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Can embryonic mortality in the dairy cow be related to oxidative stress?
Full text linkThe present thesis aimed to investigate causes and factors involved in embryo mortality (EM) in the dairy cow, in particular the role played by oxidative stress (OS).
In Chapter 4 the pregnancy-associated glycoprotein (PAG) assay, a new method for bovine pregnancy/EM diagnosis never applied in Italy before, was tested in a commercial dairy farm. The PAG test turned out to be a reliable pregnancy/EM diagnosis method although some false positive or false negative were found. For instance, some cases of PAG detection in the maternal circulation after embryonic death occurrence were found, suggesting that PAG may be a better indicator of trophoblastic function rather than embryo viability. The relationship between progesterone (P4) and PAG production was investigated in three different experiments (chapter 4.3.6, chapter 5.3.3, and chapter 6.3.4). The results obtained showed a positive effect exerted by high P4 concentrations on PAG secretion, which is particularly evident when compared to the effect of P4 levels below the threshold of 6 ng/ml in these experiments.
The relationship between OS and EM was investigated in intensive dairy production system (chapter 5) and pasture-based system (chapter 6). Very few modifications of the OS indicators were observed in case of EM suggesting a weak involvement of OS in the aetiology of EM. It is interesting the relationship observed between advanced oxidation protein products (AOPP) levels and EM which suggests a greater degree of protein oxidation in case of EM. Since AOPP seem to be a by-product of neutrophil activation during infections, it is possible that the higher levels of AOPP observed in EM may reflect subclinical inflammatory events at uterine levels that can compromise the correct embryonic development. Therefore, inflammation and not OS might be the main responsible for embryo loss.
Thus, the main contribution of this PhD thesis was to provide new knowledge about the oxidative status of dairy cows. In particular, original contributions are in relation to OS and inflammation biomarkers, as AOPP, and their involvement in EM. As a result, new questions have arisen on this area that could constitute the objective of further research programs in the future.La presente tesi intende indagare le cause e i fattori implicati nella mortalità embrionale nella bovina da latte, in particolare il ruolo rivestito dallo stress ossidativo.
Nel capitolo 4, è stato testato in un’azienda di bovine da latte il dosaggio delle pregnancy-associated glycoproteins (PAG), un nuovo metodo per la diagnosi di gravidanza/morte embrionale mai applicato in Italia prima d’ora. Il PAG test si è dimostrato efficace nel diagnosticare gravidanze e morti embrionali, nonostante si siano riscontrati alcuni falsi positivi e falsi negativi. Ad esempio, in alcuni casi le PAG sono state rilevate nel sangue materno anche dopo la morte dell’embrione, suggerendo quindi che le PAG possano essere indicatori di funzionalità del trofoblasto anziché di vitalità dell’embrione.
Il rapporto tra i livelli di progesterone (P4) e la produzione di PAG è stato studiato in tre diversi esperimenti (capitoli 4.3.6; 5.3.3; e 6.3.4). I risultati ottenuti mostrano un effetto positivo esercitato da elevate concentrazioni di P4 sulla secrezione di PAG, che risulta evidente soprattutto quando confrontato con l’effetto di livelli di P4 inferiori alla concentrazione soglia di 6 ng/ml trovata in questi esperimenti.
Il rapporto tra lo stress ossidativo e la mortalità embrionale è stato valutato sia in un sistema di produzione intensivo (capitolo 5), che in un sistema estensivo basato sul pascolo (capitolo 6). Si sono osservate scarse modificazioni degli indicatori di stress ossidativo nei casi di mortalità embrionale, che suggeriscono quindi un suo debole coinvolgimento nell’eziologia di questa patologia. É comunque interessante considerare la relazione emersa tra i livelli di advanced oxidation protein products (AOPP) e la mortalità embrionale, la quale induce a ritenere che vi sia un maggior grado di ossidazione delle proteine in caso di morte dell’embrione. Poiché gli AOPP sembrano essere prodotti dall’attivazione dei neutrofili in corso di infezione, è possibile che gli elevati livelli di AOPP, osservati nei casi di morte embrionale, riflettano eventi infiammatori subclinici a livello uterino che potrebbero, a loro volta, compromettere il corretto sviluppo dell’embrione. Di conseguenza, l’infiammazione, e non lo stress ossidativo, potrebbe essere il principale responsabile della perdita dell’embrione.
Il contributo maggiore apportato dalla presente tesi è quello di fornire nuove conoscenze in merito allo stato ossidativo della bovina da latte. In particolare, si sono ottenute rilevanti informazioni riguardanti alcuni indicatori di stress ossidativo e di infiammazione, come le AOPP, e il loro coinvolgimento nella mortalità embrionale.
Alla luce delle argomentazioni trattate in questo lavoro di indagine scientifica, sono derivati nuovi interrogativi che interessano il complesso tema della fisiopatologia della riproduzione della bovina da latte e che potrebbero costituire l’obiettivo di futuri programmi di ricerca
Relationship between oxidative status and pregnancy outcome in dairy cows
No full textIn ruminants, oxidative stress may be involved in several pathological conditions, including conditions that are relevant for ruminant reproduction, production and the general welfare (Miller et al 1993). Oxidative stress resulting from increased production of pro-oxidants, and/or a decrease in antioxidant defence, leads to damage of biological macromolecules and disruption of normal metabolism and physiology. Recent evidence has identified a potential role for oxidative stress as a trigger for cell death during luteolysis. Corpus luteum is essential for the maintenance of pregnancy and an excessive free radical generation damages luteal cell membrane and affects progesterone (P4) production (Kato et al 1997). This condition may lead to a failure in embryo development, increasing days open and calving intervals. It is possible that the metabolic stress induced by lactation makes the corpus luteum more susceptible to stress and free radicals may be involved in the decrease in fertility.
This study was designed to evaluate if the establishment of pregnancy may be affected by metabolic and oxidative status of dairy cows. We used 40 pasture fed Holstein-Friesian cows which were monitored daily for oestrous activity starting from day 50 postpartum. On the day of the artificial insemination (AI; Day 0), and on Days 30 and 42 after AI, blood samples were collected and reactive oxygen metabolites (ROMs) and biological antioxidant potential (BAP) concentrations were determined by a commercial kit (Diacron, Grosseto, Italy) on fresh blood by FRAS4 (H&D limited, Parma, Italy). Blood was centrifuged immediately and plasma was stored at -20 ̊C until assayed for its concentrations of P4, pregnancy associated glycoprotein (PAG), advanced oxidation protein products (AOPP), glucose, total proteins, urea, non-esterified fatty acid (NEFA), and glutathione (GSH). Plasma PAG concentrations at days 30 and 42 were used to perform a pregnancy diagnosis according to Zoli et al (1992). On the days of sampling, all cows were monitored for body condition score (BCS). The data gathered were analysed by logistic regression (GenStat); analysis included: between-subjects main effect of AI outcome (AI positive: AI+, AI negative: AI- and embryo mortality: EM), within-subjects main effect of postpartum period (Period 1: 120 days) and their interaction. Pearson’s correlation coefficients were calculated between the parameters measured in this study.
Overall, we observed 26 AI+ (33%), 49 AI- (62%), while EM was observed in 4 cases (5%). Cows that experienced EM had lower BCS values (2.5) compared to the AI+ (2.8) and AI- (2.9) cows (P<0.05). A reduction in BCS during early lactation doubles the rate of pregnancy losses and embryo mortality in dairy cows (Lopez-Gatius et al 2003). During the postpartum BCS increased and NEFA decreased (P<0.05) suggesting a recovery from the negative energy balance (NEB). The observation that on Day 0 plasma NEFA concentrations were higher in the AI+ (0.62 mmol/L; P<0.05) cows compared to the AI- (0.58 mmol/L) and EM (0.58 mmol/L) cows was unexpected as high serum concentrations of NEFA are known to have detrimental effects on fertility. Plasma glucose and ROMs concentrations increased during the postpartum (P<0.03 and P<0.01, respectively) with their highest values observed during Period 3. The gradual increase in glucose concentration suggests a recovery from the NEB. It is likely that the increase in ROMs concentration was due to the parallel increase in glucose, indeed the two were significantly correlated (r=0.2; P<0.05). Concentrations of BAP decreased during the postpartum (P<0.05) possibly as a consequence of parallel increase in ROMs levels; antioxidant depletion indeed, is considered the consequence and not the cause of oxidative stress. GSH and AOPP showed a similar trend: during Period 1 their concentrations were higher in EM than in AI+ and AI- (P<0.05). However, because of the low number of EM cases, this finding needs to be confirmed in further studies. It seems that plasma ROMs and BAP concentrations are not related to AI outcome. Considering that the maintenance of redox homeostasis is quite complex, further studies are required to clarify the role of oxidative status on cows’ fertility
Relationship between oxidative stress and the success of artificial insemination in dairy cows in a pasture-based system
No full textThis study was designed to evaluate whether the outcome of artificial insemination (AI) was affected by
the metabolic and oxidative status of dairy cows. Seventy-nine inseminations in 40 cows, were classified,
on the basis of blood progesterone (P4) and pregnancy-associated glycoprotein (PAG) concentrations and
clinical confirmation of pregnancy into, three categories: (1) positive (AI+, resulted in pregnancy, n = 26;
33%), (2) negative (AI, did not result in pregnancy, n = 49; 62%), and (3) embryonic mortality (EM, n = 4;
5%). Reactive oxygen metabolites, biological antioxidant potential, oxidative stress index, body condition
score, glucose, total proteins, albumin, urea, non-esterified fatty acids (NEFAs), cholesterol, triglycerides,
haptoglobin and advanced oxidative protein products (AOPPs) were measured on the day of AI (day 0),
and 30 and 42 days later.
Cows with EM had lower BCS scores (2.5) than AI+ (2.8) and AI (2.9) cows (P < 0.05). During the postpartum
period, body condition score (BCS) increased and NEFAs decreased (P < 0.05) suggesting a recovery
from the negative energy balance (NEB). The only significant differences found were that the mean
concentration of AOPPs was higher and that of albumin lower in EM cows than in AI+ and AI
(P < 0.05) animals. Plasma concentration of reactive oxygen metabolites and biological antioxidant potential
were not related to AI outcome. Further studies are required to confirm this finding and to clarify the
role of oxidative status on cows’ fertility
AI outcome and metabolic indicators in cows: a field study.
No full textIntroduction: The AI outcome can be affected by nutritional and metabolic conditions that may disturb either CL function and embryo implantation leading to return to oestrus. In this work, we investigated the relationship between AI outcome, blood metabolites and plasma progesterone from AI to pregnancy diagnosis.
Methods: The AI outcome (N=143) performed in 89 cows belonging to 3 herds was recorded for 6 months. The diagnosis of pregnancy was performed by ultrasonography at day 28-34. The outcome of 81 AI was monitored also by PAG measurement in mother blood (RIA) after day 25 to asses embryo viability. Plasma P4, urea, NH3, cholesterol, triglycerides, NEFA, OH-butyrate, and glucose were measured every week from AI to the pregnancy diagnosis. The relationship between AI outcome and metabolic indicators was analysed by ANOVA.
Results and discussion: 48.3% of cows became pregnant within 3 AI, 21.3% did not become pregnant after 3 or more AI, and only 3.4% became pregnant beyond 3 AI. 27% of animals did not become pregnant within the end of the experiment. In 14 cases, plasma PAG concentrations measured before a negative ultrasonographic diagnosis of pregnancy were compatible with the presence of a viable embryo thus suggesting a possible embrionic loss. In other 6 cases, plasma PAG were actually decreasing from day 25 onward, indicating that embryo death occurred. Plasma progesterone tended to be lower in negative AI only on day 18. Plasma triglicerydes concentration and NH3/urea molar ratio were slightly but significantly (P<0.05) lower in positive AI. It is unlikely that these differences can affect the establishment of pregnancy “per se”, but they may be the signal of more subtile mechanisms. Measurement of plasma PAG in association with the ultrasonographic diagnosis of pregnancy may be an important tool to identify embryonic death
Relationship between progesterone and pregnacy associated glycoprotein concentrations in the maternal circulation durino early pregnancy in dairy cows
No full textThe corpus luteum (CL) plays a major role for the successful establishment of pregnancy, and CL function during early embryonic development and implantation seems to be crucial for a correct foetus-maternal interaction. Pregnancy-associated glicoproteins (PAGs) represent a large family of glycoproteins expressed in the mononucleate and binucleate cells of the trophectoderm of several species. In the bovine, PAGs are detectable in the maternal circulation from D 24 after fertilization until parturition, and they are unquestionable indicators of the presence of a viable embryo. However, data about the biological function and the control of secretion of these glycoproteins are scarce. In this study, we tested the hypothesis that the progesterone (P4) secreted by the CL around the time of embryo implantation may influence PAG secretion during early pregnancy. The outcome of 84 artificial inseminations (AI) was studied in 56 pluriparous cows by measuring PAG and P4 (RIA) in maternal plasma samples taken at AI (D 0) and at 21, 28, 30, 45, 60 and 90 D after AI. Confirmation of pregnancy was performed by trans-rectal examination after D 45. Fourty-four AIs (52.4%) were positive, while embryo loss was observed in 10 cases (11.9%). On D 21, plasma P4 was higher, although not significantly, in positive AIs (6.09±0.39 ng/ml) than in AIs followed by embryo loss (4.88±0.79 ng/ml). Plasma PAG concentrations were significantly higher (P<0.01) in AIs performed before 80 days post parturition and were excluded from subsequent analysis. Plasma PAG concentrations were significantly lower in AIs followed by embryo loss (P<0.001) between D 30 and D 60. In most cases, embryo loss occurred between D 30 and D 45. Hormone data obtained from positive AIs performed after 80 days post parturition (N=40) were used to study the relationships between plasma progesterone measured on D 21 and PAG plasma concentrations. AIs were grouped on the basis of P4 concentrations observed on D 21 into Low P4 Group (P4<6.0 ng/ml at D 21) and High P4 Group (P4≥6.0 ng/ml at D 21), and the relationship between plasma P4 and PAG was analysed by ANOVA. On average, AIs of the High P4 Group showed higher levels of circulating PAG between D 30 and D 60 and, in particular, plasma PAG was significantly greater on D 60 (High P4 Group: 5.62±0.63 ng/ml; Low P4 Group: 4.00±0.57 ng/ml; P<0.05). The results of the present study confirm the hypothesis that circulating P4 around the time of embryo implantation can affect PAG secretion in early pregnancy
Embryonic mortality and plasma advanced oxidation protein products (AOPP) increase in dairy cows
No full textIntroduction
In dairy cows, embryonic mortality (EM) within day 42 post insemination brings fertilization rate about 50% and represents a major limitation to fertility (Mann and Lamming, 1999).
The causes of embryonic mortality are still poorly understood. Several papers indicate reactive oxygen species (ROS) and oxidative stress (OS) having a role in pathophysiology of mammal reproduction during (Fujii et al, 2005).
Accumulation of free radicals can occur in dairy cows exposed to metabolic stress or adverse environmental conditions (heat stress). If ROS generation exceeds body’s antioxidant production capacity, OS may develop (Castillo et al, 2005). In dairy cows, OS has been associated with several pathological conditions, which in turn may lead to a declined fertility (Miller et al, 1993).
Excessive ROS production can cause macromolecule damages (Fujii et al, 2005). ROS-mediated protein oxidation is complex and may generate multiple products. Oxidized proteins are often functionally inactive, can be more or, on the opposite, less susceptible to proteases leading to accumulation. They may activate the immune reaction and be the target of autoantibodies (Dean et al, 1997).
Advanced oxidation protein products (AOPP) are proteins damaged by OS formed mainly by chlorinated oxidants resulting from myeloperoxidase activity. In humans, they are responsible of induction of proinflammatory activities and cytokines, and increase in the circulation in some pathological situations. Moreover, they are referred to as markers of OS and neutrophil activation (Kalusova et al., 2005).
The aim of this work was to detect possible relationships between the artificial isemination (AI) outcome and AOPP as an indicator of protein oxidation to study the implication of protein oxidation in EM.
Materials and methods
This study was carried out in northern Italy over a ten months period. A total of 157 AIs were studied in 69 dairy cows. Blood samples were collected on the day of AI (d 0) and on days 15, 28, 35, 45, and 60 after AI.
Plasma concentrations of AOPP, glutathione (GSH), pregnancy-associated glycoprotein (PAG), malonyldehaldide (MDA), total proteins (TP), albumins (A), globulins (G) and urea were measured. Whey progesterone (P4) was analysed in samples taken every 3-4 days from day 15 post-partum to day 45 after each positive AI. Examination of whey P4 profiles was used to assess the beginning of ovarian activity.
The AI outcome was classified ex post in negative (AI-), positive (AI+) and EM. The diagnosis of EM was performed by the simultaneous examination of P4 in whey and PAG in maternal plasma.
Data were analysed using the general linear model procedures of SPSS (SPSS 15.0, 2005).
Results and discussion
The diagnosis of EM performed by the analysis of maternal plasma PAG was sensitive in detecting EM occurring after day 25 (Zoli et al., 1992). Therefore, return to oestrus before day 24 was classified as negative AI (AI-). In this study, 9 EM, 89 AI- and 49 AI+ were observed.
Plasma AOPP concentrations were significantly higher in EM (P<0.01; fig.1). On the contrary, plasma MDA and GSH were not affected by the AI outcome. However, AOPP showed weak but significant correlations with both MDA (R=0.323, P<0.001) and GSH (R=0.096, P<0.05), suggesting that AOPP may be used as a marker of OS. Although GSH is the most abundant non-protein thiol in mammalian cells and a good indicator of the blood oxidative scavenging capacity, it is also an important source of storage and transport of cysteine (Wu et al. 2004). Thus, total plasma GSH is likely more related to metabolic adaptations, such as sulphur amino acid sparing, rather than to oxidative stress and for this reason the relationship between AOPP and GSH is not very strong.
Plasma TP, A and G were not affected by the AI outcome, but they increased significantly during the post partum (P<0.01). Plasma urea was significantly affected by the AI outcome (P<0.01; fig.1), and it increased steadily throughout the post partum (P<0.05).
TP and A were significantly higher in cows beginning the ovarian activity before day 42 post partum (respectively P<0.05 and P<0.001).
In this study, we report for the first time the variations of AOPP in parallel with those of circulating proteins in the cow plasma. Plasma AOPP variations are independent from those of TP, A and G, suggesting a greater degree of protein oxidation in case of EM.
Since AOPP are considered marker of both OS and inflammation (Kalusova et al., 2005), it is possible that higher levels of AOPP observed in EM may reflect inflammatory events at uterine levels that can alter embryo development.
The data gathered in this study seem to indicate an association between EM and protein oxidation, which warrant further investigation.
Acknowledgements
Work supported by the Italian Ministry of University and Scientific Research (PRIN 2005).
References
Castillo C, J Hernandez, A Bravo, M Lopez-Alonso, V Pereira, JL Benedito. 2005. Oxidative status during late pregnancy and early lactation in dairy cows. The Veterinary Journal. 169: 286-292
Dean RT, S Fu, R Stocker MJ Davies. 1997. Biochemistry and pathology of radical-mediated protein oxidation. Biochemical Journal, 324: 1-18
Fujii J, Y Iuchi, F Okada. 2005. Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system. Reproductive Biology and Endocrinology, 3:43-53
Kalousova M, T Zima, V Tesar, S Dusilova-Sulkova, J Skrha. 2005. Advanced glycoxidation end products in chronic diseases-clinical chemistry and genetic background. Mutation Research, 579:37-46
Mann GE, GE Lamming. 1999. The influence of progesterone during early pregnancy in cattle. Reproduction in Domestic Animals, 34: 269-274
Miller JK, E Brzezinska-Slebodzinska, FC Madsen. 1993. Oxidative stress, antioxidants, and animal function. Journal of Dairy Science. 76: 2812-2823
SPSS 15.0. 2006. Base user’s Guide. SPSS Inc. Chicago, IL.
Yoshida Y, M Hayakawa, E Niki. 2005. Total hydroxyl-octadecadienoic acid as a marker for lipid peroxidation in vivo. Biofactors 24:7-15
Wu G, YZ Fang, S Yang, JR Lupton, ND Turner. 2004. Glutathione metabolism and its implication for health. Journal of Nutrition 134:489-492
Zoli AP, LA Guilbault, P Delahaut, WB Ortiz, JF Beckers. 1992. Radioimmunoassay of a bovine pregnancy- associated glycoprotein in serum: its application for pregnancy diagnosis. Biology of Reproduction 46: 83-9
Dehydroepiandrosterone secretion in dairy cattle is episodic and unaffected by ACTH stimulation
No full textThis paper describes the episodic release and response to adrenal stimulation of cortisol and dehydroepiandrosterone (DHEA) in cows. Observations made in samples taken every 10 min for 8 h (experiment 1) showed that plasma DHEA was significantly greater (P < 0.001) than DHEA-S, and release of these steroids was episodic and variable between animals (P < 0.01). No relationship was found between DHEA and cortisol. Significant (P < 0.001) DHEA-sulphate (DHEA-S) versus cortisol (R = -0.264) and DHEA-S versus DHEA (R = 0.200) correlations were found. DHEA and DHEA-S were not affected by a single ACTH challenge (experiment 2). In experiment 3, cortisol and DHEA secretions in response to prolonged ACTH administration (every 12 h for 6 days) were studied. On day 7, the episodic cortisol and DHEA release and response to the opioid antagonist naloxone were studied in blood samples taken every 10 min for 8 h. Animals were injected with naloxone after 4 h. A significant increase (P < 0.05) in mean circulating DHEA and DHEA pulse amplitude was observed during frequent sampling following ACTH treatment. DHEA and DHEA-S plasma concentrations were not affected following luteal regression (experiment 4). The effect of milk secretion around parturition on DHEA secretion was studied in dry and continuously milked cows (experiment 5). Plasma DHEA was significantly lower (P < 0.05) in milked cows. In the cow, ACTH is not an important DHEA secretagogue. Adrenal contribution to plasma DHEA is scarce. Likely, the placenta is the most important source of DHEA, and the lactating mammary gland can affect circulating DHEA levels. Investigation about the DHEA biological role in cows should be focused around parturition
Relationship between return to oestrus, luteal function and metabolic indicators in dairy cows.
No full textIntroduction: Many factors may disturb oocyte fertilization and embryonic development giving rise to return to oestrus. Among these factors, CL function at the time of embryo implantation has a major role, and luteal activity can be affected by nutritional and metabolic conditions. In this study, we investigated the relationship between AI outcome, CL activity and plasma metabolic indicators from AI to the time of expected embryo implantation.
Methods: Whey progesterone measured every 3-4 days by RIA was used to study the ovarian cycle characteristics in 33 dairy cows from day 15 post-partum to pregnancy diagnosis performed at day 30-35 after each AI. Animals were monitored until they became pregnant or for at least three consecutive AI. The whole observation period lasted seven months. Plasma progesterone, urea, cholesterol, triglycerides, NEFA, βOH-butyrate and glucose were measured in samples taken at AI and 7, 14 and 21 days after AI.
Result and discussion: 66.7% of cows became pregnant within 3 AI, 18.2% did not become pregnant within 3 AI and 15.1% did not become pregnant within the observation period. Mean plasma NEFA at the time of the first AI were at basal levels suggesting that cows were inseminated once overcome the period of negative energy balance. Plasma progesterone was significantly lower on day 14 in AI with negative outcome (5,11 0,68 vs 7,42 0,69 ng/ml; P<0,05). Negative AI were classified according to ovarian cycle length subsequent the AI (Physiological, N=11, 22,0 0,6 days; Long, N=14, 34,8 3,0 days). Plasma progesterone was significantly lower (P<0,05) after negative AI followed by a Physiological cycle only on day 21. Triglycerides and cholesterol were slightly but significantly higher in negative AI (P<0,01). These differences may imply the presence of metabolic signals affecting CL activity and/or embryo survival
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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