Chapter 74 Patrick Vincent1, Shelley L. Underwood1, Catherine Dolbec1, Nadine Bouchard1, Tom Kroetsch2, Patrick Blondin1 1 L’Alliance Boviteq Inc., St-Hyacinthe, Quebec, Canada 2 The Semex Alliance, Guelph, Ontario, Canada Fertility is a multiparametric phenomenon that relies on the use of semen of sufficient quality and quantity, accurate timing and method of insemination, and appropriate herd management. When using artificial insemination (AI), the dairy producer must manage a range of these factors, including heat detection, timing of insemination in relation to estrus, and correct handling of the frozen straws. However, it is the onus of the semen production centers (SPCs) to supply straws containing spermatozoa of good viability that produce acceptable conception rates if all other variables are managed correctly. To ensure acceptable fertility after AI, frozen-thawed spermatozoa must be present in sufficient number in each straw (concentration), and possess a number of characteristics important for fertilization. Accordingly, spermatozoa must survive the thawing procedure with normal morphology, an intact acrosome, DNA integrity, active mitochondria, and maintain forward progressive motility to traverse the female reproductive tract. Some or many of these characteristics are measured during post-thaw quality control procedures undertaken by SPCs prior to distribution. Quality control (QC) is the assurance that each batch of straws has undergone semen analysis to verify that the sample is likely to be fertile. Although semen analysis may seem easy to perform, meticulous attention to detail and technique is essential in order to obtain an accurate and reproducible analysis. Manual semen analysis using a light microscope has been the standard method for analysis in most SPCs. However, manual analyses can be very subjective and prone to within- and between-technician errors. Similarly, the use of fluorescence microscopy to assess spermatozoa for acrosome, membrane, and DNA integrity is markedly slow and limited due to the low number of spermatozoa analyzed from each sample and the incapacity for an extensive multiparametric analysis. To maximize accuracy in QC, SPCs are realizing the benefit of a multiparametric approach and have increased the rigor of their semen testing, moving from time-consuming basic subjective assessment of a few hundred spermatozoa for concentration, motility and morphology using microscopy, to the use of computer-assisted tracking to assess motility, and flow cytometry to analyze thousands of cells within seconds for characteristics such as viability, mitochondrial activity, acrosome, DNA and capacitation status. The topics for discussion in this review are the various tools and assays in use in cattle SPCs to determine QC values, factors to consider when using these tools, and how the efficacy of QC procedures may be maximized in order to predict field fertility. Computer-assisted sperm analysis (CASA) is a powerful tool for the objective assessment of sperm motility and is hence now frequently used for evaluating semen quality. The basic components of this technology consist of a microscope to visualize the sample, a digital camera to capture images, and a computer with specialized software to analyze the movement of the spermatozoa. The essential principle behind most microscopy-based CASA systems is that a series of successive images of motile spermatozoa within a static field of view are acquired by computer software algorithms, which then scan these image sequences to identify individual spermatozoa and trace their progression across the field of view. This involves recognizing the same cell in each image by its position, and inferring its next position by estimating the likelihood that it will only have moved a certain maximum distance between frames. CASA can also provide information about sperm concentration, morphology, viability, and index of DNA fragmentation of frozen-thawed sperm. However, these more specialized techniques are not routinely applied for regular analysis of frozen-thawed semen in SPCs. With the use of CASA, several motility parameters describing the specific movements of spermatozoa can be obtained in greater detail than possible in subjective assessment. These computerized measurements can be useful for assessing various sperm characteristics simultaneously and objectively, and are valuable for the detection of subtle changes in sperm motion that cannot be identified by conventional subjective semen analysis as reviewed elsewhere.1–3 The parameters typically collected using CASA systems are motility, velocity, linearity, and lateral displacement of spermatozoa as they progress along their trajectories in a sample. The percentages of total and progressive motility are the most important motility parameters in the evaluation of spermatozoa. Total motility refers to the fraction of spermatozoa that display any type of movement, whereas progressively motile spermatozoa swim forward in an essentially straight line. Spermatozoa that swim but in an abnormal way, such as in tight circles, are not included in the proportion of progressively motile sperm. In addition to evaluation of sperm motility, the software calculates the kinetic values of each spermatozoon, which covers the velocity of movement, the width of the sperm head’s trajectory, and frequency of the change in direction of the sperm head. The velocity values that are determined by CASA are the curvilinear velocity, straight-line velocity, and average path velocity.2 The amplitude of lateral head displacement and beat cross frequency are two other characteristics measured with CASA instruments.2 Despite the power of an objective evaluation by CASA, there are some constraints associated with this technology. Many factors are known to affect CASA results. The type of specimen chamber used for analysis can affect the movement of sperm, the accuracy of the cell count number, and therefore the percentage of motile spermatozoa.4 The temperature at which semen is analyzed is also an important factor that may affect CASA results. Independent studies showed that analyzing semen below 37°C significantly affected results.5,6 These groups performed CASA on spermatozoa maintained at 37°C with a stage warmer and compared the results with spermatozoa analyzed at room temperature or at 30°C. The data demonstrated a decrease in the motility parameters (percentage of motile spermatozoa and track speed) when spermatozoa were not analyzed at 37°C. These experiments suggest that a simple variation introduced in the analysis of sperm motility can have a considerable effect on the results. The concentration at which semen is analyzed is an essential aspect that influences CASA results. It has been established that at low semen concentrations (<20 million/mL), an overestimation of the concentration and thus an underestimation of the percentage of motile cells can occur due to the acquisition of nonspermatic particles (debris). On the other hand, at higher concentrations (>50 million/mL), a large proportion of the fast-moving cells will be excluded from analysis because of cell collisions, spermatozoa exiting the analysis area, or excluded on the basis of nearest-neighbor effects, leading to an underestimation of the motility.5,7 Sampling condition is a source of error when acquiring data with CASA. Computer and video camera equipment are continuously evolving and different CASA systems use various models of video camera. Most of the CASA systems allow 30 or 60 Hz as a frame rate to analyze sperm tracks and speed. Studies have shown the importance of the frame rate for reliability of the analysis.8–10 It is generally accepted that a higher frame rate is required to render an evaluation closer to the real path for a fast nonlinear sperm cell. To study a hyperactivated sperm cell, Mortimer and Swan11 suggested using the highest frame rate available on the system in order to have the most accurate evaluation. The type of extender in which semen is diluted is another aspect that should be taken into consideration when evaluating spermatozoa with CASA. Some extenders contain debris of a size similar to a sperm head, causing CASA software to include them in the analysis. Egg yolk- and milk-based diluents are examples of extenders containing such particles. In addition, when observing semen diluted with milk extender, the globular lipids mask the spermatozoa thus rendering CASA analysis impossible. To assess motility analysis in these conditions, samples could be washed to remove extender debris from semen. However, it has been established that washing the semen affects the motility of the spermatozoa,12 making correct evaluation more difficult. To overcome this problem, fluorescence technology allows discrimination of sperm cells from particles in the extender by staining sperm heads with a DNA-binding fluorochrome. Under fluorescent light, only DNA-containing objects will be detected by the CASA software, thus omitting the need for washes. This technique improves the accuracy of the concentration13 as well as the motility analysis14 when working with semen diluted in these extenders. Therefore, standardizing the type of chamber, the temperature, the concentration, and the type of extender is crucial to assure repeatable standard QC at an SPC. Motility is one of the most important characteristics believed to be associated with the fertilizing ability of spermatozoa. Several groups have reported a significant correlation between total15–18 and progressive19 motility of bull semen and its associated field fertility. However other groups have reported that the subjective analysis of semen motility did not correlate with fertility.20,21 CASA instruments collect a wide range of sperm motility parameters, allowing a more detailed and accurate analysis of sperm movements and track speed. Researchers have also tried to correlate the kinetic parameters with the field fertility of semen, with some groups able to show a positive correlation between straight-line velocity of spermatozoa and field fertility.18–22 Another study used a combination of several motility parameters to reach a very high correlation with bull fertility.20 Taken together, these studies show the high potential of CASA for estimating the quality of the semen and therefore becoming a powerful tool to measure sperm characteristics and predict bull fertility compared with standard semen evaluation. However, as mentioned above, standardization of conditions and parameters of all CASA analyses are key to obtain repeatable and valid correlations with fertility. Several models of CASA instruments are now available to evaluate the quality and motility of spermatozoa. Each system operates on similar principles but they differ in their parameter settings and use different algorithms to determine speed and trajectories. Parameter settings, threshold settings, video frame rate, and other variables will affect CASA results as reviewed by Davis and Katz.23 As mentioned above, new technologies and CASA software evolve quickly. Our laboratory undertook a small study to measure the aptitude of the CEROS (Hamilton-Thorne, USA) and the Sperm Class Analyzer (SCA; Microptics, Spain) in evaluating the motility and the concentration of frozen-thawed bovine spermatozoa diluted in an egg yolk-based extender (unpublished data). A total of 18 different frozen-thawed ejaculates were analyzed with both systems and the mean total and progressive motility percentages, concentration, average path velocity, curvilinear velocity, straight-line velocity, beat cross frequency, and amplitude of lateral head displacement were compared (Figure 74.1). Among all parameters analyzed, only the percentage of total motile cells was not significantly different between the systems. The discrepancies can be explained by the better capacity of the SCA to exclude egg yolk particles from the analysis. The SCA discriminated nonspermatic particles based on size in microns while the CEROS used pixels to estimate the size of the cells. Differences in the algorithms used to calculate slow, medium, and fast spermatozoa may also explain the variation in the motility and kinetic parameters observed between each system. Overall, this mini-study indicates high variability between CEROS and SCA systems in estimating sperm motility parameters. Figure 74.1 Comparison of CEROS (Hamilton-Thorne) and SCA (Sperm Class Analyzer) for determining concentration, percentage of total and progressive motility, lateral head displacement (ALH), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and beat cross frequency (BCF) from 18 different frozen-thawed bovine ejaculates. Columns represent mean values ± SEM. P-value <0.05 indicates a statistically significant difference between CEROS and SCA, Student’s paired t-test. Flow cytometry analyzes cells suspended in a stream of fluid passing at high velocity in front of one or several lasers. The light emitted by fluorochrome-bound cells is captured by photomultiplier tubes and converted into an electronic signal subsequently digitalized by cytometry software. Key features of flow cytometry are the acquisition and analysis of thousands of cells within seconds and the multiparametric potential of the technology. The most modern cytometers are routinely equipped with three lasers and at least 10 photomultiplier tubes, allowing cell labeling with several probes at the same time and thus enabling analysis of numerous parameters simultaneously. In the last few years, the multiparametric aspect of flow cytometry has allowed this technology to become a popular tool for evaluating sperm attributes.24–26 A wide range of fluorochromes has been developed to assess numerous characteristics of sperm cells. Here we review some of the fluorochromes used to study sperm cells with flow cytometry. Propidium iodide is the most popular dye used to identify dead cells. This membrane-permeable fluorochrome enters spermatozoa with damaged cellular membranes and binds to DNA where it can be excited with a 488-nm laser present on most cytometers.27–29 Propidium iodide is often used in combination with SYBR-14, another DNA-labeling probe.30,31 SYBR-14 is also excited by the 488-nm laser and is a permeant probe staining all cells. Added to the cells simultaneously, propidium iodide displaces or quenches the SYBR-14 fluorescence in damaged cells. A new fixable dye commercialized by Invitrogen under the name Live/Dead® fixable dead cell kit is now available to evaluate the viability of cells.32 This dye reacts with cellular amines on the surface of cells or inside the cytoplasm of cells with damaged membranes. Cell-surface staining of amines of viable cells will result in relatively dim staining compared with the bright staining of dead cells. This fixable dye belongs to a large family available in different wavelengths of excitation/emission, allowing its use on most cytometers. Evaluation of acrosomal status is mainly assessed by using plant lectins recognizing acrosomal ligands. Pisum sativum agglutinin binds mannose and galactose moieties of the acrosomal matrix. As Pisum sativum agglutinin cannot penetrate the intact acrosomal membrane, only spermatozoa with a reacted or damaged acrosome will be stained.21,33,34 However, it has been shown that Pisum sativum agglutinin has an affinity for egg yolk and nonspecific binding sites on the sperm cell surface.35,36 This aspect could become a problem when analyzing semen frozen in egg yolk-based extender and result in misinterpretation of the acrosomal status of sperm. Arachis hypogaea (peanut) agglutinin binds galactose moieties of the outer acrosome membrane and is the most popular lectin used to study the integrity of the acrosomal membrane with flow cytometry.37–39 In addition, Arachis hypogaea agglutinin seems the most reliable probe for identifying spermatozoa with a damaged acrosome as it displays less nonspecific binding to other areas of spermatozoa.40 Pisum sativum agglutinin and Arachis hypogaea agglutinin are usually labeled with FITC fluorochromes, allowing them to be used by all cytometers. Mitochondria are very important organelles involved primarily in the generation of the energetic substrates for the motility of the sperm cell. Rhodamine 123 was one of the first probes to monitor mitochondrial activity.41,42 Rhodamine 123 is sequestered in active mitochondria and washed out from the cell when the membrane potential is lost. This characteristic limits its use when quantification is needed or when fixation of spermatozoa is required. To overcome the fixation problem, Mitotracker® dye could become a solution. This fixable dye accumulates and stains active mitochondria and has the advantage of availability in different ranges of excitation and emission fluorescence.42–44 The most popular probe for evaluating mitochondrial activity is JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide).45–47 In spermatozoa containing mitochondria with a high membrane potential, JC-1 enters the mitochondrial matrix where it accumulates and forms J-aggregates and become fluorescent red. In spermatozoa containing mitochondria with low membrane potential, JC-1 cannot accumulate within the mitochondria and remains in the cytoplasm in a green fluorescent monomeric form. JC-1 has the advantage of being able to quantify the mitochondrial burst of the cell compared with Rhodamine 123 and Mitotracker. A disadvantage of the JC-1 probe is its dual fluorescence emission that limits its combination with other probes emitting at the green and red wavelngths. Assessment of chromatin status is important in determination of the fertility potential of spermatozoa. In recent years, the sperm chromatin structure assay developed by Evenson and Jost48 is the main technique used to evaluate chromatin integrity in spermatozoa by flow cytometry.49,50 The sperm chromatin structure assay uses the dual fluorescence emission of acridine orange depending on whether it binds to single-stranded DNA (red fluorescence) or double-stranded DNA (green fluorescence). Following a denaturation step, the sperm sample is incubated with acridine orange and then analyzed by flow cytometry. Denaturation will induce single-strand DNA formation when DNA breaks are present and generate a heterogeneous population of red and green fluorescence depending on the integrity of the chromatin. The most important result derived from the sperm chromatin structure assay is the ratio of red/green plus red fluorescence called the DNA fragmentation index, where a high DNA fragmentation index correlates with high DNA damage. The DNA fragmentation index has shown correlation with fertility in different species.51–53 The large luminal spectrum covered by acridine orange and the denaturation step required to induce single-strand DNA are two main inconveniences of the sperm chromatin structure assay for a multiparametric analysis. Acridine orange fluoresces in the green and red spectrum; that leaves few possibilities for adding other fluorochromes in these spectral areas and the denaturation step is performed with an acid/detergent solution not compatible with all probes. Another assay to assess DNA integrity developed for flow cytometry is the TUNEL assay (terminal transferase dUTP nick end labeling), which can identify DNA strand breaks.54–56
Bovine Semen Quality Control in Artificial Insemination Centers
Introduction
Objective assessment of sperm motility
Limitations of CASA instruments
Analysis of sperm function by flow cytometry
Sperm attributes analyzed by flow cytometry
Viability/mortality
Acrosome integrity
Mitochondrial activity
DNA integrity
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