Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern.

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.

One-step warming does not affect the in vitro viability and cryosurvival of cryotop-vitrified donkey embryos.

The objective of this study was to compare the effects of two warming protocols (three-step vs. one-step dilution) on embryo quality, post-warming embryo survival and embryo cell viability of donkey embryos vitrified by the Cryotop method. Twenty, Day 7-8, grade 1-2 donkey embryos were measured, morphologically evaluated and vitrified using the Cryotop technique. Embryos were then randomly warmed using two different warming procedures: (i) W3 (three-step dilution; n = 11): embryos were warmed in 1 M, 0.5 M and 0 M sucrose, and (ii) W1/0.5 (one-step dilution; n = 9): embryos were warmed directly in 0.5 M sucrose. After 3 and 24 h of warming, the embryos were measured and evaluated for their morphology, developmental stage and viability (Propidium Iodide-Hoechst 33,342 dyes). Although both treatments decreased embryo quality after warming (P < 0.05), no significant differences (P > 0.05) were observed between protocols in terms of post-warming embryo quality, diameter and embryo survival. Greater percentages of dead cells (P < 0.001) were observed when embryos were warmed directly in 0.5 M sucrose (one-step dilution) when compared to the three-step protocol. The percentage of ruptured embryos was 27.3% and 0% in W3 and W1/0.5 protocols (P = 0.0893), respectively. In conclusion, warming Cryotop-vitrified donkey embryos directly in 0.5 M sucrose had no negative effects on embryo quality and post-warming embryo survival. Moreover, one-step protocol may help to prevent rupture when donkey embryos warmed directly in 0.5 M sucrose. These results observed in vitro must be verified by embryo transfer.

The cryoprotective effect of Ficoll 70 on the post-warming survival and quality of Cryotop-vitrified donkey embryos.

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7-8, grade 1-2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P > 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P < 0.05). No significant differences (P > 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.

Cryopreservation of donkey embryos by the cryotop method: Effect of developmental stage, embryo quality, diameter and age of embryos.

Cryopreservation of embryos has the potential to become a valuable tool for the conservation of endangered donkey breeds. However, there are several factors that can affect cryosurvival of embryos. This study evaluates the effectiveness of the Cryotop method to vitrify donkey embryos and factors affecting the survival of vitrified-warmed embryos. Day 6-8 embryos were measured and morphologically evaluated. Embryos were then vitrified-warmed using the Cryotop technique. After 24 h post-warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). A total of 25 embryos were used, of which 17 were classified as Grade 1 (excellent), 5 as Grade 2 (good) and 3 as Grade 3 (fair). Based on their diameter, embryos were grouped as follows: ≤220 µm (n = 10), >220-300 µm (n = 8), and >300 µm (n = 7). Post-warming survival of vitrified embryos was similar (P > 0.05) to the control fresh embryos, regardless of embryonic diameter, developmental stage, and age of the embryos before vitrification. However, the proportion of embryos that survived vitrification procedures was numerically higher but not significantly different (P > 0.05) for Day 7 embryos (84.6%). The ability of Grade 1 (70.6%) and 2 (80%) embryos to survive vitrification procedures was higher (P = 0.0214) than those of Grade 3 (0%). The proportion of dead cells in Grade 3 embryos (56.5%) was higher (P < 0.01) than that of Grade 1 (3.2%) and 2 (1.5%) embryos. In conclusion, the Cryotop technique seems to be useful for Grade 1 and 2 donkey embryos. It is likely that donkey embryos show similar survival rates after vitrification in Cryotops irrespective of age, diameter and development stage.

Taxa de recuperação e características morfológicas de embriões muares (Equus caballus x Equus asinus) / Recovery rate and morphologic features of cross bred embryos (Equus caballus x Equus asinus)

A Transferência de Embrião (TE) contribuiu efetivamente para a produção de equinos e outras espécies. O mercado de muares tem apresentado um contínuo crescimento, entretanto, a aplicação das biotecnologias para a produção desses animais ainda é escassa. O presente estudo avaliou a taxa de recuperação embrionária e as características dos embriões provenientes do cruzamento de éguas com jumentos. Os embriões foram recuperados entre os dias 6 e 9 após a ovulação, dessa forma foi realizada a avaliação da taxa de recuperação embrionária e avaliação das características relacionadas com a idade, morfologia e diâmetro embrionário. A taxa de recuperação embrionária total foi de 55,9% (71/127), e não apresentou diferença para as colheitas realizadas em diferentes dias (D6-D9). Foram recuperados embriões nos estágios de mórula, blastocisto inicial, blastocisto e blastocisto expandido. O tamanho dos embriões variou entre 147-1688μm e a média do diâmetro de todos os embriões recuperados foi de 438,04μm. A recuperação de embriões muares pode ser realizada entre os dias 6 e 9 após a ovulação, e propicia a recuperação de embriões nos primeiros estágios de desenvolvimento.(AU)

Production biotechnologies, particularly embryo transfer (ET) has constantly been contributed to reproduce horses and other species. The mules market has shown continuous growth, however, the biotechnology for mule assisted reproduction is still scarce. The aim of this study was to evaluate the embryo recovery rate and the features of the embryos from mares bred with donkeys. The embryos recovery attempts were performed on days 6 to 9 after ovulation, in order to evaluate the embryo recovery rate and the features related to age, morphology and embryonic diameter in each day. The overall embryo recovery rate was 55,9% (71/127), and there was no significant difference (p>0,05) on different days (D6-D9). Embryos were recovered in stages of mórula, early blastocyst, blastocyst and expanded blastocyst. The diameter of the embryos ranged from 147-1688μm and the mean diameter of all the embryos collected was 438,04μm. The collection of hybrid embryos might be performed between days 6 and 9 after ovulation, and provides recovery of embryos in the early stages of development.(AU)

Serum biochemical profile of Pêga breed donkeys in the state of Minas Gerais / Perfil bioquímico sérico de jumentos da raça Pêga no estado de Minas Gerais

For the evaluation of serum biochemical parameters of Pêga breed donkeys (Equus asinus), for the different age groups and sex, blood samples of 123 animals were analyzed, of 29 males aged from 8 days to 10 years and of 94 females (15 lactating) aged from 2 days to 12 years, from two farms in the central-southern Minas Gerais, Brazil. The donkeys were divided by age into 5 groups: Group 1 (≤6 months), Group 2 (7-12 months), Group 3 (13-48 months), Group 4 (49-72 months), and Group 5 (≥73 months). According to the sex, they were divided into two groups, males and females. Serum biochemical elements: total protein, albumin, globulin, the A:G ratio, cholesterol, triglycerides, uric acid, creatinine, urea, phosphorus, calcium, Ca:P ratio, magnesium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and creatine kinase (CK), were evaluated in all animals. No significant differences were found for globulins, uric acid, urea and A:G ratio between age groups. Group 4 showed the highest values for total protein when compared with animals in Group 1 and 2. In Goup 2, the donkeys showed albumin levels lower than Group 3 and 4. Group 1 they had cholesterol levels higher than those in Group 2 and 4, and similar of the other groups. Higher phosphorus serum concentration was observed in Group 1. Calcium was significantly lower in Group 2. The Ca:P ratio was higher for Group 5. The magnesium values were significantly higher in donkeys older than 49 months (Group 4 and 5). The value of AST was lower for group 1. The ALP enzyme was significantly higher in younger animals up to 12 months, followed by gradual decrease with advancing age. The values of GGT were higher in donkeys up to 6 months, followed by decreasing values for subsequent groups. No differences were found between genders for albumin, cholesterol, creatinine, urea, uric acid, Ca:P ratio, magnesium, ALT, AST, and alkaline phosphatase. Females had higher values for total protein, globulin and triglycerides. Males showed higher values for A:G ratio, phosphorus, calcium and CK. The results showed that age and sex can influence serum biochemical values of Pêga breed donkeys.(AU)

Para a avaliação dos parâmetros bioquímicos séricos de jumentos (Equus asinus) da raça Pêga, quanto às diferentes faixas etárias e sexo, foram analisadas amostras sanguíneas de 123 animais, sendo 29 machos com idades de 8 dias a 10 anos e 94 fêmeas (15 lactantes) de 2 dias a 12 anos, de dois criatórios na região centro-sul do estado de Minas Gerais. Os animais foram divididos em 5 grupos de acordo com as idades: Grupo 1 (≤6 meses); Grupo 2 (7-12 meses); Grupo 3 (13-48 meses); Grupo 4 (49-72 meses) e Grupo 5 (≥73 meses). De acordo com o sexo, foram divididos em dois grupos, machos e fêmeas. Para todos os animais foram realizadas as análises de proteínas totais, albumina, globulinas, relação A:G, colesterol, triglicérides, ácido úrico, creatinina, ureia, fósforo, cálcio, relação Ca:P, magnésio, alanina aminotransferase (ALT), aspartato aminotransferase (AST), fosfatase alcalina (FAL), gama glutamiltransferase (GGT) e creatina quinase (CK). Não foram encontradas diferenças significativas para os elementos globulinas, ácido úrico, ureia e relação A:G entre as faixas etárias. O Grupo 4 apresentou os maiores valores para proteínas totais quando comparados aos animais dos grupos 1 e 2. Os animais do Grupo 2 mostraram valores de albumina inferiores aos Grupos 3 e 4. Os animais do Grupo 1 apresentaram valores de colesterol superiores aos do Grupo 2 e 4, e semelhante aos demais grupos. Maior concentração sérica de fósforo foi observada nos animais do grupo 1. O cálcio apresentou valor significativamente menor no Grupo 2. A relação Ca:P foi maior para o grupo 5. Os valores do magnésio foram estatisticamente superiores nos animais com idade superior a 49 meses (Grupos 4 e 5). O valor da AST foi menor para o Grupo 1.As enzimas FAL apresentaram valor significativamente maior nos animais mais jovens até 12 meses, seguida de redução gradual com o avançar da idade. Os valores da GGT foi maior nos jumentos com até seis meses de idade, seguido de valores decrescentes para os grupos subsequentes. Não foram encontradas diferenças entre os sexos para albumina, colesterol, creatinina, ureia, ácido úrico, relação Ca:P, magnésio, ALT, AST e fosfatase alcalina. As fêmeas tiveram valores maiores para proteínas totais, globulinas e triglicérides. Os machos mostraram maiores valores para relação A:G, fósforo, cálcio e CK. Pelos resultados nota-se que a idade e o sexo podem influenciar nos valores bioquímicos séricos dos jumentos da raça Pêga.(AU)

Comparison of different cryopreservation methods for horse and donkey embryos.

BACKGROUND: Few studies have been published about cryopreservation and embryo assessment in horses and donkeys. OBJECTIVES: To evaluate the viability of embryos collected from mares and jennies that were cryopreserved by slow freezing or by vitrification. STUDY DESIGN: Randomised controlled experiment. METHODS: Horse (n=19) and donkey (n=16) embryos (≤300 µm) were recovered on days 6.5-7.5 post-ovulation and assigned to control or cryopreservation protocols of slow freezing or vitrification. For slow freezing, 1.5 mol/L ethylene glycol (EG) was used. For vitrification, horse embryos were exposed to 1.4 mol/L glycerol, 1.4 mol/L glycerol + 3.6 mol/L EG and 3.4 mol/L glycerol + 4.6 mol/L EG, using Fibreplug or a 0.25 mL straw; donkey embryos were vitrified using Fibreplug with similar EG-glycerol solutions to above or 7.0 mol/L EG. Dead cells, apoptotic and fragmented nuclei, and cytoskeleton quality were assessed on thawed/warmed embryos. RESULTS: A significant decrease in embryo quality was observed after cryopreservation (P<0.05). Although the percentage of dead cells was lower (P<0.05) in control than in cryopreserved embryos, no differences were observed between freezing protocols used for horse or donkey embryos. While no differences were detected in the number of apoptotic cells in warmed horse embryos, in donkey embryos a higher incidence of apoptosis was measured after vitrification with EG-glycerol in Fibreplug (P<0.05). Vitrified horse embryos had a significantly (P<0.05) higher percentage of nonviable cells than donkey embryo. Actin cytoskeleton quality did not differ between treatments. MAIN LIMITATIONS: Difficulties in obtaining a large number of embryos meant that the number of embryos per group was low. CONCLUSIONS: Vitrified horse and donkey embryos did not show higher susceptibility to cell damage than those preserved by slow freezing, whether using straws or Fibreplug. However, Fibreplug with EG 7 mol/L resulted in fewer nonviable and apoptotic cells in donkey embryos. Donkey embryos showed lower susceptibility to vitrification than horse embryos. THE SUMMARY IS AVAILABLE IN SPANISH - SEE SUPPORTING INFORMATION.

Reproductive efficiency of asymptomatic Theileria equi carriers mares submitted to an embryo transfer program / Eficiência reprodutiva de éguas portadoras assintomáticas de Theileira equi submetidas a um programa de transferência de embriões

This study aimed to assess and evaluate the effects of Theileria equi infection on embryonic recovery, gestation and early embryonic loss. Thirteen Mangalarga Marchador Theileria equi positive donors (diagnosed through nested-PCR) and 40 embryos receptors were used. Donors were submitted to two embryo collections in two consecutive estrous cycles (GId); after, the same mares were treated with imidocarb dipropionate (1.2mg/kg IM.) in order to collect more embryos in two more estrous cycles (GIId). Receptors were divided into two groups (control and with treated) with 20 animals each, where one group was the control (GIr) and the other one (GIIr) treated with 1.2mg/kg IM of imidocarb dipropionate assessing the gestation rate at 15, 30, 45 and 60 days. After 52 embryo collections, the embryonic recovery rates were 53.84% (14/26) and 65.38% (17/26) (p> 0.05) for GId and GIId, respectively. The gestation rate was 70% (14/20) (p>0.05) at 15, 30, 45 and 60 days in group GIr and for GIIr was 85% (17/20) (p>0.05) at 15 days, 80% (16/20) (p>0.05) at 30, 45 and 60 days. The treatment with imidocarb dipropionate did not cause significant improvement in the reproductive efficiency at an ET program.

Este estudo teve por objetivo avaliar a influência da infecção por Theileria equi nas taxas de recuperação embrionária, gestação e perda embrionária precoce. Foram utilizadas 13 doadoras e 40 receptoras de embrião da raça Mangalarga Marchador, positivas para Theileria equi através da técnica de nested-PCR. Nas doadoras foram realizados duas coletas de embriões em dois ciclos estrais consecutivos (GId), em sequência, esses mesmos animais foram tratados com dipropionato de imidocarb (1,2mg/kg IM.) para realização de mais duas coletas de embriões em dois ciclos estrais (GIId). As receptoras foram divididas em dois grupos de 20 animais cada, onde um grupo foi o controle (GIr) e, o outro grupo, foi tratado (GIIr) com 1,2mg/ Kg IM de dipropionato de imidocarb, com intuito de avaliar a taxa de gestação aos 15, 30, 45 e 60 dias. Após a realização de 52 coletas de embrião, as taxas de recuperação embrionária foram de 53,84% (14/26) e 65,38% (17/26) (p> 0,05) para GId e GIId, respectivamente. A taxa de gestação foi de 70% (14/20) (p>0,05) aos 15, 30, 45 e 60 dias no grupo GIr e para o GIIr foi 85% (17/20) (p>0,05) aos 15 dias, 80% (16/20) (p>0,05) aos 30, 45 e 60 dias. O tratamento com dipropionato de imidocarb não promoveu melhora significativa na eficiência reprodutiva em um programa de TE.

Efeito da idade sobre a fertilidade e perdas embrionárias de éguas inseminadas com sêmen asinino diluído e resfriado a 5ºC por 12 horas de armazenamento / Effect of age on fertility and embryonic loss of mares inseminated with jackass semen diluted and cooled at 5ºC for 12 hours

Estudou-se o efeito da idade sobre a fertilidade de éguas inseminadas com sêmen asinino diluído, resfriado e armazenado. Os ciclos foram acompanhados por palpação transretal e rufiação, sendo as inseminações realizadas às terças, quintas e sábados, a partir da detecção de um folículo de 3,0 a 3,5cm de diâmetro, em um dos ovários, até a ovulação. O sêmen de cinco jumentos da raça Pêga foi diluído nos diluidores de leite em pó desnatado-glicose ou glicina-gema de ovo, resfriado a 5ºC e armazenado por 12 horas, sendo a dose inseminante de 400 x 106 espermatozoides móveis (no momento da diluição final, pré-resfriamento). Os resultados de 195 ciclos estrais, referentes a 141 éguas, foram agrupados em classes, de acordo com a idade das éguas: 2,5 a 6 anos, 6,5 a 10 anos, 10,5 a 14 anos e 14,5 a 19 anos. As taxas de concepção, ao primeiro ciclo, foram de 68,42%, 50,75%, 46,88% e 52,17% e, após quatro ciclos, de 69,57%, 47,92%, 46,34% e 45,71% para as faixas etárias de 2,5 a 6, 6,5 a 10, 10,5 a 14 e 14,5 a 19 anos, respectivamente (P>0,05). A idade não teve efeito sobre a fertilidade das éguas inseminadas com sêmen asinino diluído e resfriado...

The effect of the mare age on fertility of mares inseminated with diluted, cooled and stored jackass semen was studied. The females were controlled by transrectal palpation and teasing, and inseminated every Tuesday, Thursday and Saturday, since the detection of a 3.0 to 3.5cm follicle diameter, in one of the ovaries, until ovulation. The semen of five Pêga jackasses was diluted in skim milk-glucose or in egg yolk-glicine extender and cooled at 5ºC for 12 hours, with the inseminate dose of 400 x 106 motile spermatozoa (at the moment of the final dilution, before cooling). The results of 195 cycles of 141 mares were grouped, in accordance with the age: 2.5 to 6 years, 6.5 to 10 years, 10.5 to 14 years and 14.5 to 19 years. The pregnancy rates for the first cycle were 68.42%, 50.75%, 46.88% and 52.17%, and after four cycles, the pregnancy rates/cycle were 69.57%, 47.92%, 46.34% and 45.71%, respectively for 2.5 to 6, 6.5 to 10, 10.5 to 14 and 14.5 to 19 years (P>0,05). The mare age had no influence on fertility, using diluted and cooled jackass semen...

Paternally expressed genes predominate in the placenta.

The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes, of which 10 were paternally expressed. An additional 78 candidate imprinted genes identified by RNA sequencing also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase-1 (HAT1), a gene involved in chromatin modification, and lymphocyte antigen 6 complex, locus G6C, a newly identified imprinted gene in the major histocompatibility complex. The 78 candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This variability results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting.

Ultrasonographic features of the mule embryo, fetus and fetal-placental unit.

The aim of this study was to establish baseline ultrasound data concerning the mule conceptus during gestation. Ten multiparous Trotter mares were artificially inseminated with chilled semen from an Amiatino jack donkey. Daily transrectal ultrasonography was carried out from the day of ovulation until Day 50 of gestation to determine the following: first detection of the embryonic vesicle (EV), mobility phase, EV diameter, day of EV fixation, changes in EV shape, date of yolk sac regression and embryo crown-rump length. Monthly ultrasonic assessments from Day 50 of gestation to term were carried out. These assessments included an evaluation of fetal well-being and the growth of the mule conceptus, which were monitored using the following variables: cardiac activity, fetal activity and presentation, fetal fluid echogenicity, combined thickness of the utero-placenta unit and fetal orbital and aortic diameter. Mule EV first detection was observed earlier (37% at Day 8) than that observed in the equine pregnancy. EV diameter at first detection was 4.6 ± 1.1 mm. At Day 10, 75% of EVs were detected. EV fixation occurred on Day 17.1 ± 1.1, with a mean EV diameter of 2.5 ± 0.2 cm. EV growth rate was 4.04 mm/day from Days 11 to 16, 0.4 mm/day from Days 16 to 28 and 1.78 mm/day from Days 28 to 45 of pregnancy. The embryo proper was first detected on Day 19.9 ± 1.9 (average length 2.4 ± 1.4 mm), and the embryonic heartbeat was first detected on Day 24 ± 2.4. The fetal carotid pulse was observed at six months of gestation and provided a good means by which to estimate fetal cardiac activity in advanced gestation. The fetal heart rate was recorded from Month 2 of gestation to term. The mean ± SD of the combined uteroplacental thickness was assessed at the cervical-placental junction and at the ventral abdomen in mares between Months 2 and 5 until term, respectively. An abnormal fetal-placental unit and fetal inactivity was observed in association with abortion. Mule-conceptus biometric measurements correlated significantly with the gestational age, and these data were used to predict an unusually large mule fetus, which might result in dystocia. In conclusion, we can assume that early diagnosis of pregnancy failure and assessment of fetal biophysical profile and growth charts could improve the chances of gestation completion in mule-pregnant mares. The early detection of mares at risk for an abnormal pregnancy or delivery may increase the success of prompt treatments, therefore preventing costly emergency procedures and allowing proper obstetrical and neonatal assistance.

The broad ligament: a review of its anatomy and development in different species and hormonal environments.

The broad ligament is a double fold of peritoneum forming a mesentery for the human female genital tract. We investigated the anatomy of the broad ligament in different species and its hormonal regulation to determine if it had a role in gonadal positioning. The medical and veterinary literature was reviewed for descriptions of broad ligament anatomy and development. In addition, four adult female rats were dissected to compare the macroscopic anatomy of the broad ligament with any homologous structures in the male (n = 2). Detailed review was made of human males with persistent Müllerian duct syndrome (PMDS) and of bovine freemartin calves to determine the effect of abnormal hormonal environments on broad ligament development. Human and veterinary texts show variable broad ligament development between species, most being consistent with the size and shape of the uterus and uterine tubes. The broad ligament in adult female rats is a simple peritoneal fold and is homologous with the mesentery of the testis and vas deferens in males. Patients with PMDS and bovine freemartins have a broad ligament with intermediate anatomy. In PMDS the broad ligament is elongated and narrow, and not attached to the pelvic wall. The broad ligament is the mesentery of the genital ducts, and its anatomy varies with the degree of Müllerian duct fusion. The absence of a human male homologue is unusual, as the genital mesentery persists in male rodents. Apparent lack of a male homologue in the human may relate to obliteration of the processus vaginalis. The variable development of the broad ligament in pathological conditions is consistent with a role for steroid hormones in its development.

Patent urachus with subsequent joint infection in a free-living Grevy's zebra foal.

A free-living, female Grevy's zebra (Equus grevyi) foal was found lethargic, lame, with swollen joints, pyrexia, and urine dripping from the umbilicus. It died 2 days later despite intensive care. Gross examination revealed patent urachus and suppurative arthritis. Swabs were taken from the joints, the patent urachus, and urine for bacteriology. The dominant isolate was Escherichia coli. The joint infection was probably secondary to septicemia, resulting from the patent urachus. To our knowledge, this is the first report of neonatal patent urachus in a wild equid.

Ultrasonographic evaluation of the conceptus from days 10 to 60 of pregnancy in jennies.

Daily ultrasound examinations were conducted from Days 10 to 60 (ovulation = Day 0) of pregnancy to monitor the conceptus in jennies (n = 12). The embryonic vesicle was first detected on Day 11.5 +/- 0.9 (mean +/- SD; range 10 to 13 d) and was mobile until movement ceased (fixation) on Day 15.5 +/- 1.4 (range, 13 to 18 d). The vesicle was spherical from Days 10 to 18 (mean growth rate, 3.2 mm/d), non spherical (irregular) with a reduced growth rate (0.5 mm/d) from Days 19 to 29, and then grew at a moderate rate (1.6 mm/d) up to Day 46. On average, detection of the embryo proper (consistently located on the ventral aspect of the yolk sac) and embryonic heartbeat were Days 20.7 +/- 1.2 and 23.5 +/- 1.3, respectively. Formation of the allantoic sac was first detected on Day 24.4 +/- 1.7 and was complete on Day 36.8 +/- 1.6. Descent of the fetus (and formation of the umbilical cord) began on Day 37.9 +/- 1.7 and was complete on Day 44.1 +/- 2.1. Crown-rump length averaged 3.7, 15.4, 22.7, 37.5 and 59.6 mm on Days 20, 30, 40, 50 and 60, respectively. In general, morphologic features and dates of occurrence were similar to those reported previously in the mare.