254. Effect of donor age and follicle size on oocyte developmental competence in the pig

Oocytes utilised for in vitro embryo production (IVP) are typically derived from 3–8 mm ovarian follicles of slaughtered pre-pubertal pigs. Following in vitro maturation (IVM), pre-pubertal oocytes display lower developmental competence (DC) than adult oocytes. The aim of this study was to determine the proportion of follicles 3, 4, and 5–8 mm in diameter on the surface of pre-pubertal and adult ovaries, and assess DC of corresponding oocytes. Oocytes were matured for 46 h in modified medium 199. Mature oocytes from the three follicle size cohorts were activated with calcium ionophore to assess blastocyst embryo formation rate. Data were subjected to arcsine transformation, ANOVA and the Tukey post-hoc test. Compared with adult ovaries, pre-pubertal ovaries contained a higher proportion of 3 mm follicles (46 ± 4 v. 72 ± 4%, P<0.01), but a lower proportion of 4 mm (33 ± 3 v. 22 ± 3%, P<0.01) and 5–8 mm follicles (21 ± 5 v. 6 ± 2%, P<0.01). Adult oocytes from the three follicle sizes displayed similar DC (41±2% to 47±3%). DC of pre-pubertal oocytes improved with increasing follicle size (3 mm < 4 mm < 5–8 mm; 12±4%, 27±8% and 50±8%, respectively; P < 0.05). In conclusion, the predominance of 3 mm follicles accounts for the low DC of oocytes from pre-pubertal donors compared with adult donors. Further research is required to understand DC acquisition in pre-pubertal pig oocytes from the smaller follicles <5mm in diameter.

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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab205 ◽

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.

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Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

Veterinary World ◽

10.14202/vetworld.2021.3164-3169 ◽

2021 ◽

pp. 3164-3169

Author(s):

Mohamed M. M. El-Sokary ◽

Al-Shimaa Al-H. H. El-Naby ◽

Amal R. Abd El Hameed ◽

Karima Gh. M. Mahmoud ◽

T. H. Scholkamy

Keyword(s):

Embryo Culture ◽

In Vitro Maturation ◽

Culture Media ◽

Effective Concentration ◽

Developmental Competence ◽

Carnitine Supplementation ◽

Oocyte Developmental Competence ◽

High Lipid ◽

Significant Difference

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

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327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab327 ◽

2006 ◽

Vol 18 (2) ◽

pp. 271 ◽

Cited By ~ 1

Author(s):

T. S. Hussein ◽

R. B. Gilchrist ◽

J. G. Thompson

Keyword(s):

In Vitro Maturation ◽

Cumulus Cell ◽

Blastocyst Stage ◽

Developmental Competence ◽

Bovine Oocyte ◽

Paracrine Factors ◽

Cell Functions ◽

Oocyte Developmental Competence ◽

Secreted Factors

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

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Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture

Reproduction Fertility and Development ◽

10.1071/rd11118 ◽

2012 ◽

Vol 24 (5) ◽

pp. 656 ◽

Cited By ~ 28

Author(s):

Islam M. Saadeldin ◽

Ok Jae Koo ◽

Jung Taek Kang ◽

Dae Kee Kwon ◽

Sol Ji Park ◽

...

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Formation Rate ◽

Early Period ◽

Threshold Effect ◽

Developmental Competence ◽

Control Group ◽

Maturation Medium ◽

Paradoxical Effects

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus–oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10–6 M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4 × 10–6 M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine–paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

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Relationship between follicle size and oocyte developmental competence in prepubertal and adult pigs

Reproduction Fertility and Development ◽

10.1071/rd07018 ◽

2007 ◽

Vol 19 (7) ◽

pp. 797 ◽

Cited By ~ 69

Author(s):

Melanie A. Bagg ◽

Mark B. Nottle ◽

David T. Armstrong ◽

Christopher G. Grupen

Keyword(s):

In Vitro Maturation ◽

Fold Increase ◽

Cell Number ◽

Developmental Competence ◽

Blastocyst Development ◽

Parthenogenetic Activation ◽

Follicle Size ◽

Low Efficiency ◽

The Relationship

The present study compared the distribution and steroid composition of 3-, 4- and 5–8-mm follicles on the surface of prepubertal and adult ovaries, and determined the relationship between follicle size and developmental competence of oocytes following parthenogenetic activation. The effect of 1 mm dibutyryl cAMP (dbcAMP) for the first 22 h of in vitro maturation (IVM) on the embryo development of prepubertal oocytes from the three follicle size cohorts was also determined. Compared with adult, prepubertal ovaries contained a higher proportion of 3-mm follicles (46 v. 72%, respectively), but a lower proportion of 4-mm (33 v. 22%, respectively) and 5–8-mm follicles (21 v. 6%, respectively). Adult follicular fluid (FF) contained 11-fold higher levels of progesterone (P4) than prepubertal FF, with similar levels observed between all adult follicle sizes. In prepubertal FF, the P4 concentration increased with follicle size from 3 to 4 to 5–8 mm. Rates of blastocyst development following parthenogenetic activation of adult oocytes from all three follicles sizes were similar (approximately 55%), whereas rates from prepubertal oocytes increased with increasing follicle size from 3 (17%) to 4 (36%) to 5–8 mm (55%). Treatment with dbcAMP for the first 22 h of IVM led to a 1.5-fold increase in the rate of blastocyst development for prepubertal oocytes from 3-mm follicles, but had no effect on prepubertal oocytes from the 4 and 5–8 mm classes. Mean blastocyst cell number increased with follicle size in prepubertal ovaries and was similar for all follicle sizes in adult ovaries. The present study demonstrates that the low efficiency of in vitro embryo production observed using prepubertal compared with adult pig oocytes is due to a greater proportion of 3-mm follicles on prepubertal ovaries, which contain oocytes of inferior developmental competence.

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313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab313 ◽

2015 ◽

Vol 27 (1) ◽

pp. 245 ◽

Cited By ~ 1

Author(s):

N. W. Santiquet ◽

A. F. Greene ◽

W. B. Schoolcraft ◽

R. L. Krisher

Keyword(s):

Embryo Development ◽

In Vitro Maturation ◽

Subsequent Development ◽

Developmental Competence ◽

Nuclear Maturation ◽

Oocyte Developmental Competence ◽

Oocyte Collection ◽

Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

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Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows on oocyte developmental competence

Reproduction ◽

10.1530/rep-13-0383 ◽

2014 ◽

Vol 147 (1) ◽

pp. 33-43 ◽

Cited By ~ 25

Author(s):

S Asaf ◽

G Leitner ◽

O Furman ◽

Y Lavon ◽

D Kalo ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Transvaginal Ultrasound ◽

Formation Rate ◽

Developmental Competence ◽

Cortical Granule ◽

Disruptive Effect ◽

Cell Stage ◽

Oocyte Developmental Competence

Mastitis is associated with decreased fertility in dairy cows. In the current study, we created an experimental model to simulate short-term mastitis by a single intramammary administration of Gram-negative endotoxin ofEscherichia coliorigin (G−), or Gram-positive toxin ofStaphylococcus aureusorigin (G+), to examine the effect of mastitis on oocyte developmental competence. Healthy Holstein cows were synchronized, and follicular fluid (FF) of cows treated with G+ or G− and of uninfected cows (controls) was aspirated from the preovulatory follicles by transvaginal ultrasound procedure. The aspirated FF was used as maturation medium forin vitroembryo production. The distribution of matured oocytes into different cortical granule classes and meiotic stages was affected by G− administration (P<0.05) but not by G+ administration. The proportion of oocytes that cleaved to two- and four-cell stage embryos (44 h postfertilization) was lower in both G+ and G− groups than in controls (P<0.05). Blastocyst formation rate (7–8 days postfertilization) was lower in the G− group (P<0.05) and numerically lower in the G+ group compared with their uninfected counterparts. The total cell number in blastocysts did not differ among groups; however, the apoptotic index was higher in the G+ group (P<0.05), but not in the G− group, relative to controls. Examining mRNA relative abundance in oocytes and early embryos revealed mastitis-induced alterations inPTGS2(COX2),POU5F1, andHSF1but not inSLC2A1(GLUT1) orGDF9. Results indicate a differential disruptive effect of mastitis induced by G− and G+ on oocyte developmental competence in association with alterations in maternal gene expression.

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