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

[EN] Preimplantation embryo manipulations during standard assisted reproductive technologies (ART) have significant repercussions on offspring. However, few studies to date have investigated the potential long-term outcomes associated with the vitrification procedure. Here, we performed an experiment to unravel the particular effects related to stress induced by embryo transfer and vitrification techniques on offspring phenotype from the foetal period through to prepuberal age, using a rabbit model. In addition, the focus was extended to the liver function at prepuberal age. We showed that, compared to naturally conceived animals (NC), offspring derived after embryo exposure to the transfer procedure (FT) or cryopreservation-transfer procedure (VT) exhibited variation in growth and body weight from foetal life to prepuberal age. Strikingly, we found a nonlinear relationship between FT and VT stressors, most of which were already present in the FT animals. Furthermore, we displayed evidence of variation in liver function at prepuberal age, most of which occurred in both FT and VT animals. The present major novel finding includes a significant alteration of the steroid biosynthesis profile. In summary, here we provide that embryonic manipulation during the vitrification process is linked with embryo phenotypic adaptation detected from foetal life to prepuberal age and suggests that this phenotypic variation may be associated, to a great extent, with the effect of embryo transfer., This research was funded by Conselleria d'Educacio, Investigacio, Cultura i Esport, Spain, grant number AICO/2019/272. Ximo Garcia-Dominguez was supported by a research grant from the Ministry of Economy, Industry and Competitiveness of Spain (BES-2015-072429).

[EN] Effects were analysed of dextran supplementation to Me2SO and acetamide rabbit semen freezing extenders on quality characteristics of rabbit spermatozoa and reproductive performance. The final concentration of cryoprotectants in pooled semen samples was 12.4 % Me2SO for the A extenders, 10.7 % Me2SO and 2.9 % acetamide for the D extenders and 8.9 % Me2SO and 2.9 % acetamide in F extenders, with a supplementation of 1.7 % sucrose in all cases. There was not inclusion of dextran in the A0, D0, F0; while 5 % dextran was included in A5, D5, F5 and 10 % dextran in A10, D10 and F10 extenders. Sperm motility and viability rates were similar with use of the different extenders. Acrosome integrity after the freeze-thawing processes, however, was markedly greater when there was dextran supplementation of D and F extenders. Prolificacy was affected by extender composition. When there was artificial insemination (AI) using semen cryopreserved in the A extenders, number of kits born was similar to when there was AI with fresh semen when there was inclusion of 5% dextran for cryopreservation, while there was no effect on prolificacy when there was cryopreservation of semen using the D and F extenders. In conclusion, dextran supplementation of extenders containing Me2SO and acetamide resulted in greater acrosome integrity. Furthermore, when there was AI using sperm preserved in cryo-diluents containing an intermediate concentration of Me2SO, combined with inclusion of 5 % dextran, there was a marked beneficial effect on rabbit doe reproductive performance., This research was supported in part by AGL2017-85162-C2-1-R from Ministry of Economy and Competitiveness and IVIA-51919 from IVIA projects. A.M. Talavan is supported by PTA-2015-11265-I funds from Ministry of Economy and Competitiveness. English text version revised by N. Macowan English Language Service.

The present study was conducted to characterise rabbit sperm proteins focusing on the influence of the genetic origin. Six samples were recovered during two months from five males from genotype A (New Zealand White origin) and five from genotype R (California origin). Sperm proteins were extracted and subjected to in-gel digestion nano LC-MS/MS and bioinformatics analysis. The resulting library included 487 identified proteins validated with ≥95% Confidence (unused Score ≥ 1.3). All the identified proteins belonged to Oryctolagus cuniculus taxonomy. These data are available via ProteomeXchange with identifier PXD007989. Only 7 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Regarding the comparison of the sperm proteins abundance between genotypes, forty proteins were differentially expressed. Among them, 25 proteins were over-expressed in genotype A, while 15 proteins were over-expressed in genotype R. In conclusion, this study characterizes for the first time rabbit sperm proteins and provides evidence that genotype is related to a specific abundance of spermatozoa proteins.

[EN] The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, beta nerve growth factor ((beta-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as beta-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and beta-NGF, they could be used to develop new extenders with less hormone concentration. (C) 2017 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L. Casares-Crespo is supported by a scholarship from Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by funds from Instituto Valenciano de Investigaciones Agrarias (IVIA) and Ministerio de Empleo y Seguridad Social (Programa de Garantia Juvenil).

[EN] The present study was conducted to characterise rabbit seminal plasma proteins (SP proteins) focusing on the influence of the genetic origin and seasonality. In addition, ß-NGF protein quantity in SP was determined. Semen samples were recovered from January to December 2014 using 6 males belonging to genotype A and six from genotype R. For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment. A total of 24 pools (3 for each season and genetic line) were analysed. SP proteins of the two experimental groups were recovered and subjected to in-solution digestion nano LC¿MS/MS and bioinformatics analysis. The resulting library included 402 identified proteins validated with ¿95% Confidence (unused Score¿1.3). These data are available via ProteomeXchange with identifier PXD006308. Only 6 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Twenty-three proteins were differentially expressed between genotypes, 11 over-expressed in genotype A and 12 in genotype R. Regarding the effect of season on rabbit SP proteome, results showed that there is no clear pattern of protein variation throughout the year. Similar ß-NGF relative quantity was observed between seasons and genotypes. In conclusion, this study generates the largest library of SP proteins reported to date in rabbits and provides evidence that genotype is related to a specific abundance of SP proteins., This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L. Casares-Crespo is supported by a scholarship from Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by Spanish funds from IVIA and Ministerio de Empleo y Seguridad Social (Youth Guarantee Program). The authors are grateful to M. Luz Valero for her excellent technical assistance.

The present study was designed to prove new rabbit insemination extenders containing aminopeptidase inhibitors (AMIs) with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. In addition, different hormone concentrations were tested in these extenders, evaluating their in vivo effect on rabbit reproductive performance after artificial insemination. A total of 911 females were inseminated with semen diluted with the four experimental extenders (C4 group: 4 μg buserelin/doe in control medium (Tris-citric acid-glucose supplemented with bestatin 10 μM and EDTA 20 mM), C5 group: 5 μg of buserelin/doe in control medium, Q4 group: 4 μg of buserelin/doe into CS-DS nanoparticles in control medium, Q5 group: 5 μg of busereline/doe into CS-DS nanoparticles in control medium). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 versus 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied (P > 0.05). We conclude that the CS-DS nanoparticles prepared by a coacervation process as carrier for buserelin acetate allows reducing the concentration of hormone used in extenders supplemented with bestatin and EDTA without affecting the fertility and prolificacy of rabbit females.

This study was designed to develop chitosan (CS)-dextran sulphate (DS) nanoparticles containing a GnRH analogue and to study their effect on rabbit (Oryctolagus cuniculus) semen quality. Six experimental extenders were tested as follows: (control) Tris-citric acid-glucose (TCG), (1) 0.05% CS-0.05% DS (4:1), (2) 0.1% CS-0.05% DS (4:1), (3) 0.05% CS-0.05% DS (3:1), (4) 0.1% CS-0.05% DS (3:1), (5) 0.1% CS-0.05% DS (2:1). CS and DS were dissolved in TCG medium, and nanoparticles were obtained through magnetic stirring. Rabbit seminal samples were incubated up to 5 hr at 37°C in the extenders, and seminal quality was evaluated. The entrapment efficiency was 40%-50%. After 5 hr at 37°C, a 20% of the hormone was released. Results showed that the presence of CS-DS nanoparticles did not affect rabbit semen motility, viability and membrane functionality; however, acrosome integrity was significantly higher versus control (p < .001).

[EN] There is increasing interest in using rabbits for research as a laboratory model as well as for industrial production of meat, wool and fur. Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to either support genetic improvement programs, ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, pos- ology and the effect of gonadotropins used simultaneously, the donor and the environment. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (CTP), alone or sup- plemented with LH, versus a FSH standard protocol of five equal doses administered twice daily to su- perovulate rabbit does (20 per group and 29 control females). We determined: 1) the impact of this stimulation on in vitro development and mRNA expression at blastocyst stage and 2) in vivo embryo development and viability rate at birth of transferred embryos. Our outcomes showed that the ovulation rate was similar among the different ovarian stimulation groups, reaching more than fourfold the ovulation rate of a control doe. While rates of embryos developing to the blastocyst stage after 48 h of in vitro culture were similar between groups, the hatched blastocyst rate was higher for superovulated embryos from CTP group. Moreover, no significant differences among mRNA expression of OCT4, SOX2 and NANOG genes were detected. Nevertheless, embryos from ovarian stimulated does with CTP þ LH showed significantly higher implantation rates and survival at birth among the different ovarian stim- ulation groups and similar to those in the control group. In conclusion, the results of this study suggest that a single injection of long acting corifollitropin alfa can be effectively used in rabbits to elicit a more than fourfold increase in ovulation rate compared to control animals. In addition, the LH supplemen- tation allows us to obtain similar in vivo embryo development results as in the control group., This research was supported by the projects: Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (CICYT) and Prometeo II 2014/36 Generalitat Valenciana research program. English text version revised by N. Macowan English Language Service.

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.

Contents This study aims to assess the efficiency of in vivo oocyte and embryo recovery after a recombinant human FSH (rhFSH) treatment in rabbit does. Females were distributed in two experimental groups: donor does were treated with rhFSH (superovulation group) for 3 days prior to artificial insemination (embryo recovery) or ovulation induction (oocyte recovery) and does without treatment remained as the control group. Mature oocytes or embryos were collected with the laparoscopy technique 16 h after ovulation induction (oocytes) or 72 h after artificial insemination (embryos). Up to four recoveries were performed with each doe. Recovery efficiencies differed significantly between embryos (84%) and oocytes (58%). Yet, the recovery rates for the superovulation and control groups did not differ. The rhFSH group was associated with a significant increase (p

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 T, 5% CO(2), and saturated humidity for 48 It. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH + 10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH + 5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14 +/- 0.074 and 0.15 +/- 0.076 vs. 0.46 +/- 0.078 and 0.50 +/- 0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does. (C) 2009 Elsevier Inc. All rights reserved.

The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240 kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI-TOF/TOF or LC-MS/MS analysis and were the following ones: FAM115E-like (220, 113 and 59 kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72 kDa), annexin A5 (32 kDa), lipocalin allergen Ory c 4 precursor (19 kDa), and haemoglobin subunit zetalike (13 kDa) between genetic lines and FAM115E-like (113 kDa), haemoglobin subunit zetalike (13 kDa) and β-nerve growth factor (12 kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies.

[EN] The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of amino peptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 +/- 0.26 and 9.3 +/- 0.23 vs. 8.2 +/- 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size. (C) 2016 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L Casares-Crespo is supported by a scholarship from Institute Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund.

[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] Assisted reproduction technologies require ovarian stimulation to increase the number of oocytes and embryos. Currently, superstimulation is achieved by gonadotropin treatment, but the embryo yield and quality are highly variable. Commonly, commercial preparations derived from pituitary and urinary origin are used to superovulate. Hence, ovarian superstimulation protocols have usually included both FSH and LH. The appearance of recombinant gonadotropins manufactured by genetic engineering techniques has ensured high quality and batch-to-batch consistency. Moreover, this enables us to assess the importance of LH in the ovarian stimulation. The main aim of this study was to evaluate the effect of recombinant human LH supplementation (10%) on embryonic development produced by rabbit does superovulated with low or high concentration (18.75 or 37.50 IU) of recombinant human FSH (rhFSH). Females treated with rhFSH increased the ovulation rate, and it was significantly higher when the high FSH dose was supplemented with LH. The superstimulation treatment used did not significantly affect in vitro development rate until the expanded blastocyst stage. The results of this study seem to suggest that, in terms of superovulatory response, when rabbit does are treated with 37.5-IU rhFSH, the use of LH supplementation allows an increase in the number of follicles recruited and the quality of embryos, in terms of ability to develop in vitro until blastocyst, and the expression profile of OCT4, NANOG, and SOX2 genes is not affected., This research was supported in part by the Valencian regional government research project PROMETEOII/2014/036. The authors would like to thank Neil Macowan Language Services for revising the English version of the article.

SummaryOvarian stimulation protocols are used usually to increase the number of oocytes collected. The determination of how oocyte quality may be affected by these superovulation procedures, therefore, would be very useful. There is a high correlation between oocyte ATP concentration and developmental competence of the resulting embryo. The aim of this study was to evaluate the effect of follicle stimulating hormone (FSH) origin and administration protocols on oocyte ATP content. Rabbit does were distributed randomly into four groups: (i) a control group; (ii) the rhFSH3 group: females were injected, every 24 h over 3 days, with 0.6 μl of rhFSH diluted in polyvinylpyrrolidone (PVP); (iii) the pFSH3 group: females were injected every 24 h over 3 days with 11.4 μg of pFSH diluted in PVP; and (iv) the pFSH5 group: females were injected twice a day for 5 days with 11.4 μg of pFSH diluted in saline serum. Secondly, the effect of pFSH5 protocol on developmental potential was evaluated. Developmental competence of oocytes from the control and pFSH5 groups was examined. Differences in superovulation treatments were found for ATP levels. In the pFSH5 group, the ATP level was significantly lower than that of the other groups (5.63 ± 0.14 for pFSH group versus 6.42 ± 0.13 and 6.19 ± 0.15 for rhFSH3 and pFSH3, respectively; P < 0.05). In a second phase, only 24.28% of pFSH5 ova developed into hatched blastocysts compared with 80.39% for the control group. A negative effect on oocyte quality was observed in the pFSH5 group in ATP production, it is possible that, after this superovulation treatment, oocyte metabolism would be affected.

Candidiasis has become a prevalent infection in different types of immunocompromised patients. The cell wall of Candida albicans plays important functions during the host-fungus interactions. Cell wall (surface) proteins of C. albicans are major elicitors of host immune responses during candidiasis, and represent candidates for vaccine development. Groups of mice were vaccinated subcutaneously with a beta-mercaptoethanol (beta-ME) extract from C. albicans containing cell wall proteins. Vaccinated mice were then infected with a lethal dose of C. albicans. Increased survival and decreased fungal burden were observed in vaccinated mice as compared to a control group, and 75% of vaccinated mice with the beta-ME extract survived this otherwise lethal infection. We used a proteomic approach (2-DE followed by immunoblotting) to demonstrate a complex polypeptidic pattern associated with the beta-ME extract used in the vaccine formulation and to detect immunogenic components recognized by antibodies in immune sera from vaccinated animals. Reactive protein spots were identified by MALDI-TOF-MS and searches in genomic databases. As a conclusion, vaccination strategies using C. albicans cell wall proteins induce protective responses. These antigens can be identified by proteomic approaches and may be used as components of subcellular vaccines against candidiasis.

Fish sperm quality has traditionally been estimated by subjective evaluation of motility and sperm concentration. Alternative methods for evaluation of sperm quality have been developed in the last decade and enable estimation of spermatozoa head morphometry, membrane integrity and mitochondrial function. Weekly injections of human chorionic gonadotropin (hCG) induced spermiation in farmed male European eels. The milt volume increased from the 5th to 12th weeks. Sperm concentration significantly increased from the 5th week, reaching the highest values at the 8th week, while best motility results were registered at the 9th week of treatment. Coinciding with these intervals, the percentage of dead spermatozoa determined with Hoechst staining showed a reduction in the 8th to 11th weeks of treatment, while the percentage of mitochondrial functionality determined by JC-1 staining did not show a similar pattern. The automatic sperm morphology analysis (ASMA) of the spermatozoa head length, width, area and perimeter showed a significant growth from the 5th to 8th weeks. However, the analysis of isolated descriptive parameters may be difficult to understand because there is a variability in these parameters for each week, making knowledge of the growth kinetic complex. The global size of the spermatozoa head was calculated by applying principal component analysis (PCA), because this method establishes new components that make the interpretation of results easier, allowing a whole interpretation of the changes in the cell morphology. PC1 defines the global head size and shows a significant increase between the 5th and 8th weeks of treatment, showing shorter changes until 12th week. PC2 shows a significant increase in the spermatozoa width from the 5th to 7th weeks. Considering the results of the variations in the principal components defining European eel spermatozoa morphometry, it may be concluded that hCG maturative treatment produced thick cells during the first weeks of spermiation, and subsequent samplings showed an increase in cell width and length. These changes in sperm morphometry coincide with the highest sperm quality assessed as sperm motility and concentration, as well as with the best results obtained in previous studies reporting the best sperm quality between weeks 8 and 10 of hCG treatment. These results support the use of ASMA and Hoechst staining techniques as alternative methods for the evaluation of fish sperm quality.

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out its before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts: (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself, and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed. (c) 2006 Elsevier Inc. All rights reserved.

The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, it? vitro, in vivo-matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at -30 degrees C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen-thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co-incubated with 2 x 10(6) frozen-thawed spermatozoa during 4 h at 37 degrees C in Tyrode's medium under an atmosphere of 5% CO(2) in air with maximal humidity. After co-incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL 35 and 46 mu m/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 mu m, for semen frozen at -30 degrees C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at -30 degrees C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at -30 degrees C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at -30 degrees C, in LNV and fresh semen, respectively). Sperm frozen at -30 degrees C seemed to be more capacitated.

Background: The morphogenetic conversion between yeast and hyphal growth forms appears to be crucial in the pathogenesis of invasive candidiasis, and can be regulated by environmental signals such as extracellular pH. Aims: To characterise the epitope recognised by monoclonal antibody 1H4, and to evaluate the expression of its corresponding epitope in Candida albicans cells under different conditions of pH and temperature, and “in vivo”, in tissue samples from patients with human candidiasis. Methods: Monoclonal antibody 1H4 was generated against the 58 kDa cell wall mannoprotein of C albicans (mp58), and was further characterised by immunoblot analysis, periodate treatment of the antigenic preparations, and agglutination experiments of C albicans strains 3153A, SC5314, and 412, cultured under different environmental conditions (growth media and pH). An immunohistochemical study was performed in 24 human tissue samples from patients with mucocutaneous and systemic candidiasis. Results: 1H4 recognises a pH sensitive carbohydrate epitope on the surface of C albicans cells, and this epitope is not restricted to mp58, but is shared with other cell wall mannoproteins. Immunohistochemical findings indicated that expression of the 1H4 epitope on C albicans cells in tissue sections from human candidiasis correlates with tissue invasion and pH of the niche. 1H4 immunoreactivity was also found in candida remnants within macrophages. Conclusions: The fact that 1H4 epitope expression selectively identifies invasive forms of C albicans , in addition to candida remnants within macrophages, supports its potential value in the diagnosis and management of human candidiasis.

The 58-kDa surface mannoprotein of Candida albicans (mp58) elicits strong antibody responses during infection. Epitope mapping with sera from patients with candidiasis and control individuals indicated the presence of multiple IgG-reactive continuous epitopes on the protein, expanding both the amino- and carboxy-terminal domains and several internal regions. These immunoreactive regions were similar to the ones previously identified using sera from immunized animals. Two of the epitopic regions (including the C-terminal domain) showed increased reactivity with antibodies present in sera from patients with candidiasis as compared to control individuals. Patients who survived the infection displayed increased antibody reactivity towards the C-terminal epitope as compared to those succumbing to candidiasis. A monoclonal antibody directed towards this epitopic region conferred protection in serum therapy experiments in a murine model of hematogenously disseminated candidiasis. Together, these observations indicate the carboxy-terminal antibody binding domain of C. albicans mp58 represents a protective epitope during candidiasis.

The aim of this study was to assess the application of a cryopreservation program to preserve two selected rabbit lines. One of them was selected by litter size at weaning, line V (Synthetic breed). The second, line R (synthetic breed), was selected by growth rate. In this study, embryos were collected, from donor does belonging to the 7th and 15th generations of lines R and V, respectively, were vitrified and were stored from 1992-1993. Those embryos from donor does belonging to the 17th and 21st generations of lines R and V respectively, were vitrified and sotred from 1998-1999. Embryo transfers were carried out in 1999. Morphologically normal embryos at the morulae stage were cryopreserved by vitrification in a 2.8 M dimethyl-sulfoxide + 3.5 M ethylene-glycol + 0.3 g x L(-1) bovine serum albumine in Dulbecco phosphate buffered saline solution. The main problem in the cryopreservation program was the low embryo production efficiency: significant differences were obtained in recovery efficiency between lines and line R showed the lowest proportion of donor does with 55% (at least 4 normal embryos) vs. 72% in line V. However, after transfer in recipient does of line V, the fertility rate at birth (81%), the rate of alive born by pregnant recipients (43%) and the number of males and does with offspring (9 to 18 different males, 12 to 32 females) enabled the different generations from each line to be re-established and studies on the selection process genetic gain to be developed.

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] The choice of medium and supplements can affect meiotic regulation and may have an impact on the regulation of mammalian oocyte growth and embryonic cell function. The aim of the present study was to assess the effects of oxygen concentration and endogenous lanosterol on the in vitro maturation (IVM) media without serum and based on recombinant human chorionic gonadotrophin in prepubertal ewe oocytes. Firstly, the effect of varying oxygen concentrations (5% and 20%) during IVM in TCM-199 supplemented (4 mg/ml bovine serum albumin (BSA), 100 M cysteamine, 0.3 mM sodium pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH; Gonal-F (R) 75 UI, Serono, Italy), 0.1 UI/ml recombinant leuteinizing hormone (r-LH; Lhadi (R) 75 UI, Serono, Italy) and 1 g/ml estradiol-17) on subsequent nuclear maturation of oocytes examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) was investigated. Secondly, two concentrations of lanosterol (0, 10 and 50 M) were added to the IVM medium. Nuclear maturation of oocytes was examined as previously. Lipid content in oocytes, an important indicator of cytoplasmic maturity, was also measured using Nile red fluorescent stain. The results showed that low oxygen concentration affected the nuclear maturation. Similarly, a significantly higher rate of meiosis resumption was observed with 10 M (72.3%) of lanosterol compared with the control (51.8%) or 50 M of lanosterol (59.4%). A significantly higher content of lipids was also observed with 10 and 50 M of lanosterol (7.3 +/- 0.2 x 10(6) and 7.4 +/- 0.2 x 10(6) arbitrary units of fluorescence) compared with the control (6.7 +/- 0.2 x 10(6) arbitrary units of fluorescence). The results indicate that 10 M lanosterol during IVM in medium without serum and based on recombinant human chorionic gonadotrophin has a positive effect on maturation of prepubertal ewe oocytes., This work was supported by funds from INIA (project RZ2006-00004) and Universidad Politecnica de Valencia (20081166). English text version revised by N. Macowan English language service.

This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre-implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood., This work was supported by the Valencian Regional Government research programme (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). The authors would like to thank Neil Macowan Language Services for revising the English version of the manuscript.

Cooling sperm to and equilibrating the sperm at 5 degrees C require the most time in any sperm cryopreservation protocol. Reducing the time required for these phases would simplify sperm freezing protocols and allow greater number of ejaculates to be processed and frozen in a given time. This study determined how holding rabbit sperm at 5 degrees C for different lengths of time (0, 10, 15, 20, 30, or 45 minutes) affected the quality of rabbit sperm, measured by in vitro assays, and if reducing the cooling time to only 10 minutes affected the fertilizing ability of the sperm. Reducing the time sperm were held at 5 degrees C to 10 minutes did not affect the in vitro quality of the sperm (percent motile and with intact plasma membranes), although eliminating the cooling phase completely (directly freezing the sperm from room temperature) decreased in vitro assessed sperm quality (P < 0.01). However, reducing the time sperm were held at 5 degrees C, from 45 to 10 minutes, negatively affected the fertilizing ability of sperm in vivo (P < 0.05). In conclusion, completely eliminating cooling rabbit sperm to 5 degrees C before freezing is detrimental for rabbit sperm cryosurvival, and although shortening the time sperm are held at 5 degrees C to 10 minutes does not reduce in vitro sperm quality, it does reduce the fertility of rabbit sperm. Therefore, the length of time rabbit sperm equilibrate at 5 degrees C is crucial to the fertilizing ability of rabbit sperm and must be longer than 10 minutes. Currently, it is not known if holding rabbit sperm at 5 degrees C for less than 45 minutes will affect sperm fertilizing ability. (C) 2014 Elsevier Inc. All rights reserved.

[EN] This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me2SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me2SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me2SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me2SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters, This research was supported partly by the RTA2010-00117-00-00 from INIA, the European FEDER Funds and 125-2009 Prometeo Project (Generalitat Valenciana). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.

[EN] Ovulation induction in artificially inseminated rabbits by adding GnRH synthetic analogues in the seminal doses is a welfare-orientated method to induce ovulation in rabbits and could have some advantages in field practice. This study was conducted to determine the effect of male genotype on the aminopeptidase activity in rabbit seminal plasma and the effects of dilution rate of semen on availability and reproductive performance when buserelin acetate is added to the seminal dose. To study the aminopeptidase activity, 12 mature bucks belonging to a paternal line and 12 from a maternal line were used. The bucks from the paternal line were used to study the effect of dilution rate on the availability of buserelin acetate after 2 hours of dilution and on the reproductive performance of the doses after artificial insemination of 389 commercial crossbreed does. Aminopeptidase activity in seminal plasma is dependent on the male genotype. The paternal line resulted 27% more aminopeptidase activity than the maternal line (P < 0.05). On the other hand, semen diluted 1:20 exhibited a marked increase in the availability of buserelin acetate and the fertility in this group was significantly higher than females from dilution rate 1:5 group, which showed similar results to that of the negative control group (does inseminated with semen diluted 1:20 in non-GnRH-supplemented extender). We conclude that the bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases and is consequently affected by the dilution rate used to prepare the artificial insemination doses. (C) 2014 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2010-00117-00-00 from INIA and the European FEDER Funds. E. Moce is supported by funds from Ministerio de Ciencia e Innovacion from Subprograma Ramon y Cajal and the European Social Fund (ref. RYC-2010-06162; Madrid, Spain).

In experiments based on ruminants' individual dry matter intake (DMI) assessment, several external markers can be used to estimate faecal output when total faeces collection is not possible. However, preparation of the markers to be administered and analytical procedures used for marker content determination are time-consuming thus strongly limiting the number of animals involved in the experiments. In this paper, polyethylene glycol (PEG, molecular weight 6000 da) was tested as a faecal marker. Four trials were conducted on dry, non-lactating ewes kept in digestibility crates that allowed individual measurements. The overall experiment was designed to assess the major factors that could lessen the effectiveness of this method, assuming that the use of grab samples of faeces is sufficient. Trial 1 was designed to test two levels of PEG (20 and 40 g/day) administered in two equal amounts. Trial 2 was designed to test the effect of either a single morning (0800 h) dose (20 g/day) or a twice daily administration (0800 and 1600 h) of the same fractionated dose. Trial 3 was designed to test a 20 g/day dose of PEG administered once daily to ewes fed with hays of different qualities: medium (MH) and low (LH). In trial 4, a lower dose of PEG (10 g/day) was administered once a day to ewes fed with fresh oat-vetch forage. It was demonstrated that PEG could be precisely estimated (average prediction error = 3.47 g/kg) with near-infrared reflectance spectroscopy (NIRS). On the basis of the four trials, it has been proved that PEG administration (20 and 40 g/day) did not significantly affect the DMI of ewes fed dry diets (trials 1, 2 and 3), whereas there was an unexpected increase of DMI for ewes fed exclusively with green feed (trial 4) without DM digestibility modification. Providing PEG as a single dose (0800 h) or split into two equal parts (0800 and 1600 h) did not alter the estimated DMI. Considering the interest of grab sampling, there were clear variations of PEG in faeces with higher concentrations observed at 0800 and 1600 h and lower concentrations at 1400 h. Consequently, with PEG (measured with NIRS) administered once and using the grab sampling procedure (morning collection), it is possible to estimate the DMI of dry feeds with good accuracy. For green feeds, more research is needed as the estimated results are still highly variable.

Prenatal death can occur due to several genetic and environmental factors which alter normal embryo development, maternal environment to support normal fertilisation, development of embryos, placenta and foetus, or affect the necessary relationship between embryo and endometrium. The aim of this work was to study gestational losses and progesterone, 17 ß-estradiol and IGF I serum levels in a rabbit line selected for growth rate (paternal line). In this study, a maternal line well characterised in previous studies was used as a reference line. A total of 211 laparoscopies were carried out, and the number of corpora lutea and implanted embryos at 12 th days, total born and live born were recorded per female. To analyse the endocrine levels, blood serum was collected from 54 females with implanted embryos at 12 th and 24 th day of gestation (27 from each line). The paternal line showed the lowest ovulation frequency, number of implanted embryos, total born and live born (0.70, 11.3, 7.4, and 6.4 vs 0.86, 12.8, 11.1 and 10.6 for maternal line, respectively) and consequently, the highest implantation, gestational, foetal and perinatal losses (0.31, 0.60, 0.40, and 0.15, respectively). Progesterone serum levels at 12 th days of gestation were similar between lines; however, progesterone serum level at 24 th day of gestation was significantly lower in the paternal line (4.8 vs 8.2 ng/mL). Serum levels of 17ß-estradiol and IGF-I at 12 th days of gestation were different between lines (14.6 vs 26.5 pg/mL, 237 vs 149 ng/mL for paternal and maternal lines respectively). These higher gestational losses of the paternal line could be explained by differences in 17 ß-estradiol level at 12 th days of gestation and the possible effect on low progesterone serum levels at 24 th days of gestation. Further studies in steroid production and bioavailability have to be done during oestrus and pregnancy related with metabolic activity of this line. © 2012 Elsevier Inc., We are grateful to the Researcher Training Programme from Valencia Polytechnic University. This work was supported by the Spanish Research Project (CICYT AGL2008-03274). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.

Contact : debus@supagro.inra.fr; Maternal undernutrition during gestation can condition offspring adult health, with the periconceptional period pointed out as a key period. The aim of this study was to evaluate the effects of maternal periconceptional undernutrition on pregnancy and offspring growth performance in sheep. 52 Merinos d'Arles ewes were fed to requirements (control group, C), whereas 64 ewes received 50% of their dietary needs from -15 to +30 days post-conception (restricted group, R). Thereafter, both groups were fed according to needs. Maternal body weight (BW), body condition score (BCS) and Non Esterified Fatty Acids (NEFA), progesterone, leptin and cortisol plasma concentrations were monitored weekly during the restriction period and the following month, then monthly until weaning. Lambs were weighed weekly until weaning at 22 kg BW, then monthly. Plasma leptin was monitored monthly in lambs. The BW, BCS, and leptin concentrations were significantly decreased, whereas NEFA and cortisol concentrations were increased in R dams. Maximum progesterone concentration was higher in R ewes that had a high (10-25%) vs. low (0-10%) BW loss during restriction (27.9 +/- 2.59 vs. 20.8 +/- 2.00 ng/mL, P < 0.05). Overall, gestation was significantly longer in the R group (151.0 +/- 0.3 vs. 149.4 +/- 0.4 days, P < 0.001). There was no difference between groups for pregnancy rates, prolificacy, birth weight and lamb mortality, but the proportion of male lambs was significantly higher in the R group, only for singletons (16/26 vs. 9/26, P < 0.05). Lamb growth was not significantly modified by treatment. Leptin concentrations at birth were significantly lower in R vs. C males (6.15 +/- 0.13 ng/mL vs. 7.42 +/- 0.36 ng/mL, P < 0.05), whereas in females, leptin concentrations were significantly higher in R vs. C lambs at 4 mo of age (7.31 +/- 0.27 ng/mL vs. 6.41 +/- 0.29 ng/mL, P < 0.05). These results indicate that maternal periconceptional undernutrition in a hardy breed does not significantly affect lamb birth weight and growth rates, in contrast to previous reports in other breeds, suggesting that caution must be taken when extrapolating programming data between breeds and breeding conditions.

Bluetongue virus (BTV) is an economically important arbovirus of ruminants transmitted by Culicoides biting midges. Vector control using residual spraying or application to livestock is recommended by many authorities to reduce BTV transmission; however, the impact of these measures in terms of both inflicting mortality on Culicoides and subsequently upon BTV transmission is unclear. This study consisted of a standardized World Health Organization laboratory assay to determine the susceptibility of European Culicoides species to deltamethrin and a field trial based upon allowing individuals of a laboratory strain of Culicoides nubeculosus Meigen to feed upon sheep treated with Butox 7.5 pour-on (a deltamethrin-based topical formulation). Susceptibility in the laboratory trial was higher in colony C. nubeculosus (24-h LC [90]=0.00106%), than in field populations of Culicoides obsoletus Meigen (24-h LC[90]=0.00203%) or Culicoides imicola Kieffer (24-h LC[90]=0.00773%). In the field, the pour-on formulation was tested with a total of 816 C. nubeculosus specimens fed upon on the thigh of treated sheep. The study revealed a maximum mortality rate of 49% at 4 d post application, and duration of lethal effect was predicted to be as short as 10 d, despite testing being carried out with a highly susceptible strain. The reasons for this low efficacy are discussed with reference both to the potential for lack of spread of the active ingredient on the host and feeding patterns of the major potential vector species on the sheep host. Practical implications for vector control strategies during BTV incursions are also detailed.

[EN] The study evaluated a seminal effect on the ability to induce ovulation of a synthetic GnRH analogue, buserelin acetate, administered by vaginal mucosa in rabbit does. In a first experiment, 751 receptive nulliparous and multiparous non-lactating does were randomly assigned to groups of different seminal doses (6, 12, 24, 50, and 100 million total sperm in 0.5 mL). All seminal doses contained 5 mu g of buserelin acetate to induce ovulation by vaginal mucosa absorption. Two hundred and six does from 751 were laparoscopized at 12(th) days of gestation to evaluate ovulation induction, ovulation rate and implanted embryos, while pregnancy rate and total and live born were noted in all females. Results showed that the pregnancy rate was significantly affected by the seminal dose used (0.82 vs 0.72, 0.50, and 0.45, for 6, 24, 50, and 100 million of spermatozoa, respectively). Data from laparoscopized does showed significant differences between the group of 6 and 50 million sperm dose in the ovulation induction and consequently in the pregnancy rate (0.79 vs 0.52, 0.79 vs 0.48, respectively). Does from all groups had similar implanted embryos and litter sizes irrespective of seminal dose used. In a second experiment, inseminations were done without spermatozoa, 0.5 mL of two dilutions of seminal plasma (1/4 and 1/20) with 5 mu g of buserelin acetate were introduced into vagina from 71 receptive females and its results were compared to a control group (35 does) induced to ovulate with 1 mu g of buserelin acetate administered intramuscularly. Only 40% of females from 1/4 plasma dilution group became to ovulate. Consequently, the dilution rate of seminal plasma may reduce the availability rate of the GnRH analogue and the concentration needed to provoke the ovulation induction. (C) 2011 Elsevier Inc. All rights reserved., We wish to thank the collaboration of Manuel Sierra Guillamon, Felipe Lavara Garcia and Ivan Mular Puig. This research was supported in part by the project RTA2010-00117-00-00 from INIA and the European FEDER Funds.

Vitrification of embryos is being increasingly important for cryopreservation in mammals. However, damage and toxicity has to be reduced even more. The composition of cryoprotective medium used to immerse the embryos affects viability and developmental potential. The aim of this work was to assess the effect of the Polyvinylalcohol-PVA- and Dextran addition to vitrification media on the in vitro development of rabbit embryos from superovulated and non-superovulated females. Superovulation group were treated intramuscularly with 25 IU rhFSH. The vitrification media contained the same permeable cryoprotectans (Ethylene Glycol-ET- and Dimethyl Sulfoxide-Me₂SO-) and different macromolecules (PVA and Dextran) in different combinations. There was a significantly higher proportion of embryos without damages in mucin coat or zona pellucida after warming (undamaged embryos) in the control than in the superovulation group (95.8% vs. 83.2%, respectively). The proportion of undamaged embryos was significantly affected by the vitrification solution composition. The rate of undamaged embryos after warming in media containing 20% Me₂SO was significantly lower in media supplemented with PVA than in media with dextran (67.3 vs. 93.8, respectively). However, the proportion of undamaged embryos for the medium supplemented with dextran was similar for media with 15 or 20% Me₂SO. In conclusion, the addition of dextran to the vitrification media improve the preservation of rabbit embryos and permits to reduce the amount of Me₂SO for vitrification. Additionally, in vitro developmental ability of undamaged embryos were not affected by superovulation treatment nor vitrification media.

This study was conducted to investigate the predictive capacity of fertility and litter size of sperm head morphometric measurements when the ejaculates fulfilled the minimum requirements commonly used in artificial insemination (AI). Semen samples from 11 rabbits (77 ejaculates) were evaluated for sperm motility, abnormal spermatozoa and sperm head morphometry using computer automated sperm analysis system. Morphometric dimensions for length, width, area and perimeter were analysed. Only ejaculates with more than 70% of motility rate and

Contents The site where the semen is deposited appears to be one of the most important factors affecting pregnancy of inseminated goats. In Murciano–Granadina (MG) goats, post-cervical insemination is achieved in a limited number of females. An effective way to increase fertility rate could be by increasing post-cervical inseminations. Effect of exogenous oxytocin application to facilitate the cervical penetration and its effect on kidding rate and prolificacy in MG goats were investigated. Oestrus was synchronized using progesterone-impregnated sponges for 11 days. Females were randomly divided into three groups (n = 190) and received either an i.v. injection of 100 or 200 IU of oxytocin or saline solution 15 min before being inseminated. Data on semen deposition depth were recorded for each animal using a catheter scaled in centimetres (up to 4 cm). Depth of semen deposition was affected by the oxytocin treatment (p

This study was conducted to evaluate changes in ram seminal plasma composition from ejaculates obtained using artificial vagina (AV) and electroejaculation (EE). To address this question, we assessed the effect of semen collection method on volume, sperm concentration, sodium concentration, potassium concentration, sodium/potassium ratio, total protein content and protein profile using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide gel electrophoresis. The main findings from this study were: (i) similar volume was obtained, while sperm concentration was significantly lower for EE method; (ii) potassium and sodium/potassium concentration ratio were not influenced by recovery method, while sodium concentration increased significantly when semen was recovered using EE; (iii) approximately 80% of the total relative seminal plasma protein is represented by four protein fractions of molecular weights around 15, 21, 24 and 50 kDa and there were not differences and (iv) focussing the two-dimensional SDS-PAGE gel on the 10-25 kDa rank, the IMAGE ANALYSIS software detected around 22 spots with isoelectric points ranging from 5.1 to 6.1. Two protein spots (15 kDa and 5.5 and 22 kDa and 5.2 for molecular weight and isoelectric point respectively) increased significantly when semen was recovered using EE. One spot protein with molecular weight around 25 kDa and isoelectric point of 5.2 were only found in the seminal plasma from the semen recovery by AV. As it was demonstrated, ejaculates obtained with EE modify the sodium concentration, alter two proteins concentration and induced the loss of one protein in seminal plasma.

International audience; Rolland PH, de Lagausie P, Stathopoulos E, Lepretre O, Viudes G, Gorincour G, Hery G, de Magnee C, Paut O, Guys JM. Phasic hemodynamics and reverse blood flows in the aortic isthmus and pulmonary arteries of preterm lambs with pulmonary vascular dysfunction. Am J Physiol Heart Circ Physiol 295: H2231-H2241, 2008. First published September 26, 2008; doi: 10.1152/ajpheart.00410.2008. Time-domain representations of the fetal aortopulmonary circulation were carried out in lamb fetuses to study hemodynamic consequences of congenital diaphragmatic hernia (CDH) and the effects of endothelin-receptor antagonist tezosentan (3 mg/45 min). From the isthmic aortic and left pulmonary artery (PA) flows (Q) and isthmic aortic, PA, and left auricle pressures (P) on day 135 in 10 controls and 7 CDH fetuses (28 ewes), discrete-triggered P and Q waveforms were modelized as P(t) and Q(t) functions to obtain basic hemodynamic profiles, pulsatile waves [P, Q, and entry impedance (Z(e))], and P and Q hysteresis loops. In the controls, blood propelling energy was accounted for by biventricular ejection flow waves (kinetic energy) with low Z(e) and by flow-driven pressure waves (potential energy) with low Z(e). Weak fetal pulmonary perfusion was ensured by reflux (reverse flows) from PA branches to the ductus anteriosus and aortic isthmus as reverse flows. Endothelin-receptor antagonist blockade using tezosentan slightly increased the forward flow but largely increased diastolic backward flow with a diminished left auricle pre-and postloading. In CHD fetuses, the static component overrode phasic flows that were detrimental to reverse flows and the direction of the diastolic isthmic flow changed to forward during the diastole period. Decreased cardiac output, flattened pressure waves, and increased forward Z(e) promoted backward flow to the detriment of forward flow (especially during diastole). Additionally, the intrapulmonary arteriovenous shunting was ineffective. The slowing of cardiac output, the dampening of energetic pressure waves and pulsatility, and the heightening of phasic impedances contributed to the lowering of aortopulmonary blood flows. We speculate that reverse pulmonary flow is a physiological requirement to protect the fetal pulmonary circulation from the prominent right ventricular stream and to enhance blood flow to the fetal heart and brain.

The aim of this study was to evaluate the supplementation of semen extender with two synthetic GnRH analogues (buserelin and triptorelin) to induce ovulation in rabbit does submitted to artificial insemination. In a first experiment, 255 receptive multiparous does were inseminated with 0.5 mL of Tris-citrate-glucose extender supplemented or riot with two GnRH synthetic analogues. Experimental groups were: NC (not supplemented extender), PC (not supplemented extender and does treated with 1 mu g of buserelin i.m.), 132 (2 mu g per female buserelin supplemented extender), 135 (5 mu g per female buserelin supplemented extender), T2 (2 mu g per female triptorelin supplemented extender) and T5 (5 mu g per female triptorelin supplemented extender). Thirteen does of NC females ovulated, reaching an ovulation rate similar to the other groups. Ovulation rate was similar in all groups (11.4-12.5). The efficiency of ovulation induction was very low (32.5%) in NC group and showed the higher results in PC females (97.8%). Only 135 females reached similar ovulation induction response than PC group. In a second experiment, 702 receptive does were inseminated to compare fertility and prolificacy parameters from the conventional insemination technique (control group, females treated with 1 mu g per female of buserelin intramuscularly) versus a supplementation with buserelin or triptorelin (5 mu g per female) in semen extender (135 and T5 groups, respectively). Fertility and prolificacy parameters were similar among the groups (77.8% fertility rate, 73.9% kindling rate, 9.4 live born and 9.9 total born). This study demonstrate the possibility of ovulation induction in rabbits by adding two GnRH synthetic analogues in the seminal doses and open up new prospects for changing rabbit insemination procedures. (C) 2007 Elsevier Inc. All rights reserved.

The freezing step of the cryopreservation protocol negatively influences the quality and fertilising ability of rabbit spermatozoa. This study determines the effect of different rates of freezing on the quality and fertilising ability of rabbit spermatozoa cryopreserved with dimethylsulfoxide (DMSO) (1.75M) and sucrose (0.05M). Ejaculates from meat rabbit line males (n=12) were pooled and each pool (n=7) was split into four aliquots. One group of straws (control, C) was frozen in static liquid nitrogen vapour (5cm above the liquid nitrogen, 10min) and the other groups were frozen at different freezing rates (degrees Cmin(-1)) from -6 degrees C to -100 degrees C using a programmable freezer: slow (-15 degrees Cmin(-1), S), medium (-40 degrees Cmin(-1), M) or fast (-60 degrees Cmin(-1), F). After thawing (50 degrees C, 12s), the quality was highest (P

This study researches the effects of supplementation with reduced glutathione (GSH, 0.5 mm) and oxidised glutathione (GSSG, 0.5 mM) freezing extenders on different semen parameters after equilibration with DMSO preservation solution (45 min at 5 degrees C) and post-thawing. The main findings that emerged from this study are that (i) addition of GSH and GSSG to the freezing media did not result in any improvement in functional sperm tests after equilibrium phase. (ii) No differences were observed after cryopreservation in functional sperm tests and embryo recovery rate. In conclusion, the addition of 0.5 mM GSH or GSSG appears not to play an important role in sperm antioxidant defence during cooling and freezing in rabbit spermatozoa.

In this work, we investigated the effect of storage in solid-phase extender on buck semen conserved at 5 degrees C. Furthermore, we studied the effect of addition of cysteine to the extender and the washing of seminal plasma on sperm survival. In Experiment 1, milk-based extender (M) was used as a control to study the effect of solid media storage (G) and cysteine supplementation (C), and the combination of both (GC), on in vitro sperm survival of washed and non-washed semen, conserved up to 72 h at 5 degrees C. Motility, acrosome integrity (NAR) and hypo-osmotic swelling tests (HOST) were evaluated to assess in vitro sperm survival. In Experiment 2, an artificial insemination (AI) field trial was performed to compare G versus M. Solid media (G) maintained motility of spermatozoa during storage higher than any other extender (67% G versus 62% GC; 61% M and 59% C; P0.05), but there was no difference in NAR or HOST between extenders (P0.05). No improvement in sperm viability was obtained by addition of cysteine to the media. Washing of semen improved motility (65% versus 60%; P0.05), NAR (70% versus 64%; P0.05) and HOST (37% versus 28%; P0.05). No significant differences in fertility were obtained between G and M extenders (47% versus 41%; P0.05). In conclusion, washing of semen and dilution in gelatin-supplemented milk extender (solid storage) appears to be a successful method for goat semen storage at 5 degrees C.

The main aim of the present study was to investigate the effect of sperm freezing medium dilution ratio (1:1, 1:2, and 1:5 v/v), two cryoprotectants: dimethyl sulphoxide (Me(2)SO) and methanol (MeOH), and the addition of foetal bovine serum (FBS) on the cryopreservation of European eel sperm. The effect of these factors was evaluated comparing post-thawing viability with fluorescent staining (Hoechst bisbenzimide 33258) and the spermatozoa head morphometry, determined with computer-assisted morphology analysis (ASMA). The 1:5 (v/v) dilution ratio resulted in a lower viability in comparison with 1:1 and 1:2 (52.8+/-2.3% vs. 67.4+/-2.3% and 65.1+/-2.3%, respectively, p=0.0001), but without effects on the head morphology. Although the viability was not significantly different between Me(2)SO and MeOH (60.4+/-1.9 vs. 63.2+/-1.9%, respectively, p=0.305), a decrease of spermatozoa head area and perimeter was found when spermatozoa were frozen with methanol (6.19+/-0.01 vs. 6.36+/-0.01 microm(2) and 17.28+/-0.05 vs. 17.49+/-0.05 microm, for area and perimeter and MeOH and Me(2)SO, respectively, p=0.0001). Finally, a higher viability (75.1+/-1.7 vs. 48.5+/-1.7, with or without FBS, respectively, p=0.0001) and higher spermatozoa head size (6.40+/-0.01 vs. 6.15+/-0.01microm(2) and 17.88+/-0.05 vs. 16.89+/-0.05 microm, for area and perimeter, with or without FBS, respectively, p=0.0001) were found when cells were frozen-thawed in freezing media supplemented with FBS. Based on the above findings, dilution ratios lower than 1:5 (v/v) and the addition of serum improved the viability results after cryopreservation. Future studies are required in order to understand the spermatozoa membrane interchange mechanisms in response to the changes in spermatozoa head size caused by cryoprotectants and freezing media supplements.

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.

The aim of the present study was to characterise European eel spermatozoa morphometrically, as a basis for future studies on the morphological effects of methods for sperm cryopreservation and sperm quality. This characterisation was carried out measuring several spermatozoa morphology parameters (head length, width, area and perimeter) by scanning electron microscopy (SEM), in comparison with measurements developed in European eel spermatozoa with computer-assisted morphology analysis (ASMA). Spermatozoa head morphology showed differences in width (1.15+/-0.01 microm versus 1.12+/-0.01 microm), perimeter (14.68+/-0.13 microm versus 13.72+/-0.19 microm) and area (5.36+/-0.06 microm2 versus 1.12+/-0.01 microm2) for ASMA and SEM, respectively. When head length was evaluated, significant differences were found, being higher for SEM methodology (5.09+/-0.04 microm versus 4.29+/-0.03 microm). The curved and elongated spermatozoa head in eels means a problem for the ASMA system (Sperm Class Analyser), Morfo Version 1.1, Imagesp, Barcelona, Spain), causing an error in the length measurements. However, similar results were obtained by both techniques when spermatozoa head length was considered as the greater length between two points within the object (4.29+/-0.03 microm versus 4.31+/-0.04 microm for ASMA and SEM, respectively). In conclusion, this is one of the first applications of ASMA in fish and the first in this species, and confirms this system as a useful tool with wide applications in future fish spermatozoa studies. Width, perimeter and area could be used as parameters for the spermatozoa morphology evaluation, whereas the length requires a new programming of the Imagesp software.

Contents An artificial insemination programme was carried out to study the effect of factors such as depth of semen deposition, inseminator skill, farm, sire and expression of oestrus on pregnancy rate in Murciano–Granadina (MG) goats during non-breeding season and using frozen semen. Frozen-thawed semen from six males was applied by three technicians to inseminate a total of 551 goats in 17 farms distributed throughout the Mediterranean area of Spain. Pregnancy rate was determined at 6 weeks after insemination by transabdominal ecography. Overall pregnancy rate was 57%. Farm and depth of semen deposition affected pregnancy rate, whereas the sire and the technician had no effect. The deeper the semen was deposited in the genital tract, the higher was the rate of pregnancy obtained, being greater when the catheter reached the uterus. In spite of the relevant difference observed (48.2% vs 59.0%), pregnancy rate of females not coming into oestrus until 30 h after sponge removal was not significantly different, compared with those showing oestrus during the OD procedure. In conclusion, our field assay data on AI in MG goat with frozen-thawed semen showed that post-cervical insemination presented significantly greater pregnancy rate in comparison to when semen is deposited in the vagina or in the caudal part of the cervix.

The aim of this work was to evaluate the effect of different doses of eCG administered subcutaneously (0, 50 and 200 IU) and the hormonal induction of ovulation (GnRH or hCG) on embryo recovery and in vitro development of embryos post-vitrification in two selected lines of rabbit does. The two selected lines were line V (selected for the litter size at weaning) and line R (selected for growth rate). Administration of 200 IU of eCG significantly increased ovulation rate (19.2 +/- 1.2 versus 15.5 +/- 1.1 and 12.2 +/- 1.3, and the number of haemorrhagic follicles (13.8+/-1.6 versus 3.8+ /- 1.4 and 3.8 +/- 1.7), but significantly decreased recovery rate (28.8 +/- 6.3 versus 47.7 +/- 5.7 and 48.7 +/- 6.7, 200 IU versus 50 IU and 0 IU eCG, respectively), the number of normal embryos recovered per doe with at least one embryo (5.8 +/- 0.9 versus 8.2 +/- 0.9, 200 IU versus 50 IU eCG doses) and the in vitro development of embryos post-vitrification (51.9% versus 66.1%, 200 IU versus 50 IU eCG doses, respectively). Inducing ovulation with hCG significantly increased ovulation rate when compared with GnRH (17.3 +/- 0.8 versus 13.8+/-1.4), but no significant differences in embryo recovery and embryo development post-vitrification were observed between the two treatments. No significant differences were observed between the two selected lines in ovulation and recovery rates, the number of haemorrhagic follicles and the number of recovered embryos per doe. However, the post-vitrification in vitro rate of development was 59.7% for line R and 51.9% for line V (p < 0.05). It was concluded that the use of 50 IU of eCG subcutaneous with hCG or GnRH prior to embryo cryopreservation programmes in rabbits achieves the best results for embryo recovery, with the best development of recovered embryos post-vitrification.

This study was conducted to determine if different sperm characteristics correlate with the in vivo fertility of rabbit sperm. A total of 2,765 heterospermic inseminations were performed in commercial rabbitries using 50-pooled samples of fresh semen. Sperm motility and morphological evaluations were performed on each of the heterospermic pooled samples to asses the seminal quality, and the percentage of kindling rate (76.2%) and number of kits born alive (9.3) were recorded. Sperm motility parameters, assessed using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer, Microptic, Barcelona, Spain), were: average path velocity, curvilinear velocity, straight-line velocity, linearity, amplitude of lateral head displacement, beat cross-frequency, wobble and percentage of total motile spermatozoa. Morphological analyses included the percentage of sperm with a normal apical ridge, the percentage of sperm with cytoplasmatic droplets and the percentage of abnormal sperm. Significant correlations were observed between kindling rate and the percentage of total motile cells (r=0.31; P0.05), linearity index (r=-0.32; P0.05) and the percentage of abnormal sperm in the sample (r=-0.32; P0.05). Regression models including motility and the morphological parameters explained 45% of the variation in kindling rate. These results indicate that motility parameters, determined by CASA systems, in combination with sperm morphology analyses can provide some information about the fertilizing potential of rabbit sperm.