The Effect of Lysophosphatidic Acid duringIn VitroMaturation of Bovine Oocytes: Embryonic Development and mRNA Abundances of Genes Involved in Apoptosis and Oocyte Competence

In the present study we examined whether LPA can be synthesized and act duringin vitromaturation of bovine cumulus oocyte complexes (COCs). We found transcription of genes coding for enzymes of LPA synthesis pathway (ATXandPLA2) and of LPA receptors (LPAR 1–4) in bovine oocytes and cumulus cells, followingin vitromaturation. COCs were maturedin vitroin presence or absence of LPA (10−5 M) for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance ofFSTandGDF9in oocytes and decreased mRNA abundance ofCTSsin cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels ofBCL2and lower transcription levels ofBAXresulting in the significantly lowerBAX/BCL2ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhancedin vitrodevelopment until the blastocyst stage, improved oocyte competence may be relevant for subsequentin vivosurvival.

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Astaxanthin counteracts the effects of heat shock on the maturation of bovine oocytes

Reproduction Fertility and Development ◽

10.1071/rd17271 ◽

2018 ◽

Vol 30 (9) ◽

pp. 1169 ◽

Cited By ~ 7

Author(s):

J. Ispada ◽

T. A. Rodrigues ◽

P. H. B. Risolia ◽

R. S. Lima ◽

D. R. Gonçalves ◽

...

Keyword(s):

Heat Shock ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Negative Effects ◽

Cellular Mechanisms ◽

Sod Activity ◽

Oocyte Competence ◽

Maturation Medium ◽

Bovine Oocytes

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14 h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25 nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

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Relationship between apoptosis and survival molecules in human cumulus cells as markers of oocyte competence

Zygote ◽

10.1017/s0967199417000429 ◽

2017 ◽

Vol 25 (5) ◽

pp. 583-591 ◽

Cited By ~ 4

Author(s):

Liana Bosco ◽

Roberto Chiarelli ◽

Maria Carmela Roccheri ◽

Domenica Matranga ◽

Giovanni Ruvolo

Keyword(s):

Cumulus Cell ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Terminal Deoxynucleotidyl Transferase ◽

Oocyte Competence ◽

Fragmentation Index ◽

Valuable Marker ◽

First Time

SummaryTo select from a single patient the best oocytes able to reach the blastocyst stage, we searched for valuable markers for oocytes competence. We evaluated the DNA fragmentation index (DFI) and the level of some survival molecules, such as AKT, pAKT and pERK1/2, in individual cumulus cell–oocyte complexes (COC). The study included normo-responder women. The average age of the patients was 34.3. DFI in cumulus cells was evaluated using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay in situ. AKT, pAKT and pERK1/2 were measured by immunological assay and densitometric analysis of fluorescent signals using NIS-Elements BR 3.10 image software. Statistical analysis was performed using STATA SE/14.1. The study focused on 53 patients involved after informed consent. Out of 255 MII oocytes, 197 were fertilized and the derived embryos had the following evolution: 117 completed the development to blastocyst and were transferred to uterus; 57 were vitrified at the blastocyst stage; and 23 were arrested during in vitro culture at different stages of cleavage. We found a significant statistical difference between the DFI of cumulus cells of the arrested embryos and the transferred blastocysts (P = 0.004), confirming that DFI could be considered as a valuable marker of oocyte competence. In addition, the pAKT/DFI ratio was higher in cumulus cells of oocytes able to produce blastocysts, indicating that DFI is significantly lower when pAKT is higher (P = 0.043). This study demonstrates for the first time that the relationship between apoptosis and survival molecules can be used as a marker to select the best oocytes.

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PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

Journal of Animal Science ◽

10.1093/jas/skab235.602 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 327-328

Author(s):

Galina Singina

Keyword(s):

Growth Factor ◽

Cell Number ◽

Blastocyst Stage ◽

Total Cell ◽

Control Group ◽

Total Cell Number ◽

Developmental Potential ◽

Maturation Medium ◽

Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P<0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P<0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

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334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab334 ◽

2006 ◽

Vol 18 (2) ◽

pp. 275

Author(s):

H. S. Lee ◽

Y. I. Seo ◽

X. J. Yin ◽

S. G. Cho ◽

I. H. Bae ◽

...

Keyword(s):

In Vitro Maturation ◽

Uv Light ◽

Cumulus Cells ◽

Oocyte Activation ◽

Cell Stage ◽

Oocyte Competence ◽

Treatment Groups ◽

Maturation Medium ◽

Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

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165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab165 ◽

2014 ◽

Vol 26 (1) ◽

pp. 196

Author(s):

K. R. L. Schwarz ◽

R. C. Botigelli ◽

F. C. Castro ◽

M. R. Chiaratti ◽

C. L. V. Leal

Keyword(s):

Lipid Content ◽

Fetal Calf Serum ◽

Calf Serum ◽

Cumulus Cells ◽

Control Group ◽

Maturation Medium ◽

Cgmp Pathway ◽

Bovine Oocytes ◽

Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

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Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts

Reproduction ◽

10.1530/rep-09-0509 ◽

2010 ◽

Vol 139 (2) ◽

pp. 331-335 ◽

Cited By ~ 52

Author(s):

Michiko Nakai ◽

Hiroyuki Kaneko ◽

Tamas Somfai ◽

Naoki Maedomari ◽

Manabu Ozawa ◽

...

Keyword(s):

Testicular Tissue ◽

Blastocyst Stage ◽

Large Mammals ◽

Immunodeficient Mice ◽

Neonatal Piglets ◽

Porcine Oocyte ◽

Large Animals ◽

First Time

Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.

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Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽

10.1530/rep.0.1230455 ◽

2002 ◽

pp. 455-465 ◽

Cited By ~ 74

Author(s):

YH Choi ◽

CC Love ◽

LB Love ◽

DD Varner ◽

S Brinsko ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Polar Body ◽

Cumulus Cells ◽

Fertilization Rate ◽

Developmental Competence ◽

Cleavage Rate ◽

First Polar Body ◽

Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

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148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab148 ◽

2007 ◽

Vol 19 (1) ◽

pp. 191

Author(s):

K. B. Lee ◽

A. Bettegowda ◽

J. J. Ireland ◽

G. W. Smith

Keyword(s):

Blastocyst Stage ◽

Total Cell ◽

Mrna Abundance ◽

Differential Staining ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cell Stage ◽

Oocyte Competence ◽

Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P < 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P < 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

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163IMPROVEMENT OF IN VITRO PRODUCTION OF PIG EMBRYOS BY SYNCHRONISATION OF OOCYTE MEIOTIC MATURATION WITH CYCLOHEXIMIDE

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab163 ◽

2004 ◽

Vol 16 (2) ◽

pp. 204 ◽

Cited By ~ 1

Author(s):

J. Ye ◽

K.H.S. Campbell ◽

M.R. Luck

Keyword(s):

Cumulus Cells ◽

Treated Group ◽

Blastocyst Stage ◽

Developmental Competence ◽

Meiotic Maturation ◽

In Vitro Production ◽

Maturation Medium ◽

Pre Treatment ◽

Vitro Production

It is suggested that the relatively high rates of polyspermic fertilization and poor development of pig embryos produced in vitro are caused by asynchronous oocyte maturation. We have recently shown that pre-treatment of pig oocytes with cycloheximide (CHX) is an efficient way of synchronizing their meiotic maturation in vitro. However, it is not known whether this procedure affects fertilization or further development. The present study examined the effects of CHX-synchronised meiotic maturation on subsequent embryo development and the response to FSH. Pig ovaries were collected from a local abattoir. Cumulus-oocyte complexes (COCs) were aspirated from 3–5mm diameter follicles with a translucent appearance and extensive vascularization. COCs were first pre-incubated in defined maturation medium (DM; M199 with Earle’s salts, 25mM HEPES and sodium bicarbonate, 3mM L-glutamine, 0.1% (w/v) BSA, 0.57mM cysteine, 10ngmL−1 EGF, 0.2μgmL−1 pLH, 100μmL−1 penicillin and 0.1mgmL−1 streptomycin) or in DM supplemented with 50ngmL−1 pFSH (DMF) and 5μgmL−1 CHX for 12h. COCs were then further cultured in the same DM without CHX for 24–30h or in DMF for 36h. For controls, COCs were cultured conventionally in DM for 42h or DMF for 48h. After removal of cumulus cells, all cultured oocytes were inseminated with ejaculated sperm at a final concentration of 300000mL−1 for 6h. The IVF medium was modified Tris-buffered medium containing 0.1% BSA, 20μM adenosine and 0.2mM reduced glutathione. Putative embryos were cultured in NCSU23 without glucose but supplemented with 4.5mM Na lactate and 0.33 mM Na pyruvate for 2 days. Cleaved embryos were further cultured in normal NCSU23 for 4 days. IVM and IVF were performed in 5% CO2 in air and IVC in 5% CO2, 5% O2, 90% N2, all at 39°C and 95% RH. Three replicates with DM, with or without CHX, and one with DMF, with or without CHX, were performed with 30–50 oocytes in each replicate. Statistical comparisons were by t-test. The result with DM showed that the rate for normal cleavage at 2 days after insemination of CHX-treated oocytes (40.6±3.8%) was similar to that of controls (40.4±3.5%). However, the proportion developing to healthy blastocysts at Day 6 was significantly higher in the CHX-treated group (16.9±1.2%) than in controls (9.6±1.3%; P<0.05). A significantly higher number of Day 2-cleaved embryos from CHX-treated oocytes developed to the day 6 blastocyst stage compared with controls (44.7±5.0% and 22.3±2.4%, respectively; P<0.05). Supplementation of the basic maturation medium with pFSH increased the rate of cleavage in both CHX-treated oocytes (73.2%) and controls (76.9%) and increased the proportions developing to healthy blastocysts at Day 6 (CHX-treated: 39.0%; control: 11.5%). We conclude that oocytes pre-treated with CHX retain their developmental competence and that meiotic synchronization with CHX improves the efficiency of in vitro production of pig embryos. (Supported by BBSRC 42/S18810.)

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182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab182 ◽

2017 ◽

Vol 29 (1) ◽

pp. 199

Author(s):

A. W. Harl ◽

E. L. Larimore ◽

A. Al Naib ◽

L. K. Wooldridge ◽

A. D. Ealy ◽

...

Keyword(s):

Cell Expansion ◽

Cumulus Cell ◽

Prostaglandin F2α ◽

Blastocyst Stage ◽

Control Group ◽

Blastocyst Development ◽

Cumulus Expansion ◽

Oocyte Competence ◽

Maturation Medium

The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10 mm) and small follicles (≤3 mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n = 250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21 h of maturation). Other COC (n = 770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P < 0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P < 0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P < 0.05) and also less likely to develop to the blastocyst stage (P < 0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42 h after prostaglandin F2α injection. Samples from individual cows were categorized as having high oestradiol (>800,000 pg mL−1; H) or low oestradiol concentrations (<800,000 pg mL−1; L). The FF was retained for use in in vitro experiments, where it was added to maturation media (20% vol/vol). Cumulus-oocyte complexes (n = 1,775) were randomly distributed into treatments across 12 in vitro maturation/fertilization replicates (H and L, balanced within replicate; 4 replicates/cow). Each replicate included the following 3 control groups: maturation medium containing BSA without FF, maturation medium without BSA with abattoir-derived FF, and maturation medium without BSA and without FF. The COC were matured in their assigned medium for 21 h, and then all COC were subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 7 and 8 post-fertilization. Oestradiol content of the FF (H v. L) did not affect oocyte cleavage nor blastocyst rates on Day 7 or 8. The results of these studies indicate that although FF improves cumulus cell expansion during maturation in vitro, it does not result in higher rates of cleavage or blastocyst development regardless of oestradiol content.

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