Cryopreservation Of Equine Embryos Research Articles

A touch of glass: cryopreservation of equine embryos by vitrification

The cryopreservation of equine embryos larger than 300 µm in diameter has proved problematic for many decades. However, there now exist protocols to undertake vitrification of such embryos with good pregnancy rates following their subsequent warming and transfer to recipient mares. This article discusses the reasons why equine embryos have proved so difficult to cryopreserve, and looks at cryopreservation methods before focusing on embryo vitrification. It also provides a historical perspective on the development of vitrification and details the latest techniques and emerging methodologies aimed at making embryo cryopreservation more accessible for practitioners.

Colabamento manual da blastocele não foi efetivo para aumentar a viabilidade de embriões equinos vitrificados

Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos <300 μm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are >300 μm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.

Vitrification of equine expanded blastocysts following puncture with or without aspiration of the blastocoele fluid.

Historically, cryopreservation of equine embryos >300μm gave poor pregnancy rates until researchers collapsed the blastocoele cavity and aspirated the blastocoele fluid prior to vitrification. To determine if aspiration of the blastocoele fluid prior to vitrification is essential for post warming survival. In vivo experiments. Fifty embryos were recovered on day 7-8 and washed in holding medium (HM; M-199HEPES+20% FBS+antibiotics). Embryos were punctured using a micromanipulator mounted 30μm biopsy needle; following this 28 had >90% of their blastocoelic fluid actively aspirated while the remaining 22 were not-aspirated. Embryos were then vitrified using a two-step process with increasing concentrations of DMSO and ethylene glycol (7.5-15% v:v), and 0.5mol/L sucrose in the final solution before being loaded onto a Cryolock device and plunged into liquid nitrogen. The embryos were warmed by plunging the Cryolock tip into HM with 1mol/L sucrose at 37°C. After 1min, the embryos were transferred to HM+0.5mol/L sucrose at RT for 4min before transfer into HM for a further 4min prior to transfer to a recipient mare. Mean (±s.e.) embryo diameter was not significantly different between the punctured and punctured plus aspirated group (646.4±61.7 vs. 754.8±59.1μm, respectively; P=0.215). Nonaspirated and aspirated embryos gave pregnancy rates of 10/22 (45%) and 21/28 (75%) respectively (P=0.061). Sub-dividing embryos on the basis of size showed that vitrification of larger embryos (>550μm) yielded a significantly higher pregnancy rate when they were aspirated vs. not-aspirated (13/18 [72%] vs. 1/10 [10%], respectively; P=0.006), whereas there was no difference for smaller embryos (8/10 [80%] vs. 9/12 [75%], respectively; P=0.8). Group sizes are limited. Aspiration of blastocoele fluid from embryos ≤550μm is not a pre-requisite for successful vitrification. The Summary is available in Spanish - see Supporting Information.

Cryopreservation of Equine Embryos: Current State‐of‐the‐Art

During the past 15 years, embryo transfer (ET) has become increasingly widespread within the sport-horse breeding industry. At present, however, the vast majority (>95%) of horse embryos are transferred fresh or after chilled storage for up to 24 h, whereas cryopreservation is rarely employed despite its obvious potential for simplifying recipient mare management and facilitating long-term storage and international transport of embryos. A number of inter-related factors have contributed to the slow development and implementation of equine embryo cryopreservation, and these include the following: (i) the absence of commercially available products for reliably stimulating superovulation; (ii) very poor pregnancy rates following cryopreservation of embryos >300 μm in diameter; (iii) difficulty in recovering embryos at early developmental stages amenable to cryopreservation; and (iv) interembryo variation in susceptibility to cryodamage. However, acceptable success rates (>55% pregnancy) have been reported for both slow-frozen and vitrified small embryos (<300 μm), and there is renewed interest in cryopreservation, not only in the context of standard ET programmes, but also because it would facilitate pre-implantation genetic testing and allow wider access to techniques for producing embryos in vitro, such as intracytoplasmic sperm injection and nuclear transfer. This article will review the current status of equine embryo cryopreservation.

77 PREGNANCY RATES AFTER VITRIFICATION OF FRESH AND COOLED EQUINE EMBRYOS USING THE CRYOTOP METHOD

Cryopreservation of equine embryos is still not a routine procedure. Pregnancies have been obtained after transfer of vitrified embryos of less than 300 μm (Eldridge-Panuska et al. 2005). The aim of this study was to use the cryotop method (Kuwayama, 2007) to obtain pregnancies after transfer of vitrified thawed cooled and fresh embryos collected in our clinical embryo transfer programme. Embryos were assigned either to be vitrified within 3 h of collection or to be cooled for 18–24 h before vitrification. All embryos were vitrified and thawed by Cryotop Vitrification Kit® (Cryo Tech Laboratory®). Briefly, they were equilibrated in a solution containing ethylene glycol (EG), dimethylsulphoxide (DMSO) in TCM-199 for 10 to 25 min. Then they were moved to vitrification solution containing EG, DMSO, and sucrose in TCM-199 and loaded with a glass capillary onto the top of the film strip. After loading, almost all the solution was removed to leave only a thin layer covering the embryo, and the sample was quickly immersed into liquid nitrogen and covered with a protective cap. The time between entry to vitrification solution and nitrogen was from 1 to 3 min. At warming, the strip was immersed directly for 1 min into a 37°C medium containing sucrose in TCM-199. The embryo was incubated 3 min in a diluent solution, washed twice 5 min each in washing solution, and further cultured in DMEM F-12 with 10% FBS at 38.5°C 5% CO2 between 2 to 5 h. For transfer, the embryo was loaded in 0.5-mL straws. All recipient mares had ovulated 4 to 7 days before nonsurgical transfer. Pregnancies were detected 6 to 8 days later. A total of 15 embryos, grades 1 to 2, were obtained. Fresh embryos (n = 7) ranged between 250 and 800 μm, and refrigerated embryo (n = 8) diameter was between 130 and 550 μm. Pregnancy rates were 37.5% (3/8) for embryos cooled before vitrification and 28.6% (2/7) for embryos vitrified within 3 h. The overall pregnancy rate was 33.3% (5/15). Shipping cooled embryos allows maintaining a large number of recipients far away from donors, without decreasing pregnancy rate. It also makes it possible to send embryos to a specialised laboratory in order to be vitrified and preserved until recipients are available. Equine embryos collected 6 days after ovulation are generally smaller than 300 μm and have shown the highest survival rate after cryopreservation. However, the embryo recovery rate is higher when flushing is performed at Day 7 or 8. This cryopreservation protocol could provide a way to vitrify fresh and cooled embryos up to 550 μm, which would prevent the loss of valuable embryos collected in more advanced stages of development. In summary, pregnancies can be obtained after cooling for 18 to 24 h and vitrification of embryos collected 7 or 7.5 days after.

Effect of dehydration prior to cryopreservation of large equine embryos

Cryopreservation of equine embryos > 300 μm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7–8, grade 1, equine embryos 300–1350 μm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, slow freeze ( n = 21); (B) 10 min in 1.5 M glycerol, slow freeze ( n = 15); (C) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, followed by exposure to thaw solutions, then culture medium ( n = 5); (D) transferred directly to culture medium ( n = 5). Frozen embryos were thawed and subjected to a three-step cryoprotectant removal. Five embryos from each treatment were evaluated morphologically after 24 and 48 h culture (1 = excellent, 5 = degenerate/dead). All treatments had at least 4/5 embryos with a quality score ⩾ 3 at these time points except treatment B (2/5 at 24 h, 1/5 at 48 h). Subsequent embryos from treatment A ( n = 16) or B ( n = 10) were matched in sets of two for size and treatment, thawed, and immediately transferred in pairs to 13 recipients. Only two recipient mares were pregnant; one received two 400 μm embryos from treatment A, and the other one 400 and one 415 μm embryo from treatment B. There was no advantage of incorporating a 2 min dehydration step into the cryopreservation protocol for large equine embryos.

In vitro comparisons of two cryopreservation techniques for equine embryos: Slow-cooling and open pulled straw (OPS) vitrification

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO) + 7.5% ethylene glycol (EG) for 3 min and in 18% DMSO + 18% EG + 0.4 M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3 h and evaluated using 4′,6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3 h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42 ± 6 versus 46 ± 9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27 ± 5 versus 26 ± 6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.

Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods

Cryopreservation of equine embryos with conventional slow-cooling procedures has proven challenging. An alternative approach is vitrification, which can minimize chilling injuries by increasing the rates of cooling and warming. The open pulled straw (OPS) and cryoloop have been used for very rapid cooling and warming rates. The objective of this experiment was to compare efficacy of vitrification of embryos in OPS and the cryoloop to conventional slow cool procedures using 0.25 mL straws. Grade 1 or 2 morulae and early blastocysts (≤300 μm in diameter) were recovered from mares on Day 6 or 7 post ovulation. Twenty-seven embryos were assigned to three cryopreservation treatments: (1) conventional slow cooling (0.5°C/min) with 1.8 M ethylene glycol (EG) and 0.1 M sucrose, (4) vitrification in OPS in 16.5% EG, 16.5% DMSO and 0.5 M sucrose, or (3) vitrification with a cryoloop in 17.5% EG, 17.5% DMSO, 1 M sucrose and 0.25 μM ficoll. Embryos were evaluated for size and morphological quality (Grade 1 to 4) before freezing, after thawing, and after culture for 20 h. In addition, propidium iodide (PI) and Hoechst 33342 staining were used to assess percent live cells after culture. There were no differences (P > 0.1) in morphological grade or percent live cells among methods. Mean grades for embryos after culture were 2.9 ± 0.2, 3.1 ± 0.1, and 3.3 ± 0.2 for conventional slow cooling, OPS and cryoloop methods, respectively. Embryo grade and percent live cells were correlated, r = 0.66 (P > 0.004). Thus OPS and the cryoloop were similarly effective to conventional slow-cooling procedures for cryopreserving small equine embryos.

Cryopreservation of equine embryos by two vitrification methods

Cryopreservation of equine embryos by two vitrification methods

Cryopreservation of Equine Embryos

Principles and procedures for cryopreservation of equine embryos are described. Embryos less than 250 microM in diameter can be cryopreserved successfully if glycerol is used as the cryoprotectant. Cooling is takes place in such a way that most of the water leaves the cells before intracellular ice forms, and glycerol is removed after thawing without undue osmotic swelling of cells. Vitrification procedures also show promise for small embryos. Satisfactory procedures for cryopreserving embryos of more than 250 microM in diameter are not yet available.

A new procedure for the cryopreservation of equine embryos

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2×2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN 2) at −33 C or −38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to −6 C and then seeded, 0.3 C/min to −30 or −35 C and 0.1 C/min to −33 or −38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at −33 C was superior (P<0.05) to that of embryos plunged at −38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at −38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at −33 C or frozen in straws and plunged at −33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at −33 C resulted in higher quality embryos than plunging at −38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at −33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate ( 8 10 , 80%) than expanded blastocysts ( 1 7 , 14%).

Antagonistic effect of LH on FSH-induced superovulation in cattle

Equine embryo transfers have increased dramatically in the past decade, but in spite of the advantages of cryopreservation of equine embryos, this technology has not increased proportionally. Lack of a superovulation protocol for mares and the inability to freeze embryos >300 μm have been the limiting factors impeding equine embryo cryopreservation. Data from both controlled laboratory settings and commercial embryo transfer facilities have shown that small embryos can be slow cooled or vitrified and, after thawing and transfer, provide pregnancy rates of 50%–70% similar to that obtained with bovine embryos. In contrast, studies have shown that embryos >300 are damaged more during slow cooling or vitrification than those <300 μm and result in low pregnancy rates after transfer. The presence of the acellular capsule in the equine blastocyst and the large volume of blastocoele fluid were thought to be the major reason for poor survival of cryopreserved large equine embryos. However, deflating the embryo before freezing has been shown to improve the survival of cryopreserved large equine embryos dramatically. Unfortunately, this must be done using very expensive equipment. Developments that could improve the success of equine embryo cryopreservation are as follows: having hormones available for superovulation, a means of hastening the embryo into the mare's uterus to consistently collect <300 μm embryos, or the development of a simple means of collapsing embryos >300 μm.

Influence of day of collection on recovery rate in mature cycling mares

Equine embryo transfers have increased dramatically in the past decade, but in spite of the advantages of cryopreservation of equine embryos, this technology has not increased proportionally. Lack of a superovulation protocol for mares and the inability to freeze embryos >300 μm have been the limiting factors impeding equine embryo cryopreservation. Data from both controlled laboratory settings and commercial embryo transfer facilities have shown that small embryos can be slow cooled or vitrified and, after thawing and transfer, provide pregnancy rates of 50%–70% similar to that obtained with bovine embryos. In contrast, studies have shown that embryos >300 are damaged more during slow cooling or vitrification than those <300 μm and result in low pregnancy rates after transfer. The presence of the acellular capsule in the equine blastocyst and the large volume of blastocoele fluid were thought to be the major reason for poor survival of cryopreserved large equine embryos. However, deflating the embryo before freezing has been shown to improve the survival of cryopreserved large equine embryos dramatically. Unfortunately, this must be done using very expensive equipment. Developments that could improve the success of equine embryo cryopreservation are as follows: having hormones available for superovulation, a means of hastening the embryo into the mare's uterus to consistently collect <300 μm embryos, or the development of a simple means of collapsing embryos >300 μm.