Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022.

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3-8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

Random-start ovarian stimulation in an oocyte donation programme: a large, single-centre, experience.

RESEARCH QUESTION: Do live birth rates differ between recipients matched with donors using conventional ovarian stimulation compared with those using random-start protocols? DESIGN: Retrospective analysis of 891 ovarian stimulations in egg donors (January-December 2018) and clinical outcomes in matched recipients (n = 935). Donors commenced ovarian stimulation on day 1-3 of the menstrual cycle (n = 223) or in the mid/late-follicular (n = 388) or luteal phase (n = 280) under a conventional antagonist protocol. Live birth rate of matched recipients was the main outcome. RESULTS: Duration of stimulation and total gonadotrophin dose were comparable between conventional versus random-start groups. The number of collected eggs were similar (17.6 ± 8.8 versus 17.2 ± 8.5, P = 0.6, respectively). Sub-group analysis showed that stimulation length (10.2 ± 1.8 versus 9.8 ± 1.7 versus 10.4 ± 1.7, P < 0.001) and gonadotrophin consumption (2041.5 ± 645.3 versus 2003.2 ± 647.3 versus 2158.2 ± 685.7 IU, P = 0.01) differed significantly between the conventional, mid/late follicular and luteal phase groups, respectively. In matched recipients receiving fresh oocytes and undergoing fresh embryo transfer, the biochemical pregnancy (63.8% and 63.3%; P = 0.9), clinical pregnancy (54.6% and 56.1%; P = 0.8) and live birth rates (47.7% and 46.6%; P = 0.7) per embryo-transfer were similar between conventional versus random groups. Similar results were obtained in recipients receiving vitrified eggs. Euploidy rate was also comparable. CONCLUSIONS: No notable variations were found in clinical outcomes using oocytes obtained from random-start protocols and those proceeding from conventional ovarian stimulation in oocyte donation treatments. Luteal-phase stimulation seems to require longer stimulation and higher FSH consumption. Random-start stimulation strategy does not impair the potential of the oocyte yield or clinical outcomes in oocyte donation cycles.

Implicit bias in diagnosing mosaicism amongst preimplantation genetic testing providers: results from a multicenter study of 36 395 blastocysts.

STUDY QUESTION: Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? SUMMARY ANSWER: Our analysis of 36 395 blastocyst biopsies across eight genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. WHAT IS KNOWN ALREADY: Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible embryo ploidy outcomes following PGT-A. However, in the era of mosaicism, embryo selection has become increasingly complex. Biological, technical, analytical, and clinical complexities in interpreting such results have led to substantial variability in mosaicism rates across PGT-A providers and clinics. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. STUDY DESIGN, SIZE, DURATION: In this international, multicenter cohort study, we reviewed 36 395 consecutive PGT-A results, obtained from 10 035 patients across 11 867 treatment cycles, conducted between October 2015 and October 2021. A total of 17 IVF centers, across eight PGT-A providers, five countries and three continents participated in the study. All blastocysts were tested using trophectoderm biopsy and next-generation sequencing. Both autologous and donation cycles were assessed. Cycles using preimplantation genetic testing for structural rearrangements were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The PGT-A providers were randomly categorized (A to H). Providers B, C, D, E, F, G, and H all reported mosaicism, whereas Provider A reported embryos as either euploid or aneuploid. Ploidy rates were analyzed using multilevel mixed linear regression. Analyses were adjusted for maternal age, paternal age, oocyte source, number of embryos biopsied, day of biopsy, and PGT-A provider, as appropriate. We compared associations between genetic testing providers and PGT-A outcomes, including the number of chromosomally normal (euploid) embryos determined to be suitable for transfer. MAIN RESULTS AND THE ROLE OF CHANCE: The mean maternal age (±SD) across all providers was 36.2 (±5.2). Our findings reveal a strong association between PGT-A provider and the diagnosis of euploidy and mosaicism. Amongst the seven providers that reported mosaicism, the rates varied from 3.1% to 25.0%. After adjusting for confounders, we observed a significant difference in the likelihood of diagnosing mosaicism across providers (P < 0.001), ranging from 6.5% (95% CI: 5.2-7.4%) for Provider B to 35.6% (95% CI: 32.6-38.7%) for Provider E. Notably, adjusted euploidy rates were highest for providers that reported the lowest rates of mosaicism (Provider B: euploidy, 55.7% (95% CI: 54.1-57.4%), mosaicism, 6.5% (95% CI: 5.2-7.4%); Provider H: euploidy, 44.5% (95% CI: 43.6-45.4%), mosaicism, 9.9% (95% CI: 9.2-10.6%)); and Provider D: euploidy, 43.8% (95% CI: 39.2-48.4%), mosaicism, 11.0% (95% CI: 7.5-14.5%)). Moreover, the overall chance of having at least one euploid blastocyst available for transfer was significantly higher when mosaicism was not reported, when we compared Provider A to all other providers (OR = 1.30, 95% CI: 1.13-1.50). Differences in diagnosing and interpreting mosaic results across PGT-A laboratories raise further concerns regarding the accuracy and relevance of mosaicism predictions. While we confirmed equivalent clinical outcomes following the transfer of mosaic and euploid blastocysts, we found that a significant proportion of mosaic embryos are not used for IVF treatment. LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of the study, associations can be ascertained, however, causality cannot be established. Certain parameters such as blastocyst grade were not available in the dataset. Furthermore, certain platform-related and clinic-specific factors may not be readily quantifiable or explicitly captured in our dataset. As such, a full elucidation of all potential confounders accounting for variability may not be possible. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the strong need for standardization and quality assurance in the industry. The decision not to transfer mosaic embryos may ultimately reduce the chance of success of a PGT-A cycle by limiting the pool of available embryos. Until we can be certain that mosaic diagnoses accurately reflect biological variability, reporting mosaicism warrants utmost caution. A prudent approach is imperative, as it may determine the difference between success or failure for some patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Torres Quevedo Grant, awarded to M.P. (PTQ2019-010494) by the Spanish State Research Agency, Ministry of Science and Innovation, Spain. M.P., L.B., A.R.L., A.L.R.d.C.L., N.P.P., M.P., D.S., F.A., A.P., B.M., L.D., F.V.M., D.S., M.R., E.P.d.l.B., A.R., and R.V. have no competing interests to declare. B.L., R.M., and J.A.O. are full time employees of IB Biotech, the genetics company of the Instituto Bernabeu group, which performs preimplantation genetic testing. M.G. is a full time employee of Novagen, the genetics company of Cegyr, which performs preimplantation genetic testing. TRIAL REGISTRATION NUMBER: N/A.

Progesterone levels on the day of embryo transfer using a single pessary of 400mg of vaginal progesterone vs. 200mg x2 pessaries in hormonal replacement cycles.

OBJECTIVE: Does the use of 400mg pessaries of micronized progesterone provide comparable results as pessaries of 200mg x2, in terms of progesterone levels in hormonal replacement cycles for embryo transfer?. METHODS: Retrospective cohort study based on 299 embryo transfer treatments under artificial endometrial preparation carried out at Instituto Bernabeu. 131 patients received 1 pessary of 400 mg b.i.d. (group A) and 168 received 2 pessaries of 200 mg b.i.d. (group B). RESULTS: Mean serum progesterone levels were similar between groups (A: 13.64±4.47ng/mL vs. B: 13.88±7.17ng/mL). There were no differences in suboptimal progesterone levels between groups (A: 11.5% vs. B: 16.8%). In terms of patients receiving additional progesterone supplementation, there were no differences between groups (A: 26% vs. B: 35.3%.). No differences between groups were observed in clinical outcomes: pregnancy rate (PR) (A: 55% vs. B: 54.8%), biochemical pregnancy loss rate (BPLR) (A: 13.4% vs. B: 17.6%), miscarriage rate (MR) (A: 17.9% vs. B: 19.8%) and ongoing pregnancy rate (OPR) (A: 36.5% vs. B: 34.1%). CONCLUSIONS: One progesterone pessary of 400mg (Cyclogest®) twice daily appears to be non-inferior to the use of two-200mg pessaries twice daily in terms of progesterone levels in HRT cycles.

Occurrence and limited zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Balantioides coli infections in free-ranging and farmed wild ungulates in Spain.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1-3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2-6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3-1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulate-adapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate species would not presumably play a significant role as source of human infections by these pathogens. Wild ruminants do not seem to be susceptible hosts for B. coli.

Does mRNA COVID-19 vaccination in oocyte donors impact ovarian stimulation parameters or IVF outcomes for recipients?

RESEARCH QUESTION: What is the effect of mRNA severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in young oocyte donors in terms of ovarian response to stimulation, fertilization rate, embryo development and clinical outcomes in recipients? DESIGN: This retrospective, multicentre cohort study evaluated 115 oocyte donors who had undergone at least two ovarian stimulation protocols (before and after complete SARS-CoV-2 vaccination) between November 2021 and February 2022. Comparisons were made of the primary outcomes of days of stimulation, total dose of gonadotrophins and laboratory performance in ovarian stimulation in oocyte donors before and after vaccination. A total of 136 cycles in matched recipients were analysed as secondary outcomes and, from those, 110 women received a fresh single-embryo transfer, with analysis of biochemical ß-human chorionic gonadotrophin concentrations and rates of clinical pregnancy with heartbeat. RESULTS: Longer stimulation was required in the post-vaccination than pre-vaccination group (10.31 ± 1.5 versus 9.51 ± 1.5 days; P < 0.001) along with higher gonadotrophin consumption (2453.5 ± 740 versus 2235.5 ± 615 IU; P < 0.001) with a similar starting dose of gonadotrophins in both groups. More oocytes were retrieved in the post-vaccination group (16.62 ± 7.1 versus 15.38 ± 7.0; P = 0.02). However, the number of metaphase II (MII) oocytes was similar between groups (pre-vaccination 12.61 ± 5.9 versus post-vaccination 13.01 ± 6.6; P = 0.39) and the ratio of MII/retrieved oocytes favoured the pre-vaccination group (0.83 ± 0.1 versus 0.77 ± 0.2 post-vaccination; P = 0.019). In recipients with a similar number of provided oocytes, the fertilization rate, total number of obtained blastocysts, number of top-quality blastocysts, and rates of biochemical pregnancy and clinical pregnancy with heartbeat were not significantly different between groups. CONCLUSIONS: This study shows no adverse influence of mRNA SARS-CoV-2 vaccination on ovarian response in a young population.

Conventional follicular-phase ovarian stimulation vs. luteal-phase stimulation in suboptimal responders: a randomized controlled trial.

Objective: To compare the oocyte yield between follicular-phase stimulation (FPS) and luteal-phase stimulation (LPS) in suboptimal responders. Design: Prospective, randomized, crossover clinical trial. Patients: Forty-one patients with infertility according to the POSEIDON (Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number) criteria (1b/2b). Interventions: Crossover study on 2 assigned ovarian stimulations that started randomly in the follicular or luteal phase. The in vitro fertilization cycles were not consecutive but separated in time (45 days to 6 months). The random crossover design ensured that all subjects received the first treatment by chance. Main Outcome Measures: The primary objective was the number of cumulus-oocyte complexes retrieved in each cycle. Secondary objectives were number of metaphase II and fertilized oocytes, additional doses of recombinant follicle-stimulating hormone, and the duration of ovarian stimulation (days). Results: The mean number of cumulus-oocyte complexes retrieved was similar between the FPS and LPS groups (7.5 ± 4.6 vs. 7.0 ± 4.1; 95% confidence interval [CI] for the mean, 5.8-8.7 vs. 5.6-8.3, respectively; the difference between means, -0.5; 95% CI, -1.8 to +1.5). Similarly, the mean number of metaphase II oocytes retrieved was not different between the FPS and LPS groups (5.4 ± 3.6 vs. 5.2 ± 2.8; 95% CI for the mean, 4.2-6.5 vs. 4.3-6.1, respectively; the difference between means, -0.2; 95% CI, -1.2 to +1.1). Moreover, the secondary objectives were similar between FPS and LPS groups. Conclusions: In this study, the oocyte yield in LPS did not increase in suboptimal responders compared with that in FPS when the onset of LPS was separated in time from FPS. Clinical Trial Registration Number: NCT039393990 https://beta.clinicaltrials.gov/study/NCT03939390.

FSH receptor genotype and its influence on the results of donor ovarian stimulation using corifollitropin alfa.

RESEARCH QUESTION: Does the FSH receptor (FSHR) genotype influence the results of donor ovarian stimulation using corifollitropin alfa? DESIGN: A prospective cohort study was performed including 152 oocyte donor ovarian stimulations: group 1 (n = 80) using a single dose of 150 µg of corifollitropin alpha; and group 2 (n = 72) using in addition to corifollitropin alpha, continued stimulation using recombinant FSH 225 IU daily. Allelic discrimination was used to genotype the FSHR p.N680S polymorphism. Linear regression analysis was performed to study the differences between groups. RESULTS: No differences in clinical characteristics between genotypes were reported. Overall, the results of ovarian stimulation were better in oocyte donors with SN and NN genotypes compared with SS in terms of the number of retrieved oocytes (15.78 versus 10.83; P = 0.008) and retrieved metaphase II (MII) oocytes (12.34 versus 9.00; P = 0.032). Corresponding differences were also observed in group 1 for the number of retrieved oocytes (13.83 versus 7.50, P = 0.018) and retrieved MII oocytes (10.24 versus 5.42; P = 0.038). However, in group 2 no significant differences were found for oocytes retrieved (17.55 versus 13.06, P = 0.064) or MII oocytes (14.25 versus 11.39; P = 0.12). CONCLUSIONS: This study suggests that ovarian stimulation protocols with corifollitropin alfa in women with the SS genotypes could be associated with fewer oocytes and MII oocytes retrieved. Despite the fact that corifollitropin alfa has a longer half-life, the results for the SS genotype do not match those for the other genotypes, so other factors must be involved. Therefore, to tailor treatments, it would be advisable to genotype women at p.N680S of the FSHR.

Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

The fungal-related intracellular parasite Enterocytozoon bieneusi is a worldwide public health and veterinary problem. Here we demonstrated that it was present in wild boar, and wild and farmed red deer in Spain, with genotypes potentially capable of infecting humans, posing a public health risk.

Bagaza virus and Plasmodium spp. coinfection in red-legged partridges (Alectoris rufa), in Southern Spain 2019.

Flaviviruses such as West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) virus and avian malaria parasites are vector borne pathogens that circulate naturally between avian and mosquito hosts. WNV and USUV and potentially also BAGV constitute zoonoses. Temporal and spatial cocirculation and coinfection with Plasmodium spp., and West Nile virus has been documented in birds and mosquito vectors, and fatally USUV-infected passerines coinfected with Plasmodium spp. had more severe lesions. Also, WNV, USUV and BAGV have been found to cocirculate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or coinfections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain that were coinfected with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where cocirculation of BAGV, USUV and WNV was confirmed in 2011 and 2013. Partridges were found dead in early October 2019. Birds had mottled locally pale pectoral muscles, enlarged, congestive greenish-black tinged livers and enlarged kidneys. Microscopically congestion and predominantly mononuclear inflammatory infiltrates were evident and Plasmodium phanerozoites were present in the liver, spleen, kidneys, muscle and skin. Molecular testing and sequencing detected Plasmodium spp. and BAGV in different tissues of the partridges, and immunohistochemistry confirmed the presence and colocalization of both pathogens in the liver and spleen. Due to the importance of the red-legged partridge in the ecosystem of the Iberian Peninsula and as driver of regional economy such mortalities are of concern. Such outbreaks may reflect climate change related shifts in host, vector and pathogen ecology and interactions that could emerge similarly for other pathogens.

Vitrification does not affect birth weight: lessons from the oocyte donation model.

RESEARCH QUESTION: Is embryo cryopreservation a cause of high birth weight and large for gestational age (LGA) in singletons resulting from vitrified-warmed embryo transfer? DESIGN: Retrospective cohort study evaluating 670 oocyte recipients who underwent fresh (367 cycles) or vitrified-warmed embryo transfer (303 cycles) at Instituto Bernabeu between July 2017 and March 2019. All single blastocyst transfers carried out in an artificial cycle that resulted in a singleton live birth were included. RESULTS: Maternal age (42.21 ± 4.45; 42.79 ± 3.83; P = 0.519), body mass index (23.34 ± 3.69; 23.80 ± 3.78; P = 0.075), gestational age (38.96 ± 1.97; 38.77 ± 2.15; P = 0.207), maternal smoking (10.8%; 13.0%; P = 0.475), gestational diabetes (4.9%; 4.3% P = 0.854), preeclampsia (2.7%; 5.6%; P = 0.074), hypertensive disorders (3.3%; 2.3%; P = 0.494), maternal parity (multiparous 18.5%; 14.5%; P = 0.177) and liveborn gender (female 44.5%; 48.8%; P = 0.276) were not significantly different between fresh or vitrified-warmed groups. Endometrial thickness was significantly higher in the fresh versus vitrified-warmed group (8.83 ± 1.73 versus 8.57 ± 1.59; P = 0.035, respectively). Oocyte donor height was similar between the fresh versus vitrified-warmed group (163.22 ± 5.88 versus 164.27 ± 6.66 cm; P = 0.057, respectively). Mean birth weight was not significantly different (3239.21 ± 550.43; 3224.56 ± 570.83; adjusted P = 0.058). No differences were observed in macrosomia (7.1%; 6.3%; adjusted OR 0.857, 95% CI 0.314 to 2.340, P = 0.764), LGA (6.0%; 6.7%; adjusted OR 0.450, 95% CI 0.176 to 1.149, P = 0.095), pre-term birth (10.9%; 9.0% adjusted P = 0.997), very pre-term birth (0.8%; 1.3%; adjusted P = 1.000), extremely pre-term birth (0%; 1.0%; adjusted P = 0.998); underweight (10.0%; 7.0%; adjusted P = 0.050); very low weight (0.6; 1.1%; adjusted P = 1.000) and small for gestational age (1.9%; 0.7%; adjusted P = 0.974) between fresh or vitrified-warmed groups. CONCLUSION: This study eliminates potential confounders that might influence fetal growth and demonstrates that embryo vitrification and warming procedures do not affect birth weight.

Fatal cases of bovine anaplasmosis in a herd infected with different Anaplasma marginale genotypes in southern Spain.

The genus Anaplasma contains various species capable of causing disease in animals and humans. Anaplasma marginale is one of the main tick-borne pathogens of bovines in tropical and subtropical regions; however, these bacteria are now being detected more frequently in other regions of the world including Europe. In July 2017, abortions, mortality and morbidity in Retinta breed of cattle were investigated in southwestern Spain. Based on clinical signs, the provisional clinical diagnosis of bovine anaplasmosis was made. A molecular-phylogenetic approach was used to characterize A. marginale using multiple markers, including 16S rRNA, msp1a, msp4 and msp5 genes. The msp1α sequence was different from the previously described sequences from Spain as well as other countries. The isolates of A. marginale were classified as Genotype C, with two of the five tandem repeats in the amino acid sequences MSP1α being novel. The highest variability was observed in the four sequences of msp5 which was depicted in their clustering into multiple clades on a phylogenetic tree. Comparison of msp5 nucleotide sequences and the corresponding amino acid sequences revealed the co-existence of different strains in the same region. This study highlights the occurrence of clinical bovine anaplasmosis in an endemic region of Spain.

Efficacy and safety of intranasal haloperidol in an acute Psychiatry Unit: a pilot study on schizophrenic patients with mild-modedate agitation.

To study the efficacy and safety of intranasal administration of 5 mg haloperidol on mild-moderate agitated patients with schizophrenia or schizoaffective disorder in an acute psychiatry unit setting.

Eficacia y seguridad del haloperidol intranasal en una Unidad de Agudos: estudio piloto en pacientes esquizofrénicos con leve-moderada agitación / Efficacy and safety of intranasal haloperidol in an acute Psychiatry Unit: a pilot study on schizophrenic patients with mild-modedate agitation

Objetivo: Estudiar la eficacia y seguridad de la administración de 5 mg de haloperidol intranasal en pacientes conesquizofrenia y trastorno esquizoafectivo, con leve o moderada agitación, ingresados en una unidad de agudos depsiquiatría.Método. Diseño: Estudio piloto de ensayo clínico, fase IV,con evaluador ciego, unicéntrico, aleatorizado y controladode grupos paralelos, comparando la administración intranasalcon la intramuscular. Sujetos: 16 pacientes; 7 administraciónintranasal y 9 administración intramuscular. Medidas de eficacia: Escala de Síntomas Positivas y Negativos-ComponenteExcitación (PANSS-EC); Escala de Impresión Clínica Global(CGI). Medidas de seguridad: Cambios en el ECG registrados5 minutos pretratamiento y 5 minutos postratamiento.Resultados. La administración intranasal mostró mayorrapidez de acción en comparación con la intramuscular enla PANSS-EC (p = 0,042) y la CGI (p = 0,041) a los 10 minutos de la administración, con similar eficacia a los 20, 30 y60 minutos. Sin diferencias significativas en el QTc basal ypostratamiento.Conclusión. El haloperidol intranasal fue una alternativarápida, efectiva y bien tolerada para reducir la agitación leve-moderada en estos pacientes. (AU)

Aim: To study the efficacy and safety of intranasal administration of 5mg haloperidol on mild-moderate agitatedpatients with schizophrenia or schizoaffective disorder in anacute psychiatry unit setting.Method. Design: Pilot study of clinical trial, phase IV,open-label, observer-blind, single-center, randomized a haloperidol-controlled trial comparing intranasal with intramuscular administration. Subjects: 16 patients; 7 intranasaladministration, and 9 intramuscular administration. Efficacymeasurement: Positive and Negative Syndrome Scale-Excited Component (PANSS-EC); Clinical Global Impressions-Improvement Scale (CGI). Safety measurement: Changes inthe ECG registered 5 minutes pre-treatment and 5 minutespost-treatment.Results. Intranasal administration showed more quickaction compared with intramuscular on the PANSS-EC(p=0.042) and CGI (p=0.041) 10 minutes after administration, with similar efficacy up to 20, 30, and 60 minutes. There were no significant differences between QTc baseline andpost-treatment.Conclusion. Intranasal haloperidol was a rapid, effective,and well-tolerated alternative for reducing acute mild-moderate agitation. (AU)

Exogenous Melatonin Improves the Reproductive Outcomes of Yearling Iberian Red Deer (Cervus elaphus hispanicus) Hinds.

The aim of this study was to assess the effect of melatonin implants on the reproductive performance of yearling Iberian red deer (Cervus elaphus hispanicus) hinds. It also explored exogenous melatonin administration as a tool to minimize the negative effect of a low yearling hind's liveweight on their reproductive efficiency. In addition, the effect of melatonin-treated yearling hinds on non-treated hinds was studied in order to provide a practical and economical protocol to improve farms' productivity. A total of 4520 Iberian red deer hinds belonging to the same farm were included in this study. Melatonin (108 mg/hind) implants were administered three-fold every 30 days before the breeding season. Fertility rates, calves' weights and calving dates were registered for each hind. The results showed that exogenous melatonin increased significantly (p < 0.05) the calves' weight (32.39 ± 1.07 kg vs. 27.65 ± 1.11 kg for Weight 1calf (July) and 46.59 ± 1.50 kg vs. 41.79 ± 1.54 kg for Weight 2calf (August, at weaning)) and advanced the calving date by 15 days in yearling hinds compared to the non-treated group. In addition, the administration of melatonin implants before the breeding season was able to minimize the negative effect of low yearling hinds' liveweight (Weight 1hind) on their future reproductive outcomes, as the fertility rates increased by 46% and the calves' weight increased by 7 kg after the melatonin treatment, regardless of the yearlings' weight. Finally, when both experimental groups (melatonin and non-treated) were kept separate, higher fertility rates (76.73 ± 7.18% vs. 66.94 ± 7.41%) were observed for the melatonin-treated hinds compared to the non-treated hinds. However, when both groups of yearling hinds were maintained together, no significant differences were observed in their fertility outcomes (78.13 ± 21.26% vs. 78.12 ± 23.32%). Therefore, melatonin implants may be used in yearling Iberian red deer hinds as a management tool to improve their reproductive productivity.

Beneficial Effects of Melatonin in the Ovarian Transport Medium on In Vitro Embryo Production of Iberian Red Deer (Cervus elaphus hispanicus).

A major limiting factor for the development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, compared to livestock animals is the poor availability and limited access to biological material. Thus, the use of post-mortem ovaries from slaughtered animals represent a source of oocytes for the large scale production of embryos needed for research and to improve the efficiency of IVP. However, these oocytes are not as developmentally competent as their in vivo counterparts. Moreover, oocytes are usually obtained from ovaries that have been transported for long distances, which may also affect their quality. In order to overcome the issues associated with prolonged storage times of post-mortem material, in this study we examined the effect of melatonin supplementation to the ovary transport medium on oocyte quality, embryo yield, and blastocyst quality in Iberian red deer. When necessary, sheep was used as an experimental model due to the large number of samples required for analysis of oocyte quality parameters. Oocytes were in vitro matured and assessed for early apoptosis; DNA fragmentation; reactive oxygen species (ROS); reduced glutathione (GSH) content, mitochondrial membrane potential, and distribution; and relative abundance of mRNA transcript levels. After in vitro fertilization, embryo rates and blastocyst quality were also investigated. The results revealed that melatonin treatment significantly increased intracellular level of GSH in sheep oocytes. Moreover, the percentage of cleavage and blastocyst yield in red deer was greater compared to the Control group and there was lower abundance of oxidative stress- and apoptosis-related SHC1, TP53, and AKR1B1 mRNA transcripts in blastocysts for the Melatonin group. In conclusion, the supplementation of melatonin to the ovary storage medium had a positive effect on the developmental competence and quality of resulting blastocysts in Iberian red deer.

No effect of inoculation site and injection device on the skin test response of red deer to the intradermal injection of Mycobacterium avium-derived purified protein derivative (PPD).

Mycobacterial diseases are important health issues in farmed deer. The single intradermal tuberculin test is the standard test for tuberculosis testing in deer. We studied two factors which might influence the response of deer to skin testing: the inoculation site and the injection device. Deer included in this study were 2.5 years old farmed red deer (Cervus elaphus) hinds (n = 80). Two areas of 3 cm × 3 cm were shaved at the left side of the neck. Site A (SA) was situated about 10 cm caudal to the head, while site B (SB) was 10 cm caudal to SA. All hinds received at the same time two 0.1 ml inoculations of Mycobacterium avium derived purified protein derivative (aPPD). One inoculation was made by syringe and the other one with the needle-free syringe Dermojet. To test the inoculation site effect, half of the animals were inoculated by Dermojet in SA and by syringe in SB to compare with the inoculation in SA by syringe and Dermojet in SB in the other half. No differences were recorded for the injection device nor for the inoculation site. Ten hinds had a skinfold increase larger than 30 tenths of mm by any injection device and inoculation site. Seven (9%) and 6 (8%) hinds were classified as positive by syringe and Dermojet, and at the anterior or posterior inoculation site, respectively. The distribution of skinfold thickness increases did not differ by injection device. Our findings support the needle-free Dermojet syringe as a suitable tool for skin-testing in red deer and suggest no relevant effect of the position of the inoculation site along the neck in red deer.