Vitrification of early-stage bovine and equine embryos

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Theriogenology 71 (2009) 349–354 www.theriojournal.com

Vitrification of early-stage bovine and equine embryos L.F. Campos-Chillo`n, T.K. Suh, M. Barcelo-Fimbres, G.E. Seidel Jr., E.M. Carnevale * Animal Reproduction and Biotechnology Laboratory, Foothills Campus, Colorado State University, Fort Collins, CO 80523-1683, USA Received 22 February 2008; received in revised form 23 July 2008; accepted 4 August 2008

Abstract The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EGDMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1–3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient’s oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI. # 2009 Elsevier Inc. All rights reserved. Keywords: Vitrification; Bovine; Equine; Embryos; ICSI

1. Introduction Vitrification is an alternative to freezing [1]. This technique involves rapid cooling and warming rates, small volumes, high viscosity and the use of high concentrations of cryoprotectant solutions to bring about a glass physical state in which crystalline ice does not

* Corresponding author. Tel.: +1 970 491 8626; fax: +1 970 491 7005. E-mail address: [email protected] (E.M. Carnevale). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.08.001

form [2]. Vitrification is rapid, inexpensive, and has been used to cryopreserve embryos (from several mammals) at various stages of development [2]. Valuable genetics can be preserved through the cryopreservation of sperm, oocytes, or embryos. Methods to cryopreserve sperm and embryos have been successful [3]. However, despite advances in cryobiology, cryopreservation of oocytes from most species is inefficient, and results are inconsistent [4]. In humans, cryopreservation of zygotes offers a theoretical advantage, because the center of the lipid phase transition curve (Tm) is lower in zygotes than in oocytes [5]. However,

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studies are needed to compare the chilling injury of bovine and equine oocytes versus early embryos. Minimal research has been conducted on methods to cryopreserve bovine embryos in early cleavage stages. One reason is that blastocysts can be produced reliably for non-surgical transfer after in-vitro fertilization (IVF) and cultured in vitro. In contrast, although cleavagestage embryos can be produced by ICSI in the horse, embryo culture systems are inefficient, with <20% of injected oocytes reported to develop into blastocysts [6,7]. Therefore, cleavage stage embryos are often surgically transferred into recipient’s oviducts. Cryopreservation of early embryos would provide a valuable alternative in the horse, allowing a suitable recipient to be identified or embryos to be stored until a more optimal time of year. Uterine embryos have been collected from mares and successfully vitrified [8,9]. Other researchers obtained pregnancies (50%) after freezing and thawing equine blastocysts that were produced after ICSI and cultured in vitro or in sheep oviducts [10]. Foals have been produced from vitrified and warmed equine oocytes; however, only 14% of the cryopreserved oocytes resulted in pregnancies [11]. Because of the high lipid content of equine oocytes, morphological visualization of pronuclei to assess viability can be difficult. However, zygotes can be cultured for 1–3 d to evaluate viability by the number and timing of cellular divisions before cryopreservation. To our knowledge, cryopreservation of equine zygotes or early cleavage-embryos produced by ICSI has not been reported. A major determent to developing methods to cryopreserve early-stage embryos in the horse is the paucity of oocytes. Cryotolerance and lipid content of in vivorecovered bovine and equine embryos before capsule formation are similar [12]. Therefore, we anticipated that early-stage bovine embryos would provide an appropriate experimental model for equine embryos. The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) establish equine pregnancies after vitrification and warming of early embryos, using procedures that were optimized for bovine embryos. 2. Materials and methods 2.1. Production and cryopreservation media for bovine embryos All reagents used were obtained from Sigma Chemical Co. (St. Louis, MO, USA), except otherwise

indicated. Bovine embryos were produced by in-vitro fertilization in four replicates using semen from two bulls. Briefly, oocytes were aspirated from 2 to 8 mm follicles from abattoir-derived ovaries. Maturation of oocytes was done in chemically defined medium (CDM) [13], supplemented with 0.5% FAF-BSA, 15 ng/mL NIDDK-oFSH-20, 1 mg/mL USDA-LH-B5, 0.1 mg/mL E2, 50 ng/mL EGF, and 0.1 mM cysteamine for 23 h at 38.5 8C in 5% CO2 and air. Frozen semen was centrifuged through a Sperm-Talp [14] based Percoll gradient. Sperm (5  105 sperm/mL) and oocytes were co-incubated in fertilization medium (FCDM; CDM supplemented with 0.5% FAF-BSA, 2 mM caffeine and 0.02% heparin) for 18 h at 38.5 8C in 5% CO2 and air. Presumptive zygotes were cultured in CDM-1 (CDM supplemented with nonessential amino acids, 10 mM EDTA, and 0.5% FAF-BSA) for 2.5 d. For bovine embryo cryopreservation, vitrification solutions were prepared using a base medium of HCDM-1 (Hepes buffered CDM-1 containing 25 mM Hepes, 5 mM NaHCO3, 0.25% FAF-BSA, 20% FCS, and no EDTA). Vitrification procedures were done on a warming stage at 38 8C, until embryos were placed in liquid nitrogen vapor or plunged into liquid nitrogen. 2.2. Cryopreservation in 0.25-mL straws In a series of studies, presumptive zygotes (1-cell at 18 h after IVF) were cryopreserved using 0.25-mL straws and three methods of cryopreservation. In the first method [15], conventional slow-cooling of presumptive zygotes was attempted. Briefly, presumptive zygotes (n = 18) were equilibrated in 1.5 M ethylene glycol (EG) and 0.5 M galactose in HCDM-1 for 5 min; straws were seeded at 6 8C, cooled at 0.5 8C/min to 32 8C, and then plunged into liquid nitrogen. The second method was a two-step vitrification procedure [16]. Presumptive zygotes were placed in 5 M EG in HCDM-1 for 1 (n = 18), 2 (n = 15) or 3 min (n = 16) during the first step. For the second step, they were placed in 7 M EG, 0.5 M galactose, and 18% (w/v) Ficoll 70 in HCDM-1 for 45 s, and plunged into liquid nitrogen. The third method of cryopreservation was a three-step vitrification procedure [17]. Presumptive zygotes (n = 20) were exposed to 1.4 M glycerol (G) in HCDM-1 for 5 min, moved to 1.4 M G and 3.6 M EG in HCDM-1 for 5 min, and then transferred to 3.4 M G and 4.6 M EG for 45 s. Immediately, straws were placed in liquid nitrogen vapor for 1 min and then plunged into liquid nitrogen. In the first method using conventional cryopreservation, embryos were thawed by placing the 0.25-mL straws in air (24 8C) for 10 s and then in water

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at 37 8C for 30 s. For warming straws after the two-step and three-step vitrification procedures, the straws were held in air for 10 s, placed in water at 37 8C for 10 s, and flicked four to six times to mix columns. After warming, embryos were washed five times in HCDM-1 and cultured in CDM-1 for 2.5 d. Embryos were evaluated for cleavage 3 d after IVF. 2.3. vitrification with super open pulled straws (OPS) Upon review of the preliminary results using 0.25mL straws, an experiment was designed using super open pulled straws (OPS # 19050-0125, Minitube, Ingersoll, ON, Canada) for vitrification of presumptive zygotes (1-cell) and 8-cell embryos. The experimental design included two OPS vitrification methods and two embryological stages. The experiment was replicated four times with semen from two bulls. Embryos were vitrified as 1-cell embryos (presumptive zygotes at 18 h after IVF, n = 221) and 8-cell embryos (6 to 8 cells at 2.5 d after IVF, n = 231). Embryos were moved between media in 1-mL increments of medium. In the first method (EG-O) [18], embryos were held in HCDM-1; transferred into 1.5 M EG in HCDM-1 (500 mL) for 5 min; and moved into a 20-mL drop of 7 M EG and 0.6 M galactose in HCDM-1 for 30 s. While in the final vitrification solution, the embryos were loaded (by capillary action) into OPS, with approximately 1 mL of medium. The OPS containing the embryo was immediately plunged into liquid nitrogen. For the second method (EG-DMSO) [19], embryos were exposed to 1.1 M EG and 1.1 M dimethyl sulfoxide (DMSO) in HCDM-1 (500 mL) for 3 min, and transferred into a 20-mL drop of 2.5 M EG, 2.5 M DMSO, and 0.5 M galactose for 30 s. The embryos were loaded and plunged as described for EG-O. Open pulled straws containing embryos were warmed within 1 h after vitrification. Embryos in EG-O and EG-DMSO were warmed by placing the tip of the OPS into 500 mL of 1 M galactose in HCDM-1 in a four-well plate (Nalge Nunc International; Rochester, NY, USA) at 38 8C; after 3 min, the embryos were moved to 0.5 and 0.25 M galactose in HCDM-1 at 38 8C for 3 min each. Control embryos (n = 162), that had not been vitrified, were placed in culture in CDM-1 for 2.5 d. At that time, they were moved into CDM-2 (CDM without EDTA and supplemented with essential and nonessential amino acids) for 4.5 d at 38.5 8C in 5% O2, 5% CO2 and 90% N2. After vitrification and warming, 1-cell embryos were cultured as described for control embryos. Eight-cell

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embryos were cultured in CDM-2 for 4.5 d after vitrification and warming. Cleavage rates were determined for control and vitrified 1-cell embryos. Blastocyst rates were determined for all embryos. 2.4. Production of equine embryos Light-horse mares (3–7 y of age) were used as oocyte donors and embryo recipients. Reproductive examinations of mares were performed using ultrasound at intervals of 2–3 d during diestrus and daily during estrus. Equine oocytes were collected by transvaginal aspiration as previously described [20], 20 h after administration of a GnRH analog (1.5 mg, im, Deslorelin, BET Labs Lexington, KY, USA). Upon recovery, oocytes were placed in maturation medium, TCM-199 with Earle’s salts (Bio Whittaker; Walkersville, MD, USA) at pH 7.4, supplemented with 10% FCS, 0.2 mM pyruvate, and 25 mg/mL gentamicin sulfate for approximately 18 h under 6% CO2 in air at 38.5 8C. Frozen semen from one stallion was used for sperm injections, as previously described [21]. Briefly, motile sperm were recovered by cutting a section of the frozen straw (50 mL) under liquid nitrogen; the cut section was placed at the bottom of a 15-mL conical tube containing 2 mL of F-CDM at 37 8C. Cumulus cells were denuded from the oocytes by repeated pipetting in GMOPS (Vitrolife, Sweden) supplemented with 200 IU/mL of hyaluronidase. A Piezo-driven injection system (Prime Tech Inc., Japan) was used for sperm injections. Sperm injection took place in a 50-mL drop of GMOPS supplemented with 0.5% FAF-BSA. Injected oocytes were cultured in DMEM/Ham’s F12 (1:1) supplemented with 10% FCS at 38.5 8C, in 5% O2, 5% CO2 and 90% N2. Numbers of cells were evaluated 1, 2, and 3 d after injection. Eight embryos at 2- to 8-cells, were vitrified and warmed using OPS and the EG-O method as described for bovine embryos after 3 wk of storage, with the exception that H-DMEM/F12 (DMEM/F12 supplemented with 25 mM Hepes, 5 mM NaHCO3, and 20% FCS) was used for the base medium. 2.5. Transfer of equine embryos Warmed embryos (n = 8) were surgically transferred [22] into the oviducts of four recipients 2–3 d after the detection of ovulation. Recipients were selected for a distinct corpus luteum, increased cervical tone, and absence of endometrial edema. The ovary and oviduct were exteriorized through the incision; embryos (two per recipient) in <50 mL of H-DMEM/F12 were placed

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approximately 3 cm into the oviductal lumen using a fire-polished glass pipette. Recipients received phenylbutazone (Vedco Inc., St. Joseph, MO, 2 g, po) for 2 d and sulfamethoxazole and trimethoprim (Mutual Pharmaceutical CO. Inc., Philadelphia, PA, USA; 800 mg/160 mg, 30 mg/kg, po) for 7 d. Pregnancy status and diameter of vesicles were determined from ultrasound images at 10–20 d after embryo transfer, at which time mares were given cloprostenol (250 mg, im; Estrumate1, Schering-Plough Coopers Animal Health, New Zealand) to terminate pregnancies. One pregnancy was allowed to continue until 45 d of gestation to evaluate embryo development and heartbeat [23].

3.2. Vitrification of 1- and 8-cell bovine embryos in OPS Cleavage rates after vitrification and warming of 1cell embryos were similar to control zygotes that were not cryopreserved. The percentages of blastocysts per 1or 8-cell embryo were lower (P < 0.05) for embryos vitrified in EG-DMSO than EG-O or for control embryos (Table 1). The blastocyst rate was higher (P < 0.05) for control embryos than embryos that had been vitrified and warmed at the 8-cell stage, with more (P < 0.05) blastocysts formed per 8-cell embryo vitrified in EG-O than EG-DMSO (Table 1). There were differences between replicates (P < 0.05) for embryos vitrified at the 8-cell stage.

2.6. Statistical analysis Data were arc-sine transformed and analyzed by ANOVA (SAS Institute Inc., Cary, NC, USA) with replicates in the model, or by using Fisher’s protected LSD. 3. Results 3.1. Cryopreservation of presumptive bovine zygotes in 0.25-mL straws Few of the presumptive zygotes that were cryopreserved in 0.25-mL straws cleaved. Of the 18 presumptive zygotes frozen with the slow-cooling method, only one cleaved after thawing. In the second method (two-step vitrification), cleavage rates were 2/ 18, 0/15 and 0/16 when embryos were placed in 5 M EG for 1, 2, or 3 min, respectively. After the three-step vitrification procedure, 1 of 20 presumptive zygotes cleaved. Cleavage rates were not different among treatments (P > 0.01).

3.3. Vitrification of early equine embryos with super open pulled straws (OPS) After the transfer of vitrified and warmed equine embryos, five of eight (62%) embryos resulted in embryonic vesicles by Day 16. Pregnancies were obtained with 2-cell embryos (2/3, 67%) and 8-cell embryos (3/5, 60%). Mean diameters of embryonic vesicles at 13 d and 20 d of gestation were 8  2 mm and 19  2 mm, respectively. The pregnancy that was maintained until 45 d and appeared normal, with an embryo proper and heartbeat detected with ultrasonography. 4. Discussion In this study, cryopreservation methods were evaluated for early bovine and equine embryos produced in vitro. Our objective was to determine the best stage of early embryo development and method of vitrification for bovine embryos, and then utilize

Table 1 Cleavage and blastocyst rates, per oocyte and per 8-cell embryo, after vitrification of one-cell embryos (presumptive zygotes) and blastocyst rates for controls and 8-cell embryos after vitrification using super open pulled straws (OPS) Vitrification method

Embryo stage at vitrification One cell

Control EG-O* EG-DMSO ** a–c

Eight cells

Cleavage per oocyte (%)

Blastocysts per oocyte (%)

Blastocysts per 8-cell embryo (%)

Blastocysts per 8-cell embryo (%)

70/81 (86.4) 63/72 (87.5) 54/68 (79.4)

21/81 (25.9)a 17/72 (23.6)a 8/68 (11.8)b

21/52 (40.4)a 17/45 (37.8)a 8/44 (18.2) b

46/81 (56.8)a 32/75 (42.7)b 11/75 (14.7)c

Within a column, means without a common superscript differed (P < 0.05). EG-O: embryos were exposed to 1.5 M EG for 5 min, 7 M EG and 0.6 M galactose for 30 s, loaded and plunged into liquid nitrogen. ** EG-DMSO: embryos were exposed to 1.1 M EG and 1.1 M DMSO for 3 min, 2.5 M EG, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged into liquid nitrogen. *

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this knowledge for a preliminary study with equine embryos. Few experiments have been conducted on cryopreservation of bovine embryos at early stages of development. Culture systems have been established for bovine embryos, with 30 to 40% blastocysts produced per oocyte and pregnancy rates with selected blastocysts that were comparable to embryos produced in vivo [24]. In this experiment, several attempts were made to cryopreserve 1-cell bovine embryos (presumptive zygotes) in 0.25-mL straws by conventional freezing and other vitrification methods; however, cleavage rates were poor. As an alternative, embryos were vitrified in super open pulled straws; the higher cooling and warming rates in OPS offer a theoretical advantage over slow freezing [2]. In the present study, the numbers of blastocysts that resulted from 1- and 8-cell embryos after vitrification with ethylene glycol (EG-O) was promising for research applications and to our knowledge is the first report using EG-O with bovine embryos; however, commercial use would be limited since intrafallopian transfer of gametes and early-stage embryos in cattle is not a widely used technique [25]. Use of the OPS and a vitrification protocol with ethylene glycol and DMSO (EG-DMSO) resulted in similar cleavage rates for control presumptive zygotes and presumptive zygotes after vitrification, warming and culture. However, blastocyst rates for 1- and 8-cell embryos vitrified with EG-DMSO were lower than controls, as previously reported by Vajta et al. [19]. The second vitrification method (EG-O) with the OPS, has been used for cryopreservation of human zygotes and 3-d embryos and has resulted in healthy pregnancies and deliveries [18]. In the present study, EG-O resulted in higher blastocyst rates for 1- and 8cell bovine embryos (23% per oocyte and 42% per 8cell embryo, respectively) compared to EG-DMSO (11% per oocyte and 14% per 8-cell embryo, respectively). In this study, EG was superior to the combination of EG and DMSO for the durations, temperatures and embryological stages studied. Ethylene glycol has minimal toxicity and is more permeable to embryos and oocytes than glycerol, DMSO, propylene glycol, or acetamide [26]. The original formulations of EG-O [18] and EG-DMSO [19] used sucrose in the vitrification and warming solutions. In the present study, sucrose was replaced with galactose to further facilitate dilution of cryoprotectants, due to differences between the two sugars in molecular weight and viscosity [27].

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For this study, early equine embryos were produced by intracytoplasmic sperm injections, because successful methods for equine IVF have not been developed [28]. Production of early-stage equine embryos is limited by expense and oocyte numbers. Therefore, bovine experiments were used to determine the vitrification method that would have the greatest potential for success with equine embryos. The most successful vitrification procedure from our bovine experiments was EG-O, and the same method was used with equine embryos, with the exception of a different base medium. In previous studies, equine embryos cultured in DMEM/F12 supplemented with 10% FCS had higher blastocyst rates compared to other complex media formulations [6]. Embryo vitrification has been successful with a variety of base media [29]. Therefore, we opted to use H-DMEM/F12 with equine embryos. Although limited embryos were available, five of eight equine embryos resulted in pregnancies after vitrification, warming and transfer. The number of available equine embryos was not sufficient for control transfers of early equine embryos without vitrification. However, in the clinical ICSI program in our laboratory, embryos were transferred an average of 34  1.2 h after sperm injection, with a mean of 3.3  0.2 cells. The pregnancy rate at 16 d was 44% (27/62) [30]. Pregnancy rates appeared similar for the experimental (vitrified) embryos in this study and clinical embryos after transfer. However, direct comparisons are difficult, because in the clinical program, oocyte donors varied in age (4–26 y), and sperm varied in quality and type (frozen, cooled and epididymal). Further research is needed to confirm our results and determine methods for vitrification of early equine embryos that optimize success and flexibility of the procedure. Sterility has been a concern with OPS vitrification, since the embryos are in direct contact with liquid nitrogen. Potentially, contamination of embryos and disease transmission could be avoided by filtering the liquid nitrogen or packaging the OPS in sterile, sealed 0.5-mL straws [2,31]; however, the sterile container must be pre-cooled to maintain the theoretical advantages of the OPS [2]. Replacing fetal calf serum with another macromolecule in the base medium could alleviate biosecurity concerns and facilitate international trade. In conclusion, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to produce equine pregnancies after vitrification, warming, and transfer of 2- to 8-cell equine embryos produced by ICSI.

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Acknowledgements Technical assistance was provided by JoAnne Stokes, Dr. Joy Altermatt, Zell Brink and Jake Cox. L.F. Campos-Chillo`n was supported by an Abney Scholarship, and partial funding was provided by the benefactors for the Preservation of Equine Genetics Program and the Hylton Family Foundation.

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