Determination of the appropriate concentration of sodium alginate used for in vitro culture of cat preantral follicles in a serum-free medium containing FSH, EGF and IGF-I.

Culture of domestic cat preantral follicles can be a suitable technology to assist oocyte conservation strategies in the family Felidae. This research was aimed to comparatively analyse cat preantral follicular development of follicles directly seeded on growth surface or encapsulated in 0.5 or 1% of sodium alginate in a serum-free medium containing FSH, EGF and IGF-I. Preantral follicles were isolated from cat ovarian cortical tissue after ovariectomy. Alginate was dissolved at 0.5 or 1% in PBS. Follicles, 4 per well, with 0% (G-0%), 0.5% (G-0.5%) or 1% (G-1%) of sodium alginate were cultured in M199 with FSH (100 ng/mL), EGF (100 ng/mL) and IGF-I (100 ng/mL) for 7 days at 37°C, 5% CO2 and 99% humidity. Culture medium was replaced every 48 h and samples were stored at -20°C until ELISA of steroid hormones. Morphometric evaluation of follicles was performed every 24 h. G-0% follicles showed granulosa cell migration away from the oocyte and disrupted morphology, whereby they reached apparently larger diameters (203.70 ± 5.82 µm; p < .05) than G-0.5% and G-1% follicles (157.89 ± 8.47 µm and 95.23 ± 1.67 µm, respectively) which maintained three-dimensional organization, being larger in G-0.5% than in G-1% (p < .05). G-0.5% follicles attained the multi-layer preantral follicle stage on day 7 of culture, whereas G-1% follicles underwent progressive atresia. On day 6, steroid concentrations were higher (p < .05) in G-0% than in G-1%: 60 ± 19 vs 0.88 ± 0.32 pg/mL oestradiol; 2.6 ± 0.84 vs 0.04 ± 0.02 ng/mL progesterone; 1.3 ± 0.22 vs 0.61 ± 0.04 ng/mL testosterone and 1.6 ± 0.54 vs 0.22 ± 0.07 ng/mL androstenedione respectively. Steroid concentrations in G-0.5% were comprised between those of G-0% and G-1% (p > .05). In conclusion, two-layer cat preantral follicles encapsulated in 0.5% alginate cultured in medium containing FSH, EGF and IGF-I can develop up to the multi-layer preantral stage in 7 days of culture, whereas follicles directly seeded on growth surface or encapsulated in 1% alginate lost their three-dimensional organization, and experienced regression with compromised steroidogenesis, respectively.

Comparación entre angiografía convencional y mediante OCT en complicaciones isquémicas secundarias a vasculitis / Comparison between conventional angiography and OCT angiography in ischaemic complications secondary to vasculitis

No disponible

Análisis y seguimiento con angiografía por tomografía de coherencia óptica de neovascularización coroidea tipo 1 en degeneración macular tras tratamiento antiangiogénico / Analysis and follow-up of type 1 choroidal neovascularisation with optical coherence tomography-angiography after antiangiogenic treatment

Objetivo: Describir las características de la neovascularización coroidea (NVC) tipo 1 en pacientes con degeneración macular asociada a la edad (DMAE), utilizando la angiografía por tomografía de coherencia óptica (A-OCT) secuencialmente durante el transcurso de un protocolo estándar de 3 inyecciones intravítreas de fármaco anti-VEGF. Métodos: Seis ojos con DMAE neovascular no tratados previamente fueron incluidos. Se obtuvieron imágenes por A-OCT empleando AngioPlex Cirrus HD-OCT 5000 (Carl Zeiss Meditec, Inc., Dublin, EE. UU.) y DRI OCT Triton SS-OCT Angio (Topcon, Medical Systems, Inc. Oakland, NJ, EE. UU.). El área estudiada comprende un escáner macular de 3×3mm. Diferentes patrones morfológicos y aspectos cuantificables de las membranas neovasculares han sido evaluados con imágenes en proyección en face, que fueron tomadas en distintos tiempos del seguimiento de los pacientes. Resultados: El grado de respuesta al tratamiento fue estimado empleando criterios de actividad de NVC para A-OCT. Puntuaciones más altas en los ítems de actividad antes del tratamiento resultaron en mayores reducciones del área de las membranas. Los resultados finales de reducción de área oscilaron entre el 83,5 y el 1,4%. Las A-OCT realizadas a la semana de tratamiento revelaron los mayores porcentajes de reducción. Conclusiones: La A-OCT ofrece la posibilidad de analizar en profundidad las características morfológicas y estructurales en NVC de tipo 1. Los criterios de actividad permiten guiar decisiones terapéuticas y evaluar la respuesta al tratamiento. Con esta técnica puede obtenerse información útil tanto cualitativa como cuantitativa. Sin embargo, son necesarios avances en el desarrollo del software y en investigación para poder definir el papel de esta herramienta en la práctica diaria (AU)

Aim: To describe the characteristics of type 1 choroidal neovascularisation (CNV) in age-related macular degeneration (ARMD) using two different optical coherence tomography angiography (OCT-A) devices sequentially during a standard protocol of three intravitreal injections of an anti-vascular endothelial growth factor (anti-VEGF). Methods: The study included 6 eyes with naïve neovascular ARMD. Macular OCT-A images were acquired using AngioPlex Cirrus HD-OCT 5000 (Carl Zeiss Meditec, Inc., Dublin, USA) and DRI OCT Triton SS-OCT Angio (Topcon, Medical Systems, Inc. Oakland, NJ, USA). The macular OCT-A scan covered an area of 3×3mm. Distinct morphological patterns and quantifiable features of the neovascular membranes were studied on en face projection images, which were taken at different stages of the follow-up. Results: Treatment response could be estimated using the OCT-A criteria of CNV activity. Higher activity scores before treatment resulted in a greater decrease in the membrane area. The estimated net decline in area ranged from 83.5% to 1.4%. The OCT-A performed one-week after treatment revealed the greatest area reductions. Conclusions: OCT-A provides new possibilities for the non-invasive assessment of features of neovascular networks and CNV structural morphology. Newly described activity criteria can also guide therapeutic decisions, and help in evaluating responses. Quantitative and qualitative information can be provided with this technique. However, further software development and future investigation are essential to define the role of this tool on a daily basis (AU)

Analysis and follow-up of type 1 choroidal neovascularisation with optical coherence tomography-angiography after antiangiogenic treatment. / Análisis y seguimiento con angiografía por tomografía de coherencia óptica de neovascularización coroidea tipo 1 en degeneración macular tras tratamiento antiangiogénico.

AIM: To describe the characteristics of type 1 choroidal neovascularisation (CNV) in age-related macular degeneration (ARMD) using two different optical coherence tomography angiography (OCT-A) devices sequentially during a standard protocol of three intravitreal injections of an anti-vascular endothelial growth factor (anti-VEGF). METHODS: The study included 6 eyes with naïve neovascular ARMD. Macular OCT-A images were acquired using AngioPlex Cirrus HD-OCT 5000 (Carl Zeiss Meditec, Inc., Dublin, USA) and DRI OCT Triton SS-OCT Angio (Topcon, Medical Systems, Inc. Oakland, NJ, USA). The macular OCT-A scan covered an area of 3×3mm. Distinct morphological patterns and quantifiable features of the neovascular membranes were studied on en face projection images, which were taken at different stages of the follow-up. RESULTS: Treatment response could be estimated using the OCT-A criteria of CNV activity. Higher activity scores before treatment resulted in a greater decrease in the membrane area. The estimated net decline in area ranged from 83.5% to 1.4%. The OCT-A performed one-week after treatment revealed the greatest area reductions. CONCLUSIONS: OCT-A provides new possibilities for the non-invasive assessment of features of neovascular networks and CNV structural morphology. Newly described activity criteria can also guide therapeutic decisions, and help in evaluating responses. Quantitative and qualitative information can be provided with this technique. However, further software development and future investigation are essential to define the role of this tool on a daily basis.

[Azathioprine-induced hepatotoxicity in a patient with Crohn's disease]. / Hepatotoxicidad inducida por azatioprina en paciente con enfermedad de Crohn.

Immunosuppressive drugs are among the pharmacological groups with the most theoretical potential to induce adverse reactions, including hepatic reactions. We report the case of a 35-year-old patient with Crohn's disease treated with azathioprine for steroid dependence which, after a hepatic biopsy, was diagnosed with nodular regenerative hyperplasia secondary to this treatment.

[Neurocysticercosis in Murcia (Spain)]. / La neurocisticercosis en Murcia.

INTRODUCTION: Neurocysticercosis (NC) is the most prevalent parasitic infection in the CNS. Its frequency in our environment has increased with migratory movements. The clinical and demographic characteristics of patients with NC attended in a third level hospital in an area with a high prevalence of immigrant have been analyzed. PATIENTS AND METHODS: A retrospective study was done of the patients registered by the Coding Service of Virgen de la Arrixaca Hospital (January 2996 to December 2009), analyzing all the clinical histories of patients discharged with the primary or secondary diagnosis of neurocysticercosis. The most relevant epidemiological, diagnostic and therapeutic data were analyzed. RESULTS: 35 patients were included in the study. Of these, 24 were men, all coming from Latin America. The most prevalent onset was seizure episode (27 patients). The number of cases per year began to increase after 2002. Although only 21 of the cases had criteria to begin medical treatment with albendazole, it had been prescribed to 29 patients. CONCLUSION: In Murcia, NC is mainly an imported disease. An increase in the annual incidence has been observed since 2002, parallel to the rise in the number of immigrants. Approximately 25% of the patients received treatment in spite of not having antiparasitic treatment indication.

La neurocisticercosis en Murcia / Neurocysticercosis in Murcia (Spain)

Introducción. La neurocisticercosis (NC) es la enfermedad parasitaria más frecuente del sistema nervioso central. Su frecuencia en nuestro medio ha aumentado en relación con los movimientos migratorios. Se analizan las características clínicas y demográficas de los casos de NC valorados en una zona con una alta tasa de población inmigrante. Pacientes y métodos. Se revisó de forma retrospectiva a los pacientes registrados por el servicio de codificación de nuestro hospital (enero 1996 a diciembre 2009), analizando todas las historias clínicas de los pacientes dados de alta con el diagnóstico primario o secundario de NC. Se analizaron los datos epidemiológicos, diagnósticos y terapéuticos más relevantes. Resultados. Se evaluó a 35 pacientes; 24 eran varones, todos procedían de América del Sur y 27 debutaron en forma de crisis comiciales. El número de casos por año aumentó a partir de 2002. Aunque sólo 21 de los casos tenían criterios para iniciar tratamiento con albendazol, lo recibieron 29 pacientes. Conclusión. En nuestra región, la NC es una enfermedad fundamentalmente importada. Se observa un incremento del número de casos anuales a partir del año 2002 coincidiendo con un aumento de la inmigración. Cerca del 25% de los pacientes recibió tratamiento antiparasitario sin indicación(AU)

Introduction. Neurocysticercosis (NC) is the most prevalent parasitic infection in the CNS. Its frequency in our environment has increased with migratory movements. The clinical and demographic characteristics of patients with NC attended in a third level hospital in an area with a high prevalence of immigrant have been analyzed. Patients and methods. A retrospective study was done of the patients registered by the Coding Service of Virgen de la Arrixaca Hospital (January 2996 to December 2009), analyzing all the clinical histories of patients discharged with the primary or secondary diagnosis of neurocysticercosis. The most relevant epidemiological, diagnostic and therapeutic data were analyzed. Results. 35 patients were included in the study. Of these, 24 were men, all coming from Latin America. The most prevalent onset was seizure episode (27 patients). The number of cases per year began to increase after 2002. Although only 21 of the cases had criteria to begin medical treatment with albendazole, it had been prescribed to 29 patients. Conclusion. In Murcia, NC is mainly an imported disease. An increase in the annual incidence has been observed since 2002, parallel to the rise in the number of immigrants. Approximately 25% of the patients received treatment in spite of not having antiparasitic treatment indication(AU)

Seasonal endocrine changes and breeding activity in mediterranean wild ruminants.

The methods of assisted reproduction commonly used in domestic animals are currently being applied to non-domestic species. This is because of the limitation of maintaining the genetic variability in the wild, as it occurs in wild ruminants of the Mediterranean Basin. Despite the apparent progress of the technology, success of the offspring to grow and become healthy adult individuals has remained low in wild species. Difficulties usually arise from insufficient information about basic reproductive biology, such as the seasonal changes in ovarian and testicular activity. Directional adaptive evolution involves development of speciespecific physiological reproductive patterns to cope with various environmental factors. Thus, species originating and living at the same latitude display different reproductive strategies to entrain the breeding activity at an optimal time of the year. The aim of this paper was to present current knowledge on reproductive physiology of Mediterranean wild ruminants as a basic prerequisite for the successful use of assisted reproduction methods. Special emphasis is given to seasonal endocrine changes, ovarian cycles and testicular activity of Iberian wild ruminants, together with the role of social interactions on the regulation of these events.

Influence of age on the relationship between annual changes in horn growth rate and prolactin secretion in the European mouflon (Ovis gmelini musimon).

Annual variations in the growth of horns, and their correlation with seasonal changes of testicular size, and prolactin (PRL) and melatonin secretion were monitored in six pubertal mouflon rams living in their original latitude (40 degrees N). Mouflons born and maintained under captive conditions were classified in two age classes: sub-adult (2 years; n=3) and adult (> or =3 years; n=3). The rate of horn growth was greater (P <0.001) in sub-adult than in adult mouflon rams. Horn growth was influenced by season in both adult and sub-adult mouflons (P <0.05) with largest monthly growth occurring in spring and summer. Seasonal variations of plasma PRL concentrations were correlated with horn growth in adult, but not in sub-adult mouflon rams. The rate of horn growth was inversely correlated with testicular size (r=-0.5, P=0.07). Seasonal changes in the amplitude of the daily melatonin rhythm in solstices and equinoxes were observed, which were not correlated with variations in the rate of horn growth. These results provide support for a possible role of PRL in the control of growth of horns in the adult mouflon.

Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle.

The actions of prolactin (PRL) on target cells depend on the type of prolactin receptor (PRLr) predominantly expressed, particularly whether the long PRLr isoform is expressed. The aims of this study were to determine the cellular localization and the changes in expression of long and short PRLr isoforms in sheep ovary throughout the estrous cycle. Long and short PRLrs were localized mostly in the same ovarian cells. Maximum signal intensity, particularly for long PRLrs, was found in stromal cells surrounding primordial and primary follicles, and, for both PRLrs, in granulosa cells of preantral follicles and in luteal cells. Moderate signal intensity for PRLrs was found in theca cells of preantral to ovulatory follicles, and in granulosa cells of antral follicles up to the gonadotropin-dependent stage. Decreasing immunoreactivity to PRLrs was found in granulosa cells of gonadotropin-dependent to ovulatory follicles. For long PRLrs in particular, no signal was found in mural granulosa cells of gonadotropin-dependent follicles; for both isoforms, no signal was found in most granulosa cells of ovulatory follicles. In primordial to gonadotropin-dependent follicles, cellular localization of PRLr was similar on days 0, 10 and 15 of the cycle. Oocytes consistently showed positive immunostaining for PRLrs. Comparative RT-PCR analysis of long and short PRLr expression showed that the short isoform is evenly expressed throughout the estrous cycle, whereas the expression of the long form increases at the time of estrus and decreases at mid-luteal phase and at the onset of the follicular phase. Expression of long PRLrs was greater than that of short PRLrs on day 0 of cycle; expression of both isoforms was similar on day 10 and on day 15, long PRLrs expression was lower than that of short PRLrs. Our results indicate that in sheep ovary, the maximum responsiveness to PRL might occur during the preovulatory phase of the estrous cycle.

Effect of constant-release melatonin implants and prolonged exposure to a long day photoperiod on prolactin secretion and hair growth in mouflon (Ovis gmelini musimon).

The aims of this study were to examine whether mouflons exposed to constant long and short day photoperiods are able to exhibit an annual cycle of hair growth and moult, and prolactin (PRL) secretion. Mouflon ewes were assigned to three groups of treatment. Ewes were maintained, either under natural photoperiod (control, n = 9), or received a series of subcutaneous melatonin implants from December to April (n = 8), or were exposed to a constant long day photoperiod (16-h light:8-h dark; 16L:8D) during 18 months (n = 7). Blood was collected weekly to determine PRL concentrations, and hair samples were clipped weekly from the base of the neck to measure the length of predominant hair. Under constant long days and with melatonin implants, mouflons expressed an annual rhythm of PRL secretion, even though these treatments modified the times of rise or falling of PRL concentrations throughout the year. Hair growth initiation was almost coincident with the summer solstice in both control and melatonin-implanted mouflons but occurred two months earlier in long day hold mouflons (P < 0.001). Long day hold mouflons had a lower hair growth rate than control and melatonin-implanted mouflons (P < 0.001), and at the end of the experiment, a shorter hair length (3.4 +/- 0.24 cm; P < 0.01) than control (4.3 +/- 0.17 cm), and melatonin-implanted mouflons (4.2 +/- 0.12 cm). Our data support the conclusion that in mouflon, an endogenous circannual rhythm of PRL secretion exists, and that the seasonal cycle of hair growth and moult appears to depend, at least in part, on circulating levels of PRL.

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway.

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway.

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.

Nocturnal variation of prolactin secretion in the Mouflon (Ovis gmelini musimon) and domestic sheep (Ovis aries): seasonal changes.

Seasonal changes in nocturnal prolactin secretion and their relationship with melatonin secretion were monitored in wild (Mouflon, Ovis gmelini musimon) and domesticated sheep (breed Manchega, Ovis aries). Two groups of eleven adult females each, were maintained outdoors under natural photoperiod. Plasma concentrations of prolactin and melatonin were determined during the summer and winter solstices and the autumn and spring equinoxes. Blood samples were collected every 3h during the night hours, and 1h before and after the onset of darkness and sunrise. Maximum mean plasma concentrations of prolactin during the dark-phase in Mouflons were observed in the summer solstice, (P<0.001) and in the summer solstice and spring equinox in Manchega ewes (P<0.001). Mean plasma concentrations of prolactin were higher in the wild species (P<0.001) during the summer solstice. In contrast, during the spring equinox, mean levels of prolactin were higher in Manchega ewes than in Mouflons (P<0.05). Plasma prolactin concentrations showed a nocturnal rhythm in both breeds, with seasonal variations (P<0.001). The increase in plasma melatonin levels during the first hour after sunset was accompanied to increasing concentrations of PRL 1h after the onset of darkness, only in the autumn and spring equinox for the Mouflon, and in the summer solstice and spring equinox for the Manchega ewes. In Mouflons, the fall of plasma PRL concentrations about the middle dark-phase in all the periods studied, coincided with high levels of melatonin. A similar relation was observed in Manchega ewes only in the winter solstice and spring equinox. The current study shows that the nocturnal rhythm of prolactin secretion exhibits seasonal variation; differences in the patterns of prolactin secretion between Mouflon and Manchega sheep are taken to represent the effects of genotype.

Effects of bromocriptine administration during the follicular phase of the oestrous cycle on prolactin and gonadotrophin secretion and follicular dynamics in merino monovular ewes.

Two experiments using Spanish Merino ewes were conducted to investigate whether the secretion of prolactin during the follicular phase of the sheep oestrous cycle was involved in the patterns of growth and regression of follicle populations. In both experiments, oestrus was synchronized with two cloprostenol injections which were administered 10 days apart. Concurrent with the second injection (time 0), ewes (n = 6 per group) received one of the following treatments every 12 h from time 0 to 72 h: group 1: vehicle injection (control); group 2: 0.6 mg bromocriptine (0.03 mg per kg per day); and group 3: 1.2 mg bromocriptine (0.06 mg per kg per day). In Expt 1, blood samples were collected every 3 h from 0 to 72 h, and also every 20 min from 38 to 54 h to measure prolactin, LH and FSH concentrations. In Expt 2, transrectal ultrasonography was carried out every 12 h from time 0 until oestrus, and blood samples were collected every 4 h to measure prolactin, LH and FSH concentrations. Ovulation rates were determined by laparoscopy on day 4 after oestrus. Bromocriptine markedly decreased prolactin secretion, but did not affect FSH concentrations, the mean time of the LH preovulatory surge or LH concentrations in the preovulatory surge. Both doses of bromocriptine caused a similar decrease in LH pulse frequency before the preovulatory surge. The highest bromocriptine dose led to a reduction (P < 0.01) in the number of 2-3 mm follicles detected in the ovaries at each time point. However, bromocriptine did not modify the total number or the number of newly detected 4-5 mm follicles at each time point, the number of follicles > 5 mm or the ovulation rate. In conclusion, the effects of bromocriptine on gonadotrophin and prolactin secretion and on the follicular dynamics during the follicular phase of the sheep oestrous cycle indicate that prolactin may influence the viability of gonadotrophin-responsive follicles shortly after luteolysis.

Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation.

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.

Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol.

Embryos were collected on Days 5 and 6 after breeding to investigate the effectiveness of ethylene glycol (ETG) and glycerol (GLY) as cryoprotectants of sheep morulae and blastocysts and to determine their optimum stage of development for cryopreservation. Only excellent (grade 1) and good (grade 2) embryos (196 morulae and 188 blastocysts) were incubated in increasing concentrations of GLY or ETG and submitted to a slow-freezing and quick-thawing procedure. Both cryoprotectants were removed using 0.25 M sucrose solution, and then embryos were cultured or transferred to determine their viability. Freezing medium containing ETG yielded higher in vitro survival rates (P < 0.01) than medium containing GLY (64.6% vs 16.0%); the difference between cryoprotectants was greater when morulae were used (57.9% vs 4.2%, P < 0.005) as compared with blastocysts (70.4% vs 21.5%, P < 0.05). There was a strong interaction between type of cryoprotectant and embryo stage (P < 0.005). After transfer of morphologically viable embryos, the in vivo development rate of embryos frozen with ETG was also higher than that of embryos frozen with GLY (45.5% vs 27.7%, P < 0.05). There were no significant differences in the number of lambs born among procedures and embryo stage, though the lowest lambing rate was obtained with morulae frozen with GLY (21.4%). Similar lambing rates were produced when blastocysts were frozen with either GLY or ETG (36.6% vs 43.0%). The best embryo survival after thawing was observed when blastocysts were frozen with ETG as cryoprotectant.

Effects of LH administration at the end of an FSH superovulatory regimen on ovulation rate and embryo production in three breeds of sheep.

In 3 experiments, 168 ewes of Manchega (n = 72), Churra (n = 62), and Merina (n = 34) breeds were used to test the hypothesis that administration of pure LH, coincident with progestogen removal during superovulation with FSH, causes an increase in the ovulation rate and number of embryos. This administration of LH can further interact with genotype, resulting in breed differential response. In each experiment, the animals were randomly assigned to 1 of 3 treatments. Estrus in all sheep was synchronized with intravaginal sponges of 30 mg of FGA for 12 d, then 270 microg of FSH were administered in 6 injections at 12-h intervals in decreasing doses, starting 48 h before sponge removal. The FSH/LH ratio of the original preparation was 3, and remained constant throughout the treatment in the control group (C). In Treatment 1, (T1) and Treatment 2, (T2), pure LH was administered coincident with progestogen removal-5th FSH injection, and with the 6th FSH injection, at 2 dose levels: 60 and 120 microg, (T1), and 120 and 240 microg (T2). Mating occurred 36 and 48 h after the progestogen removal, and the embryos were surgically collected and morphologically evaluated on Days 7 and 8 after sponge withdrawal. Overall, the results showed that LH administration at the end of the FSH treatment did not increase the ovulation rate and number of embryos in Merino (5.9 +/- 1.4 and 5.6 +/- 1.4, respectively, T1; 7.0 +/- 1.0 and 5.7 +/- 1.2, T 2; 4.9 +/- 1.1 and 2.6 +/- 0.7, C), Churra (6.8 +/-1.4 and 5.2 +/- 1.4, T1; 8.1 +/- 1.5 and 6.3 +/- 1.4, T2; 6.1 +/- 1.5 and 5.4 +/- 1.3, C) and Manchega (6.0 +/- 1.0 and 4.4 +/- 1.0, T1; 5.0 +/- 0.8 and 4.2 +/- 0.8, T2; 4.8 +/- 1.5 and 3.8 +/- 1.0, C). Administration of LH induced a significant (P < 0.05) increase in the frequency of multiple ovulations (72.3 +/- 4.3 %, T1; 74.1 +/- 11.5 %, T2; 55.6 +/- 5.9 %, C) paralleled to a decrease in the occurrence of ewes with no ovulations (8.7 +/- 2.6 %, T1; 7.6 +/- 4.6 % T2; 17.3 +/- 3.2 %, C) or 1 to 2 ovulations (18.7 +/- 4.6 %, T1; 18.1 +/- 7.5 %, T2; 26.8 +/- 5.8 % C), regardless of breed or dose of LH. No increase in the mean number of viable embryos was observed, probably due to both the high individual variability and the lower fertilization rates observed in sheep showing multiple ovulations.

Light control of the duration of the daily melatonin signal under long and short days in the Soay ram. Role of inhibition and entrainment.

Light acts in two ways to control the duration of the nocturnal melatonin rhythm. It inhibits the production of melatonin from the pineal gland and it entrains the underlying circadian rhythm generators located in the suprachiasmatic nuclei. To investigate the role of these two mechanisms under long and short days, four experiments were carried out using groups of adult Soay rams (n = 6-8). The animals were housed in individual pens in light-controlled rooms and entrained to long (LD 16:8) or short (LD 8:16) days for at least 8 wk. The treatments were as follows: (i) dark period extended by 4 h under long days (L dark-delay), (ii) dark period advanced by 4 h under long days (L dark-advance), (iii) dark period extended by 4 h under short days (S dark-delay), and (iv) dark period advanced by 4 h under short days (S dark-advance). Each treatment was given on a single day and the animals were subsequently maintained in, or transferred to, constant dim red light (DD) for 24 h. A control group (C) was run in parallel with each treatment group. Blood samples were collected every 30 min for 6-9 h during the dark-shift to monitor the light-induced changes in the secretion of melatonin, and during DD to monitor any phase shift in the endogenous rhythm (phase markers provided by onset or offset of melatonin secretion). L dark-delay resulted in a significantly (p < 0.01, ANOVA) later offset of the melatonin peak (3.4-h delay) with no phase shift of the onset of the rhythm under DD.(ABSTRACT TRUNCATED AT 250 WORDS)