Equine reproductive endocrinology

Purpose: Diagnosis of granulosa cell tumour (GCT) in mares and cryptorchidism/testicular tissue remnants in males; post-castration evaluation ( )

Production site / physiological role:
granulosa cells of ovarian follicles (small follicles; decreased production of AMH in large preovulatory follicles)
Sertoli cells of the seminiferous tubules in the testes

Time for testing: 
absence of estrus or irregular cycles (regnancy and normal cyclic activity do not exclude GCT)
behavioural changes ranging from nymphomania to stallion-like behaviour, but often no visible changes
typically one markedly enlarged ovary, the second ovary inactive (note that tumours may be bilateral)
higher probability in mares over 10 years of age, although it may occur at any age  
absence of one or both testes in the scrotum with concurrent stallion-like behaviour
breed predisposition to cryptorchidism especially in American Quarter Horse and Percheron

Aggressive behaviour is often interpreted as a sign of a granulosa cell tumour or active testicular tissue; however, studies indicate that this correlation is weak. In such cases, laboratory-based diagnostics provide an objective tool, particularly useful when imaging or palpation findings are inconclusive.

Sample: serum 0,5 mL

Method: ELISA

Sample collection: independent of oestrous cycle stage

Advantages: rapid, routine test

Limitations: severe haemolysis – the test will be cancelled, mild haemolysis may cause result distortion

Note: if results are inconsistent with clinical findings, re-testing or confirmation with a different method is recommended.

Purpose: assessment of corpus luteum function, confirmation of ovulation, evaluation of oestrous cycle stage (oestrus vs. dioestrus), early pregnancy diagnosis 

Production site / physiological role:
corpus luteum (CL) of the ovary

Time for testing: 
absence of oestrus or irregular cycles
up to approx. 45–60 days of pregnancy when assessing CL function or making decisions regarding altrenogest supplementation

Progesterone measurement is not recommended in mid and late pregnancy.

after 100–120 days of pregnancy in mares, pregnancy maintenance is supported by placental progestogens other than progesterone

Sample: serum 0,5 mL

Method: CLIA

Sample collection: ependent on the purpose of testing; cycle or pregnancy stage must be taken into account

Interpretation:
3.2 nmol/L (1,00 ng/mL) endocrinologically active CL (dioestrus/pregnancy/persistent CL)
≥ 12.8 nmol/L (4,00 ng/mL) progesterone concentration considered safe in early pregnancy

Advantages: rapid, routine test

Limitations: interpretation difficulties – in early pregnancy, marked fluctuations in progesterone concentration may occur and have no diagnostic significance.

In cases of early embryonic death with persistent CL, progesterone concentrations may still indicate pregnancy. Results should always be interpreted in conjunction with ultrasonographic findings.

Purpose: Assessment of the stage of the estrous cycle and ovarian activity. Elevated levels are observed in pregnant mares after day 60 of gestation. A marked increase in concentration may also indicate the presence of a persistent anovulatory ovarian follicle. During a silent heat (silent oestrus), estradiol concentrations may be lower than those observed during a normal oestrus cycle.

Production site / physiological role:
ovary – preovulatory follicle

Time for testing 
absence of oestrus or irregular oestrous cycles

Sample: serum 1 mL

Method: RIA

Sample collection: it depends on the purpose of the test – the stage of the oestrous cycle or pregnancy should be taken into account.

Interpretation:
< 10 pg/mL – small follicles, corresponding to the dioestrus phase / early follicular phase / seasonal anoestrus
11–15 pg/mL – medium follicles (diameter approx. 20–30 mm), corresponding to the follicular phase 
> 15 pg/mL – large follicle (diameter ≥ approx. 30 mm; preovulatory follicle), corresponding to oestrus
> 30 pg/mL – suspicion of granulosa cell tumour or persistent anovulatory follicle

Advantages: routine examination for the detection of subclinical ovarian follicular activity. 

Limitations: The test results should be interpreted in conjunction with ultrasonographic findings. 

Purpose: confirmation of pregnancy between 40 and 120 days of gestation. Not applicable in donkeys

Production site / physiological role:
Produced in endometrial cups – foetal-derived structures embedded in the uterine mucosa of the mare, formed as a result of trophoblast cell invasion (embryonic tissue) into the endometrium during early pregnancy. Equine chorionic gonadotropin (eCG) has both FSH (predominantly) and LH activity, leading to the formation of accessory corpora lutea in mares approx. between day 40 and 45 of gestation. This mechanism ensures the maintenance of sufficiently high progesterone concentrations necessary to sustain pregnancy until it can be maintained by placental progestogens, i.e. around day 100 to 120 of gestation. Occasionally, the action of eCG may also indirectly lead to the manifestation of oestrous behaviour in pregnant mares, as eCG stimulates follicular growth, resulting in increased oestradiol production responsible for oestrous signs.

Time for testing: 
suspicion/confirmation of pregnancy between days 40 and 120 of gestation. Endometrial cups undergo regression at a later stage.

Sample: serum 1 mL (chilled)

Method: ELISA

Sample collection: etween days 40 and 120 after ovulation

Interpretation: descriptive

Advantages: routine test

Limitations: Possible false-positive results – in cases of foetal death, if endometrial cups fail to regress (persistent endometrial cups), eCG concentrations may remain at levels indicative of pregnancy. Cases of endometrial cup persistence beyond 140 days of gestation have also been reported; however, these are extremely rare (approx. 1–2% of mares). 

Purpose: Pregnancy diagnosis from day 80 in mares and day 120 in jennies. Results obtained after day 110 of gestation in mares are the most reliable and clinically useful. Additionally, they provide information on foetal viability and placental function.

Production site / physiological role:
Placenta – formed via synthesis from DHEA produced in the foetal gonads, with secretion markedly increasing between days 100 and 240 of gestation, after which it begins to decline. The placenta converts DHEA into “pregnancy” oestrogens, predominantly estrone sulfate.

Time for testing: 
suspicion/confirmation of late pregnancy (from day 80 after ovulation)
supportive in cases of: suspected impaired foetal viability or placental insufficiency

Sample: serum 1 mL

Method: RIA

Sample collection: from day 80 of gestation (after ovulation) to approx. one week prior to the expected term of foaling

Interpretation:
Between day 80 and 100 after fertilisation, values > 10 ng/mL indicate pregnancy.
After day 100 after fertilisation, values > 100 ng/mL indicate pregnancy.
In the case of a negative result after day 80 of gestation, pregnancy is unlikely (test sensitivity 95–98%).

Prior to making any important clinical decisions, a clinical examination is recommended.

Advantages: one of the few laboratory parameters useful in the assessment of foetal welfare and placental function

Limitations: relatively long turnaround time. In the assessment of placental function and foetal viability, a sudden, clearly diagnostic decrease in E1S concentration is detectable only approx. 2 days before foetal death/abortion.

Purpose: determination of the time point at which pregnancy maintenance is taken over by placental progestogens (luteo-placental shift).

Production site / physiological role:
progesterone – produced by the corpus luteum (CL), responsible for maintenance of pregnancy until approx. day 100.
DHP (5α-dihydroprogesterone) – the main progestogen produced by the placenta with high affinity for progesterone receptors and strong biological activity

Time for testing: 
To support the decision to discontinue altrenogest supplementation (between days 100 and 120 of gestation), without the risk of premature cessation of pharmacotherapy.
The result precisely indicates placental maturation.
Repeated measurements of DHP in the same mare provide additional information on normal placental function in advanced pregnancy.

Sample: serum 1 mL

Metohod: LC–MS (Liquid Chromatography – Mass Spectrometry)

Sample collection: between days 100 and 120 of gestation

Interpretation:
In non-pregnant mares or during early pregnancy, the 5α-DHP/progesterone ratio is <1. After the luteo-placental shift, around days 100–120 of gestation, the 5α-DHP to progesterone ratio is >1.

Advantages: highly accurate test.

Limitations: as a potential marker of placental inflammatory conditions, it should be reassessed through repeat measurements and interpreted alongside other parameters.