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Abstract
1 2 Effects of a blend of essential oils in milk replacer on performance, rumen fermentation, 3 blood parameters and health scores of dairy heifers 4 5 Abstract 23 The objective of this study was to evaluate how the inclusion of a blend of essential oils 24 in milk replacer (MR) affects different outcomes of dairy heifers. The outcomes 25 evaluated: feed intake, performance, body development, blood cells and metabolites, 26 insulin-like growth factor-1 (IGF-1), rumen fermentation, fecal scores and respiratory . CC-BY 4.0 : bioRxiv preprint 2 27 scores. All outcomes were evaluated during pre-weaning (4 -60 d of age), and 28 carryover effects on post-weaning (61 -90 d of age) periods. The experimental units 29 utilized were 29 newborn Holstein × Gyr crossbred dairy heifers, with genetic 30 composition of 5/8 or more Holstein and 3/8 or less Gyr and body weight (BW) at birth 31 of 32.2 ± 5.2 kg. Experimental units were randomly assigned to either a control group 32 (CON, n = 15) or a treatment group, consisting of supplementation of a blend of 33 essential oils (BEO, n = 14) with1 g/d/calf (Apex Calf, Adisseo, China). During the pre-34 weaning phase, all heifers were fed 5 L of MR/d reconstituted at 15% (dry matter basis), 35 divided into two equal meals. Water and starter were provided ad libitum. During the 36 post-weaning, animals received a maximum 3 kg of starter/d, and ad libitum corn silage, 37 divided into two meals. The outcomes feed intake, fecal and respiratory scores were 38 evaluated daily. BW was measured every three days, while body development was 39 recorded weekly. Blood samples were collected on 0, 30 and 60 d of age for total blood 40 cell count, weekly to determinate ß-hydroxybutyrate, urea and glucose, and biweekly 41 for IGF-1. Ruminal parameters (pH, volatile fatty acids, ammonia-N and 42 acetate:proprionate proportion -C2:C3) were measured each 14 days. A randomized 43 complete block design with an interaction between treatment and week was the 44 experimental method of choice to test the hypothesis of effect of BEO on all outcomes. 45 An ANOVA procedure was used for continuous outcomes and a non-parametric test 46 was used for the ordered categorical outcomes both adopting a C.I. = 95%. Results 47 indicated that there was not enough evidence to accept the alternative hypothesis of 48 effect of BEO in MR on: feed intake, performance, body development and blood 49 metabolites during both pre-weaning and post-weaning periods. However, results 50 indicated that the inclusion of BEO in MR significantly affects the proportion of C2:C3 51 during pre and post-weaning (P ≤ 0.05). Similarly, the effect is significant for basophil 52 (P ≤ 0.001) and platelet (P ≤ 0.05) counts in pre-weaning. The interaction between week . CC-BY 4.0 : bioRxiv preprint 3 53 and treatment was also significant for lymphocytes (P ≤ 0.001), revealing a long-term 54 treatment immunological effect. Lastly, the effect on fecal scores was also significant (P 55 ≤ 0.05) during pre-weaning, with lower values for BEO. BEO contributed for ruminal 56 manipulation on pre-weaning and carry over effect on post-weaning; immunity 57 improvement and a decrease morbidity of neonatal diarrhea in pre-weaning phase. 58 59 Introduction 60 A good calf-rearing program should embrace aspects that go from body 61 development, stress reduction, good feed and housing management to optimum health 62 status. Average daily gain (ADG) and body weight (BW) at weaning are important 63 metrics used to measure the success of the rearing program. It is well known that these 64 parameters are related to the heifer's future milk production, as well as to its immune 65 responses. Therefore, a bad life start can impact negatively on animal's adult 66 performance (1). Neonatal diseases, especially diarrhea, and nutritional mistakes can act 67 as stressors, lowering calf immunity, and increasing animal susceptibility to other 68 disorders (2).
The changes in intestinal and ruminal microbiota, caused by disease or 70 malnutrition, can influence animal health, mortality rates and performance (3).
71 Therefore, tools that help improving heifer development and health are essential to 72 reduce disease, mortality and morbidity as well as accelerate the calf development.
73 Additionally, since a calf is born functionally non-ruminant, it must have its digestive 74 system and other organs and tissues changed in several weeks as well to adapt to the 75 colonization of specific microbiota. The bacteria in the rumen must start the 76 fermentation of carbohydrates, so the calf can become dependent mostly on volatile 77 fatty acids (VFA) and not on lactose-driven metabolism anymore (4). : bioRxiv preprint 4 78
Since diseases have a negative impact on the heifer's future performance, 79 procedures that reduce the animal's susceptibility to pathogens and stressors may 80 improve future performance and productivity. Thus feed additives, such as growth 81 promoter antibiotics, have been used to improve both rumen development and animal 82 health (5), as well as increase performance and feed efficiency (6). However, the use of 83 antibiotics in animal production for this purpose has been under severe criticism. Due to 84 the increase of bacterial resistance and impact over human health it has been banned in 85 several countries. Furthermore, the increase of organic dairy farms in the past years 86 revealed a change in milk consumers' preference with increased demand for "natural" 87 and healthier foods. An alternative to the use of these additives is the utilization of 88 herbal products, such as natural additives, for ruminant production (7).
Essential oils are herbal plant chemical components, constituted by volatile or 90 ethereal oils that have been applied as a natural and safe alternative for antibiotics (8).
91 Some of their properties are antiseptic and antimicrobial activities, that interfere on 92 bacterial, fungal and protozoa cell functioning (9), presenting the similar efficiency to 93 treat some diseases as antibiotics (10). They also contribute for prevention of oxidative 94 stress (11), and help immune response to change leucocyte phagocytic activity and 95 inhibiting complement system (12). Lastly, essential oils have a function similar to 96 ionophores, influencing the gastrointestinal tract development, rumen microbiological 97 activity, improving feed efficiency and decreasing neonatal diseases (9,13).
Previous studies have shown that essential oil supplementation in calf solid starter 99 improves performance (14,15), rumen fermentation (16) and diarrhea severity (17).
100 However, the effects over liquid diet supplementation are scarce. The aim of this study 101 was to evaluate how adding a commercial blend of essential oils (BEO) in milk replacer 102 (MR) affects feed intake, performance, feed efficiency, body development, blood cells 103 and metabolites, insulin-like growth factor-1 (IGF-1), ruminal parameters, fecal and . CC-BY 4.0
141 box. They were then mixed with a 10 mL of milk, homogenized and incorporated in 142 0.49 L of MR (0.5 g/calf at morning meal and 0.5 g/calf at afternoon meal) to ensure 143 total ingestion of the product. After ingesting 0.5 L MR with 0.5 g of the blend of 144 essential oils, the rest of the meal was given.
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The analysis of each outcome was performed independently of all others using 286 linear mixed models (package: nlme). Each independent outcome was modeled as a 287 function of the following fixed effects: treatment, experimental week, the interaction 288 between treatment and week. Birth weight and serum Brix refractometer were tested as 289 a covariate but did not improve statistical significance. Therefore they were eliminated 290 from the model. The genetic composition of the animal was included as a blocking 291 effect. The effect of heifer within treatment was included in the models to account for 292 individual variability.
The continuous outcomes such as intakes, performance, ruminal and blood 294 parameters were analyzed with ANOVA. A 95% Confidence Interval was adopted to 295 accept or deny the null hypothesis and P-values were produced with a Fisher test. In 296 order to meet the required assumptions of this model, all outcomes were tested for . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.17.995209 doi: bioRxiv preprint 13 297 normality, and variable transformation was applied to milk replacer intakes to meet that 298 assumption.
The categorical outcomes fecal and respiratory scores were analyzed using a non-300 parametric aligned rank transformation test, implemented in the R package ARTool. A 301 95% Confidence Interval was also adopted for the non-parametric tests.
303 Results and discussion 304 Intake and heifer performance 305 Most studies evaluating essential oils or a supplement with BEO to dairy calves, 306 feed the additive in the starter, to benefit rumen development and accelerate growth.
307 However, the intake of starter in the firsts weeks of age is small (23). Due to the calf's 308 limited capability of ingesting large solid feed amounts in the first days of life, the 309 supplement intake could be compromised during pre-weaning period and possibly mask 310 any effects. Therefore, in this trial, it was decided to offer BEO in the liquid diet.
The tested BEO has a strong aroma. Nevertheless, due to the way it was offered, 312 no rejection of the mixture BEO and MR was observed. The supplemented heifers 313 consumed liquid diet equally to the control treatment, with no refusal and good 314 acceptance (Table 2 ). Some essential oils have different acceptability by animals.
315 Chapman et al. (24) testing cinnamaldehyde essential oil in weaned dairy heifers fed a 316 total mix ratio, observed that the animals preferred the taste in the control treatment, and 317 the modification of feed intake was related to problems of palatability with the essential 318 oil used in the experiment. Differences between flavor and palatability of BEOs could 319 be due to the way of supply, as well as to essential oil plant source.
. CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.17.995209 doi: bioRxiv preprint 14 321 Table 2 . Pre and post-weaning milk replacer (MR) intake, starter intake, total dry 322 matter intake (DM), total crude protein intake (CP), total gross energy and water 323 intake of heifers (n = 29) of control (CON) and supplemented with blend essential 324 oils (BEO) in milk replacer during pre-weaning. 1) . At the end of week 1 until . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.17.995209 doi: bioRxiv preprint 15 330 week 3, heifers had diarrhea and this event impacted on MR intake. Differences 331 between treatments were greater in those weeks when compared with the other weeks, 332 with lower intake for CON group. It was found an effect between fecal scores and MR 333 intake (P ≤ 0.001), besides low correlation value (-0.25). Thus, results reveled an 334 inverse association between both parameters, were greater fecal scores impacted on 335 smaller MR intake, and vice versa. Intake of starter, water, total DM, CP, and gross energy, ADG and feed efficiency 342 were not affected by treatment during pre-and post-weaning (Table 2 and (Table 3) 355 during pre and post-weaning. As was also observed for intake and ADG, a week effect Table 4 ). Since there were no differences between treatments during pre-375 weaning, the carryover effect may not be assumed to be the answer for this difference.
376 Although no differences on intake were observed, heifers' ingestion behavior might 377 justify the difference in post-weaning pH. However, this parameter was not evaluated.
378 Nevertheless, previous studies with essential oils supplementation also found low pH 379 values for pre-weaning calves, but no essential oils treatment effect (16,25,27). 19 392 VFA. Possibly, for that reason, a low pH is frequently found in pre-weaning calves.
393 Therefore, acidosis has not been reported at this phase, since calves can stand lower 394 rumen pH values when compared to adult ruminants (23).
Besides the previous cited effects, essential oils supplementation is related to 396 antimicrobial and antifungal effects (9,28). Essential oils cause hydrophobicity,
Despite this, in this study, VFA concentrations were not affected by BEO 420 supplementation during pre-and post-weaning, but increased with age (P ≤ 0.05, The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.17.995209 doi: bioRxiv preprint 22 456 (5,33). However, since there were changes in C2:C3 proportion in BEO group, the 457 increase of propionic acid could consequently impact on glucose blood concentration.
458 Since essential oils can increase of insulin sensitivity, not finding glucose differences 459 between treatments does not mean that there were no changes in glucose pathway.
460 Therefore, further investigations over theses aspects is needed.
All blood cells count were within age and specie normality. According to Benesi Evaluation of the respiratory score parameters indicated that 2 BEO animals and 1 549 of CON animals exceeded score 4, indicating respiratory disease on pre-weaning. The 550 average days with high score was 1.0 ± 0 d for BEO. No effect was found on days with 551 high respiratory score or number of affected animals. However, it was observed a week 552 and an interaction week x treatment effect on pre-weaning, with difference between 553 treatments scores and lower values for BEO group in week 2 (P ≤ 0.05, Table 7 , Fig 1) .
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Week 2 was the period in which animals had higher incidence of diarrhea. Thus, it can 555 be assumed that respiratory signs were related to previous enteric disease since a higher 556 incidence of respiratory parameters occurred after diarrhea cases. Weeks 5 and 6 557 showed lower score differeces between treatments as well as lower incidence of 558 respiratory signs. The number of treated animals was 2 for BEO only during the 559 preweaning period, with an average of treatment days of 1.3 ± 1.4, and 3 for CON with 560 an average of treatment days of 2.0 ± 0.57. Treatments occurred only in the pre-weaning 561 period using antibiotics and anti-inflammatories.
Pneumonia is normally associated to the post-weaning phase. However, it may 563 affect younger calves to have it (2). Post-weaning respiratory scores revealed higher 564 mean values, but no animals had scores above 4. There was a week effect (P ≤ 0.05), in 565 week 12, probably due to weaning and dehorning stress.
It has been reported that essential oils have an antiseptic and antimicrobial activity 567 that may be beneficial in balancing intestinal microbiota (43). Gram positive bacteria 568 are the most sensitive to the essential oils microbial activity (7,12), but it is possible that 569 gram negative bacteria and some types of parasites are also susceptible (9) to different 570 essential oils. Thus, essential oils could reduce the incidence and severity of diarrhea 571 syndrome in calves trough inhibition of coliform overgrowth (44). BEO in 1.0 µg/mL 572 concentration did not inhibit bacterial growth -both E. coli and S. tryphimurium. Thus, . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.17.995209 doi: bioRxiv preprint 28 573 at this concentration BEO did not have any antibacterial in vitro effect. However, 574 besides no direct influence found over the bacterial evaluation, BEO calves presented 575 differences on basophil (Table 6 ) and lymphocyte cell populations (Fig 2) which could 576 be associated to modulation of inflammatory immune response. Thus, outcomes found 577 on fecal and respiratory scores could be associated to indirect changes in hemato-578 biochemical parameters since essential oils have antioxidant and anti-inflammatory 579 effect. Feeding BEO to pre-weaned heifers on MR did not affect intake, performance 583 parameters, blood metabolites or IGF-1 concentration. However, it changed C2:C3 584 proportion during pre-and post-weaning periods, as well as showed signs of immunity 585 improvement and lower fecal scores in the pre-weaning phase. Further research is 586 needed to define the best rout and dosage as well as understand the essential oils 587 contribution on decrease morbidity of neonatal diseases. Therefore, essential oils are a 588 health additive option to modern production sytems, and could be used as an alternative 589 to improve calf health and performance.
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