Urinary kisspeptin as a non-invasive reproductive biomarker in crossbred cattle and black Bengal does

Kisspeptin is an upstream regulator of the hypothalamic-pituitary-ovarian axis and plays a critical role in the estrous cycle and pregnancy in domestic animals. The KISS1 gene, also known as the "metastatin" gene, has emerged as a promising diagnostic biomarker in reproductive physiology across livestock species. This study aims to explore the dynamics of urinary/plasma kisspeptin and their creatinine-normalized ratio during the bovine and caprine estrous cycle and pregnancy. Experimental animals comprised two groups (cyclic and pregnant). The cyclic group consisted of 12 cyclic crossbred cows and 12 cyclic black Bengal does, and the pregnant group consisted of 12 pregnant crossbred cows and 12 pregnant black Bengal does. Early-morning fresh urine and blood samples were collected from each cyclic animal group on Day 0, 6, 12, and 21 of the estrous cycle, and at fortnightly intervals from each pregnant animal group for estimation of urinary and plasma kisspeptin. Urinary and plasma kisspeptin were estimated using enzyme-linked immunosorbent assay, and urinary and plasma creatinine values were estimated using the alkaline picrate method. Two-way repeated-measures analysis ofvariance was used to analyze the samples, followed by regression analysis and student’s t-test to assess significant differences between non-pregnant and pregnant animals. Further receiver operating curve analysis was used to assess the diagnostic performance of urinary and plasma kisspeptin and their creatinine-normalized ratios. The results obtained for the said objective revealed that urinary kisspeptin levels showed a qualitatively similar trend to plasma kisspeptin (r=0.99) during the estrous cycle in crossbred cattle and black Bengal does. The highest concentration of UKP (2.45 ± 0.21ng/ml and 2.44 ± 0.08 ng/ml) was recorded during the proestrus stage of the estrous cycle of both crossbred cows and black Bengal does. The highest plasma kisspeptin-to-creatinine ratios were also observed on Day 21 (14.49 ± 0.91 and 16.22 ± 1.53), whereas the corresponding urinary kisspeptin-to-creatinine ratios reached 11.84 ± 0.70 and 7.47 ± 0.34, respectively, in crossbred cattle and black Bengal does. Plasma and urinary kisspeptin concentrations were found to increase significantly (P<0.05) from early through late pregnancy (4.28 ± 0.19 to 33.40 ± 0.78; 2.01± 0.10 ng/ml to 23.01 ± 0.54 ng/ml) in crossbred cows and black Bengal goats (3.49 ± 0.53 to 30.50 ± 1.26 ng/mL; 1.68 ± 0.01 to 19.84 ± 0.82 ng/mL), respectively. The PKP/PC ratio (134.9 ± 10.96; 127.30 ± 6.62) was found to be significantly higher (P < 0.01) during late pregnancy than the UKP/UC ratio (71.94 ± 10.63; 68.4 ± 6.70 ng/ml) in crossbred cattle and black Bengal goats. The results of our study revealed that kisspeptin can be detected in the urine of cyclic and pregnant domestic animals, such as crossbred cattle and black Bengal does, and that its ratio to creatinine can serve as a reliable diagnostic marker for predicting ovulation and pregnancy in crossbred cows and black Bengal does.