BRIEF RESEARCH REPORT published: 14 June 2021 doi: 10.3389/fvets.2021.639719 Phytogenic Compounds Supplemented to Gestating Hyperprolific Sows Affects the Gut Health-Related Gene Expression and Histological Responses in Neonate Piglets David Reyes-Camacho 1 , José Francisco Pérez 1 , Ester Vinyeta 2 , Tobias Aumiller 2 , Lourdes Criado-Mesas 3 , Josep Maria Folch 1,3 , Jan Dirk Van der Klis 2 and David Solà-Oriol 1* 1 Animal Nutrition and Welfare Service, Department of Animal and Food Sciences, Universitat Autònoma de Barcelona, Bellaterra, Spain, 2 Delacon Biotechnik GmbH, Engerwitzdorf, Austria, 3 Department of Animal Genomics, Centre for Research in Agricultural Genomics, Universitat Autònoma de Barcelona, Bellaterra, Spain Edited by: Pietro Celi, This research aims to determine whether a specific blend of phytogenic compounds Adisseo, France (BPC) supplemented in gestating hyperprolific sow diets can promote prenatal Reviewed by: maternal effects in terms of piglet gut function and morphology. Twenty-eight Cesar Augusto Pospissil Garbossa, University of São Paulo, Brazil (Landrace × Yorkshire) gilts and sows (parity 0 to 7) were randomly distributed by Ryan Dilger, parity number and body weight into two dietary treatments: unsupplemented Control University of Illinois at (CON) (n = 14) or CON diet supplemented with 1 g/kg feed of BPC during gestation Urbana-Champaign, United States (n = 14). The BPC supplementation during gestation of sows downregulated the *Correspondence: David Solà-Oriol neonate piglets’ jejunal genes involved in oxidation (SOD2) and nutrient transport david.sola@uab.cat (SLC16A1/MCT1, SLC11A2/DMT1, and SLC39A/ZIP4), while IFNG and CLDN4 related to immune response and barrier function, respectively, were upregulated (q < 0.10). In Specialty section: This article was submitted to addition, the jejunal villus height and the ratio of the villus height to crypt depth tended Animal Nutrition and Metabolism, to increase (p < 0.10), while goblet cell volume density was higher (p < 0.05) in BPC a section of the journal compared to CON. In conclusion, dietary supplementation of BPC in gestating diets for Frontiers in Veterinary Science hyperprolific sows influences neonatal histomorphology and expression of genes related Received: 09 December 2020 Accepted: 07 May 2021 to the intestinal function and health. Published: 14 June 2021 Keywords: hyperprolific sows, prenatal exposure, neonatal programming, phytogenic compounds, piglet gut Citation: health Reyes-Camacho D, Pérez JF, Vinyeta E, Aumiller T, Criado-Mesas L, Folch JM, Van der Klis JD and INTRODUCTION Solà-Oriol D (2021) Phytogenic Compounds Supplemented to In modern pig breeds, hyperprolific dam-line sows are characterized by a high number of fetuses Gestating Hyperprolific Sows Affects in utero compared to conventional sow lines. This has increased the litter size; however, it has also the Gut Health-Related Gene Expression and Histological increased the percentage of piglets affected with varying degrees of intrauterine growth retardation Responses in Neonate Piglets. (IUGR) and a low birth weight (LBW) (1). The IUGR characteristics may be derived from a reduced Front. Vet. Sci. 8:639719. utero-placental blood flow (2), as well as increased oxidative damage, decreased mitochondrial doi: 10.3389/fvets.2021.639719 function, impaired angiogenesis, and downregulated protein levels of glucose transporters in Frontiers in Veterinary Science | www.frontiersin.org 1 June 2021 | Volume 8 | Article 639719 Reyes-Camacho et al. Piglet Gut-Health Programming by Phytogenics placentae for LBW piglets (3). Indeed, limitations in available TABLE 1 | Composition of supplemented EO in the BPCa . nutrients supplied by the sow may slow the growth of fetuses and Component g/kg in premix their intestinal development (4), which are the major causes of morbidity and impaired growth performance in LBW piglets (5). trans-Anethole 12.17 Perinatal (fetal/neonatal) gut dysfunction in newborn mammals 1.8-Cineole 9.73 not only restricts their primary function of nutrient digestion Camphor 7.42 and absorption but also could compromise their epithelial barrier p-Cymene 2.56 function and the activation and maturation of the submucosal D-Limonene 2.31 immune system (6). In fact, among the frequent problems α-Terpineol 2.07 suffered by IUGR piglets are intestinal growth and morphology Borneol 1.83 as well as altered gene expression (7). α-Pinene 1.70 In previous research, we and other authors have shown Linalool 1.46 that phytogenic compounds (PCs) in the sow’s feed can be β-Pinene 1.34 transferred to the offspring through amniotic fluid and milk (8, 9). This prenatal and postnatal exposure to PCs, through a Total EO content was about 45 g/MT of feed with the 1 kg/MT premix dosage. nutritional programming during the perinatal period, influences the oxidative status of sows and piglets (9) and also reduces weaning-associated health and welfare problems in piglets (8, Diets and Feeding 9). However, we were not able to identify the importance and The Control diet offered in gestation was formulated to meet relevance of early effects promoted by the prenatal dietary or exceed nutrient requirements for DanBred sows (10), with treatments to counteract signs of impaired gastrointestinal adaptations based on Spanish recommendations for gestating function in neonatal piglets. In the present study, we explored sows (11). The BCP experimental diet was the CON diet plus 1 whether a blend of PCs (BPC) supplemented to the diets g/kg of BPC supplement. The BPC contained 45 g/kg essential of gestating hyperprolific sows influences the small-intestine oils (EO) (EO composition described in Table 1). Based on histomorphology and gene expression in neonate piglets. It individual body condition, sows were fed their corresponding was hypothesized that BPC supplementation would enhance the diet as follows: 2.1 kg/d from weaning to service, an average of parameters related to gut health and function in neonate piglets. 2.9 kg/d from service to day 35 of gestation, and 2.5 kg/d from day 35 to day 110 of gestation (flat line). Each gestating pen was equipped with a mechanical free-access self-closing semi-cage without pneumatic actuators (Rotecna, Lleida, Spain) to keep MATERIALS AND METHODS animals individually monitored during feeding. From day 110 of Experimental Design, Animals, and gestation, sows were fed their corresponding treatment diet with Housing ad libitum intake. Twenty-eight DanBred hybrid line (Landrace × Yorkshire) gilts and sows (parity 0 to 7) were randomly distributed by parity Sampling number and body weight (BW) into two dietary treatment Eight piglets per treatment (one piglet per liter, the animal groups (n = 14 per treatment). After breeding, sows were representing the median BW within each litter) were selected fed an unsupplemented control diet (CON) or the CON diet and euthanized at farrowing (without sucking colostrum) to supplemented with 1 kg/MT of a blend of PCs (Delacon obtain jejunum tissue samples. Before euthanasia, piglets were Biotechnik GmbH, Engerwitzdorf, Austria) during the entire anesthetized by intramuscular injection of 100 mg Telazol, 50 mg gestation (BPC). During the entire gestation, the sows were ketamine, and 50 mg xylazine per 1 ml/22 kg of BW. Thereafter, individually controlled, while pen was included as a random pigs were euthanized by jugular vein injection using sodium effect. Sows were assigned to individual cages (1.8 × 0.8 m) from pentobarbital 0.5 ml/kg of BW. The jejunum tissue obtained weaning (d 0) until confirmed gestation (day 35). On day 35, for the gene expression analysis (∼1.5 cm2 ) was placed in 1 ml sows were allotted by parity and BW into group pens of seven of RNAlater (Applied Biosystems, Foster City, CA, USA) and sows/pen (5.0 × 5.0 m) until day 110 of gestation. Thereafter, stored at room temperature (25◦ C) during the first 24 h after sows were moved to the farrowing unit, where they were placed in collection. Thereafter, the tissue samples were stored at −80◦ C individual farrowing pens (2.0 × 2.6 m) mounted over a partially until analysis. Additional jejunum tissue samples (∼2 cm2 ) were slatted floor with a heated floor pad for piglets. Water was placed in individual tubes containing formaldehyde solution for provided ad libitum through a nipple waterer, and experimental histomorphology determination. diets were provided to sows in pelleted form. In addition, the sows’ reproductive performance was monitored at farrowing. Jejunal Gene Expression Study by qPCR Piglet jejunum gene expression of 56 genes related to intestinal health and functionality was quantified by RT-qPCR using an Abbreviations: BPC, blend of phytogenic compounds; GIT, gastrointestinal tract; OpenArray Real-Time PCR Platform as specified by Reyes- IUGR, intrauterine growth retardation; LWB, low birth weight; NBW, normal Camacho et al. (9). Briefly, total RNA was extracted from birth weight; PCs, phytogenic compounds; VH, Villus height. 50 mg of jejunum tissue using the Ambion RiboPure Kit Frontiers in Veterinary Science | www.frontiersin.org 2 June 2021 | Volume 8 | Article 639719 Reyes-Camacho et al. Piglet Gut-Health Programming by Phytogenics (Life Technologies, Carlsbad, CA, USA) by following the TABLE 2 | Effects of BPC supplementation during gestation of hyperprolific sows manufacturer’s protocol. RNA concentration was measured using on jejunal histomorphology of neonate pigletsa . a NanoDrop ND-1000 spectrophotometer (NanoDrop products, Jejunum morphology Treatments SEMb p-valuec Wilmington, DE, USA), and RNA quality was checked using Agilent 2100 Bioanalyzer equipment (Agilent Technologies, CON BPC Santa Clara, CA, USA). Primers were designed by spanning Villus height (VH), µm 621 756 55.4 0.072 exon–exon boundaries using the Primer Express 2.0 software Crypt depth (CD), µm 65.4 66.2 2.12 0.746 (Applied Biosystems), and genomic DNA amplification and VH:CD ratio, µm/µm 9.8 11.9 0.72 0.060 primer dimer formation were controlled. One replicate per Goblet cell density/100 µm VH 0.76 1.02 0.11 0.033 sample was run in a TaqMan OpenArray gene expression custom Lymphocyte density/100 µm VH 0.95 0.91 0.13 0.788 plate format for gene expression with 56 assays of 48 samples per plate (OpenArray plate) in a QuantStudio 12K Flex Real- a Values are expressed as means of eight newborn pigs per treatment (n = 8). Treatments: Time PCR System (Applied Biosystems, Foster City, CA). A total CON, control diet; BPC, control plus blend of phytogenic compounds. b Standard error of the mean. Statistical significance was assumed at (p < 0.05) while of 56 genes were previously selected based on the bibliography, statistical tendency was assumed at p < 0.10 using the T-test. including four reference genes. Details regarding genes and primers can be found in previously published work by González- Solé et al. (12). RESULTS Jejunal Histomorphology Measurement The Piglets’ Jejunum Histomorphology For the histomorphology evaluation, jejunum samples were fixed The jejunal histomorphology results (Table 2) showed that, for 24–48 h in neutral-buffered 10% formalin. After dehydration compared to the CON group, BPC tended to increase the VH and embedding in paraffin wax, sections of ∼3 µm were stained (p = 0.072) and VH:CD ratio (p = 0.060) in neonate piglets. The with hematoxylin and eosin. Villus height (VH), crypt depth GC density improved in the BPC group (p = 0.033). However, (CD), number of intraepithelial lymphocyte (IEL), and number no significant differences (p > 0.05) were observed in CD or of goblet cells (GC) per 100 micrometers of villus height were IEL density. measured blinded in 10 well-oriented villi and crypts per sample by the same person using a light microscope (BHS, Olympus, The Piglets’ Jejunum Gene Expression Barcelona, Spain). The results for the jejunum gene expression analysis of neonate piglets are shown in Table 3. Although only six genes showed Statistical Analyses statistical differences between treatments (p < 0.05, q < 0.10), The individual newborn piglet was considered as the the other six genes that showed statistical differences (p < experimental unit. All data were analyzed considering the 0.05) between functional groups are presented. Compared to the treatment as the main effect. The results are presented as CON, a downregulation (q < 0.10) was observed in the BPC means with their corresponding SEM. For morphology, data treatment for the SOD2 gene from the oxidation group and the were analyzed by using the TTEST procedure of the statistical SLC16A1/MCT1, SLC11A2/DMT1, and SLC39A/ZIP4 genes from package SAS 9.4 (SAS Inst. Inc., Cary, NC, USA), and significant the nutrient transport group, while from the immune response differences were declared at p < 0.05, while 0.05 < p < 0.10 were and barrier function groups an upregulation (q < 0.10) was considered significant tendencies. observed for IFNG and CLDN4, respectively. Furthermore, there Gene expression statistical analysis was performed as specified were no significant differences between treatments (p < 0.05, q > by González-Solé et al. (12). Briefly, data were collected and 0.10) for SLC15A1/PEPT1, ALPI, IDO1, DAO1, CCK, and OCLN analyzed using the Thermo Fisher Cloud software 1.0 (Applied from nutrient transport, immune response, enzyme/hormone, Biosystems) applying the applying the 2−11Ct method for and barrier function group, respectively. relative quantification (RQ). The normal distribution of the RQ values was checked with the Shapiro.test function of R 3.5.3 DISCUSSION software, and log2 transformation was applied if required. One- way ANOVA was performed, and the Benjamini–Hochberg false Although the study did not aim to research the sow’s reproductive discovery rate (FDR) was used for multiple-testing correction of performance, our findings indicate that BPC improved both p-values, defining the model: the total number of piglets born (19.9 vs. 17.8) and the number born alive (17.2 vs. 14.7), while the neonate piglet BW Yij = µi + treatj + εij tended to decrease (1.17 vs. 1.33 kg) (Supplementary Table 1) in agreement with previous studies with a larger sample size in where Yij is each observation of the outcome variable, µi is the commercial conditions (9). LBW in piglets has been associated global mean, treatj is the main effect of the treatment, and εij is the with a certain degree of IUGR (1), with impaired intestinal experimental error term. Significant gene expression differences structure, and with a transcriptomic profile and bacterial between functional groups were accepted at p < 0.05, whereas colonization in neonatal IUGR piglets (13). In previous studies significant gene expression differences between treatments were by our group, it has been observed that at birth, the jejunum accepted at FDR (q-values) < 0.10. expression of 10 genes, involved in the immune, digestive, Frontiers in Veterinary Science | www.frontiersin.org 3 June 2021 | Volume 8 | Article 639719 Reyes-Camacho et al. Piglet Gut-Health Programming by Phytogenics TABLE 3 | Effects of BPC supplementation during gestation of hyperprolific sows on related jejunal health-function mRNA relative expression of neonate pigletsa . Function Genesb Treatments SEMd p-valuee q-valuef (FDR) CON BPCc Oxidation SOD2 1.00 0.71 0.051 0.009 0.023 Nutrient transport SLC15A1/PEPT1 1.00 0.71 0.074 0.048 0.256 SLC16A1/MCT1 1.00 0.55 0.083 0.002 0.040 SLC11A2/DMT1 1.00 0.58 0.070 0.004 0.018 SLC39A4/ZIP4 1.00 0.42 0.141 0.012 0.087 Immune response IFNG 1.00 6.18 1.393 0.003 0.043 ALPI 1.00 0.58 0.090 0.011 0.117 IDO1 1.00 3.43 0.634 0.031 0.155 Enzyme/hormone DAO1 1.00 0.67 0.068 0.008 0.106 CCK 1.00 0.66 0.082 0.032 0.238 Barrier function CLDN4 1.00 3.15 0.601 0.010 0.087 OCLN 1.00 0.82 0.046 0.040 0.250 a Data are means of eight newborn pigs per treatment (n = 8). b Genes: SOD2, superoxide dismutase-2; SLC15A1/PEPT1, solute carrier family 15 (oligopeptide transporter) member 1; SLC16A1/MCT1, solute carrier family 16 (monocarboxylate transporter 1) member 1; SLC11A2/DMT1, solute carrier family 11 (proton-coupled divalent metal ion transporter) member 2; SLC39A4/ZIP4, solute carrier family 39 (zinc transporter) member 4; IFNG, interferon gamma; ALPI, intestinal alkaline phosphatase; IDO1, indoleamine 2,3 dioxygenase; DAO1, diamine oxidase; CCK, cholecystokinin; CLDN4, claudin-4; OCLN, occludin. Treatments: CON, control diet; BPC, control plus blend of phytogenic compounds. c Gene expression means for BPC are calculated in relation to the mean from CON group. d Standard error of the mean. e p-Values come from the ANOVA test. f Significant gene expression differences between treatments were accepted at FDR (q-values) < 0.10. and stress responses, is clearly different according to the piglet transport of nutrients such as thiamin, folic acid, and glucose BW category between half-sibling piglets (light vs. average by BeWo cells are modulated by PCs, such as polyphenols littermates) (unpublished data). (18). The solute carrier (SLC) genes are a large family of In this study we explored the effects of prenatal BPC protein transporters in mammals, and their gene expression is exposure on histomorphology, and gene expression related to notably affected if there is a deprivation of nutrients such as the health and functioning of the newborn piglet jejunum from amino acids (AAs), which implies an effect on the capacity of hyperprolific dam-line sows. The jejunum plays an important the SLC to regulate intracellular nutrient concentrations and, role in nutrient uptake as well as immune system programming in addition, detect alterations in extracellular nutrient levels and metabolic programming in the pigs’ early life (14). To our (19). For instance, the gene expression of the SLC38 family knowledge, there are no studies reporting the effects of PCs on was upregulated in hypothalamic cells N25/2 of mice after AA the intestinal gene expression and morphology of neonate pigs. starvation (20). Glucose deprivation increases MCT1 protein Positive findings were observed in histomorphology in neonate expression and their interaction in oxidative tumor cells (21). piglets of the BPC group, which showed improvements in the Moreover, the hypoxia induced an increase in MCT1 plasma VH, the ratio of VH:CD, and the goblet cell density compared membrane expression in glioma cells, both in in vitro and in to newborn piglets from the CON group. The effects promoted vivo models (22). The gene expression analysis of the current by BPC appear to attenuate the early effects derived from study showed that prenatal exposure of the fetus to BPC led IUGR, which is known to affect the intestinal cell proliferation– to downregulation in the jejunum of nutrient transport-related apoptosis balance in neonate piglets (5). They also affect the genes SLC16A1/MCT1, SLC11A2/DMT1, and SLC39A/ZIP4 in intestinal growth and morphology in association with the altered newborn piglets. This may indicate that compared to BPC, gene expression of growth-related proteins (7) and modify the potential deprivation of nutrients, especially for Fe2+ (23), short- volume density and function of epithelial cell types such as goblet chain fatty acids (24), and zinc (25), respectively, induced an cells, which are important constituents of the innate defense adaptative upregulation of the abovementioned SLC genes in system (15). An increased VH and ratio VH:CD may suggest newborn piglets from the CON group. an improvement in the digestive and absorptive function of When inflammation occurs in the gastrointestinal tract (GIT), the intestine as a result of an increased absorptive surface, an this can result in a decrease in digestive efficiency and reduced expression of brush border enzymes, and nutrient transport absorption of nutrients (26). In this study, the prenatal exposure systems (16). of the fetus to BPC induced an upregulation of the immune In highly prolific sows, uterine capacity or insufficiency can response IFNG and barrier function CLDN4 genes. IFNG is affect fetal growth when competition by littermates for limited critical in increasing and mediating intestinal immunity because uterine space and nutrients becomes increasingly critical (17). of its role in recognizing and eliminating pathogens. IFNG can Evidence in human studies has shown that placental uptake and have several functions; it can exhibit its immunomodulatory Frontiers in Veterinary Science | www.frontiersin.org 4 June 2021 | Volume 8 | Article 639719 Reyes-Camacho et al. Piglet Gut-Health Programming by Phytogenics effects by controlling inflammatory response, enhancing antigen by changing the epigenetic state of the fetal genome and its processing and presentation, increasing leukocyte trafficking, gene expression (34). Although the action modes of several inducing an antiviral state, boosting the antimicrobial functions, PCs are not yet fully clarified, the main action modes of the and affecting cellular proliferation and apoptosis (27). On the major compounds used in BPC, such as trans-anethole (35), 1.8- other hand, expression of Claudin proteins such as CLDN4 cineole (36), and Camphor (37), exhibit important biological within the small intestine of newborn piglets plays a vital role in properties. These include anti-inflammatory, antioxidant, and controlling barrier function and mucosal homeostasis (epithelial antimicrobial properties that could have influenced the changes tight junctions), particularly on the apical aspect of lateral on the intestinal histomorphology and gene expression of surfaces of intestinal epithelial cells where they help regulate ion neonate piglets in this study. Therefore, the prenatal exposure and macromolecule movement across the intestinal epithelium of the fetus to PCs may indicate a positive means to attenuate (28). It has been described that transcription levels of IFNG and the intestinal dysfunction in LBW piglets, especially in terms of CLDN4 showed a lower expression in LBW than normal birth structure and functionality. weight (NBW) piglets (29). Thus, results shown in the BPC group In conclusion, BPC supplemented to hyperprolific sows suggest that upregulation of IFNG and CLDN4, together with the during gestation influenced the prenatal programming of some development in jejunal histomorphology, could help to improve intestinal biological functions. This was evidenced by the the gut health of LBW piglets. improved histomorphology and gene expression related to the In addition, the induction of mitochondrial oxidative nutrient absorption, immune response, intestinal integrity, and stress during periods of nutrient deprivation in animals has oxidative stress in the jejunum of neonate piglets. been associated with decreased metabolic requirements, higher mitochondrial membrane potential, and increased superoxide DATA AVAILABILITY STATEMENT production at the level of the complex III of the electron transport chain (30). Since SOD2 activity is regulated in response The raw data supporting the conclusions of this article will be to mitochondrial superoxide production, the upregulation of made available by the authors, without undue reservation. mitochondrial SOD activity results in an overproduction of hydrogen peroxide as a product of the disproportionation ETHICS STATEMENT reaction of superoxide anion catalyzed by SOD (31). In the present study, the SOD2 gene was downregulated in the The animal study was reviewed and approved by CEEAH- jejunum of BPC newborn piglets, which may indicate that Univesritat Autònoma de Barcelona. compared with CON, undue intrauterine oxidative stress was avoided in newborn piglets from the BPC group. In swine, AUTHOR CONTRIBUTIONS it has been described that oxidative damage to DNA (8- hydroxy-20-deoxyguanosine, 8-OHdG) and proteins (carbonyls) DR-C performed the animal trial, the statistical analyses, and measured in plasma increased after the stress period such as writing—original draft preparation. JP and DS-O were the weaning, and this coincides with a rise in enzymatic antioxidant principal investigator and contributed to conceptualization and activity including SOD. Furthermore, oxidative damage to experimental design. EP and JV conducted the conceptualization macromolecules by mitochondrial dysfunction and cellular and experimental design. TA supervised the interpretation of the oxidative stress is more important in LBW than in NBW study results and reviewed the draft. LC-M and JF contributed to piglets, as measured concentrations of 8-OHdG and carbonyls the design and set-up of the gene expression studies. All authors are significantly higher (32). Thus, it suggests that upregulation contributed to the article and approved the submitted version. of the oxidation SOD2 gene shown in CON may indicate a defense mechanism against mitochondrial dysfunction and FUNDING cellular oxidative stress conditions. It has also been reported that several dietary plants and Financial support for this research from DELACON Biotechnik their bioactive components have the potential to modulate GmbH. (Engerwitzdorf, Austria) is appreciated. human transcriptome profiles of various biological pathways involved in oxidative, immune response, glucose and fatty acid ACKNOWLEDGMENTS metabolism, and inflammatory and cell signaling pathways (33). In previous studies, we have observed that perinatal exposure The authors are grateful to the staff of the Centre de Recerca to a blend of PCs containing eucalyptol, p-cymene, linalool, en Sanitat Animal from the IRTA for support received in the anethole, and thymol influenced the overexpression of the histomorphology analysis. barrier function (MUC2), and immune response (PPARGC-α, TNF-α, TGF-β1, IDO1, and IL-10) genes in suckling piglets SUPPLEMENTARY MATERIAL (9). In this study, we have observed that these effects may be anticipated by administering BPC in the sow gestation The Supplementary Material for this article can be found diet. In terms of fetal programming, maternal nutrition status online at: https://www.frontiersin.org/articles/10.3389/fvets. during fetal development may result in a permanent imprint 2021.639719/full#supplementary-material Frontiers in Veterinary Science | www.frontiersin.org 5 June 2021 | Volume 8 | Article 639719 Reyes-Camacho et al. 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(2016) 2016:389–98. doi: 10.1007/978-3-319-41342-6_18 any commercial or financial relationships that could be construed as a potential 37. Chaturvedi T, Kumar A, Kumar A, Verma RS, Padalia RC, conflict of interest. Sundaresan V, et al. Chemical composition, genetic diversity, antibacterial, antifungal and antioxidant activities of camphor- Copyright © 2021 Reyes-Camacho, Pérez, Vinyeta, Aumiller, Criado-Mesas, Folch, basil (Ocimum kilimandscharicum Guerke). Ind Crops Prod. (2018) Van der Klis and Solà-Oriol. This is an open-access article distributed under the 118:246–58. doi: 10.1016/j.indcrop.2018.03.050 terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and Conflict of Interest: EV, TA, and JV are employees of DELACON Biotechnik the copyright owner(s) are credited and that the original publication in this journal GmbH. (Engerwitzdorf, Austria) which is a global supplier of phytogenic is cited, in accordance with accepted academic practice. No use, distribution or compounds. reproduction is permitted which does not comply with these terms. Frontiers in Veterinary Science | www.frontiersin.org 7 June 2021 | Volume 8 | Article 639719