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Open pulled straw vitrification of goat embryos at various stages of development
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Theriogenology 73 (2010) 1018–1023 www.theriojournal.com
Open pulled straw vitrification of goat embryos at various stages of development A.N. Al Yacoub, M. Gauly, W. Holtz * Department of Animal Science, Georg-August-University, Goettingen, Germany Received 18 September 2009; received in revised form 23 November 2009; accepted 29 November 2009
Abstract This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts. # 2010 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Embryo transfer; Goat; Open pulled straw; Vitrification
1. Introduction The cryopreservation of gametes and embryos can be instrumental in the preservation of genetic variability, conservation of populations threatened by extinction, propagation and transport of superior breeding stock, and, in some situations, solution of
* Corresponding author. Tel.: +49 551 395605; fax: +49 551 395587. E-mail address: [email protected] (W. Holtz). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.11.028
fertility problems. In cattle breeding, the cryopreservation of supernumerary preimplantation embryos from superovulated donors has become an important tool in reproduction management although the transfer of cryopreserved embryos generally yields somewhat lower pregnancy rates than of fresh embryos. The vitrification technique prevents cell damage caused by intracellular ice crystal formation. It implies the use of a viscous medium containing high concentrations of penetrating cryoprotectants and an extremely rapid cooling rate, bringing about a glass-like solidification of liquids without ice crystals [1,2].
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Despite the fact that vitrification is faster, cheaper, and more effective [1,3,4], conventional freezing still is the prevailing means of embryo cryopreservation in both large and small ruminants. In many parts of the world, goats are an important domestic species, supplying milk, meat, and fiber; in addition, the goat may serve as a convenient model for other ruminants. Bilton and Moore [5] were the first to successfully cryopreserve caprine embryos. The first successful vitrification of goat embryos was reported by Yuswiati and Holtz [6]. In a previous study [1], it was shown that caprine blastocysts subjected to the open pulled straw (OPS) vitrification technique established by Vajta et al. [7] produces significantly better pregnancy and embryo survival rates than those cryopreserved by conventional freezing. With conventional freezing, goat morulae are known to have poor postthaw survival rates [8,9]. There is no information available on the vitrification of goat morulae or on other stages than blastocysts.
(pLH) [12] at 12-h intervals, beginning 5 d after the dinoprost treatment. Along with the last two pFSH administrations, the does received two im doses of 5.0 mg dinoprost. To induce ovulation, 18 h after the last treatment with dinoprost the does were administered 0.004 mg of the GnRH analog buserelin im (1 mL Receptal) or 500 IU im human chorionic gonadotropin (hCG; 1 mL Chorulon; Intervet, Beaucauce´, France) or 1 mL sterile physiologic saline solution. The does were tested for estrus with an aproned male at 6-h intervals and hand mated twice daily as long as they would permit a male to mount. To counteract occasionally occurring premature corpus luteum (CL) regression, 12 h after the last mating the donors were provided with a progestogen-containing ear implant (3.3 mg norgestomet; Crestar; Intervet, Beaucauce´, France), which was removed 20 h before embryo collection together with the im administration of 5.0 mg dinoprost.
2. Materials and methods
To obtain morulae, blastocysts, and hatched blastocysts, does were flushed on Days 6, 7 to 8, and 8.5 to 9 after the last mating, respectively, applying the transcervical flushing technique described elsewhere [13,14]. The flushing medium consisted of Dulbecco’s phosphate buffered saline (PBS) supplemented with 2% bovine serum albumin (A9647; Sigma, Steinheim, Germany) at a temperature of 39 8C. The embryos were recovered from the flushings under a stereomicroscope (M8; Wild, Heerbrugg, Switzerland) and classified according to their morphologic appearance [15].
2.1. Animals The experiment was conducted on Boer goats (Capra aegagrus hircus) from our own breeding flock at Goettingen, Germany (518460 N, 98410 E), during the breeding season (October to January). The goats were group-housed in open barns with straw bedding and an outdoor concrete run. They were fed a daily ration of 600 g concentrate, consisting of equal parts of a pelleted diet for breeding ewes (16% crude protein, 10.2 megajoule metabolizable energy/kg supplemented with 43 mg/kg Se, 12 mg/kg I, and 5000 mg/kg Zn), oats, and dried sugar beet pulp and had free access to wheat or barley straw, salt lick, and water. Once daily, the complete flock was routinely tested for estrus with an aproned male. 2.2. Donor preparation and superovulation The estrous cycles of donor does with serum progesterone concentrations in excess of 5 ng/mL (assessed by ELISA [10,11]) were synchronized by treating them with dinoprost (5.0 mg im; Dinolytic; Pharmacia and Upjohn, Erlangen, Germany) followed, 7 d later, by im administration of 0.004 mg of the GnRH analog buserelin (1 mL Receptal; Intervet, Unterschleissheim, Germany). Superovulation was induced by six sc administrations of 4, 4, 2, 2, 2, and 2 Armour units of porcine Follicle Stimulating Hormone (pFSH) supplemented with 40% porcine Luteinizing Hormone
2.3. Embryo collection
2.4. Cryopreservation of embryos Morphologically intact embryos at the morula, blastocyst and hatched blastocyst stage classified as ‘‘very good’’ or ‘‘good’’ were randomly assigned to either conventional slow freezing or vitrification by the OPS method as described elsewhere [1]. Only blastocysts and hatched blastocysts were subjected to conventional freezing, whereas vitrification was conducted on morulae, blastocysts, and hatched blastocysts. Embryos to be conventionally frozen were washed three times, for 3 min at a time, in M2 medium [16]. They were then sequentially transferred for 10, 10, and 20 min to M2 medium supplemented with 0.5, 1.0, and 1.5 M ethylene glycol (E9129; Sigma), respectively. Two embryos at the same stage of development from the same donor doe were loaded into 0.25-mL straws (Minitu¨b, Tiefenbach, Germany) labeled with donor number, stage and quality of the embryos, and date. Up
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to this point, all operations were conducted at room temperature. As a next step, the straws were transferred to an alcohol bath (Haake, Karlsruhe, Germany) precooled to 6 8C. After 10 min, the straws were seeded, and after another 10 min, they were cooled at a rate of 0.5 8C/min to 35 8C. After 15 min at that temperature, straws were immersed in liquid nitrogen. For thawing, straws were removed from liquid nitrogen, waved in the air at room temperature for 8 to 10 sec, and immersed, for 7 to 8 sec in a water bath at 35 8C. Then the contents of the straws were emptied into a tissue culture dish from which the embryos were recovered and passaged, at room temperature, through sequential dishes containing 1 mL M2 medium with 0.5 M sucrose and 0.75 M ethylene glycol (3 min), 0.5 M sucrose (5 min), or no addition (10 min). Until transfer, the embryos remained in M2 medium in an incubator under a humidified atmosphere of air at 39 8C for 5 to 15 min. For the vitrification of embryos, special straws had to be prepared. An 0.25-mL straw (Minitu¨b) was softened over a hot plate at 200 8C and pulled until the thinnest point had approximately half the original diameter and wall thickness. By cutting at the thinnest point, two OPS straws were obtained. Embryos to be vitrified were equilibrated, one at a time, for 10 min in 1 mL holding medium consisting of Medium 199 (PAA Laboratories, Pasching, Germany) supplemented with 0.022 g/100 mL pyruvic acid (P5280; Sigma), 0.0146 g/100 mL L-glutamine (G5763; Sigma), and 20% heat-inactivated goat serum. Thereafter they were transferred to 1 mL holding medium supplemented with 10% ethylene glycol (E9129; Sigma) and 10% dimethyl sulfoxide (Me2SO; D2650; Sigma), and, after 1 min, to a 20-mL droplet containing 20% ethylene glycol and 20% Me2SO. Within less than 25 sec, the embryo, suspended in 1 to 2 mL of the medium, was aspirated into the thin end of a labeled drawn-out straw by capillary force. The straws then were immediately plunged into liquid nitrogen (196 8C) thin-end-first. For thawing, the straws were removed from liquid nitrogen, and the thin end was immediately dipped into warming medium (holding medium containing 0.33 M sucrose) at 39 8C while the opposite end was occluded with the tip of a finger. As the contents of the straw liquefied and the air expanded, the embryos slid out into the medium. After 1 min, the embryo was passaged, for 1 min at a time, through two sequential dishes of holding medium containing 0.33 and 0.20 M sucrose, respectively, and ended up in holding medium devoid of sucrose. Within 5 to 15 min, the embryos were transferred to the uterine horn of a recipient doe.
2.5. Preparation of recipients and embryo transfer Pluriparous does from our own breeding flock were estrus induced by im administration of 5.0 mg dinoprost [17] preceded, 7 d earlier, by im administration of 0.004 mg buserelin. Estrus detection was conducted with an aproned buck twice daily; does in estrus within 48 to 72 h after prostaglandin treatment were considered suitable to serve as recipients. Morulae, blastocysts, and hatched blastocysts were transferred 5, 6 to 7, or 7.5 to 8 d after the end of estrus, respectively. The does had been deprived of feed for 2 d and of water for 1 d. To be sure a functional corpus luteum was present, a blood sample was taken on the morning of the intended transfer to be analyzed for progesterone. The does were anesthetized by iv administration of 1 mL Seaxylan (20 mg xylazine; WDT, Garbsen, Germany) and 1 mL Ursotamin (0.1 g ketamine; Serumwerke, Bernburg, Germany) and placed on a laparoscopy cradle in dorsal recumbency. The ovaries were inspected laparoscopically [18]. A blunted uterine tenaculum forceps, 255 mm long (Possi; Aesculap, Tuttlingen, Germany) was introduced via a 3-cm incision on the linea alba about 5 cm cranial to the udder and, under laparoscopic control, the tip of the uterine horn ipsilateral to the ovary displaying at least one corpus luteum was grasped without pinching. A loop of 3 to 4 cm of the uterine horn was gently exteriorized, and with the aid of a unopette (20 mL; Becton Dickinson, Plymouth, UK), the embryos were deposited in the uterine lumen through a puncture hole made in the uterine wall with a blunted 22gauge hypodermic needle about 5 cm from the utero-tubal junction. In 39 recipients, two embryos were transferred; in the remaining nine either one (n = 7) or three (n = 2) embryos, the reason being that mixing of embryos from different donors was undesirable. After repositioning of the uterus, the incision was closed with single sutures of abdominal wall and skin. Four weeks after transfer, the does were subjected to real-time ultrasonography (Aloka SSD 500, Tokyo, Japan) with a rectal 7.5-MHz lineararray transducer, as described elsewhere [19]. At parturition, kidding rate and litter size were recorded. 2.6. Statistical analysis Differences between treatment groups for the parameters pregnancy rate, kidding rate, and embryo survival were subjected to chi-square test [20]. 3. Results The results of the experiment are summarized in Table 1. Conventional cryopreservation of morulae was
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Table 1 Transfer results of caprine embryos of various developmental stages cryopreserved either conventionally or by OPS vitrification. Unless otherwise noted, recipient does received two embryos each. Embryo stage
Method of cryopreservation
Number of recipients
Recipients pregnant
Recipients kidding n %
Average litter size
Embryo survival, %
Morula
Coventionala OPS Conventional OPS Conventional OPS
— 9 10 b 11 c 9d 9e
—— 0 0z 5 50 xy 9 82 x 3 33 yz 3 33 yz
—— 0 0z 4 40f x 9 82 y 3 33 xz 2 22 fxz
— 0 2.0 1.8 1.0 1.0
— 0z 42xy 70x 19yz 13yz
Blastocyst Hatched blastocyst a
Not included in the experiment. One recipient received only one embryo. c One recipient received one embryo and two recipients received three embryos. d Two recipients received only one embryo. e Three recipient received only one embryo. f One recipient aborted. x,y,z Within columns, values with different superscripts differ (P < 0.05). b
not attempted because, from earlier experience, it was known that chances for survival are minimal. It turned out that OPS vitrification of morulae was equally ineffectual. Blastocysts cryopreserved by conventional means gave rise to pregnancies in 50% (5 of 10) of the recipients. Due to one doe aborting, the kidding rate was 40%. Litter size averaged 2.0, and total survival of transferred blastocysts was 42%. After transfer of OPSvitrified blastocysts, 82% (9 of 11) of the recipients were pregnant and went to term, average litter size being 1.8, which is typical for the flock, and overall embryo survival 70%. All kids born were healthy and normal. The difference in kidding rate between conventionally frozen and OPS-vitrified blastocysts (40% vs. 82%) was statistically significant. The cryopreservation of hatched blastocysts was less effectual. Transfer of conventionally frozen and OPSvitrified embryos both resulted in three of nine recipients (33%) pregnant. Due to one doe aborting in the group that had received vitrified embryos, the kidding rate was only 22%, compared with 33% in the group that had received conventionally frozen embryos. Only singletons were born, leading to embryo survival rates of 19% in the group receiving conventionally frozen embryos and 13% in the group receiving OPS vitrified embryos. Pregnancy, kidding, and embryo survival rates achieved by transferring hatched blastocysts that had been cryopreserved by either technique were significantly lower than what was recorded for nonhatched blastocysts. 4. Discussion The current investigation comprises an attempt to extend the favorable results achieved with OPS
vitrification of caprine blastocysts [1] to embryos of the morula and hatched blastocyst stage. From earlier studies [8,9] it is known that the postthaw survival of caprine morulae after conventional freezing is poor. Other authors [21] came to the same conclusion with in vitro studies but, upon transfer, did not record much of a difference between morulae and blastocysts. Based on our own findings [9], in the current experiment conventional freezing of morulae was omitted. Hardly any information is available on the vitrification of caprine morulae. In the very first published attempt to vitrify goat embryos [6]—though not by the OPS protocol— one of two kids born originated from a morula. Two recent investigations on OPS vitrification of caprine embryos used ‘‘morulae and early blastocysts.’’ They arrived at pregnancy rates of 14% [22] and 33% to 51% [23], respectively, but no mention was made as to what stage the surviving embryos belonged to. In the current study, transfer of OPS vitrified morulae did not result in a pregnancy. Caprine blastocysts have repeatedly been successfully cryopreserved by conventional freezing [9,21,24,25], producing results within the range observed in the current experiment. It has also been shown that it is possible to cryopreserve morulae by culturing them to the blastocyst stage beforehand [25]. In both this and the previous study [1] on the cryopreservation of goat embryos by way of conventional freezing, 42% of the transferred blastocysts were carried to term. After OPS vitrification, embryo survival amounted to 64% in the previous and 70% in the current study. The corresponding values for the proportion of does kidding were 82% and 93%, respectively. The
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outcome of both studies thus clearly favors OPS vitrification over conventional freezing. To our knowledge, the current study is the first to report successful vitrification of hatched goat blastocysts. The transfer results, however, were modest. The same was true for the conventional freezing of hatched blastocysts, an outcome that is not quite in agreement with another report [26]. The current finding is displeasing, because it seems to imply that we have no efficient means of cryopreserving embryos devoid of a zona pellucida at our disposal. This would pertain to hatched blastocysts but also to bisected [27,28], biopsied [29], or otherwise manipulated embryos. The zona pellucida is a protective coat known to control osmotic pressure [30], transport and diffusion of nutrients and metabolites [31], and, in all likelihood, also cryoprotectants. Current methods of embryo cryopreservation were empirically devised and are tailored to embryos (primarily blastocysts) enveloped in an intact zona pellucida. The zona is not a static structure and changes its character as a result of tubal passage [32], sperm penetration [33,34], cryopreservation [35], and, possibly, other effects. This might be at least partly responsible for the poor performance of cryopreserved morulae. On the other hand, the favorable results achieved with cryopreserved blastocysts might be associated with the fact that its cells are greater in number, smaller, and, being arranged more or less in a single layer around the periphery, are exposed to cryoprotectants and low temperature in a more uniform fashion than the cells of the morula, which are few, bigger, and arranged as a solid ball. It might, therefore, be necessary to devise specific protocols for the cryopreservation of embryos at stages of development other than the blastocyst stage, in particular for embryos or segments thereof devoid of a zona. From the findings of this experiment, it may be concluded that, as shown in a previous study [1], for the cryopreservation of caprine blastocysts, the OPS vitrification technique is particularly efficient, but for other stages such as morulae or hatched blastocysts, the technique might have to be modified. Acknowledgments The authors owe thanks to the donors of Dinolytic (Pharmacia and Upjohn, Erlangen, Germany), Receptal (Intervet, Unterschleissheim, Germany), Chorulon and Crestar (Intervet, Beaucauce´, France), and pFSH and pLH (J.F. Beckers, Faculty of Veterinary Medicine, State University of Lie`ge, Belgium) and to the technical staff at the laboratory (B. Sohnrey, E. Stuewe) and
animal facility (D. Daniel, J. Koehler), as well as to G. Das who helped with the statistical analysis. References [1] El-Gayar M, Holtz W. Technical note: Vitrification of goat embryos by the open pulled-straw method. J Anim Sci 2001;79:2436–8. [2] Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987;24:387–402. [3] Van Wagtendonk-de Leeuw AM, den Daas JH, Rall WF. Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution. Theriogenology 1997;48:1071– 84. [4] Campos-Chillo`n LF, Walker DJ, de la Torre-Sanchez JF, Seidel Jr GE. In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology 2006;65: 1200–14. [5] Bilton RJ, Moore NW. In vitro culture, storage and transfer of goat embryos. J Biol Sci 1976;9:125–9. [6] Yuswiati E, Holtz W. Successful transfer of vitrified goat embryos. Theriogenology 1990;34:629–32. [7] Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53–8. [8] Li R, Cameron WN, Batt PA, Trounson AO. Maximum survival of frozen goat embryos is attained at the expanded, hatching and hatched blastocyst stages of development. Reprod Fertil Dev 1990;2:345–50. [9] Puls-Kleingeld M, Nowshari MA, Holtz W. Cryopreservation of goat embryos by the one-step or three-step equilibration procedure. In: Lokeshwar RR, editor. Recent Advances in Goat Production. Nutan Printers; 1992. p. 1388–91. [10] Van de Wiel DFM, Koops W. Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma. Anim Reprod Sci 1986;10:201–13. [11] Moeller R. Enzymimmunoassays mit zweitem Antikoerper fuer Progesteron und porcines LH [Enzymeimmunoassays with second antibody for progesterone and porcine LH]. Dissertation (Dr.sc.agr.) Department of Animal Science, Georg-August-University, Goettingen, 1991, pp. 1-125. [12] Nowshari MA, Beckers JF, Holtz W. Superovulation of goats with purified pFSH supplemented with defined amounts of pLH. Theriogenology 1995;43:797–802. [13] Holtz W, Pereira RJTA, Suyadi, Wang XL, Padilla G, Sohnrey B. Collection of goat embryos via transcervical route. L. Gruner, Y. Chabert (eds.), Proc. 7th Int. Conf. Goats, Tours, France, 2000, vol. 1 pp. 490-491. [14] Suyadi, Sohnrey B, Holtz W. Transcervical embryo collection in Boer goats. Small Rumin Res 2000;36:195–200. [15] Linder GM, Wright RW. Bovine embryo morphology and evaluation. Theriogenology 1983;20:99. [16] Hogan B, Costantine F, Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory; 1986. [17] Holtz W, Sohnrey B, Gerland M, Driancourt MA. Ovsynch synchronization and fixed-time insemination in goats. Theriogenology 2008;69:785–92. [18] Sohnrey B, Holtz W. Technical note: transcervical deep cornual insemination of goats. J Anim Sci 2005;83:1543–8.
A.N.A. Yacoub et al. / Theriogenology 73 (2010) 1018–1023 [19] Padilla-Rivas GR, Sohnrey B, Holtz W. Early pregnancy detection by real-time ultrasonography in Boer goats. Small Rumin Res 2005;58:87–92. [20] Steel GD, Torrie JH. Principles and Procedures of Statistics. McGraw-Hill; 1960. [21] Le Gal F, Baril G, Vallet JC, Leboeuf B. In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol. Theriogenology 1993;40:771–7. [22] Guignot F, Bouttier A, Baril G, Salvetti P, Pignon P, Beckers JF, et al. Improved vitrification method allowing direct transfer of goat embryos. Theriogenology 2006;66:1004–11. [23] Hong QH, Tian SJ, Zhu SE, Feng JZ, Yan CL, Zhao XM, et al. Vitrification of Boer goat morulae and early blastocysts by straw and open-pulled straw method. Reprod Domest Anim 2007;42:34–8. [24] Brebion P, Baril G, Cognie´ Y, Vallet JC. Transfer d’embryons chez les ovins et les caprins. Ann Zootech 1992;41:331–9. [25] Nowshari MA, Holtz W. In vitro culture of goat morulae to blastocysts before freezing. Theriogenology 1995;44:983–8. [26] Chemineau P, Procureur R, Cognie´ Y, Lefe´vre PC, Locatelli A, Chupin D. Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue-transmission. Theriogenology 1986;26:279–90. [27] Nowshari MA, Holtz W. Transfer of split goat embryos without zonae pellucidae either fresh or after freezing. J Anim Sci 1993;71:3403–8.
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[28] Oppenheim SM, Moyer AL, Bondurant RH, Rowe JD, Anderson GB. Successful pregnancy in goats carrying their genetically identical conceptus. Theriogenology 2000;54:629–39. [29] El-Gayar M, Holtz W. Transfer of sexed caprine blastocysts freshly collected or derived from cultured morulae. Small Rumin Res 2005;57:151–6. [30] Bronson RA, McLaren A. Transfer of the mouse oviduct of eggs with and without the zona pellucida. J Reprod Fertil 1970;22: 129–37. [31] Leoni G, Berlinguer F, Rosati I, Bogliolo L. Resumption of metabolic activity of vitrified/warmed ovine embryos. Mol Reprod Dev 2002;64:207–13. [32] Kolbe T, Holtz W. Differences in proteinase digestibility of the zona pellucida of in vivo and in vitro derived porcine oocytes and embryos. Theriogenology 2005;63:1695–705. [33] Yanagimachi R. Mammalian Fertilization. In: Knobil E, Neill JD, editors. The Physiology of Reproduction, Vol. 1. New York: Raven Press; 1988. p. 135–86. [34] Suzuki T, Boediono A, Takagi M, Saha S, Sumantri C. Fertilization and development of frozen-thawed germinal vesicle bovine oocytes by a one-step dilution method in vitro. Cryobiology 1996;33:515–24. [35] Moreira da Silva F, Metelo R. Relation between physical properties of the zona pellucida and viability of bovine embryos after slow-freezing and vitrification. Reprod Domest Anim 2005;40: 205–9.
Theriogenology 73 (2010) 1018–1023 www.theriojournal.com
Open pulled straw vitrification of goat embryos at various stages of development A.N. Al Yacoub, M. Gauly, W. Holtz * Department of Animal Science, Georg-August-University, Goettingen, Germany Received 18 September 2009; received in revised form 23 November 2009; accepted 29 November 2009
Abstract This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts. # 2010 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Embryo transfer; Goat; Open pulled straw; Vitrification
1. Introduction The cryopreservation of gametes and embryos can be instrumental in the preservation of genetic variability, conservation of populations threatened by extinction, propagation and transport of superior breeding stock, and, in some situations, solution of
* Corresponding author. Tel.: +49 551 395605; fax: +49 551 395587. E-mail address: [email protected] (W. Holtz). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.11.028
fertility problems. In cattle breeding, the cryopreservation of supernumerary preimplantation embryos from superovulated donors has become an important tool in reproduction management although the transfer of cryopreserved embryos generally yields somewhat lower pregnancy rates than of fresh embryos. The vitrification technique prevents cell damage caused by intracellular ice crystal formation. It implies the use of a viscous medium containing high concentrations of penetrating cryoprotectants and an extremely rapid cooling rate, bringing about a glass-like solidification of liquids without ice crystals [1,2].
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Despite the fact that vitrification is faster, cheaper, and more effective [1,3,4], conventional freezing still is the prevailing means of embryo cryopreservation in both large and small ruminants. In many parts of the world, goats are an important domestic species, supplying milk, meat, and fiber; in addition, the goat may serve as a convenient model for other ruminants. Bilton and Moore [5] were the first to successfully cryopreserve caprine embryos. The first successful vitrification of goat embryos was reported by Yuswiati and Holtz [6]. In a previous study [1], it was shown that caprine blastocysts subjected to the open pulled straw (OPS) vitrification technique established by Vajta et al. [7] produces significantly better pregnancy and embryo survival rates than those cryopreserved by conventional freezing. With conventional freezing, goat morulae are known to have poor postthaw survival rates [8,9]. There is no information available on the vitrification of goat morulae or on other stages than blastocysts.
(pLH) [12] at 12-h intervals, beginning 5 d after the dinoprost treatment. Along with the last two pFSH administrations, the does received two im doses of 5.0 mg dinoprost. To induce ovulation, 18 h after the last treatment with dinoprost the does were administered 0.004 mg of the GnRH analog buserelin im (1 mL Receptal) or 500 IU im human chorionic gonadotropin (hCG; 1 mL Chorulon; Intervet, Beaucauce´, France) or 1 mL sterile physiologic saline solution. The does were tested for estrus with an aproned male at 6-h intervals and hand mated twice daily as long as they would permit a male to mount. To counteract occasionally occurring premature corpus luteum (CL) regression, 12 h after the last mating the donors were provided with a progestogen-containing ear implant (3.3 mg norgestomet; Crestar; Intervet, Beaucauce´, France), which was removed 20 h before embryo collection together with the im administration of 5.0 mg dinoprost.
2. Materials and methods
To obtain morulae, blastocysts, and hatched blastocysts, does were flushed on Days 6, 7 to 8, and 8.5 to 9 after the last mating, respectively, applying the transcervical flushing technique described elsewhere [13,14]. The flushing medium consisted of Dulbecco’s phosphate buffered saline (PBS) supplemented with 2% bovine serum albumin (A9647; Sigma, Steinheim, Germany) at a temperature of 39 8C. The embryos were recovered from the flushings under a stereomicroscope (M8; Wild, Heerbrugg, Switzerland) and classified according to their morphologic appearance [15].
2.1. Animals The experiment was conducted on Boer goats (Capra aegagrus hircus) from our own breeding flock at Goettingen, Germany (518460 N, 98410 E), during the breeding season (October to January). The goats were group-housed in open barns with straw bedding and an outdoor concrete run. They were fed a daily ration of 600 g concentrate, consisting of equal parts of a pelleted diet for breeding ewes (16% crude protein, 10.2 megajoule metabolizable energy/kg supplemented with 43 mg/kg Se, 12 mg/kg I, and 5000 mg/kg Zn), oats, and dried sugar beet pulp and had free access to wheat or barley straw, salt lick, and water. Once daily, the complete flock was routinely tested for estrus with an aproned male. 2.2. Donor preparation and superovulation The estrous cycles of donor does with serum progesterone concentrations in excess of 5 ng/mL (assessed by ELISA [10,11]) were synchronized by treating them with dinoprost (5.0 mg im; Dinolytic; Pharmacia and Upjohn, Erlangen, Germany) followed, 7 d later, by im administration of 0.004 mg of the GnRH analog buserelin (1 mL Receptal; Intervet, Unterschleissheim, Germany). Superovulation was induced by six sc administrations of 4, 4, 2, 2, 2, and 2 Armour units of porcine Follicle Stimulating Hormone (pFSH) supplemented with 40% porcine Luteinizing Hormone
2.3. Embryo collection
2.4. Cryopreservation of embryos Morphologically intact embryos at the morula, blastocyst and hatched blastocyst stage classified as ‘‘very good’’ or ‘‘good’’ were randomly assigned to either conventional slow freezing or vitrification by the OPS method as described elsewhere [1]. Only blastocysts and hatched blastocysts were subjected to conventional freezing, whereas vitrification was conducted on morulae, blastocysts, and hatched blastocysts. Embryos to be conventionally frozen were washed three times, for 3 min at a time, in M2 medium [16]. They were then sequentially transferred for 10, 10, and 20 min to M2 medium supplemented with 0.5, 1.0, and 1.5 M ethylene glycol (E9129; Sigma), respectively. Two embryos at the same stage of development from the same donor doe were loaded into 0.25-mL straws (Minitu¨b, Tiefenbach, Germany) labeled with donor number, stage and quality of the embryos, and date. Up
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to this point, all operations were conducted at room temperature. As a next step, the straws were transferred to an alcohol bath (Haake, Karlsruhe, Germany) precooled to 6 8C. After 10 min, the straws were seeded, and after another 10 min, they were cooled at a rate of 0.5 8C/min to 35 8C. After 15 min at that temperature, straws were immersed in liquid nitrogen. For thawing, straws were removed from liquid nitrogen, waved in the air at room temperature for 8 to 10 sec, and immersed, for 7 to 8 sec in a water bath at 35 8C. Then the contents of the straws were emptied into a tissue culture dish from which the embryos were recovered and passaged, at room temperature, through sequential dishes containing 1 mL M2 medium with 0.5 M sucrose and 0.75 M ethylene glycol (3 min), 0.5 M sucrose (5 min), or no addition (10 min). Until transfer, the embryos remained in M2 medium in an incubator under a humidified atmosphere of air at 39 8C for 5 to 15 min. For the vitrification of embryos, special straws had to be prepared. An 0.25-mL straw (Minitu¨b) was softened over a hot plate at 200 8C and pulled until the thinnest point had approximately half the original diameter and wall thickness. By cutting at the thinnest point, two OPS straws were obtained. Embryos to be vitrified were equilibrated, one at a time, for 10 min in 1 mL holding medium consisting of Medium 199 (PAA Laboratories, Pasching, Germany) supplemented with 0.022 g/100 mL pyruvic acid (P5280; Sigma), 0.0146 g/100 mL L-glutamine (G5763; Sigma), and 20% heat-inactivated goat serum. Thereafter they were transferred to 1 mL holding medium supplemented with 10% ethylene glycol (E9129; Sigma) and 10% dimethyl sulfoxide (Me2SO; D2650; Sigma), and, after 1 min, to a 20-mL droplet containing 20% ethylene glycol and 20% Me2SO. Within less than 25 sec, the embryo, suspended in 1 to 2 mL of the medium, was aspirated into the thin end of a labeled drawn-out straw by capillary force. The straws then were immediately plunged into liquid nitrogen (196 8C) thin-end-first. For thawing, the straws were removed from liquid nitrogen, and the thin end was immediately dipped into warming medium (holding medium containing 0.33 M sucrose) at 39 8C while the opposite end was occluded with the tip of a finger. As the contents of the straw liquefied and the air expanded, the embryos slid out into the medium. After 1 min, the embryo was passaged, for 1 min at a time, through two sequential dishes of holding medium containing 0.33 and 0.20 M sucrose, respectively, and ended up in holding medium devoid of sucrose. Within 5 to 15 min, the embryos were transferred to the uterine horn of a recipient doe.
2.5. Preparation of recipients and embryo transfer Pluriparous does from our own breeding flock were estrus induced by im administration of 5.0 mg dinoprost [17] preceded, 7 d earlier, by im administration of 0.004 mg buserelin. Estrus detection was conducted with an aproned buck twice daily; does in estrus within 48 to 72 h after prostaglandin treatment were considered suitable to serve as recipients. Morulae, blastocysts, and hatched blastocysts were transferred 5, 6 to 7, or 7.5 to 8 d after the end of estrus, respectively. The does had been deprived of feed for 2 d and of water for 1 d. To be sure a functional corpus luteum was present, a blood sample was taken on the morning of the intended transfer to be analyzed for progesterone. The does were anesthetized by iv administration of 1 mL Seaxylan (20 mg xylazine; WDT, Garbsen, Germany) and 1 mL Ursotamin (0.1 g ketamine; Serumwerke, Bernburg, Germany) and placed on a laparoscopy cradle in dorsal recumbency. The ovaries were inspected laparoscopically [18]. A blunted uterine tenaculum forceps, 255 mm long (Possi; Aesculap, Tuttlingen, Germany) was introduced via a 3-cm incision on the linea alba about 5 cm cranial to the udder and, under laparoscopic control, the tip of the uterine horn ipsilateral to the ovary displaying at least one corpus luteum was grasped without pinching. A loop of 3 to 4 cm of the uterine horn was gently exteriorized, and with the aid of a unopette (20 mL; Becton Dickinson, Plymouth, UK), the embryos were deposited in the uterine lumen through a puncture hole made in the uterine wall with a blunted 22gauge hypodermic needle about 5 cm from the utero-tubal junction. In 39 recipients, two embryos were transferred; in the remaining nine either one (n = 7) or three (n = 2) embryos, the reason being that mixing of embryos from different donors was undesirable. After repositioning of the uterus, the incision was closed with single sutures of abdominal wall and skin. Four weeks after transfer, the does were subjected to real-time ultrasonography (Aloka SSD 500, Tokyo, Japan) with a rectal 7.5-MHz lineararray transducer, as described elsewhere [19]. At parturition, kidding rate and litter size were recorded. 2.6. Statistical analysis Differences between treatment groups for the parameters pregnancy rate, kidding rate, and embryo survival were subjected to chi-square test [20]. 3. Results The results of the experiment are summarized in Table 1. Conventional cryopreservation of morulae was
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Table 1 Transfer results of caprine embryos of various developmental stages cryopreserved either conventionally or by OPS vitrification. Unless otherwise noted, recipient does received two embryos each. Embryo stage
Method of cryopreservation
Number of recipients
Recipients pregnant
Recipients kidding n %
Average litter size
Embryo survival, %
Morula
Coventionala OPS Conventional OPS Conventional OPS
— 9 10 b 11 c 9d 9e
—— 0 0z 5 50 xy 9 82 x 3 33 yz 3 33 yz
—— 0 0z 4 40f x 9 82 y 3 33 xz 2 22 fxz
— 0 2.0 1.8 1.0 1.0
— 0z 42xy 70x 19yz 13yz
Blastocyst Hatched blastocyst a
Not included in the experiment. One recipient received only one embryo. c One recipient received one embryo and two recipients received three embryos. d Two recipients received only one embryo. e Three recipient received only one embryo. f One recipient aborted. x,y,z Within columns, values with different superscripts differ (P < 0.05). b
not attempted because, from earlier experience, it was known that chances for survival are minimal. It turned out that OPS vitrification of morulae was equally ineffectual. Blastocysts cryopreserved by conventional means gave rise to pregnancies in 50% (5 of 10) of the recipients. Due to one doe aborting, the kidding rate was 40%. Litter size averaged 2.0, and total survival of transferred blastocysts was 42%. After transfer of OPSvitrified blastocysts, 82% (9 of 11) of the recipients were pregnant and went to term, average litter size being 1.8, which is typical for the flock, and overall embryo survival 70%. All kids born were healthy and normal. The difference in kidding rate between conventionally frozen and OPS-vitrified blastocysts (40% vs. 82%) was statistically significant. The cryopreservation of hatched blastocysts was less effectual. Transfer of conventionally frozen and OPSvitrified embryos both resulted in three of nine recipients (33%) pregnant. Due to one doe aborting in the group that had received vitrified embryos, the kidding rate was only 22%, compared with 33% in the group that had received conventionally frozen embryos. Only singletons were born, leading to embryo survival rates of 19% in the group receiving conventionally frozen embryos and 13% in the group receiving OPS vitrified embryos. Pregnancy, kidding, and embryo survival rates achieved by transferring hatched blastocysts that had been cryopreserved by either technique were significantly lower than what was recorded for nonhatched blastocysts. 4. Discussion The current investigation comprises an attempt to extend the favorable results achieved with OPS
vitrification of caprine blastocysts [1] to embryos of the morula and hatched blastocyst stage. From earlier studies [8,9] it is known that the postthaw survival of caprine morulae after conventional freezing is poor. Other authors [21] came to the same conclusion with in vitro studies but, upon transfer, did not record much of a difference between morulae and blastocysts. Based on our own findings [9], in the current experiment conventional freezing of morulae was omitted. Hardly any information is available on the vitrification of caprine morulae. In the very first published attempt to vitrify goat embryos [6]—though not by the OPS protocol— one of two kids born originated from a morula. Two recent investigations on OPS vitrification of caprine embryos used ‘‘morulae and early blastocysts.’’ They arrived at pregnancy rates of 14% [22] and 33% to 51% [23], respectively, but no mention was made as to what stage the surviving embryos belonged to. In the current study, transfer of OPS vitrified morulae did not result in a pregnancy. Caprine blastocysts have repeatedly been successfully cryopreserved by conventional freezing [9,21,24,25], producing results within the range observed in the current experiment. It has also been shown that it is possible to cryopreserve morulae by culturing them to the blastocyst stage beforehand [25]. In both this and the previous study [1] on the cryopreservation of goat embryos by way of conventional freezing, 42% of the transferred blastocysts were carried to term. After OPS vitrification, embryo survival amounted to 64% in the previous and 70% in the current study. The corresponding values for the proportion of does kidding were 82% and 93%, respectively. The
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outcome of both studies thus clearly favors OPS vitrification over conventional freezing. To our knowledge, the current study is the first to report successful vitrification of hatched goat blastocysts. The transfer results, however, were modest. The same was true for the conventional freezing of hatched blastocysts, an outcome that is not quite in agreement with another report [26]. The current finding is displeasing, because it seems to imply that we have no efficient means of cryopreserving embryos devoid of a zona pellucida at our disposal. This would pertain to hatched blastocysts but also to bisected [27,28], biopsied [29], or otherwise manipulated embryos. The zona pellucida is a protective coat known to control osmotic pressure [30], transport and diffusion of nutrients and metabolites [31], and, in all likelihood, also cryoprotectants. Current methods of embryo cryopreservation were empirically devised and are tailored to embryos (primarily blastocysts) enveloped in an intact zona pellucida. The zona is not a static structure and changes its character as a result of tubal passage [32], sperm penetration [33,34], cryopreservation [35], and, possibly, other effects. This might be at least partly responsible for the poor performance of cryopreserved morulae. On the other hand, the favorable results achieved with cryopreserved blastocysts might be associated with the fact that its cells are greater in number, smaller, and, being arranged more or less in a single layer around the periphery, are exposed to cryoprotectants and low temperature in a more uniform fashion than the cells of the morula, which are few, bigger, and arranged as a solid ball. It might, therefore, be necessary to devise specific protocols for the cryopreservation of embryos at stages of development other than the blastocyst stage, in particular for embryos or segments thereof devoid of a zona. From the findings of this experiment, it may be concluded that, as shown in a previous study [1], for the cryopreservation of caprine blastocysts, the OPS vitrification technique is particularly efficient, but for other stages such as morulae or hatched blastocysts, the technique might have to be modified. Acknowledgments The authors owe thanks to the donors of Dinolytic (Pharmacia and Upjohn, Erlangen, Germany), Receptal (Intervet, Unterschleissheim, Germany), Chorulon and Crestar (Intervet, Beaucauce´, France), and pFSH and pLH (J.F. Beckers, Faculty of Veterinary Medicine, State University of Lie`ge, Belgium) and to the technical staff at the laboratory (B. Sohnrey, E. Stuewe) and
animal facility (D. Daniel, J. Koehler), as well as to G. Das who helped with the statistical analysis. References [1] El-Gayar M, Holtz W. Technical note: Vitrification of goat embryos by the open pulled-straw method. J Anim Sci 2001;79:2436–8. [2] Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987;24:387–402. [3] Van Wagtendonk-de Leeuw AM, den Daas JH, Rall WF. Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution. Theriogenology 1997;48:1071– 84. [4] Campos-Chillo`n LF, Walker DJ, de la Torre-Sanchez JF, Seidel Jr GE. In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology 2006;65: 1200–14. [5] Bilton RJ, Moore NW. In vitro culture, storage and transfer of goat embryos. J Biol Sci 1976;9:125–9. [6] Yuswiati E, Holtz W. Successful transfer of vitrified goat embryos. Theriogenology 1990;34:629–32. [7] Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53–8. [8] Li R, Cameron WN, Batt PA, Trounson AO. Maximum survival of frozen goat embryos is attained at the expanded, hatching and hatched blastocyst stages of development. Reprod Fertil Dev 1990;2:345–50. [9] Puls-Kleingeld M, Nowshari MA, Holtz W. Cryopreservation of goat embryos by the one-step or three-step equilibration procedure. In: Lokeshwar RR, editor. Recent Advances in Goat Production. Nutan Printers; 1992. p. 1388–91. [10] Van de Wiel DFM, Koops W. Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma. Anim Reprod Sci 1986;10:201–13. [11] Moeller R. Enzymimmunoassays mit zweitem Antikoerper fuer Progesteron und porcines LH [Enzymeimmunoassays with second antibody for progesterone and porcine LH]. Dissertation (Dr.sc.agr.) Department of Animal Science, Georg-August-University, Goettingen, 1991, pp. 1-125. [12] Nowshari MA, Beckers JF, Holtz W. Superovulation of goats with purified pFSH supplemented with defined amounts of pLH. Theriogenology 1995;43:797–802. [13] Holtz W, Pereira RJTA, Suyadi, Wang XL, Padilla G, Sohnrey B. Collection of goat embryos via transcervical route. L. Gruner, Y. Chabert (eds.), Proc. 7th Int. Conf. Goats, Tours, France, 2000, vol. 1 pp. 490-491. [14] Suyadi, Sohnrey B, Holtz W. Transcervical embryo collection in Boer goats. Small Rumin Res 2000;36:195–200. [15] Linder GM, Wright RW. Bovine embryo morphology and evaluation. Theriogenology 1983;20:99. [16] Hogan B, Costantine F, Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory; 1986. [17] Holtz W, Sohnrey B, Gerland M, Driancourt MA. Ovsynch synchronization and fixed-time insemination in goats. Theriogenology 2008;69:785–92. [18] Sohnrey B, Holtz W. Technical note: transcervical deep cornual insemination of goats. J Anim Sci 2005;83:1543–8.
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