mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages

Background The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. Methods BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct- specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Results Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. Conclusion This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered

Evaluierung der Reaktionen von bovinen primären Oviduktepithelzellen unter physiologischen und pathophysiologischen Bedingungen

The mammalian oviduct is crucial for the gamete maturation and supporting the early embryo development. The in vitro culturing of bovine oviductal epithelial cells (BOEC) allows researchers to study these cells without the systemic variation that occur in animals. Various in vitro culturing conditions require careful optimization. The pathophysiological mechanisms of bacterial species in the bovine reproductive tract leading to postpartum diseases and sub-optimal fertility have become an important topic for research. Trueperella pyogenes is a commonly isolated pathogen highly associated with abnormal vaginal discharge in infected animals. Other bacterial species such as Bacillus pumilus could be involved in the development of postpartum diseases. Little is known about pathogens or their endotoxins interaction with BOEC. The present work was set up to understand cellular responses of BOEC under different culturing conditions and further in co-culture with the mentioned bacteria. In the first part of the project, oviducts were collected at the slaughterhouse and classified into non-luteal and luteal stage. Monolayer culture of BOEC was conducted to determine cellular transcription and functional capability of BOEC 1) in transition from in vivo state to in vitro state; 2) during three consecutive cell culture passages; 3) affected by the impact of LOW (5 mM) and HIGH (25 mM) glucose content media; and 4) influenced by the effect of different phases of the estrous cycle in vitro. Total RNA was extracted from in vivo and in vitro samples and subjected to quantitative polymerase chain reaction. Moreover, collected supernatants from passage 0 (P0) and passage 3 (P3) BOEC were measured for prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin (IL) 8 release to observe the influence of in vitro culturing on BOEC functionality. Almost all candidate genes mRNA expression pattern (prostaglandin synthases, cell metabolism enzymes and mucins) were evidently changed from in vivo to in vitro. The mRNA expression pattern of microsomal prostaglandin E2 synthase 2 (PTGES2), mucin 1 (MUC1), -4 and OVGP1 was influenced by the number of cell culture passages. The mRNA expression of most candidate genes was not affected by the concentration of glucose in the culture media. The estrous cycle stage altered candidate genes mRNA expression in BOEC in vitro, notably at later passages (>P2), but not in vivo. MUC1 was the only selected gene whose mRNA expression pattern was influenced by estrous cycle stage at P1. The release rate of PGE2 and OVGP1 between P0 and P3 cells was not significantly different, yet BOEC in P3 released evident higher amounts of IL8 compared with cells in P0. The second part of the project entirely focused on inflammatory responses of in vitro cultured BOEC under the standardized milieu, based on the outcome from the initial phase of the project, during incubation with either T. pyogenes or B. pumilus. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicity of infection (MOI) of T. pyogenes or B. pumilus. Cells remained viable with T. pyogenes at a MOI of 0.01 and with B. pumilus at a MOI 1 and 10. Transcription level and released rate of candidate pro-inflammatory factors was not evidently changed in BOEC co-cultured with T. pyogenes compared with the controls. Meanwhile, higher mRNA expression of IL1A, -1B, -6, tumor necrosis factor alpha (TNFA), chemokine (C-X-C motif) ligand (CXCL) 1/2, -3, -5 and IL8 along with PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. Addition of B. pumilus released higher amount of IL8 and PGE2 from BOEC than controls. The impact of culturing condition on cellular immune response was followed up and it was noticed that viability and early pro-inflammatory response of P3 BOEC incubated with bacteria was clearly lower than from P0 BOEC. In conclusion, the present work showed that conventional oviductal cell culturing highly influences the physiological and immunological responses of the cells prior to any treatment. This is very important and has to be considered during research on BOEC at in vitro level. Furthermore, this work successfully revealed a differential inflammatory response of oviductal cells against live strains of pathogenic/potential pathogenic bacteria isolated from the bovine reproductive tract. These results can contribute to a better understanding of these microorganisms interactions with the oviductal cells.Die Ovidukte von Säugetieren spielen eine wichtige Rolle bei der Reifung der Gameten und unterstützen die Entwicklung des frühen Embryos. Die in vitro Kultivierung von bovinen Oviduktepithelzellen (BOEC) ermöglicht es Wissenschaftlern, diese Zellen zu untersuchen, ohne dass die Zellen den systemischen Einflüssen des tierischen Organismus unterliegen. Verschiedene in vitro Kultivierungsbedingungen erfordern eine sorgfältige Optimierung. Pathophysiologische Mechanismen, die durch Bakterienspezies im bovinen Reproduktionstrakt verursacht werden, führen zu nachgeburtlichen Krankheiten und zu suboptimaler Fruchtbarkeit, und sind daher zu einem wichtigen Forschungsthema geworden. Trueperella pyogenes ist ein häufig nachgewiesener Krankheitserreger, der eine hohe Korrelation zu abnormem Vaginalausfluss bei erkrankten Tieren zeigt. Andererseits können andere Bakterienspezies, wie Bacillus pumilus, bei der Entstehung von nachgeburtlichen Erkrankungen ein Rolle spielen. Wenig ist bislang bekannt über die Wechselwirkung von Krankheitserregern oder ihren Endotoxinen mit BOEC. Die vorliegende Arbeit soll zum besseren Verständnis zellulärer Antworten von BOEC bei verschiedenen Kultivierungsbedingungen und weiterführend bei Ko-Kultivierung mit den erwähnten Bakterien beitragen. Im ersten Teil des Projekts wurden Ovidukte auf dem Schlachthof gewonnen und nachfolgend in die Nicht-Luteal- bzw. Lutealphase eingeteilt. Monolayerzellkulturen wurden von BOEC angelegt, um die zelluläre Transkription und die Funktionalität der Zellen zu bestimmen 1) beim Übergang vom in vivo-Zustand in den in vitro-Zustand; 2) während drei nacheinander folgenden Zellkultur-Passagen; 3) zwischen Nährmedien mit niedrigen (LOW; 5 mM) und hohen (HIGH; 25 mM) Glukosegehalt; und 4) unter dem Einfluss verschiedener Phasen des Sexualzyklus in vitro. Die Gesamt-RNA wurde aus in vivo- und in vitro–Proben extrahiert und einer quantitativen Polymerase- Kettenreaktion zugeführt. Darüber hinaus wurden Zellkultur-Überstände von BOEC in Passage 0 (P0) und P3 gewonnen und diese auf Prostaglandin E2 (PGE2)-, oviduktspezifisches Glykoprotein 1 (OVGP1)- und Interleukin 8 (IL8)-Freisetzung bestimmt, um den Einfluss der in vitro Kultivierung auf die Funktionalität der BOEC zu untersuchen. Die mRNA-Expressionsmuster fast aller Kandidatengene (Prostaglandin-Synthasen, Enzyme des Zellmetabolismus und Muzine) änderten sich deutlich beim Übergang vom in vivo– in den in vitro- Zustand. Das mRNA-Expressionsmuster von Prostaglandin E2-Synthase 2 (PTGES2), Muzin (MUC) 1, -4 und OVGP1 wurde durch die Zahl der Zellkulturpassagen beeinflusst. Die mRNA-Expression der meisten Kandidaten-Gene wurde durch die Glukosekonzentration des Zellkulturmediums nicht beeinflusst. Die Phase des Sexualzyklus veränderte die mRNA-Expression der Kandidaten-Gene in BOEC in vitro, besonders in späten Passagen (>P2), aber nicht in vivo. MUC1 war das einzige Kandidatengen, dessen mRNA-Expression in P1 vom Stadium des Sexualzyklus beeinflusst wurde. Die Freisetzungsrate von PGE2 und OVGP1 war zwischen Zellen der P0 und P3 nicht signifikant unterschiedlich, aber BOEC setzte IL8 in höheren Mengen in Zellen der P3 frei als in P0. Der zweite Teil des Projekts befasste sich mit den Entzündungsreaktionen in den in vitro kultivierten BOEC während der Inkubation mit T. pyogenes oder B. pumilus unter den Standardbedingungen basierend auf den Ergebnissen des ersten Teils. Die Funktionalität der BOEC wurde durch Trypanblau-Färbung nach 24 h Ko- Kultivierung mit verschiedenen „Multiplicity of infection“ (MOI) von T. pyogenes oder B. pumilus untersucht. Die Zellen blieben mit T. pyogenes bei einer MOI 0.01 und mit B. pumilus bei einer MOI 1 und 10 vital. Die Transkription der Entzündungsfaktoren und deren Freisetzung änderte sich nicht signifikant in den BOEC bei Ko-Kultivierung mit T. pyogenes im Vergleich zu den Kontrollen. Dennoch wurde eine höhere mRNA-Expression von IL1A, -1B, -6, Tumornekrose-Faktor alpha (TNFA), Chemokin-(C-X-C-Motiv)-Ligand 1/2 (CXCL1/2), -3, -5 und IL8 wie auch von PG-Synthasen in BOEC bei Ko-Kultivierung mit B. pumilus beobachtet. Bei Zugabe von B. pumilus setzten BOEC höhere Mengen von IL8 und PGE2 frei als in den Kontrollen. Ebenso wurde der Einfluss der Kultivierungsbedingungen auf die zelluläre Immunreaktion verfolgt, wobei sich herausstellte, dass die Funktionalität und die Entzündungsreaktion bei mit Bakterien inkubierten BOEC in P3 offensichtlich niedriger lag als bei BOEC in P0. Zusammenfassend lässt sich feststellen, dass die vorliegende Arbeit zeigen konnte, dass konventionelle Ovidukt-Zellkulturbedingungen die physiologischen und immunologischen Zellantworten in großem Maße beeinflussen, bevor eine Behandlung der Zellen vorgenommen wird. Dies ist sehr wichtig und muss beachtet werden, wenn BOEC auf in-vitro-Ebene untersucht werden. Weiterhin konnte diese Arbeit erfolgreich eine unterschiedliche Entzündungsreaktion von Oviduktzellen gegenüber lebenden pathogenen Bakterienspezies aus dem bovinen Reproduktionstrakt zeigen. Diese Ergebnisse tragen zu einem besseren Verständnis der Wechselwirkung zwischen diesen Mikroorganismen und den Oviduktzellen bei

The use of milk Fourier transform mid-infrared spectra and milk yield to estimate heat production as a measure of efficiency of dairy cows

BACKGROUND: Transformation of feed energy ingested by ruminants into milk is accompanied by energy losses via fecal and urine excretions, fermentation gases and heat. Heat production may differ among dairy cows despite comparable milk yield and body weight. Therefore, heat production can be considered an indicator of metabolic efficiency and directly measured in respiration chambers. The latter is an accurate but time-consuming technique. In contrast, milk Fourier transform mid-infrared (FTIR) spectroscopy is an inexpensive high-throughput method and used to estimate different physiological traits in cows. Thus, this study aimed to develop a heat production prediction model using heat production measurements in respiration chambers, milk FTIR spectra and milk yield measurements from dairy cows. METHODS: Heat production was computed based on the animal’s consumed oxygen, and produced carbon dioxide and methane in respiration chambers. Heat production data included 168 24-h-observations from 64 German Holstein and 20 dual-purpose Simmental cows. Animals were milked twice daily at 07:00 and 16:30 h in the respiration chambers. Milk yield was determined to predict heat production using a linear regression. Milk samples were collected from each milking and FTIR spectra were obtained with MilkoScan FT 6000. The average or milk yield-weighted average of the absorption spectra from the morning and afternoon milking were calculated to obtain a computed spectrum. A total of 288 wavenumbers per spectrum and the corresponding milk yield were used to develop the heat production model using partial least squares (PLS) regression. RESULTS: Measured heat production of studied animals ranged between 712 and 1470 kJ/kg BW0.75. The coefficient of determination for the linear regression between milk yield and heat production was 0.46, whereas it was 0.23 for the FTIR spectra-based PLS model. The PLS prediction model using weighted average spectra and milk yield resulted in a cross-validation variance of 57% and a root mean square error of prediction of 86.5 kJ/kg BW0.75. The ratio of performance to deviation (RPD) was 1.56. CONCLUSION: The PLS model using weighted average FTIR spectra and milk yield has higher potential to predict heat production of dairy cows than models applying FTIR spectra or milk yield only

Lameness detection and scoring

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Milk intake, body anatomy and composition in calves

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