P.S.P. Gupta

Effect of Taurine and Melatonin in the Culture Medium on Buffalo In Vitro Embryo Development

This study was carried out to investigate the effect of supplementing culture medium with different concentrations of taurine and melatonin, on buffalo oocyte in vitro meiotic maturation and embryo development. In experiment 1, oocytes were matured in vitro and the cleaved embryos were cultured in the same following seven culture medium; (i) control (TCM 199 + 10% SS); (ii) control + 0.5 mM taurine; (iii) control + 1 mM taurine; (iv) control + 3 mM taurine; (v) control + 5 microM melatonin; (vi) control + 10 microM melatonin and (vii) control + 50 microM melatonin. In experiment 2, based on the results of experiment 1, to examine the synergistic effect of antioxidants, the oocytes were matured in culture medium (TCM199 + 10% SS), supplemented with both taurine at 1 mM and melatonin at 10 microM concentration and the cleaved embryos were cultured in the same medium. Supplementation of taurine at 1 mM concentration in the culture medium resulted in a higher (p < 0.05) transferable embryo (TE) yield when compared with control (20.6% vs 14.1%). Supplementation of melatonin at 10 and 50 microM concentration in the culture medium resulted in a higher (p < 0.05) meiotic maturation rate (90.3% and 88.8% respectively) and TE yield (28.4% and 27.2% respectively), than the other treatments. In experiment 2, the TE yield did not improve by supplementing the culture medium with both taurine and melatonin, when compared with melatonin alone. In conclusion, the results of this study demonstrated that, enriching the culture medium with taurine and melatonin, improves in vitro embryo production efficiency in buffaloes. In particular, a high TE yield was obtained by enriching the culture medium with 10 microM melatonin.

Effect of breeding season on in vivo oocyte recovery and embryo production in non-descriptive Indian river buffaloes (Bubalus bubalis)

The present study was carried out to examine the effect of season on in vivo oocyte recovery and embryo production in non-descriptive, Indian river buffaloes (Bubalus bubalis). Ovum pick up (OPU) was conducted twice a week for 8 weeks during peak (October-March) and low (April-September) breeding season in live buffaloes (n=6). OPU was performed using ultrasound equipment with a 5MHz transvaginal transducer, a single lumen 18-gauge, 55-cm long needle and a constant vacuum pressure of 110mmHg. The number and size of follicles was determined before puncture. The recovered oocytes were graded and only grade A and grade B oocytes were used for in vitro production (IVP) of embryos. The mean number of follicles observed per animal per session did not differed (P<0.05) between animals or between puncture sessions in both low and peak breeding seasons. Higher (P<0.05) number of follicles were observed (4.8+/-0.2 versus 3.1+/-0.3) and punctured (4.0+/-0.2 versus 2.4+/-0.2) during peak breeding season when compared to low breeding season. Oocytes recovered (1.6+/-0.1 versus 1.0+/-0.3) per animal per session were higher (P<0.05) in peak breeding season than low breeding season. During the peak breeding season, the blastocyst yield per animal per session (0.3+/-0.4 versus 0.18+/-0.4) was higher (P<0.05) than the low breeding season. However, season did not significantly affect the percentage of oocytes suitable for IVP (grade A+B) and blastocyst production rate. In conclusion, the efficiency of OPU combined with IVP was higher during the peak breeding season than the low breeding season in buffaloes.

l‐carnitine Mediated Reduction in Oxidative Stress and Alteration in Transcript Level of Antioxidant Enzymes in Sheep Embryos Produced In Vitro

The objective of this study was to find out the effect of L-carnitine on oocyte maturation and subsequent embryo development, with L-carnitine-mediated alteration if any in transcript level of antioxidant enzymes (GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2) in oocytes and developing sheep embryos produced in vitro. Different concentrations of L-carnitine (0 mm, 2.5 mm, 5 mm, 7.5 mm and 10 mm) were used in maturation medium. Oocytes matured with 10 mm L-carnitine showed significantly (p < 0.05) higher cleavage (66.80% vs 39.66, 41.76, 44.64, 64.31%), morula (48.50% vs 20.88, 26.01, 26.99, 44.72%) and blastocyst (33.22% vs 7.66, 9.19, 10.71, 28.57%) percentage as compared to lower concentrations (0 mm, 2.5 mm, 5 mm and 7.5 mm). Cleavage percentage between 10 mm and 7.5 mm L-carnitine were not significantly different. Maturation rate was not influenced by supplementation of any experimental concentration of L-carnitine. There was a significant (p < 0.05) decrease in intracellular ROS and increase in intracellular GSH in 10 mm L-carnitine-treated oocytes and embryos than control group. Antioxidant effect of L-carnitine was proved by culturing oocytes and embryos with H2O2 in the presence of L-carnitine which could be able to protect oocytes and embryos from H2O2-induced oxidative damage. L-carnitine supplementation significantly (p < 0.05) upregulated the expression of GPx and downregulated the expression of SOD2 genes, whereas the expression pattern of SOD1 and GAPDH (housekeeping gene) genes was unaffected in oocytes and embryos. It was concluded from the study that L-carnitine supplementation during in vitro maturation reduces oxidative stress-induced embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH that in turn improved developmental potential of oocytes and embryos and alters transcript level of antioxidant enzymes.

Effects of heavy metals and pesticides on buffalo (Bubalus bubalis) spermatozoa functions in vitro.

Industrial toxic metals, pollutants and bio-accumulative pesticides interfere with the male reproductive functions in farm animals. Frozen-thawed semen samples were incubated with heavy metals (cadmium and lead) and pesticides (chlorpyrifos and endosulfan) of different concentrations (0, 0.005, 0.05, 0.02, 0.1, 0.5, 1.0, 2.0 and 4.0 μg/ml) for 1 h, and various spermatozoa functional parameters and in vitro fertilization rates were assessed. Any significant effect was assessed by comparing the 1 h data between the control and treatment groups. Progressive forward motility was significantly (p < 0.05) reduced in spermatozoa exposed to lower concentrations (0.05-0.5 μg/ml) of toxic substances. The straight-line velocity (μm/s) and the average path velocity (μm/s) were significantly (p < 0.05) reduced in spermatozoa exposed to 1.0 and 0.5 μg/ml of cadmium (11.6 ± 1.9 and 16.3 ± 1.9) and chlorpyrifos (10.4 ± 1.5 and 17.1 ± 1.3), respectively, when compared to control (20.4 ± 1.4 and 28.1 ± 1.7). The acrosomal integrity was also significantly (p < 0.05) reduced at 0.05 μg/ml of chlorpyrifos (33.3 ± 1.9), 1.0 μg/ml of cadmium (36.8 ± 3.7), 1.0 μg/ml of lead (39.4 ± 2.8) and 0.5 μg/ml of endosulfan (38.3 ± 3.2), respectively. The spermatozoa chromatin decondensation was significantly (p < 0.05) affected at higher concentrations (>0.5 μg/ml) of these chemicals. The mitochondrial membrane potential (%) was significantly (p < 0.05) reduced at 0.05 μg/ml of cadmium (3.2 ± 0.2) and chlorpyrifos (4.3 ± 0.4), 0.1 μg/ml of lead (3.8 ± 0.3) and 0.5 μg/ml of endosulfan (3.2 ± 0.3) when compared to control (6.7 ± 1.0). The in vitro fertilization capabilities (cleavage percentage) of spermatozoa were significantly reduced at 1.0 μg/ml of cadmium (28.3 ± 2.4) and 2.0 μg/ml of lead (31.1 ± 2.7), chlorpyrifos (29.4 ± 2.2) and endosulfan (32.6 ± 2.5) when compared to control (59.4 ± 4.4). This study suggested that the mitochondrial membrane potential was primarily affected even with lowest doses of toxic chemicals. Cadmium when compared to lead and chlorpyrifos when compared to endosulfan were found to be more toxic to the spermatozoa.

Oocyte recovery by ovum pick up and embryo production in river buffaloes (Bubalus bubalis).

Ovum pick up (OPU) was conducted twice a week for 12 weeks in six cycling, non-descriptive (local breed), Indian buffaloes to study the efficiency of OPU on recovery of oocytes for embryo production. OPU was performed using an ultrasound equipment with a 5-MHz transvaginal transducer, a single-lumen, 18-gauge, 55-cm-long needle and a constant vacuum pressure of 110 mmHg. The number and size of follicles were determined before puncture. The recovered oocytes were graded, washed, matured for 24 h and then fertilized with frozen-thawed semen, followed by embryo culture on the oviductal monolayer. The mean number of follicles observed per animal per session did not differ between animals or between puncture sessions. A mean number of 3.62 +/- 0.32 mm follicles were observed, 2.90 +/- 0.15 mm follicles were punctured and 1.21 +/- 0.07 oocytes were recovered per animal per session, with an average recovery rate of 42%. Of the total oocytes recovered, 64% were suitable for in vitro embryo production (grade A + B) whereas 36% were classified to be of grades C + D. A mean number of 0.25 +/- 0.2 transferable embryos was produced in vitro per buffalo per session with a transferable embryo production rate of 32%. In conclusion, this study demonstrated that twice-a-week OPU could be applied repeatedly, without any adverse effects on the follicular growth and oocyte recovery and that recovered oocytes could be used for in vitro embryo production in buffaloes.

Effect of ammonia‐generating diet on ovine serum and follicular fluid ammonia and urea levels, serum oestrogen and progesterone concentrations and granulosa cell functions

This study was undertaken to elucidate the effect of ammonia-generating diet on serum and follicular fluid ammonia and urea levels, serum oestrogen and progesterone concentrations and granulosa cell growth and secretion parameters in ewes (Ovis aries). Ewes were fed with 14% CP diet (control) or ammonia-generating diet or ammonia-generating diet plus soluble sugar. The serum and follicular fluid ammonia and urea level, serum oestrogen and progesterone levels and granulosa cell (obtained from ovaries of slaughtered ewes) growth parameters and secretory activities were estimated. Ammonia-generating diet (high-protein diet) increased the serum ammonia and urea concentration. Supplementation of soluble sugar significantly reduced the ammonia concentration in serum with comparable levels as in control group; however, the urea level in the same group was higher than that observed in control group. Supplementation of soluble sugar significantly reduced the follicular fluid ammonia concentration; however, the level was significantly higher compared to control group. Supplementation of soluble sugar brought down the follicular fluid urea level comparable to that observed in control group. Oestrogen and progesterone levels remained unchanged in ewes fed with different types of diet. Oestrogen and progesterone secretion were significantly lowered from granulosa cells recovered from ewes fed with high ammonia-generating diet. Low metabolic activity and high incidence of apoptosis were observed in granulosa cells obtained from ovaries of ewes fed with ammonia-generating diet.

In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes

The present study was undertaken to investigate the oocyte morphology, its fertilizing capacity and granulosa cell functions in ewes (obese, normal, metabolic stressed and emaciated). Ewes (Ovis aries) of approximately 3 years of age (Bellary breed) from a local village were screened, chosen and categorized into a) normal b) obese but not metabolically stressed, c) Emaciated but not metabolically stressed d) Metabolically stressed based on body condition scoring and blood markers. Oocytes and granulosa cells were collected from ovaries of the ewes of all categories after slaughter and were classified into good (oocytes with more than three layers of cumulus cells and homogenous ooplasm), fair (oocytes one or two layers of cumulus cells and homogenous ooplasm) and poor (denuded oocytes or with dark ooplasm). The good and fair quality oocytes were in vitro matured and cultured with fresh semen present and the fertilization, cleavage and blastocyst development were observed. The granulosa cells were cultured for evaluation of metabolic activity by use of the MTT assay, and cell viability, cell number as well as estrogen and progesterone production were assessed. It was observed that the good and fair quality oocytes had greater metabolic activity when collected from normal and obese ewes compared with those from emaciated and metabolically stressed ewes. No significant difference was observed in oocyte quality and maturation amongst the oocytes collected from normal and obese ewes. The cleavage and blastocyst production rates were different for the various body condition classifications and when ranked were: normal>obese>metabolically stressed>emaciated. Lesser metabolic activity was observed in granulosa cells obtained from ovaries of emaciated ewes. However, no changes were observed in viability and cell number of granulosa cells obtained from ewes with the different body condition categories. Estrogen and progesterone production from cultured granulosa cells were not different in normal and obese ewes. Estrogen and progesterone secretions were less from granulosa cells recovered from metabolically stressed and emaciated ewes. The results suggested that oocyte morphology, fertilizing capacity and granulosa cell growth were dependent on body condition and feeding status of the animals.

Isolation and Culture of Ovine and Bubaline Small and Large Pre‐antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle

Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100-250 microm) and large pre-antral follicles (LPFs, 250-450 microm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 +/- 3.3 vs 12.1 +/- 2.6 and LPFs: 16.0 +/- 3.6 vs 9.2 +/- 1.8) and buffaloes (SPFs: 9.2 +/- 1.3 vs 4.1 +/- 1.0 and LPFs: 10.3 +/- 2.7 vs 5.4 +/- 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 +/- 2.7 vs 16.4 +/- 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.

Ammonia concentrations in different size classes of ovarian follicles of sheep (Ovis aries): Possible mechanisms of accumulation and its effect on oocyte and granulosa cell growth in vitro

The present study investigated the concentrations and the mechanisms of accumulation of ammonia in different sizes of ovarian follicles and the effect of ammonia on oocyte and granulosa cell growth and functions in vitro with sheep (Ovis aries) as an animal model. The effects of cyclicity, seasonality, phases of the estrous cycle, and seasons (environmental) on ammonia concentrations in follicular fluid were also investigated. The effect of ammonia on in vitro development of oocytes (maturation rate, viability rate, cleavage rate, morulae/blastocysts yield) recovered from different sizes of follicles was examined at the levels of 0, 50, 100, 150, 250, 300, and 500 μM. Same concentrations of ammonia were examined on growth parameters (metabolic activity, viability, cell number increment, monolayer formation, apoptosis rate) and hormone (progesterone, estrogen) secretion activity of granulosa cells in vitro. Results suggested as the follicle size increased, ammonia concentrations decreased. The ammonia concentrations in ovine follicular fluid were found to be 261.5 ± 32.4, 157.7 ± 19.2, and 42.9 ± 8.3 μM, respectively, for small, medium, and large follicles. The corresponding ranges were 290 to 238 μM, 184 to 142 μM, and 70 to 22 μM. The differences were due to more accumulation of fluid, less metabolic activity of granulosa cells, and elevation of protein, potassium, and chloride as the follicle size increased. The seasonality and phases of the estrous cycle did not have any effect on ammonia level in ovarian follicles. Ammonia concentrations in all size classes of follicles examined were significantly reduced in ewes during hot seasons compared to cold seasons and in acyclic animals compared to cyclic ones. Ammonia impaired oocyte development at 300 μM when the oocytes were isolated from small follicles and at 250 μM when the oocytes were isolated from medium and large follicles. In contrast, ammonia caused the negative impact on granulosa cells growth and secretary activity at 250 μM when the cells were isolated from small and medium follicles and at 150 μM when the cells were isolated from large follicles.

Post-thaw development of in vitro produced buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification

The present study was conducted to examine post-thaw in vitro developmental competence of buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification. In vitro produced embryos were incubated with a medium containing cytochalasin-b (cyto-b) in a CO2 incubator for 40 min for microfilament stabilization and were cryopreserved by a two-step vitrification method at 24℃ in the presence of cyto-b. Initially, the embryos were exposed to 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in a base medium for 4 min. After the initial exposure, the embryos were transferred to a 7 µl drop of 25% EG and 25% DMSO in base medium and 0.3 M sucrose for 45 sec. After warming, the embryos were cultured in vitro for 72 h. The post-thaw in vitro developmental competence of the cyto-b-treated embryos did not differ significantly from those vitrified without cyto-b treatment. The hatching rates of morulae vitrified without cyto-b treatment was significantly lower than the non-vitrified control. However, the hatching rate of cyto-b-treated vitrified morulae did not differ significantly from the non-vitrified control. This study demonstrates that freezing of buffalo embryos by cytoskeletal stabilization and vitrification is a reliable method for long-term preservation.