The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovula... more The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovulatory treatment protocols in sheep increases the number of smaller follicles able to grow and ovulate in response to the exogenous FSH treatment (Lopez-Alonso C et al. 2004 Reprod. Fertil. Dev. 16, 233). The aim of our study was to test if such treatment affects the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from GnRH-antagonist treated sheep during an ovarian by perstimulation protocol. Adult Sarda sheep (n = 18) were synchronized by the insertion of intravaginal sponges (Day 0) which were left in situ for 12 days; on Day 7, group A (n = 10) received a single dose of 3 mg of Antarelix (Teverelix, Europeptides, France) s.c., while group B (n = 8) served as control. All animals received 96 IU of FSH (Ovagen, ICP, New Zealand) administered in 4 equal doses given i.m. every 12 h starting on Day 10. Twelve hours after the last FSH administration oocytes were ...
Journal of Developmental Origins of Health and Disease, 2018
The present study used a sheep model of intrauterine growth restriction, combining maternal under... more The present study used a sheep model of intrauterine growth restriction, combining maternal undernutrition and twinning, to determine possible markers of early damage to the fetal kidney. The occurrence of early deviations in fetal hemodynamics which may be indicative of changes in blood perfusion was assessed by Doppler ultrasonography. A total of 24 sheep divided in two groups were fed with the same standard grain-based diet but fulfilling either their daily maintenance requirements for pregnancy (control group; n=12, six singleton and six twin pregnancies) or only the 50% of such quantity (food-restricted group; n=12; four singleton and eight twin pregnancies). All the fetuses were assessed by both B-mode and Doppler ultrasonography at Day 115 of pregnancy. Fetal blood supply was affected by maternal undernutrition, although there were still no evidences of brain-sparing excepting in fetuses at greatest challenge (twins in underfed pregnancies). However, there were early changes ...
The aim of this work was to evaluate developmental competence and gene expression of prepubertal ... more The aim of this work was to evaluate developmental competence and gene expression of prepubertal and adult ovine oocytes. GV prepubertal and adult oocytes were matured, fertilized and cultured in vitro until blastocyst stage;; the time (days) needed to reach this stage was recorded. Blastocysts developed on different days were cultured for hatching to evaluate their quality in relation to cleavage rate. Adult and prepubertal GV oocytes and blastocyst-stage embryos produced, respectively, at 6 and 7 days were compared for quantitative expression of poly(A) polymerase (polyA-P), glucose transporter I (Glut-I), desmocollin II (desmoII), plakofilin (plako) and heat shock protein 70.1 (HSP70) genes. Confirming previous results (Ledda et al., 1996 Zygote 4, 343–348), fertilized prepubertal ovine oocytes developed to blastocyst stage at lower rates than the adult ones (19.9 v. 51.3%, respectively, P<0.001) and this stage was delayed 24h in prepubertal compared to adult embryos (P<0.0...
This study determined the influence of a short-term glucogenic nutritional treatment on circulati... more This study determined the influence of a short-term glucogenic nutritional treatment on circulating concentrations of glucose, insulin, insulin-like growth factor 1 (IGF-1), nonesterified fatty acids (NEFA), and urea, and on their correspondent levels in follicular fluid (FF) collected 12 h after the end of the treatment. After estrous synchronization with intravaginal progestagen-impregnated sponges, 20 Sarda ewes were randomly allocated into two experimental groups (GLU and WAT) and, from day 7 to day 10 (day 0 = day of sponge removal), the GLU group was gavaged with a glycogenic mixture, whereas the WAT group was gavaged with water (control group). Follicular development was stimulated by FSH administration from day 8 to 10. At day 11, ovaries were collected and follicular fluid processed. Plasma changes were assessed from day 6 to 11. In GLU group, circulating concentration of glucose (P < 0.0001), insulin (P < 0.0001), and IGF-1 (P < 0.01) rose significantly, whereas NEFA and urea concentrations decreased (P < 0.0001), as compared with controls. In particular, in FF the higher glucose concentrations found in GLU ewes compared with controls (P < 0.0001) were not accompanied by any increase in insulin and IGF-1 concentrations. NEFA (P < 0.0001) and urea (P < 0.0001) were lower in FF of GLU than WAT group, although NEFA clearance in the ovary proved to be less efficient than at the systemic level. No significant difference between groups was found in FF concentrations of pregnancy-associated plasma protein A (a protease regulating the levels of free IGF-1 in follicles), glutathione, and in its total antioxidant capacity. These results suggest that glycogenic mixture administration creates a suitable follicular microenvironment for the conception period in dairy ewes.
There is general consensus that the quality of in vitro-produced embryos is inferior to that of t... more There is general consensus that the quality of in vitro-produced embryos is inferior to that of their in vivo counterparts and that along with other factors this may be responsible for the poorer cryosurvival and the lower pregnancy rates reported for these embryos. The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess whether in vitro-produced ovine embryo quality could be determined by the timing blastocoelic cavity re-expansion after vitrification, thawing, and in vitro culture. Embryos were produced in vitro from ovine ovaries collected at the local slaughterhouse following a standard protocol developed for ovine oocytes, as previously described (Berlinguer et al. 2005 Reprod. Fertil. Dev. 17, 201). Blastocysts were then vitrified/warmed according to a simple method (Leoni et al. 2002 Cryobiology 45, 204-212) and cultured in vitro in TCM-199 supplemented with 1...
Cryopreservation of oocytes is still an open problem because of their structural sensitivity to t... more Cryopreservation of oocytes is still an open problem because of their structural sensitivity to the cooling and freezing process. Meiotic spindle disorganization and chromosomal aberrations are frequently observed, possibly due to the alteration of molecules involved in the meiotic cell cycle regulation and spindle formation such as maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). In this study we treated MII ovine oocytes with caffeine before vitrification and we evaluated the effect on (1) MPF and MAPK activity and (2) oocyte spontaneous or induced parthenogenetic activation. Oocytes from slaughterhouse sheep ovaries were in vitro-matured for 21 h in TCM-199 + 10% FCS + FSH, LH + cysteamine, and then incubated for 2 h with (+) or without (-) caffeine (20 mM; Sigma-Aldrich, Milan, Italy). Thereafter, MII oocytes were vitrified by equilibration with 10% ethylene glycol (EG) + 10% DMSO (30 s), exposure to 20% EG + 20% DMSO + 0.5 M sucrose (20 s), loading...
The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMF... more The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks. Studies have been carried out on endangered Mohor gazelle sperm cryopreservation (Garde et al. 2003 Biol. Reprod. 69, 602-611), but there are no studies on oocytes in this species. The purpose of this work was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC. The study was conducted using six reproductively mature female Mohor gazelles from the breeding herd at the Estacion Experimental de Zonas Aridas. Animals were synchronized by insertion of controlled progesterone internal drug release (CIDR) devices for 14 days and removal of the devices on the day of ovum pickup (OPU). Follicular growth was stimulated by a total of 5.28 mg of oFSH (Ovagen, ICP, Auckland, New Zealand) given in four equal doses every 12 h. OPUs were performe...
In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing g... more In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing...
The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), mater... more The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), were determined in ovine oocytes and in vitro-produced preimplantation embryos. The existence of ZAR1 and MATER in ovine species has not been reported previously. Reverse transcription–polymerase chain reaction was performed on germinal vesicle and IVM MII oocytes, as well as in in vitro fertilised and cultured two-, four-, eight- and 12/16-cell embryos, morulae and blastocysts. Quantification of gene expression by real-time polymerase chain reaction showed the highest abundance of all transcripts analysed in the immature oocyte. During the following stages of preimplantation development, the mRNAs examined exhibited different patterns of expression, but often significant decreases were observed during maturation and maternal–embryonic transition. The transcription ...
Polymers have been used as a substitute for serum in vitrification solutions for embryos and oocy... more Polymers have been used as a substitute for serum in vitrification solutions for embryos and oocytes. This study was designed to replace serum with defined commercial macromolecules in vitrification solution for in vitro matured ovine oocytes. Oocytes were cryopreserved in two vitrification solutions (16.5 percent ethylene glycol + 16.5 percent dimethyl sulphoxide) supplemented with 1 percent of SuperCool X-1000 and 1 percent SuperCool Z-1000 (Ice Blockers) or 20 percent foetal calf serum (FCS). After warming, oocytes viability and developmental potential after processing for in vitro embryo production were assessed. The number of viable oocytes (87.4 percent and 85.9 percent), cleaveage rates (21.4 percent and 19.6 percent) and blastocyst development rates (4.8 percent and 4.5 percent) were similar for Ice Blockers and FCS, respectively. On the basis of these findings, it may be concluded that combined use of Ice Blockers (SuperCool X-1000 and SuperCool Z-1000) as supplementation i...
The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovula... more The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovulatory treatment protocols in sheep increases the number of smaller follicles able to grow and ovulate in response to the exogenous FSH treatment (Lopez-Alonso C et al. 2004 Reprod. Fertil. Dev. 16, 233). The aim of our study was to test if such treatment affects the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from GnRH-antagonist treated sheep during an ovarian by perstimulation protocol. Adult Sarda sheep (n = 18) were synchronized by the insertion of intravaginal sponges (Day 0) which were left in situ for 12 days; on Day 7, group A (n = 10) received a single dose of 3 mg of Antarelix (Teverelix, Europeptides, France) s.c., while group B (n = 8) served as control. All animals received 96 IU of FSH (Ovagen, ICP, New Zealand) administered in 4 equal doses given i.m. every 12 h starting on Day 10. Twelve hours after the last FSH administration oocytes were ...
Journal of Developmental Origins of Health and Disease, 2018
The present study used a sheep model of intrauterine growth restriction, combining maternal under... more The present study used a sheep model of intrauterine growth restriction, combining maternal undernutrition and twinning, to determine possible markers of early damage to the fetal kidney. The occurrence of early deviations in fetal hemodynamics which may be indicative of changes in blood perfusion was assessed by Doppler ultrasonography. A total of 24 sheep divided in two groups were fed with the same standard grain-based diet but fulfilling either their daily maintenance requirements for pregnancy (control group; n=12, six singleton and six twin pregnancies) or only the 50% of such quantity (food-restricted group; n=12; four singleton and eight twin pregnancies). All the fetuses were assessed by both B-mode and Doppler ultrasonography at Day 115 of pregnancy. Fetal blood supply was affected by maternal undernutrition, although there were still no evidences of brain-sparing excepting in fetuses at greatest challenge (twins in underfed pregnancies). However, there were early changes ...
The aim of this work was to evaluate developmental competence and gene expression of prepubertal ... more The aim of this work was to evaluate developmental competence and gene expression of prepubertal and adult ovine oocytes. GV prepubertal and adult oocytes were matured, fertilized and cultured in vitro until blastocyst stage;; the time (days) needed to reach this stage was recorded. Blastocysts developed on different days were cultured for hatching to evaluate their quality in relation to cleavage rate. Adult and prepubertal GV oocytes and blastocyst-stage embryos produced, respectively, at 6 and 7 days were compared for quantitative expression of poly(A) polymerase (polyA-P), glucose transporter I (Glut-I), desmocollin II (desmoII), plakofilin (plako) and heat shock protein 70.1 (HSP70) genes. Confirming previous results (Ledda et al., 1996 Zygote 4, 343–348), fertilized prepubertal ovine oocytes developed to blastocyst stage at lower rates than the adult ones (19.9 v. 51.3%, respectively, P<0.001) and this stage was delayed 24h in prepubertal compared to adult embryos (P<0.0...
This study determined the influence of a short-term glucogenic nutritional treatment on circulati... more This study determined the influence of a short-term glucogenic nutritional treatment on circulating concentrations of glucose, insulin, insulin-like growth factor 1 (IGF-1), nonesterified fatty acids (NEFA), and urea, and on their correspondent levels in follicular fluid (FF) collected 12 h after the end of the treatment. After estrous synchronization with intravaginal progestagen-impregnated sponges, 20 Sarda ewes were randomly allocated into two experimental groups (GLU and WAT) and, from day 7 to day 10 (day 0 = day of sponge removal), the GLU group was gavaged with a glycogenic mixture, whereas the WAT group was gavaged with water (control group). Follicular development was stimulated by FSH administration from day 8 to 10. At day 11, ovaries were collected and follicular fluid processed. Plasma changes were assessed from day 6 to 11. In GLU group, circulating concentration of glucose (P < 0.0001), insulin (P < 0.0001), and IGF-1 (P < 0.01) rose significantly, whereas NEFA and urea concentrations decreased (P < 0.0001), as compared with controls. In particular, in FF the higher glucose concentrations found in GLU ewes compared with controls (P < 0.0001) were not accompanied by any increase in insulin and IGF-1 concentrations. NEFA (P < 0.0001) and urea (P < 0.0001) were lower in FF of GLU than WAT group, although NEFA clearance in the ovary proved to be less efficient than at the systemic level. No significant difference between groups was found in FF concentrations of pregnancy-associated plasma protein A (a protease regulating the levels of free IGF-1 in follicles), glutathione, and in its total antioxidant capacity. These results suggest that glycogenic mixture administration creates a suitable follicular microenvironment for the conception period in dairy ewes.
There is general consensus that the quality of in vitro-produced embryos is inferior to that of t... more There is general consensus that the quality of in vitro-produced embryos is inferior to that of their in vivo counterparts and that along with other factors this may be responsible for the poorer cryosurvival and the lower pregnancy rates reported for these embryos. The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess whether in vitro-produced ovine embryo quality could be determined by the timing blastocoelic cavity re-expansion after vitrification, thawing, and in vitro culture. Embryos were produced in vitro from ovine ovaries collected at the local slaughterhouse following a standard protocol developed for ovine oocytes, as previously described (Berlinguer et al. 2005 Reprod. Fertil. Dev. 17, 201). Blastocysts were then vitrified/warmed according to a simple method (Leoni et al. 2002 Cryobiology 45, 204-212) and cultured in vitro in TCM-199 supplemented with 1...
Cryopreservation of oocytes is still an open problem because of their structural sensitivity to t... more Cryopreservation of oocytes is still an open problem because of their structural sensitivity to the cooling and freezing process. Meiotic spindle disorganization and chromosomal aberrations are frequently observed, possibly due to the alteration of molecules involved in the meiotic cell cycle regulation and spindle formation such as maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). In this study we treated MII ovine oocytes with caffeine before vitrification and we evaluated the effect on (1) MPF and MAPK activity and (2) oocyte spontaneous or induced parthenogenetic activation. Oocytes from slaughterhouse sheep ovaries were in vitro-matured for 21 h in TCM-199 + 10% FCS + FSH, LH + cysteamine, and then incubated for 2 h with (+) or without (-) caffeine (20 mM; Sigma-Aldrich, Milan, Italy). Thereafter, MII oocytes were vitrified by equilibration with 10% ethylene glycol (EG) + 10% DMSO (30 s), exposure to 20% EG + 20% DMSO + 0.5 M sucrose (20 s), loading...
The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMF... more The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks. Studies have been carried out on endangered Mohor gazelle sperm cryopreservation (Garde et al. 2003 Biol. Reprod. 69, 602-611), but there are no studies on oocytes in this species. The purpose of this work was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC. The study was conducted using six reproductively mature female Mohor gazelles from the breeding herd at the Estacion Experimental de Zonas Aridas. Animals were synchronized by insertion of controlled progesterone internal drug release (CIDR) devices for 14 days and removal of the devices on the day of ovum pickup (OPU). Follicular growth was stimulated by a total of 5.28 mg of oFSH (Ovagen, ICP, Auckland, New Zealand) given in four equal doses every 12 h. OPUs were performe...
In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing g... more In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing...
The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), mater... more The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), were determined in ovine oocytes and in vitro-produced preimplantation embryos. The existence of ZAR1 and MATER in ovine species has not been reported previously. Reverse transcription–polymerase chain reaction was performed on germinal vesicle and IVM MII oocytes, as well as in in vitro fertilised and cultured two-, four-, eight- and 12/16-cell embryos, morulae and blastocysts. Quantification of gene expression by real-time polymerase chain reaction showed the highest abundance of all transcripts analysed in the immature oocyte. During the following stages of preimplantation development, the mRNAs examined exhibited different patterns of expression, but often significant decreases were observed during maturation and maternal–embryonic transition. The transcription ...
Polymers have been used as a substitute for serum in vitrification solutions for embryos and oocy... more Polymers have been used as a substitute for serum in vitrification solutions for embryos and oocytes. This study was designed to replace serum with defined commercial macromolecules in vitrification solution for in vitro matured ovine oocytes. Oocytes were cryopreserved in two vitrification solutions (16.5 percent ethylene glycol + 16.5 percent dimethyl sulphoxide) supplemented with 1 percent of SuperCool X-1000 and 1 percent SuperCool Z-1000 (Ice Blockers) or 20 percent foetal calf serum (FCS). After warming, oocytes viability and developmental potential after processing for in vitro embryo production were assessed. The number of viable oocytes (87.4 percent and 85.9 percent), cleaveage rates (21.4 percent and 19.6 percent) and blastocyst development rates (4.8 percent and 4.5 percent) were similar for Ice Blockers and FCS, respectively. On the basis of these findings, it may be concluded that combined use of Ice Blockers (SuperCool X-1000 and SuperCool Z-1000) as supplementation i...
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