Interactive effects of granulosa cell apoptosis, follicle size, cumulus-oocyte complex morphology, and cumulus expansion on the developmental competence of goat oocytes: a study using the well-in-drop culture system

Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus-oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8-16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73-84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.     

Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents

Folliculogenesis is a complex process regulated by various paracrine and autocrine factors. In vitro growth systems of primordial and preantral follicles have been developed for future use of immature oocytes, as sources of fertilizable oocytes and for studying follicular growth and oocyte maturation mechanisms. Rodents were often chosen for in vitro follicular culture research and a lot of factors implicated in folliculogenesis have been identified using this model. To date, the mouse is the only species in which the whole process of follicular growth, oocyte maturation, fertilization and embryo transfer into recipient females was successfully performed. However, the efficiency of in vitro culture systems must still be considerably improved. Within the follicle, numerous events affect cell proliferation and the acquisition of oocyte developmental competency in vitro, including interactions between the follicular cells and the oocyte, and the composition of the culture medium. Effects of the acting factors depend on the stage of follicle development, the culture system used and the species. This paper reviews the action of endocrine, paracrine factors and other components of culture medium on in vitro growth of preantral follicles in rodents.     

Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development

Germinal vesicle (GV)-stage oocytes retrieved from antral follicles undergo nuclear maturation in vitro, which typically occurs prior to cytoplasmic maturation. Short-term culture with meiotic inhibitors has been applied to arrest oocytes at the GV stage aiming to synchronize nuclear and ooplasmic maturity. However, the results obtained are still far from the in vivo situation. In order to acquire competence, immature oocytes may require meiotic arrest in vitro for a more extended period. The phosphodiesterase type 3-inhibitor (PDE3-I) is a potent meiotic arrester. The effects of a prolonged culture with PDE3-I on oocyte quality prior to and after reversal from the inhibition are not known. This study tested the impact of long-term in vitro exposure of two PDE3-Is, org9935 and cilostamide, on oocytes using a mouse follicle culture model. The results showed that PDE3-I (maximum of 10 microM) during a 12-day culture of follicle-enclosed oocytes did not alter somatic cell proliferation, differentiation or follicle survival. In addition, the steroid production profile was not significantly modified by a 12-day exposure to PDE3-I. The recombinant human chorionic gonadotrophin/recombinant human epidermal growth factor stimulus induced a characteristic normal progesterone peak of luteinization and normal mucification of the cumulus cells, while the enclosed oocyte remained blocked at the GV stage. In vitro maturation of denuded or cumulus-enclosed oocytes derived from org9935- or cilostamide-exposed follicles progressed through meiosis and formed morphologically normal meiotic spindles with chromosomes properly aligned at the equator. In conclusion, long-term culture with PDE3-I was harmless to somatic cell function, differentiation, oocyte growth and maturation. Our results suggested that PDE3-I can be applied when extended oocyte culture is required to improve ooplasmic maturation.     

Dissociation of oocyte nuclear and cytoplasmic maturation by the addition of insulin in cultured bovine antral follicles

Follicles of 4-8 mm in diameter were dissected from ovaries and cultured in Waymouth culture medium in the presence or absence of insulin (5 mug/ml) at 39 degrees C in a humidified atmosphere of 45% O2, 5% CO2 and 50% N2 for 24 h. Following follicle culture, the oocytes were collected and examined for developmental potential, total protein profile and ultrastructural aspects. Oocytes aspirated directly from follicles of the same size were used as controls. Addition of insulin to the follicle culture medium significantly reduced expression of the low molecular weight insulin-like growth factor-binding proteins (IGFBPs) in the follicular fluid, and significantly reduced the cleavage rate of subsequently matured and fertilised oocytes (0.52 vs 0.61). However, there were no differences in the proportion of cleaved embryos which developed to the blastocyst stage (0.30 vs 0.28), nor embryo quality as assessed by total cell number (137 +/- 8.53 vs 124.6 +/- 6.95). The total protein profiles of immature oocytes recovered after 24 h of follicle culture were compared by PAGE. There were marked differences between the two groups, unmatured oocytes recovered from the insulin-positive follicle group showed a protein pattern similar to that of matured oocytes. In addition, examination of ultrastructural features by transmission electron microscopy indicated that oocytes from follicles cultured in the presence of insulin undergo many of the cytoplasmic changes associated with oocyte maturation. In conclusion, follicle culture in the presence of insulin is beneficial for follicular survival and significantly reduces cleavage but has no detrimental effects on the development of cultured embryos. However, many of the cytoplasmic changes associated with oocyte maturation occur prior to the induction of nuclear maturation.     

FSH withdrawal improves developmental competence of oocytes in the bovine model

Combinations of genetic, environmental, and management factors are suspected to explain the loss in fertility observed for over 20 years in dairy cows. In some cases, IVF is used. When compared with in vivo embryo production, IVF resulted in low success rates until the FSH coasting process (FSH starvation after superstimulation) was introduced in 2002. Increased competence associated with FSH withdrawal of aspirated oocyte for in vitro maturation and IVF has not been optimized nor explained yet. The goal here was to determine and characterize the optimal oocyte competence acquisition window during the coasting period by determining blastocyst rates and follicular cohort development. Commercial milking cycling cows (n=6) were stimulated with 3 days of FSH (6×40?mg NIH Folltropin-V given at 12?h intervals) followed by a coasting period of 20, 44, 68, or 92?h. Each animal was exposed to the four conditions and served as its own control. At the scheduled time, transvaginal aspirations of immature oocytes were performed followed by IVF of half the oocytes. The outcomes were as follows: i) FSH coasting was optimal at a defined period: between 44 and 68?h of coasting; ii) The best estimated coasting duration was ～54±7?h; iii) Under these conditions, the best statistical blastocyst rate estimation was ～70%; iv) Between 44 and 68?h of coasting, follicle size group proportions were similar; v) Follicle diameter was not linearly associated with competence. In conclusion, coasting duration is critical to harvest the oocytes at the right moment of follicular differentiation.     

The earliest stages of folliculogenesis in vitro

In recent years several follicle culture systems have been pioneered in different mammalian species for studying ovarian folliculogenesis and culturing immature oocytes. Applications of these in vitro techniques include fertility preservation for humans, conservation of rare animals and development of oocyte banks for research purposes. Immature female gametes in the ovarian cortex can be cryopreserved for later use if culture techniques are available afterwards to promote growth and maturation. This review focuses on biochemical and biophysical factors related to oocyte culture in mice, the only animal in which live offspring have been produced after folliculogenesis in vitro. The advantage of using mice for these studies is that, in parallel to development of follicle culture systems, essential knowledge on folliculogenesis can be obtained from knockout mouse models. Recent experiments in mice stressed the principal role of the oocyte in follicle development and the strict timing of the biological processes underlying oogenesis in vitro. In large domestic animals and humans, study of oocyte culture is confounded by the constitutively prolonged nature of ovarian follicle development. In humans, only some aspects of follicle development have been studied because of the limited availability of suitable material for experimentation, technical difficulties related to manipulation of very small structures and lack of knowledge on physiological regulation of the early stages of follicle growth. Only a few reports describe ovarian follicular growth in vitro. In this review, relevant information on hormonal and growth factor regulation of the earliest stages of follicle growth in mammals is reviewed. Techniques are becoming available for the precise isolation of distinct classes of follicle and powerful molecular biology techniques can be used in studies of ovarian tissue culture.     

Fluctuations in bovine ovarian follicular fluid composition throughout the oestrous cycle

Bovine oocyte maturation in vitro frequently results in abnormal cytoplasmic maturation and failure to acquire developmental competence. This is, in part, likely to be due to the non-physiological nutritional milieu to which oocytes are exposed. Improvements in oocyte developmental potential may be achieved by modelling nutrient profiles on those of preovulatory follicular fluid (FF). However, little is known about fluctuations in FF nutrient levels according to follicle dominance and oestrous cyclicity. This study therefore characterised the carbohydrate and amino acid profile of FF according to these parameters, and compared preovulatory FF composition with that of maturation medium. Carbohydrate concentrations (n = 121) were determined enzymatically whilst amino acid profiles (n = 40) were determined by reverse-phase HPLC. Pyruvate and glucose concentrations were unaffected by follicle dominance, whereas Stage III-IV lactate profiles were higher in non-dominant FF (P < 0.01). While most dominant FF amino acid concentrations were affected by oestrous stage, only glutamate, alanine, leucine and lysine levels fluctuated in non-dominant FF. Glucose and lactate concentrations were significantly negatively correlated, whereas most amino acids were significantly positively correlated with each other. Maturation medium had higher pyruvate and lower lactate concentrations than preovulatory FF (P < 0.001), whereas glucose level was similar. All amino acid levels (except histidine, taurine, alanine and tryptophan) differed significantly between maturation medium and preovulatory FF. These data indicated that FF composition varies throughout the oestrous cycle. Preovulatory FF nutrient profile differed from that of maturation medium, perhaps accounting for the poor developmental competence of in vitro matured oocytes. These data may contribute to the formulation of a nutritionally more physiological maturation medium.     

Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). After in vitro maturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequent in vitro culture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.     

Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence

Mitochondria are the most abundant organelles in the mammalian oocyte and early embryo. While their role in ATP production has long been known, only recently has their contribution to oocyte and embryo competence been investigated in the human. This review considers whether such factors as mitochondrial complement size, mitochondrial DNA copy numbers and defects, levels of respiration, and stage-specific spatial distribution, influence the developmental normality and viability of human oocytes and preimplantation-stage embryos. The finding that mitochondrial polarity can differ within and between oocytes and embryos and that these organelles may participate in the regulation of intracellular Ca(2+)homeostasis are discussed in the context of how focal domains of differential respiration and intracellular-free Ca(2+)regulation may arise in early development and what functional implications this may have for preimplantation embryogenesis and developmental competence after implantation.     

Offspring produced from heterotopic ovarian allografts in male and female recipient mice

Studies on human ovarian xenografts and mouse allografts indicate that the male hormonal milieu and exogenous gonadotrophin administration stimulate antral follicle growth. However, it is not known whether oocytes produced under these conditions are developmentally competent. The objective of our study was to evaluate the developmental competence of oocytes produced in heterotopic mouse ovarian grafts placed in male and female recipient mice. Gonadotrophins were 7.5 IU pregnant mare serum gonadotrophin (PMSG) alone or 7.5 IU PMSG and 7.5 IU human chorionic gonadotrophin or were not given prior to oocyte collection. The developmental competence of oocytes was assessed by performing in vitro fertilisation and embryo transfer to recipients. When no gonadotrophins were given the cleavage rate was similar for oocytes collected from ovarian grafts in male and female recipients. Gonadotrophin treatment significantly (P < 0.05) increased two-cell formation by oocytes grown in female graft recipients but not in male recipients. Implantation rates, fetal development and the birth of live young were unaffected by the sex of the graft recipient or gonadotrophin treatment. Live offspring were produced from oocytes collected from ovarian grafts in male and female recipients treated with or without gonadotrophins. In conclusion, this work has shown that the hormonal environment of male mice can support the growth of oocytes in ovarian allografts and that these oocytes can produce live offspring.     

Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30 min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.     

Effect of aging on follicular function may be relieved by exogenous gonadotropin treatment in a sheep model

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12-14 years) and young adult ewes (4-5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E(2)) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E(2) and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen.     

Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57-59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55-61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.     

Physiological temperature variants and culture media modify meiotic progression and developmental potential of pig oocytes in vitro

Ovarian follicles in vivo are cooler than surrounding abdominal and ovarian tissues. This study investigated whether typical follicular temperatures influence the maturation and developmental potential of pig oocytes in vitro. Oocytes were synchronised at the germinal vesicle (GV) stage and incubated at 39, 37 or 35.5 degrees C. When compared with 39 degrees C, which is often used for in vitro studies, lower temperatures delayed spontaneous progression to the metaphase I and II (MI and MII) stages of meiosis. The MII was delayed by about 12 h per degrees C. All oocytes had normal morphology. Oocytes reaching GV breakdown (GVBD) at 39 degrees C were subsequently unaffected by cooling, demonstrating thermal sensitivity during the pre-GVBD stage only. Simultaneous assay of maturation-controlling kinases (maturation promoting factor (MPF) and MAPK) showed that cooling delayed kinase activation, provided it was applied prior to GVBD. Activity profiles remained coupled to the stage of meiosis. Neither enzyme was directly thermally sensitive over this temperature range. Following in vitro fertilisation, fewer blastocysts developed from embryos derived from 35.5 or 37 degrees C oocytes as compared with those from 39 degrees C oocytes. Manipulation of fertilisation timings to allow for delayed maturation showed that over-maturing or aging at lower temperatures compromises subsequent embryo development, despite normal nuclear maturation; the GV stage was again the thermally sensitive period. Cleavage rates were improved by the culture of oocytes with follicle-stimulating hormone (FSH) at 37 but not at 35.5 degrees C. Inclusion of 20% follicular fluid in the oocyte medium restored the blastocyst rate to that seen at higher temperatures. Thus, FSH and follicular fluid may allow oocytes to achieve normal developmental potential at in vivo temperatures.     

Effects of follicle size and electrolytes and glucose in maturation medium on nuclear maturation and developmental competence of bovine oocytes

The concentrations of electrolytes (Na, K, Cl, Mg and Ca) and glucose in small follicle (SF) follicular fluid (SFF) and large follicle (LF) follicular fluid (LFF) from slaughterhouse-derived ovaries were studied. Oocytes were matured in medium based on synthetic oviductal fluid. The effects of various concentrations of electrolytes (Na, K, Ca and Mg) and glucose in the maturation medium on the progression of nuclear maturation and subsequent development were also studied. K in SFF was significantly greater than that in LFF. The Mg concentration in follicular fluid (FF) is 2.0-2.3 mM, which is greater than the concentration present in medium generally used for culture. The glucose concentration in FF is about 3.5-3.9 mM and rapidly decreases during the preservation of ovaries. LF oocytes resumed nuclear maturation and progressed to the M2 stage significantly faster than those collected from SF oocytes. In addition, more LF oocytes developed to blastocysts than did SF oocytes. Changing the Na/K ratio in the maturation medium from 16 to 24 did not affect either the progression of nuclear maturation or the rate of development. A low concentration of Mg (0.5 mM) combined with a low Ca concentration (0.5 mM) inhibited the rate of development, but did not affect the progression of nuclear maturation. On the other hand, increasing the Mg concentration to 2.0 mM from 0.5 mM hastened the progression of nuclear maturation and improved the rate of blastulation, irrespective of the Ca concentration. The progression of nuclear maturation was faster and the rate of development was greater with 5.56 mM glucose than with 1.5 mM glucose. The difference in time needed to progress to M2 among the experiment was about 2-3 h. Therefore, prolonging the maturation periods from 21 to 24 h did not change the rate of development. Our results show that the concentrations of Mg and glucose in the maturation medium and the follicle size enveloping the oocyte affect the progression of nuclear maturation and subsequent development. The time requirement for oocytes to reach M2 is strongly related to the developmental competence of the oocytes.     

Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus-oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2, TNFAIP6, and HAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression of CYP11A1 in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.     

Use of parentage testing to determine optimum insemination time and culture media for oocyte transfer in mares

Parentage identification was used to test the developmental competence of oocytes cultured under different conditions and fertilized in vivo after oocyte transfer. Oocytes were collected transvaginally from follicles of estrous mares approximately 22 h after administration of human chorionic gonadotropin. Oocytes were cultured for approximately 16 h in one of three media, with or without addition of hormones and growth factors. Groups of three or four oocytes, cultured in different media, were transferred into the oviduct contralateral to a recipient's own ovulation. Recipients were inseminated with semen from two different stallions at 15 h before and 2.5 h after oocyte transfer. Sixteen days after transfer, embryos were recovered from uteri and submitted for parentage testing. The percentage of oocytes resulting in embryonic vesicles was nearly identical (P >0.05) for transferred oocytes (32/44, 73%) versus ovulated oocytes of recipients (9/13, 69%). More (P <0.01) oocytes were fertilized by sperm inseminated before (35/38, 92%) versus after (3/38, 8%) oocyte transfer. Tissue culture medium (TCM)-199 was superior to equine maturation medium I (EMMI; a SOF-based medium) for culturing oocytes (P <0.05), although addition of hormones and growth factors during culture did not improve (P >0.05) development of embryos.     

Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season

The domestic cat experiences circannual variations in ovarian activity and intrafollicular oocyte quality. One result is poor nuclear and cytoplasmic maturation during in vitro maturation (IVM) conducted during the annual non-breeding season (July through November). In an attempt to overcome this seasonal phenomenon immature oocytes were collected from July through November and cultured in a conventional IVM medium (IVM1) or in IVM1 supplemented with different FSH concentrations and antioxidant (ascorbic acid or cysteine). Nuclear status of oocytes was assessed after IVM or IVF. Embryo stage and blastocyst quality were evaluated after 7 days of in vitro culture. Although the addition of antioxidant alone had no effect, the presence of 10 microg FSH ml(-1) improved nuclear maturation (75.4+/-4.1% versus 48.7+/-8.8% in IVM1; P<0.05) and fertilization success (47.9+/-3.2% versus 35.0+/-5.1% in IVM1; P<0.05). Furthermore, developmental competence of fertilized oocytes was enhanced (P<0.05) only in the presence of ascorbic acid (30.6+/-6.7%) or cysteine (33.6+/-5.1%) compared with IVM1 (8.1+/-8.8%). Consequently, blastocyst yield (17% of total oocytes cultured) was highest when oocytes were matured in medium containing higher FSH concentration and antioxidants. The results of this study demonstrate that meiotic and developmental competences are inherent to the immature cat oocyte collected during the non-breeding season. However, appropriate mechanisms (perhaps seasonal variation in FSH receptors or lack of antioxidant capacity of the cumulus-oocyte complex) are inadequate during this period of gonadal quiescence. Regardless, this compromised oocyte function during the non-breeding season can be overridden by altering in vitro culture conditions to include supplemental FSH and antioxidant.