Bacteria and antibiotic resistance detection in fractures of wild birds from wildlife rehabilitation centres in Spain.

Anatomic adaptations make birds more prone to open fractures with exposed bone parts losing vascularization. As a result of this exposure, fractures are colonized by different microorganisms, including different types of bacteria, both aerobic and anaerobic, causing osteomyelitis in many cases. For this reason, antibiotic treatment is common. However, carrying out antibiotic treatment without carrying out a previous antibiogram may contribute to increased resistance against antibiotics, especially in migratory wild birds. In this paper, bacterial counts regarding fracture type, bacterial identification and antibiotic resistance have been analysed in wild birds from wildlife rehabilitation centres in Spain. The results obtained showed that open fractures had higher bacterial counts (CFU/mL) than closed ones. Bacteria in family Enterobacteriaceae, identified were Escherichia spp., Enterobacter spp., Shigella spp., Hafnia alvei, Proteus mirabilis, Leclercia adecarboxylata and Pantoea agglomerans. Other bacteria present in wild birds' fractures were Aeromonas spp., Enterococcus spp. Bacillus wiedmannii and Staphylococcus sciuri. All species found presented resistance to at least one of the antibiotics used. Wild birds can be implicated in the introduction, maintenance and global spreading of antibiotic resistant bacteria and represent an emerging public health concern. Results obtained in this paper support the idea that it is necessary to take this fact into account before antibiotic administration to wild animals, since it could increase the number of bacteria resistant to antibiotics.

Dogs as a source of Salmonella spp. in apparently healthy dogs in the Valencia Region. Could it be related with intestinal lactic acid bacteria?

BACKGROUND: Although salmonellosis is considered one of the most important food-borne zoonotic diseases in Europe, close contact between dogs and their owners can also be a potential source of Salmonella spp. for humans. This study assessed the prevalence and antimicrobial resistance of Salmonella spp. in apparently healthy dogs in the Valencian Region, eastern Spain. Moreover, a macroscopic comparison of lactic acid bacteria in both Salmonella-positive and Salmonella-negative dogs was carried out. RESULTS: Of a total of 325 dogs sampled, 6 (1.85%) were positive for Salmonella spp. with 3 different serotypes, Havana (3), Mikawasima (2) and monophasic Typhimurium (1). All isolates were susceptible to all antimicrobials tested except monophasic S. Typhimurium, which was resistant to ampicillin. Finally, macroscopic results revealed that lactic acid bacteria had higher heterogeneity in the Salmonella-negative dogs than in the Salmonella-positive dogs. Although the results in our study showed a low prevalence of Salmonella spp., raw food has been suggested as a risk factor for bacteria in dog faeces. CONCLUSIONS: Public awareness campaigns on good hygiene practices, especially after handling canine faeces or raw food, are necessary. Furthermore, to reduce the potential transmission of bacteria, dogs should be fed food that has been properly cooked, as raw or undercooked food can be a source of zoonotic pathogens. Moreover, further studies must be performed to determine the relationship between lactic acid bacteria and Salmonella spp. in dog faeces.

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.

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 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.

Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level.

Although numerous studies have demonstrated that cryopreservation alters gene expression, less is known about those embryos that implanted successfully and continued in gestation. To raise the question of the neutrality of this technique, we examine the effects of vitrification through gestation in rabbit before and after the implantation. We monitored the distribution of losses of 569 vitrified morulae, observing that embryos which reach the last pre-implantatory stage are able to implant. However, we found that not all implanted embryos had the ability to continue with their gestation. The results reveal that vitrification decreased foetus and maternal placenta weights at mid-gestation, but led to a higher offspring birth weight. A novel finding is that while no differences in gene expression were detected in pre-implantatory embryos at day 6, vitrification affects a gene and protein expression in the placenta at day 14. Our results for first time reveal strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. On the basis of these findings, our work leaves the question open as to whether the effects we observed that cause vitrification during foetal development could give rise to some type of physiological or metabolic alteration in adulthood.

Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation.

Several studies have extensively examined structural and biochemical damage induced by cryopreservation that may lead to loss of rabbit embryo viability, but very little information is available on alterations in growth during gestation and at gene expression level. We started our work by comparing the distribution of losses of embryo and foetal development between control and vitrified rabbit morulae. Furthermore, data on foetal sack, foetal and maternal placenta and foetus size for 10-14 days of gestation were evaluated by ultrasonography. We reported that vitrification procedure causes detrimental effects on rabbit embryo and foetal development, with two major peaks of losses: one before the implantation (at day 6) and the other during the second part of gestation (after day 14). However, foetal loss may occur during the implantation process and placenta development, as there was a reduction in development of foetus produced from vitrified-warmed embryos between day 10 and 14 of gestation. For these reasons, using a recent microarray study performed in frozen-thawed rabbit embryos as a point of reference, we analysed the effects of vitrification procedure on the expression of 10 candidate genes in 6-day-old blastocysts obtained after vitrification and transfer. We observed that the relative expressions of mRNA transcripts from SCGB1A1, EMP1, ANXA3 and EGFLAM genes were significantly altered. This could help explain why a large number (29%) of vitrified embryos were successfully implanted but subsequently failed to develop to term. Further studies in subsequent embryo-foetal developmental stages, such as initiation of placenta formation, together with more sensitive high-throughput tools, should help us understand the deficiencies that hinder foetal development and identify the repairing mechanism employed by embryos to overcome vitrification effects.

Treatment with cholesterol-loaded methyl-ß-cyclodextrin increased the cholesterol in rabbit oocytes, but did not improve developmental competence of cryopreserved oocytes.

Membrane cholesterol:phospholipids ratio is an important determinant of cell chilling sensitivity. At low temperatures, major membrane destabilisation occurs when the membrane undergoes a phase transition. To increase membrane fluidity and stability during cooling and thus increase oocyte cryoresistance, cholesterol has been added to the plasma membrane. This study was conducted to determine if cholesterol could be incorporated into rabbit oocytes by incubation with cholesterol-loaded methyl-ß-cyclodextrin (CLC) and if added cholesterol could improve the developmental ability of cryopreserved oocytes after parthenogenetic activation or intracytoplasmic sperm injection. Fresh, frozen and vitrified oocytes incubated with CLC containing 20% NBD-labelled cholesterol (NBD-CLC) were evaluated using confocal microscopy. Fluorescence intensity was higher in fresh oocytes than in cryopreserved ones. Pre-treating rabbit oocytes with 1mg of NBD-CLC/mL did not improve cleavage and developmental rates after cryopreservation. Results showed that treatment with CLC increased the cytoplasmic cholesterol content, but did not improve cleavage rate and developmental competence of cryopreserved oocytes.

In vivo development of vitrified rabbit embryos: effects of vitrification device, recipient genotype, and asynchrony.

This study was designed to evaluate the effects of vitrification device, recipient genotype, and recipient asynchrony on implantation rate, offspring rate at birth, and fetal losses of rabbit embryos. Morphologically normal embryos (N = 787) recovered at 72 hours of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos in Cryotop and ministraw devices were transferred into females induced to ovulate 60 hours (asynchrony) or 72 hours (synchrony) before transfer. In addition, recipient genotypes were analyzed (maternal and paternal genotype). The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total kits born were recorded. Fetal losses were calculated as the difference between total born at birth and implanted embryos. Our data show that a combination of Cryotop device and recipient asynchrony at -12 hours provides the most successful rate of offspring at birth, although a similar implantation rate was obtained with both devices. Thus, low fetal loss rates were observed for embryos vitrified in Cryotop independently of recipient synchrony, and embryos vitrified in straws revealed a two-fold higher rate of fetal losses. Moreover, when an asynchrony between vitrified embryo and recipients was applied, higher rates of embryos developed to term were obtained regardless of the device used. Finally, we found a highly significant association of the recipient genotype with implantation rate, offspring rate at birth, and fetal losses. In conclusion, the current study findings show that Cryotop enhances offspring rate because it is associated with a lower rate of fetal loss. This study thus provides additional evidence that recipient genotype and recipient asynchrony affect offspring rate at birth and indicates that the genotype of the recipient and the recipient asynchrony have a significant effect on implantation rate and fetal losses after vitrification.

Effect of embryonic and maternal genotype on embryo and foetal survival in rabbit.

The aim of this work was to study the influence of embryonic and maternal genotype of two lines of rabbits selected by growth rate (line R) and litter size at weaning (line A) on prenatal survival. Embryos were recovered at 48 h of gestation from R and A donors (39 and 35 does, respectively) and reciprocally transferred to the oviducts of recipient does to the R (n = 15) and A (n = 14) lines. Each recipient doe received six embryos from line R into one oviduct and six embryos from line A into the other. Recipient does were examined by laparoscopy to determine implantation rate on day 14 and slaughtered on day 25 of gestation to determine the number of live foetuses and the weight of foetuses and placentas. No differences were found between lines in fertilization rate and stage of embryo development at 48 h post-insemination. Implantation rate was affected by both the embryonic and maternal genotype. While embryos from donor line A had the highest implantation rate (0.78 ± 0.032 vs 0.65 ± 0.036 for line R), recipient line R had a better implantation rate (0.78 ± 0.033 vs 0.64 ± 0.036 for line A). Foetal survival was affected by the embryonic genotype. Embryos from donor line A had a higher foetal survival rate than embryos from donor line R (0.65 ± 0.036 vs 0.53 ± 0.038, respectively) but lower foetal and placenta weights. In conclusion, while embryonic genotype influenced both implantation and foetal survival rate, R embryos had the lowest rates, maternal genotype affected the implantation rate and R recipients may show a greater uterine receptivity during implantation period. Moreover, it must be observed that foetal and placenta weights were significantly affected by embryonic genotype and heavier for R line.

Maternal exposure to high temperatures disrupts OCT4 mRNA expression of rabbit pre-implantation embryos and endometrial tissue.

We examined the effect of prolonged high heat stress on reproductive performance and its relationship with gene expression in pre-implantation embryos and endometrial tissue. In experiment 1, primiparous rabbit does were divided into two environments: control does (maintained between 14 and 22°C) and heat-treated does housed in a climatic chamber (maintained between 25 and 35°C). Females were reproducing, and the litter size and live born kits were assessed at 2nd and 3rd partum. In heat-treated does, lower litter size (9.7 ± 0.48 and 11.4 ± 0.50) and fewer live born kits (7.2 ± 0.55 and 10.2 ± 0.57) were observed, although similar ovulation rates and numbers of pre-implantation embryos were noted. In experiment 2, after 3rd partum multiparous non-lactating does from each experimental group were used to obtain pre-implantation embryos and endometrial tissue. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-É£, HSP70 and HSP90 were analysed by real-time qPCR. Higher values of OCT-4 expression were observed in embryos and endometrial tissue in females reproduced under heat conditions. Moreover, elevated temperatures have been shown to up-regulate VEGF in embryos and down-regulate Ifn-É£ in endometrial tissue. The findings suggest a deleterious temperature effect on litter size and live born kits as a consequence of variation in gene expression pattern of the pre-implantational embryo and the endometrium associated with proliferation and differentiation and probably with implantation and uterine and foetal development during gestation.