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Although many devices have been developed to reduce sample volume, with an explosion of methods appearing in the literature over the last decade, commercially available devices with simultaneous vitrification of a larger number of embryos are scarce, with the apparent gap for their use in prolific livestock species. In this study, we investigated the effectiveness of a new three-dimensional (3D)-printed device that combines minimum volume cooling vitrification with simultaneous vitrification of a larger number of rabbit embryos. Late morulae/early blastocysts were vitrified with the open Cryoeyelet ® device ( n = 175; 25 embryos per device), the open Cryotop ® device ( n = 175; 10 embryos per device), and the traditional closed French mini-straw device ( n = 125; 25 embryos per straw) and compared in terms of in vitro development and reproductive performance after transfer to adoptive mothers. Fresh embryos constituted the control group ( n = 125). In experiment 1, there was no difference in the development rate to the blastocyst hatching stage between the CryoEyelet ® and the other devices. In experiment 2, the CryoEyelet ® device showed a higher implantation rate compared with the Cryotop ® (6.3% unit of SD, p = 0.87) and French mini-straw ® (16.8% unit of SD, p = 1.00) devices. In terms of offspring rate, the CryoEyelet ® device was similar to the Cryotop ® device but superior to the French straw device. Regarding embryonic and fetal losses, the CryoEyelet ® showed lower embryonic losses compared to other vitrification devices. The analysis of bodyweight showed that all devices showed a similar outcomes-a higher birthweight but a lower body weight at puberty than those in the fresh transfer embryos group. In summary, the CryoEyelet ® device can be used for the vitrification of many late morulae or early blastocyst stage rabbit embryos per device. Further studies should be performed to evaluate the CryoEyelet ® device in other polytocous species for the simultaneous vitrification of a large number of embryos.

In order to harness the potential of metanephroi allotransplantation to the generation of a functional kidney graft on demand, we must achieve further growth post-transplantation. Sildenafil citrate (SC) is widely known as a useful inductor of angiogenesis, offering renoprotective properties due to its anti-inflammatory, antifibrotic, and antiapoptotic effects. Here, we performed a laparoscopic metanephroi allotransplantation after embedding sildenafil citrate into the retroperitoneal fat of non-immunosuppressed adult rabbit hosts. Histology and histomorphometry were used to examine the morphofunctional changes in new kidneys 21 days post-transplantation. Immunofluorescence of E-cadherin and renin and erythropoietin gene expression were used to assess the tubule integrity and endocrine functionality. After the metanephroi were embedded in a 10 µM SC solution, the new kidneys' weights become increased significantly. The E-cadherin expression together with the renin and erythropoietin gene expression revealed its functionality, while histological mature glomeruli and hydronephrosis proved the new kidneys' excretory function. Thus, we have described a procedure through the use of SC that improves the outcomes after a metanephroi transplantation. This study gives hope to a pathway that could offer a handsome opportunity to overcome the kidney shortage.

The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 °C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism.

Increasing evidence indicates that assisted reproductive technologies (ARTs) disturb skewed sex-ratio and induce sex-dimorphic postnatal effects. Undoubtedly, the combination of multiple ovulation and embryo transfer (MOET) together with the use of vitrification technique (MOVET) is currently being used in breeding programs. However, since the first case of sex skewing reported in 1991, the accumulative and long-term transmission of skewed sex-ratio to future generations has not been thoroughly evaluated. Here we test as MOVET program induce a skewed sex ratio, and we consider skewed sex ratio transmission to future generations. To this end, we first evaluated the F1 generation, demonstrating that a MOVET program causes a severe imbalance skewed secondary sex ratio (SSR) towards male by 12%. This imbalanced persist after a second MOVET program (F2 generation), with an accumulative skewed SSR towards male by 25%. Finally, using a crossbred generation derived from crossing F1 males derived from a MOVET program with naturally-conceived (NC) females, we show that the imbalance skewed SRR persist. Bodyweight comparison between MOVET animals and NC counterparts revealed significant changes at birth, weaning and adulthood. However, there was a significant interaction between F2 MOVET animals and sex, demonstrating an apparent accumulative sex-dimorphic effect. At adulthood, MOVET derived males presented a lower body weight. In conclusion, we show that the MOVET program causes a direct, accumulative and long-term transmission of skewed SSR., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Preimplantation embryo manipulations during standard assisted reproductive technologies (ART) have significant repercussions on offspring. However, few studies to date have investigated the potential long-term outcomes associated with the vitrification procedure. Here, we performed an experiment to unravel the particular effects related to stress induced by embryo transfer and vitrification techniques on offspring phenotype from the foetal period through to prepuberal age, using a rabbit model. In addition, the focus was extended to the liver function at prepuberal age. We showed that, compared to naturally conceived animals (NC), offspring derived after embryo exposure to the transfer procedure (FT) or cryopreservation-transfer procedure (VT) exhibited variation in growth and body weight from foetal life to prepuberal age. Strikingly, we found a nonlinear relationship between FT and VT stressors, most of which were already present in the FT animals. Furthermore, we displayed evidence of variation in liver function at prepuberal age, most of which occurred in both FT and VT animals. The present major novel finding includes a significant alteration of the steroid biosynthesis profile. In summary, here we provide that embryonic manipulation during the vitrification process is linked with embryo phenotypic adaptation detected from foetal life to prepuberal age and suggests that this phenotypic variation may be associated, to a great extent, with the effect of embryo 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.

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.

The advent of assisted reproductive technologies (ART) in mammals involved an extraordinary change in the environment where the beginning of a new organism takes place. Under in vitro conditions, in which ART is currently being performed, it likely fails to mimic optimal in vivo conditions. This suboptimal environment could mediate in the natural developmental trajectory of the embryo, inducing lasting effects until later life stages that may be inherited by subsequent generations (transgenerational effects). Therefore, we evaluated the potential transgenerational effects of embryo exposure to the cryopreservation-transfer procedure in a rabbit model on the offspring phenotype, molecular physiology of the liver (transcriptome and metabolome) and reproductive performance during three generations (F1, F2 and F3). The results showed that, compared to naturally-conceived animals (NC group), progeny generated after embryo exposure to the cryopreservation-transfer procedure (VT group) exhibited lower body growth, which incurred lower adult body weight in the F1 (direct effects), F2 (intergenerational effects) and F3 (transgenerational effects) generations. Furthermore, VT animals showed intergenerational effects on heart weight and transgenerational effects on liver weight. The RNA-seq data of liver tissue revealed 642 differentially expressed transcripts (DETs) in VT animals from the F1 generation. Of those, 133 were inherited from the F2 and 120 from the F3 generation. Accordingly, 151, 190 and 159 differentially accumulated metabolites (DAMs) were detected from the F1, F2 and F3, respectively. Moreover, targeted metabolomics analysis demonstrated that transgenerational effects were mostly presented in the non-polar fraction. Functional analysis of molecular data suggests weakened zinc and fatty acid metabolism across the generations, associated with alterations in a complex molecular network affecting global hepatic metabolism that could be associated with the phenotype of VT animals. However, these VT animals showed proper reproductive performance, which verified a functional health status. In conclusion, our results establish the long-term transgenerational effects following a vitrified embryo transfer procedure. We showed that the VT phenotype could be the result of the manifestation of embryonic developmental plasticity in response to the stressful conditions during ART procedures.

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.

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., Competing Interests: The authors declare no conflict of interest.

Objective: Uterine transplantation is now considered a feasible treatment for women with absolute uterine factor infertility and has been successfully performed for a woman with Asherman's syndrome (AS). The endometrium is a clinically and histologically distinct entity from the surrounding myometrium. Endometrial transplantation (ETx) may offer a less invasive option, with less immunogenic impact, to restore fertility in women with severe AS. The objective of this study was to assess the feasibility of ETx by evaluating surgical and reproductive outcomes following endometrial autotransplantation in a rabbit model., Study Design: A longitudinal study assessing surgical, biochemical, radiological, reproductive and histological outcomes following endometrial autotransplantation in ten New Zealand white rabbits., Results: Ten procedures were performed, including 8 endometrial auto-transplants (ETx) and 2 endometrial resections (ER), to control against endometrial regeneration. Eight procedures were successful, whereas two rabbits from the ETx group died intra-operatively. Three rabbits were euthanised at 48, 72 and 96 h post-operatively to assess gross and histological appearances. Two rabbits, one from the ETx group and one from the ER group, died four weeks and eight weeks post-operatively. Three rabbits subsequently underwent two cycles of in-vitro fertilization. The first cycle resulted in an implantation rate of 57% in the un-operated uteri. In two rabbits who underwent ETx, an implantation rate of 28.6% was seen. In the second cycle, an implantation rate of 61.9 % (13 implantations) was observed in the control uteri. In the two ETx females, an implantation rate of 14.3 % was seen. No pregnancies were seen in either cycle in the animals who underwent ER. Despite successful implantations in both cycles in the ETx rabbits, no livebirths were achieved. Following death or euthanasia there was gross and microscopic evidence of viable endometrium following ETx, but not following ER., Conclusion: This study has revealed, for the first time, the feasibility of ETx with gross and microscopic evidence of viable endometrium, and the demonstration of clinical pregnancies. Whilst further studies are essential, and the achievement of successful livebirths fundamental, ETx may offer a potential fertility restoring opportunity for women with severe, treatment refractory cases of AS., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus , Enterobacter aerogenes , and Aspergillus brasiliensis . Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae-early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures., Competing Interests: Abbreviations: The following abbreviations are used in this manuscript:

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.

Kidney transplantation from deceased or living human donors has been limited by donor availability as opposed to the increasing demand, and by the risk of allograft loss rejection and immunosuppressive therapy toxicity. In recent years, xenotransplantation of developed kidney precursor cells has offered a novel solution for the unlimited supply of human donor organs. Specifically, transplantation of kidney precursors in adult hosts showed that intact embryonic kidneys underwent maturation, exhibiting functional properties, and averted humoural rejection post-transplantation from non-immunosuppressed hosts. Even if supply and demand could be balanced using xenotransplants or lab-grown organs from regenerative medicine, the future of these treatments would still be compromised by the ability to physically distribute the organs to patients in need and to produce these products in a way that allows adequate inventory control and quality assurance. Kidney precursors originating from fifteen-day old rabbit embryos were vitrified using Cryotop® as a device and VM3 as vitrification solution. After 3 months of storage in liquid nitrogen, 18 kidney precursors were transplanted into non-immunosuppressed adult hosts by laparoscopy surgery. Twenty-one days after allotransplantation, 9 new kidneys were recovered. All the new kidneys recovered exhibited significant growth and mature glomeruli. Having achieved these encouraging results, we report, for the first time, that it is possible to create a long-term biobank of kidney precursors as an unlimited source of organs for transplantation, facilitating the inventory control and distribution of organs., (Copyright © 2015 Elsevier Inc. All rights reserved.)