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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access FREEZABILITY OF BUCK SPERMATOZOA TREATED WITH CHELATING AGENT FOR FIBRONECTIN TYPE II PROTEINS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) SHINY M.; Hiron M. HarshanA study to evaluate the effect of Fn2 type protein chelating agent (choline chloride) or immediate extension and washing on the freezability of buck spermatozoa was carried out at Artificial Insemination Centre, under the department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, Thrissur using 18 normal ejaculates from an adult healthy Malabari crossbred buck. The total seminal plasma protein content was estimated by Lowry’s method. The ejaculates were grouped into three with group I consisting of ejaculates collected directly into Tris extender alone, group II consisting of ejaculates collected into Tris extender containing choline chloride and group III serving as the control. The processed semen was packed in 0.25 ml French straws after extending them in Tris yolk glycerol extender and cryopreservation was carried out. The volume of buck semen samples collected under group III (control) were 0.57 ± 0.09 ml and average sperm concentration was 3971 ± 99.42 million per ml. The gross semen characteristics except volume (0.6 ± 0.09 ml and 0.32 ± 0.03 ml respectively) and sperm concentration (3683 ± 115.49 and 3905 ± 147.50 million per ml) could not be evaluated directly from semen samples of group I and group II as these were collected directly into a collection vial containing Tris extender and choline chloride solution respectively. Mean progressive motile spermatozoa of the three groups were 83.83 ± 1.25, 82.67 ± 1.26 and 84.67 ± 0.84 per cent respectively, while the mean live sperm percentage were observed to be 94.83 ± 0.83, 94.17 ± 1.08, 95.33 ± 0.61 respectively in the three groups. Average per cent of abnormal spermatozoa of the three groups were 3.00 ± 0.63, 3.00 ± 0.68 and 2.50 ± 0.56 respectively. Mean per cent of acrosomal integrity of spermatozoa were 90.50 ± 0.99, 90.17 ± 1.17 and 89.67 ± 0.95 respectively in three groups. After equilibration, the percentage progressive sperm motile spermatozoa in group I, II and III (control) were 76.17 ± 1.08, 77.17 ± 1.08 and 73.67 ± 1.15 respectively. The viable spermatozoa percentage of three groups observed at prefreeze stage were 82.67 ± 1.36, 83.33 ± 0.76 and 79.33 ±0.99 respectively which did not show any significant difference between the groups. The intact acrosome percentage at prefreeze levels were 83.67 ± 1.12 in group I, 82.00 ± 0.97 in group II and 82.67 ± 0.95 in group III. The sperm abnormality of the three groups were 3.33 ± 0.56, 3.33 ± 0.42 and 3.50 ± 0.67 respectively. The mean of membrane integrity by hypo osmotic swelling test of group I, group II and group III were 67.67 ± 2.04, 65.17 ± 2.02, 61.17 ± 1.66 respectively. Spermatozoa of group I had significantly higher HOS response than spermatozoa of group III (control). After freezing and thawing, the mean percentage of progressively motile spermatozoa were found to be 40.83 ± 0.91 in group I, 44.67 ± 1.31 in group II and 33.83 ± 1.30 in group III. The post-thaw live spermatozoa were 49.17 ± 0.91, 52.17 ± 1.30 and 44.17 ± 1.40 respectively. The values obtained in both the treatment groups differed significantly from the values observed in the control group. The values of intact acrosome in percentage were 65.83 ± 1.35 in group I, 71.00 ± 1.00 in group II and 63.33 ± 1.05 in group III. The spermatozoa of choline chloride treated group had significantly higher intact acrosome than spermatozoa of both group I and control group. The percentage of sperm abnormality obtained after thawing were 11.33 ± 0.56 in group I, 10.67 ± 0.49 in group II and 11.17 ± 0.60 group III respectively. The mean of membrane integrity by hypo osmotic swelling test of group I, group II and group III were 43.83 ± 2.50, 48.17 ± 2.30, 37.17 ± 1.89 respectively. Significant difference (p<0.05) was obtained between teh HOS response of spermatozoa of choline chloride treated group and the control group. It was observed that both immediate extension and washing of Malabari buck spermatozoa or treatment with choline chloride at the time of semen collection resulted in improved sperm post thaw characters. Of the two techniques, treating spermatozoa with choline chloride at the time of collection resulted in better post thaw motility, viability acrosome integrity and HOS response of frozen buck semen.ThesisItem Open Access GONADOTROPHINS MEDIATED SUPEROVULATORY RESPONSE AND VIABILITY OF FRESH AND VITRIFIED RABBIT EMBRYOS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) VEENA C. PHILIP; Metilda JosephSuperovulation was induced in two groups (A and B) of Newzealand White female rabbits by administration of a single dose of 150 IU of eCG intramuscularly in group A, and two 3 mg and then four 4 mg injections of p-FSH subcutaneously at 12 hour intervals, in group B. On observation of heat signs, mating of females with the same buck was done twice and to induce ovulation 150 IU hCG was given intravenously. Animals belonging to group C remained as control. In vitro collection of embryos were done 72 h post coitum. The onset and intensity of oestrum, number of ovulations, embryo recovery and quality of embryos were studied and compared with those of control animals. Eighteen transferrable embryos from each group were subjected to OPS vitrification. After a minimum storage period of 10 days, the vitrified embryos were thawed and examined for morphological characteristics and membrane integrity was assessed by Trypan blue staining. Further, viability of embryos after in vitro culture was also assessed. The mean interval from gonadotrophin administration to onset of oestrus in groups A and B were 77±5 h and 75±3.30 h, respectively. The intensity of heat was higher in treatment groups than in control. The percentage of animals which showed sexual receptivity in A, B and C groups were 66.67, 100 and 100 respectively. The mean ovulation rate in groups A, B and control were 8.50±5.22, 10.67±3.25 and 7.50±1.38, respectively. The range of corpora lutea was 0 to 33 in group A, 0 to 21 in group B and 2 to 12 in group C. Eventhough total ovulation points were more in group B followed by groups A and C, there was no significant difference in the ovulation rate between the three groups. The total number of haemorrhagic follicles in the groups A, B and control were 1.00±0.63, 10.67±4.64 and 2.00±0.58, respectively. The average number of anovulatory follicles in groups A, B and C were observed as 1.49±0.23, 2.40±0.20 and 1.76±.08, respectively. Significant difference (p<0.05) could be seen in the number of haemorrhagic and anovulatory follicles between group B and other two groups. The mean total ovarian response in the groups A, B and C were 11±6.50, 26.33±7.45 and 11.67±1.9, respectively which did not show any significant difference. 119 The average (percentage) embryo recovery rate in groups A, B and C were 5.50±3.83 (55.56%), 7.83 (66.50%) and 4.83±1.72 (53.41%), respectively. The mean fertilization rate in groups A, B and C were100, 100 and 93.55 per cent, respectively. The corresponding values for mean (percentage) transferrable embryo recovery rate in the three groups were 5.50±3.83 (100), 7.50±2.83 (95.74) and 3.33±1.20 (68.97) respectively. Overall total (percentage) of morulae and blastocysts recovered 72 h post coitum was 49 (44.95) and 60 (55.05) respectively. Percentage embryo recovery rate after OPS vitrification in groups A, B and C were 72, 77.78 and 55.56, respectively. Percentage of morphologically normal embryos recovered from animals belonging to these groups were 92.30, 71.43 and 80, respectively. After Trypan blue staining of morphologically normal embryos, the percentage of viable embryos in the three groups were as 81.8, 83.33 and 71.43, respectively. On further viability assessment of vitrified embryos after TB staining using in vitro culture revealed development of 77.78 per cent of the embryos after 24 h. Present study revealed that administration of gonadotrophins followed by mating twice at the induced heat and hCG supplementation could be effectively used for producing satisfactory superovulatory response in rabbits. The study also indicated that Open pulled straw vitrification technique can be successfully applied for cryopreservation of rabbit embryos. Trypan blue staining technique can be employed as a satisfactory method for assessing the viability of rabbit embryos.ThesisItem Open Access UTERINE INVOLUTION AND FERTILITY IN CROSSBRED COWS TREATED WITH ECBOLICS DURING PUERPERIUM(COLLEGE OF VETERINARY AND ANIMAL SCIENCE-MANNUTHY,THRISSUR, 2016) PRAVEEN, S; UNNIKRISHNAN, M PThe study was undertaken with the objective to assess the efficacy of ecbolic agents on uterine involution, resumption of ovarian cyclicity and fertility in post partum crossbred cows. A total of 36 apparently healthy cows, immediately after normal calving, were randomly allotted to four groups of nine animals each and were administered with oxytocin - 50 IU (group I); cloprostenol sodium - 500 μg (group II) and methylergometrine maleate – 5mg (group III) intra muscularly within two hours of calving. Animals in group IV were left untreated (control). Cows were subjected to detailed clinico-gynaecological and ultrasonographic evaluation at day 10 post-partum and at five day interval till day 40 or complete uterine involution, for assessing uterine involution and resumption of ovarian cyclicity. Blood samples were collected on day 10, 20, 30 and 40 post-partum, for serum progesterone estimation. Administration of ecbolics significantly reduced occurrence of RFM and mean number of days required for complete uterine involution as compared with untreated group. Uterine wall thickness among different groups significantly reduced from day 10 to 30 post-partum post-partum. By day 40 post-partum, all the animals in the treatment groups and 88.89 per cent of animals in control group completed uterine involution. The mean number of days required for complete uterine involution in group I to IV were 31.67 ± 1.67, 28.33 ± 1.67, 28.89 ± 1.62 and 32.22 ± 1.21 respectively. Calving to first observed estrus interval, calving to first AI interval, calving to conception interval were comparatively shorter in group II, although not significantly different from other groups. Conception rate in group I to IV after first service was 22.22, 44.44, 22.22 and 11.11 per cent respectively and overall conception rate (up to three AI) was 55.56, 77.78, 55.56 and 55.56 per cent, respectively, which also indicated better result in PGF2 treated group.ThesisItem Open Access FERTILITY MANAGEMENT IN CROSSBRED CATTLE DURING HEAT STRESS BY FORTIFYING LUTEAL FUNCTION(COLLEGE OF VETERINARY AND ANIMAL SCIENCE-MANNUTHY,THRISSUR, 2016) BASIL PAUL; UNNIKRISHNAN, MPA study was conducted among crossbred cattle with the objective to assess the effect of heat stress on fertility and also to evaluate the effect of fortifying luteal function by using hCG, on fertility. Apparently healthy cycling crossbred cows detected in oestrus were utilized for the study. The experiment was carried out at ULF & FRDS, Mannuthy in two phases viz. period of the year with average low THI (November- January) and period of the year with average high THI (March –May). In each phase animals were divided randomly in to treatment and control groups of 10 animals each. Cervical mucus was collected during oestrus for study of rheological properties and BCMPT; artificial Insemination (AI) was done in all the animals and those in treatment groups were administered 1500 IU hCG on day 6 post AI. Blood samples were collected on day 6, 12 and 24 post-AI for progesterone assay and pregnancy was confirmed by rectal examination on day 45 post AI in non-returned animals. The average THI of the station was calculated as per LPHSI (1990) formula using meteorological data from CAADECCS, Mannuthy. Average values during low and high THI phase were 79 and 83.5 respectively. Mean duration of standing oestrus in low THI period and high THI period did not differ significantly (24.67 ± 0.98 vs 24.70 ± 1.17 h). Contrary to the previous reports, intensity and duration of oestrus was not affected by heat stress. Transparency of oestrual mucus was not affected by heat stress Mean spinbarkeit value during heat stress was significantly higher, which was contrary to previous reports. Mean spinbarkeit value was significantly higher among conceived than non-conceived animals. Majority of animals had typical fern pattern in both the phases and heat stress did not seem to influence the fern pattern. Per cent of animals exhibiting typical fern pattern was more among conceived animals than non-conceived animals. Cervical mucus penetration distance was not affected by heat stress, but it was significantly higher among conceived animals than non-conceived. Though there was no significant difference between phases regarding progesterone level on 6th and 12th day post AI, concentration on 12th day during comfortable period was higher than that recorded during heat stress. Administration of hCG had no significant effect on subsequent luteal function Mean serum progesterone level in both the phases was significantly higher in conceived animals compared to non-conceived animals on 6th , 12th and 24th day post The pregnant animals in both phases had mean serum progesterone levels of >2ng/ml on day 24. The conception rate was better in low THI phase than during high THI phase (37.7 vs 25 per cent). Conception rate was higher in treatment group than control (40 vs 25 per cent). Even though hCG administration did not significantly increase progesterone level in treated animals, better conception was recorded among treated animals. Also, better conception was recorded during low THI phase (comfortable period).ThesisItem Open Access SURVIVAL RATE OF IMMATURE AND IN VITRO MATURED BOVINE OOCYTES VITRIFIED USING CRYOLOOP METHOD(COLLEGE OF VETERINARY AND ANIMAL SCIENCE-MANNUTHY,THRISSUR, 2016-07-05) ALICIAH MARIE KURIAKOSE; METILDA JOSEPHSuccessful cryopreservation of oocyte using vitrification ensures the availability of large number of oocytes for in vitro maturation (IVM) and in vitro fertilization (IVF). The present study was carried out to evaluate effect of meiotic stage on survival rate of bovine oocytes after cryoloop vitrification and to assess viability using morphological evaluation and trypan blue staining. Ovaries of 36 slaughtered cattle were selected and randomly allotted to three groups. Oocytes were recovered by follicular aspiration. Half of the group І culture grade oocytes (n=44) were subjected to trypan blue (0.05%) staining for viability assessment and aceto-orcein staining for evaluation of nuclear status. Culture grade oocytes in group ІІ (n=38) were matured in vitro after cryoloop vitrification and culture grade oocytes in group ІІІ (n=39) were vitrified after in vitro maturation. A total of 241 oocytes were aspirated and yield of immature oocytes per cow was 6.69. Trypan blue staining of culture grade oocytes obtained by morphological evaluation in group І yielded only 81.82 per cent live oocytes. Aceto-orcein staining of group І culture grade oocytes revealed that 54.44 per cent of oocytes were in GV stage. Morphological survival rate of mature vitrified oocytes in group ІІІ was higher than immature vitrified and matured oocytes in group ІІ (66.67% vs 52.63%). Cumulus expansion, polar body extrusion and trypan blue dye exclusion rate were also higher in group ІІІ, whereas nuclear maturation rate was almost similar. The meiotic stage at which cryoloop vitrification was carried out had no significant influence on survival rate, viability, cumulus expansion, polar body extrusion and nuclear maturation rate of bovine oocytes. Hence immature bovine oocytes can be preserved using cryoloop technique, where facilities are not available for IVM. Compared to morphological evaluation, trypan blue staining technique was found to be more reliable for assessing oocyte viability.ThesisItem Open Access EFFICACY OF MEDICAL MANAGEMENT AND SURGICAL TRANS-CERVICAL CATHETERISATION FOR CANINE CYSTIC ENDOMETRIAL HYPERPLASIA(College of Veterinary and animal Science,Mannuthy, 2016-07-05) Chinnu P, Vijayan; Shibu, SimonAs a part of the study, a preliminary survey was conducted to investigate the occurrence of cystic endometrial hyperplasia (CEH) in bitches from 2013 to 2015. Twelve bitches under six years of age with CEH were included in the study and grouped into two. Group I animals were subjected to medical management (mifepristone-cabergoline-prostaglandin combination) and Group II animals underwent surgical trans-cervical catheterization. Physical, physiological, haematological and biochemical parameters and ultrasound characters of the affected uterus were studied and compared. Progesterone concentration was estimated at weekly intervals till clinical recovery. Preliminary survey revealed that CEH was frequently reported in the Labrador (20.44%) and Rottweilers (19.95%). A negative correlation was observed between age of animals and the number of cases reported with CEH. Higher percentage of occurrence of CEH was in the age group of two and three years (27.1 %). All clinical signs subsided 14 days after commencement of treatment in both the groups. The major micro organism obtained from vaginal swab was E.coli (59%). Leucocytosis was observed in animals in both the group (range 16.5-44.2103/mm3) with neutrophilia (range 77-91 %), lymphopenia and normocytic normochromic anaemia before treatment and values were in normal physiological range by Day 21 in Group I and by Day 14 in Group II. Elevation in BUN and creatinine was observed in both the groups before treatment. Even though the level of progesterone was not abnormally high in CEH affected animals with respect to the stage of oestrous cycle however, it reduced to basal levels after treatment. Ultrasonographically mean thickness of uterine wall, trace area and diameter of largest sacculation were reduced after treatment and became normal by Day 14. Based on the clinical recovery the surgical group had an earlier recovery though it involved a time consuming surgical procedure. Till detailed studies on conception rates are available both surgical and medical management to retain breeding potential have to be considered positively.ThesisItem Open Access ENDOMETRIAL CYTOLOGY IN POSTPARTUM CROSS-BRED DAIRY COWS AND ITS CORRELATION WITH FERTILITY(College of Veterinary and animal Science,Mannuthy, 2016) Arokia Robert, M; Shibu SimonThe study was undertaken with the objectives of characterizing endometrial cytology in postpartum dairy cows and determining the prevalence of subclinical endometritis with reference to its effect on subsequent fertility. The study was conducted in KVASU farms at Mannuthy and Thumboormuzhi. Twenty-four apparently healthy postpartum cross-bred dairy cows were subjected to detailed clinico-gynaecological and vaginoscopic examination. Endometrial sampling was done using uterine cytobrush (UC) and uterine lavage (UL) on 30, 40 and 50 days postpartum (DPP). The mean (±SE) per cent of polymorphonuclear (PMN) cells by UC were 7.53 ± 1.56, 3.23 ± 1.75 and 1.35 ± 2.21 in normal group and 9.80 ± 1.84, 10.20 ± 2.07 and 17.01 ± 2.61 in endometritis group on 30, 40 and 50 DPP, respectively. Whereas by UL, the PMN cells per cent were 7.64 ± 1.76, 4.92 ± 1.41 and 1.21 ± 1.74 in normal group and 11.20 ± 2.08, 14.60 ± 1.67 and 21.70 ± 2.06 in endometritis group, respectively, on 30, 40 and 50 DPP. In normal group, significant difference between UC and UL on both 30 and 40 DPP. In the endometritis group, significant difference was observed between UC and UL on 30, 40 and 50 DPP. The endometritis group cows were treated with intrauterine infusion of 100 µg E.coli lipopolysaccharide (LPS). The mean (±SE) pH of vaginal discharge in normal and endometritis group was 7.17 ± 0.15 and 8.20 ± 0.11, respectively and pre and post intrauterine treatment of endometritis group was 8.20 ± 0.11 and 7.40 ± 0.12, respectively. The conception rates after first, second and third service (overall) observed in this study were 42.86, 64.29 and 78.57 per cent in normal group; 20, 50 and 60 per cent, respectively, in endometritis group after LPS treatment. From the results of the study, it could be inferred that endometrial cytology could be used for a presumptive diagnosis of SCE. Metricheck and disposable vaginal speculum are recommended for routine diagnosis of endometritis. The prevalance of endometritis in KVASU farms was found to be 41.67 per cent. E.coli LPS has a reasonable therapeutic effect in the management of SCE and thereby increase the conception rate.ThesisItem Open Access ULTRASONOGRAPHIC STUDIES ON PERIOVULATORY FOLLICULAR DYNAMICS AND RESPONSE TO GnRH THERAPY IN REPEAT BREEDING CATTLE WITH PROLONGED OESTRUS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2017) NIYAS, E.; Kurien, M. O.The study was carried out with the objectives of ultrasonographic (US) assessment of follicular dynamics (FD), progesterone (P4) concentration and to determine the efficacy of GnRH administration on ovulation and fertility in repeat breeding (RB) cows with prolonged oestrus. Thirty healthy postpartum crossbred dairy cows maintained under identical conditions were selected from University Livestock Farm and Fodder Research and Development Scheme (ULF & FRDS). Ten cows with normal estrous cycle and exhibiting estrous period of less than 36 h formed the control group and twenty repeat breeding animals with history of prolonged oestrus (>36 h) was classified under Group II (n=10) and Group III (n=10). The study was conducted in two phases. In Phase-I, follicular dynamics and luteal growth and regression characteristics of all the animals were studied by transrectal ultrasonography (TRUS) for one complete oestrous cycle length. Blood samples were collected for serum progesterone estimation on day 0, 5, 10 and day of next oestrus. In Phase-II, cows exhibiting oestrus in Group I and III were inseminated when the follicle attained maximum size and re-insemination after 24 h carried out if ovulation did not occur. Cows in Group II were treated with 20 μg Buserelin acetate intramuscularly when the diameter of preovulatory follicle reached the persistent dominant size and inseminated 8 h after GnRH administration. Pregnancy was diagnosed by TRUS on day 25 after insemination and was confirmed on day 60 of pregnancy. In the control group 70 per cent animals exhibited two waves per cycle and 30 per cent animals shown three wave cycles whereas in RB animals three pattern of follicular growth noticed, the patterns observed were three waves (50%), two waves (40%) and one wave (10%) respectively. The mean maximum diameter (mm) of preovulatory follicle for cows with 2 waves was 16.93± 0.63 and 14.78± 0.83 while it was 13.57± 0.96 and 14.92± 0.74 in 3 wave cycles in normal & RB respectively. Duration of persistence of ovulatory follicle was found to be longer in RB animals (>24 h in 70% animals) whereas it was less than 24 h in all normally cycling animals in the control group. The luteal growth and regression pattern between the groups varied without significant difference. The mean serum progesterone value on day 5 & 10 in RB animals were lower (1.77± 0.19 ng/ml and 3.31± 0.29 ng/ml) when compared to animals with normal oestrus (2.17± 0.19 and 4.76± 0.29 ng/ml). A significant difference in the progesterone concentration on day 0 (0.36± 0.03 ng/ml in control group and 0.67± 0.03 in RB animals) was observed. Conception rate was higher in the control group (70 per cent) and it was 60 per cent in group II (GnRH treated) and 20 per cent in group III (non-treated) animals.ThesisItem Open Access ULTRASONOGRAPHIC STUDIES ON UTERINE AND OVARIAN CHANGES DURING EARLY PREGNANCY IN BOVINES(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2017-08-16) Naveen Kumar, R. H.; Shibu SimonWith an aim to compare two methods of early pregnancy diagnosis by ultrasonography and progesterone assay a study was conducted at ULF and FRDS, Mannuthy. Twelve apparently healthy post-partum dairy cows with normal cyclicity and body condition score were selected for the study. Trans-rectal ultrasonographic examination was performed from day eight post insemination to day 30 on alternate days. The animals were grouped as pregnant and non-pregnant based on ultrasonographic findings and return to oestrus. Further, the pregnant animals were ultrasonograhically evaluated till day 60 at five days interval. Blood samples were collected on days 0, 2, 4, 8, 17, 21 and 24 for estimation of serum progesterone level. Size of CL had no significant difference between days 8 to 60 in pregnant animals. When CL changes were compared between pregnant and nonpregnant animals, no significant difference was observed between these two groups till day 16. Thereafter, significant reduction in size was observed in nonpregnant animals. The mean day of disappearance of CL cavity was 16.55 and 15.5 in pregnant and non-pregnant animals, respectively. The CL blood flow was the same in pregnant and non-pregnant animals till day 15. But, on day 16 blood flow was increased in non-pregnant animals than pregnant animals. Thereafter, it was reduced in non-pregnant animals, whereas, in pregnant animals it was maintained at same level. Earliest day on which foetal/extrafoetal structures were visualised is as follows; anechoic area (16 d), amniotic vesicle (22 d), embryo (22 d), heart (24 d), placentome (26 d), CRL (28 d), vertebral column (35 d), limb buds (40 d), BPD (40 d), liver (45 d), spleen (45 d), tail bud (45 d), BD (45 d) and UD (45 d), cloven feet (50 d), orbit (50 d) and brain ventricles (50 d), Bladder (55 d), Falx cerebri (55 d) and each vertebrae and ribs were easily distinguishable by (60 d). The PI and RI values for umbilical blood flow had no significant difference between days. Pregnancy could be detected as early as day 24 by B-mode ultrasonography, whereas, when Colour Doppler ultrasonography was combined with B-Mode ultrasonography, pregnancy can be diagnosed as early as day 18. Although based on progesterone assay pregnancy could be diagnosed as early as day 18 it may not be confirmatory as serum progesterone level could be enhanced in some pathological conditions also.