By Dr Kate May,BVSc (Pret), MMedVet(Gyn)(Pret), DipACT
In order to fully understand the application of certain hormone assays currently available in small animals, we need to briefly recap the physiology behind it all.
The overall controlling structure in all reproductive pathways is the hypothalamus. The hypothalamus is a specialized ventral portion of the brain made up of various nerve cell bodies, such as the tonic centre, the surge centre and the pre-ventricular centre. The tonic centre is responsible for episodic (basal) release of the hormone, Gonadotrophin Releasing Hormone (GnRH). The hypothalamus is connected to the anterior lobe of the pituitary gland via the hypothalamo-hypophyseal portal system. Secretion of GnRH by the hypothalamus results in release of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) from the anterior pituitary (adenohypophysis).
In the female, FSH stimulates recruitment and selection of a cohort of follicles on the ovary. In monotoccus species, once a single follicle becomes dominant, it starts to secrete inhibin and estradiol, which have negative feedback on the release of FSH from the adenohypohysis and also result in atresia of the non-dominant follicles.
Once the increasing amount of oestradiol from the dominant follicle reaches a threshold, it stimulates the surge centre in the hypothalamus to secrete a pre-ovulatory GnRH surge, which in turn causes an LH surge which results in final maturation and ovulation of the dominant follicle. Once the follicle has ovulated, the cells within the newly formed corpus luteum (CL) switch from oestradiol to progesterone secretion. The progesterone has negative feedback on both the hypothalamus and the adenohypophysis, therefore preventing further follicular development during dioestrus. The episodic secretion of FSH during dioestrus is enough to result in the recruitment of small follicles during dioestrus, however, these fail to produce enough oestradiol to cause the pre-ovulatory surge in GnRH and LH required for ovulation and these follicles soon regress.
The female animal then enters a period of dioestrus, the length of which depends on the species. In polyoestrous species like cows and horses, dioestrus will last for roughly 17 days, thereafter, if there has been no recognition of pregnancy, the endometrium will secrete prostaglandin-F2α which results in luteolysis of the CL and resumption of cyclicity. In species that do not have maternal recognition of pregnancy, for example the dog, then the CL will continue for its normal lifespan of 70-90 days before regressing.
In the male, there is no surge centre equivalent of that of the female animal. Instead, frequent, intermittent release of GnRH from the tonic centre, causes an equivalent release of LH from the adenohypophysis. The LH acts on the Leydig (Interstitial) cells within the testes to produce testosterone which is required for spermatogenesis. This episodic release of LH is important to prevent sustained release of testosterone which would result in down-regulation of the GnRH/LH feedback system. In the Sertoli cells, under the influence of FSH, testosterone is converted to dihydro-testosterone and oestradiol. Testosterone, dihydro-testosterone and oestradiol all exert a negative feedback effect on the hypothalamus and the adenohypophysis. The Sertoli cells also secrete inhibin, which further suppresses the release of FSH. The importance of inhibin and suppressed FSH release in the male is currently unclear.
In bitches, an in house LH assay (WITNESS® LH kit, available on request from Zoetis South Africa) can be used during oestrus monitoring to determine the pre-ovulatory LH surge which occurs about 48 hours prior to ovulation.1,2 The LH-surge only lasts 24-48 hours, therefore twice daily monitoring of LH levels may be indicated to identify the LH surge 3,4. In bitches, oocytes are ovulated as primary oocytes, which still need to undergo further maturation and expulsion of the polar body before they become secondary oocytes capable of being fertilized. This maturation process take roughly 48 hours.
Therefore, the optimal time for insemination when unlimited matings or AI is available is roughly 4 days after detection of the LH surge and then every 48 hours until the bitch goes into dioestrus. With frozen semen which has a limited lifespan of roughly 12-24 hours or when limited access to the stud dog dictates only one mating, then the optimal day for insemination is on day 5 or 6 post LH surge. The LH-surge is very useful for monitoring the length of gestation in the bitch as parturition will occur 65 +/- 1 day post LH-surge. Once a bitch or a queen has undergone an ovariohysterectomy/ovariectomy, the removal of the gonads and, by extension, the gonadal steroids, removes the negative feedback on the hypothalamus and the adenohypophysis. In one study, basal LH levels in ovariectomised bitches (n=6) were significantly higher (20.2 ± 3.6 µg/L) compared to unspayed bitches in anoestrus (n=6, 0.64 ± 0.04 µg/L).
A once off LH assay to determine whether or not a bitch has been ovariectomised has excellent sensitivity (98%) but moderate specificity (78%) meaning that a single low LH assay confirms that a bitch is intact, but that that a single high LH assay does not confirm that she is ovarectomised.13 Serial LH assays can be used to confirm her ovariectomised status.
In dogs, a much more practical method to monitor the oestrus cycle and determine the optimum time to breed a bitch is the use of progesterone assays. In bitches, granulosa cell production of oestradiol switches to progesterone production just prior to the pre-ovulatory LH surge. Various studies have established that the progesterone equivalent at the time of the LH surge is about 6 nmol/L 14,15 and at the time of ovulation, progesterone levels reach > 16 nmol/L and these results can be used to determine the optimal time to breed a bitch. With frozen semen AI, inseminations are performed 5 and/or 6 days after the rise in progesterone indicating the LH surge has occurred .
Semi-quantitative in-house progesterone kits (PreMate™- Camelot Farms, College Station, TX, TARGET™ Canine Ovulation Timing Test Kit – BioMetallics, Princeton, NJ) which show a colour change depending on the plasma progesterone concentration are not accurate and many laboratories use human reagents which may produce erroneous results. For accurate progesterone results a qualitative radioimmunoassay (RIA) or a chemiluminescent immunoassay must be used. Currently, Idexx South Africa send all animal samples for progesterone to the Reproduction Laboratory at Onderstepoort* for testing and interpretation.
In bitches exhibiting silent oestrous cycles where there are very few external signs of heat or where the bitch is not reacting appropriately to the male, progesterone may be used to monitor her cycle and determine the best time for AI or mating.
In bitches with a history of primary anoestrus (bitch >24 months of age without showing signs of oestrus), monthly vaginal cytology and progesterone assays can used to determine whether a bitch is beginning pro-oestrus or whether she is exhibiting a silent heat without external signs of oestrus. In older bitches exhibiting signs of secondary anoestrus (>12 months between successive oestrous cycles , serum progesterone assays can be used to determine whether she is currently in dioestrus and the owner missed the previous signs of oestrus or whether a luteal cyst is present whereby progesterone concentrations will persist beyond the 70-90 day lifespan of a normal CL.16 An ultrasonographic examination of the reproductive tract should also be performed with special attention paid to the ovaries to check for cystic structures. In a pregnant bitch, progesterone levels drop to below 5 nmol/L about 24-48h prior to partus and can be used to determine readiness for whelping. A decrease in plasma progesterone to less than 3 nmol/L in pregnant bitches may indicate impending abortion.17 Recent work by De Cramer et al will assist us in more accurately determining the optimum day for elective caesarian sections in dogs.
In dogs and cats, the hormone relaxin is secreted by the placenta and a qualitative relaxin assay (WITNESS® available on request from Zoetis South Africa ) can be used to diagnose pregnancy from about, 16-22 days after the first day of cytological dioestrous in dogs (D1) or 22-28 days after ovulation and about 31 days after mating in cats. In dogs, prior to establishment of the placenta, a false-negative result may be obtained
Testosterone can be used in the diagnosis of bilateral cryptorchidism in the dog or in unilateral cryptorchidism where only the descended testis has been removed. In “castrated” male dogs with unknown backgrounds, that are exhibiting intact male characteristics such as urine marking, aggression and interest in females, various diagnostic options are available. The undescended testis may be present anywhere from the caudal pole of the kidney to the inguinal canal and a dedicated ultrasonographic abdominal examination may be instrumental in identifying the cryptorchid testis. Cryptorchid testes remain hormonally active but lose spermatogenic capability and are therefore more likely to undergo neoplastic change with sertoli cell tumours having the higher incidence in cryptorchid testes.
Due to the episodic release of LH and therefore testosterone, as well as the low systemic concentration of testosterone, a hCG or GnRH -stimulation test with subsequent measurement of testosterone will give a better indication of whether or not testicular tissue is present. The baseline value will be more than double if testicular tissue is present.18 Owing to the overlap of resting ranges between cryptorchid and neutered males, LH is not accurate for the identification of castrated males
In the future, Anti-Mullerian Hormone (AMH) assays may be more accurate for the diagnosis of crytorchidism in dogs and are already being used in the diagnosis of cryptorchid stallions. AMH is secreted by the sertoli cells and the granulosa cells in the ovary and a measurable concentration of AMH indicates the presence of testicular or ovarian tissue.19 An AMH test would therefore also be useful to determine the reproductive status of dogs or bitches. The test is currently available at the Veterinary Population Management Laboratory at Onderstepoort, however, it is very costly at this stage.
In cats, the presence of spines on the penis indicates the presence of testosterone-secreting tissue. These spines atrophy about 6 weeks post castration and provide a very convenient means of determining the presence of cryptorchid testis/es in the tomcat prior to the use of hormone assays or ultrasonography.
Ovarian remnant syndrome
Ovarian Remnant Syndrome (ORS), is defined as functional ovarian tissue present in a previously ovariectomized patient. ORS is an iatrogenic condition that results from failure to remove all ovarian tissue during ovariohysterectomy. This is most often due to surgical error such as incomplete ovary removal or auto-transplantation of dropped ovarian tissue. It has been shown that ovarian tissue placed free in the abdomen can become re-vascularized and functional, In the majority of reports of ORS, residual ovarian tissue is found located in the region of the ovarian pedicle so incomplete ovary removal is suspected in most cases.20, 21 Another reported explanation for ORS is the presence of extra-ovarian tissue such as ectopic ovarian tissue or an accessory ovary that is not identified at the time of surgery. Accessory ovaries have been reported in cows, women and cats and in most reports, located in the proper ligament.22 Accessory ovaries have not been reported in dogs.
Clinical signs of ORS are consistent with pro-oestrus or oestrus and include vulvar discharge (serosanguinous or purulent), vulvar swelling, mammary gland enlargement, behavioural signs such as attracting males and postural behaviour in cats. 23 Polyuria and polydipsia, weight loss, alopecia and poor coat have also reported with most animals having more than one clinical sign.24 The time to clinical signs after OHE can vary greatly. One study had a range of one month to ten years with a median time to diagnosis of 17 months. Animals with neoplastic ovarian tissue, such as granulosa cell tumours, take longer to present with clinical signs with a reported median interval of 96 months. 24 Dogs with ORS have been shown to develop ovarian neoplasms (23.8%) more frequently than intact dogs (6.25%).24-26
Diagnosis of ORS can be difficult because the ovarian tissue may be very small. There are several tests to diagnose ovarian remnants in a patient with clinical signs. By far the easiest and least costly is to perform vaginal cytology during signs of oestrus and look for the presence of superficial or cornification vaginal epithelial cells which would indicate the presence of oestrogen-secreting tissue. However, exogenous oestrogen exposure (such as DES for urinary incontinence) and atypical Cushing’s disease may also cause similar changes to vaginal cytology.20,27
Progesterone can be used in the diagnosis of ovarian remnant syndrome in bitches or queens. A suspicious history of cyclic “heat” episodes, as well as superficial cells identified on a vaginal cytology can be confirmed by testing plasma progesterone levels about 2 weeks after signs of oestrus subside. Cats can be induced to ovulate using hCG or by repeated stimulation of the vagina with a cotton bud and progesterone taken 2 weeks later. High progesterone would indicate luteal tissue and confirm the diagnosis of an ovarian remnant.
Following a GnRH-stimulation test, levels of LH in suspect ovarian-remnant bitches (4.1 ± 0.7 µg/L) were significantly higher than bitches in anoestrus (0.64 ± 0.04 µg/L) but significantly lower than intact bitches (20.2 ± 3.6 µg/L ).11 However, lack of availability of a quantitative test for LH leave this in the research realm.
One of the most promising tests for determining the presence of ovarian tissue is Anti-Mullerian Hormone (AMH). Because the ovaries are the sole source of AMH, a positive test would indicate the presence of ovaries or ovarian tissue. 28,29 If the remnant is very small, it may not produce enough AMH so a negative test does not rule out ORS. The test is currently available only through Cornell University, requires one red top tube (shipped frozen or on ice) and costs $70 per sample, however enquiries can be made at the Veterinary Population Management Laboratory at Onderstepoort.