2.4. Reproductive Hormonal Profile during Postoperative Observation Period

The serum concentrations of FSH, LH, estrogen, and progesterone were measured in the spayed group and control groups using ELISA. In the spayed groups, at the day of conducting transrectal ultrasound scanning (day 1 preoperatively; 1 week, 2 weeks, and 4 weeks postoperatively) as well as the day before moving to a slaughterhouse, approximately 10 mL of blood was collected from the jugular vein in plain blood collection tubes (Becton Dickinson, Plymouth, UK). The collected blood was allowed to clot for 15 min and then centrifugated at 1000× g for 10 min at 4 °C. The isolated serum was stored in a deep freezer at −80 °C until use. The sera collected from control group with same methods before the day of slaughtering were also included in this assay. ELISA kits for measuring reproductive hormones were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). After the stored serum was thawed, a mixture of the sample, enzyme immunoassay (EIA) buffer, tracer, and antiserum was incubated for 60–90 min at room temperature (RT) for estrogen and progesterone level analysis. Thereafter, samples in the 96-well plate were reacted with development reagents (Ellman’s Reagent) for 60 min in an incubator. For FSH and LH, samples, anti-FSH (or LH)-horseradish peroxidase, and anti-FSH (or LH)-biotin-conjugate were mixed and placed into streptavidin-precoated 96-well plate. After incubation for 60 min, the wells were incubated with 3,3`,5,5`-Tetramethylbenzidine (TMB) substrate for 15 min, after which stop solution was added. The reacted 96-well plates were read at 405–450 nm wavelength using a microplate reader (Epoch, Biotek, Winooski, VT, USA). The concentration of each hormone in the serum was calculated using a four-parameter logistic fit with a free software (www.myassay.com: accessed on 2 December 2020).