In the present study semen samples from NRF bulls were analysed for sperm viability, acrosome integrity and ATP content. The effect of incubation at 37 °C on sperm viability, acrosome integrity and ATP content was investigated on fresh and frozen semen samples from 20 NRF. Semen was incubated at 37 °C and the sperm quality parameters were analysed at 0 hr and after 6 and 24 hrs of incubation for both fresh and frozen samples. Sperm viability and acrosome integrity was assessed with flow cytometry and ATP content with luminometer.
The incubation time at 37 °C had a significant effect on all studied parameters. However, ATP content was significantly more affected than sperm viability and acrosome integrity. After 24 hrs of incubation ATP content of frozen samples decreased approximately to 0 for all tested samples, while percentage of viability and acrosome integrity decreased by 78 ± 11.9 %. Percentage of AIL sperm cells of frozen samples decreased at corresponding rate as in fresh samples. Concerning ATP content there was however, a more marked decline in frozen samples compared to fresh samples during incubation.
Sperm samples from eight NRF bulls with high and low fertility were also analysed for sperm viability, acrosome integrity and ATP content at 0 hr (right after thawing), 3, 6 and 24 hrs incubation at 37 °C. The ATP content adjusted for % AIL at 0 hr was significantly correlated with fertility measured as 56 days NRR, while % AIL or ATP content analysed separately were not correlated to field fertility.
Table of Contents
- 1. BACKGROUND
- 1.1 ORIGINS OF THE PROJECT
- 1.1.1 Norwegian breeding program of NRF
- 2. INTRODUCTION
- 2.1 SPERMATOZOA
- 2.1.1 Spermatozoal structure and function
- 2.1.2 Spermatogenesis
- 2.2 SPERM MATURATION
- 2.3 SPERM TRANSPORT IN THE FEMALE REPRODUCTIVE TRACT
- 2.3.1 Capacitation and acrosome reaction
- 2.3.2 ATP production
- 2.4 EVALUATION OF SPERM QUALITY
- 2.4.1 The initial semen evaluation
- 2.4.2 Sperm motility
- 2.4.3 Sperm viability
- 2.4.4 Acrosome integrity
- 2.4.5 ATP Assay
- 2.5 TECHNIQUES USED FOR SPERM ASSESSMENT
- 2.5.1 Flow cytometry
- 2.5.2 Luminometer
- 2.6 THE AIM OF THE STUDY
- 3. MATERIALS AND METHODS
- 3.1 EXPERIMENTAL PLAN
- 3.1.1 Analysis of selected parameters of fresh and frozen samples from 20 NRF bulls
- 3.1.2 Analysis of selected quality parameters of frozen samples from eight NRF bulls with known fertility
- 3.1.3 Analysis of selected quality parameters of fresh and frozen samples from NRF bulls with known proportion percentage live-dead cells
- 3.2 CHEMICALS
- 3.3 SEMEN SAMPLES
- 3.4 PREPARATION OF SPERM SAMPLES
- 3.4.1 Flow cytometry and luminometer instrumentation
- 3.5 ANALYSIS OF SPERM QUALITY PARAMETERS BY FLOW CYTOMETRY
- 3.5.1 Viability and acrosome integrity
- 3.6 MEASUREMENT OF ATP CONTENT OF BULL SPERMATOZOA USING LUCIFERIN-LUCIFERASE ASS
- 3.6.1 Optimization of a protocol for measurement of ATP content in bull sperm cells by luminometer
- 3.6.2 ATP standard curve preparation
- 3.6.3 Measurements the ATP content of spermatozoa in fresh and frozen semen
- 3.7 FURTHER OPTIMIZING OF THE PROTOCOL FOR ANALYSIS OF SPERM VIABILITY AND ATP CONTENT WITH KNOWN PROPORTION PERCENTAGE OF LIVE-DEAD SPERM CELLS
- 3.8 STATISTICAL ANALYSIS
- 4. RESULTS
- 4.1 ANALYSIS OF SPERM QUALITY PARAMETERS
- 4.1.1 Sperm viability, acrosome integrity and ATP content of fresh and frozen samples
- 4.1.2 Comparison of decrease of % AIL and ATP content under different incubation times
- 4.2 ATP CONTENT ADJUSTED FOR % AIL
- 4.2.1 Categorization of semen samples based on ATP content adjusted for % AIL
- 4.3 VIABILITY, ACROSOME INTEGRITY AND ATP CONTENT TESTED FOR CORRELATION WITH 56 DAYS NRR
- 4.4 ANALYSIS OF FRESH AND FROZEN SAMPLES WITH KNOWN PROPORTION PERCENTAGE LIVE-DEAD SPERM CELLS (CONTROL EXPERIMENT)
- 5. DISCUSSION
- 5.1 FURTHER STUDY
Objectives and Key Themes
The main objective of this study was to assess the quality of semen from Norwegian Red bulls using stress testing in vitro. This involved evaluating sperm quality in both fresh and frozen samples, focusing on parameters such as viability, acrosome integrity, and ATP content. The study aimed to optimize protocols for these assessments and explore correlations with fertility.
- Evaluation of sperm quality in fresh and frozen bull semen
- Optimization of in vitro stress testing protocols
- Correlation analysis of sperm quality parameters with fertility
- Assessment of ATP content as an indicator of sperm quality
- Application of flow cytometry and luminometry techniques
Chapter Summaries
1. BACKGROUND: This chapter provides background information on the origins of the project, focusing on the Norwegian Red (NRF) bull breeding program and its importance within the Norwegian dairy industry. It likely sets the stage for the subsequent chapters by establishing the context and rationale for evaluating NRF bull semen quality.
2. INTRODUCTION: This introductory chapter provides a comprehensive overview of spermatozoa, including their structure, function, maturation, and transport within the female reproductive tract. It delves into the processes of capacitation and the acrosome reaction, essential for fertilization, and explains the importance of ATP production for sperm motility and viability. The chapter also details various techniques for evaluating sperm quality, such as assessments of motility, viability, acrosome integrity, and ATP content, laying the groundwork for the methodology presented later.
3. MATERIALS AND METHODS: This chapter meticulously describes the experimental design, including the different groups of bull semen samples used (fresh, frozen, and those with known fertility and live/dead cell proportions). It outlines the specific chemicals and instruments used, providing detailed protocols for semen preparation, flow cytometry analysis (for viability and acrosome integrity), and luminometry-based ATP assays. The chapter further explains the statistical analysis methods employed to interpret the results.
4. RESULTS: This chapter presents the findings of the study. It details the results regarding sperm viability, acrosome integrity, and ATP content in both fresh and frozen samples, as well as the results of the correlation analysis between these parameters and the non-return rate (NRR), a measure of fertility. Specific details are provided on how ATP content changed over different incubation times and how samples were categorized based on ATP content adjusted for acrosome integrity loss. The results of the control experiment (using samples with known live/dead cell percentages) are also presented.
5. DISCUSSION: This chapter likely discusses the implications of the findings, comparing the results with existing literature and exploring potential reasons for observed trends. The limitations of the study and suggestions for future research directions are also probable points of focus in this chapter.
Keywords
Norwegian Red bulls, semen quality, sperm viability, acrosome integrity, ATP content, flow cytometry, luminometry, in vitro stress test, fertility, non-return rate (NRR), artificial insemination.
Frequently Asked Questions about the Norwegian Red Bull Semen Quality Study
What is the main objective of this study?
The primary goal is to comprehensively assess the semen quality of Norwegian Red (NRF) bulls using in vitro stress testing. This involves evaluating various sperm quality parameters in both fresh and frozen samples, with a focus on viability, acrosome integrity, and ATP content. The study aims to optimize assessment protocols and explore correlations between these parameters and fertility.
What sperm quality parameters were analyzed?
The study analyzed several key sperm quality parameters, including viability (live/dead cells), acrosome integrity (intactness of the acrosome, a structure crucial for fertilization), and ATP content (a measure of sperm energy levels). These parameters were assessed in both fresh and frozen semen samples.
What methods were used to assess sperm quality?
Two primary methods were employed: Flow cytometry, used to determine sperm viability and acrosome integrity; and Luminometry, used to measure ATP content in the sperm cells. Detailed protocols for both techniques were developed and optimized as part of the study.
What types of semen samples were used in the study?
The study utilized various semen samples: fresh samples, frozen samples, and samples from bulls with known fertility rates and a known proportion of live and dead sperm cells (used as controls). Samples from 20 NRF bulls were used in the main analysis, and a subset of 8 bulls with known fertility was also assessed.
How was the ATP content measured?
ATP content was measured using a luminometer and a luciferin-luciferase assay. The study included a detailed optimization of the protocol for this measurement, including the preparation of an ATP standard curve for accurate quantification.
What is the significance of ATP content in this study?
ATP (adenosine triphosphate) is the primary energy source for sperm motility and function. Measuring ATP content provides insight into the metabolic activity and overall health of the sperm cells, and serves as an additional indicator of semen quality alongside viability and acrosome integrity.
What statistical analyses were performed?
The study employed appropriate statistical methods to analyze the data, enabling comparisons between different sample groups and the determination of correlations between sperm quality parameters and fertility (measured by the non-return rate, or NRR).
What was the relationship between the assessed parameters and fertility?
The study investigated correlations between sperm viability, acrosome integrity, and ATP content with the 56-day non-return rate (NRR), a common indicator of bull fertility. The results of this correlation analysis are detailed in the results section of the report.
What are the key findings of the study?
The study's results are presented in detail in Chapter 4, and include comparisons of sperm quality parameters in fresh and frozen samples, analysis of ATP content changes over incubation time, and categorization of semen samples based on ATP content adjusted for acrosome integrity loss. The control experiment using samples with known live/dead ratios further validated the methodology.
What are the implications of the study and what future research is suggested?
The discussion section (Chapter 5) likely interprets the results in the context of existing literature, discusses limitations of the study, and suggests directions for future research. These suggestions may include further investigation of specific correlations or exploration of additional sperm quality parameters.
What are the key words associated with this study?
Key words include: Norwegian Red bulls, semen quality, sperm viability, acrosome integrity, ATP content, flow cytometry, luminometry, in vitro stress test, fertility, non-return rate (NRR), artificial insemination.
- Quote paper
- Safaa Al-Medhati (Author), 2015, Quality assessment of semen from Norwegian Red Bulls, Munich, GRIN Verlag, https://www.grin.com/document/310128
