Oxidative Stress and Fertility in Dairy Animals

Fertility of dairy cow is reduced in last few decades with increased productivity. Reduced fertility may limit the profitability and sustainability of dairy farms. Fertility is a multi-factorial trait and it is affected by number of factors viz. genetic, environmental, dietary and management factors with their complex interactions which makes difficult to determine the exact reason. Stress could be one important cause of decreasing fertility viz. stress due to high milk production, abiotic stress, meeting nutrient demand and supply (oxidative stress). Various antioxidants play important role in reducing the oxidative stress in animals. Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and engulfment by antioxidants in the body. Formation of ROS is a continuous and normal process of cellular metabolism, in low concentration this is essential for oocyte formation, steroidogenesis, apoptosis, sperm capacitation and acrosome reaction but in higher concentration it affects cell membrane integrity and functions, damage DNA, lipid and protein metabolism.  Many biological reactions generate reactive oxygen species that accepts electrons from biological molecules such as fatty acids, proteins and DNA. Exposure of cells to ROS can lead to disruptions in membrane structure, reduced activity of enzymes and other proteins. Given their potential harm, biological system has a variety of homeostasis mechanisms to limit the consequences of ROS production. ROS biomarkers include –CH, -OOH, -SH, C=C and –OH which are found in both male and female reproductive tract and known to affect sperm quality and function, oocyte quality and embryo viability (Agarwal et al., 2005). Antioxidants remove ROS through direct or enzymatically-catalyzed reactions while other molecules neutralize the oxidative damage caused by ROS. The magnitude of adverse changes in physiology caused by an increase in ROS generation depend not simply on the amount of ROS produced but rather on the balance between concentrations of ROS and antioxidant systems. During stress such as transition period, post partum negative energy balance, heat stress during summer, immunity system and anti-oxidant system is not working sufficiently to protect the animals which may lead to decreased milk yield and sub-fertility in dairy animals. In human, 25-40% of semen samples in infertile men were rich in ROS (Agarwal et al., 2006) which indicate that there is close association between ROS and infertility.

Energy is generated in the cell aerobically by reducing molecular oxygen (O₂) to water. Two molecules of water are produced from reduction of molecular oxygen by accepting four electrons in oxidative phosphorylation reaction. About 98-99% of the oxygen consumed follows this process, remaining oxygen is not completely reduced and ROS such as superoxide (formed by reduction of oxygen with one electron), hydrogen peroxide (two electrons) and hydroxyl radical (three electrons) are formed. Fig 1 depicts the process of ROS production and how they are converted to water in the cell. 

Fig. 1 Reactive oxygen species production (MPO- myeloperoxidase; SOD- superoxide dismutase) 

ROS are also produced during prostaglandin synthesis and by neutrophils to kill ingested microorganisms. In neutrophils, oxygen is converted to superoxide by NADPH oxidase. O₂^- is more toxic than H₂O₂ and its rapid removal is important. This ROS is in turn reduced to hydrogen peroxide by superoxide dismutase. Hydogen peroxide generated can remove by three different pathways

For unsaturated fatty acids, oxidation by ROS in the presence of iron generates additional ROS through the Fenton reaction that leads to the formation of lipid peroxides. A chain of reaction consuming unsaturated membrane fatty acids can develop. HOCl produced by neutrophils is a strong oxidant that acts as a bactericidal agent in phagocytic cells.

Physiological status of animal and ROS production: Physiological stage that are associated with increases in metabolism, such as lactation, growth, exercise, and heat stress, would also be associated with increased ROS generation viz. amounts of glutathione peroxidase in serum and glutathione peroxidase mRNA in mammary tissue increases after the onset of lactation. Cows with higher milk yields also had higher concentrations of lipid hydroperoxides in serum (Löhrke et al., 2005). During pregnancy, corpus luteum is essential for progesterone synthesis and maintenance of pregnancy; an excessive free radical generation damages luteal cell membrane and affects progesterone production. This condition may lead to embryo mortality, increasing calving intervals resulting in huge economical loss. Production of ROS may also increased in cows with higher body condition score (BCS) before calving and with greater losses in body condition (BCS) after calving. The amount of TBARS (Thiobarbituric Acid Reactive Substances) in the blood may be higher in summer than winter perhaps due to heat stress. Generation of ROS can increase during inflammation probably because of the involvement of prostaglandins and neutrophils in inflammatory processes

Quantification of oxidative stress: The oxidative stress can be quantified by direct or indirect measurement of oxidants and antioxidants in the body. Reactive oxygen metabolites (ROMs) kit can be used to quantify oxidant level in biological fluids. Free radical analytical system (FRAS 4) technology is simple and reliable method of assessing oxidative status in dairy cows (Merlo et al., 2008) which can be performed easily under field conditions. ROS produced due to lipid peroxidation in animals is often determined by measuring malondialdehyde (MDA) through TBARS assay. MDA reacts with thiobarbituric acid and produces red pigments which can be measure in spectrophotometer (Janero, 1990). Isoprostane is best marker of lipid peroxidation which can be measured by ELISA (Milne et al., 2005). Various biomarkers of oxidative stress like FRAP (Ferric Reducing Ability of Plasma), ROMs, BAP (Biological Antioxidant Potential) TEAC (Trolox Equivalent Antioxidant Capacity) can be assay by Spectrometry and TRAP (Total Radical Antioxidant Potential) by Chemiluminescence (Celi, 2010). The oxidative stress measurement is essential to improve health and productivity of animals as well as economy of the farmers.

An antioxidant is a molecule that inhibits the oxidation. Endogenous antioxidants can be categorized as enzymatic antioxidant (SOD, GSH-Px), non enzymatic antioxidants (sulfhydryl groups of albumin) and non enzymatic low-molecular-weigh antioxidants (glutathione, α-tocopherol, β-carotene). A group of enzymes that reduce free radicals, including superoxide dismutase that convert superoxide to hydrogen peroxide and catalase; glutathione peroxidase (which uses GSH as an electron donor); and peroxiredoxin that convert hydrogen peroxide to water. Other enzymes involved in repair of damage to oxidized molecules including glutathione transferase (reduction of oxidized lipids), thioredoxin reductase (which uses an electron from thioredoxin to reduce oxidized proteins) and glutathione reductase (converts oxidized glutathione to the reduced state). These enzymes often require metal cofactors such as copper, zinc, manganese. While these trace minerals are not strictly antioxidants, their concentration and availability can affect redox status. Antioxidant can be classified on the basis of their mode of action and the cellular compartment (cytoplasmic vs. membrane) in which they reside. Some antioxidants act as free radical sinks that inactive ROS through donation of an electron. The major antioxidant in the cytoplasmic compartment of the cell is the tripeptide glutathione (GSH) which can donate the hydrogen on the sulphydryl group of its cysteine directly to ROS. Other antioxidants that act as free radical sinks include ascorbic acid (or vitamin C), also in the cytoplasm, and the membrane antioxidants are α-tocopherol (or vitamin E), β-carotene and ubiquinol (coenzyme Q). Dietary polyphenols and flavonoids react directly with ROS in water-soluble compartments. Heat stress is also associated with an increase in concentrations of superoxide dismutase and intracellular thiols in circulating erythrocytes. Supplementation of antioxidant to dairy animals would improve reproductive function probably by production of ROS in reproductive tissues in lesser concentration and by improveing the redox state in reproductive tissues.

It is said that bulls are half of the herd, so their reproductive efficiency and role in maintaining the superior herd is essential. There are many nutrients and additives that improve the efficiency of male reproduction, but abiotic stress like hot and humid condition during summer, flood and famine etc. may interfere with the normal reproduction. In that situation herdsmen needs to supplement anti-stress agent or antioxidants. Role of antioxidants in the male fertility is summarized below for easy understanding of the common readers.

Oxidative stress in dairy cows may results into reproductive disorders like retention of placenta, mastitis, udder edema. Retention of placenta seems to be connected with imbalance between antioxidant and pro-oxidant. High frequency of retention of placenta was observed when the lipid metabolism in late pregnancy is disturbed (Goff, 2009).Disturbed metabolic pathway of collgen or hyaluronic acid may result into improper separation of cotyledonary membrane. Increase in concentration of 8- iso PGF₂ α in caruncles and cotlydone of placenta may result into tissue oxidative damage and retention of placenta (Celi, 2010).  It is well known that the prepartum administration of vitamin E and selenium can reduce the incidence of retained placenta. It is observed that the frequency of retention of placenta was decreased by administration of Vitamin E or Se alone or combination. Dietary supplementation with β-carotene has also been shown to reduce incidence of retained placenta.  The beneficial effects of antioxidant supplementation depend upon the adequacy of vitamin E and selenium in the diet. Both organic and inorganic sources of selenium are effective in preventing retained placenta (Cerri et al., 2009). Antioxidant supplementation improves immune function; neutrophil function is enhanced by vitamin E and selenium supplementation prepartum (Spears and Weiss, 2008) and β-carotene can enhance lymphocyte capacity for proliferation (Michal et al., 1994).

Antioxidant supplementation would reduce the incidence of infectious disease in the uterus by improving the immune status of animal (Spears and Weiss, 2008). Reducing incidence of retained placenta may reduce uterine infections because occurrence of retained placenta predisposes cows to uterine infections (Le Blanc et al., 2008). In fact, feeding 300 or 600 mg/d β-carotene for 8 week beginning 4 week before expected calving reduced the proportion of cows diagnosed with metritis from 18% in controls to 7% (300 mg/d supplementation, Michal et al., 1994). Similarly, intramuscular injection of 3,000 mg vitamin E at 8 to 14 d before calving reduced the incidence of metritis from 9% to 4% (Erskine et al., 1997).

Administration of antioxidants could affect fertility in two ways. A reduction in incidence of retained placenta or uterine infections caused by prepartum administration of antioxidants Secondly, the oocyte and pre-implantation embryo are susceptible to damage by ROS (Moss et al., 2009) and increasing the antioxidant status of the reproductive tract in the postpartum period might improve competence of the oocyte or embryo for development. Vitamin E and selenium is affecting uterine health and cows having a second service were more likely to have an infectious uterine disease and to be responsive to antioxidant treatment.

Heat stress can have disastrous effects on lactation and fertility of cows as recent reports suggests that there is an increase in environmental temperature due to anthropogenic cause viz., rapid urbanization, industrialization, deforestation, increased human population etc. There is evidence that one of the causes for embryonic mortality in heat-stressed cows is production of ROS by embryos developing at elevated body temperatures (Hansen, 2007). Administration of melatonin, an indoleamine with antioxidant properties, may be reduced the effects of heat stress on embryonic survival. In lactating dairy cows, most antioxidant treatments proved effective in improving fertility of cows but after subjecting to heat stress to be studied in detail. This may be one of the major research area in years to come.

Conclusions

Oxidative stress is one important cause of reduced fertility in dairy animals. Assessment of oxidative stress status (OSS) of animal at different physiological state is essential to reduce economical losses due to infertility, increase calving interval and other reproductive diseases. Physiological factors like high milk yield and occurrence of heat stress, post partum negative energy balance influences redox status of the cow and the magnitude of an antioxidant function. A successful antioxidant therapy is one that reaches reproductive tissues in high enough concentrations to reduce damage caused by ROS. The effective concentration will depend upon antioxidant solubility in cytoplasmatic and membrane compartments of the cell. The route of administration of antioxidant, the degree to which the molecule escapes ruminal degradation and clearance rate from tissues and blood is very important to achieve desire level of concentration in body. All antioxidants from natural products found in plants to be tested in future not only to improve male and female reproductive efficiency but also to improve feed efficiency. Oxidative stress biomarkers should be estimated to know animal health status. Still lot of research is needed to understand the pathophysiology of oxidative stress in dairy animals and standardization of range of values of oxidative stress biomarker so as to modulate the antioxidant feeding therapy in livestock sector. 

Agarwal, A., Gupta, S. and Sharma, R.K. (2005) Role of oxidative stress in female reproduction. Reprod.Biol.Endocrinol. 3, 28.

Agarwal, A., Nandipati, K.C., Sharma, R.K., Zippe, C.D. and Raina, R. (2006) Role of oxidative stress in pathophysiological mechanism of erectile dysfunction. J.Androl.27 (3), 335-347.

Bilaspuri G.S. and Bansal, A.K. (2008) Mn²⁺ a potent antioxidant and stimulator of sperm capacitation and acrosome reaction in cross breed cattle bulls. Arcchiv fur Tierzucht, 51(2) 149-156.

Celi, P. (2010) Biomarkers of oxidative stress in ruminant medicine: Review Immunopharmacology and Immunotoxicology, 1–8. DOI: 10.3109/08923973.2010.514917.

Celi, P. (2010) The role of oxidative stress in small ruminants’ health and production. R. Bras. Zootec., v.39, p.348-363.

Cerri R.L., Rutigliano H.M., Lima F.S., Araújo D.B. and Santos J.E. (2009) Effect of source of supplemental selenium on uterine health and embryo quality in high-producing dairy cows. Theriogenology. 71:1127-1137.

Erskine, R.J., Bartlett P.C., Herdt, T and Gaston, P. (1997) Effects of parenteral administration of vitamin E on health of periparturient dairy cows. J Am Vet Med Assoc. 211:466-469.

Goff, J.P. (2006) Major advances in our understanding of nutritional influences on bovine health. J Dairy Sci. 89, 1292–1301.

Hansen, P.J. (2007) To be or not to be--determinants of embryonic survival following heat shock. Theriogenology, 68 (1):S40-S48.

Janero, D.R. (1990) Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biology and Medicine 9, 515–540.

LeBlanc, S.J., Duffield T.F., Leslie, K.E., Bateman, K.G., TenHag, J., Walton, J.S. and Johnson,W.H. (2002) The effect of prepartum injection of vitamin E on health in transition dairy cows. J Dairy Sci. 85:1416-1426.

Löhrke, B., Viergutz, T., Kanitz, W., Losand, B., Weiss, D.G. and Simko, M. (2005) Short communication: hydroperoxides in circulating lipids from dairy cows: implications for bioactivity of endogenous-oxidized lipids. J Dairy Sci. 88:1708-1710. 

Merlo, M., Celi, P., Barbato, O and Gabai, G. (2008) Relationships between oxidative status and  pregnancy outcome in dairy cows. Animal Production inAustralia27, 84.

Michal, J.J., Heirman, L.R., Wong, T.S., Chew, B.P., Frigg, M. and Volker, L. (1994) Modulatory effects of dietary β-carotene on blood and mammary leukocyte function in periparturient dairy cows. J Dairy Sci. 77:1408-1421.164.

Milne, G.L., Musiek, E.S., Morrow, J.D., 2005. F2-Isoprostanes as markers of oxidative stress in vivo: an overview. Biomarkers 10, 10–23.

Moss, J.I., Pontes, E. and Hansen P.J. (2009) Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione. Arch Toxicol, 83:1001-1007.

Spears, J.W. and Weiss, W.P. (2008) Role of antioxidants and trace elements in health and immunity of transition dairy cows. Vet J. 176:70-76.