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 Geno
1.1.2 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
6. CONCLUSION
7. REFERENCES
8. APPENDIX
Objectives and Topics
The research focuses on the quality assessment of semen from Norwegian Red (NRF) bulls, specifically aiming to develop an objective in vitro method for evaluating sperm quality that correlates with field fertility potential. The study investigates how stress factors, such as incubation time at 37 °C, affect sperm viability, acrosome integrity, and intracellular ATP content in both fresh and cryopreserved samples, with the ultimate goal of improving selection processes for artificial insemination.
- Evaluation of sperm viability, acrosome integrity, and ATP content under heat stress.
- Development of an ATP-based assay for sperm quality assessment.
- Correlation analysis between laboratory-measured sperm parameters and field fertility (56-day non-return rate).
- Impact of cryopreservation on sperm quality and metabolic function.
- Categorization of bulls based on ATP content adjusted for acrosome-intact live (AIL) sperm cells.
Excerpt from the Book
2.4.1 The initial semen evaluation
Immediately, the evaluation of in vitro semen quality is performed after collection (Senger, 2005). These assessments include a visual appraisal of colour, density, presence of dirt or other contaminants, measurements of ejaculate volume and sperm concentration (Al-Makhzoomi et al., 2008). The appearance of the normal ejaculate in bulls is typically a uniform near-white (Mocé and Graham, 2008). In practice the semen sample should be free of blood and flocculent material, as this is indicative of infection in reproductive tract (Mocé and Graham, 2008).
The volume of an ejaculate can be measured by pouring the semen into a graduated cylinder, graduated markings on the collection tube or by weighing the sample (Mocé and Graham, 2008). The sperm concentration means the number of sperm cells/ml of semen sample (Haugan et al., 2007). The sperm concentration can be determined by haemocytometer or a counting apparatus (Kocks and Broekhuijse, 2014). The ejaculate volume and concentration of spermatozoa are important parameters to determine the total number of sperm in the ejaculate by multiplying the volume with sperm concentration/ml. This determines the number of insemination doses to be produced from each ejaculate (Senger, 2005).
Summary of Chapters
1. BACKGROUND: Provides context on the collaboration with Geno SA and the importance of Norwegian Red (NRF) bulls in the national and international dairy industry.
2. INTRODUCTION: Details the biological characteristics of spermatozoa, including spermatogenesis, maturation, and transport, alongside standard methods for evaluating sperm quality.
3. MATERIALS AND METHODS: Describes the experimental design, including laboratory procedures for flow cytometry and luminometer-based ATP analysis, and the handling of semen samples.
4. RESULTS: Presents data on how incubation affects sperm viability and ATP levels, and identifies correlations between laboratory metrics and field fertility.
5. DISCUSSION: Interprets the findings regarding the effectiveness of ATP content as a fertility predictor and addresses the impact of heat stress on sperm quality parameters.
6. CONCLUSION: Reaffirms the finding that ATP content adjusted for AIL at 0 hours is a significant predictor of field fertility for NRF bulls.
Keywords
Sperm quality, NRF bulls, ATP content, Flow cytometry, Luminometer, Sperm viability, Acrosome integrity, Artificial insemination, Field fertility, Non-return rate (NRR), Cryopreservation, Incubation, Stress testing, Semen evaluation, In vitro
Frequently Asked Questions
What is the primary objective of this research?
The main objective is to find an objective in vitro laboratory method to evaluate bull sperm quality that reliably correlates with field fertility, specifically measured as the 56-day non-return rate (NRR).
Which specific sperm quality parameters were measured?
The study measured sperm viability, acrosome integrity, and intracellular ATP content using flow cytometry and luminometer-based assays.
How does incubation at 37 °C affect sperm samples?
Incubation at 37 °C acts as a stress factor, leading to a significant decrease in sperm viability, acrosome integrity, and a rapid depletion of intracellular ATP content over 6 and 24 hours.
What is the significance of the "ATP content adjusted for % AIL" metric?
This metric normalizes ATP levels against the percentage of acrosome-intact live (AIL) sperm, providing a more refined index for evaluating the functional quality of semen compared to measuring these parameters in isolation.
How was the correlation between lab results and field fertility determined?
The study compared laboratory-analyzed sperm traits (specifically ATP content adjusted for AIL at 0 hours) against the 56-day non-return rate (NRR) data provided by the breeding company, Geno.
What main conclusion was drawn regarding fertility prediction?
The study concludes that a significant positive correlation exists between ATP content adjusted for AIL at the time of thawing and field fertility, making it a potentially valuable tool for identifying high-fertility bulls.
What role does cryopreservation play in the findings?
Cryopreservation was shown to be a major stress factor that significantly reduces sperm quality and metabolic function compared to fresh samples, requiring careful evaluation before artificial insemination.
Why did the study categorize semen samples into groups?
Samples were categorized into low, moderate, and high quality based on their ATP/AIL ratios to provide a practical classification system for differentiating between bulls with varying fertility potentials.
- Arbeit zitieren
- Safaa Al-Medhati (Autor:in), 2015, Quality assessment of semen from Norwegian Red Bulls, München, GRIN Verlag, https://www.grin.com/document/310128
