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Friday, January 28, 2022

Perinatal penile adhesions

Keywords: prepuce, equine, prepuberal, colt

A frenulum joins the prepuce and penis on the ventral aspect of the penis in all domestic animals except for the equine species. Usually, the frenulum In most animals breaks down largely under the effect of androgens (perhaps attempts at erection as well) after puberty. In horses this is not the case. No frenulum is present at birth and within days of foaling, a colt is able to extend his penis completely.

During fetal development the penis is of course adherent to the prepuce, dividing itself from that structure as organogenesis proceeds. 

In rare cases, exuberant adhesion between the prepuce and penis may actually occlude the preputial opening, even preventing urination. In one such case (see reference), gentle separation of internal lamina of the Prepuce and mild traction on the penis resolve the condition successfully

The images below show the penis and prepuce of a colt late in gestation.  Note the tenuous connection between the penis and prepuce at this time. At the time of foaling, the penis will have separated completely from the prepuce. 


Figure 1: Partial adhesion between penile and preputial mucosa (solid black arrow) in a newborn colt. The penis is being drawn to the right, the prepuce to the left. Image size: 3264 x 2448px

Reference

Canisso I.F. et al. 2020. Congenital phimosis causing preputial swelling in a newborn foal. Can Vet J .
61:247-250.


Thursday, January 23, 2020

Pyometra in a pluriparous Standardbred mare.

Keywords: pyometra, equine, treatment.

Primary author: Dr Rob Lofstedt.  Dept of Health Management, Atlantic Veterinary College. 

Additional authors:  Dr Anna  Potter (primary clinician & corresponding author: apotter@upei.ca ) and Dr Martha Mellish, both of the section of Theriogenology, Dept of Health Management, Atlantic Veterinary College & Dr Shannon Martinson of the Department of Pathology and Microbiology, Atlantic Veterinary College.


Editors: Drs Rob Lofstedt (lofstedt@upei.ca) and Allan Gunn (algunn@csu.edu.au). 

A 21 year-old nulliparous Standardbred mare in a teaching herd was euthanized because of respiratory pathology, lameness and a diagnosis of pyometra. Understandably, this mare had experienced numerous per vagina examinations, uterine cultures, uterine biopsies and at least one hysteroscopy.

Early in April 2019, an enlarged, fluid-filled uterus was palpable. On transrectal ultrasonography, this fluid was partially echogenic and suspected to be pus (see figure 1). Due to its size and dependency, it was not possible to delineate the uterus. It was also not possible to palpate the ovaries. Trans-abdominal ultrasonography revealed that the uterus was highly distended and at its cranial aspect, lay adjacent to the xiphoid process. Nevertheless (and typical for mares with pyometra) the mare was bright, alert and responsive and had a normal hemogram.

The mare’s ovaries containing no luteal tissue; not an unusual finding in equine pyometra. Pyometra in mares does not necessarily develop in a progesterone dominated milieu i.e. unlike the situation in cattle, cervical closure and myometrial quiescence due to progesterone is not required for the development of pyometra. In mares it is more likely that retention of  pus within the uterus is a function of deficient myometrial activity than cervical pathology (See LeBlanc et al 1994 and Troedsson 1999), especially in older animals. Occasionally however, cervical pathology is implicated.


Figure 1. Note the accumulation of moderately echogenic pus within the uterine lumen and follicles within the ovaries. This image also serves as an excellent example of  two common ultrasonographic artifacts; reverberation and enhancement through transmission (ETT). Image Copyright: Dr Martha Mellish. mmellish@upei.ca  Image size: 862 x 947 px.

During per-vagina examination, the mare’s cervix was found to be closed tightly. Cervical patency was only established after approximately 20 minutes of digital manipulation and only then, did pus drip from the vulva lips as shown in figure 2. This suggested that cervical pathology was indeed implicated in retention of pus within the uterus, perhaps in conjunction with poor myometrial tone as discussed earlier. As seen in figure 2, a large diameter stomach tube was eventually passed through the cervix to drain the uterus. Cytology and culture were performed prior to drainage of the pus.


Figure 2. The main image shows how pus dripped from the mare’s vulva after gradual dilation of the cervical canal. The inset shows how pus was then drained from the uterus. Image Copyright: Dr Martha Mellish. mmellish@upei.ca  Image size: 1513 x 1024 px

Several days after initial drainage, the uterus was  flushed repeatedly with saline (see figure 3). Then oxytocin was administered to facilitate the expulsion of any remaining fluid. In addition, 1000 mg of prostaglandin E-1 (misoprostol) in methylcellulose was applied to the cervix and cervical canal to facilitate dilation for drainage and further treatment.


Figure 3. Serial saline flushes showing the increase in clarity with each successive flush. Copyright: Dr Anna Potter apotter@upei.ca Image size 2000 x 1215

Pending culture and sensitivity results, the uterus was lavaged daily with lactated ringers or saline solution.  Two days after the initial drainage, culture results revealed Pseudomonas aeruginosa, sensitive to gentamicin, amikacin and enrofloxacin.  Treatment was initiated with 1g of gentamicin buffered with 10ml of 8.4% sodium bicarbonate infused into the uterus after uterine lavage, every 24 hours for five days.  On the fourth day of antibiotic treatment, an additional treatment of 100ml of 90% DMSO was added into the second to last flush for three days. These treatments were followed by twice daily oxytocin treatment at appropriate intervals.

Two weeks after initial dilation of the cervix and uterine flushing, ultrasonography revealed a uterus devoid of any free fluid, multiple small follicles in both ovaries and a corpus luteum. Uterine culture at that time revealed Citrobacter koseri and Streptococcus zooepidemicus. However, approximately a month days later, the uterus had re-filled with pus and uterine culture again revealed growth of Streptococcus zooepidemicus. Six days later i.e. approximately 48 days from the initial dilation of the cervix, approximately 2.5 liters of pus was drained and treatment with saline flushes, DMSO and oxytocin treatment were re-started. On this occasion, treatment lasted for four days; the antibiotic being penicillin, not gentamicin. On day four, another uterine culture revealed growth of Citrobacter koseri.

The mare was re-examined about three and a half months after initial presentation and summer rest on pasture. At that time, three liters of pus was drained from her uterus, followed by saline lavage.

In early November, 2019, re-examination revealed that a large amount of pus had accumulated in the uterus again. The mare was then euthanized and submitted for post mortem exam. Her pus-filled uterus is shown in the inset of Figure 4.


Figure 4. The pus-filled uterus of the mare seen during postmortem examination. The appearance of pus shown here is typical for equine pyometra. Image Copyright: Dr Shannon Martinson. smartinson@upei.ca  Image size: 1500 x 911 px.



Figure 5. It is probable that the cervical lesion seen here was implicated in the development of pyometra, together with general cervical cicatrization and myometrial compromise. The cause of the lesion was unknown and could not have been caused by foaling as this was a nulliparous mare. Image Copyright: Dr Shannon Martinson. smartinson@upei.ca Image size: 2002 x 1232 px.

Bearing in mind the difficulty of cervical dilation in this case and the absence of spontaneous drainage of pus before and after treatment, cervical pathology (seen in figure 5; probably fibrosis after cervical damage) was probably important in the development of pyometra in this case.

Editor’s comments: In light of the typical recurrence and usually dismal prognosis of equine pyometra, some may be critical of the handling of this case with regard to repeated attempts at physical drainage, antibiotic and anti inflammatory treatment. Consider however, that in recent years, the use of cervical wedge resection and intra-cervical stents have provided favorable outcomes in cases of equine pyometra. This suggests that even if one presumes the presence of myometrial inadequacy, attempts to dilate the cervix both physically and hormonally (using PGE analogs) may hold merit and should be considered in some cases. Of course, wedge resection and cervical stents should be considered as well.

The bacteriology in this case also deserves comment. The significance of any of the bacteria isolated is open to question. For example, a literature search of Citrobacter koseri is devoid of examples of infertility in mares so this can be presumed to be a contaminant.  Also, Streptococcus zooepidemicus is not only a common cause of endometritis in mares, it is a common commensal and contaminant of uterine cultures as well. Also, Pseudomonas aeroginosa is commonly isolated from soil samples and it therefore likely to be present on mares at pasture. The absence or presence of bacteria in single or serial cultures in this mare is also open to discussion; bacterial cultures vary in success according to sampling or culture methods. Essentially therefore, the bacterium or bacteria responsible for pyometra in this case remains a question.

Selected references:

Aguilar, J. et al. 2006. Importance of using guarded techniques for the preparation of endometrial cytology smears in mares. Theriogenology. 66:423-430

Arnold, C.E. et al. 2015 Cervical wedge resection for treatment of pyometra secondary to transluminal cervical adhesions in six mares. J. Am Vet Med Assoc. 246: 13540-1357

Blanchard, T. et al 1981. Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares. Theriogenology 16: 541-552

Ismaïl, R. et al 2013. Methods for recovering microorganisms from solid surfaces used in the food industry: A review of the literature. Int. J. Environ. Res. Public Health. 10:6169-6183

Katila, T, 2016 Evaluation of diagnostic methods in equine endometritis. Reproductive Biol. 16:189-196

Krohn, J. et al 2019 Use of a cervical stent for long‐term treatment of pyometra in
the mare: A report of three cases  Reprod Dom Anim. 54:1155–1159.

LeBlanc M.M. et al. 1994 Scintigraphic measurement of uterine clearance in normal mares and mares with recurrent endometritis.
Equine Vet.J. 26: 109-133

Pasolini M.V. et al. 2015 Endometritis and infertility in the mare – The challenge in equine breeding industry–A review. Open access peer-reviewed chapter.

Rötting A.K. et al 2004 Total and partial ovariohysterectomy in seven mares. British Equine Vet. J. 36:29-33

Troedsson. M.H.T. 1999 Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology. 52:461-471

Tuesday, May 21, 2019

Allantoic cysts


Keywords: cysts, allantoic, mesoderm, equine, chorion

Accumulations of fluid are occasionally seen on the allantoic surface of the placenta. They vary from microscopic in size to more that 15 cm in diameter.  Figure 1 shows examples of allantoic cysts. They contain clear interstitial fluid and fluctuate on contact. Allantoic cysts can also be seen on ultrasonography.

Figure 1. Allantoic cysts viewed from the allantoic surface of the placenta. Copyright for the larger image belongs to Dr Amy Clark. Email: aclark@essexequine.net. The author has not been able to ascertain the origin of the smaller image. Image size: 1103 x 849 px

Figure 2 shows accumulation of water adjacent to placental veins after it was introduced into the umbilical vein at faucet pressure. This probably illustrates the potential for the allantoic membrane to detach from the allantois; a precursor to the formation of allantoic vesicles. With some concern that this observation was due to excessive water pressure coupled with venous autolysis, the author was interested to see that the same phenomenon has been described by Ginther (1992) using air injection adjacent to placental vessels.

Figure 2. Accumulation of fluid adjacent to veins in the placenta after infusing water at faucet pressure into the umbilical vein. Image size: 2000 x 1419 px

Ginther (1992) stated that the air in his observations had entered the exocelom between the allantois ad chorion and separated the two membrane systems. This author however, suggests that it is more specific to ascertain that water or air (as the case may be) enters the exocelom-like space created within the mesoderm, not the exocelom itself. Many readers will not be familiar with this anatomical nuance. The following text box explains the difference between the two spaces.

Using figure 3 as a guide, note that a mesodermal layer begins to cover the allantois as it grows upward. The image shown here is one of a 28 day embryo with the yolk sac only partially surrounded by the allantois at its base. The allantois will surround the yolk sac almost completely at 55 to 60 days of gestation. Note that the yolk sac lies on the inward side of the allantois and the chorion on its outside. Figure 3 shows how the mesoderm grows into the exocelom, a space between the yolk sac and chorion, bringing a blood supply to the chorion. Within the advancing mesoderm itself however, numerous cavities develop and form an exocelom-like space, separate from the exocelom itself. This space persists until foaling. It was first described by Enders, A.C. and Liu, I.K. in 2000 and is an important part in the genesis of allantoic cysts. 

Figure 3. The genesis of an exocelom-like space within the mesoderm. On occasion, this is manifested as an allantoic cyst as shown on the right hand side of the image. Image size: 2632 X1717 px

The anatomy of a partially collapse allantoic cyst is shown in figure 4. This is a very large image, allowing close examination of its sub sections.

Figure 4. The anatomy of a partially collapsed allantoic cyst. Subsections of the image can be examined in detail after the image is expanded. They include: 1. The allantoic membrane, 2. A microcotyledon, 3. A cross section of an artery, 4. A vas vasorum (subsidiary vessel) within the artery wall and the collapsed lumen of the artery itself. Note the abbreviations: allantois (All) and chorion (Ch). Image size: 6050 x 4800 px

In summary, the allantoic membrane is only loosely connected to the chorion. This allows for separation of the toxic waste product in the allantois from the nutritional and respiratory functions of the chorion. As discussed in another LORI entry, the exocelom-like space may also form part of a de facto lymphatic system in the placenta. Furthermore, it has been suggested that the exocelom-like space may allow for a degree of motility for the fetus against the chorion which must remain attached to the endometrium at all costs.  However, it is likely that the allantoic membrane can be torn away from the chorion by movement of the foal's torso or limbs against the allantochorion, especially in late gestation when uterine space is limited. This would create an enlarged exocelom-like interstitial space in which fluid could accumulate, forming an allantoic cyst.

Selected references:

Bellini, C. et al. 2012. Are there lymphatics in the placenta? Lymphology 45:34-36

Castro, E. et al. 2011. Neither normal nor diseased placentas contain lymphatic vessels. Placenta 32:310-316

Ebrahim EI-Nefiawy, N. 2017. Development of Human Umbilical Vessels in The Second Trimester of regnancy: Histological, Immunohistochemical and Morphometric study. DOI:10.21608/EJH.2017.4079

Enders, A.C. and Liu, I.K. 2000. A unique exocelom-like space during early pregnancy in the horse. Placenta 21:575-583.

Ginther, O.J. 1992. Embryology and placentation. In: Reproductive Biology of the Mare: Basic and Applied Aspects, 2nd edn., Ed: O.J. Ginther, Equiservices, Cross Plains. page 462

Morresey P.R. 2009. Allantoic vesicles: Only a problem when they are a problem? Equine Vet Edu. 21:145-146

Schlafer, D.H. 2004. Post mortem examination of the equine placenta, fetus and neonate: methods and interpretations. Proc. Am. Ass. Equine Pract. 50:144-161.

Singh, K. et al 2009. Mega allantoic vesicles of the equine placenta. Equine Vet.Ed. 21: 143-144.






Monday, May 20, 2019

Fetal-placental circulation and the question of lymphatic drainage

Keywords: blood supply, vessels, equine, placenta, umbilical, artery, vein, urachus

Figure 1 shows how the umbilical arteries on either side of the bladder course towards the umbilical cord. These arteries of course, differ from those in the postnatal animal because they carry de-oxygenated blood. Because umbilical arteries transport blood under high pressure, they have a histological structure i.e. thick walled and elastic, similar to arteries in postnatal animals.  Figure 1 also shows how the bladder lies between the umbilical arteries and merges cranially into the urachus.

Figure 1. Arterial circulation and urine flow entering the umbilical cord. Image size: 1442 x 692 px

A brief review of fetal circulation:
During gestation, oxygenated blood from the placenta enters the liver via the umbilical vein but there are species variations in that regard. In horses (and pigs) it does not course through the liver as a single major vessels (as is the case in most animals). Instead, the umbilical vein splits into several veins that perfuse the liver which then pass into the posterior vena cava. In other animals the liver is only partially perfused by the umbilical vein and then, only on one side, not the other. This for example, is true of sheep where only the left lobe of the liver is supplied by the umbilical vein, while the right lobe is supplied by portal venous branches.

With no need for oxygenation by the fetal lungs, two thirds of this blood bypasses the right ventricle through the foramen ovale, entering the left ventricle and returning to the peripheral circulation. Any blood that leaves the right heart via the pulmonary artery, is shunted into the aorta via the ductus arteriosis. The foramen ovale begins to close at about 320 days of gestation and should close completely by 4 days postpartum. After birth, the umbilical vein and both umbilical arteries collapse. The umbilical arteries form the round ligaments on either side of the bladder and the umbilical vein forms the round ligament within the falciform ligament of the liver. The latter is so called because of its resemblance to a scythe (> Latin falx meaning sickle or scythe). For this, we thank the fruitful imagination of some long lost anatomist.

For an excellent diagram of fetal circulation, see Wilsher, S. et al. 2011. 

The arborized venules and veins of the placenta converge into a few major veins then finally, into a single umbilical vein within the amnionic portion of the umbilical cord. Therefore, the umbilical cord proximal to the fetus (within the amnion) contains only three major blood vessels; two arteries and one vein. Several abnormal variations on this theme have been described in humans by various authors and in the horse by Wilsher, S. et al. 2011. In normal specimens, these vessels are shown in figure 3 and another LORI entry as well.

Figure 2 shows the umbilical vein coursing into the liver. In the fetus it delivers oxygenated blood to the fetus.

Figure 2. Fetal circulation from the placenta showing the umbilical vein entering the liver from the umbilical cord. Image size: 1367 x 647 px

The histology of vessels in the umbilical cord is interesting. As mentioned earlier, the presence or absence of oxygenated blood has little effect on their structure when compared to arteries and veins within the body of the fetus. Blood pressure appears to be the dominant factor in their structure. In figure 3, one can appreciate that situation. 

Figure 3: A cross section of an umbilical cord from within the amnion, taken approximately 12 hours after foaling. The parity of the mare is not known. Image size: 1727 x 1336 px 

In figure 3, note the two thick-walled umbilical arteries (one torn during foaling) and the much larger, thin-walled umbilical vein. Also note the voluminous urachus. The urachus is virtually absent in humans because the allantois is reduced to a vestigial structure within the umbilical cord. Finally, note the relative absence of Wharton's jelly in comparison to humans as well.  

About Wharton's jelly:
Wharton's jelly is a mucopolysaccharide substance akin to the vitreous humor in the eye. Perhaps some are aware that Wharton's jelly was named for Thomas Wharton, an English Physician of the mid 17th century. Certainly, this author was not. Even more surprising, was that another structure far from the reproductive tract also bears his eponym; the duct of the sub-mandibular salivary gland!

The tunica adventitia in all animals provides strengthening around umbilical blood vessels throughout the body. Strangely, in humans, it is only umbilical vessels that lack the tunica adventitia. In this entry, the tunica adventitia is obvious around the equine umbilical vein and arteries In humans by contrast, Wharton's jelly is thought to provide support to the blood vessels in the absence of the tunica adventitia.

Despite a comprehensive search, the author was not able to retrieve publications on Wharton's jelly in primates but presumes its presence and nature is similar to that in humans.

Wharton's jelly serves as a significant research and commercial source for fetal stem cells in humans. This may occur in veterinary medicine as well. In fact, stem cells were recently harvested from the umbilical cord of a newborn foal and used to enhance wound healing in an adult horse.

Despite the interest in harvesting stem cells from the umbilical cord, little is known about where one should harvest multi-potential cells within a cross section of the cord in either humans or animals. Data reviewed by Davies. J.E. et al. show that the area around the umbilical blood vessels may be richest in these valuable cells. Although significant structural differences exist between species and Wharton's jelly is not obvious in domestic animals, it seems logical to suggest that perivascular areas be harvested for use in horses.

Tissue was examined at high magnification from three sites as shown in figure 3.They are labelled A, B and C. See below.

Figure 3 A: Area A; a high power section of the wall of the umbilical vein.  Note the predominance of collagenous tissue, the presence of an interposing smooth muscle layer and the absence of elastic tissue. Image size: 1904 x 1168 px

Figure 3B: Area B; a high power section of the wall of the umbilical artery. Note the predominance of smooth muscle layers and elastic tissue. Collagenous tissue is present as well but does not predominate as it seen in the umbilical vein. Image size: 1904 x 1258 px

Figure 3C: Area C. Note that the urachus is lined by both simple cuboidal and transitional epithelium. One will recall that the rest of the urinary tract is mostly lined with transitional epithelium. Despite being part of the urinary tract in the fetus, the allantois is lined with simple cuboidal epithelium. Image size: 2000 x 1150 px

The image below shows the infused blood supply to the chorioallantois (allantochorion). Because of space restriction in the infusion vessel, the placenta was not laid out in the traditional "F" shape for examination. Part of the amnion was torn away before the author retrieved the specimen. An intact amnion is shown in figure 8.

Figure 4. The blood supply and drainage in a placenta from a normal foaling. The venous system has been injected with red dye and emphasized digitally for clarity. The arteries remain off-white in color. The placenta is viewed from its allantoic surface. Image size: 2459 x 1424 px

In figure 4, the umbilical vein was infused with dye for facility. This is a large vessel and easy to catheterize for infusion.  No thought was given to the fact that veins usually have valves and as such, would have prevented back-flow of the dye into the vascular network. Unexpectedly, the veins filled with dye easily, with no evidence of valvular occlusion. This suggested the absence of valves in the placental veins. Upon researching this possibility, the author was surprised to learn that placental veins in other species are indeed devoid of valves; an interesting physiological situation on which to speculate. The author dissected some major veins and tributaries. Indeed, as shown in figure 5, no valves were seen in those vessels.

Figure 5: Three sections of placental veins from the allantochorion sampled at random, showing the absence of valves. Image size: 1914 x 1156 px

Figure 6a: A network of arterioles and venules beneath the allantoic membrane viewed from within the allantois. Capillaries (5 to 10 microns in diameter) are not visible at this magnification. Image size: 1967 x 1227 px

Figure 6b: This image shows the macroscopic similarity between veins and arteries in the allantochorion. The exclamation mark next to the vein label shows that this was unexpected. Image size: 2000 x 1234 px

Placental veins carry oxygenated blood therefor one might expected them to be more red in appearance than the veins. Following the umbilical vein to its tributaries in figure 6b, this was not the case. 

In figure 6a, note how the majority of small vessels run parallel to one another. This is reminiscent of capillary architecture in villi of the microcotyledons; elegantly illustrated by Abd-Elnaeim et al. 2006 using corrosion casting and scanning EM. Parallel placement of vessels against a transfusion surface (the chorion in this case) provides for efficient transfusion of gasses, waste and nutrients. All these vessels are closely adherent to the chorion. The allantoic membrane by contrast, is loosely attached to the chorion and can easily be detached from the chorion. This can be seen around the larger vessels in figure 6, where water from the infused vessels has split the allantois from the chorion. This observation is substantiated in figure 7 sampled from a non-infused placenta.

Figure 7: A cross section of the allantochorion showing the location of major blood vessels adjacent to the chorion, not the allantois. In terms of placental function, this would be expected. In this case, placental vessels were not infused in any manner, yet the tenuous connection between allantois and chorion is obvious. This may be an important factor with regard to the formation of allantoic cysts as discussed elsewhere in LORI (pending). Image size: 1436 x 1320 px

Figure 8: A cross section of the allantoic membrane, showing the small vessels that serve that structure. There appear to be few major vessels serving the allantois. It is likely that this is the case because the allantois is a storage structure for waste products, requiring little or no transfusion system. In all probability, small vessels are required for the structural integrity of the allantoic membrane alone. Image size: 1085 x 1315 px

Figure 8: This placenta shows the expected distribution of arteries and veins in the amnioallantois (usually abbreviated to amnion). The yellow arrow indicates where urine flow would have emerged from the urachus. The large, rejected endometrial cups (allantoic polyps) are discussed elsewhere in LORI (pending). Image size: 1085 x 1315 px

As expected, the vascularity in the amnion is sparse compared to that in the allantochorion. This is perhaps even more obvious in figure 4 than figure 8. The vascular supply to the amnion serves only the integrity of two fused, and relatively simple membranes. The supply to the allantochorion serves the complex nutritional, excretory, secretory and respiratory interface between the dam and fetus. 

Lymphatic drainage from the placenta
In essence, the placentas in horses, humans and other animals lack lymphatic systems. The placenta contains neither lymphatic vessels nor lymph nodes. What then is the mechanism for draining interstitial fluid from the placenta?  

Tissues throughout the body are of course, sustained by the presence of interstitial fluid provided by capillaries. Of the total amount of interstitial fluid delivered by capillaries, approximately 85% enters the venules under the effect of osmotic pressure. The remaining fluid drains into blind-ended lymphatic vessels and immediately becomes known as lymph, although its composition is essentially the same as interstitial fluid. 

It is not known how the placenta deals with drainage of interstitial fluid. However it may be safe to assume that the absorptive character of the placenta itself (transfusing fluid from the maternal circulation) partially represents the function of the lymphatic system in the placenta. The remaining fluid in interstitial spaces may be absorbed directly into the vasa vasori of placental veins. This subject in addressed in another LORI entry (pending).

For this entry, the author wishes to acknowledge the assistance of colleagues Drs T. Muirhead and G.Wright, Dr Martha Mellish for collecting specimens and the global technical support from the AVC post mortem laboratory and quality assurance programs.

Selected references:

Abd-Elnaeim et al. 2006 Structural and hemovascular aspects of placental growth throughout gestation in young and aged mares. Placenta 27:1103-1113

Arpi, L.M.B. 2018. Histology of Umbilical Cord in Mammals http://dx.doi.org/10.5772/intechopen.80766

Bellini, C. et al. 2012. Are there lymphatics in the placenta? Lymphology 45:34-36

Castro, E. et al. 2011. Neither normal nor diseased placentas contain lymphatic vessels. Placenta 32:310-316

Davies. J.E. et al. 2017. Concise review: Wharton’s Jelly: The rich, but enigmatic, source of mesenchymal stromal cells. Stem cells translational medicine. 6:1620–1630

Ebrahim EI-Nefiawy, N. 2017. Development of Human Umbilical Vessels in The Second Trimester of regnancy: Histological, Immunohistochemical and Morphometric study. DOI:10.21608/EJH.2017.4079

Edelstone, D.I. et al. 1978 Liver and ductus venosus blood flows in fetal lambs in utero. Circulation research 42: 426-443

Faber, J.J. and Anderson, D.F. 2002. Am J Physiol Heart Circ Physiol 282:H850–H854

Girodroux, M. et al. 2019. A single umbilical artery and omphalophlebitis in an Arabian foal. Equine Vet. Educ. 31:6-12

Lanci, A. et al. 2019. Heterologous Wharton's Jelly Derived Mesenchymal Stem Cells Application on a Large Chronic Skin Wound in a 6-Month-Old Filly. Front. Vet. Sci., 30 January 2019 | https://doi.org/10.3389/fvets.2019.00009

Spivack, M. 1946. The anatomic peculiarities of the human umbilical cord and their clinical significance Am.J.Ob.Gyn. 52:387-401

Stehbens, W.E. et al. 2005. Histopathology and ultrastructure of human umbilical blood vessels.
Fetal Pediatriatric Pathol. 24:297-315.

Slater, S. 2005. Patent ductus arteriosus in a 9-day-old Grant’s zebra. Can Vet J. 46:647–648

Wilsher, S. et al. 2011. Three types of anomalous vasculature in the equine umbilical cord.Equine Vet. Educ. 23:109-118



Tuesday, February 19, 2019

The oviduct (uterine tube) revisited

Keywords: equine, mare, oviduct, uterine tube

The Nomina Anatomica Veterinaria refers to this structure as the uterine tube (to distinguish it from the oviduct of birds). However, that term is seldom used in either practice or publication. In both humans and animals, it is instead referred to as the oviduct. The oviduct in any species is amazing but even more so in mares. This entry substantiates that impression.

As shown in figure 1, the oviduct runs within the ovarian bursa, almost parallel to its margin but a full centimeter or more away.

Figure 1. The left ovary of a two year old mare, suspended under water. The ovulation fossa is invisible, pointing ventrally in the image. 2903 x 2054 px


Figure 1 is labeled above. Divisions of the oviduct (infundibulum, ampulla etc) shown here are those adopted from, and and described in: Aguilar, J.J. et al. 2012. Histological characteristics of the equine oviductal mucosa at different reproductive stages. J.Equine.Vet. Sci. 32:99-105. Note: The ovary in situ hangs from the mesovarian ligament so that the ovulation fossa faces ventrally. In this image, the ovary and bursa have been rotated as shown by the arrow in the small inset. The ovulation fossa is not yet visible despite this rotation. Image size: 1600 x 1041 px

The ovarian bursa can be likened to a lateral, low-drooping eyelid over an eye (the ovary). The long dimension of the ovoid-shaped ovary lies on a cranial-caudal axis. The cranial pole of this axis is slightly higher than the caudal pole. The infundibulum lies at the cranial pole of the ovary. As shown in figure 1, it is not attached to the ovary. In fact, it lies a remarkable distance from the ovulation fossa.

This anatomy never ceases to amaze the author. In essence, the infundibulum acts like a catcher's mitt in a baseball game, covering an ambitious area some distance from the pitcher's mound i.e. the ovulation fossa in this analogy. The baseball is of course, the oocyte. The infundibulum is well supplied with smooth muscle and engorged blood vessels during estrus, expanding the catcher's mitt. Yet the precise mechanism and magic behind the dependability of the catcher remains unknown and unseen.

For those not familiar with baseball i.e. (insert nationality here), the author suggests consulting the book "Complete idiots guide to baseball and oocytes".

It has been suggested that the fimbriae of the infundibulum sweep the surface of the ovary at the time of ovulation, picking up the oocyte in the process, moving it into the complex folds of the infundibulum. Although the frequency of loss of oocytes into the peritoneal cavity is unknown in mares, it probably does occur; it has certainly been documented in humans. Certainly, losses of oocytes into the peritoneal cavity is described in poultry, especially broiler hens. Interestingly, laying hens, selected for egg production are less prone to peritoneal loss of oocytes. In rodents and canids, oocyte-catching expertise by the infundibulim is less important than other species. This is because ovarian bursae in those animals surround their ovaries completely and are continuous with the infundibula themselves. This makes it impossible for their oocytes to escape into the peritoneal cavity.

Figure 2: An oocyte, 150µ in diameter is shown at the end of the yellow arrow. This simulates the appearance of a real oocyte shortly after ovulation. Note its size relative to the infundibulum and ostium. Also note the cloud-like mass around the oocyte. This is a simulation of the large cumulus oophorus that accompanies the oocyte into the infundibulum. The cumulus is lost within 6 to 12 hours after the oocyte enters the oviduct (personal communication; Dr Katrin Hinricks).  Image size: 1630 x 1236 px

When stretched out, the oviduct in a mare is a little longer than the human hand i.e. about 20 to 30 centimeters. It is a continuum with no distinct delineation. To facilitate functional descriptions however, it is divided into three main sections i.e. the infundibulum (L.< funnel), ampulla (L.< flask) and finally the isthmus (L.<neck of land between two seas) narrowing as it enters the uterus at a papilla that forms the uterotubal junction.

Figure 3: This figure includes a large section of the infundibulum and a smaller inset image of the isthmus. They are both at the same scale of magnification. Again, the author has modeled an oocyte in the infundibulum and in addition, an embryo in the isthmus. Both are visible beside black bars 150µ ling at the end of the yellow arrows. This was done to compare the size those structures with the histology of parts of the oviduct. The diameter of an equine oocyte is approximately 150µ; slightly larger than that a bovine oocyte (120µ). By day 6, the equine embryo is approximately 200µ in diameter. The white scale within each image is 500µ and the small bar adjacent to the oocyte and embryo is 150µ long. Image size: 4242 x 3090 px

The oviduct is of course, a conduit to transport oocytes, then embryos from the ovary to the uterus. But it is also an organ that performs the seemingly impossible task of (often simultaneously) transporting oocytes towards the uterus while promoting the ascent of spermatozoa from the uterus into the oviduct. Like other tubular organs throughout the body it has inner circular and outer longitudinal layers of smooth muscle that promote peristaltic movement. It also has within its mucosal lining, ciliated cells that also play a role in gamete transport. Presumably, peristaltic movements play a major role in transporting oocytes towards the uterus while cilia perform a major role with regard to the ascent of spermatozoa. However, the exact integration of these two propelling mechanisms has yet to be described.

It is known that spermatozoa ascend into the oviduct and bind to its epithelium, usually laying in wait for the oocyte to arrive after ovulation. It is possible for fertilization to occur when spermatozoa arrive in the oviduct up to 18 hours after ovulation (with post ovulation insemination) but it is far more common for spermatozoa to spend a day or two or even up to 7 days in the oviduct before ovulation. During that time, Ca++ fluxes within the oviduct suppress capacitation while gaseous exchange and nutrition keep spermatozoa viable. True, it is more likely that an oocyte will be fertilized with close synchrony between insemination and ovulation but the ability of the oviduct to keep spermatozoa viable for long periods of time is still remarkable; far superior than any device contrived by humans.

As amazing as sperm preservation maybe in mares, it is overshadowed by the achievement of oviducts in other species. In some fruit bats in hibernation for example, spermatozoa can survive for weeks even months within the oviduct! 

Capacitation and the release of spermatozoa from their binding sites on the oviduct epithelium is orchestrated by changes in the steroid milieu, especially increased progesterone production shortly before ovulation. The effect of the oviduct environment on spermatozoa is of critical importance in mares. Specifically (and peculiar to mares again) it is only in the oviduct that fertilization can occur. Therefore, unless spermatozoa are injected directly  into oocytes (ICSI), in-vitro fertilization in horses is seldom successful.


Figure 2: A 16 gauge blunted needle is introduced into the ostium of the oviduct from a two-year-old mare. Blue dye is then introduced to outline the convoluted shape of the oviduct. Evidence of the dye in the uterus is shown as it permeates through to the serosa at the site marked B. This image defines the oviduct clearly. However the anatomical divisions (especially the ampullary-ithmic junction) so glibly discussed in literature, are far from obvious. Image size: 3456 x 2557 px

The uterotubal junction too, is a remarkable structure in mares. Spermatozoa deposited in the uterus are swept up to the uterotubal junction and are found in the oviduct within a few minutes of insemination. Yet, it is virtually impossible in normal mares, to force either fluid or air from the uterine lumen into the oviduct. This is because the oviduct of the mare is unique amoung domestic species with respect to its uterotubal junction. In mares, the distal oviduct has a well developed muscularis which acts as a sphincter, making mechanical entry from the uterus difficult. One can introduce fine tubes into the oviduct from the uterus but otherwise the uterotubal junction in mares acts as a one-way valve preventing fluid ascent from the uterus. To some degree, this may explain the relative lack of oviduct pathology in mares compared to cattle.

Alter about six and a half days in the oviduct, incubating mainly at the ampullary-ithmic junction, embryos enter the uterus. In mares of course, single embryos are far more common than twin embryos. At that time, late morulas or early blastocysts can be collected by flushing the uterus. Before that time it is impossible to retrieve a fertilized embryo by flushing the uterus alone.

Although unfertilized oocytes are occasionally found in the uterus, this is unusual. In general, if an oocyte is not fertilized in mares, it will not reach uterus. This phenomenon is unique among equids. Therefore it is generally not important to determine if oocytes have been fertilized when they are collected for embryo transfer. It is now universally recognized that production of prostaglandin E2 by embryos (not oocytes) causes relaxation of oviduct smooth muscle. This allows transport of the embryo into the uterus. When mares are examined postmortem, it is not unusual to find flattened, degenerate oocytes from previous cycles, caught within the oviduct.

Pathology? 
Fibrinous masses that can be several mm in size, are often found in the oviducts of mares. Again, this is a phenomenon peculiar to equids. It has been suggested that they are pathological and may block the passage of  oocytes and embryos in the oviduct. However, these masses are found in 75% to 85% of mares (as reviewed by Tsutsumi, Y. 1979) therefore they are unlikely to be pathological. The origin of the masses is unknown but they may arise from fibrin discharged from follicles after ovulation, during the formation of corpora hemorrhagica. In that regard, it is also very common to see fibrin tags in and around the ovary in apparently normal mares. In fact, careful inspection of the images in this entry will reveal such tags. The author had the dubious privilege of spending many hours at an equine slaughter plant and saw such tags many times, often in young mares. The same can be said for para-ovarian (wolffian) cysts, sometimes reported as abnormal too. The vast majority of otherwise normal mares have these cysts. 

Selected references:

Allen, W.E. et al. 1979. Evaluation of uterine tube function in pony mares. Vet. Record 105: 364-366

Arnold, C.E. and Love, C.C. 2013. Laparoscopic evaluation of oviductal patency in the standing mare. Theriogenology 79: 905-910

Bennett, S. 2002. Surgical evaluation of oviduct disease and patency in the mare. Proc. AAEP 48:347-349

Betteridge, K.J. 2000. Comparative aspects of equine embryonic development. Anim. Reprod. Sci. 60: 691-702

Brinsko, S,P. 1991. The effect of uterine lavage performed four hours post insemination on pregnancy rate in mares. Theriogenology 35: 1111-1119

Dobrinski, I. et al. 1997. Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biol. Reprod. 56: 861-869

Freeman, D.A. 1991. Time of embryo transport through the mare oviduct. Theriogenology 36: 823-830

Ghazal, S et al. Glob. libr. women's med., (ISSN: 1756-2228) 2014; DOI 10.3843/GLOWM.10317

Hinrichs, K. 2010. In vitro production of equine embryos: State of the art. Reprod. Domestic Anim 45: 3-8

Hunter, R.H.F. 1999 Ovarian follicular fluid, progesterone and Ca2+ ion influences on sperm release from the Fallopian tube reservoir. Gamete biology. 54: 283-291

Hunter, R.H.F. 2008. Sperm release from oviduct epithelial binding is controlled hormonally by peri‐ovulatory graafian follicles. Molecular Reprod. Devel. Incorporating Gamete Research 75: 167-174

Inoue, Y. 2013 Hysteroscopic hydrotubation of the equine oviduct. Equine Vet J. 45:761-765

Kenney, R.M. 1993. A review of the pathology of the equine oviduct. R. M. Kenney. Equine Vet. J. 25 (S15): 42-46

Leemans, B.M. 2015. Why doesn’t conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization. Reproduction 152: R233-R245

Navara, K. J. 2015. Higher rates of internal ovulations occur in broiler breeder hens treated with testosterone. Poult Sci. 94:1346-1352

Rigby, S. et al. 2000. Oviductal sperm numbers following proximal uterine horn or uterine body insemination. Proc. AAEP. 46:332-334

Saltiel, A. et al. 1986. Pathologic findings in the oviducts of mares.  Am. J. Vet Res. 47: 594-597

Sieme, H. et al. 2003. The effects of different insemination regimes on fertility in mares. Theriogenology 60: 1153-1164

Smits, K et al. 2016 The equine embryo influences immune-related gene expression in the oviduct. Biol. Reprod. 36: 1-8

Tsutsumi, Y. 1979. Evidence of the origin of the gelatinous masses in the oviducts of mares.
J. Reprod. Fert. 57: 287-290

Weber, J.A. 1995. Relaxatory effect of prostaglandin E2 on circular smooth muscle isolated from the equine oviductal isthmus. Biol. Reprod. Monograph. series1: 125-130

Wednesday, February 6, 2019

An STL model of the equine uterus

Keywords: model, 3D, STL, equine, uterus.

The author has used a stereolithographic (STL) file to view and print equine uteruses and pelvic structures in 3D.
NEW: This, small file can even be downloaded on a cell phone. Pay no attention to dialog alluding to processing of the file. Go ahead and download it. With the App "Fast STL viewer" (see Google play store) already downloaded on your phone, click on this downloaded file (not the App itself!) and the uterus will appear on your phone as shown in the thumbnail above. Incidentally, the same file can be downloaded and viewed on your laptop. 

Viewing the model:
For STL viewing alone on your laptop the author suggests that you download this file. Click on it and your laptop (PC or Apple) will bring up a suitable program for viewing. An example of  such an interface is shown below. 
Printing the model:
Custom STL printing can be done through Axis Prototypes in Montreal after contacting the author.  Axis holds the right to a digitally engineered model of the tract and pelvis that is ready for printing. High quality prints such as those seen here are not inexpensive.  Printing a 25 cm model (caudal to cranial) printed from STS nylon will cost approximately $600 Can. ($460 US). 

Manipulating the model:
In the absence of a real uterus or a printed tract, this digital model serves to remind one of many practical aspects of equine reproduction  A short list comes to mind immediately. Some may wish to print this list so that it can be referred to as the model is manipulated. Better still, manipulate the model on a cell phone while reading the learning points on a laptop.

1.Rotate the tract for a view of the vulva lips and clitoris. Be reminded of its "winking" appearance often, but not exclusively, seen during estrus. Note the importance of the clitoris in diagnosis of CEM and the conformation of the vulva lips in infertility. 

2.Enter the vagina and move cranially until the external urethral orifice is encountered. Note that the hymen is normally found just cranial to the orifice but is absent here because this tract was modeled from that of a pluriparous mare. At this point, one should also contemplate the potential problem of urine pooling in the cranial vagina in older mares due to splanchnoptosis.

3.Traveling further cranial in the vagina, one can see the opening of the cervix, showing its dorsal frenulum. Consider the simple nature of the equine cervix compared to that in other species i.e. there are no transverse cervical rings and the cervix is easily dilated for artificial insemination, embryo collection and is even distensible enough to remove fetuses that are three or four months old. The same distensibility is certainly not encountered in ruminants and other domestic animals.

4.Turning to the left and right at a point just caudal to the cervix, one should be reminded that the vagina lies completely within the peritoneal cavity at this point. This is important because it permits one to penetrate the cranial vagina for standing ovariectomy in mares. It also means that if the cranial vagina is torn during foaling, evisceration can occur from this site.

5.Withdraw from the vagina and rotate the model so that the cervix is visible from within the peritoneal cavity. Note that the cranial portion of cervix is firmly suspended between the ilial shafts on either side, by the mesocervix i.e. the caudal portion of the mesometrium. This is significant because the cervix of a mare cannot be grasped, elevated and manipulated as it can in a cow.

6.Rotate and tilt the model so that the mesometrium, containing the uterine artery (middle uterine artery) can be seen clearly. Be reminded that a uterine artery can be forced back against the shaft of the ileum during foaling, rupturing the vessel, causing severe hemorrhage within the mesometrium. Unlike the situation in cows, there is no fremitus in this artery during pregnancy.

7.Rotate the model so that the attachment of the mesometrium the uterus along its lateral borders can be appreciated. Note in turn, how the mesometrium is attached to the abdominal wall on either side. This firm attachment affords substantial stability to the equine uterus and explains in part why uterine torsion is less common in mares than it is in cows. It also means that the equine uterus cannot be retracted during palpation as is done in cattle.

8.Rotate the uterus so that the intercornual ligament is visible. Although it is fairly well-developed. It is never used to retract the uterus  for reasons just mentioned.

9.Tilt the uterus so that its dorsal surface is visible, showing how the bifurcation of the uterine horns provides a T-shaped structure instead of the Y-shape characteristic of the bovine uterus.

10.Rotate the uterus up and down on it's transverse axis, noting the length of the uterine body in a mare compared to the very short body in ruminants. This has many implications. For example, in cattle is very important to enter a particular uterine horn for insemination or embryo collection. In mares by contrast, the long uterine body means that semen is simply deposited anywhere in the body and during embryo collection, the embryo is collected from the uterine body.

11.Rotate the tract and pan in and out over the ovaries, noting their large size in comparison to those of cows. Be reminded that although equine corpora lutea are three or four times the diameter of bovine corpora lutea, they cannot be detected by transrectal ovarian palpation. This is because equine  ovaries are surrounded by thick tunica albugineae, disguising the presence of corpora lutea. Therefore, to determine if a mare is ovulating (in the absence of progesterone assay) one must use ultrasonography

12. Noticeably absent from this model are the ovarian bursae. The author apologizes for that oversight and time allowing, will add that in his next life.





Tuesday, November 13, 2018

Unusual placentation in a twin pregnancy. 

Keywords: placenta, equine, mare, inverted, twin, complication

Companion to an entry on twinning in mares in LORI.

There is still some uncertainty as to how and why twin equine pregnancies result in abortion. Sharing the available endometrial area is certainly a significant factor. Although typical inflammatory reactions at placental interfaces do not characterize twin abortions, immunological rejection of one co-twin by another may also be an important factor. Figure 1 shows one of numerous permutations of twin placentas apposed to one another within the uterus; placental sharing of the endometrial surface may be almost equal. In others, sharing is dramatically unequal. Figure is a schematic representation of such a case. Indeed, the situation in this pregnancy.

In Figure 1 red arrows indicate an area of apposition between the two chorionic surfaces. In these placentas there was little if any, macroscopic reaction between the two conceptuses despite the intimacy of placental apposition. Approximately 40% of the chorionic surface from the smaller co-twin was invaginated into the placenta of the larger twin.

Figure 1: Apposition of twin placentas and the path of delivery for both foals. Size available: 1626 x 2172px

The green arrows in Figure 1 show where the chorioallantois of the larger twin ruptured; in the region of the cervical star. The chorioallantois containing the smaller foal (yellow arrows) appeared within the allantoic cavity of the larger foal, invaginated within a pocket of the larger foal's placenta. The smaller foal was then delivered through a rupture of two layers of chorioallantois; that of the larger foal and that of its own placenta. The lower part of Figure 1 shows the path (grey arrow) that had to be taken by the smaller foal to be born; through the placenta of the larger foal.

Figure 2. A schematic representation of the placentation in this case. In some cases, the smaller co-twin may die and become a mummified attachment to the placenta of a normal foal. Image size available: 2553 x 1886px

Often, the placentas of twin foals will lie side by side within the uterus and each foal will be born in a conventional fashion, through the rupture of independent chorioallantoic membranes adjacent to the cervix.