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The increasing prevalence of Listeria monocytogenes infections is a public health issue. Although studies have shown that ruminants constitute reservoirs of this foodborne pathogen, little is known about its epidemiology and genetic diversity within ruminant farms. Here we conducted a large-scale genomic and epidemiologic longitudinal study of Listeria spp. in dairy ruminants and their environments, comprising 19 farms monitored for three consecutive seasons (N=3251 samples). L. innocua was the most prevalent Listeria spp, followed by L. monocytogenes. L. monocytogenes was detected in 52.6% of farms (prevalence in feces samples 3.8%, in farm environment samples 2.5%) and more frequently in cattle (4.1%) and sheep (4.5%) than in goat farms (0.2%). Lineage I accounted for 69% of L. monocytogenes isolates. Among animal samples, the most prevalent sublineages (SL) and clonal complexes (CC) were SL1/CC1, SL219/CC4, SL26/CC26 and SL87/CC87, whereas SL666/CC666 was prevalent in environmental samples. 61 different L. monocytogenes CTs (cgMLST sequence types) were found, 17 of them (27.9%) common to different animals and/or surfaces within the same farms. L. monocytogenes prevalence was not affected by farm hygiene but by season: the overall prevalence of L. monocytogenes in cattle farms was higher during winter, and in sheep farms was higher during winter and spring. Cows in their second lactation had a higher probability of L. monocytogenes fecal shedding than other lactating cows. This study highlights that dairy farms constitute a reservoir for hypervirulent L. monocytogenes and the importance of continuous animal surveillance to reduce the burden of human listeriosis.IMPORTANCEListeria monocytogenes is a bacterial pathogen responsible for listeriosis, the foodborne disease with the highest hospitalization and case-fatality rate. Despite increasing evidence that dairy products and ruminant farms are important reservoirs of L. monocytogenes, little is known about the epidemiology and genetic diversity of Listeria spp. within dairy ruminant farms. We report the largest Listeria spp. longitudinal study in individual domestic animals, and the first using whole-genome sequencing for a deep isolate characterization. Here, we show that domestic ruminants can be asymptomatic carriers of pathogenic Listeria, that L. monocytogenes fecal shedding is often intermittent, and that hypervirulent L. monocytogenes clones are overrepresented in dairy farms. Moreover, we uncover the effect of seasons and lactation number on the prevalence of L. monocytogenes in ruminants. Our study highlights the need for Listeria spp. monitoring in farm animals to control the spread of hypervirulent L. monocytogenes and reduce the burden of human listeriosis.

Listeria monocytogenes is a major human and animal foodborne pathogen. However, data from environmental reservoirs remain scarce. Here, we used whole-genome sequencing to characterize Listeria species isolates recovered over 1 year from wild animals in their natural habitats in Spain. Three different Listeria spp. (L. monocytogenes [n = 19], Listeria ivanovii subsp. londoniensis [n = 4], and Listeria innocua [n = 3]) were detected in 23 animal tonsils (9 deer, 14 wild boars) and 2 feeding troughs. No Listeria species was detected in feces. L. monocytogenes was detected in tonsils of 44.4% (8 out of 18) of deer and 40.7% (11 out of 27) of wild boars. L. monocytogenes isolates belonged to 3 different core genome multilocus sequence typing (cgMLST) types (CTs) of 3 distinct sublineages (SL1, SL387, and SL155) from lineages I and II. While cgMLST type L1-SL1-ST1-CT5279 (IVb; clonal complex 1 [CC1]) occurred only in one animal, types L1-SL387-ST388-CT5239 (IVb; CC388) and L2-SL155-ST155-CT1170 (IIa; CC155) were retrieved from multiple animals. In addition, L1-SL387-ST388-CT5239 (IVb; CC388) isolates were collected 1 year apart, revealing their long-term occurrence within the animal population and/or environmental reservoir. The presence of identical L. monocytogenes strains in deer and wild boars suggests contamination from a common food or environmental source, although interhost transmission cannot be excluded. Pathogenicity islands LIPI-1, LIPI-3, and LIPI-4 were present in 100%, 5%, and 79% of the L. monocytogenes isolates, respectively, and all L. monocytogenes lineage II isolates (n = 3) carried SSI-1 stress islands. This study highlights the need for monitoring L. monocytogenes environmental contamination and the importance of tonsils as a possible L. monocytogenes intrahost reservoir. IMPORTANCEListeria monocytogenes is a foodborne bacterial pathogen responsible for listeriosis. Whole-genome sequencing has been extensively used in public health and food industries to characterize circulating Listeria isolates, but genomic data on isolates occurring in natural environments and wild animals are still scarce. Here, we show that wild animals carry pathogenic Listeria and that the same genotypes can be found at different time points in different host species. This work highlights the need of Listeria species monitoring of environmental contamination and the importance of tonsils as a possible L. monocytogenes intrahost reservoir.

Simple Summary Knowing which bacteria dominate vaginal microbiota and its variation throughout the cycle is important to study how to prevent reproductive diseases. In women, vaginal microbiota is dominated by Lactobacillus but this does not happen in other animals. Little is known about equine vaginal microbiota. The aim of this study was to describe the dynamics of equine vaginal microbiota during the ovarian cycle. Eight healthy adult Arabian mares were used to characterize vaginal microbiota by standard microbiologic and metagenomic procedures. The abundance of Lactobacillus was < 2% by both methods, meaning that equine vaginal microbiota was not dominated by these bacteria. Dominant bacteria included other genera such as Porphyromonas and Campylobacter among others. No changes in vaginal microbiota composition were found, suggesting that equine vaginal microbiota was stable throughout the ovarian cycle. Abstract Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by Porphyromonas, Campylobacter, Arcanobacterium, Corynebacterium, Streptococcus, Fusobacterium, uncultured Kiritimatiaellae and Akkermansia. Lactobacillus comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No differences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.

Este artículo se encuentra disponible en la siguiente URL: https://www.sciencedirect.com/science/article/abs/pii/S0147957120301648?via%3Dihub This is the pre-peer reviewed version of the following article: Tardón, A., Bataller, E., Llobat, L., & Jiménez-Trigos, E. (2021). Bacteria and antibiotic resistance detection in fractures of wild birds from wildlife rehabilitation centres in Spain. Comparative immunology, microbiology and infectious diseases, vol. 74 (feb.), art. 101575, which has been published in final form at https://doi.org/10.1016/j.cimid.2020.101575 Este es el pre-print del siguiente artículo: Tardón, A., Bataller, E., Llobat, L., & Jiménez-Trigos, E. (2021). Bacteria and antibiotic resistance detection in fractures of wild birds from wildlife rehabilitation centres in Spain. Comparative immunology, microbiology and infectious diseases, vol. 74 (feb.), art. 101575, que se ha publicado de forma definitiva en https://doi.org/10.1016/j.cimid.2020.101575 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 analyzed 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.

Simple Summary The use of intravaginal devices may generate retention of vaginal secretions and abnormal discharges at device withdrawal, which may create changes in the normal vaginal bacterial microbiota. These changes in vaginal microbiota could allow the growth of opportunistic pathogenic bacteria which can require antibiotic treatment. The addition of antibiotics on sponges poses a risk of milk contamination. In dairy cows, it has been observed that the use of lactic acid bacteria (LAB) confers a health benefit to the reproductive tract against bacterial infections. However, LAB use in intravaginal sponges in sheep has not been reported. The aims of this study were to describe the dynamics of the ewes’s vaginal cultivable microbiota during a classic long-term estrous synchronization protocol and to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. The present results suggest that LAB infusion in the ewe’s vagina do not affect general health status and did not reduce fertility during natural mating. Abstract Sheep estrous synchronization is mainly based on progestagen-impregnated sponges which could cause vaginitis. Several species of Lactobacillus used as probiotics are commonly used in the treatment or prevention of urogenital infections in humans. However, no studies have been performed to analyze the potential use of probiotics to prevent urogenital infections in sheep. A randomized controlled clinical trial was conducted with 21 one-year-old ewes to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. Synchronization of estrus was based on intravaginal sponges for 14 days. Bacterial communities (Enterobacteriaceae and lactic acid bacteria) were highly fluctuating over time and between animals. The safety of probiotic infusion (mix of Lactobacillus spp. 60% L. crispatus, 20% L. brevis and 20% L. gasseri) in the vagina of healthy ewes was firstly confirmed. Neutrophils were observed in 80% (8/10) of the control ewes compared to 36% (4/11) of the ewes in the probiotic group 2 days after sponge removal (p = 0.056). Fertility in the control and probiotic groups was 60% (6/10) and 91% (10/11), respectively p = 0.097. These results suggest that Lactobacillus spp. infusion in the ewe’s vagina does not affect general health status or fertility.

Este artículo se encuentra disponible en la siguiente URL: https://www.frontiersin.org/articles/10.3389/fvets.2020.00371/full#h1 The vaginal microbiota plays an important role in the health of dairy cattle, and it could be manipulated for the prevention and treatment of reproduction-related infections. The present study profiles and compares the vaginal microbiota of healthy dairy heifers during the estrous cycle focusing the results in follicular (estrus) and luteal (diestrus) phases using 16S rRNA sequencing of the V3–V4 hypervariable region. Twenty 13–16-months-old virgin dairy heifers from a single farm were included in this study. Vaginal swabs and blood samples were obtained during estrus (6–8 h before artificial insemination) and diestrus (14 days after insemination). Estrus was evaluated by an activity monitoring system and confirmed with plasma progesterone immunoassay. Results showed that the taxonomic composition of the vaginal microbiota was different during the follicular and luteal phases. At the phylum level, the most abundant bacterial phyla were Tenericutes, Firmicutes, and Bacteroidetes which comprised more than 75%of the vaginal microbiota composition. The next more abundant phyla, in order of decreasing abundance, were Proteobacteria, Actinobacteria, Fusobacteria, Epsilonbacteraeota, and Patescibacteria. Together with Tenericutes, Firmicutes, and Bacteroidetes represented more than 96% of the bacterial composition. Ureaplasma, Histophilus, f_Corynebacteriaceae, Porphyromonas, Mycoplasma, Ruminococcaceae UCG-005, were the most abundant genera or families. The results also showed that the vaginal microbiota of dairy heifers was non-lactobacillus dominant. The genus Lactobacillus was always found at a low relative abundance during the estrous cycle being more abundant in the follicular than in the luteal phase. Despite more research is needed to explore the potential use of native vaginal microbiota members as probiotics in dairy heifers, this study represents an important step forward. Understanding how the microbiota behaves in healthy heifers will help to identify vaginal dysbiosis related to disease.

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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 metanephros (M) in rabbits. Materials and methods Microscopic dissection was conducted to obtain metanephros 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 metanephros were deposited (using an epidural catheter) close to a patent blood vessel in the retroperitoneal fat. Seventy metanephros 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 metanephros and the development rate of the transplanted metanephros. Results The lower time limit for the extraction of metanephros from the rabbits was day 14. Three weeks after transplantation, only 3/24 14-day-old metanephros grew at minimal expression (12.5%). In contrast, 10/20 (50%) 15-day-old and 12/26 (46.1%) 16-day-old metanephros grew. These metanephros had differentiated sufficiently toward normal glomeruli, proximal and distal tubules and collecting ducts. 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 metanephros from embryos as a feasible and noninvasive technique. The recipients did not require immunosuppression.

[Purpose]: The 'foetal origin hypothesis' postulates that a number of organ structures and associated functions undergo programming during embryonic and foetal life and the neonatal period, which determines the set point of physiological and metabolic responses that carry into adulthood. We evaluate the relationship between high environmental temperatures and the reproductive function of male offspring to determine whether pregnant mammals and their infants are potentially vulnerable to the effects of climate change. [Methods]: Rabbit pups were exposed to high temperatures during gestation and lactation. [Results]: Foetal and postnatal exposure to high temperatures did not alter semen characteristics and was associated with a similar fertility rate and number of pups born. Moreover, males showed reduced rate of maturing and carcass traits at adulthood. [Conclusion]: Our findings suggest that male exposure during the foetal period to high temperatures did not affect sperm quality but permitted an adaptive phenotypic plasticity of growth in adulthood. © 2014 Informa UK Ltd.

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. (C) 2013 Elsevier Inc. All rights reserved.

[EN] This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development., This research was supported by the projects: UGAVIII/16/2015, VEGA 1/0611/15, by the Spanish Research project AGL2014-53405-C2-1-PComision Interministerial de Ciencia y Tecnologia (CICYT), Generalitat Valenciana research program (Prometeo II 2014/036), grant of Slovak Research and Development Agency: APVV-14-0043 and by the European Community under project no 26220220180: Building Research Centre "AgroBioTech". B. Kulikova received fellowship from a Collaborative European Network on Rabbit Genome Biology (RGB-Net) (COST-STSM-TD1101)

[EN] This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62±4.7% and 62±4.9%, respectively); however, the rates were significantly lower (P, This research was supported by the projects Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (FMJ, JSV) and Generalitat Valenciana research program (Prometeo II 2014/036, JSV, FMJ).

[EN] 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., The authors would like to thank Neil Macowan Language Services for revising the English version of the manuscript. This work was supported by the Generalitat Valenciana research programme (Prometeo 2009/125).

The aim of this study was to investigate the effect of Taxol and Cytochalasin B on the spindle, chromosome configuration and development to blastocyst stage after parthenogenesis activation of in vivo matured rabbit oocytes after vitrification. Oocytes were randomized into four groups: oocytes treated with Cytochalasin B or Taxol before vitrification, oocytes without treatment before vitrification and fresh oocytes. Oocytes were vitrified using Cryotop method, and meiotic spindle and chromosomal distribution were assessed with a confocal laser scanning microscopy. To determine oocyte competence, in vitro development of oocytes was assessed with parthenogenesis activation. There were no significant differences in the frequencies of normal spindle (33.0%, 31.0% and 32.6%, for non-treated, Taxol-treated and Cytochalasin B-treated oocytes, respectively) and chromosome (48.3%, 46.6% and 34.8%, for non-treated, Taxol-treated oocytes and Cytochalasin B-treated oocytes respectively) in vitrified groups, but significantly lower than those of fresh group (89.7% and 90.2%, for normal spindle and chromosome organization, respectively). No statistical differences were found in the cleavage and blastocyst development rates between non-treated and Taxol-treated oocytes (7.7% and 1.5% and 13.7% and 4.6%, for non-treated and Taxol-treated oocytes, respectively), although they were significantly lower than in the fresh group (42.3% and 32.1%, for cleavage and blastocyst development, respectively). Oocytes treated with Cytochalasin B failed to reach blastocyst stage. Normal spindle, chromosome configuration and blastocyst development of in vivo matured rabbit oocytes were damaged in vitrification, which was not improved by Taxol and Cytochalasin B pre-treatment before vitrification. Moreover, a detrimental effect on blastocyst development of Cytochalasin B pre-treatment before vitrification was observed.

Contents Although much progress has been made in oocyte cryopreservation since 1971, live offspring have only been obtained in a few species and in rabbits. The aim of our study was to evaluate the effect of vitrification and slow freezing on the meiotic spindle, cortical granule (CG) distribution and their developmental competence. Oocytes were vitrified in 16.84% ethylene glycol, 12.86% formamide, 22.3% dimethyl sulphoxide, 7% PVP and 1% of synthetic ice blockers using Cryotop as device or slow freezing in 1.5 m PROH and 0.2 m sucrose in 0.25 ml sterile French mini straws. Meiotic spindle and CG distribution were assessed using a confocal laser-scanning microscope. To determine oocyte competence, in vitro development of oocytes from each cryopreservation procedure was assessed using parthenogenesis activation. Our data showed that oocytes were significantly affected by both cryopreservation procedures. In particular, meiotic spindle organization was dramatically altered after cryopreservation. Oocytes with peripheral CG distribution have a better chance of survival in cryopreservation after slow-freezing procedures compared to vitrification. In addition, slow freezing of oocytes led to higher cleavage and blastocyst rates compared to vitrification. Our data showed that, in rabbits, structural alterations are more evident in vitrified oocytes than in slow-frozen oocytes, probably as a consequence of sensitivity to high levels of cryoprotectants. Slow-freezing method is currently the recommended option for rabbit oocyte cryopreservation.

[EN] 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., We thank the ASAV (Valencian Poultry Association) and the Santander bank for the financial support of the project and the staff of the CECAV (Center for Poultry and Animal Feed Quality of the Valencian Region) for their assistance. Sofia Ingresa was supported by a research grant from the Spanish Ministry of Education (programme FPU13/03306). English text version was revised by N. Macowan English Language Service.

Embryonic kidney xenotransplantation could represent a new solution to the scarcity of kidneys for transplantation.To determine the feasibility of allogeneic laparoscopic transplantation of metanephroi (M) in rabbits.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.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.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.

[EN] 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.

[EN] 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., Statement of funding: This work was supported by funds from the Generalitat Valenciana Research Programme (Prometeoll 2014/036).

[ES] Antecedentes: Una solución novedosa a la escasez de rinones ˜ para trasplante puede ser el xenotrasplante de rinones ˜ 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 alogé- nico de metanefros de embriones como una técnica factible y no invasiva. Los receptores no necesitaron inmunosupresión., [EN] 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.

[EN] 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. Guardar / Salir Siguiente >, We would like to thank the staff of the Valencian Poultry Association (ASAV) for funding this project, the Centre for Poultry Quality and Animal Feed of Valencia (CECAV) for offering us their facilities, and all his staff for their cooperation and dedication to this work. I would also like to thank the staff of the Department of Animal Science at the Polytechnic University of Valencia for their support of this study.

[EN] 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., This work was supported by funds from the Generalitat Valenciana Research Programme (PrometeoII 2014/036).

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-β-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., This work was supported by the Spanish Research Project AGL2011-30170-C02-01 and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jimenez was supported by a research Grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262). Eva Moce was supported by funds from the Ministry of Science and Innovation Ramon y Cajal sub-programme (Ref. RYC-2010-06162; Madrid, Spain).

[EN] 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. (C) 2014 Elsevier Inc. All rights reserved., This work was supported by the Spanish Research Project AGL2011-30170-C02-01 Comision Interministerial de Ciencia y Tecnologia (CICYT) and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). E. Jimenez-Trigos was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d, ACIF/2010/262). English text version was done by N. Macowan English Language Service.

Although numerous studies have demonstrated that cryopreservation alters gene expression of early embryos and questions the neutrality of the technique in adulthood, 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 the rabbit gestation 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 reveal for first time strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. Based on 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., This work was supported by the Generalitat Valenciana research program (Prometeo 2009/125) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M D Saenz-de-Juano was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254).

[EN] 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 9 h 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, This work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT) and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jimenez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262).

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 degrees C, n = 29) and does heat stressed in a climatic chamber (maintained between 32-37 degrees 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-gamma, 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., This work was supported by the Spanish Research Projects AGL2008-03274 and AGL2011-30170-C02-01. Estrella Jimenez-Trigos and Carmen Naturil-Alfonso were supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262 and ACIF/2013/296, respectively). The English text version was revised by N. Macowan English Language Service.

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‐ɣ, 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‐ɣ 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., This work was supported by the Spanish Research Projects AGL2008–03274 (CICYT). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Estrella Jiménez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262).

[EN] 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. (C) 2013 Elsevier Inc. All rights reserved., This work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT) and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jimenez-Trigos was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d.ACIF/2010/262). The english text version was revised by N. Macowan English Language Service.

Defective sperm-zona pellucida binding and penetration are the main causes of IVF failure. The purpose of this study was to evaluate the effect of zona pellucida thickness in fertilization failure and test the influence of zona pellucida thickness on implantation and birth in rabbits. Embryos and oocytes were collected from 72 females on Day 2 post-insemination. A total of 559 normal embryos were recovered; 402 embryos were transferred by laparoscopy and 157 embryos were used to measure the zona pellucida thickness using the ImageJ program. Laparoscopies were also performed on all does at Day 12 of gestation to record the number of implanted embryos. Litter size at birth was recorded. The mean zona pellucida thickness of the 157 embryos and of the 64 control group oocytes (18.3 ± 0.2 and 18.5 ± 0.3 ¿m, respectively) was significantly less than the zona pellucida thickness of the 74 failed fertilization oocytes (19.2 ± 0.3 ¿m). The probabilities of the regression coefficient being positive were 0.72 and 0.74 for implantation and birth, respectively, and the subsequent means of the coefficient were 2.92 and 0.03 for implantation and birth, respectively. In conclusion, the zona pellucida thickness has an important influence on in vivo fertilization and implantation processes, but not on birth. © 2012 Elsevier B.V., This research was supported by the Valencian Regional Government research programme (Prometeo 2009/125) and the Spanish Research Projects (CICYTAGL2008-03274). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.

In vivo fertilisation techniques such as intraoviductal oocyte transfer have been considered as alternatives to bypass the inadequacy of conventional in vitro fertilisation in rabbit. There is only one study in the literature, published in 1989, that reports live offspring from cryopreserved rabbit oocytes. The aim of the present study was to establish the in vivo fertilisation procedure to generate live offspring with frozen oocytes. First, the effect of two recipient models (i) ovariectomised or (ii) oviduct ligated immediately after transfer on the ability of fresh oocytes to fertilise were compared. Second, generation of live offspring from slow-frozen oocytes was carried out using the ligated oviduct recipient model. Throughout the experiment, recipients were artificially inseminated 9 hours prior to oocyte transfer. In the first experiment, two days after unilateral transfer of fresh oocytes, oviducts and uterine horns were flushed to assess embryo recovery rates. The embryo recovery rates were low compared to control in both ovariectomised and ligated oviduct groups. However, ligated oviduct recipient showed significantly (P, This work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT) and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jimenez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d (ACIF/2010/262). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.