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20103
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Subject: Animal Reproduction and Gynecology × Author: KAU × End date: 2012 × Start date: 2010 ×
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    ThesisItemOpen Access
    In vitro maturation of caprine follicular oocytes
    (Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2010) Ambili, John; KAU; Joseph, Mathew
    This study was designed to analyse the effect of three oocyte retrieval methods, aspiration, slicing and puncture on the yield of different quality grades of oocytes and to evaluate the in vitro maturation rate of different grades of caprine oocytes. One hundred and thirty eight ovaries of Malabari goats and its crossbreds collected from the slaughter house were subjected to the oocyte retrieval methods. The oocytes harvested were graded based on the number of cumulus cell layers and ooplasm character into A, B, C and poor quality grades. Oocytes of A, B and C grades were subjected to maturation for 24 h in TCM-199 medium under standard culture conditions. Average yield of COC per ovary by aspiration, slicing and puncture was 3.93 ± 0.11, 4.44 ± 0.06 and 3.59 ± 0.07 respectively. Yield was significantly higher in slicing method than aspiration and puncture. The percentage yield of A, B, C and poor quality grades of oocytes by aspiration method was 26.74 per cent, 26.62 per cent, 24.77 per cent and 21.87 per cent respectively. Mean yield of oocytes of each quality grade by the same method were 1.05 ± 0.05, 1.05 ± 0.08, 0.98 ± 0.07 and 0.86 ± 0.04 respectively. Slicing yielded 23.08 per cent A class, 28.15 per cent B class, 24.44 per cent C class and 23.96 per cent poor quality oocytes. Mean yield of oocytes per ovary in these classes by slicing method were 1.04 ± 0.04, 1.25 ± 0.05, 1.08 ± 0.07 and 1.06 ± 0.06 respectively. Percentage yield of A, B, C and poor quality oocytes by puncture was 29.01 per cent, 30.26 per cent, 22.07 per cent and 18.66 per cent respectively. Mean yield per ovary by puncture method was 1.04 ± 0.04, 1.08 ± 0.03, 0.79 ± 0.04 and 0.67 ± 0.03 for A, B, C and poor quality oocytes respectively. No significant difference was observed in the yield of A, B and C class oocytes between aspiration, slicing and puncture. Yield of poor quality oocytes were significantly more in slicing method. The cumulus expansion rate of A class oocytes obtained by aspiration, slicing and puncture was 77.75 per cent, 69.70 per cent and 71.49 per cent respectively. Class C oocytes exhibited a cumulus expansion rate of 63.48 per cent, 51.37 per cent and 63.39 per cent respectively when collected by aspiration, slicing and puncture method. Class C oocytes obtained by aspiration, slicing and puncture when subjected to in vitro maturation exhibited a cumulus expansion rate of 39.17, 32.57 and 37.29 per cent respectively. Retrieval method was found to have no significant effect on cumulus expansion potential of caprine oocytes, whereas the COC morphology had significant effect on cumulus expansion potential. Nuclear maturation rate of A class oocytes collected by aspiration, slicing and puncture method were respectively 40, 30 and 50 per cent and polar body extrusion rate was 30, 20 and 30 per cent respectively. Class B oocytes exhibited nuclear maturation rate of 20, 10 and 30 per cent and polar body extrusion rate of 10, 10 and 20 per cent respectively by aspiration, slicing and puncture. Ten, 10 and 20 per cent of C class oocytes retrieved by aspiration, slicing and puncture exhibited nuclear maturation and 10 per cent polar body extrusion was observed in C class oocytes retrieved by puncture. None of the C class oocytes collected by aspiration or slicing exhibited polar body extrusion. This study proved that slicing is a better method than aspiration or puncture for retrieval of oocytes from caprine ovaries as it yielded more number of oocytes per ovary. Retrieval methods had no significant effect, whereas COC morphology was found to have significant effect on cumulus expansion, nuclear maturation and polar body extrusion rates of different grades of oocytes
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    ThesisItemOpen Access
    Diagnostic and therapeutic approaches for enhancing reproductive efficiency in female dogs
    (College of Veterinary and Animal Sciences, Mannuthy, 2010) Deepthi, L; KAU; Aravinda Ghosh, K N
    The present study was undertaken for finding optimal breeding time in bitches, early pregnancy diagnosis and tackling pseudopregnancy using antiprolactin drugs. Experiment consisted of 55 apparently healthy bitches which were brought to the Small Animal Obstetrics and Gynaecology unit of Veterinary College Hospital, Mannuthy and University Veterinary Hospital, Kokkalai for getting breeding advice. Fertility rate was assessed in two different groups viz., Group I (n=30) and Group II (n=25). The conception rate was 73.3 per cent in group I and 88.0 per cent in group II. Conception rate was more in those animals, which were bred, based on vaginoscopy than those bred based upon clinico-gynaecological examination and vaginal exfoliative cytology. This supported the view that performing vaginoscopy will augment conception rate in bitches. Clinico-gynaecological examination together with vaginal exfoliative cytology was found to be useful for finding optimal breeding time in bitches under field conditions. Vaginoscopy, in addition to being a non-invasive method, had the added advantage of predicting the fertilization period, thereby breeding advice could be more accurate as it helps in the visual inspection of vaginal mucosal folds. Pregnancy diagnosis was carried out in 20 animals at 20th, 35th and 50th day of gestation. Abdominal palpation at 20 days after breeding was non-confirmatory of pregnancy but at 35 days of gestation foetus could be appreciated as tense distinct uterine swellings. By ultrasound scanning at 20 days of gestation the image of embryonic vesicles appeared as spherical structures with anechoic consistency. No foetus or foetal membranes could be visualized at this stage. By day 25 to 27, echogenic foetal mass and heart beat became detectable. By day 28 to 33, head and body of the foetus were similar in size and revealed flickering heart beats. Anatomical features of the foetus became more obvious by about 34 to 39 days of gestation. Shape and size of the foetal head became distinguishable from the body at this stage. The percentage accuracy for transabdominal palpation at 20 days of gestation was found to be 20 per cent which improved to 75 per cent by about 35 days. The percentage accuracy at 50 days was 60 per cent. This study suggested that transabdominal palpation was not reliable in early and late gestation. For ultrasound scanning, the percentage accuracy at 20 days of gestation was found to be 15 per cent which improved to 90 per cent by 35 days. The percentage accuracy improved to 100 per cent by 50 days of gestation. Thus ultrasound scanning could be used as reliable tool for assessing the foetal viability. The level of serum alkaline phosphatase at 20th, 35th and 50th day of gestation was found to be 67.90 + 2.98, 91.85 + 2.10 and 139.65 + 6.84 U/L respectively. The level of haptoglobin at 20th day of gestation was 53.10 + 3.22 mg/dl where as the level elevated to 81.12 + 3.40 and 119.44 + 3.16 mg/dl by 35 days and 50 days of gestation respectively. The level of serum globulin at 20th, 35th and 50th day of gestation was 2.43 + 0.12, 3.05 + 0.11 and 3.74 + 0.15 g/dl respectively. Statistical analysis revealed significant difference between day 20 and day 50 (P<0.05). Thus the level of serum alkaline phosphatase, serum haptoglobin and serum globulin was found to be increasing as the pregnancy advanced and this could be used as indicators of healthy pregnancy. Total erythrocyte count (TEC) at 20, 35 and 50 days of gestation was 6.07 + 0.16, 5.58 + 0.15 and 5.12 + 0.16 million/cmm respectively. Haemoglobin concentration at day 20, 35th day and 50 days of gestation were found to be 11.86 + 0.28, 10.45 + 0.27 and 9.17 + 0.30 g/dl respectively. Packed cell volume values were 40.45 + 0.83, 37.30 + 0.87and 33.40+ 1.10 percent respectively at day 20, day 35 and 50 days of gestation. Erythrocyte sedimentation rate at day 20, day 35 and 50th day of gestation were found to be 10.31 + 0.73, 15.41 + 0.54 and 21.85 + 1.04 mm/hr respectively. Haemogram studies showed significant decrease in the total erythrocyte count, haemoglobin and packed cell volume at different gestational age (P<0.05). These changes were attributed by haemodilution and increased plasma volume. But erythrocyte sedimentation rate has shown an increase which could be attributed to the endometrial implantation by the embryo. The total leucocyte count at day 20, day 35 and day 50 of gestation was found to be 13844.90 + 539.90, 15449.00 + 569.86 and 17502.50 + 780.21 cells/cmm respectively. The neutrophil count at 20th, 35th and 50th day of gestation was 67.30 + 1.11, 70.30 + 4.95 and 75.35 + 1.27 per cent respectively. The lymphocyte count was found to be 27.80 + 0.87, 31.55 + 0.88 and 36.95 + 1.03 per
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    ThesisItemOpen Access
    Factors affecting conception rate on artificial insemination in goats
    (College of Veterinary and Animal Sciences, Mannuthy, 2010) Remya Rajan, V; KAU; Metilda, Joseph
    With the objective of evaluating the factors affecting conception rate on artificial insemination in goats, a study 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 22 ejaculates collected thrice weekly from an adult Malabari crossbred buck. Semen samples for chilling were diluted in Tris-yolk buffer and preserved at 3-5oC for 72 hours. Cryopreservation was done in Tris yolk glycerol extender after the removal of seminal plasma. The semen was diluted and packed in 0.25 ml straws and each dose contained 200 million progressively motile sperms before processing.A total of 44 adult healthy does brought to the AI Centre for insemination were selected for the study after detecting heat using buck jar technique. Selected does were at random allotted to four groups with eleven animals in each group. Does belonging to Group I, II and III were inseminated using chilled semen having motility over 35 per cent and stored for 0-24 h, 24-48 h and 48-72 h respectively. Animals of Group IV were inseminated using frozen semen having a minimum of 35 per cent post thaw motility. Transcervical insemination was carried out in the does in oestrus by speculum method. The depth of penetration of AI gun was assessed using another sheath as yardstick. Pregnancy diagnosis was performed at two months of gestation by ultrasonography or at three months of gestation by abdominal palpation.Average volume, density and mass activity of buck semen were 1.25 ± 0.97 ml, DDDD and ++++ respectively. Colour of the semen was creamy with a yellowish tinge. Average semen pH and sperm concentration were 6.89 ± 0.21 and 2781.82 ± 51.69 millions per ml respectively. The mean percentage of initial motility, sperm viability, abnormality and intact acrosomes was 82.77 ± 0.33, 90.14 ± 0.53, 2.28 ± 0.12 and 92.27 ± 0.21 respectively.The percentage of sperm motility at 24, 48 and 72 h of preservation at refrigeration temperature was 70.25 ± 0.60, 61.13 ± 0.72 and 42.81 ± 0.95 respectively. The live sperm percentage dropped to 83.42 ± 1.27 at 24 h, 78.84 ± 1.47 at 48 h and 74.57 ± 1.53 at 72 h of preservation by chilling. The percentage of sperm abnormalities increased to 2.97 ± 0.13 at 24 h, 4.12 ± 0.15 at 48 h and 5.34 ± 0.11 at 72 h of preservation. The percentage of intact acrosomes was 90.27 ± 0.18 at 24 h, 87.71 ± 0.37 at 48 h and 85.09 ± 0.44 at 72 h of refrigeration storage. There was significant difference between sperm motility, viability, abnormality and acrosome integrity at various storage periods.The percentage of sperm motility of 78.50 ± 0.50 at the end of initial extension decreased to 37.67 ± 0.49 after cryopreservation. The mean live sperm percentage was 84.66 ± 0.91 at the end of initial extension which after freezing and thawing dropped to 60.36 ± 0.77. The mean percentage of sperm abnormalities increased from 3.82 ± 0.12 to 6.34 ± 0.22 at the end of cryo preservation. The mean percentage of intact acrosomes was 85.68 ± 0.72 at the end of initial extension which showed a decrease to 76.06 ± 1.41 after cryopreservation. There was significant difference (p<0.01) between semen quality of frozen semen and chilled semen at various storage periods. Predominant behavioural signs observed using “Buck jar” technique were bleating, wagging of tail, frequent urination and flehmen reaction with an average intensity of heat score 2.91 ± 0.10. The major clinical signs were vulval oedema, moistness and hyperaemia of vagina, mucus discharge and opening of cervical os. Average depth of penetration of AI gun was 20.07 ± 1.35 mm. Overall conception rate in does inseminated using chilled semen was 72.73 per cent. Conception rate in Group I, II and III were 81.82, 63.64 and 72.73 per cent respectively, which did not differ significantly. The conception rate in does inseminated using frozen semen was 27.27 per cent. The study indicated that progressive motility and fertility of buck semen decrease on freezing causing a significant (p<0.01) reduction in conception rates compared to chilled semen. Intensity of heat and depth of penetration of AI gun have significant correlation with pregnancy status of does inseminated using frozen semen. The study revealed that liquid storage of buck semen under refrigeration is a viable alternative for propagation of germplasm of superior bucks with low freezability as it ensures better conception rates.