Antioxidants: Role In Combating Heat Stress During Early Embryonic Development

Stress is a reflex reaction of animals in harsh environments and causes unfavorable consequences ranges from discomfort to death. Escalating global temperatures combined with global increases in the number of production animals and the intensification of agriculture, including (but not limited to) that in emerging economies, has resulted in heat stress becoming an important challenge facing the global dairy industry. Global average temperatures are expected to increase by about 2–13 °F (1–7 °C) by the end of the century. Even small changes in global average temperature can lead to large changes in the environment. In the scenario of global climatic change, different environmental stresses are severe threats to organisms worldwide. Among all stress conditions, elevated temperature is seen as the most serious threat to animal production. Heat stress is defined as the sum of external forces acting on an animal that causes an increase in body temperature and evokes a physiological response (Dikmen and Hansen, 2009). The condition includes heat cramp, heat exhaustion, and heat stroke. The thermal comfort zone for most animals ranges between 4 and 25 °C, and temperatures exceeding 25 °C will result in heat stress. Excessive flow of energy (in the form of unabated heat) into the body, in addition to energy depletion required for growth and lactation can lead to deteriorated living conditions,  and, in extreme cases, death, unless the animal can activate various adaptive mechanisms to increase the external net energy flow. The environmental conditions driving heat stress are presented using the temperature-humidity index (THI), a calculated index that incorporates the effects of environmental temperature with relative humidity.

In tropical countries like India, where more than 85% places experience moderate to high temperature humidity index (THI) during summer season, heat stress is a concern for farm animals particularly buffalo which forms the mainstay of dairy agriculture. In terms of THI, the value of THI more than 72 is considered as stressful and THI value above 78 is considered as very severe heat stress for buffalo. It is used to evaluate the impact of climatic conditions that contribute to heat stress on the production of livestock all over the world. The THI for dairy cattle has been calculated to be 72 (Thom, 1959), however for high producing dairy cows, this has been shifted down to 68 because of the concurrent rise of metabolic heat production which is associated with vast milk production.

Various studies in past have reported that the reproductive efficiency of the animals gets affected either directly or indirectly due to of heat stress, such as the duration of estrus, conception rate, uterine function, endocrine status, follicular growth and development, luteolytic mechanisms, early embryonic development and fetal growth has found to be altered as a response to heat stress in animals. Thermal stress leads to reduced embryo growth or failed embryo survival produced either in vivo or vitro, as the heat stress is found to interfere with protein synthesis, oxidative cell damage, reducing interferon- tau production for signaling pregnancy recognition, as well as  expression of stress-related genes associated with apoptosis.

Infertility caused due to heat is not only attributed to hormonal secretion and embryo development but also the damage it causes to the oocyte, as during IVF the rate of blastocyst development was found to be reduced in summer. Exposure of heifers to heat stress between the onset of estrus and insemination increased the proportion of abnormal and retarded embryos. This suggests that the process of oocyte maturation is susceptible to heat stress.

It is noteworthy that cellular exposure to heat stress increases the production of ROS promoting cellular oxidation events, associated with cellular hyperthermia. The extent of ROS damage to cell system depends on the balance between their production and removal rates. If the protective mechanism cannot remove the high amount of ROS or the damage caused by, the animal gets into oxidative stress condition. The oxidative stress (OS) condition occurs when the generation of ROS and other radical species exceeds the scavenging capacity by antioxidants, due to excessive production of ROS and/or inadequate supplements intake of antioxidants and/or inactivation of antioxidant enzymes.

Antioxidants, both enzymatic and non-enzymatic, provide necessary defense against oxidative stress as a result of thermal stress. Excessive amount of ROS negatively influences oocyte maturation and oocyte fertilization, together with a role in decreasing sperm motility, sperm number, and sperm-oocyte fusion, with deleterious impact on embryo development. Enzymatic and synthetic (dietary) antioxidants are the main defense factors against oxidative stress induced by free radicals. Enzymatic antioxidants include superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GSR), peroxiredoxins, and non-enzymatic antioxidants, known as natural dietary supplements, including vitamin C vitamin E, selenium, zinc beta carotene, carotene, taurine, hypotaurine and glutathione. Natural antioxidants, widely distributed in food (fruits, vegetables, cereals, mushrooms, beverages, flowers, spices, and traditional medicinal herbs), exhibit an extensive range of biological effects, such as anti-inflammatory, antibacterial, antiviral, anti-aging, and anticancer properties. In this context, different studies tested natural non-enzymatic antioxidants supplementation for its potential influence on IVF outcomes. Concerning the role in IVF, the effects of different antioxidant supplementation on the quality and cryotolerance of in vitro produced embryos, together with the positive effects on in vitro maturation of oocytes and on early embryonic development were assessed using experimental models. Some of the antioxidants studied in past to combat the effect of hest stress on gametes and embryos in vitro  are melatonin, coenzyme Q10, cysteamine, mercaptoethnol, ascorbic acid etc.

Melatonin is a pineal secretory product regulating circadian rhythms. Several studies have documented its capacity in scavenging ROS, also within ovarian follicles. In detail, melatonin supplementation has a role in improving the activity of mitochondria. The mtDNA copy number, the degree of mitochondria granulated clustering, together with the mitochondrial membrane potential, resulted an increase with melatonin supplementation in MII-stage oocytes. Melatonin also manifested the ability of protecting embryos from the damaging effects of different stressors, such as heat and H2O2.  Investigations have revealed that the effect of beta mercapthnol on oocyte maturation and embryo development is correlated with biosynthesis of intracellular glutathione (GSH) which is a tripeptide thiol that has many important roles in intracellular physiology and metabolism. One of the most important roles of GSH is to maintain the redox state in cells, protecting them against harmful effects caused by oxidative injuries. GSH synthesis is highly dependent on the availability of cysteine in the medium. Beneficial effects of cysteine in in vitro maturation of oocytes and development of embryos have been reported. Also, based on its chemical structure, vitamin C is an electron donor and therefore a reducing agent. It thus has two different biochemical roles: as an antioxidant and an enzymatic cofactor. Ascorbic acid also can regulate DNA methylation of preimplantation embryos, thereby affecting the methylation level and mRNA transcription abundance of pluripotency genes in blastocyst stage and improving the developmental quality of blastocysts which may be related to the increased activity of methylation‐related enzymes (TET3, DNMT1, and DNMT3b) after the addition of ascorbic acid

Coenzime Q10 (CoQ10), an electron carrier in mitochondria also acts as scavenger against reactive oxygen species.. The positive role of CoQ10 in ameliorating the quality of postovulatory aged oocytes, the competency, together with the fertilization capacity of aged gametes, was documented in vitro. Truong and colleagues (2017) looked at the effects of the addition of a combination of triple antioxidants (acetyl-l-Carnitine, N-acetyl-Cysteine, lipoic-acid) in the culture medium of mouse pronucleate oocytes and preimplantation embryos. L-carnitine reduces ROS levels with its antioxidant actions and acts through the regulation and transport of long chain fatty acids into mitochondria for oxidation and ATP production. As well, lipoic acid regulates mitochondrial function and ATP production and stimulates the expression of antioxidant genes involved in defense mechanisms against oxidative stress. N-acetyl cysteine acts through the up-regulation of glutathione (GSH) synthesis, protecting from oxidative stress. A beneficial effect of these combined antioxidants on embryo development has been observed, and also a positive correlation between the presence of these antioxidants and increased blastocyst cells number has been reported in past. The evidences about the efficacy of these compounds, administered alone in culture medium, in increasing the oocytes fertilization rate and in improving embryo development has also been highlighted in experimental studies which have allowed us to delineate the effects of use of antioxidants in vitro and in particular, the molecular effects of these compounds on gametes and embryos.

Singh, M.K., Saini, S., Kumar, V. and Ashutosh

National Dairy Research Institute, Karnal, Haryana-132001