Embryonic mortality and plasma advanced oxidation protein products (AOPP) increase in dairy cows

Introduction 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: 83-92