Tamil Nadu Veterinary and Animal Sciences University, Chennai
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ThesisItem Open Access Effect Of Donor Cell Type On The In Vitro Development Of Buffalo Somatic Cell Nuclear Transfer Embryos(Tamil Nadu Veterinary and Animal Sciences University, 2005) Simon, Liz; TANUVAS; Veerapandian, C; Ramadass, P; Subramanian, A; Balasubramanian, SSomatic Cell Nuclear Transfer (SCNT) is an important technology which has immense potential application in animal production and pharmaceuticals. Research works on SCNT in Bubalus bubalis were limited and with the objective of optimizing various aspects of SCNT in buffaloes, experiments were carried out on maturation of oocytes, donor cell type, fusion, activation and culture of NT embryos. A total of 2,982 oocytes retrieved by slicing method from a total of 662 buffalo ovaries collected from the Chennai slaughter house were utilized for the study. A recovery rate of 4.5 oocytes and a yield of 3.43 good quality oocytes (A,B and C grades) per ovary were recorded. The supplementation of cysteamine (100 μM) and Epidermal Growth Factor (10 ng/ml) to the maturation media, TCM 199 with FBS, FSH, LH and Estradiol, significantly (P<0.01) improved the maturation rate (mean V SE) based on extrusion of the first polar body, when compared to the control (77.59 V 1.08 vs. 63.87V2.00 per cent).In the experiment designed to standardize the electrofusion protocol, two DC pulses of 1.4 - 1.6 kV/cm for 35 μsec, for fetal fibroblasts (FF) and adult skin fibroblasts (AF) and similar pulses for 25 μsec, for cumulus (C) were found to give the best fusion and development rates. The NT embryos activated at 24 h post maturation (hpm) had better developmental rates than those activated at 30 hpm. The fusion at 24 hpm and activation at 28-30 hpm gave better fusion and development rates compared to fusion and immediate activation at 24 hpm. In another experiment, in which reconstructs were made with different donor cell types and their fusion and development rates (per cent, mean V SE) were compared, the contemporary cumulus (FC, 41.00 V 1.80) cells had significantly (P<0.05) lower fusion rates compared to FF (52.6 V 2.98) and AF (46.9 V 1.80). The cleavage rate was the highest in FF (42.27 V 3.10) followed by FC (39.3 V 3.87) and AF (35.07 V 2.93). However the reconstructs with FC showed the maximum development rates to 4, 8 and 16 cell stages (25.47 V 3.97, 11.68 V 2.38 and 2.62 V 1.05) followed by FF (24.84 V 2.84, 7.97 V 1.66 and 2.17 V 0.72) and AF (15.51 V 2.36, 2.53 V 1.06 and 0.66 V 0.46, respectively). The sex of the FF had no influence on the development rates and 2-5 cell passages in the preparation of karyoplasts were found to give better results compared to 6-12 passages. The mean cleavage and progression to further development stages were found to be the best in G 1 /G 2 , followed by TCM 199 and the least in SOFaa medium. In G 1 /G 2 alone 12.19 V 5.66 and 1.75 V 1.49 per cent (mean V SE) developed to morula and blastocyst stages respectively. The transfer of a total of 16 embryos to six healthy recipients on fourth or fifth day of estrous cycle did not result in any conception. In conclusion, the results of the present study showed that FF, AF and C cells could be successfully reprogrammed and developed to pre- implantation embryos and FC cells would be the most ideal karyoplast for SCNT in water buffaloes. An electrofusion regimen of two DC pulses of 1.4 - 1.6 kV/cm for 35 μsec and fusion at 24 hpm followed by activation at 28-30 hpm were suitable for buffalo SCNT. The development of NT embryos was best supported by G 1 /G 2 followed by TCM 199 media.ThesisItem Open Access Effect Of Synchronization Of Follicular Wave Emeregence On Superovulation And Embryo Yield In Crossbred Cows(Tamil Nadu Veterinary and Animal Sciences University, 2009) Satheshkumar, S.; TANUVAS; Subramanian, A.; Veerapandian, C; Palanisamy, A.The present study was aimed at assessing the effect of GnRH analogue in synchronizing the follicular wave emergence in Jersey crossbred cattle and to study the effect of initiating FSH treatment (normal and reduced doses) after synchronizing the emergence of follicular wave on superovulatory response and embryo yield. Six healthy, non-lactating and regularly cycling Jersey crossbred cows aged between 5-6 yrs were utilized for the study. Initially, as a control study, the normal follicular wave pattern was ultrasonographically investigated in all the six cows. In experimental group, all the animals were injected with GnRH analogue (Buserelin acetate; 10 μg i.m.) on Day 6 of the cycle (Day 0 – oestrum) and follicular wave pattern was studied. All the six animals were subjected for four superovulatory treatments. Treatment1-Conventional: FSH (400 mg - Folltropin-V) treatment was initiated on Day 10; Treatment II – Gn-D8-400: FSH treatment (400 mg) was initiated on the day of GnRH synchronized follicular wave emergence (Day 8); Treatment III – Gn-D10-400: FSH treatment (400 mg) was initiated two days after synchronized follicular wave emergence (Day 10) and Treatment IV- Gn-D10-200: Similar to Gn-D10-400 group, but superstimulated with 200 mg. Monitoring the normal follicular wave pattern revealed that 77.8, 16.7 and 5.6 per cent of oestrous cycles exhibited three, two and four follicular waves respectively. The first wave dominant follicles (DF) were in a growth phase during Days 0.8 – 6.67 of the oestrous cycle and got deviated from the subordinate follicles by 3.30 – 3.67 days after emergence irrespective of the number of follicular waves succeeding the first one. Thus characteristics of the first wave DF was much predictable than that of the subsequent waves. Three peaks of FSH levels could be appreciated in three-wavecycles preceding the emergence of each follicular wave. When GnRH was administered on Day 6 of the cycle, the DF (10.83 ± 0.38 mm) ovulated in all the animals (100 %) in a mean interval of 27.67 ± 0.21 h and a synchronized homogenous group of follicles emerged after two days (Day of 8.00 ± 0.0). With the formation of an additional luteal structure (ACL), the progesterone concentration (10.57 ± 0.61 ng / ml) was significantly (P<0.01) increased than normal cycle (5.58 ± 0.45 ng / ml) during the mid luteal phase. In Conventional group, all the animals (100%) responded for FSH, with 13.67 ± 1.80 CL and 2.00 ± 0.37 AF. However, the superovulatory response varied widely (9 - 21ovulations). In Gn-D10-400 group, the animals responded with 11.00 ± 0.63 (10 – 14) CL and 3.50 ± 0.22 (3 – 4) AF comparable to the Conventional group. In both Conventional and Gn-D10-400 groups, almost all the Class III follicles on the day of superovulatory oestrus ovulated indicating that these follicles were healthy enough to respond to endogenous LH surge. In Gn-D8-400 group, the mean number of CL and AFs were 5.00 ± 1.77 (1 – 11) and 12.83 ± 4.65 (2 - 30) respectively. More number of Class II follicles and suprabasal progesterone levels (due to incomplete luteolysis) on the day of oestrus was correlated positively with increased number of AFs. The embryo recovery rate (53.89 %) in Gn-D10-400 was comparable to that of Conventional group (55.49 ± 9.70 %), but the recovery rate (36.57 ± 16.44 %) was substantially affected in Gn-D8-400 group. In Gn-D10-400 group, a higher percentage (87.24 %) of transferable quality (Grade 1 and 2) embryos and lower incidence of arrested / degenerated embryos (3.33 %) and UFO (1.85 %) was recorded than the Conventional (79.49%, 6.72 % and 6.25 % respectively) group. However, in Gn-D8- 400 group, arrested/degenerated and UFO (62.50%) and Grade 4 embryos (20.84%) constituted the major proportion of ova recovered and transferable quality embryos accounted for only 8.34 per cent. Increased anovulatory follicular population from the day of oestrus to the day of embryo collection and thus an increased concentration of oestradiol would have resulted in a poor embryo recovery / quality in Gn-D8 group. With the reduced dose of FSH (200 mg) in Gn-D10 group, all the animals responded with a mean superovulatory response of 6.33 ± 0.99 CL (4-11) and an average of 44.24 per cent embryos / ova were recovered. When compared with Gn- D8-400 group, a non-significant increase in superovulatory response and embryo recovery rate was recorded in Gn-D10-200 group.ThesisItem Open Access Effect Of Synchronization Of Follicular Wave Emeregence On Superovulation And Embryo Yield In Crossbred Cows(Tamil Nadu Veterinary and Animal Sciences University, 2009) Satheshkumar, S; TANUVAS; Subramanian, AThe present study was aimed at assessing the effect of GnRH analogue in synchronizing the follicular wave emergence in Jersey crossbred cattle and to study the effect of initiating FSH treatment (normal and reduced doses) after synchronizing the emergence of follicular wave on superovulatory response and embryo yield. Six healthy, non-lactating and regularly cycling Jersey crossbred cows aged between 5-6 yrs were utilized for the study. Initially, as a control study, the normal follicular wave pattern was ultrasonographically investigated in all the six cows. In experimental group, all the animals were injected with GnRH analogue (Buserelin acetate; 10 μg i.m.) on Day 6 of the cycle (Day 0 – oestrum) and follicular wave pattern was studied. All the six animals were subjected for four superovulatory treatments. Treatment1-Conventional: FSH (400 mg - Folltropin-V) treatment was initiated on Day 10; Treatment II – Gn-D8-400: FSH treatment (400 mg) was initiated on the day of GnRH synchronized follicular wave emergence (Day 8); Treatment III – Gn-D10-400: FSH treatment (400 mg) was initiated two days after synchronized follicular wave emergence (Day 10) and Treatment IV- Gn-D10-200: Similar to Gn-D10-400 group, but superstimulated with 200 mg. Monitoring the normal follicular wave pattern revealed that 77.8, 16.7 and 5.6 per cent of oestrous cycles exhibited three, two and four follicular waves respectively. The first wave dominant follicles (DF) were in a growth phase during Days 0.8 – 6.67 of the oestrous cycle and got deviated from the subordinate follicles by 3.30 – 3.67 days after emergence irrespective of the number of follicular waves succeeding the first one. Thus characteristics of the first wave DF was much predictable than that of the subsequent waves. Three peaks of FSH levels could be appreciated in three-wavecycles preceding the emergence of each follicular wave. When GnRH was administered on Day 6 of the cycle, the DF (10.83 + 0.38 mm) ovulated in all the animals (100 %) in a mean interval of 27.67 + 0.21 h and a synchronized homogenous group of follicles emerged after two days (Day of 8.00 + 0.0). With the formation of an additional luteal structure (ACL), the progesterone concentration (10.57 + 0.61 ng / ml) was significantly (P<0.01) increased than normal cycle (5.58 + 0.45 ng / ml) during the mid luteal phase. In Conventional group, all the animals (100%) responded for FSH, with 13.67 + 1.80 CL and 2.00 + 0.37 AF. However, the superovulatory response varied widely (9 - 21ovulations). In Gn-D10-400 group, the animals responded with 11.00 + 0.63 (10 – 14) CL and 3.50 + 0.22 (3 – 4) AF comparable to the Conventional group. In both Conventional and Gn-D10-400 groups, almost all the Class III follicles on the day of superovulatory oestrus ovulated indicating that these follicles were healthy enough to respond to endogenous LH surge. In Gn-D8-400 group, the mean number of CL and AFs were 5.00 + 1.77 (1 – 11) and 12.83 + 4.65 (2 - 30) respectively. More number of Class II follicles and suprabasal progesterone levels (due to incomplete luteolysis) on the day of oestrus was correlated positively with increased number of AFs. The embryo recovery rate (53.89 %) in Gn-D10-400 was comparable to that of Conventional group (55.49 + 9.70 %), but the recovery rate (36.57 + 16.44 %) was substantially affected in Gn-D8-400 group. In Gn-D10-400 group, a higher percentage (87.24 %) of transferable quality (Grade 1 and 2) embryos and lower incidence of arrested / degenerated embryos (3.33 %) and UFO (1.85 %) was recorded than the Conventional (79.49%, 6.72 % and 6.25 % respectively) group. However, in Gn-D8- 400 group, arrested/degenerated and UFO (62.50%) and Grade 4 embryos (20.84%) constituted the major proportion of ova recovered and transferable quality embryos accounted for only 8.34 per cent. Increased anovulatory follicular population from the day of oestrus to the day of embryo collection and thus an increased concentration of oestradiol would have resulted in a poor embryo recovery / quality in Gn-D8 group. With the reduced dose of FSH (200 mg) in Gn-D10 group, all the animals responded with a mean superovulatory response of 6.33 + 0.99 CL (4-11) and an average of 44.24 per cent embryos / ova were recovered. When compared with Gn- D8-400 group, a non-significant increase in superovulatory response and embryo recovery rate was recorded in Gn-D10-200 group.ThesisItem Open Access Fertility In Bitches Following Intravaginal And Intrauterine Insemination With Fresh And Frozen Semen(Tamil Nadu Veterinary and Animal Sciences University, 2007) Sathiamoorthy, T; TANUVAS; Asokan, S.A.; Veerapandian, C; Kalatharan, J; Nambi, A.P.This study was conducted to compare the conception rate and litter size in bitches inseminated following intravaginal and intrauterine route with fresh and frozen semen and to study the effect of commercial semen extender on post thaw characters of dog semen. To study the efficacy of vaginoscopy to predict fertile insemination time in bitches was also taken as one of the objectives. Sixty bitches were randomly divided into six groups viz., Group I, II, III, IV, V and VI 10 each. The time of insemination was based on vaginoscopy in group I to V and on vaginal exfoliative cytology in group VI bitches. Semen was collected from seven male dogs of different breeds, evaluated and used for cryopreservation either with Tris or Tes-tris extender.Group I and II bitches were subjected to intravaginal insemination while Group III and IV bitches were subjected to intrauterine insemination using fresh and frozen semen, respectively. Group V and VI bitches were inseminated intravaginally using frozen semen supplemented with prostatic fluid and with fresh semen using vaginal exfoliative cytology to determine time of insemination, respectively. The conception rate obtained in this study was 50, 20, 50, 30, 30 and 40 per cent in group I, II, III, IV, V and VI bitches respectively. The mean post thaw motility was significantly higher for semen cryopreserved with Tris when compared to Tes-tris extender. The percentage of mean abnormality was significantly lower in semen cryopreserved with Tris when compared to Tes-tris extender. The mean percentage of plasma membrane integrity was significantly higher for Tris when compared to Tes- tris extender. Hence, it is concluded from this study that the conception rates were similar for both intravaginal and intrauterine insemination with fresh semen and higher conception rates were obtained when frozen semen was deposited intrauterine rather than intravaginal. Semen cryopreserved with Tris extender was found to gave better post thaw characters compared to Tes-tris extender. Vaginoscopy was found to be effective in assessing the optimum insemination time compared to vaginal exfoliative cytology. Addition of prostatic fluid with frozen semen had significant influence on increasing the conception rate with intravaginal insemination.ThesisItem Open Access Influence Of Different Culture Systems On The Quality Of Somatic Cell Nuclear Transfer Embryos In Buffaloes(Tamil Nadu Veterinary and Animal Sciences University, 2010) Reena, D.; TANUVAS; Veerapandian, C.; Kathiresan, D.; Subramanian, A.; Parthiban, M.Out of 1371 ovaries collected from freshly slaughtered buffaloes a total of 7,227 oocytes were retrieved by slicing, with an average yield of 5.27 ± 0.68 per ovary. The maturation rates based on cumulus expansion and nuclear maturation in the Tissue Culture Medium 199 (TCM 199) media were significantly (P<0.01) greater than that in Synthetic Oviduct fluid (SOF) media (85.16 ± 3.14 vs. 69.09 ± 2.02 and 72.96 ± 3.27 vs. 50.80 ± 3.79 percent, respectively). In the experiment on the influence of various treatments to induce donor cells into G0/G1 phase (Gap phase 0/Gap phase 1) of cell cycle, fetal fibroblast subjected to serum starvation (SS), Confluence formation (CF) and Roscovitine treatment (RT) had fusion rates of (mean ± SE) 67.50 ± 4.79, 78.06 ± 1.94 and 57.03 ± 8.23 per cent, respectively. There was significant difference (P<0.05) in the fusion rates between treatment groups. Similarly with cumulus cells as donor cells there was significant difference (P<0.05) in the fusion rates between SS, CF and RT groups (80.82 ± 3.26, 59.49 ± 2.32 and 58.23 ± 0.60 percent, respectively). However, there was no significant difference observed between groups in both fetal fibroblasts and cumulus cells. Morula derived from cumulus cell synchronized with serum starvation only progressed into blastocyst at 3.90 ±2.55 per cent. In another experiment to compare two enucleation methods, the fusion rate was significantly (P<0.01) higher after physical aspiration than that after chemical enucleation (72.13 ± 2.53 vs. 55.36 ± 2.43 per cent). Cleavage and developmental rates of NT embryos were significantly (P<0.05) higher after physical aspiration than after chemical enucleation. Activation of fused couplets by ionomycin followed by subsequent administration of 6- Dimethyl aminopurine (6-DMAP) produced significantly (P<0.05) higher rates of cleavage and developmental stages of Nuclear Transfer (NT) embryos compared to activation by ionomycin + cycloheximide, strontium + 6- DMAP, strontium + cycloheximide, strontium + cytochalasin B. Ionomycin + 6- DMAP activated embryos alone progressed to blastocyst with a mean percentage of 1.78 ± 1.18. The cleavage rate of NT embryos was significantly higher (P<0.01) in G1/G2 systems (60.11 ± 1.28 per cent) when compared to co-culture, SOF and Potassium Simplex Optimization Medium (KSOM) (31.98 ± 1.93, 19.86 ± 1.98 and 12.22 ± 1.69, respectively). There was significant difference (P<0.05) in the developmental rates also among co-culture, KSOM and G1/G2 media. When two oxygen tensions for culture were compared, 5 per cent O2 tension had significantly (P<0.01) higher cleavage and developmental rates of NT embryos than 20 per cent O2 tension. Hatching occurred in one of the blastocyst cultured under 5 per cent oxygen level with hatching rate of 1.67 ± 1.67 percent. In another experiment on the use of the agents altering the levels of epigenetic modification of reconstructs, the treatment with 5-aza-deoxy cytadine (5-aza-dC), Trichostatin A (TSA) and combination of TSA and 5-aza-dC resulted in lower cleavage rates (mean ± SE) of 20.04 ± 2.64, 20.69 ± 2.16 and 38.33 ± 3.27 per cent, respectively when compared with control (47.41 ± 3.11). Morula derived from control and combination of TSA and 5-Aza-dC treatment groups only progressed into blastocyst at 2.08 ± 2.08 per cent in both. Similarly hatching occurred in one of the blastocyst cultured with combination of TSA and 5-aza-dC and control group. It is concluded that TCM-199 with supplements was more suited for in vitro maturation; cumulus cells synchronized by SS yielded more blastocyst: combination of ionomycin and 6-DMAP produced better activation: O2 tension at 5 percent gave better results than 20 and embryo culture was best supported by G1/G2 medium in the production of Somatic Cell Nuclear Transfer (SCNT) embryos.ThesisItem Open Access Ultrasound Guided Transvaginal Follicular Aspiration And In Vitro Embryo Production In Cows(Tamil Nadu Veterinary and Animal Sciences University, 2004) Jayakumar, S; TANUVAS; Asokan, S.A.; Veerapandian, C; Subramanian, A; Kalatharan, JUltrasound guided transvaginal follicular aspiration (TVFA) and ovum pick up (OPU) to collect oocytes from live cows for in vitro embryo production has opened up greater possibilities to exploit productive and reproductive potential of the valuable donor. Experiment I was designed to study the effect of once versus twice a week OPU on size distribution of follicles, aspiration rate, oocyte recovery rate, oocyte quality and embryo development. The results of the present study showed that a highly significant (P<0.01) difference in the distribution of medium sized follicles existed between once (2.33 ± 0.37) and twice (5.48 ± 0.79) a week aspiration. The total number of follicles aspirated and oocytes recovered were significantly higher in twice (4.13 ± 0.48 and 1.25 ± 0.30) than once (2.17 ± 0.32 and 2.24 ± 0.30) a week group. The quality of A, B and C grade oocytes were significantly higher in twice than once a week aspiration group. In the second experiment, between FSH treated and untreated groups, the distribution of small and medium sized follicles (0.92 ± 0.22 vs 2.57 ± 0.68), number of follicles available for puncture (9.48 ± 0.92 vs 5.10 ± 0.67), number of follicles aspirated (7.28 ± 0.81 vs 2.24 ± 0.20) and mean number of oocytes recovered (5.48 ± 0.79 vs 1.24 ± 0.18) were significantly higher in FSH treated than untreated group. The quality of B and C grade oocytes obtained were significantly higher following FSH treatment. The mean percentage of cleavage rate and embryos reaching 4 cell stage did not differ between groups but the further development of 4 cell stage embryos to 8 to 16 cell and > 16 cell was observed only in the FSH treated group. There was no statistical difference existed between oocytes supplemented with cysteamine and with out cysteamine on maturation rate and embryo development rate. The in vitro development of embryos through IVMFC were corresponding to the time of development and DAPI staining of embryos showed a perfect nuclei: blastomere ratio. Out of 128 in vivo day 7 embryos examined, the proportion of compact morula, early blastocyst, blastocyst and expanded blastocyst were 52.34, 16.40, 28.12 and 3.12 per cent, respectively. The percentage of grade 1, 2, 3 and 4 of compact morula were 32.83, 35.82, 30.00 and 1.49; for early blastocyst were 61.90, 38.09, 0.00 and 0.00; for blastocyst were 55.55, 36.11, 8.33 and 0.00 and for expanded blastocyst were 100.00, 0.00, 0.00 and 0.00 per cent, respectively. Viability assessment of in vivo embryo showed that, the embryos classified as grade 1 (excellent), 2 (good) and 3 (fair) resulted in pregnancy rate of 21.42, 11.76 and 0 per cent, respectively.Morphologically graded fair quality embryos were found to be dead on FDA staining and DAPI staining showed less number of blastomere nuclei (18 to 31). Effect of OPU on donor cows showed that there was no pathological changes observed at the site of epidural injection, fornix vagina and ovary. In conclusion, the results of the present study showed that transvaginal follicular aspiration and OPU was an effective technique for successful recovery of oocytes from living cows. Increasing the frequency of aspiration to twice a week resulted in generation of higher percentage of good quality oocytes than once a week group. Further, FSH pretreatment of donors prior to OPU greatly enhanced the overall OPU efficiency and in vitro embryo production rate. Transfer of morphologically assessed in vivo embryos resulted in acceptable pregnancy rate and assessment of viability using in vitro culture and staining technique would further improve the pregnancy rate and the efficiency of embryo transfer technology.OtherItem Open Access சினை மாடுகள் பராமரிப்பு(TANUVAS, 1995) Mathialagan, P.