Reproductive Performance of Female Rabbits Inseminated with Extenders Supplemented with GnRH Analogue Entrapped in Chitosan-Based Nanoparticles.

Rabbit is a reflexively ovulating species. Accordingly, in the practice of artificial insemination (AI) ovulation must be induced via exogenous GnRH (Gonadotropin-Releasing Hormone) administration, which may be performed intramuscularly, subcutaneously, or intravaginally. Unfortunately, the bioavailability of the GnRH analogue when added to the extender is lower due to the proteolytic activity in the seminal plasma and the poor permeability of the vaginal mucosa. The aim of the study was to refine the practice of AI practice in rabbits by replacing parenteral GnRH analogue administration (subcutaneous, intravenous, or intramuscular injection) with intravaginal application, while reducing its concentration in the diluent. Extenders containing the buserelin acetate in chitosan-dextran sulphate and chitosan-alginate nanoparticles were designed and 356 females were inseminated. Reproductive performance of females inseminated with the two experimental extenders, receiving 4 µg of buserelin acetate intravaginally per doe, was compared with that in the control group, the does of which were inseminated with the extender without the GnRH analogue and induced to ovulate with 1 µg of buserelin acetate administered intramuscularly. The entrapment efficiency of the chitosan-dextran sulphate complex was higher than that of chitosan-alginate. However, females inseminated with both systems showed similar reproductive performance. We conclude that both nanoencapsulation systems are an efficient way of intravaginal ovulation induction, allowing a reduction in the level of the GnRH analogue normally used in seminal doses from 15-25 µg to 4 µg.

Roles of host genetics and sperm microbiota in reproductive success in healthy rabbit.

Although the effects of sperm microbiota and sperm quality have been described previously, recent studies provide evidence that female genital modifications triggered by seminal components could be of significant importance to identify some disturbances associated with fertility. So, sperm microbiota could play a key role in sperm quality, contributing to fertilisation. To understand how sperm microbiota diversity is influenced by the host genetics, the symbiotic bacteria in four inbred lines raised in the same animal facility and their effects on sperm quality and fertility were analysed. Forty healthy rabbits from four selected Spanish commercial lines were used in this research (three based on litter performance, designated A, V and LP, and one selected for daily body weight gain, called R). Significant variations in the seminal concentration, morphology and some motion parameters were found among inbred lines, but sperm motility and viability were similar among inbred lines. After mating, inbred lines selected for litter size had the same fertility rate, significantly higher than inbred line selected for body weight (82 ± 3.3%, 79 ± 3.5% and 89 ± 4.5% versus 61 ± 3.7%, for the A, V and LP vs R lines, respectively, p < 0.05). Bacteria belonging to Proteobacteria, Firmicutes, Fusobacteria and Bacteroidetes were identified in sperm microbiota. At genus level, the bacterial community composition in the sperm microbiota was influenced by host genetics. A total of 35, 16, 34, and 51 genera were accurately detected in the A, V, LP, and R lines, respectively. Moreover, Enhydrobacter, Ferruginibacter, Myroides Paracoccus, Rheinheimera, Tepidiphilus, Tetradesmus obliquus and Thauera genera were present only in the inbred lines selected for litter size. Moreover, the discriminant analysis revealed Lysinibacillus and Flavobacterium genera as potential biomarkers for fertility. Thus, these two genera may play a key role in fertility. Our results demonstrated the existence of a rabbit inbred line-specific variation in bacterial occurrence in sperm microbiota. Moreover, fertility differentials among inbred lines that were not predicted by routine semen analysis could be partly explained by the symbiotic state of the semen microbiota.

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer.

Although assisted reproduction technologies (ARTs) are recognised as safe, and most of the offspring seem apparently healthy, there is clear evidence that ARTs are associated with changes in the embryo's developmental trajectory, which incur physiological consequences during the prenatal and postnatal stages of life. The present study aimed to address the influence of early (day-3 embryos) embryo transfer and cryopreservation on embryo survival, size, and metabolome at the preimplantation stage (day-6 embryos). To this end, fresh-transferred (FT) and vitrified-transferred (VT) embryos were compared using naturally-conceived (NC) embryos as a control reference. The results show that as in vitro manipulation was increased (NC < FT < VT), both embryo survival rate (0.91 ± 0.02, 0.78 ± 0.05 and 0.63 ± 0.05, for NC, FT, and VT groups, respectively) and embryo size (3.21 ± 0.49 mm, 2.15 ± 0.51 mm, 1.76 ± 0.46 mm of diameter for NC, FT, and VT groups, respectively) were significantly decreased. Moreover, an unbiased metabolomics analysis showed overall down-accumulation in 40 metabolites among the three experimental groups, with embryo transfer and embryo cryopreservation procedures both exerting a cumulative effect. In this regard, targeted metabolomics findings revealed a significant reduction in some metabolites involved in metabolic pathways, such as the Krebs cycle, amino acids, unsaturated fatty acids, and arachidonic acid metabolisms. Altogether, these findings highlight a synergistic effect between the embryo transfer and vitrification procedures in preimplantation embryos. However, the ex vivo manipulation during embryo transfer seemed to be the major trigger of the embryonic changes, as the deviations added by the vitrification process were relatively smaller.

Rederivation by Cryopreservation of a Paternal Line of Rabbits Suggests Exhaustion of Selection for Post-Weaning Daily Weight Gain after 37 Generations.

Rabbit selection programmes have mainly been evaluated using unselected or divergently selected populations, or populations rederived from cryopreserved embryos after a reduced number of generations. Nevertheless, unselected and divergent populations do not avoid genetic drift, while rederived animals seem to influence phenotypic traits such as birth and adult weights or prolificacy. The study aimed to evaluate the effect of a long-term selection for post-weaning average daily weight gain (ADG) over 37 generations with two rederived populations. Specifically, two coetaneous populations were derived from vitrified embryos with 18 generational intervals (R19 and R37), reducing or avoiding genetic drift and environmental and cryopreservation effects. After two generations of both rederived populations (R21 vs. R39 generations), all evaluated traits showed some progress as a result of the selection, the response being 0.113 g/day by generation. This response does not seem to affect the estimated Gompertz growth curve parameters in terms of the day, the weight at the inflexion point or the adult weight. Moreover, a sexual dimorphism favouring females was observed in this paternal line. Results demonstrated that the selection programme had improved ADG without variations in adult body weight but, after 37 generations of selection, this trait seems exhausted. Given the reduction in the cumulative reproductive performance and as a consequence in the selection pressure, or possibly/perhaps due to an unexpected effect, rederivation could be the cause of this weak selection response observed from generation 18 onwards.

Long-Term Effects Following Fresh/Vitrified Embryo Transfer Are Transmitted by Paternal Germline in a Large Size Rabbit Cohort.

The concept of developmental programming suggests that the early life environment influences offspring phenotype in later life, whose effects may also be manifested in further generations. Valuable pieces of evidence come from the fields applying assisted reproductive technologies (ARTs), which deprive embryos of their optimal maternal environment and were thus associated with subsequent developmental deviations. Recently, we demonstrated that the in vitro manipulations during a vitrified embryo transfer procedure incurs a cumulative and transgenerational decline in the growth performance of the resulting offspring. Here, we provide a longitudinal study to investigate whether previous developmental deviations could be indistinctly paternally or maternally transmitted using crossbred mattings. Our findings revealed that early embryo manipulations through fresh and vitrified embryo transfer incurred paternally transmissible effects over the growth pattern and adult body weight, which seemed not inheritable via the female germline. Similar inheritable effects were observed after fresh and vitrified embryo transfer, suggesting that disturbing optimal embryo development through in vitro manipulations was the principal trigger of transmissible effects, rather than embryo cryopreservation per se.

Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model.

Nowadays, assisted reproductive technologies (ARTs) are considered valuable contributors to our past, but a future without their use is inconceivable. However, in recent years, several studies have evidenced a potential impact of ART on long-term development in mammal species. To date, the long-term follow-up data are still limited. So far, studies have mainly focused on in vitro fertilization or in vitro culture, with information from gametes/embryos cryopreservation field being practically missing. Herein, we report an approach to determine whether a vitrified embryo transfer procedure would have long-term consequences on the offspring. Using the rabbit as a model, we compared animals derived from vitrified-transferred embryos versus those naturally conceived, studying the growth performance, plus the weight throughout life, and the internal organs/tissues phenotype. The healthy status was assessed over the hematological and biochemical parameters in peripheral blood. Additionally, a comparative proteomic analysis was conducted in the liver tissue to investigate molecular cues related to vitrified embryo transfer in an adult tissue. After vitrified embryo transfer, birth weight was increased, and the growth performance was diminished in a sex-specific manner. In addition, vitrified-transferred animals showed significantly lower body, liver and heart weights in adulthood. Molecular analyses revealed that vitrified embryo transfer triggers reprogramming of the liver proteome. Functional analysis of the differentially expressed proteins showed changes in relation to oxidative phosphorylation and dysregulations in the zinc and lipid metabolism, which has been reported as possible causes of a disturbed growth pattern. Therefore, we conclude that vitrified embryo transfer is not a neutral procedure, and it incurs long-term effects in the offspring both at phenotypic and molecular levels. These results described a striking example of the developmental plasticity exhibited by the mammalian embryo.

Developmental Plasticity in Response to Embryo Cryopreservation: The Importance of the Vitrification Device in Rabbits.

In this study, we evaluated the effect of embryo vitrification using two different devices on adulthood phenotype in rabbits. In vitro development, prenatal embryo survival, body weight, growth performance, haematological and biochemical peripheral blood analysis, reproductive performance, and lactation performance traits were compared between the experimental groups. They derived from naturally-conceived embryos (NC), fresh-transferred embryos (FT), vitrified-transferred embryos using mini-straw (VTs), or vitrified-transferred embryos using Cryotop (VTc). Straw-vitrified embryos exhibited lower in vitro developmental rates and in vivo survival rates following embryo transfer compared to its Cryotop-vitrified counterparts. Moreover, the VTs group exhibited higher foetal losses than VTc, FT, and NC groups. Independently of the vitrification device, vitrified-transferred (VT) offspring showed a skewed sex ratio in favour of males, and an increased birth bodyweight. In contrast, postnatal daily growth was diminished in all ART (i.e., FT and VT) animals. In adulthood, significant differences in body weight between all groups was founded-all ART progenies weighed less than NC animals and, within ART, VT animals weighed less than FT. For VT groups, weight at adulthood was higher for the VTs group compared with the VTc group. Peripheral blood parameters ranged between common values. Moreover, no differences were found in the fertility rates between experimental groups. Furthermore, similar pregnancy rates, litter sizes, and the number of liveborns were observed, regardless of the experimental group. However, decreased milk yield occurred for VTc and FT animals compared to VTs and NC animals. A similar trend was observed for the milk composition of dry matter and fat. Concordantly, reduced body weight was found for suckling kits in the VTc and FT groups compared to VTs and NC animals. Our findings reveal that developmental changes after the embryo vitrification procedure could be associated with an exhibition of the embryonic developmental plasticity. Moreover, to our best knowledge, this study reports the first evidence demonstrating that the vitrification device used is not a trivial decision, providing valuable information about how the cooling-warming rates during vitrification can be partly responsible of the postnatal phenotypic variations.

The harmful effect of removing the extracellular vitrification medium during embryo cryopreservation using a nylon mesh device in rabbit.

During the last decades, many techniques have been developed to reduce sample volume and improve cooling and warming rates during embryo vitrification. The vast majority are based on the "minimum drop size" concept, in which the vitrification solution around embryos is reduced by aspiration, leaving a tiny part of volume surrounding embryos. However, novel cryodevices were aimed to remove the entire vitrification solution. This study was designed to compare the "minimum drop size" technique using Cryotop® with the nylon mesh as cryodevice on rabbit morula embryos. The outcomes assessed were the in vitro development rates (experiment 1) and the offspring rates at birth (experiment 2). Embryos were vitrified in a two-step procedure; equilibrium (10% EG + 10% Me2SO) for 2 min and vitrification (20% EG + 20% Me2SO) for 1 min. In experiment 1, embryos (n = 323) were warmed and subsequently in vitro cultured for 48 h to assess the embryo developmental capability to reach the hatching-hatched blastocyst stage. In experiment 2, embryos were transferred using the laparoscopic technique (n = 369) to assess the offspring rate at birth. In this context, rates of in vitro embryo development were similar between vitrified groups (0.73 ± 0.042% and 0.66 ± 0.047% for Cryotop® and nylon mesh device, respectively), but lower than in the fresh group (0.97 ± 0.016%, p < 0.05). In experiment 2, there were no significant differences in survival rates (offspring born/total embryos transferred) among the Cryotop® device group and fresh group (0.41 ± 0.049% and 0.49 ± 0.050%, respectively). But significantly lower value was obtained in the nylon mesh device group (0.18 ± 0.030%). These results indicate that nylon mesh is not suitable as cryodevice for rabbit morula vitrification, remaining those using the "minimum drop size" methodology as the best option.

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment. The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species. Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

A single injection of corifollitropin alfa supplemented with human chorionic gonadotropin increases follicular recruitment and transferable embryos in the rabbit.

Superovulation protocols are designed to achieve maximum embryo yields. Nevertheless, ovarian response control and the quality of obtained embryos are still a challenge. On the other hand, to save the superovulated embryos until their subsequent use, it is usual to cryopreserve them, so it is also crucial to assess their cryotolerance. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (FSH-CTP) alone or supplemented with human chorionic gonadotropin (hCG) and to determine the impact of this stimulation on in vitro and in vivo development of fresh or devitrified embryos. Our outcomes showed that ovulation rate and recovered embryos were significantly increased when hCG was used. In vitro development of fresh and devitrified embryos and survival at birth were not significantly affected by superstimulation treatment. Results of this study suggest that a single injection of long-acting FSH-CTP supplemented with hCG can be effectively used in rabbits to elicit an increase in ovulation rate and number of recovered embryos. Furthermore, we demonstrated that hCG supplementation had no negative effects in embryo cryosurvival and development, showing similar survival rate at birth than FSH-CTP alone group.

Correction: Overweight in young males reduce fertility in rabbit model.

[This corrects the article DOI: 10.1371/journal.pone.0180679.].

Correction: Successful re-establishment of a rabbit population from embryos vitrified 15 years ago: The importance of biobanks in livestock conservation.

[This corrects the article DOI: 10.1371/journal.pone.0199234.].

Successful re-establishment of a rabbit population from embryos vitrified 15 years ago: The importance of biobanks in livestock conservation.

Genetic resource banks (GRB) are a valuable tool for maintaining genetic variability and preserving breeds from pathogens or catastrophe, enabling us to assess and correct breeding schemes, minimizing the impact of genetic drift and facilitating diffusion. This study tests their efficiency in re-establishing two extinct populations of a synthetic rabbit line selected for daily weight gain, using vitrified embryos from two generations (18th and 36th) separated by 15 years of genetic selection. The effect of long-term storage of vitrified embryos in liquid nitrogen was also evaluated. A total of 516 vitrified embryos using the same protocol were transferred into 54 recipients. The embryos had been maintained in liquid nitrogen during 2 different periods, (i) 1 year (301 embryos and 26 transfers, 36th generation) and (ii) 15 years (259 embryos and 28 transfers, 18th generation). A total of 80.0% (8/10 to 18th) and 60.0% (9/15 to 36th) of the foundational sire families were eventually re-established. Over approximately one year, animals within each population were crossed to produce the next generation and re-establish the original population size. Our study demonstrated that our GRB of embryos vitrified 15 years ago is a successful strategy to re-establish rabbit populations to continue the breeding programme.

Cryosurvival of rabbit embryos obtained after superovulation with corifollitropin alfa with or without LH.

The efficiency of an embryo bank depends on provision of optimal conditions for recovery, cryopreservation and transfer to a breed or strain. In this sense, increasing the number of embryos available using superovulation should improve the cryobank efficiency. However, vagueness of response to conventional protocols to control or increase ovarian response and the quality of oocytes and embryos and their cryotolerance remain a challenge. The aim of our study was to evaluate the effect of corifollitropin alpha (CTP) and a recombinant human FSH (rhFSH), alone or supplemented with rhLH, on embryo cryosurvival by in vitro development and OCT4 and NANOG mRNA abundance at blastocyst stage and offspring rate. In vitro development of vitrified embryos was not significantly affected by superstimulation with or without rhLH supplementation, resulting in similar development rates to those of the control groups (fresh and vitrified embryos from non-superstimulated donor does). Blastocysts developed from vitrified embryos showed higher levels of OCT4 transcript abundance than fresh control, while NANOG transcript abundance was only higher in the blastocysts developed from vitrified embryos after superstimulation treatment in comparison with control groups. The implantation and offspring rates at birth were negatively affected by supplementation with rhLH. Both rhFSH or CTP vitrified embryo groups showed an implantation rate similar to those of the control groups, but an offspring rate lower than control. In conclusion, embryos produced using corifollitropin alpha did not compromise the cryosurvival of vitrified embryos in the rabbit. In addition, this study points out the negative effect of rhLH supplementation in terms of offspring rate on embryo vitrification.

Extra-uterine (abdominal) full term foetus in a 15-day pregnant rabbit.

BACKGROUND: While ectopic pregnancies account for 1-2% of all pregnancies, abdominal pregnancy is extremely rare, accounting for approximately 1% of ectopic pregnancies. Extrauterine abdominal pregnancy is defined as the implantation and development of an embryo in the peritoneal cavity. The present report is the first of an incidental case of abdominal pregnancy within four full-term foetus simultaneously with 2 weeks of physiological gestation in a healthy doe rabbit. CASE PRESENTATION: The doe was born on November 3, 2014 and the first partum took place on May 18, 2015. The doe had previously delivered and weaned an average of 12.0 ± 1.41 live kits at birth (no stillbirths were recorded) during 5 consecutive pregnancies. The last mating was on December 18, 2015 and the detection of pregnancy failure post breeding (by abdominal palpation) on December 31, 2015. Then, the doe was artificially inseminated on January 27, 2016, diagnosed pregnant on February 11, 2016 and subsequently euthanized to recover the foetus. A ventral midline incision revealed a reproductive tract with 12 implantation sites with 15 days old foetus and 4 term foetus in abdominal cavity. There were two foetus floating on either side of the abdominal cavity and two suspended near the greater curvature of the stomach. They were attached to internal organs by means of one or 2 thread-like blood vessels that linked them to the abdominal surfaces. CONCLUSIONS: In our opinion a systematic monitoring of rabbit breeding should be included to fully understand and enhance current knowledge of this phenomenon of abdominal pregnancy.

Overweight in young males reduce fertility in rabbit model.

Semen quality has certainly declined over the past few decades, possibly owing to modern lifestyle factors. In this sense, the role of overweight and obesity in the development of subfertility in males has generated a considerable amount of interest in recent years. However, there is no consensus on whether overweight or obesity impaired sperm quality. Thus, based on the ongoing debate about risk factors for subfertility associated with overweight and obesity in men, this study was designed to investigate the effect of overweight on sperm quality parameters and fertility success in randomized controlled trial in a rabbit model. Fourteen male rabbits were randomly assigned to a control group in which nutritional requirements were satisfied or a group fed to satiety from 12 to 32 weeks of age. At 24 weeks of age, semen samples were analysed weekly by conventional semen analysis for 8 weeks. In addition, during the trial female rabbits were artificially inseminated by each male to assess the fertility success and the number of offspring. Young males fed to satiety were associated with a significant increase in body weight (13.6% overweight) and perirenal fat thickness (5%). Male overweight presented a significant decrease in sperm concentration. There were no differences in the remaining sperm parameters. However, male overweight showed a clear and significant decrease in fertility success (control group, 64±8.9% versus fed to satiety group, 35±9.2%), but not in the number of offspring. Taken together, our findings provide new evidence on the loss of fertility induced by overweight in males.

Effect of corifollitropin alfa supplemented with or without LH on ovarian stimulation and embryo viability in rabbit.

There is increasing interest in using rabbits for research as a laboratory model as well as for industrial production of meat, wool and fur. Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to either support genetic improvement programs, ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, posology and the effect of gonadotropins used simultaneously, the donor and the environment. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (CTP), alone or supplemented with LH, versus a FSH standard protocol of five equal doses administered twice daily to superovulate rabbit does (20 per group and 29 control females). We determined: 1) the impact of this stimulation on in vitro development and mRNA expression at blastocyst stage and 2) in vivo embryo development and viability rate at birth of transferred embryos. Our outcomes showed that the ovulation rate was similar among the different ovarian stimulation groups, reaching more than fourfold the ovulation rate of a control doe. While rates of embryos developing to the blastocyst stage after 48 h of in vitro culture were similar between groups, the hatched blastocyst rate was higher for superovulated embryos from CTP group. Moreover, no significant differences among mRNA expression of OCT4, SOX2 and NANOG genes were detected. Nevertheless, embryos from ovarian stimulated does with CTP + LH showed significantly higher implantation rates and survival at birth among the different ovarian stimulation groups and similar to those in the control group. In conclusion, the results of this study suggest that a single injection of long acting corifollitropin alfa can be effectively used in rabbits to elicit a more than fourfold increase in ovulation rate compared to control animals. In addition, the LH supplementation allows us to obtain similar in vivo embryo development results as in the control group.

Embryo transfer manipulation cause gene expression variation in blastocysts that disrupt implantation and offspring rates at birth in rabbit.

OBJECTIVE: In the current study we aimed to evaluate the effect of embryo transfer on gene expression during pre-implantation development and its consequences on implantation rate, offspring rate at birth and embryonic and fetal losses in the rabbit model. STUDY DESIGN: The mRNA expressions of 8 candidate genes were compared between 6-day-old in vivo-produced embryos (non-manipulated embryos) to those of 6-day-old embryos previously recovery at the third day of development and transferred into recipient rabbit females (manipulated embryos). Furthermore, we compared between both experimental groups the implantation rate and offspring rate at birth and embryonic and fetal losses. RESULTS: Differences in transcript abundance of OCT4, C1qTNF1, EMP1 and TNFAIP6 were observed in transferred embryos. In addition, lower implantation and offspring rates at birth were obtained in transferred embryos than in the control group. In addition, embryonic losses were significantly higher in the transferred group than in the control. However, fetal losses were similar between groups. CONCLUSION: The findings of the current study show that embryo transfer manipulation influenced mRNA expression of late blastocysts prior to implantation, resulting in higher gestational losses as a consequence of faulty embryonic implantation.

Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method.

This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62 ± 4.7% and 62 ± 4.9%, respectively); however, the rates were significantly lower (P<0.05) than those of the fresh group (95 ± 3.4%). In experiment 2, vitrified embryos were transferred using laparoscopic technique. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total offspring were recorded. Embryonic and fetal losses were calculated as the difference between implanted embryos and embryos transferred and total born at birth and implanted embryos, respectively. The rate of implantation and development to term was similar between both vitrification devices (56 ± 7.2% and 50 ± 6.8% for implantation rate and 40 ± 7.1% and 35 ± 6.5% for offspring rate at birth); but significantly lower than in the fresh group (78 ± 6.6% for implantation rate and 70 ± 7.2% for offspring rate at birth, P<0.05). Likewise, embryonic losses were similar between both vitrification devices (44 ± 7.2% and 50 ± 6.8%), but significantly higher than in the fresh group (23 ± 6.6%, P < 0.05). However, fetal losses were similar between groups (10 ± 4.4%, 15 ± 4.8% and 8 ± 4.2%, for vitrified, Cryotop or CPIL and fresh, respectively). These results indicate that the CPIL device is as effective as the Cryotop device for vitrification of rabbit embryos, but at a cost of €0.05 per device.

Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy.

Very limited information on the post-implantatory effects of vitrification has been published till now. We observed in a previous study that the vitrification procedure for the cryopreservation of embryos introduced transcriptomic and proteomic modifications in the rabbit foetal placenta at the middle of gestation. Now, we have conducted a proteomic study to determine whether protein alterations in the foetal placenta induced by the vitrification procedure remain during pregnancy. In this study, we used 2D-DIGE and mass spectrometry (MALDI-TOF-TOF and LC-MS/MS analysis) to identify the protein changes during middle and late stages of gestation (Day 14 and Day 24, respectively) in rabbit foetal placenta. We identified 11 differentially expressed proteins at Day 14 and 13 proteins at Day 24. Data are available via ProteomeXchange with identifiers PXD001840 and PXD001836. In addition, we demonstrate the presence of three proteins, serum albumin, isocitrate dehydrogenase 1 [NADP+], and phosphoglycerate mutase 1, which were altered during pregnancy. We demonstrate the existence of changes in foetal placental protein during pregnancy induced by the vitrification procedure, which brings into question whether vitrification effects observed during foetal development could lead to physiological and metabolic disorders in adulthood. This effect, taken together with other effects reported in the literature, suggests that embryo cryopreservation is not neutral.