Age-associated deterioration in follicular fluid induces a decline in bovine oocyte quality

2017 ◽  
Vol 29 (4) ◽  
pp. 759 ◽  
Author(s):  
Shun Takeo ◽  
Koji Kimura ◽  
Koumei Shirasuna ◽  
Takehito Kuwayama ◽  
Hisataka Iwata
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Glycation End Products ◽  
Maternal Aging ◽  
Blastulation Rate

Maternal age affects the quality of oocytes. The present study examined whether follicular fluid (FF) is a casual factor for age-associated decline in oocyte quality. First, we measured the concentration of advanced glycation end-products (AGE) in FF derived from young (21–45 months; Young-FF) and aged (≥120 months; Aged-FF) cows and found significantly higher concentrations of AGE in Aged-FF than Young-FF. Second, oocytes were collected from ovaries of young or aged cows and cultured in maturation medium containing 10% FF derived from young or aged cows. Regardless of oocyte origin, Aged-FF accelerated nuclear maturation progression and gap junction closure between oocytes and cumulus cells, increased reactive oxygen species (ROS) content and the rate of abnormal fertilisation of oocytes and decreased blastulation rate compared with Young-FF. Furthermore, supplementation of maturation medium with AGE induced similar age-associated events in oocytes derived from young cows, in that AGE accelerated the progression of nuclear maturation, increased ROS content in oocytes, increased the rate of abnormal fertilisation and decreased blastulation rate. In conclusion, maternal aging increased the concentration of AGE in FF, and both AGE and Aged-FF accelerated nuclear maturation and reduced the developmental competence of oocytes.

206 EFFECT OF ADDITION OF FOLLICULAR FLUID OR GROWTH DIFFERENTIATION FACTOR-9 ON IN VITRO MATURATION OF PORCINE OOCYTES DENUDED 20h AFTER THE START OF IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 234
Author(s):  
P. Ferré ◽  
T. T. M. Bui ◽  
M. T. Tran ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Dapi Staining ◽  
Experimental Conditions ◽  
Nuclear Maturation ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9

The interruption of communication between oocyte and cumulus cells (CC) can trigger meiotic resumption and exogenous additives, such as follicular fluid (FF) and growth differentiation factor-9 (GDF9), can improve oocyte quality and the developmental competence. This study was undertaken to examine if the absence and presence of FF from medium follicles (MF; 3–6 mm in diameter) or recombinant human GDF9 (Biovision, Milpitas, CA, USA) during the first or/and second half of in vitro maturation (IVM) had any effects on IVM of oocytes from small follicles (SF; 0.5–2 mm in diameter) or MF when the oocytes were denuded at 20 h after the start of IVM. Cumulus-oocyte complexes (COC) were aspirated from SF or MF of slaughtered prepubertal gilt ovaries. Groups of ~30 COC were cultured in a 300-μL drop of porcine oocyte medium containing 50 µM β-mercaptoethanol (mPOM) with or without 10% (v/v) FF and/or 100 ng mL–1 GDF9 at 39°C and 5% CO2 in air. During the first 20 h after the start of IVM, the medium was supplemented with 1 mM dibutyryl c-AMP, 10 IU mL–1 eCG and 10 IU mL–1 hCG. After the first period of IVM, the CC surrounding the oocytes were removed and the denuded oocytes continued culture for IVM with or without FF or/and GDF9 in the absence of dibutyryl c-AMP and gonadotropins in the same medium for another 24 h. At the end of IVM, meiotic progression of the oocytes was examined by DAPI staining. Statistical analyses from at least 4 replicates data were performed by a 2-way ANOVA and a Tukey’s multiple comparisons test. Removal of CC 20 h after the start of IVM significantly improved the incidence of mature oocytes derived from SF (59.2–64.1% v. 41.6–43.1% in controls, P < 0.05) but not from MF (73.1–78.5% v. 70.6–71.8% in controls), whereas regardless of supplementation with FF or GDF9, the maturation rates were always significantly higher in the denuded oocytes from MF (72.4–83.6%) than SF (57.8–66.2%; P < 0.05). Despite of the origin of COC (SF or MF), maturation rates of oocytes denuded 20 h after the start of IVM were not affected by supplementation with FF or GDF9 during the first and/or second half of IVM (P > 0.05). In summary, CC removal from COC 20 h after the start of IVM promotes nuclear maturation of oocytes from SF. Exogenous additives such as GDF9 and follicular fluid from MF do not seem to affect the promotion of nuclear maturation in our experimental conditions.

Time course of the meiotic arrest in sheep cumulus–oocyte complexes treated with roscovitine

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 310-318 ◽  
Author(s):  
Letícia Ferrari Crocomo ◽  
Wolff Camargo Marques Filho ◽  
Camila Louise Ackermann ◽  
Daniela Martins Paschoal ◽  
Midyan Daroz Guastali ◽  
...  
Keyword(s):  
Exposure Time ◽  
Cell Expansion ◽  
Time Course ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Maturation Medium

SummaryTemporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus–oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

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

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 159-164 ◽  
Author(s):  
Hisataka Iwata ◽  
Shu Hashimoto ◽  
Mayuko Ohota ◽  
Koji Kimura ◽  
Kenichi Shibano ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Rate Of Development ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Follicle Size ◽  
The Difference ◽  
Oviductal Fluid ◽  
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.

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 249-259 ◽  
Author(s):  
Hisashi Nabenishi ◽  
Hiroshi Ohta ◽  
Toshihumi Nishimoto ◽  
Tetsuo Morita ◽  
Koji Ashizawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Bovine Oocytes

SummaryIn the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

Cerium oxide nanoparticles (CeO2 NPs) improve the developmental competence of in vitro-matured prepubertal ovine oocytes

2017 ◽  
Vol 29 (5) ◽  
pp. 1046 ◽  
Author(s):  
F. Ariu ◽  
L. Bogliolo ◽  
A. Pinna ◽  
L. Malfatti ◽  
P. Innocenzi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Group A ◽  
Chromatin Configuration ◽  
Development In Vitro

The present study investigated whether supplementation with different doses of cerium dioxide nanoparticles (CeO2 NPs) during in vitro maturation (IVM) of prepubertal ovine oocytes influenced their embryonic development in vitro. Cumulus–oocyte complexes derived from the ovaries of slaughtered prepubertal sheep underwent IVM with CeO2NPs (0, 44, 88 or 220 µg mL–1). Matured oocytes were fertilised in vitro and zygotes were cultured for 7 days. The results demonstrated that CeO2NPs were internalised in the cumulus cells and not in the oocyte. The treatment with CeO2NPs did not affect nuclear maturation or intracellular levels of reactive oxygen species of the oocytes. The percentage of oocytes with regular chromatin configuration and cytoskeleton structures when treated with 44 µg mL–1 CeO2NPs was similar to oocytes matured in the absence of CeO2NPs and significantly higher than those treated with 88 or 220 µg mL–1 CeO2NPs. The relative quantification of transcripts in the cumulus cells of oocytes matured with 44 µg mL–1 CeO2NPs showed a statistically lower mRNA abundance of BCL2-associated X protein (BAX), B-cell CLL/lymphoma 2 (BCL2) and superoxide dismutase 1 (SOD1) compared with the 0 µg mL–1 CeO2 NPs group. A concentration of 44 µg mL–1 CeO2NPs significantly increased the blastocyst yield and their total, inner cell mass and trophectoderm cell numbers, compared with the 0 and 220 µg mL–1 groups. A low concentration of CeO2NPs in the maturation medium enhanced in vitro embryo production of prepubertal ovine oocytes.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p &gt; 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

scholarly journals Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts fromIn VitroMaturation of Bovine Oocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jolanta Opiela ◽  
Joanna Romanek ◽  
Daniel Lipiński ◽  
Zdzisław Smorąg
Keyword(s):  
Dna Fragmentation ◽  
Oocyte Maturation ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
The Mean ◽  
Bovine Oocytes

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were maturedin vitroin control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higherBaxmRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

Improvement of the developmental competence of canine oocyte using caffeine supplementation during IVM at different maturation time

Zygote ◽  
2018 ◽  
Vol 26 (2) ◽  
pp. 162-167 ◽  
Author(s):  
Mohamed Fathi ◽  
A. Salama ◽  
Magdy R. Badr
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Free Medium ◽  
Maturation Time ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Oviductal Fluid

SummaryThe aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher (P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates (P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant (P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

172 DETERMINING THE REQUIREMENTS FOR LH AND FSH DURING SHEEP IN VITRO OOCYTE MATURATION

2014 ◽  
Vol 26 (1) ◽  
pp. 200 ◽  
Author(s):  
C. de Frutos ◽  
R. Vicente-Perez ◽  
P. J. Ross
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Mixed Logit ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Grade 3

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

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