• Energy Research

The possibility of artificially inducing activation of MII buffalo oocytes may allow us to evaluate indirectly the quality of oocytes after in vitro maturation. The aim of this work was to compare buffalo embryo development after IVF and after chemical activation by two different agents. A further goal was to evaluate the effects of aging of oocytes on post-parthenogenetic and post-fertilization development. In Experiment 1 cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After IVM the COCs were either fertilized in vitro (positive control) or activated with ethanol and ionomycin, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP) for 4 h. In vitro culture (IVC) was carried out up to the blastocyst stage. In Experiment 2 COCs were matured in vitro for 18, 21, 24, 27 and 30 h before activation was triggered with ethanol, followed by 6-DMAP. In Experiment 3 COCs were fertilized in vitro at 18, 21, 24, 27 and 30 h post-maturation. Ethanol activation gave better results than the IVF control group, with higher cleavage rate (71.4 +/- 7.8 versus 55.8 +/- 5.8, respectively; P < 0.05) and a higher proportion of oocytes developing into morulae-blastocysts (32.6 +/- 6.5 versus 22.9 +/- 7.5, respectively; P < 0.05). Within the activation groups, ethanol supported the highest development in terms of cleavage (71.4 +/- 7.8 versus 59.4 +/- 10.7; P < 0.05) and morulae-blastocysts rate (32.6 +/- 6.5 versus 25.7 +/- 8.3; n.s.). It was also demonstrated that aging negatively affects post-parthenogenetic and post-fertilization development.

The possibility of artificially inducing activation of MII buffalo oocytes may allow us to evaluate indirectly the quality of oocytes after in vitro maturation. The aim of this work was to compare buffalo embryo development after IVF and after chemical activation by two different agents. A further goal was to evaluate the effects of aging of oocytes on post-parthenogenetic and post-fertilization development. In Experiment 1 cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After IVM the COCs were either fertilized in vitro (positive control) or activated with ethanol and ionomycin, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP) for 4 h. In vitro culture (IVC) was carried out up to the blastocyst stage. In Experiment 2 COCs were matured in vitro for 18, 21, 24, 27 and 30 h before activation was triggered with ethanol, followed by 6-DMAP. In Experiment 3 COCs were fertilized in vitro at 18, 21, 24, 27 and 30 h post-maturation. Ethanol activation gave better results than the IVF control group, with higher cleavage rate (71.4 +/- 7.8 versus 55.8 +/- 5.8, respectively; P < 0.05) and a higher proportion of oocytes developing into morulae-blastocysts (32.6 +/- 6.5 versus 22.9 +/- 7.5, respectively; P < 0.05). Within the activation groups, ethanol supported the highest development in terms of cleavage (71.4 +/- 7.8 versus 59.4 +/- 10.7; P < 0.05) and morulae-blastocysts rate (32.6 +/- 6.5 versus 25.7 +/- 8.3; n.s.). It was also demonstrated that aging negatively affects post-parthenogenetic and post-fertilization development.

The aim was to establish the capacity of buffalo heifers to adapt their metabolic requirements to a low energy diet. Murrah buffalo (Bubalus bubalis) heifers undergoing regular estrous cycles were randomly assigned by age, live weight (LW) and body condition score (BCS) to a high energy group (HE, 5.8 milk forage units (MFU)/day, n=6) or low energy group (LE, 3.6 MFU/day, n=6). Circulating concentrations of metabolic substrates, metabolic hormones and reproductive hormones were determined weekly for 19 weeks. Ovarian follicular characteristics and oocyte parameters were also ascertained weekly. Heifers fed the LE diet had a better dry matter conversion than heifers fed the HE diet and the calculated daily energy provision was negative for heifers fed the LE diet (-0.248 MFU) and positive for heifers fed the HE diet (5.4 MFU). Heifers fed the HE diet had an increase in 50 kg LW over the duration of the study whereas LW remained constant for heifers fed the LE diet. The BCS of heifers fed the HE diet (4.2) was greater (P<0.05) than the BCS for heifers fed the LE diet (3.4). Heifers fed the HE diet had greater (P<0.05) circulating concentrations of metabolic substrates (glucose, total cholesterol and HDL cholesterol) and metabolic hormones (insulin, glucagon, leptin and T3) compared with heifers fed the LE diet. There were no significant differences in circulating reproductive hormones between the two groups of heifers. Ovarian follicular characteristics were similar for the two groups of heifers while heifers fed the LE diet tended to have oocytes of reduced quality compared with heifers fed the HE diet. The most notable finding was that heifers fed the LE diet had a negative calculated daily energy provision but were able to maintain LW and reproductive activity. It was concluded that buffalo heifers may potentially have the capacity to undergo metabolic adjustment and reduce their energy requirements when dietary energy is limiting. This adaptive capacity would explain why buffaloes remain productive in environments that are limiting to other ruminants.

The aim was to establish the capacity of buffalo heifers to adapt their metabolic requirements to a low energy diet. Murrah buffalo (Bubalus bubalis) heifers undergoing regular estrous cycles were randomly assigned by age, live weight (LW) and body condition score (BCS) to a high energy group (HE, 5.8 milk forage units (MFU)/day, n=6) or low energy group (LE, 3.6 MFU/day, n=6). Circulating concentrations of metabolic substrates, metabolic hormones and reproductive hormones were determined weekly for 19 weeks. Ovarian follicular characteristics and oocyte parameters were also ascertained weekly. Heifers fed the LE diet had a better dry matter conversion than heifers fed the HE diet and the calculated daily energy provision was negative for heifers fed the LE diet (-0.248 MFU) and positive for heifers fed the HE diet (5.4 MFU). Heifers fed the HE diet had an increase in 50 kg LW over the duration of the study whereas LW remained constant for heifers fed the LE diet. The BCS of heifers fed the HE diet (4.2) was greater (P<0.05) than the BCS for heifers fed the LE diet (3.4). Heifers fed the HE diet had greater (P<0.05) circulating concentrations of metabolic substrates (glucose, total cholesterol and HDL cholesterol) and metabolic hormones (insulin, glucagon, leptin and T3) compared with heifers fed the LE diet. There were no significant differences in circulating reproductive hormones between the two groups of heifers. Ovarian follicular characteristics were similar for the two groups of heifers while heifers fed the LE diet tended to have oocytes of reduced quality compared with heifers fed the HE diet. The most notable finding was that heifers fed the LE diet had a negative calculated daily energy provision but were able to maintain LW and reproductive activity. It was concluded that buffalo heifers may potentially have the capacity to undergo metabolic adjustment and reduce their energy requirements when dietary energy is limiting. This adaptive capacity would explain why buffaloes remain productive in environments that are limiting to other ruminants.

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.