Dystocia in cattle and horses: a compilation of historical artworks dedicated to Professor Gerhard Sand (1861-1921).

Professor Gerhard Sand (1861-1921) was the first professor of veterinary obstetrics at the Royal Veterinary and Agricultural University, Copenhagen, Denmark. He began teaching the theory and practice of obstetrics to veterinary students in 1887 and spent the following years until his death in 1921 developing the veterinary obstetrics teaching program. During this period, veterinary obstetrics was established as an independent discipline at the Royal Veterinary and Agricultural University. Professor Sand's teaching had a major impact on the field of veterinary obstetrics in Scandinavia. He was devoted to teaching veterinary obstetrics and produced a number of obstetrical illustrations, some of which showed different causes of dystocia, mainly fetal malpresentation in cattle and horses. Professor Sand created the illustrations with the intention of publishing a handbook of obstetrics, but due to illness and an early death, this work was never completed. This compilation of historical artworks of dystocia in cattle and horses includes some of these illustrations and is published to honour Professor Sand, with the intention of making his illustrations widely available for the teaching of veterinary obstetrics.

Ultrasonographic determination of day of parturition based on fetal biparietal diameter in pregnant bitches: Comparison of simple linear regression and mixed linear regression in breed-specific models and maternal weight grouped models.

The aim of this retrospective study was to compare simple linear regression and mixed linear regression on data grouped by breed or maternal weight group. The comparison was done to find the most accurate model for predicting day of parturition in pregnant bitches in clinical practice. The retrospective data consisted of fetal biparietal diameter determined by ultrasonography and day of parturition for all included bitches. The study population was divided into five maternal weight groups (miniature (≤5 kg), small (>5 to 10 kg), medium (>10 to 25 kg), large (>25 to 40 kg), and giant (>40 kg)) with three breeds in each group with 26 miniature-breed bitches, 13 small-breed bitches, 19 medium-breed bitches, 22 large-breed bitches, and 20 giant-breed bitches. The data was used to develop models to determine the number of days before parturition based on fetal biparietal diameter. A statistically significant effect was seen for grouping by maternal weight group (p<0.0001) and by breed (p=0.0057). Breed-specific models were derived and compared to each other within the same maternal weight group. Statistically significant differences between some miniature-breed and small-breed bitches were found using mixed linear regression analysis. The accuracies of all models were given as number of births within ±1 and ±2 days of estimated day of parturition and compared to an acceptable limit of 80% at ±2 days. All breeds and maternal weight groups except Dogue de Bordeaux and giant-breed bitches met the limit. Poor accuracies were seen when applying data from each breed to the maternal weight grouped model. Simple linear regression analyses were compared to mixed linear regression analyses. The simple linear regression analyses obtained the best accuracies for most of the breeds which is most likely to be caused by overestimation. Comparison of Chihuahua and German Shepherd to other studies showed similar accuracies between the highest reported and the two linear models. We recommend the use of breed-specific models based on mixed linear regression analysis in clinical practice. Further research is needed to analyze the differences between the two linear models and to confirm the tendency of more accurate predictions of day of parturition for medium-breed, large-breed, and giant-breed bitches when using breed-specific models.

A Diagnostic Survey of Aborted Equine Fetuses and Stillborn Premature Foals in Denmark.

Background: Loss of pregnancy in mares can have many different causes, including both infectious and non-infectious conditions. Extrapolation of findings from other studies is often uncertain as the significance of each cause varies across regions. Causes of pregnancy loss in mares have never been thoroughly studied in Denmark, so a prospective cross-sectional cohort study targeting the entire Danish population of pregnant mares was performed over a period of 13 months to obtain knowledge of the significance of individual causes. Fifty aborted or prematurely delivered stillborn fetuses were submitted for necropsy and examined by a panel of diagnostic laboratory methods. Results: Overall, a cause of fetal loss was established for 72% of the examined cases. Most cases (62%) were lost due to a non-infectious cause, of which obstruction of the feto-placental blood circulation due to severe torsion of the umbilical cord was most prevalent. Pregnancy loss due to a variety of opportunistic bacteria, including bacteria not previously associated with abortion in mares, accounted for 12%, while equid alphaherpesvirus (EHV) type 1 was the cause of pregnancy loss in 8% of the cases. EHV type 4 and Chlamydiaceae species were identified in some cases, but not regarded as the cause of fetal loss. Conclusion: Umbilical cord torsion was found to be the most prevalent cause of fetal loss in Danish mares, while infectious causes such as EHV type 1 and streptococci only accounted for a minor proportion of the losses. The study highlights the need for defined criteria for establishing an abortion diagnosis in mares, particularly in relation to EHV types 1 and 4.

Moody Mares-Is Ovariectomy a Solution?

Unwanted behaviour in mares is a commonly presenting problem to the veterinarian. This behaviour may range from the mare being uncooperative or aggressive when handled on the ground, kicking, bucking or rearing when ridden or being aggressive towards other horses. This purpose of the study was to evaluate whether bilateral ovariectomy in mares with unwanted behaviour improved the mare's behaviour and/or rideability from the owner's perspective. The mares were grouped and compared statistically based on their histological classification as having either "normal" or neoplastic ovaries. This study is a retrospective case series report of twenty-eight ovariectomized mares. A semi-quantitative value (1-10) pre- and post-ovariectomy for A) behaviour on ground/in stable and B) rideability was given, based on the owner's observations. The horses were grouped based on their histopathologic diagnosis as "Normal ovaries" or "Neoplasia". Following ovariectomy, rideability improved, with a score of ≥5 in 80% (8/10) of mares with normal ovaries and in 57% (8/14) of mares with ovarian neoplasm. A behavioural improvement of ≥5 was observed in 40% (4/10) of mares with normal ovaries, and in 43% (6/14) of mares with ovarian neoplasm. A significant difference was observed between the semi-quantitative value pre- and post-ovariectomy in both groups. No difference was observed in change in behaviour and rideability score between the group with normal ovaries and neoplastic ovaries. Mares with unwanted behaviour not obviously related to the oestrus cycle and to painful conditions may benefit from ovariectomy to alter their behaviour and rideability.

Colonization of the bovine uterus by Candida kefyr.

BACKGROUND: While fungal infections of the bovine uterus are well-known diseases in pregnant cattle, very limited knowledge exists on the presence and significance of fungi in the uterus of non-pregnant cows. Presence of fungi in the uterine lumen of postpartum (pp) cows has been reported, but little attention has been paid to this as most studies of the bovine pp uterus have focused on bacteria. CASE PRESENTATION: Microscopy of uterine lavage cytology slides of three cows from one herd revealed the presence of numerous yeast-like organisms, which were located either free in the fluid or within macrophages. Two of the cows were around 30 days pp, while the third was 7 months pp. None of the cows had been treated with antibiotics. Culturing of the flush samples was unsuccessful, but Sanger sequencing of DNA extracted from an endometrial biopsy of one of the cows revealed the presence of Candida kefyr (Kluyveromyces marxianus). Fluorescence in situ hybridization examination of endometrial tissue sections of two cows using probes targeting 18S rRNA of the K. marxianus group was performed and revealed the presence of yeast cells on the endometrium. Histology was performed and demonstrated hyphal and non-hyphal yeast-like organisms on the surface of endometrium and in the crypts. Tissue invasion was restricted to the superficial part of the epithelium and although endometrial inflammation was present, this was mild and considered as not being caused by the fungi. One of the cows became pregnant and delivered a normal calf at term, while the two others were not bred. CONCLUSIONS: Candida kefyr is commonly isolated from milk of cows with mastitis, but has not been reported in association with other diseases of cattle. The infection was present as a monoculture in all three cows, but the fungi had only colonized the uterine lumen and the endometrial surface. Only a mild non-suppurative endometrial inflammation was present, but within the uterine luminal content, many macrophages having phagocytized yeast cells were present. Re-examination of the cows did not reveal a persistent infection, so the infection probably resolved spontaneously.

Presence of bacteria in the endometrium and placentomes of pregnant cows.

Bacterial invasion of the bovine uterus during the postpartum period occurs in most cows, but the general consensus is that these bacteria are eliminated before the next pregnancy. The pregnant uterus has therefore hitherto been considered a sterile environment, but this assumption has now been challenged by recent studies in humans, which indicate that bacteria can be present in the placenta of term pregnancies without causing abortion. The aim of the present study was therefore to investigate whether bacteria are present in the uterus of pregnant cows. Specimens were taken from the inter-caruncular endometrium and from placentomes of slaughtered pregnant cows (n = 43) and subjected to histology, fluorescence in situ hybridization and massive parallel sequencing. Bacteria were observed in the tissue from 90.7% (39/43) of the cows by fluorescence in situ hybridization. Fusobacterium necrophorum, Porphyromonas levii and Trueperella pyogenes were located within the endometrium, on the endometrial surface and in the caruncular stroma, but their presence was not associated with inflammation. Data from massive parallel sequencing of the 16S rRNA gene from a subset of 15 cows indicated that the most abundant bacteria were the families Porphyromonadaceae, followed by Ruminococcaceae and Lachnospiraceae. Our results indicate that the bovine uterus is not a sterile environment during pregnancy as previously assumed and that a cow can carry a pregnancy despite the presence of a few potentially pathogenic bacteria in the uterus.

An investigation of the microbiota in uterine flush samples and endometrial biopsies from dairy cows during the first 7 weeks postpartum.

Metritis and endometritis commonly occur in dairy cows after calving. Although numerous studies have been performed to identify the causative pathogens, a complete overview has not been done. Metagenomic studies have analyzed the bacterial populations of uterine flush samples from postpartum (pp) dairy cows, but the microbiota in the uterine luminal fluid may differ from the microbiota of the endometrium itself, and important putative pathogens may have been overlooked. In the present study, we compared the microbiota of the uterine lumen and the endometrium of healthy, metritic, and endometritic cows. Samples were collected from 68 Holstein dairy cows at 1, 4, and 7 weeks pp, and the data were analyzed by deep sequencing of the V1 and V2 hypervariable regions of the 16S ribosomal RNA gene. The results showed that Porphyromonadaceae, Fusobacteriaceae, Leptotrichiaceae, and Mycoplasmataceae may be associated with uterine disease. The microbiota of the uterine flush samples and the endometrial biopsies were correlated, but the microbiota of the biopsies was more diverse. Fusobacteriaceae and Leptotrichiaceae were not observed in the biopsies at week 7, whereas they accounted for 20% and 13%, respectively, of the bacterial populations in the flush samples. The Mycoplasmataceae family was observed in much higher quantity in the flush samples than in the biopsies of the endometritis groups at weeks 4 and 7. Our findings support the observations of previous metagenomic studies and illustrate the importance of including endometrial biopsies to obtain more detailed knowledge of the pp uterine microbiota.

Risk factors associated with uterine fluid after breeding caused by Streptococcus zooepidemicus.

Infectious endometritis is a major cause of infertility in the mare and inflicts major losses on the equine breeding industry. The ability of the mare to eliminate uterine infections has been studied intensively for decades; however, despite identification of several factors contributing to the multifactorial pathogenesis and improved treatment, infectious endometritis remains a significant problem in a subpopulation of broodmares. Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most commonly isolated pathogens from the uterus of mares, suffering from infectious endometritis. Its ability to cause chronic latent infection by residing deep within the endometrial tissue has previously been described. The aim of the present study was to correlate different mare risk factors to infectious endometritis and pregnancy rates in broodmares and to investigate whether clonal or genetically distinct S. zooepidemicus strains isolated from mares with endometritis were associated with mare risk factors and the outcome of insemination. Mares (N = 152) were examined after natural cover, and 20% (31 mares) had intrauterine fluid (IUF) accumulation. Fifty-five percent (16 of 29) of the mares with IUF had infectious endometritis, and S. zooepidemicus was isolated in 81% (13 of 16) of these mares. Significantly more resting mares had IUF compared with foaling mares, and the foaling mares had the highest prevalence of positive bacterial growth from the uterine swab. The per-cycle pregnancy rate for the study was 63%. Pregnancy rate was significantly negatively affected by increased age (>12 years) and increased parity (>4), and the lowest pregnancy rate was observed in resting mares compared with foaling, barren, and maiden mares. Up to four S. zooepidemicus isolates were selected from each mare with growth of S. zooepidemicus from the uterine swab sample and further characterized by pulsed-field gel electrophoresis. In total, S. zooepidemicus isolates from 18 mares were analyzed with pulsed-field gel electrophoresis. In 13 mares, the isolates selected from each mare showed a high genetic relatedness within each individual mare, whereas two genetically distinct strains were isolated in five mares. A clonal S. zooepidemicus infection was associated with increased age, high parity, and poor vulvar conformation. Mares with clonal infection had a low pregnancy rate (38%) compared with mares with two strains isolated (80%). In conclusion, the results indicate that clonal S. zooepidemicus endometritis is associated with increased age, parity, and poor vulvar conformation in the broodmare.

Revisiting bovine pyometra--new insights into the disease using a culture-independent deep sequencing approach.

The bacteria present in the uterus during pyometra have previously been studied using bacteriological culturing. These studies identified Fusobacterium necrophorum and Trueperella pyogenes as the major contributors to the pathogenesis of pyometra. However, an increasing number of culture-independent studies have demonstrated that the bacterial diversity in most environments is underestimated in culture-based studies. Consequently, fastidious pyometra-associated pathogens may have been overlooked. Therefore, the primary purpose of this study was to investigate the diversity of bacteria in the uterus of cows with pyometra by using culture-independent 16S rRNA PCR combined with next generation sequencing. We investigated the microbial composition in the uterus of 21 cows with pyometra, which were obtained from a Danish slaughterhouse. Similar to the observations from the culture studies, Fusobacteriaceae, the family that F. necrophorum belongs to, was the operational taxonomic unit (OTU) observed in the largest quantities. By contrast, the Actinomycetaceae family, which includes T. pyogenes, constituted only 1% of the total number of reads. Thus we cannot confirm the previously reported role of species from this family in the pathogenesis of pyometra. Finally, we identified a large number of sequences representing three families of Gram-negative bacteria in the pyometra samples: Porphyromonadaceae, Mycoplasmataceae, and Pasteurellaceae. It is likely that these families comprise potential pathogenic species of a fastidious nature, which have been overlooked in previous studies. Our results increase the knowledge of the complexity of the pyometra microbiota and suggest that pathogens in addition to F. necrophorum may be involved in the pathogenesis of pyometra.

Streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group.

Streptococcus equi subsp. zooepidemicus is the pathogen most commonly isolated from the uterus of mares. S. zooepidemicus is an opportunistic pathogen and part of the resident flora in the caudal reproductive tract. The aim of this study was to investigate whether a genotypically distinct subpopulation of S. zooepidemicus is associated with endometritis in the mare, by genotyping and comparing uterine S. zooepidemicus strains with isolates from the vagina and clitoral fossa. Mares with (n=18) or without (n=11) clinical symptoms of endometritis were included. Uterine samples were obtained using a guarded endometrial biopsy punch, whereas a swab was used to recover samples from the cranial vagina and the clitoral fossa. If S. zooepidemicus was present, up to three colonies were selected from each anatomical location (max. 9 isolates per mare). Bacterial isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). S. zooepidemicus was isolated from the endometrium of 12 mares. A total of 88 isolates were analyzed by PFGE: 31 from the endometrium, 26 from the cranial vagina and 31 isolates from the clitoral fossa. For MLST 21 isolates were chosen. Results demonstrated a higher genetic similarity of the isolates obtained from infectious endometritis compared to isolates obtained from the caudal reproductive tract. In conclusion, we demonstrate for the first time that a genetically distinct group of S. zooepidemicus is associated with infectious endometritis in the mare.

Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro.

Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.