Na citrate (g) 25.95 3.7 Disodium EDTA (g) 3.699 3.699 Na bicarbonate (g) 1.2 1.2
Table 5 Composition of the Citrate-EDTA, and Glucose-EDTA media used for centrifugation and extender for freezing equine semen (Adapted from (Cochran et al 1984)).
Lactose solution (11% w:v) 50 (ml) Glucose EDTA solution (Table 5) 25 (ml)
Egg Yolk 20 (ml)
Gylcerol 5 (ml)
Equex STM 0.5 (ml)
Table 6. Composition of the Lactose-EDTA media used as an extender for freezing equine semen (Adapted from (Cochran et al 1984)).
Centrifuge media Freezing extender
UHT Skim Milk INRA 82
Egg yolk (v/v) 2% Glycerol (v/v) 2.5% Table 7 from (Vidament 2005)
Some reezing extenders and centrifugation extenders are often protected by patent and are commercially available (e.g. EZ Freezin™ “LE” (Lactose EDTA) or E-Z Freezin™ “MFR5” (modified French), Animal Reproduction Systems, Chino, US; Ghent™, Minitube, Landshut, Germany; EqcellSire A+B™, IMV, L´Áigle,. France; BotuCrio™, www.criovital.com).
Commercial extenders should be sterile, quality tested and standardized and are convenient. Because of errors in measurement and /or contamination of media we encourage the use of commercial formulations for freezing semen. It should be noted, however, that occasionally procedures that are designed to sterilise media may decrease spermatozoal motility (Aurich et al 2007).
Centrifugation. The gel free portion of the ejaculate and is diluted to (roughly) 50 X 106 spermatozoa per ml or 1:1 with centrifuge medium prewarmed to 370C. After though mixing the suspension is transferred into 50 ml centrifuge tubes and centrifuged for 15 min at approximately 1500-1900 RPM (400-600g). The exact conditions of centrifugation may have to be varied to produce a proper pellet and depends on the concentration of spermatozoa and even its motility. Low motility and concentration samples will form a firmer pellet in less time.
Centrifugation is a process that damages the sperm. An alternative is to freeze concentrated spermatozoa collected as the sperm rich fraction with an open ended AV.
Most laboratories indicate g forces that are from the bottom of the tube. The tables below clearly show the effect on g forces as the distance from the centre of the tube increases. For example if we centrifuged at 1500 rpm then the bottom of the sample would have a force 344 g and the top of the tube a force of 130 g if the tubes were filled to 50 ml. However if double the number of centrifuge tubes were used then each tube would have a force at the top of extended sample of 238 g as only half of the fluid volume would be present in each tube (25 ml in this example) at 1500rpm. Thus recovery of sperm is increased when lower volumes are used to centrifuge. The disadvantage of this technique is that more sperm can be lost when multiple tubes are used due to smaller pellet size and difficulty removing the semen without losses.
g= RCF=0.00001118rN2
g= Relative centrifuge force r= Rotational radius (cm) N= Rotating speed (rpm)
Table 9 Gravitational (g) forces listed at the bottom of the centrifuge tube using the distance from the
centre of the rotor at varying rpm
3.7cm 5.2cm 6.9cm 8.6cm 10.3cm 12cm 13.7cm G 60ml 50ml 40ml 30ml 20ml 10ml 0ml 1000rpm 41 58 77 96 115 134 153 1100rpm 58 70 93 116 140 162 185 1200rpm 60 84 111 139 166 193 221 1300rpm 70 98 130 163 195 227 259 1400rpm 81 114 152 189 227 264 301 1500rpm 93 130 174 216 259 302 344 1600rpm 106 149 197 246 295 343 395 1700rpm 120 168 223 278 333 388 443 1800rpm 134 188 250 311 373 434 496 1900rpm 149 209 278 346 416 484 553 2000rpm 165 232 309 384 460 536 612 2100rpm 182 256 340 424 508 592 675 2200rpm 200 281 373 465 557 649 741 2300rpm 219 307 408 509 608 710 810 2400rpm 238 336 444 554 663 773 882 2500rpm 259 363 482 601 719 838 957 3000rpm 372 523 694 865 1036 1207 1378 3500rpm 567 712 944 1177 1410 1642 1875 4000rpm 662 930 1234 1538 1842 2147 2450 Table 10 g forces at the specific depth of fluids (calculated for our centrifuge).
Alternatively a Nomograph can be used to calculate the g forces (These are available on the internet- Fig 26). G cm 5 6 7 8 9 10 11 12 13 14 15 1000rpm 55.9 67.08 78.26 100.6 100.6 111 122.9 134.1 145.3 156.5 167.7 1100rpm 67.5 81 94.5 108 121.5 135 148.5 162 175.5 189 202.5 1200rpm 80.5 96.6 112.7 128.8 144.9 161 177.1 193.2 209.3 225.4 241.5 1300rpm 94.5 113.4 132.3 151.2 170.1 189 207.9 226.8 245.7 264.6 283.5 1400rpm 110 132 154 176 198 220 242 264 286 308 330 1500rpm 125 150 175 200 225 250 275 300 325 350 375 1600rpm 143 171.6 200.2 228.8 257.4 286 314.6 343.2 371.8 400.4 429 1700rpm 161.5 193.8 226.1 258.4 290.7 323 355.3 387.6 419.9 452.2 484.5 1800rpm 181 217.2 253.4 289.6 325.8 362 398.2 434.4 470.6 506.8 543 1900rpm 201.1 241.3 281.6 321.8 362.0 402 442.5 482.7 522.9 563.2 603.4 2000rpm 223.5 268.2 312.9 357.6 402.3 447 491.7 536.4 581.1 625.8 670.5 2100rpm 246.5 295.8 345.1 394.4 443.7 493 542.3 591.6 640.9 690.2 739.5 2200rpm 270.5 324.6 378.7 432.8 486.9 541 595.1 649.2 703.3 757.4 811.5 2300rpm 295 354 413 472 531 590 649 708 767 826 885 2400rpm 320 384 448 512 576 640 704 768 832 896 960 2500rpm 349.5 419.4 489.3 559.2 629.1 699 768.9 838.8 908.7 978.6 1048.5 3000rpm 503 603.6 704.2 804.8 905.4 1006 1106.6 1207.2 1307.8 1408.4 1509 3500rpm 685 822 959 1096 1233 1370 1507 1644 1781 1918 2055 4000rpm 895 1074 1253 1432 1611 1790 1969 2148 2327 2506 2685
Fig 26 With a nomogram (nomograph) the relative centrifugal force (RCF) can be calculated with RPM and the radius at the bottom of the centrifuge tube. From http://aquaticpath.umd.edu/nomogram.html
Another process becoming more popular is the use of a cushion fluid deposited at the bottom of the centrifuge tube (Fig 27). The layer or cushion enables high speed centrifugation (1000 g for 20 minutes). The cushion fluid is 60% wt/vol iodixanol in water and is a significantly denser medium than the sperm extender mixture. It is marketed as Optiprep™ (Axis-Shield, Oslo, Norway); Eqcellsire™ Component B (IMV, LÁigle, France) and CushionFluid™ (Minitüb, Landshut, Germany). We have recently evaluated this for freezing of spermatozoa and have been very impressed with the differences in post centrifuge motility of semen compared to our more traditional two stage centrifuge. In addition very high recovery rates are found. It is assumed a 95% recovery rate with 1000 g for 20 minutes is achievable; however it is prudent to always examine the supernatant to assure that it is nearly sperm free. Regardless, good laboratory procedures would dictate a measurement of final spermatozoa concentration prior to loading straws. This can be achieved with a Nucleocounter (SP 100TM), a CASA with killed sperm and LejaTM slides or a haemacytometer count.
Fig 27 Layer of sperm on cushion fluid post centrifuge
The type of iodixanol cushion medium used (i.e., OptiPrep™, Eqcellsire® Component B, or Cushion Fluid™) does not impact post-centrifugation semen quality. Use of extenders with high viscosities may result in reduced sperm sedimentation rates during centrifugation. Still another possibility is the use of glass nipple-bottom tubes (Pesce Laboratory Sales, Kenneth Square, PA, USA) combined with a small volume (~30µl) of cushion fluid. This maximum recovery when low numbers of spermatozoa are provided in an ejaculate. In addition after aspiration of supernatant the resuspension of sperm pellet and cushion fluid together in extender and then AI without removal of the cushion fluid is possible. It has been reported that high speed cushioned centrifugation of stallion semen had no detrimental effect on fertility (Ecot et al 2005).
Apparently is better to use an opaque semen extender (NFDSM) rather than an optically clear extender (Waite et al 2008).
‘Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear velocity, and average-path velocity) yielded higher values following centrifugation in nipple-bottom tubes (P<0.05). Sperm recovery rate following cushioned centrifugation was similar between semen previously diluted in optically clear centrifugation extender (100%) and semen diluted in opaque centrifugation extender (100%); however, MOT and PMOT were higher in semen subjected to cushioned centrifugation in opaque extender (P<0.05). An extender by tube-type interaction was not detected for recovery rate or post-centrifugation semen quality. In Experiment 2, sperm recovery rate following cushioned centrifugation in nipple-bottom tubes was similar when forces of 400xg or 600xg were applied (90 and 90%, respectively; P>0.05), and no resulting differences in semen quality were detected between these treatment groups (P>0.05). The type of iodixanol cushion medium used (i.e., OptiPrep, Eqcellsire Component B, or Cushion Fluid did not impact post- centrifugation semen quality, based on the laboratory values measured (P>0.05). In conclusion, cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes yielded a high sperm harvest, while maintaining sperm function. An optically opaque extender, commonly used in the equine breeding industry, can be used to achieve this goal’(Waite et al 2008)
The supernatant is discarded taking care not remove any sperm from the pellet and the semen is reconstituted in the appropriate volume of freezing extender, which has been placed on the bench from the incubator (at 370C) at the beginning of centrifugation. At this time both the centrifuged sample and the extender have cooled to between 22-270C (providing centrifuge holders are prewarmed and the laboratory is thermostatically controlled). Note: For the first few times a particular stallion is frozen the concentration of spermatozoa remaining in the centrifuge medium should be used to determine the number of spermatozoa lost through processing. Select Breeder Service laboratories always count the final concentration of spermatozoa prior to loading straws (see above).We normally expect a recovery of 80% of the spermatozoa that are centrifuged at 400-600g and 95% at 1000g. A 10% reduction in spermatozoal motility associated with centrifugation is acceptable but we generally do not see this with a cushinoid centrifuge. For most laboratories the aim is to have one billion (109) (range 800 X 106 to 1 X 109) spermatozoa per insemination dose, regardless of the progressive motility. This is based on trail freezes with acceptance only of stallions with a post freeze progressive motility of >30%. At least one study (Volkmann and van Zyl 1987) has demonstrated significantly better pregnancy rates by increasing the number of PMS from 175 x 106 to 249 X 106.
Packaging. Straws should be labelled with the stallions name, the registration number (if applicable), the breed, the processing center and the date of collection. Our preferred method for sealing the 0.5 ml straws is with coloured plastic balls that are seated in about 3 mm. We use a semi-automatic straw filling and sealing machine from IMV. 0.5 ml polyvinylchloride straws can also be sealed with PVC powder or heat sealed at the opposite end to the cotton plug. 5 ml or 2.5 ml straws can be sealed with coloured plastic beads or ball bearings. When loading semen, failure to have an air bubble within the fluid column, to allow for rapid expansion of ice crystals during thawing, may result the straw exploding.
Cooling. Although not universally accepted, we find that for most stallions, slow cooling of the straws from room temperature to 50C at a rate of ~-0.1/min improves the progressive motility of the frozen/thawed spermatozoa. This is more apparent when using certain types of freezing extenders.
Freezing. The use of programmable freezers produces the most consistent freezing of straws, however straws have also been successfully frozen over a liquid nitrogen vapour. To freeze over a vapour a specific quantity of liquid nitrogen is placed in a styrofoam container, usually up to a designated line, relative to the height of the freezing rack. For 0.5ml straws, positioning the straws between 1-8 cm above the surface has been advocated. Straws remain at this position for 5-10 min and are then plunged into liquid nitrogen. Considerable experimentation with each individual stallion is necessary with each individual stallion to provide the most optimum cooling rate. Five ml straws are placed 1-3 cm above the level of liquid nitrogen for 15-20 min and then plunged. When the straws are in the liquid nitrogen vapour they can cool to -1600C if left long enough. They are stored at -1960C in the liquid nitrogen.
Mechanical freezers usually cool at a rate of 100C/min to -150C and then from 250C/min from -15 to - 1200C. Straws are held at -1200C for 2-3 min and then plunged into liquid nitrogen.
Thawing. A variety of different regimes have been proposed.
1) For 0.5 ml straws (PVC) they can either be thawed by placing then in 370C water bath for 30 seconds or >, or into 750C for exactly 7 secs then into 370C for at least 5 secs (Pickett and Amann 1993).
2) For 2.5 ml straws are recommended to be thawed for 5 min in a 370C waterbath.
3) 5 ml straws are placed in a 500C waterbath for 45 sec and then into 370C for at least 10 sec.
Post thaw evaluation. Post thaw quality of spermatozoa representing each ejaculate processed should be evaluated approximately 0-2 days after the freeze. This should prevent semen being frozen from a stallion if he has a decrease in semen quality whilst at the collection facility.
Two straws representing each freeze should be examined. The entire contents of the straw should be discharged into prewarmed extender at 370C. The extender semen ratio should be adjusted to give a final concentration for motility evaluation of ~ 20 X 106. Motility estimates should be made at 0 and 30 min after thawing (semen maintained at 370C). Criteria for acceptability is commonly 30% PMS at thaw and > 40% PMS at 30 min.
We use a CASA (computer automated sperm analysis) system from Hamilton Thorn Biosciences (CEROS) (see above). This equipment allows operators to evaluate the semen accurately without influences of prejudice.
A minimum accepted dose is 200 X 106 motile spermatozoa. The decision as to whether to accept semen of lesser quality will depend on factors such as stallion age and value of the offspring etc. Utilising the above techniques above our aim is always to provide at least between 300 X 106 PMS per insemination dose.
Facilities available for the use of artificial insemination of horses.
As frozen/thawed semen has a short half-life, insemination must take place close to ovulation. This necessitates frequent examination of the mare as often as may be required to accurately predict ovulation. It is considered that manual palpation alone will not provide sufficiently reliable evidence on when to predict ovulation and thus facilities should include the capabilities of ultrasonographic examination of follicular development.
Facilities should be available to breed the mare under cover with adequate restraint to avoid damage to the mare or personnel involved in with the procedure. Restraining devices such as stocks should be immediately adjacent to the semen handling area or laboratory and should be constructed to avoid damage to the semen by temperature fluctuation or contamination before insemination.
Semen Handling: Stallion spermatozoa are fragile, easily damaged and short lived. They are susceptible to cold-shock, direct sunlight, many antibiotics and low levels of residues that may be left on the liners of artificial vaginas, glassware and other receptacles with which the semen may come in contact. Care should be taken to assure that all contact surfaces are made of non-spermicidal material, are thoroughly cleaned and have been rinsed with de-ionised water.
Before insemination, all handling and preparation of semen should take place in a clean room close to the insemination area. This room should be heated or cooled to maintain a minimum temperature of 22- 260C and the following equipment must be available:
a) a microscope (preferably with a heated stage) for evaluating viability of spermatozoa (progressively motile sperm) at 200 x magnification and preferably with phase contrast,
b) an incubator maintaining temperature at 370C , and
c) a water bath for thawing spermatozoa which is capable of accurately maintaining the temperature specified for thawing different batches of semen.
Mare Insemination: The mare should be cleaned and prepared following normal procedures for artificial insemination. Prior to thawing semen, all equipment should be prepared and made ready. Care should be taken to avoid spermicidal preservatives in lubricants by using KY gel. If the thawed semen needs to be removed from its receptacle (ie. glass ampoule or 5 ml straws) prior to insemination, then care should be taken to avoid using syringes with rubber plungers (the lubricant in these syringes has been demonstrated to be spermicidal).
Management of mares inseminated artificially with frozen semen.
Try to assure that only mares of normal fertility are bred with frozen semen.
Pre-breeding Evaluation: The requirements for pre-breeding gynaecological examination will vary according to the veterinarian and status of the mare. Most mares should be cultured and demonstrated negative for venereal pathogens (Klebsiella pneumoniae, Pseudomonas aeruginosa and Taylorella equigenitalis). Ultrasonographic examination should be used to determine that the mare has no signs of uterine inflammation (fluid). Other examination procedures such as uterine cytology, biopsy, endoscopic exam, digital manipulation of the cervix, etc. may be necessary depending upon the history of the mare to be bred, her age and recent foaling complications etc.
Timing of insemination with frozen semen: Although the frozen thawed spermatozoa from some stallions may survive 24 hours or more after insemination, best results are achieved if the semen is inseminated in a period from 12 hours before, until a maximum of 6 hours after ovulation. Insemination greater than 6 hours after ovulation results in a decreased chance of conception and an increased incidence of early embryonic death. Because each breeding unit of frozen semen is expensive to produce, it is recommended mares are bred a maximum of 2 times per cycle, and preferably once. There are various ways that a mare can be
managed during the insemination period to optimise the time of insemination and vary according to economic considerations, facilities and veterinary experience:
A) Ultrasonographic examination every 6 hours. Mares are inseminated immediately ovulation has been detected.
B) 12-24 hourly examination. Ultrasonography is used to determine when the follicle(s) is immediately pre-ovulatory. The mare is inseminated immediately and re-checked in 12-24 hours. If ovulation has not occurred 24 hours later, the mare is re-inseminated at each inspection until ovulation has occurred. This will result in the use of more semen.
C) Ovulation induction. When ultrasonographic evaluation of a mare in early oestrus reveals the presence of 30-35 mm follicle and she is treated with 3000 IU of hCG (i.v.) then most mares will ovulated in 36 + 4 hours. Under these circumstances, the mare is examined at hCG + 12 hours then + 24 hours . The mare may be inseminated at this time and re-inseminated 24 hours later if she has not ovulated (hCG + 48 hours). An alternative method is to inseminate all mares once at hCG + 36 hours. The advantages of using hCG are that it limits the number of examinations, while still allowing flexibility in relation to time of insemination. If pregnancy is not established, then subsequent cycles may be best managed with the use of another ovulation-inducing agent (Deslorelin -GnRH) (McKinnon et al 1992) in those mares where hCG antibodies may result in a varied or less predictable response. Using Deslorelin we anticipate ovulation at 41 ± 3 hours (McKinnon et al 1997). So mares are commonly treated with Deslorelin at 7 pm. With this approach we find most mares ovulate from 6 am to 4 pm, two days after the 7 pm administration (41 hours ± two Standard Deviations or 6 hours) (Fig 28). When treated with either ovulation drug, if time and facilities permit (i.e. a referral hospital) we would examine those mares at hCG/Deslorelin Q 6 hours until ovulation is confirmed. This has the advantage of continually monitoring fluid and also avoiding breeding mares that have abnormal ovulations such as AHF’s.
Time to ovulation -2 0 2 4 6 8 10 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Hours Nu m b er of m a res ovulat ing hCG Ovuplant
Fig 28 Adapted from (McKinnon et al 1997)
Artificial insemination: The mare should be restrained and then prepared for breeding as hygienically as possible and clean water should remove all traces of spermicidal antiseptic soaps, etc. After the frozen semen has been thawed according to the suppliers recommendations, it should be inseminated immediately. When the breeding dose is contained in one 0.5 ml straw, it will be difficult to evaluate the motility of the spermatozoa from samples bred. In these cases, recommendations are to sacrifice 1-2 straws in every 10 processed to assess the motility from each ejaculate. On other occasions, when semen needs to be placed inside a syringe prior to insemination, it is possible to check the motility after breeding from residues left within the A.I. pipette or syringe. Accurate evaluation will only be possible after the semen has been warmed to 37oC for 5 minutes (use a warmed slide and cover slip). If possible, insemination should be performed as far up the uterine horn ipsilateral to the ovulatory follicle as possible.
Post-artificial insemination examination: Mares should routinely be examined 24 hours after breeding to ensure ovulation has occurred and that there is no evidence of uterine fluid which may necessitate intra- uterine or systemic anti-bacterial treatment. Post-breeding surgery such as Caslick may be performed at