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Theriogenology 76 (2011) 143152 www.theriojournal.com

Successful cryopreservation of expanded equine blastocysts

Y.H. Choia, I.C. Veleza, F.L. Rierab, J.E. Roldnb, D.L. Hartmanc, S.B. Blissc, T.L. Blancharda, S.S. Haydena, K. Hinrichsa,*
a

College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas 77843, USA b Centro de Reproduccin Equina Doa Pilar, Lincoln, Argentina c Hartman Equine Reproduction Center, Whitesboro, TX 76273, USA Received 26 September 2010; received in revised form 23 December 2010; accepted 25 January 2011

Abstract Effective cryopreservation of expanded equine blastocysts ( 300 m in diameter) has been difcult, perhaps due to the volume of blastocoele uid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrication. First, non-biopsied, small embryos ( 300 m) were vitried in ne-diameter microloader pipette tips using dimethylsulfoxidecontaining medium (DM) or ethylene glycol-containing medium (EG). A third group was vitried with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrication, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300 730 m in diameter were then biopsied and vitried; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele uid after biopsy was associated with higher survival. Therefore, an altered (Central) biopsy technique was used to aspirate blastocoele uid, followed by vitrication in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P 0.1). Finally, expanded blastocysts 407 to 565 m in diameter were biopsied from the periphery, and blastocoele uid was removed with gentle suction. After vitrication with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These ndings demonstrated that blastocoele collapse and vitrication in ne-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts. 2011 Elsevier Inc. All rights reserved.
Keywords: Embryo; Cryopreservation; Embryo transfer; Embryo freezing; Horse

1. Introduction Cryopreservation of equine embryos has extensive clinical application, but has been problematic. Although small equine embryos ( 300 m in diameter) have had acceptable pregnancy rates after cryopreservation, both by conventional freezing and by vitrica-

* Corresponding author. Tel.: 979-862-1338; fax: 979-845-6544. E-mail address: khinrichs@cvm.tamu.edu (K. Hinrichs). 0093-691X/$ see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.01.028

tion (45 67%) [1,2], rates of pregnancy after transfer of cryopreserved equine blastocysts 300 m in diameter have been low (0 to 38%) [15]. The equine blastocyst expands rapidly during its rst days in the uterus, increasing from an average diameter of 200 m at Day 6 after ovulation, to 400 m at Day 7, and over 800 m at Day 8 [6]. Because of the relatively delayed entry of the equine embryo into the uterus from the oviducts (late Day 5 after ovulation) [7], obtaining small embryos for cryopreservation en-

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tails increased monitoring of ovulation timing and adjustment of the time of embryo recovery. Lower recovery rates have been reported when uterine ush is performed on Day 6 than on later days [8 10]. Consequently, equine embryos are typically ushed from the uterus on Days 7 or 8 after ovulation. Because of the drawbacks of embryo recovery at Day 6, a focus of equine cryopreservation research has been to develop methods to successfully cryopreserve Day 7 expanded blastocysts. The equine blastocyst at Day 7 is covered with a resilient embryonic capsule [6], which starts to form soon after the equine embryo enters the uterus [11]. The difculty in cryopreserving expanded equine blastocysts may be related to the presence of the capsule, to the status of differentiation of the embryo, to the large amount of uid within the blastocoele, or just to the overall size of the embryo. Attempts to overcome these obstacles have been unsuccessful. Treatment with trypsin to dissolve the capsule did not signicantly improve viability of expanded equine blastocysts after cryopreservation [12,13]. Use of cytochalasin B in an attempt to prevent damage to the cell membrane also was not effective [13]. Removal of the capsule does not appear to be an option, as this results in failure of the blastocyst to develop after transfer [14]. Dehydration of the blastocyst before freezing did not increase pregnancy rates (i.e., one pregnancy from 16 embryos) [5]. We recently reported successful trophoblast biopsy of equine expanded blastocysts up to 1,350 m in diameter, using micromanipulation with the Piezo drill [15]. The capsule was punctured during the biopsy and the blastocyst collapsed; however, these biopsied embryos resulted in normal pregnancy rates after transfer. Perhaps similar breaching of the trophoblast and capsule, or collapse of the blastocoele, could facilitate cryopreservation of equine embryos. In human embryos, puncture of the blastocoele to allow collapse was associated with higher viability after cryopreservation [16,17]. This approach has also been utilized in pig blastocysts [18]. To the best of our knowledge, only one report on manipulation of the blastocoele uid in expanded equine embryos is available; in this report, one pregnancy was achieved with an embryo in which the blastocoele uid was aspirated and replaced with vitrication solution. Unfortunately, this pregnancy was lost before 30 d [19]. In vitrication, the speed of the cooling step is essential; in that regard, smaller volumes of medium lead to faster cooling rates. Ferret embryos, like those of the horse, have high amounts of lipid and are difcult to

cryopreserve successfully (review, [20]). In a recent report, ferret embryos vitried in ne diameter microloader pipette tips (Eppendorf, Westbury, NY, USA), had live birth rates up to 77% after thawing and transfer [20]. This method may support better viability in equine embryos after vitrication than does the 0.25 mL straw currently utilized [2]. The present study was conducted to investigate methods to establish a repeatable, effective method for cryopreservation of expanded equine blastocysts 300 m in diameter, using capsule puncture and blastocoele collapse via micromanipulation, followed by vitrication using a ne diameter pipette (microloader tip). 2. Materials and methods 2.1. Experimental design Five studies were conducted. The design of these studies is outlined below; methods used in the experiments are detailed starting in Section 2.2. 2.1.1. Study 1. Standard vitrication of biopsied embryos Study 1 was conducted to survey whether Day 6 or early Day 7 embryos ( 350 m diameter) that had been collapsed after biopsy could be successfully vitried using a standard vitrication technique using 0.25 mL straws [2]. Embryos (n 4) collected on Days 6 or 7 after ovulation were biopsied and vitried either immediately after biopsy (n 1), or after culture to allow re-expansion (3 to 6 h) before vitrication (n 3). Embryos were subsequently warmed and cultured in vitro for 48 h to assess viability. Subsequently, ve Day 6 embryos, 157 to 260 m in diameter, were biopsied and immediately vitried; thereafter, they were warmed and transferred to recipient mares. 2.1.2. Study 2. Viability of non-biopsied embryos 300 m in diameter, vitried using a ne-diameter pipette Study 2 was conducted to evaluate alternative methods for embryo vitrication, using non-biopsied embryos. In vitro-produced blastocysts ( 250 m diameter) were vitried in microloader tips by one of two methods: DM (utilizing dimethylsulfoxide) as described by Sun et al [20] (n 10); or EG (utilizing ethylene glycol) as described by Campos-Chilln et al [21] (n 8). Vitried embryos were then warmed and cultured in vitro to assess growth. An additional eight in vitro-produced blastocysts were vitried using the DM method, then transferred to recipient mares: four embryos were warmed, cultured

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in vitro for 18 h, then shipped for transfer 4 to 6 h in a warmed passive heat-exchange device (Equitainer, Hamilton Research, Inc., South Hamilton, MA, USA), and four embryos were warmed and immediately shipped for transfer 4 to 6 h in a portable incubator (MicroQ, Mesa, AZ, USA), as detailed in Section 2.6. Subsequently, an additional treatment, EG/s, vitried as for EG, but warmed in sucrose as for the DM method, was assessed. Ten in vitro-produced blastocysts were vitried then warmed, using EG/s, and cultured in vitro to assess growth. Following this, embryos recovered in vivo on D 6 after ovulation, 300 m in diameter, were vitried in either the DM (n 4) or EG/s (n 6) treatments. These embryos were warmed and transferred to recipient mares either immediately after warming, or after culture in vitro for 6 h. 2.1.3. Study 3. Viability of blastocysts 300 m in diameter after biopsy and vitrication Embryos 300 to 730 m in diameter were recovered from mares on Days 7 or 8 after ovulation, and were biopsied. The estimated percentage of blastocoele uid lost during biopsy was recorded. The embryos were vitried immediately after biopsy using either the DM method (n 16) or the EG/s method (n 13). Embryos were then warmed and were shipped in a portable incubator 4 to 6 h, or cultured in vitro 6 h, before transfer. One embryo was shipped in an Equitainer because the portable incubator was not available. 2.1.4. Study 4. Viability of embryos after central suction of blastocoele uid and cells before vitrication, and after immediate or delayed transfer In Study 4, the biopsy procedure was modied to include placement of the pipette into the center of the blastocoele, with active suction to remove the blastocoele uid and obtain cells (Central method). The effect of the 6 h post-warming culture period on pregnancy rates was also evaluated. Day 7 in vivo-produced blastocysts, 300 to 710 m in diameter, were biopsied with the Central method, then vitried using the EG/s technique. After warming, embryos were either transferred immediately to recipient mares (n 7), or were cultured in vitro for 6 h before transfer (n 8). 2.1.5. Study 5. Viability of embryos subjected to trophoblast biopsy and gentle aspiration of blastocoele uid from the trophoblast periphery before vitrication In Study 5, blastocoele uid was aspirated from Day 7 embryos, 407 to 565 m in diameter. Fluid was gently suctioned from the blastocoele with the pipette

remaining at the embryo periphery. These embryos were vitried using the EG/s method, then were warmed and shipped 4 to 6 h in a warmed Equitainer before transfer (n 7). One presumed late Day 7 embryo, 780 m in diameter, was similarly biopsied, vitried, warmed, and transferred. 2.2. Embryo production and recovery In vitro produced blastocysts were obtained from abattoir-derived oocytes, or from oocytes recovered by transvaginal ultrasound-guided follicle aspiration, as previously described [22,23]. Briey, oocytes were matured in vitro, then subjected to intracytoplasmic sperm injection, and presumptive zygotes were cultured in DMEM/F-12 (Sigma-Aldrich, St. Louis, MO, USA), with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) for 7 to 10 d in an atmosphere of 5% CO2, 5% O2, and 90% N2 [24]. For recovery of in vivo-produced blastocysts, mares reproductive tracts were evaluated by transrectal ultrasonography. On the morning that a follicle 33 mm in diameter was found, ovulation was induced by injection of human chorionic gonadotropin (Intervet, Millsboro, DE, USA), 2,000 IU i.v., and/or biorelease deslorelin (BETPharm, Lexington, KY, USA), 1.5 mg i.m., and mares were inseminated. Embryos were collected on Days 6, 7, or 8 after ovulation, by standard transcervical uterine ush. All work with mares was performed according to the United States Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training and was approved by the Laboratory Animal Care Committee at Texas A&M University. Some embryos were produced at commercial embryo transfer facilities (Hartman Equine Reproduction Center, Whitesboro, TX, USA and Centro de Reproduccin Equina Doa Pilar, Lincoln, Argentina) and all embryos were transferred at these facilities. Use of the mares at these facilities was reviewed and approved by the Clinical Research Review Committee, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University. Embryos produced at Hartman Equine Reproduction Center (n 2) were shipped to the laboratory at Texas A&M in holding medium in a shipping container with warmed coolant cans, as previously described [15]. 2.3. Embryo biopsy In Study 1, biopsy was performed in Dulbeccos PBS without calcium and magnesium (Invitrogen) with 0.05% polyvinylalcohol. In Studies 3 through 5, biopsies were performed in CZB-M with 10% FBS [25] or

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in Human Tubal Fluid (HTF; Fecunditas, Buenos Aires, Argentina). Embryo biopsy was conducted as previously reported [15]. Briey, embryos were held with a holding pipette in 50 L droplets of medium in a Petri dish on an inverted microscope. A biopsy pipette, 15 3 m external diameter and attached to a Piezo drill, was used. In embryos with a zona pellucida, the zona was breached by drilling with the Piezo drill. In embryos having only a capsule, the capsule was breached by multiple pulses with the Piezo drill at a higher setting (speed 6, intensity 7). In Studies 1 and 3, the biopsy pipette was then placed against the trophoblast layer and cells were removed by suction, with pulses of the Piezo drill applied if necessary. In Study 4, the biopsy procedure was modied (Central procedure); in this procedure the capsule and trophoblast layer were penetrated, and the pipette was advanced into the center of the blastocoele. Blastocoele uid was obtained by suction until the cavity was emptied ( 70% of uid removed), then cells were obtained from the inner aspect of the blastocyst. In Study 5, the biopsy technique was again modied. The capsule and trophoblast were penetrated, then 70% of uid in the blastocoele was removed by applying gentle suction with the pipette from the periphery of the trophoblast. Thereafter, cells were obtained from the collapsed trophoblast. 2.4. Vitrication In Study 1, biopsied embryos were vitried as described by Eldridge-Panuska et al [2] using 0.25 mL straws. Briey, embryos were washed four times in Dulbeccos PBS without calcium and magnesium, supplemented with 0.3 mM sodium pyruvate, 3.3 mM glucose, and 20% FBS (m-DPBS). Then, embryos were placed in series of media prepared using m-DPBS as the base medium: 1.4 M glycerol for 5 min, 1.4 M glycerol 3.6 M ethylene glycol for 5 min and 3.4 M glycerol 4.6 M ethylene glycol for 1 min. The embryos in the nal solution were loaded into 0.25 mL straws between two columns of dilution solution (0.5 M galactose in m-DPBS) and the straws were heat-sealed, then immediately put into a plastic goblet which was being held in liquid N2, with the opening of the goblet above the level of the N2. After holding in this manner for 1 min, the goblet and straw were plunged into liquid N2. For warming, straws were held in air for 10 s, then in a 20 C water bath for 10 s. They were icked 4 6 times to mix solutions, then held horizontally at room

temperature for 4 5 min. They were transferred to mares, or expressed from the straw, washed, and placed in culture, within 10 min after thawing. In Study 2, embryos were vitried as described by Sun et al [20] using ne-diameter microloader pipette tips, with minor modications (DM method), or as described by Campos-Chilln et al [21], but with the modication that the embryo was vitried in the microloader tip (EG method). The microloader tip was approximately 10 cm long, and had a relatively constant inner diameter of 250 m for approximately the rst 4.5 cm from the free end. The inner diameter then increased gradually, reaching 500 m at 5.8 cm from the free end. For use, the distal 3 cm of the pipette was cut off, resulting in a 7 cm pipette with an inner diameter of 250 m. Tips were cut to larger diameters for larger embryos; the diameter needed was estimated at the time the collapsed embryo was placed in the rst vitrication medium. The largest tip inner diameter used was 495 m for an embryo originally 780 m in diameter before biopsy. Measurements of embryos and of pipette diameters were obtained using a calibrated measurement device on photomicrographs (AxioVision version 4.5, Carl Zeiss MicroImaging Inc., Thornwood, NY, USA). For the DM treatment, embryos were incubated at 38.2 C in Dulbeccos PBS supplemented with 0.1% glucose, 36 mg/L pyruvate, and 0.4% BSA (s-DPBS) for 12 min, then in cryopreservation medium I (sDPBS with 20% FBS, 1.34 M ethylene glycol, and 1.05 M DMSO) for 4 min, and nally in cryopreservation medium II (s-DPBS with 20% FBS, 2.95 M ethylene glycol, 2.32 M DMSO, and 0.9 M sucrose). Within 40 s after transfer to medium II, embryos were aspirated into a microloader tip (one or two embryos per tip) in a minimal amount of medium. The tips were immersed in cryotubes lled with liquid N2, which were then capped and stored in liquid N2 until warming. Embryos were warmed by removing the microloader tips from the cryotubes, and immersing the tips in 3 mL of s-DPBS with 0.3 M sucrose in a 35-mm Petri dish at 38.2 C. The embryos were released into the dish by attaching a pipettor to the tip and evacuating the tip, and were held in the dish for 1 min. The embryos were then placed into s-DPBS containing 0.15 M sucrose for 5 min, then in s-DPBS without sucrose for 5 min. For the EG method, embryos were held in DMEM/F-12 with 20% FBS (DMEM/F-12/FBS), then transferred into 1.5 M ethylene glycol in

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DMEM/F-12/FBS for 5 min, and then moved into 7 M ethylene glycol and 0.6 M galactose in DMEM/ F-12/FBS. While in the nal vitrication solution, the embryos were loaded into a microloader tip and processed within 40 s as described above. The warming procedure was similar to that for the DM method, except that the media used were 1.0, 0.5, and 0.25 M galactose in DMEM/F-12/FBS, and the embryos were held in each medium for 3 min. For the EG/s method, the EG vitrication protocol and DM warming protocol were used. 2.5. Assessment of growth after in vitro culture of biopsied-vitried embryos In Studies 1 and 2, vitried embryos were warmed and cultured in 500 L DMEM/F-12/FBS in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.2 C for 48 to 72 h to evaluate embryo growth in vitro. Embryo growth was assessed by measuring the embryo diameter immediately before biopsy and again at the end of the culture period. For in vitro-produced embryos, this measure was modied. In vitro-produced equine embryos do not expand normally in culture, as the zona pellucida does not thin completely and the capsule does not form. Rather, these embryos expand to a certain extent, then extrude cells through the ICSI- or biopsy-induced breach in

the zona pellucida as they continue to grow (Fig. 1D). Thus, for in vitro-produced embryos that extruded cells through the zona pellucida during the culture period, the total width of the embryo (diameter of the embryo plus the distance to the farthest extent of the extrusion) was used as a measure of growth. 2.6. Embryo handling for transfer Embryos were either shipped for transfer to recipient mares (Texas) or were transferred on-site (Argentina). For shipment, blastocysts were placed in 1.1 mL of equilibrated DMEM/F-12/FBS at 38.2 C in a nominal 1 mL glass vial. The vials were either wrapped in 120 mL ballast at 38.2 C, and placed in an Equitainer in which the container coolant cans had been warmed to 38.2 C, or were placed in a portable incubator at 38.2 C. The time between packaging of shipped embryos and transfer to recipient mares was 4 to 6 h. For transfer, embryos were recovered from the vial and loaded into embryo transfer guns in the transport medium, and transferred within 5 min. Embryos transferred at the site of collection were warmed and either processed for transfer immediately or cultured for 6 h in 200 L DMEM/F-12/FBS in an atmosphere of 5% CO2, 5% O2 and 90% N2 at

Fig 1. In vitro growth of in vitro-produced equine blastocysts vitried with the EG/s technique (A) before vitrication and (B) after vitrication and warming, and at (C) 24 h and (D) 72 h of in vitro culture. Bar 100 m.

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38.2 C before transfer. For transfer, embryos were washed in holding medium (Vigro, Bioniche Animal Health USA, Inc., Pullman, WA, USA), loaded into embryo transfer guns, and transported at room temperature to the location of the recipient mares. Time between loading in the gun and transfer was 30 min to 2 h. Embryo transfer (one blastocyst per mare) was performed transcervically. Pregnancies were evaluated by transrectal ultrasonography starting 5 d after transfer. Pregnancies were monitored ultrasonographically until an embryonic heartbeat was seen (heartbeat stage; approximately D 25) or until D 30 if no embryonic heartbeat was seen, and were terminated by injection of prostaglandin F2 (Lutalyse, Pzer Animal health, Pittsburgh, PA, USA), 5 to 10 mg i.m. Five pregnancies in Study 4 were not terminated. 2.7. Statistical analysis Differences in pregnancy rates among treatments were evaluated using Fishers Exact Test. Differences in size of embryos resulting in pregnancy and not resulting in pregnancy were compared using the Mann-Whitney Rank Sum Test.

3. Results 3.1. Study 1. Standard vitrication of biopsied embryos A summary of results for all ve studies is shown (Table 1). Biopsied blastocysts were seen to collapse after the biopsy procedure was performed, as blastocoele uid was lost from the punctured trophoblast, either by aspiration during the collection of cells or by leakage. All three embryos that were cultured before vitrication re-expanded to near their original diameter within 3 h. After vitrication and warming, two of these embryos, initially 205 and 318 m in diameter, did not grow in culture, whereas the other, initially 199 m in diameter before biopsy, grew slightly to 258 m after 48 h culture. The embryo that was vitried immediately after biopsy, in its collapsed state, re-expanded and increased in size after warming and culture. This embryo was 351 m in diameter before biopsy, and developed to a diameter of 455 m at 48 h culture after vitrication and warming. Based on the above results, ve additional D-6 in vivo-recovered embryos intended for transfer to recipient mares were vitried immediately after biopsy. No pregnancies resulted from transfer of these embryos.

Table 1 Development and pregnancy rate of equine embryos vitried using various techniques. Study 1 Vitrication technique Biopsy, SVa Biopsy, culture, SV Biopsy, SV DMc EGe EG/sf DM DM EG/s Biopsy, DM Biopsy, EG/s Centralg, EG/s Central, EG/s, immed transfer Fluid removal, EG/s Fluid removal, EG/s Embryo source UFb UF UF VTd VT VT VT UF UF UF UF UF UF UF UF Embryo diameter ( m) 351 205318 300 250 250 250 250 300 300 300730 360700 300630 330710 780 407565 Culture n 1 3 10 8 10 Growth in culture, n 1 1 5 10 3 10 8 4 6 16 13 8 7 1 7 3 (38) 0 3 (50) 8 (50) 6 (46) 1 (13) 4 (57) 0 6 (86) 3 (38) 3 (50) 2 (13) 6 (46) 1 (13) 4 (57) 5 (71) 0 Transfer n Pregnancy after transfer, n (%) Heartbeat stage, n (%)

3 4 5
a b c d e f g

SV, Standard vitrication in 0.25 mL straws. UF, embryo recovered on uterine ush. DM, vitried with DMSO-based medium in a microloader tip. VT, embryo produced in vitro by ICSI and culture to the blastocyst stage. EG, vitried with ethylene glycol based medium in a microloader tip. EG/s, vitried with the EG method and warmed with sucrose-based medium as for DM. Central, the micropipette was introduced into the blastocoele, and uid and cells were aspirated.

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3.2. Study 2. Viability of non-biopsied embryos 300 m in diameter vitried using a ne-diameter pipette The proportion of embryos developing in vitro (regaining normal morphology and increasing in diameter or width) after warming were 10 of 10 for the DM technique and 3 of 8 for the EG technique after 72 h in vitro culture. In embryos that grew in culture, the average percentage increase in diameter/width was 29% (from a mean of 180 to 233 m) in the DM group, and 17% (from a mean of 195 to 229 m) in the EG group. The pregnancy rate after transfer of in vitro-produced embryos vitried by the DM technique was 3 of 8; this was 1 of 4 for embryos cultured 18 h before shipment, and 2 of 4 for embryos shipped immediately after warming. All three pregnancies developed normally to the heartbeat stage. Embryo growth and morphology after warming appeared to be better in the DM technique, in which embryos were warmed in sucrose-containing media. Thus, additional embryos already vitried by the EG technique were warmed in sucrose (EG/s technique as detailed in Section 2.4) to determine whether this would increase their viability. When evaluated after culture in vitro, 10 of 10 embryos vitried by EG/s had developed. These embryos had an increase in diameter/ width over the period of culture of 80% (from a mean of 172 m to a mean of 309 m; Fig. 1). For in vivo-recovered embryos vitried and transferred to recipient mares, pregnancy rates were 0 of 4 for the DM treatment (embryos of 165 to 260 m diameter) and 3 of 6 for the EG/s treatment (embryos of 165 to 280 m diameter); this was 1 of 3 for embryos transferred immediately after warming, and 2 of 3 for embryos transferred after 6 h culture. All three pregnancies in the EG/s treatment developed normally to the heartbeat stage. 3.3. Study 3. Viability of blastocysts 300 m in diameter after biopsy and vitrication Sixteen in vivo-recovered blastocysts, 300 to 730 m in diameter, were biopsied and vitried using the DM method, and 13 in vivo-recovered blastocysts, 360 to 700 m in diameter, were biopsied and vitried using the EG/s method. Pregnancy rates at 12 d were 8 of 16 (50%) for the DM treatment and 6 of 13 (46%) for the EG/s treatment. There was no difference in pregnancy rate between embryos transferred on-site (8 of 18, 44%) and those shipped before transfer (6 of 11, 55%). One pregnancy in the DM treatment was lost before

17 d. Of the remaining seven pregnancies in the DM treatment, two resulted in an embryo proper with an ultrasonographically visible heartbeat; these embryos were 599 and 650 m at the time of biopsy. The remaining ve pregnancies developed trophoblast only, as determined by absence of an ultrasonographically visible embryo proper or heartbeat at the expected time. All six pregnancies in the EG/s treatment developed normally to the heartbeat stage. The initial diameters of embryos producing pregnancies in the EG/s group were 360, 360, 370, 465, 570, and 620 m; the initial diameters of embryos not producing pregnancies were 480, 480, 480, 491, 570, 570, and 700 m. The diameters of embryos producing pregnancies (mean 458 m) were not signicantly different from those of embryos not producing pregnancies (mean 539 m; P 0.18). The estimated percentage of blastocoele uid lost after biopsy was recorded for the 13 embryos in the EG/s group, and this was evaluated in relationship to the pregnancy status after transfer for these embryos. The pregnancy rates were 0 of 3, 2 of 5, and 4 of 5 for embryos losing 10, 20 to 30, and 70% of their blastocoele uid after biopsy. 3.4. Study 4. Viability of embryos after central suction of blastocoele uid and cells before vitrication, and after immediate or delayed transfer The Central aspiration procedure resulted in removal of 70% of the blastocoele uid from all embryos. Pregnancy rates were 4 of 7 (57%) for embryos transferred immediately after warming. The initial diameters of embryos producing pregnancies were 330, 390, 390, and 510 m (mean 405 m); the initial diameters of embryos not producing pregnancies were 430, 600, and 710 m (mean 580 m). The difference in size between groups was not signicant (P 0.11). The pregnancy rate was 1 of 8 (13%) for embryos cultured for 6 h before transfer; the embryo producing the pregnancy was initially 420 m in diameter. The ve pregnancies produced in this study were allowed to be maintained; one was lost by 90 d (pregnancy in the cultured group) and one was lost at 8 mo (embryo 390 m); no cause of abortion was identied. The remaining three pregnancies were carried to term and produced healthy foals, two llies and one colt, born at 340, 342 and 345 days after transfer of warmed embryos. One expanded blastocyst, 403 m in diameter, was biopsied, vitried using EG/s, then warmed and cultured in vitro to demonstrate the morphology of the embryo through these stages (Fig. 2).

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Fig 2. In vitro growth of an in vivo-recovered equine blastocyst vitried with the EG/s technique (A) before and (B) after biopsy, (C) after vitrication and warming, and (D) after 24 h culture in vitro. Bar 100 m.

3.5. Study 5. Viability of embryos subjected to trophoblast biopsy and gentle aspiration of blastocoele uid from the trophoblast periphery before vitrication Trophoblast aspiration and gentle suction of blastocoele uid from the trophoblast periphery resulted in loss of 70% of the blastocoele uid in all embryos. The 780 m embryo did not produce a pregnancy. The pregnancy rate for embryos 407 to 565 m in diameter was 6 of 7 (86%). One pregnancy, from an embryo initially 412 m in diameter, was lost before 20 d. The remaining ve pregnancies developed normally to the heartbeat stage, resulting in a 71% normal pregnancy rate. The initial diameters of the embryos resulting in normal pregnancy after transfer were 407, 470, 476, 494, and 565 m. 4. Discussion From these results, we concluded that expanded equine blastocysts up to 650 m in diameter can produce normal pregnancies after cryopreservation, using the technique of blastocoele collapse before vitrication. Based on the higher pregnancy rates associated

with greater loss of uid from the blastocoele found in Studies 3 and 5, we inferred that it was a decrease in volume, rather than just penetration of the capsule and/or trophoblast, allowing cryoprotectant access, that supported the high viability after this procedure. The cryoprotectant used for vitrication affected embryo viability. In Study 2, in vitro-produced embryos did not have high survival in vitro after vitrication with the EG treatment. It should be noted that in vitro-produced embryos do not form capsules and have a lower cell number than do embryos recovered in vivo [26], which may affect their response to cryopreservation. For in vivo-recovered embryos, whereas the DM vitrication technique supported a 50% pregnancy rate after transfer of biopsied expanded blastocysts (8 of 16), only two of these pregnancies developed normally to the heartbeat stage. In contrast, of 17 pregnancies produced from expanded blastocysts after biopsy followed by the EG/s vitrication technique, one pregnancy was lost, whereas the remaining 16 developed normally to the heartbeat stage. Initially, embryos were shipped for transfer, as no recipient herd was available at the laboratory at Texas A&M. The embryos were warmed then shipped warm

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[15]; thus, shipment incurred a period of culture. This post-warming culture period was continued initially when embryos were transferred on-site (Argentina), in case this was associated with the success of the procedure. However, in Study 4 acceptable pregnancy rates (57%) were obtained without a culture period after warming. Shipment of embryos in warm Equitainers in Study 5 was associated with a high pregnancy rate (71%); thus a portable incubator was not necessary for survival of vitried-warmed embryos during shipment. The method used for blastocoele collapse may affect pregnancy rates after transfer. The Central aspiration technique (Study 4) was associated with a relatively low pregnancy rate (1 of 8 versus 4 of 5 pregnant after complete blastocoele collapse in Study 3, in embryos cultured after warming). This may have been associated with disruption of the developing endoderm lining the blastocoele, which forms early in blastocyst development in the horse [review, 27]. This was most vivid in embryos cultured after warming, indicating a possible interaction between the Central technique and culture before transfer; alternatively, there may have been a problem with the culture environment during Study 4. However, the culture system was being used concurrently for development of in vitro-produced blastocysts, with normal blastocyst rates (data not shown). Although not signicant, there was a trend for embryos producing pregnancies to be smaller than were embryos not producing pregnancies. Of ve embryos 700 m, only one, 710 m in diameter and vitried using the DM technique, produced a pregnancy and this developed trophoblast only. If this trend is veried, it may be due not only to the larger size of the embryo, but also to the need for cutting the pipette tip to a larger diameter, as this increases the volume of uid within the pipette and thus slows the speed at which cooling occurs. Use of an open vitrication system might allow successful vitrication of larger embryos. In Study 1, none of ve biopsied embryos cryopreserved with a standard vitrication method (using 0.25 mL straws) produced pregnancies after warming and transfer. The reason for the failure of these embryos to establish pregnancy was unclear. This standard method of vitrication [2] has been previously used successfully in our laboratory in non-biopsied embryos (Hinrichs and Choi, unpublished data). Now that the parameters for biopsy medium, blastocoele collapse technique, and vitrication medium are better dened, further work with standard vitrication methods is needed, as handling embryos with the

microloader tip requires technical expertise in both vitrication and warming steps. The success of cryopreservation of equine expanded blastocysts after blastocoele collapse is a major advance for equine clinical practice, and opens the door to future research in this area. The method for blastocoele collapse used in this study requires a micromanipulator. Alternative methods for blastocoele collapse should be evaluated that would allow embryos to be vitried without the need for shipment to a referral laboratory. In addition, as noted above, use of standard vitrication or slow-cooled freezing procedures with collapsed blastocysts would allow mare-side warming before transfer. Future research should focus on development of methods to simplify collapse and cryopreservation procedures for expanded equine blastocysts. Acknowledgments This work was supported by a grant from the American Quarter Horse Foundation, by the Link Equine Research Endowment, Texas A&M University, and by Ms. Kit Knotts. We thank Bioniche Animal Health for generous donation of media. References
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Y.H. Choi et al. / Theriogenology 76 (2011) 143152 at 144, 156 or 168 hours after ovulation. Equine Vet J Suppl 1997;25:60 2. Flood PF, Betteridge KJ, Diocee MS. Transmission electron microscopy of horse embryos 316 days after ovulation. J Reprod Fertil Suppl 1982;32:319 27. Legrand E, Bencharif D, Barrier-Battut I, Delajarraud H, Cornire P, Fini F, Tainturier D, Bruyas JF. Comparison of pregnancy rates for days 7 8 equine embryos frozen in glycerol with or without previous enzymatic treatment of their capsule. Theriogenology 2002;58:7213 (abstract). Maclellan LJ, Carnevale EM, Coutinho da Silva MA, McCue PM, Seidel GE Jr, Squires EL. Cryopreservation of small and large equine embryos pre-treated with cytochalasin-B and/or trypsin. Theriogenology 2002;58:71720 (abstract). Stout TA, Meadows S, Allen WR. Stage-specic formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo. Anim Reprod Sci 2005;87:269 81. Choi YH, Gustafson-Seabury A, Velez IC, Hartman DL, Bliss S, Riera FL, Roldan JE, Chowdhary B, Hinrichs K. Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction 2010;140: 893902. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrication gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2010;19:2884 8. Mukaida T, Oka C, Goto T, Takahashi K. Articial shrinkage of blastocoeles using either a mico-needle or a laser pulse prior to the cooling steps of vitrication improves survivial rate and pregnancy outcome of vitried human blastocysts. Hum Reprod 2006;21:3246 52. Lin L, Du Y, Kragh PM, Li J, Bolund L, Yang H, Zhang X, Kuwayama M, Vajta G. Induced blastocoel collapse improves survival rates of porcine blastocysts after vitrication. Reprod Fertil Dev 2008;20:121 (abstract). [19] Scherzer J, Fayrer-Hosken RA, Ray L, Hurley DJ, Heusner GL. Advancements in large animal embryo transfer and related biotechnologies. Reprod Domest Anim 2008;43:371 6. [20] Sun X, Li Z, Yi Y, Chen J, Leno GH, Engelhardt JF. Efcient term development of vitried ferret embryos using a novel pipette chamber technique. Biol Reprod 2008;79:832 40. [21] Campos-Chilln LF, Suh TK, Barcelo-Fimbres M, Seidel GE Jr, Carnevale EM. Vitrication of early-stage bovine and equine embryos. Theriogenology 2009;71:349 54. [22] Choi YH, Love LB, Varner DD, Hinrichs K. Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection. Theriogenology 2006;66: 955 63. [23] Jacobson CC, Choi YH, Hayden SS, Hinrichs K. Recovery of mare oocytes on a xed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection. Theriogenology 2010;73:1116 26. [24] Hinrichs K, Choi YH, Love LB, Varner DD, Love CC, Walckenaer BE. Chromatin conguration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence. Biol Reprod 2005;72: 114250. [25] Choi YH, Chung YG, Walker SC, Westhusin ME, Hinrichs K. In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture. Zygote 2003;11:77 86. [26] Tremoleda JL, Stout TA, Lagutina I, Lazzari G, Bevers MM, Colenbrander B, Galli C. Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation. Biol Reprod 2003;69:1895906. [27] Hinrichs K, Choi YH, Walckenaer BE, Varner DD, Hartman DL. In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture. Theriogenology 2007;68:5219.

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