Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

Protein source in maturation media affects gene expression in cumulus cells and embryo development in cattle.

We aimed to evaluate if protein source (PS) alterations during IVM affect embryo sex/development and gene expression profile in cumulus cells (CCs). Bovine oocytes were matured and cultured in the presence of FBS or BSA. Then, the PS effect during IVM on gene expression (GPC4, VCAN, GHR, PTGS2, and ALCAM) was determined. CC biopsy was removed before and after IVM treatments. After fertilization and cultured, CCs were grouped according to their fate into CCs from immature COCs, CCs from COCs that did or did not result in embryos (according to PS). Results showed that when the culture was performed in FBS presence, blastocyst rate was higher (p < 0.05) than BSA. However, when embryos were cultured with BSA, no effect (p > 0.05) of PS during IVM was observed. PS used during IVM did not affect embryos sex (p > 0.05) but changed VCAN, GHR, PTGS2, and ALCAM genes expression. No differences (p > 0.05) were observed between immature and mature CCs groups in gene expression, regardless of their fate. Only the GHR gene was related to embryo production but just with FBS on IVM. In conclusion, PS can affect embryo development when using the serum on IVM and IVC, influences CCs gene expression, and has to be considered when studying oocyte quality markers.

Using Cumulus Cell Biopsy as a Non-Invasive Tool to Access the Quality of Bovine Oocytes: How Informative Are They?

The present study aimed to determine whether cumulus cells (CC) biopsy, acquired before or after in vitro maturation (IVM), presents similar gene expression pattern and if would compromises oocyte quality. First, immature cumulus oocyte complexes (COCs) were distributed: (1) maturated in groups (control); (2) individually maturated, but not biopsied; (3) subjected to CC biopsy before maturation and individually matured; (4) individually matured and submitted to CC biopsy after maturation; (5) individually matured and CC biopsied before and after maturation. Secondly, candidate genes, described as potential markers of COCs quality, were quantified by RT-qPCR in CCs before and after IVM. After in vitro fertilization (IVF), zygotes were tracked and sorted regarding their developmental potential: fully developed to embryo, cleaved and arrested, and not-cleaved. The COC's biopsy negatively affects embryo development (p < 0.05), blastocyst cell number (p < 0.05), and apoptotic cell ratio (p < 0.05), both before and after IVM. The PTGS2, LUM, ALCAM, FSHR, PGR, SERPINE2, HAS2, and PDRX3 genes were differentially expressed (p < 0.05) on matured CCs. Only PGR gene (p = 0.04) was under-expressed on matured CCs on Not-Cleaved group. The SERPINE2 gene was overexpressed (p = 0.01) in the Cleaved group on immature CCs. In summary, none of the selected gene studies can accurately predict COC's fate after fertilization.

Transcriptome of D14 in vivo x in vitro bovine embryos: is there any difference?

It is well-established that in vitro culture affects quality, gene expression, and epigenetic processes in bovine embryos and that trophectoderm cells are the most susceptible to abnormalities. These changes have been reported as the main factors responsible for losses observed after transfer of in vitro-produced embryos. The present study aimed to investigate the effect of an in vitro system on bovine embryo transcriptional profiles on D14 of development. Two groups were used-one with embryos produced in vitro until D7 (day 7; VT group) and another with embryos produced in vivo by hormonal stimulation, with embryos collected on D7 (VV group). D7 embryos at similar developmental stages from both treatments were transferred to recipient uteri and recollected on D14. From D14 embryos of both treatments, trophoblast samples were removed by biopsy for sexing and transcriptome analyses. Embryos were sexed by polymerase chain reaction (PCR), and only males were used for RNA sequencing. In total, 29,005 transcripts were expressed, from which 900 were differentially expressed, but only 29 genes were significantly differentially expressed. In addition, 20 genes were found uniquely for VV and 27 for VT. These findings suggested that although the uterine environment minimized transcriptional differences, it was not able to make trophoblasts from the in vitro embryos similar to the in vivo ones. The few genes exhibiting differences are in control of important events that may be responsible for embryonic losses occurring during the first period of gestation.

Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates.

Assisted reproductive techniques have significantly contributed to animal breeding programs. Similarly, genomics has provided important information and tools to improve the accuracy of selection. However, the greatest benefits of those tools can only be expected when they are combined, allowing animals to be selected accurately early in life. Therefore, obtaining DNA samples from embryos without compromising their viability is essential for the consolidation of preimplantation genomic selection. We aimed to evaluate the effect on the gestation rate of conducting a biopsy of in vivo (VV) and in vitro-produced (IVP) bovine embryos. The VV and IVP embryos were distributed into two groups: VV-B (biopsied embryos; n = 380) and VV-C (intact embryos-controls; n = 229) and IVP-B (biopsied embryos; n = 91) and IVP-C (intact embryos-controls; n = 227), respectively. After biopsy, embryos from both groups VV-B and IVP-B were cultured for an additional 3 hours before being transferred to synchronized recipients. To evaluate the quality of the DNA obtained in the biopsies, this was used to determine the sex of embryos by polymerase chain reaction. No effect (P > 0.05) of the biopsy was observed for any of the treatments, the pregnancy rate at D 60 post-transfer being similar for VV-B: 206/380 (54.21%) and VV-C: 128/229 (55.89%) and for IVP-B: 24/91 (26.37%) and IVP-C: 45/227 (19.82%). Also, no effect (P > 0.05) of the embryo's stage of development was detected on percentage of pregnant recipients when in vitro embryos were transferred. From the biopsies analyzed, about 90% had the sex determined, confirming that DNA was there and it was efficiently amplified. The results indicated that biopsy does not affect the viability of IVV and IVP bovine embryos and can be used in commercial programs to associate assisted reproductive technologies with genomic selection.

Intrafollicular transfer of fresh and vitrified immature bovine oocytes.

Embryo production by intrafollicular oocyte transfer (IFOT) represents an alternative for production of a large number of embryos without requiring any hormones and only basic laboratory handling. We aimed to (1) evaluate the efficiency of IFOT using immature oocytes (IFIOT) and (2) compare embryo development after IFIOT using fresh or vitrified immature oocytes. First, six IFIOTs were performed using immature oocytes obtained by ovum pickup. After insemination and uterine flush for embryo recovery, 21.3% of total transferred structures were recovered excluding the recipient's own oocyte or embryo, and of those, 26% (5.5% of transferred cumulus-oocyte complexes [COCs]) were morula or blastocyst. In the second study, we compared fresh and vitrified-warmed immature COCs. Four groups were used: (1) fresh immature COCs (Fresh-Vitro); (2) vitrified immature COCs (Vit-Vitro), with both groups 1 and 2 being matured, fertilized, and cultured in vitro; (3) fresh immature COCs submitted to IFIOT (Fresh-IFIOT); and (4) vitrified immature COCs submitted to IFIOT (Vit-IFIOT). Cumulus-oocyte complexes (n = 25) from Fresh-IFIOT or Vit-IFIOT groups were injected into dominant follicles (>10 mm) of synchronized heifers. After excluding one structure or blastocyst, the recovery rates per transferred oocyte were higher (P < 0.05) for Fresh-IFIOT (47.6%) than for Vit-IFIOT (12.0%). Blastocyst yield per initial oocyte was higher (P < 0.05) for Fresh-Vitro (42.1%) than for Fresh-IFIOT (12.9%). Vit-Vitro presented higher (P < 0.05) embryo development (6.3%), compared to Vit-IFIOT, which did not result in any extra embryo. Although IFOT did not improve developmental competence of vitrified oocytes, we achieved viable blastocysts and pregnancies produced after IFIOT of fresh bovine immature oocytes. Further work on this technique is warranted as an option both for research studies and for clinical bovine embryo production in the absence of laboratory facilities for IVF.

Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence.

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0-3.0, 3.1-6.0, 6.1-8.0 and >or=8.1 mm. Cumulus-oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription-polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles>6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P<0.05) in oocytes from follicles>or=8.1 mm in diameter than in oocytes from follicles<6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

Quantitative expression of candidate genes for developmental competence in bovine two-cell embryos.

Only competent oocytes are able to undergo complete maturation and normal embryonic development. Therefore, the identification of genes that are differentially expressed in competent oocytes would contribute to our understanding of the factors controlling competency. It is well known that time of cleavage after insemination in vitro is highly correlated with embryonic developmental potential and this can be used to distinguish between oocytes of different quality. The main objective of this study was to identify genes associated with competency and rapid cleavage. We examined the expression of 16 candidate genes (IDH, YEAF Cathepsin B, RAD50, TCP1 NCOR1, HUEL, STK6, ZNF403, AOP2, EEF1A1, Hsp90, Hsp40, AKR1B1, PGRMC1, and DMRT2) in early and late cleaving embryos, by real time PCR. These transcripts were derived from previous study in our laboratory using cDNA coming from a suppressive subtraction hybridization (SSH) between early cleaving versus late cleaving embryos spotted on a microarray slide. Of the 16 genes evaluated, 3 (IDH, YEAF, and H2A) showed statistical difference (P < 0.05) between early and late cleaving embryos. However, some genes such as Cathepsin B (P = 0.0677), RAD50 (P = 0.0899), and TCP1 (P = 0.0824) tended to show higher expression in the early cleaving than in the late cleaving embryo. In conclusion, we have identified three genes (YEAF, IDH, H2A) that were differentially expressed in the early cleaving embryos, and their expression can be associated with greater developmental competence.

Involvement of steroid hormones on in vitro maturation of pig oocytes.

The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus-oocyte complexes (COCs). collected from follicles of 2-5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17beta (0-3000 ng/ml), progesterone (0-5000 ng/ml) and testosterone (0-300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect (P > 0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17beta or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly (P > 0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17beta and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation.

Influence of hormones and follicular fluid on maturation of pig oocytes

To evaluate the effect of follicular fluid on in vitro maturation, pig oocytes were cultured in the presence of hormones where 10 percent of fetal calf serum (FCS), 10 percent of follicular fluid from large follicles (l-pFF), 10 percent of follicular fluid from medium follicles (m-pFF) or no supplement were added. When oocytes where matured in medium containing the hormones the addition of different supplements did not affect (P<0.05) nuclear maturation. However, changing the supplement altered the cytoplasmic maturation, with higher rate (P<0.05) observed in the l-pFF group. To determine the effect of the presence of hormone and/or supplement during maturation, the oocytes were cultured either in presence of TCM-199 alone, with hormones, with 10 percent l-pFF or with hormones and 10 percent l-pFF. The highest proportion of oocytes undergoing nuclear and cytoplasmic maturation was obtained when both hormones and follicular fluid were present. Cumulus expansion had a significant (P<0.05) effect on cytoplasmic maturation with the non-expanded groups showing a lower percentage of maturation in all groups. When the adequacy of gonadotropins levels were evaluated by adding higher or lower concentrations into the maturation medium neither beneficial nor detrimental effects were observed in either nuclear or cytoplasmic maturation. These results suggest that changes in the composition of the medium can alter the percentage of oocytes completing maturation. Follicular fluid combined with hormones was found to give better conditions for pig oocyte maturation in vitro.

O presente estudo avaliou o efeito da presença do líquido folicular e hormônios durante a maturação nuclear e citoplasmática em ovócitos suínos. Para avaliar o efeito do líquido folicular, os oócitos foram maturados in vitro em TCM-199 na presença de hormônios, em que o meio foi suplementado com 10 por cento de soro fetal bovino (SFB), 10 por cento de líquido folicular de folículos grandes (l-pFF), 10 por cento de líquido folicular de folículos médios (m-pFF) ou nenhum suplemento. Quando os oócitos foram maturados no meio contendo hormônios, a adição de diferentes suplementos não afetou (P<0,05) a maturação nuclear. Entretanto, mudanças no suplemento provocaram uma alteração na maturação citoplasmática, sendo que as maiores taxas (P<0,05) foram observadas no grupo com l-pFF. Para determinar o efeito da presença de hormônios e/ou suplemento durante a maturação, os oócitos foram cultivados em TCM-199, TCM-199 com hormônios, TCM-199 com 10 por cento l-pFF ou TCM-199 com hormônio e 10 por cento l-pFF. A maior proporção de oócitos que sofreram maturação nuclear e citoplasmática ocorreu no grupo em que ambos, hormônios e líquido folicular, estavam presentes. A expansão do cumulus influenciou (P<0,05) na maturação citoplasmática, sendo que os oócitos sem expansão apresentaram menor taxa de maturação em todos os grupos. Quando diferentes concentrações de gonadotrofinas foram utilizadas durante a maturação, não foi observado nenhum efeito na maturação nuclear ou citoplasmática. Os resultados deste estudo sugerem que as mudanças na composição do meio podem afetar as taxas de maturação, sendo que o líquido folicular, combinado com hormônios, proporciona as melhores condições para maturação de oócitos suínos.

Efeito estacional sobre características ovarianas e produçäo de oócitos em vacas Bos indicus no Mato Grosso do Sul / Seasonality of ovarian characteristics and oocytes production in Bos indicus cattle in Mato Grosso do Sul

Verificou-se o efeito de duas distintas estaçöes do ano (seca e chuvosa) sobre algumas características ovarianas em vacas Bos indicus abatidas na regiäo de Campo Grande, MS. Ovários (n = 10) foram obtidos nos meses de novembro e dezembro de 1998 e de janeiro a outubro de 1999. No laboratório, os ovários foram avaliados quanto ao peso (g), volume (Vol. (cm³) = 3/4 p x comprimento/2 x largura/2 x espessura/2), número de corpos lúteos, número de folículos com > 9 mm de diâmetro, número total de folículos com menos de 9 mm, número de oócitos, oócitos viáveis e oócitos degenerados. O efeito principal da estaçäo (seca ou chuvosa) foi estimado pela análise de variância (teste t), para modelos completamente ao acaso. Utilizou-se a análise da correlaçäo simples entre as variáveis estudadas, ajustadas para o efeito da estaçäo. Os resultados revelaram que o peso dos ovários (5,1 x 6,5 g), folículos totais (10,1 x 13,7), corpos lúteos (0,32 x 0,47, p < 0,05) e a percentagem de oócitos viáveis (19,6 por cento x 35,6 por cento) sobre o total de oócitos variaram significativamente (p < 0,01) entre as estaçöes seca e chuvosa, respectivamente. A análise da correlaçäo (r) mostrou coeficientes significativos (p < 0,01) entre peso e volume (r = 0,78), peso e total de folículos (r = 0,32), peso e corpos lúteos (r = 0,41), total de folículos e oócitos viáveis (r = 57), entre outros. Concluiu-se que importantes modificaçöes na funçäo ovariana, com base na produçäo e qualidade dos oócitos, podem ser estimadas entre a estaçäo seca e chuvosa. Com base nestas características, a estaçäo chuvosa torna-se mais favorável para a implantaçäo de programas reprodutivos em rebanhos comerciais