Reproductive Hormone Assay in Domestic Animals – Clinical Uses

Reproductive phenomena of both male and female animals are regulated by hormones. Estimation of specific hormone is carried out for identifying the normal and abnormal reproductive functions in domestic animals.

Hormone analysis techniques:

Radioimmunoassay (RIA) and Enzyme linked immune sorbent assay (ELISA) are commonly used for analysis of hormones. These techniques are sensitive, specific, relatively inexpensive and require small amounts of assay material. RIA and ELISA are widely used for diagnosis of various reproductive problems and monitoring therapy in domestic animals.

Effects of storage on hormone concentration:

Blood is commonly used for estimation of different reproductive hormones. Urine, milk, saliva and feces can also be used for the assay.

i. Blood: Erythrocytesfrom cow can metabolize progesterone to other steroids. Concentration of progesterone in heparinized bovine blood samples quickly decrease (about 10% to 20% reduction/hour) due to the conversion of progesterone to 20β -hydroxylated gestagens. This decline in whole blood progesterone level can be reduced by addition of sodium fluoride. If blood samples are stored at lower temperature, decrease in the progesterone level is slower. Significant loss in concentration of reproductive hormones can be prevented by storing plasma or serum samples in frozen conditions for longer periods also.

ii. Milk: Sodium azide, potassium dichromate, or thimerosal can be used to preserve milk samples at room temperature for about 3 weeks for assay of progesterone. Milk can be stored for several months at 4 to 5°C for progesterone estimation.

Clinical uses of reproductive hormone analysis:

Concentration of hormones as well as endocrine assay varies between humans and animals. One example is the concentration of estrogen which is approximately 10-fold lower in animals compared to human. So, hormone assay systems for animals should be more accurate. Following criteria need to be fulfilled for considering hormone assay as a diagnostic technique-

  1. The release of hormone should be steady.
  2. There should be close correlation between hormone concentration at sampling and concentration of the hormone released from the endocrine gland.

Hormone analysis is only supportive. Hence, for proper diagnosis, clinical examination of animals should be carried out along with estimation of hormones.

Clinical uses of different reproductive hormones in domestic animals are as follows:

1. Progesterone:

i. Cows: Estimation of progesterone is of clinical significance in females of different domestic animals. It is used for early detection of pregnancy in cows. Progesterone level in plasma is estimated 19 to 24 days after a fertile breeding in cows for early detection of pregnancy compared to that of non-fertile breeding (Robertson and Sarda, 1971). Milk progesterone concentration can also be used for early pregnancy test.

 Non-Pregnant cowsPregnant cows
Progesterone concentration< 0.5 ng/ml (1.6 nmol/liter)4-8 ng/ml (13 to 26 nmol/liter)
Table 1: Plasma progesterone concentration in cows at 21 days post breeding

Reference: Kaneko et. al. (1997)

Progesterone estimation is helpful in understanding the clinical conditions in postpartum cows. Elevated progesterone values indicate that significant ovarian activity is present. Progesterone assay can also be used to understand the ovarian activity and for confirmation of the presence of luteal tissue associated with endometritis, pyometra and ovarian cysts.

ii. Horse: In mares, progesterone estimation is carried out to understand the ovarian activity. In case of normal folliculogenesis and ovulation in mares progesterone level is elevated. Ovarian activity in mares can be verified by estimating progesterone at an interval of 5-days over 20 days period.

iii. Sheep: Progesterone estimation is carried out in ewe for early detection of pregnancy. Progesterone level in ewes found to be increased from 6 to 13 nmol/liter to 38 to 64 nmol/liter between days 60  and 125 of pregnancy (Stabenfeldt et al. , 1972) due to increased progesterone production from the fetoplacental unit.

iv. Dog: Occurrence of ovulation in bitches can be confirmed by estimating progesterone. In the peri-ovulatory period, progesterone level increased slightly above baseline and at ovulation, the level reaches at about 15 nmol/liter (Concannon et al, 1975). Ovulatory failure in bitches can also be detected by estimating progesterone.

2. Estrone sulfate

i. Cow: Estrone sulfate concentration is found to be increased in blood of cows at about day 80 and in milk at about day 100 of pregnancy.Estimation ofestrone sulfate in the milk samples indicates pregnancy status of cows.

ii. Sheep: The fetoplacental unit produces estrone sulfate in the pregnant ewes. The plasma level of estrone sulfate in ewes is usually elevated at around day 70 after conception. (Tsang, 1978)

3. Pregnant mare’s serum gonadotropin (PMSG):

Horse: PMSG is used for diagnosis of pregnancy in mares. PMSG concentration in blood is found to be elevated in mares with normal pregnancies as well as in mares in which embryonic mortality is exibited after day 40 of gestation. Thus, PMSG estimation in mares does not confirm the presence of fetus. Sometimes, estimation of PMSG diagnoses the pregnancy in mares but they did not deliver the foal. (Jeffcott et al., 1987). To overcome this, estrone sulfate is estimated in mares after day 100 of pregnancy. Estrone sulfate estimation is a reliable test for diagnose pregnancy and eliminating false-positive diagnosis by PMSG measurement in mares.

4. Conjugated Estrogens:

Horse: Estrone is conjugated after secretion to form estrogen conjugates. Concentration of conjugated estrogens in plasma is elevated between days 35 and 40 of gestation in mares (Kindahl et al. 1982) Level of conjugated estrogens can also be measured in urine of mares to diagnose pregnancy.

5. Testosterone:

i. Horse: Concentration of testosterone in plasmadiffers in the mare according to the reproductive state. During anestrous, plasma testosterone concentration is less than 52 pmol/liter and during cyclic ovarian activity its level ranges between 69 to 139 pmol/liter. Granulosa-theca cell tumors in the mares can be diagnosed by estimating plasma testosterone. (Stabenfeldt et al., 1979) In this case, the plasma testosterone concentration is over 347 pmol/liter.

In male horses, diagnosis of cryptorchidism can be carried out by estimating plasma testosterone. Plasma testosterone concentration in most of the cryptorchid horses ranges from 693 to 3467 pmol/liter (Cox 1975).  

ii. Dog: Testosterone is commonly estimated in dogs to check the secretory status of the Leydig cells. In normal dogs, plasma testosterone level ranges from about 3.5 nmol/lite to about 35 nmol/liter. Testosterone level is observed to be declined in dogs with Sertoli cell tumors.

6. Relaxin:

Dog: In pregnant bitch, relaxin in is first detected atday 18 to 25 of gestation. During anestrous and throughout non-pregnant ovarian cycles, relaxin is undetectable in bitches. This hormone can’t be detected in male dogs also (Steinetz et al., 1996) Relaxin level is maximum by days 40 to 50 of pregnancy and slightly decreases before parturition. Canine relaxin assay kits are commercially available. These relaxin assay kits are used to diagnose of pregnancy in bitches as early as 21 days after breeding.

Subrat Kumar Dash1 and Gurpreet Singh2

1Assistant Professor, 2Associate Professor,
Department of Veterinary Physiology and Biochemistry, College of Veterinary Science, Rampura Phul Guru Angad Dev Veterinary and Animal Sciences University