Aborto infectocontagioso em éguas: uma revisão bibliográfica / Infectious-contagious abortion in mares: a literature review / Aborto infecto-contagioso en yeguas: una revisión de la literatura

O aborto infectocontagioso em éguas é um tema de grande relevância e interesse para os médicos veterinários e criadores de equinos. Além dos impactos econômicos decorrentes de perdas e redução das taxas reprodutivas, os surtos de abortos causados por doenças infectocontagiosas representam uma ameaça significativa para a saúde equina. Neste trabalho, realizamos uma revisão bibliográfica abrangente sobre as principais doenças que causam abortos infecciosos em éguas. Nosso objetivo é fornecer uma visão geral das patologias mais relevantes nesse contexto, abordando suas características clínicas, epidemiologia, diagnóstico e medidas de controle. Para isso, realizamos uma busca em bancos de dados renomados, como o PubMed e o Scopus, por artigos científicos relevantes publicados nos últimos dez anos. As informações selecionadas foram cuidadosamente analisadas, comparadas e sintetizadas, com o intuito de identificar as principais doenças e suas implicações na saúde reprodutiva das éguas. Esta revisão pretende auxiliar veterinários, pesquisadores e profissionais da área a compreenderem melhor essas doenças e desenvolverem estratégias eficazes de prevenção e controle.(AU)

El aborto infeccioso en yeguas es un tema de gran relevancia e interés para veterinarios y criadores de equinos. Además de las repercusiones económicas derivadas de las pérdidas y la reducción de las tasas reproductivas, los brotes de aborto causados por enfermedades infecciosas representan una importante amenaza para la salud equina. En este artículo, realizamos una revisión exhaustiva de la literatura sobre las principales enfermedades que causan abortos infecciosos en yeguas. Nuestro objetivo es ofrecer una visión general de las patologías más relevantes en este contexto, abordando sus características clínicas, epidemiología, diagnóstico y medidas de control. Para ello, buscamos en bases de datos de renombre como PubMed y Scopus artículos científicos relevantes publicados en los últimos diez años. La información seleccionada fue cuidadosamente analizada, comparada y sintetizada con el fin de identificar las principales enfermedades y sus implicaciones en la salud reproductiva de las yeguas. El objetivo de esta revisión es ayudar a veterinarios, investigadores y profesionales del sector a comprender mejor estas enfermedades y desarrollar estrategias eficaces de prevención y control.(AU)

Infectious abortion in mares is a topic of great relevance and interest for veterinarians and equine breeders. In addition to economic impacts from losses and reduced reproductive rates, abortion outbreaks caused by infectious diseases represent a significant threat to equine health. In this paper, we conduct a comprehensive literature review on the major diseases that cause infectious abortions in mares. Our goal is to provide an overview of the most relevant pathologies in this context, addressing their clinical features, epidemiology, diagnosis, and control measures. To this end, we searched renowned databases such as PubMed and Scopus for relevant scientific articles published in the last ten years. The selected information was carefully analyzed, compared and synthesized in order to identify the main diseases and their implications in the reproductive health of mares. This review aims to assist veterinarians, researchers, and professionals in the field to better understand these diseases and develop effective prevention and control strategies.(AU)

Genome activation in equine in vitro-produced embryos.

Embryonic genome activation is a critical event in embryo development, in which the transcriptional program of the embryo is initiated. The timing and regulation of this process are species-specific. In vitro embryo production is becoming an important clinical and research tool in the horse; however, very little is known about genome activation in this species. The objective of this work was to identify the timing of genome activation, and the transcriptional networks involved, in in vitro-produced horse embryos. RNA-Seq was performed on oocytes and embryos at eight stages of development (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst; n = 6 per stage, 2 from each of 3 mares). Transcription of seven genes was initiated at the 2-cell stage. The first substantial increase in gene expression occurred at the 4-cell stage (minor activation), followed by massive gene upregulation and downregulation at the 8-cell stage (major activation). An increase in intronic nucleotides, indicative of transcription initiation, was also observed at the 4-cell stage. Co-expression network analyses identified groups of genes that appeared to be regulated by common mechanisms. Investigation of hub genes and binding motifs enriched in the promoters of co-expressed genes implicated several transcription factors. This work represents, to the best of our knowledge, the first genomic evaluation of embryonic genome activation in horse embryos.

Allele-specific expression analysis reveals conserved and unique features of preimplantation development in equine ICSI embryos†.

Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an important role in early development, notably NANOGNB. The main actor in X-chromosome inactivation, XIST, was significantly upregulated at the 8-cell, morula, and blastocyst stages in female embryos, with high expression at the latter. Sexual dimorphism in gene expression was identified at all stages, with strong representation of the X-chromosome in females from the 16-cell to the blastocyst stage. Female embryos showed biparental X-chromosome expression at all stages after the 4-cell stage, demonstrating the absence of imprinted X-inactivation at the embryo level. The analysis of gene dosage showed incomplete dosage compensation (0.5 < X:A < 1) in MII oocytes and embryos up to the 4-cell stage, an increase of the X:A ratio at the 16-cell and morula stages after genome activation, and a decrease of the X:A ratio at the blastocyst stage, which might be associated with the beginning of X-chromosome inactivation. This study represents the first critical analysis of parent- and sex-specific gene expression in early equine embryos produced in vitro.

Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm300 µm). However, more research is required to find the best method for embryos ≥700 µm.

Equine early pregnancy endocrine profiles and ipsilateral endometrial immune cell, gene expression and protein localisation response.

We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.

Sepsis due to Yersinia enterocolitica in an aborted equine fetus: case report / Sepse por Yersinia enterocolitica em um feto equino abortado: relato de caso

Yersinia enterocolitica is a bacterium with zoonotic potential and there are no previous records of this bacteria being isolated from aborted foals. This report aims to describe a case of sepsis due to Y. enterocolitica in a seven month old aborted equine. The fequinoetus was submitted to necropsy and samples of all the organs were collected for the histological exam. Samples of liver, lung, placenta, and stomach contents were collected for bacterial culture. Macroscopically, the liver was enlarged with yellowish heterogeneous color, heart with pale myocardial areas; lungs not collapsed, heavy and shiny, thickened umbilical cord covered with fibrin and pus. Histopathologically, there was moderate multifocal necrosuppurative myocarditis and thrombosis, moderate diffuse suppurative bronchopneumonia, mild multifocal fibrinonecrotic hepatitis, and moderate diffuse necrosuppurative omphalitis with intralesional bacterial myriads and thrombosis. Mild multifocal suppurative placentitis, nephritis, myositis, cystitis, and dermatitis were also observed, in addition to mild diffuse lymphoid rarefaction. The microbiological evaluation identified Y. enterocolitica in the liver, lung, and stomach fluid. This is the first report of sepsis due to Y. enterocolitica causing an abortion in a horse. This bacterium has zoonotic importance; therefore, it should be investigated in abortion in this species, serving as a differential diagnosis in reproductive disorders.(AU)

Yersinia enterocolitica é uma bactéria com potencial zoonótico, e não há informações desse agente como causa de abortamento em equinos. O objetivo deste relato é descrever um caso de sepse por Y. enterocolitica em um feto equino abortado aos sete meses. O feto foi submetido à necropsia, e amostras de todos os órgãos foram processadas para histopatologia. Para microbiologia, foram coletadas amostras de fígado, pulmão, placenta e conteúdo estomacal. Macroscopicamente, observou-se fígado aumentado com coloração amarelada heterogênea; coração com áreas pálidas no miocárdio; pulmões não colabados, pesados e brilhantes; e cordão umbilical espessado e recoberto por fibrina e pus. Na análise histopatológica, havia miocardite necrossupurativa multifocal moderada e trombose, broncopneumonia supurativa difusa moderada, hepatite fibrinonecrótica multifocal discreta e onfalite necrossupurativa difusa moderada com miríades bacterianas intralesionais e trombose. Observou-se também placentite, nefrite, miosite, cistite e dermatite supurativa multifocal discreta, além de rarefação linfoide difusa discreta. A avaliação microbiológica identificou Y. enterocolitica no fígado, no pulmão e no líquido estomacal. Este é o primeiro relato de sepse por Y. enterocolitica causando abortamento na espécie equina. Essa bactéria tem importância zoonótica, portanto deve ser investigada em casos de abortamento nessa espécie, servindo como diagnóstico diferencial em tal distúrbio reprodutivo.(AU)

Timed artificial insemination in crossbred mares: Reproductive efficiency and costs.

Timed artificial insemination (TAI) has boosted the use of conventional artificial insemination (CAI) by employing hormonal protocols to synchronize oestrus and ovulation. This study aimed to evaluate the efficiency of a hormonal protocol for TAI in mares, based on a combination of progesterone releasing intravaginal device (PRID), prostaglandin (PGF2α ) and human chorionic gonadotropin (hCG); and compare financial costs between CAI and TAI. Twenty-one mares were divided into two groups: CAI group (CAIG; n = 6 mares; 17 oestrous cycles) and TAI group (TAIG; n = 15 mares; 15 oestrous cycles). The CAIG was subjected to CAI, involving follicular dynamics and uterine oedema monitoring with ultrasound examinations (US), and administration of hCG (1,600 IU) when the dominant follicle (DF) diameter's ≥35 mm + uterine oedema + cervix opening. The AI was performed with fresh semen (500 × 106 cells), and embryo was recovered on day 8 (D8) after ovulation. In TAI, mares received 1.9 g PRID on D0. On D10, PRID was removed and 6.71 mg dinoprost tromethamine was administered. Ovulation was induced on D14 (1,600 IU of hCG) regardless of the DF diameter's, and AI was performed with fresh semen (500 × 106 cells). On D30 after AI, pregnancy was confirmed by US. The pregnancy rate was 80.0% in TAIG and 82.3% in CAIG (p > .05). The TAI protocol resulted in 65% reduction in professional transport costs, and 40% reduction in material costs. The TAI was as efficient as CAI, provided reduction in costs and handlings, and is recommended in mares.

Oral misoprostol does not hasten oviductal transport of day-5 horse embryos.

In horses, prostaglandin E2 (PGE2) is produced by embryos around Day 5 post-ovulation; PGE2 functions directly at the oviduct promoting embryo transport into the uterus. Non-surgical collection of horse embryos for cryopreservation is recommended at Day 6.5-7 post-ovulation. It was proposed that misoprostol administered orally will hasten oviductal transport of horse embryos. In Experiment 1 (n = 15) there was comparison of time of embryo recovery (Day 6 and 6.5 post-ovulation) from mares administered misoprostol (Day 5 and 5.5) orally to that of untreated mares. On Day 6, embryo collections were attempted; if no embryo was collected, there was a second attempt on Day 6.5. In Experiment 2, (n = 16) misoprostol treatment was initiated on Day 4.5; there was the first embryo collection attempt on Day 5.5, followed by Day 6 and 6.5 if no embryo was collected. Blood samples were collected at 12 h intervals on Day 4.5 or 5, to Day 6.5. In Experiment 1, on days 6 and 6.5, respectively, there was collection of seven and one of a total of eight embryos detected at the time of collection per group (P = 1). In Experiment 2, 12 embryos were collected during 15 cycles with there being a total of three, two, and one collected from mares of both groups on Day 5.5, 6, and 6.5 post-ovulation, respectively (P = 1). Serum progesterone concentrations were not different (P ≥ 0.05). In conclusion, misoprostol, when administered orally, does not hasten oviductal transport of horse embryos.

Endothelial cells and angiogenesis in the horse in health and disease-A review.

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle.

Influência da somatotropina recombinante bovina no desenvolvimento folicular e na coleta de embriões em éguas / Influence of bovine recombinant somatotropin in the follicular development and embryo colletion in mares

Dez éguas, sem raça definida, foram submetidas a avaliações ultrassonográficas durante o intervalo interovulatório, avaliando-se folículos ≥ 5mm. Cinco éguas foram tratadas com 500mg de r-bST no primeiro e no 14º dia pós-ovulação (grupo GT), e as demais com soro fisiológico (grupo GC). Quando o folículo dominante atingiu diâmetro ≥ 40mm, foram induzidas com hCG e inseminadas 24 horas após, sendo submetidas à coleta de embrião seis dias após a ovulação. Os dados foram agrupados de acordo com o diâmetro do folículo dominante nas fases de emergência, divergência, dominância, pré-ovulatória, indução, inseminação e ovulação. Todas as éguas foram usadas duas vezes, no mesmo grupo. O GT apresentou crescimento folicular precoce para as fases de emergência, divergência, dominância e pré-ovulatória, assim como para o seu maior folículo subordinado, que cresceu mais cedo. As taxas de recuperação foram de 90% (GC) e 70% (GT), em 16 estruturas coletadas, obtendo-se uma não fecundada e um blastocisto inicial para o grupo GC; os demais, no estágio de mórula, apresentaram comportamento semelhante entre os grupos. Conclui-se que a r-bST influencia a dinâmica folicular de éguas, levando a uma antecipação do desenvolvimento folicular, que pode ser utilizada para encurtar o ciclo estral.(AU)

Ten undefined mare breeds were submitted to ultrasonographic evaluations during the interovulatory interval, evaluating follicles measuring ≥ 5mm. Five mares were treated with 500mg r-bST on the first and the 14th day after ovulation (TG group), and the others with saline (CG group). When the dominant follicle reached a diameter ≥ 40mm the ovulation was induced with hCG, and the mares were inseminated 24 hours later and submitted to embryo collection six days after ovulation. The data were grouped according to the diameter of the dominant follicle in the emergence, divergence, dominance, preovulatory, induction, insemination and ovulation phases. All mares were used twice, in the same group. The GT showed early follicular growth for the emergence, divergence, dominance and pre-ovulatory phases, as well as for its greater subordinate follicle, growing earlier. The recovery rates were 90% (CG) and 70% (TG), and 16 structures were collected, obtaining an unfertilized embryo and an initial blastocyst for the CG group, the others in the morula stage behaved similarly between the groups. It can be concluded that r-bST influences the follicular dynamics of the mares, leading to an anticipation of the follicular development that can be used to shorten the estrous cycle.(AU)

Effect of warming method on embryo quality in a simplified equine embryo vitrification system.

Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38 °C) for each of three warming systems: commercial warming solution (Kit); commercial embryo holding medium (EHM) with decreasing concentrations of sucrose (EHM + SS); or EHM alone without added sucrose. Embryos (n = 9 to 14 per treatment) were cultured in vitro for 24 h, stained with DAPI, TUNEL, and fluorophore-labelled phalloidin, and evaluated for nucleus number, mitotic rate, apoptotic rate, and actin filament distribution. In Exp. 2, to survey embryo viability in vivo, vitrified IVP blastocysts were shipped to an embryo transfer facility, then warmed immediately before transfer to recipient mares, using the warming treatments associated with the nominally best (Kit-RT, Kit-38, EHM-RT) and poorest (EHM + SS-38) assessed embryo quality in Exp. 1 (n = 7 to 8 per treatment). Subsequently, IVP blastocysts produced as part of our clinical program were vitrified and shipped, then warmed in embryo holding medium at an embryo transfer facility before transfer to recipient mares; fresh IVP embryos were shipped and transferred as controls. In Exp. 1, embryos increased significantly in diameter after culture (P < 0.01), with no difference among treatments. There was no difference (P > 0.05) in the number of viable nuclei, apoptotic rate, or microfilament distribution among treatments, or between vitrified-warmed and Control embryos. The mitotic rate was higher (P = 0.021) for Kit-RT (3.6%) when compared with the other treatment groups (1.5-2.0%). In Exp. 2, there was no difference (P > 0.05) in initial pregnancy (71.4-87.5%) or heartbeat (57.1%-85.7%) rates among warming treatments. In the clinical trial, there was no difference (P > 0.05) between vitrified-warmed and Control embryos in initial pregnancy (90.9% and 66.6%, respectively) or heartbeat (81.8% and 66.6%, respectively) rates. These results indicate that a semi-closed vitrification system using commercially-available media, and incorporating warming in the field in a single step using commercial embryo holding medium without cryoprotectants, can provide high pregnancy rates with IVP equine embryos.

Efeito de protocolo hormonal e programa de luz artificial na gestação de éguas receptoras de embrião durante o período de transição de primavera / Effect of hormonal protocol and artificial light program on the pregnancy of embryo recipient mares during spring transition period

O objetivo do estudo foi comparar o efeito das técnicas hormonais e de luz artificial nas éguas receptoras de embrião acíclicas avaliando as taxas de gestação aos 14 e 28 dias durante a fase de transição de primavera. Os 48 animais foram distribuídos aleatoriamente nos grupos: controle (CONT, n=16), éguas cíclicas na fase ovulatória; luz artificial (LUZ, n=16), éguas acíclicas submetidas ao tratamento de luz artificial; e hormônio (HORM, n= 16), éguas acíclicas submetidas ao protocolo hormonal na fase de transição. As éguas do grupo LUZ foram estimuladas por 60 dias com luz artificial durante cinco horas por dia. Nos grupos CONT e LUZ, quando observada a presença de folículo 35 mm de diâmetro e edema uterino grau II, foram administrados 1,5 mg de acetato de deslorelina e 1500 UI de hCG para induzir a ovulação. As éguas do grupo HORM foram tratadas com três doses de 1,5 mg de benzoato de estradiol e seguiram os mesmos protocolos dos Grupos CONT e LUZ. Foi avaliada a taxa de gestação por ultrassonografia aos 14 dias e confirmação aos 28 em todos os grupos experimentais. Foi realizada análise descritiva e teste Qui-quadrado (significância de 5%). Taxas de gestação aos 14 e 28 dias foram semelhantes (p>0,05) entre todos os grupos. Os tratamentos HORM e LUZ durante o período de transição inverno-primavera mostraram-se eficazes para atender ao programa de transferência de embr

The aim of this study was to compare the effect of hormonal and artificial light techniques on acyclic embryo recipient mares by assessing pregnancy rates at 14 and 28 days during the spring transition period. The 48 animals were randomly assigned to the groups: control (CONT, n = 16), cyclic mares in the ovulatory phase; artificial light (LIGHT, n = 16), acyclic mares subjected to artificial light treatment; and hormone (HORM, n = 16), acyclic mares submitted to hormonal protocol in transition phase. In the LIGHT group, mares were stimulated with artificial light for five hours a day, for 60 days. In CONT and LIGHT groups, when a follicle ≥ 35 mm in diameter and uterine edema ≥ grade II were observed, 1.5 mg of deslorelin acetate and 1500 IU hCG were administered to induce ovulation. In the HORM group, mares were treated with three doses of 1.5 mg of estradiol benzoate and followed the same protocols as the CONT and LIGHT groups. Pregnancy rate was assessed by ultrasound at 14 days and confirmation at 28 days in all experimental groups. Descriptive analysis and chi-square test (5% significance) were performed. Pregnancy rates at 14 and 28 days were similar (p> 0.05) among all groups. The HORM and LIGHT treatments during the winter-spring transition period proved to be effective during the embryo transfer programs. As it is a more natural method, the LIGHT protocol has the potential to be one more biotechnological tool in equine reproduction.

Efeito de protocolo hormonal e programa de luz artificial na gestação de éguas receptoras de embrião durante o período de transição de primavera / Effect of hormonal protocol and artificial light program on the pregnancy of embryo recipient mares during spring transition period

O objetivo do estudo foi comparar o efeito das técnicas hormonais e de luz artificial nas éguas receptoras de embrião acíclicas avaliando as taxas de gestação aos 14 e 28 dias durante a fase de transição de primavera. Os 48 animais foram distribuídos aleatoriamente nos grupos: controle (CONT, n=16), éguas cíclicas na fase ovulatória; luz artificial (LUZ, n=16), éguas acíclicas submetidas ao tratamento de luz artificial; e hormônio (HORM, n= 16), éguas acíclicas submetidas ao protocolo hormonal na fase de transição. As éguas do grupo LUZ foram estimuladas por 60 dias com luz artificial durante cinco horas por dia. Nos grupos CONT e LUZ, quando observada a presença de folículo ≥ 35 mm de diâmetro e edema uterino ≥ grau II, foram administrados 1,5 mg de acetato de deslorelina e 1500 UI de hCG para induzir a ovulação. As éguas do grupo HORM foram tratadas com três doses de 1,5 mg de benzoato de estradiol e seguiram os mesmos protocolos dos Grupos CONT e LUZ. Foi avaliada a taxa de gestação por ultrassonografia aos 14 dias e confirmação aos 28 em todos os grupos experimentais. Foi realizada análise descritiva e teste Qui-quadrado (significância de 5%). Taxas de gestação aos 14 e 28 dias foram semelhantes (p>0,05) entre todos os grupos. Os tratamentos HORM e LUZ durante o período de transição inverno-primavera mostraram-se eficazes para atender ao programa de transferência de embrião. Por ser um método mais natural, o protocolo LUZ tem potencial como mais uma ferramenta biotecnológica na reprodução de equinos.

The aim of this study was to compare the effect of hormonal and artificial light techniques on acyclic embryo recipient mares by assessing pregnancy rates at 14 and 28 days during the spring transition period. The 48 animals were randomly assigned to the groups: control (CONT, n = 16), cyclic mares in the ovulatory phase; artificial light (LIGHT, n = 16), acyclic mares subjected to artificial light treatment; and hormone (HORM, n = 16), acyclic mares submitted to hormonal protocol in transition phase. In the LIGHT group, mares were stimulated with artificial light for five hours a day, for 60 days. In CONT and LIGHT groups, when a follicle ≥ 35 mm in diameter and uterine edema ≥ grade II were observed, 1.5 mg of deslorelin acetate and 1500 IU hCG were administered to induce ovulation. In the HORM group, mares were treated with three doses of 1.5 mg of estradiol benzoate and followed the same protocols as the CONT and LIGHT groups. Pregnancy rate was assessed by ultrasound at 14 days and confirmation at 28 days in all experimental groups. Descriptive analysis and chi-square test (5% significance) were performed. Pregnancy rates at 14 and 28 days were similar (p> 0.05) among all groups. The HORM and LIGHT treatments during the winter-spring transition period proved to be effective during the embryo transfer programs. As it is a more natural method, the LIGHT protocol has the potential to be one more biotechnological tool in equine reproduction.

Evidence of five digits in embryonic horses and developmental stabilization of tetrapod digit number.

Previous work comparing the developmental mechanisms involved in digit reduction in horses with other mammals reported that horses have only a 'single digit', with two flanking metapodials identified as remnants of digit II and IV. Here we show that early Equus embryos go through a stage with five digit condensations, and that the flanking splint metapodials result from fusions of the two anterior digits I and II and the two posterior digits IV and V, in a striking parallel between ontogeny and phylogeny. Given that even this most extreme case of digit reduction exhibits primary pentadactyly, we re-examined the initial stages of digit condensation of all digit-reduced tetrapods where data are available and found that in all cases, five or four digits initiate (four with digit I missing). The persistent pentadactyl initiation in the horse and other digit-reduced modern taxa underscores a durable developmental stability at the initiation of digits. The digit evodevo model may help illuminate the biological circumstances under which organ systems become highly stabilized versus highly plastic.

Embryo survival in the oviduct not significantly influenced by major histocompatibility complex social signaling in the horse.

The major histocompatibility complex (MHC) influences sexual selection in various vertebrates. Recently, MHC-linked social signaling was also shown to influence female fertility in horses (Equus caballus) diagnosed 17 days after fertilization. However, it remained unclear at which stage the pregnancy was terminated. Here we test if MHC-linked cryptic female choice in horses happens during the first days of pregnancy, i.e., until shortly after embryonic entrance into the uterus and before fixation in the endometrium. We exposed estrous mares to one of several unrelated stallions, instrumentally inseminated them with semen of another stallion, and flushed the uterus 8 days later to test for the presence of embryos. In total 68 embryos could be collected from 97 experimental trials. This success rate of 70.1% was significantly different from the mean pregnancy rate of 45.7% observed 17 days after fertilization using the same experimental protocol but without embryo flushing. Embryo recovery rate was not significantly dependent on whether the mares had been socially exposed to an MHC-dissimilar or an MHC-similar stallion. These observations suggest that MHC-linked maternal strategies affect embryo survival mainly (or only) during the time of fixation in the uterus.

Power Doppler can detect the presence of 7-8 day conceptuses prior to flushing in an equine embryo transfer program.

In order to determine whether differences in uterine blood flow between pregnant and non-pregnant mares can be used to predict the presence of the equine embryo prior to flushing in an embryo transfer program, power Doppler ultrasonography was used on a total of 52 mares on days 7 or 8 post-ovulation. Computer analysis of Doppler images was subsequently performed using ImageJ v1.48 software. Vascular perfusion of the endometrium was analyzed using spot meter techniques, measuring mean pixel intensity and area of blood flow. Mares with positive flushings presented a higher uterine blood flow area (one embryo: 54.01 ± 2.27 mm2 or two embryos: 61.01 ± 6.73 mm2) prior to embryo recovery compared to barren mares (21.77 ± 2.22 mm2) (p≤0.05). However, significant differences in vascular perfusion were not detected between single or twin pregnancies. Blood flow area appears to be a good predictor for differentiation between pregnant and non-pregnant mares with an AUC: 0.869; p≤0.001 and an optimal cut-off value of 37.21 mm2. Both the mare's age and day of embryo recovery caused effects on uterine vascular perfusion. According to Youden's J statistics the uterine blood flow area of young pregnant mares was greater than 25.4 mm2 on day 7 (with a sensitivity of 75% and a specificity of 87.5%) and greater than 21.02 mm2 on day 8 post-ovulation (with a sensitivity of 93.8% and a specificity of 100%). The uterine blood flow area in adult pregnant mares was greater than 41.4 mm2 on day 7 (with a sensitivity of 80% and a specificity of 85.5%) and greater than 35.55 mm2 on day 8 after ovulation (with a sensitivity of 97.2% and a specificity of 85.7%). Evaluation on day 8 is therefore considered to be more reliable. Older and middle aged pregnant mares (5-18 years old) had increased uterine vascularization compared to young pregnant mares (2-5 years old) (p≤0.001). Conversely, older barren mares showed higher endometrial vascularity (35.06 ± 2.56 mm2) than young (17.21 ± 1.26 mm2) and middle aged non-pregnant mares (23.84 ± 1.50 mm2) (p≤0.05). We hypothesized that the higher blood flow area seen in older barren mares may be a consequence of a subclinical endometritis due to repeated flushing for embryo recovery. The results of the present study indicate that power Doppler ultrasound combined with computer assisted analysis of images are reliable techniques to detect early pregnancy prior to embryo recovery.

Assessing the fertility of two mares cloned from the same founder animal.

Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n = 31-39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pregnancies (5/7 for one, 11/12 for the other) were obtained at day 14 post-ovulation (D14 ), and 12 (3/7 for one; 9/12 for the other) foals were born. One cloned mare was a less efficient donor mare than the other (p < .05), In control donor mares, 623 embryo collections were performed, with a recovery rate (80%-496/623) significantly higher than for cloned mares. The recovery rate in the subpopulation of 2-5-year-old control donor mares (same age of cloned mares) (89%-127/143) and The recovery rate in the subpopulation of 12 control mares bred with the seven same stallions as clones (55%-17/31), were both higher than for cloned mare (p < .05). The success rate of transfer was not different between embryos produced by cloned mares (84%-16/19) and those produced by control donor mares (79%-392/496). However, the foaling rate per embryo collection was significantly lower for cloned mares (28%-12/43) than for control donor mares (52% - 325/623) (p < .05).

The mule (Equus mulus) as a recipient of horse (Equus caballus) embryos: Comparative aspects of early pregnancy with mares.

The aim of this study was to compare the embryonic and early fetal development of horse embryos between recipient mules and mares from day 10-60 of pregnancy, in addition to hormonal (eCG and progesterone), ovarian, and uterine characteristics for approximately 4 months. Embryo donor mares (n = 5) and two groups of recipients (acyclic mules, n = 7; cyclic mares, n = 7) were used. Donor mares were monitored daily by transrectal ultrasonography and inseminated using fresh semen. Cyclic recipient mares were synchronized with the donor's ovulation using PGF2α and deslorelin acetate. Mules were prepared for the embryo transfers with estrogen and progestagen. Embryo collection and transfer were performed 8 days after ovulation of the donor mares. Pregnancy diagnosis with ultrasonography began 1 day after embryo transfer. After pregnancy confirmation, the recipient mules received long-acting progesterone once weekly for at least 120 days. The first day of detection (day 10) of an embryonic vesicle (EV) was similar between mules and mares. A period of extensive intrauterine mobility of the embryonic vesicle was observed similarly in mules and mares from days 10-17. The day of fixation of the EV in mules tended to be 1-day earlier than in mares; however, the diameter and growth rate of the EV did not differ between the two species. The embryo proper was first detected at day 20, and the crown-rump, width, and diameter were similar between the two recipient types. The heartbeat and allantoic sac tended to be detected 1 day later in mules than in mares, while the umbilical cord was first observed around day 40 in both species. Besides the expected differences found in ovarian aspects and eCG production, similar endometrial diameter, uterine tone and echotexture, and progesterone levels were seen between the two types of recipients. In conclusion, striking ultrasound similarities in equine embryo and fetal development, and uterine characteristics were seen between mules and mares used as recipients of horse embryos.

Morphology, developmental stages and quality parameters of in vitro-produced equine embryos.

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection.

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22-27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.