Effect of plasma progesterone on oocyte recovery, oocyte quality, and early in-vitro developmental competence of embryos in Bos indicus dairy cows

The effects of heat stress on Bos taurus reproductive performance in tropical and subtropical regions are well known, and have been associated with lower oocyte developmental capacity. The aim of this study was to evaluate the developmental competence of oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) dairy cows raised in a Brazilian tropical region, located at 21°35′S latitude, 43°51′W longitude, and 435 meters altitude. Cumulus–oocyte complexes (COCs) were recovered by oocyte pickup (OPU) from mature non-lactating Holstein (n = 9) and Gyr (n = 13) donor cows between the end of spring and the beginning of autumn, with at least two OPU sessions/cow. COCs were in vitro-maturated in TCM-199 (GIBCO, Grand Island, NY, USA) with 10% inactivated estrus cow serum for 24 h under 5% CO2 at 38.5°C in air. Bos taurus and Bos indicus semen with similar cleavage rates, previously evaluated by in vitro fertilization with oocytes obtained from slaughterhouse ovaries, were used to reduce bull effect. Holstein and Gyr spermatozoa were obtained through swim-up method and co-incubated with Holstein (n = 390) and Gyr (n = 505) oocytes, respectively, in Fert-TALP medium (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180) supplemented with 10 μg/mL heparin (Sigma-Aldrich, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson et al. 1996 Theriogenology 45, 41–49) supplemented with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. On Day 7 to 8 of co-culture, Gyr and Holstein blastocysts were assessed and those classified as grade 1 (IETS Manual) were transferred to synchronized Bos indicus × Bos taurus crossbred recipients managed under the same nutritional and environmental conditions. Pregnancy diagnosis was performed between 35 and 50 days after estrus. Cleavage, blastocyst, and pregnancy rates were analyzed by chi-square test. Cleavage and blastocyst rates were greater (P < 0.05) in Gyr than in Holstein (66.7% vs. 53.1% for cleavage and 19.6% vs. 10.8% for blastocyst, respectively), but the pregnancy rate was similar (P > 0.05; 44.5% vs. 60% for Gyr and Holstein, respectively). These results show that Gyr oocytes obtained in a tropical region have greater developmental capacity than Holstein oocytes, suggesting an interaction between genotype and environment that influences the success of an in vitro embryo production program; nevertheless, the blastocyst viability after transferring to recipients is similar for both breeds.

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193 Effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab193 ◽

2020 ◽

Vol 32 (2) ◽

pp. 225

Author(s):

M. Saleem ◽

Z. Sarwar ◽

M. Saad ◽

I. Zahoor ◽

N. Ahmad ◽

...

Keyword(s):

Dairy Cattle ◽

Recovery Rate ◽

Body Condition Score ◽

Bos Indicus ◽

Oocyte Quality ◽

Developmental Competence ◽

Cleavage Rate ◽

Healthy Group ◽

Oocyte Recovery ◽

Frozen Semen

Unhygienic practices at the time of parturition or AI lead to uterine infections. The uterine infections ultimately result in genetic drain by culling the elite animals. The invivo developmental competence of embryos is compromised in clinically metritic animals. The genetic potential of problematic females could be harvested by invitro embryo production (IVEP). Therefore, the objective of the present study was to evaluate the effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle. This experiment was carried out from December 2017 to April 2018. Ovaries were collected from a local abattoir (Bos indicus; 5- to 8-year-old dairy cattle, body condition score 2.75±0.25, mixed parity). These ovaries (n=982) were divided into two groups: (1) clinically metritic (n=184), and (2) healthy (n=798), based upon the presence or absence of pus in the uterine lumen. Oocytes were aspirated from follicles using an 18G needle attached to a 10-mL syringe. Cumulus-oocyte complexes (COCs) were categorized into A, B, C, and D grades based on the number of layers of cumulus cells and integrity of ooplasm. The oocytes of grades A and B were subsequently transferred in groups (10/group) in four-well plates containing 100-μL droplets. The droplets with oocytes were covered with prewarmed mineral oil and incubated for 24h at 38.5°C, 5% CO2, and 95% relative humidity. The oocytes were evaluated for IVM on the basis of cumulus expansion. Frozen semen was thawed and prepared using the sperm swim-up procedure for each group. Spermatozoa and oocytes were incubated together for a period of 18h. The presumptive zygotes were invitro cultured for 4 days in a CO2 incubator under similar culture conditions. The cleavage rate, 4-cell, and 8-cell stages were recorded on Days 2, 3, and 4 after the day of insemination, respectively. Data on oocyte recovery, oocyte quality, IVM, cleavage rate, and 4-cell and 8-cell stages were analysed by Chi-squared test using SPSS software (version 20; IBM Corp.) for Windows. Results demonstrated that recovery rate was lower (63.8% vs. 71.7%; P<0.05) in clinically metritic compared with healthy cattle. Similarly, oocytes of grade A and B quality were lower (41.0% vs. 51.1%; P<0.05), whereas those of C and D quality were higher (59.0% vs. 48.9%; P<0.05) in clinically metritic compared with the healthy group. Moreover, 4-cell (38.2% vs. 54.8%) and 8-cell stage embryos (11.3% vs. 29.1%), were lower (P<0.05) in the clinically metritic compared with the healthy group, respectively. However, maturation rate and cleavage rate did not differ (P>0.05) between groups. In conclusion, metritis in slaughterhouse ovaries negatively affects oocyte recovery rate, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle.

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143 Effect of corpus luteum on recovery rate, quality, and in vitro developmental competence of oocytes in Bos indicus dairy cows

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab143 ◽

2019 ◽

Vol 31 (1) ◽

pp. 196

Author(s):

M. Saad ◽

Z. Sarwar ◽

M. Saleem ◽

U. Arshad ◽

M. Shahzad ◽

...

Keyword(s):

Dairy Cows ◽

Corpus Luteum ◽

Recovery Rate ◽

Bos Indicus ◽

Developmental Competence ◽

Serum Concentrations ◽

Embryo Production ◽

The Mean ◽

In Vitro Embryo Production

Assisted reproductive technologies have modernized the dairy industry for the rapid multiplication of superior genetic traits. However, the exploitation of genetics through in vitro embryo production in farm animals has been struggling during the last 3 decades. The presence or absence of a corpus luteum (CL) on ovaries from which cumulus-oocyte complexes were recovered has been shown to affect the quality of oocytes and in vitro embryo production outcome in Bos taurus cows. Similar information is lacking in Bos indicus cows. Therefore, the objectives of the present study were to determine the effect of presence or absence of a CL on recovery rate, quality, and in vitro developmental competence of oocytes in Bos indicus dairy cows. This study was conducted from December 2017 to April 2018. The ovaries were harvested from a local abattoir (Bos indicus; 5- to 8-year-old cows having mixed parity with clinically normal reproductive tracts). These ovaries (n=750) were divided into 2 groups [(1) CYCLIC (n=318) and (2) ACYCLIC (n=432)] based on the presence or absence of mature CL having follicles on either left or right ovaries of slaughtered cows. Blood samples from the jugular vein were collected at slaughter. Serum concentrations of progesterone of CYCLIC and ACYCLIC cows were measured using radioimmunoassay. Mean number of recovered oocytes per ovary and serum concentration of progesterone were compared using PROC TTEST model. However, quality, maturation, cleavage, 8-cell, 16-cell, and morula rates were analysed by PROC FREQ model of Chi-squared. All the data was analysed using SAS (SAS Institute Inc., Cary, NC, USA). Results revealed that the mean serum concentrations of progesterone (4.21±0.4v. 0.5±0.2ng mL−1; P<0.05) were higher in CYCLIC as compared with ACYCLIC cows, respectively. The mean number of oocytes recovered per ovary (6.5±4.5v. 4.0±4.4; P<0.05) was higher in CYCLIC than in ACYCLIC cows. The oocytes with grade I+II quality (55.3v. 47.6%; P<0.05) were higher, whereas oocytes with grade III+IV quality (44.5v. 52.4%; P<0.05) were lower, in CYCLIC as compared with ACYCLIC cows, respectively. Furthermore, cleavage rate (70.9v. 52.8%; P<0.05) was higher in CYCLIC than in ACYCLIC cows, respectively. Similarly, the 8-cell (38.5v. 20.8%; P<0.05) and 16-cell (20.0v. 10.9%; P<0.05) stage embryos were greater in CYCLIC as compared with ACYCLIC cows, respectively. In conclusion, the presence of CL has a beneficial effect on recovery rate, quality, and in vitro embryo production outcomes in Bos indicus dairy cows. It is implied that for ovum pickup, cyclic cows should be preferred.

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Intrafollicular conditions as a major link between maternal metabolism and oocyte quality: a focus on dairy cow fertility

Reproduction Fertility and Development ◽

10.1071/rd11901 ◽

2012 ◽

Vol 24 (1) ◽

pp. 1 ◽

Cited By ~ 54

Author(s):

J. L. M. R. Leroy ◽

D. Rizos ◽

R. Sturmey ◽

P. Bossaert ◽

A. Gutierrez-Adan ◽

...

Keyword(s):

Dairy Cows ◽

Embryo Quality ◽

Ovarian Follicle ◽

Oocyte Quality ◽

Developmental Competence ◽

Metabolic Disturbances ◽

Oocyte Growth ◽

Adverse Conditions ◽

Maternal Metabolism

Reduced oocyte and embryo quality are recognised as major factors in the problem of disappointing fertility in high producing dairy cows. This review aims to shed more light on the importance of the intrafollicular environment in the subfertility problem in dairy cows. Metabolic disturbances associated with negative energy balance (NEB) early postpartum are associated with ovarian dysfunction. Changes in the growth pattern of the ovarian follicle during a period of NEB can indirectly affect oocyte quality. Furthermore, a maternal metabolic disorder (linked with NEB or nutritionally induced) may alter the endocrine and biochemical composition of the follicular fluid, the micro-environment of the growing and maturing female gamete. The maturing oocyte is very sensitive to any perturbation in its direct environment and in vitro maturation models revealed that some of these metabolic changes reduce the oocyte’s developmental competence. Also, embryo quality is significantly reduced due to maturation in adverse conditions. Well balanced and timed oocyte metabolism and gene expression are crucial to safeguard an optimal oocyte development. In that perspective, metabolome and transcriptome parameters of the oocyte may serve to predict reproductive success rates. Finally, there is growing evidence that adverse conditions for oocyte growth and maturation may also jeopardise the health and performance of the offspring.

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

Human Reproduction ◽

10.1093/humrep/deab130.218 ◽

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 > 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

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238 CAFFEINE PREVENTS A LOSS OF CALCIUM OSCILLATORY RESPONSE ASSOCIATED WITH POSTOVULATORY AGING OF MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab238 ◽

2009 ◽

Vol 21 (1) ◽

pp. 217

Author(s):

T. Wakai ◽

N. Zhang ◽

R. A. Fissore

Keyword(s):

Subsequent Development ◽

Oocyte Quality ◽

Developmental Competence ◽

Oscillatory Activity ◽

Strontium Chloride ◽

Oocyte Activation ◽

Mouse Oocytes ◽

Oocyte Aging ◽

Postovulatory Aging

Numerous studies have demonstrated that postovulatory aging of oocytes prior to fertilization has detrimental effects on oocyte quality and developmental competence. Oocyte aging is accompanied by abnormal oocyte activation and subsequent development, suggesting a disruption of Ca2+ oscillations after fertilization. The inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in mammals is responsible for the majority of Ca2+ release during fertilization (Miyazaki S et al. 1993 Dev. Biol.). Previously, we reported that phosphorylation of IP3R1 at an MPM-2 epitope may play an important role in facilitating the induction of Ca2+ oscillations at the MII stage (Lee B et al. 2006 Development), indicating that IP3R1 phosphorylation may be a good indicator of the health of the oocyte. However, few studies have investigated the alteration of the Ca2+ signaling and IP3R1 function associated with oocyte aging. On the other hand, a previous report showed that caffeine increased MPF activity and suppressed fragmentation after parthenogenetic activation of aged oocytes (Kikuchi K et al. 2000 Biol. Reprod.). Therefore, the purpose of the present study was to examine whether and how Ca2+ oscillatory activity changes during oocyte aging and to test if caffeine prevents the negative effects of oocyte aging. MII mouse oocytes were collected 14 h after hCG injection and cultured in vitro for 8, 24 or 48 h with or without caffeine (5 or 10 mm). Oocyte quality was assessed by the occurrence of spontaneous fragmentation, monitoring of Ca2+ oscillations after exposure to 10 mm strontium chloride, Western blot analysis of IP3R1 phosphorylation and immunostaining of IP3R1. In oocytes in vitro aged for 8 h, the duration of the first Ca2+ rise was significantly decreased compared with fresh MII oocytes, although this reduction was not observed in MII oocytes treated with 5 mm caffeine. The phosphorylation of IP3R1 at the MPM-2 epitope was slightly decreased during oocyte aging in both caffeine and noncaffeine treatment. Importantly, whereas IP3R1 in MII oocytes treated for 8 h with 5 mm caffeine displayed the typical cortical cluster organization, IP3R1 in aged oocytes without caffeine became dispersed in the cytoplasm. In addition, caffeine significantly suppressed the spontaneous fragmentation that is normally observed by 48 h of in vitro culture. These results suggest that the Ca2+ oscillatory activity is compromised during oocyte aging and caffeine prevents the loss of integrity of Ca2+ signaling possibly by keeping the cortical distribution of IP3R1.

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169 THE RELATIONSHIP BETWEEN OOCYTE RECOVERY AND EMBRYO PRODUCTION IN BOS INDICUS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab169 ◽

2012 ◽

Vol 24 (1) ◽

pp. 196

Author(s):

M. P. Palhão ◽

E. R. Oliveira ◽

M. M. Gioso ◽

B. C. Carvalho ◽

L. G. B. Siqueira ◽

...

Keyword(s):

Body Condition Score ◽

Bos Indicus ◽

Pregnancy Rates ◽

Embryo Production ◽

Exact Test ◽

Oocyte Recovery ◽

Conversion Rates ◽

Condition Score ◽

The Relationship

The ovarian follicular population has been used as a parameter to evaluate fertility and also the potential of donors undergoing assisted reproductive procedures in both human medicine and animal practice. There is a high correlation between follicular population and oocyte recovery by ovum pickup (OPU), but the relationship between oocyte recovery, embryo production and pregnancy rates may not be fully understood. The aim of the present study was to evaluate whether the conversion rate of oocytes to embryos and further pregnancies could be positively related to the number of cumulus–oocyte complexes (COC) recovered after OPU in cattle. For this purpose, records of 626 OPU sections from 251 nonlactating Gyr cows (dairy Zebu breed) were analysed. The animals had a good body condition score, were kept in a good feeding pasture (Brachiaria spp.) and were supplemented with corn silage and a mixture of corn, soybeans and vitamin and minerals, according to their nutritional requirements. For each ovarian aspiration, the ovarian follicular wave was previously synchronized with an auricular implant (Norgestomet-Crestar®), IM injections of 2 mg of oestradiol benzoate (Gonadiol®) and 0.25 mg of D-cloprostenol (Sincrocio®). The OPU procedures were performed using an ultrasound device (Aquila Vet, Esaote, São Paulo, Brazil) equipped with a vaginal sector 7.5-MHz probe, disposable 20 G needles and a vacuum pressure of 80 mmHg. The cows were ranked in quartiles regarding the total number of COC recovered. To reduce bias related to the eventual fluctuation of OPU results, for the present analysis the authors used only the recorded OPU session of each cow with the highest number of COC recovered. Viable COC were fertilized with sex-sorted (X) semen of Gyr bulls previously tested for in vitro embryo production. Conversion rates (%) of the total and viable oocytes to embryos, viable oocytes to pregnancy and embryo to pregnancy were evaluated for each quartile. Differences between the first and fourth quartiles were accessed by Fisher's exact test. In the 251 OPU, 4246 total and 3173 viable COC were recovered, resulting in the production of 1001 embryos (31.5%) and 453 pregnancies (45.3%). The cows ranked in the first, second, third and fourth quartiles produced >30 (41.6 ± 10.6), 21 to 30 (25.2 ± 3.0), 12 to 20 (15.9 ± 2.6) and <12 (6.7 ± 3.1) total oocytes. The average viable oocyte (29.1 ± 11.0, 18.1 ± 5.3, 11.1 ± 3.7 and 4.5 ± 2.7, respectively) and embryo production (8.6 ± 5.7, 5.2 ± 3.6, 3.8 ± 2.8 and 1.8 ± 1.8, respectively) were different (P < 0.0001) among all quartiles. Pregnancy rates, however, did not differ (46.0, 44.9, 43.9 and 45.6%, respectively; P > 0.05). Interestingly, the conversion rates (viable oocytes to embryos and viable oocytes to pregnancies) were higher (P < 0.0001 and 0.002) in cows from the last quartile (51.1 and 31.9%) compared with those from the first quartile (23.7 and 14.7%). In conclusion, the number of COC recovered by OPU (and consequently the ovarian follicular count) can further predict the total number of embryos and pregnancies produced, but it is not directly related to the oocyte development potential. Biotran and Fapemig Project CVZ APQ 01654/09 and BPD 0007/10.

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253 OXIDATIVE STRESS MAY IMPAIR OOCYTE QUALITY IN DAIRY COWS OF REPRODUCTIVE AGE WITH A REDUCED ANTRAL FOLLICLE COUNT

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab253 ◽

2013 ◽

Vol 25 (1) ◽

pp. 274 ◽

Cited By ~ 1

Author(s):

I. Tessaro ◽

F. Franciosi ◽

V. Lodde ◽

D. Corbani ◽

A. M. Luciano ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Antral Follicle ◽

Oocyte Quality ◽

Antral Follicle Count ◽

Oxide System ◽

Reproductive Age ◽

Developmental Competence ◽

Mitochondrial Activity

In dairy cattle, oocytes isolated from ovaries with a reduced antral follicle count (AFC) have a low embryonic developmental competence. This may be related to oxidative stress, as indicated by our recent finding that ovaries with reduced AFC show a defective endothelial nitric oxide synthase/nitric oxide system. To further test this hypothesis, we evaluated whether the poor developmental competence of these oocytes was possibly due 1) to an imbalance of the reduced glutathione (GSH) system, because GSH is the major antioxidant compound stored within the oocyte and protects the zygote and early embryos from oxidative damage, and 2) to reduced mitochondrial activity. Ovaries were obtained from the abattoir, and oocytes were collected from ovaries with reduced AFC, with fewer than 10 follicles of 2 to 6 mm in diameter, and aged-matched controls, with more than 10 follicles of 2 to 6 mm in diameter. Oocyte GSH content was evaluated using the 5,5′-dithio-bis(2-nitrobenzoic acid)-GSH reductase recycling micro-GSH assay before and after in vitro maturation (IVM) in the presence or absence of 100 µM cysteamine, a GSH precursor. At the same time the developmental competence after IVF was assessed. Moreover, the mitochondrial activity during IVM was evaluated in additional oocytes from the two ovarian categories by specific MitoTracker dyes (MitoTracker FM Green and MitoTracker Orange CMTMRos, Invitrogen, Carlsbad, CA, USA) and subsequent image analysis (ImageJ software). All data were analysed by ANOVA followed by Fisher’s least significant differences test, and P-values <0.05 were considered significant. Experiments were repeated at least three times. Oocytes isolated from ovaries with a low AFC had a similar GSH content compared with oocytes isolated from control ovaries (n = 65 and 85, respectively; 4.31 ± 0.41 v. 4.51 ± 0.42 pmol oocyte–1). After IVM, oocytes from ovaries with reduced AFC showed a significantly lower GSH content compared with control oocytes (n = 55 and 65, respectively; 4.36 ± 0.31 v. 6.59 ± 0.39 pmol oocyte–1); however, cysteamine supplementation during IVM induced GSH accumulation similar to the control (n = 80 and 85, respectively; 9.88 ± 0.77 v. 10.45 ± 0.88 pmol oocyte–1). It is interesting that the increase in intracellular GSH content significantly improved the developmental competence of oocytes from ovaries with a reduced AFC (n = 196 and 201, respectively; 20.1 ± 2.9% v. 6.2 ± 1.6%), although the blastocyst rate remained lower than the control either with or without cysteamine (n = 218 and 212, respectively; 33.3 ± 3.8% and 34.2 ± 2.4%). Further, immature oocytes from ovaries with a low AFC showed a reduced mitochondrial membrane potential compared with control oocytes (n = 13 and 18, respectively; 1.74 ± 1.19 v. 2.22 ± 1.72, calculated as the ratio between the fluorescence of active and total mitochondria), whereas at the end of IVM, it declined in both categories at a comparable level (n = 17 and 24, respectively; 1.19 ± 0.10 and 1.30 ± 0.06). Our data confirmed the hypothesis that both the GSH imbalance and defective mitochondrial activity contribute to the limited developmental competence of oocytes from ovaries with a reduced AFC. This work was supported by Dote ricerca applicata-FSE, Regione Lombardia, Italy (VL, IT).

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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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143 TREATMENT WITH VITAMINS A AND E IMPROVE OOCYTE QUALITY AND IN VITRO EMBRYO DEVELOPMENT IN BOS INDICUS COWS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab143 ◽

2011 ◽

Vol 23 (1) ◽

pp. 175 ◽

Cited By ~ 2

Author(s):

J. J. F. Evangelista ◽

C. E. A. Souza ◽

M. E. A. Moraes ◽

A. A. A. Moura

Keyword(s):

Embryo Development ◽

Bos Indicus ◽

Oocyte Quality ◽

Positive Influence ◽

Early Embryo ◽

Oocyte Yield ◽

The Impact ◽

Positive Effect ◽

Paired T Test

We assessed the impact of a single intra-muscular injection of vitamins A and E on oocyte quality and yield and early embryo development in Bos indicus cows. Twenty Bos indicus cows, of Gyr, Brahman, and Nellore breeds, weighing between 450 and 600 kg were subjected to ovum pick-up (OPU). Oocytes were collected in Dulbecco’s PBS (DBPS) containing heparin and antibiotics, counted, and morphologically classified. Viable oocytes were taken to the laboratory, in vitro matured for 24 h and in vitro fertilized using 25 million sperm mL–1. After 168 h of incubation (39°C, 5% CO2), viable embryos were counted and classified. Then, after 10 days, the cows received an intra-muscular injection of 1 000 000 UI of vitamin A and 1 g of vitamin E, and, after 12 days, were again subjected to the same procedure described above. Differences in oocyte yield and embryo development were analysed using paired t-test. The 40 OPUs from 20 cows yielded a total of 520 oocytes. Nellore and Brahman cows produced more embryos/cow (P < 0.01) compared with Gyr. After vitamin treatment, the cows produced more oocytes (n = 303; P < 0.01) compared with the previous OPU (n = 217), resulting in 95 more viable oocytes (31%). Brahman, Gyr, and Nellore cows yielded 11.2 ± 1.8, 8.5 ± 1.5, and 12.0 ± 2.6 oocytes before vitamin treatment, respectively. From those oocytes, 224 embryos were obtained, 89 before vitamin injection and 135 after treatment (P < 0.02), with 36 more embryos (40%) produced. Irrespective of breed, cows responded equally to vitamin injection. A single parenteral injection of vitamins A and E had a significant positive effect on oocyte yield after OPU and in vitro embryo development on Bos indicus cows. We suggest that this effect is probably due to the positive influence of retinoids on oocyte and embryo development.

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