Maternal contributions to pregnancy success: from gamete quality to uterine environment

The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.(AU)

Effect of follicle size on oocytes recovery rate, quality, and in-vitro developmental competence in Bos indicus cows

The objective of the present study was to determine the effect of follicle size on recovery rate, quality, and in-vitro developmental competence of oocytes in Bos indicus cows. The ovaries (n = 507) of Bos indicus cows having age of 5-8 years, with mixed parity, BCS 2.75 ± 0.25, and clinically normal reproductive tracts were collected from the local abattoir. The follicles on the ovaries were divided into two groups based upon their size; 1) ≥6 mm diameter, and 2) <6 mm diameter. After initial evaluation of quality of the oocytes, the COCs were in vitro matured, fertilized, and cultured to determine the in vitro developmental competence. The oocyte recovery, quality, maturation, cleavage, 4-cell, 8-cell, and 16-cell stages were analyzed using PROC GLIMMIX procedure of SAS. However, the number of oocytes recovered per ovary was analyzed using MIXED procedure of SAS. Results revealed that the recovery of oocytes (LSM ± SEM) derived from the follicles having size <6 mm per ovary was greater (1.02 vs. 3.14 ± 0.13; P < 0. 0001). However, the percentage (n/n) recovery [69.8 (474/679) vs. 62.7% (1454/2320); P = 0.01] and grade I_+_II oocytes [68.4 (324/474) vs. 57.9% (842/1454); P < 0.0001] was greater in ≥6 mm as compared with <6 mm group, respectively. However, maturation rate did not differ [92.9 (288/310) vs. 92.2% (296/321); P = 0.98] between the groups. In contrast, cleavage rate [58.1 (180/310) vs. 47.4% (152/321); P = 0.01], the 4-cell [34.5 (107/310) vs. 18.7% (60/321); P = 0.0003], 8-cell [15.5 (48/310) vs. 7.8% (25/321); P = 0.008], and 16-cell [8.7 (27/310) vs. 2.1% (7/321); P = 0.004] stage embryos were greater in ≥6 mm group. It can be concluded that oocytes derived from follicle ≥6 mm have better in vitro developmental competence based on embryonic conversion in Bos indicus cows.

Effect of follicle size on oocytes recovery rate, quality, and in-vitro developmental competence in Bos indicus cows

The objective of the present study was to determine the effect of follicle size on recovery rate, quality, and in-vitro developmental competence of oocytes in Bos indicus cows. The ovaries (n = 507) of Bos indicus cows having age of 5-8 years, with mixed parity, BCS 2.75 ± 0.25, and clinically normal reproductive tracts were collected from the local abattoir. The follicles on the ovaries were divided into two groups based upon their size; 1) ≥6 mm diameter, and 2) <6 mm diameter. After initial evaluation of quality of the oocytes, the COCs were in vitro matured, fertilized, and cultured to determine the in vitro developmental competence. The oocyte recovery, quality, maturation, cleavage, 4-cell, 8-cell, and 16-cell stages were analyzed using PROC GLIMMIX procedure of SAS. However, the number of oocytes recovered per ovary was analyzed using MIXED procedure of SAS. Results revealed that the recovery of oocytes (LSM ± SEM) derived from the follicles having size <6 mm per ovary was greater (1.02 vs. 3.14 ± 0.13; P < 0. 0001). However, the percentage (n/n) recovery [69.8 (474/679) vs. 62.7% (1454/2320); P = 0.01] and grade I_+_II oocytes [68.4 (324/474) vs. 57.9% (842/1454); P < 0.0001] was greater in ≥6 mm as compared with <6 mm group, respectively. However, maturation rate did not differ [92.9 (288/310) vs. 92.2% (296/321); P = 0.98] between the groups. In contrast, cleavage rate [58.1 (180/310) vs. 47.4% (152/321); P = 0.01], the 4-cell [34.5 (107/310) vs. 18.7% (60/321); P = 0.0003], 8-cell [15.5 (48/310) vs. 7.8% (25/321); P = 0.008], and 16-cell [8.7 (27/310) vs. 2.1% (7/321); P = 0.004] stage embryos were greater in ≥6 mm group. It can be concluded that oocytes derived from follicle ≥6 mm have better in vitro developmental competence based on embryonic conversion in Bos indicus cows.(AU)

Expressão gênica em ovócitos suínos de diferentes classificações morfológicas / Gene expression in swine oocytes in different morphological classifications

A produção in vitro de embriões suínos tem alcançado resultados insatisfatórios: ovócitos maturados in vivo produzem uma porcentagem maior de embriões em relação aos maturados in vitro. O sucesso da maturação in vitro está diretamente relacionado com a competência ovocitária. Somente ovócitos competentes são capazes de serem fecundados e terem desenvolvimento embrionário normal. A competência ovocitária pode ser avaliada por vários parâmetros. Recentemente têm sido utilizados como parâmetro os estudos da expressão de genes associados com a competência. O presente trabalho teve por objetivo avaliar diferenças na expressão dos genes BMP15, RYBP, MATER e ZAR1 em ovócitos imaturos de diferentes classes morfológicas, sendo elas: 1, 2, 3 e 4, com a finalidade de proporcionar importantes marcadores moleculares relacionados com a capacidade ovocitária. O RNA total dos ovócitos foi extraído e utilizado como molde para a síntese da primeira fita de cDNA. Os resultados da expressão gênica foram analisados utilizando-se modelo misto, considerando os dados de expressão gênica variável dependente e as classes ovocitárias variáveis independentes. Os genes BMP15, ZAR1 e RYBP apresentaram expressão semelhante nas classes ovocitárias 1, 2 e 3; somente a categoria 4 diferiu na expressão desses genes (P<0,05). O gene MATER foi expresso de forma semelhante em todas as classes ovocitárias estudadas (P>0,05). A técnica de RT-qPCR foi eficiente para detecção desses transcritos em ovócitos de diferentes classes. No entanto, para melhor entendimento do envolvimento desses transcritos na aquisição da competência ovocitária, são necessários mais estudos avaliando ovócitos de diferentes classes morfológicas, em diferentes fases de desenvolvimento, e implicação de outros genes envolvidos com a competência ovocitária.(AU)

The in vitro production of pig embryos has achieved unsatisfactory results; in vivo matured oocytes produce a higher percentage of embryos compared to in vitro maturation. The success of in vitro maturation is directly related to oocyte competence. Only competent oocytes are capable of being fertilized and have normal embryonic development. The oocyte competence can be assessed using several parameters. Recently these parameters have been used for gene expression studies associated with competence. This work aimed to evaluate differences in gene expression BMP15, RYBP, MATER, ZAR1 as endogenous control and the constitutive gene GAPDH in immature oocytes of different morphological classes which are: 1, 2, 3 and 4, in order to provide significant molecular markers linked to the ability of development. Oocytes Total RNA was extracted and used as a template for synthesis of the first cDNA strand. The results of gene expression were analyzed using a mixed model, considering the dependent gene expression data and independent ovocitary variable classes. The genes BMP15, RYBP ZAR1 and showed similar ovocitary expression in classes 1, 2 and 3 differ only in category 4 in their expression (P<0.05). The MATER gene was similarly expressed in all ovocitary classes studied (P>0.05). The RTQ-PCR technique was effective for detection of these transcripts in oocytes from different classes. However, for better understanding of the involvement of these transcripts in the acquisition of oocyte competence more studies are needed to evaluate different morphological classes of oocytes at different stages of development and the implication of other genes involved in oocyte competence.(AU)

Molecular markers of fertility in cattle oocytes and embryos progress and challenges

In order for assisted reproduction technologies to improve, better methods to discern eggs and embryos according to their level of developmental competence are urgently required to substitute or complement the subjective morphological selection criteria still broadly in use. Objective and reliable molecular markers of viability have been studied during the last decades as robust options to select the best oocytes and embryos for embryo transfer programs. These molecular methodologies rely mostly on the novel “OMICS” technologies. Among these, transcriptomics is the primary platform applied so far in animal breeding research mainly due to the possibility to amplify small samples. In addition, biomarkers of competence have not only been instrumental to select the best oocytes and embryos for reproductive technologies, but have also shed light on the intricate molecular physiology leading to the acquisition of developmental capacity within the ovary and on how the embryo manifests this potential during culture. Nevertheless, such molecular profiling usually implies the destruction of the oocyte/ embryo hereby preventing the practical use of biomarkers in in vitro embryo production and transfer systems. Alternatively , encouraging results have been lately obtained from non-invasive technologies based on biopsies of follicular somatic cells surrounding the developing egg, as well as from metabolic analysis of follicular fluid or spent culture media. This work summarizes the achievements of recent years in the field of biomarkers of competent bovine embryos. The main challenges will be exposed, while the future guidelines will help to comprehend why biomarkers of developmental competence appear promising to take us steps forward in the amelioration of reproductive technologies.

Molecular markers of fertility in cattle oocytes and embryos progress and challenges

In order for assisted reproduction technologies to improve, better methods to discern eggs and embryos according to their level of developmental competence are urgently required to substitute or complement the subjective morphological selection criteria still broadly in use. Objective and reliable molecular markers of viability have been studied during the last decades as robust options to select the best oocytes and embryos for embryo transfer programs. These molecular methodologies rely mostly on the novel “OMICS” technologies. Among these, transcriptomics is the primary platform applied so far in animal breeding research mainly due to the possibility to amplify small samples. In addition, biomarkers of competence have not only been instrumental to select the best oocytes and embryos for reproductive technologies, but have also shed light on the intricate molecular physiology leading to the acquisition of developmental capacity within the ovary and on how the embryo manifests this potential during culture. Nevertheless, such molecular profiling usually implies the destruction of the oocyte/ embryo hereby preventing the practical use of biomarkers in in vitro embryo production and transfer systems. Alternatively , encouraging results have been lately obtained from non-invasive technologies based on biopsies of follicular somatic cells surrounding the developing egg, as well as from metabolic analysis of follicular fluid or spent culture media. This work summarizes the achievements of recent years in the field of biomarkers of competent bovine embryos. The main challenges will be exposed, while the future guidelines will help to comprehend why biomarkers of developmental competence appear promising to take us steps forward in the amelioration of reproductive technologies.(AU)

Improving oocyte quality in cows and heifers - What have we learned so far?

The major challenge in assisted reproduction technologies remains producing oocytes with optimal developmental competence. Such developmental competence can be defined as the ability of the oocyte to fulfill maturation, undergo su ccessful fertilization, reach the blastocyst stage and yi eld a viable and healthy progeny. The main follicular parameters linked to oocyte competence are presented in this review: follicle size, health/atresia status, effects of superstimulation and level of differentiation. In a commercial environment, exogenous FSH superstimulation combined with FSH starvation (referred to as the coasting period) and ovum- pick up followed by in vitro maturation and fertilization, leads to maximal blastocyst rates in Bos taurus heifers and cows. This coasting period increases the proportion of medium-to-large size follicles and may induce slight atresia in the follicular cells, which improves developmental competence. The optimal period for oocyte retrieval in this context has been recently characterized in cows and is re lated to follicular size and state of differentiation. Apoptosis related signals have been identified in various stud ies as implicated in antral folliculogenesis. Follicular somatic cell transcriptome studies are currently used to identify competence related markers. Finally, oocyte competence acquisition is a complex and dynamic process. The best oocytes are obtained from follicles of the optimal size and differentiation status, which can be manipulated with the right hormonal regimen.

Improving oocyte quality in cows and heifers - What have we learned so far?

The major challenge in assisted reproduction technologies remains producing oocytes with optimal developmental competence. Such developmental competence can be defined as the ability of the oocyte to fulfill maturation, undergo su ccessful fertilization, reach the blastocyst stage and yi eld a viable and healthy progeny. The main follicular parameters linked to oocyte competence are presented in this review: follicle size, health/atresia status, effects of superstimulation and level of differentiation. In a commercial environment, exogenous FSH superstimulation combined with FSH starvation (referred to as the coasting period) and ovum- pick up followed by in vitro maturation and fertilization, leads to maximal blastocyst rates in Bos taurus heifers and cows. This coasting period increases the proportion of medium-to-large size follicles and may induce slight atresia in the follicular cells, which improves developmental competence. The optimal period for oocyte retrieval in this context has been recently characterized in cows and is re lated to follicular size and state of differentiation. Apoptosis related signals have been identified in various stud ies as implicated in antral folliculogenesis. Follicular somatic cell transcriptome studies are currently used to identify competence related markers. Finally, oocyte competence acquisition is a complex and dynamic process. The best oocytes are obtained from follicles of the optimal size and differentiation status, which can be manipulated with the right hormonal regimen.(AU)