HOW TO INCORPORATE ASSISTED REPRODUCTIVE TECHNOLOGIES


years old), and subsequent ICSI was performed us- ing frozen-thawed semen from 147 different stal- lions. OPU was performed year round, except for July and August, at Utrecht University (the Neth- erlands) using amodified human transvaginal probe and a 12-gauge double-lumen needle. Periproce- dure antibiosis, analgesia, epidural anesthesia, and sedation were provided as described by Claes et al.4 Follicles larger than 5 mm were punctured and the fluid aspirated into collecting bottles, using a vac- uum pump adjusted to aspirate 20 mL of fluid in approximately 1 minute. After emptying, follicles were flushed 5 to 12 times (depending on the num- ber of follicles present) with a follicle-size dependent volume (0.5 to 7 mL) of either a commercial embryo flushing mediuma,b supplemented with 20,000 IU/L heparin sodium,c or a commercial oocyte recovery medium.d Recovered fluid was passed through a 75-m filter, and the contents of the filter were washed and the cumulus-oocyte-complexes located using a dissecting microscope. Recovered oocytes were packaged into 1-mL cryovials in modified Hepes-buffered Synthetic Oviductal Fluid (mH- SOF), and shipped overnight to Avantea (Cremona, Italy) at 22°C in prewarmed polystyrene boxes de- signed for the transport of organs for transplanta- tion, an Equitainere or a temperature controlled incubator.f In vitro oocyte maturation (24 hours), ICSI, and in


vitro culture of embryos were performed as de- scribed previously.4 After IVM, ICSI was per- formed on all oocytes with a visible polar body (i.e., at the metaphase II (MII) stage) using frozen- thawed semen from a stallion selected by the mare owner. A small piece (one third to one tenth) of a 0.5-mL straw was cut off and thawed by immersion in modified Tyrode’s Albumin, Lactate, Pyruvate medium at 38°C. Motile, grossly morphologically normal sperm cells were then selected either by swim-up or by centrifugation through a 45–90% Per- coll density gradient, and ICSI was performed using a Piezo drill. In vitro blastocyst formation was as- sessed daily on days 6 to 8 (day of ICSI  day 0). Resulting blastocysts were cryopreserved immedi- ately after recognition by controlled rate freezing in mH-SOF containing 10% glycerol, and stored in liq- uid nitrogen. Embryos were transported back to Utrecht in liq-


uid nitrogen. Approximately 30 to 60 minutes be- fore transfer into recipient mares, embryos were thawed and the glycerol was washed out by sequen- tial 5-minute washes in mH-SOF containing 8, 6, 4, 2, and 0% glycerol.4 Transfer to recipient mares was performed in mares sedated with 4 mg detomi- dine hydrochloride (IV) using a Polansky type spec- ulum and Wilsher forceps on days 3 to 6 after ovulation; day 4 after ovulation was considered ideal, and deviations from this were to avoid week- ends or for other practical reasons. Recipient mares received no additional treatments, and sub- sequent examination for pregnancy was carried out


7 to 10 days after transfer. If no pregnancy was seen, mares were reexamined 2 to 4 days later. In the case of pregnancy, mares were reexamined 23 and 37 days after embryo transfer (ET). A combination of ANOVA and logistic regression


was used to examine the influence of procedural, donor mare (mares submitted for OPU  5 times) and stallion (stallions used for ICSI on  5 mares) factors that influenced the success of blastocyst for- mation in terms of the likelihood of producing a blastocyst, and the mean number of blastocysts per OPU or per injected oocyte. Similar statistical ap- proaches were used to examine the influence of do- nor and recipient mare, and embryo characteristics on the likelihoods of pregnancy and pregnancy loss. To minimize any mare-related bias, when examin- ing stallion effects only the results for the first OPU per mare were included.


3. Results


An average of 24 follicles per mare were aspirated (range, 6–72), yielding a mean of 13.4 oocytes (range, 0–55; 56% recovery). Somewhat surpris- ingly, the efficiency of oocyte recovery did not im- prove markedly with experience, although the number of follicles punctured did increase during the first few months as a result of improved techni- cal proficiency. Follicle number was affected by mare age, with older mares (20 years) presenting fewer follicles (mean  SD, 20.9  7.9 follicles) and yielding fewer oocytes (9.7  4.7 oocytes), with a lower efficiency of recovery (48%). Overall, the percentage of oocytes that success-


fully matured to MII and were injected was 57%. Of these, 68.5% cleaved and 14.1% yielded a blasto- cyst on days 6 to 8; with an average of 1.09 blasto- cysts per OPU (range, 0–10), and with 56% of all OPUs yielding at least 1 embryo. For 31 OPUs, no ICSI was performed because either no oocytes were recovered (once), the oocytes were found to be con- taminated after IVM (once), no oocytes made it to MII (8 times; 0.9%), or transport of the oocytes was delayed by 24 hours and the oocytes arrived at 16°C (21 times). On other occasions, transport was delayed by 24 hours (i.e., 48 hours total transit time) but it was decided to proceed with ICSI be- cause the percentage of oocytes reaching MII was unaffected. When examining mares submitted repeatedly for


OPU (5 times) or stallions used on multiple mares (5), it became apparent that while there was a stallion effect on parameters of ICSI success, there was amore marked mare effect. In this respect, for the most frequently used stallions (n  21) and mares (n  30), the percentage of blastocysts per injected oocyte ranged from 4.4% to 30.1% (mean, 13.7%) for stallions compared to 0% to 44.4% (mean 18%) for mares; blastocysts per OPU ranged from 0.37 to 1.57 (mean, 1.08) for stallions compared to 0 to 3.29 (mean, 1.39) for mares and the percentage of OPUs yielding at least 1 embryo ranged from 25% to


AAEP PROCEEDINGS  Vol. 65  2019 371


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