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In vivo development of vitrified rabbit embryos: Effects of vitrification device, recipient genotype, and asynchrony
Theriogenology 79 (2013) 1124–1129
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In vivo development of vitrified rabbit embryos: Effects of vitrification device, recipient genotype, and asynchrony F. Marco-Jiménez*, R. Lavara, E. Jiménez-Trigos, J.S. Vicente Institute of Science and Animal Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2012 Received in revised form 8 February 2013 Accepted 12 February 2013
This study was designed to evaluate the effects of vitrification device, recipient genotype, and recipient asynchrony on implantation rate, offspring rate at birth, and fetal losses of rabbit embryos. Morphologically normal embryos (N ¼ 787) recovered at 72 hours of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos in Cryotop and ministraw devices were transferred into females induced to ovulate 60 hours (asynchrony) or 72 hours (synchrony) before transfer. In addition, recipient genotypes were analyzed (maternal and paternal genotype). The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total kits born were recorded. Fetal losses were calculated as the difference between total born at birth and implanted embryos. Our data show that a combination of Cryotop device and recipient asynchrony at 12 hours provides the most successful rate of offspring at birth, although a similar implantation rate was obtained with both devices. Thus, low fetal loss rates were observed for embryos vitrified in Cryotop independently of recipient synchrony, and embryos vitrified in straws revealed a two-fold higher rate of fetal losses. Moreover, when an asynchrony between vitrified embryo and recipients was applied, higher rates of embryos developed to term were obtained regardless of the device used. Finally, we found a highly significant association of the recipient genotype with implantation rate, offspring rate at birth, and fetal losses. In conclusion, the current study findings show that Cryotop enhances offspring rate because it is associated with a lower rate of fetal loss. This study thus provides additional evidence that recipient genotype and recipient asynchrony affect offspring rate at birth and indicates that the genotype of the recipient and the recipient asynchrony have a significant effect on implantation rate and fetal losses after vitrification. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Cryotop French straw Morulae Synchrony Embryo transfer
1. Introduction The rabbit breeding industry is increasingly using selected lines [1]. Generation and characterization of these lines require great effort and they must be kept in stock even if not needed for commercial use [2]. Embryo cryopreservation can be used as a tool in setting up genetic resource banks for biodiversity preservation in animal breeding and laboratory products (transgenics, clones), protecting against loss caused by disease or hazards. From * Corresponding author. Tel.: þ34 96 38794335; fax: þ34 96 3877439. E-mail address: [email protected] (F. Marco-Jiménez). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.02.008
a genetic standpoint, the cryopreservation of inbred strains is useful to establish control populations to study the genetic drift and gain when selection programs are applied [1,3,4]. Successful vitrification of mammalian embryos, including rabbits, has been the subject of intense research over many years [5]. The efficiency of vitrifying embryos is shown by survival rates at birth, ranging between 25% and 65% [1,6–10]. Moreover, our previous results showed that storing vitrified embryos in liquid nitrogen is an effective long-term storage option that maintains similar pregnancy rate, fertility, and survival at birth for at least 15 years [11]. Factors that influence the high variability of embryo survival among experiments include the concentration and
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composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution [12]. However, the embryo implantation rates are also attributed, among other factors, to the transfer technique, which depends on several anatomical, physiological, and mechanical aspects [13]. Specifically, the efficacy of cryopreservation programs depends on several factors, such as embryo stage or genotypes of donors and recipients [14]. Numerous studies have investigated the effect of genotype as a variable in embryo cryosurvival [15–21]. However, little consideration has been devoted to recipient genotype and its influence on transferred cryopreserved embryos. To the best of our knowledge, only Vicente and García-Ximénez [22] have reported the effect of recipient doe genotype on survival rate at birth of frozen rabbit embryos. In contrast, because of its essential importance in establishing and maintaining pregnancy, the mechanisms of synchrony have been intensively studied since the first description by Chang in 1950 [23]. However, when a cryopreservation procedure was applied, freezing and thawing induced a delay in the resumption of normal metabolic and synthetic activities in the thawed embryos, as demonstrated in frozen-thawed mouse embryos after transfer [24]. In early studies, it was suggested that the transfer of frozen-thawed rabbit embryos into asynchronous recipients could resolve this problem [25,26]. From that moment on, all studies were carried out using asynchronous recipients [8,10,11,27–29]. However, to the best of our knowledge, no studies have examined the effect of recipient asynchrony on the transfer of vitrified embryos. The latest approach to improving the vitrification system consists of minimizing the volume to allow extremely high cooling and warming rates [5,30–33]. Though the 0.25-mL volume straws limit the cooling rate to less than 2500 C/ min [12], vitrification using Cryotop increases the cooling rate approximately nine-fold (22,800 C/min). Likewise, the warming rate is 42,100 and 1300 C/min for Cryotops and straws, respectively [34]. However, in rabbit only Hochi et al. [35] obtained offspring derived from vitrified-warmed zygotes using the Cryotop method. This study was therefore designed to investigate the effect of vitrification device, recipient genotype, and recipient asynchrony on implantation rate, offspring rate at birth, and fetal losses of rabbit embryos. 2. Materials and methods All chemicals, unless otherwise stated, were reagentgrade and purchased from Sigma-Aldrich Química S.A. (Alcobendas, Madrid, Spain). The Ethics and Animal Welfare Committee of the Universidad Politécnica de Valencia approved this study. All animals were handled according to the principles of animal care published by Spanish Royal Decree 1201/2005 (BOE, 2005; BOE is the official Spanish State Gazette). 2.1. Animals Rabbit from two selected lines, maternal and paternal, were used. The maternal line was based on New Zealand
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White rabbits selected since 1980 by a family index for litter size at weaning [36]. The paternal line is a synthetic line selected since 1990 by individual selection on daily gain from weaning to slaughter age (28 and 63 days [37]). Animals were housed individually at the Polytechnic University of Valencia experimental farm under a controlled 16-hour light:8-hour dark photoperiod and fed a commercial diet. 2.2. Embryo recovery A total of 74 nulliparous female animals were used as donor female contemporaries to recipients (37 from the maternal line and 37 from the paternal line). Female animals were treated with 25 IU of eCG intramuscular (Intervet International B.V., Bowmeer, Holland) to induce receptivity. After 48 hours, female animals were artificially inseminated with a heterospermic pool of semen from male animals of the same line to randomize male effect. At artificial insemination time, female animals were administered 1 mg of buserelin acetate (Hoechst Marion Roussel S.A., Madrid, Spain) to induce ovulation and slaughtered 72 hours later. Embryos were collected at room temperature by flushing the oviducts and uterine horns with 10 mL of embryo recovery media consisting of Dulbecco phosphate buffered saline (DPBS) supplemented with 0.2% (wt/vol) bovine serum albumin (BSA), and antibiotics (penicillin G sodium 300,000 IU, penicillin G procaine 700,000 IU, and dihydrostreptomycin sulphate 1250 mg; Penivet 1; Divasa Farmavic, Barcelona, Spain). After recovery, morphologically normal embryos (morulae and blastocysts) from both lines were classified as normal (presenting homogenous cellular mass, mucin coat, and spherical zona pellucida according to International Embryo Transfer Society classification) and pooled to randomize embryo effect. 2.3. Vitrification and warming procedures Embryos were vitrified using the vitrification procedure described by Vicente et al. [8] using two devices; French ministraw (IMV, L’Aigle, France) and Cryotop (Kitazato Co., Fuji, Japan). The Cryotop consists of a flat rectangular leaf of polypropylene measuring 20 0.7 0.1 mm attached to a thin, 5-cm long handle. Embryos were vitrified in a twostep addition procedure. At vitrification time, embryos were transferred into equilibration solution consisting of 10% (vol/vol) ethylene glycol and 10% (vol/vol) dimethyl sulfoxide dissolved in base medium (BM; DPBS supplemented with 0.2% [wt/vol] BSA), at room temperature for 2 minutes. The embryos were then transferred to vitrification solution consisting of 20% (vol/vol) ethylene glycol and 20% (vol/vol) dimethyl sulfoxide in BM. Next, the embryos were loaded into 0.125-mL sterile plastic ministraws between two drops of DPBS separated by air bubbles, sealed with a sterile plug or loaded into the Cryotop device and directly plunged into liquid nitrogen within 1 minute. After storage in liquid nitrogen, embryos were warmed according to the device used. When the French ministraw device was employed, embryos were placed at 10 cm from vapor nitrogen until the vitrified fraction begin to crystallize (20–30 seconds) and warmed by submerging the straws in a water bath at 20 C for 10 seconds. To remove the vitrification media, the two-step procedure was used. Briefly,
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warmed embryos were placed in a culture dish containing 0.7 mL of 0.33 M sucrose in BM and after 5 minutes embryos were washed in BM before transfer. All samples in Cryotops were warmed by abrupt immersion of the naked Cryotop in 200-mL drops of 0.33 M sucrose at 25 C in BM; after 5 minutes, embryos were washed in BM before transfer. Warming embryos were scored and only undamaged embryos were cataloged as transferable. 2.4. Embryo transfer A total of 440 vitrified embryos (234 for Cryotop and 206 for straw) and 147 fresh embryos were transferred into 49 adult nulliparous female animals (25 from the maternal line and 24 from the paternal line). Only receptive females (determined by vulva color) were induced to ovulate by injection of 1 mg of buserelin acetate (Hoechst Marion Roussel S.A., Madrid, Spain) 72 (synchrony or 0 hours) or 60 hours (asynchrony or 12 hours) before transfer. Female animals were anesthetized using 4 mg/kg xylazine (Rompún; Bayer A.G., Leverkusen, Germany) im, after 0.4 mL/kg ketamine hydrochloride (Imalgène 500; Merial S.A., Lyon, France) iv. Embryo transfer was performed using the laparoscopic technique described by Besenfelder and Brem [38]. The number of transfer embryos was standardized to 12 (six embryos into each oviduct). 2.5. Traits Fourteen days after insemination, female animals were anesthetized following the same procedure described in section 2.4., and ventral midline laparoscopy was carried out, noting implanted embryos. At birth, total kits born were recorded. Fetal losses were calculated as the difference between total born at birth and the number of implanted embryos. 2.6. Statistical analyses A generalized linear model including as fixed effects vitrification devices (Cryotop and straw), recipient genotype (paternal and maternal line), and recipient asynchrony (0 and 12 hours) and their double interactions. The error was designated as having a binomial distribution using the probit link function. Binomial data for implantation rate, offspring rate at birth, and fetal losses were assigned a value of one if positive development had been achieved or a zero if it had not. A P value of less than 0.05 was considered to indicate a statistically significant difference. Data are presented as least square mean standard error of the mean. All statistical analyses were carried out using a commercially available software program (SPSS 16.0 software package; SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effect of vitrification device, recipient genotype, and recipient asynchrony on implantation rate The implantation rate was significantly affected by vitrification device, recipient genotype, and recipient
asynchrony; the statistically significant interactions occurred between vitrification devices and recipient genotype (Table 1). Vitrified embryos (independent of the device used) had a reduced implantation rate (0.41 0.038 and 0.48 0.040 vs. 0.86 0.034, for Cryotop and straw vs. fresh, respectively; Table 2). The maternal line had the highest implantation rate (0.66 0.028 vs. 0.55 0.047, for maternal line vs. paternal line, respectively; Table 2). As for the effect of recipient synchrony, high rates of implantation were obtained with 12 hours of asynchrony (0.46 0.039 vs. 0.74 0.033, for 0 and 12 hours, respectively; Table 2). Embryos vitrified using straws and transferred in the maternal line had the highest implantation rate (0.65 0.041 vs. 0.48 0.048, for straw vs. Cryotop, respectively; Table 3). However, embryos vitrified and transferred in the paternal line had similar implantation rates regardless of the device used (0.30 0.059 vs. 0.35 0.063, for straw vs. Cryotop, respectively; Table 3). 3.2. Effect of vitrification device, recipient genotype, and recipient asynchrony on offspring rate at birth The offspring rate at birth was significantly affected by vitrification device, recipient genotype, and recipient asynchrony; the only statistically significant interactions occurred between vitrification devices and degree of synchrony (Table 1). According to the implantation rate, vitrified embryos (independent of the device used) reduced the offspring rate at birth (0.33 0.038 and 0.25 0.038 vs. 0.63 0.047, for Cryotop and straw vs. fresh, respectively; Table 2). Regarding the effect of recipient asynchrony, high rates of implantation were obtained with 12 hours of asynchrony (0.46 0.039 vs. 0.74 0.033, for 0 and 12 hours, respectively; Table 2). As to the recipient genotype, the paternal line had the lowest implantation rate (0.27 0.039 vs. 0.53 0.029, for the paternal line vs. the maternal line, respectively; Table 2). Thus, transfers to recipients at 0 hours were much more successful when embryos were fresh (0.69 0.067; Table 4); transfers of vitrified embryos (independent of the device used) to asynchronous
Table 1 Significance of factors and interactions on implantation rate, offspring at birth, and fetal losses. Categories
Factors Vitrification devices Recipient genotype Degree of synchrony Interactions Vitrification device by recipient genotype Vitrification device by degree of synchrony Recipient genotype by degree of synchrony
Variables Implantation rate
Offspring rate at birth
Fetal lossesa
*
*
*
*
*
*
*
*
ns
*
ns
ns
ns
*
*
ns
ns
ns
Abbreviation: ns, nonsignificant. a Calculated as the difference between the number of implanted embryos and the number of offspring at birth. * P 0.05.
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Table 2 Effect of recipient genotype and degree of synchrony of vitrified rabbit embryos on implantation, offspring at birth, and fetal losses. Experimental groups Vitrification device Cryotop Straw Fresh Recipient genotype Maternal Paternal Degree of synchrony (h) 0 12
N
Implantation rate
Offspring at birth rate
Fetal lossesa
234 206 147
0.41 0.038b 0.48 0.040b 0.86 0.034c
0.33 0.038b 0.25 0.038b 0.63 0.047c
0.20 0.051b 0.51 0.072c 0.14 0.050b
395 192
0.66 0.028c 0.55 0.047b
0.53 0.029c 0.27 0.039b
0.13 0.032c 0.44 0.070c
291 296
0.46 0.039b 0.74 0.033c
0.31 0.037b 0.49 0.035c
0.25 0.056 0.28 0.038
Results are shown as least square mean SEM. a Calculated as the difference between the number of implanted embryos and the number of offspring at birth. b,c Values in the same column with different superscript letters are statistically different (P < 0.05).
recipients at 12 hours yielded poor results (0.53 0.051 and 0.38 0.058, for vitrified embryos using Cryotop and straw, respectively; Table 4). 3.3. Effect of vitrification device, recipient genotype, and recipient asynchrony on fetal losses Fetal losses were significantly affected by the vitrification device and recipient genotype; the only statistically significant interactions occurred between vitrification devices and degree of synchrony (Table 1). Embryos vitrified with straws had the highest fetal losses compared with the Cryotop device and fresh embryos (0.51 0.072 vs. 0.20 0.051 and 0.14 0.050, for straw vs. Cryotop and fresh, respectively; Table 2). Regarding the recipient genotype, maternal line had the lowest number of fetal losses (0.13 0.032 vs. 0.44 0.070, for the maternal line vs. the paternal line, respectively; Table 2). There were no significant differences in the degree of synchrony (Table 2). Thus, transfers of fresh embryos to asynchronous recipients at 12 hours had the highest number of fetal losses (0.41 0.072), and vitrified embryos (regardless of the device used) transferred to asynchronous recipients at 12 hours had the lowest number of fetal losses (0.14 0.044 and 0.34 0.074 for vitrified embryos using Cryotop and straw, respectively; Table 4). However, the Cryotop always showed fewer fetal losses compared with the straw device, independent of the asynchronous recipients (0.28 0.104 vs. 0.67 0.100 and 0.14 0.044 vs. 0.34 0.074, for Cryotop vs. straw and recipients asynchrony at 0 vs. 12 hours, respectively; Table 4). 4. Discussion In our laboratory, setting up the rabbit embryo cryobank as a fundamental tool to measure genetic gain began in the nineties with a straw device and since then more than 10,000 embryos have been vitrified from different selected lines [11]. Over the past decade, significant improvements have been achieved in cryopreservation technology with the development of vitrification-based procedures. In the vitrification approach, a series of devices has been developed to minimize the volume of vitrification solution. For example, devices like the Cryotop allow greater cooling and warming rates (69,250 C/min
and 117,000 C/min, respectively) in comparison with the straw device (1827 C/min and 2950 C/min, cooling and warming rates, respectively [39]). The advantage of these devices over the straw has not been clearly demonstrated in improved rates of offspring at birth. Does cooling and warming rate enhance pregnancy outcome of vitrified embryos? This was the subject of the present report. Our data indicate that a combination of use of the Cryotop device and recipient asynchrony at 12 hours provides the most successful rate of offspring at birth, although a similar implantation rate was obtained with both devices. Based on the outcome, the embryos appearing as morphologically good after vitrification sustain sublethal damage which has been manifested during fetal development. In this way, low fetal loss rates were observed for embryos vitrified in the Cryotop device regardless of recipient synchrony, with values according to the common reproductive performance in rabbit (at approximately 20% [40–42]). In contrast, embryos vitrified in a straw revealed a two-fold higher rate of fetal losses, which confirms a relation between sublethal cryoinjuries and device. Cryopreserved embryos induce ultrastructural lesions affecting determinant cell components, such as membranes, mitochondria, and the cytoskeleton [43–45]. Nevertheless, embryonic cells are able to rapidly restore DNA synthesis within 3 hours after thawing, and protein synthesis requires 18 to 24 hours to be similar to nonvitrified control embryos [46]. Our results suggest that the device affects the reversibility of cryoinjuries, in agreement with Succu et al. [47]. Because embryos that have overcome alterations caused by vitrification have the same ability to implant regardless of the device, though fetal loss rates were different between both devices, we suggest that Table 3 Significant interactions between vitrification devices and recipient genotype at transfer on implantation rate. Vitrification device
Cryotop Straw Fresh
Recipient genotype Maternal
Paternal
0.48 0.048a 0.65 0.041c 0.82 0.043d
0.35 0.063a,b 0.30 0.059b 0.90 0.049d
Results are shown as least square mean SEM. a–d Values in the same column with different superscript letters are statistically different (P < 0.05).
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Table 4 Significant interactions between the degree of synchrony and vitrification carriers at transfer on offspring rate at birth and fetal losses. Carriers
Fetal lossesa
Offspring rate at birth Degree of synchrony (h)
Cryotop Straw Fresh
Degree of synchrony (h)
0
12
0
12
0.17 0.044b 0.14 0.038b 0.69 0.067c
0.53 0.051c 0.38 0.058d 0.56 0.064c
0.28 0.104b,d 0.67 0.100e 0.03 0.029c
0.14 0.044d 0.34 0.074b 0.41 0.072b
Results are shown as least square mean SEM. a Calculated as the difference between the number of implanted embryos and the number of offspring at birth. b–e Values in the same column with different superscript letters are statistically different (P < 0.05).
device cooling and warming rates could entail failures in maternal–embryo cross talk and formation of the placenta before day 15 of development. The success of pregnancy depends on a receptive endometrium, a normal blastocyst, a synchronized cross talk at the maternal–fetal interface at the time of implantation, and finally a successful placentation and remodeling of uterine vasculature [48]. In the present study we found similar uterine receptivity for both devices. Thus, residual damage in embryos seems to be an erroneous fetal placenta development from 18-19th day of gestation. When the cryopreservation procedure is applied, freezing and thawing induce a delay in the resumption of normal metabolic and synthetic activities [24]. This leads to retarded embryonic development, so asynchrony between donors and recipients is a prerequisite for the development of frozen-thawed rabbit morulae [25,26]. When we applied an asynchrony between vitrified embryo and recipients, higher rates of embryos developed to term were obtained regardless of the device used. These results are in accord with the literature, although previous studies have focused on frozen embryos, and to the best of our knowledge no previous studies have examined the effect of recipient asynchrony on the transfer of vitrified embryos [8,25– 28,40]. The influence of maternal and embryonic genotype in prenatal survival is not clear. However, it has been suggested that maternal and embryonic genotype and the interaction between both genotypes affect prenatal survival [49–54]. Moreover, recipient genotype affected distribution of the fetuses [55]. Maurer and Haseman [56] found strain differences in freezing-thawing sensitivity of rabbit embryos and in maternal genotype of recipient doe. In mouse embryos, some authors have reported that after thawing viability was influenced by maternal and embryonic genotype [17–19]. A previous study demonstrated that a major factor in differential survival rate of frozen embryos between two strains of embryos was the genotype of recipient does [22]. We ruled out embryonic genotype effect, because in our study pools of embryos including both genotypes were used in each transfer. When the recipient genotype effect was evaluated, the paternal genotype does had a higher percentage of fetal losses, according to their reproductive features [40]. This reflects the fact that recipient genotype is crucial in providing an adequate uterine environment to support adhesion, embryo–uterus cross talk, and placental and fetal development to term after vitrified embryo transfer, as has been observed with fresh embryos [57].
4.1. Conclusions The findings of the current study show that use of the Cryotop enhances offspring rate because it is associated with lower rates of fetal loss. Additionally, differences in implantation, offspring rate at birth, and fetal losses were because of the recipient genotype and recipient asynchrony. Nevertheless, further studies should be done to understand the molecular mechanisms of the embryo– uterus cross talk and fetal placenta development associated with the vitrification device. Acknowledgments This work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT) and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jiménez-Trigos was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALiþd. ACIF/2010/ 262). The english text version was revised by N. Macowan English Language Service. References [1] García ML, Baselga M. Estimation of genetic response to selection in litter size of rabbits using a cryopreserved control population. Livest Prod Sci 2002;74:45–53. [2] Santacreu MA, Mocé ML, Climent A, Blasco A. Divergent selection for uterine capacity in rabbits. II. Correlated response in litter size and its components estimated with a cryopreserved control population. J Anim Sci 2005;83:2303–7. [3] Apelo CL, Kanagawa H. Pathogens associated with mammalian embryo (A Review). Jpn J Vet Res 1989;37:49–69. [4] García ML, Blumeto O, Capra G, Vicente JS, Baselga M. Vitrified embryos transfer of two selected Spanish rabbit lines to Uruguay. 7th World Rabbit Congress. Valencia, Spain: Universidad Politecnica de Valencia; 2000. A:139–142. [5] Vajta G, Kuwayama M. Improving cryopreservation systems. Theriogenology 2006;65:236–44. [6] Kasai M, Hamaguchi Y, Zhu SE, Miyake T, Sakurai T, Machida T. High survival of rabbit morulae after vitrification in an ethylene glycolbased solution by a simple method. Biol Reprod 1992;39:284–9. [7] Vicente JS, Garcia-Ximenez F. Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae. Theriogenology 1994;42:1205–15. [8] Vicente JS, Viudes-De-Castro MP, Garcia ML. In vivo survival rate of rabbit morulae after vitrification in a medium without serum protein. Reprod Nutr Dev 1999;39:657–62. [9] Lopez-Bejar M, Lopez-Gatius F. Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure. Theriogenology 2002;58:1541–52. [10] Mocé ML, Blasco A, Santacreu MA. In vivo development of vitrified rabbit embryos: effects on prenatal survival and placental development. Theriogenology 2010;73:704–10.
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In vivo development of vitrified rabbit embryos: Effects of vitrification device, recipient genotype, and asynchrony F. Marco-Jiménez*, R. Lavara, E. Jiménez-Trigos, J.S. Vicente Institute of Science and Animal Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2012 Received in revised form 8 February 2013 Accepted 12 February 2013
This study was designed to evaluate the effects of vitrification device, recipient genotype, and recipient asynchrony on implantation rate, offspring rate at birth, and fetal losses of rabbit embryos. Morphologically normal embryos (N ¼ 787) recovered at 72 hours of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos in Cryotop and ministraw devices were transferred into females induced to ovulate 60 hours (asynchrony) or 72 hours (synchrony) before transfer. In addition, recipient genotypes were analyzed (maternal and paternal genotype). The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total kits born were recorded. Fetal losses were calculated as the difference between total born at birth and implanted embryos. Our data show that a combination of Cryotop device and recipient asynchrony at 12 hours provides the most successful rate of offspring at birth, although a similar implantation rate was obtained with both devices. Thus, low fetal loss rates were observed for embryos vitrified in Cryotop independently of recipient synchrony, and embryos vitrified in straws revealed a two-fold higher rate of fetal losses. Moreover, when an asynchrony between vitrified embryo and recipients was applied, higher rates of embryos developed to term were obtained regardless of the device used. Finally, we found a highly significant association of the recipient genotype with implantation rate, offspring rate at birth, and fetal losses. In conclusion, the current study findings show that Cryotop enhances offspring rate because it is associated with a lower rate of fetal loss. This study thus provides additional evidence that recipient genotype and recipient asynchrony affect offspring rate at birth and indicates that the genotype of the recipient and the recipient asynchrony have a significant effect on implantation rate and fetal losses after vitrification. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Cryotop French straw Morulae Synchrony Embryo transfer
1. Introduction The rabbit breeding industry is increasingly using selected lines [1]. Generation and characterization of these lines require great effort and they must be kept in stock even if not needed for commercial use [2]. Embryo cryopreservation can be used as a tool in setting up genetic resource banks for biodiversity preservation in animal breeding and laboratory products (transgenics, clones), protecting against loss caused by disease or hazards. From * Corresponding author. Tel.: þ34 96 38794335; fax: þ34 96 3877439. E-mail address: [email protected] (F. Marco-Jiménez). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.02.008
a genetic standpoint, the cryopreservation of inbred strains is useful to establish control populations to study the genetic drift and gain when selection programs are applied [1,3,4]. Successful vitrification of mammalian embryos, including rabbits, has been the subject of intense research over many years [5]. The efficiency of vitrifying embryos is shown by survival rates at birth, ranging between 25% and 65% [1,6–10]. Moreover, our previous results showed that storing vitrified embryos in liquid nitrogen is an effective long-term storage option that maintains similar pregnancy rate, fertility, and survival at birth for at least 15 years [11]. Factors that influence the high variability of embryo survival among experiments include the concentration and
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composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution [12]. However, the embryo implantation rates are also attributed, among other factors, to the transfer technique, which depends on several anatomical, physiological, and mechanical aspects [13]. Specifically, the efficacy of cryopreservation programs depends on several factors, such as embryo stage or genotypes of donors and recipients [14]. Numerous studies have investigated the effect of genotype as a variable in embryo cryosurvival [15–21]. However, little consideration has been devoted to recipient genotype and its influence on transferred cryopreserved embryos. To the best of our knowledge, only Vicente and García-Ximénez [22] have reported the effect of recipient doe genotype on survival rate at birth of frozen rabbit embryos. In contrast, because of its essential importance in establishing and maintaining pregnancy, the mechanisms of synchrony have been intensively studied since the first description by Chang in 1950 [23]. However, when a cryopreservation procedure was applied, freezing and thawing induced a delay in the resumption of normal metabolic and synthetic activities in the thawed embryos, as demonstrated in frozen-thawed mouse embryos after transfer [24]. In early studies, it was suggested that the transfer of frozen-thawed rabbit embryos into asynchronous recipients could resolve this problem [25,26]. From that moment on, all studies were carried out using asynchronous recipients [8,10,11,27–29]. However, to the best of our knowledge, no studies have examined the effect of recipient asynchrony on the transfer of vitrified embryos. The latest approach to improving the vitrification system consists of minimizing the volume to allow extremely high cooling and warming rates [5,30–33]. Though the 0.25-mL volume straws limit the cooling rate to less than 2500 C/ min [12], vitrification using Cryotop increases the cooling rate approximately nine-fold (22,800 C/min). Likewise, the warming rate is 42,100 and 1300 C/min for Cryotops and straws, respectively [34]. However, in rabbit only Hochi et al. [35] obtained offspring derived from vitrified-warmed zygotes using the Cryotop method. This study was therefore designed to investigate the effect of vitrification device, recipient genotype, and recipient asynchrony on implantation rate, offspring rate at birth, and fetal losses of rabbit embryos. 2. Materials and methods All chemicals, unless otherwise stated, were reagentgrade and purchased from Sigma-Aldrich Química S.A. (Alcobendas, Madrid, Spain). The Ethics and Animal Welfare Committee of the Universidad Politécnica de Valencia approved this study. All animals were handled according to the principles of animal care published by Spanish Royal Decree 1201/2005 (BOE, 2005; BOE is the official Spanish State Gazette). 2.1. Animals Rabbit from two selected lines, maternal and paternal, were used. The maternal line was based on New Zealand
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White rabbits selected since 1980 by a family index for litter size at weaning [36]. The paternal line is a synthetic line selected since 1990 by individual selection on daily gain from weaning to slaughter age (28 and 63 days [37]). Animals were housed individually at the Polytechnic University of Valencia experimental farm under a controlled 16-hour light:8-hour dark photoperiod and fed a commercial diet. 2.2. Embryo recovery A total of 74 nulliparous female animals were used as donor female contemporaries to recipients (37 from the maternal line and 37 from the paternal line). Female animals were treated with 25 IU of eCG intramuscular (Intervet International B.V., Bowmeer, Holland) to induce receptivity. After 48 hours, female animals were artificially inseminated with a heterospermic pool of semen from male animals of the same line to randomize male effect. At artificial insemination time, female animals were administered 1 mg of buserelin acetate (Hoechst Marion Roussel S.A., Madrid, Spain) to induce ovulation and slaughtered 72 hours later. Embryos were collected at room temperature by flushing the oviducts and uterine horns with 10 mL of embryo recovery media consisting of Dulbecco phosphate buffered saline (DPBS) supplemented with 0.2% (wt/vol) bovine serum albumin (BSA), and antibiotics (penicillin G sodium 300,000 IU, penicillin G procaine 700,000 IU, and dihydrostreptomycin sulphate 1250 mg; Penivet 1; Divasa Farmavic, Barcelona, Spain). After recovery, morphologically normal embryos (morulae and blastocysts) from both lines were classified as normal (presenting homogenous cellular mass, mucin coat, and spherical zona pellucida according to International Embryo Transfer Society classification) and pooled to randomize embryo effect. 2.3. Vitrification and warming procedures Embryos were vitrified using the vitrification procedure described by Vicente et al. [8] using two devices; French ministraw (IMV, L’Aigle, France) and Cryotop (Kitazato Co., Fuji, Japan). The Cryotop consists of a flat rectangular leaf of polypropylene measuring 20 0.7 0.1 mm attached to a thin, 5-cm long handle. Embryos were vitrified in a twostep addition procedure. At vitrification time, embryos were transferred into equilibration solution consisting of 10% (vol/vol) ethylene glycol and 10% (vol/vol) dimethyl sulfoxide dissolved in base medium (BM; DPBS supplemented with 0.2% [wt/vol] BSA), at room temperature for 2 minutes. The embryos were then transferred to vitrification solution consisting of 20% (vol/vol) ethylene glycol and 20% (vol/vol) dimethyl sulfoxide in BM. Next, the embryos were loaded into 0.125-mL sterile plastic ministraws between two drops of DPBS separated by air bubbles, sealed with a sterile plug or loaded into the Cryotop device and directly plunged into liquid nitrogen within 1 minute. After storage in liquid nitrogen, embryos were warmed according to the device used. When the French ministraw device was employed, embryos were placed at 10 cm from vapor nitrogen until the vitrified fraction begin to crystallize (20–30 seconds) and warmed by submerging the straws in a water bath at 20 C for 10 seconds. To remove the vitrification media, the two-step procedure was used. Briefly,
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warmed embryos were placed in a culture dish containing 0.7 mL of 0.33 M sucrose in BM and after 5 minutes embryos were washed in BM before transfer. All samples in Cryotops were warmed by abrupt immersion of the naked Cryotop in 200-mL drops of 0.33 M sucrose at 25 C in BM; after 5 minutes, embryos were washed in BM before transfer. Warming embryos were scored and only undamaged embryos were cataloged as transferable. 2.4. Embryo transfer A total of 440 vitrified embryos (234 for Cryotop and 206 for straw) and 147 fresh embryos were transferred into 49 adult nulliparous female animals (25 from the maternal line and 24 from the paternal line). Only receptive females (determined by vulva color) were induced to ovulate by injection of 1 mg of buserelin acetate (Hoechst Marion Roussel S.A., Madrid, Spain) 72 (synchrony or 0 hours) or 60 hours (asynchrony or 12 hours) before transfer. Female animals were anesthetized using 4 mg/kg xylazine (Rompún; Bayer A.G., Leverkusen, Germany) im, after 0.4 mL/kg ketamine hydrochloride (Imalgène 500; Merial S.A., Lyon, France) iv. Embryo transfer was performed using the laparoscopic technique described by Besenfelder and Brem [38]. The number of transfer embryos was standardized to 12 (six embryos into each oviduct). 2.5. Traits Fourteen days after insemination, female animals were anesthetized following the same procedure described in section 2.4., and ventral midline laparoscopy was carried out, noting implanted embryos. At birth, total kits born were recorded. Fetal losses were calculated as the difference between total born at birth and the number of implanted embryos. 2.6. Statistical analyses A generalized linear model including as fixed effects vitrification devices (Cryotop and straw), recipient genotype (paternal and maternal line), and recipient asynchrony (0 and 12 hours) and their double interactions. The error was designated as having a binomial distribution using the probit link function. Binomial data for implantation rate, offspring rate at birth, and fetal losses were assigned a value of one if positive development had been achieved or a zero if it had not. A P value of less than 0.05 was considered to indicate a statistically significant difference. Data are presented as least square mean standard error of the mean. All statistical analyses were carried out using a commercially available software program (SPSS 16.0 software package; SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effect of vitrification device, recipient genotype, and recipient asynchrony on implantation rate The implantation rate was significantly affected by vitrification device, recipient genotype, and recipient
asynchrony; the statistically significant interactions occurred between vitrification devices and recipient genotype (Table 1). Vitrified embryos (independent of the device used) had a reduced implantation rate (0.41 0.038 and 0.48 0.040 vs. 0.86 0.034, for Cryotop and straw vs. fresh, respectively; Table 2). The maternal line had the highest implantation rate (0.66 0.028 vs. 0.55 0.047, for maternal line vs. paternal line, respectively; Table 2). As for the effect of recipient synchrony, high rates of implantation were obtained with 12 hours of asynchrony (0.46 0.039 vs. 0.74 0.033, for 0 and 12 hours, respectively; Table 2). Embryos vitrified using straws and transferred in the maternal line had the highest implantation rate (0.65 0.041 vs. 0.48 0.048, for straw vs. Cryotop, respectively; Table 3). However, embryos vitrified and transferred in the paternal line had similar implantation rates regardless of the device used (0.30 0.059 vs. 0.35 0.063, for straw vs. Cryotop, respectively; Table 3). 3.2. Effect of vitrification device, recipient genotype, and recipient asynchrony on offspring rate at birth The offspring rate at birth was significantly affected by vitrification device, recipient genotype, and recipient asynchrony; the only statistically significant interactions occurred between vitrification devices and degree of synchrony (Table 1). According to the implantation rate, vitrified embryos (independent of the device used) reduced the offspring rate at birth (0.33 0.038 and 0.25 0.038 vs. 0.63 0.047, for Cryotop and straw vs. fresh, respectively; Table 2). Regarding the effect of recipient asynchrony, high rates of implantation were obtained with 12 hours of asynchrony (0.46 0.039 vs. 0.74 0.033, for 0 and 12 hours, respectively; Table 2). As to the recipient genotype, the paternal line had the lowest implantation rate (0.27 0.039 vs. 0.53 0.029, for the paternal line vs. the maternal line, respectively; Table 2). Thus, transfers to recipients at 0 hours were much more successful when embryos were fresh (0.69 0.067; Table 4); transfers of vitrified embryos (independent of the device used) to asynchronous
Table 1 Significance of factors and interactions on implantation rate, offspring at birth, and fetal losses. Categories
Factors Vitrification devices Recipient genotype Degree of synchrony Interactions Vitrification device by recipient genotype Vitrification device by degree of synchrony Recipient genotype by degree of synchrony
Variables Implantation rate
Offspring rate at birth
Fetal lossesa
*
*
*
*
*
*
*
*
ns
*
ns
ns
ns
*
*
ns
ns
ns
Abbreviation: ns, nonsignificant. a Calculated as the difference between the number of implanted embryos and the number of offspring at birth. * P 0.05.
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Table 2 Effect of recipient genotype and degree of synchrony of vitrified rabbit embryos on implantation, offspring at birth, and fetal losses. Experimental groups Vitrification device Cryotop Straw Fresh Recipient genotype Maternal Paternal Degree of synchrony (h) 0 12
N
Implantation rate
Offspring at birth rate
Fetal lossesa
234 206 147
0.41 0.038b 0.48 0.040b 0.86 0.034c
0.33 0.038b 0.25 0.038b 0.63 0.047c
0.20 0.051b 0.51 0.072c 0.14 0.050b
395 192
0.66 0.028c 0.55 0.047b
0.53 0.029c 0.27 0.039b
0.13 0.032c 0.44 0.070c
291 296
0.46 0.039b 0.74 0.033c
0.31 0.037b 0.49 0.035c
0.25 0.056 0.28 0.038
Results are shown as least square mean SEM. a Calculated as the difference between the number of implanted embryos and the number of offspring at birth. b,c Values in the same column with different superscript letters are statistically different (P < 0.05).
recipients at 12 hours yielded poor results (0.53 0.051 and 0.38 0.058, for vitrified embryos using Cryotop and straw, respectively; Table 4). 3.3. Effect of vitrification device, recipient genotype, and recipient asynchrony on fetal losses Fetal losses were significantly affected by the vitrification device and recipient genotype; the only statistically significant interactions occurred between vitrification devices and degree of synchrony (Table 1). Embryos vitrified with straws had the highest fetal losses compared with the Cryotop device and fresh embryos (0.51 0.072 vs. 0.20 0.051 and 0.14 0.050, for straw vs. Cryotop and fresh, respectively; Table 2). Regarding the recipient genotype, maternal line had the lowest number of fetal losses (0.13 0.032 vs. 0.44 0.070, for the maternal line vs. the paternal line, respectively; Table 2). There were no significant differences in the degree of synchrony (Table 2). Thus, transfers of fresh embryos to asynchronous recipients at 12 hours had the highest number of fetal losses (0.41 0.072), and vitrified embryos (regardless of the device used) transferred to asynchronous recipients at 12 hours had the lowest number of fetal losses (0.14 0.044 and 0.34 0.074 for vitrified embryos using Cryotop and straw, respectively; Table 4). However, the Cryotop always showed fewer fetal losses compared with the straw device, independent of the asynchronous recipients (0.28 0.104 vs. 0.67 0.100 and 0.14 0.044 vs. 0.34 0.074, for Cryotop vs. straw and recipients asynchrony at 0 vs. 12 hours, respectively; Table 4). 4. Discussion In our laboratory, setting up the rabbit embryo cryobank as a fundamental tool to measure genetic gain began in the nineties with a straw device and since then more than 10,000 embryos have been vitrified from different selected lines [11]. Over the past decade, significant improvements have been achieved in cryopreservation technology with the development of vitrification-based procedures. In the vitrification approach, a series of devices has been developed to minimize the volume of vitrification solution. For example, devices like the Cryotop allow greater cooling and warming rates (69,250 C/min
and 117,000 C/min, respectively) in comparison with the straw device (1827 C/min and 2950 C/min, cooling and warming rates, respectively [39]). The advantage of these devices over the straw has not been clearly demonstrated in improved rates of offspring at birth. Does cooling and warming rate enhance pregnancy outcome of vitrified embryos? This was the subject of the present report. Our data indicate that a combination of use of the Cryotop device and recipient asynchrony at 12 hours provides the most successful rate of offspring at birth, although a similar implantation rate was obtained with both devices. Based on the outcome, the embryos appearing as morphologically good after vitrification sustain sublethal damage which has been manifested during fetal development. In this way, low fetal loss rates were observed for embryos vitrified in the Cryotop device regardless of recipient synchrony, with values according to the common reproductive performance in rabbit (at approximately 20% [40–42]). In contrast, embryos vitrified in a straw revealed a two-fold higher rate of fetal losses, which confirms a relation between sublethal cryoinjuries and device. Cryopreserved embryos induce ultrastructural lesions affecting determinant cell components, such as membranes, mitochondria, and the cytoskeleton [43–45]. Nevertheless, embryonic cells are able to rapidly restore DNA synthesis within 3 hours after thawing, and protein synthesis requires 18 to 24 hours to be similar to nonvitrified control embryos [46]. Our results suggest that the device affects the reversibility of cryoinjuries, in agreement with Succu et al. [47]. Because embryos that have overcome alterations caused by vitrification have the same ability to implant regardless of the device, though fetal loss rates were different between both devices, we suggest that Table 3 Significant interactions between vitrification devices and recipient genotype at transfer on implantation rate. Vitrification device
Cryotop Straw Fresh
Recipient genotype Maternal
Paternal
0.48 0.048a 0.65 0.041c 0.82 0.043d
0.35 0.063a,b 0.30 0.059b 0.90 0.049d
Results are shown as least square mean SEM. a–d Values in the same column with different superscript letters are statistically different (P < 0.05).
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Table 4 Significant interactions between the degree of synchrony and vitrification carriers at transfer on offspring rate at birth and fetal losses. Carriers
Fetal lossesa
Offspring rate at birth Degree of synchrony (h)
Cryotop Straw Fresh
Degree of synchrony (h)
0
12
0
12
0.17 0.044b 0.14 0.038b 0.69 0.067c
0.53 0.051c 0.38 0.058d 0.56 0.064c
0.28 0.104b,d 0.67 0.100e 0.03 0.029c
0.14 0.044d 0.34 0.074b 0.41 0.072b
Results are shown as least square mean SEM. a Calculated as the difference between the number of implanted embryos and the number of offspring at birth. b–e Values in the same column with different superscript letters are statistically different (P < 0.05).
device cooling and warming rates could entail failures in maternal–embryo cross talk and formation of the placenta before day 15 of development. The success of pregnancy depends on a receptive endometrium, a normal blastocyst, a synchronized cross talk at the maternal–fetal interface at the time of implantation, and finally a successful placentation and remodeling of uterine vasculature [48]. In the present study we found similar uterine receptivity for both devices. Thus, residual damage in embryos seems to be an erroneous fetal placenta development from 18-19th day of gestation. When the cryopreservation procedure is applied, freezing and thawing induce a delay in the resumption of normal metabolic and synthetic activities [24]. This leads to retarded embryonic development, so asynchrony between donors and recipients is a prerequisite for the development of frozen-thawed rabbit morulae [25,26]. When we applied an asynchrony between vitrified embryo and recipients, higher rates of embryos developed to term were obtained regardless of the device used. These results are in accord with the literature, although previous studies have focused on frozen embryos, and to the best of our knowledge no previous studies have examined the effect of recipient asynchrony on the transfer of vitrified embryos [8,25– 28,40]. The influence of maternal and embryonic genotype in prenatal survival is not clear. However, it has been suggested that maternal and embryonic genotype and the interaction between both genotypes affect prenatal survival [49–54]. Moreover, recipient genotype affected distribution of the fetuses [55]. Maurer and Haseman [56] found strain differences in freezing-thawing sensitivity of rabbit embryos and in maternal genotype of recipient doe. In mouse embryos, some authors have reported that after thawing viability was influenced by maternal and embryonic genotype [17–19]. A previous study demonstrated that a major factor in differential survival rate of frozen embryos between two strains of embryos was the genotype of recipient does [22]. We ruled out embryonic genotype effect, because in our study pools of embryos including both genotypes were used in each transfer. When the recipient genotype effect was evaluated, the paternal genotype does had a higher percentage of fetal losses, according to their reproductive features [40]. This reflects the fact that recipient genotype is crucial in providing an adequate uterine environment to support adhesion, embryo–uterus cross talk, and placental and fetal development to term after vitrified embryo transfer, as has been observed with fresh embryos [57].
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