Study Area and Duration The study was performed at the Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh during November 2011 to June 2012.
Seed Selection Wheat seeds sample (Variety Sotabdi) was collected from Bangladesh Agricultural Development Corporation (BADC) office, Mymensingh. The seeds had a record of about 85 percent of germination rate after 48 hours at room temperature.
Animal Selection and Management Fifty crossbred cows were selected from the Bangladesh Agricultural University Dairy Farm, Mymensingh for the present study. Among them, 25 cows were artificial inseminated (Pregnant) and 25 cows were not artificially inseminated (Nonpregnant). The age of the cows ranged from 4 to 8 years and their parity ranged between 0 and 5. The cows were fed green grasses mixed with rice straw. They also supplied mixed concentrate (Rice polish, wheat bran, broken rice, and oil cake) with limited grazing facilities.
Preparation of Instrument The petridishes, beaker, pipette are properly cleaned and sterilized by heating at 121o C for 20 minutes. The wheat seeds were taken and uniformly spread over a single layer filter paper bed in sterile petridishes.
Urine Sample Collection The urine samples used for detection of pregnancy exhibited by the inhibition of seed germination were collected from 25 cows which are artificial inseminated (AI) and 25 cows which are not artificial inseminated. The samples were collected during natural maturation as well as by induced maturation, done by continuous stroking of the skin just below the vulva. The urine of the pregnant cows was confirmed by per rectal palpation on 80th days post-AI. The urine samples were collected early in the morning on day 14, 21, 28, 35 and 45 of post-AI and were subjected to seed germination inhibition test within four to six hours of collection. Regardless of the method, clean, sterilized and dry plastic containers were used to collect the urine.
Seed Germination Inhibition Test After the collection of urine from the selected cows was transported to the laboratory as soon as possible. In the laboratory, the urine was diluted at the ratio of 1:4 with distilled water. The germination inhibition test was observed by adding three types of samples: Urine samples from AI cows (Pregnant), urine samples from the non-AI cows (Nonpregnant) and distilled water samples which were kept as control. Seventy-five (75) sterilized petridishes were divided equally into three groups (Namely group A, B and C) each of which contained 25. About 15 ml of diluted urine from AI cows (Pregnant) was added in each petridishes of group A and about 15 ml of diluted urine from non-AI cows (Nonpregnant) was added in each petridishes of group B. Similarly about 15 ml of distilled water was added in each petridishes of group C for control. Then A filter paper was placed on each petridish (75) of all groups (A, B and C) containing 15 wheat seeds on its. All the petridishes (75) of three groups were covered with the trays to avoid evaporation, whereas there was little air movement and they were undisturbed for three days.
Seed Germination Inhibition Test After the collection of urine from the selected cows was transported to the laboratory as soon as possible. In the laboratory, the urine was diluted at the ratio of 1:4 with distilled water. The germination inhibition test was observed by adding three types of samples: Urine samples from AI cows (Pregnant), urine samples from the non AI cows (Nonpregnant) and distilled water samples which kept as control. Seventy-five (75) sterilized petridishes were divided equally into three groups (Namely group A, B and C) each of which contained 25. About 15 ml of diluted urine from AI cows (Pregnant) was added in each petridishes of group A and about 15 ml of diluted urine from non AI cows (Nonpregnant) was added in each petridishes of group B. Similarly about 15 ml of distilled water was added in each petridishes of group C for control. Then A filter paper was placed on each petridish (75) of all groups (A, B, and C) containing 15 wheat seeds on its. All the petridishes (75) of three groups were covered with the trays to avoid evaporation, whereas there was little air movement and they were undisturbed for three days.