Anti-Müllerian hormone concentration in sheep and its dependence of age and independence of BMP15 genotype: an endocrine predictor to select the best donors for embryo biotechnologies.

Embryo biotechnologies contribute significantly to the genetic enhancement of livestock, although their efficiency remains limited in sheep, mainly owing to variable ovarian responses to gonadotropins. At present, anti-Müllerian hormone (AMH), which is produced by the granulosa cells of the small antral follicles, is a reliable endocrine marker of the ovarian follicle reserve in many species. The expression of AMH in granulosa cells was shown to be stimulated by bone morphogenetic proteins (BMPs) in vitro, so a mutation affecting the BMP15 gene might modulate AMH production in vivo. The present study aimed to assess plasma AMH concentrations before puberty in two groups of Rasa Aragonesa ewes that were carrying (R+) or not carrying (++) the prolific FecX(R) allele and to relate them with their AMH concentrations at adulthood. Additionally, we sought to establish in both genotypes whether AMH measurements during a laparoscopic ovum pick-up (LOPU) program could be predictive of the number of ovarian follicles (≥3 mm) and recovered cumulus-oocyte complexes (COCs). No differences in AMH were found between the R+ and ++ ewes before puberty or during the adult age. Before puberty, the AMH concentration tended to increase from 3 to 4.5 months and to decline at 6 months to levels similar to those observed later in adults (333.8 ± 73.3, 483.2 ± 135.5, and 184.1 ± 38.2 pg/mL, respectively; P < 0.1), showing a large variability between individuals and between ages. A relationship between the AMH concentrations before puberty and during adulthood was not found, likely reflecting different follicular growth dynamics. In adults, the AMH concentration at the beginning of the FSH treatment was strongly correlated with the number of punctured follicles at LOPU in R+ and ++ ewes (r = 0.75 and 0.78, respectively; P < 0.001), and it was possible to accurate determine AMH cutoff values for both genotypes to identify high-responding ewes. On average, 5.1 extra follicles and 2.7 extra COCs were expected per each 100 pg/mL increase in AMH (P < 0.0001 and P < 0.01, respectively). The repeatability of AMH concentration from session to session was 0.70 (P < 0.0001). Our results demonstrated that, regardless of age, the presence of the FecX(R) allele did not affect plasma AMH levels. During adulthood, AMH proved to be a good predictor of the ovarian response to FSH stimulation. Such an indicator could therefore be used to improve the performance of embryo biotechnologies in sheep.

Influence of the FecX(R) allele in heterozygous ewes on follicular population and outcomes of IVP and ET using LOPU-derived oocytes.

Ewes heterozygous for the FecX(R) allele (R+) in the bone morphogenetic protein 15 (BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the FecX(R) allele on follicle number and size, oocyte competence and in vitro production (IVP) remains undefined. With these aims, 8 R+ and 8 wild-type (++) ewes were subjected to 2 laparoscopic ovum pick-up (LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles >3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH-treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (-0.2 mm in untreated and -0.8 mm in FSH-treated ewes; p < 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus-oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p < 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture.

The efficiency of in vitro ovine embryo production using an undefined or a defined maturation medium is determined by the source of the oocyte.

In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.

First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning.

Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats. Oocytes were enucleated and coupled to bucardo's fibroblasts by electrofusion. Reconstructed embryos were cultured for 36h or 7d and transferred to either Spanish ibex or hybrid (Spanish ibex malex domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardo's donor cells. To our knowledge, this is the first animal born from an extinct subspecies.

[Occupational asthma]. / Asma ocupacional.

Occupational asthma is an entity caused by conditions deriving from a certain work milieu and not from stimuli found outside the workplace. Nowadays, occupational asthma is the most frequent respiratory occupational disease in the majority of the industrialised countries and it is estimated that the proportion of new cases of asthma that can be attributed to exposure at work is around 10-15%. It can be developed due to an immunological mechanism or to a non-immunological mechanism. Influential in its development are the type of agent exposed to, the level and form of exposure and genetic factors of susceptibility. In the diagnostic process there is a concurrent confirmation that the patient has bronchial asthma and that this has been caused by occupational reasons. As shown by the natural history of the disease, an early diagnosis and the consequent posterior actions result in an improved prognosis.

Asma ocupacional / Occupational asthma

Asma ocupacional es aquella entidad que se desarrollapor causas o condiciones derivadas de un determinadomedio laboral y no por estímulos que seencuentran fuera del trabajo. El asma ocupacionalconstituye hoy en día la enfermedad respiratoria ocupacionalmás frecuente en la mayoría de los paísesindustrializados y se calcula que la proporción de nuevoscasos de asma atribuibles a la exposición laboralse sitúa en torno al 10-15%.Puede desarrollarse tanto por un mecanismoinmunológico como no inmunológico. En su desarrolloinfluyen el tipo de agente al que se está expuesto, elnivel y modo de exposición y factores genéticos de susceptibilidad.En el proceso diagnóstico concurre laconfirmación de que el paciente tiene asma bronquialy la confirmación de que ésta se produce por causalaboral. Como demuestra la historia natural de la enfermedad,un diagnóstico precoz y las consiguientesacciones posteriores redundan en un mejor pronósticode la misma

Occupational asthma is an entity caused byconditions deriving from a certain work milieu and notfrom stimuli found outside the workplace. Nowadays,occupational asthma is the most frequent respiratoryoccupational disease in the majority of theindustrialised countries and it is estimated that theproportion of new cases of asthma that can beattributed to exposure at work is around 10-15%.It can be developed due to an immunologicalmechanism or to a non-immunological mechanism.Influential in its development are the type of agentexposed to, the level and form of exposure and geneticfactors of susceptibility. In the diagnostic processthere is a concurrent confirmation that the patient hasbronchial asthma and that this has been caused byoccupational reasons. As shown by the natural historyof the disease, an early diagnosis and the consequentposterior actions result in an improved prognosis