Are Essential Fatty Acids a Bottleneck to Reproduction? This presentation, and the accompanying discussion, provides the basis for recommending that high producing cows require essential fatty acids be added to their diet in rumen protected form. The Arm & Hammer Animal Nutrition Group at Church & Dwight Co., Inc. has conducted and published many research trials on the subject of ruminally protected fat in the form of MEGALAC. We have demonstrated that MEGALAC, when appropriately added to a cow’s diet provides high quality energy in the form of highly digestible fatty acids. MEGALAC has been shown to have the highest net energy value of all added dietary fats and dose not alter rumen fermentation negatively as other fat sources can do. Our process for manufacturing calcium salts of fatty acids (MEGALAC) results in a product that has the highest quantity of total fat with the smallest amount of free fat and virtually no rancid or oxidized fatty acids compared to other manufacturers. After reviewing over 50 published articles on feeding MEGALAC there appeared to be a consistent trend for improved reproduction and health among cows fed MEGALAC versus all other fat sources. We embarked upon an extensive research program to elucidate why this finding was evident. The results will be shown in this presentation. The research results lead to the creation of MEGALAC-R in North America and MEGALAC-E in Brazil. MEGALAC-R has been sold into the North American market for just over two years and has been very successful in improving reproductive performance of herds as well as improving the metabolic status of cows. It is still the only product of its kind on the market that has the high quality standards of Church & Dwight that MEGALAC has. Others have tried to copy its formulation unsuccessfully and if imitation is the highest form of flattery, then we are flattered. Elliot Block, Senior Principle Scientist ARM & HAMMER® Animal Nutrition 469 North Harrison St., Princeton, NJ 08543 Elliot.Block@churchdwight.com

Outline Summary Introduction Description of Studies Results Why and What Description of Studies Herds, Cows, Diets Results Reproduction, Milk Production Health, Economics Conclusions and Recommendations Before we get into the details of the trials that we have supported and conducted, we will first discuss what is the importance of essential fatty acids and how they impact animal performance and well-being. Nutritionists have known for many years that monogastric animals (swine, poultry and humans) have a specific requirement for essential fatty acids. Deficiencies and imbalances documented include reproductive failure, problems with the immune system and bone metabolism as well as skin lesions. Up until the recent past we have not concerned ourselves with this subject for cows as there was no noticeable problem. However, with the advent of timed artificial insemination and synchronized breeding program, cows that are hormonally treated to cycle and reproduce have not responded to these protocols as predicted. In other words, these programs will improve reproductive performance but not to the level that would have been predicted based on research. Upon closer inspection of survey data, it appears that we are losing ground on pregnancy rates in both cows and heifers that bore no relation to standard dietary nutrients (protein, amino acids, energy, carbohydrates, vitamins or minerals). We then decided to apply some basic monogastric nutritional principles and investigate the essential fatty acids.

Protein Fat Amino Acids Fatty Acids Very few nutritionists will make final decisions on a ration based on crude protein. Crude protein is a simple measurement of all nitrogen in feeds. This nitrogen can be in the form of true protein, but also measures the non-protein nitrogen in feeds. They know that what cows really require is true protein in the intestine (called Metabolizable Protein), which is made up of individual amino acids. In the past we have estimated crude fat by conducting an ether extract on feeds. Ether extract contains true fat but also contains other fat soluble components that are not fat. These include plant pigments, vitamins and waxes. These other components do have the energetic value n or contain the fatty acids that we really desire out of true fat. True fat is composed of fatty acids and glycerol. Typical fat (or triglyceride) is composed of three fatty acids connected to a glycerol molecule. Just as with protein where we desire to obtain amino acids, we want the fatty acids in fat as our nutrient source.

Protein Fat Amino Acids Fatty Acids Essential Amino Acids Acids Required in the diet (cannot be made in the body) Fatty Acids Essential Fatty Acids - Required in the diet (cannot be made in the body) Very few nutritionists will make final decisions on a ration based on crude protein. Crude protein is a simple measurement of all nitrogen in feeds. This nitrogen can be in the form of true protein, but also measures the non-protein nitrogen in feeds. They know that what cows really require is true protein in the intestine (called Metabolizable Protein), which is made up of individual amino acids. In the past we have estimated crude fat by conducting an ether extract on feeds. Ether extract contains true fat but also contains other fat soluble components that are not fat. These include plant pigments, vitamins and waxes. These other components do have the energetic value n or contain the fatty acids that we really desire out of true fat. True fat is composed of fatty acids and glycerol. Typical fat (or triglyceride) is composed of three fatty acids connected to a glycerol molecule. Just as with protein where we desire to obtain amino acids, we want the fatty acids in fat as our nutrient source. Within the group of amino acids in proteins there are certain of those considered to be essential to the animal. The reason that they are called essential is because the body cannot synthesize them yet the are needed for normal functioning of the animal. Similarly, within the group of fatty acids that comprise fat, certain of these are also considered essential to the animal. They cannot be made by the body but are essential for proper function.

Protein Fat Amino Acids Fatty Acids Essential Amino Acids Acids Required in the diet Methionine, lysine, etc. (10 total) Fatty Acids Essential Fatty Acids - Required in the diet - Linoleic acid (18:2) - Linolenic acid (18:3) (only two) Of all the amino acids, ten fall into the category of “essential”. These include the two amino acids thought to be the most limiting to milk production, Methionine and Lysine. Of all the fatty acids, only two cannot be made by the body and are considered to be “essential”. These are Linoleic acid and Linolenic acid. Nomenclature for fatty acids is usually shown as two numbers. The first number refers to the length of the carbon chain and the second number refers to the number of double bonds. Zero double bonds is called a saturated fatty acid, one double bond is called a monounsaturated fatty acid, and more than one double bond is called a polyunsaturated fatty acid. Most of the fatty acids in feeds are greater than 16 carbons in length. Palmitic acid (16:0) is saturated and found in many foods and feeds and is highly digestible. Stearic acid (18:0) is saturated and is found in tallow and animal fat. It is also produced by the rumen microbes when the cow is fed unprotected unsaturated fatty acids by a process called biohydrogenation. The problem with Stearic acid is that is not well digested by cows and has a low energy value compared to other unsaturated fatty acids that reach the intestine. This is why MEGALAC and MEGALAC-R or E have such a high energetic value. The highly digestible unsaturated fatty acids contained in these products are ruminally protected from becoming Stearic acid. Although products like soybeans have a high amount of unsaturated fatty acids, these become less digestible as the rumen microbes hydrogenate them to Stearic acid. The essential fatty acids mentioned above are Linoleic (18:2 – 18 carbons with 2 double bonds) and Linolenic (18:3 – 18 carbons with 3 double bonds).

Energy Metabolic Effects Fat Fatty Acids Fermentation, Digestion, Energy Metabolic Effects Fat Crude Fat (Ether Extract) Energy What is happening in the field of nutrition is a shift away from ether extract and crude fat towards more descriptive and precise terminology regarding fat. This new description allows us to begin separating out the energetic effect of fat from the effects of fatty acids on rumen fermentation, intestinal digestion and some of the metabolic effects of the essential fatty acids. There is a limit to the amount of free or unprotected fat that you can add to a ration. Whole oilseeds in the raw or roasted form release unsaturated fatty acids that can upset rumen fermentation. These fatty acids can be toxic to microbes at certain concentrations resulting in reduced fiber digestion, milk fat depression and a reduction in microbial protein that can lead to a protein deficit even though the original ration appeared to be sufficient. Further, as these fatty acids become hydrogenated by the rumen microbes, the resulting saturated fatty acids have a lower energy value than the fatty acids originally fed to the cow. Saturated fatty acids can be fed to the cow in the form of animal fat, hydrogenated tallow or poorly processed ruminally protected fat. These will have a smaller impact on rumen fermentation but will also have a low digestibility compared to unsaturated fatty acids reaching the intestine, as with MEGALAC products. In fact, these saturated fatty acids have 60% of the Net Energy value of MEGALAC, which makes them only slightly better energetically than ground corn.

Milk Response to Rumen Active-Fat (Hypothetical Model) II III + Milk Increase, lb/d Dr. Tom Jenkins at Clemson University put together a very descriptive model showing how to use added fat in a diet. He breaks down added fat into three distinct phases. In Phase I you can add fat to a diet from any source (tallow, oilseeds, etc.) and get improvements in milk yield due to the extra energy. At some point (this will be described later) you will exceed the amount of free fatty acids that the microbes can handle and any additional fat beyond that point begins to reduce microbial function and fermentation. This is now Phase II of adding fat. Any additional fat, therefore, increases the dietary energy density but is offset by the reduction in rumen performance and possibly feed intake. The net result is that added fat beyond that point does not increase milk production. At another point along the curve (Phase III) you see a reduction in milk production when you add more fat. In this phase, the reduction in rumen functionality and reductions in feed intake far outweigh any added energy to the diet from fat. - Added Fat, % Jenkins, 1998 44 42

Bypass Fat - Breaks the Fat Barrier MEGALAC I II III + Milk Increase, lb/d The benefit of calcium salts of fatty acids (MEGALAC) is that they are inert in the rumen and do not negatively affect rumen function. This benefit allows you to extend Phase I on the graph. In other words you can add more fat to a diet with MEGALAC than with any other fat source and continue to realize improved milk production where other fat sources would fail. Our philosophy is that the first amount of incremental fat added to a diet should be in the least expensive yet highly digestible form possible and remain in Phase I of te graph. This could be oilseeds (cotton, soy). Once you have maximized the Phase I fat addition incremental fat should be added in the form of MEGALAC. Now the question is “How do I know when I have reached the end of Phase I on fat addition?” Dr. Jenkins's model defines the amount of rumen active or free fat that you can add to a diet. It is based on the amount of fibre in the diet coupled with the degree of unsaturation of the fatty acids in the fat used.. Research has shown that as the neutral detergent fibre level (NDF) in the diet increases, the amount of free or active fat can increase without upsetting rumen fermentation to any significant degree. Research has also shown that the more unsaturated the fatty acids in the fat, the less of that fat that can be added to the diet. We have put together a fat feeding worksheet (next slide) that can help to determine how much fat can be added. - Added Fat, % Jenkins, 1998 42 44

This worksheet is a ration fat EVALUATOR. It does not balance diets. If we input the milk production, milk fat %, and dry matter intake along with the NDF level of the diet (left side of the worksheet), the model will tell you how much fat can be fed in this diet and how much of that can come from rumen active fat and how much from MEGALAC. On the right side of the screen you can input the actual ingredients that contain free or active fat with the quantities that are currently being fed. The worksheet will calculate the quantities of unsaturated fatty acids and tell you if you have exceeded the Phase I recommendations. NOTE that cells colored in blue can be inputs by the user.

Essential Fatty Acids Linoleic (18:2) Linolenic (18:3) Now that we’ve discussed the effects of fatty acids on rumen fermentation and intestinal digestion, we can move further along on the metabolic effects of these two essential fatty acids, Linoleic (18:2) and Linolenic (18:3) acids. Realize that it is only MEGALAC-R and MEGALAC-E that have data that conclusively show that these fatty acids are delivered to the cow. The problem with some of the competitive products is that, while they start with the correct raw materials that contain these unsaturated fatty acids, their production process is not elaborated enough to ensure that the resulting product has the same fatty acids that were originally used. Many competitors either start their process with poor quality raw materials and do not have controls over pressure, temperature and cooling resulting in finished product that now has saturated fatty acids and/or rancid or oxidized fatty acids. As previously mentioned, deficiencies of these essential fatty acids have been shown to cause metabolic problems with skin, reproduction and the immune system in all mammals that have been studied to date, including humans.

Reproduction Research and Fat Staples and Thatcher, University of Florida Reviewed Studies in 1997 Feeding fat: Increased number and size of ovarian follicles Conception rate 17% greater (11 of 18 studies) Fat source most studied - MEGALAC® Statistically improved pregnancy rates! Why?????????? Drs. Charles Staples and Bill Thatcher at the University of Florida reviewed the effects of feeding fat on reproductive performance of dairy cows. We already have clearly defined that negative energy balance, beginning just before calving and continuing throughout early lactation, causes reproductive inefficiencies (delayed ovulations, poor signs of estrous, etc.). Staples and Thatcher wanted to know if dietary fat had a specific effect on reproduction other than its energy contribution in alleviating this negative energy balance. In reviewing the literature, they found 18 studies that fed diets with and without added fat but also had the same energy values by using carbohydrates to make the diets isocaloric (same energy). Using these studies they concluded that added fat had a specific, positive effect on conception rates and on the number and size of ovarian follicles outside of the energy effect. Interestingly, 11 of the 18 studies showed this positive response and 10 of the 11 positive studies used MEGALAC as the fat source. The conclusions pointed to the fact that only MEGALAC was supplying some amount of unsaturated fatty acids to the cow that the other sources of fat were not.

MEGALAC®-R Essential Fatty Acids: A Source of Essential Fatty Acids Why: MEGALAC®-R Essential Fatty Acids: A Source of Essential Fatty Acids These are lost in milk of high-producing cows Supplemental Essential Fatty Acids: Increase size of ovulating follicle Improve embryo survival Increase pregnancies Have potential to improve health MEGALAC-R is a nutritional supplement that supplies essential fatty acid (EFAs) nutrients to the cow in addition to the energetic, rumen inert properties and benefits of regular MEGALAC. The reason that they are termed “essential” is because the normal biological and biochemical processes in the body cannot produce them. Previous research with MEGALAC and prototypes to MEGALAC-R have shown the benefits listed in the slide above.

Linoleic 18:2 to 20:4 (omega 6) Linolenic 18:3 to 20:5 and 22:6 One obvious question is ‘What are these fatty acids used for in the body that makes them absolutely essential to the animal?’ Linoleic and Linolenic acids cannot be produced by the mammalian system but all other fatty acids needed by metabolism can be produced. The body uses these two fatty acids to produce other fatty acids. Linoleic acid is used to produce Arachidonic acid and Linolenic acid is used to produce two other fatty acids called EPA and DHA (commonly referred to as fish oil fatty acids). The reason that these three fatty acids arising from the essential fatty acids are important is evident on the next slide.

20:4 omega 6 PGE2 PGF2 20:5 omega 3 PGE3 22:6 PGF3 These three fatty acids are what the body uses to synthesize the group of compounds called PROSTAGLANDINS. Prostaglandins are a group of signaling or messenger compounds that cause almost all reactions in the body to proceed fast, slow, or not at all. They are intimately associated with the immune response, bone metabolism and reproduction. In the reproduction process, the prostaglandins are involved with everything from the estrus cycle itself to ovulation, implantation and parturition. Inadequate production of these prostaglandins that can be caused by an essential fatty acid deficiency can and will lead to inefficient reproduction processes. As an aside, you might ask why essential fatty acids improve reproduction but not the fish oil fatty acids. This is theorized to be true because the half life of EPA and DHA in the body is short while the essential fatty acids can be stored and become part of cell membranes. When the body needs a prostaglandin produced, the essential fatty acid in the cell membrane can be used to accomplish this. If we relied on EPA and DHA for prostaglandin synthesis we would need a constant supply in the diet to assure that these fatty acids were present at the exact moment that the body wanted to produce a prostaglandin.

If we look at a cell membrane, the interior of the membrane has finger-like projections, which are individual fatty acids. Some of these fatty acids must be the essential fatty acids. When the body requires prostaglandin, the essential fatty acid is released from the membrane and is converted to the appropriate prostaglandin. The space left by this prostaglandin production has to be filled with a fatty acid. If there is an essential fatty acid deficiency, the space can be filled with any fatty acid. Te result of this would be to have a suboptimally functioning cell membrane that can no longer produce the required prostaglandins. Lipid Bilayer

How much linoleic acid (18:2) does a cow secrete per day in milk? In milk, 2 to 6% of milk fat is 18:2 100 lbs/day @ 3.5% fat and 4% 18:2 = 0.14 lbs/day 18:2 (64g/day) Assuming no other need for 18:2, this would be the requirement One of the first questions we had to answer was whether there was a case to be made for need of these fatty acids. We will use Linoliec acid (18:2) to make this case. Admittedly, unlike in poultry and swine nutrition, we do not know the specific requirement for intestinally available Linoleic and Linolenic acids in the cow. However, if we simply calculate the amount of Linoleic acid secreted in milk we have an idea of some absolute minimum that we need to replace in a day. In the example in the slide above, this cow secreting 100 pounds of milk per day is producing milk with 64 grams of Linoleic acid. If there were no other requirement in the body for this fatty acid (which is a false statement) then 64 grams would be her daily requirement to be absorbed from the intestine. Based on information derived from swine and laboratory animals the actual requirement is likely 2 to 3 times greater than this (120 to 180 grams/day). Since other cells in the body and other functions require linoleic acid (18:2), there is likely an additional requirement for 18:2 above this 64 g/day

Duodenal appearance of 18:2 ranged from 39 to 63 g/d vs. 64 g secreted Using the CPM fat sub-model simulating several (12) rations: DMI ranging from 22 to 29 kg/day Ingredients Alfalfa, Corn Silage (30:70 to 70:30) SBM (1-3.5 kg/d) Corn (6-9 kg/d) Fish meal (0-1 kg/d) MEGALAC (0-1 kg/d) The CPM (Cornell, University of Pennsylvania, Miner Research Institute) ration balancing/evaluating program is software system that uses the CNCPS (Cornell Net Carbohydrate and Protein System) equations to evaluate dairy cow performance based on a given diet. The CPM model is universally accepted as our best estimates to predict dairy performance. We used the fatty acid sub model of this system to estimate amounts of Linoleic acid delivered to the intestine of cows given 12 fictitious diets. These diets ranged from fairly typical high production diets to fairly absurd diets designed to “push” the system. The highest amounts of Linoleic acid that we could get to the cow was 63 grams per day versus the 64 grams that we previously calculated that she would secrete in milk. Therefore, we are extremely confident that there is a biological and physiological need to create supplements that would deliver more of these essential fatty acids to cows. Duodenal appearance of 18:2 ranged from 39 to 63 g/d vs. 64 g secreted

Effect of Long Chain Fatty Acids on Reproductive Tissues and Lactation Performance of Holstein Cows C.R. Staples et al. 2000 One of the first studies that we supported to determine if there were effects on reproductive parameters by essential fatty acids was conducted at the University of Florida by Drs. Charles Staples and Bill Thatcher. The results were published in the Journal of Dairy Science in 2000 as an abstract and in 2001 as a journal paper. The next few slides shows the major results of this trial.

Early Lactation Study; Dairy Science Meetings, 2001 One diet was fed to cows with treatment being four different types of supplemental fatty acids in the form of Calcium salts of fatty acids (as in MEGALAC products). Treatments were either no supplemental fatty acids or an addition of 2.2% of the dry matter as calcium-fatty acids high in Oleic acid (nonessential), Linoleic acid (essential), or the fish oil fatty acids, EPA and DHA (non-essential but are produced from the essential fatty acids). Diets were fed to non-pregnant, cycling, early lactation dairy cows. Reproductive indicators were measured. Porcine meat/bone ml

Ultrasound was used to determine if fatty acids had an effect on follicle and corpus luteum size. The size of the final ovulatory follicle is positively correlated with health and viability of the ovum. Both the Linoleic acid and EPA/DHA treatments caused the final follicles to be significantly larger than the control diet or oleic acid treatments.

Cells from the uterus and corpus luteum were isolated and evaluated for their ability to produce prostaglandins. Only cows supplemented with the essential fatty acids directly had a higher capability of producing prostaglandins in these tissues. In this case the EPA and DHA supplement was unable to achieve this.

At the end of the trial, cows were all synchronized with HCG and the progesterone profiles were followed for that induced cycle. Only the cows supplemented with Linoleic acid had maintained progesterone in the last days of the cycle. There are two important implications for maintained progesterone levels through the entire cycle. First, if the cows did become pregnant during a cycle, the high progesterone would help assure that the pregnancy is maintained during the early critical phases after implantation. Second, if cows were not pregnant, the high progesterone at the end of the cycle followed by a sharp decline in progesterone at the beginning of the next cycle would improve the expression (behavioral signs) of estrus (heat).

We also evaluated the production performance of cows fed these supplements. Milk production was increased for all the supplemental fat treatments compared to control cows. While milk fat percentage was unaffected by treatment, milk protein percentage was decreased with the fish oil fatty acids, EPA and DHA. This is not an uncommon finding as many trials reported in the literature show a milk protein depression when fish oils are fed. These other trials also usually show a decrease in dry matter intake. We did not see a significant depression in feed intake when DHA and EPA were fed, however, there certainly was a tendency for this feed intake depression to occur.

CPM /CNCPS fat submodel comparison of fat sources to deliver EFA (ADSA Abstract 2002. Univ Penn.) Researchers at the University of Pennsylvania (Moate and Chalupa) used the CPM/CNCPS model to evaluate various fat sources in their ability to deliver essential fatty acids to the intestine of cows. The results were published in the Journal of Dairy Science (2002) as an abstract. In the results above, the basal diet (formulated for a cow in early lactation producing 45kg of milk per day) was delivering 500 g of total fatty acids per day. After rumen transformations (biohydrogenation) and accounting for differential absorption for fatty acids, the basal diet delivered 48 g and 1.1 grams per day of Linoleic and Linolenic acids, respectively. Obviously not enough for the cow example requiring 130 to 180 g per day of Linoleic. Another interesting fatty acid that they evaluated was one called trans 18:1 (trans Oleic acid). It is thought that when this fatty acid exceeds 60 g per day in the intestine you can start causing milk fat depression. This basal diet supplied 29 g of trans 18:1. They then evaluated 400 g of additional fatty acids from various sources (M=MEGALAC; MR=MEGALAC-R; EB=Energy Booster-hydrogenated fatty acids; T=Tallow; RSB=Roasted Soybeans; WCS=Whole Cottonseed). Note that MEGALAC-E in South America has double the quantity of essential fatty acids that MEGALAC-R does in North America. Only MEGALAC-R yielded the highest amounts of absorbed essential fatty acids and when added to the amounts from the basal diets, approaches the estimated requirements of the cow. Even roasted soybeans with twice the essential fatty acid content as MEGALAC-R produced less of these for absorption due to rumen transformations. Additionally, notice that the RSB and WCS produced a large amount of trans 18:1 for absorption, which is why we often see milk fat depressions when these are fed at levels on excess of 2kg per day, delivering more than 400g of fatty acids.

Production trial at Miner Institute with MEGALAC-R Prototype ADSA Abst Production trial at Miner Institute with MEGALAC-R Prototype ADSA Abst. 2001 (accepted J. Feed Anim Sci Tech.) The results from a study conducted at the Miner Research Institute with our first prototype MEGALAC-R product are shown above. In this case, we were not yet certain about the formulation so we used soy fatty acids in the production of the calcium salts. The objective was to evaluate the production performance of cows fed MEGALAC-R prototype versus regular MEGALAC. Theoretically, the two products should act identically in cows with no differences between then in milk productivity. High producing Holsteins were fed MEGALAC or the MEGALAC-R prototype beginning at parturition and continued through the first 10 weeks of lactation. As expected, there were no differences in the production performance of cows. All cows performed well on the diets at over 43 kg milk per day and 4.2% fat. The only differences were in the fatty acid profiles of the milk fat. The milk fat of the cows fed the MEGALAC-R prototype had more CLA and Linoleic acid in the milk, indicating that the fatty acids were protected from rumen biohydrogenation.

Calcium Salts of Polyunsaturated Fatty Acids Deliver More Essential Fatty Acids to the Lactating Dairy Cow   M.L. Theurer*1, M.A. McGuire1, E. Block2, W.K. Sanchez2 1University of Idaho, Moscow 2ARM & HAMMER® Animal Nutrition Group, Princeton, NJ Pacific Northwest Nutrition Conference 2002 Upon finalizing the formulation for MEGALAC-R, we went to the University of Idaho to conduct a very similar trial to the one at the Miner Research Institute.

Research Objective Evaluate intake, milk production and milk composition effects of cows fed MEGALAC-R Essential Fatty Acids compared to those fed MEGALAC Rumen Bypass Fat. The objective is stated above.

Materials and Methods Experimental design and animals Three 14-d periods in a switchback design. Nineteen lactating Holstein cows (83 ± 9 DIM) 12 days diet adaptation followed by 2 days of sample collection Dietary treatments Supplements added to basal diet and fed once/d, individually through Calan gates Basal diet (DM Basis) consisted of 30% chopped alfalfa hay, 6% triticale silage, 9% whole cottonseed, 1.6% calcium salt supplement, 53% concentrate mix (rolled barley, steam rolled corn, distillers grains, soybean meal) MEGALAC (CON) or MEGALAC-R (TRT) fed at 462 g/d. Trial design is explained above.

Effect of calcium salts of polyunsaturated fatty acids on milk production and feed intake kg/d Again, comparing MEGALAC to MEGALAC-R there were no differences in dry matter intake (DMI) or milk production. Milk DMI

Fat or Protein Yield (kg/d) Effect of calcium salts of polyunsaturated fatty acids on milk components Fat or Protein Percent Fat or Protein Yield (kg/d) Fat % Protein % Fat Yield Protein Yield Milk components were not significantly affected either.

Effect of calcium salts of polyunsaturated fatty acids on palmitic and linoleic acid in milk fat % of Total Fatty Acids Linoleic acid (18:2) Palmitic acid (16:0) *** *** P < 0.0001 The fatty acid composition of the milk again showed that those cows the calcium salts of the Linoleic acid had higher concentrations of this fatty acid in milk.

Conclusions Feeding Ca-salts of polyunsaturated fatty acids did not alter milk production, milk fat percent, or dry matter intake when compared to MEGALAC. Calcium salts of polyunsaturated fatty acids limited rumen biohydrogenation of essential fatty acids and supplied more essential fatty acids to the small intestine as detected in milk fat. The conclusions as stated by the authors are shown above.

How it Started? Conducted several research trials Facilities People transition cow, early lactation, USDA drug Facilities Close-up & Fresh Cow Pens People Employees Veterinarian & Nutritionist Reproduction improvement/performance Now we had the basic information to begin field testing a product to determine if there were effects on a productive performance or health functions. We estimated that cows needed more essential fatty acids and we had the research to show that these fatty acids can be delivered to cows in their intestines. In order to have statistically valid information, we needed to recruit large dairy operations that were capable of separating their prepartum transition cows as well as their cows in early lactation. Data collection had to be as precise as if we were conducting a drug trial. This includes a high level of performance by the employees, managers, nutritionists and veterinarians involved. Finally, we had to have reliable reproduction performance data captured in a retrievable database.

Reproduction Heavy emphasis at KRU Fresh cow/ transition cow program Monitor transition cows Modified Targeted Breeding Program Solid breeding program before the MEGALAC-R trials Our first study was conducted with a MEGALAC-R prototype at the farm “Kows-R-Us” (KRU) in Idaho. The farm is a 1600-lactating cow farm with open lots and free stalls. They employ a modified target breeding program and monitor all animals on the farm. Their data collection is with DairyComp 305. We examined their records prior to starting any trial and found that their hers averaged 13,000 kg of milk and their pregnancy rate was 17%. Considering the national average for pregnancy rate is 12%, this herd is considered excellent. Since many people define pregnancy rate differently, it would be prudent here to discuss how this metric should be viewed. Pregnancy rate, for the purpose of both economic and herd performance indices, is the number of cows that were confirmed pregnant out of ALL COWS THAT WERE ELIGIBLE TO BECOME PREGNANT (all open cows) within a 21 day cycle. The mistake that people make is that they assume (for example) that if they bred 10 cows this month and 4 of those cows were confirmed pregnant 45 days later, that the pregnancy rate was 40%. This is NOT CORRECT. What was left out of the denominator were all of the cows that were eligible to be bred but never were bred. These cows may not have been considered because they were sick, had silent heats, missed heats, cystic, or for ay number of other reasons. Irrespective of the reason, they were open cows that were not bred and will therefore negatively affect the economics and overall productive efficiency of the farm. To continue with our example of 10 cows bred with 4 pregnancies, there were 15 additional cows that were eligible to be bred but were not. Now the total number of cows eligible to be bred is 25 with 4 confirmed pregnancies. The pregnancy rate for that month is 16%, not the 50% previously calculated.

First Trial Feeding MEGALAC-R (prototype) to close-up cows Trial June 2000-March 2001 ~350 cows /treatment Treated group fed 1/4 #/hd/day MEGALAC-R for 21 days pre-fresh Control group In this trial at KRU we only worked with the prepartum transition cows. These cows were split into two equivalent pens. One pen served as control and the other was the MEGALAC-R prototype treated pen. Treated cows received 115 g per cow per day of the MEGALAC-R prototype from 21 days prepartum through parturition. After calving ALL cows received the same diets, which contained regular MEGALAC. Between June 2000 and March 2001 we had a total of 700 cows (350 per treatment group) that completed the trial. Cows were fed the supplement prepartum but we had to monitor reproduction through successive reproductive cycles.

The data above was taken directly from DairyComp 305 The data above was taken directly from DairyComp 305. The data was sorted by calendar date representing 21-day cycles (all cows irrespective of days in milk). The second column is the number of cows that were eligible to be in heat (Ht Elig) during that cycle followed in the next column by the actual number of cows that were in heat (Heat). Pct is heat detection rate percentage. Pg Elig is the number of cows that were eligible to become pregnant that month (all open cows) and Preg is the number of cows that were actually confirmed pregnant 45 days post insemination. Finally, a PREGNANCY RATE (Pct) can be calculated as a percentage by dividing the pregnancies confirmed (Preg) by the number of cows eligible to become pregnant (Pg Elig). Overall, cows that received the MEGALAC-R prototype prepartum had a significantly high pregnancy rate (24%) compared to control cows (19%). Note that even the 19% rate is considered excellent.

We then re-sorted the data by days in milk (DIM) We then re-sorted the data by days in milk (DIM). This farm had a 50-day voluntary waiting period before they would breed any cows, which is why the data starts at 50 DIM. Some cows dropped out of the data set resulting in slightly different numbers from the previous slide because cows that were beyond 155 DIM or those that were by chance bred before 50 DIM were included in the previous data set (slide) but excluded here. Again, we see a major improvement in pregnancy rate in cows fed the MEGALAC-R prototype. However, we see that in addition to getting more cows successfully pregnant, that most of the improvement was occurring in the first 2 cycles. Therefore, not only does MEGALAC-R get more cows pregnant but it also gets them pregnant sooner. This is of extreme economic importance to the dairyman.

Analyzing this data by Chi-Square analysis shows that the pregnancy rate was improved by 24%, which was statistically significant (P<.02).

A New Formulation We were now prepared to go forward with a formulated product for retail sale. In North America we sell MEGALAC-R with 25% Linoleic acid and 3.5% Linolenic acid and in South America we produce MEGALAC-E with 45% Linoleic and 6% Linolenic acid.

What: The largest reproduction data set (with high producing cows on modern dairies) ever assembled! Top management, excellent reproduction > 27,000 lbs. 305 ME milk > 5,000 cows, > 14,000 eligible breedings Studies from across the U.S.A. We now had the challenge of developing field data for this product. The challenge was to have sufficient numbers for cows, breedings and locations across the USA. Secondly, we had the challenge of having herds that were already considered in the top 15% for both production and reproduction with reliable and accessible data. We set the criteria that any herd for a field trial had to have a 305 mature equivalent (ME) production of at least 12,500 kg milk with a n established pregnancy rate of greater than 12%. WE also determined that we needed at least 14,000 breedings in the data set from at least 5,000 cows to be assured that the statistical analysis would have relevance.

How: – Four Dairies 1400-cow free stall dairy - Northeast 2500-cow free stall dairy - Midwest 1600-cow open lot dairy - West 3900-cow open lot dairy - West Fed either what they currently were feeding (MEGALAC or Tallow; control) vs. MEGALAC-R in place of the control ¼ lb prepartum ½ or 1 lb postpartum We met these criteria with the four herds outlined n the slide above. The ideal trial was to have treatment cows fed 115 g/day prepartum and 450 g/d postpartum MEGALAC-R with control cows fed the same amount of regular MEGALAC. Unfortunately, due to managerial considerations and to accommodate the farm’s nutritionists, there were variations on this ideal design. Only the farm in the Northeast fed the products at this rate. The other operations fed variations on the theme (i.e., control cows getting Tallow instead of MEGALAC, feeding rates were 225g postpartum instead of 450g, all cows fed MEGALAC-R prepartum and only postpartum were they split into MEGALAC vs. MEGALAC-R). The next slide shows the results from these studies.

Presenting the data as pregnancy rates would be too confusing as, by definition, a rate is an outcome within a given time frame. Therefore, calculated the cumulative pregnancies over the number of reproductive cycles examined on each farm and divided that by the total number of animals that were enrolled in the program in the time frame (open cows that started and finished the feeding program). This resulted in a cumulative percent pregnant cows for the study. Trial #1 was that Northeast farm that conducted the trial as we designed. In that case the cumulative pregnancies increased by 19% for cows in the MEGALAC-R group. The other farms also had positive responses to MEGALAC-R but lower in magnitude due to the reduced feeding rate. Interestingly, in Trail#4, ALL cows received MEGALAC-R prepartum and were split into MEGALAC vs. MEGALAC-R only in the postpartum period. This farm still had an overall positive response to MEGALAC-R proving that it is important to feed the product both pre and post-partum to achieve maximum benefit.

The two trials with the lowest responses to MEGALAC-R were further evaluated. When we separated out the first lactation animals for the older cows, we found that these first lactation animals responded much better than older cows. This was ample evidence to us that older cows certainly need more than 225g/d of MEGALAC-R. These younger animals that are lighter in body weight, smaller, and generally produce less milk needed less essential fatty acid to improve their reproductive performance.

MEGALAC-R fed Prepartum No MEGALAC-R prepartum 6/15/01 – 11/1/01 After 11/1/01 MEGALAC-R fed Prepartum No MEGALAC-R prepartum At the conclusion of the study, two of the four farms decided not to continue MEGALAC-R feeding. In the slide above, you can see the results on pregnancy rates (Pct column following the Preg column) for each 21-day cycle after removal of the MEGALAC-R. The pregnancy rate on this farm for cows bred in their first cycle (55 DIM) went from 33% when fed MEGALAC-R to 22% when the MEGALAC-R was removed; second cycle results went from 16% to 12%; etc. Upon seeing these results this farm went back onto MEGALAC-R.

7/15/01 – 12/1/01 After 12/1/01 On Experiment Off Experiment Similarly, the other farm that stopped feeding MEGALAC-R at the conclusion of the trial showed similar results. First cycle pregnancy rates (45 DIM) declined from 20 to 12%: second cycle (66 DIM) declined from 21 to 14%; etc. This farm also recommenced feeding MEGALAC-R.

Follow-Up Trial 1/4 #/hd/day MEGALAC-R all close-ups Feed 1/2 #/hd/day MEGALAC-R lactating cows to 120-150 DIM Cows randomized into control or treated groups …at freshening moved to treated or control pen FCH & cows kept in separate strings We revisited that Idaho farm (Kows-R-Us) to repeat our most challenging trial. All cows fed 115g/day of MEGALAC-R prepartum and half the cows fed 225g/day regular MEGALAC and the other half fed 225g/day MEGALAC-R postpartum. We kept the first calf heifers (FCH) in separate strings postpartum.

21 day Preg Rate Bredsum Calculations Example Bredsum records for overall (all animals) controls and MEGALAC-R and heifers controls and MEGALAC-R. Overall First lactation heifers In the next slide we’ll show the results in the overall herd and the results for the first calf heifers alone. Remember, ALL cows received MEGALA-R prepartum, therefore, differences would prove a need for feeding this product postpartum.

Overall – Control The following slides show an example printout from the DairyComp 21-day heat trial (bredsum) from the 1600-cow dairy. This dairy was used to test the most challenging comparison – all cows were fed MEGALAC-R prepartum and the treatment animals were fed 225g/day MEGALAC-R compared to 225g/day MEGALAC during the fresh and early lactation periods (to approximately 110 – 120 days in milk). This slide shows the overall (all cows) reproductive performance for cows fed the control diet. The average pregnancy rate for 13 cycles was 20%. Note that anything near 20% represents an excellent pregnancy rate. 20

Overall – MEGALAC-R This slide shows the overall (all cows) reproductive performance for cows fed the MEGALAC-R diet. Even though the herd was doing well with a 20% average pregnancy rate, there was a large increase in overall pregnancy rates (23% vs. 20%) for all cows fed MEGALAC-R vs. those fed the control ration. 23

Control - First Lactation Heifers This slide shows the 1st lactation cows only reproductive performance for those fed the Control diet. Note that the first lactation heifers fed the control diet (MEGALAC-R prepartum and regular MEGALAC postpartum) had a better overall pregnancy rate than the rest of the herd fed the control diet (23% for these animals vs. 20% for the rest of the control cows). 23

First Lactation Heifers – MEGALAC-R This slide shows the pregnancy rates for 1st lactation cows fed the treatment diet (MEGALAC-R prepartum AND postpartum). Note the very large increase in overall pregnancy rates (30% vs. 23%) for these first lactation cows fed MEGALAC-R vs. those fed MEGALAC postpartum. We believe the 1st lactation animals responded better than the cows due to their smaller body sizes and lower milk production. Their essential fatty acid requirements were lower due to these factors. 30

Drs. James Furguson (University of Pennsylvania production medicine dept.) and Mark Kinsel created this economic evaluation program. Behind this screen are a series of complex modeling calculations that evaluate how a change, any change, in management, feeding and other costs affected the economics of the herd. On the left side of the screen you see the input data for the herd economics before we made any change (Baseline column). These were the actual numbers that the producer entered for the previous trial at Kows-R-Us. They were obtained from his financial records as well as DairyComp 305. The column in yellow (Alternative) is the input data from the producer on the actual results of this MEGALAC-R trial. Note that the cost of the supplement is included in the input. The right half of the screen shows the Expenses and Income Statement for the Baseline and Alternative data. Note that the pregnancy rate went from 20 to 23% as shown in the trial results. When expenses and income are expressed on a yearly basis, the MEGALAC-R supplement showed a net benefit (after all costs including the cost of the MEGALAC-R) of $0.14/cow/day or $50.84 per cow per year. Whether you have 50 cows or 5,000 cows, this is a significant change in profitability.

Another Large California Herd Evaluation A large dairy company in California two Dairies at 1.2 lbs of MEGALAC and put two other similar dairies on 0.6 lbs of MEGALAC and 0.6 lbs of MEGALAC-R. All managed and fed the same Evaluation began in June 2002, ended June 2003. Data from full year used to evaluate the intervention. Our final field study was conducted on a “Mega-Farm”. This is a 5,000 cow operation that houses cows in four identical barns. All feed handing is centralized so that all barns receive the same diets and ingredients. The herd average for this operation is 14,600kg milk (rolling average) and a 16% pregnancy rate (still considered far above average). The normal diet for this herd has 550g of regular MEGALAC per cow per day. We removed 275g of MEGALAC from two of the barns and replaced it with 275g of MEGALAC-R (all 4 barns still received 550g per day of total MEGALAC products). We evaluated the four barns side by side for a full year. The data from the four barns was obtained directly from the producer and entered into the economics calculator.

In this case the pregnancy rates were improved with MEGALAC-R from 17 In this case the pregnancy rates were improved with MEGALAC-R from 17.5% to 22.75%. In this case, the treated cows received MEGALAC-R both pre and post-partum, which increased the cost of treatment. In spite of the higher cost, the net benefit (accounting for the cost of treatment) was $0.21 per cow per day or $76.18 per cow per year. Notice that the cost of supplement (MEGALAC-R only) was only $.05 per cow per day higher when expressed on a yearly basis. However, the net benefit was $.21 per cow per day. THAT IS MORE THAN A 4-to-1 RETURN ON INVESTMENT.

Summary MEGALAC-R is a profitable investment Every trial showed a positive response Feeding 1 pound MEGALAC-R postpartum, showed the greatest response in both cows and first-calf heifers Feeding MEGALAC-R prepartum is critical to success MEGALAC-R reduces early embryonic death MEGALAC-R has shown other benefits (immunity related) Better understanding of future research directions

Aurora Florida Dairy #1 began Sept

Aurora Florida Dairy #1 began Sept

Aurora Florida Dairy #2 began Sept

Aurora Florida Dairy #2 began Sept

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