2009: Advances in canine semen evaluation techniques Advances in canine semen evaluation techniques 1 C. Lopate 2 Reproductive Revolutions, Inc, 1000 Wilsonville Rd #55, Newberg, OR, USA 3 Abstract 4 Veterinarians are frequently asked to evaluate dog semen for a variety of reasons, 5 including but not limited to: breeding soundness examination, shipment of fresh, cooled semen, 6 cryopreservation; after a conception failure; after an illness suspect to affect fertility; or after 7 testicular neoplasia or prostatic disease is diagnosed. Ultimately, the goal of any semen 8 evaluation is to predict how likely it is for a male to be successful when used in a breeding 9 situation. Standard semen evaluation in the dog includes determination of semen volume, 10 spermatozoal motility (both total and progressive), velocity, concentration, total 11 spermatozoa/ejaculate, and morphology. Semen morphology during a typical breeding 12 soundness examination is typically performed using one of two stains: Wright-Giemsa or eosin-13 nigrosin. In the dog, standards for normal semen parameters include a semen concentration of 14 > 10 million spermatozoa/kg bodyweight; >70% progressively motile sperm, and >70% 15 morphologically normal spermatozoa. Many times, semen quality will exceed these parameters 16 yet fertility of the dog may still be suboptimal. If infection and prostatic disease can be ruled out 17 as causes of the subfertility, the clinician is left with attempting to further evaluate the ejaculate 18 to determine the cause of the infertility. Evaluation of the sperm’s function is the next logical 19 step. 20 Male factor infertility is said to account for up to 50% of the failed pregnancy attempts in 21 humans.1 No estimates have been made for the canine, but clinical experience would tell us 22 that male factor infertility accounts for a significant portion of either non-pregnancy, early 23 embryonic death, or small litter size. When a standard semen evaluation fails to elicit a clear 24 diagnosis of infertility, additional testing of the ejaculate is desirable. This testing may include 25 additional bright field spermatozoal staining techniques which differentiate different parts of the 26 169 sperm cell; alternate microscopic evaluation of sperm morphology using differential interference 27 contrast or phase contrast morphology; electron microscopy (EM), both scanning and 28 transmission; acrosomal testing; hypo-osmotic swelling testing (HOST); sperm chromatin 29 structure analysis (SCSA); computer assisted spermatozoal analysis (CASA/ASMA); fluorescent 30 antibody staining techniques using flow cytometric analysis; anti-sperm antibody (ASA) testing; 31 assays for reactive oxygen species (ROS); chromosomal studies; and sperm function testing 32 including zona binding assays, sperm penetration assays. 33 While there is some information on the canine in this regard, much of the information 34 presented will be from human, bovine, and other domestic species research, where this topic 35 has been far more extensively evaluated. The data from other species can be extrapolated to 36 the canine although more research is needed to determine if its use is appropriate in the dog as 37 a predictor of fertility. This paper describes the use of these additional diagnostic methods of 38 spermatozoal analysis for infertility assessment. CASA will only be covered briefly as another 39 paper in this symposium is dedicated to its use. 40 Keywords: Canine, semen evaluation, male infertility 41 42 Introduction 43 In the human andrology laboratory setting strict criteria have been developed and 44 accepted by the World Health Organization (WHO) for the evaluation of sperm morphology.1-8 A 45 normal human sperm has a specified size and shape, with a smooth outline, an acrosome that 46 comprises between 40 and 70% of the sperm head, has no neck, midpiece or tail defects, and 47 has no droplets more than ½ the size of the sperm’ s head. Use of these strict criteria makes 48 morphologic examination of semen more uniform and allows for easier comparison of research 49 studies. Thus far, no such criteria have been adapted to the domestic species of animals, 50 making semen evaluation less uniform and comparison of infertility studies and application of 51 treatments more difficult. 52 170 Bright field microscopy 53 Bright field microscopy is one of the simplest forms of semen evaluation since every 54 veterinary clinic possesses the equipment to perform the evaluation. There are many different 55 stains which are available for morphologic assessment of spermatozoa. Commonly used stains 56 include eosin-nigrosin (EN), modified Wright-Giemsa, Feulgen, India ink, and Spermac. Each 57 stain has advantages and disadvantages and will be discussed individually. 58 EN stain has been a conventional stain used for semen morphology in domestic and 59 non-domestic animals for many years.8,9 Slides stained with EN have a dark background and a 60 white or pink staining spermatozoon. The equatorial ridge can be clearly seen and some 61 defects in the acrosomal cap and sperm head may be identified including knobbed or ruffled 62 acrosomes, diadem defects, and nuclear vacuoles. The midpiece can be differentiated from the 63 tail-piece or flagellum based on the change in thickness between the two structures. Midpiece 64 defects such as thickening, roughening, proximal and distal cytoplasmic droplets, distal 65 midpiece reflexes, coiling and bending may be determined. Tail defects including bending and 66 coiling as well as duplicity may be diagnosed. EN is also a vital stain. Sperm that have intact 67 membranes and normal chromatin will not allow eosin to be drawn into the cell and so stain 68 white, while sperm with damaged membranes and damaged chromatin will stain pink due to 69 eosin uptake. EN staining overestimates the number of intact membranes when compared to 70 fluorescent assays (see flow cytometry section).10 Use of live:dead information requires that 71 proper staining procedure is used including prompt staining after collection, prevention of cold 72 shock to the sperm, allowing adequate incubation time before smearing the slide, and rapid 73 drying to prevent cracking or cell death. Round cells cannot be differentiated with this stain. The 74 stain is hypotonic and this may result in stain artifact, such as coiled tails, if they are not dried 75 properly. Cracks in the stain may also be noted if the smear is too thick. 76 Modified Wright-Giemsa stain (Diff-Quik®; Siemens Healthcare Diagnostics, Deerfield, 77 IL, USA) is a simple, inexpensive, and rapid method of staining sperm.9 Alterations in head 78 171 staining may correlate with DNA defects. The sperm head stains deeply basophilic from the 79 equatorial region downward and more lightly basophilic in the area of the acrosomal cap. The 80 midpiece and tail stain eosinophilic. Details of the acrosome are not visible with this stain, so 81 only basic alterations in head size and shape are detectable. Midpiece and tail defects that can 82 be visualized include cytoplasmic droplets, distal midpiece reflexes, bent and coiled midpieces 83 and tails. This stain has the added benefit of allowing differentiation of round cells in the 84 ejaculate, making identification of WBC vs. germ cells possible. 85 Feulgen staining is a time consuming procedure that allows for better visualization of 86 sperm nuclear and head abnormalities.9 The DNA in the sperm head stains magenta allowing 87 good nuclear detail and making defects of the nucleus (vacuoles, diadem defects, etc) clearly 88 visible. The acrosome, midpiece and tail do not stain. The stain must be made fresh daily or 89 changes in the pH will affect the intensity of the stain. Slides may be evaluated with either 90 bright field or phase contrast microscopy. The use of phase contrast microscopy results in 91 greater color contrast. Feulgen staining identified more abnormalities of DNA pattern and head 92 shape in the bull sperm nucleus than standard EN stain.11 93 India ink is a one-step stain that is simple and inexpensive to use. It provides a black 94 background and a white or clear sperm outline. It does not allow for good visualization of the 95 acrosome or equatorial region. It does however allow for differentiation of alterations in head 96 size and shape, major defects of the midpiece including cytoplasmic droplets, distal midpiece 97 reflexes, bent or coiled midpieces, and bent or coiled tails. 98 Spermac® (Conception Technologies, San Diego, CA, USA) is expensive and 99 moderately time-consuming but is technically a simple staining technique.12 It is important that 100 thin smears of semen are made and are air dried for no more than 10 minutes prior to fixing. 101 Once fixed, the remaining steps in the staining procedure may be delayed indefinitely. The 102 nucleus of the cell stains dark red while the acrosome stains light green, and the midpiece and 103 tail stain dark green. This is an excellent stain for acrosomal evaluation and also allows for 104 172 good midpiece and tail piece evaluation. Assessment of the nucleus is not as accurate, but 105 head size and shape can be evaluated. Round cells can be visualized but they cannot be 106 differentiated. 107 Papanicolaou stain is commonly used in human andrology labs, but is not commonly 108 used in veterinary applications due to the complexity of the procedure.8,13 109 Phase contrast microscopy is a form of microscopy that allows small phase shifts of light 110 through a transparent cell which are converted into contrast and amplitude changes in the 111 image.14 Two light rays are focused exactly inside the opening of the condenser annular ring. 112 The two rays are refracted so that they exit the condenser in parallel. The light is minimally 113 refracted on passing through the specimen and it travels in parallel into the objective where it 114 enters the back focal plane of the objective. A phase plate is positioned in the back focal plane 115 to line up with the condenser annulus. 116 Differential interference contrast (DIC) microscopy is a system whereby a polarized light 117 source enters a prism and is diverted into two beams at 90 degree angles to each other.14 118 These rays pass through a condenser and then the sample. The beams enter and pass through 119 the sample about 0.2μm apart from each other. Since the beams pass through different parts of 120 the cell they follow different optical paths. The beams then enter the objective lens of the 121 microscope and passes through a second prism which recombines the rays into one polarized 122 beam. This recombination leads to interference (since they are on different optical paths) which 123 either brightens or darkens the image. The resultant image appears to be three-dimensional. 124 DIC microscopy provides excellent resolution and clarity of cellular structure with minimal 125 artifacts. It does require that the sperm sample is in a media of similar refractive index to the 126 cells themselves which means that evaluation of semen in skim milk or yolk extenders is 127 difficult. 128 No staining of the cell is required for either phase or DIC microscopy, but for morphology 129 assessment, the sperm are generally fixed in formol buffered saline and viewed at high power 130 173 (40 – 100x). Both of these forms of microscopy provide for more detailed visualization of all 131 parts of the sperm cell including the acrosome, nucleus, midpiece and tail piece than bright field 132 microscopy alone. 133 Sperm morphologic assessment plays an integral role in predicting success with IUI, IVF 134 and ART techniques.3-8 Determination of teratozoospermia prior to attempting advanced 135 insemination techniques will help with management choices. For example it has been shown in 136 many human studies that by increasing sperm concentration at the time of insemination, 137 patients with teratozoospermia will have a greater success rate with IUI and IVF procedures.3-5 138 In humans it has also been repeatedly demonstrated that once the number of normal sperm 139 drops below 14% infertility is a consistent result; with individuals with 0-4% normal forms have 140 the lowest success rates (45%), 5 – 14% normal forms have moderate success rates (75%), 141 while > 14% normal forms have good success rates (85%) when advanced reproductive 142 techniques like IVF and ICSI are applied.3-6 143 Acrosome stains 144 Giemsa stain makes the acrosome appear dark purple.9 It provides good detail of the 145 acrosome but needs to be made fresh for each use and does not allow for evaluation of the 146 sperm nucleus, midpiece or tail. 147 A sperm triple stain of Trypan blue stains the spermatozoa blue, Bismarck brown stained 148 the post-acrosome region light brown and rose bengal stained the acrosome light red.15 Sperm 149 can be differentiated into four groups with this stain: dead sperm with fully or partially inactivated 150 acrosomes, dead spermatozoa with missing or degenerated acrosomes, live spermatozoa with 151 reacted acrosomes, and live spermatozoa with active or normal acrosomes. This staining 152 technique has been used for humans, mouse, bull, horse, goat and boar semen. 153 A one step stain including fast green FCF, rose bengal and ethyl alcohol has been used 154 to stain the acrosome of cat spermatozoa.16 This stain allows differentiation of acrosome intact, 155 174 acrosome reacted or damaged sperm, and acrosome non-intact sperm. The slide is examined 156 using bright field microscopy at 1000x. 157 Coomassie blue stain has been used to assess acrosomal integrity in bulls, boars, and 158 stallions.17,18 This staining procedure is relatively simple and results in intense blue staining of 159 intact acrosome and lack of stain uptake in acrosome reacted sperm. The results of Coomassie 160 blue staining correlate well with fluorescent staining (see below), DIC and bright field 161 microscopy techniques following incubation with calcium ionophore to induce the acrosome 162 reaction.17 163 Fluorescent stains, like acridine orange, can be used to evaluate sperm that are 164 extenders in opaque extender, like skim milk.9 Acrosomal integrity can be evaluated using 165 fluorescent microscopy, phase-contrast or DIC microscopy. 166 Acrosome staining can be performed using bis-benzimide dye Hoescht 33 258 and a 167 FITC-pisum sativum agglutinin (FITC-PSA) after induction of the acrosome reaction by calcium 168 ionophore.19 Sperm are incubated in TALP media and then calcium ionophore A23 187 is 169 added to induce the acrosome reaction. Then the sperm suspension is permealized in 170 methanol and incubated with a lectin in order to bind the FITC-PSA probe. The Hoescht 33 258 171 is then used to stain the sperm and fluorescent microscopy is used to differentiate acrosome 172 reacted versus non-acrosome reacted sperm. It is a simple, quick technique to assess 173 acrosome status, but it does not allow for morphologic assessment of the cell itself. Samples in 174 egg yolk based extenders do not hinder using this technique. 175 Trypan blue or Congo red stain can be precipitated by neutral red and then stained with 176 Giemsa to stain bull, boar and rabbit sperm, but not stallion sperm. This is a simple and reliable 177 staining procedure that results in the stained sperm being classified as live or dead with intact 178 acrosomes, loose or damaged acrosomes, detached acrosome and detached acrosome with no 179 post acrosomal ring.20 180 Hypo-osmotic swelling test 181 175 This test is based on the concept that the normal sperm tail membrane will allow fluid to 182 pass into the cell freely under hypo-osmotic conditions. As the fluid flows into the cell, the tail 183 swells. Membrane integrity is important in sperm metabolism and changes in membrane 184 properties must occur for capacitation and the acrosome reaction to occur normally.8,21 HOST 185 not only assesses the morphologic integrity of the plasmalemma but it also assesses its function 186 and biochemical activity.8,10 In humans and bulls, there is strong correlation between the HOST, 187 the sperm penetration assay, and there is a good interrelationship between HOST and motility 188 and morphology.8, 10, 22 In bulls and humans, HOST was a good predictor of success with IVF.10 189 The HOST is simple, fast and inexpensive.8, 21 One tenth of a milliliter of spermatozoa is 190 incubated in one milliliter of 60 mOsmol fructose solution at 37 °C for 45 minutes. Then 1– 2 191 drops of this mixture is examined using phase contrast microscopy at 200 x and 400 x. Two 192 hundred sperm are counted and the percentage of sperm with curled or swollen tails is 193 determined. HOST is positively correlated to motility (r = 0.94). The premise being motile sperm 194 have normal membranes and will coil or swell when incubated in a hypo-osmotic solution. 195 As sperm are cooled for increasing amounts of time, decreasing numbers of sperm will 196 be HOS+ indicating damage to the sperm membrane with prolonged cooling.21 A similar 197 phenomenon is noted after cryopreservation due to sperm membrane damage. Prolonged 198 heating also damages the sperm membrane resulting in few HOS+ cells. Use of HOST on 199 rewarmed chilled or post-thaw frozen semen may be predictive of the highest quality samples to 200 be used for insemination by selecting for samples with the highest number of HOS+ cells. The 201 HOST may be a beneficial addition to the semen evaluation in dogs with poor fertility but a 202 normal spermiogram. 203 Acrosome assays, acrosome reaction testing and capacitation testing 204 To evaluate acrosome status, sperm must be removed from seminal plasma via 205 centrifugation and then are resuspended in capacitation mediim.23 Hyperactivation can be 206 determined through the use of CASA. The clinical relevance of hyperactivation has yet to be 207 176 determined. The acrosome reaction can be evaluated through the use of dyes, fluorescent 208 antibodies or lectins. Induction of the acrosome reaction is most readily induced following 209 incubation of the sperm first in capacitation media and then calcium ionophore (A23187) is 210 added.8,23 It can be added in high concentration and a short incubation period used (30 – 60 211 minutes) or at low concentration and a long incubation period used (3 hours). The samples are 212 then washed and re-suspended in protein free media and the cells are smeared on slides that 213 are air dried. The slides are then fixed in alcohol and are stained with peanut agglutinin (PNA), 214 Pisum sativum agglutinin (PSA) or fluorescent-labelled lectins and then evaluated with 215 fluorescent microscopy. The number of acrosome reacted cells are then counted and a 216 percentage of all cells is determined.23,24 Samples that have high numbers of prematurely 217 reacted cells or which do not respond to incubation with calcium ionophore not likely to be able 218 to complete fertilization.6,7,23,25,26 In humans, there is a high predictive power of induced 219 acrosome reaction and successful IVF outcome.26 220 Acrosome reaction can also be detected using staining with fluorescein-conjugated 221 lectins, like PSA or PNA, plus fluoresceinisothiocyanate (FITC).8,25 This combination of stains 222 evaluates damage to the acrosome while at the same time differentiating acrosome reacted 223 from acrosome intact sperm. PSA binds to the acrosomal contents while PNA binds to the outer 224 acrosomal membrane.27 Acrosomal integrity of canine sperm has been successfully assessed 225 using flow cytometry and staining with FITC conjugated PSA and PI.28 Capacitation status of 226 chilled and frozen thawed canine sperm has been assessed with a chlortetracycline (CTC) 227 assay and CASA for evidence of hyperactivation.29 Dog semen has been evaluated for its 228 cryopreservability by first inducing the acrosome reaction with calcium ionophore and then 229 staining the sperm with FITC-PNA along with the membrane impermeable DNA supravital stain 230 ethidium homodimer1 (EthD-1).30 Samples were evaluated with fluorescence microscopy and 231 flow cytometry. The number of cells that underwent the acrosome reaction via ionophore was 232 well correlated with a similar percentage of cells that had acrosome damage post 233 177 cryopreservation. Furthermore, the amount of damage to cells caused by acrosome reaction 234 from calcium ionophore was strongly negatively correlated with the number of motile sperm 235 present after freezing. 236 Bovine sperm have been incubated with calcium ionophore to stimulate the acrosome 237 reaction and then fixed in formaldehyde.31 Afterwards they are stained with naphthol yellow S 238 plus erythrosin B or with naphthol yellow S plus aniline blue. This is a permanent fixative and 239 the use of DIC microscopy is required to evaluate acrosomal status. Alternatively, bovine sperm 240 may be treated with fluoresceinated PSA to assess the acrosome reaction similar to that 241 described previously.27 242 Triple staining techniques for acrosomal evaluation have also been described but are 243 more time consuming than the above mentioned techniques and so are not routinely used in the 244 clinical or research setting.8 245 Sperm penetration assays 246 These assays asses the ability of the sperm to undergo capacitation, the acrosome 247 reaction, membrane fusion and chromatin decondensation in the presence of an oocyte.4,6-8,23,32 248 Sperm must be prepared for the assay by incubating overnight in a capacitation medium or 249 storing in a TES-tris buffer with egg yolk for 24 – 48 hours and then applying thermal shock. 250 After this processing step, the sperm are divided into microdrops and zona-free hamster eggs 251 are added. They incubate for 3 hours and the number of eggs penetrated and the number of 252 sperm/egg are counted. The count is performed by looking for swollen heads within the 253 unstained eggs using phase-contrast or phase-interference microscopy or after staining with 254 acridine orange (AO) and using fluorescence microscopy. Use of the TES-tris buffer procedure 255 was more highly correlated with fertility and successful outcomes with IVF in humans in some 256 studies4,6,7,23 while in others it’s predictive power was questionable.26 257 Hemizona assays 258 178 This test assesses the availability of the proper molecules on the sperm’s head for it to 259 bind to the zona pellucida and initiate interaction with the oocyte.8,23 A bisected zona pellucida 260 from a normal oocyte is used. Each half of the zona is incubated with sperm for 4 hours and 261 then the number of bound cells is counted. Zona pellucida binding assays have been used to 262 evaluate the fertilizing capacity of chilled and frozen-thawed canine spermatozoa.33 The test 263 allows an estimation of the damage caused by manipulation of semen on the fertilizing ability of 264 sperm. This test also demonstrates the critical interaction between the zona pellucida and the 265 sperm cell during fertilization and tests multiple sperm functions, including capacitation and 266 ligand-induced acrosome reaction.4,6-8,34,35 Of the classic sperm parameters, morphology was 267 the best predictor of the ability of sperm to bind to the zona pellucida.3,4,6,32,34 In conventional 268 IVF studies in humans, defective sperm-zona binding and zona penetration are common causes 269 of failure of fertilization.7,35 There is a high predictive power of sperm-zona pellucida binding 270 and successful outcome with IVF in humans.26,35 271 Electron microscopy – transmission (TEM) 272 The sperm rich fraction is mixed 1:2 with cacodylate-buffered 6% glutaraldehyde.8,36 273 This mixture is centrifuged, the supernatant removed and the pellet resuspended in 0.1 M 274 sodium cacodylate buffer. This sample is washed a second time and the supernatant removed. 275 The pellet is fixed in a solution of 1% osmium tetroxide in 0.1 M cacodylate buffer and is 276 centrifuged. The osmicated pellet is dehydrated through a graded series of ethanol, is then 277 rinsed in propylene oxide and is then embedded in Poly/Bed 812 or araldite. Sections are cut at 278 80 nm thickness and are then mounted on 300-mesh nickel grids and stained with uranyl 279 acetate and lead citrate for TEM. 280 TEM may identify lesions of the plasma membrane, acrosome, mitochondria, and 281 nuclear chromatin.8,36 Quantification of morphologic defects is not possible but a detailed 282 description of the defects is provided. TEM may also help identify and characterize other cells in 283 179 the ejaculate including germ cells, WBC and infectious organisms. DNA fragmentation caused 284 by oxidative stress or exposure to toxins may be identified. 285 TEM may identify defects of the tail in patients with motility issues that are not apparent 286 with light microscopy.8,37 If a single defect is present in at least 20 – 30 sections it is considered 287 a ciliary dyskinetic condition. Total or partial dyein arm deficiency occurs in 3% of human 288 patients with abnormal motility. Fragmentation of the plasma membrane and necrosis of the 289 microtubules is typical of necrospermia and is found in 23% of human patients with 290 asthenozoospermia.38 Multiple fine ultrastructural defects are noted in another 23% of human 291 patients with this condition. Missing outer microtubules, disorganized axonemes, missing 292 central microtubules, additional microtubules above and beyond 9 + 2, thickened and/or 293 disorganized fibrous sheaths, absent radial spokes and translocated tubules are other common 294 defects noted in this group. Some of these defects are noted alone and others in combination. 295 In patients whose total motility is > 30% and at least some normal tails were evident, 296 pregnancies using assisted reproductive techniques (ART) may be successfully obtained.37 297 A microtubular mass defect was noted on examination of spermatozoa from seven 298 stallions with three of these stallions descending from a single sire.38 There was subfertility in 299 four of these stallions, although it appeared to be at least partially compensable. Detailed 300 description of the ultrastructure of the bovine sperm head and midpiece are available.39,40 301 Electron microscopy is currently available for clinical cases at the veterinary colleges of 302 Auburn University, Texas A & M University, and University of Saskatchewan. 303 Antisperm antibody assay 304 In humans, antisperm antibody production is a common cause of male factor 305 infertility.8,23 There are two commercial assays (SpermMar®, Conception Technologies, and 306 Immunobead Test®, Irvine Scientific, Santa Ana, CA, USA) available for human antisperm 307 antibody assay. These tests provide semiquantitative results regarding the degree of antibody 308 binding present and detect the presence of IgA and IgG antisperm antibodies. If ≥ 20% of the 309 180 sperm bind to the beads, a sample is considered positive for the presence of antisperm 310 antibodies.23 Serum may be assayed for antibodies using a tray agglutination test. At this time, 311 the importance of antisperm antibodies in domestic animals is unclear but may be a useful test 312 for dogs with autoimmune orchitis/epididymitis. 313 Flow cytometry for DNA and morphology measurement 314 Binding of fluorescent dyes to sperm chromatin permits the identification of sperm DNA 315 abnormalities and can be measured using a flow cytometer.8,41 Sperm are stained using 316 fluorescent assay and then run through the flow cytometer to differentiate cells with normal DNA 317 integrity from abnormal. Sperm are typically oriented to be in the same plane before they are 318 excited by a laser beam to induce fluorescence and then flow past a fluorescence detector 319 which monitors exactly how much fluorescence each cell has. Sperm that have uniform head 320 size and shape display a uniform degree of fluorescence while cells with abnormal size and 321 shape have amounts of fluorescence outside the normal ranges.41 In addition to being able to 322 differentiate morphologically normal from abnormal sperm, flow cytometry can also differentiate 323 sperm with normal motility from those with decreased motility.42 One cause of decreased 324 motility in humans is a result of a break in the DNA strands of the sperm nucleus and their 325 mitochondria. Flow cytometry and TUNEL (terminal deoxy-nucleotidyl transferase-mediated 326 deoxyuridine triphosphate-nick end labeling) testing both confirm the presence of these strand 327 breaks in this population of asthenozoospermic men. 328 One of the greatest assets of using flow cytometry for sperm evaluation is the sheer 329 number of sperm that can be evaluated in a short period of time. Routine microscopic 330 assessment of sperm involves counting either 100 or preferably 200 cells. But with the use of 331 flow cytometry, thousands of cells can be assessed in a matter of minutes.41 Another 332 assessment that can be performed using flow cytometry is that of sperm viability. A dual 333 staining technique using carboxifluorescein diacetate (CFDA) and propidium iodide (PI) was 334 used to validate the ability to differentiate live canine sperm from dead sperm.43 Plasma 335 181 membrane integrity was also validated in stallion sperm using either CFDA and PI or SYBR-14 336 stain and PI.25 In these tests, live sperm fluoresce green (from CFDA or SYBR-14), dead sperm 337 fluoresce red (PI), while dying sperm fluoresce both colors.25,32 338 Tests of mitochondrial activity 339 Rhodamine 123 (Rh123) is a mitochondrial probe which is combined with the viability 340 stains of PI and carboxydimethylfluorescein diacetate (CDMFDA) to allow for the determination 341 of sperm with intact membranes (CDMFDA+) and functional mitochondria (Rh123+) from dead 342 sperm (PI+).25 Stallion sperm with functional membranes and mitochondria correlate well with 343 sperm viability and motility.25 344 Cytochemical tests for sperm chromatin integrity 345 The integrity of nuclear chromatin results from a combination of factors including sperm 346 maturation processes, damage due to oxidative stress and other endogenous factors.8,44 347 Chromatin maturation depends on proper replacement of histone with transition proteins and 348 finally basic protamines. This transition results in compact packaging of the DNA and is 349 enforced by cross-linking with protamine-disulfide bonds. In some abnormal sperm, histones 350 may be partially or completely replaced by protamines resulting in loosely packed chromatin. 351 Detection of this loose packing may be found with the aniline blue (AB) test. DNA is more 352 predisposed to denaturation by heat and low pH when chromatin is packed loosely than when it 353 is tightly packed.44 Chromatin proteins in sperm with abnormal DNA are more susceptible to 354 staining with acidic dyes like AB, acridine orange and toluidine blue.44 All three of these staining 355 protocols provide a good estimation of the number of sperm with normal vs loosely packed 356 chromatin structure. If more than 30% of sperm have loosely packed chromatin structure an 357 association with increased infertility is noted which correlates with results of SCSA (see below). 358 Sperm chromatin analysis 359 Sperm morphology is well correlated with semen quality.4,7,8,45,46 The head of the sperm 360 consists primarily of nuclear chromatin, so subtle changes in sperm head morphology may be 361 182 related to abnormalities of DNA content. Measurement of a set of parameters regarding the 362 sperm head (size and shape) and midpiece can align sperm into certain populations of sperm, 363 such that the chromatin content in each sperm head can be determined to be normal or 364 abnormal using automated sperm morphometric analysis (ASMA). Non-compensable defects 365 (those that cannot be overcome by increasing the number of sperm in a breeding dose) are 366 typically related to sperm with abnormal head morphology.4,7,45,46 Sperm head shape has been 367 correlated with fertility and resistance to cryopreservation. Sperm nuclear DNA fragmentation is 368 positively correlated with lower IVF fertilization rates, impaired implantation, increased risk of 369 abortion, and increased risk of disease in offspring, including pediatric cancer.7,8,31,47-49,51 Fertile 370 sperm have stable DNA which is able to decondense at the appropriate time during the 371 fertilization process such that the oocyte has access to this DNA for combination with its own 372 DNA complement.48,49,52 373 Sperm DNA damage may occur on several different levels.4,7,8,31,48- 50,52 Mitochondrial 374 DNA damage can occur and be manifest as deletions, point mutations and polymorphism and is 375 associated with decreased semen quality, asthenozoospermia and male infertility. Nuclear DNA 376 damage may occur as a result of oxidative stress, sperm chromatin packaging and apoptosis. 377 DNA damage may occur as a result of environmental factors, pollutants, infection, inflammation, 378 or the presence of ROS.48,49 379 For SCSA, sperm are treated with an acidic solution (pH1.2) in order to denature their 380 DNA in situ.8,46,48 Sperm with normal chromatin will not denature under these conditions while 381 abnormal DNA will denature. The sperm are then stained with acridine orange. This is a 382 metachromatic DNA stain. Chromatin which has been denatured into single stranded DNA will 383 fluoresce red, while DNA which does not denature (remains double stranded) will fluoresce 384 green. The percentage of cells with denatured DNA is determined and is called %COMP. The 385 percentage of sperm with non-detectable vs detectable DNA fragmentation is called the DNA 386 fragmentation index (DFI). The percentage of sperm with immature chromatin is called high 387 183 DNA stainability (HDS). Combining the use of SCSA and ASMA may assist in evaluating dogs 388 with normal spermiograms and poor fertility.46,48 389 DNA fragmentation may also be evaluated using alkaline single-cell gel electrophoresis 390 testing, TUNEL assay; Comet assay, in situ nick translation, and DNA breakage detection-391 fluorescent in situ hybridization assay (DBD-FISH). These assays use fluorescence 392 microscopy.7,48-50,52 Staining techniques using aniline blue, toluidine blue and chromomycin A3 393 also may be used to identify chromatin packaging defects.52 At this time, few of these assays 394 beyond SCSA are used in clinical practice as it remains to be determined what the clinical 395 relevance of negative outcomes means to fertility. There also still remains significant variability 396 in techniques between labs resulting in disparate results. Certainly in veterinary medicine, data 397 regarding most of these tests in clinical practice is lacking, although they are slowly being 398 introduced.32 399 On the other hand, SCSA, has been accepted as an important tool in the diagnosis of 400 infertility and in prognosticating human couple’s success rates with ART.48,49 In humans, DFI is 401 a strong indicator of successful pregnancy outcome.4,7,48-50 Prediction of successful outcome 402 with intrauterine insemination is well correlated with the degree of DNA fragmentation.48,49 The 403 number of sperm that have DNA strand breaks is negatively correlated with their ability to 404 fertilize an oocyte during IVF.48-50 Sperm DFI was shown to be negatively correlated with sperm 405 concentration, motility and normal morphology.50 Fertilization failure of sperm with DNA 406 fragmentation may be able to be overcome by application of ART procedures, such as 407 intracytoplasmic sperm injection (ICSI), however embryo development may be affected with 408 resultant early embryonic death (EED) or abortion.4,7,32,48-52 In humans, a DNA fragmentation 409 rates of > 30% seem to impede fertility, and in couples with high DFI, the use of ICSI will 410 improve pregnancy rates over the use of IVF.48,49,52 411 In stallions, SCSA has been evaluated and shown to be an indicator of some forms of 412 subfertility or infertility.25,53 Subfertile stallions had higher %COMP levels than did stallions with 413 184 normal fertility and there was a negative correlation between seasonal pregnancy rate and 414 %COMP, % morphologically normal sperm, and % motile sperm. In boars, there is an inverse 415 relationship between farrowing rate and numbers of pigs/litter compared to %DFI.48 Pregnancy 416 rates in bulls with high %DFI were lower than for bulls with low %DFI.48 It appears that the 417 threshold for fertility in bulls (10 – 20%) and boars (8%) is much lower than for humans).48 A 418 threshold level is not yet available for dogs. 419 SCSA is currently offered for clinical cases at SCSA Inc (Brookings, SD; www.scsa.com) 420 and at Texas A & M University College of Veterinary Medicine. 421 Assays for reactive oxygen species 422 ROS are very important during the sperm capacitation process in order for tyrosine 423 phosphorylation events to occur normally.7,8 They are normally produced at low levels, 424 however, in some cases of infertility they are produced in much higher amounts. ROS interrupt 425 sperm function by causing peroxidative damage to the plasma membrane and thereby impairing 426 motility, the acrosome reaction (exocytosis), and disrupting sperm-oocyte fusion. Oxidative 427 stress may also cause mitochondrial DNA and nuclear genome damage.7 There are 428 chemluminescent assays using redox-sensitive probes (lucigenin and luminol) for human 429 spermatozoa.7,8 High levels of chemluminesence affect the fertilizing capacity of sperm both in 430 vivo and vitro. The presence of WBCs in the ejaculate will greatly increase the amount of ROS 431 present, therefore WBCs must be removed from the samples prior to testing.7 In the presence 432 of seminal plasma, protection from ROS produced by WBC is afforded, while for ART 433 technologies, these ROS will likely have a much more significant role. 434 Fluorescence in situ hybridization (FISH) 435 This procedure allows for the analysis of chromosome numbers in individual sperm. 436 Individuals with oligoasthenoteratozoospermia are at increased risk of having chromosomal 437 abnormalities such as aneuploidy, double aneuploidy and diploidy.1,54 Sperm are fixed in a 438 methanol:acetic acid solution and then the DNA is decondensed in an acidic salt solution. 439 185 Sperm probes for specific chromosomes each fluoresce in specific color ranges are applied and 440 in situ hybridization is performed. In humans, teratozoospermia in the form of macrocephalic, 441 multi-tailed sperm have an increased incidence of aneuploidy.54 Other morphologic 442 abnormalities may also be associated with specific chromosomal abnormalities and this area 443 bears the need for further investigation.54 444 Computer assisted sperm analysis and automated sperm morphometric analysis 445 CASA is a technique employing a computerized system that tracks mean percentage of 446 motile sperm, mean percentage of progressively motile sperm, mean curvilinear velocity and 447 mean straight line velocity per microscopic field.8 The ejaculate is diluted to a specified 448 concentration and the system uses a special gridded microscope slide that accepts a constant 449 volume of semen thus providing a consistent number of sperm to be evaluated each time. The 450 pre-warmed and loaded slide is placed into a thermostatically controlled chamber for analysis. 451 The computer takes video images of the sperm and stores then for analysis. The system 452 recognizes motile from non-motile sperm and other organic debris by comparing luminosity 453 (gray-scale intensity) and size of the object. There are also preset user-defined thresholds for 454 size and luminosity that help prevent mistaking other cells and debris for non-motile sperm. 455 Computerized systems have been shown to be more accurate than subjective assessment of 456 sperm motility in human, equine, bovine and canine studies.6,8,32,55-57 CASA provides a more 457 discriminating estimation of motility than subjective evaluation with greater repeatability. 458 ASMA is now also available and provides a more accurate and repeatable evaluation of 459 general sperm morphology.4,6,25,32,46,58,59 With this automated process, as with CASA, sperm are 460 diluted in physiologic media to a specified concentration and then a fixed drop placed on a slide 461 and air dried. The slides are then stained and coverslips permanently affixed to the slide before 462 processing in the analyzer. In this way a consistent number of sperm/field may be analyzed. 463 Staining method is also important. Papanicolaou stain and Giemsa stain have been used 464 successfully for morphometric analysis in humans, stallions and dogs. The machine obtains a 465 186 variety of head measurements including length, width, area, perimeter and width/length. A 466 specified gray scale is required and allows differentiation of sperm heads from other cells and 467 debris on the slide. Midpiece width and area, distance between the major axes of the head and 468 midpiece, angle of divergence of the midpiece from the head axis can also be assessed. 469 Abnormal sperm head, midpiece and tail morphology can be detected 95% of the time with 470 these measurements.45,46,58 471 In the normal dog, a significant variation in head area, length, width and roundness 472 exists, but the analyzer still provides accurate differentiation of teratozoospermic samples.45,46,59 473 Ovalness was the least variable factor obtained while length and width had more variation 474 between dogs. Within dogs, there was less variation of any measurement. Similar variability in 475 sperm head shape and size is noted with SCSA analysis of the same dogs, indicating that 476 ASMA may be a valuable tool when assessing sperm for teratozoospermia.46 477 In vitro fertilization 478 This is the ultimate test of sperm function.8,23 In human medicine, the end point of IVF is 479 what percent of MII oocytes are fertilized and develop to the 8 cell stage by day 3 post 480 insemination.4,32 481 Sperm function tests 482 Table 1. Sperm functions and the sperm function tests that assess them23 483 Motility CASA Light microscopy Morphology ASMA Light microscopy EM Flow cytometry Capacitation IVF SPA Acrosome Reaction IVF SPA Acrosome reaction tests Acrosin assays 187 Zona pellucida binding IVF HZA Zona pellucida penetration IVF Oocyte-sperm fusion IVF SPA CASA – computer assisted sperm analysis; ASMA – automated sperm morphometric 484 analysis; EM – Electron microscopy; SPA – sperm penetration assay; IVF – in vitro fertilization; 485 HZA – hemi-zona assay 486 Summary 487 It is clear that there is much more to be evaluated regarding male fertility than the basic 488 semen evaluation. The concept that simply providing a specified number of normal appearing, 489 motile sperm at the proper time in relation to breeding will result in acceptable pregnancy rates 490 is clearly a misconception. There are many aspects of sperm function that may affect the 491 functional competence of the sperm cell, beyond its basic size, shape and motility.7 When faced 492 with a dog that has subfertility, the clinician must first rule out the most obvious causes for the 493 problem and them move on to more advanced semen diagnostic testing to exhaust all possible 494 diagnoses. In the process of making the diagnosis, the clinician may discover a method of 495 treating or correcting for the problem. Unfortunately, in some cases, even with exhaustive 496 testing, a diagnosis may remain elusive. Research in all areas discussed in this paper is needed 497 for all domestic animals including the canine. 498 References 499 1. 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