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African amphibian diversity remains underestimated with many cryptic lineages awaiting formal description. An important hotspot of amphibian diversification is the Guineo-Congolian rainforest in Central Africa, its richness attributable to present day and ancestral range fragmentation through geological barriers, habitat expansion and contraction, and the presence of steep ecological gradients. The charismatic Nectophryne tree toads present an interesting case study for diversification in this region. The two formally described species comprising this genus show nearly identical geographic distributions extending across most of the Guineo-Congolian rainforest, but show little morphological disparity. Both species harbour extensive genetic diversity warranting taxonomic revisions, and interestingly, when comparing the subclades within each, the two species show remarkably parallel diversification histories, both in terms of timing of phylogenetic splits and their geographic distributions. This indicates that common processes may have shaped the evolutionary history of these lineages.

Incubation gas atmosphere affects the development of in vitro produced embryos. In this study, there was examination of effects of two different oxygen (O2) tensions (5 % and 21 %) during in vitro maturation (M5 and M21) and/or fertilization (F5 and F21) on embryo production and quality in deer and sheep. There was assessment of the percentage of embryos with cell cleavage occurring, percentage that developed to the blastocyst stage, and analysis of the relative abundance of mRNA transcript for genes important for development to the blastocyst stage. The O2 tension treatment did not affect (P > 0.05) percentage cleavage or blastocyst development in either species. In sheep, there was a greater abundance of SHC1, GPX1, TP53, BAX and NRF1 mRNA transcript (P < 0.05) in M21 F5-derived embryos. In deer, there was a greater abundance of SOD2 mRNA transcript (P < 0.05) when oocytes had been matured under relatively lesser O2, regardless of the tension used during fertilization. There was a lesser abundance of SOX2 mRNA transcript (P < 0.05) in the M5F21 compared to the other three treatment groups. The AKR1B1 mRNA transcript was in greater abundance (P < 0.05) in M21 F21 as compared to M21 F5 and M5F21 group, and there was a greater abundance PLAC8 mRNA transcript (P < 0.05) in M21 F21, as compared to all other treatment groups. In conclusion, while O2 tension had no effect on developmental rates it did affect the relative abundance of mRNA transcript of multiple genes related to important cell functions during development., This research was supported by the Spanish Ministry of Economy and Competitiveness (AGL2013-48421-R and AGL2015-66145-R). MIC was supported by a Ministry of Economy and Competitiveness scholarship and PPF was supported by a University of Castilla-La Mancha scholarship.

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon PMSG Serum Gonadotrophin) on day 12, upon CIDR removal. They were then inseminated with one of each sperm treatment, at the following post-eCG intervals: I_1, 55:01–55:30 h; I_2, 55:31–56:00 h; I_3, 56:01–56:30 h; or, I_4, 56:31–57:00 h. Pregnancy rates were assessed at parturition. Average pregnancy rates were highest (P < 0.05) for Conventional samples (77.6%), but similar between YSS (49.8%) and Control sorted (51.3%) samples. However, when insemination interval was taken into account, pregnancy rates within the YSS group, pregnancy rates were 80 and 83.1% for I_1 and I_2, respectively were obtained. Notably, these rates were similar (P > 0.05) to the average pregnancy rates obtained with Conventional samples (77.6%). As expected, YSS sperm yielded 94% male offspring contrasting with the 57% males obtained with Conventional and Control sorted samples. Our findings support the importance of developing specific insemination timing protocols to improve pregnancy rates when using frozen-thawed sex-sorted sperm. These findings provide the foundation for further investigations in order to determine why the YSS sperm are able to fertilize the oocyte in a shorter period of time than the conventional samples., This work was supported by Spanish Ministry of Economy and Competitiveness (AGL2010-21487 and IPT-2012-1066-060000).

Sperm morphometry is one characteristic which may be useful in prediction of fertility and sperm freezability in a species. Knowledge of the sperm characteristics of the ejaculate and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. The aim of the current study was to provide a general description of the sperm from two falcon species (Peregrine falcon Falco peregrinus peregrinus/brookei and Gyrfalcon Falco rusticolus) including immature sperm, sperm head morphometric descriptors, and the existence of mature sperm subpopulations. Semen samples were collected by massage and voluntary false copulation and diluted with Lake and Ravie medium. Smears were prepared of the diluted samples, stained with Hemacolor, and subjected to: 1) morphological analysis (bright field optical microscopy), and 2) computerised morphometric analysis; each sperm head was measured for length, width, area and perimeter. In addition, in the Gyrfalcon, pooled semen was frozen in pellets using DMA as a cryoprotectant and the analyses repeated after thawing. The mean percentage of immature sperm (spermatocytes and spermatids) was similarly high in all species/subspecies: Brookei Peregrine falcon (F. p. brookei) 55.5%, European Peregrine falcon (F. p. peregrinus) 65.5% and Gyrfalcon 64.7%. Clustering analyses identified four subpopulations of mature spermatozoa with different morphometric characteristics (P, This work was funded by INIA grant RZ2012-00013-C02., and GAMESA grant Cryoraptor.

Semen collection for cryopreservation is a key step for small ruminant conservation programs. While in these species semen is mainly collected via artificial vagina (AV), electroejaculation (EE) provides a viable alternative for untrained males. Herein we investigated the effect of semen collection method on post-thaw sperm quality by comparing two small ruminant species, sheep and goats. Semen from Blanca-Celtibérica bucks and Manchega rams was collected by AV and EE on the same day and cryopreserved using a standard protocol. At thawing, sperm motion parameters were evaluated by CASA, whereas membrane stability (YO-PRO-1), sperm viability (propidium iodide, PI) and mitochondrial activity (Mitotracker Deep Red) were analyzed using flow cytometry. The semen collection method negatively affected post-thaw sperm quality in bucks but not in rams. Thus, in bucks, post-thaw sperm motility was higher for samples collected by AV as compared to those obtained via EE. Similarly, post-thaw sperm parameters evaluated by flow cytometry were worse for buck samples collected by EE than those collected by AV in the same species, or than ram samples regardless of collection method. These results suggest that ovine and caprine spermatozoa have a different response to the cryopreservation process depending upon the semen collection method used. We hypothesize that the EE procedure may lead to changes in the composition of the ejaculate in bucks that would make spermatozoa more susceptible to the cryopreservation process, whereas this procedure would have had no effect on ram spermatozoa. This assumption requires further investigation., This research was supported by INIA (RZ2008-00009-CO2). The authors thank AGRACE, their farmers and CERSYRA staff for their collaboration in the collection of the samples. Manuel Ramón is supported by the DOC-INIA program.

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1 and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation., This research was supported by Education and Science Council (JCCM [Education and Science Council in spanish], Spain) grant (PAI09-0006-3806). E. Del Olmo is supported by a grant for training of technical personnel associated with PTA2008-0858-P project (Spanish ministry of Science and Innovation, MICINN). Manuel Ramón is supported by the DOC-INIA (Doctoral-National Institute of Agricultural Research) program.

Little is known about the relationship between feed intake behaviour and cholesterol levels in humans. This can be attributed to the fact that feed intake behaviour in humans is difficult to assess. The relationships between feed intake, feed efficiency and feed intake behaviour, and cholesterol and triglyceride levels were investigated at an average age of 187 days, in a pig model consisting of 202 Duroc barrows. Feed intake and feed intake behaviour were recorded individually and daily by means of an electronic identification system. Animals with high levels of total cholesterol also had high levels of high-density lipoprotein (HDL), low-density lipoprotein (LDL) cholesterol and triglycerides. Animals with high levels of HDL also had high levels of LDL and triglycerides, and animals with high levels of LDL also had high levels of triglycerides. Animals with higher BW, higher backfat thickness, higher BW gain, higher gain of backfat deposition, higher feed intake, higher residual feed intake (RFI) and higher feed intake rate had higher levels of total, HDL and LDL plasma cholesterol. Results indicate that the relationship between feed intake and cholesterol levels is a long-term relationship, while the relationship between RFI and cholesterol levels is more of a short-term nature. The relationship between intake rate and cholesterol plasma levels disappeared after correction for the amount of feed consumed. Results indicate that feed intake independent of metabolic BW, growth and fatness, i.e. 'RFI', was positively correlated with cholesterol plasma levels. This suggests that eating food over and above the maintenance and growth requirements constitutes a health risk independent of the level of fatness.

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin–hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin–hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen–thawed ram spermatozoa. Only the medium supplemented with heparin–hypotaurine–BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin–hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa., M. Ramón was supported by the Research Recruitment Program from the National Institute for Agricultural and Food Research program.

Single Layer Centrifugation is a useful technique to select sperm with good quality. The use of selection methods such as Androcoll could become an important tool to improve the quality of sperm samples and therefore to improve other artificial reproductive techniques such as sperm sex sorting, in vitro fertilization or AI. The aim of this study was to evaluate the effect of a Single Layer Centrifugation with Androcoll-S on the sperm quality of red deer sperm samples of two different origins, electroejaculated samples and epididymal samples obtained post-mortem, after thawing and after an incubation for 2. h at 37. °C. Sperm motility, viability, membrane permeability, mitochondrial activity, acrosomal status and DNA fragmentation were determined for all samples. The samples selected by Androcoll-S showed an improvement in sperm kinematics compared to unselected samples after thawing and after incubation. The same effect was observed in parameters such as viability, mitochondrial activity or acrosomal status which were improved after the selection. In contrast, no difference was found in DNA fragmentation between selected and unselected samples within the same sperm type. We conclude that sperm selection by SLC with Androcoll-S after thawing for red deer sperm of both types is a suitable technique that allows sperm quality in both types of sperm samples to be improved, thereby improving other assisted reproductive techniques. Further studies (IVF and in vivo fertilization) are required to determine whether this improvement can increase fertility, as has been shown for other species., This work was supported by Spanish Ministry of Science and Innovation (MICINN) with the project (AGL2010-21487) and by Junta de Castilla y La Mancha (PRE123/2010) and by the Veterinary Faculty at SLU (JM Morrell).

The present study aimed to examine the behavior of ram spermatozoa subjected to a vitrification process in free-egg yolk diluents in relation with conventional diluents and cryopreservation protocol used in this species. Previously it was investigated the toxicity of cryoprotectants, sucrose and glycerol, based on different concentrations (sucrose at 0.03. M, 0.05. M, 0.15. M and 0.25. M; and glycerol at 3%, 7%, 14% and 18%) compared to a commercial extender (Biladyl® with 20% egg yolk and 7% glyerol). Cryoprotectants which reported less toxicity were chosen to perform the vitrification and results were compared with the conventional cryopreservation. Semen from three rams was collected by electroejaculation. The sperm evaluation was carried out at 0, 2 and 4 h through the incubation time at 37 °C for the experiment of toxicity and, at thawing when cryopreservation was performed. The sperm quality throughout the incubation time always resulted lower (P ≤ 0.05) for the free-egg yolk diluents in relation to Biladyl® (control), obtaining the lowest values of sperm quality with the highest concentrations of sucrose and glycerol. The vitrification was carried out with combinations of sucrose and glycerol (sucrose at 0.03 and 0.05. M with 3% and 7% of glycerol, respectively) and with Biladyl® (at different sperm concentrations). The vitrification decreased drastically (P ≤ 0.05) the sperm quality when combinations of sucrose and glycerol were used. Nevertheless, the sperm samples vitrified with Biladyl® at the lowest sperm concentration showed acceptable values of viability, acrosome integrity and DFI, although the sperm motility was strongly decreased. In conclusion, the use of vitrification with diluents based on combinations of sucrose and glycerol did not work for semen cryopreservation of ram. Promising results were obtained when diluents with egg yolk were used in the vitrification procedure, although more studies are necessary to improve this technique and the use of diluents without egg yolk., M Ramón is supported by the DOC-INIA program.

OBJECTIVE: The COPD Assessment Test (CAT) has been proposed for assessing health status in COPD, but little is known about its longitudinal changes. The objective of this study was to evaluate 1-year CAT variability in patients with stable COPD and to relate its variations to changes in other disease markers. METHODS: We evaluated the following variables in smokers with and without COPD at baseline and aft er 1 year: CAT score, age, sex, smoking status, pack-year history, BMI, modified Medical Research Council (mMRC) scale, 6-min walk distance (6MWD), lung function, BODE (BMI, obstruction, dyspnea, exercise capacity) index, hospital admissions, Hospital and Depression Scale, and the Charlson comorbidity index. In patients with COPD, we explored the association of CAT scores and 1-year changes in the studied parameters. R ESULTS: A total of 824 smokers with COPD and 126 without COPD were evaluated at baseline and 441 smokers with COPD and 66 without COPD 1 year later. At 1 year, CAT scores for patients with COPD were similar ( ± 4 points) in 56%, higher in 27%, and lower in 17%. Of note, mMRC scale scores were similar ( ± 1 point) in 46% of patients, worse in 36%, and better in 18% at 1 year. One-year CAT changes were best predicted by changes in mMRC scale scores ( β -coefficient, 0.47; P

Conservation of genetic resources from endangered breeds may be conducted through germinal banks. Preservation of healthy samples is paramount to avoid preserving pathogens shed with germinal products. The black variety of Manchega sheep (BMS), and endangered breed endemic to south-central Spain, is the subject of a conservation program; a germinal bank has been recently established. However, several pathogens circulating in BMS flocks may be shed with semen and threaten BMS preservation. Therefore, we investigated the sanitary status of BMS flocks and semen samples from 4 of the 17 flocks in which this variety is bred worldwide. A serological screening for Maedi-Visna virus, bluetongue virus, Pestivirus spp., Brucella spp., Coxiella burnetii, Chlamydophila spp., Mycobacterium tuberculosis complex, Mycobacterium avium paratuberculosis, Anaplasma spp., Mycoplasma agalactiae, Toxoplasma gondii and Neospora caninum was performed to assess for pathogens potentially shed by semen. Semen samples from 11 of the 35 BMS rams and 4 samples from coexisting rams of the white variety (WMS) were analyzed by PCR to detect Maedi-Visna virus, C. burnetii, Anaplasma marginale, Anaplasma phagocytophilum and T. gondii. Maedi-Visna virus RNA was detected in 3 semen samples (2 BMS and 1 WMS) while C. burnetii DNA was detected in 3 samples from WMS rams. Pathogens that can be transmitted by semen were present in BMS flocks, and Maedi-Visna virus and C. burnetii showed the highest potential for transmission by artificial insemination. Our results point to the need of testing semen samples kept for conservation purposes of BMS before using them for artificial insemination., This work was supported by the Spanish Ministry for the Economy and Competitiveness-MINECO (Project RZ2010-00006-C02-01). F. Ruiz-Fons acknowledges funding by MINECO through a ‘Juan de la Cierva’ contract.

The present study aims to examine the predictive value of some sperm parameters on male fertility. Semen samples from six Manchega rams were collected and cryopreserved. Sperm quality was assessed after thawing and after 2 h of incubation, either in the freezing extender (37 °C) or after dilution in Synthetic Oviductal Fluid (SOF) (38 °C, 5% CO2), attempting to mimic the physiological conditions of the female reproductive tract. The following sperm parameters were evaluated: motility and kinetic parameters by computer-assisted semen analyzer (CASA), and sperm viability (propidium iodide), mitochondrial membrane potential (JC-1), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), and intracellular calcium (fluo-3) by flow cytometry. Results showed no significant differences between incubation media neither after thawing nor after incubation. There were no significant correlations between fertility and sperm parameters assessed by flow cytometry. However, after incubation in the freezing extender, sperm samples from males with poor fertility yielded less linearity and velocity (P < 0.05) as indicated by motility parameters analyzed by CASA. These results indicate that kinematic sperm motility parameters evaluation by CASA might be useful to identify samples with poor fertility., This research was supported by Education and Science Council (JCCM, Spain) grant (PAI09-0006-3806). Enrique del Olmo is supported by a grant for training of technical personnel associated with the project PTA2008-0858-P (Spanish Ministry of Science and Innovation, MICINN). Manuel Ramón is supported by the DOC-INIA program, and Felipe Martínez-Pastor is supported by the Ramón y Cajal program (RYC-2008-02560, Spanish Ministry of Science and Innovation, MICINN)

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl ®, Andromed ® and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl ® and Andromed ® resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl ® or Andromed ® are used as freezing extenders., This research was supported by INIA (RZ2008-00009-CO2). The authors thank AGRACE, their farmers and CERSYRA staff for their collaboration in the collection of the samples. Pilar Jiménez-Rabadán is recipient of scholarship from INIA. Manuel Ramón is supported by the DOC-INIA program (Ministry of Science and Innovation of Spain).

The effect of organic species on a solar-driven photo-Fenton treatment of a mixture of pesticides (methyl-oxydemethon, methidathion, carbaryl and dimethoate) has been studied in this paper. Triethoxyisododecyl alcohol, acetophenone and ethylenediaminetetraacetic acid (EDTA) have been used as examples of surfactants, solvents and complexing agents, respectively. An inhibitory effect on mineralization as well as on the elimination of the pesticides was observed in the case of the aliphatic surfactants, most probably due to the competition between the pesticides and the added organic matter for reaction with the relatively unselective hydroxyl radical. A methodology combining chemical analyses and bioassays was tested in order to explore the applicability of coupling a photo-Fenton process with a biological treatment in the presence of the surfactant. Despite the complexity of the mixture under study, a reliable monitoring of the process was accomplished; the biocompatibility of the mixture was enhanced and the optimal irradiation intensity was achieved just after complete removal of the pesticides. © 2011 Elsevier B.V., We want to acknowledge the financial support of Spanish Ministerio de Ciencia y Tecnologia (Project CTQ2009-13459-C05-03), Generalitat Valenciana (Project GVPRE/2008/369), and Universidad Politecnica de Valencia (PAID 003-581).

[EN] The role of hydroxyl radical in different solar photocatalytic processes, namely photo-Fenton, titanium dioxide-based oxidation and organic photocatalysis (triphenylpyrylium) has been investigated. Using a methodology based on the flash photolytic generation of OH. from N-hydroxypyridine-2(1H)-thione, followed by t-stilbene trapping, the reaction rate constants of different pesticides (dimethoate, methidathion, alachlor and pyrimethanyl) with this reactive oxygen species in deaerated acetonitrile have been determined. The results obtained under photo-Fenton conditions are in reasonable agreement with the estimated rate constants; hence the assumption that the photo-Fenton reaction mainly involves participation of the hydroxyl radical seems plausible. The oxidation pattern using titanium dioxide was also investigated; however, under these conditions no clear correlation could be found due to participation of an alternative electron transfer mechanism.Finally, pyrimethanil was the most reactive pesticide when photodegradation was carried out in the presence of TPP, in spite of its low reactivity towards hydroxyl radical. Laser flash photolysis experiments evidenced that the reaction occurs via photoinduced electron transfer within a ground state complex; this is in good correlation with the relative amount of pyranyl radical formation. © 2011 Elsevier B.V., Financial support from the Spanish Government (Projects CTQ2009-13699 and CTQ2009-13459-C05-03) is gratefully acknowledged.

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter2/[4 × π × area]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds., This work was supported by the Education and Science Council of Junta de Comunidades de Castilla-La Mancha (PBC-05-008). A. Maroto-Morales and O. García-Álvarez were recipients of scholarships from Junta de Comunidades de Castilla-La Mancha and INIA, respectively. M.C. Esteso was supported by the Juan de la Cierva program, and F. Martínez-Pastor was supported by the Ramón y Cajal program (Spanish Ministry of Science and Innovation).

Los quistes del rafe medio perineal son lesiones congénitaspoco habituales en la práctica clínica diaria, siendoparticularmente infrecuentes en la infancia. Se presentandos casos pediátricos afectados de dicha lesión, en los que se requirió escisión quirúrgica. Revisamos aspectosembriológicos, diagnósticos y opciones terapéuticasque pueden ser de utilidad al pediatra o cirujano pediátricoante la presencia de un caso afectado de dichaenfermedad. : Median raphe cysts of the perineum are uncommoncongenital lesions. Congenital lesions rarely encounteredin daily clinical practice. Their diagnosis in childhood isparticularly rare. We report two children with these cystsrequiring surgical removal. We review the embryological, diagnostic and therapeutic aspects of these cysts, whichcould be of interest to pediatricians and pediatric surgeonswhen faced with this pathology in children.

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower., This work was supported by the Education and Science Council (PBI-05-011), by the Spanish Ministry of Education and Science (RZ2006-00006-C3), and by the Agriculture Council (PREG-05-004) of Junta de Comunidades de Castilla-La Mancha (JCCM). Olga García-Álvarez and Alejandro Maroto Morales were recipients of scholarships from INIA and JCCM, respectively. Felipe Martínez-Pastor, María Rocío Fernández-Santos, and Milagros C. Esteso were supported by the Juan de la Cierva program from the Spanish Ministry of Education and Science.

A heterologous in vitro fertilization (IVF) test using calf oocytes with zona pellucida was employed to assess the fertility of thawed ram sperm samples. Six males with significant differences in fertility (P = 0.003) were used. The males were classified as having high fertility (≥42%) and low fertility (≤41%). Male fertility was not influenced by number of inseminated ewes (P = 0.584), insemination technician (P = 0.156), insemination date (P = 0.323) or farm (P = 0.207). Thawed sperm samples were employed to assess several sperm parameters for each male: motility, acrosomal integrity, viability, membrane stability, membrane phospholipid disorder, mitochondrial membrane potential and chromatin stability. These samples were used to carry out a heterologous in vitro fertilization. In vitro-matured calf oocytes (n = 716) were inseminated with thawed ram semen and in vitro cultured for 40 h. Overall, at thawing, variability among males respect to sperm quality was high. Despite this variability, there were not differences (P < 0.05) between fertility groups. Yield of hybrid embryos ranged from 31 to 59% between males. There were not differences between males (P = 0.340). However, there were differences between fertility groups (high fertility: 55%; low fertility: 39%; P = 0.020). Multiple regression analysis showed that the heterologous in vitro fertility was the only predictive parameter for in vivo male fertility. Correlation between both parameters was fair (r2 = 0.760; P = 0.025). These results indicate that heterologous in vitro fertilization tests can be useful to predict the fertility of ram spermatozoa using calf oocytes with intact-zona pellucida., This work was supported by Instituto de Investigación y Tecnología Agraria y Alimentaria (INIA) (Project RTA 2005-00091-CO2) and by the Education and Science Council of Junta de Comunidades de Castilla-La Mancha (PBI-05-011). The authors thank to Frimancha Industrias Cárnicas S.A. for their collaboration in the collection of the samples used in this work. Olga García-Álvarez and Alejandro Maroto Morales were recipient of a scholarships from INIA and Junta de Comunidades de Castilla-La Mancha, respectively. Felipe Martínez-Pastor, M. Rocío Fernández-Santos and Milagros C. Esteso were supported by the Juan de la Cierva programme from Spanish Ministry of Science and Innovation.

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes–Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 °C. Then, they were diluted down to 100 × 106 sperm/mL and frozen at −20 °C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample., This study has been supported in part by Junta de Castilla y León (AB29) and Junta de Castilla-La Mancha (PREG-01-0006).

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of ∼200 × 106 spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P < 0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 °C (slow: 0.23 °C/min; rapid: 4.2 °C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P < 0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8 ± 1.4 versus 46.2 ± 1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P < 0.05), with the high values after thawing found with the use of the rapid protocol (64.5 ± 1.4 versus 59.9 ± 1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability., This study was sponsored by Grant AGL2000-0671 from the Spanish Ministry of Science and Technology. M.R. Fernández-Santos was recipient of a scholarship from the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha (Spain).

The objective of this study was to evaluate the effect of the thawing procedure on deer semen freezability. Frozen semen from the Genetic Resource Bank (GRB) of the Zoological Park of Buenos Aires (Argentina) was used. Seven mature stags (two red deer, two Père David’s deer and three fallow deer) were used as semen donors. Semen was diluted with a TRIS-egg yolk medium, packed in 0.25 ml straws and frozen in nitrogen vapour. For thawing, the frozen straws were subjected to the following procedures: (I) 70 °C, 5 s; (II) 50 °C, 8 s and (III) 37 °C, 10 s. Freeze-thaw motility percentage (FMP) and spermatozoa rating (FMR) were determined subjectively. Viability and acrosome integrity (NAR) were also assessed and the hypo-osmotic swelling test (HOST) was used to assess membrane integrity. Freeze-thaw motility percentage, FMR and NAR were assessed after an incubation of 1 h in citrate-yolk at 42 °C, and FMP and FMR after 2 h of incubation under the same conditions. The thawing procedure did not have an effect on the seminal characteristics evaluated immediately after this process. However, differences in FMP after 2 h of incubation (P, This study was sponsored by research funds of the Ministerio Español de Ciencia y Tecnologı́a (AGL2000-0671) and by Grant 190/PA-35 from the Consejerı́a de Agricultura y Medio Ambiente of the Junta de Comunidades de Castilla-La Mancha (Spain). The authors thank ALGAR SA for lending us the Père David’s deer stags, and Ma Dolores Pérez-Guzmán, for her help during the analysis of the data. Ana J. Soler is the recipient of a scholarship from the Consejerı́a de Ciencia y Tecnologı́a de la Junta de Comunidades de Castilla-La Mancha.

The non-oxidative dehydrogenation of butane over a Cr2O3/Al2O3 catalyst has been studied in a fluidized-bed reactor with separate butane and oxygen feeds. Under suitable conditions, feed segregation allows the stable creation of separated reaction and catalyst regeneration (by coke burning) zones. The effect of the main operating variables (temperature, oxygen to hydrocarbon ratio, and relative velocity) in the process performance was studied.

Publisher Summary The study of the binding of hormones to specific receptor site and the biological effect of this binding has supposed a great development in biology. Intracellular cyclic nucleotides are generally to be intermediates in the thyrotropic action of thyroid-stimulating hormone (TSH); its levels might control cell function and metabolism in the thyroid gland. Reports indicate that the immunoglobulins of Graves' disease block the TSH binding with its receptor and elicit the same effect, reflecting an stimulus on the adenylate cyclase (AC) and an increase of the cyclic 3',5'-adenosine monophosphate (cAMP). Changes has been observed in the cAMP and cyclic 3',5' guanosine monophosphate (cGMP) concentrations in reverse directions provoked by the hormonal stimulation. This phenomenon has been studied in Graves' disease and it is assumed that the same would be true for thyroid gland. The common intracellular pathway for the TSH includes activation of adenylate cyclase at the inner surface of the membrane, elevation of the intracellular concentration of cAMP, and activation of the cyclic AMP-dependent protein-kinase. The immunoglobulins of Graves' disease patients are capable of simulating TSH.

Publisher Summary Several investigators have demonstrated that t hyroid-stimulating hormone (TSH) activates cyclic adenylate cyclase in the thyroid gland, which synthesizes 3'-5' adenosine monophosphate (cAMP) from adenosine triphosphate. The immunoglobulins in Graves' disease can stimulate the thyroid adenylate cyclase system through their interaction with the TSH receptor. The experience with various assays procedures offered the hypothesis that thyroid-stimulating antibody (TSAb) is an autoantibody to human thyroid antigen. A sensitive technique for the measurement of TSAb has been developed, which is based on an increase in the concentration of cAMP in human thyroid slices incubated with the test material. The activation pattern of thyroid is observed after exposure either with sera or immunoglobulins. Data indicates that sera can be used instead of immunoglobulins in the assays of cAMP generations. A regulatory short loop inhibitory effect of thyroid hormones on the thyroid gland has been proposed.