Gonadal Testing General Guidelines

Dogs and cats

Veterinarians may be required to determine the gonadal status of animals in various circumstances including:

  • After desexing surgery to confirm complete removal of gonadal tissue
  • Supposedly desexed bitches and queens exhibiting signs of oestrus (confirmation of "ovarian remnant syndrome")
  • Suspected cryptorchid males
  • Female cats and dogs with unknown desexed in history, particularly stray animals

Tests available for confirming cryptorchidism vs castration:

  • Anti-Müllerian hormone (AMH)
  • Testosterone
  • Examination of the penile spines (cats, in-house)
  • Ultrasound

Tests available for confirming ovarian remnants and intact status in females:

  • Anti-Müllerian hormone (AMH)
  • Luteinising Hormone (LH)
  • Progesterone
  • Vaginal cytology
  • Ultrasound


Anti-Müllerian hormone

Anti-Müllerian hormone (AMH) is produced by the follicles of a sexually mature ovary and Sertoli cells in a sexually mature testes. After complete ovariectomy or castration, levels of AMH decrease significantly. Intact bitches and dogs, and cryptorchid animals, will have higher levels of AMH than completely desexed animals. A single serum test can differentiate these animals. After desexing, it is recommended to wait seven days to allow serum levels to decrease before testing to confirm ovariectomy was complete. Low levels of AMH indicate the animal is desexed, high levels indicate the animal has functional gonadal tissue. AMH is available for male or female cats and dogs.

This test is also useful in differentiating mares with ovarian granulosa cell tumours from normal mares.

Luteinising hormone

Luteinising hormone (LH) is available for female dogs and cats (not males).

In intact bitches and queens, or those with functional ovarian remnants, basal LH is low due to negative feedback from ovarian estradiol. Serum LH will only be elevated at the natural LH peak preceding ovulation. In fully ovariectomised females, LH is persistently high due to lack of this negative feedback control.

A single low LH value indicates that functional ovarian tissue is present. If a positive (high) value is recorded, the animal must be retested with a new sample at least two hours after original sampling. This is to differentiate a persistently high value (no functional ovarian tissue present) from the transient pre-ovulatory LH peak (functional ovarian tissue present).

Currently, there is no published literature supporting the use of the serum LH assay to distinguish fully castrated from intact male dogs or cats. However, one study has confirmed that fully castrated male dogs have higher LH levels than intact animals. Use of the LH test for male dogs is therefore off-label and not validated, and interpretation remains with the veterinarian if requested.

Vaginal cytology

Vaginal cytology can be performed in-house or submitted to the laboratory. Cornification of vaginal cells is oestrogen-dependent and indicates cycling activity. However, this test requires the animal to be in pro-oestrus or oestrus and may need repeat testing over a week to demonstrate the increase in cornified cells. False positives can occur if the keratinised vulval epithelium is sampled rather than vaginal epithelium. False negatives can occur if the animal is not cycling at the time of sampling.

Ultrasound for gonadal tissue

This can be performed in-house, but requires an experienced operator and sensitive ultrasound machine. Small ovarian remnants or abdominal cryptorchid testes may not be detected.


Progesterone is produced by the post-ovulatory corpus luteum. Confirmation of ovarian tissue by serum progesterone therefore requires the bitch or queen to be in dioestrus or pregnant. Serum progesterone levels in anoestrus intact queens and bitches will not be different than ovariectomised animals. Stimulation tests can be used to induce ovulation and progesterone production according to the following protocols:

Queen: in the queen, 500 IU of hCG (Chorulon, Intervet) is given intramuscularly between one and three days after the onset of oestrus behaviour. A baseline blood sample and second blood sample taken seven days later are both analysed for progesterone. Another protocol recommends the use of Buserelin, a synthetic GnRH (Receptal, Intervet). Two intramuscular doses of 0.5 ml are given 24 hours apart and blood assayed for progesterone three days later. Progesterone concentrations increase at least fivefold in ovarian remnants.

Bitches: bitches in oestrus with progesterone concentrations less than 2 ng/ml (levels greater than this can be used to confirm the presence of a functional ovary) are challenged with hCG or GnRH. The test is less reliable than in the cat, however a 7 to 14 day post injection progesterone concentration of greater than 2 ng/ml confirms ovarian remnant syndrome. The dose rates are: hCG 44 IU/kg or 400 IU per dog IM; GnRH 2 ug/kg or 50 ug per dog IM.


Testosterone can be measured, but interpretation can be hampered by naturally fluctuating levels, particularly with cryptorchidism. Stimulation with GnRH or hCG may be required to demonstrate functional gonadal tissue.

Examination of the penile spines

In male cats, penile spines are expected to be present if there is functional testicular tissue present; spines are hormone-induced and will regress following castration.



Place NJ, Hansen BS, Cheraskin J, Cudney SE, Flanders JA, Newmark AD, Barry B, and Scarlett JM (2011) Measurement of serum anti-Müllerian hormone concentration in female dogs and cats before and after ovariohysterectomy. J Vet Diagn Invest 23: 524-7

Olson PN, Mulnix JA, and Nett TM. (1992) Concentrations of luteinizing hormone and follicle-stimulating hormone in the serum of sexually intact and neutered dogs. Am J Vet Res 53(5):762-6.

Lofstedt RM & Vanleeuwen (2002) Evaluation of a commercially available luteinising hormone test for its ability to distinguish between ovariectomised and sexually intact bitches. JAVMA 220(9):1331-5

Cornell University Animal Health Diagnostic Center (2011) Canine and Feline Reproductive Function Tests http://diagcenter.vet.cornell.edu