Bovine semen has been cryopreserved since more than a half century for artificial insemination and nowadays it is being widely used all over the world. However, it is well known that the cryopreservation procedure is detrimental to sperm particularly because of chemical and physical stress factors which are occurring during this process. One important factor is oxidative stress which, in turn, affects biological membranes and DNA of sperm. Bovine sperm themselves have only few amounts of endogenous antioxidants for the protection against reactive oxygen species (ROS) and the main antioxidant source is the seminal plasma. Therefore, the development of sensitive techniques for monitoring the activity of antioxidants in seminal plasma is of clinical importance. Sensitive chemiluminescence techniques have been employed to monitor total antioxidant capacity of human seminal fluid.
Polyunsaturated fatty acids (PUFA) play an important role in regulating sperm membrane fluidity and spermatogenesis. After freezing and thawing, the portion of PUFA in sperm plasma membrane decreases significantly due to lipid peroxidation. Low portions of C20 and C22 PUFAs in sperm in old bulls were related to reductions in sperm quality and -fertilizing ability.
In various feeding experiments polyunsaturated fatty acids (PUFA) have been supplied to change the fatty acid composition of sperm membrane in order to improve sperm quality and fertility. Indeed, the fatty acid profile of sperm membranes can be modified with diet and an improvement of sperm quality was observed in a variety of livestock species including chicken, turkey, boar and stallion. However, it is possible that feeding of PUFAs reduces also the antioxidative capacity of semen which, in turn, can disturb sperm quality in the case of excessive ROS production.
The aims of this study are: (i) to determine total antioxidant capacity of bovine seminal plasma and its relationship with other antioxidants and sperm quality; (ii) to ascertain whether feeding omega-3-fatty acid reduces the antioxidative status of seminal plasma.
Contents
1 INTRODUCTION
2 REVIEW OF LITERATURE
2.1 Reactive Oxygen Species
2.1.1 Definition and existence of reactive oxygen species
2.1.2 Oxidative stress
2.1.3 Significance of oxidative stress in male reproduction
2.1.3.1 Sources of ROS in semen
2.1.3.2 Targets and pathological role of ROS in semen
Lipids of sperm plasma membrane and lipid peroxidation
Damage of DNA
Damage of proteins
Apoptosis
2.1.3.3 Physiological role of ROS in semen
2.2 Antioxidants
2.2.1 Enzymatic antioxidants
2.2.1.1 Superoxide Dismutase
2.2.1.2 Glutathione Peroxidase
2.2.1.3 Catalase
2.2.2 Non-enzymatic antioxidants
2.3 Seminal plasma and its antioxidative significance for male reproduction
2.3.1 Accessory glands and seminal plasma
2.3.2 Antioxidative properties of seminal plasma
2.4 Measurement of oxidative stress and antioxidants using chemiluminescence
3 OWN EXPERIMENTAL STUDIES
3.1 Establishment of a new assay for the determination of total antioxidative capacity of bovine seminal plasma
3.1.1 Abstract
3.1.2 Introduction
3.1.3 Materials and Methods
3.1.3.1 Chemicals
3.1.3.2 Semen collection, dilution and freezing
3.1.3.3 Handling of seminal plasma
3.1.3.4 Antioxidant assays
Total Antioxidant Capacity
Instrumentation and automated measurement of TAC SOD and GPx
Superoxide Dismutase (SOD) Assay
Glutathione Peroxidase (GPx) Assay
Determination of protein concentrations
Measurements of intra- and inter-assay Variations
3.1.3.5 Flow cytometric analyses
Plasma Membrane Integrity and Acrosomal Integrity
Lipid Peroxidation (LPO)
Sperm Chromatin Structure Assay
3.1.3.6 Statistical analysis
3.1.4 Results
3.1.4.1 Reproducibility of TAC-, SOD- and GPx assays
3.1.4.2 TAC, SOD and GPx levels in seminal plasma
3.1.4.3 Volume and sperm concentration of ejaculates
3.1.4.4 Sperm quality
3.1.4.5 Relationships between antioxidant levels and between antioxidant levels and sperm quality
3.1.5 Discussion
3.2 Effects of feeding omega-3-fatty acids on sperm quality of Holstein Friesian bulls before and after cryopreservation: Effects on seminal plasma
3.2.1 Abstract
3.2.2 Introduction
3.2.3 Materials and Methods
3.2.3.1 Bulls
3.2.3.2 Dietary supplementation of bulls
3.2.3.3 Semen collection, dilution and freezing
3.2.3.4 Handling of seminal plasma and measurement of TAC, GPx and SOD
3.2.3.5 Flow cytometric analyses
Plasma Membrane Integrity and Acrosomal Integrity
Sperm Chromatin Structure Assay
3.2.3.6 Fatty acid extraction and analysis
3.2.3.7 Statistical analysis
3.2.4 Results
3.2.4.1 Fatty acid analysis
3.2.4.2 Dietary effects on sperm quality and fatty acid composition
3.2.4.3 Effect of feeding ALA on antioxidant levels of seminal plasma
3.2.4.4 Correlation between fatty acids and antioxidants
3.2.5 Discussion
Research Objectives and Topics
The primary aim of this work is to establish a reliable assay for the determination of the total antioxidative capacity (TAC) of bovine seminal plasma and to investigate its relationship with other antioxidants and sperm quality parameters. Furthermore, the study explores the influence of dietary supplementation with omega-3 fatty acids (alpha-linolenic acid) on the antioxidative status of seminal plasma and the subsequent effects on sperm quality before and after cryopreservation.
- Total antioxidant capacity (TAC) of bovine seminal plasma
- Oxidative stress and antioxidant defense mechanisms in sperm
- Effects of omega-3 fatty acid supplementation on bull semen
- Standardization of assay methods for antioxidant enzyme activities
- Correlation between fatty acid composition and antioxidant levels
Excerpt from the book
2.1.2 Oxidative stress
All cells need O2 to produce efficient energy in mitochondria. The mitochondrial electron transport system consumes more than 85 % of all oxygen consumed by the cell. On the other hand, oxygen consumption generates by-products called ROS, because 1-3% of electrons escape from the chain of that transport system and univalent reduction of molecular oxygen results in superoxide formation (HALLIWELL 1990; HALLIWELL and CROSS 1994; HALLIWELL 2006).
Aerobic cells are normally exposed to ROS but they can survive under toxic conditions of oxygen because they have protector molecules against this oxygen toxicity, the antioxidants (HALLIWELL 2006). Normally, continuous production of ROS and activated oxygen species in the body is controlled tightly by antioxdants. If this sensitive equilibrium between oxidants and antioxidants is disordered, oxidative stress occurs which, in turn, increases the rates of cellular damage. SIES (1991; 1993) defined the oxidative stress as a disturbance in prooxidant-antioxidant balance in favor for prooxidant.
Summary of Chapters
1 INTRODUCTION: This chapter outlines the importance of cryopreservation in bovine semen and introduces the problem of oxidative stress, highlighting the role of seminal plasma antioxidants and the impact of polyunsaturated fatty acids.
2 REVIEW OF LITERATURE: This section provides a comprehensive overview of reactive oxygen species, the mechanisms of oxidative stress in male reproduction, the function of various antioxidants, and current methods for measuring oxidative stress in semen.
3 OWN EXPERIMENTAL STUDIES: This chapter details the establishment of a new assay for measuring total antioxidative capacity in bovine seminal plasma and investigates the impact of omega-3 fatty acid supplementation on bull sperm quality and seminal antioxidant levels.
Keywords
Bovine semen, cryopreservation, oxidative stress, total antioxidant capacity (TAC), seminal plasma, omega-3 fatty acids, alpha-linolenic acid, lipid peroxidation, glutathione peroxidase, superoxide dismutase, sperm motility, sperm quality, reactive oxygen species, flow cytometry, fertility
Frequently Asked Questions
What is the primary focus of this dissertation?
The dissertation focuses on investigating the antioxidative capacity of bovine seminal plasma, specifically establishing a reliable assay to measure total antioxidant capacity (TAC) and examining how it relates to sperm quality and the effects of feeding omega-3 fatty acids to bulls.
What are the key thematic areas addressed in this work?
The work covers reactive oxygen species (ROS), the role of antioxidant defense systems in semen, lipid peroxidation, the impact of fatty acid composition on sperm membranes, and the influence of dietary supplementation on semen quality after cryopreservation.
What is the main research question or goal?
The goal is to determine the total antioxidant capacity of bovine seminal plasma and its relationship with other antioxidants and sperm quality, while also determining if feeding omega-3 fatty acids reduces the antioxidative status of the seminal plasma.
Which scientific methods were employed?
The study utilized automated chemiluminescence assays for TAC, enzyme activity assays (GPx, SOD), flow cytometric analysis (using FITC-PNA, PI, and C11-BODIPY) to evaluate membrane integrity and lipid peroxidation, and gas chromatography for fatty acid analysis.
What does the main part of the work cover?
The main part covers two experimental studies: the establishment of an automated assay for TAC and antioxidant enzymes (GPx, SOD), and a feeding study to determine the effects of dietary alpha-linolenic acid (ALA) versus palmitic acid (PA) on semen quality.
Which keywords characterize this work?
Key terms include bovine semen, cryopreservation, TAC, seminal plasma, omega-3 fatty acids, antioxidant enzymes, lipid peroxidation, and sperm quality.
Does the study conclude that omega-3 supplementation affects seminal plasma antioxidants?
No, the study concluded that the feeding of neither saturated nor polyunsaturated fatty acids affected the antioxidant levels in bovine seminal plasma, though both types of fatty acids had positive effects on sperm quality after cryopreservation.
What role does the seminal plasma play in sperm protection?
The seminal plasma acts as the primary source of antioxidants for sperm, as sperm cells themselves have limited cytoplasmic space for endogenous antioxidant enzymes, making the seminal environment crucial for protecting against oxidative damage.
- Quote paper
- Oğuz Çalışıcı (Author), 2010, Investigation of antioxidative capacity in bovine seminal plasma. Effects of Omega-3 fatty acids, Munich, GRIN Verlag, https://www.grin.com/document/385556
