Follicular size affects the meiotic competence of in vitro matured prepubertal and adult oocytes in sheep

Non-atretic follicles dissected from prepubertal and adult ovaries were allocated in three groups: a) < 1 mm; b) 1-2 mm; c) > 2 mm. After 24 h of maturation, a lower percentage of adult oocytes from group a (P < 0.01) reached metaphase II than those from groups b and c (70.4 versus 89.5 and 95.5). Prepubertal oocytes showed similar results (P < 0.01; 27.2 versus 79.8 and 81.8). There was a significant difference (P < 0.01) in meiotic progression between prepubertal and adult oocytes of < 1 mm follicles. The diameter of prepubertal oocytes derived from group a was significantly lower (P < 0.01) compared to groups b and c (138.1 versus 142.1 and 145.6); adult oocytes showed similar results (P < 0.01; 142.2 versus 157.2 and 158.1 Oocytes with the same diameter derived from different follicles of prepubertal and adult ovaries showed similar meiotic progression rates. Des follicules non-atrétiques sélectionnés à partir d’ovaires d’ovins prépubères et adultes ont été répartis en trois groupes selon leur diamètre : a) < 1 mm ; b) 1-2 mm ; c) > 2 mm. Après 24 h de maturation les ovocytes dérivant des ovaires d’animaux adultes appartenant au groupe a ont atteint la MII dans des pourcentages nettement inférieurs (p < 0,01) à ceux observés pour les groupes b et c (70,4 versus 89,5 et 95,5). Des résultats identiques ont été obtenus avec les ovocytes provenant d’animaux prépubères (p < 0,01 ; 27,2 versus 79,8 et 81,8). Une grande différence (p < 0,01) dans le potentiel à reprendre la méiose existe entre les ovocytes de prépubères et des adultes provenant de follicules < 1 mm. Le diamètre des ovocytes d’agneaux provenant de follicules du groupe a était nettement inférieur (p < 0,01 ) par rapport à ceux provenant de follicules des groupes b ou c (138,1 versus 142,1 et 145,6). Des résultats identiques ont été observés pour les ovocytes provenant d’animaux adultes (p < 0,01, 142,2 versus 157,2 et 158,1). Des ovocytes de même diamètre provenant d’ovaires de prépubères et d’adultes ne diffèrent pas dans leur aptitude à reprendre la méiose

Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes

The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater ( P < .01) number of oocytes per animal (86.2 ± 7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5 ± 6.1) of the same age or with treated neonatal ewes (6.1 ± 0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs 74.24), metaphase I (89.13 vs 87.18), and metaphase II (77.91 vs 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs 73.7) and blastocyst rates (22.2 vs 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs 76.9) and the in vivo survival rates (46.1 vs 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age

M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured <i>in vitro</i> and <i>in vivo</i>

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p &lt; 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p &lt; 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU•mL–1), LH (0.1 IU•mL–1)+ progesterone (1 μg•mL–1) + oestradiol (1 μg•mL–1) + cysteamine (100 μM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P &lt; 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P &lt; 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation

Sheep embryos derived from FSH/eCG treatment have a lower in vitro viability after vitrification than those derived from FSH treatment

In the non breeding period, the effect of two superovulatory treatments (eCG/FSH in single dose or FSH alone in four decreasing doses) on the production of embryo quality following in vitro viability after vitrification procedures was investigated using forty-four adult Sarda breed ewes. In sheep treated with eCG/FSH, the mean number of corpora lutea was significantly (P &lt; 0.05) higher (11.8±4.0 vs. 8.05±3.8), although the recovery rate was significantly (P &lt; 0.01) lower (74.6 vs. 59.9) than with FSH alone. After vitrification (ethylene glycol and glycerol) was repeated three times, the rates of re-expansion at first and second warming were significantly (P &lt; 0.01) higher in embryos derived from FSH alone than in those with both gonadotrophins (94.9 and 41.9 vs. 72.8 and 18.6) and after the last vitrification the hatched blastocyst rates were 22.5 and 7.6. After differential stain, blastocysts derived from FSH alone showed a mean number of cells significantly higher than blastocysts from eCG/FSH (184.2 vs. 157.7). It was concluded that superovulatory treatment with eCG/FSH may increase the ovarian responses compared with FSH alone, but these embryos showed a reduction in viability rates after repeated vitrification. Pendant la saison non reproductive, on a évalué, chez 44 brebis adultes de race Sarde, l'effet de deux traitements de superovulation (le premier avec les eCG/FSH en une seule administration, le deuxième avec la FSH administrée en quatre doses décroissantes) sur la qualité des embryons produits et sur la viabilité in vitro après vitrification. Dans le groupe traité avec les eCG/FSH, le nombre moyen d'ovulations était plus élevé (11,8±4,0 vs. 8,05±3,8, P &lt; 0,05) tandis que le taux de récupération était plus faible (74,6 vs. 59,9, P &lt; 0,01) par rapport au groupe traité avec la FSH seule. Les embryons ont suivi 3 vitrifications successives (éthylène glycol et glycérol). Les pourcentages de réexpansion de la cavité blastocoelique après les deux premiers cycles de vitrification-décongélation étaient significativement (P &lt; 0,01) supérieurs pour les embryons dérivés du traitement avec la FSH seule par rapport à ceux dérivés du traitement effectué avec les deux gonadotropines (94,9 et 41,9 vs. 72,8 et 18,6). Après la dernière vitrification, les pourcentages d'éclosion étaient respectivement de 22,5 et de 7,6 pour les groupes FSH et eCG/FSH. Après coloration différentielle, les blastocystes dérivés du traitement avec la FSH seule avaient un nombre moyen de cellules supérieur (p &lt; 0,01) par rapport aux blastocystes dérivés du traitement avec eCG/FSH (184,2 vs. 157,7). En conclusion, le traitement superovulatoire avec eCG/FSH augmente la réponse ovarienne par rapport au traitement avec la FSH seule, mais ces embryons montrent une réduction des taux de survie en culture après les processus répétés de vitrification-décongélation

Embryo transfer and related technologies in sheep reproduction

This paper reviews the status of embryo transfer and the major technologies applied to preimplantation of embryos in sheep. Embryo production from superovulated ewes is hindered by an unpredictable response to hormonal treatment. Progress in this area should be expected by an appropriated control of follicular development with gonadotropin-releasing hormone (GnRH) agonist or antagonist prior to gonadotrophin administration. Simple protocols for the cryopreservation of sheep embryos by vitrification are already available and the development of frozen-thawed blastocysts to term is close to the fresh ones. Further research is required to identify factors able to promote the maturation in vitro of oocytes, namely those obtained from prepubertal animals. Semen and embryo sexing procedures are available in cattle although much less attention was paid to their application to sheep. Among all the reproductive technologies, cloning with embryonic and foetal cells has progressed dramatically in sheep and nuclear transfer has been used to produce transgenic animals as an alternative to pronuclear injection. The production of the first lamb cloned from a somatic cell opened new opportunities in animal breeding as well as exciting lines of basic research. The overall conclusions are that, apart from superovulation, the application of in vitro technologies is likely to evolve rapidly and once applied, a great impact on traditional and new animal productions should be expected. However, a better understanding of the changes in gene expression, induced in embryos by different in vitro manipulation procedures, is necessary to prevent abnormal foetal development. Cette revue traite du transfert d’embryons et des principales biotechnologies appliquées à l’embryon ovin. La production d’embryons, après superovulation, est limitée par la réponse non reproductible au traitement hormonal. Un progrès pourrait venir d’un contrôle approprié du développement folliculaire avec un agoniste ou antagoniste de GnRH appliqué avant le traitement gonadotrope. Des protocoles simples pour la congélation d’embryons ovins par vitrification sont disponibles et, après décongélation, le développement à terme de blastocystes congelés est proche de celui des embryons frais. De nouvelles recherches seront nécessaires pour identifier les facteurs capables de stimuler la maturation in vitro des ovocytes, en particulier ceux d’animaux prépubères. Le sexage des spermatozoïdes et des embryons est possible chez les bovins, mais peu appliqué chez les ovins. De toutes les techniques de reproduction, c’est celle du clonage à partir de cellules embryonnaires ou foetales qui a le plus progressé; le transfert nucléaire a été utilisé pour produire des animaux transgéniques, comme alternative à l’injection dans les pronoyaux. La production du premier agneau cloné à partir d’une cellule somatique a ouvert de nouvelles perspectives en élevage et en recherche fondamentale. En conclusion, excepté dans le domaine de la superovulation, les biotechnologies vont évoluer rapidement; leur application aura certainement un grand impact sur les méthodes traditionnelles et nouvelles de production. Cependant, une meilleure connaissance des effets sur l’expression de gènes embryonnaires induits par les manipulations in vitro serait nécessaire pour éviter un développement foetal anormal

Two culture systems showing a biphasic effect on ovine embryo development from the 1-2 cell stage to hatched blastocysts

This study compared the effect of using either CZB or TCM 199 media on both the development of 1-2 cell ovine embryos from superovulated ewes to the blastocyst stage (Experiment 1), and the hatching process of ovine blastocysts developed in vitro (Experiment 2). For the first 5 d, the CZB medium showed higher rates of embryo development than the TCM 199 medium (p &lt; 0.001). The embryos reaching the &gt; 16 cell stage were 79 vs 52% and 74 vs 20% with or without an oviductal monolayer, respectively, and those reaching the blastocyst stage were 71 vs 46% and 46 vs 13% with or without cells. The CZB medium was less able to support the hatching process of the blastocysts obtained in the first experiment than was the TCM-199 medium + 10% FCS (fetal calf serum) with cells (31 vs 92%; p &lt; 0.001) or without cells (13 vs 66%; p &lt; 0.001). No blastocysts completely escaped from the zona pellucida (ZP) in the CZB medium compared with 80 and 61 % in the TCM 199 medium with or without cells, respectively. In Experiment 3, 47% of the blastocysts migrated through the artificial opening of the ZP and hatched completely. After 24 h of culture in the CZB medium, however, they showed blastocoelic cavity breakdown. During the preliminary cleavages, the ovine embryos developed better in CZB medium than in TCM 199, but the latter was more efficient in promoting the hatching process of the blastocysts. Les effets d’un milieu de culture, CZB, et d’un milieu de culture, TCM 199 + 10% de sérum de veau foetal, ont été observés sur le développement in vitro d’embryons d’ovins du stade 1-2 cellules au stade blastocyste (expérience 1) et dans le processus d’éclosion des blastocystes produits lors de l’expérience précédente (expérience 2). Le milieu CZB exerce un effet positif sur le développement embryonnaire par rapport au milieu TCM 199 après culture jusqu au stade &gt; 16 cellules (79 vs 52 % et 74 vs 20%) ou jusqu’au stade blastocyste (71 vs 46% et 46 vs 13%) en présence de cellules et en l’absence de cellules tubaires respectivement. Le processus d’éclosion est plus faible dans le milieu CZB que dans le milieu TCM 199 soit en présence (31 vs 92%) soit en l’absence (13 vs 66%) de cellules tubaires respectivement. Dans le CZB (expérience 3), 47% des blastocystes éclosent par une fente artificielle dans la zone pellucide, mais, après 24 h de culture, la cavité blastocoelique se dégonfle. Ces résultats montrent que le milieu CZB est plus indiqué pour le développement précoce d’embryons ovins alors que le milieu TCM 199 est plus efficace au moment de la phase d’éclosion

Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P&#60;0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P&#60;0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P&#60;0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P&#60;0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development

Livelli di inquinanti organici persistenti in stenelle (<i>Stenella coeruleoalba</i>) spiaggiate nel nord Sardegna = Concentration of persistent organic pollutants in striped dolphin (<i>Stenella coeruleoalba</i>) stranded in North Sardinia

Long-lived apex predators, such as marine mammals, are particularly at risk from effects of persistent organic pollutants (POPs), e.g. polychlorinated biphenyls (PCBs) and dichlorodiphenylethanes (e.g. DDT), due to bioaccumulation and biomagnification. POPs are lipophilic compounds that tend to accumulate in the lipid-rich blubber. In marine mammals, POPs enter the body almost exclusively through the diet. In the present study, the levels of PCBs and DDTs in the blubber of striped dolphin stranded in North Sardinia were determined. Our results showed that pollutant concentration are related to age and sex of the individuals. Considering the well known harmful consequences of bioaccumulation of POPs in marine mammals, further studies are needed to determine which POPs might be linked to effects on health

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December – March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 μl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique