Genital tubercles, genitalia and gonads in fetal sexing
Keywords: fetus, sexing, ultrasound, male, equineThere are many reasons to determine fetal gender in horses. These include a multitude of personal decisions by owners such as preferences for stud Thoroughbred colts, the wish to retain fillies for breeding, insurance concerns, a need for female polo ponies, male draft horses and so on.
Detection and examination of the genital tubercles
First, a note on accurate age determination of an equine fetus. In general stud practice, determination of the exact duration of pregnancy is not possible. Mares are commonly examined at two day intervals or over weekend at three day intervals. When a CH is first detected, it is impossible determine its age. Therefore ovulation could have occurred shortly before one's examination or close to two or three days earlier. If one assumes a two day interval between examinations and pregnancy is timed from when the follicle was last seen, a "55 day pregnancy" could be 53 to 55 days old. If the duration of pregnancy is timed from when a CH is first seen, the pregnancy could be 55 to 57 days old. This seemingly trivial point takes on critical importance when a mare is found to be pregnant with twins at "15 days" of gestation!It is only when frozen semen is used, that the approximate time of ovulation (and the age of the fetus) is known. Even when embryo transfer is used, the exact age of the fetus is not known. Therefore all the fetal ages in this discussion must be regarded as approximate.
Early sex determination is usually done by transrectal ultrasonography, between 59 and 68 days of gestation by detecting the genital tubercle. The genital tubercle is the precursor of the penis in the male and the clitoris in the female. Around day 55 gestation it appears as a hyperechoic equal sign (=) located between the fetal hindlimbs, at an approximately equal distance between the tail and the umbilicus. As the fetus grows the relative position of the tubercle changes, becoming closer to the tail in the female and the umbilicus in the male.
The image below shows a 64 day male fetus still in its amnion. The arrow indicates the position of the genital tubercle (arrow) used for sex determination. It is highly echogenic and usually easy to locate, just caudal to the umbilical cord. In females, the genital tubercle is located under the tail i.e. in the position of the vulva in a neonate.
In this case, the remnants of the yolk (YS) can be seen, consistent with this stage of gestation.
Image size: 1000 x 717px.
The image below shows the genital tubercles (green rings) of twin fetuses aborted at approximately 60 days of gestation. The upper fetus is male, the lower one female.
Image size: 1000 x 744px.
Detection and examination of the external genitalia, mammary glands and gonads.
A wide diagnostic window for gender determination exists between 100 and 260 days gestation. During this time, the external genitalia, mammary glands and fetal gonads themselves are examined. and multiple parameters to validate diagnosis (fetal primary sex organs), but may require a combination of trans-rectal and trans-abdominal ultrasound scanning.
The external genitalia of the fetus may be clearly identified on ultrasound as early as 100 days gestation. In the male fetus, a fully comprehensive gender diagnosis includes the identification of the penis, prepuce scrotum/testicular compartments urethra and gonads. In the female fetus, the vulva and clitoris, mammary gland, nipples and again, the gonads themselves.
In the 150 day old male fetus below, the scrotum and penis are well formed but the testicles have not yet descended into the scrotum. Each enlargement seen within the scrotum in this fetus is due to the gubernaculum, not the testicle. This explains why the scrotal contents are relatively anechogenic during fetal sexing (see the image below this one).
In this transabdominal ultrasonograph, captured at seven months of gestation, the anechogenic contents of the scrotum and the penis are clearly visible. Note the relatively anechogenic scrotal contents. The penis itself is not visible here because it runs on a plane that is dorsal to the urethra and scrotum.
Image size: 820 x 614px Author and copyright. Dr Stephanie Bucca: Doha, Qatar. stefbucca@gmail.com
At four to seven months in gestation, the vulva lips, mammary gland and teats can be used to identify a female fetus.
Image size: 2050 x 1612px Author and copyright. Dr Stephanie Bucca : Doha, Qatar. stefbucca@gmail.com
In image A, the base of the fetal tail is visible with faint cross sections of some of the coccygeal vertebrae seen to the left of the tail base. Between the buttocks of the fetus one can see its anus, vulva lips and clitoris.
In image B, at just under five months of gestation, this fetus is in posterior presentation showing one half of the mammary gland and its nipple. One hind limb is extended, with the femur shown in cross section. Vernix is clearly visible in the amnion but not the allantois.
In image C, the fetus is in transverse dorsal presentation. Between its buttocks, the anus and vulva lips of the fetus are clearly visible. Essentially, this is the same view as in image A, but closer to the fetus
After about eight months or nine months of gestation, transabdominal ultrasonography for sexing is less predictable than earlier. Although the fetus becomes fixed in cranial longitudinal presentation, its hind quarters are encased in the pregnant horn and may be raised towards the ovaries. (The hind legs are often palpable per rectum in late gestation). In fact, at this time, fetal cardiac examination can be more fruitful than attempts to examine their genitals. Even with low frequency ultrasound, penetration may not be sufficient to examine genital areas of diagnostic interest.
Although male and female fetal gonads have a similar macroscopic appearance, they differ remarkably on ultrasonography, especially when examined by doppler flow ultrasonography.
On the right side of this image, the tunica albuginea and the clear demarcation between cortex and medulla is obvious. The medulla is peripheral to the cortex in equids (a quirk of embryology) and because of its blood supply, it is less echogenic than the cortex. The donut-like appearance of the fetal ovary is very different to the relatively homogeneous appearance of the fetal testicle (see the following image). This forms the basis for accurate sexing of equine fetuses. Approximate time windows for these examinations are between 4 and 5 months transrectally and 7 and 8 months transabdominally.
Image size: 2050 x 1612px Author and copyright. Dr Stephanie Bucca: Doha, Qatar. stefbucca@gmail.co
When doppler flow ultrasonography is used, the accuracy of gender determination is close to 100% accurate. This because the medulla of the ovary is well vascularized, making it an ideal target for doppler flow examination. The value of doppler flow ultrasonography is easily appreciated in the image above (the scale is the same for both sub-images).
In male fetuses examined by doppler flow ultrasonography, the donut-like appearance is absent and doppler flow variation is largely restricted to the centrally located blood vessels. That appearance is shown here:
Image size: 820 x 614px Author and copyright. Dr Stephanie Bucca: Doha, Qatar. stefbucca@gmail.co.
Some find doppler flow imaging less useful than others, deferring to the appearance of male and female gonads on B mode ultrasonography alone. This is because movement of the fetus itself can cause considerable echogenic noise during doppler flow ultrasonography.
The author wishes to acknowledge the assistance of Drs Stephanie Bucca, Juan Samper and Carlos Pinto in creating this LORI entry.
Video (click on image and decrease page size to view at optimal resolution):
Video (PENDING) size 614 x 480px. Assembled and edited by Dr Rob Lofstedt (lofstedt@upei.ca). Copyright; Dr Carlos Pinto (tubercle files) Carlos.Pinto@cvm.osu.edu and Dr Juan Samper (gonad files) jsamper@telus.net.
References:
1. Bucca S. 2005. Equine fetal gender determination from mid- to advanced-gestation by ultrasound. Theriogenology 64:568–571
2. Renaudin, C.D., Gillis C.L. and Tarantal, A.F. 1997 Transabdominal combined with transrectal ultrasonographic determination of equine fetal gender during Midgestation. AAEP proceedings. 43: 252-255.
3. Resende H.L. et al. 2013. Determination of equine fetal sex by Doppler ultrasonography of the gonads.Equine Vet J. 2013 Nov 15. doi: 10.1111/evj.12213. (Epub)
