Protein Restriction in Dairy Cattle and Its Effects on Fibroblast Growth Factor 21

Protein Restriction in Dairy Cattle and its Effects on Fibroblast Growth Factor 21 1

University of Wisconsin-Platteville

Protein Restriction in Lactating Dairy Cattle and its Effects on Fibroblast Growth Factor 21

Michael Krueger Dr. James Hampton, Advisor [Type text] [Type text] [Type text] 2

Abstract

This paper contains summaries from other research findings and information on student/faculty research performed on the hormone Fibroblast Growth Factor 21. FGF21 is a newly discovered hormone and very limited research has been performed on it, but current information tells us that it is a signal that the body sends out in response to both starvation and ketogenic diets. Previous research has looked at the effects of complete nutrient restriction, protein restriction in rodents, and its effects on animals. The research project being presented in this paper covers the methods and results of how FGF21 levels are affected when protein restricting dairy cattle. Protein Restriction in Dairy Cattle and its Effects on Fibroblast Growth Factor 21 3

Introduction

In Wisconsin, one of the most economically important areas of livestock research continues to

include factors that influence milk production. Successful reproduction, and therefore lactation, is

dependent on the nutritional state of the animal. In recent years, many novel hormonal factors have

been studied that have linked the ability of an animal to reproduce to its current nutritional state. One

such factor is fibroblast growth factor 21 (FGF21). FGF21 is a protein hormone produced by the

liver following fasting or feed deprivation in many species (Potthoff et al. 2012). One of the most

challenging aspects of dairy production is being able to feed lactating dairy cattle enough nutrients to

support high levels of milk production. In many instances, fertility and reproduction suffer from this

lack of nutrients as well. Interestingly, FGF21 transgenic female mice that over-express FGF21are

infertile and mice treated with exogenous FGF21 fail to produce a preovulatory LH surge (Owen et

al., 2013). Therefore, FGF21 may serve as the signal that prevents animals under negative energy

balance from reproducing successfully.

Consistent with studies in rodents and humans, a study has shown that following caloric

restriction, FGF21 was elevated in the plasma of lactating dairy cows within 14 days of calorie

restriction (Schoenberg et al., 2011). However, a more recent study in rodents has shown that FGF21

was increased following protein restriction alone, and not solely dependent on energy restriction

(Laeger et al., 2014). More studies have also shown us that FGF21 is present during different

lactation periods in cattle as well. It has been found that when cattle are in their peak lactation levels

of FGF21 are highest and when in dry period is basically nonexistent (Schoenberg et al., 2011). This

helps to confirm previous results that calorie restriction increases FGF21 levels (Schoenberg et al.,

2011) because of the differences in the energy usage of the cattle during different lactation phases.

When cattle are in peak lactation they are expending more energy than can be brought in via intake.

This causes a state of nutrient deprivation and thus increases the concentration of FGF21 levels [Type text] [Type text] [Type text] 4 within cattle. On the contrary, when cattle are on a dry cow diet, they are fed enough to maintain body condition and are thus in a state of nutrient equilibrium. This causes levels of FGF21 to be almost nonexistent in the cattle. These findings suggest that reproductive failure may be linked to lack of protein in an animal’s diet rather than simply dependent on gross calorie restriction.

Methods/Materials

All procedures were carried out based on the approval by the UW-Platteville Animal Care and

Use Committee. The overall plan was to recreate the protein restriction experiment performed on the rodents (Laeger et al., 2014), but instead protein restrict cattle to see if they would yield similar results of increased levels of FGF21. The purpose of this is the vital role reproduction has in the dairy industry. Without successful reproduction there is no lactation and thus no milk. When this hormone is expressed it tells the body that it is starving and switches energy usage from nonessential functions, such as reproduction, to essential functions, such as survival. If we can prove that cattle react similarly to protein restriction as mice, then we can use that information to formulate diets to help increase chances of conception thus saving the producer both time and money. The hypothesis for this experiment predicted that when cattle were fed high levels of protein, their FGF21 levels would be initially low or nonexistent, but after protein restricted we expected to see increases in FGF21.

Based on cattle availability, and space available in the facility, 2 different groups of 4 head of cattle were used. This resulted in a total of 8 head of cattle. To compensate for small number of test subjects, the cattle were used as both their own control and test group throughout this experiment.

This was accomplished by drawing blood from cattle during times of increased protein intake and decreased protein intake allowing for comparison of the both data. Figure 1 illustrates our process of when blood drawing occurred. Blood was drawn from cattle during 3 separate times; before dry off, 1 week post dry off, and 2 weeks post dry off. The purpose was that before dry off cattle are on a lactating diet which means it contains a high amount of protein which would yield low or nonexistent levels of FGF21. When cows are in dry off they are no longer producing and thus require a diet much Protein Restriction in Dairy Cattle and its Effects on Fibroblast Growth Factor 21 5 lower in protein. Predictions favor the outcome that the decrease in protein in the diets of cattle would cause an increase in FGF21 levels.

Figure 1: Blood was drawn via tail vein in cattle. First blood draw occurred while cow

was still on a lactating diet but close to dry off time. Second blood draw occurred 7 days

after cows were moved to dry cow pen and switched to dry cow diet. Third blood draw

occurred 14 days after the transition from lactating to dry cow pen.

Following blood draw samples were kept in vials and refrigerated to allow clotting to occur over night. The day after blood collection, after samples had been clotted, a centrifuge, set to 3,000 g for

30 minutes, was used to separate out the serum from the rest of the blood components. Pipettes were then used to extract the serum from the rest of the sample and put into a separate container. The serum samples were then left frozen until all samples had been collected and centrifuged from the cattle.

After all serum samples had been separated and collected the data was then analyzed. The process by which this occurred was through a BUN assay and by sending samples to Missouri. The purpose of the BUN (blood, urea, nitrogen) assay is because it is used to measure the amounts of protein in the cattle’s blood. If the initial prediction was correct, then this test should help confirm that with a high producing diet there is more protein in the blood and with a dry cow diet there is less protein in the blood. The reason samples were sent to Missouri is because there is no commercially available

FGF21 Bovine assay for purchase, but Missouri has one. This allowed measurement of FGF21 in the cattle’s blood which was predicted to be low in the first blood draw, during the lactating diet, and high in the second two blood draws, during the dry cow diet.

Data Analysis [Type text] [Type text] [Type text] 6

Results from the BUN assay, as shown in Figure 2, show that the initial hypothesis did not match the results. During the first blood draw protein levels in the blood were high due to high protein in a lactating diet, which was expected. During the second blood draw when the cow transitioned to a dry off diet, the protein levels decreased because the cow was given a lower amount of protein in its diet.

Since the cattle were still in the process of transitioning from producing to being dried off this caused the protein levels in the blood to decrease, which was also expected. The results from the third blood draw were unexpected. Despite being fed lower amounts of protein, there is more protein in the blood then there would be if the cattle were on a lactating, high protein, diet. A hypothesis for these results is that the cows had completely transitioned and were no longer lactating so did not require as high of levels of protein to keep maintained. This means that there is a higher amount of protein in a dry cows blood due to the fact that they are no longer producing which could also disprove our prediction for the results of the FGF21 assay.

Figure 2: Results from the BUN assay performed on the previously discussed blood

drawings. Day 0 represents when cattle were on a lactating diet and shows high amounts

of protein, which was predicted. Day 7 represents cattle 7 days after being transitioned.

The results show lower protein levels, which we predicted. Day 14 represents cattle 14

days after being transitioned. Results show the highest amount of protein in the system,

which we did not predict.

Data from the FGF21 assay from Missouri, as displayed in Figure 3, show similar results as the data from the BUN assay. Day 0, when cattle were on a lactating diet, there was a lower concentration of FGF21 due to being on a higher protein diet. In the seven-day period between samples, there tended to be an increase in FGF21 concentrations (P = 0.10), which was consistent with the studies in rats. This was caused because of the quick transition from lactating to dry cow diet. The sample of data that was not included in Figure 3 was the results from the 14 days post dry cow transition. The Protein Restriction in Dairy Cattle and its Effects on Fibroblast Growth Factor 21 7

results would have been similar to cattle on a lactating diet due to finally transitioning to a dry cow

diet and no longer producing.

This data provides the first steps necessary to continue studying the effects of protein restriction

and FGF21 concentrations in cattle. However, the diets used in this preliminary experiment were not

designed to exactly mimic the severity of protein restriction used in the rodent experiments and the

increase in FGF21 was most likely to overall calorie restriction as seen in previous dairy experiments

(Schoenberg et al., 2011).

FGF 21 Concentrations

19 18 17 16 15 14 13 12 11 10 0 7

Figure 3: Results from the FGF21 assay. Day 0 represents cattle on a lactating

diet. Concentrations of FGF21 are low during this time. Day 7 represents cattle 7

days after transitioning to a dry cow diet. FGF21 concentrations were high as

predicted.

Future Directions [Type text] [Type text] [Type text] 8

Plans for future testing with protein restriction and FGF21 would involve mimicking the severity of the protein restriction better as done in previous experiments with rodents. To do this it would be better to test cattle during similar lactation phases. This would be accomplished by drawing blood from cattle in the middle of lactation while still on a lactating diet. The next step would be to then protein restrict cattle to about 50% of their normal protein limit. This type of experiment would better mimic the rodent experiment cited in this paper and would give more drastic differences in FGF21 concentrations. A diagram of this sort of experiment is best illustrated in Figure 4.

Control Diet Low Protein Diet ( 1 0 0 % P r o t e i n R e q u i r e m e n t ) ( 5 0 % P r o t e i n R e q u i r e m e n t )

Transi on

Day 1 7 14 19 25 32

Blood Sample Blood Sample

Figure 4: In this version of the experiment all animals will be fed a control diet providing 100 % of their nutrient requirements (NRC, 2001) for a period of 14 days. Blood samples will be collected for subsequent analysis of FGF21 on day 7 and 14 during the control period. Following the control diet, animals will be fed a transitional diet for 4 days, each day containing decreasing amounts of crude protein. Following the transitional diet, all animals will be fed a diet containing 50% of their daily crude protein requirements (NRC, 2001), while all other nutrient requirements will be similar to the diet fed during the control period. Blood samples will be collected for subsequent analysis of

FGF21 on day 7 and 14 during the protein restriction period. Protein Restriction in Dairy Cattle and its Effects on Fibroblast Growth Factor 21 9

This project is an important first step in accumulating preliminary data that could be included in a larger research project. Based on the data collected in this experiment, several follow-up experiments will be needed to fully examine the relationship between FGF21 and protein restriction in dairy cattle.

While FGF21 has been examined in nutrient restricted dairy cattle before, the relationship between protein restriction and FGF21 has not been examined in dairy cattle making this research vital for future research. Follow-up studies could involve collecting liver samples from lactating dairy cows and quantifying differences in mRNA for FGF21 from protein-restricted cows. Therefore, this current performed experiment, has served as the foundation for several subsequent studies that may eventually link elevated FGF21 with reproductive failure in livestock. [Type text] [Type text] [Type text] 10

References Laeger T., Henagan T.M., Albarado D.C., Redman L.M., Bray G.A., Noland R.C. FGF21 is an endocrine signal of protein restriction. The Journal of Clinical Investigation. 2014 Aug 18;124(9):3913–3922. NRC, Nutrient Requirements of Dairy Cattle (7th ed.)Natl. Acad. Press, Washington, DC (2001) Owen BM, et al. FGF21 contributes to neuroendocrine control of female reproduction. Nat Med. 2013;19(9):1153–1156. Potthoff MJ, Kliewer SA, Mangelsdorf DJ. Endocrine fibroblast growth factors 15/19 and 21: from feast to famine. Genes Dev. 2012;26(4):312–324. Schoenberg KM, Giesy SL, Harvatine KJ, et al. Plasma FGF21 is elevated by the intense lipid mobilization of lactation. Endocrinology. 2011;152(12):4652–61.