Fecal Hormone Assay and Urinalysis of Pregnant Cattle

| Variation in concentrations of Thyroxin (T4), Thyroid stimulating hormone (TSH) and Follicle stimulating hormone (FSH) shed in feces may help to identify physiological states of animals, but the efficacy of the technique needs to be validated for each species. Therefore, a short term cross sectional study was performed in 120 pregnant cattle for fecal hormonal analysis and urine analysis. For this, Enzyme linked immune sorbent assay (ELISA) technique was used for TSH, T4, and FSH assay and dip strip with microscopic test for urinalysis. Variation of T4, TSH and FSH concentrations in three trimesters of pregnancy was estimated in feces of cattle. Highest T4 concentration was found in third trimester of pregnancy 165.2 ± 53.4ng/ml with TSH (μIU/ml) fluctuation of decreasing trends. TSH was highest in second trimester as 0.5 ±0.4 μIU/ml. There was no significant variation (p>0.05) for fecal T4 and TSH concentration. FSH level (mIU/ml) was sharply decreased (p< 0.01) with the advancement of pregnancy. Urinary pH turns acidic to alkaline in advance stage of pregnancy and specific gravity fluctuated among three trimesters of pregnancy. No significant variation (p>0.05) was found between variables for urine pH and specific gravity. The important identified deposits of urine were crystals, leucocytes, blood, casts, uric acid and calcium phosphate crystal in pregnant cattle. Crystalluria were a frequent finding of routine urine examination of cattle suggesting possible prone to urolithiasis, acute uric acid nephropathy induced by drugs such as sulphadiazine.

Palpation of the uterine contents rectally 40-45 days after insemination is probably the most commonly used method for pregnancy diagnosis.The main disadvantage of rectal palpation is that it cannot be performed until later in gestation than some other methods.Pregnancy can also be determined by ultrasound or by measuring pregnancy related biochemical markers (Ropstad et al., 1998).Besides that, fecal hormone analysis is now widely used to monitor reproductive hormones in captive and free ranging wild life (Pereira et al., 2006) as well as in laboratory animals (Chelini et al., 2005).Thus this technique appears like an attractive noninvasive, inexpensive, alternative tool that avoids the stress effects related to blood sampling.As estrogens are end products of steroid metabolism, the compounds in plasma and feces are rather similar (Schwarzenberger et al., 1996).In contrast, progesterone is extensively metabolized, and in ungulate species a large number of progestins is present in feces (Palme et al., 1997).Because the immunoassays used to detect excreted steroids were developed for use in human serum, plasma, or urine, it is necessary to test these assays for cross reactivity with the 5α5β-reduced metabolites present in fecal samples of ruminants (Palme et al., 1997).Regarding above background, the present study was conducted to estimate the selected hormone and their level in fecal sample and analyse various urinary constituents of pregnant cattle.
The study was conducted at two dairy farms in Chittagong metropolitan area of Bangladesh.Several types of cow breed were reared (50%, 75% Holstein Friesian (HF), Jersey and several local breed) in face in housing system.A questionnaire was made before collection of sample.120 cattle of different trimesters were selected from farm record of two-studied farm (60 from each).The pregnant cattle were sub grouped to three trimesters as the method described earlier by (Torell, 2015).Freshly void fecal and mid-stream urine samples were collected from tagged cattle.Sterile vials were used to collect the samples.Samples were then kept in ice box and transported to Physiology laboratory of Chittagong Veterinary and Animal Sciences University (CVASU).Samples were preserved in -20ºC, in Physiology laboratory of CVASU.0.2 gm fecal sample was weighted out using digital weighing balance and taken to test tube.Then 5.0 ml of 90% ethanol (4.5 ml ethanol and .5 ml distilled water) was added and vortexed briefly.Then boiling was done in hot water bath (90ºC) for 20 minutes and ethanol was added to keep from boiling dry.The volume of extract kept up to preboil level with ethanol and was centrifuged at 1500 rpm for 20 minutes.
Extract was poured into a second set of test tubes.To the remaining fecal pellets 90% ethanol was added and vortexed for 30 seconds.It was centrifuged at 1500 rpm for 15 minutes.The first and second extracts were combined and stored in cryovial and was frozen the extract in -20ºC for until analysis.Specific anti-hormone antibodies were coated onto micro-titration wells.Extracted test samples were applied.Hormones (T 4 , TSH, and FSH) with Horse radish Peroxidase Enzyme (Human ® , Germany) were added.It competes with the released fecal hormones for available binding sites on the solid phase.After incubation, the wells were washed with water to remove any unbound hormone or enzyme conjugate.On addition of the Substrate (TMB), a colour develops only on those wells in which enzyme was present.As a result, hormone molecule being "sandwiched" between the solid phase and the enzyme linked antibodies.The reaction was stopped by the addition of dilute Hydrochloric acid and the absorbance was then measured at 450nm in Erba LisaScan II TM .The mean absorbance values were used for each specimen to determine the corresponding concentration of hormone from the standard curve (Wang et al., 2013).
Urine test strip was used (model: Uric 10 CF) for chemical and physical examination of urine.The presence of chemical and physical change of urine by constituents' material was detected by colour change.Leucocytes, nitrites, urobilinogen, protein, PH, blood, specific gravity, ketone body, bilirubin, and glucose in urine were qualitatively tested from urine.Twelve random urine samples (Two from each trimester i.e 12 from two farms) were transferred to falcon tube and tagged properly.After that those were centrifuged at 3000 rpm for 30 minutes.After centrifugation, supernatant was discarded and sediment was vortexed properly.Then one drop of sediment was taken in a slide and by using cover slip various constituents was identified at 10X, 40X magnification.T 4 was 150.8 ± 32.9 ng/ml.Simultaneously, another two hormones TSH and FSH concentration were 0.3 ± 0.3 mIU/ml and 12.1 ± 7.5 mIU/ml, respectively which have shown in box plot diagram (Figure 1).After grouping the pregnant cattle according to trimester basis fluctuated level of hormone were found (Table 1).The concentration of fecal T 4 was highest in 3 rd trimester of pregnancy 165.2 ± 53.4 ng/ml.In trimester categories, no significant variation (p>0.05) for fecal T 4 concentration was observed.In feces, mean FSH metabolite concentration was 19.9 ± 3.4 mIU/ ml of extracted feces at first trimester.It started to decrease in the second trimester of pregnancy reaching 9.4 ± 2.7 mIU/ml of extracted feces and 4.2 ± 1.6 mIU/ml of extracted feces in 3 rd trimester.Highly significant difference (p<0.01) was observed among different trimester for fecal FSH concentration.Fecal concentration of TSH started to increase in 2 nd trimester to reach a peak value at this time (0.5 ± 0.4 µIU/ml of extracted feces) and then decreased sharply on the subsequent 3 rd trimester (0.1±0.14 µIU/ ml of extracted feces).There was no significant variation (p>0.05) for fecal TSH concentration.One hundred twenty urine samples were tested by dipstick.Level of leucocytes, nitrites, urobilinogen, p H , specific gravity, protein, blood, bilirubin, ketone and glucose was observed.Trace amount (15 Ca CELLS/µl) of leucocytes was found in seven samples (2 nd trimester), urobilinogen was found positive in 116 samples (1mg/dl in 97 samples and 4mg/dl found in 19 samples).Here, highest percentage of urobilinogen was found in urine sample of cattle of 3 rd trimester of pregnancy.102 samples revealed the presence of trace amount (1mg/dl) protein in urine.The presence of ketone bodies found in all samples; among them 101 samples have 15 mg/dl and another 19 had 40 mg/dl of urine.Trace amount of glucose (<250 mg/dl) was found in 9 samples in 2 nd trimester of pregnancy.We found no significant (P>0.05)association of specific gravity with corresponding urine p H and blood in urine (Table 2).No significant variation (p>0.05) was found between variables for urine P H and specific gravity.Uric acid crystal was found in majority of samples and calcium phosphate crystal was also abundant.Calcium oxalate was present in few samples.The concentration of fecal T 4 (ng/ml) was lowest in 1 st trimester of cattle pregnancy 131.4 ± 17.6 and peak in 3 rd trimester of cattle pregnancy 165.2 ± 53.4.There was no significant variation (p>0.05) for fecal T 4 concentration.(Keech et al., 2010) observed fecal T 4 concentration in steller sea lion ((Eumetopias jubatus).The average fecal concentration of T 4 was 2012 ng/g of feces and peak T 4 concentration was 2842 ng/g feces of steller sea lion (Eumetopias jubatus).Fluctuating phenomena was seen in fecal TSH (µIU/ml) concentration.It was 0.2 ± 0.2 in 1 st trimester and 0.1 ± 0.14 in third trimester but peak level of TSH was seen in 2 nd trimester of pregnancy in cattle.Highly significant difference (p<0.01) between variables for fecal FSH concentration was observed.Sangeetha and Rameshkumar (2014) examined the fecal FSH concentration of sheep (Ovis aries).Fecal FSH average concentration in Sheep during pregnancy was 0.81 ± 0.01mIU/ml, highest concentration found during estrus was 3.12 ± 0.02 mIU/ml and lowest concentration during lactation was 0.03 ± 0.01 mIU/ml.2008) also found the colour fields of strip correspond to the following acetoacetic acid values: 0 (negative), 25 (+), 100 (++) and 300 (+++) mg/dL.Ketones in the urine are caused by an abnormal carbohydrate metabolism.Urine analysis is not often part of a veterinary surgeon's diagnostic armory but recent advances in interpretation of urinary pH and macromineral content make it an available and interesting investigative tool (Husband, 2010).Maximum samples had shown the presence of calcium phosphate and uric acid crystals in urine.It may be due to, pathological conditions such as urolithiasis, acute uric acid nephropathy, ethylene glycol poisoning, and hypereosinophilic syndrome.Additionally, crystalluria can also be due to drugs such as sulphadiazine (Thamilselvan and Khan, 1997).
By evaluating T 4 , TSH, FSH, urinalysis (physical, chemical, microscopic) in pregnant cattle we found statistically significant alterations of FSH during three trimesters of cattle.Non-invasive method like hormone analysis and urinalysis may be used as tool for pregnancy diagnosis.
Author recommends further research on fecal hormone analysis for monitoring reproductive status of animal as attractive, non invasive, inexpensive alternative tool compare to blood sampling.

Figure 1 :
Figure 1: Box plot diagram for FSH, TSH and T 4 in fecal extracted samples Among the analysed extracted fecal samples (n =120) collected from two dairy farms, the average concentration of

Figure 2 :
Figure 2: Fecal T4 level during course of pregnancyTable 3: p H and specific gravity of urine in three trimesters of pregnant cattle

Figure 3 :
Figure 3: Fecal FSH level during course of pregnancy

Figure 4 :
Figure 4: Fecal TSH level during course of pregnancy

Table 1 :
Fecal hormone concentration in different trimester of pregnant cattle

Table 2 :
Association of specific gravity with corresponding urine P H and blood in urine

Table 4 :
Microscopic examination of urine samples