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

NMR characterization of animals’ follicular fluids

Follicular Fluid (FF) provides a special environment to the oocyte during its maturation in vivo. The FF is derived from the sanguineous plasma and secretions, synthesised in the follicle wall that contain a large variety of metabolites (1). These metabolites are probably involved in the physiology of the oocytes (1). The chemical composition of follicular fluids is important because it is an indicator of the secretory activities and metabolism of follicular cells and thus could be related to the follicular quality. It could also provide a useful indication of the oocyte growth and maturation (2). High Resolution Nuclear Magnetic Resonance (NMR) spectroscopy provides a unique tool for studying metabolites. Initially, NMR spectroscopy was used mainly in biomedicine but it is found now in many physiological applications (3). As the NMR spectroscopy provides opportunities for obtaining qualitative and quantitative data from body fluids, it was hypothesized that this technique could provide information on mammals’ follicular fluid and on intrafollicular changes that occur during follicular growth and ovulation. As some of these changes are probably of crucial importance for oocyte developmental competence, a better knowledge of the mammals’ follicular fluid composition by 1H NMR analysis should help to resolve some of the problems encountered during in vitro procedures in the mammals. The characterization of the chemical composition of mammals follicular fluids, namely sheep, cattle, mare and pork, and the study of the changes observed during follicular growth and maturation using NMR spectroscopy will be presented. FF samples were collected from antral follicle of different dimensions. One-dimensional (1D) 1H experiments (CPMG, DOSY) were obtained for all the FF samples. In addition, several two dimensional (2D) (homo and heteronuclear) experiments (DQF-COSY, TOCSY, JRES, 1H-13C HSQC) were performed to aid in the assignment of the signals and in the identification of the metabolites in FF. A direct evaluation of the lipids, carbohydrates and metabolites were obtained from the combination of the 1D and 2D NMR experiments

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

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

DuoStim – a reproducible strategy to obtain more oocytes and competent embryos in a short time-frame aimed at fertility preservation and IVF purposes. A systematic review

Recent evidence suggests that follicular development occurs in a wave-like model during the ovarian cycle, where up to three cohorts of follicles are recruited to complete folliculogenesis. This understanding overtakes the previous dogma stating that follicles grow only during the follicular phase of the menstrual cycle. Therefore, in in vitro fertilization (IVF), novel protocols regarding ovarian stimulation have been theorized based on the use of gonadotrophins to prompt the growth of antral follicles at any stage of the menstrual cycle. These unconventional protocols for ovarian stimulation aim at a more efficient management of poor-prognosis patients, otherwise exposed to conflicting outcomes after conventional approaches. DuoStim appears among these unconventional stimulation protocols as one of the most promising. It combines two consecutive stimulations in the follicular and luteal phases of the same ovarian cycle, aimed at increasing the number of oocytes retrieved and embryos produced in the short time-frame. This protocol has been suggested for the treatment of all conditions requiring a maximal and urgent exploitation of the ovarian reserve, such as oncological patients and poor responders at an advanced maternal age. At present, data from independent studies have outlined the consistency and reproducibility of this approach, which might also reduce the drop-out between consecutive failed IVF cycles in poor-prognosis patients. However, the protocol must be standardized, and more robust studies and cost-benefit analyses are needed to highlight the true clinical pros and cons deriving from DuoStim implementation in IVF

Tissue-specific and minor inter-individual variation in imprinting of <i>IGF2R</i> is a common feature of <i>Bos taurus</i> concepti and not correlated with fetal weight

The insulin-like growth factor 2 receptor (IGF2R) is essential for prenatal growth regulation and shows gene dosage effects on fetal weight that can be affected by in-vitro embryo culture. Imprinted maternal expression of murine Igf2r is well documented for all fetal tissues excluding brain, but polymorphic imprinting and biallelic expression were reported for IGF2R in human. These differences have been attributed to evolutionary changes correlated with specific reproductive strategies. However, data from species suitable for testing this hypothesis are lacking. The domestic cow (Bos taurus) carries a single conceptus with a similar gestation length as human. We identified 12 heterozygous concepti informative for imprinting studies among 68 Bos taurus fetuses at Day 80 of gestation (28% term) and found predominantly maternal IGF2R expression in all fetal tissues but brain, which escapes imprinting. Inter-individual variation in allelic expression bias, i.e. expression of the repressed paternal allele relative to the maternal allele, ranged from 4.6−8.9% in heart, 4.3−10.2% in kidney, 6.1−11.2% in liver, 4.6−15.8% in lung and 3.2−12.2% in skeletal muscle. Allelic bias for mesodermal tissues (heart, skeletal muscle) differed significantly (P&lt;0.05) from endodermal tissues (liver, lung). The placenta showed partial imprinting with allelic bias of 22.9−34.7% and differed significantly (P&lt;0.001) from all other tissues. Four informative fetuses were generated by in-vitro fertilization (IVF) with embryo culture and two individuals displayed fetal overgrowth. However, there was no evidence for changes in imprinting or DNA methylation after IVF, or correlations between allelic bias and fetal weight. In conclusion, imprinting of Bos taurus IGF2R is similar to mouse except in placenta, which could indicate an effect of reproductive strategy. Common minor inter-individual variation in allelic bias and absence of imprinting abnormalities in IVF fetuses suggest changes in IGF2R expression in overgrown fetuses could be modulated through other mechanisms than changes in imprinting

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