFat KxDay3 FatxDay KxFatxDay 4:0 2.86 2.94 2.71 2.82 0.129 0.10 0.02 0.84 0.34 0.02 0.72 6:0 1.77 1.81 1.54 1.63 0.11 0.06 0.01 0.48 0.01 0.01 0.67 8:0 0.89 0.90 0.73 0.78 0.06 0.13 0.01 0.32 0.01 0.01 0.52 10:0 2.01 1.97 1.58 1.68 0.14 0.50 0.01 0.10 0.01 0.01 0.53 12:0 2.55 2.41 2.08 2.11 0.15 0.21 0.01 0.05 0.01 0.01 0.61 iso 13:0 0.03 0.03 0.02 0.02 0.002 0.76 0.01 0.73 0.39 0.07 0.78 anteiso 13:0 0.04 0.03 0.05 0.04 0.006 0.01 0.01 0.52 0.49 0.23 0.24 13:0 0.11 0.10 0.10 0.12 0.01 0.69 0.63 0.14 0.89 0.20 0.56 iso 14:0 0.10 0.12 0.08 0.10 0.007 0.01 0.01 0.64 0.11 0.67 0.17 14:0 9.83 9.48 8.53 8.59 0.33 0.20 0.01 0.06 0.04 0.15 0.04 iso15:0 0.21 0.22 0.17 0.19 0.006 0.01 0.01 0.21 0.97 0.01 0.27 45 anteiso 15:0 0.51 0.51 0.42 0.45 0.01 0.02 0.01 0.06 0.31 0.01 0.31

14:1 1.27 1.12 1.12 1.03 0.09 0.01 0.01 0.29 0.45 0.08 0.86 15:0 1.06 0.98 0.91 0.88 0.05 0.01 0.01 0.21 0.01 0.01 0.92 iso 16:0 0.27 0.32 0.25 0.29 0.03 0.01 0.01 0.42 0.36 0.99 0.63 16:0 24.95 25.36 23.32 23.16 0.56 0.65 0.01 0.29 0.43 0.02 0.47 iso 17:0 0.45 0.45 0.43 0.43 0.01 0.91 0.01 0.54 0.08 0.03 0.65 C16:1 + anteiso 17:0 2.21 2.14 2.10 2.01 0.11 0.04 0.01 0.86 0.54 0.03 0.80 17:0 0.61 0.60 0.52 0.54 0.02 0.37 0.01 0.04 0.18 0.01 0.66 17:1 0.23 0.23 0.21 0.22 0.01 0.94 0.01 0.60 0.10 0.16 0.86 18:0 10.90 11.55 11.92 12.77 0.50 0.01 0.01 0.54 0.73 0.01 0.50 trans-6+8 18:1 0.73 0.59 1.01 0.82 0.05 0.01 0.01 0.21 0.40 0.89 0.94 trans-9 18:1 0.63 0.50 0.95 0.70 0.05 0.01 0.01 0.02 0.07 0.35 0.96 trans-10 18:1 2.26 1.72 3.31 2.50 0.20 0.01 0.01 0.11 0.24 0.12 0.89 trans-11 18:1 0.69 0.78 0.71 0.82 0.10 0.02 0.52 0.75 0.02 0.01 0.23 trans-12 18:1 0.41 0.35 0.54 0.88 0.22 0.53 0.14 0.36 0.41 0.44 0.37

cis-9 18:1 25.77 26.56 27.19 27.66 0.99 0.02 0.01 0.55 0.61 0.01 0.85 cis-11 18:1 0.77 0.72 0.90 0.80 0.04 0.01 0.01 0.09 0.04 0.01 0.95 18:2 4.62 4.24 5.22 4.62 0.15 0.01 0.01 0.05 0.01 0.01 0.48 20:0 0.15 0.16 0.16 0.18 0.007 0.01 0.01 0.52 0.67 0.30 0.27 20:1 0.14 0.14 0.14 0.14 0.005 0.01 0.23 0.62 0.55 0.13 0.91 18:3 0.30 0.27 0.31 0.28 0.008 0.01 0.01 0.62 0.01 0.15 0.72 cis-9, trans-11 CLA 0.58 0.61 0.64 0.67 0.04 0.22 0.01 0.91 0.01 0.01 0.34 CLA (other) 0.05 0.04 0.06 0.05 0.004 0.01 0.01 0.06 0.22 0.03 0.89 trans-10, cis-12 CLA 0.04 0.04 0.07 0.05 0.007 0.01 0.01 0.01 0.19 0.12 0.78

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Number of carbons: number of double bonds. 3Data were collected at 30 h, d 7, 14, and 21.

Table 8: Effects of treatment on milk fatty acid concentrations, 30 h after diets were first fed (n=16 cow period per treatment)

Treatment1 P< Fatty Acid, g/100g fatty acids2 LF-K LF+K HF-K HF+K SEM K Fat K*Fat 4:0 2.79 2.79 2.93 2.88 0.17 0.80 0.25 0.82 6:0 1.70 1.59 1.69 1.60 0.10 0.16 0.93 0.87 8:0 0.85 0.78 0.83 0.76 0.062 0.13 0.62 1.00 10:0 1.91 1.66 1.78 1.66 0.13 0.04 0.47 0.50 12:0 2.42 2.13 2.25 2.13 0.15 0.02 0.28 0.29 iso 13:0 0.03 0.03 0.03 0.03 0.002 0.09 0.54 0.57 anteiso 13:0 0.03 0.03 0.06 0.03 0.008 0.01 0.01 0.02 13:0 0.10 0.09 0.10 0.10 0.01 0.43 0.66 0.45 iso 14:0 0.10 0.11 0.08 0.09 0.01 0.01 0.01 0.57

47 14:0 9.35 9.00 8.73 8.57 0.41 0.18 0.01 0.61

Iso 15:0 0.20 0.21 0.18 0.18 0.01 0.03 0.03 0.63 anteiso 15:0 0.51 0.48 0.45 0.47 0.02 0.85 0.01 0.10 14:1 1.25 1.12 1.13 1.03 0.12 0.03 0.06 0.77 15:0 0.98 0.93 0.94 0.92 0.05 0.24 0.32 0.56 iso 16:0 0.28 0.31 0.24 0.28 0.03 0.07 0.10 0.69 16:0 24.0 24.5 23.5 23.2 0.49 0.86 0.01 0.11 iso 17:0 0.45 0.47 0.45 0.46 0.02 0.21 0.91 0.58 C16:1 + anteiso 17:0 2.20 2.26 2.14 2.07 0.14 0.97 0.07 0.36 17:0 0.56 0.58 0.54 0.57 0.02 0.15 0.36 0.51 17:1 0.22 0.24 0.22 0.23 0.01 0.11 0.39 0.79 18:0 11.4 11.8 11.7 12.6 0.63 0.01 0.05 0.29 trans-6+8 18:1 0.76 0.66 1.01 0.87 0.05 0.01 0.01 0.62 trans-9 18:1 0.65 0.61 0.92 0.76 0.05 0.03 0.01 0.18 trans-10 18:1 2.43 2.08 3.02 2.64 0.19 0.02 0.01 0.91

trans-11 18:1 0.59 0.52 0.90 0.75 0.12 0.25 0.01 0.68 trans-12 18:1 0.42 0.35 0.56 0.45 0.02 0.01 0.01 0.37 cis-9 18:1 27.0 26.5 28.0 27.8 0.80 0.01 0.45 0.76 cis-11 18:1 0.79 0.82 0.82 0.80 0.05 0.86 0.99 0.25 18:2 4.78 4.66 4.90 4.73 0.15 0.14 0.30 0.77 20:0 0.16 0.16 0.16 0.20 0.02 0.20 0.12 0.29 20:1 0.14 0.14 0.13 0.14 0.005 0.31 0.06 0.86 18:3 0.30 0.28 0.30 0.29 0.008 0.22 0.73 0.69 cis-9, trans-11 CLA 0.56 0.49 0.74 0.62 0.05 0.02 0.01 0.59 CLA (other) 0.05 0.05 0.06 0.05 0.004 0.14 0.30 0.76 trans-10, cis-12 CLA 0.05 0.07 0.06 0.05 0.02 0.86 0.92 0.24

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Number of carbons: number of double bonds. 48

Table 9: Effects of treatment on milk fatty acid concentrations, d 21 of treatment (n=16 cow period per treatment)

Treatment1 P< Fatty Acid, g/100g fatty acids2 LF-K LF+K HF-K HF+K SEM K Fat KxFat SCFA 23.5 24.0 18.6 20.4 1.09 0.01 0.01 0.06 4:0 2.82 3.01 2.53 2.75 0.16 0.01 0.01 0.84 6:0 1.76 1.94 1.35 1.61 0.11 0.01 0.01 0.25 8:0 0.91 1.00 0.62 0.78 0.07 0.01 0.01 0.14 10:0 2.06 2.22 1.35 1.69 0.17 0.01 0.01 0.13 12:0 2.60 2.67 1.85 2.12 0.18 0.01 0.01 0.09 iso 13:0 0.03 0.03 0.03 0.02 0.001 0.01 0.01 0.08 anteiso 13:0 0.06 0.05 0.05 0.05 0.011 0.40 0.83 0.55 13:0 0.12 0.12 0.09 0.09 0.014 0.92 0.01 0.72

49 iso 14:0 0.10 0.12 0.08 0.09 0.010 0.02 0.01 0.21

14:0 9.92 9.92 8.06 8.62 0.43 0.06 0.01 0.06 iso15:0 0.21 0.22 0.16 0.18 0.009 0.01 0.01 0.88 anteiso 15:0 0.51 0.53 0.41 0.43 0.02 0.07 0.01 0.99 14:1 1.31 1.14 1.15 1.11 0.10 0.02 0.03 0.10 15:0 1.08 1.06 0.85 0.86 0.06 0.77 0.01 0.61 iso 16:0 0.26 0.31 0.26 0.26 0.03 0.19 0.21 0.20 16:0 24.6 25.2 22.8 23.7 0.49 0.02 0.01 0.54 LCFA 51.7 50.5 58.4 55.6 1.51 0.01 0.01 0.12 iso 17:0 0.47 0.45 0.42 0.40 0.02 0.18 0.01 0.93 C16:1 + anteiso 17:0 2.23 2.06 2.22 2.11 0.14 0.06 0.79 0.65 17:0 0.62 0.62 0.49 0.52 0.02 0.06 0.01 0.26 17:1 0.23 0.22 0.22 0.22 0.01 0.33 0.35 0.33 18:0 10.7 11.5 12.0 12.3 0.58 0.05 0.01 0.41 trans-6+8 18:1 0.77 0.59 1.07 0.79 0.06 0.01 0.01 0.30

trans-9 18:1 0.67 0.47 1.02 0.66 0.08 0.01 0.01 0.28 trans-10 18:1 2.46 1.75 3.52 2.52 0.25 0.01 0.01 0.46 trans-11 18:1 0.63 0.84 0.53 0.64 0.07 0.01 0.01 0.31 trans-12 18:1 0.41 0.36 0.55 0.44 0.03 0.01 0.01 0.15 cis-9 18:1 25.7 25.6 28.5 28.3 0.98 0.69 0.01 0.88 cis-11 18:1 0.79 0.69 0.94 0.83 0.06 0.01 0.01 0.84 18:2 4.79 4.11 5.53 4.56 0.13 0.01 0.01 0.14 20:0 0.15 0.17 0.16 0.16 0.008 0.03 0.11 0.07 20:1 0.14 0.14 0.14 0.14 0.006 0.35 0.09 0.20 18:3 0.30 0.26 0.32 0.27 0.009 0.01 0.01 0.98 cis-9, trans-11 CLA 0.57 0.65 0.55 0.58 0.04 0.16 0.30 0.53 CLA (other) 0.05 0.04 0.07 0.05 0.005 0.01 0.01 0.50 trans-10, cis-12 CLA 0.04 0.04 0.09 0.05 0.01 0.02 0.01 0.12

5 1

0 Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Number of carbons: number of double bonds.

Table 10: Effect of treatment on proportion of C 18:0 and C 18:2 relative to total concentration of C 18 fatty acids (n=16 cow period per treatment)

Treatment1 P< Fatty Acid, g/100g C 18 FA2 LF-K LF+K HF-K HF+K SEM K Fat KxFat 18:0 22.4 24.6 21.9 23.6 0.75 0.01 0.17 0.68 18:2 10.1 8.8 10.1 8.8 0.30 0.01 0.93 0.86

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Number of carbons: number of double bonds.

Table 11: Effect of treatment on estimated urine excretion (L/d) (n=16 per treatment)

Treatment1 P< Liters LF-K LF+K HF-K HF+K SEM K Fat KxFat Method KxMethod FatxMethod KxFatxMethod Average 24.2 32.5 24.9 37.5 2.45 0.01 0.01 0.05 0.29 0.68 0.27 0.38 Creatinine method2 23.4 33.2 24.0 36.0 2.67 0.01 0.16 0.36 MUN method3 24.5 31.7 25.6 38.8 2.62 0.01 0.05 0.14

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Urine volume calculated as (29 mg daily creatinine excretion/kg BW)/ (analyzed creatinine in urine sample) 3Urine volume calculated as (0.0259*kg BW * MUN (mg/dL))/ (analyzed N in urine sample)

Table 12: Effect of treatment on mineral intake and excretion (g/day) (n=16 per treatment)

Treatment1 P< Mineral LF-K LF+K HF-K HF+K SEM K Fat KxFat Intake2 Phosphorus 103 94 91 91 4.8 0.01 0.01 0.01 Potassium 282 488 254 438 17 0.01 0.01 0.12 Calcium 191 505 150 407 13 0.01 0.01 0.01 Magnesium 96 94 90 88 3.8 0.04 0.01 0.93 Sulfur3 91 89 77 77 3.8 0.27 0.01 0.32 Sodium 48 53 43 46 2.1 0.01 0.01 0.14 Nitrogen 629 609 586 569 27 0.06 0.01 0.88 Milk Excretion

53 Phosphorus 28 26 27 26 2.7 0.01 0.18 0.53

Potassium 49 47 46 45 5.5 0.13 0.09 0.53 Calcium 35 33 35 34 3.4 0.02 0.56 0.82 Magnesium 3.5 3.3 3.6 3.4 0.4 0.07 0.46 0.95 Sulfur 9.6 9.2 9.3 8.9 0.9 0.02 0.07 0.92 Sodium 12 11 13 11 1.8 0.19 0.83 0.84 Urine Excretion Creatinine method4 Phosphorus 2.1 2.1 2.5 1.9 0.9 0.57 0.88 0.48 Potassium 185 351 167 355 23.5 0.01 0.51 0.34 Calcium 4.5 0.5 3.8 0.4 0.7 0.01 0.41 0.48 Magnesium 11 7.0 8.3 5.3 0.9 0.01 0.01 0.18 Sulfur 59 45 49 41 3.2 0.01 0.01 0.09 Sodium 37 20 35 33 4.3 0.01 0.09 0.03 Nitrogen 264 253 253 235 10.7 0.17 0.16 0.70

MUN method5 Phosphorus 2.2 2.0 2.7 2.0 0.9 0.40 0.67 0.63 Potassium 194 336 182 383 24 0.01 0.35 0.12 Calcium 4.9 0.4 4.4 0.4 0.9 0.01 0.74 0.69 Magnesium 12 6.6 9.1 5.9 1.0 0.01 0.02 0.13 Sulfur 63 42 53 44 4.0 0.01 0.33 0.12 Sodium 40 19 38 36 4.7 0.01 0.06 0.03 Nitrogen 273 236 268 256 12.6 0.02 0.41 0.17

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2 Calculated as grams of mineral in DM fed – grams mineral of mineral in DM refusal 3Calculated as % in diet x DMI 4 Urine volume calculated as (29 mg daily creatinine excretion/kg BW)/ (analyzed creatinine in urine sample) 5 Urine volume calculated as (0.0259*kg BW * MUN (mg/dL))/ (analyzed N in urine sample) 54

Table 13: Effect of treatment on urine mineral excretion/mineral intake (n=16 per treatment)

Treatment1 P< Mineral LF-K LF+K HF-K HF+K SEM K Fat KxFat Creatinine method2 Phosphorus 1.91 2.23 2.94 2.01 0.95 0.54 0.41 0.20 Potassium 64.9 71.4 65.2 80.9 3.44 0.01 0.02 0.02 Calcium 2.35 0.10 2.42 0.11 0.38 0.01 0.88 0.93 Magnesium 11.6 7.42 9.22 5.90 0.81 0.01 0.01 0.43 Sulfur 72.9 55.8 60.3 51.0 5.47 0.01 0.01 0.16 Sodium 80.7 44.4 76.9 69.6 9.65 0.01 0.16 0.06 Nitrogen 34.6 49.8 38.2 57.8 3.47 0.01 0.01 0.21 MUN method3 Phosphorus 2.04 2.10 3.15 2.15 1.00 0.38 0.28 0.32 Potassium 68.6 68.2 72.0 88.7 5.20 0.10 0.02 0.08 Calcium 2.54 0.08 2.87 0.11 0.56 0.01 0.69 0.74 Magnesium 12.2 6.90 10.2 6.61 1.04 0.01 0.15 0.28 Sulfur 78.3 52.1 66.0 54.3 6.11 0.01 0.28 0.12 Sodium 88.7 40.8 84.1 74.5 10.4 0.01 0.12 0.04 Nitrogen 36.3 47.5 40.9 62.9 3.80 0.01 0.01 0.08

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K). 2Calculated as (29 mg daily creatinine excretion/kg BW) / (analyzed creatinine in sample) 3Calculated as (0.0259*kg BW * MUN (mg/dL)) / (analyzed N in sample)

Table 14: Effect of treatment on mineral milk concentration, g/kg (n=16 per treatment)

Treatment1 P< Mineral LF-K LF+K HF-K HF+K SEM K Fat KxFat Phosphorus 0.85 0.83 0.89 0.86 0.02 0.05 0.01 0.55 Potassium 1.48 1.47 1.51 1.50 0.03 0.43 0.08 0.77 Calcium 1.06 1.05 1.15 1.13 0.04 0.32 0.01 0.69 Magnesium 0.11 0.11 0.12 0.11 0.003 0.19 0.01 0.40 Sulfur 0.29 0.29 0.30 0.30 0.007 0.26 0.02 0.23 Sodium 0.38 0.36 0.41 0.38 0.04 0.22 0.30 0.82

1Treatments were low fat without added K (LF-K), low fat with added K (LF+K), high fat without added K (HF-K), and high fat with added k (HF+K).

Figure 1: Effect of level of dietary fat and K on DMI by week within period (n=16 per treatment). Low Fat=4.2% LCFA; High Fat=5.2% LCFA; K-=1.2% K; K+=2.2% K.

Low Fat

High Fat DMI (kg) DMI K- 21 K+

18 1 2 3 Week Within Period

Figure 2: Effect of level of dietary fat and K on daily DMI over the period (n=16 per treatment). Low Fat=4.2% LCFA; High Fat=5.2% LCFA; K-=1.2% K; K+=2.2% K.

23.5

22.5

Low Fat 21.5

High Fat DMI (kg) DMI K- 21 K+

20.5

19.5

19 0 5 10 15 20 Day Within Period

Figure 3: Effect of level of dietary fat and K on milk production over the period (n=16 per treatment). Low Fat=4.2% LCFA; High Fat=5.2% LCFA; K-=1.2% K; K+=2.2% K.

Low Fat 32 High Fat K-

Milk ProductionMilk (kg) 31 K+

27 1 2 3 Week Within Period

Figure 4: Effect of level of dietary fat and K on milk fat yield over the period (n=16 per treatment). Low Fat=4.2% LCFA; High Fat=5.2% LCFA; K-=1.2% K; K+=2.2% K.

1.2

1.1

Low Fat 0.9 High Fat

K- Milk (kg) Fat Milk Yield K+ 0.8

0.7

0.6 0 5 10 15 20 Day Within Period

Figure 5: Effect of level of dietary fat and K on milk fat percent over the period (n=16 per treatment). Low Fat=4.2% LCFA; High Fat=5.2% LCFA; K-=1.2% K; K+=2.2% K.

3.4

3.2

Low Fat High Fat

2.8 Milk Fat Milk % K- K+

2.6

2.4

2.2 0 5 10 15 20 Day Within Period

Figure 6: Correlation of milk fat percent to trans-10 cis-12 concentration (n=16 per treatment)

Trans-10, cis-12 CLA=0.0928 – 0.014 Milk Fat %

0.6

0.5

P<0.07 0.4

12 12 CLA R² = 0.05 -

cis 0.3

10, 10, -

0.2 Trans

0.1

0 0 1 2 3 4 5 6 Milk Fat %

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