Oocyte quality and in vivo embryo survival after ovarian stimulation in nulliparous and multiparous rabbit does.

Superovulation treatments aim to stimulate multifollicular recruitment, maximizing the number of oocytes or transferable embryos produced. Factors associated with the superovulation protocol, female characteristics and many other factors are determinants in the number and quality of oocytes obtained. An accurate way to assess oocyte quality more precise than morphological appearance is genetic expression. The present study aims to compare the response of nulliparous and multiparous females to superovulatory stimulation, studying its effect on the expression of some genes associated with the activation, growth, development and oocyte-embryo transition of oocytes, as well as its impact on in vivo embryonic development and viability rate at birth. In a first experiment, the effect of stimulation treatment on the ovulation response and the expression of the MSY2, MATER, ITPR1, ITPR2, ITPR3, eIF4E, PAR1, PAPOL-A, PAPOL-G, ZAR1 and YY1 genes in nulliparous and multiparous females were determined. In a second experiment, the implantation and viability at birth of embryos from superovulated nulliparous and multiparous females were analysed. The ovulation rate was significantly higher in the superovulation groups than in the control groups. The ovulation rate was significantly increased in nulliparous females compared with multiparous does. From the eleven genes analysed, only the expression of MATER, PAPOL-A, PAPOL-G and ZAR-1 genes was shown to be different among experimental groups. Finally, in terms of implantation rate and viability at birth, the nulliparous control group showed better results than the rest of the groups. Both hyperstimulation treatment and reproductive female's history seem to alter the transcriptome of important genes related to oocyte maturation and competence acquisition, affecting in vivo embryo viability.

Maternal vaccination as a Salmonella Typhimurium reduction strategy on pig farms.

AIMS: The control of Salmonella in pig production is necessary for public and animal health, and vaccination was evaluated as a strategy to decrease pig prevalence. METHODS AND RESULTS: The study examined the efficacy of a live Salmonella Typhimurium vaccine, administered to sows on eight commercial farrow-to-finish herds experiencing clinical salmonellosis or Salmonella carriage associated with S. Typhimurium or its monophasic variants. Results of longitudinal Salmonella sampling were compared against eight similarly selected and studied control farms. At the last visit (~14 months after the start of vaccination), when all finishing stock had been born to vaccinated sows, both faecal shedding and environmental prevalence of Salmonella substantially declined on the majority of vaccinated farms in comparison to the controls. A higher proportion of vaccine farms resolved clinical salmonellosis than controls. However, Salmonella counts in positive faeces samples were similar between nonvaccinated and vaccinated herds. CONCLUSIONS: The results suggest that maternal vaccination is a suitable option for a Salmonella Typhimurium reduction strategy in farrow-to-finish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella vaccines have the potential to reduce the prevalence of Salmonella in pigs and result in a reduction of human cases attributed to pork.

Feed restriction regime in a rabbit line selected for growth rate alters oocyte maturation manifested by alteration in MSY2 gene expression.

Young rabbit females selected for growth rate may have nutritional needs, which may not be met with the common practice of feed restriction during rearing in commercial rabbit production. The aim of this study was to analyse whether two different feeding programmes: ad libitum or restricted (130 g/day) feeding, applied in young rabbit females for 1 month at the end of rearing, could modulate the origin of ovulation process and the quality of the oocytes. At 16 weeks of age, 34 females were randomly assigned to restricted or ad libitum feeding, maintaining these conditions for a month. Then, in an initial experiment, transcriptional profiling of hypothalamus-hypophysis tissue was performed to assess failure to ovulate. In the second experiment, the gene expression analysis of some candidate genes related to oocytes quality was performed. Our results demonstrated that neither of the two feeding programmes modified the transcription of hypothalamus-hypophysis tissue, while the only differences in MSYR expression were found in in vivo mature oocytes ready for successful fertilization. Specifically, MSYR was over-expressed in oocytes from females fed ad libitum. MSYR is one of the most abundant proteins in the oocyte and has proven to be a key regulator of maternal RNA transcription and translation. This finding suggests that MSYR gene is a promising gene in our understanding of the relationship between high growth rate and reproductive performance decline.

Feeding programs promoting daily feed intake stability in rabbit males reduce sperm abnormalities and improve fertility.

Feeding programs promoting daily feed intake (DFI) stability in rabbit males could be useful to ensure successful coverage of their nutritional requirements and for continued production of quality semen. To evaluate two feeding systems designed to reduce DFI variability, 115 rabbit males at age 1.2 years were randomly assigned to three different treatments for 294 days: CS, animals fed ad libitum with a control diet (127-g starch and 281-g total soluble fiber [hemicellulose + soluble fiber] kg(-1) dry matter); SF, males fed ad libitum with diet enriched in soluble fiber (86-g starch and 330-g total soluble fiber kg(-1) dry matter); and R, animals fed with CS diet but daily restricted to maintenance requirements. Feed intake, body weight, body condition, and variability of DFI were controlled every 42 days, and individual semen volume and sperm motility, concentration, acrosome status, and abnormalities every 15 days. In six commercial farms, the number of females inseminated, pregnant and kindling, as well as the number of kits born alive, was registered for 15,893 inseminations with pooled semen from each treatment. DFI was significantly lower for R males than for the other treatments (on average, -12 ± 4 g/day; P < 0.001). Daily weight gain of R males was close to zero and significantly lower than in the other groups (-1.42 g/day; P < 0.001). Variability of DFI was significantly (P < 0.01) lower for R males (7%) than for males of dietary treatments CS (13%), with SF males showing intermediate values (11%). Semen from R males presented lower sperm abnormalities (-5.9%; P < 0.05) and higher percentages of normal and motile spermatozoa (-3.4% than SF males; P < 0.05). Dietary treatments formulated to reduce DFI variability (SF and R) led to an improvement of kindling to pregnant and kindling to insemination ratio (+0.039 and + 0.060 ± 0.015, respectively; P < 0.05) compared with CS treatment. In conclusion, a moderate restriction of rabbit males may be useful to fit their needs and provide a constant daily supply of nutrients, with some sperm morphologic characteristics being improved, as well as the fertility of their pooled semen.

FIRST STEPS TOWARDS ORGAN BANKS: VITRIFICATION OF RENAL PRIMORDIAL.

BACKGROUND: Kidney transplantation from deceased or living human donors has been limited by donor availability as opposed to the increasing demand, by the risks of allograft loss rejection and immunosuppressive therapy toxicity and by limitations of organ preservation protocols, which is essential to organise staff and facilities, transport organs, and perform necessary laboratory tests. However, the cryopreservation of composite tissues poses technical challenges beyond those seen in the preservation of single tissue types or organs. OBJECTIVE: The purpose of our study was to establish a protocol for long-term storing of renal primordia, that generates new adult kidneys after transplant into a syngeneic non-immunosuppressed host. MATERIALS AND METHODS: Metanephroi from 16-days-old embryos were microdissected and vitrified following the minimum essential volume method and using Cryotop as a device and VM3 as vitrification solution. After 3 months of storage in liquid nitrogen (-196 degree C), 20 metanephroi were warmed and transplanted using minimally invasive laparoscopic surgery into retroperitoneal fat of 5-month-old immune-competent New Zealand rabbits. In the same way, 22 fresh metanephroi were transplanted. Twenty-one days after transplantation, hosts were euthanized and developed kidneys were recovered and evaluated morphologically and histologically. RESULTS: Significant growth and fully differentiated mature glomeruli and tubule were observed in all kidney graft explants recovered. In total, 5 metanephroi (25.0%) were successfully grown after vitrification. In the same way, 12 metanephroi (54.5%) were successfully grown in the fresh group. CONCLUSION: These encouraging results reported that metanephroi not only survive vitrification, but they vascularized and developed morphologically normal glomeruli after their allotransplantation. These results suggest that it's possible to create a long-term biobank of kidney precursors as an unlimited source of organs for transplantation, and open new therapeutic possibilities for the patients with chronic renal failure.

Campylobacter epidemiology from breeders to their progeny in Eastern Spain.

While horizontal transmission is a route clearly linked to the spread of Campylobacter at the farm level, few studies support the transmission of Campylobacter spp. from breeder flocks to their offspring. Thus, the present study was carried out to investigate the possibility of vertical transmission. Breeders were monitored from the time of housing day-old chicks, then throughout the laying period (0 to 60 wk) and throughout their progeny (broiler fattening, 1 to 42 d) until slaughter. All samples were analyzed according with official method ISO 10272:2006. Results revealed that on breeder farms, Campylobacter isolation started from wk 16 and reached its peak at wk 26, with 57.0% and 93.2% of positive birds, respectively. After this point, the rate of positive birds decreased slightly to 86.0% at 60 wk. However, in broiler production all day-old chicks were found negative for Campylobacter spp, and the bacteria was first isolated at d 14 of age (5.0%), with a significant increase in detection during the fattening period with 62% of Campylobacter positive animals at the end of the production cycle. Moreover, non-positive sample was determined from environmental sources. These results could be explained because Campylobacter may be in a low concentration or in a non-culturable form, as there were several studies that successfully detected Campylobacter DNA, but failed to culture. This form can survive in the environment and infect successive flocks; consequently, further studies are needed to develop more modern, practical, cost-effective and suitable techniques for routine diagnosis.

Effects of Female Dietary Restriction in a Rabbit Growth Line During Rearing on Reproductive Performance and Embryo Quality.

Maternal diet prior to mating has an effect on reproductive performance. We analysed the effect of maternal dietary restriction during rearing on reproductive performance, the embryo development and foetal growth. Females were categorized in two groups: (i) does with ad libitum access to feed or (ii) restricted. Two experiments were performed: (i) after 1 month, receptive females from both experimental groups were artificially inseminated and the reproductive performance was recorded during three reproductive cycles; at the first insemination, the body weight and perirenal fat thickness were recorded, and (ii) females from both experimental groups were inseminated, and 24 h later, embryos were recovered and transferred to recipient females from a maternal line. Later, embryonic implantation was assessed at day 14 by laparoscopy and foetal growth was monitored by ultrasound examination. In experiment 1, no differences in kindling rate was found, but prolificacy was showed to be higher in ad libitum does, which also were heavier than restricted ones. In experiment 2, no differences among does either in body weight, in perirenal fat thickness or in reproductive performance (ovulation rate and embryo recovery rate) were related to differences in feed intake. However, despite similar embryonic implantation losses, embryos from restricted females demonstrated higher foetal and gestational losses. Embryos from restricted does presented lower foetal growth than embryos from ad libitum does. Therefore, our results demonstrated that nutrition before first conception in a rabbit line selected for growth rate may impact on the embryo and results in a disturbance in gestational losses and foetal growth over all reproductive life.

Trasplante laparoscópico de metanefros: un paso inicial al xenotrasplante renal / Laparoscopic transplantation of metanephroi: A first step to kidney xenotransplantation

Antecedentes: Una solución novedosa a la escasez de riñones para trasplante puede ser el xenotrasplante de riñones embrionarios. Objetivo: Conocer la viabilidad del trasplante alogénico laparoscópico de metanefros (M) en conejos. Material y método: Se realizó disección microscópica para obtener metanefros en embriones de 14 días de edad (24 M), 15 (20 M) y 16 (26 M). Mediante laparoscopia abdominal de un puerto se insertó percutáneamente una aguja raquídea y por ella, mediante un catéter epidural, depositamos el metanefros cerca de un vaso sanguíneo patente en la grasa retroperitoneal. Setenta metanefros se trasplantaron a 18 conejos. Tres semanas después los animales fueron explorados por cirugía abierta. Se analizó la comparación de la madurez embrionaria, las variables morfométricas de los metanefros y la tasa de desarrollo de los metanefros trasplantados. Resultados: El límite temporal inferior para la extracción de metanefros en conejos es el día 14. Tres semanas postrasplante crecieron a una mínima expresión solo 3/24 M de 14 días (12,5%). Por el contrario, 10/20 (50%) de los de 15 días y 12/26 (46,1%) de los de 16 días de edad crecieron y se diferenciaron de tal manera que se habían desarrollado normalmente los glomérulos, túbulos proximales y distales y conductos colectores. No se detectaron cambios inmunológicos relevantes en sangre periférica. Conclusiones: Describimos, por primera vez en la literatura, el trasplante laparoscópico alogénico de metanefros de embriones como una técnica factible y no invasiva. Los receptores no necesitaron inmunosupresión

Background: Embryonic kidney xenotransplantation could represent a new solution to the scarcity of kidneys for transplantation. Objective: To determine the feasibility of allogeneic laparoscopic transplantation of metanephroi (M) in rabbits. Material and method: Microscopic dissection was conducted to obtain metanephroi from 14-day-old (24 M), 15-day-old (20 M) and 16-day-old (26 M) embryos. Using single-port abdominal laparoscopy, a spinal needle was inserted percutaneously, through which the metanephroi were deposited (using an epidural catheter) close to a patent blood vessel in the retroperitoneal fat. Seventy metanephroi were transplanted to 18 rabbits. Three weeks later, the animals were examined through open surgery. We compared the embryonic maturity, the morphometric variables of the metanephroi and the development rate of the transplanted metanephroi. Results: The lower time limit for the extraction of metanephroi from the rabbits was day 14. Three weeks after transplantation, only 3/24 14-day-old metanephroi grew at minimal expression (12.5%). In contrast, 10/20 (50%) 15-day-old and 12/26 (46.1%) 16-day-old metanephroi grew. These metanephroi had differentiated sufficiently for the glomeruli, proximal and distal tubules and collecting ducts to develop normally. We detected no relevant immunological changes in the peripheral blood. Conclusions: We have described for the first time in the literature the allogeneic laparoscopic transplantation of metanephroi from embryos as a feasible and noninvasive technique. The recipients did not require immunosuppression

Laparoscopic transplantation of metanephroi: A first step to kidney xenotransplantation.

BACKGROUND: Embryonic kidney xenotransplantation could represent a new solution to the scarcity of kidneys for transplantation. OBJECTIVE: To determine the feasibility of allogeneic laparoscopic transplantation of metanephroi (M) in rabbits. MATERIAL AND METHOD: Microscopic dissection was conducted to obtain metanephroi from 14-day-old (24M), 15-day-old (20M) and 16-day-old (26M) embryos. Using single-port abdominal laparoscopy, a spinal needle was inserted percutaneously, through which the metanephroi were deposited (using an epidural catheter) close to a patent blood vessel in the retroperitoneal fat. Seventy metanephroi were transplanted to 18 rabbits. Three weeks later, the animals were examined through open surgery. We compared the embryonic maturity, the morphometric variables of the metanephroi and the development rate of the transplanted metanephroi. RESULTS: The lower time limit for the extraction of metanephroi from the rabbits was day 14. Three weeks after transplantation, only 3/24 14-day-old metanephroi grew at minimal expression (12.5%). In contrast, 10/20 (50%) 15-day-old and 12/26 (46.1%) 16-day-old metanephroi grew. These metanephroi had differentiated sufficiently for the glomeruli, proximal and distal tubules and collecting ducts to develop normally. We detected no relevant immunological changes in the peripheral blood. CONCLUSIONS: We have described for the first time in the literature the allogeneic laparoscopic transplantation of metanephroi from embryos as a feasible and noninvasive technique. The recipients did not require immunosuppression.

Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential.

Assisted reproduction technologies require ovarian stimulation to increase the number of oocytes and embryos. Currently, superstimulation is achieved by gonadotropin treatment, but the embryo yield and quality are highly variable. Commonly, commercial preparations derived from pituitary and urinary origin are used to superovulate. Hence, ovarian superstimulation protocols have usually included both FSH and LH. The appearance of recombinant gonadotropins manufactured by genetic engineering techniques has ensured high quality and batch-to-batch consistency. Moreover, this enables us to assess the importance of LH in the ovarian stimulation. The main aim of this study was to evaluate the effect of recombinant human LH supplementation (10%) on embryonic development produced by rabbit does superovulated with low or high concentration (18.75 or 37.50 IU) of recombinant human FSH (rhFSH). Females treated with rhFSH increased the ovulation rate, and it was significantly higher when the high FSH dose was supplemented with LH. The superstimulation treatment used did not significantly affect in vitro development rate until the expanded blastocyst stage. The results of this study seem to suggest that, in terms of superovulatory response, when rabbit does are treated with 37.5-IU rhFSH, the use of LH supplementation allows an increase in the number of follicles recruited and the quality of embryos, in terms of ability to develop in vitro until blastocyst, and the expression profile of OCT4, NANOG, and SOX2 genes is not affected.

Embryo vitrification in rabbits: Consequences for progeny growth.

The objective of this research is to examine if there are any effects of the rederivation procedures on rabbit growth pattern and on weight of different organ in adults. For this purpose, three experiments were conducted on two different groups of animals (control group and vitrified-transferred group) to evaluate the possible effect of embryo manipulation (vitrification and transfer procedures) on future growth traits. The first experiment studies body weight from 1 to 9 weeks of age from the two groups. The second experiment describes the growth curve of progeny from experimental groups and analyzes their Gompertz curve parameters, including the estimation of adult body weight. The third experiment has been developed to study if there are any differences in different organ weight in adult males from the two experimental groups. In general, the results indicate that rederivation procedures had effect on the phenotypic expression of growth traits. The results showed that rabbit produced by vitrification and embryo transfer had higher body weight in the first four weeks of age than control progeny. Results from body weight (a parameter) and b parameter estimated by fitting the Gompertz growth curve did not show any difference between experimental groups. However, differences related with growth velocity (k parameter of the Gompertz curve) were observed among them, showing that the control group had higher growth velocity than the vitrified-transferred group. In addition, we found that liver weight at 40th week of age exhibits significant differences between the experimental groups. The liver weight was higher in the control males than in the VF males. Although the present results indicate that vitrification and transfer procedures might affect some traits related with growth in rabbits, further research is needed to assess the mechanisms involved in the appearance of these phenotypes and if these phenotypes could be transferred to the future progeny.

First steps of in vitro gastrulation in rabbit vitrified embryos.

BACKGROUND: The in vitro rabbit embryo production and their cryopreservation methodologies such as vitrification generate less viable embryos, and occasionally, with significant differences from those that are not subjected to any treatment. Besides, in vitrified rabbit embryos little information is available about exactly when and where begin to emerge the first differences that finally result in foetal losses comparing with non-vitrified embryos. OBJECTIVE: The aim of this study was to evaluate the vitrification effects on the early in vitro gastrulation events. MATERIALS AND METHODS: After oviductal transfers of vitrified and non-vitrified embryos (control) in rabbit recipients, blastocysts from 144h (6-day-old) were recovered and cultured into TCM199 supplemented with rabbit homologous serum media for 48 hours. Gastrula stage and measures of perimeter and area of blastocyst and gastrula were noted. Moreover, eight independent pools consisting of six embryos each one were generated for each experimental group (control and vitrified) and total RNA was isolated to study the OCT4 gene expression. RESULTS: Of 151 control and 164 vitrified morulae transferred, 69.5 % and 70.1 % developed in vivo to 6-day-old blastocyst respectively. After 24 hour of in vitro culture, 41.8 % of vitrified blastocyst had begun the neurulation (stage 5-) versus 22.8 % of control group. Nevertheless, the vitrified group showed the highest percentage of collapsed blastocyst at 48 hours (26.8 %). Non morphometric differences differences were observed in perimeter and area of blastocyst and gastrula between control and vitrified group at 0 and 24 hours. By contrast, perimeter and gastrula areas were slightly higher for the vitrified group than those for the control group at 48 hours of in vitro culture. CONCLUSION: The study reveal the existence of the first morphological differences in vitrified blastocysts of 7 and 8-day-old, marked by a further development of gastrulation in the vitrified group.

Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification.

Parthenote embryos offer multiple opportunities in biotechnological research, so it is important to analyse the possibilities for their cryopreservation in order to establish a biobank. The aim of this experiment was to determine the effect of culture conditions and vitrification on rabbit parthenogenetic embryos. Parthenotes were cultured under in vivo and in vitro conditions until day 3 (late morula/early blastocyst), when they were vitrified. Immediately after warming, they were newly cultured under in vivo and in vitro conditions till day 6 (blastocyst stage). Both culture conditions showed similar late morula/early blastocyst (0.39±0.056 vs. 0.46±0.043, for in vivo and in vitro, respectively) and blastocyst rates (0.12±0.068 vs. 0.13±0.070, for in vivo and in vitro, respectively). However, no parthenote was recovered when a combination of culture conditions was performed. To our best knowledge, this is the first demonstration of the ability of rabbit parthenogenetic embryos to develop after vitrification, with similar embryo development after in vivo or in vitro culture. Nevertheless, our results highlight the importance of culture conditions on the morphology of parthenote embryos. Therefore, we have described that special attention should be paid on culture conditions to generate parthenote embryos, with a view to their subsequent use, for example in embryonic stem cell production.

Comparison of different sampling types across the rearing period in broiler flocks for isolation of Campylobacter spp.

Campylobacter is the most common bacterial cause of human gastrointestinal disease in most developed countries. It is generally accepted that poultry products are a significant source of foodborne Campylobacter infections in humans. Assessing the effectiveness of any potential intervention at farm level requires monitoring of the Campylobacter status of broiler flocks, using appropriate sampling methods. The aim of this study was to assess the influence of the sample type across the rearing period for the detection of Campylobacter spp. at farm level. During this study, 21 commercial broiler farms were intensively sampled. Each farm was visited and sampled at different times during the rearing period (d 1, 7, 14, 21, 28, 35, and 42). On the first day of rearing, the status of the house and the day-old flock was evaluated, and environmental and cecal samples were collected. During rearing, 4 different sample types were collected: feces with sock swabs (sock swabs), feces directly from the litter (feces), cloacal swabs, and cecal content. All samples were analyzed according to ISO 10272-1:2006 (Annex E) and also by direct culture. The results of this study showed that Campylobacter spp. were detected in all of the sample types on d 14 of rearing. From this point on, the detection increased significantly, with a maximum detection rate by the end of rearing, regardless of the sample type. All samples that were negative upon direct culture were also negative after pre-enrichment. At the end of rearing, the percentage of samples positive for Campylobacter spp. was 71.4% for cecal samples, 61.9% for cloacal swabs, 45.2% for sock swabs, and 69.1% for fecal samples. C. jejuni was detected in all the sample types, with positive rates ranging from 67.1 to 76.0% for cecal samples and cloacal content, respectively. Cecal samples, cloacal swabs, and fecal samples cultured by direct plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA) without pre-enrichment have the same sensitivity for detection of Campylobacter spp. in broiler flocks independent of the day of rearing.

Role of embryonic and maternal genotype on prenatal survival and foetal growth in rabbit.

The aim of this work was to evaluate the influence of maternal and embryonic genotype on prenatal survival and foetal growth during pregnancy. Embryos were recovered at 48 h of gestation from two different donor lines (R = 46 and A = 40) and transferred to nulliparous recipient does (26 R and 24 A). Each recipient doe received six embryos into one oviduct from line R, and six embryos form line A into the other. Laparoscopy was performed at Day 14 to determine implantation rate. Recipient females were slaughter at Days 14, 24 and 30 (12, 24, and 14, respectively) to determine the number of live foetuses and the weight of live foetuses, foetal placenta and maternal placenta. A transcriptome analysis was performed to search for differences between foetal placentas at Days 14 and 24 of development. Prenatal survival at Days 14, and 24 was affected by embryonic genotype and determined by maternal genotype at Day 30. Foetal weight at Day 14 was influenced by both genotypes, being the weight higher for group A/A (0.29 ± 0.01 g vs 0.19 ± 0.01 g, for group R/R). However, both genotypes were determinant for foetal placenta weight at Day 24, while those genotypes affected maternal placenta weight at Day 30. Nevertheless, no differences in foetal placenta at transcriptome level and progesterone and IGF-I plasma levels in recipient does were found. In conclusion, results indicate that the influence of embryo and maternal genotype on the prenatal survival and growth seems to be changing over gestation.

Generation of live offspring from vitrified embryos with synthetic polymers SuperCool X-1000 and SuperCool Z-1000.

BACKGROUND: Ice growth and recrystallisation are considered important factors in determining vitrification outcomes. Synthetic polymers inhibit ice formation during cooling or warming of the vitrification process. OBJECTIVE: The aim of this study was to assess the effect of adding commercially available synthetic polymers SuperCool X-1000 and SuperCool Z-1000 to vitrification media on in vivo development competence of rabbit embryos. METHODS: Four hundred and thirty morphologically normal embryos recovered at 72 h of gestation were used. The vitrification media contained 20% dimethyl sulphoxide and 20% ethylene glycol, either alone or in combination with 1% of SuperCool X-1000 and 1% SuperCool. RESULT: Our results show that embryos can be successfully vitrified using SuperCool X-1000 and SuperCool Z-1000 and when embryos are transferred, live offspring can be successfully produced. CONCLUSIONS: In conclusion, our results demonstrated that we succeeded for the first time in obtaining live offspring after vitrification of embryos using SuperCool X-1000 and SuperCool Z-1000 polymers.

Use of cyclodextrins to increase cytoplasmic cholesterol in rabbit embryos and their impact on live kits derived from vitrified embryos.

BACKGROUND: Low cryotolerance in oocytes and embryos is frequently associated with lipid accumulation in the cytoplasm. OBJECTIVE: This study aimed to evaluate the effect of cyclodextrin used as a cholesterol loader to change cytoplasmic cholesterol content of embryos and raise their tolerance to cryopreservation. METHODS: In the first experiment compact morulae-early blastocysts were exposed to CLC (0.11 mM and 0.23 mM cholesterol) for 1 hour. In the second experiment, embryos were exposed to CLC (0.11 mM and 0.23 mM cholesterol) and then vitrified. RESULT: Using both concentrations, cytoplasmic cholesterol content was increased. Vitrified groups demonstrated a lower capacity for embryonic development (in vitro and in vivo) compared to the control groups. Nevertheless, live young were obtained in all groups. CONCLUSIONS: In conclusion, we have demonstrated the feasibility of using cyclodextrin as a carrier for cholesterol into rabbit embryo cytoplasm, although further studies are required to clarify the usefulness of CLC use in embryo cryopreservation.

A novel technique for oviduct occlusion to generate live births from cryopreserved rabbit oocytes after in vivo fertilisation.

Intraoviductal transfer technique in combination with in vivo fertilisation has arisen as an effective technique to assess live births after transfer of slow-frozen oocytes in the rabbit. Nevertheless, the great disadvantage of this method is the accumulation of tubal fluid in a large number of females after clamping the oviducts. In this study, we develop an alternative method to minimise damage to the oviduct and increase the birth rate. The aims of this study were (1) to evaluate the ability of cyanoacrylate tissue adhesive to occlude the oviduct for female sterilisation; (2) to evaluate the effect of oviduct occlusion immediately after transferring fresh oocytes on in vivo fertilisation; and (3) to assess this technique to generate live births from fresh and slow-frozen oocytes. In all the experiments, recipients were artificially inseminated 9h prior to occluding the oviducts. In the first experiment, the left oviduct was blocked with cyanoacrylate tissue adhesive, while the right one was used as a control. Six days later, oviducts and uterine horns were flushed to assess embryo recovery rates. While the embryo recovery rate was 79.2% in the intact oviduct, no embryos were recovered in the blocked one. In the second experiment, fresh oocytes were transferred into both oviducts, which were immediately occluded. Six days later, the in vivo fertilisation success rate was 33.7%. Finally, in the last experiment, slow-frozen oocytes were transferred and the rate of live births was 13.2±4.5%. The study shows that when using this method the generation of live births from slow-frozen oocytes increases significantly. In addition, our results suggest that in vivo environment could help improve the results of oocyte cryopreservation.

First pregnancy and live birth from vitrified rabbit oocytes after intraoviductal transfer and in vivo fertilization.

Intraoviductal oocyte transfer in combination with in vivo fertilization has arisen as an alternative method to induce pregnancies from cryopreserved oocytes in rabbits. In this study, offspring were obtained for the first time from vitrified rabbit oocytes using this technique. In all the experiments, recipients were artificially inseminated 9 hours before oocyte transfer. Cryopreserved (vitrified and slow-frozen) and noncryopreserved (fresh) oocytes were transferred into both oviducts, which were immediately closed using cyanoacrylate tissue adhesive to block the entry of the recipient's own oocytes. Three transferred group females that received vitrified oocytes became pregnant and delivered a total of nine live young naturally. The results revealed that there were no differences in the live birth rate between vitrified and slow-frozen oocytes (5.5% and 4.4%, respectively). When fresh oocytes were transferred, this rate increased to 19.2%, whereas in the control females (nontransferred) the rate of offspring obtained was 71.4%. This is the first reported result of the development to term of vitrified rabbit oocytes and suggests that an in vivo environment could help improve the results of oocyte cryopreservation.

Effect of exposure to heatwave during blastocyst formation on reproductive performance of female rabbits.

We examined the effect of female exposure to heatwave during blastocyst formation on their reproductive performance and its effect on transcriptome in blastocyst and endometrial tissue. In this study, a total of 72 rabbit does were artificially inseminated and divided into two environmental groups 2 days later: does under conventional conditions (maintained between 14-22°C, n = 29) and does heat stressed in a climatic chamber (maintained between 32-37°C, n = 43). The heat-stressed group were kept under these conditions for 3 days and returned to conventional conditions thereafter. Five days post-insemination, 48 does were slaughtered to collect blastocyst and endometrium samples. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-É£, HSP70 and HSP90 were analysed by qRT-PCR. At day 12 of gestation, 24 females were examined by laparoscopy to evaluate implanted embryos and at birth the total kits born and individual weights were recorded. Results revealed no gene expression changes in blastocyst and endometrial tissue under heatwave exposure. Moreover, our results demonstrated that rabbit embryos developed from 8-16 cells to blastocyst during a heatwave did not affect implantation rates, total number of kits born and foetal losses. In summary, these results demonstrate that heatwave period is not a critical point in the reproductive performance of rabbits during blastocyst formation.