2009: Antibiotics and other additives for semen extenders to enhance fertility Antibiotics and other additives for semen extenders to enhance fertility 1 S. P. Brinsko 2 Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Texas 3 A&M University, College Station, TX, USA 4 Abstract 5 Semen extender formulations have been evolving for over 70 years and this 6 evolution continues. Although milk was found to be suitable as a major component of 7 equine semen extenders over 60 years ago, preparation of the extenders remained tedious 8 and time consuming in part, due to the need to heat the milk before use. With the 9 publication of the formulation for a nonfat, dried skim milk–glucose (NFDSM-Gluc) in 10 the 1970s, a simple, convenient semen extender became available, prompting a dramatic 11 expansion in the use of equine artificial insemination. This basic formulation of the 12 NFDSM-Gluc extender is still in use today, with various modifications. More recent 13 work has further identified the beneficial components of milk, which has led to more 14 defined extender formulations. This paper briefly reviews the development of equine 15 semen extenders and discusses the effect of including various additives such as 16 antibiotics, cryoprotectants, and antioxidants on equine sperm survival and fertility. 17 18 Keywords: Stallion, semen extender, additives 19 20 369 Introduction 21 The search for an optimum semen extender formulation has been an ongoing 22 quest almost from the inception of artificial insemination. Despite the fact that artificial 23 insemination of mares is reported to have occurred much earlier than in cows and much 24 of the early work in the field was done in the horse, the development of effective 25 extenders for stallion semen was outpaced by the development of bull semen extenders.1,2 26 This was due in part to the lack of demand for storage of stallion semen, the belief that 27 stallion sperm lacked “the innate resistant factor necessary for storage” and the restrictive 28 attitude of many breed registries toward artificial insemination.1,3 While a thorough 29 review of the development of equine semen extenders is beyond the scope of this 30 manuscript, a brief review of early formulations should help set the stage for where we 31 are today. 32 Over the years, numerous extender formulations have been employed in attempts 33 to improve the survivability of stallion semen. Included in these formulations are various 34 combinations and levels of: egg-yolk, sugars, buffers, citric acid, electrolytes, gelatin, 35 glycerin, honey, milk products and even blood serum and follicular fluid.1,2,3 The milk 36 products have included mare’s milk, sheep and goat milk, cow’s milk, cream, skim milk, 37 buttermilk, and nonfat dried milk solids (NFDSM).1,2,3 One of the first extenders for 38 stallion semen was a glucose-sulphate-peptone formulation,4 which was later modified to 39 a tartaric-glucose-peptone formulation.5 Peptone is a water-soluble mixture of amino 40 acids and peptides derived from the partial hydrolysis of protein. Although the source of 41 the peptone used in those early experiments is not stated, it is interesting to note that the 42 370 peptone that can currently be obtained from Sigma-Aldrich (St. Louis, MO, USA) is 43 derived from the enzymatic digestion of the milk protein, casein. 44 For many years, equine semen extender formulations were either the same or very 45 similar to those used for bull semen. Results were mixed, sometimes being favorable and 46 oftentimes less than satisfactory. After the discovery that adding egg yolk to a buffer 47 improved the survival of bull sperm6 and lessened its susceptibility to temperature 48 shock,7 a number of investigators incorporated egg yolk into equine semen extenders.8,9,10 49 In 1949, Buĭko-Rogalevič reported that sperm motility was preserved for 8 to 13 days 50 when stallion semen was diluted in an egg yolk-glucose extender compared to 2.5 days 51 when diluted with glucose and that an 85.5% pregnancy rate was achieved with semen 52 stored for 12 to 42 hours in this egg yolk-glucose extender.9 In a series of experiments, 53 Kühr obtained similar results with sperm survival increasing from 8.2 hours in undiluted 54 semen to 100.8 hours in 7% glucose and to 290 hours when semen was diluted in 7% 55 glucose + 5% egg yolk.11 Other extender formulations based on successful bull semen 56 extenders were far less satisfactory for stallion semen.2] Pace and Sullivan reported that 57 the fertilizing capacity of equine semen was depressed almost immediately after mixing 58 with hydrogen ion extenders.12 Investigators from several laboratories found that even 59 though various extender formulations could maintain sperm motility, the fertility of 60 semen diluted in these extenders was poor.3,11-14 The inferiority of these extenders is 61 best exemplified by the fact that pregnancy rates were higher when similar numbers of 62 sperm were inseminated using raw semen than with extended semen.12,14,15 As a result, 63 even up through the mid 1970s, it was recommended by some to use raw semen for 64 equine artificial insemination unless the semen was to be stored or unless antibiotics 65 371 needed to be added to the semen because the stallion was shedding pathogenic 66 bacteria.3,14,16 When one examines the composition of the extenders used in many of 67 those studies, it is likely that the glycerol they contained contributed to the poor fertility 68 observed. 69 Milk-based extenders 70 Milk was used as an extender for stallion semen as early as the 1940s and boiled 71 mare’s milk was reported to yield more favorable results than sheep, goat and even cow’s 72 milk.17 One of the major drawbacks of using fresh milk in semen extenders is the need to 73 heat the milk to 92 to 95 °C for 10 minutes in order to inactivate lactenin, which is toxic 74 to sperm.18-20 Because of the heating and pasteurization involved in their manufacture, 75 use of reconstituted dried milk products is thought to alleviate the need for heating when 76 used in semen extenders. In the late 1950s and early 1960s, the Chinese established a 77 very successful equine artificial insemination program involving 40 stallions and 78 thousands of mares, using semen diluted in a powdered milk-based extender.21 In 79 comparative studies, Cheng reported that both maintenance of sperm motility and 80 pregnancy rates were higher using the powdered milk extender when compared to fresh 81 mare’s milk or sugar-based (glucose or sucrose) extenders.21,22 Following up on 82 favorable results with bull semen in the late 1950s, workers at Texas A&M evaluated 83 reconstituted buttermilk with glucose added (BMG) as an equine semen extender.1 84 Although fertility was not examined, this BMG extender was found to be far superior to 85 mare’s milk, cow’s milk and egg yolk-glucose extenders for preserving sperm motility 86 for up to four days. The dried buttermilk was an “extra grade” product prepared by a 87 company in Wisconsin and it may be that limited availability of this product precluded its 88 372 widespread use in semen extenders. However, non-fat dried skim milk had been readily 89 available for years and once Kenney and co-workers23 published the recipe for a non-fat 90 dried milk solids-glucose extender (NFDMS-Gluc), this ‘Kenney extender’ as it is 91 known, revolutionized equine artificial insemination in the western world. With the 92 availability of a convenient, reliable semen extender, the use of artificial insemination in 93 horses increased worldwide and the basic formula for this extender has remained virtually 94 unchanged since its publication in 1975. Kenney-type extenders are available from a 95 number of commercial sources, differing primarily in the type and level of antibiotic(s) 96 added to the basic formulation. 97 Antibiotics 98 Inclusion of antibiotics in semen extenders is meant to reduce or eliminate 99 bacterial growth in semen, especially when it is stored, and to help control post breeding 100 endometritis. As with many other extender components, the incorporation of antibiotics 101 was based on satisfactory methods employed with bull semen. However, it was found 102 that the levels of antibiotics commonly used for bull semen extenders were toxic to 103 stallion sperm.1 Berry and Gazder reported that inclusion of 400 I.U./mL of penicillin and 104 1 mg/mL of streptomycin in their BMG extender was effective in controlling bacterial 105 growth without adversely affecting sperm motility. The original Kenney extender 106 contained either 1,500 I.U. of crystalline penicillin/mL and 1.5 mg of crystalline 107 streptomycin/mL or 1 mg/mL of reagent grade gentamicin. Antibiotics commonly 108 included alone or in combination in equine semen extenders today are: penicillin, 109 streptomycin, polymixin-B, ticarcillin, timentin, gentamicin, and amikacin. Although less 110 commonly used, ceftiofur24 and pipericillin,25 have also been shown to be safe and 111 373 effective antibiotics to include in equine semen extenders. For some very acidic 112 antibiotics, eg. gentamicin and amikacin, buffers also need to be added to adjust pH and it 113 is important to use reagent grade rather than injectable products because the 114 preservatives in the latter can be toxic to sperm. 115 While sperm motility and fertility of stored stallion semen can generally be 116 maintained or improved by extenders containing any of the antibiotics listed above, the 117 choice of which antibiotic to include in the extender may be determined based on specific 118 needs or circumstances. For some normal stallions, certain antibiotics appear to be more 119 favorable than others for maintaining sperm motility in stored semen. Certainly, for 120 stallions that are shedding specific pathogens into their semen, the choice of antibiotic to 121 include in the extender should be based on the sensitivity pattern of the offending 122 organism(s). 123 In the 1980s, Colorado State University entered into a licensing agreement with a 124 commercial company to market a NFDSM-Glu extender. The formulation was 125 essentially the same as the Kenney extender except that 1000 IU/mL of polymixin B 126 sulfate replaced gentamicin sulfate as the antibiotic.26 For a number of years, this 127 extender (EZ –Mixin® original formula, Animal Reproduction Systems, Chino, CA, 128 USA) was used extensively in the industry for both fresh and cooled-stored equine 129 semen. Later, Colorado workers examined the effects of different antibiotics on motion 130 characteristics in stored semen.27 Reagent grade amikacin sulfate, ticarcillin disodium, 131 gentamicin sulfate and polymixin B sulfate were added to a nonfat, dried, skim milk - 132 glucose seminal extender at concentrations of 1000 or 2000 μg or IU/ml. They found that 133 overall the addition of antibiotics to extender did not significantly improve motion 134 374 characteristics of sperm over control samples but that levels of gentamicin sulfate greater 135 than 1000 μg /ml and polymixin B sulfate equal to or greater than 1000 IU/ml 136 significantly reduced sperm motility. These workers concluded that genatmicin and 137 polymixin B greater than or equal to these levels should be avoided in seminal extenders 138 used for cooled semen. Texas A&M workers performed a similar series of experiments, 139 but also evaluated the control of bacterial growth.24 Results of this study demonstrated 140 that semen stored in extender containing 1000 IU/mL of polymixin B sulfate resulted in 141 the greatest reduction in sperm motion characteristics and the poorest control of bacterial 142 growth. These workers determined that a NFDMS-Gluc extender containing potassium 143 penicillin G (1000 IU/mL) and amikacin sulfate (1000 μg/mL) yielded the best 144 combination of motility maintenance and control of bacterial growth. Individual stallion 145 effects were also noted. 146 While not an antibiotic, the inclusion of the sugar mannose into semen extenders 147 has been proposed by Illinois workers as an alternative to antibiotics for reducing post 148 breeding bacterial endometritis.28 Previous work from this laboratory has indicated that 149 this sterioisomer of glucose was able to reduce the adherence of certain bacteria to 150 endometrial tissue.29-31 Replacing up to 37 mg/mL of glucose with mannose in NFDSM-151 Gluc semen extender did not affect the fertilizing capacity of sperm when immediate 152 insemination was performed on reproductively healthy mares.28 However, whether the 153 inclusion of mannose in semen extenders can control bacterial growth in semen or 154 maintain acceptable pregnancy rates with cooled-transported semen or in susceptible 155 mares requires further study. 156 375 Variations on basic components 157 Texas A&M workers also developed another variation of the Kenney extender. 158 This formulation not only contained the penicillin-amikacin combination but also reduced 159 the level of glucose from 4.9 mg/mL to 2.65 mg/mL with the addition of sucrose at 4.0 160 mg/mL. This TAMU extender has proven to be an excellent extender for use in fresh, 161 cool-stored breeding programs and also as a base extender for frozen semen after the 162 addition of egg yolk and glycerol. 163 French workers developed a successful milk-based extender that has been widely 164 used for frozen semen. In addition to sterilized skim milk, glucose and antibiotics, the 165 base INRA 82 extender also contains lactose, raffinose, sodium citrate and potassium 166 citrate to which egg yolk and glycerol are added prior to freezing.32 More recently, 167 studies which evaluated the effects of different milk fractions on sperm survival resulted 168 in the development of a defined milk protein extender (INRA 96) for use with fresh and 169 cooled semen.33 In this extender, skim milk is replaced with the specific milk 170 component; native phosphocaseinate (NPPC) in a Hank’s salts solution supplemented 171 with HEPES, glucose, lactose (HGLL) and BSA. While no difference was detected in 172 sperm motility after 24 h storage of semen in either INRA 82 or INRA 96, fertility was 173 higher for the semen stored in INRA 96.33 This extender was also shown to be as efficient 174 at preserving sperm motility and fertility when semen was stored at 15 ºC as when stored 175 at 4 ºC.34 This extender can also be used for freezing stallion semen. A fertility trial was 176 conducted comparing INRA 82 and INRA 96 supplemented with egg yolk and glycerol. 177 Although motility parameters were significantly higher in INRA 82 than in INRA 96, the 178 376 INRA96 extender significantly improved per-cycle pregnancy rates compared with 179 INRA82 (71% versus 40%) in a total of 84 mare cycles.35 180 Japanese workers reported that the addition of 2% casein and 5% egg-yolk to a 181 boar semen extender (Modena) resulted in superior sperm viability in cooled stored 182 semen compared to Kenney extender.36 Semen stored in this extender at 5 ºC resulted in 183 14 of 22 mares becoming pregnant within 72 h of storage and 3 of 4 mares becoming 184 pregnant with semen stored within 96 to 120 hours. The problem with adding egg yolk to 185 extenders is that it compromises the ability to accurately assess sperm motion 186 characteristics if the extender is not clarified. 187 Workers in Austria, evaluated another defined milk protein extender (EquiPro®, 188 Minitüb, Tiefenbach, Germany) containing caseinate, selected whey proteins, a range of 189 different sugars and glycine.37 Interestingly, casein and glycine were components of 190 early extenders such as the CGH-27 extender described by Nishikawa in 1975.13 After 191 48 and 72 hours of storage at 5 ºC semen stored in EquiPro® extender reportedly had 192 significantly higher sperm motility than that stored in a Kenney extender. They also 193 reported that centrifugation and removal of 90% of the seminal plasma, which is replaced 194 by the defined milk protein extender, increased the longevity of sperm during storage. 195 Seminal plasma 196 The adverse effects of seminal plasma on the survival of equine sperm were 197 recognized as early as the 1930s in the investigations of semen storage.5,38,39 Many early 198 investigations not only examined various extender formulations, but also optimal dilution 199 ratios of semen in extender. More recently, Colorado workers demonstrated that when 200 using milk-based extenders, complete removal of seminal plasma resulted in significant 201 377 reductions in the sperm motion characteristics of cooled equine semen whereas 202 suspension of equine sperm in extenders containing 5 to 20% seminal plasma maintained 203 motion characteristics for over 72 hours of cooled storage.40,41 Subsequently, it has been 204 widely recommended that dilution ratios of at least three to four parts extender to one part 205 semen be used for cooled equine semen, so that the level of seminal plasma does not 206 exceed 20 to 25% by volume and the sperm concentration remains between 25 x 106 and 207 50 x 106/mL. For some stallions, whose sperm do not tolerate the rigors of cooling and 208 storage using simple dilution, centrifugation and partial removal of the seminal plasma to 209 achieve even lower levels (≤ 10 to 12%, v:v) may be necessary to optimize sperm 210 survival.4 However there are other stallions whose seminal plasma is so toxic to their 211 sperm that complete removal is necessary to avoid a rapid reduction in longevity. When 212 complete removal of seminal plasma is required, alternatives to typical milk-based 213 extenders must be employed. 214 Padilla and Foote43 demonstrated that after centrifugation and complete removal 215 of seminal plasma, the motility of cooled-stored equine sperm was greatly improved 216 when resuspended in a Kenney’s NFDSM-Gluc extender supplemented with a high-217 potassium modified Tyrode’s medium (KMT). However when KMT extender was used 218 in the presence of seminal plasma, motility was reduced, indicating an interaction 219 between seminal plasma and the extender composition. Workers at Texas A&M 220 confirmed these results, and went on further to demonstrate that fertility was maintained 221 with 13 of 17 mares becoming pregnant using semen stored for 48 h in the KMT 222 extender.44 Other work from this laboratory demonstrated that both motility and DNA 223 integrity were maintained in sperm from which seminal plasma was removed, followed 224 378 by resuspension in either Kenney extender or modified Kenney Tyrodes-type extender 225 [45]. Other investigators have shown that motion characteristics and acrosomal integrity 226 of sperm are maintained when stored 48 hours after seminal plasma removal and 227 resuspension in a Kenney extender supplemented with commercially available phosphate 228 buffered saline containing glucose and pyruvate.46 Investigators from this laboratory 229 also reported pregnancy rates of 75% (3/4) and 88% (22/25), when this extender was 230 used with semen from two poor cooling stallions in a commercial cooled-transported 231 semen program.47 232 The effects of seminal plasma are not always deleterious and appear to be stallion 233 dependent. When semen from stallions that exhibited low post-thaw sperm motility 234 (<20%) was supplemented with seminal plasma from stallions that produce semen with 235 high post-thaw motility, greater numbers of spermatozoa survived cryopreservation.48 236 Cryoprotectants 237 The discovery in the 1930s that the addition of egg yolk to suitable buffers 238 significantly increased the fertilizing capacity of stored sperm from a number of species 239 resulted in the widespread use of artificial insemination in dairy cows.6,49,50 Most equine 240 freezing extenders consist of milk, egg yolk, glycerol, various sugars, and electrolytes. 241 While chicken eggs are the most common source of yolk used in semen extenders, yolk 242 from other species has been substituted with favorable results. One study demonstrated 243 that sperm motility parameters were improved when stallion semen was frozen in lactose 244 EDTA extender supplemented with duck egg yolk rather than chicken egg yolk .51 245 379 Glycerol has been one of the most widely used cryoprotectants for frozen semen. 246 However, while a higher level of glycerol often yields better post-thaw sperm motility, 247 higher glycerol levels are also contraceptive in the mare.12,52,53 Levels of glycerol in 248 early studies ranged from as low a 1% to as high as 10%. In fact, the first reported 249 pregnancy using frozen-thawed epididymal stallion sperm was obtained using an 250 extender containing 10% glycerol (glycerin).54 Many equine semen freezing extenders 251 currently contain approximately 4% glycerol, but some European studies suggest that a 252 final glycerol concentration of 2 to 3.5% may be most appropriate for cryopreservation of 253 equine semen.55,56 However, INRA 96 with 6% glycerol was recently reported to 254 improve survivability of cryopreserved equine sperm while not adversely affecting 255 fertility.57 256 Because of the tremendous variability observed in the post-thaw motility and 257 fertility of stallion semen frozen in conventional extenders, alternative cryoprotectants to 258 glycerol have been investigated. In one study, the presence of glutamine at 50 mM was 259 not sufficient to offset the need to use glycerol.58 However, it was found that 50 mM 260 glutamine added to a 2.5% glycerol medium significantly improved sperm motility 261 compared to classical freezing medium containing 2.5% glycerol. These workers 262 concluded that glutamine has a synergistic cryoprotective effect with glycerol on 263 cryopreservation of stallion sperm, and suggested that glutamine acts at the extra-cellular 264 level, independently of glycerol. 265 Recent studies have demonstrated that both methyl formamide and dimethyl 266 formamide could protect stallion sperm from cryodamage as effectively as glycerol, and 267 it was suggested that these cryoprotectants might provide an alternative for stallions that 268 380 have poor post-thaw sperm motility when frozen in glycerol.59 A new freezing extender 269 Botu-Crio® (Biotech Botucatu, Botucatu,Sao Paulo, Brazil) has recently been made 270 commercially available. The main difference in Botu-Crio® compared to other freezing 271 extenders, is the combination of glycerol and methyl formamide as the cryoprotectant. 272 Fertility was assessed for good and poor freezing stallions in a retrospective analysis of 273 355 cycles of mares bred with semen frozen in a glucose–EDTA–lactose extender 274 containing glycerol and on 98 mare cycles for semen frozen in Botu-Crio®.60 While 275 there was no difference in fertility in the good freezing group between extenders, fertility 276 of the poor freezing group was significantly better for semen frozen in Botu-Crio®. It 277 was concluded that the Botu-Crio® extender appears to improve the post-thaw quality 278 and fertility of stallions with semen that is considered to have poor freezability. 279 Antioxidants 280 Oxidative damage to sperm during storage is thought to be a potential cause of the 281 decline in motility and fertility. Endogenous lipase activity in seminal plasma was 282 suggested to be a contributing factor in the adverse effects of seminal plasma on cooled 283 stallion sperm.61 Numerous antioxidants have been added to semen extenders, with 284 varying results, in an effort to prevent damage to equine sperm by lipid peroxidation.62-68 285 As with many other extender additives, much of the work with antioxidants has examined 286 in vitro sperm characteristics rather than fertility. 287 The addition of taurine to several different extenders was reported to consistently 288 result in better sperm motility after storage than non-taurine containing extenders.62 289 Addition of ascorbic acid was found to increase the percentage of membrane intact sperm 290 stored in a skim milk extender compared to controls.65 In contrast, another study found 291 381 that the addition of the enzymatic scavenger catalase, or a variety of water-soluble or 292 lipid-soluble antioxidants did not significantly improve the maintenance of sperm 293 motility in semen stored at 5 °C in a NFDSM-Gluc extender.64 The addition of 2 mM 294 pyruvate to a skim milk extender was beneficial in maintaining sperm motility for semen 295 stored for 48 hours, and based on embryo recovery rates, also tended to improve 296 fertility.69 Although lactate dehydrogenase activity was found to be correlated with 297 sperm motility,70 neither pyruvate nor lactate could protect sperm from a H2O2 challenge, 298 and it was suggested that beneficial effects exerted by the addition of pyruvate or lactate 299 to semen extenders were probably resulting from them acting as an energy source rather 300 than as antioxidants.67 Quercetin was recently reported to protect sperm from 301 peroxidation after challenge with xanthine-xanthine-oxidase.66 These authors also 302 suggested that addition of quercetin to NFDSM-Gluc extender could reduce lipid 303 peroxidation of sperm and thereby prevent premature capacitation of sperm while still 304 allowing the sperm to capacitate and acrosome react after insemination.68 However, this 305 latter conclusion was drawn from the ability of sperm in quercetin treated semen extender 306 to acrosome react after challenge with A23187, which is not very physiologic, and the 307 authors rightly suggested that fertility trials should be performed to determine the 308 effectiveness of quercetin on sperm storage. 309 The value of including additional antioxidants to semen extenders has been 310 challenged by results of more recent experiments, which indicate that there is not a 311 substantial increase in lipid peroxidation during semen storage and that peroxidative 312 damage to sperm membranes is not the predominant cause of reduced semen quality.71,72 313 Workers from this laboratory report that the inherent antioxidative activity in stallion 314 382 semen appears to prevent the formation of reactive oxygen species (ROS) and that the 315 simple addition of extender increases this activity further.71,72 Other workers have also 316 suggested that although equine seminal plasma contains high superoxide dismutase-like 317 activity, sperm themselves have limited glutathione peroxidase and superoxide 318 dismutase-like activity.73 They also suggest that the enzymatic antioxidant activity in 319 equine sperm appears to be predominantly derived from seminal plasma adsorbed onto 320 the sperm plasma membrane and that removal of seminal plasma during semen 321 processing may increase oxidative stress in equine sperm. Brazilian investigators reported 322 that lipid oxidation in the seminal plasma appeared to be a general indicator for sperm 323 damage and suggested that both lipid and protein oxidation may aid in the identification 324 of subfertile stallions, but only during the non-breeding season.74 They also reported that 325 ROS production levels did not appear to result in compromised sperm DNA integrity, 326 which indicated to them that either the measurements were within physiological levels 327 and/or that there is an efficient antioxidant activity in stallion sperm cells.74 328 Conclusions 329 The NFDSM-Gluc formulation, with slight variations on the basic components 330 ranging from antibiotics to sugars, remains the mainstay of equine semen extenders. 331 Inclusion of a variety of other components such as cryoprotectants and antioxidants has 332 also been attempted, with mixed results. 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