- Email: [email protected]
Cryopreservation of Equine Embryos
Accepted Manuscript Cryopreservation of Equine Embryos Edward L. Squires, Patrick M. McCue
PII:
S0737-0806(16)30055-7
DOI:
10.1016/j.jevs.2016.03.009
Reference:
YJEVS 2057
To appear in:
Journal of Equine Veterinary Science
Received Date: 26 February 2016 Revised Date:
24 March 2016
Accepted Date: 24 March 2016
Please cite this article as: Squires EL, McCue PM, Cryopreservation of Equine Embryos, Journal of Equine Veterinary Science (2016), doi: 10.1016/j.jevs.2016.03.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Cryopreservation of Equine Embryos
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Edward L. Squiresa* and Patrick M. McCueb
Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546,
USA
Equine Reproduction Laboratory Colorado State University , Ft Collins Co 80521
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b
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*Corresponding author. Tel.: +1-859-218-1176; fax +1-859-257-8542
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Abstract
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E-mail address: [email protected] (Edward L. Squires)
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 um have been the limiting factors impeding equine embryo cryopreservation. Data from both controlled laboratory settings as well as commercial embryo transfer facilities have shown that
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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
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those < 300 um 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
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embryo prior to freezing has been shown to improve the survival of cryopreserved large equine embryos dramatically .Unfortunately this must be done using very expensive equipment.
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Developments that could improve the success of equine embryo cryopreservation are : having hormones available for superovulation; a means of hastening the embryo into the mare’s uterus in order to consistently collect <300 µm embryos or; the development of a simple means of
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collapsing embryos >300 µm.
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Keywords Equine, embryo , cryopreservation , vitrification
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1. Introduction Equine embryo transfer is a technique that has been used in the equine industry since the late
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1970s and early 1980s. The majority of embryos collected during those decades were transferred as fresh embryos and the recipients and donors were on the same farm. With the development of techniques for cooled storage of embryos at 5°C (Carnevale et al. [1]) for 12-24 hr, shipment of embryos to recipient stations became a reality. This stimulated the embryo transfer business and
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resulted in the establishment of several large embryo transfer programs that receive shipped
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embryos. In contrast , the concept of cooled , transported embryos has not been embraced by the cattle industry . Generally when a donor cow is flushed the embryos are transferred into the available recipients and any extra embryos are frozen.
Equine embryo transfer numbers have increased dramatically in the last couple of decades [2] , generally driven by the change in breed regulations by the major breeds in the USA as well
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as the polo industry in Argentina and the sport horse industries in Brazil. Many breeds now allow unlimited registration of embryo transfer foals. However the number of embryos frozen has not increased proportionately. A significant difference between embryo transfer in cattle and
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horses is that a very predictable superovulation regime is available for cattle and typically six
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transferable embryos are available from each flush. In contrast , superovulation is not currently available in horses and consequently embryo recovery is based on only one ovulation and generally ranges from 50-70% embryo recovery per cycle. As a consequence implementation of cryopreservation procedures in clinical practice has been limited by the number of embryos available and , to a lesser extent, relatively low demand by the equine breeding.
Despite the
relative limited use of cryopreservation in the horse, there are some distinct advantages: 1. minimize the number of recipients and thus decrease the cost of embryo transfer; 3
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2. ability to bank embryos, especially from young mares while their performance and the genetic value is being determined;
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3. exportation or importation of embryos; 4. Transport of frozen embryos may have less health risks then importing live animals;
5. collection and cryopreservation of embryos in the off season so they can be transferred early
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the following breeding season;
6. Cryopreservation of an embryo while genetic testing or sexing is being conducted;
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7.in vitro produced embryos can be taken out of culture as a morulae or early blastocyst and cryopreserved successfully.
However, there also are some disadvantages of frozen embryos over fresh or cooled embryos. Even though pregnancy rates with small (<300 µm) embryos can be 50-65%, these
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rates are still lower than those for fresh or cooled embryos [3]. In addition since the embryo develops and increases in diameter rapidly once it reaches the uterus, it is difficult to recover small embryos unless the time of ovulation is accurately determined by frequent examination of
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the mare. Embryos >300 µm have very poor survival after cryopreservation, providing a 20-30%
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pregnancy rate [4]. As mentioned previously, without the hormones available for superovulating the mare, there are very few extra embryos for freezing. Also just recently the American Quarter Horse Association (AQHA) changed their rule stating that frozen embryos can only be used for transfer for 2 years after the death of the mare. This more than likely will discourage the idea of embryo banking. 2. Embryo size
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Size of the embryo is the major factor affecting survival after cryopreservation. When the embryo enters the uterus from the oviduct it is 150-220 µm in size and has the morphology of a morula or early blastocyst. Within 0.5 to 1.0 days the embryo will increase in diameter to >300
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µm and become a blastocyst [5]. Morula and early blastocyst are surrounded by a thick zona pellucida. However , by day 7 post-ovulation the zona pellucida has thinned out and the
embryos >300 µm do not survive freezing and thawing:
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underlying acellular glycoprotein capsule has formed [6]. There are several reasons why
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1. the capsule impedes the penetration of the cryoprotectant;
2. thickness of the capsule has been shown to be correlated to freezability of the embryo [7]: 3. small surface-area-volume ratio, 4.the large amount of blastocoele fluid.
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A discussion will follow later as to how researchers have tried to modify the large embryo to allow better survival after cryopreservation.
Needless to say, most of the equine embryos that are currently frozen are small, morulae or
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early blastocysts obtained by performing embryo recovery 6.0-6.5 days after ovulation. This
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requires that the mare be examined every 4-6 hr after hCG or GnRH agonist administration until ovulation is detected or rely on a timed-ovulation protocol and flush 8 days after injection of hCG as demonstrated by Eldridge-Panuska et al. [8]. 3. Slow cooling
The first equine embryos were frozen by a slow- cool method. A pregnancy was produced but subsequently lost [9]. Yanamoto et al., 1982 [10] reported the birth of the first frozen/thawed-embryo foal. Slade et al. [11] in our laboratory in 1985 used essentially a bovine 5
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protocol to slow- cool equine embryos. Glycerol was used as the cryoprotectant and embryos packaged in either ampules or 0.5 ml plastic straws. Embryos were cooled at 4°C/min from room temperature to -6°C, seeded at -6°C and held for 15 min then cooled at 0.3°C/min to -30°C and
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0.1°C/min to -33°C then plunged into nitrogen. Six of 23 embryos were unsuitable for transfer after thawing and the remaining 17 were transferred. Eight of 10 , with a mean diameter of 173 µm , resulted in a pregnancy, while only 1 of 7 classified as blastocysts resulted in a pregnancy.
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Lascombes and Pashen [12] as part of a commercial program used a similar slow- cooling
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procedure with glycerol added in two steps. A 56% pregnancy rate was reported after transferring 43, day 6-6.5 embryos (<220 µm). Combined these studies demonstrate that pregnancy rates following transfer of small equine embryos using a slow- cooling method provide an pregnancy rate similar to what is obtained with frozen bovine embryos. However the problem with slow- cooling small equine embryos is that the appropriate size is difficult to
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acquire, slow- cooling method takes 1-2 hr and an expensive programmable freezer is needed. This more than likely prompted the search for alternative methods such as vitrification which is
4. Vitrification
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quicker and requires no expensive equipment.
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The alternative to slow- cooling of embryos that has gained in popularity is a process called vitrification. This is an ultra-rapid cooling method that prevents ice crystals by cooling so fast that the liquid changes to a solid , glass-like phase without ice formation [2]. This has the advantage of being fast and easy but does expose the embryo to high concentrations of cryoprotectants. Thus the type of cryoprotectants and the timing of exposure to these agents becomes very important. The effect of size of the equine embryo on survival may be even more critical with vitrification than with slow- cool. 6
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Hochi et al. [13] reported on the viability following vitrification of equine embryos of various sizes: <200, 200-300, and >300 µm. Re-expansion of the small embryos after freezing and thawing was quite good for <200 (7/8), 200-300 (6/8) µm embryos but only 2 of 8 embryos
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>300 µm re-expanded.
Eldridge-Panuska [8] conducted a study to compare embryo development rates after
vitrification , warming and transfer of: (1 small (<300 µm) versus large (>300 µm) embryos; (2
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small embryos that were transferred after dilution of cryoprotectants in vitro post-thaw or by
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direct transfer with in-straw dilution; and (3 large embryos vitrified with different concentrations of ethylene glycol. Embryos were assigned to one of three groups depending on their stage of development and diameter: (1 morulae and very early blastocysts <300 µm; (2 blastocysts; and (3 blastocysts >300 µm.
Embryos were exposed to vitrification solutions at room temperature (22-24°C) and placed in
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a drop (200 µL) of 1.4 M glycerol in PBS for 5 min , moved to a 200 ul drop of 1.4 M glycerol + 3.6 M ethylene glycol for 5 min, and then transferred into 30 µL of final vitrification solution of 3.4 M glycerol + 4.6 M ethylene glycol (EG/G). The 30 µL of final vitrification solution,
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containing the embryo, was loaded into the center of a 0.25 mL, non-irradiated, polyvinyl
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chloride straw (IMV, Maple Grove, MN, USA) separated by two air bubbles from two columns (30 µL) of final vitrification solution. Straw ends were loaded with two columns (90µL) of 0.5 M galactose in base medium. The total time for exposure of the embryo to the final vitrification solution and to load the embryo into the straw was <1 min. The straw was heat-sealed and placed for 1 min into a cooled plastic goblet surrounded by liquid nitrogen vapor. After 1 min in vapor the entire goblet containing the straw was then plunged into liquid nitrogen. Embryos were warmed by holding in room temperature air for 10 s before being immersed in a 20°C water bath 7
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for 10 s. Contents of the straw were expelled into a Petri dish and stirred gently to facilitate mixture of the vitrification (EG/G) and dilution solutions (Gal). The embryo was transferred into a 200 µL drop of 0.25 M galactose in PBS for 5 min and then transferred into PBS. Within 10
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min , warmed embryos (n=3 per recipient/group) , separated by developmental stages , were nonsurgically transferred into the uteri of recipients mares that had ovulated 6 days previously.. Uteri of recipients were examined by transrectal ultrasonography at 4, 6, 8 and 10 days after transfer to
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image embryonic vesicles. Pregnant recipients were again scanned on Day 38 to determine
viability of fetuses, at which time pregnancies were terminated. Embryonic vesicles resulted
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from three morulae or early blastocysts and from one blastocyst <300 µm (4/6 embryos <300 µm). No pregnancy was observed after transfer of blastocysts >300 mm (n=3). In a second experiment , embryos <300 and >300 µm were vitrified, thawed and transferred as in Experiment 1. Some embryos <300 µm were also transferred using a direct-transfer
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procedure (DT) without taking the embryos out of the straw. Embryo development rates to Day 16 were not different for embryos <300 µm that were transferred without removing from the straw as in Experiment 1 (10/22, 46%) or transferred by DT (16/26, 62%). Embryos >300 mm
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(n=19) did not produce embryonic vesicles.
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Conclusions of this study were that : 1) ) large embryos do not survive the vitrification process well , 2) small (<300 µm) embryos can be collected 6.5 d after ovulation (8 d after hCG administration) with good recovery rates, 3) equine morulae and early blastocysts stage embryos can be vitrified and produce pregnancy rates nearly as high as non-cryopreserved embryos [3], and 4) direct transfer was successful and would allow the practitioner to transfer embryos without step-wise dilutions and without a microscope or dilution media in the field.
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Another study showed that equine embryos can be cooled for an average of 16 hr and then vitrified/thawed and transferred (Hudson et al. [14]). In this study, embryos were collected 6.5 days after ovulation and assigned to 1 of 2 groups: Group 1, cooled to 5°C in a passive cooling
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device for 12 to 19 hours before vitrification; and Group 2, vitrified immediately upon collection. There was no difference (P >0.05) between pregnancy rates of embryos that were vitrified
immediately upon collection (15/20, 75%) versus those that were cooled for an average of 15
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hours before vitrification (13/20, 65%). This study demonstrated that embryos could be collected
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in one location, cooled and subsequently shipped to a referral center for vitrification.
5. Manipulating the large embryo prior to freezing
It is clear from many studies that pregnancy rates after either slow- cooling or vitrification
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are too low to justify freezing of embryos much over 300 µm. Knowing that the potential reasons for the poor freezability of the equine blastocysts maybe a function of the acellular capsule, increased cell numbers and/ or increased blastocoele volume [15] , researchers have tried to
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modify the larger equine embryo prior to freezing. The thickness of the capsule has been shown to be correlated with freezability. [7]). Maclellan et al. [16] pre-treated embryos >300 µm with
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trypsin, cytochalasin-B and the combination of the two agents prior to slow- cooling the embryos in a two step addition of glycerol (5, 10%). Cytochalasin-B is a microfilament inhibitor used previously to improve cryosurvival in pig embryos. Trypsin was added to determine if enzymatic treatment of the capsule before cryopreservation would improve permeability of the cryoprotectant and embryo survival. This study also directly compared the viability of small and large embryos after slow- cooling/thawing and transfer. Forty-five embryos were obtained (17, <300 and 28, >300 µm). Of the small embryos, 8 were cultured in vitro for 6 hr prior to transfer 9
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and 9 transferred immediately after thawing. Day 16 pregnancy rates were similar (P >0.05): direct transfer 5/9; cultured 7/8; and non-frozen 14/20. Embryos >300 were assigned to 1 of 4 groups: 1) no treatment; 2) trypsin; 3) cytochalasin-B; and 4) combination. Embryos were slow
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cooled , frozen/thawed and stained with DAPI in SOF/Hepes at 38°C for 15 min. Four of eight embryos had >90% staining and were considered dead and the remaining had 10 to 40%
fluorescing cells. Embryos pre-treated with cytochalasin-B had similar pregnancy rates (3/7) as
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embryos frozen with no pre-treatment (4/7). However, pre-treatment with trypsin or the
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combination of trypsin and cytochalasin -B did not result in any pregnancies. A more recent study was conducted in the Netherlands [17] to compare the type and extent of cellular damage suffered by small and large embryos during cryopreservation by slow-cooling and vitrification. These authors utilized molecular techniques to evaluate cytoskeletal quality and mitochondrial activity. Embryos were collected 156-168 hr after ovulation by transcervical
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lavage of the uterus. Those graded with a quality score of 1 or 2 were divided into size classes of ≤300 or >300 µm and assigned to one of the following treatment groups: 1) control (≤300 µm, n=5; >300 µm, n=7); 2) exposure to slow-freezing (≤300 µm, n=5; >300 µm, n=5) without
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freezing; 3) exposure to vitrification solution (≤300 µm, n=5; >300 µm, n=6) without cooling; 4) cryopreservation by slow-freezing (≤300 µm, n=7; >300 µm, n=8), stored in liquid nitrogen for at
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least 7 days prior to analysis; or 5) cryopreservation by vitrification (≤300 µm, n=8; >300 µm, n=7), stored in liquid nitrogen for at least 7 days prior to analysis. Embryos were incubated for 15 min at 37°C in PBS containing 0.1 mg/l 4′,6-diamidino-2phenylindole dihydrochloride (DAPI) and examined using a conventional fluorescence microscope equipped with a digital camera, to count the dead (DAPI-stained) cells.Embryos were washed and then stained with 1 µmol/l Mitotracker Red CMHX-Ros at 37°C for 30 min to 10
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label active mitochondria. Exposure to cryoprotectants without freezing had no effect on cytoskeleton quality, but cryopreservation by either slow- cooling or vitrification reduced cytoskeletal quality of both small and large embryos; however, there were no differences due to
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cryopreservation technique. However, neither exposure to cryoprotectants nor cryopreservation of embryos markedly affected mitochondrial activity within the cells of either small or large embryos. This study used molecular techniques to confirm that larger embryos are damaged
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more than smaller embryos. As suggested previously the reasons for the poor viability with the large embryos post-thaw may be due to inadequate penetration of cryoprotectants into the inner
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cell mass due to the capsule or volume of the embryo.
Recent studies have provided some encouraging results that collapse of the blastocyts prior to freezing using a micromanipulator to penetrate the capsule [18,19] or laser [20] may improve the viability of cryopreserved equine blastocysts. Scherzer et al. [20] tested a method to enhance
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penetration of cryoprotectants across the capsule by using a laser to create a small opening and replacing blastocoele fluid with cryoprotectant solution. Embryos were then vitrified with a CryoLeaf system. Three of the four small embryos and 4 of 9 large blastocysts (>300 µm)
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resulted in a vesicle upon transfer. Unfortunately, only one of the recipients was still pregnant at 23 days of gestation. It seems likely that the majority of these vesicles were trophoblastic
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vesicles. Choi et al. [18_] demonstrated that an equine blastocyst biopsied using a piezo drill would collapse but the blastocoele cavity refilled within 3 hours. In a subsequent study by this same group [21]) they evaluated the viability of equine blastocysts after biopsy, fluid removal and vitrification. Embryos 300-730 µm biopsied and vitrified in DMSO resulted in 8/16 pregnancies at d 12 and 2/16 at d 25. This compared to 6/13 (at d 12 and 25) for similar size blastocysts biopsied and vitrified in ethylene glycol and warmed in sucrose-based medium.
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Additional studies were done where the piezo drill was used to obtain cells and fluid from the center of the embryo or from the trophoblast periphery. This resulted in removal of >70% of the blastocoele fluid. Pregnancy rates were 4/7 for the central aspiration method and 5/7 for the
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aspiration method from the trophoblast periphery. They concluded that expanded blastocysts up to 650 µm can produce pregnancies after vitrification. From these studies it was apparent that the decrease in >70% of fluid was essential for success and that just penetration of the capsule was
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not sufficient to sustain viability after vitrification. It was encouraging that the majority of these large blastocysts developed heart beats by day 25. These results demonstrated that expanded
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equine blastocysts can produce normal pregnancies after cryopreservation. The technique from their study that produced the highest pregnancy rate was 1) complete collapse of the blastocyst by aspiration of fluid from the trophoblast periphery, 2) vitrification in micropipette loader tips and 3) use of the EG’s vitrification method
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Most recently , Diaz et.al.[19] evaluated several blastocysts micromanipulation and vitrification procedures for day 8 embryos.They compared a single versus double puncture of the capsule and direct or indirect introduction of cryoprotectants.In experiment 1 , 24 day 8 embryos were
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subjected to either a single puncture or double puncture treatment. For the one puncture treatment the injection pipette was inserted through the capsule until the trophectoderm was
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penetrated while for the two puncture technique the pipette was inserted completely through the embryo . In both treatments 95-99% of the blastocoel fluid was removed. Embryos were either exposed to vitrification solution ( VS 1 solution) by a direct injection into the blastocoel cavity or indirectly by exposure .All embryos were exposed to a 3 step vitrification procedure and before plunging into liquid nitrogen they were loaded onto a open vitrification device ( Cryolock, Biotech Inc., Cumming Ga). Based on re-expansion of embryos after culture, they saw no benefit
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to a double puncture or direct injection of cryoprotectant. In fact it appeared that the two puncture system and direct injection of cryoprotectant may have caused an increased risk of capsule loss.Six day 8 embryos ( ranging in size from 448 to 1168 um) were vitrified using the
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indirect introductionof cryoprotectant method and transferred into recipients.Five of 6 recipients were pregnant at 25 days.They concluded that three factors may have been responsible for the high in vitro and in vivo embryo viability : 1, removal of 95-99% of the blastocoel fluid ; 2, use
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of a 3 step vitification procedure and ; 3 use of an open system vitrification device.
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Another approach that has been used to decrease the volume of the blastocoele fluid prior to vitrification was to use sucrose as a means of dehydrating the embryo prior to freezing (Barfield et al. [22]. Forty six, day 7-8 embryos, 300-1350 µm were exposed to the following treatments:1) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, slow cooled (n=21); 2) 10 min in 1.5 M glycerol, slow freeze (n=15); 3) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol
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followed by exposure to thaw solution and cultured (n=5); and 4) transferred directly to culture medium (n=5). Five embryos from each treatment were evaluated morphologically at 24 and 48 hr of culture. The majority of embryos had a score of >3 (1=excellent and 5=degenerate/dead).
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Embryos from treatments 1 and 2 (16 and 10, respectively) were transferred into recipients and only 2 recipients became pregnant. The pregnancies were from the smallest embryos transferred
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(400-415 µm). These authors concluded that there was no advantage of incorporating a 2 min dehydration step prior to cryopreservation. They went on to suggest that a longer dehydration period might have been more beneficial since many of the larger embryos had very little sign of collapse except a slight pulling away from the capsule. A slower cooling rate was also suggested as a means of allowing more time for dehydration prior to freezing. 6. Pregnancy rates from commercial transfer of vitrified embryos 13
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Most of the studies published to date on pregnancy rates of cryopreserved equine embryos have been in university settings and based on a small number of transfers. Unlike the data available for cooled, transported embryos there are few if any reports of large scale fertility trials
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with frozen equine embryos. Part of the reason is that there are relatively few equine embryos frozen commercially for the reasons presented previously. Most equine embryos are either
been transferred and still remain stored in liquid nitrogen.
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transferred fresh or after cooling for 12-24 hr. In addition, most equine embryos frozen have not
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Data on pregnancy rates after transfer of frozen/thawed embryos were obtained from two commercial embryo transfer facilities in the USABoth of these facilities transferred hundreds of embryos each year from donor mares flushed on the farm and embryos that were shipped to the facilities. Each of these embryo transfer stations maintained a large number of recipients. The majority of the embryos were acquired from Quarter horse type mares that were flushed 6.5 days
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after ovulation. Embryos were washed though several drops of holding medium (Equine Holding Medium, Bioniche Animal Health, Pullman, WA). Both farms used the vitrification procedure described by Eldridge-Panuska [8] using a commercial vitrification kit (Bioniche Animal
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Health, Pullman ,WA). Each farm made a slight modification to the procedure: Farm 1 used a pipette instead of the 0.25 ml straw to move the embryo between the vitrification solutions and
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transferred the embryos directly without taking them out of the straw; Farm 2 used 0.25 ml straws to move the embryo between the solutions and after thawing the embryos were placed into equine holding medium for up to an hour before transfer. The results from transfer of vitrified/thawed embryos in Farm 1 were categorized by embryo size. The 11 and 25 day pregnancy rates for embryos 150-174 µm were 69 and 63% (n=296); 64 and 59% (n=108) for embryos 175-200 µm; and 71 and 53% (n=17) for embryos 201 µm or 14
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greater. Overall based on 421 vitrified/thawed embryos transferred the pregnancy rate at 11 days was 68% and 62% at 25 days. Their pregnancy rate for fresh embryos at the same facility at 25
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days was 75% (n=3060) and 70% for cooled embryos (n=1479). Data for Farm 2 was based on ultrasound exams done at 14 and 45 days and thus cannot be directly compared to Farm 1. All of the embryos vitrified on this farm were <300 µm but size ranges were not included with the data. Pregnancy rate at 14 days was 58% (n=239) and 51% at
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45 days.
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These commercial data certainly validate that small equine embryos can be vitrified and result in good pregnancy rates. In discussions with many other veterinarians, the success of using the vitrification kits ( i.e. pregnancy rates after transfer) for cryopreservation of equine embryos has been quite variable (personal communication). It has been my experience in teaching courses on embryo vitrification that there are several potential steps where damage can occur. The most
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serious would be over exposure of the embryo to the VS3 solution, which has 3.4 M glycerol and 4.6 M ethylene glycol, which can be quite toxic to the embryo. Also since the embryo is quite small and tends to float in the vitrification solutions it can be difficult to find. Lastly, when
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moving the embryo with the 0.25 ml straw too much fluid can be transferred from VS1 to VS2 to
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VS3, thus diluting out the cryoprotectant level. 7. Cryopreservation of in vitro produced (IVP) embryos There are now several locations within the USA, Europe and South America where equine embryos are produced in vitro by ovum pick-up (OPU), in vitro culture, ICSI, embryo culture and embryo transfer. Most of these IVP embryos are cultured for 7 to 9 days then transferred as fresh embryos or transported to recipient facilities for transfer 12-24 hr later. However this requires that synchronized recipients are available. The ability to freeze IVP equine embryos 15
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would be more efficient and less costly. Galli et al. [23] reported on their commercial program of freezing IVP equine embryos. IVP embryos were cryopreserved with a standard slow- cool protocol using 10% glycerol. Non-surgical embryo transfers were performed over 5 breeding
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seasons (2001-2005). Nine pregnancies were obtained from 13 transfers (69%). At the time their manuscript was written 5 foals had been produced. Although there were no other types of
embryos transferred for comparison, it would appear the equine IVP embryo survives freezing
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quite well. Production of embryos in vitro has the advantage that one can remove the embryo at
8. Future of equine embryo freezing
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the perfect size and stage for maximal survival after freezing.
Nearly 60% of bovine embryos that collected are frozen for transfer at a later time. This certainly reduces the number of recipients that need to be maintained thus reducing the cost and increasing flexibility. For this same trend to occur in the horse industry several developments
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will have to occur. Being able to consistently superovulate mares would provide the extra embryos for freezing. Based on several studies both equine pituitary FSH [24] and recombinant FSH [25] can induce multiple ovulations such that 2-4 embryos maybe collected per flush.
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Embryos from superovulated mares have been shown to be quite viable after vitrification as long
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as they are <300 µm [14]. Unfortunately there are currently no commercial products available to the veterinarian for superovulation. Other strategies for superovulation than those based on FSH injection need to be examined. Other developments that could improve success is to either have a means of hastening the embryo into the mare’s uterus in order to consistently collect <300 µm embryos or develop a simple means of collapsing embryos >300 µm. Certainly if all three of these developments occurred and pregnancy rates with slow cooled or vitrified embryos were nearly equal fresh embryos then equine embryo cryopreservation would increase dramatically. 16
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Acknowledgements The authors would like to thank Drs Jim Bailey and Ryan Coy of Royal Vista Southwest ,
providing data on pregnancy rates with vitrified embryos . References
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Pursell , OK and Drs Steve and Cade Burns of Burns Quarter Horse Ranch , Menifee Cal for
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[5] Battut I, Colchen S, Fieni F, Tainturier D, Bruyas JF. Success rates when attempting to nonsurgically collect equine embryos at 144, 156 or 168 hours after ovulation. Equine Vet J
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[6] Betteridge KJ, Eaglesome MD, Mitchell D, Flood PF, Beriault R. Development of horse embryos up to twenty-two days after ovulation: Observations on fresh specimens. J Anat 1982; 135:191-209.
[7] Legrand E, Bencharif D, Barrier-Battut I, Delajarraud H, Corniere P, Fieni F, et al. Comparison of pregnancy rates for days 7-8 equine embryos frozen in glycerol with or without previous enzymatic treatment of their capsule. Theriogenology 2002; 58:721-3. 17
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[8] Eldridge-Panuska WD, di Brienza VC, Seidel GE , Squires EL, Carnevale EM. Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos. Theriogenology
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2005; 63(5):1308-19. [9] Griffin JL, Castleberry RS, Schneider HS Jr. Influence of day of collection on embryo recovery rates in mature cycling mares. Theriogenology 1981; 15:106(abstr).
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[10] Yanamoto Y, Oguri N, Tsutsumi Y, Hachinohe Y. Experiments in the freezing and storage of equine embryos. J Reprod Fertil Suppl 1982; 32:399-403.
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[11] Slade NP, Takeda T, Squires EL, Elsden RP, Seidel GE Jr. A new procedure for the cryopreservation of equine embryos. Theriogenology 1985; 24(1):45-58. [12] Lascombes FA, Pashen RL. Results from embryo freezing and post-ovulation breeding in a commercial embryo transfer programme. Proc 5th International Symposium on Equine Embryo
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Transfer. Havemeyer Foundation Monograph Series 3, 2000, Saari, Finland, pp 95-96. [13] Hochi S, Fujimoto T, Oguri N. Large equine blastocysts are damaged by vitrification procedures. Reprod Fertil Dev 1995; 7(1):113-7.
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[14] Hudson JJ, McCue PM, Carnevale EM, Welsh S, Squires EL. The effects of cooling and
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vitrification of embryos from mares treated with equine follicle- stimulating hormone on pregnancy rates after nonsurgical transfer. J Equine Vet Sci 2006; 26(2):51-4. [15] Bruyas JF, Battut I, Pol JM, Botrel C, Fiene F, Tainturier D. Quantitative analysis of morphological modifications of day 6.5 horse embryos after treatment with four cryoprotectants: Differential effects on the inner cell mass and trophoblast cells. Biol Reprod Monogr Series 1995; 1:329-39.
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[16] 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
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trypsin.Theriogenology 2002; 58:717-20. [17] Hendriks WK, Roelen BA, Colenbrander B, Stout TA. Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow-freezing or vitrification.
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Equine Vet J 2015; 47(6):701-7.
[18] Choi YH, Gustafson-Seabury A, Velez IC, Hartman DL, Bliss S, Riera FL, et al. Viability
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of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction 2010; 140(6):893-902.
19.Diaz Fabian , Bondiolli K , Paccamonti D , Gentry G . Cryopreservation of day 8 embryos after blastocyst micromanipulation and vitrification .Theriogenology 2016 ; 85: 894-903
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[19] Scherzer J, Davis C, Hurley DJ. Laser-assisted vitrification of large equine embryos. Reprod Domest Anim 2011; 46(6):1104-6.
[21] Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB, Blanchard TL, Hayden
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SS, Hinrichs K. Successful cryopreservation of expanded equine blastocysts. Theriogenology
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2011; 76(1):143-52.
[22] Barfield JP, McCue PM, Squires EL, Seidel GE Jr. Effect of dehydration prior to cryopreservation of large equine embryos. Cryobiology 2009 Aug; 59(1):36-41. [23] Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Anim Reprod Sci 2007 Mar; 98(1-2):39-55. 19
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[24] McCue PM, LeBlanc MM, Squires EL. eFSH in clinical equine practice. Theriogenology. 2007 Aug;68(3):429-33 [25] Meyers-Brown G, Bidstrup LA, Famula TR, Colgin M, Roser JF. Treatment with
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recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine
luteinizing hormone (reLH) increases embryo recovery in superovulated mares. Anim Reprod
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Sci. 2011 Oct;128(1-4):52-9.
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Small equine embryos can be successfully frozen Larger ( >300um ) embryos must be deflated prior to freezing Methods for superovulation are needed to enhance embryo recovery
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Vitrification and transfer of small embryos results in good pregnancy rates
PII:
S0737-0806(16)30055-7
DOI:
10.1016/j.jevs.2016.03.009
Reference:
YJEVS 2057
To appear in:
Journal of Equine Veterinary Science
Received Date: 26 February 2016 Revised Date:
24 March 2016
Accepted Date: 24 March 2016
Please cite this article as: Squires EL, McCue PM, Cryopreservation of Equine Embryos, Journal of Equine Veterinary Science (2016), doi: 10.1016/j.jevs.2016.03.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Cryopreservation of Equine Embryos
a
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Edward L. Squiresa* and Patrick M. McCueb
Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546,
USA
Equine Reproduction Laboratory Colorado State University , Ft Collins Co 80521
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b
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*Corresponding author. Tel.: +1-859-218-1176; fax +1-859-257-8542
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Abstract
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E-mail address: [email protected] (Edward L. Squires)
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 um have been the limiting factors impeding equine embryo cryopreservation. Data from both controlled laboratory settings as well as commercial embryo transfer facilities have shown that
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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
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those < 300 um 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
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embryo prior to freezing has been shown to improve the survival of cryopreserved large equine embryos dramatically .Unfortunately this must be done using very expensive equipment.
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Developments that could improve the success of equine embryo cryopreservation are : having hormones available for superovulation; a means of hastening the embryo into the mare’s uterus in order to consistently collect <300 µm embryos or; the development of a simple means of
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collapsing embryos >300 µm.
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Keywords Equine, embryo , cryopreservation , vitrification
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1. Introduction Equine embryo transfer is a technique that has been used in the equine industry since the late
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1970s and early 1980s. The majority of embryos collected during those decades were transferred as fresh embryos and the recipients and donors were on the same farm. With the development of techniques for cooled storage of embryos at 5°C (Carnevale et al. [1]) for 12-24 hr, shipment of embryos to recipient stations became a reality. This stimulated the embryo transfer business and
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resulted in the establishment of several large embryo transfer programs that receive shipped
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embryos. In contrast , the concept of cooled , transported embryos has not been embraced by the cattle industry . Generally when a donor cow is flushed the embryos are transferred into the available recipients and any extra embryos are frozen.
Equine embryo transfer numbers have increased dramatically in the last couple of decades [2] , generally driven by the change in breed regulations by the major breeds in the USA as well
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as the polo industry in Argentina and the sport horse industries in Brazil. Many breeds now allow unlimited registration of embryo transfer foals. However the number of embryos frozen has not increased proportionately. A significant difference between embryo transfer in cattle and
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horses is that a very predictable superovulation regime is available for cattle and typically six
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transferable embryos are available from each flush. In contrast , superovulation is not currently available in horses and consequently embryo recovery is based on only one ovulation and generally ranges from 50-70% embryo recovery per cycle. As a consequence implementation of cryopreservation procedures in clinical practice has been limited by the number of embryos available and , to a lesser extent, relatively low demand by the equine breeding.
Despite the
relative limited use of cryopreservation in the horse, there are some distinct advantages: 1. minimize the number of recipients and thus decrease the cost of embryo transfer; 3
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2. ability to bank embryos, especially from young mares while their performance and the genetic value is being determined;
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3. exportation or importation of embryos; 4. Transport of frozen embryos may have less health risks then importing live animals;
5. collection and cryopreservation of embryos in the off season so they can be transferred early
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the following breeding season;
6. Cryopreservation of an embryo while genetic testing or sexing is being conducted;
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7.in vitro produced embryos can be taken out of culture as a morulae or early blastocyst and cryopreserved successfully.
However, there also are some disadvantages of frozen embryos over fresh or cooled embryos. Even though pregnancy rates with small (<300 µm) embryos can be 50-65%, these
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rates are still lower than those for fresh or cooled embryos [3]. In addition since the embryo develops and increases in diameter rapidly once it reaches the uterus, it is difficult to recover small embryos unless the time of ovulation is accurately determined by frequent examination of
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the mare. Embryos >300 µm have very poor survival after cryopreservation, providing a 20-30%
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pregnancy rate [4]. As mentioned previously, without the hormones available for superovulating the mare, there are very few extra embryos for freezing. Also just recently the American Quarter Horse Association (AQHA) changed their rule stating that frozen embryos can only be used for transfer for 2 years after the death of the mare. This more than likely will discourage the idea of embryo banking. 2. Embryo size
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Size of the embryo is the major factor affecting survival after cryopreservation. When the embryo enters the uterus from the oviduct it is 150-220 µm in size and has the morphology of a morula or early blastocyst. Within 0.5 to 1.0 days the embryo will increase in diameter to >300
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µm and become a blastocyst [5]. Morula and early blastocyst are surrounded by a thick zona pellucida. However , by day 7 post-ovulation the zona pellucida has thinned out and the
embryos >300 µm do not survive freezing and thawing:
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underlying acellular glycoprotein capsule has formed [6]. There are several reasons why
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1. the capsule impedes the penetration of the cryoprotectant;
2. thickness of the capsule has been shown to be correlated to freezability of the embryo [7]: 3. small surface-area-volume ratio, 4.the large amount of blastocoele fluid.
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A discussion will follow later as to how researchers have tried to modify the large embryo to allow better survival after cryopreservation.
Needless to say, most of the equine embryos that are currently frozen are small, morulae or
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early blastocysts obtained by performing embryo recovery 6.0-6.5 days after ovulation. This
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requires that the mare be examined every 4-6 hr after hCG or GnRH agonist administration until ovulation is detected or rely on a timed-ovulation protocol and flush 8 days after injection of hCG as demonstrated by Eldridge-Panuska et al. [8]. 3. Slow cooling
The first equine embryos were frozen by a slow- cool method. A pregnancy was produced but subsequently lost [9]. Yanamoto et al., 1982 [10] reported the birth of the first frozen/thawed-embryo foal. Slade et al. [11] in our laboratory in 1985 used essentially a bovine 5
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protocol to slow- cool equine embryos. Glycerol was used as the cryoprotectant and embryos packaged in either ampules or 0.5 ml plastic straws. Embryos were cooled at 4°C/min from room temperature to -6°C, seeded at -6°C and held for 15 min then cooled at 0.3°C/min to -30°C and
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0.1°C/min to -33°C then plunged into nitrogen. Six of 23 embryos were unsuitable for transfer after thawing and the remaining 17 were transferred. Eight of 10 , with a mean diameter of 173 µm , resulted in a pregnancy, while only 1 of 7 classified as blastocysts resulted in a pregnancy.
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Lascombes and Pashen [12] as part of a commercial program used a similar slow- cooling
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procedure with glycerol added in two steps. A 56% pregnancy rate was reported after transferring 43, day 6-6.5 embryos (<220 µm). Combined these studies demonstrate that pregnancy rates following transfer of small equine embryos using a slow- cooling method provide an pregnancy rate similar to what is obtained with frozen bovine embryos. However the problem with slow- cooling small equine embryos is that the appropriate size is difficult to
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acquire, slow- cooling method takes 1-2 hr and an expensive programmable freezer is needed. This more than likely prompted the search for alternative methods such as vitrification which is
4. Vitrification
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quicker and requires no expensive equipment.
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The alternative to slow- cooling of embryos that has gained in popularity is a process called vitrification. This is an ultra-rapid cooling method that prevents ice crystals by cooling so fast that the liquid changes to a solid , glass-like phase without ice formation [2]. This has the advantage of being fast and easy but does expose the embryo to high concentrations of cryoprotectants. Thus the type of cryoprotectants and the timing of exposure to these agents becomes very important. The effect of size of the equine embryo on survival may be even more critical with vitrification than with slow- cool. 6
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Hochi et al. [13] reported on the viability following vitrification of equine embryos of various sizes: <200, 200-300, and >300 µm. Re-expansion of the small embryos after freezing and thawing was quite good for <200 (7/8), 200-300 (6/8) µm embryos but only 2 of 8 embryos
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>300 µm re-expanded.
Eldridge-Panuska [8] conducted a study to compare embryo development rates after
vitrification , warming and transfer of: (1 small (<300 µm) versus large (>300 µm) embryos; (2
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small embryos that were transferred after dilution of cryoprotectants in vitro post-thaw or by
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direct transfer with in-straw dilution; and (3 large embryos vitrified with different concentrations of ethylene glycol. Embryos were assigned to one of three groups depending on their stage of development and diameter: (1 morulae and very early blastocysts <300 µm; (2 blastocysts; and (3 blastocysts >300 µm.
Embryos were exposed to vitrification solutions at room temperature (22-24°C) and placed in
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a drop (200 µL) of 1.4 M glycerol in PBS for 5 min , moved to a 200 ul drop of 1.4 M glycerol + 3.6 M ethylene glycol for 5 min, and then transferred into 30 µL of final vitrification solution of 3.4 M glycerol + 4.6 M ethylene glycol (EG/G). The 30 µL of final vitrification solution,
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containing the embryo, was loaded into the center of a 0.25 mL, non-irradiated, polyvinyl
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chloride straw (IMV, Maple Grove, MN, USA) separated by two air bubbles from two columns (30 µL) of final vitrification solution. Straw ends were loaded with two columns (90µL) of 0.5 M galactose in base medium. The total time for exposure of the embryo to the final vitrification solution and to load the embryo into the straw was <1 min. The straw was heat-sealed and placed for 1 min into a cooled plastic goblet surrounded by liquid nitrogen vapor. After 1 min in vapor the entire goblet containing the straw was then plunged into liquid nitrogen. Embryos were warmed by holding in room temperature air for 10 s before being immersed in a 20°C water bath 7
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for 10 s. Contents of the straw were expelled into a Petri dish and stirred gently to facilitate mixture of the vitrification (EG/G) and dilution solutions (Gal). The embryo was transferred into a 200 µL drop of 0.25 M galactose in PBS for 5 min and then transferred into PBS. Within 10
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min , warmed embryos (n=3 per recipient/group) , separated by developmental stages , were nonsurgically transferred into the uteri of recipients mares that had ovulated 6 days previously.. Uteri of recipients were examined by transrectal ultrasonography at 4, 6, 8 and 10 days after transfer to
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image embryonic vesicles. Pregnant recipients were again scanned on Day 38 to determine
viability of fetuses, at which time pregnancies were terminated. Embryonic vesicles resulted
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from three morulae or early blastocysts and from one blastocyst <300 µm (4/6 embryos <300 µm). No pregnancy was observed after transfer of blastocysts >300 mm (n=3). In a second experiment , embryos <300 and >300 µm were vitrified, thawed and transferred as in Experiment 1. Some embryos <300 µm were also transferred using a direct-transfer
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procedure (DT) without taking the embryos out of the straw. Embryo development rates to Day 16 were not different for embryos <300 µm that were transferred without removing from the straw as in Experiment 1 (10/22, 46%) or transferred by DT (16/26, 62%). Embryos >300 mm
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(n=19) did not produce embryonic vesicles.
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Conclusions of this study were that : 1) ) large embryos do not survive the vitrification process well , 2) small (<300 µm) embryos can be collected 6.5 d after ovulation (8 d after hCG administration) with good recovery rates, 3) equine morulae and early blastocysts stage embryos can be vitrified and produce pregnancy rates nearly as high as non-cryopreserved embryos [3], and 4) direct transfer was successful and would allow the practitioner to transfer embryos without step-wise dilutions and without a microscope or dilution media in the field.
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Another study showed that equine embryos can be cooled for an average of 16 hr and then vitrified/thawed and transferred (Hudson et al. [14]). In this study, embryos were collected 6.5 days after ovulation and assigned to 1 of 2 groups: Group 1, cooled to 5°C in a passive cooling
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device for 12 to 19 hours before vitrification; and Group 2, vitrified immediately upon collection. There was no difference (P >0.05) between pregnancy rates of embryos that were vitrified
immediately upon collection (15/20, 75%) versus those that were cooled for an average of 15
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hours before vitrification (13/20, 65%). This study demonstrated that embryos could be collected
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in one location, cooled and subsequently shipped to a referral center for vitrification.
5. Manipulating the large embryo prior to freezing
It is clear from many studies that pregnancy rates after either slow- cooling or vitrification
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are too low to justify freezing of embryos much over 300 µm. Knowing that the potential reasons for the poor freezability of the equine blastocysts maybe a function of the acellular capsule, increased cell numbers and/ or increased blastocoele volume [15] , researchers have tried to
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modify the larger equine embryo prior to freezing. The thickness of the capsule has been shown to be correlated with freezability. [7]). Maclellan et al. [16] pre-treated embryos >300 µm with
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trypsin, cytochalasin-B and the combination of the two agents prior to slow- cooling the embryos in a two step addition of glycerol (5, 10%). Cytochalasin-B is a microfilament inhibitor used previously to improve cryosurvival in pig embryos. Trypsin was added to determine if enzymatic treatment of the capsule before cryopreservation would improve permeability of the cryoprotectant and embryo survival. This study also directly compared the viability of small and large embryos after slow- cooling/thawing and transfer. Forty-five embryos were obtained (17, <300 and 28, >300 µm). Of the small embryos, 8 were cultured in vitro for 6 hr prior to transfer 9
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and 9 transferred immediately after thawing. Day 16 pregnancy rates were similar (P >0.05): direct transfer 5/9; cultured 7/8; and non-frozen 14/20. Embryos >300 were assigned to 1 of 4 groups: 1) no treatment; 2) trypsin; 3) cytochalasin-B; and 4) combination. Embryos were slow
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cooled , frozen/thawed and stained with DAPI in SOF/Hepes at 38°C for 15 min. Four of eight embryos had >90% staining and were considered dead and the remaining had 10 to 40%
fluorescing cells. Embryos pre-treated with cytochalasin-B had similar pregnancy rates (3/7) as
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embryos frozen with no pre-treatment (4/7). However, pre-treatment with trypsin or the
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combination of trypsin and cytochalasin -B did not result in any pregnancies. A more recent study was conducted in the Netherlands [17] to compare the type and extent of cellular damage suffered by small and large embryos during cryopreservation by slow-cooling and vitrification. These authors utilized molecular techniques to evaluate cytoskeletal quality and mitochondrial activity. Embryos were collected 156-168 hr after ovulation by transcervical
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lavage of the uterus. Those graded with a quality score of 1 or 2 were divided into size classes of ≤300 or >300 µm and assigned to one of the following treatment groups: 1) control (≤300 µm, n=5; >300 µm, n=7); 2) exposure to slow-freezing (≤300 µm, n=5; >300 µm, n=5) without
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freezing; 3) exposure to vitrification solution (≤300 µm, n=5; >300 µm, n=6) without cooling; 4) cryopreservation by slow-freezing (≤300 µm, n=7; >300 µm, n=8), stored in liquid nitrogen for at
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least 7 days prior to analysis; or 5) cryopreservation by vitrification (≤300 µm, n=8; >300 µm, n=7), stored in liquid nitrogen for at least 7 days prior to analysis. Embryos were incubated for 15 min at 37°C in PBS containing 0.1 mg/l 4′,6-diamidino-2phenylindole dihydrochloride (DAPI) and examined using a conventional fluorescence microscope equipped with a digital camera, to count the dead (DAPI-stained) cells.Embryos were washed and then stained with 1 µmol/l Mitotracker Red CMHX-Ros at 37°C for 30 min to 10
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label active mitochondria. Exposure to cryoprotectants without freezing had no effect on cytoskeleton quality, but cryopreservation by either slow- cooling or vitrification reduced cytoskeletal quality of both small and large embryos; however, there were no differences due to
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cryopreservation technique. However, neither exposure to cryoprotectants nor cryopreservation of embryos markedly affected mitochondrial activity within the cells of either small or large embryos. This study used molecular techniques to confirm that larger embryos are damaged
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more than smaller embryos. As suggested previously the reasons for the poor viability with the large embryos post-thaw may be due to inadequate penetration of cryoprotectants into the inner
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cell mass due to the capsule or volume of the embryo.
Recent studies have provided some encouraging results that collapse of the blastocyts prior to freezing using a micromanipulator to penetrate the capsule [18,19] or laser [20] may improve the viability of cryopreserved equine blastocysts. Scherzer et al. [20] tested a method to enhance
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penetration of cryoprotectants across the capsule by using a laser to create a small opening and replacing blastocoele fluid with cryoprotectant solution. Embryos were then vitrified with a CryoLeaf system. Three of the four small embryos and 4 of 9 large blastocysts (>300 µm)
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resulted in a vesicle upon transfer. Unfortunately, only one of the recipients was still pregnant at 23 days of gestation. It seems likely that the majority of these vesicles were trophoblastic
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vesicles. Choi et al. [18_] demonstrated that an equine blastocyst biopsied using a piezo drill would collapse but the blastocoele cavity refilled within 3 hours. In a subsequent study by this same group [21]) they evaluated the viability of equine blastocysts after biopsy, fluid removal and vitrification. Embryos 300-730 µm biopsied and vitrified in DMSO resulted in 8/16 pregnancies at d 12 and 2/16 at d 25. This compared to 6/13 (at d 12 and 25) for similar size blastocysts biopsied and vitrified in ethylene glycol and warmed in sucrose-based medium.
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Additional studies were done where the piezo drill was used to obtain cells and fluid from the center of the embryo or from the trophoblast periphery. This resulted in removal of >70% of the blastocoele fluid. Pregnancy rates were 4/7 for the central aspiration method and 5/7 for the
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aspiration method from the trophoblast periphery. They concluded that expanded blastocysts up to 650 µm can produce pregnancies after vitrification. From these studies it was apparent that the decrease in >70% of fluid was essential for success and that just penetration of the capsule was
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not sufficient to sustain viability after vitrification. It was encouraging that the majority of these large blastocysts developed heart beats by day 25. These results demonstrated that expanded
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equine blastocysts can produce normal pregnancies after cryopreservation. The technique from their study that produced the highest pregnancy rate was 1) complete collapse of the blastocyst by aspiration of fluid from the trophoblast periphery, 2) vitrification in micropipette loader tips and 3) use of the EG’s vitrification method
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Most recently , Diaz et.al.[19] evaluated several blastocysts micromanipulation and vitrification procedures for day 8 embryos.They compared a single versus double puncture of the capsule and direct or indirect introduction of cryoprotectants.In experiment 1 , 24 day 8 embryos were
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subjected to either a single puncture or double puncture treatment. For the one puncture treatment the injection pipette was inserted through the capsule until the trophectoderm was
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penetrated while for the two puncture technique the pipette was inserted completely through the embryo . In both treatments 95-99% of the blastocoel fluid was removed. Embryos were either exposed to vitrification solution ( VS 1 solution) by a direct injection into the blastocoel cavity or indirectly by exposure .All embryos were exposed to a 3 step vitrification procedure and before plunging into liquid nitrogen they were loaded onto a open vitrification device ( Cryolock, Biotech Inc., Cumming Ga). Based on re-expansion of embryos after culture, they saw no benefit
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to a double puncture or direct injection of cryoprotectant. In fact it appeared that the two puncture system and direct injection of cryoprotectant may have caused an increased risk of capsule loss.Six day 8 embryos ( ranging in size from 448 to 1168 um) were vitrified using the
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indirect introductionof cryoprotectant method and transferred into recipients.Five of 6 recipients were pregnant at 25 days.They concluded that three factors may have been responsible for the high in vitro and in vivo embryo viability : 1, removal of 95-99% of the blastocoel fluid ; 2, use
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of a 3 step vitification procedure and ; 3 use of an open system vitrification device.
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Another approach that has been used to decrease the volume of the blastocoele fluid prior to vitrification was to use sucrose as a means of dehydrating the embryo prior to freezing (Barfield et al. [22]. Forty six, day 7-8 embryos, 300-1350 µm were exposed to the following treatments:1) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, slow cooled (n=21); 2) 10 min in 1.5 M glycerol, slow freeze (n=15); 3) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol
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followed by exposure to thaw solution and cultured (n=5); and 4) transferred directly to culture medium (n=5). Five embryos from each treatment were evaluated morphologically at 24 and 48 hr of culture. The majority of embryos had a score of >3 (1=excellent and 5=degenerate/dead).
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Embryos from treatments 1 and 2 (16 and 10, respectively) were transferred into recipients and only 2 recipients became pregnant. The pregnancies were from the smallest embryos transferred
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(400-415 µm). These authors concluded that there was no advantage of incorporating a 2 min dehydration step prior to cryopreservation. They went on to suggest that a longer dehydration period might have been more beneficial since many of the larger embryos had very little sign of collapse except a slight pulling away from the capsule. A slower cooling rate was also suggested as a means of allowing more time for dehydration prior to freezing. 6. Pregnancy rates from commercial transfer of vitrified embryos 13
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Most of the studies published to date on pregnancy rates of cryopreserved equine embryos have been in university settings and based on a small number of transfers. Unlike the data available for cooled, transported embryos there are few if any reports of large scale fertility trials
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with frozen equine embryos. Part of the reason is that there are relatively few equine embryos frozen commercially for the reasons presented previously. Most equine embryos are either
been transferred and still remain stored in liquid nitrogen.
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transferred fresh or after cooling for 12-24 hr. In addition, most equine embryos frozen have not
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Data on pregnancy rates after transfer of frozen/thawed embryos were obtained from two commercial embryo transfer facilities in the USABoth of these facilities transferred hundreds of embryos each year from donor mares flushed on the farm and embryos that were shipped to the facilities. Each of these embryo transfer stations maintained a large number of recipients. The majority of the embryos were acquired from Quarter horse type mares that were flushed 6.5 days
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after ovulation. Embryos were washed though several drops of holding medium (Equine Holding Medium, Bioniche Animal Health, Pullman, WA). Both farms used the vitrification procedure described by Eldridge-Panuska [8] using a commercial vitrification kit (Bioniche Animal
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Health, Pullman ,WA). Each farm made a slight modification to the procedure: Farm 1 used a pipette instead of the 0.25 ml straw to move the embryo between the vitrification solutions and
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transferred the embryos directly without taking them out of the straw; Farm 2 used 0.25 ml straws to move the embryo between the solutions and after thawing the embryos were placed into equine holding medium for up to an hour before transfer. The results from transfer of vitrified/thawed embryos in Farm 1 were categorized by embryo size. The 11 and 25 day pregnancy rates for embryos 150-174 µm were 69 and 63% (n=296); 64 and 59% (n=108) for embryos 175-200 µm; and 71 and 53% (n=17) for embryos 201 µm or 14
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greater. Overall based on 421 vitrified/thawed embryos transferred the pregnancy rate at 11 days was 68% and 62% at 25 days. Their pregnancy rate for fresh embryos at the same facility at 25
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days was 75% (n=3060) and 70% for cooled embryos (n=1479). Data for Farm 2 was based on ultrasound exams done at 14 and 45 days and thus cannot be directly compared to Farm 1. All of the embryos vitrified on this farm were <300 µm but size ranges were not included with the data. Pregnancy rate at 14 days was 58% (n=239) and 51% at
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45 days.
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These commercial data certainly validate that small equine embryos can be vitrified and result in good pregnancy rates. In discussions with many other veterinarians, the success of using the vitrification kits ( i.e. pregnancy rates after transfer) for cryopreservation of equine embryos has been quite variable (personal communication). It has been my experience in teaching courses on embryo vitrification that there are several potential steps where damage can occur. The most
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serious would be over exposure of the embryo to the VS3 solution, which has 3.4 M glycerol and 4.6 M ethylene glycol, which can be quite toxic to the embryo. Also since the embryo is quite small and tends to float in the vitrification solutions it can be difficult to find. Lastly, when
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moving the embryo with the 0.25 ml straw too much fluid can be transferred from VS1 to VS2 to
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VS3, thus diluting out the cryoprotectant level. 7. Cryopreservation of in vitro produced (IVP) embryos There are now several locations within the USA, Europe and South America where equine embryos are produced in vitro by ovum pick-up (OPU), in vitro culture, ICSI, embryo culture and embryo transfer. Most of these IVP embryos are cultured for 7 to 9 days then transferred as fresh embryos or transported to recipient facilities for transfer 12-24 hr later. However this requires that synchronized recipients are available. The ability to freeze IVP equine embryos 15
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would be more efficient and less costly. Galli et al. [23] reported on their commercial program of freezing IVP equine embryos. IVP embryos were cryopreserved with a standard slow- cool protocol using 10% glycerol. Non-surgical embryo transfers were performed over 5 breeding
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seasons (2001-2005). Nine pregnancies were obtained from 13 transfers (69%). At the time their manuscript was written 5 foals had been produced. Although there were no other types of
embryos transferred for comparison, it would appear the equine IVP embryo survives freezing
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quite well. Production of embryos in vitro has the advantage that one can remove the embryo at
8. Future of equine embryo freezing
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the perfect size and stage for maximal survival after freezing.
Nearly 60% of bovine embryos that collected are frozen for transfer at a later time. This certainly reduces the number of recipients that need to be maintained thus reducing the cost and increasing flexibility. For this same trend to occur in the horse industry several developments
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will have to occur. Being able to consistently superovulate mares would provide the extra embryos for freezing. Based on several studies both equine pituitary FSH [24] and recombinant FSH [25] can induce multiple ovulations such that 2-4 embryos maybe collected per flush.
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Embryos from superovulated mares have been shown to be quite viable after vitrification as long
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as they are <300 µm [14]. Unfortunately there are currently no commercial products available to the veterinarian for superovulation. Other strategies for superovulation than those based on FSH injection need to be examined. Other developments that could improve success is to either have a means of hastening the embryo into the mare’s uterus in order to consistently collect <300 µm embryos or develop a simple means of collapsing embryos >300 µm. Certainly if all three of these developments occurred and pregnancy rates with slow cooled or vitrified embryos were nearly equal fresh embryos then equine embryo cryopreservation would increase dramatically. 16
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Acknowledgements The authors would like to thank Drs Jim Bailey and Ryan Coy of Royal Vista Southwest ,
providing data on pregnancy rates with vitrified embryos . References
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Pursell , OK and Drs Steve and Cade Burns of Burns Quarter Horse Ranch , Menifee Cal for
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[16] 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
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[21] Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB, Blanchard TL, Hayden
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[22] Barfield JP, McCue PM, Squires EL, Seidel GE Jr. Effect of dehydration prior to cryopreservation of large equine embryos. Cryobiology 2009 Aug; 59(1):36-41. [23] Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Anim Reprod Sci 2007 Mar; 98(1-2):39-55. 19
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[24] McCue PM, LeBlanc MM, Squires EL. eFSH in clinical equine practice. Theriogenology. 2007 Aug;68(3):429-33 [25] Meyers-Brown G, Bidstrup LA, Famula TR, Colgin M, Roser JF. Treatment with
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Sci. 2011 Oct;128(1-4):52-9.
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Small equine embryos can be successfully frozen Larger ( >300um ) embryos must be deflated prior to freezing Methods for superovulation are needed to enhance embryo recovery
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Vitrification and transfer of small embryos results in good pregnancy rates