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The materno-placental microbiome of gravid beef cows under moderate feed intake restriction

The materno-placental microbiome of gravid beef cows under moderate feed intake restriction Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 The materno-placental microbiome of gravid beef cows under moderate feed intake restriction Gwendolynn L. Hummel, Kelly L. Woodruff, Kathleen J. Austin, Ryan M. Knuth, Jordan D. Williams, and Hannah C. Cunningham-Hollinger Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA © The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribu- tion, and reproduction in any medium, provided the original work is properly cited. Transl. Anim. Sci. 2021.5:S159–S163 https://doi.org/10.1093/tas/txab172 in the rumen fluid (RF) of both control (CON) INTRODUCTION and feed restricted (FR) cows in late gestation, Although the existence of a reproductive and compare these microbes to microbial popu- microbiome is evidenced in cattle (Ault et al., 2019; lations within various structures of the placenta, Hummel et al., 2020), the methods by which mi- including the cotyledon (COT), intercotyledonary crobes colonize the placenta remain poorly under- membrane (ICM), and allantois (AL). Swabs of stood. Mechanisms facilitating microbial transfer vaginal epithelium (VAG) were also collected and from the maternal gut have been investigated in compared to both the maternal gut and placenta cattle, which include passage through the circu- in order to evaluate the effects of feed restriction latory system to the placental environment (Jeon on this reproductive microbiome. et  al., 2017). Contributions of the fecal microbi- ome to the vaginal flora, which have themselves MATERIALS AND METHODS been suggested to ascend to and colonize the pla- Experimental protocols were approved by the cental microbiome (Goldenburg et al., 2008), have University of Wyoming International Care and also been considered (Jeon et al., 2017). Therefore, Use Committee. it is necessary to evaluate the maternal gut as a potential niche for microbes inhabiting the repro- ductive tract and placenta in late gestation. Animals and Study Design Changes to feed intake are capable of altering Multiparous Angus cross-bred cows (n  =  16) the maternal gut microbiome in ruminants (Hu were stratified by initial body weight and BCS to et al., 2018), as well as the placental microbiome either a control (CON; n = 8) group with ad lib- of humans (Antony et al., 2015). We hypothesize itum access to grass hay and alfalfa, or a 70% feed that feed intake restriction will result in a less di- intake-restricted group (FR; n = 7), although one verse rumen microbiome in the gravid cow, which cow of the FR group calved prior to sampling. will in turn result in decreased placental microbial Treatment group sorting occurred 3 mo prior to diversity. We also hypothesized that the vaginal each cow’s expected calving date, calculated from microbiome, which remains stable throughout the date of AI. pregnancy (Romero et al., 2014; Ault et al., 2019), will be unaffected. Our objectives were to iso- late and identify bacterial and archaeal species Sample Collection 1 Ten days prior to their expected calving date, Corresponding author: hcunnin6@uwyo.edu RF was collected from each cow by orally passing Received May 4, 2021. Accepted September 29, 2021. a flexible stomach tube to the rumen and applying S159 Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 Hummel et al. S160 suction via syringe until 20–30  mL RF were as- (Advanced Research Computing Center, 2018). pirated (Zhou et  al., 2009). Swabs of vaginal epi- Alpha-diversity metrics, including Shannon index thelium were also collected at this time using a and Faith’s Phylodiversity, were generated in sterile double-sheathed equine uterine culture swab QIIME2. Pairwise comparisons were made for (Jorgenson Labs, Loveland, CO) following previ- sample type by treatment using Kruskal–Wallis ously described methods (Hummel et  al., 2020). permutational multivariate analysis of variance Naturally delivered placentae were collected and (PERMANOVA). Similar pairwise comparisons COT dissected from the middle of the gravid horn. were made for phylogenetic beta-diversity indices, Excess tissue was trimmed, and COT were sec- including weighted and unweighted UniFrac dis- tioned to fit within a 2-mL tube. Sections of the tances. Statistical significance was considered when chorioallantois were separated by blunt dissection, q ≤ 0.05, and a tendency when 0.05 < q ≤ 0.10, with and samples of the ICM and AL, no greater than 2 q representing each P-value adjusted for false dis- in , were collected no more than 3 in from the um- covery rate under the Benjamini–Hochberg method. bilical insertion site. All samples were flash frozen at −80 °C until further analysis. RESULTS Microbial DNA Isolation and Sequencing The Microbiomes of the Maternal Gut and Vagina Under Feed Restriction Bead tubes containing sterilized silicon (0.1  g Shannon’s diversity index, a measure of abun- of 0.5-mm beads) and zirconia (0.3  g of 0.1-mm dance and evenness in microbiome alpha-diversity, beads) were prepared for microbial DNA extrac- revealed that the CON RF microbiome was signifi- tion of RF (0.25  g). Whole swab heads of VAG cantly greater in richness (Figure 1; q = 0.03) than were sterilely cut and placed in their respective bead FR RF. Faith’s phylodiversity, a measure of phylo- tubes. Lysis buffer (1 mL; 500 mM NaCl, 400 mM genetic diversity between microbial species, tended Tris-HCL, 50  mM EDTA, 4% SDS; Zhao et  al., to be greater in CON RF (q  =  0.09). In addition, 2015) was added to each bead tube and chemical FR RF tended toward decreased phylodiversity and mechanical lysis was performed utilizing the among those more dominant microbial taxa, repre- Precellys Evolution (Bertin Instruments, Rockville, sented by weighted UniFrac (q = 0.06). The related- MD). Placental tissue underwent the same lysis step ness and diversity of less dominant microbial taxa with bead tubes and lysis buffer provided with the was significantly decreased in FR RF, represented QIAamp PowerFecal kit (Qiagen, Germantown, by unweighted UniFrac (q = 0.05). MD), and all samples subsequently underwent a The VAG microbiome did not differ between 70 °C incubation for 10 min. All samples repeated treatments in any alpha- or beta-diversity metric (q the lysis step with 300-µL lysis buffer and centri- ≥ 0.11). fugation to precipitate (Yu and Morrison, 2004). The QIAamp PowerSoil Minikit (Qiagen) was Relationship Between the Maternal Microbiome used to further purify microbial DNA from RF and Placenta and VAG. Quality and concentration of DNA was assessed with the Nanodrop spectrophotom- The maternal gut of CON cows was richer and eter. Following the Illumina MiSeq System library more diverse than the CON AL (q ≤ 0.03), but these preparation protocol, each sample was prepared FR microbiomes did not differ in alpha-diversity (q as 30-ng/µL aliquots and pooled for sequencing of ≥ 0.26). The RF of both CON and FR cows was the V4 region of the 16S rRNA gene. Samples were similar in richness (q = 0.11) and tended to differ in sequenced at the Colorado State University MIP phylodiversity (q = 0.09) from the ICM. Although NGS Illumina Core. CON RF was similar to the COT in both richness and phylodiversity (q ≥ 0.11), FR RF tended toward dissimilarity with the COT in both alpha-diversity Bioinformatic Analysis metrics (q = 0.06). Sequence analysis was conducted in QIIME2 Unlike the maternal gut, the VAG of FR cows v.  2020.8 (Bolyen et  al., 2019) and quality fil- was similar to the AL in richness and phylodiver- tering, pairing, and denoising were accomplished sity (q ≥ 0.43), although the CON VAG tended to be through the DADA2 plugin (Callahan et al., 2016) richer than the AL (q = 0.06). Like the maternal gut, on the University of Wyoming Advanced Research the VAG of both CON and FR cows was similar Computing Center Teton computing environment in richness (q ≥ 0.11) and the CON VAG tended to Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 Reduced feed and materno-placental microbiome S161 Figure 1. Alpha diversity box plots depicting Shannon richness of sample type by feeding treatment. Samples include maternal rumen fluid (RF) and vaginal swabs (VAG), along with three placental tissues: the intercotyledonary membrane of the chorion (ICM), cotyledon (COT), and allantois (AL). Each sample type is represented within a control diet (CON) or 70% feed intake restricted diet (FR). X and y denote tendencies to differ at 0.05 < q ≤ 0.10. Figure 2. Unweighted UniFrac principle coordinate analysis (PCoA) plot of sample type by feeding treatment. The allantois (AL) in feed restriction (FR) tended toward similarity with the maternal rumen fluid (RF; q = 0.06) and vaginal (VAG; q = 0.07) microbiomes where control (CON) pregnancies differed (q = 0.01). The RF differed from VAG in both treatments (q ≤ 0.02). differ in Faith’s phylodiversity (q = 0.10) from the and unweighted UniFrac (q = 0.07). Where FR RF ICM where the FR VAG did not (q = 0.55). Finally, differed in dominant and non-dominant taxa with the VAG and COT of both treatment groups were the COT and ICM (q ≤ 0.03), it tended to be similar similar in richness and phylodiversity (q ≥ 0.11). to the AL under both UniFrac metrics (Figure 2; The RF and VAG of CON tended to share q = 0.07), as did the FR VAG (q = 0.09). Both the both dominant and nondominant microbial taxa CON VAG and RF remained different from the with the COT and ICM, represented by weighted AL in both dominant and non-dominant microbial Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 Hummel et al. S162 taxa (q ≤ 0.02). The FR VAG also differed in both reflected within the placental microbiome and UniFrac values from the FR COT and ICM (q ≤ may further influence inoculation of the fetal gut. 0.03), although the FR VAG and ICM only tended A greater understanding of the maternal gut’s influ- toward dissimilarity regarding dominant microbial ence on the reproductive microbiome throughout taxa (q = 0.06). gestation is required in order to guide nutritional decisions and management practices in beef cows DISCUSSION and to identify the optimal microbiome for repro- ductive performance. Consistent with previous findings in feed re- stricted ewes (Hu et  al., 2018), the mature bovine ACKNOWLEDGMENTS rumen is influenced by feed intake restriction, where the FR microbiome is less rich and less diverse than Funding was provided by the University of that of CON cows. The species counts of phyla that Wyoming Agricultural Experiment Station FY20 are present in a low relative abundance in the ma- Competitive Grant Program. ture rumen were most impacted, which may exert changes on VFA production, microbial lipid me- LITERATURE CITED tabolism, and overall ruminal pH (Hu et al., 2018) Advanced Research Computing Center. Teton Computing and potentially altering metabolites and microbes Environment, Intel x86_64 cluster. University of available to the gestating fetus. Wyoming. 2018. Antony,  K.  M., J.  Ma, K.  B.  Mitchell, D.  A.  Racusin, Consistent with our hypothesis, these differ- J.  Versalovic, and K.  Aagaard. 2015. The preterm pla- ences did not carry to the VAG microbiome, which cental microbiome varies in association with excess ma- is resistant to change throughout gestation (Romero ternal gestational weight gain. Am. J.  Obstet. Gynecol. et al., 2014; Ault et al., 2019). However, the FR VAG 212:653.e1–653.16. doi:10.1016/j.ajog.2014.12.041 displayed similarities in alpha-diversity with the Ault,  T.  B., B.  A.  Clemmons, T.  S.  Reese, F.  G.  Dantas, G. A. Franco, T. P. L. Smith, J. L. Edwards, P. R. Myer, maternal gut that the CON treatment did not, po- and K. G. Pohler. 2019. Bacterial taxonomic composition tentially validating a relationship between these mi- of the postpartum cow uterus and vagina prior to artificial crobial sites (Jeon et al., 2017). It remains possible insemination. J. Anim. Sci. 97:4305–4313. doi:10.1093/jas/ that a prolonged period of feed intake restriction skz212 may be capable of altering the vaginal microbiome, Bolyen,  E., J.  R.  Rideout, M.  R.  Dillon, N.  A.  Bokulich, similar to the effects of dietary changes in pregnant C. C. Abnet, G. A. Al-Ghalith, H. Alexander, E. J. Alm, M.  Arumugam, F.  Asnicar, et  al. 2019. Reproducible, women (Ravel et al., 2011). interactive, scalable and extensible microbiome data Importantly, the maternal gut shared a greater science using QIIME 2. Nat. Biotechnol. 37:852–857. degree of microbial characteristics with the COT doi:10.1038/s41587-019-0209-9 within the CON treatment. This may indicate that Callahan,  B.  J., P.  J.  McMurdie, M.  J.  Rosen, A.  W.  Han, the COT plays a role in microbial transmission to A.  J.  Johnson, and S.  P.  Holmes. 2016. DADA2: High- resolution sample inference from Illumina amplicon data. the placenta, as the COT is responsible for per- Nat. Methods. 13:581–583. doi:10.1038/nmeth.3869 fusing the placental organ with blood (Wooding Goldenburg, R. L., W. W. Andrews, A. R. Goepfert, O. Faye- and Burton, 2008), a hypothesized mode of bac- Peterson, S. P. Cliver, W. A. Carlo, and J. C. Hauth. 2008. terial transmission in cattle (Jeon et  al., 2017). The Alabama preterm birth study: Umbilical cord blood Additionally, the maternal rumen of FR preg- Ureaplasma urealyticum and Mycoplasma hominis cul- nancies shared alpha-diversity characteristics with ture in very preterm newborns. Am. J.  Obstet. Gynecol. 198:43.e1–e5. doi:10.1016/j/ajog.2007.07.033 the AL, implicating decreased microbial diversity Hu,  F., Y.  Xue, C.  Guo, J.  Liu, and S.  Mao. 2018. The re- within the FR placenta as a whole, and potentially sponse of ruminal fermentation, epithelium-associated indicating decreased microbiome diversity within microbiota, and epithelial barrier function to severe feed the fetal gut, which may be contributing a micro- restriction in pregnant ewes. J. Anim. Sci. 96:4293–4305. biome to the AL through the elimination of waste doi:10.1093/jas/sky306 Hummel,  G.  L., K.  L.  Woodruff, K.  J.  Austin, T.  L.  Smith, (Schlafer et al., 2000). and H.  C.  Cunningham-Hollinger. 2020. Evidence for the amnion-fetal gut-microbial axis in late gestation IMPLICATIONS beef calves. Transl. Anim. Sci. 4(Suppl 1):S174–S177. doi:10.1093/tas/txaa138 These data indicate a critical influence of the Jeon, S. J., F. Cunha, A. Vieira-Neto, R. C. Bicalho, S. Lima, maternal gut upon the placental microbiome, as M.  L.  Bicalho, and K.  N.  Galvão. 2017. Blood as a both sites were altered by feed intake restriction. route of transmission of uterine pathogens from the gut Feed intake restriction shaped a less diverse, less to the uterus in cows. Microbiome 5:109. doi:10.1186/ robust maternal rumen microbiome, which was s40168-017-0328-9 Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 Reduced feed and materno-placental microbiome S163 Ravel,  J., P.  Gajer, Z.  Abdo, G.  M.  Schneider, S.  S.  Koenig, Placentation: Ruminants (Ewe and Cow) In: Comparative S.  L.  McCulle, S.  Karlebach, R.  Gorle, J.  Russell, Placentation. Structures, Functions and Evolution. C.  O.  Tacket, et  al. 2011. Vaginal microbiome of repro- Springer, Berlin, Belgium. p. 133. ductive-age women. Proc. Natl. Acad. Sci. U.  S. A. 108 Yu, Z., and M. Morrison. 2004. Improved extraction of PCR- Suppl 1:4680–4687. doi:10.1073/pnas.1002611107 quality community DNA from digesta and fecal samples. Romero, R., S. S. Hassan, P. Gajer, A. L. Tarca, D. W. Fadrosh, Biotechniques 36:808–812. doi:10.2144/04365ST04 L. Nikita, M. Galuppi, R. F. Lamont, P. Chaemsaithong, Zhao,  L., Q.  Meng, L.  Ren, W.  Liu, X.  Zhang, Y.  Huo, and J. Miranda, et al. 2014. The composition and stability of Z. Zhou. 2015. Effects of nitrate addition on rumen fer- the vaginal microbiota of normal pregnant women is dif- mentation, bacterial biodiversity and abundance. Asian- ferent from that of non-pregnant women. Microbiome Australas. J.  Anim. Sci. 28:1433–1441. doi:10.5713/ 2:4. doi:10.1186/2049-2618-2-4 ajas.15.0091 Schlafer, D. H., P. J. Fisher, and C. J. Davies. 2000. The bovine Zhou,  M., E.  Hernandez-Sanabria, and L.  L.  Guan. 2009. placenta before and after birth: placental development Assessment of the microbial ecology of ruminal meth- and function in health and disease. Anim. Reprod. Sci. anogens in cattle with different feed efficiencies. Appl. 60-61:145–160. doi:10.1016/s0378-4320(00)00132-9 Environ. Microbiol. 75:6524–6533. doi:10.1128/ Wooding,  P., and G.  Burton. 2008. Synepitheliochorial AEM.02815-08. Translate basic science to industry innovation http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

The materno-placental microbiome of gravid beef cows under moderate feed intake restriction

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Copyright © 2021 American Society of Animal Science
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Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 The materno-placental microbiome of gravid beef cows under moderate feed intake restriction Gwendolynn L. Hummel, Kelly L. Woodruff, Kathleen J. Austin, Ryan M. Knuth, Jordan D. Williams, and Hannah C. Cunningham-Hollinger Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA © The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribu- tion, and reproduction in any medium, provided the original work is properly cited. Transl. Anim. Sci. 2021.5:S159–S163 https://doi.org/10.1093/tas/txab172 in the rumen fluid (RF) of both control (CON) INTRODUCTION and feed restricted (FR) cows in late gestation, Although the existence of a reproductive and compare these microbes to microbial popu- microbiome is evidenced in cattle (Ault et al., 2019; lations within various structures of the placenta, Hummel et al., 2020), the methods by which mi- including the cotyledon (COT), intercotyledonary crobes colonize the placenta remain poorly under- membrane (ICM), and allantois (AL). Swabs of stood. Mechanisms facilitating microbial transfer vaginal epithelium (VAG) were also collected and from the maternal gut have been investigated in compared to both the maternal gut and placenta cattle, which include passage through the circu- in order to evaluate the effects of feed restriction latory system to the placental environment (Jeon on this reproductive microbiome. et  al., 2017). Contributions of the fecal microbi- ome to the vaginal flora, which have themselves MATERIALS AND METHODS been suggested to ascend to and colonize the pla- Experimental protocols were approved by the cental microbiome (Goldenburg et al., 2008), have University of Wyoming International Care and also been considered (Jeon et al., 2017). Therefore, Use Committee. it is necessary to evaluate the maternal gut as a potential niche for microbes inhabiting the repro- ductive tract and placenta in late gestation. Animals and Study Design Changes to feed intake are capable of altering Multiparous Angus cross-bred cows (n  =  16) the maternal gut microbiome in ruminants (Hu were stratified by initial body weight and BCS to et al., 2018), as well as the placental microbiome either a control (CON; n = 8) group with ad lib- of humans (Antony et al., 2015). We hypothesize itum access to grass hay and alfalfa, or a 70% feed that feed intake restriction will result in a less di- intake-restricted group (FR; n = 7), although one verse rumen microbiome in the gravid cow, which cow of the FR group calved prior to sampling. will in turn result in decreased placental microbial Treatment group sorting occurred 3 mo prior to diversity. We also hypothesized that the vaginal each cow’s expected calving date, calculated from microbiome, which remains stable throughout the date of AI. pregnancy (Romero et al., 2014; Ault et al., 2019), will be unaffected. Our objectives were to iso- late and identify bacterial and archaeal species Sample Collection 1 Ten days prior to their expected calving date, Corresponding author: hcunnin6@uwyo.edu RF was collected from each cow by orally passing Received May 4, 2021. Accepted September 29, 2021. a flexible stomach tube to the rumen and applying S159 Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 Hummel et al. S160 suction via syringe until 20–30  mL RF were as- (Advanced Research Computing Center, 2018). pirated (Zhou et  al., 2009). Swabs of vaginal epi- Alpha-diversity metrics, including Shannon index thelium were also collected at this time using a and Faith’s Phylodiversity, were generated in sterile double-sheathed equine uterine culture swab QIIME2. Pairwise comparisons were made for (Jorgenson Labs, Loveland, CO) following previ- sample type by treatment using Kruskal–Wallis ously described methods (Hummel et  al., 2020). permutational multivariate analysis of variance Naturally delivered placentae were collected and (PERMANOVA). Similar pairwise comparisons COT dissected from the middle of the gravid horn. were made for phylogenetic beta-diversity indices, Excess tissue was trimmed, and COT were sec- including weighted and unweighted UniFrac dis- tioned to fit within a 2-mL tube. Sections of the tances. Statistical significance was considered when chorioallantois were separated by blunt dissection, q ≤ 0.05, and a tendency when 0.05 < q ≤ 0.10, with and samples of the ICM and AL, no greater than 2 q representing each P-value adjusted for false dis- in , were collected no more than 3 in from the um- covery rate under the Benjamini–Hochberg method. bilical insertion site. All samples were flash frozen at −80 °C until further analysis. RESULTS Microbial DNA Isolation and Sequencing The Microbiomes of the Maternal Gut and Vagina Under Feed Restriction Bead tubes containing sterilized silicon (0.1  g Shannon’s diversity index, a measure of abun- of 0.5-mm beads) and zirconia (0.3  g of 0.1-mm dance and evenness in microbiome alpha-diversity, beads) were prepared for microbial DNA extrac- revealed that the CON RF microbiome was signifi- tion of RF (0.25  g). Whole swab heads of VAG cantly greater in richness (Figure 1; q = 0.03) than were sterilely cut and placed in their respective bead FR RF. Faith’s phylodiversity, a measure of phylo- tubes. Lysis buffer (1 mL; 500 mM NaCl, 400 mM genetic diversity between microbial species, tended Tris-HCL, 50  mM EDTA, 4% SDS; Zhao et  al., to be greater in CON RF (q  =  0.09). In addition, 2015) was added to each bead tube and chemical FR RF tended toward decreased phylodiversity and mechanical lysis was performed utilizing the among those more dominant microbial taxa, repre- Precellys Evolution (Bertin Instruments, Rockville, sented by weighted UniFrac (q = 0.06). The related- MD). Placental tissue underwent the same lysis step ness and diversity of less dominant microbial taxa with bead tubes and lysis buffer provided with the was significantly decreased in FR RF, represented QIAamp PowerFecal kit (Qiagen, Germantown, by unweighted UniFrac (q = 0.05). MD), and all samples subsequently underwent a The VAG microbiome did not differ between 70 °C incubation for 10 min. All samples repeated treatments in any alpha- or beta-diversity metric (q the lysis step with 300-µL lysis buffer and centri- ≥ 0.11). fugation to precipitate (Yu and Morrison, 2004). The QIAamp PowerSoil Minikit (Qiagen) was Relationship Between the Maternal Microbiome used to further purify microbial DNA from RF and Placenta and VAG. Quality and concentration of DNA was assessed with the Nanodrop spectrophotom- The maternal gut of CON cows was richer and eter. Following the Illumina MiSeq System library more diverse than the CON AL (q ≤ 0.03), but these preparation protocol, each sample was prepared FR microbiomes did not differ in alpha-diversity (q as 30-ng/µL aliquots and pooled for sequencing of ≥ 0.26). The RF of both CON and FR cows was the V4 region of the 16S rRNA gene. Samples were similar in richness (q = 0.11) and tended to differ in sequenced at the Colorado State University MIP phylodiversity (q = 0.09) from the ICM. Although NGS Illumina Core. CON RF was similar to the COT in both richness and phylodiversity (q ≥ 0.11), FR RF tended toward dissimilarity with the COT in both alpha-diversity Bioinformatic Analysis metrics (q = 0.06). Sequence analysis was conducted in QIIME2 Unlike the maternal gut, the VAG of FR cows v.  2020.8 (Bolyen et  al., 2019) and quality fil- was similar to the AL in richness and phylodiver- tering, pairing, and denoising were accomplished sity (q ≥ 0.43), although the CON VAG tended to be through the DADA2 plugin (Callahan et al., 2016) richer than the AL (q = 0.06). Like the maternal gut, on the University of Wyoming Advanced Research the VAG of both CON and FR cows was similar Computing Center Teton computing environment in richness (q ≥ 0.11) and the CON VAG tended to Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 Reduced feed and materno-placental microbiome S161 Figure 1. Alpha diversity box plots depicting Shannon richness of sample type by feeding treatment. Samples include maternal rumen fluid (RF) and vaginal swabs (VAG), along with three placental tissues: the intercotyledonary membrane of the chorion (ICM), cotyledon (COT), and allantois (AL). Each sample type is represented within a control diet (CON) or 70% feed intake restricted diet (FR). X and y denote tendencies to differ at 0.05 < q ≤ 0.10. Figure 2. Unweighted UniFrac principle coordinate analysis (PCoA) plot of sample type by feeding treatment. The allantois (AL) in feed restriction (FR) tended toward similarity with the maternal rumen fluid (RF; q = 0.06) and vaginal (VAG; q = 0.07) microbiomes where control (CON) pregnancies differed (q = 0.01). The RF differed from VAG in both treatments (q ≤ 0.02). differ in Faith’s phylodiversity (q = 0.10) from the and unweighted UniFrac (q = 0.07). Where FR RF ICM where the FR VAG did not (q = 0.55). Finally, differed in dominant and non-dominant taxa with the VAG and COT of both treatment groups were the COT and ICM (q ≤ 0.03), it tended to be similar similar in richness and phylodiversity (q ≥ 0.11). to the AL under both UniFrac metrics (Figure 2; The RF and VAG of CON tended to share q = 0.07), as did the FR VAG (q = 0.09). Both the both dominant and nondominant microbial taxa CON VAG and RF remained different from the with the COT and ICM, represented by weighted AL in both dominant and non-dominant microbial Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/Supplement_S1/S159/6446455 by DeepDyve user on 07 December 2021 Hummel et al. S162 taxa (q ≤ 0.02). The FR VAG also differed in both reflected within the placental microbiome and UniFrac values from the FR COT and ICM (q ≤ may further influence inoculation of the fetal gut. 0.03), although the FR VAG and ICM only tended A greater understanding of the maternal gut’s influ- toward dissimilarity regarding dominant microbial ence on the reproductive microbiome throughout taxa (q = 0.06). gestation is required in order to guide nutritional decisions and management practices in beef cows DISCUSSION and to identify the optimal microbiome for repro- ductive performance. Consistent with previous findings in feed re- stricted ewes (Hu et  al., 2018), the mature bovine ACKNOWLEDGMENTS rumen is influenced by feed intake restriction, where the FR microbiome is less rich and less diverse than Funding was provided by the University of that of CON cows. The species counts of phyla that Wyoming Agricultural Experiment Station FY20 are present in a low relative abundance in the ma- Competitive Grant Program. ture rumen were most impacted, which may exert changes on VFA production, microbial lipid me- LITERATURE CITED tabolism, and overall ruminal pH (Hu et al., 2018) Advanced Research Computing Center. Teton Computing and potentially altering metabolites and microbes Environment, Intel x86_64 cluster. University of available to the gestating fetus. Wyoming. 2018. Antony,  K.  M., J.  Ma, K.  B.  Mitchell, D.  A.  Racusin, Consistent with our hypothesis, these differ- J.  Versalovic, and K.  Aagaard. 2015. The preterm pla- ences did not carry to the VAG microbiome, which cental microbiome varies in association with excess ma- is resistant to change throughout gestation (Romero ternal gestational weight gain. Am. J.  Obstet. Gynecol. et al., 2014; Ault et al., 2019). However, the FR VAG 212:653.e1–653.16. doi:10.1016/j.ajog.2014.12.041 displayed similarities in alpha-diversity with the Ault,  T.  B., B.  A.  Clemmons, T.  S.  Reese, F.  G.  Dantas, G. A. Franco, T. P. L. Smith, J. L. Edwards, P. R. Myer, maternal gut that the CON treatment did not, po- and K. G. Pohler. 2019. Bacterial taxonomic composition tentially validating a relationship between these mi- of the postpartum cow uterus and vagina prior to artificial crobial sites (Jeon et al., 2017). It remains possible insemination. J. Anim. Sci. 97:4305–4313. doi:10.1093/jas/ that a prolonged period of feed intake restriction skz212 may be capable of altering the vaginal microbiome, Bolyen,  E., J.  R.  Rideout, M.  R.  Dillon, N.  A.  Bokulich, similar to the effects of dietary changes in pregnant C. C. Abnet, G. A. Al-Ghalith, H. Alexander, E. J. Alm, M.  Arumugam, F.  Asnicar, et  al. 2019. Reproducible, women (Ravel et al., 2011). interactive, scalable and extensible microbiome data Importantly, the maternal gut shared a greater science using QIIME 2. Nat. Biotechnol. 37:852–857. degree of microbial characteristics with the COT doi:10.1038/s41587-019-0209-9 within the CON treatment. This may indicate that Callahan,  B.  J., P.  J.  McMurdie, M.  J.  Rosen, A.  W.  Han, the COT plays a role in microbial transmission to A.  J.  Johnson, and S.  P.  Holmes. 2016. DADA2: High- resolution sample inference from Illumina amplicon data. the placenta, as the COT is responsible for per- Nat. Methods. 13:581–583. doi:10.1038/nmeth.3869 fusing the placental organ with blood (Wooding Goldenburg, R. L., W. W. Andrews, A. R. Goepfert, O. Faye- and Burton, 2008), a hypothesized mode of bac- Peterson, S. P. Cliver, W. A. Carlo, and J. C. Hauth. 2008. terial transmission in cattle (Jeon et  al., 2017). The Alabama preterm birth study: Umbilical cord blood Additionally, the maternal rumen of FR preg- Ureaplasma urealyticum and Mycoplasma hominis cul- nancies shared alpha-diversity characteristics with ture in very preterm newborns. Am. J.  Obstet. Gynecol. 198:43.e1–e5. doi:10.1016/j/ajog.2007.07.033 the AL, implicating decreased microbial diversity Hu,  F., Y.  Xue, C.  Guo, J.  Liu, and S.  Mao. 2018. The re- within the FR placenta as a whole, and potentially sponse of ruminal fermentation, epithelium-associated indicating decreased microbiome diversity within microbiota, and epithelial barrier function to severe feed the fetal gut, which may be contributing a micro- restriction in pregnant ewes. J. Anim. Sci. 96:4293–4305. biome to the AL through the elimination of waste doi:10.1093/jas/sky306 Hummel,  G.  L., K.  L.  Woodruff, K.  J.  Austin, T.  L.  Smith, (Schlafer et al., 2000). and H.  C.  Cunningham-Hollinger. 2020. Evidence for the amnion-fetal gut-microbial axis in late gestation IMPLICATIONS beef calves. Transl. Anim. Sci. 4(Suppl 1):S174–S177. doi:10.1093/tas/txaa138 These data indicate a critical influence of the Jeon, S. J., F. Cunha, A. Vieira-Neto, R. C. Bicalho, S. Lima, maternal gut upon the placental microbiome, as M.  L.  Bicalho, and K.  N.  Galvão. 2017. Blood as a both sites were altered by feed intake restriction. route of transmission of uterine pathogens from the gut Feed intake restriction shaped a less diverse, less to the uterus in cows. 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Appl. 60-61:145–160. doi:10.1016/s0378-4320(00)00132-9 Environ. Microbiol. 75:6524–6533. doi:10.1128/ Wooding,  P., and G.  Burton. 2008. Synepitheliochorial AEM.02815-08. Translate basic science to industry innovation

Journal

Translational Animal Science – Oxford University Press

Published: Nov 30, 2021

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