Pregnancy diagnosis is a crucial on-farm strategy employed to improve performance and fertility of dairy cattle and minimize economic losses. Hence, this study was carried out to validate the Punyakoti test, a cheap and simple pregnancy diagnostic tool, for application in dairy cattle especially among smallholder farmers. The study involved four treatment groups: distilled water (DW), urine from artificially inseminated cows (AIC), non-inseminated cows (non-AIC), and non-inseminated heifers (non-AIH). Free-catch urine samples, from 18 randomly selected dairy animals, were collected on day 15, 22, 30, and 35 after insemination and used at a dilution rate of 1:4. Maize seeds were soaked in different treatment groups, followed by observation for germination and shoot length. Data were analysed descriptively to obtain the means, and inferential statistics including Kruskal-Wallis and Bonferroni tests were employed to explore significance between the means. The Eta coefficient test statistic evaluated the association between selected variables. The lowest means for germination (21.92±1.15) and shoot length (0.91±0.05) of seeds in AIC group were observed at day 35. The highest means on the same day were 84.00±1.95 and 3.05±0.14, for seed germination and shoot lengths in DW, respectively. The mean germination percentages of seeds in AIC differed significantly (P<0.05) between days after insemination; the mean values for other treatments did not differ significantly (P>0.05). The mean shoot lengths of seeds in AIC differed significantly (P<0.05) between days after insemination; no level of significance (P>0.05) was observed for other treatment groups. The association between treatment factor and seed germination on day 15 and 35 was 0.149 and 0.993, respectively; and was 0.166 and 0.876 on 15 and 35, respectively, with shoot lengths. In conclusion, Punyakoti test can be used for pregnancy detection, preferably towards month-end after insemination, in dairy cattle with seed germination parameter being sufficient to get reliable results.
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