Sujata Jailkhani

Changes in peripheral inhibin levels and follicular development following treatment of buffalo with PMSG and Neutra-PMSG for superovulation

Fourteen buffalo were synchronized by administration of a prostaglandin (PG) salt Lutalyse in a double injection schedule, with a single intramuscular (im) injection of 25 mg at Day -13, followed by 30 mg and 20 mg im 12 h apart on Day 0 of the experiment. The 30-mg PG injection was designated as 0 h of the experiment. Group I animals (n = 4) received saline and served as the controls, while animals in Groups II and III (n = 5 each) received PMSG (2500 IU im at -48 h. Group III animals were administered 5 ml Neutra-PMSG intravenously at 60 h. Blood samples were collected every 48 h from Day -12 to Day -4, every 24 h from Day -4 to Day 0, every 3 h from Day 1 to Day 4 and every 24 h from Day 5 to Day 10 of experiment for the measurement of peripheral plasma inhibin concentrations by RIA. The number of large follicles (> 10 mm diameter) in animals of Groups II and III was assessed by ultrasonography on Days -2, -1, 0, 1, 2, 5 and 7 of the experiment. Treatment with PMSG of Group II animals resulted in a significant increase (P < 0.05) in plasma inhibin concentrations over that of control animals of Group I at 24 to 99 h, with a peak inhibin concentration of 1.01 +/- 0.31 ng/ml at 48 h. Treatment with Neutra-PMSG in Group III animals caused a significant reduction (P < 0.05) in the peripheral inhibin concentrations at 84 to 120 h and in the number of large unovulated follicles at 168 h compared with that in Group II animals. Peripheral inhibin levels in Group III animals came down to those of Group I after 21 h of Neutra-PMSG treatment. These results suggest that treatment of buffalo with PMSG for superovulation causes a marked rise in peripheral inhibin concentrations. Administration of Neutra-PMSG after PG treatment reduces the peripheral inhibin concentrations and the number of large unovulated follicles.

Development of a simple, direct, microtitre plate enzymeimmunoassay (EIA) for progesterone determination in whole milk of buffaloes

A method of estimating progesterone in buffalo whole milk by EIA using progesterone 6 beta-OH-hemisuccinate-horseradish peroxidase as the enzyme label and an antiserum raised against progesterone-7 alpha-carboxyethyl-thioether-BSA was developed. The microtitration plates used in the assay were first coated with affinity purified sheep IgG developed against rabbit IgG. The immune reaction was performed by incubating a mixture of 1 microliter of whole milk (diluted to 20 microliters with assay buffer), 100 microliters of enzyme label and 100 microliters of antiserum for 90 min in the dark. After washing the plates, 150 microliters of the substrate solution was added. The mixture was incubated in the dark for 40 min before the reaction was stopped and the optical density was measured at 450 nm. The calibration curve was sensitive in the range 0.8-40 pg/well, corresponding to 0.8-40 ng/ml. Milk samples from cycling buffaloes were tested for progesterone concentration by running parallel EIA and RIA. A good correlation of 0.91 was obtained and the estimated values were similar using both techniques. The method has demonstrated about 10 times greater sensitivity than RIA in buffalo milk.

Assessment of superovulatory responses in terms of palpable corpora lutea and embryo recovery using milk progesterone.

Milk progesterone profiles were used to assess superovulatory responses in cyclic lactating buffalo (n = 9) in terms of the number of ovulations and the number of embryos recovered. All of the buffalo received a total of 30 ml of folltropin divided into morning and evening doses and spread over 5 days, beginning on Day 10 of the estrous cycle (day of expected estrus = Day 0). Milk samples for progesterone determination were collected on alternate days from all nine animals from Day 1 prior to the expected synchronized estrus to 5 days after flushing for embryo recovery. All animals were palpated per rectum 1 day prior to flushing in order to record the number of corpora lutea. Of an estimated 23 ovulations from the nine buffalo, only 12 embryos were recovered, of which one was an unfertilized oocyte. Milk progesterone profiles from individual buffalo suggested that a poor superovulatory response in terms of embryo recovery in some buffalo was caused by a failure to respond optimally to lutalyse treatment for the induction of estrus. It was hypothesized that ova trapping by the fimbriae of the fallopian tubes may not be efficent in this species especially in the superovulated ovaries.

Application of a sensitive, heterologous enzymeimmunoassay for progesterone determination in unextracted buffalo plasma samples collected in an embryo transfer experiment

Abstract A simple, direct enzymeimmunoassay (EIA) on microtitre plates for buffalo plasma progesterone using the second antibody coating technique and horseradish peroxidase (HRP) as the enzyme label is described and compared with the conventional progesterone radioimmunoassay (RIA). Progesterone estimates from plasma samples by EIA showed good correlation (0.84 to 0.97) with the RIA values and the levels measured in the two systems were identical. The utility of the plasma progesterone EIA in an embryo transfer experiment was determined by assaying the hormone in blood plasma samples collected frequently, before and after superovulation in follicle stimulating hormone (FSH)-primed buffaloes. A total of ten embryos were recovered from three buffaloes, of which six were from a single buffalo. Of the remaining four buffaloes from which no embryos were recovered, progesterone profiles indicated that two had not responded optimally to lutalyse treatment for estrus induction, one failed to ovulate after superovulatory treatment and one did not exhibit an optimum progesterone profile after superovulatory estrus.