SHORT-COMMUNICATION Evaluation of perfused bovine udder for gene expression studies in dairy cows

Pinto, I.S.B.; Fonseca, Ivana Machado; Brandão, Humberto M.; Gern, J. C.; Guimarães, Alexandre Silva; Carvalho, Wanessa A.; Brito, Maria Aparecida Vasconcelos Paiva; Viccini, Lyderson Facio; Martins, Marta Fonseca

doi:10.4238/gmr16019637

Cited by 6 publications

(7 citation statements)

References 14 publications

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“…The viability of the perfused udders was controlled using biochemical parameters such as lactate dehydrogenase (LDH)-activity, glucose consumption and lactate production in the perfusate (Ehinger and Kietzmann, 2000). Perfusate was sampled at 0 and 3 h post-inoculation (hpi) with S. agalactiae and indicated that the perfused bovine udders were viable throughout the experiment as reported by our research group before (Pinto et al ., 2017).…”

Section: Methodsmentioning

confidence: 99%

“…An ethical and practical issue about the use of in vivo models represents an important limitation in animal studies and the ex-vivo or extracorporeal model approach is a potentially useful alternative. This isolated perfused bovine udder approach has been reported to be suitable in studies of mammary gland metabolism (Hardwick and Linzell, 1960) and pharmacokinetics of substances after intramammary administration (Kietzmann et al ., 1993, 2010) and recently to evaluate gene expression (Pinto et al ., 2017). However, this method has not been widely used to evaluate the transcriptional response of mammary gland against pathogens.…”

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confidence: 99%

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Isolated perfused udder model for transcriptome analysis in response toStreptococcus agalactiae

Weller¹,

Fonseca

Sbardella

et al. 2019

Journal of Dairy Research

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This study aimed to evaluate the transcriptional changes occurring in isolated perfused mammary alveolar tissue in response to inoculation with S. agalactiae and to identify the most affected biological functions and pathways after 3 h. Four udders taken at slaughter from cows with healthy mammary gland were perfused ex situ with warmed and gassed Tyrode's solution. Mammary alveolar tissue samples were taken from the left fore and rear quarters (IQ-inoculated quarters) before inoculation (hour 0) and at 3 h post inoculation (hpi) and at the same times from control right fore and rear quarters (not inoculated: NIQ). A total of 1756 differentially expressed genes (DEGs) were identified between IQ and NIQ at 3 hpi using edgeR package. Within this set of DEGs, 952 were up regulated and mainly involved with innate immune response and inflammatory response, e.g., CD14, CCL5, TLR2, IL-8, SAA3, as well as in transcriptional regulation such as FOS, STAT3 and NFKBIA. Genes down-regulated (804) included those involved with lipid synthesis e.g., APOC2, SCD, FABP3 and FABP4. The most affected pathways were chemokine signaling, Wnt signaling and complement and coagulation cascades, which likely reflects the early stage response of mammary tissue to S. agalactiae infection. No significant gene expression changes were detected by RNA-Seq in the others contrasts. Real time-PCR confirmed the increase in mRNA abundance of immune-related genes: TLR2, TLR4, IL-1β, and IL-10 at 3 hpi between IQ and NIQ. The expression profiles of Casp1 and Bax for any contrasts were unaffected whereas Bcl2 was increased in IQ, which suggests no induction of apoptosis during the first hours after infection. Results provided novel information regarding the early functional pathways and gene network that orchestrate innate immune responses to S. agalactiae infection. This knowledge could contribute to new strategies to enhance resistance to this disease, such as genomic selection.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Isolated perfused udder model for transcriptome analysis in response toStreptococcus agalactiae

Weller¹,

Fonseca

Sbardella

et al. 2019

Journal of Dairy Research

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“…However, increasing concern for animal welfare is increasing the community pressure on ethics committees for animal use in experiments. As a result, ethics committees have recommended stricter protocols and the reduction or, if possible, the replacement of animals with alternative laboratory methods (Hartung, 2008; Pinto et al ., 2017). In addition, experiment length and the costs to the acquisition of animals, feeding and labour are important limitations of in vivo procedures (Doke and Dhawale, 2015).…”

Section: Introductionmentioning

confidence: 99%

In situ and in vitro techniques for estimating degradation parameters and digestibility of diets based on maize or sorghum

et al. 2020

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An experiment was conducted to evaluate: (1) the effects of ensiling maize or sorghum grains after reconstitution on readily soluble fraction (a), potentially degradable fraction in the rumen (b) and rate constant for degradation of b (c) of dry matter (DM), organic matter (OM) and starch (STA); and (2) an appropriate incubation time for in situ or in vitro procedures to estimate in vivo digestibility. Four rumen-cannulated Nellore bulls (body weight = 262 ± 19.6 kg) distributed in a 4 × 4 Latin square were used. Diets were based on dry ground maize (DGM); or dry ground sorghum (DGS); or reconstituted ground maize silage; or reconstituted ground sorghum silage. In vitro and in situ incubations of the individual grains and diets were simultaneously performed with in vivo digestibility. In general, reconstituted grains and diets based on reconstituted grains presented greater (P < 0.05) fraction a and lower (P < 0.05) fraction b of DM, OM and STA compared to dry grains and diets based on dry grain. However, the magnitude of response of the reconstitution and ensiling process on DM and OM degradability parameter was greater for maize than that for sorghum. Moreover, no differences (P > 0.05) were observed between DGM- and DGS-based diets for c estimates. The results suggest that the reconstitution process promotes grains protein matrix breakdown increasing STA availability. The incubation times required for in vivo digestibility estimations of DM, OM and STA are 24 h for in situ and 36 h for in vitro procedures.

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“…When rst experiments in udders were accomplished, study results of ex vivo infection experiments in isolated perfused udders have been published that support the feasibility to use udders as a meaningful model [18,19]. Nevertheless, we believe that the current data are still valuable, as the LPS stimulation allows a direct comparison with former in vitro and in vivo studies and helps to further evaluate the values of isolated perfused bovine udders in mastitis research.…”

Section: Discussionmentioning

confidence: 82%

Early Inflammatory Events of Mastitis - A Pilot Study With the Isolated Perfused Bovine Udder

Brand

Filor

Bäumer

2021

Preprint

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Background: Bovine mastitis is an important health and cost factor in the milk industry. To elucidate whether isolated perfused bovine udders can be used to study early inflammatory events of mastitis, 1 mg of lipopolysaccharide (LPS) was instilled into quarters of 10 isolated perfused bovine udders. Three hours and 6 hours after LPS instillation, tissue samples were taken from the gland cistern and base of the udder, subsequently stored in RNAlater and processed for the determination of inflammation-dependent gene regulation by real-time RT-qPCR. Gene expression analysis was performed using delta-delta Ct method. To translate mRNA results to protein, IL-1ß and IL-6 were determined in tissue homogenate by ELISAResults: The instillation of 1 mg LPS lead to an increased expression of pro-inflammatory cytokines and chemokines like TNF-α, CCL20, CXCL8 as well as of IL-1 ß , IL-6 and IL-10, lingual antimicrobial peptide (LAP) and S100A9. However, the degree of elevation differed slightly between gland cistern and udder base and markedly between 3 hours and 6 hours after instillation, with a distinct increase in mediator expression after 6 hours. IL-1β protein increased in a time-dependent manner, whereas IL-6 was unchanged within 6 hours of LPS instillation.Conclusion: Compared to in vivo studies with instillation of LPS into udders of living cows, a similar inflammation-dependent gene regulation profile can be mimicked in the isolated perfused bovine udder, indicating a supplementation of animal experiments.

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SHORT-COMMUNICATION Evaluation of perfused bovine udder for gene expression studies in dairy cows

Cited by 6 publications

References 14 publications

Isolated perfused udder model for transcriptome analysis in response toStreptococcus agalactiae

Isolated perfused udder model for transcriptome analysis in response toStreptococcus agalactiae

In situ and in vitro techniques for estimating degradation parameters and digestibility of diets based on maize or sorghum

Early Inflammatory Events of Mastitis - A Pilot Study With the Isolated Perfused Bovine Udder

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